Physiological responses of European sea bass (Dicentrarchus labrax) exposed to increased carbon dioxide and reduced seawater salinities.

BACKGROUND: The iono- and osmoregulatory capacities of marine teleosts, such as European sea bass (Dicentrarchus labrax) are expected to be challenged by high carbon dioxide exposure, and the adverse effects of elevated CO2 could be amplified when such fish migrate into less buffered hypo-osmotic estuarine environments. Therefore, the effects of increased CO2 on the physiological responses of European sea bass (Dicentrarchus labrax) acclimated to 32 ppt, 10 ppt and 2.5 ppt were investigated. METHODS: Following acclimation to different salinities for two weeks, fish were exposed to present-day (400 µatm) and future (1000 µatm) atmospheric CO2 for 1, 3, 7 and 21 days. Blood pH, plasma ions (Na+, K+, Cl-), branchial mRNA expression of ion transporters such as Na+/K+-ATPase (NKA), Na+/K+/2Cl- co-transporters (NKCC) and ammonia transporters (e.g. Rhesus glycoproteins Rhbg, Rhcg1 and Rhcg2) were examined to understand the iono- and osmoregulatory consequences of elevated CO2. RESULTS: A transient but significant increase in the blood pH of exposed fish acclimated at 10 ppt (day 1) and 2.5 ppt (day 21) was observed possibly due to an overshoot of the blood HCO3- accumulation while a significant reduction of blood pH was observed after 21 days at 2.5ppt. However, no change was seen at 32 ppt. Generally, Na + concentration of control fish was relatively higher at 10 ppt and lower at 2.5 ppt compared to 32 ppt control group at all sampling periods. Additionally, NKA was upregulated in gill of juvenile sea bass when acclimated to lower salinities compared to 32 ppt control group. CO2 exposure generally downregulated NKA mRNA expression at 32ppt (day 1), 10 ppt (days 3, 7 and 21) and 2.5ppt (days 1 and 7) and also a significant reduction of NKCC mRNA level of the exposed fish acclimated at 32 ppt (1-3 days) and 10 ppt (7-21 days) was observed. Furthermore, Rhesus glycoproteins were generally upregulated in the fish acclimated at lower salinities indicating a higher dependance on gill ammonia excretion. Increased CO2 led to a reduced expression of Rhbg and may therefore reduce ammonia excretion rate. CONCLUSION: Juvenile sea bass were relatively successful in keeping acid base balance under an ocean acidification scenario. However, this came at a cost for ionoregulation with reduced NKA, NKCC and Rhbg expression rates as a consequence.

Synergistic effect of nitrate exposure and heatwaves on the growth, and metabolic activity of microalgae, Chlamydomonas reinhardtii, and Pseudokirchneriella subcapitata.

Aquatic biota are threatened by climate warming as well as other anthropogenic stressors such as eutrophication by phosphates and nitrate. However, it remains unclear how nitrate exposure can alter the resilience of microalgae to climate warming, particularly heatwaves. To get a better understanding of these processes, we investigated the effect of elevated temperature and nitrate pollution on growth, metabolites (sugar and protein), oxidative damage (lipid peroxidation), and antioxidant accumulation (polyphenols, proline) in Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata. The experiment involved a 3 × 3 factorial design, where microalgae were exposed to one of three nitrate levels (5, 50, or 200 mg L-1 NO3-l) at 20 °C for 2 weeks. Subsequently, two heatwave scenarios were imposed: a short and moderate heatwave at 24 °C for 2 weeks, and a long and intense heatwave with an additional 2 weeks at 26 °C. A positive synergistic effect of heatwaves and nitrate on growth and metabolites was observed, but this also led to increased oxidative stress. In the short and moderate heatwave, oxidative damage was controlled by increased antioxidant levels. The high growth, metabolites, and antioxidants combined with low oxidative stress during the short and moderate heatwaves in moderate nitrate (50 mg L-1) led to a sustainable increased food availability to grazers. On the other hand, long and intense heatwaves in high nitrate conditions caused unsustainable growth due to increased oxidative stress and relatively low antioxidant (proline) levels, increasing the risk for massive algal die-offs.

Long-term stress induced cortisol downregulation, growth reduction and cardiac remodeling in Atlantic salmon.

Stress and elevated plasma cortisol in salmonids have been linked with pathological remodeling of the heart and deterioration of fitness and welfare. However, these associations were based on biomarkers that fail to provide a retrospective view of stress. This study is the first whereby the association of long-term stress, using scale cortisol as a chronic stress biomarker, with cardiac morphology and growth performance of wild Atlantic salmon (Salmo salar) is made. Growth, heart morphology, plasma and scale cortisol levels, and expression of genes involved in cortisol regulation of the hypothalamic-pituitary-interrenal axis of undisturbed fish (control) were compared with those of fish exposed daily to stress for 8 weeks. Though scale cortisol levels showed a time-dependent accumulation in both groups, plasma and scale cortisol levels of stress group fish were 29.1% and 25.0% lower than those of control fish, respectively. These results correlated with the overall upregulation of stress-axis genes involved in the systemic negative feedback of cortisol, and local feedback via 11ß-hydroxysteroid dehydrogenases, glucocorticoid and mineralocorticoid receptors in the stress treatment at the hypothalamus and pituitary level. These lower cortisol levels were, however, counterintuitive in terms of the growth performance as stress group fish grew 33.7% slower than control fish, which probably influenced the 8.4% increase in relative ventricle mass in the stress group. Though compact myocardium area between the treatments was comparable, these parameters showed significant linear correlations with scale cortisol levels, indicating the involvement of chronic stress in cardiac remodeling. These findings underscore the importance of scale cortisol as biomarker when associating chronic stress with long-term processes including cardiac remodeling.

Quantifying changes in umbilicus size to estimate the relative age of neonatal blacktip reef sharks (Carcharhinus melanopterus).

Sharks can incur a range of external injuries throughout their lives that originate from various sources, but some of the most notable wounds in viviparous shark neonates are at the umbilicus. Umbilical wounds typically heal within 1 to 2 months post-parturition, depending on the species, and are therefore often used as an indicator of neonatal life stage or as a relative measure of age [e.g. grouping by umbilical wound classes (UWCs), according to the size of their umbilicus]. To improve comparisons of early-life characteristics between studies, species and across populations, studies using UWCs should integrate quantitative changes. To overcome this issue, we set out to quantify changes in umbilicus size of neonatal blacktip reef sharks (Carcharhinus melanopterus) around the island of Moorea, French Polynesia, based on temporal regression relationships of umbilicus size. Here, we provide a detailed description for the construction of similar quantitative umbilical wound classifications, and we subsequently validate the accuracy of our classification and discuss two examples to illustrate its efficacy, depletion rate of maternally provided energy reserves and estimation of parturition period. A significant decrease in body condition in neonatal sharks as early as twelve days post-parturition suggests a rapid depletion of in utero-allocated energy reserves stored in the liver. Back calculations of timing of birth based on the umbilicus size of neonates determine a parturition season from September to January, with most parturitions occurring during October and November. As such, this study contributes valuable data to inform the conservation and management of young-of-the-year blacktip reef sharks, and we therefore encourage the construction and use of similar regression relationships for other viviparous shark species.

Chronic handling stress in zebrafish Danio rerio husbandry.

The zebrafish (Danio rerio, Hamilton, 1822) is one of the most important fish model species in scientific research, with millions of fish housed in laboratory animal facilities around the world. During husbandry, it is necessary to regularly handle the fish, which could cause short- and long-term stress, possibly affecting both fish welfare and experimental outcomes. In two experiments, the authors studied effects of transferring adult zebrafish, by chasing them with a net and/or exposing them to air (netting) on different endpoints: cortisol levels, reproduction parameters and behavioural parameters. They used realistic chase and air-exposure times to mimic normal zebrafish husbandry and investigated the potential to habituate to handling stressors. Finally, the potential welfare improvements of a nutritional reward after handling were studied. All types of handling induced a stress response, but the authors did not find a correlation with the intensity of the stressor. Realistic (short) handling routines also caused stress, both after the first time and after regular handling over a long period of time. Cortisol levels peaked after 15 min, were still elevated after 30 min and dropped to resting level after 60 min. This should be taken into account by researchers when carrying out measurements or behavioural trials within an hour after handling. There is a minor potential benefit of nutritional rewards that may contribute to a faster recovery of normal behaviour. They did not find evidence of habituation to chasing and netting stress. Taking the stress response after handling into consideration will improve fish welfare and health and minimise husbandry-associated sources of variation.

Adverse Outcome Pathways for Chronic Copper Toxicity to Fish and Amphibians.

In the present review, we synthesize information on the mechanisms of chronic copper (Cu) toxicity using an adverse outcome pathway framework and identify three primary pathways for chronic Cu toxicity: disruption of sodium homeostasis, effects on bioenergetics, and oxidative stress. Unlike acute Cu toxicity, disruption of sodium homeostasis is not a driving mechanism of chronic toxicity, but compensatory responses in this pathway contribute to effects on organism bioenergetics. Effects on bioenergetics clearly contribute to chronic Cu toxicity with impacts at multiple lower levels of biological organization. However, quantitatively translating these impacts into effects on apical endpoints such as growth, amphibian metamorphosis, and reproduction remains elusive and requires further study. Copper-induced oxidative stress occurs in most tissues of aquatic vertebrates and is clearly a significant driver of chronic Cu toxicity. Although antioxidant responses and capacities differ among tissues, there is no clear indication that specific tissues are more sensitive than others to oxidative stress. Oxidative stress leads to increased apoptosis and cellular damage in multiple tissues, including some that contribute to bioenergetic effects. This also includes oxidative damage to tissues involved in neuroendocrine axes and this damage likely alters the normal function of these tissues. Importantly, Cu-induced changes in hormone concentrations and gene expression in endocrine-mediated pathways such as reproductive steroidogenesis and amphibian metamorphosis are likely the result of oxidative stress-induced tissue damage and not endocrine disruption. Overall, we conclude that oxidative stress is likely the primary driver of chronic Cu toxicity in aquatic vertebrates, with bioenergetic effects and compensatory response to disruption of sodium homeostasis contributing to some degree to observed effects on apical endpoints. Environ Toxicol Chem 2022;41:2911-2927. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Distribution of metals in water, sediment and fish tissue. Consequences for human health risks due to fish consumption in Lake Hawassa, Ethiopia.

Water pollution may be a serious environmental problem for Lake Hawassa, an important fishing and recreational site as well as a drinking-water source in Ethiopia. The present study aims at determining the distribution of metals and metalloids in the lake's water, sediment and fish tissues and assessing the resulting human health and ecological risks. Metals were detected in both abiotic and biotic samples. In water, only the Hg concentration was significantly different among sampling sites. The average concentration of As, Cd, Cr, Cu, Ni, Pb, and Zn in water were below the environmental quality thresholds, thus not having potentially adverse effect on aquatic life. In sediment, significant differences in metals concentration among sites were found for As, Cd, Pb, Co, Zn and Hg (p < 0.05). Exceedances of As, Cr, Cu, Hg, Ni and Zn were found in sediment, with Cr, Ni and Zn above the probable effect concentration and being potentially toxic to aquatic life. Fish stored more metals in their liver than in their muscle. The concentration of metals in carnivorous fish (Barbus intermedius) was not higher in muscle and liver than those in herbivores fish (Oreochromis niloticus). The Bioaccumulation Factor of Cr in all fish species muscle was >1. The Biota-Sediment Accumulation Factor of all metals in all fish species muscle were <1. Positive correlations among metals in water and correlations among metals in sediment were found, indicating a potential common pollution source. Positive correlation of total organic carbon with Cd, Co and Se and clay content with Pb, As and Hg was found and may imply that metals are easily adsorbed by the organic matter and fine sediment. With respect to the measured metals no potential health risk due to consumption of fish from Lake Hawassa was observed.

Chlorpyrifos and dichlorvos in combined exposure reveals antagonistic interaction to the freshwater fish Mrigal, Cirrhinus mrigala.

Toxicity imposed by organophosphate pesticides to the freshwater cultivable fish species mrigal (Cirrhinus mrigala) was assessed under laboratory conditions. Healthy juveniles were exposed to chlorpyrifos, dichlorvos, and their equitoxic mixture in geometric series. Median lethal concentrations of chlorpyrifos were found to be 0.906 (0.689-1.179), 0.527 (0.433-0.633), 0.435 (0.366-0.517) and 0.380 (0.319-0.450) mg/L and dichlorvos were found to be 38.432 (33.625-47.866), 22.477 (19.047-26.646), 12.442 (9.619-14.196) and 11.367 (9.496-13.536) mg/L after 24 h, 48 h, 72 h and 96 h of exposure respectively. Surprisingly, the joint toxicity of these organophosphates in the binary mixture was less than additive during most of the exposure periods. Behavioral changes exhibited by individual as well as mixture pesticide treatments were loss of schooling behavior, aggregating at corners of the test chamber, elevated opercular beatings, surplus mucus secretion, slight color changes and sudden and rapid body movements before death. Loss of fish equilibrium was noticed only in chlorpyrifos treated fish, whereas sluggish behavior was noticed only in mixture pesticide treatment. Such behavioral studies can be applied as a non-invasive bio-monitoring tool for water quality assessment for fish growth and development. Despite the same mode of action of both pesticides, the antagonistic action in the binary mixture is an interesting outcome of this research that requires further investigation for a lucid understanding of the joint toxicity mechanism of such pesticides.

Modulations of blood biochemical parameters of golden mahseer, Tor putitora following exposures to single and mixed organophosphate.

Increasing pesticide application is a serious threat to human health and biodiversity. In nature, pesticides prevail in mixtures; therefore the joint effects of pesticides should be taken into consideration due to their priority in toxicity research when aiming at realistic evaluations. In this study, individual and mixture toxicity of the commonly used organophosphate insecticides- chlorpyrifos and dichlorvos was explored. Healthy and clinically active juveniles of golden mahseer (Tor putitora) were exposed to sub-lethal doses (10% of the 96 h-LC50) of the chlorpyrifos, dichlorvos, and their mixture. Blood sampling was conducted after 24 h and 96 h of exposure, followed by a 1 week recovery period. Among the examined biochemical parameters; blood glucose in dichlorvos treatment; alanine aminotransferase and alkaline phosphatase in chlorpyrifos and dichlorvos treatments; and aspartate aminotransferase and urea in mixture pesticide treatments were elevated. In contrast, blood albumin and triglycerides were diminished in mixture pesticide treatments. Vital organs like kidney and liver of the tested animals were compromised to different magnitudes in different pesticide treatments. Kidney was found to be more sensitive than liver in terms of pesticide toxicity during this short exposure experiment. This study revealed that most of the biomarkers were mainly affected at a later exposure phase (after 96 h) and steadily recovered during the depuration period.

Mercury accumulation in muscle and liver tissue and human health risk assessment of two resident freshwater fish species in Flanders (Belgium): a multilocation approach.

Detrimental effects of chemical pollution-primarily caused by human activities-on aquatic ecosystems have increasingly gained attention. Because of its hydrophobic qualities, mercury is prone to easily bioaccumulate and biomagnify through the food chain, decreasing biodiversity and eventually also affecting humans. In the present study, accumulated mercury concentrations were measured in muscle and liver tissue of perch (Perca fluviatilis) and European eel (Anguilla anguilla) collected at 26 sampling locations in Flemish (Belgian) waterbodies, allowing a comparison of these species within a variety of environmental situations. Furthermore, effects of size and weight have been assessed, expected to influence accumulation and storage of pollutants. Mercury concentrations in perch ranged up to 1.7 µg g-1 dw (median: 0.29 µg g-1 dw) in muscle and from 0.02 to 0.77 µg g-1 dw (median: 0.11 µg g-1 dw) in liver tissue. For eel, these concentrations were between 0.07 and 1.3 µg g-1 dw (median: 0.39 µg g-1 dw) and between 0.08 and 1.4 µg g-1 dw (median: 0.55 µg g-1 dw) respectively. We found a correlation of accumulated mercury with length in perch, independent of location. Furthermore, a significant difference in accumulated mercury concentrations between the targeted species was measured, with the highest mean concentrations per dry weight in eel liver and muscle tissue. In perch, higher concentrations were found in muscle compared to liver tissue, while in eel, liver tissue showed the highest concentrations. These findings were further considered with concentrations corrected for lipid content, excluding the fat compartment, which is known to a hold negligible portion of the total and methyl mercury concentrations. This confirmed our previous conclusions, except for mercury concentrations in eel. Here there was no longer a significant difference between muscle and liver concentrations. Finally, health risk analyses revealed that only frequent consumption of local eel (> 71 g day-1) could pose risks to humans.

Association between Chromosome 4 and mercury accumulation in muscle of the three-spined stickleback (Gasterosteus aculeatus).

Anthropogenic stressors, such as pollutants, act as selective factors that can leave measurable changes in allele frequencies in the genome. Metals are of particular concern among pollutants, because of interference with vital biological pathways. We use the three-spined stickleback as a model for adaptation to mercury pollution in natural populations. We collected sticklebacks from 21 locations in Flanders (Belgium), measured the accumulated levels of mercury in the skeletal muscle tissue, and genotyped the fish by sequencing (GBS). The spread of muscle mercury content across locations was considerable, ranging from 21.5 to 327 ng/g dry weight (DW). We then conducted a genome-wide association study (GWAS) between 28,450 single nucleotide polymorphisms (SNPs) and the accumulated levels of mercury, using different approaches. Based on a linear mixed model analysis, the GWAS yielded multiple hits with a single top hit on Chromosome 4, with eight more SNPs suggestive of association. A second approach, a latent factor mixed model analysis, highlighted one single SNP on Chromosome 11. Finally, an outlier test identified one additional SNP on Chromosome 4 that appeared under selection. Out of all ten SNPs we identified as associated with mercury in muscle, three SNPs all located on Chromosome 4 and positioned within a 2.5 kb distance of an annotated gene. Based on these results and the genome coverage of our SNPs, we conclude that the selective effect of mercury pollution in Flanders causes a significant association with at least one locus on Chromosome 4 in three-spined stickleback.

Better together: Cross-tolerance induced by warm acclimation and nitrate exposure improved the aerobic capacity and stress tolerance of common carp Cyprinus carpio.

Climate warming is a threat of imminent concern that may exacerbate the impact of nitrate pollution on fish fitness. These stressors can individually affect the aerobic capacity and stress tolerance of fish. In combination, they may interact in unexpected ways where exposure to one stressor may heighten or reduce the resilience to another stressor and their interactive effects may not be uniform across species. Here, we examined how nitrate pollution under a warming scenario affects the aerobic scope (AS), and the hypoxia and heat stress susceptibility of a generally tolerant fish species, common carp Cyprinus carpio. We used a 3 × 2 factorial design, where fish were exposed to one of three ecologically relevant levels of nitrate (0, 50, or 200 mg NO3- L-1) and one of two temperatures (18 °C or 26 °C) for 5 weeks. Warm acclimation increased the AS by 11% due to the maintained standard metabolic rate and increased maximum metabolic rate at higher temperature, and the AS improvement seemed greater at higher nitrate concentration. Warm-acclimated fish exposed to 200 mg NO3- L-1 were less susceptible to acute hypoxia, and fish acclimated at higher temperature exhibited improved heat tolerance (critical thermal maxima, CTMax) by 5 °C. This cross-tolerance can be attributed to the hematological results including maintained haemoglobin and increased haematocrit levels that may have compensated for the initial surge in methaemoglobin at higher nitrate exposure.

Temperature Effects During a Sublethal Chronic Metal Mixture Exposure on Common Carp (Cyprinus carpio).

The aquatic environment is the final sink of various pollutants including metals, which can pose a threat for aquatic organisms. Waterborne metal mixture toxicity might be influenced by environmental parameters such as the temperature. In the present study, common carp were exposed for 27 days to a ternary metal mixture of Cu, Zn, and Cd at two different temperatures, 10 and 20°C. The exposure concentrations represent 10% of the 96 h-LC50 (concentration lethal for the 50% of the population in 96 h) for each metal (nominal metal concentrations of Cu: 0.08 µM; Cd: 0.02 µM and Zn: 3 µM). Metal bioaccumulation and toxicity as well as changes in the gene expression of enzymes responsible for ionoregulation and induction of defensive responses were investigated. Furthermore the hepatosomatic index and condition factor were measured as crude indication of overall health and energy reserves. The obtained results showed a rapid Cu and Cd increase in the gills at both temperatures. Cadmium accumulation was higher at 20°C compared to 10°C, whereas Cu and Zn accumulation was not, suggesting that at 20°C, fish had more efficient depuration processes for Cu and Zn. Electrolyte (Ca, Mg, Na, and K) levels were analyzed in different tissues (gills, liver, brain, muscle) and in the remaining carcasses. However, no major electrolyte losses were observed. The toxic effect of the trace metal ion mixture on major ion uptake mechanisms may have been compensated by ion uptake from the food. Finally, the metal exposure triggered the upregulation of the metallothionein gene in the gills as defensive response for the organism. These results, show the ability of common carp to cope with these metal levels, at least under the condition used in this experiment.

High environmental temperature and low pH stress alter the gill phenotypic plasticity of Hoven's carp Leptobarbus hoevenii.

Climate warming and low pH environment are known to negatively impact all levels of aquatic organism from cellular to organism and population levels. For ammonotelic freshwater species, any abiotic factor fluctuation will cause disturbance to the fish, specifically at the gills which act as a multifunctional organ to support all biological processes. Therefore, this study was designed to investigate the effect of temperature (28 vs. 32°C) and pH (7.0 vs. 5.0) stress on the gill plasticity of Hoven's carp after 20 days of continuous exposure. The results demonstrated that high temperature and low pH caused severe changes on the primary and secondary lamellae as well as the cells within lamellae. An increasing trend of the proportion available for gas exchange was noticed at high temperature in both pH exposures, which resulted from a reduction of the primary lamellae width with elongated and thinner secondary lamellae compared to fishes at ambient temperature. Following exposure to high temperature and acidic pH, Hoven's carp experienced gill modifications including aneurysm, oedema, hypertrophy, curling of secondary lamellae, epithelial lifting, hyperplasia and lamellae fusion. These modifications are indicators of the coping mechanism of Hoven's carp to the changing environment in order to survive.

Joint toxicity assessment reveals synergistic effect of chlorpyrifos and dichlorvos to common carp (Cyprinus carpio).

Common carp (Cyprinus carpio) is an important aquaculture species. However, their production and health is sometimes threatened by pesticides. In common carp, extensive studies have been done for exposures of single pesticides, but effects of mixtures such as those of the commonly used chlorpyrifos and dichlorvos, are still unknown for this species. In the first phase of this work, an acute lethal exposure experiment was conducted to estimate 24 h to 96 h lethal concentrations (LC10-90) of chlorpyrifos, dichlorvos and their mixture. Compared to dichlorvos, chlorpyrifos was found to be highly toxic to the tested species. Joint toxicity assessment of these pesticides in binary mixtures was dominated by synergism. In the second experimental phase, common carp were exposed to sub-lethal concentrations (LD-10% and HD-50% 96 h-LC50) of individual pesticides and their mixture. General fish behaviors, buccal movements and feeding attempts by fish were recorded after 1 h, 24 h, 48 h, 72 h and 96 h whereas aerobic metabolism of fish was recorded for 0-24 h, 24-48 h 48-72 h and 72-96 h of exposure. All pesticide treatments elevated buccal movements and oxygen uptake in a dose dependent manner. Feeding depression was also observed by pesticide exposure. The augmented deleterious effect of these pesticides in a mixture suggests that joint toxicity assessment is critical to develop more realistic water quality standards and monitoring guidelines.

Double whammy: Nitrate pollution heightens susceptibility to both hypoxia and heat in a freshwater salmonid.

Species persistence in a changing world will depend on how they cope with co-occurring stressors. Stressors can interact in unanticipated ways, where exposure to one stressor may heighten or reduce resilience to another stressor. We examined how a leading threat to aquatic species, nitrate pollution, affects susceptibility to hypoxia and heat stress in a salmonid, the European grayling (Thymallus thymallus). Fish were exposed to nitrate pollution (0, 50 or 200 mg NO3- L-1) at two acclimation temperatures (18 °C or 22 °C) for eight weeks. Hypoxia- and heat-tolerance were subsequently assessed, and the gills of a subset of fish were sampled for histological analyses. Nitrate-exposed fish were significantly more susceptible to acute hypoxia at both acclimation temperatures. Similarly, in 18 °C- acclimated fish, exposure to 200 mg NO3- L- 1 caused a 1 °C decrease in heat tolerance (critical thermal maxima, CTMax). However, the opposite effect was observed in 22 °C-acclimated fish, where nitrate exposure increased heat tolerance by ~1 °C. Further, nitrate exposure induced some histopathological changes to the gills, which limit oxygen uptake. Our findings show that nitrate pollution can heighten the susceptibility of fish to additional threats in their habitat, but interactions are temperature dependent.

Mixed toxicity of chlorpyrifos and dichlorvos show antagonistic effects in the endangered fish species golden mahseer (Tor putitora).

Golden mahseer (Tor putitora) is an economically important but endangered fish species in many countries. Increasing pesticide application can possess a threat to this species but their sensitivity to pesticides, typically chlorpyrifos and dichlorvos, is unknown. We determined 96 h-LC50 of chlorpyrifos and dichlorvos to be 0.753 mg/L and 12.964 mg/L, respectively, indicating higher toxicity of chlorpyrifos than dichlorvos. Despite the same mode of action, their joint effect was antagonistic, with an additive index value of - 0.58 at 96 h-LC50. Moreover, to get insights in the temporal sub-lethal effects, fish were exposed to 10% and 50% of the 96 h-LC50 values of the respective pesticides. Aerobic metabolism, opercular movements, and feeding behavior were examined for sub-lethal end-points following 24 h, 48 h, 72 h and 96 h exposure. Both chlorpyrifos and dichlorvos in single exposures induced a significant drop in oxygen consumption rate; while it was significantly elevated in the mixed pesticide exposure. Accelerated opercular movements were observed in all pesticide treatment groups but were more persistent in chlorpyrifos treatments. Reduced feeding attempts were more pronounced in chlorpyrifos and mixture treatments wherein feeding attempts dropped to zero. Overall, the acute toxicity data reported in the present study can be used to assess the maximum tolerance level of golden mahseer to chlorpyrifos and dichlorvos, and their mixture. Furthermore, the sub-lethal end point responses can be applied in monitoring the environmental risk posed by these waterborne pesticides either individually or in combination to the aquatic life.

A comparative study on the effects of three different metals (Cu, Zn and Cd) at similar toxicity levels in common carp, Cyprinus carpio.

To improve our understanding of underlying toxic mechanisms, it is important to evaluate differences in effects that a variety of metals exert at concentrations representing the same toxic level to the organism. Therefore, the main goal of the present study was to compare the effects of waterborne copper (Cu(II)), zinc (Zn(II)) and cadmium (Cd (II)) on a freshwater fish, the common carp (Cyprinus carpio), at concentrations being 0%, 25%, 50% and 100% of the 96 h LC50 (the concentration which is lethal to 50% of the population in 96 h). All the exposures were performed for a period of 1 week at 20°C. Our results show a rapid increase in the amount of copper and cadmium accumulated in the gills, while zinc only started to increase by the end of the experiment. All three metal ions increased metallothionein gene expression in both gills and liver. However, clear adverse effects were mainly observed for the Cu exposed group. Cu caused a decrease in Na level in gill tissue; it altered the expression of genes involved in ionoregulation such as Na+ /K+ -ATPase and H+ -ATPase as well as the expression of oxidative stress-related genes, such as catalase, glutathione reductase and glutathione S-transferase. Zinc and cadmium exposure did not alter the ion levels in the gills. In addition, no obvious effect of oxidative stress was observed, except for a transient increase in glutathione reductase at the highest cadmium concentration.

Synergism between elevated temperature and nitrate: Impact on aerobic capacity of European grayling, Thymallus thymallus in warm, eutrophic waters.

Climate warming and nitrate pollution are pervasive aquatic stressors that endanger the persistence of fishes prevailing in anthropogenically disturbed habitats. Individually, elevated nitrate and temperature can influence fish energy homeostasis by increasing maintenance costs and impairing oxygen transport capacity. However, it remains unknown how fish respond to simultaneous exposure to elevated temperature and nitrate pollution. Hence, we examined the combined effects of nitrate and elevated temperatures on aerobic scope (AS, maximum-standard metabolic rates) and cardiorespiratory attributes (haemoglobin HB, haematocrit HCT, relative ventricle mass RVM, and somatic spleen index SSI) in a freshwater salmonid, Thymallus thymallus. A 3 × 2 factorial design was used, where fish were exposed to one of three ecologically relevant levels of nitrate (0, 50, or 200 mg NO3- l-1) and one of two temperatures (18 °C or 22 °C) for 6 weeks. Elevated temperature increased AS by 36 % and the improvement was stronger when coupled with nitrate exposure, indicating a positive synergistic interaction. HB was reduced by nitrate exposure, while HCT was independent of nitrate pollution and temperature. Stressor exposure induced remodeling of key elements of the cardiorespiratory system. RVM was 39 % higher in fish exposed to 22 °C compared to 18 °C but was independent of nitrate exposure. SSI was independent of temperature but was 85 % and 57 % higher in fish exposed to 50 and 200 mg NO3- l-1, respectively. Taken together, these results highlight that simultaneous exposure to elevated temperatures and nitrate pollution offers cross-tolerance benefits, which may be underscored by cardiorespiratory remodeling.

Toxicity and bioaccumulation of Cadmium, Copper and Zinc in a direct comparison at equitoxic concentrations in common carp (Cyprinus carpio) juveniles.

The individual toxicity and bioaccumulation of cadmium, copper and zinc for common carp juveniles was evaluated in a direct comparison in two experimental setups. First, fish were exposed for 10 days to different metal concentrations in order to link metal bioaccumulation to LC50 values (concentration lethal to 50% of the animals) and incipient lethal levels (ILL, concentration where 50% survives indefinitely). Accumulated metals showed a positive dose dependent uptake for cadmium and copper, but not for zinc. Toxicity was in the order cadmium>copper>zinc with 96h LC50 values for cadmium at 0.20±0.16 µM, for copper at 0.77±0.03 µM, and for zinc at 29.89±9.03 µM respectively. For copper, the 96h exposure was sufficient to calculate the incipient lethal level and therefore 96h LC50 and ILL levels were the same, while for cadmium and zinc 5 to 6 days were needed to reach ILL resulting in slightly lower values at 0.16 µM and 28.33 µM respectively. Subsequently, a subacute exposure experiment was conducted, where carp juveniles were exposed to 2 equitoxic concentrations (10% and 50% of LC50 96 h) of the three metals for 1, 3 and 7 days. Again a significant dose-dependent increase in gill cadmium and copper, but not in zinc, was observed during the 7-day exposure. Copper clearly affected sodium levels in gill tissue, while zinc and cadmium did not significantly alter any of the gill electrolytes. The overall histopathological effects (e.g. hyperemia and hypertrophy) of the metal exposures were mild for most of the alterations. Our study showed that copper an cadmium (but not zinc) showed dose dependent metal accumulation, however this bioaccumulation was only correlated with mortality for cadmium. Metal specific alterations were reduced gill sodium levels in copper exposed fish and oedema of the primary epithelium which typically occurred in both levels of zinc exposure.