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Investigating the effects of a sub-lethal metal mixture of Cu, Zn and Cd on bioaccumulation and ionoregulation in common carp, Cyprinus carpio
Abstract
The aquatic environment is continuously under threat because it is the final receptor and sink of waste streams. The development of industry, mining activities and agriculture gave rise to an increase in metal pollution in the aquatic system. Thus a wide occurrence of metal mixtures exists in the aquatic environment. The assessment of mixture stress remains a challenge considering that we can not predict the toxicity of a mixture on the basis of single compounds. Therefore the analysis of the effects of environmentally relevant waterborne mixtures is needed to improve our understanding of the impact of metal pollution in aquatic ecosystems. Our aim was to assess whether 10 % of the concentration of the 96 h LC50 (the concentration that is lethal to 50 % of the population in 96 h) of individual metal exposures can be considered as a "safe" concentration when applied in a trinomial mixture. Therefore, common carp were exposed to a sublethal mixture of Cu 0.07 ± 0.001 μM (4.3 ± 0.6 μg/L), Zn 2.71 ± 0.81 μM (176.9 ± 52.8 μg/L) and Cd 0.03 ± 0.0004 μM (3.0 ± 0.4 μg/L) at 20 °C for a period of one week. Parameters assessed included survival rate, bioaccumulation and physiological biomarkers related to ionoregulation and defensive mechanisms such as MT induction. Our results showed a sharp increase in Cu and Cd concentration in gills within the first day of exposure while Zn levels remained stable. The accumulation of these metals led to a Na drop in gills, liver and muscle as well as a decreased K content in the liver. Biomarkers related to Na uptake were also affected: on the first day gene expression for H+-ATPase was transiently increased while a concomitant decreased gene expression of the Na+/H+ exchanger occurred. A fivefold induction of metallothionein gene expression was reported during the entire duration of the experiment. Despite the adverse effects on ionoregulation all fish survived, indicating that common carp are able to cope with these low metal concentrations, at least during a one week exposure.
... Pollution caused by potentially toxic elements (PTE) in aquatic environments constitutes a special problem because these elements are persistent and bioaccumulate through food chains (Monferrán et al. 2016, Ali andKhan 2018). Even though some of the PTE can be classified as essential (copper [Cu], zinc [Zn]) because they are necessary for organisms, others are non-essential and can be considered harmful even at low concentrations (arsenic [As], cadmium [Cd], chrome [Cr], lead [Pb], mercury [Hg]) because they have no role in biological systems (Lemus et al. 2014, Castaldo et al. 2020. However, the toxicity of each element depends on its concentration and chemical form (Ali et al. 2021). ...
... While it is common to find low concentrations of Cr in fish (Yousafzai et al. 2017), the results of this study show higher concentrations than those reported by other authors (Čelechovská et al. 2007(Čelechovská et al. , Yousafzai et al. 2017. Finally, Cu is essential for metabolic functions, but in high concentrations it might represent a risk for liver and kidney (Maurya et al. 2019, Castaldo et al. 2020). However, Papagiannis et al. (2004) indicate that accumulation of Cu is low in freshwater fish, which agrees with the results in this study. ...
... Likewise, the liver is considered a contaminant storage since it acts as an active site of chronic effects caused by pollutants in most animals (Mziray and Kimirei 2016); also, it is the main tissue for biomonitoring elements that accumulate through food elements (Albuquerque et al. 2021). When pollutants exceed the accumulation capacity in the liver, the fish begins to store pollutants in other tissues such as muscle (Monferrán et al. 2016, Castaldo et al. 2020. ...
Environmental pollution is a major problem worldwide. One of the major health issues is the consumption of potentially toxic elements through fish intake, particularly in urban areas. Therefore, it is necessary to assess the human health risks of fish consumers. This study aims to determine concentrations of As, Cd, Cr, Cu, Hg, Pb, and Zn in the liver and muscle of freshwater carp (Cyprinus carpio) caught in El Tunal River, Durango, Mexico. Carps were collected in dry (n = 7) and rainy (n = 10) seasons. The highest concentrations of all elements were found in the liver, where As and Zn (4.83 and 6.51 µg/g w/w, respectively) were predominant. Meanwhile, As, Cr, and Zn (1.00, 1.12, and 1.09 µg/g w/w, respectively) were predominant in muscle. The concentrations of As, Cd, Cr, Pb, and Zn had significant differences between seasons (p < 0.05), showing that during the rainy season the concentration of elements in the river, and hence in fish tissues, was reduced. Elements in edible tissue were below the levels allowed in Mexico and by international agencies. The mean and total target hazard quotients were below 1, indicating a minor risk to human health. Based on this study, fish consumption does not represent a risk to human health; however, it is necessary to continue monitoring pollutants since their accumulation can increase over time.
... Based on previous results obtained in our lab (Castaldo et al., 2020b;Delahaut et al., 2020), we hypothesized on the one hand a fast Cu and Cd bioaccumulation, and on the other hand a delayed Zn accumulation occurring only after 1 week of exposure. Moreover we hypothesize that the presence of metals would trigger the response of MTs in order to mitigate possible deleterious effects. ...
... The general experimental run protocol as described by Shrivastava et al. (2017), consisted of a denaturation program (3 min at 95 • C), an amplification and quantification program repeated 40 times (15 s at 95 • C, 20 s at 60 • C) and a melting curve program (60-95 • C). Oligonucleotide primers were, β-actin (Wu et al., 2014), eEF (Sinha et al., 2012), H + -ATPase (Sinha et al., 2016), Na + /K + -ATPase (Castaldo et al., 2020b), Na + /H + exchanger (Castaldo et al., 2020b), CTR1 (Castaldo et al., 2020a), and metallothionein (Reynders et al., 2006b). Quantification cycles (Cq) values were automatically calculated on the log curve for each gene with MxPro QPCR software (Agilent Technologies, Waldbronn, Germany). ...
... The general experimental run protocol as described by Shrivastava et al. (2017), consisted of a denaturation program (3 min at 95 • C), an amplification and quantification program repeated 40 times (15 s at 95 • C, 20 s at 60 • C) and a melting curve program (60-95 • C). Oligonucleotide primers were, β-actin (Wu et al., 2014), eEF (Sinha et al., 2012), H + -ATPase (Sinha et al., 2016), Na + /K + -ATPase (Castaldo et al., 2020b), Na + /H + exchanger (Castaldo et al., 2020b), CTR1 (Castaldo et al., 2020a), and metallothionein (Reynders et al., 2006b). Quantification cycles (Cq) values were automatically calculated on the log curve for each gene with MxPro QPCR software (Agilent Technologies, Waldbronn, Germany). ...
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.
... Additionally, this species is considered to be a good bioindicator for ecotoxicological studies (Altun et al., 2017;Rajeshkumar et al., 2017) and is recommended in Organization for Economic Co-operation and Development (OECD) guidelines, as one of six fish species for regulatory testing (OECD, 2019). As a model species, common carp is used to study the impact of metals both in the lab and in the field, on for example bioaccumulation (Bervoets et al., 2009;Castaldo et al., 2020;Delahaut et al., 2019;Delahaut et al., 2020), energy status (De Boeck et al., 1995a;Kunwar et al., 2009), swimming capacity (Delahaut et al., 2019) or oxidative stress (Cortes-Diaz et al., 2017;Dugmonits et al., 2013;García-Medina et al., 2017;Pillet et al., 2019). Metal is found in every aquatic ecosystem and anthropogenic activity discharges metals directly into water from mines, industry, intensive agriculture, household waste or traffic (Burger, 2008;Coufalík et al., 2019;Stohs and Bagchi, 1995). ...
... A recent study investigating the impact of a similar environmentally relevant Cu/Cd/Zn mixtures on common carp, showed a number of effects on ion-regulation (reduced Na + transport) and detoxification functions (strong induction of metallothionein expression) (Castaldo et al., 2020), as well as oxidative stress capacities (Pillet et al., 2019) reflecting different toxicity mechanisms of the Cu/Cd/Zn mixture. The present study aimed to investigate whether these mechanisms translate into changes in whole-animal physiological performance linked to bioaccumulation of metal in tissues, and whether these are affected by temperature. ...
... However, at both temperatures, complete acclimatisation of the carp is expected by the end of the experiment. To facilitate direct comparisons with previous observations (Castaldo et al., 2020;Pillet et al., 2019), the concentrations of metal used in the present study targeted the concentrations previously used (10% of the LC 50 for single metal, as defined by Delahaut et al. (2020)), and also reflected ecologically-relevant concentrations. ...
In a natural ecosystem, fish are subjected to a multitude of variable environmental factors. It is important to analyze the impact of combined factors to obtain a realistic understanding of the mixed stress occurring in nature. In this study, the physiological performance of juvenile common carp (Cyprinus carpio) exposed for one week to an environmentally relevant metal mixture (4.8 μg/L of copper; 2.9 μg/L of cadmium and 206.8 μg/L of zinc) and to two temperatures (10 °C and 20 °C), were evaluated. After 1, 3 and 7 days, standard (SMR) and maximum metabolic rate (MMR) were measured and aerobic scope (AS) was calculated. In addition, hematocrit, muscle lactate, histology of the gills and metal accumulation in gills were measured. While SMR, MMR and AS were elevated at the higher temperature, the metal mixture did not have a strong effect on these parameters. At 20 °C, SMR transiently increased, but no significant changes were observed for MMR and AS. During metal exposure, hematocrit levels were elevated in the 20 °C group. The bioaccumulation of Cd in the gills reflected the increased metabolic rate at the higher temperature, with more accumulation at 20 °C than at 10 °C. Anaerobic metabolism was not increased, which corresponds with the lack of significant histopathological damage in the gill tissue. These results show that common carp handled these metal exposures well, although increased temperature led to higher Cd accumulation and necessitated increased hematocrit levels to maintain aerobic performance.
... The accumulation mechanism of heavy metals occurs as a result of combination of uptake, metabolism and excretion (Boudjema et al., 2022). Metals can become toxic when their uptake is not balanced by excretion processes and detoxifying systems (Castaldo et al., 2020). We agree with the findings of Boudjema et al. (2022) and Castaldo et al. (2020). ...
... Metals can become toxic when their uptake is not balanced by excretion processes and detoxifying systems (Castaldo et al., 2020). We agree with the findings of Boudjema et al. (2022) and Castaldo et al. (2020). Many reports from our laboratory indicated that most of the reports published earlier were based on individual impact analysis of heavy metals on different organisms. ...
... species exposed, body size, ration size, and body temperature) (van der Zande et al., 2020). The liver and kidneys are known to be one of the main organs in fish with a detoxification function when exposed to metal contamination via metal excretion and due to metal-binding proteins (MT) (M'kandawire et al., 2017;Castaldo et al., 2020). Moreover, the importance of cytochrome P450 and glutathione-S-transferases for metals/NPs detoxification in fish has been demonstrated by several studies (Scown et al., 2010;Carvalho et al., 2012;Teles et al., 2019). ...
... Furthermore, in agreement with Brix et al. (2017), reporting that both Cd and Cu significantly decreased Zn accumulation in rainbow trout gills exposed to a binary mixture of Zn and Cd/Cu, we found that, in the present study, ZnO NPs uptake in the ZnO NPs and GNs combination exposure was lower compared to a single exposure of ZnO NPs in all organ tissues. A similar study by Castaldo et al. (2020) found that in common carp, Zn accumulation under a co-exposure with Cd was four times lower compared to the predicted single exposure scenario. The authors suggested that a reciprocal inhibition of Zn uptake can occur in common carp. ...
In this study, we investigated the bioaccumulation and toxicokinetics of zinc oxide nanoparticles (ZnO NPs) alone and in the presence of graphene nanosheets (GNs) in the blackfish (Capoeta fusca). Blackfish were exposed via water to two ZnO NPs concentrations alone or as a combination with GNs and uptake of Zn into the gills, intestine, liver, and kidney was assessed at 7, 14 and 28 d. Zn elimination from these tissues was then assessed after a further 7, 14 and 28 d in clean water for both ZnO NPs concentrations and combined ZnO NPs/GN exposures. In the body tissues analyzed of exposed fish, the highest amounts of Zn occurred in the intestine and the lowest amount in the liver. Zn levels in blackfish after 28 d of exposure were higher in all treatment groups compared to those on 7 d (p < 0.05). For both ZnO NPs exposure concentrations, the highest amount of Zn was eliminated from the intestine, followed by the gills. Furthermore, elimination kinetics for both ZnO NPs concentrations alone and in combination with GNs showed that the shortest half-life for Zn is occurring in the intestine. Moreover, uptake rates of Zn in fish exposed to ZnO NPs + GNs followed the same pattern observed for the ZnO NP, with intestine and gills having the highest levels followed by kidney and liver. Thus, we show accumulation and elimination of Zn from ZnO NPs in blackfish depends on the tissue, exposure concentration and duration, and is dependent on the presence of GNs.
... species exposed, body size, ration size, and body temperature) (van der Zande et al., 2020). The liver and kidneys are known to be one of the main organs in fish with a detoxification function when exposed to metal contamination via metal excretion and due to metal-binding proteins (MT) (M'kandawire et al., 2017;Castaldo et al., 2020). Moreover, the importance of cytochrome P450 and glutathione-S-transferases for metals/NPs detoxification in fish has been demonstrated by several studies (Scown et al., 2010;Carvalho et al., 2012;Teles et al., 2019). ...
... Furthermore, in agreement with Brix et al. (2017), reporting that both Cd and Cu significantly decreased Zn accumulation in rainbow trout gills exposed to a binary mixture of Zn and Cd/Cu, we found that, in the present study, ZnO NPs uptake in the ZnO NPs and GNs combination exposure was lower compared to a single exposure of ZnO NPs in all organ tissues. A similar study by Castaldo et al. (2020) found that in common carp, Zn accumulation under a co-exposure with Cd was four times lower compared to the predicted single exposure scenario. The authors suggested that a reciprocal inhibition of Zn uptake can occur in common carp. ...
... Also, it causes hyperdiffusability of ions across the gill barrier, resulting in increased Na + outflow [66]. The HA-induced hypocalcemia and low pH lead to either decrease in the systemic K + content or increase in net efflux of K + [67] After recovery, serum ionized sodium, chloride, and KCllevels returned to normal, indicating a partial tolerance due to posttranslational modifications in the gene encoding particular ion transporters, similar to what was observed in common carp (Cyprinus carpio) when given trinomial metal combinations [68]. ...
Hyaluronic acid (HA) is a versatile polysaccharide with a broad range of biological, pharmacological, and therapeutic applications due to its anti-inflammatory properties, low immunogenicity, natural breakdown, compatibility with live tissue, and capacity to promote cell growth and specialization. However, the potential negative effects of HA on aquatic ecosystems, especially fish, have not been fully investigated. This study examines for the first time the potential physiological, immunological, and histological effects of HA in African catfish, as well as the long-term implications following recovery. Four groups, totaling 24 African catfish in triplicate, were subjected to varying concentrations of HA (500–100,000 µg/L) over a 15-day exposure period, followed by a 15-day recuperation phase, leading to physiological changes dependent on the dosage. The electrolyte levels in the plasma and the equilibrium of redox processes were significantly upset by exposure to HÀ. Fish exposed to high HA concentrations displayed dose-dependent declines in immunological function and antioxidant enzymes. Only the fish exposed to modest doses of HA showed complete recovery when compared to the control fish, indicating that exposure to HA resulted in irreversible alterations. The histological investigations showed a number of dosage-dependent pathological alterations in the renal and hepatic tissues, including perivascular and peritubular fibrosis, tubular necrosis, and vacuolar degeneration. The degree of these alterations varied according to the dose. Concluding that fish may have irreversible alterations as a result of high acidity. The present study provides a fundamental understanding of the detrimental effects of HA on fish. To fully evaluate the potential dangers associated with HA exposure and its long-term effects on aquatic life, more research is needed.
... Heavy metals cannot be decomposed within fish, so they accumulate in the liver over time, resulting in heightened concentrations. Additionally, gills play crucial roles in respiration, osmoregulation, excretion, and pH regulation, serving as primary conduits for metal ion exchange in water and having a large surface area that allows for rapid diffusion of metal ions [20][21][22]. Fish muscles typically have lower heavy metal content compared to other body parts, indicating a higher likelihood of heavy metal enrichment in viscera than muscles [23,24]. ...
At a time when heavy metal pollution is increasing, assessing the levels of contamination and associated health risks is crucial. Samples of water, aquatic plants, and fish were collected from four key areas of heavy metal pollution prevention and control in Zhejiang Province. The levels of elements were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES). A human health risk model was also developed. The study revealed that heavy metal pollution in the five industrial zones exceeded the national standard for Class V water. Elements like arsenic (As), cadmium (Cd), and chromium (Cr) exceeded permissible levels in aquatic plants across all industrial zones; the exception was lead (Pb). Moreover, the heavy metal concentrations in subject fish tissues collected from each industrial area exceeded safe limits, especially in the gut. According to the human health risk evaluation model, the health risk (1.12 × 10−3) and children’s health risk (1.10 × 10−3) in these prevention and control zones surpassed the maximum acceptable human risk values. In conclusion, heavy metal elements, along with other pollutants, accumulate and become concentrated in the examined aquatic plants and fish. These pollutants move through the food chain, impacting the entire aquatic ecosystem and posing a health risk to nearby populations.
... The exposed fish stopped feeding for the first two days and later the exposed fish took normal food. Metals can become toxic when their uptake is not balanced by excretion processes and detoxifying systems (Castaldo et al., 2020). The present investigation is a combined effect of effluent waste study on fishes under field and laboratory conditions. ...
... The process of trace metals bioaccumulation in organisms involves a delicate balance of absorption, metabolic processing, and elimination mechanisms, as outlined by Casas and Bacher (2006). While trace metals are vital, excessive intake, beyond an organism's excretion and detoxification capabilities, can lead to toxicity, as highlighted by Pelletier and Campbell., (2004), and Castaldo et al. (2020). Although there has been extensive focus on the impacts of individual trace metals, the toxicological interactions resulting from mixed heavy metal exposure (whether antagonistic, synergistic, or neutral) are less studied. ...
... The levels of metals in the aquatic ecosystem at any given time represent the current contamination while that found in the aquatic species show an effect of bioaccumulation resulting from a relatively exposure period [44]. Heavy metal accumulation in fish primarily results from life events such as breathing and predation via the food chain. ...
The purpose of this study was to analyze levels of heavy metals and human health risk implications associated with fish consumption from the Yangtze River. A total of 60 fish muscles were taken from six different fish species—Hypophthalmichthys molitrix, Ctenopharyngodon idellus, Blicca bjoerkna, Mylopharyngodon piceus, Carassius carassius and Pelteobagrus fulvidraco—and digested using standard protocols. Contents of lead (Pb), cadmium (Cd), zinc (Zn), aluminum (Al) cobalt (Co), manganese (Mn), chromium (Cr), and copper (Cu), were analyzed using an Atomic Absorption Spectroscophotometer ((ZEEnit 700 P Zeeman)). Based on consumer health risk indicators, the health implications to children and adults upon consuming the analyzed fish species were assessed. Findings revealed that Zn recorded the highest mean concentration of 9.87 µg/g in Carassius carassius followed by Mn (7.97 µg/g) in Pelteobagrus fulvidraco, Cu (2.07 µg/g) in Mylopharyngodon piceus, Pb (1.04 µg/g) in Hypophthalmichthys molitrix, Cr (0.63 µg/g) in Hypophthalmichthys molitrix, Cd (0.19 µg/g) in Blicca bjoerkna and Ni (0.16 µg/g) (w/w) in Pelteobagrus fulvidraco. In addition, the health risk assessments revealed that children are at heightened non-carcinogenic risk for Pb, Cd, and Co upon consuming the examined fish species.
... In addition to naturally occurring toxicants, such as poisonous food sources, venomous predators and toxic algal blooms, a wide range of chemical stressors of anthropogenic origin reach aquatic ecosystems via for instance wastewater effluents and runoff (Deblonde et al. 2011, Müller et al. 2020. Upon harmful exposure to a chemical stressor, the organism's ability to maintain homeostasis is exceeded and consequently, adverse effects arise (Adiele et al. 2011, Zeng et al. 2019, Castaldo et al. 2020. In order to cope with variations in the natural environment, various molecular defense systems have consequently evolved to relieve general chemical stress and maintain cellular homeostasis (Kültz 2003, Sulmon et al. 2015, Birnie-Gauvin et al. 2017, Wang et al. 2019a). ...
Aquatic organisms are constantly at risk of being exposed to potentially harmful chemical compounds of natural or anthropogenic origin. Biological life can for instance respond to chemical stressors by changes in gene expression, and thus, certain gene transcripts can potentially function as biomarkers, i.e. early warnings, of toxicity and chemical stress. A major challenge for biomarker application is the extrapolation of transcriptional data to potential effects at the organism level or above. Importantly, successful biomarker use also requires basal understanding of how to distinguish actual responses from background noise. The aim of this thesis is, based on response magnitude and variation, to evaluate the biomarker potential in a set of putative transcriptional biomarkers of general toxicity and chemical stress.
Specifically, I addressed a selection of six transcripts involved in cytoprotection and oxidative stress: catalase (cat), glutathione-S-transferase (gst), heat shock proteins 70 and 90 (hsp70, hsp90), metallothionein (mt) and superoxide dismutase (sod). Moreover, I used metal exposures to serve as a proxy for general chemical stress, and due to their ecological relevance and nature as sedentary filter-feeders, I used bivalves as study organisms.
In a series of experiments, I tested transcriptional responses in the freshwater duck mussel, Anodonta anatina, exposed to copper or an industrial waste-water effluent, to address response robustness and sensitivity, and potential controlled (e.g. exposure concentration) and random (e.g. gravidness) sources of variation. In addition, I performed a systematic review and meta-analysis on transcriptional responses in metal exposed bivalves to (1) evaluate what responses to expect from arbitrary metal exposures, (2) assess the influence from metal concentration (expressed as toxic unit), exposure time and analyzed tissue, and (3) address potential impacts from publication bias in the scientific literature.
Response magnitudes were generally small in relationship to the observed variation, both for A. anatina and bivalves in general. The expected response to an arbitrary metal exposure would generally be close to zero, based on both experimental observations and on the estimated impact from publication bias. Although many of the transcripts demonstrated concentration-response relationships, large background noise might in practice obscure the small responses even at relatively high exposures. As demonstrated in A. anatina under copper exposure, this can be the case already for single species under high resolution exposures to single pollutants. As demonstrated by the meta-regression, this problem can only be expected to increase further upon extrapolation between different species and exposure scenarios, due to increasing heterogeneity and random variation. Similar patterns can also be expected for time-dependent response variation, although the meta-regression revealed a general trend of slightly increasing response magnitude with increasing exposure times.
In A. anatina, gravidness was identified as a source of random variability that can potentially affect the baseline of most assessed biomarkers, particularly when quantified in gills. Response magnitudes and variability in this species were generally similar for selected transcripts as for two biochemical biomarkers included for comparison (AChE, GST), suggesting that the transcripts might not capture early warnings more efficiently than other molecular endpoints that are more toxicologically relevant. Overall, high concentrations and long exposure durations presumably increase the likelihood of a detectable transcriptional response, but not to an extent that justifies universal application as biomarkers of general toxicity and chemical stress. Consequently, without a strictly defined and validated application, this approach on its own appears unlikely to be successful for future environmental risk assessment and monitoring. Ultimately, efficient use of transcriptional biomarkers might require additional implementation of complementary approaches offered by current molecular techniques.
... The levels of metals in the aquatic ecosystem at any given time represent the current contamination while that found in the aquatic species show an effect of bioaccumulation resulting from a relatively exposure period (Castaldo, Pillet et al. 2020). Heavy metals accumulation in sh primarily results from life events such as breathing and predation via the food chain. ...
Heavy metal contamination in aquatic environments has been a hot topic in the past decades. The current study aims to analyze levels of heavy metals and human health risk implications associated with fish consumption from the Yangtze River. Muscles of 60 fish samples which comprised six different fish species: Hypophthalmichthys molitrix , Ctenopharyngodon idellus , Blicca bjoerkna , Mylopharyngodon piceus , Carassius carassius, and Pelteobagrus fulvidraco ; were analyzed for total lead (Pb), cadmium (Cd), zinc (Zn), aluminium (Al) cobalt (Co), magnesium (Mg), chromium (Cr), and copper (Cu). The health risk indicators associated with consumers’ health were estimated for both children and adults. The finding of the study revealed that of the analyzed metals, Zn recorded the highest mean concentration of 9.87 µg/g in Carassius carassius followed by Mn (7.97 µg/g) in Pelteobagrus fulvidraco , Cu (2.07 µg/g) in Mylopharyngodon piceus , Pb (1.04 µg/g) in Hypophthalmichthys molitrix , Cr (0.63 µg/g) in Hypophthalmichthys molitrix , Cd (0.19 µg/g) in Blicca bjoerkna and Ni (0.16 µg/g) in Pelteobagrus fulvidraco all measured in wet weight ( w/w ). In addition, the health risk assessments revealed that children are at heightened non-carcinogenic risk for Pb, Cd, and Co upon consuming the examined fish species. The principal component analysis revealed that the pollution of metals in the Yangtze River originates mainly from anthropogenic activities and could deteriorate the quality of fish in the Yangtze River. Therefore, this study could contribute scientific information to help in proper monitoring and regulations to protect the natural resources and human health along the Yangtze River.
... It has been reported that the expression of Rh protein increased significantly in the gill of Eriocheir sinensis, Portunus trituberculatus, and L. vannamei after abiotic stress, and ammonia excretion was inhibited after Rh protein knockdown in Caenorhabditis elegans and P. trituberculatus (Martin et al., 2011;Ren et al., 2015;Aida et al., 2016;Si et al., 2018). The Na + /H + exchanger in gill cells creates an electrochemical gradient for regulation osmotic pressure, which can help aquatic organisms balance the changes of osmotic pressure in vivo (Castaldo et al., 2020). The potential promotion of ammonia secretion also in Na + /H + exchanger (Fehsenfeld and Wood, 2018). ...
Saline-alkaline waters are stressful environments where most aquatic animals can’t survive normally, and alkalinity is one of the key limited environmental factors. Due to strong adaptability to environment, the ridgetail white prawn Exopalaemon carinicauda is a potential good species suitable for large-scale culture in saline-alkaline waters. Exploring its alkaline adaptability mechanism will help to guide more marine crustaceans to saline-alkaline culture. In this study, an integrative analysis of the gill-specific transcriptome and proteome at 0, 12, and 36 h after alkalinity stress was performed to identify important regulators and pathways involved in alkalinity adaption of E. carinicauda. A total of 3,157 differentially expressed genes (DEGs) and 443 differentially expressed proteins (DEPs) were identified at 12 and 36 h compared with 0 h. Base on the transcriptome analysis, the Gene Ontology (GO) enriched terms were mainly related to ion transport, including “calcium-transporting ATPase activity,” “ATPase coupled ion transmembrane transporter activity,” “divalent inorganic cation transmembrane transporter activity,” etc., and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways mainly refer to the processes of endocrine system at both 12, and 36 h. Based on the proteomic analysis, KEGG pathways related to lipolysis and amino acids metabolism were significantly enriched at 12 h, and carbohydrate metabolism and immune response were significantly enriched at 36 h. There were significantly up-regulated expressions of ion transport related genes including aquaporin, carbonic anhydrase, ammonium transporter Rh type A-like, Na+/H+-exchanger, etc., as well as ion transport proteins including V-type proton ATPase 116 kDa subunit a-like isoform X1, sodium-potassium ATPase beta, vesicle associated membrane protein, etc. after alkalinity exposure, which indicating their important roles in response to alkalinity stress. The results of integrated analysis between proteome and transcriptome showed that up-regulated DEG/DEP (aldehyde dehydrogenase) was significantly enriched at 12 h and the up-regulated DEG/DEP (peptidylglycine alpha) was significantly enriched at 36 h, suggesting the two molecules may be critical in response to alkalinity change. This study reveals the first time-course, gill-specific, combined transcriptomic and proteomic profiling associated with alkalinity adaption of E. carinicauda and provides new insights into the mechanisms underlying the molecular response to alkalinity stress in shrimp.
... The compositions of metal mixtures in the environment are wide-ranging, and developing models that predict the toxicity of those mixtures to aquatic organisms is challenging (Van Genderen et al. 2015). Nevertheless, recently considerable research attention has been directed towards understanding the potential ecological risks and toxicological impact for the aquatic ecosystem associated with multimetal and metalbased mixtures (Kapia et al. 2016;Castaldo et al. 2020;Cao et al. 2020;Vendrell-Puigmitja et al. 2020;Zhao et al. 2021). Therefore, to gain a more realistic approach in risk assessments for mixtures of pollutants, it is essential to consider the acute and chronic effects of mixtures on fish exposed in the range of (or at least close to) environmentally relevant concentrations (Jijie et al. 2020;Alvarado-Flores et al. 2021). ...
Sticklebacks (Gasterosteiformes) are increasingly used in ecological and evolutionary research and have become well established as role model species for biologists. However, ecotoxicology studies concerning behavioural effects in sticklebacks regarding stress responses, mainly induced by chemical mixtures, have hardly been addressed. For this purpose, we investigated the swimming behaviour (including mortality rate based on 96-h LC50 values) of two ecologically similar three-spined (Gasterosteus aculeatus) and nine-spined sticklebacks (Pungitius pungitius) to short-term (up to 24 h) metal mixture (MIX) exposure. We evaluated the relevance and efficacy of behavioural responses of test species in the early toxicity assessment of chemical mixtures. Fish exposed to six (Zn, Pb, Cd, Cu, Ni, and Cr) metals in the mixture were either singled out by the Water Framework Directive as priority or as relevant substances in surface water, which was prepared according to the environmental quality standards (EQSs) of these metals set for inland waters in the European Union (EU) (Directive 2013/39/EU). The performed behavioural analysis showed the main effect on the interaction between time, species, and treatment variables. Although both species exposed to MIX revealed a decreasing tendency in swimming activity, these species’ responsiveness to MIX was somewhat different. Substantial changes in the activity of G. aculeatus were established after a 3-h exposure to MIX solutions, which was 1.43-fold lower, while in the case of P. pungitius, 1.96-fold higher than established 96-h LC50 values for each species. This study demonstrated species-specific differences in response sensitivity to metal-based water pollution, indicating behavioural insensitivity of P. pungitius as model species for aquatic biomonitoring and environmental risk assessments.
... Following Cd exposure, the main mechanism of fish toxicity is the disturbance of Na + and Ca 2+ ion homeostasis [14,[36][37][38][39][40][41][42][43]. Whole body Na + levels in zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) are commonly used as indicators for acute and subacute Cd 2+ toxicity in fish [14,15,36]. ...
The amounts of cadmium in multiple organs and the amounts of Na+ and Ca2+ in the carcass were measured in dead and surviving southern catfish exposed to different concentrations of Cd. The 96 h median lethal concentration was 6.85 mg/L. The Cd content and Cd accumulation rate were positively correlated with Cd exposure concentrations, and there were significant differences between dead and surviving individuals, indicating that both Cd content in tissues and Cd accumulation rates were correlated with mortality. Cd levels in the liver of dead fish were saturated. A lethal threshold for Cd concentration in the whole fish was obtained. Bioconcentration factors for Cd did not decrease with increasing exposure. Acute exposure to waterborne Cd caused a significant decrease in the ion content of the fish carcass. There was a significant difference between the Na+ content of the carcass of dead fish (34.54 μmol/g wet weight) and surviving fish (59.34 μmol/g wet weight), which was not the case with the Ca2+ content, indicating that the lethal toxicity of Cd was probably related to the decrease in Na+ content. Collectively, these results suggest that whole-fish Cd concentration and carcass Na+ content can be useful indicators of fish acutely exposed to Cd.
... Common carp (Cyprinus carpio) is a common species in China's freshwater fish breeding market, which has a stronger response to pressure stress and is easily affected by environmental pollutants [21]. Therefore, common carp was selected as the experimental object to evaluate the renal toxicity of Zn and As. ...
Arsenic (As) pollution is ubiquitous in water, which shows immunotoxicity to aquatic organisms. As an indispensable regulator of gene transcription and enzymatic modification, zinc (Zn) may play a preventive and therapeutic effect on As toxicity. The purpose of this study was to investigate the interactions of As and Zn on the head kidney of common carp Cyprinus carpio. Herein the carp were treated alone or in combination with waterborne As³⁺ (2.83 mg/L) and/or Zn²⁺ (1 mg/L). Results suggested a head kidney-toxic effect of As exposure, which was manifested by the histopathological damage of the head kidney, elevation of nuclear translocation of pro-inflammatory nuclear factor-kappa light chain enhancer of B cells (NF-κB), and blockage of the anti-oxidative nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. The global activation of three endoplasmic reticulum (ER) stress pathways led to the execution of programmed cell death, including ER apoptosis mediated by C/EBP-homologous protein (CHOP), death receptor–mediated exogenous cell apoptosis, and the endogenous apoptosis executed by Caspases9. The combined application of Zn can significantly improve the histopathological damage of the head kidney, the imbalance of the antioxidant system, and the apoptosis outcomes due to ER stress. In conclusion, this study indicates that Zn has an antagonistic effect on the head kidney injury of common carp induced by sub-chronic As exposure. The results of this study provide basic data for the risk assessment of As accumulation in an aquatic environment and a reference for the use of Zn preparation in aquaculture.
... Assessment of the stress caused by chemical mixtures remains a challenge given that it cannot be predicted based on individual components. Therefore, to gain a better understanding of metal pollution in aquatic ecosystems, it is necessary to study effects of environmentally relevant metal mixtures in the aquatic environment (Castaldo et al., 2020). Such metals as copper (Cu), zinc (Zn), chromium (Cr) and nickel (Ni) are essential nutrients required for various biochemical and physiological functions (WHO/-FAO/IAEA, 1996). ...
The present study tested the biological consequences of exposure to a multimetal mixture as a multiple chemical stressor on Oncorhynchus mykiss at molecular, cellular, physiological and whole-organism levels and on biomarker responses of this fish during the depuration period. To represent environmentally relevant multiple chemical stressors, in our study, we used the mixture of Zn, Cu, Ni, Cr, Pb and Cd at the concentrations corresponding to Maximum-Permissible-Concentrations (MPCs) acceptable for the EU inland waters. This study was undertaken with a view to elucidate if changes in the MPC of the test mixture components (Ni, Pb, Cd) could cause significantly different biomarker responses in O. mykiss from those previously determined in the carnivorous and omnivorous fishes exposed to the mixture of the same metals but at different MPCs of Ni, Pb and Cd. This study has revealed that exposure to mixtures of metals at MPC produces genotoxic effects in fish blood erythrocytes and a lethargic effect on O. mykiss behaviour, and, also, significantly increases the levels of Cd, Cr and Ni accumulated in the gills tissue. O. mykiss successfully depurated Cr and Ni in less than 28 days, however, the level of Cd decreased by only approximately 40% over the same period. A significant capacity of O. mykiss to restore its DNA integrity (Comet assay) after exposure to metal mixtures was revealed. However, the 28-day recovery period proved to be insufficiently long for erythrocytes with nuclear abnormalities to recover to the unexposed level. In conclusion, changes in the MPCs of Ni, Pb and Cd in the test mixture produce biological effects similar to those previously determined in S. salar, R. rutilus and P. fluviatilis exposed to the mixture of the same metals but at lower MPCs of Ni and Pb and at higher MPC of Cd.
... When the fish is subjected to cadmium stress in the environment, the level of MT will increase significantly to combine with Cd, thereby acting as a detoxifier. However, when the amount of cadmium stress exceeds the MT binding capacity, excessive cadmium will cause damage to the fish, and further, the expression of MT decreases (Castaldo et al. 2020). The fact that the expression of the MT gene was downregulated with low Cd concentration, while the downregulation of MT was not observed in the treatments with a higher concentration of Cd. ...
An unexplored attributing molecular mechanism of Cd toxicity is interference with the epigenetic machinery, such as DNA methylation, processes that are crucial for early fetal development. In order to investigate the effects of Cd on the expression of metallothionein (MT) and Dnmts transcripts, markers of DNA methylation, and signaling pathway gene expression, zebrafish embryos were exposed during 24 hours post-fertilization (starting at maximum 8-cell stage) to 0.0089, 0.089, and 0.89 μM Cd. The results showed that the Cd accumulation in zebrafish embryo reached a stable level after 12 hpf, and the Cd accumulation at individual time points was significantly different among different concentration groups. MT mRNA fold was significantly positive with the Cd content in embryos. We observed that the expression level of DNA methyltransferase (Dnmts) in the 0.089 μM Cd exposure group was significantly up-regulated. Dnmt1 expression was significantly up-regulated in the 0.89 μM Cd exposure group, and Dnmt3s expression and global methylation levels were significantly down-regulated. Cd up-regulated ErbB-3 gene expression, down-regulated ErbB-4 gene expression, and neutralized ErbB-1 gene expression. Cd activated Ca²⁺, MAPK-JUK, p38 MAP kinase, PI3K-AKT, and VEGF signaling pathway genes, indicating these pathway genes related to Cd exposure level. The results are helpful to clarify the molecular mechanism of DNA methylation in zebrafish embryo under metal pressure and further interference with the epigenetic machinery.
... Heavy metals (HMs) are one of the most hazardous and bioaccumulative contaminants in the aquatic environment. Industrial waste, mines, agricultural fields, and wastewater treatment plants release significant amounts of Cr, Cd, and Cu exceeding the standard limit (Castaldo et al., 2020;Masindi and Muedi, 2018). The standards for Cr and Cd in utilizable water are 0.1 mg/L and 0.01 mg/L, respectively and for Cu is up to 1.3 mg/L (APHA, 2005). ...
Heavy metals (HMs) in an aquatic environment mainly affects fish, and thus, fish are convenient pollution bio-indicators. In this study, the toxic effects of HM mixture (chromium (Cr), cadmium (Cd), copper (Cu)) in 0 mg/L to 3.2 mg/L concentration range was investigated in Cyprinus carpio (28 days). HM accumulation, histopathology, oxidative stress, and gut microbial changes were evaluated. HMs accumulated in the order of Cr > Cu > Cd, primarily in the kidneys and finally scales. Reactive oxygen species generation increased in all exposure groups upto day 14, with maximum generation at 3.2 mg/L mixture, which later decreased on day 28 in all. Malondialdehydeand and superoxide dismutase levels increased from day 7 to 28 with increased HM concentrations, while total protein showed an inverse trend. Gill histopathology showed major changes such as uplifted and disintegrated primary lamella, and secondary lamella shortening. The kidneys were characterized by glomerular necrosis, Bowman’s capsule expansion, and tubular space dilatation. Proteobacteria and Firmicutes abundance increased upto 59.4% and 99.16% in 0.8 mg/L and 3.2 mg/L treatment groups, respectively. This study provided a better understanding on the physiology and gut microbiota alteration in C. carpio under multiple HM stress.
... In order to avoid the accumulation of waste products, such as ammonia, 90 % of the water was changed daily. As indicated in Castaldo et al. (2020), aerated EPA medium-hard water was prepared 24 h in advance and kept at 20°C. Conductivity (275 ± 6.2 μS/cm) and pH (8.2 ± 0.2) were measured daily. ...
In the aquatic environment, metals are present as mixtures, therefore studies on mixture toxicity are crucial to thoroughly understand their toxic effects on aquatic organisms. Common carp were used to assess the effects of short-term Cu(II) and Cd(II) mixtures, using a fixed concentration of one of the metals, representing 25 % of its individual 96h-LC50 (concentration lethal for 50 % of the population) combined with a variable concentration of the other metal corresponding to 10, 25 or 50 % of its 96h-LC50, and vice versa. Our results showed a fast Cu and Cd bioaccumulation, with the percentage of increase in the order gill > liver > carcass. An inhibitory effect of Cu on Cd uptake was observed; higher Cu concentrations at fixed Cd levels resulted in a decreased accumulation of Cd. The presence of the two metal ions resulted in losses of total Na, K and Ca. Fish tried to compensate for the Na loss through the induction of the genes coding for Na⁺/K⁺-ATPase and H⁺-ATPase. Additionally, a counterintuitive induction of the gene encoding the high affinity copper transporter (CTR1) occurred, while a downregulation was expected to prevent further metal ion uptake. An induction of defensive mechanisms, both metal ion binding protein and anti-oxidant defences, was observed. Despite the metal accumulation and electrolyte loss, the low mortality suggest that common carp is able to cope with these metal levels, at least during a one-week exposure.
Cadmium (Cd) is a highly toxic substance in the aquatic ecosystem, which can represent a high risk to fish. Fish are exposed to heavy metals through waterborne and dietary pathways, some of which are absorbed by the body and can accumulate in specific tissues without being eliminated. The accumulation varies depending on several factors such as dose, exposure route, exposure time, metal types, and biological status of the fish, and environmental parameters such as DO, salinity, pH, and metal speciation. As Cd speciation occurs in the water, the amount accumulated in the fish can vary, and consuming Cd-accumulated fish can pose a risk to human health. Cd introduced into the body of fish can directly affect blood properties through the circulatory system. Cd introduced into the circulatory system of fish can reach all tissues through the blood flow, and the accumulation of specific tissues is different depending on the blood flow by the energy and oxygen demand of each tissue. Therefore, this review aimed to determine the toxic effects of Cd exposure in fish and identify indicators to assess the extent of Cd bioaccumulation toxicity in fish induced by Cd exposure.
The present study was carried out to determine the effect of dietary cadmium exposure on growth performance, changes in manganese, zinc, copper, calcium, magnesium, iron, selenium, and cadmium metals in liver and muscle tissue, liver antioxidant enzymes, and the histology of the fish. Rainbow trout (Oncorhynchus mykiss) with weights of 39.45 ±1.13 g was used in the experiment conducted in 2 groups and three replicates. The Control group was fed a cadmium-free diet, and the Cadmium group was fed a diet containing 5.03 µg/kg of cadmium twice a day until satiation. It was determined that cadmium intake through the diet affected growth rate and the feed evaluation performance negatively. In fish exposed to cadmium, manganese, zinc, copper, calcium, magnesium, iron, selenium, superoxide dismutase, catalase and glutathione peroxidase values in both muscle and liver tissues were significantly (p<0.05) decreased, whereas cadmium levels in muscle and liver and malondialdehyde levels in liver were significantly (p<0.05) increased. The histopathological examination of the liver revealed that cadmium caused liver damage. These results showed that rainbow trout exposed to dietary cadmium were highly sensitive to the metal, and the decreased levels of metals such as copper, zinc, manganese, and selenium in the liver tissue, which are involved in the antioxidant defence system, can be considered an indicator of the weakening of the antioxidant defence system.
Different anthropogenic activities as well as natural sources contribute enormously towards various heavy metal contaminations in aquatic habitats. Cadmium (Cd) is one of most prevalent and toxic heavy metals with a long half life. Unlike terrestrial animals, exposure of Cd in fishes may happen not only through feeds but also from its habitat water. Bioaccumulation of Cd in fishes occurs in many tissues, but mainly in gill, liver, kidney, skin, and muscle. The concentrations of Cd in fish tissues depend upon the extent and duration of Cd exposure, species and age of fishes, dietary minerals and antioxidant concentrations, and habitat water quality. Specific histopathological observations in liver, kidney, and gill are useful to understand the effects of Cd, which could help to determine the ameliorating methods to be adopted. Exposure of Cd exerts several adverse effects on general growth and development, reproductive processes, osmoregulation, morphological and histological structures, stress tolerance, and endocrine system, mainly due to changes in biological functions induced by differential expressions of several genes related to oxidative stress, apoptosis, inflammation, immunosuppressions, genotoxicity, Cd chelation and carbohydrate metabolism. Chronic biomagnifications of Cd exceeding the permitted level may be harmful not only to the fishes itself but also to humans through food chains. Amelioration of such toxic heavy metal that has been categorized as a potent carcinogenic in humans is of utmost importance. Main modes of amelioration encompas reducing oxidative damages by promoting the antioxidative defenses, decreasing Cd absorption, increasing excretion through excretory system and improving the tolerance of fishes to Cd toxicity. Many amelioration measures such as use of minerals (for example, zinc, calcium, and iron), vitamins (vitamin C, A, and E), different herbs, probiotics and other agents (taurine, bentonite, chitosan, zeolite, and metallothionein) have been explored for their effective roles to reduce Cd bioaccumulation and toxicity symptoms in fishes. The present review discusses bioaccumulation of Cd, histopathological alterations, oxidative stress, synergism-antagonism, and gene regulation in different tissues, and its amelioration measures in fishes.
According to the Environmental Protection Agency in Taiwan, the common carp (Cyprinus carpio) is one species of fish for acute toxic test. It has been found to be extremely sensitive to the toxicity of Cd²⁺; Furthermore, the goldfish (Carassius auratus) has a higher resistance than common carp upon Cd²⁺ exposure, but both fish are the same family. The aim of the study was to compare the physiological and histo-pathological responses between goldfish and common carp under exposure to sublethal concentrations of Cd²⁺ in order to understand the reasons behind the Cd²⁺-resistance. Results showed that metallothionein (MT) protein levels in visceral tissues were exceptionally increased and elevated at an earlier time in goldfish than in common carp. Meanwhile, the amount of Cd²⁺ accumulation in goldfish was higher than common carp after Cd²⁺ exposure. The histo-pathological results revealed that the density of gill mucus cells and the thickness of gill epithelium in common carp were raised earlier than in goldfish, but the histo-pathological findings resemble each other. According to the data, we suggested the efficient response of MT proteins may contribute to goldfish with a higher Cd²⁺ tolerance.
Marine pollutants such as heavy metals are well-known due to their bioaccumulative nature, toxicity and persistence in the marine environment. The present research aims to evaluate the bioaccumulation of three metals (Cd, Zn and Cu), individual mean (multimetal) bioaccumulation index (IMBI) and metal pollution index (MPI) as an assessment tool in Mediterranean mussel (Mytilus galloprovincis), based on the full factorial central composite face centered (CCF) design. Mussels were exposed to different sub-lethal concentrations (0-10%-72h-LC50-72h-mg.L⁻¹) of studied heavy metals for three days. At the end of exposure cycle, the bioaccumulations of heavy metals content in mussels were determined. Multiple regressions were used to fit experimental data to a second-order polynomial equation, which was then examined using variance analysis (ANOVA). The results revealed that the indexes IMBI and MPI depend on the type and concentration of metals and also on the metals combination. Accordingly, the toxicological metal–metal interactions were found not linear meaning supra-additive (synergistic), infra-additive (antagonistic) or no/zero interaction. The optimization of experimental data was used to determine the optimal conditions for the heavy metal bioaccumulation. It was demonstrated that the CCF design combined with the concept of quantitative bioindicators can be used as appropriate tool in ecotoxicological evaluation and prediction of metals bioaccumulation.
In this study, we investigated the bioaccumulation and toxicokinetics of zinc oxide nanoparticles (ZnO NPs) alone and in the presence of graphene nanosheets (GNs) in the blackfish (Capoeta fusca). Blackfish were exposed via water to two ZnO NPs concentrations alone or as a combination with GNs and uptake of Zn into the gills, intestine, liver, and kidney was assessed at 7, 14 and 28 d. Zn elimination from these tissues was then assessed after a further 7, 14 and 28 d in clean water for both ZnO NPs concentrations and combined ZnO NPs/GN exposures. In the body tissues analyzed of exposed fish, the highest amounts of Zn occurred in the intestine and the lowest amount in the liver. Zn levels in blackfish after 28 d of exposure were higher in all treatment groups compared to those on 7 d (p < 0.05). For both ZnO NPs exposure concentrations, the highest amount of Zn was eliminated from the intestine, followed by the gills. Furthermore, elimination kinetics for both ZnO NPs concentrations alone and in combination with GNs showed that the shortest half-life for Zn is occurring in the intestine. Moreover, uptake rates of Zn in fish exposed to ZnO NPs + GNs followed the same pattern observed for the ZnO NP, with intestine and gills having the highest levels followed by kidney and liver. Thus, we show accumulation and elimination of Zn from ZnO NPs in blackfish depends on the tissue, exposure concentration and duration, and is dependent on the presence of GNs.
Copper (II) oxide nanoparticles (n-CuO) and copper (II) sulfate (CuSO4), commonly used in industrial and agricultural applications, are released in substantial amounts to the environment where they may then interact in unexpected ways with other materials such as engineered nanomaterials (ENM). In this study, we assessed the combined effects of their interaction with titanium (IV) dioxide nanoparticles (n-TiO2), a widely used ENM, on the ATP levels and cell membrane integrity of Escherichia coli in a natural aqueous medium (Lake Michigan water). Under dark conditions, we observed that n-TiO2 addition had negligible effects on copper toxicity, except in the case of higher doses of CuSO4 where n-TiO2 attenuated bacterial stress effects presumably by the adsorption of copper ions at the n-TiO2 surface. However, an opposite effect was observed under exposure to simulated solar irradiation (SSI) with synergistic stress occurring in mixtures of n-TiO2 with either CuSO4 or n-CuO. Enhanced photoactivity (as measured by methylene blue decay) in both CuSO4/n-TiO2 and n-CuO/n-TiO2 mixtures compared to single-component solutions explains the amplified stress effects of the mixtures under SSI, but we were unable to directly measure an increase in the production of reactive oxygen species (ROS) in the mixtures. Under environmental conditions that include light exposure, then, Cu/n-TiO2 mixtures exert amplified cellular stress exceeding that expected based on individual component effects.
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.
Heavy metals incidence in the aquatic environment and its accumulation in fish are under constant review. Gilthead seabream (Sparus aurata) specimens were exposed for two weeks to sediments highly concentrated in metals, collected at the Portman Bay (Murcia, Spain). The metals bioaccumulation was tested in liver, muscle and skin. The potential of the sediment exposure to induce variation of the stress biomarkers genes was conducted in liver and skin. Results revealed that sediments were highly contaminated with metals. However, following 2 weeks exposure to the sediments, Cd accumulates only in liver. Interestingly, the expression of the genes mta, hsp 70 and hsp 90 were significantly down-regulated in skin. Nevertheless, cyp1a1 gene was up-regulated only in liver. Results uphold that the stress response magnitude was organ-dependent and the skin was the most responsive tissue to metal stress conditions. These results suggest that skin should be considered as target organ for biomarkers analysis in fishes.
Freshwater fish, Oreochromis niloticus, were individually exposed to 0, 0.1, 0.5, 1.0, and 1.5 96 h (acute) and 0.05 μg/mL concentrations of the same metals for 0, 5, 10, 20, and 30 days (chronic). Following each period, metal accumulation and ion levels (Na +, K +, Ca +2, and Mg +2) were measured in the gills, kidneys, and muscles. Except for Ag +, none of the metals killed the fish within 30 days. Silver killed all the fish within 16 days. With the exceptions of Ag + and Cr 6+, as their levels were below detection limits, metal accumulation occurred in the tissues following both acute and chronic exposures. Ion levels in the tissues were altered by metal exposure, the general tendency being a decrease in Na + and K + levels and an increase in Mg 2+ and Ca 2+ levels. Acute exposure to heavy metals seemed to be more effective in altering ion levels of the tissues than chronic exposure. Na + was the most affected ion while Mg 2+ was the least affected. Results of this study emphasize that ion levels in the tissues of O. niloticus can be altered by heavy metals, both in acute and chronic exposures. This suggests that heavy metals should be monitored carefully in ecotoxicological studies in the field due to their importance in fish physiology.
Background: Cadmium (Cd) is considered as one of the most toxic heavy metal. Intake of Cd by fish has serious implications. Metal pollution from multifarious sources has adverse effects on aquatic ecosystems. In aquatic systems, Cd is most readily absorbed by organisms directly from the water in its free ionic form. In many contaminated situations with heavy metals, Cd has become an important element of concern because of its bioaccumulative nature in food webs. Therefore, fish living in polluted waters tend to accumulate heavy metals in their tissues. Objectives: To assess the influence of Cd on freshwater fish, this review briefly addresses the Cd emission sources, uptake and impacts of Cd on freshwater fish and bioaccumulation nature of Cd by emphasizing the Cd accumulation affinity in freshwater fish tissues. Results and Discussion: Many studies have been carried out on Cd thresholds in diet and tissues of freshwater fish species. Affinity of freshwater fish to Cd is diverse. Generally, Cd accumulation depends on concentration, time of exposure, way of uptake, environmental conditions and intrinsic factors. Metal accumulation primarily depends on waterborne and dietary pathways. It shows a relationship of Cd level in fish tissues with the age and size of fish. Some species of fish show the highest Cd accumulation in the liver while others in kidneys and gills. Accumulation of Cd by the body muscles is always reported as comparatively low. Cd in freshwater environments results biological and environmental implications by altering reproductive and physiological behaviors of freshwater fish and abilities which ultimately affect environmental permanence and biodiversity of the ecosystem.
Accumulation of copper, zinc and cadmium in gill, liver and muscle tissues of Oreochromis niloticus was deter-mined after exposing the animals to (ppm) 5.0 Cu, 5.0 Zn, 1.0 Cd, and their mixtures over 24, 48 and 96 h. AAS method was used in determining the tissue metal levels. Accumulation increased with increasing exposure pe-riods when exposed to metals separately or in mixture. Highest zinc and cadmium accumulation was in the order gill > liver > muscle while the order of copper accumulation was liver > gill > muscle when exposed to metals separately. Maximum accumulation of Cu was in liver while those of Zn and Cd were in gill tissues when exposed to metal mixture. Muscle accumulation was lowest in metal mix-ture-exposed fish. Effects of the metals applied singly in-creased Cu levels in liver and that of Zn in gill and muscle tissues at the tested concentrations.
Iron, copper, zinc, and eight other minerals are classified as essential trace elements because they present in minute in vivo quantities and are essential for life. Because either excess or insufficient levels of trace elements can be detrimental to life (causing human diseases such as iron-deficiency anemia, hemochromatosis, Menkes syndrome and Wilson's disease), the endogenous levels of trace minerals must be tightly regulated. Many studies have demonstrated the existence of systems that maintain trace element homeostasis, and these systems are highly conserved in multiple species ranging from yeast to mice. As a model for studying trace mineral metabolism, the zebrafish is indispensable to researchers. Several large-scale mutagenesis screens have been performed in zebrafish, and these screens led to the identification of a series of metal transporters and the generation of several mutagenesis lines, providing an in-depth functional analysis at the system level. Moreover, because of their developmental advantages, zebrafish have also been used in mineral metabolism-related chemical screens and toxicology studies. Here, we systematically review the major findings of trace element homeostasis studies using the zebrafish model, with a focus on iron, zinc, copper, selenium, manganese, and iodine. We also provide a homology analysis of trace mineral transporters in fish, mice and humans. Finally, we discuss the evidence that zebrafish is an ideal experimental tool for uncovering novel mechanisms of trace mineral metabolism and for improving approaches to treat mineral imbalance-related diseases.
Exposure of freshwater trout (Salmo gairdneri) to waterborne Cd2+ results in accumulation of the metal in the branchial epithelial cells and its appearance in the blood. Cd2+ apparently enters the cells via Ca2+ channels in the apical membrane. Transfer of Cd2+ through the basolateral membrane is probably by diffusion. Inhibition by Cd2+ of transepithelial Ca2+ influx is time-and Cd2+-concentration-dependent. The inhibition of transepithelial Ca2+ influx is accompanied by blockage of apical Ca2+ channels. In line with the assumption that cytosolic Cd2+ inhibits Ca2+ uptake by inhibiting the basolateral Ca2+ pump, we hypothesize that the blockage of Ca2+ channels is an indirect effect of Cd2+ and results from a rise in cytosolic Ca2+ level caused by inhibition of the basolateral membrane Ca2+ pump.
The aim of the present study was to investigate the distribution of selected metals (As, Cd, Pb, Hg, Cr, Cu and Zn) in tissues of the common carp (Cyprinus carpio L.) grown in ponds. A total of 125 market-ready carp (aged 3 - 4 years, weight 2312 583 g) from 10 fishponds in the Czech Republic were analysed. The tissues analysed included muscle, liver, kidney, spleen, gills, ovaria and testes. Metal concentrations were determined by the AAS, mercury was determined using a cold-vapour AAS analyse (AMA 254). Results showed different affinities of metals to different tissues. The highest concentrations of As and Hg were in muscle tissue, Cu in the liver, and Cd and Zn in the kidneys, compared with levels in other tissues (p < 0.01). The lowest metal concentrations (particularly of Cd) were found in the spleen, gills and gonads. Lead concentrations in the individual tissues were low and practically identical. Chromium concentrations were also balanced with sporadic significant differences (p < 0.05). Significantly higher concentrations of Cu, Cr (p < 0.05) and Zn (p < 0.01) were found in the ovaria compared with the testes. The study showed that meat and gonads of pond carp in the Czech Republic are safe from the point of view of contamination with the metals investigated.
Summary N-ethymaleimide-sensitive ATPase activity was measured in crude homogenates of gill tissue from rainbow trout using a coupled-enzyme ATPase assay in the presence of EGTA, ouabain and azide. This NEM-sensitive ATPase activity, determined to be about 1.5 mmolmg21 protein h21 at 15˚C for freshwater trout, is also inhibited by other H+- ATPase blockers such as DCCD, DES, PCMBS and bafilomycin. It is concluded, therefore, that the NEM-sensitive ATPase activity was generated by a proton- translocating ATPase. Since this NEM-sensitive ATPase was also sensitive to the plasma membrane ATPase inhibitor vanadate, we conclude that the H+-ATPase in fish gill is of the plasma membrane type. The major role of the H+-ATPase in the gill epithelium is to facilitate Na+ uptake from fresh water. Sodium concentration in the external medium was the primary regulator of the H +-ATPase in f ish gills, with low sodium levels being associated with high H+-ATPase activity. High external calcium concentration had a marked stimulatory effect on H+-ATPase activity in fish gills when the sodium level was low. Environmental hypercapnia induced a 70% increase in the H +-ATPase activity in fish gills. H +-ATPase activity was also elevated in freshwater fish after chronic cortisol infusion.
We assessed whether fish that tolerate higher levels of Cu exposure have a higher capacity to induce metallothionein (MT) synthesis than other, more sensitive, fish species. Furthermore, we examined if a correlation could be found between tissue Cu accumulation and MT levels. Cu accumulation and MT concentrations in gill, liver, kidney and muscle of rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio) were measured during a 1 week exposure to a sublethal Cu (1 μM).
Acutely lethal (24 h) exposure of adult rainbow trout (Oncorhynchus mykiss) to 4.9 mol copperl-1 in fresh water (pH 7.9, [Ca2+]0.8 mEql-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.
Several components of branchial copper uptake were identified in juvenile freshwater rainbow trout (Oncorhynchus mykiss) using 64Cu. On the basis of competitive interactions between sodium and copper uptake,inhibition of copper uptake by a proton pump inhibitor (bafilomycin A1, 2μmol l-1) and a Na+ channel blocker (phenamil, 100μmol l-1), it appears that a proportion of the branchial copper uptake occurs via an apical Na+ channel. This sodium-sensitive copper uptake demonstrates saturation kinetics, with a Km of 7.1 nmol l-1 and a Jmax of 21.2 pmol g-1 h-1, and is characterized by an IC50 of 104 μmol l-1 sodium. On the basis of residual copper uptake in the presence of high sodium concentrations (20 mmol l-1) and differential inhibition of sodium and copper uptake by phenamil (100 μmol l-1), a sodium-insensitive component of copper uptake is also present in trout gills. It demonstrates saturation kinetics with a comparably low Km (9.6 nmol l-1) but a lower maximum transport capacity (Jmax=3.5 pmol g-1 h-1)than the sodium-insensitive system. Sodium uptake exhibits saturation kinetics with a Km of 69 μmol l-1. Copper reduced branchial sodium transport affinity but increased the maximal sodium transport capacity.
When this book was first published in 1996, the pollution of the earth's freshwater habitats was, as it is now, a topic of major concern. This synthesis considers the effects of pollutants on aquatic animals via a series of research and review articles that present experimental evidence of sublethal and lethal effects of a range of toxicants at the physiological, cellular and subcellular levels, and that explore techniques for detection of pollution damage. Topics covered include routes of uptake of toxicants; the effect of acute and chronic exposure to toxic metal ions, particularly zinc, copper and aluminium, with emphasis on the mechanisms of toxicity and responses to chronic exposure to sublethal levels; the impact on fish biology of two chemicals of current concern, nitrites and polyaromatic hydrocarbons, which may act as oestrogenic substances or potent mutagens; and in vitro studies of the mechanisms of toxicity at the cellular and subcellular level, including damage of DNA, using cultured fish cells.
Environmental monitoring and risk assessment approaches which include a more holistic view on the effects of pollutants on biota are increasingly sought by regulators and policy makers. Therefore, caged carp juveniles (Cyprinus carpio) were transplanted for 7 weeks along a known Cd and Zn pollution gradient. Metal (Cu, Cd and Zn) accumulation in gill and liver tissue and effect biomarkers (growth, condition factor (CF), hepatosomatic index (HSI), oxygen consumption, swimming capacity, Na ⁺ /K ⁺ -ATPase activity (NKA) and metallothionein (MT) levels) were compared. Up to 10-fold higher cadmium concentrations were measured in the gills of the fish at the most polluted locations compared to the laboratory control fish. Similarly, cadmium concentrations in liver tissues of field-exposed fish were significantly higher than those measured in laboratory control fish. Cu and Zn concentrations in the gills were not significantly different between field-exposed and control organisms, whereas higher levels in liver tissues were measured in carps deployed in some locations. Effects on liver MT levels were up to 10 times greater for organisms exposed to the field, whereas no clear effect of the metal exposure on NKA in the gill tissue was observed. A decrease in muscle glycogen stores was observed for all organisms deployed in the field, while liver glycogen levels decreased only in fish exposed to two of the 5 sites compared to the laboratory control fish. Additionally, significant drops in liver protein- and lipid stores were observed. No effect on oxygen consumption rates and swimming capacity was observed. The CF and HSI of caged fish reflected the pollution gradient in the river and considerable loss of weight was observed for fish transplanted in the most polluted site. Overall, this active biomonitoring study successfully revealed differences in metal accumulation, physiological and organismal endpoints as a direct consequence of field exposure.
Analyzing effects of metal mixtures is important to obtain a realistic understanding of the impact of mixed stress in natural ecosystems. The impact of a one-week exposure to a sublethal metal mixture containing copper (4.8 μg/L), cadmium (2.9 μg/L) and zinc (206.8 μg/L) was evaluated in the common carp (Cyprinus carpio). To explore whether this exposure induced oxidative stress or whether defense mechanisms were sufficiently fitting to prevent oxidative stress, indicators of apoptosis (expression of caspase 9 [CASP] gene) and of oxidative stress (malondialdehyde [MDA] level and xanthine oxidase [XO] activity) were measured in liver and gills, as well as activities and gene expression of enzymes involved in antioxidant defense (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx], glutathione reductase [GR] and glutathione-S-transferase [GST]). The total antioxidative capacity (T-AOC) was also quantified. No proof of oxidative stress was found in either tissue but there was indication of apoptosis in the liver. CAT, GPx, GR and GST total activities were reduced after 7 days, suggesting a potential decrease of glutathione levels and risk of increased free radicals if the exposure would have lasted longer. There were no major changes in the total activities of antioxidant enzymes in the gills, but the relative expression of the genes coding for CAT and GR were triggered, suggesting a response at the transcription level. These results indicate that C. carpio is well equipped to handle these levels of metal pollution, at least during short term exposure.
Cadmium (Cd) is a heavy metal that accumulates easily in organisms and causes several detrimental effects, including tissue damage. Cd contamination from anthropogenic terrestrial sources flows into rivers, and through estuaries to the ocean. To evaluate the toxic effects of Cd on estuary crustaceans, we exposed the mud crab Scylla paramamosain to various Cd concentrations (0, 10.0, 20.0, and 40.0mg/L) for 24h. We also exposed mud crabs to a fixed Cd concentration (20.0mg/L) for various periods of time (0, 6, 12, 24, 48, and 72h). We observed that after exposure to Cd, the surfaces of the gill lamellae were wrinkled, and the morphologies of the nuclei and mitochondria in the hepatopancreas were altered. We analyzed the expression profiles of 36 stress-related genes after Cd exposure, including those encoding metallothioneins, heat shock proteins, apoptosis-related proteins, and antioxidant proteins, with quantitative reverse transcription PCR. We found that exposure to Cd altered gene expression, and that some genes might be suitable bioindicators of Cd stress. Gene expression profiles were organ-, duration-, and concentration-dependent, suggesting that stress-response genes might be involved in an innate defense system for handling heavy metal exposure. To the best of our knowledge, this study is the first one of histopathology and stress-response gene expression pattern of Scylla paramamosain after Cd exposure. Our work could increase our understanding of the effect of environmental toxins on estuary crustaceans.
Biotic Ligand Models (BLMs) for individual metals improve our ability to regulate metals in the aquatic environment by considering the effects of water quality parameters (ionic composition, pH, DOC) on metal bioavailability. However, in natural aquatic systems, organisms are often simultaneously exposed to multiple metals and these interactions are not currently considered in BLMs or most environmental regulations. Recently, several different mixture BLMs (mBLMs) have been developed to begin assessing this issue. Some of these models assume competitive interactions between all metals, while others assume only metals with similar modes of action (e.g., Na⁺ or Ca²⁺ antagonists) will competitively interact. In this study, we used standard in vivo 3-h gill metal binding assays to characterize the uptake of Cd, Pb, and Zn individually and in binary mixtures with Ag, Cd, Cu, Pb, Ni, and Zn across a range of concentrations that encompassed the 96-h LC50 for each metal. Inhibition of Cd, Pb, and Zn uptake at the gill by introduction of a second metal was consistent with mode of action in some cases, but not others. Further, contrary to expectations, inhibition was always either non-competitive or could not be defined statistically. We also observed one example of stimulated metal uptake (Ni stimulated Zn uptake). Consistent with our previous experiments on Ag, Cu, and Ni, these studies suggest that current mBLM frameworks will need revision to better reflect the mechanisms underlying metal mixture interactions.
Survival of eggs and larvae decreased with increasing concentration of metals. Abnormalities in some newly hatched larvae from eggs exposed to various concentrations of Zn and Cd are recorded. Eggs were more sensitive than larvae. -from Authors
Heavy metals are frequently encountered as mixtures of essential and non-essential elements. Therefore, evaluation of their toxic effects individually does not offer a realistic estimate of their impact on biological processes. We studied effects of exposure to mixtures of essential and toxic metals (Cr, Cd and Pb) on biochemical, immunotoxicity level and morphological characteristics of the various tissues of a biomarker freshwater fish common carp using environmentally relevant concentrations. Fish were exposed to metal mixture through tank water for 7, 15 and 30 days, under controlled laboratory conditions. Tissue accumulation of the metals was measured using Atomic Absorption Spectrophotometric techniques. Chromium, cadmium and lead accumulation in muscle, gills, liver, kidney and intestine, tissue of common carp exposed to mixture metals for 30 days increased significant compared with control group (p < 0.001). However, the activity of antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) levels was significant altered in various tissues of exposed fish. Besides, the lipid peroxidation (LPO) was significant (p < 0.001) increased. Moreover, the tumor necrosis factor - α (TNF-α), interleukin (IL-6), and interferon-γ (IFN-γ) contents in tissues of muscle, gills, liver, kidney and intestine were increased significant compared with control fish (p < 0.001). In addition, microscopic examination of the main alterations in general morphology of fish gills included spiking and fusion of secondary lamellae, formation of club-shaped filaments epithelium in the interlamellar regions and hepatocytes showed damage of central vein and rupture of irregular hepatic plate with more number of vacuoles in the fish exposed to metal mixture for a longer duration (30 days). These results of this study clearly demonstrate that concentration individual and mixtures of metals in aquatic systems will greatly influence the cytokine alterations may result in an immune suppression or excessive activation in the treated common carp as well as may cause immune dysfunction or reduced immunity. In conclusion, toxicity of multiple metal mixtures of Cr, Cd and Pb has antioxidant and immunotoxic effects on C. carpio.
In this study, we aimed to identify the toxic effects of chlorpyrifos exposure on the tissues of common carp. For this purpose, we evaluated histopathological changes in the brain, gills, liver, kidney, testis, and ovaries after 21 days of chlorpyrifos exposure. Activation of 8-OHdG, cleaved caspase-3, and iNOS were assesed by immunofluorescence assay in chlorpyrifos-exposed brain and liver tissue. Additionally, we measured the expression levels of caspase-3, caspase-8, iNOS, MT1, CYP1A, and CYP3A genes in chlorpyrifos-exposed brain tissue, as well as the expression levels of FSH and LH genes in chlorpyrifos-exposed ovaries, using qRT-PCR. We observed severe histopathological lesions, including inflammation, degeneration, necrosis, and hemorrhage, in the evaluated tissues of common carp after both high and low levels of exposure to chlorpyrifos. We detected strong and diffuse signs of immunofluorescence reaction for 8-OHdG, iNOS, and cleaved caspase-3 in the chlorpyrifos-exposed brain and liver tissues. Furthermore, we found that chlorpyrifos exposure significantly upregulated the expressions of caspase-3, caspase-8, iNOS, and MT1, and also moderately upregulated CYP1A and CYP3A in the brain tissue of exposed carp. We also noted downregulation of FSH and LH gene expressions in chlorpyrifos-exposed ovary tissues. Based on our results, chlorpyrifos toxication caused crucial histopathological lesions in vital organs, induced oxidative stress, inflammation, and apoptosis in liver and brain tissues, and triggered reproductive sterility in common carp. Therefore, we can propose that chlorpyrifos toxication is highly dangerous to the health of common carp. Moreover, chlorpyrifos pollution in the water could threaten the common carp population. Use of chlorpyrifos should be restricted, and aquatic systems should be monitored for chlorpyrifos pollution.
Because of the ever increasing complexity of environmental contamination profiles, there are limitations to the use of analytical pollutant measurements for monitoring and prioritization of watercourses. The potential of biomarkers has been debated for many years, especially in laboratory settings, but there is a need for studies evaluating these approaches in the field. We evaluated the usefulness of a selection of biomarkers, mostly indicators of general physiological status and common stress responses such as oxidative stress, to discriminate among environmental pollution profiles, with the aim of prioritizing contaminated watercourses for targeted remediation efforts. To this end, juvenile common carp (Cyprinus carpio Lin.) were exposed in cages in the field to Flemish watercourses with varying pollution profiles. After six weeks of exposure, the bioaccumulation of key pollutants was measured, and a set of organismal, biochemical and transcriptional endpoints was determined in several tissue types. After data integration a discrete set of 14 parameters was identified, that could successfully distinguish all watercourses from each other. We show that an integrated biomarker approach, mainly targeting common stress responses, can offer the resolving power to discriminate among environmentally relevant exposure scenarios, and a means to prioritize watercourses for targeted remediation.
The Acute toxicity of copper on three life stages of common carp, Cyprinus Carpio Var. Communis. viz. 30 day, 60 day and 90 day were investigated. The 96h LC50 along with the 95% confidence limits were 0.04 (0.0389 - 0.041), 0.1234 (0.1208 - 0.1261) and 0.1577 (0.1535 - 0.162) mg Cu/l for the 30 day, 60 day and the 90 day old fish respectively. It was observed that the toxicity of copper decreases with the age of fish and that in the experiments, mortalities occurred more during the initial hours than at the end hours of the experimental period.
The effects of increased endogenous cortisol levels were compared with those of sublethal copper exposure in the freshwater common carp, Cyprinus carpio. Fish were exposed to either increased levels of endogenous cortisol (200 ng/ml) or sublethal copper (1.9 muM) alone or were pretreated by elevating plasma cortisol levels prior to copper exposure to assess whether interactions between both treatments occurred. Effects induced by increased cortisol levels included increased Na+/K+-adenosine triphosphate (ATPase) activity and increased plasma Na+ and plasma osmolarity, while copper exposure induced anaerobic metabolism, gill damage, decreasing Na+/K+-ATPase activity, decreasing plasma ion levels, and blood thickening. Pretreatment of copper-exposed fish with cortisol partially protected these fish by reducing the copper-induced decrease in Na+/K+-ATPase activity. Overall, the results obtained in this study argue against a major role for cortisol as an intermediate for the toxic effects of copper.
The main objective of this study was to understand the mode of interaction between waterborne copper (Cu) and high environmental ammonia (HEA) exposure on freshwater fish, and how they influence the toxicity of each other when present together. For this purpose, individual and combined effects of Cu and HEA were examined on selected physiological and ion-regulatory processes and changes at transcript level in the common carp (Cyprinus carpio). Juvenile carp were exposed to 2.6μM Cu (25% of the 96h LC50value) and to 0.65mM ammonia (25% of the 96h LC50value) singly and as a mixture for 12h, 24h, 48h, 84h and 180h. Responses such as ammonia (Jamm) and urea (Jurea) excretion rate, plasma ammonia and urea, plasma ions (Na(+), Cl(-) and K(+)), muscle water content (MWC) as well as branchial Na(+)/K(+)-ATPase (NKA) and H(+)-ATPase activity, and branchial mRNA expression of NKA, H(+)-ATPase, Na(+)/H(+) exchanger (NHE-3) and Rhesus (Rh) glycoproteins were investigated under experimental conditions. Results show that Jamm was inhibited during Cu exposure, while HEA exposed fish were able to increase excretion efficiently. In the combined exposure, Jamm remained at the control levels indicating that Cu and HEA abolished each other's effect. Expression of Rhcg (Rhcg-a and Rhcg-b) mRNA was upregulated during HEA, thereby facilitated ammonia efflux out of gills. On the contrary, Rhcg-a transcript level declined following Cu exposure which might account for Cu induced Jamm inhibition. Likewise, Rhcg-a was also down-regulated in Cu-HEA co-exposed fish whilst a temporary increment was noted for Rhch-b. Fish exposed to HEA displayed pronounced up-regulation in NKA expression and activity and stable plasma ion levels. In both the Cu exposure alone and combined Cu-HEA exposure, ion-osmo homeostasis was adversely affected, exemplified by the significant reduction in plasma [Na(+)] and [Cl(-)], and elevated plasma [K(+)], along with an elevation in MWC. These changes were accompanied by a decline in NKA activity. Gill H(+)-ATPase mRNA levels and activities were not affected by either Cu or HEA or both. Likewise, NHE-3 expression remained unaltered but tended to be numerically higher during HEA exposure. Overall, these data suggest that at equitoxic concentrations (25% of 96h LC50), the individual effect of Cu is more harmful while HEA induces quicker adaptive responses. Our findings also denote a competitive mode of interaction, exemplified by the inhibition of HEA -mediated adaptive responses in the presence of Cu.
The present text provides a review of waterborne as well as dietary copper (Cu) toxicity and Cu homeostasis in fish, and leads to suggestions for further research in this area. Copper, although essential for life, is a potent toxicant and as such, delicate homeostatic controls have evolved at the organismal and cellular level. During exposure to elevated levels of Cu in the water or the diet, homeostatic systems may become overwhelmed such that cellular Cu levels increase to a point where protein function becomes impaired. A range of potential cellular targets for Cu manifest in altered physiology and toxicity, at the organ and organismal level during Cu exposure; these targets and organismal responses are discussed. Copper toxicity is not simply a matter of ambient concentrations as water chemistry greatly influences not only the bioavailability of this metal but also the physiology and thus susceptibility of fish. This complexity has recently been realized and is being considered in current environmental regulations for Cu. Prolonged exposure to Cu elicits an acclimation response which includes a compensatory response of the functions impaired by Cu and adjustments in the homeostatic control of the metal, enabling fish to survive despite continued exposure and to tolerate subsequent exposures to higher concentrations. Although not quantified, these acclimation responses must occur at a cost to the organism, possibly explaining observations of reduced growth, reproductive output, and swimming performance. The literature review forming the basis for this chapter was completed by December 2010.
Cadmium (Cd) is a naturally occurring ubiquitous element of environmental concern. Concentrations of Cd in the geosphere are generally low except for enrichment in association with Zn, Pb, and Cu sulfidic ore deposits, as well as some phosphate rock formations. Uses of Cd include batteries, pigments, stabilizers, coatings, and some alloys. The toxicity of Cd to aquatic species depends on speciation, with the free ion, Cd 2+ concentration being proportional to bioavailability. Toxicity is reduced via complexation of Cd 2+ by inorganic and organic anions and through competitive interactions between Ca 2+ and Cd 2+ for uptake sites. Cd 2+ acute toxicity involves disruption of ion homeostasis, particularly Ca, but also Na and Mg. Uptake at the fish gill occurs apically via a lanthanum-sensitive voltage-independent epithelial Ca 2+ channel located in the mitochondria-rich chloride cells and basolaterally via Ca-ATPase and an Na/Ca exchanger. Chronic exposure also involves ionoregulatory disturbance and disruptions to growth, reproduction, the immune system, endocrine, development, and behavior; gill, liver, and kidney histopathologies also develop. Cadmium accumulates primarily in the kidney and liver. Accumulation has been linked to oxidative damage, which may be a mechanism linking the many different chronic effects. Uptake across gastrointestinal and olfactory surfaces is not well understood but evidence exists for gut Cd uptake via L-type voltage-gated Ca 2+ channels, a high-affinity Ca 2+-ATPase and a divalent metal transporter-1. Acclimation to chronic Cd exposure involves a variety of defense and detoxification processes, such as antioxidants (catalase), metallothionein, glutathione, and heat shock proteins, making the development of bioaccumulation-based impact prediction models for Cd a challenge.
Cadmium and copper accumulations in gills of zebrafish were measured during a 48 h exposure to 0.025 mu M Cd-106 and 0.05 or 0.5 mu M Cu-65 as a single metal or their mixtures. The gill transcript levels of genes involved in the transport of Cu (CTR1 and ATP7A), Na (NHE-2), Ca (ECaC), divalent metals (DMT1), and Zn (ZIP8) were also compared between treatments at 24 and 48 h. Cd uptake was significantly suppressed in the presence of Cu, indicating interaction between Cu and Cd at uptake sites, but Cu uptake was unaffected by Cd. The decrease in Cd accumulation rates in the presence of Cu was associated with an increase in transcript abundance of ECaC at 24 h and DMT1 at 48 h and a decrease in Zip8 transcript levels, all known as routes for Cd uptake. Fish exposed to 0.5 mu M Cu-65 show an increase in gill ATP7a transcript abundance, suggesting that Cu is removed from the gill and is transferred to other organs for detoxification. A reduction in gill CTR1 transcript abundance was observed during the Cu-Cd exposure; this may be a regulatory mechanism to reduce Cu loading if Cu is entering the gills by other uptake routes, such as ECaC and DMT1.
Metal binding to fish gills forms the basis of the biotic ligand model (BLM) approach, which has emerged as a useful tool for conducting site-specific water quality assessments for metals. The current BLMs are designed to assess the toxicity of individual metals, and cannot account for the interactive effects of metal mixtures to aquatic organisms including fish. The present study was designed mainly to examine the interactive effects of waterborne metals (Cd, Zn, Cu, Ag, and Ni) in specific binary combinations on short-term (3h) gill-metal binding and essential ion (Ca(2+) and Na(+)) uptake (a physiological index of toxicity) in fish, using juvenile freshwater rainbow trout (Oncorhynchus mykiss) as the model species. We hypothesized that binary mixtures of metals that share a common mode of uptake and toxicity (e.g., Cd and Zn - Ca(2+) antagonists, Cu and Ag - Na(+) antagonists) would reduce the gill binding of each other via competitive interactions and induce less than additive effects on ion transport. In addition, the mixture of metals that have different modes of uptake and toxicity (e.g., Cd and Cu, or Cd and Ni) would not exhibit any interactive effects either on gill-metal binding or ion transport. We found that both Zn and Cu reduced gill-Cd binding and vice versa, however, Ni did not influence gill-Cd binding in fish. Surprisingly, Ag was found to stimulate gill-Cu binding especially at high exposure concentrations, whereas, Cu had no effect on gill-Ag binding. The inhibitory effect of Cd and Zn in mixture on branchial Ca(2+) uptake was significantly greater than that of Cd or Zn alone. Similarly, the inhibitory effect of Cu and Ag in mixture on branchial Na(+) uptake was significantly greater than that of Cu or Ag alone. The inhibitory effects of Cd and Zn mixture on Ca(2+) uptake as well as Cu and Ag mixture on Na(+) uptake were found to follow the principles of simple additivity. In contrast, no significant additive effect on either Ca(2+) or Na(+) uptake was recorded in fish exposed to the mixture of Cd and Cu. Overall, we found that although the effects of metal mixture interactions on gill-metal binding did not always match with our original assumptions, the effects of metal mixtures on toxicity in fish were generally consistent with our predictions. The findings of the present study have important implications for improving the BLM approach to assess metal mixture toxicity in fish.
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The present work evaluates the effects of various levels of dietary choline on antioxidant defenses and gene expressions of Nrf2 signaling molecule in spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed with six different experimental diets containing graded levels of choline at 165 (choline-deficient control), 310, 607, 896, 1167 and 1820 mg kg(-1) diet for 65 days. At the end of the feeding trail, fish were challenged with Aeromonas hydrophila and mortalities were recorded over 17 days. Dietary choline significantly decreased malondialdehyde and protein carbonyl contents in spleen and head kidney. However, anti-superoxide anion and anti-hydroxyl radical activities in spleen and head kidney also decreased. Interestingly, activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) in spleen, GPx activity in head kidney, and glutathione contents in spleen and head kidney were decreased with increase of dietary choline levels up to a certain point, whereas, activities of SOD, GST and GR in head kidney showed no significantly differences among groups. Similarly, expression levels of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b and GR gene in spleen and head kidney were significantly lower in group with choline level of 607 mg kg(-1) diet than those in the choline-deficient group. The relative gene expressions of Nrf2 in head kidney and Keap1a in spleen and head kidney were decreased with increasing of dietary choline up to a certain point. However, the relative gene expression of Nrf2 in spleen were not significantly affected by dietary choline. In conclusion, dietary choline decreased the oxidant damage and regulated the antioxidant system in immune organs of juvenile Jian carp.
Rainbow trout (Salmo gairdneri) were held in metabolizable energy chambers at Standard Environmental Temperature (15°C) for 72h following a single feeding of a semi-purified test diet containing tracer quantities of a radioisotope of zinc ((65)Zn) and different combinations of dietary calcium level and zinc source. Gill wastes, urine, and feces were separately collected. After 72h, the fish were killed, and samples of the following tissues removed: eyes, skin, muscle, blood, bone, liver, bile, kidney, gill, spleen, stomach, pyloric caeca, intestine, gonad, and remaining carcass. Radioactivity in the tissues and wastes was determined and the body distribution of the ingested zinc was quantified. Approximately 58% of the administered dose of(65)Zn was recovered. Of the recovered dose, 43.2% was present in the gastro-intestinal tract, 27% in the feces, 14% in the gill water, 16% in the body of the fish, and less than 1% in the urine. Of individual tissues, the gill, liver, kidney, and spleen had concentrations of(65)Zn higher than blood, while the remaining tissues had lower concentrations. Body and tissue levels were increased but not significantly by feeding(65)Zn as an amino acid chelate, compared to feeding as inorganic(65)Zn, while dietary calcium level had no effect. The results of this study indicate that the gills play a major role in excretion of dietary zinc, while the urine plays a minor role.
Rainbow trout were exposed for 3 wk to copper at 0.09, 0.18, 0.29, 0.40, or 0.59 of the mean control incipient lethal level (ILL) of copper. Whole body copper concentration increased with both exposure concentration and time. Mean residues stabilized in the range of 34 to 37 μg/g dry weight after 2 to 3 wk of exposure at 0.29 to 0.59 ILL.After 3 wk at 0.29 ILL of copper, most fish survived a subsequent exposure at 1.7 ILL, during which their elevated body concentration of copper did not change appreciably (35 to 33 μg/g). In contrast, control fish died rapidly, their body concentration rising from 3.8 to 7.4 μg/g).Sublethal copper exposure increased the protein content of a low-molecular-weight hepatoprotein fraction. There was also greater incorporation into that protein of 14C from intraperitoneally administered [14C]leucine. Fish exposed to cadmium showed a similar increase in hepatoprotein but not increased 14C incorporation. This suggests different, metal-specific proteins. Results are discussed with respect to a previously reported acclimation to copper by the fish, acclimation which significantly altered lethal tolerance of copper.
Metallothionein, a low-molecular-weight protein whose apoprotein, thionein, is induced by exposure to cadmium, copper, mercury, and zinc, plays an important role in the transport and storage of heavy metals; it also provides a protective role against the toxic effects of these metals by sequestering and thus reducing the amount of the free metal ions. Interest in the detoxification role of metallothionein in fisheries research and aquatic toxicology has increased because of the increasing contamination of aquatic habitats by heavy metals. Reports of laboratory and field investigations have shown that metallothionein synthesis is induced in fishes during chronic and acute exposures to metals. There is also evidence of the presence in fish of low-molecular-weight inorganic-binding proteins that are different from metallothionein, as well as evidence that cadmium is not sequestered by metallothionein in rainbow trout Salmo gairdneri. The sequestering of metal contaminants by metallothionein and subsequent toxic effects in fish and other animals has been described by the “spillover” hypothesis, which states that once metallothionein is saturated by metals, excess metal ions spill over into other cellular compartments and cause pathological lesions. We review the literature on metallothionein in fish, and emphasize the protein's role in the detoxification and binding of metal contaminants.
A sublethal effect of zinc exposure in freshwater fish is the inhibition of calcium uptake. However, calcium also inhibits zinc uptake and this inhibition has been found to be competitive (Spry D.J. & Wood C.M. (1989). J. Exp. Biol., 142, 425–446).The subject of the acclimation of aquatic animals to metals is well researched, but there is, as yet, no simple answer as to how this acclimation is accomplished. The objectives of the present study were to characterize the interactions of zinc on calcium uptake kinetics, and to investigate if a restoration of calcium influx was associated with zinc acclimation. In addition, the possible involvement of metallothionein and a reduced zinc influx in the acclimation process was studied.
The effects of a metal mixture on Cd bioavailability and uptake in the freshwater mussel Pyganodon grandis (formerly Anodonta grandis grandis) were investigated in a limnocorral experiment in a Precambrian Shield lake during the summer of 1992. Differences in the partitioning of Cd in water, sediment, and mussels were identified between limnocorrals treated with Cd alone or with Cd and a mixture of metals (Cu, Zn, Pb, and Ni) at three concentration levels. Loss of Cd from the water column was slower in treatments with the metal mixture (22- to 34-day half-life) than in the treatment with Cd alone (11-day half-life). Despite the higher concentrations of Cd in the water column in treatments with the mixture of metals, the mussels accumulated proportionally less Cd as the metal concentrations increased. These relationships were observed in mussels exposed for 40 days ([Cd] <4.4 µg·L-1) and 80 days ([Cd] = 4-14 µg·L-1). The uncoupling of the effects of the metal mixture on Cd bioavailability and uptake suggests that laboratory studies may be appropriate for characterizing metal uptake in mussels exposed to mixtures of metals under nonequilibrium conditions. The significant deviation in the behavior of Cd in the presence of the metal mixture emphasizes the need to further investigate regulatory approaches that focus on individual contaminants.
Environmental conditions such as oxygen concentration, temperature, hardness, salinity and presence of other metals may modify metal toxicity to fish. Hypoxic conditions, temperature increase, and acidification usually render the fish more susceptible to intoxication. On the contrary, increase in mineral content (hardness and salinity) reduce metal toxicity. Interactions among various metals present in the water may modify their toxicity, synergistic, additive or antagonistic effects may occur.
Iron, copper and cadmium are determined in brine shrimp specimens using graphite furnace atomic absorption spectrometry. Four different digestion procedures, (i.e., dry ashing, hot-block, high pressure and microwave heating digestion) are compared. It was concluded that microwave heating digestion in polyethylene autosampler cups is excellent for the rapid dissolution of sub-milligram amounts of biological material. Applying platform atomisation and peak-area integration, a calibration against acid standards can be used for the determination of the three metals. The use of the ammonium phosphate-magnesium nitrate sample modification for the determination of cadmium allows a maximum pre-treatment temperature of 1000 °C. Continuum source background correction does not cause correction errors and background-corrected analyte absorbance signals appear undistorted. There is good agreement between the results obtained from furnaces and flames, confirming that the chemical and spectral interferences have been overcome.
Incubation of fertilized eggs in water containing 0.001-0.05 ppm of cadmium resulted in decreased swelling of the eggs, extended development time (delayed hatching) and reduced survival of embryos compared with the control. The proportion of normal, live hatched fry also was reduced in Cd'+ solutions (8-35%) with respect to the control (8648%). Acute toxicity tests revealed high individual variability of fish tolerance to cadmium; 96-h LC,, results were 0.002 ppm for fish under 10 days, 0.005 ppm lo-20 days, and 0.007 ppm of cadmium over 20 days.
Waterborne ammonia has become a persistent pollutant of aquatic habitats. During certain periods (e.g. winter), food deprivation may occur simultaneously in natural water. Additionally, under such stressful circumstances, fish may be enforced to swim at a high speed in order to catch prey, avoid predators and so on. Consequently, fish need to cope with all these stressors by altering physiological processes which in turn are controlled by their genes. In this present study, toxicogenomic analyses using real time PCR was used to characterize expression patterns of potential biomarker genes controlling growth, ion regulation and stress responses in common carp subjected to elevated ammonia (1 mg/L; Flemish water quality guideline for surface water) following periods of feeding (2% body weight) and fasting (unfed for 7 days prior to sampling). Both feeding groups of fish were exposed to high environment ammonia (HEA) for 0 h (control), 3h, 12h, 1 day, 4 days, 10 days, 21 days and 28 days, and were sampled after performing swimming at different speeds (routine versus exhaustive). Results show that the activity and expression of Na(+)/K(+)-ATPase, an important branchial ion regulatory enzyme, was increased after 4-10 days of exposure. Effect of HEA was also evident on expression patterns of other ion-regulatory hormone and receptor genes; prolactin and cortisol receptor mRNA level(s) were down-regulated and up-regulated respectively after 4, 10 and 21 days. Starvation and exhaustive swimming, the additional challenges in present study significantly further enhanced the HEA effect on the expression of these two genes. mRNA transcript of growth regulating hormone and receptor genes such as Insulin-like growth factor I, growth hormone receptor, and the thyroid hormone receptor were reduced in response to HEA and the effect of ammonia was exacerbated in starved fish, with levels that were remarkably reduced compared to fed exposed fish. However, the expression of the growth hormone gene itself was up-regulated under the same conditions. Expression of somatolactin remained unaltered. Stress representative genes, cytochrome oxidase subunit 1 showed an up-regulation in response to HEA and starvation while the mRNA level of heat shock protein 70 was increased in response to all the three stressors. The expression kinetics of the studied genes could permit to develop a "molecular biomarker system" to identify the underlying physiological processes and impact of these stressors before effects at population level occur.
If the modes of action of toxicants were better understood, we could more accurately predict their effects as pollutants; much knowledge already exists in medical sciences, and could be transferred.Sublethal changes within fish have been demonstrated by many sensitive histopathological and biochemical techniques. Unfortunately, ecological significance often remains obscure for these effects and others mentioned below. Growth should be routinely measured in all chronic experiments since it is easy, but it is not always a sensitive indicator of toxicity. Swimming speed is less often affected by toxicants than might be expected; clear-cut effects are usually related to impaired respiration. A toxicant might be assessed by testing whether it reduced the fish's scope for activity (active minus standard oxygen uptake). Behavioural change is often a very sensitive response, although significance in nature may be uncertain. Feeding behaviour of fish is badly affected by some pollutants. Avoidance reactions to many pollutants have been demonstrated in the laboratory, but no pattern for predicting avoidance in nature is yet available. Effects on other aspects of behaviour such as social interactions should be explored.Reproduction seems to be one of the most sensitive of chronic or sublethal responses which is clearly meaningful in nature. Reproductive success should be tested whenever feasible. It is sometimes sharply affected through inhibition of spawning or through toxicity to fish fry.Perfect bioassays are studies of production in communities of artificial or experimental streams, being equivalent to small controlled ecological experiments. Such studies are especially meaningful if they cover one or more reproductive cycles.Laboratory bioassays are inadequate if they do not explain the results of field bioassays in polluted waters. The ultimate method of bioassay is an ecological survey of a polluted habitat.Current estimates of “safe” levels of pollutants for fish are mostly 0.01–0.4 of the lethal concentration. The higher values of 0.1–0.4 are obtained from distribution of fishes in polluted rivers, and from extrapolations from lethal levels to negligible mortality. Lower values of 0.01–0.05 arise mostly from consideration of chronic, sublethal, and cumulative toxicity.There is a continuing need for critical reviews on individual pollutants.
The widespread distribution of metallothioneins (MTs) in aquatic animals is firmly established, having been reported for at least 80 species of fish and invertebrates. To date. MTs of only eight species have been characterized to the extent that the primary structure has been determined. These include three fish. one echinoderm. two molluscs (both bivalves), and two crustaceans (both decapods). MTs of major taxa remain to be characterized. Information on other groups, especially invertebrates, will help to clarify phylogenetic relationships among MTs of diverse taxa. The occurrence of multiple forms of MTs in a single species indicates that a better understanding of MT in aquatic animals will require consideration of the regulation and function of the individual forms since it appears that specific biological functions will be attributable to the various forms. The toxicological significance of MT induction has been supported by the numerous studies that have been conducted with aquatic organisms. The notion that metal-binding by MT is protective of cellular function is supported by these studies, although the specific intracellular interactions remain to be clarified. A better understanding of both the normal and toxicological functions of MTs will be useful in this regard. From a practical view. the premise that MTs can be used for routine assessment of metalexposed aquatic animals represents a still-to-be realized promise. In a number of cases. MT induction or increased levels of MT-bound metals in individuals in metal-contaminated environments can be used to justify continued development and testing of diagnostics markers of metal exposure based on MT function. An extension of such observations to include a relationship with cellular or organismal well-being will benefit from an approach that considers MT induction together with protected functional responses.
Levels of sodium, potassium and calcium ions and the activities of Na+K+, Mg2+ and Ca2+ ATPases were estimated in the muscle of the fry and fingerlings of an economically important edible freshwater fish, , at 1,2,3 and 4 days on exposure to the lethal, 4.3 mg/L and 17.1 mg/L respectively for the fry and fingerlings,and 1,7,15 and 30 days on exposure to sublethal, 0.86 mg/L and 3.42 mg/L, concentrations of cadmium. All the ion levels and ATPase activities progressively decreased over time of exposure, in the order day 1 < 2 < 3 < 4, in the muscle of both fry and fingerlings on exposure to lethal concentrations; the magnitude of decrease was higher in the fry than the fingerlings. In sublethal concentrations, an initial decrease in these parameters of the fry was noticed at day 1, but on further exposure they increased over control, however, the order was day 7 > 15 > 30 in sodium ions and Na+K+ and Mg2+ ATPase activities and 7 < 15 > 30 in potassium and calcium ions and Ca2+ ATPase activity. In fingerlings, all the parameters decreased at days 1 and 7 (day 1 < 7) but increased at days 15 and 30 (day 15 < 30). All these changes in iono-regulatory pattern of the fish were discussed in the light of available literature.
Due to prolonged use of silver in many applications, it enters into the freshwater and affects the aquatic organisms. Fingerlings of Cirrhinus mrigala were exposed to acute and sublethal concentrations of silver nitrate and the alterations of gill Na(+)/K(+)-ATPase, plasma electrolytes and biochemical parameters were assessed. The median lethal concentration of silver nitrate to the fish C. mrigala for 96 h was found to be 0.107 mg/l (with 95% confidence limits). 1/10th of LC 50 96 h value (0.0107 mg/l) was selected for sublethal study. During acute treatment branchial Na(+)/K(+)-ATPase activity was inhibited approximately 44.34% after 96 h of exposure. In sublethal treatment, silver nitrate could not produce a significant change in the activity of the enzyme at the end of 7th day. However, after 14th day, significant (p < 0.05) decrease was noted showing 22.52%-49.11% in rest of the study period. Silver intoxication resulted hyponatremia, hypokalemia, hypochloremia, and hypoproteinemia in both the treatments. Despite the decrease in these parameters, plasma glucose level was found to be increased in both the treatments to endure the silver toxicity. We suggest that the alterations in branchial Na(+)/K(+)-ATPase activity, plasma electrolytes, and biochemical parameters of fish may be useful in environmental biomonitoring and to assess the health of fish in freshwater habitat contaminated with silver.
The glutathione S-transferases (GSTs) are a multifunctional family of phase II enzymes that detoxify a variety of environmental chemicals, reactive intermediates, and secondary products of oxidative damage. GST mRNA expression and catalytic activity have been used as biomarkers of exposure to environmental chemicals. However, factors such as species differences in induction, partial analyses of multiple GST isoforms, and lack of understanding of fish GST gene regulation, have confounded the use of GSTs as markers of pollutant exposure. In the present study, we examined the effect of exposure to cadmium (Cd), a prototypical environmental contaminant and inducer of mammalian GST, on GST mRNA expression in coho salmon (Oncorhynchus kisutch) liver, gill, and olfactory tissues. GST expression data were compared to those for metallothionein (MT), a prototypical biomarker of metal exposure. Data mining of genomic databases led to the development of quantitative real-time PCR (qPCR) assays for salmon GST isoforms encompassing 9 subfamilies, including alpha, mu, pi, theta, omega, kappa, rho, zeta and microsomal GST. In vivo acute (8-48 h) exposures to low (3.7 ppb) and high (347 ppb) levels of Cd relevant to environmental scenarios elicited a variety of transient, albeit minor changes (<2.5-fold) in tissue GST profiles, including some reductions in GST mRNA expression. In general, olfactory GSTs were the earliest to respond to cadmium, whereas, more pronounced effects in olfactory and gill GST expression were observed at 48 h relative to earlier time points. Although evaluation of GSTs reflected a cadmium-associated oxidative stress response, there was no clear GST isoform in any tissue that could serve as a reliable biomarker of acute cadmium exposure. By contrast, metallothionein (MT) mRNA was consistently and markedly induced in all three tissues by cadmium, and among the tissues examined, olfactory MT was the most sensitive marker of cadmium exposures. In summary, coho salmon exhibit a complex GST tissue profile consisting of at least 9 isoforms, all of which are present in the peripheral olfactory system. Short-term exposure to environmental levels of Cd causes transient changes in salmon GST consistent with oxidative stress, and in some cases, includes a loss of GST. In a biomarker context, however, monitoring of tissue MT mRNA expression, especially in the peripheral olfactory system, may be of greater utility for assessing short-term environmental exposures to cadmium.
The influence of metal-metal interactions on uptake, accumulation, plasma transport and chronic toxicity of dietary Cu, Cd and Zn in rainbow trout (Oncorhynchus mykiss) was explored. Juvenile rainbow trout were fed diets supplemented with (μg/g) 500 Cu, 1000 Zn and 500 Cd singly and as a ternary mixture at 2.5% body weight daily ration for 28 days. Complex interactions among the metals dependent on the tissue/organ, metals ratios and duration of exposure were observed. While Zn did not accumulate, whole-body Cd and Cu concentrations increased following linear and saturation patterns, respectively. Early enhanced whole-body Cu accumulation in fish exposed to the metals mixture was correlated with reduced Cd concentration whereas late enhancement of Cd accumulation corresponded with elevated Cd concentration. This suggests early mutual antagonism and late cooperation between Cd and Cu probably due to interactions at temporally variable metal accumulation sites. At the level of uptake, Cd and Cu were either antagonistic or mutually increased the concentrations of each other depending on the duration of exposure and section of the gut. At the level of transport, enhanced Cd accumulation in plasma was closely correlated with reduced concentrations of both Zn and Cu indicating competitive binding to plasma proteins and/or antagonism at uptake sites. Compared to the Cu alone exposure, Cu concentrations were either lower (gills and carcasses) or higher (liver and kidney) in fish exposed to the metals mixture. On the other hand, Cd accumulation was enhanced in livers and carcasses of fish exposed to the mixture compared to those exposed to Cd alone, while Zn stimulated Cu accumulation in gills. Chronic toxicity was demonstrated by elevated malondialdehyde levels in livers and reduced concentrations of Zn and Cu in plasma. Overall, interactions of Cd, Cu and Zn are not always consistent with the isomorphous competitive binding theory.
This is the first study to implement a genomic approach to ascertain both transcriptional and functional end points of chronic Cu toxicity in fish associated with experimentally manipulated water chemistries. Over 21 d, zebrafish acclimated to softwater (Na(+) = 0.06 mM, Ca(2+) = 0.08 mM, Mg(2+) = 0.03 mM) were exposed to the following: soft-water (Ctrl); 12 microg L(-1) Cu (Cu); 3.3 mM Na(+) (Na); 3.3 mM Na(+) + 12 microg L(-1) Cu (Na + Cu); 3.3 mM Ca(2+) (Ca); or 3.3 mM Ca(2+) + 12 microg L(-1) Cu (Ca + Cu). Although effective at reducing Cu load in all tissues, Na(+) in the presence of Cu did not decrease the degree of oxidative damage, particularly in the gill and gut. In contrast, Ca + Cu treatment decreased Cu accumulation in gill, but not liver or gut, with no reduction in oxidative damage. Transcriptional analysis of candidate genes (atp7a, ctr1, ECaC, esr1) showed principally a down regulation of transcripts with the Cu only treatment, while Ca + Cu treatment restored some of the genes to control levels. Conversely, the Na + Cu treatment had a strong, opposing affect when compared to that of Cu alone. Zebrafish Affymetrix GeneChips revealed significantly clustered patterns of expression. Changes in expression induced by Cu appeared to be opposite to the majority of the other treatments. Our data on the preventative or enhancing effects of Na(+) and Ca(2+) both alone and in the presence of Cu, may, in the future, facilitate the incorporation of gene expression end points into a biotic ligand model predicting chronic Cu toxicity in this tropical model species of genomic importance.
Recent studies indicate that elasmobranch fish respond differently to metal exposure than marine teleosts. Accumulation rates can be high, which despite the fact that normal background levels for metals in the marine environment are low, is worrying due to the long life span and late fecundity of most shark. The goals of the present study were to examine differences in accumulation rates and toxicity of a range of metals at equimolar concentrations (10microM) in the Mediterranean or spotted dogfish, Scyliorhinus canicula. For this purpose, we exposed the dogfish to Ni (587microg/L), Cd (1124microg/L), Pb (2072microg/L), Cu (635microg/L), and Ag (1079microg/L and two additional exposures at 10microg/L and 1microg/L) for one week and measured total metal accumulation, metallothionein induction, and parameters related to osmoregulation. Our study confirms the high toxicity and accumulation rates of Ag for elasmobranch fish, even at levels 100 to 1000 times lower than exposure levels of other metals. Also Pb accumulated readily in all organs, but did not cause any osmoregulatory disturbance at the exposure levels used. Ni and Cd seem to accumulate primarily in the kidney while Cu mainly accumulated in liver. In contrast to Ni and Cd, the three other metals Ag, Cu and Pb accumulated in the rectal gland, an important organ for osmoregulation and possible target organ for metal toxicity. Only Cu succeeded in initiating a protective response by inducing MT synthesis in liver and gills.
The uptake of essential (Cu, Ni, and Zn) and nonessential (Cd and Pb) metals in the gills and whole body of zebrafish exposed to a mixture of trace elements at environmentally relevant concentrations was investigated using a stable isotope technique. Negative and positive interactions as well as nonlinear responses were observed. The Cd and Pb uptake processes were influenced the most by other metals. The uptake of Cd was inhibited by Cu, Pb, and Zn and enhanced in the presence of Ni at concentrations above 0.1 microM. Pb uptake rates were consistently increasing in the presence of Cd, Ni, and Zn in both gills and the whole body, except in one case of decreased whole body Pb uptake in the presence of Cd. The addition of Cu resulted in more complex nonlinear variations in Pb uptake rates. The addition of Pb, in turn, facilitated Cu uptake with a more pronounced effect in the gills, while Zn had a stimulating effect on the whole body level. Uptake of Ni continuously decreased with the addition of Zn, and some decline in whole body Ni accumulation was observed in the presence of Cd. In contrast Cu increased the Ni uptake rates in both gills and the whole body. The results demonstrate the complexity of the uptake processes occurring in media containing a mixture of metals at environmentally relevant concentrations. These interactions may be of key significance in understanding and predicting metal uptake, accumulation, and toxicity in multimetal exposure scenarios.