Effects of diclofenac on sentinel species and aquatic communities in semi-natural conditions.

Due to the potential hazard of diclofenac on aquatic organisms and the lack of higher-tier ecotoxicological studies, a long-term freshwater mesocosm experiment was set up to study the effects of this substance on primary producers and consumers at environmentally realistic nominal concentrations 0.1, 1 and 10 µg/L (average effective concentrations 0.041, 0.44 and 3.82 µg/L). During the six-month exposure period, the biovolume of two macrophyte species (Nasturtium officinale and Callitriche platycarpa) significantly decreased at the highest treatment level. Subsequently, a decrease in dissolved oxygen levels was observed. High mortality rates, effects on immunity, and high genotoxicity were found for encaged zebra mussels (Dreissena polymorpha) in all treatments. In the highest treatment level, one month after the beginning of the exposure, mortality of adult fish (Gasterosteus aculeatus) caused effects on the final population structure. Total abundance of fish and the percentage of juveniles decreased whereas the percentage of adults increased. This led to an overall shift in the length frequency distribution of the F1 generation compared to the control. Consequently, indirect effects on the community structure of zooplankton and macroinvertebrates were observed in the highest treatment level. The No Observed Effect Concentration (NOEC) value at the individual level was < 0.1 µg/L and 1 µg/L at the population and community levels. Our study showed that in more natural conditions, diclofenac could cause more severe effects compared to those observed in laboratory conditions. The use of our results for regulatory matters is also discussed.

Usability of the bivalves Dreissena polymorpha and Anodonta anatina for a biosurvey of the neurotoxin BMAA in freshwater ecosystems.

The environmental neurotoxin ß-methylamino-L-alanine (BMAA) may represent a risk for human health in case of chronic exposure or after short-term exposure during embryo development. BMAA accumulates in freshwater and marine organisms consumed by humans. It is produced by marine and freshwater phytoplankton species, but the range of producers remains unknown. Therefore, analysing the phytoplankton composition is not sufficient to inform about the risk of freshwater contamination by BMAA. Filter-feeders mussels have accumulation capacities and therefore appear to be relevant to monitor various pollutants in aquatic ecosystems. We investigated the suitability of the freshwater mussels Dreissena polymorpha and Anodonta anatina for monitoring BMAA in water. Both species were exposed to 1, 10, and 50 µg of dissolved BMAA/L daily for 21 days, followed by 42 days of depuration in clean water. On days 0, 1, 7, 14, and 21 of exposure and 1, 7, 14, 21 and 42 of depuration, whole D. polymorpha and digestive glands of A. anatina were sampled, and the total BMAA concentration was measured. D. polymorpha accumulated BMAA earlier (from day 1 at all concentrations) and at higher tissue concentrations than A. anatina, which accumulated BMAA from day 14 when exposed to 10 µg BMAA/L and from day 7 when exposed to 50 µg BMAA/L. As BMAA accumulation by D. polymorpha was time and concentration-dependent, with a significant elimination during the depuration period, this species may be able to reflect the levels and dynamics of water contamination by dissolved BMAA. The species A. anatina could be used for monitoring water concentrations above 10 µg BMAA/L.

Differentiation of sympatric zebra and quagga mussels in ecotoxicological studies: A comparison of morphometric data, gene expression, and body metal concentrations.

The zebra mussel is among the best studied freshwater molluscs in ecotoxicology, but information on the quagga mussel is lacking. Considering its potential spread, we selected a river in France in which zebra and quagga mussels coexisted, and then we used genetic markers to differentiate the two species and compared morphological parameters. cDNA sequencing assays of ten genes already used in zebra mussels were performed on quagga mussels to obtain functional specific primers. Then we analyzed the expression of genes involved in cellular metabolic activities (Cytochrome-c-oxidase - cox, and ATP synthase - atp), detoxification processes (Glutathione-S-Transferase - gst), oxidative stress (Catalase - cat), and digestive functions (Amylase - amy) on the two species. Whereas morphometric analysis underlined similarities in shape between the two species, relative gene expression profiles and metal concentrations evidenced strong differences. Quagga mussels notably presented half as high concentrations in Cd and Pb, two particularly toxic elements, as zebra mussels. These results imply that i) particular attention should be paid to properly distinguish the two species considering their similar external appearance, and ii) zebra mussels cannot be replaced by quagga mussels in ecotoxicological studies without preliminary investigations on biomarker response patterns. To our knowledge, this study is the first to have undertaken such an approach in gene expression analysis in quagga mussels, and more generally to have compared such biomarker responses of zebra and quagga mussels in the field.

Aerobic and anaerobic energy production in juvenile roach (Rutilus rutilus): regulation of glycolytic process by ethofumesate at two temperatures.

The aim of this study was to evaluate the coupled impact of an herbicide, ethofumesate, and temperature on the cellular energy metabolism of juvenile roach, especially on the glycolysis pathway. Juvenile roach were exposed to 0, 0.5, 5, and 50 µg/L of ethofumesate for 7 days in laboratory conditions at two temperatures (10 and 17 °C). The energy reserves (carbohydrate, lipid, and protein) were quantified, since the availability of substrates regulates the glycolysis. Then, the glycolysis was studied at the biochemical level by the measurement of the glycolytic flux and at the molecular level with the measurement of the relative expression of four genes encoding for glycolysis enzymes. This study revealed different effect of ethofumesate on the glycolysis pathway according to the temperature of exposure. Indeed, at 10 °C, it appeared that only the molecular regulation level was affected, whereas, at 17 °C, ethofumesate acted on the biochemical level. The differences observed between the two exposures imply the establishment of different strategies in order to maintain to cope with stress according to the temperature of exposure.

Variations in gene expression levels in four European zebra mussel, Dreissena polymorpha, populations in relation to metal bioaccumulation: A field study.

The present study was performed to validate the suitability of using gene expression in zebra mussels, Dreissena polymorpha, for biomonitoring of freshwater environment. Mussels were collected in four French rivers (Meuse, Moselle, Oise and Vilaine) in spring and autumn. Relative gene expression of 9 candidate genes involved in cellular metabolic activities (Cytochrome-c-oxidase - cox, and ATP synthase - atp), detoxification process (Metallothionein - mt and Glutathion-S-Transferase - gst), oxidative stress (Catalase - cat, Superoxyde Dismutase - sod and Glutathion peroxidase - gpx) and digestive functions (Amylase - amy and Cellulase - ghf) were measured in digestive gland. Metal bioaccumulation in tissues and morphometric parameters were also analyzed to interpret molecular responses. All our results are consistent with different physiological reactions to environmental condition between zebra mussel populations. In spring, the levels of mt, sod, gpx, cat, atp, amy and ghf relative expression were significantly higher in mussels with the lowest metal bioaccumulation (the Meuse) compared to at least one of the other sites. In autumn, this higher expression levels in Meuse River were still observed for gpx, cat, atp and amy. This study has also pointed out different sources of variability in gene expression (individual size, season, trophic resources and origin of mussels) which are inevitable in natural fluctuant environment. This underlines the importance to take them into account in field study to propose a correct interpretation of biomarker responses.

Zebra mussel as a new tool to show evidence of freshwater contamination by waterborne Toxoplasma gondii.

AIMS: The objective of this study was to evaluate if freshwater bivalves can be used to detect the presence of Toxoplasma gondii in water bodies. METHODS AND RESULTS: Zebra mussels (Dreissena polymorpha) were caged for 1 month upstream and downstream of the discharge points of wastewater treatment plants (WWTPs). Physiological status was assessed to assure good health of bivalves during transplantation. The presence of T. gondii was investigated in mussel tissues by qPCR. In autumn, T. gondii was detected in mussels caged downstream of the discharge points of two WWTPs. In spring, it was detected upstream of one WWTP. CONCLUSIONS: For the first time, T. gondii DNA has been shown in a continental mollusc in environmental conditions. This highlights the interest of an active approach that could be applied independently of the presence or accessibility of autochthonous populations, and underlines the presence of T. gondii in natural waters under pressure of WWTP discharge at a certain time of the year. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that transplanted zebra mussels could be used as biosamplers to reveal contamination of freshwater systems by T. gondii.

Assessment of Toxoplasma gondii levels in zebra mussel (Dreissena polymorpha) by real-time PCR: an organotropism study.

Water quality is a public health concern that calls for relevant biomonitoring programs. Molecular tools such as polymerase chain reaction (PCR) are progressively becoming more sensitive and more specific than conventional techniques to detect pathogens in environmental samples such as water and organisms. The zebra mussel (Dreissena polymorpha) has already been demonstrated to accumulate and concentrate various human waterborne pathogens. In this study, first, a spiking experiment to evaluate detection levels of Toxoplasma gondii DNA in zebra mussel organs using real-time PCR was conducted. Overall, lower DNA levels in the hemolymph, digestive gland, and remaining tissues (gonad and foot) were detected compared to mantle, muscle, and gills. Second, an in vivo experiment with 1000 T. gondii oocysts per mussel and per day for 21 consecutive days, followed by 14 days of depuration time in protozoa-free water was performed. T. gondii DNA was detected in all organs, but greatest concentrations were observed in hemolymph and mantle tissues compared to the others organs at the end of the depuration period. These results suggest that (i) the zebra mussel is a potential new tool for measuring T. gondii concentrations and (ii) real-time PCR is a suitable method for pathogen detection in complex matrices such as tissues.

Detection of immunotoxic effects of estrogenic and androgenic endocrine disrupting compounds using splenic immune cells of the female three-spined stickleback, Gasterosteus aculeatus (L.).

Today, the list of endocrine disrupting compounds (EDCs) in freshwater and marine environments that mimic or block endogenous hormones is expanding at an alarming rate. As immune and reproductive systems may interact in a bidirectional way, some authors proposed the immune capacities as attractive markers to evaluate the hormonal potential of environmental samples. Thus, the present work proposed to gain more knowledge on direct biological effects of natural and EDCs on female fish splenic leucocyte non-specific immune activities by using ex vivo assays. After determining the optimal required conditions to analyze splenic immune responses, seven different EDCs were tested ex vivo at 0.01, 1 and 100nM over 12h on the leucocyte functions of female three-spined stickleback, Gasterosteus aculeatus. In summary, we found that natural hormones acted as immunostimulants, whilst EDCs were immunosuppressive.

Towards a better understanding of biomarker response in field survey: a case study in eight populations of zebra mussels.

In order to provide reliable information about responsiveness of biomarkers during environmental monitoring, there is a need to improve the understanding of inter-population differences. The present study focused on eight populations of zebra mussels and aimed to describe how variable are biomarkers in different sampling locations. Biomarkers were investigated and summarised through the Integrated Biomarker Response (IBR index). Inter-site differences in IBR index were analysed through comparisons with morphological data, proteomic profiles and genetic background of the studied populations. We found that the IBR index was a good tool to inform about the status of sites. It revealed higher stress in more polluted sites than in cleaner ones. It was neither correlated to proteomic profiles nor to genetic background, suggesting a stronger influence of environment than genes. Meanwhile, morphological traits were related to both environment and genetic background influence. Together these results attest the benefit of using biological tools to better illustrate the status of a population and highlight the need of consider inter-population difference in their baselines.

Effects of aluminium and bacterial lipopolysaccharide on oxidative stress and immune parameters in roach, Rutilus rutilus L.

Aluminium is used in diverse anthropogenic processes at the origin of pollution events in aquatic ecosystems. In the Champagne region (France), high concentrations of aluminium (Al) are detected due to vine-growing practices. In fish, little is known about the possible immune-related effects at relevant environmental concentrations. The present study analyzes the simultaneous effects of aluminium and bacterial lipopolysaccharide (LPS), alone and in combination, on toxicological biomarkers in the freshwater fish species Rutilus rutilus. For this purpose, roach treated or not with LPS were exposed to environmental concentrations of aluminium (100 µg/L) under laboratory-controlled conditions for 2, 7, 14 and 21 days. After each exposure time, we assessed hepatic lipoperoxidation, catalase activity, glutathione reductase activity and total glutathione content. We also analyzed cellular components related to the LPS-induced inflammatory response in possible target tissues, i.e. head kidney and spleen. Our results revealed a significant prooxidant effect in the liver cells and head kidney leukocytes of roach exposed to 100 µg of Al/L for 2 days. In liver, we observed more lipoperoxidation products and lower endogenous antioxidant activity levels such as glutathione reductase activity and total glutathione content. These prooxidant effects were associated with a higher oxidative burst in head kidney leukocytes, and they were all the more important in fish stimulated by LPS injection. These findings demonstrate that environmental concentrations of Al induce oxidative and immunotoxic effects in fish and are associated to an immunomodulatory process related to the inflammatory response.

Bioaccumulation of human waterborne protozoa by zebra mussel (Dreissena polymorpha): interest for water biomonitoring.

Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii are ubiquitous pathogens, which waterborne transmission has been largely demonstrated. Since they can be found in various watercourses, interactions with aquatic organisms are possible. Protozoan detection for watercourses biomonitoring is currently based on large water filtration. The zebra mussel, Dreissena polymorpha, is a choice biological model in ecotoxicological studies which are already in use to detect chemical contaminations in watercourses. In the present study, the zebra mussel was tested as a new tool for detecting water contamination by protozoa. In vivo exposures were conducted in laboratory experiments. Zebra mussel was exposed to various protozoan concentrations for one week. Detection of protozoa was realized by Taqman real time qPCR. Our experiments evidenced C. parvum, G. duodenalis and T. gondii oocyst bioaccumulation by mussels proportionally to ambient contamination, and significant T. gondii prevalence was observed in muscle tissue. To our knowledge, this is the first study that demonstrates T. gondii oocyst accumulation by zebra mussel. The results from this study highlight the capacity of zebra mussels to reveal ambient biological contamination, and thus to be used as a new effective tool in sanitary biomonitoring of water bodies.

Temporal patterns of digestive enzyme activities and feeding rate in gammarids (Gammarus fossarum) exposed to inland polluted waters.

The aim of this study was to use digestive enzyme activities in Gammarus fossarum as biomarkers during active biomonitoring. Standardised gammarids were transplanted for 7 days to five sites in the Riou Mort watershed contaminated by polymetallic pollution. This experiment was conducted on seven different dates from February 2009 to June 2010. Feeding rates were tracked, along with amylase, cellulase and trypsin activities. We found that feeding rate and digestive capacity were reduced in the most polluted site, "Joany," in comparison with the reference site "Up.Lot". The results suggested that trypsin was more sensitive than the other two carbohydrases. In the four other sites, seasonal differences were observed during the 2yr but no clear pattern can be established. This study highlights the ability of G. fossarum to demonstrate environmental disturbances and suggests the use of a caging process in certain seasons. Caging organisms and feeding ad libitum is advantageous, as it reduces inter-individual variability and removes dependence on the native food fluctuations. However, confounding factors other than temperature were present, and the interpretation of digestive enzyme activities is complex.

Effect of water quality and confounding factors on digestive enzyme activities in Gammarus fossarum.

The feeding activity and subsequent assimilation of the products resulting from food digestion allow organisms to obtain energy for growth, maintenance and reproduction. Among these biological parameters, we studied digestive enzymes (amylase, cellulase and trypsin) in Gammarus fossarum to assess the impact of contaminants on their access to energy resources. However, to enable objective assessment of a toxic effect of decreased water quality on an organisms' digestive capacity, it is necessary to establish reference values based on its natural variability as a function of changing biotic and abiotic factors. To limit the confounding influence of biotic factors, a caging approach with calibrated male organisms from the same population was used. This study applied an in situ deployment at 23 sites of the Rhone basin rivers, complemented by a laboratory experiment assessing the influence of two abiotic factors (temperature and conductivity). The results showed a small effect of conductivity on cellulase activity and a significant effect of temperature on digestive enzyme activity but only at the lowest temperature (7 °C). The experimental conditions allowed us to define an environmental reference value for digestive enzyme activities to select sites where the quality of the water impacted the digestive capacity of the organisms. In addition to the feeding rate, this study showed the relevance of digestive enzymes as biomarkers to be used as an early warning tool to reflect organisms' health and the chemical quality of aquatic ecosystems.

Protozoa interaction with aquatic invertebrate: interest for watercourses biomonitoring.

Toxoplasma gondii, Cryptosporidium parvum, and Giardia duodenalis are human waterborne protozoa. These worldwide parasites had been detected in various watercourses as recreational, surface, drinking, river, and seawater. As of today, water protozoa detection was based on large water filtration and on sample concentration. Another tool like aquatic invertebrate parasitism could be used for sanitary and environmental biomonitoring. In fact, organisms like filter feeders could already filtrate and concentrate protozoa directly in their tissues in proportion to ambient concentration. So molluscan shellfish can be used as a bioindicator of protozoa contamination level in a site since they were sedentary. Nevertheless, only a few researches had focused on nonspecific parasitism like protozoa infection on aquatic invertebrates. Objectives of this review are twofold: Firstly, an overview of protozoa in worldwide water was presented. Secondly, current knowledge of protozoa parasitism on aquatic invertebrates was detailed and the lack of data of their biological impact was pointed out.

One-year monitoring of core biomarker and digestive enzyme responses in transplanted zebra mussels (Dreissena polymorpha).

A 12-month active biomonitoring study was performed in 2008-2009 on the Vesle river basin (Champagne-Ardenne, France) using the freshwater mussel Dreissena polymorpha as a sentinel species; allochthonous mussels originating from a reference site (Commercy) were exposed at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle river basin. Selected core biomarkers (acetylcholinesterase (AChE) activity, glutathione-S transferase (GST) activity, metallothionein concentration), along with digestive enzyme activities (amylase, endocellulase) and energy reserve concentrations (glycogen, lipids), were monitored throughout the study in exposed mussels. At the Fismes and Ardre sites (downstream basin), metallic and organic contamination levels were low but still high enough to elicit AChE and GST activity induction in exposed mussels (chemical stress); besides, chemical pollutants had no apparent deleterious effects on mussel condition. At the Bouy and Sept-Saulx sites (upstream basin), mussels obviously suffered from adverse food conditions which seriously impaired individual physiological state and survival (nutritional stress); food scarcity had however no apparent effects on core biomarker responses. Digestive enzyme activities responded to both chemical and nutritional stresses, the increase in energy outputs (general adaptation syndrome-downstream sites) or the decrease in energy inputs (food scarcity-upstream sites) leading to mid- or long-term induction of digestive carbohydrase activities in exposed mussels (energy optimizing strategy). Complex regulation patterns of these activities require nevertheless the use of a multi-marker approach to allow data interpretation. Besides, their sensitivity to natural confounding environmental factors remains to be precised.

One-year monitoring of reproductive and energy reserve cycles in transplanted zebra mussels (Dreissena polymorpha).

A 12-month active biomonitoring study was performed in 2008-2009 on a northern French river system using the freshwater mussel Dreissena polymorpha as a sentinel species. Allochtonous mussels originating from a reference site (Commercy) were caged at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle River basin. The main objective of the study was to characterize the influence of biotic (sex, food availability) and abiotic (temperature, chemicals) factors on the reproductive and energy reserve (glycogen, lipids) cycles of exposed mussels. Both cycles were markedly disturbed at the Bouy and Sept-Saulx sites where the lowest chlorophyll a levels were recorded during the study. At these sites, mussels obviously faced a negative energy balance, as confirmed by the impairment of their physiological state and byssal attachment. At other exposure sites, reproductive and energy reserves cycles were less impacted but were still dependent on the nutritional state of mussels. The latter appeared as a significant natural confounding factor in ecotoxicological survey performed in low polluted areas.

Lessons from a transplantation of zebra mussels into a small urban river: An integrated ecotoxicological assessment.

It is often difficult to evaluate the level of contamination in small urban rivers because pollution is mainly diffuse, with low levels of numerous substances. The use of a coupled approach using both chemical and biological measurements may provide an integrated evaluation of the impact of micro-pollution on the river. Zebra mussels were transplanted along a metal and organic pollution gradient in spring 2008. For two months, mussels and water samples were collected from two sites every two weeks and analyzed for metal and PAH content as well as water physicochemical parameters. Diffusive gradients in thin film (DGT) were also used to assess levels of labile metals. Exposure of mussels to contaminants and potential impact were evaluated using physiological indices and various biomarkers including condition index (CI), defense mechanisms (glutathione-S-transferase: GST), digestive enzymes (amylase and cellulase) and genotoxicity (micronucleus test: MN and comet assay: CA). For most contaminants, the water contamination was significantly higher downstream. Bioaccumulation in zebra mussels was related to water contamination in the framework of the biodynamic model, which allowed us to take into account the biological dilution that was caused by the growth of soft tissue downstream. Thus, metal influxes were on average two times higher downstream than upstream in particular for Zn, Cr, Cu and Cd. Significant differences in condition index were observed (final CI was 0.42 ± 0.03 downstream and 0.31 ± 0.03 upstream) reflecting a better food availability downstream. Moreover a significant decrease of GST activity and digestive enzymes activity in the cristalline style was observed downstream. Interpreting this decrease requires considering not only micro-pollution but also the trophic status related to the water's physicochemistry. The MN test and the CA on gill cells highlighted genotoxicity in mussels transplanted downstream compared to upstream.

Effects of metals on feeding rate and digestive enzymes in Gammarus fossarum: an in situ experiment.

The feeding activity and afterward the assimilation of the products resulting of the food digestion, allow organisms to obtain energy useful for growth, maintenance and reproduction. These biological parameters may be studied to assess the impact of contaminants on the energy metabolism of organisms, which could induce potential effects at an individual level. The studied species was an amphipod Gammarus fossarum, which has a high ecological relevance since it is widespread in European streams and plays a major role in the breakdown of leaf litter. Thus some G. fossarum were transplanted in four sites of a river characterized by metal contamination (Amous River, France). The following parameters were studied: digestive enzymes activities (esterase, beta-glucosidase, beta-galactosidase, amylase and endoglucanase), feeding rate, metal bioaccumulation and survival. Results showed a strong relationship between digestive enzymes activities, feeding rate and metal contents.

Physiological and behavioural responses of Gammarus pulex (Crustacea: Amphipoda) exposed to cadmium.

The aim of this study was to investigate the effects of cadmium on physiological and behavioural responses in Gammarus pulex. In a first experiment, cadmium LC50s for different times were evaluated in 264 h experiment under continuous mode of exposure (LC50(96 h)=82.1 microgL(-1), LC50(120 h)=37.1 microgL(-1), LC50(168 h)=21.6 microgL(-1), LC50(264 h)=10.5 microgL(-1)). In a second experiment, the physiological and behavioural responses of the amphipod exposed to cadmium (0, 7.5 and 15 microgL(-1)) were investigated under laboratory conditions. The mortality and the whole body cadmium concentration of organisms exposed to cadmium were significantly higher than in controls. Concerning physiological responses, cadmium exposure exerted a significant decrease on osmolality and haemolymph Ca(2+) concentration, but not on haemolymph Na(+) and Cl(-) concentrations, whereas the Na(+)/K(+)-ATPase activity was significantly increased. Behavioural responses, such as feeding rate, locomotor and ventilatory activities, were significantly reduced in Cd exposed organisms. Mechanism of cadmium action and consequent energetic reallocation in favour of maintenance functions (i.e., osmoregulation) are discussed. The results of this study indicate that osmolality and locomotor activity in G. pulex could be effective ecophysiological/behavioural markers to monitor freshwater ecosystem and to assess the health of organisms.

Bioaccumulation of metals in sediment elutriates and their effects on growth, condition index, and metallothionein contents in oyster larvae.

The bioavailability of Cd, Cu, Zn, and Pb from two metal-contaminated sediments (Bidassoa and Dunkerque) was studied using Crassostrea gigas larvae exposed to sediment elutriates. The metal contents within the sediments, the larvae and larval growth, the condition index, and the induction of metallothionein in the larvae were measured. The larval growth and condition index were only affected after exposure to the highest elutriates concentration (5 to 25%) from the most contaminated sediment (Dunkerque). Bioaccumulation of all metals was observed in larvae exposed to Dunkerque elutriatre; only Cu bioaccumulation was observed in the Bidassoa elutriate. The results from larvae exposed to both sediment elutriates show a strong correlation between bioaccumulated metal considered individually or in combination and the metallothionein level in larvae presenting no detrimental effect. On the other hand, in the case of larvae exposed to the highest Dunkerque elutriate concentration and showing the highest metal body burden, we observed a drop in the metallothionein level. These results indicate that metallothionein is a more sensitive indicator of heavy metal pollution than physiological endpoints taken into account in bioassays and could be proposed as an early biomarker of metal exposure in larvae. However, care must be taken with "fault control" due to the toxicological effect on larvae metabolism in the case of substantial contaminant exposure.