Interaction of Silver Nanoparticles and Silver Ions with Soil, Plant and Earthworm Aporrectodea caliginosa : A dissertation submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

Silver nanoparticles (AgNPs) are frequently synthesised for use in consumer products and appliances because of their antimicrobial properties and ease of incorporation into plastics, industrial materials and solutions. Currently, there are no regulations for the use of AgNPs in consumer products or their disposal. Therefore, there is a need to evaluate the impact of AgNPs in the environment. Because of their chemistry and slow dissociation to reactive Ag ions (Ag+) over time, AgNPs have been shown in both prokaryote and eukaryote models to be cytotoxic. Ionic Ag could bio-accumulate in the environment, thereby causing toxic effects on soil microbes and other organisms, plants, and animals. This thesis examined (1) the mobility of AgNPs and Ag+ (as silver nitrate, AgNO3) in soil and (2) the uptake of Ag by various plants. In particular, it evaluated (3) the chronic toxic effects on sunflower plants, and (4) the acute and chronic toxic effects on Aporrectodea caliginosa earthworms. In the soil mobility study, at a specific pH, the KD value (distribution of Ag between solid and solution phases) for AgNPs was 10-fold higher than for Ag+, indicating that Ag+ is more mobile and more toxic than AgNPs. This could be due to saturation of binding sites in soil. KD increased at higher pHs because of increased sorption to variable charged surfaces in soil. In soil incubation studies conducted at 20°C and 35°C, the solubility of AgNPs/Ag+ was greater at the higher soil temperature. The sorption of AgNPs to soil decreased over time, because all AgNPs were transformed into Ag+ due to degradation over time. Plant uptake of Ag in different parts (root cf. shoot) of perennial ryegrass (Lolium perenne L.) grown in soil amended with different concentrations (ranging from 0.0019% to 1%) of AgNPs and Ag+ indicated that Ag+ was 10-fold more soluble than AgNPs. Soil pH had a significant effect on sorption of AgNPs/Ag+ by plants, with sorption increasing with increased pH. Exposure of ryegrass to various AgNPs/Ag+ concentrations showed that concentrations < 10 mg/kg (dry matter) had a stimulatory effect on plant growth but >200 mg/kg of AgNPs/Ag+ reduced plant growth. In an Ag uptake study of nine vegetables (spinach, parsley, radish, lettuce, rocket, carrot, silver beet, leek, beetroot) grown in Templeton Silt Loam soil amended with 70 mg/kg AgNPs/Ag+, Ag uptake by control plants from naturally present Ag in soil was low (0.5 mg/kg) compared to a 3.5 to 3.8 mg/kg concentration in plants grown in soils spiked with AgNPs/Ag+ respectively. The highest concentration factor (5–9) occurred in carrot, silverbeet and spinach. A more detailed study on Ag concentrations in sunflower following 53-days’ exposure to soil amended with 150 mg/kg AgNPs/Ag+ showed that Ag accumulated in the roots > leaves. In the sunflower, Ag+ > AgNPs increased the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) in a dose- and time dependent manner and also increased the lipid peroxides, ascorbate oxidase, pyrogallol peroxidase and guaiacol peroxidase. Total carotenoids were lower only in the plants exposed to Ag+. Chlorophyll A but not chlorophyll B was significantly inhibited (P<0.05) in plants exposed to AgNP/Ag+. Total protein and total soluble carbohydrate significantly declined in sunflower exposed to the two Ag compounds, but the total phenolic compounds, urease enzyme activity and vitamins A, E and C were increased. The LD50 of AgNPs and Ag+ on earthworms was determined as 2,649 and 305 mg/kg soil respectively (unpublished data). A short-term (24 and 48 h) filter paper study and a more extensive long-term (4-week) soil sub-chronic toxicity study in earthworms on the effects of AgNPs and Ag+ showed a dose- and time-dependent enhancement of lipid peroxidation, and several-fold increase in the activities of a range of antioxidant enzymes (including SOD, CAT, GPx and GST), with Ag+ more toxic than AgNPs. A study comparing the toxicity of AgNPs and Ag+ to earthworms by measuring pharmacokinetic parameters showed that when exposed to soil amended with 20 mg/kg AgNPs/Ag+, Ag+ was more toxic than AgNP as shown by terminal half-life, mean residence time, area under the curve, maximum concentration, bio-concentration factor, and the rate of elimination. Thus, Ag+ > AgNPs caused dose- and time-dependent growth inhibition and oxidative stress in both A. caliginosa earthworms and plants, with resultant increases in lipid peroxidation and antioxidant enzymes. In conclusion, this study of AgNPs and Ag+ effects on Ag mobility in soil, accumulation of Ag in nine vegetable types, and effects on two plants (sunflower, ryegrass) and an earthworm (A. caliginosa) showed accumulation of Ag, increased lipid peroxidation, and elevation of antioxidant enzyme (CAT, SOD, GPx, GST) activities, in addition to changes in a series of other parameters in sunflower and ryegrass that correlated with their adverse effects. The multi-level effect approach adopted in this study, including an earthworm pharmacokinetic study, provided a better understanding of the potential risk of AgNPs to non-target plants and earthworms, although Ag+ proved to be more toxic than AgNPs. This study shows that evaluating several lower tier biomarkers offered a meaningful and an informative assessment of potential effects of AgNPs on plants and earthworms.