Acute and chronic toxicity of the fungicide carbendazim to the earthworm Eisenia fetida under tropical versus temperate laboratory conditions.

Research efforts into the potential side-effects of pesticides on beneficial organisms have focused on temperate test species and conditions. There is thus a need for studies into the ecotoxicity of a vaster range of pesticides under tropical conditions. The present study therefore aimed to compare the acute and chronic toxicity of the fungicide carbendazim to the earthworm Eisenia fetida under tropical and temperate conditions. To this end, laboratory toxicity tests were conducted with a tropical and European strain of E. fetida, using different artificial (OECD and TAS) and natural (LUFA and TNS) soils, and under different test temperatures (20 °C and 28 °C). In the acute lethality tests with artificial soils, the tropical strain of E. fetida was three to four order of magnitude less sensitive than the European strain, which is ascribed to the higher test temperature and (hence) higher microbial activity/pesticide degradation. The tropical strain was particularly sensitive in the tropical natural soil, which was attributed to the low pH (3.9) of this soil. The chronic toxicity tests overall also showed a lower sensitivity of the tropical strain on reproduction. These findings thus support the use of toxicity data generated under temperate conditions in tropical pesticide effect assessments. However, intensive agricultural practices in the tropics may dictate that exposure levels (and hence potentially also risks) are higher.

Ecotoxicity of boric acid in standard laboratory tests with plants and soil organisms.

To verify the continuous sensitivity of ecotoxicological tests (mainly the test organisms), reference substances with known toxicity are regularly tested. Ideally, this substance(s) would lack specificity in its mode action, be bioavailable and readily attainable with cost-effective means of chemical characterization. Boric acid has satisfied these criteria, but has most recently been characterized as a substance of very high concern, due to reproductive effects in humans, thus limiting its recommendation as an ideal reference toxicant. However, there is probably no other chemical for which ecotoxicity in soil has been so intensively studied; an extensive literature review yielded lethal (including avoidance) and sublethal data for 38 taxa. The ecotoxicity data were evaluated using species sensitivity distributions, collectively across all taxa, and separately according to species type, endpoints, soil type and duration. The lack of specificity in the mode of action yielded broad toxicity among soil taxa and soil types, and provided a collective approach to assessing species sensitivity, while taking into consideration differences in test methodologies and exposure durations. Toxicity was species-specific with Folsomia candida and enchytraied species demonstrating the most sensitivity; among plants, the following trend occurred: dicotyledonous (more sensitive) ≫ monocotyledonous ≫ gymnosperm species. Sensitivity was also time and endpoint specific, with endpoints such as lethality and avoidance being less sensitive than reproduction effects. Furthermore, given the breadth of data and toxicity demonstrated by boric acid, lessons learned from its evaluation are discussed to recommend the properties required by an ideal reference substance for the soil compartment.

Analysis and dissipation of the antiparasitic agent ivermectin in cattle dung under different field conditions.

Cattle treated with the veterinary parasiticide ivermectin fecally excrete residues. The authors report the exposition and dissipation characteristics of these residues in dung of ivermectin-treated cattle and in soil beneath this dung on pastures in Canada, France, Switzerland, and The Netherlands. Residues were quantified for dung collected from cattle after 3 d, 7 d, 14 d, and 28 d posttreatment and subsequently exposed in the field for up to 13 mo. The authors optimized a high-performance liquid chromatography-fluorescence detection method to detect ivermectin residues in dung and soil matrices. They showed that a solid phase extraction and purification step generally can be eliminated to reduce the time and cost of these analyses. They also found that the addition of water to relatively dry samples improves the extraction efficiency of residues. They then analyzed the field samples to document differences in ivermectin dissipation in cattle dung among sites, with 50% dissipation times of up to 32 d and 90% dissipation times >396 d. They further showed that the dissipation characteristics of residues are comparable between dung of ivermectin-treated cattle and dung to which ivermectin has been added directly. Lastly, they report the first use of a desorption electrospray ionization-high-resolution-mass spectrometric method to detect residues of metabolites in a dung matrix. Environ Toxicol Chem 2016;35:1924-1933. © 2016 SETAC.

Nontarget effects of ivermectin residues on earthworms and springtails dwelling beneath dung of treated cattle in four countries.

The authorization of veterinary medicinal products requires that they be assessed for nontarget effects in the environment. Numerous field studies have assessed these effects on dung organisms. However, few studies have examined effects on soil-dwelling organisms, which might be exposed to veterinary medicinal product residues released during dung degradation. The authors compared the abundance of earthworms and springtails in soil beneath dung from untreated cattle and from cattle treated 0 d, 3 d, 7 d, 14 d, and 28 d previously with ivermectin. Study sites were located in different ecoregions in Switzerland (Continental), The Netherlands (Atlantic), France (Mediterranean), and Canada (Northern Mixed Grassland). Samples were collected using standard methods from 1 mo to 12 mo after pat deposition. Ivermectin concentrations in soil beneath dung pats ranged from 0.02 mg/kg dry weight (3 mo) to typically <0.006 mg/kg dry weight (5-7 mo). Earthworms were abundant and species-rich at the Swiss and Dutch sites, less common with fewer species at the French site, and essentially absent at the Canadian site. Diverse but highly variable communities of springtails were present at all sites. Overall, results showed little effect of residues on either earthworms or springtails. The authors recommend that inclusion of soil organisms in field studies to assess the nontarget effects of veterinary medicinal products be required only if earthworms or springtails exhibit sensitivity to the product in laboratory tests. Environ Toxicol Chem 2016;35:1959-1969. © 2015 SETAC.

A four-country ring test of nontarget effects of ivermectin residues on the function of coprophilous communities of arthropods in breaking down livestock dung.

By degrading the dung of livestock that graze on pastures, coprophilous arthropods accelerate the cycling of nutrients to maintain pasture quality. Many veterinary medicinal products, such as ivermectin, are excreted unchanged in the dung of treated livestock. These residues can be insecticidal and may reduce the function (i.e., dung-degradation) of the coprophilous community. In the present study, we used a standard method to monitor the degradation of dung from cattle treated with ivermectin. The present study was performed during a 1-yr period on pastures in Canada, France, The Netherlands, and Switzerland. Large effects of residue were detected on the coprophilous community, but degradation of dung was not significantly hampered. The results emphasize that failure to detect an effect of veterinary medicinal product residues on dung-degradation does not mean that the residues do not affect the coprophilous community. Rather, insect activity is only one of many factors that affect degradation, and these other factors may mask the nontarget effect of residues. Environ Toxicol Chem 2016;35:1953-1958. © 2015 SETAC.

Validation of a standard field test method in four countries to assess the toxicity of residues in dung of cattle treated with veterinary medical products.

Registration of veterinary medical products includes the provision that field tests may be required to assess potential nontarget effects associated with the excretion of product residues in dung of treated livestock (phase II, tier B testing). However, regulatory agencies provide no guidance on the format of these tests. In the present study, the authors report on the development of a standardized field test method designed to serve as a tier B test. Dung was collected from cattle before and up to 2 mo after treatment with a topical application of a test compound (ivermectin). Pats formed of dung from the different treatments were placed concurrently in the field to be colonized by insects. The abundance, richness, and diversity of insects developing from egg to adult in these pats were compared across treatments using analysis of variance tests. Regression analyses were used to regress abundance, richness, and diversity against residue concentrations in each treatment. Results of the regression were used to estimate mean lethal concentration (LC50) values. The robustness of the method and the repeatability of its findings were assessed concurrently in 4 countries (Canada, France, Switzerland, and The Netherlands) in climatically diverse ecoregions. Results were generally consistent across countries, and support the method's formal adoption by the European Union to assess the effects of veterinary medical product residues on the composition and diversity of insects in dung of treated livestock. Environ Toxicol Chem 2016;35:1934-1946. © 2015 Crown in the right of Canada. Published by Wiley Periodicals Inc., on behalf of SETAC.

Evaluation of eco-toxicological effects of the parasiticide moxidectin in comparison to ivermectin in 11 species of dung flies.

A standardized bioassay previously developed with ivermectin for the yellow dung fly (Scathophagidae) and the face fly (Muscidae) was applied to test the response of 11 dung fly species to the presumably less toxic parasiticide moxidectin. The results were compared to existing data for the same species tested with ivermectin, albeit two new species (Scathophaga suilla and Musca domestica) were tested here with both the substances. Estimated lethal effect concentrations LC50 at which 50% of the flies died ranged more than tenfold from 0.012 mg moxidectin/kg fresh dung for Sepsis neocynipsea (Sepsidae) to 0.140 mg moxidectin/kg fresh dung for the house fly Musca domestica (Muscidae). In most of the species, we additionally revealed sub-lethal effects at lower moxidectin concentrations in terms of retarded growth and development and reduced body size. Mortality thresholds were about ten times higher for moxidectin than for ivermectin, hence moxidectin is indeed less toxic than ivermectin in absolute terms. Crucially, we obtained strong correlations among the 11 tested fly species in both lethal and sub-lethal responses to the two substances, such that species relatively sensitive to ivermectin were also relatively sensitive to moxidectin. Such correlations are expected if the two substances are structurally related and function in the same manner by disturbing ion channel transport. Methodologically speaking, all species used proved suitable for toxicological testing of parasiticides.

Standardized laboratory tests with 21 species of temperate and tropical sepsid flies confirm their suitability as bioassays of pharmaceutical residues (ivermectin) in cattle dung.

Veterinary pharmaceuticals excreted in the dung of treated livestock can have strong non-target effects on the dung organism community. We report results of ecotoxicological tests with ivermectin for 21 species of temperate (Europe, North America) and tropical (Asia, Central America) black scavenger flies (Diptera: Sepsidae), using standardized methods developed previously for the yellow dung fly and the face fly. Our study documents great variation in ivermectin sensitivity of more than two orders of magnitude among species and even populations within species: estimated lethal effect concentrations LC(50) (at which 50% of the flies died) ranged from 0.05 to 18.55 µg/kg dung fresh weight (equivalent to 0.33-132.22 µg/kg dung dry weight). We also show that controlled laboratory tests can--within reasonable limits-be extended to the field or to laboratory settings without climate control, as obtained LC(50) were roughly similar. In addition to lethal effects, our study revealed relevant sub-lethal effects at lower ivermectin concentrations in terms of prolonged development, smaller body size and reduced juvenile growth rate. Finally, oviposition choice experiments showed that females generally do not discriminate against dung containing ivermectin residues. We conclude that sepsid flies are well suited test organisms for pharmaceutical residues in the dung of livestock due to their ease and speed of rearing and handling, particularly in the tropics, where high-tech laboratory equipment is often not available.

Improving ecological risk assessment in the Mediterranean area: selection of reference soils and evaluating the influence of soil properties on avoidance and reproduction of two oligochaete species.

A current challenge in soil ecotoxicology is the use of natural soils as test substrates to increase ecological relevance of data. Despite the existence of six natural reference soils (the Euro-soils), some parallel projects showed that these soils do not accurately represent the diversity of European soils. Particularly, Mediterranean soils are not properly represented. To fill this gap, 12 natural soils from the Mediterranean regions of Alentejo, Portugal; Cataluña, Spain; and Liguria, Italy, were selected and used in reproduction and avoidance tests to evaluate the soil habitat function for earthworms (Eisenia andrei) and enchytraeids (Enchytraeus crypticus). Predictive models on the influence of soil properties on the responses of these organisms were developed using generalized linear models. Results indicate that the selected soils can impact reproduction and avoidance behavior of both Oligochaete species. Reproduction of enchytraeids was affected by different soil properties, but the test validity criteria were fulfilled. The avoidance response of enchytraeids was highly variable, but significant effects of texture and pH were found. Earthworms were more sensitive to soil properties. They did not reproduce successfully in three of the 10 soils, and a positive influence of moisture, fine sand, pH, and organic matter and a negative influence of clay were found. Moreover, they strongly avoided soils with extreme textures. Despite these limitations, most of the selected soils are suitable substrates for ecotoxicological evaluations.

The effects of the insecticide lambda-Cyhalothrin on the earthworm Eisenia fetida under experimental conditions of tropical and temperate regions.

Plant Protection Products can affect soil organisms and thus might have negative impacts on soil functions. Little research has been performed on their impact on tropical soils. Therefore, the effects of the insecticide lambda-Cyhalothrin on earthworms were evaluated in acute and chronic laboratory tests modified for tropical conditions, i.e. at selected temperatures (20 and 28°C) and with two strains (temperate and tropical) of the compost worm Eisenia fetida. The insecticide was spiked in two natural soils, in OECD artificial soil and a newly developed tropical artificial soil. The effects of lambda-Cyhalothrin did rarely vary in the same soil at tropical (LC50: 68.5-229 mg a.i./kg dry weight (DW); EC50: 54.2-60.2 mg a.i./kg DW) and temperate (LC50: 99.8-140 mg a.i./kg DW; EC50: 37.4-44.5 mg a.i./kg DW) temperatures. In tests with tropical soils and high temperature, effect values differed by up to a factor of ten.

Results of an international ring test with the dung fly Musca autumnalis in support of a new OECD test guideline.

A standardized bioassay using the face fly, Musca autumnalis L. (Diptera: Muscidae), was developed to test the lethal and sublethal toxicity of parasiticide residues in livestock dung. The repeatability of this test was assessed for the parasiticide ivermectin in seven tests performed in four laboratories in Germany and France. Additional results of limit tests were provided by two laboratories from the UK. Test results had an acceptable range of heterogeneity. The calculated effect concentration at which 50% emergence was observed (EC50) averaged 4.65+/-2.17 (Standard Deviation (SD) microg ivermectin/kg fresh dung (range: 1.20-7.7)). Effects on emergence were, with one exception, not observed below the No Observed Effect Concentration (NOEC) ranging between 1.11 and 3.33microg ivermectin/kg. No effect on development time was observed. We conclude that the face fly is suitably sensitive, and the methods sufficiently repeatable, to support use of this standardized bioassay by the international community in the registration of new veterinary pharmaceuticals. Following these considerations, this species was accepted as a possible test organism in a recently published OECD Guideline (No. 228).

Lethal and sublethal toxic effects of a test chemical (ivermectin) on the yellow dung fly (Scathophaga stercoraria) based on a standardized international ring test.

A standardized bioassay using the yellow dung fly, Scathophaga stercoraria L. (Diptera: Scathophagidae), was developed to test the lethal and sublethal toxicity of parasiticide residues in livestock dung. The repeatability of the bioassay was assessed for the parasiticide ivermectin in 13 tests performed by seven laboratories in Germany, the United Kingdom, Switzerland, and Canada. Test results had an acceptable range of heterogeneity. The calculated median effective concentration for 50% (EC50) egg-to-adult mortality was 20.9 +/- 19.1 microg ivermectin/kg dung fresh weight (FW) (mean +/- standard deviation; range, 6.33-67.5 microg/kg). Mortality was not observed below a calculated no-observable-effect concentration (NOEC) of 8.1 +/- 7.7 microg/kg FW. However, prolonged development time (and, in a subset of tests, reduced body size) was observed above a calculated NOEC of 0.8 +/- 0.8 microg/kg FW. An oviposition site choice test revealed that yellow dung fly females do not discriminate among dung of different ivermectin concentrations. Thus, the yellow dung fly is suitably sensitive, and the methods are sufficiently repeatable, to support use of this standardized bioassay by the international community in the registration of new veterinary pharmaceuticals.

Effects of three pesticides on the avoidance behavior of earthworms in laboratory tests performed under temperate and tropical conditions.

Little research has been performed on the impact of pesticides on earthworms under tropical conditions. Taking into consideration the often-limited resources in tropical countries, simple screening tests are needed. Therefore, it was investigated whether three pesticides relevant for the Brazilian Amazon (benomyl, carbendazim, lambda-cyhalothrin) affect the avoidance behavior of the earthworm Eisenia fetida. The tests were performed for two days according to ISO guideline 17512 but were adapted to tropical conditions (i.e. test substrate, test organism and temperature). The results indicate that this test gives reproducible and reliable results. Toxicity values (NOEC, EC50) are lower than those determined in 14 day-acute mortality tests and are approximately in the same range such as those found in 56 day-chronic reproduction tests with the same earthworm species, which were performed in parallel. Therefore, the use of the earthworm avoidance tests is recommended as a screening tool for the risk assessment of pesticides.

Effects of the fungicide benomyl on earthworms in laboratory tests under tropical and temperate conditions.

Soil organisms play a crucial role in the terrestrial ecosystem. Plant protection products (PPPs) are known to affect soil organisms and might have negative impacts on soil functions influenced by these organisms. Little research has been performed to date on the impact of PPPs on tropical soil ecosystems. Therefore, in this study it was investigated whether the effects of the fungicide benomyl (chosen as a model substance) differ between tropical and temperate regions and whether data generated under temperate conditions can be used for the Environmental Risk Assessment (ERA) in tropical regions. The effect of benomyl on earthworms was evaluated in acute and chronic laboratory tests modified for tropical conditions. These tests were performed at two temperatures (20 degrees C and 28 degrees C) and with two strains (temperate and tropical) of the compost worm Eisenia fetida. The fungicide was spiked in two natural and two artificial soils. In addition to the organization for economic cooperation and development (OECD) artificial soil, a tropical artificial soil (TAS), containing a tropical fern product (xaxim) instead of peat, was developed in this study. The results from the laboratory tests and a literature review showed that the effects of benomyl were, on average, lower under tropical conditions (LC(50): 450-630 mg active ingredient (a.i.)/kg; EC(50): 0.8-12.9 mg a.i./kg) than under temperate conditions (LC(5)0: 61-67 mg a.i./kg; EC(50): 1.0-1.6 mg a.i./kg) by a maximum factor of 10.3 (acute tests) and 12.9 (chronic tests). This result might be caused by an increased degradation of benomyl, and/or its first metabolite carbendazim, at higher temperatures, but a different sensitivity of the two worm strains cannot be ruled out. Despite the lower toxicity under tropical conditions and assuming comparable application rates, a preliminary assessment confirms the risk of benomyl to soil invertebrates under both conditions.

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

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

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

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