The physiological responses of freshwater rainbow trout,Oncorhynchus mykiss,during acutely lethal copper exposure

Acutely lethal (24 h) exposure of adult rainbow trout (Oncorhynchus mykiss) to 4.9 mol copper·l^-1 in fresh water (pH 7.9, [Ca²⁺]0.8 mEq·l^-1) caused a rapid decline of plasma Na⁺ and Cl^- and arterial O₂ tension, and initially a pronounced tachycardia. The internal hypoxia probably resulted from histopathologies observed in the gills of fish exposed to copper, such as cell swelling, thickening and curling of the lamellae, and haematomas. Copper cannot therefore be considered purely as an ionoregulatory toxicant during acutely lethal conditions. Mortality during exposure to copper could not simply be explained by the plasma ionic dilution, nor by the internal hypoxia, since arterial O₂ content remained relatively unchanged. Secondary to the ionoregulatory and respiratory disturbances were a number of deleterious physiological responses which included a massive haemoconcentration (haematocrit values as high as 60%) and a doubling of the mean arterial blood pressure. The time-course of these changes suggest that cardiac failure was the final cause of death. In this respect copper exposure resembles low pH exposure in freshwater trout (Milligan and Wood 1982). Copper and H⁺ appear to be similar in both the primary site of their toxic action (the gills) and the secondary physiological consequences which result from acutely lethal exposures. Furthermore, the acute toxicity syndrome observed may be common to many metals which cause ionoregulatory and/or respiratory problems in freshwater fish.Abbreviations C _aO₂ arterial oxygen content - FR water flow rate - Hb haemoglobin - Hct haematocrit - H _m ⁺ net metabolic acid load - IU international unit - MABP mean arterial blood pressure - MCHC mean corpuscular haemoglobin content - MO₂ rate of oxygen consumption - P _aCO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen partial pressure - T _amm total ammonia (=NH₃+NH ₄ ⁺ ) - TCO₂ total carbon dioxide - TOC total organic carbon - %Hb–O₂ percentage of haemoglobin saturated with oxygen     

Developmental regulation of metallothionein mRNA, zinc and copper levels in rainbow trout, Salmo gairdneri

The metallothionein (MT) gene expression profile was followed in rainbow trout during early embryo development and in liver and gonads during the period of sexual maturation. The hepatic MT mRNA levels increase at the end of sexual maturation in both male and female rainbow trout. Although both isoforms of MT mRNA accumulate in the liver, there is a preferential increase in MT-A in the female liver. Concomitantly with this increase in MT there is a redistribution of zinc and copper to MT. In the juvenile female there is an abundance of MT mRNA in the ovaries. This is correlated to high levels of zinc in the MT fraction upon Sephadex G-75 chromatography. During ovary development the MT mRNA levels and the MT-bound zinc levels drop, with an increase in zinc being bound to high-molecular-mass proteins. At ovulation most of the zinc is found in the membrane portion upon centrifugation. In contrast to the ovaries, there are no apparent changes in either trace metal distribution or MT mRNA levels during testis development. In the developing embryo there is an increase in MT-bound copper at gastrulation. This is accompanied by an increase in both isoforms of MT mRNA. At hatch both the copper and zinc levels increase in the MT fraction, with a concomitant increase in mainly MT-A mRNA. These findings indicate that the variations in MT mRNA levels during development are closely associated with metal regulation.     

Application of the biotic ligand model to predicting zinc toxicity to rainbow trout,fathead minnow,and Daphnia magna

The Biotic Ligand Model has been previously developed to explain and predict the effects of water chemistry on the toxicity of copper, silver, and cadmium. In this paper, we describe the development and application of a biotic ligand model for zinc (Zn BLM). The data used in the development of the Zn BLM includes acute zinc LC50 data for several aquatic organisms including rainbow trout, fathead minnow, and Daphnia magna. Important chemical effects were observed that influenced the measured zinc toxicity for these organisms including the effects of hardness and pH. A significant amount of the historical toxicity data for zinc includes concentrations that exceeded zinc solubility. These data exhibited very different responses to chemical adjustment than data that were within solubility limits. Toxicity data that were within solubility limits showed evidence of both zinc complexation, and zinc-proton competition and could be well described by a chemical equilibrium approach such as that used by the Zn BLM.     

Comparative proteomics of copper exposure and toxicity in rainbow trout,common carp and gibel carp

Species specific differences in transporters, chaperones, metal binding proteins and other targets are important in metal toxicity. Therefore, we have studied the effects of copper exposure on the proteome of gill tissue from Oncorhynchus mykiss, Cyprinus carpio and Carassius auratus gibelio, which have different sensitivities toward copper. Fish were exposed to the Flemish water quality standard for surface waters, being 50 μg/L, for 3 days. Sampled gill tissue was subjected to a 2D-Dige and an iTRAQ analysis. While gibel carp showed more positive responses such as increased apolipoprotein A-I, transferrin and heat shock protein 70, common carp's gill tissue on the other hand displayed a changed actin cytoskeleton, and indications of a changed metabolism. These last two traits were evident in rainbow trout as well, together with decreased expressions of transferrin and albumin. urthermore, the Weighted Gene Co-Expression Network Analysis of rainbow trout data revealed a network of 98 proteins related to Cu accumulation in gill, of which the occurrence of proteins related to oxidative stress, such as superoxide dismutase and cytochrome c were promising. Additionally, the outcome of the different proteomics techniques demonstrates the usefulness of iTRAQ analysis compared to 2D-Dige and the need for fully annotated genomes.     

The distribution of zinc and copper in plasma, erythrocytes and erythrocyte membranes of rainbow trout (Salmo gairdneri)

1. The zinc and copper concentration of plasma was determined in rainbow trout, lake trout, walleye and whitefish. 2. These fish had mean plasma zinc concentrations ranging from 9.3 to 15.1 ppm and copper concentrations from 0.6 to 1.3 ppm. 3. In rainbow trout, the concentration of zinc and copper is greater in the erythrocyte membrane than in the total erythrocyte. 4. Ultrafilterable plasma zinc and copper concentration in rainbow trout was determined to be 0.03 and 0.019 ppm, respectively. 5. Dialysis of rainbow trout plasma against 20 mM EDTA results in removal of 99% of the zinc and 88% of the copper from plasma proteins.     

A lead-gill binding model to predict acute lead toxicity to rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss, approximately 2 g) were exposed to 0.6-1.0 microM Pb (125-200 microgl(-1)) for 3 h in ion-poor water. Complexing ligands (citrate, ethylenediamine, organic matter (OM)) or competing cations (Ca, Mg, Na) were added to the water. After exposure, trout gills were removed and analyzed for accumulated Pb. From these exposures, a conditional equilibrium binding constant (K) for Pb-gill binding was calculated (log K(Pb-gillPb)=6.0), plus conditional binding constants for cationic competition at the Pb binding sites and for Pb binding to OM in the water. These log K values were entered into the MINEQL+ aquatic chemistry equilibrium program, to calculate binding of Pb by trout gills. Two versions of the Pb-gill binding model were generated, one of which took into account OM quality as indicated by a simple measure of OM aromaticity, the specific absorption coefficient. The two model versions were tested against acute Pb toxicity (as the time to reach 50% fish mortality; LT50) during 1-week exposures of trout to 3.9 microM Pb in water collected from across southern Ontario. Both versions of the model generated highly significant correlations between the LT50 values and gill Pb concentrations calculated from measured exposure water chemistry, with the OM quality version correlating slightly better. Water pH also correlated well with the LT50 values, because the Pb exposures were in the pH range (7-8) where there is a nearly linear relationship between water pH and inorganic complexation of Pb. Advantages of the Pb-gill binding model include its completeness and the flexibility inherent in its conceptual framework, for example the inclusion of competition by Ca and H(+) for Pb binding sites on gills, and inclusion of complexation of Pb in the water column by natural OM and by carbonate.     

Effects of copper on the acute cortisol response and associated physiology in rainbow trout

The aim of this study was to determine the effects of chronic waterborne copper (Cu) exposure on the acute stress-induced cortisol response and associated physiological consequences in rainbow trout (Oncorhynchus mykiss). Trout were exposed to 30 μg Cu/L in moderately hard water (120 mg/L as CaCO(3)) for 40 days, following which time the acute cortisol response was examined with a series of stressors. At 40 days, a 65% increase in Cu was observed in the gill, but no accumulation was observed in the liver, brain or head kidney. Stressors such as air exposure or confinement did not elicit an increase in circulating cortisol levels for Cu-exposed fish, in contrast to controls. However, this inhibitory effect on the acute cortisol response appeared to have few implications on the ability of Cu-exposed fish to maintain ion and carbohydrate homeostasis. For example, plasma Na(+), Ca(2+) and glucose levels as well as hepatic glycogen levels were the same post-stress in control and Cu-exposed fish. Trout were also challenged with exposure to 50% seawater for 48 h, where Cu-exposed trout maintained plasma Na(+), glucose and hepatic glycogen levels. However, Cu-exposed fish experienced decreased plasma K(+) levels throughout the Cu exposure and stress tests. In conclusion, chronic Cu exposure resulted in the abolition of an acute cortisol response post-stress. There was no Cu accumulation in the hypothalamus-pituitary-interrenal axis (HPI axis) suggesting this was not a direct toxic effect of Cu on the cortisol regulatory pathway. However, the lack of an acute cortisol response in Cu-exposed fish did not impair the ability of the fish to maintain ion and carbohydrate homeostasis. This effect on cortisol may be a strategy to reduce costs during the chronic stress of Cu exposure, and not endocrine disruption as a result of toxic injury.     

Changes in circulating blood cell levels of rainbow trout, Salmo gairdneri Richardson, following acute and chronic exposure to copper

Significant leucopenia, predominantly the result of a large reduction in lymphocyte concentration, was observed in rainbow trout exposed to 301 μg1⁻¹ copper for 24h. A strong trend ( P = 0–085) toward reduced erythrocyte concentration was also apparent. The pattern of haematological change after chronic exposure (118 μg 1⁻¹ copper for 16 weeks) was substantially different. With the exception of a significant increase in neutrophil concentration (neutrophilia), all blood cell concentrations had returned to control levels.     

The toxicity to Clarias lazera of copper and zinc applied jointly

1. The acute toxicity to juvenile Clarias lazera of a mixture of copper and zinc over a 96 hr exposure period was determined. Fish were exposed to the summation of half the 96 hr TLm value of each toxicant. 2. Percentage survival was much reduced indicating that the metals potentiate each others lethal action. 3. Comparison between metal residues in fish exposed to copper and zinc or to their mixture showed that the uptake of one metal was decreased by the presence of the other. 4. Toxic effects of the mixture on the physiological parameters studied were mainly attributable to copper, indicating that the presence of zinc did not influence the mode of action of copper.     

Effect of hardness on bioavailability and toxicity of cadmium to rainbow trout

Abstract

Water quality characteristics affecting toxicity of metals to aquatic life include pH, inorganic and organic ligands (negatively charged ions and molecules), and water hardness. Ligands control the ability of natural waters to bind metals which could adversely affect aquatic life. Bioavailability of metals in natural waters is primarily controlled by alkalinity. Hardness does not affect metal complexation but can reduce acute toxicity through antagonistic mechanisms. In most natural waters, concentrations of alkalinity and hardness are similar, but they may be very different in some waters.

Most toxicity studies have not distinguished between reduced toxicity resulting from effects of hardness and that resulting from complexation of metals by ligands. A series of acute and long-term experiments were conducted to assess these relationships while exposing rainbow trout (Oncorhynchus mykiss) to cadmium (Cd) in waters of low alkalinity (30 mg L^?1) and hardnesses of 400, 200, and 50 mg L^?1 adjusted with magnesium sulfate (MgSO⁴). These tests did not show a strong antagonistic influence of Mg hardness on Cd toxicity. At Mg hardnesses of 50, 200, and 400 mg L^?1, 96-h LC50s were 3.02, 6.12, and 5.70 μg Cd L^?1, differing by a factor of only 1.8. Similarly, chronic values derived from 100-day experiments in waters with the same range of hardness were 1.47, 3.57, and 3.64 μg L^?1, respectively. With an eight-fold difference in Mg hardness, chronic values differed by a factor of only 2.5. Antagonistic properties of hardness are primarily controlled by Ca with Mg playing a minor role. The long-term role of Ca in reducing metal toxicity will require further investigation.     

Uptake and accumulation of zinc in juvenile rainbow trout,Salmo gairdneri

Synopsis The toxicity of zinc to rainbow trout was determined and the 72 h median lethal concentration was found to be 2.00 mg l^–1 in freshwater, hardness 7.50 mg l^–1 as calcium. An insignificant increase in zinc concentration of internal tissues occurred in fish exposed to 1.52 mg l^–1 in freshwater for 72 h. However, there was a significant uptake of zinc by gills and the body surface. Fish exposed to 10 mg l^–1 zinc for 72 h in two-thirds sea water showed significant zinc uptake by liver, rectum and muscle, when compared to control fish. Drinking rate decreased from 1.43 to 0.26 ml kg^–1 h^–1 when zinc sulphate was added to freshwater. Trout adapted to two-thirds sea water showed no decrease in drinking, about 7 ml kg^–1 h^–1 when zinc was added to the water.     

Acclimation-induced changes in toxicity of arsenic and cyanide to rainbow trout, Salmo gairdneri Richardson

Pre-exposure of rainbow trout, Salmo gairdneri to sublethal levels of arsenic or cyanide, resulted in significant changes in the incipient lethal levels of the respective toxicants. Fish gradually increased their tolerance to arsenic (as arsenite) by 47% during a three week preexposure to 0.22 of the incipient lethal level. Mean whole-body aresenic concentrations increased from 2 to 27 μg g⁻¹ dry weight during the first week, then decreased to a stable level of 15 after two and three weeks. Fish decreased their tolerance to cyanide (as HCN) by 32 and 15% after one and two weeks of pre-exposure to 0–35 of the incipient lethal level. This initial sensitization disappeared after three weeks. Renal tubular parenchymatous oedema was observed after two and three weeks of cyanide pre-exposure. Neither arsenic nor cyanide affected growth at these levels of pre-exposure.     

In vitro evidence for the ionoregulatory role of rainbow trout mucus in acid, acid/aluminium and zinc toxicity

Rainbow trout body mucus dialysed with acidified distilled water at pH 7,5 and 3 experienced ion depletion which was greatest at pH 3 and minimal between pH 7 and 5. Mucus Na⁺ loss is exacerbated in the presence of 1 mg I⁻¹ aluminium as A1₂(SO₄), at pH 5 and 7. Al₂(SO₄), causes greater depletion of Na⁺ from mucus than A1C1₃. A lethal level of zinc (2 mg 1⁻¹) does not deplete mucus Na⁻ or K⁺, unlike a lethal level of aluminium (1 mg 1⁻¹) at pH 7. The results are discussed in terms of the ionoregulatory role of mucus in heavy metal and acid toxicity.     

The interactions of water hardness and pH with the acute toxicity of zinc to the brown trout, Salmo trutta L.

Exposure of brown trout, Salmo trutta , to zinc under continuous flow conditions over 96 h showed that both water hardness and pH exert major influences on the toxicity of the metal. 96-h LC50 values for total zinc ranged from <0.14mg 1⁻¹ in alkaline soft water (pH 8; lOmg 1⁻¹ as CaCO₃) to 3.20 mg 1⁻¹ in acidic hard water (pH 5; 204 mg 1⁻¹ as CaCO₃). A variable reduction in zinc toxicity in hard water compared with soft water over the pH range 4–9 was attributed to high external calcium. Zinc toxicity was positively correlated with decreasing acidity over the pH range 5–7, the metal being most toxic at pH 8–9 where metal complexes predominate. Below pH 5 metal toxicity also increased, irrespective of hardness. Water hardness and pH interacted with zinc toxicity in a complex manner, apparently dependent on physical and chemical transformations of the metal, and as changes in uptake. detoxification and excretion by the fish.     

The effect of copper, hardness and pH on the growth of rainbow trout, Salmo gairdneri

Rainbow trout ( Salmo gairdneri ) were held at a ked ration and activity regime and were exposed to a number of copper, pH and hardness combinations. Growth rate, appetite and gross conversion efficiency were determined over three consecutive 10-day periods of exposure. Growth rate was most affected during the first 10 days of exposure and partial or complete recovery was observed thereafter. For a given pH, less copper was required to reduce growth by a given amount at low than at high levels of hardness. At a given hardness, copper-induced depressions in growth rate were more pronounced and recovery slower in a low than in a high pH. No distinction could be made among total soluble or extractable copper but predicted concentrations of six specific cupric ions varied with pH and hardness. Regression analysis indicated that only Cu²⁺ and CuOH⁺ could be significantly correlated with growth rate.     

Some effects of exposing rainbow trout (Salmo gairdneri Richardson) to phenol solutions

Phenol (C₆H₅OH) at non-lethal concentrations in hard water had no effect on the urine flow rate or haematocrit of rainbow trout for exposure times of 24 h. Phenol was detected in the urine in a non-conjugated form and unchanged phenol was also extracted from muscle, blood and brain. Uptake of phenol into tissue was found to be rapid with an equilibrium concentration being reached in 3 h. Loss of phenol after exposure was as rapid. The equilibrium concentration for muscle was similar to the phenol concentration to which the fish were exposed. Blood and brain contained smaller amounts. Close to or above the lethal threshold concentration (48-h lc₅₀9 mg 1^-l; 15°C) the fish had higher than ambient concentrations in their tissues most notably in the brain. Above the lethal threshold there is evidence of a large uptake of phenol by erythrocytes.     

Cadmium toxicity to rainbow trout, Salmo gairdneri Richardson: a study of eggs and alevins

Cadmium toxicity to rainbow trout was evaluated at two cadmium concentrations (0.01, 0.10 mg1⁻¹) and in water without added cadmium using eggs reared through the larval (alevin) phase to swim-up fry. Exposure to 0.1 mg Cd 1⁻¹ promoted premature hatching, retarded growth, increased rates of mortality and the occurrence of developmental abnormalities such as spinal curvature and blood clots. Three methods of holding the eggs and alevins during the toxicity test were compared. Hatching time and success, alevin mortality, growth and development were not influenced by the method of containment but of the three methods, a subdivided perforated box system proved particularly useful for monitoring the responses of specific animals throughout the toxicity test.