Determination of water quality standards for chemical mixtures: extension of a methodology developed for herbicides to a group of insecticides and a group of pharmaceuticals.

Monitoring surveys throughout America and Europe have demonstrated the widespread presence of organic synthetic substances such as pesticides and pharmaceuticals in surface water. To avoid deleterious effects on the aquatic system, many countries determine water quality criteria for pesticides. For each substance, the comparison of the concentration measured in water with its criterion gives an indication of the pressure this substance put on the aquatic system. However, in the environment, aquatic organisms are not only exposed to single pesticides but typically to mixtures of these substances. It is therefore of particular importance to take mixtures into account when defining water quality criteria, which is rarely done yet. We recently developed a method to define consistent and comparable water quality criteria for mixtures of herbicides having a similar mode of action. These criteria are calculated based on species sensitivity curves; the method assumes that these curves are parallel for substances having similar mode of action. The aim of this study was to apply our method to six organophosphates (insecticides) and to three ß-blockers (pharmaceuticals), other groups of compounds commonly detected in surface water. We found some evidence that the developed methodology gives consistent results for these groups too. The hypothesis of parallelism was accepted in 2/3 (ß-blockers) and 2/6 of the cases (organophosphates) for both species sensitivity curves based on effect concentrations 50% and on no-observed effect concentrations. The use of water quality criteria for mixture is illustrated by two case studies, which show the importance of taking mixtures into account in water quality legislation.

Multigeneration effects of insect growth regulators on the springtail Folsomia candida.

Multigeneration tests are very useful for the assessment of long term toxicity of pollutants such as endocrine disruptor compounds. In this study, multigeneration reproduction tests adapted from the ISO standard 11267 were conducted with the Collembola Folsomia candida. Springtails were exposed to artificial soil contaminated with four insect growth regulators (methoprene, fenoxycarb, teflubenzuron, and precocene II) according to two different experimental set-ups. In the first set-up, the parental generation (F(0)) of Collembola was exposed to a pollutant for 28 days. Juveniles from the F(1) generation were transferred to uncontaminated soil for another 28-day period to generate the F(2) generation. In the second set-up, the F(0) generation was exposed to a pollutant for 10 days before being transferred to uncontaminated soil to reproduce. After 18-28 days, juveniles from the F(1) were transferred to clean soil to generate the F(2) generation. An effect on the number of hatched juveniles of the F(2) generation was observed for methoprene after exposure of the F(0) for 28 days and hatching of F(1) in contaminated soil. For methoprene and teflubenzuron, significant effects were even observed on the F(2) generation with the second experimental set-up, when only the F(0) generation was exposed for 10 days. This shows that the impact of these substances is transgenerational, which can have important consequences for the population of these or other organisms. No effect on the F(2) generation was observed with fenoxycarb and precocene II with the 10-day exposure experiment. Our results show that the developed experimental procedures are appropriate to assess the long term effects of endocrine disrupting compounds on the reproduction of the non-target species F. candida. Another important finding is that two substances with the same predicted mode of action (i.e., the two juvenile hormone analogues fenoxycarb and methoprene) do not necessarily affect the same endpoints in F. candida.

Effects of dinoseb on energy reserves in the soil arthropod Folsomia candida.

This study evaluated the usefulness of energy reserves as an early warning system in Folsomia candida when exposed to the pesticide dinoseb. After different exposure times, survival, reproduction, growth (weight and length), lipid and protein content of the organisms were determined. After six days of exposure at 15-30microg of dinoseb/g dry soil, the weight, lipid, and protein content of the exposed organisms were higher than the controls. This stimulation seems to indicate that Collembola adopt a strategy of increasing their growth in order to improve their reproduction. This hypothesis was confirmed by the number of eggs laid which was greater in exposed organisms. After 21 days, all measured parameters decreased. The results show that after having produced an effort to increase growth and reproduction, lethality increases. The selected energy reserves are not more sensitive than the classically measured parameters such as reproduction, but can be more predictive for a pollutant stress encountered by the organisms.

Modeling the concentration-response function of the herbicide dinoseb on Daphnia magna (survival time, reproduction) and Pseudokirchneriella subcapitata (growth rate).

Models describing dose-response relationships are becoming increasingly popular in ecotoxicology. They allow simple and thorough evaluations of toxicity test results, including inter- and extrapolations to concentrations or exposure times other than those tested. Simple parametric regression models are of particular interest because their parameters may be attributed mechanistic meanings and they can be applied without sophisticated mathematical and computational support. We recently proposed a four-parameter logistic regression model to fit the survival data of Daphnia magna under dinoseb stress. The model parameters are the maximum survival time, the minimum time required for an individual to die, effect concentration, EC(50), and a curve shape parameter. This model has now been applied to compare the lethality and reproduction toxicity of D. magna and the growth inhibition of Pseudokirchneriella subcapitata under dinoseb stress. It can be fitted adequately to all the measured data and the parameters can be attributed biological meanings in any of the three endpoints. A comparison of the modeled concentration-response functions of all three endpoints for dinoseb toxicity shows that the range of ECs with respect to both D. magna and algae is steep (a decrease of between 0.1 and 0.6 mg/L). The survival and reproduction of D. magna exhibit similar characteristic concentration-response functions and toxicities. The statistical no-effect concentration (SNEC) is 0.14 (survival) and 0.11 (reproduction)mg/L, respectively. On the other hand, algae seem to be less sensitive to dinoseb than D. magna (SNEC: 0.48 mg/L). However, further investigations of individual algae may lead to a more suitable comparison. We speculate that the four parameters of the model function can be related to specific properties of chemicals and organisms. Characterization of these properties would allow simple and appropriate estimation of the toxic effects of these chemicals.

Tropical ecotoxicity testing with Ceriodaphnia cornuta.

The Sai Gon-Dong Nai river system in southern Vietnam is of great social and economic importance yet receives a large amount of industrial, domestic, and agricultural discharges. Toxicity assessment has started to become an issue in Vietnam, and it is important to employ a test system that is appropriate for typical Vietnamese conditions with a species that is representative of the invertebrates living in its aquatic ecosystems. The aim of this study was to develop and to validate an ecotoxicity test with an autochthonous organism. The microcrustacean Ceriodaphnia cornuta (Cladocera) was isolated from the Sai Gon River. A protocol was developed for the culturing of the organisms, and satisfactory results were obtained for long-term reproduction of C. cornuta. Quality control criteria were established. The toxicity of two relevant pesticides (diazinon and methyl parathion) and two metals (chromium and mercury) to C. cornuta was evaluated and compared to the standard organisms for ecotoxicological testing: Daphnia magna and Vibrio fischeri. Nonlinear regression models were applied to estimate such parameters as EC(50). The results of acute toxicity tests showed that C. cornuta was less tolerant than D. magna and V. fischeri to salinity and that C. cornuta was more sensitive than D. magna and V. fischeri to potassium dichromate, diazinon, methyl parathion, and mercury.

Toxic effects of Irgarol 1051 on phytoplankton and macrophytes in Lake Geneva.

Irgarol 1051 is a recent herbicidal compound, inhibitor of photosynthesis, used in antifouling paints. This toxic is persistent in aquatic environments, with low abiotic and biotic degradation, highly phytotoxic, and has already been detected in estuaries and coastal areas, with suspected negative impacts on non-target organisms (aquatic plants and algae). We measured the toxicity of Irgarol 1051 to macrophytes and phytoplankton from Lake Geneva (between Switzerland and France) by determining chlorophyll fluorescence yield, and phytoplankton primary production. Long-term toxicity for phytoplankton was estimated in a microcosm study, and growth inhibition tests were performed with isolated algal strains. The concentration of Irgarol 1051 was analysed in the water, and the most polluted site showed a higher level (up to 135 ng/L) than the lowest observed effect concentration for phytoplankton (8-80 ng/L), while the macrophytes appeared to be more tolerant to Irgarol 1051 in short-term tests. The microcosm study showed that phytoplankton structure might be even more sensitive to Irgarol 1051.

Hsp70 instability and induction by a pesticide in Folsomia candida.

The heat shock protein Hsp70 has been shown to be a promising biomarker in aquatic and terrestrial organisms. However, its analysis in the soil insect Folsomia candida (Collembola) poses many problems as the protein is particularly unstable in this species. Western blotting has shown that the principal degradation fragment has a size of 48 kDa. We have developed a Western blot method that avoids the degradation of Hsp70 and was successful in detecting the protein in the springtail F. candida after a heat shock (12, 18 and 24 h at 32 degrees C). In the second part of the study the organisms were exposed to artificial compressed soil contaminated with the dinitrophenol dinoseb (10, 15 and 20 micrograms g-1 dry weight [DW]). Hsp70 was analysed in pooled samples (40 to 150 collembola according to age) after 1, 2, 3, 4, 5, 6, 7, 11 and 14 days. The only significant induction was observed after 5 days at 20 micrograms g-1 DW of dinoseb. The induction patterns over time were dissimilar for the different concentrations and a relatively high variability between the replicates was observed. Our results show that we must be cautious when interpreting biomarker results, especially those for Hsp70.

Effects of dinoseb on the life cycle of Daphnia magna: modeling survival time and a proposal for an alternative to the no-observed-effect concentration.

Risk assessment is in urgent need of more accurate toxic effect endpoints than those currently in use, especially for low concentrations. Often such endpoints are estimated by analysis of variance, linear interpolation, or smoothing. As these statistical methods are not always satisfactory, some authors have proposed to describe the entire dose-response curves by fully formalized parametric regression models whose parameters have toxicological meaning. These models allow a better evaluation of pollutant effects, including inter- and extrapolation to any other than the measured effect values. Following this line, a four-parameter logistic regression model (standard model) was fitted to survival data of Daphnia magna under pesticide (dinoseb) stress. The heterogeneity of the variance was taken into account with a both-sides logarithmic transformation. Besides the standard model, a hormesis and a threshold model were tested too. These two others models have been described in the literature and might better represent the dose-response function we are looking for. All three models showed a good fit to our data, and the statistics gave no hints as to which model is the most appropriate. As no evidence was seen for hormesis or for the existence of a threshold concentration, we used the simplest, namely, the standard model, for most of our calculations. Model calculations allow the quantification of the effects on individuals' longevity as well as on mean survival time of the population. We used them to define a no-effect value, the statistical-no-effect concentration (SNEC). The SNEC is based on the confidence bands of the modeled regression and represents the highest value for which an effect is statistically not different from the control. The SNEC is an alternative to classical endpoints, like the no-observed-effect concentration (NOEC) or the low-effect concentrations (e.g., EC10, EC5, EC1).