The Use of Cholinesterase Activity in Flounder (Platichthys flesus) Muscle Tissue as a Biomarker of Neurotoxic Contamination in UK Estuaries

Abstract

This study investigated the occurrence and levels of neurotoxic contamination in UK estuaries by the determination of cholinesterase (ChE) activity in the muscle of the flounder (Platichthys flesus). Samples were obtained from 16 sites in the estuaries of the rivers Humber, Mersey, Tamar, Tees and Tyne. ChE activity levels were compared to those at a reference site- the River Alde, Suffolk. Twelve sites exhibited significantly (p<0.05) reduced ChE activity. The Tyne estuary showed a strong gradient of effect that suggested a source of neurotoxic-contamination upstream of the sampled sites. Representative water samples from several of the estuaries were analysed for the presence of 32 organophosphate (OP) and 20 carbamate (C) pesticides from which residues of nine OPs and six Cs were detected. Correlation analysis of ChE activity with gender, size, GSI, HSI and condition factor suggested that these variables did not influence neural activity in this study. It was concluded that the reduction in ChE activity was contaminant mediated and that OP and C pesticides were probable contributors. Influences on ChE activity, higher order implications and its use as a monitoring tool, including reactivation techniques, are discussed.

Introduction

The estuarine environment is unique in comparison to other water bodies, due to the physico-chemical dynamism associated with the mixing of fresh and marine waters. These characteristics have resulted in the development of rich and diverse ecological systems which are potentially under threat as they act as the conduit for a wide range of anthropogenic inputs to the marine environment from terrestrial and riverine sources. It is known that many UK estuaries are contaminated with a wide range of pollutants including: metals (Laslett, 1995), polycyclic aromatic hydrocarbons (PAH) (Law et al., 1997), pesticides (MAFF, 1995), polychlorinated biphenyls (PCBs) (CEFAS, 1997) and surfactants (Blackburn and Waldock, 1995). Furthermore, it has been demonstrated that these contaminants can exist at potentially harmful concentrations in the water Matthiessen et al., 1993, Kirby et al., 1998 and more especially in sediments (Matthiessen et al., 1998a).

Conventional bioassay methods and chemical analysis techniques have permitted the assessment of the concentrations of specific pollutants in water and sediment and the potential biological effects of these on factors such as survival, growth and reproduction. However, the need to detect and assess the effects of contamination at low concentrations and in complex mixtures has led to the development of a wide range of sub-lethal indicators of exposure to, and effects of, contaminants and other environmental stressors (Livingstone et al., 1997). These indicators are collectively referred to as `biomarkers' and can be either specific (monitoring for the presence/effects of specific chemical classes) or general.

The extent of estuarine contamination and its possible effects on estuarine organisms has been investigated using a number of techniques including fish abundance fluctuations (Pomfret et al., 1991), disease occurrence in fish (Vethaak and Jol., 1996) and the acute toxicity of water (Kirby et al., 1998) and sediment (Matthiessen et al., 1998a). However, these approaches are relatively insensitive and whilst they offer important tools for looking at integrated effects they do not offer the diagnostic ability to investigate potential problems caused by specific contaminant classes. Certain biomarker techniques offer a more focussed approach; metallothionein for metals (Sulaiman et al., 1991), plasma vitellogenin for oestrogenic substances Lye et al., 1997, Allen et al., 1997, Allen et al., 1999, Matthiessen et al., 1998b and ethoxyresorufin-o-deethylase (EROD) induction for PAH/PCBs (Kirby et al., 1999b).

Pesticides have been highlighted as important estuarine contaminants (Matthiessen, 1988). The use of most organochlorine pesticides has been restricted in recent years due to their environmental persistence and this has led to a great increase in the use of organophosphate (OP) and carbamate (C) alternatives (Ceron et al., 1996). Although less persistent than their predecessors, they are generally more toxic and have been responsible for fish kills in the past (Zinkl et al., 1991). Organophosphate and carbamate insecticides are potent neurotoxins and exert their toxicity by blocking the breakdown of acetylcholine (ACh) by the enzyme, acetylcholinesterase (AChE). Acetylcholine is the primary neurotransmitter in the sensory and neuromuscular systems of fish. The activity of this system is vital to normal behaviour and muscular function (Payne et al., 1996) and it represents a prime target on which some toxicants can realize a detrimental effect. The levels of ACh at a neuro-junction must be carefully regulated, and this role is fulfiled by the enzyme AChE, which degrades the ACh into the inactive products choline and acetic acid. These are reabsorbed and used as raw materials for the continued production of ACh. Inhibition of the AChE enzyme will result in a build up of ACh causing a continuous and excessive stimulation of the nerve/muscle fibres which will result in tetany, paralysis and eventual death.

Organophosphate and carbamate pesticides have the potential to exhibit neurotoxic activity at low concentrations. Zinkl et al. (1991) cite examples of median lethal concentrations of OPs (parathion and azinphos-methyl) to fish as low as 10 μg l⁻¹ and sub-lethal effects on olfactory function in Atlantic salmon, which is essential in normal endocrine and reproductive activity, were discovered for the OP diazinon at concentrations as low as 1 μg l⁻¹ (Moore and Waring, 1996). Chemical analysis of surface waters in estuaries and rivers has shown concentrations of fenitrothion up to 0.3 μg l⁻¹ (House et al., 1997), and Virtue and Church (1993) reported short-term peaks of the OP diazinon as high as 200 μg l⁻¹. It is possible that these pesticides in combination are present in UK estuaries at levels causing toxic effects. The degradation of OPs and Cs is relatively rapid (Ferrando et al., 1992) and their persistence in the environment is thought to be relatively short (Murty and Ramani, 1992), and so toxic concentrations are thought to be transient. However, it is also possible that continuous seasonal inputs, which could occur in estuarine environments, could maintain semi-stable concentrations at neurotoxic levels. Furthermore, surveys of fish muscle AChE levels along a pollution gradient in the North Sea (Galgani et al., 1992a) and in marine environments associated with effluents in Canada (Payne et al., 1996) provide strong evidence that significant suppression of neural activity is occurring in some areas.

This study was designed to ascertain whether evidence of neurotoxicity was apparent in UK estuaries using the measurement of ChE activity in flounder (Platichthys flesus) muscle. Flounder is an estuarine/coastal species that resides for the majority of the year in estuaries, only migrating to deeper water in the winter months (Hylland et al., 1996), and as such it represents a sentinel for medium-term exposure. Its relative abundance and broad geographical distribution qualify it as a good target species for biological effects monitoring, and its suitability for such applications has been endorsed by Goksøyr et al. (1996). A closely related species, dab (Limanda limanda), has been used previously to monitor the presence of marine neurotoxicity Galgani et al., 1992b, Bocquené et al., 1993. Although ChE activity has previously been measured in the European flounder before Stieger et al., 1989, Sturm et al., 1999, this appears to be the first time that this species has been used for the environmental monitoring of ChE inhibition.

Chemical analysis of estuarine water from representative sites in the estuaries of the Alde, Humber, Mersey, Tamar, Tees and Tyne was also undertaken for a range of OP and C pesticides in an attempt to link the ChE levels observed to the presence of these potent neurotoxic contaminants. In total, 32 OP and 20 C compounds were selected for analysis on the basis of their scale of use in the UK.