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.

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.

Evaluation of the genotoxic, mutagenic and oxidant stress potentials of municipal solid waste incinerator bottom ash leachates.

Triplicate aqueous leachates of a municipal solid waste incineration bottom ash (MSWIBA) were produced according to a European standardised method. Leachates analysis showed relatively low concentrations (less than 1 mg.l(-1)) for four metals (iron, cadmium, lead and copper). No mutagenic activity was revealed after performing the Salmonella/microsome assay with and without microsomal activation. With the Vicia root tip micronucleus assay, a significant increase in micronucleated cells was observed between 3.4% and 100% leachate concentration. Significant and elevated antioxidant stress enzyme activities, e.g., superoxide dismutase (SOD), catalase (CAT), peroxidase (PER) and glutathione reductase (GR), were detected in Vicia root tissues even at the lowest tested leachate concentration (i.e., 0.3%), whereas this was not always the case in leaf tissues, which showed tissue specificity for the tested enzymes. At the lowest concentration (i.e., 0.3%), a higher increase was observed (respectively 197% and 45% compared to the control) for root glutathione reductase and peroxidase activities over those of other enzymes (superoxide dismutase and catalase). Our results suggest that MSWIBA aqueous leachates need to be formally tested with genotoxic sensitive tests before recycling and support the hypothesis that plant genotoxicity is related to the cellular production of reactive oxygen species (ROS).

Biological fluxes conversion and SXRF experiment with a new active biomonitoring tool for atmospheric metals and trace element deposition.

A new active biomonitoring tool, keeping alive mosses for 2 months, had demonstrated the buffering action of the water presence on the biological activity of three moss species (chlorophyll fluorescence induction method on Pleurozium schreberi, Scleropodium purum, Eurhynchium praelongum). The two more resistant mosses were exposed on four different sites with parallel exposure of monthly bulk collectors during three successive periods of 2 months. The coarse and sedimentable particles of bulk collectors were separated into different size classes (> 1 mm; 1000-200 microm; 200-40 microm; 40-20 microm). Dry depositions and mosses were analysed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for a stock (microg g(-1)- fluxes (microg m(-2) t(-1)) conversion. The "moss plate" allowed relative site (Ba, Cs, Sb, Sr, U) and species (Pb, Sb, Ti, V) comparisons. Two months were enough for a significant enrichment (Ba, Cd, Pb, Sb, Ti, U, V). Good explicative models were elaborated for Ba, Pb, Sb and Ti (r2 > 70%) including categorical (sites, moss species) and quantitative variables (fluxes of particles size classes). Entire and intact shoots of Scleropodium purum were analysed by detected synchrotron radiation induced X-ray fluorescence (SXRF). The in vivo distribution of the multi-elemental short term enrichment along the moss stem was mainly localized in the plant older parts (Pb, Ti, Cl, Se).

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.

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.

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.

Effects of algae frozen at different temperatures on chronic assessment endpoints observed with Daphnia magna.

The Daphnia magna 21-day juvenile production test is not yet fully standardized because of many sources of variation. One is the diet provided to daphnids: the ration must be sufficient and the quality of algal cells must be optimal for achieving the required number of offspring defined by the new OECD guidelines. The experiments reported herein first examined the effects of Raphidocelis subcapitata after it had been maintained under four different conditions of storage (4, -20, -80, and -196 degrees C) on the survival, reproductive performance (over 21 days), and growth (ascertained by dry weight) of individually held D. magna for three generations. Under all of the four regimes tested, daphnids survived and reproduced in a manner which fulfilled the current OECD guidelines for a valid test, but the best results were obtained with fresh algae and algae frozen at -80 degrees C. Second, although D. magna has been widely used to determine toxicity of chemical substances, there are no reports in the literature that describe a rigorous study of the nutritional quality of the algae given to daphnids. Therefore, cell number, optical density, amount of organic carbon, and esterasic activity (assessed by intracellular breakdown of FDA to fluorescein) of algae that have been preserved at 4, -20, -80, and -196 degrees C were investigated. This part of the study indicated that freezing had no effect on cell numbers, in contrast to optical density, amount of organic carbon, and esterasic activity. First, it was found that esterasic activity was closely correlated to the reproductive performance of daphnids. It appears important, therefore, to consider the inclusion of this enzymatic activity as part of the routine quality control given to this microinvertebrate chronic procedure.

The environmental risks of industrial waste disposal: an experimental approach including acute and chronic toxicity studies.

The toxicity of 15 leachates of various solid industrial wastes accepted in an engineered landfill has been studied. A cost-effective battery of tests allowing evaluation of acute and chronic toxicity, as well as genotoxicity, and investigations on different trophic levels in the aquatic environment has been used. Acute toxicity was tested on bacteria (Microtox assay with Photobacterium phosphoreum) and microcrustaceans (Daphnia magna immobilization assay). A growth inhibition test of microalgae was carried out on Raphidocelis subcapitata. A 28-day chronic test with Daphnia magna was used to detect effects on reproduction. Genotoxicity was evaluated by means of the Ames test conducted on the crude aqueous phase and also on the concentrated fractions of water-extractable micropollutants (liquid-liquid and freeze-dried extracts). Chemical analyses of leachates were carried out simultaneously. The toxicity varied greatly between the different wastes. Toxic effects were observed in the short and/or in the long term. Four samples were potentially genotoxic. In most cases, toxicity registered could not be correlated with results of the chemical analyses. This study demonstrates the usefulness of associating a toxicological monitoring with chemical analyses in waste management.

An attempt to improve the SOS chromotest responses.

The SOS Chromotest was carried out on leachates of ten industrial wastes with the standard procedure and a miniaturized version with microplates. The two methods gave identical results in nine samples (eight negative and one positive). A simple additional manipulation is described for the identification of the false positive response that is frequently observed with complex mixtures. It involves challenging the SOS Chromotest bacteria with samples (having previously shown a positive genotoxic response) just before the enzymatic activities (i.e. beta-galactosidase and alkaline phosphatase) are estimated colorimetrically. This additional step eliminates discrepancies between the results for the standard and the miniaturized procedures.

An example of interaction between environmental pollutants: modification of thiram toxicity to freshwater organisms by nitrites or nitrates in relation to nitrosamine synthesis.

Thiram, a dithiocarbamate fungicide, is known to evolve to dimethylnitrosamine (DMNA) when associated with nitrites. Conditions of appearance of that carcinogenic compound have been studied in short-term experiments by association of the fungicide, nitrates or nitrites, and species representative of freshwater biota. DMNA has been estimated by GLC equipped with a specific detector. Chlorella vulgaris can rapidly produce nitrites from nitrates and DMNA is obtained in presence of thiram. Daphnia magna can also synthesize DMNA but nitrites have to be added to the medium. Increased toxicity of thiram is observed. The same results are obtained on Cyprinus carpio and for a part on Brachydanio rerio. When the species are associated in a 15-day experimental food chain, and intoxicated algae feed the two other levels, no significant transfer is observed. Nevertheless, some DMNA hazard may exist for particular species exposed to thiram associated with nitrites or even nitrates if algae are present.

[Study model of the mid-range ecotoxicity of chemical substances]. / Modèle d'étude de l'écotoxicité à moyen terme des substances chimiques.

Semi-chronic ecotoxic effects are measured by studying mortality and reproduction of daphnids, grown in toxic medium and fed with toxic chlorellae during 4 weeks. Toxic biosorption is controlled analytically. Toxicity of Cd++ at a concentration of an order of magnitude of by liter is revealed after 14 days. Cr6+ appears non toxic at concentrations under 25 micrograms/l.

Interest of dynamic tests in acute ecotoxicity assessment in algae.

Sorption of toxics by algae may be important and occurs very early. Thus, a decrease of the experimental toxic concentrations in the medium results in understating toxicity when tests are conducted under static conditions. In this work, two different methods of exposure of algae (Chlorella vulgaris) are studied, the static test and the pseudodynamic test. Acute effects (biological and analytical effects) of inorganic compounds (Cu2+, Cd2+, Pb2+, Cr6+) have been evaluated for 96 hr of exposure; in each case, IC50 is much lower in the dynamic condition than in the static one. The percentage of reduction varies from 55 to 75% after 96 hr. Accumulation of metal by chlorellae is greater when testing by the pseudodynamic way, with Cu2+ and Pb2+. But in the case of Cd2+ and Cr6+, the concentration factors are similar in the two kinds of exposure. These results point out the advantage of the pseudodynamic test, of which the methodology is very easy, for a more realistic assessment of acute ecotoxicity in these organisms.

Accumulation of cadmium in a freshwater food chain experimental model.

The study of food chain contamination in nature or in the laboratory requires precise definitions of some terms. A simplified model of a freshwater food chain has been elaborated to try to point out hazards related to the presence of cadmium in the aquatic environment. Three different species belonging to various trophic levels, but to the same aquatic systems were used. The required biological and analytical criteria are described. The time course of the experiment was 34 days starting with a 10-day contamination of the first trophic level (algae). The algae were then separated, washed with fresh medium, and given to the second trophic level (daphnids) to eat for 20 days. The third trophic level (fish) was then introduced for 4 days as a scavenger. The methodology was designed to determine the accumulation of cadmium by daphnids and then fish from the algal source. The results show a high direct bioaccumulation of cadmium in algae. The indirect bioaccumulation of daphnids was of the same order of magnitude, but for fish it was smaller. Nevertheless the cadmium transfer from daphnids to fish was obvious.

[Interest of some biological criteria in studying daphnids contaminated by cadmium nitrate (author's transl)]. / Utilisation de quelques critères d'études biologiques dans les intoxications à moyen terme de daphnies par du nitrate de cadmium.

Three biological criteria were studied on daphnids after 28 days of contamination with three different concentrations of cadmium (5, 10, 15 micrograms/l), such as mortality, dry weight and reproduction rate relative to general metabolic state of the crustacea. The value of Cl50-28 exceed 15 micrograms/l for dry weight and mortality determinations, on the other hand it is only 10 micrograms/l for reproduction rate. The latter appears as the most sensible criteria.