Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema cos tatum) 

PMRA Submission Number { ... .. ... } EPA MRID Number 48790910 

Data Requirement: PMRA OAT A CODE 

EPA DP Barcode 

OECD Data Point 

EPAMRID 

EPA Guideline 

{ ............ } 

401566 

{ ............ } 

48790910 

850.4500 

Test material: Nicosulfuron (DPX-V9360) Technical Purity: 94.7% 

Common name: Nicosulfron 

Chemical name: IUPAC: 2-[(4,6-Dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl]-N,N-dimethylnicotinamide 

CAS name: 2-[[[[ ( 4,6-dimethoxy-2-pyrimidinyl)amino ]carbonyl]amino ]sulfony 1]-N, N-dimethyl-3-

pyridinecarboxamide 

CAS No.: 111991-09-4 

Synonyms: DPX-V9360-184 

Primary Reviewer: Mia Howard 

Environmental Scientist, CDM Smith 

Secondary Reviewer: Teri S. Myers 

Environmental Scientist, CDM Smith 

Primary Reviewer: Geoffrey Sinclair, Biologist 

EP A/OPP/EFED/ERB-5 

Secondary Reviewer(s): { ...................................... } 

{EPA/OECD/PMRA} 

Reference/Submission No.: { ..................... } 

Company Code 

Active Code 

Use Site Category: 

EPA PC Code 

{ ........ .. .. } 

{ .......... .. } 

{ ............ } 

129008 

[For PMRA] 

[For PMRA] 

[ForPMRA] 

Date Evaluation Completed: {dd-mm-yyyy} 

Signature: ~~waJ2 

Date: 02/21/13 

Signature: ~ '. ·.s mAh/Loo~ Date: 08/14/13 ~ . . ')'",_ 

Date: 8/22/13 

Date: { ............ } 

CITATION: Arnie, J.R., Porch, J.R., Kendall, T.Z., Krueger, H.O. 2011. Nicosulfuron (DPX-V9360) Technical: A 

96-Hour Toxicity Test with the Marine Diatom (Skeletonema costatum). An unpublished study performed by 

Wildlife International, Ltd., Easton, Maryland. Laboratory project ID 112A-395. Study sponsored by E.I. du 

Pont de Nemours and Company. Sponsor study DuPont-32607. Study completed 21 December 2011. 

DISCLAIMER: This document provides guidance for EPA and PMRA reviewers on how to complete a data 

evaluation record after reviewing a scientific study concerning the acute toxicity of a pesticide to aquatic 

nonvascular plants. It is not intended to prescribe conditions to any external party for conducting this study nor to 

establish absolute criteria regarding the assessment of whether the study is scientifically sound and whether the 

study satisfies any applicable data requirements. Reviewers are expected to review and to determine for each study, 

on a case-by-case basis, whether it is scientifically sound and provides sufficient information to satisfy applicable 

data requirements. Studies that fail to meet any ofthe conditions may be accepted, if appropriate; similarly, studies 

that meet all of the conditions may be rejected, if appropriate. In sum, the reviewer is to take into account the 

totality of factors related to the test methodology and results in determining the acceptability of the study. 

Page 1 of 15 
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 1 of 15

Data Requirement: PMRA DATA CODE {............}

EPA DP Barcode 401566

OECD Data Point {............}

EPA MRID 48790910

EPA Guideline 850.4500

Test material: Nicosulfuron (DPX-V9360) Technical Purity: 94.7%

Common name: Nicosulfron

Chemical name: IUPAC: 2-[(4,6-Dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl]-N,N-dimethylnicotinamide

CAS name: 2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-N,N-dimethyl-3-

pyridinecarboxamide

CAS No.: 111991-09-4

Synonyms: DPX-V9360-184

Primary Reviewer: Mia Howard Signature: 

Environmental Scientist, CDM Smith Date: 02/21/13

Secondary Reviewer: Teri S. Myers Signature: 

Environmental Scientist, CDM Smith Date: 08/14/13 

Primary Reviewer: Geoffrey Sinclair, Biologist Date: 8/22/13

Secondary Reviewer(s): {......................................} Date: {............}

{EPA/OECD/PMRA}

Reference/Submission No.: {.....................}

Company Code {............} [For PMRA]

Active Code {............} [For PMRA] 

Use Site Category: {............} [For PMRA]

EPA PC Code 129008

Date Evaluation Completed: {dd-mm-yyyy}

CITATION: Arnie, J.R., Porch, J.R., Kendall, T.Z., Krueger, H.O. 2011. Nicosulfuron (DPX-V9360) Technical: A 

96-Hour Toxicity Test with the Marine Diatom (Skeletonema costatum). An unpublished study performed by 

Wildlife International, Ltd., Easton, Maryland. Laboratory project ID 112A-395. Study sponsored by E.I. du 

Pont de Nemours and Company. Sponsor study DuPont-32607. Study completed 21 December 2011.

DISCLAIMER: This document provides guidance for EPA and PMRA reviewers on how to complete a data 

evaluation record after reviewing a scientific study concerning the acute toxicity of a pesticide to aquatic 

nonvascular plants. It is not intended to prescribe conditions to any external party for conducting this study nor to 

establish absolute criteria regarding the assessment of whether the study is scientifically sound and whether the 

study satisfies any applicable data requirements. Reviewers are expected to review and to determine for each study, 

on a case-by-case basis, whether it is scientifically sound and provides sufficient information to satisfy applicable 

data requirements. Studies that fail to meet any of the conditions may be accepted, if appropriate; similarly, studies 
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 2 of 15

that meet all of the conditions may be rejected, if appropriate. In sum, the reviewer is to take into account the 

totality of factors related to the test methodology and results in determining the acceptability of the study.

EXECUTIVE SUMMARY:

In a 96-hour acute toxicity study, cultures of marine diatom were exposed to nicosulfuron technical at nominal 

concentrations of 0 (negative control) 6.3, 13, 25, 50, and 100 mg ai/L under static conditions. Mean measured 

concentrations were <3.00 (<LOD, negative control), 6.2, 13, 25, 49, and 98 mg ai/L. The NOAEC value based on 

yield, growth rate, and biomass was 98 mg ai/L based on mean measured concentrations. The EC50 values based on 

yield, growth rate, and biomass were all >98 mg ai/L. The % growth inhibition of cell density in the treated algal 

culture as compared to the control ranged from -44 to 13%. 

There were no compound-related phytotoxic effects.

This toxicity study is scientifically sound and satisfies the guideline requirement for an acute algal (marine diatom, 

Skeletonema costatum) toxicity study. It does not reach a definitive endpoint and is therefore classified as 

supplemental quantitative. 

Results Synopsis

Test Organism: Marine diatom, Skeletonema costatum

Test Type (Flow-through, Static, Static Renewal): Static

Yield

EC05: 43 mg ai/L 95% C.I.: N/A to 134 mg ai/L

EC50: > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L

Probit Slope: N/A

Growth rate

EC05: > 98 mg ai/L 95% C.I.: N/A

EC50: > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L

Probit Slope: N/A

Biomass

EC05: 41.1 mg ai/L 95% C.I.: N/A to 121 mg ai/L

EC50: > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L

Probit Slope: N/A

Endpoint(s) Effected: None
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 3 of 15

I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: The study was performed based on the procedures outlined in (1) the U.S. 

EPA Series 850—Ecological Effects Test Guidelines OPPTS 850.5400; (2) 

the OECD Guideline for Testing of Chemicals, 201; and (3) the ASTM 

Standard Guide 1218-90E. The following deviations from the U.S. EPA 

OCSPP 850.4500 (2012) guidelines are noted:

1. Three replicates were used per exposure level (and 6 for the control). A minimum of 4 replicates per 

level is required.

2. Calculations were based on nominal cell densities at time zero which could have some impact on the 

determination growth rates.

3. The test vessels were continuously shaken with a mechanical shaker at 100 rpm. It is recommended 

that cultures of S. costatum be shaken manually 1-2 times per day rather than continuously shaken. If 

clumping is not experienced, test vessels may be shaken continuously at approximately 60 rpm.

4. The pH and total organic carbon and particulate matter concentrations of the dilution water were not 

reported.

5. At test termination the coefficient of variation for mean control yield was 39.7%. The coefficient of 

variation should be less than 15% and it should be less than 15% for average specific growth rate,

Deviations 1 through 4 were not deemed to affect the validity of the study. Deviation 5, however, made

interpretations of the comparisons of the negative control and the treatment levels difficult. There was a 9.5 %

and 13 % reduction that was not detected due to the high variability throughout the study. Because the 

reductions were not strictly dose dependent and variability occurred over all 

treatment levels a definitive endpoint was not attainable in this study.

COMPLIANCE: Signed and dated GLP, Quality Assurance and No Data Confidentiality 

statements were provided. The study was conducted in accordance with 

the GLP principles of the U.S. EPA (40 CFR Part 160), which are 

compatible with the GLP principles of the OECD (ENV/MC/CHEM (98) 

17) and Japan MAFF (11 NohSan, Notification No. 6283) with one 

exception: periodic analyses of well water for potential contaminants were 

not performed according the GLP standards, but were performed using a 

certified laboratory and standard U.S. EPA analytical methods.

A. MATERIALS:

1. Test material Nicosulfuron (DPX-V9360) technical

Description: Solid

Lot No./Batch No. : JUN10MA218

Purity: 94.7%

Stability of compound 

under test conditions: Stable. Mean measured concentrations ranged 98 to 100% of nominal.

(OECD recommends water solubility, stability in water and light, pKa, Pow, and vapor pressure of test 

compound)

Storage conditions of 
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 4 of 15

test chemicals: Ambient 

Physicochemical properties of nicosulfuron technical.

Parameter Values Comments

Water solubility at 20ΕC Not reported

Vapor pressure Not reported

UV absorption Not reported

pKa Not reported

Kow Not reported

2. Test organism: 

Name: Marine diatom, Skeletonema costatum

EPA requires a nonvascular species: For tier I testing, only one species, S. capricornutum, to be 

tested; for tier II testing, S. costatum, A. flos-aquae, S. capricorntum, and a freshwater diatom is 

tested. 

OECD suggests the following species are considered suitable: S. capricornutum, S. subspicatus, and 

C. vulgaris. If other species are used, the strain should be reported

Strain: Not reported

Source: In-house cultures originally obtained from the Provasoli-Guillard National 

Center for Culture of Marine Phytoplankton (CCMP)

Age of inoculum: 3 days

Method of cultivation: Cultured and tested in saltwater algal medium

B. STUDY DESIGN:

1. Experimental Conditions

a. Range-finding study: A range-finding test was used to select test concentrations for the definitive 

test based on consultation with study sponsor but was not presented in this study.

b. Definitive Study

Table 1: Experimental Parameters
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 5 of 15

Parameter Details Remarks

Criteria

Acclimation period:

Culturing media and conditions: 

(same as test or not)

Health: (any mortality observed)

Continuously cultured in-house

Same as test

Algae were taken from actively 

growing cultures

EPA recommends two week acclimation 

period.

OECD recommends an amount of algae 

suitable for the inoculation of test 

cultures and incubated under the 

conditions of the test and used when still 

exponentially growing, normally after 

an incubation period of about 3 days. 

When the algal cultures contain 

deformed or abnormal cells, they must 

be discarded.

Test system

Static/static renewal

Renewal rate for static renewal

Static

N/A

EPA expects the test concentrations to 

be renewed every 3 to 4 days (one 

renewal for the 7 day test, 3-4 renewals 

for the 14 day test). 

Incubation facility

Temperature-controlled 

environmental chamber

Duration of the test 96 hours

EPA requires: 96-120 hours

OECD: 72 hours 

Test vessel

Material: (glass/stainless steel)

Size:

Fill volume:

Glass

250 mL

100 mL OECD recommends 250 ml conical 

flasks are suitable when the volume of 

the test solution is 100 ml or use a 

culturing apparatus.

Details of saltwater algal medium
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 6 of 15

Parameter Details Remarks

Criteria

pH at test initiation:

pH at test termination:

Chelator used:

Carbon source:

Salinity (for marine algae):

7.6 to 8.0

8.4 to 8.6

Na2EDTAx2H2O

Not reported

30 ppt

OECD recommends the medium pH 

after equilibration with air is ~8 

with less than .001 mmol/l of 

chelator if used.

EPA recommends 20X-AAP and 

chelating agents (e.g. EDTA) in the 

nutrient medium for optimum cell 

growth. Lower concentrations of 

chelating agents (down to one-third 

of the normal concentration 

recommended for AAP medium) may 

be used in the nutrient medium used 

for test solution preparation if it is

suspected that the chelator will 

interact with the test material. 

ASTM reference, E1415-91and D

3978-80 (reapproved 1987).

If non-standard nutrient medium 

was used, detailed composition 

provided (Yes/No)

Yes
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 7 of 15

Parameter Details Remarks

Criteria

Dilution water 

source/type:

pH:

salinity (for marine algae):

water pretreatment (if any):

Total Organic Carbon:

particulate matter:

metals:

pesticides:

chlorine:

Artificial saltwater made with 

Wildlife International, Ltd. Well 

water

Adjusted to 8.0 with 10% HCl

30 ppt

Purified

Not reported

Not reported

Some detected (see remarks)

None detected

Chloride: 3.5 mg/L

Water analyses were performed on 

samples collected on 29 December 

2010

Metals (mg/L)

Calcium (35.1)

Magnesium (13.0)

Potassium (6.77)

Sodium (18.5)

EPA pH: Skeletonema costatum= ~8.0 

Others = ~7.5 from beginning to end of 

the test. EPA salinity: 30-35 ppt. EPA is 

against the use of dechlorinated water.

OECD: pH is measured at beginning of 

the test and at 72 hours, it should not 

normally deviate by more than one unit 

during the test.

Indicate how the test material is 

added to the medium (added 

directly or used stock solution)

Used a 100 mg/L stock solution 

prepared in saltwater algal medium. 

The stock solution was sonicated for 

20 minutes and inverted to mix, and 

stirred with a stir bar while all 

subsequent dilutions were made.

Aeration or agitation Continuously shaken with a 

mechanical shaker at 100 rpm

Initial cells density 10,000 cells/mL This is nominal densities. Initial cell 

density was not quantified but 

estimated from inoculums volume 

(which is reasonable assuming a 

well mixed parental population). 

Despite this, the concentrations 

measured at time 1 were similar 

(with expected variability) and high 

variability throughout the study like 

does not make this a major 

deviation.
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 8 of 15

Parameter Details Remarks

Criteria

EPA requires an initial number of 3,000 

- 10,000 cells/mL. For Anabaena flosaquae, cell counts on day 2 are not 

required.

OECD recommends that the initial cell 

concentration be approximately 10,000 

cells/ml for S. capricornutum and S. 

subspicatus. When other species are 

used the biomass should be 

comparable.

Number of replicates

Control:

Solvent control:

Treatments:

6

N/A

3

EPA requires a negative and/or solvent 

control with 3 or more replicates per 

doses. Navicula sp.tests should be 

conducted with four replicate.

OECD preferably three replicates at 

each test concentration and ideally twice 

that number of controls. When a vehicle 

is used to solubilize the test substance, 

additional controls containing the 

vehicle at the highest concentration used 

in the test.

Test concentrations

Nominal:

Measured:

0 (control), 6.3, 13, 25, 50, and 100 

mg ai/L

<3.00 (<LOQ), 6.2, 13, 25, 49, and 

98 mg ai/L

EPA requires at least 5 test 

concentrations, with each at least 60% 

of the next higher one. 

OECD recommends at least five 

concentrations arranged in a geometric 

series, with the lowest concentration 

tested should have no observed effect on 

the growth of the algae. The highest 

concentration tested should inhibit 

growth by at least 50% relatively to the 

control and, preferably, stop growth 

completely. 

Solvent (type, percentage, if used) N/A
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 9 of 15

Parameter Details Remarks

Criteria

Method and interval of analytical 

verification

The concentrations of the test 

solutions were analytically verified 

at 0 and 96 hours using HPLC with 

UV detection.

Test conditions 

Temperature:

Photoperiod:

Light intensity and quality:

20.4 to 21.4oC

14 hours light/10 hours dark

4100 to 4720 lux, cool-white 

fluorescent light

EPA temperature: Skeletonema: 20ΕC, 

Others: 24-25ΕC; EPA photoperiod: S. 

costatum 14 hr light/ 10 hr dark, 

Others: Continuous; EPA light: 

Anabaena: 2.0 Klux (±15%), Others: 4 -

5 Klux (±15%)

OECD recommended the temperature in 

the range of 21 to25o

C maintained at ± 

2o

C and continuous uniform illumination 

provided at approximately 8000 Lux 

measured with a spherical collector.

Reference chemical (if used)

name:

concentrations:

N/A

Other parameters, if any N/A

2. Observations: 

Table 2: Observation parameters

Parameters Details Remarks

Criteria

Parameters measured including the 

growth inhibition/other toxicity 

symptoms

Cell density

Growth rate

EPA recommends the growth of the algae 

expressed as the cell count per mL, biomass 

per volume, or degree of growth as 

determined by spectrophotometric means.
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 10 of 15

Parameters Details Remarks

Criteria

Measurement technique for cell 

density and other end points

Cell density was determined 

using an electronic particle 

counter (Coulter Electronics, 

Inc.) Growth rate was 

calculated from cell density 

using a logarithmic equation.

EPA recommends the measurement 

technique of cell counts or chlorophyll a

OECD recommends the electronic particle 

counter, microscope with counting 

chamber, fluorimeter, spectrophotometer, 

and colorimeter. (note: in order to provide 

useful measurements at low cell 

concentrations when using a 

spectrophotometer, it may be necessary to 

use cuvettes with a light path of at least 4 

cm).

Observation intervals Every 24 hours

EPA and OECD: every 24 hours.

Other observations, if any Cells were microscopically 

examined for morphological 

abnormalities and signs or 

aggregation and adherence to 

the test chamber.

Cells appeared normal.

Indicate whether there was an 

exponential growth in the control

Yes, after 96 hours, the mean 

cell density of the negative 

control was 93.3x104 cells/mL, 

exceeding the 30-fold increase 

required by the guideline.

EPA requires control cell count at 

termination to be 2X initial count or by a 

factor of at least 16 during the test.

OECD: cell concentration in control 

cultures should have increased by a factor 

of at least 16 within three days.

Were raw data included? Yes.

II. RESULTS and DISCUSSION:

A. INHIBITORY EFFECTS:

After 96 hours, the mean cell density of the negative control was 93.3x104 cells/mL, yielding inhibitions 

relative to the negative control of -23, -2, -44, 10, and 13% for mean measured exposure concentrations of 6.2, 

13, 25, 49, and 98 mg ai/l, respectively. The NOAEC and EC50 values calculated by the study author based on 

cell density were 98 and >98 mg ai/L, respectively.

After 96 hours, the mean growth rate of the negative control was 0.0467/hour, yielding inhibitions relative to 

the negative control of -5, -1, -9, 2, and 2% for mean measured exposure concentrations of 6.2, 13, 25, 49, and 

98 mg ai/l, respectively. The NOAEC and EC50 values calculated by the study author based on growth rate 

were 98 and >98 mg ai/L, respectively.
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 11 of 15

After 96 hours, the yield of the negative control was 92.4x104 cells/mL, yielding inhibitions relative to the 

negative control of -23, -2, -45, 10, and 13% for mean measured exposure concentrations of 6.2, 13, 25, 49, and 

98 mg ai/l, respectively. The study author did not calculate or analyze yield values.

After 96 hours, the mean biomass of the negative control was 2211, yielding inhibitions relative to the negative 

control of -15, 1, -32, 14, and 13% for mean measured exposure concentrations of 6.2, 13, 25, 49, and 98 mg 

ai/l, respectively. 

There were no compound related phytotoxic effects. There was not a major change in pH during the study.
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 12 of 15

Table 3: Effect of nicosulfuron technical on algal growth (marine diatom, Skeletonema costatum)

Treatment

Nominal (and 

mean measured)

mg a.i./L

Initial cell

density (x104

cells/mL)

Cell density (x104 cells/mL) at

48 hours 72 hours 96 hours

cell count % inhibitiona

Negative control 1 12.4 32.6 93.3 --

6.3 (6.2) 1 11.1 37.1 114.5 -23

13 (13) 1 10.9 31.4 95.5 -2

25 (25) 1 10.5 41.7 134.8 -44

50 (49) 1 9.2 27.6 84.4 10

100 (98) 1 9.1 30.1 81.3 13

Reference 

chemical (if used)

N/A

a Calculated by the study author relative to the negative control

Table 4: Effect of nicosulfuron technical on algal growth (marine diatom, Skeletonema costatum)

Treatment

Nominal (and 

mean measured)

mg a.i./L

Initial cell

density

(x104

cells/mL)

Mean growth rate

(hour-1)

Yield (x104 cells/mL)1 Biomass (area under the 

curve)1

0-96 hours

% 

inhibitiona 0-96 hours

% 

inhibitionb 0-96 hours

% 

inhibitionb

Negative control 1 0.0467 N/A 92.4 N/A 2211 N/A

6.3 (6.2) 1 0.0488 -5 113.5 -23 2551 -15

13 (13) 1 0.0471 -1 94.5 -2 2188 1

25 (25) 1 0.0511 -9 133.9 -45 2916 -32

50 (49) 1 0.0459 2 83.6 10 1901 14

100 (98) 1 0.0455 2 90.3 13 1934 13

Reference chemical 

(if used) N/A

1 Yield and biomass were calculated by the reviewer. Yield was calculated as final minus initial cell density. 

Biomass was calculated as the area under the cell density growth curve.

a Calculated by the study author relative to the negative control

b Calculated by the reviewer relative to the negative control

Table 5: Statistical endpoint values.* (calculated by the study author based on mean measured 

concentrations)

Statistical Endpoint Cell density Growth rate Yield1 Biomass1
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 13 of 15

NOAEC or EC05 (mg a.i./L) 98 98 Not reported Not reported

EC50 (mg a.i./L) >98 >98 Not reported Not reported

Reference chemical, if used

NOAEC

IC50/EC50

N/A

* Do not use this table, if the study was deemed unacceptable.

1 Yield and biomass were not assessed by the study author

B. REPORTED STATISTICS:

The study author analyzed the endpoints for cell density and growth rate. The data assessed for normality and 

homogeneity of variance using Shapiro-Wilk’s and Levene’s tests, respectively. The data met the assumptions 

of homogeneity of variance, but not normality and log transformation of the data failed to correct the issue. 

Because Dunnett’s test is considered to be robust even in slight departures of normality and therefore the data 

were analyzed using ANOVA followed by Dunnett’s tests. The EC50 values empirically estimated.
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 14 of 15

C. VERIFICATION OF STATISTICAL RESULTS:

Statistical Method: The reviewer statistically analyzed the endpoints for yield, growth rate, and biomass using 

CETIS version 1.8.7.4 statistical software. All analyses were based on mean measured exposure 

concentrations. The data were assessed for normality and homogeneity of variance using Shapiro-Wilk’s and 

Bartlett’s tests, respectively. The biomass data were confirmed to be normally distributed and have 

homogeneous variances and were therefore analyzed using Dunnett’s test. The yield and growth rate data had 

homogeneous variances, but were not normally distributed and were therefore analyzed using Mann-Whitney U 

tests. The ECx values for yield and biomass were determined using nonlinear regression (Bruce-Versteeg). 

EC50 values were not reported because 95% confidence intervals were not calculable, so they were empirically 

determined to be greater than the highest test concentration.

Yield

EC05: 43 mg ai/L 95% C.I.: N/A to 134 mg ai/L

EC50: >98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L

Probit Slope: N/A

Growth rate

EC05: > 98 mg ai/L 95% C.I.: N/A

EC50: > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L

Probit Slope: N/A

Biomass

EC05: 41.1 mg ai/L 95% C.I.: N/A to 121 mg ai/L

EC50: >98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L

Probit Slope: N/A

D. STUDY DEFICIENCIES:

Deviations 1 through 4 were not deemed to affect the validity of the study. Deviation 5, was concerning

Because it indicated a lack of control in the experiment. The 40% coefficient of variation in the negative 

control makes the conclusions of this study difficult to interpret since there was a 9.5 % and 13 % reduction in 

the highest two treatment levels. Eliminating the two samples in the control that were responsible for driving

the variability in the control did not change the conclusions of the test given the high degree of variability that

was observed throughout the test. Therefore, while concerning deviation 5 was not determined in impact the 

conclusions of the study. 

E. REVIEWER’S COMMENTS:

The reviewer’s results for growth rate agree with those of the study author. The reviewer statistically analyzed 

the endpoints for yield and biomass, whereas the study author did not. The reviewer calculated replicate yield 

values as final minus initial cell density. The reviewer calculated replicate biomass values as the area under the 

cell density growth curve. 

All test solutions were clear and colorless at test initiation.
Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX-V9360) Technical to 

Algae (Skeletonema costatum)

PMRA Submission Number {........} EPA MRID Number 48790910

Page 15 of 15

The in-life phase of the definitive test was conducted from 20 to 24 October 2011.

F. CONCLUSIONS: 

This study had a high degree of variability in the control groups as indicated in the deviation section. This 

could have potentially influenced the conclusions of the study especially since other freshwater taxa are 

sensitive to Nicosulfuron. Despite the high variability within all treatment groups any responses visually 

determined did not appear to dose responsive. Therefore even with less variability it is unlikely that meaningful 

differences would have been detected that would have classified this as more than slightly toxic to Skeletonema 

costatum. Nicosulfuron in this study would be classified as practically nontoxic to the marine diatom 

Skeletonema costatum.

Yield

EC05: 43 mg ai/L 95% C.I.: N/A to 134 mg ai/L

EC50: > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L

Probit Slope: N/A

Growth rate

EC05: > 98 mg ai/L 95% C.I.: N/A

EC50: > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L

Probit Slope: N/A

Biomass

EC05: 41.1 mg ai/L 95% C.I.: N/A to 121 mg ai/L

EC50: > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L

Probit Slope: N/A

Endpoint(s) Effected: None

III. REFERENCES: 

1 Organisation for Economic Cooperation and Development. 2006. O BCD

Guidelines for Testing of Chemicals, 201: Freshwater Alga and Cyanobacteria,

Growth Inhibition Test. Adopted 23 March 2006.

2 U.S. Environmental Protection Agency. 1996. Series 850- Ecological Effects

Test Guidelines (draft), OPPTS Number 850.5400: Algal Toxicity, Tiers I and II.

3 ASTM Standard Guide 1218-90E. 1990. Standard Guide for Conducting Static

96-Hour Toxicity Tests with Microalgae. American Society for Testing and

Materials. Philadelphia, Pennsylvania.

4 The SAS System for Windows. 2001. Version 8.2. S AS Institute, Inc., Cary,

North Carolina

5 Bruce, Robert D. and Donald J. Versteeg. 1992. A Statistical Procedure for

Modeling Continuous Toxicity Data. Environmental Toxicology and Chemistry.

11: 1485-1494.
CETIS Summary Report Report Date: 26 Feb-13 08:01 (p 1 of 2)

Test Code: 129008 48790910 | 05-6853-3502

OCSPP 850.4500 Algal Toxicity Wildlife International

Batch ID: 17-4152-1554

Start Date: 20 Oct-11

Ending Date: 24 Oct-11

Test Type: Algal Cell Growth (96-h)

Duration: 96h

Protocol: OCSPP 850.4500 Aquatic Plant (Algae) Diluent: Algal Culture Media

Species: Skeletonema costatum Brine:

Source: Lab In-House Culture

Analyst:

Age: 3 d

Sample ID: 02-7963-9338

Sample Date: 20 Oct-11

Receive Date:

Code: 48790910

Sample Age: NA

Source: E.I. du Pont de Nemours and Company

Station:

Client: CDM Smith

Material: Nicosulfuron Project:

Nicosulfron technical 129008 48790910

Nicosulfuron technical 129008 48790910

Batch Note:

Sample Note:

Comparison Summary

Analysis ID Endpoint NOEL LOEL TOEL PMSD TU Method

07-2826-1757 96h AUC 98 >98 NA 43.9% Dunnett Multiple Comparison Test

00-1569-8600 96h AUC 98 >98 NA 32.7% Williams Multiple Comparison Test

11-9950-9277 96h Cell Density 98 >98 NA NA Jonckheere-Terpstra Step-Down Test

09-8118-6844 96h Cell Density 98 >98 NA 48.7% Mann-Whitney U Two-Sample Test

13-7173-3830 96h Growth Rate 98 >98 NA NA Jonckheere-Terpstra Step-Down Test

03-9274-6426 96h Growth Rate 98 >98 NA 9.61% Mann-Whitney U Two-Sample Test

95% LCL 95% UCL

Point Estimate Summary

Analysis ID Endpoint Level mg ai/L TU Method

09-1793-4392 96h AUC IC5 41.1 N/A 121 Nonlinear Regression

IC10 59.9 N/A 143

IC15 77.2 N/A 152

IC20 94.4 N/A 189

IC25 112 N/A 288

IC40 174 N/A N/A

IC50 226 N/A N/A

05-8496-9589 96h Cell Density IC5 43 N/A 134 Nonlinear Regression

IC10 58.9 N/A 160

IC15 72.8 N/A 163

IC20 86.1 N/A 172

IC25 99.5 N/A 220

IC40 143 N/A N/A

IC50 178 N/A N/A

000-503-186-3 CETISTM v1.8.7.4 Analyst:________ QA:________
CETIS Summary Report Report Date: 26 Feb-13 08:01 (p 2 of 2)

Test Code: 129008 48790910 | 05-6853-3502

OCSPP 850.4500 Algal Toxicity Wildlife International

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

96h AUC Summary

95% LCL 95% UCL %Effect

0 Negative Contro 6 2210 1480 2940 1680 3260 284 697 31.5% 0.0%

6.2 3 2550 902 4200 1980 3280 383 664 26.0% -15.3%

13 3 2190 847 3530 1860 2810 312 540 24.7% 1.08%

25 3 2920 2620 3220 2780 3020 69.8 121 4.15% -31.9%

49 3 1900 934 2870 1540 2320 225 389 20.5% 14.0%

98 3 1930 994 2870 1700 2370 218 378 19.6% 12.5%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

96h Cell Density Summary

95% LCL 95% UCL %Effect

0 Negative Contro 6 92.4 53.8 131 66.4 151 15 36.7 39.7% 0.0%

6.2 3 114 -7.86 235 82.9 170 28.2 48.9 43.0% -22.9%

13 3 94.5 8.72 180 74 134 19.9 34.5 36.5% -2.29%

25 3 134 102 166 122 147 7.42 12.9 9.6% -45.0%

49 3 83.6 17.6 150 62.8 113 15.3 26.6 31.8% 9.52%

98 3 80.3 18 143 65.2 109 14.5 25.1 31.2% 13.1%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

96h Growth Rate Summary

95% LCL 95% UCL %Effect

0 Negative Contro 6 0.0467 0.0427 0.0506 0.0439 0.0523 0.00152 0.00373 8.0% 0.0%

6.2 3 0.0488 0.0386 0.059 0.0461 0.0536 0.00238 0.00412 8.44% -4.61%

13 3 0.0471 0.0384 0.0558 0.045 0.0511 0.00202 0.00351 7.45% -0.89%

25 3 0.0511 0.0486 0.0535 0.0501 0.0521 0.00057 0.000988 1.93% -9.42%

49 3 0.0459 0.0381 0.0537 0.0433 0.0494 0.00181 0.00314 6.84% 1.63%

98 3 0.0455 0.038 0.053 0.0437 0.049 0.00174 0.00301 6.62% 2.47%

C-mg ai/L Control Type

96h AUC Detail

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6

0 Negative Contro 1890 1700 1810 2940 1680 3260

6.2 2400 3280 1980

13 2810 1890 1860

25 2940 3020 2780

49 1540 2320 1840

98 1730 1700 2370

C-mg ai/L Control Type

96h Cell Density Detail

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6

0 Negative Contro 69.8 73.1 67.8 126 66.4 151

6.2 82.9 170 87.8

13 134 74 75.1

25 122 147 132

49 62.8 113 74.4

98 65.2 66.4 109

C-mg ai/L Control Type

96h Growth Rate Detail

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6

0 Negative Contro 0.0444 0.0449 0.0441 0.0505 0.0439 0.0523

6.2 0.0461 0.0536 0.0467

13 0.0511 0.045 0.0451

25 0.0501 0.0521 0.051

49 0.0433 0.0494 0.045

98 0.0437 0.0439 0.049

000-503-186-3 CETISTM v1.8.7.4 Analyst:________ QA:________