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Effect of Temperature and Pirimiphos Methyl on Biochemical Biomarkers in Chironomus riparius Meigen
Ecotoxicology and Environmental Safety 52, 128}133 (2002) Environmental Research, Section B doi:10.1006/eesa.2002.2160, available online at http://www.idealibrary.com on
Effect of Temperature and Pirimiphos Methyl on Biochemical Biomarkers in Chironomus riparius Meigen Amanda Callaghan,* Thomas C. Fisher,* - Albania Grosso,- Graham J. Holloway,* and Mark Crane*Division of Zoology, School of Animal and Microbial Sciences, The University of Reading, Whiteknights, P.O. Box 228, Reading, Berkshire RG6 6AJ, United Kingdom; and -School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 OEX, United Kingdom Received April 24, 2001
Fourth-instar Chironomus riparius Meigen larvae were exposed to the organophosphate (OP) insecticide pirimiphos methyl (0, 0.1, 1.0, and 10 g/L) for 48, 72, or 96 h at three temperatures (3, 12, or 223C). Two biochemical biomarkers, acetylcholinesterase (AChE) and glutathione S-transferase (GST), were measured in individual larvae from each treatment. AChE activity was inhibited by the OP in a dose-responsive fashion. This response remained similar at all three temperatures, demonstrating that AChE is a robust and speci5c biomarker. Exposure duration had little e4ect on AChE activity. In contrast, GST activity was induced at the highest OP insecticide concentration, but induction was also evident at 33C. There was a signi5cant e4ect of exposure duration, with an overall decline in GST activity over time. This result agrees with previous work suggesting that GSTs are not particularly suitable for use as a biomarker of pesticide exposure or e4ect in Chironomus. 2002 Elsevier Science (USA)
Key Words: Chironomus riparius; acetylcholinesterase; glutathione S-transferase; biomarker; organophosphate insecticide; temperature.
INTRODUCTION
Biomarkers that employ enzyme activity measurements to detect low levels of pollution are increasingly used in ecological risk assessments of aquatic ecosystems. These biomarkers have the potential to identify the incidence of exposure to, and e!ects caused by, xenobiotics such as pesticides, so providing an early warning of potentially damaging e!ects at higher levels of biological organization (McCarthy and Shugart, 1990). The de"nition of what constitutes a biomarker is open to debate (Peakall and Walker, 1996; Van Gestel and Van Brummelen, 1996), but a working de"nition of &&biochemical biomarker'' is &&biochemical sublethal changes resulting from individual exposure to To whom correspondence should be addressed. Fax: 0118 931 0180. E-mail: [email protected].
xenobiotics'' (Lagadic et al., 1994). Biochemical changes include the induction of detoxi"cation enzymes that are capable of degrading the xenobiotic or the reduction in activity of enzymes sensitive to inhibition by the xenobiotic. Acetylcholinesterase (AChE) is a carboxylesterase enzyme important in the maintenance of normal nerve function. AChE is the primary target of neurotoxic pesticides such as organophosphates (OPs) and carbamates designed to control invertebrate pests (Hassall, 1990). Inhibition resulting from irreversible binding at the AChE active site leads to the accumulation of acetylcholine in the synapse, resulting in the disruption of normal function (Habig and DiGiulio, 1991). The measurement of AChE activity to detect inhibition resulting from pesticide binding has been used successfully in the past with various invertebrates (Karnak and Collins, 1974; Day and Scott, 1990; Crane et al., 1995; Ibrahim et al., 1998) including Chironomus riparius (Detra and Collins, 1991; Sturm and Hansen, 1999; Fisher et al., 2000; Olsen et al., 2001; Callaghan et al., 2001; Crane et al., in press). Glutathione S-transferases (GSTs) are also a popular biochemical biomarker that can detect the presence of various pesticides through the measurement of induction (Ladagic et al., 1994; Motoyama and Dauterman, 1977; Usui et al., 1977; Clark et al., 1986). GST genes represent a family of detoxi"cation enzymes that catalyze the conjugation of reduced glutathione (GSH) with a group of compounds having electrophilic centers. These can include nitrocompounds (Usui et al., 1977), polycyclic aromatic hydrocarbons (Cheung et al., 2001), organophosphates (Motoyama and Dauterman, 1977; Usui et al., 1977), and organochlorines (Clark et al., 1986). The GSH conjugation products become less toxic and more water soluble, so allowing easier excretion from cells after further metabolism. Unlike the AChE biomarker, GSTs are far less speci"c and are therefore an ideal partner to help detect pesticides that do not bind to the AChE active site, such as organochlorines.
128 0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
BIOCHEMICAL BIOMARKERS IN CHIRONOMIDS
Chironomus riparius Meigen larvae are widely used in toxicity tests. They are easy to culture, sensitive to many pollutants, and have a short life cycle (Ingersoll and Nelson, 1990). Their ability to burrow into sediments makes them good biological indicators for toxicants that may be adsorbed to sediments. The size of the third- and fourth-instar larvae is su$ciently large to allow measurement of enzyme activity in individuals. There is a distinct advantage to using individual, rather than batches of organisms, for this kind of analysis because the large set of data points produced allows for more powerful statistical analyses and the investigation of interindividual variability. The objective of this study was to expose larvae of C. riparius to an organophosphate pesticide at di!erent temperatures through time and measure subsequent e!ects on biomarkers. This was to test the speci"city of each biomarker to the pesticide and determine the extent to which variation in activity can be related to di!erences in environmental temperature. MATERIALS AND METHODS
Exposure of Chironomid Larvae to the Insecticide C. riparius larvae, initially obtained from WRc plc (Medmenham, UK), were cultured according to standard methods (ASTM, 1999). One fourth-instar larva was added at random to each of 192 100-ml acid-washed beakers until each beaker contained 10 larvae. Sixty-four beakers were placed in a 33C room, 64 in a 123C room, and 64 in a 223C room. The pH (Whatman pH -sensor), temperature, and dissolved oxygen concentration (Jenway 9071 DO meter) were determined before organisms were added to the beakers. The insecticide pirimiphos methyl (o-2-diethylamino-6-methylpyrimidin-4-yl o,o-dimethyl phosphorothioate, CAS No. 29232-93-7, kindly provided by Zeneca Agrochemicals, Jealott's Hill, Berks, UK) was added to the water at nominal concentrations of 0, 0.1, 1, and 10 g/L. Beakers were arranged at random on a bench and covered with polyethylene "lm (Sainsbury food wrap) to reduce evaporation. After 48, 72, and 96 h, mortality, pH, temperature, and dissolved oxygen concentration were determined in each vessel. Two replicate beakers of 10 larvae were removed at each time point for each treatment and larvae frozen individually in Eppendorf tubes in liquid nitrogen for subsequent biomarker analysis. Eppendorf tubes were stored at !803C. Acetylcholinesterase Analysis Each snap-frozen larva was homogenized in 0.02 M icecold phosphate bu!er, pH 8 (PB), containing 1% triton X-100. Tubes were centrifuged (Biofuge, Heraeus instruments) at 13,000g and 43C for 4 min.
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The AChE assay was based on the method of Ellman et al. (1961) modi"ed for use in a microtiter plate (Fisher et al., 2000). The 96-well microtiter plate (MPO1, Life Science (UK) Ltd, Greiner) was loaded with 100 l of 8 mM di-thionitrobenzene (Sigma D-8130) in PB and 0.75 mg/ml NaHCO . Three replicates of 50 l of assay blank (PB, pH 8, containing 0.1% triton x-100) or quality control enzyme (eel cholinesterase, Sigma C-3389 made up on ice in a cold room as a nominal 0.5 unit/ml in ice-cold PB), or homogenate supernatant were added. This was followed by the addition of 50 l of 16 mM acetylthiocholine iodide (Sigma A-5751) in PB. The microtiter plate was inserted into the plate reader (Anthos Labtec, Salzburg) and incubated for 5 min at 303C. There was a premeasurement mix for 2.5 min at 1150 rpm. The activity rate was measured as change in OD/min at 412 nm. The molar extinction coe$cient of 8160 for a light path of 0.6 cm was used for activity calculation. Glutathione S-transferase Analysis GST analysis was based on the method of Habig et al. (1974), modi"ed for use in microtiter plates. The microtiter plate was loaded in ice with three replicates of 50 l of assay blank (PB containing 0.1% triton X-100 and 0.1% (v/v) phenylmethylsulfonyl#oride (PMSF, Sigma P-7626)), standard enzyme (1 unit/ml of Equine GST (Sigma G-6511)) in ice-cold phosphate bu!er, pH 6.5, containing 1 mg/ml bovine serum albumin (BSA, fraction V, 96}99% (Sigma A-2153)), and samples (diluted with two volumes of PB adjusted to produce a "nal pH of 6.5, containing 0.15% (v/v) PMSF). The plate was then incubated for 5 min at 303C in the plate reader. Meanwhile the substrate mixture was made by adding 7 ml of 20 mM reduced glutathione (Sigma G4251) in 0.1 M potassium dihydrogen phosphate KH PO (anhydrous, 99% purity, Sigma P5379), 1 mM ethylenediamine tetraacetic acid (Sigma ED4SS) to 12.5 ml 0.02M sodium hydrogen phosphate bu!er, pH 6.5. After a 5-min incubation of the solution at 303C in a thermostatic bath (Grant Instruments), 1.4 ml of 40 mM 1-chloro-2,4-dinitrobenzene (Sigma C-6396) in 95% ethanol was added; 50 l of the substrate mixture was then added to the microtiter plate and the plate was incubated for a further 2 min at 303C accompanied by intermittent shaking at 1150 rpm. The measurement period was 10 min at 303C. The rate was measured as a single wavelength kinetic change in OD/min at 340 nm. The molar extinction coe$cient of 5760 for a light path of 0.6 cm was used for the activity calculation. The activities of both GST and AChE were expressed as activity per unit protein. The protein assay was a microtiter plate version of the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL).The BCA working reagent mixture was incubated in a water bath at 303C for 10 min prior to the addition of 200 to 20 l of supernatant homogenate or
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standard protein solutions (0, 100, 200, 400, 600, 800, 1100, 1500 g/L of BSA in PB, pH 8, containing 0.1% triton x-100). The plate was then inserted into the preheated plate reader and the reaction run for 10 min at 303C. The rate was measured as change in OD/min at 550 nm. Protein concentration was calculated in relation to a regression line drawn through points obtained from the standard curve. Statistical Analyses Analysis of variance (ANOVA) was used to detect di!erences between treatments. Survival data were arcsine-transformed before analysis. Exposure duration, temperature, and pesticide concentration were the three factors and either AChE or GST activity was the dependent variable. A Student}Newman}Keuls (SNK) test was then used to identify di!erences between treatment groups. Analyses were performed using Unistat 4.5 software (Unistat Ltd., 1996). RESULTS
Physical Data The pH of the test water did not vary from 7.0 throughout the experiment. The mean temperature in vessels in each of the three test rooms was 3, 12, and 223C. Dissolved oxygen concentrations did not fall below 65% air saturated volume. Mortality There was a signi"cant main e!ect on chironomid survival of both temperature (F "5.27, P"0.0062) and time (F "26.51, P(0.001), and an interaction be tween these two factors (F "4.83, P"0.0002). This is because animals died faster at 3 and 123C compared to 223C and also, mortality increased after 72 and 96 h of exposure (Fig. 1). There was neither a signi"cant e!ect of pesticide concentration on mortality, nor any other twoway or three-way interaction between factors. AChE Data Figure 2 illustrates mean AChE activity in larvae exposed to three di!erent temperatures and four concentrations of pirimiphos methyl over 96 h. There was a signi"cant interaction between exposure duration, temperature, and pesticide concentration (F "2.035, P"0.019). A concentration}response relationship was evident, with signi"cant reductions in AChE activity found between 0.1, 1, and 10 g/L treatments (SNK test, P(0.05). There were no signi"cant di!erences between the control and the 0.1 g/L treatment. The SNK test was unable to detect signi"cant di!erences between treatments for the other two main factors, exposure duration and temperature, and Fig. 2 suggests that interactions between these factors and
FIG. 1. Survival of C. riparius exposed to pirimiphos methyl (0, 0.1, 1.0, and 10 g/L) at three temperatures (3, 12, and 223C) for 24, 48, 72, and 96 h.
concentration of pirimiphos methyl were of little biological signi"cance. Glutathione S-transferase Data GST activities in larvae exposed to three di!erent temperatures and at four concentrations of pirimiphos methyl over 96 h are presented in Fig. 3. There were no signi"cant interactions between the factors when the GST data were analyzed. Instead, each factor had a signi"cant main e!ect: exposure duration (F "5.522, P"0.004), temper ature (F "33.889, P(0.0001), and pesticide concen tration (F "5.763, P"0.0007). This is because GST activity was signi"cantly higher in animals sampled after 48 h compared with those sampled at 72 and 96 h (SNK, P(0.05), signi"cantly higher (SNK, P(0.05) in animals maintained at 33C when compared with those kept at 12 and 223C, and signi"cantly lower (SNK, P(0.05) in animals maintained at 223C when compared with those kept at 12 and 33C, and signi"cantly higher (SNK, P(0.05) in animals exposed to the 10 g/L treatment.
BIOCHEMICAL BIOMARKERS IN CHIRONOMIDS
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pesticide poisoning but relatively insensitive to environmental variation. These results demonstrate that AChE inhibition is caused by organophosphates but remains invariable over changing temperatures. AChE inhibition in chironomids is a very robust biomarker. Olsen et al. (2001) found that organic carbon concentrations at 13 sites had no signi"cant in#uence on AChE activity in "eld-deployed C. riparius larvae. A previous study of AChE activity in C. riparius following a 48-h exposure to pirimiphos methyl found that although activity was associated with reduced larval weight and reduced adult fecundity after several days, it was not in#uenced by food ration (Crane et al., in press). This suggests that measurement of C. riparius AChE in "eld situations, where food ration may vary considerably, is a useful method for monitoring exposure to organophosphate insecticides, and one that is predictive of e!ects on individual health. This latter fact is supported by correlations between AChE inhibition by chlorpyrifos in C. riparius larvae and development time, adult size, and burrowing behavior (Callaghan et al., 2001). Although GSTs are induced by both organophosphate and organochlorine insecticides in the house#y (Hayaoka
FIG. 2. Acetylcholinesterase activities (nmol/min/g protein) in C. riparius larvae after exposure to pirimiphos methyl (0, 0.1, 1.0, and 10 g/L) at three temperatures (3, 12, and 223C) for 48, 72, and 96 h. Error bars are standard errors of the mean.
DISCUSSION
Temperature is an important environmental determinant for ectothermic organisms since it a!ects many biological processes. Many organisms operate optimally within a very narrow temperature range. Deviations from this range can be stressful, especially during development (Parsons, 1987, 1989; Imasheva et al., 1997). At the molecular level, temperature stress directly a!ects biological molecules such as enzymes, which are crucial for the survival of the organism, whereas at the population level it may result in chronic e!ects within the genetic architecture of the population (Imasheva et al., 1997). Whereas temperature can be a general stressor, xenobiotic compounds such as pirimiphos methyl are often more speci"c because they generally a!ect individual pathways within the organism (Parsons, 1990). Biomarkers can be applied to detect both forms of stress and ideally relate exposure to e!ect. The measurement of AChE activity is a fairly well established biomarker of organophosphate poisoning in C. riparius (Fisher et al., 2000, Callaghan et al., 2001; Detra and Collins, 1991; Ibrahim et al., 1998). However, if AChE is to be useful as a real tool to help detect low levels of organophosphate pollution, it should be very speci"c to
FIG. 3. Glutathione-S-transferase activities (nmol/min/g protein) in C. riparius larvae after exposure to pirimiphos methyl (0, 0.1, 1.0, and 10 g/L) at three temperatures (3, 12, and 223C) for 48, 72, and 96 h. Error bars are standard errors of the mean.
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and Dauterman, 1982; Clark et al., 1986), Lepidoptera (Lagadic et al., 1994), and the American cockroach (Usui et al., 1977), no such e!ect has been found in C. riparius until now. However, in this study a statistically signi"cant GST induction following pirimiphos methyl exposure occurred at the highest test concentration (10 g/L) only. Negative results have previously been found following exposure to lindane and pirimiphos methyl in C. riparius larvae (Hirthe et al., 2001, Crane et al., in press) and the use of GSTs as biomarkers of xenobiotic exposure has been questioned by other authors. Various pollutants have failed to induce GSTs in invertebrates (Stegeman et al. 1992; Fitzpatrick et al., 1995; Bond and Bradley, 1997) and vertebrates (Fenet et al., 1998). In addition, variation in GST activity related to seasonal variation in temperature and oxygen levels has been found (Power and Sheehan, 1996). The only signi"cant inductions of GST activity found in the current experiment were at low temperature and in the 10 g/L OP treatment. Although signi"cant, the induction associated with this latter treatment was largely driven by results from all exposure durations at 123C and 48-h exposure in the 223C treatment, as seen in Fig. 3. This rather inconsistent pattern of induction would be di$cult to interpret in a "eld situation. GSTs represent a family of genes (Clark et al., 1973; Motoyama and Dauterman, 1977; Wang et al., 1991). Clark et al. (1986) puri"ed GST from two strains of house#y and found that both had two forms, each with speci"city toward certain pesticides. It is possible that the substrate used in the current method detects only one of these genes optimally, the one that coincidentally is not induced to a large extent by either OPs or organochlorines. Alternatively, GST genes in C. riparius may not generally be sensitive to induction by these pesticides. CONCLUSION
The inhibition of AChE activity in C. riparius larvae as a biomarker of OP pesticide has again proved to be sensitive and robust under di!erent environmental conditions. These results support an increasing body of data that demonstrate the suitability of this assay in C. riparius to identify and monitor OP pollution. GST induction was less sensitive as a biomarker of OP pollution and was in#uenced by temperature, with induction evident at 33C. There was a signi"cant e!ect of exposure duration, with an overall decline in GST activity over time. This result agrees with previous work suggesting that GSTs are not particularly suitable for use as a biomarker of pesticide exposure or e!ect in Chironomus.
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ACKNOWLEDGMENTS
Habig, C. and DiGiulio, T. (1991). Biochemical characteristics of cholinesterase in aquatic organisms. In Cholinesterase Inhibiting Insecticides: ¹heir Impact on =ildlife and the Environment (P. Mineau, Ed.), pp. 19}33. Elsevier Science, Amsterdam.
This project was funded by the NERC Environmental Diagnostics Thematic Programme (GST 02/1558).
Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974). Glutathione S-transferase: The "rst enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130}7139.
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Olsen, T., Ellerbeck, L., Fisher, T., Callaghan, A., and Crane, M. (2001). Variability in acetylcholinesterase and glutathione S-transferase activities in Chironomus riparius Meigen deployed in situ at uncontaminated "eld sites. Environ. ¹oxicol. Chem. 20. Parsons, P. A. (1987). Evolutionary rates under environmental stress. Evol. Biol. 21, 311}347. Parsons, P. A. (1989). Environmental stresses and conservation of natural populations. Annu. Rev. Ecol. Syst. 20, 29}49. Parsons, P. A. (1990). Fluctuating asymmetry: An epigenetic measure of stress. Biologic. Rev. 65, 131}145. Peakall, D. B. and Walker, C. H. (1996). Commentary. Ecotoxicology 5, 227. Power, A. and Sheehan, D. (1996). Seasonal variation in the anitoxidant defence systems of gill and digestive gland of the Blue Mussel Mytilus edulis. Comp. Biochem. Physiol. 114, 99}103. Stegeman, J., Brouwer, M., DiGuilio, R., Forlin, L., Fowler, B., Sanders, B., and VanHeld P. (1992). Molecular response to environmental contamination: Enzymes and protein system as indicators of chemical exposure and e!ect. In Biomarkers: Biological, Physiological and Histological Markers of Anthropogenic Stress (R. Huggett, R. Kimerele, P. J. Mehrle, and H. Bergman, Eds.), pp. 273}275. Lewis, Boca Raton, FL. Sturm, A. and Hansen, P. D. (1999). Altered cholinesterase and monooxygenase levels in Daphnia magna and Chironomus riparius exposed to environmental pollutants. Ecotoxicol. Environ. Saf. 42, 9}15. Usui, K., Fukami, J., and Shishido, T. (1997). Insect glutathione S-transferase: Separation of transferases from fat bodies of American cockroaches active on organophosphorus triesters. Pestic. Biochem. Physiol. 7, 249}260. Van Gestel, C. A. M. and van Brummelen, T. C. (1996). Incorporation of the biomarker concept in ecotoxicology calls for a rede"nition of terms. Ecotoxicology 5, 217}225. Wang, J. Y., McCommas, S., and Sylvanen, M. (1991). Molecular cloning of a glutathione S-transferase overproduced in an insecticide-resistant strain of the house#y (Musca domestica). Mol. Gen. Genet. 227, 260}266.
Effect of Temperature and Pirimiphos Methyl on Biochemical Biomarkers in Chironomus riparius Meigen Amanda Callaghan,* Thomas C. Fisher,* - Albania Grosso,- Graham J. Holloway,* and Mark Crane*Division of Zoology, School of Animal and Microbial Sciences, The University of Reading, Whiteknights, P.O. Box 228, Reading, Berkshire RG6 6AJ, United Kingdom; and -School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 OEX, United Kingdom Received April 24, 2001
Fourth-instar Chironomus riparius Meigen larvae were exposed to the organophosphate (OP) insecticide pirimiphos methyl (0, 0.1, 1.0, and 10 g/L) for 48, 72, or 96 h at three temperatures (3, 12, or 223C). Two biochemical biomarkers, acetylcholinesterase (AChE) and glutathione S-transferase (GST), were measured in individual larvae from each treatment. AChE activity was inhibited by the OP in a dose-responsive fashion. This response remained similar at all three temperatures, demonstrating that AChE is a robust and speci5c biomarker. Exposure duration had little e4ect on AChE activity. In contrast, GST activity was induced at the highest OP insecticide concentration, but induction was also evident at 33C. There was a signi5cant e4ect of exposure duration, with an overall decline in GST activity over time. This result agrees with previous work suggesting that GSTs are not particularly suitable for use as a biomarker of pesticide exposure or e4ect in Chironomus. 2002 Elsevier Science (USA)
Key Words: Chironomus riparius; acetylcholinesterase; glutathione S-transferase; biomarker; organophosphate insecticide; temperature.
INTRODUCTION
Biomarkers that employ enzyme activity measurements to detect low levels of pollution are increasingly used in ecological risk assessments of aquatic ecosystems. These biomarkers have the potential to identify the incidence of exposure to, and e!ects caused by, xenobiotics such as pesticides, so providing an early warning of potentially damaging e!ects at higher levels of biological organization (McCarthy and Shugart, 1990). The de"nition of what constitutes a biomarker is open to debate (Peakall and Walker, 1996; Van Gestel and Van Brummelen, 1996), but a working de"nition of &&biochemical biomarker'' is &&biochemical sublethal changes resulting from individual exposure to To whom correspondence should be addressed. Fax: 0118 931 0180. E-mail: [email protected].
xenobiotics'' (Lagadic et al., 1994). Biochemical changes include the induction of detoxi"cation enzymes that are capable of degrading the xenobiotic or the reduction in activity of enzymes sensitive to inhibition by the xenobiotic. Acetylcholinesterase (AChE) is a carboxylesterase enzyme important in the maintenance of normal nerve function. AChE is the primary target of neurotoxic pesticides such as organophosphates (OPs) and carbamates designed to control invertebrate pests (Hassall, 1990). Inhibition resulting from irreversible binding at the AChE active site leads to the accumulation of acetylcholine in the synapse, resulting in the disruption of normal function (Habig and DiGiulio, 1991). The measurement of AChE activity to detect inhibition resulting from pesticide binding has been used successfully in the past with various invertebrates (Karnak and Collins, 1974; Day and Scott, 1990; Crane et al., 1995; Ibrahim et al., 1998) including Chironomus riparius (Detra and Collins, 1991; Sturm and Hansen, 1999; Fisher et al., 2000; Olsen et al., 2001; Callaghan et al., 2001; Crane et al., in press). Glutathione S-transferases (GSTs) are also a popular biochemical biomarker that can detect the presence of various pesticides through the measurement of induction (Ladagic et al., 1994; Motoyama and Dauterman, 1977; Usui et al., 1977; Clark et al., 1986). GST genes represent a family of detoxi"cation enzymes that catalyze the conjugation of reduced glutathione (GSH) with a group of compounds having electrophilic centers. These can include nitrocompounds (Usui et al., 1977), polycyclic aromatic hydrocarbons (Cheung et al., 2001), organophosphates (Motoyama and Dauterman, 1977; Usui et al., 1977), and organochlorines (Clark et al., 1986). The GSH conjugation products become less toxic and more water soluble, so allowing easier excretion from cells after further metabolism. Unlike the AChE biomarker, GSTs are far less speci"c and are therefore an ideal partner to help detect pesticides that do not bind to the AChE active site, such as organochlorines.
128 0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
BIOCHEMICAL BIOMARKERS IN CHIRONOMIDS
Chironomus riparius Meigen larvae are widely used in toxicity tests. They are easy to culture, sensitive to many pollutants, and have a short life cycle (Ingersoll and Nelson, 1990). Their ability to burrow into sediments makes them good biological indicators for toxicants that may be adsorbed to sediments. The size of the third- and fourth-instar larvae is su$ciently large to allow measurement of enzyme activity in individuals. There is a distinct advantage to using individual, rather than batches of organisms, for this kind of analysis because the large set of data points produced allows for more powerful statistical analyses and the investigation of interindividual variability. The objective of this study was to expose larvae of C. riparius to an organophosphate pesticide at di!erent temperatures through time and measure subsequent e!ects on biomarkers. This was to test the speci"city of each biomarker to the pesticide and determine the extent to which variation in activity can be related to di!erences in environmental temperature. MATERIALS AND METHODS
Exposure of Chironomid Larvae to the Insecticide C. riparius larvae, initially obtained from WRc plc (Medmenham, UK), were cultured according to standard methods (ASTM, 1999). One fourth-instar larva was added at random to each of 192 100-ml acid-washed beakers until each beaker contained 10 larvae. Sixty-four beakers were placed in a 33C room, 64 in a 123C room, and 64 in a 223C room. The pH (Whatman pH -sensor), temperature, and dissolved oxygen concentration (Jenway 9071 DO meter) were determined before organisms were added to the beakers. The insecticide pirimiphos methyl (o-2-diethylamino-6-methylpyrimidin-4-yl o,o-dimethyl phosphorothioate, CAS No. 29232-93-7, kindly provided by Zeneca Agrochemicals, Jealott's Hill, Berks, UK) was added to the water at nominal concentrations of 0, 0.1, 1, and 10 g/L. Beakers were arranged at random on a bench and covered with polyethylene "lm (Sainsbury food wrap) to reduce evaporation. After 48, 72, and 96 h, mortality, pH, temperature, and dissolved oxygen concentration were determined in each vessel. Two replicate beakers of 10 larvae were removed at each time point for each treatment and larvae frozen individually in Eppendorf tubes in liquid nitrogen for subsequent biomarker analysis. Eppendorf tubes were stored at !803C. Acetylcholinesterase Analysis Each snap-frozen larva was homogenized in 0.02 M icecold phosphate bu!er, pH 8 (PB), containing 1% triton X-100. Tubes were centrifuged (Biofuge, Heraeus instruments) at 13,000g and 43C for 4 min.
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The AChE assay was based on the method of Ellman et al. (1961) modi"ed for use in a microtiter plate (Fisher et al., 2000). The 96-well microtiter plate (MPO1, Life Science (UK) Ltd, Greiner) was loaded with 100 l of 8 mM di-thionitrobenzene (Sigma D-8130) in PB and 0.75 mg/ml NaHCO . Three replicates of 50 l of assay blank (PB, pH 8, containing 0.1% triton x-100) or quality control enzyme (eel cholinesterase, Sigma C-3389 made up on ice in a cold room as a nominal 0.5 unit/ml in ice-cold PB), or homogenate supernatant were added. This was followed by the addition of 50 l of 16 mM acetylthiocholine iodide (Sigma A-5751) in PB. The microtiter plate was inserted into the plate reader (Anthos Labtec, Salzburg) and incubated for 5 min at 303C. There was a premeasurement mix for 2.5 min at 1150 rpm. The activity rate was measured as change in OD/min at 412 nm. The molar extinction coe$cient of 8160 for a light path of 0.6 cm was used for activity calculation. Glutathione S-transferase Analysis GST analysis was based on the method of Habig et al. (1974), modi"ed for use in microtiter plates. The microtiter plate was loaded in ice with three replicates of 50 l of assay blank (PB containing 0.1% triton X-100 and 0.1% (v/v) phenylmethylsulfonyl#oride (PMSF, Sigma P-7626)), standard enzyme (1 unit/ml of Equine GST (Sigma G-6511)) in ice-cold phosphate bu!er, pH 6.5, containing 1 mg/ml bovine serum albumin (BSA, fraction V, 96}99% (Sigma A-2153)), and samples (diluted with two volumes of PB adjusted to produce a "nal pH of 6.5, containing 0.15% (v/v) PMSF). The plate was then incubated for 5 min at 303C in the plate reader. Meanwhile the substrate mixture was made by adding 7 ml of 20 mM reduced glutathione (Sigma G4251) in 0.1 M potassium dihydrogen phosphate KH PO (anhydrous, 99% purity, Sigma P5379), 1 mM ethylenediamine tetraacetic acid (Sigma ED4SS) to 12.5 ml 0.02M sodium hydrogen phosphate bu!er, pH 6.5. After a 5-min incubation of the solution at 303C in a thermostatic bath (Grant Instruments), 1.4 ml of 40 mM 1-chloro-2,4-dinitrobenzene (Sigma C-6396) in 95% ethanol was added; 50 l of the substrate mixture was then added to the microtiter plate and the plate was incubated for a further 2 min at 303C accompanied by intermittent shaking at 1150 rpm. The measurement period was 10 min at 303C. The rate was measured as a single wavelength kinetic change in OD/min at 340 nm. The molar extinction coe$cient of 5760 for a light path of 0.6 cm was used for the activity calculation. The activities of both GST and AChE were expressed as activity per unit protein. The protein assay was a microtiter plate version of the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL).The BCA working reagent mixture was incubated in a water bath at 303C for 10 min prior to the addition of 200 to 20 l of supernatant homogenate or
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standard protein solutions (0, 100, 200, 400, 600, 800, 1100, 1500 g/L of BSA in PB, pH 8, containing 0.1% triton x-100). The plate was then inserted into the preheated plate reader and the reaction run for 10 min at 303C. The rate was measured as change in OD/min at 550 nm. Protein concentration was calculated in relation to a regression line drawn through points obtained from the standard curve. Statistical Analyses Analysis of variance (ANOVA) was used to detect di!erences between treatments. Survival data were arcsine-transformed before analysis. Exposure duration, temperature, and pesticide concentration were the three factors and either AChE or GST activity was the dependent variable. A Student}Newman}Keuls (SNK) test was then used to identify di!erences between treatment groups. Analyses were performed using Unistat 4.5 software (Unistat Ltd., 1996). RESULTS
Physical Data The pH of the test water did not vary from 7.0 throughout the experiment. The mean temperature in vessels in each of the three test rooms was 3, 12, and 223C. Dissolved oxygen concentrations did not fall below 65% air saturated volume. Mortality There was a signi"cant main e!ect on chironomid survival of both temperature (F "5.27, P"0.0062) and time (F "26.51, P(0.001), and an interaction be tween these two factors (F "4.83, P"0.0002). This is because animals died faster at 3 and 123C compared to 223C and also, mortality increased after 72 and 96 h of exposure (Fig. 1). There was neither a signi"cant e!ect of pesticide concentration on mortality, nor any other twoway or three-way interaction between factors. AChE Data Figure 2 illustrates mean AChE activity in larvae exposed to three di!erent temperatures and four concentrations of pirimiphos methyl over 96 h. There was a signi"cant interaction between exposure duration, temperature, and pesticide concentration (F "2.035, P"0.019). A concentration}response relationship was evident, with signi"cant reductions in AChE activity found between 0.1, 1, and 10 g/L treatments (SNK test, P(0.05). There were no signi"cant di!erences between the control and the 0.1 g/L treatment. The SNK test was unable to detect signi"cant di!erences between treatments for the other two main factors, exposure duration and temperature, and Fig. 2 suggests that interactions between these factors and
FIG. 1. Survival of C. riparius exposed to pirimiphos methyl (0, 0.1, 1.0, and 10 g/L) at three temperatures (3, 12, and 223C) for 24, 48, 72, and 96 h.
concentration of pirimiphos methyl were of little biological signi"cance. Glutathione S-transferase Data GST activities in larvae exposed to three di!erent temperatures and at four concentrations of pirimiphos methyl over 96 h are presented in Fig. 3. There were no signi"cant interactions between the factors when the GST data were analyzed. Instead, each factor had a signi"cant main e!ect: exposure duration (F "5.522, P"0.004), temper ature (F "33.889, P(0.0001), and pesticide concen tration (F "5.763, P"0.0007). This is because GST activity was signi"cantly higher in animals sampled after 48 h compared with those sampled at 72 and 96 h (SNK, P(0.05), signi"cantly higher (SNK, P(0.05) in animals maintained at 33C when compared with those kept at 12 and 223C, and signi"cantly lower (SNK, P(0.05) in animals maintained at 223C when compared with those kept at 12 and 33C, and signi"cantly higher (SNK, P(0.05) in animals exposed to the 10 g/L treatment.
BIOCHEMICAL BIOMARKERS IN CHIRONOMIDS
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pesticide poisoning but relatively insensitive to environmental variation. These results demonstrate that AChE inhibition is caused by organophosphates but remains invariable over changing temperatures. AChE inhibition in chironomids is a very robust biomarker. Olsen et al. (2001) found that organic carbon concentrations at 13 sites had no signi"cant in#uence on AChE activity in "eld-deployed C. riparius larvae. A previous study of AChE activity in C. riparius following a 48-h exposure to pirimiphos methyl found that although activity was associated with reduced larval weight and reduced adult fecundity after several days, it was not in#uenced by food ration (Crane et al., in press). This suggests that measurement of C. riparius AChE in "eld situations, where food ration may vary considerably, is a useful method for monitoring exposure to organophosphate insecticides, and one that is predictive of e!ects on individual health. This latter fact is supported by correlations between AChE inhibition by chlorpyrifos in C. riparius larvae and development time, adult size, and burrowing behavior (Callaghan et al., 2001). Although GSTs are induced by both organophosphate and organochlorine insecticides in the house#y (Hayaoka
FIG. 2. Acetylcholinesterase activities (nmol/min/g protein) in C. riparius larvae after exposure to pirimiphos methyl (0, 0.1, 1.0, and 10 g/L) at three temperatures (3, 12, and 223C) for 48, 72, and 96 h. Error bars are standard errors of the mean.
DISCUSSION
Temperature is an important environmental determinant for ectothermic organisms since it a!ects many biological processes. Many organisms operate optimally within a very narrow temperature range. Deviations from this range can be stressful, especially during development (Parsons, 1987, 1989; Imasheva et al., 1997). At the molecular level, temperature stress directly a!ects biological molecules such as enzymes, which are crucial for the survival of the organism, whereas at the population level it may result in chronic e!ects within the genetic architecture of the population (Imasheva et al., 1997). Whereas temperature can be a general stressor, xenobiotic compounds such as pirimiphos methyl are often more speci"c because they generally a!ect individual pathways within the organism (Parsons, 1990). Biomarkers can be applied to detect both forms of stress and ideally relate exposure to e!ect. The measurement of AChE activity is a fairly well established biomarker of organophosphate poisoning in C. riparius (Fisher et al., 2000, Callaghan et al., 2001; Detra and Collins, 1991; Ibrahim et al., 1998). However, if AChE is to be useful as a real tool to help detect low levels of organophosphate pollution, it should be very speci"c to
FIG. 3. Glutathione-S-transferase activities (nmol/min/g protein) in C. riparius larvae after exposure to pirimiphos methyl (0, 0.1, 1.0, and 10 g/L) at three temperatures (3, 12, and 223C) for 48, 72, and 96 h. Error bars are standard errors of the mean.
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and Dauterman, 1982; Clark et al., 1986), Lepidoptera (Lagadic et al., 1994), and the American cockroach (Usui et al., 1977), no such e!ect has been found in C. riparius until now. However, in this study a statistically signi"cant GST induction following pirimiphos methyl exposure occurred at the highest test concentration (10 g/L) only. Negative results have previously been found following exposure to lindane and pirimiphos methyl in C. riparius larvae (Hirthe et al., 2001, Crane et al., in press) and the use of GSTs as biomarkers of xenobiotic exposure has been questioned by other authors. Various pollutants have failed to induce GSTs in invertebrates (Stegeman et al. 1992; Fitzpatrick et al., 1995; Bond and Bradley, 1997) and vertebrates (Fenet et al., 1998). In addition, variation in GST activity related to seasonal variation in temperature and oxygen levels has been found (Power and Sheehan, 1996). The only signi"cant inductions of GST activity found in the current experiment were at low temperature and in the 10 g/L OP treatment. Although signi"cant, the induction associated with this latter treatment was largely driven by results from all exposure durations at 123C and 48-h exposure in the 223C treatment, as seen in Fig. 3. This rather inconsistent pattern of induction would be di$cult to interpret in a "eld situation. GSTs represent a family of genes (Clark et al., 1973; Motoyama and Dauterman, 1977; Wang et al., 1991). Clark et al. (1986) puri"ed GST from two strains of house#y and found that both had two forms, each with speci"city toward certain pesticides. It is possible that the substrate used in the current method detects only one of these genes optimally, the one that coincidentally is not induced to a large extent by either OPs or organochlorines. Alternatively, GST genes in C. riparius may not generally be sensitive to induction by these pesticides. CONCLUSION
The inhibition of AChE activity in C. riparius larvae as a biomarker of OP pesticide has again proved to be sensitive and robust under di!erent environmental conditions. These results support an increasing body of data that demonstrate the suitability of this assay in C. riparius to identify and monitor OP pollution. GST induction was less sensitive as a biomarker of OP pollution and was in#uenced by temperature, with induction evident at 33C. There was a signi"cant e!ect of exposure duration, with an overall decline in GST activity over time. This result agrees with previous work suggesting that GSTs are not particularly suitable for use as a biomarker of pesticide exposure or e!ect in Chironomus.
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ACKNOWLEDGMENTS
Habig, C. and DiGiulio, T. (1991). Biochemical characteristics of cholinesterase in aquatic organisms. In Cholinesterase Inhibiting Insecticides: ¹heir Impact on =ildlife and the Environment (P. Mineau, Ed.), pp. 19}33. Elsevier Science, Amsterdam.
This project was funded by the NERC Environmental Diagnostics Thematic Programme (GST 02/1558).
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