The use of pesticides in the agricultural industry has continuously increased. In 2017, the total amount of pesticides employed in agriculture use was more than 4.1 million tons (Food and Agriculture Organization of the United Nations (FAO)). The wide use of pesticides and their potential effects upon entering into environments and subsequent hazards on the balance of ecosystems are drawing more attention (Bishop et al. 2020, Fang et al. 2019, Utami et al. 2020). Thorough knowledge of biochemical responses and combined effects of pesticides in the environment is essential for proper risk assessment (Sakthiselvi et al. 2020, Zhou et al. 2020).
Neonicotinoid pesticides are one of the most commonly used classes of insecticides and affect nicotinic acetylcholine receptors to influence insects’ nervous systems, ultimately resulting in their paralysis and death. Because of their efficient insecticidal activity and low toxicity to vertebrates and most invertebrates, neonicotinoids are widely used in crop applications, seed treatment, soil, and control of pests on household pets, underlying the world-leading sales of this class of pesticides among all others (Cimino et al. 2017, Douglas &Tooker 2015). In China, 8000 tons of acetamiprid (ACE) and 14,000 tons of imidacloprid (IMI) are used every year (Xusheng et al. 2013). Owning to their high solubility and relatively low degradation in soil, water, and other chemical reagents, the environmental and health effects of neonicotinoids have gained research attention. In recent years, the negative effects of neonicotinoids in organisms have been studied, including their associated underlying risk to human health (Hallmann et al. 2014). Neonicotinoids have been reported to reduce the reproduction of bees and increase their mortality via inhibiting their homing ability (Rundlof et al. 2015, Stanley et al. 2015, Woodcock et al. 2017). A previous study showed that serum concentrations of IMI and ACE are 0.65 and 0.06 µg/L, respectively, in people over sixty years old in Saudi Arabia (Li et al. 2020). The metabolism and dissipation of IMI and ACE are relatively safe. However, differences in environmental conditions, microbial activity, and soil properties influence IMI and ACE dissipation in soil (Castillo Diaz et al. 2016, Xu et al. 2020). Additionally, neonicotinoid insecticides are assimilated by plants and transported to organs, including roots, thus contaminating soil and under-ground organisms. It is crucial to understand the impact of IMI and ACE on the soil organisms to enable proper risk assessment and safety.
Chlorpyrifos (CRF) is a typical organophosphorus pesticide and neurotoxic insecticide, as an acetylcholinesterase inhibitor that overstimulates the nervous system. It is extensively applied to seeds, lawns, and crops. In particular, the total production of CRF exceeds 200,000 tons, with rates of use increasing by 10 % annually in China (John &Shaike 2015). However, CRF contamination has aroused the concerns of researchers since 1989. Previous studies have reported that CRF increased the population of fungi and the number of bacteria, while decreasing nitrogen fixation and microorganism levels in soil (John &Shaike 2015, Pandey &Singh 2004, Singh et al. 2015). In addition, in 2019, CRF was noted to have potential developmental toxicity, neurotoxicity, and genotoxicity, which increased attention on its human health impacts by the European Food Safety Authority (EFSA)((FSN) 2019). Thus, reasonable and efficient risk assessment of CRF could help farmers to use this pesticide safely.
ABM is generated by actinomycete fungi (Streptomyces avermitilis), belonging to the family of avermectins, and it is used to eliminate nasal bots, gastrointestinal nematodes and lung worms in sheep and cattle (Campbell 2012). ABM is a mixture containing less than 20% avermectin B1b and more than 80% avermectin B1a. It interferes with neurophysiological activities and stimulates the release of γ-amino butyric acid (GABA), which has an inhibitory effect on the nerve conduction of arthropods, resulting in their paralysis, and death. ABM is slightly solubility in water and has a lipophilic character that makes it difficult for mammals to metabolize, 80—98% of the chemical dose is eliminated in stools and transferred to soil (Sun et al. 2005). It is essential to evaluate the toxicities of ABM using soil organisms.
The global population of earthworms has decreased gradually owing to threats of environmental pollutants, habitat loss and reductions of microbial communities. Earthworms (Eisenia fetida) are the most populous terrestrial soil animal species. They have a critical role in breaking down organic matter and thus contribute to soil fertility and soil formation (Mattsson et al. 2017). Earthworms inhabit moist soil, where they come in contact with pesticides via inhalation, swallowing soil, gut absorbing and skin contact. In recent decades, earthworms have been used as a typical model of invertebrate species in soil toxicology studies. They have been selected for research because they are easy to culture in laboratory settings, have a high reproduction rate, short life cycle, and small body size, and can be used as bioindicators of soil pollutants. However, earthworms are sensitive to environmental contaminants. Despite the abundance of earthworms, their population size and diversity have both declined. The wide use of pesticides could be a serious threat to earthworm survival. Therefore, earthworms are suitable for the toxicological assessment of environmental pollutants.
Despite IMI, ACE, CRF, and ABM having different modes of action, their applications all induce severe damage to soils (Hasenbein et al. 2015, Wang et al. 2015b). Pesticides may disturb soil ecosystems and impact soil invertebrate structure (Yang et al. 2018). As a result, the wide use of insecticides is being questioned on environmental grounds in a number of countries, leading to usage restrictions. However, in spite of growing research efforts to understand insecticide use and its potential effects on a variety of organisms, we still lack assessments of the combined effects of insecticide pollutants in the environments, especially in soil, in order to evaluate their potential risks to underground organisms. To fill such a knowledge gap, we measured the individual and joint toxic effects of IMI, ACE, CRF, and ABM on earthworms, including effects on the activities of acetylcholinesterase (AChE) and cellulase. AChE is the target enzyme for pest control by insecticides, such as neonicotinoids and organophosphate pesticides. In earthworms, cellulase activity indicates relatively high activities of endoglucanase more broadly (Ikarashi et al. 2016).
In order to assess the toxic effects of pesticides to earthworms, artificial soil test protocols have been used (OECD 1984, 2004). Artificial soil tests simulate the natural environment of earthworms. In this type of assay, earthworms absorb pollutants through their guts (Brami et al. 2017). Many studies could use this approach to more efficiently assess the toxicities of contaminants to earthworms.