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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16/May/2001 to 18/May/2001
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The work reported was performed carefully, the results reasonable, and the conclusions conservative. Data was generated according to generally valid and internationally accepted testing guidelines.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:

Test material

Details on test material:
- Name of test material (as cited in study report: Perfluoropropionic Acid (PFPA, CF3CF2COOH), CAS# 422-64-0.
- Substance type: The test substance was generated from the hydrolysis of perfluoro-2-methyl-3-pentanone (C6-ketone) in deionized water. The concentration of product in the hydrolysis solution was measured by the Sponsor Laboratory as 4.75 %, w/w. No other hydrolysis products were present in the solution. The following describes the solution that is a result of that hydrolysis process:
- Physical state: Liquid
- Analytical purity:4.75% PFPA in water
- Stability under test conditions: The test substance was demonstrated to be stable under test
conditions by the analysis of samples collected during the definitive toxicity test.
- Storage condition of test material: Room temperature in the dark in a 4-liter amber glass bottle.

Sampling and analysis

Analytical monitoring:
Details on sampling:
Analytical determination of test substance concentration (active ingredient) was performed with samples collected from the approximate center of each replicate test vessel after 0 and 48 hours. The samples from replicate test vessels were maintained as individual samples (not pooled) and stored in 60 mL polypropylene bottles.

The 0 hour samples were accompanied by a laboratory control sample* prepared at 400 mg/L test substance in dilution water. The 48 hour samples were accompanied by a 400 mg/L laboratory control sample* and two matrix spike samples* prepared at 400 mg/L in water from a control vessel. Samples were refrigerated until they were shipped for analysis. They were shipped to the Sponsor in a cooler with ice by overnight courier.

* Though not specifically stated, these samples are used for determining if any abiotic processes for removal or interference were present.

Test solutions

Details on test solutions:
The test substance contained a small amount of acetone (169 mg/L as determined by NMR), used to aid in the creation of the test substance from the parent material, CF3CF2-CO-CF(CF3)2. This is an impurity, not a carrier in this study. The concentration of residual acetone is such that at a concentration of 1000 mg/L of the test substance in the test solution, the concentration of acetone would be 3.6 mg/L.

A stock solution with a nominal concentration of 1000 mg/L was prepared by bringing 42.1 grams of test substance (4.75% PFPA) to a total volume of 2000 mL with dilution water in a class A glass volumetric flask. The pH was adjusted from 2.7 to 7.2 with 6N NaOH, concentrated HCl, 0.10N NaOH, and 0.10N HCl. Appropriate amounts of the 1000 mg/L stock solution were added directly to dilution water to prepare the test concentrations.

A 1000 mg/L acetone stock solution was prepared and acetone control solutions were prepared by bringing 1.2 mL of this stock solution up to 330 mL with dilution water to produce 3.6 mg/L acetone solutions (the same concentration of acetone present in the 1000 mg/L test substance solutions).

The definitive toxicity test utilized 5 concentrations of test substance (150, 250, 400, 600, and 1000 mg/L nominal), a dilution water control, and an acetone control (3.6 mg acetone/L) at a temperature of 20+/-1C.

The acetone control was utilized at 3.6 mg/L to match the acetone concentration in the 1000 mg/L test substance solutions.

Mean measured concentrations of the test substance were: <0.0121 (control), <0.00511 (acetone control), 178, 285, 430, 619, 1080 mg/L. Mean measured concentrations were used for all toxicity determinations. No insoluble material was observed during the definitive study.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water flea
- Source: Original culture purchased from Aquatic Biosystems, Inc, Fort Collins, Colorado, and received by T.R. Wilbury Laboratories on April 10, 2001. Parent organisms were reared at T.R. Wilbury Laboratories in 3800 mL glass jars that contained aerated, filtered dilution water under static renewal conditions. Temperature was maintained between 19.2 and 21.0 C, dissolved oxygen was >/= 7.9 mg/L, and the pH was 7.1 – 8.3.
- Age at study initiation: Neonates (< 24 h) collected 22 days after initial appearance of neonates in parent culture.
- Weight at study termination (control organisms): 0.20 mg (average wet weight[blotted dry])
- Feeding during test Not described.

- Culturing / Acclimation period: 21 days rearing. Study utilized <24 hour old neonates
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: The animals were fed a mixture of green alga (Pseudokirchneriella subcapitata), yeast, alfalfa, and trout chow (Lot #DC03). Amount not given.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): Disease, injury, and abnormalities were not seen and ephippia were not being produced by the parent daphnids.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
Not noted.

Test conditions

160-180 mg/L as CaCO3
Test temperature:
19.0-20.9 ºC
7.2-7.3 at initiation of study; 7.8-7.9 at 48 h
Dissolved oxygen:
9.3-9.5 mg/L at initiation of study; 8.6-8.7 mg/L at 48 h.
Nominal and measured concentrations:
Test vessel Nominal PFPA Conc. (mg/L) Mean Measured PFPA Conc. (mg/L) (SD)
Control 0 <0.0121*
Acetone control 0 <0.00511**
Exposure vessels 150 178 (7)
250 285 (19)
400 430 (14)
600 619 (49)
1000 1080 (140)

Limit of quantitation (LOQ) was 0.005 mg/L

*Reported as the mean across replicate sample analysis, all non-detect at 0 hours and one non-detect and one detection of 0.0121 mg PFPA/L at 48 hours.

**Reported as the mean across replicate sample analysis, all non-detect at 0 hours and one non-detect and one detection of 0.00511 mg PFPA/L at 48 hours.
Details on test conditions:
Range finding was performed under static conditions with a control, an acetone control (3.6 mg/L), and five nominal concentrations of test substance at 0.1, 1.0, 10, 100, and 1000 mg/L. One replicate, containing five daphnids, was used for each test concentration. After 48 hours 100% survival was observed in all control and in the 0.1, 1.0, and 10 mg/L test solutions, 80% survival at 100 mg/L, and 60% survival at 1000 mg/L. After 48 h of exposure, two of the three surviving daphnids in the 1000 mg/L solution were immobile.


- Test vessel: 16 oz. (474 mL) glass jars
- Type: Loosely covered
- Material, size, headspace, fill volume: Glass, 16 oz = 474 mL, headspace not measured but each contained 270 mL of control or test solution, media depth approximately 7 cm.
- Aeration: Not noted
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate:
-Dilution water: Carbon filtered deionized water, adjusted to a hardness of 160 to 180 mg/L as CaCO3, aerated and recirculated through particle filters, activated carbon, and an ultraviolet sterilizer. Stored in polyethylene tanks. A sample of dilution water collected at the start of the definitive test had a hardness of 160 mg/L as CaCO3. Water quality was analyzed as part of a standard biannual quality testing. See attached table titled "Water testing results for Daphnia magna test on PFPA by TR Wilbury.

- Adjustment of pH: Prior to use in the test, the stock solution pH was adjusted from 2.7 to 7.2 as described in "Details on Test Solutions".

- Conductivity: Controls: 480 - 490 µmho/cm; Test solutions (1080 mg/L): 1010-1040 µmho/cm

- Photoperiod: 16 hour light with 8 hour dark photoperiod, including 15 minute transition period between dark and light.

- Light intensity and quality: Cool white fluorescent lights providing and intensity of approximately 58 foot-candles (note, this is equivalent to 1.7 W/sq. cm at 555 nm).
Reference substance (positive control):
not required

Results and discussion

Effect concentrations
48 h
Dose descriptor:
Effect conc.:
> 1 080 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
No insoluble material was observed during the definitive test. Nominal concentrations of the test substance were: 0 mg/L (control and acetone control), 150, 250,400,600, and 1000 mg/L. Mean measured concentrations of the test substance were: <0.0121 mg/L (control), <0.00511 mg/L (acetone control), 178, 285, 430, 619, and 1080 mg/L. Mean measured concentrations were 103 to 119% of nominal values and they were stable during the test.

Exposure of daphnids (Daphnia magna) to the test substance at mean measured concentrations of 178, 285, 430, 619, and 1080 mg/L resulted in >50% survival. NOTE: the immobilization data is not dose-responsive.

In addition to immobilization, one daphnid exposed to 619 mg/L in replicate 1 for 24 hours, one daphnid exposed to 619 mg/L in replicate 2 for 48 hours, two daphnids exposed to 1080 mg/L in replicate 1 for 48 hours, and three daphnids exposed to 1080 mg/L in replicate 2 for 48 hours were lethargic.
Reported statistics and error estimates:
Results of the toxicity test could not be interpreted by standard statistical techniques because more than 50% of the daphnids exposed to each of the five tested concentrations of the test substance were alive and unaffected at the end of the test.

Any other information on results incl. tables

Summary of Analytical Results for Concentration of PFPA in Daphnia Acute Toxicity Testing
Time Point Group # and
Nominal Concentration (ppm)
Analytical Sample Name Test Sample ID PFPA Concentration
Beginning of in-life phase
(0 - Hours exposure)
Group 0
0 mg/L
2206th-051601-01 Blank <LOQ
2206th-051601-02 Control 1 <LOQ
2206th-051601-03 Control 2 <LOQ
2206th-051601-04 Acetone Control 1 <LOQ
2206th-051601-05 Acetone Control 2 <LOQ
Group 1
150 mg/L
2206th-051601-06 1 - 1 180
2206th-051601-07 1 - 2 186
Group 2
250 mg/L
2206th-051601-08 2 - 1 293
2206th-051601-09 2 - 2 271
Group 3
400 mg/L
2206th-051601-10 3 - 1 455
2206th-051601-11 3 - 2 436
2206th-051601-16 Lab Control Spike 436
Group 4
600 mg/L
2206th-051601-12 4 - 1 579
2206th-051601-13 4 - 2 690
Group 5
1000 mg/L
2206th-051601-14 5 - 1 926
2206th-051601-15 5 - 2 1160
End of in-life phase
(48 - Hours exposure)
Group 0
0 mg/L
2206th-051801-01 Blank <LOQ
2206th-051801-02 Control 1 0.0121
2206th-051801-03 Control 2 <LOQ
2206th-051801-04 Acetone Control 1 0.00511
2206th-051801-05 Acetone Control 2 <LOQ
2206th-051801-19 Effluent 0.185
Group 1
150 mg/L
2206th-051801-06 1 - 1 167
2206th-051801-07 1 - 2 177
Group 2
250 mg/L
2206th-051801-08 2 - 1 265
2206th-051801-09 2 - 2 312
Group 3
400 mg/L
2206th-051801-10 3 - 1 409
2206th-051801-11 3 - 2 419
2206th-051801-16 Lab Control Spike 425
2206th-051801-17 Matrix Spike 417
2206th-051801-18 Matrix Spike Duplicate 434
Group 4
600 mg/L
2206th-051801-12 4 - 1 640
2206th-051801-13 4 - 2 567
Group 5
1000 mg/L
2206th-051801-14 5 - 1 1260
2206th-051801-15 5 - 2 953

PFPA Daphnia Acute Test Dose response table
Mean Meas
conc., mg/L
Percent dead and immobilized
24-hr 48-hr
Control 0 0
acetone control 0 0
178 0 5
285 5 5
430 30 20
619 5 40
1080 10 20

Applicant's summary and conclusion

Validity criteria fulfilled:
For the PFPA in daphnids (Daphnia magna), the 48-hour effective concentration (EC50) is greater than 1080 mg/L (EC50 > 1080 mg/L). The 48-hour median lethal concentration (LC50) is greater than 1080 mg/L (LC50 > 1080 mg/L).
Executive summary:

The 48–hr-acute toxicity of PFPA to Daphnia magna was studied under static conditions.  Daphnids were exposed to 150, 250, 400, 600, and 1000 mg/L, nominal concentrations; 178, 285, 430, 619, 1080 mg/L, mean measured concentrations for 48 hr.  Mortality/immobilization and sublethal effects were determined daily until test termination.  The 48– hour LC50/EC50 was >1080 mg a.i./L (measured).  The NOEC was not determined.  The sublethal effects included were lethargy.  

Based on the results of this study, PFPA would  be classified as not acutely toxic to Daphnia magna in accordance with the classification system of the Globally Harmonized System.  

This toxicity study is classified as acceptable and satisfies the guideline requirements for an OECD Guideline 202, acute toxicity study with freshwater invertebrates.  

Results Synopsis  

Test Organism Age/weight: Neonates ( 24 h) / At study termination (control organisms): 0.20 mg (average wet weight[blotted dry]) Test Type: Static  

EC50:  >1080 mg/L                            95% C.I. :  NA

NOEC:  Not determined                     Probit Slope:  NA