Altered quantities and in vivo activities of cholinesterase from Daphnia magna in sub-lethal exposure to organophosphorus insecticides

Ecotoxicology and Environmental Safety 80 (2012) 118–125

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Altered quantities and in vivo activities of cholinesterase from Daphnia magna in sub-lethal exposure to organophosphorus insecticides Hongcui Liu, Bingqiang Yuan, Shaonan Li n Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 January 2012 Received in revised form 18 February 2012 Accepted 20 February 2012 Available online 20 March 2012

For investigating relationship between activity of cholinesterase (ChE) and ambient concentration of anticholinesterases, Daphnia magna had been exposed for 21 day to sub-lethal concentrations, i.e. 1/6 EC50, 1/36 EC50, and 1/216 EC50, of either triazophos or chlorpyrifos. Samples were taken at different points of time for measuring total activity and immunoreactive content of ChE and actual concentrations of the anticholinesterases. A type of antigen formerly developed by immunizing mice with purified ChE was utilized in this study to establish an indirect non-competitive ELISA for measuring immunoreactive content of ChE in Daphnia. Studies showed that for apparent activity, i.e. activity that was scaled with total protein, the insecticides caused 5.2–6.9 percent inhibition and 17.0–17.7 percent inductions during the 21 d exposure, whereas for inherent activity, i.e. activity that was scaled with immunoreactive protein, no induction was detected during the exposure. Accompanied by up to 65.9 percent and 68.0 percent promotion in terms of the immunoreactive content, up to 42.8 percent and 44.6 percent inhibition in terms of the inherent activity was indicated, respectively, for triazophos and chlopyrifos. Judged by measured concentrations, the inherent activity recovered faster than the rate of dissipation of the anticholinesterases. Result of the study suggested that the inherent activity was more sensitive than the apparent one in predicting sub-lethal and/or long-term stress of anticholinesterases. It also suggested that apart from promotion in terms of content of the ChE, the Daphnia developed capacities to block bio-concentration of anticholinesterases, and these capacities would make it liable to underestimate ambient concentration of anticholinesterases along with the time of exposure. & 2012 Elsevier Inc. All rights reserved.

Keywords: Daphnia magna Triazophos Chlorpyrifos Insecticide Inherent activity Immunoreactive ChE ELISA

1. Introduction Anticholinesterases such as organophosphorus (OPs) and carbamate (CBs) are among the most commonly used pesticides for insect pests management. Despite the high efficiencies, they tend to be toxic to non-target organisms such as fish and many species of aquatic invertebrates. It is known that the principal mechanism by which OPs and CBs elicit their toxicity comes down to the inhibition of cholinesterase (ChE) (Carvalho et al., 2003). In this sense, a number of studies had been performed to urge the enzyme as a biomarker for prediction of anticholinesterases (Sanchez-Hernandez et al., 1998; Stien et al., 1998; Cajaraville et al., 2000; Den Besten et al., 2001; Abdel-Halim et al., 2006). Anticholinesterases may affect ChE in multi aspects in vivo. It is common to see promotion in terms of activity of the enzyme in exposures (Frawley et al., 1952; Frawley and Fuyat, 1957; Genina, 1974; Hackenberger et al., 2008), a phenomenon that can be regard as a type of hormesis featured by conversion from

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low-dose stimulation to high-dose inhibition (Calabrese, 2002; Chapman, 2002). This promotion is probably result from acceleration in terms of synthesis of the enzyme in order to rectify functional disable in condition that part of the enzyme was inhibited. The point is that the anticholinesterase- caused promotion oftentimes disturbs the detection of ‘‘actual’’ inhibition of the enzyme so as to bring about difficulty for determining exposure of anticholinesterases in field (Khattab et al., 1994; Khattab and Ali, 2007). It is necessary, therefore, to develop method for measuring relative content of ChE so as to scale the ‘‘actual’’ activity of the enzyme in exposures. Enzyme-linked immunosorbent assay (ELISA), which combined efficiency and specificity, is capable of achieving the task. Daphnia magna is a species of zooplankton that belongs to Phylum of Arthropoda, Order of Crustacea, and Family of Daphniidae. It consumes algae and other small-sized phytoplankton and in turn serves as prey for freshwater fish and macro-sized aquatic invertebrates. Due to its world-wide distribution and its adaptability to laboratory culture, Daphnia has long been employed as representative species for testing of chemicals (OECD (Organization for Economic Co-operation and Development), 2004, 2008; USEPA (U.S. Environmental Protection Agency), 1996a, 1996b). Some papers had

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been published to reveal relationship between in vivo activity of ChE and ambient concentrations of anticholinesterases (Barata et al., 2001; Printes and Callaghan, 2003; Carvalho et al., 2003; Barata et al., 2004; Dama´sio et al., 2007; Printes et al., 2008). Results of the studies confirmed that, to behave the same as fish and many other aquatic organisms, Daphnia had its enzyme obviously inhibited at concentrations adjacent to the lethal ones, conflict results were obtained, however, in case that the Daphnia were exposed to concentrations far below the lethal ones. Triazophos and chlorpyrifos are two kinds of OPs commonly used nowadays in China and southeast of Asia. Considering the physico-chemical properties and application rates, there is large possibility for them to enter water-sediment systems and impose acute and/or chronic hazard to non-target aquatic organisms. A former study indicated a maximal of 76.5 percent and 60.5 percent promotion in terms activity of the ChE, as the Daphnia were exposed, in semi-static condition, to either triazophos or chlorpyrifos for 21 day (Li and Tan, 2010). This suggested accelerated synthesis and consequent promotion in quantity of the enzyme. In a study conducted with Japanese quail Coturnix coturnix, a maximal of 40 percent promotion in quantity of immunoreactive ChE was detected in serum in case the bird was orally administered with monocrotophos of 20 mg kg  1 (Khattab et al., 1994). Another study conducted with red-legged partridges Alectoris rufa cross revealed a maximal of 26.5 percent rise in quantity of immunoreactive ChE in serum in case the bird was orally administered with malathion of 167 mg kg  1 (Khattab and Ali, 2007). Up to of 50 percent increase in quantity of the enzyme was detected in brain of Psudorasbora parva in case the fish were exposed to sub-lethal concentrations of triazophos (Li et al., 2005). It is logical, therefore, to infer that anticholinesterases such as triazophos and chlorpyrifos could result in promotion in terms of quantity of the ChE in Daphnia. A type of antigen that was formerly developed by purified ChE from Daphnia was utilized in this study for measuring content of ChE in Daphnia being exposed for 21 d to various concentrations of either of triazophos or chlorpyrifos. The aim of this study was: (1) to clarify extent and pattern of promotion in terms of quantity of ChE in Dphnia along with time and concentrations, (2) to explore relationship between in vivo activity of ChE and ambient concentrations of anticholinesterases. Result of the study would help to learn advantages and disadvantages of in vivo ChE in detecting sub-lethal and/or long-term exposure of anticholinesterases and would finally benefit in situ application of the biomarker.

2. Material and methods 2.1. Reagents Triazophos (with purity of 96.6 percent) and chlorpyrifos (with purity of 99.5 percent) were obtained from the National Standards Company (Beijing, China). Horseradish peroxidase (HRP) labeled goat anti-mouse immunoglobulin G (IgG) with the enzyme of immunoassay grade purity, Coomassie Brilliant Blue G-250, propionylthiocholine iodide, and bovine serum albumin (BSA, with MW of 67,000) were obtained from Sigma-Aldrichs (Steinheim, Germany). Tramethylbenzidine (TMB), which consists of solution A and B, were purchased from Yingchuang Company (Huzhou, China). Amplexs Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217, which contains Amplex Red reagent, dimethylsulfoxide, HRP, hydrogen peroxide, and choline oxidase from Alcaligenes sp., etc.) were bought from Molecular Probes, Inc. (Eugene, Oregen,USA). Defatted milk was purchased from Shanghai Chemical Reagents Company (Shanghai, China). All of the other chemicals were of analytical grade unless stated specially.

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was cultured in semistatic M4 medium (Elendt and Bias, 1990) and fed with unicellular algae Scemedesmus subspicatus from synchronized culture at a density of 2.0–3.0  l05 cells mL  1 Dapnhia  1 day  1. The water temperature, the pH, the light intension, and the light cycle were set as 227 1 1C, 7.8 70.2, 1500–2500 lx, and 16 h light to 8 h dark, respectively.

2.3. Acute immobilization test It is essential to perform acute immobilization test to set concentrations for 21d exposure. The test was carried out in accordance with the guideline of OECD (2004) and International Standard Oranization (ISO) (1996). The experiment started with neonates of less than 24 h. Individuals were exposed to either triazophos with nominal concentrations from 5.0 to 25.0 mg L  1 (i.e. 5.0, 8.0, 12.0, 20.0, 25.0 mg L  1) or chlorpyrifos from 2.5 to 16.0 mg L  1 (i.e. 2.5, 4.0, 6.0, 10.0, 16.0 mg L  1). The dilutions were prepared by spiking acetone solution of various concentrations into the M4 medium of 40 mL. The spiking rate was 45 mL L  1 for triazophos and it was 50 mL L  1 for chlorpyrifos. The treatment group (including the solvent control) had five replicates and each of the replicate consisted of five daphnids. The temperature, the pH, the light intension, and the light cycle were set as 22 71 1C, 7.87 0.2, 1500–2500 lx, and 16 h light to 8 h dark, respectively. The number and state of the individuals were recorded at 48 h after start of the exposures.

2.4. Chronic exposure The dilutions were obtained by spiking acetone solutions of various concentrations into the M4 medium of 400 mL. The spiking rate was 13 mL L.  1 Concentration of the dilutions were set to be 1/6, 1/36, and 1/216 of 48 h EC50 of two the anticholinesterases, respectively, which supposed to influence activity of the enzyme but caused no mortality to daphnids. Ten replicates were set for each concentration including the solvent control. Each replicate consisted of about 100 Daphnia of 14 d old. The Daphnia were kept in 22 71 1Cunder a regime of 16 h light to 8 h dark. The feed (i.e. Scemedesmus subspicatus from synchronizing culture) were provided once a day during the exposure. The Daphnia were taken randomly from each of the ten replications at 2nd, 6th, 10th, 14th, 18th, and 21st d after dosing, respectively. Those that were not able to be analyzed timely were stored at  80 1C. Maximal duration of the storage was 23 d.

2.5. Enzyme activity measurement 2.5.1. Crude enzyme preparation The collected Daphnia were homogenized in 500 mL ice-cold Tris–HCl buffer (pH 7.5) that contained 0.25 percent (V/V) Triton X-100, 1 mM EDTA, and 50 mM Tris. The homogenates were centrifuged for 30 min at 4 1C in acceleration of 10,000 g. The supernatants were collected as source of the crude enzyme.

2.5.2. Protein content determination Protein concentration of crude enzyme was quantified in accordance with the method of Bradford’s (1976) using bovine serum albumin (BSA) as the reference. Light absorbencies were measured with an ultraviolet-visible spectrophotometer (UV–vis-7504PC, a product of Xinmao Co., Ltd., Shanghai, China).

2.5.3. Measurement of total activity The total activity of ChE was measured by a relatively sensitive method previously described by Zhou et al. (2000) using propionylthioncholine iodide as substrate. That was to pipe ChE-containing sample (100 mL) into a well of the polystyrene black fluorescence microplate (High Binding Plates, Catalog no. 3925, Corning, Cambridge, MA, USA). The reaction was started by adding 100 mL working solution of Amplex Red reagent/ HRP/ choline oxidase/ propionylthioncholine iodide that contained 2 U mL  1 HRP, 0.2 U mL  1choline oxidase, and 100 mM propionylthioncholine iodide. The microplate was incubated for 30 min in dark at room temperature (about 25 1C). The Reaction Buffer employed to dilute the samples and the working solution of Amplex Red reagent/HRP/choline oxidase/ propionylthioncholine iodide was 50 mM Tris–HCl with pH of 8.0. The fluorescence was measured by a fluorescence microplate reader (Tecan Genios Plus, Tecan Trading Co., Ltd. Shanghai, China) at excitation of 530 nm and emission of 590 nm. Positive control was prepared by diluting the eel acetylcholinesterase of 100 U mL  1 with the Reaction Buffer of 1X to produce a working solution of 0.2 U mL  1. Negative control was prepared using the Reaction Buffer of 1X instead of the enzyme. Total activity of the enzyme was calculated by follow formula:

2.2. Daphnia magna Total activity ¼ ½ðF x F 0 Þ  0:2=ðF 1 F 0 Þ Daphnia magna employed in this study was a pure breed of 62 D.M. obtained from Chinese Center for Disease Control and Prevention (Beijing, China). The breed

where, F1 was fluorescence of the positive control, F0 was fluorescence of the negative control, and Fx was fluorescence of a sample.

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Specific activity of the ChE was expressed as apparent and inherent one, where the former was the ratio between the total activity and the mass of total protein in supernatant and the latter was the ratio between the total activity and the content of immunoreactive protein in supernatant.

2.7. Determination of actual concentration of triazophos and chlorpyrifos in dilutions

2.6. Indirect non-competitive ELISA

2.7.1. Sample collection and pretreatment Sub-sample of 10 mL was taken from each of the ten replications at 0, 2nd, 6th, 10th, 14th, 18th, and 21st d after dosing, respectively. The replications that belonged to the same point of time were pooled. Those not able to be timely analyzed were stored at  20 1C. Maximal duration of the storage was 25 d. The pooled sample of 100 mL was spiked with sodium chloride of 10 g. The mixture was extracted twice with petroleum ether/ethylacetate (5/1, v/v) of 70 mL. Remained moisture in the organic phase was eliminated by anhydrous sodium sulfate. The solvent was then evaporated in reduced pressure at 45 1C. The volume was fixed to 1 mL with acetone. The extract was filtrated with membrane of 0.2 mm before it was appealed to analysis. Each of the above procedure was performed in triplicate.

2.6.1. Antibody The Antibody used in this study was anti-Daphnia ChE immunoglobulin G (IgG) obtained from serum of immunized BALB/c mice. The antigen employed was of electrophoretic purity. It was extracted from Daphnia magna by a 4-step procedure, i.e. ultrasonic cell disruption, Triton X-100 extraction, ammonium sulfate precipitation, and DEAE-SepharoseTM-Fast-Flow chromatography. The antibody had been proved to have low rate of cross-reaction with ChEs from invertebrates such as Chironomus kiinensis, Milleri kiser, Brachydanio rerio, Bombyx mori, Apis mellifera L, Eisenia foetida, and amphibians Xenopus laevis (Liu et al., In press).

2.6.2. Standard curve for determination of immunoreactive content of ChE The standard curve for indirect non-competitive ELISA was established according to method described in detail previously (Liu et al., In press). In brief, antigens were quantified by its capacity to bind to the antibodies absorbed on the solid phase using horseradish peroxidase complex as an indicator. The relationship between the signal and the concentration of the ChE in standard dilutions follows the equation of Y¼ a(1  e  bx) with a¼ 1.628, b¼ 0.1401, correlation coefficient of 0.9962, and standard error of 0.0547 (Fig. 1).

2.6.3. Relative content of immunoreactive ChE in 21 d exposure The content of immunoreactive ChE in Daphnia magna was measured by indirect non-competitive ELISA. The assay was performed in triplicate. To start the protocol, each well of the ELISA microtiter plate (a product of COSTAR Inc, New York, U.S.A.) was filled with enzyme solution of 100 uL. The solution was prepared in advance by diluting the crude enzyme with 0.1 M sodium carbonate/bicarbonate buffer of pH 9.6 to obtain concentration of 2.5 mg protein mL  1. Three wells were filled with the 0.1 M sodium carbonate/bicarbonate buffer of 100 mL as blank control. The plate was covered and then incubated for 2 h at 37 1C to allow the antigen bind to it. The liquid was then removed and the wells were washed three times with PBST using a DEM plate washer (a product of Beijing Tuopu Analytical Instruments, China). The buffer contained 8.03 mM Na2HPO4, 1.97 mM KH2PO4, 137 mM NaCl, 2.68 mM KCl, and 0.05 percent Tween-20, and with pH of 7.4. Next to the washes, the plate was blocked with 2 percent defatted milk in PBS for 30 min. After another washing step, antibody serum of 100 mL (pre-diluted to a ratio of 1:8000) was added to each well. The plate was then incubated for 1 h at 37 1C. After the incubation, the wells were washed and then filled with 100 mL peroxidase-labeled goat anti-mouse IgG pre-diluted to a ratio of 1:5000. The plate was incubated for another 1 h at 37 1C. After that, the wells were washed again and TMB solution of 100 mL was then added to each well to start the enzyme reaction. The reaction was stopped after 15 min by adding 2 M sulfuric acid of 100 uL. The absorbance was read at 450 nm using a plate reader (Model 680, a product from Bio-Rad Inc, Hercules, CA).

The actual concentration of triazophos and chlorpyrifos in dilutions was determined according to a method described in detail previously (Wang, 2010).

2.7.2. Analysis The concentration of pesticides in dilutions was measured by gas chromatograph of GC-2010 equipped with a flame photometric detector (FPD) (Shimadzu, Japan) and a RtxR-5 cross-linked fused silica capillary column (30 m  0.32 mm ID  0.25 mm). Samples were injected in splitless mode at 80 1C. The column was maintained at 80 1C for 1 min in initial. It was then heat to 200 1C at speed of 50 1C min  1 and was kept at the temperature for 1 min. After that, the temperature was programmed from 200 1C to 250 1C at speed of 10 1C min  1 and maintained at 250 1C for 5 min. The detector and injection port were maintained at 280 1C and 230 1C, respectively. Ultrahigh-purity nitrogen (with purity of 99.999%) was used as carrier with the column head pressure of 180.0 kPa. The constant flow of hydrogen and air was 6120 mL min  1 and 100 mL min  1, respectively. The injection volume was 1 mL. 2.7.3. Calibration curves The calibration curve was obtained from a gas chromatographic run of the standard solutions of triazophos and chlorpyrifos (both with concentration of 0.005, 0.01, 0.05, 0.5, 1 mg L  1). The linear correlation coefficient (r2) between concentrations and the cube root of peak areas was always over 0.99. 2.7.4. Percentage recovery The recoveries of the pesticides were determined by spiking M4 medium with different concentrations of triazophos or chlorpyrifos. The recovery was calculated by comparing the measured concentrations with the spiked ones. 2.8. Data analysis One-way analysis of variance (ANOVA) and the consequent comparison among treatments were conducted by data processing software DPS& following the route of ‘‘Experimental statistics—Completely randomized design—One-way ANOVA—Duncan Method’’. To take time as independent variable (x) and measured concentration of the test substances as dependent variable (y) to establish degradation equations by DPS& following the route of ‘‘Modeling—Nonlinear regression—Exponential’’. Parameters such as degradation coefficient (k) and half-life of were obtained from the equations. To take nominal concentration of the test substances as independent variable (x) and percentage immobilization relating the concentration as dependent variable (y) to establish ‘‘dose—response’’ equations by DPS& following the route of ‘‘Speciality—Bioassay—Probit analysis’’. Parameters such as regression coefficient, concentration that caused 50 percent inhibition in immobilization (EC50), and 95 percent confidence limit of the EC50 were obtained from the equations.

3. Results 3.1. Acute bioassays

Fig. 1. Standard curve for determining concentration of imunoreactive ChE in crude enzyme preparation. The assay was conducted by using the antibodies that were produced previous and the purified ChE from Daphnia. The curve was set up by plotting the ODs at 450 nm against the concentrations of imunoreactive ChE. The results were the mean of triplicate determinations after subtracting the absorbance of the blanks.

In acute bioassays, the 48h EC50 were found to be 12.92 mg L  1 and 7.12 mg L  1, respectively, with 95 percent confidence limit of 10.55–16.23 mg L  1 and 5.79–9.07 mgL  1 for triazophos and chlorpyrifos, respectively (Table 1). This indicated that the Daphnia were more sensitive to chlorpyrifos than to triazophos. Nominal concentrations employed in 21 d exposure were thus selected to be 0.06, 0.35, and 2.10 mg L  1 for triazophos, and they were selected to be 0.03, 0.20, and 1.19 mg L  1 for chlorpyrifos. The values corresponded to 1/216 EC50, 1/36 EC50, and 1/6 EC50 of the two

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Table 1 Acute toxicity of triazophos and chlorpyrifos to Daphnia magna. Insecticides

48hEC50 (mg  L  1)

Triazophos 12.92 Chlorpyrifos 7.12

‘‘Doseresponse’’ equation

Correlation coefficient

y¼ 3.22x þ 1.43 0.9967 y¼ 2.29x þ 3.18 0.9932

95 percent confidence limit (mg  L  1) 10.55–16.23 5.79–9.07

Fig. 3. Apparent activity of ChE in Daphnia magna dosed with chlorpyrifos of 0 (i.e. the solvent control), 1.19, 0.20, and 0.03 mg L  1, respectively. The samples were taken at 2nd, 6th, 10th, 14th, 18th, and 21st d after dosing. The activity was expressed as the ratio of total activity and total protein in supernatant. The column marked by asterisk is significantly different from that of control (i.e. po 0.05). Bars represent standard divisions of the means.

Fig. 2. Apparent activity of ChE in Daphnia magna dosed with triazophos of 0 (i.e. the solvent control), 2.10, 0.35, and 0.06 mg L  1, respectively. The samples were taken at 2nd, 6th, 10th, 14th, 18th, and 21st d after dosing. The activity was expressed as the ratio of total activity and total protein in supernatant. The column marked by asterisk is significantly different from that of control (i.e. p o0.05). Bars represent standard divisions of the means.

anticholinesterases, respectively. No obvious mortality was found at any of the concentrations. 3.2. Apparent activity of ChE in 21 d exposure Data diagramed in Fig. 2 and Fig. 3 showed that in comparison with the control, apparent activity, i.e. activity that was scaled with total protein in supernatant, changed significantly as the Daphnia were exposed to relative high concentration of either triazophos or chlorpyrifos. Obvious depression was detected at 2nd d of the exposures. For triazophos the rate was found to be 5.2 percent and 5.6 percent at 2.10 mg L  1 and 0.35 mg L  1, respectively (Fig. 2), and for chlorpyrifos it was found to be 6.9 percent at 2.10 mg L  1 (Fig. 3). Induction could be detected after 6th d. The maximal rate was found to be 17.0 percent for triazophos at 0.35 mg L  1 and 14th d (Fig. 2), and it was found to be 17.7 percent for chlopyrifos at 1.19 mg L  1 and 18th d (Fig. 3). The rate tended to decline close to end of the exposure. No significant difference in terms of the activity was detected, for example, between the treated and the control at end of the 21 d exposure to triazophos (Fig. 2). 3.3. Inherent activity of ChE in 21 d exposure Inherent activity, also named as ‘‘actual specific activity’’ by Khattab and Ali (2007), i.e. activity that was scaled with

Fig. 4. Inherent activity of ChE in Daphnia magna dosed with triazophos of 0 (i.e. the solvent control), 2.10, 0.35, and 0.06 mg L  1, respectively. The samples were taken at 2nd, 6th, 10th, 14th, 18th, and 21st d after dosing. The activity was expressed as the ratio of total activity of ChE and immunoreactive protein in supernatant. The column marked by asterisk is significantly different from that of control (i.e. p o 0.05). Bars represent standard divisions of the means.

immunoreactive protein in supernatant, was found depressed after 2nd d. It was at the same day that the rate of depression reached its maximal. For triazophos the value was found to be 42.8 percent at 2.10 mg L  1 (Fig. 4), and for chlopyrifos it was 44.6 percent at 1.19 mg L  1 (Fig. 5). Recovery occurred along with the time. For triazophos difference in terms of the activity of ChE was not found between the treated and the control at 0.35 mg L  1 and 0.06 mg L  1after 18th d (Fig. 4), and for chlorpyrifos the difference

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Fig. 5. Inherent activity of ChE in Daphnia magna dosed with chlorpyrifos of 0 (i.e. the solvent control), 1.19, 0.20, and 0.03 mg L  1, respectively. The samples were taken at 2nd, 6th, 10th, 14th, 18th, and 21st d after dosing. The activity was expressed as the ratio of total activity of ChE and immunoreactive protein in supernatant. The column marked by asterisk is significantly different from that of control (i.e. p o 0.05). Bars represent standard divisions of the means.

Fig. 7. Relative content of immunoreactive protein in Daphnia magna dosed with chlorpyrifos of 0 (i.e. the solvent control), 1.19, 0.20, and 0.03 mg L  1, respectively. The samples were taken at 2nd, 6th, 10th, 14th, 18th, and 21st d after dosing. The value was expressed as the ratio of content of immunoreactive ChE and that of total protein in supernatant. The column marked by asterisk is significantly different from that of control (i.e. p o 0.05). Bars represent standard divisions of the means.

Table 2 Recovery of triazophos from dilutions. Spiked levels (mg/L  1)

Mean recovery (percent)

RSD

0.05 0.5 1

103.77 98.1 102

7.33 9.55 7.00

a b

a

(percent)

nb 3 3 3

RSD ¼ relative standard deviation. n ¼number of replicates.

1.19 mg L  1 for chlopyrifos (Fig. 7). The rate declined as the time lasting. For triazophos there was no significant difference between the treated and the control at 0.06 mg L  1and 0.35 mg L  1 at end of the exposure (Fig. 6), and for chlorpyrifos the difference was not found at all of the three concentrations at end of the exposure (Fig. 7). 3.5. Quantity analysis of triazophos and chlorpyrifos

Fig. 6. Relative content of immunoreactive protein in Daphnia magna dosed with triazophos of 0 (i.e. the solvent control), 2.10, 0.35, and 0.06 mg L  1, respectively. The samples were taken at 2nd, 6th, 10th, 14th, 18th, and 21st d after dosing. The value was expressed as the ratio of content of immunoreactive ChE and that of total protein in supernatant. The column marked by asterisk is significantly different from that of control (i.e. p o0.05). Bars represent standard divisions of the means.

was not found at all the three concentrations after 18th d (Fig. 5). No induction in terms of the activity was found during the exposure. 3.4. Relative content of immunoreactive ChE in 21 d exposure As it is shown in Fig. 6 and Fig. 7, induction in terms of immunoreactive content of ChE was detected in majority period of the exposure and the maximal induction was detected at 2nd d. The value was detected to be 65.9 percent at 2.10 mg L  1 for triazophos (Fig. 6), and it was detected to be 68.0 percent at

3.5.1. Percentage recoveries of the anticholinesterases The percentage recovery was measured to be 98.1 percent– 103.77 percent for triazophos (Table 2) and it was 88.89 percent– 93.17 percent for chlorpyrifos (Table 3), with RSD ranging from 7.33 percent–9.55 percent and from 5.7 percent–7.8 percent, respectively, which were high enough to meet the criteria for residue analysis. 3.5.2. Actual concentration of the anticholinesterases in dilutions Measured concentrations of triazophos and chlorpyrifos at different point of time were listed in Tables 4 and 5. The data showed that concentration of the two anticholinesterases decreased obviously during the 21 d exposure. For triazophos, the final rate was found to be 67.2 percent, 72.9 percent, and 70.8 percent, respectively, at 0.06 mg L  1, 0.35 mg L  1, and 2.10 mg L  1, and for chlorpyrifos it was found to be 39.3 percent, 39.8 percent, 39.0 percent, respectively, at 0.03 mg L  1, 0.20 mg L  1, and 1.19 mg L  1. For triazophos, the half-life was calculated to be

H. Liu et al. / Ecotoxicology and Environmental Safety 80 (2012) 118–125

Table 3 Recovery of chlorpyrifos from dilutions. Spiked levels (mg/L  1)

Mean recovery (percent)

RSD

0.05 0.5 1

93.17 89.47 88.89

5.9 7.8 5.7

a b

a

(percent)

n

b

3 3 3

RSD ¼ relative standard deviation. n¼ number of replicates.

Table 4 Nominal and measured concentrations of triazophos at different time points. Time (d)

Concentration

a

0.06 0 2nd 6th 10th 14th 18th 21st

0.061 0.052 0.037 0.033 0.029 0.025 0.020

(mg  L  1) 0.35

(0.003) (0.004) (0.001) (0.001) (0.001) (0.001) (0.001)

0.347 0.306 0.235 0.203 0.165 0.134 0.094

2.10 (0.012) (0.029) (0.017) (0.011) (0.008) (0.010) (0.006)

2.140 1.819 1.596 1.100 0.950 0.844 0.625

(0.075) (0.044) (0.120) (0.035) (0.071) (0.024) (0.034)

a The results are expressed as the mean of three determinations (i.e. n¼ 3). Enclosed in the blanket is standard deviation (SD).

Table 5 Nominal and measured concentrations of chlorpyrifos at different time points. Time (d)

Concentration 0.03

0 2nd 6th 10th 14th 18th 21st

0.028 0.028 0.026 0.023 0.022 0.019 0.017

a

(mg  L  1) 0.20

(0.001) (0.001) (0.002) (0.001) (0.002) (0.001) (0.001)

0.216 0.192 0.181 0.168 0.136 0.135 0.13

1.19 (0.014) (0.009) (0.006) (0.004) (0.003) (0.011) (0.006)

1.187 1.17 1.01 0.986 0.933 0.821 0.724

(0.050) (0.060) (0.033) (0.066) (0.076) (0.069) (0.042)

a The results are expressed as the mean of three determinations (i.e. n¼ 3). Enclosed in the blanket is standard deviation (SD).

13.2 d, 12.5 d, 12.3 d, with p value of 0.0001, 0.0000, 0.0000, respectively, at 0.06, 0.35, and 2.10 mg L  1, and for chlorpyrifos it was calculated to be 30.3 d, 27.8 d, 32.3 d, with p value of 0.0001, 0.0001, 0.0001, respectively, at 0.03 mg L  1, 0.20 mg L  1, and 1.19 mg L  1.

4. Discussion 4.1. Acute toxicities Previous studies indicated that the 48 h EC50 of chlorpyrifos for Daphnia magna was 0.74 mg L  1 (Palma et al., 2008), 0.8 mg L  1 (Robinson, 1999), 0.19 mg L  1 (Kikuchi et al., 2000), and 3.17 pM (i.e. 1.11 mg L  1) (Printes and Callaghan, 2003), respectively, which was in some degree lower that derived from this study (i.e. 7.12 mg L  1). Not much data is published for triazophos except a paper in which the 48 h EC50 was reported to be 13.8 mg L11 (Tan et al., 2004). According to Barata et al. (2001), a clone of Daphnia magna collected from an area frequently sprayed with OPs was 4 to 10 times more tolerant than a clone from laboratory. This suggested that genetic difference might be a cause for sensitive diversity among studies (Baird and Barata, 1998; Barata et al., 2001).

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4.2. Fluctuation of apparent activity, inherent activity, and immunoreactive content of ChE in daphnia being exposed to the anticholinesterases Apparent activity of ChE was found inhibited at relatively high concentrations at beginning of the exposure. As the exposure continued, the activity tended to rise. Some of the values increased to points higher than that of the controls. These results, as they were diagramed in Figs. 2 and 3, were roughly in accord with previous study conducted by Li and Tan, 2010, where a maximal of 176.5 percent induction in terms of the activity was detected in case that the Daphnia were exposed to triazophos of 0.5 mg L  1 and a maximal of 160.5 percent induction in terms of the activity was detected in case that they were exposed to chlorpyrifos of 0.1 mg L  1. No induction in terms of inherent activity of ChE was detected at any point of time and at all of the three concentrations to be tested (Figs. 4 and 5). This result was similar to that derived from former study conducted with freshwater fish Pseudorasbora parva, where activity of brain ChE, as it was scaled with immunoreactive content of the enzyme, never rose to point higher than that of control (Li et al., 2005). Data diagramed in Figs. 2–5 indicated that concentration that resulted in certain degree of inhibition in terms of inherent activity did not cause the same inhibition in terms of apparent activity. Inhibition of 31.6 percent and 39.1 percent in terms of the inherent activity was detected, for example, in Daphnia being exposed for 2 d in chlorpyrifos of 0.03 mg L  1 and 0.20 mg L  1, respectively (Fig. 5). In spite of that, no obvious inhibition in terms of apparent activity was detected at either of the two concentrations (Fig. 3). Marked inhibition was detected, in another example, for both inherent and apparent activity at nominal concentration of 1.19 mg L  1 but the extent was more distinct in former case (Figs. 3 and 5). This result supports the opinion that the inherent activity is more sensitive than the apparent one in predicting sub-lethal exposure of anticholinesterases. Data diagramed in Figs. 6 and 7 indicated that content of immunoreactive ChE elevated significantly as the Daphnia were exposed to concentrations of either of the two anticholinesterases. According to Kaufer et al. (1999) the elevation might result from an increase in amount of ChE-related mRNA. The increase might lead to accelerated synthesis of the enzyme and ultimately helped the Daphnia to mitigate hurt caused by anticholinesterases. In spite of that the elevation was proofed not to result in prosperity of the species at organismal or population level, or to say, it had nothing to do with fecundity of Daphnia population (Li and Tan, 2010). Comparing data diagramed in Figs. 6 and 7 with that diagramed in Figs. 4 and 5, one can see that immunoreactive content of the ChE changed in negative accord, in the rough, with inherent activity of the enzyme. This suggested that acceleration in synthesis of the enzyme was a quick response towards sublethal exposure of anticholinesterases. 4.3. Relationship between inherent activity and ambient concentration of anticholinesterases Result of the chemical analysis showed that the dissipation half-life was 12.3–13.2 d and 27.8–32.3 d, respectively, for triazophos and chlorpyrifos. This suggested that chlorpyrifos was more stable in dilutions. Data cited in PPDB of International Union of Pure and Applied Chemistry (IUPAC) indicates that aqueous hydrolysis DT50 is 140 d and 25.5 d, respectively, for triazophos and chlorpyrifos (http://sitem.herts.ac.uk/aeru/iupac/). The DT50 was reported to be 5.37–41.25 d for triazophos (Zhao et al., 2004;

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Xiao et al., 2005; Sun et al., 2007; Qin et al., 2009) and it was 25.6–63.9 d for chlorpyrifos (Shi et al., 2000; Wu et al., 2001). Nonidentity in conditions for incubation might be the reason for incoordinations among studies. Inherent activity of ChE changed, in the rough, in concentration-dependant manner as it was measured at one time, and the activity tended to recover as exposures continued. In spite of that, further comparison indicated it is not comparable between the inherent activity and the measured concentration of anticholinesterases. To take triazophos as example, as the Daphnia were exposed (for 21 d) to measured concentration of 0.625 mg L  1, there existed 16.4 percent inhibition in terms of the inherent activity, whereas in case the Daphnia were exposed (for 2 d) to measured concentration of 0.306 mg L  1, there existed 39.9 percent inhibition in terms of the activity (Figs. 4 and 6). Such kind of inaccordance was also being for chlorpyrifos (Figs. 5 and 7). In general the inherent activity recovered faster in speed as it was compared with the rate of declination in terms of measured concentrations. This suggested that apart from quantity promotion in terms of the target enzyme, the Daphnia developed other capacities to mitigate stresses caused by the anticholinesterases. It is the residue in target, rather than the ambient concentration that ultimately determine the inherent activity of ChE in vivo. Due to the small in size of the Daphnia, there are difficulties to inspect the exact residue of either triazophos or chlorpyrifos in target tissues. From the fact that the inherent activity recovered faster than the rate of declination in terms of measured concentrations, it is reasonable to infer the development of other capacities to block bio-concentration of the anticholinesterases in target. It has long been noticed that there might be more than one type of ChEs in individual, some are responsible for maintaining normal status of neurotransmission, and others serves as scavengers to keep the anticholinesterases from reaching the targets (Villatte and Bachmann, 2002; Lenz et al., 2005). According to Diamantino et al. (2003), ChE of Daphnia magna shows characteristics of both acetylcholinesterases (AchE, EC 3.1.1.7) and pseudocholinesterases (PchE, EC 3.1.1.8). It remains uncertain if the Daphnia contain one type of untypical ChE, or they contain both AchE and PchE. Provided that the second case was being, the Daphnia might contain isozyme(s) that refuse to react with the antigen employed in this study and it was the isozyme(s) that scavenged the anticholinesterases so as to prevent their interring into the target. Unfortunately, the existence of the assumptive isozyme(s) made it unreliable to link the inherent activity to measured concentration of anticholinesterases without knowing the history of exposure. Anyhow, it is necessary to clarify, in further studies, types and characters of ChEs in Daphnia, especially the characters concerning immunoreactive specificity and affinity to anticholinesterases.

5. Conclusions In case the Daphnia.magna were exposed to either triazophos or chlorpyrifos at nominal concentration of 1/6EC50, 1/36 EC50, and 1/216 EC50, inherent activity of the ChE, i.e. the activity that was scaled with immunoreactive protein, was found inhibited while the immureactive content of the enzyme was found increased. The increase resulted in obvious promotion in terms of total activity of ChE at different combinations of time and concentration. Result of the study indicated that the inherent activity was more sensitive than the apparent one in predicting sub-lethal and/or long-term exposure of anticholinesterases. Judged by measured concentrations, the inherent activity recovered faster than the rate of declination in terms of the

concentrations. Unfortunately, this character made it is unreliable to link the level of inhibition to ambient concentration of the anticholinesterases. The concentration would be underestimated along with the time of exposure.

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