Testing Overview, Mixing Herbicides

Introduction to Bioassay 

Testing and Mixing 

Herbicide Solutions 

Michael Lovelace, Ph.D. 

Agronomist, USDA, AMS 

Seed Regulatory and 

Testing Branch 

Gastonia, NC 

http://www.ams.usda.gov/seed 

Agricultural Marketing Service 

Marketing and Regulatory Programs

FSA Requirements for 

Labeling Biotech- 

Derived Seed 

• No Federal Requirements 

• Section 201.8 of FSA Regulations 

– The label may contain information in 

addition to that required by the Act, 

provided such information is not 

misleading. 



Biotech-Derived Varieties 

• After release, treated the 

same as traditional varieties 

under the labeling covered by 

the FSA. 

• FDA – non-toxic, non-allergenic 

• EPA – no threat to the 

environment 

• APHIS – completed the prerelease 

program 



Common Methods for 

Detecting the Presence 

of Biotech-Derived Seed 

• DNA 

• Protein (Immunoassay) 

• Bioassay testing 



What is Bioassay Testing? 

• Bioassay: appraisal of the biological activity 

of a substance by testing its effect on an 

organism and comparing the result with 

some agreed standard. 

• Substance: Herbicide (Roundup). 

• Organism: Crop sample of interest. 

• Agreed Standard: Check sample / 

Known resistant and 

susceptible seed samples. 



Detecting Biotech-Derived 

Traits Using Bioassay Testing 

• Bioassay testing of biotech derived seeds. 

– Seed soak test 

– Towel soak test 

– Spray test 

• Provides quantifiable 

data which can be 

subjected to tolerances. 



Bioassay Test Determination 

• It is important to determine the test that 

best fits your needs. 

• Time restraints 

• Clarity of test results 

• Seed soak / Kimpak tests work best for 

soybeans and corn. 

• Towel soak tests or spray tests work 

best for cotton. 



Bioassay Setup 

• Acquire sample seeds from purity 

test and count out the number of 

seeds needed to conduct the test. 

– Sample in question 

• Roundup Treatment 

• Water Check 

– Know susceptible sample 



– Known resistant sample 





Prepare Working Solution 

• A working solution is the herbicide 

mixture used for seed soaking or 

wetting the towels. 

• Determine the concentration of the 

working solution from the test 

directions and gather information from 

pesticide label. 



Preparation of Herbicide 

Working Solutions 

Prepare calculations for 

your herbicide working 

solution. 

Calculate concentration in 

PPM (Parts Per Million). 

or 

Calculate concentration in 

molarity (g / L). 



Short Quiz 

• What is a PPM? 

• How do you calculate PPM? 

• What are the units of PPM? 

• Why is PPM unit-less? 



Gathering Pertinent Information 

• Locate the area on the 

container that contains the 

pertinent information 

• Identify the active ingredients 

and corresponding 

concentrations. 

• Know what information is 

useful. 



How much glyphosate is in 1 

Liter of Roundup? 

• Container indicates 50.2% 

glyphosate. 

• 50% of 1 liter is 500 ml, thus 

there are 500 ml of 

glyphosate in 1 L. 

• The container indicates there 

are 600 ml of glyphosate in 1 

L. 

• Where did we go wrong? 



Why Not Calculate 

Based on Percentages? 

• Existing bioassay methods 

indicate concentration 

calculations should be 

based on percentage. 

• This can lead to problems 

because the label usually 

does not specify if 

percentages are based on 

weight or volume. 

• Why does it matter? 



Importance of Percentage Type 

50% V / V (volumetric) 

1.4 g / cm 3 volume 

1.4 g / cm 3 weight 

50% g / g (gravimetric) 

Solvent 

If the 

solution is 

mixed on a 

Solvent 

If the 

solution is 

mixed on a 

basis, 

there will 

be about 

basis, 

there will 

be about 

glyphosate 10% more 

glyphosate 10% more 

1.7 g / cm 3 glyphosate 

1.7 g / cm 3 solvent on 

on a weight 

a volume 

basis. 

basis. 

If we assume that the percentage of glyphosate is 50% by volume 

and glyphosate is actually 50% by weight, we would be adding 10% 

less roundup to our working solution than we would anticipate.

Why Not Calculate 

Based on Percentages? 

• So, is it volumetric or 

gravimetric?? 

• We don’t always know!! 

• Instead, use g ai / Liter 

or lb ai / gal 



Calculating Working Solutions 

Determine the concentration of 

your working solution and how 

much you will need for the 

experiment. 

Examine herbicide container to 

establish concentration of stock 

solution. 

1000 PPM; 

1 Liter 

600 g / Liter 

Calculate the dilution of your 

stock solution to make your 

desired working solution. 

??? 



Calculating Working Solutions in PPM 

1,000 parts 

999,000 parts 

1 part 

999 parts 

1 g 

999 g 

= = 

= 

1 ml 

999 ml 

Roundup Ultra Max = 600 g ai 

1000 ml 

Must dilute roundup to acquire 

desired concentration. 

600 g ai 

1000 ml 

= 1 g ai 

X 

cross multiply 

X = 

1 g ai X 1000 ml 

600 g ai 

X = 1.67 ml 

Water 

1 ml = 1 g (assumption) 

Add the 1.67 ml of herbicide solution to 998.33 ml of water to make a 

1000 PPM working solution. 

Are we done?? Not so fast!! There is still more to consider!! 




• Lets revisit the label. 

• The label indicates glyphosate is in 

the form of isopropylamine salt. 

• Why is this important? 

• The isopropylamine salt portion of 

the formulation has no herbicidal 

activity. Furthermore, different 

formulations of glyphosate have 

different salts. 

• In order to avoid any confusion, 

our calculations need reflect the 

amount of glyphosate free acid 

(portion without the salt) in the 

solution.

Formulation Comparisons 

O 

O 

HO 

N 

P 

O - 

+ H3 N 

Glyphosate Free Acid (169 g / mole) 

Isopropylamine Salt (59 g / mole) 

• Glyphosate free acid = 169 g / mole 

• IPA salt of glyphosate = 228 g / mole 

• DAM salt of glyphosate = 201 g / mole 

• TMS salt of glyphosate = 244 g / mole 




1,000 parts 

999,000 parts 

1 part 

999 parts 

1 g 

999 g 

= = 

= 

1 ml 

999 ml 

Roundup = 600 g IPA salt of glyphosate 

Ultra Max 1000 ml herbicide solution 

Roundup = 0.445 g glyphosate free acid 

Ultra Max ml herbicide solution 

X 

169 g glyphosate free acid 

228 g IPA salt of glyphosate 

0.445 g ai 

1 ml herb = 1 g ai 

X 

cross multiply 

X = 

1 g ai X 1 ml 

0.445 g ai 

X = 2.25 ml herb 

Water 

1 ml = 1 g 

Herb solution 

1 ml ≠ 1 g 

Due to the different densities of water and herbicide solution, it may not 

be appropriate to add 2.25 ml of herbicide solution to 997.75 ml of water. 

Instead, it would be more appropriate to tare a beaker on a balance. Add 

about half the quantity of water desired. Add your herbicide solution. 

Add water to the beaker until the scale reads 1000 grams.

Use of Molarity 

• Use g ai / Liter or lb ai / gal 

• Use molecular weight (169.07) 

of the glyphosate molecule. 

• Percentage, density of 

solution, and makeup of inert 

ingredients is irrelevant in 

calculation. 

• Calculations are accepted SI 

units, whereas PPM is not. 



Calculation Conclusions 

• Things are not always as 

easy as they seem. 

• We need to rethink the way 

we do our calculations. 

• A common worksheet may 

standardize our herbicide 

solutions. 

• Molarity is a great alternative 

because there is no 

confusion with units. 



Interpreting Results 

• Count the number of seedlings in the treated 

sample and water check. 

– Normal seedlings. 

– Note differences between abnormals and seedlings 

appearing susceptible to glyphosate (may use test 

strips for clarification). 

– Ungerminated seeds. 

• Interpret results 

– Test analysis only on germinated seeds. 

– Differentiate between abnormals and seedlings 

appearing treated with glyphosate(may use test 

strips for clarification). 

• Apply results to tolerance tables.

Example Results 

• Test 1 

• 100 seed test on 

pure seed 

• Label – 98% pure 

seed 

• Test results – 4 

biotech-derived 

seedlings (4%). 

• Test 2 

• 400 seed test on 

pure seed 

• Label – 98% pure 

seed 

• Test results – 16 

biotech derived 

seedlings (4%) 



Purity Tolerance Table 

Seed, 

Seedling, or 

Plant Count 

Present 

Number of seeds, seedlings, or plants in tests 

30 50 75 100 150 200 400 800 1000 

100 or 0 0 0 0 0 0 0 0 0 0 

98 or 2 6.0 4.6 3.8 3.3 2.7 2.3 1.6 1.2 1.0 

96 or 4 8.3 6.4 5.3 4.6 3.7 3.2 2.3 1.7 1.5 

94 or 6 10.1 7.8 6.4 5.5 4.5 3.9 2.9 2.1 1.9 

92 or 8 11.5 8.9 7.3 6.3 5.2 4.5 3.4 2.4 2.2 

96 + 4.6 = 100.6 96 + 2.3 = 98.3 

90 or 10 12.8 9.9 8.1 7.0 5.7 4.9 3.8 2.8 2.4 

88 or 12 13.8 10.7 8.7 7.6 6.2 5.4 4.1 3.0 2.7 

86 or 14 14.7 11.4 9.3 8.1 6.6 5.7 4.5 3.2 2.9 

Sec 201.62. Tests for determination of percentages of kind, variety, type, hybrid, or off-type.– Tolerances for tests for determination of percentages of 

kind, variety, type, hybrid, or off-type shall be those set forth in this table, added to ½ the required pure seed tolerances determined in accordance with 

section 201.60, except that ½ the pure seed tolerance will not be applied in determining tolerances for hybrids labeled on the basis of percentage of 

pure seed which is hybrid 



Interpretation of Results 

• Test 1 

• In tolerance. 

• No violation of 

FSA. 

• Action – none. 

• Test 2 

• Out of tolerance. 

• Violation of FSA. 

• Possible re-test. 

• Action – begin 

investigation of 

shipment. 



Conclusions 

• Tolerances are applied to all seed, including 

biotech derived seed. 

• Presence of biotech derived seeds in a seed 

lot does not put it out of tolerance. 

• Currently in the U.S., the presence of biotech 

derived seeds in conventional seed lots does 

not warrant any special labeling.

Questions or Comments

Testing Overview, Mixing Herbicides

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