Effects of silver nanoparticles on bacteria and earthworms

Abstract

A foreseeably huge increase in the use of nanoparticles (NPs) could potentially result in increasing exposure of humans to environmental NPs. In order to mitigate, eliminate or avoid risks of exposure to these NPs in the environment, it is essential to develop an understanding of their mobility, reactivity, ecotoxicity and persistency. Due to the expected increased usage of silver nanoparticles (AgNP), the models predict an exponential increase in exposure, which would no doubt exacerbate concerns about ecological risks. In this research, the effects of AgNP were evaluated at two trophic levels: Escherichia coli (E. coli) bacteria, which is the most common bacteria used in commercial toxicity assays, and the earthworm Aporrectodea caliginosa (A. caliginosa), which is the most abundant species in New Zealand pastures. In order to compare the toxicity of AgNP with an inorganic and a better studied silver compound, silver nitrate (AgNO₃) was used as an aqueous counterpart. In this research, the E. coli bacteria toxicity study was accomplished by application of the SciToxTM mediated Direct Toxicity Assessment (DTA) Assay, developed by Lincoln Ventures Limited. The DTA Assay is a rapid and sensitive catalytic microbial method. In addition to the conventional standard DTA assay protocol (Method I), a modified protocol (Method II) with an additional centrifuge step to Method I was developed to eliminate the influence of AgNP on the electrodes used in the assay. Two toxicants – commercial-standard 2,4-dichlorophenol (2,4-DCP) and AgNO₃ – were applied to E. coli in the DTA assay. Clear and accurate dose-response curves were obtained with both AgNO₃ and 2,4-DCP using Methods I and II. However, no dose-dependent response in the DTA assay was observed with either Method I or II on exposure of E. coli to AgNP. Hence a growth inhibition assay was conducted by exposing E. coli to different concentrations of AgNP. A linear inhibition in bacterial growth was observed after 16 h incubation of bacteria to AgNP. A genotoxicity study was carried out with A. caliginosa earthworms. Three separate acute toxicity studies were conducted to determine the LD50 values of AgNP and AgNO₃. LD50 values were established for AgNO₃in a classical Petri-dish filter-paper study (571 ppm) and a soil-exposure study (418), but AgNP did not appear to be toxic to A. caliginosa earthworms even at 2000 ppm. Hence an exposure study was conducted using aqueous AgNP and AgNO₃. Based on the results from that study, A. caliginosa were exposed to 0, LD5, LD10, LD15, LD20, and LD25 concentrations of aqueous solutions of AgNP and AgNO₃. Two classical earthworm genotoxicity tests (the Micronucleus Test and Comet Assay) were performed. In the Micronucleus Test, the frequencies of coelomocyte micronuclei (MN) and binucleate (BN) cells were used to assess chromosomal aberrations and inhibition of cytokinesis, respectively. The frequency of MN at most concentrations was higher than for BN on exposure to both AgNP and AgNO₃. The frequencies (i.e. the sum) of BN and MN in coelomocytes were significantly higher (P < 0.05) with both AgNP and AgNO₃ at concentrations of LD15 upwards comparedwith the controls. Thus it appears that both AgNP and AgNO₃ are capable of causing chromosomal aberrations and cytokinesis failure at higher exposure concentrations. In this study, no significant changes were observed in the frequency of the three major types of coelomocytes, i.e. eleocytes, acidophils, and basophils, in the earthworms exposed to AgNP and and AgNO₃. In the Comet Assay, the comets formed due to DNA single-strand breaks in earthworm coelomocytes were scored for the comet parameters, Tail DNA (TD), DNA Tail Length (TL), and DNA Tail Moment (TM). Results of TD, TL and TM on exposure to both AgNP and AgNO₃ were not significant indicating that neither of these two chemicals at the exposure concentrations tested induces DNA damage. A functional ecotoxicogenomics study was performed using polymerase chain reaction (PCR) and reverse transcriptase quantitative PCR (RT-qPCR). In standard PCR, the superoxide dismutase (SOD) gene was for the first time detected in A. caliginosa earthworms. Primers were designed from conserved areas identified by multiple alignment of the SOD gene from Lumbricus rubellus, Eisenia fetida and the blue mussel (Mytilis edulis). A partial sequence homologous to the SOD gene was obtained using the standard PCR protocol. This gene sequence showed 90% similarity to the SOD gene in both L. rubellus and L. terrestris and 88% similarity to that in E. fetida . Following SOD gene detection and sequencing, a study of SOD gene expression in A. caliginosa earthworms exposed to AgNP was performed using RT-qPCR. A higher gene expression was observed in A. caliginosa exposed to AgNO₃ compared with AgNP. In conclusion, it is apparent that AgNP used in this study was not as toxic as AgNO₃ to either E. coli bacteria or A. caliginosa earthworms. This may be because of the poor solubility of AgNP.