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

204 Pesticides in the Modern World - Risks and Benefits

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.

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

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

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.

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


well-being or long-term health status of an animal (Paterson & Spanoghe, 1997).

**2.3 Biomarkers** 

disruption.

**2.3.1 Haematological parameters** 

Similarity, a decrease in the pH and HCO3

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

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 BChE, respectively (Sultatos, 2005).

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 conducive to maintaining the population.

#### **2.3.3 Stress proteins**

)

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

Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 207

is, by increasing or decreasing their excretion rates and/or biotransformation in the liver,

Neurosecretory structures (X-organ–sinus gland) in the eyestalk are the most important components of the neuroendocrine system of the stalk-eyed crustaceans. The main hormones secreted by the sinus gland are the following: MIH (moult-inhibiting hormone), GIH (gonad-inhibiting hormone), MOIH (mandibular-organ-inhibiting hormone), CHH (crustacean hyperglycaemic hormone), several colour change hormones (controlling pigment migration) and NDH (neurodepressing hormone). Some of these hormones have a second endocrine gland as their target (MIH, GIH, MOIH), while the others have somatic tissues as targets. MIH, GIH, MOIH and CHH belong to a single family of peptides (Fingerman et al., 1998; Chang, 2001). These neuropeptides, synthesised in the XO (X-organ), a cluster of neuron perikarya located in the medulla terminalis of the eyestalk, are transported to and stored in the axon terminals, forming a neurohaemal organ named SG (sinus gland) and released by exocytosis into the haemolymph

The CHH have been shown to regulate carbohydrate metabolism in the shore crab, *Carcinus maenas*; the kumuran prawn, *Penaeus japonicus*; the lobster, *Homarus americanus*; the freshwater crab, *Oziotelphusa senex senex*; and the fiddler crab, *Uca triangularis* (Kegel et al., 1989; Lorenzon 2005; Purna Chandra Nagaraju et al., 2005). The neurotransmitter, 5-HT (serotonin), plays a fundamental role in hormone (CHH) modulation, and at the same time, pollutants can alter their level and function. Therefore, 5-HT has been known to have a potent hyperglycaemic effect with increases in the glucose haemolymphatic concentration resulting mainly from the stimulation of glycogen breakdown in the hepatopancreas (Fingerman et al., 1998). Hyperglycaemia is a typical response of several crustacean species to chemical stressors, including some pesticides, hydrocarbons and heavy metals. However, several reports have shown that an increased haemolymphatic level of glucose alone does not necessarily prove that there was a disruptive effect on the endocrine system. Because CHH is released to raise glycaemia as an adaptive response to several stimuli (such as emersion, starvation, critical temperatures and others), this hormone has been proposed as

Crustaceans are considered as carrying a simple and primitive immune system (Fig. 5). The hepatopancreas is known as the detoxification site and also as a sensitive organ to stress, as

The hepatopancreas is essentially composed of branched tubules and of 4 types of epithelial cells: embryonic cells (E-cells), fibrillenzellen cells (F-cells), restzellen cells (R-cells) and blasenzellen cells (B-cells). E-cells are the only ones showing mitotic activity, being important in dead cell replacement. R-cells have absorptive functions supported by the presence of lipid droplets in the cytoplasm. These cells are involved in the delivery of nutrients to other organs via the haemolymph; the nutrient reserves are mobilised through R-cells to provide energy to the rest of the body. In addition, R-cells are interpreted as sites of intracellular waste deposition characterised by autophagosomes and residual bodies. These cells detoxify heavy metals and other lipophilic compounds by their accumulation in a soluble form in the cytoplasm, followed by excretion. F-cells are where protein synthesis

functioning as a crustacean stress hormone (Chang, 2001).

it quickly responds to exposure to noxious compounds.

and enzyme production occurs (Sousa et al., 2005).

hepatopancreas or other organs.

(Lorenzon, 2005).

**2.4 Histological effects** 

degradation of denatured proteins (Feige & Polla, 1995). Induction of stress protein synthesis by pesticides is reported to be highly tissue-specific in aquatic animals. Among the tissues analysed (gill, skeletal muscle and hepatopancreas) by Selvakumar et al. (2005) in *Macrobrachium malcolmsonii*, induction of hsp70 synthesis was recorded only in the gill tissue of prawns that had been exposed to sublethal concentrations of endosulfan. In contrast, exposure of prawns to sublethal concentrations of carbaryl failed to elicit hsp70 synthesis in any of the three tissues analysed.
