The effect of food on the acute toxicity of silver nitrate to four freshwater test species and acute-to-chronic ratios.

Acute silver toxicity studies were conducted with and without food for four common freshwater test species: Daphnia magna, Ceriodaphnia dubia, Pimephales promelas (fathead minnow-FHM), and Oncorhynchus mykiss (rainbow trout-RBT) in order to generate acute-to-chronic ratios (ACR). The studies were conducted similarly (i.e., static-renewal or flow-through) to chronic/early-life stage studies that were previously performed in this laboratory. The acute toxicity (EC/LC50 values) of silver without food ranged from 0.57 µg dissolved Ag/l for C.dubia to 9.15 µg dissolved Ag/l for RBT. The presence of food resulted in an increase in EC/LC50 values from 1.25× for RBT to 22.4× for C. dubia. Invertebrate food type was also shown to effect acute silver toxicity. Food did not affect EC/LC50s or ACRs as greatly in fish studies as in invertebrate studies. ACRs for both invertebrate species were <1.0 when using acute studies without food but were 1.22 and 1.33 when using acute studies with food. ACRs for FHMs ranged from 4.06 to 7.19, while RBT ACRs ranged from 28.6 to 35.8 depending on whether food was present in acute studies. The data generated from this research program should be useful in re-determining a final ACR for silver in freshwater as well as in risk assessments.

Chronic toxicity of silver nitrate to Ceriodaphnia dubia and Daphnia magna, and potential mitigating factors.

We investigated the chronic toxicity of Ag, as silver nitrate, using two freshwater aquatic cladoceran species, Ceriodaphnia dubia and Daphnia magna, to generate data for the development of a chronic ambient water quality criterion for Ag. Preliminary studies with C. dubia showed variable results which were related to the equilibration time between food and silver. Follow-up testing was conducted using a 3h equilibration time, which stabilized dissolved Ag concentrations and the toxicity of Ag(+). Results with C. dubia conducted individually (1 per cup, n=10) and in mass (30 per chamber, n=2) gave similar results once similar standardized equilibration times were used. The maximum acceptable toxicant concentration (MATC) of Ag to C. dubia and D. magna was 9.61 and 3.00microg dissolved Ag/L, respectively. The chronic toxicity of Ag(+) to C. dubia was also evaluated in the presence of: (1) dissolved organic carbon (DOC) and (2) sulfide. The addition of DOC (0.4mg/L) resulted in a approximately 50% decrease in toxicity while the addition of sulfide (75.4nM) deceased toxicity by 42%. Whole-body Ag concentration in D. magna was positively correlated with increased levels of Ag exposure, however; we observed a non-statistical decrease in whole-body Na levels, an estimator of sodium homeostasis.

Reprint of "Chronic toxicity of silver nitrate to Ceriodaphnia dubia and Daphnia magna, and potential mitigating factors".

We investigated the chronic toxicity of Ag, as silver nitrate, using two freshwater aquatic cladoceran species, Ceriodaphnia dubia and Daphnia magna, to generate data for the development of a chronic ambient water quality criterion for Ag. Preliminary studies with C. dubia showed variable results which were related to the equilibration time between food and silver. Follow-up testing was conducted using a 3 h equilibration time, which stabilized dissolved Ag concentrations and the toxicity of Ag(+). Results with C. dubia conducted individually (1 per cup, n=10) and in mass (30 per chamber, n=2) gave similar results once similar standardized equilibration times were used. The maximum acceptable toxicant concentration (MATC) of Ag to C. dubia and D. magna was 9.61 and 3.00 microg dissolved Ag/L, respectively. The chronic toxicity of Ag(+) to C. dubia was also evaluated in the presence of: (1) dissolved organic carbon (DOC) and (2) sulfide. The addition of DOC (0.4 mg/L) resulted in a approximately 50% decrease in toxicity while the addition of sulfide (75.4 nM) deceased toxicity by 42%. Whole-body Ag concentration in D. magna was positively correlated with increased levels of Ag exposure, however; we observed a non-statistical decrease in whole-body Na levels, an estimator of sodium homeostasis.

Comparison of short-term chronic and chronic silver toxicity to fathead minnows in unamended and sodium chloride-amended waters.

The chronic (early life stage [ELS]) and short-term chronic (STC) toxicity of silver (as silver nitrate) to fathead minnows (FHM) was determined concurrently in flow-through exposures (33 volume additions/d). Paired ELS (approximately 30 d) and STC (7 d) studies were conducted with and without the addition of 60 mg/L Cl (as NaCl). The paired studies in unamended water were later repeated using standard flow conditions (9 volume additions/d). The purpose of the paired studies was to determine if short-term chronic endpoints can be used to predict effects in ELS studies. For each experiment, a "split-chamber" design (organisms were held in a common exposure chamber) allowed the direct comparison between short-term and chronic exposures. It appeared that the chronic toxicity of silver was mitigated to some extent by NaCl addition. The maximum acceptable toxicant concentration for growth in the ELS study was 0.53 microg dissolved Ag/L under standard flow conditions. Early life stage and STC endpoints in all three studies typically agreed within a factor of two. Whole-body sodium and silver concentrations measured in individual fathead minnows during these studies showed an increase in silver body burdens and a decrease in sodium concentration. These results indicate that the STC study could be used as a surrogate test to estimate chronic toxicity and that the mechanism of chronic silver toxicity may be the same as for acute toxicity.

Chronic toxicity of silver to the sea urchin (Arbacia punctulata).

The chronic toxicity of silver to the sea urchin (Arbacia punctulata) was determined in 30 per thousand salinity seawater during a three-part study: A fertilization test (1-h sperm exposure), a 48-h embryo test, and a 30-d adult test. Combined data from the three tests resulted in a lowest-observed-effect concentration of 19 microg/L, a no-observed-effect concentration of 8.6 microg/L, and a maximum acceptable toxicant concentration of 13 microg/L, based on measured concentrations of dissolved silver. The 96-h median effective concentration was 40 microg/L, and the acute to chronic toxicity ratio was 3.1. During the tests, measured concentrations of free ionic silver (Ag+) were only 0.0027 to 0.0046% of dissolved silver concentrations, as predicted by ion-speciation theory. Some measured Ag+ concentrations were lower than predicted, indicating the presence of other ligands in the seawater test media. These strong sulfide ligands were exuded by the exposed sea urchins into the seawater (where Ag-sulfide complexes formed) in amounts that increased in direct proportion to the silver concentration during the toxicity test. This suggests a toxicity-defense mechanism that functioned by modifying the chemistry of the surrounding external medium.

Influence of salinity and organic carbon on the chronic toxicity of silver to mysids (Americamysis bahia) and silversides (Menidia beryllina).

Tests were conducted with mysids (Americamysis bahia) and silversides (Menidia beryllina) to evaluate the influence of salinity and organic carbon on the chronic toxicity of silver. During 7- and 28-d tests conducted at 10, 20, and 30% per hundred salinity, higher concentrations of dissolved silver generally were required to cause a chronic effect as the salinity of the seawater was increased. The 28-d mysid and silverside 20%-effective concentration values (expressed as dissolved silver) ranged from 3.9 to 60 and from 38 to 170 microg/L, respectively, over the salinity range. This pattern was not observed when the same test results were evaluated against the concentrations of free ionic silver (measured directly during toxicity tests), as predicted by the free-ion activity model. Increasing the concentration of dissolved organic carbon from 1 mg/L to the apparent maximum achievable concentration of 6 mg/L in seawater caused a slight decrease in chronic toxicity to silversides but had no effect on the chronic toxicity to mysids. The possible additive toxicity of silver in both food and water also was investigated. Even at the maximum achievable foodborne concentration, the chronic toxicity of silver added to the water was not affected when silver was also added to the food, based on the most sensitive endpoint (growth). However, although fecundity was unaffected at all five tested concentrations during the test with silver in water only, it was significantly reduced at the two highest waterborne silver concentrations (12 and 24 microg/L) during the test with silver dosed into food and water.

Evaluation of the protective effects of reactive sulfide on the acute toxicity of silver to rainbow trout (Oncorhynchus mykiss).

Acute 96-h toxicity tests were performed with juvenile rainbow trout (Oncorhynchus mykiss) exposed to AgNO3 in either the absence or the presence of 100 nM reactive sulfide to evaluate the protective effect of aqueous sulfides against ionic Ag toxicity. The sulfide was presented in the form of zinc sulfide (ZnS) clusters under oxic conditions. Silver was lost from the water column during the course of the experiment, so mean measured Ag concentrations were used to generate all median lethal concentration (LC50) data. The system was complicated in that Ag2S precipitated because of the need for large amounts of Ag to obtain lethal effects in the presence of ZnS. Some of the losses of Ag could be explained by complexation with ZnS and formation of solid Ag2S. Other losses were probably the result of partial adsorption to exposure-chamber walls or to complexation with ligands or functional groups within organic material produced by the fish. The LC50 (95% confidence interval) values generated using measured concentrations for total Ag were 139 (122-162) nM in the absence of sulfide and 377 (340-455) nM in the presence of 100 nM sulfide. The LC50 values generated using measured concentrations from filtered (pore size, 0.45 microm) water samples were 122 (105-145) nM in the absence of sulfide and 225 (192-239) nM in the presence of 100 nM sulfide. These results suggest a stoichiometric protection of sulfides up to a 2:1 ratio of Ag:sulfide. Greater accumulation of Ag at the gills was measured in fish exposed to AgNO3 in the presence of sulfide.

Evaluation of the effect of reactive sulfide on the acute toxicity of silver (I) to Daphnia magna. Part 1: description of the chemical system.

Experiments were designed to assess the potential protective effect of the presence of sulfide against the acute (48-h) toxicity of silver(I) to Daphnia magna. Tests were conducted in borosilicate glass beakers (250 ml) in moderately hard synthetic water. Toxicity solutions were replaced after 24 h by static renewal method. This paper describes the chemical system, and the acute toxicity results are presented in a companion paper. Sulfide was below detection limit (<5 nM) in controls with no sulfide added. Sulfide, added as zinc sulfide clusters at approximately 35- or approximately 350-nM concentration, dropped in concentration to approximately 25 and 250 nM, respectively, over the 24-h period of measurements. Silver also decreased in concentration during the experiment (up to 59%), and the rate of loss was greater in the absence of sulfide compared with the presence of sulfide. A filtration experiment indicated a 1:1 binding ratio of silver to sulfide and a conditional stability constant for the Ag(I)-zinc sulfide complex of log K' = 8.9. The losses of sulfide and silver during the experiments highlighted the need for regular monitoring of the important chemical components of the system, even during short (48-h) toxicity tests.

Synthesis and characterization of metal sulfide clusters for toxicological studies.

Zinc sulfide clusters were synthesized and characterized at low micromolar concentrations to assess the effect of metal-sulfide ligands on metal toxicity to aquatic organisms in oxic environments. Recommended preparation times are greater than 2 h initial reaction of equimolar sodium sulfide and zinc nitrate, followed by oxic aeration for 3 d. Ionic strength, pH, and anoxic stabilization time were found to be relatively unimportant in controlling the final yield. Adsorptive losses of zinc sulfide (ZnS) clusters to surfaces, however, were significant for a variety of vessel materials and membrane filters. Ionic strength and pH were found to be important factors controlling the extent of adsorptive losses with minimal loss for pHs greater than 9 and for soft waters. The Ag(I), Cu(II), and Hg(II) as metal sulfides completely suppress the analysis of sulfide, whereas Pb(II), Mn(II), and Co(II) partially suppress the analysis of sulfide by the methylene blue technique. Ultraviolet and fluorescence spectra are shown for synthesized ZnS clusters.

Evaluation of the effect of reactive sulfide on the acute toxicity of silver (I) to Daphnia magna. Part 2: toxicity results.

The protective effect of reactive sulfide against AgNO3 toxicity to Daphnia magna neonates was studied. Acute (48-h) toxicity tests were performed in the absence (<5 nM) and presence of low (approximately 25 nM) and high (approximately 250 nM) concentrations of zinc sulfide clusters under oxic conditions. In both the presence and the absence of sulfide, lower mean lethal concentration (LC50) values were observed when measured as opposed to nominal silver concentrations were used in calculations. This reflected the fact that measured total silver concentrations were lower than nominal concentrations due to losses of silver from solution observed during the experiment. High concentration (approximately 250 nM) of sulfide completely protected against toxicity up to the highest silver concentration tested (2 microg/L [19 nM]) with measured silver data. In the presence of environmentally realistic levels of sulfide (approximately 25 nM) in receiving waters, acute silver toxicity was reduced by about 5.5-fold. However, when filtered (0.45 microm) silver concentrations alone were considered, toxicity (48-h LC50) was similar in the absence (0.22 microg/L) and presence (0.28 microg/L) of sulfide. The difference between measured total and filtered silver was attributed to chemisorption of the metal sulfide onto the membrane filter and provides evidence that the toxic fraction of silver is that which is unbound to sulfide. Accumulation of silver was greater in daphnids exposed to silver in the presence of sulfide than in its absence, even though a toxic effect was not observed under these conditions. In this case, silver appears to be incorporated by daphnids rather than merely adsorbed on the surface. Our results point out the need to incorporate sulfide into the acute biotic ligand model and to assess its potentially large role in preventing chronic toxicity.

Effects of Cu ions and explicit water molecules on the copper binding domain of amyloid precursor protein APP(131-189): a molecular dynamics study.

Amyloid precursor protein (APP) is a cell-surface trans-membrane glycoprotein that appears to play an important role in in vivo Cu ion homeostasis. This protein includes a copper-binding-domain (CuBD) fragment consisting of residues 124-189, of which His147, His151, Tyr168, and possibly Met170 comprise the explicit Cu-binding site (CuBS). Molecular dynamics (MD) simulations are carried out on Cu-free and Cu-bound APP models, based on crystal structures including residues 131-189 obtained from the Protein Data Bank, to confirm the site of Cu-ion binding and to elucidate the effects of the oxidation state of the Cu ions (default GROMACS parameters modeled only the electrostatic binding to the Cu ions at the CuBS) and explicit water molecules on the conformational properties of the 131-189 residue portion of the CuBD. MD trajectory analysis demonstrated a conformational change of Met170. The sulfur atom of Met170 moves closer to the Cu(II) ion and away from Cu(I), and this change may play an important role in the reduction of Cu(II) and the release of Cu(I). Two explicit water molecules were included in the MD simulations. These water molecules that bind strongly to the Cu ions via their lone pair electrons result in a significant modification of the binding interactions with the other residues at the CuBS.

Displacement of Cu(II) by Ag(I) in solvated metal sulfides. A DFT and AIM computational study.

The substitution of Cu2+ by Ag+ in hydrated CuIIS and (CuII)3S3 was modeled computationally by density functional theory quantum theory of atoms in molecules, and solvent field methods. The coordination, first-shell and partly second-shell molecular structures, and thermochemical data for solvated Cu2+, Ag+, CuIIS, (CuII)3S3, AgCu2S3 and their reactions were obtained. The thermochemical data showed that displacement of Cu2+ and Cu+ from CuIIS and (CuII)3S3 by Ag+, while unfavorable in the gas phase, is facilitated in an aqueous environment. Several covalently bonded species were examined as intermediates in the substitution reactions.

Protonolysis of the Hg-C bond of chloromethylmercury and dimethylmercury. A DFT and QTAIM study.

Possible mechanisms for degrading chloromethylmercury (CH(3)HgCl) and dimethylmercury [(CH3)2Hg] involving thiol and ammonium residues were investigated by DFT and atoms-in-molecules (QTAIM) calculations. Using H2S and HS- as models for thiol and thiolate groups RSH and RS-, respectively, we obtained transition states and energy barriers for possible decomposition routes to Hg(SH)2 based on a model proposed by Moore and Pitts (Moore, M. J.; Distefano, M. D.; Zydowsky, L. D.; Cummings, R. T.; Walsh, C. T. Acc. Chem. Res. 1990, 23, 301. Pitts, K. E.; Summers, A. O. Biochemistry 2002, 41, 10287). Demethylation was found to be a multistep process that involved initial substitution of Cl- by RS-. We found that successive coordination of Hg with thiolates leads to increased negative charge on the methyl group and facilitates the protonolysis of the Hg-C bond by H-SH. This was also found to be the case for (CH3)2Hg. We found that NH4(+) readily protonolyzes the Hg-C bond of these thiolate complexes, suggesting that ammonium residues of protonated amino acids might also act as effective proton donors.

Density functional theory and QT atoms-in-molecules study on the hydration of CuI and AgI ions and sulfides.

The hydrates of Cu+, Ag+, CuS-, AgS-, Cu2S, and Ag2S were investigated with density functional theory (DFT), solvent field, and atoms-in-molecules (QTAIM) calculations. We found that covalent bonding of the first-shell water molecules to the metals plays a significant role in the total solvation energy. Molecular graphs were obtained and the bonding characterized by analysis of the electron density and its laplacian at bond critical points. Long-range electrostatic interactions between solute and the bulk solvent, quantified by solvent-field calculations, are more important for hydrated anions CuS- and AgS- than for Cu+ and Ag+ as well as for the neutral species Cu2S and Ag2S. Computed enthalpies of formation for hydrated Cu+ and Ag+ correlated well with experimentally determined values and allowed us to characterize the structures of several hydrates studied in the gas phase. We found that the stability of the hydrates is leveled in the water solvent field. The reactions of dissociation and substitution of metal sulfides in the gas phase and in solution were compared. A decrease in the of energy of the reactions in going from the gas phase to solution is explained on the basis of the higher coordination of metal atoms in the first hydration shell.

Silver speciation during chronic toxicity tests with the mysid, Americamysis bahia.

A 28-day chronic toxicity test and two 7-day chronic estimation toxicity tests were conducted with silver nitrate (AgNO(3)) and the marine invertebrate, Americamysis bahia, in 20 per thousand (parts-per thousand) salinity seawater. One 7-day test was initiated with 7-day-old mysids and the second was initiated with <24-h-old mysids. There was very good agreement between the three toxicity tests. The no-observed-effect concentration (NOEC) values from the 28-day test, the 7-day test initiated with 7-day-old mysids, and the 7-day test initiated with <24-h-old mysids were 34, 65 and 38 microg/l silver, respectively. The 96-h LC50 values from the 28-day toxicity test and the 7-day toxicity test initiated with 7-day old mysids were 260 microg/l, and the 96-h LC50 value from the 7-day toxicity test initiated with <24-h-old mysids was 280 microg/l. Free ionic silver, Ag(+), concentrations measured with a silver electrode were in good agreement with concentrations calculated using total dissolved silver and chloride concentrations. Mean measured concentrations of Ag(+) in the test solutions ranged from 0.99 to 25 ng/l for the dissolved silver concentrations that ranged from 34 to 410 microg/l silver, indicating that free ionic silver varied from 0.003 to 0.006% of the silver dissolved in the 20 per thousand salinity seawater. Understanding the relationship of salinity and silver speciation, and the effect of this relationship on chronic invertebrate toxicity, will be useful for development of a marine biotic ligand model (BLM) and a water quality criterion for silver. This model could provide an important tool for improving the relationship of laboratory toxicity test results and predicted effects in natural environments, where variations in salinity may act to modify the toxicity of silver and other metals.

Metal speciation in natural waters with emphasis on reduced sulfur groups as strong metal binding sites.

The proper application of biotic ligand models to predict metal toxicity depends on accurate prediction of metal binding to sites on natural organic matter (NOM) which compete with the 'biotic' ligand for available metal. A hard and soft metal classification along with associated ligand groups (carboxyl, phenolic, amino, sulfidic) are used as a basis to predict metal speciation in the presence of aqueous organic matter. Compilation of conditional metal formation constants (log K') are made for each ligand type using model ligands. Model ligands were chosen to reflect those found in NOM and bio-organic media. Total ligand concentration (L(T)) estimates for different natural settings and log K' values are then used to generate a L(T)-log K' distributions for a specific metal. A plot for Cu(II) gives a similar trend as a compilation of measured data for natural environments. A log K'-L(T) plot for Ag(I) shows a much more discrete binding pattern than for Cu(II). Estimation of speciation of a specific metal in a specific environmental setting and to design speciation and toxicological experiments requires accurate knowledge of the functional groups in NOM.

The biotic ligand model and a cellular approach to class B metal aquatic toxicity.

The biotic ligand model (BLM) and a cellular molecular mechanism approach represent two approaches to the correlation of metal speciation with observed toxicity to aquatic organisms. The two approaches are examined in some detail with particular reference to class B, or soft metals. Kinetic arguments are presented to suggest situations that can arise where the BLM criterion of equilibrium between all metal species in the bulk solution and the biotic ligand may not be satisfied and what might the consequences be to BLM predictive capability. Molecular mechanisms of toxicity are discussed in terms of how a class B metal might enter a cell, how it is distributed in a cell, and how the cell might respond to the unwanted metal. Specific examples are given for copper as an organism trace essential metal, which is toxic in excess, and for silver, a non-essential metal. As class B metals all bind strongly to sulfur, regulation of these metals requires that all S(II-) species be accounted for in aquatic systems, even under oxic conditions.

Determination of strong ligand sites in sewage effluent-impacted waters by competitive ligand titration with silver.

A competitive ligand titration, employing Ag+, is used to determine the binding capacity of the small amounts of strong ligands (SL) in natural water samples. Strong ligands are defined here as high-affinity binding sites for group 11 and 12 metals such as Cu(I), Hg(II), and Ag(I). In addition, the conditional binding strength (log K') is determined for Ag- and SL. Diethyldithiocarbamate (DEDC) is the competitive ligand employed. The system is set at constant pH (8.1), ionic strength (0.1 M), and excess-fixed DEDC (10 microM) to determine SLs with log K' for Ag+ of >10. Silver was chosen as the titrant metal because it binds predominantly with S(-II) versus other ligands and reduced sulfur is thought to comprise the majority of SLs in natural waters. A two-phase system, water and 1,2-dichloroethane (DCE), is required due to the insolubility of Ag-DEDC in water. Added silver partitions into Ag+ and Ag-SL in the aqueous phase and into Ag-DEDC in the DCE phase. An automated system is used to add aliquots of silver and measure Ag-DEDC by UV absorbance in the DCE phase and [Ag+] by specific ion electrode in the aqueous phase. Excess addition of silver and a "Gran's" analysis gives the binding capacity of SL. The stability constant can also be determined for each addition of silver for an overall one-site SL assumption. Cysteine was used to test the method, and urban waters revealed SL capacities from about 50 to 150 nM and log K'(Ag) of 11-12. An independent analysis of chromium-reducible sulfide correlates well with the SL capacity.