Pierre-Emmanuel Baurand

First evidence of fish genotoxicity induced by heavy metals from landfill leachates: The advantage of using the RAPD-PCR technique

Municipal leachates are loaded with heavy metals that can contaminate surface water before discharge into a receiving body of water. The aim of this study is to evaluate the genotoxic effects of heavy metals generated by domestic waste on the common roach Rutilus rutilus in the last of the four interconnected ponds at the Etueffont landfill. We used random amplified polymorphic DNA (RAPD) since it has been shown to be a powerful means of detecting a broad range of DNA damage due to environmental contaminants. Our results show the ability of RAPD analysis to detect significant genetic alterations in roach DNA, after contamination with a set of metals contained in the landfill leachates in comparison to a roach from a non-polluted reference pond. Analysis of electrophoresis profiles indicates apparent changes such as the appearance of new bands or disappearance of bands as compared to the control. In fact, mixed smearing and laddering of DNA fragments in muscle samples support the genotoxic effects of metal deposits in the roach. This study is the first evidence found via the RAPD-PCR technique in the detection of pollutant impacts on fish exposed to landfill leachates.

Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene.

Influence of landscape composition and diversity on contaminant flux in terrestrial food webs: A case study of trace metal transfer to European blackbirds Turdus merula

Although understanding the influence of the spatial arrangement of habitats and interacting communities on the processes of pollutant flux and impacts is critical for exposure and risk assessment, to date few studies have been devoted to this emergent topic. We tested the hypothesis that landscape composition and diversity affect the transfer of trace metals to vertebrates. Bioaccumulation of Cd and Pb in blood and feathers of European blackbirds Turdus merula (n=138) was studied over a smelter-impacted area (Northern France). Landscape composition (type and occurrence of the different habitats) and diversity (number of different habitat types and the proportional area distribution among habitat types) were computed around bird capture locations. Diet composition and contamination were assessed. No sex-related differences were detected, while age-related patterns were found: yearlings showed a sharper increase of tissue residues along the pollution gradient than older birds. Factors determining bird exposure acted at nested spatial scale. On a broad scale, environmental contamination mainly influenced metal levels in blackbirds, tissue residues increasing with soil contamination. At a finer grain, landscape composition and soil properties (pH, organic matter, clay) influenced metal transfer, while no influence of landscape diversity was detected. Landscape composition better explained metal transfer than soil properties did. Diet composition varied according to landscape composition, but diet diversity was not influenced by landscape diversity. Surprisingly, metal accumulation in some insect taxa was as high as in earthworms (known as hyper-accumulators). Results strongly suggested that variations in diet composition were the drivers through which landscape composition influenced metal transfer to blackbirds. This study shows that landscape features can affect pollutant transfer in food webs, partly through ecological processes related to spatial and foraging behavior of birds, and brings evidences underpinning the need to better consider landscape in environmental risk assessment and management of contaminated lands.

Coupling of Random Amplified Polymorphic DNA Profiles Analysis and High Resolution Capillary Electrophoresis System for the Assessment of Chemical Genotoxicity

Cadmium (Cd) can be toxic to terrestrial snails, but few data are available about its genotoxic effects on early life stages (ELS). The aim of this study was to investigate the genotoxic potential of Cd in embryos of Helix aspersa using a new approach that couples Random Amplified Polymorphic DNA (RAPD) and a high-resolution capillary electrophoresis system (HRS). Clutches of H. aspersa were exposed to Cd solutions (2, 4, and 6 mg/L) from the beginning of their embryonic development. In addition to a dose-dependent effect of Cd on hatching rate, DNA fragmentation was observed in embryos that were exposed to 6 mg Cd/L. The analysis of RAPD products with HRS showed differences between the profiles of exposed and nonexposed embryos, starting at 2 mg Cd/L. In comparison to the profiles of the control samples, all profiles from the exposed snails exhibited an additional 270 bp DNA fragment and lacked a 450 bp DNA fragment. These profile modifications are related to the genotoxic effect of Cd on the ELS of H. aspersa . Our study demonstrates the efficacy of coupling RAPD and HRS for a rapid and efficient screening of the effects of chemicals on DNA.

An assessment of the embryotoxicity of cadmium in the terrestrial mollusk Cantareus aspersus: from bioaccumulation to impacts at different levels of biological organization.

This study aims to determine various parameters that allow the evaluation of the toxicity of chemicals to embryos of the ubiquitous land snail Cantareus aspersus. For this purpose, we investigated morphological and physiological endpoints in control embryos and in embryos exposed to a solution of 6mg Cd/L (CdCl2) in a liquid phase bioassay: size at days 3, 6 and 10, heart rate at 7 days, delay in hatching, states of development of non-hatched eggs after 17 days and the fresh mass of newly hatched embryos. The kinetics of Cd accumulation in eggs and DNA fragmentation were also measured. The first detectable sign of adverse effects appeared after 7 days of development, when the heart rate decreased in Cd-exposed embryos compared with the control. After 10 days of exposure, Cd-exposed hatchlings exhibited a lower fresh mass than control individuals. The majority (75 percent) of non-hatched embryos at 17 days was dead and presented signs of disaggregation or malformations. The hatching of Cd-exposed eggs was delayed 4 days, and DNA fragmentation was later detected after 20 days of Cd exposure. The measurement of Cd in the eggs showed that concentrations are relatively stable during the exposure period from 3 days (20-27µg Cd/g DW) to the end of exposure. The present study completes the range of endpoints that can be used to study the effects of contaminants and provides new parameters that are readily measured throughout the embryonic development of a terrestrial mollusk.

First amplification of Eimeria hessei DNA from the lesser horseshoe bat (Rhinolophus hipposideros) and its phylogenetic relationships with Eimeria species from other bats and rodents.

Although coccidian parasites of the genus Eimeria are among the best-documented parasites in bats, few Eimeria species found in bats have been characterised using molecular tools, and none of the characterised species are found in European countries. Phylogenetic relationships of Eimeria species that parasitise bats and rodents can be related to the morphology of oocysts, independently from host range, suggesting that these species are derived from common ancestors. In the present study, we isolated a partial sequence of the Eimeria hessei 18S rRNA gene from the lesser horseshoe bat (Rhinolophus hipposideros), a European bat species. Droppings from lesser horseshoe bats were collected from 11 maternity roosts located in France that were positive for the presence of the parasite. Through morphological characterisation, the oocysts detected in the lesser horseshoe bat droppings were confirmed to be E. hessei. The unique E. hessei sequence obtained through molecular analysis belonged to a clade that includes both rodent and bat Eimeria species. However, the E. hessei oocysts isolated from the bat droppings did not show morphological similarities to rodent Eimeria species.

Genotoxicity assessment of pesticides on terrestrial snail embryos by analysis of random amplified polymorphic DNA profiles.

The study explores the relevance of coupling Random Amplified Polymorphic DNA (RAPD) and a High-Resolution capillary electrophoresis System (HRS) method for assessing the genotoxic potential of the wide variety commercial formulations of pesticides. Using this technique, the genotoxic potential of a glyphosate-based herbicide (Roundup Flash(®) (RU)) and two fungicide formulations based on tebuconazole and copper (Corail(®) and Bordeaux mixture (BM), respectively) was evaluated on terrestrial snail embryos. Clutches of Cantareus aspersus were exposed during their entire embryonic development to a range of concentration around the EC50 values (based on hatching success) for each compound tested. Three primers were used for the RAPD amplifications of pesticides samples. RAPD-HRS revealed concentration-dependent modifications in profiles generated with the three primers in RU(®)-exposed embryos from 30 mg/L glyphosate. For Corail(®)-exposed embryos, only two of the three primers were able to show alterations in profiles from 0.05 mg/L tebuconazole. For BM-exposed embryos, no signs of genotoxicity were observed. All changes observed in amplification profiles have been detected at concentrations lower than the recommended doses for vineyard field applications. Our study demonstrates the efficiency of coupling RAPD and HRS to efficiently screen the effect of pesticide formulations on DNA.