Evaluation of a battery of biotests to improve waste ecotoxicity assessment (HP 14), using incineration bottom ash as a case study.

The assessment of waste ecotoxicity (hazardous property HP14 in the European Union) is fundamental for proper waste classification and safe application/disposal. Biotests are relevant for evaluating waste complex matrices, but their efficiency is crucial to encourage their adoption at the industrial level. This work aims at evaluating possibilities of improving the efficiency of a biotest battery previously suggested in the literature, regarding test selection, duration, and/or laboratory resources optimization. Fresh incineration bottom ash (IBA) was the case study. The test battery analysed included standard aquatic (bacteria, microalgae, macrophytes, daphnids, rotifers, fairy shrimp) and terrestrial (bacteria, plants, earthworms, collembolans) organisms. The assessment followed an Extended Limit Test design (three dilutions of eluate or solid IBA) and the Lowest Ineffective Dilution (LID-approach) for ecotoxicity classification. The results emphasize the importance of testing different species. It was also evidenced that tests with daphnids and earthworms may be shortened to 24 h; the miniaturization of tests is suitable as e.g. differential sensitivity of microalgae and macrophytes was captured with low variability; alternative testing kits can be used when methodological difficulties are found. Microalgae were more sensitive than macrophytes. Similar results were found for the Thamnotoxkit and daphnids test for eluates with natural pH, so the former may be used as an alternative. B. rapa was the most sensitive organism, suggesting that it may be tested as the only terrestrial plant species and that minimum test duration is appropriate. F. candida does not appear to add information to the battery. The differences in sensitivity of A. fischeri and E. fetida compared to the remaining species were not significant enough to exclude them from the battery. Thus, this work suggests a biotest battery to test IBA comprising aquatic tests - Aliivibrio fischeri, Raphidocelis subcapitata (miniaturised test), and Daphnia magna (24 h when clear deleterious effects are observed) or Thamnocephalus platyurus (toxkit) - and terrestrial tests - Arthrobacter globiformis, Brassica rapa (14 d), and Eisenia fetida (24 h). Testing waste with natural pH is also recommended. The Extended Limit Test design considering the LID-approach seems useful in waste testing, particularly for the industry, involving low effort, test material requirements, and few laboratory resources. The LID-approach allowed for differentiating ecotoxic from non-ecotoxic effects and captured different sensitivities between species. Ecotoxicological assessment of other waste may benefit from these recommendations, but caution should be taken given the properties of each waste type.

On the importance of longer-term exposure to stressors - A critical review and proposal for multigenerational testing in standard soil invertebrates.

Standard laboratory tests to describe the impact of stressors (most notably: chemicals) on organisms offer a good compromise between feasibility and outcome, i.e., they should be reproducible and provide robust results. However, these tests may underestimate the potential effects of prolonged exposures, particularly for persistent contaminants. Within the last years, we have observed an increase in studies aiming to target prolonged exposure, e.g., via an extended test duration or by multigenerational (MG) exposure. Seemingly, both reduced and increased impacts have been observed in these studies, but it is also clear that no unique test setup was used, and test designs vary widely among studies. To better describe long term effects, MG is a highly relevant aspect which deserves more consideration at various testing and assessment levels. Therefore, we conducted a literature review focusing on available studies performed with soil invertebrates, exposed to stressors for periods longer than in standard laboratory tests, i.e., full life cycle tests, as well as extensions to standard and MG tests. So far, it has been recommended that such studies should cover more than one generation, but this statement is probably too vague. In this contribution, we summarize and critically discuss the information provided in the literature, and we provide suggestions for future research. The currently available test results from long-term studies have produced clear evidence to recommend the implementation of long-term tests in existing regulatory testing requirements (e.g., for pesticides), in particular for persistent substances and also for delayed effects. Consequently, we recommend the inclusion of such longer exposure test designs (e.g., as annexes) in current OECD and ISO guidelines. However, when doing so, the long-term test designs proposed so far have to be critically adapted for a selected set of representative soil invertebrate test species.

Novel egg life-stage test with Folsomia candida - A case study with Cadmium (Cd).

Toxicity of pollutants is known to have a different impact depending on the organisms' life stage. Standard tests are often based on one life stage, i.e. effects could be underestimated. We aimed here to develop and optimize a test system using eggs of Folsomia candida (4-5 days) instead of the juveniles (10-12 days old) required by the OECD standard test guideline No. 232 (2009). Accordingly, the exposure time and thus the test duration was extended. Tests with "standard" juveniles (10-12 days old) and, adults (21 and 28 days old) were also performed. Cadmium (Cd) was used as test substance. The extension to the test guideline starts as follows: 1) synchronization of eggs in a thin soil layer on plaster of Paris, 2) selection of viable eggs, 3) burying these eggs in groups of 5 in soil. Afterwards, the test procedure will follow the standard procedure as described in the OECD standard test. Cadmium caused ca. 50% effects on reproduction at 60 mg Cd/kg soil dry weight (DW) when exposing juveniles or adults. There was no significant impact of Cd on the eggs, the hatching process or the latter life stages until ca. 250 mg Cd/kg DW (Cd is stable during this exposure period). Hence, Cd seems to affect reproduction before egg laying, i.e., during egg formation or during juvenile-adult stages. In order to clarify whether other chemicals do act in a similar way testing with different chemicals is highly recommended. Testing of different life stages does provide insight on the mechanisms and effects of contaminants and offers important insight.

Temperature influences the toxicity of deltamethrin, chlorpyrifos and dimethoate to the predatory mite Hypoaspis aculeifer (Acari) and the springtail Folsomia candida (Collembola).

In order to assess the influence of temperature on pesticide toxicity to soil fauna, specimens of the predatory mite Hypoaspis aculeifer and the springtail Folsomia candida were exposed in artificial soil spiked with different concentrations of three pesticides (dimethoate, chlorpyrifos and deltamethrin) at 20°C vs 28°C for the mites and 20°C vs 26°C for the springtails. All tests were carried out according to OECD guidelines. In the mite tests, the toxic effects of dimethoate and chlorpyrifos on survival was about two orders of magnitude more at 28°C than at 20°C. Mite reproduction decreased in the tests with chlorpyrifos and deltamethrin by about four to five orders of magnitude at 28°C than at 20°C. (EC5028°C =1.42 and 2.52mg/kg vs EC5020°C=6.18 and 10.09mg/kg) In the collembolan tests, the toxicity of dimethoate on survival was higher at 26°C than at 20°C (LC5026°C =0.17mg/kg vs LC5020°C =0.36mg/kg), while the opposite was detected for deltamethrin (LC5026°C =11.27mg/kg vs LC5020°C =6.84mg/kg). No difference was found in the test with chlorpyrifos. Effects of dimethoate and chlorpyrifos on reproduction were higher at 26°C than at 20°C (EC5026°C =0.11 and 0.018mg/kg vs EC5020°C =0.29 and 0.031mg/kg respectively), but in the case of deltamethrin the opposite was observed (EC5026°C =12.85mg/kg vs EC5020°C =2.77mg/kg). A preliminary risk assessment of the three pesticides at the two temperature regimes based on the Toxicity Exposure Ratio (TER) approach of the European Union, shows that in general there are few different outcomes when comparing data gained at different temperatures. However, in the light of the few comparisons made data gained in temperate regions should be used with caution in the tropics.

New approach to the ecotoxicological risk assessment of artificial outdoor sporting grounds.

Artificial surfaces for outdoor sporting grounds may pose environmental and health hazards that are difficult to assess due to their complex chemical composition. Ecotoxicity tests can indicate general hazardous impacts. We conducted growth inhibition (Pseudokirchneriella subcapitata) and acute toxicity tests (Daphnia magna) with leachates obtained from batch tests of granular infill material and column tests of complete sporting ground assemblies. Ethylene propylene diene monomer rubber (EPDM) leachate showed the highest effect on Daphnia magna (EC(50) < 0.4% leachate) and the leachate of scrap tires made of styrene butadiene rubber (SBR) had the highest effect on P. subcapitata (EC(10) = 4.2% leachate; EC(50) = 15.6% leachate). We found no correlations between ecotoxicity potential of leachates and zinc and PAH concentrations. Leachates obtained from column tests revealed lower ecotoxicological potential. Leachates of column tests of complete assemblies may be used for a reliable risk assessment of artificial sporting grounds.

A new OECD test guideline for the predatory soil mite Hypoaspis aculeifer: results of an international ring test.

Predatory mites are considered important biological indicators to assess potential effects of plant protection products. Toxicity testing of terrestrial mite species is required for authorisation of plant protection products in the European Union in cases where testing of leaf dwelling mites is not relevant, i.e. for defoliating herbicides, or when persistence of the chemical in soil is a concern. Since a standardised guideline for soil mites was not available in the past, an international working group developed a soil ecotoxicity test with the gamasid mite Hypoaspis aculeifer. This paper outlines the guideline development process and the principles of the protocol, and presents the results of an international ring test from which the validity criteria for the final guideline were derived. The protocol, which was published as OECD guideline 226 in 2008, is suitable for routine regulatory testing and can be used to generate data for risk assessment of soil inhabiting arthropods.

Boric acid as reference substance: pros, cons and standardization.

Boric acid (BA) has been successfully used as reference substance in some standard test guidelines. Due to the fact that previously selected reference substances present a significant risk to human health and/or are banned for environmental reasons, BA is being discussed for broader adoption in OECD or ISO guidelines. To provide input on BA data and contribute to the discussion on its suitability as a reference substance, in the present study BA was tested with two standard soil organisms, Enchytraeus albidus and Folsomia candida, in terms of survival, reproduction and avoidance. Additionally, published data on other organisms was analysed to derive the most sensitive soil dwelling invertebrate (hazard concentration-HC5). Results showed that BA affected the tested organisms, being two times more toxic for collembolans (LC50 = 96; EC50 = 54 mg/kg) than for enchytraeids (LC50 = 325; EC50 = 104 mg/kg). No avoidance behaviour occurred despite the fact that BA affects earthworms. Actually, it is the recommended reference substance for the earthworm avoidance test. Clearly, the suitable performance of BA in one species should not be generalized to other species. Absolute toxicity is not an important criterion for the selection of a reference substance, but it has been proposed that effects should occur within a reasonable range, i.e. <1,000 mg/kg. We could confirm, compiling previous data that for most soil invertebrates, the EC50 is expected to be below 1,000 mg/kg. From these data it could be derived that the most sensitive soil dwelling invertebrate (HC5, 50%) is likely to be affected (EC10) at 28 (8-53) mg H(3)BO(3)/Kg, equivalent to 4.6 (1.4-8.7) mg boron/kg.

Effects of the veterinary pharmaceutical ivermectin on soil invertebrates in laboratory tests.

As part of the risk assessment of veterinary pharmaceuticals, the potential impact of these chemicals on soil ecosystems has to be determined according to European law. However, almost no data from standardized laboratory tests are available. Therefore, in the EU FP6 ERAPharm, the effects of the widely used veterinary pharmaceutical ivermectin, an anthelminthic, were studied in chronic laboratory tests performed according to OECD (Organisation for Economic Co-operation and Development) and ISO (International Organization for Standardization) guidelines. In detail, three soil invertebrate species--the earthworm Eisenia fetida, the springtail Folsomia candida, and the predatory mite Hypoaspis aculeifer--were tested. The nominal concentrations of ivermectin mixed into the test substrate artificial soil was verified using residue analysis, which indicated that the test substance is persistent for at least up to 28 days. As expected when considering the mode of action of this substance, survival and reproduction of collembolans were clearly affected [LC(50) = 8.4 mg/kg soil dry weight (dw); NOEC(repro) = 0.3 mg/kg soil (dw)]. Predatory mites reacted less sensitively [LC(50) > or = 31.6 mg/kg soil (dw); NOEC(repro) = 3.2 mg/kg soil (dw)]. Earthworm survival and reproduction were affected in the same order of magnitude as the predatory mites [LC(50) > or = 10 mg/kg soil (dw); NOEC(repro) = 2.5 mg/kg soil (dw)]. These results are in good agreement with the few data known from nonstandardized tests for the same or related soil invertebrate species. The results of these tests indicate that the effects of ivermectin on soil invertebrates--in particular, collembolans--cannot be excluded at field-relevant concentrations, as determined in a risk assessment according to VICH guidelines. More sophisticated higher-tier tests (e.g., in multispecies or semifield test systems) are recommended in order to assess the potential risk more accurately.

Assessing the toxicity of contaminated soils using the nematode Caenorhabditis elegans as test organism.

In this study, nine uncontaminated reference soils and 22 contaminated soils with different physico-chemical properties and contamination patterns were tested with a standardized toxicity test, using the nematode, Caenorhabditis elegans, as test organism. Fertility, growth and reproduction of C. elegans in the soils were compared with the exposure in standard soil Lufa St.2.2. C. elegans showed 100% fertility and a very low variability of growth in the reference soils. Although, reproduction varied considerably between the various reference soils, validity criteria (>30 offspring per test organism) were met in all reference soils. Moreover, Lufa St. 2.2 turned out to be a suitable and representative control soil. In order to clearly classify the effects of the polluted soils on C. elegans, toxicity thresholds were derived for nematode fertility (20% inhibition), growth (10% inhibition) and reproduction (40% inhibition) on the basis of the test inherent variability (MDD=minimal detectable difference), as well as their variability between the uncontaminated reference soils (MTI=maximal tolerable inhibition). The contaminated soils showed clear toxic effects on the nematodes, whereas the toxicity was better correlated to organic than to heavy metal contamination in bulk soil. Interestingly, the results of the nematode toxicity test were not well correlated with those of tests with oligochaetes, collembolans and plants, performed with the same soils, showing that the results are not redundant. The toxicity test using C. elegans turned out to be suitable for testing the toxicity of field collected soils and might by a valuable addition to soil test batteries.

Ecotoxicological characterisation of 12 incineration ashes using 6 laboratory tests.

In the European Waste List (2000/532/EC as amended) the ash of municipal waste incineration is defined as a so called mirror entry. This waste can be classified as hazardous or non-hazardous depending on the content of hazardous substances and other risk properties. For the assignment of waste in mirror entries, 14 criteria are defined. One of the criteria is H14 "ecotoxic". In the presented study, the ecotoxicological potential of 12 ashes from different incineration plants has been assessed using biological test systems. The test battery included aquatic tests with eluates (algae, daphnids, and luminescent bacteria) and terrestrial tests with solid waste (plants, earthworms and bacteria). The test results revealed a clear ecotoxicological hazard potential for some of the MWI ashes. Despite the fact that fresh ashes were several times more toxic than aged ashes both groups did not differ consistently in terms of toxicity. The results show also that there is no correlation between the biological effects and the analyzed chemical compounds of the ash samples.

Environmental risk assessment of veterinary pharmaceuticals: development of a standard laboratory test with the dung beetle Aphodius constans.

The environmental risk assessment of veterinary pharmaceuticals for dung beetles is required if the substance is an anti-parasiticide for the treatment of pasture animals. However, the demonstration of the environmental safety of those substances for dung fauna is hampered by the fact that no standardized laboratory test system is currently available. Here a test system using the temperate dung beetle species Aphodius (Agrilinus) constans (Scarabaeidae: Aphodiinae) Duftschmidt is described. The survival of first instar larvae of A. constans exposed to a model substance, dimethoate, spiked into formulated (i.e. dried, formulated and re-wetted) or fresh dung was measured over a period of three weeks. Larvae performed better in formulated dung which also proved to be more suitable for mixing-in test substances homogenously. Dimethoate caused significant larval mortality with LC50 values within a range of 1.3-2.8 mg a.s./kg dung (d.w.), depending on the dung type. Based on the data presented here, it is recommended to incorporate this new test system in the risk assessment process for veterinary pharmaceuticals. However, an international ringtest should to be performed beforehand to ensure adequate validation of the method.

The effect of tributyltin-oxide on earthworms, springtails, and plants in artificial and natural soils.

Chemical bioavailability in Organisation for Economic Co-operation and Development (OECD) artificial soil can contrast with bioavailability in natural soils and produce ecotoxicologic benchmarks that are not representative of species' exposure conditions in the field. Initially, reproduction and growth of earthworm and Collembolan species, and early seedling growth of a dicotyledonous plant species, in nine natural soils (with a wide range of physicochemical properties) and in OECD soil were evaluated. Soils that supported reproduction and growth of the test species were then used to investigate the toxicity of tributyltin-oxide (TBT-O). Natural soils caused greater toxicity of TBT-O to earthworms (EC(50) values varied from 0.5 to 4.7 mg/kg soil dry weight [dw]) compared with toxicity in OECD soil (EC(50) = 13.4 mg/kg dw). Collembolans were less sensitive to TBT-O than earthworms in natural soils, with EC(50) values ranging from 23.4 to 177.8 mg/kg dw. In contrast, the toxicity of TBT-O to collembolans in OECD soil (EC(50) = 104.0 mg/kg dw) was within the range of EC(50) values in natural soils. Phytotoxicity tests revealed even greater difference between the effects in natural soils (EC(50) values ranged from 10.7 to 189.2 mg/kg dw) and in OECD soil (EC(50) = 535.5 mg/kg dw) compared with results of the earthworm tests. Studies also showed that EC(50) values were a more robust end point compared with EC(10) values based on comparisons of coefficients of variation. These results show that toxicity testing should include studies with natural soils in addition to OECD soil to better reflect exposure conditions in the field.

Effects of different soil types on the Collembolans Folsomia candida and Hypogastrura assimilis using the herbicide Phenmedipham.

Soil ecotoxicology studies are usually performed in standard soils such as Organization for Economic Cooperation and Development artificial soil or LUFA ST. 2.2, a natural soil. When assessing the toxic effects in the environment, soil properties are often different from those in standard soils, which might lead to different exposure situation for the test species and therefore to misleading conclusions. Selected to cover a broad range of properties and based on the Euro-Soils concept, 17 different soils were studied regarding their suitability to two tes species: Folsomia candida and Hypogastrura assimilis (Collembola). In reproduction tests, the test species reacted differently to the soils. F. candida was less affected by soil properties: 500 to 1200 juveniles/vessel were found in untreated soils (i.e. controls). These differences can be attributed to normal interindividual variability. H. assimilis showed a significant correlation with maximum water-holding capacity and also a tendency to lower the reproductive output in soils with a low pH (<4). Therefore, some soils were revealed to be inappropriate for tests with H. assimilis. In the main tests, the effect of the reference test substance Phenmedipham (formulation Betosyp) was studied in those soils where sufficient reproduction was determined beforehand. Clearly, the chronic end point was more sensitive than survival when testing Phenmedipham. In H. assimilis, because of high variability and low effects of tested dosages, no conclusions could be drawn. In F. candida, different soils caused different toxic effects: Juveniles preferred soils with high C-to-N ratios. Higher microbial activity might support a quicker metabolization of the test substance. In general, the toxic response is caused by a synergistic action of several soil properties with each of them exerting an effect too small to be clarified with the available set of data.

The use of earthworms in ecological soil classification and assessment concepts.

Without doubt, earthworms are the most important soil invertebrates in most soils worldwide, in terms of both biomass and activity. Several species are even considered to be ecosystem engineers. Earthworms are also known to influence soil structure, soil chemistry, and, in particular, processes like organic matter decomposition. In addition, standardized sampling methods are available and their taxonomy is well known (even the first PC-aided keys have been developed). For these reasons, earthworms were recognized as a part of ecological classification and assessment schemes early on. However, due to the relatively small number at many sites, they have to be part of a battery approach. By use of examples from The Netherlands (biological indicator of soil quality) and Germany (soil biological site classification), the practicability of the use of earthworms is demonstrated in determining the influence of different anthropogenic land use forms. In these cases, the structure of the earthworm community, as well as their abundance and biomass, were used as endpoints.

The use of enchytraeids in ecological soil classification and assessment concepts.

In many soils worldwide, enchytraeids (Annelida: Oligochaeta) play a major role in soil functions like the decomposition of organic matter. In addition, standardized sampling methods are available and taxonomic as well as ecological knowledge is rapidly increasing (in particular, ecological profiles of the most abundant species). For these reasons, enchytraeids were recognized as a part of ecological classification and assessment schemes. In this contribution, the most important of these schemes are presented and it is shown that enchytraeids are most valuable as part of a battery approach. By use of examples from The Netherlands (BISQ), and Germany (BBSK), the practicability of the use of enchytraeids is demonstrated in identifying the influence of different anthropogenic land use forms. In these cases, the structure of the enchytraeid community and their abundance and biomass were used as endpoints. However, automation of the identification process can still be improved.

Leaching behaviour of pharmaceuticals in soil-testing-systems: a part of an environmental risk assessment for groundwater protection.

The leaching behaviour of six selected pharmaceuticals was tested in different soils. Leaching experiments are a part of environmental risk assessment to estimate the distribution and fate of these pharmaceuticals in the environment. Based on the results of this assessment their mobility in soil and their potential to contaminate groundwater was evaluated. When assessing the leaching behaviour of these compounds, the influence of the properties (e.g. grain size distribution, pH, Corg) of different soils has to be taken into account. The test results indicated that the leaching potential found could be rated as low for diazepam, ibuprofen, ivermectin and carbamazepine. Therefore, contamination of the groundwater with these substances seems to be unlikely if the groundwater level is covered with sufficient layers of the soils investigated. Clofibric acid and iopromide were very mobile under the experimental conditions and thus, groundwater contamination would be possible if the soil is exposed to these pharmaceuticals, i.e. wastewater irrigation. These results are more or less in agreement with groundwater monitoring data found in the literature for ibuprofen and diazepam which were in general not present in groundwater, while clofibric acid and iopromide were frequently detected. However, a discrepancy was found for carbamazepine, since it occurs very often in groundwater. This discrepancy might be explained by the fact that the leaching tests were performed with soil, whereas in reality the groundwater contamination occurs mainly over river sediments and sub soil from receiving waters.

Validating the enchytraeid reproduction test: organisation and results of an international ringtest.

In this paper the experiences concerning the organisation and results of the enchytraeid reproduction test (ERT) ringtest are summarised (for details see J. Römbke, T. Moser, Organisation and Performance of an International Ringtest for the Validation of the Enchytraeid Reproduction Test, vols. I and II. UBA-Texte 4/99, 1999, 150, 223 pp). The performance of this ringtest was in line with requirements published by OECD. It was sponsored by the German Federal Environmental Agency (Umweltbundesamt; UBA, Berlin). The UBA was also actively engaged (together with the European Chemicals Bureau, Ispra) in a scientific task force, which consisted of seven scientists experienced in terrestrial ecology and ecotoxicology. 29 institutions (mainly from universities and contract research laboratories) from 15 countries of Europe and North America participated actively in the ringtest. The co-ordinating laboratory centrally distributed the test chemicals, guidance papers and test organisms to all participants. In addition, several training courses were organised. Most participants performed two tests with the fungicide Carbendazim and two with 4-nitrophenol. For each chemical, one test was designed according to an ECx approach and the other according to an NOEC approach. Several aspects of the test protocol were modified based on the experiences gained during the ringtest. A major aspect of the project was the detailed statistical evaluation of the test results (for details see A. Weyers, J. Römbke, T. Moser, T. Ratte, Results of and statistical implications from the enchytraeid reproduction ringtest, 2001, submitted), leading to recommendations for an optimised ecotoxicological test design. A total of 92 tests were performed according to the protocol which is among the highest number ever performed in a ringtest. About 72% met the validity criteria (control mortality < 20%, > 25 juveniles per 10 adults). The effects of the two test chemicals on enchytraeid reproduction were in the range expected from data in the literature on oligochaete toxicity. However, statistical evaluation of the data was sometimes difficult as a result of the high variability in the number of juveniles. This variability was caused by several reasons, including hormesis effects or lack of experience of some participants. Comparison of the data from the NOEC and ECx approaches pointed to a clear advantage in favour of the latter. In most cases EC10 values were lower than the NOEC values determined in the same test. For details see A. Weyers et al. (loc. cit.). Reproducibility of the test data and practicability of the ERT ringtest were of the same order of magnitude as other ringtests recently performed. As a result of the ringtest, the ERT draft guideline was significantly improved. The new version is currently being standardised by OECD, ISO and ASTM.