Jean-François Férard

Comet assay in genetic ecotoxicology: A review

The Comet assay, also called the single cell gel electrophoresis (SCGE) assay or microgel electrophoresis (MGE) assay, primarily measures DNA strand breakage in single cells. Since the protocol was published by Singh et al. [1988], its use has increased in different topic areas: clinical applications, human monitoring, radiation biology, genetic toxicology, and genetic ecotoxicology. This study is a review of the investigations that have involved the alkaline version of the Comet assay in genetic ecotoxicology. It focuses mainly on the type of organisms (plants, worms, molluscs, fish, amphibians, and mammalians) but also on the type of cells that have been used for ecotoxicological studies. In the 23 papers published since 1993 and presented here, the original test procedure may have been slightly modified according to the cell type. In vitro and in vivo experiments as well as in situ studies have been carried out in various environments (water, soil, and air). Although the Comet assay is able to detect genotoxic effects of chemical and physical agents, only chemical substances and environmental complex mixtures will be considered in this review. Environ. Mol. Mutagen. 34:246–255, 1999

Ecotoxicity of selected nano-materials to aquatic organisms.

Present knowledge concerning the ecotoxic effects of nano‐materials is very limited and merits to be documented more fully. For this purpose, we appraised the toxicity of nine metallic nano‐powders (copper zinc iron oxide, nickel zinc iron oxide, yttrium iron oxide, titanium dioxide, strontium ferrite, indium tin oxide, samarium oxide, erbium oxide, and holmium oxide) and of two organic nano‐ powders (fullerene‐C60 and single‐walled carbon nanotube or SWCNT). After a simple process where nano‐powders (NPs) were prepared in aqueous solution and filtered, they were then bioassayed across several taxonomic groups including decomposers (bacteria), primary producers (micro‐algae), as well as primary and secondary consumers (micro‐invertebrates and fish). Toxicity data generated on the 11 NPs reflected a wide spectrum of sensitivity that was biological level‐, test‐, and endpoint‐specific. With all acute and chronic tests confounded for these 11 NPs, toxicity responses spanned over three orders of magnitude: >463 mg/L (24 h LC50 of the invertebrate Thamnoplatyurus platyurus for fullerene‐C60) ÷ 0.3 mg/L (96 h EC50 of the invertebrate Hydra attenuata for indium tin oxide), that is a ratio of 1543. On the basis of the MARA (Microbial Array for Risk Assessment) assay toxic fingerprint concept, it is intimated that NPs may have different modes of toxic action. When mixed in a 1:1 ratio with a certified reference material (CRM) sediment, two solid phase assays and an elutriate assay, respectively, showed that five NPs (copper zinc iron oxide, samarium oxide, erbium oxide, holmium oxide, and SWCNT) were able to increase both CRM sediment toxicity and its elutriate toxicity. This initial investigation suggests that chemicals emerging from nanotechnology may pose a risk to aquatic life in water column and sediment compartments and that further studies on their adverse effects are to be encouraged.

Assessment of the genotoxicity of contaminated soil with the Allium/Vicia-micronucleus and the Tradescantia-micronucleus assays

The present study concerns the genotoxicity of contaminated soil near Metz, France. Three plant bioassays, the Vicia faba (broad bean), the Allium cepa (white onion) and the Tradescantia (spiderwort) micronuclei tests were used to evaluate for genotoxicity. Two soil samples were tested: soil sample A (from an industrial waste site) and soil sample B (from a cokeworks waste site). Maleic hydrazide was used as the positive control. Aqueous extracts of the soil samples were used to treat the roots of Vicia and Allium, and plant cuttings of Tradescantia according to the standard protocol for these plant assays established by the International Program on Chemical Safety and the World Health Organization. The results of these tests showed differential sensitivity in the three different bioassays. Soil sample A was more toxic than soil sample B.

Statistical analysis of regulatory ecotoxicity tests

ANOVA-type data analysis, i.e.. determination of lowest-observed-effect concentrations (LOECs), and no-observed-effect concentrations (NOECs), has been widely used for statistical analysis of chronic ecotoxicity data. However, it is more and more criticised for several reasons, among which the most important is probably the fact that the NOEC depends on the choice of test concentrations and number of replications and rewards poor experiments, i.e., high variability, with high NOEC values. Thus, a recent OECD workshop concluded that the use of the NOEC should be phased out and that a regression-based estimation procedure should be used. Following this workshop, a working group was established at the French level between government, academia and industry representatives. Twenty-seven sets of chronic data (algae, daphnia, fish) were collected and analysed by ANOVA and regression procedures. Several regression models were compared and relations between NOECs and ECx, for different values of x, were established in order to find an alternative summary parameter to the NOEC. Biological arguments are scarce to help in defining a negligible level of effect x for the ECx. With regard to their use in the risk assessment procedures, a convenient methodology would be to choose x so that ECx are on average similar to the present NOEC. This would lead to no major change in the risk assessment procedure. However, experimental data show that the ECx depend on the regression models and that their accuracy decreases in the low effect zone. This disadvantage could probably be reduced by adapting existing experimental protocols but it could mean more experimental effort and higher cost. ECx (derived with existing test guidelines, e.g., regarding the number of replicates) whose lowest bounds of the confidence interval are on average similar to present NOEC would improve this approach by a priori encouraging more precise experiments. However, narrow confidence intervals are not only linked to good experimental practices, but also depend on the distance between the best model fit and experimental data. At least, these approaches still use the NOEC as a reference although this reference is statistically not correct. On the contrary, EC50 are the most precise values to estimate on a concentration response curve, but they are clearly different from the NOEC and their use would require a modification of existing assessment factors.

Induction of oxidative stress biomarkers associated with heavy metal stress in Fontinalis antipyretica Hedw.

The aquatic bryophyte Fontinalis antipyretica Hedw. was exposed to different heavy metals (Cd, Cu, Pb, Zn) at 0, 0.1, 1, 10, 100 and 1000muM concentrations. Lipid peroxidation and anti-oxidative responses in apices were evaluated after 2 and 7 days of exposure. Most treated plants showed increased levels of lipid peroxidation and enzyme activities (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GRD), ascorbate (APX) and guaiacol (GPX) peroxidases, compared to control (untreated) plants. Most of our results suggest that plants possess antioxidant enzymes which operates either unspecifically (SOD and APX) or depending on the nature of the contamination (CAT, GPX, GRD). However, for the highest metal concentration tested, these cellular defence systems seemed overwhelmed since MDA levels increased significantly. These results confirm the important role of antioxidant defences in the mechanisms of plant-resistance to heavy metal stress. Moreover, two types of concentration-response trends were identified: clear monotonous relationships were often found for SOD and MDA while bell-shaped trends were usually observed for APX, GPX and GRD. These tendencies are discussed with emphasis on possible use of these responses as plant biomarkers for freshwater biomonitoring surveys.

Environmental impact of sunscreen nanomaterials: Ecotoxicity and genotoxicity of altered TiO2 nanocomposites on Vicia faba

Mineral sunscreen nanocomposites, based on a nano-TiO(2) core, coated with aluminium hydroxide and dimethicone films, were submitted to an artificial ageing process. The resulting Altered TiO(2) Nanocomposites (ATN) were then tested in the liquid phase on the plant model Vicia faba, which was exposed 48 h to three nominal concentrations: 5, 25 and 50 mg ATN/L. Plant growth, photosystem II maximum quantum yield, genotoxicity (micronucleus test) and phytochelatins levels showed no change compared to controls. Oxidative stress biomarkers remained unchanged in shoots while in roots, glutathione reductase activity decreased at 50 mg ATN/L and ascorbate peroxidase activity decreased for 5 and 25 mg ATN/L. Nevertheless, despite the weak response of biological endpoints, ICP-MS measurements revealed high Ti and Al concentrations in roots, and X-ray fluorescence micro-spectroscopy revealed titanium internalization in superficial root tissues. Eventual long-term effects on plants may occur.

Toxicity of copper oxide nanoparticle suspensions to aquatic biota

Toxicity effects induced by nanosuspensions of CuO (<50 nm; Sigma-Aldrich) on macrophytic algae cells of Nitellopsis obtusa (96-h median lethal concentration [LC50]), microphytic algae Chlorella (30-min median inhibitory concentration [IC50]), shrimp Thamnocephalus platyurus (24-h LC50), and rotifer Brachionus calyciflorus (24-h LC50) were investigated. No substantial differences between the effects of nonsonicated and sonicated nCuO suspensions were observed. The particle size distribution analysis accomplished by the laser diffraction technique at suspension concentration from 3 to 100 mg/L revealed rapid (within 5 min) reagglomeration of the particles after the sonication. The observed adverse effects on N. obtusa cells may be attributed to nanoparticles per se, but not to ionic Cu, because neither chemical analysis nor biological testing (algae survival in the supernatants of suspensions) confirmed the presence of cupric ions in toxic amounts. Contrary to ionic Cu form, nCuO delayed the initial phase of N. obtusa cell membrane depolarization. Lethality tests with rewash demonstrated that the least used 5-min exposure in 100 mg/L nCuO sonicated suspension induced 70% mortality in charophyte cells after 8 d, whereas the rewash after a short exposure to a noticeably toxic concentration of Cu(2+) prevented cell mortality. The obtained data suggested the possible influence of a thick charophyte cell wall on the dynamics of nanotoxicity effects.

Biological Air Quality Monitoring: a Review

Air quality monitoring is currently ensured by captors measuring few physicochemical parameters, then they cannot evaluate unforeseen compounds and the interaction effects of pollutants. On the contrary, the use of bioindicators can answer these problems. Among them, plants are used in two ways:1) Passive bioindicators: trees, lichens and mosses are used to determine the distribution and even the contaminants long-term effects on vast zones and, possibly, the concentrations of pollutants to which they were exposed.2) Active bioindicators: tobacco and mosses make available the defective zones in plants and enable determination of deposition fluxes.Moreover, techniques based on biomarkers identification should increase the answers sensitivity. However, the plants present the disadvantage of being unable to provide a response in a time lapse comparable with that of the on-line analysers. Lastly, the development of tools using animal cells should allow, in the near future, to measure the total polluting load under conditions close to those of the routine techniques. Then, the real impact of the contaminant on public health could be approached.

Antioxidant enzyme activities as affected by trivalent and hexavalent chromium species in Fontinalis antipyretica Hedw.

The detoxification mechanisms of the aquatic moss, Fontinalis antipyretica Hedw., exposed to Cr was analyzed. In addition, the influence of Cr salts (as Cr nitrate, chloride and potassium bichromate) on these mechanisms has also been studied. The activity of antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1.), catalase (EC 1.11.1.6.), ascorbate peroxidase (APX, EC 1.11.1.11.), guaiacol peroxidase (GPX, EC 1.11.1.7.) and glutathione reductase (GR, EC 1.6.4.2.) increased in plants treated with Cr concentrations ranging from 6.25x10(-5) to 6.25mM when given as Cr(NO3)3. Antioxidant enzymes responded to the other two salts CrCl3 and K2Cr2O7 only with Cr concentrations higher than 6.25x10(-2)mM. Glutathione level and GSSG/GSH ratio also responded to Cr exposure but no dose-effect relationship could be observed. Moreover, two unknown thiol compounds were observed in mosses exposed to the highest Cr concentrations. Effects on chlorophyll contents and chlorophyll a/b ratios were also shown even at low Cr concentrations. Our results indicated that environmentally realistic concentrations of Cr could lead to impairment of the cellular activity towards F. antipyretica and that Cr(III), when present as a nitrate salt, was as harmful as Cr(VI).

Effects of deltamethrin (pyrethroid insecticide) on growth, reproduction, embryonic development and sex differentiation in two strains of Daphnia magna (Crustacea, Cladocera)

Acute and different chronic ecotoxic effects of deltamethrin have been investigated on two strains (coming from two different laboratories) of Daphnia magna. The effective concentrations immobilizing 50% of daphnids (EC50s) after 24 h and 48 h were 9.40 and 0.32 μg L(-1), 8.86 and 0.63 μg L(-1) for first strain (strain 1) and second strain (strain 2), respectively. Thus, there was an increase of deltamethrin ecotoxicity with time of exposure as confirmed by chronic studies. After 21 days of exposure to deltamethrin, daphnids have showed significant effects on survival at deltamethrin concentrations of 0.16 μg L(-1) and 0.31 μg L(-1) for strains 1 and 2, respectively. Eleven other endpoints were examined: body length, population growth rate and various reproductive parameters (days to first brood, number of broods, number of cumulative molts and number of neonates), embryotoxicity and appearance of males. IC10 values related to the number of juveniles per live adult were 11 and 46 ng L(-1) for strains 1 and 2, respectively. Furthermore, an increase in embryo deformities was observed at the highest concentrations tested for both strains. Following deltamethrin exposure, undeveloped second antennae, curved or unextended shell spines, and curved post abdomen spines were observed in live neonates. The production of male juveniles was only registered with strain 1 at 0.16 μg L(-1). Results suggest that deltamethrin could act as an endocrine disruptor in D. magna as it interferes with sex determination and development abnormality but there is a difference in sensitivity between the two tested strains.