The impact of imidacloprid and its metabolites on long-term neurodevelopmental and neurodegenerative disorders in a Zebrafish model

Abstract

This research aimed to assess the impact of lower dose, longer duration exposure of imidacloprid (IMI) on neurodevelopmental and neurodegenerative disorders in comparison to acute exposure within an in vivo zebrafish model. Sensitive, selective, accurate and efficient liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed to quantitate bioaccumulation of IMI and its primary metabolites, as well as changes in neurotransmitter concentrations following IMI exposure. This study also assessed liver damage in response to imidacloprid administration as a measure of acute and chronic toxicity in a zebrafish larval model. Introduction: IMI [1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is a systemic neonicotinoid insecticide belonging to the chloronicotinyl nitroguanidine chemical family produced to replace the organophosphorus pesticides in commercial agriculture (Crosby EB et al. Neurotoxicol Teratol [Internet]. 2015;49:81–90) (Kumar A et al, Egypt J Forensic Sci [Internet]. 2013;3(4):123–6). The extensive use of these pesticides increases the risk to biodiversity of freshwater aquatic surroundings due to their potential bioaccumulation and toxicity (Muazzam B et al, J Water Supply Res Technol-AQUA. 2019;68(8):718–30). While pesticide usage has economic benefits in large-scale agriculture, exposure can lead to detrimental effects on humans. Researchers studying the effect of IMI on human nicotinic receptors (α4β2) have reported that IMI may have more substantial side effects on humans than originally anticipated (Li P et al, J Neurosci Res. 2011;89(8):1295–301). Zebrafish larvae were exposed to IMI at 4 and 5 days post fertilisation to determine the no observed adverse effect level (NOAEL) of IMI. Behavioural activity tracking of the zebrafish larvae was monitored using Daniovision software. Oil red O was used to stain for fatty liver accumulation (steatosis) which is indicative of hepatotoxicity following IMI exposure. After this, adult zebrafish were exposed to the NOAEL concentration for 21 days. Key endpoints included developmental effects, behaviour indicative of neuropathology, neurodegeneration and possible bioaccumulation in the adult zebrafish tissues (brain and liver). Neurotransmitter concentrations were measured in the adult zebrafish brain tissue at the end of the treatment period to evaluate changes in neurotransmitter signalling and potential neurodevelopmental effects using a LC-MS/MS method. Bioaccumulation of IMI and its metabolites in zebrafish brain and liver was evaluated using LC-MS/MS. The NOAEL of IMI in zebrafish larvae was determined to be 2.5 μg/L. Hepatotoxicity in the form of steatosis was observed in larvae subsequent to a 5-day treatment period. Adult zebrafish were exposed to the NOAEL IMI concentration for 21 days. No significant behavioural abnormalities were observed in the adult zebrafish during the treatment period. The calibration curve for the neurotransmitters fits a quadratic (weighted 1/C) regression over the concentration range of 3.906–1000 ng/mL, 7.813–1000 ng/mL, 15.625–1000 ng/mL and 31.250–1000 ng/mL for acetylcholine, serotonin, gamma-aminobutyric acid and dopamine, respectively. The calibration curve for IMI and its metabolites fits a quadratic (weighted 1/C) regression over the concentration range of 0.098–12.5 ng/mL, 0.195–12.5 ng/mL, 0.391–12.5 ng/mL and 0.781–12.5 ng/mL for desnitro-imidacloprid, imidacloprid, imidacloprid-urea and 5-hydro-imidacloprid, respectively. Robust LC-MS/MS methods were developed for the quantitation of IMI, desnitro-imidacloprid, imidacloprid-urea and 5-hydro-imidacloprid, as well as serotonin, dopamine, acetylcholine and gamma-aminobutyric acid neurotransmitters in 200 μl zebrafish brain and liver homogenate. These methods were used to observe changes in neurotransmitter signalling in response to low-level, long-term exposure to imidacloprid, even in the absence of behavioural changes. Furthermore, together with the evaluation of bioaccumulation of imidacloprid and its metabolites in the brain and liver of zebrafish, this study provides indications of impact on human health following chronic neonicotinoid exposure.