Molecular docking: A potential tool to aid ecotoxicity testing in environmental risk assessment of pharmaceuticals

A cocktail of human pharmaceuticals pollute aquatic environments and there is considerable scientific uncertainty about the effects that this may have on aquatic organisms. Human drug target proteins can be highly conserved in non target species suggesting that similar modes of action (MoA) may occur. The aim of this work was to explore whether molecular docking offers a potential tool to predict the effects of pharmaceutical compounds on non target organisms. Three highly prescribed drugs, diclofenac, ibuprofen and levonorgestrel which regularly pollute freshwater environments were used as examples. Their primary drug targets are cyclooxygenase 2 (COX2) and progesterone receptor (PR). Molecular docking experiments were performed using these drugs and their primary drug target homologues for Danio rerio, Salmo salar, Oncorhynchus mykiss, Xenopus tropicalis, Xenopus laevis and Daphnia pulex. The results show that fish and frog COX2 enzymes are likely to bind diclofenac and ibuprofen in the same way as humans but that D. pulex would not. Binding will probably lead to inhibition of COX function and reduced prostaglandin production. Levonorgestrel was found to bind in the same binding pocket of the progesterone receptor in frogs and fish as the human form. This suggests implications for the fecundity of fish and frogs which are exposed to levonorgestrel. Chronic ecotoxicological effects of these drugs reported in the literature support these findings. Molecular docking may provide a valuable tool for ecotoxicity tests by guiding selection of test species and incorporating the MoA of drugs for relevant chronic test end points in environmental risk assessments.     

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.     

Distribution and ecotoxicity of chlorotriazines in the Scheldt Estuary (B-Nl)

As part of the Endis-Risks project, the current study describes the occurrence of the chlorotriazine pesticides atrazine, simazine and terbutylazine in water, sediment and suspended matter in the Scheldt estuary (B-Nl) from 2002 to 2005 (3 samplings a year, 8 sampling points). Atrazine was found at the highest concentrations, varying from 10 to 736 ng/l in water and from 5 up to 10 ng/g in suspended matter. Simazine and terbutylazine were detected at lower concentrations. Traces of the targeted pesticides were also detected in sediments, but these were below the limit of quantification. As part of an ecotoxicological assessment, we studied the potential effect of atrazine on molting of Neomysis integer (Crustacea:Mysidacea), a resident invertebrate of the Scheldt Estuary and a proposed test organism for the evaluation of endocrine disruption. Following chronic exposure ( approximately 3 weeks), atrazine did not significantly affect mysid molting at environmentally relevant concentrations (up to 1 microg/l).     

Evaluation of antimicrobial agents for veterinary use in the ecotoxicity test using microalgae

The influence of antimicrobial agents approved as veterinary drugs in Japan on the growth of green algae, Selenastrum capricornutum and Chlorella vulgaris, was studied in accordance with the OECD guidelines for testing chemicals. Among the agents tested, growth inhibitory activity was very varied, i.e. erythromycin showed the strongest activity (EC₅₀, 50% effective concentration, =0.037 mg/l), sulfa drugs had activity to some extent (EC₅₀s of sulfamethoxazole, sulfadiazine, and sulfadimethoxine were 1.5, 2.2, and 2.3 mg/l, respectively), but ampicillin and cefazolin did not inhibit growth (EC₅₀s > 1000 mg/l). We also investigated synergistic effect of combining sulfa drugs with trimethoprim or pyrimethamine, which are commonly used as a combined drug. By adding trimethoprim, the growth inhibitory activity of sulfamethoxazole and sulfadiazine was significantly enhanced. Growth inhibition by sulfa drugs was reduced by the addition of folic acid, indicating that they inhibit folate synthesis in green algae.     

Aquatic ecotoxicity assessment of a new natural formicide

Background, aim and scope

Agrochemicals could reach aquatic ecosystems and damage ecosystem functionality. Natural formicide could be an alternative to use in comparison with the more toxic formicides available on the market. Thus, the objective of this study was to assess the ecotoxicity of the new natural formicide Macex? with a battery of classical aquatic ecotoxicity tests.

Material and methods

Bacteria (Aliivibrio fischeri), algae (Pseudokirchneriella subcapitata), hydra (Hydra attenuata), daphnids (Daphnia magna), and fish (Danio rerio) tests were performed in accordance with international standardized methodologies.

Results

In the range of formicide concentrations tested (0.03 to 2.0?g?L^?1) EC₅₀ values varied from 0.49 to >2.0?g?L^?1, with P. subcapitata being the most sensitive species and H. attenuata and D. rerio the most tolerant species to this product in aqueous solutions.

Conclusions

This new formicide preparation can be classed as a product of low toxicity compared to the aquatic ecotoxicity of the most common commercialized formicides.     

Biodegradability and ecotoxicity of amine oxide based surfactants

The aerobic and anaerobic biodegradability as well as the aquatic toxicity of two fatty amine oxides and one fatty amido amine oxide were investigated. Aerobic biodegradation was evaluated using the CO(2) headspace test (ISO 14593) and biodegradation under anaerobic conditions was assessed employing a standardised batch test. The three amine oxide based surfactants tested were readily biodegradable under aerobic conditions but only the alkyl amido amine oxide was found to be easily biodegradable under anaerobic conditions. Toxicity to Photobacterium phosphoreum and Daphnia magna was evaluated. Bacteria (EC(50) from 0.11 to 11 mg l(-1)) proved to be more sensitive to the toxic effects of the amine oxide based surfactants than crustacea (IC(50) from 6.8 to 45 mg l(-1)). The fatty amido amine oxide showed the lowest aquatic toxicity.     

Characterisation of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos to Artemia salina and Artemia parthenogenetica

In this study, the acute toxicity of the organophosphorous pesticides dichlorvos and chlorpyrifos to two different species of Artemia (A. salina and A. parthenogenetica) was evaluated. In addition, the in vivo effect of these two pesticides on cholinesterase (ChE) activity of both A. salina and A. parthenogenetica was also determined. The characterisation of the ChE, using different substrates and specific inhibitors, and the normal range of activity in non-exposed individuals were previously investigated for both species. The results obtained indicate that the ChE of A. salina is different from that of A. parthenogenetica and that both enzymes cannot be classified neither as acetylcholinesterase nor as butyrylcholinesterase since they show intermediary characteristics between the two vertebrate forms. The range of normal ChE activity was 2.65+/-0.15 U/mg protein for A. salina, and 3.69+/-0.17 U/mg protein for A. parthenogenetica. Significant in vivo effects of both pesticides on Artemia ChE activity were found, at concentrations between 5.38 and 9.30 mg/l for dichlorvos and between 1.85 and 3.19 mg/l for chlorpyrifos. Both Artemia species are resistant to these pesticides and they are able to survive with more than 80% ChE inhibition. However, A. parthenogenetica is more resistant than A. salina, with about a 95% reduction in its ChE activity respect to the control for nauplii exposed to the median lethal concentrations (LC50), without lethal effects after 24 h of exposure.     

Toxicity and ecotoxicity of sulfonic acids: Structure-activity relationship

Currently available data on toxicological and ecotoxicological endpoints for sulfonic acids have been compiled by the BUA. This compilation reveals a common toxicological profile for the sulfonic acids when all the properties of these substances are taken into consideration. In contrast, the sulfonic acids show a much less uniform pattern with respect to their ecotoxicological properties.     

Use of freshwater plants for phytotoxicity testing: a review

Phytotoxicity data for aquatic plants have served a relatively minor role in regulatory decisions concerning the environmental hazard of most potential contaminants. A variety of phytotoxicity tests have been conducted with freshwater green algae, duckweed, blue-green algae, diatoms and rooted macrophytes (whole plants and seeds). Several test methods have been standardized for microalgae which are used primarily with chemicals, effluents, contaminated sediment elutriates and hazardous waste leachates. Current scientific understanding concerning the phytotoxic effects of these contaminants is based mostly on results for a few green algae. The greatest limitation of these results is their uncertain environmental relevance due to the large interspecific variation in response of standard algal test species and the unrealistic experimental test conditions. Results of the few field validation toxicity tests conducted to resolve this uncertainty have been chemical-specific and unpredictable. Aquatic vascular plants have been used less frequently than algae as test species. Duckweeds have been used more often than rooted submersed species but the uncertain nature of their sensitivities relative to animal and other plant species has limited their use. Regulatory interest in wetland protection, contaminated sediment evaluations and sediment quality criteria development will result in increased use of whole rooted plants and their seeds as test species. Overall, regardless of the test species, if phytotoxicity data are to be more available and effective in the hazard assessment process, additional information concerning species sensitivity, and environmental relevance of the results will be needed.     

Exposure-based ecotoxicity assessment of Co3O4 nanoparticles in marine microalgae

Environmental Science and Pollution Research - The exposure-effect study was conducted to evaluate the effect of Co3O4 nanoparticles on Tetraselmis suecica. The growth suppressing effect has been...     

Statistical uncertainty in hazardous terrestrial concentrations estimated with aquatic ecotoxicity data

Since chemicals’ ecotoxic effects depend for most soil species on the dissolved concentration in pore water, the equilibrium partitioning (EP) method is generally used to estimate hazardous concentrations (HC50) in the soil from aquatic toxicity tests. The present study analyzes the statistical uncertainty in terrestrial HC50s derived by the EP-method. For 47 organic chemicals, we compared freshwater HC50s derived from standard aquatic ecotoxicity tests with porewater HC50s derived from terrestrial ecotoxicity tests. Statistical uncertainty in the HC50s due to limited species sample size and in organic carbon–water partitioning coefficients due to predictive error was treated with probability distributions propagated by Monte Carlo simulations. Particularly for specifically acting chemicals, it is very important to base the HC50 on a representative sample of species, composed of both target and non-target species. For most chemical groups, porewater HC50 values were approximately a factor of 3 higher than freshwater HC50 values. The ratio of the porewater HC50/freshwater HC50 was typically 3.0 for narcotic chemicals (2.8 for nonpolar and 3.4 for polar narcotics), 0.8 for reactive chemicals, 2.9 for neurotoxic chemicals (4.3 for AChE agents and 0.1 for the cyclodiene type), and 2.5 for herbicides–fungicides. However, the statistical uncertainty associated with this ratio was large (typically 2.3 orders of magnitude). For 81% of the organic chemicals studied, there was no statistical difference between the hazardous concentration of aquatic and terrestrial species. We conclude that possible systematic deviations between the HC50s of aquatic and terrestrial species appear to be less prominent than the overall statistical uncertainty.     

Spatial variability of sediment ecotoxicity in a large storm water detention basin

Detention basins are valuable facilities for urban storm water management, from both the standpoint of flood control and the trapping of pollutants. Studies performed on storm water have shown that suspended solids often constitute the main vector of pollutants (heavy metals, polycyclic aromatic hydrocarbon (PAH), etc.). In order to characterise the ecotoxicity of urban sediments from storm water detention basins, the sediments accumulated over a 6-year period were sampled at five different points through the surface of a large detention basin localised in the east of Lyon, France. A specific ecotoxicological test battery was implemented on the solid phase (raw sediment) and the liquid phase (interstitial water of sediments). The results of the study validated the method formulated for the ecotoxicological characterization of urban sediments. They show that the ecotoxicological effect of the sediments over the basin is heterogeneous and greater in areas often flooded. They also show the relationship between, on one hand, the physical and chemical characteristics of the sediments and, on the other hand, their ecotoxicity. Lastly, they contribute to a better understanding of the dynamics of the pollution close to the bottom of detention basins, which can be useful for improving their design. The results of this research raise particularly the issue of using oil separators on the surface of detention basins.     

Acute ecotoxicity and genotoxicity assessment of two wastewater treatment units

Environmental Science and Pollution Research - Water contamination by discharge of untreated or poorly treated wastewater into water bodies is a current issue that may cause harm to humans. Water...     

Degradation in soil and water and ecotoxicity of rimsulfuron and its metabolites.

The degradation and ecotoxicity of sulfonylurea herbicide rimsulfuron and its major metabolites were examined in batch samples of an alluvial sandy loam and in freshwater. An HPLC-DAD method was adapted to simultaneously identify and quantify rimsulfuron and its metabolites, which was successfully validated by GC-MS analysis. In aqueous solutions, pure rimsulfuron was rapidly hydrolyzed into metabolite 1 (N-(4,6-dimethoxypyrimidin-2-yl)-N-(3-(ethylsulfonyl)-2-pyridinylurea)), which itself was transformed into the more stable metabolite 2 (N-((3-(ethylsulfonyl)-2-pyridinyl)-4,6-dimethoxy-2-pyrimidineamine)), with half-life (t(1/2)) values of 2 and 2.5 days, respectively. Hydrolysis was instantaneous under alkaline conditions (pH = 10). In aqueous suspensions of the alluvial soil (pH = 8), formulated rimsulfuron had a half-life of 7 days, whereas that of metabolite 1 was similar to that in water (about 3.5 days). The degradation of the two major metabolites was also studied in soil suspensions with the pure compounds at concentrations ranging from 1 to 10 mg l(-1). The half-life of metabolite 1 ranged from 3.9 to 5 days, close to the previous values. Metabolite 2 was more persistent and its degradation is strongly dependent on the initial concentration (C0): half-life values ranged from 8.1 to 55 days at 2-10 mg l(-1), respectively. These values are higher than those determined from the kinetics of metabolite 1 transformation into metabolite 2 (t(1/2) = 8-19 days). The ecotoxicity of the three chemicals was evaluated through their effect on Daphnia magna and Vibrio fischeri (Microtox bioassay). No effect was observed on D. magna with 24 and 48 h acute toxicity tests. Similarly, no toxic effect was observed with the Microtox test for the three chemicals in the range of concentrations tested that included the field application dose. Thus, being of low persistence and lacking acute toxicity, these chemicals present a low environmental risk. However, chronic effects should be studied in order to confirm the safety of rimsulfuron and its major metabolites.     

NAPL migration and ecotoxicity of conventional and renewable fuels in accidental spill scenarios

Fuels derived from non-petroleum renewable resources have raised interest due to their potential in replacing petroleum-based fuels, but information on their fate and effects in the terrestrial and aquatic environments in accidental spill scenario is limited. In this study, migration of four fuels (conventional diesel, conventional gasoline, renewable diesel NExBTL, and ethanol-blended gasoline RE85 containing maximum 85 % ethanol) as non-aqueous phase liquids (NAPL) in soil was demonstrated in a laboratory-scale experiment. Ecotoxicity data was produced for the same fuels. There was no significant difference in migration of conventional and renewable diesel, but gasoline migrated 1.5 times deeper and 7–9 times faster in sand than diesel. RE85 spread horizontally wider but not as deep (p?Eisenia fetida followed by ethanol-blended gasoline (LC50 1,643 mg/kg THC) and conventional diesel (LC50 2,432 mg/kg THC), although gasoline evaporated fast from soil. For comparison, the toxicity of the water-accommodated fractions (WAF) of the fuels was tested with water flea Daphnia magna and Vibrio fischeri, also demonstrating groundwater toxicity. The WAF of conventional gasoline and RE85 showed almost similar toxicity to both the aquatic test species. EC50 values of 1:10 (by volume) WAF were 9.9 %WAF (gasoline) and 9.3 %WAF (RE85) to D. magna and 9.3 %WAF (gasoline) and 12.3 %WAF (RE85) to V. fischeri. Low solubility decreased toxicity potential of conventional diesel in aquatic environment, but direct physical effects of oil phase pose a threat to organisms in nature. Renewable diesel NExBTL did not show clear toxicity to any test species.     

Microbial ecotoxicity and mutagenicity of 1-hydroxypyrene and its photoproducts.

1-Hydroxypyrene (1-HP) is a carcinogenic and slightly water-soluble polycyclic aromatic hydrocarbon. Ecotoxicity and mutagenicity of 1-HP and its photoproducts, and the effect of Mn2+ and Cu2+ on their mutagenicity were measured with microbial assay in this study. The assay includes spread plate counting, direct counting, microbial mineralization of 14C-UL-D-glucose and Mutatox Test. At the concentration examined (0.8 microM), the photoproducts (after 1.5 h solar irradiation) of 1-HP inhibited microbial glucose mineralization activity (by 64%) after microbial assemblages of a local reservoir site were exposed for 1 day. However, heterotrophic bacteria were able to utilize 1-HP photoproducts as the growth substrates and increase viability counts by up to 4.75-folds. 1-HP exhibited positive response to Mutatox Test in both direct medium and S-9 medium, with the lowest observable effective concentration of 0.625 microM in the test with direct medium. After photolysis, 1-HP decreased its mutagenicity. Mn2+ (312.5 microM-5 mM) and Cu2+ (6.25-100 microM) themselves are not mutagenic. However, addition of the metal ions before or after photolysis modifies the light readings of 1-HP during the test. Therefore, presence of metal ions could affect the genotoxicity of 1-HP in aquatic environments, depending on timing of the addition.     

Terrestrial ecotoxicity and effect factors of metals in life cycle assessment (LCA)

Life cycle impact assessment aims to translate the amounts of substance emitted during the life cycle of a product into a potential impact on the environment, which includes terrestrial ecosystems. This work suggests some possible improvements in assessing the toxicity of metals on soil ecosystems in life cycle assessment (LCA). The current available data on soil ecotoxicity allow one to calculate the chronic terrestrial HC50(EC50) (hazardous concentration affecting 50% of the species at their EC50 level, i.e. the level where 50% of the individuals of the species are affected) of nine metals and metalloids (As(III) or (V), Be(II), Cr(III) or (VI), Sb(III) or (V), Pb(II), Cu(II), Zn(II) and Ni(II)). Contrarily to what is generally advised in LCIA, the terrestrial HC50 of metals shall not be extrapolated from the aquatic HC50, using the Equilibrium Partitioning method since the partition coefficient (K(d)) of metals is highly variable. The experimental ecotoxicology generally uses metallic salts to contaminate artificial soils but the comparison of the EC50 or NOEC obtained for the same metal with different salts reveals that the kind of salt used insignificantly influences these values. In contrast, depending on the metallic fraction of concern, the EC50 may vary, as for cadmium: the EC50 of Folsomia candida, expressed as free Cd in pore water is almost 2.5 orders of magnitude lower than that expressed as total metal. A similar result is obtained with Eisenia fetida, confirming the importance of metals speciation in assessing their impact on soils. By ranking the metals according to the difference between their terrestrial and aquatic HC50 values, two groups are distinguished, which match the hard soft acids and bases (HSAB) concept. This allows to estimate their affinity for soil components and potential toxicity according to their chemical characteristics.     

Spatial distribution of trace elements and ecotoxicity of bottom sediments in Rybnik reservoir,Silesian-Poland

Environmental Science and Pollution Research - The aim of study was to integrate chemical analyses and toxicity bioassays in order to assess the environmental risk connected with the presence of...     

A protocol to select high quality datasets of ecotoxicity values for pesticides

The key to any QSAR model is the underlying dataset. In order to construct a reliable dataset to develop a QSAR model for pesticide toxicity, we have derived a protocol to critically evaluate the quality of the underlying data. In developing an appropriate protocol that would enable data to be selected in constructing a QSAR, we concentrated on one toxicity end point, the 96 h LC50 from the acute rainbow trout study. This end point is key in pesticide regulation carried out under 91/414/EEC. The dataset used for this exercise was from the US EPA-OPP database.     

Bisphenol A migration from plastic materials: direct insight of ecotoxicity in Daphnia magna

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) whose migration from food packaging is recognized worldwide. However, the real overall food contamination and related consequences are yet largely unknown. Among humans, children’s exposure to BPA has been emphasized because of the immaturity of their biological systems. The main aim of this study was to assess the reproductive impact of BPA leached from commercially available plastic containers used or related to child nutrition, performing ecotoxicological tests using the biomonitoring species Daphnia magna. Acute and chronic tests, as well as single and multigenerational tests were done. Migration of BPA from several baby bottles and other plastic containers evaluated by GC-MS indicated that a broader range of foodstuff may be contaminated when packed in plastics. Ecotoxicological test results performed using defined concentrations of BPA were in agreement with literature, although a precocious maturity of daphnids was detected at 3.0 mg/L. Curiously, an increased reproductive output (neonates per female) was observed when daphnids were bred in the polycarbonate (PC) containers (145.1?±?4.3 % to 264.7?±?3.8 %), both in single as in multigenerational tests, in comparison with the negative control group (100.3?±?1.6 %). A strong correlated dose-dependent ecotoxicological effect was observed, providing evidence that BPA leached from plastic food packaging materials act as functional estrogen in vivo at very low concentrations. In contrast, neonate production by daphnids cultured in polypropylene and non-PC bottles was slightly but not significantly enhanced (92.5?±?2.0 % to 118.8?±?1.8 %). Multigenerational tests also revealed magnification of the adverse effects, not only on fecundity but also on mortality, which represents a worrying trend for organisms that are chronically exposed to xenoestrogens for many generations. Two plausible explanations for the observed results could be given: a non-monotonic dose–response relationship or a mixture toxicity effect.