Evaluation of lipophosphoramidates-based amphiphilic compounds on the formation of biofilms of marine bacteria.

The bacteriostatic and/or bactericidal properties of few phosphoramide-based amphiphilic compounds on human pathogenic bacteria were previously reported. In this study, the potential of two cationic (BSV36 and KLN47) and two zwitterionic (3 and 4) amphiphiles as inhibitors of marine bacterial growth and biofilm formation were investigated. Results showed that the four compounds have little impact on the growth of a panel of 18 selected marine bacteria at a concentration of 200 µM, and up to 700 µM for some bacterial strains. Interestingly, cationic lipid BSV36 and zwitterionic lipids 3 and 4 effectively disrupt biofilm formation of Paracoccus sp. 4M6 and Vibrio sp. D02 at 200 µM and to a lesser extent of seven other bacterial strains tested. Moreover, ecotoxicological assays on four species of microalgae highlighted that compounds 3 and 4 have little impact on microalgae growth with EC50 values of 51 µM for the more sensitive species and up to 200 µM for most of the others. Amphiphilic compounds, especially zwitterionic amphiphiles 3 and 4 seem to be promising candidates against biofilm formation by marine bacteria.

Main conclusions and perspectives from the collective scientific assessment of the effects of plant protection products on biodiversity and ecosystem services along the land-sea continuum in France and French overseas territories.

Preservation of biodiversity and ecosystem services is critical for sustainable development and human well-being. However, an unprecedented erosion of biodiversity is observed and the use of plant protection products (PPP) has been identified as one of its main causes. In this context, at the request of the French Ministries responsible for the Environment, for Agriculture and for Research, a panel of 46 scientific experts ran a nearly 2-year-long (2020-2022) collective scientific assessment (CSA) of international scientific knowledge relating to the impacts of PPP on biodiversity and ecosystem services. The scope of this CSA covered the terrestrial, atmospheric, freshwater, and marine environments (with the exception of groundwater) in their continuity from the site of PPP application to the ocean, in France and French overseas territories, based on international knowledge produced on or transposable to this type of context (climate, PPP used, biodiversity present, etc.). Here, we provide a brief summary of the CSA's main conclusions, which were drawn from about 4500 international publications. Our analysis finds that PPP contaminate all environmental matrices, including biota, and cause direct and indirect ecotoxicological effects that unequivocally contribute to the decline of certain biological groups and alter certain ecosystem functions and services. Levers for action to limit PPP-driven pollution and effects on environmental compartments include local measures from plot to landscape scales and regulatory improvements. However, there are still significant gaps in knowledge regarding environmental contamination by PPPs and its effect on biodiversity and ecosystem functions and services. Perspectives and research needs are proposed to address these gaps.

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

The 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 microg l(-1) of epoxiconazole, no effects could be demonstrated using TTGE (Temporal Temperature Gradient gel Electrophoresis), flow cytometry or HPLC. When exposed to 10 microg 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 microg l(-1) of epoxiconazole. When exposed to 100 microg 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.

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.

Demonstrating the need for chemical exposure characterisation in a microplate test system: toxicity screening of sixteen pesticides on two marine microalgae.

Pesticides used in viticulture create a potential risk for the aquatic environment due to drift during application, runoff and soil leaching. The toxicity of sixteen pesticides and one metabolite were evaluated on the growth of two marine microalgae, Tisochrysis lutea and Skeletonema marinoi, in 96-h exposure assays conducted in microplates. For each substance, concentrations of stock solutions were analytically measured and abiotic assays were performed to evaluate the chemical stability of pesticides in microplates. For two chemicals, microalgae exposures were run simultaneously in microplates and culture flasks to compare EC50 calculated from the two exposure systems. Results from chemical analyses demonstrated the low stability of hydrophobic pesticides (log KOW > 3). For such chemicals, EC50 values calculated using measured pesticide concentrations were two-fold lower than those first estimated using nominal concentrations. Photosystem II inhibitors were the most toxic herbicides, with EC50 values below 10 µg L-1 for diuron and around double this for isoproturon. Chlorpyrifos-methyl was the only insecticide to significantly affect the growth of T. lutea, with an EC50 around 400 µg L-1. All fungicides tested were significantly toxic to both species: strobilurins showed low overall toxicity, with EC50 values around 400 µg L-1, whereas quinoxyfen, and spiroxamine, showed high toxicity to both species, especially to T. lutea, with an EC50 below 1 µg L-1 measured for spiroxamine in culture flasks. This study highlights the need to perform chemical analyses for reliable toxicity assessment and discusses the advantages and disadvantages of using microplates as a toxicity screening tool.

Combined effects of antifouling biocides on the growth of three marine microalgal species.

The toxicity of the antifouling compounds diuron, irgarol, zinc pyrithione (ZnPT), copper pyrithione (CuPT) and copper was tested on the three marine microalgae Tisochrysis lutea, Skeletonema marinoi and Tetraselmis suecica. Toxicity tests based on the inhibition of growth rate after 96-h exposure were run using microplates. Chemical analyses were performed to validate the exposure concentrations and the stability of the compounds under test conditions. Single chemicals exhibited varying toxicity depending on the species, irgarol being the most toxic chemical and Cu the least toxic. Selected binary mixtures were tested and the resulting interactions were analyzed using two distinct concentration-response surface models: one using the concentration addition (CA) model as reference and two deviating isobole models implemented in R software; the other implementing concentration-response surface models in Excel®, using both CA and independent action (IA) models as reference and three deviating models. Most mixtures of chemicals sharing the same mode of action (MoA) were correctly predicted by the CA model. For mixtures of dissimilarly acting chemicals, neither of the reference models provided better predictions than the other. Mixture of ZnPT together with Cu induced a strong synergistic effect on T. suecica while strong antagonism was observed on the two other species. The synergy was due to the transchelation of ZnPT into CuPT in the presence of Cu, CuPT being 14-fold more toxic than ZnPT for this species. The two modelling approaches are compared and the differences observed among the interaction patterns resulting from the mixtures are discussed.

The influence of natural dissolved organic matter on herbicide toxicity to marine microalgae is species-dependent.

Microalgae, which are the foundation of aquatic food webs, may be the indirect target of herbicides used for agricultural and urban applications. Microalgae also interact with other compounds from their environment, such as natural dissolved organic matter (DOM), which can itself interact with herbicides. This study aimed to evaluate the influence of natural DOM on the toxicity of three herbicides (diuron, irgarol and S-metolachlor), singly and in ternary mixtures, to two marine microalgae, Chaetoceros calcitrans and Tetraselmis suecica, in monospecific, non-axenic cultures. Effects on growth, photosynthetic efficiency (Ф'M) and relative lipid content were evaluated. The chemical environment (herbicide and nutrient concentrations, dissolved organic carbon and DOM optical properties) was also monitored to assess any changes during the experiments. The results show that, without DOM, the highest irgarol concentration (I0.5: 0.5 mg.L-1) and the strongest mixture (M2: irgarol 0.5 µg.L-1 + diuron 0.5 µg.L-1 + S-metolachlor 5.0 µg.L-1) significantly decreased all parameters for both species. Similar impacts were induced by I0.5 and M2 in C. calcitrans (around -56% for growth, -50% for relative lipid content and -28% for Ф'M), but a significantly higher toxicity of M2 was observed in T. suecica (-56% and -62% with I0.5 and M2 for growth, respectively), suggesting a possible interaction between molecules. With DOM added to the culture media, a significant inhibition of these three parameters was also observed with I0.5 and M2 for both species. Furthermore, DOM modulated herbicide toxicity, which was decreased for C. calcitrans (-51% growth at I0.5 and M2) and increased for T. suecica (-64% and -75% growth at I0.5 and M2, respectively). In addition to the direct and/or indirect (via their associated bacteria) use of molecules present in natural DOM, the characterization of the chemical environment showed that the toxic effects observed on microalgae were accompanied by modifications of DOM composition and the quantity of dissolved organic carbon excreted and/or secreted by microorganisms. This toxicity modulation in presence of DOM could be explained by (i) the modification of herbicide bioavailability, (ii) a difference in cell wall composition between the two species, and/or (iii) a higher detoxification capacity of C. calcitrans by the use of molecules contained in DOM. This study therefore demonstrated, for the first time, the major modulating role of natural DOM on the toxicity of herbicides to marine microalgae.

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.

Multigenerational exposure of the microalga Tetraselmis suecica to diuron leads to spontaneous long-term strain adaptation.

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.

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

Microcosms, 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.

In-situ microcosms, a tool for assessment of pesticide impacts on oyster spat (Crassostrea gigas).

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.

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

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 6m 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 microg L(-1) (ANOSIM, p=0.055; R=0.53), and 10 microg 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 microg L(-1). This study demonstrates that a disturbance was caused to the marine microbial community exposed to 1 microg L(-1) Roundup concentration, a value typical of those reported in coastal waters during a run-off event.