A novel method for the quantification, characterisation and speciation of silver nanoparticles in earthworms exposed in soil

Currently, metal engineered nanoparticles (ENPs) in tissues are generally quantified based on total concentrations after acid digestion of samples. Electron microscopy has also been used for non-quantitative characterisation of NPs in situ, and can be enhanced with tissue-processing methods that can extract NPs with minimal destruction. For a proper risk assessment, it is essential to quantify and characterise the ENPs in both exposure media and organisms. For this, we developed a method using a combination of enzymatic tissue processing, followed by single particle inductively coupled plasma–mass spectrometry (sp-ICP-MS) to characterise and quantify AgNPs in tissues of earthworms after in vivo exposure in soil to 50-nm AgNPs or AgNO3. Tissue concentration of Ag in worms exposed to 250 mg AgNP kg–1 soil (dry weight) was 0.502 ± 0.219 mg kg–1 (dry weight) reflecting a bioaccumulation factor of 0.002. In both AgNP- and AgNO3-treated groups, the metal-rich granule fraction contained the highest Ag concentrations (77 and 64 % respectively). Total Ag contained in the earthworm tissue of the AgNP- and AgNO3-treated groups comprised ~34 and <5 % particulate Ag respectively. Average particle size of AgNPs extracted from tissues was consistent with exposure material (44 v. 43 nm respectively). High resolution field-emission gun scanning electron microscopy in combination with energy-dispersive X-ray (FEG-SEM/EDX) identified individual AgNPs in tissue extracts with corresponding spectral elemental peaks, providing further evidence of tissue particle uptake and composition.