**3. Lethal effect of pesticides on marine bivalves**

Several studies have been conducted in various marine bivalve species in order to define LC50 for different pesticides including DDT, diuron, atrazine or lindane.

Chung et al. (2007) evaluated the sensitivity of the juvenile hard clam, *Mercenaria mercenaria*, to DDT (organochlorine pesticide) by exposure to contaminated sediments (10 day) and seawater (24-h). The aqueous LC50 (24h) value was defined at 0.61 mg L-1 DDT. and the LC50 (10 day) value for sediment toxicity tests was 5.8 mg kg-1 DDT. The authors concluded that

Effects of Pesticides on Marine Bivalves: What Do We Know and What Do We Need to Know? 231

Wessel et al. (2007) investigatied embryotoxic and genotoxic effects of the organochlorine pesticide, endosulfan, on *Crassostrea gigas* embryos. Embryotoxicity and genotoxicity in

Siu et al. (2008) used green-lipped mussels (*Perna viridis*) in order to study the bioaccumulation of organic pollutants, including organochlorine pesticides. Micronuclei and DNA strand breaks were observed in mussels transplanted in different sites and collected

Revankar and Shyama (2009) explored genotoxic effects of monocrotophos, an organophosphorous pesticide, at different time periods, 2, 3, 7 and 14 days. A significant increase of micronuclei in a dose dependant manner was observed indicaring possible

**5. Immunotoxicity in marine bivalves and susceptibilty to infectious diseases**  The impact of contaminants and other environmental factors on the immune system of bivalves is an issue of ecological and economical concern, because it may result in clinical pathology and disease, by increasing the susceptibility of affected organisms to pathogens. Contaminants known to induce alterations of immune functions including pesticides (Vial et al., 1996; Banerjee et al., 1996; Banerjee et al., 2001) are present in almost all coastal areas. Among physiological processes possibly disturbed by pollutants, the immune system is likely to be one of the more sensitive (Baier-Anderson & Anderson, 2000; Fournier et al.,

In contrast to the vertebrate immune system which consists of innate and acquired mechanisms, invertebrate immunity relies only on innate defence mechanisms. The fact that invertebrates represent more than 90% of the total number of species living on earth demonstrates the efficiency of their «primitive» host defence systems. It becomes more and more obvious that some of these innate mechanisms are conserved in invertebrates and vertebrates (Medzhitov et al., 1997; Means et al., 2000). Thus, the fundamental importance of the toxically-induced modulation of non-specific immune functions has increasingly been

Bivalve immunity is mainly supported by hemocytes and participate directly in eliminating pathogens by phagocytosis (Cheng, 1981; Feng, 1988). In addition, hemocytes produce compounds including lysosomal enzymes and antimicrobial molecules which contribute to

Investigating the effects of pesticides on hemocyte functions and immunity in bivalves has been based on the monitoring of several biomarkers (Pipe & Coles, 1995). As an example, Gagnaire et al. (2006) tested the effect of 23 pollutants on Pacific cupped oyster haemocytes by flow cytometry monitoring different cell parameters and demonstrated that 3 pesticides (2,4D, paraoxon, and chlorothalonil) induced a modulation of hemocyte activities. However,

Triforine, a fungicide, induced decreased hemocyte viability in the eastern oyster, *Crassostrea virginica* (Alvarez & Friedl, 1992). Cytotoxic effects were also observed in adult Pacific cupped oyster, *C. gigas*, hemocytes: the mean cell viability was significantly decreased at 1.0 mg L-1 of lindane (gamma-hexachlorocyclohexane) after 12 day exposure period (Anguiano et al., 2006). Alteration in cell viability was also reported in the blue mussel, *Mytilus edulis*, exposed to 0.1 mg L-1 azamethiphos, an organophosphate pesticide

terms of DNA strand breaks were observed for 300 nM and 150 nM.

chromosomal damages induced by monocrotophos.

the destruction of pathogens (Coles & Pipe, 1994).

biomarkers used differ very often between published studies.

after 4, 8, 12, 16 and 30 days.

2000).

perceived.

based on comparisons to toxicity data for other marine species, the hard clam, *Mercenaria mercenaria*, is one of the more sensitive species to contaminants.

No significant mortalities were reported after two months of exposition to 100 mg L-1 of diuron while with 100 µg L-1of isoproturon, 60% of mortalitites were observed (Moraga & Tanguy, 2000). Isoproturon has shown to be present at lower concentrations than diuron on aquatic environments (Munaron, 2006).

Lawton et al. (2010) studied the effects of atrazine on the hard clam, *Mercenaria mercenaria,* in aqueous and sediment laboratory assays. Through an acute aqueous bio-assay, these authors determined a 96h LC50 for the juvenile clams at 5608 µg L-1. They conducted also a chronic aqueous bio-assay at low atrazine concentrations and a chronic sediment bioassay over a 10 day exposure period to examine both lethal and sublethal (dry mass, shell size, and condition index) endpoints (Lawton et al., 2010). On the basis of their results, the authors suggested that atrazine is not directly toxic to *M. mercenaria* at environmentally relevant concentrations.

Bouilly et al. (2003) reported similar results for the Pacific cupped oyster, *Crassostrea gigas*:, in adult and juvenile animals subjected to 2 different concentrations of atrazine (46.5 nM and 465 nM). These authors did not observed any effect on mortality.

In vivo in laboratory assays testing 10 different concentrations (0 to 10 mg L-1) of lindane (gamma-hexachlorocyclohexane [gamma-HCH]) allowed to define the median lethal concentration (LC50) after a 12 day period as 2.22 mg L-1 in the Pacific cupped oyster, *Crassostrea gigas* (Anguiano et al., 2006). Lindane and isoproturon tested at concentrations of up to 10 mg L-1 for a 9 day esposure period showed negative effects on survival and growth of Pacific cupped oyster, *Crassostrea gigas,* larvae (Hiss & Seaman, 1993).

Domart-Coulon et al. (2002) assessed the acute cytotoxicity of an organic molluscicide, Mexel-432, used in antibiofouling treatments in industrial cooling water systems on primary cell cultures derived from 2 marine bivalve species, the Pacific cupped oyster, *Crassostrea gigas,* and the carpet clam, *Ruditapes decussatus*.

#### **4. Genotoxicity in marine bivalves**

Results reported by Jha et al. (2002) suggested that tributyltin oxide is both cytotoxic (proliferation rate index) and genotoxic (sister chromatid exchanges and chromosomal aberrations) to embryo-larval stages in the blue mussel, *Mytilus edulis*.

Bouilly et al. (2003) researched potential genotoxic effects of atrazine in the Pacific cupped oyster, *Crassostrea gigas*. Adult and juvenile oysters were subjected to 2 concentrations of atrazine: 46.5 nM, representing a realistic potential exposure (peak value found in polluted environment) and 465 nM. These authors reported significant differences in aneuploidy after atrazine treatments in comparion to control: 9% in control oysters, 16% at 46.5 nM and 20% at 465 nM atrazine. Similar aneuploidy levels were observed in adults and juveniles.

Bouilly et al. (2007) showed that the herbicide diuron induced also aneuploidy in adult Pacific cupped oysters after a 11 week exposure period at 300 ng L-1 and 3 µg L-1. The induced aneuploidy observed appeared to be transmitted to the next generation as offspring exhibited significantly higher aneuploidy levels when their parents had been exposed to diuron (Bouilly et al., 2007).

Genotoxicity induced by lindane at 0.7 mg L-1 was also demonstrated in Pacific oyster, *Crassostrea gigas*, hemocytes after a 12 day contamination period (Anguiano et al., 2006).

based on comparisons to toxicity data for other marine species, the hard clam, *Mercenaria* 

No significant mortalities were reported after two months of exposition to 100 mg L-1 of diuron while with 100 µg L-1of isoproturon, 60% of mortalitites were observed (Moraga & Tanguy, 2000). Isoproturon has shown to be present at lower concentrations than diuron on

Lawton et al. (2010) studied the effects of atrazine on the hard clam, *Mercenaria mercenaria,* in aqueous and sediment laboratory assays. Through an acute aqueous bio-assay, these authors determined a 96h LC50 for the juvenile clams at 5608 µg L-1. They conducted also a chronic aqueous bio-assay at low atrazine concentrations and a chronic sediment bioassay over a 10 day exposure period to examine both lethal and sublethal (dry mass, shell size, and condition index) endpoints (Lawton et al., 2010). On the basis of their results, the authors suggested that atrazine is not directly toxic to *M. mercenaria* at environmentally

Bouilly et al. (2003) reported similar results for the Pacific cupped oyster, *Crassostrea gigas*:, in adult and juvenile animals subjected to 2 different concentrations of atrazine (46.5 nM

In vivo in laboratory assays testing 10 different concentrations (0 to 10 mg L-1) of lindane (gamma-hexachlorocyclohexane [gamma-HCH]) allowed to define the median lethal concentration (LC50) after a 12 day period as 2.22 mg L-1 in the Pacific cupped oyster, *Crassostrea gigas* (Anguiano et al., 2006). Lindane and isoproturon tested at concentrations of up to 10 mg L-1 for a 9 day esposure period showed negative effects on survival and growth

Domart-Coulon et al. (2002) assessed the acute cytotoxicity of an organic molluscicide, Mexel-432, used in antibiofouling treatments in industrial cooling water systems on primary cell cultures derived from 2 marine bivalve species, the Pacific cupped oyster, *Crassostrea* 

Results reported by Jha et al. (2002) suggested that tributyltin oxide is both cytotoxic (proliferation rate index) and genotoxic (sister chromatid exchanges and chromosomal

Bouilly et al. (2003) researched potential genotoxic effects of atrazine in the Pacific cupped oyster, *Crassostrea gigas*. Adult and juvenile oysters were subjected to 2 concentrations of atrazine: 46.5 nM, representing a realistic potential exposure (peak value found in polluted environment) and 465 nM. These authors reported significant differences in aneuploidy after atrazine treatments in comparion to control: 9% in control oysters, 16% at 46.5 nM and 20% at 465 nM atrazine. Similar aneuploidy levels were observed in adults and juveniles. Bouilly et al. (2007) showed that the herbicide diuron induced also aneuploidy in adult Pacific cupped oysters after a 11 week exposure period at 300 ng L-1 and 3 µg L-1. The induced aneuploidy observed appeared to be transmitted to the next generation as offspring exhibited significantly higher aneuploidy levels when their parents had been exposed to

Genotoxicity induced by lindane at 0.7 mg L-1 was also demonstrated in Pacific oyster, *Crassostrea gigas*, hemocytes after a 12 day contamination period (Anguiano et al., 2006).

*mercenaria*, is one of the more sensitive species to contaminants.

and 465 nM). These authors did not observed any effect on mortality.

of Pacific cupped oyster, *Crassostrea gigas,* larvae (Hiss & Seaman, 1993).

aberrations) to embryo-larval stages in the blue mussel, *Mytilus edulis*.

*gigas,* and the carpet clam, *Ruditapes decussatus*.

**4. Genotoxicity in marine bivalves** 

diuron (Bouilly et al., 2007).

aquatic environments (Munaron, 2006).

relevant concentrations.

Wessel et al. (2007) investigatied embryotoxic and genotoxic effects of the organochlorine pesticide, endosulfan, on *Crassostrea gigas* embryos. Embryotoxicity and genotoxicity in terms of DNA strand breaks were observed for 300 nM and 150 nM.

Siu et al. (2008) used green-lipped mussels (*Perna viridis*) in order to study the bioaccumulation of organic pollutants, including organochlorine pesticides. Micronuclei and DNA strand breaks were observed in mussels transplanted in different sites and collected after 4, 8, 12, 16 and 30 days.

Revankar and Shyama (2009) explored genotoxic effects of monocrotophos, an organophosphorous pesticide, at different time periods, 2, 3, 7 and 14 days. A significant increase of micronuclei in a dose dependant manner was observed indicaring possible chromosomal damages induced by monocrotophos.
