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

Sediment toxicity

Administrative data

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Study report refers to tested substance as perfluorobutane sulfonate and is unclear whether substance was tested as free acid or as a salt. PFBS is a strong acid that will exist predominantly in ionic form in the environment; accordingly, organisms 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 sediment toxicity.

Data source

Reference

Reference Type:

publication

Title:

Evolutionary ecotoxicology of perfluoralkyl substances (PFASs) inferred from multigenerational exposure: a case study with Chironomus riparius (Diptera, Chironomidae)

Author:

Stefani F, Rusconi M, Valsecchi S, Marziali L

Year:

2014

Bibliographic source:

Aquatic Toxicology Vol. 156, pp. 41-51.

Report date:

2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 233 (Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment)

Deviations:

yes

Remarks:

test extended to 10 generations at low concentration

GLP compliance:

no

Remarks:

data published in scientific literature

Test material

Test material information

Constituent 1

Reference substance name:

1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulphonic acid

EC Number:

206-793-1

EC Name:

1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulphonic acid

Cas Number:

375-73-5

Molecular formula:

C4HF9O3S

IUPAC Name:

1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulfonic acid

Specific details on test material used for the study:

Substance (perfluorobutane sulfonate) obtained from Sigma-Aldrich, purity not specified

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

No information on sampling. Aliquots of test solutions were injected directly, no purification.

Test substrate

Vehicle:

no

Details on sediment and application:

PREPARATION OF SPIKED WATER
- Details of spiking: stock solution of PFBS (plus perfluorooctane sulfonate and perfluorooctanoic acid, all tested separately) were made at 1 g/L in methanol. 150 µL of stock solution were diluted to 15 liters test water to make a test solution.
- Controls: No positive control, composition of negative control not specified
- Concentration of vehicle in final test solution: 10 µL methanol per liter

Test organisms

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Source: native population collected from the Lambro river, Milan, Lombardy, Italy, during April 2012
- Details on collection: PFAA concentration was measured at the collection site five times between April 2012 and April 2013. PFBS concentration was < 2 ng/L but the two other studied PFAAs could be detected.
- Age of parental stock: Larvae at L3 stage
- Breeding conditions: 20 °C until emergence and egg deposition. Egg ropes were collected on the day of maximum egg production rate and held until hatching. L1 larvae were collected and pooled for the test.
- Age of animals at beginning of exposure: Larvae at L1 stage.
- Feeding during test: yes, as per OECD233

Study design

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

yes

Exposure duration

Duration:

300 d

Exposure phase:

total exposure duration

Remarks:

approximate exposure time, study followed ten generations until emergence of adults in generation 10

Test conditions

Test temperature:

20 ± 1 °C

pH:

7.8 - 8.2

Dissolved oxygen:

>66% of air saturation

Conductivity:

350 µS/cm (beginning); 590 µS/cm (end)

Nominal and measured concentrations:

-Nominal: blank, 10 µg/L
-Measured: nd, 7.7 µg/L (PFBS)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass container, 19 cm x 19 cm x 18 cm.
- Depth of sediment and overlying water: 1 cm sediment depth. Water volume was one liter. Volume would fill an empty container to a depth of ca. 3 cm.
- Aeration: per OECD TG233
- Replacement of evaporated test water, if any: yes, with demineralized water

EXPOSURE REGIME
- No. of organisms per container (treatment): 60
- No. of replicates per treatment group: ten (two cages with five test chambers. Each cage was considered a replicate for genetic testing)
- No. of replicates per control / vehicle control: ten (two cages with five test chambers. Each cage was considered a replicate for genetic testing)
- Feeding regime: per OECD TG233
- Other: Study was multigenerational. To start the next generation, egg masses from each cage (five test vessels as one replicate) were collected during the 48 hours of maximum egg production rate and held until hatching. L1 larvae were collected and pooled and used to populate newly-made test chambers under that cage.

OVERLYING WATER CHARACTERISTCS
- Type of water: reconsitituted test water per an uncited US EPA method.

CHARACTERIZATION OF FORMULATED SEDIMENT
- Formulation: 75% aquarium sand, 250-300 µm grain size; 25% sieved and sterilized natural sediment, 63 - 250 µm grain size
- Contamination history of site: Natural sediment was taken from an unimpacted river.
- Maturation of artificial substrate (if any): Test chambers were allowed to condition seven days before addition of larvae.

OTHER TEST CONDITIONS
- Photoperiod: 16h light: 8 h darkness
- Light intensity: 500 - 1000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Number eggs per egg rope, each generation (A)
Number L1 larvae, each generation (B)
Number emerged adults, each generation (C)
Number emerged adult males, each generation (D)
Emergence ratio (C/B), each generation
Sex ratio (D/C), each generation
Microsatellite analysis, even generations only.

Microsatellite analysis: After emergence of the first adults, six larvae at L4 stage per vessel from each cage (30 total) were collected, weighed, and fixed in ethanol. DNA was extracted using AchievePure DNA kits (Eppendorf) following the protocol for fixed tissues. The microsatellite loci had been identified for C. riparius previously ((Nowak et al., 2006, Mol. Ecol. Notes Vol. 6, pp. 915–917). Variability at the microsatellite loci was analyzed using PCR amplification of the specific regions, with simultaneous (i.e., multiplex) amplification of loci and use one fluorescent dye-labelling primer to facilitate the analysis. Amplified fragments were typed using an Applied Biosystems ABI 3730XL sequencer with manual fragment reading. Estimation of allele frequencies, mean number of alleles, heterozygosity, and test of departure from the Hardy–Weinberg equilibrium were calculated by using the software Arlequin 3. 5 (Excoffier and Lischer, 2010. Mol. Ecol. Resour. Vol. 10, pp. 564 - 567). Evolutionary effects (mutation rate, effective population size, probability distribution of heterozygosity and mean number of alleles) were assesed using simulated datasets and Approximate Bayesian Calculation within DIYABC v.2.0 (Cornuet et al, 2014. Bioinformatics Vol. 30, pp. 1187 - 1189). Simulated datasets were assembled assuming three levels of effective population size (all assumed some level of reduction) and three mutation rates.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

300 d

Dose descriptor:

NOEC

Remarks:

approximate duration for ten generation study

Basis for effect:

emergence rate

Remarks on result:

not determinable

Remarks:

No effect at only concentration tested (10 µ/L nominal)

Effect concentrations 2

Duration:

300 d

Dose descriptor:

NOEC

Remarks:

approximate duration for ten generation study

Basis for effect:

sex ratio

Remarks on result:

not determinable

Remarks:

No effect at only concentration tested (10 µg/L nominal)

Effect concentrations 3

Duration:

300 d

Dose descriptor:

NOEC

Remarks:

approximate duration for ten generation study

Basis for effect:

reproduction

Remarks:

as eggs per egg rope

Remarks on result:

not determinable

Remarks:

No effect at only concentration tested (10 µg/L nominal)

Details on results:

- Emergence ratios for all generations: See attachment 1. Emergence ratio for PFBS v. control was significantly reduced in the second generation, but was not statistically different in any other generation.
- Ratio male/ female for all generations: No statistical difference was seen in sex ratio for any generation.
- Characteristics of egg ropes: See attachment 2. Number of eggs per egg rope showed statistically significant reductions in two generations only, which was not considered biologically relevant.
- Other biological observations: Genetic variability, expressed as mean number of alleles and expected heterozygosity rapidly decreased throughout the experiment (See attachment 3), which was a result of test conditions. None of the exposures had a statistically significant effect v. controls. At the allelic level, no temporal trend in accumulation of significant deviations from Hardy-Weinberg equilibrium was seen. Approximate Bayesian Calculation (Attachments 4 & 5) suggested an increase in mutation rate for all PFAAs tested including PFBS, based on minimization of significant differences between observed results and nine simulated cases. The relevance of the genetic component of this experiment towards aquatic toxicity of PFBS is impossible to determine at this time.

Reported statistics and error estimates:

Statistical analyses appear to have been implemented directly within the software cited.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Perfluorobutane sulfonate had no notable effects on C. riparius after 10 generations of exposure at 10 µg/L.

Executive summary:

Long-term effects of perfluorobutane sulfonate were examined in a ten-generation test of Chironomus riparius based on the OECD 233 test guideline. A natural population was gathered and exposed continuously until eggs were deposited. Eggs were then collected and used to establish the next set of test vessel. Ten vessels were established for each substance (two other perfluoroalkyl acid substances were tested in parallel), segregated into two cages containing five test vessels. Emergence ratio, sex ratio, and number of eggs per egg rope were determined for each generation. In addition, a genetic analysis was done every even generation of each cage as a population. Genetic analysis was done on five microsatellite loci previously identified for C. riparius. Differences were seen in organism effects at some generations, but no long-term changes were observed. Genetic analysis revealed substantial loss of diversity among all populations, with no temporal trends in heterozygosity, allele number, or divergence from Hardy-Weinberg equilibrium at the allele level. Simulated results suggests that the exposure to perfluorobutane sulfonate resulted in a more stringent effect on the population coupled to a higher mutation rate. It should be noted that the study does not precisely state the test substance used, but any perfluorobutane sulfonate material used at the pH range of this experiment (range, 7.8 - 8.1) would be entirely ionized. Testing is therefore equivalent to PFBSK+.

The study was based on established guidelines but was not GLP compliant. In addition, only one concentration was tested. The implications of the genetic analysis to ecotoxicological endpoints cannot be determined at this time. The study is considered reliable with restrictions, and may be used in a weight of evidence in Risk Assessment and PBT Analysis.