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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Conducted according to guideline in effect at time of study conduct
GLP compliance:

Test material

Constituent 1
Details on test material:
- Purity: 28 wt%

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: All test concentrations, including the dilution water control. Newly prepared test solution on days 0 and 2 of the test. Old test solution on days 2 and 4 of the test. In addition, test substance samples diluted 4x into methanol for analysis at each sampling period.
- Sample storage conditions before analysis: All samples were stored refrigerated when not in use.

Test solutions

Details on test solutions:
- Method: Direct addition to testing laboratory's well water in the test chambers. The appropriate amount of test material was weighed out and added to the test chambers, and the test solutions stirred for approximately 90 minutes (which included approximately 10 minutes of sonication.) Fish were added after mixing was completed.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The dilution water control and nominal 13.1 mg total fluorinated solids/L test solutions were clear and colourless with no visible precipitate. The nominal 26.2 and 52.3 mg total fluorinated solids/L test solutions were clear with undissolved test material present and bubbles at the surface. The nominal 105 mg total fluorinated solids/L test solution was clear with suspended substance and undissolved test material present, and bubbles at the surface. The nominal 209 mg total fluorinated solids/L test solution was cloudy with suspended substance and undissolved test material present with bubbles at the surface.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Thomas Fish Company, Anderson, California
- Age at study initiation: 28 days
- Length: standard length of dilution water control fish was 2.3 to 2.7 cm in (mean 2.4 cm), at test end. Standard length of the longest fish was not more than twice the length of the shortest fish in the control.
- Weight: dilution water control fish: 0.12 to 0.18 g wet weight, blotted dry (mean 0.14 g) at test end.
- Feeding during test: fish were not fed approximately 27 hours prior to nor during the test

- Acclimation period: 17 days
- Acclimation conditions (same as test or not): The fish were held in a 272-L, circular, fibreglass holding tank (84-cm diameter, 49-cm deep) for 17 days in continuously-flowing well water. During the 7-day period preceding the test, water temperatures ranged from 10.7 to 12.0°C (mean 11.6°C).
- Type and amount of food: freshly-hatched, live brine shrimp (San Francisco Bay Brand, Newark, California) and AquaMax® Starter Fingerling 300 5D03 (PMI® Nutrition International, LLC).
- Feeding frequency: 3 times weekly
- Health during acclimation (any mortality observed): Sickness, injury, and visible abnormalities were not present in fish in the holding tank or in the fish used for testing. Mortality did not exceed 3% during the 48 hours prior to use for testing.

Study design

Test type:
other: static-renewal
Water media type:
Limit test:
Total exposure duration:
96 h

Test conditions

total alkalinity at initiation: 79 to 83 mg/L as CaCO3/L
total hardness at initiation: 158 to 159 mg/L as CaCO3/L
Test temperature:
mean temperature = 12.5°C with a range of 12.3 to 12.9°C
7.6 to 8.3
Dissolved oxygen:
6.0 to 9.4 mg/L
Nominal and measured concentrations:
Nominal concentrations (not adjusted for % fluorinated solids): 0 (control), 44.5, 89.0, 178, 356, and 712 mg/L mg test substance/L
Nominal concentrations (based on a total fluorinated solids content of 29.4%): 0 (control) 13.1, 26.2, 52.3, 105, and 209 mg total fluorinated solids/L
Mean measured total fluorinated solids concentrations: 0 (control), 11.4, 22.6, 42.4, 87.4, and 120 mg total fluorinated solids/L

Total fluorinated solids includes 28.0% amphoteric fluorinated surfactant, 1.4% impurity 1, and < 0.2% impurity 2.
Mean, measured total fluorinated solids concentration was calculated as:
(Day 0 New centrifuged + Day 2 Old centrifuged + Day 2 New centrifuged + Day 4 Old centrifuged)/4.
Mean measured concentrations ranged from 57 to 87% of the nominal concentrations adjusted for 29.4% solids by analysis.
The level of fluorinated solids in the dilution water control was below the limit of quantitation of 1.68 mg/L after application of a 160x dilution factor.
Details on test conditions:
- Test vessel: 4-L glass beakers, which held approximately 3.75 L of test solution (approximately 21-cm test solution depth). Each chamber was covered with a glass plate during the test.
- Aeration: No
- Renewal: Test solutions were renewed after approximately 48 hours.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: Dilution water control loading at test end was 0.38 g/L.

- Source/preparation of dilution water: Test facility well water adjusted to approximately 100-140 mg/L as CaCO3 by the flow-proportioned addition of CaCl2. Then aerated, passed through a green sand filter to remove iron, and filtered through 50-, 10-, and 0.45-µm filters to remove particulates.
- Conductivity: 265 to 295 µmhos/cm

- Photoperiod: 16 hours light / 8 hours darkness, included 30 minutes of transitional light preceding and following the 16-hour light interval.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality and sublethal effects (at test start, every 24 hours thereafter, and at test end).

A 96-hour static rangefinding study was conducted at nominal concentrations of 2, 20, and 200 mg total fluorinated solids/L. No mortality or sublethal effects were observed in the dilution water control or at any test concentration. Test solutions were prepared in stainless steel tanks with T-line stirrers. After agitation of the test solution during mixing, the 200 mg total fluorinated solids/L solution was unobservable due to an approximately 4-inch thick layer of foam at the test solution surface. Therefore, a second 200 mg total fluorinated solids/L chamber was prepared in a 4-L glass beaker. The solution was stirred on a magnetic stir plate and, due to decreased agitation, resulted in a solution which was clear and colourless with suspended substance and bubbles at the solution surface. Based on solution observations during the rangefinding study, a flow-through test design was impractical due to the agitation which would occur in the proportional diluter system. Instead, a static-renewal design utilizing glass beakers as test chambers was employed. The test concentrations used in the definitive study were based on the results of the range-finding test concentration.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
96 h
Dose descriptor:
Effect conc.:
> 712 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: no mortality
Remarks on result:
other: highest concentration tested
96 h
Dose descriptor:
Effect conc.:
> 120 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: total fluorinated solids
Basis for effect:
other: no mortality
Remarks on result:
other: highest concentration tested
Details on results:
No mortality or sublethal effects were seen in the dilution water control fish. Exposure of rainbow trout to mean, measured concentrations of 11.4, 22.6, 42.4, 87.4, and 120 mg total fluorinated solids/L resulted in 0, 0, 0, 0, and 0% mortality respectively, at the end of 96 hours. No sublethal effects were seen in any test group.
Reported statistics and error estimates:
The 96-hour LC50 values were not calculated because survival was greater than 50% in all test solutions.

Applicant's summary and conclusion

Validity criteria fulfilled:
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
96-h LC50 based on nominal concentrations > 712 mg/L
96-h LC50 based on mean measured concentrations (total fluorinated solids) > 120 mg/L
Executive summary:

The test substance was assessed for acute toxicity to unfed fingerling rainbow trout, Oncorhynchus mykiss, in an unaerated, static-renewal, 96-hour test. Nominal test substance concentrations (not adjusted for % fluorinated solids) were 44.5, 89, 178, 356, and 712 mg/L. Mean, measured concentrations of total fluorinated solids were 11.4, 22.6, 42.4, 87.4, and 120 mg/L, respectively. A dilution water control was used in this study. The 96-hour LC50values, based on mortality and nominal concentrations or mean, measured concentrations of total fluorinated solids, were greater than 712 mg/L and greater than 120 mg total fluorinated solids/L, respectively.