the measurement of cholinesterase activities as a biomarker
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The measurement of cholinesterase activities as a biomarkerin chub (Leuciscus cephalus): the fish length should not
be ignored
P. Flammarion*, P. Noury, J. Garric
Unite de Recherche Biologie des Ecosyste`mes Aquatiques, Cemagref, 3 bis Quai Chauveau, 69336 Lyon Cedex 9, France
Received 5 October 2001; accepted 17 January 2002
Capsule: The fish 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 fish muscle is a biomarker of the exposure to organophosphosphates and carbamates pesticides.
As such it has been used in numerous field 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 fish length, the larger fish having the lower ChE activ-
ities. We concluded that the great majority of the significant differences in ChE activities between sites could be due in fact to
differences in fish length between field samples. We stress the importance of taking into account the fish length whenever differences
in ChE levels between field sites must be interpreted. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Fish; Biomonitoring; Biomarker; ChE; Fish length
1. Introduction
Considerable efforts have been made for the last two
decades to develop and validate measurements of bio-
logical parameters to complement the information given
by the chemical analysis of contamination. Among bio-
chemical biomarkers, the measurement of fish cholines-
terase (ChE) activities has become a tool of
biomonitoring in marine (Galgani et al., 1992; Boc-
quene et al., 1993) and continental waters (Payne et al.,
1996; Sturm et al., 1999). It enables to assess the expo-sure of aquatic organisms to some pesticides (carba-
mates and organophosphates) and other molecules with
a neurotoxic action (including heavy metals).
Few studies of ChE activities have included cyprini-
formes, principally in the laboratory (Szabo et al., 1992;
Bertrand et al., 1998; Flammarion et al., 1998b). The
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 fish
ChE activities in the field. To prevent any mis-
interpretation due to potential factors of variability (e.g.
sex, temperature of water), authors generally aim tosample homogenous fish (length). Yet, despite the
abundance of chub in French rivers, we sometimes have
difficulties 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.
0269-7491/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
P I I : S 0 2 6 9 - 7 4 9 1 ( 0 2 ) 0 0 1 3 7 - 9
Environmental Pollution 120 (2002) 325330
www.elsevier.com/locate/envpol
* Corresponding author. Tel.: +33-4-72208785; fax: +33-4-
78477875.
E-mail address: [email protected] (P. Flammarion).
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2. Materials and methods
2.1. Data collection
Chub (N=335) were sampled from wild populations
by electrofishing in 1999 in 20 sites. The sampling sites
were located in the Moselle, the Rhone and the Seinewatersheds (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,
Arde` che 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
electrofishing, fish were immediately sacrificed, 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 buffer 0.1
M pH 7.8 and centrifuged at 9000 g. ChE activities of
the supernatant (S9) were measured at 405 nm by a
modified 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 butyrylcho-
linesterase (BChE) and acetylcholinesterase (AChE) are
likely to be present in fish body muscle, particularly in
marine species (Lundin, 1962; Sturm et al., 2000). Even
if it has been observed in many freshwater fish thatmuscle may contain exclusively AChE (Lundin, 1962), it
was chosen to use cholinesterase (ChE) in the paper.
Acetylthiocholine iodide (ATCI) and 5,50-dithiobis-2-
nitrobenzoic 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 lin-
ear relationship between the within group variability
[SD(ChE)] and the level of ChE (arithmetic mean):SD(ChE)=0.36mean(ChE) (R2=0.89; P
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confidence intervals (CI95) calculated from the log-
transformed distributions using the critical values of
Students t-distributions.
3. Results
We generally observed a statistical relationship
between ChE activities and fish length, the larger fish
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 per-
formed 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 difference
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 first rela-ted to the length variability among samplings.
In fish from the reference sites (Ain, Arde` che and
Drome), we confirmed that no significant difference
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
represents the ChE activities that can be expected from
unexposed fish in the field.
For each polluted sites, considering the reference
length-ChE curve (Fig. 3), values for the expected ChE
activities were estimated:
1. By knowing the fish length, a value for the expec-ted 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 significantly different (P
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the fish 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 fish as a reference for determining the normal
activity of the selected fish (Weiss, 1959, 1961; Baslowand Nigrelli, 1964; Holland et al., 1967). Lundin (1962)
interpreted the muscle ChE-length relationships as con-
sistent with simple assumptions (ChE activity propor-
tional 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 coefficient (0.87)
is [1.1; 0.7] thus containing 1. A non-linear regres-
sion found the following curve: ChE=37240/Length
(N=84, R2=0.43), consistent with the assumptions of
Lundin (1962), yet the determination coefficient (R2)was lower in that case than with the loglog relation-
ships, in addition the homogeneity of the variance is
violated when using ChE instead of Ln(ChE).
The absence of sex effect on the ChE activities sup-
ports previous results. The variability of ChE activities
is not related to sex or reproduction period in most
fishes (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 different 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 con-
sistent 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 sig-
nificantly lower ChE activities than the reference ChE:Moselle 1 and 2 (Fig. 4). Considering the low inhibition
percentages (50%) this would predict severe neurotoxic
effects in fish from these sites since fish with less than
80% normal ChE activity are considered to have suf-
fered from the exposure to some anti-cholinesterase
substances (Holland et al., 1967; Sturm et al., 1999).
The effects 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 twoMoselle sites is available. Hence, it cannot be concluded
anything about the exposure to some anti-cholinesterase
compounds in these two sites.
Besides, Ardie` res site is known as a highly contam-
inated 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 fish 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 significant increase of ChE activities was
observed in chub from the Cance site (Fig. 4). Such
effect may derive from the influence of another natural
factor that has not been taken into account in this study
yet. Indeed, the temperature of the environment may
have a significant effect on the ChE activities (Bocquene
et al., 1990). The effect 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 tempera-
ture might indicate that differences in temperature
between sites and or samplings were too low (max.
range=1323 C; data not shown) to result in sig-
nificant differences 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 fish populations
Table 1
ChE data (nmol/min/mg protein) and length (mm) in chub from 20
sites
Sites Sampling month n ChE Length
Ain September 16 170 (132; 219) 20386
Arde` che September 10 163 (117; 226) 22379
Drome March 20 235 (204; 271) 16819June 28 237 (201; 280) 16265
September 10 219 (181; 265) 18836
Ardie` res September 10 210 (189; 233) 16610
Bourbre September 11 171 (127; 229) 21138
Cance September 11 312 (275; 354) 16915
Rhone 1 October 12 148 (116; 189) 22073
Rhone 2 October 10 172 (135; 220) 23865
Rhone 3 October 10 118 (77; 182) 26372
Rhone 4 October 27 172 (140; 212) 21875
Saone 1 September 11 224 (169; 296) 18957
Saone 2 September 10 182 (132; 251) 23751
Turdine June 30 148 (125; 177) 20136
Seine 1 September 10 306 (255; 367) 1458
Seine 2 September 10 185 (154; 223) 18728
Moselle 1 July 20 74 (62; 90) 26442Moselle 2 July 13 70 (57; 86) 34463
Moselle 3 July 20 132 (99; 175) 25579
Moselle 4 July 16 147 (108; 201) 23377
Moselle 5 July 20 167 (128; 218) 22747
ChE values are given as geometric mean with 95% confidence inter-
vals in parentheses. Length values are given as means and standard
deviations.
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Fig. 4. Geometric means of ChE activities (with or without adjustment with fish length) Deviations are expressed as % of ChE activities in reference
sites. * Denotes significant difference (P
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might also result from genetic differences with the fish
populations from the other sites.
5. Conclusion
In summary, this work stresses the importance oftaking into account the fish length whenever differences
between field 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 con-
ducted. The routine use of such biomarker in biomoni-
toring 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 specifi-
city, this biomarker should be used within a battery of
biomarkers to help to identify and differentiate between
contaminants fish are exposed to in the field.
Acknowledgements
The field 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 official fishguards from the
Conseil Supe rieur 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.
References
Baslow, M.H., Nigrelli, R.F., 1964. The effect of thermal acclimation
on brain cholinesterase activity of the killifsh, Fundulus heteroclitus.
Zoologica 49, 4151.
Bertrand, C., Cousin, X., Haubruge, E., Toutant, J.P., Chatonnet, A.,
1998. Lace tylcholineste rase des poissons, cible des organopho-
sphores et des carbamates. Caracterisation du gene et des formes
mole culaires de lenzyme chez Danio rerio. Effets des anti-
cholineste rasiques. Bulletin Francais de la Peche et de la Pisci-
culture, pp. 350351, 535546.
Bocquene, G., Galgani, F., Truquet, P., 1990. Characterization and
assay conditions for use of AChE activity from several marine spe-
cies in pollution monitoring. Marine Environmental Research 30,7589.
Bocquene, G., Galgani, F., Burgeot, T., Le Dean, L., Truquet, P.,
1993. Acetylcholinesterase levels in marine organisms along French
coasts. Marine Pollution Bulletin 26, 101106.
Ellman, G.L., Courtney, K.D., Andres, V., 1961. A new and rapid
colorimetric determination of acetylcholinesterase activity. Bio-
chemical Pharmacology 7, 8895.
Flammarion, P., Garric, J., 1997. Cyprinids reference EROD levels in
low contaminated rivers : a relevant statistical approach to estimate
reference levels for EROD biomarker. Chemosphere 35, 23752388.
Flammarion, P., Garric, J., 1999. A statistical approach for classifying
the extent of EROD induction of fish sampled in clean and con-
taminated waters. Water Research 33, 26832689.
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., Morfin, P., Garric, J., 1998b.
Effect of methidathion on the cytochrome P-450 1A in the cyprinid
fish 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 e cosyste` mes hydriques dulca-
quicoles et de ses effets 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 fish
vitellogenin and alterations in testicular structure: preliminary
results of estrogenic effects in chub (Leuciscus cephalus). Ecotox-
icology 9, 127135.
Galgani, F., Bocquene, G., Cadiou, Y., 1992. Evidence of variation in
cholinesterase activity in fish 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
specific 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 fish brain
acetylcholinesterase to monitor pollution by organophosphorus
pesticides. Bulletin of Environmental Contamination and Toxicol-
ogy 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 fishes. Journal of Cell Comparative Physiology 59, 93105.
Olson, D.L., Christensen, G.M., 1980. Effects of water pollutants and
other chemicals on fish acetylcholinesterase (in vitro). Environ-
mental Research 21, 327335.
Payne, J.F., Mathieu, A., Melvin, W., Fancey, L.L., 1996. Acet-
ylcholinesterase, an old biomarker with a new future? Field trials in
association with two urban rivers and a paper mill in Newfound-
land. 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 (Gas-
terosteus aculeatus) and relation to pollution. Environmental Tox-
icology and Chemistry 18, 194200.
Sturm, A., Wogram, J., Segner, H., Liess, M., 2000. Different sensi-
tivity to organophosphates of acetylcholinesterase and butyry-
lcholinesterase 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 effect of pesticides on Carp (Cyprinus carpio L).
Acetylcholinesterase and its biochemical characterization. Ecotox-
icology and Environmental Safety 23, 3945.Tomassone, R., Dervin, C., Masson, J.P. (Eds.), 1993. Biome trie.
Mode lisation de phe nome` nes biologiques. Masson, Paris.
Vigano, L., Arillo, A., Melodia, F., Arlati, P., Monti, C., 1998. Bio-
marker 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., Gar-
ric, J., 1993. Ethoxyresorufin-O-deethylase induction in fish from a
watershed exposed to a non-point source pollution of agricultural
origin. Water Research 27, 449455.
Weiss, C.M., 1959. Response of fish to sub-lethal exposures of organic
phosphorus insecticides. Sewage and Industrial Wastes 31, 580593.
Weiss, C.M., 1961. Physiological effect of organic phosphorus insecti-
cides on several species of fish. Transactions of the American Fish-
eries Society 90, 143152.
330 P. Flammarion et al. / Environmental Pollution 120 (2002) 325330