Risk from pesticide mixtures - The gap between risk assessment and reality.

Pesticide applications in agricultural crops often comprise a mixture of plant protection products (PPP), and single fields face multiple applications per year leading to complex pesticide mixtures in the environment. Restricted to single PPP, the current European Union PPP regulation, however, disregards the ecological risks of pesticide mixtures. To quantify this additional risk, we evaluated the contribution of single pesticide active ingredients to the additive mixture risk for aquatic risk indicators (invertebrates and algae) in 464 different PPP used, 3446 applications sprayed and 830 water samples collected in Central Europe, Germany. We identified an average number of 1.3 different pesticides in a single PPP, 3.1 for complete applications often involving multiple PPP and 30 in stream water samples. Under realistic worst-case conditions, the estimated stream water pesticide risk based on additive effects was 3.2 times higher than predicted from single PPP. We found that in streams, however, the majority of regulatory threshold exceedances was caused by single pesticides alone (69% for algae, 81% for invertebrates). Both in PPP applications and in stream samples, pesticide exposure occurred in repeated pulses each driven by one to few alternating pesticides. The time intervals between pulses were shorter than the 8 weeks considered for ecological recovery in environmental risk assessment in 88% of spray series and 53% of streams. We conclude that pesticide risk assessment should consider an additional assessment factor to account for the additive, but also potential synergistic simultaneous pesticide mixture risk. Additionally, future research and risk assessment need to address the risk from the frequent sequential pesticide exposure observed in this study.

Better define beta-optimizing MDD (minimum detectable difference) when interpreting treatment-related effects of pesticides in semi-field and field studies.

The minimum detectable difference (MDD) is a measure of the difference between the means of a treatment and the control that must exist to detect a statistically significant effect. It is a measure at a defined level of probability and a given variability of the data. It provides an indication for the robustness of statistically derived effect thresholds such as the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) when interpreting treatment-related effects on a population exposed to chemicals in semi-field studies (e.g., micro-/mesocosm studies) or field studies. MDD has been proposed in the guidance on tiered risk assessment for plant protection products in edge of field surface waters (EFSA Journal 11(7):3290, 2013), in order to better estimate the robustness of endpoints from such studies for taking regulatory decisions. However, the MDD calculation method as suggested in this framework does not clearly specify the power which is represented by the beta-value (i.e., the level of probability of type II error). This has implications for the interpretation of experimental results, i.e., the derivation of robust effect values and their use in risk assessment of PPPs. In this paper, different methods of MDD calculations are investigated, with an emphasis on their pre-defined levels of type II error-probability. Furthermore, a modification is suggested for an optimal use of the MDD, which ensures a high degree of certainty for decision-makers.

Toxic masking and synergistic modulation of the estrogenic activity of chemical mixtures in a yeast estrogen screen (YES).

BACKGROUND, AIM AND SCOPE: Estrogenic and non-estrogenic chemicals typically co-occur in the environment. Interference by non-estrogenic chemicals may confound the assessment of the actual estrogenic activity of complex environmental samples. The aim of the present study was to investigate whether, in which way and how seriously the estrogenic activity of single estrogens and the observed and predicted joint action of estrogenic mixtures is influenced by toxic masking and synergistic modulation caused by non-estrogenic chemical confounders. MATERIALS AND METHODS: The yeast estrogen screen (YES) was adapted so that toxicity and estrogenicity could be quantified simultaneously in one experimental run. Mercury, two organic solvents (dimethyl sulfoxide (DMSO) and 2,4-dinitroaniline), a surfactant (LAS-12) and the antibiotic cycloheximide were selected as toxic but non-estrogenic test chemicals. The confounding impact of selected concentrations of these toxicants on the estrogenic activity of the hormone 17ss-estradiol was determined by co-incubation experiments. In a second step, the impact of toxic masking and synergistic modulation on the predictability of the joint action of 17ss-estradiol, estrone and estriol mixtures by concentration addition was analysed. RESULTS: Each of the non-estrogenic chemicals reduced the apparent estrogenicity of both single estrogens and their mixtures if applied at high, toxic concentrations. Besides this common pattern, a highly substance- and concentration-dependent impact of the non-estrogenic toxicants was observable. The activity of 17ss-estradiol was still reduced in the presence of only low or non-toxic concentrations of 2,4-dinitroaniline and cycloheximide, which was not the case for mercury and DMSO. A clear synergistic modulation, i.e. an enhanced estrogenic activity, was induced by the presence of slightly toxic concentrations of LAS-12. The joint estrogenic activity of the mixture of estrogens was affected by toxic masking and synergistic modulation in direct proportion to the single estrogens, which allowed for an adequate adaptation of concentration addition and thus unaffected predictability of the joint estrogenicity in the presence of non-estrogenic confounders. DISCUSSION: The modified YES proved to be a reliable system for the simultaneous quantification of yeast toxicity and estrogen receptor activation. Experimental results substantiate the available evidence for toxic masking as a relevant phenomenon in estrogenicity assessment of complex environmental samples. Synergistic modulation of estrogenic activity by non-estrogenic confounders might be of lower importance. The concept of concentration addition is discussed as a valuable tool for estrogenicity assessment of complex mixtures, with deviations of the measured joint estrogenicity from predictions indicating the need for refined analyses. CONCLUSIONS: Two major challenges are to be considered simultaneously for a reliable analysis of the estrogenic activity of complex mixtures: the identification of known and suspected estrogenic compounds in the sample as well as the substance- and effect-level-dependent confounding impact of non-estrogenic toxicants. RECOMMENDATIONS AND PERSPECTIVES: The application of screening assays such as the YES to complex mixtures should be accompanied by measures that safeguard against false negative results which may be caused by non-estrogenic but toxic confounders. Simultaneous assessments of estrogenicity and toxicity are generally advisable.

5-Point programme for sustainable plant protection.

This position paper intends to stimulate a profound rethinking of contemporary agricultural practice. We criticise the current intensity of chemical plant protection in Germany as ecologically unsustainable and thus threatening the achievement of key targets of environmental protection and nature conservation policies. In the first part of the paper, we provide background information on the use of plant protection products (PPP) in German agriculture, the role of agricultural policy, European pesticide legislation, the principles of and framework for environmental risk assessment and risk management of PPP, as well as environmental effects of PPP. The second part is presented against the backdrop of the European "Sustainable Use Directive" (2009/128/EC). This directive requires that "Member States shall adopt National Action Plans to set up their quantitative objectives, targets, measures, and timetables to reduce risks and impacts of pesticide use on human health and the environment and to encourage the development and introduction of integrated pest management and of alternative approaches or techniques to reduce dependency on the use of pesticides." Reflecting on the corresponding debate in Germany, we suggest the following five key principles for a sustainable use of PPP and provide recommendations for their implementation: (1) minimising use; (2) identifying, quantifying, and communicating risks; (3) optimising risk management; (4) compensating for unavoidable effects; (5) internalising external costs.

Ecotoxicological evaluation of in situ bioremediation of soils contaminated by the explosive 2,4,6-trinitrotoluene (TNT).

To evaluate the environmental relevance of in situ bioremediation of contaminated soils, effective and reliable monitoring approaches are of special importance. The presented study was conducted as part of a research project investigating in situ bioremediation of topsoils contaminated by the explosive 2,4,6-trinitrotoluene (TNT). Changes in soil toxicity within different experimental fields at a former ordnance factory were evaluated using a battery of five bioassays (plant growth, Collembola reproduction, soil respiration, luminescent bacteria acute toxicity and mutagenicity test) in combination to chemical contaminant analysis. Resulting data reveal clear differences in sensitivities between methods with the luminescent bacteria assay performed with soil leachates as most sensitive toxicity indicator. Complete test battery results are presented in so-called soil toxicity profiles to visualise and facilitate the interpretation of data. Both biological and chemical monitoring results indicate a reduction of soil toxicity within 17 months of remediation.

Soil microbial parameters and luminescent bacteria assays as indicators for in situ bioremediation of TNT-contaminated soils.

In situ bioremediation is increasingly being discussed as a useful strategy for cleaning up contaminated soils. Compared to established ex situ procedures, meaningful and reliable approaches for monitoring the remediation processes and their efficiency are of special importance. The subject of this study was the significance of two bioassays for monitoring purposes. The work was performed within the scope of a research project on the in situ bioremediation of topsoil contaminated with 2,4,6-trinitrotoluene (TNT). To evaluate changes within different experimental fields during a 17-month remediation period, the results of soil microbial assays and luminescent bacteria assays were compared with chemical monitoring data. The luminescent bacteria assays showed a significant reduction of the water-soluble soil toxicants in the treated fields. This bioassay proved to be a sensitive screening indicator of toxicity and may effectively aid the ecotoxicological interpretation of chemical monitoring data. Microbial biomass (C(mic)), the metabolic quotient (qCO2), and the ratio of microbial to organic carbon (C(mic)/C(org)) showed a highly significant correlation with total concentrations of TNT in the soil. But, in contrast to luminescent bacteria assays, this approach did not reveal any recovery of the soil at the end of the remediation period. There is clear evidence for persistent adverse effects of chronic TNT contamination on the site-specific microbial community and the local carbon cycle in the soil. The study clearly exhibits the differences between, as well as the complementary value of both bioassay approaches for monitoring short-term and long-term effects of soil contamination and the efficiency of remediation.

Identification, assessment and management of "endocrine disruptors" in wildlife in the EU substance legislation--discussion paper from the German Federal Environment Agency (UBA).

A discussion paper was developed by a panel of experts of the German Federal Environment Agency (UBA) contributing to the on-going debate on the identification, assessment and management of endocrine disruptors with a view to protect wildlife according to the EU substance legislation (plant protection products, biocides, industrial chemicals). Based on a critical synthesis of the state-of-the-art regarding regulatory requirements, testing methods, assessment schemes, decision-making criteria and risk management options, we advise an appropriate and consistent implementation of this important subject into existing chemicals legislation in Europe. Our proposal for a balanced risk management of endocrine disruptors essentially advocates transparent regulatory decision making based on a scientifically robust weight of evidence approach and an adequate risk management consistent across different legislations. With respect to the latter, a more explicit consideration of the principle of proportionality of regulatory decision making and socio-economic benefits in the on-going debate is further encouraged.

Environmental effect assessment for sexual endocrine-disrupting chemicals: Fish testing strategy.

Current standard testing and assessment tools are not designed to identify specific and biologically highly sensitive modes of action of chemicals, such as endocrine disruption. This information, however, can be important to define the relevant endpoints for an assessment and to characterize thresholds of their sublethal, population-relevant effects. Starting a decade ago, compound-specific risk assessment procedures were amended by specifically addressing endocrine-disrupting properties of substances. In 2002, the Conceptual Framework, agreed upon by OECD's Task Force on Endocrine Disrupters Testing and Assessment, did not propose specific testing strategies, and appropriate testing methods had not yet been developed and approved. In the meantime, the OECD Test Guidelines Programme has undertaken important steps to revise established and to develop new test methods, which can be used to identify and quantify effects of endocrine-disrupting chemicals on mammals, birds, amphibians, fish, and invertebrates. For fish testing of endocrine-disrupting chemicals, the first Test Guidelines have recently been adopted by the OECD and validation of further test systems is under progress. Based on these test systems and the experience gained during their validation procedures, we propose a 3-step fish testing strategy: 1) Weight-of-evidence approach for identifying potential sexual endocrine-disrupting chemicals; even after advanced specification of systematic criteria, this step of establishing initial suspicion will still require expert judgment; 2) in vivo evaluation of sexual endocrine-disrupting activity in fish by applying in vivo fish screening assays; sufficient data are available to diagnose the aromatase-inhibition and estrogen-receptor agonist mechanisms of action by indicative endpoints (biomarkers), whereas the ability of the respective biomarkers in the screening assay to identify the estrogen-receptor antagonists and androgen-receptor agonists and antagonists requires further validation; 3) characterization of sexual endocrine-mediated adverse effects including threshold concentrations; in cases when the most sensitive population-relevant endpoints and the most sensitive time window for exposure are known for the mechanisms of action, the fish full life-cycle or 2-generation test, which are the normal definitive tests, might be abbreviated to, e.g., the fish sexual development test. In the European Union, the measurement of indicative endpoints in the definitive test might be crucial for the authorization procedure under REACH and plant-protection products. The results of the definitive tests can be used in existing schemes of compound-specific environmental risk assessments.

Screening for soil toxicity and mutagenicity using luminescent bacteria--a case study of the explosive 2,4,6-trinitrotoluene (TNT).

The presented study explored the suitability of aquatic bioassays based on the marine luminescent bacterium Vibrio fischeri as screening indicators for soil toxicity and mutagenicity. The study consists of two parts: (i) determination of the bacterial toxicity and mutagenicity of the single substance 2,4,6-trinitrotoluene (TNT) and its primary reduced metabolites using three different luminescent bacteria assays and (ii) determination of the water-extractable toxicity and mutagenicity of soil samples taken at a former production plant for TNT showing complex contamination (TNT, metabolites of TNT, PAHs, and heavy metals). Resulting data indicate TNT to be predominantly responsible for the observed biological effects of soil leachates. A strategy for soil toxicity screening based on luminescent bacteria is proposed which may especially be applicable for the case of bioremediation of TNT-contaminated soils. Potentials and restrictions of this approach to soil toxicity assessment are discussed.