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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 1 of 24 Data Requirement: PMRA DATA CODE {............}

EPA DP Barcode 386906

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

EPA MRID 48376401

EPA Guideline 850.4550

Test material: Nicosulfuron (DPX

- V9360) 75WG plus dry surfactant (DPX

- GFJ47)

Purity: Nicosulfuron 75WG contains 74.8% ai

Common name

Chemical name: IUPAC : not reported

CAS name : 2

- [[[[(4,6

- Dimethoxy

- 2

- pyrimidinyl)amino]carbonyl]amino]sulfonyl]

- N,N

- dimethyl

3

- pyridinecarboxamide monohydrate (for nicosulfuron (DPX

- V9360) active ingredient)

CAS No. : 111991

- 09

- 4 (for nicosulfuron active ingredient)

Synonyms : Nicosulfuron (DPX

- V9360) 75WG plus Dry Surfactant (DPX

- GFJ47) (1 :6)

, Nicosulfuron 75WG plus DPX

- GFJ47

, DPX

- V9360 75WG (Paste Extruded Granule)

, DPX

- V9360

- 123 (Batch Number)

, DPX

- V9360

- 122 (Batch Number)

, DPX

- GFJ47

- 001 (Batch Number)

, Nicosulfuron (DPX

- V9360) 75WG

, Nicosulfuron 75WG

, 3

- Pyridinecarboxamide, 2

- [[[[(4,6

- dimethoxy

- 2

- pyrimidinyl)amino]carbonyl] amino]sulfonyl]

- N,Ndimethyl, monohydrate (inverted

CAS name for nicosulfuron active ingredient) DPX

- GFJ47 (Paste Extruded Granule)

Primary Reviewer: Mia Howard Signature:

Staff Scientist, Cambridge Environmental, Inc. Date: 10/05/12

Secondary Reviewer: Teri S. Myers Signature:

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

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

EPA/ OPP/EFED/ERB5

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

- V9360) 75WG plus Dry Surfactant (DPX

- GFJ47) (1:6):

Influence on Growth and Growth rate of the Blue

- Green Alga Anabaena flos

- aquae. An unpublished study

performed by E.I. du Pont de Nemours and Company, Haskell Laboratory for Health and Environmental

Sciences, Newark, Delaware. Laboratory project ID: DuPont

- 13989. Study sponsored by E.I. du Pont de

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 2 of 24 Nemours and Company, Wilmington, De laware . Study initiated 06 February 2004 and completed 11 M ay

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 external party for conducting this study nor to

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

st udy 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. Stu dies 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 meth odology and results in determining the acceptability of the study.

EXECUTIVE SUMMARY :

In a 96

- hour acute toxicity study, cultures of blue

- green alga ( Anabaena flos

- aquae ) were exposed to Nicosulfuron

75WG plus dry surfactant (DPX

- GFJ47) at nominal total formulation concentrations of 0 (negative control), 43.75,

87.5, 175, 350, and 700 mg/L under static conditions. Nominal Nicosulfuron 75WG concentrations were 0 (negative

control), 6.25, 12.5, 25.0, 50, and 100 mg/L. Nominal DPX

- GFJ47 surfac tant concentrations were 0 (negative

control), 37.5, 75, 150, 300, and 600 mg/L. Nominal nicosulfuron active ingredient concentrations were 0 (negative

control), 4.68, 9.35, 18.7, 37.4, and 74.8 mg ai/L. Mean measured nicosulfuron active ingredient concent rations

were <LOD (negative control), 4.95, 9.96, 20.15, 40, and 77.95 mg ai/L. The NOAEC and EC 50 /IC 50 values based

on cell density were <4.95 and 18 mg a.i/L, respectively. The NOAEC and EC 50 /IC 50 values based on area under the

curve were <4.95 and 18 mg a.i/L, respectively. The NOAEC and EC 50 /IC 50 values based on growth rate were

<4.95 and 39.91 mg a.i/L, respectively. The % growth inhibition of cell density in the treated algal culture as

compared to the control ranged from 11 to 100%

. The EC50 der ived from cell density would classify nicosulfuron

as slightly toxic to Anabaena flos

- aquae

.

No morphological abnormalities were noted.

This toxicity study is classified as scientifically sound or and satisfies the guideline requirement for an acute alg al

( Anabaena flos

- aquae ) toxicity study. It is therefore classified as acceptable.

Results Synopsis

Test Organism: Blue

- green algae, Anabaena flos

- aquae Test Type (Flow

- through, Static, Static Renewal): Static

Toxicity values based on mean measured concentrations of nicosulfuron:

Cell density

EC 05 : 7.7 mg ai/L 95% C.I.: 5.2 to 11 mg ai/L

EC 50 : 18 mg ai/L 95% C.I.: 15 to 22 mg ai/L

NOAEC: <4.95 mg ai/L

Probit Slope: 4.31 ± 0.541

Area under the curve

EC 05 : 6.6 mg ai/L 95% C.I.: 4.6 to 9.4 mg ai/L

EC 50 : 18 mg ai/L 95% C.I.: 15 to 22 mg ai/L

NOAEC: <4.95 mg ai/L

Probit Slope: 3.75 ± 0.365

Growth rate

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 3 of 24 EC 05 : Not calculable 95% C.I.: N/A

EC 50 : 39.91mg ai/L * 95% C.I.: 37.68 to 42.14 mg ai/L *

NOAEC: <4.95 mg ai/L**

Probit Slo pe: N/A

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

*The EC50 and 95% confidence interval based on growth rate were not verified by the reviewer, due to a poor fit of

the probit model. The values reported here are those calculated by the study author based on nominal nicosulfuron

active ingredient concentrations.

**This value was based on the study author’s analyses. The study author originally reported the NOAEC based on

growth rate as <4.68 mg ai/L, based on nominal n icosulfuron exposure concentrations. The reviewer converted the

NOAEC to the equivalent mean measured nicosulfuron concentration.

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 4 of 24 I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: The study as conducted according to the US EPA OPPTS Ecological

Effects Test Guidelines. The following deviations from the US EPA

OCSPP Ecological Effects Test Guidelines number 850.4550 (2012) were

noted:

1. Three replicates were used per level. A minimum of four replicates is required.

2. Test vessels were shaken on a shaking table at 105 rpm. It is recommended that test vessels be shaken

by hand once or twice per day rather than continuously shaken.

3. The pH of the test medium was 6 .28 to 7.41 at test initiation. It is recommended that the pH of the

medium be 7.5 ± 0.1. Lower initial pH values were associated with higher test concentrations.

4. The storage conditions of the test substance were not reported.

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

dilution water we re not reported.

6. The age of the algal inoculum at test initiation was not reported. A logarithmically growing culture,

typically 3 to 7 days old, is recommended.

7. The concentrations of test substance were not low enough to determine a NOAEC value.

These d eviations did not affect the validity of the study. While cyanobacteria may be influenced in changes in

pH the trends observed in this study did not appear to be pH dependent. Three replicates provided sufficient

statistical power to result treatment d ifferences and thus arrive at appropriate endpoints and conclusions.

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

statements were provided. The study was conducted in compliance with the

US EPA FIFRA (40 CFR part 160) GLP standards with one exception: no

characterization was performed on the blend used in this study. However,

Nicosulfuron 75WG was characterized.

A. MATERIALS:

1. Test material Nicosulfuron (DPX

- V9360) 75WG plus dry surfactant (DPX

- GFJ47)(1:6)

Description: Nicosulfuron 75WG is a light brown granule. The dry surfactant (DPX

GFJ47) was a brown solid.

Lot No./Batch No. :

Purity: Nicosulfuron 75WG contains 74.8% active ingredient.

Stability of compound

under test conditions: Stable. Day 4 measured nicosulfuron concentrations ranged from 105

- 109% of day 0 measured concentrations.

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

compound)

Storage conditions of

test chemicals: Not reported

Physicochemical properties of nicosulfuron.

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 5 of 24 Parameter Values Comments

Water solubility at 20 Ε C Not reported 99.9 mg

nicosulfuron/L (132

mg/L Nicosulfuron

75 WG) in 20X

AAP medium with

785 mg/L DPX

GFJ47 dry

surfactant

Vapor pressure Not reported

UV absorption Not reported

pKa Not reported

Kow Not reported

2. Test organism :

Name: Blue

- green algae, Anabaena flos

- aquae

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 Department of Botany

, Culture Collection of Alg ae, The University of Austin, Austin, Texas

Age of inoculum : Not reported

Method of cultivation: Cultured and tested in AAP nutrient medium

B. STUDY DESIGN:

1. Experimental Conditions

a. Range

- finding study. A 96

- hour range

- finding study was performed in which algae were exposed to

a negative control and nominal concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 mg ai/L (it was not

specified if the concentrations were total formulation, Nicosulfuron 75WG, or active ingredient

concentrations). Inhibiti ons of cell density relative to the negative control were

- 15,

- 26,

- 17,

- 9,

- 22,

and 99% for concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 mg ai/L, respectively.

b. Definitive Study

Table 1: Experimental Parameters

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 6 of 24 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

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 growing, normally after

an incubation period of about 3 days.

When the algal cultures contain

deformed or abnormal cell s, 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 on a shaker

table

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

50 mL Sterile Erlenmeyer flaks fitted with

foam stoppers

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

pH at test initiation: 6.28 to 7.41 The pH of the medium should be 7.5

±0.1 at the beginning of the test.

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 7 of 24 Parameter Details Remarks

Criteria

pH at test termination:

Chelator used:

Carbon source:

Salinity ( for marine algae ): 7.20 to 7.83

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

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

s uspected 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) 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

Deionized

Not reported

Not reported

Not reported

Not reported

Not reported 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 700 mg formulation/L stock

solution prepared in AAP nutrient

medium.

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 8 of 24 Parameter Details Remarks

Criteria

Aeration or agitation Shaken at 105 rpm OCSPP (2012) guidelines

recommend that test vessels be

shaken by hand once or twice per

day rather than continuously shaken

in order to prevent clumping of the

cells.

Initial cells density 10,000 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 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: 3

N/A

3 OCSPP (2012) guidelines require a

minimum of four replicates per level

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 total formulation :

Nominal Nicosulfuron 75WG: 0 (negative control), 43.75, 87.5,

175, 350, and 700 mg/L

0 (negative control), 6.25, 12.5, *Mean measured nicosulfuron

conc entrations were calculated by

the reviewer as the arithmetic mean

of the measured nicosulfuron

concentrations at days 0 and 4.

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 9 of 24 Parameter Details Remarks

Criteria

Nominal DPX

- GFJ47:

Nominal nicosulfuron active

ingredient:

M ean m easured * nicosulfuron

active ingredient : 25.0, 50.0, and 100 mg/L

0 (negative control), 37.5, 75, 150,

300, and 600 mg/L

0 (negative control), 4.68, 9.35,

18.7, 37.4, and 74.8 mg ai/L

<0.0097 (<LOD, negative control),

4.95, 9.96, 20.15, 40.00, and 77.95

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

Method and interval of analytical

verification The concentration of nicosulfuron in

the test solutions was measured on

days 0 and 4 using high performance

liquid chromatography

Test conditions

Temperature:

Photoperiod:

Light intensity and quality: 24.8 to 25.0°C

Continuous

1830 to 2070 lux , cool

- 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 unifo rm 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:

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 10 of 24 Table 2: Observation parameters

Parameters Details Remarks

Criteria

Parameters measured including the

growth inhibition/other toxicity

symptoms Healthy cell count

Area under the growth curve

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.

Measurement technique for cell

density and other end points Cell density was determined

using a hemacytometer and

compound microscope. 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 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

u seful 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 visually observed for

abnormalities . A recovery study

was performed subsequently to

the definitive test in order to

determine if the effect of the test

substance was algistatic or

algicidal.

Indicate whether there was an

exponential growth in the control Yes. After 96 hours, the cell

density of the negative control

was 656.0x10 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 96 hours, the cell density of the negative control was 656.0x10 4 cells/mL, yielding inhibitions compared

to the negative control of 11, 40, 52, 95, and 100% for nominal nicosulfuron active ingredient concentrations of

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 11 of 24 4.86, 9.35, 18.7, 37.4, and 74.8 mg ai/L, respectively. The NOAEC and E C50 values calculated by the study

author based on nominal nicosulfuron active ingredient concentrations were <4.68 and 14.77 mg ai/L,

respectively

.

After 96 hours, the growth rate of the negative control was 0.0676, yielding inhibitions compared to the

negative control of 2, 8, 12, 46, and 100% for nominal nicosulfuron active ingredient concentrations of 4.86,

9.35, 18.7, 37.4, and 74.8 mg ai/L, respectively. The NOAEC and EC50 values calculated by the study author

based on nominal nicosulfuron active i ngredient concentrations were <4.68 and 39.9 mg ai/L, respectively.

After 96 hours, the area under the curve of the negative control was 112.120 x10 6 , yielding inhibitions compared

to the negative control of 12, 47, 53, 92, and 100 % for nominal nicosulfuro n active ingredient concentrations of

4.86, 9.35, 18.7, 37.4, and 74.8 mg ai/L, respectively. The NOAEC and EC50 values calculated by the study

author based on nominal nicosulfuron active ingredient concentrations were <4.68 and 15.48 mg ai/L,

respectivel y.

No morphological abnormalities were noted. There was an increase in pH during the study. From the results of

the recovery test, the effects of the test substance were determined to be algistatic within 144 hours for nominal

nicosulfuron concentrations < 7 4.8 mg/L.

Table 3: Effect of Nicosulfuron 75WG plus dry surfactant (DPX

- GFJ47) on algal growth ( blue

- green algae,

Anabaena flos

- aquae )

Treatment

Total formulation

(nominal

Nicosulfuron 75

WG) [nominal

DPX

- GFJ47

surfactant]

{nominal

nicosulfuron active

ingredient} <mean

measured

nicosulfuron active

ingredient>

concentrations

mg/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

Negative control 1 26.7 109.3 656.0 N/A

43.75 (6.25) [37.5]

{4.86} <4.95> 1 22.7 93.3 585.3 11

87.5 (12.5 ) [75]

{9.35} <9.96> 1 14.7 89.3 390.6 40

175 (25.0) [150]

{18.7} <20.15> 1 93.3 56.0 312.0 52

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 12 of 24 350 (50) [300]

{37.4} <40.00> 1 8.0 14.7 34.7 95

700 (100) [600]

{74.8} <77.95> 1 2.7 0 0 100

Reference chemical

(if used) N/A

Table 4: Effect of Nicosulfuron 75WG plus dry surfactant (DPX

- GFJ47) on algal growth (blue

- green algae,

Anabaena flos

- aqaue )

Treatment

Total formulation

(nominal

Nicosulfuron 75

WG) [nominal

DPX

- GFJ47

surfactant]

{nominal

nicosulfuron active

ingredient} <mean

measured

nicosulfuron active

ingredient>

concentrations

mg/L Growth rate

(0

- 96 hour) %

inhibition* Area under

the growth

curve (x10 6 ) %

inhibition*

Negative control 0.0676 N/A 112.120 N/A

43.75 (6.25) [37.5]

{4.86} <4.95> 0.0664 2 98.680 12

87.5 (12.5) [75]

{9.35} <9.96> 0.0622 8 71.120 37

175 (25.0) [150]

{18.7} <20.15> 0.0598 12 53.000 53

350 (50) [300]

{37.4} <40.00> 0.0365 46 9.480 92

700 (100) [600]

{74.8} <77.95> 0.000 100 0.400 100

*Calculated by the reviewer relative to the control , using mean values

Table 5 : Statistical endpoint values.* (calculated by the study author)

Statistical Endpoint Nominal total Nominal Nominal DPX

- Nominal

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 13 of 24 formulation

concentration

(mg/L) Nicosulfuron 75

WG concentration

(mg/L) GFJ47 surfactant

(mg/L) nicosulfuron active

ingredient (mg

ai/L)

Cell density

NOAEC

EC50 (95% C.I.) <43.75

138.25 (115.78 to

160.72) <6.25

19.75 (16.54 to

22.96) <37.5

118.5 (99.24 to

137.76) <4.68

14.77 (12.37 to

17.17)

Area under the growth curve

NOAEC

EC50 (95% C.I.) <43.75

144.83 (127.61 to

161.98) <6.25

20.69 (18.23 to

23.14) <37.5

124.14 (109.38 to

138.84) <4.68

15.48 (13.64 to

17.31)

Growth rate

NOAEC

EC50 (95% C.I.) <43.75

373.52 (352.66 to

394.38) <6.25

53.36 (50.38 to

56.34) <37.5

320.16 (302.28 to

338.04) <4.68

39.91 (37.68 to

42.14)

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

B. REPORTED STATISTICS:

The study author statistically analyzed the endpoints for cell density, area under the growth curve, and growth

rate using nominal total formulation, Nicosulfuron 75WG, DPX

- GFJ47, and nicosulfuron active ingredient

concentrations. T he EC50 values and 95% confidence intervals 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 based on cell density and area under the c urve were determined using Jonckheere

- Terpstra tests.

The NOAEC value based on growth rate was determined using a non

- parametric multiple comparison test.

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 14 of 24 C. VERIFICATION OF STATISTICAL RESULTS:

Statistical Method: The reviewer analyzed the endpoints for cell density, area under the curve, and growth rate

using mean measured nicosulfuron (active ingredient) concentrations. The reviewer calculated the mean

measured concentrations as the arithmetic mean of the day 0 and day 4 measured 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 all assumptions of normality and homogeneity of variance and

therefore the NOAEC values based on these endpoints were determined using ANOVA followed by William’s

tests. The growth rate data met the assumptions of homogeneity of variance, but not normality, and

transformation of the data did not correc t the issue. Therefore, Wilcoxon’s Rank Sum Test was used to

calculate the NOAEC value based on growth rate. The preceding analyses were all performed using Toxstat 3.5

statistical software. Despite 100% inhibition at the highest test level, Wilcoxon’s test failed to detect any

significant differences in growth rate compared to the negative control. Because of the inhibition observed, the

reviewer deferred to the NOAEC value calculated by the study author in the Executive Summary and

Conclusions section s of this report. ECx values for cell density and area under the growth curve were calculated using probit analysis via Nuthatch statistical software. The ECx value based on growth rate could

not be determined using the probit method and therefore the re viewer deferred to the study author’s calculated

EC50 value in the Executive Summary and Conclusions sections

Cell density

EC 05 : 7.7 mg ai/L 95% C.I.: 5.2 to 11 mg ai/L

EC 50 : 18 mg ai/L 95% C.I.: 15 to 22 mg ai/L

NOAEC: <4.95 mg ai/L

Probit Slope: 4.31 ± 0.541

Area under the curve

EC 05 : 6.6 mg ai/L 95% C.I.: 4.6 to 9.4 mg ai/L

EC 50 : 18 mg ai/L 95% C.I.: 15 to 22 mg ai/L

NOAEC: <4.95 mg ai/L

Probit Slope: 3.75 ± 0.365

Growth rate

EC 05 : Not calculable 95% C.I.: N/A

EC 50 : Not calculable 95% C.I.: N/A

NOAEC: 77.95 mg ai/L

Probit Slope: N/A

D. STUDY DEFICIENCIES:

The deviations did not affect the validity of the study. While cyanobacteria may be influenced in changes in

pH the trends observed in this study did not appear to be pH dependent. Three replicates provided sufficient

statistical power to result treatment differences and thus arrive at appropriate endpoints and conclusions.

E. REVIEWER ’ S COMMENTS:

The results of the reviewer generally agree with those of the study author. The reviewer based all analyses on

mean measured nicosulfuron concentrations, whereas the study author used nominal concentrations only. The

statistical methods of the reviewer and study author also differed. Therefore, the results of the reviewer and

study author differ slightly.

The study author did not specify how the NOAEC based on growth rate was determined. It was only noted that

“appropriate” non

- parametric multiple comparison methods were used.

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 15 of 24 The in

- life phase of the definitive test was conducted from 16

- 20 February 2004.

F. CONCLUSIONS:

This study is scientifically sound and generally meets the guideline requirements for a toxicity test with a

freshwater cyanobacteria. It is therefore classified as acceptable

. The most sensitive endpoints based on mean

measured concentrations were cell density and area under the growth curve, with NOAEC and EC50 values of

<4.95 and 18 mg ai/L, respectively.

Cell density

EC 05 : 7.7 mg ai/L 95% C.I.: 5.2 to 11 mg ai/L

EC 50 : 18 mg ai/L 95% C.I.: 15 to 22 mg ai/L

NOAEC: <4.95 mg ai/L

Probit Slope: 4.31 ± 0.541

Area under the curve

EC 05 : 6.6 mg ai/L 95% C.I.: 4.6 to 9.4 mg ai/L

EC 50 : 18 mg ai/L 95% C. I.: 15 to 22 mg ai/L

NOAEC: <4.95 mg ai/L

Probit Slope: 3.75 ± 0.365

Growth rate

EC 05 : Not calculable 95% C.I.: N/A

EC 50 : 39.91mg ai/L * 95% C.I.: 37.68 to 42.14 mg ai/L *

NOAEC: <4.95 mg ai/L**

Probit Slope: N/A

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

*The EC50 and 95% confidence interval based on growth rate were not verified by the reviewer, due to a poor fit of

the probit model. The values reported here are those calculated by the study author based on nominal nicosulfuron

active ingredient concentrations.

**This value was based on the study author’s analyses. The study author originally reported the NOAEC based on

growth rate as <4.68 mg ai/L, based on nominal nicosulfuron exposure concentrations. The reviewer converted the

NOAEC to the equivalent mean measured nicosulfuron concentration.

III. REFERENCES :

1. DuPont Haskell Laboratory (2004). Solubility of Nicosulfiiron in Algal Media Solution

(20X AAP) of Nicosulfiiron (DPX

- V9360) 75WG plus Dr y Surfactant (DPX

- GFJ47) (1:6).

Unpublished report, DuPont

- 14106.

2. Miller, W.E.; Greene, 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.

3. 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.

4. Environmental Protection Agency, National Eutrophication Research Program (1971). Algal

Assay Procedure: Bottle Test (AAP:BT). National Environmental Research Center,

Corvallis, OR 9330.

5. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods. 6th edition. The I owa State

University Press, Ames.

6. Bruce, R.D. and Versteeg, D.J. (1992). A statistical procedure for modeling continuous

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 16 of 24 toxicity data. Environ. Toxicol. Chem. 11:1485

- 1494.

7. Hochberg, Y. and. Tamhane, A.C. (1987). Multiple Comparison Procedures, Wile y, New

York.

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

- free k

- sample test against ordered alternatives,

Biometrika41, 133.

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

10. SAS Software Version 9.1, SAS Institute, Cary, NC, 2004.

11. Slob, W . (2002). Dose

- Response Modeling of Continuous Endpoints, Toxicological

Sciences 66, 298

- 312.

12. Terpstra, T.J. (1952). The Asymptotic Normality and Consistency of Kendall's Test Against

Trend, When Ties are Present in One Ranking, Indag. Math. 14, 327

- 33 3.

13. U.S.EPA. (1995). The Use of the Benchmark Dose Approach in Health Risk Assessment,

Risk Assessment Forum, EPA/630/R

- 94/007, United States Environmental Protection

Agency, Washington, DC. Principal authors: K. Crump, B. Allen, E. Faustman.

14. Westfa ll, P.H., Tobias R.D., Rom D., Wolfmger 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+SURFACTANT A.FLOS

- AQUAE CELL DENSITY

File: 6401C Transform: NO

TRANSFORMATION

Shapiro

- Wilk's Test for Normality

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

-

D = 694400.0000

W = 0.9481

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+SURFACTANT A.FLOS

- AQUAE CELL DENSITY

File: 6401C Transform: NO

TRANSFORMATION

Levene's Test for Homogeneity of Variance

ANOVA Table

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

-

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 17 of 24 SOURCE DF SS MS F

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

-

Between 5 126577.7778 25315.5556 0.7978

Within (Error) 12 380800.0000 31733.3333

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

-

Total 17 507377.7778

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

- (p

- value = 0.5719)

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+SURFACTANT A.FLOS

- AQUAE CELL DENSITY

File: 6401C Transform: NO

TRANSFORMATION

ANOVA Table

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

-

SOURCE DF SS MS F

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

-

Between 5 111479111.1111 22295822.2222 385.2965

Within (Error) 1 2 694400.0000 57866.6667

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

-

Total 17 112173511.1111

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

- (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: N ICOSULFURON+SURFACTANT A.FLOS

- AQUAE CELL DENSITY

File: 6401C Transform: NO

TRANSFORMATION

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 18 of 24 William's Test

- TABLE 1 OF 2 Ho: Control<Treatment

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

-

ORIGINAL TRANSFORMED ISOTONIZED

GROUP IDENTIFICATION N MEAN MEAN MEAN

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

-

1 NEG CONTROL 3 6560.0000 6560.0000 6560.0000

2 4.95 3 5853.3333 5853.3333 5853.3333

3 9.96 3 3906.6667 3906.6667 3906.6667

4 20.15 3 3120.0000 3120.0000 3120.0000

5 40 3 346.6667 346.6667 346.6667

6 77.95 3 0.0000 0.0000 0.0000

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

-

Title: NICOSULFURON+SURFACTANT A.FLOS

- AQUAE CELL DENSITY

File: 6401C 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 6560.0000

4.95 5853.3333 3.5979 * 1.7800 k= 1,

v=12

9.96 3906.6667 13.5090 * 1.8700 k= 2,

v=12

20.15 3120.0000 17.5142 * 1.9000 k= 3,

v=12

40 346.6667 31.6341 * 1.9200 k= 4,

v=12

77.95 0.0000 33.3991 * 1.9300 k= 5,

v=12

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

-

s = 240.5549

------------------------------------------------------------------------ Estimates of EC%

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

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 19 of 24 Parameter Estimate 95% Bounds Std.Err. Lower Bound

Lower Upper /Estimate

EC5 7.7 5.2 11. 0.079 0.68

EC10 9.3 6.7 13. 0.069 0.71

EC25 13. 9.9 17. 0.053 0.77

EC50 18. 15. 22. 0.037 0.83

Slope = 4.31 Std.Err. = 0.541

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

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

6401C : NICOSULFURON+SURFACTANT A.FLOS

- AQUAE CELL DENSITY

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

Observed vs. Predicted Treatment Group Means

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

Mean Mean

- Pred. % Control

0.00 3.00 6.56e+03 5.85e+03 713. 100. 0.00

4.95 3.00 5.85e+03 5.81e+03 46.1 99.3 0.684

9.96 3.00 3.91e+03 5.13e+03

- 1.22e+03 87.6 12.4

20.1 3.00 3.12e+03 2.5 5e+03 569. 43.6 56.4

40.0 3.00 347. 436.

- 88.9 7.45 92.6

78.0 3.00 0.00 20.8

- 20.8 0.356 99.6

AREA UNDER THE CURVE

Title: NICOSULFURON+SURFACTANT A.FLOS

- AQUAE AREA UNDER CURVE

File: 6401A Transform: NO

TRANSFORMATION

Shapiro

- Wilk's Test for Normality

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

-

D = 151.3920

W = 0.9909

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

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

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

-

Da ta PASS normality test (alpha = 0.01). Continue analysis.

Title: NICOSULFURON+SURFACTANT A.FLOS

- AQUAE AREA UNDER CURVE

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 20 of 24 File: 6401A Transform: NO

TRANSFORMATION

Levene's Test for Homogeneity of Variance

ANOVA Table

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

-

SOURCE DF SS MS F

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

-

Between 5 24.3616 4.8723 0.6156

Within (Error) 12 94.9824 7.9152

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

-

Total 17 119.3440

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

- (p

- value = 0.6906)

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+SURFACTANT A.FLOS

- AQUAE AREA UNDER CURVE

File: 6401A Transform: NO

TRANSFORMATION

ANOVA Table

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

-

SOURCE DF SS MS F

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

-

Between 5 31353.6448 6270.7290 497.0457

Within (Error) 12 151.3920 12.6160

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

-

Total 17 31505.0368

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

- (p

- value = 0.0000)

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

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

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 21 of 24 Since F > Critical F REJECT Ho: All equal (alpha = 0.05)

Title: NICOSULFURON+SURFACTANT A.FLOS

- AQUAE ARE A UNDER CURVE

File: 6401A 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 3 112.1200 112.1200 112.1200

2 4.95 3 98.6800 98.6800 98.6800

3 9.96 3 71.1200 71.1200 71.1200

4 20.15 3 53.0000 53. 0000 53.0000

5 40 3 9.4800 9.4800 9.4800

6 77.95 3 0.4000 0.4000 0.4000

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

-

Title: NICOSULFURON+SURFACTANT A.FLOS

- AQUAE AREA UNDER CURVE

File: 6401A 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 112.1200

4.95 98.6800 4.6343 * 1.7800 k= 1,

v=12

9.96 71.1200 14.1374 * 1.8700 k= 2,

v=12

20.15 53.0000 20.385 4 * 1.9000 k= 3,

v=12

40 9.4800 35.3917 * 1.9200 k= 4,

v=12

77.95 0.4000 38.5226 * 1.9300 k= 5,

v=12

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

-

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 22 of 24 s = 3.5519

------------------------------------------------------------------------ Estimates of EC%

----------------------------------------------------------------------- - Parameter Estimate 95% Bounds Std.Err. Lower Bound

Lower Upper /Estimate

EC5 6.6 4.6 9.4 0.071 0.70

EC10 8.3 6.1 11. 0.063 0.74

EC25 12. 9.5 15. 0.048 0.79

EC50 18. 15. 22. 0.034 0.85

Slope = 3.73 Std.Err. = 0.365

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

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

6401A : NICOSULFURON+SURFACTANT A.FLOS

- AQUAE AREA UNDER CURVE

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

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

Mean Mean

- Pred. %Control

0.00 3.00 112. 102. 9.67 100 . 0.00

4.95 3.00 98.7 101.

- 1.98 98.3 1.74

9.96 3.00 71.1 85.7

- 14.5 83.6 16.4

20.1 3.00 53.0 44.6 8.36 43.6 56.4

40.0 3.00 9.48 10.4

- 0.948 10.2 89.8

78.0 3.00 0.400 0.958

- 0.558 0.936 99.1

GROWTH RATE

Title: NICOSULFURON+SURFACTANT A.FLOS

- AQUAE GROWTH RATE

File: 6401G Transform: NO

TRANSFORMATION

Shapiro

- Wilk's Test for Normality

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

-

D = 0.0000

W = 0.7892

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

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 23 of 24 W = 0.8970 (alpha = 0.05 , N = 18)

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

-

Data FAIL normality 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+SURFACTANT A.FLOS

- AQUAE GROWTH RATE

File: 6401G Transform: NO

TRANSFORMATION

Levene's Test for Homogeneity of Variance

ANOVA Table

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

-

SOURCE DF SS MS F

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

-

Between 5 0.0000 0.0000 2.5136

Within (E rror) 12 0.0000 0.0000

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

-

Total 17 0.0000

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

- (p

- value = 0.0886)

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+SURFACTANT A.FLOS

- AQUAE GROWTH RATE

File: 6401G Transform: NO

TRANSFORMATION

Wilcoxon's Rank Sum Test w/ Bonferroni Adjustment Ho: Control<Treatment

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

-

MEAN IN RANK CRIT. SIG

GROUP IDENTIFICATION ORIGINAL UNITS SUM VALUE REPS 0.05

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

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

- V9360) 75WG plus

Dry Surfactant (DPX

- GFJ47) (1:6) to Algae, Anabaena flos

- aquae

PMRA Submission Number 

### {........} EPA MRID Number 48376401

Page 24 of 24 1 NEG CONTROL 0.0676

2 4.95 0.0664 6.00 None 3

3 9.96 0.0622 6.00 None 3

4 20.15 0.0598 6.00 None 3

5 40 0.0365 6.00 None 3

6 77.95 0.0000 6.00 None 3

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

- Critical values are 1 tailed ( k = 5 )

WARNING

- There are no critical values for this combination

of groups and replicates.