Amphoteric Fluorinated Surfactant

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Remarks:

Study conducted according to guideline in effect at time of study conduct

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Deviations:

no

Remarks:

Study conducted according to guideline in effect at time of study conduct

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Purity: 28 wt%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations Sampled: All test samples were analysed.
- Sampling Times: One sample plus a back-up sample from each test concentration, including the dilution water control, before and after addition of the diet solution, on day 0 of the test before the test solutions were poured into the replicate test chambers. In addition to the neat test solution samples, one sample plus a back-up sample diluted 4× into methanol (5 mL test solution + 15 mL methanol) from each test concentration, including the dilution water control, before and after addition of the diet solution, on day 0 of the test before the test solutions were poured into the replicate test chambers. One sample plus a back-up sample (new solutions) from each test concentration, including the dilution water control, after addition of the diet solution, on days 2, 9, 16, and 19 of the test before the test solutions were poured into the replicate test chambers. One sample plus a back-up sample (old solutions) from 2 of the 10 replicate chambers at each test concentration, including the dilution water control, on days 5, 12, 19, and 21 of the test.
- Sample storage conditions before analysis: Samples and back-up samples were stored refrigerated when not in use.

Test solutions

Vehicle:

no

Details on test solutions:

Test Solution Preparation
Calculations were based on initial reported solids concentrations of 28.1%, the purity information available at study initiation. The COA was later revised to purity of 29.4% solids. On day 0 and on each renewal day (Monday, Wednesday, and Friday), a primary stock solution of nominal 106.8 mg/L (equivalent to nominal 31.4 mg total fluorinated solids/L) was prepared by adding approximately 0.32 grams of the test material and bringing this up to 3 L in testing laboratory well water in a 4-L glass beaker, stirring for approximately 5-10 minutes, sonicating for approximately 7-13 minutes, and stirring again for approximately 18-27 minutes. At preparation, the stock solution was clear and colourless with no visible precipitate and bubbles on the surface. Nominal test concentrations of 3.38, 6.76, 13.5, 26.7, and 53.4 mg/L (which correspond to nominal total fluorinated solids concentrations of 0.994, 1.99, 3.97, 7.85, and 15.7 mg total fluorinated solids/L, respectively) were prepared by diluting appropriate amounts of the primary stock solution with dilution water. At test start and at each renewal, each test concentration and the dilution water control received 2.1 mL of algae per litre of test solution (final algal concentration of approximately 62,500 cells/mL in test solutions) and 2.1 mL of yeast, cereal leaves and trout chow mixture (YCT) per litre of test solution. Final test solutions were stirred for approximately 21 minutes before addition of algae and YCT and approximately 5 minutes after addition of algae and YCT. At test start, the test solutions were cloudy and pale green in colour with particles of YCT visible, except for the 15.7 mg total fluorinated solids/L test solution which also had bubbles on the surface.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna
- Source: reared at the testing laboratory in 1000-mL glass beakers (10 per beaker) which contained 1000 mL of aerated, filtered testing laboratory well water held at approximately 20.0°C.
- Age of parental stock (mean and range, SD): Neonates used in this test were less than 24 hours old and were collected from the 4th brood of 15-day old parent daphnids.
- Feeding during test: yes
- Food type and amount: 3 mL/L of a yeast, cereal leaves and trout chow (YCT) mixture and the green alga, Pseudokirchneriella subcapitata, at a rate of approximately 62,500 cells/mL of culture media. The YCT mixture was standardized to 1700-2100 mg/L solids. The combination of YCT and alga is equivalent to approximately 0.1-0.2 mg total organic carbon per daphnid per day. YCT is analysed periodically to assure that contaminants are not present at levels that would be expected to affect the scientific integrity of the study. Green algae are not routinely analysed for contaminants because they are cultured in a defined laboratory medium prepared using reagent grade chemicals and deionised water.
- Frequency: Daily
- Health (any mortality observed): Sickness, injury, and abnormalities were not seen and ephippia were not being produced by the parent daphnids. No adult immobility was seen during the 48-hour pre-test period in the culture used to supply neonates for testing.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

149-164 mg/L as CaCO3 (dilution water control).
160-176 mg/L as CaCO3 (highest test concentration with surviving organisms, 13.5 mg solids/L)

Test temperature:

18.9 and 21.1°C.

pH:

The pH ranged from 8.0 to 8.3 in new solutions and from 7.9 to 8.3 in old solutions but did not deviate by more than 0.5 units over a 48- or 72-hour period between renewals within the same replicate at all concentration levels.

Dissolved oxygen:

Overall minimum dissolved oxygen concentration for the new and old test solutions, including the dilution water control, was 7.3 mg/L or 80.2% of saturation (based on an air saturation value of 9.1 mg/L at 20°C) throughout the study.

Nominal and measured concentrations:

Nominal: 0 (control), 3.38, 6.76, 13.5, 26.7, and 53.4 mg/L.
Corrected nominal total fluorinated solids: 0.994, 1.99, 3.97, 7.85, and 15.7 mg/L (based on 29.4% solids content)
Mean, measured total fluorinated solids concentrations: 0.619, 1.50, 3.18, 6.42, and 13.5 mg/L

Mean, measured total fluorinated solids concentration was calculated as:
[(Day 0 New after diet addition, cent.) + Day 2 New + (Day 5 Old Mean) + Day 9 New + (Day 12 Old Mean) + Day 16 New + Day 19 New + (Day
19 Old Mean) + (Day 21 Old Mean)]/9
Mean measured concentrations ranged from 62% to 82% of the targeted 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 0.0420 mg/L after application of a 4x dilution factor.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (250 mL) containing 200 mL of test solution (6.5 cm depth); covered with a glass plate during the test.
- Renewal rate of test solution (frequency/flow rate): Test solutions were renewed every Monday, Wednesday, and Friday during the study. Daphnids were transferred to new test solutions using a glass pipette.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

OTHER TEST CONDITIONS
- Adjustment of pH: No data
- Photoperiod and light intensity: A photoperiod of 16 hours light (approximately 125 - 155 Lux) and 8 hours darkness was employed, which included approximately 30 minutes of transitional light (approximately 7 - 26 Lux) between the light and dark intervals.

TEST MEDIUM / WATER PARAMETERS:
- Source/preparation of dilution water: Test laboratory 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: 300 to 330 µmhos/cm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations were made daily on the number of surviving adult daphnids, occurrence of sublethal effects, and production of live or immobile young. The criterion for immobility was the lack of response to application of a gentle stimulus. Immobile daphnids (adults and young) were discarded on renewal days. Live or immobile young were counted and removed on renewal days. Length and dry weight of surviving adult daphnids were determined at test end.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.18 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: Total fluorinated solids

Basis for effect:

other: Total number of live young produced per surviving female at the end of the study

Effect concentrations 2

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

6.42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: Total fluorinated solids

Basis for effect:

other: Total number of live young produced per surviving female at the end of the study

Effect concentrations 3

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 13.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: Total fluorinated solids

Basis for effect:

other: Adult survival

Details on results:

See Table 1

Reported statistics and error estimates:

Analyses were conducted using SAS Version 8.2. All statistical tests were calculated at a significance level of p = 0.05. No statistical analysis was performed for adult survival to determine the EC50 values since there was never more than a 20% effect at any test concentration. The data for adult survival were quantal data and thus were analysed by the step-down Cochran-Armitage test, provided the data were consistent with a monotone concentration-response, and by Fisher’s Exact test otherwise. Adult mortality was observed only in the high test concentration at 21 days. The 21-day NOEC value for adult survival was 6.42 mg total fluorinated solids/L, respectively, by the Cochran-Armitage test. EC50 values could not be estimated for these data, as only 20% mortality was observed at 21 days, and no model (probit, moving average angle, or binary) could be fit to the data. The conclusion was that the 21-day EC50 value, based on adult survival, was greater than the high test concentration. The Jonckheere-Terpstra trend test was used to determine the NOEC and LOEC values for all continuous parameters (number of live young produced per surviving adult attest end, number of immobile young produced per surviving adult at test end, first day of reproduction, surviving adult length, and surviving adult weight). The 21-day EC50 value for live young produced were obtained by the Bruce-Versteeg regression Model. The 21-day EC50 value for immobile young produced per surviving adult could not be calculated as no models converged, and this was supported further by a NOEC value at the high test concentration.

Any other information on results incl. tables

Table 1

		Mean (Standard Deviation)
Mean, Measured Concentrations (mg total fluorinated solids/L)	Percent Adult Survival^a	First Reproduction Day^b	Total Live Young^c	Total Immobile Young^d	Adult Length (mm)^e	Adult Dry Weight (mg)^e
Dilution Water Control	100%	8.8 (1.1)	154.9 (15.6)	0 (0)	4.2 (0.1)	0.6 (0.1)
0.619	100%	8.6 (1.0)	146.8 (26.1)	0 (0)	4.3 (0.2)	0.7 (0.1)
1.50	100%	8.2 (0.4)	143.8 (11.3)	0 (0)	4.3 (0.1)	0.6 (0.1)
3.18	100%	8.8 (1.1)	138.2 (26.3)	0 (0)	4.3 (0.1)	0.7 (0.2)
6.42	100%	9.4 (1.1)	136.5 (30.8)	0 (0)	4.3 (0.1)	0.8 (0.2)
13.5	80%	9.2 (1.7)	119.9 (37.4)	2.6 (3.9)	4.2 (0.2)	0.7 (0.1)

a Percent of adult daphnids alive at the end of the test

b First day that reproduction was observed

c Sum of live young produced per surviving adult in 21 days

d Sum of immobile young produced per surviving adult in 21 days

e Per surviving adult

Applicant's summary and conclusion

Conclusions:

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
21-day NOEC = 3.18 mg total fluorinated solids/L, based on mean, measured concentrations of total fluorinated solids and the total number of live young produced per surviving female at the end of the study.
21-day LOEC = 6.42 mg total fluorinated solids/L, based on mean, measured concentrations of total fluorinated solids and the total number of live young produced per surviving female at the end of the study.
21-day EC50 for adult survival > 13.5 mg total fluorinated solids/L

Executive summary:

The growth and reproduction of the cladoceran, Daphnia magna, were evaluated when exposed to test substance total fluorinated solids for 21 days. Parameters evaluated were adult survival, live young produced per surviving adult, immobile young produced per surviving adult, first reproduction day, adult length, and adult weight. The 21-day NOEC (no-observed-effect concentration) using Daphnia magna neonates exposed for 21 days under static-renewal conditions was 3.18 mg total fluorinated solids/L, based on mean, measured concentrations of total fluorinated solids and the total number of live young produced per surviving female at the end of the study. The LOEC (lowest-observed-effect concentration) was 6.42 mg total fluorinated solids/L. The EC₅₀ for adult survival was greater than 13.5 mg total fluorinated solids/L.