### Data Evaluation Report on the acute t oxicity of TGAI and TEP to fresh water
invertebrates - Daphnia sp.

EPA MRID Number 000103297 & 40098001

Page 2 of 11 The following template illustrates the critical elements and the level of detail required by the PMRA and the EPA in a study review of the acute toxicity to freshwater invertebrate s. It is not meant to be a "fill in the blanks" exercise although it may be appropriate in some cases. The standard format of the template should, however, be followed in preparing the study review.



### EXECUTIVE SUMMARY :

The 48-hr acute toxicity of fluridone to Daphnia magna was studied under static conditions for a technical grade active ingredient (TGAI) and an end- use product (EP), Sonar 4 AS, at two hardness levels 44 and 280 mg/L as calcium carbonate (CaCO 3 

### ). For each of these tests daphnids were exposed to a negative control, a solvent control (TGAI tests only), and fluridone at nominal concentrations of 10.0, 5.6, 3.2, 1.8, and 1.0 mg a.i./L for 48 hours. Anal ytical results confirmed exposure levels were within target levels and were within stability standards. Mortality/immobilization and signs of sublethal effects (loss of equilibrium, immobility, surfacing, labored r espiration, rapid respiration, pigmentation) were observed daily. Reviewer calculated 48-hr EC 50 

### values based on immobilization/mortality are shown in Table 1 and range from 3.0 – 5.2 mg a.i./L. Confiden ce intervals of all tests overlapped, indicating that
the formulation and hardness did not influence the 48-hour EC 50 

### values.

Based on the results of this study, fluridone is classified as moderately toxic to Daphnia magna in accordance with EFED’s acute toxicity classification system.

This study is classified as acceptable and satisfies the gui deline standards for an acute toxicity study with
freshwater invertebrates.

Results Synopsis Test Organism Age: 1 st 

### instar Test Type: Static

Table 1. Results* Calculated by Reviewer Using TOXANAL (Toxwin2009.bat), the Effect was immobility/mortality.

Observation Period Obs 1, TGAI, Hardness 44 mg/L Obs 2, TGAI, Hardness 280 mg/L Obs 3, EP, Hardness 40 mg/L Obs 4, EP, Hardness 280 mg/L Statistical Value

24 hrs 48 hrs 24 hrs 48 hrs 24 hrs 48 hrs 24 hrs 48 hrs
EC 50 (95%
Confidence Interval) in mg
a.i./L 6.3 (1.6-11) 5.2 (3.2-10) 9.1 (7.1-16) 5.2 (3.2-10) 7.3 (4.6-23) 3.0 (1.8-5.6) 10.5 (7.2-56) 4.0 (3.2-5.0)

Probit Slope (SE) 9.1 (7.1-16) NA 6.3 (1.6-11) NA 2.1 (0.8-3.4) NA 3.4 (0.8-6.0) 8.9 (3.6-14)
EC 50 Method Probit Binomial Probit Binomial Probit Binomial Probit Probit *Based on nominal concentrations, since the actua l measured concentrations were not reported.

### Data Evaluation Report on the acute t oxicity of TGAI and TEP to fresh water
invertebrates - Daphnia sp.

EPA MRID Number 000103297 & 40098001

Page 3 of 11 

### I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: The protocol was based on methods recommended by the Committee on Methods for Toxicity Tests with Aquatic Organisms (1975). Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. U.S. EPA. Ecol. Res. Serv. EPA-660/3-75-009, 61p, and standard protocols of the National Fisheries Research Laboratory

Study Deficiencies
1. The physiochemical properties of the test material were not reported. However the
information was obtained from other sources.
2. A GLP statement was not provided but protocols for recording and documenting test results, study investigators, and similar requirements to GLP were in place.
3. There was only one replicate with 10 individuals at each treatment level, but this did not
affect the determination of the EC 50 

### , confidence in estimates may be reduced.
4. Total organic carbon and particulate matter con centrations were not reported but distilled water, which should result in particulate and organic carbon levels to be within acceptable levels, was used for reconstituted water. The well water is assumed to have been within 1975 guideline standards which was cited as being followed.
5. Dissolved oxygen (DO) concentrations were not reported but control mortality was within acceptable levels, indicating DO was not likely an issue.

These deviations are considered minor for the reasons provided and do not affect the scientific soundness of this study.

COMPLIANCE : A statement of compliance with GLP procedures was not provided. The studies were completed prior to the GLP requirements. However, protocols for recording and documenting test results, study investigators, and similar requirements to GLP were in place.

A. MATERIALS :

1. Test Material Χ TGAI: Technical grade Fluridone (purity: 98% active)
Χ EP : Sonar 4 AS , 4 lb a.i./gallon field formulation (purity: 48% fluridone)

Description: TGAI: powder EP: liquid

Lot No./Batch No. : TGAI: D36-Y93-107 (Page 389) EP: X-36153 (Page 388)

Purity: TGAI: 98%

### Data Evaluation Report on the acute t oxicity of TGAI and TEP to fresh water
invertebrates - Daphnia sp.

EPA MRID Number 000103297 & 40098001

Page 4 of 11 

### EP: 48%

Stability of Compound
Under Test Conditions: Analytical results indicate that actual fluridone concentrations

average ≥ 79% of the nominal concentrations used in the study (Page 404), results within acceptable measurement error. Sampling was conducted on all treatment levels at 2 sampling intervals (p 342), this would be consistent with sampling at test initiation and termination with gui dance. Taken together these
results indicate that the test material concentrations were stable within current guideline standards. ( EPA test guidance recommends that
test material concentration not decline to less than 80% of the initial (day-0) measured concentration and the ratio of the highest c oncentration to the lowest concentration

should be at or less than 1.5:1 among replicates of the same treatment level.) 

### ] Storage conditions of
test material: Not reported

Table 2. Physicochemical properties of fluridone.

Parameter Values Comments

Water solubility at 20oC 12.3 mg/L at 25 o C Footprint Database @ 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, 2009 Aquatic photolysis half-life 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..) 

### The study report did not report physical-chemical properties of fluridone. * Therefore, the unionized form should be present within the environmentally relevant pH range.



### 2. Test organism :

Species: Daphnia magna
( EPA test guideline preferred species is Daphnia magna; OECD test guideline is Daphnia magna or any other suitable Daphnia species) 

### Age at test initiation: First Instar, <43 hours old
(EPA/OECD guideline test age # 24 hrs old)

### Data Evaluation Report on the acute t oxicity of TGAI and TEP to fresh water
invertebrates - Daphnia sp.

EPA MRID Number 000103297 & 40098001

Page 5 of 11 

### Source: In-house laboratory cultures (p 322)

B. STUDY DESIGN :

1. Experimental Conditions

a) Range-finding Study: Results for three range-finding tests were included in the raw data.

b) Definitive Study:

Table 3. Experimental Parameters

Remarks

Parameter Details Guidance
Acclimation:
Period: Conditions: (same as test or not)

Feeding: Health: (any mortality observed) 1 day
Same as test

Not fed (p 350) Not reported EPA/OECD test guidance recommends a minimum of 48 hours for brood daphnids prior to test initiation. EPA/OECD test guidance is for test organisms not to be fed during the study.

Duration of the test 48 hours
EPA/OECD test guidance is 48 hours Test condition : Static/Static Renewal/Flow Through

Type of dilution system for flow through method.

{Flow rate for flow through or renewal rate for static renewal} Static

N/A EPA recommends a minimum flow rate of 5 volume additions per 24 hours. To document stability (+ 10%) meter systems should be calibrated before the study and checked twice daily during the test period.

Aeration, if any None, p 350
A single air bubble can get under the carapace of the daphnid and kill it, or float the daphnid to the surface where it will get trapped therefore EPA guidance is for no
aeration in test cham bers during testing. Test vessel : Material: (glass/stainless steel)
Size:
Fill volume: glass beaker
200 mL
150 mL EPA guidance is 250 ml beaker with 200 ml of fill solution. Recommended biomass loading rate is < 0.8 g/L at < 17 o C and < 0.5 g/L at > 17 o C for static condition and < 1 g/L/day for flow-through condition.

### Data Evaluation Report on the acute t oxicity of TGAI and TEP to fresh water
invertebrates - Daphnia sp.

EPA MRID Number 000103297 & 40098001

Page 6 of 11 Remarks Parameter Details Guidance
Source of dilution water Reconstituted water for soft water exposures and well water aerated before use for very hard water exposure.

Reconstituted water was made using deionized water of at
least 10 6 ohms resistivity and reagent grade chemicals (USEPA 1975 guidance) EPA prefers soft reconstituted water or water from a natural source not dechlorinated tap water. However, use of dechlorinated tap water may be supportable if the biological responses for the organisms and chemical analyses of residual chlorine meet conditions in the Agency’s 850.1010 guidance for dilution water (http://www.epa.gov/opptsfrs/ publications/OPPTS_Harmonized/ 850_Ecological_Effects_Test_Guidelines/ Drafts/850-1010.pdf) Water parameters : Hardness (as CaCO 3 ) pH Dissolved oxygen Temperature Total organic carbon
and Particulate matter

Metals
Pesticides
Chlorine Alkalinity Conductivity Chemical Oxygen Demand (when testing cationic materials)

Intervals of water quality measurement 2 tests each at 40 and 280 mg/L
7.4 Not reported
18 o C Not reported, but deionized
water should be within acceptable levels, well water
assumed to be within cited guidance levels (USEPA 1975)

Within acceptable levels <0.01 mg/L Not applicable Well water: 237 mg/L 1.0 mg/L Not applicable

Laboratory water source report for metals, chlorine, and alkalinity. Not reported for pH and
hardness EPA test guidance consists of:
hardness : <250 mg/l as CaCO 3 (40 – 48 mg/l CaCO 3 for metals) (OECD guidance is 140 – 250 mg/l CaCO 3 ) pH: 6.0 – 8.5 (OECD guidance is 6-9) Temperature : 18 – 22 o C and selected temperature should not vary >1 o C during study (same as OECD guidance) Dissolved oxygen : between 60 and 100% saturation (OECD guidance is > 2 mg/l)

Treatment concentrations : Nominal (corrected for purity):

Measured {Initial, Test Termination, Time-Averaged}: For each test five treatment levels (10.0, 5.6, 3.2, 1.8, 1.0 mg a.i./L) and a negative control, additionally a solvent
control was added for each
TGAI test

Individual values not reported, average values ≥ 79% nominal EPA/OECD guidance for a definitive test is at least 5 treatment levels in a geometric
series which bracket the desired EC 50 plus a dilution control and a solvent control, if a
solvent was used.

### Data Evaluation Report on the acute t oxicity of TGAI and TEP to fresh water
invertebrates - Daphnia sp.

EPA MRID Number 000103297 & 40098001

Page 7 of 11 Remarks Parameter Details Guidance Solvent (type and concentration, if used) Acetone for TGAI; 0.1 ml/l
None used for EP EPA/OECD recommends that solvents
should not exceed 0.1 ml/l Number of replicates: Control (dilution water):
Solvent control:
Treatments: No replicates
EPA recommends a minimum of 2; OECD recommends a minimum of 4.

The ran a single replicate with a
recommended number of 10 animals in the single test vessel. Number of organisms per replicate : Control (dilution water):
Solvent control:
Treatments: 10
10
10 EPA/OECD guidance is exposure of at least 20 daphnids per treatment level. Lighting 16 hours light:8 hours dark Laboratory lighting using a
combination of Duro-test and wide-spectrum Growlux bulbs reported for chronic testing and may have been used in acute testing as well. Lighting parameters were not reported; however, the lighting set up
should be consistent with the ASTM 1980 testing parameters
and the Committee on Methods for Toxicity Tests with Aquatic Organisms (1975). EPA recommended photoperiod regime is selected from 12 – 16 hours of light with the selected regime constant during test; OECD recommends 16 hours of light but complete darkness is acceptable for some test
materials

Recovery of test material: Method recoveries and frequency of
determination

% of Nominal Level of Quantitation

Level of Detection A coefficient of variation of 1.8 (N=8) and 3.5% (N=8) was obtained for control pond water fortified with 1.0 ppm and 0.05 ppm of fluridone, respectively

Average of ≥ 79% of nominal. The method was reportedly
able to detect ~0.005 – 0.001 mg fluridone/L (p392) Positive control {if used, indicate the
reference test material and concentrations} Not applicable

Other parameters, if any

### Data Evaluation Report on the acute t oxicity of TGAI and TEP to fresh water
invertebrates - Daphnia sp.

EPA MRID Number 000103297 & 40098001

Page 8 of 11 

### 2. Observations :

Table 4. Observations

Remarks

Parameters Details Guidance Parameters measured including
the sublethal effects Immobility/Survival

Observation intervals 24-hrs

EPA/OECD recommends that observation intervals be a minimum of every 24 hours. Were raw data included? Not in the submission. Data were
submitted in Federal Lab Data Other observations, if any



### II. RESULTS AND DISCUSSION

A. MORTALITY/IMMOBILITY :

No mortality/immobility was observed in any of the cont rols. No replicates were available to determine NOAEC values statistically. Immobility was observed in all tests at some treatment level. ( EPA/OECD test
guidance is # 10% control mortality at end of study ) 

### ]

Table 5. Effect of Fluridone on immobilization of Daphnia magna

. Immobilization at Observation Period

24-Hrs 48-hrs Treatment (mg a.i./L) [nominal conc. used] No. of Organisms

at Test
Initiation Cumulative No.
Affected % Affected Cumulative No.
Affected % Affected

Observation 1, TGAI 98%, Reco nstituted Water, Hardness 44 mg/L Control (dilution water only) 10 0 0 0 0

Solvent control 10 0 0 1 10

10.0 10 6 60 10 100

5.6 10 1 10 6 60

3.2 10 0 0 0 0

1.8 10 0 0 0 0

1.0 10 0 0 0 0 Observation 2, TGAI 98%, Well Water, Hardness 280 mg/L Control (dilution water only) 10 0 0 0 0

Solvent control 10 0 0 0 0

10.0 10 7 70 10 100

5.6 10 2 20 6 60

3.2 10 0 0 3 30

1.8 10 0 0 2 20

1.0 10 0 0 0 0

### Data Evaluation Report on the acute t oxicity of TGAI and TEP to fresh water
invertebrates - Daphnia sp.

EPA MRID Number 000103297 & 40098001

Page 9 of 11 

### Table 5. Effect of Fluridone on immobilization of Daphnia magna

. Immobilization at Observation Period

24-Hrs 48-hrs Treatment (mg a.i./L) [nominal conc. used] No. of Organisms

at Test
Initiation Cumulative No.
Affected % Affected Cumulative No.
Affected % Affected

Observation 3, TEP 48%, Reconstituted Water, Hardness 44 mg/L Control (dilution water only) 10 0 0 0 0

10.0 10 5 50 10 100

5.6 10 4 40 10 100

3.2 10 5 50 6 60

1.8 10 0 0 0 0

1.0 10 0 0 0 0 Observation 4, TEP 48%, Well Water, Hardness 280 mg/L Control (dilution water only) 10 0 0 0 0

10.0 10 4 40 10 100

5.6 10 3 30 9 90

3.2 10 0 0 2 20

1.8 10 0 0 0 0

1.0 10 0 0 0

Footnotes: Data were obtained from Federal Lab Data submitted to support the Mayer and Ellersieck Data.



### B. SUB-LETHAL TOXICITY ENDPOINTS:

No sublethal effects were observed in the studies.

C. REPORTED STATISTICS : The method used to determine the LC 50 

### values, confidence limits, and slopes were the Litchfield a nd Wilcoxon (1949) method (see Page 351).

Table 6. Statistics Reported in Study Report, the Effect was immobility.

Observation Period

Statistical Value Obs 1, TGAI, Hardness 44 mg/L Obs 2, TGAI,
Hardness 280 mg/L Obs 3, TEP,
Hardness 40 mg/L Obs 4, TEP,
Hardness 280 mg/L

EC 50 (95% Confidence Interval) in mg a.i./L 4.4 (3.0-6.4) 4.4(3.0-6.6) 3.9 (3.0-5.1) 3.9 (2.9-5.0)

Probit Slope (SE) 2.40 2.40 1.56 1.55

EC 50 Method Litchfield and
Wilcoxon Litchfield and
Wilcoxon Litchfield and
Wilcoxon Litchfield and
Wilcoxon
Positive control EC 50 , if tested (Reference EC 50 and QC bounds) Not reported Not reported Not reported Not reported

Data reported in Table 1 on Page 331 of study report.

### Data Evaluation Report on the acute t oxicity of TGAI and TEP to fresh water
invertebrates - Daphnia sp.

EPA MRID Number 000103297 & 40098001

Page 10 of 11 

### D. VERIFICATION OF STATISTICAL RESULTS BY THE REVIEWER : TOXANAL (TOXWIN2009.bat) was used to process the data for 24 hour and 48 hour EC 50 

### s. The probit method was used when possible and the binomial method when ther e were less than two concentrations with percent dead or immobilized between 0 and 100%. The output f iles from the analysis are attached at the end of this evaluation report.

Table 7. Results* Calculated by Reviewer Using TOXANAL (Toxwin9.0.bat), the Effect was immobility/mortality.

Observation Period Obs 1, TGAI, Hardness 44 mg/L Obs 2, TGAI, Hardness 280 mg/L Obs 3, EP, Hardness 40 mg/L Obs 4, EP, Hardness 280 mg/L
Statistical Value 24 hrs 48 hrs 24 hrs 48 hrs 24 hrs 48 hrs 24 hrs 48 hrs

EC 50 (95%
Confidence Interval) in mg
a.i./L 6.3 (1.6-11) 5.2 (3.2-10) 9.1 (7.1-16) 5.2 (3.2-10) 7.3 (4.6-24) 3.0 (1.8-5.6) 10.5 (7.2-56) 4.0 (3.2-5.0)

Probit Slope (SE) 9.1 (7.1-16) N/A 6.3 (1.6-11) NA 2.1 (0.8-3.4) NA 3.4 (0.8-6.0) 8.9 (3.6-14)

EC 50 Method Probit Binomial Probit Binomial Probit Binomial Probit Probit
Positive control
EC 50 , if tested (Reference EC 50 and QC bounds)
*Based on nominal concentrations, since the actual m easured concentrations were not reported.



### E. STUDY DEFICIENCIES : 

### 1. The physiochemical properties of the test material were not reported. However the
information was obtained from other sources. 

### 2. A GLP statement was not provided but protocols for recording and documenting test results, study investigators, and similar requirements to GLP were in place. 

### 3. There was only one replicate with 10 individuals at each treatment level , but this did
not affect the determination of the EC 50 

### , confidence in estimates may be reduced



### . 4. Total organic carbon and particulate matter con centrations were not reported but distilled water, which should result in particulate and organic carbon levels to be within acceptable levels, was used for reconstituted water. The well water is assumed to have been within 1975 guideline standards which was cited as being followed.


### 5. Dissolved oxygen (DO) concentrations were not reported but control mortality was within acceptable levels, indicating DO was not likely an issue.



### These deficiencies are considered minor for the reasons mentioned and the study results may be used quantitatively.

F. REVIEWER’S COMMENTS :

### Data Evaluation Report on the acute t oxicity of TGAI and TEP to fresh water
invertebrates - Daphnia sp.

EPA MRID Number 000103297 & 40098001

Page 11 of 11 

### G. CONCLUSIONS : The study is scientifically sound and is classified as acceptable. Confidence intervals overlapped for all tests performed; therefore, hardness and the formulation did
influence the 48-hr EC 50 

### (immobility) of fluridone to Daphnia magna . See Table 7 for the study results. In three of the tests, only binomial 48-hr EC 50 

### values were available because partial mortality was only observed at one treatment level.

III. REFERENCES : Committee on Methods for Toxicity Tests with Aquatic Organisms (1975). Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. U.S. EPA. Ecol. Res. Serv. EPA-660/3-75-009. 61p.

Litchfield Jr., J. T., & Wilcoxon, F. 1949. A simplif ied method of evaluating dose-effect experiments. J. Pharmacol. Therap., 96 , 99-113.