Long-term effects of neonicotinoids on reproduction and offspring development in the copepod Acartia tonsa

Highlights

• Neonicotinoids are potent interferents of copepods' reproduction and offspring development.

• First-generation compounds acetamiprid and thiacloprid were the most toxic toward Acartia tonsa.

• Acetamiprid inhibited egg production, hatching, larval survival and larval development.

• Thiacloprid impaired significantly inhibited hatching and larval development.

• Clothianidin affected only larval development.

Abstract

Neonicotinoids (NEOs) are neurotoxic pesticides acting as nicotinic acetylcholine receptor agonists. NEOs' efficacy against pest insects has favoured their spreading use in agriculture, but their proven effectiveness against non-target insects in terrestrial and aquatic ecosystems also raised concern over their environmental impact. Crustaceans were often studied for the impacts of NEOs due to their economic values and nervous' system similarity with insects. However, most studies on crustaceans focused on acute effects or exposure of early-life stages, while long-term effects were seldom explored. The present study aimed to assess the potential long-term effects of four commercially available NEOs on the reproduction and offspring of the calanoid copepod Acartia tonsa, a key species in the food webs of several coastal and estuarine environments. NEOs were confirmed as potent interferents of copepod reproduction. The first-generation compound acetamiprid significantly inhibited egg production and hatching ratio at 10 ng L⁻¹, while larval survival and development were affected at 81 ng L⁻¹. Similarly, the first-generation compound thiacloprid significantly inhibited the hatching ratio and larval development at 9 ng L⁻¹, while it did not affect egg production and larval survival. Second-generation compounds were less toxic than acetamiprid and thiacloprid: clothianidin affected significantly only larval development of the offspring at 62 ng L⁻¹, while thiamethoxam was not toxic at both the tested concentrations (8 ng L⁻¹ and 84 ng L⁻¹). These data evidenced that effects on copepods may occur at concentrations below the chronic aquatic life benchmarks reported by USEPA for acetamiprid (2100 ng L⁻¹) and thiacloprid (970 ng L⁻¹), suggesting that long-term effects of NEOs have been underestimated. A comparison with environmental concentrations evidenced that NEO-mediated effects on copepods are more liable in coastal areas receiving discharge from wastewater treatment plants or diffuse inputs from agricultural land during pesticide application periods.

Introduction

Neonicotinoids (NEOs) are neurotoxic pesticides that disrupt synaptic transmission by acting as nicotinic acetylcholine receptors (nAChRs) agonists (Goulson, 2013). In particular, NEOs have a greater binding affinity for insects' nAChRs than vertebrate receptors; once bound with insects’ nAChRs, they may cause membrane depolarisation, ion channels activation, and propagation of the action potential (Tomizawa and Casida, 2005).

Selectivity towards insects, water solubility, systemic action, and versatile application methods are all properties that made NEOs one of the most relevant chemical classes of insecticides introduced to the global market (Anderson et al., 2015; Jeschke et al., 2011). Conversely, the same properties increased the concern about NEOs' presence in the environment (Bartlett et al., 2018). Water solubility favoured NEOs’ spread into surface and groundwaters through runoff, leaching, and spray drift (Goulson, 2013), while resistance to hydrolysis and, to some extent, photolysis conferred them environmental persistence (Bonmatin et al., 2015). Consequently, NEOs are widely diffused in surface waters (Li et al., 2019; Miller et al., 2021; Morrissey et al., 2015), raising concern regarding the potential effects on non-target species, mainly terrestrial and aquatic insects (Anderson et al., 2015; Raby et al., 2018a). In this context, the research has focused chiefly on freshwater invertebrates, including insects and crustaceans (Bartlett et al., 2018; Finnegan et al., 2017; Maloney et al., 2018, 2021; Raby et al., 2018a, 2018b), while effects on marine invertebrates were less studied.

Crustaceans have been the marine invertebrates more frequently exploited for studying the effects of NEOs on marine species due to their ecological and economic relevance (Butcherine et al., 2021; Williams et al., 1994) and the nervous system's similarity with insects (Osorio et al., 1997; Smarandache-Wellmann, 2016). However, studies addressing the effects of NEOs on crustaceans primarily considered acute exposures (Butcherine et al., 2021; Hano et al., 2019) or effects on early-life stages (Moeris et al., 2021; Picone et al., 2022). Chronic effects were seldom explored, although chronic exposure is a more frequent condition in nature than acute exposure, and chronic effects are more ecologically relevant than lethal and short-term effects.

Copepods are suitable indicators for assessing chronic effects since they 1) are ecologically relevant for the marine and brackish ecosystems due to their grazing activity on phytoplankton and their role as food for fish and other invertebrates, 2) are easily cultured under laboratory conditions, and 3) have a short life cycle allowing the study of transgenerational effects (Kusk and Wollenberger, 2007; Picone et al., 2018; Turner, 2004; Urban-Malinga, 2014). Furthermore, they are sensitive to a wide range of organic and inorganic chemicals (Andersen et al., 2001; Forget-Leray et al., 2005; Moeris et al., 2021; Wollenberger et al., 2005).

The present study aimed to assess the potential chronic effects of four commercially available synthetic NEOs on the reproduction and offspring of the calanoid copepod Acartia tonsa. The species is susceptible to pesticides and other organic chemicals acting as endocrine and nervous transmission disruptors (Breitholtz et al., 2003; Kusk et al., 2011; Picone et al., 2021). Moreover, its worldwide distribution, easy culturing, short generation times and ecological relevance make A. tonsa a suitable bioindicator organism for assessing the effects of toxic substances, including NEOs (Picone et al., 2022). The selected active compounds are the first-generation NEOs acetamiprid (ACE) and thiacloprid (THI) and the second-generation NEOs clothianidin (CLO) and thiamethoxam (TMX). These pesticides have expanded rapidly in the world market in the past decade (Jeschke et al., 2011). However, restrictions on their uses were established by the United States Environmental Protection Agency (USEPA), which cancelled the authorization for 12 products based on TMX and CLO (USEPA, 2019), and the European Union (EU), which first limited the use of TMX and CLO in 2013 (Regulation 485/2013/EU) and then banned all outdoor uses in 2018 (Regulations 784/2018/EU and 785/2018/EU). Furthermore, the approval for THI in the EU was not renewed in 2020 (Commission Implementing Regulation 23/2020/EU). Conversely, no actions were implemented for ACE. Nonetheless, NEOs are still used for plant and seed protection since CLO, TMX, and THI bans were often bypassed by applications for ‘emergency’ authorizations and NEOs are regularly shipped and commercialized in other parts of the world due to a lack of legislation (Dowler, 2021; Dowler and Clarke, 2020; Klingelhöfer et al., 2022).

Fertilised eggs produced by in-house laboratory cultures were exposed to NEOs to assess the effects on egg production, hatching, and the survival and development of the hatched larvae (generation F₁). The exposure was performed at sublethal concentrations, bracketing the onset of adverse effects on the larval development of A. tonsa (Picone et al., 2022).