In vivo effect confirmation of anti-androgenic compounds in sediment contact tests with Potamopyrgus antipodarum.

In order to investigate the sensitivity of Potamopyrgus antipodarum to anti-androgenic compounds, three spiked sediment tests were performed. The substances benzanthrone (7H-benz[de]anthracen-7-one), traseolide (ATII) and androstenone (5α-Androst-16-en-3-one) were previously identified in an effect-directed analysis study of the river Schijn in the north of Belgium. Although, in previous studies, all of the three compounds exhibited anti-androgenic activities in vitro, only the oxy-PAH benzanthrone had significant stimulating effects on the snails' reproduction. The reproduction of P. antipodarum was significantly stimulated, following a sigmoidal dose response curve, whereby an EC(50) of 10 ng/g dry sediment was calculated. Mortality was significantly increased at the highest concentration (69 ng/g dry sediment). The results indicate different relative potencies for the in vivo test with P. antipodarum and the in vitro anti-AR-CALUX assay, performed in a previous study. This highlights the importance of combined in vitro and in vivo assays for the effect assessment of field sediments.

Sediment contact test with Potamopyrgus antipodarum in effect-directed analyses-challenges and opportunities.

BACKGROUND AND SCOPE: Effect-directed analysis is increasingly used for the identification of key toxicants in environmental samples and there is a growing need for in vivo biotests as diagnostic tools. Within this study, we performed an in vivo sediment contact test, applicable on both native field samples and their extracts or fractions, in order to be able to compare the results from both field and laboratory studies. MATERIAL AND METHODS: A sediment contact test with the prosobranch snail, Potamopyrgus antipodarum, was carried out on extracts and fractions of field sediments from three European river basins. The results were compared with previous results of the native field samples. RESULTS: In contrast to the native sediments, the extracts of the samples led to an overall decrease in reproduction. Even the chosen reference sites had an adverse effect on the snails' reproduction. It appeared that a higher bioavailability in the organic extracts, together with a changing composition of compounds could have lead to this change in effects. The fractionation of the extracts partly led to a more differentiated picture, but the resolution was not high enough to see any distinct effects on the snails' reproduction. DISCUSSION AND CONCLUSION: Our results highlight the importance of the use of in vivo biotests and point out the relevance of bioavailability in native sediments. For further fractionation studies, a more realistic extraction procedure, together with a higher resolution fractionation, would be appropriate in order to separate individual bioavailable compounds more efficient.

A new risk assessment approach for the prioritization of 500 classical and emerging organic microcontaminants as potential river basin specific pollutants under the European Water Framework Directive.

Given the huge number of chemicals released into the environment and existing time and budget constraints, there is a need to prioritize chemicals for risk assessment and monitoring in the context of the European Union Water Framework Directive (EU WFD). This study is the first to assess the risk of 500 organic substances based on observations in the four European river basins of the Elbe, Scheldt, Danube and Llobregat. A decision tree is introduced that first classifies chemicals into six categories depending on the information available, which allows water managers to focus on the next steps (e.g. derivation of Environmental Quality Standards (EQS), improvement of analytical methods, etc.). The priority within each category is then evaluated based on two indicators, the Frequency of Exceedance and the Extent of Exceedance of Predicted No-Effect Concentrations (PNECs). These two indictors are based on maximum environmental concentrations (MEC), rather than the commonly used statistically based averages (Predicted Effect Concentration, PEC), and compared to the lowest acute-based (PNEC(acute)) or chronic-based thresholds (PNEC(chronic)). For 56% of the compounds, PNECs were available from existing risk assessments, and the majority of these PNECs were derived from chronic toxicity data or simulated ecosystem studies (mesocosm) with rather low assessment factors. The limitations of this concept for risk assessment purposes are discussed. For the remainder, provisional PNECs (P-PNECs) were established from read-across models for acute toxicity to the standard test organisms Daphnia magna, Pimephales promelas and Selenastrum capricornutum. On the one hand, the prioritization revealed that about three-quarter of the 44 substances with MEC/PNEC ratios above ten were pesticides. On the other hand, based on the monitoring data used in this study, no risk with regard to the water phase could be found for eight of the 41 priority substances, indicating a first success of the implementation of the WFD in the investigated river basins.

Effect directed analysis of riverine sediments--the usefulness of Potamopyrgus antipodarum for in vivo effect confirmation of endocrine disruption.

In vivo tests are not commonly used in effect directed analysis (EDA) approaches. In the present study, a novel methodology was developed whereby Potamopyrgus antipodarum, which is known to be sensitive to endocrine disrupting compounds, was used as test organism. Field sediments from a polluted site in the north of Belgium were extracted and fractionated using three coupled and automatically switched normal-phase HPLC columns. Part of the fractions were spiked to artificial sediments and tested in a sediment contact test with P. antipodarum. The other part was used for an in vitro effect confirmation with the ER-LUC and anti-AR CALUX assays. Two of the six tested fractions stimulated the reproduction of the snails, while two others inhibited the reproduction. The fractions that caused an increase in reproduction also showed an increased estrogenic potency in the ER-LUC assay. Chemical analysis revealed that one of the most prominent compounds in those fractions was bisphenol-A, which has already been reported to have a stimulating effect on the reproduction of P. antipodarum by other authors. Due to the fact that previous studies have shown that this snail is also present in the field at this certain site, it was possible to directly link the results with effects that were observed in the field. This study indicates that effect directed analyses, supported by in vivo biotests, are very useful tools in order to identify the compounds that cause adverse effects on organisms or even population level.

In situ cage experiments with Potamopyrgus antipodarum--a novel tool for real life exposure assessment in freshwater ecosystems.

In situ experiments are an important tool within ecotoxicological research but there is a lack of suitable methodologies especially for freshwater invertebrate species. Within this study, a novel in situ methodology with Potamopyrgus antipodarum was developed. Snails were inserted into cages, made of Plexiglas measuring 7 × 9 × 7 cm(3) and fixed with stainless steel pins into the sediment at the relevant sampling sites. During the experiment physico-chemical properties of the water and concentrations of metals, PAHs and PCBs were measured in the sediment. The growth and survival of the snails was not affected, but the reproduction increased significantly at one of the most polluted sites. The increase in reproduction was neither correlated with physico-chemical parameters, nor with the concentrations of the different compounds, but maybe related to certain groups of estrogenic compounds. The study demonstrates the excellent applicability of this novel in situ test.

Modeling the chemical and toxic water status of the Scheldt basin (Belgium), using aquatic invertebrate assemblages and an advanced modeling method.

Self-Organizing Maps have been used on monitoring sites in several Scheldt sub-basins to identify the main aquatic invertebrate assemblages and relate them to the physico-chemical and toxic water status. 12 physico-chemical variables and 2 estimates of toxic risk were available for a dataset made up of a total of 489 records. Two of the five defining clusters reflecting a relatively clean environment were composed by very well diversified functional feeding groups and sensitive taxa. The cleanest assemblage was mainly linked to the sites from the Nete sub-basin. The three other clusters were inversely described with a dominance of oligochaetes and deposit feeders as well as a bad water quality. Such an analysis can be used to support ecological status assessment of rivers and thus might be useful for decision-makers in the evaluation of chemical and toxic water status, as required by the EU Water Framework Directive.

Characterizing field sediments from three European river basins with special emphasis on endocrine effects - A recommendation for Potamopyrgus antipodarum as test organism.

The assessment of endocrine disrupting potentials of field sediments has until now been mostly limited to classical chemical analysis, in vitro assays and in vivo bioassays performed with vertebrates. There is an urgent need for easy, cheap and reproducible invertebrate tests which may be applied in certain monitoring activities. Since the mudsnail Potamopyrgus antipodarum is known to be tolerant to natural stressors, but also sensitive to endocrine disrupting chemicals, it is very likely that this organism could be suitable for the assessment of endocrine effects of e.g. field sediments. Within this study the endocrine potential of sediments in three European river basins was assessed. The yeast estrogen screen (YES) and a sediment contact test with P. antipodarum were performed. Furthermore, analyses of physico-chemical properties and concentrations of heavy metals, PAHs, organotins, natural steroids and alkylphenols were done. In the sediment contact test, the reproduction of the snail was promoted by a part of the sediments. This phenomenon could not be explained by their physico-chemical properties. However, at some of those sites a high estrogenic activity was detected in the YES, leading to the assumption that endocrine disrupting compounds could be responsible for those effects. This assumption could be confirmed to some extent with partially high concentrations of xeno-estrogens (e.g. nonylphenol) at the certain sites. Our study demonstrates the applicability of the test with P. antipodarum for a variety of sediments and once again points out the need of suitable in vivo biotests for the risk assessment of field sediments.

Assessment of stream biological responses under multiple-stress conditions.

BACKGROUND, AIM AND SCOPE: Due to the numerous anthropogenic stress factors that affect aquatic ecosystems, a better understanding of the adverse consequences on the biological community of combined pressures is needed to attain the objectives of the European Water Framework Directive. In this study we propose an innovative approach to assess the biological impact of toxicants under field conditions on a large spatial scale. MATERIALS AND METHODS: Artificial Neural Network (ANN) analyses, focusing on impacts at the community level, were carried out to identify the relative importance of environmental and toxic stress factors on the patterns observed in the aquatic invertebrate fauna from the Scheldt basin (Belgium). RESULTS AND DISCUSSION: Our results show that the use of the backpropagation algorithm of the ANN is a promising method to highlight the relationship between environmental pollution and biological responses. This method allows the effects of chemical exposure to be distinguished from the effects caused by other stressors in running waters. Moreover, the use of an overall estimate for toxic pressure in predictive models enables the links between toxicants and community alterations in the field to be clarified. The ANN correctly predicts 74% of samples with an area under the curve of 0.89 and a Cohen's kappa coefficient of 0.64. Organic load, oxygen availability, water temperature and the nitrate concentration appeared important factors in predicting aquatic invertebrate assemblages. On the other hand, toxic pressure did not seem relevant for these assemblages, suggesting that the water quality characteristics were therefore more important than exposure to toxicants in the water phase for the aquatic invertebrate communities in the study area. However, we suggest that the high organic load encountered in the Scheldt basin may lead to an underestimation of the impact of toxicity.

Diagnosis of ecosystem impairment in a multiple-stress context--how to formulate effective river basin management plans.

The Water Framework Directive (WFD) of the European Union requires member states to attain a good ecological status for all water bodies by the year 2015. This implies that the bioecological protection endpoint itself is upfront, next to abiotic chemical quality standards, as tools to protect those endpoints. Within the requirements of the Directive, ecological status and abiotic conditions will be monitored extensively. Based on the analysis of the monitoring data, authorities are required to derive Programs of Measures (PoMs) for impacted sites. Optimization of these programs requires diagnosis, to provide site-specific or catchment-specific information on the causes of observed deviations from a good ecological status. This article shows one pilot analysis of monitoring data (Scheldt River, Belgium) compiled in the scope of the EU MODELKEY project. Ecological, ecotoxicological, and statistical models are combined to quantify local ecological impact magnitudes and to identify site-specific factors that are associated with those impacts. Results show significant ecological effects in terms of taxa loss at study sites, which are highly variable among sites, with variable combinations of environmental factors associated with those effects. The results of the diagnostic approach are discussed, which appear to be complementary to the assessment of chemical status required by the Directive. Both types of assessment are useful to assist in the derivation of optimized PoMs. In addition, it could be concluded that the acute toxic pressure parameter relates to reduced taxon abundance for more than half of the studied taxa and that this parameter relates to the fraction of taxa lost under field conditions. Finally, various lessons for the execution of monitoring programs are derived because the Scheldt (bio)monitoring data set has its weaknesses, although it can be seen as typical for current monitoring programs.

Toward an integrated assessment of the ecological and chemical status of European river basins.

Here, recommendations to improve ecological and chemical status assessments in accordance with the European Water Framework Directive (WFD) are made on the basis of experience gained from the MODELKEY project database, linking existing biological and chemical monitoring data of 3 case study river basins (Elbe, Scheldt, and Llobregat). The data analysis within and across river basins revealed major obstacles to be tackled, including scarcity of matching ecological and chemical monitoring sites for cause-effect relationships as well as a general lack of stressor-specific metrics for single biological quality elements (BQE) to enable a comprehensive risk assessment of all predominant stressors, including toxicity. An example of such a metric, which is recommended for the BQE of benthic macroinvertebrates, is the trait-based species-at-risk index (SPEAR) that correlated well with a respective measure for toxic stress, referred to as toxic units, based on simple mixture toxicity concepts. Surprisingly, the assessment of chemical status of a total of 695 monitoring sites for 2000 to 2004 showed that environmental quality standards (EQSs) were exceeded for at least 1 of the currently 41 priority pollutants (PPs) in 92% to 98% of the cases in all 3 of the river basins, which, according to definition, indicates potential effects on ecological status. A comparison of compliance with EQSs for 41 PPs with a respective effect threshold (derived for benthic macroinvertebrates) revealed that the rather conservative concept of chemical status is most likely not protective in all cases. Furthermore, to account for the many other compounds that are detected frequently in European surface waters and that may also have ecotoxicological effects, we introduced a provisional predicted no-effect concentration that is in accordance with the EQS methodology and is suggested to identify potential emerging compounds for which no or insufficient toxicity data exist. In conclusion, this study aims to support the implementation of the WFD by drawing conclusions from the analysis of heterogeneous data sets of various member states and by introducing new tools to move toward an integrated European assessment of ecological and chemical status.

Water quality indices across Europe--a comparison of the good ecological status of five river basins.

The European Water Framework Directive (WFD) requires the definition of near-natural reference conditions to determine the extent of water bodies' deviation from "good ecological status" caused by stress gradients. However, the classification of ecological quality depends on the assessment method applied and the stressor concerned. While assessment methods that are generally applicable would be favourable, many European countries employ the locally developed water quality metrics that assess the impact of organic pollution (including eutrophication) and the associated decrease in dissolved oxygen. These indices do not specifically address stress from organic toxicants, such as pesticides. The aim of this study was to examine the performance of presently used assessment methods to identify reference conditions of non-contaminated streams in five selected European river basins, covering the geographical region from Spain to Finland, as a crucial prerequisite to indicate toxic gradients. The analysis comprised the Belgium biotic index (BBI), the biological monitoring working party (BMWP) scoring system and the revised German saprobic index. For comparison, we included an adaptation of the recently developed SPEAR index. In two previous field studies, this metric highly correlated with measured pesticide gradients. In this study, SPEAR was the only indicator that was generally applicable to all monitoring data and capable of determining "high ecological status" of reference conditions in all basins. Thus, based upon previous and own results, the authors suggest the species at risk (SPEAR) index to be potentially useful as a European-wide index to address deviations from "good ecological status" due to organic toxicants and recommend it for consideration in integrated water-resource evaluations under the WFD.

MODELKEY. Models for assessing and forecasting the impact of environmental key pollutants on freshwater and marine ecosystems and biodiversity.

BACKGROUND: Triggered by the requirement of Water Framework Directive for a good ecological status for European river systems till 2015 and by still existing lacks in tools for cause identification of insufficient ecological status MODELKEY (http:// www.modelkey.org), an Integrated Project with 26 partners from 14 European countries, was started in 2005. MODELKEY is the acronym for 'Models for assessing and forecasting the impact of environmental key pollutants on freshwater and marine ecosystems and biodiversity'. The project is funded by the European Commission within the Sixth Framework Programme. OBJECTIVES: MODELKEY comprises a multidisciplinary approach aiming at developing interlinked tools for an enhanced understanding of cause-effect-relationships between insufficient ecological status and environmental pollution as causative factor and for the assessment and forecasting of the risks of key pollutants on fresh water and marine ecosystems at a river basin and adjacent marine environment scale. New modelling tools for risk assessment including generic exposure assessment models, mechanistic models of toxic effects in simplified food chains, integrated diagnostic effect models based on community patterns, predictive component effect models applying artificial neural networks and GIS-based analysis of integrated risk indexes will be developed and linked to a user-friendly decision support system for the prioritisation of risks, contamination sources and contaminated sites. APPROACH: Modelling will be closely interlinked with extensive laboratory and field investigations. Early warning strategies on the basis of sub-lethal effects in vitro and in vivo are provided and combined with fractionation and analytical tools for effect-directed analysis of key toxicants. Integrated assessment of exposure and effects on biofilms, invertebrate and fish communities linking chemical analysis in water, sediment and biota with in vitro, in vivo and community level effect analysis is designed to provide data and conceptual understanding for risk arising from key toxicants in aquatic ecosystems and will be used for verification of various modelling approaches. CONCLUSION AND PERSPECTIVE: The developed tools will be verified in case studies representing European key areas including Mediterranean, Western and Central European river basins. An end-user-directed decision support system will be provided for cost-effective tool selection and appropriate risk and site prioritisation.