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.

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.

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.

Metal accumulation and metallothionein induction in the spotted dogfish Scyliorhinus canicula.

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.

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.

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.

Accumulation and effects of metals in caged carp and resident roach along a metal pollution gradient.

Metal accumulation and effects on plasma Ca(2+), alanine transaminase (ALT) and fish condition factor were examined in caged carp (Cyprinus carpio) and resident roach (Rutilus rutilus) at four locations along the Grote Nete River system (Belgium). Cadmium and zinc accumulation were found in carp and roach, with highest concentrations at the most contaminated site (dissolved Cd: 1.82 microg/l, Zn: 967 microg/l). On the tissue level, highest cadmium concentrations were measured in kidneys of carp and roach, followed by gills, intestine and liver, while low concentrations were observed in carcass and muscle. For zinc, a similar pattern was observed (intestine>kidney>gills>liver>carcass>muscle). Comparison between species showed higher cadmium concentrations in feral roach, while zinc levels were lower, owing to the high zinc concentrations in control carp. Furthermore, comparison of metal concentrations between two sampling periods (2005 and 2000-2001) revealed a drastic decrease in cadmium concentration in gills, liver and muscle of roach, similar to the reduction in waterborne cadmium concentrations, while differences for zinc were much less pronounced. In addition to metal accumulation, increased metallothionein concentrations (approximately 2x) were found in carp and roach, while no metal-related effects were found on ALT, Ca(2+)or condition factor. However, negative effects on fish community structure, as assessed by the index of biotic integrity (IBI), were found along the pollution gradient and indicated long-term adverse effects of metal pollution.

Influence of tidal regime on the distribution of trace metals in a contaminated tidal freshwater marsh soil colonized with common reed (Phragmites australis).

A historical input of trace metals into tidal marshes fringing the river Scheldt may be a cause for concern. Nevertheless, the specific physicochemical form, rather than the total concentration, determines the ecotoxicological risk of metals in the soil. In this study the effect of tidal regime on the distribution of trace metals in different compartments of the soil was investigated. As, Cd, Cu and Zn concentrations in sediment, pore water and in roots were determined along a depth profile. Total sediment metal concentrations were similar at different sites, reflecting pollution history. Pore water metal concentrations were generally higher under less flooded conditions (mean is (2.32+/-0.08)x10(-3) mg Cd L(-1) and (1.53+/-0.03) x 10(-3) mg Cd L(-1)). Metal concentrations associated with roots (mean is 202.47+/-2.83 mg Cd kg(-1) and 69.39+/-0.99 mg Cd kg(-1)) were up to 10 times higher than sediment (mean is 20.48+/-0.19 mg Cd kg(-1) and 20.42+/-0.21 mg Cd kg(-1)) metal concentrations and higher under dryer conditions. Despite high metal concentrations associated with roots, the major part of the metals in the marsh soil is still associated with the sediment as the overall biomass of roots is small compared to the sediment.

Metal exposure and biological responses in resident and transplanted blue mussels (Mytilus edulis) from the Scheldt estuary.

The Western Scheldt river and estuary is known to be highly polluted as it receives industrial, agricultural and domestic effluents from one of the most populated and industrialised zones in Europe. Aquatic organisms are exposed to pollutants, specifically metals that are present in different environmental phases, e.g. dissolved, suspended material or sediment phases. The objective of this study was to study the relationship that exists between environmental metal levels, the degree of metal uptake by aquatic organisms with the concomitant biological responses. For this purpose the bivalve mollusk, Mytilus edulis, was selected as bioaccumulation indicator organism. Environmental (water and sediment) and mussel samples were collected during the late winter (March 2000) from sampling sites in the Scheldt estuary. Sites were selected to represent a salinity gradient from the mouth of the estuary to the furthest distribution area of mussels upstream in the system. Together with standard water quality parameters (e.g. salinity, dissolved oxygen, dissolved organic carbon, etc.) concentrations of twelve metals were analysed in the water (dissolved and suspended matter) and sediments. Levels of these metals were also measured in the soft tissue of M. edulis, together with concomitant biomarker responses in resident mussel populations at each site. The biomarkers that were included in this study were condition index, scope for growth, survival in air, cell membrane stability, DNA damage, and metallothioneins. Data were subjected to multivariate statistical analysis. The physico-chemical parameters and metals in the environmental samples clustered the sites to reflect the distribution based on the salinity gradient. Bioaccumulation results revealed increased metal uptake along a pollution gradient with the highest metal bioaccumulation occurring at the upstream most sites and therefore closest to the in the industrial activities. However, the biomarker responses clustered the sites in a manner that reflected the influence of combination of internal exposure (bioaccumulation) and external exposure (physico-chemical conditions). These differences in biomarker responses clearly demonstrated were attributed to abiotic factors other than metal pollution alone e.g. localized short-term increases in increased suspended sediment concentrations and decreased dissolved oxygen concentrations.

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.

Assimilation efficiencies of Cd and Zn in the common carp (Cyprinus carpio): effects of metal concentration, temperature and prey type.

The impact of several factors on the assimilation efficiency (AE) of Cd and Zn from food in the common carp (Cyprinus carpio) was studied. Tested prey species were midge larvae (Chironomus riparius), zebra mussels (Dreissena polymorpha) and oligochaetes (Tubifex tubifex). The Cd load of the larvae did not affect the Cd AE in the carp. The Zn AE however, was negatively related to the Zn load of the prey. Food quantity and starvation of the carp did not significantly affect the Cd AE. For Zn, a significant decrease in AE was found when carp were fed ad libitum. Decreasing the temperature from 25 degrees C to 15 degrees C did not influence the Cd AE, while for Zn a significant decrease of the AE was measured. Carp assimilated Cd from both zebra mussels and oligochaetes with a significantly lower efficiency in comparison to the midge larvae, although Zn AEs was prey independent.

Subcellular distribution of Cd in the aquatic oligochaete Tubifex tubifex, implications for trophic availability and toxicity.

We studied the compartmentalization of cadmium and zinc in the oligochaete Tubifex tubifex. The subcellular distribution was followed over time and levels of metallothionein-like proteins were measured. The impact of the speciation on the trophic transfer was studied by calculating the assimilation efficiencies of metals from Tubificidae fed to carp. It was found that carp were able to assimilate 9.8% of the cadmium. The expected assimilated amount of cadmium, based on the subcellular fractions which are thought to be trophically available, is however 72%. The zinc assimilation results suggest that the debris fraction is at least partially available to predators. Differential centrifugation techniques provide information about the tissue compartmentalization in aquatic organisms but it is not straightforward to directly link internal speciation in prey items to the actual assimilation in the predator. The possible impact that the compartmentalization of cadmium in T. tubifex will have on the toxicity to the organism is also discussed.

Flow cytometric cell cycle analysis allows for rapid screening of estrogenicity in MCF-7 breast cancer cells.

The quantitative measurement of individual cells and their characteristics by means of flow cytometry is already for many years of great value for clinical studies. However, its potential as a tool in (eco)toxicology has only recently been discovered. Analysis of cell cycle kinetics with DNA-staining dyes can offer a valuable alternative to detect effects of chemicals on cell proliferation, an important endpoint in screening estrogen-like properties of chemicals. In the present study, flow cytometric cell cycle analysis in growth arrested MCF-7 cells exposed to five xenoestrogens correspond well with cell proliferation results of the conventionally used E-screen assay. Moreover, re-induction of proliferation in MCF-7 cells, indicated by the percentage of cells in S(ynthesis)-phase, is most pronounced after 24 h exposure, thus allowing a faster screening of xenoestrogens. This flow cytometric proliferation assay confirms that the estrogenic activity of structurally analogous parabens is mediated by the estrogen receptor pathway and is proportional to the alkyl chain length. Moreover, the ER-mediated mode of action of two fluorotelomer alcohols (6:2 FTOH and 8:2 FTOH), recently reported as xenoestrogenic, could be elucidated. These results support the potential of flow cytometric cell cycle kinetics as a screening assay for estrogen-like properties of chemicals.

Swimming performance and energy metabolism of rainbow trout, common carp and gibel carp respond differently to sublethal copper exposure.

We compared the effects of sublethal waterborne copper exposure on swimming performance and respiration rates in rainbow trout, Oncorhynchus mykiss, with those in less sensitive cyprinid species such as common carp, Cyprinus carpio, and gibel carp, Carassius auratus gibelio. These cyprinids are considerably more resistant to Cu intoxication, and differ from trout in swimming performance and respiratory behaviour. Critical swimming speed (U(crit)), oxygen consumption, plasma ammonia and muscle ammonia, lactate and pH were measured during a 28-day sublethal exposure to 1 microM Cu. U(crit) decreased with 48, 31 and 13% within the first 12-24 h for rainbow trout, common and gibel respectively. Gibel carp recovered quickly and experienced no further reduction in swimming performance. Recovery of swimming capacity in rainbow trout and common carp was only partial. All three species displayed similar plasma ammonia peaks in the first hours to days, and a more gradual muscle ammonia accumulation over time. Whereas no signs of respiratory stress occurred in rainbow trout, common carp experienced a transient reduction in oxygen consumption combined with anaerobic metabolism after 24 h of exposure. At the same time, oxygen consumption was also reduced in gibel carp, but no signs of anaerobic metabolism were detected. Cu accumulated quickly to similar levels (36-39 microg g(-1) dry weight at day 3) in the gills of all three species, after which accumulation leveled off. Liver tissue of rainbow trout had a high Cu level from the start, and Cu concentration did not show any additional accumulation. In contrast, common carp liver showed a significant Cu accumulation from day 3 onwards, while accumulation in gibel livers was much slower and was significant from day 7 onwards. Interestingly, Cu accumulation patterns in plasma and kidney revealed a possibly important role for the kidney in Cu homeostasis of gibel carp.

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.