Méthodes d'évaluation de l'impact de pesticides sur le phytoplancton marin et le naissain d'huître creuse

The main goal of this work was the study of pesticide effects on natural communities of coastal phytoplankton and oyster spat, using in situ microcosms. Microbial communities (- Temporal Temperature Gradient gel Electrophoresis (TTGE) provides prokaryote (16S DNA) and eukaryote (18S DNA) community genetic fingerprints;- flow cytometry allows the discrimination of photosynthetic populations according to their scatter and fluorescence signals;- High Pressure Liquid Chromatography (HPLC) provides pigment fingerprints of the photosynthetic communities.Microscopic observations were carried out for some experiments in order to provide further information. The main results suggest TTGE as the most sensitive method to detect pesticide effects on the whole prokaryote and eukaryote community structure, compared with the two others methods that only focus on photosynthetic communities.No effect was detected when communities were exposed to the lowest concentration (0.1 µg/L) of each six pesticides. However, the three herbicides Basamaïs (bentazon), Roundup (glyphosate), Frontier (dimethenamid), and the insecticide Dursban 4 (chlorpyrifos) are likely to represent a danger for microbial communities from1 µg/L.Young oyster spat was exposed in situ to the herbicide Basamaïs and the fongicide Opus separately at 10 and 70 µg/L, and to a mixture of Basamaïs and Opus at 10 µg/L each. The results show a synergistic effect of the mixture on spat growth, with treated spat growth being half the control value, when no effects were detected when both pesticides were tested separately at 10 µg/L. This work shows that some pesticides can generate possible adverse effects in coastal areas. Roundup effects must be pointed out as they were detected at environmentally realistic concentrations. The results observed using oyster spat raise the issue of interactions between chemicals in the natural environment. Therefore, the relevance of reglementary toxicity thresholds, that do not include such possible substance interactions, should be re-examined.L'objectif principal de cette thèse était d'étudier l'impact de pesticides sur les communautés naturelles de phytoplancton côtier et sur le naissain d'huître creuse, en microcosmes in situ. Les communautés microbiennes (- la « Temporal Temperature Gradient gel Electrophoresis » (TTGE), ou gel d'éléctrophorèse avec gradient de température dans le temps, permettant d'obtenir les empreintes génétiques des communautés procaryotes (avec l'ADN codant pour l'ARN ribosomal 16S) et eucaryotes (avec l'ADN codant pour l'ARN ribosomal 18S) ;- la cytométrie en flux, qui permet de discriminer certaines populations photosynthétiques en fonction, notamment, de leurs signaux d'autofluorescence ;- la Chromatographie Liquide Haute Pression (HPLC), permettant d'obtenir les empreintes pigmentaires des communautés photosynthétiques.Des observations microscopiques ont complété l'analyse de certaines expériences. Les principaux résultats obtenus suggèrent une meilleure sensibilité de la TTGE pour la détection d'effets sur la structure de l'ensemble des communautés eucaryotes ou procaryotes, comparée aux deux autres méthodes qui concernent plus spécifiquement les communautés photosynthétiques. Parmi les six formulations commerciales testées à 0.1 µg/L, aucun effet n'est détecté. Par contre, quatre formulations sont susceptibles de représenter un danger pour les communautés microbiennes à partir de 1 µg/L : les herbicides Basamaïs (bentazone), Roundup (glyphosate), Frontier (diméthénamide), ainsi que l'insecticide Dursban 4 (chlorpyrifos).L'herbicide Basamaïs et le fongicide Opus ont été testés sur le naissain d'huître creuse, seuls à 10 et 70 µg/L, et en mélange à 10 µg/L chacun, au cours d'une expérimentation in situ. Les résultats obtenus démontrent un effet synergique des deux pesticides en mélange à 10 µg/L : la croissance du naissain est réduite de moitié, alors qu'elle n'est pas affectée par les pesticides seuls à 10 µg/L. Cette thèse permet d'illustrer les dangers potentiels représentés par certains pesticides en milieu côtier, en particulier par le Roundup dont les effets apparaissent pour des concentrations testées proches des concentrations environnementales. Les résultats obtenus sur le naissain posent la question des interactions entre molécules et donc de la pertinence des seuils de toxicité réglementaires pour les substances seules, alors même que ces seuils peuvent être différents lorsque des interactions se produisent dans l'environnement

Méthodes d évaluation de l impact de pesticides sur le phytoplancton marin et le naissain d huître creuse

L objectif était d étudier l impact de pesticides sur les communautés naturelles de phytoplancton côtier et sur le naissain d huître creuse, en microcosmes in situ. Les communautés microbiennes ont été exposées in situ à 6 pesticides (4 herbicides, 1 insecticide et 1 fongicide à 0.1, 1, 10 et 100 g/L de substance active. Le contenu des microcosmes a été analysé par 3 méthodes: (i) la Temporal Temperature Gradient gel Electrophoresis (TTGE), permettant d obtenir les empreintes génétiques des communautés procaryotes et eucaryotes (ii) la cytométrie en flux, qui permet de discriminer certaines populations photosynthétiques (iii) la Chromatographie Liquide Haute Pression (HPLC), permettant d obtenir les empreintes pigmentaires des communautés photosynthétiques. Parmi les 6 formulations testées à 0.1 g/L, aucun effet n est détecté. Par contre, 4 formulations peuvent représenter un danger pour les communautés microbiennes à partir de 1 g/L : les herbicides Basamaïs, Roundup, Frontier et l insecticide Dursban 4. Le Basamaïs et le fongicide Opus ont été testés sur le naissain d huître creuse lors dl expérimentation in situ. Un effet synergique des pesticides est obtenu en mélange à 10 g/L : la croissance du naissain est réduite de moitié, alors qu elle n est pas affectée par les pesticides seuls à 10 g/L. Ce travail illustre les dangers potentiels représentés par certains pesticides en milieu côtier, surtout par le Roundup dont les effets apparaissent aux concentrations environnementales. Les résultats obtenus sur le naissain posent la question des interactions entre molécules et de la pertinence des seuils de toxicité réglementaires pour les substances seules.The goal of this work was the study of pesticide effects on natural communities of coastal phytoplankton and on oyster spat, using in situ microcosms. Microbial communities were exposed in situ to 6 pesticides (4 herbicides, 1 insecticide and 1 fongicide) at 4 concentrations (0.1, 1, 10 and 100 g/L of active substance). The microcosm content was analysed using 3 methods: (i) Temporal Temperature Gradient gel Flectrophoresis (TTGE) provides prokaryote and eukaryote community genetic fingerprints; (ii) flow cytometry allows the discrimination of photosynthetic populations; iii) High Pressure Liquid Chromatography (HPLC) provides pigment fingerprints of thephotosynthetic communities. No effect was detected when communities were exposed to 0.1 g/L of each 6 pesticides. However, the 3 herbicides Basamaïs, Roundup Frontier, and the insecticide Dursban 4 are likely to represent a danger for microbial communities froml g/L. Young oyster spat was exposed in situ to Basamaïs and to the fongicide Opus. The results show a synergistic effect of the mixture at 10 g/L on spat growth, with treated spat growth being half the control value. No effects were detected when both pesticides were tested separately at 10 g/L. This work shows that some pesticides can generate possible adverse effects in coastal areas. Roundup effects must be pointed out as they were detected at environmentally realistic concentrations. The results observed using oyster spat raise the issue of interactions between chemicals in the natural environment. Therefore, the relevance of reglementary toxicity thresholds, that do not include such possible substance interactions, should be re-examined.BREST-BU Droit-Sciences-Sports (290192103) / SudocPLOUZANE-Bibl.La Pérouse (290195209) / SudocSudocFranceF

Evaluation of the partial renewal of in situ phytoplankton microcosms and application to the impact assessment of bentazon and dimethenamid.

International audienceMicrocosms, each consisting of 2L natural surface seawater maintained in 2.3-L glass bottles, were immersed at a depth of 6m. The renewal of 10% of microcosm volumes was carried out every other day. Phytoplankton-containing seawater was used for renewal (previously filtered through 25-, 50- or 200-μm cut-off). Phytoplankton community pigment analysis (by HPLC) and flow cytometry analysis were performed. After 13 days, data exhibited phytoplankton characteristics in microcosms in the same range as that of the natural surrounding sea water over the same period. Furthermore, in these microcosms, a negative correlation was observed between the filtration cut-off used for renewal water, and the total cell count. Herbicides were tested as commercial mixtures at 1, 10 and 100 μgL(-1) active substance. Both Frontier® (dimethenamid) and Basamais® (bentazon) induced significant modifications of the phytoplankton populations at every concentration tested. Such results suggest a possible disturbance in polluted coastal areas

Projet Squale

Il s’agissait i) de mettre en place un réseau de suivi de la qualité de l’eau, ii) de déterminer la qualité de chaque élevage larvaire dans le but de mettre en évidence un lien éventuel entre la qualité physico-chimique de l’eau de mer et la production conchylicole en écloserie, iii) mettre en évidence, l'influence de l’eau utilisée pour l’élevage, à court-terme, sur la « qualité » physiologique de l’algue fourrage T-iso utilisée pour nourrir les larves et à plus long-terme vérifier si ses capacités de croissance ainsi que sa «qualité» physiologique sont susceptibles d’être modifiées en fonction de la qualité de l’eau issue du polder. Les résultats obtenus ont mis en évidence la présence permanente de contaminants chimiques dans les eaux du polder. Les variations de concentrations observées étant essentiellement liées aux phénomènes météorologiques (pluie). L'influence du Diuron (molécule e référence, présente dans les eaux du polder) a pu être mise en évidence sur la vitesse de croissance de Tiso et sur sa composition lipidique. La pérennisation du suivi (plus léger) de la qualité des eaux semble être justifiée

Comparative study of three analysis methods (TTGE, flow cytometry and HPLC) for xenobiotic impact assessment on phytoplankton communities

International audienceThe impacts of the fungicide Opus® (epoxiconazole) on marine phytoplankton communities were assessed in a 12-day field experiment using in situ microcosms maintained underwater at 6 m depth. Three community analysis methods were compared for their sensitivity threshold in fungicide impact detection. When phytoplankton communities were exposed to 1 μg l−1 of epoxiconazole, no effects could be demonstrated using TTGE (Temporal Temperature Gradient gel Electrophoresis), flow cytometry or HPLC. When exposed to 10 μg l−1, TTGE fingerprints from PCR amplified 18S rDNA of communities exhibited significant differences compared with controls (ANOSIM, P = 0.028). Neither flow cytometry counts, nor HPLC pigment profiles allowed to show significant differences in microcosms exposed to 10 μg l−1 of epoxiconazole. When exposed to 100 μg l−1, all three methods allowed to detect significant differences in treated microcosms, as compared to controls. The TTGE analysis appears in this study as the most sensitive method for fungicide impact assessment on eukaryote microbial communities

Impact of Roundup on the marine microbial community, as shown by an in situ microcosm experiment

International audienceThe effects of the herbicide Roundup® (glyphosate) on natural marine microbial communities were assessed in a 7-day field experiment using microcosms. Bottles were maintained underwater at 6 m depth, and 10% of their water content was changed every other day. The comparison of control microcosms and surrounding surface water showed that the microcosm system tested here can be considered as representative of the natural surrounding environment. A temporal temperature gradient gel electrophoresis (TTGE) was run on 16S and 18S rDNA-amplified extracts from the whole microbial community. Cluster analysis of the 16S gel showed differences between control and treatment fingerprints for Roundup at 1 μg L−1 (ANOSIM, p = 0.055; R = 0.53), and 10 μg L−1 (ANOSIM, p = 0.086; R = 0.40). Flow cytometry analysis revealed a significant increase in the prasinophyte-like population when Roundup concentration was increased to 10 μg L−1. This study demonstrates that a disturbance was caused to the marine microbial community exposed to 1 μg L−1 Roundup concentration, a value typical of those reported in coastal waters during a run-off event

Toxicity of binary mixtures of pesticides to the marine microalgae Tisochrysis lutea and Skeletonema marinoi: Substance interactions and physiological impacts

This study screened binary mixtures of pesticides for potential synergistic interaction effects on growth of the marine microalgae Tisochrysis lutea and Skeletonema marinoi. It also examined the single and combined effects of three of the most toxic substances on microalgal physiology. Single substances were first tested on each microalgal species to determine their respective EC50 and concentration-response relationships. The toxicity of six and seven binary mixtures was then evaluated in microplate experiments on the growth of T. lutea and S. marinoi, respectively, using two mixture modelling approaches: isobolograms and the MIXTOX tool, based on Concentration Addition (CA) or Independent Action (IA) models. Significant cases of antagonism (for both species) and synergism (for S. marinoi) were observed for the mixtures of isoproturon and spiroxamine, and isoproturon and metazachlor, respectively. These two mixtures, together with that of isoproturon and diuron, for which additivity was observed, were further studied for their impacts on the physiology of each species. Exposures were thus made in culture flasks at three concentrations, or concentration combinations for mixtures, selected to cause 25%, 50% and 75% growth rate inhibition. The effects of the selected pesticides singly and in combination were evaluated at three perceived effect concentrations on esterase metabolic activity, relative lipid content, cytoplasmic membrane potential and reactive oxygen species (ROS) content by flow cytometry, and on photosynthetic quantum yield (ϕ’M) by PAM-fluorescence. Isoproturon and diuron singly and in mixtures induced 20–40% decreases in ϕ’M which was in turn responsible for a significant decrease in relative lipid content for both species. Spiroxamine and metazachlor were individually responsible for an increase in relative lipid content (up to nearly 300% for metazachlor on S. marinoi), as well as cell depolarization and increased ROS content. The mixture of isoproturon and metazachlor tested on S. marinoi caused a 28–34% decrease in ϕ’M that was significantly higher than levels induced by each of substances when tested alone. This strong decrease in ϕ’M could be due to a combined effect of these substances on the photosynthetic apparatus, which is likely the cause of the synergy found for this mixture

Microalgal sensitivity varies between a diuron-resistant strain and two wild strains when exposed to diuron and irgarol, alone and in mixtures

A wild strain of Chaetoceros calcitrans and wild and diuron-resistant strains of Tetraselmis suecica, were exposed to the PSII inhibitor herbicides diuron and irgarol, individually and in mixtures. The effects of three concentrations of diuron and irgarol and four binary mixtures were evaluated on doubling time, relative reactive oxygen species and lipid content by flow cytometry, and on photosynthetic efficiency by pulse amplitude modulated fluorescence. In both wild strains, significant effects were observed for each molecule at the highest concentration tested: at irgarol 0.5 μg L−1, C. calcitrans was shown to be more sensitive than T. suecica (+52% and +19% in doubling time, respectively), whereas at diuron 5 μg L−1, T. suecica was more affected (+125% in doubling time) than C. calcitrans (+21%). Overall, irgarol had a higher toxicity at a lower concentration than diuron (no effect at diuron 0.5 μg L−1) for both wild strains. The strongest mixture (irgarol 0.5 μg L−1 + diuron 5 μg L−1) increased doubling time by 356% for T. suecica, thus showing amplified effects when the two compounds were mixed. Sequencing of the diuron-resistant strain demonstrated a single mutation in the psbA gene coding sequence. Although resistance of this strain to diuron was confirmed with no effect at the highest diuron concentration, no resistance to irgarol was shown. In addition, the mutant strain exposed to the strongest mixture showed a 3.5-fold increase in doubling time compared with irgarol alone, thereby supporting the hypothesis of a biochemical interaction between these two compounds