Comet assay in phytoplankton as biomarker of genotoxic effects of environmental pollution.

The alkaline comet assay was tested on different microalgae: the dinoflagellates, Karenia mikimotoi and Alexandrium minutum, and the diatom, Chaetoceros gracilis. The microalgae were exposed during their exponential growth to the model direct genotoxicant, hydrogen peroxide (1h, 5 and 100muM H2O2). Following H2O2 exposure, the comet assay was validated only for K. mikimotoi for which genotoxicity was observed from the lowest tested concentration of 5 microM with a concentration-dependent effect. C. gracilis was too small in size (4 microm) to be correctly analysed. For A. minutum, our lysis buffer was not strong enough to digest the cellulosic thecal plates. For K. mikimotoi, the comet assay was thus applied for the study of the genotoxic effects of different pesticides: epoxiconazole (as Opus formulation), chlorpyriphos-ethyl (as Dursban formulation) and endosulfan at 1, 10 and 100 microg of active substance/L for 24h. Exposure to epoxiconazole in formulation resulted in an increase in the extent of DNA strand breaks at the highest tested concentration icro/L. Endosulfan exposure resulted in DNA damage for K. mikimotoi nuclei. Genotoxicity was observed from 1 microg/L of endosulfan and was not concentration dependent.

Comparative efficiencies of different non-toxic microalgal diets in detoxification of PSP-contaminated oysters (Crassostrea gigas Thunberg).

Experimental PSP contamination of adult Pacific oysters (Crassostrea gigas) by the toxic dinoflagellate Alexandrium minutum Halim (120 cells.mL-1 continuously maintained in each flume) was carried out in a recirculated seawater system to obtain toxin levels above the safety threshold. In these conditions, 150 to 300 micrograms STX.eq.100 g-1 of shellfish tissues were produced at 16 degrees C within 8 to 15 days, corresponding to field values observed along French coasts. Diets based on non-toxic flagellates or diatoms were then used to detoxify the contaminated oysters. Despite large individual variations in toxin levels at the end of the contamination period, detoxification times were of the same order of magnitude (3 to 4 days), reaching a toxin level equal to or less than the safety threshold. These variations were most likely related to marked individual variability in valve and/or clearance activities. No significant differences in detoxification rates were found when oysters were fed Isochrysis galbana, Tetraselmis suecica, Thalassiosira weissflogii, or Skeletonema costatum. The different biochemical compositions of each algal species appeared to have no significant effect on detoxification rates. GTX2/GTX3 were the dominant compounds found in shellfish tissues during depuration, whereas C toxins were quite low (< 2 micrograms STX.eq.100 g-1) and STX or NeoSTX undetectable. These results do not suggest any bioconversion of paralytic toxins but indicate good correlation between the toxin composition of Alexandrium and oyster tissues.

Bioaccumulation of mycotoxins by shellfish: contamination of mussels by metabolites of a Trichoderma koningii strain isolated in the marine environment.

To determine whether toxic metabolites produced by fungi could cause shellfish toxicities, mussels were contaminated in laboratory conditions by sterile filtrates of a liquid culture of a strain of the fungus Trichoderma koningii previously isolated from a shellfish, the cockle (Cerastoderma edule). Mussels were kept in aerated natural seawater and fed with a culture of the microalga Isochrysis galbana, to which a filtrate of liquid fungal culture was added. Mussels were exposed to contamination for 7 days at 16 or 20 degrees C and extractions were then performed and their activity tested on blowfly larvae. The same toxicity was found in the fungal filtrate and the shellfish, indicating bioaccumulation. The digestive gland was the most toxic part of the mussel, confirming contamination by filtration. Treated mussels produced a mucus which appeared to be a means of eliminating toxic metabolites.

Kinetics of Alexandrium minutum Halim toxin accumulation in mussels and clams.

Mussels (Mytilus edulis) and clams (Ruditapes philippinarum) were contaminated experimentally using cultures of Alexandrium minutum, a toxic dinoflagellate isolated from French coastal waters. Experiments were carried out in continually flushed and open-circuit systems using Alexandrium densities of 100 to 700 cells/ml delivered to tanks containing the shellfish. All experiments indicated an inversion of the relative proportions of gonyautoxins (GTX2 and GTX3) in shellfish meat during decontamination, whereas saxitoxin (STX) only accumulated during mussel depuration. However, in mussels a density as low as 100 cells/ml led within 10 days to bioaccumulation of paralytic shellfish poisoning (PSP) toxins above the public health threshold. Similar results were observed in clams subjected to fivefold higher cell densities, indicating a less effective assimilation of the dinoflagellate than by mussel. Decontamination experiments on PSP toxin-contaminated mussels (360 micrograms STX eq./100 g or lower uptake) fed two nontoxic diets (1,000 and 10,000 cells/ml of Tetraselmis suesica) showed an appreciable reduction in the time needed to decrease toxin concentration below the accepted threshold for human consumption. We suggest that a simple relation can be established between initial toxicity, the concentration of nontoxic alga available, and the time required for depuration once decontamination kinetics becomes linear and corresponds to the inverse of contamination kinetics.

Influence of initial toxicity and extraction procedure on paralytic toxin changes in the mussel.

Farmed mussels were artificially contaminated with a pure culture of an Alexandrium tamarense toxic strain (MOG 835), to assess the effect of initial toxicity on paralytic toxin change during the depuration process. As previously observed in mussel, gonyautoxin GTX2 is eliminated more slowly than other gonyautoxins. A toxic level (1300 micrograms PSP per 100 g meat) is required to produce a drastic change in the depuration course, i.e. a 'fast' depuration rate followed by a 'slow' one. Below this threshold, decontamination becomes slower as the proportion of GTX2 increases over the time course. Although GTX2 is slowly eliminated during the depuration process, it is also formed in increasing quantities during the contamination phase. It remains to be determined whether changes in GTX2/GTX3 ratios are due to chemical or biological transformation.

Patterns of experimental contamination by Protogonyaulax tamarensis in some French commercial shellfish.

As a result of the proliferation of toxic marine dinoflagellates along European coasts and the recent discovery of paralytic poisons in French shellfish, experimental studies were conducted on four species of shellfish from the Brittany coasts. Contamination rates of a culture of toxic Protogonyaulax tamarensis, were determined for Mytilus edulis, Crassostrea gigas, Pecten maximus and Ruditapes philippinarum. Mussels and scallops were very rapidly contaminated showing high toxin accumulation rates, whereas rates for oysters and clams were low. During the decontamination phase, two stages were observed in mussels and scallops: a fast decrease in toxin, of the same order of magnitude as the accumulation, followed by a slow decrease, with the toxic rate remaining above the quarantine level of 80 micrograms/100 g. Toxin analysis, both in the culture and in the shellfish, was performed using high performance liquid chromatography. GTX3 and GTX8/epiGTX8 were the dominant toxins in the early stage of the decontamination phases, whereas GTX2 was the predominant compound during the slow phase of decontamination.