DNA damage detection by Comet Assay on Daphnia magna: Application in freshwater biomonitoring.

Monitoring of water genotoxicity still remains underexploited in risk assessment. The present study aimed at standardizing and evaluating the sensitivity and applicability of the Comet Assay adapted for Daphnia magna in genotoxicological investigations in freshwater environments. Two sampling campaigns (2014-2015) were performed in the watercourses of a pilot basin located in the Parma district (Italy). Fourteen sampling stations with different Ecological Status and/or EBI values were selected, all with a good Chemical Status according to the EU-Water Framework Directive 2000/60. The Alkaline Comet Assay was performed on 48 h-aged daphnids exposed (24 h) to 23 water samples. In parallel, the acute toxicity test was carried out. Daphnids exposed to samples, collected upstream the main watercourses in non-impacted areas, showed low DNA migration (Tail Intensity percentage - TI% - in the range 2.97-13.21), similar to laboratory controls. An increase in genotoxicity (TI% in the range 20-40) proceeding from the mountain towards the plain area was observed, in agreement with the land uses and the ES/EBI values of the stations. The highest genotoxic damage was observed after exposure to samples from watercourses of the minor hydrographic network in the plain area and waterbodies receiving wastewater treatment plant outflows. A modified version of the Comet Assay able to identify the presence of genotoxins inducing DNA oxidative damage, after standardization, was applied to daphnids treated with waters from 4 selected monitoring stations. The presence of oxidative contaminants was detected downstream a wastewater treatment plant outflow. The Comet Assay on D. magna has proven to be sensitive and able to discriminate among differently impacted areas and might be applied routinely. The FPG-Comet proved to be able to highlight the presence of contaminants causing oxidative stress. In our knowledge, this is the first time that Comet Assay on Daphnia magna is successfully applied for freshwater monitoring.

Early and late maternal effects on hatching phenology of Heterocypris incongruens (Crustacea: Ostracoda).

In ephemeral ponds, the hatching asynchrony of resting eggs may be adaptive and the result of a maternal bet-hedging strategy. A mother can influence the progeny phenology through conditions experienced during life cycle even in early development stages. We investigated the consequences of a hatching delay for offspring and compared early and late maternal effects in a clonal lineage of Heterocypris incongruens. We used females from genetically identical, 40 months old, resting eggs that hatched, asynchronically, after a first (FI) or a second (SI) inundation event. Maternal origin (FI or SI) was considered an early effect involving the maternal response to hatching stimuli during the embryological dormant stage. Maternal age at deposition and egg size were considered late effects that account for maternal conditions during active stage. We compared size and development time of eggs produced by FI and SI females under laboratory condition (24°C 12:12 L:D photoperiod). Maternal origin affected development time to adulthood which was later in FI than in SI females, and fecundity that was higher in FI than in SI females. SI eggs were smaller than FI eggs: size was affected by maternal age at deposition and was directly related to the egg development time. Development time varied from 1 to 117 days and was shorter in SI eggs than in FI eggs. Our results showed that maternal response during embryological stage affects the performance in successive active stages and suggested that hatching asynchrony may be considered a risk spread strategy.

Comet Assay on Daphnia magna in eco-genotoxicity testing.

Detection of potentially hazardous compounds in water bodies is a priority in environmental risk assessment. For the evaluation and monitoring of water quality, a series of methodologies may be applied. Among them, the worldwide used toxicity tests with organisms of the genus Daphnia is one of the most powerful. In recent years, some attempts were made to utilize Daphnia magna in genotoxicity testing as many of the new environmental contaminants are described as DNA-damaging agents in aquatic organisms. The aim of this research was to develop a highly standardized protocol of the Comet Assay adapted for D. magna, especially regarding the isolation of cells derived from the same tissue (haemolymph) from newborn organisms exposed in vivo. Several methods for haemolymph extraction and different Comet Assay parameters were compared. Electrophoretic conditions were adapted in order to obtain minimum DNA migration in cells derived from untreated organisms and, at the same time, maximum sensitivity in specimens treated with known genotoxicants (CdCl2 and H2O2). Additional tests were performed to investigate if life-history traits of the cladoceran (such as the age of adult organisms that provide newborns, the clutch size of origin, the number of generations reared in standard conditions) and the water composition as well, might influence the response of the assay. This study confirms the potential application of the Comet Assay in D. magna for assessing genotoxic loads in aqueous solution. The newly developed protocol could integrate the acute toxicity bioassay, thus expanding the possibility of using this model species in freshwater monitoring (waters, sediment and soil elutriates) and is in line with the spirit of the EU Water Framework Directive in reducing the number of bioassays that involve medium-sized species.