Data Evaluation Report on the acute toxicity 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 invertebrates. 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 (CaCO3). 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. Analytical 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 respiration, rapid respiration, pigmentation) were 

observed daily. Reviewer calculated 48-hr EC50 values based on immobilization/mortality are shown in 

Table 1 and range from 3.0 – 5.2 mg a.i./L. Confidence intervals of all tests overlapped, indicating that 

the formulation and hardness did not influence the 48-hour EC50 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 guideline standards for an acute toxicity study with 

freshwater invertebrates. 

Results Synopsis

Test Organism Age: 1st 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 

EC50 (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) 

EC50 Method Probit Binomial Probit Binomial Probit Binomial Probit Probit 

*Based on nominal concentrations, since the actual measured concentrations were not reported. 
Data Evaluation Report on the acute toxicity 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 EC50, confidence in estimates may be reduced. 

4. Total organic carbon and particulate matter concentrations 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 toxicity 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 guidance. 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 concentration 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 oC 

 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, 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 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 vapor 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 toxicity 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 chambers 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 toxicity 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 106

 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 CaCO3) 

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 

18o

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 CaCO3 (40 – 48 

mg/l CaCO3 for metals) (OECD guidance is 

140 – 250 mg/l CaCO3) 

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 EC50 plus a 

dilution control and a solvent control, if a 

solvent was used.
Data Evaluation Report on the acute toxicity 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 toxicity 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 controls. 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 

Treatment 24-Hrs 48-hrs 

(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%, Reconstituted 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 toxicity 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 

Treatment 24-Hrs 48-hrs 

(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 LC50 values, confidence limits, 

and slopes were the Litchfield and 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 

EC50 (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 

EC50 Method Litchfield and 

Wilcoxon 

Litchfield and 

Wilcoxon 

Litchfield and 

Wilcoxon 

Litchfield and 

Wilcoxon 

Positive control EC50, if tested 

(Reference EC50 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 toxicity 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 EC50s. The probit method was 

used when possible and the binomial method when there were less than two concentrations with percent 

dead or immobilized between 0 and 100%. The output files 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 

EC50 (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) 

EC50 Method Probit Binomial Probit Binomial Probit Binomial Probit Probit 

Positive control 

EC50, if tested 

(Reference EC50

and QC bounds) 

 

*Based on nominal concentrations, since the actual measured 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 EC50, confidence in estimates may be reduced. 

4. Total organic carbon and particulate matter concentrations 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 toxicity 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 EC50 (immobility) of fluridone to Daphnia magna. See Table 7 for the 

study results. In three of the tests, only binomial 48-hr EC50 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 simplified method of evaluating dose-effect experiments. J. 

Pharmacol. Therap., 96, 99-113.