Ecotoxicology and Environmental Safety 97 (2013) 131–138

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Characterization of plasma cholinesterase from the White stork

(Ciconia ciconia) and its in vitro inhibition by
anticholinesterase pesticides
Ana-Lourdes Oropesa a,n, Carlos Gravato b, Susana Sánchez c, Francisco Soler a
a
Toxicology Area, School of Veterinary, University of Extremadura, Avda. de la Universidad, s/n, 10003 Cáceres, Spain
b
CIIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Laboratory of Ecophysiology, Rua dos Bragas, 289, 4050-123
Porto, Portugal
c
Wildlife Rehabilitation Center (Los Hornos), Consejería de Agricultura, Desarrollo Rural, Medio Ambiente y Energía del Gobierno de Extremadura,
Sierra de Fuentes, Cáceres, Spain

art ic l e i nf o a b s t r a c t

Article history: Blood plasma cholinesterase (ChE) activity is a sensitive biomarker of exposure to organophosphorus
Received 6 May 2013 (OP) and carbamate (CB) insecticides in vertebrates. Several studies indicate that more than one ChE
Received in revised form form may be present in blood of birds. In this study the predominant ChE activity (acetylcholinesterase –
12 July 2013
AChE– or butyrylcholinesterase – BChE–), the range of ChE activity as well as ChE age-dependent changes
Accepted 15 July 2013
Available online 17 August 2013
in non-exposed individuals of White stork (Ciconia ciconia) have been established. The in vitro sensitivity
of ChE to OP and CB insecticides such as paraoxon-methyl, carbofuran and carbaryl was also investigated.
Keywords: Plasma ChE was characterised using three substrates (acetylthiocholine iodide, propionylthiocholine
Biomarker iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulphate, BW284C51 and iso-
Cholinesterase inhibition
OMPA). The results indicated that propionylthiocholine was the preferred substrate by plasma
Ciconia ciconia
cholinesterase followed by acetylcholine and butyrylcholine and the predominant enzymatic activity
White storks
Anticholinesterase insecticides in plasma of White storks was BChE. Normal plasma BChE activity in White stork was 0.32 70.01 μmol/
min/ml for adults and 0.28 7 0.03 μmol/min/ml for juveniles. So, the age had no significant effect on the
range of BChE activity. The study on the in vitro inhibitory potential of tested anticholinesterase
pesticides on plasma ChE activity revealed that paraoxon-methyl is the most potent inhibitor followed by
carbofuran and finally by carbaryl. The percentage of in vitro plasma ChE inhibition was observed to be
similar between adults and juveniles.
& 2013 Elsevier Inc. All rights reserved.

1. Introduction
The White stork (Ciconia ciconia) is included in the IUCN Red
List of Threatened Species as of “Least Concern”. It is a widespread
species in Europe with both resident and migrant populations in
the Iberian Peninsula. According to Molina (2005), regions located
in the Southwest of Spain, such as Extremadura, host the largest
colonies. This emphasises the importance of its protection in our
country. The knowledge on the impact of long-term exposure to
pollution in wild populations is still scarce, probably due to
difficulties in working with this species, particularly in that
concerning to the collection of non-invasive samples in wild
animals. However, in relation to its conservation in the Iberian
Peninsula, these studies are needed since this is a long-living
n
Correspondence to: Toxicology Area, School of Veterinary, University of
Extremadura, Avda. de la Universidad s/n, P.O. Box 643, 10003-Caceres, Spain.
Fax: +34 927257110.
E-mail address: aoropesa@unex.es (A.-L. Oropesa).
species (up to 33 years) (del Hoyo et al., 1992) and a considerable
number of colonies are established in agricultural areas. These
birds can be chronically exposed to agrochemical contamination in
the case of resident populations or they can be exposed for several
months, usually during the breeding season, in the case of
migratory populations. Exposure to pesticides which occurs
mainly through the ingestion of contaminated preys (mainly fish,
amphibian, earthworms and snails) (Pinowski et al., 1991), seeds
treated with pesticides or poisoned bait, inhalation and contact
with contaminated air, soil and water is of particular concern due
to the toxic properties of these chemicals. Extremadura is mainly
an agricultural region with important natural spaces and therefore
it offers a suitable habitat for these birds, which are largely
resident. Pollutants present in their tissues and blood are mainly
due to local sources of exposure, mainly to the pesticides used in
agricultural activities or the presence of poisoned bait in the field.
Thus, in addition to the high interest of assessing the risks for local
populations inherent to their exposure to pesticides, the White
stork is also a convenient bioindicator of the quality of these

0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ecoenv.2013.07.022
132 A.-L. Oropesa et al. / Ecotoxicology and Environmental Safety 97 (2013) 131–138
ecosystems since it is very abundant all through the region
(Molina, 2005) where it lives all through the year because the
most of populations are resident (Alonso et al., 1992).
Agricultural pesticides are known to compromise bird survival
contributing to declining populations (Mineau, 2005), and it is
possible that pesticides might be a contributing factor to the
decline in storks in some locations. Organophosphorous (OP) and
carbamate (CB) pesticides are commonly used in agricultural
practices as insecticides because they are more degradable and
have less persistence in the environment than organochlorine
pesticides and in some occasion they are also used to make
poisoned baits to fight against vermin into hunting or livestock
areas. Consistently, they are of ecological concern since they are
toxic to wild non-target species such as raptors (Hooper et al.,
1989; Wilson et al., 1991; Goldstein et al., 1999). The toxicity of
these pesticides is mainly due to the inhibition of acetylcholines-
terase (AChE) activity, the enzyme which degrades the neuro-
transmitter acetylcholine in cholinergic synapses. The inhibition of
AChE provokes an accumulation of acetylcholine at the nerve
synapses and disruption of the nerve function (Peakall, 1992) a
toxicity mechanism that may lead to mortality. The inhibition of
cholinesterases (ChEs) is appropriate for evaluation of expo-
sure to OP and CB pesticides in avian because these are rapidly
degraded and excreted of the organisms and therefore they are not
easily detectable by chemical analysis (Hill and Fleming, 1982;
Hill, 1995; Fairbrother, 1996). Blood (ChEs), including AChE and a
less specialized enzyme commonly designed by pseudocholines-
terase or butyrylcholinesterase (BChE), are also inhibited by these
substances being a widely used non-destrutive biomarker to
diagnose the exposure of anticholinesterase agents (Sanchez
et al., 1997). On the other hand, carboxilesterases are a group of
non-specific esterase enzymes which can also be present in the
plasma of birds and to be inhibited by these groups of substances
(Thompson, 1999).
Since birds are very sensitive to OP and CB pesticides and are
frequently key species in their ecosystems, blood ChE activity has
been widely used to assess the exposure and effects of these
agrochemicals in populations inhabiting agricultural areas (Westlake
et al., 1981a, b; Gard and Hooper, 1993; Soler-Rodríguez et al., 1998;
Parsons et al., 2000; Mayack and Martin, 2003; Rendón-von Osten
et al., 2005; Roy et al., 2005). The results obtained showed the
suitability of birds ChE as a biomarker for OP and CB pesticides. In
birds, both AChE and BChE are found in blood plasma with
wide interspecies differences (Walker and Thompson, 1991;
McInnes et al., 1996). For both ethical and conservational reasons,
the use of biomarkers to investigate the exposure to pollutant in
populations of the White stork is mostly adequate since they can
be determined in a non-destructive way and in some occasions
provide early indications of toxic effects. Blood is in fact the best
biological material for non-destructive biomarker analysis (Fossi
et al., 1994). Plasma cholinesterase activities have been used to
monitor exposure to anticholinesterase pesticides in many bird
species (Dieter, 1975; Dieter and Ludke, 1975; Ludke et al.,
1975; Westlake et al., 1981a,b; Hooper et al., 1989; Wilson
et al., 1991; Rainwater et al., 1995; Goldstein et al., 1999; Parsons
et al., 2000; Parker and Goldstein, 2000), but to the best of our
knowledge there is not similar studies in White storks. Changes in
the activity of cholinesterase enzymes circulating in the plasma
observed in these studies have been considered by those authors
as an indirect measure of pesticide exposure and residue accumu-
lation. Before ChE being used as biomarker in biomonitoring
studies with wild populations and to avoid bias in the interpreta-
tion of results it is important to characterise the enzymes present
in the species and tissue to be analysed and to know the range of
variability in non-exposed individuals (Thompson, 1999; García
et al., 2000). Therefore, objectives of the current study were:
– to characterise the ChE enzymes present in the plasma of the
White stork from wild populations of the Extremadura region
(Spain) by studying their activity towards different substrates
(acetylthiocholine, propyonilthiocholine and buthyrylthiocho-
line) and their sensitivity to selective inhibitors (eserine sul-
phate as inhibitor of all ChE enzymes, iso-OMPA as inhibitor of
BChE and BW284C51 as inhibitor of AChE in vertebrates).
– to determine the range of ChE activity in the plasma in non-
exposed White stork.
– to document the influence of age in ChE activity in the plasma
of the White stork.
– to investigate the in vitro sensitivity of these enzymes to OP and
CBs. Paraoxon-methyl was used as model substance for OP,
carbofuran and carbaryl were used as model substances
for CBs.
2. Material and methods
2.1. Chemicals
Acetylthiocholine iodide, butyrylthiocholine iodide, propionylthiocholine iodide,
iso-OMPA (tetraisopropyl pyrophosphoramide), eserine sulphate, 5,5′-dithiobis(2-
nitrobenzoic acid) (DTNB), BW284C51 (1,5-bis(4-allyldimethyammoniumphenyl)
pentan-3-one dibromide), paraoxon-methyl (O,O-Dimethyl O-(4-nitrophenyl)
phosphate; Purity: 98%), carbaryl (1-Naphthyl-N-methylcarbamate; Purity: 97%)
and carbofuran (2,3-Dihydro-2,2-dimethyl-7-benzofuranol N-methylcarbamate;
Purity: 98%) were purchased from Sigma-Aldrich Química SA, Portugal.
2.2. Animals
The study was performed on a total of 31 birds classified in two groups: adult
(n ¼13) and juvenile (n¼ 18) individuals that were being rehabilitated in the Los
Hornos Wildlife Rehabilitation Centre in the Extremadura region, Spain, following
physical injuries (e.g. electrocution or fall from the nest). Adults were more than
1-year old and juveniles were approximately 2 months old. They were allocated
outdoor into a flight cage (20m  20m  5m), their food consisted of a diet based in
cut up chicken, one day dead chick and water ad libitum throughout recovery
period. The White stork is included in the List of Specially Protected Wild Species
(Real Decreto 139/2011 de 4 de febrero para el desarrollo del Listado de Especies
Silvestres en Régimen de Protección Especial y del Catálogo Español de Especies
Amenazadas) in Spain. We were authorised by Consejería de Medio Ambiente
(local government) to carry out the blood sampling.
2.3. Collection and preparations of samples
Blood sampling was conducted after the complete recovery of the animals
which was achieved in a range of 15 days to 1 month depending on the case. The
state of recovery of the birds was established by veterinarians of Wildlife
Rehabilitation Centre on the basis of appropriate medical criteria (clinical symp-
toms and haematological and biochemical determinations). The birds were
considered recovered when clinical symptoms had disappeared and the haemato-
logical (packed cell volume, haemoglobin, red blood cells and white blood cells)
and biochemical (total proteins, glucose, aspartate amino-transferase AST–, crea-
tine kinase –CK–, lactate dehydrogenase –LDH–) parameters measured were within
the physiological range established for the species by different authors (adults:
Szabó et al., 2010; and juveniles: Montesinos et al., 1997; Blázquez et al., 2006).
Blood sampling was conducted after total recovery of the birds and immediately
before their release in the wild. Blood was sampled in the morning, to avoid error
due to circadian variations in enzyme activities or other blood parameters, and in
the spring season. Whole blood was taken by puncture of the brachial vein using a
syringe with heparin (25-gauge) and was mixed immediately in heparinized tubes
(1.5 mg/dl). Plasma was obtained by centrifugation (2000g, for 5 min at 4 1C) and
immediately stored at  20 1C until further analysis.
2.4. Catalytic properties
The substrates preferences of plasma ChE were investigated by determining
the activity of ChE in the White stork plasma at increasing concentrations of
acetylthiocoline (ASCh), butyrylthiocoline (BSCh) and propionylthiocoline (PSCh)
(from 0.02 to 20.48 mM, incubation concentrations) in independent experiments,
using Ellman′s technique (Ellman et al., 1961) adapted to microplate following the
general procedure indicated in Guilhermino et al. (1996). Briefly, the ChE activity
 A.-L. Oropesa et al. / Ecotoxicology and Environmental Safety 97 (2013) 131–138 133

was determined at 25 1C using 0.050 ml of plasma and 0.250 ml of the reaction

mixture [19.96 ml potassium-phosphate buffer (0.1 M, pH ¼7.2), 1 ml of reagent
5,5′-dithiobis-(2-nitrobenzoic acid) 10 mM (DTNB) (acid dithiobisnitobenzoate and
sodium hydrogen carbonate in phosphate buffer) and 5.120 ml of substrate
0.075 M] and a wavelength of 412 nm. The ChE activity was measured (optical
density changes) for 5 min on a microplate reader (BIO-TEK Power-Wave 340). The
enzyme activity was determined in quadruplicate and expressed as μmol of
substrate hydrolised per minute per ml of plasma. According to Galgani and
Bocquene (1991) the following values of enzymatic parameters were estimated using
Lineweaver–Burk plots: Michaelis–Menten constant (Km), maximal velocity (Vmax) and
the ratio (Vmax/Km) that indicates the catalytic efficiency of the enzyme(s).

2.5. Specific inhibitors and anticholinesterase agents

Eserine sulphate, iso-OMPA and BW284C51 were used in this study as specific
inhibitors of all ChE, BChE and AChE, respectively. Stock solutions of iso-OMPA in
ethanol and BW284C51 and eserine sulphate in water were prepared at 16, 8, 2, 0.5,
0.125, 0.0313, 0.0078 mM for iso-OMPA; 800, 200, 50, 12.5, 3.13, 0.078 μM for
BW284C51 and 800, 200, 50, 12.5, 3.13, 0.78, 0.195, 0.048 μM for eserine sulphate.
Paraoxon-methyl, carbofuran and carbaryl were selected as representative of OP
and CB insecticides. For OP and CBs, solutions were prepared in ultrapure water
(paraoxon-methyl) and ethanol (carbofuran and carbaryl) at 30, 15, 7.5, 3.75, 1.875
and 0.9375 mg/l.
The effect of specific inhibitors and anticholinesterase agents on the activity of ChE
was determined after an incubation period of 30 min at 25 1C as follows: for each
chemical, 0.005 ml of each stock solution was added to 0.495 ml of White stork
plasma. Controls were incubated with 0.005 ml of ultrapure water. Additional controls
incubated with 0.005 ml of ethanol were included when appropriate. Four replicates
per treatment per each sample of plasma were used. ChE activity was determined
immediately after the end of the incubation period as previously indicated.

2.6. Statistical analysis

Results are expressed as mean7 standard error of the mean (S.E.M.). Assump-
tions of data normality and variance homogeneity were checked using the
Kolmogorov–Smirnov and Levene′s tests, respectively. As the ANOVA assumptions
were not met, the non-parametric Kruskal–Wallis test was used. The Mann–
Whitney U-test was subsequently applied for pairwise comparisons between
groups (Zar, 1996). The significance level was set at 0.05. All statistical analyses Fig. 1. Plasma cholinesterase activity of adult (A) and juveniles (B) of the White
were carried out using the statistical software SPSS v19.0 for Windows 2010. stork (Ciconia ciconia) as a function of the substrates acetylthiocholine iodide
(ASCh), S-butyrylthiocholine iodide (BSCh) and propionylthiocholine iodide (PSCh).
Values are the mean of 13 adults and 18 juveniles of the White stork (four
enzymatic determinations per bird) with corresponding standard error bars.
3. Results U¼ 1 μmol of substrate hydrolysed per minute.

3.1. ChE characterisation

Adults and juveniles′ plasma enzymes show a certain degree of

preference for PSCh. So, the maximum activity of ChE for both
groups of age was measured with PSCh and the lowest activity was
obtained with BSCh (Fig. 1A and B). The Km′s for ASCh, BSCh and
PSCh both for adult and juvenile of White storks were between
4.23  10  2 and 6.47  10  2 μM. The substrate concentrations of
0.075 M used in the assays were sufficiently high to achieve zero
order kinetics. The ratio Vmax/Km, which indicates the enzymatic
catalytic efficiency, showed the enzyme preferences: PSCh (5.70
and 5.81 for adults and juveniles, respectively)4BSCh (4.46 and
3.61 for adults and juveniles, respectively) 4ASCh (3.66 and 3.49
for adults and juveniles, respectively).
Eserine sulphate, a strong inhibitor of all the ChE enzymes, Fig. 2. Effect of eserine sulphate and BW284C51 on plasma cholinesterase activity
of adults and juveniles of the White stork (Ciconia ciconia) using acetylthiocholine
significant inhibited the enzyme activity of both adults and
as substrate. Values are the mean of 13 adults and 16 juveniles of the White stork
juveniles, with more than 90% of inhibition at 0.048 μM or higher (four enzymatic determinations per bird) with corresponding standard error bars.
concentrations (po 0.05; LOEC≤0.048 μM for both groups) (Fig. 2). U¼ 1 μmol of substrate hydrolysed per minute. (*) Indicates significant differences
These results indicate that almost all the activity measured is from from the control group mean (p o 0.05).
both ChEs (AChE and BChE). Fig. 3 shows the effects of iso-OMPA
on juvenile and adult′s plasma ChE activity. In both cases, Therefore, the NOEC and LOEC values for BW284C51 were esti-
significant inhibitions (p o0.05) were found from the lowest mated to be higher than 800 μM for adults and juveniles.
concentrations of exposure, with LOEC≤0.0078 mM for both
groups (adults and juveniles). No significant effect of the solvent 3.2. Range of activity in non-exposed individuals
ethanol on ChE activity was observed. On the other hand, plasma
ChE activity of adults and juveniles was not significantly affected The range of ChE activity in non-exposed White stocks was
by BW284C51 up to concentration of 800 μM (p4 0.05; see Fig. 2). 0.32 70.01 μmol/min/ml for adults and 0.28 7 0.03 μmol/min/ml
134 A.-L. Oropesa et al. / Ecotoxicology and Environmental Safety 97 (2013) 131–138
Fig. 3. Effect of iso-OMPA on plasma cholinesterase activity of adults and juveniles
of the White stork (Ciconia ciconia) using acetylthiocholine as substrate. Values are
the mean of 13 adults and 17 juveniles of the white stork (four enzymatic
determinations per bird) with corresponding standard error bars. U¼ 1 μmol of
substrate hydrolysed per minute. (*) Indicates significant differences from the
control group mean (po 0.05).
for juveniles using BSCh as substrate. No significant differences
(p 40.05) were found between the ChE activity of the two groups
of age.
3.3. ChE in vitro tests
Paraoxon-methyl significantly inhibited the plasma ChE activity
of adult and juveniles (po 0.05). An inhibition of more than 96%
was observed at concentrations equal to or higher than 0.94 mg/l
for both groups (Fig. 4A and B). According to the statistic analysis,
significant in vitro inhibition was obtained from lowest concentra-
tion tested for both carbofuran and carbaryl (p o0.05, respec-
tively). However, the potency of inhibition was different. So, an
inhibition greater than 79% and 87% was observed in plasma ChE
activity of adult and juvenile of White storks, respectively, at
concentrations equal to and greater than 0.94 mg/l carbofuran.
Inhibitions of more than 46% and 52% for adults and juveniles,
respectively, were obtained from the lowest concentration of
carbaryl tested.
4. Discussion
The relative inhibition of ChE activity in animals suspected to
be exposed to OP and CB insecticides can be determined by
comparison with the activity in control animals of the same
species. However, animal age can be a crucial aspect for making
suitable comparisons (Bennett and Bennett, 1991). Therefore, the
importance of the current study of establishing the range of ChE
activity in non-exposed individuals of White stork and to deter-
mine its age-dependent changes is of great interest. Moreover,
detection of low level exposure, resulting from pesticide applica-
tion in agricultural activities, by using plasma ChE may be a best
indicator of exposure because it is usually inhibited more rapidly
and to a larger degree than its activity in brain (Hill and Fleming,
1982; Sanchez et al., 1997; Soler-Rodríguez et al., 1998). The use of
plasma of White stork allows sampling without killing the animal
and may also allow repeated sampling of plasma using the same
individuals.
ChE in plasma of White stork showed slight preference for PSCh
at the concentrations tested (0.02–20.48 mM). ASCh and BSCh
were hydrolysed almost at the same rate. The ratio Vmax/Km, which
indicates the enzymatic catalytic efficiency, also showed the
enzyme preferences PSCh4 BSCh 4ASCh in adults and juveniles
of White stork.
Fig. 4. In vitro effects of anticholinesterase insecticides (methyl-paraoxon, carbo-
furan and carbaryl) on plasma cholinesterase activity of adults (A) and juveniles
(B) of the White stork (Ciconia ciconia). Values are relative (%) to the respective
control group with corresponding standard error bars and correspond to the mean
of six adults and eight juveniles of the White storks. The mean in all insecticide
treatments was significantly different from the respective control mean (p≤0.05).
The contribution of nonspecific esterases was estimated using
the compound eserine sulphate, which is considered a specific
inhibitor of ChE at low concentrations in the 10  6–10  5 M range
(Eto, 1974). In the present study, the enzymatic activity measured
in plasma of adults and juveniles of White stork was almost
completely inhibited by eserine sulphate (more than 90%), which
is considered typical for ChE. This result indicates that the activity
determined in our experimental conditions is mainly ChE. Plasma
ChE activity in adults and juveniles of White stork was sensitive to
iso-OMPA, an inhibitor of BChE but not of AChE (Barahona and
Sánchez-Fortún, 1999), from 0.008 mM. On the contrary, plasma
ChE activity in both groups was not sensitive to BW284C51, a
selective reversible inhibitor of AChE (Xu and Bull, 1994).
In base on effects of the specific inhibitors, the predominant
activity present in plasma of White storks is BChE. This conclusion
agrees with a recent study by Santos et al. (2012) also in White
stork. In birds, the proportion of plasma ChE that is BChE or AChE
varies for different species and these have several age dependent
patterns of AChE or BChE activity (McInnes et al., 1996). The
identification of BChE activity in plasma of White storks is in
agreement with study performed by Roy et al. (2005) in European
raptors. This study concluded that BChE was the predominant
activity in most species studied except in falconidae (Table 1). In
the same way, Gard and Hooper (1993) reported that BChE was the
main cholinesterase in plasma of adult of two altricial passerine
 A.-L. Oropesa et al. / Ecotoxicology and Environmental Safety 97 (2013) 131–138 135

Table 1
Summary of previous results on normal plasma cholinesterase activity (BChE) in several species of birds.

Species Sample Age (months) BChEa Units of enzymatic Reference

size (n) activity
Mean 7 (SEMb or SDc)
Min  max

Buzzard (Buteo buteo) 6 n.d. 1983.40 796.60 nmol/min/l García-Rodríguez et al. (1987)
Mallard (Anas platyrhynchos) 26 2.8 7447 32b μmol/min/l Bennett and Bennett (1991)
489–1.071
African whitebacked vulture 33 41 (≃40 days) 1795.30 7 9158.23c nmol/min/l Van Wyk et al. (1998)
(Pseudogyps africanus)
Peregrine falcon (Falco peregrinus) 32 o1 1184.007 74.99c nmol/min/ml Lanzarot et al. (2001)
Kestrel (Falco tinnunculus) Female 46 2–3 0.283 μmol/min/ml Roy et al. (2005)
0.123–0.413
Kestrel (Falco tinnunculus) Male 46 2–3 0.292 μmol/min/ml Roy et al. (2005)
0.163–0.525
Kestrel (Falco tinnunculus) Wild 39 ≥12 0.278 μmol/min/ml Roy et al. (2005)
0.139–0.507
Kestrel (Falco tinnunculus) Wild 20 ≥12 0.330 μmol/min/ml Roy et al. (2005)
nonreleasable 0.206–0.598
Barn owl (Tyto alba) Captive bred 104 2–3 2.309 μmol/min/ml Roy et al. (2005)
1.516–3.380
Barn owl (Tyto alba) Wild 25 2–3 2.202 μmol/min/ml Roy et al. (2005)
1.547–2.925
Barn owl (Tyto alba) Wild adults 11 12 2.412 μmol/min/ml Roy et al. (2005)
1.684–3.365
Tawny owl (Strix aluco) Young 81 2–3 2.507 μmol/min/ml Roy et al. (2005)
0.449–4.821
Tawny owl (Strix aluco) Adults 16 12 3.131 μmol/min/ml Roy et al. (2005)
1.146–4.324
Sparrowhawk (Accipiter nisus) 20 o 12 1.509 μmol/min/ml Roy et al. (2005)
Young 0.999–2.229
Sparrowhawk (Accipiter nisus) 7 12 1.485 μmol/min/ml Roy et al. (2005)
Adults 0.834–1.951
Egyptian vulture (Neophron 3 n.d. 0.441 μmol/min/ml Roy et al. (2005)
percnopterus) 0.306–0.570
Bonelli′s eagle (Hieraaëtus 8 n.d. 0.514 μmol/min/ml Roy et al. (2005)
fasciatus) 0.436–0.602
Booted eagle (Hieraaëtus 4 n.d. 0.381 μmol/min/ml Roy et al. (2005)
pennatus) 0.297–0.454
Marsh harrier (Circus aeruginosus) 1 n.d. 0.788 μmol/min/ml Roy et al. (2005)
Hen harrier (Circus cyaneus) 1 n.d. 0.930 μmol/min/ml (Roy et al., 2005)
Long-legged buzzard (Buteo 4 n.d. 0.905 μmol/min/ml Roy et al. (2005)
rufinus) 0.785–0.950
Buzzard (Buteo buteo) 124 n.d. 1.075 μmol/min/ml Roy et al. (2005)
0.505–2.105
Black kite (Milvus migrans) 2 n.d. 1.001 μmol/min/ml Roy et al. (2005)
0.830–1.171
Goshawk (Accipiter gentilis) 2 n.d. 1.071 μmol/min/ml Roy et al. (2005)
0.883–1.258
Honey buzzard (Pernis apivorus) 4 n.d. 3.620 μmol/min/ml Roy et al. (2005)
2.835–4.132
Lanner falcon (Falco biarmicus) 1 n.d. 0.082 μmol/min/ml Roy et al. (2005)
Hobby (Falco subbuteo) 3 n.d. 0.320 μmol/min/ml Roy et al. (2005)
0.254–0.389
Merlin (Falco columbarius) 1 n.d. 0.300 μmol/min/ml Roy et al. (2005)
Short-eared owl (Asio flammeus) 1 n.d. 1.338 μmol/min/ml Roy et al. (2005)
Long-eared owl (Asio otus) 8 n.d. 1.660 μmol/min/ml Roy et al. (2005)
1.363–1.968
Little owl (Athene noctua) 7 n.d. 3.989 μmol/min/ml Roy et al. (2005)
3.139–5.975
King Quail (Excalfactoria 9 n.d. (adults) 2.23 7 0.05c mmol/min/ml Fildes et al. (2009)
chinensis)
c
Budgerigar (Melopsittacus 6 n.d. (adults) 0.885 7 0.19 mmol/min/ml Fildes et al. (2009)
undulates)
White-plumed Honeyeater 7 n.d. (adults) 1.1307 0.30c mmol/min/ml Fildes et al. (2009)
(Lichenstomus penicillatus)
Yellow-throated Miner (Manorina 3 n.d. (adults) 0.926 7 0.53c mmol/min/ml Fildes et al. (2009)
flavigula)
c
Willie Wagtail (Rhipidura 3 n.d. (adults) 0.508 7 0.15 mmol/min/ml Fildes et al. (2009)
leucophrys)
Australian Reed-Warbler 1 n.d. (adults) 0.901 mmol/min/ml Fildes et al. (2009)
(Acrocephalus australis)
Brown Songlark (Cincloramphus 10 n.d. (adults) 1.067 0.29c mmol/min/ml Fildes et al. (2009)
cruralis)
Double-barred Finch (Taeniopygia 3 n.d. (adults) 0.417 0.05c mmol/min/ml Fildes et al. (2009)
bichenovii)
136 A.-L. Oropesa et al. / Ecotoxicology and Environmental Safety 97 (2013) 131–138
Table 1 (continued )
Species Sample Age (months)
size (n)
Australasian Pipit (Anthus 3 n.d. (adults)
Novaeseelandiae)
White stork (Ciconia ciconia) 4 –
Grey heron (Ardea cinerea) 4 –
Northern gannet (Morus 4 –
bassanus)
n.d.: not detected.
a
BChE: butyrylcholinesterase.
b
SEM: standard error mean.
c
SD: standard deviation.
species, the eastern bluebirds (Sialia sialis) and the European
starlings (Sturnus vulgaris). Similarly, Wolfe and Kendall (1998)
confirmed that, in the European starling, the plasma ChE activity is
represented by the BChE activity. Fildes et al. (2009) showed that
the BChE fraction in plasma is higher than AChE in native
Australian birds.
Gee et al. (1981) measured plasma enzyme levels in several
endangered avian species to provide baseline data for the study of
captive and wild birds. This and other related studies (Gard and
Hooper 1993; Montesinos et al., 1997; Mayack and Martin 2003;
Rendón-von Osten et al., 2005; Roy et al., 2005; Fildes et al., 2009;
Santos et al., 2012) demonstrated the need for control data on the
levels of variability of appropriate indicator enzymes in brain and
plasma of different species, to provide a baseline against which the
effects of agricultural chemicals may be assessed. Thus, in our
study using BSCh as substrate, plasma ChE activity of White stork
from non-exposed organisms was 0.32 70.01 μmol/min/ml for
adults and 0.28 70.03 μmol/min/ml for juveniles. These values
are in the same order of magnitude than those reported in the
literature for White stork (Table 1) although they used only
4 individuals. The present study revealed that developmental
stage of White stork had no significant effect on BChE activity in
plasma. This same result was obtained by Roy et al. (2005) in the
barn owl (Tyto alba) and kestrel, where no significant difference
was noted between wild adult and young birds. In contrast, this
and other authors pointed out that the age has an effect on plasma
BChE activity of several species for birds such as eastern bluebirds,
starlings and tawny owls (Strix aluco). They demonstrated an
increase in BChE activity with age in plasma, thus the BChE
activity for these species was higher in adults than in young birds
(Gard and Hooper, 1993; Roy et al., 2005).
The in vitro inhibitory potential of pesticides such as paraoxon-
methyl, carbofuran and carbaryl to ChE activity in plasma of White
storks has been studied in this work. Paraoxon-methyl was the
most potent in vitro inhibitor causing more than 96% of inhibition
at concentrations equal or greater than 0.94 mg/l. The same
sensitivity of the ChE activity in plasma to paraoxon-methyl was
found in adults and juveniles. Paraoxon-methyl is the active
metabolite of the OP insecticide parathion used to control many
insects and mites. This is converted in the body in part to
paraoxon, to carry out its mechanism of action (Hartley and
Kidd, 1983; Worthing, 1987). Flickinger et al. (1991) reported
about mortality of Canada geese (Branta canadensis) after exposure
to parathion applied in a field to control Russian wheat aphids
(Diuraphis noxia). Ludke et al. (1975) suggested that an inhibition
of brain ChE activity greater than 50% is enough to diagnose
that the pesticide is responsible for death of birds. Carbofuran
(2,3-dihydro-2,2-dimethylbenzofuran-7-yl methyl carbamate) and
BChEa Units of enzymatic Reference
activity
Mean 7 (SEMb or SDc)
Min  max
1.0757 0.10c mmol/min/ml Fildes et al. (2009)
c
0.39 7 0.12 μmol/min/ml Santos et al. (2012)
0.157 0.04c μmol/min/ml Santos et al. (2012)
0.487 0.11c μmol/min/ml Santos et al. (2012)
carbaryl (1-napthyl methylcarbamate) are broad spectrum methyl-
carbamate insecticides and acaricides (Barberá, 1989). The present
study also showed that ChE activity in plasma of White stork was
inhibited by in vitro exposure to both CBs. Thus, for carbofuran, a
ChE inhibition greater than 79% and 87% was obtained for adults
and juveniles, respectively, from the lowest concentration tested
(0.94 mg/l), whereas, for carbaryl, the inhibition at a concentration
of 0.94 mg/l varied between 46% and 52% for adults and juveniles,
respectively. A slightly greater sensitivity to both CBs in juveniles
was found when compared to adults. Thus, plasma ChE activity
can be used as a biochemical biomarker of exposure to OP and CB
compounds because this enzymatic activity can be inhibited by
these chemicals. The decreasing order of in vitro toxicity, deter-
mined as percentage of inhibition of plasma ChE, was paraoxon-
methyl4carbofuran4carbaryl. This result is in agreement with
the classification provided for these pesticides by the World
Health Organization, being extremely (toxic class Ia), highly (toxic
class Ib) and moderately hazardous (toxic class II), respectively
(WHO, 2010). These chemicals are among the most toxic pesticide
known to birds and several studies have shown in vivo effects of
these pesticides on wildlife birds. Thus, parathion is extremely
toxic to birds such as ducks (LD50 ¼0.188 mg/kg) (Hudson et al.,
1984), pigeons (LD50 ¼2.52 mg/kg) (Tucker and Haegele, 1971) or
quails (LD50 ¼5.86 mg/kg) (Dieter and Ludke, 1975), whereas it is
less toxic to pheasants (LD50 ¼12.4 mg/kg) (Tucker and Haegele,
1971). Carbofuran has been reported to cause death in gulls (Larus
californicus) and waterfowl after their feeding with vegetation and
grasshoppers, respectively, previously exposed to this pesticide
(Leighton et al., 1987; U.S. EPA, 1989). Regarding its acute toxicity it
has been reported a LD50 of 0.37 mg/kg for ducks (Hudson et al.,
1972), 7 mg/kg for pigeons (Hill and Camardese, 1984) and
4.15 mg/kg for pheasants (Hudson et al., 1984). On the other hand,
carbaryl shows a minor toxicity than previous pesticides, i.e.,
LD5042564 mg/kg for ducks, 2000 mg/kg for pigeons, 707 mg/kg
for pheasants and 2290 mg/kg for quails (Hudson et al., 1984).
In one incident, a single morning dove (Zenaida macroura) died after
a homeowner had applied carbaryl to the lawn in the vicinity of a
bird feeder, apparently contaminating the food (U.S. EPA, 2004).
In conclusion, the predominant cholinesterase activity present
in plasma of White stork was BChE. In addition, age-dependent
differences in the values of the plasma BChE activity in non-
exposed individuals were not detected and the percentage of
in vitro plasma ChE inhibition was similar between adults and
juveniles for the tested pesticides. So, the blood from White stork
chicks is a non-destructive sample that can be easily obtained,
without stress, from the animal in the nest, being useful to assess
the exposure and effects of the OF and CB pesticides in this species
by measuring the ChE activity.
 A.-L. Oropesa et al. / Ecotoxicology and Environmental Safety 97 (2013) 131–138
Acknowledgments
This study was supported by Consejería de Economía, Comercio
e Innovación de la Junta de Extremadura (Spain) and by EU (FEDER
programme).
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