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.

328 P. Flammarion et al. / Environmental Pollution 120 (2002) 325330


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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.

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The Measurement of Cholinesterase Activities as a Biomarker