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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jan 2001 - 2 Feb 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1035 (Mysid Acute Toxicity Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: ASTM Standard E729-88a Standard Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA, Standard Evaluation Procedure (Acute toxicity test for estuarine and marine organisms)

Principles of method if other than guideline:

Method based on draft version (as of 2001) of EPA OPPTS 850.1035

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:

- Concentrations: All; sampled at test initiation, 48 hours, and at test termination.
- Sampling method: Samples were collected at mid-depth from each replicate test chamber of each treatment and the control.
- Sample storage conditions before analysis: Samples were collected in plastic 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: Primary stock (1000 mg a.i./L) was prepared in dilution water, using an electronic mixer for approximately 22 minutes to aid in the solubilization of the test substance. All materials which came in contact with the test solution during formulation were made of plastic or stainless steel. Dilution of primary stock with dilution water to obtain lower concentrations.
- Controls: Blank test medium
- Evidence of undissolved material: All solutions appeared clear and colorless.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Mysid shrimp
- Source: Cultures maintained by the test laboratory with known history
- Age at study initiation: juveniles, from parental mysids of more than two weeks old.
- Culturing: Adult mysids were cultured in water from the same source and at approximately the same temperature as used during the test. Adult mysids in the cultures were held for at least 14 days prior to collection of the juveniles for testing. The adults showed no signs of disease or stress during the holding and acclimation periods. During the 14-day period preceding the test, water temperatures ranged from 24.6 to 25.9 °C. The pH of the water ranged from 8.0 to 8.2, dissolved oxygen ranged from 6.6 to 7.0 mg/L, and salinity ranged from 20 to 21 parts per thousand.
FEEDING DURING TEST
- Food type: fed live brine shrimp (Artemia sp.) nauplii daily during the test to prevent cannibalism

Test type:

static

Water media type:

brackish water

Remarks:

Natural seawater diluted to a salinity of approximately 20 parts per thousand with well water.

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23.0 - 25.5 °C, from continuous measurement during the test

pH:

8.0 - 8.3

Dissolved oxygen:

6.5 - 7.7 mg/L

Salinity:

20 parts per thousand for the 0-hour dilution water (negative control)

Nominal and measured concentrations:

Nominal: 0 mg/L, 31 mg/L, 63 mg/L, 125 mg/L, 250 mg/L, 500 mg/L, 1000 mg/L
Measured: < LOQ, 32 mg/L, 64 mg/L, 127 mg/L, 269 mg/L, 554 mg/L, 1071 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 2-L polyethylene containers 1500-mL fill volume, depth ca. 8.8 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
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural seawater collected at Indian River Inlet, Delaware, USA. Freshly collected seawater was passed through a sand filter and pumped into a 37800-L storage tank. The filtered seawater was diluted to a salinity of approximately 20 parts per thousand with well water from a 40-m deep well located at testing lab, and aerated with spray nozzles. For use, the aerated water was passed through a 0.45 µm filter to remove microorganisms and fine particles.
- Other: At test initiation, organisms were collected and transferred indiscriminately, one or two at a time, into the test chambers until each chamber contained 10 mysids.

Saltwater used for testing subject to periodic water analysis for metals, organics and pesticides, Attachments 1 and 2.
- Pesticides: all < LOQ
- 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, 159 lux at surface of representative chamber at test initiation
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality and other clinical sings of toxicity at approximately 4 h, 24 h, 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

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

372 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

95% CI, 314 - 440 mg a.i./L

Details on results:

- Mortality: See Table 2
- Mortality of control: All control organisms appeared normal during the test.

Reported statistics and error estimates:

LC50 and 95% CI calculated using the probit method.

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
	4 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
32	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
64	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
127	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
269	0 / 20 (20 AN)	0 / 20 (20 AN)	1 / 20 (19 AN)	3 / 20 (17 AN)	3 / 20 (17 AN)	15
554	1 / 20 (19 AN)	5 / 20 (13 AN, 1 E, 1 C)	9 / 20 (9 AN, 2 E)	13 / 20 (6 C, 1 E)	18 / 20 (2 C)	90
1071	1 / 20 (1 AN, 18 C)	20 / 20	20 / 20	20 / 20	20 / 20	100

¹Observations: AN = Appears Normal; C = Lethargic; E= Erratic Swimming

Validity criteria fulfilled:

yes

Remarks:

the validity criteria of EPA 850.1035 (finalized version) were fulfilled. There are five criteria for the method, and all were fulfilled for this test.

Conclusions:

The 96-hour LC50 of PFBSK+ to Americamysis bahia (previous name: Mysidopsis bahia) was 372 mg/L (EPA 850.1035, draft version at time of test; ASTM Standard E729-88a; EPA, Standard Evaluation Procedure (Acute toxicity test for estuarine and marine organisms).

Executive summary:

The acute toxicity of PFBSK+ to Mysidopsis bahia was examined in a 96-hour static test based on EPA 850.1035 (then-current draft version of method); ASTM Standard E729-88a; EPA, Standard Evaluation Procedure (Acute toxicity test for estuarine and marine organisms). The test medium was brackish water prepared from natural seawater diluted to a salinity of 20 parts per thousand with well water. Analytically determined concentrations were <LOQ, 32 mg/L, 64 mg/L, 127 mg/L, 269 mg/L, 554 mg/L, and 1071 mg/L. The 96 hour LC50 is 372 mg/L (95% CI, 314 - 440 mg/L).

The test was based on a draft version of an accepted test guideline, 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 aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15 - 25 Jan 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 test initiation, 24 hours, and at test termination.
- Sampling method: not described
- Sample storage conditions before analysis: Samples were prepared for analysis on the day the samples were collected, and stored within autosampler vials. Samples were analyzed on the day collected, or the following day.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stirring of primary stock (4000 mg a.i./L) by inversion and and stirring. Dilution of primary stock with dilution water to obtain lower concentrations.
- Controls: Blank test medium
- Evidence of undissolved material: All solutions appeared clear and colorless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Cladoceran; Water flea
- Source: Cultures maintained by the test laboratory with known history
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old, which were observed to have no neonates present less than 24 hours prior to test initiation
- Culturing: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. Adult daphnids in the cultures were held for at least 14 days prior to collection of the juveniles for testing. The adults showed no signs of disease or stress during the holding period. During the 14-day period preceding the test, water temperatures ranged from 19.1 to 20.2°C. The pH of the water ranged from 8.2 to 8.5, and dissolved oxygen ranged from 8.0 to 8.6 mg/L. Daphnids in the cultures were fed a mixture of yeast, Cerophyl®, and trout chow, as well as a suspension of the freshwater green alga, Selenastrum capricornutum. The adults were fed prior to test initiation, but neonates were not fed during the test.
FEEDING DURING TEST
- Food type: Not fed during test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

132 - 136 mg CaCO3/L

Test temperature:

19.5 - 20.5 °C, from continuous measurement during the test

pH:

7.9 - 8.5

Dissolved oxygen:

8.3 - 8.6 mg/L

Conductivity:

290 - 295 μmhos/cm

Nominal and measured concentrations:

Nominal: 0 mg/L, 250 mg/L, 500 mg/L, 1000 mg/L, 2000 mg/L, 4000 mg/L
Measured: < LOQ, 234 mg/L, 470 mg/L, 886 mg/L, 1707 mg/L, 3767 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300-mL polyethylene beakers 225-mL fill volume, depth ca. 5.5 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
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: 184 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, 144 and 143 lux at surface of representative chamber at test initiation and termination, respectively
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality and immobilization at approximately 4 h, 24h, and 48 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

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2 183 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks:

The study reported dead and immobilized daphnids separately. For the purpose of calculating the EC50, these effects are combined.

Remarks on result:

other: 95% CI, 1707 - 3767 mg a.i./L

Details on results:

- Mobility: See Table 2
- Mobility of control: At 48 hours, one daphnid was immobilized (5%), all other control organisms appeared normal during the test.

Reported statistics and error estimates:

EC50 and 95% CI calculated using binomial probability method with nonlinear interpolation.

Table 2, Cumulative percent mortality/immobility and treatment-related effects

Mean measured concentration (mg/L)	Cumulative # Immobilized and/or Dead/ # Originally Exposed (Observations)¹	Cumulative # Immobilized and/or Dead/ # Originally Exposed (Observations)¹	Cumulative # Immobilized and/or Dead/ # Originally Exposed (Observations)¹	Cumulative percent immobilized (and/or Dead)
	4 hours	24 hours	48 hours
Negative Control	0 / 20 (20 AN)	0 / 20 (20 AN)	1 / 20 (19 AN, 1 D)	5
234	0 / 20 (20 AN)	0 / 20 (20 AN)	1 / 20 (19 AN, 1M)	5
470	0 / 20 (20 AN)	0 / 20 (20 AN)	0 / 20 (20 AN)	0
886	0 / 20 (20 AN)	0 / 20 (20 AN)	1 / 20 (19 AN, 1 M)	5
1707	0 / 20 (20 AN)	0 / 20 (20 AN)	4 / 20 (16 AN, 3 D, 1 M)	20
3767	20 / 20 (20 D)	20 / 20 (20 D)	20 / 20 (20 D)	100

¹ Observations: AN = Appears Normal; D = Dead; M = Immobilized

Validity criteria fulfilled:

yes

Remarks:

the validity criteria of OECD TG 202 were fulfilled. Control immobilization was < 10% at the end of the test. The dissolved oxygen concentration was ≥ 3 mg/L in the control and test vessels at the end of the test.

Conclusions:

The 48-hour EC50 of PFBSK+ to Daphnia magna was 2183 mg/L (EPA 850.1010 and OECD TG 202).

Executive summary:

The 48-hour EC50 of PFBSK+ to Daphnia magna was examined in a static test conducted according to EPA 850.1010 and OECD TG 202. Analytically determined concentrations were < LOQ, 234 mg/L, 470 mg/L, 886 mg/L, 1707 mg/L, and 3767 mg/L. No statistically significant levels of immobilization were observed at 234, 470, and 886 mg/L; 20% cumulative immobilization was observed at 1707 mg/L, and 100% cumulative immobilization was observed at 9433 mg/L. The 48-hour EC50 is 2183 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 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 - 27 Mar 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC Part C, Method 2 'Acute toxicity for Daphnia'

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

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 and 48 hours (all concentrations). Samples for the 0 hour time point were stored in a refrigerator at 4°C for 24 hours before analysis. Other samples were analyzed the day of sampling. Test samples were analyzed in same way as analytical samples.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 105.2 mg of the test item were added to 1 liter of dilution water and treated for 1 hour in an ultrasonic bath.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea (Daphnia magna STRAUS, parthenogenetic females)
- Source (laboratory, culture collection): Strain dreived from Bundesgesundheitsamt, Berlin, Germany.
- Maintenance and acclimatisation: A population of parthenogenetic females of synchronized age structure had been maintained for more than 15 years in the test facility under constant temperature conditions (20 +1- 1 °C) at a 16 : 8 hour light-dark photoperiod (illumination: < 1000 lux). M4 medium was partly renewed once a week. The Daphnia were exclusively fed with unicellular green algae (Desmodesmus subspicatus) ad libitum. Mortalities of parent Daphnia during the culture period were recorded daily in a semi-quantitative way. The neonates were separated from their parent Daphnia by filtration prior to the acute test.
FEEDING DURING TEST
- Food type: Not fed during test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

258.83 mg/L CaCO3 at test start

Test temperature:

19.6 °C at test termination

pH:

7.8 at test termination

Dissolved oxygen:

90% - 94% of saturation at test termination

Nominal and measured concentrations:

Nominal: 0 mg/L, 100 mg/L
Measured (mean): < 5 mg/L (LOQ), 108.5 mg/L at 0 hours, 105 mg/L at 48 hours

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL glass beakers, 20 mL fill volume
- Aeration: none
- No. of organisms per vessel: 10
- No. of vessels per concentration: two
- No. of vessels per control: two
GROWTH MEDIUM
- Standard medium used: M4 medium
OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 16 h:8h
- Light intensity: < 1000 lux
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): observations made on swimming ability and immobilization at 24h, and 48h
TEST CONCENTRATIONS
-Limit Test, 0 mg/L, 100 mg/L

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Remarks:

(original study report reported result as EC0)

Effect conc.:

>= 106.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

no immobilization of test organisms observed in control and test solutions

Details on results:

No evidence of toxicity to Daphnia magna was observed in a limit test after 48 hours of exposure at 100 mg/L (nominal), 106.8 mg/L (mean measured) concentration of PFBSK+.

Reported statistics and error estimates:

none

Validity criteria fulfilled:

yes

Remarks:

the validity criteria of OECD TG 202 were fulfilled. Control immobilization was < 10% at the end of the test. The dissolved oxygen concentration was ≥ 3 mg/L in the control and test vessels at the end of the test.

Conclusions:

The 48-hour EC50 of PFBSK+ to Daphnia magna was ≥ 106.8 mg/L (EEC Annex to Directive 92/69/EEC Part C, Method 2 'Acute toxicity for Daphnia').

Executive summary:

The 48-hour EC50 of PFBSK+ to Daphnia magna was examined in a static test conducted according to EEC Annex to Directive 92/69/EEC Part C, Method 2 'Acute toxicity for Daphnia', and equivalent to OECD TG 202. The limit test was conducted with a negative control, and at 100 mg/L nominal concentration with analytically determined concentrations of < LOQ, and 106.8 mg/L. No immobilization was observed at the tested concentrations. The 48-hour EC50 is ≥ 106.8 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 aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Remarks:

Xenobiotic efflux pump activity

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Effect of substance on xenobiotic active transport enzyme.
- Short description of test conditions: Tissue samples were treated with test substance, followed by exposure to a fluorescent dye normally excluded from tissues.
- Parameters analysed / observed: Fluorescence of dye in lyzed tissue samples.

GLP compliance:

no

Specific details on test material used for the study:

Factor purity 88%

Analytical monitoring:

yes

Remarks:

test substance analysis for uptake experiment only

Details on sampling:

Tissue accumulation was studied in a test independant of xenobiotic transport systems. Whole gills were incubated in 10 mL filtered sea water (FSW) with the substance of interest added in a small volume of DMSO (DMSO concentration was always <0.25%) at 12 °C with gentle agitation.
- Concentrations: 100 nM
- Sampling method/storage before analysis: After exposure rinsed vigorously three times in 500 mL FSW. Whole gills were frozen at -80 ° until analysis. No separate sampling step.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Implicitly, test substances and rhodamine B dye stock solutions were dissolved in DMSO. No more than 0.5% of exposure solution was DMSO.
- Controls: Solvent, Verapamil as positive control.
- Other test substances:
Perfluoropentanoic acid (PFPnA, 99.1%)
Perfluorohexanoic acid (PFHxA, 98.8%)
Perfluoroheptanoic acid (PFHpA, 99.8%)
Perfluorooctanoic acid (PFOA, 99.9%)
Perfluorononanoic acid (PFNA, 99.2%)
Perfluorodecanoic acid (PFDA, 99.9%)
Perfluoroundecanoic acid (PFUnA, 102.2%)
Potassium perfluorohexanesulfonate (PFHxS, 97%)
Potassium perfluorooctanesulfonate (PFOS, 98%)
Ammonium perfluorodecanesulfonate (PFDS, 25%)

Test organisms (species):

other aquatic mollusc: Mytilus californianus

Details on test organisms:

TEST ORGANISM
- Common name: Marine mussel
- Strain/clone: wild caught
- Justification for species other than prescribed by test guideline: Species known to have high activity of enzyme transport system of interest
- Source: Rocky intertidal zone at Hopkins Marine Station, Pacific Grove, CA
- Size at capture: Valve length range: 60-85 mm

QUARANTINE (wild caught)
- Duration: Running seawater (12 °C) for 1-5 days before experiments.

Test type:

static

Water media type:

saltwater

Limit test:

yes

Total exposure duration:

90 min

Test temperature:

12 °C

Nominal and measured concentrations:

Nominal concentration only for PFBSK+ and other substances, 50 µM

Details on test conditions:

Gill tissue was excised from mussels for use in the test. A biopsy punch was used to obtain uniform, 5-mm disks for the transporter activity tests. Rhodamine B is a substrate for p-glycoprotein and is used as a marker for xenobiotic efflux transporter activity. Inhibition of the transporter results in increased fluorescence of rhodamine B within the cell. Test solutions were prepared in polystyrene dishes with 5 mL filtered seawater (FSW), 1 μM rhodamine B, and test chemicals or solvent only (0.5% DMSO). Gill tissue disks were incubated with rhodamine B and a test chemical for 90 min at 12°C, washed twice in FSW, immediately frozen on dry ice, and stored at -35°C until analysis. The amount of rhodamine B in the tissue was determined by extracting 3 discs per tested chemical in 200 μL of hypotonic lysis buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM Tris HCl, pH 7.4) by sonication. The homogenate of 3 combined discs was centrifuged at 13,000 × g for 6 min, and the supernatants were halved into separate, black 96-well microplates for measurement in a fluorescence microplate reader (CytoFluor II, PerSeptive Biosystems, Framingham, MA; emission: 590 nm, excitation: 530 nm). Standard test-substance concentration was 50 µM.

In additional experiments involving related test substances, p-gp activity in a set of tissue disks, each taken from the same individual, was measured immediately or 24 or 48 hours after a 2-hour exposure to one of four substances: 50 μM PFNA, 50 μM PFDA, 10 μM verapamil (positive control), or 0.5 % DMSO (negative control). Levels of p-gp enzyme were measured using a Western blot technique on cellular extracts taken at the same time points under the same test conditions. The activity assay was done three times and the Western blot was done twice.

Reference substance (positive control):

yes

Remarks:

verapamil

Duration:

90 min

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: fluorescence

Remarks on result:

not determinable

Remarks:

No effect at only concentration tested (50 µM)

Details on results:

PFBSK+ neither caused statistically significant inhibition of the p-glycoprotein efflux transporter at a nominal concentration of 50 µM or notable accumulation in gill tissues at 100 nM. In contrast, other perfluoroalkyl acid substances, C8, C9, and C10 carboxylates (PFOA, PFNA, PFDA), as well as perfluorohexanesulfonate (PFHxS), caused p-gp inhibition, although PFOS did not. There was no clear correlation between the extent of tissue accumulation and the magnitude of the effect on the transporter. Exposure to PFNA and PNDA caused an immediate inhibition of p-gp activity; however, inhibition had recovered by 24 hours post exposure. Inhibition by the positive control had not significantly declined at 24 hours post exposure. Conversely, levels of p-gp protein in tissues 48 hours after exposure to PFNA were elevated 50% relative to control. Response of p-glycoprotein levels to PFDA was not consistent, and no information was presented on response of enzyme expression to verapamil.

Reported statistics and error estimates:

Transporter activities in treated gill discs were compared to untreated controls for each mussel using Dunnett’s test (JMP software, version 6, SAS Institute Inc., Cary, NC).

Validity criteria fulfilled:

not applicable

Conclusions:

PFBSK+ at 50 µM did not have an effect on p-glycoprotein, a xenobiotic efflux pump, in Mytilus californianus gill tissues, and was not accumulated to high levels therein. This contrasts with several other perfluoroalkyl acid substances (perfluorohexane sulfonate, perfluorooctanoic acid, perfluorononanoic acid, and perfluorodecanoic acid).

Executive summary:

The p-glycoprotein is an active transport enzyme (efflux pump) in the multxenobiotic resistance mechanism of the marine mussel Mytilus californianus. Substances that inhibit the system are considered to be chemosensitizers, since they increase the toxicity of other substances that would otherwise be transported from cells and prevented from exerting their effects. p-Glycoprotein (p-gp) activity was measured using Rhodamine B exclusion. Tissue samples from M. californianus gill were exposed to several perfluoroalkyl acid substances (PFAAs), including PFBSK+, concurrently with Rhodamine B dye, after which fluorescence of the dye was measured in tissue lysates. Treatment with PFBSK+ at 50 µM for did not inhibit p-gp activity gill tissues, although several other PFAAs showed effects. These effects did not clearly correlate with tissue levels, and PFBSK+ did not accumulate to notable levels when tissues were exposed at 100 nM. While p-gp activity after exposure to the two most efficacious of these substances (perfluorononanoic acid, perfluorodecanoic acid) had recovered within 24 hours, expression of p-glycoprotein as measured by a Western blot increased to 150% of control at 48 hours after perfluorononanoic acid exposure. Other PFAAs are considered chemosensitizers. PFBSK+ does not share these effects and is not considered a chemosensitizer.

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 invertebrates 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 Americamysis bahia (previous name: Mysidopsis bahia) was 372 mg/L, as determined in a test based on EPA 850.1035 (draft version at time of test).

Additional information

In the key study, the acute toxicity of PFBSK+ to Mysidopsis bahia in brackish water was examined in a 96-hour static test based on EPA 850.1035 (then-current draft version of method); ASTM Standard E729-88a; and EPA, Standard Evaluation Procedure (Acute toxicity test for estuarine and marine organisms). The test was conducted in 2001, based on a draft version of an accepted test guideline, it was GLP compliant, and is considered reliable without restriction. Two supporting studies were conducted using Daphnia magna. A study from 2001 measured a 48-hr EC50 of 2183 mg/L, while a limit test from 2003 resulted in a 48-hr EC50 of ≥ 106.8 mg/L. The tests were carried out according to EPA 850.1010 and EEC Annex to Directive 92/69/EEC Part C, Method 2 'Acute toxicity for Daphnia', respectively. The tests were GLP compliant and conducted according to internationallyaccepted test guidelines, therefore they are considered reliable without restriction.

Another study, reported in 2006, examined the effect of PFBSK+ on p-glycoprotein from the marine mussel, Mytilus californianus. P-glycoprotein is an active transport (efflux pump ) enzyme for xenobiotic compounds, and substances which inhibit the enzyme are considered to be chemosensitizers which may increase the toxicity of other substances. Gill tissue samples from the organism were exposed to PFBSK+ and inhibition of the enzyme was measured. PFBSK+ at 50 μM did not have an effect on p-glycoprotein in Mytilus californius gill tissues, and it was not accumulated to high levels in the tissues. The study followed valid scientific principals and was published in a peer reviewed scientific journal. However, the direct application of the data to short-term toxicity in invertebrates is unclear and the study is given a Klimisch 3 score due to the suitability of the test in meeting the data requirement.