Data Evaluation Report on the Toxicity of Fluridone TGAI 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-measured treatment concentration was 0.094 ppm a.i. 

In the TGAI study no treatment-related effects on percent hatch, time to first hatch, time to first swim-up, fry 

survival, or overall survival were observed. No treatment-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 and 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, histopathological 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 data on daily numbers that hatched, daily swim-up numbers 

does not allow calculation of rate of development for these 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 SUPPLEMENTAL. 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 study and is classified as SUPPLEMENTAL and is not 

upgradeable. 

Results Synopsis

Test Organism Size/Age (mean Weight or Length): 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 TGAI 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 procedures of the U.S. EPA Public Draft 

OPPTS 850.1400 and Standard Operating Procedures of ABC 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 evaluation by the reviewer and the development rate for 

hatching and swim-up could therefore not be calculated and no statistics for these endpoints could be 

determined. Time to first-hatch, days to 95% hatch, and time to first swim-up were reported but the 

analysis is incomplete. 

This deviation affects the validity of the study because 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 stock 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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 stored 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 oC 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, 4th Ed. 2002 

UV absorption Not reported 

pKa 1.73* R. Grover and A. J. Cessna. 1991. Environmental Chemistry of 

Herbicides Volume II, CRC Press 

pKb 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. 

Kow 74.13 

Log Kow 1.87 from Footprint @ 

http://sitem.herts.ac.uk/aeru/footprint/en/, accessed on April 28, 

2009 

Aquatic 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 vapor 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 10o

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 TGAI 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 TGAI 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 specific 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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 reverse 

osmosis. 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 CaCO3

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 TGAI 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 CaCO3 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 were 

thinned 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 concentrations 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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? Yes 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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-hatch 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 survival, 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 treatment 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 0.98 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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 significantly reduced at the 1.71 ppm a.i. treatment group as 

compared to the control and no treatment-related differences 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 also 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 LOAEC values determined for length and weight by the 

reviewer are summarized in Table 8. 

No treatment related behavioral or morphological abnormalities were observed following hatch, spinal curvature 

was noted in one fish from the control and floridone treatments 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 hypertrophy 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 vacuolization (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 (Table 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 development (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 TGAI 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 using William’s test for length and Dunnett’s for 

wet weight. Study authors’ NOAEC values for 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 hatch 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 Fisher’s exact test with a Hochberg adjustment. The NOEC 

values for terminal lengths and wet weights were determined 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 TGAI 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 first 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 control 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. Histological findings were analyzed using the Fishers Exact 

Test. 

Comparison of the TGAI and EP nominal 0.10 treatment groups 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 relationship 

Wet 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 treatment 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 control 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 TGAI 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 significant 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, establishing a lower NOAEC for gill histopathology (0.222 ppm) 

than for growth (0.430 ppm). Differences between the study author’s and the reviewer’s results are due to the 

different statistical methods used. The more conservative 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 result, the Sonar PR treatment did not receive the test 

substance for approximately 14 hours. On Day 44, the concentration 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 deviations 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 data on daily numbers that hatched, daily swim-up numbers 

does not allow calculation of rate of development for these 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 SUPPLEMENTAL. 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 study 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 TGAI 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 Insecticide, Fungicide and Rodenticide Act (FIFRA); Good 

Laboratory Practice Standards; Final Rule (40 CFR, 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. Pesticide 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. 20th

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. 2nd ed. Prentice-Hall, Englewood Cliffs, N.J. 

Hochberg, Y. 1988. A Sharper Bonferroni Procedure 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. 2nd 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 TGAI 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 = 0.05 

 

 

 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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 = 0.05 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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 17.333 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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 variance 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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 TGAI 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 1.065 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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 1.128 
Data Evaluation Report on the Toxicity of Fluridone TGAI 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 TGAI 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 TGAI 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 TGAI 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 2.447
Data Evaluation Report on the Toxicity of Fluridone TGAI 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.