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Paper Claudio CB MAZ
Paper Claudio CB MAZ
Paper Claudio CB MAZ
Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aqtox
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 general lower than 1 μg L−1 (Schulz, 2004). However, it has been re-
ported that the concentrations found in the environment immediately
Carbaryl (1-naphthyl-N-methylcarbamate, CB) is one of the most after application can reach levels higher than 1700 μg L−1 (Norris et al.,
frequently used carbamate insecticides. CB is used to control a broad 1983; Walters et al., 2003).
spectrum of insects in over 120 different crops and as insecticide in In general, pesticides are found in aquatic environments as part of
gardens (Ware, 2000). Despite its low persistence in the environment complex mixtures. In this regard, Loewy et al. (2011) observed the
and its rapid hydrolysis in alkaline medium, CB can persist in aquatic coexistence of CB and azinphos-methyl (AZM) in drainage water from
ecosystems for over a year after application (Bacchetta et al., 2008). In flood-irrigated areas where apple crops were grown. These authors
general, pesticide concentrations in water are highly variable in time reported that AZM (O,O-dimethyl S-[4-oxo-1,2,3-benzotriazin-3(4H)-
and location, and monitoring data usually do not capture the peak yl) methyl]triazin-3-ylmethyl) was the most widely used organopho-
concentrations. Non-target species might be exposed to a wide range of sphate pesticide to control Carpocapsa pomonella in the Neuquén River
CB concentrations due to different exposure scenarios. Concentrations Valley, Argentina. This insect is the most critical pest affecting apple
of CB reported in surface waters due to agricultural practice are in 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. Aquatic Toxicology 199 (2018) 276–284
periods of application from October to March for the crop years 2008/ of organic substances metabolized by relevant enzymes may increase or
09 and 2009/10, the highest detection frequencies in surface and sub- decrease the toxicity of the individual compounds when mixed (Dorne
surface water was found for AZM and chlorpyrifos (CPF) (> 70%), et al., 2007a,b). For example, a significant increase in CB toxicity in
followed by CB (> 40%). The highest concentration levels were found red-legged partridges following the administration of the OP malathion
in shallow groundwater, with 22.5 μg L−1 for AZM and 45.7 μg L−1 for has been attributed to inhibition of CB metabolism (Johnston, 1995). In
CB (Loewy et al., 2011). AZM has also been used for pest control on a the same line, coincubation of CB and CPF with human liver micro-
number of food crops in many parts of the world, and concentrations up somes strongly inhibited CB metabolism (Tang et al., 2002). To our best
to 14.9 mg L−1 have been reported in runoff and river surface waters knowledge there are no studies addressing the modulation of CB toxi-
adjacent to agricultural fields (Castro et al., 2017; Shayeghi et al., city by AZM and thus far, there have been no studies about CYP-
2012). mediated metabolization of CB and AZM in P. corneus.
The efficacy of CB as an insecticide is due to the inhibition of Some molluscs have been proposed as suitable biological experi-
acetylcholinesterase (AChE), causing the accumulation of acetylcholine mental models to assess sublethal impacts of contaminants and mix-
in the synaptic cleft. AChE is the key target for both organophosphate tures of contaminants in aquatic ecosystems (Rittschof and McClellan-
(OP) and carbamate insecticides, and their main toxicological effects Green, 2005). The gastropod P. corneus is a hermaphroditic snail that
are very similar. However, while OPs are able to phosphorylate the usually inhabits small temporary ponds and streams. This species be-
serine residues of AChE irreversibly, the carbamylation of the same longs to the Planorbidae family, the largest family of aquatic pulmonate
residues is less stable (Sogorb and Vilanova, 2002; Van Dyk and gastropods, and has a global distribution (Jopp, 2006). P. corneus has
Pletschke, 2011). Since AZM has a thiophosphoryl bond (P = S) instead already been used as a model organism for toxicity studies (Pavlica
of a phosphoryl bond (P = O), it possesses minimal or no antic- et al., 2000; Otludil et al., 2004; Clarke et al., 2009; Benstead et al.,
holinesterase activity and requires metabolic activation by cytochrome 2011; Cacciatore et al., 2012, 2013a, 2015).
P450 (CYP) enzymes to its oxon analog to inhibit ChE activity (Tang The aims of this study were (1) to evaluate the inhibitory effects of
et al., 2006; Crane et al., 2012). CB on the B-esterases activities in soft tissues and in the hemolymph of
In addition to the CYP-mediated metabolism of OPs, several authors P. corneus; (2) to establish whether binary mixtures of the carbamate CB
have concluded that detoxification enzyme activities such as carbox- and the OP AZM depart or not from a concentration addition model on
ylesterases (CES) must also be considered when evaluating the effects of ChE activity inhibition in soft tissues; (3) to determine the kinetics of
combined exposure to OPs (Karanth et al., 2004; Richardson et al., absorption and elimination of CB and AZM pesticides alone or in binary
2001; Jansen et al., 2009; Cacciatore et al., 2012, 2013a). Besides, CES mixtures.
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 2. Materials and methods
(Sogorb and Vilanova, 2002; Sanchez-Hernandez et al., 2014).
Since anticholinesterase agents inhibit AChE, it is expected that the 2.1. Chemicals
effects of mixtures of these pesticides on this activity would follow a
concentration addition model, i.e., the concentration of each pesticide Acetylthiocholine iodide (AcSCh), p-nitrophenyl acetate (p-NPA), p-
may be replaced by an equivalent effective concentration of one of nitrophenyl butyrate (p-NPB), 5,5‘-dithio-2-bis-nitrobenzoate (DTNB),
them (Bosgra et al., 2009). However, there are several documented azinphos-methyl (AZM) PESTANAL® (CAS No. 86-50-0, 97.2% pure),
cases where mixtures of anticholinesterase pesticides do not follow this and carbaryl (CB) PESTANAL® (CAS No. 63-25-2, 99% pure) were
additive model. Previously, we have observed in homogenates of the purchased from Sigma–Aldrich. All other chemicals used were of ana-
freshwater snail Planorbarius corneus that several mixtures of AZM-oxon lytical reagent grade.
and CPF-oxon resulted in cumulative in vitro ChE inhibitions higher
than those expected on a concentration addition basis (Cacciatore et al., 2.2. Snails
2012). Similar results were found in vivo after exposure to binary
mixtures of AZM and CPF (Cacciatore et al., 2013a). Adult P. corneus snails were purchased from Discus Moron S.R.L.,
Despite the well-characterized acute toxicity of CB and AZM, few Buenos Aires, Argentina. Afterwards the snails were reared in our la-
studies exist on the bioconcentration of these pesticides in invertebrates boratory in aerated glass aquaria (17–20 L), at a temperature of
(Ramírez Mora et al., 2000; Klosterhaus et al., 2003). It is often as- 22 ± 2 °C, and under a 14:10 (L:D) h artificial photoperiod regime.
sumed that bioconcentration of anticholinesterase pesticides by aquatic Animals were fed lettuce leaves ad libitum. For all the experiments,
organisms means no risk for the ecosystem. In their natural environ- adult snails of similar size (10 ± 2 mm of shell length) were used.
ment, the exposure would almost invariably involve sublethal con- Water quality characteristics were as follows: total hard-
centrations of pesticides, due to their low partition coefficients and/or ness = 67 ± 3 mg CaCO3 L−1; alkalinity = 29 ± 2 mg CaCO3 L−1; pH
high biotransformation rates. These factors could prevent these che- 7.0 ± 0.2 and conductivity = 250 ± 17 μS cm−1.
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- 2.3. Inhibition studies
danger the survival of the species (Sancho et al., 1998; Kristoff et al.,
2011). The general experimental design consisted of 1 L glass vessels con-
In contrast to most non-polar organics, many carbamate and OP taining 600 mL of each treatment condition, i.e. different pesticide
insecticides can be rapidly metabolized in fish. Therefore, correlations concentration, solvent control and solvent-free control. Animals were
between Bioconcentration Factor (BCF) and octanol-water partition not fed during the treatments. All bioassays were performed at a tem-
coefficient (Kow) are not applicable to these compounds (Keizer et al., perature of 22 ± 2 °C, and under a photoperiod of 14:10 h light/dark
1991) and little is known whether metabolism also affects BCFs of without aeration. No mortality was observed either in control animals
anticholinesterase agents in invertebrates (Legierse et al., 1998). Even or in any of the treatments. Carbon filtered dechlorinated tap water was
less is known about the toxicokinetics of anticholinesterase insecticides used as the test medium.
in binary mixtures. Several toxicokinetic studies have highlighted how In all experiments, the concentration of acetone was kept at 0.05%
substances can inhibit, induce or compete with the activity of the CYP and solvent (acetone 0.05%) and solvent-free (dechlorinated tap water
system. Altering the biotransformation rates resulting from co-exposure only) controls were included.
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L.C. Cacciatore et al. Aquatic Toxicology 199 (2018) 276–284
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 p r2 EC50 (mg L−1) EC50 (μM)
Soft tissue
AcSCh 273 ± 10 57 ± 34 0.75 ± 0.33 0.87 ± 0.23 0.9968 1.4 ± 0.3 4.8 ± 1.0
p-NPA 123 ± 4 11 ± 27 0.17 ± 0.15 0.45 ± 0.21 0.9932 0.18 ± 0.16 0.99 ± 0.88
p-NPB 166 ± 2 4±2 0.03 ± 0.01 0.52 ± 0.07 0.9990 0.04 ± 0.01 0.19 ± 0.01
Hemolymph
AcSCh 508 ± 9 136 ± 17 0.69 ± 0.07 1.32 ± 0.15 0.9957 1.2 ± 0.1 4.2 ± 0.4
p-NPA 223 ± 4 27 ± 7 0.23 ± 0.03 0.90 ± 0.10 0.9944 0.32 ± 0.04 1.8 ± 0.2
p-NPB 120 ± 2 2±5 0.13 ± 0.02 0.57 ± 0.07 0.9998 0.13 ± 0.02 0.62 ± 0.10
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).
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L.C. Cacciatore et al. Aquatic Toxicology 199 (2018) 276–284
continuously at 412 nm and specific activity was expressed as nmol Reversed phase chromatography was performed on a Shimadzu Class-
min−1 mg protein−1. The enzymatic activity was corrected for sponta- VP model with isocratic pump (LC-10AT VP), a variable wavelength
neous hydrolysis of the substrate and non-specific reduction of the programmable UV–vis detector (SPD-10A VP) and a Supelcosil LC-18
chromogen by tissues extracts. (250 mm × 4.6 mm, 5 μm particle size) column and Supelcosil LC-18
CES activity was determined using two different substrates: p-ni- (20 mm × 4.6 mm, 5 μm particle size) Guard Column. In the case of CB,
trophenyl acetate (p-NPA) and p-nitrophenyl butyrate (p-NPB). Each the operating conditions were: mobile phase: acetonitrile/water (45:55,
supernatant or hemolymph dilution was assayed for CES activity in v/v), flow rate: 1 mL min−1, temperature: 40 °C, run time: 10 min, and
duplicate. Hydrolysis of p-NPA and p-NPB by CES was determined ac- detection wavelength: 277 nm. Linearity was observed in the con-
cording to Kristoff et al. (2010). Reactions were performed in 2.5 mL centration range of 0.10–10.6 mg L−1 with regression equation
100 mM phosphate buffer pH 8.0 containing 5% acetone and 1 mM p- y = 41,117 x (mg L−1) + 5523. In the case of AZM, the operating
NPA or p-NPB. Activity was continuously recorded at 400 nm using a conditions were: mobile phase: acetonitrile/water (45:55, v/v), flow
Shimadzu UV-160A double-beam UV–visible spectrophotometer (Shi- rate: 1 mL min−1, temperature: 40 °C, run time: 17 min, and detection
madzu, Kyoto, Japan). Specific activity was calculated using the molar wavelength: 220 nm. Linearity was observed in the concentration range
extinction coefficient for p-nitrophenol (18.6 mM−1 cm−1). of 0.17-54.6 mg L−1 with regression equation y = 118,736.73x
Protein content was determined according to the method of Lowry (mg L−1) + 974.10.
et al. (1951), using bovine serum albumin as standard.
2.5.1. Water samples
2.4. Experiment III. Toxicokinetic parameters The concentration of the exposure solutions was confirmed by
analysis of 1 mL water samples collected in duplicate. In the 48 h ex-
Uptake and elimination experiments were conducted under static posure experiments, the concentrations measured were always within
conditions in 1 L glass vessels containing 600 mL of test solution. the range 95–102% of the nominal values. In the uptake experiments,
Carbon-filtered dechlorinated tap water was used as the test medium. the 0-h–12-h average measured pesticide concentration was in all
The following physico-chemical parameters were recorded: total hard- cases > 93% of the initial concentration. Aqueous medium was also
ness = 67 ± 3 mg CaCO3 L−1; alkalinity = 29 ± 2 mg CaCO3 L−1; pH sampled at the end of the depuration phase and the presence of mea-
7.0 ± 0.2 and conductivity = 250 ± 17 μS cm−1. surable concentrations of the parent compounds of the pesticides was
For the uptake phase, snails were divided into 4 groups (water analyzed by HPLC. Results showed that parent compounds concentra-
control, CB, AZM, mixture). CB and AZM concentrations were set at tions were below the limit of detection due to pesticide metabolization
1.4 mg L−1 and 1.8 mg L−1, respectively; the binary mixture was com- and to the fact that the ratio of water to organisms was large. Therefore,
posed of 1.4 mg L−1 CB + 1.8 mg L−1 AZM. Aqueous solutions con- it was assumed that reuptake of the parent compounds excreted by the
taining the pesticide CB or AZM or the mixture CB + AZM were pre- organisms during the depuration phase was negligible because con-
pared by dissolving the pesticides in acetone, and diluted with an centration levels were very low compared to the exposure phase
appropriate amount of dechlorinated tap water. Final acetone con- (< 10%).
centration in exposure vessels was 0.05%. The organisms used as ne-
gative control were placed in the water vessels.
2.5.2. Tissue samples
For each treatment condition, 4 glass vessels with 7 snails per vessel
Tissues of each snail were homogenized with pure acetonitrile, final
were used. Seven gastropods were placed in the glass vessels at time
volume of 1.0 mL. The homogenate was centrifuged in Eppendorf tubes
zero (0 h). One snail exposed to each one of the pesticides or the mix-
at 4 °C (14,000×g, 5 min) and immediately placed on ice. The super-
ture was removed with a histological clamp at 0.5; 1; 2; 4; 6; 9; and
natants obtained were kept for later use. Twenty microlitres of the re-
12 h. Four replicates were performed on 4 consecutive days. Therefore,
sulting solution were injected directly into the chromatographic system.
a total of 112 specimens were used for this experiment and 4 in-
For the calculation of pesticide recovery, tissues of each snail were
dependent samples were analyzed per treatment group (n = 4) at each
spiked with 2, 3 and 5 μg of CB or AZM or CB +AZM, and after equi-
time point. Sampling involved a quick rinse in distilled water to ensure
libration for 1 h at room temperature they were extracted and analysed
total cleanup of the outer surface of pesticides. The animals were placed
according to the procedure described above. Average recoveries of CB
on ice for 4–6 min. The shells were carefully removed and the soft tis-
and AZM were 98% ± 5% (n = 16) and 99% ± 6% (n = 16), re-
sues isolated at 4 °C. All soft parts of each snail were placed on filter
spectively.
paper to drain extra fluids and weighed, average weight being
132 ± 18 mg. Exposure concentrations in soft tissues were confirmed
by chemical analysis. 2.6. First-order uptake and elimination kinetics
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 Bioconcentration was analyzed using a one-compartment model
12 h. The depuration phase was initiated after 12 h of exposure by re- (Rosen and Lotufo, 2007), which describes the concentration of a
moving the animals from the uptake aquaria, quickly rinsing them in chemical in an aquatic organism as the result of first-order uptake and
distilled water, and then placing them into clean glass aquaria con- elimination kinetics:
taining 600 mL of carbon filtered dechlorinated tap water. Invertebrates dCorg/dt = k1Cw − k2Corg (2)
were then sampled at the following intervals: 0; 0.25; 0.5; 1.0; 2.0; and
−1 −1
3.0 h, and prepared for solvent extraction and chemical analysis in the In this equation, k1 is the uptake rate constant (mL g h ) and k2
same way as in the uptake phase. Four replicates (n = 4 for each con- is the elimination rate constant (h−1); Cw is the exposure concentration
dition) were performed on consecutive days. in water (μg mL−1) and Corg is the concentration of the compound in
The studies were conducted without aeration and without food the organism (μg g−1 wet weight).
supply. All the tests were performed at a temperature of 22 ± 2 °C, and Since exposure concentration was constant over time (experimen-
under a photoperiod of 14:10 h light/dark. No mortality was observed tally confirmed) and Corg was not detectable at t = 0, Eq. (2) was in-
either in control animals or in any of the treatments. tegrated as:
k1
2.5. Chemical analyses Corg = C w (1 − e–k2t)
k2 (3)
Pesticide concentration was tested using HPLC with UV detector. The constants k1 and k2 were then estimated simultaneously from
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L.C. Cacciatore et al. Aquatic Toxicology 199 (2018) 276–284
Eq. (3) using nonlinear iterative regression analysis (OriginPro 7.5). with p-NPB than ChE, in the hemolymph and soft tissues, respectively
The experimentally measured elimination rate (k2(m)) was de- (Table 1).
termined by fitting the data from the depuration experiments to the
first-order decay in Eq. (4) (Legierse et al., 1998; Rosen and Lotufo, 3.2. Experiment II. B-esterase activities in snails simultaneously exposed to
2007): mixtures of CB and AZM
Corg = Cºorg e–k2(m) t (4)
The effects of two combinations of CB and AZM on ChE and CES
where C°org is the concentration of the compound in the organism at activities were studied. In all cases, the concentrations in the mixtures
t = 0 of depuration. were based on the EC50 values for inhibition of ChE activity derived
The biological half-life of the pesticides alone or in binary mixture from the assays conducted with single pesticides in soft tissues (Fig. 1A
(t1/2) was determined following Eq. (5) (Legierse et al., 1998; Rosen and see Section 2.3.2).
and Lotufo, 2007):
3.2.1. ChE activity
t½ = 0.693/k2 (5)
To study the effects of pesticide mixtures on ChE activity in soft
Bioconcentration factors (BCFs) were calculated kinetically using tissues, the concentration of each pesticide was normalized to the re-
Eq. (6) (Legierse et al., 1998; Rosen and Lotufo, 2007): spective EC50 (normalized EC50) and the results were combined and
fitted with a single nonlinear regression (Fig. 2A). This curve (y = 100/
k1
BCF = (1 + x0.87), r2 = 0.94288) with its 95% prediction interval was used as
k2 (6)
a basis for determining whether specific combinations of pesticides
departed from a concentration addition model in soft tissues. For ex-
2.7. Statistical analysis ample, in a mixture containing two pesticides each at 0.5 EC50, con-
centration-addition occurs if the cumulative ChE inhibition is equiva-
Results were expressed as mean values ± S.D. Data were analyzed lent to 1 EC50. This result would fall on the curve. Similarly, in a
by one-way ANOVA followed by Tukey HSD post-test by using mixture containing two pesticides each at 0.75 EC50, concentration-
VassarStats (http://vassarstats.net/). The level of significance used was addition occurs if the cumulative ChE inhibition is equivalent to 1.5
0.05. Prior to ANOVA, data were tested for normality and homogeneity EC50.
of variance using the Shapiro–Wilk and Levene’s tests, respectively, by The results obtained showed that ChE activity in soft tissues of snails
using OriginPro 7.5. 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
3. Results 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%
3.1. Experiment I: B-esterase activities in snails exposed to CB prediction interval. These results indicate that concentration addition
on ChE activity inhibition occurred when mixtures 1 and 2 were used.
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 3.2.2. CES activities
and CES activities, in soft tissues and in the hemolymph of P. corneus Fig. 3 represents the CES activity after 48 h exposure of snails either
(Fig. 1). to the pesticides alone, or to the binary combinations of CB and AZM
Table 1 shows the values corresponding to EC50 values calculated used for determining ChE activity. CES activities were evaluated using
for CB and the parameters of the nonlinear regression equation. In the p-NPA (Fig. 3A) and p-NPB (Fig. 3B) as substrates. Interestingly, in all
case of ChE activity, the values corresponding to the EC50 were very the experimental conditions, a strong inhibition of the CES activity was
similar in both matrices: 1.4 ± 0.3 mg L−1 (4.8 ± 1.0 μM) and found, approximately 75% to 88%.
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- 3.3. Experiment III. Toxicokinetics of CB alone or in binary mixture with
fect of CB than ChE activity both in soft tissues and hemolymph. Thus, AZM
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 3.3.1. Bioconcentration studies
sensitive with the substrate p-NPA and 6.8 and 25 times more sensitive Fig. 4 shows the uptake kinetics and the apparent steady state
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).
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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 free medium.
AZM (Fig. 4B) and a mixture of 1.4 mg L−1 CB + 1.8 mg L−1 AZM The analysis of the results presented in Table 2 shows that the ab-
(Fig. 4C) of P. corneus. The concentrations used for single pesticides sorption kinetics (k1) for both pesticides alone or in the mixture are
represent the EC50 at 48 h of ChE activity. The size of the snails used virtually identical and that the apparent steady state for CB alone or in
was similar in all trials, corresponding to adult specimens with an the mixture is reached at 2 h while for AZM alone or in the mixture is
average soft tissue wet weight of 132 ± 18 mg. Thus, absorption rates reached at 6 h. The relation of the estimated elimination kinetics (k2)
were not biased due to the size of the organism. for the pesticides alone or in the mixture shows that the CB is elimi-
Experimental data were fitted by a nonlinear regression analysis nated about 3.5 times faster than the AZM. The half-life (t1/2) estimated
iterative to equation Corg = k1/k2 Cw (1 − e−k2t), and the values of k1 from the k2 model was 0.47 h for CB and 1.6 h for AZM alone or in the
and k2 were calculated. The exponential function used, y = a mixture.
(1 − e−bx) makes it possible to calculate the best fit, by successive The analysis of the relations BCFAZM/BCFCB shows that the bio-
iterations, for parameters a and b, where a = k1/k2 Cw, b = k2, and concentration of AZM is 3.5 and 3.2 higher than that of the CB in in-
Cw = concentration of the pesticide in the water, which was held dividual exposure to the pesticides alone and in the mixture, respec-
constant during exposure and was determined experimentally (see tively.
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 3.3.2. Depuration studies
k2 value theoretical to distinguish it from and compare it with the ex- Fig. 5 shows the logarithmic representation of the first-order ex-
perimental k2(m) value obtained from the elimination phase of the ponential decay curves for CB (Fig. 5A) and AZM concentrations
parent compound when the snails go from 12 h exposure to a pesticide- (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).
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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)
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 Kristoff et al. (2010) determined that ChE activity measured in soft
parent compounds in soft tissues when transferred to a pesticide-free tissues of the freshwater gastropod Biomphalaria glabrata was inhibited
medium after 12 h of exposure. The slopes of the lines represent the in vivo by exposure to CB. P. corneus and B. glabrata belong to the same
k2(m) from which the t1/2 are calculated, as shown in Table 2. family of gastropods. However, present results show that P. corneus is
The elimination kinetics determined experimentally were between more sensitive to inhibition of ChE activity by CB than B. glabrata, with
1.9 and 2.7 times higher than those estimated by the exponential an EC50 value about 2.5 times lower.
function y = a (1 − e−bx). As in the k2 estimated by the model, no In P. corneus, the CES activities were between 2.3 and 25 times more
significant differences were observed between the k2(m) for each of the sensitive to CB than ChE activity, depending on the substrate and the
pesticides alone or in the binary mixture. tissues tested. The greatest inhibition of CES activity was observed in
soft tissues using the substrate p-NPB. Besides, the degree of inhibition
4. Discussion 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
Exposure to increasing concentrations of CB for 48 h produced B. glabrata that CES activity on the substrate p-NPB showed greater
concentration-dependent inhibitions of ChE activity. Inhibition of ChE inhibition at lower concentrations of CB than ChE activity. These results
activity was similar in both soft tissues and hemolymph, with EC50 demonstrate the usefulness of monitoring CES activities to assess car-
values of 1.4 and 1.2 mg L−1, respectively. Both values fall within the baryl exposure in these species. Conversely, in the terrestrial snail
range of CB concentrations found in some freshwater samples. For ex- Xeropicta derbentina, CES activity was not inhibited by the carbamates
ample, Walters et al. (2003) reported 1.74 mg L−1 in a rain runoff CB and carbofuran (Laguerre et al., 2009).
sample collected from a drain adjacent to a sprayed site. Furthermore, Exposure of P. corneus to binary mixtures of CB + AZM for 48 h
transient peak concentrations were reported in aquatic habitats im- showed that all the values fell in the regression curve or within the 95%
mediately after agricultural applications that reached CB levels above prediction interval, like the controls with individual pesticides, con-
4.8 mg L−1 (Norris et al., 1983). 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).
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