QualiPlate™ Kit for Cholinesterase - EnviroLogix

Highlights: 

• Assay range from 0.2 to 6 

parts per billion 

• Results in under two hours 

Contents of Kit: 

• 1 uncoated microwell plate, 

labeled Cholinesterase - test 

plate 

• Cholinesterase - oxidizing 

agent concentrate (Reagent 1) 

• Cholinesterase - oxidizing 

agent diluent (Reagent 2) 

• 1 small bottle, labeled 

Cholinesterase - microwell 

solution (Reagent A) 

• Cholinesterase - neutralizing 

agent (Reagent C) 

• Cholinesterase - enzyme 

(Reagent D) 

• Cholinesterase - substrate 

(Reagent E) 

• Cholinesterase - chromogen 

(Reagent F) 

• Cholinesterase - stopping 

solution (Reagent G) 

• Negative Control - (Reagent H)* 

• Pesticide control I - (Low 

Level) (Reagent I)* 

• Pesticide control II - (High 

Level) (Reagent J)* 

* These Reagents are replaced 

with a single Pesticide Stock 

Solution in EP 014 DF kits 

There are no washing steps 

required in the 

Cholinesterase Screening Test 

Rev. 12-12-07 

Catalog Number EP 014 and EP 014 DF 

QualiPlate Kit for 

Cholinesterase 

Intended Use 

The EnviroLogix Cholinesterase Screening Test (EP 014) is intended as a 

screening test for the detection of organophosphate or carbamate residues in a 

variety of matrices. The test kit can be used in the laboratory without extensive 

training. 

Each application for the use of the EnviroLogix Cholinesterase Screening Test is 

different. For new customer convenience, and for those ordering the EP 014 DF 

Kit for use in dried fruit, an Application Guide for Detection of Organophosphate 

and Carbamate Residues in Dried Vine and Tree Fruit has been attached as an 

Appendix. For new users, it is meant as an example of one extraction method. 

Specific extraction methods will have to be optimized for each application. The 

levels of pesticides in the pesticide standards will also be application and 

pesticide specific. Please contact EnviroLogix for any information we may have 

on applications other than the detection of organophosphates and carbamate 

residues on dried vine and tree fruit. 

Test Principles 

The Cholinesterase Screening Test is based on the use of an enzyme isolated 

from insect head. This enzyme is sensitively inhibited by a wide range of 

organophosphate and carbamate insecticides. Many "organophosphates" are 

actually phosphorothioate "pro-insecticides" and require oxidation to the 

insecticidally active "oxon" form for optimal detection sensitivity. In the absence 

of appropriate pesticide in the test sample, action of cholinesterase on its 

acetylthiocholine substrate releases thiocholine which reacts with Ellman's 

reagent (5,5'- dithio-bis-(2-nitrobenzoate), DTNB) to form a bright yellow 

product. Inhibition of cholinesterase by pesticide in the sample leads to a 

decrease in color development. 

Action of cholinesterase Acetylthiocholine 

⇓ Inhibited by pesticide 

Thiocholine + acetate 

⇓ + DTNB 

Yellow product forms 

1. The sample extract or pesticide standard is added to a microwell, followed 

by oxidizing reagent. 

2. After 5 minutes, excess oxidizing agent is inactivated using the neutralizing 

reagent. 

3. Cholinesterase enzyme is added, and mixed 30 minutes at room temperature. 

4. Substrate and chromogen are then added and incubated 15 minutes. In the 

presence of active cholinesterase, color developer turns yellow. 

5. Color development is stopped by addition of cholinesterase stopping agent. 

Since the same concentration of cholinesterase is added to every microwell, a 

sample which contains no organophosphate or carbamate pesticide enables the 

cholinesterase to act on the added enzyme substrate present in the color

Items Not Provided 

• distilled water (or equivalent) 

for preparation of calibrators 

• application sheet pertaining 

to sample type 

• microplate reader 

• microwell plate mixer 

• test tube for diluting 

oxidizing agent 

• automatic pipettes (1-20, 

20-200, 200-1000, 

1000-5000 µL) 

• 12-channel pipette 

(20-200 µL) 

• marking pens for labeling 

• timer or wristwatch 

a Since extraction methods very often 

involve a dilution of sample into 

extraction solvent, this dilution must 

be taken into account when 

determining pesticide levels in the 

sample. To calculate the 

concentration of pesticide on the 

sample, multiply the concentration of 

pesticide in the sample extract by any 

dilution factor incurred during 

extraction. The matrix used for these 

values was 100% methanol. 

b Water samples containing 

organophosphates must be collected 

in the presence of a minimum of 10% 

methanol. 

Rev. 12-12-07 

QualiPlate Kit for Cholinesterase 

Page 2 of 6 

developer. Therefore, in the absence of organophosphate or carbamate a dark 

yellow color forms. Significant levels of the pesticides will inhibit the action of 

the enzyme, resulting in a paler yellow sample. 

As with all competitive immunoassays, sample concentration is inversely 

proportional to color development. 

Darker color = Lower concentration Lighter color = Higher concentration 

Performance Characteristics 

The Cholinesterase Screening Test is sensitive to a wide range of 

organophosphate and carbamate pesticides; other insecticides, fungicides for 

powdery or downy mildew or botrytis control, and herbicides are not detected. It 

is important to recognize that different compounds are detected with differing 

sensitivities (refer to Table I). Thus, unless the particular pesticide used or 

suspected to be used on the sample being tested is known, it is important to 

confirm positive results using instrumental (gas chromatography) analyses to 

enable identification of the residue present. Where it has been established that 

only a single organophosphate or carbamate is present, the test can be used in 

conjunction with appropriate standards for semi-quantitative testing. 

Of the major insecticides used or potentially used, the test detects chlorpyrifos, 

methidathion and diazinon with high sensitivity, carbaryl with moderate 

sensitivity, and carbofuran residues with lower sensitivity. If samples are being 

screened for carbamates only (carbaryl, carbofuran), the oxidizing and 

neutralizing agent steps can be deleted. Under these conditions, organo(thio)phosphates 

are not detected. 

Limit of Detection 

Limits of detection (15 % inhibition of color development, after oxidizing step) 

when organophosphate or carbamate residues are in sample extract or water as 

indicated are listed below. See Appendix or specialized product application 

guide for detection limits in specific samples. 

Sensitivity 

in Sample 

Extract 

(ppb) a 

Sensitivity 

in water 

(ppb) b 

Sensitivity 

in Sample 

Extract 

(ppb) a 

Sensitivity 

in water 

(ppb) b 

Organophosphates 

acephate >100,000 methacrifos 500 

azinphos-ethyl 5 methamidophos 500 

azinphos-methyl 50 10 methidathion 50 

chlorfenvinphos 100 mevinphos 50 

chlorpyrifos 0.5 0.5 parathion 500 500 

chlorpyr-methyl 0.5 parathion-methyl 500 

coumaphos 5 phorate 500 

diazinon 10 5 phosmet 50 

dichlorvos 5 pirimiphos-ethyl 50 100 

dimethoate 50,000 pirimiphos-methyl 50 

ethion 1000 propetamphos >1000 

fenitrothion 200 quinalphos 5 

malathion 100 100 triazophos 0.5 

Carbamates 

carbaryl 1000 100 carbofuran 100 100

Rev. 12-12-07 

Preparation of Solutions 


Page 3 of 6 

Sample extraction and preparation of standards in sample matrix 

An application sheet specific for the sample type to be tested must be obtained 

from EnviroLogix. For new customer convenience, the Application Sheet for 

Detection of Organophosphate and Carbamate Residues in Dried Vine and Tree 

Fruit has been attached as an Appendix. Contact Technical Services for your 

customized application sheet. 

Dilution of Oxidizing agent concentrate (1 % Br2 in 1 M NaBr) 

(Caution, this reagent is caustic, handle with care) 

On the day of use, for each plate, dilute 0.3 mL of Oxidizing agent (Reagent 1) in 

2.7 mL oxidizing agent diluent (Reagent 2). Label the diluted oxidizing agent 

Reagent B. Discard unused diluted oxidizing agent as it is only stable for 24 

hours. If less than one plate will be run in one day, dilute only enough oxidizing 

agent for the number of strips to be used in one day. The volume required per 

strip is 0.2 mL (dilute 20 μL in 180 μL) or the required multiple of this dilution 

depending on the number of strips to be used. 

Chromogen solution (Reagent F) 

This reagent may have a yellow precipitate. Just prior to pipetting this reagent, 

mix the vial by swirling or inverting to resuspend the precipitate. Re-mix while 

pipetting as necessary. 

Note: Water used for reagents and effect of ambient temperature 

The quality of the water and significant variation in laboratory temperature can 

influence the total color produced in the assay. The key factor affecting assay 

color is the concentration of the enzyme reagent. Please consult EnviroLogix 

before altering assay conditions. 

Pesticide Controls 

When an extraction application has not yet been established, three pesticide 

controls are provided with this kit. A Negative Control (Reagent H) that contains 

only methanol with no pesticide, low pesticide control that contains 10 ppb 

diazinon (Reagent I) and a high pesticide control that contains 50 ppb diazinon 

(Reagent J). These controls are provided for new customers who may not have 

negative and positive samples containing known levels of organophosphate or 

carbamate pesticides, but would like to see how the assay works. These controls 

are not intended to be used in the analysis of sample extracts. In order to properly 

analyze sample extracts, pesticide standards, both negative and positive must be 

prepared using negative extracts of the matrix to be tested. See below for more 

information concerning the preparation of sample specific pesticide standards 

and the extraction of samples. 

Preparation of Pesticide Standards 

and Extraction of Samples 

Each application for the use of the EnviroLogix Cholinesterase Screening Test is 

different. For new customer convenience, the Application Sheet for Detection of 

Organophosphate and Carbamate Residues in Dried Vine and Tree Fruit has been 

attached as an Appendix. The inclusion of this application sheet is meant only as 

an example of how pesticides are extracted from fruit and how to prepare sample

NC = Negative Control 

LO = Reagent I, low level control 

HI = Reagent J, high level control 

S = Sample extracts 

Rev. 12-12-07 


Page 4 of 6 

specific pesticide standards. The application sheet explains how to make 

pesticide standards from fruit known to be free of organophosphate and 

carbamate residues using a pesticide stock that can be obtained from 

EnviroLogix. The dried fruit application sheet also describes how to extract 

organophosphate and carbamate residues from fruit. Specific extraction methods 

will have to be optimized for each application. The levels of pesticides in the 

pesticide standards will also be application and pesticide specific. Please contact 

EnviroLogix for any information we may have on applications other than the 

detection of organophosphates and carbamate residues on dried vine and tree 

fruit. 

How to Perform the Test 

NOTE: If microwell plate mixer is not used, gently mix the microwell plate by 

swirling in a circular motion 20-30 seconds after the addition of oxidizing agent 

(Reagent B), enzyme solution (Reagent D) and chromogen (Reagent F) and then 

incubate for designated time on bench top. 

1. Allow all reagents to reach ambient temperature (at least 22°C) prior to 

starting the assay. Cold reagents will result in low OD readings. Remove 

the plate from the plastic bag. Any strips that will not be used should be 

removed from the holder and returned to the plastic bag. (This is best done 

by pressing strip up from the bottom of the holder.) 

2. Add 120 µL of microwell solution (Reagent A) to each well. 

3. Add 20 µL of extract containing pesticide standard or pesticide controls 

(Reagents H, I and J) and sample extracts to each microwell, in duplicate, 

using Figure 1 as a guide. Mix microwell contents by swirling for 2-3 

seconds. 

Figure 1. Example of a typical plate setup: 

1 2 3 4 5 6 7 8 9 10 11 12 

A NC NC S5 S5 S13 S13 S21 S21 S29 S29 S37 S37 

B LO LO S6 S6 S14 S14 S22 S22 S30 S30 S38 S38 

C HI HI S7 S7 S15 S15 S23 S23 S31 S31 S39 S39 

D NC NC S8 S8 S16 S16 S24 S24 S32 S32 S40 S40 

E S1 S1 S9 S9 S17 S17 S25 S25 S33 S33 NC NC 

F S2 S2 S10 S10 S18 S18 S26 S26 S34 S34 LO LO 

G S3 S3 S11 S11 S19 S19 S27 S27 S35 S35 HI HI 

H S4 S4 S12 S12 S20 S20 S28 S28 S36 S36 NC NC 

4. Make sure that you have diluted the orange oxidizing agent concentrate 

(Reagent 1) in oxidizing agent diluent (Reagent 2), immediately before use. 

Immediately add 20 µL of DILUTED oxidizing agent (Reagent B) to each 

microwell. Gently mix by swirling the contents of the plate for 2-3 seconds. 

5. Mix the plates on the microwell plate mixer for 5 minutes, then add 20 µL 

neutralizing reagent (Reagent C) to each microwell. Mix gently. (NOTE: 

If carbamates are the only pesticides being screened the oxidation and 

neutralization step may be eliminated.) 

6. Immediately add 20 µL cholinesterase enzyme solution (Reagent D). Mix 

gently on the microwell plate mixer for 30 min at room temperature. 

7. Add 20 µL of substrate (Reagent E) and 20 µL of chromogen (Reagent F) 

to each microwell. Swirl the plate gently for a few seconds, then mix on the

Precautions and 

Notes 

• Store all QualiPlate Kit 

components at 4°C to 8°C (39°F 

to 46°F) when not in use (except 

for microtiter plate, store this at 

room temperature). 

• Do not store test kit components 

for more than 4 hours at ambient 

temperatures. 

• Do not expose QuantiPlate Kit 

components to temperatures 

greater than 37°C (99°F) or 

less than 2°C (36°F). 

• Allow all reagents to reach 

ambient temperature (18°C to 

27°C or 64°F to 81°F) before 

use. 

• Do not use kit components 

after the expiration date. 

• Do not use reagents or test 

well strips from one QualiPlate 

Kit with reagents or test well 

strips from a different 

QualiPlate Kit. 

• Do not expose Substrate to 

sunlight during pipetting or 

while incubating in the test 

wells. 

• As with all tests, it is 

recommended that results be 

confirmed by an alternate 

method when necessary. 

• The assay has been optimized 

to be used with the protocol 

provided in the kit. Deviation 

from this protocol may 

invalidate the results of the 

test. 

• Observe any applicable 

regulations when disposing of 

samples and kit reagents. 

Rev. 12-12-07 


Page 5 of 6 

microwell plate mixer for 15 minutes. A yellow color develops. 

8. After 15 minutes have elapsed, add 20 µL of stopping solution (Reagent G) 

to each microwell and mix well. This will arrest the yellow color 

development. Read the microwells at 415 nm (ideal) or 405 nm in a 

microplate reader. Read the microwells within 30 minutes of stopping the 

assay. 

Important: At no stage of the procedure are components tipped out of the 

microwells or are the microwells washed. Washing will invalidate the 

procedure. 

Sample Results and Interpretation 

If a yellow color does not develop in the pesticide-free fruit extract microwells 

within 5 minutes and you have added color developer, the test is invalid and must 

be repeated. 

Note that this test does not provide the identity of the pesticide in the sample, but 

rather an indication that the sample contains an organophosphate or carbamate 

pesticide in excess of the "low standards". For identification of the particular 

residues present, subsequent analysis of the sample by gas-liquid 

chromatography (GLC) is required. 

The interpretation of results is specific to the each application. For an example 

of a detailed discussion of how to interpret results, see the Sample results and 

interpretation section of the attached Application Sheet for the detection of 

organophosphate and carbamate residues in dried vine and tree fruit. 

Below is sample data for the pesticide controls (Reagents H, I and J). 

Figure 2. Illustrative calculations 

Sample Absorbance 

% Inhibition 

of Negative 

Control Pesticide Concentration 

Negative 

Control 

1.040 0 

No pesticide present, the highest 

absorbance possible in the assay 

Low 

Control 

0.720 30.8 

Low pesticide standard (10 ppb 

diazinon in standard extract) 

High 

Control 

0.173 83.4 

High pesticide standard (50 ppb 

diazinon in standard extract) 

Actual values may vary; this data is for demonstration purposes only.

For Technical Support 

Contact Us At: 

EnviroLogix 

500 Riverside Industrial 

Parkway 

Portland, ME 04103-1486 

USA 

Tel: (207) 797-0300 

Toll Free: 866-408-4597 

Fax: (207) 797-7533 

e-mail: 

info@envirologix.com 

website: 

www.envirologix.com 

Rev. 12-12-07 

LIMITED WARRANTY 


Page 6 of 6 

EnviroLogix Inc. (“EnviroLogix”) warrants the products sold hereunder (“the Products”) 

against defects in materials and workmanship when used in accordance with the 

applicable instructions for a period not to extend beyond a product’s printed expiration 

date. If the Products do not conform to this Limited Warranty and the customer notifies 

EnviroLogix in writing of such defects during the warranty period, including an offer by 

the customer to return the Products to EnviroLogix for evaluation, EnviroLogix will repair 

or replace, at its option, any product or part thereof that proves defective in materials or 

workmanship within the warranty period. 

ENVIROLOGIX MAKES NO OTHER WARRANTIES, EXPRESSED OR IMPLIED, 

INCLUDING BUT NOT LIMITED TO ANY IMPLIED WARRANTIES OF 

MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. The warranty 

provided herein and the data, specifications and descriptions of EnviroLogix products 

appearing in EnviroLogix published catalogues and product literature are EnviroLogix’ 

sole representations concerning the Products and warranty. No other statements or 

representations, written or oral, by EnviroLogix’ employees, agents or representatives, 

except written statements signed by a duly authorized officer of EnviroLogix Inc., are 

authorized; they should not be relied upon by the customer and are not a part of the 

contract of sale or of this warranty. 

EnviroLogix does not warrant against damages or defects arising in shipping or handling, 

or out of accident or improper or abnormal use of the Products; against defects in products 

or components not manufactured by EnviroLogix, or against damages resulting from such 

non-EnviroLogix made products or components. EnviroLogix passes on to customer the 

warranty it received (if any) from the maker thereof of such non-EnviroLogix made 

products or components. This warranty also does not apply to Products to which changes 

or modifications have been made or attempted by persons other than pursuant to written 

authorization by EnviroLogix. 

THIS WARRANTY IS EXCLUSIVE. The sole and exclusive obligation of EnviroLogix 

shall be to repair or replace the defective Products in the manner and for the period 

provided above. EnviroLogix shall not have any other obligation with respect to the 

Products or any part thereof, whether based on contract, tort, strict liability or otherwise. 

Under no circumstances, whether based on this Limited Warranty or otherwise, shall 

EnviroLogix be liable for incidental, special, or consequential damages. 

This Limited Warranty states the entire obligation of EnviroLogix with respect to the 

Products. If any part of this Limited Warranty is determined to be void or illegal, the 

remainder shall remain in full force and effect. 

EnviroLogix, the EnviroLogix logo, and QualiPlate are trademarks of EnviroLogix Inc. 

Acknowledgments 

Product licensed from CSIRO Plant Industry, Canberra, Australia. Test 

development was partly funded by the Dried Fruits R&D Council and the Grape 

& Wine R&D Corporation of Australia. 

This assay is produced and distributed under an exclusive technology license 

from CSIRO Plant Industry (Australia). 

© EnviroLogix Inc. 2007

~ APPENDIX ~ 

Application Guide 


Application Guide for the Detection of Organophosphate and 

Carbamate Residues in Dried Vine and Tree Fruit 

Intended Use 

This application guide describes the procedure for preparing negative control fruit extracts, fruit extract pesticide standards 

and the extraction of pesticide residues from fruit. Directions for the interpretation of assay results are also included in this 

guide. This Kit varies slightly from the main QualiPlate Kit for Cholinesterase in that a Pesticide Stock Solution is supplied 

instead of Reagents H, I and J. Follow the instructions in the main product insert to perform the assay. 

Before You Start 

You will need several items: 

pesticide free fruit (of the same type to be tested) 

pesticide standard stock (Supplied by EnviroLogix) 

blender for fruit (optional) 

orbital shaker 

balance for weighing fruit 

automatic pipettes (1-20, 20-200) 

marking pens for bottles of fruit extract 

timer or wristwatch 

glass bottles/ conical flasks for extracting fruit 

methanol (analytical reagent grade) for extraction of the pesticide from the fruit 

100 mL and 1 L measuring cylinders 

How to prepare fruit extract pesticide standards and extract the pesticide 

from fruit 

Introduction 

Prior to performing the EnviroLogix Organophosphate Screening Test, fruit extract pesticide standards must be prepared 

and pesticides must be extracted from fruit samples. Fruit extract pesticide standards must be prepared from the same type 

of fruit being tested. 

Pesticide free fruit to be used in the preparation of pesticide fruit standards are extracted using exactly the same method 

(and extraction solvent) as the samples to be tested. You will need to prepare a negative, low and high pesticide standard 

for each fruit type being tested. 

Preparation of pesticide standards and extraction of dried vine fruit 

Where both the organochlorine and the cholinesterase tests are to be run on the same vine fruit sample, extract the vine fruit 

and prepare vine fruit pesticide standards with 100 % methanol. This extracts 70 - 90 % of the organochlorine but only 40- 

60 % of the organophosphate or carbamate from vine fruit (which is still sufficient for screening purposes). If the test is to 

be performed for organophosphates and carbamates only, 80 % methanol should be used for both vine fruit extract pesticide 

standards and extracting organophosphates from vine fruit samples. This extracts over 80% of the target residues from the 

sample. 

Rev. 12-12-07 A-1

APPENDIX Application Guide~Dried Vine & Tree Fruit 

There are two extraction methods that may be used in making the vine fruit extract pesticide standards and extracting 

pesticides from vine fruit samples: an overnight soak method or a faster omnimixer technique. The fruit sample size, 

extraction method and equipment to be used depends on the particular time and performance requirements of the user. 

Preparation of pesticide standards (dried vine fruit) 

The Pesticide Stock Solution containing diazinon (“Diazinon”, “Gesapon”) is supplied with this kit. A low fruit extract 

standard (low level of organophosphate) and a high fruit extract standard (higher level of organophosphate) are prepared 

from the Pesticide Stock Solution. A negative control (negative fruit extract) will also need to be prepared. The fruit 

extract standards and negative control are made in the following manner: 

1. Add 20 grams of pesticide-free vine fruit (grapes, sultanas, currants or raisins) to each of three conical flasks. (If the 

faster omnimixer method of extract preparation is to be used, chop fruit as described in step 6B below.) 

2. To prepare the low level standard, add 20 μL of the pesticide standard stock to the fruit in the first conical flask. This 

will produce a final concentration that represents 0.1 ppm diazinon in fruit. 

3. To prepare the high level standard, add 200 μL of the pesticide standard stock to fruit in a second available flask . This 

will produce a final concentration that represents 1.0 ppm diazinon in fruit. The Australian MRL for diazinon in grapes 

is currently 0.5 ppm. 

4. Add no pesticide standard to the third conical flask; this will be the negative pesticide control. 

5. Add 100 mL of 80% or 100% methanol (see explanation of methanol concentration above) to each conical flask. (To 

prepare 1 L of 80% methanol, fill a 1L measuring cylinder to the 800 mL mark with methanol. Add water to the 1 L 

mark.) 

6. One of two methods may be used to prepare fruit extract pesticide standards: 

A. Overnight soaking of the sultanas is the simplest method. Whole sultanas (20 g) are shaken 30-60 min in 100 mL 

of 80 % or 100 % methanol, swirled by hand for 1 minute, then incubated (no shaking) 18-24 hours (overnight) at 

room temperature. 

B. Rapid blending of sultanas. Sultanas (20 g) are lightly chopped with a sharp knife and blended in 100 mL of 80 % 

or 100 % methanol for 2 minutes using an Omnimixer. 


(and extraction solvent) as the samples to be tested. If covered to prevent evaporation, the extracts of the fruit containing 

pesticide standards are stable for 48 hours at room temperature. These standards should be prepared and analysed at the 

same time as the sample grape, sultana or currant extracts being tested. 

Extracts should not require filtration before use, but just settling. Do not disturb the sediment in the bottom of extraction jar 

when pipetting extract. Note that other solvents such as methylated spirits should not be used as they interfere with the test, 

and produce erroneous results. 

Since action levels vary between markets, results obtained should be interpreted with respect to the target end-market. 

Extraction of pesticides from dried vine fruit samples 

Before they can be analysed in the test, organophosphates and carbamates must be extracted from the sultanas using either 

80 or 100% methanol (see explanation of methanol concentration in introduction section). Use exactly the same method of 

extraction of pesticides from fruit samples as was used to prepare fruit extract pesticide standards. Choose one of the two 

methods listed below. 

1. Overnight soaking of the sultanas is the simplest method. Whole sultanas (20 g) are shaken 30-60 min in 100 mL of 80 

% or 100 % methanol, swirled by hand for 1 minute, then incubated (no shaking) 18-24 hours (overnight) at room 

temperature. 

2. Rapid blending of sultanas. Sultanas (20 g) are lightly chopped with a sharp knife and blended in 100 mL of 80 % or 

100 % methanol for 2 minutes using an Omnimixer. 

Note : Role of fruit type Samples should be compared to zero, low and high-pesticide standards of the same fruit type (e.g. 

Carina currant extracts must be compared against standards prepared in Carina currant extract). 

Rev. 12-12-07 A-2


There are five main groups: 

- dipped sultanas, dipped raisins, - natural sultanas/ raisins, Zante currants, Carina currants 

Carina currants are larger and darker than Zante currants; the color of the methanol extract is darker and a purple color. For 

more accurate results these should be distinguished from one another and separate standards run for each type of fruit. 

Although the sensitivity of the assay is similar in each dried fruit type, the color produced in pesticide-free controls (i.e. 

zero-pesticide standards) can vary slightly but significantly. For example, Carina currants may produce 15 % higher color in 

pesticide-free standards than Zante currants, while sultanas produce even lower color. The percentage inhibition of color 

produced by the low and high pesticide standard is usually slightly lower (5-10 %) with currants and naturals than with 

dipped sultanas. 

Preparation of pesticide standards and extraction of dried tree fruit 

Preparation of pesticide standards (dried tree fruit) 

The Pesticide Stock Solution containing diazinon (“Diazinon”, “Gesapon”) is supplied with this kit. A low fruit extract 

standard (low level of organophosphate) and a high fruit extract standard (higher level of organophosphate) are prepared 

from the Pesticide Stock Solution. A negative control (negative fruit extract) will also need to be prepared. The fruit 

extract standards and negative control are made in the following manner: 

1. Add 40 grams of pesticide-free tree fruit to each of three conical flasks. 

2. To prepare the low level standard, add 40 μL of the pesticide standard stock to the fruit in the first conical flask. This 


3. To prepare the high level standard, add 400 μL of the pesticide standard stock to fruit in a second available flask . This 


4. Add no pesticide standard to the third conical flask; this will be the negative pesticide control. 

5. The dried tree fruit must be swelled with distilled (or equivalent) water by soaking 40 g fruit in 40 mL water for four to 

six hours. 

6. After swelling, add 160 mL of methanol and extract overnight while shaking. 


(and extraction solvent) as the samples to be tested. If covered to prevent evaporation, the extracts of the fruit containing 

pesticide standards are stable for 48 hours at room temperature. These standards should be prepared and analysed at the 

same time as the sample dried tree fruit extracts being tested. 

Extracts should not require filtration before use, but just settling. Do not disturb the sediment in the bottom of extraction jar 

when pipetting extract. Note that other solvents such as methylated spirits should not be used as they interfere with the test, 

and produce erroneous results. 

Since action levels vary between markets, results obtained should be interpreted with respect to the target end-market. 

Extraction of pesticides from dried tree fruit samples Before they can be analysed in the test, organophosphates and 

carbamates must be extracted from the dried tree fruit using the procedure listed in steps 5 and 6 above. 

Sample results and interpretation 

If a yellow color does not develop in the pesticide-free fruit extract microwells within 5 minutes after you have added color 

developer, the test is invalid and must be repeated. 

1. Measure and record the absorbance of each microwell. Average the absorbance of each pair of duplicate wells. 

Compare the means of absorbances obtained with each sample to that of the means of the negative control (pesticidefree 

fruit extract), and to the pesticide standards. 

2. If the Absorbance of the sample is less than the Absorbance of the chosen pesticide standard by at least 0.1 unit, the 

sample contains organophosphate or carbamate greater than or equal to the limits shown in Table I (page 5) for dried 

vine fruit and Table II (page 6) for dried tree fruit in this insert. It is recommended that fruit providing absorbance 

values below that provided by the low pesticide standard be considered as potentially contaminated fruit. 

Rev. 12-12-07 A-3


3. If fruit is to be retested, it is best that it be re-extracted for testing. If this is not possible, and the extract is to be retested 

the next day, it is recommended that the liquid extract be taken off the fruit and stored in a sealed glass container. This 

is especially important with currants, as the extract continues to get darker with time. 

4. Note that this test does not provide the identity of the pesticide in the sample, but rather an indication that the sample 

contains an organophosphate or carbamate pesticide in excess of the "low standards". For identificaton of the particular 

residues present, subsequent analysis of the sample by gas-liquid chromatography (GLC) is required. 

Sample Data 

Sample Absorbance Interpretation 

Control 1.06 zero pesticide-in-fruit standard - mean of 4 controls 

Low standard 0.67 low pesticide standard (0.1 ppm diazinon-in-fruit) 

High standard 0.38 high pesticide standard (1 ppm diazinon-in-fruit) 

S1 0.97 Since the difference between the sample absorbance and the control 

is less that 0.1, this is considered to be a negative test. 

Residues are within acceptable limits. 

S2 0.60 Concentration is more than the low pesticide standard - positive test. 

Notes • The “action” inhibition level must be determined by the industry. The low calibrator contains 0.1 ppm diazinon, 

which is 20 % of the Australian MRL for that compound. 

Table I: Limits of detection (15 % inhibition of color development, after oxidizing step) in grapes and sultanas of 

organophosphates and carbamate insecticides used in Australia. 

Compound Sensitivity: limit of detection in grapes and sultanas (ppm) a 

Aust. MRL Grapes Sultanas Currants 

Major compounds 


Chlorpyrifos (Lorsban) 0.01 b 0.0002 0.001 0.002 

Diazinon (Basudin) 0.5 0.05 0.05 0.05 

Methidathion (Supracide) 0.5 0.1 0.1 0.2 

Carbamates 

Carbaryl (Bugmaster, Sevin) 5 0.5 0.5 0.5 

Methomyl (Lannate, Nudrin, Marlin) 2 1 1 1.5 

Minor use compounds 

Azinphos-methyl 2 

Dimethoate 2 1.5 1 1 

Fenitrothion (Folithion, Sumithion) 0.5 0.2 0.5 0.5 

Maldison (Malathion, Hymal) 8 0.1 0.1 0.2 

Methiocarb (Baysol, Mesurol) 0.1 

Parathion 0.5 0.1 0.1 0.2 

Parathion-methyl 1 0.5 0.5 

Promecarb (Carbamult) 0.2 0.02 0.1 

Prothiofos (Tokuthion) 2 

Trichlofon 0.1 

a This assumes extraction of over 80 % of the target residues from the sample, using 80 % methanol. If 100 % methanol is 

used as the extractant, the sensitivity is approximately halved. 

b Recently increased for grapes and sultanas 

Rev. 12-12-07 A-4


Table II: Limits of detection (20 % inhibition of color development, after oxidizing step) in dried tree fruit of 

organophosphates and carbamate insecticides used in Australia. 

Compound Sensitivity: limit of detection in dried tree fruit (ppm) 

Major compounds 

Peaches Prunes Apricot Pears 


Diazinon (Basudin) 0.04 0.06 0.1 0.02 

Carbamates 

Carbaryl (Bugmaster, Sevin) 0.2 0.5 1.0 0.5 

Rev. 12-12-07 A-5

QualiPlate™ Kit for Cholinesterase - EnviroLogix

Create successful ePaper yourself

Delete template?

Save as template?