The minimum detectable difference (MDD) and the interpretation of treatment-related effects of pesticides in experimental ecosystems.

In the European registration procedure for pesticides, microcosm and mesocosm studies are the highest aquatic experimental tier to assess their environmental effects. Evaluations of microcosm/mesocosm studies rely heavily on no observed effect concentrations (NOECs) calculated for different population-level endpoints. Ideally, a power analysis should be reported for the concentration-response relationships underlying these NOECs, as well as for measurement endpoints for which significant effects cannot be demonstrated. An indication of this statistical power can be provided a posteriori by calculated minimum detectable differences (MDDs). The MDD defines the difference between the means of a treatment and the control that must exist to detect a statistically significant effect. The aim of this paper is to expand on the Aquatic Guidance Document recently published by the European Food Safety Authority (EFSA) and to propose a procedure to report and evaluate NOECs and related MDDs in a harmonised way. In addition, decision schemes are provided on how MDDs can be used to assess the reliability of microcosm/mesocosm studies and for the derivation of effect classes used to derive regulatory acceptable concentrations. Furthermore, examples are presented to show how MDDs can be reduced by optimising experimental design and sampling techniques.

International round-robin study on the Ames fluctuation test.

An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate-incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round-robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9-mix. Validity criteria (acceptable spontaneous and positive control-induced mutation counts) were fulfilled by 92-100%, depending on the test conditions. A two-step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine-square-root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%.

Outdoor Terrestrial Model Ecosystems are suitable to detect pesticide effects on soil fauna: design and method development.

Terrestrial Model Ecosystems (TME) were developed as one higher-tier option to detect and assess effects of pesticides on soil communities in a 1 year study using lindane (gamma-HCH) as a persistent and toxic reference pesticide. TME contained intact soil cores (diameter 300 mm, height 400 mm) including indigenous soil communities of undisturbed grassland. Forty units were placed outdoors between spring 2005 and 2006. The TME experiment was designed to provide data that fulfill the requirements of the revised European regulation on plant protection products (regulation 1107/2009/EEC replacing guideline 91/414/EC) with a focus on structural endpoints such as soil organisms and their community structure in case higher-tier evaluation is triggered. The key objective was to evaluate the dynamics and stability of species-diverse microarthropod communities of undisturbed grassland over at least 1 year after application. In grassland soils, less selection pressure towards insensitive species compared to arable land was presumed. Sufficient numbers of organisms and numerous TME replicates ensured that a statistical evaluation could be performed to estimate the sensitivity of the organisms upon application of lindane applied at high rates of 7.5 and 75 kg ai/ha. The application rates resulted in nominal concentrations of 10 and 100 mg ai/kg dry soil referred to the top 5 cm soil layer of 10 TME each; 20 untreated TME served as controls and were used to study the natural dynamics and the variability of populations under field conditions. Results showed that the grassland from which the soil cores were sampled contained communities of soil organisms marked by typical diversity of improved grassland. Lindane applied at excessive rates caused clear dose-related and long-lasting effects on the communities of microarthropods. On the contrary, lumbricids, the total feeding activity (bait lamina) and the growth of plant biomass were not affected up to 1 year after application. Based on the results of this study using a toxic reference insecticide, the methodology seems to be suitable for use in the regulatory context of the assessment of pesticides once protection goals, data requirements and the conceptual framework are defined.

The potential of individual based population models to extrapolate effects measured at standardized test conditions to relevant environmental conditions--an example for 3,4-dichloroaniline on Daphnia magna.

In current risk assessment ecotoxicological biotests (e.g.Daphnia reproduction test) are used to assess the potential impact of xenobiotics on ecosystems. The effects of chemicals and pesticides on populations of non-target organisms in the field depend not only on the exposure and the toxicity, but also on other factors such as life history characteristics. The effects of 3,4-dichloroaniline (3,4-DCA) measured with standardized test procedures, namely the Daphnia immobilisation test (OECD 202) and Daphnia reproduction test (OECD 211), were extrapolated to the population level using an individual-based Daphnia magna population model (IDamP) integrating only the effects on mortality and reproduction. The application of IDamP to extrapolate the effects on population levels was tested on two different population datasets, differing in the start population as well as in the feeding regime. The simulation results were compared to data derived from population experiments under semi-static and flow-through conditions. The IDamP model with an integrated toxicity module was able to predict the effects of 3,4-DCA on the population level under constant laboratory conditions for both datasets. This modelling approach was used to establish concentration-response relationships for 3,4-DCA on the population level. For this purpose two endpoints, the population capacity and the extinction probability, were calculated for different food levels. It turned out that the concentration-response relationship of the population capacity was less influenced by food supply, whereas for daphnid populations exposed to 3,4-DCA the extinction risk was twice as high with lower (environmental relevant) food supply. For both endpoints the lowest EC(50) was calculated to be 25 and 35 µg l(-1). The calculation of concentration-effect relationships on the population level by using a modelling approach provides a tool to extrapolate from effects derived from lab experiments to effects on the population level and can improve the ecological risk assessment of chemicals and pesticides.

Variability of sediment-contact tests in freshwater sediments with low-level anthropogenic contamination--determination of toxicity thresholds.

Freshwater sediments with low levels of anthropogenic contamination and a broad range of geochemical properties were investigated using various sediment-contact tests in order to study the natural variability and to define toxicity thresholds for the various toxicity endpoints. Tests were performed with bacteria (Arthrobacter globiformis), yeast (Saccharomyces cerevisiae), nematodes (Caenorhabditis elegans), oligochaetes (Lumbriculus variegatus), higher plants (Myriophyllum aquaticum), and the eggs of zebrafish (Danio rerio). The variability in the response of some of the contact tests could be explained by particle size distribution and organic content. Only for two native sediments could a pollution effect not be excluded. Based on the minimal detectable difference (MDD) and the maximal tolerable inhibition (MTI), toxicity thresholds (% inhibition compared to the control) were derived for each toxicity parameter: >20% for plant growth and fish-egg survival, >25% for nematode growth and oligochaete reproduction, >50% for nematode reproduction and >60% for bacterial enzyme activity.

Assessing the elimination of estrogenic activity in advanced wastewater treatment with a reporter gene-based bioassay.

Estrogen-like chemicals, so-called xenoestrogens, have become a topic of concern because they are potentially capable of disturbing the hormonal balance of wildlife and humans. Effluents of wastewater treatment plants (WWTPs) are presumably the major source of xenoestrogens in the aquatic environment. In this study, we investigated eight WWTPs with respect to their input, elimination efficiency, and output of estrogenic activity by means of a reporter gene-based bioassay. All WWTPs employed activated sludge treatment with nitrification/denitrification and tertiary treatment (second nitrification and/or filtration). Estradiol equivalents (EEQs) in the influents of the WWTPs were between 5.7 and 65.8 ng/L. The greatest inputs were found in plants treating pure domestic sewage and in samples collected in winter. Process waters either had no estrogenic activity or EEQs in the range of raw sewage, depending on the source of the process water. EEQs of effluents ranged from mostly below quantification limit (0.8 ng/L) to a maximum of 5.4 ng/L in secondary and 1.4 ng/L in tertiary effluents. These findings demonstrate the elimination efficiency of the activated sludge treatment and the further improvement by additional tertiary treatment. However, several concentrated effluents elicited little, but detectable estrogenic responses in the bioassay.

The combined effects of temperature and food consumption on body weight, egg production and developmental time in Chaoborus crystallinus De Geer (Diptera: Chaoboridae) : Some new evidence for the adaptive value of vertical migration.

Chaoborus crystallinus fourth-instar larvae were reared individually at 14°, 17° and 20° C under different food conditions. Daphnia magna of 1.25 mm average length served as prey. The following were measured: amount of prey ingested, larval weight gain, duration of fourth instar, body weight of the adults, and egg number per female. At a given temperature, the body weight, egg-number and developmental rate increased with food consumption. At a given food consumption, higher temperatures caused a decrease in body weight and egg number, and an increase in developmental rate. Gross production efficiencies for fourth-instar larvae were highest at temperatures around 17° C. The results clearly indicate that from an energetic point of view higher temperatures are disadvantageous. In C. crystallinus vertical migration is evidently a way of lowering the temperature to which the animals are exposed and hence optimizing food conversion into biomass and offspring production, especially if prey densities are below the saturation level.