Testing the response of benthic diatom assemblages to common riverine contaminants.

Benthic diatoms constitute keystone assemblages in riverine ecosystems, and their structure is used to support regulatory water quality assessment. However, no standard ecotoxicological tests exist using integrated responses of the benthic diatom assemblages. This work aimed to assess whether benthic diatom assemblages are responsive to different riverine contaminants through a previously developed rapid toxicity test, supporting future attempts towards its standardization and integration in both prospective and retrospective Environmental Risk Assessment (ERA) schemes. We selected two benthic diatoms assemblages likely responding similarly to pollution (similar IPS diatom index score), collected from two rivers in Northern-Central Portugal (sites: Palhal and Cabreia). Fresh whole diatom assemblages were exposed for 48 h to five model contaminants (glyphosate, imidacloprid, SDS, CuSO4, and Pb). At the end of the test, changes induced by the exposures in overall yield and in the yield of each diatom genus were assessed. The assemblage collected at Palhal was invariably more responsive and sensitive than that collected at Cabreia, both considering overall and genus-specific yields, regardless of the tested contaminant. Achnanthes, Fragilaria and Navicula were the most responsive genus, regardless of the tested contaminant or assemblage. The distinct response profiles observed for the two assemblages to the same contaminants at the same concentration ranges suggest that using this test method to support prospective ERA is inadequate. However, the method can be an asset supporting retrospective ERA, as the responses seem to be shaped by the interplay of resilience drivers promoted by the local conditions, e.g. adaptive changes in assemblage structure.

Can parasites adapt to pollutants? A multigenerational experiment with a Daphnia × Metschnikowia model system exposed to the fungicide tebuconazole.

There is increasing evidence about negative effects of fungicides on non-target organisms, including parasitic species, which are key elements in food webs. Previous experiments showed that environmentally relevant concentrations of fungicide tebuconazole are toxic to the microparasite Metschnikowia bicuspidata, a yeast species that infects the planktonic crustacean Daphnia spp. However, due to their short-term nature, this and other experimental studies were not able to test if parasites could potentially adapt to these contaminants. Here, we tested if M. bicuspidata parasite can adapt to tebuconazole selective pressure. Infected D. magna lineages were reared under control conditions (no tebuconazole) and environmentally realistic tebuconazole concentrations, for four generations, and their performance was compared in a follow-up reciprocal assay. Additionally, we assessed whether the observed effects were transient (phenotypic) or permanent (genetic), by reassessing parasite fitness after the removal of selective pressure. Parasite fitness was negatively affected throughout the multigenerational exposure to the fungicide: prevalence of infection and spore load decreased, whereas host longevity increased, in comparison to control (naive) parasite lineages. In a follow-up reciprocal assay, tebuconazole-conditioned (TEB) lineages performed worse than naive parasite lineages, both in treatments without and with tebuconazole, confirming the cumulative negative effect of tebuconazole. The underperformance of TEB lineages was rapidly reversed after removing the influence of the selective pressure (tebuconazole), demonstrating that the costs of prolonged exposure to tebuconazole were phenotypic and transient. The microparasitic yeast M. bicuspidata did not reveal potential for rapid evolution to an anthropogenic selective pressure; instead, the long-term exposure to tebuconazole was hazardous to this non-target species. These findings highlight the potential environmental risks of azole fungicides on non-target parasitic fungi. The underperformance of these microbes and their inability to adapt to such stressors can interfere with the key processes where they intervene. Further research is needed to rank fungicides based on the hazard to non-target fungi (parasites, but also symbionts and decomposers), towards more effective management and protective legislation.

Concentration and timing of application reveal strong fungistatic effect of tebuconazole in a Daphnia-microparasitic yeast model.

Given the importance of pollutant effects on host-parasite relationships and disease spread, the main goal of this study was to assess the influence of different exposure scenarios for the fungicide tebuconazole (concentration×timing of application) on a Daphnia-microparasitic yeast experimental system. Previous results had demonstrated that tebuconazole is able to suppress Metschnikowia bicuspidata infection at ecologically-relevant concentrations; here, we aimed to obtain an understanding of the mechanism underlying the anti-parasitic (fungicidal or fungistatic) action of tebuconazole. We exposed the Daphnia-yeast system to four nominal tebuconazole concentrations at four timings of application (according to the predicted stage of parasite development), replicated on two Daphnia genotypes, in a fully crossed experiment. An "all-or-nothing" effect was observed, with tebuconazole completely suppressing infection from 13.5µgl-1 upwards, independent of the timing of tebuconazole application. A follow-up experiment confirmed that the suppression of infection occurred within a narrow range of tebuconazole concentrations (3.65-13.5µgl-1), although a later application of the fungicide had to be compensated for by a slight increase in concentration to elicit the same anti-parasitic effect. The mechanism behind this anti-parasitic effect seems to be the inhibition of M. bicuspidata sporulation, since tebuconazole was effective in preventing ascospore production even when applied at a later time. However, this fungicide also seemed to affect the vegetative growth of the yeast, as demonstrated by the enhanced negative effect of the parasite (increasing mortality in one of the host genotypes) at a later time of application of tebuconazole, when no signs of infection were observed. Fungicide contamination can thus affect the severity and spread of disease in natural populations, as well as the inherent co-evolutionary dynamics in host-parasite systems.

Progressive acclimation alters interaction between salinity and temperature in experimental Daphnia populations.

Environmental stressors rarely act in isolation, giving rise to interacting environmental change scenarios. However, the impacts of such interactions on natural populations must consider the ability of organisms to adapt to environmental changes. The phenotypic adaptability of a Daphnia galeata clone to temperature rise and salinisation was investigated in this study, by evaluating its halotolerance at two different temperatures, along a short multigenerational acclimation scenario. Daphniids were acclimated to different temperatures (20°C and 25°C) and salinities (0gL(-1) and 1gL(-1), using NaCl as a proxy) in a fully crossed design. The objective was to understand whether acclimation to environmental stress (combinations of temperature and salinity) influenced the response to the latter exposure to these stressors. We hypothesize that acclimation to different temperature×salinity regimes should elicit an acclimation response of daphniids to saline stress or its interaction with temperature. Acute (survival time) and chronic (juvenile growth) halotolerance measures were obtained at discrete timings along the acclimation period (generations F1, F3 and F9). Overall, exposure temperature was the main determinant of the acute and chronic toxicity of NaCl: daphniid sensitivity (measured as the decrease of survival time or juvenile growth) was consistently higher at the highest temperature, irrespective of background conditions. However, this temperature-dependent effect was nullified after nine generations, but only when animals had been acclimated to both stressors (high salinity and high temperature). Such complex interaction scenarios should be taken in consideration in risk assessment practices.