Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Data Requirement: PMRA Data Code

EPA DP Barcode

OECD Data Point

EPAMRID

EPA Guideline

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

361296,358877,358880,358882,358885

{ }

47643007

850.1300

Test material:

Common name:

Chemical name:

IN L5300-78 Purity: 79.8% ai

Tribenuron-methyl (ai)

IUPAC: methyl 2-[4-methoxy-6-methyl-1 ,3,5-triazin-2-yl(methyl)

carbamoylsulfamoyI]benzoate

CAS name: methyI2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)

methylamino]carbonyl]amino]-sulfonyl]benzoate

CAS No.: 101200-48-0 (ai)

Synonyms: Haskell No. 17,738-03

Primary Reviewer: Christie E. Padova

Staff Scientist, Dynamac Corporation

Secondary Reviewer: Teri S. Myers

Senior Scientist, Cambridge Environmental Inc.

Reviewer: Christina Wendel

USEPA/OCSPP/OPP/ERBII/Biologist

Reference/Submission No.: {. }

Signature: t . ..p~ Date: 06/16/2011

Signature: ~'Sll~

Date: 07/13/2011

Signature:~ wA

Date: 12/06/2011

Company Code

Active Code

Use Site Category:

EPA PC Code

{ } [For PMRA]

{ } [For PMRA]

{ } [For PMRA]

128887

Date Evaluation Completed: 12/06/2011

CITATION: Hutton, D.G. 1989. Chronic Toxicity of DPX-L5300-78 to Daphnia magna.

Unpublished study performed by E.!. du Pont de Nemours and Company, Inc., Newark, DE,

and sponsored by E.!. du Pont de Nemours and Company, Wilmington, DE. Laboratory

Study No. 555-89. Study initiated July 14,1989 and completed October 18, 1989.

Page 1 of30

1•••1•••11

2086465

TEXT SEARCHABLE DOCUMENT 2011
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

EXECUTIVE SUMMARY:

The 21-day-chronic toxicity of DPX-L5300-78 (a formulation of Tribenuron-methyl, 79.8% ai)

to Daphnia magna was studied under static-renewal conditions. Daphnids were exposed to

DPX-L5300-78 at nominal concentrations of

°(negative control), 31, 62, 125, 250, 500, and

1000 mg/L. Mean-measured concentrations were <3.13 «LOQ, control), 28, 54, 110, 220, 400,

and 790 mg ai/L. The reviewer concluded that a NOAEC could not be determined in this study

due to significant (p<0.05) treatment-related delays in time to first brood release at the 28, 54,

and 110 mg ai/L levels and significant (p<0.05) treatment-dependent reductions in adult length at

all levels tested. Additionally, offspring production (both total and young/day per surviving

adult) was significantly reduced (p<0.05) at the 54, 110, and 220 mg ai/L levels. Immobility of

adults was significantly (p<0.05) affected at the 220, 400, and 790 mg ai/L levels, with complete

immobility occurring at the 400 and 790 mg ai/L levels. The 21-day ECso (with 95% c.1.) for

immobilization was 216 (184-252) mg ai/L. The 21-day ECso based on reproduction parameters

was 100 (63-170) mg ai/L for total offspring and 170 (81-380) mg ai/L for offspring

produced/day per surviving adult.

Following 21 days of exposure, survival averaged 90% in the control group, and 98, 90, 80, 72,

0, and 0% in the mean-measured 28, 54, 110, 220, 400, and 790 mg ai/L treatment levels,

respectively. The differences were statistically-reduced (p<0.05) compared to the negative

control at the 220, 400 and 790 mg ai/L levels.

The mean time to first brood release was 11.4 days for the negative control, and 13.0, 13.3, 14.4,

and 12.7 days for the mean-measured 28, 54, 110, and 220 treatment levels, respectively (no

offspring were produced at the 400 and 790 mg ai/L levels). Delays were statistically-significant

(p<0.05) compared to the control at the 28, 54 and 110 mg ai/L levels.

For the control, and mean-measured 28, 54, 110, and 220 mg ai/L levels, the total numbers of

young produced were 383,333,274, 135, and 150, respectively, and the number of offspring per

surviving parent averaged 96,85,74,41, and 52, respectively. For both parameters, differences

were statistically-significant (p<0.05) compared to the control at the 54, 110, and 220 mg ai/L

levels. No males, ephippia, or immobilized young were observed in the control group or any

treatment level during the study, and no eggs were observed in the bottom of any test vessel.

Growth was measured as length at study termination. Mean lengths were 3.7, 3.7, 3.6, 3.2, and

3.1 mm for the control and mean-measured 28, 54, 110, and 220 mg ai/L levels, respectively.

Reductions for this parameter followed a dose-dependent pattern and differences were

statistically-significant (p<0.05) compared to the control at all treated levels.

Although this study is scientifically sound it does not satisfy the guideline requirement for a

chronic toxicity study with freshwater invertebrates due to the inability to establish a definitive

NOAEC «28 mg ai/L, the lowest test concentration). Based on this, the study is classified as

Supplemental and is not repairable.

Page 2 of 30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp,

PMRA Submission Number {........} EPA MRID No. 47643007

Results Synopsis

Test Organism Age (eg. 151 instar): First instar, <24 hours old

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

21-day ECso(mortality/immobility): 216 mg ai/L

21-day ECso(reproduction): 100 mg ai/L

Slope: 1.42±0.331

NOAEC: <28 mg ai/L

LOAEC: 28 mg ai/L

95% C.I.: 184-252 mg ai/L

(moving average method)

95% C.l.: 63-170 mg ai/L

Endpoints affected: Survival, time to first brood release, total young produced, young/day

per surviving adult, and growth (length)

Most sensitive endpoint(s): Time to first brood release and growth (length)

I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted following guidelines outlined in

OECD Guideline No. 202 (1984) and U.S. EPA Standard

Evaluation Procedure, Daphnia magna Life-Cycle (21-Day

Renewal) Chronic Toxicity Test" (1986).

Deviations from OPPTS 850.1300 (draft, 1996) guidance included:

1. A NOAEC could not be determined in this study due to treatment-related effects on first

day of brood production and adult length at the lowest treatment level.

2. The source ofthe dilution water used in the toxicity test was filtered well water that had

been previously used for fathead minnow culturing in a flow-through system within the

test laboratory, and filtered through a 0.8f.lm filter prior to use in the daphnid toxicity

study. The water was not tested for the presence of ammonia, therefore it is not known if

that may have had a negative impact on the health ofthe test organisms.

3. The type and quantity offood offered at each renewal period was not reported.

These deviations do affect the acceptability ofthe study.

COMPLIANCE:

A. MATERIALS:

1. Test Material:

Signed and dated GLP (1989), Quality Assurance (1989), and Data

Confidentiality claims (2008) statements were provided.

DPX-L5300-78 (formulation of Tribenuron-methyl)

Page 3 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Description: Not reported

Lot No./Batch No.: Not reported

Purity: 79.8% ai

Stability of compound

under test conditions: The stability of Tribenuron-methyl was verified analytically at all

levels weekly throughout the study. For all levels, replicate results

were within ±21%.

Storage conditions of

test chemicals: Not reported

lySICOC emrcaI properties 0 fTrr·benuron-methy.I

Parameter Values Comments

Water solubility at 20°C Not reported

Vapor pressure Not reported

UVabsorption Not reported

pKa Not reported

Kow Not reported

Ph . h

(GECD recommends water solubility, stability in water and light, pKa, Pow, and vapor

pressure oftest compound)

2. Test Organism:

Species: Daphnia magna, first instar, <24 hours old

EPA and GECD recommend Daphnia magna

Age of the parental

stock: 21 days old

EPA recommends that young daphnids ~24 hours oldfrom a

separate parental culture be used

Source: Haskell Laboratory bred culture

EPA requires all test organisms must be producedfrom laboratory

reared culture that has been maintainedfor at least 21 days at test

conditions in dilution water with renewal ofthe culture medium at

least three times per week.

Page 4 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

B. STUDY DESIGN:

1. Experimental Conditions

a. Range-finding Study: Nominal test levels for the definitive study were based upon

acute toxicity data determined prior to study initiation. The acute, static ECso

(immobilization) was>1000 mg/L for unfed Daphnia magna.

b. Definitive Study

Table 1: Experimental Parameters

Remarks

Parameter

Parental acclimation:

Period:

Conditions: (same as test or not)

Feeding:

Health (any mortality observed):

Test condition:

static renewal/flow-through:

Type of dilution system- for flow

through method.

Renewal rate for static renewal

Details

21 days

Same as test

Trout chow (Glencoe) and

yeast (Fleischmann's)

No sickness, injuries, or

abnormalities were noted in

the test organisms prior to

initiating the test.

Static-renewal

N/A

Three times per week

Page 5 of30

Criteria

Daphnia were cultured in 2-L

beakers containing ca. 1.8 L of

water and 25 daphnia per beaker.

EPA recommends that prior to

testing, daphnids that are at least

10-12 days old (those that have

had at least one brood) should be

separatedfrom the culture, put in

separate container and

maintainedfor at least 21 days to

insure that good health

conditions are present

The parent Daphnia were

transferred into fresh test

solutions containing food three

times per week.

(EPA requires consistentflow

rate of5-10 vol/24 hours, meter

systems calibrated before study

and checked twice daily during

test period)
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Remarks

Parameter

Aeration, if any

Duration ofthe test

Test vessel

Material: (glass/stainless steel)

Size (for growth and

reproduction/survival test):

Fill volume:

Source of dilution water

Details

No supplemental aeration

was provided.

21 days

Glass

250 mL (6.1-cm diameter)

Ca. 200 mL (6.8-cm depth)

The dilution water used for

testing was filtered fish tank

water (laboratory well water

that had been used for

fathead minnow culturing in

a flow-through system, then

filtered through a 0.8-/lm

filter). Water was originally

obtained from a 350-foot

encased (in bedrock), and

aerated and filtered (10 um)

prior to use.

Criteria

EPA recommends test chambers

should not be aerated

Recommended duration is 21

days.

1. Recommended Material:

Glass, No. 316 stainless steel, or

perfluorocarbon plastics

2. Recommended Size: 250 ml

with 200 ml fill volume; 100 ml

with 80 ml fill volume GECD

guideline recommends that

parent animals be maintained

individually; one per vessel, with

50 - 100 ml ofmedium in each

vessel.

Results from periodic analysis of

the well water were provided;

water quality measurements of

the dilution water were not taken

after use in the fish culturing.

The use of old fish culture water

as the dilution water is a notable

deviation. It is unknown if the

water contained ammonia as

measurements were not taken.

Therefore it is not known if

ammonia was present in the

dilution water used in the

Daphnia toxicity test; or if it may

have had a negative impact on

the health ofthe test organisms. IL- --l.- ----' -------------

Page 6 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Remarks

Parameter Details - - - - - - - - - - -- -------- - - - - - - - -- - ----------

Criteria

Recommended source ofdilution

water includes unpolluted well or

spring water that has been tested

for contaminants, or appropriate

reconstituted water (see ASTM

for details).

Water parameters: DO was ::::60% (equivalent to

Hardness 76 ± 3 mg/L as CaC03 ::::5.5 mg/L at 20°C; reviewercalculated) throughout the study.

pH Fresh soln.: 7.1 to 8.6

Aged soln.: 7.1 to 8.1 Alkalinity was 83 ± 3 mg/L as

CaC03, and conductivity was

Dissolved oxygen Fresh soln.: 8.3 to 8.8 mg/L 165 ± 8 umhos/cm.

Aged soln.: 5.5 to 8.3 mg/L

Raw water chemistry data were

Temperature 19.5 to 20.l°C not provided. However, the

chemical characteristics ofthe

Total Organic Carbon Not reported Haskell Laboratory Well Water

Particulate matter Not reported are as follows: TOC: <1.5 mg/L;

Inorganic analytes Not reported COD: <17mg/L; Solids, total

Pesticides Not reported suspended (TSS): <4 mg/L;

Chlorine Not reported Pesticides: None detected (but

detection level was not reported);

Frequency of measurements Dissolved oxygen (DO) was Chloride: 3.7 mg/L

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

Page 7 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Remarks

Parameter Details --- -------------------------------------

Criteria

measured at each renewal Recommended hardness: 160 to

period in one old and one 180 mg/L as CaCG3; GECD

new test solution from all recommends>140 mg/L as

levels. The pH was CaCG3

measured at each renewal RecommendedpH: 7. 6 to 8.0 pH

period in one old and one should not deviate by more than

new test solution from the 1.0 unitfor more than 48 hours.

control, 31 (low), 250 GECD recommends that pH

(medium), and 1000 (high) range be 6 - 9 and does not vary

treatment levels. The more than 1.5 units in anyone

temperature was test.

continuously monitored (not Recommended dissolved

further specified) and oxygen: renewal should not

manually checked each drop below 50%for more than

workday. The hardness, 48 hours.

total alkalinity, and Recommendedflow-through:

conductivity ofthe control ?-60% throughout test.

dilution water were Recommended temperature:

measured once weekly 20°C ± 2°e.; should not deviate

during the test. from 20°C by more than 5°Cfor

more than 48 hours. GECD

recommends a range of18 -

22°C; temperature should not

vary more than ± 2°C

GECD guideline recommends

that total organic carbon <2

mg/L

Page 8 of 30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Remarks

Parameter Details ----------------------------------------

Criteria

Number of replicates: There were six toxicant levels

For growth and reproduction: 10 replicates per level spaced by a separation factor of

2.

For survival test: (same) ---- ---- ------ ---- -- -- -- ---------- -- ----

Number ofreplicates should

include a control(s) and at least

5 test concentrations; dilution

factor should not be greater than

50%.

DECD recommends that at least

5 test concentrations be used in a

geometric series with a

separation factor not exceeding

3.2.

Number of organisms: Study design follows that

For growth and reproduction: Four organisms per replicate described in OPPTS guidance for

a flow-through study. All

For survival test: (same) replicates were observed for

survival, reproduction, and

growth.

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

Recommended number of

organisms include 22

daphnids/test concentration;

7 test chambers should contain 1

daphnid each, and 3 test

chambers contain 5 daphnids

each.

DECD recommends holding a

minimum of10 daphnids

individuallyfor static tests.

For flow-through tests, 40

animals should be divided into 4

groups of10 animals at each test

concentration.

Page 9 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Remarks

Parameter Details -----------------------------------------

Criteria

Treatment Concentrations: It was not reported ifthe nominal

nominal:

°(negative control), 31, 62, concentrations were corrected for

125, 250, 500, and 100O purity ofthe test substance.

mg/L

Actual concentrations of

mean-measured: <3.13 «LOQ, control), 28, Tribenuron-methyl were

54, 110, 220, 400, and 790 determined in fresh solutions (all

mg ai/L levels) on Days 0, 7, and 14 and

in aged ('old') solutions (all

levels) on Days 7, 14, and 2l.

Replicate results were generally

maintained within 20% for all

levels, with one exception:

analytical variation was 21% at

the nominal 62 mg/L level.

Mean-measured concentrations

were rounded to two significant

figures by the study authors.

Test organisms were transferred

to new test solutions three times

a week; however, concentrations

were measured on day 0, 7, and

14 only.

Solvent (type, percentage, ifused) N/A -------------------- --------------------

Solvent concentration should not

exceed 0.5 ml/Lfor static tests or

0.1 millforflow-through tests.

Recommended solvents include

dimethylformamide, triethylene

glycol, methanol, acetone and

ethanol.

GEeD recommends ~0.1 mL/l of

solvent.

Page 10 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Remarks

Parameter Details ----- --- - - - - - - - - - - - -- - ------------- -- - --

Criteria

Lighting 16-hours light, 8-hours dark;

light intensity was ca. 560 ----------------------------------------

lux (not further specified) Recommendedphotoperiod is 16

hours light and 8 hours ofdark.

Recovery of chemical: 75 to 101% of nominal Reviewer-calculated from

replicate results oftest solutions.

Frequency of measurement: Days 0, 7, 14, and 21

LaD: Not reported

LOQ: 3.13 mg ai/L

Positive control {if used, indicate Nat assessed

the chemical and concentrations}

Other parameters, if any Daphnia were provided food Trout chow (Glencoe) and yeast

Feeding: in their freshly-prepared test (Fleischmann's) were fed to the

solutions at each renewal daphnids during culturing; it is

interval; however, the type unclear if the same food was

and quantity were not used during the definitive test.

reported.

2. Observations:

Table 2: Observations

Remarks

Parameters Details -----------------------------------------------

Criteria

Data endpoints measured - Survival

(list) - Days to first brood - ------------------------------ -- - - -- - - - -------

- Total no. young produced Recommended endpoints measured:

- No. young/surviving adult - Survival offirst-generation daphnids,

- Growth (terminal length) - Number ofyoung producedper

female,

- Dry weight (required) and length

(optional) ofeach first generation

daphnid alive at the end ofthe test,

- Observations ofother effects or

clinical signs.

Page 11 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Remarks

Parameters Details ------- ------ - ----------------- ------- -----

Criteria

Observation intervals Survival and reproduction were

observed at each renewal period

(three times per week). Growth

was determined at Day 21 (test

termination).

Were raw data included? Yes (biological data)

Other observations, if any None assessed.

II. RESULTS AND DISCUSSION

A. MORTALITY:

Following 21 days of exposure, survival averaged 90% in the control group, and 98, 90, 80,

72,0, and 0% in the mean-measured 32,56, 120,250,480, and 940 mg ai/L treatment levels,

respectively. The study author reported that differences were statistically-reduced (p<0.05)

compared to the negative control at the 400 and 790 mg ai/L levels. The 21-Day ECso for

immobilization (with 95% C.l.) was reported by the study author to be 230 (160 to 350) mg

ai/L. The NOAEC for survival was reported to be 220 mg ai/L.

Table 3: Effect of DPX-L5300-78 (formulation of Tribenuron-methyl) on Survival,

R d epro ucfron, an row d G th f 0 D h . ap. nta sp.

Treatment

Mean-Measured Survival, % Days to First No. Young Mean (and Nominal) No. Young

Cone. Day 21 Brood per Parent I Length, mm

I

mglL

Control 90 11.4 383 96 3.7

(dilution water only)

28 (31) 98 13.0 333 85 3.7

54 (62) 90 13.3* 274* 74 3.6*

110 (125) 80 14.4* 135* 41* 3.2*

Page 12 of 30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Treatment

Mean-Measured Survival, % Days to First No. Young Mean (and Nominal) No. Young

Cone. Day 21 Brood per Parent Length, mm

mglL

220 (250) 72 12.7 150* 52* 3.1*

400 (500) 0* --- 0* 0* ---

790 (1000) 0* --- 0* 0* ---

NOAEC, mg ai/L 220 28 28 28

LOAEC, mg ai/L 400 54 54 54

* Significantly different (p<0.05) compared to the control using Dunnett's Test.

B. EFFECTS ON REPRODUCTION AND GROWTH:

The mean time to first brood release was 11.4 days for the negative control, and 13.0, 13.3,

14.4, and 12.7 days for the mean-measured 28,54, 110, and 220 treatment levels,

respectively (no offspring were produced at the 400 and 790 mg ai/L levels). Differences

were statistically-significant (p<0.05) compared to the control at the 54 and 110 mg ai/L

levels. Although no significant difference was indicated for the 220 mg ai/L level, the study

author considered the statistical differences observed at the 54 and 110 levels to be treatmentrelated. Thus, the NOAEC for time to first brood release was reported to be 28 mg ai/L.

For the control, and mean-measured 28, 54, 110, and 220 mg ai/L levels, the total numbers of

young produced were 383, 333, 274, 135, and 150, respectively, and the number of offspring

per surviving parent averaged 96,85, 74, 41, and 52, respectively. For total offspring,

differences were statistically-significant (p<0.05) compared to the control at the 54, 110, and

220 mg ai/L levels. When the data were corrected for surviving parent, differences remained

statistically-significant for the study author's analysis at only the 110 and 220 mg ai/L levels.

Based upon total offspring (the more sensitive ofthe two reproductive endpoints), the study

authors reported that the NOAEC for offspring production was 28 mg ai/L. It was reported

that the 21-Day ECsofor reproduction (with 95% C.I.) was 230 (160 to 350) mg ai/L.

No males, ephippia, or immobilized young were observed in the control group or any

treatment level during the study, and no eggs were observed in the bottom of any test vessel.

Growth was measured as length (mm) at study termination. Mean lengths were 3.7, 3.7, 3.6,

3., and 3.1 mm for the control and mean-measured 28,54, 110, and 220 mg ai/L levels,

respectively. Differences were statistically-significant (p<0.05) for the study author's

analysis compared to the control at the 54, 110, and 220 mg ai/L levels, respectively. The

Page 13 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

NOAEC for growth was reported to be 28 mg ai/L.

C. REPORTED STATISTICS:

Data that were statistically analyzed included organism survival, time to first brood release,

total number of offspring, number of offspring produced per surviving adult and total body

length. Survival data were analyzed using Fisher's Exact test and the Cochran-Armitage

trend test. Reproductive and growth endpoints were analyzed using a one-way analysis of

variance (ANOVA) followed by Dunnett's procedure.

The 21-day ECso for immobilization (and presumably reproduction) was calculated by Probit

analysis. All results were reported in terms ofmean-measured concentrations.

Parental Survival

ECso: 230 mg ai/L

Probit slope: Not reported

NOAEC: 220 mg ai/L

LOAEC: 400 mg ai/L

Time to First Brood

NOAEC: 28 mg ai/L

LOAEC: 54 mg ai/L

Total offspring

NOAEC: 28 mg ai/L

LOAEC: 54 mg ai/L

Offspring/surviving adult

ECso: 230 mg ai/L

Probit slope: Not reported

NOAEC: 54 mg ai/L

LOAEC: 110 mg ai/L

Total Length

NOAEC: 28 mg ai/L

LOAEC: 54 mg ai/L

95% C.l.: 160 to 350 mg ai/L

95% C.l.: Not reported

95% C.l.: 160 to 350 mg ai/L

95% C.L: Not reported

D. VERIFICATION OF STATISTICAL RESULTS:

Statistical Method(s): The reviewer determined the NOAEC for immobility using Fisher's

Exact Test. Reproduction (time to first brood, total offspring, offspring produced/day per

surviving adult) and growth (length) endpoint data were determined to be normally

distributed using the Shapiro-Wilk's or Chi-square test and variances were confirmed to be

Page 14 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp,

PMRA Submission Number { } EPA MRID No. 47643007

homogeneous using Levene's test. The NOAEC and LOAEC for these endpoints were

determined using ANOVA, followed by Bonferroni's, Dunnett's or William's test. These

analyses were conducted using Toxstat 3.5. The reviewer determined the 21-day ECsofor

reproductive endpoints (total offspring production and offspring produced/day per surviving

adult) using the probit method via Nuthatch statistical software. The 21-day ECsofor

immobility was determined using the moving average method; the probit method did not

provide a calculable 95% confidence interval.

Parental Survival

ECso: 216 mg ai/L

NOAEC: 110 mg ai/L

LOAEC: 220 mg ai/L

Time to First Brood

NOAEC: <28 mg ai/L

LOAEC: 28 mg ai/L

Total Offspring produced

ECso: 100 mg ai/L

Probit slope: 1.42±0.331

NOAEC: 28 mg ai/L

LOAEC: 54 mg ai/L

95% C.I.: 184-252 mg ai/L

95% C.I.: 63-170 mg ai/L

Offspring/day per surviving adult

ECso: 170 mg ai/L 95% C.I.: 81-380 mg ai/L

Probit slope: 1.08±0.390

NOAEC: 28 mg ai/L

LOAEC: 54 mg ai/L

Total Length

NOAEC: <28 mg ai/L

LOAEC: 28 mg ai/L

E. STUDY DEFICIENCIES:

This study is considered scientifically sound; however, the source ofthe dilution (fish culture

water) could have contained ammonia in the water, which was neither measured nor reported

in the water quality measurements. After analysis ofthe data the reviewer concluded that a

NOAEC could not be determined for this study due to significant (p<0.05) treatment effects

on first day of brood production and adult length at the lowest treatment level.

Page 15 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

F. REVIEWER'S COMMENTS:

The reviewer's conclusions differed from the study author's. The study author did not

indicate significant effects for time to first brood production and adult length at the lowest

level, which the reviewer's analysis detected. Additionally, the reviewer's analysis detected

a lower NOAEC for offspring produced/day per surviving adult than the study author's

analysis, as well as for adult immobility. The reviewer's more conservative conclusions

regarding time to first brood production and length are presented in the Executive Summary

and Conclusions sections.

All OPPTS 850.1300 validity requirements were fulfilled. Specifically, control mortality

was 10% (no more than 20% permissible), control daphnids each produced an average of96

young (minimum of 60 required), and no ephippia were produced by control daphnids.

The study authors repeatedly stated in error that the first day of brood release, the number of

offspring per surviving adult, and length were the three most sensitive endpoints; whereas,

the three most sensitive endpoints indicated by the study author's analysis was the day of

first brood release, the total number of offspring, and length.

Test samples collected for analytical verification were stored refrigerated prior to analysis.

Aliquots were analyzed for Tribenuron-methyl either directly or following dilution with

water using high-performance liquid chromatography (HPLC) with UV detection (230 urn).

The experimental phase ofthe definitive study was conducted from July 14 to August 4,

1989.

G. CONCLUSIONS:

This study is scientifically sound, however; the study is not acceptable for use in risk

assessment. Based upon treatment-related effects on the time to first brood release and

terminal lengths ofsurviving daphnia at the 28 mg ai/L treatment level (the most sensitive

endpoints), the overall NOAEC for the study could not be determined (i.e., <28 mg ai/L). A

treatment-related reduction in offspring production (total and offspring/day per surviving

adult) was also indicated at the 54, 110 and 220 mg ai/L levels, and immobility (i.e.,

mortality) due to exposure was observed at the 220,400 and 790 mg ai/L levels (with

complete immobility at the 400 and 790 mg ai/L levels). The ECso value (with 95% c.I.) for

immobility was 216 (184 - 252) mg ai/L and was 100 (63 - 170) mg ai/L for reproduction.

Based on treatment related treatment-related effects seen in the lowest test concentration, this

study is classified as Supplemental (not repairable).

Page 16 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

21-day ECso (mortality/immobility): 216 mg ai/L 95% c.r.: 184-252 mg ai/L

(moving average method)

21-day ECso(reproduction): 100 mg ai/L 95% C.r.: 63-170 mg ai/L

Slope: 1.42±0.331

NOAEC: <28 mg ai/L

LOAEC: 28 mg ai/L

Endpoints affected: Survival, time to first brood release, total young produced, young/day

per surviving adult, and growth (length)

Most sensitive endpoint(s): Time to first brood release and growth (length)

III. REFERENCES:

Hutton, D.G. 1989. "Short-term Study Protocol Sheet", MR-4581-665, Haskell Number 15,527,

February 20,1989.

"Chronic Toxicity Test on Compounds with Daphnia magna", Haskell laboratory Aquatic Test

SOP-T23.

OECD. 1984. "OECD Guideline for Testing of Chemicals: 202",4 April 84.

Rexrode, M, and T.M. Armitage. 1986. "Standard Evaluation Procedure, Daphnia magna LifeCycle (21-Day Renewal) chronic Toxicity Test". EPA 540/9-86-141, U.S. EPA, Office of

Pesticide programs, Washington, DC, July 1986.

Hutton, D.G. 1989. "Static Acute 48-Hour EC50 of DPX-L5300-78 to Daphnia magna",

Haskell Laboratory Report No. 179-89. May 3, 1989.

Miller, R.J. 1981. Simultaneous Statistical Inference, 2nd ed., Springer Verlag, New York.

Cochran, W.G. 1954. "Some Methods for Strengthening the Common X2 Tests", Biometrics,

10, pp. 417-451.

Armitage, P. 1955. "Tests for Linear Trends in Proportions and Frequencies", Biometrics, 11,

pp. 375-386.

Finney, D.l 1971. Probit Analysis, 3rd Ed. Cambridge University Press. 333 pp.

Peace, K.E. 1988. Biopharmaceutical Statisticsfor Drug Development, Marcel Dekker, Inc.,

New York, NY, pp. 242-243.

Page 17 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Haskell Laboratory MR-5659.

United States Geological Society (USGS). 1998. Dissolved Oxygen Saturation Tables

(Originators: Radtke, D.B., White, A.F., Davis, J.V., and Wilde, F.D.). Release date: 1998

http://water.usgs.gov/software/DOTABLES/.

Page 18 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp,

PMRASubmission Number { } EPAMRID No. 47643007

APPENDIX I. OUTPUT OF REVIEWER'S STATISTICAL VERIFICATION:

Summary of Fisher's Exact Tests

GROUP IDENTIFICATION

NUMBER

EXPOSED

NUMBER

DEAD

SIG

0.05

1

2

3

4

5

6

CONTROL

28

54

110

220

400

790

40

40

40

40

40

40

40

4

1

4

8

11

40

40

*

*

*

NOTE: BECAUSE THERE WAS CONTROL MORTALITY, AND NONE

OF THE LOWER CONCENTRATIONS PRODUCED ZERO MORTALITY,

THE DATA HAS BEEN SUBJECTED TO ABBOTT'S CORRECTION.

************************************************************************

CONC. NUMBER NUMBER PERCENT BINOMIAL

EXPOSED DEAD DEAD PROB. (PERCENT)

790 36 36 100 0

400 36 36 100 0

220 36 7.000001 19.4444 0

110 36 4 11.1111 0

54 36 0 0 0

28 40 1 2.5 0

BECAUSE THE NUMBER OF ORGANISMS USED WAS SO LARGE, THE 95 PERCENT

CONFIDENCE INTERVALS CALCULATED FROM THE BINOMIAL PROBABILITY ARE

UNRELIABLE. USE THE INTERVALS CALCULATED BY THE OTHER TESTS.

AN APPROXIMATE LC50 FOR THIS SET OF DATA IS 265.0979

RESULTS CALCULATED USING THE MOVING AVERAGE METHOD

SPAN G LC50 95 PERCENT CONFIDENCE LIMITS

4 3.503224E-02 215.5241 184.1108 252.206

GOODNESS OF FIT PROBABILITY

o

IS LESS THAN 0.001.

THE PROBIT METHOD

H

64.42461

MEANS THAT IT

RESULTS CALCULATED USING

ITERATIONS G

7 .812128

A PROBABILITY OF 0

SINCE THE PROBABILITY IS LESS THAN 0.05, RESULTS CALCULATED

USING THE PROBIT METHOD PROBABLY SHOULD NOT BE USED.

SLOPE 4.070066

95 PERCENT CONFIDENCE LIMITS =-7.305843 AND 15.44597

INTERCEPT=-9.599594

LC50 = 228.3413

Page 19 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

95 PERCENT CONFIDENCE LIMITS = 0 AND +INFINITY

LC25 = 155.9058

95 PERCENT CONFIDENCE LIMITS = 0 AND +INFINITY

LC10 = 110.586

95 PERCENT CONFIDENCE LIMITS = 0 AND +INFINITY

LC05 = 90.04065

95 PERCENT CONFIDENCE LIMITS = 0 AND +INFINITY

*************************************************************************

Title:

File:

Time to First Brood

3007b Transform:

Shapiro - Wilk's Test for Normality

NO TRANSFORMATION

D = 92.8222

W = 0.9622

Critical W

W

0.9290 (alpha = 0.01 , N = 49)

0.9470 (alpha = 0.05 , N = 49)

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

Title:

File:

Time to First Brood

3007b Transform: NO TRANSFORMATION

Levene's Test for Homogeneity of variance

ANOVA Table

SOURCE

Between

Within (Error)

Total

DF

4

44

48

SS

2.2961

51.6222

53.9184

MS

0.5740

1.1732

F

0.4893

(p-value = 0.7435)

Critical F = 3.7784

= 2.5837

Since F < Critical F

(alpha = 0.01, df = 4,44)

(alpha = 0.05, df = 4,44)

FAIL TO REJECT Ho: All equal (alpha 0.01)

Title:

File:

Time to First Brood

3007b Transform:

ANOVA Table

NO TRANSFORMATION

SOURCE

Between

Within (Error)

Total

DF

4

44

48

SS

43.1778

92.8222

136.0000

MS

10.7944

2.1096

F

5.1168

Page 20 of30

(p-value = 0.0018)
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Critical F = 3.7784

= 2.5837

Since F > Critical F

(alpha

(alpha

REJECT

0.01, df = 4,44)

0.05, df = 4,44)

Ho: All equal (alpha 0.05)

Title:

File:

Time to First Brood

3007b

Bonferroni t-Test

Transform:

TABLE 1 OF 2

NO TRANSFORMATION

Ho: Control>Treatment

TRANSFORMED MEAN CALCULATED IN SIG

GROUP IDENTIFICATION MEAN ORIGINAL UNITS t STAT 0.05

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

1 Control 11.4444 11.4444

2 28 13.0000 13 .0000 2.3309 * 3 54 13.3000 13.3000 2.7805 * 4 110 14.4000 14.4000 4.4288 * 5 220 12.7000 12.7000 1.8814

Bonferroni t critical value = 2.3207 (1 Tailed, alpha = 0.05, df = 4,44)

Title:

File:

Time to First Brood

3007b

Bonferroni t-Test

Transform:

TABLE 2 OF 2

NO TRANSFORMATION

Ho: Control>Treatment

NUM OF MIN SIG DIFF % OF DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 Control 9

2 28 10 1.5487 13.5 1.5556

3 54 10 1.5487 13.5 1.8556

4 110 10 1.5487 13.5 2.9556

5 220 10 1.5487 13.5 1.2556

Title:

File:

Time to First Brood

3007b

William's Test

Transform:

- TABLE 1 OF 2

NO TRANSFORMATION

Ho: Control>Treatment

ORIGINAL TRANSFORMED ISOTONIZED

GROUP IDENTIFICATION N MEAN MEAN MEAN

------ -------------------- -------'---- ----------- -----------

1 Control 9 11.4444 11. 4444 11. 4444

2 28 10 13.0000 13 .0000 13.0000

3 54 10 13.3000 13 .3000 13.3000

4 110 10 14.4000 14.4000 13.5500

5 220 10 12.7000 12.7000 13.5500

Page 21 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Title:

File:

Time to First Brood

3007b

William's Test

Transform:

- TABLE 2 OF 2

NO TRANSFORMATION

Ho: Control>Treatment

COMPARED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEANS WILLIAMS 0.05 WILLIAMS FREEDOM USED

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

Control 11.4444

28 13.0000 2.3309 * 1.6800 k= 1, v=40

54 13.3000 2.7805 * 1.7600 k= 2, v=40

110 13.5500 3.1551 * 1.7900 k= 3, v=40

220 13.5500 3.1551 * 1.8000 k= 4, v=40

s = 1.4524

WARNING: Procedure has used isotonized means which differ from original

(transformed) means.

Title:

File:

Total Offspring

30070 Transform:

Chi-Square Test for Normality

NO TRANSFORMATION

INTERVAL < -1. 5

Actual and Expected Frequencies

-1.5 to <-0.5 -0.5 to 0.5 >0.5 to 1.5 >1. 5

EXPECTED

OBSERVED

3.3500

3

12.1000

12

19.1000

19

12.1000

13

3.3500

3

Chi-Square = 0.1414

Critical Chi-Square = 13 .277

9.488

(p-value = 0.9976)

(alpha = 0.01 , df = 4)

(alpha = 0.05 , df = 4)

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

Title:

File:

Total Offspring

30070 Transform: NO TRANSFORMATION

Levene's Test for Homogeneity of Variance

ANOVA Table

SOURCE

Between

Within (Error)

Total

DF

4

45

49

SS

43508.6800

187098.2000

230606.8800

MS

10877.1700

4157.7378

F

2.6161

(p-value = 0.0475)

Critical F = 3.7674

= 2.5787

Since F < Critical F

(alpha = 0.01, df = 4,45)

(alpha = 0.05, df = 4,45)

FAIL TO REJECT Ho: All equal (alpha

Page 22 of30

0.01)
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp,

PMRA Submission Number { } EPA MRID No. 47643007

Title:

File:

Total Offspring

30070 Transform:

ANOVA Table

NO TRANSFORMATION

SOURCE

Between

Within (Error)

Total

DF

4

45

49

SS

484423.0800

385411.9000

869834.9800

MS

121105.7700

8564.7089

F

14.1401

Critical F = 3.7674

= 2.5787

Since F > Critical F

(alpha = 0.01, df = 4,45)

(alpha = 0.05, df = 4,45)

REJECT Ho: All equal (alpha

(p-value = 0.0000)

0.05)

Title:

File:

Total Offspring

30070

Dunnett's Test

Transform:

TABLE 1 OF 2

NO TRANSFORMATION

Ho:Control<Treatment

TRANSFORMED MEAN CALCULATED IN SIG

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT 0.05

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

1 Control 383.3000 383.3000

2 28 333.4000 333.4000 1.2057

3 54 273.7000 273.7000 2.6481 * 4 110 135.1000 135.1000 5.9970 * 5 220 149.6000 149.6000 5.6466 *

Dunnett critical value = 2.2300 (1 Tailed, alpha = 0.05, df [used] 4,40)

(Actual df = 4,45)

Title:

File:

Total Offspring

30070

Dunnett's Test

Transform:

TABLE 2 OF 2

NO TRANSFORMATION

HO:Control<Treatment

NUM OF MIN SIG DIFF !l-0 OF DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 Control 10

2 28 10 92.2946 24.1 49.9000

3 54 10 92.2946 24.1 109.6000

4 110 10 92.2946 24.1 248.2000

5 220 10 92.2946 24.1 233.7000

Page 23 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Title:

File:

Total Offspring

30070

William's Test

Transform:

- TABLE 1 OF 2

NO TRANSFORMATION

Ho: Control<Treatment

ORIGINAL TRANSFORMED ISOTONIZED

GROUP IDENTIFICATION N MEAN MEAN MEAN

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

1 Control 10 383.3000 383.3000 383.3000

2 28 10 333.4000 333.4000 333.4000

3 54 10 273.7000 273.7000 273.7000

4 110 10 135.1000 135.1000 142.3500

5 220 10 149.6000 149.6000 142.3500

Title:

File:

Total Offspring

30070

William's Test

Transform:

- TABLE 2 OF 2

NO TRANSFORMATION

Ho: Control<Treatment

COMPARED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEANS WILLIAMS 0.05 WILLIAMS FREEDOM USED

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

Control 383.3000

28 333.4000 1.2057 1.6800 k= 1, v=40

54 273.7000 2.6481 * 1.7600 k= 2, v=40

110 142.3500 5.8218 * 1.7900 k= 3, v=40

220 142.3500 5.8218 * 1.8000 k= 4, v=40

s = 92.5457

WARNING: Procedure has used isotonized means which differ from original

(transformed) means.

Estimates of EC%

Parameter Estimate 95% Bounds Std. Err. Lower Bound

Lower Upper /Estimate

EC5 7.1 1.5 33. 0.33 0.22

EC10 13 . 3.6 46. 0.27 0.28

EC25 34. 15. 80. 0.18 0.43

EC50 1.0E+02 63. 1.7E+02 0.11 0.61

Slope 1. 42 Std.Err. = 0.331

!! lPoor fit: p 0.019 based on DF= 2.0 45.

Page 24 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

30070: Total Offspring

Observed vs. Predicted Treatment Group Means

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

Mean Mean -Pred. %Control

0.00 10.0 383. 393. -9.79 100. 0.00

28.0 10.0 333. 310. 23.6 78.8 21. 2

54.0 10.0 274. 257. 16.7 65.4 34.6

110. 10.0 135. 190. -54.7 48.3 51. 7

220. 10.0 150. 125. 24.1 31. 9 68.1

! ! !Warning: EC5 not bracketed by doses evaluated.

! ! !Warning: EC10 not bracketed by doses evaluated.

Title:

File:

Young/day per surviving parent

3007y Transform:

Chi-Square Test for Normality

NO TRANSFORMATION

INTERVAL <-1.5

Actual and Expected Frequencies

-1.5 to <-0.5 -0.5 to 0.5 >0.5 to 1.5 >1. 5

EXPECTED

OBSERVED

3.3500

3

12.1000

12

19.1000

20

12.1000

12

3.3500

3

Chi-Square = 0.1172

Critical Chi-Square = 13.277

9.488

(p-value = 0.9983)

(alpha = 0.01 , df = 4)

(alpha = 0.05 , df = 4)

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

Title:

File:

Young/day per surviving parent

3007y Transform:

Levene's Test for Homogeneity

ANOVA Table

NO TRANSFORMATION

of Variance

SOURCE

Between

Within (Error)

Total

DF

4

45

49

SS

2907.7200

13482.3000

16390.0200

MS

726.9300

299.6067

F

2.4263

(p-value = 0.0616)

Critical F = 3.7674

= 2.5787

Since F < Critical F

(alpha = 0.01, df = 4,45)

(alpha = 0.05, df = 4,45)

FAIL TO REJECT Ho: All equal (alpha

Page 25 of30

0.01)
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Title:

File:

Young/day per surviving parent

3007y Transform:

ANOVA Table

NO TRANSFORMATION

SOURCE

Between

Within (Error)

Total

DF

4

45

49

SS

20692.6800

28135.8000

48828.4800

MS

5173.1700

625.2400

F

8.2739

Critical F = 3.7674

= 2.5787

Since F > Critical F

(alpha = 0.01, df = 4,45)

(alpha = 0.05, df = 4,45)

REJECT Ho: All equal (alpha

(p-value = 0.0000)

0.05)

Title:

File:

Young/day per surviving parent

3007y Transform:

Dunnett's Test TABLE 1 OF 2

NO TRANSFORMATION

Ho:Control<Treatment

TRANSFORMED MEAN CALCULATED IN SIG

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT 0.05

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

1 Control 95.7000 95.7000

2 28 85.2000 85.2000 0.9390

3 54 73.6000 73.6000 1.9763

4 110 40.8000 40.8000 4.9095 * 5 220 52.3000 52.3000 3.8811 *

Dunnett critical value = 2.2300 (1 Tailed, alpha = 0.05, df [used] 4,40)

(Actual df = 4,45)

Title:

File:

Young/day per survlvlng parent

3007y Transform:

Dunnett's Test TABLE 2 OF 2

NO TRANSFORMATION

Ho:Control<Treatment

NUM OF MIN SIG DIFF % OF DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 Control 10

2 28 10 24.9369 26.1 10.5000

3 54 10 24.9369 26.1 22.1000

4 110 10 24.9369 26.1 54.9000

5 220 10 24.9369 26.1 43.4000

Page 26 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

Title:

File:

Young/day per surviving parent

3007y Transform:

William's Test - TABLE 1 OF 2

NO TRANSFORMATION

Ho: Control<Treatment

ORIGINAL TRANSFORMED ISOTONIZED

GROUP IDENTIFICATION N MEAN MEAN MEAN

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

1 Control 10 95.7000 95.7000 95.7000

2 28 10 85.2000 85.2000 85.2000

3 54 10 73.6000 73.6000 73.6000

4 110 10 40.8000 40.8000 46.5500

5 220 10 52.3000 52.3000 46.5500

Title:

File:

Young/day per surviving parent

3007y Transform:

William's Test - TABLE 2 OF 2

NO TRANSFORMATION

Ho: Control<Treatment

COMPARED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEANS WILLIAMS 0.05 WILLIAMS FREEDOM USED

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

Control 95.7000

28 85.2000 0.9390 1.6800 k= 1, v=40

54 73.6000 1.9763 * 1.7600 k= 2, v=40

110 46.5500 4.3953 * 1.7900 k= 3, v=40

220 46.5500 4.3953 * 1.8000 k= 4, v=40

s = 25.0048

WARNING: Procedure has used isotonized means which differ from original

(transformed) means.

Estimates of EC%-

Parameter Estimate 95% Bounds Std. Err. Lower Bound

Lower Upper /Estimate

EC5 5.2 0.38 73. 0.57 0.072

EC10 11. 1.4 93. 0.45 0.12

EC25 41. 12. 1.5E+02 0.27 0.28

EC50 1.7E+02 81. 3.8E+02 0.16 0.47

Slope = 1. 08 Std. Err. = 0.390

!! lPoor fit: p = 0.023 based on DF= 2.0 45.

3007Y : Young/day per surviving parent

Observed vs. Predicted Treatment Group Means

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

Mean Mean -Pred. %-Control

0.00 10.0 95.7 97.7 -2.02 100. 0.00

28.0 10.0 85.2 78.6 6.56 80.5 19.5

54.0 10.0 73.6 69.3 4.30 70.9 29.1

Page 27 of30
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

110.

220.

! ! !Warning:

l ! ! Warning:

10.0 40.8 57.3 -16.5

10.0 52.3 44.7 7.62

EC5 not bracketed by doses evaluated.

EC10 not bracketed by doses evaluated.

58.6

45.7

41.4

54.3

Title:

File:

Total Length

30071 Transform:

Chi-Square Test for Normality

NO TRANSFORMATION

INTERVAL <-1. 5

Actual and Expected Frequencies

-1.5 to <-0.5 -0.5 to 0.5 >0.5 to 1.5 >1.5

EXPECTED

OBSERVED

11.5240

20

41.6240

27

65.7040

72

41.6240

44

11.5240

9

Chi-Square = 12.6638

Critical Chi-Square = 13.277

9.488

(p-value = 0.0130)

(alpha = 0.01 , df = 4)

(alpha = 0.05 , df = 4)

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

Title:

File:

Total Length

30071 Transform: NO TRANSFORMATION

Levene's Test for Homogeneity of Variance

ANOVA Table

SOURCE

Between

Within (Error)

Total

DF

4

167

171

SS

0.2135

3.5565

3.7700

MS

0.0534

0.0213

F

2.5057

(p-value = 0.0441)

Critical F = 3.4335

= 2.4258

Since F < Critical F

(alpha = 0.01, df = 4,167)

(alpha = 0.05, df = 4,167)

FAIL TO REJECT Ho: All equal (alpha 0.01)

Title:

File:

Total Length

30071 Transform:

ANOVA Table

NO TRANSFORMATION

SOURCE DF SS MS F

Between

Within (Error)

Total

4

167

171

10.1568

7.1949

17.3516

2.5392

0.0431

58.9373

Critical F 3.4335

2.4258

(alpha

(alpha

0.01, df

0.05, df

Page 28 of30

4,167)

4,167)

(p-value = 0.0000)
Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

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

Title:

File:

Total Length

30071

Bonferroni t-Test

Transform:

TABLE 1 OF 2

NO TRANSFORMATION

Ho: Control<Treatment

TRANSFORMED MEAN CALCULATED IN SIG

GROUP IDENTIFICATION MEAN ORIGINAL UNITS t STAT 0.05

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

1 Control 3.7472 3.7472

2 28 3.6564 3.6564 1.8930

3 54 3.6056 3.6056 2.8957 * 4 110 3.2000 3.2000 10.8513 * 5 220 3.1414 3.1414 11.6977 *

Bonferroni t critical value = 2.2618 (1 Tailed, alpha = 0.05, df = 4,167)

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Bonferroni t-Test

Transform:

TABLE 2 OF 2

NO TRANSFORMATION

Ho: Control<Treatment

NUM OF MIN SIG DIFF % OF DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 Control 36

2 28 39 0.1085 2.9 0.0908

3 54 36 0.1107 3.0 0.1417

4 110 32 0.1141 3.0 0.5472

5 220 29 0.1171 3.1 0.6058

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William's Test

Transform: NO TRANSFORMATION

- TABLE 1 OF 2 Ho: Control<Treatment

'.. ORIGINAL TRANSFORMED ISOTONIZED

GROUP IDENTIFICATION N MEAN MEAN MEAN

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

1 Control 36 3.7472 3.7472 3.7472

2 28 39 3.6564 3.6564 3.6564

3 54 36 3.6056 3.6056 3.6056

4 110 32 3.2000 3.2000 3.2000

5 220 29 3.1414 3.1414 3.1414

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Data Evaluation Record on the Chronic Toxicity of DPX-L5300-78 (formulation of

Tribenuron-methyl) to Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643007

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William's Test

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- TABLE 2 OF 2 Ho: Control<Treatment

COMPARED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEANS WILLIAMS 0.05 WILLIAMS FREEDOM USED

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

Control 3.7472

28 3.6564 1.8930 * 1.6600 k= 1, v=120

54 3.6056 2.8957 * 1.7300 k= 2, v=120

10 3.2000 10.8513 * 1.7500 k= 3, v=120

220 3.1414 11.6977 * 1.7700 k= 4, v=120

s = 0.2076

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