Potassium 1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulphonate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 - 21 Dec 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Source and lot/batch No.of test material: L-7038 Lot 2
- Purity: 97.3%
- Expiration date of the lot/batch: 17 Jan 2002
- Storage condition of test material: Ambient room temperature
- Stability under test conditions: Stable

Analytical monitoring:

yes

Details on sampling:

Details on sampling
- Concentrations: All; sampled at 0, 48 and 96 hours.
- Sampling method:Samples were collected at mid-depth in each test chamber.
- Sample storage conditions before analysis: Samples were placed in plastic
vials and analyzed immediately

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stirring of primary stock (3263 mg a.i./L) with electric mixer. Dilution of primary stock with test medium to obtain lower concentrations. All materials which came in contact with the test solution during formulation were made of plastic or stainless steel.
- Controls: Blank test medium
- Evidence of undissolved material: All solutions appeared clear and colorless.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Cultures maintained at contract lab
- Age at study initiation: ca. 8 months
- Length at study termination: 35 mm (range, 30 - 40 mm) among negative controls
- Weight at study termination: 0.32 g (range, 0.22 - 0.47 g) among negative controls

ACCLIMATION
- Acclimation period: 52 hours
- Acclimation conditions: same as test
- Type and amount of food during acclimation: Not fed during acclimation.
- Health during acclimation: No signs of disease or stress

FEEDING DURING TEST
- Food type: Not fed during test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

128 mg CaCO3/L

Test temperature:

20.7 - 22.8 °C

pH:

8.3 - 8.5

Dissolved oxygen:

7.5 - 8.5 mg/L

Conductivity:

340 µmhos/cm

Nominal and measured concentrations:

Nominal: 0 mg/L, 204 mg/L, 408 mg/L, 816 mg/L, 1632 mg/L, 3263 mg/L
Measured: < LOQ, 220 mg/L, 437 mg/L, 888 mg/L, 1655 mg/L, 3341 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 25-L polyethylene aquaria, 15-L fill volume, depth ca. 16.5 cm
- Aeration: dilution water aerated prior to filling.
- No. of organisms per vessel: 10
- No. of vessels per concentration: two
- No. of vessels per control: two
- Biomass loading rate: 0.21 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 40-m deep well located at testing lab. Water was passed through a sand filter and pumped into a storage tank and aerated with spray nozzles. Water was filtered to 0.45 µm prior to testing. Periodic water analysis, Attachments 1 and 2.
- Pesticides: all < LOQ
- Alkalinity: 182 mg/L as CaCO3
- Ca/Mg ratio: 2.6 (mass basis)
- Culture medium different from test medium: No
- Intervals of water quality measurement: most recent analysis on samples collected Oct 14 and 15, 1999.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h:8h with 30-min transitions
- Light intensity: Colortone ® 50 fluorescent lights, 222 lux at surface of representative chamber

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and behavioral effects at approximately 2 h, 24h, 48 h, 72 h, and 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- Range finding study: Yes, no further details provided
- Results used to determine the conditions for the definitive study: Yes

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1 938 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI, 888 - 3341 mg a.i./L

Details on results:

- Behavioural abnormalities: Minnows in the 220 and 888 mg a.i./L treatment groups appeared normal and healthy during the test. One fish in the 437 mg a.i./L treatment group was observed to be lying on the bottom after 2 hours of exposure. However, throughout the remainder of the test all fish in the 437 mg a.i./L treatment group appeared normal. After 96-hours of exposure, mortality in the 1655 and 3341 mg a.i./L treatment groups was 30% and 100%, respectively.
- Mortality of control: None. Controls appeared normal and healthy during the test.

Reported statistics and error estimates:

LC50 and 95% CI calculated using binomial probability method with nonlinear interpolation.

Sublethal observations / clinical signs:

Mean measured concentration (mg/L)	Cumulative # Dead / # Originally Exposed - Observations¹	Cumulative # Dead / # Originally Exposed - Observations¹	Cumulative # Dead / # Originally Exposed - Observations¹	Cumulative # Dead / # Originally Exposed - Observations¹	Cumulative # Dead / # Originally Exposed - Observations¹	Cumulative percent mortality
	2 hours	24 hours	48 hours	72 hours	96 hours
Negative Control	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
220	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
437	0 / 20 (19 AN, 1 R)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
888	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
1655	0 / 20 (20 AN)	3 / 20 (17 AN)	5 / 20 (15 AN)	6 / 20 (14 AN)	6 / 20 (14 AN)	30
3341	0 / 20 (20 AN)	20 / 20	20 / 20	20 / 20	20 / 20	100

¹ Observations: AN = Appears Normal; R = Lying on Bottom

Validity criteria fulfilled:

yes

Remarks:

the validity criteria of OECD TG 203 were fulfilled. Control mortality < 10% at the end of the test. Constant conditions were maintained, including dissolved oxygen > 60% of saturation, and test substance > 80% of nominal, throughout the test.

Conclusions:

The 96-hour LC50 of PFBSK+ to Pimephales promelas was 1938 mg/L (EPA 850.1075 and OECD 203)

Executive summary:

The 96-hour LC50 of PFBSK+ to Pimephales promelas was examined in a static test conducted according to EPA 850.1075 and OECD 203. Analytically determined concentrations were < LOQ, 220 mg/L, 437 mg/L, 888 mg/L, 1655 mg/L, and 3341 mg/L. No mortalities were observed at 220, 437, and 888 mg/L, and 30% cumulative mortality was observed at 1655 mg/L, and 100% cumulative mortality was observed at 3341 mg/L. The 96-hour LC50 is 1938 mg/L. The test was conducted according to internationally accepted test guidelines and was GLP compliant. It is reliable without restriction and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 - 28 Mar 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: K100
- Sample no./year: 1069024/2002
- Purity: 98.7%
- Expiration date of the lot/batch: August 12, 2004

Analytical monitoring:

yes

Details on sampling:

Duplicate samples taken at 0, 24, 48, 72, and 96 hours. Samples prefiltered to 0.45 µm to remove particulate matter prior to analysis. In once case, samples were stored 24 h at 4 °C before analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct addition and 1 hour sonication
- Controls: Blank medium

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: Di Mamma (Netherlands)
- Age at study initiation: 136 days (birth date 08 Nov 2002)
- Length at study initiation: 3.61 cm (SD 0.22 cm)

ACCLIMATION
- Acclimation period: 69 days. Fish held at test conditions since arrival date (14 Jan 2003)
- Acclimation conditions: Same as test
- Type and amount of food during acclimation: Commercial fish food
- Feeding frequency during acclimation: Daily until 24 hours before test initiation
- Health during acclimation (any mortality observed): <5% mortality

FEEDING DURING TEST: No feeding during test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

235.6 mg CaCO3/L

Test temperature:

21.1 - 21.8 °C

pH:

7.4 - 8.1

Dissolved oxygen:

7.7 - 8.9 mg/L

Nominal and measured concentrations:

Nominal: 0 mg/L, 100 mg/L
Measured:

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria, five liter fill volume
- Aeration: Gentle aeration through narrow glass tubes
- No. of organisms per vessel: Ten
- No. of vessels per concentration: One
- No. of vessels per control: One
- Biomass loading rate: 0.95 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Per ISO7346
- Total organic carbon: <5 mg/L DOC in controls

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 light:dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and other sublethal effects at 2, 24, 48, 72, and 96 hours

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 105 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: No mortality observed

Details on results:

- Behavioural abnormalities: None noted
- Observations on body length and weight: None noted
- Mortality of control: None
- Other adverse effects control: None noted
- Effect concentrations exceeding solubility of substance in test medium: No

Sublethal observations / clinical signs:

Table, DOC analysis of test solutions

Analysis	0 h	24 h	48 h	72 h	96 h
Control repl. 1 DOC	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
Control repl. 2 DOC	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ
100 mg/L repl. 1 DOC (mg/L)	15	15	15	16	15
100 mg/L repl. 2 DOC (mg/L)	14	14	16	15	15
100 mg/L mean DOC (mg/L)	14.5	14.5	15.5	15.5	15
100 mg/L PFBSK+ concentration (mg/L)	101.5	101.5	108.5	108.5	105
Cumulative mean PFBSK+ concentration (mg/L)	101.5	101.5	103.8	105.0	105.0

Validity criteria fulfilled:

yes

Conclusions:

The EC50 of PFBSK+ to Danio rerio is >105 mg/L (EU method C.1)

Executive summary:

Short-term toxicity of PFBSK+ to fish was addressed in a limit test on Zebrafish (Danio rerio) done per EU test method C.1. Ten fish were exposed for 96 hours to a nominal concentration of 100 mg/L PFBSK+. Actual concentrations were determined daily by DOC analysis, with a cumulative average of 105 mg/L PFBSK+ for the entire test period. No mortality was observed. The LC50 was ≥105 mg/L.

The test was done under an internationally-accepted guideline under GLP criteria. Measured concentrations over time within 5% of nominal and demonstrated maintainence of stable exposure levels. The study is deemed reliable without restrictions and is suitable for Risk Assessment, Classification & Labelling, and PBT Analysis.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18 - 22 Dec 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Source and lot/batch No.of test material: L-7038 Lot 2
- Purity: 97.3%
- Expiration date of the lot/batch: 17 Jan 2002
- Storage condition of test material: Ambient room temperature
- Stability under test conditions: Stable

Analytical monitoring:

yes

Details on sampling:

Details on sampling
- Concentrations: All; sampled at 0, 48 and 96 hours.
- Sampling method:Samples were collected at mid-depth in each test chamber.
- Sample storage conditions before analysis: Samples were collected in glass vials and analyzed as soon as possible without storage.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stirring of primary stock (9433 mg a.i./L) with electric mixer. Dilution of primary stock with test medium to obtain lower concentrations. All materials which came in contact with the test solution during formulation were made of plastic or stainless steel.
- Controls: Blank test medium
- Evidence of undissolved material: All solutions appeared clear and colorless.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill
- Source: Osage Catfisheries, Inc. Osage Beach Missouri
- Age at study initiation: Juveniles, held for approximately 173 days prior to testing
- Length at study termination: 44 mm (range, 33 - 53 mm) among negative controls
- Weight at study termination: 1.0 g (range, 0.39 - 1.6 g) among negative controls

ACCLIMATION
- Acclimation period: 48 hours
- Acclimation conditions: same as test
- Type and amount of food during acclimation: Not fed during acclimation.
- Health during acclimation: No signs of disease or stress

FEEDING DURING TEST
- Food type: Not fed during test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

148 mg CaCO3/L

Test temperature:

22.0 - 23.9 °C

pH:

8.0 - 8.4

Dissolved oxygen:

5.4 - 8.2 mg/L

Conductivity:

350 µmhos/cm

Nominal and measured concentrations:

Nominal: 0 mg/L, 612 mg/L, 1224 mg/L, 2448 mg/L, 4895 mg/L, 9790 mg/L
Measured: < LOQ, 629 mg/L, 1311 mg/L, 2715 mg/L, 5252 mg/L, 9433 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 25-L polyethylene aquaria, 15-L fill volume, depth ca. 16.1 cm
- Aeration: dilution water aerated prior to filling.
- No. of organisms per vessel: 10
- No. of vessels per concentration: two
- No. of vessels per control: two
- Biomass loading rate: 0.70 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 40-m deep well located at testing lab. Water was passed through a sand filter and pumped into a storage tank and aerated with spray nozzles. Water was filtered to 0.45 µm prior to testing. Periodic water analysis, Attachments 1 and 2.
- Pesticides: all < LOQ
- Alkalinity: 178 mg/L as CaCO3
- Ca/Mg ratio: 2.6 (mass basis)
- Culture medium different from test medium: No
- Intervals of water quality measurement: most recent analysis on samples collected Oct 14 and 15, 1999.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h:8h with 30-min transitions
- Light intensity: Colortone ® 50 fluorescent lights, 220 lux at surface of representative chamber

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and behavioral effects at approximately 3 h, 24h, 48 h, 72 h, and 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- Range finding study: Yes, no further details provided
- Results used to determine the conditions for the definitive study: Yes

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

6 452 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI, 5252 - 9433 mg a.i./L

Details on results:

- Behavioral abnormalities: After 3 hours of exposure, 14 fish were lethargic and 6 were lying on the bottom of the test chamber in the 9433 mg a.i./L treatment group. All other surviving fish appeared to be normal. See Table 2 for details.
- Mortality of control: None. Controls appeared normal during the test.

Reported statistics and error estimates:

LC50 and 95% CI calculated using binomial probability method with nonlinear interpolation.

Sublethal observations / clinical signs:

Table 2, Cumulative Percent Mortality and Treatment-Related Effects

Mean measured concentration (mg/L)	Cumulative # Dead / # Originally Exposed  (Observations)¹	Cumulative # Dead / # Originally Exposed (Observations)¹	Cumulative # Dead / # Originally Exposed (Observations)¹	Cumulative # Dead / # Originally Exposed (Observations)¹	Cumulative # Dead / # Originally Exposed (Observations)¹	Cumulative percent mortality
	3 hours	24 hours	48 hours	72 hours	96 hours
Negative Control	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
629	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
1311	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
2715	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
5252	0 / 20 (20 AN)	2 / 20 (18 AN)	2 / 20 (18 AN)	3 / 20 (17 AN)	3 / 20 (17 AN)	15
9433	0 / 20 (14 C, 6 R)	15 / 20 (5 AN)	20 / 20	20 / 20	20 / 20	100

¹ Observations: AN = Appears Normal; C = Lethargic; R = Lying on Bottom

Validity criteria fulfilled:

yes

Remarks:

the validity criteria of OECD TG 203 were fulfilled. Control mortality < 10% at the end of the test. Constant conditions were maintained, including dissolved oxygen > 60% of saturation, and test substance > 80% of nominal, throughout the test.

Conclusions:

The 96-hour LC50 of PFBSK+ to Lepomis macrochirus was 6452 mg/L (EPA 850.1075 and OECD TG 203)

Executive summary:

The 96-hour LC50 of PFBSK+ to Lepomis macrochirus was examined in a static test conducted according to EPA 850.1075 and OECD TG 203. Analytically determined concentrations were < LOQ, 629 mg/L, 1311 mg/L, 2715 mg/L, 5252 mg/L, and 9433 mg/L. No mortalities were observed at 629, 1311, and 2715 mg/L, and 15% cumulative mortality was observed at 5252 mg/L, and 100% cumulative mortality was observed at 9433 mg/L. The 96-hour LC50 is 6452 mg/L.

The test was conducted according to internationally accepted test guidelines and was GLP compliant. It is reliable without restriction and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Remarks:

concerning membrane fluidity in fish leukocytes.

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Determination of membrane fluidity
- Short description of test conditions: fish leukocytes incubated with substance for 15 minutes before assay.
- Parameter observed: Fluorescence of a dye normally immobilized within cell membranes.

GLP compliance:

no

Specific details on test material used for the study:

Test report distinguishes between perfluorooctane sulfonic acid and perfluorobutane sulfonate. Since the substance was provided by a registrant, it is presumed to be PFBSK+ rather than another sulfonate

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Study also included perfluorooctane sulfonic acid and perfluorohexane sulfonate. Stock solutions were made at 10 mM in DMSO.
- Controls: 1% pentanol, blank, solvent control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none

Test organisms (species):

other: Carp, species not identified

Details on test organisms:

Carp (species not identified) were anesthetized with MS-222 and blood was drawn from the caudal vein into a heparinized syringe. Blood from 3-4 fish (ca 2.5 mL each) was pooled on each sampling occasion. Three mL of Histopaque-1077 (Sigma, St. Louis, MO) was allowed to come to room temperature and three mL of collected fish blood was layered over the top. After centrifugation (30 min at 400 x g), the upper serum layer was discarded and the opaque interface transferred to a new tube. Cells were gently resuspended in ten mL phosphate buffered saline (PBS), after which suspensions were centrifuged 10 min at 250 x g. The supernatant was discarded, cells were resuspended in 5 mL PBS, and suspensions centrifuged 10 min at 250 x g. The final cell pellet was initially resuspended in 0.5 mL PBS and counted in a hemacytometer. Final cell concentration was adjusted to 1e+05 to 1.5e+06 cells per 200 µL.

Test type:

static

Water media type:

other: Phosphate-buffered saline (PBS)

Limit test:

no

Total exposure duration:

15 min

Test temperature:

25 °C

pH:

7.4

Salinity:

0.89%

Nominal and measured concentrations:

Nominal only: blank, 1 µM, 3 µM, 10 µM, 30 µM, 100 µM
Article states that PFBSK+ was tested at the same concentration range as for PFOS. PFOS concentrations were reported in both micromolar and mg/L units, but since all stocks were made at 10 mM it is presumed that all dilutions were on a molar basis.

Details on test conditions:

Pyrene excimers are formed upon collision of a photoexcited pyrene structure with pyrene in the ground state. Eximer fluorescence is at a longer wavelength than for the monomeric pyrene structure. The rate of the excimer formation in this assay is directly dependent on the translational diffusion rate of pyrenedecanoic acid dye molecules incorporated into the cell membrane prior to exposure to test substance(s). The ratio of excimer fluorescence to monomer fluorescence intensities (IE/IM) is proportional to membrane fluidity.

Aliquots of cell suspension (300 µL) were incubated with 100 µL pyrenedecanoic acid (30 µM in 0.1 M phosphate buffer, pH 7.4, with 0.03% ethanol) and 100 µL JC-1 (15 µM in DMSO. JC-1 is a mitochondrion-specific fluorescent dye not used in PFBSK+ analysis) for 15 minutes at 25 °C to incorporate pyrenedecanoic acid into cell membranes. Excess label was then removed by two washes with PBS, and final cell volume was adjusted to 1 mL. Test solution, solvent control, or reference substance (0.1% pentanol) were added. The article is not specific regarding the volume of test material added. It is assumed that aliquots of the 10 mM perfluoroalkylsulfonate stock solutions were added directly to the 1-mL cell suspension in volumes required to reach the intended final concentration (i.e., 0.1 µL, 0.3 µL, 1.0 µL, 3.0 µL, or 10 µL). Similarly, 10 µL pentanol would have been added to attain 1% final concentration. Suspensions were then analyzed using flow cytometry. At least 10,000 cells were counted in each sample. Fluorescence of pyrenedecanoic acid was determined using a FACS Vantage flow cytometer (Becton Dickinson, San Jose, CA USA) equipped with a 365 nm argon laser (excitation) and bypass filters of 400±15 nm (monomer emission) and 450±30 nm (eximer emission). Cell scattering was shown as contour plot of FSC (forward scatter) and SSC (side scatter) to screen for cells with the correct morphology. Fluorescence intensities were recorded as histograms with event number (cell count) vs. channel number (fluorescence intensity). The raw data from each histogram was extracted, and copied to a Microsoft Excel spreadsheets for further analysis. Total fluorescence intensity for each wavelength was calculated as sum of event number times channel number. Fluorescence ratios were calculated as the ratio of the total fluorescence intensities.

Reference substance (positive control):

yes

Remarks:

pentanol

Key result

Duration:

15 min

Dose descriptor:

NOEC

Effect conc.:

100 µmol/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: membrane fluidity

Remarks on result:

other: no effect at highest concentration tested

Details on results:

PFBSK+ had no effect at the highest concentration tested. The positive control (1% pentanol) and 100 µM perfluorooctane sulfonic acid (PFOS) each had roughly the same effect level (ratio of total fluorescence at 450 nm and 400 nm, ca. 1.15 v. 0.9 in solvent controls). Additional biochemical/pharmacological tests were done with PFOS, but not PFBSK+ or perfluorohexane sulfonate (neither showed an effect in the membrane fluidity test).

Reported statistics and error estimates:

Results were analyzed by ANOVA with Dunnett’s test using DMSO-exposed cells as control.

Conclusions:

In an in vitro study, PFBSK+ at up to 100 µM had no effect on carp leukocyte membrane fluidity after 15 minutes at 25 °C.

Executive summary:

In an in vitro study, impact of PFBSK+, perfluorooctanesulfonic acid (PFOS), and perfluorohexanesulfonate on carp leukocyte membrane fluidity was examined by flow cytometry. Fluidity was determined using pyrenedecanoic acid as a membrane-bound fluorescent dye. Increased membrane fluidity allows formation eximers by energy transfer between excited and ground-state pyrene moieties, with a longer-wavelength emission by the eximer relative to the excited pyrene unit (450 nm v. 400 nm). The ratio of total fluorescence at the two wavelengths is taken as in indicator of membrane fluidity. After labelling with the dye, cells were incubated at 25 °C for 15 minutes with up to 100 µM of each substance, or 1% pentanol as postive control. PFBSK+ at up to 100 µM had no effect on carp leukocyte membrane fluidity after 15 minutes at 25 °C.

The study follows valid scientific principals and was published in a peer reviewed scientific journal. However, the direct application of the data to short-term toxicity in fish is unclear. With further research such information may be incorporated into adverse outcome pathways. At present, the study is given a Klimisch 3 score due to the suitability of the test in meeting the data requirement.

Description of key information

The 96-hour LC50 of PFBSK+ to Pimephales promelas is 1938 mg/L from a study conducted according to EPA 850.1075 and OECD TG 203 guidelines.

Additional information

The key study was conducted in 2000 according to GLP and OECD 203 and EPA 850.1075 guidelines and is considered to be reliable without restriction. A supporting study from 2003 was conducted as a limit test with Danio rerio (zebrafish) according to EU method C.1 and reported a result of 96 -hour LC50 > 105 mg/L. This test was conducted under GLP according to an internationally accepted guideline and is considered to be reliable without restriction. Another supporting study, conducted in 2000 according to GLP and OECD 203 and EPA 850.1075 guidelines, reported a result of 96 -hr LC50 = 6452 mg/L with Lepomis macrochirus. This test was also conducted under GLP according to internationally accepted guidelines and is considered to be reliable without restriction.

In another study, published in 2003, the impact of PFBSK+ on carp leukocyte membrane fluidity was examined in-vitro by flow cytometry. With carp leucocytes incubated at 25 °C for 15 minutes, with up to 100 μM of PFBSK+, no effect on cell membrane fluidity was observed. The study follows valid scientific principals and was published in a peer reviewed scientific journal. However, the direct application of the data to short-term toxicity in fish is unclear. The study is given a Klimisch 3 score due to the lack of suitability of the test in meeting the data requirement.