Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 2 of 27 EXECUTIVE SUMMARY:

The chronic toxicity of Fluridone to the early life-stage of Chinook Salmon ( Oncorhynchus tshawytscha) was studied under flow-through conditions for 75 days (14-day hatching period and 61 days post-hatch). Fertilized eggs (100 embryos/treatment), 34 days old (fully eyed stage), were exposed to Fluridone at nominal concentrations of 0 (negative control), 0.10, 0.20, 0. 40, 0.80, and 1.6 ppm a.i. Mean-measured treatment concentrations were <0.0556 (control) , 0.107, 0.222, 0.430, 0.848, and 1.71 ppm a.i. An additional treatment with the end-use product Sonar PR was included in the study. The nominal Fluridone concentration in the Sonar PR treatment was 0.10 ppm a.i.; the mean-meas ured treatment concentration was 0.094 ppm a.i.

In the TGAI study no treatment-related effects on percent ha tch, time to first hatch, time to first swim-up, fry survival, or overall survival were observed. No treatme nt-related behavioral or appearance sub-lethal effects were observed, spinal curvature was noted in one fish in the control and Floridone treatment groups which is concluded not to be treatment related. Compared to the control treatment-related reduction in standard length was observed at the 0.848 and 1.71 ppm a.i. test groups a nd reduction in wet weight at the 1.71 ppm a.i. test group. Lengths averaged 44.2 mm in the negative control, and 44.5, 44.0, 43.8, 42.8, and 42.0 mm in the 0.107, 0.222, 0.430, 0.848, and 1.71 ppm a.i. treatment groups, respectively. Wet weights averaged 1.127 g in the negative control and 1.127, 1.096, 1.101, 1.065, and 0.999 g in the 0.107, 0. 222, 0.430, 0.848, and 1.71 ppm a.i. treatment groups, respectively. In addition, histopathologi cal examination of the brain, liver, and gills revealed a dose-dependent increase in the incidence and severity of diffuse hypertrophy of the gill lamellar epithelium in fish from the ≥ 0.430 ppm a.i. groups, and in the incidence and severity of decreased hepatocellular vacuolization in the 1.71 ppm a.i. group. Based on the primary toxicity endpoints, the overall study NOAEC and LOAEC are 0.430 ppm a.i. and 0.848 ppm a.i., respectively.

No formulation treatment effect was noted between the Sonar PR 0.10 ppm a.i. nominal treatment group and the TGAI treatment group. Both groups were not significantly different from the control for any of the primary toxicity endpoints or histopathology.

This study is scientifically sound but the lack of raw da ta on daily numbers that hatched, daily swim-up numbers does not allow calculation of rate of development for thes e parameters and therefore the study is considered to not fulfill data requirements for an early life stage toxicity study with the Chinook Salmon [OPPTS 850.1400] exposed to the TGAI and is classified SUPPLEMENTA L. The study is upgradeable with the submittal and subsequent analysis of the data. The study information on the EP is scientifically sound but does not fulfill data requirements for an EP early life stage toxicity st udy and is classified as SUPPLEMENTAL and is not upgradeable.

Results Synopsis

Test Organism Size/Age (mean Weight or Lengt h): Embryos, 34 days old, fully eyed stage Test Type (Flowthrough, Static, Static Renewal): Flow-through

Primary Toxicity Overall Study NOAEC : 0.430 ppm a.i. LOAEC : 0.848 ppm a.i.

Hatching Success NOEC: > 1.71 ppm a.i.

Days to first hatch NOEC: > 1.71 ppm a.i. Days to 95% hatch NOEC: > 1.71 ppm a.i.

Days to first swim-up NOEC: > 1.71 ppm a.i.Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 3 of 27 Fry Survival (Post-hatch Days 0-61) NOEC: > 1.71 ppm a.i.

Overall Survival (Day 0 - 75) NOEC: > 1.71 ppm a.i.

Standard Length NOEC: 0.430 ppm a.i. LOEC: 0.848 ppm a.i. Wet Weight NOEC: 0.848 ppm a.i. LOEC: 1.71 ppm a.i. Endpoint(s) Affected: Primary toxicity endpoints: Length, wet weight; Sublethal effects: gill and liver histopathology

Most sensitive endpoint(s): Primary toxicity endpoint(s): Length (gill histopathology changes were observed at levels below the primary toxicity endpoints)

I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: The study protocol was based on proce dures of the U.S. EPA Public Draft

OPPTS 850.1400 and Standard Operating Procedures of A BC Laboratories, Inc. Deviations from U.S. EPA FIFRA Guideline §72-4a and Public Draft OPPTS 850.1400 included:

Guideline Deviations Noted by Reviewer 1. Raw data on daily number of organisms that hatched, daily survival and daily swim-up numbers were not reported which does not allow an independent evalua tion by the reviewer and the development rate for hatching and swim-up could therefore not be calcula ted and no statistics for these endpoints could be determined. Time to first-hatch, days to 95% ha tch, and time to first swim-up were reported but the analysis is incomplete.

This deviation affects the validity of the study becau se not all primary toxicity endpoints could not be determined. The study is upgradeable with the submittal of the data, which were indicated in the study protocol to have been collected.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.

A. MATERIALS:

1. Test Material TGAI: Fluridone EP: Sonar PR

Description: TGAI: Solid/white round pellets of various sizes EP: Solid, brown cylindrical pellets of uniform size

Lot No./Batch No. : TGAI: 8ANZ0012 EP: 73AGW1124-C

Purity: TGAI: 99.40% EP: 5%

Stability of Compound Under Test Conditions: Aqueous stock solution was prepared daily. Except for a sample collected prior to study initiation, the test material was documented to be stable in the aqueous stock solution for 24 hours; ~24 hour old stockData Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 4 of 27 solution concentrations ranged from 100-113% of initial (0-hour) levels (N=11). Weekly to biweekly samples were within + 15%. The test material was stable during the 75-day test, with mean-measured concentrations of 106-111% of nominal concentrations.

Storage conditions of test chemicals: TGAI: The test chemical was stor ed at room temperature in the dark. EP: Stored at room temperature

Table 1. Physicochemical properties of fluridone. Parameter Values Comments
Water solubility at 20oC 12.3 mg/L at 25 o C Footprint @ http://sitem.herts.ac.uk/aeru/footprint/en/ , accessed on April 28, 2009
Vapor pressure 9.77 Η 10 -8
<1 Η 10 -7 USEPA 2007c Weed Science, 4 th Ed. 2002 UV absorption Not reported
pK a 1.73* R. Grover and A. J. Cessna. 1991. Environmental Chemistry of Herbicides Volume II, CRC Press

pK b 12.3* Mackay, D., Shiu, W. Y., Ma, K. C., & Lee, S. C. 2006. Handbook of Physical-Chemical Properties and Environmental Fate for Organic Chemicals (Second ed. Vol. IV). Boca Raton, FL: CRC Press.

K ow 74.13 Log K ow 1.87 from Footprint @ http://sitem.herts.ac.uk/aeru/footprint/en/ , accessed on April 28, 2009Aquatic photolysis halflife 0.9-2.3 days MRID # 103326

(EPA/OECD guidance recommends reporting water solubility under test conditions, stability in water and light, pKa, Pow, and vap or pressure of test material..) * Therefore, the unionized form should be present within the environmentally relevant pH range.

2. Test organism :

Species: Chinook Salmon ( Oncorhynchus tshawytscha)

Age/embryonic stage at test initiation: Fertilized embryos, 34 days old, fully eyed stage. The guideline preference is for within 24 hours post-fertilization. However for trout the guideline identifies that embryos used may be as old as the eyed stage. At 10 o C, Chinook salmon should be fully eyed at 33 days and can be shocked and removed from incubation trays. Therefore, 34-day eyed Chinook embryos are satisfactory for use in this test.

Method of collection of the fertilized eggs: Fertilized by the supplier on October 23, 2002.

Source: Iron Gate Hatchery (California Department of Fish and Game).Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 5 of 27 B. STUDY DESIGN:

1. Experimental Conditions

a. Range-finding study: No range-finding study was conducted.

b. Definitive Study

Table 2: Experimental Parameters Remarks Parameter Details Criteria Parental acclimation, if any Period:

Conditions: (same as test or not)

Feeding (type, source, amount given, frequency):

Health: (any mortality observed) N/A

Number of fertilized eggs/embryos in each treatment at test initiation 100 embryos/treatment, divided into 25 embryos per replicate with four replicates per treatment EPA requires minimum of 20 embryos per replicate cup. Minimum of 30 fish per treatment for post-hatch exposure Mean-measured concentrations were determined weekly. Concentration of test material: nominal:

measured: TGAI: 0 (negative control), 0.10, 0.20, 0.40, 0.80, and 1.6 ppm a.i.

EP: 0.10 ppm a.i.

TGAI: <0.0556 (control), 0.107, 0.222, 0.430, 0.848, and 1.71 ppm a.i.

EP: 0.094 ppm a.i.

TGAI: A target concentration of 6.3 ppm a.i. stock solutions were prepared daily EPA requires a minimum of 5 concentrations and a control, all replicated, plus solvent control if appropriate. - Toxicant conc. must be measured in one tank at each toxicant level every week. - One concentration must adversely affect a life stage and one concentration must not affect any life stage. OECD requires 5 concentrations spaced by a constant factor not exceeding 3.2; concentrations of test substance in solution must be within ± 20% of the mean measured values.Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 6 of 27 Remarks Parameter Details Criteria Solvent (type, percentage, if used) Aqueous stock solutions EPA requires that solvent should not exceed 0.1 ml/L in a flow-through system. Following solvents are acceptable: dimethylformamide, triethylene glycol, methanol, acetone, ethanol. OECD requires that solvent must have no effect on survival nor produce any other adverse effects;concentration should not be greater than 0.1 ml/L. Number of replicates control: solvent control: treated ones: 4 N/A 4 EPA standard is 4 replicates per concentration EPA/OECD when a solvent has been used a solvent control needs to also be included. Mixing cells were used for each toxicant level. The flow rate was 66 L/day. Flow rates and delivery split accuracies were checked at test initiation and twice daily. Test condition : Static renewal/flow through:

Type of dilution system for flow through method: Flow rate:

Renewal rate for static renewal: Flow-through

Proportional diluter system

6.6 volume additions in a 24 hour period

N/A Intermittent flow proportional diluters or continuous flow serial diluters should be used. A minimum of 5 toxicant concentrations with a dilution factor not greater than 0.5 and controls should be used. Toxicant Mixing: 1) Mixing chamber is recommended but not required; 2) Aeration should not be used for mixing; 3) It must be demonstrated that the test solution is completely mixed before intro. into the test system; 4) Flow splitting accuracy must be within 10%. Aeration, if any No aeration was provided. Dilution water should be aerated to insure DO concentration at or near 100% saturation. Test tanks and embryo cups should not be aerated. Duration of the test 75 days: 14-day development and hatching period and 61 days post-hatch EPA post-hatch duration is 28-32 days for warm water and 60 days cold water, species specificData Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 7 of 27 Remarks Parameter Details Criteria Egg cups were oscillated vertically with a rocker arm apparatus (low rpm). Embryo cups , if used type/material: (glass/stainless steel)

size:

fill volume: Glass jars with Nitex screen (16-mesh) bottom.

9-cm diameter

Not specified EPA requires 120 ml glass jars with bottoms replaced with 40 mesh stainless steel or nylon screen.

Aquaria covered with a stainless steel screen to prevent loss of fish.

Biomass loading ranged from 0.216 g/L/day/L to 0.278 g/L/day. Test vessel type/material: (glass/stainless steel)

size:

fill volume: Glass aquaria

15 cm width Η 31 cm length

10 L (22-cm depth) EPA/OECD requires all glass or glass with stainless steel frame. Weekly analyses of total hardness, total alkalinity, and conductivity, monthly analyses of TOC and TSS, and yearly chemical characterization. Source of dilution water The dilution water was moderately hard fresh well water mixed with well water which was de-mineralized by reverseosmosis. The water was aerated, prechilled, filtered, and UV irradiated. EPA requires natural or reconstituted water; natural water should be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants

. OECD accepts any water in which the test species show control survival at least as good as presented in SEP. Water parameters: hardness: pH: TOC:

dissolved oxygen: temperature: photoperiod: 142-156 mg/L as CaCO 3 7.34-8.64 <2 mg/L (p. 21)

7.8-10.8 mg/L (76-105% saturation) 9.4-12.5 ° C; Semi-darkness until approximately 1 The TSS, TOC, and DOC were measured in the incoming dilution water at test initiation, monthly thereafter, and test termination.Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 8 of 27 Remarks Parameter Details Criteria

salinity (for marine or estuarine species):

other measurements:

interval of water quality
measurements: week post-hatch and 16:8 light/dark hours for remainder of test.

N/A

Total suspended solids: 0.4-1.0 mg/L; Metals: within acceptable levels Pesticides: <LOD.

DO, pH, and temperature were measured in all replicates at test initiation, weekly throughout the test, and at test termination. Temperature was also measured continuously in one control replicate. Total hardness and alkalinity were measured in one replicate of the control and treatment groups at test initiation, monthly thereafter, and at test termination. EPA requires hardness of 40 to 48 mg a.i./L as CaCO 3 and pH of 7.2 to 7.6 is recommended. DO must be measured at each conc. at least once a week; freshwater parameters in a control and one concentration must be analyzed once a week

. Temperature depends upon test species; should not deviate by more than 2 ° C from appropriate temperature. OECD requires DO concentration between 60 - 90% saturation

. As a minimum DO, salinity (if relevant) and temperature should be measured weekly, and pH and hardness at the beginning and end of the test. Temperature should be measured continuously.

All criteria were met for hatching success in control replicates. Post-hatch details: when the post-hatch period began:

number of hatched eggs (alevins)/ treatment released to the test chamber:

day that alevins were released from the incubation cups to the test chamber: Day 14; >95% of the embryos in the control had hatched

The number of fry per test chamber werethinned to 15 on Day 16 (2 days posthatch).

Day 14 EPA requires % of embryos that produce live fry must be > 50% in each control; % hatch in any control embryo cup must be no more than 1.6 times that in another control cup.

Post-hatch Feeding: start date:

type/source of feed:

amount given:

frequency of feeding: Day 29 (Day 15 post-hatch)

Live brine shrimp nauplii ( Artemia sp.) was provided and a commercially prepared salmon starter was added to diet on Day 40 (26 days post hatch).

Fed ad libitum

Three times a day, except during final 24 hours of testing. Stability of chemical in the test system Diluter stock solution was prepared daily. Verified by weekly analytical measurements: mean-measured concentrations averaged 106 to 111% of nominal test concentrationsData Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 9 of 27 Remarks Parameter Details Criteria Recovery of chemical:

Frequency of measurement:

LOD: LOQ: Generally 92-119% of nominal fortifications, with two outliers

Not reported

Not reported 0.0556 ppm a.i. Based on QC samples fortified at 0.0910, 18.2, 31.2, or 36.4 ppm and run concurrently with sample analyses (p. 16 and Table 3, p. 25).

Positive control {if used, indicate the chemical and concentrations} N/A

Fertilization success study , if
any number of eggs used:

on what day the eggs were removed to check the embryonic development: None conducted.

Other parameters, if any N/A

2. Observations:

Table 3: Observations
Criteria Details Remarks/Criteria Parameters measured including the sublethal effects/toxicity symptoms - Time to hatch - Hatching success - Time to swim-up - Fry survival - Measurement of growth (total and standard length and wet weights) - Sub-lethal effects - Histology/pathology (brain, gill, liver)—10 fish per treatment - Any abnormal physical changes in the embryos and/or fry as well as abnormal behaviors were noted. EPA minimally requires: - Number of embryos hatched; - Time to hatch; - Mortality of embryos, larvae, and juveniles; - Time to swim-up (if approp.); - Measurement of growth; - Incidence of pathological or histological effects; - Observations of other effects or clinical signs

. Observation intervals/dates for: egg mortality: no. of eggs hatched: mortality of fry ( e.g .alevins): swim-up behavior: growth measurements: embryonic development: other sublethal effects Daily Daily through 2 days post-hatch Daily Daily Day 75 (test termination) Not determined Daily Water quality was acceptable (Yes/No) Yes Were raw data included? YesData Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 10 of 27 Criteria Details Remarks/Criteria Other observations, if any Histology/pathology analysis was conducted for 10 randomly-selected fish from each treatment at test termination.

II. RESULTS AND DISCUSSION A. MORTALITY:

No treatment-related effect on hatching success, post-hatc h survival, or overall survival were observed. The mean percent hatch, fry survival (Day 75), and overall survival (Day 0 – 75) were 97 - 100% for the control and all treatment groups (Table 4). The NOEC was > 1.71 ppm a.i. for hatching success, fry survival, and overall survival.

No formulation effect on hatching success, post-hatch su rvival, or overall survival was observed between the nominal 10 ppm a.i. TGAI and EP treatment groups, nor was their a significant difference from the control group. Overall survival was 98% for both tr eatment groups and the control (Table 5).

Table 4: Effect of Fluridone TGAI on hatching success, fry survival and overall ELS survival of Chinook Salmon ( O. tshawytscha )
Percent Egg Hatch Percent Fry Survival

Treatment, ppm a.i. measured (and nominal) concentrations No. of eggs at study initiation Day 16 No. of thinned Alevins Day 75 Overall Survival

Negative control 100 98 60 100 0.98 0.107 (0.10) 100 98 60 100 0.98 0.222 (0.20) 100 99 60 98 0.97 0.430 (0.40) 100 99 60 98 0.97 0.848 (0.80) 100 99 60 100 0.99 1.71 (1.6) 100 100 59 98 0.98 NOAEC, ppm a.i. > 1.71 > 1.71 > 1.71 LOAEC, ppm a.i. -- -- --

Table 5: Comparison between 0.10 ppm a.i. Fluridone TGAI and EP on hatching success, fry survival and overall ELS survival of Chinook Salmon ( O. tshawytscha )

Percent Egg Hatch Percent Fry Survival Overall Survival Treatment, ppm a.i. measured (and nominal) concentrations No. of eggs at study initiation Day 16 No. of thinned Alevins Day 75 Day 0 through 75
Negative control 100 98 60 100 0.98 TGAI 0.107 (0.10) 100 98 60 100 0.98 EP 0.094 (0.10) 100 100 59 98 0.98Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 11 of 27 B. SUB-LETHAL TOXICITY AND OTHER CHRONIC EFFECTS:

No treatment-related effects on the time to hatch or time to swim-up were observed. The mean first day to hatch was 10 to 11 days, and 95% hatch occurred in all groups by Day 14 (Table 6). The mean time to first swim-up was 17-18 days. The NOAEC for these endpoints was > 1.71 ppm a.i., the highest concentration tested.

The study authors found terminal length to be significan tly reduced at the 1.71 ppm a.i. treatment group as compared to the control and no treatment-related differen ces were observed in wet weights. The study authors’ NOAEC values were 0.848 for length, and > 1.71 for weight. The reviewer used William’s over Dunnett’s for the length which assumes a monotonic relationship with increasing concentration, which resulted in a lower NOAEC than what the authors reported. Williams was al so used for wet weight as was Dunnett’s, both tests indicated the highest concentration as significantly different. Do not understand why Dunnett’s performed by authors resulted in different answer. The NOAEC and L OAEC values determined for length and weight by the reviewer are summarized in Table 8.

No treatment related behavioral or morphological abnor malities were observed following hatch, spinal curvature was noted in one fish from the control and floridone treatme nts that disappeared by the end of the test. Ten fish per group were analyzed for histology/pathology of the brain, gills, and liver. There was a dose-dependent increase in the incidence and severity of diffuse hypert rophy of the gill lamellar epithelium (mild to moderate) in fish of the 0.430, 0.848 and 1.71 ppm a.i. groups when compared to the control group. In addition, the incidence and severity of decreased hepatocellular v acuolization (minimal to moderate) was significantly increased in the 1.71 ppm a.i. group when compared to the control group. No histopathological findings were noted for brain tissue.

There was no formulation effect on days to first hatch, 95% hatch, and swim-up, between the nominal 10 ppm a.i. TGAI and EP treatment groups (Tab le 7). There was also no formulation effect to length or wet weight as compared to the TGAI (Table 9). There were no significant difference in histopathological findings between the
two.

Table 6: Effect of Fluridone on developm ent (mean±SD) of juvenile Chinook Salmon ( Oncorhynchus tshawytscha ). Treatment, ppm a.i. measured (and nominal) concentrations Days to First Hatch Days to ≥ 95% Hatch Days to First Swim-Up (Post-Hatch) Negative control 10.5 ±1.0 13.5 ± 0.58 17.5 ± 1.00 0.107 (0.10) 11.0 ± 0 14.0 ± 0 16.5 ± 1.29 0.222 (0.20) 10.5 ± 0.58 14.0 ± 0.82 17.2 ± 0.25 0.430 (0.40) 10.5 ± 0.58 13.5 ± 0.58 17.2 ± 0.25 0.848 (0.80) 10.0 ± 0 13.5 ± 0.58 16.8 ± 0.25 1.71 (1.6) 10.8 ± 0.50 13.0 ± 0.82 17.8 ± 0.48 NOAEC, ppm a.i. > 1.71 > 1.71 > 1.71 LOAEC, ppm a.i. -- -- --Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 12 of 27 Table 7: Comparison between 0.10 ppm a.i. Fluridone TGAI and EP on Days to First Hatch, 95% Hatch and Swim-Up of Chinook Salmon ( O. tshawytscha ). Treatment, ppm a.i. measured (and nominal) concentrations Days to First Hatch Days to 95% Hatch Days to First Swim-Up (Post-Hatch) Negative control 10.5 ±1.0 13.5 ± 0.58 17.5 ± 1.00 TGAI 0.107 (0.10) 11.0 ± 0 14.0 ± 0 16.5 ± 1.29 EP 0.094 (0.10) 10.8 ± 0.50 14.0 ± 0 17.5 ± 1.29

Table 8: Effect (1) of Fluridone on growth (mean±SD) of juvenile Chinook Salmon ( Oncorhynchus tshawytscha ). Treatment, ppm a.i.measured (and nominal) concentrations Length (mm) Wet Weight (g) Negative control 44.2 ± 0.5 1.127 ± 0.067 0.107 (0.10) 44.5 ± 0.6 1.128 ± 0.037 0.222 (0.20) 44.0 ± 1.8 1.097 ± 0.112 0.430 (0.40) 43.8 ± 1.0 1.098 ± 0.068 0.848 (0.80) 42.8 ± 1.0* 1.065 ± 0.085 1.71 (1.6) 42.0 ± 0.8* 1.000 ± 0.038* NOAEC, ppm a.i. 0.430 0.848 LOAEC, ppm a.i. 0.848 1.71

(1) Statistical results shown are based on the reviewer’s reanalysis of the data usi ng William’s test for length and Dunnett’s for wet weight. Study authors’ NOAEC values fo r length was 0.848 ppm a.i. and for weight > 1.71 ppm a.i. * Statistically different from the control (p ≤ 0.05).

Table 9: Comparison between 0.10 ppm a.i. Fluridone TGAI and EP on length and wet weight of Chinook Salmon ( O. tshawytscha ) young of year. Treatment, ppm a.i.measured (and nominal) concentrations Length (mm) Wet Weight (g) Negative control 44.2 ± 0.5 1.127 ± 0.067 TGAI 0.107 (0.10) 44.5 ± 0.6 1.128 ± 0.037 EP 0.094 (0.10) 43.8 ± 1.5 1.106 ± 0.101

C. REPORTED STATISTICS:

Endpoints that were analyzed statistically included ha tch and post-hatch survival, and standard length and blotted wet weights. The statistical analyses were conducted using SAS software. The hatch and post-hatch survival NOAEC values were determined using the Fishe r’s exact test with a Hochberg adjustment. The NOEC values for terminal lengths and wet weights were dete rmined using one-way analysis of variance (ANOVA) and a one-tailed Dunnett’s test. The Shapiro-Wilk’s test for normality and Levene’s test for homogeneity of variance were conducted prior to the Dunnett’s test. Mean-measured concentrations were used in all comparisons.Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 13 of 27 D. VERIFICATION OF STATISTICAL RESULTS:

Day to first hatch, 95% hatch, and firs t swim-up, length, and wet weight data were statistically analyzed for fish exposed to fluridone TGAI. The NOAEC and LOAEC for hatching success, fry survival, and overall survival could be visually determined, as no reductions from cont rol were observed. The statistical methods for the NOAEC and LOAEC determinations by the reviewer are summarized in Table 10. These analyses were conducted using TOXSTAT statistical software. Histologi cal findings were analyzed using the Fishers Exact Test.

Comparison of the TGAI and EP nominal 0.10 treatment gr oups were made using the t-test with assumption of equal variance for length, weight, days to first swim-up, and Fishers Exact test for gill and liver histology. No statistical comparison was performed for hatching success, fry survival, or overall survival as survival was within acceptable control limits; overall survival was 98% in both treatment groups. No statistical analysis was performed on days to first hatch or days to 95% hatch as both treatment groups had the same mean.

Table 10: Statistical methods used by the reviewer. Endpoint Normality Homogeneity of Variance Multiple Comparison Method

Hatching Success Did not perform a statistical analysis, all results were within acceptable control mortality levels, with no treatment related increase displayed. Days to First Hatch ShapiroWilks—Fail N/A Steels Many One Rank Test—Non parameteric, equal replicates

Days to 95% Hatch ShapiroWilks—Pass Bartlett’s Test—Fail Steels Many One Rank Test—Non parameteric, equal replicates

Days to SwimUp ShapiroWilks—Pass Bartlett’s Test—Pass Dunnett’s Test—Parametric, equal replicates

Fry Survival Did not perform a statistical analysis, all results were within acceptable control mortality levels, with no treatment related increase displayed. Overall Survival Did not perform a statistical analysis, all results were within acceptable control mortality levels, with no treatment related increase displayed. Standard Length ShapiroWilks—Pass Bartlett’s Test—Pass Williams Test—Parametric, equal replicates, assumes monotonic decreasing relationshipWet Weight ShapiroWilks—Pass Bartlett’s Test—Pass Williams Test—Parametric, equal replicates, assumes monotonic decreasing relationship Gill, diffuse hypertrophy N/A N/A Fishers Exact Test

Liver, Hepatocellular Vacuolization N/A N/A Fishers Exact Test

Brain histology No effects observed in any treatme nt group, therefore did not perform statistics

E. STUDY DEFICIENCIES:

None were noted by the study authors’ but a major one was noted by this reviewer see Section I Methods and Materials of this DER under Guideline

Fluridone was detected at 0.449 ppm a.i. in the contro l sample on Day 21 (Table 1, p. 23). This value was attributed to a sampling/preparation error of the control sample. The reviewer agrees with this determination.Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 14 of 27 F. REVIEWER’S COMMENTS:

The reviewer’s conclusions differed slightly from those of the study author. The reviewer’s analysis detected significant adverse effects on length at the 0.848 and 1.71 ppm treatment levels and wet weight at 1.71 ppm a.i., whereas the study author’s analysis only detected signi ficant reductions in length at the 1.71 ppm treatment level for length and none for wet weight. The study author reported significant effects on the gill histopathology at levels equal to and exceeding 0.430 ppm, estab lishing a lower NOAEC for gill histopathology (0.222 ppm) than for growth (0.430 ppm). Differences between the st udy author’s and the reviewer’s results are due to the different statistical methods used. The more cons ervative NOAEC and LOAEC values (from both assessments) are reported in the Executive Summary and Conclusions sections.

Two technical errors occurred during the study (p. 15). On Day 28, the delivery line for the Sonar PR diluter stock solution was not properly connected, and as a resu lt, the Sonar PR treatment did not receive the test substance for approximately 14 hours. On Day 44, the c oncentration of the freshly-prepared Sonar PR diluter stock solution was incorrectly calculated. This error resulted in the Sonar PR treatment receiving 50% of the required stock solution needed for approximately 3 days. As a result of these errors, additional sampling of the diluter and/or test solutions was conducted. These devia tions in exposure were considered to not be significant for the overall duration of the study and the information on the EP is supplemental, and was not conducted as a definitive NOAEC study for the EP.

G. CONCLUSIONS:

This study is scientifically sound but the lack of raw da ta on daily numbers that hatched, daily swim-up numbers does not allow calculation of rate of development for thes e parameters and therefore the study is considered to not fulfill data requirements for an early life stage toxicity study with the Chinook Salmon [OPPTS 850.1400] exposed to the TGAI and is classified SUPPLEMENTA L. The study is upgradeable with the submittal and subsequent analysis of the data. The study information on the EP is scientifically sound but does not fulfill data requirements for an EP early life stage toxicity st udy and is classified as SUPPLEMENTAL and is not upgradeable.

Results Synopsis Hatching success NOAEC: > 1.71 ppm a.i.

Time to first hatch NOEC: > 1.71 ppm a.i.

Time to 95% hatch NOEC: > 1.71 ppm a.i.

Time to swim-up NOAEC: > 1.71 ppm a.i.

Fry survival (Day 75; Day 61 post-hatch) NOAEC: > 1.71 ppm a.i.

Overall survival (Day 0 to 75) NOAEC: > 1.71 ppm a.i.

Length NOAEC: 0.430 ppm a.i. LOAEC: 0.848 ppm a.i.

Wet Weight NOAEC: 0.848 ppm a.i. LOAEC: 1.71 ppm a.i.

Gill histopathology NOAEC: 0.222 ppm a.i. LOAEC: 0.430 ppm a.i.

Liver histopathology NOAEC: 0.848 ppm a.i. LOAEC: 1.71 ppm a.i.

Brain histopathology NOAEC: > 1.71 ppm a.i.Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 15 of 27 Endpoint(s) Affected: Primary toxicity endpoints: Length, wet weight; Sublethal effects: gill and liver histopathology Most sensitive endpoint(s): Primary toxicity endpoint(s): Length (gill histopathology changes were observed at levels below the primary toxicity endpoints)

III. REFERENCES:

U.S. Environmental Protection Agency. 1989. Federal In secticide, Fungicide and Rodenticide Act (FIFRA); Good Laboratory Practice Standards; Final Rule (40 CF R, Part 160). Federal Register, August 17, 1989.

U.S. Environmental Protection Agency. Toxic Substances Control Act; Good Laboratory Practice Standards; Final Rule (40 CFR, Part 792). Federal Register

. Organization for Economic Cooperation and Development. 1992. OECD Guideline for the Testing of Chemicals. Method No. 210, “Fish, Early-life Stage Toxicity Test”.

U.S. Environmental Protection Agency. 1982. Pesticid e Assessment Guidelines, FIFRA, Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. EPA-540/9- 85-024. Office of Pesticide Programs. Series 72-4, Fish Early Life-Stage Test.

Mount, D.I., and W.A. Brungs. 1967. A simplified dosing apparatus for fish toxicological studies. Water Res. 1: 21-29.

American Public Health Association. 1998. Standard Methods for the Examination of Water and Wastewater. 20 th ed. Washington, DC. pp. 1-17.

The SAS System for Windows, Release 6.12. Copyright 1989-1996 by SAS Institute Inc., Cary, North Carolina, 27513, USA.

Zar, J.H. 1984. Biostatistical Analysis. 2 nd ed. Prentice-Hall, Englewood Cliffs, N.J.

Hochberg, Y. 1988. A Sharper Bonferroni Pr ocedure for Multiple Test of Significance. Biometrika . 75:800-802.

Dunnett, C.W. 1955. A Multiple Comparison Procedure for Comparing Several Treatments with a Control. J. Amer. Stat. Assoc. 50: 1096-1121.

Conover, W.J. Practical Nonparametric Statistics. 1980. 2 nd Ed.

Millikan, G.A., and D.E. Johnson. Analysis of Messy Data . 1984, Vol. 1, p. 22.Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 16 of 27 **APPENDIX 1: OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION :** 



## Days to first hatch

SUMMARY STATISTICS TABLE 1 of 2

------------------------------------------------------------------------------ GRP IDENTIFICATION N MIN MAX MEAN

--- ---------------- ---- ---------- ---------- ---------- 1 Control 4 9.000 11.000 10.500 2 0.107 4 11.000 11.000 11.000 3 0.222 4 10.000 11.000 10.500 4 0.430 4 10.000 11.000 10.500 5 0.848 4 10.000 10.000 10.000 6 1.71 4 10.000 11.000 10.750

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

SUMMARY STATISTICS TABLE 2 of 2

------------------------------------------------------------------------------ GRP IDENTIFICATION VARIANCE SD SEM

--- ---------------- -------------- ---------- ---------- 1 Control 1.000 1.000 0.500 2 0.107 0.000 0.000 0.000 3 0.222 0.333 0.577 0.289 4 0.430 0.333 0.577 0.289 5 0.848 0.000 0.000 0.000 6 1.71 0.250 0.500 0.250

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

Shapiro Wilks test for normality

------------------------------------------------------------------------------ D = 5.750 W = 0.847 Critical W (P = 0.05) (n = 24) = 0.916 Critical W (P = 0.01) (n = 24) = 0.884

------------------------------------------------------------------------------ **Data FAIL normality test** . Try another transformation.

STEELS MANY-ONE RANK TEST - Ho:Control<Treatment

---------------------------------------------------------------------------- TRANSFORMED RANK CRIT. GROUP IDENTIFICATION MEAN SUM VALUE df SIG

----- -------------------- ----------- ------- ------ ----- --- 1 Control 10.500 2 0.107 11.000 20.00 10.00 4.00 3 0.222 10.500 17.00 10.00 4.00 4 0.430 10.500 17.00 10.00 4.00 5 0.848 10.000 14.00 10.00 4.00 6 1.71 10.750 18.50 10.00 4.00

---------------------------------------------------------------------------- Critical values use k = 5, are 1 tailed, and alpha = 0.05Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 17 of 27 

## Days to 95% hatch

File: O95hatch.txt Transform: NO TRANSFORMATION

SUMMARY STATISTICS TABLE 1 of 2

------------------------------------------------------------------------------ GRP IDENTIFICATION N MIN MAX MEAN

--- ---------------- ---- ---------- ---------- ---------- 1 Control 4 13.000 14.000 13.500 2 0.107 4 14.000 14.000 14.000 3 0.222 4 13.000 15.000 14.000 4 0.430 4 13.000 14.000 13.500 5 0.848 4 13.000 14.000 13.500 6 1.71 4 12.000 14.000 13.000

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

SUMMARY STATISTICS TABLE 2 of 2

------------------------------------------------------------------------------ GRP IDENTIFICATION VARIANCE SD SEM

--- ---------------- -------------- ---------- ---------- 1 Control 0.333 0.577 0.289 2 0.107 0.000 0.000 0.000 3 0.222 0.667 0.816 0.408 4 0.430 0.333 0.577 0.289 5 0.848 0.333 0.577 0.289 6 1.71 0.667 0.816 0.408

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

Shapiro Wilks test for normality

------------------------------------------------------------------------------ D = 7.000 W = 0.925 Critical W (P = 0.05) (n = 24) = 0.916 Critical W (P = 0.01) (n = 24) = 0.884

------------------------------------------------------------------------------ **Data PASS normality** test at P=0.01 level. Continue analysis.

Hartley test for homogeneity of variance Bartletts test for homogeneity of variance

------------------------------------------------------------------------------ These two tests can not be performed because at least one group has zero variance.

**Data FAIL to meet homogeneity** of variance assumption. Additional transformations are useless.

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

STEELS MANY-ONE RANK TEST - Ho:Control<Treatment

---------------------------------------------------------------------------- TRANSFORMED RANK CRIT. GROUP IDENTIFICATION MEAN SUM VALUE df SIG

----- -------------------- ----------- ------- ------ ----- --- 1 Control 13.500 2 0.107 14.000 22.00 10.00 4.00 3 0.222 14.000 21.00 10.00 4.00 4 0.430 13.500 18.00 10.00 4.00 5 0.848 13.500 18.00 10.00 4.00 6 1.71 13.000 15.00 10.00 4.00

---------------------------------------------------------------------------- Critical values use k = 5, are 1 tailed, and alpha = 0.05Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 18 of 27 

## Days to First Swim-Up

File: oswim.txt Transform: NO TRANSFORMATION

SUMMARY STATISTICS DATA TABLE 1 of 2

------------------------------------------------------------------------------ GRP IDENTIFICATION N MIN MAX MEAN

--- ---------------- ---- ---------- ---------- ---------- 1 Control 4 17.000 19.000 17.500 2 0.107 4 15.000 18.000 16.500 3 0.222 4 17.000 18.000 17.250 4 0.430 4 17.000 18.000 17.250 5 0.848 4 16.000 17.000 16.750 6 1.71 4 17.000 19.000 17.750

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

SUMMARY STATISTICS TABLE 2 of 2

------------------------------------------------------------------------------ GRP IDENTIFICATION VARIANCE SD SEM

--- ---------------- -------------- ---------- ---------- 1 Control 1.000 1.000 0.500 2 0.107 1.667 1.291 0.645 3 0.222 0.250 0.500 0.250 4 0.430 0.250 0.500 0.250 5 0.848 0.250 0.500 0.250 6 1.71 0.917 0.957 0.479

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

Shapiro Wilks test for normality

------------------------------------------------------------------------------ D = 13.000 W = 0.936 Critical W (P = 0.05) (n = 24) = 0.916 Critical W (P = 0.01) (n = 24) = 0.884

------------------------------------------------------------------------------ **Data PASS normality** test at P=0.01 level. Continue analysis.

Bartletts test for homogeneity of variance

------------------------------------------------------------------------------ Calculated B statistic = 4.73 Table Chi-square value = 15.09 (alpha = 0.01) Table Chi-square value = 11.07 (alpha = 0.05)

Average df used in calculation ==> df (avg n - 1) = 3.00 Used for Chi-square table value ==> df (#groups-1) = 5

------------------------------------------------------------------------------ **Data PASS homogeneity** test at 0.01 level. Continue analysis.

ANOVA TABLE

------------------------------------------------------------------------------ SOURCE DF SS MS F

------------------------------------------------------------------------------ Between 5 4.333 0.867 1.201 Within (Error) 18 13.000 0.722

------------------------------------------------------------------------------ Total 23 17.333Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 19 of 27 ------------------------------------------------------------------------------
Critical F value = 2.77 (0.05,5,18) Since F < Critical F FAIL TO REJECT Ho:All groups equal

DUNNETTS TEST - TABLE 1 OF 2 Ho:Control<Treatment

---------------------------------------------------------------------------- TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

----- -------------------- ----------- ------------------ ------ --- 1 Control 17.500 17.500 2 0.107 16.500 16.500 1.664 3 0.222 17.250 17.250 0.416 4 0.430 17.250 17.250 0.416 5 0.848 16.750 16.750 1.248 6 1.71 17.750 17.750 -0.416

---------------------------------------------------------------------------- Dunnett table value = 2.41 (1 Tailed Value, P=0.05, df=18,5)

DUNNETTS TEST - TABLE 2 OF 2 Ho:Control<Treatment

---------------------------------------------------------------------------- NUM OF Minimum Sig Diff % of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

----- -------------------- ------- ---------------- ------- ------------ 1 Control 4 2 0.107 4 1.448 8.3 1.000 3 0.222 4 1.448 8.3 0.250 4 0.430 4 1.448 8.3 0.250 5 0.848 4 1.448 8.3 0.750 6 1.71 4 1.448 8.3 -0.250

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

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

---------------------------------------------------------------------------- GROUP ORIGINAL TRANSFORMED ISOTONIZED IDENTIFICATION N MEAN MEAN MEAN

------ -------------------- --- ----------- ----------- ----------- 1 Control 4 17.500 17.500 17.000 2 0.107 4 16.500 16.500 17.000 3 0.222 4 17.250 17.250 17.083 4 0.430 4 17.250 17.250 17.083 5 0.848 4 16.750 16.750 17.083 6 1.71 4 17.750 17.750 17.750

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


## Standard Length

Shapiro Wilks test for normality

------------------------------------------------------------------------------ D = 19.250 W = 0.977

Critical W (P = 0.05) (n = 24) = 0.916 Critical W (P = 0.01) (n = 24) = 0.884

------------------------------------------------------------------------------ **Data PASS normality test** at P=0.01 level. Continue analysis.

Bartletts test for homogeneity of varianceData Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 20 of 27 ------------------------------------------------------------------------------ Calculated B statistic = 6.01 Table Chi-square value = 15.09 (alpha = 0.01) Table Chi-square value = 11.07 (alpha = 0.05)

Average df used in calculation ==> df (avg n - 1) = 3.00 Used for Chi-square table value ==> df (#groups-1) = 5

------------------------------------------------------------------------------ **Data PASS homogeneity test** at 0.01 level. Continue analysis.

SUMMARY STATISTICS TABLE 1 of 2

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

GRP IDENTIFICATION N MIN MAX MEAN

--- ---------------- ---- ---------- ---------- ---------- 1 Control 4 44.000 45.000 44.250 2 0.107 4 44.000 45.000 44.500 3 0.222 4 42.000 46.000 44.000 4 0.430 4 43.000 45.000 43.750 5 0.848 4 42.000 44.000 42.750 6 1.71 4 41.000 43.000 42.000

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

SUMMARY STATISTICS TABLE 2 of 2

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

GRP IDENTIFICATION VARIANCE SD SEM

--- ---------------- -------------- ---------- ---------- 1 Control 0.250 0.500 0.250 2 0.107 0.333 0.577 0.289 3 0.222 3.333 1.826 0.913 4 0.430 0.917 0.957 0.479 5 0.848 0.917 0.957 0.479 6 1.71 0.667 0.816 0.408

------------------------------------------------------------------------------ ANOVA TABLE

------------------------------------------------------------------------------ SOURCE DF SS MS F

------------------------------------------------------------------------------ Between 5 18.708 3.742 3.500 Within (Error) 18 19.250 1.069

------------------------------------------------------------------------------ Total 23 37.958

------------------------------------------------------------------------------ Critical F value = 2.77 (0.05,5,18) Since F > Critical F **REJECT Ho:All groups equal**

DUNNETTS TEST - TABLE 1 OF 2 Ho:Control<Treatment

---------------------------------------------------------------------------- TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

----- -------------------- ----------- ------------------ ------ --- 1 Control 44.250 44.250 2 0.107 44.500 44.500 -0.342 3 0.222 44.000 44.000 0.342 4 0.430 43.750 43.750 0.684 5 0.848 42.750 42.750 2.052 6 1.71 42.000 42.000 3.078 *

---------------------------------------------------------------------------- Dunnett table value = 2.41 (1 Tailed Value, P=0.05, df=18,5)Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 21 of 27 DUNNETTS TEST - TABLE 2 OF 2 Ho:Control<Treatment

---------------------------------------------------------------------------- NUM OF Minimum Sig Diff % of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

----- -------------------- ------- ---------------- ------- ------------ 1 Control 4 2 0.107 4 1.762 4.0 -0.250 3 0.222 4 1.762 4.0 0.250 4 0.430 4 1.762 4.0 0.500 5 0.848 4 1.762 4.0 1.500 6 1.71 4 1.762 4.0 2.250

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

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

---------------------------------------------------------------------------- GROUP ORIGINAL TRANSFORMED ISOTONIZED IDENTIFICATION N MEAN MEAN MEAN

------ -------------------- --- ----------- ----------- ----------- 1 Control 4 44.250 44.250 44.375 2 0.107 4 44.500 44.500 44.375 3 0.222 4 44.000 44.000 44.000 4 0.430 4 43.750 43.750 43.750 5 0.848 4 42.750 42.750 42.750 6 1.71 4 42.000 42.000 42.000

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

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

---------------------------------------------------------------------------- ISOTONIZED CALC. SIG TABLE DEGREES OF IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

-------------------- ----------- ----------- ----- ----------- ------------- Control 44.375 0.107 44.375 0.171 1.73 k= 1, v=18 0.222 44.000 0.342 1.82 k= 2, v=18 0.430 43.750 0.684 1.85 k= 3, v=18 0.848 42.750 2.051 * 1.86 k= 4, v=18 1.71 42.000 3.077 * 1.87 k= 5, v=18

---------------------------------------------------------------------------- s = 1.034 Note: df used for table values are approximate when v > 20.



## TITLE: Chinook wet weight

SUMMARY STATISTICS TABLE 1 of 2

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

GRP IDENTIFICATION N MIN MAX MEAN

--- ---------------- ---- ---------- ---------- ---------- 1 Control 4 1.075 1.222 1.127 2 0.107 4 1.075 1.156 1.128 3 0.222 4 0.998 1.222 1.097 4 0.43 4 1.023 1.188 1.098 5 0.848 4 0.983 1.181 1.065Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 22 of 27 6 1.71 4 0.949 1.040 1.000

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

SUMMARY STATISTICS TABLE 2 of 2

------------------------------------------------------------------------------ GRP IDENTIFICATION VARIANCE SD SEM

--- ---------------- -------------- ---------- ---------- 1 Control 0.004 0.067 0.033 2 0.107 0.001 0.037 0.019 3 0.222 0.013 0.112 0.056 4 0.43 0.005 0.068 0.034 5 0.848 0.007 0.085 0.043 6 1.71 0.001 0.038 0.019

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

ANOVA TABLE

------------------------------------------------------------------------------ SOURCE DF SS MS F

------------------------------------------------------------------------------ Between 5 0.046 0.009 1.800 Within (Error) 18 0.095 0.005

------------------------------------------------------------------------------ Total 23 0.142

------------------------------------------------------------------------------ Critical F value = 2.77 (0.05,5,18) Since F < Critical F **FAIL TO REJECT Ho:All groups equal**

DUNNETTS TEST - TABLE 1 OF 2 Ho:Control<Treatment

---------------------------------------------------------------------------- TRANSFORMED MEAN CALCULATED IN GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

----- -------------------- ----------- ------------------ ------ --- 1 Control 1.127 1.127 2 0.107 1.128 1.128 -0.020 3 0.222 1.097 1.097 0.600 4 0.43 1.098 1.098 0.580 5 0.848 1.065 1.065 1.250 6 1.71 1.000 1.000 2.540 *

---------------------------------------------------------------------------- Dunnett table value = 2.41 (1 Tailed Value, P=0.05, df=18,5)

DUNNETTS TEST - TABLE 2 OF 2 Ho:Control<Treatment

---------------------------------------------------------------------------- NUM OF Minimum Sig Diff % of DIFFERENCE GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

----- -------------------- ------- ---------------- ------- ------------ 1 Control 4 2 0.107 4 0.120 10.7 -0.001 3 0.222 4 0.120 10.7 0.030 4 0.43 4 0.120 10.7 0.029 5 0.848 4 0.120 10.7 0.063 6 1.71 4 0.120 10.7 0.127

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

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

---------------------------------------------------------------------------- GROUP ORIGINAL TRANSFORMED ISOTONIZED IDENTIFICATION N MEAN MEAN MEAN

------ -------------------- --- ----------- ----------- ----------- 1 Control 4 1.127 1.127 1.128Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 23 of 27 2 0.107 4 1.128 1.128 1.128 3 0.222 4 1.097 1.097 1.098 4 0.43 4 1.098 1.098 1.098 5 0.848 4 1.065 1.065 1.065 6 1.71 4 1.000 1.000 1.000

----------------------------------------------------------------------------
WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

---------------------------------------------------------------------------- ISOTONIZED CALC. SIG TABLE DEGREES OF IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

-------------------- ----------- ----------- ----- ----------- ------------- Control 1.128 0.107 1.128 0.010 1.73 k= 1, v=18 0.222 1.098 0.573 1.82 k= 2, v=18 0.43 1.098 0.573 1.85 k= 3, v=18 0.848 1.065 1.214 1.86 k= 4, v=18 1.71 1.000 2.467 * 1.87 k= 5, v=18

---------------------------------------------------------------------------- s = 0.073 Note: df used for table values are approximate when v > 20.



## %Lamellar Epithelium Diffuse Hypertrophy (%LEH)

FISHERS EXACT TEST

============================================================================ NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL w/LEH TOTAL ANIMALS

-------------------------- ----- ----- --------------- CONTROL 10 0 10 0.107 10 0 10

---------------------------------------------------------------------------- TOTAL 20 0 20

============================================================================ CRITICAL FISHERS VALUE (10,10,10) (p=0.05) IS 6. b VALUE IS 10. Since b is greater than 6 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.

FISHERS EXACT TEST

============================================================================ NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL W/LEH TOTAL ANIMALS

-------------------------- ----- ----- --------------- CONTROL 10 0 10 0.222 9 1 10

---------------------------------------------------------------------------- TOTAL 19 1 20

============================================================================ CRITICAL FISHERS VALUE (10,10,10) (p=0.05) IS 6. b VALUE IS 9. Since b is greater than 6 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.

FISHERS EXACT TEST

============================================================================Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 24 of 27 NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL w/LEH TOTAL ANIMALS

-------------------------- ----- ----- --------------- CONTROL 10 0 10 0.43 6 4 10

---------------------------------------------------------------------------- TOTAL 16 4 20

============================================================================ CRITICAL FISHERS VALUE (10,10,10) (p=0.05) IS 6. b VALUE IS 6. Since b is less than or equal to 6 there is a significant difference between CONTROL and TREATMENT at the 0.05 level.

FISHERS EXACT TEST

============================================================================ NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL w/LEH TOTAL ANIMALS

-------------------------- ----- ----- --------------- CONTROL 10 0 10 0.848 4 6 10

---------------------------------------------------------------------------- TOTAL 14 6 20

============================================================================ CRITICAL FISHERS VALUE (10,10,10) (p=0.05) IS 6. b VALUE IS 4. Since b is less than or equal to 6 there is a significant difference between CONTROL and TREATMENT at the 0.05 level.

FISHERS EXACT TEST

============================================================================ NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL w/LEH TOTAL ANIMALS

-------------------------- ----- ----- --------------- CONTROL 10 0 10 1.71 1 9 10

---------------------------------------------------------------------------- TOTAL 11 9 20

============================================================================

CRITICAL FISHERS VALUE (10,10,10) (p=0.05) IS 6. b VALUE IS 1. Since b is less than or equal to 6 there is a significant difference between CONTROL and TREATMENT at the 0.05 level.

SUMMARY OF FISHERS EXACT TESTS

---------------------------------------------------------------------------- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED w/LEH (P=.05)

----- ------------------------- ----------- ----------- ------- CONTROL 10 0 1 0.107 10 0 2 0.222 10 1 3 0.43 10 4 * 4 0.848 10 6 * 5 1.71 10 9 *

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


## %Hepatocellular Vacuolization (%HV)Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 25 of 27 FISHERS EXACT TEST

============================================================================ NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL W/HV TOTAL ANIMALS

-------------------------- ----- ----- --------------- CONTROL 8 2 10 0.107 4 6 10

---------------------------------------------------------------------------- TOTAL 12 8 20

============================================================================ CRITICAL FISHERS VALUE (10,10,8) (p=0.05) IS 3. b VALUE IS 4. Since b is greater than 3 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.

FISHERS EXACT TEST

============================================================================ NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL W/HV TOTAL ANIMALS

-------------------------- ----- ----- --------------- CONTROL 8 2 10 0.222 2 8 10

---------------------------------------------------------------------------- TOTAL 10 10 20

============================================================================ CRITICAL FISHERS VALUE (10,10,8) (p=0.05) IS 3. b VALUE IS 2. Since b is less than or equal to 3 there is a significant difference between CONTROL and TREATMENT at the 0.05 level.

FISHERS EXACT TEST

============================================================================ NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL W/HV TOTAL ANIMALS

-------------------------- ----- ----- --------------- CONTROL 8 2 10 0.43 4 6 10

---------------------------------------------------------------------------- TOTAL 12 8 20

============================================================================ CRITICAL FISHERS VALUE (10,10,8) (p=0.05) IS 3. b VALUE IS 4. Since b is greater than 3 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.

FISHERS EXACT TEST

============================================================================ NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL W/HV TOTAL ANIMALS

-------------------------- ----- ----- --------------- CONTROL 8 2 10 0.848 6 4 10

---------------------------------------------------------------------------- TOTAL 14 6 20

============================================================================ CRITICAL FISHERS VALUE (10,10,8) (p=0.05) IS 3. b VALUE IS 6. Since b is greater than 3 there is no significant difference between CONTROL and TREATMENT at the 0.05 level. FISHERS EXACT TEST

============================================================================Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 26 of 27 NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL W/HV TOTAL ANIMALS

-------------------------- ----- ----- --------------- CONTROL 8 2 10 1.71 0 10 10

---------------------------------------------------------------------------- TOTAL 8 12 20

============================================================================ CRITICAL FISHERS VALUE (10,10,8) (p=0.05) IS 3. b VALUE IS 0. Since b is less than or equal to 3 there is a significant difference between CONTROL and TREATMENT at the 0.05 level.

SUMMARY OF FISHERS EXACT TESTS

---------------------------------------------------------------------------- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED W/HV (P=.05)

----- ------------------------- ----------- ----------- ------- CONTROL 10 2 1 0.107 10 6 2 0.222 10 8 * 3 0.43 10 6 4 0.848 10 4 5 1.71 10 10 *

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


## Comparison of Nominal 0.10 ppm a.i. TGAI and EP – Days to First Swim-Up

fluridone 0.10 Sonar PR 0.10 nominal t-Test: Two-Sample Assuming Equal Variances 18 19 15 17 fluridone 0.10 Sonar PR 0.10 nominal 16 16 Mean 16.5 17.5 17 18 Variance 1.67 1.67 mean 16.5 17.5 Observations 4 4 std dev 1.29 1.290994 Pooled Variance 1.666666667 Hypothesized Mean Difference 0 df 6 t Stat -1.095 P(T<=t) one-tail 0.158 t Critical one-tail 1.943 P(T<=t) two-tail 0.315 t Critical two-tail 2.447Data Evaluation Report on the Toxicity of Fluridone TG AI and EP to the Early Life Stage of Chinook Salmon ( Oncorhynchus tshawytscha ) PMRA Submission Number {......} EPA MRID Number 45915601



### Page 27 of 27 

## Comparison of Nominal 0.10 ppm a.i. TGAI and EP – Wet Weight fluridone 0.10 Sonar PR 0.10 nominal t-Test: Two-Sample Assuming Equal Variances 1.151 1.224

1.13 0.999 fluridone 0.10 Sonar PR 0.10 nominal 1.075 1.152 Mean 1.128 1.106 1.156 1.049 Variance 0.00138 0.0102 mean 1.128 1.106 Observations 4 4 std dev 0.04 0.101 Pooled Variance 0.005811 Hypothesized Mean Difference 0 df 6 t Stat 0.40815 P(T<=t) one-tail 0.34866 t Critical one-tail 1.94318 P(T<=t) two-tail 0.69733 t Critical two-tail 2.44691



## Comparison of Nominal 0.10 ppm a.i. TGAI and EP – Standard Length fluridone 0.10 Sonar PR 0.10 nominal t-Test: Two-Sample Assuming Equal Variances 45 45 44 42 fluridone 0.10 Sonar PR 0.10 nominal 44 45 Mean 44.5 43.75 45 43 Variance 0.333 2.25 mean 44.5 43.75 Observations 4 4 std dev 0.58 1.5 Pooled Variance 1.29167 Hypothesized Mean Difference 0 df 6 t Stat 0.9333 P(T<=t) one-tail 0.1934 t Critical one-tail 1.9432 P(T<=t) two-tail 0.3867 t Critical two-tail 2.4469



## Comparison of Nominal 0.10 ppm a.i. TGAI and EP – Hepatocellular Vacuolization

FISHERS EXACT TEST

============================================================================ NUMBER OF

---------------------------------------- IDENTIFICATION NORMAL w/HV TOTAL ANIMALS

-------------------------- ----- ----- --------------- TGAI 0.107 8 2 10 EP 0.094 5 5 10

---------------------------------------------------------------------------- TOTAL 13 7 20

============================================================================ CRITICAL FISHERS VALUE (10,10,8) (p=0.05) IS 3. b VALUE IS 5. Since b is greater than 3 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.