Aquatic Toxicology 199 (2018) 276–284

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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aqtox

Toxicokinetic and toxicodynamic studies of carbaryl alone or in binary T

mixtures with azinphos methyl in the freshwater gastropod Planorbarius
corneus
⁎
Luis Claudio Cacciatore, Noemí Rosario Verrengia Guerrero, Adriana Cristina Cochón
Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química Biológica, Ciudad Universitaria, Nuñez, 1428, Buenos Aires, Argentina

A R T I C LE I N FO A B S T R A C T

Keywords: Carbamate insecticides such as carbaryl and organophosphates such as azinphos-methyl share the ability to
Anticholinesterase insecticides inhibit the activity of B-esterases. This study aimed to (1) assess the inhibitory effects of carbaryl on B-esterase
B-esterases activity in soft tissues and hemolymph of Planorbarius corneus; (2) establish whether binary mixtures of carbaryl
Planorbarius corneus and azinphos-methyl depart or not from a model of concentration addition on the inhibition of cholinesterase
Mixtures
activity; (3) determine the bioconcentration and elimination of the pesticides. The results showed that exposure
Toxicokinetics
of gastropods to increasing concentrations of carbaryl (0.1–5 mg L−1) for 48 h inhibited cholinesterase activity in
a concentration-dependent manner, with an EC50 of 1.4 ± 0.3 mg L−1 and 1.2 ± 0.1 mg L−1 for soft tissue and
hemolymph, respectively. Carboxylesterase activity, measured with the substrates p-nitrophenyl butyrate and p-
nitrophenyl acetate, was between 2.3 and 25 times more sensitive to carbaryl inhibition than cholinesterase
activity. Binary mixtures corresponding to 0.5 EC50 carbaryl + 0.5 EC50 azinphos-methyl and 0.75 EC50 car-
baryl + 0.75 EC50 azinphos-methyl produced inhibitions of cholinesterase activity similar to those of individual
pesticides, following a model of concentration addition. Bioconcentration was analyzed using a one-compart-
ment model. The absorption kinetics (k1) for both pesticides alone (1.4 mg L−1 of carbaryl or 1.8 mg L−1 of
azinphos-methyl) or mixed (1.4 mg L−1 of carbaryl + 1.8 mg L−1 of azinphos-methyl) were similar. The elim-
ination kinetics ratio (k2) estimated for the pesticides alone or in the mixtures showed that carbaryl was
eliminated 3.5 times faster than azinphos-methyl. These results suggest that exposure of Planorbarius corneus to
binary mixtures of carbaryl and azinphos-methyl for 48 h follow a concentration addition model on inhibition of
cholinesterase activity and that the pesticide mixtures do not change the toxicokinetic parameters of the parent
compounds.

1. Introduction
Carbaryl (1-naphthyl-N-methylcarbamate, CB) is one of the most
frequently used carbamate insecticides. CB is used to control a broad
spectrum of insects in over 120 different crops and as insecticide in
gardens (Ware, 2000). Despite its low persistence in the environment
and its rapid hydrolysis in alkaline medium, CB can persist in aquatic
ecosystems for over a year after application (Bacchetta et al., 2008). In
general, pesticide concentrations in water are highly variable in time
and location, and monitoring data usually do not capture the peak
concentrations. Non-target species might be exposed to a wide range of
CB concentrations due to different exposure scenarios. Concentrations
of CB reported in surface waters due to agricultural practice are in
general lower than 1 μg L−1 (Schulz, 2004). However, it has been re-
ported that the concentrations found in the environment immediately
after application can reach levels higher than 1700 μg L−1 (Norris et al.,
1983; Walters et al., 2003).
In general, pesticides are found in aquatic environments as part of
complex mixtures. In this regard, Loewy et al. (2011) observed the
coexistence of CB and azinphos-methyl (AZM) in drainage water from
flood-irrigated areas where apple crops were grown. These authors
reported that AZM (O,O-dimethyl S-[4-oxo-1,2,3-benzotriazin-3(4H)-
yl) methyl]triazin-3-ylmethyl) was the most widely used organopho-
sphate pesticide to control Carpocapsa pomonella in the Neuquén River
Valley, Argentina. This insect is the most critical pest affecting apple
production. In particular, Loewy et al. (2011) reported that during the

Abbreviations: AChE, acetylcholinesterase; AcSCh, acetylthiocholine iodide; AZM, azinphos-methyl; BCF, bioconcentration factor; CB, carbaryl; CPF, chlorpyrifos; CES, carbox-
ylesterases; ChE, cholinesterase; CYP, cytochrome P450; DTNB, 5,5′-dithio-2-bis-nitrobenzoate; p-NPA, p-nitrophenyl acetate; p-NPB, p-nitrophenyl butyrate; OPs, organophosphates
⁎
Corresponding author at: Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Departamento de Química Biológica, Ciudad Universitaria, Pab. II. 4to piso, 1428
Buenos Aires, Argentina.
E-mail address: adcris@qb.fcen.uba.ar (A.C. Cochón).

https://doi.org/10.1016/j.aquatox.2018.04.005
Received 11 July 2017; Received in revised form 27 December 2017; Accepted 10 April 2018
Available online 14 April 2018
0166-445X/ © 2018 Elsevier B.V. All rights reserved.
L.C. Cacciatore et al.
periods of application from October to March for the crop years 2008/
09 and 2009/10, the highest detection frequencies in surface and sub-
surface water was found for AZM and chlorpyrifos (CPF) (> 70%),
followed by CB (> 40%). The highest concentration levels were found
in shallow groundwater, with 22.5 μg L−1 for AZM and 45.7 μg L−1 for
CB (Loewy et al., 2011). AZM has also been used for pest control on a
number of food crops in many parts of the world, and concentrations up
to 14.9 mg L−1 have been reported in runoff and river surface waters
adjacent to agricultural fields (Castro et al., 2017; Shayeghi et al.,
2012).
The efficacy of CB as an insecticide is due to the inhibition of
acetylcholinesterase (AChE), causing the accumulation of acetylcholine
in the synaptic cleft. AChE is the key target for both organophosphate
(OP) and carbamate insecticides, and their main toxicological effects
are very similar. However, while OPs are able to phosphorylate the
serine residues of AChE irreversibly, the carbamylation of the same
residues is less stable (Sogorb and Vilanova, 2002; Van Dyk and
Pletschke, 2011). Since AZM has a thiophosphoryl bond (P = S) instead
of a phosphoryl bond (P = O), it possesses minimal or no antic-
holinesterase activity and requires metabolic activation by cytochrome
P450 (CYP) enzymes to its oxon analog to inhibit ChE activity (Tang
et al., 2006; Crane et al., 2012).
In addition to the CYP-mediated metabolism of OPs, several authors
have concluded that detoxification enzyme activities such as carbox-
ylesterases (CES) must also be considered when evaluating the effects of
combined exposure to OPs (Karanth et al., 2004; Richardson et al.,
2001; Jansen et al., 2009; Cacciatore et al., 2012, 2013a). Besides, CES
are important contributors to the stoichiometric detoxification of many
oxons. Thus, CES enzymes can reduce the amount of pesticide available
for ChE inhibition by direct binding and sequestration of the oxons
(Sogorb and Vilanova, 2002; Sanchez-Hernandez et al., 2014).
Since anticholinesterase agents inhibit AChE, it is expected that the
effects of mixtures of these pesticides on this activity would follow a
concentration addition model, i.e., the concentration of each pesticide
may be replaced by an equivalent effective concentration of one of
them (Bosgra et al., 2009). However, there are several documented
cases where mixtures of anticholinesterase pesticides do not follow this
additive model. Previously, we have observed in homogenates of the
freshwater snail Planorbarius corneus that several mixtures of AZM-oxon
and CPF-oxon resulted in cumulative in vitro ChE inhibitions higher
than those expected on a concentration addition basis (Cacciatore et al.,
2012). Similar results were found in vivo after exposure to binary
mixtures of AZM and CPF (Cacciatore et al., 2013a).
Despite the well-characterized acute toxicity of CB and AZM, few
studies exist on the bioconcentration of these pesticides in invertebrates
(Ramírez Mora et al., 2000; Klosterhaus et al., 2003). It is often as-
sumed that bioconcentration of anticholinesterase pesticides by aquatic
organisms means no risk for the ecosystem. In their natural environ-
ment, the exposure would almost invariably involve sublethal con-
centrations of pesticides, due to their low partition coefficients and/or
high biotransformation rates. These factors could prevent these che-
micals from accumulating through food chains in higher organisms (De
Bruijn and Hermens, 1991; Sancho et al., 1998). However, different
physiological and behavioral responses can be affected which may en-
danger the survival of the species (Sancho et al., 1998; Kristoff et al.,
2011).
In contrast to most non-polar organics, many carbamate and OP
insecticides can be rapidly metabolized in fish. Therefore, correlations
between Bioconcentration Factor (BCF) and octanol-water partition
coefficient (Kow) are not applicable to these compounds (Keizer et al.,
1991) and little is known whether metabolism also affects BCFs of
anticholinesterase agents in invertebrates (Legierse et al., 1998). Even
less is known about the toxicokinetics of anticholinesterase insecticides
in binary mixtures. Several toxicokinetic studies have highlighted how
substances can inhibit, induce or compete with the activity of the CYP
system. Altering the biotransformation rates resulting from co-exposure
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Aquatic Toxicology 199 (2018) 276–284
of organic substances metabolized by relevant enzymes may increase or
decrease the toxicity of the individual compounds when mixed (Dorne
et al., 2007a,b). For example, a significant increase in CB toxicity in
red-legged partridges following the administration of the OP malathion
has been attributed to inhibition of CB metabolism (Johnston, 1995). In
the same line, coincubation of CB and CPF with human liver micro-
somes strongly inhibited CB metabolism (Tang et al., 2002). To our best
knowledge there are no studies addressing the modulation of CB toxi-
city by AZM and thus far, there have been no studies about CYP-
mediated metabolization of CB and AZM in P. corneus.
Some molluscs have been proposed as suitable biological experi-
mental models to assess sublethal impacts of contaminants and mix-
tures of contaminants in aquatic ecosystems (Rittschof and McClellan-
Green, 2005). The gastropod P. corneus is a hermaphroditic snail that
usually inhabits small temporary ponds and streams. This species be-
longs to the Planorbidae family, the largest family of aquatic pulmonate
gastropods, and has a global distribution (Jopp, 2006). P. corneus has
already been used as a model organism for toxicity studies (Pavlica
et al., 2000; Otludil et al., 2004; Clarke et al., 2009; Benstead et al.,
2011; Cacciatore et al., 2012, 2013a, 2015).
The aims of this study were (1) to evaluate the inhibitory effects of
CB on the B-esterases activities in soft tissues and in the hemolymph of
P. corneus; (2) to establish whether binary mixtures of the carbamate CB
and the OP AZM depart or not from a concentration addition model on
ChE activity inhibition in soft tissues; (3) to determine the kinetics of
absorption and elimination of CB and AZM pesticides alone or in binary
mixtures.
2. Materials and methods
2.1. Chemicals
Acetylthiocholine iodide (AcSCh), p-nitrophenyl acetate (p-NPA), p-
nitrophenyl butyrate (p-NPB), 5,5‘-dithio-2-bis-nitrobenzoate (DTNB),
azinphos-methyl (AZM) PESTANAL® (CAS No. 86-50-0, 97.2% pure),
and carbaryl (CB) PESTANAL® (CAS No. 63-25-2, 99% pure) were
purchased from Sigma–Aldrich. All other chemicals used were of ana-
lytical reagent grade.
2.2. Snails
Adult P. corneus snails were purchased from Discus Moron S.R.L.,
Buenos Aires, Argentina. Afterwards the snails were reared in our la-
boratory in aerated glass aquaria (17–20 L), at a temperature of
22 ± 2 °C, and under a 14:10 (L:D) h artificial photoperiod regime.
Animals were fed lettuce leaves ad libitum. For all the experiments,
adult snails of similar size (10 ± 2 mm of shell length) were used.
Water quality characteristics were as follows: total hard-
ness = 67 ± 3 mg CaCO3 L−1; alkalinity = 29 ± 2 mg CaCO3 L−1; pH
7.0 ± 0.2 and conductivity = 250 ± 17 μS cm−1.
2.3. Inhibition studies
The general experimental design consisted of 1 L glass vessels con-
taining 600 mL of each treatment condition, i.e. different pesticide
concentration, solvent control and solvent-free control. Animals were
not fed during the treatments. All bioassays were performed at a tem-
perature of 22 ± 2 °C, and under a photoperiod of 14:10 h light/dark
without aeration. No mortality was observed either in control animals
or in any of the treatments. Carbon filtered dechlorinated tap water was
used as the test medium.
In all experiments, the concentration of acetone was kept at 0.05%
and solvent (acetone 0.05%) and solvent-free (dechlorinated tap water
only) controls were included.
L.C. Cacciatore et al.
2.3.1. Experiment I. Single-chemical studies
Snails were exposed for 48 h to 7 nominal CB concentrations (0.1;
0.25; 0.5; 1; 1.5; 3; and 5 mg L−1). A total of 162 specimens were di-
vided into 9 groups of 18 snails per group (solvent control, solvent-free
control and those exposed to the different CB concentrations).
Triplicate vessels were used for each treatment condition and six ran-
domly selected snails were placed in each vessel. At the end of the
experiment, the hemolymph and whole soft tissues from 3 snails per
vessel were pooled together as one hemolymph sample and one soft
tissue sample. Therefore, the number of samples per treatment group
was six (n = 6). Both ChE and CES activities were measured in each
sample.
Absolute and relative EC50 values of ChE and CES inhibitor were
calculated with the 4-parameter logistic model using OriginPro 7.5
(OriginLab, Northampton, MA). This model can be written as an
equation that defines the response as a function of concentration and 4
parameters (A1, A2, p, x0). The model equation is specified by:
(A1 − A2)
y = A2 +
1 + (x/x 0) p
In this equation, “y” is the enzyme activity (expressed as nmol
substrate hydrolyzed min−1 mg protein−1) at concentration “x” of the
inhibitor. “A1” denotes the value of “y” for the maximal curve asymp-
tote (theoretically, the level of response in the absence of pesticide).
“A2” denotes the value of “y” for the minimal curve asymptote (theo-
retically, the level of response produced by an infinitely high con-
centration of the inhibitor). “p” denotes the steepness of the dose–r-
esponse curve (Motulsky and Christopoulos, 2004). The relative EC50 is
the parameter “x0” and represents the concentration corresponding to a
response midway between the estimates of the top (A1) and the bottom
(A2) plateaus. The 4 parameters were calculated with the software
OriginPro 7.5 as the best-fit values in the model by an iterative pro-
cedure. Absolute EC50 is defined as the concentration giving exactly a
50% response (Laguerre et al., 2009) and was calculated from the lo-
gistic Eq. (1) considering y = half control activity level.
2.3.2. Experiment II. Binary-mixture studies
The effects of two combinations of CB and AZM on ChE and CES
activities were analyzed. In all cases, the concentration of each pesti-
cide in the mixture tests was based on the EC50 values for ChE inhibi-
tion in soft tissue derived from tests conducted with individual pesti-
cides. An EC50 for CB of 1.4 mg L−1 was calculated in the present study
(Table 1). EC50 for AZM (1.8 mg L −1) has been previously obtained in
our laboratory under equal experimental conditions and has already
been reported (Cacciatore et al., 2013a).
Exposures to two 50:50 combinations of CB and AZM were per-
formed: mixture 1: 0.5 EC50 units of CB + 0.5 EC50 units of AZM, and
mixture 2: 0.75 EC50 units of CB + 0.75 EC50 units of AZM. At the same
time, vehicle control and single pesticide controls consisting of con-
centrations corresponding to 1.0 EC50, and 1.5 EC50 were also
Table 1
Concentrations of carbaryl (CB) causing inhibitions of 50% (EC50) on cholinesterase and carboxylesterases activities in soft tissue and hemolymph, and the four
parameter values of the non-linear regressions (A1, A2, X0 and p) shown in Fig. 2A–C.
Substratea A1 A2 X0
Soft tissue
AcSCh 273 ± 10 57 ± 34 0.75 ± 0.33
p-NPA 123 ± 4 11 ± 27 0.17 ± 0.15
p-NPB 166 ± 2 4±2 0.03 ± 0.01
Hemolymph
AcSCh 508 ± 9 136 ± 17 0.69 ± 0.07
p-NPA 223 ± 4 27 ± 7 0.23 ± 0.03
p-NPB 120 ± 2 2±5 0.13 ± 0.02
a
Cholinesterase activity was assayed using acetylthiocholine (AcSCh) as substrate and carboxylesterase activity was assayed using p-nitrophenyl acetate (p-NPA)
and p-nitrophenyl butyrate (p-NPB).
Aquatic Toxicology 199 (2018) 276–284
performed.
A total of 72 specimens were divided into 6 groups of 12 snails per
group (vehicle control, solvent-free control, CB control, AZM control,
mixture 1 and mixture 2). Four vessels were used for each treatment
condition and 3 randomly selected snails were placed in each vessel. At
the end of the experiment, the whole soft tissues from 3 snails per vessel
were pooled together as one sample. Therefore, 4 independent samples
were analyzed per treatment group (n = 4). Both ChE and CES activ-
ities were measured in each sample.
To analyze whether the effects of mixtures 1, and 2 of the pesticides
on ChE activity were additive, less than additive or more than additive,
the method described by Laetz et al. (2009) was used. In brief, each
pesticide concentration was divided by the concentration estimated to
produce a 50% decrease in ChE activity relative to controls. The EC50-
normalized data for the two pesticides were subsequently plotted in the
same graph and jointly fit with a single regression to Eq. (1) using
OriginPro 7.5 (OriginLab, Northampton, MA). When the software fits a
curve with nonlinear regression, it can superimpose on the graph pre-
diction bands. Therefore, a single curve with a 95% prediction interval
(1)
was obtained. The 95% prediction interval encloses the area that is
expected to enclose 95% of future data points. If the con-
centration–effect curves of the two anticholinesterase pesticides are
parallel, this curve can be used for detecting interactions between them
in mixtures. If concentration addition occurs, ChE inhibition for a
mixture would fall on the curve or within the 95% prediction interval
for the regression. Results falling significantly above the curve (less
than expected inhibition) would be less than additive, and results
falling significantly below the curve (more than expected inhibition)
would be more than additive. In this way, the curve fit to the data from
single-chemical trials can be used as a basis for detecting interactions
between anticholinesterase pesticides in mixtures (Laetz et al., 2009,
2013; Cacciatore et al., 2012, 2013a,b,c).
2.3.3. Enzymatic determinations
Animals were placed on ice for 6–8 min. The shells were dried with
absorbent tissue paper and the hemolymph was obtained by gently
craking the snaiĺs shell and allowing it to drain into the bottom half of a
Petri dish (Sherma and Fried, 2011). Afterwards the hemolymph was
collected with a pipette, placed into Eppendorf tubes and diluted 1:2
with cold 20 mM Tris/HCl buffer, pH 7.5, plus 0.5 mM EDTA. After
removing the shell, the entire soft body was washed in distilled water to
remove debris, placed on filter paper to drain extra fluids, and weighed.
Soft tissues were homogenized (1:10, w:v) in cold 20 mM Tris/HCl
buffer, pH 7.5, plus 0.5 mM EDTA. Homogenates were centrifuged at
11,000×g for 20 min at 4 °C and the supernatants were immediately
used in enzymatic assays.
ChE activity was measured in 100 mM phosphate buffer, pH 8.0,
0.2 mM DTNB, and 1.5 mM AcSCh as substrate according to the method
of Ellman et al. (1961). Each supernatant or hemolymph dilution was
assayed for ChE activity in duplicate. Activity was recorded
p r2 EC50 (mg L−1) EC50 (μM)
0.87 ± 0.23 0.9968 1.4 ± 0.3 4.8 ± 1.0
0.45 ± 0.21 0.9932 0.18 ± 0.16 0.99 ± 0.88
0.52 ± 0.07 0.9990 0.04 ± 0.01 0.19 ± 0.01
1.32 ± 0.15 0.9957 1.2 ± 0.1 4.2 ± 0.4
0.90 ± 0.10 0.9944 0.32 ± 0.04 1.8 ± 0.2
0.57 ± 0.07 0.9998 0.13 ± 0.02 0.62 ± 0.10
278
L.C. Cacciatore et al.
continuously at 412 nm and specific activity was expressed as nmol
min−1 mg protein−1. The enzymatic activity was corrected for sponta-
neous hydrolysis of the substrate and non-specific reduction of the
chromogen by tissues extracts.
CES activity was determined using two different substrates: p-ni-
trophenyl acetate (p-NPA) and p-nitrophenyl butyrate (p-NPB). Each
supernatant or hemolymph dilution was assayed for CES activity in
duplicate. Hydrolysis of p-NPA and p-NPB by CES was determined ac-
cording to Kristoff et al. (2010). Reactions were performed in 2.5 mL
100 mM phosphate buffer pH 8.0 containing 5% acetone and 1 mM p-
NPA or p-NPB. Activity was continuously recorded at 400 nm using a
Shimadzu UV-160A double-beam UV–visible spectrophotometer (Shi-
madzu, Kyoto, Japan). Specific activity was calculated using the molar
extinction coefficient for p-nitrophenol (18.6 mM−1 cm−1).
Protein content was determined according to the method of Lowry
et al. (1951), using bovine serum albumin as standard.
2.4. Experiment III. Toxicokinetic parameters
Uptake and elimination experiments were conducted under static
conditions in 1 L glass vessels containing 600 mL of test solution.
Carbon-filtered dechlorinated tap water was used as the test medium.
The following physico-chemical parameters were recorded: total hard-
ness = 67 ± 3 mg CaCO3 L−1; alkalinity = 29 ± 2 mg CaCO3 L−1; pH
7.0 ± 0.2 and conductivity = 250 ± 17 μS cm−1.
For the uptake phase, snails were divided into 4 groups (water
control, CB, AZM, mixture). CB and AZM concentrations were set at
1.4 mg L−1 and 1.8 mg L−1, respectively; the binary mixture was com-
posed of 1.4 mg L−1 CB + 1.8 mg L−1 AZM. Aqueous solutions con-
taining the pesticide CB or AZM or the mixture CB + AZM were pre-
pared by dissolving the pesticides in acetone, and diluted with an
appropriate amount of dechlorinated tap water. Final acetone con-
centration in exposure vessels was 0.05%. The organisms used as ne-
gative control were placed in the water vessels.
For each treatment condition, 4 glass vessels with 7 snails per vessel
were used. Seven gastropods were placed in the glass vessels at time
zero (0 h). One snail exposed to each one of the pesticides or the mix-
ture was removed with a histological clamp at 0.5; 1; 2; 4; 6; 9; and
12 h. Four replicates were performed on 4 consecutive days. Therefore,
a total of 112 specimens were used for this experiment and 4 in-
dependent samples were analyzed per treatment group (n = 4) at each
time point. Sampling involved a quick rinse in distilled water to ensure
total cleanup of the outer surface of pesticides. The animals were placed
on ice for 4–6 min. The shells were carefully removed and the soft tis-
sues isolated at 4 °C. All soft parts of each snail were placed on filter
paper to drain extra fluids and weighed, average weight being
132 ± 18 mg. Exposure concentrations in soft tissues were confirmed
by chemical analysis.
For the elimination phase, 6 snails were placed in 1 L glass vessels
with 600 mL of the CB or AZM solution or the mixture CB + AZM for
12 h. The depuration phase was initiated after 12 h of exposure by re-
moving the animals from the uptake aquaria, quickly rinsing them in
distilled water, and then placing them into clean glass aquaria con-
taining 600 mL of carbon filtered dechlorinated tap water. Invertebrates
were then sampled at the following intervals: 0; 0.25; 0.5; 1.0; 2.0; and
3.0 h, and prepared for solvent extraction and chemical analysis in the
same way as in the uptake phase. Four replicates (n = 4 for each con-
dition) were performed on consecutive days.
The studies were conducted without aeration and without food
supply. All the tests were performed at a temperature of 22 ± 2 °C, and
under a photoperiod of 14:10 h light/dark. No mortality was observed
either in control animals or in any of the treatments.
2.5. Chemical analyses
Pesticide concentration was tested using HPLC with UV detector.
279
Aquatic Toxicology 199 (2018) 276–284
Reversed phase chromatography was performed on a Shimadzu Class-
VP model with isocratic pump (LC-10AT VP), a variable wavelength
programmable UV–vis detector (SPD-10A VP) and a Supelcosil LC-18
(250 mm × 4.6 mm, 5 μm particle size) column and Supelcosil LC-18
(20 mm × 4.6 mm, 5 μm particle size) Guard Column. In the case of CB,
the operating conditions were: mobile phase: acetonitrile/water (45:55,
v/v), flow rate: 1 mL min−1, temperature: 40 °C, run time: 10 min, and
detection wavelength: 277 nm. Linearity was observed in the con-
centration range of 0.10–10.6 mg L−1 with regression equation
y = 41,117 x (mg L−1) + 5523. In the case of AZM, the operating
conditions were: mobile phase: acetonitrile/water (45:55, v/v), flow
rate: 1 mL min−1, temperature: 40 °C, run time: 17 min, and detection
wavelength: 220 nm. Linearity was observed in the concentration range
of 0.17-54.6 mg L−1 with regression equation y = 118,736.73x
(mg L−1) + 974.10.
2.5.1. Water samples
The concentration of the exposure solutions was confirmed by
analysis of 1 mL water samples collected in duplicate. In the 48 h ex-
posure experiments, the concentrations measured were always within
the range 95–102% of the nominal values. In the uptake experiments,
the 0-h–12-h average measured pesticide concentration was in all
cases > 93% of the initial concentration. Aqueous medium was also
sampled at the end of the depuration phase and the presence of mea-
surable concentrations of the parent compounds of the pesticides was
analyzed by HPLC. Results showed that parent compounds concentra-
tions were below the limit of detection due to pesticide metabolization
and to the fact that the ratio of water to organisms was large. Therefore,
it was assumed that reuptake of the parent compounds excreted by the
organisms during the depuration phase was negligible because con-
centration levels were very low compared to the exposure phase
(< 10%).
2.5.2. Tissue samples
Tissues of each snail were homogenized with pure acetonitrile, final
volume of 1.0 mL. The homogenate was centrifuged in Eppendorf tubes
at 4 °C (14,000×g, 5 min) and immediately placed on ice. The super-
natants obtained were kept for later use. Twenty microlitres of the re-
sulting solution were injected directly into the chromatographic system.
For the calculation of pesticide recovery, tissues of each snail were
spiked with 2, 3 and 5 μg of CB or AZM or CB +AZM, and after equi-
libration for 1 h at room temperature they were extracted and analysed
according to the procedure described above. Average recoveries of CB
and AZM were 98% ± 5% (n = 16) and 99% ± 6% (n = 16), re-
spectively.
2.6. First-order uptake and elimination kinetics
Bioconcentration was analyzed using a one-compartment model
(Rosen and Lotufo, 2007), which describes the concentration of a
chemical in an aquatic organism as the result of first-order uptake and
elimination kinetics:
dCorg/dt = k1Cw − k2Corg (2)
−1 −1
In this equation, k1 is the uptake rate constant (mL g h ) and k2
is the elimination rate constant (h−1); Cw is the exposure concentration
in water (μg mL−1) and Corg is the concentration of the compound in
the organism (μg g−1 wet weight).
Since exposure concentration was constant over time (experimen-
tally confirmed) and Corg was not detectable at t = 0, Eq. (2) was in-
tegrated as:
k1
Corg = C w (1 − e–k2t)
k2 (3)
The constants k1 and k2 were then estimated simultaneously from
L.C. Cacciatore et al.
Eq. (3) using nonlinear iterative regression analysis (OriginPro 7.5).
The experimentally measured elimination rate (k2(m)) was de-
termined by fitting the data from the depuration experiments to the
first-order decay in Eq. (4) (Legierse et al., 1998; Rosen and Lotufo,
2007):
Corg = Cºorg e–k2(m) t
where C°org is the concentration of the compound in the organism at
t = 0 of depuration.
The biological half-life of the pesticides alone or in binary mixture
(t1/2) was determined following Eq. (5) (Legierse et al., 1998; Rosen
and Lotufo, 2007):
t½ = 0.693/k2
Bioconcentration factors (BCFs) were calculated kinetically using
Eq. (6) (Legierse et al., 1998; Rosen and Lotufo, 2007):
k1
BCF =
k2
2.7. Statistical analysis
Results were expressed as mean values ± S.D. Data were analyzed
by one-way ANOVA followed by Tukey HSD post-test by using
VassarStats (http://vassarstats.net/). The level of significance used was
0.05. Prior to ANOVA, data were tested for normality and homogeneity
of variance using the Shapiro–Wilk and Levene’s tests, respectively, by
using OriginPro 7.5.
3. Results
3.1. Experiment I: B-esterase activities in snails exposed to CB
Exposure of the snails for 48 h to increasing concentrations of CB
(0.1–5 mg L−1) produced a concentration-dependent inhibition of ChE
and CES activities, in soft tissues and in the hemolymph of P. corneus
(Fig. 1).
Table 1 shows the values corresponding to EC50 values calculated
for CB and the parameters of the nonlinear regression equation. In the
case of ChE activity, the values corresponding to the EC50 were very
similar in both matrices: 1.4 ± 0.3 mg L−1 (4.8 ± 1.0 μM) and
1.2 ± 0.1 mg L−1 (4.2 ± 0.4 μM), in soft tissues and hemolymph, re-
spectively. The CES enzymes were more sensitive to the inhibitory ef-
fect of CB than ChE activity both in soft tissues and hemolymph. Thus,
analysis of the molar relations between the EC50 of ChE and CES ac-
tivity inhibition showed that CES were between 2.3 and 4.8 times more
sensitive with the substrate p-NPA and 6.8 and 25 times more sensitive
Fig. 1. Effects of increasing concentration of carbaryl in soft tissue and hemolymph on cholinesterase (ChE) activity (A) and on carboxilesterases (CES) activities (B
and C). ChE activity was assayed using (A) acetylthiocholine iodide (AcSCh) as substrate. CES activities were assayed using (B) p-nitrophenyl acetate (p-NPA), and
(C) p-nitrophenyl butyrate (p-NPB), as substrates. Data points show mean values ± S.D. (n = 6). ChE and CES activities from the solvent and solvent-free controls
did not differ significantly (p > 0.05). Therefore, only data from the solvent control (acetone 0.05%) is shown. The curves are fits of the logistic sigmoid equation
y = A2 + (A1–A2)/(1 + (x/x0)p), to the data using OriginPro version 7.5. The asterisk (*) denotes the values that are statistically different from controls (p < 0.05).
Aquatic Toxicology 199 (2018) 276–284
with p-NPB than ChE, in the hemolymph and soft tissues, respectively
(Table 1).
3.2. Experiment II. B-esterase activities in snails simultaneously exposed to
mixtures of CB and AZM
(4)
The effects of two combinations of CB and AZM on ChE and CES
activities were studied. In all cases, the concentrations in the mixtures
were based on the EC50 values for inhibition of ChE activity derived
from the assays conducted with single pesticides in soft tissues (Fig. 1A
and see Section 2.3.2).
3.2.1. ChE activity
(5)
To study the effects of pesticide mixtures on ChE activity in soft
tissues, the concentration of each pesticide was normalized to the re-
spective EC50 (normalized EC50) and the results were combined and
fitted with a single nonlinear regression (Fig. 2A). This curve (y = 100/
(1 + x0.87), r2 = 0.94288) with its 95% prediction interval was used as
(6)
a basis for determining whether specific combinations of pesticides
departed from a concentration addition model in soft tissues. For ex-
ample, in a mixture containing two pesticides each at 0.5 EC50, con-
centration-addition occurs if the cumulative ChE inhibition is equiva-
lent to 1 EC50. This result would fall on the curve. Similarly, in a
mixture containing two pesticides each at 0.75 EC50, concentration-
addition occurs if the cumulative ChE inhibition is equivalent to 1.5
EC50.
The results obtained showed that ChE activity in soft tissues of snails
exposed to single pesticides at concentrations of 1.0 EC50, and 1.5 EC50
(Fig. 2B) or in the mixtures 1 (0.5 EC50 units of CB + 0.5 EC50 units of
AZM) and 2 (0.75 EC50 units of CB + 0.75 EC50 units of AZM) (Fig. 2C)
fell on the combined curve y = 100/(1 + x0.87) or within the 95%
prediction interval. These results indicate that concentration addition
on ChE activity inhibition occurred when mixtures 1 and 2 were used.
3.2.2. CES activities
Fig. 3 represents the CES activity after 48 h exposure of snails either
to the pesticides alone, or to the binary combinations of CB and AZM
used for determining ChE activity. CES activities were evaluated using
p-NPA (Fig. 3A) and p-NPB (Fig. 3B) as substrates. Interestingly, in all
the experimental conditions, a strong inhibition of the CES activity was
found, approximately 75% to 88%.
3.3. Experiment III. Toxicokinetics of CB alone or in binary mixture with
AZM
3.3.1. Bioconcentration studies
Fig. 4 shows the uptake kinetics and the apparent steady state
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L.C. Cacciatore et al. Aquatic Toxicology 199 (2018) 276–284

Fig. 2. Cholinesterase (ChE) inhibition in soft tissue by single pesticides and 50:50 binary mixtures of carbaryl (CB) and azinphos-methyl (AZM) after normalization
to their respective EC50. (A) Plot of the concentration-response data shown in Fig. 1A and in a previous publication (Cacciatore et al., 2013a) after normalization to
their respective EC50 concentrations and collectively fitted with the logistic sigmoid equation y = 100/(1 + x0.87). Data points show mean values ± S.D. (n = 6) and
are expressed as percentage of solvent control. The solid line shows the result from non-linear regression and dashed lines are the 95% prediction interval. This curve
was subsequently used as a basis for determining whether specific combinations of pesticides departed or not from a concentration addition model. For example, in a
mixture containing two pesticides each at 0.5 EC50, concentration-addition occurs if the cumulative ChE inhibition is equivalent to 1 EC50. This result would fall on
the curve or within the 95% prediction interval. (B) Plot of ChE activity of snails exposed to single pesticide controls at concentrations corresponding to 1.0 EC50 (1.4
or 1.8 mg L−1, for CB and AZM, respectively), and 1.5 EC50 (2.1 or 2.7 mg L−1, for CB or AZM, respectively). Lines show the curve (y = 100/(1 + x0.87)) and the 95%
prediction interval depicted in A and triangles represent individual determinations (n = 4) expressed as percentage of solvent control. These assays were performed
at the same time as the assays with the binary mixtures shown in C. (C) Effects of 50:50 binary mixtures of CB and AZM on ChE activity. Mixture 1: 0.5 EC50 units of
CB + 0.5 EC50 units of AZM, and mixture 2: 0.75 EC50 units of CB + 0.75 EC50 units of AZM. Lines show the curve and the 95% prediction interval depicted in A.
Triangles represent individual determinations (n = 4) expressed as percentage of solvent control.

reached by constant exposure to 1.4 mg L−1 CB (Fig. 4A), 1.8 mg L−1
AZM (Fig. 4B) and a mixture of 1.4 mg L−1 CB + 1.8 mg L−1 AZM
(Fig. 4C) of P. corneus. The concentrations used for single pesticides
represent the EC50 at 48 h of ChE activity. The size of the snails used
was similar in all trials, corresponding to adult specimens with an
average soft tissue wet weight of 132 ± 18 mg. Thus, absorption rates
were not biased due to the size of the organism.
Experimental data were fitted by a nonlinear regression analysis
iterative to equation Corg = k1/k2 Cw (1 − e−k2t), and the values of k1
and k2 were calculated. The exponential function used, y = a
(1 − e−bx) makes it possible to calculate the best fit, by successive
iterations, for parameters a and b, where a = k1/k2 Cw, b = k2, and
Cw = concentration of the pesticide in the water, which was held
constant during exposure and was determined experimentally (see
Section 2.6).
The model used makes it possible to estimate k2 from the ex-
ponential equation with the data from the exposure phase; we call this
k2 value theoretical to distinguish it from and compare it with the ex-
perimental k2(m) value obtained from the elimination phase of the
parent compound when the snails go from 12 h exposure to a pesticide-
free medium.
The analysis of the results presented in Table 2 shows that the ab-
sorption kinetics (k1) for both pesticides alone or in the mixture are
virtually identical and that the apparent steady state for CB alone or in
the mixture is reached at 2 h while for AZM alone or in the mixture is
reached at 6 h. The relation of the estimated elimination kinetics (k2)
for the pesticides alone or in the mixture shows that the CB is elimi-
nated about 3.5 times faster than the AZM. The half-life (t1/2) estimated
from the k2 model was 0.47 h for CB and 1.6 h for AZM alone or in the
mixture.
The analysis of the relations BCFAZM/BCFCB shows that the bio-
concentration of AZM is 3.5 and 3.2 higher than that of the CB in in-
dividual exposure to the pesticides alone and in the mixture, respec-
tively.
3.3.2. Depuration studies
Fig. 5 shows the logarithmic representation of the first-order ex-
ponential decay curves for CB (Fig. 5A) and AZM concentrations
(Fig. 5B) and for the pesticides in the binary mixture (Fig. 5C) in the
soft tissues of P. corneus in terms of depuration time. Time zero of

Fig. 3. Soft tissue carboxylesterases (CES) inhibition by single pesticides and different binary combinations of carbaryl (CB) and azinphos-methyl (AZM). CES activity
was assayed using (A) p-nitrophenyl acetate (p-NPA) and (B) p-nitrophenyl butyrate (p-NPB) as substrates. Each bar represents the mean ± S.D. (n = 4). The
asterisk (*) denotes the values that are statistically different from controls (p < 0.05).

281
L.C. Cacciatore et al. Aquatic Toxicology 199 (2018) 276–284

Fig. 4. Bioconcentration of carbaryl (CB) (A), azinphos-methyl (AZM) (B) and in binary mixture of both pesticides (C), in P. corneus. Triangles represent individual
determinations (n = 4).

Table 2
Comparative toxicokinetics in P. corneus by exposure and subsequent elimination of carbaryl (CB) and azinphos-methyl (AZM) alone or in binary mixture.
Pesticide Apparent steady state (h) K1 (ml g−1 h−1) K2 (h−1) BCF = K1/K2 (ml g−1 h−1) K2 (experimental) (h−1) t1/2 (h)

CB 2 5.39 ± 0.20 1.50 ± 0.18 3.59 ± 0.17 3.80 ± 0.13 0.18

AZM 6 5.30 ± 0.12 0.42 ± 0.03 12.60 ± 0.10 0.80 ± 0.07 0.87
CB (mixture) 2 5.72 ± 0.43 1.52 ± 0.45 3.76 ± 0.38 4.06 ± 0.16 0.17
AZM (mixture) 6 5.21 ± 0.15 0.43 ± 0.03 12.10 ± 0.10 0.83 ± 0.03 0.84

The concentrations of pesticide exposure were: 1.4 mg L−1 CB, 1.8 mg L−1 AZM, and 1.4 mg L−1 CB + 1.8 mg L−1 AZM, in the mixture. BCF: Bioconcentration factor.
There are no significant differences between the means (k1, k2) for each single pesticide exposure and the mixture (p > 0.05).

depuration corresponds to natural logarithm of the concentration of the
parent compounds in soft tissues when transferred to a pesticide-free
medium after 12 h of exposure. The slopes of the lines represent the
k2(m) from which the t1/2 are calculated, as shown in Table 2.
The elimination kinetics determined experimentally were between
1.9 and 2.7 times higher than those estimated by the exponential
function y = a (1 − e−bx). As in the k2 estimated by the model, no
significant differences were observed between the k2(m) for each of the
pesticides alone or in the binary mixture.
4. Discussion
Exposure to increasing concentrations of CB for 48 h produced
concentration-dependent inhibitions of ChE activity. Inhibition of ChE
activity was similar in both soft tissues and hemolymph, with EC50
values of 1.4 and 1.2 mg L−1, respectively. Both values fall within the
range of CB concentrations found in some freshwater samples. For ex-
ample, Walters et al. (2003) reported 1.74 mg L−1 in a rain runoff
sample collected from a drain adjacent to a sprayed site. Furthermore,
transient peak concentrations were reported in aquatic habitats im-
mediately after agricultural applications that reached CB levels above
4.8 mg L−1 (Norris et al., 1983).
Kristoff et al. (2010) determined that ChE activity measured in soft
tissues of the freshwater gastropod Biomphalaria glabrata was inhibited
in vivo by exposure to CB. P. corneus and B. glabrata belong to the same
family of gastropods. However, present results show that P. corneus is
more sensitive to inhibition of ChE activity by CB than B. glabrata, with
an EC50 value about 2.5 times lower.
In P. corneus, the CES activities were between 2.3 and 25 times more
sensitive to CB than ChE activity, depending on the substrate and the
tissues tested. The greatest inhibition of CES activity was observed in
soft tissues using the substrate p-NPB. Besides, the degree of inhibition
of CES activity by CB, AZM or their mixtures was very high (75% to
88%). In agreement with our results, Kristoff et al. (2010) observed in
B. glabrata that CES activity on the substrate p-NPB showed greater
inhibition at lower concentrations of CB than ChE activity. These results
demonstrate the usefulness of monitoring CES activities to assess car-
baryl exposure in these species. Conversely, in the terrestrial snail
Xeropicta derbentina, CES activity was not inhibited by the carbamates
CB and carbofuran (Laguerre et al., 2009).
Exposure of P. corneus to binary mixtures of CB + AZM for 48 h
showed that all the values fell in the regression curve or within the 95%
prediction interval, like the controls with individual pesticides, con-
firming the hypothesis of a concentration addition model. Similarly,

Fig. 5. Elimination kinetics of carbaryl (CB) (A), azinphos-methyl (AZM) (B) and in binary mixture of both pesticides (C), in P. corneus. Triangles represent mean
values ± S.D. (n = 4).

282
L.C. Cacciatore et al.
Syberg et al. (2008) observed concentration addition in immobilization
experiments with Daphnia magna on exposure to binary mixtures of the
OP dimethoate and the carbamate pirimicarb.
In exposures to individual pesticides, absorption and elimination of
the pesticides was fast in P. corneus. The absorption rate of CB was equal
to that of AZM, while the apparent steady state was reached three times
faster by the carbamate. Bioconcentration of anticholinesterase agents
in aquatic organisms occurs mainly by means of passive diffusion
through the gills, epithelial tissues, or the gastrointestinal tract (Katagi,
2010). P. corneus is a non-operculate snail, so the body surface was in
contact with the solution of the OPs during testing. Invertebrates have a
high relative body surface area/volume, which facilitates the passive
diffusion of OPs. Ramírez Mora et al. (2000) were able to verify in the
operculate snail Pomacea patula with the same sublethal concentration
of CB used in P. corneus, a rapid absorption phase of CB from the water
to the hemolymph, and then a gradual distribution of the xenobiotic to
the tissues, with a rapid bioconcentration in the digestive gland.
Cacciatore et al. (2013b) observed that exposures to 5 mg L−1 of AZM
and up to the first two hours of absorption, B. glabrata showed high and
relatively constant concentrations of AZM in its hemolymph, which
were slightly lower than the exposure concentration, also suggesting a
rapid mechanism of absorption and of distribution of AZM from the
hemolymph to the tissues.
In P. corneus, when the snails were transferred to a pesticide-free
medium, elimination of CB and AZM was found to be rapid, with half-
lives of 10 and 52 min for the CB and AZM, respectively. Ramírez Mora
et al. (2000) observed that the transfer of P. patula to CB free water after
72 h of exposure was followed by rapid monophasic elimination in di-
gestive gland with a half-life of 1.0 h. Rapid kinetics of AZM elimination
with half-lives close to 45 min were observed in B. glabrata (Cacciatore
et al., 2013b).
Exposure of P. corneus to a mixture of 1.4 mg L−1 of CB and
1.8 mg L−1 of AZM did not change the toxicokinetic parameters of the
parent compounds in relation to the individual exposure to both pes-
ticides. This suggests that the unaltered toxicokinetic parameters of the
carbamate and the organophosphate could be at least partly responsible
for the concentration additions on the inhibition of the ChE activity
observed. In contrast, upon exposure of P. corneus to a mixture of
1.8 mg L−1 of AZM and 0.1 mg L−1 of CPF in the same experimental
conditions, the toxicokinetics of AZM was modified with respect to
individual exposure (Cacciatore et al., 2013c). This suggests that tox-
icokinetic interactions of both OPs may be partially responsible for the
more than additive effects on the inhibition of the ChE activity found in
certain combinations of pesticides (Cacciatore et al., 2013a).
5. Conclusions
The results of this study contribute to improve the knowledge on the
sensitivity of Type B-esterases to carbamate and organophosphate
pesticides in freshwater gastropods. CES enzymes proved to be more
sensitive than ChE inhibition by CB. Therefore, it is advisable to include
CES inhibition in the battery of biomarkers to be used to assess con-
tamination by anticholinesterase compounds. Besides, it was proved
that binary mixtures of CB and AZM did not change the toxicokinetic
parameters of the parent compounds in P. corneus, and that the effects
of these mixtures on ChE activity could be predicted using the model of
concentration addition.
Acknowledgments
This work was supported in part by a grant from Universidad de
Buenos Aires (20020110100070). The authors thank the Hospital
Nacional Prof. Dr. Alejandro Posadas the use of the chromatographic
system.
283
Aquatic Toxicology 199 (2018) 276–284
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