Tolclofos-methyl Summary of Analytical Chemistry and Residue Data 

Page 1 of 18

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND

POLLUTION PREVENTION 

MEMORANDUM

DATE: February 29, 2012

SUBJECT: Tolclofos-methyl: Non-food Use For Seed Treatment on Various 

Crops

PC Code: 128905 DP Barcodes: D395999 and D397276

Decision No.: 443921 Registration No.: NA

Petition No.: NA Regulatory Action: NA

Risk Assessment Type: Single chemical Case No.: NA

TXR No.: NA CAS No.: 57018-04-9

MRID No.: 48341956, 48627101 and 

48341957

40 CFR: NA

REVIEWER:Sheila Piper, Chemist

Risk Assessment Branch VI

Health Effects Division (HED, 7509P)

THROUGH: Felecia Fort, Chief

Risk Assessment Branch VI

Health Effects Division (HED, 7509P)

TO: Lisa Jones/Mary Waller, Risk Management Team 21

Fungicide Branch

Registration Division

Risk Management Division

Executive Summary

Tolclofos-methyl is a seed treatment agent to control a number of soil-borne fungi 

including Rhizoctonia solani in cotton and other crops. In this study, the radiolabeled test 

substance, [phenyl14C]tolclofos-methyl, was applied to soybean, corn, lettuce, cucumber, 

radish, and cotton seeds. The study was conducted to determine if the seed treatment use 

of tolclofos-methyl could be designated for non-food.
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 2 of 18

The registrant, Valent U.S.A. Corporation met with EPA on October 2, 2008 to discuss 

tolclofos-methyl radiotracer studies which were conducted to support the non-food 

classification and the required toxicology data requirements which were based on 

Valent’s review of existing data and their interpretation of the 40 CFR Part 158 

requirements for nonfood uses. Subsequently, HED’s Chemistry Science Advisory

Council (ChemSAC) met on December 3, 2008 to discuss Valent’s proposal for non-food 

use classification of the seed treatment uses of tolclofos-methyl on various crops and crop 

groups. ChemSAC concluded if all TRR results are less than 5 ppb (based on 

memorandum of Chemistry SAC-HED -7509C titled “Classification of Seed Treatments 

as Food or Nonfood Uses” dated Oct 28, 1999), then the proposed seed treatment uses of 

tolclofos-methyl on various crops can be classified as non-food.

To determine the total radioactive residue (TRR), aliquots of samples were combusted in 

a biological oxidizer followed by liquid scintillation counting (LSC) of the aliquot vials. 

HPLC was used to determine the radiochemical purity of the test substance before and 

after seed treatment. The limit of quantification (LOQ) is 0.003 ppm. The method was 

adequate for data collection based on acceptable concurrent method recoveries.

Freezer storage stability data for soybean, corn, cotton, cucumber, lettuce and radish are 

adequate. The dates that the samples were planted, collected and analyzed were less than 

30 days; therefore, a freezer storage stability study is not needed. The freezer storage 

period before the extraction analysis of treated radish tops was 13.7 months. 

The submitted TRR data on various crops are adequate to satisfy data requirements and 

supports the seed treatment uses being classified as non-food uses. The results showed 

that the soybean, corn, lettuce, cucumber, radish and cotton plants grown for seed treated 

with [phenyl14C]tolclofos-methyl at 10 or 50 g.ai/100kg did not take up radiolabeled test 

substance residue ( < 5 ppb). The radish tops showed residue levels above 5 ppb at the 

rate of 50 g/100 kg. Only the radish top samples grown from seeds showed TRR residues 

above 0.005 ppm. The radish tops grown from seeds contained residues at 0.02 – 0.03 

ppm. Since the radish tops grown for seeds showed residues above 0.005 ppm, additional 

characterization of the residues was provided. Two treated and one control crop samples 

were extracted and analyzed. The treated samples were radish tops (TRR = 0.029 ppm 

and TRR = 0.020 ppm) from replicate plots TR1 and TR2, respectively. The control 

radish tops grown from untreated seeds were spiked with radiolabeled tolclofos-methyl 

and analyzed to determine the efficiency of the extraction method. The radish tops grown 

for seeds were extracted and analyzed by HPLC. Both acetonitrile and aqueous extracts 

showed some polar radioactive residues, but did not contain any detectable amount of 

tolclofos-methyl.

There are currently no established or proposed tolerances for tolclofos-methyl in the 

United States as the proposed seed treatment uses have been deemed non-food uses. 

There are currently no established MRLs in Canada; however, there are Codex MRLs for 

lettuce, potato, and radish. Since there are no U.S. tolerances for tolclofos-methyl, there 

are no issues of compatibility with respect to the Codex MRLs.
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 3 of 18

Regulatory Recommendations and Residue Chemistry Deficiencies

HED has examined the residue chemistry database for tolclofos-methyl. There are no 

residue chemistry issues that would preclude granting the tolclofos-methyl seed treatment

as non-food uses (40 CFR Part 158) and no food or feed tolerances are required. Table 1 

shows the proposed seed treatment use of tolclofos-methyl as a non-food use on corn, 

soybean, cucumber, lettuce and radish can be translated to the following crop groups. The 

chemical structure, nomenclature and pohysicochemical properties of tolclofos-methyl 

are presented in Table 2. 

Table 1: Translation of Tolclofos-methyl Seed Treatment as a Nonfood Uses

Translation of Tolclofos-methyl Seed Treatment as a Nonfood Uses

Crop Crop Group Number Crop Group Name

Corn* 15 Cereal grains

16 Forage, fodder and straw 

Soybean* 6 Legume vegetable

7 Foliage 

20 Oilseeds

Cucumber* 8 Fruiting vegetable (except 

cucurbits)

9 Cucurbit

Lettuce* 4 Leafy vegetable (except Brassica 

vegetables)

5 Brassica cole leafy vegetables

Radish** 1 Root and tuber vegetables (except 

potatoes)

2 Leaves of root and tuber vegetables 

(human food and animal feed)

3 Bulb vegetables

Cotton**

*application rate: 10 g ai/100 kg seed

**application rate: 50 g ai/100 kg seed
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 4 of 18

Table 2: Nomenclature and Physicochemical Properties of Radiolabeled Tolclofosmethyl

Parameter Structure/Name/Value

Compound Structure

Common Name [Phenyl14C]tolclofos-methyl (Radiolabeled)

Code Names V-10178

IUPAC Name O-2,6-dichloro-p-tolyl O,O-dimethyl phosphorothioate

CAS Name O-(2,6-dichloro-4-methylphenyl) O,O-dimethyl 

phosphorothioate

CAS Registry No. 57018-04-9

Molecular Formula C9H11Cl2O3PS

Molecular Weight 305.0 (Radiolabeled, Inv No. 398)

Lot No.

CFQ15407 (Valent Radiochemical Inv. No. 398) 

The test substance received from Sumitomo Chemical 

Co. was purified and isotopically diluted with tolclofosmethyl T.G. to prepare Inventory No. 398A for seed 

treatment

Specific Activity 4.51 GBq/mmol or 887,200 dpm/μg (Inv. No. 398)

1.23 MBq/mg or 73,500 dpm/μg (Inv. No. 398A)

Radiochemical Purity 98.6% (390A)

Position of Radiolabel Phenyl ring

n-Octanol/Water Partition 

Coefficient, log POW

4.56 at 25°C

Water Solubility 1.1 ppm

Vapor Pressure 57 mPa at 20°C

O

Cl

Cl

H3C

P

S

OCH3

OCH3

*
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 5 of 18

Residue Analytical Method

Purification of Radiolabeled Test Substance

 

To determine the total radioactive residue (TRR), aliquots of samples were combusted in 

a biological oxidizer followed by liquid scintillation counting (LSC) of the aliquot vials.

The radiolabeled test substance received from Sumitomo Chemical Co. in solid form, was 

dissolved with acetonitrile for LSC and HPLC analysis. The test substance was purified 

as described in the following procedure.

Field Corn, Cotton and Soybean

Batches of field corn, cotton and soybean seeds (one batch for each of the 3 dose levels 

and one batch for the untreated control per crop) were coated with treated with the 

appropriate amount of test substance, [phenyl14C]tolclofos-methyl. The target treatment 

rates were 2.5, 5.0, and 10.0g/ 100kg of seeds for the field corn and soybean, and 10, 25, 

50g/100 kg of seeds for cotton. The actual mean treatment rates were 2.37+0.24, 

5.01+0.06, and 10.19+0.15 g of the test substance per 100kg of field corn seeds, 

10.32+0.21, 22.39+0.63, and 51.20+1.14g/100 kg of cotton seeds, and 2.82+0.09, 

5.76+0.30, 10.33+0.04g/100kg of soybean seeds (See summary Table 3a).

The radiochemical stock solution was rotary evaporated to complete dryness (water bath 

temp at ~ 30°C). The flask was kept under vacuum for an additional 30 min to allow 

traces of toluene to evaporate from the sample. The test substance was dissolved in 0.5 

mL of acetonitrile, and then 0.2 mL of water was added to the solution to lower the 

acetonitrile concentration to 71%. The solution was divided equally into 3 portions and 

each portion was loaded onto a strata-X 1g/12 mL Giga Tube column prepared earlier by

passing 1 bed-volume each of methanol and acetonitrile, followed by 4 bed-volumes of 

water. The flask containing the test substance was subsequently rinsed with 0.4 mL of 

50% acetonitrile and 0.5 mL of 40% acetonitrile and the rinses were loaded onto the 

columns. The columns were eluted subsequently with step gradients of 20 – 71% 

acetonitrile in water. The collected column fractions were analyzed by LSC and HPLC 

to locate the test substance band. 

Radish, Lettuce and Cucumber

Batches of radish, lettuce and cucumber seeds were treated with appropriate amounts of 

the test substance, [phenyl14C]tolclofos-methyl, at the target rate of 50 and 200 g/100kg 

of seeds for radish, and 10 g/100 kg of seeds for lettuce and cucumber. The actual mean 

treatment rates were 51.6+ 0.3 and 194+ 7.6 g of the test substance per 100 kg of radish 

seeds, and 9.91+ 0.39 and 10.23+1.08 g/100 kg of lettuce and cucumber seeds, 

respectively. The actual rates were 97.0-103.2% of the target rates (see summary Table 

3b)
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 6 of 18

The radiochemical stock solution was concentrated to 1.0 mL under a flow of nitrogen at 

room temperature. The solution was loaded onto a strata-X 33 μm 1g/12 mL column 

prepared earlier by passing 2 column-volumes of acetonitrile, followed by 4 columnvolumes of water. After the test solution entered the column, two rinses of the test 

substance vial with 0.5 mL of acetonitrile was loaded onto the column. The columns were 

eluted with step gradients of 0 – 73% acetonitrile in water. The collected column 

fractions were analyzed by LSC and HPLC to locate the test substance band. 

Table 3a: Total [Phenyl14C]Tolclofos-Methyl Applied to Seeds and the Treatment 

Rates

Crop

Type

Dose

Level

Target

Rate

g/100 kg

Total

Applied

dpm

Final

Treatment

dpm

Actual

Treatment

Rate

g/100 kg

CV 

%

% of

Target

Rate

Field Corn

Low 2.5 3,179,562 3,130,422 2.37 10.2 95.0

Medium 5.0 6,950,225 6,603,275 5.01 1.2 100.2

High 10.0 14,204,616 13,514,016 10.19 1.5 101.9

Cotton

Low 10.0 4,723,724 4,707,495 10.32 2.1 103.2

Medium 25.0 11,255,999 10,246,959 22.39 2.8 89.5

High 50.0 24,121,406 23,153,086 51.20 2.2 102.4

Soybean

Low 2.5 1,644,133 1,627,878 2.82 3.2 112.8

Medium 5.0 3,305,600 3,284,245 5.76 5.3 115.3

High 10.0 6,139,779 5,919,522 10.33 0.4 103.3

Table 3b: Total [Phenyl14C]Tolclofos-Methyl Applied to Seeds and the Treatment Rates 

Crop

Type

Target

Rate

g/100 kg

Analyzed

Subsample

No.

Applied

dpm/g of 

seeds

Mean

Applied

dpm/g of 

seeds

Applied

Treatment

Rate

g/100 kg

CV 

%

% of

Target

Rate

Radish

50

1 38,176,246

2 37,668,537 37,926,566 51.6 0.7 103.2

3 37,934,915

200

1 142,696,560

2 136,965,195 142,616,028 194.0 3.9 97.0

3 148,186,329

Lettuce 10

1 7,054,295

2 7,180,130 7,280,646 9.91 4.0 99.1

3 7,607,512

Cucumber 10 1 8,010,039 7,516,276 10.23 10.6 102.3
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 7 of 18

2 7,939,973

3 6,598,815

[cv=coefficient of variation; dpm=disintegrations per minute]

HED Conclusions

An adequate data collection method is available for tolclofos-methyl residues in/on crops, 

liquid scintillation counting (LSC) and HPLC. Batches of seeds were treated with 

appropriate amounts of the test substance, [phenyl14C]tolclofos-methyl. The target 

treatment rates were 2.5, 5.0 and 10.0 g/100 kg of seeds for the field corn, cotton and 

soybean, and 10, 50 and 200 g/100 kg of seeds for radish, lettuce, and cucumber. The 

percentage of target rate ranged from 89.5- 115.3.

Storage Stability

Crop samples were collected at their proper growth stage. The treated batches of seeds 

were placed in labeled 30 or 50 mL glass vials fitted with caps and shipped at ambient 

temperature to Research for Hire via commercial carriers on a next-day delivery

schedule. All RAC samples were shipped frozen on Dry Ice from Research for Hire by 

commercial carriers on overnight delivery schedules to Valent Technical Center in 

Dublin CA. The samples were analyzed within 30 days with the exception of soybean 

forage (stored for 41 days) (Table 4). Additionally, samples of radish tops were stored 

for up to 13.7 months before extraction (Table 5).

Table 4: Harvest and TRR Analysis Dates of Radish, Cucumber and Lettuce Samples

Sample 

Description

Date 

Harvested

Date 

Shipped

Date 

Processed

Date of 

TRR 

analysis

Storage 

period (days) 

before TRR 

analysis

Radish 07/10/09 07/13/09 07/20/09 07/28/09 18

Cucumber 08/10/09 08/12/09 8/18/09 08/31/09 21

Lettuce 07/22/09 08/04/09 8/12/09 08/19/09 28

Soybean forage 07/30/07 07/31/07 09/04/07 09/10/07 41

Soybean seed 

and hay 09/21/07 09/25/07 10/04/07 10/11/07 16
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 8 of 18

Field corn 

forage 08/17/07 08/21/07 08/28/07 09/07/07 17

Field corn grain 

and stover 09/14/07 09/17/07 09/21/07 09/25-26/07 8-9

Cotton seed 10/18/07 10/23/07 11/01/07 11/12/07 20

Cotton gin trash 10/18/07 10/23/07 11/06/07 11/14/07 22

Table 5: Harvest and TRR Analysis Dates of Radish Top Samples

Sample Description Date Harvested and 

Storage Started

Date Extracted 

for Analysis

Storage period 

(days) before 

extraction analysis

Treated Radish 

Tops 07/10/09 08/24/10 410 (13.5 months)

Spiked Control 

Radish Tops 07/10/09 08/30/10 416 (13.7 months)

To determine the stability of radish tops under frozen storage, the registrant also 

submitted a freezer storage stability study for tolclofos-methyl in/on lettuce (MRID 

201000183, 3/14/02). The study examined the stability of tolclofos-methyl fortified at 

0.1 mg/kg in lettuce stored frozen at <-18oC for 0, 3, 6, 12 and 18 months. The overall 

percent recovery of the method for lettuce freshly fortified with 0.1 mg/kg of tolclofosmethyl ranged from 83-102%. Lettuce were extracted with acetone: water (2:1 v:v). For 

liquid-liquid partition ethyl acetate/ cyclohexane and sodium chloride was added and 

after repeatedly mixing excess water was separated. The residue containing fraction is 

concentrated and analyzed for residues of tolclofos-methyl by gas chromatography using 

a flame photometric detector (FPD, phosphorus mode). The LOQ was 0.01 ppm.

HED Conclusions

The available storage stability data for soybean, corn, cotton, cucumber, lettuce, and 

radish is adequate. The dates that the samples were planted, collected and analyzed were 

less than 30 days except for soybean forage which was stored for 41 days. The freezer 

storage period before the extraction analysis of treated radish tops was 13.7 months. The 

petitioner submitted the results of storage stability for lettuce that showed tolclofosmethyl was stable for up to 18 months.
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 9 of 18

Radiolabeled Field Trials

Field corn, cotton and soybean seeds were planted in outdoor field plots (Greenville, MS) 

following normal agricultural practices. Each plot was approximately 30 × 30 inches and 

had one row of plants grown from treated seeds of one dose level at 3 inches apart from 

each other. The boundary of each plot was surrounded by a plastic edge liner buried in 

soil with a minimum of about 6 inches below ground. Five seeds from each dose level 

were planted per plot, and 3 plants of normal crop density were selected for sampling to 

provide triplicate samples for each RAC. 

Radish, lettuce and cucumber seeds were also planted in outdoor field plots (Stockton, 

CA) following the normal agricultural practices. Each plot was approximately 2 ft × 2 ft, 

in which seeds were planted at appropriate crop density for the respective plant. The 

boundary of each plot was surrounded by a plastic-lined wooden frame, which was buried 

in soil about 6 inches below ground. Duplicate plots were planted with treated seeds 

from each dose level and approximately 30 seeds were planted per plot for radish, 20 

seeds per plot for lettuce and 10 seeds per plot for cucumber. A total of 5 plots (1 for 

untreated control, 2 for low dose rate and 2 for high dose rate) were used for the radish 

crop, and 3 plots (1 control and 2 treated) were planted with each of the lettuce and 

cucumber crops. 

Treatment of Field Corn Seed

Field corn forage, stover and grain were harvested from the untreated control plot and 

three treatment plots which were planted with seeds treated with 14C-tolclofos methyl at 

the nominal dose rates of 2.5, 5.0, and 10.0 g/100 kg of seeds. 

Approximately 0.2 mg, 0.3 mg and 0.6 mg of Rizolex 50FL Formulation were transferred 

to 3 separate 25 mL flasks marked for low, medium and high dose, respectively. After 

adding 43 μL of water to each flask, the formulation blank and water were mixed by

sonication. The batches of 15 field corn seeds were then transferred to the respective 

flask and swirled gently and briefly to coat them evenly with the formulation blank. The 

seeds were then transferred onto labeled weighing-trays and allowed to dry at room 

temperature. Each of the 15 seeds of the low dose batch was individually spiked with 1.0 

μL of the test substance stock solution 390A and 1.0 μL of the dilution solution 390AD1. 

Each of the 15 seeds of the medium dose batch was individually spiked with 3.0 μL of 

390A only. Finally, each of the 15 seeds of the high dose batch was individually spiked 

with 6.0 μL of 390A and 1.0 μL of the dilution solution 390AD3. Identical triplicate 

aliquots of the stock and dilution solution used in each batch as the dose were transferred 

to empty vials, diluted to 5.0 mL with acetonitrile, and subaliquots of these dilution 

solutions were counted by LSC for dose verification. Three treated seeds from each 

batch were rinsed individually with 5.0 mL of acetonitrile. The radioactivity applied to 

each seed was verified by LSC analysis of the rinse and combustion analysis of the rinsed 
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 10 of 18

seed. The remaining 12 treated seeds were allowed to dry at room temperature, then 

placed individually in labeled glass vials and shipped to Research for Hire for planting. 

All of the corn forage, stover, and grain samples showed TRR levels below the LOQ 

(0.0003 ppm), and the majority of the corn samples had radioactivity level below the 

limit of detection (0.0001 ppm). The total radioactive residues in field corn are presented 

in Table 6. 

Treatment of Cotton Seeds

Cotton seed and gin trash were harvested from the untreated control plot and three 

treatment plots which were planted with seeds treated with 14C-tolclofos methyl at the 

nominal dose rates of 10, 25, and 50 g/100 kg of seeds. 

Approximately 0.2 mg, 0.5 mg and 1.1 mg of Rizolex 50FL formulation blank were 

transferred to 3 separate 25 mL capacity flasks marked for low, medium and high dose, 

respectively. After adding 28 μL of water to each flask, the formulation blank and water 

were mixed by sonication. The batches of 15 cotton seeds were then transferred to the 

respective flask and swirled gently and briefly to coat them evenly with the formulation 

blank. The seeds were then transferred onto labeled weighing-trays and allowed to dry at 

room temperature. Each of the 15 seeds of the low dose batch was individually spiked 

with 2.0 μL of the test substance stock solution 390A and 1.0 μL of the dilution solution 

390AD2. Each of the 15 seeds of the medium dose batch was individually spiked with 

5.0 μL of 390A and 1.0 μL of 390AD3. Finally, each of the 15 seeds of the high dose 

batch was individually spiked with 10.0 μL of 390A and 1.0 μL of 390AD4. Identical 

triplicate aliquots of the stock and dilution solution used in each batch as the dose were 

transferred to empty vials, diluted to 5.0 mL with acetonitrile, and subaliquots of these 

dilution solutions were counted by LSC for dose verification. Three treated seeds from 

each batch were rinsed individually with 5.0 mL of acetonitrile. The radioactivity applied 

to each seed was verified by LSC analysis of the rinse and combustion analysis of the 

rinsed seed. The remaining 12 treated seeds were allowed to dry at room temperature, 

then placed individually in labeled glass vials and shipped to Research for Hire for 

planting. 

All of the cotton seed meal, seed hull and gin trash samples (except one replicate of high 

dose gin trash sample) showed TRR levels below the LOQ (0.00035 ppm), and the 

majority of the cotton samples had radioactivity level below the limit of detection (0.0001 

ppm). The total radioactive residues in cotton are presented in Table 7. 

Treatment of Soybean Seeds

Soybean forage, hay and seed were harvested from the untreated control plot and three 

treatment plots planted with seeds treated with 14C-tolclofos methyl at the nominal dose 

rates 2.5, 5.0, and 10 g/100 kg of seeds.

Approximately 0.1 mg and 0.3 mg of Rizolex 50FL formulation blank were transferred to 

3 separate 25 mL capacity flasks marked for low, medium and high dose, respectively. 
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 11 of 18

After adding 10 μL of water to each flask, the formulation blank and water were mixed 

by sonication. The batches of 15 soybean seeds were then transferred to the respective 

flask and swirled gently and briefly to coat them evenly with the formulation blank. The 

seeds were then transferred onto labeled weighing-trays and allowed to dry at room 

temperature. Each of the 15 seeds of the low dose batch was individually spiked with 2.0 

μL of the dilution solution 390AD5. Each of the 15 seeds of the medium dose batch was 

individually spiked with 1.0 μL of the test substance stock solution 390A and 1.0 μL of 

390AD5. Finally, each of the 15 seeds of the high dose batch was individually spiked 

with 2.0 μL of 390A and 2.0 μL of 390AD5. Identical triplicate aliquots of the stock and 

dilution solution used in each batch as the dose were transferred to empty vials, diluted to 

5.0 mL with acetonitrile, and subaliquots of these dilution solutions were counted by LSC 

for dose verification. Three treated seeds from each batch were rinsed individually with 

5.0 mL of acetonitrile. The radioactivity applied to each seed was verified by LSC 

analysis of the rinse and combustion analysis of the rinsed seed. The remaining 12 treated 

seeds were allowed to dry at room temperature, then placed individually in labeled glass 

vials and shipped to Research for Hire for planting.

Three additional batches of soybean seeds (10 in each batch) were treated for replanting 

after some soybean seeds from the first treatment failed to germinate (the germination 

rate of soybean seeds was generally low in all dosing levels including the untreated 

control). The repeat treatment procedure was similar to the one described above, except 

that the treated seeds were not analyzed in addition to the analysis of the dosing aliquots.

All of the soybean forage and seed samples showed TRR level below the LOQ (0.00034 

ppm), and the majority of them had radioactivity level below the limit of detection 

(0.0001 ppm). The low and high dose soybean hay samples showed mean TRR levels of 

0.00049ppm and 0.00067, respectively. The total radioactive residues in soybean are 

presented in Table 8. 

Treatment of Radish Seed

Two batches (5.0 g each) of radish seeds were weighed into separate containers. Into a 30 

mL capacity glass vial, 216 μL of the test substance solution in acetonitrile, 398A, was 

transferred. The solution was evaporated to dryness under nitrogen at room temperature, 

and the residue was redissolved in 50 μL of acetonitrile. An aliquot of 3.59 μL of the 

formulation blank and 77 μL of water were added to the radiolabeled solution in the vial. 

The first 5.0 g batch of the radish seeds was transferred to the vial. The seeds were 

mixed with the slurry by gentle shaking until no liquid was visible in the vial. The seeds 

were poured out of the vial and discarded. New slurry was prepared in the emptied vial 

as described above, using 216 μL of 398A (concentrated to 50 μL), 3.59 μL of 

formulation blank, and 77 μL of water. The slurry was mixed well using the vortex 

mixer, and spread around the inner wall of the vial. The second 5.0 g batch of the radish 

seeds was transferred to the vial. The seeds were mixed with the slurry by gentle shaking 

until no liquid was visible in the vial. Three subsamples (0.25 g each) of these treated 

seeds were withdrawn from the vial for analysis. Seeds in each subsample were rinsed 

with 2 lots of acetonitrile (5 mL each), and the pooled seed rinse (total volume, 10 mL) 
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 12 of 18

was analyzed by LSC. The rinsed seeds were air-dried at room temperature before 

combustion analysis. A dose rate of 200 g/100kg of radish was also analyzed.

Only the radish root samples grown from seeds treated at the rate of 51.6 g/100 kg 

showed TRR level below the LOQ. All other radish samples showed residues above 

0.005 ppm. The radish tops grown from seeds treated at the rates of 51.6 g/100 kg and 

194.0 g/100 kg contained 0.02 - 0.03 ppm and 0.04 - 0.06 ppm, respectively. The radish root 

from the higher dose rateshowed residue levels of ~0.03 ppm. The total radioactive residues 

in radish are presented in Table 9. 

Treatment of Lettuce Seeds

Two batches (5.0 g each) of lettuce seeds were weighed into separate containers. Into a 

30 mL capacity glass vial, 0.72 μL of the formulation blank, 43 μL of the test substance 

solution in acetonitrile, 398A, and 87 μL of water were transferred. The slurry was 

mixed well using a vortex mixer, and spread around the inner wall of the vial. The first 

5.0 g batch of the lettuce seeds was transferred to the vial and the seeds were mixed with 

the slurry by gentle shaking until no liquid was visible in the vial. The seeds were poured 

out of the vial and discarded. New slurry was prepared in the emptied vial as described 

above, using 0.72 μL of formulation blank, 43 μL of 398A, and 87 μL of water. The 

slurry was mixed well using the vortex mixer, and spread around the inner wall of the 

vial. The second 5.0 g batch of the lettuce seeds was transferred into the vial and the 

seeds were mixed with the slurry by gentle shaking until no liquid was visible in the vial. 

Three subsamples (ca 0.25 g each) of these treated seeds (designated as LT) were 

withdrawn from the vial for analysis. Seeds in each subsample were rinsed with 2 lots of 

acetonitrile (5 mL each), and the pooled seed rinse (total volume, 10 mL) was analyzed 

by LSC. The rinsed seeds were air-dried at room temperature before combustion 

analysis.

The lettuce samples grown from seeds treated at the rate of 9.91 g/100 kg showed TRR level 

below the minimum quantification limit. The control lettuce samples also showed no 

radioactivity above background. The total radioactive residues in lettuce are presented in 

Table 9. 

Treatment of Cucumber Seeds

Two batches (10 g each) of cucumber seeds were weighed into separate containers. Into a 

50 mL capacity glass vial, 1.43 μL of the formulation blank, 86 μL of the test substance 

solution in acetonitrile, 398A, and 173 μL of water were transferred. The slurry was 

mixed well using a vortex mixer, and spread around the inner wall of the vial. The first 

10 g batch of the cucumber seeds was transferred to the vial and the seeds were mixed 

with the slurry by gentle shaking until no liquid was visible in the vial. The seeds were 

poured out of the vial and discarded. New slurry was prepared in the emptied vial as 

described above, using 1.43 μL of formulation blank, 86 μL of 398A, and 173 μL of 

water. The slurry was mixed well using the vortex mixer, and spread around the inner 

wall of the vial. The second 10 g batch of the cucumber seeds was transferred into the 

vial and the seeds were mixed with the slurry by gentle shaking until no liquid was 
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 13 of 18

visible in the vial. Three subsamples (ca 0.25 g each) of these treated seeds (designated 

as CT) were withdrawn from the vial for analysis. Seeds in each subsample were rinsed 

with 2 lots of acetonitrile (5 mL each), and the pooled seed rinse (total volume, 10 mL) 

was analyzed by LSC. The rinsed seeds were air-dried at room temperature before 

combustion analysis. The total radioactive residues in field corn are presented in Table 6.

The cucumber samples grown from seeds treated at the rate of 10.23 g/100 kg showed TRR 

level below the minimum quantification limit. The control cucumber samples also showed no 

radioactivity above background. The total radioactive residues in lettuce are presented in 

Table 9. 

Table 6: Total Radioactive Residue in Field Corn RAC Samples

Sample

Description

 

Dose level

[g/100 kg]

Sample

No.

Rep.

No.

Mean 

Aliquot wt., 

g

Mean

Aliquot

dpm

TRR

Mean

dpm/g

ppm

μg/g

Field Corn Forage

Control

[0.0]

62 1 0.266 11 <126 <0.00030

63 2 0.262 5 <35 <0.00008

64 3 0.274 4 <34 <0.00008

Low

[2.5]

65 1 0.278 10 <121 <0.00028

66 2 0.264 4 <35 <0.00008

67 3 0.258 4 <36 <0.00008

Medium

[5.0]

68 1 0.261 17 <129 <0.00030

69 2 0.262 3 <35 <0.00008

70 3 0.270 8 <34 <0.00008

High

[10.0]

71 1 0.260 12 <129 <0.00030

72 2 0.266 3 <35 <0.00008

73 3 0.265 5 <35 <0.00008

Field Corn Stover

Control

[0.0]

74 1 0.283 10 <116 <0.00027

75 2 0.259 4 <35 <0.00008

76 3 0.251 1 <36 <0.00008

Low

[2.5]

77 1 0.256 11 <129 <0.00030

78 2 0.247 3 <37 <0.00008

79 3 0.258 5 <35 <0.00008

Medium

[5.0]

80 1 0.260 12 <127 <0.00030

81 2 0.262 4 <34 <0.00008

82 3 0.249 5 <36 <0.00009

High

[10.0]

83 1 0.253 11 <130 <0.00030

84 2 0.259 6 <35 <0.00008

85 3 0.267 7 <34 <0.00008

Field Corn Grain

Control

[0.0]

86 1 0.286 10 <119 <0.00008

87 2 0.295 5 <32 <0.00027

88 3 0.284 2 <33 <0.00007

Low

[2.5]

89 1 0.283 13 <120 <0.00028

90 2 0.276 6 <34 <0.00007

91 3 0.289 6 <32 <0.00008

Medium 92 1 0.293 21 <116 <0.00027
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 14 of 18

[5.0] 93 2 0.279 25 <122 <0.00028

94 3 0.280 21 <121 <0.00028

High

[10.0]

95 1 0.292 5 <32 <0.00007

96 2 0.284 3 <33 <0.00008

97 3 0.300 3 <31 <0.00007

Table 7: Total Radioactive Residue in Cotton RAC Samples

Sample

Description

 

Dose level

[g/100 kg]

Sample

No.

Rep.

No.

Mean 

Aliquot wt., 

g

Mean

Aliquot

dpm

TRR

Mean

dpm/g

ppm

μg/g

Cotton

Seed Meal

Control

[0.0]

38P 1 0.248 2 <41 <0.00009

39P 2 0.255 5 <39 <0.00009

40P 3 0.247 3 <41 <0.00009

Low

[10.0]

41P 1 0.254 1 <40 <0.00009

42P 2 0.264 3 <38 <0.00009

43P 3 0.246 0 <41 <0.00010

Medium

[25.0]

44P 1 0.254 8 <40 <0.00009

45P 2 0.248 2 <48 <0.00009

46P 3 0.264 1 <38 <0.00009

High

[50.0]

47P 1 0.259 7 <39 <0.00009

48P 2 0.251 4 <40 <0.00009

49P 3 0.260 5 <39 <0.00009

Cotton

Seed Hull

Control

[0.0]

38H 1 0.257 2 <36 <0.00008

39H 2 0.247 2 <38 <0.00009

40H 3 0.243 19 <139 <0.00033

Low

[10.0]

41H 1 0.257 4 <36 <0.00008

42H 2 0.248 3 <38 <0.00009

43H 3 0.249 2 <37 <0.00009

Medium

[25.0]

44H 1 0.235 7 <40 <0.00009

45H 2 0.248 4 <38 <0.00009

46H 3 0.244 3 <38 <0.00009

High

[50.0]

47H 1 0.255 7 <37 <0.00009

48H 2 0.257 6 <36 <0.00008

49H 3 0.253 5 <37 <0.00009

Cotton

Gin Trash

Control

[0.0]

50 1 0.246 1 <40 <0.00009

51 2 0.244 0 <41 <0.00010

52 3 0.255 1 <39 <0.00009

Low

[10.0]

53 1 0.263 8 <38 <0.00009

54 2 0.263 2 <38 <0.00009

55 3 0.255 4 <39 <0.00009

Medium

[25.0]

56 1 0.259 13 <138 <0.00032

57 2 0.267 8 <37 <0.00009

58 3 0.251 2 <40 <0.00009

High

[50.0]

59 1 0.239 25 <150 <0.00035

60 2 0.251 21 <142 <0.00033

61 3 0.248 37 151 0.00035
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 15 of 18

Table 8: Total Radioactive Residue in Soybean RAC Samples

Sample

Description

 

Dose level

[g/100 kg]

Sample

No.

Rep.

No.

Mean 

Aliquot wt., 

g

Mean

Aliquot

dpm

TRR

Mean

dpm/g

ppm

μg/g

Soybean Forage

Control

[0.0]

2 1 0.266 13 <129 <0.00030

3 2 0.254 1 <37 <0.00009

4 3 0.277 1 <34 <0.00008

Low

[2.5]

5 1 0.268 12 <127 <0.00030

6 2 0.268 5 <35 <0.00008

7 3 0.263 2 <36 <0.00008

Medium

[5.0]

8 1 0.268 14 <127 <0.00030

9 2 0.269 7 <35 <0.00008

10 3 0.266 4 <35 <0.00008

High

[10.0]

11 1 0.249 15 <137 <0.00032

12 2 0.249 5 <38 <0.00009

13 3 0.265 7 <36 <0.00008

Soybean

Hay

Control

[0.0]

14 1 0.222 15 <151 <0.00035

15 2 0.209 12 <161 <0.00038

16 3 0.195 13 <172 <0.00040

Low

[2.5]

17 1 0.221 43 194 0.00045

18 2 0.206 50 200 0.00047

19 3 0.212 42 162 <0.00038

Medium

[5.0]

20 1 0.209 32 <160 <0.00038

21 2 0.200 27 <168 <0.00039

22 3 0.209 23 <160 <0.00038

High

[10.0]

23 1 0.209 40 188 0.00044

24 2 0.198 35 176 0.00041

25 3 0.194 97 497 0.00116

Soybean

Seed

Control

[0.0]

26 1 0.255 5 <39 <0.00009

27 2 0.263 0 <38 <0.00009

28 3 0.261 0 <38 <0.00009

Low

[2.5]

29 1 0.260 7 <38 <0.00009

30 2 0.247 2 <40 <0.00009

31 3 0.254 2 <39 <0.00009

Medium

[5.0]

32 1 0.244 7 <41 <0.00010

33 2 0.252 3 <39 <0.00009

34 3 0.249 3 <40 <0.00009

High

[10.0]

35 1 0.258 11 <138 <0.00032

36 2 0.258 10 <139 <0.00032

37 3 0.249 13 <143 <0.00034

Table 9: Total Radioactive Residue Levels in Radish, Lettuce and Cucumber Samples 

Grown from Seeds Treated with [Phenyl14C]Tolclofos-Methyl 
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 16 of 18

Sample

Description

 

Dose Rate

g/100 kg

Plot

ID

Mean 

Aliquot wt., 

g

Mean

Aliquot

dpm

CV%

TRR

Mean

dpm/g

ppm

μg/g

Radish Tops

0.0 CR1 0.2387 1 nm <245 <0.003

51.6

TR1 0.2384 508 34.7a2,133 0.029

TR2 0.2415 344 36.6b1,435 0.020

194.0

TR3 0.2447 1,140 8.9 4,655 0.063

TR4 0.2439 682 7.3 2,796 0.038

Radish Roots

0.0 CR1 0.2427 0 nm <241 <0.003

51.6

TR1 0.2362 61 nm <250 <0.003

TR2 0.2401 32 nm <243 <0.003

194.0

TR3 0.2407 563 17.1b2,343 0.032

TR4 0.2401 560 11.5b2,331 0.032

Lettuce

0.0 CL1 0.2375 0 nm <226 <0.003

9.9

TL2 0.2406 2 nm <223 <0.003

TL3 0.2442 0 nm <220 <0.003

Cucumber

0.0 CF1 0.2542 0 nm <211 <0.003

10.2

TF1 0.2555 0 nm <210 <0.003

TF2 0.2535 0 nm <212 <0.003

[a: mean of 15 aliquots; b: mean of 9 aliquots, all others: mean of 3 – 6 aliquots]

Characterization of Radish Seeds

Since the radish tops grown for seeds treated at the rate of 51.6 g/100 kg, showed 

residues above 0.005 ppm, additional characterization was provided. Two treated and 

one control crop samples were extracted and analyzed. The treated samples were radish 

tops (TRR = 0.029 ppm and TRR = 0.020 ppm) from replicate plots TR1 and TR2, 

respectively. The control radish tops grown from untreated seeds were spiked with 

radiolabeled tolclofos-methyl and analyzed to determine the efficiency of the extraction 

method.

To investigate the nature of these low level residues further and to quantify the test 

substance, tolclofos-methyl was extracted with aqueous acetonitrile, which was separated 

into acetonitrile and aqueous extracts. The replicate samples showed similar total 

radioactive residue (TRR) distribution in the extracts and in the post-extraction solids 

(PES). Approximately 81 and 80% of TRR (0.023 and 0.016 ppm) was extractable from 

samples TR1 and TR2, respectively, while about 19 and 20% (0.005 and 0.004 ppm) 

remained bound to PES. The major portion of the extractable residue (60% TRR, 0.017 

ppm in TR1, and 56% of TRR, 0.011 ppm in TR2) was in the aqueous extract. 

Approximately 22% of TRR (0.006 ppm) in TR1 and 24% (0.005 ppm) in TR2 remained 

soluble in the acetonitrile extract. The extraction TRR levels in TR1 and TR2, 
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 17 of 18

respectively, were 96 and 102% of the levels determined by combustion analysis in the 

original study. Extraction of approximately 100 g of untreated control radish tops spiked 

with [14C]tolclofos-methyl under similar conditions showed that the entire spiked 

radioactivity was extractable (97% TRR in acetonitrile extract, and 3% in aqueous 

extract).

The acetonitrile extracts were purified using strataX column chromatography, and finally

analyzed by HPLC. Both acetonitrile and aqueous extracts of treated radish tops 

samples, TR1 and TR2, showed multiple components of polar low level radioactive 

residues, but did not contain any detectable amount of tolclofos-methyl. Acetonitrile 

extract of control radish tops spiked with [14C]tolclofos-methyl, which was subjected to 

identical purification procedure, showed a single peak of [14C]tolclofos-methyl in the 

HPLC analysis. The extraction and HPLC results are presented in Table 10.

Table 10: Extraction and HPLC Results for Tolclofos-methyl in Radish Tops 

Extract/Residue Fraction

Radish Tops Sample

Spiked Control Treated TR1 Treated TR2

% TRR ppm % TRR ppm % TRR ppm

Acetonitrile Extract

Tolclofos-methyl 97.3 0.027 ND ND ND ND

Unknowns (multiple components) ND ND 21.7 0.006 23.8 0.005

Aqueous Extract

Unknowns (multiple components) 2.7 <0.001 59.5 0.017 56.2 0.011

Total extractable 100.0 0.027 81.2 0.023 80.0 0.016

Total bound in PES 0.0 0.0 18.8 0.005 20.0 0.004

Total radioactive residue 100.0 0.027 100.0 0.028 100.0 0.020

[ND= not detected]

HED Conclusions

Batches of radish, lettuce, cucumber, corn, soybean, and radish seeds were treated with 

formulated [phenyl14C]tolclofos-methyl. The target treatment rates were 50 g/100 kg of 

seeds for cotton and radish, and 10 g/100 kg of seeds for corn, soybean, lettuce and 

cucumber. The results showed that the seeds treated with [phenyl14C]tolclofos-methyl 

did not take up the radiolabeled test substance residue (<5 ppb). Only the radish top 

samples grown from seeds treated at the rate of 51.6 g/100 kg showed TRR level residues 

above 0.005 ppm. The radish tops grown from seeds contained residues at 0.02 – 0.03 

ppm. Since the radish tops grown for seeds showed residues above 0.005 ppm, additional 
Tolclofos-methyl Summary of Analytical Chemistry and Residue Data D395999

Page 18 of 18

characterization of the residues was provided. Two treated and one control crop samples 

were extracted and analyzed. The treated samples were radish tops (TRR = 0.029 ppm 

and TRR = 0.020 ppm) from replicate plots TR1 and TR2, respectively. The control 

radish tops grown from untreated seeds were spiked with radiolabeled tolclofos-methyl 

and analyzed to determine the efficiency of the extraction method. The radish tops grown 

for seeds were extracted and analyzed by HPLC. Both acetonitrile and aqueous extracts 

showed some polar radioactive residues, but did not contain any detectable amount of 

tolclofos-methyl.

REFERENCES

DP#: 362358

Subject: Tolclofos-methyl: Non-food Use for Seed Treatment

From: S. Piper

To: C. Giles-Parker 

Date: 02/28/09

MRIDs: NA