Comparative in vivo toxicokinetics of silver powder, nanosilver and soluble silver compounds after oral administration to rats.

Silver (Ag; massive, powder and nanoform) and Ag compounds are used in industrial, medical and consumer applications, with potential for human exposure. Uncertainties exist about their comparative mammalian toxicokinetic ('TK') profiles, including their relative oral route bioavailability, especially for Ag massive and powder forms. This knowledge gap impedes concluding on the grouping of Ag and Ag compounds for hazard assessment purposes. Therefore, an in vivo TK study was performed in a rat model. Sprague-Dawley rats were exposed via oral gavage for up to 28 days to silver acetate (AgAc; 5, 55, 175 mg/kg(bw)/d), silver nitrate (AgNO3; 5, 55, 125 mg/kg(bw)/d), nanosilver (AgNP; 15 nm diameter; 3.6, 36, 360 mg/kg(bw)/d) or silver powder (AgMP; 0.35 µm diameter; 36, 180, 1000 mg/kg(bw)/d). Total Ag concentrations were determined in blood and tissues to provide data on comparative systemic exposure to Ag and differentials in achieved tissue Ag levels. AgAc and AgNO3 were the most bioavailable forms with comparable and linear TK profiles (achieved systemic exposures and tissue concentrations). AgMP administration led to systemic exposures of about an order of magnitude less, with tissue Ag concentrations 2-3 orders of magnitude lower and demonstrating non-linear kinetics. The apparent oral bioavailability of AgNP was intermediate between AgAc/AgNO3 and AgMP. For all test items, highest tissue Ag concentrations were in the gastrointestinal tract and reticuloendothelial organs, whereas brain and testis were minor sites of distribution. It was concluded that the oral bioavailability of AgMP was very limited. These findings provide hazard assessment context for various Ag test items and support the prediction that Ag in massive and powder forms exhibit low toxicity potential.

European freshwater silver monitoring data do not suggest a potential European-wide risk.

European legislations frequently focus on substances that are of potential concern to human or environmental health, such as "priority substances" under the Water Framework Directive 2000/60/EC ("WFD") that are identified as substances posing a significant risk to or via the aquatic environment. The EU REACH regulation also requires the assessment of the environmental risks of chemicals put on the EU market. To properly assess the potential risk of a substance, high-quality representative monitoring data should be compared with a safe threshold concentration. The objective of this article is to evaluate different publicly available freshwater monitoring data sets for silver and investigate them for a potential European-wide risk according to the methodology used by the European Commission. Most available silver monitoring data sets contain a large proportion of undetected samples with a reported concentration below the limit of quantification (LOQ) of the analytical technique, leading to considerable uncertainty in the data set. For silver, this LOQ is often at or above the safe threshold concentration, and the method used to handle undetected samples during the data processing considerably impacts the data assessment. We demonstrate that for large data sets covering many European countries (and often a wide range of LOQs), the uncertainty in the data set does not allow us to make a general conclusion about European-wide risk. However, by examining the data sets in more detail and assessing three additional country-specific monitoring data sets, we show that silver does not pose a risk to the freshwater environment in several countries. We conclude that the available data sets need careful assessment to account for the values that are below the LOQ, and that there is currently no reliable evidence indicating a European-wide risk for silver in the aquatic environment, meaning it should not be selected as  priority substance under the WFD. Integr Environ Assess Manag 2023;19:1110-1119. © 2022 European Precious Metals Federation. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Setting a Protective Threshold Value for Silver Toward Freshwater Organisms.

Driven by Regulation (EC) No. 1272/2008 and the European Water Framework Directive 2000/60/EC, we have re-evaluated the available chronic freshwater ecotoxicity data for ionic silver (Ag) using strict data quality criteria. In addition, we generated new chronic ecotoxicity data for species potentially sensitive to Ag (the rotifer Brachionus calyciflorus, the cyanobacteria Anabaena flos-aquae, and the aquatic plant Lemna minor) using Ag nitrate as the test substance. The 10% effect concentrations for the most sensitive endpoint per test species were 0.31 µg dissolved Ag/L for B. calyciflorus (population size), 0.41 µg dissolved Ag/L for A. flos-aquae (growth rate), and 1.40 µg dissolved Ag/L for L. minor (root length). We included these values in the set of reliable chronic freshwater data, subsequently covering a total of 12 taxonomic groups and 15 species. Finally, we applied a species sensitivity distribution approach to the data set using various models. The best-fitting model (Rayleigh distribution) resulted in a threshold value protective for 95% of the species of 0.116 µg dissolved Ag/L. This value is considered reliable and conservative in terms of species protection and can be used as a solid basis for setting thresholds for Ag in freshwater after application of an appropriate assessment factor. Furthermore, this value represents reasonable worst-case conditions for bioavailability in European Union surface waters (low hardness and low dissolved organic carbon). Environ Toxicol Chem 2021;40:1678-1693. © 2021 European Precious Metals Federation. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effects of Silver Nitrate are a Conservative Estimate for the Effects of Silver Nanoparticles on Algae Growth and Daphnia magna Reproduction.

Silver (Ag) salts have been shown to be highly toxic to freshwater organisms. There is nevertheless still a high level of uncertainty as to the aquatic effects of Ag nanoparticles (AgNPs), and how these relate to the effects of soluble Ag salts. As part of the substance evaluation for Ag of the European Union Registration, Evaluation, Authorisation, and Restriction of Chemicals regulation, we have generated new data to justify read-across from soluble Ag salts to AgNPs. The aquatic toxicity to algae growth and Daphnia magna reproduction, fate, and behavior of AgNO3 versus AgNPs were tested and compared. Chloride salts in the test media were replaced with equimolar concentrations of nitrate salts. Total Ag, "conventionally" dissolved Ag (0.45 µm), and "truly" dissolved Ag (3 kDa) were determined. Algae were the most sensitive test species to AgNO3 (10% effect concentration [EC10] 0.10 µg Ag/L) when expressed as conventionally dissolved Ag. The corresponding value for AgNPs was 0.26 µg/L. For D. magna reproduction, the lowest EC10 values were 3.49 µg Ag/L for AgNO3 and 33.4 µg Ag/L for AgNPs. Using measured Ag concentrations, AgNO3 was experimentally shown to be more toxic than AgNPs for all Ag fractions. We explain these observations by a different dissolution behavior of AgNO3 versus AgNPs. The results provide experimental confirmation that AgNO3 can be used as a conservative estimate for the aquatic effects of AgNPs at comparable Ag concentrations. Environ Toxicol Chem 2019;38:1701-1713. © 2019 SETAC.

Transformation-dissolution reactions partially explain adverse effects of metallic silver nanoparticles to soil nitrification in different soils.

Risk assessment of metallic nanoparticles (NPs) is critically affected by the concern that toxicity goes beyond that of the metallic ion. The present study addressed this concern for soils with silver nanoparticles (AgNPs) using the Ag-sensitive nitrification assay. Three agricultural soils (A, B, and C) were spiked with equivalent doses of either AgNP (diameter = 13 nm) or AgNO3 . Soil solution was isolated and monitored over 97 d with due attention to accurate Ag fractionation at low (∼10 µg L-1 ) Ag concentrations. Truly dissolved (<1 kDa) Ag in the AgNO3 -amended soils decreased with reaction half-lives of 4 to 22 d depending on the soil, denoting important Ag-aging reactions. In contrast, truly dissolved Ag in AgNP-amended soils first increased by dissolution and subsequently decreased by aging, the concentration never exceeding that in the AgNO3 -amended soils. The half-lives of AgNP transformation-dissolution were approximately 4 d (soils A and B) and 36 d (soil C). The Ag toxic thresholds (10% effect concentrations, milligrams of Ag per kilogram of soil) of nitrification, evaluated at 21 or 35 d after spiking, were similar between the 2 Ag forms (soils A and B) but were factors of 3 to 8 lower for AgNO3 than for AgNP (soil C), largely corroborating dissolution differences. This fate and bioassay showed that AgNPs are not more toxic than AgNO3 at equivalent total soil Ag concentrations and that differences in Ag dissolution at least partially explain toxicity differences between the forms and among soils. Environ Toxicol Chem 2018;37:2123-2131. © 2018 SETAC.

Experimental parameters affecting sensitivity and specificity of a yeast assay for estrogenic compounds: results of an interlaboratory validation exercise.

In vitro assays are considered as the first step in a tiered approach to compound screening for hormonal activity. Although many new assays have been developed in recent years, little attention has been paid towards assay validation. Our objective was to identify critical experimental parameters in a yeast estrogen screen (YES) that affect its sensitivity and specificity. We investigated the role of incubation time, solvent type, yeast inoculum growth stage and concentration on the outcome of the YES. Compounds tested included new and established agonists, antagonists and negative controls, and results were evaluated according to prefixed statistical criteria. In addition, we assessed the assay's performance in a blind interlaboratory validation exercise (IVE). An incubation time of five days was necessary to positively identify the estrogenic properties of all agonists tested, when dissolved in DMSO. Longer incubation times were required when using an ethanol protocol. Similar estrogenic activity was reported for benzyl butyl phthalate, bisphenol-A, methoxychlor, permethrin and genistein in the IVE. One out of the three laboratories did not classify alpha,beta-endosulfan, dissolved in DMSO, as an estrogen. The same was true for 4,4'-DDE and lindane, dissolved in ethanol, a result that might be attributable to an inappropriate yeast start concentration and/or growth stage. These validation experiments show that under appropriate experimental conditions the YES yields sensitive, specific and reliable results. Therefore it fulfills the requirements as a first step screening assay to evaluate the capacity of chemicals to interact with the estrogen receptor.

Flame retardants, surfactants and organotins in sediment and mysid shrimp of the Scheldt estuary (The Netherlands).

Sediment and mysids from the Scheldt estuary, one of the largest and most polluted estuaries in Western Europe, were analyzed for a number of contaminants that have been shown to possess endocrine-disrupting activity, i.e. organotins, polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD), tetrabromobisphenol A (TBBPA), nonylphenol ethoxylates (NPE) and transformation products, nonylphenol (NP) and nonylphenol ether carboxylates (NPEC). In addition, in vitro estrogenic and androgenic potencies of water and sediment extracts were determined. Total organotin concentrations ranged from 84 to 348 ng/g dw in sediment and 1110 to 1370 ng/g dw in mysid. Total PBDE (excluding BDE-209) concentrations ranged from 14 to 22 ng/g dw in sediment and from 1765 to 2962 ng/g lipid in mysid. High concentrations of BDE-209 (240-1650 ng/g dw) were detected in sediment and mysid (269-600 ng/g lipid). Total HBCD concentrations in sediment and mysid were 14-71 ng/g dw and 562-727 ng/g lipid, respectively. Total NPE concentrations in sediment were 1422 ng/g dw, 1222 ng/g dw for NP and 80 ng/g dw for NPEC and ranged from 430 to 1119 ng/g dw for total NPE and from 206 to 435 ng/g dw for NP in mysid. Significant estrogenic potency, as analyzed using the yeast estrogen assay, was detected in sediment and water samples from the Scheldt estuary, but no androgenic activity was found. This study is the first to report high levels of endocrine disruptors in estuarine mysids.

In vitro and in vivo estrogenicity and toxicity of o-, m-, and p-dichlorobenzene.

The estrogenicity of o-, m-, and p-dichlorobenzene (DCB) was evaluated with a yeast estrogen screen (YES) and zebrafish (Danio rerio) vitellogenin (VTG) assays. With the YES, p-DCB and m-DCB were found to be estrogenic in a concentration-responsive manner. The relative potency measured with the YES (relative to 17beta-estradiol) was 2.2 x 10(-7) for p-DCB and 1.04 x 10(-8) for m-DCB. Following acute toxicity tests with the zebrafish, plasma VTG production was measured to examine the in vivo estrogenic activity of the three compounds after a 14-d exposure. Adult zebrafish were exposed to different concentrations of o-, m- and p-DCB, ranging from 0.1 to 32 mg/L; ethynylestradiol ([EE2]; 5 ng/L, 10 ng/L, 50 ng/L, and 100 ng/L) was used as a positive control. After exposure, blood samples were taken and protein electrophoresis was performed to determine the relative VTG content. Gonadosomatic indices (GSI) and condition factors (CF) were also calculated. Elevated VTG levels and decreased female GSIs were found in fish exposed to > or = 5 ng EE2/L and in fish exposed to > or = 10 mg p-DCB/L. Low GSIs coincided with high levels of VTG in the blood of female zebrafish. This relation was not only found in fish exposed to EE2 but also in controls and fish exposed to DCB. Therefore, a direct or indirect effect of VTG on the GSI is suggested rather than a direct toxic effect of the tested compounds on the gonads.

Induction of vitellogenesis in 17alpha-ethinylestradiol-exposed rainbow trout (Oncorhynchus mykiss): a method comparison.

Juvenile rainbow trout, Oncorhynchus mykiss, were exposed to the synthetic estrogen 17alpha-ethinylestradiol (EE(2)) through injection (1, 10, 25 and 50 microg EE(2)/g fish/week) and via water exposure (1, 10 and 100 ng EE(2)/l). After seven (injection and water exposure) and 14 days (only for water exposure), blood and plasma vitellogenin concentrations were quantified using indirect endpoints, i.e. plasma alkaline-labile phosphorus (ALP), plasma protein and plasma calcium. In addition, the relative gonad (GSI) and liver weight (HSI) were recorded. Actual plasma vitellogenin concentrations were measured with an enzyme immunoassay. Only fish injected with 50 microg EE(2)/g fish had a significantly higher gonad weight. No concentration-dependent changes in the HSI were detected in fish exposed via the water, but a significant dose-dependent increase of the HSI was observed in fish injected with EE(2). Exposure of rainbow trout to EE(2) had a significant effect on all tested plasma parameters. Plasma protein, phosphoprotein and calcium concentrations were significantly higher after two weeks exposure to 100 ng EE(2)/l. Fish injected with 10, 25 and 50 microg EE(2)/g fish exhibited increased plasma protein concentrations after 1 week. Compared to the controls, plasma ALP and calcium levels were significantly higher in all injected fish. A significant and positive correlation was observed between all three plasma parameters and between these indirect parameters and the actual plasma vitellogenin concentrations. These findings indicate that both the plasma ALP and the plasma calcium assay have a similar sensitivity as that of available antibody-based assays (EIA), at least in EE(2) exposure studies, and thus these assays can provide a rapid, simple and cost-effective alternative to available immunoassays.