Research Article: Ecologically relevant biomarkers reveal that chronic effects of nitrate depend on sex and life stage in the invasive fish Gambusia holbrooki

Date Published: January 28, 2019

Publisher: Public Library of Science

Author(s): Oriol Cano-Rocabayera, Adolfo de Sostoa, Francesc Padrós, Lorena Cárdenas, Alberto Maceda-Veiga, Amitava Mukherjee.


Agricultural intensification and shifts in precipitation regimes due to global climate change are expected to increase nutrient concentrations in aquatic ecosystems. However, the direct effects of nutrients widely present in wastewaters, such as nitrate, are poorly studied. Here, we use multiple indicators of fish health to experimentally test the effects of three ecologically relevant nitrate concentrations (<10, 50 and 250 mg NO3-/l) on wild-collected mosquitofish (Gambusia holbrooki), a species widely introduced for mosquito biocontrol in often eutrophic waters. Overall, biomarkers (histopathology, feeding assays, growth and caloric content and stable isotopes as indicators of energy content) did not detect overt signs of serious disease in juveniles, males or females of mosquitofish. However, males reduced food intake at the highest nitrate concentration compared to the controls and females. Similarly, juveniles reduced energy reserves without significant changes in growth or food intake. Calorimetry was positively associated with the number of perivisceral fat cells in juveniles, and the growth rate of females was negatively associated with δ15N signature in muscle. This study shows that females are more tolerant to nitrate than males and juveniles and illustrates the advantages of combing short- and long-term biomarkers in environmental risk assessment, including when testing for the adequacy of legal thresholds for pollutants.

Partial Text

Nutrient pollution results in man-made eutrophication, which is amongst the most pernicious forms of global change affecting aquatic ecosystems around the world [1,2]. Human causes of eutrophication are the inefficient use of fertilizers, aquaculture and urban outflows and atmospheric nitrogen deposition from combustion [3–5]. The ecological effects of eutrophication are well-known, including toxic algal blooms and high mortality of animals due to dissolved oxygen depletion at night [6–8]. Water authorities attempt to mitigate eutrophication by establishing safe nutrient concentrations (e.g. OECD, 1982; Directive 91/676/ECC [9,10]). However, the direct toxicity of nutrients to wildlife under chronic exposure is still poorly studied [11,12]. Considering agricultural intensification continues unabated and water purification is costly [13], there is the pressing need to get better insight into the health effects that environmentally relevant nutrient concentrations have on wildlife.

Mosquitofish were evenly distributed by size and sex amongst treatments and fish did not differ in size amongst aquaria at Time 0, either for males (F = 0.54; P = 0.90) or females (F = 1.22; P = 0.28). However, females (Mean ± S.E. = 37.6 ± 0.42 mm) were significantly bigger than males (25.9 ± 0.18 mm; t = 25.8; P < 0.001). There was no mortality in males due to nitrate and only minor mortality was recorded for juveniles (3.6%) and females (3.3%). Nonetheless, mortality did not differ significantly between treatments (Nitrate: χ2 = 1.59, P = 0.66) or sexes (Sex: χ2 = 0.43, P = 0.93). This is the first comprehensive study examining the chronic effects of nitrate on a widely introduced fish species, as exemplified by the eastern mosquitofish (Gambusia holbrooki) [28]. Moreover, this is one of the few ecotoxicological studies using short- and long-term biomarkers (e.g. growth, histopathology, feeding assays) in females, males and juveniles of the same species. Overall, we did not find overt clinical signs of disease, which supports the prevailing idea that many invasive species, including mosquitofish, have wide tolerance to changes in water quality [28,56]. However, the fact that nitrate altered food intake or energetic reserves in males and juveniles suggests that concentrations >50 mg NO3-/l cannot be considered completely safe [11,21].




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