Multi-generational impacts of organic contaminated stream water on Daphnia magna: A combined proteomics, epigenetics and ecotoxicity approach.

The present study aimed to elucidate the mechanisms of organismal sensitivity and/or physiological adaptation in the contaminated water environment. Multigenerational cultures (F0, F1, F2) of Daphnia magna in collected stream water (OCSW), contaminated with high fecal coliform, altered the reproductive scenario (changes in first brood size timing, clutch numbers, clutch size etc.), compromised fitness (increase hemoglobin, alteration in behavior), and affected global DNA methylation (hypermethylation) without affecting survival. Using proteomics approach, we found 288 proteins in F0 and 139 proteins in F2 that were significantly differentially upregulated after OCSW exposure. The individual protein expressions, biological processes and molecular functions were mainly related to metabolic processes, development and reproduction, transport (protein/lipid/oxygen), antioxidant activity, increased globin and S-adenosylmethionine synthase protein level etc., which was further found to be connected to phenotype-dependent endpoints. The proteomics pathway analysis evoked proteasome, chaperone family proteins, neuronal disease pathways (such as, Parkinson's disease) and apoptosis signaling pathways in OCSW-F0, which might be the cause of behavioral and developmental alterations in OCSW-F0. Finally, chronic multigenerational exposure to OCSW exhibited slow physiological adaptation in most of the measured effects, including proteomics analysis, from the F0 to F2 generations. The common upregulated proteins in both generations (F0 & F2), such as, globin, vitellinogen, lipid transport proteins etc., were possibly play the pivotal role in the organism's physiological adaptation. Taken together, our results, obtained with a multilevel approach, provide new insight of the molecular mechanism in fecal coliform-induced phenotypic plasticity in Daphnia magna.

Validation of a two-generational reproduction test in Daphnia magna: An interlaboratory exercise.

Effects observed within one generation disregard potential detrimental effects that may appear across generations. Previously we have developed a two generation Daphnia magna reproduction test using the OECD TG 211 protocol with a few amendments, including initiating the second generation with third brood neonates produced from first generation individuals. Here we showed the results of an inter-laboratory calibration exercise among 12 partners that aimed to test the robustness and consistency of a two generation Daphnia magna reproduction test. Pyperonyl butoxide (PBO) was used as a test compound. Following experiments, PBO residues were determined by TQD-LC/MS/MS. Chemical analysis denoted minor deviations of measured PBO concentrations in freshly prepared and old test solutions and between real and nominal concentrations in all labs. Other test conditions (water, food, D. magna clone, type of test vessel) varied across partners as allowed in the OECD test guidelines. Cumulative fecundity and intrinsic population growth rates (r) were used to estimate "No observed effect concentrations "NOEC using the solvent control as the control treatment. EC10 and EC-50 values were obtained regression analyses. Eleven of the twelve labs succeeded in meeting the OECD criteria of producing >60 offspring per female in control treatments during 21days in each of the two consecutive generations. Analysis of variance partitioning of cumulative fecundity indicated a relatively good performance of most labs with most of the variance accounted for by PBO (56.4%) and PBO by interlaboratory interactions (20.2%), with multigenerational effects within and across PBO concentrations explaining about 6% of the variance. EC50 values for reproduction and population growth rates were on average 16.6 and 20.8% lower among second generation individuals, respectively. In summary these results suggest that the proposed assay is reproducible but cumulative toxicity in the second generation cannot reliably be detected with this assay.