EVALUATION OF SENSITIVITY AND SPECIFICITY OF TWO CRUSTACEAN BIOCHEMICAL BIOMARKERS

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1 Environmental Toxicology and Chemistry, Vol. 19, No. 8, pp , SETAC Printed in the USA /00 $ EVALUATION OF SENSITIVITY AND SPECIFICITY OF TWO CRUSTACEAN BIOCHEMICAL BIOMARKERS NATHAN MCLOUGHLIN, DAQIANG YIN, LORRAINE MALTBY,* ROBERT M. WOOD, and HONGXIA YU Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, United Kingdom Department of Environmental Science and Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Nanjing University, Nanjing , Peoples Republic of China (Received 25 August 1999; Accepted 11 January 2000) Abstract Biochemical biomarkers are increasingly used for environmental assessment. Although the emphasis has been on vertebrate biomarkers, invertebrates biomarkers have been developed as well. This study evaluated the usefulness of biomarker responses of freshwater invertebrates by comparing the sensitivity and specificity of endpoints at three levels of biological organization: biochemical, physiological, and individual. The study focused on the epibenthic amphipod Gammarus pulex L., and the end points were cholinesterase (ChE) and glutathione-s-transferase (GST) activity, feeding inhibition, and mortality. Chemicals representing five major classes of toxic chemicals were assessed, including zinc, linear alkylbenzene sulphonate (LAS; surfactant), lindane (organochlorine), pirimiphos-methyl (organophosphorus), and permethrin (pyrethroid). Lethality was the least sensitive endpoint, with 96-h LC50 values ranging from 2.78 g/l for permethrin to 6.31 mg/l for LAS. Comparison of the biochemical biomarkers and the sublethal feeding rate assay indicated that whereas ChE inhibition was a specific indicator of organophosphate exposure, the biochemical assay was more than 13-fold less sensitive than the feeding rate assay. The GST biomarker performed with greater sensitivity but with lower specificity compared with the ChE biomarker. However, only on exposure to lindane did the GST biomarker marginally outperform the feeding rate assay in terms of sensitivity. Feeding inhibition is both a general and a sensitive (LC50, 3%) indicator for exposure to a range of chemicals. The Gammarus sp. ChE biomarker may have utility in providing a diagnostic and rapid indicator of organophosphate exposure, but evidence from this and other studies questions the sensitivity of this biomarker in predicting sublethal, higher-order effects. The GST biomarker may provide a rapid and sensitive indicator for toxicant exposure, but it has limited use as a diagnostic tool and provides only limited improvement in sensitivity over more ecologically relevant sublethal end points (e.g., feeding rate, growth rate). Keywords Biomarkers Cholinesterase Glutathione Feeding inhibition Gammarus pulex INTRODUCTION During the last decade, interest has increased regarding the use of biomarkers, particularly biochemical biomarkers, in environmental assessment [1 3]. For instance, inhibition of acetylcholinesterase activity has been used in the diagnosis of pesticide poisoning of wildlife [4], and it is an integral part of the Wildlife Incident Investigation Scheme in England and Wales [5]. The utility of a biochemical approach is based, in part, on the assumption that low toxicant levels cause biochemical responses within individual organisms before those effects are observed at higher levels of biological organization. Additionally, a study of biochemical effects may indicate potential causal agents of toxicity, because specific enzymatic systems may be affected by particular xenobiotics [2]. The initial development of the biomarker approach was strongly driven by research within vertebrates [6]. However, biochemical biomarkers have been developed using invertebrates as well [7], and given the diversity, abundance, and key ecological role of invertebrates within all ecosystems, the potential role of invertebrate biomarkers in environmental assessment is considerable [8]. Two commonly studied biochemical biomarkers in invertebrates are cholinesterase (ChE) and glutathione-s-transferase (GST) activity. Inhibition of ChE activity has been proposed as an effective tool for monitoring the exposure of organisms to organophosphorus and carbamate pesticides [9,10], whereas * To whom correspondence may be addressed (l.maltby@sheffield.ac.uk). induction of GST can be suggestive of exposure to organochlorine compounds [11,12]. However, recent evidence suggests that the specificity of ChE as an indicator of exposure to organophosphorus and carbamate pesticides should be questioned, because heavy metals, surfactants, and pyrethroid pesticides can inhibit ChE activity [13,14]. Although the utility of a biomarker approach may be reduced because of the lack of specificity regarding contaminants, biomarkers may still hold value if the information generated can lead to improved risk assessments. Consequently, the sensitivity of a given biochemical biomarker can be critical in gauging the application value of that assay [15]. Biochemical biomarkers may provide early detection of contaminant exposure if they are more sensitive than non-biochemical assays. Moreover, they may provide an early indication of the potential effects at higher levels of biological organization if they respond rapidly. Some indication regarding the utility of biomarker-based toxicity assessments can be gained by comparing the performance of biochemical biomarkers to alternative bioassay techniques. The simplest approach for evaluating toxicity is to examine lethal effects. However, lethality is a rather coarse measure of toxicity, and given the low concentrations of many xenobiotics discharged into the environment, use of sublethal physiological endpoints to gauge toxicity has gained increasing popularity. The feeding rate of test organisms is a sensitive indicator of toxic stress in both freshwater and marine species [16 19]. Furthermore, toxicant-induced reductions in feeding 2085

2 2086 Environ. Toxicol. Chem. 19, 2000 N. McLoughlin et al. rate have been correlated with changes in growth and reproduction [20,21], clearly demonstrating the ecological relevance of feeding rate as a toxicity endpoint. This study evaluated the usefulness of biochemical responses of freshwater invertebrates in ecological risk assessments by comparing the sensitivity and specificity of four biological endpoints: ChE activity, GST activity, mortality, and feeding inhibition. Chemicals representing five major classes of toxic chemical (i.e., metals, surfactants, organochlorine, organophosphorus, and pyrethroid pesticides) were assessed. The test species was the epibenthic amphipod Gammarus pulex L., which is widespread and common in freshwaters of the United Kingdom and is an important prey item for fish [22,23]. A detritivore, G. pulex feeds primarily on coarse particulate material such as autumn-shed leaves and, consequently, plays a key role in detritus processing and nutrient cycling [24,25]. Collection of test species MATERIALS AND METHODS Adult male G. pulex L. (length, 5 mm) were collected from Crags Stream (National Grid Reference SK ; Clowne, Derbyshire, UK) and maintained in the laboratory for one week before use in experiments. Animals were maintained at 15 1 C with a photoperiod of 12 h of light and 12 h of dark in an artificial pond water [26]. Animals were fed ad libitum on fungally conditioned alder leaves (Alnus glutinosa L.) [16]. Chemicals and reagents Pirimiphos-methyl (o-2-diethylamino-6-methylpyrimidin- 4-yl-oo-dimethylphosphorothioate) and permethrin (3-phenoxybenzyl(IRS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate) were obtained from Zeneca (Bracknell, UK). Zinc sulphate (ZnSO 4 7H 2 O) was purchased from BDH Laboratory Supplies (Poole, UK). Linear alkylbenzene sulphonate (LAS) with a chain length of 10.3% (w/w) C 10, 34.6% (w/w) C 11, 32.7% (w/w) C 12, 21.6 % (w/w) C 13, and 0.9% (w/w) C 14 was supplied by Unilever (Port Sunlight, UK). Lindane ( -isomer of 1,2,3,4,5,6-hexachlorocyclohexane) and the following reagents were obtained from Sigma Chemical (St. Louis, MO, USA): 5,5 -Ditho-bis(2-nitrobenzoic acid), acetylthiocholine iodide, phenylmethylsulfonylfluoride, 1- chloro-2,4-dinitrobenzene, reduced glutathione, bovine serum albumen, and ethylenediaminetetraacetic. Triton X-100 was purchased from BDH Laboratory Supplies, and Bio-Rad protein assay dye reagent concentrate was obtained from Bio-Rad Laboratories (Munchen, Germany). All test chemicals and reagents had a purity of 99% or greater. Experiment 1: mortality Five test concentrations and a control were established for each test chemical. Nominal test concentrations were 2, 5, 10, 20, and 30 g/l for lindane; 0.04, 0.08, 0.16, 0.3, and 0.6 g/ L for permethrin; 1, 2.5, 5, 10, and 15 g/l for pirimiphosmethyl; 1, 2, 4, 6, and 8 mg/l for zinc; and 1.5, 3, 6, 12, and 18 mg/l for LAS. Gammarus were held individually, without food, in glass tubes (length, 80 mm; diameter, 15 mm) fitted with a 1-mm mesh base. Each treatment consisted of a set of 30 tubes suspended in a 3-L glass aquarium containing 2 L of test solution or artificial pond water (control). Animals were observed at exposure times of 24, 48, 72, 96, 120, and 144 h, and dead animals were recorded and removed at each exposure. Animals were considered to be dead if no appendage movement was visible during a 20-s observation period. The test solutions were changed daily, and all tests were conducted at 15 1 with a photoperiod of 12 h of light and 12 h of dark. Dissolved oxygen, ph, and conductivity were determined daily for each chemical treatment and control using handheld meters (Models 9071, 3310, and 4071; Jenway, Dunmow, UK). Concentrations of lindane, permethrin, and pirimiphos-methyl in test solutions were determined using gas chromatography/mass spectrometry analysis (Model 5988; Hewlett-Packard, Avondale, PA, USA) with detection limits of 0.01 g/l for lindane, 0.04 g/l for permethrin, and 0.02 g/l for pirimiphos-methyl. Zinc concentrations were determined by atomic absorbance spectrophotometry using whole water samples (Perkin-Elmer 2100; Perkin-Elmer, Norwalk, CT, USA) with a detection limit of 0.4 g/l, and LAS concentrations were determined using a methylene blue spectrophotometric method with a detection limit of 0.02 mg/l [27]. Experiment 2: biomarkers Utilizing an design identical to that of the mortality test described earlier, two sets of 30 animals held individually in glass tubes were exposed to each test chemical concentration. One set of 30 animals was exposed for 24 h; the other set was exposed for 48 h. After exposure, live animals were removed from the solutions and lightly blotted with a clean paper tissue to remove excess water before being placed into 1.5-ml polythene Eppendorf tubes. The Eppendorf tubes were then immersed in liquid nitrogen ( 196 C) for 20 s to freeze the animals, which were then stored at 70 C until analysis. Each individual was analyzed for ChE and GST activity. The test solutions were changed daily, and all tests were conducted at 15 1 C with a photoperiod of 12 h of light and 12 h of dark. Dissolved oxygen, ph, and conductivity were determined daily, and test solutions were analyzed as described earlier. Analysis of ChE activity Cholinesterase activity was determined according to the method of Ellman et al. [28] as modified for a 96 well plate reader [29]. The head capsules of test animals were removed and used to prepare samples for ChE analysis. Each frozen head was homogenized for 30 s at 4 C in30 l of 0.02 M phosphate buffer at a ph of 8.0 (containing 1.0% Triton X- 100). The initial homogenate was diluted with 270 l of phosphate buffer with a ph of 8.0 and centrifuged at 4 C at g for three minutes. Two-hundred microliters of the supernatant was diluted to 1 ml with phosphate buffer with a ph of 8.0 (containing 0.1% Triton X-100) and then analyzed for ChE activity and protein content. Reagents were added to a 96-well microtiter plate for ChE analysis in the following order: 100 l of 8 mm 5,5 -dithiobis(2-nitrobenzoic acid), 50 l of diluted supernatant, and 50 l of 16 mm acetylthiocholine iodide. The plate was then preincubated for three minutes at 30 C before being read at 405 nm using an Anthos Lab-tech HTIII plate reader (Anthos LabTech, Salzberg, Austria). The assay was run at 30 C and read every 30 s during a 10-min period. Four independent measurements of ChE activity were made for each individual Gammarus organism, and an average activity calculated. The protein content of each sample was analyzed by adding 200 l of Bio-Rad protein analysis solution to 80 l of supernatant diluted with 720 l of phosphate buffer (ph, 8.0). The samples were left at room temperature for five minutes to allow color

3 Biomarker specificity and sensitivity Environ. Toxicol. Chem. 19, development, after which the absorbance was read at 620 nm. The protein sample absorbance was then compared with a standard curve prepared from bovine serum albumen. The final value for ChE activity was expressed as nmol/min/ g protein. Analysis of GST activity Glutathione-S-transferase activity was determined using a microtiter plate version of the method reported by Habig et al. [30] and Warwick [31] and 1-chloro-2,4-dinitrobenzene as substrate. Samples for GST analysis were prepared from the decapitated bodies of test animals. Each frozen body was homogenized for 30 s at 4 C in 100 l of 0.02 M phosphate buffer with a ph of 6.5 (containing 1.0% Triton X-100 and 1.0% phenylmethylsulfonyl fluoride). The homogenate was diluted with 900 l of phosphate buffer with a ph of 6.5 (containing 1.0% phenylmethylsulfonyl fluoride) and centrifuged at 4 C and 14,000 g for three minutes. One-hundred microliters of the supernatant was diluted to 1 ml using phosphate buffer with a ph of 6.5 (containing 0.1% Triton X-100 and 1.0% phenylmethylsulfonyl fluoride) and then analyzed for GST activity or protein content. Reagents were added to a 96-well microtiter plate for GST analysis in the following order: 50 l of diluted supernatant sample, and 150 l of assay mixture consisting of five parts 20 mm glutathione (containing 1 mm ethylenediaminetetraacetic) solution, nine parts 0.02 M phosphate buffer (ph, 6.5), and one part 40 mm 1-chloro-2,4-dinitrobenzene solution. The 1-chloro-2,4-dinitrobenzene and glutathione solutions were brought to room temperature just before use. The microtiter plate was preincubated for three minutes at 30 C before being read at 340 nm using an Anthos Lab-Tech HTIII plate reader (Anthos Lab-Tech, Salzburg, Austria). The assay was run at 30 C and read every 30 s for 10 min. For each individual Gammarus organism, four independent measurements of GST activity were made, and an average was calculated. The protein content of each sample was analyzed as described earlier but using a phosphate buffer with a ph of 6.5 rather than 8.0. The final value for GST activity was expressed as nmol/min/ g protein. Experiment 3: feeding rate Three test concentrations and a control were established for each test chemical. The nominal test concentrations were 0.4, 2, and 10 g/l for lindane; 0.005, 0.05, and 0.5 g/l for pirimiphos-methyl; 0.01, 0.04, and 0.08 g/l for permethrin; 0.1, 0.3, and 0.9 mg/l for zinc; and 0.1, 0.5, and 1 mg/l for LAS. Twenty G. pulex per treatment were randomly allocated to individual, two-chambered plastic vessels containing 180 ml of test solution or artificial pond water (control). The chambers of each vessel were separated by 1-mm mesh. and the animals were placed in the upper chamber with five Cladosporium sp. inoculated alder leaf disks (diameters, 16 mm) of known dry weight [16]. The leaf disks had been soaked in artificial pond water for 24 h before use. Five two-chambered vessels containing leaf material but no animals were also established for each treatment and used to control for autogenic leaf weight change. The test solutions were changed daily, and all tests were conducted at 15 1 C with a photoperiod of 12 h of light and 12 h of dark. Dissolved oxygen, ph, and conductivity were determined daily, and test solutions were analyzed as described earlier. After six days of exposure, live animals and remaining food material were removed, rinsed in clean water, dried in an oven at 60 C for four days and then weighed. Feeding rate (C), expressed as milligrams (dry wt) of food consumed per milligram (dry wt) of animal per day, was calculated using Equation 1: (L1 C L) L2 C (1) W 6 where L 1 is the dry weight of the food material initially supplied (mg), L 2 is the dry weight of the leaf material remaining after six days (mg), W is the dry weight of G. pulex, and C L is the leaf weight change correction factor given by Equation 2: (C 2 /C 1 ) CL (2) N where C 1 is the initial dry weight of the control leaves (mg), C 2 is the final dry weight of the control leaves (mg), and N is the number of control leaf sets used in each treatment. Statistical analysis All data were checked for normality using normal probability plots and Anderson-Darling normality tests, and feeding rate data were log transformed. Feeding rate and biomarker data were analyzed using one-way analysis of variance, and differences among means were determined using the Tukey test. The LC50 values were calculated using probit analysis, and effective concentration estimates were calculated using linear interpolation methods [32]. All statistical analyses were based on actual exposure concentrations and conducted at a significance level of RESULTS Mortality No significant differences were found in dissolved oxygen, ph, or conductivity between treatments (F 5,30 0.9, p 0.05). The mean values standard deviations across all treatments were mg O 2 /L, ph units, and S/cm, respectively. For all test chemicals, the LC50 was still decreasing with increasing exposure time, even after 144 h (Table 1). The 144-h LC50 values (95% CL) ranged from 0.17 (0.03) g/l for permethrin to 3.85 (0.53) mg/l for LAS. ChE and GST activity No significant differences were found in dissolved oxygen, ph, or conductivity between treatments (F 5, , p 0.05). The mean values standard deviations across all treatments were mg O 2 /L, ph units, and S/cm, respectively. Cholinesterase activities across all control groups were not significantly different (F 4, , p 0.65), with a mean enzyme activity of 0.25 nmol/min/ g protein (coefficient of variation 18.5%). Only pirimiphos-methyl caused a change in the ChE activity of Gammarus, with significant reductions in enzyme activity occurring after both 24- and 48-h exposures to 1.92 and 0.77 g/l, respectively (F 4, , p 0.001). At the highest concentration tested (11.5 g/l), the inhibition of ChE relative to controls was 87% (Fig. 1). Glutathione-S-transferase activities across all control groups were not significantly different (F 4, , p 0.21), with a mean enzyme activity of 0.20 nmol/min/ g protein (coefficient of variation 23.5%). Both lindane and permethrin caused a change in GST activity in Gammarus, with

4 2088 Environ. Toxicol. Chem. 19, 2000 N. McLoughlin et al. Table 1. Median lethal concentrations (95% Cl) for Gammarus pulex exposure for between 24 and 144 h to five chemicals Exposure time (h) Test chemical Pirimiphos-methyl ( g/l) Permethrin ( g/l) Lindane ( g/l) Zinc (mg/l) LAS a (mg/l) (3.63) (1.39) (1.41) (2.52) 3.82 (1.16) (0.55) 8.27 (1.91) 2.78 (1.14) 0.44 (0.03) (3.33) 2.15 (0.57) 6.31 (0.55) 2.14 (0.83) 0.26 (0.03) (0.25) 1.60 (0.36) 4.64 (0.51) 1.45 (0.28) 0.17 (0.03) (0.17) 1.38 (0.31) 3.85 (0.53) a LAS linear alkylbenzene sulphonate. significant increases in enzyme activity occurring after 48-h exposure to lindane at 6.14 g/l or to permethrin at 0.12 g/ L(F 5, , p 0.001). Lindane also caused a significant increase in GST activity after 24-h exposure at 12.3 g/l (F 5, , p 0.001). At the highest concentrations tested (permethrin, 0.45 g/l; lindane, g/l), induction relative to controls was 39.1% for permethrin (Fig. 2a) and 127.3% for lindane (Fig. 2b). Feeding rate No significant differences were found in dissolved oxygen, ph, or conductivity between treatments (F 3, , p 0.05). The mean values standard deviations across all treatments were mg O 2 /L, ph units, and 577 plusmn; 11 S/cm, respectively. Feeding rates did not differ significantly between control groups (F 4, , p 0.47), and the mean control feeding rate was 0.26 mg/mg/d (coefficient of variation 24.2%). All test chemicals produced a significant reduction in the feeding rate of G. pulex (F 3, , p 0.05). Feeding rates of animals exposed to the highest test concentrations were between 30 and 70% of control values (Fig. 3). measured over 144 h was fold more sensitive to pirimiphos-methyl than to ChE activity and 2.11-fold more sensitive to permethrin than to GST activity measured over 48 h. For lindane, feeding rate inhibition was moderately less sensitive than GST activity. DISCUSSION This investigation compared the sensitivity of four established biological endpoints of toxicity (LC50, ChE and GST enzyme activity, and feeding rate) within a single species to five classes of chemicals. After 144-h exposure to all test chemicals, the LC50 was still decreasing, indicating that a steady-state toxicant equilibrium had not been reached in any treatment. However, the rank order of chemical toxicities observed in this study agrees well with previously published data for G. pulex. Reported 96-h LC50 values are 5.9 to 34 g/l for lindane [33,34], 1.0 to 1.9 mg/l for zinc [35], and 3.1 to Comparison of endpoints For both the lethal bioassay and the sublethal feeding rate bioassay, the predicted rank order of chemicals, from most toxic to least toxic, after 144-h exposure was permethrin pirimiphos-methyl lindane zinc LAS (Tables 1 and 2). Sublethal endpoints showed between and fold greater sensitivity compared with median lethal estimates determined during the same exposure period (Table 3). The feeding rate Fig. 1. Mean cholinesterase (ChE) activity ( one standard error of the mean) of Gammarus pulex exposed for 24 h ( and solid line) and 48 h ( and dashed line) to pirimiphos-methyl. Symbols (*) denote treatments with enzymatic activities significantly different from the associated controls. Fig. 2. Mean glutathione-s-transferase(gst) activity ( one standard error of the mean) of Gammarus pulex exposed for 24 h ( and solid line) and 48 h ( and dashed line) to permethrin and lindane. Symbols (*) denote treatments with enzymatic activities significantly different from the associated controls.

5 Biomarker specificity and sensitivity Environ. Toxicol. Chem. 19, Fig. 3. Feeding rate of Gammarus pulex exposed for 144 h to five test chemicals. Values for test chemicals represent actual exposure concentrations. Solid and dashed lines represent mean feeding rate of pooled controls two standard errors of the mean, respectively. Note that all statistical analysis was conducted using unpooled control feeding rates. Symbols (*) denote treatments with feeding rates significantly less than that of controls. 3.4 mg/l for LAS [36]. Results from studies regarding median survival times of G. pulex on exposure to permethrin suggest a 96-h LC50 of less than 1 g/l [37]. To our knowledge, lethality data for pirimiphos-methyl are limited, but Crane et al. [38] reported 30% mortality in G. pulex exposed to a concentration of 5.0 g/l. The feeding rate of G. pulex was significantly reduced after six days of exposure to 10 g/l of lindane, 0.1 mg/l of LAS, 0.06 g/l of permethrin, 0.6 g/l of pirimiphos-methyl, or 0.14 mg/l of zinc. The results for lindane compare favorably with the significant reduction in G. pulex feeding rate observed after four days of exposure to 8.4 g/l in the laboratory [39] or seven days of exposure to 11 g/l in outdoor stream mesocosms [40]. Significant decreases in feeding rate have been previously observed in Gammarus organisms after 48-h exposure to lindane at 5.0 g/l [41]. The 144-h EC10 for feeding rate was 3.7 g/l for lindane, which compares to a lowestobserved-effect concentration for Gammarus population density and drift during a 28-d stream mesocosm study of 3.1 g/ L [40] and a lowest-observed-effect concentration for 14-d juvenile growth of 6.1 g/l [42]. To our knowledge, no previous studies have focused on the effect of LAS, permethrin, or pirimiphos-methyl on the Gammarus sp. feeding rate, but the zinc results are similar to those previously reported [17,35]. Both biochemical biomarker bioassays demonstrated a high degree of specificity and sensitivity compared with the lethality assay. Effects on ChE activity were only observed at exposure to pirimiphos-methyl, with significant inhibition of enzyme activity occurring after 24-h exposure to 1.9 g/l or 48-h exposure to 0.77 g/l. Significant reductions in G. pulex ChE activity have been observed after 24-h exposure to fenitrothion at 1 g/l, parathion at 1 g/l, and malathion at 0.1 g/l [35,43]. In addition, Day and Scott [44] reported that 24-h exposure to azinphosmethyl at 20 g/l significantly inhibited ChE activity in the amphipod Hyallela azteca. However, no evidence from the present study, or from previous studies, suggests that ChE activity in G. pulex is inhibited by chemicals other than organophosphorus pesticides, which contrasts with recent studies by Guilhermino et al. [13] on the bivalve Mytilus galloprovincialis and by Ibrahim et al. [14] on the insect Chironomus riparius. The GST activity was less specific than ChE activity, with significant induction of activity resulting from 24-h exposure to lindane at 12.3 g/l or 48-h exposure to lindane at 6.16 g/l lindane or permethrin at 0.12 g/l. Lindane-induced increase in G. pulex GST activity has been observed previously [31], but to our knowledge, the present study is the first to report increased GST activity in crustaceans on exposure to pyrethroid pesticides (i.e., permethrin). Induction of GST activity by pyrethroids has been reported previously for the insect Spodoptera fugiperda [45]. The sensitivities of the median lethal concentrations of the four assays were between and fold higher than the sublethal concentrations determined for the feeding rate and Table 2. The concentrations (95% CI) of test chemicals resulting in 10% change in cholinesterase (ChE) activity, glutathione-s-transferase (GST) activity, and feeding rate in Gammarus pulex exposed for 24, 48 or 144 h EC10 ChE activity GST acitivity Feeding rate Test chemical 24 h 48 h 24 h 48 h 144 h Pirimiphos-methyl ( g/l) Permethrin ( g/l) Lindane ( g/l) Zinc (mg/l) LAS b (mg/l) 0.94 ( ) a 0.65 ( ) 5.84 ( ) ( ) 2.30 ( ) ( ) ( ) 3.70 ( ) 0.04 ( ) ( ) a no observed effect. b LAS linear alkylbenzene sulphonate.

6 2090 Environ. Toxicol. Chem. 19, 2000 N. McLoughlin et al. Table 3. Relative sensitivities of four biological end points measured in Gammarus pulex exposed to pirimiphos-methyl, permethrin, and lindane Test chemical Pirimiphos-methyl [48-h LC50]/[48-h ChE EC10] a [144-h LC50]/[144-h feeding rate EC10] [48-h ChE EC10]/[144-h feeding rate EC10] Permethrin [48-h LC50]/[48-h GST EC10] b [144-h LC50]/[144-h feeding rate EC10] [48-h GST EC10]/[144-h feeding rate EC10] Lindane [48-h LC50]/[48-h GST EC10] [144-h LC50]/[144-h feeding rate EC10] [48-h GST EC10]/[144-h feeding rate EC10] a ChE cholinesterase. b GST glutathione-s-transferase. Sensitivity ratio the biomarker assays determined during the same exposure period. Feeding rate was the most general indicator of toxicant exposure, with all chemicals tested resulting in reduced ingestion. Biochemical biomarkers responded in a more chemical-specific manner. Cholinesterase inhibition was only detected on exposure to the organophosphate pirimiphos-methyl, with ChE inhibition at the median lethal concentration estimated to be greater than 70%. In most studies, exposure to median lethal concentrations of organophosphates usually results in greater than 50% inhibition of ChE [46 48]. A comparison of an acute endpoint (i.e., immotility) and ChE inhibition in Daphnia and Chironomus sp. indicated that the ChE biomarker was between two- and sixfold more sensitive to organophosphate exposure than lethal endpoints [49]. In similar studies with fish, no-observed-effect concentrations for ChE inhibition were between five-and 15-fold lower compared with survival and growth effect concentrations [50]. The LC50: EC10 ratio of 8.43 found in the present study demonstrates that the sensitivity of inhibited G. pulex ChE activity as an indicator of organophosphate exposure is comparable with that of other aquatic species. The G. pulex GST biomarker was less specific, with detection of significant induction on exposure to both organochlorine (i.e., lindane) and pyrethroid (i.e., permethrin) pesticides. However, the GST biomarker was a more sensitive indicator of exposure compared with the ChE biomarker, with EC10s being more than 20-fold lower than the corresponding LC50s. These results compare well with those of a previous study using G. pulex, in which increased GST activity was detected after 24-h exposure to alcohol ethoxylate and lindane at concentrations 36- and 46-fold less than 96-h LC50 concentrations [31]. Although both biomarkers were more sensitive than the lethal assay, the same was not true when compared with the sublethal feeding rate assay. Inhibition of feeding rate was approximately 30-fold more sensitive than mortality to pirimiphos-methyl exposure. In contrast, inhibition of ChE activity was only eightfold times more sensitive. Comparing the two sublethal endpoints, the EC10 for ChE inhibition was more than 13-fold higher than the EC10 for feeding rate inhibition. Studies with both vertebrates and invertebrates indicate that sublethal endpoints such as growth and reproduction may be more than 200-fold more sensitive than ChE activity as an indicator of exposure [49,51]. However, under field conditions, researchers have reported a reverse in G. pulex ChE sensitivity compared to feeding rate, with significant inhibition of ChE but no observed change in the feeding rate of animals deployed at a malathion-contaminated site [29]. The relative sensitivity of ChE inhibition and feeding rate inhibition may, therefore, depend on the specific organophosphorus pesticide to which the animal is exposed. Biomarker sensitivity relative to feeding rate inhibition improved when comparing GST induction to feeding rate inhibition for animals exposed to lindane or permethrin. Inhibition of feeding rate was six- and 19-fold more sensitive than mortality to lindane exposure and permethrin exposure, respectively, compared with a more than 21-fold difference between GST induction and mortality. The GST EC10 was approximately twice that of the feeding rate EC10 on exposure to permethrin and 1.6-fold lower than that on exposure to lindane. The 48-h EC10 for GST induction was also 2.6-fold lower than the lindane concentration reported to depress the growth of juvenile G. pulex [42]. Induction of GST in G. pulex is, therefore, as sensitive, or slightly more sensitive, to organochlorine and pyrethroid exposure than feeding rate inhibition. Short-term or rapid bioassays have the potential advantage over longer-term, chronic testing (e.g., G. pulex sp. feeding rate test) in identifying potential problems before irreversible damage has been caused to the environment. However, for biomarkers to have a pre-emptive potential in environmental monitoring, the endpoints must be both sensitive and at least correlated with effects at higher levels of biological organization. Few studies have investigated the link between effects at the biochemical level and the integrated effects at the individual, population, or community levels. However, the present study shows that even small changes observed in biochemical biomarkers can be associated with significant reductions in energy intake at the individual level. Whereas sublethal tests are generally considered to be an improvement over traditional lethal test methods, arguments over the most appropriate sublethal endpoint to measure will continue if responses across multiple levels of biological complexity are not examined. Biochemical biomarkers may offer the potential for rapid environmental assessment, but failure to detect toxicant exposure at concentrations that cause detrimental higher-order effects limits their utility. In conclusion, both the biochemical biomarkers and the feeding rate assays were more sensitive than standard lethal tests. However, ChE inhibition in G. pulex, although specific, is a rather insensitive indicator of exposure to organophosphorus pesticides compared with the G. pulex feeding rate assay. The GST biomarker is a marginally more sensitive assay compared with alternative sublethal tests, but it is also a less specific response. Feeding inhibition is a general and sensitive indicator of exposure to a range of chemicals. The G. pulex ChE biomarker may have utility in providing a diagnostic and rapid indicator of organophosphate exposure, but evidence from the present study, and from other studies, questions the sensitivity of this biomarker in predicting sublethal higherorder effects. The GST biomarker may provide a rapid and sensitive indicator of toxicant exposure, but it has limited use as a diagnostic tool and provides only limited improvement in sensitivity over more ecologically relevant sublethal endpoints (e.g., feeding rate, growth rate).

7 Biomarker specificity and sensitivity Environ. Toxicol. Chem. 19, Acknowledgement This work was supported by the UK Natural Environment Research Council (GST/02/1558) and the Environment Agency (National Centre for Ecotoxicology and Hazardous substances) and was conducted while H. Yu and D. Yin were taking part in an exchange visit funded by the Department for International Development. REFERENCES 1. McCarthy JF, Shugart LR, eds Biomarkers of Environmental Contamination. Lewis, Boca Raton, FL, USA. 2. Peakall DB, ed Animal Biomarkers as Pollution Indicators. Chapman & Hall, London, UK. 3. Travis CC, ed Use of Biomarkers in Assessing Health and Environmental Impacts of Chemical Pollutants. Plenum, New York, NY, USA. 4. Mineau P, ed Cholinesterase Inhibiting Insecticides. Their Impact on Wildlife and the Environment. Elsevier, Amsterdam, The Netherlands. 5. Greig-Smith PW Use of cholinesterase measurements in surveillance of wildlife poisoning in farmland. In Mineau P, ed, Cholinesterase Inhibiting Insecticides. Their Impact on Wildlife and the Environment. Elsevier, Amsterdam, The Netherlands, pp National Research Council Committee on biological markers. Environ Health Perspect 74: Lagadic LT, Caquet T, Ramade F The role of biomarkers in environmental assessment. V. Invertebrate populations and communities. Ecotoxicology 3: Depledge MH, Fossi MC The role of biomarkers in environmental assessment. II. Invertebrates.Ecotoxicology 3: Hassall KA The Biochemistry and Uses of Pesticides, 2nd ed. Macmillan, London, UK. 10. Kumar A, Chapman JC Profenofos toxicity to the rainbow fish (Melanotaenia duboulayi). Environ Toxicol Chem 17: Tanaka K, Nakajima M, Kurihara N The mechanism of resistance to lindane and hexadeuterated lindane in the third Yumenoshima strain of housefly. Pestic Biochem Physiol 16: Hans RK, Khan MA, Farooq M, Beg MU Glutathione-Stransferase activity in an earthworm (Pheretima posthuma) exposed to three insecticides. Soil Biol Biochem 25: Guilhermino L, Barros P, Silva MC, Soares AMVM Should the use of inhibition of cholinesterases as a specific biomarker for organophosphate and carbamate pesticides be questioned? Biomarkers 3: Ibrahim H, Kheir R, Helmi S, Lewis J, Crane M Effects of organophosphorus, carbamate, pyrethroid and organochlorine pesticides, and a heavy metal on survival and cholinesterase activity of Chironomus riparius Meigen. Bull Environ Contam Toxicol 60: Shugart LR Molecular markers to toxic agents. In Newman MC, Jagoe CH, eds, Ecotoxicology: A Heirarchical Treatment. Lewis, Boca Raton, FL, USA, pp Naylor C, Maltby L, Calow P Scope for growth in Gammarus pulex, a freshwater detritivore. Hydrobiologia 188/189: Maltby L, Naylor C, Calow P Effect of stress on a freshwater benthic detritivor: Scope for growth in Gammarus pulex. Ecotoxicol Environ Saf 19: Butler R, Roddie D, Mainstone CP The effects of sewage sludge on two life history stages of Mytilus edulis. Chem Ecol 4: Roddie BD, Redshaw CJ, Nixon S Sub-lethal biological effects monitoring using the common mussel (Mytilus edulis): Comparison of laboratory and in situ effects of an industrial effluent discharge. In Tapp JF, Hunt SM, Wharfe JR, eds, Toxic Impacts of Wastes on the Aquatic Environment. Royal Society of Chemistry, Cambridge, UK, pp Maltby L, Naylor C Preliminary observations on the ecological relevance of the Gammarus scope for growth assay: Effect of zinc on reproduction. Funct Ecol 4: Maltby L Stress, shredders and streams: Using Gammarus energetics to assess water quality. In Sutcliffe DW, ed, Water Quality and Stress in Marine and Freshwater Systems; Linking Levels of Organisation. Freshwater Biological Association, Cumbria, UK, pp Welton JS Life-history and production of the amphipod Gammarus pulex in a Dorset chalk stream. Freshwater Biol 9: Crane M Population characteristics of Gammarus pulex (L.) from five English streams. Hydrobiologia 281: Kaushik NK, Hynes HBN The fate of dead leaves that fall into streams. Arch Hydrobiol 68: Willoughby LG, Sutcliffe DW Experiments on the feeding and growth of the amphipod Gammarus pulex (L.) related to its distribution in the River Duddon. Freshwater Biol 6: Health and Safety Executive Methods for the determination of ecotoxicity. COP8. Her Majesty s Stationery Office, London, UK. 27. Standing Committee of Analysts Analysis of surfactants in waters, wastewaters and sludges. Methods for the examination of waters and associated materials. Her Majesty s Stationery Office, London, UK. 28. Ellman LG, Courtney KD, Anders V, Featherstone RM A new and rapid colormetric determination of acetylcholinesterase activity. Biochem Pharmacol 7: Crane M, Delaney P, Watson S, Parker P, Walker C The effect of malathion-60 on Gammarus pulex (L) below watercress beds. Environ Toxicol Chem 14: Habig WH, Pabst MJ, Jakoby W Glutathione-S-transferase (the first enzymatic step in mercapturic acid formation). J Biol Chem 249: Warwick O The use of a biomarker to assess the effect of xenobiotic exposure on the freshwater invertebrate Gammarus pulex. PhD thesis. University of Sheffield, Sheffield, UK, pp Norberg-King TJ, ed An interpolation estimate for chronic toxicity: The IC p approach. Technical Report O5 88. U.S. Environmental Protection Agency, Duluth, MN. 33. Abel PD Toxicity of -hexachlorocyclohexane (lindane) to Gammarus pulex: Mortality in relation to concentration and duration of exposure. Freshwater Biol 10: Stephenson RR Effects of water hardness, water temperature, and size of the test organism on the susceptibility of the freshwater shrimp, Gammarus pulex (L.) to toxicants. Bull Environ Contam Toxicol 31: Crane M Effect of zinc on four populations and two generations of Gammarus pulex (L). Freshwater Biol 33: Lewis MA, Suprenant D Comparative acute toxicities of surfactants to aquatic invertebrates. Ecotoxicol Environ Saf 7: Abel PD, Garner SM Comparisons of median survival times and median lethal exposure times for Gammarus pulex exposed to cadmium, permethrin and cyanide. Water Res 20: Crane M, Attwood C, Sheahan D, Morris S Toxicity and bioavailability of the organophosphorus insecticide pirimiphosmethyl to the freshwater amphipod Gammarus pulex L. in laboratory and mesocosm systems. Environ Toxicol Chem 18: Blockwell SJ, Taylor EJ, Jones I, Pascoe D The influence of fresh water pollutants and interaction with Asellus aquaticus (L.) on the feeding activity of Gammarus pulex (L.). Arch Environ Contam Toxicol 34: Mitchell GC, Bennett D, Pearson N Effects of lindane on macroinvertebrates and periphyton in outdoor artificial streams. Ecotoxicol Environ Saf 25: Malbouisson JFC, Young TWK, Bark AW Use of feeding rate and re-pairing of precopulatory Gammarus pulex to assess toxicity of gamma-hexachloro-cyclohexane (lindane). Chemosphere 30: Blockwell SJ, Pascoe D, Taylor EJ Effects of lindane on the growth of the freshwater amphipod Gammarus pulex (L.). Chemosphere 32: Streit B, Kuhn K Effects of organophosphorous insecticides on autochthonous and introduced Gammarus species. Water Sci Technol 29: Day K, Scott IM Use of acetylcholinesterase activity to detect sublethal toxicity in stream invertebrates exposed to low concentrations of organophosphate insecticides. Aquat Toxicol 18: Punzo F Detoxification enzymes and the effects of tem-

8 2092 Environ. Toxicol. Chem. 19, 2000 N. McLoughlin et al. perature on toxicity of pyrethroids to the fall armyworm Spodoptera fugiperda (Lepidoptera: Noctuidae). Comp Biochem Physiol C 105: Ludke JL, Hill EF, Dieter MP Cholinesterase (ChE) response and related mortality among birds fed ChE inhibitors. Arch Environ Contam Toxicol 3: Grue CE, Hart ADM, Mineau P Biological consequences of depressed brain cholinesterase activity in wildlife. In Mineau P, ed, Cholinesterase Inhibiting Insecticides. Their Impact on Wildlife and the Environment. Elsevier, Amsterdam, The Netherlands, pp Zinkl JG, Kockhart W, Kenny SA, Ward FJ The effects of cholinesterase inhibiting insecticides on fish. In Mineau P, ed, Cholinesterase Inhibiting Insecticides. Their Impact on Wildlife and the Environment. Elsevier, Amsterdam, The Netherlands, pp Sturm A, Hansen PD Altered cholinesterase and monooxygenase levels in Daphnia magna and Chironomus riparius exposed to environmental pollutants. Ecotoxicol Environ Saf 42: Beyers DW, Sikoski PJ Acetylcholinesterase inhibition in federally endangered Colorado squawfish exposed to carbaryl and malathion. Environ Toxicol Chem 13: Matz AC, Bennett RS, Landis WG Effects of azinphosmethyl on northern bobwhite: A comparison of laboratory and field results. Environ Toxicol Chem 17: