**4. Xenobiotic-induced shift in haemocyte density and morphological damages of blood cells**

Haemocytes are the immunocompetent cells which are functionally responsive to various xenobiotics present in the aquatic environment. Homeostasis of total haemocyte density of aquatic invertebrates within the permissible physiological limit may be considered as an important immunological parameter [8] of cell-mediated immune response of molluscs [2]. Chakraborty et al. (2008) reported suppression in the total count of haemocytes of the fresh‐ water edible mollusc, *L. marginalis* under the sublethal exposure of sodium arsenite, an aquatic pollutant [9]. Mukherjee et al. (2006) reported modulation in the total cell density of the same specimen under the sublethal concentrations of azadirachtin, a neem-based pesticide, a common contaminant of pond water [10]. According to them, toxin-induced alteration in the total cell dynamics may lead to a gradual decline of this species in its natural habitat. The total count of haemocyte of *Villorita cyprinoides* was found to decrease under the exposure of copper [11]. Ray et al. (2013b) reported dynamics of the total haemocyte density of *B. bengalensis* and *L. marginalis* exposed to environmentally realistic sublethal concentrations of cypermethrin and fenvalerate, respectively [2]. Authors reported a significant increase in total haemocyte count following exposure to experimental concentrations of cypermethrin and fenvalerate to *B. bengalensis* and *L. marginalis*, respectively. Russo and Madec (2007) reported effect of

PAGE 2

cells

syringe (Figure 2). The mollusc was under scientific surveillance and the activity of the mollusc

was photodocumented. After two hours of injection, hypersecretion of mucus was recorded by

the experimental P. globosa within a time span of approximately thirty minutes. The expelled

mucus contained more than ninety-five per cent of yeast particles, indicating the efficacy of

Figure 2. Rapid elimination of foreign particulates entrapped in the mucus of aquatic

<H1>Xenobiotic-induced shift in haemocyte density and morphological damages of blood

Haemocytes are the immunocompetent cells which are functionally responsive to various

xenobiotics present in the aquatic environment. Homeostasis of total haemocyte density of

aquatic invertebrates within the permissible physiological limit may be considered as an

important immunological parameter [8] of cell-mediated immune response of molluscs [2].

Chakraborty et al. (2008) reported suppression in the total count of haemocytes of the freshwater

edible mollusc, L. marginalis under the sublethal exposure of sodium arsenite, an aquatic

pollutant [9]. Mukherjee et al. (2006) reported modulation in the total cell density of the same

specimen under the sublethal concentrations of azadirachtin, a neem-based pesticide, a common

contaminant of pond water [10]. According to them, toxin-induced alteration in the total cell

dynamics may lead to a gradual decline of this species in its natural habitat. The total count of

haemocyte of Villorita cyprinoides was found to decrease under the exposure of copper [11]. Ray

et al. (2013b) reported dynamics of the total haemocyte density of B. bengalensis and L.

marginalis exposed to environmentally realistic sublethal concentrations of cypermethrin and

fenvalerate, respectively [2]. Authors reported a significant increase in total haemocyte count

following exposure to experimental concentrations of cypermethrin and fenvalerate to B.

invertebrate, P. globosa.

pollutant fomesafen pesticide on the haemocyte density in the snail, *Lymnaea stagnalis* [12]. Authors reported the increase in total number of circulating haemocytes under the exposure of fomesafen as "facilitated cell turnover." The differential cell count, on the other hand, is also considered as an important immunotoxicological marker of environmental pollution [13]. Moreover, heterogenous populations of invertebrate blood cells have been reported to perform diverse physiological activities including nonself recognition, encapsulation and generation of cytotoxic molecules. Chakraborty et al. (2008) reported that haemocyte density can be considered as a potential biomarker of toxicity of sodium arsenite, a vital pollutant of the Sunderbans estuary of India [9]. Their experimental findings revealed the variation in the relative densities of granulocytes, agranulocytes, blast-like cells, hyalinocytes and asterocytes under the exposure of sodium arsenite. Ray et al. (2013b) reported a non-linear fluctuation in the differential cell density of *B. bengalensis* and *L. marginalis* following sublethal exposure of cypermethrin and fenvalerate, respectively [2]. According to them, aquatic pesticide-induced alteration in the differential haemocyte density was indicative to stress-induced loss of cell balance, which might alter the physiological homeostasis of these species distributed in pesticide-contaminated habitat. Qubella et al. (1993) proposed that fluctuation in the differ‐ ential haemocyte density may be a result of reversible migration of haemocytes from tissues to haemolymph or *vice versa* [14].

Mukherjee et al. (2008) reported an increase in relative percentage of pro-haemocytes subpo‐ pulation in aquatic mollusc *L. marginalis* under the sublethal exposure of azadirachtin [15], a contaminant of water bodies. According to them, pesticide-induced depletion in the densities of granulocytes, agranulocytes, hyalinocytes and asterocytes was indicative to a possible impairment of haemocytic function of mussels distributed in polluted environment. Ray et al. (2013b) flow cytometrically identified (Figure 5a) five discrete subpopulations of haemocytes, that is, blast-like cells, round hyalinocytes, spindle hyalinocytes, round granulocytes, and granular asterocytes in the freshwater molluscs, *B. bengalensis* and *L. marginalis* [2]. Experi‐ mental exposure of 0.5 ppm of cypermethrin for 96 hours resulted in significant increase in the per cent populations of blast-like cells and round hyalinocytes and significant decrease in the subpopulation of granular asterocyte in *B. bengalensis* as compared to control. Exposure of 3 ppm of fenvalerate for 96 hours resulted in significant increase in the relative percentage of round granulocyte and significant decrease in the subpopulation of round hyalinocyte in *L. marginalis* as compared to control. Das et al. (2012) reported alteration of relative densities of haemocyte subpopulations of *L. marginalis* under sublethal exposures of cypermethrin [16]. Exposure to sublethal concentrations of cypermethrin yielded decrease in densities of agra‐ nulocytes and asterocytes and increase in densities of blast-like cells and granulocytes. Pyrethroids-induced alterations in the differential cell density of freshwater molluscs may lead to impairment of blood cell homeostasis in the experimental species distributed in polluted environment.

**Figure 2.** Rapid elimination of foreign particulates entrapped in the mucus of aquatic invertebrate, *P. globosa*.

**4. Xenobiotic-induced shift in haemocyte density and morphological**

Haemocytes are the immunocompetent cells which are functionally responsive to various xenobiotics present in the aquatic environment. Homeostasis of total haemocyte density of aquatic invertebrates within the permissible physiological limit may be considered as an important immunological parameter [8] of cell-mediated immune response of molluscs [2]. Chakraborty et al. (2008) reported suppression in the total count of haemocytes of the fresh‐ water edible mollusc, *L. marginalis* under the sublethal exposure of sodium arsenite, an aquatic pollutant [9]. Mukherjee et al. (2006) reported modulation in the total cell density of the same specimen under the sublethal concentrations of azadirachtin, a neem-based pesticide, a common contaminant of pond water [10]. According to them, toxin-induced alteration in the total cell dynamics may lead to a gradual decline of this species in its natural habitat. The total count of haemocyte of *Villorita cyprinoides* was found to decrease under the exposure of copper [11]. Ray et al. (2013b) reported dynamics of the total haemocyte density of *B. bengalensis* and *L. marginalis* exposed to environmentally realistic sublethal concentrations of cypermethrin and fenvalerate, respectively [2]. Authors reported a significant increase in total haemocyte count following exposure to experimental concentrations of cypermethrin and fenvalerate to *B. bengalensis* and *L. marginalis*, respectively. Russo and Madec (2007) reported effect of

**damages of blood cells**

154 Emerging Pollutants in the Environment - Current and Further Implications

Morphological alterations of haemocytes under the exposure of environmental toxins play an important role in cell-mediated immune response of molluscs (Figure 3). Ray et al. (2013b) identified cytoplasmic hypervacuolation, rounding up of cell, alteration in cell shape, hyper‐ granulation, increased cytoplasmic spreading, membrane disintegration and membrane

PAGE 2

cells

blebbing as principal aberrations in the haemocytes of *B. bengalensis* and *L. marginalis* under the exposure of pollutant like pyrethroid [2]. Cypermethrin- and fenvalerate-induced mor‐ phological damages in the circulating haemocytes of freshwater edible molluscs may lead to possible impairment of the functioning of cells and their immunological reactivity. Chakra‐ borty and Ray (2009) reported dose- and time-dependent increase in the frequency of bi‐ nucleated and micronucleated haemocytes and gill cells in the freshwater bivalve, *L. marginalis*, under the exposure of sodium arsenite [6]. Authors claimed arsenic-induced nuclear anomalies may be used as a biomarker of arsenic toxicity in freshwater ecosystems.

**Figure 3.** Photomicrographs of normal haemocytes of aquatic molluscs, *P. globosa* (a) and *L. marginalis* (c). Cypermeth‐ rin treatment (1.5 ppm/7 days) yielded intense cytoplasmic vacuolation (cv) (b) in the haemocytes of *P. globosa* and nu‐ clear disintegration (d) in the haemocytes of *L. marginalis.*

### **5. Nonself surface adhesion and aggregation response of haemocytes**

Nonself surface adhesion is considered as an important immunological mechanism which is fundamentally related to self–nonself discrimination. Cell adhesion and interaction between cell and the substratum play a pivotal role in the development, maintenance and immune recognition in multicellular animals. Mukherjee et al. (2007) studied the glass surface adhesion efficacy of haemocytes of *L. marginalis* under the sublethal concentrations of azadirachtin [17]. Authors reported a decrease in activity of surface adhesion property of haemocyte under prolonged exposure of pesticide. According to them, the data were indicative to impairment of immunological response of *L. marginalis* in its natural habitat leading to a possible dwindling of this biofilter species from aquatic ecosystem. Ghosh et al. (2008) studied the kinetics of nonself surface adhesion in the haemocytes of mud whelk, *Telescopium telescopium* exposed to diesel in the Sunderbans biosphere reserve [18]. Vehicle diesel is reported [19] to be a serious pollutant of Indian estuary. Authors reported diesel-induced inhibition in the nonself surface recognition efficacy and a shift in the kinetics of adhesion. Saha et al. (2008a) reported modulation in the nonself surface adhesion efficacy of haemocytes of edible mud crab, *Scylla serrata*, under the sublethal exposures of sodium arsenite [20]. Ray et al. (2012) reported arsenicinduced glass surface adhesion of haemocytes of juvenile mud crab [21]. According to them, arsenic-induced alteration in the reactivity of haemocyte may render the juvenile mud crab to become immunologically impaired in the parasite- and pathogen-contaminated natural habitat.

blebbing as principal aberrations in the haemocytes of *B. bengalensis* and *L. marginalis* under the exposure of pollutant like pyrethroid [2]. Cypermethrin- and fenvalerate-induced mor‐ phological damages in the circulating haemocytes of freshwater edible molluscs may lead to possible impairment of the functioning of cells and their immunological reactivity. Chakra‐ borty and Ray (2009) reported dose- and time-dependent increase in the frequency of bi‐ nucleated and micronucleated haemocytes and gill cells in the freshwater bivalve, *L. marginalis*, under the exposure of sodium arsenite [6]. Authors claimed arsenic-induced nuclear anomalies may be used as a biomarker of arsenic toxicity in freshwater ecosystems.

156 Emerging Pollutants in the Environment - Current and Further Implications

**Figure 3.** Photomicrographs of normal haemocytes of aquatic molluscs, *P. globosa* (a) and *L. marginalis* (c). Cypermeth‐ rin treatment (1.5 ppm/7 days) yielded intense cytoplasmic vacuolation (cv) (b) in the haemocytes of *P. globosa* and nu‐

Nonself surface adhesion is considered as an important immunological mechanism which is fundamentally related to self–nonself discrimination. Cell adhesion and interaction between

**5. Nonself surface adhesion and aggregation response of haemocytes**

clear disintegration (d) in the haemocytes of *L. marginalis.*

In the dynamic freshwater ecosystem, the inhabitants often compete for niche for their better survival and propagation. Overlapping in the niche leads to a state of acute predation and fighting among animals. As a result, the animals may encounter acute competition and subsequent physical damage and loss of body fluid. Cellular aggregation is a functional attribute offered by the haemocytes of invertebrates [22] to prevent the accidental blood loss by formation of biological plug at the site of wound and resist the entry of pathogenic microorganism. Selected pollutants like sodium arsenite, washing soda, pyrethroid, azadir‐ achtin are reported to affect the aggregation response of many aquatic invertebrates (Figure 4). Hence, cell–cell aggregation is considered as an immunological response for host defence. Aggregation of haemocytes around invaded microorganisms is termed as "encapsulation response" and is considered as an important immunological response [23]. When successful encapsulation occurs, a host animal can restrict the proliferative and invasive property of a pathogen. Encapsulation reaction is mediated by specific population of immunoactive blood cells and is effective in cell-mediated immunity of invertebrates. Mukherjee et al. (2011) reported anticoagulant and carbohydrate-induced interference of cellular aggregation of mussel, *L. marginalis*, under experimental exposure of azadirachtin [24]. Persistent exposure of azadirachtin inhibited the cellular aggregation response in freshwater mussel. Workers apprehend such scenario in the natural environment may lead to a decline in the population of freshwater mussel and loss of freshwater biodiversity of India. Furthermore, a drastic increase in the occurrence of free cells was recorded against ethylene diamine tetraacetic acid and manure treatment, which was suggestive to possible role of these chemical agents as inhibitor of cellular aggregation. Ray et al. (2012) reported sodium-arsenite-induced inhibition in aggregation of haemocytes of juvenile mud crab, *S. serrata* [21]. According to them, arsenicinduced alteration of immune status may impart a state of vulnerability in juvenile crab inhabiting the arsenic-polluted environment.

**Figure 4.** Typical aggregation response of haemocytes of a freshwater gastropod, *Pila globosa* (a). Cypermethrin is a contaminant of the habitat of *P. globosa* which inhibits the degree of haemocyte aggregation (b).

#### **6. Phagocytic response in the face of environmental stressors**

PAGE 2

Phagocytosis, in general, is considered a classical innate immune response reported in the majority of the invertebrate Phyla. It is an established immunological response and is consid‐ ered as a biomarker of aquatic pollution [13]. Phagocytic response enables invertebrates to combat against invading pathogens and pollutants of known and unknown chemistry. Haemocyte-mediated phagocytosis of nonself particles provides natural immunity in the bivalves [25]. Chakraborty et al. (2009) reported the inhibitory effect of sodium arsenite on the phagocytic response of *L. marginalis* under the challenge of yeast at various sublethal concen‐ trations [7]. Inhibition in phagocytic response was also recorded under similar laboratory condition for the haemocytes of arsenic-treated *L. marginalis* when challenged with human red blood corpuscles [26]. According to them, impairment in the phagocytic potential of the haemocytes of arsenic-treated mussels may lead to compromisation of the immune status of the animals distributed in the contaminated habitat. Mukherjee et al. (2011) reported azadir‐ achtin-induced suppression in the phagocytic potential of haemocytes of *L. marginalis* under the experimental challenge of charcoal particulate [27]. Azadirachtin-induced inhibition in the phagocytic response indicated a state of immune suppression of mussels. Ray et al. (2012) screened the immunotoxicological reactivity of haemocytes of juvenile mud crab, *S. serrata* of the Sunderbans biosphere reserve and reported sodium-arsenite-induced inhibition in the phagocytic potential under the challenge of yeast [21]. Arsenic-induced altered reactivity of haemocytes may affect the propagation and survival of mud crab population by increasing its vulnerability to higher rate of disease and parasite attack. Sponges, on the other hand, are nonselective filter feeders which depend on phagocytosis for the purposes of feeding and digestion (Figure 5b, c). Phagocytosis, in sponge, plays a dual physiological attribute in the form of food procurement and innate immune response [28]. Mukherjee et al. (2015b) reported washing-

Figure 4. Typical aggregation response of haemocytes of a freshwater gastropod, Pila globosa

interference of cellular aggregation of mussel, L. marginalis, under experimental exposure of

azadirachtin [24]. Persistent exposure of azadirachtin inhibited the cellular aggregation response

in freshwater mussel. Workers apprehend such scenario in the natural environment may lead to a

decline in the population of freshwater mussel and loss of freshwater biodiversity of India.

Furthermore, a drastic increase in the occurrence of free cells was recorded against ethylene

diamine tetraacetic acid and manure treatment, which was suggestive to possible role of these

chemical agents as inhibitor of cellular aggregation. Ray et al. (2012) reported sodium-arsenite-

induced inhibition in aggregation of haemocytes of juvenile mud crab, S. serrata [21]. According

to them, arsenic-induced alteration of immune status may impart a state of vulnerability in

Phagocytosis, in general, is considered a classical innate immune response reported in the

majority of the invertebrate Phyla. It is an established immunological response and is considered

as a biomarker of aquatic pollution [13]. Phagocytic response enables invertebrates to combat

against invading pathogens and pollutants of known and unknown chemistry. Haemocyte-

mediated phagocytosis of nonself particles provides natural immunity in the bivalves [25].

Chakraborty et al. (2009) reported the inhibitory effect of sodium arsenite on the phagocytic

response of L. marginalis under the challenge of yeast at various sublethal concentrations [7].

Inhibition in phagocytic response was also recorded under similar laboratory condition for the

haemocytes of arsenic-treated L. marginalis when challenged with human red blood corpuscles

[26]. According to them, impairment in the phagocytic potential of the haemocytes of arsenic-

treated mussels may lead to compromisation of the immune status of the animals distributed in

the contaminated habitat. Mukherjee et al. (2011) reported azadirachtin-induced suppression in

the phagocytic potential of haemocytes of L. marginalis under the experimental challenge of

charcoal particulate [27]. Azadirachtin-induced inhibition in the phagocytic response indicated a

state of immune suppression of mussels. Ray et al. (2012) screened the immunotoxicological

reactivity of haemocytes of juvenile mud crab, S. serrata of the Sunderbans biosphere reserve

and reported sodium-arsenite-induced inhibition in the phagocytic potential under the challenge

of yeast [21]. Arsenic-induced altered reactivity of haemocytes may affect the propagation and

survival of mud crab population by increasing its vulnerability to higher rate of disease and

degree of haemocyte aggregation (b).

juvenile crab inhabiting the arsenic-polluted environment.

<H1>Phagocytic response in the face of environmental stressors

(a). Cypermethrin is a contaminant of the habitat of P. globosa which inhibits the

soda-induced inhibition in the phagocytic potential of cells of freshwater sponge, *E. carteri*, under the challenge of yeast [5]. According to them, decrease in phagocytic potential is suggestive to a possible impairment of both food capture efficiency and innate immune status, leading to suppression of immunological status of *E. carteri* distributed in detergent-contami‐ nated natural habitat.

**Figure 5.** Flow cytometry of isolated cells of sponge, *E. carteri*, depicting their relative size and granularity (a)*.* Phago‐ cytosis of yeasts (Y) by the cells of *E. carteri* (b, c) exposed to washing soda. N = Nucleus.

## **7. Cytotoxicity of blood cells as an effective immune strategy**

**Figure 4.** Typical aggregation response of haemocytes of a freshwater gastropod, *Pila globosa* (a). Cypermethrin is a

Phagocytosis, in general, is considered a classical innate immune response reported in the majority of the invertebrate Phyla. It is an established immunological response and is consid‐ ered as a biomarker of aquatic pollution [13]. Phagocytic response enables invertebrates to combat against invading pathogens and pollutants of known and unknown chemistry. Haemocyte-mediated phagocytosis of nonself particles provides natural immunity in the bivalves [25]. Chakraborty et al. (2009) reported the inhibitory effect of sodium arsenite on the phagocytic response of *L. marginalis* under the challenge of yeast at various sublethal concen‐ trations [7]. Inhibition in phagocytic response was also recorded under similar laboratory condition for the haemocytes of arsenic-treated *L. marginalis* when challenged with human red blood corpuscles [26]. According to them, impairment in the phagocytic potential of the haemocytes of arsenic-treated mussels may lead to compromisation of the immune status of the animals distributed in the contaminated habitat. Mukherjee et al. (2011) reported azadir‐ achtin-induced suppression in the phagocytic potential of haemocytes of *L. marginalis* under the experimental challenge of charcoal particulate [27]. Azadirachtin-induced inhibition in the phagocytic response indicated a state of immune suppression of mussels. Ray et al. (2012) screened the immunotoxicological reactivity of haemocytes of juvenile mud crab, *S. serrata* of the Sunderbans biosphere reserve and reported sodium-arsenite-induced inhibition in the phagocytic potential under the challenge of yeast [21]. Arsenic-induced altered reactivity of haemocytes may affect the propagation and survival of mud crab population by increasing its vulnerability to higher rate of disease and parasite attack. Sponges, on the other hand, are nonselective filter feeders which depend on phagocytosis for the purposes of feeding and digestion (Figure 5b, c). Phagocytosis, in sponge, plays a dual physiological attribute in the form of food procurement and innate immune response [28]. Mukherjee et al. (2015b) reported washing-

contaminant of the habitat of *P. globosa* which inhibits the degree of haemocyte aggregation (b).

158 Emerging Pollutants in the Environment - Current and Further Implications

**6. Phagocytic response in the face of environmental stressors**

PAGE 2

Cytotoxic molecules generated by the immunocompetent cells of invertebrates are reported to play an important role in the destruction and deactivation of foreign engulfed pathogens [29]. Authors reported superoxide anion, nitric oxide and phenoloxidase as established cytotoxic molecules of freshwater molluscs affected by various environmental pollutants. Contamina‐ tion of natural habitat by toxic metals, metalloids, pesticides and washing soda results in a significant alteration in the cytotoxic status of invertebrates. Cytotoxic molecules are consid‐ ered as an effective component of innate immune defence of invertebrates distributed in polluted environment. Nappi and Ottaviani (2000) reported nitric oxide and superoxide anions as potential "killing agents" of invertebrates [30]. Generation of reactive oxygen intermediate and reactive nitrogen intermediate by the phagocytic cells are mediated by NADPH oxidase and nitric oxide synthase (NOS), respectively. According to them, generation of superoxide anions can be correlated with increased respiratory burst activity in phagocytic cells. Mukher‐ jee et al. (2012) reported a dose-dependent increase in the generation of superoxide anion in the cells of freshwater edible bivalve, *L. marginalis* under the exposure of azadirachtin, a neembased pesticide [31]. According to them, this response of haemocytes may be considered as cellular stress under the sublethal and environmentally realistic concentrations of azadirach‐ tin. Nitric oxide is considered as a signalling molecule generated in the biological system by the immunocytes of invertebrates as a potent cytotoxic agent for killing of invading microor‐

Figure 4. Typical aggregation response of haemocytes of a freshwater gastropod, Pila globosa

interference of cellular aggregation of mussel, L. marginalis, under experimental exposure of

azadirachtin [24]. Persistent exposure of azadirachtin inhibited the cellular aggregation response

in freshwater mussel. Workers apprehend such scenario in the natural environment may lead to a

decline in the population of freshwater mussel and loss of freshwater biodiversity of India.

Furthermore, a drastic increase in the occurrence of free cells was recorded against ethylene

diamine tetraacetic acid and manure treatment, which was suggestive to possible role of these

chemical agents as inhibitor of cellular aggregation. Ray et al. (2012) reported sodium-arsenite-

induced inhibition in aggregation of haemocytes of juvenile mud crab, S. serrata [21]. According

to them, arsenic-induced alteration of immune status may impart a state of vulnerability in

Phagocytosis, in general, is considered a classical innate immune response reported in the

majority of the invertebrate Phyla. It is an established immunological response and is considered

as a biomarker of aquatic pollution [13]. Phagocytic response enables invertebrates to combat

against invading pathogens and pollutants of known and unknown chemistry. Haemocyte-

mediated phagocytosis of nonself particles provides natural immunity in the bivalves [25].

Chakraborty et al. (2009) reported the inhibitory effect of sodium arsenite on the phagocytic

response of L. marginalis under the challenge of yeast at various sublethal concentrations [7].

Inhibition in phagocytic response was also recorded under similar laboratory condition for the

haemocytes of arsenic-treated L. marginalis when challenged with human red blood corpuscles

[26]. According to them, impairment in the phagocytic potential of the haemocytes of arsenic-

treated mussels may lead to compromisation of the immune status of the animals distributed in

the contaminated habitat. Mukherjee et al. (2011) reported azadirachtin-induced suppression in

the phagocytic potential of haemocytes of L. marginalis under the experimental challenge of

charcoal particulate [27]. Azadirachtin-induced inhibition in the phagocytic response indicated a

state of immune suppression of mussels. Ray et al. (2012) screened the immunotoxicological

reactivity of haemocytes of juvenile mud crab, S. serrata of the Sunderbans biosphere reserve

and reported sodium-arsenite-induced inhibition in the phagocytic potential under the challenge

of yeast [21]. Arsenic-induced altered reactivity of haemocytes may affect the propagation and

survival of mud crab population by increasing its vulnerability to higher rate of disease and

degree of haemocyte aggregation (b).

juvenile crab inhabiting the arsenic-polluted environment.

<H1>Phagocytic response in the face of environmental stressors

(a). Cypermethrin is a contaminant of the habitat of P. globosa which inhibits the

ganisms. It is generated during the conversion of L-arginine to L-citrulline by nitric oxide synthase. Saha et al. (2008b) reported sodium-arsenite-induced increase in the generation of the intracellular nitric oxide in estuarine mud crab, *S. serrata* [32]. According to them, dosedependent increase in the generation of nitric oxide may be considered as a biomarker of arsenic pollution in the Sunderbans delta of India. Chakraborty et al. (2009) reported inhibition in generation of nitric oxide in *L. marginalis* under prolonged exposure of arsenic [7]. Authors claimed this studied parameter as possible biomarker of arsenic toxicity in aquatic environ‐ ment. Phenoloxidase, on the other hand, is considered as another cytotoxic molecule and is involved in the process of nonself recognition, phagocytosis and melanisation. The production of toxic quinoid derivative by phenoloxidase is an early step of biosynthesis of melanin for host defence. Chakraborty et al. (2010a) reported depletion in generation of cytotoxic molecule like nitric oxide and activity of phenoloxidase in the gill of freshwater bivalve, *L. marginalis*, under the sublethal exposures of sodium arsenite [33]. According to them, sodium-arseniteinduced alteration in the cytotoxic status in gill indicated a state of immunocompromisation in the animal. Chakraborty et al. (2013) reported inhibition in the generation of superoxide anions and nitric oxide and activity of phenoloxidase in the digestive gland of the same experimental bivalve under prolonged exposure of arsenic [34]. Generation of cytotoxic molecules like superoxide anions and nitric oxide and activity of phenoloxidase are important immunological responses offered by the cells of sponge under the exposure of pathogens and toxins [5]. Authors reported washing-soda-induced alteration in the generation of superoxide anion, nitric oxide and activity of phenoloxidase in the dissociated cells of freshwater sponge, *E. carteri.* Washing-soda-induced alteration in cytotoxic response of *E. carteri* may lead to an undesirable shift in the immune status of the animal distributed in detergent contaminated natural habitat.

### **8. Lysosomal membrane stability and activity of phosphatases**

Lysosome is an important subcellular organelle involved in the process of degradation of foreign engulfed particulate. After phagocytosis, the phagocytic vacuole with engulfed foreign particles fuses with lysosome to form phagolysosome. As lysosome plays an important role in secretion of various digesting enzymes, maintenance of lysosomal membrane integrity has been gaining special scientific attention from the immunological point of view. Chakraborty and Ray (2009) reported impairment of lysosomal membrane integrity by neutral red retention assay in the haemocytes of *L. marginalis* exposed to sodium arsenite [6]. According to them, arsenic-induced fragility of lysosomal membrane may lead to leakage of lysosomal enzymes into the cytosol and subsequent destruction of the adjoining self cell or tissue. Ray et al. (2013b) reported lysosomal membrane integrity of haemocytes of *B. bengalensis* and *L. marginalis* under the experimental exposures of cypermethrin and fenvalerate, respectively [2]. Authors reported pyrethroid-induced lysosomal membrane fragility of haemocytes suggesting substantial damage and destabilization of lysosomal membrane (Figure 6). Screening of lysosomal membrane integrity of molluscan haemocytes by neutral red retention assay is considered as a possible biological marking of arsenic toxicity [6] and as an early warning tool

ganisms. It is generated during the conversion of L-arginine to L-citrulline by nitric oxide synthase. Saha et al. (2008b) reported sodium-arsenite-induced increase in the generation of the intracellular nitric oxide in estuarine mud crab, *S. serrata* [32]. According to them, dosedependent increase in the generation of nitric oxide may be considered as a biomarker of arsenic pollution in the Sunderbans delta of India. Chakraborty et al. (2009) reported inhibition in generation of nitric oxide in *L. marginalis* under prolonged exposure of arsenic [7]. Authors claimed this studied parameter as possible biomarker of arsenic toxicity in aquatic environ‐ ment. Phenoloxidase, on the other hand, is considered as another cytotoxic molecule and is involved in the process of nonself recognition, phagocytosis and melanisation. The production of toxic quinoid derivative by phenoloxidase is an early step of biosynthesis of melanin for host defence. Chakraborty et al. (2010a) reported depletion in generation of cytotoxic molecule like nitric oxide and activity of phenoloxidase in the gill of freshwater bivalve, *L. marginalis*, under the sublethal exposures of sodium arsenite [33]. According to them, sodium-arseniteinduced alteration in the cytotoxic status in gill indicated a state of immunocompromisation in the animal. Chakraborty et al. (2013) reported inhibition in the generation of superoxide anions and nitric oxide and activity of phenoloxidase in the digestive gland of the same experimental bivalve under prolonged exposure of arsenic [34]. Generation of cytotoxic molecules like superoxide anions and nitric oxide and activity of phenoloxidase are important immunological responses offered by the cells of sponge under the exposure of pathogens and toxins [5]. Authors reported washing-soda-induced alteration in the generation of superoxide anion, nitric oxide and activity of phenoloxidase in the dissociated cells of freshwater sponge, *E. carteri.* Washing-soda-induced alteration in cytotoxic response of *E. carteri* may lead to an undesirable shift in the immune status of the animal distributed in detergent contaminated

160 Emerging Pollutants in the Environment - Current and Further Implications

**8. Lysosomal membrane stability and activity of phosphatases**

Lysosome is an important subcellular organelle involved in the process of degradation of foreign engulfed particulate. After phagocytosis, the phagocytic vacuole with engulfed foreign particles fuses with lysosome to form phagolysosome. As lysosome plays an important role in secretion of various digesting enzymes, maintenance of lysosomal membrane integrity has been gaining special scientific attention from the immunological point of view. Chakraborty and Ray (2009) reported impairment of lysosomal membrane integrity by neutral red retention assay in the haemocytes of *L. marginalis* exposed to sodium arsenite [6]. According to them, arsenic-induced fragility of lysosomal membrane may lead to leakage of lysosomal enzymes into the cytosol and subsequent destruction of the adjoining self cell or tissue. Ray et al. (2013b) reported lysosomal membrane integrity of haemocytes of *B. bengalensis* and *L. marginalis* under the experimental exposures of cypermethrin and fenvalerate, respectively [2]. Authors reported pyrethroid-induced lysosomal membrane fragility of haemocytes suggesting substantial damage and destabilization of lysosomal membrane (Figure 6). Screening of lysosomal membrane integrity of molluscan haemocytes by neutral red retention assay is considered as a possible biological marking of arsenic toxicity [6] and as an early warning tool

natural habitat.

**Figure 6.** Cypermethrin-induced fragility of lysosome membrane of haemocytes of aquatic mollusc as evident from neutral red dye retention assay. Dye neutral red concentrated in the lysosomal compartments at zero minute of the assay (a). Complete diffusion of neutral red in the cytoplasm of cypermethrin-exposed haemocytes has been designat‐ ed as "end point" of the assay (b).

of environmental pollution [35]. Phosphatases are principal lysosomal enzymes which are involved in pathogen destruction and are reported as markers of environmental stress [36]. Chakraborty et al. (2010a) reported suppression in the activity of hydrolytic enzymes, acid and alkaline phosphatases in the gill of freshwater edible bivalve, *L. marginalis* under the exposure of sodium arsenite [33]. According to them, inhibition in the activity of these enzymes might cripple the immune status and nutrient mobility in the gill of *L. marginalis.* Chakraborty et al. (2013) reported inhibition in the activity of phosphatases in the haemocyte and digestive tissue of edible bivalve under similar toxic insult by environmental arsenic [34]. Workers apprehend a state of immunocompromisation of the organisms under persistent exposure of arsenic. Saha et al. (2009) reported inhibition in the activity of phosphatases in the haemocytes of estuarine mud crab, *S. serrata* under the exposure of arsenic [37]. According to them, this situation in the natural environment might result in impairment of immunological activity and opportunistic invasion of parasite and pathogen into the body of the organism.
