Chemicals in the Environment - ACS Publications - American

Chapter 16

Highly Sensitive Assay for Anticholinesterase Compounds Using 96 Well Plate Format 1

2,3

1,*

Downloaded by FUDAN UNIV on January 26, 2017 | http://pubs.acs.org Publication Date: January 24, 2002 | doi: 10.1021/bk-2002-0806.ch016

Nirankar N. Mishra , Joel A. Pedersen , and Kim R. Rogers 1

U.S. Environmental Protection Agency, National Exposure Research Laboratory, Las Vegas, N V 89119 U.S. Environmental Protection Agency, Region 9, San Francisco, CA 94105-3901 Environmental Science and Engineering Program, University of California, Los Angeles, CA 90095 2

3

The rapid and sensitive detection of organophosphorus insecticides using a 96 well plate format is reported. Several features of this assay make it attractive for development as a laboratory-based or field screening assay. Acetylcholinesterase (AChE) was stabilized in a gelatin film. The remarkable properties of the dry immobilized A C h E preparation include its stability to prolonged storage at room temperature as well as its stability to short term elevated temperatures (60°C). The enzyme could be maintained in dry gel form for 365 days at room temperature without substantial loss of activity. The absorbance assay used to measure enzyme activity was evaluated using several solvent systems including water, phosphate buffer, hexane, methanol and ethanol. The microwell assay includes a procedure to oxidize less potent P=S organophosphorus compounds to their more inhibitory oxon forms. The use of this assay to analyze field samples contaminated with mixtures of organophosphorus insecticides is also reported.

© 2002 American Chemical Society Lipnick et al.; Chemicals in the Environment ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

289


290 One means of reducing uncertainties in human exposure assessment is to better characterize concentrations of hazardous compounds present in the immediate environment of receptor populations. A significant limitation to this approach, however, is that sampling and laboratory analysis of contaminated environmental and biological samples can be slow and expensive; thus, limiting the number of samples that can be analyzed within time and budget constraints. Faster, simpler, and more cost-effective field screening methods can increase the amount of information available concerning the location, source and concentration of pollutants present in the environment (/). Among the compounds of interest for human exposure assessment are pesticides (2), particularly insecticides from the organophosphorus (OP) and carbamate classes which are widely used in agricultural and residential settings. Due to the relatively high toxicity of some of these compounds, a significant number of poisonings occurs each year (5). In addition, these compounds pose a hazard not only to the primary user, but in some cases to emergency healthcare workers as well (4). The toxic effects of OP and carbamate insecticides are mediated primarily through disruption of cholinergic neurotransmission by inhibition of A C h E (5). In addition to their acute toxicity due to inhibition of A C h E , these compounds have also been implicated with long term neurological problems such delayed neurotoxic effect (6). OP and carbamate insecticides vary considerably in their overall toxic behavior due to many factors, including absorption, metabolism and interaction of parent compounds and transformation products with various target and non-target proteins. Metabolic activation is particularly important for the phosphorothioates, a subclass of OP compounds characterized by sulfur (P=S) attached to the central phosphorous atom. For this class of insecticides, the parent compound typically shows little anticholinesterate activity whereas the oxidative transformation product (the oxon, P=0) is often highly potent (5). Although a variety of chromatographic methods have been applied for detection of OP and carbamate insecticides, these techniques are typically expensive and time-consuming. A wide variety of bioanalytical and biosensor methods based on A C h E inhibition have also been reported over the past decade (7). Although these methods show considerable promise, they are not well suited for screening large numbers of environmental samples. These assays are relatively simple and particularly sensitive to specific compounds or their transformation products, however, a number of problems hinder their widespread adoption and use. These challenges include such issues as high throughput formats, long-term stabilization of A C h E , requirement for organic extraction solvents, oxidative activation of parent compounds and the ability to derive useful information for samples containing mixtures of OP and carbamate compounds. Although significant progress has been reported in such areas as stabilization of immobilized enzymes (£), oxidative activation of parent compounds (9), high throughput

Lipnick et al.; Chemicals in the Environment ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

291 formats (JO), and the use of environmental matrices (11), the integration of these concepts and the demonstration of a simple assay format relevant to environmental samples is still of considerable importance to progress in this area. We present here a simple, sensitive, versatile and inexpensive assay format for detection of OP and carbamate insecticides. In addition, we suggest the use of paraoxon equivalents for screening environmental samples contaminated with mixtures of these insecticides.


Experimental Methods Materials Acety 1 cholinesterase from an electric eel, (EC 3.1.1.7,1000U/mg), acetylthiocholine chloride, 5,5'-dithiobis (2-nitrobenzoic acid), D (+) trehalose dihydrate, D (+) glucose, pyridostigmine bromide, and neostigmine bromide were from Sigma Chemical/Aldrich. Aldicarb, carbaryl, carbofuran, chlorpyrifos, chlorpyrifos-oxon, dichlorvos, methomyl, malathion, malaoxon, naled, paraoxon, parathion, trichlorfon, azinphos-methyl, diclofenthion, dimethoate, dimethoateoxon, terbufos, phosmet, and fenthion were obtained from Chem Service Corp. A l l others chemicals used were reagent grade. Deionized water (DI) was used for the preparation of all solutions. Enzyme Immobilization and O P and Carbamate Assay Protocol A C h E was dissolved in a solution containing 5% D-(+)-trehalose dihydrate, 5% D-(+)glucose, 0.1% of gelatin, 1 % sodium chloride and 0.002% sodium azide (TGG) and distributed into individual wells of the microtiter plate. The enzyme was dried under a stream of air for 24 hrs at 25°C, after which it was ready for use in the OP inhibition assay. Immediately prior to the assay, the A C h E was dissolved in phosphate buffered saline (PBS) solution containing 10 m M sodium phosphate, 100 m M NaCl, p H 7.4. A C h E activity was measured using the Ellman method (12). The reaction medium contained 75 \iL of 1 m M acetylthiocholine chloride, 75 \ih of 1 m M 5,5'-dithiobis (2-nitrobenzoic acid), 25 μι of A C h E (2.86 ng) and 25 \ih of DI water at p H 7.4. For the inhibition assay without prior oxidation of the inhibitors, 25 μΐ, of A C h E was incubated for 20 min with 25 μΣ of the inhibitor at concentrations ranging from 100 μΜ - 1 p M . For the assay variation that employed oxidation of pesticides containing P=S groups, the inhibitors (25 μι) were first incubated with 5 μι of 0.001% B r solution for 20 min followed by the addition of ethanol to a final concentration of 5% prior to the incubation with AChE. 2

Assay Procedure for Samples Surface runoff samples collected from four agricultural fields were extracted and concentrated by E P A method 35IOC. Solvent extracts were stored at 4°C prior to analysis by gas chromatography and


292 use in the assay. Sample extracts were analyzed on a Hewlett-Packard 6890 Series capillary gas chromatograph equipped with a flame photometric detector following U S E P A Method 1657. Prior to use in the assay, samples were solvent exchanged into hexane, taken to dryness under a stream of N then dissolved in the same volume of DI water. Dilutions of the samples were then analyzed using the bromine oxidation variation of the A C h E inhibition assay. 2

Data Reduction and Analysis For inhibition profiles, the means of triplicate data points (at individual inhibitor concentrations) were plotted. I C values were determined using either a log-logit or four parameter fit. (Correlation coefficients were determined from the best fit of these data.) Error bars representing standard deviations (SD) are only presented in selected inhibition profiles. Paraoxon equivalence (%), PE, was determined using the following relationship:


50

PE = (IC paraoxon/IC compared compound) χ 100 50

50

Results and Discussion Enzyme Stability Drawbacks for the use of enzyme-based assays in environmental monitoring include storage requirements (e.g., typically below 4°C) and limited shelf life. We report that the use of trehalose, glucose, gelatin, sodium chloride and sodium azide (TGG) for dried A C h E preparations dramatically stabilizes activity of this enzyme (Table I). Table I. Stabilization of Acetylcholinesterase Storage Media

Storage Temp

Storage Time

Activity

DI Water DI Water PBS PBS T G G (soin) T G G (dried) T G G (dried) T G G (dried) T G G (dried)

25°C 25°C 25°C 25°C 25°C 25°C 25°C 25°C 60°C

10 min 3hr 10 min 3hr 3 days 15days 60 days 365 days 10 min

100 0 100 0 75 100 100 100 100

(%)



293 The enzyme activity rapidly degraded in water or PBS solution. Although T G G allowed the maintenance of activity in solution for several days, A C h E dried in this mixture remained active for extended periods of time (i.e., 1 year) at 25° C or for short periods at 60 °C. Although the enzyme immobilization method and stabilization results described here are similar to those previously reported by Nguyen et al. (S), the demonstration of preservation of A C h E activity over extended storage periods at 25 °C and at high temperature are unique to our study. Another hindrance in the use of enzymes in environmental assays is that many insecticides display limited solubility in aqueous buffers. Consequently, we explored the use of various organic solvents with the A C h E assay (Table II). We observed that aqueous systems and nonpolar solvents such as hexane do not effect enzyme activity whereas the use of polar organic solvents such as methanol and ethanol significantly degrade the enzyme activity.

Table II. Effect of Organic Solvents on A C h E Activity

Solvents

Activity (% control) 1

DI Water PBS Hexane Ethanol Methanol 5% Ethanol 5% Methanol TGG 2

1

2

2

100 100 100 12 10 98 100 100

Activity after 20 min incubation Aqueous solution

A 5% aqueous solution of these alcohols, however, could be used to increase the solubility of pesticides without significantly decreasing enzyme activity. The effect of organic solvents on the activity of enzymes such as tyrosinase and A C h E has been well characterized (13, 14). In general, nonpolar organic solvents such as hexane do not inhibit tyrosinase, but polar organic solvents such as alcohols can cause significant inhibition. Further, Mionette et al. (15) observed effects of organic solvents on acetylcholinesterase assays similar to those we report here.



294 Inhibition Profiles Inhibition curves were determined for a number of anticholinesterase compounds over micromolar (μΜ) to picomolar (pM) concentration ranges. Compounds analyzed using this assay included the strongly inhibitory carbamates physostigmine, pyridostigmine and neostigmine (Figure 1) and moderately inhibitory carbamates such as aldicarb, carbofuran, carbaryl and methomyl (Figure 2). The inhibition curves for physostigmine and pyridostigmine showed a typical sigmoidal shape. A C h E activity in the presence of neostigmine, however, did not drop below 20% even at the highest concentrations. The relative order of inhibitory potency was neostigmine > physostigmine > pyridostigmine. The rank order of these inhibitors for A C h E is the same as previously reported (17). Curves for the moderately inhibitory carbamates again showed the characteristic sigmoidal shape with the following inhibitory potencies carbofuran ~ carbaryl > aldicarb > methomyl (Figure 2). Again, the inhibition profiles were similar in shape and position (except for methomyl) to previous reports using colorimetric or biosensor (16) assays. Inhibition profiles were determined for phosphorothioate OP insecticides such as parathion, malathion, and diazinon (Figure 3). Because these compounds were only weakly inhibitory, the measured concentration range extended from 0.1 n M to 100 μΜ. The relative order of potency was malathion > diazinon > parathion. The commercially available oxidative transformation products of parathion and malathion (i.e., paraoxon and malaoxon) as well as dichlorvos, were also measured using this assay (Figure 4). The oxidative transformation products were significantly more potent A C h E inhibitors than the parent compounds and showed inhibitory profiles comparable to dichlorvos. The cholinesterase inhibition assay yielded similar I C values for each of these compounds. Indeed, these compounds are typically reported to have inhibition constants within an order of magnitude of each other (16, 17). 50

I C Values Because of its stability, strong inhibitory effect and commercial availability, paraoxon has often been used as a reference compound for cholinesterase inhibition (7). As a result of the widespread use of paraoxon as a reference inhibitor, we elected to compare the relative potency of compounds assayed to this inhibitor in the form of paraoxon equivalence. I C values and % inhibition relative to paraoxon (paraoxon equivalence) were determined for a wide range of cholinesterase inhibitors (Table III). The extent to which the data fit a four parameter curve fit or log-logit fit are also included as correlation coefficients. The compounds are placed in ascending order of calculated I C values. A wide range of bioanalytical assays based on cholinesterase inhibition have been reported over the past decade. I C values (molar concentration yielding 10% inhibition of the control activity and typically considered as the detection limit) that have been reported for paraoxon using A C h E -based inhibition assays vary over the 5 0

50

50

10


295

100

ULI

Ο < 15 - • - Control Physostigmine - A — Pyridostigmine - • - Neostigmine


5

ίο-

3

10'

10

2

10°

1

10

10

1

10

2

3

Conœntration (nM)

Figure I. Inhibition profiles for strongly inhibitory carbamates. Data points are means, η = 3.

100

LU

Ο < Ο

60

£

40

i

10*

- Control - Aldicarb - Carbofuran - Carbaryl - Methomyl

10'

2

10*

1

10°

10

1

10

2

10

3

Concentration (nM)

Figure 2. Inhibition profiles for moderately inhibitory carbamates. Data points are means, η = 3.



296

1

10'

10°

1

10

2

10

3

10

4

10

s

10

Concentration(nM)

Figure 3. Inhibition profiles for phosphorothioate insecticides. Data points are means, n- 3.

Concentration (nM)

Figure 4. Inhibition profiles for oxon-containing organophosphorus insecticides. Data points are means, η = 3.


297 following range: 90 n M (20), 10 n M (16, 21), 0.3 n M (22), and 0.1 n M (11). The I C value for paraoxon (0.15 nM) reported here is similar to the lowest I C values reported using biosensors (7), flow injection analysis (23), or soluble enzyme assays (16). The rank order for these compounds is similar but not identical to those previously reported in the literature. For example, chlorpyrifos-oxon has been reported to be a more potent inhibitor of cholinesterase than paraoxon (18,19) and methomyl has been reported to be a more potent inhibitor than aldicarb (16). A number of factors may account for differences that we observe. The magnitude of difference between paraoxon and chlorpyrifos-oxon I C values has been shown to be dependent on both the species and tissue from which the cholinesterase is obtained. In addition, assay factors such as contact time (for the enzyme and inhibitor) as well as the removal of excess inhibitor prior to the addition of substrate are critical factors which differ among reported studies. Nevertheless, taken in context of the screening application we propose for this assay, differences in the rank order for various insecticides between our assay and various reports would likely not be a critical issue. 50

10


50

Oxidation of Phosphorothioates The oxidative transformation products of many organophosphorus insecticides are significantly more potent inhibitors of A C h E than their parent compounds. In order to make this assay more sensitive with respect to the potential use for screening environmental samples, the phosphorothioate OP insecticides (P=S) were converted to their oxon (P=0) derivatives. We found pretreatment of phosphorothioates by exposure to bromine resulted in relatively rapid and efficient conversion to the oxonate and did not inhibit enzyme activity. More specifically, exposure of samples to 0.001% B r followed by 5% ethanol (to inactivate the bromine) significantly increased the inhibitory potential of parent phosphorothioate OP compounds with no measurable damage to enzyme activity. Inhibition profiles for chlorpyrifos (parent compound), chlorpyrifos-oxon (commercial standard) and bromine oxidized chlorpyrifos are shown in Figure 5. Although bromine oxidation of the parent compound did not appear to be complete, it facilitated lowering the I C value for chlorpyrifos into the n M range. The bromine oxidation step for chlorpyrifos yielded an I C value approximately eight times higher than for the commercially available chlorpyrifos-oxon, but 270 times lower than for the parent compound. The inhibition curves for chlorpyrifosoxon and bromine oxidized chlorpyrifos display the typical sigmoidal shape above 0.1 n M . Below this concentration, however, they appear biphasic in that A C h E is inhibited (to some extent) even at low inhibitor concentrations. Standard deviation error bars were included in this figure to better clarify the anomalies of these curves. 2

50

50


298 The inhibition profiles for dimethoate (parent compound), dimethoate-oxon and the bromine oxidized parent compound are shown in Figure 6. In this case, the inhibition profiles for the commercially available dimethoate-oxon and bromine oxidized parent compound were similar, suggesting that the bromine oxidation was nearly complete. It is interesting to note that dimethoate-oxon did not show the sigmoidal shape characteristic of most compounds but rather showed an almost loglinear profile. Table III. Inhibitory Characteristics of Selected Anticholinesterases


Compound

IC (nM) 50

Paraoxon Equivalence

Correlation Coefficien

(%) Naled Malaoxon Paraoxon Neostigmine Dichlorvos Physostigmine Chlorpyrifos-oxon Pyridostigmine Carbaryl Carbofuran Aldicarb Methomyl Dimethoate-oxon Malathion Parathion Diazinon Fenthion Chlorpyrifos Trichlorfon Terbufos Phosmet Azinphos-methyl Diclofenthion Dimethoate

0.019 0.093 0.15 0.25 0.26 1.8 3.2 3.9 8,7 9.7 19.2 162 455 458 689 760 6,240 6,740 7,780 7,860 8,120 9,230 9,890 11,900

789 161 100 60 58 8 4.7 4 1.7 1.5 0.8 0.1 0.02 0.03 0.02 0.02 ND ND ND ND ND ND ND ND

0.800 0.989 0.999 0.999 0.965 0.998 0.995 0.999 0.993 0.968 0.991 0.974 0.978 0.981 0.873 0.969 0.999 0.998 0.999 0.986 0.987 0.979 0.816 0.969



299

3

2

10"

1

10"

10"

10°

1

2

10

3

10

10

Concentration (nM)

Figure 5. Inhibition profiles for chlorpyrifos and its oxidative transformation products. Data points are means, η = 3.

Ar

0 10" "I

3

Dimethoate (Bromine Oxidized) «>

tl 2

10"

",l 1

10'

10°

»»l 1

10

2

10

3

10

Concentration (nM)

Figure 6. Inhibition profiles for dimethoate and its oxidative transformation products. Data points are means, η = 3.


300 I C Values for Oxidized O P Compounds Table IV shows I C values and paraoxon equivalence values for selected phosphorothioate OP insecticides that were assayed with and without prior bromine oxidation. In addition, where commercially available, the oxon derivatives are also compared. The bromine oxidation protocol significantly increased the sensitivity of this assay to many of these insecticides. In about half of the cases the I C values were decreased between 20 and 300 times, however, several of the compounds showed only 5 0

50

50

Table I V . Oxidation and Assay of Selected Phosphorothioate Insecticides


Compound

Parathion Parathion* Paraoxon Chlorpyrifos Chlorpyrifos* Chlorpyrifos-oxon Malathion Malathion* Malaoxon Dimethoate Dimethoate* Dimethoate-oxon Diazinon Diazinon* Terbufos Terbufos* Fenthion Fenthion* Trichlorfon Trichlorfon* Phosmet Phosmet* Azinphos-methyl Azinphos-methyl* Diclofenthion Diclofenthion*

ic

50

Paraoxon Equivalence

(nM)

(%)

689 11.9 0.15 6,740 25.1 3.2 458 22.1 0.093 11,900 685 455 760 140 7,860 2,286 6,240 2,300 7,780 119 8,120 8,120 9,230 1,120 9,890 1,890

0.02 1.3 100 ND 0.6 5 0.03 0.7 161 ND 0.02 0.02 0.02 0.10 ND ND ND ND ND ND ND ND ND ND ND ND

* Parent compound assayed using the bromine oxidation protocol


301 moderate or no change due to oxidation (e.g., fenthion, phosmet). In the cases of parathion, chlorpyrifos, malathion and dimethoate, the use of commercially available oxon derivatives allowed the estimation of tne relative efficiency of the bromine oxidation step. These results show that differences in the inhibitory behavior for phosphorothioate insecticides and their oxon derivatives vary over a broad range. More specifically, the I C for malathion and its oxidative transformation product differ by almost 5000 times, whereas in the case of dimethoate, there is only a 20 fold increase in sensitivity between the parent compound and oxon derivative. Consequently, these results indicate that the efficiency of the oxidation was not the sole factor influencing the magnitude of the observed increase in sensitivity for the bromine oxidation step. The kinetics and products of bromine oxidation of phosphorothioate insecticides warrants further evaluation.


50

Mixtures of O P Compounds In many cases of environmental contamination with OP pesticides where a rapid screening assay would be of value, samples may be expected to contain multiple pesticides. These compounds may be present as parent compounds as well as various oxidative transformation products. Data from Table IV suggest that for mixtures of OP and carbamate insecticides, the most potent A C h E inhibitors would dominate the results of the inhibition assay. In an ideal circumstance, the inhibition curves for the compounds of interest would be identically shaped and placed along the abscissa (log concentration scale) at various positions depending on their relative I C values. Although the inhibition profiles for different compounds are often similar in shape, we have observed that a number of these curves do not show characteristic sigmoidal shape or similar slopes at their I C concentrations. Consequently, we investigated the inhibition profile for a mixture of five commonly used insecticides. A mixture of equimolar concentrations of chlorpyrifos, diazinon, dichlorvos, malathion and parathion (which sum to the plotted concentrations) was analyzed by this assay using the bromine oxidation protocol. Shown in Figure 7 are the inhibition profiles for paraoxon and the previously described mixture. Over a limited concentration range (i.e., 0.1 n M and above), the inhibition profile of the mixture is similar in shape to the paraoxon curve with paraoxon showing greater inhibition than the mixture. A t concentrations below 0.1 n M , the curve deviates from its typical shape and the mixture appears to inhibit to a greater extent than paraoxon. This feature in the inhibition profile (i.e., the plateau in the activity at about 80% of maximum at low compound concentrations) was also observed with chlorpyrifos-oxon (see Figure 5). 50

50

Environmental Samples Dichloromethane extracts from agricultural runoff samples which were contaminated with mixtures of up to six OP insecticides were



302 analyzed using the assay. Surface runoff water was extracted and contaminants concentrated using E P A Method 3510C. After solvent exchange into hexane, extracts were evaporated, dissolved in DI water, and inhibition profiles obtained using the bromine oxidation protocol after serial dilution (Figure 8). Sample 111 ON was the most potent inhibitor, yielding 50% inhibition at a 300-fold dilution followed by sample 1030N at a 20-fold dilution. Samples 930N and onion field behaved nearly the same as each other, yielding 50% inhibition at a 3-fold dilution and sample 945N showed 50% inhibition when undiluted. Analysis of the dichloromethane extracts by GC-FPD yielded the concentrations of chlorpyrifos, diazinon, dichlorvos, dimethoate, malathion and parathion (methyl) shown in Table V . Each of the samples were contaminated with at least two compounds and two of the samples (11 ION and onion field) were contaminated with five compounds. One might anticipate that the most potent inhibitors would contribute the majority of the observed response. In the case of these samples the rank order for I C values taken from Tables III and IV for the bromine oxidized compounds is dichlorvos > parathion (ethyl) > malathion > chlorpyrifos > diazinon > dimethoate. Viewed from this perspective, for samples 11 ΙΟΝ, 1030N and onion field, the dichlorvos concentrations would be expected to determine the observed inhibition. In the case of sample 11 ION the observed assay response underestimated the dichlorvos concentration by a factor of five. In the cases of samples 103 ON and onion field, the assay underestimated the inhibition expected from dichlorvos by a factor of 100. For sample 930N, chlorpyrifos would be expected to dominate the inhibition of A C h E . The observed assay response, in this case, underestimated the contribution from chlorpyrifos by a factor of three. For sample 945N, again chlorpyrifos would be expected to determine the inhibition and, in this case, the assay underestimated the response from chlorpyrifos present in the sample by a factor of five. In summary, the A C h E assay reported here underestimated (to various extents based on inhibition profiles in laboratory buffers) the concentrations of OP compounds present in environmental samples that were expected to result in A C h E inhibition. Factors that may have contributed to this result include incomplete recovery of the compounds (into the aqueous phase) from the solvent extract, the association of OP insecticides with other organic molecules in the extracts preventing inhibition, or the "bromine demand" exerted by other compounds in the sample matrix reducing oxidation efficiency. The role of sample matrix effects on assay response requires further evaluation. Nevertheless, as a screening tool the A C h E inhibition assay (rapidly and inexpensively) identified anticholinesterase activity in all of the extracts from contaminated environmental runoff samples. 50



303

Concentration (nM)

Figure 7. Inhibition profiles for paraoxon and organophosphorus insecticide mixture. Data points are means, η = 3

100

η

| III... • . ,. E

10

I.I S

10

I

I....... . . . I 4

10

, I

I I 3

10

III.!. I t 2

10

.

I.UI .,1 l,„i 1

10

1

10°

Sample Dilution

Figure 8. Inhibition profiles for dilutions of environmental samples contaminated with anticholinesterase compounds. Data points are means, η = 3.


304 Table V . G C - F P D Analysis of Runoff Water Extracts Sample Chlorpyrifos Diazinon Dichlorvos Dimethoate Malathion Parathion (nM) (nM) (nM) (nM) (nM) (nM)


O.Field 930N 11 ION 1030N 0945N

142 262 185 0 137

605 434 430 0 0

443 0 416 489 0

0.86 2.70 3.20 0.56 0

224 0 303 206 0

0 0 0 0 24

Conclusions This assay is not intended to yield the identities or exact concentrations of the carbamate and OP insecticides present, but rather the relative anticholinesterase activity. More specifically, this screening assay is intended to flag samples which, from a potential risk perspective, warrant further examination for specific carbamate and organophosphorus insecticide contamination. In this respect, the assay performed exceptionally well using standards in laboratory buffers. Although the assay underestimated the inhibition expected from laboratory standards, the AChE-based assay detected anticholinesterase activity present in all of the environmental samples. Due to its high sensitivity and simplicity in the detection of carbamate and organophosphorus insecticides such as those commonly used in agricultural and residential settings, this assay is a promising candidate for further development as a laboratory or field assay for screening of environmental samples related to human exposure assessment. Notice: The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded this research and approved this manuscript for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation of these products by the E P A . N N M is currently a National Research Council Fellow. Environmental samples were collected and analyzed under a cooperative agreement with I.H. (Mel) Suffet at the University of California Los Angeles.


305

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4.

5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

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