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The earthworm (Eisenia foetida) is an attractive sentinel species for detecting genotoxicity in soil. In this study, an improved single-cell gel electrophoresis (SCGE) assay was developed for detecting DNA damage in the coelomocytes (lymphocytes) of earthworms. Coelomocytes were obtained from the coelomic fluid using a modified extrusion medium that did not include the mucolytic agent guaiacol. The extruded coelomocytes contained at least three types of cells: eleocytes (75% of the total), amoebocytes, and granulocytes. The DNA migration parameters were determined for untreated cells of each type in order that the assay could be performed with minimum inter- and intra-individual variation. In addition, lysis time was reduced to 10 min, and only one neutralization step was used. DNA damage was detected in isolated eleocytes treated with hydrogen peroxide and cadmium, and in eleocytes from earthworms exposed for up to 21 days to soil containing polycyclic aromatic hydrocarbons. The SCGE assay using earthworm eleocytes appears to be a sensitive biomarker for evaluating exposure to genotoxic compounds.

Widespread pollution from agriculture is one of the major causes of the poor freshwater quality currently observed across Europe. Several studies have addressed the direct impact of agricultural pollutants on freshwater biota by means of laboratory bioassays; however, as far as copepod crustaceans are concerned, the ecotoxicological research is scarce for freshwater species and almost nonexistent for the hypogean ones. In this study, we conducted a comparative analysis of the available literature data on the sensitivity of freshwater copepods to agricultural pollutants. We also assessed the acute and chronic sensitivity of a hypogean and an epigean species, both belonging to the Crustacea Copepoda Cyclopoida Cyclopidae, to two N-fertilizers (urea and ammonium nitrate) and two herbicides (ARIANE TM II from Dow AgroSciences LLC, and Imazamox), widely used for cereal agriculture in Europe. According to the literature review, freshwater copepods are sensitive to a range of pesticides and N-fertilizers. Ecotoxicological studies on hypogean species of copepods account only one study. There are no standardized protocols available for acute and chronic toxicity tests for freshwater copepods, making comparisons about sensitivity difficult. From our experiments, ionized ammonia proved to be more toxic than the herbicide Imazamox, in both short and chronic bioassays. Urea was the less toxic chemical for both species. The hypogean species was more sensitive than the epigean one to all chemicals. For both species and for all tested chemicals, acute lethality and chronic lethality were induced at concentrations higher than the law limits of good water body quality in Europe, except for ionized ammonia, which provoked the chronic lethality of the hypogean species at a lower concentration. The hazardous concentration (HC) of unionized ammonia for 5 % of freshwater copepods, obtained by a species sensitivity distribution, was 92 μg l −1 , significantly lower than the HC computed for traditional test species from freshwater environments.

Aquatic environments located in areas cultivated with biotech soybean were studied. Water and sediment samples were analyzed for insecticides, acute toxicity, genotoxicity, detoxification biomarkers, and fish diversity. Samples were taken in the core area of soybean cultivation in Argentina; all measures were related to the distance between the crops and the streams sampled. Endosulfan (alpha + beta) concentrations as high as 553.33 microg/kg were found in sediments from environments located at 0.15 m from treated fields. Ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P4501A1 (CYP1A1) gene expression in fish showed the highest correlation with the environmental concentration of endosulfan. These biomarkers and mortality of amphipods significantly correlated with the concentration of endosulfan in water and sediment, which correlates inversely with the distance between the crop and streams. The differences with respective controls disappear at distances greater than 5 m. The fish diversity was significantly lower from distances between the margin of the stream and soybean crops, not exceeding 2 m.

The alkaline single-cell gel electrophoresis assay (comet assay) was used for the study of the genotoxic effects of insecticide Chlorpyrifos and fungicide Tebuconazole (commercial formulations) on two freshwater green algae species, Pseudokirchneriella subcapitata and Nannocloris oculata, after 24 h of exposure. The percentage of DNA in tail of migrating nucleoids was taken as an endpoint of DNA impairment. Cell viability was measured by fluorometric detection of chlorophyll "a" in vivo and the determination of cell auto-fluorescence. Only the higher concentration of Chlorpyrifos tested resulted to affect significantly the cell viability of P. subcapitata, whereas cells of N. oculata were not affected. Tebuconazole assayed concentrations (3 and 6 mg/l) did not affect cell viability of both species. The results of comet assay on P. subcapitata showed that Chlorpyrifos concentration evaluated (0.8 mg/l) exerted a genotoxic effects; while for the other specie a concentration of 10 mg/l was needed. Tebuconazole was genotoxic at 3 and 6 mg/l for both species. The comet assay evidenced damage at the level of DNA simple strains molecule at pesticide concentrations were cytotoxicity was not evident, demonstrating that algae are models to take into account in ecological risk assessments for aquatic environments.

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