**6. Other effects of pesticides on marine bivalves**

232 Pesticides in the Modern World - Risks and Benefits

(Cantry et al., 2007). Moreover, a mix of herbicides containing atrazine, diuron and isoproturon showed an effect on *C. gigas* hemocyte aggregation (Auffret et Oubella., 1997). Chlordan, an insecticide, demonstrated effects on *C. virginica* hemocyte phagocytosis at 250 µM *in vitro* (Larson et al., 1989). A decreased phagocytosis activity was observed after a triforine exposure in the eastern oyster, *C. virginica* (Alvarez and Friedl, 1992). A pesticide mixture (alachor, metolachlor, terbutylazine, glyphosate, diuron, atrazine, carbaryl and fosteyl aluminium) representative for surface waters of the Marennes-Oleron Basin (Charente Maritime, France, 0.25 nM to 4 nM) induced a decrease of phagocytic activity (Gagnaire et al., 2007). Moreover, Cantry et al. (2007) reported a decrease in phagocytic index in the blue mussel, *Mytilus edulis*, after a short exposure to 0.1 mg L-1 azamethiphos. This result suggests that azamethiphos can modulate haemocyte function in mussels at

At the contrary, Gagnaire et al. (2003) reported no effect on cell viability, cell cycle and cellular activities except for peroxidase activity for Pacific cupped oyster haemocytes

Pentachlorophenol decreased the production of ROS by the inhibition of NADPH production in the eastern oyster, *Crassostrea virginica* (Baier-Anderson & Anderson, 1996). Dieldrin, tested *in vitro* on *C. virginica* hemocytes induced a decrease of chemiluminescence at concentrations ranging from 3 to 300 µM (Larson et al., 1989). Hemocytes of *C. virginica* exposed to chlorothalonil (fungicide) for 20 h at concentrations between 4 nM and 2 µM showed no modification of cell mortality and phagocytosis, but a decrease of ROS

In the past decades, the emergence of infectious diseases has been reported in marine species and disease outbreaks have also increased (Harvell et al., 1999). According to Snieszko (Snieszko, 1974), the development of an infectious disease results from an unbalance between the host and the pathogen due to external factors (including pollutants) and/or internal factors of both protagonists (virulence of the pathogen, susceptibility of the host). Animals presenting impaired defence mechanisms may be more susceptible to

Demonstration of the relationship between pollution and increase of susceptibility to infectious diseases exist in vertebrates (Fournier et al., 1988; Van Levoren et al., 2000; Jepson et al., 2005), a few of studies was carried out in invertebrates (Galloway & Depledge, 2001). Rare studies have attempted to link contaminant presence and susceptibility to infectious diseases in marine molluscs and demonstrated harmful effects of pollutants in bivalves. Contamination of the eastern oyster, *Crassostrea virginica*, by polluted sediment and tributyltin increased the intensity of *Perkinsus marinus* infection, but no cellular or humoral parameters were modulated (Anderson et al., 1996; Chu et al., 2002). Anderson et al. (1981) demonstrated previously that the hard clam, *Mercenaria mercenaria,* exposed to PCP were unable to kill injected bacteria. Kim et al. (2008) studied the relationship of parasite detection to contaminant body burden in sentinel bivalves through a 'Mussel Watch' Program. These authors showed that correlations between parasites/pathologies and pesticides were

The Pacific cupped oyster, *Crassostrea gigas*, has been also used to evaluate the impact of a pesticide mixture (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) on some immune-related parameters and to demonstrate a relationship between infectious diseases, defence capacities and pollutants.

environmentally relevant concentrations.

exposed to atrazine in *in vitro* and *in vivo* assays.

production (Baier-Anderson & Anderson, 2000).

frequent in mussels and oysters (Kim et al., 2008).

infectious diseases.

The evaluation of acetylcholinesterase activity in marine organisms has been and is at present time extensively used as a biomarker of exposure to neurotoxic agents such as organophosphorus and carbamate pesticides. Indeed, organophosphorous compounds and carbamates including paraoxon and carbaryl are known to inhibit acetylcholinesterase (AChE) and carboxylesterase (CE) (Cooreman et al., 1993).

Paraoxon inhibited the activity of AChE in the hepatopancreas of the blue mussel, *Mytilus edulis,* in vitro at concentrations ranging from 1 µM to 1 mM (Ozretic and Krajnovic-Ozretic, 1992). Inhibition by carbaryl was less distinct. AChE from *M. edulis* hemocytes was inhibited in vitro by 0.1-3 mM paraoxon, eserine and DFP (Galloway et al., 2002). Cantry et al. (2007) showed that exposure of the blue mussel, *M. edulis*, to 0.1 mg L-1 azamethiphos, an organophosphate pesticide used to combat sea lice infestations in farmed salmonids, for periods of up to 24h caused a significant reduction in acetylcholinesterase activity in both the haemolymph and the gill. However, cholinesterases found in the Pacific cupped oyster, *Crassostrea gigas,* appeared to be insensitive to organophosphorous insecticides (Bocquene et al., 1997).

Anguiano et al. (2006) showed that after a 4 h exposure to lindane (gammahexachlorocyclohexane), filtration rates of adult Pacific cupped oysters, *Crassostrea gigas*, were significantly reduced compared with controls at concentrations of 0.3 and 0.7 mg L-1. However, a short term exposure of the blue mussel, *Mytilus edulis*, to azamethiphos did not change the feeding rate (Chantry et al., 2007). Studies carried out in adult Pacific cupped oysters revealed that diuron induces partial spawning and atrophy of the digestive epithelium after 1 week of exposure at 1 µg L-1 (Buisson et al., 2008).

Greco et al. (2010) investigated effects of a mixture of herbicides on the physiological status of the soft clam, *Mya arenaria*. Clams were exposed for 28 days to 0.01 mg L-1 of a pesticide mixture: dichlorophenoxyacetic acid (2,4-D), 2-(2-methyl-4-chlorophenoxy) propionic acid (mecoprop), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). Although a gradual sexual maturation was reported in both sexes during the course of the experiment, females demonstrated a higher sensitivity to pesticides compared to males.

Favret and Lynn (2010) during the course of a study monitoring sperm viability by flow cytometry in the eastern oyster, *Crassostrea virginica*, after exposure to a pesticide (Bayluscide) reported effects on mitochondrial membrane potential and plasma membrane in the sperm. Buisson et al. (2008) studied impact of pesticides in the cupped Pacific oyster, *C. gigas,* and reported partial spawning and atrophy of the digestive tubule epithelium in relation to pesticides.

A study with a mix of herbicides containing atrazine, diuron and isoproturon revealed effects on gene expression in the Pacific cupped oyster, *Crassostrea gigas* (Tanguy et al., 2005). Gagnaire et al. (2007) studied also the impact of pesticides on *C. gigas*, monitoring

Effects of Pesticides on Marine Bivalves: What Do We Know and What Do We Need to Know? 235

adverse effects need to be more documented through experiments carried out using

Among studies focused on pesticides, most of them have been carried out by exposing animals to relatively long periods of time (Auffret et Oubella., 1997; Tanguy et al., 2005; Bouilly et al., 2007, Buisson et al., 2008) giving an insight on the effects of chronic exposures

Nevertheless, it is well known that in natural waters, uneven concentrations of pesticides are found in the water mass because of different factors such as seasonal agricultural practices, weathering processes and peak concentrations are often found in the aquatic environment for short periods of time (Munaron, 2006; Hyne et al., 2008). Thus, long-term studies may be not so predictive of what could happen on a natural environment. Shortterm exposures of herbicides under laboratory controlled conditions have shown to exert an effect on aquatic organisms (Bretaud et al., 2000; Saglio et al., 2002). They can give an insight

In order to assess the impact of persistent pollutants on the marine ecosystem a suite of biomarkers are being extensively used worldwide (Ozretic & Krajnovic-Ozretic, 1992; Lowe & Fossato, 2000). These biomarkers are being used to evaluate exposure of various species of sentinel marine organisms (e.g. mussels, clams, oysters.) to and the effect of various pesticides using different molecular approaches (Wong et al., 1992; Cajaraville et al., 1996;

As an example, Matozzo et al. (2010) developed a multibiomarker approach in order to assess effects of environmental contaminants in the Manila clam, *Ruditapes philippinarum*, collected in 8 sites of the Lagoon of Venice (Italy). The authors used several biomarkers includig total haemocyte count and lysozyme activity, acetylcholinesterase activity in gills, vitellogenin-like protein levels in both digestive gland and cell-free haemolymph, and survival-in-air widely used to evaluate general stress conditions. In addition, different pollutants were also measured in collected animals. Results showed that the selected integrated approach between biomarkers and chemical analyses is a useful tool in

Different compounds including different pesticides have been found simultaneously in aquatic environments, underlining that experimental approaches with toxicant mixtures are needed. Most of the studies evoked before, have been carried out in adults, but juvenile organisms are known to be generally more sensitive to environmental stress than adults (Perdue et al., 1981). Additional residue-effects data on sublethal endpoints, early life

Finally, research in ecotoxicology needs also to fill the gap existing between sub-organismal responses to toxicants and effects occurring at higher levels of biological organisation (e.g.

Alvarez, M.R. & Friedl, F.E. (1992). Effects of a fungicide on *in vitro* hemocyte viability,

Anderson, R.S.; Giam, C.S.; Ray, L.E. & Tripp, M.R. (1981). Effects of environmental

impaired bacterial clearance. *Aquatic Toxicology*. Vol.1, pp. 187-195

phagocytosis and attachment in the American oyster, *Crassostrea virginica*.

pollutants on immunological competency of the clam *Mercenaria mercenaria*:

stages, and a wider range of legacy and emergent contaminants will be needed.

of the potential effect of contaminants in organisms in the natural environment.

pesticides at environmentally relevant concentrations.

on physiological functions of the organism.

Galloway et al., 2002).

biomonitoring ( Matozzo et al., 2010).

population) (Tlili et al., 2010).

*Aquaculture*. Vol.107, pp. 135-140

**8. References** 

gene expression in hemocytes by real-time PCR. The expression of genes involved in *C. gigas* hemocyte functions was up-regulated in pesticide-treated oysters compared to untreated oysters after a bacterial challenge. The authors hypothesized that gene over-expression could lead to an injury of host tissues, resulting in higher mortality rates.

Collin et al. (2011) explored under experimental conditions the effects of a cocktail of three pesticides (lindane, metolachlor and carbofuran) on physiological functions of the Pacific cupped oyster, *C. gigas,* using the suppression subtractive hybridisation technique. The authors reported a site and organ-specific response to the pesticides. Effects of imidacloprid and thiacloprid, 2 neonicotinoid insecticides, at transcriptomic and proteomic levels in the marine mussel, *Mytilus galloprovincialis*, were also reported by Dondero et al. (2010).

Tlili et al. (2010) compared the size-distribution of the intra-sedimentary bivalve *Donax trunculus* collected in two sites in Tunisia, a polluted site and a comparatively reference site The auhors showed that the size-distribution from the polluted site consisted of 4 cohorts.whereras 5 cohorts were observed in the comparatively reference site. Moreover, the mean total length size and the growth rate of cohorts were significantly reduced in the impacted site compared to the reference site. These results suggest effects of pollutants on marine bivalves at a population level with an ecological relevance.

Pariseau et al. (2010) studied haemic neoplasia in the soft-shell clam *Mya arenaria,* in relation to exposure to fungicides, chlorothalonil and mancozeb, without demonstrating a link.

#### **7. Conclusions and perspectives**

The results obtained through the cited studies alerts to the negative effects of pesticides in bivalves and may be important to initiate and implement programs to protect the bivalve estuaries. These studies bring scientific evidence regarding the biological effects of pesticides on the animals inhabiting contaminated estuaries and the potential effect of the contaminates on shellfish and on human health if the seafood is consumed.

The great variability of response (depending on duration of exposure, toxicant concentration, test species or experimental conditions) is a reminder that the effects of pollutants on the marine environment cannot be assessed by simple methods (e.g. shortterm bioassays with one or two test species). As an example, Greco et al. (2010) investigating effects of a mixture of herbicides on the physiological status of the soft clam, *Mya arenaria*, showed that in clams kept at 18°C, pesticides appeared to induce minor effects compared with animals kept at 7°C. They concluded that increased temperature may modify the response of *Mya arenaria* to pesticides.

It is recognized that bivalve habitats may differ in environmental parameters. Thus, animals may be exposed to numerous variables that include other pollutants, different temperatures, salinities, amounts of dissolved oxygen, and changes in pH. In this context, a better understanding of the possible interactions between pesticides and other abiotic environmental factors (temperature, salinity,) and biotic factors associated with the physiological status of bivalves is necessary.

As it is possible to evaluate only a limited number of environmental factors in laboratory assays, it appears difficult to investigate all of the potential environmental factors that also may affect bivalve physiology.

A lot of studies concerning effects of pesticides in bivalves have been carried out using high pollutant concentrations. However, levels of pesticides measured in superficial waters generally range below lethal concentrations for aquatic species. In this context, sub-lethal adverse effects need to be more documented through experiments carried out using pesticides at environmentally relevant concentrations.

Among studies focused on pesticides, most of them have been carried out by exposing animals to relatively long periods of time (Auffret et Oubella., 1997; Tanguy et al., 2005; Bouilly et al., 2007, Buisson et al., 2008) giving an insight on the effects of chronic exposures on physiological functions of the organism.

Nevertheless, it is well known that in natural waters, uneven concentrations of pesticides are found in the water mass because of different factors such as seasonal agricultural practices, weathering processes and peak concentrations are often found in the aquatic environment for short periods of time (Munaron, 2006; Hyne et al., 2008). Thus, long-term studies may be not so predictive of what could happen on a natural environment. Shortterm exposures of herbicides under laboratory controlled conditions have shown to exert an effect on aquatic organisms (Bretaud et al., 2000; Saglio et al., 2002). They can give an insight of the potential effect of contaminants in organisms in the natural environment.

In order to assess the impact of persistent pollutants on the marine ecosystem a suite of biomarkers are being extensively used worldwide (Ozretic & Krajnovic-Ozretic, 1992; Lowe & Fossato, 2000). These biomarkers are being used to evaluate exposure of various species of sentinel marine organisms (e.g. mussels, clams, oysters.) to and the effect of various pesticides using different molecular approaches (Wong et al., 1992; Cajaraville et al., 1996; Galloway et al., 2002).

As an example, Matozzo et al. (2010) developed a multibiomarker approach in order to assess effects of environmental contaminants in the Manila clam, *Ruditapes philippinarum*, collected in 8 sites of the Lagoon of Venice (Italy). The authors used several biomarkers includig total haemocyte count and lysozyme activity, acetylcholinesterase activity in gills, vitellogenin-like protein levels in both digestive gland and cell-free haemolymph, and survival-in-air widely used to evaluate general stress conditions. In addition, different pollutants were also measured in collected animals. Results showed that the selected integrated approach between biomarkers and chemical analyses is a useful tool in biomonitoring ( Matozzo et al., 2010).

Different compounds including different pesticides have been found simultaneously in aquatic environments, underlining that experimental approaches with toxicant mixtures are needed. Most of the studies evoked before, have been carried out in adults, but juvenile organisms are known to be generally more sensitive to environmental stress than adults (Perdue et al., 1981). Additional residue-effects data on sublethal endpoints, early life stages, and a wider range of legacy and emergent contaminants will be needed.

Finally, research in ecotoxicology needs also to fill the gap existing between sub-organismal responses to toxicants and effects occurring at higher levels of biological organisation (e.g. population) (Tlili et al., 2010).

#### **8. References**

234 Pesticides in the Modern World - Risks and Benefits

gene expression in hemocytes by real-time PCR. The expression of genes involved in *C. gigas* hemocyte functions was up-regulated in pesticide-treated oysters compared to untreated oysters after a bacterial challenge. The authors hypothesized that gene over-expression

Collin et al. (2011) explored under experimental conditions the effects of a cocktail of three pesticides (lindane, metolachlor and carbofuran) on physiological functions of the Pacific cupped oyster, *C. gigas,* using the suppression subtractive hybridisation technique. The authors reported a site and organ-specific response to the pesticides. Effects of imidacloprid and thiacloprid, 2 neonicotinoid insecticides, at transcriptomic and proteomic levels in the

Tlili et al. (2010) compared the size-distribution of the intra-sedimentary bivalve *Donax trunculus* collected in two sites in Tunisia, a polluted site and a comparatively reference site The auhors showed that the size-distribution from the polluted site consisted of 4 cohorts.whereras 5 cohorts were observed in the comparatively reference site. Moreover, the mean total length size and the growth rate of cohorts were significantly reduced in the impacted site compared to the reference site. These results suggest effects of pollutants on

Pariseau et al. (2010) studied haemic neoplasia in the soft-shell clam *Mya arenaria,* in relation to exposure to fungicides, chlorothalonil and mancozeb, without demonstrating a link.

The results obtained through the cited studies alerts to the negative effects of pesticides in bivalves and may be important to initiate and implement programs to protect the bivalve estuaries. These studies bring scientific evidence regarding the biological effects of pesticides on the animals inhabiting contaminated estuaries and the potential effect of the

The great variability of response (depending on duration of exposure, toxicant concentration, test species or experimental conditions) is a reminder that the effects of pollutants on the marine environment cannot be assessed by simple methods (e.g. shortterm bioassays with one or two test species). As an example, Greco et al. (2010) investigating effects of a mixture of herbicides on the physiological status of the soft clam, *Mya arenaria*, showed that in clams kept at 18°C, pesticides appeared to induce minor effects compared with animals kept at 7°C. They concluded that increased temperature may modify the

It is recognized that bivalve habitats may differ in environmental parameters. Thus, animals may be exposed to numerous variables that include other pollutants, different temperatures, salinities, amounts of dissolved oxygen, and changes in pH. In this context, a better understanding of the possible interactions between pesticides and other abiotic environmental factors (temperature, salinity,) and biotic factors associated with the

As it is possible to evaluate only a limited number of environmental factors in laboratory assays, it appears difficult to investigate all of the potential environmental factors that also

A lot of studies concerning effects of pesticides in bivalves have been carried out using high pollutant concentrations. However, levels of pesticides measured in superficial waters generally range below lethal concentrations for aquatic species. In this context, sub-lethal

marine mussel, *Mytilus galloprovincialis*, were also reported by Dondero et al. (2010).

could lead to an injury of host tissues, resulting in higher mortality rates.

marine bivalves at a population level with an ecological relevance.

contaminates on shellfish and on human health if the seafood is consumed.

**7. Conclusions and perspectives** 

response of *Mya arenaria* to pesticides.

physiological status of bivalves is necessary.

may affect bivalve physiology.


Effects of Pesticides on Marine Bivalves: What Do We Know and What Do We Need to Know? 237

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**13** 

*Spain* 

**for Aquaculture** 

*Faculty of Biology, University of Murcia,* 

**Immunotoxicological Effects of Environmental** 

**Contaminants in Teleost Fish Reared** 

Alberto Cuesta, José Meseguer and M. Ángeles Esteban

*Fish Innate Immune System Group, Department of Cell Biology and Histology,* 

Contamination is one of the major problems associated with the environmental sciences. Many of the environmental pollutants affect to the different aquatic animals to certain degree depending on the toxic substance, concentration, self-life and animal behaviour and biology. Direct ingestion of environmental contaminants and bioaccumulation of toxic substances in bivalves, crustaceans, molluscs or fish for human supply is a serious task to consider in human nutrition. Furthermore, it is known that to provide the necessary proteins that need and will need the world's population must intensify efforts in production of both proteins of plant origin and animal origin. Among the latter is predicted that aquaculture will be one of the fields over the coming years will increase. In this regard, aquaculture is trying for some decades to compensate this negative balance for human consumption. Among the important issues to consider in the aquaculture business the impact of the environmental contaminants in the species produced for humans need to be controlled by the farmer. In this specific field, most of studies have evaluated the toxic effects in terms of fish viability or induction of tumors using different fish models. However, relevant fish species for aquaculture are less used in these experiments. Moreover, the impact of the environmental contaminants in the immune response of these fish, and

Fish are the first group of vertebrate animals with both innate and adaptive immune responses and are essential for proper understanding of the immune system and its evolution. The fish adaptive immune responses are less effective than in mammals because they are poikilotherms and completely dependent on the environmental temperature. Therefore, the importance of the innate immune response is more relevant, but not exclusive, in the fish disease resistance to pathogens. Overall, the mechanisms and molecules involved in the immune response are quite well conserved during the immune system evolution. However, there are major differences in terms of haematopoietic organs

structure and function as well as in leucocyte distribution and function (Figure 1).

consequently in the disease resistance, have received much less attention.

**2. Overview of the teleost fish immune response** 

**1. Introduction** 

metabolites on natural phototrophic biofilms using a pollution-induced community tolerance (PICT) approach. *Aquatic Toxicology*. Vol.99, No.4, pp. 492-499

