Experimentally derived acute and chronic copper Biotic Ligand Models for rainbow trout.

We evaluated the effects of varying water chemistry ([Ca2+]=0.2-3mM, [Mg2+]=0.05-3mM, dissolved organic matter (DOM, natural, from maple leaves)=0.3-10mg of CL-1, pH=5.0-8.5) on the acute (96-h, unfed fish) and chronic (30-d, fed fish) toxicity of waterborne Cu to juvenile rainbow trout (Oncorhynchus mykiss) exposed in flow-through conditions. Acute and chronic Biotic Ligand Models (BLMs) were developed from the obtained toxicity data-sets, using the Visual MINTEQ software. Our results indicate that Cu is predominantly an acute toxicant to rainbow trout, as there were no observable growth effects and the 96-h and 30-d LC50 values were similar, with mortality mostly occurring within the first few days of exposure. Calcium and DOM were greatly protective against both acute and chronic Cu toxicity, but Mg seemed to only protect against chronic toxicity. Additional protection by pH 5.0 in acute exposure and by pH 8.5 in chronic exposure occurred. In the range of conditions tested, the observed 96-h LC50 and 30-d LC20 values varied by a factor of 39 and 27 respectively. The newly developed acute and chronic BLMs explained these variations reasonably well (i.e. within a 2-fold error), except at pH≥8 where the high observed acute toxicity could not be explained, even by considering an equal contribution of CuOH+ and Cu2+ to the overall Cu toxicity. The 96-h LC50 values of 59% of 90 toxicity tests from 19 independent studies in the literature were reasonably well predicted by the new acute BLM. The LC20 predictions from the new chronic BLM were reasonable for 7 out of 14 toxicity tests from 6 independent chronic studies (with variable exposure durations). The observed deviations from BLM predictions may be due to uncertainties in the water chemistry in these literature studies and/or to differences in fish sensitivity. A residual pH effect was also observed for both the acute and the chronic data-sets, as the ratio of predicted vs. observed LC values generally increased with the pH. Additional mechanistic studies are required to understand the influence of pH, Na, and Mg on Cu toxicity to trout. The present study presents the first experimentally developed chronic Cu BLM for the rainbow trout. To the best of our knowledge, it also presents the first acute Cu BLM that is based on a published data-set for trout. These newly developed BLMs should contribute to improving the risk assessment of Cu to fish in freshwater.

Interactions of waterborne and dietborne Pb in rainbow trout, Oncorhynchus mykiss: Bioaccumulation, physiological responses, and chronic toxicity.

In Pb-contaminated environments, simultaneous exposure to both waterborne and dietborne Pb is likely to occur. This study examined the potential interactive effects of these two pathways in juvenile rainbow trout that were exposed to Pb in the water alone, in the diet alone, and in combination for 7 weeks. The highest waterborne Pb concentration tested (110µgL(-1)) was approximately equivalent to the 7-week LC20 (97µgL(-1)) measured in a separate trial, while the lowest was a concentration often measured in contaminated environments (8.5µgL(-1)). The live diet (10% daily ration on a wet mass basis) consisted of oligochaete worms (Lumbriculus variegatus) pre-exposed for 28days to the same waterborne Pb concentration, and the highest dietary dosing rate to the trout was 12.6µg Pb g fish(-1)day(-1). With waterborne exposure, whole body Pb burden increased to a greater extent in the worms than in the fish. Nonetheless, in trout waterborne exposure still resulted in 20-60-fold greater Pb accumulation compared to dietborne Pb exposure. However, combined exposure to both waterborne and dietborne Pb reduced the whole body accumulation extensively at waterborne Pb>50µgL(-1), with similar antagonistic interaction in liver and carcass (but not gill or gut) at a lower threshold of 20µgL(-1). Growth effects in trout were minimal with marginal reductions in the dietborne and combined exposures seen only at 110µgL(-1). Chronic Pb exposure reduced lipid and carbohydrates level in the worms by 50% and 80% respectively, while protein was unchanged, so growth effects in trout may have been of indirect origin. After 7 weeks, Ca(2+) homeostasis in the trout was unaffected, but there were impacts on Na(+). Blood Na(+) was reduced in waterborne and dietborne exposures, while gut Na(+)/K(+) ATPase activities were reduced in waterborne and combined exposures. This study is the first, to our knowledge to examine the interaction of waterborne and dietborne Pb exposure in fish. While physiological impacts of Pb were observed in both worms and fish, higher concentrations of dietborne Pb actually protected fish from waterborne Pb bioaccumulation and these effects. The impacts of metals on diet quality should not be neglected in future dietborne toxicity studies using live prey.

Use of whole-body and subcellular Cu residues of Lumbriculus variegatus to predict waterborne Cu toxicity to both L. variegatus and Chironomus riparius in fresh water.

We tested the use of whole-body and subcellular Cu residues (biologically-active (BAM) and inactive compartments (BIM)), of the oligochaete Lumbriculus variegatus to predict Cu toxicity in fresh water. The critical whole-body residue associated with 50% mortality (CBR(50)) was constant (38.2-55.6 µg g(-1) fresh wt.) across water hardness (38-117 mg L(-1) as CaCO(3)) and exposure times during the chronic exposure. The critical subcellular residue (CSR(50)) in metal-rich granules (part of BIM) associated with 50% mortality was approximately 5 µg g(-1) fresh wt., indicating that Cu bioavailability is correlated with toxicity:subcellular residue is a better predictor of Cu toxicity than whole-body residue. There was a strong correlation between the whole-body residue of L. variegatus (biomonitor) and survival of Chironomus riparius (relatively sensitive species) in a hard water Cu co-exposure. The CBR(50) in L. variegatus for predicting mortality of C. riparius was 29.1-45.7 µg g(-1) fresh wt., which was consistent within the experimental period; therefore use of Cu residue in an accumulator species to predict bioavailability of Cu to a sensitive species is a promising approach.

Mechanisms of waterborne Cu toxicity to the pond snail Lymnaea stagnalis: physiology and Cu bioavailability.

We examined the mechanisms of toxicity of waterborne Cu to the freshwater pond snail Lymnaea stagnalis. The snail is one of the most sensitive species to acute Cu exposure (96 h LC(50), LC(20): 24.9, 18.0 µgl(-1)); they are not protected by the water quality criteria of the US EPA. Tissue Na and Ca were also reduced by Cu in the acute exposure. In contrast, during 28 d chronic exposures to Cu in the presence of food, which resulted in higher DOC concentrations, there was no significant mortality but an inhibition of growth, which may reflect a re-allocation of resources to detoxification. Cu detoxification was evidenced in chronic exposure by increases in metallothionein-like protein concentrations and Cu binding to metal-rich granules, decreases in thiobarbituric acid-reactive substances, and changes in the subcellular distribution in the soft tissues. Our results demonstrated that apart from external Cu bioavailability, compartmentalization of metals within the cells can alter toxicity of Cu to the snails.

Can the biotic ligand model predict Cu toxicity across a range of pHs in softwater-acclimated rainbow trout?

This study examined the effects of pH (5.0-8.5) on the toxicity of waterborne Cu to juvenile rainbow trout (Oncorhynchus mykiss) in soft water under flow-through conditions. Relationships between 96 h or 30 day Cu toxicity and 24 h lethal Cu accumulation on the gills (24 h LA(50-Acute) or 24 h LA(50-Chronic)) were examined in the context of predictions made using the biotic ligand model (BLM). Acute toxicity was relatively constant across pHs except for a 2- to 3-fold higher LC(50) at pH 5.0. In the chronic exposure, the fish had similar tolerance to Cu from pH 5.0 to 8.0, but were 3- to 4-fold more tolerant at pH 8.5. This pattern was not captured by the current BLM which predicts that acute and chronic LC(50) values should increase progressively from pH 5.0 to 8.5, with much greater values than those observed at the higher pH range. BLM-based water quality criteria would not be protective for trout at pH 8.0 or 8.5 in acute exposure to Cu in soft water. The measured 24 h LA(50-Acute) and LA(50-chronic) at pH 8.5 were higher, and 24 h LA(50-Chronic) at pH 5.0 was lower than those at the other pHs. This study indicates that gill Cu bioaccumulation does not explain toxicity at high or low pH, and the BLM needs revision to adequately predict Cu toxicity to trout in soft water.

Cadmium accumulation and in vitro analysis of calcium and cadmium transport functions in the gastro-intestinal tract of trout following chronic dietary cadmium and calcium feeding.

Juvenile rainbow trout (Oncorhynchus mykiss) were fed diets made from Lumbriculus variegatus containing environmentally relevant concentrations of Cd (approximately 0.2 and 12 microg g(-1) dry wt) and/or Ca (1, 10, 20 and 60 mg g(-1) dry wt) for 4 weeks. Ten fish per treatment were removed weekly for tissue metal burden analysis. In all portions of the gastro-intestinal tract (GIT) (stomach, anterior, mid, and posterior intestine), chronic exposure to elevated dietary Ca decreased Cd tissue accumulation to varying degrees. At week five, the GITs of the remaining fish were subjected to an in vitro gut sac technique. Pre-exposure to the different treatments affected unidirectional uptake and binding rates of Cd and Ca in different manners, dependent on the specific GIT section. Ca and Cd uptake rates were highly correlated within all sections of the GIT, and the loosely binding rate of Cd to the GIT surfaces predicted the rate of new Cd absorption. Overall, this study indicates that elevated dietary Ca is protective against Cd uptake from an environmentally relevant diet, and that Ca and Cd uptake may occur through both common and separate pathways in the GIT.

Does dietary Ca protect against toxicity of a low dietborne Cd exposure to the rainbow trout?

We examined the toxicity of Cd, provided in a natural diet and at an environmentally relevant concentration ( approximately 12microgg(-1) dry wt.), to the juvenile rainbow trout (Oncorhynchus mykiss). In addition, we tested the protection by elevated dietary Ca against both the accumulation and toxicity of dietary Cd from this natural diet (background Ca approximately 1mgg(-1) dry wt.). Food pellets were made from blackworms (Lumbriculus variegatus), and spiked with Cd and either no additional Ca or elevated ( approximately 60mgg(-1) dry wt.) concentrations for each of the treatment diets. Survival was unaffected for trout fed diet with 12microgg(-1) dry wt. Cd for a month, but growth was potentially reduced. Tissue burden analysis revealed that the stomach, liver and kidney accumulated the most Cd, with concentrations progressively increasing in the liver and kidney over the whole exposure period. Cd concentrations in the plasma and red blood cells were unaffected by the different treatments, but subcellular fractionation analysis indicated that a higher concentration of Cd was associated with the metal-sensitive fractions of red blood cells of the fish that were exposed to the dietborne Cd. Dietary Cd exposure also caused potential toxicity to cells of the stomach in that they bound more Cd to heat-denaturable proteins. However, detoxification appeared to take place in the Cd-exposed fish because more Cd was bound to metallothionein-like proteins by week 4 of exposure. Elevated Ca in the Cd diet generally protected against accumulation and toxicity of dietborne Cd. The protection against Cd accumulation was almost complete at the gills, robust in the stomach and whole body (> or =50% reductions), but not significant in the liver, kidney, carcass, plasma, or red blood cells. Elevated dietary Ca also reduced Cd accumulation in the organelles of the fish stomach and red blood cells. In addition, dietborne Ca not only reduced the uptake of Cd by the cells, but also altered how the cells handled Cd intracellularly. In general, our results have demonstrated the need to use diets with natural compositions for dietary toxicity studies.

Decoupling of cadmium biokinetics and metallothionein turnover in a marine polychaete after metal exposure.

This study investigated the kinetics of Cd bioaccumulation, detoxification, subcellular distribution, and efflux in the nereid polychaete Perinereis aibuhitensis after Cd pre-exposure. Cd pre-exposure increased the Cd body burden in the worms, but did not affect the overall Cd uptake and efflux rates and metallothionein-like protein (MTLP) concentrations. During short-term exposure to dissolved Cd, Cd in the cytosolic fraction increased after Cd pre-exposure, and this fraction also increased during the Cd efflux period, indicating that the insoluble fraction of Cd was presumably lost at a faster rate than the loss of cytosolic Cd. Even though the MTLP concentration remained comparable after Cd pre-exposure, both the MTLP synthesis rate and the degradation rate increased, thus leading to a high MTLP turnover in the Cd-exposed worms. However, Cd uptake and efflux into different protein size fractions did not follow the patterns of MTLP synthesis and degradation, strongly suggesting that Cd kinetics is decoupled from the MTLP kinetics in the worms. Our study adds to an increasing body of evidence on the complicated relationship between metal biokinetics and MTLP kinetics in different groups of marine invertebrates which have strong contrasts in their metal handling strategies.

Trophic transfer and dietary toxicity of Cd from the oligochaete to the rainbow trout.

Dietary toxicity of metals on fish is often studied using commercial pellet food, and there is a lack of investigation on the toxicity of metals that are biologically incorporated into the natural food from the aquatic environment. In this study, we investigated the toxicity of dietborne Cd from the oligochaete Lumbriculus variegatus to the rainbow trout Oncorhynchus mykiss. The oligochaete worms were exposed to waterborne Cd (0.1, 5, 20, and 200 microgL(-1)) for 1 week and the fish were fed this food exclusively (daily ration=3.5% body wet weight) for 1 month. Cd concentrations in the worms averaged 0.1, 0.6, 2.2, and 30.3 microgg(-1) wet weight respectively, whereas the whole fish accumulated 0.002, 0.005, 0.019, and 0.387 microg Cd g(-1) wet weight respectively, after feeding upon control or Cd-contaminated worms for 4 weeks. Highest concentrations of Cd were retained in the gut, followed by the kidney and liver of the fish, with the latter two increasing over time; however, gut tissue accounted for >80% of whole body Cd burdens at all times. The trophic transfer efficiency of Cd was low (0.9-6.4%) although higher than in previous studies using Cd-spiked commercial diets, and was only weakly correlated to the internal Cd storage in the worms. The level of Cd in the contaminated worms did not affect Cd trophic transfer efficiency, but was reduced over the dietary exposure period. Dietborne Cd did not interfere with whole body Ca uptake from the water or alter plasma [Ca], but reduced growth by 50% in the trout exposed to the highest Cd dose. Cd stored in the metallothionein-like proteins of the fish gut tissue increased while that in the heat-denaturable proteins was reduced, suggesting detoxification over time. This study suggests a higher bioavailability and toxicity of Cd from the natural diets than from the commercial diets used in previous studies.

Interactions of silver, cadmium, and copper accumulation in green mussels (Perna viridis).

Metal interaction is vital for assessing the use of aquatic organisms in monitoring metal contamination. The present study examined the interactions between Ag and Cd accumulation and between Ag and Cu accumulation in the green mussel (Perna viridis). Accumulation of Ag and Cd in the whole tissue of green mussels exposed to 5 microg/L of Ag and 20 microg/L of Cd for two weeks was independent; however, interaction was observed at the subcellular level. Approximately 25% of Ag shifted from the insoluble fraction (IF) to the metallothionein-like protein in the presence of Cd, which probably resulted from the competition of Cd on IF in the Ag-Cd coexposure. On the other hand, coexposure of the mussels to Ag (5 microg/L) and Cu (30 microg/L) for two weeks increased the Ag and Cu concentrations in the tissue synergistically (two- to fivefold), but Ag and Cu subcellular distributions were similar in the coexposed and the singly exposed mussels. Exposure to Ag alone increased the dietary uptake of Ag by 30%, but the effect was reduced in the presence of Cd. No interaction, however, was observed between uptake rates of metals from the dissolved phase. To conclude, a significant interaction was observed for total Ag and Cu accumulation, but not for total Ag and Cd accumulation, in the mussels. Metal interaction is more likely to be observed at the subcellular level than at the whole-tissue level.

Subcellular cadmium distribution, accumulation, and toxicity in a predatory gastropod, thais Clavigera, fed different prey.

Bioaccumulation and toxicity of Cd were investigated in a marine predatory whelk, Thais clavigera, after being fed with the rock oyster, Saccostrea cucullata, or the herbivorous snail, Monodonta labio, for up to four weeks. The oysters and snails had different subcellular Cd distributions and concentrations in their bodies given their different metal-handling strategies and were exposed to dissolved Cd for two weeks before being fed to the whelks. After four weeks of dietary exposure, the Cd body concentrations in T. clavigera increased from 3.1 microg/g to between 22.9 and 41.8 microg/g and to between 22.7 and 24.1 microg/g when they were fed with oyster and snail prey, respectively. An increasing proportion of Cd was found to be distributed in the metallothionein (MT)-like proteins and organelle fractions, whereas the relative distribution in the metal-rich granules fraction decreased when the whelks were fed Cd-exposed prey. At the highest Cd dosage, more Cd was distributed in the pool of metal-rich granules when the whelks were fed the oysters than when they were fed the snails. Among all the biomarkers measured (MT induction, condition index, lipid peroxidation, and total energy reserve including carbohydrate, lipid, and protein), only MT showed a significant difference from the control treatments, and MT was the most sensitive biomarker for dietary Cd exposure. No toxicity was found in the whelks fed different Cd-exposed prey as revealed by various biomarkers at the different biological levels. Our results imply that metal fractionation in prey can alter the subsequent subcellular metal distribution in predators and that dietary Cd toxicity to the whelks was low, even when the accumulated Cd body concentrations were high.

Modeling of cadmium bioaccumulation in two populations of the green mussel Perna viridis.

This study attempted to quantify differences in Cd biokinetics from two populations of green mussels (Perna viridis) from two sites (eastern and western) in Hong Kong with contrasting hydrological conditions. Body Cd concentrations were modeled using a simple biokinetic model coupled with measurements of dissolved Cd concentrations at each site. Mussels collected from the western site had three to six times higher Cd tissue concentration than the eastern population collected during two seasons (summer wet and winter dry), but the salinity was only lower in the western site during the summer. More Cd was distributed in the metallothionein-like and heat-sensitive proteins in the western population than the eastern population, and Cd predominantly was distributed in the insoluble fraction during summer. The Cd uptake rate constant from the dissolved phase was higher in the western population during summer due to a much lower salinity, but was comparable during winter. Dietary uptake of Cd was similar in both populations, and assimilation was lower from ingested radiolabeled seston than from diatoms. Efflux of Cd remained comparable between the two populations from two seasons (0.02-0.03 /d). Kinetic modeling demonstrates that the faster influx of Cd from aqueous phase caused the higher body Cd concentrations in the western population. The predicted Cd concentrations in mussels were comparable to those observed in the field. Our study highlights differences in Cd accumulation kinetics in different populations of mussels likely caused by the different physical environments.

Dynamics of metal subcellular distribution and its relationship with metal uptake in marine mussels.

We examined the dynamics of subcellular distribution of metals (Cd, Ag, and Zn) in the marine green mussel Perna viridis by partitioning the metals into the insoluble fraction (IF), heat-sensitive proteins (HSP), and metallothionein-like proteins (MTLP) during metal uptake and elimination. Variations in metal uptake and elimination then were correlated with the subcellular distributions of these metals. The IF and HSP were the first ligands to bind with the metals during the dissolved exposure, and more metals were found in the HSP when the metal influx rate was higher. However, to minimize toxicity, metals were redistributed from HSP to MTLP afterwards. The subcellular distribution of metals was dependent of the exposure route in the mussels. During dietary metal exposure, the metals attained equilibrium before they were assimilated and the metal assimilation efficiency was independent of the metal partitioning in different subcellular fractions. During the efflux, metals in the soluble fraction mediated depuration, whereas metals in the insoluble fraction acted as a final storage pool. Redistribution also may occur between the metal-sensitive and inactive pools without significant depuration as a secondary protective mechanism. We further demonstrated that the higher efflux rate of Ag and Cd was related to a higher partitioning in the MTLP and a lower partitioning in the IF. Our study shows that subcellular pools other than MTLP were involved in immediate metal handling in the bivalves. The wide dynamics of subcellular metal distribution suggests that the relevance of individual subcellular fractions is dependent on the exposure pathway.