Amphoteric Fluorinated Surfactant

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

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

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

Remarks:

Conducted according to guideline in effect at time of study conduct

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Remarks:

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: One sample plus a back-up sample from each test concentration, including the blank control, 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 4x into methanol (5 mL test solution + 15 mL methanol) from each test concentration, including the blank control, on day 0 of the test before the test solutions were poured into the replicate test chambers. One sample plus a back-up sample from the pooled replicates at each test concentration, including the blank control, and the single abiotic control test chamber at test end (day 4).
- Sample storage conditions before analysis: All samples and back-up samples were stored refrigerated when not in use.

Test solutions

Vehicle:

no

Details on test solutions:

Test Solution Preparation
A primary stock solution with a nominal concentration of 40 mg total fluorinated solids/L test substance was prepared by adding approximately 68 mg of the test substance to 500 mL filter-sterilized AAP nutrient medium and stirring for 23 minutes. Test solutions were prepared by adding the appropriate volume of the primary stock solution to filter-sterilized AAP nutrient medium to make nominal concentrations of 2.5, 5, 10, and 20 mg total fluorinated solids/L test substance, and stirring each for approximately 12 minutes. Aliquots of the primary stock solution were used for the nominal 40 mg total fluorinated solids/L test concentration solution and the abiotic (stability) control solution. Aliquots of the filter-sterilized AAP nutrient medium were used for the blank control solution. The blank control was clear and colourless with no visible precipitate at test start. The 2.5, 5, 10, and 20 mg total fluorinated solids/L test substance solutions were clear and colourless, were slightly bubbly on the surface, and lacked precipitate at test start. The 40 mg total fluorinated solids/L test substance solution and the abiotic control were clear and colourless, had bubbles on the surface, and lacked precipitate at test start.
AAP Nutrient Medium Preparation
The 2.5 litre volume required for the definitive and recovery tests was prepared based on the following proportions. To prepare one litre of AAP nutrient medium, 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution are added to approximately 800 mL of Milli-Q (deionised) water with mixing after each addition. Additional Milli-Q water was added to bring the medium to its final volume. The nutrient medium was adjusted to a pH of 7.49 with 1.0 N hydrochloric acid and filter-sterilized through 0.22-μm cellulose acetate filters into sterile containers. The containers with the resulting filter-sterilized AAP nutrient medium were stored in the refrigerator in the dark at approximately 4°C and acclimated to ambient temperature prior to use.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata, a freshwater, unicellular, non-motile, green alga, used in this study was cultured and maintained at the testing laboratory. The original culture source was the Department of Botany - Culture Collection of Algae - The University of Texas, Austin, Texas.

The culture method for P. subcapitata was based on published literature. Prior to the study, cultures were maintained under photoperiod, shaking speed, and temperature conditions similar to those used in the study. Illumination was maintained at 5005 ± 805 lux. The organisms were cultured in sterilized 250-mL Erlenmeyer flasks containing approximately 50 mL of filter-sterilized synthetic algal-assay procedure (AAP) nutrient medium and were aseptically transferred to fresh medium every 3 to 7 days. The flasks were fitted with sterilized foam stoppers to permit gas exchange. The P. subcapitata culture used to inoculate test vessels was aseptically transferred to fresh medium 4 days prior to use.

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

96 h

Test conditions

Test temperature:

air temperature in the environmental chamber: 23 ± 2°C
solution temperature: 23°C

pH:

7.57 to 8.20

Nominal and measured concentrations:

Nominal concentrations: 0, 8.5, 17, 34, 68, 136 mg/L and abiotic control (136 mg/L)
Nominal concentrations: 0, 2.5, 5, 10, 20, and 40 mg total fluorinated solids/L and biotic control (40 mg/L)
Mean, measured concentrations: 0, 1.80, 3.43, 7.19, 15.8, 31.6, and 31.6 mg/L total fluorinated solids/L mg at 96 hours and abiotic control (31.6 mg/L)

Total fluorinated solids content is defined as 28.0% active substance + 1.4% impurity 1 + <0.2% impurity 2.
Mean, measured total fluorinated solids concentration was calculated as (Day 0 centrifuged + Day 4 centrifuged)/2.

Details on test conditions:

The study was conducted with a blank control, 5 concentrations of test substance, and an abiotic control at a mean lighting intensity of 4478 lux (range of 4210 to 4730 lux), air temperature of 23±2°C, a liquid temperature of 23°C, and a shaking speed of 100 rpm.

Definitive Test:
The nominal concentrations of 2.5, 5, 10, 20, and 40 mg total fluorinated solids/L were chosen for the definitive test based on the results of a preliminary range-finding test and an initial definitive test. It was not possible to calculate a NOEC from this initial definitive test, therefore a second definitive test was initiated to ensure determination of a NOEC. Test organisms were exposed for 96 hours without test medium renewal.
The blank control was tested as 6 replicates and each test concentration was tested as 3 replicates. The abiotic control was tested as a single flask (no replicates). Each flask, excluding the abiotic control, was randomly assigned a number to position the test flasks on the shaker table and to eliminate bias while counting. The abiotic control flask was placed on the shaker table after the other flasks had been positioned. A sign was placed near the shaker table identifying the definitive test by its unique study description, which included the work request and service code. The individual flasks, excluding the abiotic control, were labelled with concentration, replicate, and random number. The abiotic control was labelled as “abiotic control”. All flasks, excluding the abiotic control, were inoculated with approximately 10,000 P. subcapitata cells/mL by aseptically transferring 0.635 mL of algal inoculum from a precounted, logarithmically growing stock culture.
Measurements of pH were conducted at test start in an aliquot taken directly from the appropriate mixing vessel and at 96 hours in an aliquot taken after pooling all replicates, where appropriate, of each control or test concentration. The pH of the abiotic control was taken from the single flask.

Recovery Test:
At the end of the 96-hour exposure period, the blank control, and those test concentrations exhibiting a 50% or greater inhibition of healthy cell counts relative to the blank control (nominal 20 and 40 mg total fluorinated solids/L) were selected for the recovery test.
Each blank control and test concentrations was tested as a single flask containing untreated filter-sterilized AAP nutrient medium for 3 days without test medium renewal. Recovery test flasks were prepared by diluting an approximate 0.5-mL aliquot from a single, randomly selected replicate of the blank control or the combined approximate 0.5-mL aliquots from each of the replicate flasks of the nominal 20 and 40 mg total fluorinated solids/L concentrations to a total of approximately 50 mL with fresh nutrient medium, resulting in a concentration that theoretically would not inhibit algal growth and growth rate based on visual observations during termination of the definitive test. Biomass was determined on day 3. If cell growth was evident (based on a 16x increase in cell density prior to 10 days), the recovery test was terminated and the test substance concluded to be algistatic. If cell growth was not evident, the recovery test was terminated and the test substance concluded to be algicidal.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass, yield, and growth rate, each based on cell count

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.43 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: total fluorinated solids

Basis for effect:

other: biomass, yield, and growth rate, each based on cell count

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 136 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 31.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: total fluorinated solids

Basis for effect:

growth rate

Details on results:

The mean, measured total fluorinated solids concentrations of test concentration solutions and the nominal 40 mg total fluorinated solids/L abiotic control solution at 96 hours ranged from 69 to 79% of nominal concentrations. Biomass increased in the blank control by at least a factor of 16 in 72 hours, the coefficient of variation of average specific growth rates during the 0-72 hour exposure period in the blank control replicates did not exceed 7%, and the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the blank control replicates did not exceed 35%, thereby satisfying the appropriate OECD test acceptance criteria for a 72-hour test. Biomass increased in the blank control by at least a factor of 16 in 96 hours and the coefficient of variation of average specific growth rates in the blank control replicates did not exceed 7% during the 96-hour exposure period.
Recovery data indicated that the effect of the test substance at mean, measured total fluorinated solids concentrations equal to or below 31.6 mg total fluorinated solids/L was algistatic (reversible).

Reported statistics and error estimates:

Analyses are based on nominal test substance concentrations and mean, measured total fluorinated solids concentrations and were conducted using SAS Version 8.2. All statistical tests were calculated at a significance level of p = 0.05.
The 72-hour and 96-hour data for biomass, yield, and growth rate, each based on cell count, were determined to be normally distributed (Shapiro-Wilk test) with equal variances (Levene’s test). Outliers, as determined by the Tukey outlier rule, were found in the data for biomass and yield. Since the data were normally distributed with equal variances, outliers were not considered. No outliers were found in the data for growth rate. Massive ties, as determined by exact permutation data analysis methods, were present in the data for biomass, yield, and growth rate, and consequently, exact permutation data analysis methods were used. The Jonckheere-Terpstra test was used to determine the NOEC and LOEC values. The 72-hour and 96-hour EbC50, EyC50, and ErC50 values (and 95% confidence intervals) for biomass, yield, and growth rate, respectively, each based on cell count were obtained by the Bruce-Versteeg regression model.

Any other information on results incl. tables

Parameter	Biomass, Cell Count	Yield, Cell Count	Growth Rate, Cell Count
72-hour EC50(mg/L, nominal concentration) (95% confidence interval)	50.0 (35.2 to 71.0)	49.6 (34.9 to 70.4)	>136
72-hour EC50(mg total fluorinated solids/L, mean measured concentration) (95% confidence interval)	11.1 (7.70 to 16.0)	11.0 (7.63 to 15.8)	>31.6
96-hour EC50(mg/L, nominal concentration) (95% confidence interval)	56.4 (43.1 to 73.8)	56.2 (43.0 to 73.6)	>136
96-hour EC50(mg total fluorinated solids/L, mean measured concentration) (95% confidence interval)	12.6 (9.54 to 16.6)	12.5 (9.50 to 16.6)	>31.6
72-Hour NOEC (mg/L nominal concentration)	17	17	17
72-Hour NOEC (mg total fluorinated solids/L, mean measured concentration)	3.43	3.43	3.43
96-Hour NOEC (mg/L nominal concentration)	17	17	17
96-Hour NOEC (mg total fluorinated solids/L, mean measured concentration)	3.43	3.43	3.43
72-Hour LOEC (mg/L nominal concentration)	34	34	34
72-Hour LOEC (mg total fluorinated solids/L, mean measured concentration)	7.19	7.19	7.19
96-Hour LOEC (mg/L nominal concentration)	34	34	34
96-Hour LOEC (mg total fluorinated solids/L, mean measured concentration)	7.19	7.19	7.19

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The 72-hour NOEC and LOEC values based on nominal test substance concentrations and biomass, yield, and growth rate, each based on cell count, were determined to be 17 mg/L and 34 mg/L, respectively. The 72-hour EbC50, EyC50, and ErC50 values (and 95% confidence intervals) based on nominal test substance concentrations and biomass, yield, and growth rate, each based on cell count, respectively, were 50.0 mg/L (35.2 to 71.0 mg/L), 49.6 mg/L (34.9 to 70.4 mg/L), and >136 mg/L, respectively.
The 72-hour NOEC and LOEC values based on mean, measured total fluorinated solids concentrations and biomass, yield, and growth rate, each based on cell count, were determined to be 3.43 mg total fluorinated solids/L and 7.19 mg total fluorinated solids/L, respectively. The 72-hour EbC50, EyC50, and ErC50 values (and 95% confidence intervals) based on mean, measured total fluorinated solids concentrations and biomass, yield, and growth rate, each based on cell count, respectively, were 11.1 mg total fluorinated solids/L (7.70 to 16.0 mg total fluorinated solids/L), 11.0 mg total fluorinated solids/L (7.63 to 15.8 mg total fluorinated solids/L), and >31.6 mg total fluorinated solids/L, respectively.
The 96-hour NOEC and LOEC values based on nominal test substance concentrations and biomass, yield, and growth rate, each based on cell count, were determined to be 17 mg/L and 34 mg/L, respectively. The 96-hour EbC50, EyC50, and ErC50 values (and 95% confidence intervals) based on nominal test substance concentrations and biomass, yield, and growth rate, each based on cell count, respectively, were 56.4 mg/L (43.1 to 73.8 mg/L), 56.2 mg/L (43.0 to 73.6 mg/L), and >136 mg/L, respectively.
The 96-hour NOEC and LOEC values based on mean, measured total fluorinated solids concentrations and biomass, yield, and growth rate, each based on cell count, were determined to be 3.43 mg total fluorinated solids/L and 7.19 mg total fluorinated solids/L, respectively. The 96-hour EbC50, EyC50, and ErC50 values (and 95% confidence intervals) based on mean, measured total fluorinated solids concentrations and biomass, yield, and growth rate, each based on cell count, respectively, were 12.6 mg total fluorinated solids/L (9.54 to 16.6 mg total fluorinated solids/L), 12.5 mg total fluorinated solids/L (9.50 to 16.6 mg total fluorinated solids/L), and >31.6 mg total fluorinated solids/L, respectively.
The test substance was determined to be algistatic at mean, measured concentrations less than or equal to 31.6 mg total fluorinated solids/L.

Executive summary:

The toxicity of the test substance to the green algae, Pseudokirchneriella subcapitata, was determined in a 96-hour, static toxicity test. The test substance contained 29.4% total fluorinated solids (total fluorinated solids includes 28.0% amphoteric fluorinated surfactant, 1.4% impurity 1, and < 0.2 % impurity 2) by analysis. The study was conducted with a blank (culture medium) control and 5 concentrations of test substance at a mean lighting intensity of 4478 lux (range of 4210 to 4730 lux), air temperature of 23±2°C, a liquid temperature of 23°C, and a shaking speed of 100 rpm. Synthetic algal-assayprocedure (AAP) nutrient medium was used as the test diluent and blank control. Test solutions were not renewed. Six replicates were used for the blank control, and 3 replicates were used per test concentration. A single test flask was used for the abiotic (stability) control that was included to demonstrate the stability of the test substance in AAP nutrient medium under the test conditions without the presence of algae. Biomass, yield, and growth rate, all based on cell count, were determined at 24-hour intervals over the 96-hour test.

The mean, measured total fluorinated solids concentrations of test substance test concentration solutions and the nominal 40 mg total fluorinated solids/L abiotic control solution at 96 hours ranged from 69 to 79% of nominal concentrations. Biomass increased in the blank control by at least a factor of 16 in 72 hours, the coefficient of variation of average specific growth rates during the 0-72 hour exposure period in the blank control replicates did not exceed 7%, and the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the blank control replicates did not exceed 35%, thereby satisfying the appropriate OECD test acceptance criteria for a 72-hour test. Biomass increased in the blank control by at least a factor of 16 in 96 hours and the coefficient of variation of average specific growth rates in the blank control replicates did not exceed 7% during the 96-hour exposure period.

The reductions in biomass, yield, and growth rate, each based on cell count, for Pseudokirchneriella subcapitata at 96 hours indicated a concentration-dependent response for exposure to the test substance.

The 72-hour NOEC and LOEC values based on nominal test substance concentrations and biomass, yield, and growth rate, each based on cell count, were determined to be 17 mg/L and 34 mg/L, respectively. The 72-hour EbC50, EyC50, and ErC50 values (and 95% confidence intervals) based on nominal test substance concentrations and biomass, yield, and growth rate, each based on cell count, respectively, were 50.0 mg/L (35.2 to 71.0 mg/L), 49.6 mg/L (34.9 to 70.4 mg/L), and >136 mg/L, respectively.

The 72-hour NOEC and LOEC values based on mean, measured total fluorinated solids concentrations and biomass, yield, and growth rate, each based on cell count, were determined to be 3.43 mg total fluorinated solids/L and 7.19 mg total fluorinated solids/L, respectively. The 72-hour EbC50, EyC50, and ErC50 values (and 95% confidence intervals) based on mean, measured total fluorinated solids concentrations and biomass, yield, and growth rate, each based on cell count, respectively, were 11.1 mg total fluorinated solids/L (7.70 to 16.0 mg total fluorinated solids/L), 11.0 mg total fluorinated solids/L (7.63 to 15.8 mg total fluorinated solids/L), and >31.6 mg total fluorinated solids/L, respectively.

The 96-hour NOEC and LOEC values based on nominal test substance concentrations and biomass, yield, and growth rate, each based on cell count, were determined to be 17 mg/L and 34 mg/L, respectively. The 96-hour EbC50, EyC50, and ErC50 values (and 95% confidence intervals) based on nominal test substance concentrations and biomass, yield, and growth rate, each based on cell count, respectively, were 56.4 mg/L (43.1 to 73.8 mg/L), 56.2 mg/L (43.0 to 73.6 mg/L), and >136 mg/L, respectively.

The 96-hour NOEC and LOEC values based on mean, measured total fluorinated solids concentrations and biomass, yield, and growth rate, each based on cell count, were determined to be 3.43 mg total fluorinated solids/L and 7.19 mg total fluorinated solids/L, respectively. The 96-hour EbC50, EyC50, and ErC50 values (and 95% confidence intervals) based on mean, measured total fluorinated solids concentrations and biomass, yield, and growth rate, each based on cell count, respectively, were 12.6 mg total fluorinated solids/L (9.54 to 16.6 mg total fluorinated solids/L), 12.5 mg total fluorinated solids/L (9.50 to 16.6 mg total fluorinated solids/L), and >31.6 mg total fluorinated solids/L, respectively.

The test substance was determined to be algistatic at mean, measured concentrations less than or equal to 31.6 mg total fluorinated solids/L.