### 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 An unpublished study performed by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) Technic al 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 s cientific 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 t he 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 inform ation 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) Technic al 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 accou nt 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

conce ntrations 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 EC 50 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 compare d to the control ranged from

- 44 to 13%.

There were no compound

- related phytotoxic effects.

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

Skeletonema costatum ) toxicity study. I t does not reach a definitive endpoint and is th erefore classified as supplemental quantitative

.

Results Synopsis

Test Organism: Marine diatom

, Skeletonema costatum Test Type (Flow

- through, Static, Static Renewal): Static

Yield

EC 05 : 43 mg ai/L 95% C.I.: N/A to 134 mg ai/L

EC 50 : > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L Probit Slope: N/A

Growth rate

EC 05 : > 98 mg ai/L 95% C.I.: N/A

EC 50 : > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L Probit Slope: N/A

Biomass

EC 05 : 41.1 mg ai/L 95% C.I.: N/A to 121 mg ai/L

EC 50 : > 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) Technic al 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 OPPT S 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 vess els 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,

D eviations 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 varia bility occurred over all treatment levels a definitive endpoint was not attainable in this study.

COMPLIANCE: S igned 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) tec hnical

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) Technic al to Algae ( Skeletonema costatum )

PMRA Submission Number 

### {........} EPA MRID Number 48790910

Page 4 of 15 test chemicals: Ambient

Physicochemic al properties of nicosulfuron technical

.

Parameter Values Comments

Water solubility at 20 Ε C 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 consid ered 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) Technic al 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) Technic al 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

Na 2 EDTAx2H 2 O 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

c helating 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) Technic al 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 throughou t the study like does not make this a major

deviation.

### Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX

- V9360) Technic al 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 flos

aquae, 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 ve hicle 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 t o 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) Technic al 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, c ool

- white fluorescent light EPA temperature: Skeletonema : 20 Ε C,

Others: 24

- 25 Ε C; 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 to25 o C maintained at ±

2 o 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) Technic al 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 necessar y 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 93.3x10 4 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 93.3x10 4 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

cel l 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) Technic al 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 92.4x10 4 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 negati v e 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 compou nd 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) Technic al to Algae ( Skeletonema costatum )

PMRA Submission Number 

### {........} EPA MRID Number 48790910

Page 12 of 15 Tabl e 3: Effect of nicosulfuron technical on algal growth ( marine diatom, Skeletonema costatum )

Treatment

Nominal (and mean measured)

mg a.i./L Initial cell density (x10 4

cells/mL) Cell density (x10 4 cells/mL) at

48 hours 7 2 hours 96 hours

cell count % inhibition a

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

(x10 4

cells/mL) Mean growth rate (hour - 1 ) Yield (x10 4 cells/mL) 1 Biomass (area under the curve) 1

0

- 96 hours %

inhibition a 0

- 96 hours %

inhibition b 0

- 96 hours %

inhibition b

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 Yield 1 Biomass 1

### Data Evaluation Report on the Acute Toxicity of Nicosufuron (DPX

- V9360) Technic al to Algae ( Skeletonema costatum )

PMRA Submission Number 

### {........} EPA MRID Number 48790910

Page 13 of 15 NOAEC or EC 05 (mg a.i./L) 98 98 Not reported Not reported

EC 50 (mg a.i./L) >98 >98 Not reported Not reported

Reference chemical, if used

NOAEC

IC 50 /EC 50 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 t herefore 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) Technic al 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 homogenei ty 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 homogen eous 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)

. EC 50 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

EC 05 : 43 mg ai/L 95% C.I.: N/A to 134 mg ai/L

EC 50 : >98 mg ai/L 95% C.I.: N/A

NOAEC : 98 mg ai/L Probit Slope: N/A

Growth rate

EC 05 : > 98 mg ai/L 95% C.I.: N/A

EC 50 : > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L Probit Slope: N/A

Biomass

EC 05 : 41.1 mg ai/L 95% C.I.: N/A to 121 mg ai/L

EC 50 : >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

B ecause 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 th at were res ponsible 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) Technic al 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

EC 05 : 43 mg ai/L 95% C.I.: N/A to 134 mg ai/L

EC 50 : > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L Probit Slope: N/A

Growth rate

EC 05 : > 98 mg ai/L 95% C.I.: N/A

EC 50 : > 98 mg ai/L 95% C.I.: N/A

NOAEC: 98 mg ai/L Probit Slope: N/A

Biomass

EC 05 : 41.1 mg ai/L 95% C.I.: N/A to 121 mg ai/L

EC 50 : > 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 To xicity Tests with Microalgae. American Society for Testing a nd Materials. Philadelphia, Pennsylvania.

4 The SAS System for Windows. 2001. Versi on 8.2. S AS Institute, Inc., C ary, 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

Brine: Species: Skeletonema costatum

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

Project: Material: Nicosulfuron

Nicosulfron technical 129008 48790910

Nicosulfuron technical 129008 48790910 Batch Note:

Sample Note:

Comparison Summary

NOEL LOEL TOEL

Endpoint Analysis ID Method PMSD TU

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

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

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

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

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

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

95% LCL 95% UCL Point Estimate Summary

Endpoint Analysis ID Method mg ai/L TU Level

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

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

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

### 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

Control Type Mean Min Max Std Dev

Count CV% Std Err C-mg ai/L 96h AUC Summary

95% LCL 95% UCL %Effect

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

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

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

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

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

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

Control Type Mean Min Max Std Dev

Count CV% Std Err C-mg ai/L 96h Cell Density Summary

95% LCL 95% UCL %Effect

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

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

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

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

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

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

Control Type Mean Min Max Std Dev

Count CV% Std Err C-mg ai/L 96h Growth Rate Summary

95% LCL 95% UCL %Effect

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

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

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

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

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

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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

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