Enhanced trophic transfer of chlorpyrifos from resistant Hyalella azteca to inland silversides (Menidia beryllina) and effects on acetylcholinesterase activity and swimming performance at varying temperatures

Abstract

Chlorpyrifos, an organophosphate (OP) insecticide, is prevalent in aquatic systems globally and is often implicated in aquatic toxicity during storm events. Chlorpyrifos induces toxicity by inhibition of acetylcholinesterase (AChE) activity, which has been related to alterations to fish swimming performance. Resistance to organophosphate insecticides, including chlorpyrifos, is prevalent in populations of the epibenthic amphipod Hyalella azteca in areas with known OP exposure. Previous studies have demonstrated an elevated bioaccumulation potential of insecticide-resistant prey items, however the potential for trophic transfer of chlorpyrifos from OP-resistant prey items and associated neurotoxic effects in fish predators has not been studied. Consequently, the present study aimed to determine the potential for trophic transfer of chlorpyrifos from OP-resistant H. azteca to a known predator, the inland silverside, Menidia beryllina at two temperatures (18 and 23 °C) to simulate temperature changes associated with global climate change (GCC). Fish were fed either ¹⁴C-chlorpyrifos-dosed H. azteca or control animals for 7 d, after which total bioaccumulation, percent parent chlorpyrifos, brain AChE activity and swimming performance (ramp-U_crit) were determined. Fish fed chlorpyrifos-dosed H. azteca bioaccumulated chlorpyrifos ranging from 29.9 to 1250 ng/g lipid, demonstrating the potential for trophic transfer. Lower bioaccumulation and greater biotransformation were observed in M. beryllina at 23 °C as compared to 18 °C, though this was not statistically significant. A significant 36.5% reduction in brain AChE activity was observed in fish fed chlorpyrifos-dosed H. azteca at 23 °C only, which may be attributed to increased biotransformation of parent chlorpyrifos to more potent AChE-inhibiting metabolites. Dietary chlorpyrifos exposure had no significant effect on swimming performance in M. beryllina, though ramp-U_crit was significantly increased by 25% at 23 as compared to 18 °C. These findings confirm the potential for trophic transfer of chlorpyrifos from OP-resistant prey to fish predators and the potential for elevated temperatures to exacerbate the neurotoxic effects of chlorpyrifos.

Introduction

Residues from both legacy and current use insecticides have been shown to have a host of effects on aquatic systems, including selection for pesticide-resistant genotypes (Weston et al., 2013), sub-lethal impacts on non-target organisms (Harwood et al., 2009) and alterations to population dynamics (Jergentz et al., 2004). Insecticide residues typically enter aquatic systems via agricultural and urban run-off, with concentrations found in creeks draining from highly populated areas frequently exceeding toxicity thresholds for resident macroinvertebrates (Amweg et al., 2006; Weston et al., 2005). The organophosphate (OP) insecticide, chlorpyrifos, has been in use for both agricultural and urban applications in the United States for over 60 years (US EPA, 2000) and is often associated with aquatic toxicity after storm events (Bailey et al., 2000). Chlorpyrifos elicits toxicity in target organisms by binding to acetylcholinesterase (AChE) and inhibiting the breakdown of acetylcholine in neuronal synapses, leading to neuronal effects and death (Aldridge, 1950).

Selective pressure arising from chlorpyrifos exposure has led to the development of adaptive pesticide resistance in non-target aquatic invertebrates, including the epibenthic amphipod, Hyalella azteca (Major et al., 2020), a commonly used model organism in toxicity testing. In a survey of H. azteca collected from sites with varying pesticide usage patterns, up to 1000-fold resistance to chlorpyrifos was documented in organisms inhabiting highly contaminated areas relative to laboratory animals (Major et al., 2020). Similar studies have demonstrated resistance to pyrethroid insecticides in H. azteca (Weston et al., 2013), with the associated potential for greater bioaccumulation of pyrethroids and trophic transfer to fish predators, including the inland silverside Menidia beryllina (Muggelberg et al., 2017; Derby et al., 2021). The potential for increased chlorpyrifos bioaccumulation in OP-resistant H. azteca has recently been shown (Johanif et al., 2021), but subsequent trophic transfer of chlorpyrifos to predators has not been demonstrated.

Concurrent with pesticide exposure, global climate change (GCC) is anticipated to lead to significant changes in abiotic parameters of aquatic systems including thermal ranges, salinity and pH (Durack et al., 2012) with interactive effects on contaminant biotransformation and toxicity (Hooper et al., 2013). In the Sacramento-San Joaquin Delta, maximum water temperatures are anticipated to increase by up to 5 °C by 2099 (Wagner et al., 2011) with ecological consequences expected for native fish species. M. beryllina is a euryhaline species, invasive to, and commonly found in Californian waterways and is a frequently used model organism in toxicology studies (Brander et al., 2016; Moreira et al., 2018). Alterations to temperature have been demonstrated to modify the effects of pesticides in M. beryllina, with a 10 °C increase exacerbating the effects of the herbicide diuron on the growth and condition of M. beryllina juveniles (Moreira et al., 2018). Similarly, temperatures of 28 °C were found to increase the effects of bifenthrin on fecundity in M. beryllina as compared to fish maintained at 22 °C (Decourten and Brander, 2017). Comparatively, the influence of temperature on the sublethal impacts of chlorpyrifos on fish are less well known, with studies largely focusing on derivation of acute toxicity parameters under different temperatures (Patra et al., 2015; Philippe et al., 2018).

Due to its role in AChE inhibition, exposure to chlorpyrifos has been shown to induce neurological effects including hyperactivity and paralysis in fish as reviewed in Fulton and Key (2001). At sublethal concentrations, chlorpyrifos exposure can induce a range of effects including alterations to social behavior (Khalil et al., 2013), lipid metabolism (Greer et al., 2019), inhibition of antioxidant enzyme capacity (Marigoudar et al., 2018) and reductions in swimming performance (Tierney et al., 2007). Swimming performance is an important measure of neuromuscular function in fish, which has implications for a range of processes including feeding, migration, and predator avoidance (Goulding et al., 2013). A number of studies have linked chlorpyrifos-induced AChE inhibition to alterations in swimming performance of fish. For example, Tierney et al. (2007) recorded a reduction in two measures of swimming performance in Coho salmon, Oncorhynchus kisutch, exposed to 20 and 40 μg/L chlorpyrifos, with critical swimming speed values reduced to 86.4 and 83.6% of controls, respectively. However, a decrease in brain AChE was recorded at lower concentrations (10 μg/L, 52.2% of control value) as compared to effects on swimming, suggesting a threshold between AChE inhibition and resultant effects on swimming performance.

Though many studies have focused on the lethal and sublethal effects of chlorpyrifos on fish, bioaccumulation and the effects of abiotic parameters, such as temperature on the uptake and biotransformation of chlorpyrifos, remain poorly understood. Biotransformation of chlorpyrifos in fish is catalyzed by the cytochrome P450 and glutathione-s-transferase enzyme families, with five major metabolites recorded; chlorpyrifos-oxon, 3,5,6-trichloropyridinol (TCP), the glucuronide conjugate of TCP, TCP-GA, diethyl phosphate (DEP) and diethyl thiophosphate (DETP) (Bonansea et al., 2017). Barron et al. (1993) studied the distribution of chlorpyrifos in the tissues of channel catfish and the presence of metabolites in the blood, urine and bile of the channel catfish, Ictalurus punctatus, following 24 h exposure to 12 μg/L. The authors found the highest concentrations of parent chlorpyrifos in fat, with the lowest recorded in muscle tissue. TCP was the major metabolite recorded in blood (∼40% of the total residue), with TCP-GA being the dominant compound in both bile (>90% of residue) and urine (60–90% of residue). However, limited studies have quantified the percentage of chlorpyrifos metabolites in fish following exposure, with most studies focusing on activity of biotransformation enzymes as a measure of contaminant biotransformation. Furthermore, to the authors’ knowledge, no studies have focused on the effects of temperature on biotransformation and formation of chlorpyrifos metabolites in fish.

Considering these knowledge gaps, the aims of the present study were as follows: a) to determine the potential for trophic transfer of parent chlorpyrifos and its metabolites from OP-resistant H. azteca to M. beryllina; b) to determine the effects of temperature on bioaccumulation and biotransformation of chlorpyrifos in M. beryllina; and c) to assess the impacts of dietary chlorpyrifos exposure and temperature on AChE inhibition and swimming performance in M. beryllina. To achieve these aims, M. beryllina were fed ¹⁴C-chlorpyrifos-dosed resistant H. azteca at temperatures of 18 and 23 °C for seven days. Following the exposure period, lipid-normalized bioaccumulation, percent parent and metabolites, swimming performance (ramp-U_crit) and brain AChE activity were determined.