Recommendations for ecotoxicity testing with stygobiotic species in the framework of groundwater environmental risk assessment.

As a consequence of the growing global dependence on groundwater resources, environmental risk assessments (ERA) for groundwater are increasingly required and, with that, ecotoxicological studies with groundwater fauna (stygofauna). However, the literature on the ecotoxicological studies with stygobiotic species (i.e. species that complete their life cycle exclusively in groundwater) has not expanded significantly since the first paper published in this field. The limitations regarding the use of stygobiotic species for ecotoxicological testing are clear and consistent across the globe; stygobiotic species are often 1) naturally present in low numbers, 2) difficult to collect, and 3) difficult to culture under laboratory conditions. This paper reviews the methods used in ecotoxicological studies performed with stygobiotic species, and provides ten recommendations for Good Laboratory Practice (GLP) for such tests. The recommendations focused on the following 10 points: 1) the taxonomic identification, the life stage/size and gender of the test organisms; 2) collection methodology of the organisms, including collection location, conditions and methods; 3) holding and acclimation conditions in the laboratory; 4) exposure conditions such as test set up and exposure time, number of replicates and densities of organisms in tests and in test vessels; 5) range-finding test set up and schedule; 6) final test design, including details of controls and treatments, and replication options; 7) incubation conditions, specifying temperature, pH and oxygenation levels throughout the test; 8) test duration; 9) observations and endpoints; 10) test validity criteria and compliance. The recommendations were developed for the purpose of supporting future short-term ecotoxicological testing with stygofauna through providing consistency in the protocols. A discussion of the potential implications for groundwater managers and decision-makers committed to ERA for groundwater is included.

The role of freshwater copepods in the environmental risk assessment of caffeine and propranolol mixtures in the surface water bodies of Spain.

In this study we aimed at assessing: (i) the environmental risk posed by mixtures of caffeine and propranolol to the freshwater ecosystems of Spain; (ii) the sensitivity of freshwater copepod species to the two compounds; (iii) if the toxicity of caffeine and propranolol to freshwater copepods contributes to the environmental risk posed by the two compounds in the freshwater bodies of Spain. The environmental risk was computed as the ratio of MECs (i.e. the measured environmental concentrations) to PNECs (i.e. the respective predicted no-effect concentrations). The effects of caffeine and propranolol on the freshwater cyclopoid Diacyclops crassicaudis crassicaudis were tested both individually and in binary mixtures. Propranolol posed an environmental risk in some but not in all the surface water ecosystems of Spain investigated in this study, while caffeine posed an environmental risk to all the investigated freshwater bodies, both as single compound and in the mixture with propranolol. Propranolol was the most toxic compound to D. crassicaudis crassicaudis, while caffeine was non-toxic to this species. The CA model predicted the toxicity of the propranolol and caffeine mixture for this species. D. crassicaudis crassicaudis was much less sensitive than several other aquatic species to both compounds. The sensitivity of D. crassicaudis crassicaudis does not increase the environmental risk posed by the two compounds in the freshwater bodies of Spain, however, further testing is recommended since the effect of toxicants on freshwater copepods can be more pronounced under multiple stressors and temperature increasing due to climate change.

Genotoxic effects of commercial formulations of Chlorpyrifos and Tebuconazole on green algae.

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.