Jérôme Cachot

Embryotoxic and genotoxic effects of heavy metals and pesticides on early life stages of Pacific oyster (Crassostrea gigas)

This study evaluated embryotoxicity and genotoxicity of two dissolved metals copper and cadmium (Cu and Cd) and two pesticides (metolachlor and irgarol) occurring in Arcachon Bay (SW France) in Pacific oyster (Crassostrea gigas) larvae and investigated the relationship between those two endpoints. Embryotoxicity was measured by calculating the percentage of abnormal D-shaped larvae and genotoxicity was evaluated with DNA strand breaks using the comet assay. After 24h exposure, significant increases of the percentage of abnormal D-larvae and the DNA strand breaks were observed from 0.1 μg L⁻¹ for Cu, 10 μg L⁻¹ for Cd and 0.01 μg L⁻¹ for both irgarol and metolachlor in comparison with the controls. A strong positive relationship between embryotoxicity and genotoxicity was recorded for Cu, Cd and metolachlor. The current study suggests that copper, irgarol and metolachlor can induce larval abnormalities and DNA damage in a population of exposed oysters at environmentally relevant concentrations.

Effects of copper and cadmium spiked-sediments on embryonic development of Japanese medaka (Oryzias latipes)

Because of their high capacity to accumulate contaminants such as persistent organic pollutants and heavy metals, aquatic sediments are considered as a long-term source of contamination for aquatic organisms. In compliance with the increasing interest both for sediment quality evaluation and the use of fish early life stage (ELS) toxicity assays, we proposed an embryo-larval test to evaluate embryotoxicity and genotoxicity of sediment-bound contaminants. Pre-blastula stage medaka (Oryzias latipes) embryos were exposed by static sediment contact to two model heavy metals (cadmium and copper) at environmental concentrations during the whole 10-day embryonic development. Lethal and sub-lethal effects were recorded in both embryos and larvae for 20 days post fertilisation (dpf) using several global toxicity and phenotypic endpoints. The comet assay was also performed on medaka prolarvae to evaluate genotoxic effects of the tested chemicals. Environmental concentrations of cadmium (Cd) and copper (Cu) did not affect embryo and larval survival. However, both heavy metals significantly induced morphological abnormalities, particularly spinal and cardiovascular deformities. Cd but not Cu induced tachycardia. Both heavy metals induced a significant increase in DNA damage at all tested concentrations. Resulting LOEC values for Cd and Cu corresponded to 1.9 and 8.5 μg/g d.w. sediment, respectively. Although metal bioavailability is probably lower for naturally contaminated sediments, the relatively low toxicity thresholds for both Cd and Cu raise the question of possible risk for fish embryos developing in direct contact to sediments. This study demonstrates the applicability, sensitivity and relevance of the Japanese medaka embryo-larval assay (MELA) to evaluate sediment hazardous potency at environmental concentrations of heavy metals.

A new spiked sediment assay using embryos of the Japanese medaka specifically designed for a reliable toxicity assessment of hydrophobic chemicals

Despite their low water solubility, hydrophobic pollutants are widespread in the aquatic environment and could represent a threat for living organisms. EU regulations on chemicals require accurate and reliable data on chemical toxicity. Current normalised fish toxicity assays, in particular those advocated by OECD guidelines, do not allow reliable toxicity assessment of hydrophobic compounds due to their low water solubility. In order to accurately evaluate the toxicity of this kind of compounds, a new spiked sediment assay using embryos of the Japanese medaka was developed. It consists of directly exposing fertilised eggs, during their entire embryonic development, onto the reference sediment spiked with the test compound. A large set of lethal or sublethal effects in embryos and newly hatched larvae, including non-invasive endpoints is analysed in order to maximise the sensitivity of the test. The approach was validated using four model pollutants with different modes of action: DMBA, PCB126, PCB153 and 4-nonylphenol (NP). All compounds, except PCB153, induced a dose-dependent increase in toxic effects. In fact, lethal effects only occurred at the highest tested concentration. In contrast, sub-lethal effects including skeletal deformations, cardiac activity modulation, body length reduction and hatching delay were observed at low to moderate concentrations of DMBA and PCB126. NP induced subtle effects in embryos, altering cardiac activity and hatching success but only at high concentrations. Although a few more improvements would make it a fully standardised assay, this spiked sediment assay using medaka embryos proves to be sensitive enough to measure hydrophobic chemical toxicity using an environmentally realistic mode of exposure.

Zebrafish Models for Human Acute Organophosphorus Poisoning

Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

Exploration of Daphnia behavioral effect profiles induced by a broad range of toxicants with different modes of action

Behavior is increasingly reported as a sensitive and early indicator of toxicant stress in aquatic organisms. However, the systematic understanding of behavioral effects and comparisons between effect profiles is hampered because the available studies are limited to few chemicals and differ in the exposure conditions and effect parameters examined. The aims of the present study were 1) to explore behavioral responses of Daphnia magna exposed to different toxicants, 2) to compare behavioral effect profiles with regard to chemical modes of action, and 3) to determine the sensitivity and response time of behavioral parameters in a new multi-cell exposure system named Multi-DaphTrack compared with currently utilized tests. Twelve compounds covering different modes of toxic action were selected to sample a wide range of potential effect profiles. Acute standard immobilization tests and 48 h of behavioral tracking were performed in the customized Multi-DaphTrack system and a single-cell commercialized biological early warning system. Contrasting behavioral profiles were observed for average speed (i.e., intensity, time of effect onset, effect duration), but no distinct behavioral profiles could be drawn from the chemical mode of action. Most compounds tested in the Multi-DaphTrack system induced an early and significant average speed increase at concentrations near or below the 10% effective concentration (48 h) of the acute immobilization test, demonstrating that the Multi-DaphTrack system is fast and sensitive. To conclude, behavior endpoints could be used as an alternative or complement to the current acute standard test or chemical analysis for the predictive evaluation of ecotoxic effects of effluents or water bodies.

Genetic polymorphism and its potential relation to environmental stress in five populations of the European flounder Platichthys flesus, along the French Atlantic coast.

In this study, new DNA markers were explored for the flounder Platichthys flesus. cDNA and genomic sequences of the genes encoding the glyceraldehyde-3-phosphate-deshydrogenase (GAPDH), the cytosolic creatine kinase (CK), the prostaglandin D synthase (PGDS) and the betaine homocysteine methyltransferase (BHMT) were characterized. The tumour suppressor p53 gene structure was already described. A PCR-SSCP (Single Strand Conformation Polymorphism) analysis was finally conducted to study the genetic polymorphism of different populations of flounders collected along the French Atlantic coast. Four highly contaminated French estuaries (Seine, Vilaine, Loire and Gironde) were sampled and compared to a reference estuary (Ster) to explore possible selective effect of the environment on specific allelic frequencies. Our results showed that two loci p53 and PGDS, could be potential markers of chemical stress: p53A allele frequency increased in contaminated systems compared to the reference system. In the Vilaine estuary, PGDS polymorphism could be related to pesticide stress.

Assessment of pollution in the Bizerte lagoon (Tunisia) by the combined use of chemical and biochemical markers in mussels, Mytilus galloprovincialis.

In order to assess the environmental quality of the Bizerte lagoon (Tunisia), biomarker and contaminant levels were measured in Mediterranean mussels (Mytilus galloprovincialis) from five selected sites. Persistent organic pollutants (POPs) were quantified in whole body and enzyme activities such as acetylcholinesterase (AChE), catalase (CAT) and glutathione S-transferase (GST) in gills. Despite the relatively low levels of organic contaminants, the selected biomarkers responded differently according to the pollution level at the different sites. GST and AChE activities were correlated with the amount of DDTs in mussel tissues. These two enzymatic activities were also correlated to temperature and pH. No significant difference was observed for CAT activity. Principal component analysis showed a clear separation of sampling sites in three different assemblages which is consistent with POP body burden in mussels. Our results confirmed the usefulness of combining biomarker and chemical analyses in mussels to assess chemical pollution in the Bizerte lagoon.

Development of a larval bioassay using the calanoid copepod, Eurytemora affinis to assess the toxicity of sediment-bound pollutants

Hydrophobic pollutants, in particular sediment-sorbed organic compounds, are widespread in the aquatic environment and could represent a threat to living organisms. Estuarine species, which live in turbulent ecosystems, are particularly exposed to this mode of contamination. For precise evaluation of the toxicity of hydrophobic contaminants desorbed from particles, a new larval assay using nauplii of the estuarine calanoid copepod Eurytemora affinis was developed. It consists of the direct exposure of copepods during naupliar development to elutriates of an unpolluted sediment spiked with different model contaminants. This bioassay measures the toxicity of the bioavailable fraction of particle-sorbed pollutants on the naupliar stage of copepods. Mortality and growth (non-invasive endpoints) in nauplii were analysed after six days of exposure. This approach was validated using six pollutants with different modes of action: benzo[a]pyrene (BaP), dimethylbenzo[a]anthracene (DMBA), phenanthrene (PHE), polychlorinated biphenyls (PCB 126, PCB 153) and 4-nonylphenol (4-NP). All these compounds induced a dose-dependent increase in toxic effects. Lethal effects only occurred at the highest tested concentrations: 58,541 and 6092 ng g(-1) dry weight sediment (dws), for PHE and DMBA, respectively. Sublethal effects (growth inhibition) were observed at lower concentrations for all tested compounds except PCB 153, from 8, 142, 297, 6092 and 8453 ng g(-1) dws for PCB 126, BaP, PHE, DMBA and 4-NP, respectively.

Comparative responses of sperm cells and embryos of Pacific oyster (Crassostrea gigas) to exposure to metolachlor and its degradation products.

Metolachlor is one of the most intensively used chloroacetanilide herbicides in agriculture. Consequently, it has been frequently detected in coastal waters as well as its major degradation products, metolachlor ethane sulfonic acid (MESA) and metolachlor oxanilic acid (MOA) which are encountered at higher concentrations than metolachlor. Although a few studies of metolachlor toxicity have been conducted on marine organisms, little is known about the environmental toxicity of metolachlor degradation products. In this study, the deleterious effects of metolachlor and its degradation products on spermatozoa and embryos of Crassostrea gigas have been compared using biomarkers of developmental defects, DNA damage and gene transcription levels. After 24h exposure, significant increases in the percentage of abnormal D-larvae and DNA damage were observed from 0.01 μg L(-1) for S-metolachlor and 0.1 μg L(-1) for MESA and MOA. Results showed that S-metolachlor was more embryotoxic and genotoxic than its degradation products. Oyster sperm was also very sensitive to metolachlor exposure and followed the pattern: metolachlor (0.01 μg L(-1))>MOA (0.1 μg L(-1))>MESA (1 μg L(-1)). Metolachlor and MESA mainly triggered variations in the transcription level of genes encoding proteins involved in oxidative stress responses (mitochondrial superoxide dismutase and catalase). Overall, no significant variation in transcription levels could be detected in C. gigas embryos exposed to MOA. This study demonstrates that metolachlor and its main degradation products have the potential to impact several steps of oyster development and therefore recruitment in coastal areas exposed to chronic inputs of pesticides.

Toxicity assessment of water-accommodated fractions from two different oils using a zebrafish (Danio rerio) embryo-larval bioassay with a multilevel approach

Petroleum compounds from chronic discharges and oil spills represent an important source of environmental pollution. To better understand the deleterious effects of these compounds, the toxicity of water-accommodated fractions (WAF) from two different oils (brut Arabian Light and Erika heavy fuel oils) were used in this study. Zebrafish embryos (Danio rerio) were exposed during 96h at three WAF concentrations (1, 10 and 100% for Arabian Light and 10, 50 and 100% for Erika) in order to cover a wide range of polycyclic aromatic hydrocarbon (PAH) concentrations, representative of the levels found after environmental oil spills. Several endpoints were recorded at different levels of biological organization, including lethal endpoints, morphological abnormalities, photomotor behavioral responses, cardiac activity, DNA damage and exposure level measurements (EROD activity, cyp1a and PAH metabolites). Neither morphological nor behavioral or physiological alterations were observed after exposure to Arabian Light fractions. In contrast, the Erika fractions led a high degree of toxicity in early life stages of zebrafish. Despite of defense mechanisms induced by oil, acute toxic effects have been recorded including mortality, delayed hatching, high rates of developmental abnormalities, disrupted locomotor activity and cardiac failures at the highest PAH concentrations (∑TPAHs=257,029±47,231ng·L(-1)). Such differences in toxicity are likely related to the oil composition. The use of developing zebrafish is a good tool to identify wide range of detrimental effects and elucidate their underlying foundations. Our work highlights once more, the cardiotoxic action (and potentially neurotoxic) of petroleum-related PAHs.