Application of Adverse Outcome Pathway framework in assessing nanogold exposure to Daphnia magna
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Nanotechnology has found its way into almost all aspects of technology, science and every-day life and is a promising field for future advancements. Nanomaterials (NMs) are regarded as materials where one dimension has a size range between 1- 100 nm, and they may occur in several different elemental cores and shapes with different surface coatings. The developmental of gold nanomaterials (nAu) has become advantageous as it is applicable for medicinal diagnosis (rapid test kits), imaging and as a drug delivery vector. Once NMs are released into the aquatic environment, at all stages of their product lifecycle, they have potential to cause harmful effects on aquatic organisms. However, there is limited information regarding harmful effects of these NMs especially at the molecular level. Daphnia magna is a planktonic crustacean known to be a filter feeder ingesting particles, microorganisms, and many other unicellular and filamentous algae species found in aquatic environment. Daphnia magna is an internationally recognized test organism thereby making them an important species in bioassays and have internationally standardized Organisation for Economic Co-operation and Development (OECD) protocols for toxicity testing. The adverse outcome pathway (AOP) consists of a molecular initiating event (MIE), collective key events (KEs) that can be measured which lead to an adverse outcome (AO), that needs to have a regulatory consequence. The AOP framework will be utilized in this study to address adverse effects of nAu (CTAB capped rod shaped nAu and citrate capped spherical nAu) and their ionic bulk Au on D. magna at different levels of biological organisation. The first aim of this study was to determine the acute and chronic toxicity of nAu and ionic Au using D. magna as indicator organism. The first objective was to determine the physico-chemical characteristics of the two nAu’s and ionic Au in the environmental media to explain the acute and chronic responses observed in D. magna. Secondly, to determine the LC10, LC20 and LC50 of nAu and ionic Au based on mortality data using the OECD202 protocol. The second aim was to use the AOP framework to determine the sub-lethal effects of nAu and ionic Au on D. magna. The objectives were as follow: To determine the MIE at the molecular level (metabolomics), KE at the whole organism level (physiological changes based on swimming behaviour, heart rate, and respiration) and AO at community level (functional response and reproduction). To propose an AOP for nAu and ionic Au based on the MIE, KEs and AO, D. magna were exposed to sub lethal concentrations of CTAB capped rod shaped nAu [LC10 (2 μg/L) & LC20 (4 μg/L)] and citrate capped nAu [LC10 (2 mg/L) & LC20 (20 mg/L)] and ionic Au [LC10 (1 μg/L) & LC20 (4 μg/L)], following OECD protocol. Metabolites and associated pathways disrupted were quantified using Gas chromatography coupled with time of flight mass spectrometry (GCxGC-TOF-MS). Multivariate analyses was done using MetaboAnalyst software to statistically identify the unique metabolites produced following exposure to the nAu and ionic Au and determine which metabolic pathways (MIE) were influenced The KEs were measured as follows: After 48 h exposure video recordings were taken and the heartbeat of D. magna was counted manually by playing the video clip in slow motion including the use of a pen and paper to tap the beats, respiration was measured using a 24 well sealed Loligo Systems® microplate chamber with PreSens precision sensing to measure oxygen consumption. CytoViva® dark field hyperspectral imaging was conducted in this study to examine the accumulation of nAu and physical biological damage of ionic Au exposure of daphnids. Individual and community behavioural recordings were taken at 0 h and at 48 h using a Basler monochrome GigE video camera and videos were analysed using Ethiovision X14 software. The exposed daphnids response to predation was quantified using functional response with fish (Danio rerio) used as a predator fed different prey densities and monitored for 30 minutes. CTAB capped rod shaped nAu had a size of ± 40 nm with an LC50 of 12.1 μg/L and had the highest acute toxicity of all compounds tested. The reproduction of D. magna was significantly influenced with decrease amino acids, therefore increasing energy requirement reserves resulting D. magna to have adverse effects (e.g. decline in reproduction) affecting physiological changes (heart rate, respiration, and behaviour). Citrate capped nAu had a size of ± 20 nm and were spherical in shape with a LC50 of >100 mg/L while ionic Au had a LC50 of 57 μg/L. The study revealed different physiological responses between exposure groups compared to the control. Only the highest exposure concentrations (LC20) resulted in significant (p<0.05) physiological changes with increased heart rate and corresponding decrease in oxygen consumption in D. magna. Both exposure concentrations of the nAu groups increased significantly (p <0.05) when compared to the control. However, ionic Au exposure groups showed a significant decrease (p < 0.05) when compared to the control. This study revealed that a nano-specific response can be observed and the AOP framework can be utilized to determine the effect of two different nAu and ionic Au in an aquatic ecosystem using D. magna as a model organism.