### Data Evaluation Report on the Acute Toxicity of Nicosulfuron (DPX- V9360) to Algae

(Pseudokirchneriella subcapitata)

PMRA Submission Number {

........ } EPA MRID Number 46370301

Data Requirement: PMRA DATA CODE

EPA DP Barcode

OECD Data Point

EPAMRID

EPA Guideline 

### {

............ 

### }

386906 

### {

............ 

### }

46370301

850.4500

Test material: Nicosulfuron (DPX-V9360) technical Purity: 93.2%

Common name Nicosulfuron

Chemical name: IUP AC: not reported

CAS name: 2-[[[[ ( 4,6-Dimethoxy- 2-pyrim idinyl)amino ]carbonyl]amino ]sulfonyi]-N ,N-dimethyl-3-

pyridinecarboxamide monohydrate

CAS.No. 111991-09-4

Synonyms: Nicosulforn, DPX- V9360-121, 2-Pyridinecarboxamide, 2-[[[[(4,6-dimethoxy-2-

pyrimidinyl)amino]carbonyl]amino]sulfonyi]-N,N-dimethyl, monohydrate (inverted CAS name)

Primary Reviewer: Mia Howard

Staff Scientist, Cambridge Environmental, Inc.

Secondary Reviewer: Teri S. Myers

Environmental Scientist, CDM Smith

Primary Reviewer: Geoffrey Sinclair, Biologist

EPA/OPP/EFED/ERB 5

Secondary Reviewer(s): {

...................................... }

{EPA/OECDIPMRA}

Reference/Submission No.: {.

.................... }

Company Code

Active Code

Use Site Category:

EPA PC Code 

### {

............ 

### }

{

............ 

### }

{

............ 

### }

129008 [For PMRA]

[For PMRA]

[For PMRA]

Date Evaluation Completed: {dd-mm-yyyy} 

### S

. t

# ~~~ 1gna ure:

Date: 10/01/12

Signature:**~ '"'5/YJAh,.,~~**  Date: 08/14/13**~** · · 

### 'i''r

Date: 8/26/13

Date: {

............ }

CITATION: Sloman, T.L. 2004. Nicosulfuron (DPX-V9360) Technical: Influence on Growth and Growth Rate of the

Green Alga Selenastrum capricornutum. An unpublished study performed by E.l. du Pont de Nemours and

Company, Haskell Laboratory for Health and Environmental Sciences, Newark, Delaware. Laboratory study

·

DuPont-13342. Study sponsored by E.I. duPont de Nemours and Company, Wilmington, Delaware. Study initiated

22 March 2004 and completed 15 July 2064.

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

Page 1 of23

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 1 of 23 Data Requirement: PMRA DATA CODE {............}

EPA DP Barcode 386906

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

EPA MRID 46370301

EPA Guideline 850.4500

Test material: Nicosulfuron (DPX

- V9360 ) technical Purity: 93.2%

Common name Nicosulfuron

Ch emical name: IUPAC : not reported

CAS name : 2

- [[[[(4,6

- Dimethoxy

- 2

- pyrimidinyl)amino]carbonyl]amino]sulfonyl]

- N,N

- dimethyl

- 3

- pyridinecarboxamide monohydrate

CAS No. 111991

- 09

- 4

Synonyms : Nicosulforn, DPX

- V9360

- 121, 2

- Pyridinecarboxamide, 2

- [[[[(4,6

- dimetho xy

- 2

- pyrimidinyl)amino]carbonyl]amino]sulfonyl]

- N,N

- dimethyl, monohydrate (inverted CAS name)

Primary Reviewer: Mia Howard Signature:

Staff Scientist, Cambridge Environmental, Inc. Date: 10/01/12

Secondary Revie wer: Teri S. Myers Signature:

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

Primary Reviewer: Geoffrey Sinclair, Biologist Date: 8/26/13

EPA/ OPP/EFED/ERB 5

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

{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 Co mpleted: {dd

- mm

- yyyy}

CITATION : Sloman, T.L. 2004. Nicosulfuron (DPX

- V9360) Technical: Influence on Growth and Growth Rate of the

Green Alga Selenastrum capricornutum . An unpublished study performed by E.I. du Pont de Nemours and

Company, Haskell Laborat ory for Health and Environmental Sciences, Newark, Delaware. Laboratory study

DuPont

- 13342. Study sponsored by E.I. du Pont de Nemours and Company, Wilmington, Delaware. Study initiated

22 March 2004 and completed 15 July 2004.

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 exte rnal party for conducting this study nor to establish

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 2 of 23 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 determi ne 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 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 72

- hour acute toxicity study, cultures of freshwater green alga, Pseudokirchneriella subcapitata

, were exposed to

nicosulfuron at nominal concentrations of 0 (negative control), 7.5, 15, 30, 60, and 120 mg/L under static conditions.

Mean measured concentra tions were <0.0015 (<LOD, negative control), 7.39, 14.3, 28.35, 56.3, and 98.05 mg ai/L. The

NOAEC and EC 50 /IC 50 values based on cell density were 7.39 and 24 mg a.i/L, respectively. The NOAEC and EC 50 /IC 50 values based on area under the growth curve wer e <7.39 and 25 mg a.i/L, respectively. The NOAEC and EC 50 /IC 50 values based on growth rate were 56.3 mg ai/L and 71.17 mg/L, respectively. The % growth inhibition of cell density in

the treated algal culture as compare d to the control ranged from 5 to 10 0%

. Based on the endpoints derived for cell

density this compound would be classified as slightly toxic.

There were no compound related phytotoxic effects reported.

This toxicity study is classified as scientifically sound or and does satisfy the gui deline requirement for an acute algal

( Pseudokirchneriella subcapitata ) toxicity study. It is therefore classified as acceptable. Based on the endpoints derived

for cell density, Nicosulfuron would be classified as slightly toxic to Pseudokirchneriella subcapitata.

Results Synopsis

Test Organism: Freshwater green alga, Pseudokirchneriella subcapitata

Test Type (Flow

- through, Static, Static Renewal): Static

Cell density

EC 05 : 7.5 mg a.i./L 95% C.I.: 4.9 to 11 mg a.i./L

EC 50 : 24 mg a.i./L 95% C.I. : 19 to 29 mg a.i./L

NOAEC: 7.39 mg a.i./L

Probit Slope: 3.30 ± 0.338

Area under the growth curve

EC 05 : 7.6 mg a.i./L 95% C.I.: 4.7 to 12 mg a.i./L

EC 50 : 25 mg a.i./L 95% C.I.: 20 to 31 mg a.i./L

NOAEC: <7.39 mg a.i./L

Probit Slope: 3.20 ± 0. 369

Growth rate

EC 05 : Not calculable 95% C.I.: Not calculable

EC 50 *: 71.17 mg/L 95% C.I.: 65.46 to 76.88 mg/L

NOAEC: 56.3 mg a.i./L

Probit Slope: N/A

Endpoint(s) Effected: cell density, area under the growth curve, growth rate

*The EC50 value ba sed on growth rate was not able to be statistically verified using probit analysis via Nuthatch

statistical software. Therefore, the reviewer deferred to the value reported by the study author, based on

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 3 of 23 nominal formulation concentrations. All other toxic ity values were verified by the reviewer and based on mean

measured active ingredient concentrations.

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 4 of 23 I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: The study was designed to comply with the Organization for Economic

Cooperation and Development (OECD) Gu ideline for the Testing of

Chemicals: 201 (1984) and the European Economic Communities Methods

for the Determination of Toxicity Directive 92/69/EEC Annex V, part C.3

(1992). The following deviations from the US EPA OCSPP 850.4500

(2012) guidelines are no ted:

1. The duration of the test was 72 hours. OCSPP guidelines require a duration of 96 hours.

2. Three replicates were used per test level. OCSPP guidelines require a minimum of four replicates per

level.

3. The pH of the test medium ranged from 5.45 to 7.41 a t test initiation and 5.24 to 7.48 at test

termination. OCSPP guidelines state that the initial pH of freshwater algal medium should be 7.5 ± 0.1.

4. The age of the inoculum used in the study was not reported. OCSPP guidelines recommend

logarithmically grow ing stock cultures (typically 3 to 7 days old).

5. The pH and total organic carbon, particulate mater, metal, pesticide, and chlorine contents of the

dilution water were not reported.

6. OCSPP guidlelnes state that cultures should be shaken at 60 cycles per minu te. This study agitates

cultures ate 101 rpm.

The 72 hour vs 96 hour durations may impact the derivation of the EC50. The controls increased by greater

than 100 times as required by OPPTS 4500 guidelines so the duration of 72 hours to derive accurate e ndpoints

is acceptable. The pH of 5.45 is a bit acidic and well under guideline requirements. Based on the performance

of algae however, this deviation did not appear to influence the results of the study . The experimental

design is scientifically sou nd however and despite these deviations do not affect the validity of the study.

COMPLIANCE: S igned and dated GLP, Quality Assurance and Data Confidential ity

statements were provided. The study was performed in compliance with US

EPA FIFRA (40 CFR pa rt 160) GLP standards.

A. MATERIALS:

1. Test material Nicosulfuron (DPX

- V9360) Technical

Description: White powder

Lot No./Batch No. : DPX

- V9360

- 121 (batch)

Purity: 93.2%

Stability of compound

under test conditions: Stable. Ini tial measured concentrations ranged from 96

- 103% of nominal.

Final measured concentrations ranged from 79

- 108% of nominal.

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

compound)

Storage c onditions of

test chemicals: Ambient (for purity measurement)

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 5 of 23 Physicochemical properties of nicosulfuron

Parameter Values Comments

Water solubility at 20 Ε C 750 mg/L After 25 hours of

stirring

Vapor pressure not repor ted

UV absorption not reported

pKa not reported

Kow not reported

2. Test organism :

Name: Freshwater green alga, Pseudokirchneriella subcapitata (also known as

Selenastrum capricornutum )

EPA requires a nonvascular species: For ti er 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. subspi catus, and

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

Strain: Not reported

Source: In

- house cultures originally obtained from the Department of Botany

Culture Collection of Algae

- The University of Texas at Austin, Aust in,

Texas

Age of inoculum : Not reported

Method of cultivation: Cultured in AAP nutrient medium

B. STUDY DESIGN:

1. Experimental Conditions

a. Range

- finding study. A 72

- hour range

- finding study in which algae were exposed to a negative control

and nom inal concentrations of 0.0012, 0.012, 0.12, 1.2, 12, and 120 mg/L of nicosulfuron was performed.

Inhibition of cell density compared to the negative control was 6, 15, 10, 3, 20, and 100% for treatments of

0.0012, 0.012, 0.12, 1.2, 12, and 120 mg/L of ni cosulfuron, respectively.

b. Definitive Study

Table 1: Experimental Parameters

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 6 of 23 Parameter Details Remarks

Criteria

Acclimation period:

Culturing media and conditions:

(same as test or not)

Health: (any mortality observed) Continuously cultured in the lab

Same as test

None reported 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 growin g, 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 Environmental chamber with a

shaker table

Duration of the test 72 hours

EPA requires: 96

- 120 hours

OECD: 72 hours

Test vessel

Material: (glass/stainless steel)

Size:

Fill volume: Glass

250 mL

50 mL Sterile Erlenmeyer flasks

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 AAP nutrient medium

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 7 of 23 Parameter Details Remarks

Criteria

pH at test initiation:

pH at test termination:

Chelator used:

Carbon source:

Salinity ( for marine algae ): 5.45 to 7.41

5.24 to 7.48

Na 2 EDTAx2H 2 O

NaHCO 3

N/A 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

chela ting 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

- 91 and D

3978

- 80 (reapproved 1987).

If non

- standard nutrient medium

was used, detailed composition

provided (Yes/No) A standard medium was used and a

detailed composition was also

provided

Dilution water

source/ type:

pH:

salinity ( for marine algae ):

water pretreatment (if any):

Total Organic Carbon:

particulate matter:

metals:

pesticides:

chlorine: Milli

- Q (deionized) water

Not reported

N/A

D eionized

Not reported

Not reported

Not reported

Not reported

Not report ed 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 deviat e by more than one unit

during the test.

Indicate how the test material is

added to the medium (added

directly or used stock solution) A 120 mg/L nicosulfuron working

stock solution prepared in AAP

nutrient medium was used

Aeration or agitation Shaken on a shaker table set at 101

rpm

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 8 of 23 Parameter Details Remarks

Criteria

Initial cells density 1x10 4 cells/mL

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 recommen ds 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: 3

N/A

3 OCSPP (2012 ) guidelines require a

minimum of four replicates per level

A single abiotic control was also

tested

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 pr eferably 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 concent rations

Nomina l (formulation): 0 (negative control), 7.5, 15, 30, 60,

and 120 mg/L *Calculated by the reviewer as the

arithmetic mean of the measured

concentrations at days 0 and 3

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 9 of 23 Parameter Details Remarks

Criteria

Nominal (corrected for purity):

M ean m easured * : 0 (negative control), 6.99, 14.0,

28.0, 55.9, and 112 mg ai/L

<0.0015 (<LOD, negative control),

7.39, 14.3, 28.35, 56. 3, and 98.05

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 an d, preferably, stop growth

completely.

Solvent (type, percentage, if used) N/A

Method and interval of analytical

verification The concentration of the test

substance in the test solutions were

verified on days 0 and 3 using high

performance liquid chro matography.

Test conditions

Temperature:

Photoperiod:

Light intensity and quality: 24.6 to 24.8°C

Continuous

6580 to 8750 lux , cool

- white

fluorescent light EPA temperature: Skeletonema : 20 Ε C,

Others: 24

- 2 5 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

illuminat ion provided at approximately

8000 Lux measured with a spherical

collector.

Reference chemical (if used)

name:

concentrations: N/A

Other parameters, if any N/A

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 10 of 23 2. Observations:

Tab le 2: Observation parameters

Parameters Details Remarks

Criteria

Parameters measured including the

growth inhibition/other toxicity

symptoms Cell density

Area under the growth curve

Growth rate EPA re commends the growth of the algae

expressed as the cell count per mL, biomass

per volume, or degree of growth as

determined by spectrophotometric means.

Measurement technique for cell

density and other end points Cells were counted us ing a

compound microscope and a

hemacytometer. The area under

the growth curve was calculated

based on cell density. The

growth rate was calculated using

a logarithmic equation based on

cell density. EPA recommends the measureme nt

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 A 96

- hour recovery test was

pe rformed on the negative

control and test concentrations

exhibiting >50% inhibition of

cell counts.

Indicate whether there was an

exponential growth in the control Yes. After 72 hours, the cell

density of the negative control

was 349.7x10 4 cells/mL. 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 72 hours, the cell density of the negative control was 349.7x10 4 cells/mL, yielding inhibitions compared to the

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 11 of 23 negative control of 5,39, 55, 88, and 100% for nominal exposure concentrations of 7.5, 15, 30, 60, and 120 mg/L.

The EC50 and NOAEC values calculated by the study author using nominal formulation concentrations were 24.75

and 7.5 mg/L, respectively.

After 72 hours, the area under the growth curve of the negative control was 6648.0x10 4 , yielding in hibitions

compared to the negative control of 14, 39, 55, 85, and 100% for nominal exposure concentrations of 7.5, 15, 30, 60,

and 120 mg/L. The EC50 and NOAEC values calculated by the study author using nominal formulation

concentrations were 23.48 and < 7.5 mg/L, respectively.

After 72 hours, the growth rate of the negative control was 0.0813, yielding inhibitions compared to the negative

control of 1, 8, 14, 36, and 100% for nominal exposure concentrations of 7.5, 15, 30, 60, and 120 mg/L. The EC50

and NOAEC values calculated by the study author using nominal formulation concentrations were 71.17 and 7.5

mg/L, respectively.

No morphological abnormalities were reported. There was not a major change in pH during the study.

The res ults from the recovery test suggest that the effects of nicosulfuron are algistatic within 96 hours for

concentrations < 120 mg/L.

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 12 of 23 Table 3: Effect of nicosulfuron on algal growth (freshwater green alga, Pseudokirchneriella subcapitat a )

Treatment

Nominal

formulation (and

nominal active

ingredient) [and

mean measured

active ingredient*]

concentrations

(mg/L) Initial cell

density (x10 4

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

24 hours 48 hours 72 hours

cell count % inhibition

Negative control 1 10.7 94.0 349.7 N/A

7.5 (6.99) [7.39] 1 8.0 58.0 333.7 5

15 (14.0) [14.3] 1 8.3 55.7 123.3 39

30 (28.0) [28.35] 1 9.3 38.7 155.7 55

60 (55.9) [58.6] 1 7.7 15.0 41.7 88

120 (112) [98.05] 1 0.7 1.3 0.7 100

Reference chemical

(if used ) N/A

*Calculated by the reviewer as the arithmetic mean of the measured concentrations at days 0 and 3

Table 4: Effect of nicosulfuron on algal growth (freshwater green alga, Pseudokirchneriella subcapitata )

Treatment

Nominal

formulation (and

nominal active

ingredient) [and

mean measured

active ingredient*]

concentrations

(mg/L) Area under

the growth

curve, 0

- 72 h

(x10 4 ) % inhibition Growth rate % inhibition

Negative control 6648.0 N/A 0.0813 N/A

7.5 (6.99) [7.39] 5528.0 17 0.0807 1

15 (14.0) [14.3] 4036.0 39 0.0745 8

30 (28.0) [28.35] 2960.0 55 0.0699 14

60 (55.9) [58.6] 987.0 85 0.0518 36

120 (112) [98.05] 8.0 100 0.0000 100

*Calculated by the reviewer as the arithmetic mean of the measured concentrations at days 0 and 3

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 13 of 23 Table 5 : Statistical endpoint values.* (calculated by the study author based on nominal formulation

concentrations)

Statistical Endpoint Cell density Area under the

growth curve Growth rate

NOAEC (mg /L) 7.5 <7.5 7.5

IC 50 or EC 50 (mg /L) (95% C.I.) 24.75 (20.68 to

29. 62) 23.48 (19.80 to 27.16) 71.17 (65.46 to 76.88)

Reference chemical, if used

NOAEC

IC 50 /EC 50 N/A

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

B. REPORTED STATISTICS:

The study author performed all calculations based on nominal ex posure concentrations. The EC50 values were

determined using inverse weighted least

- squares non

- linear regression. The EC50 value based on cell density was

calculated using probit analysis. The NOAEC values were determined using Jonckheere

- Terpstra test s.

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 14 of 23 C. VERIFICATION OF STATISTICAL RESULTS:

Statistical Method: The reviewer statistically analyzed the endpoints for cell density, area under the growth

curve, and growth rate using mean measured active ingredient concentrations. The data were assessed for

normality and homogeneity of variance using Shapiro

- Wilk’s and Levene’s tests, respectively. The cell density

and area under the curve data met the assumptions of normality and homogeneity of variance, and therefore the

NOAEC values based on these tw o endpoint s were calculated using ANOVA followed by William’s tests. The

growth rate data were confirmed to have homogeneous variance, but not meet the assumptions of normality.

Therefore, the NOAEC value based on growth rate was determined using a Krusk al

- Wallis test. The preceding

analyses were all performed using Toxstat 3.5 statistical software. The ECx values based on cell density and

area under the growth curve were determined using probit analysis via Nuthatch statistical software. The

reviewer w as unable to calculate ECx values using probit analysis for growth rate because the probit model was

unsuitable for the data.

Cell density

EC 05 : 7.5 mg a. i./L 95% C.I.: 4.9 to 11 mg a.i./L

EC 50 : 24 mg a.i./L 95% C.I.: 19 to 29 mg a.i./L

NOAEC: 7.39 mg a.i./L

Probit Slope: 3.30 ± 0.338

Area under the growth curve

EC 05 : 7.6 mg a.i./L 95% C.I.: 4.7 to 12 mg a.i./L

EC 50 : 25 mg a.i./L 95% C.I.: 20 to 31 mg a.i./L

NOAEC: <7.3 9 mg a.i./L

Probit Slope: 3.20 ± 0.369

Growth rate

EC 05 : Not calcu lable 95% C.I.: Not calculable

EC 50 : Not calculable 95% C.I.: Not calculable

NOAEC: 56.3 mg a.i./L

Probit Slope: N/A

D. STUDY DEFICIENCIES:

The 72 hour vs 96 hour durations may impact the derivation of the EC50. The controls increased by great er

than 100 times as required by OPPTS 4500 guidelines so the duration of 72 hours to derive accurate endpoints

is acceptable. The pH of 5.45 is a bit acidic and well under guideline requirements. Based on the performance

of algae however, this deviat ion did not appear to influence the results of the study . The experimental

design is scientifically sound however and despite these deviations do not affect the validity of the study.

E. REVIEWER ’ S COMMENTS:

The reviewer based all analyses on mean m easured active ingredient concentrations, whereas the study author used

nominal concentrations that were not corrected for purity. The reviewer used probit analysis to calculate EC 50 values, whereas the study author used probit analysis to calculate the E C 50 based on cell density and used inverse

weighted least

- squares non

- linear regression to calculate the other EC 50 values. The reviewer was unable to

calculate an EC 50 value using probit analysis for the growth rate data and therefore deferred to the val ue calculated

by the study author in the Executive Summary and Conclusions sections. The reviewer used a Kruskal

- Wallis test to

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 15 of 23 determine the NOAEC based on growth rate, whereas the study author used a Jonckheere

- Terpstra test. Therefore

, the results rep orted by the reviewer and the study author do no t agree completely.

All test solutions appeared clear and colorless except for the 120 mg/L solutions, which was slightly cloudy with

some visibly insoluble material.

The in

- life phase of the definitive te st was conducted from 5

- 12 April 2004.

F. CONCLUSIONS:

This study is scientifically sound. It is thus classified as acceptable

. A fter 72 hours, the most sensitive endpoint was

cell density , with NOAEC and EC 50 values of <7.39 and 24 mg ai/L, respect ively. With an EC50 of 24 mg ai /L

Nicosulfuron would be classified as slightly toxic to Pseudokirchneriella subcapitata.

Cell density

EC 05 : 7.5 mg a.i./L 95% C.I.: 4.9 to 11 mg a.i./L

EC 50 : 24 mg a.i./L 95% C.I.: 19 to 29 mg a.i./L

NOAEC: 7.39 mg a.i./L

Probit Slope: 3.30 ± 0.338

Area under the growth curve

EC 05 : 7.6 mg a.i./L 95% C.I.: 4.7 to 12 mg a.i./L

EC 50 : 25 mg a.i./L 95% C.I.: 20 to 31 mg a.i./L

NOAEC: <7.39 mg a.i./L

Probit Slope: 3.20 ± 0.369

Growth rate

EC 05 : Not calculabl e 95% C.I.: Not calculable

EC 50 *: 71.17 mg/L 95% C.I.: 65.46 to 76.88 mg/L

NOAEC: 56.3 mg a.i./L

Probit Slope: N/A

Endpoint(s) Effected: cell density, area under the growth curve, growth rate

*The EC50 value based on growth rate was not able to be statistically verified using probit analysis via Nuthatch

statistical software. Therefore, the reviewer deferred to the value reported by the study author, based on

nominal formulation concentrations. All other toxicity values were verified by the revie wer and based on mean

measured active ingredient concentrations.

III. REFERENCES :

DuPont Haskell Laboratory (1997). Stability and Solubitility of DPX

- V9360

- 77 in Aqueous Solutions. Unpublished

report, HA

- 97

- 165.

Miller, W.E.; Green, J.C.; Shiroyama, T. The Selenastrum capricornutum Printz Algal Assay Bottle Test; U.S.

Environmental Protection Agency; U.S. Government Printing Office: Washington, DC, 1978; EPA

- 600/9

- 78

- 018.

American Society for Testing and Materials (ASTM). (1990). “Standard Guide for Conducting Static 96

- h Toxicity Tests

with Microalgae” in ASTM Annual Book of Standards, E1218

- 90. Vol. 11.04, Philadelphia, PA.

Sokal, R.R., and Rohlf, F.J. Biometry, 2 nd ed., W.H. Freeman and Co., New York, 859 pp, 1981.

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 16 of 23 Bruce, R.D. and Versteeg, D.J. (1992), A statistical procedure for modeling continuous toxicity data. Environ. Toxicol.

Chem. 11: 1485

- 1494.

Hochberg, Y. and Tamhane, A.C. (1987); Multiple Comparison Procedures, Wiley, New York.

Jonckheere, A.R., (1954); A distribution

- free k

- sample test against ordered alternatives, Biometricka 41, 133.

SAS Software Version 8.2, SAS Institute, Cary, NC, 1999.

Slob, W. (2002); Dose

- Response Modeling of Continuous Endpoints, Toxicological Sciences 66, 298

- 312.

Terpstra, T.J. (1952); The Asymptotic N ormality and Consistency of Kendall’s Test Against Trend, When Ties are

Present in One Ranking, Indag. Math. 14, 327

- 333.

U.S. EPA (1995)

- The Use of Benchmark Dose Approach in Health Risk Assessment, Risk Assessment Forum,

EPA/630/R

- 94/007, United States Environmental Protection Agency, Washington, DC. Principal authors: Crump, K.

Allen, B., and Faustman, E.

Westfall, P.H., Tobias, R.D., Rom, D., Wolfinger, R.D., and Hochberg, Y. (1999)

- Multiple comparisons and multiple

tests, SAS Institute, Cary, North Carolina.

APPENDIX I. OUTPUT OF THE REVIEWER’S STATISTICAL VERIFICATION

CELL DENSITY

Title: NICOSULFURON P.SUBCAPITATA 72HR CELL DENSITY MEAN MEAS

File: 0301c Transform: NO TRANSFORMATION

Shapiro

- Wilk's Test for Normality

----------------------------------------------------------------------------

D = 3970.0000

W = 0.9616

Critical W = 0.8580 (alpha = 0.01 , N = 18)

W = 0.8970 (alpha = 0.05 , N = 18)

----------------------------------------------------------------------------

Data PASS normality test (alpha = 0.01). Continue analysis.

Title: NICOSULFURON P.SUBCAPITATA 72HR CELL DENSITY MEAN MEAS

File: 0301c Transform: NO TRANSFORMATION

Levene's Test for Homogeneity of Variance

ANOVA Table

----------------------------------------------------------------------------

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 17 of 23 SOURCE DF SS MS F

----------------------------------------------------------------------------

Between 5 856.4444 171.2889 1.2841

Within (Error) 12 1600.6667 133.3889

----------------------------------------------------------------------------

Total 17 2457.1111

---------------------------------------------------------------------------- (p

- value = 0.3329)

Critical F = 5.0643 (alpha = 0.01, df = 5,12)

= 3.1059 (alpha = 0.05, df = 5,12)

Since F < Critical F FAIL TO REJECT Ho: All equal (alpha = 0.01)

Title: NICOSULFURON P.SUBCAP ITATA 72HR CELL DENSITY MEAN MEAS

File: 0301c Transform: NO TRANSFORMATION

ANOVA Table

----------------------------------------------------------------------------

SOURCE DF SS MS F

----------------------------------------------------------------------------

Between 5 316092.4444 63218.4889 191.0886

Within (Error) 12 3970. 0000 330.8333

----------------------------------------------------------------------------

Total 17 320062.4444

---------------------------------------------------------------------------- (p

- value = 0.0000)

Critical F = 5.0643 (alpha = 0.01, df = 5,12)

= 3.1059 (alpha = 0.05, df = 5,12)

Since F > Critical F REJECT Ho: All equal (alpha = 0.05)

Title: NICOSULFURON P.SUB CAPITATA 72HR CELL DENSITY MEAN MEAS

File: 0301c Transform: NO TRANSFORMATION

William's Test

- TABLE 1 OF 2 Ho: Control<Treatment

------------------------------------------------------------------ ----------

ORIGINAL TRANSFORMED ISOTONIZED

GROUP IDENTIFICATION N MEAN MEAN MEAN

------ -------------------- --- ----------- ----------- -----------

1 NEG CONTROL 3 349.6667 349.6667 349.6667

2 7.39 3 333.6667 333.6667 333.6667

3 14.3 3 213.3333 213.3333 213.3333

4 28.35 3 155.6667 155.6667 155.6667

5 56.3 3 41.6667 41.6667 41.6667

6 98.05 3 0.6667 0.6667 0.6667

---------------------------------------------------------- ------------------

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 18 of 23 Title: NICOSULFURON P.SUBCAPITATA 72HR CELL DENSITY MEAN MEAS

File: 0301c Transform: NO TRANSFORMATION

William's Test

- TABLE 2 OF 2 Ho: Control<Treatment

------------- ---------------------------------------------------------------

COMPARED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEANS WILLIAMS 0.05 WILLIAMS FREEDOM USED

-------------------- ----------- ----------- ----- ----------- ------------

NEG CONTROL 349.6667

7.39 333.6667 1.0774 1.7800 k= 1, v=12

14.3 213.3333 9.1800 * 1.8700 k= 2, v=12

28. 35 155.6667 13.0630 * 1.9000 k= 3, v=12

56.3 41.6667 20.7392 * 1.9200 k= 4, v=12

98.05 0.6667 23.4999 * 1.9300 k= 5, v=12

------------------------------------- ---------------------------------------

s = 18.1888

------------------------------------------------------------------------

Estimates of EC%

--------------------------------------------------- ---------------------

Parameter Estimate 95% Bounds Std.Err. Lower Bound

Lower Upper /Estimate

EC5 7.5 4.9 11. 0.086 0.65

EC10 9.6 6.6 14. 0.076 0.69

EC25 15. 11. 20. 0.059 0.75

EC50 24. 19. 29. 0.041 0.82

Slope = 3.30 Std.Err. = 0.338

!!!Poor fit: p = 0.0014 based on DF= 3.0 12.

----- -------------------------------------------------------------------

0301C : NICOSULFURON P.SUBCAPITATA 72HR CELL DENSITY MEAN MEAS

------------------------------------------------------------------------ Observed vs. Predicted Treatment Group Means

------------------------------------------------------------------------ Dose #Reps. Obs. Pred. Obs. Pred. %Change

Mean Mean

- Pred. %Control

0.00 3.00 350. 339. 11.1 100. 0.00

7.39 3.00 334. 322. 11.5 95.2 4.83

14.3 3.00 213. 258.

- 44.8 76.2 23.8

28.4 3.00 156. 134. 22.1 39.4 60.6

56.3 3.00 41.7 35.6 6.04 10.5 89.5

98.0 3.00 0.667 6.86

- 6.19 2.03 98.0

AREA UNDER THE GROWTH CURVE

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 19 of 23 Title: NICOSULFURON P.SUBCAPITATA 72HR BIOMASS MEAN MEAS CONC

File: 0301B Transform: NO TRANSFORMATION

Shapiro

- Wilk's Test for Normality

----------------------------------------------------------------------------

D = 154636800.0000

W = 0.9832

Cr itical W = 0.8580 (alpha = 0.01 , N = 18)

W = 0.8970 (alpha = 0.05 , N = 18)

----------------------------------------------------------------------------

Data PASS normality test (alpha = 0.01). Continue analysis.

Title: NI COSULFURON P.SUBCAPITATA 72HR BIOMASS MEAN MEAS CONC

File: 0301B Transform: NO TRANSFORMATION

Levene's Test for Homogeneity of Variance

ANOVA Table

------------- ---------------------------------------------------------------

SOURCE DF SS MS F

----------------------------------------------------------------------------

Between 5 24637 600.0000 4927520.0000 0.6523

Within (Error) 12 90643200.0000 7553600.0000

----------------------------------------------------------------------------

Total 17 115280800.0000

------------------------ ---------------------------------------------------- (p

- value = 0.6656)

Critical F = 5.0643 (alpha = 0.01, df = 5,12)

= 3.1059 (alpha = 0.05, df = 5,12)

Since F < C ritical F FAIL TO REJECT Ho: All equal (alpha = 0.01)

Title: NICOSULFURON P.SUBCAPITATA 72HR BIOMASS MEAN MEAS CONC

File: 0301B Transform: NO TRANSFORMATION

ANOVA Table

---- ------------------------------------------------------------------------

SOURCE DF SS MS F

----------------------------------------------------------------------------

Between 5 9902826400.0000 1980565280.0000 153.6942

Within (Error) 12 154636800.0000 12886400.0000

----------------------------------------------------------------------------

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 20 of 23 Total 17 10057463200.0000

--------------- ------------------------------------------------------------- (p

- value = 0.0000)

Critical F = 5.0643 (alpha = 0.01, df = 5,12)

= 3.1059 (alpha = 0.05, df = 5,12)

Sin ce F > Critical F REJECT Ho: All equal (alpha = 0.05)

Title: NICOSULFURON P.SUBCAPITATA 72HR BIOMASS MEAN MEAS CONC

File: 0301B Transform: NO TRANSFORMATION

William's Test

- TABLE 1 OF 2 Ho: Control<Treatment

----------------------------------------------------------------------------

ORIGINAL TRANSFORMED ISOTONIZED

GROUP IDENTIFICATION N MEAN MEAN MEAN

------ -------------------- --- ----------- ----------- -----------

1 NEG CONTROL 3 66480.0000 66480.0000 66480.0000

2 7.39 3 55280.0000 55280.0000 55280.0000

3 14.3 3 40360.0000 40360.0000 40360.0000

4 28.35 3 29600.0000 29600.0000 29600.0000

5 56.3 3 9840.0000 9840.0000 9840.0000

6 98.05 3 80.0000 80.0000 80.0000

----------------------------------------------------------------------------

Title: NICOSULFURON P.SUBCAPITATA 72HR BIOMASS MEAN MEAS CONC

File: 0301B Transform: NO TRANSFORMA TION

William's Test

- TABLE 2 OF 2 Ho: Control<Treatment

----------------------------------------------------------------------------

COMPARED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEANS WILLIAMS 0.05 WILLIAMS FREEDOM USED

-------------------- ----------- ----------- ----- ----------- ------------

NEG CONTROL 66480.0000

7.39 55280.0000 3.8212 * 1.7800 k= 1, v=12

14.3 40360.0000 8.9115 * 1.8700 k= 2, v=12

28.35 29600.0000 12.5826 * 1.9000 k= 3, v=12

56.3 9840.0000 19.3243 * 1.9200 k= 4, v=12

98.05 8 0.0000 22.6542 * 1.9300 k= 5, v=12

----------------------------------------------------------------------------

s = 3589.7632

------------------------------------------------------------------------

Estimates of EC%

------------------------------------------------------------------------

Parameter Estimate 95% Bounds Std.Err. Lower Bound

Lower Upper /Estimate

EC5 7.6 4.7 12. 0.10 0.61

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 21 of 23 EC10 9.9 6.4 15. 0.087 0.65

EC25 15. 11. 21. 0.067 0.72

EC50 25. 20. 31. 0.047 0.79

Slope = 3. 20 Std.Err. = 0.369

!!!Poor fit: p < 0.001 based on DF= 3.00 12.0

------------------------------------------------------------------------

0301B : NICOSULFURON P.SUBCAPITATA 72HR BIOMASS MEAN MEAS CONC

-------------------------------------- ---------------------------------- Observed vs. Predicted Treatment Group Means

------------------------------------------------------------------------ Dose #Reps. Obs. Pred. Obs. Pred. %Change

Mean Mean

- Pred. %Control

0.00 3.00 6.65e+04 6.08e+04 5.66e+03 100. 0.00

7.39 3.00 5.53e+04 5.80e+04

- 2.74e+03 95.4 4.60

14.3 3.00 4.04e+04 4.74e+04

- 7.00e+03 77 .9 22.1

28.4 3.00 2.96e+04 2.60e+04 3.63e+03 42.7 57.3

56.3 3.00 9.84e+03 7.76e+03 2.08e+03 12.8 87.2

98.0 3.00 80.0 1.71e+03

- 1.63e+03 2.81 97.2

GROWTH RATE

Title: NIC OSULFURON P.SUBCAPITATA 72HR GROWTH RATE MEAN MEAS

File: 0301G Transform: NO TRANSFORMATION

Shapiro

- Wilk's Test for Normality

----------------------------------------------------------------- -----------

D = 1.4335

W = 0.7555

Critical W = 0.8580 (alpha = 0.01 , N = 18)

W = 0.8970 (alpha = 0.05 , N = 18)

----------------------------------------------------------------------------

Data FAIL norma lity test (alpha = 0.01). Try another transformation.

Warning

- The first three homogeneity tests are sensitive to non

normality

and should not be performed with this data as is.

Title: NICOSULFURON P.SUBCAPITATA 72HR GROWTH RATE MEAN MEAS

File: 0301G Transform: NO TRANSFORMATION

Levene's Test for Homogeneity of Variance

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 22 of 23 ANOVA Table

-------------------------------------------------- --------------------------

SOURCE DF SS MS F

----------------------------------------------------------------------------

Between 5 0.5759 0.1152 1.872 4

Within (Error) 12 0.7382 0.0615

----------------------------------------------------------------------------

Total 17 1.3141

------------------------------------------------------------- --------------- (p

- value = 0.1732)

Critical F = 5.0643 (alpha = 0.01, df = 5,12)

= 3.1059 (alpha = 0.05, df = 5,12)

Since F < Critical F FAIL TO REJECT Ho: All eq ual (alpha = 0.01)

Title: NICOSULFURON P.SUBCAPITATA 72HR GROWTH RATE MEAN MEAS

File: 0301G Transform: NO TRANSFORMATION

Kruskal

- Wallis' ANOVA by Ranks

- TABLE 1 OF 2

------------------------- ---------------------------------------------------

TRANSFORMED MEAN CALCULATED IN RANK

GROUP IDENTIFICATION MEAN ORIGINAL UNITS SUM

----- -------------------- ----------- ------ ------------ ------------

1 NEG CONTROL 8.1333 8.1333 45.0000

2 7.39 8.0667 8.0667 42.0000

3 14.3 7.4467 7.4467 30.0000

4 28.35 7.3233 7.3233 33.0000

5 56.3 5.1767 5.1767 15.0000

6 98.05 0.0000 0.0000 6.0000

--------------------------- -------------------------------------------------

H Value = 13.6881 Critical H = 11.0705 (alpha = 0.05 , df = 5)

(p

- value 0.0177)

Since Calc H > Crit H REJECT Ho:All groups are equal at alph a = 0.05

Title: NICOSULFURON P.SUBCAPITATA 72HR GROWTH RATE MEAN MEAS

File: 0301G Transform: NO TRANSFORMATION

Dunn's Multiple Comparison

- Kruskal

- Wallis

- TABLE 2 OF 2

------------------------ ----------------------------------------------------

GROUP

TRANSFORMED ORIGINAL 0 0 0 0 0 0

GROUP IDENTIFICATION MEAN MEAN 6 5 4 3 2 1

----- --------------- ----------- --------- - - - - - -

6 98.05 0.0000 0.0000 \

5 56.3 5.1767 5.1767 . \

4 28.35 7.3233 7.3233 . . \

3 14.3 7.4467 7.4467 . . . \

2 7.39 8.0667 8.0667 . . . . \

1 NEG CONTROL 8.1333 8.1333 * . . . . \

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

- V9360) to Algae

( Pseudokirchneriell a subcapitata )

PMRA Submission Number 

### {........} EPA MRID Number 46370301

Page 23 of 23 ----------------------------------------------------------------------------

* = significant difference (alpha = 0.05) . = no significant difference

Table q value = 2.9360 (0.05, 6) SE = 4.3499