We achieved a 90% recovery for both low and high concentrations of Ag, so there was no concern that the Ag concentration in the soil might be reduced by any means during the 56-d test period. According to the guidance document on aquatic ecotoxicology 25, the nominal concentration can be used to express toxicity if the measured concentration has a recovery >80%. Because equivalent guidelines are not available for terrestrial tests, the procedure described for aquatic tests was used.
Our objective was to provide insight into the ecotoxicity of Ag, using the earthworm reproduction test and addressing the following three endpoints: mortality, biomass increase, and number of offspring. Natural soil was used for our tests so that the results were relevant to environmental conditions. The results indicated that Ag-NPs and Ag nitrate do not induce statistically significant mortality in earthworm populations at Ag concentrations up to 200 mg/kg soil. However, we observed a statistically significant increase in the biomass of the adult worms exposed to Ag-NPs and Ag nitrate. The earthworms attempted to avoid the contaminated soil during the first 24 h of the test and then preferred to remain in the food layer spread on top of the soil, a behavior that persisted until the adult worms were removed. Our observations support earlier studies in which earthworms attempted to avoid soil contaminated with Ag-NPs and Ag nitrate at concentrations of 6.92 to 7.42 mg/kg soil, although the particles were larger and were coated with polyvinylpyrrolidone (PVP) 26. No differences between the effects of Ag-NPs and Ag nitrate were observed, confirming that earthworms appear to sense the presence of Ag+ in soil. The increase in biomass can therefore be explained by the tendency of the earthworms to favor the food layer, which leads to the ingestion of more food and an increase in biomass. The avoidance behavior and the resulting increase in biomass can be reduced by spreading the food in a thin layer on the soil surface.
After 56 d, we observed significant differences in the number of offspring at an Ag-NP concentration of 30 mg/kg soil and at a Ag nitrate concentration of 15 mg/kg soil (the lowest concentration we tested). There was only a marginal difference between the toxicity of Ag-NPs and Ag nitrate, yielding EC50 values of 74 to 80 mg/kg in comparison with the negative control for NM-300 K and 42 to 47 mg/kg for Ag nitrate. In one test with Ag-NPs, the dispersant also inhibited reproduction. Several tests with earthworms and other organisms in the presence of the dispersant were carried out, with no evidence of toxic effects (data not shown). We are therefore unable to explain why the dispersant had an inhibitory effect in one of the tests but none of the others. The calculated EC50 value based on the negative control was similar in both tests with Ag-NPs, so we focus on the results compared with the negative control in the following discussion.
Few studies have considered the different issues that influence the toxicity of Ag-NPs to the earthworm E. fetida. A limit test using PVP-coated Ag-NPs and Ag nitrate at a concentration of 1,000 mg/kg soil in a natural soil resulted in 97.5% survival for earthworms exposed to Ag-NP and 2.5% survival for those exposed to Ag nitrate 15. The number of cocoons was used as an indicator of reproduction, but the surviving earthworms produced no cocoons even in the Ag-NP test, in which most of the earthworms survived. Our data also show that reproduction is a sensitive endpoint and is strongly affected by both Ag-NPs and Ag nitrate.
Another study focusing on the influence of surface coatings on the bioaccumulation of Ag-NPs and reproduction toxicity in E. fetida was carried out with artificial soil 16. Silver nanoparticles coated with PVP and oleic acid, with a nominal particle size of 30 to 50 nm, were tested at nominal concentrations of 10, 100, and 1,000 mg/kg against Ag nitrate at nominal concentrations of 10 and 100 mg/kg. In these tests, neither the Ag nitrate nor the Ag-NPs coated with PVP and oleic acid affected growth and mortality. However, there was a significant effect on earthworm reproduction at 773.3 mg/kg for PVP-coated Ag-NPs, 727.6 mg/kg for oleic acid-coated Ag-NPs, and 94.12 mg/kg for Ag nitrate. The coated Ag-NPs were approximately 10 times less toxic than our uncoated Ag-NPs with a primary particle size of 15 nm, whereas the results for Ag nitrate were in a comparable range. However, cocoon production was again used as the parameter to measure reproduction in the investigation discussed above, whereas in the present study the juveniles were counted, which limits direct comparisons. Nevertheless, it can be assumed that particle properties, for example, size and coating, play an important role in the toxicity of Ag-NPs to earthworms.
A further study focused on the role of particle size and soil type in the toxicity of Ag-NPs to earthworms 17. Two types of soil were tested to determine the influence of soil composition, a sandy loam soil comparable to our test soil and an artificial soil. The study also included two types of Ag-NPs, one with a small particle size (10 nm) and another with particles of 30 to 50 nm, both coated with PVP. There were no differences in toxicity between the two types of particles. Growth and reproduction (expressed as “juveniles and worms”) were significantly affected in the natural soil by 7.413 mg/kg Ag nitrate, but the Ag-NPs had no significant effect on any of the tested endpoints. However, only a nominal concentration of 10 mg/kg was tested in the natural soil. The higher toxicity observed in our study may reflect differences in the organic matter content of the soils (0.93% in our case but 1.77% in an earlier study 17). The organic matter content affects the fate of Ag-NPs in soil, and all types of Ag are more mobile in mineral soils compared with soils rich in organic matter 27.
The studies discussed above indicate that the toxicity of Ag-NPs is strongly dependent on the properties of test medium (e.g., organic matter) and the coating. We also used smaller particles compared with previous investigations, which may also increase the toxicity of Ag-NPs.
Silver content of earthworms
In addition to the typical endpoints considered in the standardized reproduction test, we also investigated the uptake and accumulation of Ag in adult earthworms. As specified in OECD guideline 222 18, adult earthworms were removed after 28 d. Worms were used to determine the concentration of Ag after the gut had been purged. A concentration-dependent effect on reproduction above the lowest test concentrations (15 mg/kg) was observed, but, although the lowest and highest test concentrations differed by a factor of 13, the Ag concentrations in the earthworms were comparable (and were higher in earthworms exposed to Ag-NPs than in those exposed to Ag nitrate). We therefore assume that a steady state of Ag uptake is already achieved at 30 mg/kg soil. It is unclear whether the measured Ag is located in the tissues or whether residues remain in the gut because of incomplete purging. The comparable concentrations in earthworms exposed to soil concentrations greater than 30 mg/kg and the concentration-dependent inhibition of reproduction at concentrations of 30 to 200 mg/kg soil indicate that the Ag content in the worms is not responsible for the observed effects. It can be assumed that the fertility of adults is not affected, but the development of cocoons and the survival of juveniles in soil are sensitive life stages.
In a previous study 16, a concentration-dependent increase in the levels of Ag in earthworm tissues was observed, although none of the BAFs exceeded 1, suggesting that there was no bioaccumulation of Ag-NPs. The BAFs for Ag-NPs and Ag nitrate at 10 and 100 mg/kg soil in the study cited above were comparable to our results at 15 and 120 mg/kg soil.
As with earthworms, nematodes are also exposed to Ag via soil pore water, so studies considering the uptake of Ag-NPs in the nematode Caenorhabditis elegans are also relevant to this discussion. These studies have shown that Ag-NPs are taken up into cells from the gut lumen and that transgenerational Ag-NP transfer is possible 28. Citrate-coated Ag-NPs with a particle size of 50.6 nm induced epidermal fissuring and serious epidermal burst effects in a concentration-dependent manner at concentrations of 10 and 100 mg/L 29. However, both studies were carried out using aqueous media rather than soil and might not reliably predict the influence of Ag-NPs on earthworm guts and tissues or on juveniles or cocoons.
The concentration of Ag+ in pore water was determined in addition to the total silver concentration using the DGT approach, which showed that only 0.0001% of the nominal Ag concentration exists as freely available Ag+ in the soil pore water. There was no statistically significant difference between Ag-NPs and Ag nitrate in our tests.
In tests with C. elegans 29, the measurement of dissolved Ag+ in K-medium after 24 h of incubation revealed a concentration equivalent to 0.001% of the nominal concentration, which may differ from our results because of the different incubation conditions. In aqueous K-medium, the equilibrium between bulk Ag and Ag+ may differ from that in soil, resulting in the lower concentration of Ag+ observed in this investigation. Other studies have focused on the interactions between Ag+ and other environmentally relevant compounds. The release of Ag+ may also be modified by interactions between Ag-NPs and sulfide ions or dissolved organic matter (DOM). A study with nitrifying bacteria in a wastewater treatment plant showed that sulfide reduces the toxicity of Ag-NPs by promoting the formation of AgxSy complexes or precipitates 30. Furthermore, if the concentration of DOM is higher than that of metal ions in water, then DOM binds to the dissolved metal ions, which may influence the Ag+ content detectable by DGTs 31.
We observed a concentration-dependent increase of Ag+ in the soil pore water at least at the first two measurement points. Furthermore, the comparable concentrations of Ag+ detected via DGT in soil pore water from soils spiked with Ag-NPs and Ag nitrate match the similar effect of these substances on earthworm reproduction. Therefore, it can be assumed that Ag+ is responsible for the inhibition of earthworm reproduction. Because the effect of Ag-NPs and Ag nitrate is in a similar range, we can conclude that the locally increased ion concentration on the surface of NPs compared with evenly distributed Ag nitrate is not relevant for earthworm toxicity. This may not be the case for single-cell organisms, which may be exposed to high ion concentrations in the vicinity of AgNPs. Yang et al. 32 note that Ag-NPs are more toxic than the equivalent mass of dissolved Ag or the generation of reactive oxygen species in studies with single-cell systems (e.g., nitrifying bacteria, human cells). In multicellular organisms (e.g., nematodes, fish), toxicity is caused predominantly by the dissolution of Ag-NPs.