Sunita R. Nadella

Toxicity of dissolved Cu, Zn, Ni and Cd to developing embryos of the blue mussel (Mytilus trossolus) and the protective effect of dissolved organic carbon.

Marine water quality criteria for metals are largely driven by the extremely sensitive embryo-larval toxicity of Mytilus sp. Here we assess the toxicity of four dissolved metals (Cu, Zn, Ni, Cd) in the mussel Mytilus trossolus, at various salinity levels while also examining the modifying effects of dissolved organic carbon (DOC) on metal toxicity. In 48 h embryo development tests in natural seawater, measured EC50 values were 6.9-9.6 microg L(-1) (95% C.I.=5.5-10.8 microg L(-1)) for Cu, 99 microg L(-1) (86-101) for Zn, 150 microg L(-1) (73-156) for Ni, and 502 microg L(-1) (364-847) for Cd. A salinity threshold of >20 ppt (approximately 60% full strength seawater) was required for normal control development. Salinity in the 60-100% range did not alter Cu toxicity. Experimental addition of dissolved organic carbon (DOC) from three sources reduced Cu toxicity; for example the EC50 of embryos developing in seawater with 20 mg C L(-1) was 39 microg Cu L(-1) (35.2-47.2) a 4-fold increase in Cu EC50. The protective effects of DOC were influenced by their distinct physicochemical properties. Protection appears to be related to higher fulvic acid and lower humic acid content as operationally defined by fluorescence spectroscopy. The fact that DOC from freshwater sources provides protection against Cu toxicity in seawater suggests that extrapolation from freshwater toxicity testing may be possible for saltwater criteria development, including development of a saltwater Biotic Ligand Model for prediction of Cu toxicity.

The relative sensitivity of sperm, eggs and embryos to copper in the blue mussel (Mytilus trossulus).

Copper, an essential element, is toxic at elevated concentrations, and as a result of anthropogenic activities is becoming increasingly prevalent in marine environments. In this study, we examined the effects of copper on early life stages of the blue mussel, Mytilus trossulus. We assessed the impacts of increasing copper concentrations on embryo development, egg viability, sperm fertilization capacity and, in particular, on sperm swimming speed using computer-assisted sperm analysis. Sensitivity to copper followed the pattern: embryos > sperm > eggs. A dramatic increase in abnormal embryo development was observed following exposure to copper concentrations exceeding 10 microg/L. Sperm swimming speeds decreased significantly when exposed to 100 microg/L of copper, but lower doses did not influence sperm swimming speed. Copper exposure (at any tested concentration) did not affect sperm flagellum length, or alter egg viability. Based on our results, we suggest that exposure of sperm to copper may interfere with mitochondrial activity, which reduces sperm swimming speed during the extended duration of sperm motility in blue mussel.

Mechanisms of dietary Cu uptake in freshwater rainbow trout: evidence for Na-assisted Cu transport and a specific metal carrier in the intestine

Copper (Cu) is both a vital nutrient and a potent toxicant. The objective of this study was to analyze the mechanistic nature of intestinal Cu transport in rainbow trout using radiolabeled Cu (64Cu) and an in vitro gut sac technique. Reduction of mucosal NaCl levels inhibited Cu transport while increase caused stimulation; Na2SO4 had an identical effect, implicating Na+ rather than the anion. These responses were unrelated to solvent drag, osmotic pressure or changes in transepithelial potential. The presence of elevated luminal Ag stimulated Cu and Na+ uptake. Phenamil caused a partial inhibition of both Cu and Na+ uptake while hypercapnia stimulated Na+ and Cu transport. Cu uptake was sensitive to luminal pH and inhibited by a tenfold excess of Fe and Zn. These factors had no effect on Na+ uptake. On the basis of these results we propose a novel Na+-assisted mechanism of Cu uptake wherein the Na+ gradient stimulates an increase in the H+ concentration of the brushborder creating a suitable microenvironment for the effective transport of Cu via either DMT1 or Ctr1.

Post-prandial metabolic alkalosis in the seawater-acclimated trout: the alkaline tide comes in.

SUMMARY The consequences of feeding and digestion on acid–base balance and regulation in a marine teleost (seawater-acclimated steelhead trout; Oncorhynchus mykiss) were investigated by tracking changes in blood pH and [HCO3–], as well as alterations in net acid or base excretion to the water following feeding. Additionally the role of the intestine in the regulation of acid–base balance during feeding was investigated with an in vitro gut sac technique. Feeding did not affect plasma glucose or urea concentrations, however, total plasma ammonia rose during feeding, peaking between 3 and 24 h following the ingestion of a meal, three-fold above resting control values (∼300 μmol ml–1). This increase in plasma ammonia was accompanied by an increase in net ammonia flux to the water (∼twofold higher in fed fish versus unfed fish). The arterial blood also became alkaline with increases in pH and plasma [HCO3–] between 3 and 12 h following feeding, representing the first measurement of an alkaline tide in a marine teleost. There was no evidence of respiratory compensation for the measured metabolic alkalosis, as PaCO2 remained unchanged throughout the post-feeding period. However, in contrast to an earlier study on freshwater-acclimated trout, fed fish did not exhibit a compensating increase in net base excretion, but rather took in additional base from the external seawater, amounting to ∼8490 μmol kg–1 over 48 h. In vitro experiments suggest that at least a portion of the alkaline tide was eliminated through increased HCO3– secretion coupled to Cl– absorption in the intestinal tract. This did not occur in the intestine of freshwater-acclimated trout. The marked effects of the external salinity (seawater versus freshwater) on different post-feeding patterns of acid–base balance are discussed.

Sublethal mechanisms of Pb and Zn toxicity to the purple sea urchin (Strongylocentrotus purpuratus) during early development

In order to understand sublethal mechanisms of lead (Pb) and zinc (Zn) toxicity, developing sea urchins were exposed continuously from 3h post-fertilization (eggs) to 96 h (pluteus larvae) to 55 (±2.4) μgPb/L or 117 (±11)μgZn/L, representing ~ 70% of the EC50 for normal 72 h development. Growth, unidirectional Ca uptake rates, whole body ion concentrations (Na, K, Ca, Mg), Ca(2+) ATPase activity, and metal bioaccumulation were monitored every 12h over this period. Pb exhibited marked bioaccumulation whereas Zn was well-regulated, and both metals had little effect on growth, measured as larval dry weight, or on Na, K, or Mg concentrations. Unidirectional Ca uptake rates (measured by (45)Ca incorporation) were severely inhibited by both metals, resulting in lower levels of whole body Ca accumulation. The greatest disruption occurred at gastrulation. Ca(2+) ATPase activity was also significantly inhibited by Zn but not by Pb. Interestingly, embryos exposed to Pb showed some capacity for recovery, as Ca(2+)ATPase activities increased, Ca uptake rates returned to normal intermittently, and whole body Ca levels were restored to control values by 72-96 h of development. This did not occur with Zn exposure. Both Pb and Zn rendered their toxic effects through disruption of Ca homeostasis, though likely through different proximate mechanisms. We recommend studying the toxicity of these contaminants periodically throughout development as an effective way to detect sublethal effects, which may not be displayed at the traditional toxicity test endpoint of 72 h.

Characterization of dietary Ni uptake in the rainbow trout, Oncorhynchus mykiss

We characterized dietary Ni uptake in the gastrointestinal tract of rainbow trout using both in vivo and in vitro techniques. Adult trout were fed a meal (3% of body mass) of uncontaminated commercial trout chow, labeled with an inert marker (ballotini beads). In vivo dietary Ni concentrations in the supernatant (fluid phase) of the gut contents averaged from 2 micromoll(-1) to 24 micromoll(-1), and net overall absorption efficiency of dietary Ni was approximately 50% from the single meal, similar to that for the essential metal Cu, adding to the growing evidence of Ni essentiality. The stomach and mid-intestine emerged as important sites of Ni uptake in vivo, accounting for 78.5% and 18.9% of net absorption respectively, while the anterior intestine was a site of net secretion. Most of the stomach uptake occurred in the first 4h. In vitro gut sac studies using radiolabeled Ni (at 30 micromoll(-1)) demonstrated that unidirectional uptake occurred in all segments, with area-weighted rates being highest in the anterior intestine. Differences between in vivo and in vitro results likely reflect the favourable uptake conditions in the stomach, and biliary secretion of Ni in the anterior intestine in vivo. The concentration-dependent kinetics of unidirectional Ni uptake in vitro were biphasic in nature, with a saturable Michaelis-Menten relationship observed at 1-30 micromoll(-1) Ni (K(m) - 11 micromoll(-1), J(max) - 53 pmolcm(-2)h(-1) in the stomach and K(m) - 42 micromoll(-1), J(max) - 215 pmolcm(-2)h(-1) in the mid-intestine), suggesting mediation by a channel or carrier process. A linear uptake relationship was seen at higher concentrations, indicative of simple diffusion. Ni uptake (at 30 micromoll(-1)) into the blood compartment was significantly reduced in the stomach by high Mg (50 mmoll(-1)), and in the mid-intestine by both Mg (50 mmoll(-1)) and Ca (50 mmoll(-1)). In both regions, kinetic analysis demonstrated reductions in J(max) with unchanged K(m), suggesting non-competitive interactions. Therefore the Mg and Ca content of the food will be an important consideration affecting the availability of Ni.

Toxicity of lead and zinc to developing mussel and sea urchin embryos: Critical tissue residues and effects of dissolved organic matter and salinity

Lead (Pb) EC50 values in the very sensitive early development phases (48-72h post-fertilization) of the mussels Mytilus galloprovincialis and Mytilus trossolus and sea urchin Strongylocentrotus purpuratus in 100% sea water were: M. trossolus - 45 (95% C.I.=22-72) μgL(-1); M. galloprovincialis - 63 (36-94) μgL(-1); S. purpuratus - 74 (50-101) μgL(-1). Salinity thresholds for normal development varied: M. trossolus>21ppt; M. galloprovincialis>28ppt; S. purpuratus≥30ppt. Addition of two spectroscopically distinct dissolved organic matters (DOM) from fresh water (Nordic Reservoir) and sea water (Inshore) moderately decreased the toxicity of Pb to both mussels, but not in a concentration-dependent fashion, with only an approximate doubling of EC50 over the range of 1.4-11.2mgCL(-1). Independent Pb binding capacity determinations for DOC explained the lack of a relationship between DOM concentration and toxicity. Salinity had no effect on Pb toxicity down to 21ppt in M. trossolus, and low salinity (21ppt) did not enhance the protective effect of DOC. Both DOMs increased the toxicity of Pb in developing sea urchin embryos, in contrast to mussels. Relative to Pb, the organisms were 6-9 fold less sensitive to Zn on a molar basis in 100% seawater with the following Zn EC50s: M. trossolus - 135 (103-170) μgL(-1); M. galloprovincialis - 172 (126-227) μgL(-1), S. purpuratus - 151 (129-177) μgL(-1). Nordic Reservoir and Inshore DOM (2-12mgCL(-1)) had no significant effect on Zn toxicity to mussels, in accord with voltammetry data showing an absence of any strong ligand binding for Zn by DOMs. As with Pb, DOMs increased Zn toxicity to urchin larvae. Critical Tissue Residues (CTR) based on whole body concentrations of Pb and Zn were determined for M. galloprovincialis at 48h and S. purpuratus at 72h. The median lethal CTR values (LA50s), useful parameters for development of saltwater Biotic Ligand Models (BLMs), were approximately 4-fold higher on a molar basis for Zn than for Pb. The latter were not altered by DOM exposure, despite increased EC50 values, in accord with the tenets of the BLM.

Interactive effects of waterborne metals in binary mixtures on short-term gill-metal binding and ion uptake in rainbow trout (Oncorhynchus mykiss).

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.

Zinc bioaccumulation and ionoregulatory impacts in Fundulus heteroclitus exposed to sublethal waterborne zinc at different salinities

Exposure of Fundulus heteroclitus to an environmentally relevant Zn concentration (500 μg L⁻¹) at different salinities (0, 3.5, 10.5, and 35 ppt) revealed the following effects: (i) plasma [Zn] doubled after exposure at 0 ppt, a response which was eliminated at 35 ppt. Tissue [Zn] also increased in gill, liver, intestine, and carcass at 0 ppt. (ii) Both branchial and intestinal Ca2⁺ ATPase activities decreased in response to Zn at 0 ppt and were elevated at 35 ppt. Plasma [Ca] decreased by 50% at 0 ppt and by 30% at 3.5 ppt and increased by 20% at 35 ppt. Gill [Ca] decreased by 35% at 0 ppt and increased by about 30% at all higher salinities. (iii) Branchial Na⁺,K⁺ ATPase activity decreased by 50% at 0 ppt, increased by 30% and 90% at 10.5 and 35 ppt respectively. Intestinal Na⁺,K⁺ ATPase activity was reduced by 30% at 0 ppt. (iv) Plasma [Na] decreased by 30% at 0 ppt in Zn-exposed. Zn exposure also disturbed the homeostasis of tissue cations (Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺) in a tissue-specific and salinity-dependent manner. (v) Drinking rate was not altered by Zn exposure. In toxicity tests, acute Zn lethality (96-h LC50) increased in a close to linear fashion from 9.8 mg L⁻¹ at 0 ppt to 75.0 mg L⁻¹ at 35 ppt. We conclude that sublethal Zn exposure causes pathological changes in both Ca⁺⁺ and Na⁺ homeostases, and that increasing salinity exerts protective effects against both sublethal and lethal Zn toxicities.

Ionic status, calcium uptake, and Ca2 +-ATPase activity during early development in the purple sea urchin (Strongylocentrotus purpuratus)

Ionic status during early development was investigated in the purple sea urchin. Whole body cation concentrations (Ca(2+), Na(+), K(+), Mg(2+)), unidirectional Ca(2+) uptake rates measured with (45)Ca(2+), Ca(2+)-ATPase activity, and growth were examined at 12h intervals over the first 96h of development. Whole body Ca(2+) concentration was low initially but increased steadily by >15-fold through to the pluteus stage. Whole body Mg(2+), K(+) and Na(+) levels exhibited diverse patterns, but all increased at 72-96h. Ca(2+) uptake rates were low during initial cell cleavages at 12h but increased greatly at blastulation (24h) and then again at gastrulation (48h), declining thereafter in the pluteus stage, but increasing slightly at 96h. Ca(2+)-ATPase activity was initially low but increased at blastulation through gastrulation (24-48h) but declined thereafter in the pluteus stage. Embryonic weights did not change over most of development, but were significantly higher at 96h. Overall, the gastrulation stage displayed the most pronounced changes, as Ca(2+) uptake and accumulation and Ca(2+)-ATPase levels were the highest at this stage, likely involved in mineralization of the spicule. Biomarkers of Ca(2+) metabolism may be good endpoints for potential future toxicity studies.