Fluorescence in situ hybridisation (FISH) is a powerful molecular biological tool to detect and enumerate harmful microorganism in the marine environment. Different FISH methods are available, and especially in combination with automated counting techniques, the po- tential for a routine monitoring of harmful marine micro- algae is attainable. Various oligonucleotide probes are developed for detecting harmful microalgae. However, FISH-based methods are not yet regularly included in mon- itoring programmes tracking the presence of harmful marine microalgae. A limitation factor of the FISH technique is the currently available number of suited fluorochromes attached to the FISH probes to detect various harmful species in one environmental sample at a time. However, coupled automated techniques, like flow cytometry or solid-phase cytometry, can facilitate the analysis of nu- merous field samples and help to overcome this draw- back. A great benefit of FISH in contrast to other molecular biological detection methods for harmful ma- rine microalgae is the direct visualisation of the hybridised target cells, which are not permitted in cell free formats, like DNA depending analysis methods. Therefore, an additional validation of the FISH-generated results is simultaneously given.

Fluorescence in situ hybridisation (FISH) is a powerful molecular biological tool to detect and enumerate harmful microorganism in the marine environment. Different FISH methods are available, and especially in combination with automated counting techniques, the potential for a routine monitoring of harmful marine microalgae is attainable. Various oligonucleotide probes are developed for detecting harmful microalgae. However, FISH-based methods are not yet regularly included in monitoring programmes tracking the presence of harmful marine microalgae. A limitation factor of the FISH technique is the currently available number of suited fluorochromes attached to the FISH probes to detect various harmful species in one environmental sample at a time. However, coupled automated techniques, like flow cytometry or solid-phase cytometry, can facilitate the analysis of numerous field samples and help to overcome this drawback. A great benefit of FISH in contrast to other molecular biological detection methods for harmful marine microalgae is the direct visualisation of the hybridised target cells, which are not permitted in cell free formats, like DNA depending analysis methods. Therefore, an additional validation of the FISH-generated results is simultaneously given.

The dumping of dredged sediments represents a major stressor for coastal ecosystems. The impact on the ecosystem function is determined by its complexity not easy to assess. In the present study, we evaluated the potential of bacterial community analyses to act as ecological indicators in environmental monitoring programmes. We investigated the functional structure of bacterial communities, applying functional gene arrays (GeoChip 4.2). The relationship between functional genes and environmental factors was analysed using distance-based multivariate multiple regression. Apparently, both the function and structure of the bacterial communities are impacted by dumping activities. The bacterial community at the dumping centre displayed a significant reduction of its entire functional diversity compared with that found at a reference site. DDX compounds separated bacterial communities of the dumping site from those of un-impacted sites. Thus, bacterial community analyses show great potential as ecological indicators in environmental monitoring.

The dumping of dredged sediments represents a major stressor for coastal ecosystems. The impact on the ecosystem function is determined by its complexity not easy to assess. In the present study, we evaluated the potential of bacterial community analyses to act as ecological indicators in environmental monitoring programmes. We investigated the functional structure of bacterial communities, applying functional gene arrays (GeoChip4.2). The relationship between functional genes and environmental factors was analysed using distance-based multivariate multiple regression. Apparently, both the function and structure of the bacterial communities are impacted by dumping activities. The bacterial community at the dumping centre displayed a significant reduction of its entire functional diversity compared with that found at a reference site. DDX compounds separated bacterial communities of the dumping site from those of un-impacted sites. Thus, bacterial community analyses show great potential as ecological indicators in environmental monitoring.

In recent decades, many toxicological tests based on in vivo or in vitro models, mainly from mammalian (rat-mouse) and fish species, were used to assess the risks raised by contact or ingestion of molecules of pharmaceutical, agricultural, or natural origin. But no, or few, in vitro tests using other non-mammalian models such as bird have been explored despite their advantages: the embryonic gonads of birds have a high plasticity of development sensitive to estrogen, and sperm production is nearly two times faster than in rodents. Hence, we have established an in vitro culture of germ cells and somatic cells from chicken post-natal testis, and we have evaluated the sensitivity against the endocrine disruptor compound mono-(2-ethylhexyl) phthalate (MEHP) in comparison to previous studies using rodent and human models. After 96 h of exposure in presence of 10 μM MEHP, chicken seminiferous tubules cultures present a structural alteration, a reduction in cell proliferation and in germ cells population. Apoptosis of germ and somatic cells increases in presence of 1 μM MEHP. Furthermore, MEHP does not affect inhibin B and lactate production by Sertoli cells. These results are in accordance with previous studies using rat, mice, or human culture of testicular cells and in similar range of exposures or even better sensitivity for some "end-points" (biological parameters). In conclusion, the establishment of this postnatal testicular cells culture could be considered as an alternative method to in vivo experiments frequently used for evaluating the impact on the terrestrial wildlife species. This method could be also complementary to mammal model due to the limiting number of animals used and its elevated sensitivity.

In recent decades, many toxicological tests based on in vivo or in vitro models, mainly from mammalian (rat–mouse) and fish species, were used to assess the risks raised by contact or ingestion of molecules of pharmaceutical, agricultural, or natural origin. But no, or few, in vitro tests using other non-mammalian models such as bird have been explored despite their advantages: the embryonic gonads of birds have a high plasticity of development sensitive to estrogen, and sperm production is nearly two times faster than in rodents. Hence, we have established an in vitro culture of germ cells and somatic cells from chicken post-natal testis, and we have evaluated the sensitivity against the endocrine disruptor compound mono-(2-ethylhexyl) phthalate (MEHP) in comparison to previous studies using rodent and human models. After 96 h of exposure in presence of 10 μM MEHP, chicken seminiferous tubules cultures present a structural alteration, a reduction in cell proliferation and in germ cells population. Apoptosis of germ and somatic cells increases in presence of 1 μM MEHP. Furthermore, MEHP does not affect inhibin B and lactate production by Sertoli cells. These results are in accordance with previous studies using rat, mice, or human culture of testicular cells and in similar range of exposures or even better sensitivity for some “end-points” (biological parameters). In conclusion, the establishment of this postnatal testicular cells culture could be considered as an alternative method to in vivo experiments frequently used for evaluating the impact on the terrestrial wildlife species. This method could be also complementary to mammal model due to the limiting number of animals used and its elevated sensitivity.

The triazole fungicide epoxiconazole is extensively used to control fungi on crops and may present some potential risk from runoff on coastal ecosystems located close to agricultural areas. Phytotoxicity assessments were conducted on the marine diatom Chaetoceros calcitrans using both the active ingredient and its formulated product (Opus). The 3-day EC50 using cell count was 2.31 mg/L for epoxiconazole active ingredient and 2.9 mu g/L for epoxiconazole-formulated. The fungicide produced an increase of cellular volume, pigment (chlorophylls a, c, and carotenoids) content, ATP synthesis, and rates of photosynthesis and respiration. Progressive algal cell recovery from epoxiconazole effects occurred after 3 days, with the increasing cell density. Differences in cell age, light, and nutrient composition induced changes in epoxiconazole sensitivity. Since these parameters affect cellular division rates, the cellular density is an important parameter in toxicity tests.

The triazole fungicide epoxiconazole is extensively used to control fungi on crops and may present some potential risk from runoff on coastal ecosystems located close to agricultural areas. Phytotoxicity assessments were conducted on the marine diatom Chaetoceros calcitrans using both the active ingredient and its formulated product (Opus). The 3-day EC₅₀ using cell count was 2.31 mg/L for epoxiconazole active ingredient and 2.9 μg/L for epoxiconazole-formulated. The fungicide produced an increase of cellular volume, pigment (chlorophylls a, c, and carotenoids) content, ATP synthesis, and rates of photosynthesis and respiration. Progressive algal cell recovery from epoxiconazole effects occurred after 3 days, with the increasing cell density. Differences in cell age, light, and nutrient composition induced changes in epoxiconazole sensitivity. Since these parameters affect cellular division rates, the cellular density is an important parameter in toxicity tests.

This study aimed to compare the contamination levels of various organohalogenated compounds in two migratory fish species in the Vilaine River in western France. Organochlorinated pesticides, polychlorinated biphenyls (PCBs), dioxins (polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/Fs)), and polybrominated diphenyl ethers (PBDEs) were analyzed in two diadromous species from the Vilaine estuary, the grey mullet (Liza ramada)-an amphihaline species, and the allis shad (Alosa alosa)-an anadromous species. Fish were collected in spring 2004 and spring 2005, upstream and downstream of the Arzal Dam. PCB contamination varied from 27 to 200 ng g(-1) dry weight (d.w.). PCDDs/Fs, expressed in toxicity equivalent quantity (TEQ) varied from 0.4 to 2.8 pg g(-1) d.w. Dioxins and dioxin-like PCBs expressed in total TEQ varied from 1.4 to 18.8 pg g(-1) d.w. PBDE47 was present at around 2-10 ng g(-1) d.w. and concentrations of the insecticide dichlorodiphenyltrichloroethane breakdown product p,p'-dichlorodiphenylchloroethylene varied from 1 to 14 ng g(-1) d.w. For both species, specimens collected upstream were more contaminated. The grey mullet specimens were less contaminated than the allis shad when taken downstream of the dam but were more contaminated upstream. The allis shads presented intermediate contaminant concentrations with a less pronounced difference between upstream and downstream specimens. However, it is thought that shads do not feed when they spawn in the upstream parts of rivers, which should modify the contaminant concentrations. However, measurements in upstream shad samples show an unexpected increase of the contamination, which remains unexplained.

This study aimed to compare the contamination levels of various organohalogenated compounds in two migratory fish species in the Vilaine River in western France. Organochlorinated pesticides, polychlorinated biphenyls (PCBs), dioxins (polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/Fs)), and polybrominated diphenyl ethers (PBDEs) were analyzed in two diadromous species from the Vilaine estuary, the grey mullet (Liza ramada)—an amphihaline species, and the allis shad (Alosa alosa)—an anadromous species. Fish were collected in spring 2004 and spring 2005, upstream and downstream of the Arzal Dam. PCB contamination varied from 27 to 200 ng g⁻¹ dry weight (d.w.). PCDDs/Fs, expressed in toxicity equivalent quantity (TEQ) varied from 0.4 to 2.8 pg g⁻¹ d.w. Dioxins and dioxin-like PCBs expressed in total TEQ varied from 1.4 to 18.8 pg g⁻¹ d.w. PBDE47 was present at around 2–10 ng g⁻¹ d.w. and concentrations of the insecticide dichlorodiphenyltrichloroethane breakdown product p,p′-dichlorodiphenylchloroethylene varied from 1 to 14 ng g⁻¹ d.w. For both species, specimens collected upstream were more contaminated. The grey mullet specimens were less contaminated than the allis shad when taken downstream of the dam but were more contaminated upstream. The allis shads presented intermediate contaminant concentrations with a less pronounced difference between upstream and downstream specimens. However, it is thought that shads do not feed when they spawn in the upstream parts of rivers, which should modify the contaminant concentrations. However, measurements in upstream shad samples show an unexpected increase of the contamination, which remains unexplained.

Multi-biomarker approaches are used to assess ecosystem health and identify impacts of environmental stress on organisms. However, exploration of large datasets by environmental managers represents a major challenge for regulatory application of this tool. Several integrative tools were developed to summarize biomarker responses. The aim of the present paper is to update calculation of the "Integrated Biological Response" (IBR) described by Beliaeff and Burgeot (Environ Toxicol Chem 21:1316-1322, 2002) to avoid weaknesses of this integrative tool. In the present paper, a novel index named "Integrated Biological Responses version 2" based on the reference deviation concept is presented. It allows a clear discrimination of sampling sites as for the IBR, but several differences are observed for contaminated sites according to up- and downregulation of biomarker responses. This novel tool could be used to integrate multi-biomarker responses not only in large-scale monitoring but also in upstream/downstream investigations.

Multi-biomarker approaches are used to assess ecosystem health and identify impacts of environmental stress on organisms. However, exploration of large datasets by environmental managers represents a major challenge for regulatory application of this tool. Several integrative tools were developed to summarize biomarker responses. The aim of the present paper is to update calculation of the “Integrated Biological Response” (IBR) described by Beliaeff and Burgeot (Environ Toxicol Chem 21:1316–1322, 2002) to avoid weaknesses of this integrative tool. In the present paper, a novel index named “Integrated Biological Responses version 2” based on the reference deviation concept is presented. It allows a clear discrimination of sampling sites as for the IBR, but several differences are observed for contaminated sites according to up- and downregulation of biomarker responses. This novel tool could be used to integrate multi-biomarker responses not only in large-scale monitoring but also in upstream/downstream investigations.

A 2-year study was implemented to characterize the contamination of estuarine continuums in the Bay of Vilaine area (NW Atlantic Coast, Southern Brittany, France) by 30 pesticide and biocide active substances and metabolites. Among these, 11 triazines (ametryn, atrazine, desethylatrazine, desethylterbuthylazine, desisopropyl atrazine, Irgarol 1051, prometryn, propazine, simazine, terbuthylazine, and terbutryn), 10 phenylureas (chlortoluron, diuron, 1-(3,4-dichlorophenyl)-3-methylurea, fenuron, isoproturon, 1-(4-isopropylphenyl)-3-methylurea, 1-(4-isopropylphenyl)-urea, linuron, metoxuron, and monuron), and 4 chloroacetanilides (acetochlor, alachlor, metolachlor, and metazachlor) were detected at least once. The objectives were to assess the corresponding risk for aquatic primary producers and to provide exposure information for connected studies on the responses of biological parameters in invertebrate sentinel species. The risk associated with contaminants was assessed using risk quotients based on the comparison of measured concentrations with original species sensitivity distribution-derived hazardous concentration values. For EU Water Framework Directive priority substances, results of monitoring were also compared with regulatory Environmental Quality Standards. The highest residue concentrations and risks for primary producers were recorded for diuron and Irgarol 1051 in Arzal reservoir, close to a marina. Diuron was present during almost the all survey periods, whereas Irgarol 1051 exhibited a clear seasonal pattern, with highest concentrations recorded in June and July. These results suggest that the use of antifouling biocides is responsible for a major part of the contamination of the lower part of the Vilaine River course for Irgarol 1051. For diuron, agricultural sources may also be involved. The presence of isoproturon and chloroacetanilide herbicides on some dates indicated a significant contribution of the use of plant protection products in agriculture to the contamination of Vilaine River. Concentration levels and associated risk were always lower in estuarine sites than in the reservoir, suggesting that Arzal dam reduces downstream transfer of contaminants and favors their degradation in the freshwater part of the estuary. Results of the additional monitoring of two tidal streams located downstream of Arzal dam suggested that, although some compounds may be transferred to the estuary, their impact was probably very low. Dilution by marine water associated with tidal current was also a major factor of concentration reduction. It is concluded that the highest risks associated to herbicides and booster biocides concerned the freshwater part of the estuary and that its brackish/saltwater part was exposed to a moderate risk, although some substances may sometimes exhibit high concentration but mainly at low tide and on an irregular basis.

A 2-year study was implemented to characterize the contamination of estuarine continuums in the Bay of Vilaine area (NW Atlantic Coast, Southern Brittany, France) by 30 pesticide and biocide active substances and metabolites. Among these, 11 triazines (ametryn, atrazine, desethylatrazine, desethylterbuthylazine, desisopropyl atrazine, Irgarol 1051, prometryn, propazine, simazine, terbuthylazine, and terbutryn), 10 phenylureas (chlortoluron, diuron, 1-(3,4-dichlorophenyl)-3-methylurea, fenuron, isoproturon, 1-(4-isopropylphenyl)-3-methylurea, 1-(4-isopropylphenyl)-urea, linuron, metoxuron, and monuron), and 4 chloroacetanilides (acetochlor, alachlor, metolachlor, and metazachlor) were detected at least once. The objectives were to assess the corresponding risk for aquatic primary producers and to provide exposure information for connected studies on the responses of biological parameters in invertebrate sentinel species. The risk associated with contaminants was assessed using risk quotients based on the comparison of measured concentrations with original species sensitivity distribution-derived hazardous concentration values. For EU Water Framework Directive priority substances, results of monitoring were also compared with regulatory Environmental Quality Standards. The highest residue concentrations and risks for primary producers were recorded for diuron and Irgarol 1051 in Arzal reservoir, close to a marina. Diuron was present during almost the all survey periods, whereas Irgarol 1051 exhibited a clear seasonal pattern, with highest concentrations recorded in June and July. These results suggest that the use of antifouling biocides is responsible for a major part of the contamination of the lower part of the Vilaine River course for Irgarol 1051. For diuron, agricultural sources may also be involved. The presence of isoproturon and chloroacetanilide herbicides on some dates indicated a significant contribution of the use of plant protection products in agriculture to the contamination of Vilaine River. Concentration levels and associated risk were always lower in estuarine sites than in the reservoir, suggesting that Arzal dam reduces downstream transfer of contaminants and favors their degradation in the freshwater part of the estuary. Results of the additional monitoring of two tidal streams located downstream of Arzal dam suggested that, although some compounds may be transferred to the estuary, their impact was probably very low. Dilution by marine water associated with tidal current was also a major factor of concentration reduction. It is concluded that the highest risks associated to herbicides and booster biocides concerned the freshwater part of the estuary and that its brackish/saltwater part was exposed to a moderate risk, although some substances may sometimes exhibit high concentration but mainly at low tide and on an irregular basis.

[Departement_IRSTEA]Eaux [TR1_IRSTEA]BELCA | International audience | Estuarine areas represent complex and highly changing environments at the interface between freshwater and marine aquatic ecosystems. Therefore, the aquatic organisms living in estuaries have to face highly variable environmental conditions. The aimof this work was to study the influence of environmental changes from either natural or anthropogenic origins on the physiological responses of Mytilus edulis. Mussels were collected in theVilaine estuary during early summer because this season represents a critical period of active reproduction in mussels and of increased anthropogenic inputs from agricultural and boating activities into the estuary. The physiological status of the mussel M. edulis was evaluated through measurements of a suite of biomarkers related to: oxidative stress (catalase, malondialdehyde), detoxication (benzopyrene hydroxylase, carboxylesterase), neurotoxicity (acetylcholinesterase), reproductive cycle (vitelline, condition index, maturation stages), immunotoxicity (hemocyte concentration, granulocyte percentage, phagocytosis, reactive oxygen species production, oxidative burst), and general physiological stress (lysosomal stability). A selection of relevant organic contaminant (pesticides, (polycyclic aromatic hydrocarbons, polychlorobiphenyls) was measured as well as environmental parameters (water temperature, salinity, total suspended solids, turbidity, chlorophyll a, pheopigments) and mussel phycotoxin contamination. Two locations differently exposed to the plume of the Vilaine River were compared. Both temporal and inter-site variations of these biomarkers were studied. Our results show that reproduction cycle and environmental parameters such as temperature, organic ontaminants, and algal blooms could strongly influence the biomarker responses. These observations highlight the necessity to conduct integrated environmental approaches in order to better understand the causes of biomarker variations.