Environmental Pollution 120 (2002) 325330 www.elsevier.

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The measurement of cholinesterase activities as a biomarker in chub (Leuciscus cephalus): the sh length should not be ignored
P. Flammarion*, P. Noury, J. Garric
`mes Aquatiques, Cemagref, 3 bis Quai Chauveau, 69336 Lyon Cedex 9, France Unite de Recherche Biologie des Ecosyste Received 5 October 2001; accepted 17 January 2002

Capsule: The sh length should be considered within the statistical analysis of ChE activities in chub species.
Abstract Biomarkers are early warning systems of the exposure of aquatic organisms to pollutants. Among them, the measurement of the cholinesterase (ChE) activities in sh muscle is a biomarker of the exposure to organophosphosphates and carbamates pesticides. As such it has been used in numerous eld studies both in marine and continental waters. Cyprinids (chub, Leuciscus cephalus) were sampled in river sites (France) in relatively clean and polluted areas. We performed the statistical analysis of the ChE activities and we generally observed a statistical relationship between ChE activities and sh length, the larger sh having the lower ChE activities. We concluded that the great majority of the signicant dierences in ChE activities between sites could be due in fact to dierences in sh length between eld samples. We stress the importance of taking into account the sh length whenever dierences in ChE levels between eld sites must be interpreted. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Fish; Biomonitoring; Biomarker; ChE; Fish length

1. Introduction Considerable eorts have been made for the last two decades to develop and validate measurements of biological parameters to complement the information given by the chemical analysis of contamination. Among biochemical biomarkers, the measurement of sh cholinesterase (ChE) activities has become a tool of biomonitoring in marine (Galgani et al., 1992; Bocquene et al., 1993) and continental waters (Payne et al., 1996; Sturm et al., 1999). It enables to assess the exposure of aquatic organisms to some pesticides (carbamates and organophosphates) and other molecules with a neurotoxic action (including heavy metals). Few studies of ChE activities have included cypriniformes, principally in the laboratory (Szabo et al., 1992; Bertrand et al., 1998; Flammarion et al., 1998b). The

* Corresponding author. Tel.: +33-4-72208785; fax: +33-478477875. E-mail address: patrick.ammarion@cemagref.fr (P. Flammarion).

chub (Leuciscus cephalus) is a common freshwater cyprinid species in Western Europe, in both clean and polluted rivers. It is already used for biomonitoring of other biomarkers (Flammarion and Garric, 1997, 1999; Flammarion et al., 1998a, 2000; Vigano et al., 1998). However, to our knowledge, no results of ChE activities in chub have ever been published. Indeed, this species could prove to be suitable for the biomonitoring of sh ChE activities in the eld. To prevent any misinterpretation due to potential factors of variability (e.g. sex, temperature of water), authors generally aim to sample homogenous sh (length). Yet, despite the abundance of chub in French rivers, we sometimes have diculties in completing suitable homogenous sampling within a site and among sites. As a consequence, to obtain a reliable detailed mapping of the extent of ChE inhibitions, it is essential to separate the variations due to pollution from the natural variabilities. In this study, our objective was to examine the length dependency of the ChE activities in chub sampled in 1999 in French river areas in the Moselle, Rhone and Seine watersheds.

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2. Materials and methods 2.1. Data collection Chub (N=335) were sampled from wild populations by electroshing in 1999 in 20 sites. The sampling sites were located in the Moselle, the Rhone and the Seine watersheds (Fig. 1). All sites belong to the French water quality network with several chemical and biological indicators that are measured every year. Three sites were in areas of low pollution (Ain, ` Ardeche and Drome rivers, reference areas; Flammar ion and Garric, 1997). Four sites were in highly polluted areas downstream large industrial and urban areas (Rhone 3, Rhone 4, Seine 1, Seine 2). After capture by electroshing, sh were immediately sacriced, weighed, measured, and dissected. White skeletal muscle were removed and frozen in liquid nitrogen and kept for several days at 80  C before enzymatic assay. 2.2. ChE assay The muscle was homogenised in Phosphate buer 0.1 M pH 7.8 and centrifuged at 9000 g. ChE activities of the supernatant (S9) were measured at 405 nm by a modied Ellman et al. (1961) procedure on a 96-well microplate (Flammarion et al., 1998b): Phosphate 0.1

M pH 7.8, 0.2 mM DTNB and 2 mM ATCI. Enzymatic activities were reported on S9 protein concentrations (method of Lowry et al., 1951). Both butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are likely to be present in sh body muscle, particularly in marine species (Lundin, 1962; Sturm et al., 2000). Even if it has been observed in many freshwater sh that muscle may contain exclusively AChE (Lundin, 1962), it was chosen to use cholinesterase (ChE) in the paper. Acetylthiocholine iodide (ATCI) and 5,50 -dithiobis-2nitrobenzoic acid (DTNB) were purchased from Sigma Chemicals Co (St. Louis, MI, USA). All other chemicals were of the highest available commercial grade. 2.3. Data analysis Statistica software (StatSoft Inc., Tulsa, OK, USA) was used for all statistical analyses. We observed a linear relationship between the within group variability [SD(ChE)] and the level of ChE (arithmetic mean): SD(ChE)=0.36mean(ChE) (R2=0.89; P < 0.01). In such a case, a logarithmic transformation can stabilise the variance and obtain a normal distribution (Tomassone et al., 1993). Log-transformed ChE data conformed to the normality test (2 test for normality) and to the homogeneity of variance (Levenes test). Results were displayed with geometric means and 95%

Fig. 1. Map of the sampling sites in 1999 in the French rivers.

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condence intervals (CI95) calculated from the logtransformed distributions using the critical values of Students t-distributions.

represents the ChE activities that can be expected from unexposed sh in the eld. For each polluted sites, considering the reference length-ChE curve (Fig. 3), values for the expected ChE activities were estimated: 1. By knowing the sh length, a value for the expected activity can be estimated from the reference curve. 2. Deviations are expressed as % of the normal ChE activities. Without length adjustment, 8/17 sites were observed to be signicantly dierent (P < 0.05) from the reference sites, whereas with length adjustment only three sites were signicantly higher (Cance) or lower (Moselle 1 and Moselle 2; Fig. 4).

3. Results We generally observed a statistical relationship between ChE activities and sh length, the larger sh having the lower ChE activities (Fig. 2). This was the case for both females and males, and this was observed within each site. ANCOVA was then performed on Ln(ChE) with Ln(length) as covariate and site and sex as factors. Ln(length) was chosen rather than length because the regression with Ln(ChE) shows a higher R2 with Ln(length) (R2=0.62) than with length (R2=0.51). ANCOVA analysis showed no dierence between males and females whatever the sampling. The large inter-station variability in ChE activities (Table 1), even in the three reference sites, could then be rst related to the length variability among samplings. ` In sh from the reference sites (Ain, Ardeche and Drome), we conrmed that no signicant dierence could be observed between males and females (ANCOVA, P=0.66) and between sites (ANCOVA, P=0.21). A chub ChE-length relationships for the reference sites was then derived (Fig. 3). This line

4. Discussion This is the rst published work with ChE biomarker in the chub species. Similar results for ChE-length relationships have been obtained with other species. Sturm et al. (1999) examined the muscle ChE activities in the three spined stickleback and observed a highly signicant variation due to

Fig. 2. Relationships between ChE activities (nmol/min/mg protein) and sh length (mm) in female and male chub. The graph shows all the individual ChE/length data (N=335).

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Table 1 ChE data (nmol/min/mg protein) and length (mm) in chub from 20 sites Sites Ain ` Ardeche Drome Sampling month September September March June September September September September October October October October September September June September September July July July July July n 16 10 20 28 10 10 11 11 12 10 10 27 11 10 30 10 10 20 13 20 16 20 ChE 170 163 235 237 219 210 171 312 148 172 118 172 224 182 148 306 185 74 70 132 147 167 (132; 219) (117; 226) (204; 271) (201; 280) (181; 265) (189; 233) (127; 229) (275; 354) (116; 189) (135; 220) (77; 182) (140; 212) (169; 296) (132; 251) (125; 177) (255; 367) (154; 223) (62; 90) (57; 86) (99; 175) (108; 201) (128; 218) Length 20386 22379 16819 16265 18836 16610 21138 16915 22073 23865 26372 21875 18957 23751 20136 1458 18728 26442 34463 25579 23377 22747

` Ardieres Bourbre Cance Rhone 1 Rhone 2 Rhone 3 Rhone 4 Saone 1 Saone 2 Turdine Seine 1 Seine 2 Moselle 1 Moselle 2 Moselle 3 Moselle 4 Moselle 5

ChE values are given as geometric mean with 95% condence intervals in parentheses. Length values are given as means and standard deviations.

the sh length. Earlier results had found a negative relationships between brain AChE activity and the brain weight, and these authors used the brain weight of individual sh as a reference for determining the normal activity of the selected sh (Weiss, 1959, 1961; Baslow and Nigrelli, 1964; Holland et al., 1967). Lundin (1962) interpreted the muscle ChE-length relationships as consistent with simple assumptions (ChE activity proportional to the cell surface, proportional to the body length. . .). They observed such relationship in several species including cyprinids: the ChE activity was divided by 10 when the length was multiplied by 10 whatever the cyprinid species. This inverse relationship is consistent with ours: the CI (95%) of the linear coecient (0.87) is [1.1; 0.7] thus containing 1. A non-linear regression found the following curve: ChE=37240/Length (N=84, R2=0.43), consistent with the assumptions of Lundin (1962), yet the determination coecient (R2) was lower in that case than with the loglog relationships, in addition the homogeneity of the variance is violated when using ChE instead of Ln(ChE). The absence of sex eect on the ChE activities supports previous results. The variability of ChE activities is not related to sex or reproduction period in most shes (Galgani et al., 1992). We had some knowledge on another cyprinid species in the laboratory: gudgeon (Gobio gobio; Flammarion et al., 1998b): female ChE activities were not statistically dierent from male ChE activities whatever the treatment by methidathion,

an organophosphate, resulting in a high inhibition of the ChE activities. In addition, the ChE activities of control gudgeon [Ln(ChE) between 5 and 6] are consistent with what is observed with chub in the same range of length [Ln(length) between 4.6 and 4.8] (Fig. 3). After length adjustment, only two sites had signicantly lower ChE activities than the reference ChE: Moselle 1 and 2 (Fig. 4). Considering the low inhibition percentages (50%) this would predict severe neurotoxic eects in sh from these sites since sh with less than 80% normal ChE activity are considered to have suffered from the exposure to some anti-cholinesterase substances (Holland et al., 1967; Sturm et al., 1999). The eects observed may indicate the presence of organophosphorous or carbamates molecules or heavy metals (Olson and Christensen, 1980). In Moselle 1 sediment, high levels of Cr, Pb and Hg were detected (Flammarion et al., 1999), but the bioconcentrated levels in chub muscle were low. Unfortunately no information on pesticide contamination in these two Moselle sites is available. Hence, it cannot be concluded anything about the exposure to some anti-cholinesterase compounds in these two sites. ` Besides, Ardieres site is known as a highly contaminated site with pesticides, including anti-cholinesterase substances, due to vineyards (Vindimian et al., 1993). However, we did not observe any ChE inhibition (Fig. 4). Indeed, pesticides concentrations in this site are highly variable within the year and a better knowledge on the concentrations is needed before the sh sampling date (early September). As a matter of fact reversibility of brain AChE inhibition was shown by Weiss (1961) after 30 days or more. A surprising signicant increase of ChE activities was observed in chub from the Cance site (Fig. 4). Such eect may derive from the inuence of another natural factor that has not been taken into account in this study yet. Indeed, the temperature of the environment may have a signicant eect on the ChE activities (Bocquene et al., 1990). The eect of thermal acclimation on brain ChE activity was studied: the alterations in activity vary inversely with the temperature of acclimation (Baslow and Nigrelli, 1964) in Fundulus heteroclitus, while Hogan (1970) conversely observed a linear increase of brain AChE of bluegills according to water temperature (when temperature increases from 10 to 20  C, AChE activity is increased by +23%). In our study, the lack of correlation between ChE activities and water temperature might indicate that dierences in temperature between sites and or samplings were too low (max. range=1323  C; data not shown) to result in signicant dierences in ChE activities, as already observed by Sturm et al. (1999). Concerning the Cance site, it would be interesting to know more about the evolution of the temperature over the year of interest. Higher than normal ChE activities in sub sh populations

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` Fig. 3. LnLn linear regression between ChE activities and sh length in the reference sites (Ain, Ardeche and Drome rivers).

Fig. 4. Geometric means of ChE activities (with or without adjustment with sh length) Deviations are expressed as % of ChE activities in reference sites. * Denotes signicant dierence (P <0.05) with the reference sites.

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P. Flammarion et al. / Environmental Pollution 120 (2002) 325330 Flammarion, P., Migeon, B., Garric, J., 1998a. Statistical analysis of cyprinids EROD data in a large French watershed. Ecotoxicology and Environmental Safety 40, 144153. Flammarion, P., Migeon, B., Urios, S., Morn, P., Garric, J., 1998b. Eect of methidathion on the cytochrome P-450 1A in the cyprinid sh gudgeon (Gobio gobio). Aquatic Toxicology 42, 93102. Flammarion, P., Camus, J.C., Devaux, A., Garric, J., Lafont, M., Migeon, B., Mouthon, J., Noury, P., 1999. Validation dindicateurs ` biologiques de la contamination des ecosystemes hydriques dulca quicoles et de ses eets toxiques. Biomarqueurs et bioindicateurs, mesures dans la Moselle amont. Campagnes 1998 et 1999. Projet PNETOX. Cemagref/ENTPE. Flammarion, P., Brion, F., Palazzi, X., Babut, M., Garric, J., Migeon, B., Noury, P., Thybaud, E., Tyler, C.R., 2000. Induction of sh vitellogenin and alterations in testicular structure: preliminary results of estrogenic eects in chub (Leuciscus cephalus). Ecotoxicology 9, 127135. Galgani, F., Bocquene, G., Cadiou, Y., 1992. Evidence of variation in cholinesterase activity in sh along a pollution gradient in the North Sea. Marine Ecology Progress Series 91, 7782. Hogan, J.W., 1970. Water temperature as a source of variation in specic activity of brain acetylcholinesterase of bluegills. Bulletin of Environmental Contamination and Toxicology 5, 347353. Holland, H.T., Coppage, D.L., Butler, P.A., 1967. Use of sh brain acetylcholinesterase to monitor pollution by organophosphorus pesticides. Bulletin of Environmental Contamination and Toxicology 2, 156162. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenolreagent. Journal of Biology and Chemistry 193, 265275. Lundin, S.J., 1962. Comparative studies of cholinesterases in body muscle of shes. Journal of Cell Comparative Physiology 59, 93105. Olson, D.L., Christensen, G.M., 1980. Eects of water pollutants and other chemicals on sh acetylcholinesterase (in vitro). Environmental Research 21, 327335. Payne, J.F., Mathieu, A., Melvin, W., Fancey, L.L., 1996. Acetylcholinesterase, an old biomarker with a new future? Field trials in association with two urban rivers and a paper mill in Newfoundland. Marine Pollution Bulletin 32, 225231. Sturm, A., Wogram, J., Hansen, P.D., Liess, M., 1999. Potential use of cholinesterase in monitoring low levels of organophosphates in small streams: natural variability in three-spined stickleback (Gasterosteus aculeatus) and relation to pollution. Environmental Toxicology and Chemistry 18, 194200. Sturm, A., Wogram, J., Segner, H., Liess, M., 2000. Dierent sensitivity to organophosphates of acetylcholinesterase and butyrylcholinesterase from three-spined stickleback (Gasterosteus aculeatus): application in biomonitoring. Environmental Toxicology and Chemistry 19, 16071615. Szabo, A., Nemcsok, J., Asztalos, B., Rakonczay, Z., Kasa, P., Hieu, L.H., 1992. The eect of pesticides on Carp (Cyprinus carpio L). Acetylcholinesterase and its biochemical characterization. Ecotoxicology and Environmental Safety 23, 3945. Tomassone, R., Dervin, C., Masson, J.P. (Eds.), 1993. Biometrie. ` Modelisation de phenomenes biologiques. Masson, Paris. Vigano, L., Arillo, A., Melodia, F., Arlati, P., Monti, C., 1998. Biomarker responses in cyprinids of the middle stretch of the river Po, Italy. Environmental Toxicology and Chemistry 17, 404411. Vindimian, E., Namour, P., Munoz, J.F., Gril, J.J., Migeon, B., Garric, J., 1993. Ethoxyresorun-O-deethylase induction in sh from a watershed exposed to a non-point source pollution of agricultural origin. Water Research 27, 449455. Weiss, C.M., 1959. Response of sh to sub-lethal exposures of organic phosphorus insecticides. Sewage and Industrial Wastes 31, 580593. Weiss, C.M., 1961. Physiological eect of organic phosphorus insecticides on several species of sh. Transactions of the American Fisheries Society 90, 143152.

might also result from genetic dierences with the sh populations from the other sites.

5. Conclusion In summary, this work stresses the importance of taking into account the sh length whenever dierences between eld sites in ChE levels are to be interpreted. To more fully validate the use of chub in the assessment of ChE inhibition, laboratory studies could be conducted. The routine use of such biomarker in biomonitoring will be crucial to validate the reference curve calculated in the present study, and to better take into account potential confounding factors (temperature, behaviour, age, food, etc.). Of course, due to its specicity, this biomarker should be used within a battery of biomarkers to help to identify and dierentiate between contaminants sh are exposed to in the eld.

Acknowledgements The eld assistance of J. Belliard, P. Boet, M. Bray, F. Brion, A. Devaux, G. Gorge, J. Laroche, B. Migeon, S. Niels, P. Roger, E. Tales and ocial shguards from the Conseil Superieur de la Peche is gratefully acknowl edged. This work was supported in part by the French Ministry of Environment and the Seine-Normandie Water Agency.

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