**2. Biocides in organisms: internal actions**

#### **2.1 Uptake at the tissue and cellular level**

The toxicants direct absorption from the water by the integument and gills of crustaceans and/or through the ingestion of contaminated food via the gastrointestinal tract can cause serious toxicity to normal biological functions at the tissue, cellular, and molecular levels. However, the permeability of biological barriers and the rate of transport of chemicals into an organism are affected by the metabolic activity of the animal and, indirectly, by factors influencing this activity (water temperature, pH, hardness, the presence of other chemicals). The metabolic activity of the animal is influenced by its body size, growth rate, physical activity, and physiological state (juvenile or mature, moulting, feeding) (Zitko, 1980).

As the mechanisms of the toxic action of many pesticides usually occur on the surface of or inside the cells (Fent, 2004), the movement of these xenobiotics across membranes depends

The variety of active ingredients used as biocides and their commercial formulations, solvents and coadjuvants or related chemicals is immense. All of them are used by application with agricultural aircraft, sprayers, hand-held units, or trucks that carry the spraying equipment, according to the extension land, application protocols, crop types and soil characteristics. Studies on native fauna are scarce, and only for very few taxa have the biological effects of biocides been studied. Studies on the interrelationships among the fauna components in relation to pesticide use have also been scarce. The actions of each biocide cause different biological responses, e.g., cypermethrin provokes an increase in metabolic activity and glyphosate a decrease (Collins et al., in press). The action of each pesticide is different, and the scarce information in their effects makes it very difficult to recognise the magnitude of the harm caused by these biocides on non-target species and on aquatic environments. The studies that have been conducted have focused on assays involving the active ingredient; however, it is not only the active ingredients that cause damage to the environment but also those compounds that are in the formulation and are considered inert. These compounds can increase the toxicity of the active ingredient, facilitating its ingression in biological systems, or may be toxic by themselves. It is therefore necessary for studies not to ignore commercial formulations, because they may include several compounds that can

Fig. 3. Structure of typical area more affect by biocides through of sprayer with airplane,

The toxicants direct absorption from the water by the integument and gills of crustaceans and/or through the ingestion of contaminated food via the gastrointestinal tract can cause serious toxicity to normal biological functions at the tissue, cellular, and molecular levels. However, the permeability of biological barriers and the rate of transport of chemicals into an organism are affected by the metabolic activity of the animal and, indirectly, by factors influencing this activity (water temperature, pH, hardness, the presence of other chemicals). The metabolic activity of the animal is influenced by its body size, growth rate, physical

activity, and physiological state (juvenile or mature, moulting, feeding) (Zitko, 1980).

As the mechanisms of the toxic action of many pesticides usually occur on the surface of or inside the cells (Fent, 2004), the movement of these xenobiotics across membranes depends

runoff after rain or groundwater potentially contaminated

**2. Biocides in organisms: internal actions** 

**2.1 Uptake at the tissue and cellular level** 

**1.3 Biocides** 

affect aquatic systems.

on the chemical nature of the pesticide involved. Matsumura (1977) summarised the specific properties that influence uptake into aquatic organisms: lipid and water solubility, chemical stability against degradative action by biological systems (biotransformation), and the molecular weight of the chemical. These physicochemical properties determine the affinities of toxic compounds for the materials comprising the arthropod cuticle and plasma membrane of the cell (Hartley & Graham-Bryce, 1980).

Because lipids constitute a substantial part of the plasma membrane, lipid solubility is a very significant factor determining the rate of penetration of many toxic compounds (such as organochlorine pesticides) by passive diffusion through the non-polar portion of the membranes. Lipid solubility is usually characterised by the octanol/water partition coefficient (Kow). In other cases, both facilitated diffusion and active transport are required for the passage of toxic into the cell through channel proteins and via their association with carrier proteins, respectively (Newman & Unger, 2003). The passage through a protein channel occurs down a concentration gradient that may be subject to saturation kinetics, and it is influenced by the size of the molecule, which determines a lower permeability of the membrane with increasing molecular size (Zitko, 1980). Moreover, the uptake of several pesticide compounds requires an active process with an expenditure of metabolic energy in living tissue. Through these pathways, toxicants enter cells and cause alterations in the physicochemical properties of the cytoplasm and the pH of the medium, destruction of the membranes of the organelles, disruption of the normal functioning of the cell proteins, and inhibition of the actions of the enzymes (Sohna et al., 2004; Collins, 2010).

Because in multicellular organisms the distribution of toxicants occurs in more than one compartment, within the crustacean body, haemolymph circulation may be involved in the transport of these chemicals to their sites of action and even more so if it is an open system that flows around the organs. In other arthropods, such as insects, Brooks (1974) reported that phosphoric acid penetrates the cuticle more rapidly than organochlorine insecticides, and having passed this barrier, the toxicant enters the haemolymph and may be transported to all parts of the organism in solution, if water soluble, or bound to proteins or dissolved in lipid particles, if lipophilic. The relatively hydrophilic molecules are much more likely to remain in this circulatory fluid than small, hydrophobic molecules, which are rapidly distributed in several organs and stored in lipid tissue (Hartley & Graham-Bryce, 1980).

#### **2.2 Toxicity and biotransformation**

The adverse effects of toxic products on crustaceans depend on its concentration and affinity, activity (intrinsic toxicity, which is function of molecular structure) and chemical biotransformations (James, 1987) and the acclimation responses of the individual (Klerks, 1999). For biocides, such as organophosphates and carbamate anticholinesterases (anti-ChEs), intrinsic toxicity can be judged by measuring the inhibition of cholinesterase and propagation of action potentials on synaptic transmission (see biomarkers section).

While some organic compounds are sufficiently water-soluble (hydrophilic) for excretion and can be eliminated rapidly, many lipophilic components cannot be directly excreted and would accumulate if not processed to more polar derivatives. Because the unaltered toxicant and any of its transformation products (metabolites) may be excreted, excretion represents a possible protective mechanism against the toxicant (Newman & Unger, 2003). Usually, organic pesticides are subject to modifications through enzyme-catalysed biotransformations leading to *detoxification* or *activation* (Figure 4). Chemical

Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 203

Geraldine, 2001). Conjugation of xenobiotics with reduced glutathione (GSH), catalysed by glutathione S-transferase (GSH S-transferase), is an important physiological process in the elimination of toxic substances from the body. These authors suggest that the activation of such a mechanism probably confers cytoprotection against endosulfan-induced cellular

There are some toxicants in which biotransformation through either Phase I or Phase II can produce a highly reactive chemical, for example, the organophosphorus compounds. Although many of the insecticides in other chemical classes are toxic in their original parent forms, this is not true for many of the organophosphorus insecticides, especially those of the phosphorothioate configuration (such as parathion, chlorpyrifos, and diazinon), characterised by a P=S group. The insecticides possessing a P--S group are usually not very potent anti-ChEs, and they require bioactivation of their P=O metabolites, called oxons, to display appreciable anti-ChE potency (Tang et al., 2005). This bioactivation (reaction of desulfuration) is mediated by cytochrome P450-dependent monooxygenases through an attack on the sulphur by oxygen to create an unstable phosphooxythiiran intermediate (a three-membered ring composed of P, O, and S) that subsequently decomposes to the oxon (P---O) metabolite plus an active form of S (S:). In addition, the S is reactive in the tissues and is capable of damaging some proteins, including the cytochrome P450-dependent

Fig. 4. Different process that can occur in decapods when the animals are affected by some

Other modifications to the toxic action of xenobiotics in crustaceans may occur via the phenomenon of physiological acclimation. In this case, an individual organism that becomes exposed to a specific contaminant may be less severely affected by this contaminant if it had been previously exposed to it. This effect is generally the result of the induction of a detoxification mechanism, as cytochrome P450, in response to the initial exposure (Tang & Garside, 1987; Stuhlbacher et al., 1992). Klerks (1999) observed (in the shrimp, *Palaemonetes pugio*) that acclimation results in an increased resistance at only a limited range of concentrations, with generally no change in resistance at lower pre-exposure levels and a

stress.

monooxygenases.

biocide.

biotransformation in animals occurs via Phase I (functionalisation) and Phase II (conjugation) reactions, which are more readily excreted than the parent compound (Brooks, 1974; Oesch & Arand, 1999).

	- 1. The oxidoreductases include the quantitatively most important superfamily of xenobiotic-metabolising enzymes, the cytochrome P450-dependent monooxygenases (CYP), flavin-containing monooxygenases (FMO), monoamine oxidases (MAO), and cyclooxygenases (COX), all of which introduce oxygen into or remove electrons from their substrates, with a few exceptions.
	- 2. The dehydrogenases and reductases, such as alcohol dehydrogenases, aldehyde dehydrogenases, and carbonyl reductases, add or remove hydrogen atoms to or from the target molecule. The hydrolases comprise families of enzymes specialised in the hydrolysis of esters, amides, epoxides, or glucuronides (Oesch & Arand, 2005).

The predominant functions of Phase I reactions are the conversion of polar, lipophilic compounds into more polar, more hydrophilic compounds and the introduction or liberation of functional groups that can be used for conjugation in the subsequent Phase II of xenobiotic metabolism.

	- 1. Electrophilic substrates are taken over by the glutathione S-transferases (GSH Stransferase).
	- 2. Nucleophilic substrates (i.e., those with hydroxyl, sulfhydryl, amino, or carboxyl groups) are metabolised by UDP-glucuronosyltransferases (UGT), sulfotransferases (SULT), acetyltransferases (AT), acyl-CoA amino acid N-acyltransferases, and methyltransferases.

Phase II involves reactions such as glycosylation, sulfation, mercapturic acid formation, amino acid conjugation, and acetylation. Carboxylic acid groups in xenobiotics can be conjugated with amino acids prior to excretion (Tang et al., 2005). Metabolites formed by conjugation reactions are usually less toxic than the unconjugated compound, although there are notable exceptions to this rule (James, 1987). In addition, the metabolic events that increase the water solubility of a chemical usually cause a significant reduction in its biological half-life by making it more readily excreted (Brooks, 1974).

However, the patterns of activity of key enzymes involved in the detoxification of pesticides can be modified by the same toxic effect of xenobiotics. An elevation in glutathione Stransferase (GSH S-transferase) levels in the hepatopancreas and gills was reported for freshwater prawns (*Macrobrachium malcolmsonii*) and crabs (*Paratelphusa hydrodromus*) exposed to endosulfan, reflecting the formation of glutathione (GSH) and endosulfan complexes as a means of detoxification/elimination (Yadwad, 1989; Saravana Bhavan &

biotransformation in animals occurs via Phase I (functionalisation) and Phase II (conjugation) reactions, which are more readily excreted than the parent compound (Brooks,

1. **Phase I reactions.** In this phase, several enzymes introduce a polar reactive group to the molecule, making it more water soluble while also increasing the possibility of further metabolism by Phase II enzymes. Two major groups of enzymes involved in Phase I metabolism include oxidoreductases and hydrolases that are located in the endoplasmic

1. The oxidoreductases include the quantitatively most important superfamily of xenobiotic-metabolising enzymes, the cytochrome P450-dependent monooxygenases (CYP), flavin-containing monooxygenases (FMO), monoamine oxidases (MAO), and cyclooxygenases (COX), all of which introduce oxygen into or

2. The dehydrogenases and reductases, such as alcohol dehydrogenases, aldehyde dehydrogenases, and carbonyl reductases, add or remove hydrogen atoms to or from the target molecule. The hydrolases comprise families of enzymes specialised in the hydrolysis of esters, amides, epoxides, or glucuronides (Oesch & Arand,

The predominant functions of Phase I reactions are the conversion of polar, lipophilic compounds into more polar, more hydrophilic compounds and the introduction or liberation of functional groups that can be used for conjugation in the subsequent Phase II of

2. **Phase II reactions.** Phase II enzymes often conjugate the polar groups produced by Phase I enzymes to introduce more bulky hydrophilic substituents, such as sugars, sulphates, or amino acids, into the molecule. This conjugation substantially increases the water solubility of a chemical, making it more easily excreted. The conjugation of

1. Electrophilic substrates are taken over by the glutathione S-transferases (GSH S-

2. Nucleophilic substrates (i.e., those with hydroxyl, sulfhydryl, amino, or carboxyl groups) are metabolised by UDP-glucuronosyltransferases (UGT), sulfotransferases (SULT), acetyltransferases (AT), acyl-CoA amino acid N-acyltransferases, and

Phase II involves reactions such as glycosylation, sulfation, mercapturic acid formation, amino acid conjugation, and acetylation. Carboxylic acid groups in xenobiotics can be conjugated with amino acids prior to excretion (Tang et al., 2005). Metabolites formed by conjugation reactions are usually less toxic than the unconjugated compound, although there are notable exceptions to this rule (James, 1987). In addition, the metabolic events that increase the water solubility of a chemical usually cause a significant reduction in its

However, the patterns of activity of key enzymes involved in the detoxification of pesticides can be modified by the same toxic effect of xenobiotics. An elevation in glutathione Stransferase (GSH S-transferase) levels in the hepatopancreas and gills was reported for freshwater prawns (*Macrobrachium malcolmsonii*) and crabs (*Paratelphusa hydrodromus*) exposed to endosulfan, reflecting the formation of glutathione (GSH) and endosulfan complexes as a means of detoxification/elimination (Yadwad, 1989; Saravana Bhavan &

reticulum of the cell in many organs and tissues (James, 1987).

the xenobiotic metabolism is carried out by transferases.

biological half-life by making it more readily excreted (Brooks, 1974).

remove electrons from their substrates, with a few exceptions.

1974; Oesch & Arand, 1999).

2005).

xenobiotic metabolism.

transferase).

methyltransferases.

Geraldine, 2001). Conjugation of xenobiotics with reduced glutathione (GSH), catalysed by glutathione S-transferase (GSH S-transferase), is an important physiological process in the elimination of toxic substances from the body. These authors suggest that the activation of such a mechanism probably confers cytoprotection against endosulfan-induced cellular stress.

There are some toxicants in which biotransformation through either Phase I or Phase II can produce a highly reactive chemical, for example, the organophosphorus compounds. Although many of the insecticides in other chemical classes are toxic in their original parent forms, this is not true for many of the organophosphorus insecticides, especially those of the phosphorothioate configuration (such as parathion, chlorpyrifos, and diazinon), characterised by a P=S group. The insecticides possessing a P--S group are usually not very potent anti-ChEs, and they require bioactivation of their P=O metabolites, called oxons, to display appreciable anti-ChE potency (Tang et al., 2005). This bioactivation (reaction of desulfuration) is mediated by cytochrome P450-dependent monooxygenases through an attack on the sulphur by oxygen to create an unstable phosphooxythiiran intermediate (a three-membered ring composed of P, O, and S) that subsequently decomposes to the oxon (P---O) metabolite plus an active form of S (S:). In addition, the S is reactive in the tissues and is capable of damaging some proteins, including the cytochrome P450-dependent monooxygenases.

Fig. 4. Different process that can occur in decapods when the animals are affected by some biocide.

Other modifications to the toxic action of xenobiotics in crustaceans may occur via the phenomenon of physiological acclimation. In this case, an individual organism that becomes exposed to a specific contaminant may be less severely affected by this contaminant if it had been previously exposed to it. This effect is generally the result of the induction of a detoxification mechanism, as cytochrome P450, in response to the initial exposure (Tang & Garside, 1987; Stuhlbacher et al., 1992). Klerks (1999) observed (in the shrimp, *Palaemonetes pugio*) that acclimation results in an increased resistance at only a limited range of concentrations, with generally no change in resistance at lower pre-exposure levels and a

Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 205

ventilation in the branchial cavity due to the inhibitory action of the pesticide on the nervous system. In contrast, Saravana Bhavan & Geraldine (2001) observed in the prawn, *M. malcolmsonii,* an increase in the content of total free amino acid in the haemolymph as a result of protein degradation. In addition, the accumulation of soluble protein suggests that this was necessary to serve as a compensatory pool to restore enzymes lost to tissue necrosis and to provide prawns with the energy required to cope with the stress of exposure to

Cholinesterases are serine hydrolase enzymes and degrade the neurotransmitters in cholinergic synapses. The toxicity of some pesticides, such as organophosphates and carbamate insecticides, is mainly caused by the inhibition of ChE activity of vertebrates and invertebrates. This inhibition leads to the accumulation of acetylcholine in the synaptic terminals and therefore to a change in the normal transmission of the nervous impulse. This interference may result in neurological manifestations, such as irritability, restlessness, muscular twitching, and convulsions, that may end in the respiratory failure and death of the animal (WHO, 1986). Consequently, most studies describe the use of ChE levels as a biomarker of exposure and/or the effect of several pesticide compounds in aquatic species. However, distinct enzyme isoforms with different sensitivities towards anticholinergic contaminants may exist, depending on the species. These isoforms are usually divided into two broad classes: acetylcholinesterases (AChE) and butyrylcholinesterases (BChE), which

In crustaceans, published studies have also shown mixed results with regard to substrate preference. Fulton & Key (2001) reported that AChE in *Palaemonetes pugio* hydrolyses acetylcholine iodide (ACTH) and acetyl-b-methylthiocholine iodide (AMTH) much faster than other choline esters (such as propionylcholine) and is inactive on butyrylcholine. In contrast, BChE not only hydrolyses butyrylcholine but may also hydrolyse acetylcholine. The two enzyme isoforms may also be distinguished by their susceptibility to selective inhibitors; 1,5-bis-(4-allydimethyl-aminoniumphenyl)-pentan-3-one dibromide (BW284c51) and tetraisopropyl pyrophosphoramide (*iso*-OMPA) are selective inhibitors for AChE and

Organophosphates are generally irreversible inhibitors because the dephosphorylation rate of the bound enzyme proceeds at an insignificant rate. Therefore, the inhibitory effects of organophosphate exposure may be long lasting, with recovery depending on new enzyme synthesis (Habig & Di Giulio, 1991). Several studies with prawn, crab, and lobster species have shown that AChE inhibition in the animals still occurred days after exposure had ended (Reddy & Rao, 1988; McHenery et al*.,* 1991; Abdullah et al*.,* 1994; Key & Fulton, 2002). A slow time course for recovery of depressed AChE levels may cause exposed organisms to be susceptible to other anthropogenic or natural hazards or to exhibit behaviours not

The most abundant and widely studied group of stress proteins is the hsp70 (heat shock protein 70) protein family. The cellular functions of these proteins include the stabilisation of unfolded protein precursors before assembly, translocation of proteins into organelles, rearrangement of protein oligomers, dissolution of protein aggregates, and refolding or

are distinguished primarily based on substrate specificity (Sultatos, 2005).

endosulfan.

**2.3.2 Cholinesterase (ChE) activity (inhibition)** 

BChE, respectively (Sultatos, 2005).

conducive to maintaining the population.

**2.3.3 Stress proteins** 

decreased resistance at higher pre-exposure concentrations that are stressful or result in a significant increase in contaminant body burdens. Such resistance occurs for some contaminants but not for others, and a lack of acclimation to complex mixtures occurs because positive responses to one contaminant are offset by negative responses to another contaminant. According to this observation, this can be explained by the fact that the energetic costs resulting from exposure to one contaminant (either for damage-repair functions or for detoxification processes, such as the production of P450 oxygenases) would compete with the energetic requirements associated with exposure to the other contaminant.

#### **2.3 Biomarkers**

To evaluate effects of pollutants on animal populations, communities and ecosystems, various methods have been developed, ranging from the (sub)cellular to the ecosystem level of biological responses. However, the predictive ability of measurements at higher levels of biological organisation is limited because ecologically important effects (e.g., death or impaired organismal function) have already occurred before they can be detected at population and community levels. In recent decades, biomarkers at suborganismal levels of organisation (biochemical components or processes, physiological functions, and histological structures) have been considered to be viable measures of responses to stressors (Hansen, 2003). These indicators of stress responses are useful in assessing the short-term well-being or long-term health status of an animal (Paterson & Spanoghe, 1997).

Metabolic changes observed in crustaceans exposed to pesticide pollution create widespread disturbances in general physiological processes, such as enzymatic activities, oxygen consumption, and changing energetic requirements. Some of the standardised types of biomarkers are those linked to disturbance to osmoregulation and water balance/ionhomeostasis, cholinesterase inhibition activity, protein stress, oxidative stress, and endocrine disruption.

#### **2.3.1 Haematological parameters**

Alterations in the haemolymph protein, haemocyanin, osmolality, ion compositions, total haemocyte counts, differential haemocyte counts, total free amino acid, nucleic acids (concentrations of DNA and RNA), phenoloxidase (PO) activity, and superoxide anion (O2 - ) may occur in crustaceans as a result of toxicant expositions. Yeh et al. (2005) reported a significant depression in haemolymph osmolality that mainly resulted from a decrease in the haemolymph chloride concentration (Cl-1) in the prawn, *Macrobrachium rosenbergii,* after 8 days of exposure to sublethal concentrations of trichlorfon. However, a decrease in haemolymph pO2 was found among these prawns, which may be related to decreased ventilation and impeded respiratory gas exchange, leading to respiratory disturbances via the inhibition of respiratory mechanisms and damage to respiratory organ epithelial cells. Similarity, a decrease in the pH and HCO3- of the haemolymph induced an increase in the pCO2 level, benefiting the excretion of CO2 in the haemolymph and resulting in a decrease in TCO2, suggesting that trichlorfon disturbs the extracellular acid–base balance of prawns.

In crustaceans, gill lamellae and epipodites are involved in osmoregulation, and the histopathological changes in these structures (haemocytic congestion, gill lamellae necrosis, and the accumulation of particles surrounding the gill lamellae) were observed with lethal concentrations of fenitrothion (Lignot et al., 1997). According to these authors, the presence of particles surrounding the gill lamellae may have been a consequence of a lack of ventilation in the branchial cavity due to the inhibitory action of the pesticide on the nervous system. In contrast, Saravana Bhavan & Geraldine (2001) observed in the prawn, *M. malcolmsonii,* an increase in the content of total free amino acid in the haemolymph as a result of protein degradation. In addition, the accumulation of soluble protein suggests that this was necessary to serve as a compensatory pool to restore enzymes lost to tissue necrosis and to provide prawns with the energy required to cope with the stress of exposure to endosulfan.
