Final Report Summary - PARA-TOX (Assessing the Toxicity of nitrates in a host-Parasite system) The Marie Curie fellowship Para-Tox examined the impact of nitrate pollution on species interactions, including host-parasite relationships in a series of long-term toxicity experiments using a range of aquatic organisms: water fleas (Daphnia magna), two crayfish species (Orconectes virilis and Pacifastacus leniusculus) and fishes (guppies, Poecilia reticulata and three-spined sticklebacks, Gasterosteus aculeatus). The selected host sentinels are keystone species in freshwater ecosystems; detected alterations in their life-history traits can have major implications for ecosystem function. Also, the three-spined stickleback is listed as endangered in national legislation (Spain) and the crayfish as invasive (Europe) so studies on the effects of nitrates on these species are of conservation interest. Further, two of our selected taxa (crayfish and guppies) are captive reared for food consumption or the aquarium trade, so examining how these species respond to changes in water quality is central to optimise culture conditions. Experimentally induced infections revealed that nitrate concentrations ranging from 50 mg/l to 250 mg/l are lethal for the parasite Gyrodactylus turnbulli on guppies. Also, G. turnbulli infection intensity increased on naïve guppies following a 30 days pre-exposure to 50 mg/L and 250 mg/l nitrate, providing evidence that nitrate increases fish susceptibility to disease. Histological changes were detected in the epidermis of fish exposed to nitrate or parasites, as is typical following exposure to chemicals or other erosive stressors. At the highest nitrate level, reduced host survivorship was even recorded suggesting that nitrate concentrations within the values observed in European nitrate vulnerable zones can be lethal for fish. Thus, our data provide empirical evidence to policy makers of the risk posed by nitrates to wild fish. To validate our finding of nitrate toxicity, a similar experiment was conducted using a naturally infected fish population. In this case we examined the abundance of the ectosymbionts Trichodina spp. and Gyrodactylus spp. on three-spined sticklebacks in clean and nitrate polluted waters. Since the guppy experiment revealed lethal effects at 250 mg/l, we only used a 50 mg/L nitrate exposure for this experiment. The nitrate treatment did not significantly alter the abundance of the two ectosymbiont species on sticklebacks compared to controls, which highlights the difficulties of determining safe water quality when there is such variation between taxa. Together with nitrate pollution, the release of exotic species into natural bodies is another major threat to freshwater biodiversity and crayfish, described as ecosystem engineers, are one of the most introduced taxa. We found that nitrate levels below 200 mg/L altered the foraging and agonistic behaviours (e.g. fights, avoidance behaviour) of two widely introduced crayfish species, Orconectes virilis and Pacifastacus leniusculus. Thus, nitrate pollution might explain O. virilis spreading in designed European nitrate vulnerable areas such as the River Lee, UK, where these two crayfish species occur in sympatry.In addition to altering susceptibility to disease and animal behaviour, nitrate pollution can result in cultural eutrophication. Under these conditions, algae and microbes proliferate causing major ecological impacts, reducing the aesthetic appearance of water and may even represent a public health concern. Cladocerans including Daphnia magna naturally top-down control microbes and phytoplankton. A microcosm experiment with D. magna interacting with natural microbial communities indicated the grazing pressure of D. magna was seriously compromised under nitrate pollution and/or warming. Also, under the same environmental conditions, individual assays indicated that D. magna experienced reduced body size, filtering rate and fecundity under nitrate and/or warming. Given the key role of Daphnia in nutrient cycling, our results increase our knowledge of how nitrate pollution under climate change may affect freshwater ecosystems. Results from this study have also re-defined the tolerance thresholds of protists and micro-metazoans, which are used as sentinel taxa in monitoring studies of freshwater ecosystems and sewage treatment plant performance. Since some of the observed taxa (e.g. Vorticella spp.) can be D. magna epibionts, our results suggest that nitrate pollution may increase the epibiont abundance on D. magna in contrast to our observations on guppies and sticklebacks.Research into eutrophication at Cardiff University were complemented with studies on metal bioaccumulation and trophic interactions in fish communities of the eutrophicated Lake Titicaca. Also, the fellow validated a new body condition index for fish, and reported that eutrophication seemed to increase the body condition of the Iberian red-fin barbel (Barbus haasi). On this species, two species of the ciliate Trichodina were also reported for the first time in the Iberian Peninsula. In order to monitor individual variation, it is often necessary to mark aquatic animals but the consequences of this procedure are relatively understudied in small body-size fish species. Therefore, an additional experiment was performed during the fellowship on three-spined sticklebacks to determine whether standard practices in fish monitoring such as fin-clipping could increase fish susceptibility to disease. Our results indicated that this practice did not affect the abundance of the two ectosymbionts species on sticklebacks in clean or nitrate polluted waters. Thus, our experiment supports fin-clipping as a non-lethal sampling procedure for marking or tissue collection even in small fish. Finally, a collaboration with an aquarium retailer enabled determining the efficacy of sea salt bath, metronidazole and malachite/formalin against a white spot disease outbreak on a molly stock (Poecilia sphenops).In conclusion, nitrate levels equal or higher than the legal safe threshold (50 mg/l) can be toxic for fish and/or their parasites. Further, nitrate levels within the values found in European nitrate vulnerable areas (below 250 mg/l) can alter the foraging behaviour of important keystones in freshwater ecosystems, including two invasive crayfish species. A better understanding of how nitrate pollution alters individuals’ competitive abilities can increase the accuracy of predictive risk maps regarding how eutrophication might affect the distributional range of species. Besides a conservation interest per se for native species, such basic information can help identify areas vulnerable to invasions. Moreover, in aquaculture the rate of water exchange required to maintain safe nitrate levels for fish in recirculating water systems has ecological (i.e. water is a valuable and limited commodity) and economic (i.e. productivity, electric consumption) repercussions.Scientific outputs of this project are disseminated on his profile at Cardiff University research team (http://cripescardiff.co.uk/people/alberto-maceda-veiga/). He now continues his research at the University of Barcelona where he co-supervises a student examining the effects of nitrates on the invasive Gambusia holbrooki and how these effects observed at the individual level translate into multi-trophic impacts. Other collaborations established by the fellow while at Cardiff University, particularly those on invasive species, also equipped Dr Maceda-Veiga with the necessary skills for his next postdoctoral position at Doñana Biological Station, Spain.