An integrated multidisciplinary approach to study the effects of copper and osmotic stress in fish

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel UniversitySince many estuarine zones are impacted by copper contamination, there is an on-going effort to develop Biotic Ligand Models (BLMs) predicting copper toxicity in transitional environments. In the first stage of this project, a critical analysis of the BLM framework identified some aspects of the model that required further investigation. In particular, a BLM for estuaries needed (a) a better characterization of the dissolved organic matter (DOC) and its effect on copper availability, and (b) the inclusion in the model’s equation of a salinity-correction factor modulating the relationship between copper accumulation on the biotic ligand and toxicity. The first issue was addressed by modelling the data produced using a Chelex resin method to determine the labile fraction of copper in samples of mixed riverine and estuarine waters. A refined and simplified BLM equation was then presented, accounting for both the DOC characteristics and the relevance of the osmotic gradient in modulating the relationship between copper accumulation and toxicity. A critical analysis of the literature on copper toxicity and salinity led to the hypothesis that copper-exposed fish are more sensitive to osmotic stresses, as copper interferes with their osmoregulatory pathways. In particular, the cytosolic isoform-2 of the enzyme carbonic anhydrase (CA2) was identified as an osmotic effector protein targeted by copper and involved in osmotic stress response pathways, hence representing a mechanistic link between the combined effects of copper exposure and osmotic stress. To test this hypothesis, two in vivo studies were performed, using the euryhaline fish sheepshead minnow (Cyprinodon variegatus) and applying different rates of salinity changes as a way of dosing osmotic stress. The results showed a disturbance in plasma ion homeostasis after the salinity transitions, but notably the magnitude of the disturbance was greater in the copper-exposed individuals, suggesting a sensitizing effect of copper on the responses of fish to osmotic stress. Gene expression data demonstrated that CA2 is targeted by copper and confirmed the role of the enzyme in osmoregulatory pathways, as further supported by a promoter analysis of the gene coding for zebrafish CA2, which revealed the presence of osmotic-stress related elements. Overall, these results suggest that CA2 is an osmotic effector protein whose response can be activated by a medium level of osmotic stress through a combination of transcriptional and post-translational control circuits

The sublethal physiological effects of exposure to copper and silver mixtures on rainbow trout (Oncorhynchus mykiss)

The mechanisms behind metal-metal interactions in freshwater environments are currently not well understood. Freshwater environments consist of many different types of metals, from those naturally present such as copper (Cu) and those that originate from anthropogenic sources like silver (Ag). Both Cu and Ag use apical sodium (Na+) channels for uptake into the gills of freshwater fish. In the gills, the mechanisms of Cu2+ and Ag+ toxicity appear to be similar to one another, which is by inhibiting Na+ /potassium (K+)- adenosine triphosphatase (NKA) and carbonic anhydrase (CA). Inhibition of NKA and CA results in ionoregulatory disturbances where branchial Na+ and chloride (Cl-) uptake is reduced and can result in mortality. The overall goal of this research was to build a better understanding of the interactions between Cu and Ag in the context of sub-chronic impacts of metal mixtures on the rainbow trout (Onchorhynchus mykiss). Juvenile rainbow trout were exposed for 10 and 14 days to Cu-only (10-day: 1.0 µM Cu2+, 14-day: 0.35 µM Cu2+), Ag-only (0.04 µM Ag+) or a Cu 2+ + Ag+ mixture (10-day: 1.0 µM Cu2+ + 0.04 µM Ag+; 14-day: 0.35 µM Cu2+ + 0.04 µM Ag+). The effects of Cu-Ag interactions were assessed by measuring bioaccumulation in whole gill, liver and kidney samples, subcellular distribution in the gills and liver, and plasma Na+ and Cl- content. Mortalities were dose dependent and greatest in the mixture exposures. Significant accumulation of Cu and Ag in the gills and kidney were a result of a more than additive affect by metal interactions. Cu accumulation in the liver was also more than additive but no effect was observed on hepatic Ag. Subcellular distribution of Cu mainly occurred in metal sensitive fractions (MSF) while Ag accumulated mainly in biologically detoxified fractions (BDF). Fish exposed to the mixture for 10-days experienced more than additive disruption in plasma Na+ but not in plasma Cl-. During 14-days, mixture-exposed fish experienced a more than additive disruption on ion regulation where plasma Na+ and Cl- were significantly less than Cu2+ or Ag+-only exposed fish. Overall, the effects of metal interactions on bioaccumulation, physiological effects and mortalities were based on exposure concentration

The Combined Impacts of Ocean Acidification and Copper on the Physiology of European Sea Bass (Dicentrarchus labrax) and Shore Crabs (Carcinus maenas)

The following thesis explores the physiological effects on European sea bass (Dicentrarchus labrax) and shore crabs (Carcinus maenas) resulting from the dissolution of anthropogenic carbon dioxide (CO2) into seawater: known as ocean acidification. It assesses how ocean acidification, characterised by elevated seawater pCO2 (1200 µatm) and lowered pH (~7.7), affect the internal chemistry of these animals through the homeostatic process of acid-base regulation. Control conditions used for comparison were close to current ocean average values for CO2 (~400 µatm) and pH (8.2). The proficiency and magnitude of these compensatory mechanisms was explored. Both sea bass and shore crabs were found to be highly effective acid-base regulators and employed the same strategy to compensate the hypercapnia-induced respiratory acidosis: namely an elevation of extracellular bicarbonate (HCO3-). It then considers how these regulatory mechanisms both affect, and are affected by, simultaneous exposure to a ubiquitous coastal metal contaminant, copper. Evidence for a hitherto undocumented protective effect of elevated HCO3- against copper-induced DNA damage was found to be afforded to both sea bass and shore crab cells. DNA damage was used as a sensitive toxicity marker and blood cells were used as proxies for other internal tissues. Erythrocytes exposed in vitro (2 h) to copper (45 µg/L) showed significant DNA damage under control [HCO3-] (6 mM) but were completely protected when exposed under high [HCO3-] (12 mM). A similar protective effect was apparent in crabs under in vivo exposure (14 d) to 10 µg/L waterborne copper. Conversely, during exposure to higher waterborne copper concentrations (sea bass: 80 µg/L, shore crabs: 40 µg/L), animals showed a severe or total inhibition of acid-base regulatory ability in the face of simultaneously elevated seawater CO2 (1200 µatm). The downstream effects of longer-term (28 d) exposure to high CO2 and copper, both individually and in combination was assessed. Food conversion efficiency (FCE), growth and copper accumulation were quantified in juvenile sea bass as economically relevant endpoints. Growth and FCE remained unaffected by either stressor and copper was not accumulated in the muscle tissue: pertinent to human consumption. As a bi-product of this longer term study assessment of gut calcium carbonate production rates in these animals was possible, providing some of the first evidence of excretion rates in fish fed on naturally high calcium diets. A directly proportional influence of feeding rate on gut carbonate excretion rates as a result of increased dietary calcium was observed, and novel evidence provided of the proportional contribution of dietary and seawater calcium to excreted carbonate. Both findings have considerable application to global models of fish contribution to the oceanic carbon cycle.University of Exeter, Centre for Environment, Fisheries and Aquaculture Science (Cefas

Estudo comparativo da toxicidade e dos mecanismos de ação tóxica de nanopartículas de cobre e cobre em duas espécies de peixes da Amazônia: Apistogramma agassizii e Paracheirodon axlerodi

Copper oxide nanoparticles (nCuO) are widely used in boat antifouling paints, and are released into the environment, inducing toxicity to aquatic organisms. The present study aimed to understand the effects of nCuO and dissolved copper on two ornamental Amazon fish species: Apistogramma agassizii and Paracheirodon axelrodi. Fish were exposed to 50% nCuO LC50 (A. agassizii 375 μg L-1 and P. axelrodi 460 μg L-1) and 50% Cu LC50 (A. agassizii 20 mg L-1 and P. axelrodi 22.9 μg L-1) for 24, 48, 72 and 96 hours. Metabolic rate (MO2), gill osmorregulatory processes, gill mitochondria oxidative phosphorylation capacity and ROS generation, oxidative stress defense and morfological damages were evaluated. Our results showed a strong increase in MO2 and a higher impairment in its gill’s morphology in P. axelrodi after the copper exposures. Differently, A. agassizii presented an increased proton leak (i.e. uncoupling between respiration and ATP production) in response to nCuO and Cu exposure, thus decreasing their Respiratory Control Rate (RCR). Interestingly, this uncoupling was directly related to an increase in ROS levels. Our findings reveal that the metabolic responses of these two species in response to nCuO and Cu, which are probably caused by the differences between species natural histories, indicating that different mechanisms of toxic action of the contaminants are associated to differences in the sensibility of these two species.Nanopartículas de óxido de cobre (nCuO) são amplamente utilizadas como componentes na fabricação de tintas anti-incrustantes para revestimento de barcos, navios e estruturas submersas e, quando liberadas para o ambiente, podem promover toxicidade para organismos aquáticos. O presente trabalho teve como objetivo avaliar os efeitos das nCuO e do cobre dissolvido em duas espécies de peixes da Amazônia: Apistogramma agassizii e Paracheirodon axelrodi. Primeiramente foi determinada a toxicidade dos compostos às duas espécies (CL50-96h), e então os peixes foram expostos a 50% da CL50 nCuO (A. agassizii 375 μg L-1 e P. axelrodi 460 μg L-1) e 50% da CL50 Cu (A. agassizii 20 μg L-1 e P. axelrodi 22,9 μg L-1) por 24, 48, 72 e 96 horas. A taxa metabólica (MO2), respostas osmorregulatórias, capacidade de fosforilação oxidativa e geração de ROS nas mitocôndrias foram avaliadas, além das respostas de defesa antioxidante e danos morfológicos no epitélio branquial das espécies estudadas. Os resultados mostraram um forte aumento do MO2 e um maior efeito histopatológico na morfologia das brânquias em P. axelrodi expostos ao cobre. Embora o consumo oxigênio não tenha sido alterado após a exposição ao cobre em A. agassizii, um aumento evidente foi observado após 48h de exposição ao nCuO. Além disso, estes animais apresentaram aumento no extravasamento de prótons da membrana mitocondrial em resposta à exposição à nCuO e Cu, indicando um desbalanço entre a respiração celular e a produção de ATP, diminuindo assim a Taxa de Controle Respiratório (RCR). Curiosamente, esse desacoplamento foi diretamente relacionado com um aumento da produção de ROS. Nossos resultados revelam diferentes estratégias metabólicas entre as duas espécies estudadas em resposta ao nCuO e Cu, indicando que diferentes mecanismos de ação tóxica dos contaminantes estão associados à sensibilidade intrínseca dessas duas espécies

Identification and Localization of H+-ATPase, NHE2 and NHE3 in the gills of the southern Appalachian brook trout, Salvelinus fontinalis

Southern brook trout (Salvelinus fontinalis) native to the Great Smoky Mountains are found in headwater streams. These habitats experience increased instances of stream acidification, lowering stream pH on average ~1.0 pH unit. It is well documented that lowering environmental pH results in fish experiencing systemic acidosis. We hypothesize that during acidification events, brook trout suffer acid/base perturbation. Previous studies on other species of freshwater fishes have demonstrated an alteration in the relative expression of H+-ATPase, NHE2 and NHE3 in gill tissue during pH disturbances. Due to a lack of physiological information available, the mechanism utilized by S. fontinalis to regulate systemic pH is unknown. We hypothesized that S. fontinalis will utilize H+-ATPase, NHE2 and NHE3 to excrete excess protons when experiencing episodic stream acidification. To date, we cloned ORFs for H+-ATPase, NHE2, and NHE3 which are homologous to rainbow trout (87, 94 and 92% identical respectively). Immunohistochemical analysis demonstrated H+-ATPase, NHE2 and NHE3 expression is localized to the apical membrane and sub-apical regions of the MRCs in gill epithelia. Also, the relative expression of H+-ATPase decreased along an elevation gradient, whereas NHE3 does not

THE EFFECTS OF LAMPRICIDE 3-TRIFLUOROMETHYL-4-NITROPHENOL TOXICITY ON THE GILLS OF LARVAL SEA LAMPREY AND NON-TARGET RAINBOW TROUT AND LAKE STURGEON

The pesticide, 3-trifluoromethyl-4-nitrophenol (TFM), is widely used in the Great Lakes to control invasive sea lampreys (Petromyzon marinus) populations, but much about its sub-lethal effects remains unknown. A better understanding of its toxicity is needed to improve TFM effectiveness and to protect non-target organisms from its potential adverse effects. The objectives of this thesis were to test the following two hypotheses: (1) impairment of mitochondrial ATP production by TFM interferes with ATP-dependent ion-uptake by fish, leading to altered electrolyte balance, and (2) perturbations of gill function by TFM are exacerbated in soft, ion poor water (SW; 40 mg CaCO3 L-1). Accordingly, larval lamprey, juvenile rainbow trout (Oncorhynchus mykiss), and juvenile sturgeon (Acipenser fulvescens) were exposed to TFM concentrations toxic to larval sea lamprey for 12 h. In lamprey, the toxicity of TFM was 10-fold greater in SW compared to hard water (HW; 450 mg CaCO3 L-1). The activity and expression of Na+/K+-ATPase (NKA) and H+-ATPase (V-ATPase) remained unchanged during TFM exposure in both HW and SW. However a 28% decrease in plasma Na+ in HW and a 10% decrease in plasma Cl- in SW was found in sea lamprey during recovery from TFM exposure. In trout, TFM led to initial Na+ losses, which was compensated by increases in NKA and Total ATPase activity by 6 h in both HW and SW. These initial Na+ loses may be due to an “osmo-respiratory compromise”, where TFM-induced increases in O2 consumption may have lead to increased gill-perfusion and surface area, elevating ion-losses. There was no effect on enzyme activity or expression in sturgeon exposed to TFM in HW, but plasma Na+ dropped by 20% with a 22% increase in plasma Cl- following recovery. No significant effects of TFM on gill structure were observed in all three species. The greater sensitivity of lamprey in SW versus HW can partially be explained by greater susceptibility to ionic disturbances, but other factors are also likely involved. It is concluded that the effects of TFM on gill-mediated ion exchange are minimal and do not translate into substantive disturbances to internal electrolyte balance. However, there is a potential that these effects may persist post-TFM exposure

Resilience to an acid-base disturbance and the development and plasticity of acid-base regulatory pathways in estuarine teleosts

Since the industrial revolution marine environments have displayed marked increases in CO₂ levels. Changes in ocean chemistry – collectively termed ocean acidification (OA) – are predicted to have numerous effects on marine fish, including critical behavioral endpoints and survival, with the most severe impacts hypothesized to occur in the vulnerable early life stages. However, many regions around the globe routinely have CO₂ levels in excess of those predicted by climate change, including the estuaries in the Gulf of Mexico, which are essential habitat for many ecologically and commercially important fish species. We hypothesize that the species that inhabit these environments contain physiological traits that confer resilience to OA. Thus, it is imperative that we understand the resilience of these species as well as the underlying mechanisms that may inform on the adaptive capacity of fish in general. The underlying physiological cause for many of the outcomes of OA is a systemic acid-base disturbance, which causes an elevated arterial pCO₂ and plasma [HCO₃⁻]. Because embryos and early life stage fish lack gills and practice cutaneous gas exchange, acid-base disturbances could be exacerbated in these life stages. Furthermore, little is known about the ontogeny, development, plasticity of acid-base regulatory mechanisms in early life stage marine fish, raising many questions about their ability to compensate for disturbance. Here we present work that shows tolerance of early life stage estuarine species to elevated CO₂ levels, including survival, standard length, and yolk size. However, we did observe significant heart rates in both studied estuarine species, and although we saw no immediate impacts, the long-term impacts of elevated heart rate are of keen interest. We also observed that alterations in acid-base regulatory machinery were the largely the result of development, rather than in response to exposure to an acid-base disturbance, including an acidosis and alkalosis. Although we did observe significant increases in NHE2 and VHA as the result of exposure to elevated CO₂ levels. Of interest is that H⁺ excretion was significant elevated in response to exposure to hypercapnia, and we hypothesize that this H⁺ excretion is the result of both the NHE and VHA pathway, from data obtained via the scanning ion-selective electrode technique (SIET) and pharmacological inhibition, but this must be further investigatedMarine Scienc

The Influence of Water Chemistry and Gill Physiology on the Uptake of the Lampricide TFM by Lake Sturgeon (Acipenser fulvescens)

Application of 3-trifluoromethyl-4-nitrophenol (TFM) to control invasive sea lamprey (Petromyzon marinus) within the Laurentian Great Lakes seldom causes non-target mortality. However, under certain conditions, TFM can harm species such as the lake sturgeon (Acipenser fulvescens). Lake sturgeon less than 100mm in length are particularly vulnerable to TFM-induced mortality, and are more sensitive to TFM toxicity with increasing water alkalinity as compared to sea lamprey. The objectives of this study were to evaluate the influence of pH and alkalinity on the uptake of TFM by juvenile sturgeon using radio-labeled TFM (14C-TFM). An additional objective was to resolve why younger (YOY; young of the year) lake sturgeon were more vulnerable to TFM than older (1+; 1 year or older) animals under the same conditions. Inverse relationships were observed between the rates of TFM uptake with water pH. These pH effects support the hypothesis that greater TFM toxicity at low pH is likely a direct result of increasing concentrations of the un-ionized, more lipophilic form of TFM at lower pH, leading to greater rates of uptake via passive diffusion across the gills. Uptake of TFM was also reduced as water alkalinity increased from low (50 CaCO3 L-1) to moderate alkalinity (150 mg CaCO3 L-1), but further reductions in TFM uptake were negligible at higher alkalinities. The reductions in TFM uptake between low and moderate alkalinity were likely due to a higher capacity of the water to buffer acidic equivalents (H+ and CO2) excreted across the gill, resulting in less acidification of the gill microenvironment and therefore the formation of less un-ionized TFM. Measurements of Na+/K+-ATPase and V-ATPase activity, as well as western blotting and immunohistochemical staining, demonstrated that TFM had no adverse effects on the ionoregulatory machinery of the gills. Regardless of water chemistry conditions, the rates of TFM uptake were greatest in the YOY sturgeon than in the 1+ fish, likely due to the higher mass specific metabolic rates of the smaller fish. In conclusion, the inverse relationship between body size and TFM uptake contributes to the greater sensitivity of YOY sturgeon to TFM. Alkalinity is also protective against TFM toxicity, but the protective effects of alkalinity are negligible in waters of high alkalinity. To minimize the risk of non-target mortality in lake sturgeon, it would be prudent to conduct treatments in the fall when sturgeon are larger and have lower rates of TFM uptake. Water chemistry also has pronounced effects on TFM uptake by lake sturgeon, and should be considered prior to TFM applications to protect them from the adverse of effects of TFM, without compromising sea lamprey control efforts