Daniel Gomez Isaza

The acceleration of anthropogenic activity has caused novel or extreme environmental challenges for species to contend with. Species must now contend with complex combinations of environmental threats which include habitat degradation, pollution, and climate change. Yet, the lack of available data on how species cope when confronted with multiple environmental challenges poses a significant challenge to conservation. Nutrient pollution is regarded as one of the most common and widespread forms of habitat degradation. Anthropogenic activities have caused a rampant increase in nitrate (NO3-) concentrations, peaking at concentrations above 100 mg NO3- L-1. Aquatically respiring organisms (amphibians, amphipods, fish) are particularly vulnerable to elevated nitrate concentrations, causing significant physiological and behavioural alterations. These alterations may be exacerbated by the presence of additional threats, but information on the interactive effects of nitrate and other environmental stressors is scarce. Therefore, the first aim of this thesis was to quantify the impacts of elevated nitrate exposure on key fitness related traits using meta-analytical tools and examine whether organismal survival is affected by nitrate and its interaction with other stressors. Across studies, exposure to elevated nitrate decreased the activity, growth, and survival of aquatic taxa. Further, antagonistic interactions between nitrate and other stressors were most predominant, indicating that future research should focus on interacting stressors that act on the same physiological mechanism (e.g. pH, elevated temperature and hypoxia) as they represent the greatest likelihood for “ecological surprises”. The meta-analysis also revealed that data on fish and crustaceans is limited and these taxa were therefore the focus of subsequent experimental chapters. Environmental pH is one factor that may modify the toxicity of nitrate by exacerbating its uptake and disrupting key physiological performance traits. To test this prediction, blueclaw crayfish (Cherax destructor) were exposed to one of two pH levels (pH 5.0 and 7.0) and three nitrate concentrations (0, 50 and 100 mg NO3- L-1). Aerobic scope (maximal minus standard oxygen uptake rates) was measured at six time points and crayfish performance (chelae strength and righting response) was assessed after 28 days. Aerobic scope was compromised by the interaction between low pH and nitrate and resulted in prolonged elevations of standard oxygen uptake. Declines in aerobic scope corresponded with a lowering of chelae strength and righting capacity. Similarly, combined exposure to nitrate and low pH reduced the aerobic scope and swimming performance of spangled perch (Leiopotherapon unicolor), an effect underpinned by an accumulation of nitrate within the blood and reductions to blood-oxygen carrying capacity. Nitrate-induced reductions to oxygen transport were expected to lower species’ tolerance of, and impede their capacity to compensate for prolonged exposure to, elevated temperatures. To test this prediction, silver perch (Bidyanus bidyanus) were exposed to 28 or 32oC and simultaneously exposed to one of three nitrate concentrations (0, 50 or 100 NO3- L-1). Indicators of performance, aerobic scope and upper thermal tolerance (CTMAX) were assessed after 8-weeks. The aerobic scope of 28oC-acclimated fish declined with increasing temperature, and the effect was more pronounced in nitrate-exposed individuals. Declines in aerobic scope corresponded with poorer swimming performance and a 0.8oC decrease in CTMAX. In contrast, acclimation to 32oC masked the effects of nitrate; swimming performance was thermally insensitive, aerobic scope was maintained, and CTMAX was increased by ~1oC. These results are suggestive of a cross-tolerance interaction and potential mechanisms underlying this interaction were explored by measuring attributes of the heart, gills and blood. Plasticity of the ventricle (increased myocardial thickness) and gill structures (decreased lamellar thickness, interlamellar cell mass) following high temperature acclimation were uncovered, which potentially provide overlapping protection to elevated nitrate concentrations. Lastly, the impact of elevated nitrate on behavioural and physiological responses of silver perch to acute hypoxia were investigated. Fish were exposed to one of three nitrate treatments (0, 50 or 100 mg NO3- L-1) for three weeks, then, behavioural avoidance and aquatic surface respiration (ASR) responses to progressive hypoxia were quantified. Physiological changes evoked under progressive hypoxia were also assessed, including haematological changes, ventilation frequency (VF) and swimming performance. Exposure to elevated nitrate did not alter behavioural avoidance of low oxygen but nitrate-exposed fish did utilise ASR at a higher PO2 threshold during progressive hypoxia. Nitrate exposure had small impacts on key physiological responses; haemoglobin and haematocrit levels were reduced and the VF of nitrate-exposed fish were elevated both at rest and under hypoxic conditions. These physiological disturbances during nitrate exposure had pronounced effects on the swimming performance and hypoxia tolerance of fish and indicate that nitrate pollution is likely to increase the susceptibility of fish to aquatic hypoxia. Overall, presence of nitrate and additional stressors impaired energy homeostasis, such that aerobic scope is reduced and compromised the functioning of aerobically supported traits (e.g. swimming, righting, growth), due to disruptions of the blood-oxygen carrying capacity. Physiological compensation can offset the effects of nitrate, possibly explaining the predominance for antagonistic interactions. This body of work highlights the unpredictability of stressor interactions and underscores the importance of experimental assessments in addressing the eco-physiological constraints of species in our ever-changing world.