Common carp exposed to binary mixtures of Cd(II) and Zn(II): A study on metal bioaccumulation and ion-homeostasis.

The aquatic environment receives a wide variety of contaminants that interact with each other, influencing their mutual toxicity. Therefore, studies of mixtures are needed to fully understand their deleterious effects on aquatic organisms. In the present experiment, we aimed to assess the effects of Cd and Zn mixtures in common carp during a one-week exposure. The used nominal waterborne metal levels were 0.02, 0.05 and 0.10 µM for Cd and 3, 7.5 and 15 µM for Zn. Our results showed on the one hand a fast Cd increase and on the other hand a delayed Zn accumulation. In the mixture scenario an inhibition of Cd accumulation due to Zn was marked in the liver but temporary in the gills. For Zn, the delayed accumulation gives an indication of the efficient homeostasis of this essential metal. Between the different mixtures, a stimulation of Zn accumulation by Cd rather than an inhibition was seen in the highest metal mixtures. However, when compared to an earlier single Zn exposure, a reduced Zn accumulation was observed. Metallothionein gene expression was quickly activated in the analysed tissues suggesting that the organism promptly responded to the stressful situation. Finally, the metal mixture did not alter tissue electrolyte levels.

Physiological performance of common carp (Cyprinus carpio, L., 1758) exposed to a sublethal copper/zinc/cadmium mixture.

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.

Antagonistic bioaccumulation of waterborne Cu(II) and Cd(II) in common carp (Cyprinus carpio) and effects on ion-homeostasis and defensive mechanisms.

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 (Cyprinus carpio) 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.

Investigating the effects of a sub-lethal metal mixture of Cu, Zn and Cd on bioaccumulation and ionoregulation in common carp, Cyprinus carpio.

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 ±â€¯0.001 µM (4.3 ±â€¯0.6 µg/L), Zn 2.71 ±â€¯0.81 µM (176.9 ±â€¯52.8 µg/L) and Cd 0.03 ±â€¯0.0004 µM (3.0 ±â€¯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.

Limited oxidative stress in common carp (Cyprinus carpio, L., 1758) exposed to a sublethal tertiary (Cu, Cd and Zn) metal mixture.

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.

The impact of temperature on metal mixture stress: Sublethal effects on the freshwater isopod Asellus aquaticus.

Chemical and natural factors have been demonstrated to interact and potentially change the toxicity of the individual stressors. Yet, while there exists a multitude of papers studying the temperature-dependent toxicity of single chemicals, little research exists on the impact of temperature on chemical mixtures. This paper investigates the effect of temperature on environmentally-relevant mixtures of Cd, Cu and Pb. We linked the effects on respiration, growth, feeding rate and activity of Asellus aquaticus to the free ion activities, as a measure for the bioavailability of the metals, and the body concentrations. We observed interactions of temperature and metal body concentrations on all sublethal endpoints, except activity. Mixture effects on accumulation and feeding rate were observed as well and even an interaction between metal body burden, mixture and temperature treatment was revealed for the feeding rate of Pb exposed isopods. This research adds to a growing body of evidence that the current chemical-based monitoring is insufficient to estimate chemical toxicity in aquatic ecosystems and must, therefore, be complemented with effect-based tools.

Combined effects of metal mixtures and predator stress on the freshwater isopod Asellus aquaticus.

Biotic stressors have been demonstrated to change the toxicity of pollutants. While the combined effects of predator cues and pesticides are well documented, the interaction of predator stress with metals is a topic that has remained largely unexplored. In this laboratory experiment, the freshwater isopod Asellus aquaticus is exposed to predator cues and metal mixtures of Cd, Cu and Pb. We examined the effects on growth, respiration and, as behavioral parameters, feeding rate and activity. These were linked to the free ion activities (FIAs) in the water and the metal body concentrations. The findings reveal that Cu accumulation significantly influenced the growth rate, the feeding rate and the activity of isopods exposed to predator stress. Furthermore, we found a concentration-dependent interaction of the Cd + Pb mixtures on the feeding rate and a lower feeding rate for Cd and Pb predator exposed asellids. As several interactions were found between metals and predator stress, it demonstrates the importance of investigating how organisms and whole ecosystems respond to multiple stressors. A better understanding of these interactions will undoubtedly improve risk assessment and management.

Relationships between lines of evidence of pollution in estuarine areas: Linking contaminant levels with biomarker responses in mussels and with structure of macroinvertebrate benthic communities.

Data obtained in a pollution survey performed in estuarine areas were integrated using multivariate statistics. The sites selected for the study were areas affected by treated and untreated urban discharges, harbours or industrial activities as well as reference sites. Mussels were transplanted to each site and after different times of exposure, samples of water, sediments and mussels were collected. Biomarkers were analysed on mussels after 3 and 21 days of transplant whereas concentrations of contaminants were measured in water, sediments and mussels after 21 days of transplant. The structure of macroinvertebrate benthic communities was studied in sediment samples. Studied variables were organised into 5 datasets, each one constituting a line of evidence (LOE): contaminants in water, contaminants in sediments, contaminants accumulated by transplanted mussels, biomarkers in transplanted mussels and changes in the structure of macroinvertebrate benthic communities of each sampling site. Principal Component Analysis (PCA) identified the variables of each LOE best explaining variability among sites. In order to know how LOEs relate to each other, Pearson's correlations were performed. Contaminants in sediments were not correlated with the rest of LOEs. Contaminants in water were significantly correlated with contaminants and biomarkers in mussels and with structure of macroinvertebrate benthic communities. Similarly, significant correlations were found between contaminants and biomarkers in mussels and between biomarkers in mussels and structure of macroinvertebrate benthic communities. In conclusion, biomarker responses give relevant information on pollution in estuarine areas and provide a link between chemical and ecological statuses of water bodies in the context of the Water Framework Directive.

Uptake and toxicity of Cd, Cu and Pb mixtures in the isopod Asellus aquaticus from waterborne exposure.

The present study evaluated interactions of waterborne Cd, Cu and Pb mixtures on metal uptake rates in the isopod Asellus aquaticus and related this to mixture effects on toxicity. Secondly, it was assessed whether observed mixture effects were better related to isopod body concentrations compared to exposure concentrations. Isopods were exposed for 10 days to single, binary and tertiary mixtures including five different concentrations of Cd (0.107 to 277 µg L(-1)), Cu (3.35 to 2117 µg L(-1)) and Pb (0.782 to 443 µg L(-1)). Mortality was assessed every day while isopod body concentrations, growth (biomass) and energy reserves (glycogen, lipid and protein reserves) were assessed at the end of the experiment. Synergistic interactions of combined Cd and Pb exposure on Cd and Pb uptake as well as on growth rates and mortality rates were observed. Mixture effects of combined Cd and Pb exposure on toxicity endpoints were directly related to increased Cd uptake in the Cd+Pb treatment. No mixture interactions of Cu on Cd or Pb uptake (and vice versa), nor on toxicity endpoints were observed. All toxicity endpoints were related to body concentrations. However, mixture effects disappeared when growth and mortality rates were expressed on body concentrations instead of exposure concentrations. By combining information of mixture effects on metal uptake with mixture toxicity data, the present study provides more insight in the way metal mixtures interfere with aquatic organisms and how they can induce toxic effects.

Uranium and cadmium provoke different oxidative stress responses in Lemna minor L.

Common duckweed (Lemna minor L.) is ideally suited to test the impact of metals on freshwater vascular plants. Literature on cadmium (Cd) and uranium (U) oxidative responses in L. minor are sparse or, for U, non-existent. It was hypothesised that both metals impose concentration-dependent oxidative stress and growth retardation on L. minor. Using a standardised 7-day growth inhibition test, the adverse impact of these metals on L. minor growth was confirmed, with EC50 values for Cd and U of 24.1 ± 2.8 and 29.5 ± 1.9 µm, respectively, and EC10 values of 1.5 ± 0.2 and 6.5 ± 0.9 µm, respectively. The metal-induced oxidative stress response was compared through assessing the activity of different antioxidative enzymes [catalase, glutathione reductase, superoxide dismutase (SOD), ascorbate peroxidase (APOD), guaiacol peroxidase (GPOD) and syringaldizyne peroxidase (SPOD)]. Significant changes in almost all antioxidative enzymes indicated their importance in counteracting the U- and Cd-imposed oxidative burden. However, some striking differences were also observed. For activity of APODs and SODs, a biphasic but opposite response at low Cd compared to U concentrations was found. In addition, Cd (0.5-20 µm) strongly enhanced plant GPOD activity, whereas U inhibited it. Finally, in contrast to Cd, U up to 10 µm increased the level of chlorophyll a and b and carotenoids. In conclusion, although U and Cd induce similar growth arrest in L. minor, the U-induced oxidative stress responses, studied here for the first time, differ greatly from those of Cd.

Ecological impact assessment of sediment remediation in a metal-contaminated lowland river using translocated zebra mussels and resident macroinvertebrates.

The present study investigated to what extent accumulated metal levels in aquatic invertebrates can reflect environmental contamination and how these tissue levels can be related to alterations in macroinvertebrate communities in the dredged River Dommel. Metal accumulation was measured in translocated zebra mussels (Dreissena polymorpha) and resident Chironomidae. Furthermore, macroinvertebrate community composition was assessed. Our results indicated that trends of total metal concentrations in surface water of the Dommel in time are reflected well by metal levels in tissue of D. polymorpha. In contrast, sediment-bound metals were the most dominant exposure route for Chironomidae. Alterations in macroinvertebrate community composition were observed during dredging and significant relations between metal levels in invertebrate tissues and ecological responses were found. Our results demonstrated that metal accumulation in both zebra mussels and Chironomidae can be used as an integrated measure of metal bioavailability and to predict ecological effects of metal toxicity on macroinvertebrate communities.

The impact of increased oxygen conditions on metal-contaminated sediments part II: effects on metal accumulation and toxicity in aquatic invertebrates.

The present study evaluated the effect of increasing oxygen concentrations in overlying surface water on the accumulation and toxicity of sediment-bound metals in the aquatic invertebrates Lumbriculus variegatus, Asellus aquaticus and Daphnia magna. A 54 days experiment using three experimental treatments (90% O(2) in overlying surface water, 40% O(2) and a non-polluted control) was conducted. At 6 different time points (after 0, 2, 5, 12, 32 and 54 days) acid volatile sulfides (AVS), simultaneously extracted metals (SEM) and total organic carbon (TOC) were measured in the superficial sediment layer (0-1 cm). At each time point, accumulated metal levels as well as the available energy stores were measured in L. variegatus and A. aquaticus and each time D. magna was exposed to surface water in a 24 h toxicity test. Additionally metallothionein-like protein (MTLP) induction was quantified in L. variegatus. Oxygen induced changes in sediment AVS resulted in faster accumulation of metals from contaminated sediments in A. aquaticus, while no differences in toxicity in this species were observed. Ag, Cr, As and Co accumulation as well as toxicity in water exposed D. magna were clearly enhanced after 54 days, caused by oxidation of metal-sulfide complexes. Due to their feeding and burrowing behaviour, metal accumulation and toxicity in L. variegatus was not influenced by geochemical characteristics. Nevertheless, a rapid induction of MTLP was observed in both the 90% O(2) and the 40% O(2) treatment. The present study showed that elevated oxygen concentrations in overlying surface water can directly enhance metal accumulation and toxicity in aquatic invertebrates, however this is highly dependent on the organisms ecology and most dominant metal exposure route (water vs. sediment).

The impact of increased oxygen conditions on metal-contaminated sediments part I: effects on redox status, sediment geochemistry and metal bioavailability.

In order to evaluate the effect of improved oxygen concentrations in overlying surface water on the redox status, sediment geochemistry and metal bioavailability in metal-polluted sediments a 54 days lab experiment with two different experimental treatments was conducted (90% and 40% O(2)). Changes in redox potential (Eh) in the sediment were monitored over time. At 6 different time points (after 0, 2, 5, 12, 32 and 54 days) and at 4 sediment depths (0-1, 1-4, 4-8 and 8-15 cm), acid volatile sulfides (AVS), simultaneously extracted metals (SEM) and total organic carbon (TOC) were measured and metal release to overlying surface water was determined. Labile metal species in both water and sediment were measured using Diffusive Gradients in Thin films (DGT). Our results showed that elevated oxygen levels in overlying surface water led to an Eh increase in the sediment of the 90% O(2) treatment from 0 to ± 200 mV while AVS concentrations in the upper sediment layer decreased by 70%. Following AVS oxidation metal availability in the pore water was highly elevated after 54 days. However, Cu remained strongly bound to the sediment during the whole experiment. Only a limited metal release to the overlying surface water was noticed, which was due to the fact that SEM(tot) concentrations in the sediment did not yet exceeded AVS levels ([SEM(tot) - AVS]/f(OC) = 0) after 54 days. Additionally, adsorption on Fe and Mn hydroxides and particulate organic carbon also slowed down any potential metal release. Our results indicated that increasing oxygen concentrations due to general water quality improvements can enhance the mobility of trace metals which may result in the leaching of sediment-bound metals to overlying surface water, even in undisturbed watercourses.

Potential of an in vitro toolbox combined with exposure data as a first step for the risk assessment of dietary chemical contaminants.

In vitro risk assessment of dietary contaminants has become a priority in human food safety. This paper proposes an in vitro approach associating different complementary tools in an original toolbox and aims to improve the assessment of the toxicological impact of dietary contaminants at realistic human exposure levels, with a special focus on the intestinal compartment. The system is based on the use of four complementary cellular tools, namely stress gene induction in transgenic strains of Escherichia coli, modulation of the activity of key biotransformation enzymes (cytochrome P-450 (CYP) 1A1 and 3A4) in a human intestinal cell line, and activation of aryl hydrocarbon receptor (AhR) and oestrogenic receptor (ER)-dependent genes in agonistic and antagonistic assays with luciferase reporter cells. It was applied to four chosen model molecules: ochratoxin A (OTA) and deoxynivalenol (DON), two common food-borne mycotoxins, and imazalil (IMA) and benomyl (BEN), two fungicides widely occurring in foodstuffs. All these assays were performed at or around a realistic intestinal concentration, determined through a deterministic approach based on the calculation of a theoretical maximum daily intake (TMDI). Using the four model molecules, it is clearly highlighted that induction of CYP1A1 activity and inhibition of CYP3A4 activity occurred in Caco-2 cells at a realistic intestinal concentration of IMA. Furthermore, some bacterial stress genes were induced in a range of realistic concentrations, following exposure to DON and IMA. In addition, BEN clearly provoked an ER agonistic activity in a human oestrogen sensitive reporter cell line. All these results are in accordance with the literature, suggesting that the in vitro toolbox constitutes an interesting approach in order to obtain a first 'fingerprint' of dietary contaminants at realistic human exposure for further risk assessment.

Metallothioneins failed to reflect mercury external levels of exposure and bioaccumulation in marine fish--considerations on tissue and species specific responses.

The suitability of metallothioneins (MT) in fish as biomarker of exposure to mercury has been questioned. Therefore, this study aimed at investigating the relationship between external levels of exposure, mercury accumulation and MT content, assessing species and tissue specificities. Two ecologically different fish species--Dicentrarchus labrax and Liza aurata--were surveyed in an estuary historically affected by mercury discharges. Total mercury (T-Hg) and MT content were determined in gills, blood, liver, kidney, muscle and brain. All tissues reflected differences in T-Hg accumulation in both species, although D. labrax accumulated higher levels. Regarding MT, D. labrax revealed a depletion in brain MT content and an incapacity to induce MT synthesis in all the other tissues, whereas L. aurata showed the ability to increase MT in liver and muscle. Tissue-specificities were exhibited in the MT inducing potential and in the susceptibility to MT decrease. L. aurata results presented muscle as the most responsive tissue. None of the investigated tissues displayed significant correlations between T-Hg and MT levels. Overall, the applicability of MT content in fish tissues as biomarker of exposure to mercury was uncertain, reporting limitations in reflecting the metal exposure levels and the subsequent accumulation extent.

Environmental pollutants and type 2 diabetes: a review of mechanisms that can disrupt beta cell function.

The prevalence of diabetes mellitus is currently at epidemic proportions and it is estimated that it will increase even further over the next decades. Although genetic predisposition and lifestyle choices are commonly accepted reasons for the occurrence of type 2 diabetes, it has recently been suggested that environmental pollutants are additional risk factors for diabetes development and this review aims to give an overview of the current evidence for this. More specifically, because of the crucial role of pancreatic beta cells in the development and progression of type 2 diabetes, the present work summarises the known effects of several compounds on beta cell function with reference to mechanistic studies that have elucidated how these compounds interfere with the insulin secreting capacity of beta cells. Oestrogenic compounds, organophosphorus compounds, persistent organic pollutants and heavy metals are discussed, and a critical reflection on the relevance of the concentrations used in mechanistic studies relative to the levels found in the human population is given. It is clear that some environmental pollutants affect pancreatic beta cell function, as both epidemiological and experimental research is accumulating. This supports the need to develop a solid and structured platform to fully explore the diabetes-inducing potential of pollutants.

A kinetic model for multicomponent wastewater substrate removal by partial ozonation and subsequent biodegradation.

A kinetic model for multicomponent substrate removal by the partial ozonation process is presented. The model consists of a component describing the co-evolution of COD and BOD as a function of ozone dosage and a mass transfer based component describing ozone dosage as a function of time. A multiple zero order reaction concept is used to describe the multicomponent kinetic behaviour. The model has been verified experimentally by comparing stoichiometric ratio and ozone reaction rate of conventional partial ozonation processes and partial ozonation processes with intermittent biodegradation. The model is found to effectively describe the change in stoichiometry and reaction rate that typically occurs during ozonation processes. Intermittent biodegradation is found to have no effect on the instantaneous stoichiometric ratio or the instantaneous ozone reaction rate. This implicates that intermittent BOD removal results in an additional decrease in required ozonation time and ozone demand compared to the conventional partial ozonation process and in addition to the expected decrease resulting from BOD removal.

Wastewater treatment plant modeling supported toxicity identification and evaluation of a tank truck cleaning effluent.

The aim of this work is the Toxicity Identification Evaluation (TIE) of highly toxic tank truck cleaning wastewater effluent. Conventional TIE, using EDTA and activated carbon addition, revealed organic compounds as main source of toxicity. Additional toxicant characteristics could be derived from hydraulic wastewater treatment plant simulation being high intake frequency, low biodegradability and high acute toxicity ratio between Pseudokirchneriella subcapitata and Daphnia magna. The risk probability of compounds present in the influent wastewater was simulated using USEPA Estimation Program Interface (EPI) software. Compound toxicity, solubility and removal rate in a wastewater treatment plant were incorporated into one risk number indicative for the probability of a compound to cause toxicity in the effluent. The herbicide acetochlor was deducted from these TIE procedures as major toxicant and this was confirmed by chemical measurements, concentrations in the effluent samples ranged from 3.73+/-0.52 ppm to 7.8+/-2.1 ppm acetochlor equivalents.