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

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to fish, other

Remarks:

extended fish embryo toxicity test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

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. However, test solutions had pH 7.4 ± 0.6, demonstrating that PFBS was entirely ionized during the test. Therefore, toxicity testing on the acid is equivalent to testing on the potassium salt.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 236 (Fish Embryo Acute Toxicity Test)

Version / remarks:

Guideline was modified to extend duration of exposure and add sub-lethal endpoints

Deviations:

not applicable

GLP compliance:

no

Remarks:

Academic research publication

Specific details on test material used for the study:

Factor purity >98%

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions prepared in [moderately hard] reconstituted [fresh] water as defined in ISO 7346-3
- Controls: Negative control only. Six other perfluoroalkyl acids were tested at the same time.
- Evidence of undissolved material: Stock solutions were prepared below substance solubility limits.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Strain AB
- Source: Internal stock

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: 2 females plus 3 males in each breeding group.
- Method of collection of fertilised eggs: On the evening before collection, breeding groups were placed in 10-L glass aquaria equipped with spawning nets. Eggs were collected 30 minutes after onset of illumination on the day of collection.
- Subsequent handling of eggs: Eggs were rinsed to remove debris and examined under a stereomicroscope. Only normally-developed, fertilized eggs in four-cell stage or later were selected for the test. These were randomly distributed to wells in the test plates within 15 minutes of collection.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

144 h

Hardness:

6.7° dH

Test temperature:

26 ± 1 °C

pH:

7.2 - 7.6

Conductivity:

468 µS/cm

Nominal and measured concentrations:

Blank, 10 mg/L, 30 mg/L, 100 mg/L, 300 mg/L, 1000 mg/L, 3000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Flat-bottom 48-well microplates (four each per substance tested)
- Type: closed (covered with parafilm)
- Material, size, headspace, fill volume: polystyrene, 750 µL test medium per well
- Aeration: None
- No. of fertilized eggs per vessel: Six per well
- No. of vessels per concentration (replicates): Four (distributed to separate plates)
- No. of vessels per control (replicates): Four (distributed to separate plates)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Moderately hard reconstituted fresh water per ISO7346-3 (1996)

OTHER TEST CONDITIONS
- Adjustment of pH: Not mentioned. However, given a highest concentration tested of 3000 mg/L, pH adjustment would have been necessary to maintain the measured pH ca. 7.4 at end of the exposure period.
- Photoperiod: 14 h light: 10 h dark

EFFECT PARAMETERS MEASURED (with observation intervals as hours post fertilization, hpf) :
Mortality
Embryo coagulation, 24, 48, 144 hpf
Lack of heart beat, 48, 144 hpf

Sublethal effects (combined for analysis)
Categorical sublethal variables
Tail, head, or eye malformations, 24, 48, 144 hpf
Reduced pigmentation, 48 hpf
Presence of edema, 48, 144 hpf
Lack of circulation, 48 hpf
Tremor, 48, 144 hpf
Side-laying, 144 hpf
Unhatched viable embryos, 144 hpf
Continuous sublethal variables
Number movements in one minute, 24 hpv
heart rate, 48 hpv
hatching time, 48-144 hpv

Mortality and sublethal effects observed by stereomicroscopy.

Neurobehavioral effects, 144 hpv after stereomicroscopy

For neurobehavioral effects, plates were read using a theViewpoint Zebrabox® behavioral recording system (ViewPointLife Sciences, Lyon, France). The insttrument contains an automated video camera and visible and infrared light sources for light and dark observations. Plates were acclimated in light for 10 minutes, after which activity was monitored in four consecutive, 10-minute light or dark phases. Activities were integrated over one-minute intervals in each ten minute observation period. For analysis of movement response variables, only active larvae were considered. Variables considered were the activity counts (number of movement events during the integration period, for active and non-active larvae), swimming time, and swimming distance, which were combined into average swimming speed, active swimming speed, and relative swimming time. The observations dataset was compared to the dataset of explanatory variables (chemical identity, with seven perfluoroalkyl acids tested; concentration expressed qualititatively as low (< EC50), medium (dose immediately above and below EC50), or high (>EC50); length of carbon chain; polar functionality (sulfonate v. carbonate); and observation period (1st dark interval through 2nd light). Comparison was by multivariate analysis of variance via permutation. The three characteristics (chemical, observation period, and qualitative concentration) were tested in a three-factor fully orthogonal analysis, with significance determined using 10,000 permutations. The permutation tests used the DISTLM version 5 program (Dept. Statistics, University of Auckland,New Zealand). The effects of the different explanatory characteristics on zebrafish behavior were also analyzed by Redundancy Analyses using CANOCO v. 4.54 program (Biometris, Wageningen, Netherlands).

It should be noted that due to excess mortality, no PFBS data was used from the "High" exposed samples.

Reference substance (positive control):

no

Duration:

144 h

Dose descriptor:

LC50

Effect conc.:

1 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

based on embryo coagulation or lack of heart beats

Remarks on result:

other: 95% CI, 1100 - 1900 mg/L

Duration:

144 h

Dose descriptor:

EC50

Effect conc.:

450 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Combined lethal and non-lethal effects

Remarks on result:

other: 95% CI, 350 - 600 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

300 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: heart rate

Duration:

144 h

Dose descriptor:

NOEC

Basis for effect:

time to hatch

Remarks on result:

other: no effect to maximum test concentration

Details on results:

Due to inadequate statistical power among individual sublethal endpoints, a combined EC50 was determined for lethal and non-lethal effects. NOECs were determined for heart rate and time to hatch, but not for combined lethal and sublethal effects. It should be noted that edema was a highly prevalent effect in this study, showing results for this substance as well as trifluoroacetic acid, perfluorobutanoic acid, and perfluorooctane sulfonic acid. In contrast, spinal curvature was noticed for other substances but not for PFBS.

In the neurobehavioral analysis, it was found that perfluoroalkyl acid substances (PFAAs) in general affect behavior as well as embryonic development, in both cases at concentrations higher than commonly measured in surface waters. Among the seven PFAAs tested, PFBS had the highest impact on behavioral endpoints relative to its toxicity, with a positive effect on swimming speed and a negative effect on other endpoints. There was a reduction in the overall activity at the highest concentrations relative to control, and the general tendency of D. rerio larvae to become more active after a sudden transition to darkness was supressed in these cases. It should be noted that PFBS had an intermediate EC50 and was tested in a higher concentration range than many of the other PFAAs. While the overall qualitative dose-response for the seven PFAAs was significant at the level P = 0.0001, no summary statistics were provided for dose-response of individual substances.

Reported statistics and error estimates:

The analyses were based on individual embryos as experimental units. The continuous data (heart rate, time to hatch, # movements counted in one minute) were analyzed using one-way ANOVA with two-sided Dunnett’s post hoc test. EC50 values with 95% confidence intervals were calculated for categorical data (all other endpoints) using probit analysis and defined as the concentration when 50% of the embryos displayed sublethal or lethal effects. Lowest observed effect concentration (LOEC) and no observed effect concentration
(NOEC) parameters were determined on the basis of Dunnett‘s test. Significance level was p < 0.05. All analyses of microscopy data were performed in Minitab® 16. Analysis of neurobehavioral data were as described above.

Validity criteria fulfilled:

not applicable

Conclusions:

The 144-hour LC50 of PFBS to D. rerio embryos was 1500 mg/L. The 144-hour EC50 for mortality plus sublethal effects was 450 mg/L. The 48-hour NOEC for heart rate was 300 mg/L. Effects were observed on swimming activity and speed at the highest concentrations tested. The test method was similar to OECD Guideline 236, but with an extended exposure period and with additional sublethal endpoint observations. The results are directly applicable to PFBSK+.

Executive summary:

Long-term toxicity of PFBS to fish was assessed in a static test conducted on Danio rerio. The method was similar to OECD Guideline 236, but with an extended exposure period and with additional sublethal endpoint investigated. Fertilized eggs were incubated in medium hard reconsitituted fresh water containing 0, 10, 30, 100, 300, 1000, or 3000 mg/L of PFBS. Six other perfluoroalkyl acid substances were tested in parallel. After the conclusion of a 144-hour exposure period during which embryos and larvae were examined by stereomicroscopy, fish swimming behavior was examined with an automated video system. The LC50 at 144 hours was 1500 mg/L. Individual sublethal endpoints did not provide adequate statistical power for correlation with concentration, but an EC50 of 450 mg/L was derived from the sum of all lethal and sublethal effects. A NOEC of 300 mg/L after a 48-hour exposure could be determined. Swimming behavior was also affected at higher concentrations, with a reduction of normal swimming activity on transition to darkness and an increase in swimming speed. PFBS is a strong acid that will exist predominantly in ionic form at environment pH-values, and in particular the range (7.4 ± 0.6) measured in this test. Therefore, embryos were exposed to PFBS- anions, and the study is equivalent to a test done with PFBSK+. Therefore, this study is relevant to the examination of long-term toxicity to fish and is directly applicable to PFBSK+.

The experiments described followed a method similar to an internationally-accepted and were published in peer-reviewed scientific journals. The method is generally considered to be an invitro fish acute test and a surrogate for live-animal testing. However, the exposure time was extended and additional observations on sublethal effects were added. The results are considered reliable with restrictions and suitable for use in a weight of evidence for long-term toxicity to fish.

Reference 2

Endpoint:

long-term toxicity to fish, other

Remarks:

extended fish embryo toxicity test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

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:

other: Draft Guideline for Fish Embryo Acute Toxicity Test

Version / remarks:

Draft Guideline was basis for current OECD Guideline 236. Study modified Draft to extend duration of exposure and add sub-lethal endpoints

Deviations:

not applicable

GLP compliance:

no

Remarks:

Academic research publication

Specific details on test material used for the study:

Factor purity ≥97%

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions prepared in medium-hard reconstituted fresh water as defined in OECD 203
- Controls: Negative and positive controls. Three other perfluoroalkyl acids were tested at the same time.
- Evidence of undissolved material: None, all chemicals were soluble in water for the tested concentrations.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Wild type
- Source:Commercial supplier (Aqua hobby, Heist-op-den-berg, Belgium)
METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: Eight aquaria with 4 females plus 4 males in each breeding group (64 total). Fish were maintained in aerated and biologically filtered medium hard reconstituted fresh water (MHRFW) at 26 ± 0.5 °C, 14 h / 10 h light/darkness, with two feedings per day of either micro feed pellets (Microgran, Sera, Heinsberg, Germany) or thawed Artemia nauplii. Water was renewed and aquaria cleaned once weekly.
- Method of collection of fertilised eggs: Spawn traps with a mesh bottom were used to prevent adults from eating eggs, and artificial plants were used as spawning stimulants. Spawning commenced within 30 minutes after onset of illumination and eggs were collected within 30 minutes after spawning.
- Subsequent handling of eggs: Eggs were rinsed in clean MHRFW to remove debris and examined under a stereo microscope. Only fertilized eggs without anomalies or damaged membranes were used. These were randomly distributed to individual wells in the test plates within 60 minutes after spawning.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

120 h

Test temperature:

26 ± 0.3 °C

pH:

7.2 - 7.5

Nominal and measured concentrations:

Nominal concentration only: control, 50 mg/L, 100 mg/L, 250 mg/L, 500 mg/L, 1000 mg/L, 3000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 24-well microplates (two each per test substance concentration)
- Type (delete if not applicable): closed (covered with self-adhesive foil)
- Material, size, headspace, fill volume: polystyrene, 2 mL per well
- Aeration: None
- No. of fertilized eggs/embryos per vessel: one per well. Each plate contained twenty exposed embryos at one concentration and four negative controls, 24 total per plate
- No. of vessels per concentration (replicates): Forty
- No. of vessels per control (replicates): Forty-eight (assumed using four control eggs per plate, two plates per test substance concentration, six concentrations)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Medium-hard reconsituted freshwater per OECD 203
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod:14 h:10 h light:darkness
- Other: Dead embryos and discarded chorion were removed after hatching

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : See Table 1. All determinations were made by stereomicroscopy. Abnormal embryo development was scored using standard development descriptions published by Kimmel et al., Dev. Dyn. 203, 253–310 (1995). A digital photograph was taken of each larva and ImageJ software (US National Institutes of Health, available at http://rsbweb.nih.gov/ij/) was used to estimate length.

Reference substance (positive control):

yes

Remarks:

3,4-dichloroaniline at 3.7 mg/L

Key result

Duration:

120 h

Dose descriptor:

NOEC

Effect conc.:

250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mortality and/or malformations

Remarks on result:

other: It is unclear from description of data analysis whether NOEC was based on an individual effect or aggregated mortality plus malformations

Duration:

120 h

Dose descriptor:

LC50

Effect conc.:

> 3 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

1 530 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Combined mortality and malformations

Remarks on result:

other: 95% CI, 1320 - 1780 mg/L

Details on results:

- Days to hatch and numbers hatched: No effect on hatching at highest concentration tested
- Data for length and weight of surviving fish: No effect on length at highest concentration tested
- Type of and number with morphological abnormalities: Malformations of embryos were apparent only at 96 h and 120 h. Pericardial and yolk sack edema were not observed for PFBS, but head and tail malformations were observed. In addition, uninflated swim bladder was observed among all test substances but was not used for scoring.
- Other biological observations: Due to low mortality, LC50 values could not be calculated for PFBS. Further, indicators of teratogenicity could not be calculated since LC50 values were not available. Significant (p<0.01) effects were found on heart rate at 48 h (pre-hatching, significant increase) and 72 h (post-hatching, significant decrease)
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

<10% mortality among negative controls. Positive control (3,4-dichloroaniline at 3.7 mg/L) showed 72% to 92% mortality.

Reported statistics and error estimates:

Mortality was corrected for percent mortality among controls. EC50 for combined mortality and malformations were calculated using the trimmed Spearman-Karber method using USEPA's trimmed Spearman-Karber v.1.5 software. One-way analysis of variance (ANOVA) followed by Dunnett’s test was used to determine the NOEC values based on all studied parameters. One-way ANOVA was used to determine significance of effects on heart rate and length and two-way ANOVA to determine effects on hatching. A Bonferroni correction was used to adjust for multiple testing of the data on heart rate, length and hatching. Data were considered significantly different when p-values were <0.05. All statistical tests were performed using GraphPad Prism 5 (GraphPad Software, San Diego, California, USA).

Validity criteria fulfilled:

not applicable

Conclusions:

The 120-hour LC50 of PFBSK+ to D. rerio embryos was >3000 mg/L. The 120-hour EC50 for mortality plus malformations was 1530 mg/L. The 120-hour NOEC was 250 mg/L. Effects were observed on heart rate at the highest concentrations (3000 mg/L) tested. The test method was similar to OECD Guideline 236, but with an extended exposure period and with additional sublethal endpoint observations.

Executive summary:

Long-term toxicity of PFBSK+ to fish was assessed in a static test conducted on Danio rerio. The method was similar to OECD Guideline 236, but with an extended exposure period and with additional sublethal endpoint investigated. Fertilized eggs were incubated in medium hard reconsitituted fresh water containing 0, 50, 100, 250, 500, 1000, or 3000 mg/L of PFBSK+. Three other perfluoroalkyl acid substances were tested in parallel. The LC50 at 144 hours was >3000 mg/L, with an EC50 of 1530 mg/L derived from the sum of all mortality and malformations scored. A 120-hour NOEC of 250 mg/L was also determined. Heart rate had showed significant effects at 48-h (prehatch) and 72-h (posthatch) at the highest concentration tested (3000 mg/L).

The experiments described followed a method similar to an internationally-accepted test guideline and were published in a peer-reviewed scientific journal. The method is generally considered to be an in-vitro fish acute test and a surrogate for live-animal testing. However, the exposure time was extended and additional observations on sublethal effects were added. The results are considered reliable with restrictions and suitable for use in a weight of evidence for long-term toxicity to fish.

Description of key information

The NOEC is expected to be in the range of 250-338 mg/L.

Additional information

Long-term toxicity of PFBSK+ to fish was addressed in two studies. Both were based on the Fish Embryo Toxicity test, which is normally considered a surrogate for short-term toxicity testing on adult fish. However, in both cases the duration of the test was extended and sublethal endpoints were added. Chapter R7b of the Guidance on Information Requirements notes that the then-current 48-h FET should be extended up to six days (144 hours) if there is evidence of delayed toxicity. The results of these tests are therefore accepted as long-term fish toxicity values. The first test, which was extended to 120 hours and included an explicit positive control, showed a NOEC of 250 mg/L, as well as an EC50 of 1530 mg/L for combined embryo mortality and malformations. The second test was extended to 144 hours and used the free acid form (PFBS) was used as test substance. Since test solution pH was measured and remained 7.4 ± 0.6, the test is equivalent to testing with PFBSK+. No positive control was used in the second test. However, the second test (as well as the first) included a number of perfluoroalkyl acid substances, and clear toxicity was shown for other the test substances. Therefore, lack of positive control is not a barrier to acceptance of the result. In the second test, an EC50 for combined mortality and sublethal effects of 450 mg/L PFBS was reported, however the equivalent NOEC was not provided (EC50 is equivalent to 507 mg/L PBSFK+ on a molecular weight basis). Specific NOECs of 300 mg/L PFBS for 48-h heart rate (equivalent to 338 mg/L PFBSK+) and 1000 mg/L for 144-h hatching time (1130 mg/L PFBSK+) were provided. In addition, effects on swimming activity were reported at 144 hours at the higher test concentrations (≥1000 mg/L PFBS). By a weight of evidence, PFBSK+ is expected to show little long-term toxicity to fish.