Behavioural endpoints in earthworm ecotoxicology

Background, Aims and Scope  Endpoints in earthworm ecotoxicology scheduled in guidelines are mortality and reproduction rates. However, not only the direct influence of pollutants on population parameters but also changes in behaviour such as substrate avoidance can have an important impact on soil ecosystems. In practice two different avoidance response tests are applied in earthworm ecotoxicology: (i) a six-chamber test system and (ii) a two-chamber test system. Both avoidance response-test systems were compared to establish their respective advantages and disadvantages in order to advance the standardisation of behavioural tests. The earthworm avoidance-response tests were applied in addition to the standard acute and chronic earthworm toxicity tests (ISO 11268) in order to compare the sensitivity of the test endpoints. Methods  Test substrates were contaminated with crude oil and 2,4,6-Trinitrotoluene (TNT), respectively. The test species wasEisenia fetida. The earthworms were exposed to the contaminated substrates and their mortality (14 d), reproduction rates (number of cocoons after 28 d, juvenile hatching after 56 d), and substrate preference (48 h) determined. Results and Discussion  Whereas 1000 mg/kg TPH (Total Petroleum Hydrocarbons) did not show any lethal effects, 100% mortality occurred in soil with comparable TNT concentration. The acute tests consistently produced the highest effect concentrations whereas reproduction and substrate avoidance were the more sensitive test parameters. Both behavioural test systems, when compared, showed similar substrate avoidance after an incubation time of 48 h. The six-chamber test system provides the potential to test six different substrates/concentrations at one time. It was observed, however, that earthworms did not migrate among all test chambers within a test unit in order to select the most appropriate substrate. Orientation was observed only between directly neighbouring test compartments, which complicates the interpretation of the test results. Conclusion  Substrate avoidance and reproduction variables were clearly more sensitive test endpoints than mortality. Therefore avoidance-response tests proved to be useful test methods in detecting effects of sublethal concentrations of pollutants on earthworms. The test duration of the avoidance tests is much shorter compared to the standard acute and chronic earthworm toxicity tests, which makes them a quick screening tool for identifying potential soil toxicity. Both avoidance-response test systems showed comparable results regarding the test sensitivity. Nonetheless, the incomplete substrate use in the six-chamber avoidance test due to the reduced migration possibilities (orientation only to neighbouring chambers) might reduce the distinctness of test results as it allows only reliable information on the most avoided and therefore most toxic substrate but not on 1 a clear dose-response pattern. Thus, to gain valid results, the number of replicates and the arrangement of the different substrates must be adopted. The two-chamber test system is less time-consuming due to easy handling and test results can be quantified more easily. Recommendations and Outlook  In consequence of the better validity of test results, lower expenses for test containers and less time for handling, the use of the two-chamber system is preferred over the six-chamber test system to assess the toxicity of polluted soil. Because of the ecosystem consequences of behavioural effects and the fact that avoidance response tests can reveal the toxic potential of pollutants in low concentrations, such tests should be included into ecotoxicological test protocols.     

Comparison of the effects of zinc nitrate-tetrahydrate and tributyltin-oxide on the reproduction and avoidance behavior of the earthworm Eisenia andrei in laboratory tests using nine soils

The aim of this contribution was to evaluate whether the sensitivity of the earthworm avoidance test is comparable to that of the earthworm reproduction test (standard test guidelines for both are available). The objective was to determine if relatively simple short-term tests are useful as an initial screening step for the environmental risk assessment (ERA) of potentially contaminated soils prior to performing long-term, elaborate tests. Therefore, the effects of two model substances, zinc nitrate-tetrahydrate and tributyltin-oxide (TBT-O) on the reproduction and the avoidance behavior of the earthworm Eisenia andrei were compared using Organization for Economic Co-operation and Development (OECD) artificial soil as well as eight natural soils, including LUFA St. 2.2 soil, covering a wide range of pH-values, organic matter content and texture. Almost all tests fulfilled the validity criteria defined in the two standard guidelines published by the International Organization for Standardization (ISO), despite the fact that the properties of natural soils differed clearly from those of OECD artificial soil. The median effective concentration (EC50) values estimated for zinc nitrate-tetrahydrate in 48-h avoidance tests and in 56-day reproduction tests were often similar when using the same soil (seven out of nine soils). However, in the case of TBT-O, the outcome was more complex: in two test soils the avoidance EC50 values were by a factor >3 higher than the reproduction EC50 values. In one of the test soils it was the other way around and the remaining soils showed comparable EC50 values in both tests. Summarizing the results obtained here and according to experiences reported in the available literature, the earthworm avoidance test may be recommended as an initial screening tool in the ERA of potentially contaminated soils. However, further research is needed to understand the factors that contribute to the difference in sensitivity between reproduction and avoidance tests.     

Avoidance test with<Emphasis Type="Italic">Eisenia fetida</Emphasis> as indicator for the habitat function of soils: Results of a laboratory comparison test

Intention, Goal, Scope, Background. The habitat function of soils is often assessed using the reproduction test withEisenia fetida. As this test is rather labour-intensive, an alternative is needed which is less cost-intensive in terms of duration and workload, but gives reasonable results. The avoidance test withE. fetida is a suitable screening test meeting these criteria. However, before a novel test system can be generally recommended it has to be ensured that comparable results are acquired from different laboratories on the basis of the respective test guideline.

Objective

The avoidance test withE. fetida was performed as laboratory comparison test. The results were compared with those of the earthworm acute and reproduction tests carried out with the same soils.

Methods

The three tests were performed by three laboratories using eight contaminated soils and three control soils. The contaminated soils were mixed with the control soils to obtain different concentrations of the contamination.

Results and Discussion

The results of the avoidance test show that despite the partially considerable standard deviations a 72% agreement in the assessment of soils was reached with a toxicity criterion of >80% avoidance response. The sensitivity is at least that for the reproduction test and considerably surmounts the sensitivity of the acute test.

Recommendation and Outlook

The avoidance test is considered to be a suitable screening test for assessing the habitat function of soils. The whole test design could be improved by reducing the standard deviations among parallel test batches. With regard to standardization it is recommended to use control soils which have the same properties as the soils described in respective guidelines (e.g. ISO 11269-2, OECD 216, 217).

     

Duration of the standard earthworm avoidance test: Are 48 h necessary?

Ecotoxicological tests are often recommended for the environmental risk assessment (ERA) of contaminated soils. In comparison to chemical residue analysis that focuses on individual contaminants, ecotoxicological tests are able to integrate the effects of the overall contamination, including interactions between individual contaminants, as well as between the contaminants and the various soil properties. However, their use is limited by the fact that the most sensitive tests are chronic tests such as the earthworm reproduction test which lasts 56 days. In order to promote the use of ecotoxicological tests for the routine assessment of contaminated test soils, the usage of short-term earthworm avoidance tests were investigated in the German project ERNTE. According to the International Organization for Standardization (ISO) guideline no. 17512-1, such a test has a duration of just 48 h which, although less than chronic tests, is still quite long in comparison to modern methods of chemical analysis whereby results are often available within 24 h (“on-site analysis”). Therefore, we assessed the effects of shortening the duration of the earthworm avoidance test from 48 to 24 h on the resulting ecotoxicological data. Eight uncontaminated natural soils and 22 contaminated natural soils containing a wide range of chemicals were tested concurrently for 24 and 48 h. Additionally, seven of the uncontaminated natural soils (one was excluded due to its low pH) plus Organization for Economic Co-operation and Development (OECD) artificial soil or LUFA (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany) St. 2.2 soil were spiked with two model chemicals (zinc nitrate-tetrahydrate and tributyltin-oxide (TBT-O)) and tested accordingly. It was also investigated whether the results would differ when using either standard LUFA St. 2.2 or OECD artificial soil as a control when contaminated natural soils were tested. Statistical analysis of the data indicates that a decrease in test duration in general did not result in a different assessment of the test soils. In view of the fact that an ERA of contaminated soils is increasingly starting with an on-site analysis (i.e. data are available within 24 h) it is recommended to change the existing ISO guideline, i.e. to decrease the test duration from 48 to 24 h. In doing so, the use of a multi-concentration design increases the robustness of the test results. Both OECD and LUFA St. 2.2 soils are equally suitable as controls.     

Earthworm avoidance test for soil assessments

For ecotoxicological assessments of contaminated or remediated soils pointing to the habitat function of soils for biocenoses, standardized tests with earthworms (acute test, reproduction test) are available among others. Tests used for routine applications should be sensitive and indicate impacts on test organisms after short test periods. The usually applied earthworm tests do not satisfactorily fulfil these criteria. Therefore, in the present work, a behavioural test with earthworms (test criterion: avoidance) was investigated in detail using uncontaminated, artificially contaminated and originally contaminated soils. It was demonstrated that the avoidance behaviour is primarily determined by pollutants, and not by chemical-physical soil properties. The sensitivity of the presented test reaches the sensitivity of established tests. For waste sites, a considerably higher sensitivity was determined. An avoidance behaviour of at least 80% of the worms leaving the soil to be assessed is proposed as a criterion for toxicity.     

Technical Recommendations for the Update of the ISO Earthworm Field Test Guideline (ISO 11268-3) (5 pp)

Background, Aim and Scope   Background, Aims and Scope. The earthworm field test firstly was developed by the German Federal Biological Research Centre for Agriculture and Forestry (BBA) for testing of pesticides and later internationally standardised by the International Organization for Standardization (ISO) as a tool for characterizing soil quality. It is mainly used for the assessment of effects of pesticides on earthworms under field conditions. Growing experience has shown that the practical performance of the test can be improved. Main Features: In two meetings organised by the Federal Biological Research Centre for Agriculture and Forestry (Braunschweig, 2002) and later the German Federal Agency for Consumer Protection and Food Safety (Lille, 2005) an ad-hoc working group of experts from various countries and institutions discussed the practical performance and recommendations that should be taken into account in a revised draft following the current ISO guideline after the proposed revision has been approved by voting in the periodical review. Results: The outcome of the discussions and the background of the recommendations of the ad-hoc working group are briefly summarised in this paper. These are mainly methodological details in order to improve the test performance (e.g. by decreasing variability or increasing practicability). Discussion: These recommendations are proposed to be considered for the next update of the current ISO guideline. They should be seen as technical amendments still waiting for their approval by standardisation organisations. Conclusions: Although this method focuses on the assessment of effects of pesticides on earthworms, the recommendations will also be valuable for testing contaminated soils. The recommendations presented may also be a first step for splitting the current guideline into two fields of application, one for testing of chemicals and pesticides (i.e. within the scope of OECD) and another one for testing soil quality (i.e. within the scope of ISO). Perspectives: Nevertheless, there is still a major need for guidance concerning the interpretation of effects determined in such complex field tests.     

Effects-based Assays in the Earthworm Aporrectodea caliginosa: Their Utilisation for Evaluation of Contaminated Sites before and after Remediation (8 pp)

Goal, Scope and Background   In a preliminary ecological risk assessment, potential adverse effects of contaminants are often evaluated by measuring chemical residues and comparing these with regulatory guidelines. However limitations with this approach with regards to establishing actual effects have resulted in the increasing usage of sublethal effects-based assays, including biomarkers, to evaluate the hazard posed by contaminants in the environment. In this study a number of effects-based endpoints in the earthworm Aporrectodea caliginosa were evaluated to determine their comparative sensitivity for assessment of adverse effects of soil contaminated with petroleum hydrocarbons. Methods   Adult and juvenile earthworms were exposed for 4 weeks to sublethal concentrations of soil collected before and after remediation of a petroleum-contaminated site. A suite of endpoints were measured in these earthworms, including mortality, fecundity, growth, and juvenile maturation, and two less traditional endpoints, the biomarker, the neutral red retention assay (NRRA) and an avoidance behaviour test. Results and Discussion   Cocoon viability in this species is not a reliable parameter to measure, due to low viability in controls and a high coefficient of variation. Growth in adult earthworms was a more sensitive parameter than cocoon production. Maturation and growth of juveniles have been proposed as more sensitive endpoints than adult cocoon production and growth respectively. This was not apparent in the growth parameters, but maturation of juveniles did appear to be more sensitive than cocoon production by adults. The NRRA was a more sensitive parameter than cocoon production, and the NRRA and growth were both affected at the lowest concentration tested. The NRRA response appeared to be more sensitive than growth, but NRRT was only evaluated in one soil only, while the other parameters were assessed in two soils. However, the NRRA has previously been found to be more sensitive than growth after exposure to a number of contaminants. The avoidance behaviour assay exhibited similar sensitivity to growth and fecundity and could therefore be useful as a simple pre-screening test. Conclusion   The chronic endpoints, growth, cocoon production, and juvenile maturation parameters, were all sensitive endpoints for detecting exposure to the petroleum-hydrocarbon-contaminated soil. The NRRA was the most sensitive of the endpoints assessed and could be used as an early-warning indicator to predict adverse impacts. Avoidance behaviour could be used as a simple pre-screening test to evaluate contaminated soils prior to more extensive and invasive testing. Recommendations and Perspective   Measuring chemical concentrations in environmental samples is not always useful, as the toxicological impacts of exposure to these concentrations are not always discernible. However, the use of effects-based endpoints, either in situ or in the laboratory using laboratory-reared earthworms, can account for the bioavailability of chemicals in the soil, and can therefore provide information on the toxicological impacts of exposure. The assays tested in this research were sensitive indicators of exposure, and therefore can be used to determine potential ecological risks at contaminated sites and to monitor the progress of remediation at these sites.     

Earthworm avoidance of biochar can be mitigated by wetting

Biochar has a great potential for enhancing soil fertility and carbon sequestration while enabling beneficial waste disposition. Because of the potential for widespread application, it is essential to proactively assess and mitigate any unintended consequences associated with soil biochar amendment. We conducted soil avoidance tests, growth and reproduction tests, and oxidative stress assays with the earthworm Eisenia foetida to assess the potential toxicity of soil amended with biochar produced from apple wood chips. Earthworms avoided soils containing 100 and 200 g/kg dry biochar at statistically significant levels (p < 0.05), and after 28-day incubation, these earthworms lost more weight than those in control (unamended) soil. However, biochar did not affect the reproduction of earthworms. We investigated whether the observed avoidance was due to nutrition deficiency, desiccation, or the presence of toxic polynuclear aromatic hydrocarbons (PAHs) formed during biochar production by pyrolysis. Nutrition deficiency was excluded by the lack of earthworm avoidance to soil amended with nutrient-deficient sand instead of biochar. Although traces of PAH were detected in the tested biochar (e.g., 25.9 μg/kg fluorene, 3290 μg/kg naphthalene, and 102 μg/kg phenanthrene), the lack of lipid peroxidation and no increase in superoxide dismutase activity in biochar-exposed earthworms suggests that presence of toxic compounds was not a likely reason for avoidance. Furthermore, wetting the biochar to its field capacity resulted in statistically undetectable avoidance relative to control soil, indicating that insufficient moisture could be a key factor affecting earthworm behavior in soil amended with dry biochar. To avoid desiccation of invertebrates and enable their beneficial ecosystem services, we recommend wetting biochar either before or immediately after soil application.     

Terrestrial Ecotoxicity of Eight Chemicals in a Systematic Approach (7 pp)

Background and Objective   Terrestrial ecotoxicity data are required for many research purposes. The data are derived either from the literature or elaborated by own investigations. As the terrestrial toxicity tests are usually time-consuming and labour intensive, the experiments are performed with a limited number of test organisms and soils. In the context of a project sponsored by CEFIC-LRI (European Chemical Industry Council – Long-Range Research Initiative), EC50-values were systematically elaborated for eight chemicals with a wide range of logKow-values (CdCl2, Trinitrotoluene, 3,4-dichloroaniline, 2,4-dichlorophenol, Tributyltinchloride, Pentachlorophenol, Benzo(a)pyrene, p,p-dichloro-2,2-diphenyl-1,1,1-trichloroethane). The substances were selected covering a broad range of physico-chemical and ecotoxicological properties. As toxicity endpoints, microbial activities, plant germination and growth as well as reproduction of earthworms and collembola were determined. As such systematic investigations are rarely performed and for some substances no data existed, the data pool is made available to the scientific community.Methods   All toxicity tests were conducted on three different soil types (sandy soil, silty soil, loamy soil), according to ISO and OECD guidelines Results, Discussion, Conclusion and Outlook   The different toxicities of the chemicals, the influence of soil properties on bioavailability as well as different sensitivities of test organisms and test parameters are reflected by the EC50-values. The results showed that the EC50-values calculated on the basis of nominal concentrations can significantly vary from EC50-values derived from analytical concentrations for some substances. To avoid false conclusions, this has to be considered especially when concentrations determined in the field are compared with toxicity data obtained from the literature or calculated on the basis of nominal concentrations. Moreover, the results indicate that terrestrial ecotoxicological tests should be accompanied by chemical analyses, to be sure to derive sound EC50-values. As the tests were performed according to international guidelines, the comprehensive data pool relevant for the terrestrial ecotoxicity of eight chemicals is considered to be of great value for further research and discussions dealing with the habitat function of soils.     

Invertebrates in risk assessment development of a test battery and of short term biotests for ecological risk assessment of soil

4 Conclusion  In general, all biotests used in the ring test are applicable for risk assessment of field soil and contaminated or remediated soil material. Of these, the most sensitive tests were selected as a integral part of the test battery [4]. This selection did not consider either the duration of the tests, which is crucial for the acceptance of tests by the remediation industry, and the effects of pollutants on biomarkers, which indicate the departure of an organism from its normal biochemical or physiological status. Therefore, the proposed test battery has to be supplemented with biotests of short duration. The best choice for a short test using the endpoint reproduction is the biotest withCaenorhabditis elegans which characterises the habitat function of soil. The appropriate screening test for the retention function is the ETr (electron transfer) inhibition test with submitochondrial particles, which provides reliable results in less than 15 min. For screening the habitat function, avoidance tests with soil invertebrates like annelids or enchytraeids are optimal, as both annelids are exposed to the bioavailable pollutants in the soil pore water. The effect of environmental chemicals at the individual level and below can be analysed with the Celegans ToxChip, which gives information of the status of biomarkers on the molecular and biochemical level. Preliminary SOPs for these biotests are formulated. They have still to be evaluated by international ring tests.     

A Chronic Plant Test for the Assessment of Contaminated Soils. Part 2: Testing of contaminated soils (10 pp)

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PREAMBLE According to the German Federal Soil Protection Act (BBodSchG 1998), the habitat function of soils must be protected. Despite the fact that in the Federal Soil Protection Ordinance (BBodSchV 1999) it has not been established how this goal can be reached reliably, it is clear that such a biological function can only adequately be assessed using biological test methods. This is especially true when a soil is contaminated by a mixture of often unknown chemicals. In such a case the use of chemical analysis aiming at a small range of known substances is not sufficient and must therefore be supplemented by biological methods. For this reason, several standardised test methods are available (e.g. using earthworms, collembolans or plants; Römbke and Knacker 2003; ISO 2003). Since acute tests are usually not sensitive enough for the assessment of potentially contaminated soils (e.g. Hund-Rinke et al. 2002), chronic tests like the earthworm reproduction tests (ISO 1998) are recommended for this purpose.

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A chronic plant test for the determination of phytotoxicity was missing until quite recently. The term phytotoxicity is understood here as the capacity of a compound or a contaminated soil to cause temporary or long-lasting damage to plants (EPPO 1997). Therefore, the German Ministry for Education and Research sponsored a project (1997 – 1999) in which – based on existing standardised methods – such a chronic plant laboratory test was developed and partly validated (Kalsch and Römbke 2000). The new test can be used for the evaluation of single chemicals (see Part 1 of this mini-series) as well as for the assessment of contaminated or remediated soils (see Part 2 of this mini-series).

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ABSTRACT Background and Scope. A new chronic plant test system which is based on experiences with various acute plant tests (e.g. published by OECD or ISO) and existing North American Plant-Life-Cycle Bioassays was standardised in a project sponsored by the German government. Characteristic properties of the test system, which can be performed either with Brassica rapa (turnip rape) or Avena sativa (oat), are described in Part I of this mini-series.

Methods

This new test was used to assess the effects of natural soil samples contaminated with TNT (2,4,6-Trinitrotoluene) or PAHs (poly-aromatic hydrocarbons). The soils were tested after taken from the field as well as after being remediated. Different control and reference soils were used to evaluate the test results. In addition, they were compared with the results of tests in which either TNT or Pyrene was spiked to field and standard soils (see Part I of this mini-series).

Results

All contaminated soils showed clear effects in the Chronic Plant Test (usually B. rapa was more sensitive than A. sativa). LUFA 2.2 standard soil and OECD artificial soil are well-suited as control and mixture substrates, while reference soils collected at uncontaminated sites were several times phytotoxic. In most of the latter cases, soil properties could be identified as the main cause of these effects (e.g. the pH value). While in general the sensitivity of the reproduction and biomass endpoints did not differ much, it is recommended to measure different endpoints (i.e. biomass and reproduction) due to the different mode-of-action of contaminants. In the case of TNT a good agreement between the results of single chemical tests and tests with TNT-contaminated soils was found (due to the minimal effects of Pyrene the same statement is not possible for PAHs).

Outlook

The results of this work have been used to prepare a draft test guideline, which has recently been standardised by the International Organisation for Standardisation (ISO). Based on these experiences, recommendations are given how this chronic test system could be used as part of a test battery for the assessment of contaminated soils.     

Applying a GLM-based approach to model the influence of soil properties on the toxicity of phenmedipham to Folsomia candida

Purpose

Soil properties are the main explanation to the different toxicities obtained in different soils due to their influence on chemical bioavailability and the test species performance itself. However, most prediction studies are centred on a few soil properties influencing bioavailability, while their direct effects on test species performance are usually neglected. In our study, we develop prediction models for the toxicity values obtained in a set of soils taking into account both the chemical concentration and their soil properties.

Materials and methods

The effects on the avoidance behaviour and on reproduction of the herbicide phenmedipham to the collembolan Folsomia candida is assessed in 12 natural soils and the Organisation for Economic Co-operation and Development (OECD) artificial soil. The toxicity outcomes in different soils are compared and explanatory models are constructed by generalised linear models (GLMs) using phenmedipham concentrations and soil properties.

Results and discussion

At identical phenmedipham concentrations, the effects on reproduction and the avoidance response observed in OECD soil were similar to those observed in natural soils, while effects on survival were clearly lower in this soil. The organic matter and silt content explained differences in the avoidance behaviour in different soils; for reproduction, there was a more complex pattern involving several soil properties.

Conclusions

Our results highlight the need for approaches taking into account all the soil properties as a whole, as a necessary step to improve the prediction of the toxicity of particular chemicals to any particular soil.     

A Chronic Plant Test for the Assessment of Contaminated Soils. Part 1: Method development (9 pp)

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PREAMBLE According to the German Federal Soil Protection Act (BBodSchG 1998), the habitat function of soils must be protected. Despite the fact that in the Federal Soil Protection Ordinance (BBodSchV 1999) it has not been established how this goal can be reached reliably, it is clear that such a biological function can only adequately be assessed using biological test methods. This is especially true when a soil is contaminated by a mixture of often unknown chemicals. In such a case the use of chemical analysis aiming at a small range of known substances is not sufficient and must therefore be supplemented by biological methods. For this reason, several standardised test methods are available (e.g. using earthworms, collembolans or plants; Römbke and Knacker 2003; ISO 2003). Since acute tests are usually not sensitive enough for the assessment of potentially contaminated soils (e.g. Hund-Rinke et al. 2002), chronic tests like the earthworm reproduction tests (ISO 1998) are recommended for this purpose.

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A chronic plant test for the determination of phytotoxicity was missing until quite recently. The term phytotoxicity is understood here as the capacity of a compound or a contaminated soil to cause temporary or long-lasting damage to plants (EPPO 1997). Therefore, the German Ministry for Education and Research sponsored a project (1997 – 1999) in which – based on existing standardised methods – such a chronic plant laboratory test was developed and partly validated (Kalsch and Römbke 2000). The new test can be used for the evaluation of single chemicals (see Part 1 of this mini-series) as well as for the assessment of contaminated or remediated soils (see Part 2 of this mini-series).

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ABSTRACT Background and Scope. As part of the efforts to improve the biological testing of contaminated soils, the German government sponsored the standardisation of a chronic plant bioassay. This new test is based on experiences with various acute plant tests (e.g. published by OECD or ISO) and existing North American Plant-Life-Cycle Bioassays. In this contribution the characteristic properties of the test are described.

Methods

The test can be performed either with Brassica rapa (turnip) or Avena sativa (oat). Its duration is 35 to 64 days with OECD artificial soil and a German standard field soil acting as controls. Water and nutrients are provided by an automatic wick irrigation system. Besides measuring biomass and shoot length, the number of pods, seeds and flowers are applied as chronic measurement endpoints. During the development of the test, TNT (2,4,6-trinitrotoluene) and Pyrene were used as model test substances.

Results

Pyrene did not affect B. rapa (turnip) in concentrations of up to 10.000 mg/kg soil (due to the often low sensitivity of A. sativa (oat) no further test with this substance was performed). Depending on the endpoint the results varied in the tests with TNT. With few exceptions, the NOEC (No Observed Effect Concentration) values were determined as 55.5 mg TNT/kg soil for B. rapa (turnip) and as 75 (unfertilised) and 150 (fertilised) mg TNT/kg soil for A. sativa (oat). The EC50-values varied between 96.3 and 207.2 mg TNT/kg soil for B. rapa (turnip) and 183.1 – 505.6 mg TNT/kg soil for A. sativa (oat), depending on the endpoint.

Outlook

The results of this work have been used to prepare a draft test guideline, which has recently been standardised by the International Organisation for Standardisation (ISO). Practical experiences with this test system are described in Part II of this mini series.     

Tolerance of genetically characterized <Emphasis Type="Italic">Folsomia candida</Emphasis> strains to phenmedipham exposure

Background, Aims, and Scope  The springtail Folsomia candida is a commonly used model species in ecotoxicological soil testing. The species reproduces parthenogenetically and, thus, laboratories use different clonal lineages. In this study, we investigated if genetic divergence between F. candida strains impacts the reaction to chemical stress and may thus affect the outcome of toxicity tests. Methods  In two exposure assays (life-cycle reproduction test and avoidance behaviour test), three laboratory strains of F. candida from Portugal (PTG), Spain (SPN) and Denmark (DNK) were exposed to different concentrations of the reference chemical phenmedipham. Genetic divergence among strains was estimated based on mitochondrial COI sequence data. Results  No significant differences between tolerance towards phenmedipham exposure were observed in a reproduction test. In contrast, one strain (SPN) showed a decreased susceptibility to phenmedipham compared to the other strains (PTG and DNK) in the avoidance assay. Discussion  A phylogenetic tree based on mitochondrial COI sequences revealed clear genetic differentiation between both ‘reaction types’. Thus, we found a potential lineage dependent stress reaction in avoidance behaviour towards the pesticide. Conclusions  Our findings have implications for the comparability of test results among laboratories. Reproduction tests seem to be more robust towards interclonal genetic differentiation than avoidance tests. Recommendations and Perspectives  We recommend the use of molecular tools for simple and cost effective genetic characterization of F. candida strains used in chemical avoidance tests. Closer investigations concerning the relation between genetic relatedness and chemical response will provide a more detailed and comprehensive picture on the role of intraspecific genetic differentiation in stress tolerance. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

Stress protein response (Hsp70) and avoidance behaviour in Eisenia fetida, Aporrectodea caliginosa and Lumbricus terrestris when exposed to imidacloprid

Purpose  

The earthworm species Eisenia fetida and Eisenia andrei are commonly used in ecotoxicological standard tests. In the present study, we compared the sensitivity of E. fetida with that of two soil-dwelling earthworm species (Aporrectodea caliginosa and Lumbricus terrestris) in order to evaluate the capacity of E. fetida to predict effects of the insecticide imidacloprid. Responses were compared using two endpoints, a biochemical (changes in heat shock protein level (hsp70)) and a behavioural (avoidance behaviour).     

Using earthworm avoidance behaviour to assess the toxicity of formulated herbicides and their active ingredients on natural soils

Background, aim, and scope

Soil quality has been threatened by intensive agricultural practises, namely those relying on the application of pesticides, such as herbicides. Among the non-target terrestrial organisms exposed to such scenarios, earthworms are key ecological receptors widely used in ecotoxicological studies. As such, this work aims to assess the effects of two herbicide active ingredients (a.i.)—sulcotrione and penoxsulam—and their respective commercial formulations—MIKADO® and VIPER® (referred as Mikado and Viper)—on the avoidance behaviour of Eisenia andrei. In an attempt to enhance the ecological relevance of the generated toxicity data, the avoidance tests were run with standard (LUFA 2.2; L) and natural soils (from corn and rice fields), as long as their habitat function did not constrain the earthworm behaviour.

Methodology

Earthworms were bred in the lab before test conductance. The natural soils used as substrates were collected before the cropping season on corn (C) and rice (R) fields, which are integrated in a wide area exploited for agriculture. Their physico-chemical characterization evolved the determination of pH (H₂O, KCl), conductivity, organic matter (OM) and clay/silt contents, and water-holding capacity (WHC). The avoidance tests intended to ascertain (1) the random distribution of earthworms in the natural soils C and R (dual-control tests), (2) the habitat function of natural soils against each other and against L soil, (3) the effect of active ingredients and formulated herbicides on E. andrei behaviour. Avoidance tests with the a.i.s were only performed in L soil. Data evaluation followed ISO (2005) guidelines.

Results

C and R soils presented higher OM (5.1% and 4.5%, respectively) and clay/silt (53.3 and 43.1, respectively) contents and WHC (107.2 and 109.9%, respectively) than L soil (4.1, 21.4 and 48.0%, correspondingly). Earthworms distributed randomly in dual-control tests, but preferred R soil significantly, relative to L or C soils. The LOEC and EC₅₀ values calculated for sulcotrione (>1,000.0 and 1,263.3 mg a.i. kg^-1, respectively) and Mikado (1,012.8 and 1,301.3 mg a.i. kg^-1, respectively) were much higher than those calculated for penoxsulam (100 and 80.6 mg a.i. kg^-1, respectively) or Viper (52.7 and 51.5 mg a.i. kg^-1, respectively), when L soil was used as substrate. Moreover, the habitat function of L soil contaminated with the formulated herbicide Viper was more constrained relative to that of the a.i. penoxsulam. Viper induced higher % avoidance on E. andrei exposed to the contaminated L soil compared to that under the R soil.

Discussion

The response of earthworms to R (attraction) and C (avoidance) soils could be related, not only to the quantity of OM content, but also to the quality of organic and inorganic fractions of soil, beyond other intrinsic properties of soils. Both Mikado and sulcotrione impacted the behaviour of E. andrei only slightly. This endpoint was more affected under penoxsulam or Viper exposures on L soil, being the latter-formulated product even more repellent for E. andrei than the a.i. The effect of adjuvants added to the commercial formulation of Viper, may have increased the toxicity of the a.i. Thereby, our results reinforce the need for a careful assessment of the impacts of formulated products. Furthermore, since there was a reduction in earthworm % avoidance under Viper exposures on the natural soil R, it was possible that pesticide bioavailability had been reduced by its sorption to OM and clay mineral sorption sites.

Conclusions

Though the standard L soil should be used for reproducibility and comparison means, other natural soils should be added to the assessment of chemicals, for sake of ecological relevance. Both herbicides induced avoidance behaviour on E. andrei, albeit stronger effects were denoted by penoxsulam and its respective formulated product, Viper. Overall, avoidance tests provided a sensitive, valuable and feasible response either to compare the habitat function of different standard and agricultural natural soils or to test the effect of herbicides.

Recommendations and perspectives

An effort should be made to enlarge the terrestrial ecotoxicological database as a way to fulfil the huge lack of information available for this ecosystem. In this context, additional research congregating a potential linkage between physiological activities sustaining the regular metabolism of earthworms and their avoidance behaviour or even their reproductive effects would be welcomed, especially in what regards formulated pesticides. Such approach would provide a robust and comprehensive understanding of chemical effects. Furthermore, it is encouraged that natural soils should be used to improve the reliability of chemical testing.     

Boric acid as alternative reference substance for earthworm field tests

Purpose  

Boric acid was applied in an earthworm field test according to ISO 11268-3 as a possible alternative for the currently used reference substances that may no longer be available in the near future.     

The use of sewage sludge as soil amendment. The need for an ecotoxicological evaluation

Background, aim, and scope  Sewage sludge use in agriculture should be limited by the presence of metals and other persistent environmental pollutants. The present study aims to contribute for the definition of a test battery of ecotoxicological assays that allows a proper ecotoxicological characterization of sludges, providing information on their potential hazard and identified “safe” application levels. Materials and methods  Three sludges from distinct sources (urban, olive-processing, and electroplating industries) were tested using avoidance and reproduction tests with earthworms (Eisenia andrei) and springtails (Folsomia candida) and plant growth tests with turnips (Brassica rapa) and oats (Avena sativa). Different soil–sludge mixture concentrations mimicking recommended/realistic field dosages were tested. Results  Only the sludge from the electroplating industry induced an avoidance response from the earthworms (EC₅₀ = 0.4 t/ha) and collembolans (no observed effect concentration (NOEC) = 15 t/ha). This sludge was the only sludge responsible for any effect on the reproductive output of the earthworms (EC₅₀ = 7.74 t/ha). Regarding collembolans, none of the sludges tested caused any significant decrease in reproduction. In higher plant tests, the two industrial sludges were toxic, causing a decrease growth in both species. The EC₂₀ values determined for B. rapa were 20.3 and 24.2 t/ha and for A. sativa 14.7 and 16.2 t/ha for sludges from olive-processing and electroplating industries, respectively. Discussion  The metal loadings of the different test sludges could partially explain the results obtained. The toxicity of the test sludge from electroplating industry observed on the tested invertebrates and plants could be explained by the high amount of total chromium from which 22.3% was in the most toxic oxidation state—Cr(VI). However, the toxicity caused by the sludge from the olive-processing industry in the test plants could be attributed to the presence of other compounds (not measured in this study) since the metal content was not high enough to induce such an effect. The absence of toxicity showed by the urban test sludge was in agreement with its low levels of metals. Conclusions  The response of the different test organisms and end points varied according to the sludge type. The urban sludge was non-toxic whereas the sludge from the electroplating industry caused a toxic effect on almost all parameters measured (avoidance behavior of both test organisms, reproduction of earthworms, and growth of both plant species). Sludge from the olive-processing industry only caused a toxic effect on growth of both plant species. By analyzing the sensitivity of the different parameters for the most toxic sludge, it was found that avoidance and reproduction were more sensitive than plant growth, whereas plant seed germination was not sensitive at all. Recommendations and perspectives  The ecotoxicological evaluation of wastes can be used as an environmental safety control of sludge use in agriculture. A tiered approach could be adopted for this purpose, incorporating avoidance tests in the first tier (screening level) and reproduction and plant growth tests in a second tier. But more evidence aiming to define the most suitable ecotoxicological test battery for specific sludges with a different contamination profile is still needed.     

Adaptation of the enchytraeid toxicity test for use with natural soil types

Assessments of chemical toxicity to soil invertebrates have been traditionally performed in formulated Standard Artificial Soil, or in standard LUFA 2.2 natural soil. Physical and chemical properties of these standard soils often do not represent the diversity of properties of natural soils and can be inadequate surrogates of exposure conditions of soil biota in the field. We investigated performance of three species of the genus Enchytraeus (E. albidus, E. crypticus, E. luxuriosus) using soil types covering a wide range of European and North American soil properties. Results showed that adult survival and juvenile production by E. albidus and E. luxuriosus were inhibited in acidic soils with pH ≤ 5, while less acidic soils having properties within following ranges: 2.5–8.0% OM, 6–26% clay, and 4–80% sand, could sustain survival and reproduction at or near validity criteria levels of enchytraeid toxicity test ISO/16387. Performance of E. crypticus in North American soils having properties within following ranges: 1.2–42% OM, 1–29% clay, and 4.4–8.2 pH, met or exceeded all validity criteria. Data established in our studies can be used for amending the validity criteria of current ISO/16387 when used with E. crypticus and OECD soil.     

Effects of liming on survival and reproduction of two potentially invasive earthworm species in a northern forest Podzol

During the last several decades, colonization of soil by exotic earthworms and their effects on soil properties and biodiversity have been reported in forests of North America. In some northern hardwood stands, acid soils or harsh climate may have prevented earthworm colonization. However, climatic change and the increasing use of liming to restore the vigor of declining sugar maple (Acer saccharum Marsh.) stands, situated on base-poor soils in USA and Canada, could make many of these sites more suitable for earthworm colonization. We tested survival and reproduction of two exotic earthworm species (Lumbricus terrestris and Amynthas hawayanus) in unlimed and limed soils at the northern limit of the northern hardwood forest distribution in Canada. Improving soil parameters of base-poor, acidic soils by liming positively influenced activity, survivability and reproductive output of L. terrestris in this northern hardwood forest. In contrast, the high mortality and low vigor of L. terrestris observed in the unlimed plots show that soils in this area with a pH of 4.3 are not favorable to this species. Our results suggest that A. hawayanus was very active prior to winter at both soil pHs, but was not able to complete its life cycle during one year at this latitude. Both earthworm species significantly reduced organic C and total N, and increased the C/N ratio of the forest floor. Given that forest liming activities are increasing in proximity to human activities, there is high probability that some earthworm species, such as L. terrestris, will invade limed northern hardwood forests in the next decades, with possible consequences for soil organic matter turnover, nutrient cycling and forest biodiversity and dynamics.