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

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jul - 31 Aug 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1730 (Fish Bioconcentration Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 41-2600-8442-5, lot #2, TCR-000 17-7 1
- Expiration date of the lot/batch: 17 Jan 2002
- Purity: 97.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient conditions
- Stability under test conditions: stable

Details on sampling:

- Sampling intervals/frequency for test organisms: Tissues samples were collected on days 0 (0 and 4 hours), 1, 3, 7, 14, 21, and 28 during the uptake phase of the test, and on days 1, 3, 7, 10 and 14 of the depuration phase.
- Sampling intervals/frequency for test medium samples: Water samples were collected on days -2 and -1 during the pre-uptake phase of the test, on days 0 (0 and 4 hours), 1, 3, 7, 14, 21, and 28 during the uptake phase of the test, and on days 1, 3, 7, 10 and 14 of the depuration phase.
- Sample storage conditions before analysis: Tissue samples were extracted immediately, or stored frozen at -14 °C. Water samples were analyzed as soon as possible after collection without storage.
- Details on sampling and analysis of test organisms and test media samples:

Water samples: All water samples were collected from mid-depth of each test chamber using a glass pipette. At each water sampling interval, two water samples were collected from the negative control and three samples were collected from each of the two PFBS treatment groups. One negative control sample and two samples from each of the PFBS treatment groups were analyzed for PFBS. The remaining samples were held in reserve as backup samples.

Tissue samples: Fish were impartially removed from the test chambers and euthanized by severing the spinal cord above the opercular region. A sufficient number of fish were collected to provide two replicate samples of negative control fish and four replicate samples of each PFBS treatment group. The fish were blotted dry and measured for total length and wet weight within approximately 15 minutes of collection, when possible. Each fish was then rinsed with dilution water, blotted dry again and dissected into edible and nonedible tissue fractions. Dissection was accomplished by making an incision from just posterior to the base of the pectoral fin dorsally through the spinal cord. The head, fins and viscera were removed from the body and were considered to be nonedible tissue. The remaining tissue was considered the edible tissue. Tissue samples were transferred to tared scintillation vials and weighed prior to extraction.

Vehicle:

no

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Overhead mixing. Stock solutions were made at approximately weekly intervals during the uptake phase of the test. The stock solutions were injected into the diluter mixing chambers (at a rate of 0.350 mL/minute) where they were mixed with dilution water (at a rate of 350 mL/minute) to achieve the desired test concentrations.
- Controls: Medium blank only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc): After mixing, the stock solutions appeared clear and colorless.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Source: Osage Catfisheries, Osage Beach, Missouri, USA
- Length at study termination: 55 mm (range 50-61 mm) for 10 negative control fish
- Weight at study termination: 1.81 g (range 1.37-2.76) for 10 negative control fish
- Health status: During the holding and acclimation periods prior to testing the fish showed no signs of disease or stress.
- Description of housing/holding area: Test chambers were 106-L stainless steel aquaria filled with approximately 80 L of test solution. The depth of the test water in a representative chamber was approximately 19 cm. Test chambers were indiscriminately positioned in a temperature-controlled water bath designed to maintain a constant temperature.
- Feeding during test
- Food type: Flake food and brine shrimp nauplii
- Frequency: Once daily

ACCLIMATION
- Acclimation period: 48 hours (following 20-day holding period)
- Acclimation conditions (same as test or not): yes
- Type and amount of food: none during acclimation, daily during holding

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

16 d

Hardness:

119 (104 – 128 mg/L)

Test temperature:

21.9 – 22.1 °C

pH:

8.0 - 8.5

Dissolved oxygen:

7.4 - 8.8 mg/L

Conductivity:

324 (300 – 330 umhos/cm)

Nominal and measured concentrations:

Nominal: negative control, 0.50 mg/L, 5.0 mg/L
Mean measured:

Reference substance (positive control):

no

Conc. / dose:

0.53 mg/L

Temp.:

22 °C

pH:

8.1

Type:

BCF

Value:

0.38 dimensionless

Basis:

whole body w.w.

Time of plateau:

7 d

Calculation basis:

steady state

Conc. / dose:

0.53 mg/L

Temp.:

22 °C

pH:

8.1

Type:

BCF

Value:

1.1 dimensionless

Basis:

whole body w.w.

Time of plateau:

4.2 d

Calculation basis:

kinetic

Conc. / dose:

5.2 mg/L

Temp.:

22 °C

pH:

8.1

Type:

BCF

Value:

0.3 dimensionless

Basis:

whole body w.w.

Time of plateau:

3 d

Calculation basis:

steady state

Conc. / dose:

5.2 mg/L

Temp.:

22 °C

pH:

8.1

Type:

BCF

Value:

0.36 dimensionless

Basis:

whole body w.w.

Time of plateau:

7 d

Calculation basis:

kinetic

Elimination:

yes

Parameter:

DT50

Remarks:

whole fish

Depuration time (DT):

1.3 d

Remarks on result:

other: 0.53 mg/L test concentration

Elimination:

yes

Parameter:

DT50

Remarks:

whole fish

Depuration time (DT):

2.1 d

Remarks on result:

other: 5.2 mg/L test concentration

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Value:

0.6

Remarks on result:

other: 0.53 mg/L test concentration

Rate constant:

overall depuration rate constant (d-1)

Value:

0.54

Remarks on result:

other: 0.53 mg/L test concentration

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Value:

0.12

Remarks on result:

other: 5.2 mg/L test concentration

Rate constant:

overall depuration rate constant (d-1)

Value:

0.33

Remarks on result:

other: 5.2 mg/L test concentration

Details on kinetic parameters:

- Computation / data analysis: BIOFAC model was used to estimate kinetic parameters

Details on results:

- Mortality of test organisms: One mortality at 5.2 mg/L test concentration.
- Behavioural abnormalities: None
- Observations on feeding behavior: No effect
- Observations on body length and weight: No effect
- Reproduction during test period: No
- Mortality and/or behavioural abnormalities of control: None
- Loss of test substance during test period: No

Table 1, Concentrations in Water Samples During the Uptake Phase

Nominal concentration	Concentration on day of study (mg/L)
(mg/L)	0 (0 hours)	0 (4 hours)	1	3	7	14	21	28	Mean	% nominal
Negative Control	< LOQ	< LOQ	<LOQ	<LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
0.5	0.515	0.519	0.478	0.542	0.525	0.519	0.593	0.515	0.53	106
	0.518	0.525	0.49	0.542	0.559	0.514	0.567	0.516
5	5.24	5.35	5.02	5.08	5.17	5.36	5.04	5.45	5.2	104
	5.42	5.43	5.12	5.12	5.22	5.32	5.21	5.4

The Limit of Quantitation (LOQ) was 0.125 mg/L.

Concentrations in Individual Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 0.53 mg/L

	Edible tissue conc (mg/kg)	Edible tissue wt (g)	Non-edible tissue conc (mg/kg)	Non-edible tissue wt (g)	Whole fish conc (mg/kg)
0 (4 h)	<0.0530	0.9446	<0.0630	1.2556	<LOQ
0 (4 h)	<0.0585	0.8512	<0.0729	1.0863	<LOQ
0 (4 h)	<0.0393	1.2723	<0.0439	1.8064	<LOQ
0 (4 h)	<0.0590	0.8483	<0.0749	1.0570	<LOQ
1	0.0382	1.4848	0.0786	1.6591	0.0595
1	<0.0428	1.1682	0.0894	1.5313	<LOQ
1	<0.0915	0.5462	0.112	0.7838	<LOQ
1	<0.0650	0.7672	0.0730	1.1590	<LOQ
3	0.0874	0.9636	0.180	1.2489	0.140
3	0.0636	2.0468	0.138	2.1180	0.101
3	0.0911	0.6166	0.173	0.8193	0.138
3	0.0704	0.9082	0.141	1.3600	0.113
7	0.116	1.2395	0.265	1.0629	0.185
7	0.136	0.7690	0.293	0.6806	0.210
7	0.101	1.2201	0.263	0.9194	0.171
7	0.133	1.0375	0.307	1.0075	0.219
14	<0.0880	0.5688	0.235	0.8077	<LOQ
14	<0.0715	0.6971	0.181	1.1184	<LOQ
14	0.0869	1.2892	0.236	1.6895	0.171
14	0.105	0.5636	0.179	1.0733	0.154
21	0.136	1.4931	0.341	1.6291	0.243
21	0.125	1.2111	0.314	1.5265	0.230
21	0.213	0.9709	0.579	1.2477	0.419
21	0.114	0.9750	0.323	1.1193	0.226
28	0.0682	0.9166	0.185	1.2116	0.135
28	0.103	1.3823	0.262	1.7309	0.191
28	0.0619	0.9593	0.153	1.4567	0.117
28	0.0871	0.8614	0.243	1.1446	0.176
Depuration day
1	1.19	0.9400	0.207	1.4752	0.590
1	3.31	0.8289	1.15	1.3630	1.97
1	0.209	0.8374	0.537	1.3587	0.412
1	3.98	0.7705	6.31	1.1058	5.35
3	0.0758	1.2572	0.156	1.5573	0.120
3	0.0571	1.2397	0.145	1.5719	0.106
3	<0.0341	1.4670	<0.0457	1.7321	<LOQ
3	<0.0488	1.0255	0.0934	1.3102	<LOQ
7	<0.0401	1.2473	0.092	1.4536	<LOQ
7	<0.0476	1.0515	<0.0515	1.5374	<LOQ
7	<0.0414	1.2077	<0.0562	1.4110	<LOQ
7	<0.0915	0.5453	<0.0982	0.8047	<LOQ
10	<0.0459	1.0910	<0.0634	1.2482	<LOQ
10	0.108	1.0374	<0.0620	1.2777	<LOQ
10	<0.0580	0.8632	<0.0645	1.2273	<LOQ
10	<0.0595	0.8421	<0.0755	1.0490	<LOQ
14	<0.0499	1.0021	0.0796	1.3124	<LOQ
14	<0.0520	0.9644	<0.0543	1.4587	<LOQ
14	<0.0630	0.7932	<0.0808	0.9847	<LOQ
14	<0.0364	1.3735	<0.0490	1.6148	<LOQ

Concentrations in Individual Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 5.2 mg/L

Uptake day	Edible tissue conc (mg/kg)	Edible tissue wt (g)	Non-edible tissue conc (mg/kg)	Non-edible tissue wt (g)	Whole fish conc (mg/kg)
0 (4 h)	0.127	0.8480	0.233	1.0907	0.187
0 (4 h)	0.0975	0.8819	0.173	1.2291	0.141
0 (4 h)	0.0811	1.1497	0.178	1.3879	0.134
0 (4 h)	0.127	0.6880	0.247	0.9672	0.197
1	0.240	0.8780	0.726	1.2479	0.525
1	0.273	0.9052	0.730	1.1803	0.532
1	0.311	0.5573	0.702	0.8573	0.548
1	0.234	0.8420	0.598	1.3474	0.458
3	0.675	1.2035	1.49	1.2976	1.10
3	0.487	0.8812	1.35	1.1688	0.979
3	0.611	1.4409	2.08	1.4651	1.35
3	0.448	1.4412	1.29	1.4124	0.865
7	1.07	1.6347	3.10	1.1526	1.91
7	1.19	1.6604	2.95	1.2130	1.93
7	0.701	1.4695	2.04	1.2203	1.31
7	1.50	0.9706	3.68	0.8058	2.49
14	0.438	0.6935	0.960	1.2983	0.778
14	1.12	0.9984	3.28	1.2129	2.31
14	1.15	0.7593	3.42	1.0165	2.45
14	0.748	2.1590	2.22	2.3433	1.51
21	0.596	1.0974	1.56	1.3836	1.13
21	0.593	1.1861	1.66	1.6043	1.21
21	1.50	1.1840	4.06	1.4418	2.91
21	0.590	1.3253	1.64	1.6631	1.17
28	0.764	1.1961	1.66	1.4016	1.25
28	0.907	0.7393	2.02	1.1143	1.58
28	0.565	0.9953	2.05	1.3411	1.46
28	0.837	0.8194	2.22	1.2026	1.66
Depuration day
1	0.718	1.1689	2.09	1.4841	1.49
1	0.622	0.8329	2.32	1.1449	1.61
1	1.05	0.8862	3.29	1.1917	2.34
1	0.467	0.5526	1.17	1.1047	0.922
3	0.235	1.0013	0.676	1.4174	0.493
3	0.197	0.8749	0.529	1.2464	0.392
3	0.601	1.5729	1.57	1.8050	1.12
3	0.413	1.1804	1.36	1.6609	0.967
7	0.196	1.2959	0.691	1.5717	0.467
7	0.151	0.9239	0.530	1.0888	0.356
7	0.124	0.9014	0.330	1.0053	0.233
7	<0.0879	0.5686	<0.0808	0.9801	<LOQ
10	0.0791	1.0421	0.181	1.1765	0.133
10	<0.0740	0.6779	<0.0800	0.9876	<LOQ
10	<0.0815	0.6133	<0.0887	0.8965	<LOQ
10	<0.0930	0.5384	<0.0903	0.8802	<LOQ
14	<0.0410	1.2193	<0.0494	1.6027	<LOQ
14	<0.0454	1.1015	<0.0602	1.3159	<LOQ
14	<0.0685	0.7300	<0.0773	1.0248	<LOQ
14	<0.0705	0.7080	<0.0847	0.9371	<LOQ

Validity criteria fulfilled:

yes

Conclusions:

PFBSK+ does not bioaccumulate in fish (kinetic BCF 1.1 in an OECD 305 test)

Executive summary:

PFBSK+ bioconcentration was examined in a test conducted under OECD 305 guidelines. Fish (Lepomis macrochirus) were exposed for 28 days with a 14-day depuration period. Kinetic constants were estimated using BIOFAC. Average measured test substance concentrations were 0.53 mg/L and 5.2 mg/L. Whole-organisms steady-state BCFs were ca 0.3 and were attained in ≤ 7 days. Kinetic BCF values were 0.36 at 5.2 mg/L and 1.1 at 0.53 mg/L. BIOFAC estimates for time to reach 50% clearance were 1.3 days at 0.53 mg/L and 2.9 days at 5.2 mg/L. PBSFK+ does not bioaccumulate in fish.

This study was conducted under internationally accepted test guidelines under GLP. It is considered reliable without restrictions and is suitable for Risk Assessment, Classification & Labelling, and PBT Analysis.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test

Version / remarks:

No guideline cited, publication describes a flow-through method

GLP compliance:

no

Specific details on test material used for the study:

Potassium perfluorobutane sulfonate standard at 99.9% purity

Details on sampling:

- Sampling intervals/frequency for test medium samples: During the uptake period, at 0.25, 4.5, 12, 18, 36, 72, 144, 197, 244, and 288 h. During depuration period, at 48 and 96 hours.
- Sampling intervals/frequency for test organisms: During uptake period, at 4.5, 9, 18, 36, 72, 144, and 288 hours. During depuration period, at 4.5, 9, 18, 36, 72, 144, 288, 456, and 792 hours. Three fish were sampled from exposed tanks and one from controls.
- Sample storage conditions before analysis: Samples analyzed when collected

- Details on sampling and analysis of test organisms and test media samples:
Medium samples were analyzed directly after filtration through a 0.2 µm nylon syringe filter.

Sampled fish were anesthetized with MS-222, a blood sample (50–200 ml) was drawn, and fish were subsequently euthanized. An incision
was made along the ventral surface from the anus to the gills, and the entire liver was removed for analysis. The GI tract was removed but not analyzed. The blood, liver, and carcass samples were analyzed separately at each sampling time to determine the kinetics of uptake and depuration.

In addition, immature rainbow trout (30–48 g) were exposed for 12 days under the same conditions. Three fish were sampled for tissue distribution analysis. Blood samples were drawn under anesthesia, after which the fish were euthanized and dissected. Tissues collected were spleen, heart, liver, gall bladder, gonads, gills (bones removed), adipose tissue (separated from pyloric ceca), gut (including esophagus, stomach, intestine, and pyloric ceca), and kidney. A small sample of white muscle was also removed adjacent to the dorsal fin of each fish and separated from the skin.

Liver and blood samples were homogenized in 15-ml plastic (polypropylene copolymer) centrifuge tubes containing 3 mL of Na2CO3 (0.25 M), 1 ml of water, 1 ml of the ion-pairing agent tetrabutylammonium hydrogensulfate (TBAS, 0.5 M adjusted to pH 10), and 100 µl (25 ng) of the internal standard perfluorononanoic acid (PFNA). Other tissue samples were ground under liquid nitrogen to a fine powder and then homogenized in 10-20 mL NaCO3. An aliquot of the homogenate was then transferred to a separate tube containing 1 ml of water, 1 ml of TBAS sulfate, and 100 µl of PFNA before extraction.

For extraction, homogenates were shaken vigorously for 10 minutes with 5 mL methyl-t-butyl ether (MTBE), followed by centrifugation to separate the organic phase. The upper MTBE layer was removed to a separate tube and the extraction was repeated, combining supernatants. The solvent was then removed under a stream of high-purity nitrogen gas, and the analytes were taken up in 1 to 2 ml of 50:50 (v/v) water:methanol. Solutions were filtered through a 0.2 µm nylon syringe filter before analysis.

Vehicle:

no

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solution contained several perfluorinated carboxylates and sulfonates, each at ca. 2 µg/L nominal concentration. All substances were dissolved in a small amount of methanol and diluted in 30L of reverse osmosis-purified water. A precipitate was removed by filtration with glass microfiber (GF/C) filters.
- Controls: water
- Chemical name of vehicle: None

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Rainbow Springs (Thamesford, ON, Canada)
- Weight at study initiation: 5-10 g, mean initial mass: 7.3 g (exposed), 7.9 g (control)
- Feeding during test: daily at a rate of 1.5% body weight per day, corrected for growth throughout the experiment by checking weight of total biomass every two to three days.
- Food type: Trout feed from Martin Mills (Tavistock, ON, Canada).

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): yes

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

other: dechlorinated and carbon-filtered tap water

Total exposure / uptake duration:

12 d

Total depuration duration:

33 d

Test temperature:

12 °C

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria lined with polypropylene bags, 45-L fill volume
- Type of flow-through (e.g. peristaltic or proportional diluter): Dilution water was gravity fed to each aquarium at 500 mL/min, and a peristaltic pump delivered the stirred stock solution into the dilution stream of the treatment tank at 0.5 mL/min.
- No. of vessels per concentration: one
- No. of vessels per control / vehicle control: one
- Biomass loading rate: 8g/L in exposure vessel, 2.5 g/L in control vessel
- Other: Fish growth was monitored by weighing the total biomass every 2 to 3 d throughout the course of the experiment.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 h

Nominal and measured concentrations:

Nominal concentration: ca. 2 µg/L
Measured concentration: 1.4 ± 0.14 µg/L

Key result

Remarks on result:

not determinable

Remarks:

PBSFK+ could not be detected in tissue samples

Details on results:

- Mortality of test organisms: 1 mortality (2%) in exposed fish
- Observations on body length and weight: Growth rate 4.9 mg/d
- Other biological observations: Liver somatic index (LSI) 1.1 ± 0.03%
- Mortality and/or behavioural abnormalities of control: No mortalities in control fish, growth rate 5.1 mg/d, LSI 1.1 ± 0.07%. Controls health was essentially the same as exposed fish health.

Validity criteria fulfilled:

not applicable

Conclusions:

PFBSK+ could be detected in exposure water but not exposed organisms in a bioconcentration experiment. PFBSK+ is not expected to bioconcentrate in fish

Executive summary:

PFBSK+ bioconcentration potential was assessed in a flow-through study with simultaneous exposure to several perfluorinated carboxylic and sulfonic acids. Rainbow trout (Oncorhynchus mykiss) were exposed at a nominal concentration of ca. 2 µg/L of each acid. Mean measured concentration of PFBSK+ was 1.4 ± 0.14 µg/L in the test medium. PFBSK+ could not be detected in exposed organisms.  PFBSK+ is not expected to bioconcentrate in fish.

This study did not follow an explicit guideline, but followed acceptable scientific principles including analytical determination of media concentrations. The results were published in a peer-reviewed journal. The study is deemed reliable with restrictions and is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test

Version / remarks:

No guideline cited, publication describes a feeding method

GLP compliance:

no

Specific details on test material used for the study:

Potassium perfluorobutane sulfonate standard at 99.9% purity

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test medium samples: No details available on sampling of treated food. Aquarium water was sampled to verify lack of contamination on day 30 of exposure (of 34).
- Sampling intervals/frequency for test organisms: During uptake period, on days 4, 7, 14, 21, 28, and 34. During depuration period, on days 7, 14, 21, 28, 34, and 41. Three fish were sampled from exposed tanks and one from controls except on uptake day 4, on which six exposed and two control fish were taken. Fish were always sampled ca. 24-hours after final feeding.
- Sample storage conditions before analysis: Samples analyzed when collected.
- Details on sampling and analysis of test organisms and test media samples:
No details are available on analysis of treated food.

Sampled fish were anesthetized with MS-222 and euthanized. An incision was made along the ventral surface from the anus to the gills, and the entire liver was removed for analysis. The GI tract was removed but not analyzed. The remaining carcass samples were analyzed separately.

Liver samples were homogenized in 15-ml plastic (polypropylene copolymer) centrifuge tubes containing 3 mL of Na2CO3 (0.25 M), 1 ml of water, 1 ml of the ion-pairing agent tetrabutylammonium hydrogensulfate (TBAS, 0.5 M adjusted to pH 10), and 100 μl (25 ng) of the internal standard perfluorononanoic acid (PFNA). Carcass samples were ground under liquid nitrogen to a fine powder and then homogenized in 10-20 mL NaCO3. An aliquot of the homogenate was then transferred to a separate tube containing 1 ml of water, 1 ml of TBAS sulfate, and 100 μl of PFNA before extraction. For extraction, homogenates were shaken vigorously for 10 minutes with 5 mL methyl-t-butyl ether (MTBE), followed by centrifugation to separate the organic phase. The upper MTBE layer was removed to a separate tube and the extraction was repeated once more, combining supernatants. The solvent was then removed under a stream of high-purity nitrogen gas, and the analytes were taken up in 1 to 2 ml of 50:50 (v/v) water:methanol. Solutions were filtered through a 0.2 μm nylon syringe filter before analysis.

For water samples, 25-mL aliquots were amended with 0.6 g Na2CO3, 1 g TBAS, and 100 µL PFNA, after which samples were extracted twice with MTBE as with other samples.

Vehicle:

no

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION OF SPIKED FISH FOOD
- Details on fish food (source, fat content as supplied, etc): Multiple perfluorinated carboxylic and sulfonic acids were tested simultaneously. Spiked food was prepared by adding starter food (Martin Mills, Tavistock, ON, Canada) to a round-bottom flask containing a solution of all the test compounds in methanol. The flask was placed on a rotary evaporator and the mixture was slowly dried under vacuum. The resulting spiked food was further dried in an oven at 60 °C for 3 h to remove excess methanol. Control food was prepared in the same manner, minus test compounds.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Rainbow Springs (Thamesford, ON, Canada)
- Weight at study initiation: 2 - 5 g, mean initial mass: 2.54 g (exposed), 2.51 g (control)
- Feeding during test: daily at a rate of 1.5% body weight per day, corrected for growth throughout the experiment
- Food type: Trout feed from Martin Mills (Tavistock, ON, Canada).
ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): yes

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

other:

Total exposure / uptake duration:

34 d

Total depuration duration:

41 d

Test temperature:

12 °C

Details on test conditions:

TEST SYSTEM
- Test vessel: Fish tank
- No. of vessels per concentration: two
- No. of vessels per control / vehicle control: one
- Biomass loading rate: ca. 2 g/L in exposure vessel, 1.1 g/L in control vessel
- Other: Fish growth was monitored by weighing the total biomass every 2 to 3 d throughout the course of the experiment.
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 h
- Overall daily feeding rate used in the study: 1.5% body weight adjusted for growth
- Number of feeds per day: One feeding per day.
- Other: To minimize transfer of test compounds from spiked food to the water column, pellets were slowly sprinkled into the treatment tanks in several stages, allowing the fish to ingest each quantity before additional food was added. Fish always fed voraciously, and food was typically consumed within 5 s of being offered. PFBSK+ was not detected in test chamber water on day 30 of exposure.

Nominal and measured concentrations:

Measured concentration: 0.32 μg/ feed

Key result

Remarks on result:

not determinable

Remarks:

PBSFK+ could not be detected in tissue samples at sufficient time points to calculated uptake rate or kinetic BAF

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

3.3 d

Remarks on result:

other: depuration for liver

Key result

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Liver depuration rate constant

Value:

0.21

Remarks on result:

other: Rate constant could not be calculated in carcass

Details on kinetic parameters:

PFBSK+ could not be detected in carcass samples. It was detected in liver samples on the last three uptake sampling intervals and at the first sampling time of the depuration phase. Only depuration rate could be estimated from the data.

Details on results:

Mortality of test organisms: none
- Observations on body length and weight: Growth rate 13.0 mg/d
- Other biological observations: Liver somatic index (LSI) 1.4 ± 0.04%
- Mortality and/or behavioural abnormalities of control: No mortalities in control fish, growth rate 12.1 mg/d,
LSI 1.2 ± 0.06%. Controls health was no significantly differnt to exposed fish health.

Reported statistics:

Fish weight (FW) was best predicted by the exponential growth model, FW = a · exp(g · t), where a is a constant, g is the growth rate, and t is the time. All tissue concentrations were corrected for growth dilution by determining the percent increase in FW at each sampling interval, relative to t = 0, by using the predicted exponential growth rate equation. The depuration rate constants (kd) were determined by linear regression after fitting the growth-corrected depuration concentrations (Cfish(t)) to the first-order decay model Cfish(t) = a · exp(-kd · t), where a is a constant. Depuration half-life was calculated by the formula ln(2)/kd.

Validity criteria fulfilled:

not applicable

Conclusions:

PFBSK+ could not be detected in exposed organisms at most time points in a bioaccumulation (feeding) experiment. PFBSK+ is not expected to bioconcentrate in fish

Executive summary:

PFBSK+ bioaccumulation potential was assessed in a feeding study with simultaneous exposure to several perfluorinated carboxylic and sulfonic acids. Rainbow trout (Oncorhynchus mykiss) were fed once daily for 33 days at a rate of 1.5% of biomass/day, followed by a 41 day depuration period. Measured concentration of PFBSK+ in treated feed was 0.32 µg/g. PFBSK+ could be detected in liver but not gutted carcass, with detection only on exposure days 21, 28 and 34 plus depuration day 7.  A depuration rate constant from liver corresponding to a half-life of 3.3 days could be calculated, but uptake kinetics could not be calculated. PFBSK+ is not expected to bioconcentrate in fish.

This study did not follow an explicit guideline, but followed acceptable scientific principles including analytical determination of media concentrations. The results were published in a peer-reviewed journal. The study is deemed reliable with restrictions and is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.

Reference 4

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Study is of the free acid form of PFBS. For pH values within the range 6.0 to 8.5 as specified by OECD TG305, bioconcentration testing on the acid is equivalent to testing on the potassium salt.

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Deviations:

not specified

GLP compliance:

no

Specific details on test material used for the study:

Perfluorobutane sulfonic acid CF3(CF2)3SO3H

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test medium samples: Every other day during the uptake period.
- Sampling intervals/frequency for test organisms: During uptake period, at 1d, 5 d, 10 d, 15 d, 20 d, 22 d and 24 d. During depuration period, at 25 d, 32 d, 40 d and 48 d. Five fish each were pooled into three replicates for each treatment (control, low concentration, high concentration). Samples of liver, muscle, ovary, fecal matter, and plasma were collected.
- Sample storage conditions before analysis: Samples were stored at -20 °C until analysis.

- Details on sampling and analysis of test organisms and test media samples:
50 mL samples were spiked with isotopically labeled surrogate standards prior to solid phase extraction (SPE) with Phenomenex X-AW 33u polymeric weak anion cartridges (200 mg, 6 mL), coupled to a vacuum manifold. SPE cartridges were conditioned with 4 mL of 0.01 N KOH in methanol (KOH/MeOH), followed by 4 mL of methanol and 4 mL of Milli-Q water, with a flow rate of 5 mL/min. Water samples were loaded on the cartridges at a flow rate of 1 drop/s. The cartridges were washed with 4mL of 25mM sodium acetate, then eluted with 3 mL of 0.01 N KOH/MeOH. Extracts were evaporated to dryness under a gentle nitrogen stream. 200 μL of methanol:Milli-Q water (2:3 v/v) was used to rinse and transfer concentrated extracts to polypropylene vials. Lastly, 2.45 ng of 13C8-PFOA (injection internal standard), used to quantify recovery of internal surrogate compounds, was spiked prior to LC-ESI-MS/MS analysis.

For analysis of tissues, subsamples (liver ca. 0.1 g; muscle ca. 0.5 g; ovary ca. 0.5 g; fecal matter ca. 0.2 g) were thawed and weighed directly into 15-mL polypropylene centrifuge tubes. 0.5 mL Milli-Q water was added, then samples were homogenized via sonication. 5 mL KOH/MeOH was added, along with internal surrogate compounds. Samples were then extracted via sonication for 30 min, then centrifuged at 10,000 rpm, 4 °C for 5 min. Supernatant was transferred to another 15-mL tube. This process was repeated a second time. Duplicate extracts (10 mL) were combined. A 1 mL aliquot of this extract was diluted with 50 mL Milli-Q water, then extracted via SPE as for water samples

For plasma samples, 20 μL of thawed plasma (obtained from 5 pooled fish) was added to a 2-mL centrifuge tube and spiked with isotopically labeled internal surrogate compounds. 0.5 mL KOH/MeOH was added. Samples were extracted via sonication for 30 min, followed by centrifugation at 12,000 rpm, 4 °C for 5 min. Supernatant was transferred to another 2-mL tube. This was repeated a second time. The duplicate extracts were combined, evaporated under a gentle nitrogen stream to near dryness and spiked with recovery standard (13C8-PFOA, 2.45 ng).

In all, 21 substances were included in the exposure solution, and ten internal surrogates were identified to standardize analysis. 18O2-perfluorohexanesulfonic acid (18O2-PFHxS) was used as the internal surrogate for PFBS at 12 ng in the spike.

Vehicle:

no

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solution contained 21 perfluorinated organic acids, with low- and high-exposure groups.
- Controls: water
- Chemical name of vehicle: None

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Mainland tropical fish farm, Singapore
- Initial mass (mean): 0.8032 - 0.8308 mg
- Feeding during test: twice daily.
- Food type: Artemia.

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): yes

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

other: dechlorinated and carbon-filtered tap water

Total exposure / uptake duration:

24 d

Total depuration duration:

24 d

Test temperature:

25 ± 2 °C

Details on test conditions:

OTHER TEST CONDITIONS
- Photoperiod: 16 h:8 h light:darkness

Nominal and measured concentrations:

Measured concentrations: < MDL, 2.15 ± 0.28 µg/L, 17.8 ± 1.28 µg/L

Reference substance (positive control):

no

Conc. / dose:

2.15 µg/L

Temp.:

25 °C

Type:

BCF

Value:

19.5 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Conc. / dose:

17.8 µg/L

Temp.:

25 °C

Type:

BCF

Value:

27.5 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Details on results:

- Mortality of test organisms: 2 mortalities (0.66%) in low-exposed fish, 1 mortality (0.33%) in high exposed fish.
- Observations on body length and weight: Growth rate 9.9 mg/d in low-exposed fish, 11.9 mg/d in high exposed fish.
- Other biological observations: Liver somatic index (LSI) 2.55% in low-exposed fish, 2.68% in high exposed fish.
- Mortality and/or behavioural abnormalities of control: 1 mortality in control fish (0.32%), growth rate 8.9 mg/d, 2.56%. Controls health was essentially the same as exposed fish health.
- Other: The whole body BCF was estimated by calculating a whole-body burden. The body burden was calculated as the sum of tissue concentrations times the wet weight of the respective tissues. The average weight of plasma, liver, muscle and ovary per fish are 0.0045 g, 0.02 g, 0.25 g and 0.15 g, respectively in this study, i.e. whole body BCFss= (Cplasma * 0.0045 + Cliver * 0.02 + Cmuscle * 0.25 + Covary * 0.15) / (0.0045 + 0.02 + 0.25 + 0.15) [* Cwater]. It should be noted that this calculation ignores all other tissues in estimating the whole-body burden.

Reported statistics:

Growth-corrected concentrations were calculated using the exponential growth model to predict the fish weight (FW), i.e FW = a · exp(g · t), where a is a
constant, g is the growth rate, and t is the time. All tissue concentrations were corrected for growth dilution by determining the percent increase in FW at each sampling interval, relative to t = 0, by using the predicted exponential growth rate equation. The depuration rate constants (kd) were determined by linear regression after fitting the growth-corrected depuration concentrations (Cfish(t)) to the first-order decay model Cfish(t) = a · exp(-kd · t), where a is a constant. Depuration half-life was calculated by the formula ln(2)/kd. Uptake rate constants (ku) were determined by iterative nonlinear regression (Origin, Version 9.0, North Hampton, MA, USA) after fitting the growth-corrected concentrations to the integrated form of the kinetic rate equation for constant aqueous exposure, i.e. Cfish(t) = [(ku)(Cwater)/(kd)] · [1 - exp(-kd · t)], where kd is a fixed parameter.

Table 1, Tissue-specific kinetic and BCF parameters

Tissue and exposure level	ku (L/kg/d)¹	kd (1/d)¹	Half-life (d)²	log BCFk (L/kg ww)²	log BCFss (L/kg ww)²
Plasma, low	23.4 ± 14.5 (0.75)	0.194 ± 0.003 (0.94)	3.6 ± 0.1	2.08 ± 0.27 ³	1.60 ± 0.12 ³
Plasma, high	19.6 ± 0.7 (0.88)	0.129 ± 0.004 (0.92)	5.4 ± 0.2	2.18 ± 0.02 ³	1.41 ± 0.03 ³
Liver, low	32.1 ± 20.6 (0.75)	0.233 ± 0.006 (0.69)	3.0 ± 0.1	2.14 ± 0.28	1.24 ± 0.09
Liver, high	7.7 ± 2.9 (0.89)	0.139 ± 0.005 (0.89)	5.0 ± 0.2	1.74 ± 0.17	1.27 ± 0.05
Muscle, low (<MDL in tissue)	─	─	─	─	─
Muscle, high	0.3 ± 0.2 (0.79)	0.212 ± 0.001 (1.00)	3.3 ± 0.0	0.18 ± 0.21	0.66 ± 0.10
Ovary, low	5.6 ± 2.0 (0.84)	0.191 ± 0.002 (0.98)	3.6 ± 0.0	1.47 ± 0.16	1.72 ± 0.06
Ovary, high	8.6 ± 2.5 (0.90)	0.099 ± 0.006 (0.85)	7.0 ± 0.4	1.94 ± 0.13	1.82 ± 0.04

1, rate constants are shown as (constant ± standard error (r²))

2, values shown as (parameter ± standard error)

3, BCF values for plasma are dimensionless

Validity criteria fulfilled:

not applicable

Conclusions:

PFBS had a whole-body BCFss of 19.5 - 27.5 in a flow-through test conducted per OECD TG305. Results are directly applicable to PFBSK+.

Executive summary:

PFBS bioconcentration potential was assessed in a flow-through study conducted on Zebrafish (Danio rario) according to OECD guideline 305, with simultaneous exposure to 21 perfluorinated organic acids. Two levels of PFBS exposure were used, and were analytically determined to average 2.15 and 17.8 µg/L. Exposure was for 24 days, followed by 24 days depuration. Fish were collected at several times and dissected. Resulting tissues (plasma, liver, ovary, and muscle) were pooled into three replicates of five fish per sample time. Tissue concentrations were used to develop kinetic rate constants for depuration and uptake, as well as kinetic BCF and steady-state BCF at the end of the exposure period. Whole-body BCF values were calculated by weighting tissue specific values by mass of the tissue. PFBS had a whole-body BCFss of 19.5 - 27.5. Because PFBS is a strong acid and will exist entirely in anionic form at pH values mandated by the test guideline, results are equivalent to testing on the salt. Therefore, results for PFBS are directly applicable to PFBSK+. PFBSK+ is not expected to bioconcentrate in fish.

While details are scarce, this study followed an accepted guideline. The results were published in a peer-reviewed journal. Exposure to multiple substances simultaneously is not expected to have an impact on bioconcentration of each substance, and the use of the free acid form of the substance is equivalent to the potassium salt in the pH range necessary for health of the fish. The study is deemed reliable with restrictions and is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.

Reference 5

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Study is of the tetrabutylammonium salt of PFBS. Because both the tested substance and the registered substance will dissociate completely in solution, and because only the anion would be analyzed in either case, bioconcentration testing is equivalent to testing on the potassium salt.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test

GLP compliance:

no

Specific details on test material used for the study:

tetrabutylammonium nonafluorobutanesulfonate at a chemical purity of sulfonic acids ≥ 98% (mixture of linear and branched isomers) as standards was obtained from Sigma-Aldrich Chemie.

Radiolabelling:

no

Details on sampling:

Details on sampling
- Sampling intervals/frequency for test medium samples: Twice per week starting at test initiation.
- Sampling intervals/frequency for test organisms: During uptake period, at test initiation, 7 d, 14 d and 28 d. During depuration period, at 3 d, 7 d, 14 d and 28 d (overall study days 31, 35, 42, and 56). Four to five fish were taken on each sampling date. Animals were anesthetized and blood drawn by heart puncture. Afterward, animals were euthanized and dissected. Muscle, skin, liver, kidney and gill were collected. In addition, the remaining carcass of each animal was washed to remove treated feed from the gut, and the pyloric caecum was removed. Four animals (two from day 0, one each from accumulation days 7 and 14) were cleaned and frozen undissected for comparative whole fish analysis.
- Sampling intervals for feed: 3 representative samples of feed from each week's coating batch.
- Sample storage conditions before analysis: Samples were stored at -18 °C until analysis.
- Details on sampling and analysis of test organisms and test media samples:
All sample types except blood were homogenized before extraction. One-gram aliquots of homogenate were spiked with 50 µL isotopically labeled surrogate standards (100 µg/L in each), then received 2 mL each acetonitrile and water, were shaken vigorously by hand for 30s, and then were shaken 15 minutes on a mechanical mixer. After the addition of 1.5 g of a salt mixture (QuEChERS-Mix) containing 62% magnesium sulfate, 15% sodium chloride, 8% disodium hydrogen citrate sesquihydrate, and 15% trisodium citrate dihydrate, the samples were shaken for 30 s by hand and centrifuged for 5 min at 1800 x g. The salt solution allowed a clear separation of the organic phase. The organic layer was transferred to a new tube and diluted with 2 mL purified water prior to purification by solid-phase extraction (SPE) using OASIS WAX polymeric weak anion cartridges (60 mg, 2 mL, Waters, Eschborn, Germany). SPE cartridges were conditioned with 2 mL of water:formic acid 100:2, then with 2 mL of methanol. Samples extracts were loaded on the cartridges, washed with 2 mL of water:formic acid 1000:1, then with 2 mL of methanol, and then eluted with 2 mL of 1:1000 NH4OH/MeOH. Extracts were evaporated to dryness under a gentle nitrogen stream and redissoved in 250 μL of methanol:water (1:1 v/v). Redissolved extracts were filtered using 0.45 µm Chromafil® (Macherey-Nagel, Düren, Germany) polyester disposable syringe filters.

For analysis of water samples, 100 ml samples were amended with 50 μL of the internal standard solution (concentration: 10 μg/l per analyte) and concentrated by solid-phase extraction using a Strata XAW cartridge (60 mg sorbent, 3 ml volume) from Phenomenex (Aschaffenburg, Germany). Before applying the sample, the cartridges were conditioned with 2 ml methanol followed by 2 ml demineralized water. After the samples were added, the cartridges were washed with 1 ml methanol and the analytes were eluted with 2 ml 0.1% ammonia in methanol. The eluate was concentrated to dryness under a nitrogen stream at 39°C and the residue dissolved in 250 µL methanol-water (50:50 v/v) using a vortex mixer. To remove any impurities, the dissolved residue was filtered through 0.2 μm syringe filters Chromafil® (Macherey-Nagel, Düren, Germany).

In all 5 substances were included in the exposure solution, and three internal surrogates were used to standardize analysis. 13C-perfluorooctanesulfonic acid (13C-PFOS) was used as the internal surrogate for PFBS.

Vehicle:

no

Details on preparation of test solutions, spiked fish food or sediment:

The test diet was commercial, extruded trout feed (4-mm pellet size, Milkivit, F-2P B40; Skretting) with the 5 test substances (PFBS, PFHxS, PFOS, PFOA, and PFNA) at a nominal concentration of 500 mg/kg dry weight each. Two 3-kg batches were prepared. An aliquot of stock solution was diluted in acetone:dichloromethane (1:1, v/v). This solution was applied evenly to the surface of the feeding pellets using a pneumatic spray gun, with continuous mixing of the feed pellets during application. The spiked feed was then dried overnight at room temperature to drive off residual solvent. Spiked pellets were then coated with alginate gel to reduce leaching of test substance(s) from the pellets after distribution over water. Ccoating procedure was done general according Duis et al. (Aquacult Res [1995] 26:549–556) but with a 50% reduction of alginate gel. Coated pellets were prepared weekly during the study to prevent spoilage of the experimental diet. The test diet was stored at 4 °C in the dark.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Eyed eggs were purchased from a fish farm and reared at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME Schmallenberg, Germany)
- Age at study initiation (mean and range, SD): ca. 1 year
- Weight at study initiation (mean and range, SD): 314 ± 21 (300 - 350) g
- Weight at termination (mean and range, SD): 808 ± 62 g
- Feeding during test
- Food type: Trout feed as prepared
- Amount: 2.6% of average body weight per day
- Frequency: Twice daily

ACCLIMATION
- Acclimation period: 18 days
- Acclimation conditions (same as test or not): same as test

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

other: purified tap water

Total exposure / uptake duration:

28 d

Total depuration duration:

28 d

Test temperature:

14 - 16 °C

Dissolved oxygen:

≥80% saturation

Details on test conditions:

TEST SYSTEM
- Test vessel: Open 2000-L stainless steel tank, 1400 L fill volume.
- Aeration: In-tank ventilation, aeration of return water
- Renewal rate of test solution (frequency/flow rate): 173 L/h
- No. of organisms per vessel: 35
- No. of vessels per concentration: one
- No. of vessels per control / vehicle control: none
- Biomass loading rate: ca. 8 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Preconditioned tap water with activated carbon filtration. In addition to renewal, tank water was purified by carbon filtration at a rate of 1700 L/h
- Other: uneated food and fecal material removed throughout the test to reduce exposure through leached test substance.

OTHER TEST CONDITIONS
- Photoperiod: 12 h:12 h light:dark
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall daily feeding rate used in the study: 2.6% of fish weight per day. 4 or 5 representative fish from the basin were weighed weekly calculate the average live weight.
- For OECD 305 part III (dietary exposure fish bioaccumulation), number of feeds per day (number of feeds daily ration split between): two feeds. First feed continued until feed acceptance visibly slowed, remainder given in second feeding.

Nominal and measured concentrations:

Nominal, 500 µg/kg dw in feed. Measured, 185 ± 26 µg/kg dw

Reference substance (positive control):

no

Remarks on result:

not measured/tested

Remarks:

BMFs not corrected for lipid contend due to low partition into lipids

Key result

Conc. / dose:

0.185 µg/g food

Temp.:

>= 14 - <= 16 °C

Type:

BMF

Value:

0.02 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic, corrected for growth

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

10.8 d

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.064

Details on kinetic parameters:

- Fish fed at 2.53% of body weight per day had an assimilation efficiency of 5.98% for PFBS. Tissue specific depuration rates are reported in Table 1.
- Specific growth rate: 0.0204/d during accumulation, 0.0133/d during depuration, 0.0169/day overall
- Feed conversion ratio: 1.14 during accumulation, 1.30 during depuration, 1.17 overall.

Details on results:

In all, only 0.89% of administered PFBS was detected in fish tissues (parameter combines assimilation with depuration as of day 28). The highest concentrations on day 28 were in liver and blood, with the largest mass (0.89% of administered dose) being recovered from carcass remaining after dissection (see Table 1).

Table 1, Tissue-specific parameters

Tissue	Day maximum reached	Concentration on day 28 (µg/kg ww)¹	Depuration half-life (d)
Liver	28²	110 ± 8.29	6.5
Muscle	14	7.62 ± 6.94	4.6
Skin	7	12.5 ± 4.721	2.5
Gill	7	11.6 ± 1.17	4.8
Kidney	14	3.00 ± 1.43	─
Blood	14	31.6 ± 10.7	6.3
Carcass	7	5.14 ± 1.44	2.2
Whole body	─	7.03 ± 1.52	10.8

1, concentrations are shown as (mean ± absolute standard deviation)

2, liver concentration was 80% of maximum by day 7

Validity criteria fulfilled:

not applicable

Conclusions:

The tetrabutylammonium salt of PFBS had a whole-body BMFkinetic of 0.02 in a feeding study of market-sized rainbow trout (Oncorhyncus mykiss). Results are directly applicable to PFBSK+.

Executive summary:

PFBS (as the tetrabutylammonium salt) bioaccumulation potential was assessed in a feeding study with simultaneous exposure to several perfluorinated carboxylic and sulfonic acids. Market sized (ca. 300 gram at study initiation) rainbow trout (Oncorhynchus mykiss) were fed twice daily for 28 days at a rate of 2.53% of body mass/day, followed by a 28 day depuration period. Measured concentration of PFBS in treated feed was 0.185 μg/g. Samples were taken at exposure days 0, 7, 14, and 28, and at depuration days 3, 7, 14, and 28. All tissues except liver had attained maximum concentration by day 14. Whole-body BMF from kinetic parameters was 0.02, with an depuration half-life of 10.8 days. Tissue-specific half-lives were individually shorter. Because PFBS is a strong acid, both the tested substance and the registered substance will dissociate completely in solution, and because only the anion would be analyzed in either case, bioconcentration testing is equivalent to testing on the potassium salt. Therefore, results for PFBS as the tetrabutylammonium salt are directly applicable to PFBSK+. PFBSK+ is not expected to bioconcentrate in fish.

This study did not follow an explicit guideline, but followed acceptable scientific principles including analytical determination of media concentrations. The results were published in a peer-reviewed journal. Exposure to multiple substances simultaneously is not expected to have an impact on bioconcentration of each substance, and the use of the tetrabutylammonium salt of the acid is equivalent to the potassium salt with regard to bioconcentration of the anion. The study is deemed reliable with restrictions and is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.

Reference 6

Endpoint:

bioaccumulation in sediment species: invertebrate

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The study examines biomagnification of the anion PFBS- using environmentally-contaminated sediment in a laboratory setting. As PFBS is a strong acid that will exist predominantly in ionic form in the environment, invertebrates would be exposed to PFBS- anions regardless of the original material (PFBS acid v. potassium salt v. another ionic material). Therefore, this study is relevant to the examination of biomagnification in aquatic invertebrates

Qualifier:

according to guideline

Guideline:

other: EPA/600/R–99/064 Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates, 2nd ed.

Version / remarks:

Updates EPA/600/R-94/024

Deviations:

not specified

GLP compliance:

not specified

Specific details on test material used for the study:

Study used naturally-contaminated river sediments and analyzed for PFBS as anion by LCMS. No information is available regarding the material originally contaminating the sediment.

Radiolabelling:

no

Details on sampling:

- Sampling intervals for test organisms: Test organisms were collected at the end of the exposure period (28 days)
- Sampling intervals for test medium samples: No information. It is assumed that that sediments were analyzed only before the sample chambers were assembled.
- Sample storage conditions before analysis: Invertebrate samples were stored at -20 °C until analysis.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):

Test organisms: Organisms were separated from sediment by sieving and left in clean water for eight to nine hours to purge gut contents. They were then combined and frozen until analysis. Samples were thawed and composites weighing >2 grams were split into two subsamples for analysis. 0.5-1 mL purified water (18 MΩ/cm) was added to each composite with 30s vortex mixing to homogenize the organisms. Internal recovery standard (100 μL of 10 ng/g 13C8-PFOA) was added after which samples were vortex mixed and reweighed. Solvent extraction was by addition of 4.0 mL 0.25 M tetrabutylammonium hydrogen sulfate, followed by the addition of 5.0 mL of methyl tert-butyl ether (MTBE). Samples were then rotated for 15 to 24 hrs on a rotisserie mixer, centrifuged 5 minutes at 650*g and 18 to 22 °C, and placed in the freezer until the aqueous phase solidified. The MTBE layer was transferred to a fresh vial and stored in the freezer. The aqueous phase was thawed and reextracted twice more with 3 mL of MTBE using the same approach. The combined MTBE extracts were evaporated to dryness in a 5-psi vacuum under a stream of filtered air and resuspended in 60:40 acetonitrile:water with a mixture matrix internal standard added.

Sediments: The following is derived from supporting information and the methods paper Washington et al 2008. Supporting information indicates that the rotisserie mixer was substituted for a shaker table, but Washington et al makes no mention of a shaker table. Sediments were tested for moisture content. Mixing steps were by vortex mixer, and samples were reweighed after essentially all additions or manipulations (indicated by '*'). Aliquots (1 gram) of each sediment were adjusted to 50% moisture using 18MΩ/cm water and mixed to homogenize*. Internal recovery standard was added*, followed by 200 µL 2.0M sodium hydroxide*. Samples were mixed and allowed to react for 30 minutes. Acetonitrile was added to achieve a 60:40 acetonitrile:water ratio, and solutions were mixed to homogenize*. 200 µL 2.0M HCl was then added to neutralize and samples were mixed*. Samples were then sonicated for one hour. Internal recovery standard was added* after which samples were evaporated to dryness* and resuspended in 4 mL 0.25M tetrabutylammonium bisulfate/0.125M sodium carbonate by mixing*. Methyl t-butyl ether (MTBE, 5 mL) was added and mixed*, after which samples were sonicated 60 minutes. MTBE layers were then transfered to fresh flasks*. The supplementary information states that the acetonitrile extraction step was repeated three more times and the four extracts combined before evaporation. Solvent was removed under filtered air*, the extracts resuspended in 60:40 acetonitrile:water*, and an aliquot of matrix internal standard added.

Controls: Field blanks were made by bringing a sample of clean sand to the field, pouring the sand over all utensils used to collect and homogenize sediments, collecting it, and extracting with sediments collected at that site. Tissue blanks were taken from composites of the initial pool of animals before exposure. Empty tubes were carried through the extraction processes to act as procedural blanks.

Vehicle:

no

Details on preparation of test solutions, spiked fish food or sediment:

Test used contaminated sediment downstream of industrial sites. Sediments were characterized but were not further processed with regard to exposure concentration.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

TEST ORGANISM
- Source: Organisms were obtained from in-house cultures
- Length at study initiation: 1-2 mm, chose to minimize asexual framentation during the exposure period.
- Feeding during test: No, organism subsisted on native organic matter in the sediments.

Route of exposure:

sediment

Test type:

semi-static

Water / sediment media type:

natural sediment

Remarks:

overlying water was moderately hard reconstituted laboratory water

Total exposure / uptake duration:

28 d

Hardness:

100-150 mg CaCO3/L

Test temperature:

23 °C

Dissolved oxygen:

>2.5 mg/L

Conductivity:

360 - 460 µS/cm

Details on test conditions:

TEST SYSTEM
- Test vessel: 3-L polycarbonate test chamber containing ca. 1 L of sediment sample covered with 2 L overlying water
- Renewal rate of test solution (frequency/flow rate): 50% of overlying water three times per day.
- Biomass loading rate: based on organic carbon content of sediment, at least 50:1 carbon:worm on a dry weight basis

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: moderately hard reconstituted laboratory water
- Alkalinity: 75 - 125 mg/L
- Total ammonia: 0 - 2.6 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 16:8 light:dark

Nominal and measured concentrations:

Twenty-four sediments from eight sites were tested. Mean concentrations are provided in Table 2

Key result

Conc. / dose:

<= 0.22 µg/kg sediment dw

Temp.:

23 °C

Type:

BSAF

Value:

0.31 dimensionless

Basis:

whole body w.w.

Remarks:

CV = 42

Calculation basis:

other: tissue concentrations at end of exposure period

Details on results:

- Mortality of test organisms: Only 70% of organisms could be recovered from text chambers. Since this was also true of the relatively less contaminated upstream sites 1 and 2, it is not considered to have a negative impact on the study.

Reported statistics:

Concentrations of PFBS in sediment were not significantly related to organic carbon, clay, iron, or iron hydroxides content (Table 3). Pearson coefficients for relationship between sediment and tissue concentrations of PFBS were similarly not significant. However, normalization of the sediment/tissue relationship to iron hydroxide was significant, while normalizations to organic carbon or clay were not (Table 3)

Table 2, Mean concentrations (ng/g) of PFBS (with coefficient of variation, n = 3) in sediments and tissues of Lumbriculus variegatus by site

Matrix v. Site	Site 1	Site 2	Site 3	Site 4	Site 5	Site 6	Site 7	Site 8
Sediment (dry weight basis)	0.05 (87)	< MDL	0.11 (73)	0.17 (16)	0.09 (14)	0.22 (38)	0.20 (15)	0.10 (16)
Tissue (wet weight basis)	1.91 (37)	1.66 (28)	3.30 (15)	3.52 (25)	2.61 (11)	3.03 (22)	2.63 (22)	2.36 (17)

Table 3, Pearson correlation coefficients (r) with alpha values (n = 18) for specified relationships involving concentrations of PFBS

Relationship	Unnormalized	Organic C	Total Fe	Fe oxides	Clay
Sediment concentration v. sediment characteristic	─	0.39	0.21	0.31	0.28
(alpha value)	─	0.1072	0.4130	0.2079	0.2656
Tissue concentration v. sediment concentration normalized to sediment characteristic	0.29	0.31	0.49	0.66	0.43
(alpha value)	0.2504	0.2149	0.0381	0.0034	0.0783

Validity criteria fulfilled:

not specified

Conclusions:

PFBS did not bioacculate in the tissues of Lumbriculus variegatus after a 28-day exposure in contaminated sediments. The results are directly applicable to PFBSK+

Executive summary:

PFBS bioaccumulation potential was assessed in a semi-static test conducted on Lumbriculus variegatus according to USEPA guideline EPA/600/R–99/064. Organism were exposed to sediment obtained upstream and downstream from a land-application site for sewage sludge. A number of perfluorinated acids were present, and no additional test substances were added. PFBS was analyzed as the anion. After 28 days' exposure, an average BSAF(wet weight) of 0.31 (CV = 42) was calculated. Association of tissue concentration with sediment concentration was not improved by normalizing to organic carbon content of the sediment; however, iron hydroxide normalization was significantly improved the relationship. PFBS is not expected to bioaccumulated in sediment invertebrates. The nature of the substance(s) present in the sediment cannot be assigned since it results from historical contamination. However, PFBS is a strong acid that will exist predominantly in ionic form in the environment. Therefore, invertebrates would be exposed to PFBS- anions regardless of the original material (PFBS acid v. potassium salt v. another ionic material).  Therefore, this study is relevant to the examination of biomagnification in aquatic invertebrates and is directly applicable to PFBSK+. PFBSK+ is not expected to bioaccumulation in sediment invertebrates.

The study followed an accepted guideline. The results were published in a peer-reviewed journal. Exposure to multiple substances simultaneously in historically-contaminated sediments is not expected to have an impact on bioconcentration of each substance, and nature of the substance initially contaminating the samples is of little relevance do to dissociation and respeciation of the materials in the sediment. The study is deemed reliable with restrictions and is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis.

Description of key information

PFBSK+ does not bioaccumulate in aquatic organisms.

Additional information

Bioconcentration and bioaccumulation are available from laboratory studies where the water, sediment, or food was dosed. Laboratory studies were nearly all given a Klimisch reliability ranking of 2, except for those studies conducted under Good Laboratory Practices (GLP) which were given a Klimisch ranking of 1. Due to the high water solubility (46,200 – 56,600 mg/L) and low sorption of PFBSK+, the relevant route of environmental exposure is the water column, not sediment or food.  The most relevant studies to compare against the REACH “B” criteria (Annex XIII of EC1907/2006) are three laboratory fish BCF studies. These three studies yielded BCF values ranging from 0.30 to 27.5, several orders of magnitude below the REACH criteria of 2000 and 5000 for “B” and “vB”, respectively.  Data from fish feeding studies (dietary biomagnification) are also available. The ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.11, PBT/vPvB V2.0 notes that “A relevant BMF or TMF value higher than 1 […] can also be considered as an indication of very high bioaccumulation”. There are two laboratory feeding studies which reported BMF values for juvenile and adult rainbow trout, in which whole fish BMF values ranged from <<1 to 0.02.  A laboratory study evaluating bioaccumulation in worms from sediment is also available. This study had an appropriate number of replicates and good sample sizes.  A sediment BSAF of 0.31 (wet wt.) was derived based on non-normalized concentrations in sediment and tissue.  Correlation to lipid content and fraction organic carbon in sediment did not correlate with BSAF across several sediment samples.