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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 - 15 Jan 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: Commission Directive 88/302/EEC; Official Journal ofthe EC L 133 Part C (1988)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

not specified

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:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: aeration tank of a waste water treatment plant treating predominantly domestic sewage (Wupper area water authority, Germany)
- Date of collection: 2003-01-14
- Pretreatment: aeration of the activated sludge; daily feed (once) with synthetic medium
- pH of the suspension before application: 7.1
- Test concentration of the activated sludge: 480 mg/L suspended solids

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

18.5-18.9 °C

pH:

7.3-8.4

Nominal and measured concentrations:

Nominal: 100, 1000, 10000 mg/L

Details on test conditions:

TEST SYSTEM
- Aeration: Yes
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Notes on procedure: Because of strong respiration of the activated sludge only 0.48 g/L suspended solids were used. The test item has been added to about 130 ml deionized water and stirred overnight before testing (equilibration phase).

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

See Table 1

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: The EC50 was within the 5 to 30 mg/L range considered acceptable for the test.

Table 1. Respiration rates and Percent Inhibition

Test Concentration (mg/L)	Respiratory rate test item (mg/L·h)	Inhibition (%)	O2 start (mg O2/L)	O2 end (mg O2/L)	Exposure time (min.)
100	32.4	0.0	5.6	2.9	5
1000	32.0	0.0	4.3	2.7	3
10000	26.0	16.1	3.8	2.5	3
Control, mean	31.0
Control 1	30.0		4.7	3.7	2
Control 2	32.0		4.7	3.1	3
Reference substance (5 mg/L)	30.0	3.2	4.0	3.0	2
Reference substance (10 mg/L)	18.0	41.9	4.9	4.3	2
Reference substance (20 mg/L)	10.5	66.1	6.2	5.5	4

Validity criteria fulfilled:

yes

Remarks:

All validity criteria of the test method were met: respiratory rates of the 2 controls differ less than 15%, respiratory rate of the controls < 60 mg O2/L·h, EC50 of the reference substance 3,5-dichlorophenol is in the range 5- 30 mg/L

Conclusions:

The 3-hour EC50 (respiration) of PFBSK+ to activated sludge is > 10000 mg/L and the corresponding NOEC is 1000 mg/L (Commission Directive 88/302/EEC; Official Journal of the EC L 133 Part C).

Executive summary:

The toxicity of PFBSK+ to activated sludge was assessed according to the Commission Directive 88/302/EEC; Official Journal of the EC L 133 Part C. The validity criteria were met based on a difference between the duplicate controls of 5%. The 3-hour EC50 (respiration) of PFBSK+ to activated sludge is > 10000 mg/L and the corresponding NOEC is 1000 mg/L. The study was performed in accordance with national standards under GLP. The study is deemed reliable without restriction and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

27 - 30 Nov 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition 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: Lot 2
- Expiration date of the lot/batch: March 2010
- Purity: 97.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient room temperature

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Test substance stock solutions were prepared at nominal concentrations of 1000 and 2000 mg a.i./L in high quality water (Nanopure®).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Denton Wastewater Treatment Plant (Denton, MD, USA)
- Preparation of inoculum for exposure: The sludge was sieved using a 2 mm screen and then settled for approximately 30 minutes. The supernatant above the settled solids was drained and the total suspended solids (TSS) concentration of the settled sludge was determined. The concentration of the sludge was adjusted to 4000 mg/L solids (± 10%) by dilution with Nanopure® water. Sludge was then maintained in the laboratory for three days prior to use. Approximately 50 mL of synthetic sewage was added to each liter of activated sludge and the sludge was continuously aerated. Before use, the pH and total suspended solids concentration of the activated sludge were determined.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21-22 °C

pH:

7.7

Nominal and measured concentrations:

1, 3, 10, 30, 100, 300 and 1000 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Concentrations: 1, 3, 10, 30, 100, 300 and 1000 mg a.i./L
- Sample conditions before analysis: Control, reference, and treatment test mixtures were incubated at 20 ± 2 °C and aerated for 3 hours at a rate sufficient to maintain solids in suspension. The mixtures were prepared and aerated in 500mL plastic Erlenmeyer flasks and then transferred into a 300 mL Biochemical Oxygen Demand (BOD) bottle to the conduct dissolved oxygen (DO) measurements.
- Details on procedure: Test mixtures were prepared at 15 minute intervals starting with the first control. The control contained 9.6 mL of synthetic sewage, 120 mL of inoculum, and enough NANOpure@ water to bring the total volume up to 300 mL. The mixture was promptly aerated at a rate sufficient to provide aerobic conditions and keep the solids in suspension. Subsequent mixtures contained 9.6 mL of synthetic sewage, 120 mL of inoculum, the appropriate amount of test substance or volume of reference substance stock solution, and enough NANOpure@ water to bring the total volume up to 300 mL. Finally, a second control was prepared. All mixtures were aerated for three hours.
- Aeration: Yes
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Nutrients provided for bacteria: 16.0g peptone, 11.0 g meat extract, 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl2 2H2O, 0.2 g MgSO4 7H2O, 2.8 g K2HPO4 (dissolved in 1 L of Nanopure® water)

EFFECT PARAMETERS MEASURED: Dissolved oxygen concentration after 3 hours exposure. DO readings were recorded every 10 seconds for 10 minutes or until the DO dropped below 1.0 mg/L using a YSI Model 50B Dissolved Oxygen Meter. A respiration rate was calculated for each test mixture and expressed in mg O2/L/hour. The rate was calculated using DO values between approximately 6.5 mg O2/L and 2.5 mg O2/L, or over a 10 minute period if the DO did not reach approximately 2.5 mg O2/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Details on results:

Although the key study did not report a NOEC, at 1000 mg/L there was 8.2% inhibition, but 17.5% inhibition was reported at 300 mg/L, -6.4% inhibition at 100 mg/L and 15.3% inhibition at 30 mg/L. There was no dose-responsive relationship. Therefore, 1000 mg/L can be considered the NOEC.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 15.6 mg/L The EC50 was within the 5 to 30 mg/L range considered acceptable for the test.

Table 1. Respiration Rates and Percent Inhibitions

Treatment/Nominal Concentration	Respiration Rate (mg O2/L/hour)	Percent Inhibition
Control 1	43.6	NA
Control 2	45.9	NA
3,5-dichlorophenol 3 mg/L	42.4	5.3
3,5-dichlorophenol 15 mg/L	24.4	45.5
3,5-dichlorophenol 50 mg/L	4.3	90.4
Perfluorobutanesulfonate (PFBS) 1 mg a.i./L	45.8	-2.3
Perfluorobutanesulfonate (PFBS) 3 mg a.i./L	45.0	-0.6
Perfluorobutanesulfonate (PFBS) 10 mg a.i./L	40.0	11.0
Perfluorobutanesulfonate (PFBS) 30 mg a.i./L	37.9	15.3
Perfluorobutanesulfonate (PFBS) 100 mg a.i./L	47.6	-6.4
Perfluorobutanesulfonate (PFBS) 300 mg a.i./L	36.9	17.5
Perfluorobutanesulfonate (PFBS) 1000 mg a.i./L	41.1	8.2

Validity criteria fulfilled:

yes

Remarks:

Control respiration rates within 15%; reference substance EC50 in range 5-30 mg/L

Conclusions:

The 3-hour EC50 (respiration) of PFBSK+ to activated sludge is > 1000 mg a.i./L. The corresponding 3-hour NOEC is 1000 mg a.i./L. Test conducted according to OECD 209.

Executive summary:

The toxicity of PFBSK+ to activated sludge was assessed according to the OECD 209 method. The validity criteria were met based on a difference between the duplicate controls of 5%. The 3-hour EC50 (respiration) of PFBSK+ to activated sludge is > 1000 mg a.i./L. The corresponding 3-hour NOEC is 1000 mg a.i./L. The study was performed in accordance with internationally-accepted test guidelines under GLP. The study is deemed reliable without restriction and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis.

Reference 3

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: ISO 11348-3 (Water Quality-Determination of the Inhibitory Effect of Water Samples on the Light Emission of Vibrio fischeri (Luminescent Bacteria Test))

Deviations:

not specified

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: provided by the 3M Company

OTHER SPECIFICS:
- Other substances tested in parallel: Perfluorooctane sulfonate (PFOS) potassium salt, perfluorooctanoic
acid (PFOA), docusate sodium, triclosan, 2,4,6-trichlorophenol (TCP), and Polyfox 656 (PF-656).

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Vibrio fisheri

Details on inoculum:

The bacterial assay used Biofix Lumitest (Macherey–Nagel, Germany) in which the bacterial reagent is supplied freeze-dried (V. fischeri NRRL-B 11177), reconstituted and incubated at 3 °C for 5 min before use.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

15 min

Details on test conditions:

The analysis media was 0.34 M NaCl (2% w/v) and tests were performed at 18 °C and the measurements of light were made using a microplate luminometer.

Reference substance (positive control):

no

Key result

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

17 520 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: luminescence inhibition

Remarks on result:

other: 95% CI: 16850-18200 mg/L

Validity criteria fulfilled:

not specified

Conclusions:

The 15-min EC50 (luminescence inhibition) of PFBSK+ to Vibrio fischeri was 17520 mg/L (95% CI: 16850 - 18200 mg/L) per ISO 11348-3 standard protocol.

Executive summary:

The toxicity of PFBSK+ to Vibrio fischeri was assessed in a 15-minute test according to ISO 11348 -3 standard protocol. The 15-min EC50 (luminescence inhibition) of PFBSK+ to Vibrio fischeri was 17520 mg/L (95% CI: 16850 - 18200 mg/L). The study followed a national standard method but with minimal details; therefore, the study is deemed reliable with restrictions and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis.

.

Reference 4

Endpoint:

toxicity to microorganisms, other

Remarks:

paramecium, avoidance behavior

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Remarks:

backward swimming behavior in paramecium

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Determination of avoidance behavior
- Short description of test conditions: organisms were exposed in Tris-buffered KCl/CaCl2 solution and then transferred to a high-KCl solution to observe reaction.
- Parameters analysed / observed: Duration of net backward swimming

GLP compliance:

no

Specific details on test material used for the study:

Factor purity >95%

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Stock solutions were made at 10 mM in DMSO. Prior to the test, the stock solutions were diluted to obtain the necessary concentrations. From context, exposure was in both the reference (low-KCl) and test (high-KCl) solutions.

Test organisms (species):

Paramaecium caudatum

Details on inoculum:

Paramecium caudatum strain KNZ 82 was cultured in 0.5% wheat grass powder inoculated with Klebsiella pneumoniae two days before introduction of P. caudatum. Single cells were isolated and transferred to fresh culture medium 500 µL wells of a depression slide. After four days, paramecia in late log-phase or stationary phase were transferred to low-KCl reference medium for at least 30 minutes prior to exposure.

Test type:

static

Water media type:

other: buffered salts

Limit test:

no

Total exposure duration:

60 min

Post exposure observation period:

Exposure continued with transfer to high-KCl test medium. In some tests, calcium-channel inhibitors were added 60 s after transfer.

Test temperature:

20-24 °C

pH:

7.2

Nominal and measured concentrations:

Nominal concentrations only. For PFBSK+, only control (0 µM), 20 µM, 100 µM, and 200 µM were used. Other fluorinated and non-fluorinated surfactants were tested at a wider range of concentrations.

Details on test conditions:

TEST SYSTEM
Details on performance of the test are limited. Although not described in detail, it was determined from context that cells were first held 60 minutes in a reference solution which contained 1 mM KCl, 1 mM CaCl2, and 1 mM Tris-HCl (pH 7.2), plus the toxicant at the specified concentration. Cells were then transferred to the test solution containing 1 mM CaCl2, 1 mM Tris-HCl (pH 7.2), the toxicant at the same concentration as in the reference solution, and KCl at 20 mM (i.e., a potassium challenge). The paramecia were monitored by stereomicroscopy. After transfer, organisms swam backwards, gyrated in place, and eventually resumed forward-swimming. The net time spent swimming backwards was summed to determine duration of backwards swimming.

The mechanism of swimming reversal is controled by an increase in ciliary Ca2+ in association with a calcium-dependent depolarizing action potential at the membrane. An effect on backwards swimming duration may be caused by alteration in calcium ion transport. Nifedipine and other substances inhibit calcium transport and reduce the duration of backwards swimming. To confirm whether effects in this experiment were due to changes in calcium transport, in some experiments with surfactants other than PFBS, cadmium(II) (250 µM) or nifedipine (500 µM) was added to the test solution 60 s after transfer. Both agents are calcium channel blockers.

Critical micelle concentrations (CMCs) for anion surfactants were obtained from the literature. For anion surfactants showing significant prolongation of backwards swimming, logarithm of the ratio of backwards swimming duration (exposed/control) was plotted versus the logarithm of CMC.

Reference substance (positive control):

no

Key result

Duration:

60 min

Dose descriptor:

NOEC

Effect conc.:

100 µmol/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: backwards swimming behavior

Details on results:

PFBSK+ at 100 µM did not cause a change in duration of backwards swimming, but the next higher concentration (ca 200 µM) caused a significant (p < 0.05) increase in duration of backward swimming (See Illustration). No further testing was done with PFBSK+ and no EC50 could be calculated. Other surfactants were also tested. Perfluorooctane sulfonate was more effective than PFBSK+ in causing increased backwards swimming behavior, with an EC50 of 29.8 ± 4.1 µM. Similarly, sodium dodecyl sulfate had an EC50 of 17.1 ± 1.2 µM.

In all, thirteen anionic surfactants were tested (see Table 1). Among these, critical micelle concentration had a strong relationship with backwards swimming (R² = 0.8008, see Attachment 1). It should be noted that cationic surfactant exposure caused a reduction in backwards swimming duration, and the non-ionic surfactant Tween 20 had no effect on backwards swimming duration. Two fluorinated dicarboxylic acids were also tested and showed no significant effect at 100 µM.

In tests involving calcium channel blockers cadmium (Cd2+) and nifedipine, addition of the channel blocker to PFOS-exposed organisms 60 seconds after transfer to the high-K+ test solutions reduced the duration of backwards swimming (see Attachment 2). Only PFOS was tested in this way.

It is reasonable to conclude that the effects seen in this experiment are due to excess calcium channel activation, in a non-specific, surfactant-mediated mechanism. PFBS does not show appreciable surfactant strength and also does not show the same level of effect on backwards swimming as the other surfactants.

Reported statistics and error estimates:

The NOEC for PFBS was determined by ANOVA followed by Williams' test. Generally, dose-response data were assessed using ANOVA followed by Williams' test or Dunnett's test. Student's t-tests were used to evaluate the inhibitory experiments. Values of p < 0.05 were considered statistically significant. The Hill equation was used to calculate EC50 values.

Conclusions:

The NOEC for PFBSK+ on Paramecium caudatum backwards swimming (an avoidance response) was 100 µM (ca. 30 mg/L) on 60 minutes exposure.

Executive summary:

Effects of PFBSK+ on avoidance behavior (backwards swimming) in Paramecium caudatum were examined in conjunction with a variety of fluorinated and non-fluorinated surfactants. Organisms were exposed to a surfactant for 60 minutes in a low-potassium reference solution, and then transferred to a high-potassium challenge solution. Net backwards swimming time was determined after transfer. The NOEC for PFBSK+ was 100 µM (ca. 30 mg/L). For other anion surfactants, critical micelle concentration was correllated with an index of backwards swimming (R² = 0.8008), and backwards swimming was inhibited by addition of a calcium channel blocker. Backwards swimming thus appears to be affected by a non-specific effect of anionic surfactants on calcium transport. PFBSK+ is limited in its surface activity, and had less effect on behavior than the other anionic surfactants tested.

Details of the study are sparse. The tests followed valid scientific principals but the result was published in rapid-response journal with limited peer review. The relevance of this study to the endpoint is not clear. It is therefore assigned a Klimish 3 score. With other information, the result may be used in consideration of Adverse Outcome Pathways.

Description of key information

The 3-hour EC50 (respiration) of PFBSK+ to activated sludge is > 10000 mg/L and the corresponding NOEC is 1000 mg/L (Commission Directive 88/302/EEC; Official Journal of the EC L 133 Part C).

Additional information

In the key study, the toxicity of PFBSK+ to activated sludge was assessed according to the Commission Directive 88/302/EEC; Official Journal of the EC L 133 Part C. The study was performed in accordance with national standards under GLP and is deemed reliable without restriction and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis. It was chosen as the key study due to the clarity of the reported results.

In a supporting study, the toxicity of PFBSK+ to activated sludge was assessed according to the OECD 209 method. The 3-hour EC50 (respiration) of PFBSK+ to activated sludge is > 1000 mg a.i./L. The study was performed in accordance with internationally-accepted test guidelines under GLP and is deemed reliable

without restriction and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis. A second supporting study resulted in a 15-min EC50 (luminescence inhibition) of PFBSK+ to Vibrio fischeri of 17520 mg/L (95% CI: 16850 - 18200 mg/L) per ISO 11348-3 standard protocol. The study followed a national standard method but with minimal details; therefore, the study is deemed reliable with restrictions and suitable for use in Risk Assessment, Classification & Labeling, and PBT Analysis. In an additional study, effects of PFBSK+ on avoidance behavior (backwards swimming) in Paramecium caudatum were examined in conjunction with a variety of fluorinated and non-fluorinated surfactants. The 60-minute NOEC for PFBSK+ was 100 μM (ca. 30 mg/L). Details of the study are sparse. The tests followed valid scientific principals but the result was published in rapid-response journal with limited peer review. The relevance of this study to the endpoint is not clear. It is therefore assigned a Klimish 3 score and was not used to draw conclusions.