Toxicology Group, Wageningen University, P.O. Box 8000, 6700 EA Wageningen, The Netherlands
Received May 5, 2000; accepted July 25, 2000
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ABSTRACT |
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Key Words: organophosphate insecticides; carbamates; rainwater; esterase; N-methylindoxylacetate; Apis mellifera.
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INTRODUCTION |
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In order to carry out a hazard assessment of the complex mixture of pesticides in rainwater, knowledge of the concentration and the consequent toxicity of an individual pesticide are insufficient, because combination effects such as synergism or antagonism are neglected. Furthermore, unexpected toxic pesticides or metabolites of pesticides are missed when only the parent compounds expected to be present are analyzed. Therefore, a better approach could be to measure the integrated toxic potency of the complex mixture using a bioassay that enables identification and quantification of relevant toxic endpoints. The toxicity of organophosphate and carbamate insecticides is based on their ability to block esterases, thus inhibiting the hydrolysis of the neurotransmitter acetylcholine by acetylcholinesterase (AChE). Since the end of the 1940s, esterase inhibition has been measured in the monitoring of organophosphate compounds (O'Brien, 1960), by manometric (Metcalf and March, 1949
) and spectrophotometric (Hestrin, 1949
) techniques. Esterase activity was often determined in mammalian erythrocytes or in crude homogenates made from insects or vertebrate tissues after in vivo or in vitro exposure to inhibitors. Sensitivity, applicability and simplicity were considerably improved by the introduction of a new colorimetric technique using acetylthiocholine as a specific substrate for AChE (Ellman et al., 1961
). Brogdon and Barber (1987) successfully downscaled this method to a microtiter plate assay, allowing quantification at concentrations in the order of 0.110 µM.
The objective of the current study was to develop a more sensitive small-volume assay for measuring esterase inhibition, enabling quantification at lower concentrations of inhibitors. To select the optimum test conditions, inhibition kinetics were studied for the hydrolysis of two substrates (i.e., specific S-acetylthiocholine-iodide or nonspecific N-methylindoxylacetate) by two types of esterases, i.e., purified AChE from electric eel (Electrophorus electricus), which is commercially available, and a homogenate of honeybee heads (Apis mellifera). The latter type of esterase was chosen because honeybees have long been known to be sensitive to organophosphate and carbamate insecticides (e.g., Anderson and Atkins, 1966; Anderson et al., 1968; Atkins et al., 1970a,b; Johansen, 1969). Dichlorvos was used as a model organophosphate inhibitor allowing quantification of the mixture toxicity of the rainwater extract into dichlorvos equivalent concentrations. Solid-phase extraction (SPE) of rainwater was optimized using methanol, dichloromethane and hexane for subsequent elution of extracted pesticides.
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MATERIALS AND METHODS |
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Homogenate of honeybee heads.
Worker honeybees (Apis mellifera) were obtained from the Laboratory of Entomology (Wageningen University). Bees were anaesthetized with CO2, frozen to death at 20°C and decapitated. With a potter instrument, 18 heads were homogenized six times in 2 ml of P-buffer. After each homogenization step, the potter tube was put on ice, allowing the chitinous parts of the crushed bee heads to sink down. Next, the crude homogenate was collected, leaving the crushed bee heads; 2 ml of fresh P-buffer was added to the remaining parts of the crushed heads and homogenization was repeated. After the last step all crude homogenate collected (about 12 ml) was centrifuged (5 min; 4388 x g). The supernatant was divided into small batches (250 and 500 µl) and frozen at 20°C. Protein levels were measured by the method of Bradford (1976) and ranged from 1.3 to 1.5 g/l.
Esterase inhibition assay.
All experiments were performed in P-buffer in a microtiter plate. ATC was used as a substrate to determine specific cholinesterase activity. ATC hydrolysis was measured as an increase in OD412 (15 s time intervals; SpectraMax 340 platereader [Molecular Devices Corporation]) caused by the reaction of thiocholine with DTNB to produce the yellow 5-thio-2-nitro-benzoic acid anion (Ellman et al., 1961). MIA was used as a substrate to determine nonspecific esterase activity. MIA hydrolysis was measured as an increase in fluorescence (70 s time intervals; CytoFluorTM 2350 platereader [Millipore]) caused by the hydrolysis product N-methylindoxyl (
ex = 418 nm;
em = 500 nm).
In all microtiter plates, one pesticide or sample was tested in several concentrations. First P-buffer was added to reach final volumes of 300 and 200 µl in all wells using ATC and MIA as a substrate, respectively. Next, 50 µl of inhibitor was added and incubation was started at time t0 by adding 50 µl of enzyme solution. To correct for nonenzymatic hydrolysis, some wells received P-buffer instead of enzyme. With ATC as a substrate, enzyme concentrations added were 75 µl bee homogenate per ml. With MIA, concentrations added were 15.2 µl bee homogenate per ml or 102 mU eel AChE concentrations per ml.
Two different protocols have been used. In protocol I experiments, one single concentration of substrate was added to the different columns of the microtiter plate after 4 different incubation periods (t1t4), enabling following of progressing irreversible inhibition. In protocol II experiments, 4 different substrate concentrations were added to the different columns of the microtiter plate immediately after addition of the inhibitor, enabling measurement of instant reversible inhibition. Treatments were measured in triplicate.
Applying protocol I with ATC as a substrate, 100 µl of DTNB (5mM in P-buffer) and 100 µl of ATC (0.795 mM in P-buffer) were added at time points t1t4 and with MIA as a substrate, 10 µl of MIA (5.3 mM in methanol) was added. Increases in absorption and fluorescence, respectively, were measured immediately after substrate addition.
Using ATC as a substrate in protocol II, 100 µl of DTNB and 100 µl of 0.143, 0.239, 0.398 and 0.795 mM ATC in P-buffer were added immediately after incubation had started and increase in absorbance was measured. Using MIA as a substrate in protocol II, 10 µl of 0.954, 1.59, 2.65 and 5.3 mM MIA in methanol were added immediately after incubation had started and fluorescence was measured.
Rainwater sampling.
Rainwater was sampled in a natural conservation area (De Regulieren near Culemborg), which is considered to be a relative background area for the Dutch situation. Rainwater samples were collected at 1.5 m above ground level in 10 open samplers. Each sampler consisted of a clean amber glass bottle (2.5 l), with a polyester funnel (24 cm in diameter) on top. Samples were collected within 2 days after major showers: April 29, May 7, May 16, and June 13, 1997. By then, bottles had remained in the field for a period of 6, 8, 9 and 23 days, respectively.
Sample processing.
At arrival at the laboratory, pH was measured in each bottle and samples were stored in the dark at 4°C. Out of the total sample, a 6 l sub-sample was filtered over a glass fiber filter (Schleicher & Schuell GF50, 47mm in diameter). Filters were replaced when clogged. On the average, 24 filters were needed to filter the sub-sample. Filters were dried and pH of the sub-sample was set at 5.0 (± 0.1) with 1N HCl or NaOH. Solid phase extraction was performed using an extraction disk, made of rigid glass fiber material with polystyrene divinylbenzene as active phase (Ztek Accu.FloTM SDVB Fiber SPE Disks). The disk was pre-washed with methanol, dichloromethane and hexane (3*10 ml per solvent). After drying, the disk was conditioned with 10 ml of methanol for 15 min, after which 300 ml of demineralized water was pulled through the disk to wash away the methanol using a vacuum pump. Finally, the sub-sample of 6 l was pulled through the disk. After extraction, the 24 glass fiber filters were put upon the dried disk and both disk and filters were eluted successively with methanol, dichloromethane and hexane (3*2 ml per solvent). Extracts were separated by solvent, collected, evaporated under a gentle nitrogen stream at 37°C, and dissolved in 10 µl of 1-propanol as carrier. Extracts were kept in the dark at 4°C and were freshly diluted into 2 ml of P-buffer (4°C) on each experimental day. Next, the diluted extract was further diluted into a linear concentration series, with dilution factors depending on the esterase inhibiting potency of the sample and with each dilution having a final volume of 650 µl, allowing to add 12*50 µl to the microtiter plate. For each sample, 1-propanol concentrations were standardized, with a maximum final concentration of 0.07% in the microtiter plate.
Spike analysis for recovery assessment.
A batch of 6 l demineralized water was spiked with the pesticide mixture (100 µg of each constituent). The spiked sample and a control sample of 6 l demineralized water were extracted similar to the rainwater samples. The pesticide mix was chosen to represent a general mixture of OPs that may be present in rainwater, containing not only oxon-forms, but also less potent thio-forms. Recovery was calculated by comparing the esterase inhibiting potency (protocol II) of the 100-fold diluted extract of the spiked batch (final volume = 1 ml) to the potency of a 100 µg/ml solution prepared directly from the pesticide mixture itself.
Data analysis and statistics.
In the present study, special emphasis was placed on expressing the overall toxic potency of a mixture of compounds into a reliable single value, which is easy to interpret. Esterase inhibiting potencies were recalculated into equivalent concentrations of dichlorvos (with 95% confidence interval) based on the reaction kinetics of esterase mediated hydrolysis. Both substrates (such as ATC and MIA) and inhibitors (such as organophosphate and carbamate insecticides) are hydrolyzed by esterases according to the same ubiquitous reaction scheme (Main, 1964; Aldridge and Reinier 1972
):
![]() | (1) |
First, the substrate or inhibitor AB quickly binds to the free esterase EH to form a reversible Michaelis-Menten complex (EHAB). Next, the enzyme is irreversibly carboxylated (in the case of MIA or ATC as substrate) or phosphorylated or carbamylated (in the case of organophosphates or carbamates, respectively, as inhibitors) into EA, and BH is split off simultaneously. Finally, EA is hydrolyzed into EH and AOH. As the rate of hydrolysis (k+3) is much slower for inhibitors than for substrates, they inhibit esterases irreversibly.
In view of the results, most emphasis was given to the quantification of the reversible inhibition of honeybee homogenate mediated MIA hydrolysis by small concentrations of OP esters, which could be described by Michaelis-Menten kinetics for competitive inhibition:
![]() | (2) |
where v0 is the initial rate of the enzyme catalyzed reaction immediately after addition of the substrate to the enzyme, Vmax is the maximum initial reaction rate, KM is the Michaelis constant, Ki is the inhibitor constant, [S] is the substrate concentration, and [I] is the inhibitor concentration. KM and Ki are both constants which can be interpreted as typical concentrations: KM is the substrate concentration at which the initial reaction rate (v0) in the absence of an inhibitor ([I] = 0) is equal to Vmax and Ki is the inhibitor concentration for which twice as much substrate is needed to reach an initial reaction rate (v0) of
Vmax than in an uninhibited situation. Thus, esterase inhibiting capacity increases with decreasing Ki values.
To recalculate the esterase inhibiting potency of a sample into equivalents of a standard organophosphate insecticide (i.e., dichlorvos), Ki values of both samples and dichlorvos were calculated by performing a reciprocal transformation of the Michaelis equation (2) into the Lineweaver-Burk equation
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This again was rewritten into
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Multiple linear regression was performed on 1/[S] and [I]/[S], with least squares being weighed by (v0)2 (SPSS for Windows, version 7.5.2). Ki was calculated by dividing the regression coefficients:
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A 95% confidence interval for Ki was calculated applying Fieller's theorem (Fieller, 1954; Collett, 1994
):
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where L is either the upper or lower limit of the confidence interval (depending on ±) and
![]() | (7A) |
![]() | (7B) |
![]() | (7C) |
Variation of Ki was calculated using the following estimation (Chatfield, 1983)
![]() | (8) |
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where CV is the coefficient of variance, and E the estimated value.
This procedure was applied to calculate Ki values of both dichlorvos and rainwater extracts. Total inhibitor concentrations [I] in the extracts were expressed as dimensionless concentration factors of the rainwater [ml rainwater per ml reaction mixture], whereas concentrations [I] of the dichlorvos had dimensions of [nM]. Since both Ki values represent an inhibitor concentration with the same characteristics, the toxic potency of the rainwater sample could be expressed in terms of nmoles dichlorvos per liter rainwater by
![]() | (9) |
where DCVEq is the toxic equivalent of the rainwater expressed in [nmoles dichlorvos per liter rainwater], Ki(DCV) is the Ki value of dichlorvos and Ki(ex) is the Ki value of the extract. A 95% confidence interval for DCVEq was again calculated by Fieller's theorem (equation 5).
The progressive inhibition of honeybee homogenate mediated ATC hydrolysis and AChE mediated MIA hydrolysis was described by
![]() | (10) |
(Main, 1964; Main and Iverson, 1966
; Aldridge and Reinier, 1972
), where V0 and Vt are the initial reaction rates of substrate hydrolysis at t = 0 and at time t after starting incubation of the enzyme with inhibitor, and [I] is the concentration of the inhibitor. KA is the affinity constant (k1/k+1) for the formation of the reversible complex, and k+2 is the rate of the progressing irreversible phosphorylation. Bimolecular inhibition constants (ki = k+2/KA; Main, 1964) were calculated by nonlinear regression based on equation 10
.
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RESULTS |
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No such intercepts were found for the progressive inhibition curves of MIA-hydrolysis by electric eel AChE incubated with dichlorvos, except for the highest dichlorvos concentration tested (1000 µg/l). Despite the fact that enzyme concentrations were used with similar MIA-hydrolyzing capacities (i.e., 15.2 µl of bee homogenate per ml 102 mU of eel AChE per ml), progressive inhibition of AChE mediated MIA-hydrolysis was found only at dichlorvos concentrations higher than the highest concentration tested in Figure 1B
. A bimolecular inhibition constant ki was estimated of 6.3*105 M1 min1.
For greatest sensitivity of the assay it was decided to further apply bee esterases and MIA as the enzyme source and its substrate, respectively. As the reversible enzyme inhibitor complex was formed at low concentrations of inhibitor, quantification of the kinetics of this complex was further studied in reversible inhibition experiments with dichlorvos (Protocol II).
Reversible Inhibition Experiments with Dichlorvos
The initial reaction rate of the honeybee esterase catalyzed hydrolysis of MIA immediately decreased with increasing concentrations of dichlorvos. As expected, no such dose-response relationship was found for ATC hydrolysis, because no intercept was observed in Figure 1A. After reciprocal transformation of the initial reaction rate [v0] and the concentration of MIA [S], the obtained Lineweaver-Burk plot (Figure 2
) was best described by the multiple regression model based on competitive inhibition (Equation 3
; Table 1
). The model is characterized by a common intercept 1/Vmax on the vertical 1/v0-axis for all dichlorvos concentrations tested and by slopes increasing proportionally with higher concentrations of inhibitor. This model for competitive inhibition kinetics was further applied to quantify the esterase inhibiting potency of rainwater extracts, because alternative regression models based on uncompetitive or mixed inhibition kinetics were less appropriate. Besides, competitive inhibition of MIA hydrolysis catalyzed by honeybee homogenate occurred at dichlorvos concentrations about one order of magnitude lower than progressive inhibition (cf. Figures 1 and 2
).
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Reversible Inhibition Experiments with Rainwater Extracts
For all four rainwater samples tested, MIA hydrolysis by honeybee esterases was most inhibited by the first elution fraction with methanol. Consecutive elution with dichloromethane and hexane yielded fractions with much less esterase inhibiting potency, and no inhibition was detected after exposure to pure or diluted rainwater. The inhibition of MIA hydrolysis by the methanol fraction (Figure 3) and the dichloromethane and hexane fraction of a rainwater extract show clear dose-response relationships.
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DISCUSSION |
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Irrespective of the enzyme source and substrate used, progressive inhibition by dichlorvos was very slow at these concentrations. Using higher concentrations of dichlorvos, bimolecular inhibition constants of 6.3*105 and 1.1*105 M1 min1 were determined for honeybee esterase mediated ATC hydrolysis and AChE mediated MIA hydrolysis, respectively. These Ki values are within the same range as reported by De Bruijn and Hermens (1993) and Xu and Bull (1994), who used ATC as a substrate for purified AChE and determined Ki values in the range of 104106 M1 min1 for 21 of all 23 oxon-analogues of organophosphates studied. In practice this means that for more than 90% of the inhibitors tested, concentrations ranging from 707000 nM are required to reach 50% of inhibition within 10 min of incubation (IC5010min). To quantify the reversible inhibition complex, a maximum dichlorvos concentration of 16 nM was tested (Figure 2), which is 4.4440 times less than this range of IC5010min values. Considering the goal of the present study to select optimum test conditions for measuring low concentrations of esterase inhibitors in small samples, it was decided to quantify the toxic potency of rainwater by the reversibly inhibited hydrolysis of the substrate MIA by esterases present in honeybee homogenate.
Substrate Specificity of Esterases
The homogenate of honeybee heads contains not only AChE, but also many unspecific cytosolic carboxylesterases. Apparently, using both specific ATC and nonspecific MIA as a substrate allows discrimination between the activity of acetylcholinesterase and carboxylesterases in the honeybee homogenate, because different combinations of substrates and enzymes have different inhibition kinetics. As ATC hydrolysis by honeybee homogenate showed similar kinetics of progressive inhibition by dichlorvos as ATC hydrolysis by purified AChE (cf. Figure 1A and De Bruijn and Hermens, 1993), it is hypothesized that ATC is actually hydrolyzed by the acetylcholinesterase present in honeybee homogenate, and not by the nonspecific carboxyl-esterases. This is confirmed by results from Bitondi and Mestriner (1983), who found no cholinesterase activity for 6 different isozymes of carboxylesterases isolated from homogenate of Apis mellifera using ATC as substrate.
For organophosphate compounds, reversible inhibition of carboxylesterases from honeybee homogenates with nonspecific substrates has been described previously. Frohlich et al. (1990) found mixed mechanisms of competitive and uncompetitive inhibition for the alfalfa leafcutting bee Megachile rotundata and Spoonamore et al. (1993) found that inhibition mechanisms were highly competitive in nature (explicitly for dichlorvos) for the honeybee Apis mellifera. In both studies, p-nitrophenylacetate was used as a nonspecific substrate. In the study of Bitondi and Mestriner (1983), this same substrate was exclusively hydrolyzed by only one carboxylesterase isozyme (number 3) of the 6 isolated isozymes of carboxylesterases (Figure 4). This isozyme 3 also showed highest hydrolysis activity for the substrates indoxyl acetate and bromo-indoxyl acetate, which are closely related to MIA. Based on the similarity between the results of Spoonamore et al. (1993) and our study, regarding reversible competitive inhibition kinetics, it can be hypothesized that MIA hydrolysis is mainly catalyzed by the unspecific carboxylesterases present in the homogenate rather than by acetylcholinesterases (Figure 4
). Apparently, these carboxylesterases are also progressively inhibited at high concentrations of inhibitor, but have different inhibition kinetics than AChE at low inhibitor concentrations, due to a higher affinity constant KA.
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Application of the Esterase Inhibition Assay to Rainwater Samples
Already in the first elution step of the extraction disk with methanol, most esterase-inhibiting potency of the rainwater was collected (Table 2). The dichloromethane fraction contained much less potency and the hexane fraction contained negligibly amounts. Therefore, elution with methanol and dichloromethane is sufficient to collect all toxic potency from the extraction disk.
The lowest estimated concentration of 2.0 ng dichlorvos equivalents per liter rainwater (hexane elution fraction of June 13, 1997, Table 2) was calculated by applying equation 9
, with Ki values of 0.79 mg/l and 404 ml/ml for dichlorvos and rainwater, respectively. Based on the latter value, 2 ng/l in the rainwater is equivalent to 0.8 µg/l in the microtiter plate. Assuming this to be the highest concentration required in the concentration series and taking into account that final 1-propanol concentrations should be smaller than 0.09%, the sensitivity of the assay can be increased by reducing the dilution volume of the extract from 2 to 0.4 ml and by reducing the volume of 1-propanol as a carrier from 10 to 5 µl. Thus, the limit of detection is decreased to 1.4 ng/l dichlorvos equivalents in 6 l samples and to 14 ng/l in 0.6 l samples.
The maximum dichlorvos-equivalent concentration of 125 ng per liter of rainwater determined in this study (Table 2) exceeded 179 times the Maximum Permissible Concentration (MPC) of dichlorvos in surface water (0.7 ng/l; Crommentuijn et al., 1997
). However, this concentration is still 3.236 times lower than the maximum concentrations of the individual compound dichlorvos previously reported in rainwater in The Netherlands collected in different locations and in different seasons (Fleverwaard, 1993
; Province of South-Holland, 1994
; Baas and Duyser, 1997
). Further validation of the assay with chemical analyses of the rainwater samples has been performed, and good correlations have been found both in time and in place for chemically analyzed organophosphate compounds and esterase inhibiting potencies in rainwater extracts (Hamers et al., manuscript submitted).
Expressing the esterase inhibiting potency of the extracts in dichlorvos equivalent concentrations assumes that the complex mixture of inhibitors in the extract behave as a virtual single compound with its own specific Ki value. The proportional increase (R2>0.98) of the slopes of the Lineweaver-Burk plots with increasing concentrations of inhibitor mixture (i.e., rainwater extract or the mixture of 20 organophosphate insecticides) supports this assumption.
Conclusions
N-methylindoxylacetate (MIA) and carboxylesterases from honeybee homogenate is the most sensitive combination of substrate and enzyme, respectively, to determine the esterase inhibiting potency of a mixture of organophosphate and carbamate insecticides. In combination with solid-phase extraction, a bioassay based on the inhibited hydrolysis of MIA by carboxylesterases allows the rapid quantification of the esterase inhibiting potency in rainwater as low as 2 ng dichlorvos equivalents per liter. The sensitivity of carboxylesterases from honeybee homogenate is attributed to the instant formation of a reversible Michaelis-Menten complex between the inhibitor and the esterases, which is not formed between the inhibitor and purified AChE. Based on the inhibitor constants (Ki) of the model inhibitor dichlorvos and the rainwater extracts, the esterase inhibiting potency of the rainwater can be recalculated into an equivalent concentration of dichlorvos. Most inhibitory capacity is present in the relatively hydrophilic fraction of rainwater extracts. In the 4 rainwater samples tested, honeybee esterase inhibiting potency was equivalent to dichlorvos concentrations of 12 to 125 ng/l.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Anderson, L. D., and Atkins Jr., E. L. (1966). 1965 Research on the effect of pesticides on honey bees. Am. Bee J. 106, 206208.
Anderson, L. D., Atkins Jr., E. L., Todd, F. E., and Levin, M. D. (1968). Research on the effect of pesticides on honey bees 196667. Am. Bee J. 108, 277279.
Atkins Jr., E. L., Anderson, L. D., and Greywood, E. A. (1970a). Research on the effect of pesticides on honey bees 196869. Part I. Am. Bee J. 110, 387389.
Atkins Jr., E. L., Anderson, L. D., and Greywood, E. A. (1970b). Research on the effect of pesticides on honey bees 196869. Part II. Am. Bee J. 110, 426429.
Baas, J., and Duyser, J. H. (1997). Pesticidenconcentraties in lucht en regenwater Pilotstudie naar concentraties pesticiden in lucht en regenwater in Naaldwijk, de Zilk en meetpost Noordwijk. TNO. Report TNO-MEPR 97/113, Apeldoorn.
Bitondi, M. M. G., and Mestriner, M. A. (1983) Esterase isozymes of Apis mellifera: substrate and inhibition characteristics, developmental ontogeny and electrophoretic variability. Biochem. Genet. 21, 985-1002.[ISI][Medline]
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72, 248254.[ISI][Medline]
Brogdon, W. G., and Barber, A. M. (1987). Microplate assay of acetylcholinesterase inhibition kinetics in single-mosquito homogenates. Pest. Biochem. Physiol. 29, 252259.[ISI]
Brouwer, F. M., and Van Berkum, S. (1996). CAP and environment in the European Union: Analysis of the effects of the CAP on the environment and assessment of existing environmental conditions in policy. Wageningen Press, Wageningen, the Netherlands.
Chatfield, C. (1983). Statistics for technology a course in applied statistics (3rd ed.). Chapman & Hall, London.
Collett, D. (1994). Modeling binary data. Chapman & Hall, London.
Crommentuijn, T., Kalf, D. F., Polder, M. D., Posthumus, R., and Van de Plassche, E. J. (1997) Maximum Permissible Concentrations and Negligible Concentrations for pesticides RIVM Report 601501 002. National Institute of Public Health and The Environment (RIVM), Bilthoven, The Netherlands.
De Bruijn, J., and Hermens, J. (1993). Inhibition of acetylcholinesterase and acute toxicity of organophosphorous compounds to fish: a preliminary structure-activity analysis. Aquat. Toxicol. 24, 257274.[ISI]
Draaijers, G. P. J., Berdowski, J. J. M., Bloos, J. P. J., Van der Brugh, J. H. A. M., Coenen, P. W. H. G., and Evers, C. W. A. (1998). Emission data for The Netherlands 1996 and estimates for 1997. Publication Series Emission Inventory Nr. 45. Ministry of Housing, Spatial Planning and the Environment (VROM), The Hague.
Ellman, G. L., Courtney, K. D., Andres Jr., V., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 8895.[ISI][Medline]
Fieller, E. C. (1954). Some problems in interval estimation. J. Roy. Statist. Soc. Ser. B 16, 175185.[ISI]
Fleverwaard (1993). Bestrijdingsmiddelen in neerslag en in oppervlaktewater. Fleverwaard Water Regulatory Board, Lelystad.
Frohlich, D. R., Boeker, E. A., and Brindley, W. A. (1990). The kinetics and inhibition of p-nitrophenylacetate-hydrolysing esterases in a solitary bee, Megachile rotundata (Fab.). Xenobiotica 20, 481487.[ISI][Medline]
Hestrin, S. (1949). The reaction of acetylcholine and other carboxylic acid derivates with hydroxylamine, and its analytical application. J. Biol. Chem. 180, 249261.
Johansen, C. (1969). The bee poisoning hazard from pesticides. Washington Agricultural Experiment Station Bulletin 709.
Main, A. R. (1964). Affinity and phosphorylation constants for the inhibition of esterases by organophosphates. Science 144, 992993.[ISI][Medline]
Main, A. R., and Iverson, F. (1966). Measurement of the affinity and phosphorylation constants governing irreversible inhibition of cholinesterases by di-isopropyl phosphorofluoridate. Biochem. J. 100, 525531.[ISI][Medline]
Metcalf, R. L., and March, R. B. (1949). Studies of the mode of action of parathion and its derivatives and their toxicity to insects. J. Econ. Entomol. 42, 721728.[ISI][Medline]
O'Brien, R. D. (1960). Toxic phosphorus esters: Chemistry, metabolism, and biological effects. Academic Press, New York and London.
Province of South-Holland (1994). Bestrijdingsmiddelen in neerslag in Zuid-Holland Province of South-Holland-DWM, The Hague.
RIVM/CBS (1999). Milieucompendium 1999 Het milieu in cijfers Samson BV, Alphen aan de Rijn, The Netherlands.
Spoonamore, J. E., Frohlich, D. R., and Wells, M. A. (1993). p-Nitrophenylacetate hydrolysis by honey bee esterases: kinetics and inhibition. Xenobiotica 23, 279284.[ISI][Medline]
Xu, G., and Bull, D. L. (1994). Acetylcholinesterase from the horn fly (Diptera: Muscidae) II: Biochemical and Molecular properties. Arch. Insect Biochem. Physiol. 27, 109121.[ISI]