**2. Materials and methods**

#### **2.1. Site description**

not epidemiologic studies to detect chronic effects of the pesticides; those should exist at least for the agricultural journeymen and for vulnerable groups, since they lack elementary

The exposure doses can be small but persistent, causing chronic health problems [8]. DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane, also called dichlorodiphenyl trichloro‐ ethane); was first used to protect military areas and personnel against malaria, typhus, and other vector-terminal diseases [9]. In Mesoamerica (Mexico, Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua and Panama) DDT was used until the year 2000; Mexi‐ co and Nicaragua being the last nations that applied the insecticide in agriculture and for the control of malaria (the amount used for Mexico is approximately 69,545 tons be‐

Technical-grade DDT contains 65-80% p,p'-DDT, 15-21% o,p'-DDT, and up to 4% p,p'-DDD (bis[4-chlorophenyl]-1,1,-dichloroethane)[11]. When sprayed, DDT can drift, sometimes for long distances. In the soil, the compound can evaporate or attach to wind-blown dust. In the environment, DDT breaks down to p,p'-DDE (bis[4-chlorophenyl]-1,1-dichloroethene) [12], an extremely stable compound that resists further environmental breakdown or metabolism by organism; DDE is the form usually found in human tissue in the highest concentration, especially in areas where there has been no recent use of the parent compound [9]. DDT and DDE can also be transferred from the placenta and breast milk to fetuses and infants. Al‐ though some ingested DDT is converted to DDA (bis[4-chlorophenyl]-acetic acid) and ex‐ creted, any non-metabolized DDT and any DDE produced is stored in fat, as is all absorbed DDE, which cannot be metabolized. DDT and DDE are highly soluble in lipid; their concen‐ trations are much higher in human adipose tissues (about 65% fat) than in breast milk (2.5-4% fat), and higher in breast milk than in blood or serum (1% fat) [13]. DDT and DDE

The use of DDT in Central and South America, Mexico, Africa, and some Asian coun‐ tries where this has been used for vector control in the past 5-10 years, DDT concentra‐ tions in human tissues remain high. For example, in Mexico, the total DDT concentration in breast milk fat was 5.7 µg/g in 1994-95 and 4.7 µg/g in 1997-98 [15]. Others Mexican data where workers used DDT to control mosquitoes, have very high DDT concentra‐ tions. Mexican data revealed that the geometric mean of total DDT was 104.48 µg/g in adipose tissue of 40 DDT sprayers in 1996 [16]; however in Finland, USA, and Canada, the value was less than 1 µg/g in adipose tissue in the general population [17]. In anoth‐ er Mexican study, the serum concentration of p,p'-DDE was much higher in DDT spray‐ ers (188 µg/L) than in children (87 µg/L); also in adults (61 µg/L) who lived in sprayed

The organophosphate insecticides have the advantage of low environmental persistence over the chlorinated pesticide compounds. However, studies carried out in individuals with exposure to insecticides in Mexico, and other countries, associate the pesticide exposure with adverse health effects, as much in the humans as in experimental animals. These dam‐ ages can be evident by the presence of certain biochemical indicators in the different biologi‐

protection, and don't have the correct information about pesticide toxicity.

312 Insecticides - Development of Safer and More Effective Technologies

tween 1957 and 2000) [10].

concentrations increase with age [14].

houses, but were not otherwise exposed to DDT [3].

Sonora State territory has 179,355 km2 and it is located in Sierra Madre Occidental; geo‐ graphically it is north 32° 29', to south 26° 18' of north latitude, to east 108° 25', to west 115° 03'. The weather in coastal of Sonora is dry. The average annual temperature is around 22°C, being the average maximum 38°C (June and July) and the average mini‐ mum temperature is 5°C (January). Only 7% of the land is appropriate for agricultural use and ninety five percent of this area is irrigated. In July and August the rain reaches 450 mm. The weather in Sonora State restricts the agricultural activities. However, in vil‐ lages Yaqui and Mayo, valleys of Hermosillo, Caborca and Guaymas the major crops with irrigation are wheat, cotton, safflower, watermelon, sesame, garbanzo, sorghum, corn and vine [23]. The agriculture in the south of Sonora is based on 90% of crops such as corn, wheat, oleaginous and cotton [24].

#### **2.2. Population study**

*Group 1, field workers*. The municipal headboards included in this group are localized in the following coordinates: Obregon city 27º 29' north latitude and 109º39' west latitude with a height of 10 m above sea level and Navojoa 27º 05' north latitude and 109º39' west latitude with 40 m above sea level [23].

The town council Cajeme has a population of 175,177 men, from this population, 6,983 live in Yaqui town. In Huatabampo there are 38,563 men, specifically in "Jupare" (1,026 men). Navojoa has a population of 69,341 from this population, 445 men live in 5 de Ju‐ nio cooperative [25]. In this study participated 37 men from Yaqui town, 19 from "Ju‐ pare" and 21 from "5 de Junio" cooperative, this is 0.53%, 1.85% and 4.7% of total population of men, respectively.

*2.5.2. Blood analysis*

(SOD, RandoxMR).

*2.5.3. Urine analysis*

*2.5.4. Semen analysis*

crystals, epithelial cells, etc.

*2.5.5. Insecticides extraction*

Blood samples were analyzed in laboratory of General Hospital SS in Cd. Obregon and the laboratory of General Hospital of Navojoa. Blood analysis included hematic biometry using an analyzer Sysmex K-4500, blood chemistry test (glucose, urea, creatinine, uric acid, choles‐ terol and triglycerides) and total proteins (albumin and globulin) using an analyzer HITA‐ CHI 911 and also determination of enzymes in serum such as levels of plasma cholinesterase (RandoxMR), alkaline phosphatase (RocheMR), transaminases, and superoxide dismutase

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Urine was analyzed by two types of analysis; biochemical and microscopic analysis. Bio‐ chemical analysis included glucose, proteins, bilirubin, kenotic bodies, urobilinogen (combo test-10). The macroscopic analysis included number of bacteria, erythrocytes, leucocytes,

Analysis of semen was carried out by using international standardized techniques [26]. Mac‐ roscopic and microscopic analyses were included; in the first one liquefaction, aspect, vis‐ cosity, pH and volume were determined. In the microscopic analysis motility, viability, presence of leucocytes, erythrocytes, germinal cells, dendrites, agglutination (specific and

*Blood, urine and semen.* Samples were analyzed following the methodology proposed in [27]. Briefly, 0.5 mL of sample were taken and added 4 mL hexane; the mixture was shaken for 15 seconds and then centrifuged for 2.30 minutes at 2500 rpm. The supernant was transferred to a tube and added 1 mL of 5% K2CO3 and 4 mL hexane. Centrifuged for 2.30 minutes at 2500 rpm, supernant was transferred to a tube and evaporated to dryness. Extract was dis‐ solved with 100 µL hexane and analyzed by gas chromatography. *Breast milk* was analyzed

The analytical standards were from Chem Service (West Chester, PA). Quantitation of insec‐ ticides was by comparison of five –point calibration curve. The detection ranges used for cal‐ ibration curves were 50-0.1 µg/L. The average percent recoveries for organochlorine pesticides were p,p'-DDD 95%, p,p'-DDE 98% and p,p'DDT 105%, for organophosphates

Quantitative analyses were performed by gas chromatography (GC) using a Varian model CP-3800 equipped with an electron capture detector (ECD)(USA). The insecticides were sep‐ arated using VA-1701 (Varian, 30 m x 0.25 mm) capillary column. The injection volume was

unspecific), number of spermatozoa and morphology were determined.

were diazinon 99%, clorpyrifos 91%, malathion 106% and parathion 92%.

using a matrix solid-phase dispersion technique [28].

**2.6. Insecticides residues analyses in body fluids**

#### **2.3. Group 2, nursing mothers**

The women that participated in this study were located at Pesqueira community. The com‐ munity is located between 28 and 30º parallel north latitude in Sonora, Mexico [23]. The weather in this region is dry with rain in summer [23]. The women are dedicated to cultivat‐ ing and packing table grape. When this study was conducted the population in Pesqueira was 3,648 residents; 47.8% women, from this percentage only 10% were in reproductive age [25]. In the Health center there was a record of 20% women included in the breastfeeding program. However, 26% women in reproductive age (exposed or working with pesticides) participated in the present study, being 1.4% of total population. It is probably that perma‐ nent residents of agricultural areas are chronically exposed to chemical residues through wind, drinking water and even clothing from field workers.

#### **2.4. Participation and surveys**

Men and women (nursing period) that voluntarily participated in this study, signed a for‐ mat according to the norms of Mexican Secretary of Health (SS). Participants filled up a sur‐ vey; they provided demographic data (age, marital status, residence, residence time) and also information related to work history, pesticides exposure, clinical history, issues related to sexuality, pathology and drugs addiction (alcohol, tobacco, cocaine and marihuana, among others). People with drugs addictions or health issues that could have influence with the biochemical determinations of the body fluids were excluded from this research.

#### **2.5. Blood, urine and semen sampling**

Blood, urine and semen samples were collected from members of group 1. For hematic bio‐ metry analysis, blood chemistry and biochemical indicators, samples were taken with empty stomach. Blood samples were taken by venous puncture and collected in to vacuum tubes (VacutainerMR) with and without anticoagulant. Once blood was coagulated, it was centri‐ fuged and supernant was transferred in to a new tube for analysis.

Urine samples were collected in a sterile container and kept at 4 °C until analysis. Se‐ men samples were collected in sterile container (including a code, date and time when were collected) by the participant at home. Samples were analyzed no more than 24 h af‐ ter sampling.

#### *2.5.1. Breast milk sampling*

Breast milk was collected either manually or with a breast milk collector in to a 50 mL coni‐ cal glass tubes (wrapped with aluminum foil). Samples were kept at -20ºC until analysis.

#### *2.5.2. Blood analysis*

nio cooperative [25]. In this study participated 37 men from Yaqui town, 19 from "Ju‐ pare" and 21 from "5 de Junio" cooperative, this is 0.53%, 1.85% and 4.7% of total

The women that participated in this study were located at Pesqueira community. The com‐ munity is located between 28 and 30º parallel north latitude in Sonora, Mexico [23]. The weather in this region is dry with rain in summer [23]. The women are dedicated to cultivat‐ ing and packing table grape. When this study was conducted the population in Pesqueira was 3,648 residents; 47.8% women, from this percentage only 10% were in reproductive age [25]. In the Health center there was a record of 20% women included in the breastfeeding program. However, 26% women in reproductive age (exposed or working with pesticides) participated in the present study, being 1.4% of total population. It is probably that perma‐ nent residents of agricultural areas are chronically exposed to chemical residues through

Men and women (nursing period) that voluntarily participated in this study, signed a for‐ mat according to the norms of Mexican Secretary of Health (SS). Participants filled up a sur‐ vey; they provided demographic data (age, marital status, residence, residence time) and also information related to work history, pesticides exposure, clinical history, issues related to sexuality, pathology and drugs addiction (alcohol, tobacco, cocaine and marihuana, among others). People with drugs addictions or health issues that could have influence with

Blood, urine and semen samples were collected from members of group 1. For hematic bio‐ metry analysis, blood chemistry and biochemical indicators, samples were taken with empty stomach. Blood samples were taken by venous puncture and collected in to vacuum tubes (VacutainerMR) with and without anticoagulant. Once blood was coagulated, it was centri‐

Urine samples were collected in a sterile container and kept at 4 °C until analysis. Se‐ men samples were collected in sterile container (including a code, date and time when were collected) by the participant at home. Samples were analyzed no more than 24 h af‐

Breast milk was collected either manually or with a breast milk collector in to a 50 mL coni‐ cal glass tubes (wrapped with aluminum foil). Samples were kept at -20ºC until analysis.

the biochemical determinations of the body fluids were excluded from this research.

fuged and supernant was transferred in to a new tube for analysis.

population of men, respectively.

314 Insecticides - Development of Safer and More Effective Technologies

wind, drinking water and even clothing from field workers.

**2.3. Group 2, nursing mothers**

**2.4. Participation and surveys**

**2.5. Blood, urine and semen sampling**

ter sampling.

*2.5.1. Breast milk sampling*

Blood samples were analyzed in laboratory of General Hospital SS in Cd. Obregon and the laboratory of General Hospital of Navojoa. Blood analysis included hematic biometry using an analyzer Sysmex K-4500, blood chemistry test (glucose, urea, creatinine, uric acid, choles‐ terol and triglycerides) and total proteins (albumin and globulin) using an analyzer HITA‐ CHI 911 and also determination of enzymes in serum such as levels of plasma cholinesterase (RandoxMR), alkaline phosphatase (RocheMR), transaminases, and superoxide dismutase (SOD, RandoxMR).

#### *2.5.3. Urine analysis*

Urine was analyzed by two types of analysis; biochemical and microscopic analysis. Bio‐ chemical analysis included glucose, proteins, bilirubin, kenotic bodies, urobilinogen (combo test-10). The macroscopic analysis included number of bacteria, erythrocytes, leucocytes, crystals, epithelial cells, etc.

#### *2.5.4. Semen analysis*

Analysis of semen was carried out by using international standardized techniques [26]. Mac‐ roscopic and microscopic analyses were included; in the first one liquefaction, aspect, vis‐ cosity, pH and volume were determined. In the microscopic analysis motility, viability, presence of leucocytes, erythrocytes, germinal cells, dendrites, agglutination (specific and unspecific), number of spermatozoa and morphology were determined.

#### *2.5.5. Insecticides extraction*

*Blood, urine and semen.* Samples were analyzed following the methodology proposed in [27]. Briefly, 0.5 mL of sample were taken and added 4 mL hexane; the mixture was shaken for 15 seconds and then centrifuged for 2.30 minutes at 2500 rpm. The supernant was transferred to a tube and added 1 mL of 5% K2CO3 and 4 mL hexane. Centrifuged for 2.30 minutes at 2500 rpm, supernant was transferred to a tube and evaporated to dryness. Extract was dis‐ solved with 100 µL hexane and analyzed by gas chromatography. *Breast milk* was analyzed using a matrix solid-phase dispersion technique [28].

#### **2.6. Insecticides residues analyses in body fluids**

The analytical standards were from Chem Service (West Chester, PA). Quantitation of insec‐ ticides was by comparison of five –point calibration curve. The detection ranges used for cal‐ ibration curves were 50-0.1 µg/L. The average percent recoveries for organochlorine pesticides were p,p'-DDD 95%, p,p'-DDE 98% and p,p'DDT 105%, for organophosphates were diazinon 99%, clorpyrifos 91%, malathion 106% and parathion 92%.

Quantitative analyses were performed by gas chromatography (GC) using a Varian model CP-3800 equipped with an electron capture detector (ECD)(USA). The insecticides were sep‐ arated using VA-1701 (Varian, 30 m x 0.25 mm) capillary column. The injection volume was 1 µL. Nitrogen (purity 99.999 %) was used as the carrier gas at a flow of 1.5 mL/min. The injector temperature was 180°C and the detector temperature was 300°C. The temperature program was as follows: initial temperature 220°C, increasing temperature at 9°C min-1 until the final temperature of 300°C was reached. Data was analyzed using a program Star Chro‐ matography Workstation 5.51.

In the case group, a total of 77 men participated; the majority maintained contact with orga‐ nophosphate pesticides; the average age of this group was 40 years and 11 years of residen‐ cy in the site of the study with a maximum of 45 years. The average work years with pesticide exposure was 28, with a maximum of 50 years working in agriculture. Based on data from work history a 62% of the cases had contact with pesticides; from this percentage 43% applied them, 27% works in the places where they were applied and 30% works where they are applied. Only 18% of the field workers uses protection while applying pesticides (like gloves, special clothing for welding fumes, paint fumes o foundry fumes). These results suggest that field workers are chronically exposed to pesticides due to few safety precau‐ tions are taken to handle them. There for, it is of importance that the field workers receive

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317

In the case group besides being in contact with pesticides during work activities they are al‐ so in their place of residency; considering this background, the time of exposure is 16 years in average and 65% of them apply insecticides in their homes; 27% are applied with an an‐ nual frequency, 23% are applied semiannually, while 10% every 3 months and 8% monthly. Some factors can exacerbate the toxicological effects caused by pesticide exposure, such as the consumption of alcohol and drugs. The present study found that 69% of the individuals of the case group consume alcohol with a monthly frequency, 16% consume less than 5 cigarettes dai‐ ly, 8% and 6% less than 20 cigarettes and the rest only 1 cigarette, 4% consumes cocaine, none of the cases consumes marihuana, nor intravenous drugs. Erection and ejaculation problems (4%) were found in case group. Case and control groups had problems having children (6%). Unlike the control group, the case group presented sexual transmission diseases; around 4% had gon‐ orrhea. Both groups have children with congenital health problems (approximately 6%).

Some of the reported symptoms in the case group were cramps (61%), tiredness and weakness (53%), blurred vision (45%), sweating (45%), tearing (43%), nervousness (38%), dizziness (37%) and tingling in the extremities (37%). According to literature it can be considered that pesticide intoxication is nonspecific and produce the subclinical symptoms identified in the present study in addition to anorexia, insomnia, digestive alterations and itching of skin and mucous [11]. Mostly the symptoms caused by pesticides exposure are diagnosed as common cold or flu [29]. This symptomatology is not produced at the same time because every chemical product acts in a different way and will differ in each of the persons with a chronic exposure. In the present study, during the physical auscultation, the average weight and height in the case group and control group was 82 and 81 kg and 1.72 m, respectively. The vital signs were normal for both groups; 70 and 80 pulsations/min (normal value 70-80 pulsations/min), respiratory rate 20 breaths/min

*Group 2.* Nursing women, description based on surveys. A total of 51 surveys were made to nursing women, 79% of them have been living in this community for more than five years. The average age was 24 years while the median was 23. The highest age was 39 and the low‐ est 16. The average body weight of women was 82 kg (±15.9), with an average height of 1.72 m (±0.07). The average number of children was three, 98% were married (including the ones that live in free union) and only 2% were single (this includes also the ones that are di‐ vorced). The 72% of the women were housewives, but 92% of them mentioned to have worked in agriculture. The 77% of the participants were in contact with pesticides; 69% ap‐

(normal 12-20 breaths /min) and blood pressure 120/180 (normal 120/180).

training to be aware of the possible health issues related to pesticides exposure.

#### **2.7. Design and analysis of the studies**

The design of the study 1 was of the type "Case/Control", where 77 men integrated case group and 17 the control group. Participants of both groups were the same ages (18 to 70 years old). Control group did not have evidence of pesticides exposure. The sample size for the cases group represented approximately 5% of the total male population's in the range of ages selected in the study.

The study 2 was integrated by 39 nursing mothers between 17 and 39 years old, selected randomly among those that accepted to participate. All the other characteristics were similar to study 1.

The nominal data were analyzed by group (in study 1) using contingency tables and Chisquare statistics. Continuous numeric data (age, height, weight, etc.) were reported with de‐ scriptive statistics (minimum, maximum, mean, median, and standard deviation). In study 1 there were analysis of variance comparing groups (cases *vs* controls) for the numeric and nominal variables. Several exposure indicators (reported by the literature, erythrocytes, VCM, and RDW) were analyzed by linear regression versus time of exposure, and pesticide amount on a particular body fluid. Also, multiple correlation coefficients were estimated be‐ tween several biological indicators.

For the study 2 beside of the descriptive statistics, some relationships were evaluated such as the use of protective gear, age, number of years of exposure, children's number, among others. The pesticide residues in breast milk were compared with the maximum residual limits stated by international organizations.
