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} 

S1gna . t 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 An unpublished study performed by E.l. du Pont de Nemours and 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 

(Pseudokirchneriella 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

Chemical 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-dimethoxy-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 Reviewer: 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): {......................................} 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) 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 Laboratory 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 external party for conducting this study nor to establish 
Data Evaluation Report on the Acute Toxicity of Nicosulfuron (DPX-V9360) to Algae 

(Pseudokirchneriella 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 determine for each study, on a case-bycase 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 concentrations were <0.0015 (<LOD, negative control), 7.39, 14.3, 28.35, 56.3, and 98.05 mg ai/L. The 

NOAEC and EC50/IC50 values based on cell density were 7.39 and 24 mg a.i/L, respectively. The NOAEC and EC50/IC50

values based on area under the growth curve were <7.39 and 25 mg a.i/L, respectively. The NOAEC and EC50/IC50

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 compared to the control ranged from 5 to 100%. 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 guideline 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

EC05: 7.5 mg a.i./L 95% C.I.: 4.9 to 11 mg a.i./L

EC50: 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

EC05: 7.6 mg a.i./L 95% C.I.: 4.7 to 12 mg a.i./L

EC50: 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

EC05: Not calculable 95% C.I.: Not calculable

EC50*: 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 
Data Evaluation Report on the Acute Toxicity of Nicosulfuron (DPX-V9360) to Algae 

(Pseudokirchneriella subcapitata)

PMRA Submission Number {........} EPA MRID Number 46370301

Page 3 of 23

nominal formulation concentrations. All other toxicity 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 

(Pseudokirchneriella 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) Guideline 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 noted:

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 at 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 growing 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 minute. 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 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 deviation 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.

COMPLIANCE: Signed and dated GLP, Quality Assurance and Data Confidentiality 

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

EPA FIFRA (40 CFR part 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. Initial 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 conditions of 

test chemicals: Ambient (for purity measurement)
Data Evaluation Report on the Acute Toxicity of Nicosulfuron (DPX-V9360) to Algae 

(Pseudokirchneriella subcapitata)

PMRA Submission Number {........} EPA MRID Number 46370301

Page 5 of 23

Physicochemical properties of nicosulfuron

Parameter Values Comments

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

stirring

Vapor pressure not reported

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 tier I testing, only one species, S. capricornutum, to be 

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

tested. 

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

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

Strain: Not reported

Source: In-house cultures originally obtained from the Department of BotanyCulture Collection of Algae-The University of Texas at Austin, Austin, 

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 nominal 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 nicosulfuron, respectively.

b. Definitive Study

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

(Pseudokirchneriella 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 growing, normally after 

an incubation period of about 3 days. 

When the algal cultures contain 

deformed or abnormal cells, they must 

be discarded.

Test system

Static/static renewal

Renewal rate for static renewal

Static

N/A

EPA expects the test concentrations to 

be renewed every 3 to 4 days (one 

renewal for the 7 day test, 3-4 renewals 

for the 14 day test). 

Incubation facility

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 

(Pseudokirchneriella 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

Na2EDTAx2H2O

NaHCO3

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 

chelating agents (e.g. EDTA) in the 

nutrient medium for optimum cell 

growth. Lower concentrations of 

chelating agents (down to one-third 

of the normal concentration 

recommended for AAP medium) may 

be used in the nutrient medium used 

for test solution preparation if it is

suspected that the chelator will 

interact with the test material. 

ASTM reference, E1415-91and D 

3978-80 (reapproved 1987).

If non-standard nutrient medium 

was used, detailed composition 

provided (Yes/No)

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)

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 

(Pseudokirchneriella subcapitata)

PMRA Submission Number {........} EPA MRID Number 46370301

Page 8 of 23

Parameter Details Remarks

Criteria

Initial cells density 1x104 cells/mL

EPA requires an initial number of 3,000 

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

required.

OECD recommends that the initial cell 

concentration be approximately 10,000 

cells/ml for S. capricornutum and S. 

subspicatus. When other species are 

used the biomass should be 

comparable.

Number of replicates

Control:

Solvent control:

Treatments:

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 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 (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 

(Pseudokirchneriella subcapitata)

PMRA Submission Number {........} EPA MRID Number 46370301

Page 9 of 23

Parameter Details Remarks

Criteria

Nominal (corrected for purity):

Mean measured*:

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 and, 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 chromatography.

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: 20ΕC, 

Others: 24-25ΕC; EPA photoperiod: S. 

costatum 14 hr light/ 10 hr dark, 

Others: Continuous; EPA light: 

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

5 Klux (±15%)

OECD recommended the temperature in 

the range of 21 to25o

C maintained at ± 

2o

C and continuous uniform 

illumination provided at approximately 

8000 Lux measured with a spherical 

collector.

Reference chemical (if used)

name:

concentrations:

N/A

Other parameters, if any N/A
Data Evaluation Report on the Acute Toxicity of Nicosulfuron (DPX-V9360) to Algae 

(Pseudokirchneriella subcapitata)

PMRA Submission Number {........} EPA MRID Number 46370301

Page 10 of 23

2. Observations: 

Table 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 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

Cells were counted using 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 measurement 

technique of cell counts or chlorophyll a

OECD recommends the electronic particle 

counter, microscope with counting 

chamber, fluorimeter, spectrophotometer, 

and colorimeter. (note: in order to provide 

useful measurements at low cell 

concentrations when using a 

spectrophotometer, it may be necessary to 

use cuvettes with a light path of at least 4 

cm).

Observation intervals Every 24 hours

EPA and OECD: every 24 hours.

Other observations, if any A 96-hour recovery test was 

performed 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 349.7x104 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 349.7x104 cells/mL, yielding inhibitions compared to the 
Data Evaluation Report on the Acute Toxicity of Nicosulfuron (DPX-V9360) to Algae 

(Pseudokirchneriella 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 6648.0x104

, yielding inhibitions 

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 results 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 

(Pseudokirchneriella subcapitata)

PMRA Submission Number {........} EPA MRID Number 46370301

Page 12 of 23

Table 3: 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)

Initial cell

density (x104

cells/mL)

Cell density (x104 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

(x104

)

% 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 

(Pseudokirchneriella 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

IC50 or EC50 (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

IC50/EC50

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

(Pseudokirchneriella 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 two endpoints 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 Kruskal-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 was unable to calculate ECx values using probit analysis for growth rate because the probit model was 

unsuitable for the data.

Cell density

EC05: 7.5 mg a.i./L 95% C.I.: 4.9 to 11 mg a.i./L

EC50: 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

EC05: 7.6 mg a.i./L 95% C.I.: 4.7 to 12 mg a.i./L

EC50: 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

EC05: Not calculable 95% C.I.: Not calculable

EC50: 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 greater 

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 deviation 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 measured active ingredient concentrations, whereas the study author used 

nominal concentrations that were not corrected for purity. The reviewer used probit analysis to calculate EC50

values, whereas the study author used probit analysis to calculate the EC50 based on cell density and used inverse 

weighted least-squares non-linear regression to calculate the other EC50 values. The reviewer was unable to 

calculate an EC50 value using probit analysis for the growth rate data and therefore deferred to the value 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 

(Pseudokirchneriella 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 reported by the reviewer and the study author do not 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 test was conducted from 5-12 April 2004.

F. CONCLUSIONS: 

This study is scientifically sound. It is thus classified as acceptable. After 72 hours, the most sensitive endpoint was 

cell density, with NOAEC and EC50 values of <7.39 and 24 mg ai/L, respectively. With an EC50 of 24 mg ai /L 

Nicosulfuron would be classified as slightly toxic to Pseudokirchneriella subcapitata.

Cell density

EC05: 7.5 mg a.i./L 95% C.I.: 4.9 to 11 mg a.i./L

EC50: 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

EC05: 7.6 mg a.i./L 95% C.I.: 4.7 to 12 mg a.i./L

EC50: 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

EC05: Not calculable 95% C.I.: Not calculable

EC50*: 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 reviewer 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, 2nd ed., W.H. Freeman and Co., New York, 859 pp, 1981.
Data Evaluation Report on the Acute Toxicity of Nicosulfuron (DPX-V9360) to Algae 

(Pseudokirchneriella 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 Normality 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 

(Pseudokirchneriella 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.SUBCAPITATA 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.SUBCAPITATA 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 

(Pseudokirchneriella 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 

(Pseudokirchneriella 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

 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 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 24637600.0000 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 < Critical 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 

(Pseudokirchneriella 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)

 Since 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 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 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 80.0000 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 

(Pseudokirchneriella 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: NICOSULFURON 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 normality test (alpha = 0.01). Try another transformation.

 Warning - The first three homogeneity tests are sensitive to nonnormality

 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 

(Pseudokirchneriella 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 1.8724

 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 equal (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 alpha = 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 

(Pseudokirchneriella 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