Internal redistribution of radiolabelled silver among tissues of rainbow trout (Oncorhynchus mykiss) and European eel (Anguilla anguilla): the influence of silver speciation

Influence of water Ag(I) speciation on pharmacokinetics of Ag(I) during a post-exposure period was investigated in rainbow trout (Oncorhynchus mykiss) and European eel (Anguilla anguilla). The rainbow trout is sensitive to waterborne ionic Ag(+) whereas the eel is tolerant. The fish were acclimated to either of two chloride concentrations, 10 or 1200 microM, in synthetic soft water and then exposed to a sublethal 24-h pulse of 1.3 microgl(-1) of 110mAg(I) added as 110mAgNO(3) in these waters. The protocol provided exposures to mainly the free ion Ag(+) (low chloride water) or mainly AgCl(aq) (high chloride water). Contents and concentrations of 110mAg(I) in tissues and body fluids were then monitored over a 67-day post-exposure period in Ag(I)-free water of the same chloride levels. Changing the speciation of Ag(I) in the water had no effect on the whole body load of 110mAg(I), but did result in differences in internal distribution. In trout, changing water Ag(I) speciation significantly altered elimination or accumulation of Ag(I) in several body compartments. Notably, trout exposed to AgCl(aq) eliminated 110mAg(I) from the kidney more quickly than trout exposed to Ag(I) primarily as Ag(+). This elimination was matched by higher concentrations of 110mAg in liver of trout exposed to Ag(I) primarily as AgCl(aq). In eel, shifting speciation from Ag(+) to AgCl(aq) hastened elimination of 110mAg(I) from mid and posterior intestine and increased 110mAg(I) retention in kidney. While there was no difference between the two fish species in whole body 110mAg(I) load, most internal body compartments of trout had higher 110mAg(I) concentrations than those in eel early in the experiment. Because tissue-specific elimination times were longer in eel than in trout, these differences were generally cancelled by the end of the 67-day depuration period. The only exception was the liver, which in trout continued to accumulate 110mAg(I) throughout the experiment but in eel remained unchanged. The combined effect of 110mAg(I) movements in the two species was that trout retained all their accumulated 110mAg(I) through the 67-day period, whereas the body burden of 110mAg(I) in eel was reduced to half initial values by day 67.     

A nose-to-nose comparison of the physiological effects of exposure to ionic silver versus silver chloride in the European eel (Anguilla anguilla) and the rainbow trout (Oncorhynchus mykiss)

Physiological mechanisms of silver toxicity (as silver nitrate) to the sensitive rainbow trout (Oncorhynchus mykiss) (96 h LC50: 10.2 μg silver l(-1), in soft, low chloride water) and the more tolerant European eel (Anguilla anguilla)(96 h LC50: 34.4 μg silver l(-1), in the same water) were investigated during acute exposure to silver, using concentrations varying from 3 to 22 μg silver l(-1). Silver was present either predominantly in the form of ionic silver, or in the form of silver chloride complexes (AgCl(aq)). Inhibition of the branchial Na(+),K(+)-ATPase enzyme activity and the active influx of Na(+) leading to net Na(+) loss were the key toxic effect in both species. In the rainbow trout, but not in the European eel, Cl(-) influx was also impaired during silver exposure. However, even under control conditions, Cl(-) influx was negligible in the eel. Water Cl(-) clearly protected against the silver-induced physiological disturbance in rainbow trout, presumably by changing the speciation of silver from ionic silver to AgCl complexes. However, such a protective effect was not observed in the European eel. Differences in whole body Na(+) turnover rates between the two species (1.1% per day in the European eel versus 19% per day in the rainbow trout) together with the lack of effect of silver exposure on Cl(-) homeostasis in the European eel are hypothesized to be the main reasons for the different silver tolerance observed in the two species.     

Bioaccumulation and distribution of silver in four marine teleosts and two marine elasmobranchs: influence of exposure duration, concentration, and salinity

The bioaccumulation of waterborne silver (added as AgNO(3)) was compared amongst drinking (teleosts: rainbow trout, tidepool sculpin, plainfin midshipmen, and English sole) and non-drinking marine fish (elasmobranchs: Pacific spiny dogfish and long nose skate) exposed to 14.5 μg/l Ag for 21 days in 30-ppt seawater. In addition, 21-day exposures were performed on trout, midshipmen, and sculpin at 0 (control), 1.5, 14.5, and 50 μg/l Ag to evaluate the effect of silver concentration, and on sculpins acclimated to 18 and 30 ppt salinity and sampled periodically up to 21 days to evaluate the effects of salinity and exposure duration. A 48-h acute exposure (250 μg/l Ag) was also carried out on sculpins at 10, 18, 24, and 30 ppt. The 1.5-and 14.5-μg/l Ag levels are of regulatory importance, but are several orders of magnitude higher than normal environmental levels. Silver uptake occurred in all exposures, but internal accumulations were less than proportional to exposure concentration (1.5-50.0 μg/l Ag), and tended to saturate over time, suggesting that physiological regulation occurred. Control (non-exposed) fish exhibited measurable levels of silver in all tissues (10-200 μg Ag/kg wet weight), suggesting that they accumulate silver from the natural environment throughout their lifetimes. After 21-day exposure to 14.5 μg/l Ag, silver levels increased 2-20-fold in most tissues of all species, with the greatest concentrations occurring in the livers of teleosts (order: liver>gills>/=intestines>white muscle) and the gills of elasmobranchs (order: gills>liver>white muscle>intestines). Rainbow trout accumulated more silver than the other teleosts, and were the only species to suffer significant mortality, effects likely associated with added salinity stress. Accumulations were fairly uniform amongst the other teleosts. Similar concentrations in gills and intestines suggested that both branchial and intestinal uptake occurred, with the latter potentially dominant; indeed sole exhibited no silver build-up in the gills. The two elasmobranchs exhibited no silver build-up in intestines but much higher levels in gills, indicating that in the absence of drinking, only branchial uptake occurs. Nevertheless, based on whole liver content, the elasmobranchs accumulated silver 5-15-fold faster than the three teleosts. Over 21-day exposures (1.5-50.0 μg/l Ag) in sculpin, salinity markedly affected silver accumulation, with tissue-specific levels approximately 6-fold higher at 18, than at 30 ppt. However, there was negligible effect of salinity on silver accumulation during 48 h at 250 μg/l Ag. Silver bioaccumulation appears to be markedly affected by speciation. At lower salinities, or higher [Ag], a neutral charged AgCl(aq) complex exists in the water, allowing for increased bioaccumulation to occur. At higher salinity, only less bioavailable, negatively-charged AgCl(n)(1-n) complexes are present (AgCl(2)(-), AgCl(3)(2-), AgCl(4)(3-)).     

The changes to apical silver membrane uptake, and basolateral membrane silver export in the gills of rainbow trout (Oncorhynchus mykiss) on exposure to sublethal silver concentrations

Juvenile rainbow trout acclimated to softwater were exposed to 0 or 8.3 nM Ag (added as silver nitrate) for 21 days. On days 1, 7 and 21 gill, kidney and liver levels of silver; branchial Na+ influx, efflux and net flux rate; gill and kidney K+ -dependent p-nitrophenol phosphatase activity; and gill and liver accumulation of "new" Ag were measured. In addition, the concentration-dependent uptake of Ag by gill basolateral membrane vesicles (BLMV) was assessed in control fish and those exposed to 8.3 nM Ag for 7 days. Ag induced a significant increase in Na+ efflux following 1 day of exposure that resulted in an increase in net loss of Na+ and a reduction in Na+ influx. By day 21 this perturbation to Na+ balance had been corrected, but kidney K+ -dependent p-nitrophenol phosphatase activity was significantly reduced. Unexpectedly, the Ag concentrations in the liver of Ag exposed fish only significantly increased (two-fold) following 7 days of exposure and were not elevated when compared to controls on day 21. In contrast, the gill and kidney accumulated significant concentrations of Ag (20-fold increase) following 7 days of exposure, and the Ag concentration in these tissues remained similar on day 21. The gills of Ag exposed fish accumulated significantly less "new" Ag than the controls on days 7 and 21 following exposure, suggesting a down-regulation of branchial Ag uptake. The BLMV of Ag exposed fish showed a significant increase in V(max) [control fish BLMV V(max) = 2811.9+/-190.8 pmol (110 m)Ag/(mg protein x min) and Ag exposed fish BLMV V(max) = 3688.3+/-659.8 pmol (110 m)Ag/(mg protein x min) (P = 0.033)], suggesting that they are able to increase export of Ag from the gills on exposure to Ag. The results from this study demonstrate a complex array of physiological processes that control the bioreactive concentrations of Ag in the gills, including: cytoplasmic sequestration, a down-regulation of apical entry and potentially an increase in basolateral membrane extrusion.     

Is Cl- protection against silver toxicity due to chemical speciation?

In freshwater teleosts, the primary mechanism of acute silver toxicity is inhibition of Na(+)/K(+) ATPase and carbonic anhydrase at the gill, leading to net Na(+) and Cl(-) loss due to the continued diffusion of these ions into the hypoosmotic external environment. External Cl(-) has been shown to protect rainbow trout (Oncorhychus mykiss) against silver toxicity presumably by complexation to form AgCl. However, Cl(-) does not appear to greatly influence silver toxicity to at least two other species, the European eel (Anguilla Anguilla) and the fathead minnow (Pimephales promelas). We hypothesized that differences in protective effects of Cl(-) at the gill were due to differing requirements or mechanisms for Cl(-) uptake among fish species. To test this hypothesis, we exposed Fundulus heteroclitus, which does not take up Cl(-) across the gills, and Danio rerio and P. promelas, which do rely on Cl(-) uptake across the gills, to Ag(+) in waters of varying Cl(-) concentration. The 96-h LC50s of F. heteroclitus exposed to Ag(+) in soft water with 10 microM Cl(-), 1mM KCl, and 0.5mM MgCl(2) were 3.88, 1.20, and 3.20 microg/L, respectively, and not significantly different. The 96-h LC50s for D. rerio exposed to Ag(+) in soft water with 10 microM Cl(-) and 1mM KCl were 10.3 and 11.3 microg/L, respectively and P. promelas exposed under the same conditions were 2.32 and 2.67 microg/L, respectively. Based on these results, increasing external Cl(-) concentration by as much as 1mM (35.5mg/L) did not offer protection against Ag(+) toxicity to any fish species tested. Although previous results in our laboratory have demonstrated that P. promelas do take up Cl(-) at the gill, a mechanism of uptake has not been identified. Additional experiments, investigating the mechanisms of Na(+) and Cl(-) influx at the gill of P. promelas and the influence of silver, demonstrated that Cl(-) uptake in P. promelas acclimated to soft water occurs through both a Na(+):K(+):2Cl(-) co-transporter and a Cl(-)/HCO(3)(-) exchanger, but is not dependent on carbonic anhydrase. Further, acclimation water chemistry was found to greatly influence subsequent branchial silver accumulation, but Cl(-) uptake was not sensitive to 10 microg/L Ag(+).     

Bioavailability of silver and its relationship to ionoregulation and silver speciation across a range of salinities in the gulf toadfish (Opsanus beta)

Silver is taken up as a Na(+) analog (Ag(+)) by freshwater organisms, but little is known about its bioavailability in relation to salinity. Adult Opsanus beta were acclimated to 2.5, 5, 10, 20, 40, 60, 80, and 100% seawater (Cl(-)=545 mM) and exposed for 24 h to 2.18 microg L(-1) silver as (110m)Ag-labelled AgNO(3), a concentration close to the U.S. EPA marine criterion and less than 0.1% of the acute 96-h LC50 in seawater. Plasma osmolality, Na(+), and Cl(-) remained approximately constant from 100% down to 20-40% seawater, thereafter declining to 89% (osmolality) and 82% (Na(+), Cl(-)) of seawater values at the lowest salinity (2.5% seawater), while plasma Mg(2+) was invariant. Ionic measurements in intestinal fluids and urine supported the view that above the isosmotic point (about 32% seawater), toadfish drink the medium, absorb Na(+), Cl(-), and water across the gastrointestinal tract, actively excrete Na(+) and Cl(-) across the gills, and secrete Mg(2+) into the urine. Below this point, toadfish appear to stop drinking, actively take up Na(+) and Cl(-) at the gills, and retain ions at the kidney. Silver accumulation varied greatly with salinity, by nine-fold (whole body), 26-fold (gill tissue), and 18-fold (liver), with the maxima occurring in 2.5% seawater, the minima in 40% seawater (close to the isosmotic point), and slightly greater values at higher salinities. Highest silver concentrations occurred in liver, second highest in gills, intermediate concentrations in kidney, spleen, and gastrointestinal tissues, and lowest in swim bladder and white muscle, though patterns changed with salinity. There were substantial biliary but minimal urinary levels of silver. The salinity-dependent pattern of silver accumulation best correlated with the abundance of the neutral complex AgCl(0), though the presence of small amounts of Ag(+) at the lowest salinities may also have been important. In contrast, silver accumulation in the esophagus-stomach was greatest in 100% seawater and least at the isosmotic salinity (five-fold variation), a pattern probably explained by drinking and silver uptake into the blood through the gills. Models of silver bioavailability across salinity must consider the presence of silver-binding ligands on both gills and gastrointestinal tract, changing silver speciation, and the changing ionoregulatory physiology of the organism.     

The time course of silver accumulation in rainbow trout during static exposure to silver nitrate: physiological regulation or an artifact of the exposure conditions?

The pattern of gill silver accumulation in rainbow trout during waterborne silver exposure has been reported to be unusual, reaching a peak in the first few hours of silver exposure followed by a marked decline with continued exposure. The potential causes of the pattern were investigated. Rainbow trout (1-5g) were exposed in a static system to 110mAg labeled AgNO(3) at a total concentration of 1.92microg Agl(-1) for 24h in synthetic soft water. Periodically throughout the exposure, gill and body 110mAg accumulation, gill and body 24Na uptake (from which whole body Na(+) uptake was calculated), gill Na(+)K(+)-ATPase activity, plus water silver (total and dissolved), Cl(-) and total organic carbon (TOC) concentrations were measured. Gill silver levels rapidly increased, peaked at 3h of exposure and then decreased until a plateau was reached at 12h of exposure. Body (minus gills) silver levels increased steadily over the exposure period until 18h of exposure. Whole body Na(+) uptake decreased, was maximally inhibited by 3h of exposure but recovered by 12h despite continued silver exposure. Gill Na(+)K(+)-ATPase activity was not inhibited until 5h of exposure. The water dissolved silver concentration declined by approximately 70% over the 24h exposure period and the TOC content of the water increased over three-fold during the first 2h of exposure. There was a decrease in the calculated contribution of Ag(+) (from 20.9 to 2.5%) and an increase in the calculated contribution of Ag-TOC complexes (from 77 to 97.3%) to the total water silver concentration over the first 2h of exposure. Apical silver uptake into the gills decreased over the initial 2.5h of exposure while basolateral silver export out of the gills to the body remained constant throughout the exposure. The results of this study suggest that: (1) physiological regulation of silver movement may explain the pattern of gill silver accumulation observed in rainbow trout, although not by a mechanism coupled to Na(+)K(+)-ATPase inhibition as originally proposed; (2) alternatively or additionally, a decreased bioavailability of silver, due to the static exposure conditions, may explain the pattern of gill accumulation; (3) the early inhibition of whole body Na(+) uptake observed during silver exposure occurs via a mechanism other than Na(+)K(+)-ATPase inhibition; and (4) gill silver accumulation may be an appropriate endpoint for biotic ligand modeling.     

Protection by two complexing agents, thiosulphate and dissolved organic matter, against the physiological effects of silver nitrate to rainbow trout (Oncorhynchus mykiss) in ion-poor water

Adult rainbow trout (Oncorhynchus mykiss) were exposed in ion-poor water ( approximately 50 microM Ca) to silver added as AgNO(3) or to AgNO(3) plus either thiosulphate (Na(2)S(2)O(3)) or dissolved organic matter (DOM). The effects of these exposures were assessed through repetitive blood sampling over 4 days. Trout exposed to 0.1 microM AgNO(3) alone accumulated large amounts of Ag on their gills and in their plasma, showed progressive losses of plasma Na and Cl, and had elevated concentrations of plasma glucose. In one set of exposures trout exposed to AgNO(3) alone also had increased cough rates, slightly higher ventilation rates, somewhat lower arterial oxygen tensions, and increased blood lactate concentrations. In contrast, trout exposed to 0.1 microM AgNO(3) plus 5 microM thiosulphate or 35 mg C l(-1) DOM accumulated less Ag on their gills and in their plasma, and showed no adverse ionoregulatory or respiratory effects due to Ag. These results demonstrate ionoregulatory and sometimes respiratory effects in fish exposed to ionic Ag(+) in ion-poor water, depending on water chemistry, and demonstrate the protective effects of synthetic and natural complexing agents through a reduction in the amount of ionic Ag(+) available to bind at the gills.     

The physiological effects of a biologically incorporated silver diet on rainbow trout (Oncorhynchus mykiss)

Silver was biologically incorporated into a diet by exposing rainbow trout for 7 days to 100 mg/l of waterborne silver as silver thiosulphate. These fish were processed into a fine powder (trout meal) and pelleted to form a nutritionally balanced feed which was then fed to juvenile rainbow trout (Oncorhynchus mykiss). Fish were fed either a diet containing 3.1 microg/g biologically incorporated silver (an environmentally relevant concentration), or one of three control diets containing approximately 0.05 microg/g Ag for 128 days. All dietary treatments were fed to satiation once daily. Dietary silver did not significantly affect mortality, growth, food consumption, or food conversion efficiency. Furthermore, ion regulation (plasma Na(+) levels and Na(+) influx rates), hematological parameters (hematocrit, plasma protein, hemoglobin levels), plasma glucose, metabolism (oxygen consumption, ammonia and urea excretion rates) and intestinal Na/K-ATPase and amylase activities were all unaffected. Based on the physiological parameters investigated here, this dietary silver exposure appeared to be physiologically benign to rainbow trout. However, silver concentrations in the livers of the silver-fed fish were significantly elevated at day 16, and reached a steady-state level of approximately 20 microg/g Ag by day 36. The concentration specific accumulation rate in the livers of fish fed biologically incorporated silver was about 4.6 orders of magnitude greater than when fed dietary silver sulfide, indicating much greater bioavailability. Despite this increase, hepatic metallothionein concentrations remained unchanged, in contrast to waterborne exposures, indicating that bioaccumulated silver behaves differently depending on whether it is taken up from the diet or from the water. Apart from a significant reduction in hepatic Cu at day 16, liver concentrations of Cu and Zn were not affected by dietary silver. Silver concentrations were also significantly elevated (relative to control fish) in the kidneys of the silver-treated fish on days 88 and 126, and in the gills and plasma at day 126.     

An in vitro biotic ligand model (BLM) for silver binding to cultured gill epithelia of freshwater rainbow trout (Oncorhynchus mykiss)

"Reconstructed" gill epithelia on filter supports were grown in primary culture from dispersed gill cells of freshwater rainbow trout (Oncorhynchus mykiss). This preparation contains both pavement cells and chloride cells, and after 7-9 days in culture, permits exposure of the apical surface to true freshwater while maintaining blood-like culture media on the basolateral surface, and exhibits a stable transepithelial resistance (TER) and transepithelial potential (TEP) under these conditions. These epithelia were used to develop a possible in vitro version of the biotic ligand model (BLM) for silver; the in vivo BLM uses short-term gill binding of the metal to predict acute silver toxicity as a function of freshwater chemistry. Radio-labeled silver ((110m)Ag as AgNO(3)) was placed on the apical side (freshwater), and the appearance of (110m)Ag in the epithelia (binding) and in the basolateral media (flux) over 3 h were monitored. Silver binding (greater than the approximate range 0-100 mug l(-1)) and silver flux were concentration-dependent with a 50% saturation point (apparent K(d)) value of about 10 mug l(-1) or 10(-7) M, very close to the 96-h LC50 in vivo in the same water chemistry. There were no adverse effects of silver on TER, TEP, or Na(+), K(+)-ATPase activity, though the latter declined over longer exposures, as in vivo. Silver flux over 3 h was small (<20%) relative to binding, and was insensitive to water chemistry. However, silver binding was decreased by elevations in freshwater Na(+) and dissolved organic carbon (humic acid) concentrations, increased by elevations in freshwater Cl(-) and reductions in pH, and insensitive to elevations in Ca(2+). With the exception of the pH response, these effects were qualitatively and quantitatively similar to in vivo BLM responses. The results suggest that an in vitro BLM approach may provide a simple and cost-effective way for evaluating the protective effects of site-specific waters.     

Influence of salinity and organic matter on silver accumulation in Gulf toadfish (Opsanus beta)

To help extend the freshwater based biotic ligand model for silver (Ag) into brackish and saltwater conditions, 50g Gulf toadfish (Opsanus beta) were acclimated to 2.5%, 5%, 10%, 20%, 40%, 80%, or 100% salt water and exposed for 6d to 1.0microM AgNO(3), with or without 10mg C/L organic matter. Suwannee River natural organic matter collected by reverse osmosis was used. Silver accumulation in toadfish gills and plasma decreased as salinity increased, indicating low bioavailability of AgCl complexes. Complexation of Ag by organic matter, normally important in freshwater conditions, was less important as salinity increased. Although relatively little intestinal Ag uptake was observed, both liver and bile accumulated Ag from water imbibed past the isosmotic salinity point ( approximately 1/3 salt water). Toadfish also produced intestinal carbonate pellets, minerals which did not influence Ag accumulation. Our results further stress the importance of Ag speciation, physiological mechanisms, and intestinal Ag uptake when modelling Ag uptake and toxicity beyond freshwater conditions.     

Acute and chronic physiological effects of silver exposure in three marine teleosts

This study evaluated the physiological effects of waterborne silver (added as AgNO(3)) on seawater fish, using acute (48-72 h) high level exposures (250-650 microg/l Ag) on tidepool sculpins (Oligocottus maculosus), and chronic (up to 21 day) low level exposures (1.5-50 microg/l Ag) on tidepool sculpins, plainfin midshipmen (Porichthys notatus), and rainbow trout (Oncorhynchus mykiss). Sculpins were tested at different salinities. Acclimation to lower salinity (18 vs 30 ppt) led to altered physiology, with higher ammonia excretion (J(Amm)), lower oxygen consumption, and lower branchial and intestinal Na(+)/K(+)-ATPase activities, but no difference in drinking rate. Short-term exposure to high silver levels tended to stimulate M(O(2)), J(Amm), and drinking rate. However, long-term exposure to low levels of silver depressed both J(Amm) and M(O(2)), and also led to decreased drinking rates. Both inhibition and stimulation of Na(+)/K(+)-ATPase activity occurred, dependent upon length and concentration of exposure, salinity (18 vs 30 ppt), tissue (gill vs intestine), and fish species (sculpin vs midshipmen vs rainbow trout). While the effects were variable, due to differing balances between inhibitory and compensatory responses, chronic silver exposure significantly altered Na(+)/K(+)-ATPase activity levels in almost all tests. In total, these findings reinforce the view that intestinal osmoregulatory function (drinking, Na(+)/K(+)-ATPase activity) is an important site of toxic impact for waterborne silver, that gill Na(+)/K(+)-ATPase activity is also a site of impact, and that chronic exposures at silver concentrations (1.5, 14.5 microg/l Ag) close to current or proposed water quality guidelines (albeit much higher than normal environmental levels), exert a variety of sublethal effects on marine teleosts.     

Silver accumulation in Daphnia magna in the presence of reactive sulfide

Previously, we demonstrated a higher silver body burden when Daphnia magna were exposed to silver in the presence of environmentally relevant concentrations (25 nM) of reactive sulfide, but the explanation was unclear. In the present study, D. magna were exposed to AgNO3 (0.93 microg Ag/L=8.6 nM as a mixture of cold Ag and (110m)Ag) in synthetic water in either the presence or absence of 25 nM sulfide as zinc sulfide clusters. After 1-h exposure, daphnids were transferred to clean water for up to 5-h depuration. At different times of Ag exposure and depuration, daphnids were randomly sampled for whole body silver burden. Also, after 1 h, daphnids were sampled for silver accumulation in "gills" (small organs on the thoracic appendages), digestive tract, and carcass. Other groups were exposed to the same silver and sulfide concentrations for 1 h and then sampled for whole-body autoradiography. Silver body burden was about two-fold higher in the presence of sulfide. A two-fold increase in silver burden in "gills" and digestive tract, but not in carcass, was also observed in the presence of sulfide. Absolute differences due to sulfide were greatest in digestive tract and explained most of the difference in whole body burden. Transfer to clean water caused a significant drop in silver concentration in whole body and all compartments to similar levels in the two groups after 5-h depuration. These results indicate that the higher silver body burden observed in the presence of sulfide is mainly due to sulfide-bound silver in the digestive tract of the daphnids. This conclusion is supported by autoradiography, which showed a high concentration of silver in the digestive tract of daphnids exposed to Ag/sulfide.     

Effects of water hardness on the physiological responses to chronic waterborne silver exposure in early life stages of rainbow trout (Oncorhynchus mykiss)

Early life stages of rainbow trout were exposed to 0, 0.1 and 1 microg/L Ag (as AgNO(3)) in very soft water (2mg/L CaCO(3)), moderately hard water (150 mg/L CaCO(3)) and hard water (400mg/L CaCO(3)) of low dissolved organic carbon concentration (0.5mg C/L) from fertilization to swim-up (64 days) under flow-through conditions, and monitored for whole embryo/larval silver accumulation, Na(+) and Cl(-) concentrations, Na(+) uptake and Na(+)K(+)-ATPase activity. The objective of the study was to investigate potential protective effects of water hardness on the physiological responses to chronic silver exposure. In the absence of silver, there was little effect of hardness on the ionoregulatory parameters studied, though higher hardness did improve survival post-hatch. At all three water hardness levels, whole embryo/larval Na(+) uptake was low and relatively constant prior to 50% hatch, but dramatically increased following 50% hatch, whereas Na(+)K(+)-ATPase activity steadily increased over development. Whole embryo/larval Na(+) and Cl(-) concentrations were low and constant prior to 50% hatch, but following 50% hatch Na(+) concentration increased, while Cl(-) concentration decreased. Following 50% hatch, exposure to 0.1 and 1 microg/L Ag resulted in a decrease in whole embryo/larval Na(+) concentration, Cl(-) concentration, Na(+) uptake and Na(+)K(+)-ATPase activity, indicating that the mechanism of chronic silver toxicity involves an ionoregulatory disturbance, and is similar to the mechanism of acute silver toxicity. An increase in water hardness reduced or eliminated the effect of silver on these parameters while enhancing survival, suggesting that the nature of the protective effect of hardness involves effects on the ionoregulatory disturbance associated with silver exposure. An increase in water hardness did not fully protect against the accumulation of silver associated with silver exposure. These results suggest that it may be possible to model chronic silver toxicity using a biotic ligand type model, and that a physiologically based model may be more appropriate because Na(+)K(+)-ATPase activity or Na(+) uptake is an endpoint for prediction rather than whole embryo or larval silver accumulation.     

Sensitivity of the spiny dogfish (Squalus acanthias) to waterborne silver exposure

The physiological effects of waterborne silver exposure (added as AgNO(3)) on spiny dogfish, Squalus acanthias, were evaluated at 30, 200 and 685 microg silver per l in 30 per thousand seawater. These concentrations cover the toxic range observed for freshwater teleosts, where silver is extremely toxic, to seawater teleosts which tolerate higher silver concentrations. However, these levels are considerably higher than those that occur in the normal environment. At 685 microg l(-1), dogfish died within 24 h. Causes of death were respiratory as well as osmoregulatory failure. Arterial P(a)O(2) rapidly declined below 20 Torr, and blood acidosis (both respiratory and metabolic) occurred. Urea excretion increased dramatically and plasma urea dropped from 340 to 225 mM. There were pronounced increases in plasma Na(+), Cl(-), and Mg(2+), indicative of ionoregulatory failure due to increased diffusive permeability as well as inhibited NaCl excretion. At 200 microg l(-1), fish died between 24 and 72 h of silver exposure. The same physiological events occurred with a small time delay. At 30 microg l(-1), effects were much less severe, although slight mortality (12.5%) still occurred. Respiratory alkalosis occurred, together with moderate elevations in plasma Na(+) and Cl(-) levels. Silver accumulated to the highest concentrations on gills, with only low levels in the intestine, in accord with the virtual absence of drinking. Na(+)/K(+)-ATP-ase activities of gill and rectal gland tissue were impaired at the highest silver concentration. Normal gill function was impaired due to swelling and fusion of lamellae, lamellar aneurism and lifting of the lamellar epithelium. Our results clearly indicate that this elasmobranch is much more sensitive (about 10-fold) to silver than marine teleosts, with silver's toxic action exerted on the gill rather than on the intestine, in contrast to the latter.     

Depuration and anatomical distribution of the amnesic shellfish poisoning (ASP) toxin domoic acid in the king scallop Pecten maximus

The depuration kinetics of the domoic acid of four body fractions (digestive gland, adductor muscle, gonad+kidney and gills+mantle) of the scallop Pecten maximus was studied over 295 days. The scallops, which had acquired the toxins during a Pseudo-nitzschia australis episode that took place the week before the beginning of the experiment, were maintained in tanks with running seawater. All the body fractions, except the adductor muscle, decreased their domoic acid burden throughout the experiment. The amount of toxin in the muscle dropped sharply at the start of the experiment but increased again at the end, to levels that were higher than the initial ones. Several dynamic models of depuration kinetics, which included the depuration of each fraction (excluding the adductor muscle) and the transfers between them, were constructed, implemented and fitted to the data to obtain their parameters. The estimated depuration rates were very low, both considering and not considering the transfer of toxin between organs or the effect of weight loss. There were strong differences in the domoic acid burden of the body fractions studied but not between their depuration rates. No net transfer from the digestive gland, the tissue with highest domoic acid concentration, to the other fractions was found, as the inclusion of these processes in the models produced only a marginally better fit to the data. The depuration of domoic acid was slightly, but significantly, affected by biomass. Weight loss induced domoic acid loss, suggesting that part of the depuration may be produced by the direct loss of bivalve cells. The concentration or dilution effect, due to decreases or increases in biomass, documented for other species and toxins, has little importance in Pecten maximus.     

Toxicity of silver to two freshwater algae, Chlamydomonas reinhardtii and Pseudokirchneriella sub-capitata, grown under continuous culture conditions: influence of thiosulphate

In a test of the biotic ligand model (BLM), the uptake and toxicity of silver, in the absence or presence of the inorganic ligand, thiosulphate, were assessed for two freshwater green algae, Chlamydomonas reinhardtii and Pseudokirchneriella sub-capitata, using turbidostat continuous cultures. In the initial experiments, run in the absence of thiosulphate, the influent Ag concentration was varied from 0 to 75 nM in steps; for each influent concentration, silver uptake was calculated and the algal growth rate was determined. Silver uptake rates at low Ag concentrations were similar for both algae (e.g., 14-19 nmolm(-2)h(-1), for influent Ag(+) concentrations of approximately 9 nM) but at higher exposures uptake by P. sub-capitata exceeded that of C. reinhardtii. Despite this higher uptake rate, in the absence of thiosulphate P. sub-capitata was not more sensitive to free silver; 50% growth inhibition was reached at influent free Ag(+) concentrations of 15+/-7 and 22+/-13 nM for C. reinhardtii and P. sub-capitata, respectively. In the second series of experiments, the free Ag(+) concentration was held constant ( approximately 9 nM in the influent; 2-3 nM in the effluent) while the concentration of the silver thiosulphate complex, AgS(2)O(3)(-), was increased from 9 to 90 nM in steps. Under such conditions, the BLM would predict that silver uptake and toxicity should remain constant. On the contrary, both silver uptake and silver toxicity increased, indicating that the anionic silver thiosulphate complex enters the algal cells via a membrane-bound sulphate transporter and contributes to uptake and toxicity. However, for both algae there were indications that silver assimilated in this manner was somewhat less toxic to the algal cell than silver that entered via cation transport only. Physiological indicators of stress revealed possible different intracellular targets for these two freshwater algae, proteins and enzymes for C. reinhardtii and the photosynthetic apparatus for P. sub-capitata.     

Reduced cytotoxicity of silver ions to mammalian cells at high concentration due to the formation of silver chloride

Silver-containing antimicrobial agents are used in various medical products. However, their toxicity to mammalian cells has not been sufficiently evaluated. Numerous studies have unveiled evidence of significant antimicrobial properties associated with Ag ions. In cell culture media or human body fluids, the free Ag⁺ has rich opportunities to complex with Cl^?. Surprisingly, studies on the toxicity of solid form AgCl_(s) to mammalian cells are quite limited. In this study, we evaluated the cytotoxicity of Ag ions and silver chloride colloids on red blood cells and human mesenchymal stem cells (hMSCs). The adverse effects of silver chloride on red blood cells and hMSC were viewed by SEM and LIVE/DEAD viability staining, respectively. Among different tested chemical forms of silver, AgCl was identified to be the least cytotoxic. Moreover, a decline in the cytotoxicity of AgCl at significantly high concentrations was observed. We attributed the reduced cytotoxicity to aggregated AgCl which limited the bioavailability of free Ag⁺ ions.     

Contrasting effects of chloride on the toxicity of silver to two green algae, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii

Short-term silver toxicity was determined for two freshwater algae, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii, in the presence and absence of chloride. Silver speciation in the exposure media was controlled and algal growth was measured over 6 h. For P. subcapitata, an alga with low Ag uptake fluxes, silver toxicity could be predicted on the basis of the free Ag+ concentration, in the presence or absence of significant complexation by chloride ions, as predicted by the biotic ligand model (BLM). For C. reinhardtii, an alga with high Ag uptake fluxes, silver toxicity was better predicted by the concentration of all labile dissolved Ag species than by free silver, a result that is consistent with diffusion through the unstirred layer surrounding the cell surface being the rate-limiting step in silver uptake. For both species, growth inhibition could be predicted on the basis of the Ag intracellular quota in the presence or absence of chloride, indicating that silver toxicity is a direct result of intracellular accumulation rather than cell surface interactions.     

Uptake and effects of manufactured silver nanoparticles in rainbow trout (Oncorhynchus mykiss) gill cells

Nanoparticles are already widely used in technology, medicine and consumer products, but there are limited data on their effects on the aquatic environment. In this study the uptake and effect of citrate (AgNP_CIT) and polyvinylpyrrolidone (AgNP_PVP) coated manufactured silver nanoparticles, as well as AgNO₃ (Ag⁺) were tested using primary gill cells of rainbow trout (Oncorhynchus mykiss). Prior to use, the nanoparticles were characterized for size, surface charge and aggregation behavior. Gill cells were cultured either as monolayers on solid support, or as multilayers on a permeable support cell culturing system, enabling transport studies. The uptake of silver nanoparticles and Ag⁺ after exposure to 10 mg L⁻¹ was determined with microscopical methods and inductively coupled plasma mass spectrometry (ICP-MS). Cytotoxicity, in terms of membrane integrity, as well as oxidative stress (depletion of reduced glutathione) was tested at silver concentrations ranging from 0.1 mg L⁻¹ to 10 mg L⁻¹. Results show that AgNP_CIT nanoparticles are readily taken up into gill cell monolayers while uptake was less for AgNP_PVP. In contrast, it appears that the slightly smaller AgNP_PVP were transported through cultured multilayers to a higher extent, with transport rates generally being in the ng cm⁻² range for 48 h exposures. Transport rates for all exposures were dependent on the epithelial tightness. Moderate cytotoxic effects were seen for all silver treatments. Levels of reduced glutathione were elevated in contrast to control groups, pointing on a possible overcompensation reaction. Taken together silver nanoparticles were taken up into cells and did cause silver transport over cultured epithelial layers with uptake and transport rates being different for the two nanoparticle species. All silver treatments had measurable effects on cell viability.