Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Data Requirement: PMRA Data Code

EPA DP Barcode

OECD Data Point

EPAMRID

EPA Guideline

{ }

361296,358877,358880,358882,358885

{ }

47643006

850.1300

IN L5300-20 Purity: 95.8-96.8%

Tribenuron-methyl

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

carbamoylsulfamoyl]benzoate

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

methylamino]carbonyl]amino]-sulfonyl]benzoate

CAS No.: 101200-48-0

Synonyms: DPX-L5300-20; Haskell No. 15,527

Test material:

Common name:

Chemical name:

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

Signature: {!~ t. PJ..nr--

Date: 06/16/2011

Signature: ~'5

Date: 07/12/2011.

Signature: W

Date: 12/06/2011

Reference/Submission No.: {. }

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 IN L5300-20 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Unpublished study performed by E.!.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. 164-89. Study initiated February 22,1989 and completed April 10, 1989.

Page 1 of29

'1111.1111

2086464

TEXT SEARCHABLE DOCUMENT 2011
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

EXECUTIVE SUMMARY:

The 21-day-chronic toxicity of IN L5300-20 (Tribenuron-methyl) to Daphnia magna was

studied under static-renewal conditions. Daphnids were exposed to IN L5300-20 at nominal

concentrations of

°(negative control), 31, 62, 125, 250, 500, and 1000 mg/L. Mean-measured

concentrations were <17.5 «LOQ, control), 32, 56, 120,250,480, and 940 mg ai/L. The 21-day

ECso for immobilization was >940 mg ai/L, the highest concentration tested. The reviewer

concluded that a NOAEC could not be determined in this study due to suggestive treatmentrelated effects at the lowest treatment level (32 mg ai/L). Significant reductions (p<0.05) were

detected for total offspring production at the 32, 120, 250, 480, and 940 mg ai/L treatment levels

and for adult length at the 32, 250, 480, and 940 mg ai/L levels; while the responses were not

entirely linear, both endpoints were significantly reduced at the lowest level suggesting that the

effect may have been treatment-related. A treatment-related delay of first brood release was

additionally indicated at the 940 mg ai/L level and the number of offspring produced/day per

surviving adult was adversely affected at the 250, 480, and 940 mg ai/L treatment levels. The

21-day ECso based on reproduction parameters was 380 (330 -440) mg ai/L for total offspring

and 410 (350 - 470) mg ai/L for offspring produced/day per surviving adult.

Following 21 days of exposure, survival averaged 85, 95, 80, 85, 78, 82, and 70% in the control

and mean-measured 32, 56, 120, 250, 480, and 940 mg ai/L treatment levels, respectively. The

study author's analysis detected a significant difference (p<0.05) compared to the negative

control at the 940 mg ai/L level, while the reviewer's analysis did not detect any differences for

this endpoint.

The mean time to first brood release was 9.3 days for the negative control, and 10.4, 10.1, 10.0,

9.7, 8.8, and 15.6 days for the mean-measured 32, 56, 120,250,480, and 940 mg ai/L treatment

levels, respectively. The mean number of offspring per surviving parent averaged 116, 85, 115,

98, 89, 36, and 2 for the control and mean-measured 32, 56, 120, 250, 480, and 940 mg ai/L

levels, respectively. Mean lengths were 3.7, 3.6, 3.6, 3.6, 3.6, 3.5, and 3.3 mm for the control,

and mean-measured 32, 56, 120, 250, 480, and 940 mg ai/L levels, respectively. 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.

This study is scientifically unsound and does not satisfy the guideline requirement for a chronic

toxicity study with freshwater invertebrates. The dissolved oxygen was <60% (equivalent to 5.5

mg/L at 20°C; reviewer-calculated) for an unspecified period oftime at all levels during the

study. In addition, the resulting analysis ofthe data indicated that a NOAEC could not be

determined, as treatment-related effects were seen in the lowest test concentration. Therefore,

based on these analyses, the results from this study are not reliable, and are not acceptable for

use within ecological risk assessments. This study is classified as invalid.

Page 2 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Results Synopsis

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

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

21-day ECso (mortality/immobility): >940 mg ai/L

21-day ECso (reproduction): 380 mg ai/L

Slope: 5.07±0.724

NOAEC: <32 mg ai/L

LOAEC: 32 mg ai/L

95% C.L: N/A

95% C.L: 330-440 mg ai/L

Endpoints affected: Survival, time to first brood release, offspring production, and growth

(length)

Most sensitive endpoint(s): Total offspring production 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:

I. A NOAEC could not be determined in this study due to suggestive adverse effects on

total offspring production and adult length at the lowest treatment level.

2. Dissolved oxygen in the test vessels of aged solutions was <60% (equivalent to 5.5 mg/L

at 20°C; reviewer-calculated) for an unspecified period oftime at all levels during the

study.

3. 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 (l.Sum 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.

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

5. Although a I N NaOH solution was used to enhance solubility ofthe test substance, a

solvent control group was not included in the study. Furthermore, it was unknown if

concentrations ofNaOH were the same for all levels.

These deviations do affect the acceptability ofthe study.

Page 3 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

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

Confidentiality claims (2008) statements were provided.

A. MATERIALS:

1. Test Material IN L5300-20 (Tribenuron-methyl)

Description: Not reported

Lot No./Batch No. : Not reported

Purity: 95.8 - 96.8%

Stability of compound

under test conditions: The stability oftribenuron-methyl was verified analytically at

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

results were within ±20%.

Storage conditions of

test chemicals: Not reported

IYSICOC emicaI propertires 0 fTrr'benuron-methryr,I

Parameter Values Comments

Water solubility at 20°C Not reported

Vapor pressure Not reported

UV absorption 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: 25 days old

EPA recommends that young daphnids ~24 hours oldfrom a

separate parental culture be used

Page 4 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Source:

B. STUDY DESIGN:

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.

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 720 mg/L for unfed Daphnia magna and>1000 mg/L for fed

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):

Details

25 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.

Page 5 of29

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

maintained/or at least 21 days to

insure that good health conditions

are present
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Remarks

Parameter Details

Criteria

Test condition:

static renewal/flow-through:

Type of dilution system- for flow

through method.

Renewal rate for static renewal

Aeration, if any

Duration ofthe test

Static-renewal

N/A

Three times per week

No supplemental aeration

was provided.

21 days

The parent Daphnia were

transferred into fresh test solutions

containing food three times per

week.

(EPA requires consistent.flow rate

of5-10 vol124hours, meter

systems calibrated before study

and checked twice daily during

test period)

EPA recommends test chambers

should not be aerated

Recommended duration is 21

days.

Test vessel

Material: (glass/stainless steel)

Size (for growth and

reproduction/survival test):

Fill volume:

Glass -------------------------------------------

1. Recommended Material:

Glass, No. 316 stainless steel, or

250 mL (6.1-cm diameter) per.fluorocarbon plastics

2. Recommended Size: 250 ml

Ca. 200 mL (6.8-cm depth) with 200 mlfillvolume;100 ml

with 80 ml fill volume DECD

guideline recommends that parent

animals be maintained

individually; one per vessel, with

50 - 100 ml ofmedium in each

vessel.

Page 60f29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Remarks

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

Criteria

Source of dilution water The dilution water used for Results from periodic analysis of

testing was filtered fish tank the well water were provided;

water (laboratory well water water quality measurements ofthe

that had been used for dilution water were not taken after

fathead minnow culturing in use in the fish culturing.

a flow-through system, then

filtered through a 0.8-f.lm The use of old fish culture water

filter). Water was originally as the dilution water is a notable

obtained from a 350-foot deviation. It is unknown ifthe

encased (in bedrock), and water contained ammonia as

aerated and filtered (IO urn) measurements were not taken.

prior to use in fish culturing. 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. --- ----- - - - - - ------- - - - - - - ------ - - - - -------

Recommended source ofdilution

water includes unpolluted well or

spring water that has been tested

for contaminants, or appropriate

reconstituted water (see ASTMfor

details).

Water parameters: The pH offresh solutions

Hardness 75 ± 2 mg/L as CaC03 increased with increasing test

concentrations.

pH Fresh soln.: 7.2 to 8.8

Aged soln.: 7.1 to 8.2 DO was <60% (equivalent to 5.5

mg/L at 20°C; reviewerDissolved oxygen Fresh soln.: 8.2 to 8.7 mglL calculated) for an unspecified

Aged soln.: 4.6 to 7.4 mglL period oftime at all levels during

the study.

Temperature 19.7 to 20.4°C

Alkalinity was 82 ± 2 mglL as

Total Organic Carbon Not reported CaC03, and conductivity was 169

Particulate matter Not reported ± 2 umbos/em.

Inorganic ana1ytes Not reported

Pesticides Not reported Raw water chemistry data were

Chlorine Not reported not provided. However, the

chemical characteristics ofthe - - - - - -- ---- -- - - - - -------- -- - - -- -- ---- - - --

Page 7 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

I

Parameter

I

Details I... n .n ••R;:::;....... n. n ··1

Frequency of measurements Dissolved oxygen (DO) was Haskell Laboratory Well Water

measured at each renewal are as follows: TOC: <1.5 mg/L;

period in one old and one COD: <17mg/L; Solids, total

new test solution from all suspended (TSS): <4 mg/L;

levels. The pH was Pesticides: None detected (but

measured at each renewal detection level was not reported);

period in one old and one Chloride: 3.7 mg/L

new test solution from the -- --- - - - - - - - - - - - - - - - - - - - - ---- - - - - - - - - - - -

control, 31 (low), 500 Recommended hardness: 160 to

(medium), and 1000 (high) 180 mg/L as CaC03; OECD

treatment levels. The recommends>140 mg/L as

temperature was CaC03

continuously monitored (not RecommendedpH: 7. 6 to 8.0 pH

further specified) and should not deviate by more than

manually checked each 1.aunit for more than 48 hours.

workday. The hardness, OECD recommends that pH range

total alkalinity, and be 6 - 9 and does not vary more

conductivity of the control than 1.5 units in anyone test.

dilution water were Recommended dissolved oxygen:

measured once weekly renewal should not drop below

during the test. 50%for more than 48 hours.

Recommendedflow-through:

?,60% throughout test.

Recommended temperature:

20 DC ± rc; should not deviate

from 20 DC by more than 5 DCfor

more than 48 hours. OECD

recommends a range of18 - 22°C;

temperature should not vary more

than ± 2°C

OECD guideline recommends that

total organic carbon <2 mg/L

Number ofreplicates: 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%.

OECD recommends that at least 5

Page 8 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Remarks

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

Criteria

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.

OEeD 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 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

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 1000 purity ofthe test substance.

mg/L

Concentrations of Tribenuronmean-measured: <17.5 «LOQ, control), 32, methyl were determined in fresh

56, 120,250,480, and 940 solutions (all levels) on Days 0, 7,

mg ai/L and 14 and in aged ('old')

solutions (all levels) on Days 7,

14, and 21 using HPLC. Replicate

results were maintained within

20% for all levels.

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) I N NaOH, concentration Used to enhance solubility ofthe

was not reported. test substance.

A solvent control group was not

included in the study.

It is not known if concentrations

ofNaOH were the same for all

levels.

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

Solvent concentration should not

exceed 0.5 mllLjor static tests or

0.1 mllLjorflow-through tests.

Recommended solvents include

dimethylformamide, triethylene

glycol, methanol, acetone and

ethanol.

DEeD recommends ::::'0.1 mL/L oj

solvent.

Page 10 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Remarks

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

Criteria

Lighting l6-hours light, 8-hours dark;

light intensity was ca. 590 -------------------------------------------

lux (not further specified) Recommendedphotoperiod is 16

hours light and 8 hours ofdark.

Recovery of chemical: 83 to 102% of nominal Reviewer-calculated from

replicate results oftest solutions.

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

LaD: Not reported

LOQ: 17.5 mg ai/L

Positive control {if used, indicate Not 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 ifthe same food was used

and quantity were not 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 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

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 85% in the control group, and 95, 80, 85,

78,82, and 70% in the mean-measured 32,56, 120,250,480, and 940 mg ai/L treatment

levels, respectively. The difference was statistically-reduced (p<0.05) compared to the

negative control at the 940 mg ai/L level. The 21-Day ECso for immobilization was >940 mg

ai/L, the highest mean-measured concentration. The NOAEC for survival was 480 mg ai/L.

Table 3: Effect ofIN L5300-20 (Tribenuron-methyl) on Survival, Reproduction, and

Growth 0 fDap,h . nta sp.

Treatment

Mean-Measured Survival, % Days to First No. Young No. Young Mean

(and Nominal) Day 21 Brood per Parent Length, mm

Cone. (mg/L)

Control 85 9.3 443 116 3.7 (dilution water only)

32 (31) 95 10.4* 320* 85 3.6

56 (62) 80 10.1* 373 115 3.6

120 (125) 85 10.0 340 98 3.6

250 (250) 78 9.7 282* 89 3.6*

480 (500) 82 8.8 126* 36* 3.5*

Page 12 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Treatment

Mean-Measured Survival, % Days to First No. Young No. Young Mean

; (and Nominal) Day 21 Brood per Parent Length, mm

Cone. (mgIL)

940 (1000) 70* 15.6* 6* 2* 3.3*

NOAEC, mg ai/L 480 480 250 120

LOAEC, mg ai/L 940 940 480 250

* 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 9.3 days for the negative control, and 10.4, 10.1,

10.0,9.7,8.8, and 15.6 days for the mean-measured 32,56, 120,250,480, and 940 mg ai/L

treatment levels, respectively. Differences were statistically-significant (p<0.05) compared

to the control at the 32, 56, and 940 mg ai/L levels. However, the differences observed for

the 32 and 56 mg ai/L levels were deemed to be not biologically significant due to the

nonlinearity and heteroscedasticity ofthe data. Thus, the NOAEC for time to first brood

release was 480 mg ai/L.

For the control, and mean-measured 32,56, 120,250,480, and 940 mg ai/L levels, the total

numbers of young produced were 443,320,373,340,282, 126, and 6, respectively, and the

number of offspring per surviving parent averaged 116, 85, 115, 98, 89, 36, and 2,

respectively. For total offspring, differences were statistically-significant (p<0.05) compared

to the control at the 32, 250, 480, and 940 mg ai/L levels. However, when the data were

corrected for surviving parent, differences remained statistically-significant only at the 480

and 940 mg ai/L levels. Thus, the NOAEC for offspring production was 250 mg ai/L. It was

reported that the observed ECso for reproduction (calculated using failure to produce eggs as

the observation criterion) was ca. 900 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, measured as length at study termination, was the most sensitive endpoint. Mean

lengths were 3.7,3.6,3.6,3.6,3.6,3.5, and 3.3 mm for the control, and mean-measured 32,

56, 120,250,480, and 940 mg ai/L levels, respectively. Differences were statisticallysignificant (p<0.05) compared to the control at the 250, 480, and 940 mg ai/L levels,

respectively. The NOAEC for growth was 120 mg ai/L.

C. REPORTED STATISTICS:

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

Page 13 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

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 (ANOYA) followed by Dunnett's procedure.

During this study, no concentration caused 2:50% immobilization; therefore, the 21-day ECso

was empirically estimated to be greater than the highest tested concentration. The 21-day

ECso for reproduction was calculated by Probit analysis using failure to produce eggs as the

observation criterion. All results were reported in terms of mean-measured concentrations.

Parental Survival

ECso: > 940 mg ai/L

NOAEC: 480 mg ai/L

LOAEC: 940 mg ai/L

Time to First Brood

NOAEC: 480 mg ai/L

LOAEC: 940 mg ai/L

Offspring/surviving adult

ECso: ca. 900 mg ai/L

Probit slope: Not reported

NOAEC: 250 mg ai/L

LOAEC: 480 mg ai/L

Total Length

NOAEC: 120 mg ai/L

LOAEC: 250 mg ai/L

95% C.l.: N/A

95% C.l.: Not reported

95% C.I.: 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 Chi-square test and variances were confirmed to be homogeneous using

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

ANOYA, followed by Dunnett'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.

Parental Survival

ECso: > 940 mg ai/L 95% C.l.: N/A

Page 14 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number {........} EPA MRID No. 47643006

NOAEC: 940 mg ai/L

LOAEC: >940 mg ai/L

Time to First Brood

NOAEC: 480 mg ai/L

LOAEC: 940 mg ai/L

Total Offspring produced

ECso: 380 mg ai/L

Probit slope: 5.07±0.724

NOAEC: <32 mg ai/L

LOAEC: 32 mg ai/L

95% c.r. 330-440 mg ai/L

Offspring/day per surviving adult

ECso: 410 mg ai/L 95% C.l.: 350-470 mg ai/L

Probit slope: 5.64±0.920

NOAEC: 120 mg ai/L

LOAEC: 250 mg ai/L

Total Length

NOAEC: <32 mg ai/L

LOAEC: 32 mg ai/L

E. STUDY DEFICIENCIES:

This study is considered scientifically unsound, as the dissolved oxygen was <60%

(equivalent to 5.5 mg/L at 20°C; reviewer-calculated) for an unspecified period oftime at all

levels during the study. In addition, 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. The combination ofthese multiple stressors may have impacted the

test organisms, and after analysis ofthe data the reviewer concluded that a NOAEC could not

be determined for this study due to significant (p<0.05) suggestive treatment effects on total

offspring production and adult length at the lowest treatment level. Furthermore, the

following notable deviations from OPPTS 850.1300 guidance were observed: dissolved

oxygen in the test vessels of aged solutions was <60%; and although a 1 N NaOH solution

was used to enhance solubility ofthe test substance, a solvent control group was not included

in the study. Additionally, it was unknown what the concentrations ofNaOH were and if

those concentrations were the same for all treatment levels.

F. REVIEWER'S COMMENTS:

The reviewer's conclusions differed from the study author's. While both analyses detected

significant reductions in total offspring production at the lowest treatment level, the study

Page 15 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number {........} EPA MRID No. 47643006

author dismissed the effect because overall it was not dose-dependent. The study author did

not note a similar reduction for 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, but the reviewer's analysis

did not detect any effect on adult immobility. The reviewer's more conservative conclusions

regarding reproduction and length are presented in the Executive Summary and Conclusions

sections.

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

was 15% (no more than 20% permissible), control daphnids each produced an average of 116

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

It was reported that a 1 N sodium hydroxide (NaOH) solution was used to bring the pH ofthe

stock solutions up to ca. 9 before testing in order to increase solubility ofthe test substance.

No further details were provided, so it was unknown if concentrations ofNaOH were the

same for all levels. A solvent control group was not included in the study design.

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 nm).

The experimental phase ofthe definitive study was conducted from February 22 to March 15,

1989.

G. CONCLUSIONS:

This study is scientifically unsound and is thus not acceptable. The dissolved oxygen was

<60% (equivalent to 5.5 mg/L at 20°C; reviewer-calculated) for an unspecified period of

time at all levels during the study, and the source ofthe dilution water (fish culture water)

could have contained ammonia. The combination ofthese multiple stressors may have

negatively impacted the test organisms, and the resulting analysis ofthe data indicated that a

NOAEC could not be determined «32 mg ai/L, the lowest test concentration). Based upon a

treatment-related reduction in offspring/day per surviving adult and in terminal lengths of

surviving daphnia at the 32 mg ai/L treatment level (the most sensitive endpoints), the overall

NOAEC for the study could not be determined. A treatment-related reduction in offspring

production/day per surviving adult was also indicated at the 250, 480 and 940 mg ai/L levels,

and a treatment-related delay offirst brood release was indicated at the 940 mg ai/L level.

The ECsovalues for immobility and reproduction (total offspring) were >940 and 380 mg

ai/L, respectively. Based on the major deviations that were noted above, the results from this

study are not reliable, and are not acceptable for use. Therefore, this study is classified as

invalid.

21-day ECso(mortality/immobility): >940 mg ai/L

21-day ECso (reproduction): 380 mg ai/L

Page 16 of29

95% C.L: N/A

95% C.L: 330-440 mg ai/L
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp,

PMRA Submission Number { } EPA MRID No. 47643006

Slope: 5.07±O.724

NOAEC: <32 mg ai/L

LOAEC: 32 mg ai/L

Endpoints affected: Survival, time to first brood release, offspring production, and growth

(length)

Most sensitive endpoint(s): Total offspring production 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. 1984. "48-Hour EC50 to Daphnia magna", Haskell Laboratory Report No. 461-

84, Revised November 15, 1984.

Hutton, D.G. "Static Acute 48-Hour EC50 of IN L5300-20 to Fed Daphnia magna", Haskell

Laboratory Report to be issued.

Siegel, S. 1956. Nonparametric Statisticsfor the Behavioral Sciences. McGraw-Hill, New

York, pp. 96-104.

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

Steel, R.G.D., and J.H. Torrie. 1980. Principles and Procedures a/Statistics. 2nd Edition,

McGraw-Hill, New York.

Milliken, G.A., and D.E. Johnson. 1984. Analysis 0/Messy Data, Volume I, Lifetime Learning

Publications, Belmont, CA.

Haskell Laboratory MR-5659.

Page 17 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

APPENDIX I. OUTPUT OF REVIEWER'S STATISTICAL VERIFICATION:

Summary of Fisher's Exact Tests

NUMBER NUMBER SIG

GROUP IDENTIFICATION EXPOSED DEAD 0.05

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

CONTROL 40 6

1 32 40 2

2 56 40 8

3 120 40 6

4 250 40 9

5 480 40 7

6 940 40 12

Title:

File:

First Day of Brood

3006b 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

4.6900

1

16.9400

20

26.7400

30

16.9400

16

4.6900

3

Chi-Square = 4.5145

Critical Chi-Square = 13.277

9.488

(p-value = 0.3408)

(alpha = 0.01 , df = 4)

(alpha = 0.05 , df = 4)

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

Title:

File:

First Day of Brood

3006b Transform: NO TRANSFORMATION

Levene's Test for Homogeneity of Variance

ANOVA Table

SOURCE

Between

Within (Error)

Total

DF

6

63

69

SS

53.0857

106.0000

159.0857

MS

8.8476

1.6825

F

5.2585

Critical F 3.1028 (alpha

2.2464 (alpha

0.01, df

0.05, df

6,63)

6,63)

(p-value = 0.0002)

Since F > Critical F REJECT Ho: All equal (alpha

Page 18 of29

0.01)
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Title:

File:

First Day of Brood

3006b Transform:

ANOVA Table

NO TRANSFORMATION

SOURCE

Between

Within (Error)

Total

DF

6

63

69

SS

313.7714

283.5000

597.2714

MS

52.2952

4.5000

F

11.6212

Critical F = 3.1028

= 2.2464

Since F > Critical F

(alpha = 0.01, df = 6,63)

(alpha = 0.05, df = 6,63)

REJECT Ho: All equal (alpha

(p-value = 0.0000)

0.05)

Title:

File:

First Day of Brood

3006b

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 9.3000 9.3000

2 32 10.4000 10.4000 1.1595

3 56 10.1000 10.1000 0.8433

4 120 10.0000 10.0000 0.7379

5 250 9.7000 9.7000 0.4216

6 480 8.8000 8.8000 -0.5270

7 940 15.6000 15.6000 6.6408 *

Dunnett critical value = 2.3400 (1 Tailed, alpha = 0.05, df [used] 6,60)

(Actual df = 6,63)

Title:

File:

First Day of Brood

3006b

Dunnett's 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 10

2 32 10 2.2199 23.9 1.1000

3 56 10 2.2199 23.9 0.8000

4 120 10 2.2199 23.9 0.7000

5 250 10 2.2199 23.9 0.4000

6 480 10 2.2199 23.9 -0.5000

7 940 10 2.2199 23.9 6.3000

Page 19 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Title:

File:

First Day of Brood

3006b

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 9.3000 9.3000 9.3000

2 32 10 10.4000 10.4000 9.8000

3 56 10 10.1000 10.1000 9.8000

4 120 10 10.0000 10.0000 9.8000

5 250 10 9.7000 9.7000 9.8000

6 480 10 8.8000 8.8000 9.8000

7 940 10 15.6000 15.6000 15.6000

Title:

File:

First Day of Brood

3006b

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 9.3000

32 9.8000 0.5270 1.6700 k= 1, v=60

56 9.8000 0.5270 1.7500 k= 2, v=60

120 9.8000 0.5270 1.7700 k= 3, v=60

250 9.8000 0.5270 1.7800 k= 4, v=60

480 9.8000 0.5270 1.7900 k= 5, v=60

940 15.6000 6.6408 * 1.7900 k= 6, v=60

s = 2.1213

WARNING: Procedure has used isotonized means which differ from original

(transformed) means.

Title:

File:

Total Offspring produced

30060 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

4.6900

1

16.9400

23

26.7400

25

16.9400

14

4.6900

7

Chi-Square = 6.8323

Critical Chi-Square = 13.277

9.488

(p-value = 0.1450)

(alpha = 0.01 , df = 4)

(alpha = 0.05 , df = 4)

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

Page 20 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Title:

File:

Total Offspring produced

30060 Transform:

Levene's Test for Homogeneity

ANOVA Table

NO TRANSFORMATION

of Variance

SOURCE DF SS MS F

Between

Within (Error)

6

63

65694.0857

221621.9000

10949.0143

3517.8079

3.1125

Total 69 287315.9857

(p-value = 0.0098)

Critical F = 3.1028

= 2.2464

Since F > Critical F

(alpha = 0.01, df = 6,63)

(alpha = 0.05, df = 6,63)

REJECT Ho: All equal (alpha 0.01)

Title:

File:

Total Offspring produced

30060 Transform:

ANOVA Table

NO TRANSFORMATION

SOURCE DF SS MS F

Between

Within (Error)

6

63

1388497.2857

572605.3000

231416.2143

9088.9730

25.4612

Total 69 1961102.5857

(p-value = 0.0000)

Critical F = 3.1028

= 2.2464

Since F > Critical F

(alpha = 0.01, df = 6,63)

(alpha = 0.05, df = 6,63)

REJECT Ho: All equal (alpha 0.05)

NO TRANSFORMATION

Ho:Control<Treatment

Transform:

TABLE 1 OF 2

Total Offspring produced

30060

Dunnett's Test

Title:

File:

TRANSFORMED MEAN CALCULATED IN SIG

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT 0.05

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

1 Control 442.8000 442.8000

2 32 319.9000 319.9000 2.8826 * 3 56 373.1000 373.1000 1.6348

4 120 339.9000 339.9000 2.4135 * 5 250 282.0000 282.0000 3.7715 * 6 480 125.5000 125.5000 7.4421 * 7 940 5.5000 5.5000 10.2567 *

Dunnett critical value = 2.3400 (I Tailed, alpha = 0.05, df [used] 6,60)

(Actual df = 6,63)

Page 21 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Title:

File:

Total Offspring produced

30060 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 32 10 99.7673 22.5 122.9000

3 56 10 99.7673 22.5 69.7000

4 120 10 99.7673 22.5 102.9000

5 250 10 99.7673 22.5 160.8000

6 480 10 99.7673 22.5 317.3000

7 940 10 99.7673 22.5 437.3000

Title:

File:

Total Offspring produced

30060

William's Test

Transform:

- TABLE 1 OF 2

NO TRANSFORMATION

Ho: Control<Treatment

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

ORIGINAL TRANSFORMED ISOTONIZED

GROUP IDENTIFICATION N MEAN MEAN MEAN

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

1 Control 10 442.8000 442.8000 442.8000

2 32 10 319.9000 319.9000 346.5000

3 56 10 373.1000 373.1000 346.5000

4 120 10 339.9000 339.9000 339.9000

5 250 10 282.0000 282.0000 282.0000

6 480 10 125.5000 125.5000 125.5000

7 940 10 5.5000 5.5000 5.5000

Title:

File:

Total Offspring produced

30060

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 442.8000

32 346.5000 2.2587 * 1.6700 k= 1, v=60

56 346.5000 2.2587 * 1.7500 k= 2, v=60

120 339.9000 2.4135 * 1.7700 k= 3, v=60

250 282.0000 3.7715 * 1.7800 k= 4, v=60

480 125.5000 7.4421 * 1.7900 k= 5, v=60

940 5.5000 10.2567 * 1.7900 k= 6, v=60

s = 95.3361

WARNING: Procedure has used isotonized means which differ from original

(transformed) means.

Page 22 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

30060 : Total Offspring produced

Williams Test

[One-Sided Test for Decrease, alpha = 0.050000 ]

Dose Isotone

Means

T-bar P-value Significance

o

32

56

120

250

480

940

443

347

347

340

282

126

5.5

2.259

2.259

2.413

3.771

7.442

10.26

0.014

0.016

0.011

<0.005

<0.005

<0.005

*

*

*

*

*

*

"*"=Significant; "N.S."=Not Significant.

Estimates of EC%

Parameter Estimate 95% Bounds Std.Err. Lower Bound

Lower Upper /Estimate

EC5 1.8E+02 1.3E+02 2.5E+02 0.072 0.72

EC10 2.1E+02 1.6E+02 2.8E+02 0.062 0.75

EC25 2.8E+02 2.3E+02 3.5E+02 0.047 0.81

EC50 3.8E+02 3.3E+02 4.4E+02 0.032 0.86

Slope = 5.07 Std.Err. = 0.724

Goodness of fit: p = 0.055 based on DF= 4.0 63.

30060 : Total Offspring produced

Observed vs. Predicted Treatment Group Means

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

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

Mean Mean -Pred. %Control

0.00 10.0 443. 367. 76.1 100. 0.00

32.0 10.0 320. 367. -46.8 100. 2.42e-06

56.0 10.0 373. 367. 6.40 100. 0.00119

120. 10.0 340. 365. -24.8 99.5 0.542

250. 10.0 282. 302. -20.4 82.5 17.5

480. 10.0 126. 113. 12.7 30.8 69.2

940. 10.0 5.50 8.71 -3.21 2.37 97.6

Page 23 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Title:

File:

Young/Day/Surviving Parent

3006y 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

4.6900

2

16.9400

23

26.7400

28

16.9400

9

4.6900

8

Chi-Square = 9.8278

Critical Chi-Square = 13.277

9.488

(p-value = 0.0434)

(alpha = 0.01 , df = 4)

(alpha = 0.05 , df = 4)

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

Title:

File:

Young/Day/Surviving Parent

3006y Transform:

Levene's Test for Homogeneity

ANOVA Table

NO TRANSFORMATION

of Variance

SOURCE

Between

Within (Error)

Total

DF

6

63

69

SS

7134.5337

23405.2440

30539.7777

MS

1189.0890

371.5118

F

3.2007

(p-value = 0.0083)

Critical F = 3.1028

= 2.2464

Since F > Critical F

(alpha = 0.01, df = 6(63)

(alpha = 0.05, df = 6,63)

REJECT Ho: All equal (alpha 0.01)

Title:

File:

Young/Day/Surviving Parent

3006y Transform:

ANOVA Table

NO TRANSFORMATION

SOURCE

Between

Within (Error)

Total

DF

6

63

69

SS

109122.2480

58272.0840

167394.3320

MS

18187.0413

924.9537

F

19.6627

(p-value = 0.0000)

Critical F = 3.1028

= 2.2464

Since F > Critical F

(alpha = 0.01, df = 6(63)

(alpha = 0.05, df = 6,63)

REJECT Ho: All equal (alpha

Page 24 of29

0.05)
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Transform:

TABLE 1 OF 2

Title:

File:

Young/Day/Surviving Parent

3006y

Dunnett's Test

NO TRANSFORMATION

Ho:Control<Treatment

TRANSFORMED MEAN CALCULATED IN SIG

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT 0.05

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

1 Control 115.5000 115.5000

2 32 84.5000 84.5000 2.2792

3 56 114.9000 114.9000 0.0441

4 120 97.9000 97.9000 1.2940

5 250 89.2000 89.2000 1.9337

6 480 35.7000 35.7000 5.8672 * 7 940 1.8600 1.8600 8.3552 *

Dunnett critical value = 2.3400 (1 Tailed, alpha = 0.05, df [used] =6,60)

(Actual df = 6,63)

Transform:

TABLE 2 OF 2

Title:

File:

Young/Day/Surviving Parent

3006y

Dunnett's Test

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 32 10 31.8266 27.6 31.0000

3 56 10 31.8266 27.6 0.6000

4 120 10 31.8266 27.6 17.6000

5 250 10 31.8266 27.6 26.3000

6 480 10 31.8266 27.6 79.8000

7 940 10 31.8266 27.6 113.6400

Title:

File:

Young/Day/Surviving Parent

3006y Transform:

William's Test - TABLE 1 OF 2

NO TRANSFORMATION

Ho: Control<Treatment

ORIGINAL TRANSFORMED ISOTONIZED

GROUP IDENTIFICATION N MEAN MEAN MEAN

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

1 Control 10 115.5000 115.5000 115.5000

2 32 10 84.5000 84.5000 99.7000

3 56 10 114.9000 114.9000 99.7000

4 120 10 97.9000 97.9000 97.9000

5 250 10 89.2000 89.2000 89.2000

6 480 10 35.7000 35.7000 35.7000

7 940 10 1.8600 1.8600 1.8600

Page 25 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Title:

File:

Young/Day/Surviving Parent

3006y 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 115.5000

32 99.7000 1. 1617 1.6700 k= 1, v=60

56 99.7000 1. 1617 1.7500 k= 2, v=60

120 97.9000 1.2940 1.7700 k= 3, v=60

250 89.2000 1.9337 * 1.7800 k= 4, v=60

480 35.7000 5.8672 * 1.7900 k= 5, v=60

940 1.8600 8.3552 * 1.7900 k= 6, v=60

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

s = 30.4131

WARNING: Procedure has used isotonized means which differ from original

(transformed) means.

Williams Test

[One-Sided Test for Decrease, alpha =

Dose Isotone T-bar P-value

Means

0.050000 ]

Significance

0 116

32 99.7 1.162 N.S.

56 99.7 1.162 N.S.

120 97.9 1.294 N.S.

250 89.2 1.934 0.036 *

480 35.7 5.867 <0.005 *

940 1. 86 8.355 <0.005 *

"*"=Significant; "N.S."=Not Significant.

Estimates of EC%

Parameter

EC5

EC10

EC25

EC50

Estimate

2.1E+02

2.4E+02

3.1E+02

4.1E+02

95% Bounds

Lower Upper

1.5E+02 2.9E+02

1.8E+02 3.2E+02

2.5E+02 3.8E+02

3.SE+02 4.7E+02

Std. Err.

0.073

0.063

0.048

0.033

Lower Bound

/Estimate

0.72

0.75

0.80

0.86

Slope = 5.64 Std.Err. = 0.920

Goodness of fit: p = 0.17 based on DF= 4.0 63.

3006Y : Young/Day/Surviving Parent

Observed vs. Predicted Treatment Group Means

Page 26 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

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

Mean Mean -Pred. %Control

0.00 10.0 116. 103. 12.5 100. 0.00

32.0 10.0 84.5 103. -18.5 100. 2.43e-08

56.0 10.0 115. 103. 11.9 100. 6.11e-05

120. 10.0 97.9 103. -4.92 99.9 0.142

250. 10.0 89.2 90.8 -1.63 88.2 11. 8

480. 10.0 35.7 35.0 0.729 34.0 66.0

940. 10.0 1. 86 2.02 -0.165 1. 97 98.0

Title: Total length

File: 30061 Transform: NO TRANSFORMATION

Chi-Square Test for Normality

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

15.4100

18

55.6600

52

87.8600

83

55.6600

63

15.4100

14

Chi-Square = 2.0418 (p-value = 0.7281)

Critical Chi-Square 13.277

9.488

(alpha = 0.01 , df = 4)

(alpha = 0.05 , df = 4)

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

Title:

File:

Total length

30061 Transform: NO TRANSFORMATION

Levene's Test for Homogeneity of Variance

ANOVA Table

SOURCE

Between

Within (Error)

Total

DF

6

223

229

SS

0.0994

2.8039

2.9033

MS

0.0166

0.0126

F

1.3178

(p-value = 0.2501)

Critical F = 2.8837

= 2.13 94

Since F < Critical F

(alpha = 0.01, df = 6,223)

(alpha = 0.05, df = 6,223)

FAIL TO REJECT Ho: All equal (alpha

Page 27 of29

0.01)
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp,

PMRA Submission Number { } EPA MRID No. 47643006

Title:

File:

Total length

30061 Transform:

ANOVA Table

NO TRANSFORMATION

SOURCE DF SS MS F

Between

Within (Error)

Total

6

223

229

3.1652

6.9435

10.1087

0.5275

0.0311

16.9423

Critical F = 2.8837

= 2.1394

Since F > Critical F

(alpha = 0.01, df = 6,223)

(alpha = 0.05, df = 6,223)

REJECT Ho: All equal (alpha

(p-value = 0.0000)

0.05)

Title:

File:

Total length

30061

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 3.7118 3.7118

2 32 3.6000 3.6000 2.6831 * 3 56 3.6344 3.6344 1.7807

4 120 3.6353 3.6353 1.7868

5 250 3.5516 3.5516 3.6547 * 6 480 3.5485 3.5485 3.7866 * 7 940 3.2929 3.2929 9.3026 *

Bonferroni t critical value = 2.4122 (1 Tailed, alpha = 0.05, df = 6,223)

Title:

File:

Total length

30061

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 34

2 32 38 0.1005 2.7 0.1118

3 56 32 0.1048 2.8 0.0774

4 120 34 0.1032 2.8 0.0765

5 250 31 0.1057 2.8 0.1602

6 480 33 0.1040 2.8 0.1633

7 940 28 0.1086 2.9 0.4189

Page 28 of29
Data Evaluation Record on the Chronic Toxicity of IN L5300-20 (Tribenuron-methyl) to

Freshwater Invertebrates - Daphnia sp.

PMRA Submission Number { } EPA MRID No. 47643006

Title:

File:

Total length

30061

William's Test

Transform:

- TABLE 1 OF 2

NO TRANSFORMATION

Ho: Control<Treatment

ORIGINAL TRANSFORMED ISOTONIZED

GROUP IDENTIFICATION N MEAN MEAN MEAN

------ ------~------------- ----------- ----------- -----------

1 Control 34 3.7118 3.7118 3.7118

2 32 38 3.6000 3.6000 3.6221

3 56 32 3.6344 3.6344 3.6221

4 120 34 3.6353 3.6353 3.6221

5 250 31 3.5516 3.5516 3.5516

6 480 33 3.5485 3.5485 3.5485

7 940 28 3.2929 3.2929 3.2929

Title:

File:

Total length

30061

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 3.7118

32 3.6221 2.1522 * 1.6600 k= 1, v=120

56 3.6221 2.0628 * 1.7300 k= 2, v=120

120 3.6221 2.0948 * 1.7500 k= 3, v=120

250 3.5516 3.6547 * 1.7700 k= 4, v=120

480 3.5485 3.7866 * 1.7700 k= 5, v=120

940 3.2929 9.3026 * 1.7800 k= 6, v=120

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

s = 0.1765

WARNING: Procedure has used isotonized means which differ from original

(transformed) means.

Page 29 of29