Effects of transgenic corn and Cry1Ab protein on the nematode, Caenorhabditis elegans

The effects of the insecticidal Cry1Ab protein from Bacillus thuringiensis (Bt) on the nematode, Caenorhabditis elegans, were studied with soil from experimental fields cultivated with transgenic Bt corn (MON810) and with trypsinized Cry1Ab protein expressed in Escherichia coli. The content of Cry1Ab protein was above the detection limit of an ELISA test in only half of the soil samples obtained from transgenic plots, ranging from 0.19 to 1.31 ng g⁻¹ dry weight. In a laboratory bioassay, C. elegans was exposed to rhizosphere and bulk soil from fields with isogenic or transgenic corn or to solutions of Cry1Ab protein (0, 24, 41, 63, 118, and 200 mg l⁻¹) over a period of 96 h, with growth and reproduction serving as the test parameters. Nematode reproduction and growth were significantly reduced in rhizosphere and bulk soil of Bt corn compared with soil from isogenic corn and were significantly correlated with concentrations of the Cry1Ab protein in the soil samples. Moreover, the toxicity of pure Cry1Ab protein to the reproduction and growth of C. elegans was concentration-dependent. As significant inhibition occurred at relatively high concentrations of the Cry1Ab protein (41 mg l⁻¹), the effects of the soil samples from Bt corn could not be assigned directly to the toxicity of the Cry1Ab protein. The results demonstrate that bioassays with the nematode, C. elegans, provide a promising tool for monitoring the potential effects of Bt toxins in aqueous medium and soils.     

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.     

A soil toxicity test using the nematode Caenorhabditis elegans and an effective method of recovery

A new method for recovering nematodes from soils in an efficient, reproducible, and non-destructive manner has been developed. It was used to conduct short-term soil toxicity tests using the soil-dwelling nematode Caenorhabditis elegans and several different soil types spiked with copper chloride. The recovery method, which involves centrifugation through a colloidal silica suspension, allows the nematodes to be extracted from the soil matrix so that lethality can be assessed. The nematodes are unharmed by the recovery procedure, and both live and dead individuals are recovered with high efficiency (well over 80%), allowing reproducible concentration-response curves to be made after a 24-h exposure. The LC₅₀s for copper were increased about tenfold by the presence of soil, and different soils had significantly different effects on toxicity. Toxicity of copper ion was also influenced by the concentration of sodium chloride and potassium chloride in the test solution, and the presence of bacteria increased the toxicity of copper ion in some soils. The LC₅₀s in soil were close to the LC₅₀ for the 2-week earthworm soil toxicity test, suggesting that a 24-h nematode toxicity test may be comparable to the 2-week earthworm test in terms of sensitivity.     

A semi-fluid gellan gum medium improves nematode toxicity testing

This study examined an alternative test medium for nematodes that use gellan gum as the gelling agent instead of agar. The semi-fluid consistency of the gel-like component nematode growth gellan gum (CNGG) supports three-dimensional distribution of the nematodes and food bacteria, but still allows free movement of the former. Moreover, flexible preparation of the medium and easy recovery of the test organisms are possible. Here, the effects of the nematicides ivermectin (pharmaceutical) and aldicarb (pesticide) and of the metal cadmium on the growth and reproduction of the free-living nematodes Caenorhabditis elegans and Panagrolaimus cf. thienemanni were studied in CNGG media. Results were compared to those obtained with the standard liquid test media in order to evaluate the applicability of CNGG for nematode toxicity testing. The sensitivity of P. cf. thienemanni to all three substances was found to be higher than that of C. elegans, but both nematodes showed the highest sensitivity to ivermectin exposure. This raises concerns about the risk posed by the pharmaceutical to non-target nematodes. In contrast to ivermectin bioassays carried out in CNGG medium, those conducted in liquid medium resulted in wide-ranging variability between and within replicates. Thus, CNGG seems to be particularly valuable for testing hydrophobic substances with a high sorption affinity as it favors their sorption to food bacteria and minimizes contact with the surfaces of the test vessels. However, the medium was less suitable for deriving toxicity thresholds for cadmium and may likewise not be an appropriate choice for testing other metals. The medium introduced herein was shown to be appropriate for sublethal nematode toxicity testing and likely provides a convenient environment for testing other nematode species. Besides improved testing of hydrophobic substances, CNGG also offers advantages for long-term studies, such as full life-cycle experiments, in which fresh medium is regularly needed. Moreover it may be beneficial for testing other poorly soluble or insoluble substances, such as nanoparticles.     

Availability of metals to the nematode Caenorhabditis elegans: toxicity based on total concentrations in soil and extracted fractions

Current regulation of metals in soils is based on total metal concentrations rather than on actual exposure concentrations. Considering the extreme variation in soil physicochemical properties, total concentrations are not reflective of the availability and resultant toxicity of metals in different soils. In this study, the availability of Cd, Cu, Ni, Pb, and Zn to the free-living soil nematode Caenorhabditis elegans was assessed after 24-h exposures in three soils using a sequential soil extraction procedure. Albany soil, sampled from southern Georgia, USA, is characterized by a high sand content, whereas Cecil soil from the Piedmont region of Georgia contains higher fractions of clay and organic matter. The final soil was an American Society for Testing and Materials (ASTM) artificial medium composed of peat, kaolin clay, sand, and calcium carbonate. Based on their composition, ASTM medium would sorb metals most strongly and Albany soil the least strongly. In fact, 24-h lethal concentrations to 50% (LC50s) of nematodes for the five metals as determined by the total metal concentration followed this trend. In addition, water-extractable metals were lowest in ASTM medium and highest in Albany soil when spiked at the same concentrations. Our data show the need to consider soil type when performing toxicological tests and establishing site-specific allowable metal concentrations in soil.     

Utility of Caenorhabditis elegans for Assessing Heavy Metal Contamination in Artificial Soil

There is an increasing need for the development of soil bioassay protocols. Currently the only internationally standardized soil test organism is the lumbricid earthworm Eisenia fetida. Many alternate soil test organisms have been proposed. This work compares Caenorhabditis elegans to several other test organisms, including E. fetida, for heavy metals in soil. In this evaluation, such factors as ease of testing and culturing, duration of testing, soil volume needed, and the sensitivity of the organism were considered. Results show that C. elegans is more sensitive than most other organisms evaluated and is similar in response to E. fetida. The second issue compares C. elegans LC₅₀ values to heavy metals criteria specified in the US EPA regulations for land application of sewage sludge. Currently, the regulations are set on total metals in the soil and do not consider bioavailability of the metals. Regulations do not consider soil physiochemical properties, such as organic matter content, clay content, and cation exchange capacity, which have been shown to affect the availability of metals to soil organisms. While the C. elegans LC₅₀ values are above standard values in artificial soil, work in our lab indicates that the LC₅₀s are below regulation values for other soil types. Due to the ease of culturing and testing, good sensitivity, along with the wealth of biological information and ecological relevance, C. elegans is a good organism for use in soil bioassays. Received: 2 June 1999/Accepted: 13 January 2000     

Tolerance of the Nematode Caenorhabditis elegans to pH,Salinity, and Hardness in Aquatic Media

The toxicity of many chemicals depends on the physical conditions of the test environment, and any change or adjustment made to the tests can alter the results. Therefore it is important to establish the sensitivity of the test organism over a range of test conditions to determine when it is necessary to make adjustment and to what extent. In this study, we established the tolerance range of the nematode Caenorhabditis elegans for pH, salinity and hardness using 24- (without food source) and 96-h (with food source) aquatic toxicity tests. The tests were performed in two media: K-medium and moderately hard reconstituted water (MHRW). C. elegans has high tolerance under these test conditions. In K-medium worms survived a pH range of 3.1 to 11.9 for 24 h and 3.2 to 11.8 for 96 h without significant (p > 0.05) lethality. In MHRW the pH range was 3.4 to 11.9 for 24 h and 3.4 to 11.7 for 96 h. Salinity tolerance tests were approximated with NaCl and KCl individually. Up to 15.46 g/L NaCl and 11.51 g/L KCl were tolerated by C. elegans in K-medium without significant lethality (p > 0.05). In MHRW higher salt concentrations were tolerated; about 20.5 g/L NaCl and 18.85 g/L KCl did not show any adverse effect compared to control. Hardness tolerance was tested by adding NaHCO₃. The nematode could tolerate 0.236 to 0.246 g/L of NaHCO₃. The high tolerance of C. elegans to these test conditions (pH, salinity, and hardness) allows more versatility than other organisms commonly used in aquatic toxicity tests. It also allows the monitoring of effluents and receiving waters from freshwater or estuarine sources without dilution or adjustment. Received: 14 February 1996/Revised: 20 June 1996     

Ag2Se quantum dots damage the nervous system of nematode Caenorhabditis elegans

Silver selenide quantum dots (Ag₂Se QDs), as a novel type of QDs, are valuable in the biomedical application due to their low-toxic and excellent optical property in near infrared region, but the biosafety assessment of Ag₂Se QDs is rare. In this study, the findings suggested that the accumulation of Ag₂Se QDs in the body of nematodes decreased the lifespan and damaged normal neurobehaviors of Caenorhabditis elegan (C. elegans). Furthermore, Ag₂Se QDs caused excessive reactive oxygen species (ROS) productions and altered expressions of several genes associated with redox equilibrium, which might contribute to neurotoxic outcomes in nematode C. elegans. According to this study, it is necessary and important for researchers to pay attention to the biosafety assessment of presumed low-toxic nanomaterials, like Ag₂Se QDs, especially on sensitively toxic targets, i.e. the nervous system.

     

Toxicity of four nitrogen-heterocyclic polyaromatic hydrocarbons (NPAHs) to soil organisms

The aims of this study were: (i) to investigate the toxicity of N-heterocyclic polyaromatic hydrocarbons (NPAHs) quinoline, acridine, phenazine, and 1,10-phenanthroline to the soil invertebrates Eisenia fetida, Enchytraeus crypticus, Folsomia candida, and Caenorhabditis elegans, (ii) to compare the toxicity of four NPAHs and the species sensitivity, and (iii) to discuss possible risks of these compounds in soils. Different toxicities were found for the tested NPAHs which might be partially explained by their structure and properties. Effect concentrations expressed as soil pore-water concentrations were related to log K_ow, which indicated narcosis as the most probable mode of toxic action. The species sensitivity decreased in the rank: springtails >enchytraeids=earthworms> nematodes. Predicted no-effect concentration (PNEC) values were calculated for all tested species giving values from 0.5 to 6.8 mg/kg. It is unlikely that there is a risk for soil organisms in natural soils where lower NPAHs concentrations are expected.     

Delivery of alpha-tocopherol through soluble dietary fibre-based nanofibres for improving the life span of Caenorhabditis elegans

The effect of alpha-tocopherol (α-TOC) delivered by soluble dietary fibre-based nanofibres (α-TOC-SDNF) on the life span of nematode Caenorhabditis elegans N2 (wild type) and TK22 (mev-1 mutants) with and without heat shock was investigated. Without heat shock, the wild-type and mev-1 mutants maintained in the 100?µg/mL of α-TOC-SDNF had longer life spans than their respective blank control groups. With heat shock, the wild-type N2 in the 200?µg/mL of α-TOC-SDNF had a survival rate of 5% at day 49, while no nematodes survived in the blank control group. An increased pharyngeal pumping rate was observed in the α-TOC-SDNF treated mev-1 mutants worms compared to the blank control group. Encapsulating α-TOC in SDNF yielded protective effects and the life span and pumping rate of C. elegans was increased with α-TOC delivered by SDNF.     

Carbofuran effects in soil nematode communities: using trait and taxonomic based approaches

This work intends to implement the use of native soil nematode communities in ecotoxicological tests using a model pesticide and two geographically nematode communities (Mediterranean and sub-tropical) in order to obtain new perspectives on the evaluation of the toxic potential of chemical substances.The environmental condition of the nematode communities was described using a trait-based approach (grouping the organisms according to their feeding traits) and a traditional taxonomic method (identification to family level). Effects on total nematode abundance, number of families and abundance of nematode feeding groups as well as potential shifts in both trophic and family structure were assessed.Agricultural soils from Curitiba (Brazil) and Coimbra (Portugal) were sampled and the corresponding nematode communities were extracted. Part of the collected soil was defaunated and spiked with four doses of a carbofuran commercial formulation. Afterwards each of the replicates was inoculated with a nematode suspension containing ≈200 or 300 nematodes. After 14 and 28 d of exposure the nematodes were extracted, counted and identified at family level and separately classified according to their feeding traits. The patterns of nematode responses revealed a decrease in the total abundance and a reduction in the number of families. Despite the similar effects observed for both communities, statistically significant toxic effects were only found within the Portuguese community. The total nematode abundance was significantly reduced at the highest carbofuran concentrations and significant shifts in the family structure were detected. However, the trophic structure, i.e., the contribution of each feeding group for the overall community structure, did not significantly change along the contamination gradient. Results showed that using such a trait-based approach may increase the ecological relevance of toxicity data, by establishing communalities in the response to a chemical from two different taxonomic communities, although with potential loss of information on biodiversity of the communities.     

Comparison of the sensitivity of three nematode species to copper and their utility in aquatic and soil toxicity tests

Nematodes are useful organisms for aquatic and soil toxicity testing because of their abundance and diversity as well as their ease of culturing and maintenance in the laboratory. The nematode Caenorhabditis elegans has been used extensively in toxicity testing, but its sensitivity to metal exposures in relation to other nematodes remains unclear. In this study, we compare the sensitivity and ease of use of two other rhabditid nematodes, Panagrellus redivivus and Pristionchus pacificus, to C. elegans. Toxicity endpoints were chosen to investigate the effects of Cu on the survival of these nematodes after soil exposures and on the survival, reproduction, movement, and feeding behavior of nematodes after exposures in aquatic medium. In all lethality testing, P. pacificus was the most sensitive, C. elegans exhibited intermediate sensitivity, and P. redivivus was the least sensitive. Reproduction and movement of C. elegans and reproduction of P. pacificus were decreased 50% by similar concentrations of Cu (EC50s approximately 2 mg/L), but P. pacificus movement was less sensitive to Cu exposures (EC50 = 8 mg/L). Although all nematodes may be useful in lethality assays, using P. redivivus in toxicity tests is complicated by the presence of two sexes and difficulties in obtaining age-synchronized cultures. Pristionchus pacificus is an ideal acute toxicity-testing organism because of its sensitivity and ease of culturing. However, C. elegans appears to be more sensitive and therefore most useful in behavioral assays. Future studies of the relative sensitivities of nematodes in toxicity testing should continue to investigate additional toxicants, nematode species, and quantifications of sublethal effects after soil exposures.     

Toxicity Assessment of Two Soils from Jales Mine (Portugal) Using Plants: Growth and Biochemical Parameters

Contaminants in soils can enter food chains through primary producers. Bioavailable contaminants can induce growth, and reproductive or biochemical changes in plants. To evaluate the bioavailability of heavy metals in two soils from Jales mine surroundings, bioassays with the plants Brassica rapa (RCBr) and Avena sativa were performed. Biochemical parameters (protein and malondialdehyde [MDA] content, and catalase and peroxidase activities) were also measured. The soils had different heavy metal contents: JNC soil contained low heavy metal concentrations, whereas JC soil had high heavy metal contents. Results stressed the difference between species sensitivity, with A. sativa showing no toxicity effects when exposed to both soils. On the other hand, B. rapa presented a decrease in growth parameters when exposed to JNC soil and no changes when exposed to JC soil. A Life Cycle Bioassay confirmed this trend for B. rapa exposed to JNC soil, but also evidenced that JC soil was affecting B. rapa in terms of flower and seed pod production. Biochemical assays showed that plants affected by heavy metals also displayed oxidative stress, with an increase in MDA production, reduction of protein content, and reduction of catalase and peroxidase activities. All bioassays revealed that JNC soil, although with a lower heavy metal content, had a higher bioavailable fraction when compared to JC soil, which consequently increased its toxicity to plants.     

A plant life-cycle bioassay for contaminated soil,with comparison to other bioassays: Mercury and zinc

Bioassays using rapid-cycling plants allow measurement of multiple endpoints and assessment of impacts on both growth and reproduction. Selections of Brassica rapa develop rapidly in a broad range of soils and are very consistent in production of flower and seed. Their sensitivity to variation in growth conditions was investigated to define the variables that most affect performance. Yield differences between soils were substantial, indicating the need for careful selection and use of control treatments. The sensitivity to contaminants was investigated with applications of mercury (Hg) and zinc (Zn) to three soils. In a sand soil, bloom initiation was slowed by <10 mg Hg kg^–1 soil and <50 mg Zn kg^–1 soil. In contrast, lettuce emergence and earthworm survival were less sensitive to these metals in this soil. Survival of Daphnia magna and the Microtox^® assay in soil extracts were more sensitive to Hg than bloom initiation, but less sensitive to Zn. A similar relationship among the bioassays was observed for two finer-textured soils, although for these, effects were usually apparent only at soil metal concentrations >200 mg kg^–1. Enzyme assays were included for comparison, but were not sensitive to Hg contamination. Rapid-cycling B. rapa selections are suitable for routine bioassays, and are representative of several widely distributed and utilized species.     

Stress-Response Protein Expression and DAF-16 Translocation were Induced in Tributyltin-Exposed <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Exposure to tributyltin (TBT) with graded sublethal doses (0, 1, 10, 50 and 200 nM) resulted in the release of reactive oxygen species (ROS) and DNA damage in the nematode Caenorhabditis elegans. After the worms carrying transgenic reporters were exposed to TBT, the expressions of superoxide dismutase (SOD-3), glutathione S-transferase (GST-4) and heat shock proteins (HSP-4, HSP-16.2 and HSP-70) were semi-quantified after exposure. The results indicated that TBT caused dose-dependent induction of SOD-3, GST-4, HSP-4 and HSP-70. Furthermore, TBT exposure also induced DAF-16 translocation from cytoplasm to nucleus. The results implicated that C. elegans might be a potential animal model for TBT level monitoring and toxicity assessment.     

Effect of Single and Paired Metal Inputs in Soil on a Stress-Inducible Transgenic Nematode

A toxicity test using a transgenic strain of the free-living soil nematode Caenorhabditis elegans carrying a stress-inducible β-galactosidase reporter has been adapted for use in soil biomonitoring. High concentrations (250 μg · g⁻¹) of cadmium are required to induce the stress response in worms exposed to Lufa 2.2 soil. Even at relatively high concentrations, the response to copper and zinc additions alone is minimal, yet combinations of cadmium and copper in the test soil induce a larger response than with cadmium alone at the same concentration. In contrast, the addition of both zinc and cadmium induces a lower response than cadmium additions alone. Analysis of the interstitial water suggests that there is preferential occupation by copper of sorption sites in the soil, allowing more cadmium to remain in solution. Conversely, cadmium and zinc would appear to interact similarly with the soil constituents, resulting in an increase of both metals in solution with increased additions to the soil. Aquatic tests mimic the results of the soil test, so it is not increased cadmium availability alone that causes an increased stress response when both cadmium and copper are present. The presence of other metals could reduce the amount of cadmium available, which may be one factor in the zinc moderation of the stress response to cadmium. Intracellular mechanisms may also contribute to the copper enhancement of the stress response to cadmium. Received: 30 November 1998/Accepted: 12 May 1999     

Assessment of Toxicity Reduction in Wastewater Effluent Flowing Through a Treatment Wetland Using Pimephales promelas, Ceriodaphnia dubia, and Vibrio fischeri

Effluent toxicity is regularly assessed with Ceriodaphnia dubia short-term chronic and Vibrio fischeri toxicity tests. Condition factor and hemotocrit of fish have recently been used to assess fish health following exposure to xenoestrogens in complex municipal effluents. To assess the ability of a treatment wetland to reduce or remove toxicity of a municipal effluent, we compared C. dubia and V. fischeri bioassays to Pimephales promelas responses in situ. Final whole effluent was diverted to a constructed wetland and effluent samples were taken daily from four sites, at incremental distances from the inflow, for a 3-week study. Overlapping 7-day C. dubia tests and V. fischeri assays were conducted with samples from each wetland site concurrent with a 3-week fish exposure. C. dubia survival and fecundity were significantly (< 0.0001) reduced at the inflow, but steadily improved with distance from the inflow. Fish condition and hematocrit were lower (α < 0.05) at wetland sites closer to the inflow than other wetland sites and laboratory controls. However, effluent toxicity was not detected by V. fischeri bioassays. Our findings indicate that 7-day C. dubia bioassays were most sensitive to effluent toxicity and suggest that other bioassays should be used concurrent with V. fischeri assays for municipal effluent toxicity testing. Received: 20 November 2000/Accepted: 18 June 2001     

Effects of Zinc Contamination on a Natural Nematode Community in Outdoor Soil Mesocosms

The effect of zinc on the indigenous nematode fauna of a sandy soil was determined in an experimentally contaminated outdoor field plot. The aims of the study were to describe and quantify the changes in density of separate nematode taxa and total nematodes, and the changes in the number of taxa, species diversity, community maturation, and species composition in response to zinc exposure with time, and to compare the observed responses with benchmark concentrations for soil as derived from the species sensitivity distribution (SSD) for zinc toxicity. Speciation of zinc in pore water was considered and CaCl₂-exchangeable zinc concentrations were used as a measure of the bioavailable zinc fraction. After contamination, a reduction of total zinc and an increase of labile sorbed zinc over time occurred, concurrent with various changes in soil and biological characteristics. Data analyses on the nematode species revealed different sensitivity levels for several population and community endpoints to zinc exposure. Based on no observed effect concentration (NOEC) values, the most sensitive community-level response was obtained with principal response curve (PRC) analysis, which incorporates all density data in a single analysis. The PRC-based community NOECs were 56, 100, and 100 mg total Zn/kg dry soil after 3, 10, and 22 months of exposure, respectively. Based on 0.01 M CaCl₂-exchangeable zinc, the community response appeared to increase, as NOECs were 4.9, 4.4, and 0.67 mg exchangeable Zn/kg dry weight. Total density was least sensitive, followed by diversity of taxa and the Shannon-Wiener index. NOECs for separate species covered a broad range from sensitive to tolerant species. This range of sensitivities was similar to the one found for other species groups tested in the field plot soil. A comparison was made between benchmark concentrations HC5 and HC50 derived from the general SSD of soil organisms for zinc and the nematode response data. These comparisons roughly confirm the predictions of the SSD model, that is, the community NOEC is in agreement with the benchmark that should protect the soil ecosystem's integrity, and large adverse effects were found at the benchmark derived for setting remediation urgency. Received: 9 November 2000/Accepted: 2 August 2001     

Using earthworms to test the efficiency of remediation of oil-polluted soil in tropical Mexico

This study focuses on the medium-term effects of soil bioremediation on mortality and reproduction rates of Eisenia fetida (laboratory experiment) and of the tropical earthworm Polypheretima elongata (field experiment). We compared soils restored with the two bioremediation technologies landfarming (LF) and compost-bioremediation (BI) with control soils and with soils contaminated with 1% and 2% of petroleum. Control and restored soils both were fertile and showed low hydrocarbon contents. The mortality of E. fetida was not influenced by soil restoration and by contamination with 1% petroleum; it only increased in soils contaminated with 2% petroleum. However, the reproduction rate of E. fetida was significantly lower in the soils restored with LF and in those contaminated with 1% crude oil and significantly higher in the soils restored with BI. P. elongata showed the same reaction as E. fetida. We conclude that it is important to include reproduction or other sub-lethal tests for earthworms when estimating the efficiency of restoration techniques.