The 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 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.

To investigate the ability of microalgae to develop stable, long-term resistance to herbicides, the marine microalga Tetraselmis suecica was exposed to the herbicide diuron (5 μg/L) for a 43-generation exposure period followed by a 12-generation depuration phase. During the first 25 generations, diuron-exposed cultures showed doubling times ranging from 1.95 to 2.6 days, which was 2 to 2.5-fold longer than control cultures. Between generations 25 and 38, during diuron exposure, two out of the three exposed cultures exhibited a spontaneous drop in doubling time. These results provided evidence of culture adaptation to diuron. To assess persistence of the diuron adaptation observed on growth performance, one of the adapted cultures (D3) was maintained for 12 months in unexposed conditions and then tested by a second, short-term exposure to diuron 5 μg/L, in parallel with a control culture (C1) for six generations. Flow cytometry analyses were used to monitor cell density, viability, morphology, relative chlorophyll content and intracellular reactive oxygen species (ROS) level. Under these conditions, diuron induced a strong increase of doubling time in exposed-C1 cultures (2.5-fold longer than unexposed-C1 cultures), but no significant increase occurred in exposed D3-cultures compared with unexposed D3- and unexposed C1-cultures, showing the persistence of adaptation in the previously-exposed strain D3. Intracellular ROS level showed the same trend. Significant differences were observed between these strains, with weaker effects of diuron on strain D3 compared with strain C1: forward scatter (FSC), representing relative cell size, decreased in exposed cultures (67.8% and 95% of the controls for C1 and D3, respectively), whereas FL3 as relative chlorophyll content increased in exposed cultures (115.6% and 108.6% of the controls for C1 and D3, respectively). Results of second exposure to diuron revealed that the adaptation of strain D3 had persisted after 12 months of depuration, as no growth impairment was observed. This study demonstrates the possible appearance of stable diuron resistance in microalgae in cases of strong, multigenerational chronic exposure to this herbicide in polluted environments.

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.

Effects of the herbicide Basamaïs (bentazon) and the fungicide Opus (epoxiconazole) on oyster spat (Crassostrea gigas) were assessed using in-situ microcosms in a field experiment lasting 13 days. Six-week-old hatchery spat (mean size 1.1 mm), previously collected on PVC plates, was immersed in glass bottles filled with 200 mum filtered seawater. Bottles were maintained underwater at 6 m depth and their water content changed every other day. Growth, measured as shell area index increase, was 126 +/- 4% in the control bottles. While no growth differences were observed between control and individual pesticide treatments at 10 microg l(-1), oysters treated with a mix of 10 microg l(-1) Opus and 10 microg l(-1) Basamaïs showed a 50% growth reduction compared with the control (P < 0.0001), suggesting a synergistic effect of these contaminants. Laboratory controls in microcosms maintained in a water bath with filtered natural light, were not significantly different from in-situ microcosm controls in the field, for organic weight content or growth. This in-situ experiment in microcosms allowed us to conclude that: (1) oyster spat can achieve significant growth in bottles immersed in situ without supplementary food; (2) this microcosm system is reliable and easy to use for environmental toxicity tests with C. gigas spat; (3) such microcosm systems can also be run in a laboratory water bath instead of more technically difficult immersed field experiments; (4) the synergistic effect observed here, at a concentration simulating a peak agricultural runoff event, suggests that the impacts of pesticides could be a real threat for oysters in estuarine areas.