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.