**2.3.4 Oxidative stress**

Under normal conditions, equilibrium exists between the amounts of free radicals generated and antioxidants available to quench or scavenge them, thereby protecting the organism against the deleterious effects of pollutants. However, oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted as a result of the depletion of antioxidants or excessive accumulation of the reactive oxygen species (ROS), or both, leading to damage to macromolecular components (Scandalios, 2005). Many xenobiotics, such as pesticides, may cause oxidative stress, leading to the generation of ROS and alterations in antioxidants or free oxygen radicals scavenging enzyme systems in aquatic animals (Dettbarn et al., 2005). However, the cells of crustaceans possess a variety of chemical and enzymatic mechanisms to protect them from oxidative damage. These mechanisms include an enzymatic antioxidant defence system comprising enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione Stransferase (GSH S-transferase) and non-enzymatic antioxidants like glutathione (GSH), ascorbic acid (vitamin C) and a-tocopherol (vitamin E), which are capable of neutralising or scavenging the reactive oxygen species (Vijayavel & Balasubramanian, 2009). These authors showed that the toxicity of fenvalerate to the prawn, *Penaeus monodon,* led to a significant induction of lipid peroxidation and GSH S-transferase activity in the hepatopancreas, muscle and gills. On the contrary, the activities of SOD, CAT, glutathione peroxidase, vitamin C, vitamin E and GSH were reduced in prawns exposed to sublethal concentrations of fenvalerate.

#### **2.3.5 Neuroendocrine systems**

Toxicity induced by a pesticide is the result of interaction of the compound or one of its metabolites with the biochemical events involved in the homeostatic control of a physiological process (Newman & Unger, 2003). Physiological processes are mostly coordinated by hormones. Therefore, the effects of organic compounds on functions regulated by hormones in crustaceans could be used as biomarkers of environmental pollutants.

According to Rodríguez et al. (2007), endocrine disruption can take place at different physiological levels: 1) altering (inhibiting or stimulating) the secretion of hormones; this possible effect is related to mechanisms that control both the release of hormones from endocrine cells and the synthesis of these hormones; 2) interfering with hormone-receptor interaction; in this sense, endocrine-disrupting compounds (EDCs) can act as agonists or antagonists by directly binding to a hormone receptor. Indirectly, however, an EDC could interfere via several mechanisms at any step of the transductional pathway of a hormone, therefore altering its final effect; 3) modifying the metabolism of circulating hormones, that is, by increasing or decreasing their excretion rates and/or biotransformation in the liver, hepatopancreas or other organs.

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 (Lorenzon, 2005).

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

#### **2.4 Histological effects**

206 Pesticides in the Modern World - Risks and Benefits

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

Under normal conditions, equilibrium exists between the amounts of free radicals generated and antioxidants available to quench or scavenge them, thereby protecting the organism against the deleterious effects of pollutants. However, oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted as a result of the depletion of antioxidants or excessive accumulation of the reactive oxygen species (ROS), or both, leading to damage to macromolecular components (Scandalios, 2005). Many xenobiotics, such as pesticides, may cause oxidative stress, leading to the generation of ROS and alterations in antioxidants or free oxygen radicals scavenging enzyme systems in aquatic animals (Dettbarn et al., 2005). However, the cells of crustaceans possess a variety of chemical and enzymatic mechanisms to protect them from oxidative damage. These mechanisms include an enzymatic antioxidant defence system comprising enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione Stransferase (GSH S-transferase) and non-enzymatic antioxidants like glutathione (GSH), ascorbic acid (vitamin C) and a-tocopherol (vitamin E), which are capable of neutralising or scavenging the reactive oxygen species (Vijayavel & Balasubramanian, 2009). These authors showed that the toxicity of fenvalerate to the prawn, *Penaeus monodon,* led to a significant induction of lipid peroxidation and GSH S-transferase activity in the hepatopancreas, muscle and gills. On the contrary, the activities of SOD, CAT, glutathione peroxidase, vitamin C, vitamin E and GSH were reduced in prawns exposed to sublethal concentrations

Toxicity induced by a pesticide is the result of interaction of the compound or one of its metabolites with the biochemical events involved in the homeostatic control of a physiological process (Newman & Unger, 2003). Physiological processes are mostly coordinated by hormones. Therefore, the effects of organic compounds on functions regulated by hormones in crustaceans could be used as biomarkers of environmental

According to Rodríguez et al. (2007), endocrine disruption can take place at different physiological levels: 1) altering (inhibiting or stimulating) the secretion of hormones; this possible effect is related to mechanisms that control both the release of hormones from endocrine cells and the synthesis of these hormones; 2) interfering with hormone-receptor interaction; in this sense, endocrine-disrupting compounds (EDCs) can act as agonists or antagonists by directly binding to a hormone receptor. Indirectly, however, an EDC could interfere via several mechanisms at any step of the transductional pathway of a hormone, therefore altering its final effect; 3) modifying the metabolism of circulating hormones, that

any of the three tissues analysed.

**2.3.4 Oxidative stress** 

of fenvalerate.

pollutants.

**2.3.5 Neuroendocrine systems** 

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 it quickly responds to exposure to noxious compounds.

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

Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 209

imbalance and its histological effects may eventually cause the death of the affected individual because of acidosis or progressive asphyxia (Vonk, 1960, Schmidt Nielsen, 1997).

Fig. 5. Histological pesticide effects in crustacean organs and its results. Crab image

Another effect of hypoxia is a decrease in locomotion. Crustaceans regulate their oxygen consumption within a range of dissolved oxygen concentrations. The minimal dissolved oxygen concentration within this range is called the critical oxygen concentration, below which crustaceans are not able to regulate their oxygen consumption. Given this situation of hypoxia, many crustaceans reduce their movements as a way of reducing the oxygen consumed by muscles, using the available oxygen instead of using it for metabolism (Zou et al., 1992, Zou & Stueben, 2006). The same response occurs if an animal has a deficiency in oxygen uptake, as in both situations the oxygen concentration in haemolymph is unsaturated. The oxygen deficiency causes a decrease in locomotive activities, with several effects. Animals are unable to escape from the contaminated area, exacerbating the effects of the contaminants. At the same time, a decrease in movement, as mentioned above makes

In addition to their respiratory function, gills play an important role in nitrogen compound excretion. Crustaceans are ammoniotelic animals, i.e., their nitrogenous metabolic end products are mainly excreted in the form of ammonia. The antennal gland plays the key role in body water and divalent cation regulation, but it plays a minor role in ammonia excretion, as in some cases less than 2% of the total ammonia is excreted in the urine via the

The high lipid solubility of ammonia makes it more diffusible through phospholipid bilayers. The mechanism supporting ammonia excretion in crustaceans is the simple diffusion of the non-ionic NH3 along a concentration gradient and the partial excretion of

Several aquatic crab species possess an excretion system based on the ionised form of ammonia, NH4+, a water soluble compound which effluxes through the gill epithelium. Freshwater crabs have tighter gill epithelia than their marine relatives, developed to avoid ionic efflux and tolerate a hyposmotic environment. This epithelium is much less permeable by NH4+, and freshwater crabs release their nitrogen compounds mainly as ammonia

+, whose release through diffusion is facilitated because of its

modified from Collins et al. (2004).

animals more susceptible to predators.

hydrophobicity (Weihrauch et al., 1999, 2004).

the ionised form NH4

(Weihrauch et al., 1999).

antennal gland system (Parry, 1960; Cameron & Batterton, 2004).

Exposure to pesticides causes an imbalance in epithelial cells. Among the effects found, biocides cause an increase in R- and F-cells and an inhibition in E-cells. An R-cell increase in response to noxious compounds may be related to two different strategies. More R-cells may increase the detoxification rate because a higher number of cells increases detoxification. However, noxious compounds cause effects not only in the hepatopancreas but also in gills, gonads, and other organs. As other body parts require energy to recover from deleterious effects, the R-cell number increases for transporting energetic resources, i.e., lipids. When submitted to pesticides, F-cells increase for the production of more enzymes as a way of deactivating toxic compounds. R- and F-cells increase because both cellular types play roles in detoxification, and each one develops a different action for the same purpose. A decrease in E-cells becomes important if we consider that pesticides cause necrosis and increase cellular apoptosis. These cells replace dead cells with new ones, trying to mitigate cell loss. Exposure to pesticides also causes haemocytic infiltration in the interstitial sinus, abnormal lumen of the tubules, separation of necrotic cells from basal laminae, thickened basal laminae, necrotic tubules containing tissue debris, melanisation and coagulation in the thickened basal laminae and walling off of the tubules by haemocytes around the thickened basal laminae. All these effects combined may cause deficiencies in hepatopancreas function, with in turn may cause death (Saravana Bhavan & Geraldine, 2000, Bianchini & Monserrat 2007, Collins 2010).

In gills, one of the most important intake sites, biocide exposure also causes several histological damages, which in turn may cause functional deficiencies. Haemocytic infiltration in the haemocoelic space, swelling of the gill lamellae, lifting of lamellar epithelium, fusion of lamellae, abnormalities in the histoarchitecture, necrosis and other malformations are some the effects produced by pesticides in freshwater prawns and crabs. Gills are related to the transport of respiratory gases, their obvious function, and also with ammonia excretion, as the majority of waste nitrogenous compound excretion occurs through the gill epithelium. Gill damage may also cause difficulties in oxygen intake, eventually asphyxia, and disrupt osmoregulatory function. A decrease in oxygen consumption may cause progressive internal hypoxia, with several effects such as metabolism shifts and locomotive difficulties. Crustaceans generally maintain an aerobic metabolism as a way of obtaining energy from food reserves. Aerobic metabolism, through the Krebs cycle, provides more energy than anaerobic metabolism. However, this kind of cellular "respiration" requires enough oxygen to be developed. When the amount of oxygen needed for the maintenance of aerobic metabolism in not achieved, crustaceans obtain energy by glycolysis, an anaerobic metabolism of carbohydrates. This type of metabolism, although it allows individuals to obtain energy for vital actions, has two serious effects: lactic acid release and an underutilisation of the energy accumulated. While in aerobic metabolism, animals obtain 36 mol of ATP from 1 mol of glucose, in anaerobic metabolism, they obtain only 2 mol of ATP from 1 mol of glucose, with the production of 2 mol of lactic acid (Schmidt-Nielsen, 1997). In animals with sporadic hypoxia, lactic acid is used as a substrate for further oxidation, completing the Krebs cycle and gaining the full energy value of the original carbohydrate substrate. However, in animals with oxygen intake decreased by histological damage, hypoxia may be not temporary; if they continue to be exposed to the aggressor agent, gills are not able to reconstitute itselves, or recuperation time is not quick enough to supply the oxygen demand. The continuous internal hypoxia may provoke a constant release of lactic acid as metabolic waste, with the consequent acid imbalance. This

Exposure to pesticides causes an imbalance in epithelial cells. Among the effects found, biocides cause an increase in R- and F-cells and an inhibition in E-cells. An R-cell increase in response to noxious compounds may be related to two different strategies. More R-cells may increase the detoxification rate because a higher number of cells increases detoxification. However, noxious compounds cause effects not only in the hepatopancreas but also in gills, gonads, and other organs. As other body parts require energy to recover from deleterious effects, the R-cell number increases for transporting energetic resources, i.e., lipids. When submitted to pesticides, F-cells increase for the production of more enzymes as a way of deactivating toxic compounds. R- and F-cells increase because both cellular types play roles in detoxification, and each one develops a different action for the same purpose. A decrease in E-cells becomes important if we consider that pesticides cause necrosis and increase cellular apoptosis. These cells replace dead cells with new ones, trying to mitigate cell loss. Exposure to pesticides also causes haemocytic infiltration in the interstitial sinus, abnormal lumen of the tubules, separation of necrotic cells from basal laminae, thickened basal laminae, necrotic tubules containing tissue debris, melanisation and coagulation in the thickened basal laminae and walling off of the tubules by haemocytes around the thickened basal laminae. All these effects combined may cause deficiencies in hepatopancreas function, with in turn may cause death (Saravana Bhavan & Geraldine, 2000, Bianchini &

In gills, one of the most important intake sites, biocide exposure also causes several histological damages, which in turn may cause functional deficiencies. Haemocytic infiltration in the haemocoelic space, swelling of the gill lamellae, lifting of lamellar epithelium, fusion of lamellae, abnormalities in the histoarchitecture, necrosis and other malformations are some the effects produced by pesticides in freshwater prawns and crabs. Gills are related to the transport of respiratory gases, their obvious function, and also with ammonia excretion, as the majority of waste nitrogenous compound excretion occurs through the gill epithelium. Gill damage may also cause difficulties in oxygen intake, eventually asphyxia, and disrupt osmoregulatory function. A decrease in oxygen consumption may cause progressive internal hypoxia, with several effects such as metabolism shifts and locomotive difficulties. Crustaceans generally maintain an aerobic metabolism as a way of obtaining energy from food reserves. Aerobic metabolism, through the Krebs cycle, provides more energy than anaerobic metabolism. However, this kind of cellular "respiration" requires enough oxygen to be developed. When the amount of oxygen needed for the maintenance of aerobic metabolism in not achieved, crustaceans obtain energy by glycolysis, an anaerobic metabolism of carbohydrates. This type of metabolism, although it allows individuals to obtain energy for vital actions, has two serious effects: lactic acid release and an underutilisation of the energy accumulated. While in aerobic metabolism, animals obtain 36 mol of ATP from 1 mol of glucose, in anaerobic metabolism, they obtain only 2 mol of ATP from 1 mol of glucose, with the production of 2 mol of lactic acid (Schmidt-Nielsen, 1997). In animals with sporadic hypoxia, lactic acid is used as a substrate for further oxidation, completing the Krebs cycle and gaining the full energy value of the original carbohydrate substrate. However, in animals with oxygen intake decreased by histological damage, hypoxia may be not temporary; if they continue to be exposed to the aggressor agent, gills are not able to reconstitute itselves, or recuperation time is not quick enough to supply the oxygen demand. The continuous internal hypoxia may provoke a constant release of lactic acid as metabolic waste, with the consequent acid imbalance. This

Monserrat 2007, Collins 2010).

imbalance and its histological effects may eventually cause the death of the affected individual because of acidosis or progressive asphyxia (Vonk, 1960, Schmidt Nielsen, 1997).

Fig. 5. Histological pesticide effects in crustacean organs and its results. Crab image modified from Collins et al. (2004).

Another effect of hypoxia is a decrease in locomotion. Crustaceans regulate their oxygen consumption within a range of dissolved oxygen concentrations. The minimal dissolved oxygen concentration within this range is called the critical oxygen concentration, below which crustaceans are not able to regulate their oxygen consumption. Given this situation of hypoxia, many crustaceans reduce their movements as a way of reducing the oxygen consumed by muscles, using the available oxygen instead of using it for metabolism (Zou et al., 1992, Zou & Stueben, 2006). The same response occurs if an animal has a deficiency in oxygen uptake, as in both situations the oxygen concentration in haemolymph is unsaturated. The oxygen deficiency causes a decrease in locomotive activities, with several effects. Animals are unable to escape from the contaminated area, exacerbating the effects of the contaminants. At the same time, a decrease in movement, as mentioned above makes animals more susceptible to predators.

In addition to their respiratory function, gills play an important role in nitrogen compound excretion. Crustaceans are ammoniotelic animals, i.e., their nitrogenous metabolic end products are mainly excreted in the form of ammonia. The antennal gland plays the key role in body water and divalent cation regulation, but it plays a minor role in ammonia excretion, as in some cases less than 2% of the total ammonia is excreted in the urine via the antennal gland system (Parry, 1960; Cameron & Batterton, 2004).

The high lipid solubility of ammonia makes it more diffusible through phospholipid bilayers. The mechanism supporting ammonia excretion in crustaceans is the simple diffusion of the non-ionic NH3 along a concentration gradient and the partial excretion of the ionised form NH4+, whose release through diffusion is facilitated because of its hydrophobicity (Weihrauch et al., 1999, 2004).

Several aquatic crab species possess an excretion system based on the ionised form of ammonia, NH4+, a water soluble compound which effluxes through the gill epithelium. Freshwater crabs have tighter gill epithelia than their marine relatives, developed to avoid ionic efflux and tolerate a hyposmotic environment. This epithelium is much less permeable by NH4 +, and freshwater crabs release their nitrogen compounds mainly as ammonia (Weihrauch et al., 1999).

Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 211

Gonads are characterised by fewer but bigger oocytes, with more energetic reserves for the extended embryonic stage. In subtropical regions, gonad development occurs during late winter, spring and summer, the same period when pesticide applications. The drift and runoff provoke the migration of biocides to aquatic environments, causing a continuous

Ovary growth in crustaceans has two different periods: endogenous vitellogenesis (vitellogenesis I) and exogenous vitellogenesis (vitellogenesis II). The first period is characterised by an autosynthesis of lipovitellin and slow oocyte growth. The second period is characterised by the input of exogenous vitellogenin (a vitellin precursor) from outside of the ovary, mainly from the hepatopancreas, and rapid oocytic growth. Along with all the compounds provided by the hepatopancreas, lipophilic pesticides migrate to the ovaries (Lubzens et al., 1995). The effects of these biocides include abnormalities in shape, as the loss of the typical spherical shape of ovarian follicles; abnormal oocyte area increase or decrease, depending on pesticide type; and oocyte atresia (Rodriguez et al., 1994, Lee et al., 1996). The abnormal development of the ovaries causes a reduction in the available oocytes for fecundation, with the consequent reduction in eggs and the future brood, decreasing the

Once fecundation occurs, females carry their eggs in their pleon until juveniles or mysis hatch. If these females live in contaminated areas, the exposure to biocides causes different effects in eggs and embryos. The easiest observable effect is death, but embryo death may occur at relatively high pesticide concentrations. Eggs are surrounded by the chorion, which isolates them from the environment. In the case of freshwater decapods, the chorion is thicker than that present in marine decapods because it has to protect the embryo from the osmotic stress caused by the environment. This thicker chorion also isolates the embryos from biocides and other compounds (Lindley et al., 1999; Varó et al., 2006). This protective effect makes embryos more resistant to toxicants, in some cases more resistant than juveniles, with a median lethal concentration similar to adults in several cases (Key et al., 2003; Li et al., 2006). Furthermore, embryos are more sensitive to pesticides when they are close to hatching because of the thinning of the chorion, which allows more pesticide to enter into the egg. This effect is also observable in prawns exposed to different salinity levels, as embryos are more sensitive to osmotic stress when

In addition to lethality, constant exposure to pesticides may cause differences in incubation periods and several abnormalities in embryos. Among these abnormalities, biocides may cause hydropsy, abnormal eye spots and several atrophies in the eyes, the pleon and the dorsal spine (Rodriguez & Pisanó, 1993; Lee & Oshima, 1998). All these abnormalities provoke the death of the juvenile, either from internal malformations of organs or from the incapability to moult successfully. Additionally, abnormalities in pleopods and pereiopods

Constant exposure to pesticides causes a reduction in functional oocytes, resulting in fewer eggs, a reduction in surviving embryos and a decrease in juveniles that will reach the adult stage, which in turn provokes effects on populations, the community and the ecosystem.

Freshwater environments impose a severe osmotic stress to the animals living there. Marine crustacean reproduction is characterised by a large brood, which hatches as larvae and

contact with females during gonad maturation.

population over the short and medium term.

they are close to hatching (Ituarte et al., 2005).

cause the inability to eat, find food or avoid predators.

**3. Reproduction effects** 

Concentrations of NH3 in the environment are kept low as a result of bacterial nitrification of ammonia to nitrite and nitrate, followed by the absorption of autotrophic organisms. This kind of environment favours ammonia excretion as a passive process driven by diffusion along a gradient. This process applies only to pelagic animals, generally prawns, colonising the water column, where the dilution and nitrification processes of aquatic biota keep ammonia concentrations really low. Benthic animals, such as crabs and crayfishes, are often faced with higher ambient concentrations of ammonia, present especially in anoxic, deep, stagnant water. Some species take refuge by hiding in riparian rocks and vegetation. Other species bury themselves in mud or, in the case of burrowing species, build extensive caves, in some cases more than 1 metre deep, with aerial or aquatic entry holes. They live in the bottom, where they find protection from predators and where the water is stagnant for hours. The very low water exchange rate, along with the fact that animals produce and excrete metabolic ammonia, increases the ammonia concentration and difficult simple diffusion.

The process that these crustaceans uses to eliminate ammonia is active excretion. Ammonia excretion rates are correlated with Na+ absorption (Pressley et al., 1981, Harris et al., 2001). NH4+ substitutes for K+ in the activation of the ouabain-sensitive Na+/K+-ATPase, which is located in the basolateral membranes of the gill epithelium cells (Towle et al., 1981; Towle & Kays, 1986). This Na+/K+-ATPase is synergistically stimulated by NH4+ and K+. In freshwater decapods, at high NH4+ concentrations, the pump exposes a new binding site for NH4+ that modulates the activity of the Na+/K+-ATPase independently of K+ ions (Romano & Zeng, 2007, 2010).

Histological damage in gills, and the mucus segregation observed in prawns exposed to pesticides, may hinder ammonia excretion. These effects are especially relevant in freshwater benthic crustaceans, mainly crabs and crayfishes. As mentioned above, the passive efflux of ammonium (NH4+) is difficult because of the thickened gill epithelium, while ammonia excretion (NH3) is difficult because of the environmental concentration. Nitrogenous waste compounds are eliminated by active efflux, and histological damage provoked by pesticides hinders this excretion process. When decapods are not able to eliminate the ammonia produced by nitrogen compound metabolism, it accumulates in the haemolymph, with several effects on individuals. Ammonia modifies the release of cytokines and increases the activity of lysosomal hydrolases. Ammonia toxicity is mediated by the excessive activation of *N*-methyl-*D*-aspartate (NMDA)-type glutamate receptors in the brain. As a consequence, cerebral ATP is depleted, while intracellular Ca2+ increases, with subsequent increases in intracellular K+ and, finally, cell death (Weihrauch et al., 1999, 2004).

The intensity of the observed effects is related to pesticide concentration and animal resistance. Nevertheless, many of the described effects were achieved at concentrations that usually occur in the environment after aerial or terrestrial pesticide applications. The constant aggression provoked by biocides induces malfunctions in this vital organ, which eventually may cause the death of an individual.

#### **2.4.1 Histopathological effects on female gonads**

Freshwater decapods modified their reproductive strategy when they conquered freshwater environments. Larval stages were abbreviated or suppressed, and females invest their energy in fewer but more expensive progeny, which hatch at a more advanced stage.

Concentrations of NH3 in the environment are kept low as a result of bacterial nitrification of ammonia to nitrite and nitrate, followed by the absorption of autotrophic organisms. This kind of environment favours ammonia excretion as a passive process driven by diffusion along a gradient. This process applies only to pelagic animals, generally prawns, colonising the water column, where the dilution and nitrification processes of aquatic biota keep ammonia concentrations really low. Benthic animals, such as crabs and crayfishes, are often faced with higher ambient concentrations of ammonia, present especially in anoxic, deep, stagnant water. Some species take refuge by hiding in riparian rocks and vegetation. Other species bury themselves in mud or, in the case of burrowing species, build extensive caves, in some cases more than 1 metre deep, with aerial or aquatic entry holes. They live in the bottom, where they find protection from predators and where the water is stagnant for hours. The very low water exchange rate, along with the fact that animals produce and excrete metabolic ammonia, increases the ammonia concentration and difficult simple

The process that these crustaceans uses to eliminate ammonia is active excretion. Ammonia excretion rates are correlated with Na+ absorption (Pressley et al., 1981, Harris et al., 2001). NH4+ substitutes for K+ in the activation of the ouabain-sensitive Na+/K+-ATPase, which is located in the basolateral membranes of the gill epithelium cells (Towle et al., 1981; Towle & Kays, 1986). This Na+/K+-ATPase is synergistically stimulated by NH4+ and K+. In freshwater decapods, at high NH4+ concentrations, the pump exposes a new binding site for NH4+ that modulates the activity of the Na+/K+-ATPase independently of K+ ions (Romano

Histological damage in gills, and the mucus segregation observed in prawns exposed to pesticides, may hinder ammonia excretion. These effects are especially relevant in freshwater benthic crustaceans, mainly crabs and crayfishes. As mentioned above, the passive efflux of ammonium (NH4+) is difficult because of the thickened gill epithelium, while ammonia excretion (NH3) is difficult because of the environmental concentration. Nitrogenous waste compounds are eliminated by active efflux, and histological damage provoked by pesticides hinders this excretion process. When decapods are not able to eliminate the ammonia produced by nitrogen compound metabolism, it accumulates in the haemolymph, with several effects on individuals. Ammonia modifies the release of cytokines and increases the activity of lysosomal hydrolases. Ammonia toxicity is mediated by the excessive activation of *N*-methyl-*D*-aspartate (NMDA)-type glutamate receptors in the brain. As a consequence, cerebral ATP is depleted, while intracellular Ca2+ increases, with subsequent increases in intracellular K+ and, finally, cell death (Weihrauch et al., 1999,

The intensity of the observed effects is related to pesticide concentration and animal resistance. Nevertheless, many of the described effects were achieved at concentrations that usually occur in the environment after aerial or terrestrial pesticide applications. The constant aggression provoked by biocides induces malfunctions in this vital organ, which

Freshwater decapods modified their reproductive strategy when they conquered freshwater environments. Larval stages were abbreviated or suppressed, and females invest their energy in fewer but more expensive progeny, which hatch at a more advanced stage.

eventually may cause the death of an individual.

**2.4.1 Histopathological effects on female gonads** 

diffusion.

2004).

& Zeng, 2007, 2010).

Gonads are characterised by fewer but bigger oocytes, with more energetic reserves for the extended embryonic stage. In subtropical regions, gonad development occurs during late winter, spring and summer, the same period when pesticide applications. The drift and runoff provoke the migration of biocides to aquatic environments, causing a continuous contact with females during gonad maturation.

Ovary growth in crustaceans has two different periods: endogenous vitellogenesis (vitellogenesis I) and exogenous vitellogenesis (vitellogenesis II). The first period is characterised by an autosynthesis of lipovitellin and slow oocyte growth. The second period is characterised by the input of exogenous vitellogenin (a vitellin precursor) from outside of the ovary, mainly from the hepatopancreas, and rapid oocytic growth. Along with all the compounds provided by the hepatopancreas, lipophilic pesticides migrate to the ovaries (Lubzens et al., 1995). The effects of these biocides include abnormalities in shape, as the loss of the typical spherical shape of ovarian follicles; abnormal oocyte area increase or decrease, depending on pesticide type; and oocyte atresia (Rodriguez et al., 1994, Lee et al., 1996). The abnormal development of the ovaries causes a reduction in the available oocytes for fecundation, with the consequent reduction in eggs and the future brood, decreasing the population over the short and medium term.

Once fecundation occurs, females carry their eggs in their pleon until juveniles or mysis hatch. If these females live in contaminated areas, the exposure to biocides causes different effects in eggs and embryos. The easiest observable effect is death, but embryo death may occur at relatively high pesticide concentrations. Eggs are surrounded by the chorion, which isolates them from the environment. In the case of freshwater decapods, the chorion is thicker than that present in marine decapods because it has to protect the embryo from the osmotic stress caused by the environment. This thicker chorion also isolates the embryos from biocides and other compounds (Lindley et al., 1999; Varó et al., 2006). This protective effect makes embryos more resistant to toxicants, in some cases more resistant than juveniles, with a median lethal concentration similar to adults in several cases (Key et al., 2003; Li et al., 2006). Furthermore, embryos are more sensitive to pesticides when they are close to hatching because of the thinning of the chorion, which allows more pesticide to enter into the egg. This effect is also observable in prawns exposed to different salinity levels, as embryos are more sensitive to osmotic stress when they are close to hatching (Ituarte et al., 2005).

In addition to lethality, constant exposure to pesticides may cause differences in incubation periods and several abnormalities in embryos. Among these abnormalities, biocides may cause hydropsy, abnormal eye spots and several atrophies in the eyes, the pleon and the dorsal spine (Rodriguez & Pisanó, 1993; Lee & Oshima, 1998). All these abnormalities provoke the death of the juvenile, either from internal malformations of organs or from the incapability to moult successfully. Additionally, abnormalities in pleopods and pereiopods cause the inability to eat, find food or avoid predators.

Constant exposure to pesticides causes a reduction in functional oocytes, resulting in fewer eggs, a reduction in surviving embryos and a decrease in juveniles that will reach the adult stage, which in turn provokes effects on populations, the community and the ecosystem.

#### **3. Reproduction effects**

Freshwater environments impose a severe osmotic stress to the animals living there. Marine crustacean reproduction is characterised by a large brood, which hatches as larvae and

Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World 213

Fig.6. Pesticide effects in crustaceans' behavior and its results in population. Crustacean

The eggs of ovigerous females that live in contaminated areas may be resistant to pollutants, but toxicants may cause the death of juveniles after hatching, when they are not protected by a chorion. Moreover, moult events are a critical period for crustaceans, as their exoskeletons become softer and they are more vulnerable to external contaminants such as pesticides. In juveniles, the intermoult period is short, and the lethal effects of pesticides are

Growth is an interesting aspect in decapods in that it includes both internal and external factors. The intermoult period and increase in size are affected by different factors, such as diet (mainly protein, and lipid level variation), interspecific interactions (searching for agonistic behaviour and hierarchical conditions), temperature, biocides (or xenobiotic elements). Moreover, the growth in many species shows isometry and/or allometry variations in the ontogeny, and thus growth pattern can be affected. The study methods are different according to a study's objectives. In some cases, the animals are evaluated in groups, e.g., with diets; in other cases, a study is conducted with isolated animals to observe

The capacity of an organism for survival, growth, and reproduction involves competition for energy resources at the individual level (Schmidt-Nielsen, 1997). Toxicant-induced shifts in energy allocations to these life-history activities will have important consequences on population. For example, higher respiration rates of estuarine crustaceans sublethally exposed to a variety of pesticides reduced juvenile growth by lowering growth efficiency rates, suggesting that increased metabolic demands lowered the amount of assimilated energy available for production of new tissue (McKenney & Hamaker, 1984; McKenney & Matthews, 1990). The assessment of changes in growth and energy stores of toxicantsensitive life stages have a direct link to ecological consequences of environmental stress and can be useful as biomarkers to diagnose early damage in aquatic populations (Newman

image modified from Collins et al. 2004.

increased during that critical period.

the xenobiotic effects on growth through chronic assays.

**4. Growth** 

& Unger, 2003).

undergoes several stages up to the juvenile stage. Freshwater environments impose a severe osmotic stress on unprotected eggs and the free larvae stage. In the same way, developing embryos must be protected against this stress. When they conquered these environments, decapods developed different strategies to protect eggs and embryos. The primitive pelagic larval phases were suppressed; larval stages occur inside the egg, and the offspring hatch as mysis or juveniles. This internal development (i.e., inside the egg) imposes a greater protection to embryos against environmental pressures, especially in the susceptible larval stages. To support these internal stages, eggs increased in size and energy resources, mainly lipoproteins, because embryos grow inside the eggs and use their internal energy resources. Freshwater decapod females carry their eggs in the pleon, protecting them until the larvae or juveniles hatch. Because of their increased size, the number of eggs that a female can carry decreased, resulting in a concomitant decrease in the number of offspring (Ruppert & Barnes, 1994; Lee & Bell, 1999).

Several pesticides are highly lipophilic and are accumulated mainly in lipid reserves. During ovary development, oocytes accumulate lipids and lipoproteins, mainly lipovitellin, forming the vellum, which in turn will be used by the embryo as an energetic resource (the embryo "feeds" on the vellum). Attached to the lipovitellins, pesticides enter to the oocytes and accumulate on them. One explanation for the relatively greater resistance of females to organic pollutants is the distribution of these toxicants in the ovary, decreasing their concentrations in vital organs such as the hepatopancreas and delaying death (Sheridan, 1975; Menone et al., 2000; Wirth et al., 2001; Menone et al., 2004, 2006; Santos de Souza et al., 2008). The presence of pesticides in the oocytes implies that the embryo, beginning with fertilisation, is exposed to pesticides. Embryos grow and feed on the lipid reserves present on the vellum, with the consequent intake of pesticides. This may provoke not only the death of the embryos, with the release of dead eggs by the female, but also sublethal effects, such as abnormal size in eggs; deformation of embryos, such as tissue dropsy, atrophy, abnormal or depigmented eyes; and abnormalities in the pleon, telson, and spine, pereiopods and pleopods (Rodriguez et al., 1994; Saravana Bhavan & Geraldine, 2001). Deformation may cause difficulties in hatching or in brood survival, as activities such as swimming and searching for prey or escaping from predators may be hampered, and even moulting may not be successfully completed (Fig. 6).

In the case of freshwater decapods, the amount of vittelins and the time that embryos spend inside the eggs are greater than found in their marine relatives. This provokes an extended exposure time to different concentrations of pesticides, which depends on the exposure of females during gonad development and pesticide concentration in the ovaries.

Because of the osmotic stress that freshwater environments present, freshwater decapods possess a thicker chorion for protecting embryos from external aggressions. This chorion also protects them from biocides, making eggs as resistant as adults in some freshwater prawns and crabs, leaving juveniles as the most vulnerable (Key et al., 2003; Li et al., 2006). When the embryo is close to hatching, the chorion narrows to allow embryos to hatch, also allowing external agents to come into contact with embryos, making them more vulnerable to external agents, as observed in the prawn, *Palaemonetes argentinus* (Ituarte et al., 2005).

Fig.6. Pesticide effects in crustaceans' behavior and its results in population. Crustacean image modified from Collins et al. 2004.

The eggs of ovigerous females that live in contaminated areas may be resistant to pollutants, but toxicants may cause the death of juveniles after hatching, when they are not protected by a chorion. Moreover, moult events are a critical period for crustaceans, as their exoskeletons become softer and they are more vulnerable to external contaminants such as pesticides. In juveniles, the intermoult period is short, and the lethal effects of pesticides are increased during that critical period.
