The use of the multivariate Principal Response Curve (PRC) for community level analysis: a case study on the effects of carbendazim on enchytraeids in Terrestrial Model Ecosystems (TME).

The effects of the fungicide carbendazim (formulation Derosal) on enchytraeids were determined in Terrestrial Model Ecosystem (TME) tests. TMEs consisted of intact soil columns (diameter 17.5 cm; length 40 cm) taken from three grassland sites (Amsterdam (The Netherlands), Bangor (Wales, England) and Flörsheim (Germany)) or an arable site (Coimbra (Portugal)). Results for each TME site were evaluated using the multivariate Principal Response Curve (PRC) method. The resulting No-Observable Effect Concentrations (NOECs) for the community were compared with the NOECs generated by univariate statistical methods. Furthermore, the EC(50)s (median effect concentrations) for the three taxa with the highest taxon weights determined by the PRC were compared with EC(50)s for the other endpoints. In eight out of 16 cases the PRC revealed the lowest NOEC for the enchytraeid species community. The lowest EC(50)s with the closest 95% confidence limits were calculated for the abundance of the three taxa with the highest taxon weights identified by the PRC. The EC(50)s ranging from 0.19-2.79 mg carbendazim/kg soil are similar to values from laboratory toxicity studies reported in the literature. Therefore, PRC is a useful instrument to analyse microcosm and mesocosm experiments; it allows for determination of NOECs for the species community (NOEC(community)), the evaluation of the taxa with the most pronounced treatment-related decrease in abundance and of the calculation of meaningful EC(50) values for those. The resulting NOEC(community) and EC(50) values offer a comprehensive tool for the risk assessment of chemicals at the ecosystem level.

Derivation of soil values for the path 'soil-soil organisms' for metals and selected organic compounds using species sensitivity distributions.

BACKGROUND, AIMS AND SCOPE: According to the German Federal Soil Protection Act, the natural function of soil as a habitat for human beings, animals, plants and soil organisms is, among other things, to be protected by deriving soil values for important chemicals regarding their amounts in the environment, their persistence and/or their toxicity. This contribution presents the results of the mathematical derivation of such values for nine metals and ten organic substances from soil ecotoxicological effect values available in the literature for microbial processes, plants and soil invertebrates. MATERIAL AND METHODS: Ecotoxicological data were mostly extracted from published papers and reports and had to originate from valid studies that were performed according to internationally standardised guidelines (e.g. ISO) or were otherwise well documented, plausible and performed according to accepted laboratory practice. As test results, both structural (i.e., effects on mortality, growth or reproduction) and functional (i.e., effects on microbial activity or organic matter breakdown) parameters were included. The derivation of soil values was performed using the distribution based extrapolation model (DIBAEX) and EC(50)s (Effective Concentration) as input data. RESULTS: For 19 compounds, soil values could be calculated. In 18 of these 19 cases clear laboratory ecotoxicological effects (i.e., EC50 values) below the calculated soil value have been found in the literature. DISCUSSION: In those few cases where a comparison with field studies is possible, effects have been observed in the same order of magnitude as the calculated soil values. A comparison with other similar approaches confirmed the plausibility of the calculated values. CONCLUSIONS: The DIBAEX-method is a feasible and widely accepted method for deriving soil values from ecotoxicological input data. Data availability was already satisfactory for some substances, but other substances, especially organics, were only poorly covered. The soil values presented here were based on EC50 input data. However, depending on the protection level aimed at by using soil values in legislation, it might be appropriate to use other input data such as NOECs in the derivation process. RECOMMENDATIONS AND PERSPECTIVES: It is recommended to generate an appropriate number of data for further relevant substances by means of a test battery or multi-species approaches such as terrestrial model ecosystems. These tests should also consider the influence of the bioavailability of substances. A final recommendation for legally binding soil values demands a plausibility check of the mathematically derived values. This should include a comparison with natural background concentrations, soil values for other pathways and soil values used in legislation of other countries. Finally, expert judgement always has to be considered.

New screening test to predict the potential impact of ivermectin-contaminated cattle dung on dung beetles.

According to European Union recommendations, a test method has been developed to evaluate the effects of veterinary pharmaceuticals on dung feeding insects. This test method was evaluated with the dung beetle Aphodius constans by using fecal residues of ivermectin after a pour-on administration. Dung of different age (and thus containing different concentrations of ivermectin) as well as mixtures of highly-contaminated spiked dung with untreated control dung were studied in five test runs in two laboratories. The concentration of ivermectin (active substance; a.s.) in the dung samples was verified analytically. The main test endpoint was the survival of first instar larvae. The LC50 using dung directly obtained from treated cattle ranged from 470 to 692 microg a.s. kg(-1) dung (dry weight; d.w.) and 67 to 97 microg a.s. kg(-1) dung (fresh weight; f.w.). Using mixtures, the outcome of two tests was almost identical: 770 to 781 microg a.s. kg(-1) dung (d.w.); 109 to 132 microg a.s. kg(-1) dung (f.w.). In comparison to the LC50 values obtained when ivermectin was spiked in control dung at several concentrations (LC50 880-985 microg a.s. kg(-1) dung (d.w.)), the LC50 values were again very similar. Three conclusions can be drawn from these results. The proposed test method seems to be robust and allows for the initiation of an international validation process (including ringtesting). Because of only small differences found in tests in which the test substance was spiked into control dung and those in which dung from treated cattle was applied, the use of a standard test method is proposed. The effects of ivermectin on ecologically relevant dung beetles obtained in a standardised test method reflect the results from field studies and are in the range of environmentally relevant concentrations.

Improvement of the applicability of ecotoxicological tests with earthworms, springtails, and plants for the assessment of metals in natural soils.

The environmental risk assessment of metals in the soil compartment is based mainly on tests performed in Organization for Economic Cooperation and Development (OECD) artificial soil, but ecologically, the use of natural soils would be more relevant. In this contribution, the reproduction and growth of three standard species (an earthworm, a collembolan, and a dicotyledonous plant, respectively) was evaluated in nine natural soils (covering a wide range of pH values, organic matter content, texture, and so on) and in OECD artificial soil. Afterward, the effects of the model chemical zinc nitrate were assessed in all soils that were identified as being suitable for these species. The test results indicate that the toxicity of zinc nitrate can be higher by a factor of approximately four compared to artificial soil for invertebrates (earthworms and collembolans), whereas plants are only slightly more sensitive in some natural soils than in artificial soil. When comparing the different endpoints, it could be confirmed that the median effective concentration (EC50) is the most robust compared to the highly uncertain 10% effective concentration. Decreasing toxicity of zinc nitrate to collembolans was significantly correlated with an increase in soil pH but not with cation exchange capacity (CEC) or organic carbon (OC) content. No significant correlation was found between the toxicity of zinc nitrate to earthworms or plants and soil pH, CEC, or OC content. Possible consequences of the results are discussed, such as the testing of natural soils in addition to the OECD artificial soil or the inclusion of an additional safety factor to use the EC50 in current risk assessment schemes focusing on no-observed-effect concentrations.

Toxicity of four veterinary parasiticides on larvae of the dung beetle Aphodius constans in the laboratory.

The environmental risk assessment of veterinary pharmaceuticals for dung beetles is strongly hampered because no standardized test method is available so far. Therefore, a test with the temperate dung beetle species Aphodius constans was developed. The survival of beetle larvae was determined after exposure to four veterinary parasitical pharmaceuticals (ivermectin, moxidectin, dicyclanil, and praziquantel) representing different treatment regimes, modes of action, and effect levels. The test was performed in the laboratory (three week duration) with fresh dung, as well as formulated (dried, ground, and rewetted) dung as test substrate (i.e., at least one range-finding test, two definitive test runs per pharmaceutical). Ivermectin was the most toxic substance (median lethal concentration [LC50] = 0.88-0.98 mg of active substance per kilogram of dung dry weight [mg a.s./kg dung (dry wt)] followed by dicyclanil (LC50 = 1.5-6.0 mg a.s./kg dung [dry wt]) and moxidectin (LC50 = 4.0-5.4 mg a.s./kg dung [dry wt]), whereas praziquantel showed very low toxicity (LC50 > 1,000 mg a.s./kg dung [dry wt]). The toxicity in fresh and formulated dung differed by a factor of between 1.1 and 4. The comparison with literature data on toxic effects of these substances on dung beetles in the laboratory or in the field is difficult because no results for praziquantel and dicyclanil have been published so far. With the use of data from ivermectin and moxidectin, the test results are on the same order of magnitude as those known from other studies. On the basis of the experiments reported here, it is recommended that this test be standardized in an international ring test so that it can be incorporated into the risk assessment process as described in the respective international guidelines for the registration of veterinary pharmaceuticals.

Effect of soil properties and aging on the toxicity of copper for Enchytraeus albidus, Enchytraeus luxuriosus, and Folsomia candida.

In the present study, the effect of the heavy-metal salt copper chloride (CuCl2.2H2O) in soils freshly spiked (3 d) and aged (70 +/- 10 d; mean +/- SD) was studied in the test species Enchytraeus albidus, E. luxuriosus, and Folsomia candida. Up to nine soils were used: Besides the Organisation for Economic Co-operation and Development (OECD) artificial soil and the Agricultural Testing and Research Agency (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany) 2.2 natural standard soils, the others were selected based on the EURO Soil approach, taking into account the effect of different soil parameters (pH, organic matter, grain size distribution, and carbon to nitrogen ratio). Additionally, the effect of the chloride ions was studied separately. The results revealed the following: First, a soil effect was observed; for example, in F. candida, median effective concentrations (EC50s) varied between 262 mg/kg in a sample from the same site as the original EURO Soil 5 soil and greater than 1,000 mg/kg in OECD soil. Second, an aging effect was observed, mainly in F. candida. For example, toxicity of offspring survival was increased twofold in the OECD soil and approximately eightfold with aging in the EURO Soil 7 soil, whereas the enchytraeid species did not react differently after aging. Third, an effect of chloride ions on reproduction of the animals was found; however, this effect was independent of the aging period. Fourth, species variation was seen in terms of sensitivity (EC50), decreasing in the following order: E. luxuriosus > E. albidus >> F. candida. Differences in toxicity of offspring survival between enchytraeids and F. candida might be explained by the different routes of uptake.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: effects of carbendazim on nematodes.

The effects of the fungicide carbendazim (applied in the formulation Derosal) on nematodes was determined in Terrestrial Model Ecosystem (TME) tests and field-validation studies. TMEs consisted of intact soil columns (diameter 17.5 cm; length 40 cm) taken from a grassland or, in one case, from an arable site. The TMEs were taken from the same site where the respective field study was performed. The tests were performed in Amsterdam (The Netherlands), Bangor (Wales, England), Coimbra (Portugal) and Flörsheim (Germany). Differences concerning nematode overall abundance, the number of nematode families, the trophical structure of the nematode cenosis and the maturity index (MI) were not found between the controls of TME tests and the respective field-validation studies. Effects caused by the chemical treatment, however, were observed on the number of nematode families, on the trophical structure of the nematode cenosis and on the maturity index (MI). Effects on the relative abundance of the omnivorous nematodes were most pronounced, whereas the overall nematode abundance was not affected. The observed effects appear not to differ between the TME tests and the respective field-validation studies. All measurement endpoints in both TMEs and field, showed rather large variations. Therefore, NOEC-values were often equal or higher than the highest treatment level and EC50-values were calculated only for the omnivorous nematodes. NOEC- and EC50-values derived from the TME ring-test and the field-validation study indicate that the reproducibility (i.e. the variation between the partners) was reasonable, although different soils from different sites were used. The EC50-values determined for the effect of carbendazim on the relative abundance of the omnivorous nematodes ranged between 0.93 and 7.24 kg a.i./ha (1.24-9.63 mg/kg). Due to the higher sensitivity of the relative abundance of the omnivorous nematodes compared to the other measurement endpoints it is recommended to use this parameter as the main endpoint.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: effects of carbendazim on nutrient cycling.

The effect of the fungicide carbendazim (applied in the formulation Derosal) on nutrient cycling in soil was determined in Terrestrial Model Ecosystem (TME) tests and corresponding field-validation studies, which were performed in four different countries (United Kingdom, Germany, Portugal, and The Netherlands). The tests used different soil types, and lasted for 16 weeks. On three of the four sites, grassland soils were used while the fourth site had an arable soil. TMEs consisted of intact soil columns (diameter 17.5 cm; length 40 cm) and were taken from the site where the field study was performed. In the first series of TME tests, carbendazim applied at dosages unto 77.8 kg a.i./ha did not affect sulfate and phosphate concentrations in the top 15 cm soil layers. These nutrients were therefore no longer included in the second series of TME tests and the field-validation studies. Ammonium concentrations in the top soil layers of TMEs and field plots, and in the leachates of the TME columns did not show any effect of carbendazim treatment. Nitrate concentrations in soils and leachates did show some reduction at the highest treatment levels (77.8 kg a.i./ha in the first TME tests, 87.5 kg a.i/ha in the second TME tests and the field-validation studies). Since nitrate concentrations in both soils and leachates were correlated with soil moisture content, these effects could however, mainly be attributed to variations in soil moisture contents, and in some cases also indirectly to effects on earthworm activity. It is concluded that carbendazim, even at dosages as high as 87.5 kg a.i./ha, does not have a significant impact on soil nutrient cycling processes. Nutrient levels in TME tests and the field-validation studies generally showed similar patterns, thus confirming the predictive value of the TME test system.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: conceptual approach and study design.

During spring and summer 1999 a ring-test and field-validation study with an open, intact Terrestrial Model Ecosystem (TME) was conducted at four different European sites (Amsterdam, The Netherlands; Bangor, U.K.; Coimbra, Portugal; Flörsheim, Germany). The objective of the study was to establish a standardised method which allows the impact of chemical stressors on terrestrial compartments at ecosystem level to be investigated and possible uses of such data in existing Environmental Risk Assessments (ERAs) for chemicals to be evaluated. This issue of Ecotoxicology presents in a series of papers the results of the TME ring-test and field-validation study. Additionally, results derived from an open-homogeneous terrestrial microcosm (Integrated Soil Microcosm, ISM) are included in this series as a separate paper. In this first paper of the series background information on the planning and organisation of the study are given. The conceptual approach and the design of the study with TMEs are briefly outlined, based on the scientific discussion on the use of terrestrial microcosms in ecology and applied environmental sciences during the last 25 years. Further, some suggestions are presented on the selection of measurement endpoints to quantify structural and functional aspects of terrestrial ecosystems. Finally, the main results of the TME-study are summarised and conclusions are drawn on the technical feasibility of TMEs, their comparability with field studies and the potential use of TMEs in ERA.