Inge Werner

Mixtures of Chemical Pollutants at European Legislation Safety Concentrations: How Safe Are They?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.

Benchmarking Organic Micropollutants in Wastewater, Recycled Water and Drinking Water with In Vitro Bioassays

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Effect-Based Tools for Monitoring and Predicting the Ecotoxicological Effects of Chemicals in the Aquatic Environment

Ecotoxicology faces the challenge of assessing and predicting the effects of an increasing number of chemical stressors on aquatic species and ecosystems. Herein we review currently applied tools in ecological risk assessment, combining information on exposure with expected biological effects or environmental water quality standards; currently applied effect-based tools are presented based on whether exposure occurs in a controlled laboratory environment or in the field. With increasing ecological relevance the reproducibility, specificity and thus suitability for standardisation of methods tends to diminish. We discuss the use of biomarkers in ecotoxicology including ecotoxicogenomics-based endpoints, which are becoming increasingly important for the detection of sublethal effects. Carefully selected sets of biomarkers allow an assessment of exposure to and effects of toxic chemicals, as well as the health status of organisms and, when combined with chemical analysis, identification of toxicant(s). The promising concept of “adverse outcome pathways (AOP)” links mechanistic responses on the cellular level with whole organism, population, community and potentially ecosystem effects and services. For most toxic mechanisms, however, practical application of AOPs will require more information and the identification of key links between responses, as well as key indicators, at different levels of biological organization, ecosystem functioning and ecosystem services.

The European technical report on aquatic effect-based monitoring tools under the water framework directive

The Water Framework Directive (WFD), 2000/60/EC, requires an integrated approach to the monitoring and assessment of the quality of surface water bodies. The chemical status assessment is based on compliance with legally binding Environmental Quality Standards (EQSs) for selected chemical pollutants (priority substances) of EU-wide concern. In the context of the mandate for the period 2010 to 2012 of the subgroup Chemical Monitoring and Emerging Pollutants (CMEP) under the Common Implementation Strategy (CIS) for the WFD, a specific task was established for the elaboration of a technical report on aquatic effect-based monitoring tools. The activity was chaired by Sweden and co-chaired by Italy and progressively involved several Member States and stakeholders in an EU-wide drafting group. The main aim of this technical report was to identify potential effect-based tools (e.g. biomarkers and bioassays) that could be used in the context of the different monitoring programmes (surveillance, operational and investigative) linking chemical and ecological status assessment. The present paper summarizes the major technical contents and findings of the report.

Correlation between heat-shock protein induction and reduced metabolic condition in juvenile steelhead trout (Oncorhynchus mykiss) chronically exposed to elevated temperature

The metabolic condition, stress response, and growth rates of juvenile steelhead trout (Oncorhynchus mykiss) were assessed throughout a 10-week exposure to 15 and 20 °C, each at full and half food ration. Metabolic measurements included white muscle phosphocreatine (PCr), ATP, ADP and AMP, hepatic glycogen, and global metabolite profiling of muscle and liver using 1H nuclear magnetic resonance (NMR) spectroscopy. Stress was assessed via induction of heat shock proteins (hsps) 63, 72, 78 and 89, and growth was determined from otolith measurements. Exposure to 20 °C induced hsp synthesis, particularly hsp72. Principal components analyses revealed positive correlations between increased stress (elevated hsp72 and hsp89) and decreased metabolic condition (decreased PCr, ATP and glycogen). However, no effect on growth was observed, suggesting that 20 °C is the upper limit of the steelhead trouts preferred range. The NMR analysis of multiple metabolite classes not only identified a decrease in PCr, ATP and glycogen in temperature exposed fish, but also provided a more integrated description of the biochemical response to thermal stress.

Isomer selectivity in aquatic toxicity and biodegradation of bifenthrin and permethrin

Synthetic pyrethroids are widely used insecticides, and contamination of surface aquatic ecosystems by pyrethroid residues from runoff is of particular concern because of potential aquatic toxicity. Pyrethroids also are chiral compounds consisting of multiple stereoisomers. In the present study, we evaluated the diastereomer and enantiomer selectivity of cis-bifenthrin (cis-BF) and permethrin (PM) in their aquatic toxicity and biodegradation. The 1R-cis enantiomer was the only enantiomer in cis-BF showing toxicity against Ceriodaphnia dubia. Incubation with pesticide-degrading bacteria showed that the trans diastereomer of PM was selectively degraded over the cis diastereomer, whereas the 1S-cis enantiomer in cis-BF or cis-PM was preferentially degraded over the corresponding 1R-cis enantiomer. The enantioselectivity was significantly greater for cis-PM than for cis-BF and also varied among different strains of bacteria. Isomer selectivity may be a common phenomenon in both aquatic toxicity and biodegradation of pyrethroids, and this should be considered when assessing ecotoxicological risks of these compounds in sensitive ecosystems.

Sublethal toxicity of commercial insecticide formulations and their active ingredients to larval fathead minnow (Pimephales promelas).

Toxic effect concentrations of insecticides are generally determined using the technical grade or pure active ingredient. Commercial insecticide formulations, however, contain a significant proportion (>90%) of so-called inert ingredients, which may alter the toxicity of the active ingredient(s). This study compares the sublethal toxicity of two insecticides, the pyrethroid bifenthrin, and the phenylpyrazole fipronil, to their commercial formulations, Talstar and Termidor. Both insecticides are used for landscape treatment and structural pest control, and can be transported into surface water bodies via stormwater and irrigation runoff. We used larval fathead minnow (Pimephales promelas), to determine effects on growth and swimming performance after short-term (24h) exposure to sublethal concentrations of pure insecticides and the respective formulations. Significantly enhanced 7d growth was observed at 10% of the 24h LC(10) (53 microg L(-)(1)) fipronil. Swimming performance was significantly impaired at 20% of the 24h LC(10) (0.14 microg L(-)(1)) of bifenthrin and 10% of the 24h LC(10) of Talstar (0.03 microgL(-)(1)). Fipronil and Termidor led to a significant impairment of swimming performance at 142 microgL(-)(1) and 148 microgL(-)(1) respectively, with more pronounced effects for the formulation. Our data shows that based on dissolved concentrations both formulations were more toxic than the pure active ingredients, suggesting that increased toxicity due to inert ingredients should be considered in risk assessments and regulation of insecticides.

Linking mechanistic and behavioral responses to sublethal esfenvalerate exposure in the endangered delta smelt; Hypomesus transpacificus (Fam. Osmeridae)

BackgroundThe delta smelt (Hypomesus transpacificus) is a pelagic fish species listed as endangered under both the USA Federal and Californian State Endangered Species Acts and considered an indicator of ecosystem health in its habitat range, which is limited to the Sacramento-San Joaquin estuary in California, USA. Anthropogenic contaminants are one of multiple stressors affecting this system, and among them, current-use insecticides are of major concern. Interrogative tools are required to successfully monitor effects of contaminants on the delta smelt, and to research potential causes of population decline in this species. We have created a microarray to investigate genome-wide effects of potentially causative stressors, and applied this tool to assess effects of the pyrethroid insecticide esfenvalerate on larval delta smelt. Selected genes were further investigated as molecular biomarkers using quantitative PCR analyses.ResultsExposure to esfenvalerate affected swimming behavior of larval delta smelt at concentrations as low as 0.0625 μg.L-1, and significant differences in expression were measured in genes involved in neuromuscular activity. Alterations in the expression of genes associated with immune responses, along with apoptosis, redox, osmotic stress, detoxification, and growth and development appear to have been invoked by esfenvalerate exposure. Swimming impairment correlated significantly with expression of aspartoacylase (ASPA), an enzyme involved in brain cell function and associated with numerous human diseases. Selected genes were investigated for their use as molecular biomarkers, and strong links were determined between measured downregulation in ASPA and observed behavioral responses in fish exposed to environmentally relevant pyrethroid concentrations.ConclusionsThe results of this study show that microarray technology is a useful approach in screening for, and generation of molecular biomarkers in endangered, non-model organisms, identifying specific genes that can be directly linked with sublethal toxicological endpoints; such as changes in expression levels of neuromuscular genes resulting in measurable swimming impairments. The developed microarrays were successfully applied on larval fish exposed to esfenvalerate, a known contaminant of the Sacramento-San Joaquin estuary, and has permitted the identification of specific biomarkers which could provide insight into the factors contributing to delta smelt population decline.

Acute, sublethal exposure to a pyrethroid insecticide alters behavior, growth, and predation risk in larvae of the fathead minnow (Pimephales promelas)

The present study determined the effects of environmentally relevant, short-term (4-h) exposure to the pyrethroid insecticide esfenvalerate on mortality, food consumption, growth, swimming ability, and predation risk in larvae of the fathead minnow (Pimephales promelas). Acute effect concentrations were determined, and in subsequent experiments, fish were exposed to the following measured sublethal concentrations: 0.072, 0.455, and 1.142 microg/L of esfenvalerate. To measure growth rates (% dry wt/d), 8-d-old fathead minnows were exposed to esfenvalerate for 4 h, then transferred to control water and held for 7 d. Food consumption and abnormal swimming behavior were recorded daily. Additional behavioral experiments were conducted to evaluate how esfenvalerate affects the optomotor response of the fish. To quantify predation risk, esfenvalerate-exposed fathead minnow larvae were transferred to 9.5-L aquaria, each containing one juvenile threespine stickleback (Gasterosteus aculeatus). Sticklebacks were allowed to feed for 45 min, after which the number of surviving minnows was recorded. No mortality occurred during 4-h exposures to esfenvalerate, even at nominal concentrations of greater than 20 microg/L. Delayed mortality (50%) was observed at 2 microg/L after an additional 20 h in clean water. Fish exposed to 0.455 and 1.142 microg/L of esfenvalerate exhibited impaired swimming and feeding ability as well as reduced growth compared to fish exposed to 0.072 microg/L and controls. Predation risk also was significantly increased for larvae exposed to 0.455 and 1.142 microg/L of esfenvalerate. These results demonstrate that larval fish experiencing acute exposures to sublethal concentrations of this insecticide exhibit significant behavioral impairment, leading to reduced growth and increased susceptibility to predation, with potentially severe consequences for their ecological fitness.

Triclosan Impairs Swimming Behavior and Alters Expression of Excitation-Contraction Coupling Proteins in Fathead Minnow (Pimephales promelas)

Triclosan (TCS), a high volume chemical widely used in consumer products, is a known aquatic contaminant found in fish inhabiting polluted watersheds. Mammalian studies have recently demonstrated that TCS disrupts signaling between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR), two proteins essential for excitation-contraction (EC) coupling in striated muscle. We investigated the swimming behavior and expression of EC coupling proteins in larval fathead minnows (Pimephales promelas) exposed to TCS for up to 7 days. Concentrations as low as 75 μg L(-1) significantly altered fish swimming activity after 1 day; which was consistent after 7 days of exposure. The mRNA transcription and protein levels of RyR and DHPR (subunit CaV1.1) isoforms changed in a dose and time dependent manner. Crude muscle homogenates from exposed larvae did not display any apparent changes in receptor affinity toward known radioligands. In nonexposed crude muscle homogenates, TCS decreased the binding of [(3)H]PN20-110 to the DHPR and decreased the binding of [(3)H]-ryanodine to the RyR, demonstrating a direct impact at the receptor level. These results support TCSs impact on muscle function in vertebrates further exemplifying the need to re-evaluate the risks this pollutant poses to aquatic environments.