Earthworm Avoidance Test Research Articles

Copper toxicity on Eisenia fetida in a vineyard soil: a combined study with standard tests, genotoxicity assessment and gut metagenomic analysis

Copper (Cu) toxicity is a pressing concern for several soils, especially in organic viticulture. The objective of this work was to assess Cu toxicity on the non-target organism Eisenia fetida, employing both traditional and novel tools for early identification of Cu-induced damages. In addition to traditional tests like avoidance and reproductive toxicity experiments, other tests such as the single cell gel electrophoresis (SCGE) and gut microbiome analysis were evaluated to identify early and more sensitive pollution biomarkers. Four sub-lethal Cu concentrations were studied, and the results showed strong dose-dependent responses by the earthworm avoidance test and the exceeding of habitat threshold limit at the higher Cu doses. An inverse proportionality was observed between reproductive output and soil Cu concentration. Bioaccumulation was not detected in earthworms; soil concentrations of potentially bioavailable Cu were not affected by E. fetida presence or by time. On the contrary, the SCGE test revealed dose-dependent genotoxicity for the ‘tail length’ parameter already at the second day of Cu exposition. Gut microbiome analysis a modulation of microbial composition, with the most aboundant families being Pectobateriaceae, Comamonadaceae and Microscillaceae. Bacillaceae increased over time and showed adaptability to copper up to 165 mg/kg, while at the highest dose even the sensitive Acetobacteriaceae family was affected. The research provided new insights into the ecotoxicity of Cu sub-lethal doses highlighting both alterations at earthworms’ cellular level and changes in their gut microbiota.

Ecotoxicological Tests of Metal-Contaminated soils

The raw steel production plays a key role in the economic development of Slovakia. Unfortunately, the technology of steel production, agricultural and industrial activities contribute to contamination of the soil in the region Eastern Slovakia. The purpose of this study was to investigate the effect of heavy metals in urban soils from the Košice area, using bioassays on earthworms and phytotoxicity. An earthworm avoidance test has potential advantages for use in evaluation of hazardous soils sites and proved as a quick approach to determining the presence of contaminants. The earthworm (Dendrobaena veneta) takes up and retains metals from soil containing of heavy metals. Assessment of soil phytotoxicity was based on germination and seedling growth of the terrestrial plant (mustard Sinapis alba). The level of Cr, Co, Cd and Hg were assessed in 8 industrial soils (4 agricultural areas and 4 grass-plot areas) from the area U.S. Steel Košice. The highest levels contaminations of chromium (278mg/kg), cobalt (39mg/kg), cadmium (21mg/kg) and mercury (0.80mg/kg) were determined for the grass-plot soils (main gate of the U. S. Steel-plant). Phytotoxkit results for the agricultural soils from around Košice showed that, the potential toxicity values are lowly, represented by a lower percentage of inhibition in germination range between 5 and 33 % and the average percentage of growth inhibition was 12-39 % for Sinapis alba. The results for the grass-plot areas soils from around Košice showed the percentage of inhibition in germination range between 13 and 47 % and the average percentage of growth inhibition was 19-49 % for Sinapis alba. Low mortality effects were recorded in the tests with Dendronaena veneta. The distribution of the worms found in the double control was within the range 10–100 % for all areas of the agricultural soils and grass-plot soils, for after 48h. The significant (P<0.05) avoidance by Dendronaena veneta were 100% in soils of areas Gomboš and from main gate of the U. S. Steel-plant, Košice. The above results reflect that not only may the worms be able to detect metals, but in fact they may also change their behavioural response over time.

Ecotoxicity of imidacloprid to soil invertebrates in two tropical soils with contrasting texture.

Imidacloprid is one of the most commercialized insecticides in agriculture in the world, with a broad spectrum of action. However, little is known about the effects of commercial formulations containing this active ingredient (a.i.) on non-target organisms in tropical soils. Our objective was to assess the toxicity based on the predicted environmental concentration (PEC) of imidacloprid, in the avoidance behaviour of earthworms and collembolans as well as in the reproduction of collembolans, in two representative soils of the Brazilian Cerrado with contrasting texture (clayey Oxisol and sandy Entisol). Ecotoxicity tests were carried out according to ISO protocols to assess the avoidance behaviour of earthworms (Eisenia andrei) and avoidance and reproduction of collembolans (Folsomia candida). In the earthworm's avoidance test, more than 80% of the individuals were found in the control, in all tested concentrations, indicating a possible habitat function loss in both soils. The avoidance behaviour of collembolans was observed in both soils, being more expressive (up to 75% of escape) in Oxisol. In Entisol, only the two highest concentrations were avoided (up to 63%). There was a negative effect on the reproduction of collembolans in both soils, with a higher EC50 value (0.255 mg kg-1) in Oxisol than in Entisol (0.177 mg kg-1), demonstrating higher toxicity in the sandy soil. These differences were attributed to the contrasting texture of the studied soils, probably due to lower retention of the a.i. in the sandy soil, causing an increased bioavailability. This study demonstrated that imidacloprid can be highly toxic to soil invertebrates, even in soil concentrations lower than those expected from recommended dose, causing an impact on the edaphic organisms and, consequently, compromising its functions in the soil ecosystem.

Biochars produced from coconut palm biomass residues can aid regenerative agriculture by improving soil properties and plant yield in humid tropics

Lignin-rich recalcitrant biomass residues of coconut palms viz. (i) mature coconut husk, (ii) tender (immature or green) coconut husk (iii) coconut leaf petiole and (iv) coir-pith were successfully pyrolysed using a simple charring kiln into carbon-rich, black, light weight and porous biochars. High alkalinity and good ash content made them fit for remediating acid soils. High potassium content in these biochars could help reduce the use of inorganic K. Thermogravimetric analysis showed the mass loss phases of husk and coconut leaf petiole biochars to be similar. However, all four biochars gave smooth curves indicating thermal stability of the product. Positive seed germination and earthworm avoidance tests proved their potential as soil amendment. Soil incubation studies with coconut biochars in graded doses, alone or in combination with coconut leaf vermicompost, increased the pH, organic carbon and potassium contents, and promoted plant-beneficial microbiota and enzyme activities. Pot studies with tender coconut husk biochar and coconut leaf vermicompost enhanced the dry weight of cowpea plants accompanied with increased arbuscular mycorrhizal sporulation and root colonization, and root nodule dry weight. A field trial resulted in higher chilli yields with tender coconut husk biochar + coconut leaf vermicompost addition. The results from our studies highlight the potential of pyrolysis as an innovative technology for quick recycling of highly recalcitrant coconut palm biomass residues to biochars as a local source of soil amendment to aid regenerative agriculture in humid tropics.

Agro-Environmental Benefit and Risk of Manure- and Bone Meal-Derived Pyrogenic Carbonaceous Materials as Soil Amendments: Availability of PAHs, PTEs, and P

The worldwide boom of biochar and pyrogenic carbonaceous material application as a potential soil additive has brought about not only agricultural benefits such as enhanced crop yield, nutrients supply (P), and soil organic carbon increase, but also, on the other hand, environmental risk of organic (polycyclic aromatic hydrocarbons (PAHs)) and potentially toxic element (PTE) penetration into arable soils. Therefore, our study assessed pyrogenic carbonaceous materials (PCM) produced from the P-rich feedstocks—chicken manure (CM) and bone meal (BM)—as promising and safe alternatives for inorganic P fertilizers. Pyrogenic materials produced in the process of slow pyrolysis at residence time 2 h, 400 and 500 °C, were characterized by determination of pH, electrical conductivity (EC), elemental analysis of total C, H, N, S scanning electron microscopy (SEM), total content of P, selected potentially toxic elements (PTEs), and available forms of PTEs and P by diethylenetriaminepentaacetic acid (DTPA) and calcium-acetate-lactate (CAL) extractions. CMPCM4, CMPCM5, BMPCM4, and BMPCM5 were characterized by determination of total 16 US-EPA (U.S. Environmental Protection Agency) PAHs by toluene extraction protocol and available concentrations by Tenax resin approach. Additionally, CMPCM4, CMPCM4, BMPCM4, and BMPCM5 were tested in earthworm avoidance test with Eisenia foetita and short-term rye-seedling germination test. Obtained results showed decreasing of total carbon in the order of BM &gt; BMPCM4 &gt; BMPCM5 and increasing in the order of CM &lt; CMPCM4 &lt; CMPCM5. Total phosphorus content increased from 56.8 ± 1.7 g kg−1 (BM) to 85.2 ± 4.2 g kg−1 (BMPCM4) to 110.5 ± 7.0 g kg−1 (BMPCM5). In the case of chicken manure-derived pyrogenic materials, total phosphorus content increased in the order of CM (22.9 ± 2.0 g kg−1) &lt; CMPCM4 (37.0 ± 4.5 g kg−1) &lt; CMPCM5 (40.0 ± 3.4 g kg−1). Availability of selected PTEs and P decreased in pyrogenic materials compared to feedstock. Total concentration of ∑16-US-EPA PAHs in BMPCM4 and BMPCM5 was 3.92 mg kg−1; CMPCM4, 7.33 mg kg−1; and CMPCM, 6.69 mg kg−1. The Tenax-available ∑16-PAHs showed concentrations of 0.53 mg kg−1 for BMPCM4, 0.26 mg kg−1 for BMPCM5, 1.13 mg kg−1 for CMPCM4, and 0.35 mg kg−1 for CMPCM5. Total P concentrations determined in rye aboveground tissues showed the highest accumulation ability in the case of CMPCM5 compared to other samples. Pyrogenic carbonaceous materials produced from chicken manure and bone meal at 400 and 500 °C have the potential to be P slow release fertilizers and may be ecologically safe.

Can Local Enhancement in Earthworms Affect the Outcome of the Standard Earthworm Avoidance Test?

Earthworms exhibit clumping behaviour in and out of the soil. However, it remains unknown if such social behaviour ultimately influences the outcome of ecotoxicological experiments in the laboratory. We performed several overnight avoidance tests to determine whether social behaviour (i.e., local enhancement) is a factor in pollution avoidance behaviour in the earthworm Eisenia fetida. The results showed that there was no clear influence of social behaviour on the choice or avoidance of Cd contaminated soils, although we suspect that 50mgCd/kg might not have been high enough to elicit a significant avoidance response. Nevertheless, when offered a choice between clean undisturbed soil and previously inhabited soil, the worms preferred the previously inhabited soil (p < 0.01). While the level of metal pollution investigated in this study did not disrupt or help predict social dynamics, local enhancement, perhaps driven by some sort of habitat imprinting, was successfully documented in Eisenia fetida.

Effects of waste materials on Caenorhabditis elegans (Nematoda) using the ISO standard soil toxicity test.

The ecotoxicological characterization of waste according to the European Waste List (HP14) is part of its hazard classification, which is based on 15 different hazardous properties and should include toxicity tests representing the aquatic and terrestrial compartment. Besides established soil toxicity tests with bacteria, plants, and earthworms, the standardized test with the nematode Caenorhabditis elegans (ISO 10872:2010) is suitable for testing soils and wastes, however, has never been validated for the purpose of waste toxicity assessment. Therefore, 23 different waste samples were tested for their toxicity on growth and reproduction of C. elegans to validate the suitability of ISO 10872 (ISO 2010) for assessing the ecotoxicological hazard of waste and to compare the results with those of other ecotoxicological test systems. C. elegans showed a comparable response to the waste samples as the earthworm avoidance test, however, with single samples where the nematodes indicated a higher toxicity than the E. fetida, thus, providing non-redundant information to the ecotoxicological hazard assessment of wastes. Also due to the short duration (4 days) and small-scale test set-up, the soil toxicity test with C. elegans turned out to be a valuable addition to already existing test batteries, for assessing the ecotoxicity of wastes in the soil compartment.

Uso de testes de fuga com minhocas Eisenia andrei e Eisenia fetida para identificação da toxidade de agrotóxicos no Brasil: Uma breve revisão da literatura

The trade of pesticides in Brazil in 2017 was 539,944.95 tons of active ingredients. Tests that evaluate the avoidance behavior in soil fauna, such as earthworms, indicate the loss of soil habitat function and the risks of these pesticides to the environment. These organisms have chemoreceptors and sensory tubers in the body which make them highly sensitive to chemicals in the environment. The objective of the present study was to review the literature on the use of the Eisenia fetida e Eisenia andrei earthworm avoidance test in the evaluation of pesticides in Brazil. The databases PubMed, Web of Science and Google Scholar were used for bibliographic research using the terms: earthworm, avoidance, Brazil or agrotóxicos, teste de fuga/evitamento, Eisenia fetida e Eisenia andrei. We found 13 studies, totaling 25 evaluations and 17 active ingredients, which evaluated the avoidance behavior in Eisenia fetida e Eisenia andrei in Brazil. The most tested pesticide, with eight evaluations, was the Glyphosate herbicide which is the most commercialized in Brazil. The avoidance behavior was observed in 17 evaluations. The use of this earthworm avoidance test is a sensitive tool to evaluate the ecotoxicological risks of pesticides.

Toxicological assessment of pesticide contaminated soils with use of biotests

The paper presents the results of experiments on ecological toxicity assessment performed for 12 soil samples collected at 3 obsolete pesticide “tombs” in Poland, before their final disposal. Bioavailability of the main pollutants: p,p’-DDT, lindane and methoxychlor was assessed for selected samples by consecutive solid phase extraction using a Tenax TA sorbent. Several toxicity bioassays were also carried out, including: reducers (Microtox Soild Phase), producers (Phytotox) and consumers (tests of avoidance, acute toxicity and reproduction with use of the earthworm Eisenia foetida). Data from toxicity tests were discussed against the results of the chemical analysis of a wide range of pesticides determined by GC-ECD and GC-NPD. This part of the study enabled the evaluation of the applicability of the aforementioned bioassays in the assessment of pesticide soil pollution. Results of toxicity tests showed a slight to severe impairment of habitat function for all of the contaminated samples, which was only partially reflected by the analytical data. The most sensitive biotest was earthworm reproduction, followed by Phytotox and earthworm acute toxicity. Earthworm avoidance and Microtox tests were found to be of rather limited usability.

Proposed modification to avoidance test with Eisenia fetida to assess metal toxicity in agricultural soils affected by mining activities

Use of avoidance tests is a quick and cost-effective method of assessing contaminants in soils. One option for assessing earthworm avoidance behavior is a two-section test, which consists of earthworms being given the choice to move between a test soil and a control substrate. For ecological relevance, tested soils should be field-contaminated soils. For practical reasons, artificial soils are commonly used as the control substrate. Interpretation of the test results compromised when the test soil and the artificial substrate differ in their physico-chemical properties other than just contaminants. In this study we identified the physico-chemical properties that influence avoidance response and evaluated the usefulness of adjusting these in the control substrate in order to isolate metal-driven avoidance of field soils by earthworms. A standardized two-section avoidance test with Eisenia fetida was performed on 52 uncontaminated and contaminated (Cu >155mgkg−1, As >19mgkg−1) agricultural soils from the Aconcagua River basin and the Puchuncaví Valley in Chile. Regression analysis indicated that the avoidance response was determined by soil organic matter (OM), electrical conductivity (EC) and total soil Cu. Organic matter content of the artificial substrate was altered by peat additions and EC by NaCl so that these properties matched those of the field soils. The resultant EC80 for avoidance (indicative of soils of “limited habitat”) was 433mg Cu kg−1 (339 – 528mgkg−1 95% confidence intervals). The earthworm avoidance test can be used to assess metal toxicity in field-contaminated soils by adjusting physico-chemical properties (OM and EC) of the artificial control substrate in order to mimic those of the field-collected soil.

Ecotoxicological assessment of a biochar-based organic N-fertilizer in small-scale terrestrial ecosystem models (STEMs)

Small-scale Terrestrial Ecosystem Models (STEMs) are valuable tools in soil ecotoxicological approaches. We assessed the effects of topsoil incorporation (0–15cm) of a biochar-based organic N-fertilizer on the weight and vertical distribution of Eisenia andrei (epigeic earthworm) and on germination and growth of Brassica rapa (turnip) in STEMs. Single-species earthworm avoidance behaviour (ISO 17512-1:2005) and plant germination (based on ISO 11269-2:2012) tests were also performed for contextualization.A 28day exposure in STEMs showed no variation in earthworm fresh weight. However, increasing biochar concentrations led to an increased earthworm migration from the biochar amended topsoil to deeper soil layers. Such a migration reached 100% at a lower biochar application rate (25t/ha) compared to the standard earthworm avoidance tests (50t/ha), making the STEMs approach potentially more conservative for this endpoint. Plant germination in STEMs significantly declined at 25t/ha, with no germination at 75 and 100t/ha. Alongside it, a significant decrease in total plant biomass also occurred in STEMs (10t/ha) before that in traditional plant germination tests (100t/ha), suggesting a possible interaction between both species. Further, the coefficients of variation (ratios) for the earthworm and plant endpoints in STEMs were generally comparable to that of the single-species tests. By combining two interacting organisms in a more representative exposure scenario and accounting for an ecologically relevant endpoint (earthworm vertical distribution) that is overlooked in traditional single-species tests, the STEMs approach was suitable for higher-tier biochar ecotoxicological assessment.

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 48h which, although less than chronic tests, is still quite long in comparison to modern methods of chemical analysis whereby results are often available within 24h (“on-site analysis”). Therefore, we assessed the effects of shortening the duration of the earthworm avoidance test from 48 to 24h 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 48h. 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 24h) it is recommended to change the existing ISO guideline, i.e. to decrease the test duration from 48 to 24h. 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.

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.

Simple Biotoxicity Tests for Evaluation of Carbonaceous Soil Additives: Establishment and Reproducibility of Four Test Procedures

Biochar derived from pyrolysis has received much attention recently as a soil additive to sequester carbon and increase soil fertility. Hydrochar, a brown, coal-like substance produced via hydrothermal carbonization, has also been suggested as a beneficial soil additive. However, before soil application, both types of char need to be tested for potential toxic effects. The aim of this study was to develop simple, inexpensive, and easy-to-apply test procedures to identify negative effects of chars but not to provide false-negative results. The following tests, based partly on ISO norm biotoxicity test procedures, were chosen: (i) cress germination test for gaseous phytotoxic emissions; (ii) barley germination and growth test; (iii) salad germination test; and (iv) earthworm avoidance test for toxic substances. Test reproducibility was ensured by carrying out each test procedure three times with the same biochar. Several modifications were necessary to adapt the tests for biochars/hydrochars. The tested biochar did not induce negative effects in any of the tests. In contrast, the beet-root chip hydrochar showed negative effects in all tests. In an extension to the regular procedure, a regrowth of the harvested barley shoots without further nutrient additions yielded positive results for the hydrochar, which initially had negative effects. This implies that the harmful substance(s) must have been degraded or they were water soluble and leached. Tests with a biochar and hydrochar showed that the proposed modified quick-check test procedures provide a fast assessment of risks and effects of char application to soils within a short period of time (<2 wk).

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.

Adapting ecotoxicological tests based on earthworm behavior to assess the potential effectiveness of forest soil liming

Two laboratory experiments were carried out to determine the potential effects of lime application on the behavior of earthworms inoculated in an acidified forest soil from Vosges Mountains. Several field soils were studied: (i) a non-limed soil that had received decades of atmospheric acid depositions ( pH H 2 O = 3.9 ) , (ii) an in situ limed soil that had been limed 6 years before at 2.5 t ha −1 ( pH H 2 O = 4.2 ) and (iii) in vitro limed soils composed of non-limed soil mixed with several lime amounts in the laboratory corresponding to field rates of 1, 2.5, 5, 10 and 20 t ha −1 . Firstly, we adapted the earthworm avoidance test ( ISO, 2006 ) by using Eisenia fetida as the model organism and Lumbricus castaneus , a local species, to determine earthworm preference between non-limed and limed soils. Secondly, a cast production (CP) study was performed according to Capowiez et al. (2009) with Lumbricus terrestris , in seven different treatments (non-limed soil, in situ limed and five in vitro limed soils with pH from 5 to 6.3). Both species avoided the non-limed soil in favour of in situ and in vitro limed soils for both species. Results of the CP bioassay showed that the bioturbation behavior of L. terrestris significantly increased with the increase of soil pH, following a dose–response curve. However, in the long term (>6 years), the application of lime was insufficient to significantly improve soil conditions for enhancing earthworm activity. In conclusion, we may recommend this kind of earthworm laboratory tests with field soils to assess any changes in soil quality over time due to liming application.

From waste to soil—carbon contents, respiration rates and ecotoxicological effects of an uncovered landfill site after 50 years

Purpose In this study, the ecotoxicological effects of the soil contaminations at an uncovered landfill site are assessed with two biological tests (earthworm avoidance test and luminous bacteria test). Furthermore, the state of rotting of the organic substance is estimated. Therefore, total organic carbon (TOC) contents and basal respiration rates are measured.

Eisenia fetida avoidance behavior as a tool for assessing the efficiency of remediation of Pb, Zn and Cd polluted soil

Remediation by means of soil leaching with ethylenediaminetetraacetic acid (EDTA) is capable of extracting the most labile soil fractions, leaving the residual metals in biologically non-available forms. We evaluated the feasibility of the standardized earthworm ( Eisenia fetida) avoidance test for assessing the efficiency of soil remediation of Pb, Zn and Cd polluted soil. Chemical extraction tests (six-step sequential extraction, toxicity characteristic leaching procedure, physiologically based extraction test, diethylenediaminepentaacetic acid extraction) indicated that the mobility, oral bioaccessibility and phytoavailability of Pb, Zn and Cd were consistently reduced. However, the avoidance test showed no significant avoidance of polluted soil in favor of that which had been remediated. Pb, Zn and Cd accumulation in E. fetida mirrored the decreasing pattern of metal potential bioavailability gained by leaching the soil with increasing EDTA concentrations. The calculated bioaccumulation factors indicated the possibility of underestimating the metal bioavailability in soil using chemical extraction tests.

Ecotoxicity of wastes in avoidance tests with Enchytraeus albidus, Enchytraeus crypticus and Eisenia fetida ( Oligochaeta)

Contact bioassays are important for testing the ecotoxicity of solid materials. However, survival and reproduction tests are often not practical due to their duration which may last for several weeks. Avoidance tests with soil invertebrates may offer an alternative or extension to the classic test batteries due to their short duration (days rather than weeks) and due to a sensitive sub-acute endpoint (behavior). The aims of our study were: (a) to evaluate the effects of three solid industrial wastes (incineration ash, contaminated wood chips and contaminated soil) on three Oligochaeta species (enchytraeids Enchytraeus albidus, Enchytraeus crypticus and earthworm Eisenia fetida) in avoidance tests; (b) to compare the sensitivity among the species and to compare results of avoidance test to reproduction tests; (c) to elucidate if measuring the weight in the earthworm avoidance test could be reasonable additional endpoint. Avoidance mostly increased with the increasing percent of waste in the mixture showing a dose–response curve. E. fetida was the most sensitive species and E. crypticus the least one. An additional endpoint, (changes in weight after two-day exposure) was not found to be more sensitive than avoidance reaction, but it confirmed that earthworms staying in the highest concentrations of the waste mixture were affected showing apparent weight reduction. Our results indicate that avoidance tests with earthworms and enchytraeids are feasible for waste testing.

Comparative sensitivity of Eiseniaandrei and Perionyxexcavatus in earthworm avoidance tests using two soil types in the tropics

Terrestrial avoidance behavior is proposed as a fast and cost-effective method for assessing effects of pesticides on earthworms. Tropical species however, have rarely been used in avoidance tests. Avoidance tests were performed with Perionyx excavatus, a tropical species, and Eisenia andrei as the standard species, using chlorpyrifos and carbofuran in artificial and natural soil. Earthworms were exposed to concentrations of 1–900 (chlorpyrifos) and 1–32 (carbofuran) mg a.i. kg −1 dry soil in a two-chamber system under tropical conditions (26 ± 2 °C, 48 h). No significant difference was found in the control tests comparing the two soils used, suggesting soil type did not affect the distribution of the worms. The results suggest a higher sensitivity of E. andrei, with EC 50S for the effect on avoidance behavior for both pesticides being a factor of 2–3 lowers than for P. excavatus. Earthworm avoidance tests with local species should therefore be used with caution when applied as a tool for pesticide risk assessment in the tropics. Endpoints generated through avoidance tests in this study are shown to be less sensitive than reproduction and more sensitive than survival. This was further confirmed by literature data available. Earthworm avoidance tests therefore can only replace survival tests as an initial screening tool for risk assessment.