Régine Maury-Brachet

Gold-mining activities and mercury contamination of native amerindian communities in French Guiana: key role of fish in dietary uptake.

In 1994, the French National Public Health Network reported significant mercury exposure of native Amerindians in French Guiana. In 1997, a study was conducted in the Wayana community to quantify the dietary intake and to identify the fish species contributing the most to the contamination. The study was completed by an impregnation analysis based on Hg determination in hair samples. The methodology used was a detailed familial dietary study associated with Hg measurements in fish and some game. The study was conducted over 7 days in two different seasons in the four most populated Wayana villages on the upper part of the Maroni River (521 people; 70% of the Wayana population in French Guiana). Analysis was based on data on consumption obtained from 165 people in a 1-14 day period (i.e., 940 persons [times] days) and involved 270 fish samples from 48 species. Total Hg and monomethylmercury (MMHg) were also determined in hair samples (235 samples for total Hg). The results confirm mercury exposure of the Wayana population related to a diet rich in fish, which are relatively highly contaminated for certain species (up to 1.62 mg/kg fresh weight or 8.1 mg/kg dry weight in skeletal muscle). Results from hair samples showed that 57% of the Amerindians had Hg levels above the World Health Organization (WHO) safety limit (10 microg/g); all those over 1 year of age had a Hg intake greater than the WHO safety limit (200 microg MMHg/week for a 60-kg male). Hg concentrations in fish muscle were closely linked to the feeding regime and position of fish in the food webs. Overall, 14.5% of the fish collected exceeded the 0.5 mg/kg (fresh weight) safety limit. Four carnivorous species accounted for no less than 72% of the metal ingested by the Wayana families, although these represented only 28% of the consumed fish biomass. In conclusion, this study revealed excessive exposure to mercury in the Wayana population in French Guiana related to the consumption of contaminated fish.

Contamination by Heavy Metals (Cd, Zn, Cu, and Hg) of Eight Fish Species in the Gironde Estuary (France)

The Gironde estuary, one of the largest in Europe, is considerated as a reference ecological system, with all the western European diadromous fish species present. The national biomonitoring program on the coastal marine environment has revealed since 1979 severe metal pollution (mostly cadmium [Cd]) in oysters collected from the estuary. No data are available on metal contamination levels in fish, despite their ecological and economic importance. We present the results from a detailed study based on 4 metals (Cd, zinc [Zn], copper [Cu], and mercury [Hg]) measured in 4 organs (gills, dorsal skeletal muscle, liver, and kidneys) from 8 fish species illustrating several ecological combinations: European eel (Anguilla anguilla), twaite shad (Alosa fallax), bass (Dicentrarchus labrax), meagre (Argyrosomus regius), flounder (Platichthys flesus),, thin-lippid grey mullet (Liza ramada), sole (Solea vulgaris), and Canary drum (Umbrina canariensis). The results show very marked differences between species and organs, as well as very significant variations between the 4 metals. Although metal concentrations measured in fish muscle are low, except in the case of Hg for theA. fallax, high levels of Cu and Cd were observed in the kidneys and livers ofL. ramada andA. anguilla. A multifactorial analysis based on rank ordered metal concentrations for the 8 fish species clearly shows 4 clusters of species assigned to the different degrees of metal contamination, from the lowest contaminated (A. regius, D. labrax, S. vulgaris, andU. canariensis), to the most contaminated group (L. ramada). The most contaminated species (L. ramada, A. angailla, andP. flesus) are characterized by long residence times in the estuary, between 3.5 and 14 yr. ForL. ramada, biofilms with high metal storage capacities would be the principal uptake route; the two other species are benthic with a carnivorous regime. Comparisons between our data and four estuaries (Seine, France; Mersey, U.K.; La Plata, Argentina; Guadalquivir, Spain), on a limited number of common species, metals and fish organs, clearly reveal higher Cd bioaccumulation levels in the Gironde estuary.

Serial analysis of gene expression in the skeletal muscles of zebrafish fed with a methylmercury-contaminated diet.

Mercury (Hg) is a widespread environmental contaminant and its organic form, methylmercury (MeHg), has been known as a potent neurotoxic since the Minamata tragedy. In the Amazonian basin, gold mining leads to MeHg biomagnification all along the food web, culminating in piscivorous fish, ultimately responsible for contamination of human beings through fish consumption. In order to assess the biological impact of dietary MeHg on fish at the genome scale, we contaminated zebrafish with MeHg-contaminated food for 25 days (13.5 microg of Hg/g of food). A serial analysis of gene expression (SAGE) was conducted on the skeletal muscle because this tissue does not perform MeHg demethylation, and 19171 SAGE tags were sequenced from the control library versus 22 261 from the MeHg-contaminated library, corresponding to 5280 different transcripts. Among those identified, 60 genes appeared up-regulated and 15 down-regulated by more than 2 times. A net impact of MeHg was noticed on 14 ribosomal protein genes, indicating a perturbation of protein synthesis. Several genes involved in mitochondrial metabolism, the electron transport chain, endoplasmic reticulum (ER) function, detoxification, and general stress responses were differentially regulated, suggesting an onset of oxidative stress and ER stress. Several other genes for which expression varied with MeHg contamination could be clustered in various compartments of the cells life, such as lipid metabolism, calcium homeostasis, iron metabolism, muscle contraction, and cell cycle regulation. This study reveals the effectiveness of the SAGE approach to acquire a better understanding of the MeHg global effects. Furthermore, this is the first time that the SAGE was used to characterize the effect of a toxicant at the genome scale in an aquatic organism.

Mercury methylation rates of biofilm and plankton microorganisms from a hydroelectric reservoir in French Guiana.

The Petit-Saut ecosystem is a hydroelectric reservoir covering 365km(2) of flooded tropical forest. This reservoir and the Sinnamary Estuary downstream of the dam are subject to significant mercury methylation. The mercury methylation potential of plankton and biofilm microorganisms/components from different depths in the anoxic reservoir water column and from two different sites along the estuary was assessed. For this, reservoir water and samples of epiphytic biofilms from the trunk of a submerged tree in the anoxic water column and from submerged branches in the estuary were batch-incubated from 1h to 3 months with a nominal 1000ng/L spike of Hg(II) chloride enriched in (199)Hg. Methylation rates were determined for different reservoir and estuarine communities under natural nutrient (reservoir water, estuary freshwater) and artificial nutrient (culture medium) conditions. Methylation rates in reservoir water incubations were the highest with plankton microorganisms sampled at -9.5m depth (0.5%/d) without addition of biofilm components. Mercury methylation rates of incubated biofilm components were strongly enhanced by nutrient addition. The results suggested that plankton microorganisms strongly contribute to the total Hg methylation in the Petit-Saut reservoir and in the Sinnamary Estuary. Moreover, specific methylation efficiencies (%Me(199)Hg(net)/cell) suggested that plankton microorganisms could be more efficient methylating actors than biofilm consortia and that their methylation efficiency may be reduced in the presence of biofilm components. Extrapolation to the reservoir scale of the experimentally determined preliminary methylation efficiencies suggested that plankton microorganisms in the anoxic water column could produce up to 27mol MeHg/year. Taking into account that (i) demethylation probably occurs in the reservoir and (ii) that the presence of biofilm components may limit the methylation efficiency of plankton microorganisms, this result is highly consistent with the annual net MeHg production estimated from mass balances (8.1mol MeHg/year, Muresan et al., 2008a).

Biofilm and mercury availability as key factors for mercury accumulation in fish (Curimata cyprinoides) from a disturbed amazonian freshwater system

The Petit-Saut hydroelectric reservoir was filled in 1994 on the Sinnamary River in French Guiana (Amazonian basin). Flooding of the equatorial rain forest led to anoxia in most of the water column and enhanced mercury methylation in the reservoir hypolimnion. We selected the benthivorous/omnivorous fish species Curimata cyprinoides to investigate total mercury and methylmercury (MeHg) bioavailability and bioaccumulation capacities in the reservoir and downstream in the Sinnamary River. Mercury concentrations in the dorsal skeletal muscle were 10-fold higher in fish from the downstream zone. Stomach contents and stable nitrogen and carbon isotope ratios showed that biofilms and the associated invertebrate communities represented important food sources at the two sites. The delta 13C measurements indicated that biofilms in the flooded forest zone of the reservoir consist of endogenous primary producers; downstream, they are based on exogenous organic matter and microorganisms, mainly from the anoxic layers of the reservoir. Total mercury and MeHg concentrations in the biofilms and associated invertebrates were much higher at the downstream site compared to concentrations at the reservoir. Our results clearly show the importance of MeHg export from the anoxic layers of this tropical reservoir. We conclude that differences between biofilm composition and MeHg concentrations in the ingested food could explain the marked differences observed between mercury levels in fish.

Remediation of a watershed contaminated by heavy metals: a 2-year field biomonitoring of periphytic biofilms.

This study focuses on an industrial contamination site subjected to remediation processes since 2007 in the Riou-Mort watershed (southwest France). The purpose was to assess the first impacts of remediation on periphytic biofilms, and was performed during two years of biomonitoring. Periphytic biofilms were collected on glass slides immersed 24 days at different sites along the contamination gradient for 12 colonisation cycles. Metal contaminations (Cd and Zn) were analysed in biofilms and the evolution of diatom communities was assessed, integrating teratology quantifications. Despite remediation work initiated at the industrial site, this study demonstrated the persistence of metal contamination in water, as well as in biofilms. In addition, our data, showed that the remediation process was initially marked by an increase in metal contamination in the river, with increasing diatom community shifts. Metal-contaminated biofilms presented decreasing species diversities and were dominated by metal-resistant species such as Eolimna minima, whom abundances increased in 2010 reaching 57.2±10%. No significant decrease in metal accumulation was observed and total Cd content in biofilms collected downstream the industrial site ranged from 772.7±88 in July 2009 to 636.9±20 μg/gDW in July 2010. Results obtained on artificial substrates were compared with those of natural substrates and showed similar diatom communities and abundances of deformed diatoms but lower diversities. This ensured that glass slide subtrates gave a good representation of periphytic biofilm health. Finally, results were compared to studies performed before the remediation process and this did not reveal a decrease of metal accumulation in biofilms nor shifts in taxonomic composition of the communities, rather the remaining dominance of metal resistant species such as E. minima was confirmed.

Mercury and methylmercury concentrations in high altitude lakes and fish (Arctic charr) from the French Alps related to watershed characteristics

Total mercury (THg) and methylmercury (MeHg) concentrations were measured in the muscle of Arctic charr (Salvelinus alpinus) and in the water column of 4 lakes that are located in the French Alps. Watershed characteristics were determined (6 coverage classes) for each lake in order to evaluate the influence of watershed composition on mercury and methylmercury concentrations in fish muscle and in the water column. THg and MeHg concentrations in surface water were relatively low and similar among lakes and watershed characteristics play a major role in determining water column Hg and MeHg levels. THg muscle concentrations for fish with either a standardized length of 220mm, a standardized age of 5 years or for individualuals did not exceed the 0.5mg kg(-1) fish consumption advisory limit established for Hg by the World Health Organization (WHO, 1990). These relatively low THg concentrations can be explained by watershed characteristics, which lead to short Hg residence time in the water column, and also by the short trophic chain that is characteristic of mountain lakes. Growth rate did not seem to influence THg concentrations in fish muscles of these lakes and we observed no relationship between fish Hg concentrations and altitude. This study shows that in the French Alps, high altitude lakes have relatively low THg and MeHg concentrations in both the water column and in Arctic charr populations. Therefore, Hg does not appear to present a danger for local populations and the fishermen of these lakes.

Incidence of invasive macrophytes on methylmercury budget in temperate lakes: Central role of bacterial periphytic communities

Several studies demonstrated high mercury (Hg) methylation and demethylation in the periphyton associated with floating roots in tropical ecosystems. The importance of aquatic plants on methylmercury production in three temperate ecosystems from south-western France was evaluated through Hg species concentrations, and Hg methylation/demethylation activities by using stable isotopic tracers ((199)Hg(II), Me(201)Hg). Hg accumulation and high methylation and demethylation yields were detected in plant roots and periphyton, whereas results for sediment and water were low to insignificant. The presence of sulfate reducing prokaryotes was detected in all compartments (T-RFLP based on dsrAB amplified through nested PCR) and their main role in Hg methylation could be demonstrated. In turn, sulfate reduction inhibition did not affect demethylation activities. The estimation of net MeHg budgets in these ecosystems suggested that aquatic rhizosphere is the principal location for methylmercury production and may represent an important source for the contamination of the aquatic food chain.

Mercury bioaccumulation along food webs in temperate aquatic ecosystems colonized by aquatic macrophytes in south western France

Mercury (Hg) is considered as an important pollutant for aquatic systems as its organic form, methylmercury (MeHg), is easily bioaccumulated and bioamplified along food webs. In various ecosystems, aquatic periphyton associated with macrophyte was identified as an important place for Hg storage and methylation by microorganisms. Our study concerns temperate aquatic ecosystems (South Western France) colonized by invasive macrophytes and characterized by high mercury methylation potentials. This work establishes original data concerning Hg bioaccumulation in organisms (plants, crustaceans, molluscs and fish) from five contrasting ecosystems. For low trophic level species, total Hg (THg) concentrations were low (from 27±2ngTHgg(-1)dw in asiatic clam Corbicula fluminea to 418±114ngTHgg(-1)dw in crayfish Procambarus clarkii). THg concentrations in some carnivorous fish (high trophic level) were close to or exceeded the International Marketing Level (IML) with values ranging from 1049±220ngTHgg(-1)dw in pike perch muscle (Sander lucioperca) to 3910±1307ngTHgg(-1)dw in eel muscle (Anguilla Anguilla). Trophic levels for the individuals were also evaluated through stable isotope analysis, and linked to Hg concentrations of organisms. A significant Hg biomagnification (r(2)= 0.9) was observed in the Aureilhan lake, despite the absence of top predator fish. For this site, Ludwigia sp. periphyton, as an entry point of Hg into food webs, is a serious hypothesis which remains to be confirmed. This study provides a first investigation of Hg transfer in the ecosystems of south western France and allows the assessment of the risk associated with the presence of Hg in aquatic food webs.

Specific Effects of Dietary Methylmercury and Inorganic Mercury in Zebrafish (Danio rerio) Determined by Genetic, Histological, and Metallothionein Responses

A multidisciplinary approach is proposed here to compare toxicity mechanisms of methylmercury (MeHg) and inorganic mercury (iHg) in muscle, liver, and brain from zebrafish (Danio rerio). Animals were dietary exposed to (1) 50 ng Hg g(-1), 80% as MeHg; (2) diet enriched in MeHg 10000 ng Hg g(-1), 95% as MeHg; (3) diet enriched in iHg 10000 ng Hg g(-1), 99% as iHg, for two months. Hg species specific bioaccumulation pathways were highlighted, with a preferential bioaccumulation of MeHg in brain and iHg in liver. In the same way, differences in genetic pattern were observed for both Hg species, (an early genetic response (7 days) for both species in the three organs and a late genetic response (62 days) for iHg) and revealed a dissimilar metabolization of both Hg species. Among the 18 studied genes involved in key metabolic pathways of the cell, major genetic responses were observed in muscle. Electron microscopy revealed damage mainly because of MeHg in muscle and also in liver tissue. In brain, high MeHg and iHg concentrations induced metallothionein production. Finally, the importance of the fish origin in ecotoxicological studies, here the seventh descent of a zebrafish line, is discussed.