Population 9,910,000 9,880,000 9,860,000\*

**3. Prevalence of carbon monoxide poisoning in Hungary**

circumstances [2].

355 in 2015.

2013, 2014, and 2015.

\*Source: Hungarian Central Statistics Office.

**Table 1.** Rates of CO poisoning and deaths from 2013 to 2015.

CO enters human body via breathing and gas exchange, furthermore CO is also generated endogenously in small amounts during oxygen consumption in healthy subjects. The hemoxidase enzyme, responsible for the catabolism of hemoglobin into biliverdine, is responsible for endogenous CO production.

CO has higher affinity in binding to hemoglobin then oxygen, thus development of carboxyhemoglobin (CO-Hgb) is responsible for the major signs and symptoms, and late complications associated with CO intoxication such as cardiovascular and neurological complications due to hypoxia which are often lethal.

The aim of this chapter is to summarize the toxicokinetics, epidemiology, pathophysiology, signs and symptoms, diagnosis, differential diagnosis and treatment options of carbon monoxide poisoning. In doing so, a case report involving a whole family that suffered severe carbon monoxide intoxication will be presented including the story of a successful management of a pregnant woman (one of the family members) who underwent an urgent cesarean section to protect the baby from carbon monoxide intoxication. This will help to elaborate on treatment options to be carried out pre- and during hospitalization; and discuss the merits of hyperbaric oxygen therapy.

## **2. Toxicokinetics of carbon monoxide**

CO is formed as a by-product of burning organic compounds; that is why poisoning by CO is common during power outages due to storms, as a result of the improper use of gasoline-powered portable generators to provide electricity and indoor use of charcoal briquettes for cooking and heating. Other sources of CO include improperly vented gas water heaters, kerosene space heaters, charcoal grills, malfunctioning or obstructed exhaust systems stoves, portable heaters, fires, cigarette smoke, and automobile exhausts.

In a healthy adult alveolar CO concentration, during one cigarette smoke is increased to 400–500 ppm. Nonsmoker individual in the same room is also exposed to CO, resulting 25–100 ppm alveolar concentration [1].

CO is absorbed and eliminated through the lungs. The amounts inhaled and exhaled are dependent on the alveolar-capillary pressure gradient of oxygen and alveolar diffusion. CO intoxication also occurs by inhalation of methylene chloride vapors, a volatile liquid found in degreasers, solvents, and paint removers. The liver metabolizes as much as onethird of inhaled methylene chloride to CO. A significant percentage of methylene chloride is stored in the tissues, and continued release results in elevated CO levels for at least twice as long as with direct CO inhalation. The half-time of CO in a healthy adult takes 4 hours breathing on air, 1.5 hours breathing on 100% oxygen, and 20 minutes in hyperbaric oxygen circumstances [2].

## **3. Prevalence of carbon monoxide poisoning in Hungary**

gas. CO diffuses through general building constructions (brick and wood). Generally, atmospheric concentration of CO is low, however, in urban and industrial regions it may be elevated. Poisoning usually occurs via impaired operating heating and mechanical systems and

74 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

CO enters human body via breathing and gas exchange, furthermore CO is also generated endogenously in small amounts during oxygen consumption in healthy subjects. The hemoxidase enzyme, responsible for the catabolism of hemoglobin into biliverdine, is responsible for

CO has higher affinity in binding to hemoglobin then oxygen, thus development of carboxyhemoglobin (CO-Hgb) is responsible for the major signs and symptoms, and late complications associated with CO intoxication such as cardiovascular and neurological complications

The aim of this chapter is to summarize the toxicokinetics, epidemiology, pathophysiology, signs and symptoms, diagnosis, differential diagnosis and treatment options of carbon monoxide poisoning. In doing so, a case report involving a whole family that suffered severe carbon monoxide intoxication will be presented including the story of a successful management of a pregnant woman (one of the family members) who underwent an urgent cesarean section to protect the baby from carbon monoxide intoxication. This will help to elaborate on treatment options to be carried out pre- and during hospitalization; and discuss the merits of

CO is formed as a by-product of burning organic compounds; that is why poisoning by CO is common during power outages due to storms, as a result of the improper use of gasoline-powered portable generators to provide electricity and indoor use of charcoal briquettes for cooking and heating. Other sources of CO include improperly vented gas water heaters, kerosene space heaters, charcoal grills, malfunctioning or obstructed exhaust systems stoves, portable heaters, fires, cigarette smoke, and automo-

In a healthy adult alveolar CO concentration, during one cigarette smoke is increased to 400–500 ppm. Nonsmoker individual in the same room is also exposed to CO, resulting

CO is absorbed and eliminated through the lungs. The amounts inhaled and exhaled are dependent on the alveolar-capillary pressure gradient of oxygen and alveolar diffusion. CO intoxication also occurs by inhalation of methylene chloride vapors, a volatile liquid found in degreasers, solvents, and paint removers. The liver metabolizes as much as onethird of inhaled methylene chloride to CO. A significant percentage of methylene chloride

fire emergencies.

endogenous CO production.

hyperbaric oxygen therapy.

bile exhausts.

**2. Toxicokinetics of carbon monoxide**

25–100 ppm alveolar concentration [1].

due to hypoxia which are often lethal.

Carbon monoxide intoxication cases have been documented in Hungary from 2012. Hence, as shown in **Figure 1**, the total number of cases was, respectively, 235 in 2013, 375 in 2014, and 355 in 2015.

**Figure 1.** Prevalence of carbon monoxide poisoning in Hungary. Figure shows total number of cases and lethal cases in 2013, 2014, and 2015.


**Table 1.** Rates of CO poisoning and deaths from 2013 to 2015.

These figures translate to 2.37–3.80 cases per 100,000 people per year. Since, the number of deaths from CO poisoning were, respectively, 14 in 2013, 13 in 2014, and 12 in 2015, the case fatality rates have been decreasing from 5.96 in 2013 to 3.38 in 2015 (**Table 1**). These figures when compared to other countries such the USA, they appear to below [3].

The decrease in the case of fatality rates can be explained not only by the decreasing population, but also by the efforts of the Hungarian National Ambulance that successfully initiated a widespread campaign to increase awareness of CO intoxication. In this project, education focuses on safe handling of household heating systems, importance of regular, controlled, and authorized servicing of equipment and the use of CO level detector devices.

## **4. Pathophysiology**

Carbon monoxide poisoning leads to impaired oxygen delivery and utilization at the cellular level. In doing so, it affects several organs including the brain, heart, and other organs with the highest oxygen requirement. It causes cellular hypoxia by impedance of oxygen delivery as it reversibly binds hemoglobin, resulting in relative functional anemia. Because it binds hemoglobin 230–270 times more avidly than oxygen, even small concentrations can result in significant levels of carboxyhemoglobin (HbCO).

to myoglobin than oxygen. This results in more severe peripheral myogenic ischemia and

**Figure 2.** Effects of CO poisoning on hemoglobin-oxygen dissociation curve. In response to CO intoxication the Hgb-O<sup>2</sup>

dissociation curve is shifted to the left, physiological sigmoid shape of the curve is impaired, and tissue O2

%: oxygen saturation, PaO2

compensate tissue hypoxia are lacking. CO also binds to the cytochrome oxidase and NADPH systems at high concentration or long time exposure poisoning. This causes impaired mito-

Signs and symptoms of CO intoxication depend on the CO concentration, thus the CO-Hgb levels. **Table 2** provides a list of general symptoms depending on gas concentration and CO-Hgb levels. Generally, tissue hypoxemia dominates and causes the most severe complications in the cardiovascular and central nervous system. Physical signs of CO intoxication lack unique features. CO-Hgb may cause cherry-pink color on skin, however it only occurs in high levels of CO-Hgb, when the accompanied cyanosis usually lighten cherry-pink sign. Further

Co-Hgb and CO-myoglobin impair myocardial oxygen delivery. Ischemic myocardium is a severe complication in CO poisoning. Clinical features include decreased inotrope function of the left ventricle, accompanied by hypotension and hemodynamic instability. Furthermore, arrhythmogenic complication occurs in one-third of CO-intoxicated patients. Myocardial tissue hypoxia can trigger atrial and ventricular arrhythmias. Malignant ventricular arrhythmia (ventricular tachycardia or ventricular fibrillation) may occur at high CO-Hgb levels, which

pressure is normal, thus forced respiratory responses to

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: partial oxygen pressure.

delivery is

77

also myocardial ischemia. Partial O2

decreased. CO: carbon monoxide, SpO2

**5. Signs and symptoms**

chondrial activity and cellular energy development.

organ specific signs and symptoms are discussed below.

**5.1. Cardiovascular signs and symptoms**

Several studies have indicated that CO may cause brain lipid peroxidation and leukocytemediated inflammatory changes in the brain, a process that may be stopped by hyperbaric oxygen therapy. Studies have demonstrated release of nitric oxide free radicals from platelet and vascular endothelium, following exposure to CO concentrations of 100 ppm. One study suggests a direct toxicity of CO on myocardium that is separate from the effect of hypoxia [4].

It should be noted that the severity of CO poisoning depends on Ref. [5]:


Exogenous CO intake is regulated via alveolar-capillary diffusion rate. Alveolar-capillary diffusion is dependent on the permeability of the alveolar membrane and from the hemoglobin concentration of alveolar capillaries. Diffusion efficacy of CO in the alveoli is 80%, similarly as for oxygen. The hemoglobin binding efficacy of CO is 200–250 times greater than those of oxygen. Thus, in the presence of CO, oxygen-hemoglobin binding is impaired, however partial oxygen pressures are normal. Main pathological step behind CO intoxication and the related organ dysfunction is the severe tissue hypoxia, caused by elevated levels of CO-Hgb. The hemoglobin-oxygen dissociation curves are shifted to the left, meaning that less oxygen can bind to Hgb at the same partial oxygen pressure levels and CO-Hgb cannot deliver oxygen to the peripheral tissues effectively (**Figure 2**). Furthermore, CO also has higher binding efficacy

**Figure 2.** Effects of CO poisoning on hemoglobin-oxygen dissociation curve. In response to CO intoxication the Hgb-O<sup>2</sup> dissociation curve is shifted to the left, physiological sigmoid shape of the curve is impaired, and tissue O2 delivery is decreased. CO: carbon monoxide, SpO2 %: oxygen saturation, PaO2 : partial oxygen pressure.

to myoglobin than oxygen. This results in more severe peripheral myogenic ischemia and also myocardial ischemia. Partial O2 pressure is normal, thus forced respiratory responses to compensate tissue hypoxia are lacking. CO also binds to the cytochrome oxidase and NADPH systems at high concentration or long time exposure poisoning. This causes impaired mitochondrial activity and cellular energy development.

## **5. Signs and symptoms**

These figures translate to 2.37–3.80 cases per 100,000 people per year. Since, the number of deaths from CO poisoning were, respectively, 14 in 2013, 13 in 2014, and 12 in 2015, the case fatality rates have been decreasing from 5.96 in 2013 to 3.38 in 2015 (**Table 1**). These figures

The decrease in the case of fatality rates can be explained not only by the decreasing population, but also by the efforts of the Hungarian National Ambulance that successfully initiated a widespread campaign to increase awareness of CO intoxication. In this project, education focuses on safe handling of household heating systems, importance of regular, controlled, and authorized servicing of equipment and the use of CO level detector

Carbon monoxide poisoning leads to impaired oxygen delivery and utilization at the cellular level. In doing so, it affects several organs including the brain, heart, and other organs with the highest oxygen requirement. It causes cellular hypoxia by impedance of oxygen delivery as it reversibly binds hemoglobin, resulting in relative functional anemia. Because it binds hemoglobin 230–270 times more avidly than oxygen, even small concentrations can result in

Several studies have indicated that CO may cause brain lipid peroxidation and leukocytemediated inflammatory changes in the brain, a process that may be stopped by hyperbaric oxygen therapy. Studies have demonstrated release of nitric oxide free radicals from platelet and vascular endothelium, following exposure to CO concentrations of 100 ppm. One study suggests a direct toxicity of CO on myocardium that is separate from the effect of hypoxia [4].

Exogenous CO intake is regulated via alveolar-capillary diffusion rate. Alveolar-capillary diffusion is dependent on the permeability of the alveolar membrane and from the hemoglobin concentration of alveolar capillaries. Diffusion efficacy of CO in the alveoli is 80%, similarly as for oxygen. The hemoglobin binding efficacy of CO is 200–250 times greater than those of oxygen. Thus, in the presence of CO, oxygen-hemoglobin binding is impaired, however partial oxygen pressures are normal. Main pathological step behind CO intoxication and the related organ dysfunction is the severe tissue hypoxia, caused by elevated levels of CO-Hgb. The hemoglobin-oxygen dissociation curves are shifted to the left, meaning that less oxygen can bind to Hgb at the same partial oxygen pressure levels and CO-Hgb cannot deliver oxygen to the peripheral tissues effectively (**Figure 2**). Furthermore, CO also has higher binding efficacy

It should be noted that the severity of CO poisoning depends on Ref. [5]:

• Alveolar-capillary diffusion parameters (e.g., general pulmonology status).

• Accompanied illnesses and general condition of the patient.

when compared to other countries such the USA, they appear to below [3].

76 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

devices.

**4. Pathophysiology**

• The time of the exposure.

• The concentration of inhaled CO gas.

significant levels of carboxyhemoglobin (HbCO).

Signs and symptoms of CO intoxication depend on the CO concentration, thus the CO-Hgb levels. **Table 2** provides a list of general symptoms depending on gas concentration and CO-Hgb levels. Generally, tissue hypoxemia dominates and causes the most severe complications in the cardiovascular and central nervous system. Physical signs of CO intoxication lack unique features. CO-Hgb may cause cherry-pink color on skin, however it only occurs in high levels of CO-Hgb, when the accompanied cyanosis usually lighten cherry-pink sign. Further organ specific signs and symptoms are discussed below.

#### **5.1. Cardiovascular signs and symptoms**

Co-Hgb and CO-myoglobin impair myocardial oxygen delivery. Ischemic myocardium is a severe complication in CO poisoning. Clinical features include decreased inotrope function of the left ventricle, accompanied by hypotension and hemodynamic instability. Furthermore, arrhythmogenic complication occurs in one-third of CO-intoxicated patients. Myocardial tissue hypoxia can trigger atrial and ventricular arrhythmias. Malignant ventricular arrhythmia (ventricular tachycardia or ventricular fibrillation) may occur at high CO-Hgb levels, which


Pathophysiology of CO intoxication of the fetus includes:

**3.** Fetal-Hgb binds CO 172 times more than O2

**7. Diagnosis and differential diagnosis**

higher than maternal CO-Hgb.

transport via the placenta is impaired in correlation with the elevated CO-Hgb

(lower affinity than adult Hgb), albeit CO bind-

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measure-

**2.** CO enters fetal circulation via passive diffusion or facilitated diffusion through the placenta and forms fetal-CO-Hgb, further worsening fetal hypoxemia. Placental CO diffusion is dependent on gestation age and weight of the fetus. In later stage pregnancy, placental diffusion capacity increases and CO intoxication are more severe on the fetus. Many cases presented in the literature lethal, in utero CO intoxication in pregnancies close to terminus [11].

ing capacity is longer in time than in adult Hgb. Usually fetal CO-Hgb levels are 10–15%

Given the vagueness and the broad spectrum of complaints, misdiagnosis of carbon monoxide (CO) toxicity is common. Hence, it is necessary to specifically inquire about possible exposures when considering the diagnosis. In some nonfire-related incidents, the most common symptoms were headaches (37%) followed by dizziness (18%), and nausea (17%) [10]. When a patient has a history compatible with CO exposure and when more than one patient in a group or household presents with similar complaints, the following symptoms have been noted: malaise, flu-like symptoms, fatigue, dyspnoea on exertion, chest pain, palpitations, lethargy, confusion, hallucination, agitation, visual disturbance, syncope, seizure, and neu-

It should be noted that chronic exposure to CO may produce the above symptoms; but what is more common is the gradual-onset neuropsychiatric symptoms such as memory disturbance including retrograde and anterograde amnesia, impaired judgment and psychosis. Some patients may develop delayed neuropsychiatric symptoms, often after severe intoxications associated with coma. After recovery from the initial incident, patients present several days to weeks later with neuropsychiatric symptoms such as those just described. Two-third of

The gold standard diagnosis of CO poisoning can be accomplished only by detecting the level of circulating carboxyhemoglobin (CO-Hgb). The measurement of CO-Hgb can be

is required. Either arterial or venous blood can be used for testing. The analysis of CO-Hgb requires direct spectrophotometric measurement in specific blood-gas analyzers. It is noteworthy that the latest emergency defibrillator-monitor systems are equipped with CO-Hgb sensors. Equally, it is important to underline that even in case of severe CO-Hgb intoxi-

oxy- and CO-Hgb. Thus, diagnosis in emergency pre-hospital care may be challenging. Even

sensors are not able to detect CO-Hgb, a special equipment

levels can be sensed, due to similar spectrophotometry waveform of

performed via blood-gas analyses or by spectrophotometry by performing SpO2

**1.** Maternal O2

level of the mother

rological symptoms [5].

patients eventually recover completely.

ments. However, general SpO2

cation, normal SpO2

**Table 2.** Symptoms of CO intoxication in relation with CO concentration and CO-Hgb levels.

may be lethal. Myocardial ischemia may also cause chest pain, angina pectoris and can trigger vegetative reflexes (nausea, vomiting, sweating, and vertigo).

#### **5.2. Neurological signs and symptoms**

Loss of consciousness and acute neurological symptoms are due to neuronal hypoxemia. However, after surviving acute intoxication, major studies reported late-onset neuropsychiatric syndrome related to CO poising [6, 7]. This includes change in personality, depressive disorders, cognitive deficits, and psychomotor imbalance. Exact signaling pathways are not yet detailed, general brain hypoxemia/anoxia tends to be responsible for long-term neurological complications.

## **6. CO poisoning in pregnancy**

CO poisoning is rare condition in pregnancy. When occurs, it is a critical condition, the mother and the fetus may suffer severe long-term complications. Due to fetal hemoglobin transport system, the intoxication of the fetus is usually more severe than it is in the mother [8]. Fetal hemoglobin desaturation has higher levels in tissue transport, than adult hemoglobin, even more the fetal gas exchange via the placenta eliminates CO slowly [9]. CO poisoning in pregnancy can cause severe hypoxic/anoxic damage in all fetal organs and tissues. Prenatal injury of the central nervous system may lead to life-long mental and somatic dysfunction, attention deficit disorder or behavioral disorders, due to high sensitivity for hypoxic attacks. Mainly, fetal cortical region and basal ganglions suffer in case of tissue hypoxia [10].

Pathophysiology of CO intoxication of the fetus includes:


## **7. Diagnosis and differential diagnosis**

may be lethal. Myocardial ischemia may also cause chest pain, angina pectoris and can trigger

30–50%

**CO concentration Symptoms CO-Hgb level** 35 ppm Mild headache, vertigo 5–20%

200 ppm Previous listed above + altered reflexes and cognitive alert 400 ppm Previous listed above + nausea, vomiting, tachycardia, tachypnea, palpitation, angina pectoris

78 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

800 ppm Previous listed above + arrhythmia, convulsion, respiratory insufficiency, loss of consciousness

3200 ppm Convulsions, loss of consciousness, lethal in 30 minutes

12,800 ppm Loss of consciousness after 2–3 breaths, lethal in minutes

1600 ppm Convulsions, loss of consciousness, lethal in 2 hours 50–66%

6400 ppm Loss of consciousness, lethal in 20 minutes >66%

**Table 2.** Symptoms of CO intoxication in relation with CO concentration and CO-Hgb levels.

Loss of consciousness and acute neurological symptoms are due to neuronal hypoxemia. However, after surviving acute intoxication, major studies reported late-onset neuropsychiatric syndrome related to CO poising [6, 7]. This includes change in personality, depressive disorders, cognitive deficits, and psychomotor imbalance. Exact signaling pathways are not yet detailed, general brain hypoxemia/anoxia tends to be responsible for long-term neuro-

CO poisoning is rare condition in pregnancy. When occurs, it is a critical condition, the mother and the fetus may suffer severe long-term complications. Due to fetal hemoglobin transport system, the intoxication of the fetus is usually more severe than it is in the mother [8]. Fetal hemoglobin desaturation has higher levels in tissue transport, than adult hemoglobin, even more the fetal gas exchange via the placenta eliminates CO slowly [9]. CO poisoning in pregnancy can cause severe hypoxic/anoxic damage in all fetal organs and tissues. Prenatal injury of the central nervous system may lead to life-long mental and somatic dysfunction, attention deficit disorder or behavioral disorders, due to high sensitivity for hypoxic attacks. Mainly, fetal cortical region and basal ganglions suffer in case of tissue

vegetative reflexes (nausea, vomiting, sweating, and vertigo).

**5.2. Neurological signs and symptoms**

100 ppm Headache, vertigo, fatigue

**6. CO poisoning in pregnancy**

logical complications.

hypoxia [10].

Given the vagueness and the broad spectrum of complaints, misdiagnosis of carbon monoxide (CO) toxicity is common. Hence, it is necessary to specifically inquire about possible exposures when considering the diagnosis. In some nonfire-related incidents, the most common symptoms were headaches (37%) followed by dizziness (18%), and nausea (17%) [10]. When a patient has a history compatible with CO exposure and when more than one patient in a group or household presents with similar complaints, the following symptoms have been noted: malaise, flu-like symptoms, fatigue, dyspnoea on exertion, chest pain, palpitations, lethargy, confusion, hallucination, agitation, visual disturbance, syncope, seizure, and neurological symptoms [5].

It should be noted that chronic exposure to CO may produce the above symptoms; but what is more common is the gradual-onset neuropsychiatric symptoms such as memory disturbance including retrograde and anterograde amnesia, impaired judgment and psychosis. Some patients may develop delayed neuropsychiatric symptoms, often after severe intoxications associated with coma. After recovery from the initial incident, patients present several days to weeks later with neuropsychiatric symptoms such as those just described. Two-third of patients eventually recover completely.

The gold standard diagnosis of CO poisoning can be accomplished only by detecting the level of circulating carboxyhemoglobin (CO-Hgb). The measurement of CO-Hgb can be performed via blood-gas analyses or by spectrophotometry by performing SpO2 measurements. However, general SpO2 sensors are not able to detect CO-Hgb, a special equipment is required. Either arterial or venous blood can be used for testing. The analysis of CO-Hgb requires direct spectrophotometric measurement in specific blood-gas analyzers. It is noteworthy that the latest emergency defibrillator-monitor systems are equipped with CO-Hgb sensors. Equally, it is important to underline that even in case of severe CO-Hgb intoxication, normal SpO2 levels can be sensed, due to similar spectrophotometry waveform of oxy- and CO-Hgb. Thus, diagnosis in emergency pre-hospital care may be challenging. Even more, differential diagnose of CO poisoning includes wide range of acute and chronic medical conditions. Early signs and symptoms are atypical, thus it is extremely important to be aware and keep in mind the possibility of CO intoxication, when the circumstances of the scenario suggest. In severe cases, every medical condition that may cause loss of consciousness should be ruled out.

**8.1. Pre-hospital care**

clinical status.

**First medical contact** • Detecting CO

**In-hospital treatment**

• Assessing all possible intoxicated patients • 100% oxygen ASAP to all poisoned

• Evaluating CO-Hgb levels and severity • Assessing peripheral organ dysfunction • Assessing central nervous complication • Preventing further hypoxic tissue damage • Considering special treatment options

• Neuropsychiatric follow-up

• Assessing the severity of signs and symptoms • Assessing special needs: children, pregnancy

Pre-hospital care includes the following:

• Instituting cardiac monitoring.

gen therapy with a nonrebreather mask.

ventilatory support and provide 100% oxygen therapy.

• Obtaining an estimate of exposure time, if possible.

ondary SIRS (systemic inflammatory response syndrome).

**Textbox 1.** Summarizes key elements of first medical contact and in-hospital treatment.

• Avoid exertion to limit tissue oxygen demand.

• Promptly removing the patient from continued exposure and immediately institute oxy-

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• Performing intubation for the comatose patient for airway protection, or if necessary, for

• Alerting the emergency department of the up-coming comatose or unstable patients be-

• Drawing early blood samples for accurate correlation between CO-Hgb measurements and

In CO intoxication, the most important approach is to remove the patient from CO-intoxicated gas area as soon as safely possible and provide high flow 100% oxygen inhale by nonrebreathing masks. The respiratory, hemodynamic status, and consciousness level defines whether the patient needs further respiratory (mechanical ventilation) or hemodynamic support. Controlled hyperventilation provides faster CO elimination. These should be administered as soon as possible, even in pre-hospital first medical contact. For those patients, who suffered CO intoxication in fire event, care should be taken on burns and burn-syndrome-related sec-

cause rapid or direct transfer to a hyperbaric center may be indicated.

The main differential diagnoses considerations include ruling out:


During in-hospital diagnostics several nonspecific tests can be performed, which help clinicians to determine and quantify the severity of organ specific tissue damage. In this concept, cardiac biomarkers can estimate myocardial ischemia: for example, troponin I, T, and creatine kinase. Peripheral myogenic injury may be estimated via myoglobin urine levels. NT-pro-BNP is able to mirror elevated left ventricular filling pressure and heart failure, especially in the case of severe myocardial ischemia and related acute heart decompensation. Further laboratory tests are able to estimate renal (BUN, creatinine) and hepatic function (liver transaminase). Blood count, ion levels, and glucose levels are also measured routinely. To follow-up metabolic status, blood-lactate should be investigated. Elevated lactate levels suggest more severe CO poisoning and are the predictor of worse outcome [8].

In the most severe cases, radiology scanning of the central nervous system is required to estimate damages. Hence computer tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography (PET) scans are advisable to detect organic neurological complications, mainly bilateral ischemic focus in basal ganglions and subcortical white matter damages. Rarely, haemorrhagic complication can also occur if there is a severe central nervous hypoxia/anoxia in high metabolic demand areas of the brain.

### **8. Treatment options**

Treatment protocol should follow the ACBDE rule, as for every emergency scenario. Airway, breathing, circulation, disability, and exposure should be assessed and treated in respective order. In short, it means that the clinical team whether first responders or people first attending to the patient, must ensure that the airways are clear from any obstruction, that the breathing is present or is restored and efficient, that blood circulation is assured; that further complications and disability are prevented, and that the victim is removed or shielded from further exposure. In sequence, the treatment options include pre-hospital care and hospital care in emergency departments.

#### **8.1. Pre-hospital care**

more, differential diagnose of CO poisoning includes wide range of acute and chronic medical conditions. Early signs and symptoms are atypical, thus it is extremely important to be aware and keep in mind the possibility of CO intoxication, when the circumstances of the scenario suggest. In severe cases, every medical condition that may cause loss of conscious-

• other intoxications such as by ethyl- or methyl-alcohol, cyanide, drugs of abuse, or medications;

• metabolic disorders such as hypoglycemia, hepatic coma, hypothyroid coma, diabetic keto-

During in-hospital diagnostics several nonspecific tests can be performed, which help clinicians to determine and quantify the severity of organ specific tissue damage. In this concept, cardiac biomarkers can estimate myocardial ischemia: for example, troponin I, T, and creatine kinase. Peripheral myogenic injury may be estimated via myoglobin urine levels. NT-pro-BNP is able to mirror elevated left ventricular filling pressure and heart failure, especially in the case of severe myocardial ischemia and related acute heart decompensation. Further laboratory tests are able to estimate renal (BUN, creatinine) and hepatic function (liver transaminase). Blood count, ion levels, and glucose levels are also measured routinely. To follow-up metabolic status, blood-lactate should be investigated. Elevated lactate levels suggest more

In the most severe cases, radiology scanning of the central nervous system is required to estimate damages. Hence computer tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography (PET) scans are advisable to detect organic neurological complications, mainly bilateral ischemic focus in basal ganglions and subcortical white matter damages. Rarely, haemorrhagic complication can also occur if there is a severe central

Treatment protocol should follow the ACBDE rule, as for every emergency scenario. Airway, breathing, circulation, disability, and exposure should be assessed and treated in respective order. In short, it means that the clinical team whether first responders or people first attending to the patient, must ensure that the airways are clear from any obstruction, that the breathing is present or is restored and efficient, that blood circulation is assured; that further complications and disability are prevented, and that the victim is removed or shielded from further exposure. In sequence, the treatment options include pre-hospital care and hospital

The main differential diagnoses considerations include ruling out:

80 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

• infective neurological disorders such as encephalitis or meningitis;

severe CO poisoning and are the predictor of worse outcome [8].

nervous hypoxia/anoxia in high metabolic demand areas of the brain.

ness should be ruled out.

acidosis, and lactic acidosis;

• psychiatric conditions.

**8. Treatment options**

care in emergency departments.

Pre-hospital care includes the following:


In CO intoxication, the most important approach is to remove the patient from CO-intoxicated gas area as soon as safely possible and provide high flow 100% oxygen inhale by nonrebreathing masks. The respiratory, hemodynamic status, and consciousness level defines whether the patient needs further respiratory (mechanical ventilation) or hemodynamic support. Controlled hyperventilation provides faster CO elimination. These should be administered as soon as possible, even in pre-hospital first medical contact. For those patients, who suffered CO intoxication in fire event, care should be taken on burns and burn-syndrome-related secondary SIRS (systemic inflammatory response syndrome).

#### **First medical contact**


#### **In-hospital treatment**


**Textbox 1.** Summarizes key elements of first medical contact and in-hospital treatment.

#### **8.2. Hospital care**

In-hospital management requires thorough assessment and support of all damaged organs. Pre- and intra-hospital care should take careful consideration on myocardial ischemia. ECG recording should be carefully analyzed and actions should be taken to decrease myocardial injury and prevent further myocardial ischemia. High-flow oxygen therapy, is needed to prevent myocardial and central nervous ischemic injury, to support renal function, and to compensate metabolic disturbances. The administration of hyperbaric oxygen therapy is the mainstay therapy for CO intoxication. Hyperbaric oxygen therapy initiates CO-Hgb elimination from the human body, and increase partial oxygen pressure and oxygen delivery of circulating blood. During hyperbaric oxygen therapy, the Hgb–oxygen dissociation curve is shifted to the right, thus oxygen binding of Hgb and tissue oxygen delivery is increased. Myoglobin and cytochrome systems bind CO in lower rate. Every patient with CO intoxication may benefit from hyperbaric oxygen therapy, however availability and costs tailor patient population for this treatment. Patients should be selected via the following criteria [9, 12]:

**9. Case report**

ence any longer convulsions.

This case report is provided from the Hungarian National Pediatric Ambulance Service. The ambulance got a call from a family member who said that their 6-year-old child suffered from convulsions while in the bathroom; but regained consciousness spontaneously, though he still had impaired mental function and could not answer simple questions. It is important to note that convulsion in a 6-year-old child may result from several conditions; hence a skillful

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Fortunately, the Hungarian pediatric ambulance services are equipped with the latest emergency care machines including CO detectors. When the ambulance arrived at the scene, the CO detectors alarmed a high concentration of CO (335 ppm). Beside the 6-year-old child, there were also five adults in the flat. The child was conscious, with altered mental status. All people were immediately evacuated from the flat to fresh air and the fire service was called on scene, to detect source of CO. The pediatric ambulance with its LifePak®15 ECG-defibrillator system could measure SpCO via spectrophotometry detectors. The SpCO measurements showed more than 30% CO-Hgb levels in all people who were in the flat. Hence, high-flow oxygen therapy using nonrebreathing oxygen masks was provided to all victims. All of them were in a stable condition based on the vital signs (heart rate, blood pressure, ECG monitor, Neurological evaluation (Glasgow Come Scale)), though adults had moderate symptoms of fatigue, dizziness, and vertigo. With the use of the removal from further exposure and the administration of oxygen, the mental status of the child also improved and he did not experi-

With regard to the pregnant woman among the victims, it should be noted that she was one of the adult patients was pregnant woman. She was 26-year old and in the 39th week of her pregnancy. Her initial SpCO assessment showed a 28% CO-Hgb level, a severe CO intoxication. Her management was continued in the hospital, her CO-Hgb levels had decreased to 14.5% at the time of hospital admission. Given the high level of CO-Hgb levels, an emergency cesarean section was carried out in order to maintain fetal oxygenation. This was performed within 1 hour after hospital admission. This procedure was needed because there was only one hyperbaric oxygen chamber available in Hungary, so organizing an emergency hyperbaric oxygen therapy would have been difficult and time-consuming lost time. The fetal CO-Hgb level was 5% at the time of birth, which is the level of a smoking adult. The new-born was otherwise healthy and received high-flow oxygen support until total elimination of CO-Hgb. The mother also eliminated her remaining CO-Hgb in the following 1.5 hours. The mother and the

The lessons learnt from the above case were that ambulance services with latest diagnosis equipment and devices are critical in recognizing a diagnosis of CO quickly and facilitation medical care secondly, the administration of oxygen must be performed as soon as possible; thirdly, that an efficient triage should be performed by the providers who first see the victims in order to identify those who are in critical situations and who should be prioritized such as children and pregnant women; finally, the performance of an urgent cesarean section has been effective in preventing accumulation of CO in fetal and thus saved the life of the baby.

questioning is necessary to establish the most likely cause.

baby were discharged from the hospital being in good health.


Hyperbaric oxygen therapy is proven to decrease late neurological complications and deficit in CO intoxication. Neuropsychiatric tests are able to estimate loss of function, these may help finding those patients who benefit the most from rarely available and expensive therapy [13, 14]. Hyperbaric oxygen therapy should be administered as soon as possible, but at least in 6 hours to exposure time. It is cautioned here that, although some studies have reported major reductions in delayed neurologic sequelae, cerebral oedema, pathologic central nervous system (CNS) changes, and reduced cytochrome oxidase impairment as a result of hyperbaric oxygen therapy, some systematic reviews have not revealed a clear benefit of HBO, so no clear guidelines for its use have been determined. [15, 16].

#### **8.3. CO intoxication treatment in pregnancy**

The major cornerstone of treating a pregnant woman with CO intoxication is to decrease fetal CO-Hgb level. Fetal oxygenation must be provided; noting that in pregnancy, hyperbaric oxygen therapy is superior to urgent cesarian section [15]. However, hyperbaric oxygen therapy is rarely available, thus in many cases, urgent cesarean section is the only option to support fetal oxygenation. Fetal CO-Hgb levels cannot be directly measured but can be estimated. Moreover, fetal ultrasound or MRI scan, if available, can be used to check for central nervous system damage and guide further therapy for the new-born if a cesarian section was performed [16].

## **9. Case report**

**8.2. Hospital care**

the following criteria [9, 12]:

• CO-Hgb level above 25%.

• Severe metabolic imbalance (lactatemia).

use have been determined. [15, 16].

**8.3. CO intoxication treatment in pregnancy**

• Loss of consciousness.

• Pregnancy.

performed [16].

In-hospital management requires thorough assessment and support of all damaged organs. Pre- and intra-hospital care should take careful consideration on myocardial ischemia. ECG recording should be carefully analyzed and actions should be taken to decrease myocardial injury and prevent further myocardial ischemia. High-flow oxygen therapy, is needed to prevent myocardial and central nervous ischemic injury, to support renal function, and to compensate metabolic disturbances. The administration of hyperbaric oxygen therapy is the mainstay therapy for CO intoxication. Hyperbaric oxygen therapy initiates CO-Hgb elimination from the human body, and increase partial oxygen pressure and oxygen delivery of circulating blood. During hyperbaric oxygen therapy, the Hgb–oxygen dissociation curve is shifted to the right, thus oxygen binding of Hgb and tissue oxygen delivery is increased. Myoglobin and cytochrome systems bind CO in lower rate. Every patient with CO intoxication may benefit from hyperbaric oxygen therapy, however availability and costs tailor patient population for this treatment. Patients should be selected via

82 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

• Evidence of organ ischemic function (e.g., renal or myocardial injury).

Hyperbaric oxygen therapy is proven to decrease late neurological complications and deficit in CO intoxication. Neuropsychiatric tests are able to estimate loss of function, these may help finding those patients who benefit the most from rarely available and expensive therapy [13, 14]. Hyperbaric oxygen therapy should be administered as soon as possible, but at least in 6 hours to exposure time. It is cautioned here that, although some studies have reported major reductions in delayed neurologic sequelae, cerebral oedema, pathologic central nervous system (CNS) changes, and reduced cytochrome oxidase impairment as a result of hyperbaric oxygen therapy, some systematic reviews have not revealed a clear benefit of HBO, so no clear guidelines for its

The major cornerstone of treating a pregnant woman with CO intoxication is to decrease fetal CO-Hgb level. Fetal oxygenation must be provided; noting that in pregnancy, hyperbaric oxygen therapy is superior to urgent cesarian section [15]. However, hyperbaric oxygen therapy is rarely available, thus in many cases, urgent cesarean section is the only option to support fetal oxygenation. Fetal CO-Hgb levels cannot be directly measured but can be estimated. Moreover, fetal ultrasound or MRI scan, if available, can be used to check for central nervous system damage and guide further therapy for the new-born if a cesarian section was This case report is provided from the Hungarian National Pediatric Ambulance Service. The ambulance got a call from a family member who said that their 6-year-old child suffered from convulsions while in the bathroom; but regained consciousness spontaneously, though he still had impaired mental function and could not answer simple questions. It is important to note that convulsion in a 6-year-old child may result from several conditions; hence a skillful questioning is necessary to establish the most likely cause.

Fortunately, the Hungarian pediatric ambulance services are equipped with the latest emergency care machines including CO detectors. When the ambulance arrived at the scene, the CO detectors alarmed a high concentration of CO (335 ppm). Beside the 6-year-old child, there were also five adults in the flat. The child was conscious, with altered mental status. All people were immediately evacuated from the flat to fresh air and the fire service was called on scene, to detect source of CO. The pediatric ambulance with its LifePak®15 ECG-defibrillator system could measure SpCO via spectrophotometry detectors. The SpCO measurements showed more than 30% CO-Hgb levels in all people who were in the flat. Hence, high-flow oxygen therapy using nonrebreathing oxygen masks was provided to all victims. All of them were in a stable condition based on the vital signs (heart rate, blood pressure, ECG monitor, Neurological evaluation (Glasgow Come Scale)), though adults had moderate symptoms of fatigue, dizziness, and vertigo. With the use of the removal from further exposure and the administration of oxygen, the mental status of the child also improved and he did not experience any longer convulsions.

With regard to the pregnant woman among the victims, it should be noted that she was one of the adult patients was pregnant woman. She was 26-year old and in the 39th week of her pregnancy. Her initial SpCO assessment showed a 28% CO-Hgb level, a severe CO intoxication. Her management was continued in the hospital, her CO-Hgb levels had decreased to 14.5% at the time of hospital admission. Given the high level of CO-Hgb levels, an emergency cesarean section was carried out in order to maintain fetal oxygenation. This was performed within 1 hour after hospital admission. This procedure was needed because there was only one hyperbaric oxygen chamber available in Hungary, so organizing an emergency hyperbaric oxygen therapy would have been difficult and time-consuming lost time. The fetal CO-Hgb level was 5% at the time of birth, which is the level of a smoking adult. The new-born was otherwise healthy and received high-flow oxygen support until total elimination of CO-Hgb. The mother also eliminated her remaining CO-Hgb in the following 1.5 hours. The mother and the baby were discharged from the hospital being in good health.

The lessons learnt from the above case were that ambulance services with latest diagnosis equipment and devices are critical in recognizing a diagnosis of CO quickly and facilitation medical care secondly, the administration of oxygen must be performed as soon as possible; thirdly, that an efficient triage should be performed by the providers who first see the victims in order to identify those who are in critical situations and who should be prioritized such as children and pregnant women; finally, the performance of an urgent cesarean section has been effective in preventing accumulation of CO in fetal and thus saved the life of the baby.

## **10. Concluding remarks**

The key learning points from this chapter include that CO is silent killer; fatal CO intoxication can occur and present with plain, nonspecific signs and symptoms. Prevention is a key to avoid severe intoxication: education, safe heating systems, and use of CO detectors. Furthermore, the first medical contact has an important role in recognizing CO intoxication with the use of diagnosing devices and sometimes, based on the signs and symptoms. It is important to perform a differential diagnosis based on information gathered from patients and relatives. With regard to actual management of CO intoxication, the administration of high flow, 100% oxygen therapy must be administered as soon as possible in pre-hospital care. Later in-hospital management should include the evaluation, treatment, and prevention of further organ damage and the neurological deficits. More importantly, prompt care and interventions are required for pregnant women because children and fetus are prone to suffer more severe CO intoxication. Therefore, hyperbaric oxygen therapy should be initiated as soon as possible when available or an emergency cesarean section should be performed to save the fetus from CO intoxication.

[5] Ernst A, Zibrak JD. Carbon monoxide poisoning. New England Journal of Medicine.

Carbon Monoxide Intoxication: Experiences from Hungary

http://dx.doi.org/10.5772/intechopen.70010

85

[6] Thom SR, Taber RL, Mendiguren II, Clark JM, Hardy KR, Fisher AB. Delayed neuropsychologic sequelae after carbon monoxide poisoning: Prevention by treatment with

[7] Kwon OY, Chung SP, Ha YR, Yoo IS, Kim SW. Delayed postanoxic encephalopathy after

[8] Hutter CD, Blair ME. Carbon monoxide – Does foetal exposure cause sudden infant

[9] Greginor JL, Tosi JM, Ruhlmann és mtsai S. Acute carbon monoxide intoxication during pregnancy. One case report and review of the literature. Emergency Medicine Journal.

[10] Wu L, Wang R. Carbon monoxide, endogenous production, physiological functions, and pharmacological applications. Pharmacological Reviews. 2005;**57**(4):585-630

[11] Farrow JR, Davis GJ, Roy és mtsai TM. Fetal death due to nonlethal maternal carbon

[12] Brown DB, Mueller GL, Golich FC. Hyperbaric oxygen treatment for carbon monoxide poisoning in pregnancy: A case report. Aviation, Space, and Environmental Medicine.

[13] Devine SA, Kirkley SM, Palumbo CL. MRI and neuropsychological correlates of carbon monoxide exposure: A case report. Environmental Health Perspectives. 2002;**110**(10):1051-1055

[14] Pepe G, Castelli M, Nazerian és mtsai P. Delayed neuropsychological sequelae after carbon monoxide poisoning: Predictive risk factor in the emergency department. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine. 2011 Mar 17;

[15] Gabrielli A, Layon AJ. Carbon monoxide intoxication during pregnancy: A case presentation and pathophysiologic discussion, with emphasis on molecular mechanisms.

[16] Koren G, Sharav T, Pastuszak és mtsai A. A multicenter, prospective study of fetal outcome following accidental carbon monoxide poisoning in pregnancy. Reproductive

monoxide poisoning. Journal of Forensic Sciences. 1990;**35**:1448-1452

hyperbaric oxygen. Annals of Emergency Medicine. 1995;**25**(4):474

death syndrome? Medical Hypotheses. 1996;**46**:1-4

Journal of Clinical Anesthesia. 1995;**7**(1):82-87

carbon monoxide poisoning. Emergency Medicine Journal. 2004;**21**(2):250

1998;**339**(22):1603

2001;**18**(5):399-401

1992;**63**(11):1011-1014

Toxicology. 1991;**5**:397-403

**19**:16

## **Acknowledgements**

Eva Gesztes Dr, Gabor Zacher Dr and Richard Doroszlai for the management of the child and the pregnant woman in the case report and providing their clinical data for this chapter.

### **Author details**

#### Edit Gara

Address all correspondence to: gara.editgara@gmail.com

Semmelweis University, Budapest, Hungary

## **References**


[5] Ernst A, Zibrak JD. Carbon monoxide poisoning. New England Journal of Medicine. 1998;**339**(22):1603

**10. Concluding remarks**

save the fetus from CO intoxication.

Address all correspondence to: gara.editgara@gmail.com

Semmelweis University, Budapest, Hungary

Gynecology. 2000;**107**:833-838

of Neurological Sciences. 2007;**262**:122-130

childhood. Pediatric Emergency Care. 2015 Jul 14;**37**(1):1-7

**Acknowledgements**

**Author details**

Edit Gara

**References**

The key learning points from this chapter include that CO is silent killer; fatal CO intoxication can occur and present with plain, nonspecific signs and symptoms. Prevention is a key to avoid severe intoxication: education, safe heating systems, and use of CO detectors. Furthermore, the first medical contact has an important role in recognizing CO intoxication with the use of diagnosing devices and sometimes, based on the signs and symptoms. It is important to perform a differential diagnosis based on information gathered from patients and relatives. With regard to actual management of CO intoxication, the administration of high flow, 100% oxygen therapy must be administered as soon as possible in pre-hospital care. Later in-hospital management should include the evaluation, treatment, and prevention of further organ damage and the neurological deficits. More importantly, prompt care and interventions are required for pregnant women because children and fetus are prone to suffer more severe CO intoxication. Therefore, hyperbaric oxygen therapy should be initiated as soon as possible when available or an emergency cesarean section should be performed to

84 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

Eva Gesztes Dr, Gabor Zacher Dr and Richard Doroszlai for the management of the child and the pregnant woman in the case report and providing their clinical data for this chapter.

[1] Aubard Y. Carbon monoxide poisoning in pregnancy. British Journal of Obstetrics and

[2] Prockop LD, Chichkova RI. Carbon monoxide intoxication: An updated review. Journal

[3] Hungarian Central Statistical Office Database. Available from: www.ksh.hu/engstadat [4] Damlapinar R, Arikan FI, Sahin S, Dallar Y. Lactate level is more significant than carboxyhemoglobin level in determining prognosis of carbon monoxide intoxication of


**Chapter 5**

**Provisional chapter**

**Toxicity of β-Lactam Antibiotics: Pathophysiology,**

**Molecular Biology and Possible Recovery Strategies**

**Toxicity of β-Lactam Antibiotics: Pathophysiology,** 

DOI: 10.5772/intechopen.70199

**Molecular Biology and Possible Recovery Strategies**

Beta (β)-lactam antibiotics are wide-spectrum antibiotics used for various bacterial infections. The aim of this chapter is to summarize the knowledge about the toxicity of β-lactam antibiotics and issues associated to their inappropriate use. This review has highlighted that β-lactam antibiotics are a group of products that have a chemical structure characterized by a β-lactam ring and are one of the most common antibacterial agents. However, due to the inappropriate use including abuse and misuse, resistance to the β-lactam antibiotics is currently a global crisis. Moreover, even when used appropriately, they have been linked to triggering allergic reactions like urticaria, bronchoconstriction, also severe conditions like immune-mediated haemolytic anaemia and intravascular haemolysis. It is known that some β-lactam antibiotics are neurotoxic, some are nephrotoxic, some are genotoxic and some are toxic to urogenital system. Several factors are involved in the occurrence of toxic effects including the dosage and renal status. Several strategies are possible to overcome β-lactam antibiotics-triggered toxicity, including rational prescribing, substitution combination and phage therapy which seems promising. Public health education for clinical teams and patients is essential in ensuring that this group of antibi-

**Keywords:** β-lactam antibiotics, β-lactamase, toxicity, side effects, treatment strategies

Beta (β)-lactam antibiotics are one of most commonly used class of antimicrobial agents around the world. β-lactam or β-lactamase inhibitor antibiotics are substances that disrupt bacterial cell-wall formation via penicillin-binding proteins (PBPs) where they bind covalently at the terminal step of peptidoglycan cross-linking in bacteria [1]. The first generation

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Elif Bozcal and Melih Dagdeviren

Elif Bozcal and Melih Dagdeviren

http://dx.doi.org/10.5772/intechopen.70199

otics are retained in therapeutics.

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**

## **Toxicity of β-Lactam Antibiotics: Pathophysiology, Molecular Biology and Possible Recovery Strategies Molecular Biology and Possible Recovery Strategies**

**Toxicity of β-Lactam Antibiotics: Pathophysiology,** 

DOI: 10.5772/intechopen.70199

Elif Bozcal and Melih Dagdeviren Elif Bozcal and Melih Dagdeviren Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70199

#### **Abstract**

Beta (β)-lactam antibiotics are wide-spectrum antibiotics used for various bacterial infections. The aim of this chapter is to summarize the knowledge about the toxicity of β-lactam antibiotics and issues associated to their inappropriate use. This review has highlighted that β-lactam antibiotics are a group of products that have a chemical structure characterized by a β-lactam ring and are one of the most common antibacterial agents. However, due to the inappropriate use including abuse and misuse, resistance to the β-lactam antibiotics is currently a global crisis. Moreover, even when used appropriately, they have been linked to triggering allergic reactions like urticaria, bronchoconstriction, also severe conditions like immune-mediated haemolytic anaemia and intravascular haemolysis. It is known that some β-lactam antibiotics are neurotoxic, some are nephrotoxic, some are genotoxic and some are toxic to urogenital system. Several factors are involved in the occurrence of toxic effects including the dosage and renal status. Several strategies are possible to overcome β-lactam antibiotics-triggered toxicity, including rational prescribing, substitution combination and phage therapy which seems promising. Public health education for clinical teams and patients is essential in ensuring that this group of antibiotics are retained in therapeutics.

**Keywords:** β-lactam antibiotics, β-lactamase, toxicity, side effects, treatment strategies

## **1. Introduction**

Beta (β)-lactam antibiotics are one of most commonly used class of antimicrobial agents around the world. β-lactam or β-lactamase inhibitor antibiotics are substances that disrupt bacterial cell-wall formation via penicillin-binding proteins (PBPs) where they bind covalently at the terminal step of peptidoglycan cross-linking in bacteria [1]. The first generation

of β-lactam antibiotics were penicillins, followed by cephalosporins; later carbapenems and monocyclic β-lactams that have been recently introduced and are currently in service for the treatment of infectious disease caused by pathogenic bacteria [2]. However, following the occurrence of resistance to the penicillin, new β-lactam antibiotics are being researched for enhancing the spectrum efficiency against the β-lactam-resistant bacteria [3]. In terms of lifethreatening infections, β-lactam antibiotics are almost indispensable for therapies in intensive care units (ICUs). However, the therapy with β-lactam antibiotics remains unresolved since β-lactamase resistance is disseminating rapidly among pathogenic bacteria. The most serious types of β-lactamase resistance today are extended-spectrum β-lactamases (ESBLs), carbapenemases and metallo-β-lactamases (MBLs) [1, 4, 5].

of β-lactam antibiotics and issues associated to their inappropriate use. It is hoped that a good understanding of the structures, mechanisms of action and risk factors leading to resistance to β-lactam antibiotics will assist both clinicians and researchers in the design of

Toxicity of β-Lactam Antibiotics: Pathophysiology, Molecular Biology and Possible Recovery...

http://dx.doi.org/10.5772/intechopen.70199

89

Beta (β)-lactam antibiotics are a class of antibiotics that contain a β-lactam ring in their molecular structures. Generally, β-lactam antibiotics have a common function: inhibition on cellwall biosynthesis of the peptidoglycan layer in the bacteria. And this makes β-lactams the most extensively used antibiotics [19]. For over 70 years, penicillin and related antibiotics have been used extensively for the control and treatment of bacterial infections. Improving the efficiency of these antibiotics is still important, and it captivates increasing attention of researchers to overcome the infections depending on highly resistant bacteria. Development of new β-lactam group of antibiotics generally depends on enhancing the spectrum efficiency against the pathogenic bacteria. Besides, resistance mechanisms with special features may be

Over the years, countless penicillin derivatives have been produced including penicillins, cephalosporins, carbapenems, oxapenems, oxacephams as well as monocyclic, spirocyclic and multicyclic ring systems [2]. The first β-lactam antibiotic was 'penicillin G' in the beginning of 1940s [1]. Afterwards, naturally occurring penicillin was 'penicillin V' which was an oral formulation, still in use for the therapeutic purposes. Followed by the occurring resistance to the penicillin, semi-synthetic penicillins were developed such as methicillin [20]. Moreover, some of the significant penicillins from the beginning up to date are the following: oxacillin, cloxacillin, ampicillin, nafcillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, termocillin

Cephalosporins are another subgroup of β-lactam antibiotics, and the first penicillinase-stable cephalosporin was discovered during 1950s [21]. It was efficient for the treatment of infectious diseases mainly caused by penicillinase-producing pathogenic bacteria [22]. Numerous cephalosporins are in use nowadays including cefotaxime, ceftriaxone, cefepime, ceftazidime and cefuroxime [23]. A new addition has been made to the cephalosporin family with a siderophore-substituted cephalosporin (S-649266) that contains a catechol segment which facilitates entry into bacterial cells through iron transportation system. Moreover, this cephalosporin is

Furthermore, carbapenems are also members of the β-lactam antibiotics which act by binding to penicillin-binding proteins and lead to the inhibition of bacterial cell-wall synthesis. However, they have broader spectrum than other cephalosporins and have been proven successful against *Enterobacteriaceae* including ESBLs [25]. Carbapenem-related agents and other carbapenems that have extended-spectrum activity include meropenem, imipenem, ertape-

anti-resistance interventions.

targeted [1].

and mecillinam [20].

stable against several carbapenemases [24].

nem and doripenem which have been used widely [1].

**2. Classes of beta (β)-lactam antibiotics**

The large amount of use and misuse of β-lactam antibiotics has been inducing the β-lactam resistance for decades; besides β-lactam antibiotics have many side or adverse effects including allergy and toxicity [6, 7]. Since beta-lactam rings are different in their structure, they can be recognized by the immune system and this leads to hypersensitivity in some patients [8]. For example, cephalosporin can induce a range of hypersensitivity reaction and anaphylaxis in patients with IgE-mediated allergy [9]. Also, β-lactams are the most known causes of druginduced fevers [10]. β-lactam antibiotics are neurotoxic, nephrotoxic, genotoxic and some are reproductive toxic. The nephrotoxic effects of β-lactams lead to proximal tubular necrosis [11]. Some are toxic to reproductive system; tazobactam/piperacillin has a toxic effect on reproductive systems and also developmental toxicity reported that tazobactam has an influence on maternal toxicity [12]. Another significant point for the side effects of β-lactams is the toxicity on central nervous system. It can be observed after the administration of β-lactam antibiotics such as penicillin; hence, clinical data have reported disorientation, twitching, somnolence and myoclonus [7]. β-lactam antibiotics such as imipenem and cephaloridine have been reported to cause an irreversible injury to the renal anionic substrate uptake and respiration [13]. Since this effect is dose-dependent, it can be resolved through dosing reduction based on renal function test results [6].

To overcome the resistance and toxicity of β-lactam antibiotics, many attempts have been reported. Developing more stable and more effective strategies is the key factor. For instance, ampicillin/sulbactam, amoxicillin/clavulanate, ticarcillin/clavulanate and piperacillin/tazobactam are the most used β-lactamase inhibitor combinations for clinicians [2]. Moreover, the selection of right β-lactam antibiotics for patients who have an antibiotic allergy is important. To overcome this issue, some of allergy tests are available, for example, penicillin skin testing [14]. Fighting with bacteria via the phage therapy is another option to overcome toxicity/allergy of β-lactams and it reduces the resistance risk of β-lactams. A unique and effective phage against metallo-β-lactamase producing *Pseudomonas aeruginosa* (*P. aeruginosa*) has been used to treat a catfish infection. However, this phage is not useful for the treatment of infections in human [15]. By the way, combination of phages and antibiotics is possible; it is called phage–antibiotic synergy (PAS) and has been proven successful in some bacteria normally resistant to β-lactam antibiotics such as cefotaxime [16]. Interestingly, some studies have reported that phages were not toxic on mammalian cells [17, 18]. Thus, the aim of this chapter is to summarize the knowledge about the toxicity of β-lactam antibiotics and issues associated to their inappropriate use. It is hoped that a good understanding of the structures, mechanisms of action and risk factors leading to resistance to β-lactam antibiotics will assist both clinicians and researchers in the design of anti-resistance interventions.

## **2. Classes of beta (β)-lactam antibiotics**

of β-lactam antibiotics were penicillins, followed by cephalosporins; later carbapenems and monocyclic β-lactams that have been recently introduced and are currently in service for the treatment of infectious disease caused by pathogenic bacteria [2]. However, following the occurrence of resistance to the penicillin, new β-lactam antibiotics are being researched for enhancing the spectrum efficiency against the β-lactam-resistant bacteria [3]. In terms of lifethreatening infections, β-lactam antibiotics are almost indispensable for therapies in intensive care units (ICUs). However, the therapy with β-lactam antibiotics remains unresolved since β-lactamase resistance is disseminating rapidly among pathogenic bacteria. The most serious types of β-lactamase resistance today are extended-spectrum β-lactamases (ESBLs), carbapen-

88 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

The large amount of use and misuse of β-lactam antibiotics has been inducing the β-lactam resistance for decades; besides β-lactam antibiotics have many side or adverse effects including allergy and toxicity [6, 7]. Since beta-lactam rings are different in their structure, they can be recognized by the immune system and this leads to hypersensitivity in some patients [8]. For example, cephalosporin can induce a range of hypersensitivity reaction and anaphylaxis in patients with IgE-mediated allergy [9]. Also, β-lactams are the most known causes of druginduced fevers [10]. β-lactam antibiotics are neurotoxic, nephrotoxic, genotoxic and some are reproductive toxic. The nephrotoxic effects of β-lactams lead to proximal tubular necrosis [11]. Some are toxic to reproductive system; tazobactam/piperacillin has a toxic effect on reproductive systems and also developmental toxicity reported that tazobactam has an influence on maternal toxicity [12]. Another significant point for the side effects of β-lactams is the toxicity on central nervous system. It can be observed after the administration of β-lactam antibiotics such as penicillin; hence, clinical data have reported disorientation, twitching, somnolence and myoclonus [7]. β-lactam antibiotics such as imipenem and cephaloridine have been reported to cause an irreversible injury to the renal anionic substrate uptake and respiration [13]. Since this effect is dose-dependent, it can be resolved through dosing reduction based on

To overcome the resistance and toxicity of β-lactam antibiotics, many attempts have been reported. Developing more stable and more effective strategies is the key factor. For instance, ampicillin/sulbactam, amoxicillin/clavulanate, ticarcillin/clavulanate and piperacillin/tazobactam are the most used β-lactamase inhibitor combinations for clinicians [2]. Moreover, the selection of right β-lactam antibiotics for patients who have an antibiotic allergy is important. To overcome this issue, some of allergy tests are available, for example, penicillin skin testing [14]. Fighting with bacteria via the phage therapy is another option to overcome toxicity/allergy of β-lactams and it reduces the resistance risk of β-lactams. A unique and effective phage against metallo-β-lactamase producing *Pseudomonas aeruginosa* (*P. aeruginosa*) has been used to treat a catfish infection. However, this phage is not useful for the treatment of infections in human [15]. By the way, combination of phages and antibiotics is possible; it is called phage–antibiotic synergy (PAS) and has been proven successful in some bacteria normally resistant to β-lactam antibiotics such as cefotaxime [16]. Interestingly, some studies have reported that phages were not toxic on mammalian cells [17, 18]. Thus, the aim of this chapter is to summarize the knowledge about the toxicity

emases and metallo-β-lactamases (MBLs) [1, 4, 5].

renal function test results [6].

Beta (β)-lactam antibiotics are a class of antibiotics that contain a β-lactam ring in their molecular structures. Generally, β-lactam antibiotics have a common function: inhibition on cellwall biosynthesis of the peptidoglycan layer in the bacteria. And this makes β-lactams the most extensively used antibiotics [19]. For over 70 years, penicillin and related antibiotics have been used extensively for the control and treatment of bacterial infections. Improving the efficiency of these antibiotics is still important, and it captivates increasing attention of researchers to overcome the infections depending on highly resistant bacteria. Development of new β-lactam group of antibiotics generally depends on enhancing the spectrum efficiency against the pathogenic bacteria. Besides, resistance mechanisms with special features may be targeted [1].

Over the years, countless penicillin derivatives have been produced including penicillins, cephalosporins, carbapenems, oxapenems, oxacephams as well as monocyclic, spirocyclic and multicyclic ring systems [2]. The first β-lactam antibiotic was 'penicillin G' in the beginning of 1940s [1]. Afterwards, naturally occurring penicillin was 'penicillin V' which was an oral formulation, still in use for the therapeutic purposes. Followed by the occurring resistance to the penicillin, semi-synthetic penicillins were developed such as methicillin [20]. Moreover, some of the significant penicillins from the beginning up to date are the following: oxacillin, cloxacillin, ampicillin, nafcillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, termocillin and mecillinam [20].

Cephalosporins are another subgroup of β-lactam antibiotics, and the first penicillinase-stable cephalosporin was discovered during 1950s [21]. It was efficient for the treatment of infectious diseases mainly caused by penicillinase-producing pathogenic bacteria [22]. Numerous cephalosporins are in use nowadays including cefotaxime, ceftriaxone, cefepime, ceftazidime and cefuroxime [23]. A new addition has been made to the cephalosporin family with a siderophore-substituted cephalosporin (S-649266) that contains a catechol segment which facilitates entry into bacterial cells through iron transportation system. Moreover, this cephalosporin is stable against several carbapenemases [24].

Furthermore, carbapenems are also members of the β-lactam antibiotics which act by binding to penicillin-binding proteins and lead to the inhibition of bacterial cell-wall synthesis. However, they have broader spectrum than other cephalosporins and have been proven successful against *Enterobacteriaceae* including ESBLs [25]. Carbapenem-related agents and other carbapenems that have extended-spectrum activity include meropenem, imipenem, ertapenem and doripenem which have been used widely [1].

Finally, at molecular level, it is worth noting that there is a peptidoglycan layer which is critical for bacterial cell-wall structural wholeness and stability, particularly in Grampositive organisms, being the outermost and primary component of the wall [26]. During the final stage formation of the peptidoglycan, there is a transpeptidation step catalysed by DD-transpeptidases which are PBPs. β-lactam antibiotics inhibit these PBPs and ultimately lead to cell lysis [27]. It should be noted that PBPs are classified according to their molecular mass: the first category is low-molecular-mass PBPs that are monofunctional, for example, D-Ala-D-Ala carboxypeptidases; the second category is high-molecular-mass PBPs that are bifunctional enzymes containing a transpeptidase (D-Ala-D-Ala-dependent) and a transglycosylase [1, 28, 29].

devices like urinary catheters are also prone to antibiotic-resistant bacteria [38]. Moreover, the length of hospital stay is another risk factor; patients who have stayed more than 3 days in hospitals and who have been previously treated with β-lactam antibiotics might be considered as a risk factor for the acquisition of β-lactamase resistance [39]. Furthermore, it has been reported in Italy that the risk factors for ESBL-producing *S. marcescens* and *K. pneumoniae* acquisition in neonatal ICU include low birthweight, gestational period and the use of invasive devices [40].

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Further observations have noted that clinical isolates that have ESBL-produced *E. coli* strains occur generally in hospitalized patients exposed to invasive procedures [41]. It is also noted that when antibiotics are cheap and accessible, this encourages their overuse and subsequent resistance [42]. It has been reported that there is a positive relationship between antibiotic consumption and the emergence and spreading of resistant bacterial strains. One of the most significant reasons of this is the lack of enforcement of legislation which result in the sale of

**4. Factors and mechanisms involved in resistance to β-lactam antibiotics**

Clinicians can prescribe beta-lactams without a real need; yet the health condition could have been treated by diet or rest. Sometimes, the consumers can take drugs without a medical advice or can take more doses than prescribed doses because of mental illnesses like Alzheimer's disease or dementia. Though some people know that antibiotics are used against bacterial infections, few are aware that antibiotics are not useful for viral infections [44]. When antibiotics are used in viral infections, they can trigger bacterial resistance while infections are not cured because of their viral background. Clinicians, surgeons, patients, consumers and caretakers as well require up-to-date information about the appropriate usage of β-lactam antibiotics because their misuse can cause severe conditions including bacterial resistance or

It should be remembered that resistance to penicillin was noted in early 1940s; this finding has no clinical significance until the 1970s [20]. However, β-lactam resistance became a global crisis nowadays [46]. Resistance to the β-lactams usually occurs by three different mechanisms: decreased access of antimicrobials to the target PBPs (efflux pumps), altered PBPs (affinity of

Although efflux pumps are found in almost all bacterial species, the β-lactamase production is the most efficient of the three mechanisms. This resistance mechanism generally depends on plasmids that include various virulence genes consisting of multiple β-lactamases of different classes in this way. This is why β-lactamase resistance can sprawl among various bacterial species. There are two types of β-lactamases: (a) serine-β-lactamases and (b) MBLs. Serine-β-lactamases comprise ESBLs and carbapenemases that hydrolyse carbapenem antibiotics and cephalosporins [2, 20]. We can suffice to say that the most common mechanism for drug resistance to β-lactam antibiotics is bacterial synthesis of β-lactamases. Many bacteria synthesize beta-lactamases that degrade beta-lactam antibiotics before they reach the cell wall. Gram-positive bacteria that produce beta-lactamase excrete the enzyme into the

antibiotics without a prescription in many countries [43].

allergic/toxic side effects [45].

binding decreased) and β-lactamase production [1].

#### **3. Specific cases of resistance to β-lactam antibiotics**

In most instances, particularly in case of life-threatening bacterial infections, antibiotics are the core of treatment [19]. Two of the main goals of β-lactam antibiotics are prevention and treatment of bacterial infections occasioned by susceptible bacteria [1]. Antimicrobial resistance has a greater risk for critically ill patients. For example, it is well known that ICUs are facing a major problem with β-lactam antibiotics [30]. Moreover, many case studies have been reported issues intensively with regard to resistance to the β-lactam antibiotics. A recent case was reported about extensively drug-resistant (particularly β-lactams) *Escherichia coli (E. coli),* which was isolated from the urine of a 63-year-old man from Phetchabun, Thailand. A craniotomy resulted that it is also difficult to treat infections like this via β-lactam antibiotics [31]. Another case was reported from an 87-year-old woman, who had clinical signs, for example, fever, dysuria and suprapubic pain. Urine culture produces a positive result for *Klebsiella pneumoniae* and *E. coli,* both resistant to multiple antibiotics including β-lactams [32]. β-lactam resistance can be seen in serious infections such as cystic fibrosis. Pollini and coworkers reported that a metallo-βlactamase-producing *P. aeruginosa* identified in a cystic fibrosis patient was resistant to carbapenems [33]. Furthermore, New Delhi β-lactamase-1 (NDM-1) producing *Enterobacteriaceae* infections have been found in patients suffering from type 2 diabetes mellitus infections [34]. Bacteria that produce NDM-1 have been dealt with resistance also in other classes of antimicrobials and virulently restrict treatment options [34].

It is important to note that the clinical outcomes in patients with *P. aeruginosa* infection are poor, with a case fatality rate being higher in patients with MBL-producing *P. aeruginosa* [35]. A recently approved antibiotic is ceftolozane (cephalosporin), which is a combination with tazobactam, has shown a potent activity and has been used successfully for treatment of the urinary tract and intra-abdominal infections [36]. Furthermore, there was a report from an organ transplant unit where a 61-year-old lung transplant patient in Chicago had *Serratia marcescens (S. marcescens)* infection with imipenem resistance. Since beta-lactam antibiotics could not be used, several antibiotics were prescribed instead such as trimethoprim-sulfamethoxazole, itraconazole, ceftriaxone, cefepime and levofloxacin; this is clearly a costly exercise [37].

There is an observation that patients at a high risk for developing colonization with β-lactam resistance include both the severely ill and well-on patients. Likewise, patients with medical devices like urinary catheters are also prone to antibiotic-resistant bacteria [38]. Moreover, the length of hospital stay is another risk factor; patients who have stayed more than 3 days in hospitals and who have been previously treated with β-lactam antibiotics might be considered as a risk factor for the acquisition of β-lactamase resistance [39]. Furthermore, it has been reported in Italy that the risk factors for ESBL-producing *S. marcescens* and *K. pneumoniae* acquisition in neonatal ICU include low birthweight, gestational period and the use of invasive devices [40].

Finally, at molecular level, it is worth noting that there is a peptidoglycan layer which is critical for bacterial cell-wall structural wholeness and stability, particularly in Grampositive organisms, being the outermost and primary component of the wall [26]. During the final stage formation of the peptidoglycan, there is a transpeptidation step catalysed by DD-transpeptidases which are PBPs. β-lactam antibiotics inhibit these PBPs and ultimately lead to cell lysis [27]. It should be noted that PBPs are classified according to their molecular mass: the first category is low-molecular-mass PBPs that are monofunctional, for example, D-Ala-D-Ala carboxypeptidases; the second category is high-molecular-mass PBPs that are bifunctional enzymes containing a transpeptidase (D-Ala-D-Ala-dependent) and a transgly-

In most instances, particularly in case of life-threatening bacterial infections, antibiotics are the core of treatment [19]. Two of the main goals of β-lactam antibiotics are prevention and treatment of bacterial infections occasioned by susceptible bacteria [1]. Antimicrobial resistance has a greater risk for critically ill patients. For example, it is well known that ICUs are facing a major problem with β-lactam antibiotics [30]. Moreover, many case studies have been reported issues intensively with regard to resistance to the β-lactam antibiotics. A recent case was reported about extensively drug-resistant (particularly β-lactams) *Escherichia coli (E. coli),* which was isolated from the urine of a 63-year-old man from Phetchabun, Thailand. A craniotomy resulted that it is also difficult to treat infections like this via β-lactam antibiotics [31]. Another case was reported from an 87-year-old woman, who had clinical signs, for example, fever, dysuria and suprapubic pain. Urine culture produces a positive result for *Klebsiella pneumoniae* and *E. coli,* both resistant to multiple antibiotics including β-lactams [32]. β-lactam resistance can be seen in serious infections such as cystic fibrosis. Pollini and coworkers reported that a metallo-βlactamase-producing *P. aeruginosa* identified in a cystic fibrosis patient was resistant to carbapenems [33]. Furthermore, New Delhi β-lactamase-1 (NDM-1) producing *Enterobacteriaceae* infections have been found in patients suffering from type 2 diabetes mellitus infections [34]. Bacteria that produce NDM-1 have been dealt with resistance also in other classes of antimicro-

It is important to note that the clinical outcomes in patients with *P. aeruginosa* infection are poor, with a case fatality rate being higher in patients with MBL-producing *P. aeruginosa* [35]. A recently approved antibiotic is ceftolozane (cephalosporin), which is a combination with tazobactam, has shown a potent activity and has been used successfully for treatment of the urinary tract and intra-abdominal infections [36]. Furthermore, there was a report from an organ transplant unit where a 61-year-old lung transplant patient in Chicago had *Serratia marcescens (S. marcescens)* infection with imipenem resistance. Since beta-lactam antibiotics could not be used, several antibiotics were prescribed instead such as trimethoprim-sulfamethoxazole, itraconazole, ceftriaxone, cefepime and levofloxacin; this is clearly a costly exercise [37]. There is an observation that patients at a high risk for developing colonization with β-lactam resistance include both the severely ill and well-on patients. Likewise, patients with medical

**3. Specific cases of resistance to β-lactam antibiotics**

90 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

bials and virulently restrict treatment options [34].

cosylase [1, 28, 29].

Further observations have noted that clinical isolates that have ESBL-produced *E. coli* strains occur generally in hospitalized patients exposed to invasive procedures [41]. It is also noted that when antibiotics are cheap and accessible, this encourages their overuse and subsequent resistance [42]. It has been reported that there is a positive relationship between antibiotic consumption and the emergence and spreading of resistant bacterial strains. One of the most significant reasons of this is the lack of enforcement of legislation which result in the sale of antibiotics without a prescription in many countries [43].

## **4. Factors and mechanisms involved in resistance to β-lactam antibiotics**

Clinicians can prescribe beta-lactams without a real need; yet the health condition could have been treated by diet or rest. Sometimes, the consumers can take drugs without a medical advice or can take more doses than prescribed doses because of mental illnesses like Alzheimer's disease or dementia. Though some people know that antibiotics are used against bacterial infections, few are aware that antibiotics are not useful for viral infections [44]. When antibiotics are used in viral infections, they can trigger bacterial resistance while infections are not cured because of their viral background. Clinicians, surgeons, patients, consumers and caretakers as well require up-to-date information about the appropriate usage of β-lactam antibiotics because their misuse can cause severe conditions including bacterial resistance or allergic/toxic side effects [45].

It should be remembered that resistance to penicillin was noted in early 1940s; this finding has no clinical significance until the 1970s [20]. However, β-lactam resistance became a global crisis nowadays [46]. Resistance to the β-lactams usually occurs by three different mechanisms: decreased access of antimicrobials to the target PBPs (efflux pumps), altered PBPs (affinity of binding decreased) and β-lactamase production [1].

Although efflux pumps are found in almost all bacterial species, the β-lactamase production is the most efficient of the three mechanisms. This resistance mechanism generally depends on plasmids that include various virulence genes consisting of multiple β-lactamases of different classes in this way. This is why β-lactamase resistance can sprawl among various bacterial species. There are two types of β-lactamases: (a) serine-β-lactamases and (b) MBLs. Serine-β-lactamases comprise ESBLs and carbapenemases that hydrolyse carbapenem antibiotics and cephalosporins [2, 20]. We can suffice to say that the most common mechanism for drug resistance to β-lactam antibiotics is bacterial synthesis of β-lactamases. Many bacteria synthesize beta-lactamases that degrade beta-lactam antibiotics before they reach the cell wall. Gram-positive bacteria that produce beta-lactamase excrete the enzyme into the extracellular space. Gram-negative bacteria excrete beta-lactamase into the periplasmic space located between the cytoplasmic membrane and the outer membrane, where the cell wall is located; while the genes that encode beta-lactamases can be located on either (a) the bacterial chromosome; (b) plasmids; or (c) transposable elements which enhance the spread of betalactamases among different bacterial species [47].

ESBLs are implicated in serine β-lactamases [48]. ESBLs are able to hydrolyse extended-spectrum cephalosporin; thereof they are active against the β-lactam antibiotics such as ceftazidime, ceftriaxone and oxyimino-monobactams. ESBLs are generally produced by Gram-negative bacteria including *Enterobacteriaceae* such as *E. coli* and *K. pneumoniae* [57]. The types of ESBLs are TEM- β-lactamases, SHV-β-lactamases and CTX-M-type β-lactamases. TEM-type ESBLs are originated from TEM-1 and TEM-2; however, the number of TEM-type ESBLs is over 100. The most prevalent TEM-type ESBLs were detected in *E. coli* and *K. pneumoniae* [58]. SHV-type ESBLs are more widespread according to the TEM-type ESBLs and more than 100 SHV-type ESBLs are known around the world mainly reported from *Enterobacteriaceae*, *P. aeruginosa* and *Acinetobacter* spp. CTX-M-type ESBLs have the ability to hydrolyse cefotaxime and cefepime. In contrast to TEM and SHV enzymes, CXT-M-type ESBLs have no point mutations in their structures [58]. Until today, 128 different types of CTX-M were reported such as CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25. Likewise, the type of CTX-M-15 is the most

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The class B MBLs can be divided into four subclasses according to their structure: (a) B1aVIM, imipenemase (IMP), DIM, SPM; (b) B1b: NDM, (c) B2: cphA, (d) B3: LI and AIM [37, 60]. Moreover, Tripoli metallo-β-lactamase (TBM-1) was also included in MBLs [58, 61]. The earlier MBLs were reported in Japan in 1980s called imipenemase in *P. aeruginosa* [62]*.* After that, several varieties of IMP were reported such as Verona integron-encoded metallo-β-lactamase (VIM) and SPM-type enzymes and it was reported that VIM-producing bacteria are distributed intensely [63]. The New Delhi metallo-β-lactamase is a globally distributed enzyme discovered of late. NDM was reported for the first time in year 2009 and referred to as NDM-1. NDM-1 can bind and hydrolyse all beta-lactams with the exception of aztreonam. There are 13 different NDMs reported: NDM-1 to 14. The variation of these NDM types results from mutation within the gene encoding the β-lactamase [64]. #NDM-1 has drawn attention since the gene encoding these MBLs is located in a mobile genetic element and the pattern of spread proves to be more complex [65]. NDM can coexist with other antibiotic-resistant genes. Recently, plasmid-mediated NDM was reported in Thailand and coexisted with colistin resistance [31]. Co-carriage of ESBL, AmpC and NDM-1 genes among carbapenem-resistant *Enterobacteriaceae* in India [66] was reported. There are several MBL types reported occasionally. For instance, GIM-1 MBL was reported in a clinical isolate (*P. aeruginosa*) in Germany in year 2002, firstly. In recent years, GIM-1 was started to be reported in other bacterial species such as *S. marcescens*, *E. cloacae* and *Acinetobacter pittii* [60, 67]. Similarly, SIM-1 was obtained uncommonly and an integron-encoded blaSIM-1 was reported from *Acinetobacter baumannii* in Korea firstly [68]. Moreover, TMB-1 was reported in an *Achromobacter xylosoxidans* strain

There are several β-lactam antibiotics, and they have various side effects (**Table 1**). Although it is very hard to gather data on all the adverse effects and present them in this section, a description of toxic side effects known to be associated with the use of this category of antibi-

isolated from a hospital environment sample in Tripoli, Libya [69].

**5. Toxicity of β-lactam antibiotics**

otics is subsequently presented.

prevalent in *E. coli* strains [59].

Carbapenemases are distinct among the β-lactamases; they are able to hydrolyse most of the penicillins, cephalosporins and carbapenems. In 1980s and 1990s, carbapenems were considered as the 'last resort antibiotics' which used primarily against ESBL- or AmpC-producing bacteria. Molecular classes of A, B and D β-lactamases are known as carbapenemases. The carbapenemase resistance generally occurs in bacteria involving OprD porin loss, overexpression of efflux systems, overproduction of AmpC-type β-lactamase and acquisition of carbapenemase-encoding genes [46]. A and D enzymes are the group of carbapenemases having serine-based hydrolytic mechanisms; however, the group of B carbapenemases are known as MBLs [48]. Also, MBLs are inhibited by chelate-divalent cations like EDTA. The group A carbapenemases include members of the *S. marcescens enzym*e (SME), imipenem-hydrolysingβ-lactamase (IMI), not metallo-enzyme carbapenemase (NMC), Guiana-extended-spectrum (GES) and *K. pneumoniae* carbapenemases (KPC) families and their hydrolytic mechanism requires their active serine site at position 70 [37]. This feature gives them the ability to hydrolyse many β-lactam antibiotics like carbapenems, cephalosporins, penicillins and aztreonam, and all are inhibited by clavulanate and tazobactam [37]. SME-1 was first reported in England from two *S. marcescens* isolates. SME-1 has identical features with SME-2 and SME-3. SME-3 β-lactamases differentiate from SME-1 gene by a single amino acid substitution of tyrosine for histidine at position 105 [49], and SME-1 is encoded by chromosome in *blaSME−1* gene [37]. Since chromosomally encoded *blaSME−1* gene was not detected in any plasmids, mobile genetic elements can be concluded that there is a limitation of SME-1 enzyme distribution [50]. The IMI and NMC-A enzymes have been found in clinical isolates of *Enterobacter cloacae* from the United States, Croatia, Finland and France. Most *blaIMI−1* genes are located on chromosome and it is related to *imi-R* gene that limits their dissemination and their expression at a high level [51]. NMC-A and IMI-1 have 97% amino acid similarity and they are similar to the SME-1, with approximately 70% of amino acid identity encoded by bacterial genome B [52]. The GES family enzymes consist of 26 variants of GES. For example, GES-1 was reported from *K. pneumoniae* strain in year 2000. The other GES enzymes are the following: GES-2, GES-4, GES-5, GES-6, GES-11, GES-14 and GES-18 are able to hydrolyse imipenem. Among these enzymes, GES-2 and GES-5 have carbapenemase activity. *K. pneumoniae* carbapenemases are another carbepenemases, identified in *K. pneumoniae*. These enzymes are known as one of the most significant enzymes due to their effectiveness to the carbapenems [53].

OXA-type β-lactamases include Group D carbapenemases and OXA referred to oxacillinases since they hydrolyse isoxazolyl penicillin oxacillin [51]. Nowadays, OXA-type enzymes contain over 400 enzymes. With regard to their amino acid sequences, OXA-type enzymes have 12 subgroups accommodating OXA-23, OXA-24/40, OXA-48, OXA-51, OXA-58, OXA-134a, OXA-143, OXA-211, OXA-213, OXA-214, OXA-229 and OXA-235 [54]. In spite of OXA enzymes that were generally detected in *Acinetobacter* species, these enzymes were started to be reported in other *Enterobacteriaceae* members such as *Salmonella* spp. and *P. aeruginosa;* this showed that OXA-type β-lactamases can spread to the *Enterobacteriaceae* members [54–56].

ESBLs are implicated in serine β-lactamases [48]. ESBLs are able to hydrolyse extended-spectrum cephalosporin; thereof they are active against the β-lactam antibiotics such as ceftazidime, ceftriaxone and oxyimino-monobactams. ESBLs are generally produced by Gram-negative bacteria including *Enterobacteriaceae* such as *E. coli* and *K. pneumoniae* [57]. The types of ESBLs are TEM- β-lactamases, SHV-β-lactamases and CTX-M-type β-lactamases. TEM-type ESBLs are originated from TEM-1 and TEM-2; however, the number of TEM-type ESBLs is over 100. The most prevalent TEM-type ESBLs were detected in *E. coli* and *K. pneumoniae* [58]. SHV-type ESBLs are more widespread according to the TEM-type ESBLs and more than 100 SHV-type ESBLs are known around the world mainly reported from *Enterobacteriaceae*, *P. aeruginosa* and *Acinetobacter* spp. CTX-M-type ESBLs have the ability to hydrolyse cefotaxime and cefepime. In contrast to TEM and SHV enzymes, CXT-M-type ESBLs have no point mutations in their structures [58]. Until today, 128 different types of CTX-M were reported such as CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25. Likewise, the type of CTX-M-15 is the most prevalent in *E. coli* strains [59].

The class B MBLs can be divided into four subclasses according to their structure: (a) B1aVIM, imipenemase (IMP), DIM, SPM; (b) B1b: NDM, (c) B2: cphA, (d) B3: LI and AIM [37, 60]. Moreover, Tripoli metallo-β-lactamase (TBM-1) was also included in MBLs [58, 61]. The earlier MBLs were reported in Japan in 1980s called imipenemase in *P. aeruginosa* [62]*.* After that, several varieties of IMP were reported such as Verona integron-encoded metallo-β-lactamase (VIM) and SPM-type enzymes and it was reported that VIM-producing bacteria are distributed intensely [63]. The New Delhi metallo-β-lactamase is a globally distributed enzyme discovered of late. NDM was reported for the first time in year 2009 and referred to as NDM-1. NDM-1 can bind and hydrolyse all beta-lactams with the exception of aztreonam. There are 13 different NDMs reported: NDM-1 to 14. The variation of these NDM types results from mutation within the gene encoding the β-lactamase [64]. #NDM-1 has drawn attention since the gene encoding these MBLs is located in a mobile genetic element and the pattern of spread proves to be more complex [65]. NDM can coexist with other antibiotic-resistant genes. Recently, plasmid-mediated NDM was reported in Thailand and coexisted with colistin resistance [31]. Co-carriage of ESBL, AmpC and NDM-1 genes among carbapenem-resistant *Enterobacteriaceae* in India [66] was reported. There are several MBL types reported occasionally. For instance, GIM-1 MBL was reported in a clinical isolate (*P. aeruginosa*) in Germany in year 2002, firstly. In recent years, GIM-1 was started to be reported in other bacterial species such as *S. marcescens*, *E. cloacae* and *Acinetobacter pittii* [60, 67]. Similarly, SIM-1 was obtained uncommonly and an integron-encoded blaSIM-1 was reported from *Acinetobacter baumannii* in Korea firstly [68]. Moreover, TMB-1 was reported in an *Achromobacter xylosoxidans* strain isolated from a hospital environment sample in Tripoli, Libya [69].

## **5. Toxicity of β-lactam antibiotics**

extracellular space. Gram-negative bacteria excrete beta-lactamase into the periplasmic space located between the cytoplasmic membrane and the outer membrane, where the cell wall is located; while the genes that encode beta-lactamases can be located on either (a) the bacterial chromosome; (b) plasmids; or (c) transposable elements which enhance the spread of beta-

Carbapenemases are distinct among the β-lactamases; they are able to hydrolyse most of the penicillins, cephalosporins and carbapenems. In 1980s and 1990s, carbapenems were considered as the 'last resort antibiotics' which used primarily against ESBL- or AmpC-producing bacteria. Molecular classes of A, B and D β-lactamases are known as carbapenemases. The carbapenemase resistance generally occurs in bacteria involving OprD porin loss, overexpression of efflux systems, overproduction of AmpC-type β-lactamase and acquisition of carbapenemase-encoding genes [46]. A and D enzymes are the group of carbapenemases having serine-based hydrolytic mechanisms; however, the group of B carbapenemases are known as MBLs [48]. Also, MBLs are inhibited by chelate-divalent cations like EDTA. The group A carbapenemases include members of the *S. marcescens enzym*e (SME), imipenem-hydrolysingβ-lactamase (IMI), not metallo-enzyme carbapenemase (NMC), Guiana-extended-spectrum (GES) and *K. pneumoniae* carbapenemases (KPC) families and their hydrolytic mechanism requires their active serine site at position 70 [37]. This feature gives them the ability to hydrolyse many β-lactam antibiotics like carbapenems, cephalosporins, penicillins and aztreonam, and all are inhibited by clavulanate and tazobactam [37]. SME-1 was first reported in England from two *S. marcescens* isolates. SME-1 has identical features with SME-2 and SME-3. SME-3 β-lactamases differentiate from SME-1 gene by a single amino acid substitution of tyrosine for histidine at position 105 [49], and SME-1 is encoded by chromosome in *blaSME−1* gene [37]. Since chromosomally encoded *blaSME−1* gene was not detected in any plasmids, mobile genetic elements can be concluded that there is a limitation of SME-1 enzyme distribution [50]. The IMI and NMC-A enzymes have been found in clinical isolates of *Enterobacter cloacae* from the United States, Croatia, Finland and France. Most *blaIMI−1* genes are located on chromosome and it is related to *imi-R* gene that limits their dissemination and their expression at a high level [51]. NMC-A and IMI-1 have 97% amino acid similarity and they are similar to the SME-1, with approximately 70% of amino acid identity encoded by bacterial genome B [52]. The GES family enzymes consist of 26 variants of GES. For example, GES-1 was reported from *K. pneumoniae* strain in year 2000. The other GES enzymes are the following: GES-2, GES-4, GES-5, GES-6, GES-11, GES-14 and GES-18 are able to hydrolyse imipenem. Among these enzymes, GES-2 and GES-5 have carbapenemase activity. *K. pneumoniae* carbapenemases are another carbepenemases, identified in *K. pneumoniae*. These enzymes are known as one of the most

lactamases among different bacterial species [47].

92 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

significant enzymes due to their effectiveness to the carbapenems [53].

OXA-type β-lactamases include Group D carbapenemases and OXA referred to oxacillinases since they hydrolyse isoxazolyl penicillin oxacillin [51]. Nowadays, OXA-type enzymes contain over 400 enzymes. With regard to their amino acid sequences, OXA-type enzymes have 12 subgroups accommodating OXA-23, OXA-24/40, OXA-48, OXA-51, OXA-58, OXA-134a, OXA-143, OXA-211, OXA-213, OXA-214, OXA-229 and OXA-235 [54]. In spite of OXA enzymes that were generally detected in *Acinetobacter* species, these enzymes were started to be reported in other *Enterobacteriaceae* members such as *Salmonella* spp. and *P. aeruginosa;* this showed that OXA-type β-lactamases can spread to the *Enterobacteriaceae* members [54–56].

There are several β-lactam antibiotics, and they have various side effects (**Table 1**). Although it is very hard to gather data on all the adverse effects and present them in this section, a description of toxic side effects known to be associated with the use of this category of antibiotics is subsequently presented.


was given in normal dosing ranges but it induced a retinal toxicity; fortunately, the resulting visual loss was recovered after a week [73]. Neurotoxicity induced by β-lactams can be a result of renal failure, which increases the amount of the antibiotic in the circulating blood. Hence, even in normal dosing ranges, β-lactam antibiotics pose risks in case of renal failure [74]. The relationship between the nervous system and β-lactams is not just the toxicity, but the molecular interaction may also have positive consequences. β-lactams may have neuroprotective roles in some instances [75]. The molecular glutamate mechanism takes place for that protection [76]. Also, β-lactams can help in treating ischaemic rat brain during the acute phase [77]. The effect of β-lactams on the glutamate receptors affects the lab animals' behaviour. Rats' dependence on alcohol and morphine may be decreased with the β-lactam

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Nephrotoxicity is a very serious side effect of antibiotics generally. β-lactam antibiotics are both dangerous as a mono therapy or as a combination therapy agent [81]. These chemicals induce toxicity in kidneys via a couple of molecular mechanisms. Tubular cells are under threat because of the excess active transport from blood to these cells; however, less efflux and accumulation happens. The other mechanism is acylation of target proteins, which cause respiratory arrest by inactivation of mitochondrial anionic substrate carriers in cells. The other nephrotoxic side effects occur via lipid peroxidation of renal cortex [11, 82]. For critically ill patients, it is very dangerous to use antibiotics such as tazobactam with piperacillin because

Genotoxic effects of some β-lactams have been shown in some studies done in vitro. Ceftazidime is toxic to bone marrow stromal cells via DNA polymerase inhibition [84]. Ceftriaxone genotoxicity was shown in human peripheral blood lymphocytes, while amoxicillin genotoxicity was studied with both human lymphocytes and gastric mucosa cells result-

Also, β-lactam antibiotics have toxic effects on the urogenital system. A synthetic β-lactam caused urothelial hyperplasia in rats but scientist suggested that chemical is not toxic to human [87]. In a case study, it has been reported that penicillin-G induced haemorrhagic cystitis, but the patient recovered in 8 days [88]. Some of the β-lactams toxicity grades and

After any toxic reaction in an organism, the resulting defects could be very severe and cannot be reversed; hence, there is a need for special care for recovery. When it is established that there is no recovery, substituting the toxic chemical or product can help reduce the risk of

The potential strategies to overcome β-lactam antibiotic-triggered toxicity are as follows:

**6. Strategies to overcome β-lactam antibiotic-triggered toxicity**

**1.** Replacing the toxic β-lactam with a non-allergic/toxic one,

**2.** Using phage therapy instead of chemicals,

application [78–80].

side effects.

of the toxic effect on renal tubule [83].

ing in β-lactams having genotoxicity risk [85, 86].

possible adverse effects were shown in **Table 1**.

**Table 1.** Illustrative adverse effects of β-lactam antibiotics.

The use of β-lactam antibiotics has been linked to triggering allergic reactions like urticaria, bronchoconstriction, also severe conditions like immune-mediated haemolytic anaemia and intravascular haemolysis [70]. It is known that some β-lactam antibiotics are neurotoxic, some are nephrotoxic, some are genotoxic and some are toxic to urogenital system.

Neurotoxic side effects of β-lactam antibiotics are well-known conditions for decades. The administration route and the dose of antibiotic are important factors that determine whether a neurological dysfunction would occur. β-lactam antibiotics can trigger epilepsy or seizures because of their chemical structures of β-lactams that make them capable of binding to the gamma-aminobutyric acid (GABA) receptors in the brain. Some of the β-lactams are GABA receptor antagonists [7]. This is the reason why penicillin injection can be used as an epilepsy model in rats [71]. In a case report, it was proposed that β-lactams triggered a tardive seizure in a patient after an electroconvulsive therapy [72]. It has been noted also that the retina is a target for neurotoxic pathologies. In a case report after the cataract surgery, cefuroxime was given in normal dosing ranges but it induced a retinal toxicity; fortunately, the resulting visual loss was recovered after a week [73]. Neurotoxicity induced by β-lactams can be a result of renal failure, which increases the amount of the antibiotic in the circulating blood. Hence, even in normal dosing ranges, β-lactam antibiotics pose risks in case of renal failure [74]. The relationship between the nervous system and β-lactams is not just the toxicity, but the molecular interaction may also have positive consequences. β-lactams may have neuroprotective roles in some instances [75]. The molecular glutamate mechanism takes place for that protection [76]. Also, β-lactams can help in treating ischaemic rat brain during the acute phase [77]. The effect of β-lactams on the glutamate receptors affects the lab animals' behaviour. Rats' dependence on alcohol and morphine may be decreased with the β-lactam application [78–80].

Nephrotoxicity is a very serious side effect of antibiotics generally. β-lactam antibiotics are both dangerous as a mono therapy or as a combination therapy agent [81]. These chemicals induce toxicity in kidneys via a couple of molecular mechanisms. Tubular cells are under threat because of the excess active transport from blood to these cells; however, less efflux and accumulation happens. The other mechanism is acylation of target proteins, which cause respiratory arrest by inactivation of mitochondrial anionic substrate carriers in cells. The other nephrotoxic side effects occur via lipid peroxidation of renal cortex [11, 82]. For critically ill patients, it is very dangerous to use antibiotics such as tazobactam with piperacillin because of the toxic effect on renal tubule [83].

Genotoxic effects of some β-lactams have been shown in some studies done in vitro. Ceftazidime is toxic to bone marrow stromal cells via DNA polymerase inhibition [84]. Ceftriaxone genotoxicity was shown in human peripheral blood lymphocytes, while amoxicillin genotoxicity was studied with both human lymphocytes and gastric mucosa cells resulting in β-lactams having genotoxicity risk [85, 86].

Also, β-lactam antibiotics have toxic effects on the urogenital system. A synthetic β-lactam caused urothelial hyperplasia in rats but scientist suggested that chemical is not toxic to human [87]. In a case study, it has been reported that penicillin-G induced haemorrhagic cystitis, but the patient recovered in 8 days [88]. Some of the β-lactams toxicity grades and possible adverse effects were shown in **Table 1**.

## **6. Strategies to overcome β-lactam antibiotic-triggered toxicity**

After any toxic reaction in an organism, the resulting defects could be very severe and cannot be reversed; hence, there is a need for special care for recovery. When it is established that there is no recovery, substituting the toxic chemical or product can help reduce the risk of side effects.

The potential strategies to overcome β-lactam antibiotic-triggered toxicity are as follows:


The use of β-lactam antibiotics has been linked to triggering allergic reactions like urticaria, bronchoconstriction, also severe conditions like immune-mediated haemolytic anaemia and intravascular haemolysis [70]. It is known that some β-lactam antibiotics are neurotoxic, some

Neurotoxic side effects of β-lactam antibiotics are well-known conditions for decades. The administration route and the dose of antibiotic are important factors that determine whether a neurological dysfunction would occur. β-lactam antibiotics can trigger epilepsy or seizures because of their chemical structures of β-lactams that make them capable of binding to the gamma-aminobutyric acid (GABA) receptors in the brain. Some of the β-lactams are GABA receptor antagonists [7]. This is the reason why penicillin injection can be used as an epilepsy model in rats [71]. In a case report, it was proposed that β-lactams triggered a tardive seizure in a patient after an electroconvulsive therapy [72]. It has been noted also that the retina is a target for neurotoxic pathologies. In a case report after the cataract surgery, cefuroxime

are nephrotoxic, some are genotoxic and some are toxic to urogenital system.

**Antibiotic Adverse/side effects Toxicity grade Populations-**

94 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

Regenerative anaemia

**Table 1.** Illustrative adverse effects of β-lactam antibiotics.

L-695,256 is a novel 2-fluorenonyl carbapenem

L-695,256 is a novel 2-fluorenonyl carbapenem

FCE 22891 a penem

antibiotic

**circumstances**

Mild Squirrel monkeys Experimental [70]

Haemolytic anaemia Moderate Rhesus monkeys Experimental [70]

Hemorrhagic cystitis Moderate Infection patient adult Case [88]

ECT taken

ECT taken

infectious patients

infectious patients

Genotoxicity Moderate Human lymphocytes Experimental [85]

Urotoxicity Severe Rats Experimental [87]

Cephaloglycin Nephrotoxicity Moderate Rabbit kidney extracts Experimental [13] Imipenem Nephrotoxicity Severe Rabbit kidney extracts Experimental [13]

Penicillin Epileptic seizures Moderate Rats Experimental [71]

Piperacillin Nephrotoxicity Moderate 1200 patients Randomized

Cefotiam Convulsion Mild Schizophrenic adult,

Cefazolin Nephrotoxicity Mild Hospitalized adult

Ceftriaxone Nephrotoxicity Mild Hospitalized adult

Convulsion Mild Schizophrenic adult,

Cefuroxime Retinal toxicity Mild Cataract patient adult Case [73]

Amoxicillin Genotoxicity Severe Human gastric cells Experimental [86]

**Type of study References**

control trial

Case [72]

Case [72]

Case-control [81]

Case-control [81]

[83]


In implementing the substitution strategy, one way is to use other available β-lactam antibiotics for clinical use called ESBLs such as cephalosporins, carbapenem, imipenem, monobactam and aztreonam [2]. Another strategy is designing/choosing a β-lactamase inhibitor which makes it possible to use a smaller dose of or a mild β-lactam. Clavulanic acid, tazobactam and sulbactam are known inhibitors that are used for this purpose [2, 89].

are neurotoxic, some are nephrotoxic, some are genotoxic and some are toxic to urogenital system. Several factors are involved in the occurrence of toxic effects including the dosage and the renal status. Several strategies to overcome β-lactam antibiotics triggered toxicity include rational prescribing, substitution combination and phage therapy which seems promising. Public health education for clinical teams and patients is essential in ensuring that this group

Toxicity of β-Lactam Antibiotics: Pathophysiology, Molecular Biology and Possible Recovery...

http://dx.doi.org/10.5772/intechopen.70199

97

1 Department of Biology, Faculty of Science, Basic and Industrial Microbiology Section,

2 Department of Biology, Faculty of Science, General Biology Section, Ege University, Izmir,

[1] Bush K, Bradford PA. Beta-lactams and beta-lactamase inhibitors: An overview. Cold Spring Harbor Perspectives in Medicine. 2016;**6**: pii: a025247. DOI: 10.1101/cshperspect.a025247 [2] Konaklieva MI. Molecular targets of beta-lactam-based antimicrobials: Beyond the usual

[3] Watkins RR, Papp-Wallace KM, Drawz SM, Bonomo RA. Novel beta-lactamase inhibitors: A therapeutic hope against the scourge of multidrug resistance. Frontiers in

[4] Cai XF, Sun JM, Bao LS, Li WB. Risk factors and antibiotic resistance of pneumonia caused by multidrug resistant *Acinetobacter baumannii* in pediatric intensive care unit. World Journal of Emergency Medicine. 2012;**3**(3):202-207. DOI: 10.5847/wjem.j.i

[5] Mereuta AI, Tuchilus C, Badescu AC, Iancu LS. Metallo-beta-lactamase-mediated resistance among carbapenem-resistant *Pseudomonas aeruginosa* clinical isolates. Revista medico-chirurgicala a Societatii de Medici si Naturalisti din Iasi. 2011;**115**(4):1208-1213 [6] Lagace-Wiens P, Rubinstein E. Adverse reactions to beta-lactam antimicrobials. Expert Opinion on Drug Safety. 2012;**11**(3):381-399. DOI: 10.1517/14740338.2012.643866

[7] Chow KM, Hui AC, Szeto CC. Neurotoxicity induced by beta-lactam antibiotics: From bench to bedside. European Journal of Clinical Microbiology & Infectious Diseases.

suspects. Antibiotics. 2014;**3**:128. DOI: 10.3390/antibiotics3020128

Microbiology. 2013;**4**:392. DOI: 10.3389/fmicb.2013.00392

2005;**24**(10):649-653. DOI: 10.1007/s10096-005-0021-y

of antibiotics are retained in therapeutics.

\* and Melih Dagdeviren<sup>2</sup>

Istanbul University, Istanbul, Turkey

ssn.1920-8642.2012.03008

\*Address all correspondence to: elif.bozcal@istanbul.edu.tr

**Author details**

Elif Bozcal<sup>1</sup>

Turkey

**References**

With regard to phage therapy, it has always been an alternative to the antibiotics; however, the concerns about the production cost have made it difficult. The phage therapy has been kept on the shelves for decades: a less toxic option which was as effective as antibiotics [90]. But it is nowadays on the table as a potential replacement for β-lactam antibiotics in the near future. It has been successfully applied to cultured African catfish, which were infected by *P. aeruginosa* and positive results have evaluated, while there was a resistance for β-lactam antibiotics [15], and phage therapy may be a logical substitute for β-lactam antibiotics in the near future.

With regard to combination strategy, β-lactam antibiotics have been used with aminoglycosides and they created a synergistic effect, which helped to reduce the doses required for both groups. This strategy has been able to increase the efficiency of the β-lactam antibiotics [91–94]. But clinicians should be aware that, because of a possibility of unexpected adverse effects, dialysis facilities should be available [95].

With regard to rational prescribing, clinical teams including doctors, nurses and pharmacists need to work hand in hand in order to select, purchase, control, restrict and ensure that patients are prescribed β-lactam antibiotics only when needed and that the patients must be counselled on the appropriate use thereof. Considering preventive medicine perspective, it is cheaper trying to limit the misuse/abuse of β-lactam antibiotics.

## **7. Concluding remarks**

The above review has highlighted that β-lactam antibiotics are a group of products that have a chemical structure characterized by a β-lactam ring and are one of the most common antibacterial agents. However, due to inappropriate use including abuse and misuse, resistance to the β-lactam antibiotics is currently a global crisis. It usually occurs by three different mechanisms: decreased access of antimicrobials to the target PBPs (efflux pumps), altered PBPs (affinity of binding decreased) and β-lactamase production.

Moreover, even when used appropriately, they have been linked to triggering allergic reactions like urticaria, bronchoconstriction, also severe conditions like immune-mediated haemolytic anaemia and intravascular haemolysis [70]. It is known that some β-lactam antibiotics are neurotoxic, some are nephrotoxic, some are genotoxic and some are toxic to urogenital system. Several factors are involved in the occurrence of toxic effects including the dosage and the renal status. Several strategies to overcome β-lactam antibiotics triggered toxicity include rational prescribing, substitution combination and phage therapy which seems promising. Public health education for clinical teams and patients is essential in ensuring that this group of antibiotics are retained in therapeutics.

## **Author details**

**3.** Using β-lactamase inhibitors,

**4.** Using other chemicals in combination with β-lactams,

96 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

**6.** Rational drug prescribing and treatment monitoring.

sulbactam are known inhibitors that are used for this purpose [2, 89].

In implementing the substitution strategy, one way is to use other available β-lactam antibiotics for clinical use called ESBLs such as cephalosporins, carbapenem, imipenem, monobactam and aztreonam [2]. Another strategy is designing/choosing a β-lactamase inhibitor which makes it possible to use a smaller dose of or a mild β-lactam. Clavulanic acid, tazobactam and

With regard to phage therapy, it has always been an alternative to the antibiotics; however, the concerns about the production cost have made it difficult. The phage therapy has been kept on the shelves for decades: a less toxic option which was as effective as antibiotics [90]. But it is nowadays on the table as a potential replacement for β-lactam antibiotics in the near future. It has been successfully applied to cultured African catfish, which were infected by *P. aeruginosa* and positive results have evaluated, while there was a resistance for β-lactam antibiotics [15], and phage therapy may be a logical substitute for β-lactam antibiotics in the near future.

With regard to combination strategy, β-lactam antibiotics have been used with aminoglycosides and they created a synergistic effect, which helped to reduce the doses required for both groups. This strategy has been able to increase the efficiency of the β-lactam antibiotics [91–94]. But clinicians should be aware that, because of a possibility of unexpected adverse

With regard to rational prescribing, clinical teams including doctors, nurses and pharmacists need to work hand in hand in order to select, purchase, control, restrict and ensure that patients are prescribed β-lactam antibiotics only when needed and that the patients must be counselled on the appropriate use thereof. Considering preventive medicine perspective, it is

The above review has highlighted that β-lactam antibiotics are a group of products that have a chemical structure characterized by a β-lactam ring and are one of the most common antibacterial agents. However, due to inappropriate use including abuse and misuse, resistance to the β-lactam antibiotics is currently a global crisis. It usually occurs by three different mechanisms: decreased access of antimicrobials to the target PBPs (efflux pumps), altered PBPs

Moreover, even when used appropriately, they have been linked to triggering allergic reactions like urticaria, bronchoconstriction, also severe conditions like immune-mediated haemolytic anaemia and intravascular haemolysis [70]. It is known that some β-lactam antibiotics

**5.** Performing a dialysis (for very severe cases),

effects, dialysis facilities should be available [95].

**7. Concluding remarks**

cheaper trying to limit the misuse/abuse of β-lactam antibiotics.

(affinity of binding decreased) and β-lactamase production.

Elif Bozcal<sup>1</sup> \* and Melih Dagdeviren<sup>2</sup>

\*Address all correspondence to: elif.bozcal@istanbul.edu.tr

1 Department of Biology, Faculty of Science, Basic and Industrial Microbiology Section, Istanbul University, Istanbul, Turkey

2 Department of Biology, Faculty of Science, General Biology Section, Ege University, Izmir, Turkey

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**Chapter 6**

**Provisional chapter**

**Acute Poisoning with Neonicotinoid Insecticide**

**Acute Poisoning with Neonicotinoid Insecticide**

DOI: 10.5772/intechopen.72004

Neonicotinoids are a class of insecticides considered less toxic to humans than organophosphates, carbamates, organochloride and pyrethroids. The purpose of this chapter was to systematize existing data in the literature on acute intoxication with neonicotinoids to help practitioners. Clinical manifestations vary across different human systems. Gastrointestinal symptoms consist of nausea, vomiting, abdominal pain and corrosive lesions. In the central nervous system, headaches, agitation, confusion, fasciculations, seizures or coma may occur, while tachycardia or bradycardia, hypertension, hypotension and palpitations occur in the cardiovascular system. Respiratory effects are dyspnea, aspiration pneumonia or respiratory failure. Solvents that drive the insecticide also have an important role in the toxic effects. There are no specific biological tests of neonicotinoid intoxication, and their dosing is not routinely available. Treatment is symptomatic. Mortality is less than 3%, well below the poisoning with anticholinesterase insecticides,

**Keywords:** neonicotinoids, insecticides, poisoning, cardiovascular symptoms

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Poisoning with insecticides is a public health problem in many countries [1]. Organophosphates and carbamates are the principal cause of serious poisonings, sometimes leading to death. Due to the high toxicity of these compounds, new insecticides called neonicotinoids (syn-

The term "neonicotinoid" was initially used by Izuru Yamamoto for imidacloprid and related insecticides to differentiate the new insecticidal active compounds of n-AChRs from older

Nicolai Nistor, Otilia Elena Frăsinariu and

Nicolai Nistor, Otilia Elena Frăsinariu and

Additional information is available at the end of the chapter

like organophosphates and carbamates.

thetic analogs of nicotine) have been created [2].

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72004

Violeta Ştreangă

**Abstract**

**1. Introduction**

nicotine insecticides [3].

Violeta Ştreangă

**Provisional chapter**

## **Acute Poisoning with Neonicotinoid Insecticide**

**Acute Poisoning with Neonicotinoid Insecticide**

DOI: 10.5772/intechopen.72004

Nicolai Nistor, Otilia Elena Frăsinariu and Violeta Ştreangă Violeta Ştreangă Additional information is available at the end of the chapter

Nicolai Nistor, Otilia Elena Frăsinariu and

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72004

#### **Abstract**

Neonicotinoids are a class of insecticides considered less toxic to humans than organophosphates, carbamates, organochloride and pyrethroids. The purpose of this chapter was to systematize existing data in the literature on acute intoxication with neonicotinoids to help practitioners. Clinical manifestations vary across different human systems. Gastrointestinal symptoms consist of nausea, vomiting, abdominal pain and corrosive lesions. In the central nervous system, headaches, agitation, confusion, fasciculations, seizures or coma may occur, while tachycardia or bradycardia, hypertension, hypotension and palpitations occur in the cardiovascular system. Respiratory effects are dyspnea, aspiration pneumonia or respiratory failure. Solvents that drive the insecticide also have an important role in the toxic effects. There are no specific biological tests of neonicotinoid intoxication, and their dosing is not routinely available. Treatment is symptomatic. Mortality is less than 3%, well below the poisoning with anticholinesterase insecticides, like organophosphates and carbamates.

**Keywords:** neonicotinoids, insecticides, poisoning, cardiovascular symptoms

## **1. Introduction**

Poisoning with insecticides is a public health problem in many countries [1]. Organophosphates and carbamates are the principal cause of serious poisonings, sometimes leading to death. Due to the high toxicity of these compounds, new insecticides called neonicotinoids (synthetic analogs of nicotine) have been created [2].

The term "neonicotinoid" was initially used by Izuru Yamamoto for imidacloprid and related insecticides to differentiate the new insecticidal active compounds of n-AChRs from older nicotine insecticides [3].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Neonicotinoids are used in agriculture, horticulture and forestry to combat various pests. They are also used to combat fleas in domestic animals, as well as against household pests. After spraying, they act by direct contact with the insects, or by ingestion, when the insects pierce or consume the vegetative parts of plants [4]. Neonicotinoids are developed and continue to be launched on the market, often in the absence of direct human toxicity data. The human toxicity of these insecticides is often extrapolated from studies on animals, whose relevance is unclear. Therefore, more studies on acute intoxications with this class of insecticides are needed. The information resulting from these studies can contribute to the risk assessment and management of patients with neonicotinoid intoxications and support the decisions of the regulatory agencies for these substances [5]. The insecticidal activity of neonicotinoids results mainly from the agonist effect on the insects' postsynaptic nicotinic receptors for acetylcholine. They are attached to the nicotinic receptors of the postsynaptic membranes from both nerve and muscle cells and thus disrupt the transmission of the nervous influx into the central and peripheral nervous system. Additionally, this interaction with the nicotinic receptors determines their desensitization, leading to a loss of synaptic transmission of the nervous impulse [6, 7]. In recent years, some studies have suggested that neonicotinoids have a negative impact on bees near crops exposed to neonicotinoids. It is known that exposure to thiamethoxam may cause bees to be disoriented [8, 9]. In 2013, the European Food Safety Agency published a report confirming that neonicotinoids pose a risk to bees and pollinators. For this reason, under the precautionary principle, the European Commission has decided to temporarily suspend the use of three neonicotinoid substances (imidacloprid, clothianidin and thiamethoxam) for seed treatment in the agriculture of all EU member States [10]. However, in some countries, based on derogations from the ministry, they are still used.

**2. Physicochemical properties of neonicotinoids**

**Figure 1.** Common names and molecular structures of the neonicotinoids [14].

Neonicotinoids are classified by the EPA as both Class II and Class III agents and are labeled with the signal word "Warning" or "Caution." Imidacloprid, the first neonicotinoid insecticide discovered in 1994 in Japan, is a structural analogue of nicotine derived from N-nitroguanidine

Acute Poisoning with Neonicotinoid Insecticide http://dx.doi.org/10.5772/intechopen.72004 109

In a large study conducted recently in three countries in Europe (the United Kingdom, Germany and Hungary), the results were contradictory: in the United Kingdom and Hungary, neonicotinoids had a negative impact on bees, while in Germany they did not seem to have affected their health status [11]. The findings of another recent Canadian study, conducted in Ontario and Quebec, are that neonicotinoids have negative effects on bees, including a 23% decrease in their life span [12]. It is therefore necessary to continue the studies on this subject in order to reach a clear and definitive scientific conclusion. Neonicotinoid insecticides are considered to have low toxicity in humans because they interact much less with nicotinic receptors in vertebrates than in insects and penetrate less the blood-brain barrier. Provided that they are less toxic in humans, neonicotinoid insecticides have become increasingly used throughout the world. However, the ingestion of large amounts of these insecticides has been associated with the occurrence of severe poisoning [13].

Hence, this review was performed in order to clarify some aspects of the diagnosis and treatment of poisonings with neonicotinoids, useful for practitioners who face such cases and maybe helping improve management of these intoxications. The physicochemical properties, toxicokinetics, experimental data and mechanism of action of neonicotinoids, clinical symptoms, diagnosis, treatment and prognosis of acute intoxication with this relatively new class of insecticides are discussed below.

## **2. Physicochemical properties of neonicotinoids**

Neonicotinoids are used in agriculture, horticulture and forestry to combat various pests. They are also used to combat fleas in domestic animals, as well as against household pests. After spraying, they act by direct contact with the insects, or by ingestion, when the insects pierce or consume the vegetative parts of plants [4]. Neonicotinoids are developed and continue to be launched on the market, often in the absence of direct human toxicity data. The human toxicity of these insecticides is often extrapolated from studies on animals, whose relevance is unclear. Therefore, more studies on acute intoxications with this class of insecticides are needed. The information resulting from these studies can contribute to the risk assessment and management of patients with neonicotinoid intoxications and support the decisions of the regulatory agencies for these substances [5]. The insecticidal activity of neonicotinoids results mainly from the agonist effect on the insects' postsynaptic nicotinic receptors for acetylcholine. They are attached to the nicotinic receptors of the postsynaptic membranes from both nerve and muscle cells and thus disrupt the transmission of the nervous influx into the central and peripheral nervous system. Additionally, this interaction with the nicotinic receptors determines their desensitization, leading to a loss of synaptic transmission of the nervous impulse [6, 7]. In recent years, some studies have suggested that neonicotinoids have a negative impact on bees near crops exposed to neonicotinoids. It is known that exposure to thiamethoxam may cause bees to be disoriented [8, 9]. In 2013, the European Food Safety Agency published a report confirming that neonicotinoids pose a risk to bees and pollinators. For this reason, under the precautionary principle, the European Commission has decided to temporarily suspend the use of three neonicotinoid substances (imidacloprid, clothianidin and thiamethoxam) for seed treatment in the agriculture of all EU member States [10]. However, in some countries, based on derogations

108 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

In a large study conducted recently in three countries in Europe (the United Kingdom, Germany and Hungary), the results were contradictory: in the United Kingdom and Hungary, neonicotinoids had a negative impact on bees, while in Germany they did not seem to have affected their health status [11]. The findings of another recent Canadian study, conducted in Ontario and Quebec, are that neonicotinoids have negative effects on bees, including a 23% decrease in their life span [12]. It is therefore necessary to continue the studies on this subject in order to reach a clear and definitive scientific conclusion. Neonicotinoid insecticides are considered to have low toxicity in humans because they interact much less with nicotinic receptors in vertebrates than in insects and penetrate less the blood-brain barrier. Provided that they are less toxic in humans, neonicotinoid insecticides have become increasingly used throughout the world. However, the ingestion of large amounts of these insecticides has been

Hence, this review was performed in order to clarify some aspects of the diagnosis and treatment of poisonings with neonicotinoids, useful for practitioners who face such cases and maybe helping improve management of these intoxications. The physicochemical properties, toxicokinetics, experimental data and mechanism of action of neonicotinoids, clinical symptoms, diagnosis, treatment and prognosis of acute intoxication with this relatively new class

from the ministry, they are still used.

of insecticides are discussed below.

associated with the occurrence of severe poisoning [13].

Neonicotinoids are classified by the EPA as both Class II and Class III agents and are labeled with the signal word "Warning" or "Caution." Imidacloprid, the first neonicotinoid insecticide discovered in 1994 in Japan, is a structural analogue of nicotine derived from N-nitroguanidine

**Figure 1.** Common names and molecular structures of the neonicotinoids [14].


Thiamethoxam is converted to clothianidin, which is more active than the parent molecule, mostly by CYP 3A4 and to a lesser extent by CYP 2C19 and 2B6, and is demethylated by 2C19. Clothianidin is demethylated by CYP 3A4, 2C19 and 2A6 [18]. There is no accumulation of neonicotinoids in the body: over 90% are eliminated in less than 24 h and totally in 48 h [15, 19].

**Figure 2.** Sites of metabolite attack on IMI and 6-chloronicotinic acid (CNA) for phase I and phase II reactions [16].

Neonicotinoids are neither irritating to eyes and skin (rabbit) nor sensitizing (guinea pigs) [15]. Their acute toxicity to mammals is variable depending on the type of neonicotinoid. The amount of toxic product that kills 50% of the experimental animals, called the lethal dose (LD

With an LD 50 of 425–475 mg/kg administered orally in rats, the toxicity of imidacloprid is mild to moderate. A single 42 mg/kg dose has no effect. After 2–6 h, poisoned animals present

Neonicotinoids have been designed to be effective as insecticides by contact or ingestion [21]. In both insects and humans, neonicotinoids behave as postsynaptic acetylcholine receptor agonists, which are neurotransmitters of the central nervous system, the parasympathetic nervous system and some of the sympathetic system. Their irreversible link-

inhibit the transmission of the nervous influx. Their high insect toxicity is explained by the predominance of nicotinic receptors in the central nervous system of these species, by

/K+

Acute Poisoning with Neonicotinoid Insecticide http://dx.doi.org/10.5772/intechopen.72004 111

channels and

with apathy, trembling with ataxia, hypothermia and respiratory arrest [19, 20].

ages with these receptors initially stimulate, then rapidly block the Na+

**Common name Local effects Rat oral LD50 Rabbit dermal LD50**

Acetamiprid Absent 450 >2000 Clothianidin Absent >5000 >2000 Dinotefuran Absent 2000 >2000 Imidacloprid Absent 4870 >2000 Thiamethoxam Absent >5000 >2000

**Table 2.** Neonicotinoid pesticides mammalian toxicities (mg/kg of body weight) [7].

**4. Experimental data and mechanisms of action**

50), is illustrated in **Table 2** for the main neonicotinoids.

**Table 1.** The physical state of neonicotinoids.

and is the best sold worldwide. Currently, besides imidacloprid, the neonicotinoid family includes thiamethoxam, clothianidin, thiacloprid, acetamiprid, dinotefuran, nitenpyram, nitiazine, imidaclothiz, flonicamid, sulfoxaflor and cycloxaprid, the chemical structure of which is shown in **Figure 1** [14]. Currently, neonicotinoids are homologated in agriculture in more than 120 countries, being sold under various commercial names [14] (**Table 1**).

### **3. Toxicokinetics**

Most human toxicity data available are about imidacloprid. Penetration through the skin (predominantly in the agricultural environment) of neonicotinoid insecticides is not quantified in humans. Intoxication through the respiratory tract is negligible given that these molecules are nonvolatile. However, there may be a secondary swallowing of the inhaled aerosol microparticles. The first prospective study conducted in Sri Lanka by Mohamed F. et al. on 68 patients (61 with voluntary ingestion and 7 with cutaneous exposure) showed that the mean plasma concentration of imidacloprid was 10.58 ng/l, IQR: 3.84–15.58 ng/l, range: 0.02–51.25 ng/l. In seven patients, the plasma concentration remained elevated for 10–15 h postingestion, suggesting that absorption and/or elimination may be prolonged at high doses. The time-concentration profiles have demonstrated a rapid initial absorption [5]. The plasma peak is reached within 2 h. There is no preferential distribution in fat-rich tissues [15]. In insects, mammals and plants, neonicotinoids undergo phase I and phase II biotransformation (**Figure 2**).

In vitro studies on the metabolism of neonicotinoids have indicated the importance of cytochrome P450s (CYP) in their oxidation and reduction. Through a variety of human CYP isoenzymes, imidacloprid is oxidized to 5-hydroxyimidacloprid and imidacloprid olefin and reduced to nitrosoguanidine, aminoguanidine and urea imidacloprid. The most active CYP isoenzyme for oxidation of the imidacloprid residue is CYP 3A4 (CYP most abundant in humans), followed by CYP 2C19, 2A6 and 2C9. For nitroreduction, the most active CYP are: CYP 1A2, 2B6, 2D6 and 2E1 [16, 17].

**Figure 2.** Sites of metabolite attack on IMI and 6-chloronicotinic acid (CNA) for phase I and phase II reactions [16].

Thiamethoxam is converted to clothianidin, which is more active than the parent molecule, mostly by CYP 3A4 and to a lesser extent by CYP 2C19 and 2B6, and is demethylated by 2C19. Clothianidin is demethylated by CYP 3A4, 2C19 and 2A6 [18]. There is no accumulation of neonicotinoids in the body: over 90% are eliminated in less than 24 h and totally in 48 h [15, 19].

## **4. Experimental data and mechanisms of action**

and is the best sold worldwide. Currently, besides imidacloprid, the neonicotinoid family includes thiamethoxam, clothianidin, thiacloprid, acetamiprid, dinotefuran, nitenpyram, nitiazine, imidaclothiz, flonicamid, sulfoxaflor and cycloxaprid, the chemical structure of which is shown in **Figure 1** [14]. Currently, neonicotinoids are homologated in agriculture in more

Sulfoxaflor White solid Cycloxaprid Wettable powder

Second-generation neonicotinoids Thiamethoxam Slightly creamy crystalline powder, odorless

Nitenpyram Pale yellow crystals

Acetamiprid White crystals, white fine powder, odorless

Thiacloprid Yellow crystalline powder, odorless

Clothianidin Clear colorless solid powder, odorless

Most human toxicity data available are about imidacloprid. Penetration through the skin (predominantly in the agricultural environment) of neonicotinoid insecticides is not quantified in humans. Intoxication through the respiratory tract is negligible given that these molecules are nonvolatile. However, there may be a secondary swallowing of the inhaled aerosol microparticles. The first prospective study conducted in Sri Lanka by Mohamed F. et al. on 68 patients (61 with voluntary ingestion and 7 with cutaneous exposure) showed that the mean plasma concentration of imidacloprid was 10.58 ng/l, IQR: 3.84–15.58 ng/l, range: 0.02–51.25 ng/l. In seven patients, the plasma concentration remained elevated for 10–15 h postingestion, suggesting that absorption and/or elimination may be prolonged at high doses. The time-concentration profiles have demonstrated a rapid initial absorption [5]. The plasma peak is reached within 2 h. There is no preferential distribution in fat-rich tissues [15]. In insects, mammals

and plants, neonicotinoids undergo phase I and phase II biotransformation (**Figure 2**).

In vitro studies on the metabolism of neonicotinoids have indicated the importance of cytochrome P450s (CYP) in their oxidation and reduction. Through a variety of human CYP isoenzymes, imidacloprid is oxidized to 5-hydroxyimidacloprid and imidacloprid olefin and reduced to nitrosoguanidine, aminoguanidine and urea imidacloprid. The most active CYP isoenzyme for oxidation of the imidacloprid residue is CYP 3A4 (CYP most abundant in humans), followed by CYP 2C19, 2A6 and 2C9. For nitroreduction, the most active CYP are:

than 120 countries, being sold under various commercial names [14] (**Table 1**).

**Generation of neonicotinoids Type of neonicotinoid Physical state**

110 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

First-generation neonicotinoids Imidacloprid Clear crystals or beige powder

Third-generation neonicotinoids Dinotefuran White crystalline solid, odorless

**3. Toxicokinetics**

**Table 1.** The physical state of neonicotinoids.

CYP 1A2, 2B6, 2D6 and 2E1 [16, 17].

Neonicotinoids are neither irritating to eyes and skin (rabbit) nor sensitizing (guinea pigs) [15]. Their acute toxicity to mammals is variable depending on the type of neonicotinoid. The amount of toxic product that kills 50% of the experimental animals, called the lethal dose (LD 50), is illustrated in **Table 2** for the main neonicotinoids.

With an LD 50 of 425–475 mg/kg administered orally in rats, the toxicity of imidacloprid is mild to moderate. A single 42 mg/kg dose has no effect. After 2–6 h, poisoned animals present with apathy, trembling with ataxia, hypothermia and respiratory arrest [19, 20].

Neonicotinoids have been designed to be effective as insecticides by contact or ingestion [21]. In both insects and humans, neonicotinoids behave as postsynaptic acetylcholine receptor agonists, which are neurotransmitters of the central nervous system, the parasympathetic nervous system and some of the sympathetic system. Their irreversible linkages with these receptors initially stimulate, then rapidly block the Na+ /K+ channels and inhibit the transmission of the nervous influx. Their high insect toxicity is explained by the predominance of nicotinic receptors in the central nervous system of these species, by


**Table 2.** Neonicotinoid pesticides mammalian toxicities (mg/kg of body weight) [7].

the absence of the blood-brain barrier and by their affinity for some insect-specific receptor subtypes, in particular α4β2 [19]. In mammals, the predominant receptor subtype is α4p2, which is found to have the highest density in the thalamus. In the developing brain, this subtype is involved in proliferation, apoptosis, migration, differentiation, synapse formation and neuronal circuits [22]. Given that in mammals nicotinic receptors have a wider distribution (neuromuscular junction), the neonicotinoid affinity for these receptors is lower and the central action is reduced because of poor intracerebral penetration [17]. The toxicity of imidacloprid is very low in dermal exposure and is moderate in case of ingestion. In case of inhalation, the toxicity is variable: as dust it is considered to be slightly toxic, but as aerosols it is very toxic [23].

## **5. Epidemiology of acute poisoning with neonicotinoids in humans**

Despite the widespread use of neonicotinoids, there are few reports in the literature on human toxicity. The first case report was made in 2001 by Wu IW et al. Clinical manifestation included drowsiness, disorientation, dizziness, oral and gastroesophageal erosions, hemorrhagic gastritis, productive cough, fever, leukocytosis and hyperglycemia. The patient recovered without complications with supportive treatment and was discharged 4 days after ingestion [24]. Most cases were reported in USA (Texas), France, China and Sri Lanka [25, 26, 13]. The epidemiological aspects resulting from these studies are summarized in **Table 3**.

The prevalence of voluntary intoxication was different from one study to another: only 2% in the study in Texas and 4.9% in the study in France, as opposed to the studies in China and Sri Lanka, where 81% and 82%, respectively, were classified as an attempt of suicide [25, 26, 13]. There are also differences in childhood poisoning frequency: in the study conducted in Texas, 37% were patients under the age of 19, and in France 36.6% were children under the age of 14, whereas the study in China recorded only two children with accidental poisoning. The study

Sri Lanka The dosing was done in laboratories in Australia

**Studies Cohort of the study Country of study Epidemiological findings**

China • Dramatic increase in the number of

poisoning • 67 males

out of the total of 70 • Two children with accidental

cases between 2003 and 2007: 58 cases

Acute Poisoning with Neonicotinoid Insecticide http://dx.doi.org/10.5772/intechopen.72004 113

• 48 cases were suicidal attempts, of which eight consumed ethanol • Toxic: imidacloprid (64 cases), acetamiprid (4) and clothianidin (2). • Two deaths (mortality rate 2.9%)

• 61 out of 68 patients (91%) were intoxi-

• 82% were voluntary poisonings

cated by ingestion

70 cases of acute intoxication reported by the Taiwan National Poison Center between 1987 and 2007 (retrospective study)

68 cases of acute poisoning with imidacloprid between March 2002 and March 2007 (prospective study)

**Table 3.** The main epidemiological aspects of neonicotinoid intoxication studies.

Regarding the manner of intoxication, in the US study only 51% of poisoning occurred through ingestion, compared to 91% (61/68 cases) in Sri Lanka and 81% (57/70) in China.

In addition to these studies, several sporadic cases have been reported. Thus, eight cases were reported in India [27–33], two in Turkey [34, 35], two in Portugal [36], two in Colombia [37], two in Japan [38], one case in Saudi Arabia [39], Iran [40], Poland [41] and four cases in Taiwan

Symptoms of neonicotinoid poisoning appear to be less severe in humans than in insects, because their affinity for human nicotinic receptors is lower and they do not cross the bloodbrain barrier. Clinical picture is better known for intoxication with imidacloprid, which is the oldest neonicotinoid used as an insecticide. A large study conducted in Texas between 2000 and 2012 on 1142 patients with neonicotinoid exposure found that the main symptoms are: eye irritation and dermatitis, nausea, vomiting, corrosive oral mucosal lesions,

in Sri Lanka does not specify the age of the patients.

**6. Clinical symptoms of poisoning by neonicotinoids**

[41–45].

Phua DH et al, 2009 [13]

Mohamed F et al., 2009 [5]



**Table 3.** The main epidemiological aspects of neonicotinoid intoxication studies.

the absence of the blood-brain barrier and by their affinity for some insect-specific receptor subtypes, in particular α4β2 [19]. In mammals, the predominant receptor subtype is α4p2, which is found to have the highest density in the thalamus. In the developing brain, this subtype is involved in proliferation, apoptosis, migration, differentiation, synapse formation and neuronal circuits [22]. Given that in mammals nicotinic receptors have a wider distribution (neuromuscular junction), the neonicotinoid affinity for these receptors is lower and the central action is reduced because of poor intracerebral penetration [17]. The toxicity of imidacloprid is very low in dermal exposure and is moderate in case of ingestion. In case of inhalation, the toxicity is variable: as dust it is considered to be slightly

112 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

**5. Epidemiology of acute poisoning with neonicotinoids in humans**

**Studies Cohort of the study Country of study Epidemiological findings**

Despite the widespread use of neonicotinoids, there are few reports in the literature on human toxicity. The first case report was made in 2001 by Wu IW et al. Clinical manifestation included drowsiness, disorientation, dizziness, oral and gastroesophageal erosions, hemorrhagic gastritis, productive cough, fever, leukocytosis and hyperglycemia. The patient recovered without complications with supportive treatment and was discharged 4 days after ingestion [24]. Most cases were reported in USA (Texas), France, China and Sri Lanka [25, 26, 13]. The epidemiological aspects resulting from these studies are summarized in **Table 3**.

USA • 77% with imidacloprid

• 17% with dinotefuran

• 32 serious cases (2.9%)

aged 6–19 years • 97% accidental

France • Increase from 8 cases in 1999 to 34 cases in 2012

5–14 years

of intoxication

intensive care

• Intoxication mode: ingestion (51%), cutaneous (44%) and ocular (11%) • 28% children aged <5 years and 9%

• 120 cases (28%) were children <5 years

• 389 poisonings were accidental, 24 voluntary, and in 15 cases it was not possible to specify the circumstances

• Seven cases were serious, requiring

• 37 cases (8.6%) children aged

• 64% female

toxic, but as aerosols it is very toxic [23].

1142 cases of acute

intoxication reported by Texas Intoxication Control Centers over the period 2000–2012 (retrospective study)

482 cases of acute poisoning with imidacloprid between January 1999 and December 2012 (retrospective study)

Forrester M et al., 2014 [25]

Boels D, Chataigner D, 2014 [26]

The prevalence of voluntary intoxication was different from one study to another: only 2% in the study in Texas and 4.9% in the study in France, as opposed to the studies in China and Sri Lanka, where 81% and 82%, respectively, were classified as an attempt of suicide [25, 26, 13].

There are also differences in childhood poisoning frequency: in the study conducted in Texas, 37% were patients under the age of 19, and in France 36.6% were children under the age of 14, whereas the study in China recorded only two children with accidental poisoning. The study in Sri Lanka does not specify the age of the patients.

Regarding the manner of intoxication, in the US study only 51% of poisoning occurred through ingestion, compared to 91% (61/68 cases) in Sri Lanka and 81% (57/70) in China.

In addition to these studies, several sporadic cases have been reported. Thus, eight cases were reported in India [27–33], two in Turkey [34, 35], two in Portugal [36], two in Colombia [37], two in Japan [38], one case in Saudi Arabia [39], Iran [40], Poland [41] and four cases in Taiwan [41–45].

## **6. Clinical symptoms of poisoning by neonicotinoids**

Symptoms of neonicotinoid poisoning appear to be less severe in humans than in insects, because their affinity for human nicotinic receptors is lower and they do not cross the bloodbrain barrier. Clinical picture is better known for intoxication with imidacloprid, which is the oldest neonicotinoid used as an insecticide. A large study conducted in Texas between 2000 and 2012 on 1142 patients with neonicotinoid exposure found that the main symptoms are: eye irritation and dermatitis, nausea, vomiting, corrosive oral mucosal lesions,


**Symptomatology**

**Benign forms**

**Moderate forms**

**Severe forms**

**Severe symptoms that threaten vital prognosis**

• Intense pain, extreme rigidity

• Extensive cramps

• Extended, diffuse fasciculations

• Rhabdomyolysis with complications

• CPK > 10,000 ui/l

• Compartment syndrome

• Kidney failure, anuria

• Serum creatinine >500 μmol/l

• Massive hemolysis

• Methemoglobinemia >50%

• Coagulation disorders with bleeding

• Anemia, leukopenia, severe

thrombocytopenia

• ASAT, ALT >50 times normal

• Affecting clotting factors

• Clinical signs of liver failure

Acute Poisoning with Neonicotinoid Insecticide http://dx.doi.org/10.5772/intechopen.72004 115

**Pronounced or prolonged symptoms or signs**

**Minor, mild symptoms that** 

**spontaneously regress**

• Slight or moderate pain

• Pain, stiffness, cramps

• Fasciculations

• Rhabdomyolysis

• CPK: 1500–10,000 ui/l

• Sensitivity to palpation

• Rhabdomyolysis

• CPK: 250–1500 ui/l

Kidney

Blood

• Minor hemolysis

• Methemoglobinemia ranging from

10 to 30%

Liver

• ASAT, ALT: 2–5 times normal

• ASAT, ALAT: 5–50 times normal

• Without obvious clinical signs of liver

dysfunction

**Table 4.** The symptomatology of imidacloprid poisoning depending on severity [26].

• Proteinuria and/or minimal

• Proteinuria and/or massive hematuria

• Oliguria, polyuria

• Serum creatinine: 200–500 mmol/l

• Hemolysis

• Methemoglobinemia ranging from 30 to 50%

• Coagulation disorders without bleeding

• Anemia, leukopenia, thrombocytopenia

hematuria

Muscle


**Symptomatology**

**Benign forms**

**Moderate forms**

**Severe forms**

**Severe symptoms that threaten vital prognosis**

• Deep coma with inappropriate or absent

response to pain

• Glasgow score 3–7

• Depression or respiratory failure

• Extreme agitation

• Generalized seizures

• Convulsive state, opisthotonus

• Generalized paralysis or paralysis that affects

114 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

vital functions

• Blindness, deafness

**Pronounced or prolonged symptoms or signs**

• Disturbances of consciousness with delayed

response to pain

• Glasgow score 8–11

• Apnea, bradypnea

• Confusion, agitation, hallucinations, delirium

• Localized or generalized seizures (rarely)

• Marked extrapyramidal symptoms

• Noticeable cholinergic/anticholinergic

symptoms

• Isolated paralysis without affecting vital

functions

• Visual and auditory disturbances

• Marked irritation

• Corneal ulcer

• Corneal perforations

• Definitive sequelae

• Second-degree burns >50% BSA to adult and

>30% BSA to child

• Third-degree burns >2% BSA

• Extensive edema comprising the adjacent

member and the adjacent parts

• Critical location of edema with danger to the

integrity of the upper airways

• Limited, circumscribed corneal involvement

• Punctual keratitis

Eye

• Irritation, conjunctival hyperemia,

tears

• Minor eyelid edema

Skin

• Irritation, first-degree burns

• Second-degree burns with 10–50% BSA to adult

and 10–30% BSA to child

• Third-degree marks <2% BSA

• Localized edema involving the entire member

• Localized necrosis

• Moderate pain

• Second-degree burns and <10% body

surface area (BSA)

Bites/stinging

• Edema, localized pruritus

• Discrete pain

**Minor, mild symptoms that** 

**spontaneously regress**

Neurological

• Sleepiness, vertigo, ataxia, tinnitus

• Glasgow score 12–14

• Slight agitation

• Minor extrapyramidal symptoms

• Minor cholinergic/anticholinergic

symptoms

• Paresthesia

• Minor visual and hearing

impairments

**Table 4.** The symptomatology of imidacloprid poisoning depending on severity [26]. dizziness, hypertension and tachycardia [25]. The French Toxicity Co-ordination Committee analyzed 428 cases of exposure to imidacloprid between 1999 and 2012, of which over 27% were children under 5 years of age. As a result of this study, the symptomatology was better outlined according to the severity of the intoxication (mild, moderate or severe), as illustrated in **Table 4** [26].

There was reported a case with concomitant intoxication with imidacloprid and alcohol ingestion, resulting in multiple organ failure [45]. Another case was reported by Agarwal and Srinivas, manifesting severe neuropsychiatric disorders and rhabdomyolysis [31]. In Colombia, Estrada et al. signaled two cases admitted with digestive manifestations, coma (Glasgow score 6 and 3, respectively) and respiratory failure with 70–75% Sa O2 . One of the patients also presented dormant miosis and was thus given atropine [37]. In Saudi Arabia, there was a case with generalized erythematous maculopapular rash typical of leukoclastic vasculitis, which was confirmed by biopsy. It also associated hepatic and renal dysfunction, requiring dialysis [40]. Another case reported in Taiwan presented fatal ventricular fibrillation [45].

The main symptoms of acetamiprid poisoning are severe nausea, vomiting, muscle weakness, hypothermia and convulsions [46]. Clinical manifestations include tachycardia, hypotension, electrocardiogram changes, hypoxia and thirst in the case of the highest serum concentration of acetamiprid. The symptoms were partly similar to acute organophosphate intoxication [30]. In one case, ventricular fibrillation that lasted 11 h after ingestion was described [45]. Similar to imidacloprid poisonings, there was reported a case of severe multiple organic dysfunction [43]. Another case of acetamiprid intoxication was by suicidal ingestion, where symptoms included prolonged muscle weakness, similar to the intermediate syndrome in organophosphorus intoxication. The case resolved in about 3 weeks [35].

Depending on the intensity and duration of cardiovascular, respiratory and digestive symp-

**Forms Cardiovascular symptoms Respiratory symptoms Digestive symptoms**

• Airways irritation • Coughing, breathlessness

• Slight dyspnea • Slight bronchospasm • Abnormal thoracic radiography with or without minor

symptoms

• Dyspnea

symptoms

• Prolonged cough • Stridor, bronchospasm

• Hypoxia requiring oxygen administration • Abnormal pulmonary radiography with moderate

• HCO3

• K: 2.5–2.9 or 6–6.9 mmol/l • Severe hypoglycemia: 0.3–0.5 g/l or 1.7–2.8 mmol/l • Prolonged hyperthermia

: 10–14 sau > 40 mmol/l • pH: 7.16–7.24 sau 7.60–7.69

• HCO3

• pH < 7.15 or >7.7

• K: <2.5 or >7 mmol/l

or <1.7 mmol/l • Malignant hyperthermia/ hypothermia

> • Vomiting • Diarrhea • Abdominal pain • Minor oral ulceration • Endoscopy: erythema, stage I edema

• Pronounced or prolonged

• Endoscopy: stage IIa transient ulcerative lesions

vomiting • Diarrhea • Abdominal pain • First-grade burns of a critical area or II and III grade burns on limited

areas • Dysphagia

< 10 mmol/l

Acute Poisoning with Neonicotinoid Insecticide http://dx.doi.org/10.5772/intechopen.72004 117

• Severe hypoglycemia <0.3 g/l

toms, three degrees of severity can be distinguished (**Table 6**):

**Table 5.** Metabolic disorders depending on the severity of intoxication [26].

**Clinical forms Mild Moderate Severe**

Acidobasic disorders

Electrolytic disorders

• HCO3

: 15–20 or 30–40 mmol/l • pH: 7.25–7.32 or 7.50–7.59

• K: 3–3.4 or 5.2–5.9 mmol/l • Moderate hypoglycemia: 0.5–0.7 g/l or 2.8–3.9 mmol/l • Short-term hyperthermia

**3.** Severe: severe symptoms that may influence the vital prognosis.

**1.** Benign: mild symptoms that spontaneously regress;

• Discrete, transient hypotension • Transient, discrete hypertension

○ Adults 40–50 b/min ○ Children 60–80 b/min ○ New born 80–90 b/min • Frequent premature beats • Atrial flutter/fibrillation • AVB grade I or II • Prolonged QRS and QTc • Repolarization modifications • Myocardial ischemia • Hypo/hypertension

Benign • Isolated extrasystoles

Moderate • Sinus bradycardia:

**2.** Moderate: pronounced or prolonged symptoms/signs;

Symptoms of thiamethoxam intoxication are less known, and cases are rarely reported. Vinod et al. noted a case manifesting nausea, vomiting, agitation and multiple episodes of generalized tonic-clonic seizures within the first 2 h of ingestion of thiamethoxam. Subsequently, coma, hypotension, renal failure, metabolic acidosis and rhabdomyolysis occurred, with fatal outcome 36 h after ingestion [29].

Solvents used in neonicotinoid insecticide solutions can also play an important part in poisoning symptoms. Although not all solvents contained by neonicotinoid insecticides are known, most of them use N-methylpyrrolidone. Ingestion of a large amount of this substance irritates the upper gastrointestinal tract and causes oral ulceration, nausea, vomiting, dysphagia, odynophagia and abdominal pain [24].

## **7. Diagnosis of poisoning incidents**

The diagnosis is based on anamnesis and clinical symptoms. There are no specific abnormalities of acute poisoning with neonicotinoids [5]. There may be metabolic disturbances (**Table 5**). Dosage of neonicotinoids is not routinely available, its interest being purely medicolegal. Depending on the symptomatology of each case, additional investigations may be necessary.


**Table 5.** Metabolic disorders depending on the severity of intoxication [26].

dizziness, hypertension and tachycardia [25]. The French Toxicity Co-ordination Committee analyzed 428 cases of exposure to imidacloprid between 1999 and 2012, of which over 27% were children under 5 years of age. As a result of this study, the symptomatology was better outlined according to the severity of the intoxication (mild, moderate or severe), as illus-

There was reported a case with concomitant intoxication with imidacloprid and alcohol ingestion, resulting in multiple organ failure [45]. Another case was reported by Agarwal and Srinivas, manifesting severe neuropsychiatric disorders and rhabdomyolysis [31]. In Colombia, Estrada et al. signaled two cases admitted with digestive manifestations, coma (Glasgow score

sented dormant miosis and was thus given atropine [37]. In Saudi Arabia, there was a case with generalized erythematous maculopapular rash typical of leukoclastic vasculitis, which was confirmed by biopsy. It also associated hepatic and renal dysfunction, requiring dialysis

The main symptoms of acetamiprid poisoning are severe nausea, vomiting, muscle weakness, hypothermia and convulsions [46]. Clinical manifestations include tachycardia, hypotension, electrocardiogram changes, hypoxia and thirst in the case of the highest serum concentration of acetamiprid. The symptoms were partly similar to acute organophosphate intoxication [30]. In one case, ventricular fibrillation that lasted 11 h after ingestion was described [45]. Similar to imidacloprid poisonings, there was reported a case of severe multiple organic dysfunction [43]. Another case of acetamiprid intoxication was by suicidal ingestion, where symptoms included prolonged muscle weakness, similar to the intermediate syndrome in

Symptoms of thiamethoxam intoxication are less known, and cases are rarely reported. Vinod et al. noted a case manifesting nausea, vomiting, agitation and multiple episodes of generalized tonic-clonic seizures within the first 2 h of ingestion of thiamethoxam. Subsequently, coma, hypotension, renal failure, metabolic acidosis and rhabdomyolysis occurred, with fatal

Solvents used in neonicotinoid insecticide solutions can also play an important part in poisoning symptoms. Although not all solvents contained by neonicotinoid insecticides are known, most of them use N-methylpyrrolidone. Ingestion of a large amount of this substance irritates the upper gastrointestinal tract and causes oral ulceration, nausea, vomiting, dysphagia, ody-

The diagnosis is based on anamnesis and clinical symptoms. There are no specific abnormalities of acute poisoning with neonicotinoids [5]. There may be metabolic disturbances (**Table 5**). Dosage of neonicotinoids is not routinely available, its interest being purely medicolegal. Depending on the symptomatology of each case, additional investigations

[40]. Another case reported in Taiwan presented fatal ventricular fibrillation [45].

organophosphorus intoxication. The case resolved in about 3 weeks [35].

. One of the patients also pre-

6 and 3, respectively) and respiratory failure with 70–75% Sa O2

116 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

trated in **Table 4** [26].

outcome 36 h after ingestion [29].

nophagia and abdominal pain [24].

may be necessary.

**7. Diagnosis of poisoning incidents**

Depending on the intensity and duration of cardiovascular, respiratory and digestive symptoms, three degrees of severity can be distinguished (**Table 6**):




Sometimes, organophosphorus intoxication may also be associated. In this case, acute poisoning may be manifested by miosis, bradycardia, hypersalivation and bronchorrhea. Because of these symptoms, atropine and oximes may be improperly used as an antidote. It is unknown whether these drugs are effective or may worsen the outcome of neonicotoid insecticide poisonings. In cases with life-threatening muscarinic manifestations (e.g., bronchorrhea with airway compromise), the use of atropine may be justified in neonicotinoid-poisoned patients [13, 44]. Oximes (e.g., pralidoxime) are usually either ineffective or contraindicated. In the absence of organophosphorus pesticides, oximes have a weak inhibitory effect on acetylcholinesterase activity and therefore may increase nicotinic effects (tachycardia, hypertension and muscle weakness) [5]. Because the severity of poisoning is not proportional to the plasma concentration of neonicotinoids, hemofiltration is ineffective

Acute Poisoning with Neonicotinoid Insecticide http://dx.doi.org/10.5772/intechopen.72004 119

The numbers of neonicotinoid poisonings have increased in the last decade, given that neonicotinoid insecticide is highly used. Respiratory, cardiovascular and certain neurological presentations (dyspnea/apnea, coma, tachycardia, hypotension, mydriasis and bradycardia) are symptoms of severe neonicotinoid intoxication [43]. Biochemical abnormalities and rhabdomyolysis have been reported as potentially serious complications that might lead to

Mortality through imidacloprid poisoning ranges from 0% to 4.2% in various studies. In a study in Taiwan, neonicotinoid poisoning mortality was 2.9%, inferior to that with organophosphates (12.3%) or carbamates (7.3%), but close to that with synthetic pyrethroids (3.1%) [47]. In the study in France conducted on 428 patients with acute neonicotinoid poisoning, six deaths were recorded [26]. Another study in Korea on 24 cases shows a mortality rate of 4.2% [48]. In addition to these studies on larger cohorts of patients, sporadic cases of acute neonic-

**Authors, year of the study Place of study The type of neonicotinoid Death numbers**

in increasing their elimination [13, 43].

mortality [5].

**9. Prognosis and comparative mortality rates**

otinoid poisoning deaths are also reported in the literature (**Table 7**).

**Table 7.** Deaths by acute poisoning with neonicotinoids reported in the literature.

Proença P et al., 2005 [36] Portugal Imidacloprid 2 Huang NC et al.,2006 [45] Taiwan Imidacloprid 1 Shadnia S et al., 2008 [40] Iran Imidacloprid 1 Yeh IJ et al., 2010 [44] China Imidacloprid 1 Iyyodurai R et al., 2010 [30] India Imidacloprid 1 Harish J et al., 2011 [49] India Imidacloprid 1 Fuke C et al., 2014 [49] Japan Imidacloprid 1 Vinod KV et al., 2015 [29] India Thiacloprid 1

**Table 6.** Degrees of severity according to intensity and duration of cardiovascular, respiratory and digestive symptoms [26].

Differential diagnosis should take into account intoxications with other pesticides. This poisonings can mimic light forms of organophosphorus or carbamates intoxications. Also, many cases reported involved combinations of multiple pesticides and ethanol [5, 13].

## **8. Management of poisoning incidents**

Management of acute neonicotinoid insecticide poisoning is mainly symptomatic and supportive. Dermal and mucosal exposures should be decontaminated as soon as possible [13].

In case of coma and respiratory distress, intubation and assisted ventilation associated with hemodynamic support are required. The presence of solvents in liquid neonicotinoid formulas makes the activated charcoal and gastric lavage or vomiting ineffective, due to the risk of inhalation pneumonia [13]. Gastric lavage and activated charcoal should be avoided if corrosive injuries of the oral and gastrointestinal mucosa are found. Activated charcoal may hinder endoscopic evaluation of patients with corrosive lesions [13]. Prudent aspiration of the gastric contents can be considered, with respiratory protection, and if the ingested volume is high (over 100 ml), the ingestion period is short (less than 1 h) [13, 15]. The respiratory effects of neonicotinoids should be carefully monitored, particularly hypoventilation and respiratory failure. Patients with upper airway injuries such as hoarseness and stridor caused by irritant and corrosive effects of the solvent should probably undergo endoscopic assessment of vocal cordons [13].

Sometimes, organophosphorus intoxication may also be associated. In this case, acute poisoning may be manifested by miosis, bradycardia, hypersalivation and bronchorrhea. Because of these symptoms, atropine and oximes may be improperly used as an antidote. It is unknown whether these drugs are effective or may worsen the outcome of neonicotoid insecticide poisonings. In cases with life-threatening muscarinic manifestations (e.g., bronchorrhea with airway compromise), the use of atropine may be justified in neonicotinoid-poisoned patients [13, 44]. Oximes (e.g., pralidoxime) are usually either ineffective or contraindicated. In the absence of organophosphorus pesticides, oximes have a weak inhibitory effect on acetylcholinesterase activity and therefore may increase nicotinic effects (tachycardia, hypertension and muscle weakness) [5]. Because the severity of poisoning is not proportional to the plasma concentration of neonicotinoids, hemofiltration is ineffective in increasing their elimination [13, 43].

## **9. Prognosis and comparative mortality rates**

Differential diagnosis should take into account intoxications with other pesticides. This poisonings can mimic light forms of organophosphorus or carbamates intoxications. Also, many

**Table 6.** Degrees of severity according to intensity and duration of cardiovascular, respiratory and digestive symptoms

**Forms Cardiovascular symptoms Respiratory symptoms Digestive symptoms**

118 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

• Respiratory failure: severe bronchospasm, dyspnea, airway obstruction, ARDS, pulmonary edema, glottis edema, bronchopneumonia, pneumopathy, pneumothorax • Abnormal pulmonary radiography with severe symptoms

• Severe digestive hemorrhage • Digestive perforation • Enlarged II and III grade

burns • Severe dysphagia • Endoscopy: transmural ulcer lesions, circumferential lesions, perforations stage IIB, III and IV

Management of acute neonicotinoid insecticide poisoning is mainly symptomatic and supportive. Dermal and mucosal exposures should be decontaminated as soon as possible [13]. In case of coma and respiratory distress, intubation and assisted ventilation associated with hemodynamic support are required. The presence of solvents in liquid neonicotinoid formulas makes the activated charcoal and gastric lavage or vomiting ineffective, due to the risk of inhalation pneumonia [13]. Gastric lavage and activated charcoal should be avoided if corrosive injuries of the oral and gastrointestinal mucosa are found. Activated charcoal may hinder endoscopic evaluation of patients with corrosive lesions [13]. Prudent aspiration of the gastric contents can be considered, with respiratory protection, and if the ingested volume is high (over 100 ml), the ingestion period is short (less than 1 h) [13, 15]. The respiratory effects of neonicotinoids should be carefully monitored, particularly hypoventilation and respiratory failure. Patients with upper airway injuries such as hoarseness and stridor caused by irritant and corrosive effects of the sol-

cases reported involved combinations of multiple pesticides and ethanol [5, 13].

vent should probably undergo endoscopic assessment of vocal cordons [13].

**8. Management of poisoning incidents**

Severe • Severe sinus bradycardia:

○ Adults <40 b/min ○ Children <60 b/min ○ New born <80 b/min • Severe sinus tachycardia ○ Adults >180 b/min ○ Children >190 b/min ○ New born >200 b/min • Ventricular dysrhythmia with

vital prognosis • AVB grade III • Asystole

• Myocardial infarction

• Malignant hypertensive

• Shock

[26].

disorder

The numbers of neonicotinoid poisonings have increased in the last decade, given that neonicotinoid insecticide is highly used. Respiratory, cardiovascular and certain neurological presentations (dyspnea/apnea, coma, tachycardia, hypotension, mydriasis and bradycardia) are symptoms of severe neonicotinoid intoxication [43]. Biochemical abnormalities and rhabdomyolysis have been reported as potentially serious complications that might lead to mortality [5].

Mortality through imidacloprid poisoning ranges from 0% to 4.2% in various studies. In a study in Taiwan, neonicotinoid poisoning mortality was 2.9%, inferior to that with organophosphates (12.3%) or carbamates (7.3%), but close to that with synthetic pyrethroids (3.1%) [47]. In the study in France conducted on 428 patients with acute neonicotinoid poisoning, six deaths were recorded [26]. Another study in Korea on 24 cases shows a mortality rate of 4.2% [48]. In addition to these studies on larger cohorts of patients, sporadic cases of acute neonicotinoid poisoning deaths are also reported in the literature (**Table 7**).


**Table 7.** Deaths by acute poisoning with neonicotinoids reported in the literature.

### **10. Concluding remarks**

Neonicotinoids act quite selectively on insects, but they are not free of human toxicity. Several cases of acute intoxication with such insecticides, sometimes severe, resulting in death, have been reported in the literature. The above review has highlighted the consequences of poisoning with these newer pesticides, not very well known at the moment. Therefore such information is valuable for clinicians, regulatory authorities and the public at large. Given the fact that these insecticides are increasingly being used in agriculture, horticulture and fish farming, but also for combating domestic pests, more studies on the human health effects of neonicotinoids exposure are needed and maybe some awareness programs about its toxicity should be implemented.

[7] Fishel FM. Pesticide Toxicity Profile Neonicotinoid Pesticides. University of Florida,

Acute Poisoning with Neonicotinoid Insecticide http://dx.doi.org/10.5772/intechopen.72004 121

[8] Henry M, Béguin M, Requier F, et al. A common pesticide decreases foraging success and survival in honey bees. Science. 2012;**336**:348-350. DOI: 10.1126/science.1215039

[9] Fischer J, Müller T, Spatz A-K, et al. Neonicotinoids interfere with specific components of navigation in honeybees. PLoS One. 2014;**9**:e91364. DOI: 10.1371/journal.pone.0091364

[10] Proposition de resolution relative à la préservation des insectes pollinisateurs, de l'environnement et de la santé et à un moratoire sur les pesticides de la famille des néo-

[11] Woodcock BA, Bullock JM, Shore RF, Heard MS, Pereira MG, Redhead J, Ridding L, et al. Country-specific effects of neonicotinoid pesticides on honey bees and wild bees.

[12] Tsvetkov N, Samson-Robert O, Sood K, Patel HS, Malena DA, Gajiwala PH, Maciukiewicz P, et al. Chronic exposure to neonicotinoids reduces honey bee health near corn crops.

[13] Phua DH, Lin CC, ML W, et al. Neonicotinoid insecticides: An emerging cause of acute pesticide poisoning. Clinical Toxicology. 2009;**47**:336-341. DOI: 10.1080/15563650802644533

[14] Kasiotis KM, Machera K. Neonicotinoids and their metabolites in human biomonitoring: A review. Hellenic Plant Protection Journal. 2015;**8**:33-45. DOI: https://doi.org/10.1515/

[15] Testud F. Insecticides néonicotinoïdes. EMC—Pathologie professionelle et l'environ-

[16] Swenson TL. Neonicotinoid Insecticide Metabolism and Mechanisms of Toxicity in

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[18] Shi X, Dick RA, Ford AK, Casida JE. Enzymes and inhibitors in Neonicotinoid insecticide metabolism. Journal of Agricultural and Food Chemistry. 2009;**97**(11):4861-4866.

[19] Sheets LP. Imidacloprid: A neonicotinid insecticide. In: Krieger RI, editor. Handbook of Pesticide Toxicology. 2nd ed. San Diego: Academic Press; 2001. pp. 1123-1130

[20] Gibbsons D, Morrissey C, Mineau P. A review of the direct and indirect effects of neonicotinoids and fipronil on vertebrate wildlife. Environmental Science and Pollution

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Florida; 2016. Available from: http://edis.ifas.ufl.edu/pi117

### **Author details**

Nicolai Nistor1,2, Otilia Elena Frăsinariu1,2\* and Violeta Ştreangă1,2

\*Address all correspondence to: otiliafrasinariu@gmail.com

1 "Grigore T. Popa" University of Medicine and Pharmacy Iasi, Iași, Romania

2 Regional Center of Toxicology, "St. Mary" Children's Emergency Clinic Hospital Iasi, Iași, Romania

#### **References**


[7] Fishel FM. Pesticide Toxicity Profile Neonicotinoid Pesticides. University of Florida, Florida; 2016. Available from: http://edis.ifas.ufl.edu/pi117

**10. Concluding remarks**

**Author details**

Romania

**References**

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Neonicotinoids act quite selectively on insects, but they are not free of human toxicity. Several cases of acute intoxication with such insecticides, sometimes severe, resulting in death, have been reported in the literature. The above review has highlighted the consequences of poisoning with these newer pesticides, not very well known at the moment. Therefore such information is valuable for clinicians, regulatory authorities and the public at large. Given the fact that these insecticides are increasingly being used in agriculture, horticulture and fish farming, but also for combating domestic pests, more studies on the human health effects of neonicotinoids exposure are needed and maybe some awareness programs about its toxicity should be implemented.

Nicolai Nistor1,2, Otilia Elena Frăsinariu1,2\* and Violeta Ştreangă1,2

120 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

1 "Grigore T. Popa" University of Medicine and Pharmacy Iasi, Iași, Romania

2 Regional Center of Toxicology, "St. Mary" Children's Emergency Clinic Hospital Iasi, Iași,

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ijrms20140573


**Chapter 7**

Provisional chapter

**Occupational Risk Factors for Acute Pesticide Poisoning**

DOI: 10.5772/intechopen.72006

Occupational Risk Factors for Acute Pesticide Poisoning

Types of pesticides are used in farming to increase the productivity and protection of crops or to control pests. However, exposure to acute pesticide poisoning is one of the most important occupational risk factors among farmers all over the world. They are directly exposed to high levels of pesticide poisoning when involved in the handling, spraying, mixing, or preparing of it. The low educational level, lack of information, training, judgment or experience, and the inability to read on pesticide safety are playing an important role for farmers' acute poisoning. On the other side, poor technology, inadequate personal protection, inappropriate type of clothing and equipment also leads to farmers' acute poisoning. Hence, this paper focuses on occupational risk factors for acute pesticide poisoning among farmers and their occupational safety. Discovering risk factors is also crucial for investigating health problems of farmers and its inevitable effects on their body. As it is seen from previous studies instead of field research such as deep interview on farmers' acute poisoning, data were mostly collected from hospitals. This study tries to emphasize the importance of field study to discover the risk factors for acute pesticide poisoning among farmers and their occu-

Keywords: occupational risk factors, farmers, acute pesticide poisoning, occupational

According to the World Health Organization, pesticides are chemical compounds that are used to kill pests, insects, rodents, fungi, and unwanted weeds. By their nature, pesticides are potentially toxic to humans which can also cause harm to human health [1]. The incorrect and unsafe use of pesticides can be a threat to human health [2]. They are mostly used for crop

> © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**among Farmers in Asia**

among Farmers in Asia

Emine Selcen Darçın, Murat Darçın, Murat Alkan and Gürdoğan Doğrul

Emine Selcen Darçın, Murat Darçın, Murat Alkan and Gürdoğan Doğrul

http://dx.doi.org/10.5772/intechopen.72006

Abstract

pational safety in Asia.

safety

1. Introduction

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Provisional chapter

## **Occupational Risk Factors for Acute Pesticide Poisoning among Farmers in Asia** Occupational Risk Factors for Acute Pesticide Poisoning

DOI: 10.5772/intechopen.72006

Emine Selcen Darçın, Murat Darçın, Murat Alkan and Gürdoğan Doğrul Emine Selcen Darçın, Murat Darçın,

among Farmers in Asia

Additional information is available at the end of the chapter Murat Alkan and Gürdoğan Doğrul

http://dx.doi.org/10.5772/intechopen.72006 Additional information is available at the end of the chapter

#### Abstract

Types of pesticides are used in farming to increase the productivity and protection of crops or to control pests. However, exposure to acute pesticide poisoning is one of the most important occupational risk factors among farmers all over the world. They are directly exposed to high levels of pesticide poisoning when involved in the handling, spraying, mixing, or preparing of it. The low educational level, lack of information, training, judgment or experience, and the inability to read on pesticide safety are playing an important role for farmers' acute poisoning. On the other side, poor technology, inadequate personal protection, inappropriate type of clothing and equipment also leads to farmers' acute poisoning. Hence, this paper focuses on occupational risk factors for acute pesticide poisoning among farmers and their occupational safety. Discovering risk factors is also crucial for investigating health problems of farmers and its inevitable effects on their body. As it is seen from previous studies instead of field research such as deep interview on farmers' acute poisoning, data were mostly collected from hospitals. This study tries to emphasize the importance of field study to discover the risk factors for acute pesticide poisoning among farmers and their occupational safety in Asia.

Keywords: occupational risk factors, farmers, acute pesticide poisoning, occupational safety

## 1. Introduction

According to the World Health Organization, pesticides are chemical compounds that are used to kill pests, insects, rodents, fungi, and unwanted weeds. By their nature, pesticides are potentially toxic to humans which can also cause harm to human health [1]. The incorrect and unsafe use of pesticides can be a threat to human health [2]. They are mostly used for crop

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

protection and to increase productivity in agriculture. Due to its widespread use in agriculture, occupational pesticide poisoning is likely to become a major health problem among agricultural workers all over the world. Since they directly contact with pesticides through spraying, mixing, handling, and preparing, farmers are at risk of exposure to pesticides. Hence, exposure to pesticides poisoning is an important occupational risk among farmers [3–5].

Farmers' low education level, lack of information and training on pesticide safety, poor spraying technology, and inadequate personal protection during pesticide use are the main reasons of poisoning [6]. These unsafe and misuses of pesticides can result in serious short-term or longterm health problems [7, 8]. Some of the symptoms of poisoning could affect the skin, nervous system, eyes, respiratory system, cardiovascular system, and gastrointestinal tract [9–11].

Discovering risk factors for acute pesticide poisoning of farmers is a very important issue for occupational safety. It will lead to decrease the number of poisoning cases. However, there have been very few researches that studied the risk factors for acute pesticide poisoning of farmers in agriculture. This chapter describes what the general risk factors of acute pesticide poisoning for farmers in Asia and the importance of their occupational safety are. Below, we present a description of pesticides and their mechanisms of actions.

• Fungicides (to kill mold or fungi): when applied to wood, they are called wood preservatives.

Oral Dermal

http://dx.doi.org/10.5772/intechopen.72006

127

Occupational Risk Factors for Acute Pesticide Poisoning among Farmers in Asia

WHO class LD50 for the rat (mg/kg body weight)

Ia. Extremely hazardous <5 <50 Ib. Highly hazardous 5–50 50–200 II. Moderately hazardous 50–2000 200–2000 III. Slightly hazardous Over 2000 Over 2000

• Other products such as algaecides (to kill algae), miticides (to kill moths), and acaricides

Secondly, pesticides are also responsible for thousands of acute pesticide poisoning and human injuries. Many people in developed and developing countries are affected by the negative effects of pesticides. This is mostly due to several reasons such as the lack of proper regulations, low

The WHO issued classification of pesticides by hazard as seen in Table 1. The classification distinguishes between the more and the less hazardous forms of each pesticide in that it is based on the toxicity of the technical compound and on its formulations. Its classification ranges from extremely hazardous (class Ia) to slightly hazardous (class III) to unlikely to present acute hazard

Acute pesticide poisoning has become a main health problem especially in developing countries with more than 300,000 deaths in a year. This is due to the poorer regulation, lack of surveillance systems, lack of experience or judgment, less enforcement, the inability to read, lack of training, and inadequate access to information systems [19]. Besides, the easy availability of highly toxic

It is not hard to diagnose symptoms of acute pesticide poisoning in humans due to their diversity and resemblance to other conditions. These symptoms could be classified as [9–11]:

• Skin: skin discomfort, rashes, blistering, burns, sweating, contact dermatitis, jaundice

pesticides in farmers' homes has made pesticides the preferred means of suicide [20, 21].

• Rodenticides to kill mice, rats, moles, and other rodents.

U. Unlikely to present acute hazard 5000 or higher

education levels, little experience, and easy availability in markets [16].

• Fumigants, which kill most organisms.

(to kill ticks) [17].

Table 1. Classification of pesticides [18].

3. Acute pesticide poisoning

(class U) [18].

## 2. What are pesticides?

Food and Agriculture Organization of the United Nations (FAO) defines that pesticide means any substance or mixture of substances intended for preventing, destroying, or controlling any pest. It also destroys vectors of human or animal disease; unwanted plants or animals causing harm during or otherwise interfering with the production, processing, storage, transport or marketing of food; agricultural commodities; wood and wood products or animal feedstuffs; or substances which may be administered to animals for the control of insects, arachnids, or other pests in or on their bodies. The term also comprises substances anticipated for use as a plant growth regulator, defoliant, desiccant or agent for thinning fruit or inhibiting the premature fall of fruit, and substances applied to crops either before or after harvest to protect the commodity from deterioration during storage and transport [12].

First of all, pesticides are designed to kill, prevent, destroy, repel, or mitigate living organisms such as pests (insects, mites, nematodes, weeds, and rats) [13], including insecticide, herbicide, fungicide, and various other substances used to control pests [14]. Pesticides are also used for crop protection, preservation of food and materials, and prevention of vector-borne diseases [15] like malaria, dengue, yellow fever dengue, leishmaniasis, and Japanese encephalitis [15, 16]. Briefly, it should be noted that:



Table 1. Classification of pesticides [18].

protection and to increase productivity in agriculture. Due to its widespread use in agriculture, occupational pesticide poisoning is likely to become a major health problem among agricultural workers all over the world. Since they directly contact with pesticides through spraying, mixing, handling, and preparing, farmers are at risk of exposure to pesticides. Hence, exposure

Farmers' low education level, lack of information and training on pesticide safety, poor spraying technology, and inadequate personal protection during pesticide use are the main reasons of poisoning [6]. These unsafe and misuses of pesticides can result in serious short-term or longterm health problems [7, 8]. Some of the symptoms of poisoning could affect the skin, nervous system, eyes, respiratory system, cardiovascular system, and gastrointestinal tract [9–11].

Discovering risk factors for acute pesticide poisoning of farmers is a very important issue for occupational safety. It will lead to decrease the number of poisoning cases. However, there have been very few researches that studied the risk factors for acute pesticide poisoning of farmers in agriculture. This chapter describes what the general risk factors of acute pesticide poisoning for farmers in Asia and the importance of their occupational safety are. Below, we

Food and Agriculture Organization of the United Nations (FAO) defines that pesticide means any substance or mixture of substances intended for preventing, destroying, or controlling any pest. It also destroys vectors of human or animal disease; unwanted plants or animals causing harm during or otherwise interfering with the production, processing, storage, transport or marketing of food; agricultural commodities; wood and wood products or animal feedstuffs; or substances which may be administered to animals for the control of insects, arachnids, or other pests in or on their bodies. The term also comprises substances anticipated for use as a plant growth regulator, defoliant, desiccant or agent for thinning fruit or inhibiting the premature fall of fruit, and substances applied to crops either before or after harvest to protect the

First of all, pesticides are designed to kill, prevent, destroy, repel, or mitigate living organisms such as pests (insects, mites, nematodes, weeds, and rats) [13], including insecticide, herbicide, fungicide, and various other substances used to control pests [14]. Pesticides are also used for crop protection, preservation of food and materials, and prevention of vector-borne diseases [15] like malaria, dengue, yellow fever dengue, leishmaniasis, and Japanese encephalitis [15, 16].

• Insecticides (for killing insects) such as organochlorines, organophosphates, and carbamates. This category also includes insect repellents such as diethyltoluamide (DEET) and

• Herbicides or weed killers include substances such as paraquat, glyphosate, and propanil.

to pesticides poisoning is an important occupational risk among farmers [3–5].

126 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

present a description of pesticides and their mechanisms of actions.

commodity from deterioration during storage and transport [12].

2. What are pesticides?

Briefly, it should be noted that:

citronella.


Secondly, pesticides are also responsible for thousands of acute pesticide poisoning and human injuries. Many people in developed and developing countries are affected by the negative effects of pesticides. This is mostly due to several reasons such as the lack of proper regulations, low education levels, little experience, and easy availability in markets [16].

The WHO issued classification of pesticides by hazard as seen in Table 1. The classification distinguishes between the more and the less hazardous forms of each pesticide in that it is based on the toxicity of the technical compound and on its formulations. Its classification ranges from extremely hazardous (class Ia) to slightly hazardous (class III) to unlikely to present acute hazard (class U) [18].

## 3. Acute pesticide poisoning

Acute pesticide poisoning has become a main health problem especially in developing countries with more than 300,000 deaths in a year. This is due to the poorer regulation, lack of surveillance systems, lack of experience or judgment, less enforcement, the inability to read, lack of training, and inadequate access to information systems [19]. Besides, the easy availability of highly toxic pesticides in farmers' homes has made pesticides the preferred means of suicide [20, 21].

It is not hard to diagnose symptoms of acute pesticide poisoning in humans due to their diversity and resemblance to other conditions. These symptoms could be classified as [9–11]:

• Skin: skin discomfort, rashes, blistering, burns, sweating, contact dermatitis, jaundice

• Nervous system: poor concentration, feelings of weakness, headache, dizziness, mood disturbances, depression, stupor, muscle twitching, seizures, paralysis, loss of consciousness, coma, excessive sweating, impaired vision, tremors, panic attacks, cramps

4. Risk factors for acute pesticide poisoning among farmers in Asia

hygiene such as washing their hands or their clothes after spraying [6, 22, 30].

farmers (3%), and other miscellaneous agricultural workers (19%) [31].

According to a study, approximately 75% of pesticide usage in the United States occurs in agriculture. As such, agricultural workers are at greater risk of pesticide exposure than nonagricultural workers. The findings proved that acute pesticide poisoning in the agricultural industry would continue to be a crucial issue. Of the 3271 cases included in the analysis, 2334 (71%) were employed as farmworkers. The remaining cases were employed as processing/packing plant workers (12%),

Risk factors for acute pesticide poisoning among agricultural workers are also an important issue. A study explored work-related risk factors of acute occupational pesticide poisoning among male farmers in South Korea in 2011. In this study, a total of 1958 male farmers were interviewed. It suggested that the risk of acute occupational pesticide poisoning increased with lifetime days of pesticide application (OR, 1.74; 95% CI, 1.32–2.29). Reasons were not wearing personal protective equipment such as gloves (OR, 1.29) or masks (OR, 1.39), not following pesticide label instructions (OR, 1.61), applying the pesticide in full sun (OR, 1.48), and applying

the pesticide upwind (OR, 1.54) which greatly increased risk of pesticide poisoning [32].

According to a research conducted in China, pesticide poisoning is an important health problem among Chinese farm workers. One thousand farmers were chosen from two villages. Farmers who reported risky behaviors such as not having personal protective equipment, having had a leaky knapsack, not avoiding physical contact with liquid pesticides, or continuing to apply pesticides when feeling ill had greatly higher risk of acute pesticide poisoning than farmers who did not report these behaviors (all p ≤ 0.01) [33]. Data collection from 482 rice farmers in Thailand about risk factors for acute pesticide poisoning gave almost same results.

Pesticides play an important role in farming to increase the productivity of agriculture. However, globally acute pesticide poisoning is a major problem for those who are directly involved in the handling of pesticides, particularly farm workers in many developing countries. They routinely are exposed to high levels of pesticides. Farmers' exposure to acute pesticide poisoning occurs during the preparation of the pesticide spray. There are other ways that farmers are exposed to pesticide poisoning. One of them is during cleaning up of pesticide spraying equipment. Farmers who mix, load, and spray pesticides can be exposed to these toxins due to spills and splashes. Farmers can also be exposed to neighboring fields or by direct contact with pesticide residues on the crop or soil [28]. However, the unsafe, unconscious, indiscriminate, and extensive use of pesticides represents health problems [21]. There are many reasons of agricultural workers' exposure to acute pesticide poisoning. One of them is unsafe use or misuse of pesticides by farmers. Others are poor knowledge of storage, handling, and disposal [29]. Mostly hand pump is used as a spraying equipment by farmers. They complain of poor maintenance and leaks in the sprays. However, despite the fact that protective equipment, gear, and appropriate clothing were provided, farm workers did not use protective gear, as it was uncomfortable in the hot and humid conditions. Farm sprayers did not practice common

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Sometimes, symptoms of acute pesticide poisoning in humans do not become apparent for a long time. Age, body weight, sex, and metabolism all impact the health hazards at different levels. Due to its late effects, they can cause chronic illnesses. Besides general population, both male and female farm workers and children are at high risk to acute pesticide poisoning due to consumer goods or pesticide drift from farm fields. Mostly, women are the major workforce on plantations. They are exposed to pesticides due to mixing, handling, and spraying pesticides in the farm. It is well known that the harmful effects of toxins affect women workforce more than men due to their physiological makeup. According to a study carried on women sprayers, effects of pesticide exposure among them were fatigue, back pain, giddiness, difficulty in breathing, skin problems, nausea, eye irritation, headache, tight feeling in the chest, and swelling. They also stated that some side effects such as bleeding nose occurred initially upon spraying pesticides [22].

According to another study in Pakistan, it was reported upon the use of pesticides in cotton fields; all female workers suffered from headache, nausea, and vomiting [23]. While a study in Bangladesh reported the chronic illnesses on farm workers, about 85% of farm workers reported suffering gastrointestinal disorders during and after spraying, whereas 61, 63, and 47 of them reported, respectively, skin discomfort, eye problems, and feeling of weakness [24].

Moreover, children tend to be involved in more intense contact with their environment than adults. Therefore, they unintentionally ingest pesticides and are poisoned more often than grown-ups [25]. This incident generally occurs because of the unsafe storage and the different behaviors of young children who tend to put everything in their mouths and no ability to read labels and respond appropriately [26].

There have been several researches conducted in developing countries on acute pesticide poisoning. One of them is a study by Hoek and Konradsen in a rural area of Sri Lanka [27]. This study was conducted in two rural hospitals in Sri Lanka where 239 acute pesticide poisoning cases were examined. It reported that the large majority (84%) of incidents were intentional self-poisoning, with a case fatality rate of 18% due to endosulfan and paraquat. Ten of the non-intentional poisoning cases were due to exposure to the poison while spraying pesticides in the fields. Whereas 13% of the patients (31 out of the 239) reported a previous admission for pesticide poisoning, 20% reported a previous suicide attempt.

## 4. Risk factors for acute pesticide poisoning among farmers in Asia

• Nervous system: poor concentration, feelings of weakness, headache, dizziness, mood disturbances, depression, stupor, muscle twitching, seizures, paralysis, loss of conscious-

• Respiratory system: sore throat, runny nose, cough, pulmonary edema, difficulty breath-

Sometimes, symptoms of acute pesticide poisoning in humans do not become apparent for a long time. Age, body weight, sex, and metabolism all impact the health hazards at different levels. Due to its late effects, they can cause chronic illnesses. Besides general population, both male and female farm workers and children are at high risk to acute pesticide poisoning due to consumer goods or pesticide drift from farm fields. Mostly, women are the major workforce on plantations. They are exposed to pesticides due to mixing, handling, and spraying pesticides in the farm. It is well known that the harmful effects of toxins affect women workforce more than men due to their physiological makeup. According to a study carried on women sprayers, effects of pesticide exposure among them were fatigue, back pain, giddiness, difficulty in breathing, skin problems, nausea, eye irritation, headache, tight feeling in the chest, and swelling. They also stated that some side effects such as bleeding nose occurred initially upon

According to another study in Pakistan, it was reported upon the use of pesticides in cotton fields; all female workers suffered from headache, nausea, and vomiting [23]. While a study in Bangladesh reported the chronic illnesses on farm workers, about 85% of farm workers reported suffering gastrointestinal disorders during and after spraying, whereas 61, 63, and 47 of them reported, respectively, skin discomfort, eye problems, and feeling of weakness [24]. Moreover, children tend to be involved in more intense contact with their environment than adults. Therefore, they unintentionally ingest pesticides and are poisoned more often than grown-ups [25]. This incident generally occurs because of the unsafe storage and the different behaviors of young children who tend to put everything in their mouths and no ability to read

There have been several researches conducted in developing countries on acute pesticide poisoning. One of them is a study by Hoek and Konradsen in a rural area of Sri Lanka [27]. This study was conducted in two rural hospitals in Sri Lanka where 239 acute pesticide poisoning cases were examined. It reported that the large majority (84%) of incidents were intentional self-poisoning, with a case fatality rate of 18% due to endosulfan and paraquat. Ten of the non-intentional poisoning cases were due to exposure to the poison while spraying pesticides in the fields. Whereas 13% of the patients (31 out of the 239) reported a previous

admission for pesticide poisoning, 20% reported a previous suicide attempt.

ness, coma, excessive sweating, impaired vision, tremors, panic attacks, cramps

• Eyes: tearing, irritation, conjunctivitis

• Cardiovascular system: cardiac arrhythmias

• Gastrointestinal tract: nausea, vomiting, diarrhea, abdominal pain

128 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

ing, respiratory failure

spraying pesticides [22].

labels and respond appropriately [26].

Pesticides play an important role in farming to increase the productivity of agriculture. However, globally acute pesticide poisoning is a major problem for those who are directly involved in the handling of pesticides, particularly farm workers in many developing countries. They routinely are exposed to high levels of pesticides. Farmers' exposure to acute pesticide poisoning occurs during the preparation of the pesticide spray. There are other ways that farmers are exposed to pesticide poisoning. One of them is during cleaning up of pesticide spraying equipment. Farmers who mix, load, and spray pesticides can be exposed to these toxins due to spills and splashes. Farmers can also be exposed to neighboring fields or by direct contact with pesticide residues on the crop or soil [28]. However, the unsafe, unconscious, indiscriminate, and extensive use of pesticides represents health problems [21]. There are many reasons of agricultural workers' exposure to acute pesticide poisoning. One of them is unsafe use or misuse of pesticides by farmers. Others are poor knowledge of storage, handling, and disposal [29]. Mostly hand pump is used as a spraying equipment by farmers. They complain of poor maintenance and leaks in the sprays. However, despite the fact that protective equipment, gear, and appropriate clothing were provided, farm workers did not use protective gear, as it was uncomfortable in the hot and humid conditions. Farm sprayers did not practice common hygiene such as washing their hands or their clothes after spraying [6, 22, 30].

According to a study, approximately 75% of pesticide usage in the United States occurs in agriculture. As such, agricultural workers are at greater risk of pesticide exposure than nonagricultural workers. The findings proved that acute pesticide poisoning in the agricultural industry would continue to be a crucial issue. Of the 3271 cases included in the analysis, 2334 (71%) were employed as farmworkers. The remaining cases were employed as processing/packing plant workers (12%), farmers (3%), and other miscellaneous agricultural workers (19%) [31].

Risk factors for acute pesticide poisoning among agricultural workers are also an important issue. A study explored work-related risk factors of acute occupational pesticide poisoning among male farmers in South Korea in 2011. In this study, a total of 1958 male farmers were interviewed. It suggested that the risk of acute occupational pesticide poisoning increased with lifetime days of pesticide application (OR, 1.74; 95% CI, 1.32–2.29). Reasons were not wearing personal protective equipment such as gloves (OR, 1.29) or masks (OR, 1.39), not following pesticide label instructions (OR, 1.61), applying the pesticide in full sun (OR, 1.48), and applying the pesticide upwind (OR, 1.54) which greatly increased risk of pesticide poisoning [32].

According to a research conducted in China, pesticide poisoning is an important health problem among Chinese farm workers. One thousand farmers were chosen from two villages. Farmers who reported risky behaviors such as not having personal protective equipment, having had a leaky knapsack, not avoiding physical contact with liquid pesticides, or continuing to apply pesticides when feeling ill had greatly higher risk of acute pesticide poisoning than farmers who did not report these behaviors (all p ≤ 0.01) [33]. Data collection from 482 rice farmers in Thailand about risk factors for acute pesticide poisoning gave almost same results. Pesticide exposure mostly caused from the misuse of pesticides including erroneous beliefs of farmers about pesticide toxicity, the use of faulty spraying equipment or lack of proper maintenance of spraying equipment, and protective gear and appropriate clothing during handling of pesticides [34].

## 5. Interventions for reducing risk factors for farmers' exposure to acute pesticide poisoning

Farmers are the major users of pesticides in agriculture. For this reason, they are more vulnerable to acute pesticide poisoning. There are many ways to reduce the farmers' exposure to acute pesticide poisoning. First of all, farmers must be aware of pesticide risks and its effect on health. The duty of national and international organizations, governments and pesticide industry is to disseminate educational materials of all types to pesticide users, farmers, farmer organizations, agricultural workers, unions, and other interested parties. Thus, pesticide users should learn educational materials before applying pesticides and should follow procedures [12]. Special educational programs can be set up by governments for farm workers prior to pesticide application. This procedure will provide knowledge on risks of pesticide poisoning use and also will decrease the pesticide exposure of farmers [35]. On the other side, protective and appropriate type of clothing and equipment must be used by farmers to prevent exposure to pesticides in all stages of pesticide spraying, mixing, and handling [28]. The United Nations Systems encourage international treaties to restrict export and sale of the most toxic pesticides. Many countries have signed them, but practically they are not implementing these agreements [16]. For this, all governments should develop a legal framework for the control of pesticides [36]. Lack of education, insufficient management, and inappropriate legal framework are the proximal causes of acute pesticide poisoning. Studies which were mentioned below illustrate the situation.

A study that was designed to assess participants' knowledge on the safety use of pesticides involved 300 farm workers working in India. As shown in Table 2, good knowledge on the safety use of pesticides was greatly influenced by the education level of farm workers. It is also seen that farmers lacked seriousness to practice safety measures despite their sufficient knowledge on the safety use of pesticides [35].

In the same study, there was a significant correlation (r, 0.525; p < 0.001) between the knowledge score and the practice score on protective measures.

Table 3 shows that 71.3% of the participants were reported wearing protective clothes and special gloves; 86% were reported wearing of special face mask; 46.1% of farmers were reported wearing face mask while working in the farm; 81.3% of them were reported not eating, drinking, and smoking during spraying; 66.7% of them were reported reading label instructions, 84.7% of the them were mentioned that they used leftover pesticide solutions on the same day, and 55.0% of the participants were reported knowing not to keep the leftover pesticide in a drinking container for later use while only 35% are following. Almost all (100%) participants stated that they wash hands after pesticide application [35].

Another research is carried out in Kuwait with a total of 250 participated farmers. According to the study, the majority (58%) of the farmers did not use any personal protective equipment when mixing or spraying pesticides. Farmers mentioned the reasons for not using personal

Education N Mean Std. deviation F-value p-value

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131

Occupational Risk Factors for Acute Pesticide Poisoning among Farmers in Asia

Never Sometimes Always

5.6% 51.9% 42.5% 71.3%

15.9% 38.0% 46.1% 86.0%

8.2% 38.9% 52.9% 81.3%

10.0% 42.0% 48.0% 66.7%

1.6% 65.4% 33.1% 84.7%

36.7% 18.0% 45.3% 100.0%

12.7% 52.1% 35.2% 55.0%

23.7% 26.3% 50.0% 100.0%

Knowledge on the safety use of pesticides Up to primary 98 78.316 12.3771 6.328\*\* 0.002

Practice on the safety use of pesticides Up to primary 98 52.870 17.1966 0.233 0.793

Safety practice Level of practicing safety measures Total

Wearing of protective clothes and gloves 12 111 91 214

Wearing of special face mask 41 98 119 258

Not eating, drinking, and smoking during the application of pesticides 20 95 129 244

Reading and following label instructions 20 84 96 200

Using leftover pesticide solution in the same day 4 166 84 254

Washing hands after pesticide application 110 54 136 300

Not keeping the leftover pesticide in drinking container 21 86 58 165

Washing contaminated clothes separately 71 79 150 300

Table 3. Safety practices in relation to pesticide knowledge [35].

Table 2. Knowledge and practice on the safety use of pesticides based on their education [35].

\*Significant @ 5% level. \*\*Significant @ 1% level. Up to secondary 126 79.934 8.6792 10th and above 76 83.929 13.6800 Total 300 81.083 12.3728

Up to secondary 126 52.034 10.3631 10th and above 76 51.563 10.2635 Total 300 52.188 12.9429


\*Significant @ 5% level.

Pesticide exposure mostly caused from the misuse of pesticides including erroneous beliefs of farmers about pesticide toxicity, the use of faulty spraying equipment or lack of proper maintenance of spraying equipment, and protective gear and appropriate clothing during handling

130 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

5. Interventions for reducing risk factors for farmers' exposure to acute

Farmers are the major users of pesticides in agriculture. For this reason, they are more vulnerable to acute pesticide poisoning. There are many ways to reduce the farmers' exposure to acute pesticide poisoning. First of all, farmers must be aware of pesticide risks and its effect on health. The duty of national and international organizations, governments and pesticide industry is to disseminate educational materials of all types to pesticide users, farmers, farmer organizations, agricultural workers, unions, and other interested parties. Thus, pesticide users should learn educational materials before applying pesticides and should follow procedures [12]. Special educational programs can be set up by governments for farm workers prior to pesticide application. This procedure will provide knowledge on risks of pesticide poisoning use and also will decrease the pesticide exposure of farmers [35]. On the other side, protective and appropriate type of clothing and equipment must be used by farmers to prevent exposure to pesticides in all stages of pesticide spraying, mixing, and handling [28]. The United Nations Systems encourage international treaties to restrict export and sale of the most toxic pesticides. Many countries have signed them, but practically they are not implementing these agreements [16]. For this, all governments should develop a legal framework for the control of pesticides [36]. Lack of education, insufficient management, and inappropriate legal framework are the proximal causes of acute pesticide poisoning. Studies which were mentioned below illustrate

A study that was designed to assess participants' knowledge on the safety use of pesticides involved 300 farm workers working in India. As shown in Table 2, good knowledge on the safety use of pesticides was greatly influenced by the education level of farm workers. It is also seen that farmers lacked seriousness to practice safety measures despite their sufficient knowl-

In the same study, there was a significant correlation (r, 0.525; p < 0.001) between the knowl-

Table 3 shows that 71.3% of the participants were reported wearing protective clothes and special gloves; 86% were reported wearing of special face mask; 46.1% of farmers were reported wearing face mask while working in the farm; 81.3% of them were reported not eating, drinking, and smoking during spraying; 66.7% of them were reported reading label instructions, 84.7% of the them were mentioned that they used leftover pesticide solutions on the same day, and 55.0% of the participants were reported knowing not to keep the leftover pesticide in a drinking container for later use while only 35% are following. Almost all (100%)

of pesticides [34].

the situation.

edge on the safety use of pesticides [35].

edge score and the practice score on protective measures.

participants stated that they wash hands after pesticide application [35].

pesticide poisoning

\*\*Significant @ 1% level.

Table 2. Knowledge and practice on the safety use of pesticides based on their education [35].


Table 3. Safety practices in relation to pesticide knowledge [35].

Another research is carried out in Kuwait with a total of 250 participated farmers. According to the study, the majority (58%) of the farmers did not use any personal protective equipment when mixing or spraying pesticides. Farmers mentioned the reasons for not using personal protective equipment such as lack of availability when needed (35%), personal protective equipment being uncomfortable in the local hot and humid climate (90%), too expensive (65%), and slowing you down (29%). Younger and educated farmers were more likely to use personal protective equipment compared to older farmers. Farmers were asked if they take safety measures to reduce their risk of exposure to pesticides. The majority of respondents reported not eating (72%), drinking (65%), or smoking (59%) when mixing or applying pesticides. Over 70% of respondents, however, did not wash work clothing used while mixing or spraying pesticides separately from other cloths. Similarly, 46% of respondents reported that they did not consider wind direction when spraying pesticides [37].

Author details

References

07-2017]

287–294

223-228

Emine Selcen Darçın<sup>1</sup>

1 Gazi University, Ankara, Turkey

Essays. 2009;4:945-949

pesticides. Toxicology 2003;192:249–261

Environment International. 2009;35:273-278

orst.edu/rmpp.htm Accessed: 12-08-2017

spectives. 2004;112:142-147

\*, Murat Darçın2

2 Gendarmerie and Coast Guard Academy, Ankara, Turkey

\*Address all correspondence to: darcin@gazi.edu.tr

, Murat Alkan<sup>2</sup> and Gürdoğan Doğrul2

Occupational Risk Factors for Acute Pesticide Poisoning among Farmers in Asia

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133

[1] WHO. Improving Availability of Information about Human Exposures to Pesticides [Internet]. 2017. Available from: http://www.who.int/ipcs/poisons/pesticides/en/ [Accessed: 20-

[2] Damalas CA. Understanding benefits and risks of pesticide use. Scientific Research and

[3] Wesseling C, Aragón A, Castillo L, Corriols M, Chaverri F, de la Cruz E, Keifer M, Monge P, Partanen TJ, Ruepert C, van Wendel de Joode B. Hazardous pesticides in Central America. International Journal of Occupational and Environmental Health 2001;7:

[4] Konradsen F, van der Hoek W, Cole DC, Hutchinson G, Daisley H, Singh S, et al. Reducing acute poisoning in developing countries—Options for restricting the availability of

[5] Coronado GD, Thompson B, Strong L, Griffith WC, Islas I. Agricultural task and exposure to organophosphate pesticides among farm workers. Environmental Health Per-

[6] Hurtig AK, Sebastian MS, Soto A, Shingre A, Zambrano D, Guerrero W. Pesticide use among farmers in the Amazon Basin of Ecuador. Archives of Environmental Health. 2003;14(58):

[7] Soares W, Almeida RMVR, Moro S. Rural work and risk factors associated with pesticide

[8] Remor AP, Totti CC, Moreira DA, Dutra GP, Heuser VD, Boeira JM. Occupational exposure of farm workers to pesticides: Biochemical parameters and evaluation of genotoxicity.

[9] Reigart JR, Roberts JR, editors. Recognition and Management of Pesticide Poisoning. 5th ed. Washington: Environmental Protection Agency [Internet]; 1999. Available from: http://npic.

use in Minas Gerais, Brazil. Cadernos de Saúde Pública. 2003;19:1117-1127

The above findings suggest that governments and pesticide-producing industries should develop and promote the use of pesticide application methods and equipment that decreases risks to human health and expose to pesticide poisoning. Therefore, governments, pesticide industries, NGOs, and international organizations should work together to prevent the pesticide poisoning [12].

The initiative, Agricultural Worker Protection Standard (WPS), for example, is aimed at reducing the risk of pesticide poisoning among agricultural workers. It also offers occupational protections to over two million agricultural workers and pesticide handlers. It is intended to decrease the pesticide exposure incidents among farmworkers and their family members. The requirements in the WPS are intended to inform workers and handlers about pesticide safety and provide protections from potential exposure to pesticides. It keeps workers out of areas being treated with pesticides [38]. It is therefore a laudable intervention that should be continued and evaluated in order to learn from it and improve it.

## 6. Conclusion

Unsafe pesticide use and acute pesticide poisoning are major problems which affect the health of many farm workers all around the world. There are many reasons why acute pesticide poisonings are common among farmers. Some of main risk factors which results to occupational pesticide poisoning of farmers are an insufficient level of knowledge about pesticide use, wearing inappropriate personal protective equipment during spraying, and poor technology. Further studies should specially focus on occupational pesticide poisoning among farmers in order to determine possible short-term and long-term health effects and to develop preventive measures for it.

It is concluded from the existing studies that there are few studies that looked into the risk factors for occupational acute pesticide poisoning. More studies and researches are required in this field to create awareness among governments especially in developing countries. Governments and NGOs must be encouraged and asked for financial support by academicians for better and deep researches on occupational poisoning. It is also the duty of the governments to arrange special educational training programs for the farmers to increase their knowledge on the safety use of pesticide. This educational program will also help farmers to learn more about risk factors of poisoning and occupational safety. In addition, instead of obtaining from hospitals, data must be obtained from quantitative research methods to learn more about the real reasons of occupational poisoning.

## Author details

protective equipment such as lack of availability when needed (35%), personal protective equipment being uncomfortable in the local hot and humid climate (90%), too expensive (65%), and slowing you down (29%). Younger and educated farmers were more likely to use personal protective equipment compared to older farmers. Farmers were asked if they take safety measures to reduce their risk of exposure to pesticides. The majority of respondents reported not eating (72%), drinking (65%), or smoking (59%) when mixing or applying pesticides. Over 70% of respondents, however, did not wash work clothing used while mixing or spraying pesticides separately from other cloths. Similarly, 46% of respondents reported that

The above findings suggest that governments and pesticide-producing industries should develop and promote the use of pesticide application methods and equipment that decreases risks to human health and expose to pesticide poisoning. Therefore, governments, pesticide industries, NGOs, and international organizations should work together to prevent the pesti-

The initiative, Agricultural Worker Protection Standard (WPS), for example, is aimed at reducing the risk of pesticide poisoning among agricultural workers. It also offers occupational protections to over two million agricultural workers and pesticide handlers. It is intended to decrease the pesticide exposure incidents among farmworkers and their family members. The requirements in the WPS are intended to inform workers and handlers about pesticide safety and provide protections from potential exposure to pesticides. It keeps workers out of areas being treated with pesticides [38]. It is therefore a laudable intervention that should be contin-

Unsafe pesticide use and acute pesticide poisoning are major problems which affect the health of many farm workers all around the world. There are many reasons why acute pesticide poisonings are common among farmers. Some of main risk factors which results to occupational pesticide poisoning of farmers are an insufficient level of knowledge about pesticide use, wearing inappropriate personal protective equipment during spraying, and poor technology. Further studies should specially focus on occupational pesticide poisoning among farmers in order to determine possible short-term and long-term health effects and to develop preventive measures for it.

It is concluded from the existing studies that there are few studies that looked into the risk factors for occupational acute pesticide poisoning. More studies and researches are required in this field to create awareness among governments especially in developing countries. Governments and NGOs must be encouraged and asked for financial support by academicians for better and deep researches on occupational poisoning. It is also the duty of the governments to arrange special educational training programs for the farmers to increase their knowledge on the safety use of pesticide. This educational program will also help farmers to learn more about risk factors of poisoning and occupational safety. In addition, instead of obtaining from hospitals, data must be obtained from quantitative research methods to learn more about the real reasons of occupa-

they did not consider wind direction when spraying pesticides [37].

132 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

ued and evaluated in order to learn from it and improve it.

cide poisoning [12].

6. Conclusion

tional poisoning.

Emine Selcen Darçın<sup>1</sup> \*, Murat Darçın2 , Murat Alkan<sup>2</sup> and Gürdoğan Doğrul2

\*Address all correspondence to: darcin@gazi.edu.tr


## References


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[24] Miah SJ, Hoque A, Paul A, Rahman A. Unsafe use of pesticide and its impact on health of farmers: A case study in Burichong Upazila, Bangladesh. IOSR Journal of Environmental

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[25] UNEP. Childhood Pesticide Poisoning: Information for Advocacy and Action. Châtelaine, Switzerland: United Nations Environment Programme [Internet]; 2004. Available from: http://www.who.int/ceh/publications/en/pestpoisoning.pdf [Accessed 20-07-2017] [26] David Suzuki Foundation. Northern Exposure: Acute Pesticide Poisonings in Canada [Internet]. 2007. Available from: http://www.davidsuzuki.org/publications/downloads/

[27] van der Hoek W, Konradsen F. Risk factors for acute pesticide poisoning in Sri Lanka.

[28] Damalas CA, Koutroubas SD. Farmers' exposure to pesticides: Toxicity types and ways of

[29] Karunamoorthi K, Mohammed M, Wassie F. Knowledge and practices of farmers with reference to pesticide management: Implications on human health. Archives of Environ-

[30] Damalas CA, Georgiou EB, Theodorou MG. Pesticide use and safety practices among Greek tobacco farmers: A survey. International Journal of Environmental Health Research.

[31] Calvert GM, Karnik J, Mechler L, Beckman J, Morissey B, Sievert J, Barrett R, Lackovic M, Mabee L, Schwartz A, Mitchell Y, Moraga-McHaley S. Acute pesticide poisoning among agricultural workers in the United States, 1998–2005. American Journal of Industrial

[32] Kim JH, Kim J, Cha ES, Ko Y, Kim DH, Kim DH, Lee WJ. Work-related risk factors by severity for acute pesticide poisoning among male farmers in South Korea. International

[33] Zhang X, Zhao W, Jing R, Wheeler K, Smith GA, Stallones L, Xiang H. Work-related pesticide poisoning among farmers in two villages of southern China: A cross-sectional

[34] Raksanam B, Taneepanichskul S, Siriwong W, Robson M. Factors associated with pesticide risk behaviors among rice farmers in rural community, Thailand. Journal of Environ-

[35] Kumari PL, Reddy KG. Knowledge and practices of safety use of pesticides among farm

[36] Food and Agriculture Organization of the United Nations (FAO). Guidelines for Legislation on the Control of Pesticides [Internet]. 1989. Available from: http://www.fao.org/ fileadmin/templates/agphome/documents/Pests\_Pesticides/Code/Old\_guidelines/LEGIS.

Journal of Environmental Research and Public Health. 2013;10:1100-1112

workers. Journal of Agriculture and Veterinary Science. 2013;6(2):1-8

Science, Toxicology and Food Technology. 2014;8(1):57-67

2007/DSF-pesticide-poisoning.pdf [Accessed 12-07-2017]

prevention. Toxics. 2016;4(1):1-10

2006;16(5):339-348

Medicine. 2008;51(12):883-898

mental & Occupational Health. 2012;67:109-116

survey. BMC Public Health. 2011;11:429-436

ment and Earth Science. 2012;2(2):32-39

pdf [Accessed: 01-10-2017]

Tropical Medicine & International Health 2005;10(6):589–596


[24] Miah SJ, Hoque A, Paul A, Rahman A. Unsafe use of pesticide and its impact on health of farmers: A case study in Burichong Upazila, Bangladesh. IOSR Journal of Environmental Science, Toxicology and Food Technology. 2014;8(1):57-67

[10] Bödeker W. Zur Häufigkeit tödlicher und nichttödlicher Pestizidvergiftungen: eine Betractung nationaler und internationaler Morbiditäts- und Mortalitätsstatistiken. En: Bödeker W, Dümmler C., Hrsg. Pestizide und Gesundheit. Karlsruhe, Alemania: Verlag

[11] Alavanja MCR, Hoppin JA, Kamel F. Health effects of chronic pesticide exposure: Cancer

[12] FAO. International Code of Conduct on the Distribution and Use of Pesticides: Guidelines on Compliance and Enforcement of a Pesticide Regulatory Programme [Internet]. 2006. Available from: www.fao.org/WAICENT/FAOINFO/AGRICULT/AGP/ AGPP/

[13] Watts M. Highly Hazardous Pesticides in the Pacific, National Toxics Network, New Zealand [Internet]. 2016. Available from: http://www.ntn.org.au [Accessed: 20-07-2017]

[14] EPA. What is a Pesticide [Internet]. 2017. Available from: https://www.epa.gov/ minimum-

[15] National Resource Council. Pesticides in the Diets of Infants and Children. Washington,

[16] Jers E. Pesticides: Pesticides Are Responsible for Millions of Human Poisonings and Hundreds of Thousands Deaths each Year [Internet]. 2014. Available from: http://www. globalhealthminders.dk/ghm-emerging-issue-brief-pesticides/ [Accessed: 20-07-2017]

[17] WHO. Children's Health and the Environment. WHO Training Package for the Health Sector [Internet]. 2008. Available from: http://www.who.int/ceh/capacity/Pesticides.pdf

[18] WHO. The WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification. Geneva: World Health Organization [Internet]; 2009. Available from: http://www.who.int/ipcs/publications/pesticides\_hazard/en/ [Accessed: 28-07-2017] [19] Thundiyil JG, Stober J, Besbelli N, Pronczuk J. Acute pesticide poisoning: A proposed classification tool. Bulletin of the World Health Organization. 2008;86(3):205-212

[20] Konradsen F. Acute pesticide poisoning—A global public health problem. Danish Medi-

[21] Jeyaratnam J. Acute pesticide poisoning: A major global health problem. World Health

[22] Pesticide Action Network (PAN) Asia and the Pacific. Poisoned and Silenced: A Study of Pesticide Poisoning in the Plantations [Internet]. 2002. Available from: www.panap.net

[23] Tahir S, Anwar T. Assessment of pesticide exposure in female population living in cotton growing areas of Punjab, Pakistan. Bulletin of Environmental Contamination and Toxi-

and neurotoxicity. Annual Review of Public Health. 2004;25(1):155-197

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Pesticid/Code/ Download/Compliance06.pdf [Accessed: 20-07-2017]

risk-pesticides/what-pesticide [Accessed: 20-07-2017]

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[37] Jallow MF, Awadh DG, Albaho MS, Devi VY, Thomas BM. Pesticide knowledge and safety practices among farm workers in Kuwait: Results of a survey. International Journal of Environmental Research and Public Health. 2017;14(4):3-15

**Chapter 8**

Provisional chapter

**Activating Carbon Fibers and Date Pits for Use in Liver**

DOI: 10.5772/intechopen.71891

Activating Carbon Fibers and Date Pits for Use in Liver

Acute liver failure (ALF) is a rare, potentially fatal complication of severe hepatic illness. It is a syndrome that triggers a cascade of events, leading to multiple organ failures and often death. The work aimed at demonstrating the usefulness of activated raw date pits and carbon fiber reinforced polymers (CFRP) in the management of ALF. The activated carbons produced are used for adsorption of albumin bound toxins from the liver of patients with ALF. The liver is not cured, however, patients are given the time they need to find a suitable donor. Initially, date pits are milled and epoxy is removed from the CFRP. Both materials then undergo pyrolysis and activation treatments. The activated carbon fiber (ACF) and powdered activated carbon (PAC) resulting are tested using FTIR and TGA analysis. FTIR spectrums provide information about functional groups present in the samples and TGA graphs illustrate weight loss as treatment temperature increases. From the data analysis carried out, it appears that the process of recycling both; date pits and CFRP was successful. This confirms the ability of PAC and ACFs to adsorb toxins and as potential candidates for consideration in the search for effective

Acute liver failure (ALF) is a rare, potentially fatal complication of severe hepatic illness resulting from various causes; it is a devastating syndrome that triggers a cascade of events, leading to multiple organ failure and often death. The work presented below aimed at demonstrating the

> © The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Toxin Adsorption**

Toxin Adsorption

Ali Hilal-Alnaqbi

and Ali Hilal-Alnaqbi

Abstract

1. Introduction

Ameereh Seyedzadeh, Asel Mwafy,

Ameereh Seyedzadeh, Asel Mwafy,

http://dx.doi.org/10.5772/intechopen.71891

treatment options for liver failure.

Keywords: carbon fiber, pyrolysis, chitosan, adsorption, activation

Waleed Khalil Ahmed, Kamala Pandurangan and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Waleed Khalil Ahmed, Kamala Pandurangan

[38] United States Environmental Protection Agency (EPA). Agricultural Worker Protection Standard (WPS) [Internet]. 2017. Available from: https://www.epa.gov/pesticide-workersafety/agricultural-worker-protection-standard-wps [Accessed: 20-07-2017]

Provisional chapter

## **Activating Carbon Fibers and Date Pits for Use in Liver Toxin Adsorption** Activating Carbon Fibers and Date Pits for Use in Liver

DOI: 10.5772/intechopen.71891

Ameereh Seyedzadeh, Asel Mwafy, Waleed Khalil Ahmed, Kamala Pandurangan and Ali Hilal-Alnaqbi Ameereh Seyedzadeh, Asel Mwafy, Waleed Khalil Ahmed, Kamala Pandurangan

Additional information is available at the end of the chapter and Ali Hilal-Alnaqbi

http://dx.doi.org/10.5772/intechopen.71891 Additional information is available at the end of the chapter

#### Abstract

Toxin Adsorption

[37] Jallow MF, Awadh DG, Albaho MS, Devi VY, Thomas BM. Pesticide knowledge and safety practices among farm workers in Kuwait: Results of a survey. International Journal

[38] United States Environmental Protection Agency (EPA). Agricultural Worker Protection Standard (WPS) [Internet]. 2017. Available from: https://www.epa.gov/pesticide-worker-

safety/agricultural-worker-protection-standard-wps [Accessed: 20-07-2017]

of Environmental Research and Public Health. 2017;14(4):3-15

136 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

Acute liver failure (ALF) is a rare, potentially fatal complication of severe hepatic illness. It is a syndrome that triggers a cascade of events, leading to multiple organ failures and often death. The work aimed at demonstrating the usefulness of activated raw date pits and carbon fiber reinforced polymers (CFRP) in the management of ALF. The activated carbons produced are used for adsorption of albumin bound toxins from the liver of patients with ALF. The liver is not cured, however, patients are given the time they need to find a suitable donor. Initially, date pits are milled and epoxy is removed from the CFRP. Both materials then undergo pyrolysis and activation treatments. The activated carbon fiber (ACF) and powdered activated carbon (PAC) resulting are tested using FTIR and TGA analysis. FTIR spectrums provide information about functional groups present in the samples and TGA graphs illustrate weight loss as treatment temperature increases. From the data analysis carried out, it appears that the process of recycling both; date pits and CFRP was successful. This confirms the ability of PAC and ACFs to adsorb toxins and as potential candidates for consideration in the search for effective treatment options for liver failure.

Keywords: carbon fiber, pyrolysis, chitosan, adsorption, activation

### 1. Introduction

Acute liver failure (ALF) is a rare, potentially fatal complication of severe hepatic illness resulting from various causes; it is a devastating syndrome that triggers a cascade of events, leading to multiple organ failure and often death. The work presented below aimed at demonstrating the

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

usefulness of activated raw date pits and carbon fiber reinforced polymers (CFRP) in the management of acute liver failure. Date pits are the seeds of Phoenix dactylifera L. (Arecaceae) that have been used in Arabic traditional medicine for centuries to treat diabetes. In the following pages, an overview is presented with a focus on artificial liver support systems including the roles of bilirubin toxin, activated charcoal and purified water.

the presence of higher albumin concentrations played a role in decreasing the bilirubin adsorption. This indicates that in devices similar to MARS, the problem of adsorbing albumin over bilirubin might occur. However, in such a study the influence of changing AC concentrations on the efficiency of bilirubin adsorption was not considered. This means that the negative effect of increasing the albumin concentrations on bilirubin adsorption can be solved. Using higher concentration of AC is one of the possible solutions in which the effect of albumin concentrations will no longer affect the efficiency of bilirubin adsorption. Another very effective method could

Activating Carbon Fibers and Date Pits for Use in Liver Toxin Adsorption

http://dx.doi.org/10.5772/intechopen.71891

139

In addition, previous studies showed that granular AC does not have a high adsorption capacity for bilirubin toxin. Thus, preparing AC in powder form by grinding it will play a vital role in increasing the adsorption capacity of bilirubin toxin [11]. Using granular AC has its own drawbacks in which it does not utilize its full capabilities. This is due to the very small surface area and pore structure of the granular AC as opposed to the powdered AC which has such a high adsorption capacity. By comparing the granular and powdered AC, it can be clearly seen that the adsorption properties depend on two very essential and important ele-

Nikolaev et al. [12] studied the efficiency of adsorbing bilirubin toxin of different types of AC. The different types of AC which were used in this research are Nitrogen based granular carbons, AC based on pyrolysis and fibrous AC. The tests were carried out for different particle sizes ranging between 7 and 9 μm of fibrous AC and 0.5–1.0 mm for activated carbon

Furthermore, a previous study used surface modified chitosan beads to examine their binding affinities for bilirubin in buffer solutions as compared to AC. Throughout conducting this study, it was observed that chitosan beads adsorbed a bilirubin average of 1.18 mg/g of chitosan beads whereas AC adsorbed 0.74 mg/g [13]. Based on the information provided, it can be clearly concluded that combining powdered AC which has a porous structure and large surface area as well as high adsorption capacity with chitosan's high binding affinities for bilirubin could provide a large adsorption capacity for bilirubin. In other words, combining both could play an important role in increasing and enlarging the adsorption capacity for bilirubin than each functional alone. This method is still under testing in order to use it for the

To test for bilirubin adsorption using AC, an albumin-bound bilirubin solution is prepared and mixed with PBS solution to form a stock which is left to stabilize for six hours. PBS is a solution similar to blood plasma which will provide an in lab alternative to using blood for testing. Bilirubin binds to high and low affinity sites on albumin. The AC is expected to adsorb the bilirubin bound to the low affinity sites. The stock will then be serially diluted into different concentrations at a PH of 7.4. a control is taken from each concentration as well as samples containing different amounts of AC. The samples are retained in amber bottles to avoid photo degradation of the bilirubin and are placed in a shaking water bath set at 140 rpm and 37C. A spectrophotometer is then used to test for the albumin and bilirubin present in each sample every hour for the first four hours then after 16 hrs. Between readings, bottles are kept in a shaking water bath. The readings are taken for two wavelengths; 416 nm for bilirubin testing

be coating the AC samples with high binding affinity solutions such as chitosan gel.

ments which are the particle size and internal surface area.

which was prepared using the pyrolysis technique.

adsorption of protein bound toxins from the liver.

#### 1.1. Liver failure

Acute liver failure occurs when patients' livers cease to remove all the toxins in the blood stream therefore an increase in the level of toxins becomes an issue. Some of these toxins bind to certain proteins in order to travel through the blood stream without poisoning other cells and tissues [1]. Due to this strong bond between proteins, such as albumin serum and toxins, it is hard to eliminate them using conventional dialysis [2]. This is because dialysis is only capable of removing water-soluble toxins. However, date seeds are listed in folk remedies for treatment of various infectious diseases, diabetes, hypertension and cancer due to their hepatoprotective effects attributed to the antioxidant and free radical scavenging activities. In the absence of effective means to remove toxins from the liver, liver support devices are utilized to provide patients with the stability they need to either recover or the time they need for transplantation [3]. Liver support devices are categorized into Artificial liver support devices (ALSD) and Bio-Artificial liver support devices (BLSD). The difference being that ALSD are purely mechanical, or in other words; not cell based, while BLSD include a cell element allowing the device to act as a replacement for some of the most important functions in the liver. Payable to this, ALSD act as a bridge to transplantation and do not have the ability to allow the existing liver to recover. On the other hand, BLSD, while acting as a detoxifier also provides the ability to salvage the liver [4].

An example of an ALSD is MARS (Molecular Adsorbent Recirculating System), which is a device used to remove albumin-bound toxins by selectively adsorbing them through membranes in the machine. The toxins are adsorbed onto the surfaces of materials like activated carbon through the membranes [5]. Removing toxins in this manner is crucial for patients with end-stage liver failure due to the single option being liver transplant but lack of donors and the need for a suitable donor with the right blood type in time makes the situation life threatening [6]. The ability to remove these toxins is a temporary fix which gives patients the time they need to find a suitable donor [7]. Carbon is treated to form activated carbon (AC), which is characterized by low-volume pores and a large surface area. AC is one of the best adsorbents due to its large surface area that can be greater than 1000 m2 /g, leading to its broad uses in many purification and medical applications.

#### 1.2. Bilirubin toxin

Bilirubin toxin binds to a certain protein called albumin to travel through the blood stream. Such a bond is hard to break [8]. Thus, many researchers studied the effect of increasing the albumin concentrations on bilirubin adsorption [9]. This was accomplished by many different group studies. Annesi and his team [10] tested for bilirubin as well as tryptophan toxin adsorption using several AC samples with a particle size of 0.3 - 0.5mm. Results showed that the presence of higher albumin concentrations played a role in decreasing the bilirubin adsorption. This indicates that in devices similar to MARS, the problem of adsorbing albumin over bilirubin might occur. However, in such a study the influence of changing AC concentrations on the efficiency of bilirubin adsorption was not considered. This means that the negative effect of increasing the albumin concentrations on bilirubin adsorption can be solved. Using higher concentration of AC is one of the possible solutions in which the effect of albumin concentrations will no longer affect the efficiency of bilirubin adsorption. Another very effective method could be coating the AC samples with high binding affinity solutions such as chitosan gel.

usefulness of activated raw date pits and carbon fiber reinforced polymers (CFRP) in the management of acute liver failure. Date pits are the seeds of Phoenix dactylifera L. (Arecaceae) that have been used in Arabic traditional medicine for centuries to treat diabetes. In the following pages, an overview is presented with a focus on artificial liver support systems including the roles of

Acute liver failure occurs when patients' livers cease to remove all the toxins in the blood stream therefore an increase in the level of toxins becomes an issue. Some of these toxins bind to certain proteins in order to travel through the blood stream without poisoning other cells and tissues [1]. Due to this strong bond between proteins, such as albumin serum and toxins, it is hard to eliminate them using conventional dialysis [2]. This is because dialysis is only capable of removing water-soluble toxins. However, date seeds are listed in folk remedies for treatment of various infectious diseases, diabetes, hypertension and cancer due to their hepatoprotective effects attributed to the antioxidant and free radical scavenging activities. In the absence of effective means to remove toxins from the liver, liver support devices are utilized to provide patients with the stability they need to either recover or the time they need for transplantation [3]. Liver support devices are categorized into Artificial liver support devices (ALSD) and Bio-Artificial liver support devices (BLSD). The difference being that ALSD are purely mechanical, or in other words; not cell based, while BLSD include a cell element allowing the device to act as a replacement for some of the most important functions in the liver. Payable to this, ALSD act as a bridge to transplantation and do not have the ability to allow the existing liver to recover. On the other hand, BLSD, while acting as a detoxifier also

An example of an ALSD is MARS (Molecular Adsorbent Recirculating System), which is a device used to remove albumin-bound toxins by selectively adsorbing them through membranes in the machine. The toxins are adsorbed onto the surfaces of materials like activated carbon through the membranes [5]. Removing toxins in this manner is crucial for patients with end-stage liver failure due to the single option being liver transplant but lack of donors and the need for a suitable donor with the right blood type in time makes the situation life threatening [6]. The ability to remove these toxins is a temporary fix which gives patients the time they need to find a suitable donor [7]. Carbon is treated to form activated carbon (AC), which is characterized by low-volume pores and a large surface area. AC is one of the best adsorbents

Bilirubin toxin binds to a certain protein called albumin to travel through the blood stream. Such a bond is hard to break [8]. Thus, many researchers studied the effect of increasing the albumin concentrations on bilirubin adsorption [9]. This was accomplished by many different group studies. Annesi and his team [10] tested for bilirubin as well as tryptophan toxin adsorption using several AC samples with a particle size of 0.3 - 0.5mm. Results showed that

/g, leading to its broad uses in

bilirubin toxin, activated charcoal and purified water.

138 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

provides the ability to salvage the liver [4].

many purification and medical applications.

1.2. Bilirubin toxin

due to its large surface area that can be greater than 1000 m2

1.1. Liver failure

In addition, previous studies showed that granular AC does not have a high adsorption capacity for bilirubin toxin. Thus, preparing AC in powder form by grinding it will play a vital role in increasing the adsorption capacity of bilirubin toxin [11]. Using granular AC has its own drawbacks in which it does not utilize its full capabilities. This is due to the very small surface area and pore structure of the granular AC as opposed to the powdered AC which has such a high adsorption capacity. By comparing the granular and powdered AC, it can be clearly seen that the adsorption properties depend on two very essential and important elements which are the particle size and internal surface area.

Nikolaev et al. [12] studied the efficiency of adsorbing bilirubin toxin of different types of AC. The different types of AC which were used in this research are Nitrogen based granular carbons, AC based on pyrolysis and fibrous AC. The tests were carried out for different particle sizes ranging between 7 and 9 μm of fibrous AC and 0.5–1.0 mm for activated carbon which was prepared using the pyrolysis technique.

Furthermore, a previous study used surface modified chitosan beads to examine their binding affinities for bilirubin in buffer solutions as compared to AC. Throughout conducting this study, it was observed that chitosan beads adsorbed a bilirubin average of 1.18 mg/g of chitosan beads whereas AC adsorbed 0.74 mg/g [13]. Based on the information provided, it can be clearly concluded that combining powdered AC which has a porous structure and large surface area as well as high adsorption capacity with chitosan's high binding affinities for bilirubin could provide a large adsorption capacity for bilirubin. In other words, combining both could play an important role in increasing and enlarging the adsorption capacity for bilirubin than each functional alone. This method is still under testing in order to use it for the adsorption of protein bound toxins from the liver.

To test for bilirubin adsorption using AC, an albumin-bound bilirubin solution is prepared and mixed with PBS solution to form a stock which is left to stabilize for six hours. PBS is a solution similar to blood plasma which will provide an in lab alternative to using blood for testing. Bilirubin binds to high and low affinity sites on albumin. The AC is expected to adsorb the bilirubin bound to the low affinity sites. The stock will then be serially diluted into different concentrations at a PH of 7.4. a control is taken from each concentration as well as samples containing different amounts of AC. The samples are retained in amber bottles to avoid photo degradation of the bilirubin and are placed in a shaking water bath set at 140 rpm and 37C. A spectrophotometer is then used to test for the albumin and bilirubin present in each sample every hour for the first four hours then after 16 hrs. Between readings, bottles are kept in a shaking water bath. The readings are taken for two wavelengths; 416 nm for bilirubin testing and 350 nm for albumin testing. The albumin readings are expected to remain stable since AC adsorbs bilirubin and not albumin. Bilirubin on the other hand is expected to decrease with time in the bottles containing the AC.

pregnancy as well as lowering cholesterol levels. Several studies have been carried out on the possibility of utilizing ACF in the medical field. It was found that ACF can be used as an antiflatulent in the abdominal radiography process. Other than that, activated carbon in general is a significant ingredient for stomach remedies. It helps in controlling diarrhea and flatulence, as

Activating Carbon Fibers and Date Pits for Use in Liver Toxin Adsorption

http://dx.doi.org/10.5772/intechopen.71891

141

Water makes up more than two thirds of human body weight and the earth's surface. This is evidence that water is one of the most essential substances on earth. In fact, if there was no water there would be no life on earth. However, that same water might be harmful to the human body if it was not purified. Thus, water purification is an important industry requirement in which water undergoes a number of treatments to be usable and drinkable. Water purifiers are designed to remove impurities and contaminants from water. Several materials are used in water purifiers to help removing these contaminants. ACF can be used as a media for water purification. It is a porous material with a high adsorption capacity by which it could adsorb the pollutants perfectly. Recently some studies have shown that ACF is popular in water purification industry and that is due to removing heavy metals like lead, as well as

There are three possible methods of recycling CFRP which include mechanical, thermal and chemical recycling. Mechanical recycling mainly involves size reduction methods, such as cutting, trimming or shredding. Thermal recycling comprises of material treatment at very high temperatures. The type of thermal recycling of interest in this work is pyrolysis, where the material is heated in a furnace in the presence of Nitrogen gas. Furthermore, the chemical recycling process

Carbon fiber reinforced polymers (CFRPs) are composite materials that are often coated by epoxy layers. These epoxy layers add to the carbon fiber properties in which it has been proved through different studies that epoxy contributes in the enhancement of the chemical resistance, strength and durability of the composite materials [27]. Although, the use of epoxy resin is beneficial in many aspects, it originates difficulties related to the removal of this very strong resin. Therefore, many methods are followed in order to remove epoxy from carbon fiber [28]. Using acetone as a solvent to dissolve the epoxy is one of the approaches that is followed during the curing process. A certain amount of acetone is added to the carbon fiber to dissolve an epoxy resin. In other words, acetone/epoxy solutions of different concentrations are prepared. The solution is then sonicated and stirred for an hour. After that, the mixture is exposed to heating at a certain temperature. Lastly, hardener is added to the mixture. Other studies

revolves around the removal of the matrix using chemical dissolution reagents [26].

well as lowering the toxin levels throughout the body.

dissolving chemicals and some types of parasites.

2.2. Removing epoxy from carbon fiber

2. Methods of recycling CFRP and removing epoxy

1.4.2. Purification of water

2.1. Recycling CFRP

#### 1.3. Activated carbon

#### 1.3.1. Date pit activated carbon

AC can be prepared from many different raw materials depending on availability, surface area, pore size distribution and porous texture [14]. Some examples are wood, coal, coconut, rice husk, shells of plants, stones of fruits, asphalt, metal carbides, carbon blacks, and polymer scraps [15]. Due to the abundance of date palm biomass in the UAE, it can be used to prepare AC. Date pits have a mass varying between 10 and 15% of the total date fruit mass. Payable to their high nutritional value, date pits' utilization is highly requested by the date processing industries in raising their value-added products [16].

#### 1.3.2. Activated carbon fiber

Carbon fiber reinforced polymers (CFRP) are widely used in many industries, most important of which are the aerospace and automobile industries [17]. This is due to their lightweight, very high strength and ability to endure impact [18]. Around 20% of yearly prepreg carbon fiber production goes to use in the aerospace field as well as 30% being facilitated to the automobile industry [19]. An increasing number of aircraft structures have become highly dependent on the use of these materials. Some of which are the Boeing 787 Dreamliner and Airbus A380 and A350 [20].

With an increase in demand for CFRP comes a larger amount of scrap generated. Scrap sources include end of roll, trim, out-timed and out-of-spec waste. Many manufacturers deal with this waste by disposing of it into landfills [21]. This is not only harmful for the environment but is also costly for manufacturers [22]. Therefore, methods of recycling these CFRP are being researched and some are being implemented on an industrial scale. Recycling CFRP can either be done thermally or chemically [23].

#### 1.4. Applications of activated carbon fiber

In today's world, activated carbon fiber (ACF) is a highly versatile material which has been a pioneer within a broad range of applications [24]. What was stated as an exotic material many years ago is nowadays playing an intrinsic role in our daily lives [25]. As outlined below, several applications of ACF will be discussed.

#### 1.4.1. Medical applications

In the medical field, activated carbon fiber has many remarkable uses. It is normally linked with the adsorption of organic toxins in which it is widely used as an antitoxin in the treatment of kidney and liver disease. In other words, for blood dialysis in the treatment of liver and kidney disease, ACF is used as a filtering medium that adsorbs toxins from the blood stream of patients' bodies. Other uses in the medical field include the treatment of cholestasis during pregnancy as well as lowering cholesterol levels. Several studies have been carried out on the possibility of utilizing ACF in the medical field. It was found that ACF can be used as an antiflatulent in the abdominal radiography process. Other than that, activated carbon in general is a significant ingredient for stomach remedies. It helps in controlling diarrhea and flatulence, as well as lowering the toxin levels throughout the body.

#### 1.4.2. Purification of water

and 350 nm for albumin testing. The albumin readings are expected to remain stable since AC adsorbs bilirubin and not albumin. Bilirubin on the other hand is expected to decrease with

140 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

AC can be prepared from many different raw materials depending on availability, surface area, pore size distribution and porous texture [14]. Some examples are wood, coal, coconut, rice husk, shells of plants, stones of fruits, asphalt, metal carbides, carbon blacks, and polymer scraps [15]. Due to the abundance of date palm biomass in the UAE, it can be used to prepare AC. Date pits have a mass varying between 10 and 15% of the total date fruit mass. Payable to their high nutritional value, date pits' utilization is highly requested by the date processing

Carbon fiber reinforced polymers (CFRP) are widely used in many industries, most important of which are the aerospace and automobile industries [17]. This is due to their lightweight, very high strength and ability to endure impact [18]. Around 20% of yearly prepreg carbon fiber production goes to use in the aerospace field as well as 30% being facilitated to the automobile industry [19]. An increasing number of aircraft structures have become highly dependent on the use of these materials. Some of which are the Boeing 787 Dreamliner and

With an increase in demand for CFRP comes a larger amount of scrap generated. Scrap sources include end of roll, trim, out-timed and out-of-spec waste. Many manufacturers deal with this waste by disposing of it into landfills [21]. This is not only harmful for the environment but is also costly for manufacturers [22]. Therefore, methods of recycling these CFRP are being researched and some are being implemented on an industrial scale. Recycling CFRP can either

In today's world, activated carbon fiber (ACF) is a highly versatile material which has been a pioneer within a broad range of applications [24]. What was stated as an exotic material many years ago is nowadays playing an intrinsic role in our daily lives [25]. As outlined below,

In the medical field, activated carbon fiber has many remarkable uses. It is normally linked with the adsorption of organic toxins in which it is widely used as an antitoxin in the treatment of kidney and liver disease. In other words, for blood dialysis in the treatment of liver and kidney disease, ACF is used as a filtering medium that adsorbs toxins from the blood stream of patients' bodies. Other uses in the medical field include the treatment of cholestasis during

time in the bottles containing the AC.

industries in raising their value-added products [16].

1.3. Activated carbon

1.3.1. Date pit activated carbon

1.3.2. Activated carbon fiber

Airbus A380 and A350 [20].

1.4.1. Medical applications

be done thermally or chemically [23].

1.4. Applications of activated carbon fiber

several applications of ACF will be discussed.

Water makes up more than two thirds of human body weight and the earth's surface. This is evidence that water is one of the most essential substances on earth. In fact, if there was no water there would be no life on earth. However, that same water might be harmful to the human body if it was not purified. Thus, water purification is an important industry requirement in which water undergoes a number of treatments to be usable and drinkable. Water purifiers are designed to remove impurities and contaminants from water. Several materials are used in water purifiers to help removing these contaminants. ACF can be used as a media for water purification. It is a porous material with a high adsorption capacity by which it could adsorb the pollutants perfectly. Recently some studies have shown that ACF is popular in water purification industry and that is due to removing heavy metals like lead, as well as dissolving chemicals and some types of parasites.

## 2. Methods of recycling CFRP and removing epoxy

#### 2.1. Recycling CFRP

There are three possible methods of recycling CFRP which include mechanical, thermal and chemical recycling. Mechanical recycling mainly involves size reduction methods, such as cutting, trimming or shredding. Thermal recycling comprises of material treatment at very high temperatures. The type of thermal recycling of interest in this work is pyrolysis, where the material is heated in a furnace in the presence of Nitrogen gas. Furthermore, the chemical recycling process revolves around the removal of the matrix using chemical dissolution reagents [26].

#### 2.2. Removing epoxy from carbon fiber

Carbon fiber reinforced polymers (CFRPs) are composite materials that are often coated by epoxy layers. These epoxy layers add to the carbon fiber properties in which it has been proved through different studies that epoxy contributes in the enhancement of the chemical resistance, strength and durability of the composite materials [27]. Although, the use of epoxy resin is beneficial in many aspects, it originates difficulties related to the removal of this very strong resin. Therefore, many methods are followed in order to remove epoxy from carbon fiber [28]. Using acetone as a solvent to dissolve the epoxy is one of the approaches that is followed during the curing process. A certain amount of acetone is added to the carbon fiber to dissolve an epoxy resin. In other words, acetone/epoxy solutions of different concentrations are prepared. The solution is then sonicated and stirred for an hour. After that, the mixture is exposed to heating at a certain temperature. Lastly, hardener is added to the mixture. Other studies showed how adding acetone might impact the characteristics of epoxy. According to these authors, the occurrence of residual solvent might lead to some significant changes in the mechanical and physical properties as well as the thermal degradation.

Further studies will later be conducted regarding the use of the different types of charcoal produced for the adsorption of albumin-bound bilirubin. Batch tests will be carried out on different samples to test for the effect of coated AC dosage on the adsorption capacity of the materials. Moreover, the contact time between the coated AC and bilirubin toxin will be tested to determine whether increasing the contact time will increase the amount of bilirubin adsorbed.

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The aim of this work is to use thermal treatment by pyrolysis to remove the epoxy from the CFRP and prepare the material for activation. According to literature, some drawbacks to pyrolysis may be faced, including the loss of mechanical properties such as tensile strength and elasticity. Furthermore, the fibers released are likely to be covered in char, a black residue

Several materials were utilized in the preparation, pyrolysis and activation processes of the

To prepare the CFRP sheets, obtained from the aerospace industry, for pyrolysis, the sheets of material are shredded into strips. This is to enable their placement in the furnace to carry out the thermal treatments. The steps toward activated carbon fiber are pyrolysis and activation, both carried out in a furnace (GSL – 1500X). the samples of CFRP are separately placed in the steel roll inside the furnace. For the pyrolysis process, nitrogen gas (N2) is passed through the sample and for activation, carbon dioxide (CO2) is passed into the steel roll. In order to control the operation temperatures inside the furnace, it is connected to a laptop with the PT software installed. The temperature program to be run is added to this software and the temperature

Following pyrolysis, samples of the treated carbon fiber are immersed in acetone to indicate the presence or absence of epoxy. In accordance to later tests, if the acetone turned into a

The carbon fiber reinforced polymer is waste product obtained from the aerospace industry. It is attained in sheets, which are shredded before carrying out treatment and tests. The method followed for activating the material is physical activation to avoid contamination or poisoning in case the activated carbon fiber is to be used for purification or medical purposes. Physical

The aim of pyrolysis is to remove the epoxy in the material as well as provide an inert atmosphere for activation to take place. The process involves passing nitrogen gas through

yellow color, this is an indication of the presence of epoxy in the sample.

activation involves a two-step process; pyrolysis and activation.

3. Experimental processing

formed during the thermal degradation of the resin.

rises and falls are monitored on the screen.

3.2. Procedural setups

3.2.1. Pyrolysis

3.1. Materials

CFRP.

In contrast, FTIR results showed that the presence of acetone does not affect the chemical properties of the material. According to the literature, it can be seen that a more efficient method of removing epoxy has been applied. This technique is based on using thermal processes in order to remove the epoxy from the carbon fiber. Thermal processes could include more than one method and technique such as pyrolysis. Pyrolysis is one of the most studied thermal processes due to its popularity in the commercial scale [29]. The process operates at different temperature ranges depending on what type of resin is used. For example, epoxy resin tends to be stronger than other types of resin which means it might require higher temperatures to be fully removed. One of the main drawbacks of using this technique is the remaining resin residue such as char. This char contaminates the fibers and thus a post treatment is needed to burn it. However, exposing this composite material to high temperatures during the treatment might lead to a significant reduction in the properties of the material. Different group works have studied the effect of raising the temperature on the properties of the carbon fiber. The results showed that for some types of fiber such as glass fibers a temperature of 1300C is needed in order to remove the char completely and to produce a perfectly clean one but a reduction by up to 85% of the tensile strength was observed [30]. Thus, in order to produce an acceptable strength for the carbon fibers a maximum pyrolysis temperature in the range of 500–550C is advisable.

Chitosan can be used in many different applications. In biomedical applications, Chitosan can act as an interaction site in order to increase the adsorption capacity [31]. Therefore, in this work chitosan was used in the coating process in which both activated carbon fiber (ACF) and date pit activated carbon (DPAC) samples were coated with Chitosan [32]. The chitosan used in coating both materials contains amino (NH2) and hydroxyl (OH) groups on its chains [33]. These groups accomplished the main benefit of using chitosan. In other words, these groups helped in forming AC with a large adsorption capacity [34].

Chitosan is a natural source that is available abundantly in nature. It can be produced and found naturally in the cell walls of fungi, the shells of crustaceans and the shells of insects [35]. It is produced commercially in many different forms. Chitosan powder is one of the forms which can be used and found in the market [36]. Thus, in this work the chitosan powder is used in making chitosan gel which will be used to coat the samples of ACF and DPAC.

This work focuses on removing the epoxy from CFRP and activating, both date pit powder and CFRP at different temperatures. This will allow a comparison of the results under different conditions to find the optimum activating temperature for the materials. Chitosan gel is then prepared by mixing chitosan powder and dilute acetic acid. The resulting solution is used to coat both types of charcoal. It is expected that coating will increase the adsorption efficiency of the materials by providing a high binding affinity for carbon fiber. The resulting samples are all compared to conclude which material has a higher bilirubin adsorption capacity and the optimum activation temperature for the DPAC and ACF.

Further studies will later be conducted regarding the use of the different types of charcoal produced for the adsorption of albumin-bound bilirubin. Batch tests will be carried out on different samples to test for the effect of coated AC dosage on the adsorption capacity of the materials. Moreover, the contact time between the coated AC and bilirubin toxin will be tested to determine whether increasing the contact time will increase the amount of bilirubin adsorbed.

## 3. Experimental processing

#### 3.1. Materials

showed how adding acetone might impact the characteristics of epoxy. According to these authors, the occurrence of residual solvent might lead to some significant changes in the

In contrast, FTIR results showed that the presence of acetone does not affect the chemical properties of the material. According to the literature, it can be seen that a more efficient method of removing epoxy has been applied. This technique is based on using thermal processes in order to remove the epoxy from the carbon fiber. Thermal processes could include more than one method and technique such as pyrolysis. Pyrolysis is one of the most studied thermal processes due to its popularity in the commercial scale [29]. The process operates at different temperature ranges depending on what type of resin is used. For example, epoxy resin tends to be stronger than other types of resin which means it might require higher temperatures to be fully removed. One of the main drawbacks of using this technique is the remaining resin residue such as char. This char contaminates the fibers and thus a post treatment is needed to burn it. However, exposing this composite material to high temperatures during the treatment might lead to a significant reduction in the properties of the material. Different group works have studied the effect of raising the temperature on the properties of the carbon fiber. The results showed that for some types of fiber such as glass fibers a temperature of 1300C is needed in order to remove the char completely and to produce a perfectly clean one but a reduction by up to 85% of the tensile strength was observed [30]. Thus, in order to produce an acceptable strength for the carbon fibers a maximum pyrolysis temperature in

Chitosan can be used in many different applications. In biomedical applications, Chitosan can act as an interaction site in order to increase the adsorption capacity [31]. Therefore, in this work chitosan was used in the coating process in which both activated carbon fiber (ACF) and date pit activated carbon (DPAC) samples were coated with Chitosan [32]. The chitosan used in coating both materials contains amino (NH2) and hydroxyl (OH) groups on its chains [33]. These groups accomplished the main benefit of using chitosan. In other words, these

Chitosan is a natural source that is available abundantly in nature. It can be produced and found naturally in the cell walls of fungi, the shells of crustaceans and the shells of insects [35]. It is produced commercially in many different forms. Chitosan powder is one of the forms which can be used and found in the market [36]. Thus, in this work the chitosan powder is used in making chitosan gel which will be used to coat the samples of ACF and DPAC.

This work focuses on removing the epoxy from CFRP and activating, both date pit powder and CFRP at different temperatures. This will allow a comparison of the results under different conditions to find the optimum activating temperature for the materials. Chitosan gel is then prepared by mixing chitosan powder and dilute acetic acid. The resulting solution is used to coat both types of charcoal. It is expected that coating will increase the adsorption efficiency of the materials by providing a high binding affinity for carbon fiber. The resulting samples are all compared to conclude which material has a higher bilirubin adsorption capacity and the

groups helped in forming AC with a large adsorption capacity [34].

optimum activation temperature for the DPAC and ACF.

mechanical and physical properties as well as the thermal degradation.

142 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

the range of 500–550C is advisable.

The aim of this work is to use thermal treatment by pyrolysis to remove the epoxy from the CFRP and prepare the material for activation. According to literature, some drawbacks to pyrolysis may be faced, including the loss of mechanical properties such as tensile strength and elasticity. Furthermore, the fibers released are likely to be covered in char, a black residue formed during the thermal degradation of the resin.

Several materials were utilized in the preparation, pyrolysis and activation processes of the CFRP.

To prepare the CFRP sheets, obtained from the aerospace industry, for pyrolysis, the sheets of material are shredded into strips. This is to enable their placement in the furnace to carry out the thermal treatments. The steps toward activated carbon fiber are pyrolysis and activation, both carried out in a furnace (GSL – 1500X). the samples of CFRP are separately placed in the steel roll inside the furnace. For the pyrolysis process, nitrogen gas (N2) is passed through the sample and for activation, carbon dioxide (CO2) is passed into the steel roll. In order to control the operation temperatures inside the furnace, it is connected to a laptop with the PT software installed. The temperature program to be run is added to this software and the temperature rises and falls are monitored on the screen.

Following pyrolysis, samples of the treated carbon fiber are immersed in acetone to indicate the presence or absence of epoxy. In accordance to later tests, if the acetone turned into a yellow color, this is an indication of the presence of epoxy in the sample.

#### 3.2. Procedural setups

#### 3.2.1. Pyrolysis

The carbon fiber reinforced polymer is waste product obtained from the aerospace industry. It is attained in sheets, which are shredded before carrying out treatment and tests. The method followed for activating the material is physical activation to avoid contamination or poisoning in case the activated carbon fiber is to be used for purification or medical purposes. Physical activation involves a two-step process; pyrolysis and activation.

The aim of pyrolysis is to remove the epoxy in the material as well as provide an inert atmosphere for activation to take place. The process involves passing nitrogen gas through the sample. The shredded CFRP is added to the steel roll, which is then inserted into the furnace. The steel roll is positioned correctly using a marked rod and the furnace is closed from both ends with tubes through which the nitrogen gas will flow. The gas enters and leaves the furnace passing through the CFRP sample. To ensure that approximately the right amount of gas is passing through the sample, the outlet tube is connected to a tilted water bottle and the bubbles created are observed. To change the gas flow rate, the valve on the nitrogen gas cylinder is controlled.

capacity of the activated carbon fiber will be tested and compared with the adsorption capacity

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Chitosan gel is used to coat a sample of the DPAC to allow for comparisons with the ACF and uncoated DPAC. To prepare the chitosan gel, 198 ml of water are mixed with 2 ml of acetic acid in a reagent bottle to form dilute acetic acid. The outcome is a 1% concentrated acetic acid by volume. 100 ml of the solution is then measured using a measuring cylinder and poured into a beaker with a magnet placed inside and put on a magnetic stirrer to stir and heat the solution first. 0.5 g of chitosan powder is gradually added to the stirring diluted acid, to avoid splashes, clumping and achieve a more even distribution. The magnetic stirrer is set at a temperature around 45C. Approximately an hour later, when the chitosan gel has reached the desired consistency, 5 g of the activated carbon are weighed and added gradually to the gel after turning off the heater so the mixture is only stirring. The carbon is then left to coat overnight as it stirs (for about 24 hours). The coating procedure is carried out three times while oven

drying between each coating. This is done to ensure that the DPAC is fully coated [8].

For determining the characterization of activated carbon fiber, FTIR and TGA analysis are

The main benefits of using a Fourier Transform Infrared (FTIR) spectroscopy is that it is quick and takes measurements in a matter of seconds. It also has a very high sensitivity due to the extremely delicate detectors employed in the device. Infrared spectroscopy gives information about the chemical structure and functional groups of raw materials and the prepared activated carbon. The data obtained presented information about the interaction and polymeric association between chitosan and carbon. The Infra-red transmission spectra were recorded with a Perkin Elmer spectrophotometer ranging from 400 to 4000 cm<sup>1</sup> using the KBr technique.

TGA (Thermogravimetric analysis) is an essential method used in materials characterization. It is a technique in which the mass of a substance is measured as a function of temperature or time as the sample is monitored under a controlled atmosphere. This technique provides useful information about chemical phenomena such as solid–gas reactions and thermal decomposition, as well as physical phenomena including phase transitions, absorption and desorption. A typical TGA analyzer consists of a sample pan that is backed by a precision balance inside a furnace. The temperature inside the furnace is programmed under a controlled temperature. The mass of the sample is monitored during the experiment. This experiment may occur under different conditions in which it could be conducted under a variety of atmospheres including: inert gas, carburizing gases, ambient air, vapors and vacuum. Also, the experiment might take place under a variety of pressures such as high vacuum, high pressure,

of activated date pit powder.

3.2.4. Chitosan gel preparation and coating

3.3. Experimental techniques used

3.3.1. FTIR (Fourier transform infrared spectroscopy)

conducted.

3.3.2. TGA test

Passing an inert gas through the sample aims at removing impurities, such as hydrogen and oxygen to create a more stable and heat resistant compound composed mainly of carbon. The pyrolysis treatment is carried out by increasing the temperature inside the furnace while passing the gas through the steel roll. The temperature is kept at its peak for a specific period of time before it is dropped back to room temperature. To control the temperature rises and falls, the furnace is connected to a laptop with the PT software installed where a simple program is entered for the various temperatures and durations.

To find the optimum temperature for the pyrolysis and activation processes, they are executed at various temperatures; 600, 800 and 1000�C. Tests are later used to decide which treatment temperature is most effective.

#### 3.2.2. Acetone test

One of the biggest challenges faced in this work is the removal of the epoxy in the material. This is because the exact nature of the epoxy is unknown due to restrictions from the aircraft manufacturers. Therefore, acetone testing is used after the pyrolysis process as a step to ensure that the epoxy has been eliminated from the material. Approximately 20 ml of acetone are added to a beaker and a sample of the material which has undergone pyrolysis treatment is added to the beaker and left to soak overnight. It is expected that the acetone of the sample containing epoxy turns yellow, as opposed to clear for the sample which contains no epoxy. This test is done on samples treated at all temperatures prior to activation.

#### 3.2.3. Activation

During the next stage, carbon dioxide gas is passed through the sample to allow some carbon to react with it producing carbon monoxide gas, as shown in Eq. (1), which is then removed from the system through the water bottle with the excess gas. This process is known as gasification and develops porosity in the material by removing some carbon atoms. The furnace is operated in a similar way to the pyrolysis process with the only difference being the gas utilized. Since pyrolysis treatments will be carried out at three different temperatures of 600, 800 and 1000�C, the same will be done with the activation treatment.

$$\text{C} + \text{CO}\_2 \rightarrow 2\text{CO} \tag{1}$$

It is expected, according to previous literature, that the carbon fiber will have a larger adsorption capacity of toxins than powdered activated carbon. In order to prove this, the adsorption capacity of the activated carbon fiber will be tested and compared with the adsorption capacity of activated date pit powder.

#### 3.2.4. Chitosan gel preparation and coating

the sample. The shredded CFRP is added to the steel roll, which is then inserted into the furnace. The steel roll is positioned correctly using a marked rod and the furnace is closed from both ends with tubes through which the nitrogen gas will flow. The gas enters and leaves the furnace passing through the CFRP sample. To ensure that approximately the right amount of gas is passing through the sample, the outlet tube is connected to a tilted water bottle and the bubbles created are observed. To change the gas flow rate, the valve on the nitrogen gas

Passing an inert gas through the sample aims at removing impurities, such as hydrogen and oxygen to create a more stable and heat resistant compound composed mainly of carbon. The pyrolysis treatment is carried out by increasing the temperature inside the furnace while passing the gas through the steel roll. The temperature is kept at its peak for a specific period of time before it is dropped back to room temperature. To control the temperature rises and falls, the furnace is connected to a laptop with the PT software installed where a simple

To find the optimum temperature for the pyrolysis and activation processes, they are executed at various temperatures; 600, 800 and 1000�C. Tests are later used to decide which treatment

One of the biggest challenges faced in this work is the removal of the epoxy in the material. This is because the exact nature of the epoxy is unknown due to restrictions from the aircraft manufacturers. Therefore, acetone testing is used after the pyrolysis process as a step to ensure that the epoxy has been eliminated from the material. Approximately 20 ml of acetone are added to a beaker and a sample of the material which has undergone pyrolysis treatment is added to the beaker and left to soak overnight. It is expected that the acetone of the sample containing epoxy turns yellow, as opposed to clear for the sample which contains no epoxy.

During the next stage, carbon dioxide gas is passed through the sample to allow some carbon to react with it producing carbon monoxide gas, as shown in Eq. (1), which is then removed from the system through the water bottle with the excess gas. This process is known as gasification and develops porosity in the material by removing some carbon atoms. The furnace is operated in a similar way to the pyrolysis process with the only difference being the gas utilized. Since pyrolysis treatments will be carried out at three different temperatures

It is expected, according to previous literature, that the carbon fiber will have a larger adsorption capacity of toxins than powdered activated carbon. In order to prove this, the adsorption

C þ CO2 ! 2CO (1)

This test is done on samples treated at all temperatures prior to activation.

of 600, 800 and 1000�C, the same will be done with the activation treatment.

program is entered for the various temperatures and durations.

144 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

cylinder is controlled.

temperature is most effective.

3.2.2. Acetone test

3.2.3. Activation

Chitosan gel is used to coat a sample of the DPAC to allow for comparisons with the ACF and uncoated DPAC. To prepare the chitosan gel, 198 ml of water are mixed with 2 ml of acetic acid in a reagent bottle to form dilute acetic acid. The outcome is a 1% concentrated acetic acid by volume. 100 ml of the solution is then measured using a measuring cylinder and poured into a beaker with a magnet placed inside and put on a magnetic stirrer to stir and heat the solution first. 0.5 g of chitosan powder is gradually added to the stirring diluted acid, to avoid splashes, clumping and achieve a more even distribution. The magnetic stirrer is set at a temperature around 45C. Approximately an hour later, when the chitosan gel has reached the desired consistency, 5 g of the activated carbon are weighed and added gradually to the gel after turning off the heater so the mixture is only stirring. The carbon is then left to coat overnight as it stirs (for about 24 hours). The coating procedure is carried out three times while oven drying between each coating. This is done to ensure that the DPAC is fully coated [8].

#### 3.3. Experimental techniques used

For determining the characterization of activated carbon fiber, FTIR and TGA analysis are conducted.

#### 3.3.1. FTIR (Fourier transform infrared spectroscopy)

The main benefits of using a Fourier Transform Infrared (FTIR) spectroscopy is that it is quick and takes measurements in a matter of seconds. It also has a very high sensitivity due to the extremely delicate detectors employed in the device. Infrared spectroscopy gives information about the chemical structure and functional groups of raw materials and the prepared activated carbon. The data obtained presented information about the interaction and polymeric association between chitosan and carbon. The Infra-red transmission spectra were recorded with a Perkin Elmer spectrophotometer ranging from 400 to 4000 cm<sup>1</sup> using the KBr technique.

#### 3.3.2. TGA test

TGA (Thermogravimetric analysis) is an essential method used in materials characterization. It is a technique in which the mass of a substance is measured as a function of temperature or time as the sample is monitored under a controlled atmosphere. This technique provides useful information about chemical phenomena such as solid–gas reactions and thermal decomposition, as well as physical phenomena including phase transitions, absorption and desorption. A typical TGA analyzer consists of a sample pan that is backed by a precision balance inside a furnace. The temperature inside the furnace is programmed under a controlled temperature. The mass of the sample is monitored during the experiment. This experiment may occur under different conditions in which it could be conducted under a variety of atmospheres including: inert gas, carburizing gases, ambient air, vapors and vacuum. Also, the experiment might take place under a variety of pressures such as high vacuum, high pressure, and constant pressure. The data collected from the thermal reactions which occur inside the TGA are represented into a plot. That plot is indicating the thermal curve which is displayed as time or temperature in the x-axis and as weight (mg) or weight percent (%) in the y-axis. The TGA thermal curve provides different useful information about the characteristics of the materials. The purity of the sample is one of the characteristics that can be determined using the TGA thermal curve. This can be accomplished by calculating the formula weight through substituting the atomic mass in the formula and then comparing the measured values with the calculated ones. To further characterize the sample, an extrapolated onset temperature that represents the temperature at which the weight loss starts can be calculated. Another useful calculation that illustrates one of the characterization of the sample is the peak calculation of the first derivative of the weight loss curve. The peak of the 1st derivative shows the inflection point which is basically represents the point of greatest rate of change on the weight loss curve. The temperature range at which thermolysis would be performed is determined by analyzing the samples thermogravimetrically.

## 4. Results and discussion

#### 4.1. FTIR analysis

#### 4.1.1. Date pits activated carbon

FTIR Analysis, was conducted for further identification of the functional groups present in activated carbon. Figure 1 depicts the corresponding FTIR range of the date pit activated carbon that primarily concludes that phenols, carboxylic acids, and carbonyl functional groups are the ones present, all of which are categorized as typical acidic functional groups.

to the hydroxyl (OH) group and the peak at 2329 cm<sup>1</sup> is related to the C C stretching as the data in Table 1 presents. Furthermore, the frequency observed at a wavelength of 1843 cm<sup>1</sup> is due to the CHO stretching. 1450–1479 cm<sup>1</sup> are related to the vibrations of the CH bonds. Peaks at 1068–1148 cm<sup>1</sup> and 1565cm<sup>1</sup> are attributed to the vibrations at CdOdC and N-H stretching respectively. Moreover, the non-symmetric vibration absorption of C]O is illus-

OH bending vibration frequency respectively. According to the data illustrated for the ACF, it is observed that the spectrum at different temperatures portrays similar patterns but the intensity of the absorption bands is increased as the treatment temperature is increased.

Thermogravimetric analysis (TGA) and differential thermogravimetric analysis (DTG) profiles of the DP-AC are shown in Figure 3. The TGA curve gives an approximation about the weight loss of the sample with respect to temperature, while the DTG curve indicates the decomposition temperature as well as the oxidation temperature of the sample. The first decomposition stage occurred between 21.5 C ≈ 99C where a weight loss of 10.4% was observed. This loss was due to the release of surface bounded water and volatile matters. The highest rate of degradation in this stage was indicated by the peak on the DTG curve at a maximum rate of decomposition temperature of 54C. The next stage showed a slower rate of weight loss with only 10.6% change over a wide range of temperature between 100C up to 900C. Most of the weight loss in this stage was due to the decomposition of hemicellulose and cellulose constituents. While for temperatures above ≈ 450C, the weight loss was due to decomposition of

, as well as the stretching vibration of the C]O or C]N present

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. The peaks at around 1403 and 650 cm<sup>1</sup> are due to the CH2 bending and

trated in the peak at 1840 cm<sup>1</sup>

4.2.1. Date pits activated carbon

4.2. TGA (Thermogravimetric analysis)

Figure 1. FTIR spectrum of the date pit activated carbon.

at 1750–1640 cm<sup>1</sup>

Furthermore, the spectrum consisted of an IR band of around 3427 cm<sup>1</sup> which is assigned to a vibration stretch of hydroxyl group denoted as OH. Additionally, C]O group frequency is observed at a wavelength of 1720 cm<sup>1</sup> while aliphatic groups at around 2923 and 2855 cm<sup>1</sup> , along with 1033 cm<sup>1</sup> which corresponds to CdO stretching, as tabulated in Table 1. In elucidating chitosan coated carbon's FTIR spectra that merely represent the adsorption of both chitosan and carbon, it has been noted, as Figure 1 demonstrates, that the stretching vibration of OdH and NdH functional groups are observed at a frequency of 3433 cm<sup>1</sup> . The bands at around 2923 and 2855 cm<sup>1</sup> correspond to the asymmetric and symmetric stretching. Moreover, the peaks in the range of 1630, 1157, 1381, 1017 and 890 cm<sup>1</sup> assigned to NdH bending, CdN stretching, OdH in plane bending, CdCdC Skeletal in the backbone and CH3dCdOH stretching respectively as recorded in Table 1. Bands around 647 and 496 cm<sup>1</sup> indicate the presence of OH and CdC bending vibrations respectively [19].

#### 4.1.2. Activated carbon fiber

Fourier Transform Infrared Spectroscopy (FTIR) analysis is done in order to identify the functional groups associated with the activated carbon fiber. The Figure 2 illustrates the FTIR specterum obtained from the carbon fiber before and after activation at different temperatures of 500, 600, 800 and 1000C. The spectrum shows an IR band of 3600 and 3200 cm<sup>1</sup> is assigned

Figure 1. FTIR spectrum of the date pit activated carbon.

and constant pressure. The data collected from the thermal reactions which occur inside the TGA are represented into a plot. That plot is indicating the thermal curve which is displayed as time or temperature in the x-axis and as weight (mg) or weight percent (%) in the y-axis. The TGA thermal curve provides different useful information about the characteristics of the materials. The purity of the sample is one of the characteristics that can be determined using the TGA thermal curve. This can be accomplished by calculating the formula weight through substituting the atomic mass in the formula and then comparing the measured values with the calculated ones. To further characterize the sample, an extrapolated onset temperature that represents the temperature at which the weight loss starts can be calculated. Another useful calculation that illustrates one of the characterization of the sample is the peak calculation of the first derivative of the weight loss curve. The peak of the 1st derivative shows the inflection point which is basically represents the point of greatest rate of change on the weight loss curve. The temperature range at which thermolysis would be performed is determined by analyzing

146 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

FTIR Analysis, was conducted for further identification of the functional groups present in activated carbon. Figure 1 depicts the corresponding FTIR range of the date pit activated carbon that primarily concludes that phenols, carboxylic acids, and carbonyl functional groups

Furthermore, the spectrum consisted of an IR band of around 3427 cm<sup>1</sup> which is assigned to a vibration stretch of hydroxyl group denoted as OH. Additionally, C]O group frequency is observed at a wavelength of 1720 cm<sup>1</sup> while aliphatic groups at around 2923 and 2855 cm<sup>1</sup>

along with 1033 cm<sup>1</sup> which corresponds to CdO stretching, as tabulated in Table 1. In elucidating chitosan coated carbon's FTIR spectra that merely represent the adsorption of both chitosan and carbon, it has been noted, as Figure 1 demonstrates, that the stretching vibration

around 2923 and 2855 cm<sup>1</sup> correspond to the asymmetric and symmetric stretching. Moreover, the peaks in the range of 1630, 1157, 1381, 1017 and 890 cm<sup>1</sup> assigned to NdH bending, CdN stretching, OdH in plane bending, CdCdC Skeletal in the backbone and CH3dCdOH stretching respectively as recorded in Table 1. Bands around 647 and 496 cm<sup>1</sup>

Fourier Transform Infrared Spectroscopy (FTIR) analysis is done in order to identify the functional groups associated with the activated carbon fiber. The Figure 2 illustrates the FTIR specterum obtained from the carbon fiber before and after activation at different temperatures of 500, 600, 800 and 1000C. The spectrum shows an IR band of 3600 and 3200 cm<sup>1</sup> is assigned

are the ones present, all of which are categorized as typical acidic functional groups.

of OdH and NdH functional groups are observed at a frequency of 3433 cm<sup>1</sup>

indicate the presence of OH and CdC bending vibrations respectively [19].

the samples thermogravimetrically.

4. Results and discussion

4.1.1. Date pits activated carbon

4.1.2. Activated carbon fiber

4.1. FTIR analysis

to the hydroxyl (OH) group and the peak at 2329 cm<sup>1</sup> is related to the C C stretching as the data in Table 1 presents. Furthermore, the frequency observed at a wavelength of 1843 cm<sup>1</sup> is due to the CHO stretching. 1450–1479 cm<sup>1</sup> are related to the vibrations of the CH bonds. Peaks at 1068–1148 cm<sup>1</sup> and 1565cm<sup>1</sup> are attributed to the vibrations at CdOdC and N-H stretching respectively. Moreover, the non-symmetric vibration absorption of C]O is illustrated in the peak at 1840 cm<sup>1</sup> , as well as the stretching vibration of the C]O or C]N present at 1750–1640 cm<sup>1</sup> . The peaks at around 1403 and 650 cm<sup>1</sup> are due to the CH2 bending and OH bending vibration frequency respectively. According to the data illustrated for the ACF, it is observed that the spectrum at different temperatures portrays similar patterns but the intensity of the absorption bands is increased as the treatment temperature is increased.

#### 4.2. TGA (Thermogravimetric analysis)

#### 4.2.1. Date pits activated carbon

,

. The bands at

Thermogravimetric analysis (TGA) and differential thermogravimetric analysis (DTG) profiles of the DP-AC are shown in Figure 3. The TGA curve gives an approximation about the weight loss of the sample with respect to temperature, while the DTG curve indicates the decomposition temperature as well as the oxidation temperature of the sample. The first decomposition stage occurred between 21.5 C ≈ 99C where a weight loss of 10.4% was observed. This loss was due to the release of surface bounded water and volatile matters. The highest rate of degradation in this stage was indicated by the peak on the DTG curve at a maximum rate of decomposition temperature of 54C. The next stage showed a slower rate of weight loss with only 10.6% change over a wide range of temperature between 100C up to 900C. Most of the weight loss in this stage was due to the decomposition of hemicellulose and cellulose constituents. While for temperatures above ≈ 450C, the weight loss was due to decomposition of


Table 1. Functional groups associated with wavelength bands.

lignin. At temperature 900C, the amount of ash residual was 79% which is considered as indication for the thermal stability of the material (date pits activated carbon) under investigation over a broad range of temperatures.

#### 4.2.2. Activated carbon fiber

Thermogravimetric analyses (TGA) were conducted in a TA (schimadzu Q40) equipment. The samples of the activated carbon fiber were heated up to 600C at a heating rate of 10C/min under nitrogen atmosphere (flow rate 100 mL/min). Using the data obtained, TGA curves were prepared as shown in Figure 4. The TGA curve produced shows the weight loss versus time curves for several temperatures. The temperatures at which the activated carbon fibers were prepared during the pyrolysis are indicated in Figure 4. Based on Figure 4, it can be clearly

seen that the TGA test was done for several samples of activated carbon fiber at different temperatures. These temperatures are 500, 600, 800 and 1000C. TGA curve shows a weight loss due to the release of moisture at 200C. Only one prominent weight loss peak around 300

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149

Figure 2. FTIR spectrum of the activated carbon fiber.

Figure 3. TGA profile of date pit activated carbon.

Activating Carbon Fibers and Date Pits for Use in Liver Toxin Adsorption http://dx.doi.org/10.5772/intechopen.71891 149

Figure 2. FTIR spectrum of the activated carbon fiber.

Figure 3. TGA profile of date pit activated carbon.

lignin. At temperature 900C, the amount of ash residual was 79% which is considered as indication for the thermal stability of the material (date pits activated carbon) under investiga-

Aliphatic Groups 2923 cm<sup>1</sup> – 2855 cm<sup>1</sup>

Functional Group Wavelength

148 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

<sup>C</sup>C stretching 2329 cm<sup>1</sup> CHO stretching 1843 cm<sup>1</sup>

N-H stretching 1565 cm<sup>1</sup> C=O 1840 cm<sup>1</sup>

CH2 bending 1403 cm<sup>1</sup> OH bending vibration 650 cm<sup>1</sup>

N-H bending 1630 cm<sup>1</sup> C-N stretching 1157 cm<sup>1</sup> O-H bending 1381 cm<sup>1</sup> C-C-C 1017 cm<sup>1</sup> CH3-C-OH 890 cm<sup>1</sup> OH 647 cm<sup>1</sup> C-C 496 cm<sup>1</sup>

OH 3427 cm<sup>1</sup> C=O 1720 cm<sup>1</sup> C-O 1033 cm<sup>1</sup>

OH 3600 cm<sup>1</sup> – 3200 cm<sup>1</sup>

CH bonds 1450 cm<sup>1</sup> – 1479 cm<sup>1</sup> C-O-C vibrations 1068 cm<sup>1</sup> – 1148 cm<sup>1</sup>

C=N 1750 cm<sup>1</sup> – 1640 cm<sup>1</sup>

Thermogravimetric analyses (TGA) were conducted in a TA (schimadzu Q40) equipment. The samples of the activated carbon fiber were heated up to 600C at a heating rate of 10C/min under nitrogen atmosphere (flow rate 100 mL/min). Using the data obtained, TGA curves were prepared as shown in Figure 4. The TGA curve produced shows the weight loss versus time curves for several temperatures. The temperatures at which the activated carbon fibers were prepared during the pyrolysis are indicated in Figure 4. Based on Figure 4, it can be clearly

tion over a broad range of temperatures.

Table 1. Functional groups associated with wavelength bands.

4.2.2. Activated carbon fiber

Activated Carbon Fiber

Chitosan Coated AC

Date Pit AC

seen that the TGA test was done for several samples of activated carbon fiber at different temperatures. These temperatures are 500, 600, 800 and 1000C. TGA curve shows a weight loss due to the release of moisture at 200C. Only one prominent weight loss peak around 300

These findings also confirm the ability of date pits AC and CFRPs as adsorbent of toxins and potential candidates for consideration in the search for effective treatment options for liver

Activating Carbon Fibers and Date Pits for Use in Liver Toxin Adsorption

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151

The authors would like to thank UAEU for their support of the project. This work has been

Ameereh Seyedzadeh, Asel Mwafy, Waleed Khalil Ahmed, Kamala Pandurangan and

Department of Mechanical Engineering, College of Engineering, United Arab Emirates

[1] Annesini M, Piemonte V, Turchetti L. Removal of albumin-bound toxins from albumincontaining solutions: Tryptophan fixed-bed adsorption on activated carbon. Chemical

[2] Magosso E, Ursino M, Colì L, Baraldi O, Bolondi L, Stefoni S. A modeling study of bilirubin kinetics during molecular adsorbent recirculating system sessions. Artificial

[3] Ding W, Zou L, Sun S, Li W, Gao D. A new method to increase the adsorption of proteinbound toxins in artificial liver support systems. Artificial Organs. 2014;38(11):954-962 [4] McKenzie TJ, Lillegard JB, Nyberg SL, editors. Artificial and Bioartificial Liver Support.

[6] Shinke K, Ando K, Koyama T, Takai T, Nakaji S, Ogino T. Properties of various carbon nanomaterial surfaces in bilirubin adsorption. Colloids and Surfaces B: Biointerfaces.

[7] Nieuwoudt MJ. Bio-Artificial Liver Support System: An Evaluation of Models Used in Demonstrating or Improving Metabolic and Clinical Efficacy. University of Pretoria; 2010

[8] Asano T, Tsuru K, Hayakawa S, Osaka A. Bilirubin adsorption property of sol-gel-derived titania particles for blood purification therapy. Acta Biomaterialia. 2008;4(4):1067-1072

Seminars in Liver Disease. Thieme Medical Publishers. 2008;28(02):210-217

financially funded by the SURE program at UAE University, Al Ain.

Engineering Research and Design. 2010;88(8):1018-1023

[5] Puri P, Anand AC. Liver support devices. MEDICINE. 2012;22

\*Address all correspondence to: alihilal@uaeu.ac.ae

failure.

Acknowledgements

Author details

Ali Hilal-Alnaqbi\*

References

University, Al Ain, UAE

Organs. 2006;30(4):285-300

2010;77(1):18-21

Figure 4. TGA profile of activated carbon fiber.

to 550C was observed in the derivative mass loss curve. The corresponding weight loss between 300 to 550C is related to the pyrolysis of the material. Similar patterns were observed for all the samples at various temperatures.

FTIR results give information about the functional groups present on the two materials; DPAC and ACF. This test allows for comparisons between the composition and makeup of the activated carbon. The TGA tests allow observations related to the material behavior under temperature conditions and drawing conclusions based on the variation of the activated material. Consequently, these assessments acknowledge important aspects of the ACs and provide a means for their comparisons. However, the adsorption capacity for bilirubin was not tested and therefore, direct conclusions regarding which material is a better adsorbent for bilirubin could not be drawn. Therefore, this test will be studied and conducted in further research.

#### 5. Conclusion

The results presented above show that activation of date pits and CFRP was successful. The preparation of ACs from date pits and CFRPs and their characterization pave the way for their applications in the field of clinical practice, notably, for the adsorption of bilirubin, which is considered a toxin secreted by patients suffering from liver failure.

Data generated from the processes of activation and characterization showed that the rates of absorption for activated carbon fiber materials, as expected, were higher as compared to the date pits AC. This was noticed by the visibly larger peak frequencies on the FTIR graphs indicating higher absorption of the CFs. Furthermore, TGA graphs illustrated higher thermal properties meaning that the CF is a better absorbent.

These findings also confirm the ability of date pits AC and CFRPs as adsorbent of toxins and potential candidates for consideration in the search for effective treatment options for liver failure.

## Acknowledgements

The authors would like to thank UAEU for their support of the project. This work has been financially funded by the SURE program at UAE University, Al Ain.

## Author details

Ameereh Seyedzadeh, Asel Mwafy, Waleed Khalil Ahmed, Kamala Pandurangan and Ali Hilal-Alnaqbi\*

\*Address all correspondence to: alihilal@uaeu.ac.ae

Department of Mechanical Engineering, College of Engineering, United Arab Emirates University, Al Ain, UAE

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to 550C was observed in the derivative mass loss curve. The corresponding weight loss between 300 to 550C is related to the pyrolysis of the material. Similar patterns were observed

FTIR results give information about the functional groups present on the two materials; DPAC and ACF. This test allows for comparisons between the composition and makeup of the activated carbon. The TGA tests allow observations related to the material behavior under temperature conditions and drawing conclusions based on the variation of the activated material. Consequently, these assessments acknowledge important aspects of the ACs and provide a means for their comparisons. However, the adsorption capacity for bilirubin was not tested and therefore, direct conclusions regarding which material is a better adsorbent for bilirubin could not be

The results presented above show that activation of date pits and CFRP was successful. The preparation of ACs from date pits and CFRPs and their characterization pave the way for their applications in the field of clinical practice, notably, for the adsorption of bilirubin, which is

Data generated from the processes of activation and characterization showed that the rates of absorption for activated carbon fiber materials, as expected, were higher as compared to the date pits AC. This was noticed by the visibly larger peak frequencies on the FTIR graphs indicating higher absorption of the CFs. Furthermore, TGA graphs illustrated higher thermal properties

drawn. Therefore, this test will be studied and conducted in further research.

150 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

considered a toxin secreted by patients suffering from liver failure.

for all the samples at various temperatures.

Figure 4. TGA profile of activated carbon fiber.

meaning that the CF is a better absorbent.

5. Conclusion


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33-37


**Section 3**

**Forensic Toxicology Topics**

**Forensic Toxicology Topics**

**Chapter 9**

**Provisional chapter**

**Simplified Analysis of Toxic Gaseous Substance in**

**Simplified Analysis of Toxic Gaseous Substance in** 

DOI: 10.5772/intechopen.70029

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Toxicological examination in forensic practice is important for the proper diagnosis of acute poisoning [1]. The forensic pathologist requests toxicological analysis to forensic toxicologist

Toxicological examination in forensic practice is important for the proper diagnosis of acute poisoning. We have discussed the properties and features of poisoning incidents due to gaseous substances and elaborated on the simplified analytical techniques and apparatus used for their identification and quantitation for forensic purposes. Briefly, we have explained the simplified analysis of toxic gaseous substances such as carbon monoxide, hydrogen cyanide, hydrogen sulfide, and helium in blood. The techniques used include color testing, gas chromatography, detector tube, oximeter, and spectrophotometric method. In doing so, we have shared our experiences and highlighted the fact that the analysis of gaseous substances can be performed using readily available laboratory tools and equipment. We have emphasized the need and usefulness of the reference data tables for guiding forensic diagnosis. We hope that the above overview will assist other colleagues to implement such simplified techniques for the advancement of

**Keywords:** toxicological examination, toxic gaseous substances, simplified analysis

**Forensic Practice: Experiences from Japan**

**Forensic Practice: Experiences from Japan**

Hiroshi Kinoshita, Naoko Tanaka, Ayaka Takakura, Mostofa Jamal, Asuka Ito, Mitsuru Kumihashi,

Shoji Kimura, Kunihiko Tsutsui, Shuji Matsubara and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Hiroshi Kinoshita, Naoko Tanaka, Ayaka Takakura, Mostofa Jamal, Asuka Ito, Mitsuru Kumihashi, Shoji Kimura, Kunihiko Tsutsui,

Shuji Matsubara and Kiyoshi Ameno

http://dx.doi.org/10.5772/intechopen.70029

forensic medicine practice.

Kiyoshi Ameno

**Abstract**

**1. Introduction**

**Provisional chapter**

## **Simplified Analysis of Toxic Gaseous Substance in Forensic Practice: Experiences from Japan Forensic Practice: Experiences from Japan**

**Simplified Analysis of Toxic Gaseous Substance in** 

DOI: 10.5772/intechopen.70029

Hiroshi Kinoshita, Naoko Tanaka, Ayaka Takakura, Mostofa Jamal, Asuka Ito, Mitsuru Kumihashi, Shoji Kimura, Kunihiko Tsutsui, Shuji Matsubara and Kiyoshi Ameno Takakura, Mostofa Jamal, Asuka Ito, Mitsuru Kumihashi, Shoji Kimura, Kunihiko Tsutsui, Shuji Matsubara and Kiyoshi Ameno Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Hiroshi Kinoshita, Naoko Tanaka, Ayaka

http://dx.doi.org/10.5772/intechopen.70029

#### **Abstract**

Toxicological examination in forensic practice is important for the proper diagnosis of acute poisoning. We have discussed the properties and features of poisoning incidents due to gaseous substances and elaborated on the simplified analytical techniques and apparatus used for their identification and quantitation for forensic purposes. Briefly, we have explained the simplified analysis of toxic gaseous substances such as carbon monoxide, hydrogen cyanide, hydrogen sulfide, and helium in blood. The techniques used include color testing, gas chromatography, detector tube, oximeter, and spectrophotometric method. In doing so, we have shared our experiences and highlighted the fact that the analysis of gaseous substances can be performed using readily available laboratory tools and equipment. We have emphasized the need and usefulness of the reference data tables for guiding forensic diagnosis. We hope that the above overview will assist other colleagues to implement such simplified techniques for the advancement of forensic medicine practice.

**Keywords:** toxicological examination, toxic gaseous substances, simplified analysis

### **1. Introduction**

Toxicological examination in forensic practice is important for the proper diagnosis of acute poisoning [1]. The forensic pathologist requests toxicological analysis to forensic toxicologist

in case of poisoning or poisoning suspected cases. It will usually consist of two-stage testing at autopsy [2]. The first step is usually performed as a screening test. The second step is required for the identification and quantification of its causative agent following the confirmatory test. Toxic gaseous substances are one of the targets for toxicological examination in a daily forensic practice. Simplified analysis of gaseous substances involves both first and second step of toxicological examination in forensic practice.

**3. Specific substances and its simplified analysis methods**

and it causes insufficient oxygen supply in tissues [12, 18, 19].

CO is an odorless, nonirritable, and colorless gas and is slightly lighter than air (specific gravity for the air; 0.97). It is mainly produced by incomplete combustion of fuels or carbon compounds [8, 12]. Its common sources are vehicle exhaust, smoke from fire, and improperly maintained heating systems. CO is the leading cause of poisoning death in Japan [13–15], and also a common cause of poisoning in the United States [16, 17]. The annual number of victims by CO poisoning is about 2000–4000 in Japan, including accidental or suicidal cases [13–15]. CO is absorbed from the lung into the bloodstream. As the affinity of CO for hemoglobin is 230–270 times greater than that of oxygen, it binds to hemoglobin in erythrocyte, and forms carboxyhemoglobin (CO-Hb) [12, 18, 19]. The formation of CO-Hb (represented as a percentage of the total hemoglobin) in blood depends on various factors such as the concentration of inspired CO, duration of CO exposure, pulmonary ventilation, exercise, and health status [12, 18]. The toxicity of CO is thought to be tissue hypoxia due to the formation of CO-Hb. Its binding is a reversible process; however, as the binding between CO and hemoglobin is strong, the CO elimination half-life is long, about 4–5 hours under room air ventilation for a resting adult at sea level. The formation of CO-Hb decreases the capacity of oxygen transport,

Simplified Analysis of Toxic Gaseous Substance in Forensic Practice: Experiences from Japan

http://dx.doi.org/10.5772/intechopen.70029

159

The hypoxia due to CO-Hb formation causes signs and symptoms. Clinical symptoms roughly correlate with CO-Hb levels (**Table 1**). The CO-Hb concentration of nonsmoking healthy subjects is 1–3%, and around 5–8% in smokers. No symptom is observed below 10% of CO-Hb levels. Neurological symptoms such as headache, dizziness, nausea, and weakness are observed in CO-Hb level from 10 to 30%. Increase of respiration and heart rate, syncope, and confusion are observed in 30–50% of CO-Hb level. When the level of CO-Hb exceeds 50%, it becomes life-threatening. It is noteworthy that the value of CO-Hb is important for the diagnosis of CO-poisoning or fire-related death [12, 18, 19]. In addition to hemoglobin, CO

**3.1. Carbon monoxide (CO)**

**CO-Hb (%) Clinical symptom** 0–10 No symptom

10–20 Headache, ear ringing, fatigue

70+ Respiratory failure, death

20–30 Headache, weakness, nausea, vomiting

30–40 Severe headache, dizziness, nausea, vomiting

50–60 Coma, convulsions, depressed respiration

40–50 Syncope, confusion, increased respiration and heart rate, muscle weakness

60–70 Coma, convulsions, cardiorespiratory depression, often fatal

**Table 1.** Correlation of carboxyhemoglobin (CO-Hb) levels to clinical symptoms.

In the present chapter, we share our experiences about the analysis of gaseous substances such as carbon monoxide, hydrogen sulfide, cyanide, and helium.

## **2. Autopsy and subsequent toxicological examination in gas-related poisoning incidents**

Gaseous substances can cause acute poisoning. They get absorbed into the body by inhalation. Most of them do not produce specific symptoms but they can induce dizziness, lethargy, headaches, and suffocation. There is no specific finding at autopsy in most poisoning cases [1, 2]. Most of gaseous substances cause little or no tissue damages. However, there will be observed unique findings in some poisoning cases, such as cherry red appearance of postmortem lividity in carbon monoxide poisoning, bright pink lividity with bitter almond odor in cyanide poisoning, and dark green coloration and rotten egg smell in hydrogen sulfide poisoning [2]. Nonspecific findings such as generalized organ congestion and pulmonary edema may be observed in most cases [1]. The presence of gaseous poisons is usually indicated by the circumstances of the incident, and involvement of gaseous substances is sometimes indicated by circumstantial evidence [3].

Although detailed management is out of scope of this chapter, it is sufficient to mention that the management of poisoning by gaseous substances involves the use of antidotes when available; decontamination; artificial respiration with demand-valve resuscitators, bag-valve-mask devices; administration of hyperbaric oxygen; performance of cardiopulmonary resuscitation (CPR); and close observation and monitoring of vital signs.

In our laboratories, toxicological examination is routinely performed on a daily basis—from the screening test using immunoassay kits to subsequent identification and quantification using gas chromatography mass spectrometry or liquid chromatography mass spectrometry techniques. Since the physiological effects of most gaseous substances correlate with the concentration in blood, it becomes the best indicator of toxicity [4]. As numerous reference tables for fatal levels of chemicals have been reported [5–9], forensic diagnosis is made in reference to the values reported in the data tables. In addition, several other factors have to be considered for toxicological evaluation; these include the properties of the sample, diffusion and redistribution, degradation, and metabolism [1, 2, 10, 11]. In the light of the above, we have instituted simplified analysis techniques for gaseous substances; these techniques provide a lot of information promptly that aid forensic diagnosis. In the following paragraphs, we describe these techniques for each of the main gaseous substances.

## **3. Specific substances and its simplified analysis methods**

#### **3.1. Carbon monoxide (CO)**

in case of poisoning or poisoning suspected cases. It will usually consist of two-stage testing at autopsy [2]. The first step is usually performed as a screening test. The second step is required for the identification and quantification of its causative agent following the confirmatory test. Toxic gaseous substances are one of the targets for toxicological examination in a daily forensic practice. Simplified analysis of gaseous substances involves both first and second step of

In the present chapter, we share our experiences about the analysis of gaseous substances

Gaseous substances can cause acute poisoning. They get absorbed into the body by inhalation. Most of them do not produce specific symptoms but they can induce dizziness, lethargy, headaches, and suffocation. There is no specific finding at autopsy in most poisoning cases [1, 2]. Most of gaseous substances cause little or no tissue damages. However, there will be observed unique findings in some poisoning cases, such as cherry red appearance of postmortem lividity in carbon monoxide poisoning, bright pink lividity with bitter almond odor in cyanide poisoning, and dark green coloration and rotten egg smell in hydrogen sulfide poisoning [2]. Nonspecific findings such as generalized organ congestion and pulmonary edema may be observed in most cases [1]. The presence of gaseous poisons is usually indicated by the circumstances of the incident, and involvement of gaseous substances is sometimes indicated

Although detailed management is out of scope of this chapter, it is sufficient to mention that the management of poisoning by gaseous substances involves the use of antidotes when available; decontamination; artificial respiration with demand-valve resuscitators, bag-valve-mask devices; administration of hyperbaric oxygen; performance of cardiopulmonary resuscitation

In our laboratories, toxicological examination is routinely performed on a daily basis—from the screening test using immunoassay kits to subsequent identification and quantification using gas chromatography mass spectrometry or liquid chromatography mass spectrometry techniques. Since the physiological effects of most gaseous substances correlate with the concentration in blood, it becomes the best indicator of toxicity [4]. As numerous reference tables for fatal levels of chemicals have been reported [5–9], forensic diagnosis is made in reference to the values reported in the data tables. In addition, several other factors have to be considered for toxicological evaluation; these include the properties of the sample, diffusion and redistribution, degradation, and metabolism [1, 2, 10, 11]. In the light of the above, we have instituted simplified analysis techniques for gaseous substances; these techniques provide a lot of information promptly that aid forensic diagnosis. In the following paragraphs,

**2. Autopsy and subsequent toxicological examination in gas-related** 

toxicological examination in forensic practice.

**poisoning incidents**

by circumstantial evidence [3].

such as carbon monoxide, hydrogen sulfide, cyanide, and helium.

158 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

(CPR); and close observation and monitoring of vital signs.

we describe these techniques for each of the main gaseous substances.

CO is an odorless, nonirritable, and colorless gas and is slightly lighter than air (specific gravity for the air; 0.97). It is mainly produced by incomplete combustion of fuels or carbon compounds [8, 12]. Its common sources are vehicle exhaust, smoke from fire, and improperly maintained heating systems. CO is the leading cause of poisoning death in Japan [13–15], and also a common cause of poisoning in the United States [16, 17]. The annual number of victims by CO poisoning is about 2000–4000 in Japan, including accidental or suicidal cases [13–15]. CO is absorbed from the lung into the bloodstream. As the affinity of CO for hemoglobin is 230–270 times greater than that of oxygen, it binds to hemoglobin in erythrocyte, and forms carboxyhemoglobin (CO-Hb) [12, 18, 19]. The formation of CO-Hb (represented as a percentage of the total hemoglobin) in blood depends on various factors such as the concentration of inspired CO, duration of CO exposure, pulmonary ventilation, exercise, and health status [12, 18]. The toxicity of CO is thought to be tissue hypoxia due to the formation of CO-Hb. Its binding is a reversible process; however, as the binding between CO and hemoglobin is strong, the CO elimination half-life is long, about 4–5 hours under room air ventilation for a resting adult at sea level. The formation of CO-Hb decreases the capacity of oxygen transport, and it causes insufficient oxygen supply in tissues [12, 18, 19].

The hypoxia due to CO-Hb formation causes signs and symptoms. Clinical symptoms roughly correlate with CO-Hb levels (**Table 1**). The CO-Hb concentration of nonsmoking healthy subjects is 1–3%, and around 5–8% in smokers. No symptom is observed below 10% of CO-Hb levels. Neurological symptoms such as headache, dizziness, nausea, and weakness are observed in CO-Hb level from 10 to 30%. Increase of respiration and heart rate, syncope, and confusion are observed in 30–50% of CO-Hb level. When the level of CO-Hb exceeds 50%, it becomes life-threatening. It is noteworthy that the value of CO-Hb is important for the diagnosis of CO-poisoning or fire-related death [12, 18, 19]. In addition to hemoglobin, CO


**Table 1.** Correlation of carboxyhemoglobin (CO-Hb) levels to clinical symptoms.

combines with heme-proteins such as myoglobin and cytochrome oxidase, and it may cause the impairment of cardiac and neurological functions [12, 18].

The most characteristic appearance of the body in poisoning case is a cherry red color of the skin. It is usually observed in cases where CO-Hb exceeds 30% [1]. At autopsy, the common findings include discoloration of blood, organs, and muscle that become cherry red color, as a result of CO-Hb and carboxymyoglobin. Other autopsy findings such as pulmonary edema and generalized organ congestion are also observed [1].

With regard to the identification and quantification of CO, several methods and techniques have been reported [20]. Spectrophotometric methods and gas chromatography techniques are widely used. The CO-Hb is relatively stable under storage in cool and dark conditions [18, 21–23]. It is important to note that postmortem production of CO has been reported in some conditions, and therefore, it is recommended not to use body cavity fluids such as pleural effusion for the measurement of CO in severe putrefied case [24–28].

For identification purposes, the qualitative test for CO includes color test and microdiffusion tests. Color test is a simple procedure where a blood sample mixed with 0.01 M ammonia solution (1:20) [29] or a few drops of blood are added to some 10% sodium hydroxide solution [1]. This test is based on the fact that CO-Hb is relatively tolerant to alkaline condition. However, as other simple methods have been established, color testing for CO poisoning is now rarely required and not recommended [1, 29]. The microdiffusion test using Conway cell [30] or on the filter paper [31] have been reported. It is based on reaction with palladium chloride. This is still the most widely used method since it was invented by Conway in 1944.

The detector tube method is widely used for the determination of various gaseous substances [46, 47]. It is also applied for the quantitation of CO in blood [48]. This apparatus consists of a CO-separator tube, CO-detector tube, and aspirating pump. The CO-separator tube is packed with silica gel particles coated with ferricyanide [48]. The CO-detector tube is packed with silica gel particles coated with sulfite palladium potassium [46, 49]. These tubes and pump are connected in series. The CO in blood is released following the injection of blood sample (200 μL) in CO-separator tube, and the released CO gas is detected by the CO-detector tube, followed by the aspirating of the pump. As the detector tube is easy to carry at the scene where an incident has taken place or to a point-of-care testing, it is applied to not only screening test, but also for

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**Figure 1.** Spectra of blood sample from CO-Hb: 0, 68, and 95%, respectively.

Oximeter is routinely used for laboratory test [50, 51], and it is also applied in forensic medicines [52–64]. This instrument uses seven wavelengths in the visible region for the determination of various hemoglobin species, such as oxyhemoglobin, CO-Hb, reduced hemoglobin, and methemoglobin. It automatically analyzes the proportion of each species of hemoglobin and oxygen contents. This oximeter system (**Figure 2**) requires 50 μL of blood for a single measurement, and it may be a valid option in case of difficult blood sampling due to severe blood loss. As there are many advantages such as no necessity of sample preparation, easy handling, and portability, it is suitable for forensic practice. In a recent study, it has been reported that squeezed splenic blood can be used as an alternative specimen for CO-Hb measurement using oximeter [65].

Hydrogen cyanide (molecular weight, 27; boiling point, 25.7°C) is a colorless gas or liquid, with a bitter almond like odor [8, 12]. Cyanide is used for various purposes, such as fumigate,

quantitation.

**3.2. Cyanide**

With regard to quantification, spectrophotometric method, gas chromatography, detection tube, and oximeter are used. The spectrophotometric method is the most popular, and various assay procedures have been reported. CO-Hb could be determined by the changes of absorption spectrum in either Soret (410–425 nm) [32–34] or visible region (500–600 nm) [30, 35–38]. In our laboratories, we perform the measurement of the spectrum of blood sample by adding sodium hydrosulfite. The addition of sodium dithionite reduces oxyhemoglobin without affecting the CO-Hb. This procedure is simple, and it does not need an extraction from the sample. **Figure 1** shows the spectra of blood samples from a normal nonsmoker (CO-Hb: 0%) and CO poisoning victim (CO-Hb: 68 and 95%, respectively). Twin-peaked spectrum was observed in CO poisoning sample.

The CO is extracted and introduced in gas chromatography. Various methods and apparatus have been reported for its extraction [39–44]. And the released CO is detected by the thermal conductivity detector (TCD) [39–42, 44], or the flame ionization detector with the catalytic reduction of the CO to methane [43]. As this method is a direct measurement of CO contents in the sample as well as a measurement of the hemoglobin, two measures represent the percentage of CO-Hb. Application of gas chromatography equipped with semiconductor detector has been reported for forensic practice [45]. This gas chromatography system (sensor gas chromatography, sGC) is highly sensitive for CO and has some advantages such as portability and easy handling. This apparatus does not need a gas cylinder as it uses the room air as the career gas. Although it is not commonly in use, further application in the field of forensic medicine would be expected.

**Figure 1.** Spectra of blood sample from CO-Hb: 0, 68, and 95%, respectively.

The detector tube method is widely used for the determination of various gaseous substances [46, 47]. It is also applied for the quantitation of CO in blood [48]. This apparatus consists of a CO-separator tube, CO-detector tube, and aspirating pump. The CO-separator tube is packed with silica gel particles coated with ferricyanide [48]. The CO-detector tube is packed with silica gel particles coated with sulfite palladium potassium [46, 49]. These tubes and pump are connected in series. The CO in blood is released following the injection of blood sample (200 μL) in CO-separator tube, and the released CO gas is detected by the CO-detector tube, followed by the aspirating of the pump. As the detector tube is easy to carry at the scene where an incident has taken place or to a point-of-care testing, it is applied to not only screening test, but also for quantitation.

Oximeter is routinely used for laboratory test [50, 51], and it is also applied in forensic medicines [52–64]. This instrument uses seven wavelengths in the visible region for the determination of various hemoglobin species, such as oxyhemoglobin, CO-Hb, reduced hemoglobin, and methemoglobin. It automatically analyzes the proportion of each species of hemoglobin and oxygen contents. This oximeter system (**Figure 2**) requires 50 μL of blood for a single measurement, and it may be a valid option in case of difficult blood sampling due to severe blood loss. As there are many advantages such as no necessity of sample preparation, easy handling, and portability, it is suitable for forensic practice. In a recent study, it has been reported that squeezed splenic blood can be used as an alternative specimen for CO-Hb measurement using oximeter [65].

#### **3.2. Cyanide**

combines with heme-proteins such as myoglobin and cytochrome oxidase, and it may cause

The most characteristic appearance of the body in poisoning case is a cherry red color of the skin. It is usually observed in cases where CO-Hb exceeds 30% [1]. At autopsy, the common findings include discoloration of blood, organs, and muscle that become cherry red color, as a result of CO-Hb and carboxymyoglobin. Other autopsy findings such as pulmonary edema and

With regard to the identification and quantification of CO, several methods and techniques have been reported [20]. Spectrophotometric methods and gas chromatography techniques are widely used. The CO-Hb is relatively stable under storage in cool and dark conditions [18, 21–23]. It is important to note that postmortem production of CO has been reported in some conditions, and therefore, it is recommended not to use body cavity fluids such as pleural effusion for the mea-

For identification purposes, the qualitative test for CO includes color test and microdiffusion tests. Color test is a simple procedure where a blood sample mixed with 0.01 M ammonia solution (1:20) [29] or a few drops of blood are added to some 10% sodium hydroxide solution [1]. This test is based on the fact that CO-Hb is relatively tolerant to alkaline condition. However, as other simple methods have been established, color testing for CO poisoning is now rarely required and not recommended [1, 29]. The microdiffusion test using Conway cell [30] or on the filter paper [31] have been reported. It is based on reaction with palladium chloride. This is still the most widely used method since it was invented by Conway in 1944.

With regard to quantification, spectrophotometric method, gas chromatography, detection tube, and oximeter are used. The spectrophotometric method is the most popular, and various assay procedures have been reported. CO-Hb could be determined by the changes of absorption spectrum in either Soret (410–425 nm) [32–34] or visible region (500–600 nm) [30, 35–38]. In our laboratories, we perform the measurement of the spectrum of blood sample by adding sodium hydrosulfite. The addition of sodium dithionite reduces oxyhemoglobin without affecting the CO-Hb. This procedure is simple, and it does not need an extraction from the sample. **Figure 1** shows the spectra of blood samples from a normal nonsmoker (CO-Hb: 0%) and CO poisoning victim (CO-Hb: 68 and 95%, respectively).

The CO is extracted and introduced in gas chromatography. Various methods and apparatus have been reported for its extraction [39–44]. And the released CO is detected by the thermal conductivity detector (TCD) [39–42, 44], or the flame ionization detector with the catalytic reduction of the CO to methane [43]. As this method is a direct measurement of CO contents in the sample as well as a measurement of the hemoglobin, two measures represent the percentage of CO-Hb. Application of gas chromatography equipped with semiconductor detector has been reported for forensic practice [45]. This gas chromatography system (sensor gas chromatography, sGC) is highly sensitive for CO and has some advantages such as portability and easy handling. This apparatus does not need a gas cylinder as it uses the room air as the career gas. Although it is not commonly in use, further application in the field of forensic medicine

Twin-peaked spectrum was observed in CO poisoning sample.

would be expected.

the impairment of cardiac and neurological functions [12, 18].

160 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

generalized organ congestion are also observed [1].

surement of CO in severe putrefied case [24–28].

Hydrogen cyanide (molecular weight, 27; boiling point, 25.7°C) is a colorless gas or liquid, with a bitter almond like odor [8, 12]. Cyanide is used for various purposes, such as fumigate,

cyanide. This method is user-friendly and highly sensitive for cyanide. However, since it is difficult to store the guaiac-cupper paper for a long period, we prepare it immediately before examination. Other color test or commercially available test tube methods and test papers have also been used to test cyanide in blood samples [29, 66]. The microdiffusion test using Conway cell (pyridine-pyrazolone method) have been reported [67]. Although this method is also highly sensitive for cyanide, and also used as a quantitative examination, its main drawback is

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For quantitative testing, gas chromatography and detection tube are employed in our laboratories. To do so, cyanide is extracted from the sample by adding the concentrated phosphoric acid or sulfuric acid, and detected by nitrogen phosphorus detector (NPD) or flame thermoionic detector (FTD), equipped with a gas chromatography device [69, 70]. Application of sGC has also been reported for cyanide measurement [71]. The sGC system is also highly sensitive for cyanide and has some advantages such as it is easy to operate and portable. Further

The detector tube is also used for the quantitation of cyanide in blood [46, 47, 72]. This apparatus consists of a cyanide-separator tube, cyanide-detector tube, and aspirating pump. These tubes and the pump are connected in series. The cyanide-separator tube is packed with silica gel particles coated with sulfuric acid, and the released cyanide gas is detected by the cyanide-detector tube, followed by the aspirating of the pump (**Figure 4**). The cyanide-detector tube is packed with silica gel particles coated with mercuric chloride and pH indicator [46], and the hydrochloric acid formation by the reaction between cyanide and mercuric chloride was observed. Because the detector tube is easy to handle and portable, it can be carried to the scene of accident or poisoning and at the point of care, and it is well applied in forensic medicine practice.

application would be expected in the field of forensic medicine (**Figure 3**).

**Figure 3.** Equipment of the sensor gas chromatography for hydrogen cyanide quantification.

that it is relatively time-consuming.

**Figure 2.** Portable oximeter (AVOX 4000) and its operation. Sample cartridge is shown in lower left-hand corner.

fungicide, insecticide, metal polishes, and electroplating. Hydrogen cyanide is present in fire smoke from burning nitrogen-containing plastics. Poisoning occurs by hydrogen cyanide gas inhalation or ingestion of cyanide salts. It is a highly toxic substance with a rapid onset of toxic effects as it is absorbed quickly from lung or the stomach [12]. Cyanide binds to heme iron in cytochrome complex, and it inhibits cellular respiration. The symptoms of acute poisoning include headaches, tachypnea, dizziness, coma, seizure, and death within 10–20 minutes in severe cases [12].

At autopsy, the appearance of the body is slightly bright pink. This is thought to be due to the presence of excess oxyhemoglobin [1]. Moreover, because cyanide inhibits cellular respiration, tissue oxygen consumption would be decreased. The stomach wall is damaged by the alkaline nature of stomach contents in case of cyanide salt ingestion [1]. It is well known that almond-like odor is one of the characteristics of cyanide poisoning [1, 8, 12, 19]. However, it can be detectable only approximately in one-third to half of the victims [19, 66], as this characteristic depends on the genetic trait [1]. Other autopsy findings are nonspecific in cyanide poisoning [1].

Cyanide level in blood is useful to confirm its toxicity [12]. The blood sample should be taken from the peripheral sites in case of cyanide salt ingestion, to exclude the effect of postmortem diffusion [66]. The normal blood cyanide level is 0.016 μg/mL for nonsmoker and 0.041 μg/mL for smoker. Cyanide concentration of less than 0.2 μg/mL in blood does not usually elicit any symptom [12]. Fatal concentration of cyanide in blood has been reported to be not lower than 3–5 μg/mL [67]. Lethal dose of potassium cyanide ingestion for adults is 200–300 mg.

For identification purposes, various methods have been devised. Color and microdiffusion tests are the most common [67, 68]. The Schöenbein-Pagenstecher method, using guaiac-cupper paper, is employed as a preliminary test [68]. This coated paper turns blue in the presence of cyanide. This method is user-friendly and highly sensitive for cyanide. However, since it is difficult to store the guaiac-cupper paper for a long period, we prepare it immediately before examination. Other color test or commercially available test tube methods and test papers have also been used to test cyanide in blood samples [29, 66]. The microdiffusion test using Conway cell (pyridine-pyrazolone method) have been reported [67]. Although this method is also highly sensitive for cyanide, and also used as a quantitative examination, its main drawback is that it is relatively time-consuming.

For quantitative testing, gas chromatography and detection tube are employed in our laboratories. To do so, cyanide is extracted from the sample by adding the concentrated phosphoric acid or sulfuric acid, and detected by nitrogen phosphorus detector (NPD) or flame thermoionic detector (FTD), equipped with a gas chromatography device [69, 70]. Application of sGC has also been reported for cyanide measurement [71]. The sGC system is also highly sensitive for cyanide and has some advantages such as it is easy to operate and portable. Further application would be expected in the field of forensic medicine (**Figure 3**).

The detector tube is also used for the quantitation of cyanide in blood [46, 47, 72]. This apparatus consists of a cyanide-separator tube, cyanide-detector tube, and aspirating pump. These tubes and the pump are connected in series. The cyanide-separator tube is packed with silica gel particles coated with sulfuric acid, and the released cyanide gas is detected by the cyanide-detector tube, followed by the aspirating of the pump (**Figure 4**). The cyanide-detector tube is packed with silica gel particles coated with mercuric chloride and pH indicator [46], and the hydrochloric acid formation by the reaction between cyanide and mercuric chloride was observed. Because the detector tube is easy to handle and portable, it can be carried to the scene of accident or poisoning and at the point of care, and it is well applied in forensic medicine practice.

fungicide, insecticide, metal polishes, and electroplating. Hydrogen cyanide is present in fire smoke from burning nitrogen-containing plastics. Poisoning occurs by hydrogen cyanide gas inhalation or ingestion of cyanide salts. It is a highly toxic substance with a rapid onset of toxic effects as it is absorbed quickly from lung or the stomach [12]. Cyanide binds to heme iron in cytochrome complex, and it inhibits cellular respiration. The symptoms of acute poisoning include headaches, tachypnea, dizziness, coma, seizure, and death within 10–20 minutes in

**Figure 2.** Portable oximeter (AVOX 4000) and its operation. Sample cartridge is shown in lower left-hand corner.

162 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

At autopsy, the appearance of the body is slightly bright pink. This is thought to be due to the presence of excess oxyhemoglobin [1]. Moreover, because cyanide inhibits cellular respiration, tissue oxygen consumption would be decreased. The stomach wall is damaged by the alkaline nature of stomach contents in case of cyanide salt ingestion [1]. It is well known that almond-like odor is one of the characteristics of cyanide poisoning [1, 8, 12, 19]. However, it can be detectable only approximately in one-third to half of the victims [19, 66], as this characteristic depends on the genetic trait [1]. Other autopsy findings are nonspecific

Cyanide level in blood is useful to confirm its toxicity [12]. The blood sample should be taken from the peripheral sites in case of cyanide salt ingestion, to exclude the effect of postmortem diffusion [66]. The normal blood cyanide level is 0.016 μg/mL for nonsmoker and 0.041 μg/mL for smoker. Cyanide concentration of less than 0.2 μg/mL in blood does not usually elicit any symptom [12]. Fatal concentration of cyanide in blood has been reported to be not lower than

For identification purposes, various methods have been devised. Color and microdiffusion tests are the most common [67, 68]. The Schöenbein-Pagenstecher method, using guaiac-cupper paper, is employed as a preliminary test [68]. This coated paper turns blue in the presence of

3–5 μg/mL [67]. Lethal dose of potassium cyanide ingestion for adults is 200–300 mg.

severe cases [12].

in cyanide poisoning [1].

**Figure 3.** Equipment of the sensor gas chromatography for hydrogen cyanide quantification.

As a qualitative test for H<sup>2</sup>

cedure is easy to perform [66].

matography method measures extracted H<sup>2</sup>

The detector tube is also used for the quantitation of H<sup>2</sup>

point of care, and it is well suited for forensic practice.

used as one of the simplified analytical methods.

identification and quantitation for forensic purposes.

S-separator tube, H<sup>2</sup>

previously, these tubes and pump are connected in series. The H<sup>2</sup>

with silica gel particles coated with phosphoric acid, and the released H<sup>2</sup>

S-detector tube, followed by the aspirating of the pump. The H<sup>2</sup>

packed with silica gel particles coated with lead acetate [46, 49], and the indicator range is then observed. As this method is easy to operate and portable, it can be carried to the scene or

Helium (He), a colorless and odorless inert gas, acts as a simple asphyxiant agent. It causes oxygen depletion by the replacement of the inspired air [77]. It has highly diffusive properties and low solubility in water. It is used as a career gas for party balloons or cryogenic liquids. Medically, mixture gas of He and oxygen improve the oxygen flow in patient with upper

The identification and quantification of helium in forensic samples is usually performed using a headspace gas chromatography with TCD detector or gas chromatography mass spectrometry [79–84]. It has been reported that lung tissue, intratracheal, and stomach gas are suitable matrices for the analysis of the inert gases [79–84]. The gas sampling at the time of autopsy is relatively easy, it is a good practice to consider gas sampling in case helium exposure is

There are a lot of toxic gases that cause tissue damages, such as ammonia gas or chlorine gas [19]. These gases are widely used as industrial chemicals and cause irritation and inflammation at the points of contact. They may cause tissue necrosis in severe cases. Although the incidence of poisoning cases by these gases is relatively low, the detector tube method is often

We have discussed the properties and features of poisoning incidents due to gaseous substances and elaborated on the simplified analytical techniques and apparatus used for their

As a quantitative test for H<sup>2</sup>

tus consist of a H<sup>2</sup>

by the H<sup>2</sup>

**3.4. Helium**

suspected.

airway obstruction [78].

**3.5. Other toxic gases**

**4. Conclusion**

S, color testing is commonly used. The lead acetate paper is used as

Simplified Analysis of Toxic Gaseous Substance in Forensic Practice: Experiences from Japan

S, gas chromatography and detection tube are used. The gas chro-

S using flame photometric detector (FPD) [74, 75].

S-detector tube, and aspirating pump. As explained

S in blood [46, 47, 76]. This appara-

http://dx.doi.org/10.5772/intechopen.70029

S-separator tube is packed

S gas is detected

S-detector tube is

S. This pro-

165

a preliminary test [66, 68]. The sample is mixed with sulfuric acid and heated, then the lead

acetate paper is suspended; if the paper turns black, it indicates the presence of H<sup>2</sup>

**Figure 4.** Procedure of the measurement of hydrogen cyanide in blood using detector tube (A). The detector tube is connected to the aspirating pump and separator tube (B).

#### **3.3. Hydrogen sulfide (H<sup>2</sup> S)**

The H<sup>2</sup> S is a gas with a rotten egg smell. It is colorless, flammable, and heavier than air (specific gravity for the air, 1.19) [8, 12]. It is formed as a by-product of the chemical industry. It occurs in volcanic gases and hot springs, and it is also formed at the process during putrefaction of organic substances. It is highly toxic and causes cellular asphyxia by the inhibition of cytochrome oxidase, like cyanide. Its toxicity depends on various factors such as its concentration in air and duration of exposure [12]. An odor is detectable at a concentration of 0.2 ppm in air, but olfactory paralysis is observed at 100–150 ppm. Inflammatory conditions such as rhinitis, pharyngitis, bronchitis, and pulmonary edema are also observed as a result of its irritant properties [12]. The systemic toxicity is shown by headaches, nausea, vomiting, dizziness, loss of consciousness, and respiratory failure that are observed following high levels of H2 S exposure (above 500 ppm); unfortunately, most fatalities occur at the scene [12].

Nonspecific findings such as generalized organ congestion and pulmonary edema can be observed. The dark-greenish discoloration of cerebral gray matter, organ, and skin with rotten egg smell has been reported [1, 12].

The blood sulfide concentration is less than 0.05 μg/mL in normal healthy subjects [8]. Fatal concentration of sulfide in blood has been reported to be not lower than 0.13–0.45 μg/mL [73]. Since the formation of sulfide by postmortem degradation of protein has been reported, the interpretation of the results requires caution and expertise [12].

As a qualitative test for H<sup>2</sup> S, color testing is commonly used. The lead acetate paper is used as a preliminary test [66, 68]. The sample is mixed with sulfuric acid and heated, then the lead acetate paper is suspended; if the paper turns black, it indicates the presence of H<sup>2</sup> S. This procedure is easy to perform [66].

As a quantitative test for H<sup>2</sup> S, gas chromatography and detection tube are used. The gas chromatography method measures extracted H<sup>2</sup> S using flame photometric detector (FPD) [74, 75]. The detector tube is also used for the quantitation of H<sup>2</sup> S in blood [46, 47, 76]. This apparatus consist of a H<sup>2</sup> S-separator tube, H<sup>2</sup> S-detector tube, and aspirating pump. As explained previously, these tubes and pump are connected in series. The H<sup>2</sup> S-separator tube is packed with silica gel particles coated with phosphoric acid, and the released H<sup>2</sup> S gas is detected by the H<sup>2</sup> S-detector tube, followed by the aspirating of the pump. The H<sup>2</sup> S-detector tube is packed with silica gel particles coated with lead acetate [46, 49], and the indicator range is then observed. As this method is easy to operate and portable, it can be carried to the scene or point of care, and it is well suited for forensic practice.

#### **3.4. Helium**

**3.3. Hydrogen sulfide (H<sup>2</sup>**

egg smell has been reported [1, 12].

The H<sup>2</sup>

H2

**S)**

164 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

connected to the aspirating pump and separator tube (B).

S is a gas with a rotten egg smell. It is colorless, flammable, and heavier than air (specific gravity for the air, 1.19) [8, 12]. It is formed as a by-product of the chemical industry. It occurs in volcanic gases and hot springs, and it is also formed at the process during putrefaction of organic substances. It is highly toxic and causes cellular asphyxia by the inhibition of cytochrome oxidase, like cyanide. Its toxicity depends on various factors such as its concentration in air and duration of exposure [12]. An odor is detectable at a concentration of 0.2 ppm in air, but olfactory paralysis is observed at 100–150 ppm. Inflammatory conditions such as rhinitis, pharyngitis, bronchitis, and pulmonary edema are also observed as a result of its irritant properties [12]. The systemic toxicity is shown by headaches, nausea, vomiting, dizziness, loss of consciousness, and respiratory failure that are observed following high levels of

**Figure 4.** Procedure of the measurement of hydrogen cyanide in blood using detector tube (A). The detector tube is

S exposure (above 500 ppm); unfortunately, most fatalities occur at the scene [12].

interpretation of the results requires caution and expertise [12].

Nonspecific findings such as generalized organ congestion and pulmonary edema can be observed. The dark-greenish discoloration of cerebral gray matter, organ, and skin with rotten

The blood sulfide concentration is less than 0.05 μg/mL in normal healthy subjects [8]. Fatal concentration of sulfide in blood has been reported to be not lower than 0.13–0.45 μg/mL [73]. Since the formation of sulfide by postmortem degradation of protein has been reported, the Helium (He), a colorless and odorless inert gas, acts as a simple asphyxiant agent. It causes oxygen depletion by the replacement of the inspired air [77]. It has highly diffusive properties and low solubility in water. It is used as a career gas for party balloons or cryogenic liquids. Medically, mixture gas of He and oxygen improve the oxygen flow in patient with upper airway obstruction [78].

The identification and quantification of helium in forensic samples is usually performed using a headspace gas chromatography with TCD detector or gas chromatography mass spectrometry [79–84]. It has been reported that lung tissue, intratracheal, and stomach gas are suitable matrices for the analysis of the inert gases [79–84]. The gas sampling at the time of autopsy is relatively easy, it is a good practice to consider gas sampling in case helium exposure is suspected.

#### **3.5. Other toxic gases**

There are a lot of toxic gases that cause tissue damages, such as ammonia gas or chlorine gas [19]. These gases are widely used as industrial chemicals and cause irritation and inflammation at the points of contact. They may cause tissue necrosis in severe cases. Although the incidence of poisoning cases by these gases is relatively low, the detector tube method is often used as one of the simplified analytical methods.

## **4. Conclusion**

We have discussed the properties and features of poisoning incidents due to gaseous substances and elaborated on the simplified analytical techniques and apparatus used for their identification and quantitation for forensic purposes.

In doing so, we have shared our experiences and highlighted the fact that the analysis of gaseous substances can be performed using readily available laboratory tools and equipment. We have emphasized the need and usefulness of the reference data tables for guiding forensic diagnosis.

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ed. Geneva: World Health Organization; 1999

Research and Practice in Forensic Medicine. 1985;**28**:185-190

tem blood samples. Journal of Analytical Toxicology. 2000;**24**:572-578

We hope that the above overview will assist other colleagues to implement such simplified techniques for the advancement of forensic practice.

## **Acknowledgements**

This work was supported by JSPS KAKENHI Grant-in-Aid for Scientific Research (C) Number 15K08873.

## **Author details**

Hiroshi Kinoshita<sup>1</sup> \*, Naoko Tanaka<sup>1</sup> , Ayaka Takakura<sup>1</sup> , Mostofa Jamal<sup>1</sup> , Asuka Ito<sup>1</sup> , Mitsuru Kumihashi<sup>1</sup> , Shoji Kimura<sup>1</sup> , Kunihiko Tsutsui<sup>2</sup> , Shuji Matsubara<sup>3</sup> and Kiyoshi Ameno<sup>1</sup>

\*Address all correspondence to: kinochin@med.kagawa-u.ac.jp

1 Department of Forensic Medicine, Faculty of Medicine, Kagawa University, Kagawa, Japan

2 Health Sciences, Faculty of Medicine, Kagawa University, Kagawa, Japan

3 Community Health Care Education Support Center, Postgraduate Clinical Education Center, Kagawa University Hospital, Kagawa, Japan

## **References**


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In doing so, we have shared our experiences and highlighted the fact that the analysis of gaseous substances can be performed using readily available laboratory tools and equipment. We have emphasized the need and usefulness of the reference data tables for guiding forensic diagnosis. We hope that the above overview will assist other colleagues to implement such simplified

This work was supported by JSPS KAKENHI Grant-in-Aid for Scientific Research (C)

, Ayaka Takakura<sup>1</sup>

, Kunihiko Tsutsui<sup>2</sup>

1 Department of Forensic Medicine, Faculty of Medicine, Kagawa University, Kagawa, Japan

[2] Skopp G. Postmortem toxicology. Forensic Science Medicine and Pathology. 2010;**6**:314-325 [3] Moffat AC, Osselton MD, Widdop B. Forensic toxicology. In: Moffat AC, Osselton MD, Widdop B, editors. Clarke's Analysis of Drugs and Poisons in Pharmaceuticals, Body Fluids and Postmortem Materials. London: Pharmaceutical Press; 2004. pp. 80-93

[4] Poklis A. Forensic toxicology. In: Eckert WG, editor. Introduction to Forensic Sciences.

[5] Stead AH, Moffat AC. A collection of therapeutic, toxic and fatal blood drug concentra-

[6] Winek CL, Wahba WW, Winek CL Jr, Balzer TW. Drug and chemical blood-level data

3 Community Health Care Education Support Center, Postgraduate Clinical Education

, Mostofa Jamal<sup>1</sup>

, Shuji Matsubara<sup>3</sup>

, Asuka Ito<sup>1</sup>

and

,

techniques for the advancement of forensic practice.

166 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

\*, Naoko Tanaka<sup>1</sup>

Center, Kagawa University Hospital, Kagawa, Japan

, Shoji Kimura<sup>1</sup>

\*Address all correspondence to: kinochin@med.kagawa-u.ac.jp

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2 Health Sciences, Faculty of Medicine, Kagawa University, Kagawa, Japan

**Acknowledgements**

Number 15K08873.

**Author details**

Hiroshi Kinoshita<sup>1</sup>

Kiyoshi Ameno<sup>1</sup>

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Mitsuru Kumihashi<sup>1</sup>


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**Chapter 10**

**Provisional chapter**

**Optical Express Methods of Monitoring of Pathogens**

Hygienic standards to the quality of potable water require continuous monitoring of the absence of pathogenic microorganisms directly in water flow. Despite a great number of laboratory devices for checking the quality of potable water, there are no express analyzers for monitoring of pathogenic organisms, which could be embedded directly into the automatic checkout systems. The reasons of it are low concentration of pollutions and the presence of additional effects, which impede automatic data processing. The new method, the express analysis of pathogens in water, was developed. It shall be mentioned that the proposed method of express diagnostics allows detection of infectious agents in the water in minutes based on nonlinear effects. This research has, hopefully, laid the foundation for development of a prototype for determination of the content of the genetically modified soy in meat products. The inventive methods can be recommended for DNA diagnostics in medicine ,veterinary sciences and insanitation. The main advantage of this method is that there is no need for DNA isolation. It is sufficient to create a suspen-

**Keywords:** express diagnostics, viruses, pathogens, laser monitoring, colloidal solution

Hygienic standards to the quality of potable water require continuous monitoring of the absence of pathogenic microorganisms directly in water flow. Despite a great number of laboratory devices for checking the quality of potable water, there are no express analyzers for monitoring of pathogenic organisms, which could be embedded directly into the automatic checkout systems. The reasons of it are low concentration of pollutions and the presence of additional effects, which impede automatic data processing. At present, monitoring

**Optical Express Methods of Monitoring of Pathogens** 

DOI: 10.5772/intechopen.70236

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**in Drinking Water and Water-Based Solutions**

**in Drinking Water and Water-Based Solutions**

Tatiana Moguilnaya and Aleksey Sheryshev

Additional information is available at the end of the chapter

Tatiana Moguilnaya and Aleksey Sheryshev

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70236

sion of the product by centrifugation.

**Abstract**

**1. Introduction**


**Provisional chapter**

## **Optical Express Methods of Monitoring of Pathogens in Drinking Water and Water-Based Solutions in Drinking Water and Water-Based Solutions**

**Optical Express Methods of Monitoring of Pathogens** 

DOI: 10.5772/intechopen.70236

Tatiana Moguilnaya and Aleksey Sheryshev Tatiana Moguilnaya and Aleksey Sheryshev Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70236

#### **Abstract**

[80] Auwaerter V, Grosse Perdekamp M, Kempf J, Schmidt U, Weinmann W, Pollak S. Toxicological analysis after asphyxia suicide with helium and a plastic bag. Forensic

[81] Tanaka N, Kinoshita H, Jamal M, Kumihashi M, Tobiume T, Tsutsui K, Ameno K. Usefulness of intratracheal gas analysis in an autopsy case of helium inhalation.

[82] Tanaka N, Takakura A, Jamal M, Kumihashi M, Ito A, Ishimoto S, Tsutsui K, Kimura S, Ameno K, Kinoshita H. Stomach gas as a useful matrix for detecting ante-mortem gas exposure. A case of asphyxia by helium inhalation. Romanian Journal of Legal Medicine.

[83] Oosting R, van der Hulst R. Peschier L, Verschraagen M. Toxicological findings in three cases of suicidal asphyxiation with helium. Forensic Science International. 2015;**256**:38-41

[84] Malbranque S, Mauillon D, Turcant A, Rouge-maillart C, Mangin P. Quantification of fatal helium exposure following self-administration. International Journal of Legal

Science International. 2007;**170**:139-141

2016;**24**:21-22

Medicine. 2016;**130**:1535-1539

Romanian Journal of Legal Medicine. 2013;**21**:237-238

172 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

Hygienic standards to the quality of potable water require continuous monitoring of the absence of pathogenic microorganisms directly in water flow. Despite a great number of laboratory devices for checking the quality of potable water, there are no express analyzers for monitoring of pathogenic organisms, which could be embedded directly into the automatic checkout systems. The reasons of it are low concentration of pollutions and the presence of additional effects, which impede automatic data processing. The new method, the express analysis of pathogens in water, was developed. It shall be mentioned that the proposed method of express diagnostics allows detection of infectious agents in the water in minutes based on nonlinear effects. This research has, hopefully, laid the foundation for development of a prototype for determination of the content of the genetically modified soy in meat products. The inventive methods can be recommended for DNA diagnostics in medicine ,veterinary sciences and insanitation. The main advantage of this method is that there is no need for DNA isolation. It is sufficient to create a suspension of the product by centrifugation.

**Keywords:** express diagnostics, viruses, pathogens, laser monitoring, colloidal solution

## **1. Introduction**

Hygienic standards to the quality of potable water require continuous monitoring of the absence of pathogenic microorganisms directly in water flow. Despite a great number of laboratory devices for checking the quality of potable water, there are no express analyzers for monitoring of pathogenic organisms, which could be embedded directly into the automatic checkout systems. The reasons of it are low concentration of pollutions and the presence of additional effects, which impede automatic data processing. At present, monitoring

of microbiological parameters of the composition of drinking water is of great importance. Water supply systems are the key to ensuring the livelihoods of cities, in connection with which the issue of realizing the control of drinking water parameters directly in the pipeline flow in real time is becoming increasingly acute. To solve this problem, it is necessary to implement continuous monitoring, including super-toxicants in drinking water. At the same time, much attention is paid to the development of fundamentally new and highly effective detection and control systems for the spread of pathogenic microorganisms. The problem is complicated by the fact that the maximum permissible concentration of pathogenic microorganisms can be within even a few molecules.

**2. Methods and findings**

**Method Need for sample** 

1. Agglutination method

4. Method of a fixation of the complement

5. Phagocytic activity

6. Immune-enzyme

7. Neutralization method

8. Method of the luminescence

9. The method of stimulated luminescence and SBS

test

analysis

**preparation**

**Table 1.** Comparison of methods by duration and sophistication.

As a preliminary to a detailed description, a comparative analysis of the methods is given in **Table 1**. This comparative analysis is shown to highlight the benefits of our method, which will allow automated monitoring of pathogens. It should be noted that the certification of the method was carried out on the laboratory basis of the State Research Center for Applied Microbiology and Biotechnology (Obolensk, Moscow Region) and at the All-Russian Research Institute of Veterinary Sanitation, Hygiene and Ecology of the Russian Academy of Agricultural Sciences. Approbation of the device was carried of the double-blind method. The double-blind method is an experimental procedure in which neither the representatives of the testing organization nor the researchers know what is in the tested samples. The double-blind method is used to avoid the appearance of the effects of the experimenter's prejudice relative to the studied characteristic (the Rosenthal effect) and to eliminate the possibility of distorting the results of the experiment of the knowledge effect of what is required of the subject.

Optical Express Methods of Monitoring of Pathogens in Drinking Water and Water-Based...

http://dx.doi.org/10.5772/intechopen.70236

175

In **Table 2**, results of comparison of tests by a double-blind method against a standard test method are shown. It should be noted also that we studied transmission IR spectra of a number of pathogen (salmonella, viruses of herpes genitaler, hepatitis A and C, grippe A and B) solutions and luminescence of nanomarkers. In our experiments, the laser radiation passed through a quartz cell with water solutions of the pathogens, nanosilver,

Yes Yes Yes 1 h 1 day

Yes Yes Yes 1 h 1 day

Yes Yes Yes 24 h 2 days

Yes Yes Yes 2–4 h 1 day

Yes Yes Yes 4–7 days 5–8 days

No Yes No 15 min 30 min

No No No 2 min 1 min

**Use of reagents Duration of the** 

**measurement**

**Duration of result processing**

**Special laboratory conditions**

2. Immune deposition Yes Yes Yes 20–60 min 1 day 3. Immunobloom Yes Yes Yes 2–4 h 1 day

To date, the control is performed by the laboratory methods. That process takes from several hours to several days, depending on the method. In addition, these tests must be conducted in laboratory conditions by highly qualified personnel. That necessitates the development of a device that can be integrated into an automated line for monitoring drinking water. For example, the PCR method is an excellent diagnostic method, but it requires preliminary DNA isolation. The method of luminescent analysis using fluorescent markers requires very specialized sample preparation.

Thus, the problem of real-time assessing quality of drinking water to guarantee the absence of hazardous biological and chemical substances, even in small and ultrasmall concentrations, is becoming increasingly important. The content of pathogenic microorganisms in water can vary from 10−8 up to 105 mg/m3 . Although there are a great number of laboratory devices for checking the quality of potable water, there are no express analyzers for monitoring of pathogenic microorganisms that could be embedded directly into the automatic checkout systems. The optical phenomena of stimulated scattering in water solutions, which contain DNA, are investigated insufficiently.

Yet such research would be of great interest for the development of automatic control systems of potable water. Laser methods are widely applied to the analysis of structures of multicomponent liquids and are thus useful as a tool for the determination of low concentrations. In this chapter we describe new methods of express monitoring of viruses and pathogenic organisms in water and aqueous solutions based on nonlinear effects. We also describe a modified PCR method of diagnosis of genetically modified foods. The proposed methods can be recommended for the diagnosis of DNA, in the field, in medicine and veterinary sciences, in sanitary epidemiological studies to detect agents of dangerous infections in the event of potential bioterrorist attacks

#### **1.1. Aim and objectives of our studies**

The purpose and objectives of our research are to determine in spectra what kind the optical parameters allows us to diagnostic patogens with a probability of not less than 95%. To do this, no less than 1500 spectra were obtained for each of the pathogens studied. A large amount of experimental material allowed to create software that automatically determines the type of pathogen and signals its presence.

## **2. Methods and findings**

of microbiological parameters of the composition of drinking water is of great importance. Water supply systems are the key to ensuring the livelihoods of cities, in connection with which the issue of realizing the control of drinking water parameters directly in the pipeline flow in real time is becoming increasingly acute. To solve this problem, it is necessary to implement continuous monitoring, including super-toxicants in drinking water. At the same time, much attention is paid to the development of fundamentally new and highly effective detection and control systems for the spread of pathogenic microorganisms. The problem is complicated by the fact that the maximum permissible concentration of pathogenic microor-

174 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

To date, the control is performed by the laboratory methods. That process takes from several hours to several days, depending on the method. In addition, these tests must be conducted in laboratory conditions by highly qualified personnel. That necessitates the development of a device that can be integrated into an automated line for monitoring drinking water. For example, the PCR method is an excellent diagnostic method, but it requires preliminary DNA isolation. The method of luminescent analysis using fluorescent markers requires very spe-

Thus, the problem of real-time assessing quality of drinking water to guarantee the absence of hazardous biological and chemical substances, even in small and ultrasmall concentrations, is becoming increasingly important. The content of pathogenic microorganisms in water can

checking the quality of potable water, there are no express analyzers for monitoring of pathogenic microorganisms that could be embedded directly into the automatic checkout systems. The optical phenomena of stimulated scattering in water solutions, which contain DNA, are

Yet such research would be of great interest for the development of automatic control systems of potable water. Laser methods are widely applied to the analysis of structures of multicomponent liquids and are thus useful as a tool for the determination of low concentrations. In this chapter we describe new methods of express monitoring of viruses and pathogenic organisms in water and aqueous solutions based on nonlinear effects. We also describe a modified PCR method of diagnosis of genetically modified foods. The proposed methods can be recommended for the diagnosis of DNA, in the field, in medicine and veterinary sciences, in sanitary epidemiological studies to detect agents of dangerous infections in the event of

The purpose and objectives of our research are to determine in spectra what kind the optical parameters allows us to diagnostic patogens with a probability of not less than 95%. To do this, no less than 1500 spectra were obtained for each of the pathogens studied. A large amount of experimental material allowed to create software that automatically determines the type of

. Although there are a great number of laboratory devices for

ganisms can be within even a few molecules.

cialized sample preparation.

vary from 10−8 up to 105 mg/m3

investigated insufficiently.

potential bioterrorist attacks

**1.1. Aim and objectives of our studies**

pathogen and signals its presence.

As a preliminary to a detailed description, a comparative analysis of the methods is given in **Table 1**. This comparative analysis is shown to highlight the benefits of our method, which will allow automated monitoring of pathogens. It should be noted that the certification of the method was carried out on the laboratory basis of the State Research Center for Applied Microbiology and Biotechnology (Obolensk, Moscow Region) and at the All-Russian Research Institute of Veterinary Sanitation, Hygiene and Ecology of the Russian Academy of Agricultural Sciences. Approbation of the device was carried of the double-blind method. The double-blind method is an experimental procedure in which neither the representatives of the testing organization nor the researchers know what is in the tested samples. The double-blind method is used to avoid the appearance of the effects of the experimenter's prejudice relative to the studied characteristic (the Rosenthal effect) and to eliminate the possibility of distorting the results of the experiment of the knowledge effect of what is required of the subject.

In **Table 2**, results of comparison of tests by a double-blind method against a standard test method are shown. It should be noted also that we studied transmission IR spectra of a number of pathogen (salmonella, viruses of herpes genitaler, hepatitis A and C, grippe A and B) solutions and luminescence of nanomarkers. In our experiments, the laser radiation passed through a quartz cell with water solutions of the pathogens, nanosilver,


**Table 1.** Comparison of methods by duration and sophistication.


The ratio of the number of emitted photons to the total number of elementary events is called the quantum yield of the reaction. The quantum yields of bioluminescence, unlike most chemiluminescent reactions, are very high, reaching values of 0.1–1. Chemiluminescence is rarely carried out for the reaction processes in aqueous solutions at neutral pH at such a quantum yield Bioluminescent process power radiation depends on the difference in energy between the oxidized forms of the ground and excited states of luciferin. This power is related to the radiation frequency by the relation ΔE = hv; On half the width of the radiation band, which

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177

The stimulated Brillouin scattering (SBS) is a nonlinear process that can occur in liquid media at large intensity. The mechanism of the appearance of Brillouin radiation arises from the photon-phonon interaction. The basic mechanism of SBS phenomenon is illustrated in **Figure 1b**. In scattering effects, energy gets transferred from one light wave to another wave at a longer wavelength or lower energy. The lost energy is absorbed by the molecular vibrations, or phonons, in the medium. Stimulated scattering is affected by the threshold level. The SRS is a nonlinear parametric interaction between light and molecular vibrations. Optical phonon participates in SRS, but acoustic photon participates in SBS. Due to SRS power transferred from shorter wavelength channels to the longer wavelength channels. SRS occurs in both directions, either forward or backward direction. The input signal from the laser source is the pump signal and generates a new wave due to scattering in the medium. This wawe is called the Stokes wave. SBS occurs only in the backward direction; for example, when input power exceeds threshold power, Stokes power shifted to the backward direction. Pump wave losses power, while Stokes wave gains power. The pump wave creates acoustic wave in transmission medium through a process called electrostriction. The interaction between pump wave and acoustic wave creates the generation of back propagating optical wave which is called Stokes wave. When acoustic waves travel through the transparent liquid media, they induce spatially periodic local compressions and expansions which in turn cause local increases and decreases in the refractive index. This phenomenon is known as photoelastic effect altered to a degree such that a significant portion of the optical signal is back-scattered. So, we can say that the acoustic wave alters the optical properties of the fiber, including the refractive index. This fluctuation of refractive index scatters the incident wave and creates Stoke wave which

**Figure 1.** Comparison of the phenomenon of luminescence (a) and stimulated Brillouin scattering (b).

has a width usually аbout 50 nm (**Figure 1a**) [1].

*2.1.2. Overview on stimulated Brillouin scattering*

**Table 2.** Comparison of results by methods used by duration and false positive.

or nanomarkers. We found that exciting radiations with wavelengths 1017 and 810 nm induce the stimulated Brillouin scattering (SBS) in spectra of the water containing DNA. We believe that the power threshold for the onset of this effect is achieved by adding a forced radiation luminescence and laser radiation. A comparative analysis of the methods is given in **Table 1**. Comparative analysis shows that our method (9) is promising for automated monitoring of pathogens.

#### **2.1. Basic concepts of methods for monitoring pathogens in water**

#### *2.1.1. Overview of bioluminescence*

Bioluminescence is a form of chemiluminescence where light energy is released by a chemical reaction. The reaction can occur either inside or outside the cell. Chemiluminescence occurs like many chemical reactions, for example, by recombination of free radicals or the oxidation reaction. In this case, as in the bioluminescence reaction, released energy is not dissipated as heat, as is the case during most of the exothermic chemical reactions, and consumed in the formation of one of the reaction products in an excited electronic state. Emitting light during the chemiluminescent reaction is necessary to satisfy at least two conditions: Firstly, the energy released during the reaction must exceed about 41–71.5 kcal/mol, and, secondly, the energy difference between the ground and excited states that the reaction product should be less than the enthalpy of the chemical reaction. Under these conditions, it can be formed with a sufficiently high transition of oxidized luciferin in the excited state and the subsequent transition of it to the ground state with the emission of photons

The ratio of the number of emitted photons to the total number of elementary events is called the quantum yield of the reaction. The quantum yields of bioluminescence, unlike most chemiluminescent reactions, are very high, reaching values of 0.1–1. Chemiluminescence is rarely carried out for the reaction processes in aqueous solutions at neutral pH at such a quantum yield Bioluminescent process power radiation depends on the difference in energy between the oxidized forms of the ground and excited states of luciferin. This power is related to the radiation frequency by the relation ΔE = hv; On half the width of the radiation band, which has a width usually аbout 50 nm (**Figure 1a**) [1].

#### *2.1.2. Overview on stimulated Brillouin scattering*

or nanomarkers. We found that exciting radiations with wavelengths 1017 and 810 nm induce the stimulated Brillouin scattering (SBS) in spectra of the water containing DNA. We believe that the power threshold for the onset of this effect is achieved by adding a forced radiation luminescence and laser radiation. A comparative analysis of the methods is given in **Table 1**. Comparative analysis shows that our method (9) is promising for auto-

Bioluminescence is a form of chemiluminescence where light energy is released by a chemical reaction. The reaction can occur either inside or outside the cell. Chemiluminescence occurs like many chemical reactions, for example, by recombination of free radicals or the oxidation reaction. In this case, as in the bioluminescence reaction, released energy is not dissipated as heat, as is the case during most of the exothermic chemical reactions, and consumed in the formation of one of the reaction products in an excited electronic state. Emitting light during the chemiluminescent reaction is necessary to satisfy at least two conditions: Firstly, the energy released during the reaction must exceed about 41–71.5 kcal/mol, and, secondly, the energy difference between the ground and excited states that the reaction product should be less than the enthalpy of the chemical reaction. Under these conditions, it can be formed with a sufficiently high transition of oxidized luciferin in the excited state and the subsequent

**2.1. Basic concepts of methods for monitoring pathogens in water**

**Table 2.** Comparison of results by methods used by duration and false positive.

transition of it to the ground state with the emission of photons

mated monitoring of pathogens.

**Pathogen/the method and result of research**

*E. coli* 12571 agglutination method

*Enterococcus faecalis*

Shigella flex 3 immunobloom

Method of a fixation of the complement

Kolifag MS-2 phagocytic activity test

*Staphylococcus aureus* by immune-enzyme analysis

**Test/ positive** **Device/ positive** **Test/ negative**

176 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

**Device /negative duration of the measurement**

20 yes 20 yes 20 yes 1 h 0 0 0 0

20 yes 20 yes 20 yes 20–60 min 0 0 0 01

10 yes 10 yes 10 yes 2–4 h 0 0 0 01

10 yes 10 yes 10 yes 1 h 0 0 0 01

5yes 5yes 10yes 24 h 0 0 0 01

5 yes 5 yes 5 yes 2–4 h 0 0 0 02

**Test/false negative**

**Device/false negative**

**Test/false positive**

**Device/false positives**

*2.1.1. Overview of bioluminescence*

The stimulated Brillouin scattering (SBS) is a nonlinear process that can occur in liquid media at large intensity. The mechanism of the appearance of Brillouin radiation arises from the photon-phonon interaction. The basic mechanism of SBS phenomenon is illustrated in **Figure 1b**. In scattering effects, energy gets transferred from one light wave to another wave at a longer wavelength or lower energy. The lost energy is absorbed by the molecular vibrations, or phonons, in the medium. Stimulated scattering is affected by the threshold level. The SRS is a nonlinear parametric interaction between light and molecular vibrations. Optical phonon participates in SRS, but acoustic photon participates in SBS. Due to SRS power transferred from shorter wavelength channels to the longer wavelength channels. SRS occurs in both directions, either forward or backward direction. The input signal from the laser source is the pump signal and generates a new wave due to scattering in the medium. This wawe is called the Stokes wave. SBS occurs only in the backward direction; for example, when input power exceeds threshold power, Stokes power shifted to the backward direction. Pump wave losses power, while Stokes wave gains power. The pump wave creates acoustic wave in transmission medium through a process called electrostriction. The interaction between pump wave and acoustic wave creates the generation of back propagating optical wave which is called Stokes wave. When acoustic waves travel through the transparent liquid media, they induce spatially periodic local compressions and expansions which in turn cause local increases and decreases in the refractive index. This phenomenon is known as photoelastic effect altered to a degree such that a significant portion of the optical signal is back-scattered. So, we can say that the acoustic wave alters the optical properties of the fiber, including the refractive index. This fluctuation of refractive index scatters the incident wave and creates Stoke wave which

**Figure 1.** Comparison of the phenomenon of luminescence (a) and stimulated Brillouin scattering (b).

propagates in the opposite direction. The magnitude of the photoelastic effect increases with increasing input optical power. If the input power reaches the threshold level of SBS and the refractive index is variable, then an acoustic wave appears. Modern research shows that water has a quasi-crystalline structure. Similar effects were observed in the liquid and solid [2].

This radiation was registered in short-range IR. Experiments were repeated, at least, 40 times for statistical significance. Spectral characteristics were measured for a range of concentra-

developed the coherent spectroscopic method for the monitoring of the pathogenic organisms directly in water pipeline, genetically modified products, and nanostructured materials in colloidal solution. The method is based on an analysis of spectral characteristics of stimulated radiation, passed through the solution. Spectra were analyzed by the spectrum analyzer "Agilent" (USA) with spectral resolution 0.5 nm or by the spectrum analyzer "AQ6370C," which provides spectral resolution, ±0.01 nm and 0.02 nm; maximum input power, +20 dBm; and a sensitivity level of input power, −90 dBm. The input-output waveguides bring the exciting and passing radiations from laser to the cell and from cell to the analyzer, respectively.

We found that exciting radiations with wavelengths 1017 and 810 nm induce the stimulated Brillouin scattering in spectra of the water containing pathogen DNAs. We revealed that peak positions and widths of "fingerprints" for pathogens under study and optical densities of these bands were proportional pathogen content, if their content was less 15%. Thus, Stokes and anti-Stokes bands of the stimulated Brillouin scattering can be used to recognize the pathogens. Nonlinear effects like SBS arise due to the density of radiation in this solution [3]. The similar density of radiation is reached due to a combination of the luminescence fields and laser radiation [2] giving the characteristic peak. The modal frequency of this peak depends on the type of DNA accordingly. The strains of viruses were obtained from the Museum of the State Scientific Center. We investigated the inelastic scattering spectra from water solutions of many pathogens (**Table 3**). Traditional methods for detection of microorganisms are based on the enumeration of bacterial cells after their cultivation on a nutrient media. This method is sensitive, inexpensive, and simple, but it takes at least several days for completion. Two known methods for reducing the detection time—immunoassay and polymerase chain reaction—are complicated. At the present time, the problem of automatic monitoring of characteristics of drinking water directly within a water steam is particularly acute. Two resonance control techniques could be particularly instrumental in finding solution of that problem—spectroscopy of Raman scattering and stimulated Brillouin scattering. The utilization of Raman scattering spectroscopy technique requires transferring an enormous amount of energy for extracting an informative signal, which is considerably lower than the noise [4]. At present laser-based technology requires no consumables, or reagents are currently being developed [2]. A new method for detection of live or dead pathogens in water is described below. It is based on the diagnostics of nonlinear effects, which comprises two phenomena: an induced luminescence of DNA under the influence of laser radiation and a stimulated Brillouin scattering (SBS) [5]. The following model parameters were selected for the identification: of the peaks positions of the spectral line, corresponding to a pathogen and the difference between two wavelengths corresponding, respectively, to the laser mode maximum and of the peak of a spectral line of Stokes . Previously, we studied the spectra of virus [6]. The present work is focused on studying spectra of bacteria. The induced luminescence of DNA under the influence of laser radiation differs from ordinary luminescence in their characteristics [1]. Luminescence phenomenon was observed if the laser power is below the

(cells/ml). The data were placed into a spectra database. We

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Optical Express Methods of Monitoring of Pathogens in Drinking Water and Water-Based...

tions of pathogens, from 10 to 109

threshold of the Brillouin scattering effect.

Our investigations showed the occurrence of luminescence mainly in solutions containing DNA or fluorescent markers. Brillouin scattering was observed in solutions containing low concentrations of particles, including nanoscale. Based on these investigations, we developed the method of monitoring nanoparticle sizes, composition, chemical reactions, and transition processes occurring in solutions. At present we are creating series of devices for monitoring MPC (maximum permissible concentration) in tap water by using the proposed method.

#### **2.2. Description of the monitoring of pathogens in water by resonance laser spectroscopy techniques**

We used a specially designed testing bench and the scheme of experimental setup shown in **Figure 1**. We analyzed about 700 spectra for each of the pathogens. Pathogens that we studied are shown in **Table 1**. We excited viruses and bacteria suspensions with a laser beam having wavelengths of 810 and 1017 nm and then evaluated the forward-scattering spectra. The testing bench consists of the following components: (1) the block of the three of semiconductor laser sources with wavelengths λ1 = 1017 nm, λ2 = 810 nm, and λ3 = 670 nm; (2) the quartz cuvette; (3) spectrum analyzer "Agilent" with the spectral resolution 0.05 nm, equipped with the microcomputer for processing spectra; (4) and (5) waveguides for the radiation input-output ; and (6) the computer equipped with original software for processing spectra (**Figure 1a**, **b**). The laser radiation passed through a waveguide into the cuvette with the sample. Sometimes, we used an additional laser with a wavelength λ4 = 532 nm. That source enhanced the intensity of luminescence. The radiation, which passed through the cuvette and the output waveguide, was analyzed by a spectrum analyzer (**Figure 2**).

**Figure 2.** (a) The experimental setup: (1) the block of the three of the semiconductor laser sources with exciting wavelengths λ<sup>1</sup> = 1017 nm, λ<sup>2</sup> = 810 nm, and λ<sup>3</sup> = 670 nm; (2) the quartz cuvette; (3) spectrum analyzer "Agilent"; and (4) computer equipped with original software for processing spectra.; (b) photo.

This radiation was registered in short-range IR. Experiments were repeated, at least, 40 times for statistical significance. Spectral characteristics were measured for a range of concentrations of pathogens, from 10 to 109 (cells/ml). The data were placed into a spectra database. We developed the coherent spectroscopic method for the monitoring of the pathogenic organisms directly in water pipeline, genetically modified products, and nanostructured materials in colloidal solution. The method is based on an analysis of spectral characteristics of stimulated radiation, passed through the solution. Spectra were analyzed by the spectrum analyzer "Agilent" (USA) with spectral resolution 0.5 nm or by the spectrum analyzer "AQ6370C," which provides spectral resolution, ±0.01 nm and 0.02 nm; maximum input power, +20 dBm; and a sensitivity level of input power, −90 dBm. The input-output waveguides bring the exciting and passing radiations from laser to the cell and from cell to the analyzer, respectively.

propagates in the opposite direction. The magnitude of the photoelastic effect increases with increasing input optical power. If the input power reaches the threshold level of SBS and the refractive index is variable, then an acoustic wave appears. Modern research shows that water has a quasi-crystalline structure. Similar effects were observed in the liquid and solid [2].

178 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

Our investigations showed the occurrence of luminescence mainly in solutions containing DNA or fluorescent markers. Brillouin scattering was observed in solutions containing low concentrations of particles, including nanoscale. Based on these investigations, we developed the method of monitoring nanoparticle sizes, composition, chemical reactions, and transition processes occurring in solutions. At present we are creating series of devices for monitoring MPC (maximum permissible concentration) in tap water by using the proposed method.

**2.2. Description of the monitoring of pathogens in water by resonance laser spectroscopy** 

We used a specially designed testing bench and the scheme of experimental setup shown in **Figure 1**. We analyzed about 700 spectra for each of the pathogens. Pathogens that we studied are shown in **Table 1**. We excited viruses and bacteria suspensions with a laser beam having wavelengths of 810 and 1017 nm and then evaluated the forward-scattering spectra. The testing bench consists of the following components: (1) the block of the three of semiconductor laser sources with wavelengths λ1 = 1017 nm, λ2 = 810 nm, and λ3 = 670 nm; (2) the quartz cuvette; (3) spectrum analyzer "Agilent" with the spectral resolution 0.05 nm, equipped with the microcomputer for processing spectra; (4) and (5) waveguides for the radiation input-output ; and (6) the computer equipped with original software for processing spectra (**Figure 1a**, **b**). The laser radiation passed through a waveguide into the cuvette with the sample. Sometimes, we used an additional laser with a wavelength λ4 = 532 nm. That source enhanced the intensity of luminescence. The radiation, which passed through the cuvette and

**Figure 2.** (a) The experimental setup: (1) the block of the three of the semiconductor laser sources with exciting

= 670 nm; (2) the quartz cuvette; (3) spectrum analyzer "Agilent"; and (4)

the output waveguide, was analyzed by a spectrum analyzer (**Figure 2**).

**techniques**

wavelengths λ<sup>1</sup>

= 1017 nm, λ<sup>2</sup>

= 810 nm, and λ<sup>3</sup>

computer equipped with original software for processing spectra.; (b) photo.

We found that exciting radiations with wavelengths 1017 and 810 nm induce the stimulated Brillouin scattering in spectra of the water containing pathogen DNAs. We revealed that peak positions and widths of "fingerprints" for pathogens under study and optical densities of these bands were proportional pathogen content, if their content was less 15%. Thus, Stokes and anti-Stokes bands of the stimulated Brillouin scattering can be used to recognize the pathogens. Nonlinear effects like SBS arise due to the density of radiation in this solution [3]. The similar density of radiation is reached due to a combination of the luminescence fields and laser radiation [2] giving the characteristic peak. The modal frequency of this peak depends on the type of DNA accordingly. The strains of viruses were obtained from the Museum of the State Scientific Center. We investigated the inelastic scattering spectra from water solutions of many pathogens (**Table 3**). Traditional methods for detection of microorganisms are based on the enumeration of bacterial cells after their cultivation on a nutrient media. This method is sensitive, inexpensive, and simple, but it takes at least several days for completion. Two known methods for reducing the detection time—immunoassay and polymerase chain reaction—are complicated. At the present time, the problem of automatic monitoring of characteristics of drinking water directly within a water steam is particularly acute. Two resonance control techniques could be particularly instrumental in finding solution of that problem—spectroscopy of Raman scattering and stimulated Brillouin scattering. The utilization of Raman scattering spectroscopy technique requires transferring an enormous amount of energy for extracting an informative signal, which is considerably lower than the noise [4]. At present laser-based technology requires no consumables, or reagents are currently being developed [2]. A new method for detection of live or dead pathogens in water is described below. It is based on the diagnostics of nonlinear effects, which comprises two phenomena: an induced luminescence of DNA under the influence of laser radiation and a stimulated Brillouin scattering (SBS) [5]. The following model parameters were selected for the identification: of the peaks positions of the spectral line, corresponding to a pathogen and the difference between two wavelengths corresponding, respectively, to the laser mode maximum and of the peak of a spectral line of Stokes . Previously, we studied the spectra of virus [6]. The present work is focused on studying spectra of bacteria. The induced luminescence of DNA under the influence of laser radiation differs from ordinary luminescence in their characteristics [1]. Luminescence phenomenon was observed if the laser power is below the threshold of the Brillouin scattering effect.


The gradient was prepared by laminating successive to each other 50, 40, 30, 20, and 10% sucrose solution in amounts of 6 ml each tube to nitrocellulose followed by diffusion at 4 C for 12 h. The solution containing the virus was layered gently onto the surface gradient in the volume of 1 ml in each tube. We carried out the study of the dynamics of change in the intensity of luminescence. To do this, we utilized the laser, with the power being two times lower

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181

We continuously measured the spectral characteristics by the analyzer. The reference time started after the cuvette with a given concentration of the bacteria was placed in the optical path. The saved spectra retained their timing marks. We found that frequency of mode has a time drift (**Figure 3**).We observed two peaks in 1–2 s; then both peaks merged into one, and

We investigated scattering, arising at the passage of laser radiation through colloidal solution (water, alcohol, physical solution) and fine-dispersed phase. We studied transmission spectra

Two shifted and one unshifted frequency components relative to laser frequency were detected in these spectra. The center of our attention was placed on the shifted component study, due to the fact that the analysis of processes in the fine-dispersed phase information is simple. We observed that the shifted components occur as in the Stokes and anti-Stokes ranges. On our opinion spectra of some samples under study are the spectra of Brillouin scattering as the distance between the central and the shift components does not depend on the excitation source wavelength. In other spectra we observed the spectral distributions of induced super fluorescence in liquids. In this case, the intensity of the shifted spectral distribution was higher than the intensity of the spectral distribution of laser radiation by several orders of magnitude, and the width of the spectral peak was decreased sharply. In both cases the width of the Lorentzian component, selected from such shifted spectral distribution, is proportional to the

of a substance containing DNA: solutions of viruses and microorganisms.

size of the particles, even if the solution contains impurities mainly proteinaceous.

**Figure 3.** Intensity of stimulated luminescence of hepatitis C in the physiological solution from time (a) 30 s, (b) 1 min,

than the SBS phenomena threshold.

after 3–4 s, the luminescence disappeared.

*2.3.2. Spectra of viruses*

and (c) 2 min.

**Table 3.** Pathogens included in the experiment.

#### **2.3. Investigation of luminescence in the water solution**

#### *2.3.1. Description of the experiment*

We studied the luminescence of the hepatitis C virus. For this we used a source with a power below the threshold. The virus was diluted in the different media (physiology liquor, water, alcohol) in proportions of the greatest possible (10<sup>10</sup> cells/ml) concentration, intermediate (10<sup>5</sup> cells/ml) concentration, and minimum concentration (10<sup>1</sup> cells/ml) of a virus in liquor. We used a strain of HCV genotype 1b isolated from a patient with hepatitis C. Then we cleaned and Concentration of the hepatitis virus in solution . At the initial stage of the purification of the culture medium containing the virus was removed from the cell pellet using low-speed centrifugation. To reduce the loss of infectivity and minimize proteolysis of proteins, centrifugation was performed at 4 C. The supernatant was completely removed, and the precipitate was resuspended by adding the virus into each centrifuge cup 1 ml NTE buffer pH 7.4. Then, the contents were combined, and the resulting cup material was further resuspended in a Dounce homogenizer (Dounce Tissue Homogenizers). Then, for destruction of virus aggregates, resulting suspension was sonicated for 1 min at setting Soniprep 150 (Sanyo).

To obtain a viral suspension of high purity, zonal ultracentrifugation method was used in a sucrose density gradient. Two solutions were prepared by sucrose (Sigma-Aldrich, USA) in NTE buffer at pH 7.4 at a concentration of 10 and 50%. For this we used three basic solutions Sucrose with a concentration of 20%, 30, 40%.

The gradient was prepared by laminating successive to each other 50, 40, 30, 20, and 10% sucrose solution in amounts of 6 ml each tube to nitrocellulose followed by diffusion at 4 C for 12 h. The solution containing the virus was layered gently onto the surface gradient in the volume of 1 ml in each tube. We carried out the study of the dynamics of change in the intensity of luminescence. To do this, we utilized the laser, with the power being two times lower than the SBS phenomena threshold.

We continuously measured the spectral characteristics by the analyzer. The reference time started after the cuvette with a given concentration of the bacteria was placed in the optical path. The saved spectra retained their timing marks. We found that frequency of mode has a time drift (**Figure 3**).We observed two peaks in 1–2 s; then both peaks merged into one, and after 3–4 s, the luminescence disappeared.

#### *2.3.2. Spectra of viruses*

**2.3. Investigation of luminescence in the water solution**

11 *Compylobacter jejuni* **Fungus and parasites**

**Viruses Bacteria**

cells/ml) concentration, and minimum concentration (10<sup>1</sup>

Sucrose with a concentration of 20%, 30, 40%.

We studied the luminescence of the hepatitis C virus. For this we used a source with a power below the threshold. The virus was diluted in the different media (physiology liquor, water, alcohol) in proportions of the greatest possible (10<sup>10</sup> cells/ml) concentration, intermediate (10<sup>5</sup>

1 Herpes simplex virus 17 *Mycobacterium tuberculosis avium* 2 Herpes zoster virus 18 *Mycobacterium tuberculosis bovis*

9 Hepatitis D virus 25 Streptococcus ß-hemolytischer 10 AIDS virus 26 *Streptococcus pneumoniae* **Bacteria** 27 *Streptococcus viridans*

 Herpes genitaler virus 19 *Mycoplasma hominis* Epstein-Barr virus 20 *Neisseria gonorrhoeae* Cytomegalovirus 21 *Neisseria meningitidis* Hepatitis A virus 22 *Peptostreptococcus anaerobius*

180 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

7 Hepatitis B virus 23 *Proteus mirabilis* 8 Hepatitis C virus (genotype 1B) 24 *Staphylococcus aureus*

12 *Chlamydia psittaci* 28 *Candida albicans*

13 *Chlamydia trachomatis* 29 *Gardia lamblia* intestinalis 14 *Enterococcus* 30 *Plasmodium malariae* 15 *Helicobacter pylori* 31 *Trichomonas vaginalis*

used a strain of HCV genotype 1b isolated from a patient with hepatitis C. Then we cleaned and Concentration of the hepatitis virus in solution . At the initial stage of the purification of the culture medium containing the virus was removed from the cell pellet using low-speed centrifugation. To reduce the loss of infectivity and minimize proteolysis of proteins, centrifugation was performed at 4 C. The supernatant was completely removed, and the precipitate was resuspended by adding the virus into each centrifuge cup 1 ml NTE buffer pH 7.4. Then, the contents were combined, and the resulting cup material was further resuspended in a Dounce homogenizer (Dounce Tissue Homogenizers). Then, for destruction of virus aggre-

gates, resulting suspension was sonicated for 1 min at setting Soniprep 150 (Sanyo).

To obtain a viral suspension of high purity, zonal ultracentrifugation method was used in a sucrose density gradient. Two solutions were prepared by sucrose (Sigma-Aldrich, USA) in NTE buffer at pH 7.4 at a concentration of 10 and 50%. For this we used three basic solutions

cells/ml) of a virus in liquor. We

*2.3.1. Description of the experiment*

16 *Mycobacterium tuberculosis* hominis

**Table 3.** Pathogens included in the experiment.

We investigated scattering, arising at the passage of laser radiation through colloidal solution (water, alcohol, physical solution) and fine-dispersed phase. We studied transmission spectra of a substance containing DNA: solutions of viruses and microorganisms.

Two shifted and one unshifted frequency components relative to laser frequency were detected in these spectra. The center of our attention was placed on the shifted component study, due to the fact that the analysis of processes in the fine-dispersed phase information is simple. We observed that the shifted components occur as in the Stokes and anti-Stokes ranges. On our opinion spectra of some samples under study are the spectra of Brillouin scattering as the distance between the central and the shift components does not depend on the excitation source wavelength. In other spectra we observed the spectral distributions of induced super fluorescence in liquids. In this case, the intensity of the shifted spectral distribution was higher than the intensity of the spectral distribution of laser radiation by several orders of magnitude, and the width of the spectral peak was decreased sharply. In both cases the width of the Lorentzian component, selected from such shifted spectral distribution, is proportional to the size of the particles, even if the solution contains impurities mainly proteinaceous.

**Figure 3.** Intensity of stimulated luminescence of hepatitis C in the physiological solution from time (a) 30 s, (b) 1 min, and (c) 2 min.

We find out that the peak intensity of shifted distribution does not always have the logarithmic dependence on composition (**Figure 4**). We created autocorrelation functions of such dependencies. Our investigations showed the occurrence of super luminescence mainly in solutions containing DNA or fluorescent markers. Brillouin scattering was observed in solutions containing low concentrations of particles, including nanoscale.

respectively, of 1:3, 1:1, and 3:1. We also used mixes of *E. coli* and latex particles with the same

The spectra of bacteria, unlike viruses [4], often have several maxima (**Figure 4**). However, as a rule, only one peak has an intensity maximum at the same frequency in different solutions, such as water or physiological solution. And, the position of only one peak depends on the

The logarithm of the intensity at that frequency is a linear function of the concentration up to a

depend on a power of IR source (**Figure 5**). At the same time, for the developed method, the difference between the frequencies (wavelengths) of the maximum of the Lorenz component of a laser mode and the maximum of the Lorenz component of a Stokes component was found to be an informative parameter [1]. However, the Stokes component exists no more than 20 min in the spectrum. Obviously, pathogen DNA will be destroyed during this time. For a practical application, we can use the difference between the frequency maximum of the laser mode and the

its respective peak appeared in the anti-Stokes region. We examined the forward-scattering spectra for the mixes containing two or more pathogens (**Figure 6**). The average difference between wavelength of the peak maximum of the laser mode and the peak of Stokes components was equal to 1 ± 0.03 nm, which considerably exceeded the spectral resolvability of the

To do this, we utilized the laser, with the power being two times lower than the SBS phenomena threshold. We continuously measured the spectral characteristics by the analyzer. The reference time started after the cuvette with a given concentration of the bacteria was placed in the optical path. The saved spectra retained their timing marks. We found that frequency of mode has a time drift. This process was continued until the intensity of the Stokes component had been reduced to the level of noise. Obviously, this effect was caused by DNA damage. It has been found that Stokes component appeared much earlier, within 25–30 seconds after the start

centrations. We believe that at low concentrations the scattering of a signal delay is caused by a

**Figure 5.** Example of distributions of the logarithm of the spectral intensity (dBm) for *B. subtilis var. niger*: (a) concentration

cells/ml.

to 106

, the signal existed longer than in the case of higher con-

cells/ml. In the case of low

frequency maximum of the fundamental mode of the bacteria (**Figure 6**) exceeded 10<sup>2</sup>

device. The signal from pathogen organism was observed in the limited time interval.

of cells/ml. However, the intensity is not an informative parameter, because it will

Optical Express Methods of Monitoring of Pathogens in Drinking Water and Water-Based...

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cells/ml;

183

concentrations and the same respective ratio concentration.

of irradiation of the object, at moderate concentrations of 10<sup>4</sup>

cells/ml and (b) bacteriophage (coliphage) concentration of 10<sup>3</sup>

solutions with concentrations, up to 106

concentration.

level of 105

of 103

#### *2.3.3. Spectra of bacteria*

We studied the inelastic scattering spectra from water solutions, containing *Escherichia coli* with concentration from 10 cells/ml to 109 cells/ml, as well as from a mix of *E*. *coli*, *B. subtilis var. niger*, *Shigella flexneri*, and the latex beads. The strains of *E. coli*, *B. subtilis var. niger*, and *Sh. flexneri* were obtained from the Museum of the State Scientific Center of Applied Microbiology and Biotechnology .The bacterial cultures were grown at 37°C for 48 h under aerobic conditions on a solid nutrient media (agar Hottinger, pH 7.2). Suspensions with bacterial cells (10<sup>9</sup> cells/ml) were prepared in 2 ml of 0.9% NaCl solution by using industry standard samples with 10-unit turbidity. Then, suspensions were diluted with distilled water to final concentrations ranging from 1 × 10<sup>8</sup> to 1 × 102 cells/ml. Every sample was tested by placing 0.1 ml of bacterial suspension into the appropriate solid nutrient medium. And then, the number of colonies was calculated after 48 h incubation period at 37 ± 1°C. At least 30–50 colonies must be growing on the agar medium. The suspensions of bacterial cell (*B. subtilis var. niger* and *Shigella flexneri*) were prepared for the analysis as 2 ml of 0.9% NaCl samples with the concentrations of 105 and 103 cells/ml. For assessing the sensitivity, we used bacterial suspensions of live microorganisms with seven concentrations —1 × 10<sup>2</sup> , 1 × 103 , 1 × 104 , 1 × 105 , 1 × 106 , 1 × 107 , 1 × 10<sup>8</sup> cells/ml—and the suspensions of *E*. *coli* microorganisms with the same concentrations but inactivated for 30 min at 1000C. For assessing the specificity, we used the mixes of *E. coli* and *Sh. flexneri* microorganisms with the concentration of 1×104 cells/ml and with the ratios,

**Figure 4.** Example of distributions of the logarithm of the spectral intensity (dBm) of different viruses: (a) laser, (b) mixture grippe A+B, (c) herpes 1, and (d) herpes II.

respectively, of 1:3, 1:1, and 3:1. We also used mixes of *E. coli* and latex particles with the same concentrations and the same respective ratio concentration.

We find out that the peak intensity of shifted distribution does not always have the logarithmic dependence on composition (**Figure 4**). We created autocorrelation functions of such dependencies. Our investigations showed the occurrence of super luminescence mainly in solutions containing DNA or fluorescent markers. Brillouin scattering was observed in solu-

We studied the inelastic scattering spectra from water solutions, containing *Escherichia coli*

*var. niger*, *Shigella flexneri*, and the latex beads. The strains of *E. coli*, *B. subtilis var. niger*, and *Sh. flexneri* were obtained from the Museum of the State Scientific Center of Applied Microbiology and Biotechnology .The bacterial cultures were grown at 37°C for 48 h under aerobic conditions on a solid nutrient media (agar Hottinger, pH 7.2). Suspensions with bacterial cells (10<sup>9</sup> cells/ml) were prepared in 2 ml of 0.9% NaCl solution by using industry standard samples with 10-unit turbidity. Then, suspensions were diluted with distilled water to final concentra-

bacterial suspension into the appropriate solid nutrient medium. And then, the number of colonies was calculated after 48 h incubation period at 37 ± 1°C. At least 30–50 colonies must be growing on the agar medium. The suspensions of bacterial cell (*B. subtilis var. niger* and *Shigella flexneri*) were prepared for the analysis as 2 ml of 0.9% NaCl samples with the concen-

 cells/ml—and the suspensions of *E*. *coli* microorganisms with the same concentrations but inactivated for 30 min at 1000C. For assessing the specificity, we used the mixes of *E. coli*

**Figure 4.** Example of distributions of the logarithm of the spectral intensity (dBm) of different viruses: (a) laser, (b)

cells/ml, as well as from a mix of *E*. *coli*, *B. subtilis* 

cells/ml. Every sample was tested by placing 0.1 ml of

, 1 × 104

, 1 × 105

, 1 × 106

cells/ml and with the ratios,

, 1 × 107 ,

cells/ml. For assessing the sensitivity, we used bacterial suspensions of

, 1 × 103

tions containing low concentrations of particles, including nanoscale.

182 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

to 1 × 102

live microorganisms with seven concentrations —1 × 10<sup>2</sup>

and *Sh. flexneri* microorganisms with the concentration of 1×104

*2.3.3. Spectra of bacteria*

tions ranging from 1 × 10<sup>8</sup>

and 103

mixture grippe A+B, (c) herpes 1, and (d) herpes II.

trations of 105

1 × 10<sup>8</sup>

with concentration from 10 cells/ml to 109

The spectra of bacteria, unlike viruses [4], often have several maxima (**Figure 4**). However, as a rule, only one peak has an intensity maximum at the same frequency in different solutions, such as water or physiological solution. And, the position of only one peak depends on the concentration.

The logarithm of the intensity at that frequency is a linear function of the concentration up to a level of 105 of cells/ml. However, the intensity is not an informative parameter, because it will depend on a power of IR source (**Figure 5**). At the same time, for the developed method, the difference between the frequencies (wavelengths) of the maximum of the Lorenz component of a laser mode and the maximum of the Lorenz component of a Stokes component was found to be an informative parameter [1]. However, the Stokes component exists no more than 20 min in the spectrum. Obviously, pathogen DNA will be destroyed during this time. For a practical application, we can use the difference between the frequency maximum of the laser mode and the frequency maximum of the fundamental mode of the bacteria (**Figure 6**) exceeded 10<sup>2</sup> cells/ml; its respective peak appeared in the anti-Stokes region. We examined the forward-scattering spectra for the mixes containing two or more pathogens (**Figure 6**). The average difference between wavelength of the peak maximum of the laser mode and the peak of Stokes components was equal to 1 ± 0.03 nm, which considerably exceeded the spectral resolvability of the device. The signal from pathogen organism was observed in the limited time interval.

To do this, we utilized the laser, with the power being two times lower than the SBS phenomena threshold. We continuously measured the spectral characteristics by the analyzer. The reference time started after the cuvette with a given concentration of the bacteria was placed in the optical path. The saved spectra retained their timing marks. We found that frequency of mode has a time drift. This process was continued until the intensity of the Stokes component had been reduced to the level of noise. Obviously, this effect was caused by DNA damage. It has been found that Stokes component appeared much earlier, within 25–30 seconds after the start of irradiation of the object, at moderate concentrations of 10<sup>4</sup> to 106 cells/ml. In the case of low solutions with concentrations, up to 106 , the signal existed longer than in the case of higher concentrations. We believe that at low concentrations the scattering of a signal delay is caused by a

**Figure 5.** Example of distributions of the logarithm of the spectral intensity (dBm) for *B. subtilis var. niger*: (a) concentration of 103 cells/ml and (b) bacteriophage (coliphage) concentration of 10<sup>3</sup> cells/ml.

spectra for solutions containing live bacteria and dead cells. The analysis of these dynamics demonstrated that dead cells do not produce luminescence. The peak of a Stokes component of dead cell is formed by the energy transfer from the laser mode. Central peak frequency of a Stokes component at a concentrations above 102 cells/ml is displaced into the anti-Stokes region (1010 nm), which utilized laser with λ1 = 1017 nm. On the basis of these results, the method and the corresponding device have been developed. That device was

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185

Accurate determination of live and dead bacteria is important in many microbiology applications. Traditionally, viability in bacteria is synonymous with the ability to form colonies on solid growth medium. Alternative methods, such as fluorescent in situ hybridization (FISH); nucleic acid amplification techniques such as real-time quantitative PCR (RT-qPCR or qPCR), reverse transcriptase PCR (RT-PCR), and propidium monoazide-PCR cannot be used for continuous monitoring, as they require complex sample preparation [9]. These traditional methods are time-consuming, and they do not provide real-time results or timely information required in the industrial applications. We have developed a method of detection of live and dead cells of pathogenic organisms. Separately, we have studied the luminescence of live and dead cells. To do this, we took the spectral distribution at a pump power slightly below the threshold. Linear relationship between intensity and the concentration for luminescence was not observed.

We conducted studies that determine the difference in spectra between live and dead pathogens, and detoxification of organism suspensions of microorganisms was disinfected by boiling for 30 min. We have found that the dead cells do not produce luminescence. We researched the difference in time dynamics of appearing spectral line for live and dead pathogens. In the first 2 or 3 min, we did not observe Stokes component for dead cells (**Figure 9**). Over time, the peak in the Stokes region appeared, and the intensity of the laser mode decreased (**Figures 8** and **10**). The peak of the Stokes component is a result of energy transfer from the laser mode. The fall of the laser intensity modes was observed in lasers with both employed wavelengths: λ2 = 840 nm and λ1 = 1017 nm. However, in the second case, the drop in intensity was stronger, and the frequency maximum of the second mode was observed in anti-Stokes region (1010 nm). Due to this study, it was possible to identify live and dead *E. coli* cells in the solu-

At the present time, the areas under the transgenic crops are being increased all over the world. Thus, 60% of all the areas allocated for cultivation of soy are occupied by the plants containing the transgenic lines of this product. Genetically modified organisms are the live organisms, which due to introduction of alien genes acquired new phenotypic properties. Usage of living modified organisms (viable crops) in the territory of the Russian Federation is forbidden by the law, and only import of the vegetative raw materials processed in appropri-

tions with previously undetected pathogenic components.

ate way is permitted

**2.5. Spectroscopic methods for monitoring genetically modified products**

tested on a water supply pipeline.

**2.4. Detection of live and dead bacteria**

**Figure 6.** The logarithm of the intensity of the laser mode (1) and Stokes component (2) vs. laser exposure time for the living *E. coli*: laser with λ2 = 810 nm; log of intensity Stokes component (2); – concentration 103 cells/ml; – concentration live 105 cells/ml; – concentration live 107 cells/ml; log of intensity laser mode (1) in the presence of *E. coli*; – concentration live 103 cells/ml; – concentration live 107 cells/ml.

nonuniform distribution of contaminated DNA in the volume of a tested specimen. The rapid decay indicates that fewer cells are rapidly destroyed by the action of coherent radiation. In the case of extremely high concentrations, we believe that the increase in the threshold in a signal generation was due to an increase in the required power of the exciting radiation of the pump. Most likely, that was because the destroyed cells were also absorbing the stimulating radiation, but they were not contributing to the formation of resonance scattering. For more details, please refer to [7].

Spectra from suspensions of the mix of pathogens with the latex beads have two Stokes components. One of them appears without delay in time, and its intensity was independent of the concentration of the latex beads. The intensity of bead component has several orders of magnitude lower than one of the pathogens. However, we did not detect the anti-Stokes component of the peaks corresponding to the dispersion of latex beads. In this case, we did not observe any change in the magnitude of this component in time.

To identify the main informative parameters of the method, the experimental samples were statistically processed. Initial comparative analysis was based on Student's t-test [6].

We developed a proprietary software for the diagnostics of the pathogens [8]. The informative parameters for diagnostics were (a) the reference spectra previously registered in the database and preprocessed based on statistical analysis of the spectral distribution of the pathogen; (b) the difference between the lengths of the laser mode high waves and Stokes (anti-Stokes component); and (c) the number of peaks in the distribution of the Stokes spectrum. Statistical analysis indicated that the above algorithm of recognition is capable of detecting the presence of bacteria spectrum with 0.95 probability [6, 9]. The pathogen monitoring method has developed. We obtained a large amount of experimental data and conducted statistical analysis aiming to determine the probability of detection of spectra by Student's t-test. We have studied the dynamics of the spectra for a number of direct dispersion of bacteria. In addition, we have studied the dynamics of changes in the spectra for solutions containing live bacteria and dead cells. The analysis of these dynamics demonstrated that dead cells do not produce luminescence. The peak of a Stokes component of dead cell is formed by the energy transfer from the laser mode. Central peak frequency of a Stokes component at a concentrations above 102 cells/ml is displaced into the anti-Stokes region (1010 nm), which utilized laser with λ1 = 1017 nm. On the basis of these results, the method and the corresponding device have been developed. That device was tested on a water supply pipeline.

#### **2.4. Detection of live and dead bacteria**

nonuniform distribution of contaminated DNA in the volume of a tested specimen. The rapid decay indicates that fewer cells are rapidly destroyed by the action of coherent radiation. In the case of extremely high concentrations, we believe that the increase in the threshold in a signal generation was due to an increase in the required power of the exciting radiation of the pump. Most likely, that was because the destroyed cells were also absorbing the stimulating radiation, but they were not contributing to the formation of resonance scattering. For more details, please

**Figure 6.** The logarithm of the intensity of the laser mode (1) and Stokes component (2) vs. laser exposure time for the

cells/ml; log of intensity laser mode (1) in the presence of *E. coli*; – concentration

cells/ml; – concentration

living *E. coli*: laser with λ2 = 810 nm; log of intensity Stokes component (2); – concentration 103

184 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

cells/ml.

Spectra from suspensions of the mix of pathogens with the latex beads have two Stokes components. One of them appears without delay in time, and its intensity was independent of the concentration of the latex beads. The intensity of bead component has several orders of magnitude lower than one of the pathogens. However, we did not detect the anti-Stokes component of the peaks corresponding to the dispersion of latex beads. In this case, we did not

To identify the main informative parameters of the method, the experimental samples were

We developed a proprietary software for the diagnostics of the pathogens [8]. The informative parameters for diagnostics were (a) the reference spectra previously registered in the database and preprocessed based on statistical analysis of the spectral distribution of the pathogen; (b) the difference between the lengths of the laser mode high waves and Stokes (anti-Stokes component); and (c) the number of peaks in the distribution of the Stokes spectrum. Statistical analysis indicated that the above algorithm of recognition is capable of detecting the presence of bacteria spectrum with 0.95 probability [6, 9]. The pathogen monitoring method has developed. We obtained a large amount of experimental data and conducted statistical analysis aiming to determine the probability of detection of spectra by Student's t-test. We have studied the dynamics of the spectra for a number of direct dispersion of bacteria. In addition, we have studied the dynamics of changes in the

statistically processed. Initial comparative analysis was based on Student's t-test [6].

observe any change in the magnitude of this component in time.

refer to [7].

live 105

live 103

cells/ml; – concentration live 107

cells/ml; – concentration live 107

Accurate determination of live and dead bacteria is important in many microbiology applications. Traditionally, viability in bacteria is synonymous with the ability to form colonies on solid growth medium. Alternative methods, such as fluorescent in situ hybridization (FISH); nucleic acid amplification techniques such as real-time quantitative PCR (RT-qPCR or qPCR), reverse transcriptase PCR (RT-PCR), and propidium monoazide-PCR cannot be used for continuous monitoring, as they require complex sample preparation [9]. These traditional methods are time-consuming, and they do not provide real-time results or timely information required in the industrial applications. We have developed a method of detection of live and dead cells of pathogenic organisms. Separately, we have studied the luminescence of live and dead cells. To do this, we took the spectral distribution at a pump power slightly below the threshold. Linear relationship between intensity and the concentration for luminescence was not observed.

We conducted studies that determine the difference in spectra between live and dead pathogens, and detoxification of organism suspensions of microorganisms was disinfected by boiling for 30 min. We have found that the dead cells do not produce luminescence. We researched the difference in time dynamics of appearing spectral line for live and dead pathogens. In the first 2 or 3 min, we did not observe Stokes component for dead cells (**Figure 9**). Over time, the peak in the Stokes region appeared, and the intensity of the laser mode decreased (**Figures 8** and **10**). The peak of the Stokes component is a result of energy transfer from the laser mode. The fall of the laser intensity modes was observed in lasers with both employed wavelengths: λ2 = 840 nm and λ1 = 1017 nm. However, in the second case, the drop in intensity was stronger, and the frequency maximum of the second mode was observed in anti-Stokes region (1010 nm). Due to this study, it was possible to identify live and dead *E. coli* cells in the solutions with previously undetected pathogenic components.

#### **2.5. Spectroscopic methods for monitoring genetically modified products**

At the present time, the areas under the transgenic crops are being increased all over the world. Thus, 60% of all the areas allocated for cultivation of soy are occupied by the plants containing the transgenic lines of this product. Genetically modified organisms are the live organisms, which due to introduction of alien genes acquired new phenotypic properties. Usage of living modified organisms (viable crops) in the territory of the Russian Federation is forbidden by the law, and only import of the vegetative raw materials processed in appropriate way is permitted

Today, the most effective control method is the method of PCR (polymerase chain reaction), allowing not only to detect the presence of GMO in products but also to determine their quantity. Medical practice widely employs invasive methods of infectious disease detection based on the analysis of blood samples with the help of polymerase chain reaction and immuneenzyme analysis (IEA). The use of these methods for express diagnostics in mass screening implies a number of problems: the need of a special laboratory equipment according to sanitary and epidemiological standards, expensive test systems, and time that is necessary to get results (from several hours to several days). These factors hamper rapid epidemiological interventions in case of spreading infections as viral hepatitis and immunodeficiency virus and call for updating of the present and development of new diagnostic methods. Improvement of express diagnostics includes possibility to use the method in field conditions, its economical efficiency, high capacity, and quick results.

depends on the composition of the primers and is usually 4–5°C below the temperature of their fusion. Duration of the stage is 0.5–2 min. A wrong choice of a temperature results either in a bad linkage of the primers with a matrix (if the temperature is too high) or in a linkage in

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187

By using a primer, the DNA polymerase makes a copy of a matrix chain. This is a stage of elongation. The polymerase begins a synthesis of the second chain from the primer's 3′-end, which has bonded a matrix, and moves along the matrix in the direction from 3′ to 5′. The temperature of the elongation depends on a polymerase. The frequently used polymerases Taq and Pfu are most active at 72°C. The period of elongation depends on both the kind of DNA polymerase and on the length of an amplificated fragment. Usually, the period of elongation is accepted as equal to 1 min per each thousand of base pairs. When all the cycles are finished, an additional stage of the final elongation is often done to complete the construction of all the one-chained fragments. This stage lasts 7–10 min. The quantity of the specific product of the reaction (limited by primers) theoretically increases proportionally to 2n, where n is the

DNA markers are based on PCR primers. For real-time analysis, special DNA amplifiers with an optical unit are used to detect fluorescence within the reaction tube during the reaction. For real-time analysis, special DNA amplifiers with an optical unit are used to detect fluorescence within the reaction tube during the reaction [7]. These amplifiers are known as markers. Now, carrying out of an express analysis is essentially impossible, because a preliminary extraction of a DNA of an investigated product is necessary. This can be done only in the conditions of a special laboratory. This is due to the fact that, if the lab ware or reactants are polluted by the products of the other reactions or other protein molecules, the results will be incorrect. Special premises are necessary for cleaning of the nucleic acids and for the analysis of the products of amplification. Considerable time is also necessary for a periodic heating of the tests. If the latter problem is solved, the division of DNA and cleaning of the nucleic acids essentially do not allow us to develop an express analyzer working directly with a product.

This experiment was conducted in order to develop a prototype of an express analyzer of a genetically modified soy. To do so, a certain number of problems are needed to be solved such as ensuring optical control of the parameters of soy in a multicomponent environment containing parasitic parameters of DNA, optimizing heat exchange system in a real-time mode, and developing an optical scheme for reading of the informative parameters and a signal processing system with account of the errors. With the above aims in mind, we undertook a research of the markers applied for carrying out of the reaction. In order to obtain a stimulated luminescence, the light-emitting diode pumping was replaced with a laser pumping, which resulted in an increase of the marker's own luminescence by more than an order. We replaced the lamp in the optical unit with a laser in order to reduce noise. The equipment for removing and analyzing the spectral characteristics and spectra of the inelastic scattering of marker are shown in **Figure 7**. We modified the experimental setup described in Part 2 a little.

a wrong place and appearance of nonspecific products (if the temperature is too low).

number of reaction cycles.

*2.5.2. Description of the experiment*

Unfortunately, the PCR method of analysis has its drawbacks. It requires difficult, long preparation of tests, expensive equipment, and specially equipped premises. All this does not allow us to use the PCR method for a mass express screening of products.

#### *2.5.1. Overview on PCR method*

Polymerase chain reaction (PCR) is an experimental method of the molecular biology, allowing us to achieve a substantial growth of the small concentrations of certain fragments of nucleic acids (DNA) in a biological material (test). The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase, which the method is named after, are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. PCR is not generally considered to be a recombinant DNA method, as it does not involve cutting and pasting DNA, only amplification of existing sequences.

In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered, and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions. PCR amplifies a specific region of a DNA strand (the DNA target). The PCR was carried out in an amplifier, a device, which ensures a periodic cooling and heating of the test tubes, usually with the accuracy not less than 0.1°C. Modern amplifiers allow us to set complex programs, including with a possibility of "a hot start." For real-time PCR, special devices are produced equipped with a fluorescent detector.

When chains are disengaged, the temperature is lowered, so that the primers could contact a one-chained matrix. This stage is called hybridization. The temperature of the hybridization depends on the composition of the primers and is usually 4–5°C below the temperature of their fusion. Duration of the stage is 0.5–2 min. A wrong choice of a temperature results either in a bad linkage of the primers with a matrix (if the temperature is too high) or in a linkage in a wrong place and appearance of nonspecific products (if the temperature is too low).

By using a primer, the DNA polymerase makes a copy of a matrix chain. This is a stage of elongation. The polymerase begins a synthesis of the second chain from the primer's 3′-end, which has bonded a matrix, and moves along the matrix in the direction from 3′ to 5′. The temperature of the elongation depends on a polymerase. The frequently used polymerases Taq and Pfu are most active at 72°C. The period of elongation depends on both the kind of DNA polymerase and on the length of an amplificated fragment. Usually, the period of elongation is accepted as equal to 1 min per each thousand of base pairs. When all the cycles are finished, an additional stage of the final elongation is often done to complete the construction of all the one-chained fragments. This stage lasts 7–10 min. The quantity of the specific product of the reaction (limited by primers) theoretically increases proportionally to 2n, where n is the number of reaction cycles.

DNA markers are based on PCR primers. For real-time analysis, special DNA amplifiers with an optical unit are used to detect fluorescence within the reaction tube during the reaction. For real-time analysis, special DNA amplifiers with an optical unit are used to detect fluorescence within the reaction tube during the reaction [7]. These amplifiers are known as markers.

Now, carrying out of an express analysis is essentially impossible, because a preliminary extraction of a DNA of an investigated product is necessary. This can be done only in the conditions of a special laboratory. This is due to the fact that, if the lab ware or reactants are polluted by the products of the other reactions or other protein molecules, the results will be incorrect. Special premises are necessary for cleaning of the nucleic acids and for the analysis of the products of amplification. Considerable time is also necessary for a periodic heating of the tests. If the latter problem is solved, the division of DNA and cleaning of the nucleic acids essentially do not allow us to develop an express analyzer working directly with a product.

#### *2.5.2. Description of the experiment*

Today, the most effective control method is the method of PCR (polymerase chain reaction), allowing not only to detect the presence of GMO in products but also to determine their quantity. Medical practice widely employs invasive methods of infectious disease detection based on the analysis of blood samples with the help of polymerase chain reaction and immuneenzyme analysis (IEA). The use of these methods for express diagnostics in mass screening implies a number of problems: the need of a special laboratory equipment according to sanitary and epidemiological standards, expensive test systems, and time that is necessary to get results (from several hours to several days). These factors hamper rapid epidemiological interventions in case of spreading infections as viral hepatitis and immunodeficiency virus and call for updating of the present and development of new diagnostic methods. Improvement of express diagnostics includes possibility to use the method in field conditions, its economical

Unfortunately, the PCR method of analysis has its drawbacks. It requires difficult, long preparation of tests, expensive equipment, and specially equipped premises. All this does not allow

Polymerase chain reaction (PCR) is an experimental method of the molecular biology, allowing us to achieve a substantial growth of the small concentrations of certain fragments of nucleic acids (DNA) in a biological material (test). The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase, which the method is named after, are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. PCR is not generally considered to be a recombinant DNA method, as it does not involve cutting and pasting DNA, only amplification of existing

In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered, and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions. PCR amplifies a specific region of a DNA strand (the DNA target). The PCR was carried out in an amplifier, a device, which ensures a periodic cooling and heating of the test tubes, usually with the accuracy not less than 0.1°C. Modern amplifiers allow us to set complex programs, including with a possibility of "a hot start." For real-time PCR, special devices are produced

When chains are disengaged, the temperature is lowered, so that the primers could contact a one-chained matrix. This stage is called hybridization. The temperature of the hybridization

us to use the PCR method for a mass express screening of products.

186 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

efficiency, high capacity, and quick results.

*2.5.1. Overview on PCR method*

equipped with a fluorescent detector.

sequences.

This experiment was conducted in order to develop a prototype of an express analyzer of a genetically modified soy. To do so, a certain number of problems are needed to be solved such as ensuring optical control of the parameters of soy in a multicomponent environment containing parasitic parameters of DNA, optimizing heat exchange system in a real-time mode, and developing an optical scheme for reading of the informative parameters and a signal processing system with account of the errors. With the above aims in mind, we undertook a research of the markers applied for carrying out of the reaction. In order to obtain a stimulated luminescence, the light-emitting diode pumping was replaced with a laser pumping, which resulted in an increase of the marker's own luminescence by more than an order. We replaced the lamp in the optical unit with a laser in order to reduce noise. The equipment for removing and analyzing the spectral characteristics and spectra of the inelastic scattering of marker are shown in **Figure 7**. We modified the experimental setup described in Part 2 a little. We used an additional laser with a wavelength of 532 nm to enhance luminescence of the marker and achieve the SBS threshold.

As our previous research has shown, there is a protein luminescence (meat, milk) in the visible spectral range. The protein luminescence is absent in the near-IR range radiations. Also, the power of a luminescent marker is insignificant, and it is necessary to carry out rather many PCR reactions. The luminescence of an extraneous protein does not allow us to carry out diagnostics because of an inadmissible signal/noise ratio. Thus, if we analyze the spectrum of the investigated object in the near infrared region, we can confine ourselves to centrifuging the sample for uniform distribution in solution.

Also, spectral distributions during excitation of the markers by two types of lasers, with the wavelength corresponding to the wavelength exciting a marker and the wavelength lying within the range of the registration of the radiation, are obtained. An example of the of spectral intensity distribution (dBm) marker HEX-1030 with laser pumping with an operating wavelength λ3 = 1016nm and laser pumping with an operating wavelengths λ3 = 1016nm and λ4 = 532 nm is shown in **Figure 8a** and **b** accordingly. The research also demonstrated that in the frequency range of about 1.01 microns, a peak of the inelastic scattering appears, proportional to keeping of a marker in a solution. Since luminescence in this area should fall, the reasons for occurrence of this peak are most likely connected with SBS effect. The maximum peak power increases by more than 1 dBm, if we used laser pumping of two lasers. We assumed that the difference in the position of the laser maximum and Stokes intensities in the spectra of the inelastic light scattering in the natural and modified soy is connected with the change of the molecular structure of the genetically modified samples, which in turn leads to a change of the electronic power levels of the biological objects (**Figure 9**).

The maximum peak power increases by more than 1 dBm, if we use laser pumping of two lasers. However, for practice, only one laser can be used. The spectrum analyzer can be replaced with a photodetector, if you control only one component. This allows you to develop cheap devices. We developed an experimental sample of a device using one laser in the near-

= 532 nm or 488 nm, 633 nm λ<sup>1</sup>

= 1016nm.

Optical Express Methods of Monitoring of Pathogens in Drinking Water and Water-Based...

= 674 nm, λ<sup>2</sup>

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= 1016 nm; and (b) laser pumping with

= 810 nm,

189

The spectrometer was replaced by a receiver. We did not isolate soy DNA. We received a soybean mixture with water and centrifuged. Intensity of the inelastic scattering of the natural and genetically modified soy in absolute and relative figures is shown in **Figure 9a, b**. So, we

field region (**Figure 10**).

an operating wavelengths λ<sup>3</sup>

and λ<sup>3</sup>

**Figure 8.** The equipment analyzing the spectral characteristics λ<sup>4</sup>

= 1010 nm HEX-1030 laser with an operating wavelength λ<sup>3</sup>

**Figure 9.** Marker HEX-1030 (a) laser pumping with an operating wavelength λ<sup>3</sup>

=532 nm.

= 1016 nm and λ<sup>4</sup>

Thus, the registration of the position of the maximum and the intensity of the inelastic diffused light in the field of inelastic scattering of light in the field of 850–1100 nm can be used as informative parameters for determination of the genetically modified soy. The spectral distributions of the inelastic scattering can serve as informative parameters for determination of the presence of a genetically modified product.

**Figure 7.** An example of the spectral intensity distributions (dBm); a(2) live *E. coli*; b(3) dead *E. coli*. Arrow "a" shows the peak corresponding to indicate *E. coli* laser mode; arrow "b."

Optical Express Methods of Monitoring of Pathogens in Drinking Water and Water-Based... http://dx.doi.org/10.5772/intechopen.70236 189

We used an additional laser with a wavelength of 532 nm to enhance luminescence of the

As our previous research has shown, there is a protein luminescence (meat, milk) in the visible spectral range. The protein luminescence is absent in the near-IR range radiations. Also, the power of a luminescent marker is insignificant, and it is necessary to carry out rather many PCR reactions. The luminescence of an extraneous protein does not allow us to carry out diagnostics because of an inadmissible signal/noise ratio. Thus, if we analyze the spectrum of the investigated object in the near infrared region, we can confine ourselves to centri-

Also, spectral distributions during excitation of the markers by two types of lasers, with the wavelength corresponding to the wavelength exciting a marker and the wavelength lying within the range of the registration of the radiation, are obtained. An example of the of spectral intensity distribution (dBm) marker HEX-1030 with laser pumping with an operating wavelength λ3 = 1016nm and laser pumping with an operating wavelengths λ3 = 1016nm and λ4 = 532 nm is shown in **Figure 8a** and **b** accordingly. The research also demonstrated that in the frequency range of about 1.01 microns, a peak of the inelastic scattering appears, proportional to keeping of a marker in a solution. Since luminescence in this area should fall, the reasons for occurrence of this peak are most likely connected with SBS effect. The maximum peak power increases by more than 1 dBm, if we used laser pumping of two lasers. We assumed that the difference in the position of the laser maximum and Stokes intensities in the spectra of the inelastic light scattering in the natural and modified soy is connected with the change of the molecular structure of the genetically modified samples, which in turn leads to

Thus, the registration of the position of the maximum and the intensity of the inelastic diffused light in the field of inelastic scattering of light in the field of 850–1100 nm can be used as informative parameters for determination of the genetically modified soy. The spectral distributions of the inelastic scattering can serve as informative parameters for determination

**Figure 7.** An example of the spectral intensity distributions (dBm); a(2) live *E. coli*; b(3) dead *E. coli*. Arrow "a" shows the

a change of the electronic power levels of the biological objects (**Figure 9**).

marker and achieve the SBS threshold.

fuging the sample for uniform distribution in solution.

188 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

of the presence of a genetically modified product.

peak corresponding to indicate *E. coli* laser mode; arrow "b."

**Figure 8.** The equipment analyzing the spectral characteristics λ<sup>4</sup> = 532 nm or 488 nm, 633 nm λ<sup>1</sup> = 674 nm, λ<sup>2</sup> = 810 nm, and λ<sup>3</sup> = 1010 nm HEX-1030 laser with an operating wavelength λ<sup>3</sup> = 1016nm.

The maximum peak power increases by more than 1 dBm, if we use laser pumping of two lasers. However, for practice, only one laser can be used. The spectrum analyzer can be replaced with a photodetector, if you control only one component. This allows you to develop cheap devices. We developed an experimental sample of a device using one laser in the nearfield region (**Figure 10**).

The spectrometer was replaced by a receiver. We did not isolate soy DNA. We received a soybean mixture with water and centrifuged. Intensity of the inelastic scattering of the natural and genetically modified soy in absolute and relative figures is shown in **Figure 9a, b**. So, we

**Figure 9.** Marker HEX-1030 (a) laser pumping with an operating wavelength λ<sup>3</sup> = 1016 nm; and (b) laser pumping with an operating wavelengths λ<sup>3</sup> = 1016 nm and λ<sup>4</sup> =532 nm.

have also established the new method for rapid analysis of genetically modified soy in meat products detected by a modified PCR fluorescent method.

The PCR Stafipol-RV kit is designed to quantitatively detect the in vitro DNA of the causative agent of *Staphylococcus aureus* in a biological material by polymerase chain reaction with a

Optical Express Methods of Monitoring of Pathogens in Drinking Water and Water-Based...

http://dx.doi.org/10.5772/intechopen.70236

191

Another set of reagents is used to quantify the hepatitis C virus (HCV) RNA by polymerase chain reaction (PCR) with hybridization-fluorescent detection in real-time mode "AmpliSens HCV-Monitor-FL." The use of various consumables for different pathogens makes this method very expensive. Therefore, at the present time, the problem of automatic monitoring of characteristics of drinking water directly within a water steam is

Two resonance control techniques could be particularly instrumental in finding solution of that problem—spectroscopy of Raman scattering and stimulated Brillouin scattering. The utilization of Raman scattering spectroscopy technique requires transferring an enormous amount of energy for extracting an informative signal, which is considerably lower than the

Laser-based technology requires no consumables, or reagents are currently being developed [10]. A new method for detection of pathogens in water and water-based solutions is described below. It is based on the diagnostics of nonlinear effects, which comprises two phenomena: an induced luminescence of DNA under the influence of laser radiation and a stimulated Brillouin scattering (SBS) [11]. The following model parameters were selected for the identification of the peak positions of a spectral line corresponding to a pathogen and the difference between two wavelengths corresponding, respectively, to a laser mode maximum

We studied transmission IR spectra of a number of pathogen (salmonella, viruses of herpes genitaler, hepatitis A and C, grippe A and B) solutions and luminescence of nanomarkers. In our experiments, the laser radiation passed through a quartz cell with water solutions of the pathogens, nanosilver, or nanomarkers. We found that exciting radiations with wavelengths 1017 and 810 nm induce the stimulated Brillouin scattering (SBS) in spectra of the water containing DNA. We believe that the power threshold for the onset of this effect is achieved by

The new method, the express analysis of pathogens in water, was developed. It shall be mentioned that the proposed method of express diagnostics allows detection of infectious agents in the water in minutes based on nonlinear effects. This research has, hopefully, laid the foundation for development of a prototype for determination of the content of the genetically modified soy in meat products. The inventive methods can be recommended for DNA diagnostics in medicine ,veterinary sciences and in sanitation . The main advantage of this method is that there is no need for DNA isolation. It is sufficient to create a suspension of the product

fluorescent detection of the result in real time.

and to a peak of a spectral line of a pathogen.

adding a forced radiation luminescence and laser radiation.

particularly acute.

**4. Conclusions**

by centrifugation.

noise [3].

#### *2.5.3. Comparison of standard PCR method and a modified PCR method*

**Table 4** compares the two methods. Analysis shows that our method can be the basis of devices working outside the laboratory, directly in shops and cafes.

**Figure 10.** Distribution of signals in the presence of genetically modified soybean: (1) laser signal and (2) marker signal.


**Table 4.** Comparison of standard and modified PCR methods.

## **3. Discussions**

Traditional methods for detection of microorganisms are based on the enumeration of bacterial cells after their cultivation on a nutrient media. This method is sensitive, inexpensive, and simple, but it takes at least several days for completion.

Two known methods for reducing the detection time—immune assay and polymerase chain reaction—are complicated. In addition to the fact that this method requires the isolation of DNA, it also uses various sets of reagents to identify and differentiate the tested pathogen. For example, there are the set of reagents for the detection and differentiation of DNA from bacteria of the genus *Shigella* spp. and enteroinvasive *E. coli* (EIEC), *Salmonella* (*Salmonella* spp.), and thermophilic *Campylobacter* spp., modified with specific primers to *B. subtilis var. nigger*). In environmental objects and clinical material by polymerase chain reaction (PCR) with hybridization-fluorescent detection, "AmpliSens® *Shigella* spp. and EIEC/*Salmonella* spp./*Campylobacter* spp.-FL" and "AmpliSens® *Bacillus* anthracis-FRT" are often used in practice.

The PCR Stafipol-RV kit is designed to quantitatively detect the in vitro DNA of the causative agent of *Staphylococcus aureus* in a biological material by polymerase chain reaction with a fluorescent detection of the result in real time.

Another set of reagents is used to quantify the hepatitis C virus (HCV) RNA by polymerase chain reaction (PCR) with hybridization-fluorescent detection in real-time mode "AmpliSens HCV-Monitor-FL." The use of various consumables for different pathogens makes this method very expensive. Therefore, at the present time, the problem of automatic monitoring of characteristics of drinking water directly within a water steam is particularly acute.

Two resonance control techniques could be particularly instrumental in finding solution of that problem—spectroscopy of Raman scattering and stimulated Brillouin scattering. The utilization of Raman scattering spectroscopy technique requires transferring an enormous amount of energy for extracting an informative signal, which is considerably lower than the noise [3].

Laser-based technology requires no consumables, or reagents are currently being developed [10]. A new method for detection of pathogens in water and water-based solutions is described below. It is based on the diagnostics of nonlinear effects, which comprises two phenomena: an induced luminescence of DNA under the influence of laser radiation and a stimulated Brillouin scattering (SBS) [11]. The following model parameters were selected for the identification of the peak positions of a spectral line corresponding to a pathogen and the difference between two wavelengths corresponding, respectively, to a laser mode maximum and to a peak of a spectral line of a pathogen.

We studied transmission IR spectra of a number of pathogen (salmonella, viruses of herpes genitaler, hepatitis A and C, grippe A and B) solutions and luminescence of nanomarkers. In our experiments, the laser radiation passed through a quartz cell with water solutions of the pathogens, nanosilver, or nanomarkers. We found that exciting radiations with wavelengths 1017 and 810 nm induce the stimulated Brillouin scattering (SBS) in spectra of the water containing DNA. We believe that the power threshold for the onset of this effect is achieved by adding a forced radiation luminescence and laser radiation.

## **4. Conclusions**

have also established the new method for rapid analysis of genetically modified soy in meat

**Table 4** compares the two methods. Analysis shows that our method can be the basis of

**Figure 10.** Distribution of signals in the presence of genetically modified soybean: (1) laser signal and (2) marker signal.

Traditional methods for detection of microorganisms are based on the enumeration of bacterial cells after their cultivation on a nutrient media. This method is sensitive, inexpensive, and

Two known methods for reducing the detection time—immune assay and polymerase chain reaction—are complicated. In addition to the fact that this method requires the isolation of DNA, it also uses various sets of reagents to identify and differentiate the tested pathogen. For example, there are the set of reagents for the detection and differentiation of DNA from bacteria of the genus *Shigella* spp. and enteroinvasive *E. coli* (EIEC), *Salmonella* (*Salmonella* spp.), and thermophilic *Campylobacter* spp., modified with specific primers to *B. subtilis var. nigger*). In environmental objects and clinical material by polymerase chain reaction (PCR) with hybridization-fluorescent detection, "AmpliSens® *Shigella* spp. and EIEC/*Salmonella* spp./*Campylobacter* spp.-FL" and

simple, but it takes at least several days for completion.

**Table 4.** Comparison of standard and modified PCR methods.

"AmpliSens® *Bacillus* anthracis-FRT" are often used in practice.

products detected by a modified PCR fluorescent method.

**3. Discussions**

*2.5.3. Comparison of standard PCR method and a modified PCR method*

190 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

devices working outside the laboratory, directly in shops and cafes.

**Standard PCR Modified PCR method**

Reaction time (1 h) Reaction time (1 min)

The need for DNA extraction Diagnosis is made immediately in the sample The need to organize specialized laboratories The need to organize specialized laboratories

> The new method, the express analysis of pathogens in water, was developed. It shall be mentioned that the proposed method of express diagnostics allows detection of infectious agents in the water in minutes based on nonlinear effects. This research has, hopefully, laid the foundation for development of a prototype for determination of the content of the genetically modified soy in meat products. The inventive methods can be recommended for DNA diagnostics in medicine ,veterinary sciences and in sanitation . The main advantage of this method is that there is no need for DNA isolation. It is sufficient to create a suspension of the product by centrifugation.

## **Author details**

Tatiana Moguilnaya\* and Aleksey Sheryshev

\*Address all correspondence to: mogilnay@mail.ru

Moscow Aviation Institute (National Research University), Moscow, Russia

#### **References**

[1] Bioluminescence: Wikipedia. February 2017. https://en.wikipedia.org/wiki/Bioluminescence

**Chapter 11**

**Provisional chapter**

**The Aflatoxin Quicktest™—A Practical Tool for**

**The Aflatoxin Quicktest™—A Practical Tool for** 

DOI: 10.5772/intechopen.71331

Contamination of corn, peanuts, milk and dairy products with aflatoxins is a worldwide problem, particularly in subtropical regions where the climatic conditions are ideal for the growth of *Aspergillus flavus*, the fungi that produces these toxins. Developing countries have major difficulties in marketing these products abroad due to the stringent international regulations concerning this carcinogenic toxin. Adding to the problem is the analytical cost involved in monitoring the produce, which require sophisticated instrumentation and qualified personnel, neither of which are available for field testing. The development of a rapid Aflatoxin Quicktest™ provides an effective, reliable and cheaper option for screening levels of aflatoxin above the regulatory thresholds in such produce. The test consists of a lateral flow device (LFD) coated with antibodies specific to aflatoxin B1, although it detects other aflatoxins (i.e. G and M) with high cross-reactivity. Its high sensitivity allows analysis of these toxins in the range 2–40 μg/kg of sample in 15 minutes, plus the time for extraction, which varies among different products. Quantification of the test results is done using a Quick Reader, by comparing the readings of individual tests against a standard curve of the analytes in the same manner as it is done with any other analytical equipment. A validation study was carried out using peanuts from Australia and peanuts and corn from Timor-Leste to assess the performance of the Aflatoxin Quicktest™. Results obtained with the LFD showed a good correlation with the standard

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

above 0.90 for all cases), indicating

and reproduction in any medium, provided the original work is properly cited.

the Aflatoxin Quicktest™ is capable of measuring levels of aflatoxins accurately and reliably. Given their ease of use, low cost and fast processing time, the Aflatoxin Quicktest™ can be used for screening agricultural produce in countries that cannot afford the costly

**Keywords:** mycotoxins, food, analysis, lateral flow devices, peanuts, maize

**Ensuring Safety in Agricultural Produce**

Robert Williams, Graeme Wright, Ivan R. Kennedy

**Ensuring Safety in Agricultural Produce**

Francisco Sánchez-Bayo, Luis de Almeida,

Francisco Sánchez-Bayo, Luis de Almeida,

Additional information is available at the end of the chapter

analytical measurements by HPLC-fluorescence (r2

alternative of using specialised personnel and equipment.

Additional information is available at the end of the chapter

Robert Williams, Graeme Wright, Ivan R. Kennedy and Angus Crossan

and Angus Crossan

**Abstract**

http://dx.doi.org/10.5772/intechopen.71331


**Chapter 11**

**Provisional chapter**

## **The Aflatoxin Quicktest™—A Practical Tool for Ensuring Safety in Agricultural Produce Ensuring Safety in Agricultural Produce**

**The Aflatoxin Quicktest™—A Practical Tool for** 

DOI: 10.5772/intechopen.71331

Francisco Sánchez-Bayo, Luis de Almeida, Robert Williams, Graeme Wright, Ivan R. Kennedy and Angus Crossan Robert Williams, Graeme Wright, Ivan R. Kennedy and Angus Crossan Additional information is available at the end of the chapter

Francisco Sánchez-Bayo, Luis de Almeida,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71331

#### **Abstract**

**Author details**

**References**

Tatiana Moguilnaya\* and Aleksey Sheryshev

\*Address all correspondence to: mogilnay@mail.ru

service US-AB; 2011. ISBN 978-91-7415-844-1

414;(2013):012023 DOI: 10.1088/1742-6596/414/1/12023

Systems and Devices. 2014;**12**:9-13

2003;**5621**:18-23

pomoch-ov.php

Moscow Aviation Institute (National Research University), Moscow, Russia

192 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

[1] Bioluminescence: Wikipedia. February 2017. https://en.wikipedia.org/wiki/Bioluminescence [2] Starunov VS, Fabelinsky IL. Stimulated Mandelstam-Brillouin scattering and stimulated

[3] Abdelsalam M. Theoretical Studies of Raman Scattering. Stockholm, Sweden: Universitets-

[4] Tomilin VI, Mogilnaya Yu T, Kononenko AB. Research of dynamics of signal detection of the laser device at control of pathogenic microorganisms by the SBS-method. Ecological

[5] Moguilnaya Yu T, Saguitova E, Botikov AG. Instrumentation for non-invasive expressdiagnostics bacteriophages and viruses by optical method. Smart Medical and Biomedical Sensor Technology. Providence, Rhode Island, USA: Proceedings of SPIE;

[6] Tomilin VI, Moguilnaya Yu T. Study of stocksic and antistocksic components of laser device's signal for controlling of drinking water parameters. Vestnik of the Bauman

[7] Moguilnay Yu T, Tomilin VA, Bobkov PV. Monitoring of toxicants by SBS in a turbulent flow of water. International Journal Laser Physics. Journal of Physics: Conference Series

[8] Mogilnaya T, Bobkov P, Toiling V, Agibalov A. Development of devices for rapid analy-

[9] http://www.diss.seluk.ru/m-biologiya/30006763-1-moskva-2012-osnovi-polimeraznoycepnoy-reakcii-pcr-sostavitelya-cel-posobiya-osnovi-polimeraznoy-cepnoy-reakcii-pcr-

[10] The BioSentry™ Offers State-of-the-Art, Laser-Based Technology to Provide Continuous, Online, Real-time Monitoring for Microorganisms in Water Systems. http://www.inter-

[11] Cheglov VA. Can I. Create a Laser on the Genetic Structure? Raman Scattering. // 70 Years

line.nl/media/1000116/biosentry\_wms\_v3.2.pdf [Accessed: January 25, 2017]

of Research. Lebedev Physical Institute. Moscow 2004 year p. 250

sis of the presence of toxins in drinking water. Prybory. 2013(1):45-51

Moscow State Technical University. Instrumentation Engineering. 2015;**N2**:83-91

entropy (temperature) scattering of light. Physics-Uspekhi. 1969;**98**:441-491

Contamination of corn, peanuts, milk and dairy products with aflatoxins is a worldwide problem, particularly in subtropical regions where the climatic conditions are ideal for the growth of *Aspergillus flavus*, the fungi that produces these toxins. Developing countries have major difficulties in marketing these products abroad due to the stringent international regulations concerning this carcinogenic toxin. Adding to the problem is the analytical cost involved in monitoring the produce, which require sophisticated instrumentation and qualified personnel, neither of which are available for field testing. The development of a rapid Aflatoxin Quicktest™ provides an effective, reliable and cheaper option for screening levels of aflatoxin above the regulatory thresholds in such produce. The test consists of a lateral flow device (LFD) coated with antibodies specific to aflatoxin B1, although it detects other aflatoxins (i.e. G and M) with high cross-reactivity. Its high sensitivity allows analysis of these toxins in the range 2–40 μg/kg of sample in 15 minutes, plus the time for extraction, which varies among different products. Quantification of the test results is done using a Quick Reader, by comparing the readings of individual tests against a standard curve of the analytes in the same manner as it is done with any other analytical equipment. A validation study was carried out using peanuts from Australia and peanuts and corn from Timor-Leste to assess the performance of the Aflatoxin Quicktest™. Results obtained with the LFD showed a good correlation with the standard analytical measurements by HPLC-fluorescence (r2 above 0.90 for all cases), indicating the Aflatoxin Quicktest™ is capable of measuring levels of aflatoxins accurately and reliably. Given their ease of use, low cost and fast processing time, the Aflatoxin Quicktest™ can be used for screening agricultural produce in countries that cannot afford the costly alternative of using specialised personnel and equipment.

**Keywords:** mycotoxins, food, analysis, lateral flow devices, peanuts, maize

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1. Introduction**

Aflatoxins constitute a major group of mycotoxins produced by certain fungi, mainly *Aspergillus flavus* and *Aspergillus parasiticus*, which grow in soil, decaying vegetation, hay and grains. These fungi can infect various crops ─ frequently peanuts, maize, other cereals as well as tree nuts ─ either prior to harvest or under moist conditions in stored agricultural produce, leading to their contamination with aflatoxins [1–3]. Contamination levels can sometimes exceed thousands of parts per billion (ppb, either μg/kg or μg/L) in individual kernels of peanuts or other grains, but given the uneven distribution within a batch of produce an intensive, strategic sampling is required to assess the extent of the contamination [4].

Contamination of the food supply with aflatoxins poses a serious problem not only for the health effects it causes in people and livestock, but also for trade. Strict regulations have been enacted by the World Health Organisation to prevent trading aflatoxin contaminated produce among countries. The maximum residue limits (MRLs) in grain for human consumption are in the range 2–20 ppb, and for animal feed in the range 20–300 ppb, depending on the country, whereas for milk they can be as low as 0.05 μg/L [10]. In order to meet the international regulations on aflatoxin, countries have to adopt expensive monitoring programs in order to screen their agricultural produce. Screening to detect aflatoxin contamination often relies on fluorescence and has been achieved by reference standards using thin layer chromatography, adsorption on minicolumns [11], high-performance liquid chromatography (HPLC) or liquid chromatography coupled to mass spectrometry (LC-MS) [12, 13]. Unfortunately, many developing countries lack the infrastructure necessary to comply with such regulations even though they often recognise the problem [14]. For example, aflatoxin has been identified as a threat to human health in Timor-Leste [15], and yet some of their agricultural produce could

The Aflatoxin Quicktest™—A Practical Tool for Ensuring Safety in Agricultural Produce

http://dx.doi.org/10.5772/intechopen.71331

195

Specific antibodies to aflatoxins have provided an alternative means to conduct ELISA immunoassays [16], which can accurately measure the levels of contamination in grains and nuts. ELISA technologies are more affordable than instrumental analysis such as HPLCfluorescence or LC-MS, but require a level of analytical skills that may not be found in many developing countries, where the aflatoxin problem is most prevalent [14, 17]. Based on the same immunoassays principles, lateral flow devices (LFD) employing gold nanoparticles have been recently developed [18–20] to be used as rapid methods for screening aflatoxins in

One such device [19], the Aflatoxin QuickTest™, provides an effective, reliable and low cost option for screening levels of aflatoxins and meets the regulatory thresholds of agricultural produce. Quantification, as described here, is achieved using a suitable reader and standard curve of the analytes in the same manner as it is done with other analytical equipment. A validation study for quantification using peanuts and maize from Australia and Timor-Leste was carried out in order to assess the performance of the Aflatoxin QuickTest™ and it is presented

This chapter reviews the past research that led to the development of the current Aflatoxin QuickTest™, its use and applications. But before describing how it works, a description of this

A polyclonal antibody specific to aflatoxin B1 (AFB1) was developed by Lee et al. [16] using a conjugate of aflatoxin B1-bovine serum albumin (BSA) as antigen. The antibodies were specific to aflatoxin B1, detecting this compound in a mixture of four aflatoxins (B1, B2, G1 and G2),

**2. Development of antibodies for detection of aflatoxin**

not be marketed in recent years for lack of compliance.

food commodities.

here for the first time.

novel technology is required.

**2.1. Antibodies for aflatoxin**

Four main types of aflatoxins are recognised, namely B1, B2, G1 and G2, with aflatoxin B1 and G1 as well as their metabolic product M1 being the most commonly found in agricultural produce (**Figure 1**). In terms of acute toxicity, the oral lethal dose (LD50) of aflatoxin B1 for monkeys is 1.75 mg/kg, and for ducks 1.70–2.45 mg/kg. However, these toxins are also carcinogenic because once ingested they are metabolised by the liver to a reactive epoxide intermediate; as a result, chronic ingestion of small amounts of aflatoxins (i.e. in the ppb range) typically produce liver cancer, so they are classified as strong carcinogens by the International Agency for Research on Cancer (IARC). For example, it has been estimated that 12% of cancer occurrence in Indonesia is in the liver, which is linked to consumption of aflatoxin contaminated food [5]. Livestock animals suffer the same effects, with pigs and chicken being particularly susceptible to these mycotoxins. However, animals can also transform aflatoxins B and G into the M metabolites by hydroxylation in the liver, and these transformation products can appear in eggs as well as in poultry meat [6]. Both aflatoxin M1 and M2 are even more toxic, with LD50 for ducks in the range 0.28–0.32 mg/kg. Ruminant animals fed contaminated fodder are more tolerant, but can also pass M aflatoxins into milk and other dairy products [7–9].

**Figure 1.** Chemical structures of common aflatoxins found in nuts and grains (B1, B2, G1, G2) or milk (M1) as a by-product.

Contamination of the food supply with aflatoxins poses a serious problem not only for the health effects it causes in people and livestock, but also for trade. Strict regulations have been enacted by the World Health Organisation to prevent trading aflatoxin contaminated produce among countries. The maximum residue limits (MRLs) in grain for human consumption are in the range 2–20 ppb, and for animal feed in the range 20–300 ppb, depending on the country, whereas for milk they can be as low as 0.05 μg/L [10]. In order to meet the international regulations on aflatoxin, countries have to adopt expensive monitoring programs in order to screen their agricultural produce. Screening to detect aflatoxin contamination often relies on fluorescence and has been achieved by reference standards using thin layer chromatography, adsorption on minicolumns [11], high-performance liquid chromatography (HPLC) or liquid chromatography coupled to mass spectrometry (LC-MS) [12, 13]. Unfortunately, many developing countries lack the infrastructure necessary to comply with such regulations even though they often recognise the problem [14]. For example, aflatoxin has been identified as a threat to human health in Timor-Leste [15], and yet some of their agricultural produce could not be marketed in recent years for lack of compliance.

Specific antibodies to aflatoxins have provided an alternative means to conduct ELISA immunoassays [16], which can accurately measure the levels of contamination in grains and nuts. ELISA technologies are more affordable than instrumental analysis such as HPLCfluorescence or LC-MS, but require a level of analytical skills that may not be found in many developing countries, where the aflatoxin problem is most prevalent [14, 17]. Based on the same immunoassays principles, lateral flow devices (LFD) employing gold nanoparticles have been recently developed [18–20] to be used as rapid methods for screening aflatoxins in food commodities.

One such device [19], the Aflatoxin QuickTest™, provides an effective, reliable and low cost option for screening levels of aflatoxins and meets the regulatory thresholds of agricultural produce. Quantification, as described here, is achieved using a suitable reader and standard curve of the analytes in the same manner as it is done with other analytical equipment. A validation study for quantification using peanuts and maize from Australia and Timor-Leste was carried out in order to assess the performance of the Aflatoxin QuickTest™ and it is presented here for the first time.

This chapter reviews the past research that led to the development of the current Aflatoxin QuickTest™, its use and applications. But before describing how it works, a description of this novel technology is required.

## **2. Development of antibodies for detection of aflatoxin**

#### **2.1. Antibodies for aflatoxin**

**1. Introduction**

Aflatoxins constitute a major group of mycotoxins produced by certain fungi, mainly *Aspergillus flavus* and *Aspergillus parasiticus*, which grow in soil, decaying vegetation, hay and grains. These fungi can infect various crops ─ frequently peanuts, maize, other cereals as well as tree nuts ─ either prior to harvest or under moist conditions in stored agricultural produce, leading to their contamination with aflatoxins [1–3]. Contamination levels can sometimes exceed thousands of parts per billion (ppb, either μg/kg or μg/L) in individual kernels of peanuts or other grains, but given the uneven distribution within a batch of produce an intensive,

Four main types of aflatoxins are recognised, namely B1, B2, G1 and G2, with aflatoxin B1 and G1 as well as their metabolic product M1 being the most commonly found in agricultural produce (**Figure 1**). In terms of acute toxicity, the oral lethal dose (LD50) of aflatoxin B1 for monkeys is 1.75 mg/kg, and for ducks 1.70–2.45 mg/kg. However, these toxins are also carcinogenic because once ingested they are metabolised by the liver to a reactive epoxide intermediate; as a result, chronic ingestion of small amounts of aflatoxins (i.e. in the ppb range) typically produce liver cancer, so they are classified as strong carcinogens by the International Agency for Research on Cancer (IARC). For example, it has been estimated that 12% of cancer occurrence in Indonesia is in the liver, which is linked to consumption of aflatoxin contaminated food [5]. Livestock animals suffer the same effects, with pigs and chicken being particularly susceptible to these mycotoxins. However, animals can also transform aflatoxins B and G into the M metabolites by hydroxylation in the liver, and these transformation products can appear in eggs as well as in poultry meat [6]. Both aflatoxin M1 and M2 are even more toxic, with LD50 for ducks in the range 0.28–0.32 mg/kg. Ruminant animals fed contaminated fodder are more tolerant, but can also pass M aflatoxins into milk and other dairy products [7–9].

strategic sampling is required to assess the extent of the contamination [4].

194 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

B1 B2 M1

O

O O

O O

OCH3

O

OCH3

O

O

O O

O

O

G1 G2

**Figure 1.** Chemical structures of common aflatoxins found in nuts and grains (B1, B2, G1, G2) or milk (M1) as a by-product.

O

O O

O

O

O O

O O

O

O

OH

O O

<sup>O</sup> OCH3

OCH3

<sup>O</sup> OCH3

A polyclonal antibody specific to aflatoxin B1 (AFB1) was developed by Lee et al. [16] using a conjugate of aflatoxin B1-bovine serum albumin (BSA) as antigen. The antibodies were specific to aflatoxin B1, detecting this compound in a mixture of four aflatoxins (B1, B2, G1 and G2), but showed significant cross-reaction with aflatoxin G1 (57–61%) when an individual compound was tested (**Table 1**). This is fortunate, as both aflatoxin B1 and G1 are the two most common aflatoxins found in contaminated produce. Sensitivity of short competitive ELISA assays (15 minutes) showed median inhibition concentration (IC50) values of 21.6 ± 2.7 ppb after a 5-fold dilution of the sample extract – a necessary step to minimise the negative effect of solvent on the antibodies – and a detection range from 4.2 to 99.9 μg/kg sample. This ELISA was able to detect and quantify levels of aflatoxins in peanut, corn, soybean and pistachio samples without significant matrix effects [16].

nanoparticles conjugated to the specific antibodies of the target analyte (AuNP-IgG); at the other end of the strip there is an absorbent pad (**Figure 2**). The strips are contained within small plastic cassettes that have a well for placing drops of the sample solution and a window to visualise the T and C lines. Conjugates and antibodies on the LFD are usually stable for months when the strips are kept under dry and dark conditions at room temperature, enabling

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Some authors have successfully described LFDs for aflatoxins that work well with corn [18], grains and feedstuffs [25, 26] and milk [27], but require strip treatment before use. A new LFD for aflatoxins that did not require special pre-treatment was developed by Masinde et al. [19] and has been commercialised as the Aflatoxin Quicktest™ by QuickTest Technologies. The T line in the Aflatoxin Quicktest™ contains aflatoxin-conjugate (AFB1-C) and the C line a non-specific goat anti-rabbit antibody (G-IgG); the sample pad contains gold nanoparticles (~10–15 micron) conjugated to the specific antibodies (AuNP-IgG) developed by Lee et al. for

For running a test, two drops of sample extract are placed over the sample pad, dissolving the AuNP-IgG nanoparticles, which run laterally over the strip towards the absorbent pad. Any aflatoxin present in the sample extract will compete with the AuNP-IgG particles at the T line, where the excess conjugated antigen will bind to them and produce a coloured line. The remaining AuNP-IgG particles will continue moving towards the other end and will bind to the G-IgG at the C line, also producing colour. The time for the competing targets, aflatoxin and AuNP-IgG, to reach an equilibrium is about 15 minutes, although 5 minutes may be suf-

The interpretation of the assay is straightforward: an absence of colour at the T line indicates a high concentration of aflatoxins in the sample extract, as it has outcompeted the gold nanoparticles, whereas a full coloured T line indicates the absence of aflatoxins in the sample. A faded line indicates the presence of some aflatoxin in such a way that the less the colour development, the more aflatoxin is present. The coloured C line confirms that the test is valid,

**Figure 2.** Schematic diagram of a lateral flow device (LFD). A nitrocellulose strip is coated with a solution of a particular target analyte (T line) and a general antibody (C line). The sample pad contains gold nanoparticles conjugated to the specific antibodies of the analyte (AuNP-IgG). The absorbent pad at the other end captures the excess solution flowing

that is, when no colour appears at the C line, the test is invalid or the strip is faulty.

easy transport and storage for use at a later time.

aflatoxin (AFB1).

across the strip.

ficient for initial visual detection [19].

Furthermore, a validation of the SUNQuik ELISA, which uses the AFB1 antibodies, was carried out using 12 peanut samples that were also analysed by standard HPLC-fluorescence. Levels of total aflatoxins measured by the two analytical methods showed an excellent correlation (r2 = 0.938) over a concentration range 0–1200 μg/kg sample, with no false negatives [21].

#### **2.2. Lateral flow devices for aflatoxins**

Whilst the ability of the polyclonal AFB1 antibodies to quantify levels of aflatoxins in grains and nuts was demonstrated, the application of ELISA assays to monitoring surveys requires certain laboratory conditions and appropriate skills by qualified personnel. A simpler method was needed that could be used in field settings by less skilled operators.

In recent years, more convenient procedures based on immunochemistry have been sought for organic contaminants in food and the environment. The lateral flow device (LFD) has been most popular because of its simplicity in design and its automatic function, each LFD unit requiring only a few drops of sample solution for operation. Comprehensive reviews [22–24] have described the lateral flow assay system in depth, covering a broad range of immunoassay procedures and including nucleic acid applications. Many successful ELISAs can be readily converted to LFDs using the same or similar immuno-reagents; important properties established for ELISAs such as the plots of concentration giving 50% inhibition (IC50) perform in a similar manner in LFDs. In essence, a competitive LFD consists of an impervious nitrocellulose strip coated transversal with two lines that contain either a particular target analyte (test line, T) or a general antibody (control line, C). In addition, a sample pad contains colloidal gold


**Table 1.** Cross reactivity of aflatoxins and metabolites in polyclonal AFB1 assays (after Lee et al. [16]).

nanoparticles conjugated to the specific antibodies of the target analyte (AuNP-IgG); at the other end of the strip there is an absorbent pad (**Figure 2**). The strips are contained within small plastic cassettes that have a well for placing drops of the sample solution and a window to visualise the T and C lines. Conjugates and antibodies on the LFD are usually stable for months when the strips are kept under dry and dark conditions at room temperature, enabling easy transport and storage for use at a later time.

but showed significant cross-reaction with aflatoxin G1 (57–61%) when an individual compound was tested (**Table 1**). This is fortunate, as both aflatoxin B1 and G1 are the two most common aflatoxins found in contaminated produce. Sensitivity of short competitive ELISA assays (15 minutes) showed median inhibition concentration (IC50) values of 21.6 ± 2.7 ppb after a 5-fold dilution of the sample extract – a necessary step to minimise the negative effect of solvent on the antibodies – and a detection range from 4.2 to 99.9 μg/kg sample. This ELISA was able to detect and quantify levels of aflatoxins in peanut, corn, soybean and pistachio

196 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

Furthermore, a validation of the SUNQuik ELISA, which uses the AFB1 antibodies, was carried out using 12 peanut samples that were also analysed by standard HPLC-fluorescence. Levels of total aflatoxins measured by the two analytical methods showed an excellent correlation (r2 = 0.938) over a concentration range 0–1200 μg/kg sample, with no false negatives [21].

Whilst the ability of the polyclonal AFB1 antibodies to quantify levels of aflatoxins in grains and nuts was demonstrated, the application of ELISA assays to monitoring surveys requires certain laboratory conditions and appropriate skills by qualified personnel. A simpler method

In recent years, more convenient procedures based on immunochemistry have been sought for organic contaminants in food and the environment. The lateral flow device (LFD) has been most popular because of its simplicity in design and its automatic function, each LFD unit requiring only a few drops of sample solution for operation. Comprehensive reviews [22–24] have described the lateral flow assay system in depth, covering a broad range of immunoassay procedures and including nucleic acid applications. Many successful ELISAs can be readily converted to LFDs using the same or similar immuno-reagents; important properties established for ELISAs such as the plots of concentration giving 50% inhibition (IC50) perform in a similar manner in LFDs. In essence, a competitive LFD consists of an impervious nitrocellulose strip coated transversal with two lines that contain either a particular target analyte (test line, T) or a general antibody (control line, C). In addition, a sample pad contains colloidal gold

 **(ppb) %CR**

was needed that could be used in field settings by less skilled operators.

Aflatoxin B1 0.8–6.5 100.0 Aflatoxin G1 1.4–10.8 57.1–60.5 Aflatoxin B2 13.5–55.0 5.9–11.8 Aflatoxin G2 50.1–83.0 1.6–7.8 Aflatoxin P1 >250 <0.3–1.0 Aflatoxin M1 >250 <0.3 to <2.6

**Table 1.** Cross reactivity of aflatoxins and metabolites in polyclonal AFB1 assays (after Lee et al. [16]).

samples without significant matrix effects [16].

**2.2. Lateral flow devices for aflatoxins**

**Compound IC50\***

IC50 = median inhibition concentration.

\*

Some authors have successfully described LFDs for aflatoxins that work well with corn [18], grains and feedstuffs [25, 26] and milk [27], but require strip treatment before use. A new LFD for aflatoxins that did not require special pre-treatment was developed by Masinde et al. [19] and has been commercialised as the Aflatoxin Quicktest™ by QuickTest Technologies. The T line in the Aflatoxin Quicktest™ contains aflatoxin-conjugate (AFB1-C) and the C line a non-specific goat anti-rabbit antibody (G-IgG); the sample pad contains gold nanoparticles (~10–15 micron) conjugated to the specific antibodies (AuNP-IgG) developed by Lee et al. for aflatoxin (AFB1).

For running a test, two drops of sample extract are placed over the sample pad, dissolving the AuNP-IgG nanoparticles, which run laterally over the strip towards the absorbent pad. Any aflatoxin present in the sample extract will compete with the AuNP-IgG particles at the T line, where the excess conjugated antigen will bind to them and produce a coloured line. The remaining AuNP-IgG particles will continue moving towards the other end and will bind to the G-IgG at the C line, also producing colour. The time for the competing targets, aflatoxin and AuNP-IgG, to reach an equilibrium is about 15 minutes, although 5 minutes may be sufficient for initial visual detection [19].

The interpretation of the assay is straightforward: an absence of colour at the T line indicates a high concentration of aflatoxins in the sample extract, as it has outcompeted the gold nanoparticles, whereas a full coloured T line indicates the absence of aflatoxins in the sample. A faded line indicates the presence of some aflatoxin in such a way that the less the colour development, the more aflatoxin is present. The coloured C line confirms that the test is valid, that is, when no colour appears at the C line, the test is invalid or the strip is faulty.

**Figure 2.** Schematic diagram of a lateral flow device (LFD). A nitrocellulose strip is coated with a solution of a particular target analyte (T line) and a general antibody (C line). The sample pad contains gold nanoparticles conjugated to the specific antibodies of the analyte (AuNP-IgG). The absorbent pad at the other end captures the excess solution flowing across the strip.

The Aflatoxin Quicktest™ can detect aflatoxins at 0.1 μg/L in water or liquid samples that do not require dilution, that is, milk. For solid samples that require extraction (e.g. grain, nuts) with methanol or ethanol, a 10-fold dilution must be applied to avoid serious inactivation of the antibodies; in this case, the limit of detection is at least 1 μg/L or more, depending on the extraction method.

## **3. Quantification of aflatoxin by the Aflatoxin Quicktest™**

The conjugated AuNPs-IgG in the LFD compete with the sample analyte for the same target at the T line in a similar way as in a competitive ELISA assay [21]. Therefore, the amount of immuno-gold attached to the T line is inversely proportional to the level of analyte in the sample and this simple relationship can be used to estimate the analyte concentration when an optical reader that measures reflectance of immuno-gold is available. Reading is typically done for one LFD unit at a time, but instruments with multiple slots for reading test devices are also available, allowing greater sample throughput speed. In practice, readers operate by estimating the ratio of the area under the peaks corresponding to the test and the control line (T/C), because no two strips contain exactly the same amount of gold nanoparticles. A ratio around 1 indicates absence of the analyte, whereas lower values indicate its presence and zero values indicate levels of analyte above the range of detection.

Given the operation of the law of mass action in binding of analytes by specific antibodies, the volume or number of drops added to the sample pad of the LFD is not critical for analysis as the reaction with AuNP-IgG nanoparticles is concentration dependent; the majority of the analyte molecules remain in solution, given the small number of antibody molecules. However, it is preferable to standardise the number of drops to induce reliable lateral flow, with 2 drops being optimal for the Aflatoxin Quicktest™.

As with any other analytical technique, the ratio readings must be compared to a standard curve established beforehand using known concentrations of the target analyte. An example for the Aflatoxin Quicktest™ is presented in **Figure 3**. It should be noted that the shape of the curve is best described by an exponential function, which becomes sigmoidal (with a straight section between two bend ends) when plotted against the logarithm of the aflatoxin concentrations. As in ELISA assays, extreme ratio values either at near zero or at some maximum of the range in LFDs must also be rejected, since accurate estimates can only be made in the straight region of the curve, which is usually found between ratio values of 0.15 and 0.85. Consequently, the working range of Aflatoxin Quicktest™ is in the 0.1–2.0 ppb region for direct sample analysis, but 2.0–40 ppb for solid samples requiring solvent extraction and dilution 10-fold. Repeated measurements of the standard solutions using the Aflatoxin Quicktest™ show good reliability of the assays, with a coefficient of variation of 6.4% within this working range.

Samples that contain aflatoxin levels above the range of detection are diluted and reanalysed. More than one dilution may be needed when the contamination levels are very high. Care is needed to extract only well-mixed grain samples given the extremely uneven distribution of aflatoxin among kernels.

**Figure 3.** Establishment of standard curves using LFDs. (A) Aflatoxin QuickTest™ cassette strips used at different concentrations of an aflatoxin mixture; (B) standard curve from the reflectance ratio of test- and control-lines. Error bars

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indicate the 95% confidence intervals of duplicate measurements (D1 and D2) by the optical reader.

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The Aflatoxin Quicktest™ can detect aflatoxins at 0.1 μg/L in water or liquid samples that do not require dilution, that is, milk. For solid samples that require extraction (e.g. grain, nuts) with methanol or ethanol, a 10-fold dilution must be applied to avoid serious inactivation of the antibodies; in this case, the limit of detection is at least 1 μg/L or more, depending on the

The conjugated AuNPs-IgG in the LFD compete with the sample analyte for the same target at the T line in a similar way as in a competitive ELISA assay [21]. Therefore, the amount of immuno-gold attached to the T line is inversely proportional to the level of analyte in the sample and this simple relationship can be used to estimate the analyte concentration when an optical reader that measures reflectance of immuno-gold is available. Reading is typically done for one LFD unit at a time, but instruments with multiple slots for reading test devices are also available, allowing greater sample throughput speed. In practice, readers operate by estimating the ratio of the area under the peaks corresponding to the test and the control line (T/C), because no two strips contain exactly the same amount of gold nanoparticles. A ratio around 1 indicates absence of the analyte, whereas lower values indicate its presence and zero

Given the operation of the law of mass action in binding of analytes by specific antibodies, the volume or number of drops added to the sample pad of the LFD is not critical for analysis as the reaction with AuNP-IgG nanoparticles is concentration dependent; the majority of the analyte molecules remain in solution, given the small number of antibody molecules. However, it is preferable to standardise the number of drops to induce reliable lateral flow,

As with any other analytical technique, the ratio readings must be compared to a standard curve established beforehand using known concentrations of the target analyte. An example for the Aflatoxin Quicktest™ is presented in **Figure 3**. It should be noted that the shape of the curve is best described by an exponential function, which becomes sigmoidal (with a straight section between two bend ends) when plotted against the logarithm of the aflatoxin concentrations. As in ELISA assays, extreme ratio values either at near zero or at some maximum of the range in LFDs must also be rejected, since accurate estimates can only be made in the straight region of the curve, which is usually found between ratio values of 0.15 and 0.85. Consequently, the working range of Aflatoxin Quicktest™ is in the 0.1–2.0 ppb region for direct sample analysis, but 2.0–40 ppb for solid samples requiring solvent extraction and dilution 10-fold. Repeated measurements of the standard solutions using the Aflatoxin Quicktest™ show good reliability

Samples that contain aflatoxin levels above the range of detection are diluted and reanalysed. More than one dilution may be needed when the contamination levels are very high. Care is needed to extract only well-mixed grain samples given the extremely uneven distribution of

of the assays, with a coefficient of variation of 6.4% within this working range.

**3. Quantification of aflatoxin by the Aflatoxin Quicktest™**

198 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

values indicate levels of analyte above the range of detection.

with 2 drops being optimal for the Aflatoxin Quicktest™.

aflatoxin among kernels.

extraction method.

**Figure 3.** Establishment of standard curves using LFDs. (A) Aflatoxin QuickTest™ cassette strips used at different concentrations of an aflatoxin mixture; (B) standard curve from the reflectance ratio of test- and control-lines. Error bars indicate the 95% confidence intervals of duplicate measurements (D1 and D2) by the optical reader.

## **4. Validation of the Aflatoxin Quicktest™**

Two separate studies were carried out to validate the performance of the Aflatoxin Quicktest™ in analysing peanuts and maize samples for aflatoxin contamination. The first study comprised peanuts samples from Australian growers in Queensland, which were collected during the 2015 and 2016 growing seasons and maize kernels infected with *A. flavus* in a laboratory trial. The second study involved a comprehensive survey of peanuts and maize from markets in Timor-Leste, carried out during 2014 and 2015. In both studies, extracts of the raw samples were analysed using the Aflatoxin Quicktest™ as well as the standard methods of analytical laboratories, that is, HPLC with fluorescence detector or LC-MS/MS, so their results could be compared.

#### **4.1. Australian validation study**

From late April 2015, from every load of peanuts delivered into the Kingaroy intake of the Peanut Company of Australia (PCA), duplicate 80% methanol extracts were collected as part of the normal aflatoxin mini-column test conducted at intake. For each load, a separate extraction was performed, such that a duplicate methanol extract was collected for both HPLC (15 ml) and Aflatoxin Quicktest™ (5 ml) aflatoxin analysis. For the 2015 season, about 170 extracts were collected and stored in the fridge (5°C) until the validation study commenced in June of that year.

All 15 ml extracts were analysed by HPLC-fluorescence at the PCA Technical Centre using the company's standard method accredited by National Association of Testing Authorities (NATA, Australia), which included addition of 5% Holaday salt solution to the methanol extract. A selected subset of 13 positive and 12 negative samples randomly chosen was then used in the validation study.

The corresponding subset of 5 ml extracts (n = 25) were analysed in June by the Aflatoxin Quicktest™. For this analysis, 200 μl were taken into an Eppendorf tube and 1.8 ml of phosphate buffer solution (PBS – 50 mM, pH 7.4) added so as to reduce the concentration of methanol to less than 10%. Two drops of the solution were placed on the strip well and left to develop colour for 15 minutes on the laboratory bench, after which time the strips were immediately read using an LFD Quick Reader (Tianjin Jiuding Diagnostics Ltd., China). Samples that produced results above the detection range (2–40 ppb) were diluted further in PBS and reanalysed until their readings fell within this range; all results were calculated taking into account the dilutions factors used for each sample.

Results from the 2015 peanut validation study are shown in **Figure 4A**, where it can be seen the excellent correlation between the HPLC and Aflatoxin Quicktest™ analyses for total aflatoxins (r2 = 0.934 on the logarithmic transformed data).

The same procedure was repeated in 2016, but this time 45 peanut extracts were used in the validation study, which was conducted in October of that year. The results for 2016 (**Figure 4B**) showed also a good correlation between the two analyses (r2 = 0.956), even though a number of samples were well above of the detection range. The results of the second year confirm the validity of the Aflatoxin Quicktest™ as a tool for detecting and measuring total aflatoxin levels in peanuts.

**Figure 4.** Validation of the Aflatoxin QuickTest™ for peanut samples collected by the Peanut Company of Australia in

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2015 (A) and 2016 (B).

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**4. Validation of the Aflatoxin Quicktest™**

200 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

**4.1. Australian validation study**

in June of that year.

used in the validation study.

aflatoxin levels in peanuts.

account the dilutions factors used for each sample.

toxins (r2 = 0.934 on the logarithmic transformed data).

Two separate studies were carried out to validate the performance of the Aflatoxin Quicktest™ in analysing peanuts and maize samples for aflatoxin contamination. The first study comprised peanuts samples from Australian growers in Queensland, which were collected during the 2015 and 2016 growing seasons and maize kernels infected with *A. flavus* in a laboratory trial. The second study involved a comprehensive survey of peanuts and maize from markets in Timor-Leste, carried out during 2014 and 2015. In both studies, extracts of the raw samples were analysed using the Aflatoxin Quicktest™ as well as the standard methods of analytical laboratories, that is, HPLC with fluorescence detector or LC-MS/MS, so their results could be compared.

From late April 2015, from every load of peanuts delivered into the Kingaroy intake of the Peanut Company of Australia (PCA), duplicate 80% methanol extracts were collected as part of the normal aflatoxin mini-column test conducted at intake. For each load, a separate extraction was performed, such that a duplicate methanol extract was collected for both HPLC (15 ml) and Aflatoxin Quicktest™ (5 ml) aflatoxin analysis. For the 2015 season, about 170 extracts were collected and stored in the fridge (5°C) until the validation study commenced

All 15 ml extracts were analysed by HPLC-fluorescence at the PCA Technical Centre using the company's standard method accredited by National Association of Testing Authorities (NATA, Australia), which included addition of 5% Holaday salt solution to the methanol extract. A selected subset of 13 positive and 12 negative samples randomly chosen was then

The corresponding subset of 5 ml extracts (n = 25) were analysed in June by the Aflatoxin Quicktest™. For this analysis, 200 μl were taken into an Eppendorf tube and 1.8 ml of phosphate buffer solution (PBS – 50 mM, pH 7.4) added so as to reduce the concentration of methanol to less than 10%. Two drops of the solution were placed on the strip well and left to develop colour for 15 minutes on the laboratory bench, after which time the strips were immediately read using an LFD Quick Reader (Tianjin Jiuding Diagnostics Ltd., China). Samples that produced results above the detection range (2–40 ppb) were diluted further in PBS and reanalysed until their readings fell within this range; all results were calculated taking into

Results from the 2015 peanut validation study are shown in **Figure 4A**, where it can be seen the excellent correlation between the HPLC and Aflatoxin Quicktest™ analyses for total afla-

The same procedure was repeated in 2016, but this time 45 peanut extracts were used in the validation study, which was conducted in October of that year. The results for 2016 (**Figure 4B**) showed also a good correlation between the two analyses (r2 = 0.956), even though a number of samples were well above of the detection range. The results of the second year confirm the validity of the Aflatoxin Quicktest™ as a tool for detecting and measuring total

**Figure 4.** Validation of the Aflatoxin QuickTest™ for peanut samples collected by the Peanut Company of Australia in 2015 (A) and 2016 (B).

In addition to the peanut extracts, 16 samples of maize kernels that had been infected with *A. flavus* in the laboratory were also used for validating the Aflatoxin Quicktest™. The kernels were extracted with a mixture of 80% methanol and 4% Holaday salt solution and analysed first by HPLC-fluorescence at the PCA Technical Centre. The levels of aflatoxins in the kernels were sometimes very high, with the highest reaching 111 mg/kg (ppm). Most of the aflatoxin was found to be G1 (63 ± 27%) and B1 (26 ± 23%), whereas both G2 and B1 were usually below 3%.

Aliquots of the extracts (100 μl) were taken into 900 μl of PBS solution for direct analysis by Aflatoxin Quicktest™, and diluted further in PBS if the readings were above the detection range.

In spite of the high levels of contamination in the maize kernels, which required dilutions of the extracts up to 60,000-fold, a comparison of results by the two analytical methods showed an acceptable correlation for all the aflatoxins in the samples (**Figure 5**, r<sup>2</sup> = 0.89). This correlation improved when the results of the Aflatoxin Quicktest™ were compared to the levels of aflatoxin B1 and B2 (r2 = 0.97 and 0.93, respectively) as determined by HPLC, whereas those of aflatoxins G1 and G2 showed lower correlations (r2 = 0.86 and 0.76, respectively). This is in agreement with the differential sensitivity of the antibodies present in the commercial Aflatoxin Quicktest™, which are 100% specific to aflatoxin B1 but less specific to the other forms (see **Table 1**). Aflatoxins B1 and B2 are commonly found in tropical and subtropical regions of the world, whereas aflatoxins G1 and G2 are commonly produced by *A. parasiticus*, a soil species that is rare or absent in South East Asia [28].

**Figure 5.** Validation of the Aflatoxin QuickTest™ for maize kernels infected with high levels of aflatoxins: AFB1, AFB2, AFG1, AFG2 and total aflatoxins (AF).

**Figure 6.** Validation of the Aflatoxin QuickTest™ for peanut (A) and maize (B) samples from Timor-Leste market surveys.

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**Figure 6.** Validation of the Aflatoxin QuickTest™ for peanut (A) and maize (B) samples from Timor-Leste market surveys.

**Figure 5.** Validation of the Aflatoxin QuickTest™ for maize kernels infected with high levels of aflatoxins: AFB1, AFB2,

In addition to the peanut extracts, 16 samples of maize kernels that had been infected with *A. flavus* in the laboratory were also used for validating the Aflatoxin Quicktest™. The kernels were extracted with a mixture of 80% methanol and 4% Holaday salt solution and analysed first by HPLC-fluorescence at the PCA Technical Centre. The levels of aflatoxins in the kernels were sometimes very high, with the highest reaching 111 mg/kg (ppm). Most of the aflatoxin was found to be G1 (63 ± 27%) and B1 (26 ± 23%), whereas both G2 and B1 were usually below 3%. Aliquots of the extracts (100 μl) were taken into 900 μl of PBS solution for direct analysis by Aflatoxin Quicktest™, and diluted further in PBS if the readings were above the detection range. In spite of the high levels of contamination in the maize kernels, which required dilutions of the extracts up to 60,000-fold, a comparison of results by the two analytical methods showed an acceptable correlation for all the aflatoxins in the samples (**Figure 5**, r<sup>2</sup> = 0.89). This correlation improved when the results of the Aflatoxin Quicktest™ were compared to the levels of aflatoxin B1 and B2 (r2 = 0.97 and 0.93, respectively) as determined by HPLC, whereas those of aflatoxins G1 and G2 showed lower correlations (r2 = 0.86 and 0.76, respectively). This is in agreement with the differential sensitivity of the antibodies present in the commercial Aflatoxin Quicktest™, which are 100% specific to aflatoxin B1 but less specific to the other forms (see **Table 1**). Aflatoxins B1 and B2 are commonly found in tropical and subtropical regions of the world, whereas aflatoxins G1 and G2 are commonly produced by *A. parasiticus*, a soil species that is rare or absent in South

202 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

AFG1, AFG2 and total aflatoxins (AF).

East Asia [28].

#### **4.2. Timor-Leste validation study**

Surveys were conducted in 2013, 2014 and 2015 to collect maize and peanut kernels from markets, seed producers and households in 42 districts of Timor-Leste. A small subset of 33 peanut and 30 maize samples from the two latter years were used for the validation study.

certified laboratories in Indonesia or Australia. Moreover, they can now check the raw produce on site without having to wait weeks until they receive the results from the laboratories. No doubt, this potentially gives them significant competitive advantage in the markets. It is anticipated that application of this technology may allow rapid, accurate and low cost screen-

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By measuring the levels of aflatoxin in their produce farmers can now manage the problem, whereas lack of awareness of this contamination will only spread the contamination with these toxins among the local population [29], leading to unpleasant health consequences for consumers [5], and probably to a rejection of the products in the market place. Only what can be measured can be managed. In this regard, monitoring of agricultural produce should be followed by practical training sessions where farmers are instructed how to avoid aflatoxin

So far only grain and nuts have been tested using the Aflatoxin QuickTest™, but it is obvious that other agricultural commodities such as milk and dairy products can also be analysed using this technology. In the case of milk, no extraction may be required, so samples can be used directly for detecting levels of this contaminant in the range 0.1–2.0 μg/L or higher, after

The current Aflatoxin QuickTest™ discussed here uses polyclonal antibodies specific for aflatoxin B1 and G1 (**Table 1**). Different antibodies have also been developed that have variable sensitivities towards other forms of aflatoxin and can be marketed in the near future in accor-

, Robert Williams2

, Graeme Wright<sup>3</sup>

,

ing of Timorese agricultural produce.

contamination in the first place [1, 30, 31].

diluting by a given factor.

**Author details**

Ivan R. Kennedy4

Australia

**References**

dance with specific market needs.

Francisco Sánchez-Bayo1,4\*, Luis de Almeida2

and Angus Crossan4

4 QuickTest Technologies Ltd, Eveleigh, NSW, Australia

Agriculture. 2002;**42**(5):595-605

\*Address all correspondence to: francisco.sanchez-bayo@sydney.edu.au

2 Ministry of Agriculture and Fisheries (MAF), Dili, Timor-Leste 3 Peanut Company of Australia (PCA), Kingaroy, QLD, Australia

1 School of Life and Environmental Sciences, The University of Sydney, Eveleigh, NSW,

[1] Rachaputi N, Krosch S, Wright GC. Management practices to minimise pre-harvest aflatoxin contamination in Australian peanuts. Australian Journal of Experimental

Samples of well-mixed kernels (100 g) were ground using a commercial blender and the meal thus obtained was extracted with 200 ml of 80% methanol containing 4% NaCl in a blender for 3 minutes. The extracts were filtered and 4 ml of supernatant collected for subsequent analysis by both Aflatoxin Quicktest™ and standard analytical methods. All peanut samples were analysed by HPLC-fluorescence at the PCA analytical facilities (Kingaroy, Queensland), whereas only 15 maize samples were analysed by LC-MS/MS at the National Measurement Institute (Sydney, Australia).

Aliquot of the extracts (200 μl) were diluted in phosphate buffer solution (ratio 1:10) to make it ready for Aflatoxin Quicktest™ analysis. Two drops of this solution were added to each strip and allowed to develop colour in 15 minutes. The strips were then read using the Quick Reader, and the results printed and recorded. For readings above 40 ppb, the sample extracts were further diluted and reanalysed again.

The results by both analytical methods were compared in order to validate the Aflatoxin Quicktest™ procedure. The majority of samples showed levels of total aflatoxin below 100 μg/kg. Regression analysis on the sets of peanut (n = 33) and maize samples (n = 15) showed coefficients of determination (r2 ) of 0.989 for peanuts (71% aflatoxin B1) and 0.942 for maize (91% aflatoxin B1) (**Figure 6A** and **B**).

In summary, both validation studies were successful, demonstrating the accuracy of the Aflatoxin Quicktest™, which renders results comparable to those obtained by the standard analytical methods in certified laboratories.

## **5. Advantages of the Aflatoxin QuickTest™**

Advantages of the LFD technology are the ease of use, rapid development time, no need for dangerous chemicals, straightforward reading of test results and low cost of the strips. Moreover, very little and inexpensive equipment is required other than a Quick Reader and solvents for the extractions.

This technology is designed to help primary producers screen their produce before selling it in the market. Because the test is very simple and easy to understand, users only require a basic training to become proficient. Thus, local co-operatives, small companies and even farmers can learn it and apply it in their own facilities.

Based on the demonstrated performance of the Aflatoxin QuickTest™ in evaluating aflatoxin contamination in maize and peanuts, the government of Timor-Leste is supporting its use for screening these and other agricultural produce that may be contaminated with this toxin. Local companies may now use this technology to meet international food safety standards for the export market, instead of resorting to the expensive alternative of sending samples to certified laboratories in Indonesia or Australia. Moreover, they can now check the raw produce on site without having to wait weeks until they receive the results from the laboratories. No doubt, this potentially gives them significant competitive advantage in the markets. It is anticipated that application of this technology may allow rapid, accurate and low cost screening of Timorese agricultural produce.

By measuring the levels of aflatoxin in their produce farmers can now manage the problem, whereas lack of awareness of this contamination will only spread the contamination with these toxins among the local population [29], leading to unpleasant health consequences for consumers [5], and probably to a rejection of the products in the market place. Only what can be measured can be managed. In this regard, monitoring of agricultural produce should be followed by practical training sessions where farmers are instructed how to avoid aflatoxin contamination in the first place [1, 30, 31].

So far only grain and nuts have been tested using the Aflatoxin QuickTest™, but it is obvious that other agricultural commodities such as milk and dairy products can also be analysed using this technology. In the case of milk, no extraction may be required, so samples can be used directly for detecting levels of this contaminant in the range 0.1–2.0 μg/L or higher, after diluting by a given factor.

The current Aflatoxin QuickTest™ discussed here uses polyclonal antibodies specific for aflatoxin B1 and G1 (**Table 1**). Different antibodies have also been developed that have variable sensitivities towards other forms of aflatoxin and can be marketed in the near future in accordance with specific market needs.

## **Author details**

**4.2. Timor-Leste validation study**

Institute (Sydney, Australia).

were further diluted and reanalysed again.

showed coefficients of determination (r2

maize (91% aflatoxin B1) (**Figure 6A** and **B**).

analytical methods in certified laboratories.

solvents for the extractions.

**5. Advantages of the Aflatoxin QuickTest™**

farmers can learn it and apply it in their own facilities.

Surveys were conducted in 2013, 2014 and 2015 to collect maize and peanut kernels from markets, seed producers and households in 42 districts of Timor-Leste. A small subset of 33 peanut and 30 maize samples from the two latter years were used for the validation study.

204 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

Samples of well-mixed kernels (100 g) were ground using a commercial blender and the meal thus obtained was extracted with 200 ml of 80% methanol containing 4% NaCl in a blender for 3 minutes. The extracts were filtered and 4 ml of supernatant collected for subsequent analysis by both Aflatoxin Quicktest™ and standard analytical methods. All peanut samples were analysed by HPLC-fluorescence at the PCA analytical facilities (Kingaroy, Queensland), whereas only 15 maize samples were analysed by LC-MS/MS at the National Measurement

Aliquot of the extracts (200 μl) were diluted in phosphate buffer solution (ratio 1:10) to make it ready for Aflatoxin Quicktest™ analysis. Two drops of this solution were added to each strip and allowed to develop colour in 15 minutes. The strips were then read using the Quick Reader, and the results printed and recorded. For readings above 40 ppb, the sample extracts

The results by both analytical methods were compared in order to validate the Aflatoxin Quicktest™ procedure. The majority of samples showed levels of total aflatoxin below 100 μg/kg. Regression analysis on the sets of peanut (n = 33) and maize samples (n = 15)

In summary, both validation studies were successful, demonstrating the accuracy of the Aflatoxin Quicktest™, which renders results comparable to those obtained by the standard

Advantages of the LFD technology are the ease of use, rapid development time, no need for dangerous chemicals, straightforward reading of test results and low cost of the strips. Moreover, very little and inexpensive equipment is required other than a Quick Reader and

This technology is designed to help primary producers screen their produce before selling it in the market. Because the test is very simple and easy to understand, users only require a basic training to become proficient. Thus, local co-operatives, small companies and even

Based on the demonstrated performance of the Aflatoxin QuickTest™ in evaluating aflatoxin contamination in maize and peanuts, the government of Timor-Leste is supporting its use for screening these and other agricultural produce that may be contaminated with this toxin. Local companies may now use this technology to meet international food safety standards for the export market, instead of resorting to the expensive alternative of sending samples to

) of 0.989 for peanuts (71% aflatoxin B1) and 0.942 for

Francisco Sánchez-Bayo1,4\*, Luis de Almeida2 , Robert Williams2 , Graeme Wright<sup>3</sup> , Ivan R. Kennedy4 and Angus Crossan4

\*Address all correspondence to: francisco.sanchez-bayo@sydney.edu.au

1 School of Life and Environmental Sciences, The University of Sydney, Eveleigh, NSW, Australia


4 QuickTest Technologies Ltd, Eveleigh, NSW, Australia

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The Aflatoxin Quicktest™—A Practical Tool for Ensuring Safety in Agricultural Produce

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208 Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

## *Edited by Ntambwe Malangu*

This book, which is the result of contributions from a team of international authors, presents a collection of materials that can be categorized into two groups. The first group of papers deals with clinical toxicology topics including poisoning by anticoagulant rodenticides, food toxins, carbon monoxide, the toxicity of betalactam antibiotics, acute neonicotinoid poisoning, occupational risk factors for acute pesticide poisoning, activating carbon fibers, and date pits for use in liver toxin adsorption. The second group of papers deals with forensic or analytical toxicology topics such as simplified methods for the analysis of gaseous toxic agents, rapid methods for the analysis and monitoring of pathogens in drinking water and waterbased solutions, as well as the linkages between clinical and forensic toxicology. Each chapter presents new information on the topic discussed based on authors' experience while summarizing existing knowledge. As such, this book will be a good teaching aid and can be a prescribed or recommended reading for postgraduate students and professionals in the fields of public health, medicine, pharmacy, nursing, biology, toxicology, and forensic sciences.

Poisoning - From Specific Toxic Agents to Novel Rapid and Simplified Techniques for Analysis

Poisoning

From Specific Toxic Agents to Novel Rapid

and Simplified Techniques for Analysis

*Edited by Ntambwe Malangu*

Photo by sakhorn38 / iStock