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REACH

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

EC number: 249-616-3 | CAS number: 29420-49-3

Life Cycle description
Uses advised against

Environmental fate & pathways

Bioaccumulation: terrestrial

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference 1

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

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- in a laboratory setting using soils amended with a commercial PFOS mixture containing PFBS. 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 bioaccumulation in terrestrial invertebrates

Qualifier:

no guideline followed

Principles of method if other than guideline:

Worms were exposed for 28 days to soil which had been previously been amended with commercial chemical mixtures and then used to grow corn (Zea mays)

GLP compliance:

Specific details on test material used for the study:

Study used a commercial PFOS mixture as source, identified as perfluorooctanesulfonate, and analyzed for PFBS as anion by LCMS. It is unclear whether the free acids or salts were used in this test.

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: worms were analyzed at the end of the test period.
- Sampling intervals/frequency for test medium samples: soils were analyzed at the beginning of the earthworm exposure period (end of the corn-growing period).
- Details on sampling and analysis of test organisms and test media samples:

Test matrix: Soils were air-dried before processing. Samples (0.1 g) were spiked with 0.5 µL of N-d3-MeFOSA and 0.5 µL of N-d5-EtFOSA, and extracted with methanol in a polypropylene tube (10 min agitation at room temperature, 30 min ultrasonication at 40 °C) and centrifuged for 15 min at 3000 rpm. Ultrasonication and centrifugation were repeated with fresh methanol. Supernatant methanol layers were combined and cleaned by passage through EnviCarb cartridges (500 mg, 6 mL). Extractes were diluted to one liter with Milli-Q and loaded in Oasis WAX cartridges (500 mg, 6 mL). Eluent and volume were not stated. Eluate volume was reduced to 140 µL under a gentle stream of nitrogen. 240 µL of methanol and 240 µL of 2 mM ammonium acetate were added to the final extract spiked with 0.5 µL of 13C9-PFNA solution prior to HPLC-MS/MS injection.

Tissue samples: Earthworms were held 24 hours on clean substrate to depurate particulate matter, and then freeze-dried at ca. −50 °C for 24 h in a lyophilizer. Earthworm samples (0.2 g) were spiked with 0.5 µL of N-d3-MeFOSA and 0.5 µL of N-d5-EtFOSA were extracted with acetonitrile (vortex-mixing, 10 min shaking, 30 min ultrasonication at 40ºC) and centrifuged for 15 min at 3000 rpm. Extraction was repeated once, and extracts were combined and evaporated to 2 mL under nitrogen. 100 µL of acetic acid was added, extracts were centrifuged 5 min at 2000 rpm, and supernatant passed through EnviCarb SPE cartridges. The purified extract was then reduced according to the procedure previously described for soils.

Vehicle:

Details on preparation and application of test substrate:

- Method of mixing into soil: Soils received either a commercial PFOS (perfluorooctanesulfonate) mixture at 50 mg PFOS/kg soil, or the commercial PFOS mixture at 50 mg PFOS/kg soil with a DecaBDE fire retardant at 5 mg DecaBDE/kg soil, and potted. Corn plants were grown in the soil for 28 days. After removal of the plants, the pots were used for the earthworm experiment.
- Controls: Untreated soil

Test organisms (species):

Eisenia andrei

Total exposure / uptake duration:

28 d

Total depuration duration:

1 d

Test temperature:

21 ± 1 °C

pH:

7.5

TOC:

3.0 %

Moisture:

irrigation at 100 mL/day, ambient air humidity 55-60%

Details on test conditions:

TEST SYSTEM
- No. of organisms per container (treatment): Ten
- No. of replicates per treatment group: Six
- No. of replicates per control / vehicle control: Six

SOURCE AND PROPERTIES OF SUBSTRATE
- Soil texture
- % sand: 82.2%
- % silt: 3.8% coarse (0.02- % clay:8.4 %
- Soil taxonomic classification: Loamy sand
- pH soil: 7.5
- Organic carbon (%): 3.0 %

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

Nominal and measured concentrations:

Measured, 0.04 ± 0.01 µg/g dw at end of exposure period

Key result

Type:

BCF

Value:

ca. 2.33 - ca. 2.75 dimensionless

Basis:

whole body d.w.

Calculation basis:

steady state

Remarks on result:

other: Results for two treaments. Values are Bioaccumulation Factors (BAFs), without correction for organism lipid or soil organic carbon content

Validity criteria fulfilled:

not applicable

Conclusions:

PFBS has a BAFs of 2.33 and 2.75 (results for two individual treatments) on a dry weight basis for Eisenia andrei in treated soil. Results are directly applicable to PFBSK+.

Executive summary:

PFBS bioaccumulation potential was assessed in a test conducted on earthworm (Eisenia andrei). Soils were amended with a commercial mixture of perfluorooctane sulfonate (PFOS) containing PFBS or with the PFOS mixture plus commerical DecaBDE fire retardant. Perfluorohexanesulfonate was also present in the mixture. It is ambiguous from the published materials whether the materials were added as free acids or as salt mixtures. PFBS was analyzed as the anion. Soils were first used to grow corn (Zea mays), after which plant materials were removed for analysis and earthworms added. After 28 days' exposure, and 24 hours' depuration, BAFs of 2.33 and 2.75 (dry weight basis) were calculated. PFBS is not expected to bioaccumulate in soil invertebrates. PFBS is a strong acid that will exist predominantly in ionic form in the environment, especially at the native pH (7.5) of the soil, and 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 terrestrial invertebrates and is directly applicable to PFBSK+. PFBSK+ is not expected to bioaccumulate in soil invertebrates.

While details are scarce, this study followed an accepted scientific procedures. 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 nature of the substance initially containing the samples is of little relevance due to dissociation and respeciation of the materials in the soil. The study is deemed reliable with restrictions and is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis as part of a weight of evidence approach.

Reference 2

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 soils amended with multiply-contaminated sewage sludge 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 bioaccumulation in terrestrial invertebrates

Qualifier:

no guideline followed

Principles of method if other than guideline:

Worms were exposed for 21 days to soil in microcosms amended with contaminated biosolids

GLP compliance:

Specific details on test material used for the study:

Study used contaminated biosolids as soil amendment, and analyzed for PFBS as anion by LCMS. No information is available regarding the original substance(s).

Radiolabelling:

Details on sampling:

Vehicle:

Details on preparation and application of test substrate:

- Method of mixing into soil: Soils were amended with one of four biosolids which had previously been analyzed. Amendment rate was linked to nitrogen requirement of the seeded plants and ranged from 0.12 to 0.56 kg per treatment in 8 kg total soil.
- Controls: Untreated soil

Test organisms (species):

Eisenia andrei

Total exposure / uptake duration:

21 d

Total depuration duration:

1 d

Test temperature:

21 ± 1 °C

Moisture:

irrigation at 100 mL/day, ambient air humidity 55-60%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): PVC cylinders, 30 cm high, 20 cm internal diameter, closed with nylon mesh at bottom to minimize soil loss.
- Amount of soil or substrate: 8 kg
- No. of organisms per container (treatment): 20
- No. of replicates per treatment group: Three
- No. of replicates per control / vehicle control: Three
- Other: Microcosms additionally were seeded with thirty seeds of Triticum aestivum, Brassica rapa and Vicia sativa

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- History of site: Typical agricultural soil of known history
- Treatments with pesticides or fertilizers: not in ten years prior to test
- Depth of sampling: upper 20 cm of soil profile
- Other: Soil sieved (2 mm) and homogenized before use. Four biosolids were used for amendment: 1) aerobically digested municipal solid waste (MSW) compost; 2) anaerobically digested thermal drying sludge; 3) aerobically digested composted sewage sludge; 4) anaerobically digested MSW compost. While these biosolids had the highest aggregate concentrations of substances of interest among 16 potential sources evaluated, only biosolid 4) contained detectable concentrations of PFBS (2.05 ± 0.36 µg/kg dw)

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

Nominal and measured concentrations:

PFBS concentrations were

Key result

Type:

BCF

Basis:

whole body d.w.

Calculation basis:

other:

Remarks on result:

not determinable

Remarks:

Substance tissue concentrations

Details on results:

Biosolid samples were contaminated with multiple fluorinated and halogenated materials. PFOS could be detected and quantified in all soil samples at levels similar to PFBS, and could also be quantified in tissue samples.

Validity criteria fulfilled:

not applicable

Conclusions:

PFBS could not be detected in Eisenia andrei reared for 21 days in treated soil. Results are directly applicable to PFBSK+.

Executive summary:

PFBS bioaccumulation potential was assessed in a test conducted on earthworm (Eisenia andrei). Microcosm soils were amended with one of four multiply-contaminated biosolids samples in triplicate (15 systems including controls). Perfluorooctanesulfonate was also present in the biosolids. The nature of the substance(s) present in the biosolids cannot be assigned since it results from multiple sources. PFBS was analyzed as the anion. Microcosms were simultaneous seeded with three plant varieties and provided with 20 earthworms. After 21 days' exposure, and 24 hours' depuration, PFBS could be detected in three of four microcosms but could not be detected in earthworm tissue. BAFs could not be calculated. PFBS is a strong acid that will exist predominantly in ionic form in the environment, and 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 terrestrial invertebrates and is directly applicable to PFBSK+.

While details are scarce, this study followed acceptable scientific procedures. 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 nature of the substance initially contaminating the samples is of little relevance due to dissociation and respeciation of the materials in the soil. The study is deemed reliable with restrictions and is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis as part of a weight of evidence approach.

Reference 3

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 report is unclear whether substance was tested as free acid or salt. 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 bioaccumulation in terrestrial invertebrates

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 317 (Bioaccumulation in Terrestrial Oligochaetes)

Deviations:

not applicable

GLP compliance:

Specific details on test material used for the study:

PFBS was purchased from TCI at 98% purity. Substance was identified as perfluorobutane sulfonate.

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: Worms sampled on exposure days 2, 4, 6, 8, 12, 16, 20, 24, 28 and 30 and depuration days 2, 4, 6, 8, 12, 16, 20, 24, 28.
- Sampling intervals/frequency for test medium samples: Soil appears not to have been analyzed. Rather, nominal concentrations were used where needed.
- Sample storage conditions before analysis: Frozen at -20 °C after lyophilization and grinding.
- Details on sampling and analysis of test organisms and test media samples: On sampling days, worms were removed from soil and kept on clean paper for 24 hours to depurate gut contents. Worms were then pooled, washed with tap water and then distilled water, then dried with clean towel paper and weighed immediately. Worms were then frozen at -20 °C for 12 h, freeze-dried for 48 h, and weighed again. Dried earthworms were ground in a methanol-washed mortar and pestle under liquid nitrogen and stored until extraction.

For extraction, ca. 0.1 g dried earthworm sample was extracted 3 times with 5 mL of 10 mM NaOH in methanol (vortex-mixing, 16 h shaking at 250 RPM, and 15 min centrifugation at 3500 RPM). Supernatant liquid was transferred to a fresh tube, combining replicate extracts from the samples, and evaporated to 2 mL under a gentle stream of nitrogen. Extracts were then diluted in 100 mL water and purified using solid phase extraction (SPE). Clearnert PEP (Polar Enhanced Polymer) SPE columns (500 mg/6 mL, Agela Technologies) were preconditioned with 10 mL of methanol, followed by 10 mL of water. Extracts were then loaded to the SPE columns, which were then dried under vacuum. Columns were eluted with 10 mL of methanol, which was evaporated under a gentle stream of nitrogen to ca. 1mL and centrifuged at 12,000 rpm for 30 min.

Vehicle:

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): Ten perfluoroalkylated substances (PFASs) were dissolved in methanol. Prepared soil was weighed out. A small portion of pre-weighed soil was spiked with one mL of stock solution of 10 PFASs dissolved mixed thoroughly. Solvent was removed by placing the soil in a fume hood for 24 h. Aliquots of the remaining untreated soil were added to the spiked soil and then mixed thoroughly, repeating addition until all weighed-out soil was incorporated. Soils were then shaken five times per day for six days, and stored in darkness at room temperature until use (4 days). Three spiked soils were prepared in this fashion, at 100 µg/kg, 200 µg/kg, and 500 µg/kg in each PFAS
- Controls: untreated soil

Test organisms (species):

Eisenia fetida

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: local earthworm farm in Tianjin, China
- Age at test initiation: mature
- Weight at test initiation: ca. 3 grams over 10-13 worms
- Weight at termination: worms lost weight over the study period, amount of weight loss not specified.

ACCLIMATION
- Acclimation period: >14 days

Total exposure / uptake duration:

30 d

Total depuration duration:

28 d

Test temperature:

22 ± 2 °C

pH:

7.67

TOC:

4.88%

Moisture:

30%

Details on test conditions:

TEST SYSTEM
- Test container: 250 mL glass beakers
- Amount of soil or substrate: 100 g
- No. of organisms per container (treatment): 10 to 13
- No. of replicates per treatment group: three
- No. of replicates per control / vehicle control: three
- Biomass loading rate: 3 g / vessel

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site: about 20 km southwest of Tianjin, China
- Other: No detectable PFAS contamination
- Depth of sampling: 0 - 10 cm
- Soil texture
- % sand: 12%
- % silt: 64%
- % clay: 24%
- Soil taxonomic classification: loam
- pH soil: 7.67
- Organic carbon (%): 4.88%
- Cation exchange capacity: 38.47 cmol/kg
- Moisture (%): 1.03% in air dried soil, brought to 30% for experiment
- Preparation: Soil was air-dried for two weeks, ground, and sieved to 2 mm

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous darkness
- Feeding: none

VEHICLE CONTROL PERFORMED: no

Nominal and measured concentrations:

Nominal, 0 µg/kg, 100 µg/kg, 200 µg/kg, 500 µg/kg

Key result

Type:

BSAF

Value:

0.048 other: grams oc/grams dw

Basis:

whole body d.w.

Remarks:

not corrected for lipid content

Calculation basis:

kinetic

Remarks on result:

other:

Remarks:

at 200 µg/kg nominal

Type:

BSAF

Value:

0.021 other: gram dw/gram ww

Basis:

whole body w.w.

Remarks:

not corrected for lipid content

Calculation basis:

steady state

Remarks on result:

other: at 100 µg/kg nominal

Type:

BSAF

Value:

0.011 other: gram dw / gram ww

Basis:

whole body w.w.

Remarks:

not corrected for lipid content

Calculation basis:

steady state

Remarks on result:

other: at 200 µg/kg nominal

Type:

BSAF

Value:

0.008 other: gram dw/gram ww

Basis:

whole body w.w.

Remarks:

not corrected for lipid content

Calculation basis:

steady state

Remarks on result:

other: at 500 µg/kg nominal

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

9.6 d

Kinetic parameters:

- Uptake rate constant k(s): 0.003 ± 0.001 gram oc/gram dw day
- Depuration rate constant k(e):0.072 ± 0.010 /day
- Time to reach 90% of steady state: 31.9 days

Details on results:

- Mortality of test organisms: none
- Observations on feeding behavior: worms were not fed during test.
- Observations on body length and weight: worms lost weight by the end of the test period
- Other biological observations: despite weight loss, worms from spiked soils appeared no different than worms from control soils. All worms appeared to be in good health.
- Mortality and/or behavioural abnormalities of control: no

Reported statistics:

Uptake and elimination constants (ku and ke) were calculated using non-linear regression within Origin v.8.0 software. First-order models were used in both cases. Elimination rate constant was calculated using C = Co * e^(-ke * t), where C is concentration at time t, and Co is initial concentration. Uptake rate constant followed C = [(ku * Cs)/ke] * [1-e^(-ke * t)], where Cs is the organic carbon-normalized concentration of PFBS on a dry weight basis.

Validity criteria fulfilled:

not applicable

Conclusions:

PFBS has a BSAF (kinetic) 0.048 on a tissue dry weight basis, normalized to soil organic carbon content, for Eisenia fetida in treated soil. Results are directly applicable to PFBSK+.

Executive summary:

PFBS bioaccumulation potential was assessed in a test conducted on earthworm (Eisenia fetida). Soils were amended with a mixture of ten perfluoroalkyl substances including PFBS. It is ambiguous from the published materials whether the materials were added as free acids or as salt mixtures. PFBS was analyzed as the anion. Concentrations in worm tissue were followed over a 30-day exposure period and a 28-day depuration period. Worms were allowed 24 hours on clean tissue to depurate treated soil before extraction and analysis. A kinetic BSAF of 0.048 was calculated based on tissue dry weight concentrations and soil organic carbon content. PFBS is not expected to bioaccumulate in soil invertebrates. PFBS is a strong acid that will exist predominantly in ionic form in the environment, especially at the native pH (7.67) of the soil, and 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 terrestrial invertebrates and is directly applicable to PFBSK+. PFBSK+ is not expected to bioaccumulate in soil invertebrates.

Reference 4

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

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- in a laboratory setting using soils amended with a commercial PFOS mixture containing PFBS. As PFBS is a strong acid that will exist predominantly in ionic form in the environment, plants 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 bioaccumulation in terrestrial plants.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Corn plants (Zea mays) were grown for 28 days in soil which had been previously been amended with one of two commercial chemical mixtures.
Spinach (Spinacia oleracea) was grown for 28 days in soil which had been amended with biosolids.
Tomato (Solanum lycopersicum L.) was grown for 6 months in soil which had been amended with biosolids.

GLP compliance:

Specific details on test material used for the study:

Study used a variety of mixtures as sources, and analyzed for PFBS as anion by LCMS. It is unclear whether the free acids or salts were used in this test. At neutral pH, the testing is equivalent to PFBSK+

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: plant material was analyzed at the end of the test period.
- Sampling intervals/frequency for test medium samples: soils were analyzed at the beginning and end of the plant-growing period.
- Details on sampling and analysis of test organisms and test media samples:

Test matrix: Soils were air-dried before processing. Samples (0.1 g for corn, 5 g for spinach and tomato) were spiked with 0.5 µL of N-d3-MeFOSA and 0.5 µL of N-d5-EtFOSA, and extracted with methanol in a polypropylene tube (10 min agitation at room temperature, 30 min ultrasonication at 40 °C) and centrifuged for 15 min at 3000 rpm. Ultrasonication and centrifugation were repeated with fresh methanol. Supernatant methanol layers were combined and cleaned by passage through EnviCarb cartridges (500 mg, 6 mL). Extracts were diluted to one liter with Milli-Q water and loaded in Oasis WAX cartridges (500 mg, 6 mL). Eluent and volume were not stated. Eluate volume was reduced to 140 µL under a gentle stream of nitrogen. 240 µL of methanol and 240 µL of 2 mM ammonium acetate were added to the final extract spiked with 0.5 µL of 13C9-PFNA solution prior to HPLC-MS/MS injection.

Tissue samples: Plant material was freeze-dried at ca. −50 °C for 24 h in a lyophilizer. Mass and type of extracted tissue varied by species: spinach: 1 g whole plant; tomato: 1.5 g root, 5 g stem, 5 g leaf, 4-8 g of ripe fruit; corn: 0.01 g root, 0.1-0.4 g leaf. Tissue samples were spiked with 0.5 µL of N-d3-MeFOSA and 0.5 µL of N-d5-EtFOSA were extracted with acetonitrile (vortex-mixing, 10 min shaking, 30 min ultrasonication at 40ºC) and centrifuged for 15 min at 3000 rpm. Extraction was repeated once, and extracts were combined and evaporated to 2 mL under nitrogen. 100 µL of acetic acid was added, extracts were centrifuged 5 min at 2000 rpm, and supernatant passed through EnviCarb SPE cartridges. The purified extract was then reduced according to the procedure previously described for soils.

Vehicle:

Details on preparation and application of test substrate:

- Method of mixing into soil:
Tomato, Spinach: Soils received one of two biosolids at a rate corresponding to plant nitrogen requirements. Biosolids were analyzed and contained multiple perfluorinated acid substances as well as other halogenated materials.
Corn: Soils received either a commercial PFOS (perfluorooctanesulfonate) mixture at 50 mg PFOS/kg soil, or the commercial PFOS mixture at 50 mg PFOS/kg soil with a DecaBDE fire retardant at 5 mg DecaBDE/kg soil, and potted.
- Controls: Untreated soil

Test organisms (species):

other: Zea mays, Solanum lycopersicum L, Spinacia oleracea

Total exposure / uptake duration:

28 d

Test temperature:

21 ± 1 °C

pH:

7.5

TOC:

3.0 %

Moisture:

irrigation at 100 mL/day, ambient air humidity 55-60%

Details on test conditions:

TEST SYSTEM
- No. of replicates per treatment group: Six (corn) or eight (tomato, spinach)
- No. of replicates per control / vehicle control: Six (corn) or eight (tomato, spinach)

SOURCE AND PROPERTIES OF SUBSTRATE
- Soil texture
- % sand: 82.2%
- % silt: 3.8% coarse (0.02- % clay:8.4 %
- Soil taxonomic classification: Loamy sand
- pH soil: 7.5
- Organic carbon (%): 3.0 %

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

Nominal and measured concentrations:

Corn: 0.04 ± 0.01 µg/g dw at end of exposure period
Tomato: Treatment 1, not detected in biosolids nor in soil at initiation or at end of exposure period
Treatment 2, 2.05±0.36 ng/g dw biosolids, 0.13±0.03 ng/g dw at initiation, 0.20±0.09 ng/g dw at end of exposure period
Spinach: Treatment 1, not detected in biosolids or in soil at initiation, 0.20 ng/g dw at end of exposure period
Treatment 2, 2.05±0.36 ng/g dw biosolids, not detected in soil at initiation or at end of exposure period

Key result

Type:

BCF

Value:

ca. 3.75 dimensionless

Basis:

other: Root tissue d.w.

Calculation basis:

other: tissue conc. to final soil conc.

Remarks on result:

other: Results for Z. mays, Treatment 1. Values are Bioaccumulation Factors (BAFs), without correction for tissue lipid or soil organic carbon content

Key result

Type:

BCF

Value:

ca. 3 - ca. 33 dimensionless

Basis:

other: Leaf tissue d.w.

Calculation basis:

other: tissue conc. to final soil conc.

Remarks on result:

other: Results for Z. mays, Treatments 1&2. Values are Bioaccumulation Factors (BAFs), without correction for tissue lipid or soil organic carbon content

Type:

BCF

Remarks on result:

not determinable

Remarks:

Details on results:

PFBS was detected in corn root and leaf tissue.
In treatment 1, root concentration was 0.15±0.05 µg/g d.w., and leaf concentration was 0.12±0.03 µg/g d.w.
In treatment 2, root concentration was Transfer factors (bioaccumulation factors) were calculated for this summary based on final soil concentration

PFBS was not detected in spinach tissue (<0.269 ng/g d.w). No transfer factor (Bioaccumulation factor could be determined).

PFBS was not detected in tomato tissues (root, <0.406 ng/g d.w.; stem, <0.233 ng/g d.w.; leaf, <0.801 ng/g d.w; fruit, <0.047 ng/g d.w.). No transfer factor (Bioaccumulation factor could be determined).

Validity criteria fulfilled:

not applicable

Conclusions:

PFBS had a BAFof 3.75 in corn (Zea mays) root tissue and range of ca. 3.0 - 33 in leaf tissue on a dry weight basis after 28 days' growth in soil spiked with commercial perfluorooctane sulfonate mixtures. Spinach (Spinacia oleracea) and tomato (Solanum lycopersicum L.) plants showed no detectable tissue levels after growth in soils treated with biosolids. PFBS could not be detected at most time point in the biosolids-treated soil.

Executive summary:

PFBS bioaccumulation potential was assessed in a test conducted on corn (Zea mays) spinach (Spinacia oleracea) and tomato (Solanum lycopersicum L.). Soils for the corn trial were amended with a commercial mixture of perfluorooctane sulfonate (PFOS) containing PFBS or with the PFOS mixture plus commerical DecaBDE fire retardant. The remaining trials were done with biosolids amendment. Biosolids contained multiple perfluorinated acid and other halogenated substances. Perfluorobutanesulfonate ion was the analyte, and it is ambiguous from the published materials whether the materials were added as free acids or as salt mixtures. PFBS is a strong acid that will exist predominantly in ionic form in the environment, especially at the native pH (7.5) of the soil, and plants 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 plants and is directly applicable to PFBSK+. PFBS anions were seldom detected in biosolids-amended soils and were not detected in tissues of spinach (28 day exposure) or tomato (6 months exposure). PFBS was detected in spiked soils used for corn, but BAFs were ca 3.0 to 33 after 28 days' exposure. PFBSK+ is not expected to bioaccumulate in terrestrial plants.

Reference 5

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

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- in a laboratory setting using soils amended with biosolids containing a variety of perfluoroalkyl acid substances. 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 bioaccumulation in terrestrial plants.

Qualifier:

no guideline available

Principles of method if other than guideline:

Plants were grown from seed until maturation in biosolids-amended soil. Concentration of PFBS anion was measured in soil and edible tissue.

GLP compliance:

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: Lettuce leaves were sampled at maturity (56 days after sprouting for greenhouse phase, ca. 45 days for field phase.
- Sampling intervals/frequency for test medium samples: Duplicate samples at end of exposure period.
- Sample storage conditions before analysis: Samples frozen at -20 °C before analysis
- Details on sampling and analysis of test organisms and test media samples:
Plant material was homogenized in a food processor prior to extraction. Homogenized plant tissue (0.5−2 g) was transferred to a 50 mL polypropylene vial, to which a surrogate spiking solution containing 2 ng each of isotopically labeled surrogate standards was added. 7 mL of solvent mixture (50/50 dichloromethane (DCM) and 99:1 (v/v) methanol (MeOH):ammonium hydroxide) was added to the sample and heated (30 °C) in a sonication bath for 30 min followed by shaking for 1 hour. The sample was centrifuged at 1467xg (RCF) for 20 min, and the extract was decanted into a separate 50 mL tube. This procedure was done for a total of three extraction cycles. The combined extract was evaporated at 50 °C under nitrogen to dryness. The extract was dissolved and given an oxidative clean-up with 1 mL of a basic hydrogen peroxide solution (20 μL ammonium hydroxide and 980 μL 30% hydrogen peroxide), vortexed, and sonicated in a heated (30 °C) bath for 2 h. An additional aliquot (7 mL) of the basic DCM/MeOH mixture was added to each oxidized extract, vortexed, and heated in a sonication bath for 30 min. The extract was centrifuged at 1467 xg for 20 minutes and decanted into a glass 20 mL scintillation vial. This reextraction procedure was done for a total of three cycles. The combined extract was evaporated at 50 °C under nitrogen to dryness and reconstituted with 1 mL of 99:1 (v/v) MeOH and acetic acid. The extract was run through a cleanup column packed with 100 mg of diamino and 100 mg of ENVICarb. For analysis, 105 μL of the cleaned extract was transferred to an autosampler vial and diluted with 1350 μL of pure water plus 45 μL of 0.01% aqueous ammonium hydroxide.

Separate aliquots of plant tissue were dried overnight at 70 °C to determine dry weight of the material. Concentration was expressed on a dry weight basis.

For soil extraction, 1 g aliquots were weighed into 50-mL polypropylene vials to which a solution containing isotopically-labeled surrogate standard was added prior to sequential extraction via sonication with basic methanol. All extracts were combined, evaporated to dryness, reconstituted in acidic methanol, subjected to a dispersed ENVI-Carb™ clean-up, and analyzed by LC-MS/MS.

Separate aliquots of soil were dried overnight at 105°C to determine moisture content. All results are reported on a dry weight basis.

Vehicle:

Details on preparation and application of test substrate:

Test materials were not added to soils as pure materials but as mixtures in biosolid preparations. Two soils were used. The control soil had no reported history of biosolids amendment. The same soil was amended with 10% composted biosolids on a mass basis to create an 'industrial' soil. The biosolids were from a small, municipal STP impacted by PFAA manufacturing. The second ('municipal') soil came from a reclamation site in Illinois, USA, where municipal biosolids had been applied at reclamation rates for 20 years. The two sites were in the same vicinity. No additional test substance(s) was added.

Test organisms (species):

other: Lactuca sativa

Details on test organisms:

Lettuce (Lactuca sativa)

Total exposure / uptake duration:

56 d

Total depuration duration:

0 d

Test temperature:

Daytime: 18 °C - 21 °C
Nighttime: 10 °C - 13 °C

pH:

6.4 - 7.6

TOC:

1.45 - 6.34%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 15 cm squat pots containing 1 kg dw soil. Soils were sieved to 6.3 mm for homogeneity before filling the pots.
- No. of organisms per container (treatment): Two seeds per pot, each pot considered a single replicate
- No. of replicates per treatment group: Five
- No. of replicates per control / vehicle control: Five

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site: Illinois, USA
- History of site: For the reclamation site, municipal biosolids had been applied at reclamation rates over the previous 20 years, reaching the cumulative biosolids application rate of 1654 Mg/ha. The control soil, which was also intentionally mixed with biosolids to make an "industrial" soil, was taken from nearby fields and had experienced similar cropping systems.
- Vegetation cover: This field had been planted with rotations of cereal crops such as corn, wheat, and sorghum.
- Treatments with pesticides or fertilizers: The control soil had received only commercial fertilizers. No information was given on pesticide usage.
- Soil taxonomic classification: Both were classified as Lenzburg silt loams (See Table 1).
- Moisture: Drip irrigation was supplied based on crop needs and seasonal demand (2-3 times per day for 1-2 min, totalling ca 100-600 mL/day)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours total of either daylight or full-spectrum supplemental lighting

OTHER
In addition, a limited-scale field study was conducted in the Midwestern U.S. Plots (3.0 m × 4.6 m) were established and planted with lettuce (Lactuca sativa ‘Black-Seeded Simpson’) and tomato (Lycopersicon lycopersicum ‘Burpee Big Boy Hybrid’). Fertilization via biosolids occurred at four application rates (plus control). Treatments were one-half of the agronomic rate of biosolids application to meet crop nitrogen (N) requiremen (0.5×), agronomic rate (1×), two times the agronomic rate (2×), and four times the agronomic rate (4×). Each treatment (and control) was done in triplicate. Crops were grown and harvested following normal agricultural practices.

Key result

Type:

BCF

Value:

> 4.22 - < 14.5 dimensionless

Basis:

other: leaf tissue concentration

Calculation basis:

other: final concentrations at tissue maturity

Remarks on result:

other: result from greenhouse experiment with two different soils

Type:

BCF

Value:

<= 2.02 dimensionless

Basis:

other: leaf tissue concentration

Calculation basis:

other: final concentrations at tissue maturity

Remarks on result:

other: results of field experiments at four application rates

Table 2, Concentrations of PFBS in soil and leaf tissue during greenhouse and field trials

Trial	Soil concentration (ng/g dw)	Tissue concentration (ng/g dw)	Bioaccumulation factor
Greenhouse test
Control	<0.10¹	<0.01	─
"Industrial" soil	48.58±2.49	205.24±18.06	4.22±0.37
Municipal soil	0.21±0.01	3.03±0.80	14.5±3.84
Field test
Control	0.20±0.02	<0.04	─
0.5X sludge application²	0.20±0.04	<0.04	─
1X sludge application	0.30±0.04	<0.04	─
2X sludge application	0.31±0.04	<0.04	─
4X sludge application	0.80±0.09	1.62±0.26	2.02±0.32

1, concentration indicated as 'less-than' are <LOQ for that matrix

2, sludge applied at half (0.5X) the agronomic application rate for the crop in this soil.

Validity criteria fulfilled:

not applicable

Conclusions:

The Bioaccumulation Factor of PFBS in soils impacted by biosolids is <14.5 for lettuce (Lactuca sativa) leaves grown to maturity. The results are applicable to PFBSK+

Executive summary:

PFBS bioaccumulation potential was assessed in a test conducted on lettuce (Lactuca sativa). Soils were either amended in triplicate with a biosolids mixture containing multiple contaminants, or else were grown in soil with a 20-year history of biosolids application. The nature of the substance(s) present in the biosolids cannot be assigned since it results from multiple sources. PFBS was analyzed as the anion. Lettuce plants were grown to maturity and the leaves were analyzed as well as soil samples. PFBS was detected in both soil and leaf tissue in several treatments, with a maximum Bioaccumulation Factor of 14.5. PFBS is a strong acid that will exist predominantly in ionic form in the environment, and plants 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 terrestrial plants and is directly applicable to PFBSK+.

While details are scarce, this study followed acceptable scientific procedures. 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 nature of the substance initially contaminating the samples is of little relevance due 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 as part of a weight of evidence approach.

Reference 6

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

Qualifier:

no guideline available

Principles of method if other than guideline:

Plants were grown from seed until maturation in biosolids-amended soil. Concentration of PFBS anion was measured in soil and edible tissue.

GLP compliance:

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: Tomato fruit were sampled at maturity (84 - 112 days after sprouting for greenhouse phase, ca. 100 days for field phase.
- Sampling intervals/frequency for test medium samples: Duplicate samples at end of exposure period.
- Sample storage conditions before analysis: Samples frozen at -20 °C before analysis
- Details on sampling and analysis of test organisms and test media samples:
Plant material was homogenized in a food processor prior to extraction. Homogenized plant tissue (0.5−2 g) was transferred to a 50 mL polypropylene vial, to which a surrogate spiking solution containing 2 ng each of isotopically labeled surrogate standards was added. 7 mL of solvent mixture (50/50 dichloromethane (DCM) and 99:1 (v/v) methanol (MeOH):ammonium hydroxide) was added to the sample and heated (30 °C) in a sonication bath for 30 min followed by shaking for 1 hour. The sample was centrifuged at 1467xg (RCF) for 20 min, and the extract was decanted into a separate 50 mL tube. This procedure was done for a total of three extraction cycles. The combined extract was evaporated at 50 °C under nitrogen to dryness. The extract was dissolved and given an oxidative clean-up with 1 mL of a basic hydrogen peroxide solution (20 μL ammonium hydroxide and 980 μL 30% hydrogen peroxide), vortexed, and sonicated in a heated (30 °C) bath for 2 h. An additional aliquot (7 mL) of the basic DCM/MeOH mixture was added to each oxidized extract, vortexed, and heated in a sonication bath for 30 min. The extract was centrifuged at 1467 xg for 20 minutes and decanted into a glass 20 mL scintillation vial. This reextraction procedure was done for a total of three cycles. The combined extract was evaporated at 50 °C under nitrogen to dryness and reconstituted with 1 mL of 99:1 (v/v) MeOH and acetic acid. The extract was run through a cleanup column packed with 100 mg of diamino and 100 mg of ENVICarb. For analysis, 105 μL of the cleaned extract was transferred to an autosampler vial and diluted with 1350 μL of pure water plus 45 μL of 0.01% aqueous ammonium hydroxide.

Separate aliquots of plant tissue were dried overnight at 70 °C to determine dry weight of the material. Concentration was expressed on a dry weight basis.

For soil extraction, 1 g aliquots were weighed into 50-mL polypropylene vials to which a solution containing isotopically-labeled surrogate standard was added prior to sequential extraction via sonication with basic methanol. All extracts were combined, evaporated to dryness, reconstituted in acidic methanol, subjected to a dispersed ENVI-Carb™ clean-up, and analyzed by LC-MS/MS.

Separate aliquots of soil were dried overnight at 105°C to determine moisture content. All results are reported on a dry weight basis.

Vehicle:

Details on preparation and application of test substrate:

Test organisms (species):

other: Lycopersicon lycopersicum

Details on test organisms:

Tomato (Lycopersicon lycopersicum)

Total exposure / uptake duration:

> 84 - < 112 d

Total depuration duration:

0 d

Test temperature:

Daytime: 18 °C - 21 °C
Nighttime: 10 °C - 13 °C

pH:

6.4 - 7.6

TOC:

1.45 - 6.34%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 7.6-L pots containing 5 kg dw soil. Soils were sieved to 6.3 mm for homogeneity before filling the pots.
- No. of organisms per container (treatment): Five seeds per pot, thinned to one plant at cotyledon stage
- No. of replicates per treatment group: Five
- No. of replicates per control / vehicle control: Five

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site: Illinois, USA
- History of site: For the reclamation site, municipal biosolids had been applied at reclamation rates over the previous 20 years, reaching the cumulative biosolids application rate of 1654 Mg/ha. The control soil, which was also intentionally mixed with biosolids to make an "industrial" soil, was taken from nearby fields and had experienced similar cropping systems.
- Vegetation cover: This field had been planted with rotations of cereal crops such as corn, wheat, and sorghum.
- Treatments with pesticides or fertilizers: The control soil had received only commercial fertilizers. No information was given on pesticide usage.
- Soil taxonomic classification: Both were classified as Lenzburg silt loams (See Table 1).
- Moisture: Drip irrigation was supplied based on crop needs and seasonal demand (2-3 times per day for 1-2 min, totalling ca 100-600 mL/day)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours total of either daylight or full-spectrum supplemental lighting

OTHER
In addition, a limited-scale field study was conducted in the Midwestern U.S. Plots (3.0 m × 4.6 m) were established and planted with lettuce (Lactuca sativa ‘Black-Seeded Simpson’) and tomato (Lycopersicon lycopersicum ‘Burpee Big Boy Hybrid’). Fertilization via biosolids occurred at four application rates (plus control). Treatments were one-half of the agronomic rate of biosolids application to meet crop nitrogen (N) requiremen (0.5×), agronomic rate (1×), two times the agronomic rate (2×), and four times the agronomic rate (4×). Each treatment (and control) was done in triplicate. Crops were grown and harvested following normal agricultural practices.

Key result

Type:

BCF

Value:

<= 0.42 dimensionless

Basis:

other: fruit tissue concentration

Calculation basis:

other: final concentrations at tissue maturity

Remarks on result:

other: result from greenhouse experiment with two different soils

Type:

BCF

Remarks on result:

not determinable

Remarks:

tissue concentration below limit of detection in field experiments at four application rates

Table 2, Concentrations of PFBS in soil and leaf tissue during greenhouse and field trials

Trial	Soil concentration (ng/g dw)	Tissue concentration (ng/g dw)	Bioaccumulation factor
Greenhouse test
Control	<0.10¹	<0.65	─
"Industrial" soil	48.58±2.49	19.38±3.26	0.42±0.08
Municipal soil	0.21±0.01	<0.65	─
Field test
Control	0.20±0.02	<0.07	─
0.5X sludge application²	0.20±0.04	<0.07	─
1X sludge application	0.30±0.04	<0.07	─
2X sludge application	0.31±0.04	<0.07	─
4X sludge application	0.80±0.09	<0.07	─

1, concentration indicated as 'less-than' are <LOQ for that matrix

2, sludge applied at half (0.5X) the agronomic application rate for the crop in this soil.

Validity criteria fulfilled:

not applicable

Conclusions:

The Bioaccumulation Factor of PFBS in soils impacted by biosolids is <0.42 for tomato (Lycopersicon lycopersicum) fruit grown to maturity. The results are applicable to PFBSK+

Executive summary:

PFBS bioaccumulation potential was assessed in a test conducted on tomato (Lycopersicon lycopersicum). Soils were either amended in triplicate with a biosolids mixture containing multiple contaminants, or else were grown in soil with a 20-year history of biosolids application. The nature of the substance(s) present in the biosolids cannot be assigned since it results from multiple sources. PFBS was analyzed as the anion. Tomato plants were grown to maturity and the fruit were analyzed as well as soil samples. PFBS was detected in both soil and fruit tissue in several treatments, with a maximum Bioaccumulation Factor of 0.42. PFBS is a strong acid that will exist predominantly in ionic form in the environment, and plants 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 terrestrial plants and is directly applicable to PFBSK+.

While details are scarce, this study followed acceptable scientific procedures. 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 nature of the substance initially contaminating the samples is of little relevance due 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 as part of a weight of evidence approach.

Reference 7

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

Qualifier:

no guideline available

Principles of method if other than guideline:

Plants were grown from seed until maturation in biosolids-amended soil. Concentration of PFBS anion was measured in soil as well as edible and non-edible tissue.

GLP compliance:

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: Plant specimens were sampled at crop maturity (tomato, 162 days; radish, 67 days; celery, 224 days; sugar snap pea, 129 days). Tissues sampled were below-ground root, above ground stem and leaves ('shoot' samples), and fruit (tomato and pea only).
- Sampling intervals/frequency for test medium samples: Duplicate samples at end of exposure period.
- Sample storage conditions before analysis: Samples frozen at -20 °C before analysis
- Details on sampling and analysis of test organisms and test media samples:
Plant material was homogenized in a food processor prior to extraction. Homogenized plant tissue (0.5−2 g) was transferred to a 50 mL polypropylene vial, to which a surrogate spiking solution containing 2 ng each of isotopically labeled surrogate standards was added. 7 mL of solvent mixture (50/50 dichloromethane (DCM) and 99:1 (v/v) methanol (MeOH):ammonium hydroxide) was added to the sample and heated (30 °C) in a sonication bath for 30 min followed by shaking for 1 hour. The sample was centrifuged at 1467xg (RCF) for 20 min, and the extract was decanted into a separate 50 mL tube. This procedure was done for a total of three extraction cycles. The combined extract was evaporated at 50 °C under nitrogen to dryness. The extract was dissolved and given an oxidative clean-up with 1 mL of a basic hydrogen peroxide solution (20 μL ammonium hydroxide and 980 μL 30% hydrogen peroxide), vortexed, and sonicated in a heated (30 °C) bath for 2 h. An additional aliquot (7 mL) of the basic DCM/MeOH mixture was added to each oxidized extract, vortexed, and heated in a sonication bath for 30 min. The extract was centrifuged at 1467 xg for 20 minutes and decanted into a glass 20 mL scintillation vial. This reextraction procedure was done for a total of three cycles. The combined extract was evaporated at 50 °C under nitrogen to dryness and reconstituted with 1 mL of 99:1 (v/v) MeOH and acetic acid. The extract was run through a cleanup column packed with 100 mg of diamino and 100 mg of ENVICarb. For analysis, 105 μL of the cleaned extract was transferred to an autosampler vial and diluted with 1350 μL of pure water plus 45 μL of 0.01% aqueous ammonium hydroxide.

Separate aliquots of plant tissue were dried overnight at 70 °C to determine dry weight of the material. Concentration was expressed on a dry weight basis.

For soil extraction, 1 g aliquots were weighed into 50-mL polypropylene vials to which a solution containing isotopically-labeled surrogate standard was added prior to sequential extraction via sonication with basic methanol. All extracts were combined, evaporated to dryness, reconstituted in acidic methanol, subjected to a dispersed ENVI-Carb™ clean-up, and analyzed by LC-MS/MS.

Separate aliquots of soil were dried overnight at 105°C to determine moisture content. All results are reported on a dry weight basis.

Vehicle:

Details on preparation and application of test substrate:

Test organisms (species):

other: Lycopersicon lycopersicum, Raphanus sativus, Apium graveolens var. dulce, Pisum sativum var. macrocarpon

Details on test organisms:

Tomato (Lycopersicon lycopersicum): seeds (‘Stupice’) obtained from Lake Valley Seed (Boulder, CO, USA)

Radish (Raphanus sativus): seeds (‘Ricardo’) obtained from Paramount Seeds Inc. (Stuart, FL, USA)

Celery (Apium graveolens var. dulce): seeds ('Tall Utah 52/70 Improved') were obtained from Botanical Interests (Broomfield, CO)

Snap pea (Pisum sativum var. macrocarpon): seeds were obtained from Botanical Interests (Broomfield, CO)

Total exposure / uptake duration:

> 67 - < 224 d

Test temperature:

Daytime: 18 °C - 21 °C
Nighttime: 10 °C - 13 °C

pH:

6.4 - 7.6

TOC:

1.51 - 6.34%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Tomato, 7.6-L pots containing 5 kg dw soil. All others, 15 cm pots containing 2.5 kg dw soil. Soils were sieved to 6.3 mm for homogeneity before filling the pots.
- No. of organisms per container (treatment): Tomato, five seeds per pot, thinned to one plant at cotyledon stage. Radish, three seeds per pot. Celery, five seens per pot. Snap pea, 2 seed per pot, thinned to one per pot after germination.
- No. of replicates per treatment group: Five
- No. of replicates per control / vehicle control: Five

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site: Illinois, USA
- History of site: For the reclamation site, municipal biosolids had been applied at reclamation rates over the previous 20 years, reaching the cumulative biosolids application rate of 1654 Mg/ha. The control soil, which was also intentionally mixed with biosolids to make an "industrial" soil, was taken from nearby fields and had experienced similar cropping systems.
- Vegetation cover: This field had been planted with rotations of cereal crops such as corn, wheat, and sorghum.
- Treatments with pesticides or fertilizers: The control soil had received only commercial fertilizers. No information was given on pesticide usage.
- Soil taxonomic classification: Both were classified as Lenzburg silt loams (See Table 1).
- Moisture: Drip irrigation was supplied based on crop needs and seasonal demand (2-3 times per day for 1-2 min, totalling ca 100-600 mL/day)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours total of either daylight or full-spectrum supplemental lighting. Pots were randomly arranged to account for light variation within the greenhouse

Key result

Type:

BCF

Value:

1.27 dimensionless

Basis:

other: radish root concentration

Remarks:

std error, 0.40

Calculation basis:

other: final concentrations in edible tissue at maturity

Remarks:

67 days

Key result

Type:

BCF

Value:

2.21 dimensionless

Basis:

other: celery shoot concentration

Remarks:

std error, 0.60

Calculation basis:

other: final concentrations in edible tissues at maturity

Remarks:

224 days

Key result

Type:

BCF

Value:

0.33 dimensionless

Basis:

other: pea fruit concentration

Remarks:

std error, 0.05

Calculation basis:

other: final concentration in edible tissues at maturity

Remarks:

129 days

Kinetic parameters:

Kinetic parameters were not determined as concentrations were only measured at crop maturity

Details on results:

Concentration data for edible tissue is reported for all three soil type (control, artificially amended "industrial" soil, and soil from the municipal sludge reclamation site) in Table 2. BAFs were calculated using data for crops grown in the artificially contaminated "industrial" soil only (Table 3). Plant tissue BAFs were calculated based on soil concentration. Due to PFBS ionization at environmental pH, entry into plants from the air via the stomata was assumed to be insignificant compared with uptake through the roots.

Reported statistics:

Statistical significance was determined by Analysis of Variance (ANOVA) with Tukey’s Test (α = 0.05); homogeneity of variance was assessed by
Levene’s Test (α = 0.05). Statistical analyses were calculated using OriginPro 9.0.

Table 2, Mean concentrations of PFBS in soil and edible plant tissue (ng/g dw)

Trial	Control soil	"Industrial" soil	Municipal soil
Soil concentration	<0.10¹	48.58±2.49²	0.21±0.01
Radish root	22.36±2.74	61.89±19.35	23.88±2.10
Celery shoot	<0.07	107.13±29.18	4.49±2.80
Pea fruit	<0.07	16.18±2.55	<0.07

1, Concentration indicated as 'less-than' are <LOQ for that matrix

2, Mean ± standard error (n = 3 to 5)

Table 3, Mean concentrations (ng/g dw) and BAFs for PFBS in plant tissue

Plant\Tissue	Root	Shoot	Fruit
Tomato concentration	34.40±15.08¹	177.10±21.78	reported in prior study²
Tomato BAF	0.71±0.31	3.65±0.45	0.42±0.08
Radish concentration	61.89±19.35	164.23±13.36	─
Radish BAF	1.27±0.40	3.38±0.27	─
Celery concentration	122.62±23.86	107.13±29.18	─
Celery BAF	2.52±0.49	2.21±0.60	─
Pea concentration	43.02±10.63	200.09±20.13	16.18±2.55
Pea BAF	0.89±0.22	4.12±0.41	0.33±0.05

1, Mean ± standard error (n = 3 to 5)

2, Result reported elsewhere in this dossier

Validity criteria fulfilled:

not applicable

Conclusions:

The Bioaccumulation Factor of PFBS in soils impacted by biosolids is less than ca. 4 for tissues from a variety of plants. The results are applicable to PFBSK+.

Executive summary:

PFBS bioaccumulation potential was assessed in a test conducted on tomato (Lycopersicon lycopersicum), Radish (Raphanus sativus), Celery (Apium graveolens var. dulce), and Sugar snap pea (Pisum sativum var. microcarpon). Soils were either amended in triplicate with a biosolids mixture containing multiple contaminants, or else were grown in soil with a 20-year history of biosolids application. The nature of the substance(s) present in the biosolids cannot be assigned since it results from multiple sources. PFBS was analyzed as the anion. Plants were grown to maturity. Root, above-ground stem & leaf, and fruit (if present) tissues were analyzed, as were soil samples. PFBS was detected in both soil and plant tissue in several treatments. Concentrations were generally highest in above-ground stem & leaf tissue, with a maximum Bioaccumulation Factor of ca. 4 in plant tissues. PFBS is a strong acid that will exist predominantly in ionic form in the environment, and plants 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 terrestrial plants and is directly applicable to PFBSK+.

While details are scarce, this study followed acceptable scientific procedures. 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 nature of the substance initially contaminating the samples is of little relevance due 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 as part of a weight of evidence approach.

Reference 8

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Uptake by plant tissues
- Short description of test conditions: Hydroponic plant growth
- Parameters analysed / observed: Concentrations in growth medium and plant tissues

GLP compliance:

Specific details on test material used for the study:

Perfluorobutanesulfonic acid potassium salt (K-PFBS), ≥98%,from Sigma Aldrich (Zwijndrecht, Netherlands).
12 other perfluoroalkyl acid substances (PFAAs) were tested simultaneously:
Perfluorobutyric acid
Perfluoropentanoic acid
Perfluorohexanoic acid
Perfluorooctanoic acid
Perfluorononanoic acid
Perfluorodecanoic acid
Perfluoroundecanoic acid
Perfluorododecanoic acid
Perfluorotridecanoic acid
Perfluorohexanesulfonate, potassium salt
linear Perfluorooctanesulfonate, potassium salt
branched Perfluorooctanesulfonate, potassium salt

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: End of experiment only
- Sampling intervals/frequency for test medium samples: 3-5 days
- Sample storage conditions before analysis: Plant samples were stored at -20 °C until analysis
- Details on sampling and analysis of test organisms and test media samples:
Plant samples: Plants were collected at the end of the exposure period, divided into roots and leaves, and stored. For extraction, samples were washed with demineralized water, dried superficially and homogenized with a household blender. Homogenate (10 g) was weighed into a 50 mL polypropylene tube (PP) and spiked with internal standard solution. Six mL of 0.4 M NaOH was then added with vortex mixing, after which the samples were left in the refrigerator overnight. Four mL of a 0.5 M tetrabutylammoniumhydrogensulfate solution and 8 mL of a 0.25 M Na2CO3/NaHCO3 buffer were then added. The homegenate was then extracted with 10 mL of methyl t-butyl ether (MTBE) with 1 min vortex mixing followed by 10 min in an ultrasonic bath, and then centrifugation for 10 min at 3000 rpm. The supernatant was transferred to a 15 mL PP tube and reduced under a gentle stream of nitrogen. The extraction was repeated twice more with 5 mL of MTBE. The MTBE phases were combined, evaporated to 1 mL, and passed through a pre-conditioned Florisil SPE cartridge. Cartridges were washed once with 10 mL of MTBE before eluting the analytes with 10 mL of MeOH/MTBE (30:70, v:v). Eluates were concentrated to 1 mL, and foliage samples were further purified with ENVI-Carb to reduce green coloration. ENVI-Carb cleanup was per Powley et al (2005). Anal. Chem. 77 (19), 6353−6358.

Water samples: Water samples were extracted with pre-conditioned weak anion-exchange SPE cartridges. Test solutions were spiked with internal standards and loaded onto the SPE cartridges at a rate of ca. 1−2 drops per second. The cartridges were then washed with 40:60 methanol:water and dried under vacuum before eluting the analytes two times with 500 μL of 2% NH4OH in Methanol (v:v).

The final extracts of water and plant samples were filtered through 0.2 µm Acrodisc LC 13 GHP filters (Pall Corp, Washington, NY USA) into 2 mL polypropylene vials, both precleaned with methanol, and were stored at 4 °C until analysis. Purified extracts and calibration standards were diluted 1:1 with water prior to analysis.

Since plant samples were not dried, concentrations are expressed on a fresh weight basis.

Vehicle:

Details on preparation and application of test substrate:

Stock solutions of the PFAAs used in this experiment were made, but the solvent was not identified. Aliquots were loaded to the nutrient solution at a rate of 100 µL/L.

Test organisms (species):

other: Lactuca sativa, var. Attraction

Details on test organisms:

TEST ORGANISM
- Common name: Lettuce
- Source: Plants grown internally. Seeds were germinated and pregrown for two weeks in soil, explanted, rinsed to remove residual soil, and transferred to the hydroponic system.

Total exposure / uptake duration:

40 d

Test temperature:

25 °C

pH:

6.0

Details on test conditions:

Nominal and measured concentrations:

nominal concentrations: Blank, 10 ng/L, 100 ng/L, 1 μg/L, and 10 μg/L
measured concentrations: 0.33±0.13 ng/L, 15.8±2.6 ng/L, 80±24 ng/L, 0.845±0.095 µg/L, 7.315±1.393 µg/L

Key result

Type:

BCF

Value:

> 0.6 - < 2 other: mL/g

Basis:

other: leaf tissue concentration

Time of plateau:

40 d

Calculation basis:

other: tissue concentration at end of exposure period

Type:

BCF

Value:

> 2 - < 8 other: mL/g

Basis:

other: root tissue concentration

Time of plateau:

40 d

Calculation basis:

other: tissue concentration at end of exposure period

Details on results:

Root tissue concentrations were linearly correlated with medium concentrations (Fig. 1). Foliage-root concentration factors of 0.15 - 0.30 may be calculated from the available data.

During the exposure period plants grew from less than <1 g to >300 g total biomass on average. No effects were observed on growth of plants, nor were effects on plant health (discoloration, spots) observed. Average volume of total transpired water was 10.96 mL/g biomass at end of the experiment.

Transpiration stream concentration factor was estimated by dividing total mass of PFBSK+ in leaves by the total mass of PFBSK+ available for transport into the plant according to the formula:

TSCF = (foliage concentration X total mass of foliage) / (medium concentration X total volume of transpired medium)

This approach to calculating TSCF assumes:
1) No degradation in the plant,
2) No physical loss from plant leaves (volatilization to air or translocation back to root),
3) Uptake only through roots.

The TSCF calculated in this way was in the range of 0.05 - 0.1 for PFBSK+. This indicates inhibition of uptake in leaves.

Table 1, Measured concentrations and Bioconcentration Factors.

Nominal concentration	Medium conc. (ng/L)	Root tissue conc. (ng/g)	Root BCF¹	Leaf tissue conc. (ng/g)	Leaf BCF
Control	0.33 ± 0.13	0.13 ± 0.11	─	<0.023	─
10 ng/L	15.8 ± 2.6	0.13 ± 0.15	8	0.03 ± 0.02	2
100 ng/L	80 ± 24	0.35 ±0.29	4	0.05 ± 0.02	0.6
100 ng/L	845 ± 95	1.78 ± 0.36	2	0.54 ± 0.12	0.6
10000 ng/L	7315 ± 1393	16.5 ± 4.2	2	4.5 ± 1.3	0.6

1, BCF = concentration in tissue (ng/g) / concentration in medium (ng/mL). Note change in medium concentration unit between tabulated values and BCF calculation.

Validity criteria fulfilled:

not applicable

Conclusions:

The bioconcentration factors of PFBSK+ in lettuce (Lactuca sativa var. Attraction) grown hydroponically over an exposure period of 40 days was 2-8 for root tissue and 0.6 - 2 for leaf tissue. Foliage-Root Concentration Factors were 0.15-0.30. Transpiration Stream Concentration Factors were in the range of 0.05 - 0.1.

Executive summary:

PFBSK+ bioaccumulation potential was assessed in a test conducted on lettuce (Lactuca sativa var. Attraction) grown hydroponically in half-strength Hoaglund's medium. A variety of perfluoroalkyl acid substances were tested simultaneously. Plant weight and weight of medium transpired were measured over a 40-day exposure period. Plants were collected, divided into root and leaf samples, and analyzed. Media samples were also analyzed. Tissue-specific bioconcentration factors were calculated, demonstrating a low potential to bioaccumulate in lettuce tissues.

The study followed acceptable scientific procedures. 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. The study is deemed reliable with restrictions and is suitable for Risk Assessment, Classification & Labeling, and PBT Analysis as part of a weight of evidence approach.

Reference 9

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 Jul 2010 - 7 Jun 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Uptake by plant tissues
- Short description of test conditions: Hydroponic plant growth
- Parameters analysed / observed: Concentrations in growth medium and plant tissues

GLP compliance:

Specific details on test material used for the study:

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: Tomato and zucchini fruit were harvested when ripe. Other tissues were collected to end the experiment when all plants had at least one ripe fruit. Cabbage heads were harvested when cracked, with other tissues collected at the same time.
- Sampling intervals/frequency for test medium samples: Not specified.
- 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:
Plant samples: At the end of the exposure period, plants were collected and divided into tissue types (root, stem, twig (tomato and zucchini only), leaf and head (cabbage only)) for storage. For extraction, samples were washed with demineralized water, dried superficially and homogenized with a household blender. Homogenate (10 g) was weighed into a 50 mL polypropylene (PP) tube and spiked with internal standard solution. Five mL of 0.4 M NaOH were then added with vortex mixing, after which the samples were left in the refrigerator overnight. Four mL of a 0.5 M tetrabutylammoniumhydrogensulfate solution and 5 mL of a 0.25 M Na2CO3/NaHCO3 buffer were then added. The homegenate was then extracted with 10 mL of methyl t-butyl ether (MTBE) with 1 min vortex mixing followed by 10 min in an ultrasonic bath, and then centrifugation for 10 min at 3000 rpm. The supernatant was transferred to a 50 mL PP tube and the extraction was repeated twice more, combining extracts. Extracts were then evaporated to 2 mL, and passed through a pre-conditioned 1000 mg Florisil SPE cartridge (Applied Separations, Allentown, PA, USA) containing a 1-gram sodium sulfate prelayer. Cartridges were washed once with 10 mL of MTBE before eluting the analytes with 10 mL of MeOH/MTBE (30:70, v:v). Eluates were concentrated to 1 mL, and when necessary extracts were further purified with ENVI-Carb 120/140 (Supelco, Bellefonte, PA, USA) to reduce coloration. ENVI-Carb cleanup was per Powley et al (2005). Anal. Chem. 77 (19), 6353−6358.

Water samples: Water samples were extracted using pre-conditioned 60 mg Oasis weak anion-exchange SPE cartridges (Waters, Wexford, Ireland). Test solutions were spiked with internal standards and loaded onto the SPE cartridges at a rate <2 drops per second. The cartridges were then washed with 40:60 methanol:water and dried under vacuum before eluting the analytes two times with 500 μL of 2% NH4OH in Methanol (v:v). Samples of tomato nutrient solution loaded at 10 µg/L were analyzed directly without extraction.

Plant tissue concentrations were expressed on a fresh weight basis.

All final extracts were passed through an Acrodisc LC 13 GHP 0.2 µm nylon filter (Pall Inc., Port Washington, NY, USA) into 2 mL PP vials and stored at 4 °C until analysis.

Vehicle:

Details on preparation and application of test substrate:

Stock solutions of the PFAAs used in this experiment were made, but the solvent was not identified. Aliquots were loaded to the nutrient solution at a rate of 100 µL/L.

Test organisms (species):

other: Tomato (Solanum lycopersicum var. Moneymaker), zucchini (Cucurbita pepo var. Black Beauty) and cabbage (Brassica oleracea convar. capitata var. alba)

Details on test organisms:

TEST ORGANISM
- Common name: Tomato, Zucchini, Cabbage
- Source: Plants grown internally. Plants were pregrown in soil until cotyledons were fully developed, explanted, rinsed to remove residual soil, and transferred to the hydroponic system.

Total exposure / uptake duration:

> 71 - < 143 d

Test temperature:

ca. 25 °C

pH:

6.0

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 10L polypropylene buckets. Plants were set in a mesh basket containing clay beads to support the roots, with baskets suspended above the growth medium. Only plant roots were in contact with the medium.
- Amount of soil or substrate: 8L half-strength Hoagland's nutrient solution.
- No. of organisms per container (treatment): one
- No. of replicates per treatment group: six (tomato, cabbage) or four (zucchini)
- No. of replicates per control / vehicle control: two
- Renewal of test medium: test solutions replaced at several intervals during exposure period by replacing the entire bucket with one containing freshly prepared medium.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 14 h light: 10h darkness. Plants were randomly repositioned until too large to move.
- Other: tomato plants were pruned at the tips for height and to remove side shoots. The mass of cuttings was much less than the total biomass of the plant. Only lower parts of the plant were sampled.

VEHICLE CONTROL PERFORMED: no

PARAMETERS MEASURED: Plant weight and mass of water transpired. Two buckets without plants were set up to allow measurement of background evaporation rate.

Plants exclude PFAAs to a greater or lesser extent during transpiration, leading to increasing PFAA concentrations in the medium as the remaining volume decreased. An average concentration was determined as noted below (see "Any other information on materials and methods incl. table").

Nominal and measured concentrations:

nominal concentrations, tomato: Blank, 10 ng/L, 100 ng/L, 1 μg/L, and 10 μg/L
nominal concentrations, others: Blank, 10 ng/L, 100 ng/L, 500 ng/L, 1 μg/L
average bulk concentrations, tomato: NR, 18.4 ng/L, 180 ng/L, 1.801 μg/L, and 18.799 μg/L
average bulk concentrations, zucchini: NR, 22.1 ng/L, 228 ng/L, 1.110 µg/L, 2.139 μg/L
average bulk concentrations, cabbage: NR, 19.9 ng/L, 193 ng/L, 0.967 µg/L, 1.793 μg/L

Key result

Type:

BCF

Value:

> 0.1 - < 0.2 other: mL/g

Basis:

edible fraction

Remarks:

tomato

Time of plateau:

96 d

Calculation basis:

other: fruit concentration (fresh weight) at approximate harvest date

Key result

Type:

BCF

Value:

> 0.3 - < 0.4 other: mL/g

Basis:

edible fraction

Remarks:

zucchini

Time of plateau:

62 d

Calculation basis:

other: tissue concentration (fresh weight) at approximate harvest date

Key result

Type:

BCF

Value:

> 0.3 - < 0.5 other: mL/g

Basis:

edible fraction

Remarks:

cabbage

Time of plateau:

112 d

Calculation basis:

other: tissue concentration (fresh weight) at harvest

Details on results:

No effects were observed on growth of plants, nor were effects on plant health (discoloration, spots) observed. A number of cabbage plants died during the experiment, but losses were not correlated with treatment level and appeared to be due to excessive temperature (>25 °C) during the experiment.

Average volume of total transpired water was 46.7 L for tomato, 41.4 L for zucchini, and 24.6 L for cabbage. Transpiration stream concentration factor was estimated by dividing total mass of PFBSK+ in leaves by the total mass of PFBSK+ available for transport into the plant according to the formula:

TSCF = (foliage concentration X total mass of foliage) / (medium concentration X total volume of transpired medium)

This approach to calculating TSCF assumes:
1) No degradation in the plant,
2) No physical loss from plant leaves (volatilization to air or translocation back to root),
3) Uptake only through roots.

The TSCF calculated in this way was in the range of 0.05 - 0.25 for PFBSK+. This indicates inhibition of uptake in leaves.

Table 1, Concentrations in medium and tomato tissues

Nominal loading (ng/L)	Medium (ng/L)	Root tissue (ng/g)	Stem tissue (ng/g)	Twig tissue (ng/g)	Leaf tissue (ng/g)	Fruit tissue (ng/g)
10	18.4 ± 0.99	<0.001	0.057 ± 0.055	0.17 ± 0.018	0.65 ± 0.37	<0.001
100	180 ± 8.59	1.34 ± 0.88	0.56 ± 0.11	1.68 ± 0.41	6.96 ± 1.84	0.040 ± 0.011
1000	1801 ± 29.2	9.21 ± 9.12	2.05 ± 1.41	18.3 ± 5.30	126 ± 42.0	0.27 ± 0.11
10000	18799 ± 1487	78.7 ± 67.4	36.7 ± 23.9	102 ± 62.6	522 ± 192	2.73 ± 1.71

Table 2, Tissue specific BCFs (mL/g) for tomato

Nominal	Root	Stem	Twig	Leaf	Fruit
10	─	3.10	9.24	35.3	─
100	7.44	3.11	9.33	38.7	0.222
1000	5.11	1.14	10.2	70.0	0.150
10000	4.19	1.95	5.43	27.8	0.145

Table 3, Concentrations in medium and zucchini tissues

Nominal loading (ng/L)	Medium (ng/L)	Root tissue (ng/g)	Stem tissue (ng/g)	Twig tissue (ng/g)	Leaf tissue (ng/g)	Fruit tissue (ng/g)
10	22.1 ± 1.70	0.21 ± 0.13	<0.001	<0.001	0.47 ± 0.02	<0.001
100	228 ± 7.13	2.59 ± 1.18	0.34 ± 0.16	0.15 ± 0.035	4.81 ± 0.80	0.07 ± 0.04
500	1110 ± 41.3	13.91 ± 11.0	1.38 ± 0.69	0.93 ± 0.41	22.8 ± 11.9	0.36 ± 0.11
1000	2139 ± 58.8	19.04 ± 5.95	2.69 ± 0.92	1.48 ± 0.62	41.1 ± 1.60	0.74 ± 0.11

Table 4, Tissue specific BCFs (mL/g) for zucchini

Nominal	Root	Stem	Twig	Leaf	Fruit
10	9.50	─	─	21.3	─
100	11.4	1.49	0.66	21.1	0.31
500	12.5	1.24	0.84	20.5	0.32
1000	8.90	1.26	0.69	19.2	0.35

Table 5, Concentrations in medium and cabbage tissues

Nominal loading (ng/L)	Medium (ng/L)	Root tissue (ng/g)	Stem tissue (ng/g)	Leaf tissue (ng/g)	Head tissue (ng/g)
10	19.9 ± 1.23	0.30 ± 0.16	<0.002	0.21 ± 0.047	<0.001
100	193 ± 13.7	3.84 ± 1.96	0.87 ± 0.042	1.96 ± 0.67	0.078 ± 0.029
500	967 ± 58.6	11.6 ± 1.56	0.31 ± 0.063	10.1 ± 2.67	0.3 ± 0.10
1000	1793 ± 75.6	31.5 ± 10.3	0.90 ± 0.18	21.4 ± 7.67	0.88 ± 0.28

Table 6, Tissue specific BCFs (mL/g) for cabbage

Nominal	Root	Stem	Leaf	Head
10	15.1	─	10.6	─
100	19.9	4.51	10.2	0.40
500	12.0	0.32	10.4	0.31
1000	17.6	0.50	11.9	0.49

Validity criteria fulfilled:

not applicable

Conclusions:

The Bioaccumulation Factors of PFBSK+ in edible portions of tomato (Solanum lycopersicum var. Moneymaker), zucchini (Cucurbita pepo var. Black Beauty) and cabbage (Brassica oleracea convar. capitata var. alba) grown hydroponically were 0.1-0.2 for tomato, 0.3-0.4 for zucchini, and 0.3-0.5 for cabbage. Transpiration Stream Concentration Factors were in the range of 0.05 - 0.25.

Executive summary:

PFBSK+ bioaccumulation potential was assessed in a test conducted on tomato (Solanum lycopersicum var. Moneymaker), zucchini (Cucurbita pepo var. Black Beauty) and cabbage (Brassica oleracea convar. capitata var. alba) grown hydroponically in half-strength Hoaglund's medium. A variety of perfluoroalkyl acid substances were tested simultaneously. Plant weight and weight of medium transpired were measured over the exposure period. Plants were collected, divided into root, above-ground, and edible samples, and then analyzed. Media samples were also analyzed. Leaf tissues generally contained the highest concentration. However, total mass of PFBSK+ in leaves was less than the total mass available from the volume of transpired medium as indicated by the Transpiration Stream Concentration Factors. Concentrations in edible portions (tomato and zucchini fruit, cabbage head) were less than concentrations in growth medium. PFBSK+ showed limited potential to bioconcentrate in this experiment.

Reference 10

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Uptake by plant tissues
- Short description of test conditions: Hydroponic plant growth
- Parameters analysed / observed: Concentrations in growth medium and plant tissues

GLP compliance:

Remarks:

Academic research publication

Specific details on test material used for the study:

Perfluorobutanesulfonic acid potassium salt (K-PFBS), ≥98%,from Campro Scientific (Berlin, Germany)
Nine other perfluoroalkyl acid substances (PFAAs) were tested simultaneously:
Perfluorobutyric acid
Perfluoropentanoic acid
Perfluorohexanoic acid
Perfluorooctanoic acid
Perfluorononanoic acid
Perfluorodecanoic acid
Perfluorotridecanoic acid
Perfluorohexanesulfonate, sodium salt
Perfluorooctanesulfonate, sodium salt

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: Tissue samples were collected at five days, when sufficient material became available.
- Sampling intervals/frequency for test medium samples: Not analyzed.
- Sample storage conditions before analysis: Samples not stored before extraction.
- Details on sampling and analysis of test organisms and test media samples:
Plant samples: At the end of the exposure period, plants were collected and divided into tissue types (root, shoot) for extraction. A portion was dried to constant weight to obtain dry weight. For extraction, root material was washed 2x with 1 mM CaSO4 and once with deionized water to remove adsorbed test substance. Plant materials were homogenized with a mortar and pestle under liquid nitrogen. Homogenate (0.2 g) was weighed into a centrifuge tube and spiked with internal standard solution. Two mL each of acetonitrile and demineralized water were then added and the tubes shaken vigorously by hand for 30 s and on a mechanical shaker for 30 min. 1.0 g of a mineral salts mixture (15% trisodium citrate dihydrate, 8% disodium citrate sesquihydrate, 15% NaCl, 62% anh. MgSO4) were then added with 30 s shaking, after which the samples were centrifuged 5 min at 1800 xg. The mineral salts allows formation of an organic supernatant from the water:acetonitrile solution. The organic layer was then mixed with two mL of demineralized water and extracted using preconditioned 60 mg Oasis WAX (weak anion exchange, Waters, Eschborn, Germany) cartridges. Preconditioning was with 2 mL 0.1% formic acid followed by 2 mL methanol. The organic extracts were applied and washed with 1 mL 2% formic acid followed 1 mL methanol. Cartridges were eluted with 2 mL 0.1% ammonia in methanol and the eluates evaporated to dryness under a stream of nitrogen. Extracts were then taken back up in 0.25 ml 50:50 methanol:water mixture and filtered through polyester filters (pore size 0.2 µm, Macherey & Nagel, Düren, Germany).

Plant tissue concentrations were expressed on a dry weight basis.

Vehicle:

yes

Remarks:

ethanol

Details on preparation and application of test substrate:

- Controls: Vehicle control only
- Chemical name of vehicle: ethanol as cosolvent
- Concentration of vehicle in test medium: Stock solutions were 200 mg/L test substance with 220 mL/L absolute ethanol. Test solutions contained 440 µL/L
- Evaporation of vehicle before use: No

Test organisms (species):

other: Maize (Zea mays)

Details on test organisms:

TEST ORGANISM
- Common name: Maize
- Strain: Zea mays L cv. Amadeo
- Source: Plants grown internally
- Germination: Seeds were soaked 18 h in 1 mM CaSO4 and held between two pieces of filter paper moistened with 1 mM CaSO4 in darkness at 26 °C for 3 d. After 6 days plants were transferred to a polyethylene container with 40 L of 0.25x growth medium. One day later the medium was changed to 0.5x. Two days later the medium was changed to full strength at the desired pH (5, 6 or 7) and the plants held for six days. An aliquot of stock solution was then added to begin the experiment.

Total exposure / uptake duration:

5 d

Test temperature:

26 °C

pH:

pH 4.90 ± 0.18, pH 6.02 ± 0.08, or pH 7.06 ± 0.05

Moisture:

Relative humidity 50%

Details on test conditions:

TEST SYSTEM
- Test container: polyethylene
- Amount of soil or substrate: 40 L of a growth medium containing:
2.5 mM Ca(NO3)2,
0.9 mM K2SO4,
0.1 mM KH2PO4,
0.1 mM K2HPO4,
0.6 mM MgSO4,
5.0 mM CaCl2,
0.2 mM Fe-EDTA,
1.0 µM H3BO3,
2.0 µM MnSO4,
0.5 µM ZnSO4,
0.3 µM CuSO4,
0.005 µM (NH4)6Mo7O24

- No. of replicates per treatment group: three
- No. of replicates per control / vehicle control: three

OTHER TEST CONDITIONS
- Adjustment of pH: pH held at setpoint through automatic titration with H2SO4
- Photoperiod: 16 h light: 8h darkness at 500 µE/m²*s

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

nominal concentrations: Blank, 100 μg/L
No analytical confirmation of medium concentration

Key result

Type:

BCF

Value:

ca. 5.5 dimensionless

Basis:

other: root:shoot transfer factor

Time of plateau:

5 d

Calculation basis:

other: final concentrations in root and shoot tissue

Remarks on result:

other: transfer factors were not significantly different at pH 5, pH 6 or pH 7

Kinetic parameters:

- Uptake rate constant k(s): 1.04 µg/(g root dw * d) at 100 µg/L. Uptake rate constants were not significantly different at pH 5, pH 6 or pH 7

Details on results:

Control medium pH was not provided. Total plant weight and root weight were not significantly different among controls or exposed plants at pH 5, pH 6, or pH 7. Shoot weight was not significantly different between controls and exposed plants, although shoot weight was significantly higher (p ≤ 0.05) for plants grown in pH 5 medium than pH 7 medium. However, PFBSK+ transfer factors and uptake rates were not significantly different by pH, and data were pooled for all pH values to determine final values.

Due to short exposure period, an overall bioaccumulation factor would have been of limited value. Uptake of PFBSK+ was <0.06% of total material over the five exposure days

Reported statistics:

Statistical evaluation was done using the program IBM SPSS Statistics (Version 20). Homogeneity of variance was examined using the Levene test and was found to be p > 0.1. Normality of data (n = 12) was evaluated using the Kolmogorov–Smirnov test. PFBSK+ data were non-normal. Variance statistical evaluation was done using Dunnett-T3 when variance was non-homogeneous, and using Tukey HSD whe variance was homogeneous.

Validity criteria fulfilled:

not applicable

Conclusions:

Uptake rate of PFBSK+ by maize (Zea mays) roots grown hydroponically was 1.04 µg / (g root dry weight * d) and showed no effect due to pH. Root:shoot transfer factor was ca. 5.5 and showed no effect due to pH.

Executive summary:

PFBSK+ bioaccumulation potential was assessed in a test conducted on maize (Zea mays tomato L. cv. Amadeo). Seedlings were exposed for five days after adaptation to the growth medium. Ten perfluoroalkyl acid substances were tested simultaneously. Exposure concentration was 100 µg/L for each substance, and medium pH was adjusted to pH 5, pH 6, or pH 7. Plants were collected, divided into root and shoot samples, and then analyzed. Bioconcentation factors were not reported in this experiment but would not have been meaningful given the short duration of the exposure. Less than 0.06% of available PFBSK+ was taken up by plants over the exposure period, with most being transferred to shoot tissues.

Reference 11

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Uptake by plant tissues
- Short description of test conditions: plant growth in amended subsurface soil
- Parameters analysed / observed: Concentrations in plant tissues

GLP compliance:

Remarks:

Academic research publication

Specific details on test material used for the study:

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: straw and kernels were harvested at the end of the exposure period
- Sampling intervals/frequency for test medium samples: Not analyzed

- Details on sampling and analysis of test organisms and test media samples:
Plant samples: At the end of the exposure period, plants were harvested and divided into tissue types. Plant materials were then dried to steady state at 39 °C and ground to <1.0 mm in a hammer mill. Ground material (0.2 g for straw, 0.5 g for kernel) was weighed into a centrifuge tube and spiked with 50 µL of internal standard solution. Two mL each of acetonitrile and demineralized water were then added and the tubes shaken vigorously by hand for 30 s and on a mechanical shaker for 30 min. 1.0 g of a mineral salts mixture (15% trisodium citrate dihydrate, 8% disodium citrate sesquihydrate, 15% NaCl, 62% anh. MgSO4) were then added with 30 s shaking, after which the samples were centrifuged 5 min at 1800 xg. The mineral salts allows formation of an organic supernatant from the water:acetonitrile solution. The organic layer was then mixed with two mL of demineralized water and extracted using preconditioned 60 mg Oasis WAX (weak anion exchange, Waters, Eschborn, Germany) cartridges. Preconditioning was with 2 mL 0.1% formic acid followed by 2 mL methanol. The organic extracts were applied and washed with 2 mL 2% aqueous formic acid followed 2 mL methanol. Cartridges were eluted with 2 mL 0.1% (v/v) ammonia in methanol and the eluates evaporated to dryness under a stream of nitrogen at 39 °C. Extracts were then taken back up in 0.25 ml 50:50 methanol:water mixture and filtered through polyester filters (pore size 0.45 µm, Macherey & Nagel, Düren, Germany). A procedural blank was done at the same time.

Plant tissue concentrations were expressed on a dry weight basis.

Vehicle:

yes

Remarks:

ethanol

Details on preparation and application of test substrate:

Test organisms (species):

other: Maize (Zea mays)

Details on test organisms:

TEST ORGANISM
- Common name: Maize
- Strain: Zea mays L cv. Amadeo
- Source: Seed from Kleinwanzlebener Saatzucht (Einbeck, Germany)

ACCLIMATION
- Acclimation period: Exposure began 24 days after seed was sown

Total exposure / uptake duration:

128 d

pH:

7.2

TOC:

0.274%

Moisture:

60% of field capacity

Details on test conditions:

TEST SYSTEM
- Test container: 12-L Ahr pot
- Amount of soil or substrate: 12 kg
- No. of replicates per treatment group: four
- No. of replicates per control / vehicle control: four
- Biomass loading rate: one plant per pot

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Other: Nutrient-poor subsurface soil air-dried for five days at room temperature and sieved to 10 mm. All tested substances were - Soil characteristics
% sand: 48
% silt: 34
% clay: 18
carbon (total) 0.274%
nitrogen (total) 0.024%
sulfur (total) 0.023%
phosphorus by Calcium-lactate-acetate (CAL) extraction 5.94 mg/(kg soil)
potassium (CAL) 38.8 mg/(kg soil)
magnesium (CAL) 166 mg/(kg soil)
copper by Diethylenetriaminepentaacetic acid (DTPA) extraction 0.60 mg/(kg soil)
iron (DTPA) 34.6 mg/(kg soil)
manganese (DTPA) 11.0 mg/(kg soil)
zinc (DTPA) 1.11 mg/(kg soil)
maximum number of free cation binding sites (pH = 8.1) 8.9 cmol/(kg soil)
- Fertilizer application: 24 g of ground PFAA-free (- Irrigation: Pots were watered with deionized, PFAA-free water to maintain 60% of field capacity

OTHER TEST CONDITIONS
- Adjustment of pH: no

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

nominal concentrations: Blank, 0.25 mg/kg, 1.00 mg/kg
No analytical confirmation of soil concentration

Key result

Type:

BCF

Value:

> 1.84 - < 3.85 dimensionless

Basis:

other: straw concentration

Remarks:

(dry weight basis)

Time of plateau:

128 d

Calculation basis:

other: final concentrations in straw, nominal soil concentration

Remarks on result:

other: range is for 1.00 and 0.25 mg/kg, values not significantly different (α, 0.05)

Key result

Type:

BCF

Value:

> 0.005 - < 0.008 dimensionless

Basis:

other: kernel concentration

Remarks:

(dry weight basis)

Time of plateau:

128 d

Calculation basis:

other: final concentration in kernel, nominal soil concentration

Remarks on result:

other: range is for 1.00 and 0.25 mg/kg, values not significantly different (α, 0.05)

Details on results:

PFBSK concentrations:
straw, 0.25 mg/kg soil: 962 ± 720 mg/kg dw
straw, 1.00 mg/kg soil: 1843 ± 687 mg/kg dw
kernel, 0.25 mg/kg soil: 1.93 ± 0.16 mg/kg dw
kernel, 1.00 mg/kg soil: 5.44 ± 1.90 mg/kg dw
While measured concentrations for tissue type were significantly different (α, 0.05) between soil concentrations, BCFs for straw or kernel were not significantly different between soil concentrations.

Reported statistics:

Statistical evaluation was done using the program IBM SPSS Statistics (Version 20). Homogeneity of variance was examined using the Levene test. Differences between concentrations of PFAAs in straw and kernel were evaluated by single-factor analysis of variance (ANOVA) and the Tukey-HSD test when variance was homogeneous. Where variances were inhomogeneous, the data were first subjected to logarithmic transformation (log10(x)), with the results of the comparison of means applied to the original data.

Validity criteria fulfilled:

not applicable

Conclusions:

The Bioaccumulation Factors of PFBSK+ in maize (Zea mays tomato L. cv. Amadeo) were 0.005-0.008 for kernel (edible portion) and 1.84-3.85 for stalk (animal feed portion).

Executive summary:

PFBSK+ bioaccumulation potential was assessed in a test conducted on maize (Zea mays tomato L. cv. Amadeo). Seeds were germinated for 24 days in soil, after which the soil was amended with test substances delivered in aqueous solution. Ten perfluoroalkyl acid (PFAA) substances were tested simultaneously. Exposure concentrations were 0.25 mg/kg dw and 1.00 mg/kg dw for each substance. During the exposure period, moisture was replenished with PFAA-free water. Plants were collected, divided into stalk and kernel samples, and then analyzed. Soils were not analyzed, with accumulation factors calculated using nominal soil concentrations. Because of the irrigation protocol and uptake of test substance by plants, average soil concentration during the exposure period may have been somewhat less than nominal concentrations. Calculated accumulation factors may slightly underestimate actual values.

Reference 12

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Uptake by plant tissues
- Short description of test conditions: Hydroponic plant growth
- Parameters analysed / observed: Time-dependent concentrations in growth medium and plant tissues

GLP compliance:

Remarks:

Academic research publication

Specific details on test material used for the study:

perfluorobutane sulfonate (PFBS) potassium salt, 98%, was purchased from Sigma-Aldrich (St. Louis, MO USA)

Nine other perfluoroalkyl acid substances (PFAAs) were tested simultaneously:
Perfluorobutanoic acid
Perfluoropentanoic acid
Perfluorohexanoic acid
Perfluoroheptanoic acid
Perfluorooctanoic acid
Perfluorononanoic acid
Perfluorodecanoic acid
Perfluorohexanesulfonate, potassium salt
Perfluorooctanesulfonate, potassium salt

Radiolabelling:

Details on sampling:

- Sampling intervals/frequency for test organisms: every one to three days during exposure for kinetic experiment, on depuration days 1, 2 and 5 during depuration phase, on day 11 for test of uptake as a function of concentration
- Sampling intervals/frequency for test medium samples: as above
- Sample storage conditions before analysis: Roots and shoots were stored separately in polypropylene vials, weighed, and immediately frozen at -80 °C.

- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Plants were grown hydroponically so that a portion of the root tissue was held in agar medium. Roots were cut from the bottom of the agar for each biological replicate, rinsed and blotted dry before storage. Only the portion of roots in direct contact with medium was collected. The upper 1 cm was discarded. The entire upper portion of each plant was cut away and stored separately.

Plant tissue was lyophilized prior to extraction, with dry weight recorded. Samples up to 30 mg were extracted directly. Larger samples were homogenized and a 30 mg subsample taken. Coarsely homogenized material was weighed into 2-mL safe-lock vials to which a stainless steel bead, 10 µL internal standard solution, and 0.75 mL methanol were added. Tubes were then shaken for 7 min at 30/s on a Retch MM400 mixer mill to homogenize, followed by 10 min sonication at 40 °C and 15 min centrifugation at 14000 x g. The supernatant liquid was transferred to another vial and the extraction was repeated twice more. The combined extract was then cleaned using pre-conditioned (5 mL dichloromethane, 5 mL methanol) 250 mg Supelclean ENVI-Carb columns (Sigma-Aldrich). The extract was transferred to the cartridge and drawn through under vacuum. The original sample vial was rinsed with 0.5 mL methanol which was then drawn through the column, and finally the column was rinsed with an additional 0.5 mL methanol. The combined methanolic eluates were concentrated to 0.5 mL under a nitrogen stream and diluted with 0.5 mL water.

Media samples were analyzed by direct injection, with no extraction or concentration step.

Vehicle:

yes

Remarks:

methanol

Details on preparation and application of test substrate:

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): methanol
- Concentration of vehicle in test medium: <0.5% in final test solution
- Evaporation of vehicle before use: No

Test organisms (species):

other: Arabidopsis thaliana

Details on test organisms:

TEST ORGANISM
- Common name: cress
Wild-type A. thaliana seeds were surface-sterilized in batches with a solution of 20% bleach and 0.1% Tween 20 surfactant. Approximately 10 µL of seeds were treated with 1mL of sterilization solution under 5 min of continuous shaking. The solution was removed, and the seeds were washed 4 times with sterile water. The cleaned seeds were then stored up to 3 days at 4 °C in the dark for stratification. Seeds were allowed to germinate on a layer of agar medium overlaying a container of growth medium. The roots penetrated the agar layer within three days, and seedlings were allowed an additional 10 or 13 days before exposure. At start of exposure, plants had at least 4 rosette leaves, and roots were ca. 2 to 4 cm long. Basal growth medium during exposure was same as the initial growth period.

Total exposure / uptake duration:

> 10 - < 14 d

Total depuration duration:

5 d

Test temperature:

22 °C

pH:

5.7

Moisture:

50% relative humidity

Details on test conditions:

TEST SYSTEM
- Test container (material, size): plastic box filled with 315 mL growth medium. Plants were germinated on a layer of agar medium subdivided by a rack into an 8x12 array, with two seeds in every other position. The agar was directly in contact with the growth medium. The box and rack were covered by a plastic lid closed with breathable tape.
- No. of organisms per container (treatment): 96
- No. of replicates per treatment group: three to four per time point

SOURCE AND PROPERTIES OF SUBSTRATE:
Plants were grown a medium consisting of 4.43 g of Murashige and Skoog basal medium, 0.5 g of 2-(N-morpholino) ethanesulfonic acid hydrate buffer, and 5 g of sucrose per liter of Milli-Q filtered water (Millipore), adjusted to pH 5.7 with NaOH, and filter sterilized.

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

VEHICLE CONTROL PERFORMED: No

To initiate the uptake experiment, plants which had been grown on blank medium for twelve or fifteen days were transferred as a rack to a new box containing medium spiked with 2 µg/L of each test substance. For the uptake rate experiment, subsamples were taken at one to three day intervals until day 14 or day 10. The number of plants in each subsample (biological replicates) cannot be determined from the text, nor the total number of boxes in the uptake phase. For the depuration phase, four extra boxes were assembled. These boxes were subdivided into three chambers containing 24 plants each. On day 11 of the uptake experiment, the racks were transferred to fresh medium without PFAAs. On each of depuration days 1, 2, and 5, three chambers were sampled at random from the four boxes. Material from each biological replicate was combined after each plant was individually harvested.

In addition, a sorption experiment was conducted in which plant boxes were set up as for the uptake experiment, but with six different exposure levels. Boxes were sampled at a single time, at 11 days of exposure. Medium concentrations at this time point were also determined.

Nominal and measured concentrations:

Nominal concentrations, uptake: blank and 2 µg/L
Nominal concentration, sorption: 0.2 µg/L, 0.5 µg/L, 1.0 µg/L, 2.0 µg/L, 10 µg/L, and 20 µg/L
Measured concentrations were not reported but used to calculate sorption

Key result

Type:

BCF

Value:

7.2 L/kg

Basis:

other: shoot tissue

Time of plateau:

10 d

Calculation basis:

steady state

Remarks on result:

other: log BCF, 0.86 ± 0.04

Key result

Type:

BCF

Value:

12.6 L/kg

Basis:

other: root tissue

Time of plateau:

4 d

Calculation basis:

steady state

Remarks on result:

other: log BCF, 1.10 ± 0.03

Type:

BCF

Value:

6.3 L/kg

Basis:

other: shoot tissue

Time of plateau:

11 d

Calculation basis:

other: intercept of sorption isotherm

Remarks on result:

other: log BCF, 0.8 ± 0.2

Type:

BCF

Value:

10 L/kg

Basis:

other: root tissue

Time of plateau:

11 d

Calculation basis:

other: intercept of sorption isotherm at several concentrations.

Remarks on result:

other: log BCF, 1.0 ± 0.2

Elimination:

yes

Remarks:

root tissue

Parameter:

DT50

Depuration time (DT):

0.33 d

Remarks on result:

other: Root concentrations were fitted with non-linear one-phase decay equation having a non-zero plateau concentration

Elimination:

yes

Remarks:

shoot tissue

Remarks on result:

other: slope of elimination curve not significantly different than zero (p = 0.811)

Kinetic parameters:

Root tissue:
- Uptake rate constant k(1): 1.5 ± 0.5 /d
- Equilibrium concentration: 27 ± 2 ng/g dw
- Depuration rate constant k(2): 2.085 /d at Co = 47 ng/g dw. R² = 0.899

- Indication of bi- or multiphasic kinetics: in root tissue, a plateau concentration of 10 ng/g dw was observed

Shoot tissue:
- Uptake rate constant k(1): 0.4 ± 0.1 /d
- Equilibrium concentration: 16 ± 1 ng/g dw
- Depuration rate constant k(2): depuration not significantly different than zero (p = 0.811)

- Computation / data analysis: for the uptake phase (see Illustration), root and shoot concentrations were fitted with a first order kinetic model: C(t)=Ceq x (1- e^(-k1 x t)). For the elimination phase, only root tissue showed significant loss of test material. The root concentrations were fit with a non-linear one-phase decay model assuming a final plateau concentration: C(t)=[(Co-Cplateau) x e^(-k2 x t)] + plateau

Reported statistics:

Statistical tests (e.g., Student t tests) as well as linear and nonlinear regressions were performed with Prism 6.0e (GraphPad Software).

Validity criteria fulfilled:

not applicable

Conclusions:

The Bioaccumulation Factors of PFBSK+ in roots and shoots of cress (Arabidopsis thaliana) were 12.6 and 7.2, respectively. Steady state was achieved within ten days. Uptake rate constant was 1.5 ± 0.5 /d for root tissue and 0.4 ± 0.1 /d for shoot tissue. Elimination rate constant was 2.085 /d for root tissue, with a plateau concentration of 10 ng/g dw. Elimination of PFBSK+ from shoot tissue was not significantly different from zero.

Executive summary:

PFBSK+ bioaccumulation potential was assessed in a test conducted on cress (Arabidopsis thaliana) grown hydroponically. A variety of perfluoroalkyl acid substances were tested simultaneously. Exposure period was up to 14 days, with an elimination period of five days thereafter for selected test containers. Plants were collected, divided into root and aerial samples, and then analyzed. Media samples were also analyzed. Steady-state was achieved within the exposure period. Root tissues contained the highest concentrations of PFBSK+. During depuration, root tissue concentrations declined quickly to a plateau level. No significant elimination was observed from shoots. In batch accumulation experiment done at several concentrations with an 11-day exposure period, accumulation factors were similar to those observed in the kinetic experiment. PFBSK+ showed limited potential to bioconcentrate in this study.

Reference 13

Endpoint:: bioaccumulation: terrestrial
Type of information:: experimental study
Adequacy of study:: other information
Reliability:: 3 (not reliable)
Rationale for reliability incl. deficiencies:: unsuitable test system
Justification for type of information:: The study examines biomagnification of the anion PFBS- in a laboratory setting using soils reclaimed water containing a variety of perfluoroalkyl acid substances. 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 bioaccumulation in terrestrial plants.
Qualifier:: no guideline available
Principles of method if other than guideline:: Plants were grown from seed until maturation under irrigation with reclaimed wastewater. Concentration of PFBS anion was measured in edible and non-edible tissue.
GLP compliance:: no
Radiolabelling:: no
Details on sampling:: - Sampling intervals/frequency for test organisms: Lettuce leaf and strawberry fruit specimens were sampled at crop maturity. Lettuce leaf samples were extracted from each plant. Strawberry fruits were composited for each treatment. In addition, root and shoot (i.e., stem and leaf) specimens were taken from whole strawberry plants once sufficient fruit mass was obtained (three treatment levels only).
- Sampling intervals/frequency for test medium samples: Soil concentrations were not measured during the test, but were measured separately in batch adsorption experiments and used to determine distribution coefficients.
- Sample storage conditions before analysis: Samples frozen at -20 °C before analysis
- Details on sampling and analysis of test organisms and test media samples:
Plant material was homogenized in a food processor prior to extraction. Homogenized plant tissue (0.5−2 g) was transferred to a 50 mL polypropylene vial, to which a surrogate spiking solution containing 2 ng each of isotopically labeled surrogate standards was added. 7 mL of solvent mixture (50/50 dichloromethane (DCM) and 99:1 (v/v) methanol (MeOH):ammonium hydroxide) was added to the sample and heated (30 °C) in a sonication bath for 30 min followed by shaking for 1 hour. The sample was centrifuged at 1467xg (RCF) for 20 min, and the extract was decanted into a separate 50 mL tube. This procedure was done for a total of three extraction cycles. The combined extract was evaporated at 50 °C under nitrogen to dryness. The extract was dissolved and given an oxidative clean-up with 1 mL of a basic hydrogen peroxide solution (20 μL ammonium hydroxide and 980 μL 30% hydrogen peroxide), vortexed, and sonicated in a heated (30 °C) bath for 2 h. An additional aliquot (7 mL) of the basic DCM/MeOH mixture was added to each oxidized extract, vortexed, and heated in a sonication bath for 30 min. The extract was centrifuged at 1467 xg for 20 minutes and decanted into a glass 20 mL scintillation vial. This reextraction procedure was done for a total of three cycles. The combined extract was evaporated at 50 °C under nitrogen to dryness and reconstituted with 1 mL of 99:1 (v/v) MeOH and acetic acid. The extract was run through a cleanup column packed with 100 mg of Chromabond diamino (Macherey-Nagel Inc., Bethlehem, PA USA) and 100 mg of Supelco ENVICarb (Sigma-Aldrich, St. Louis, MO USA). For analysis, 105 μL of the cleaned extract was transferred to an autosampler vial and diluted with 1350 μL of pure water plus 45 μL of 0.01% aqueous ammonium hydroxide.

Separate aliquots of plant tissue were dried overnight at 70 °C to determine dry weight of the material. Concentration was expressed on a dry weight basis.

For soil extraction, 1 g aliquots were weighed into 50-mL polypropylene vials to which a solution containing isotopically-labeled surrogate standard was added prior to sequential extraction via sonication with basic methanol. All extracts were combined, evaporated to dryness, reconstituted in acidic methanol, subjected to a dispersed ENVI-Carb™ clean-up, and analyzed by LC-MS/MS. Soil concentrations were not reported as such although soil-water distribution coefficients were (Table 1).

Water sample preparation and analysis were done as per Sepulvado JG, Blaine AC, Hundal LS, and Higgins CP (2011) Occurrence and fate of perfluorochemicals in soil following the land application of municipal biosolids. Environ. Sci. Technol. Vol. 45, No.19, pp. 8106−8112.
Vehicle:: no
Details on preparation and application of test substrate:: Test substance was applied as a mixture of perfluoroalkyl acid substance in reclaimed wastewater. The wastewater was obtained from a pilot-scale sewage treatment plant receiving waste from a student apartment complex. Average properties (standard deviation) were pH 6.6 (0.4), conductivity 750 µS/cm (130), alkalinity 54 mg/L CaCO3 (21), COD 17.5 mg/L (4.7), total nitrogen 9.1 mg/L (7.5), nitrate 5.9 mg/L (6.6), total phosphorus 9.2 mg/L PO4 (6.5). Loading rates PFBS and each other PFAA to the reclaimed water were 0.2, 0.4, 1, 2, 4, 10, 20, and 40 μg/L.
Test organisms (species):: other: Lactuca sativa and Fragaria ananassa
Details on test organisms:: Lettuce (Lactuca sativa var ‘Multy’) seeds were obtained from Paramount Seeds, Inc. (Stuart, FL USA) and started in seed starter plugs with tap water irrigation. Plants at four-true-leaf stage were collected, gently submerged in tap water to remove soil debris, and transplanted bare-root. Exposure duration is approximate and derived from a related study.

Strawberry (Fragaria ananassa ‘Albion’) plants were obtained bare-root from Sakuma Brothers Farm (Burlington, WA USA) and planted directly. Treatment began when all plants had at least one leaf.
Total exposure / uptake duration:: ca. 50 d
Test temperature:: Daytime: 18 °C - 21 °C
Nighttime: 10 °C - 13 °C
TOC:: 0.4 - 6 %
Details on test conditions:: TEST SYSTEM
- Test container (material, size): Lettuce, 15 cm squat pots containing 1.5 kg dw soil. Strawberry, 15 cm standard pots containing 5 kg soil.
- No. of organisms per container (treatment): 3 to 5 plants per pot
- No. of replicates per treatment group: Five
- No. of replicates per control / vehicle control: Five

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site: Most pots were filled with topsoil obtained from a local nursery (Golden, CO USA) and blended 3:1 with sand to attain 0.4% organic carbon content. Two other soils were obtained from Agvise Labs (Northwood, ND USA).
- Treatments with pesticides or fertilizers: A single application of slow-release Osmocote (nitrogen−phosphorus−potassium: 19−6−12) was mixed into each pot before planting at a rate of ca. 5 g/plant.
- Soil taxonomic classification: The topsoil was not classified. Also tested were a sandy loam (2% organic carbon; mineral content 62% sand, 19% silt, 19% clay) and a loam (6% organic carbon; mineral content 45% sand, 36% silt, 19% clay).
- Moisture: Lettuce, 100 mL per pot 3 times weekly. Strawberry, 200 mL per pot 3 times weekly. All watering was done by hand to avoid contaminating plant with irrigation water.
- Other: The sandy loam and loam soils were used only in an experiment with lettuce plants treated at 10 µg/L in reclaimed water.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours total of either daylight or full-spectrum supplemental lighting (Plantmax 1000W bulbs). Pots were randomly arranged to account for light variation within the greenhouse.
Nominal and measured concentrations:: Nominal:Tap water control, reclaimed water control, 0.2, 0.4, 1, 2, 4, 10, 20, 40 μg/L
Measured: <0.025, <0.025, 0.216, 0.399, 1.05, 2.00, 3.46, 10.9, 20.9, 31.3 µg/L
: Key result
Type:: BCF
Value:: 102 dimensionless
Basis:: other: lettuce leaf tissue
Remarks:: std error, 22.9
Calculation basis:: other: final concentrations in edible tissue at maturity
Remarks:: ca. 50 days
Remarks on result:: other: soil with 0.4% organic carbon content
: Key result
Type:: BCF
Value:: 316 dimensionless
Basis:: other: lettuce leaf tissue
Remarks:: std error, 44.1
Calculation basis:: other: final concentrations in edible tissues at maturity
Remarks:: ca. 50 days
Remarks on result:: other: soil with 2% organic carbon content
: Key result
Type:: BCF
Value:: 58.3 dimensionless
Basis:: other: lettuce leaf tissue
Remarks:: std error, 17.6
Calculation basis:: other: final concentration in edible tissues at maturity
Remarks:: ca. 50 days
Remarks on result:: other: soil with 6% organic carbon content
Kinetic parameters:: Kinetic parameters were not determined as concentrations were only measured at crop maturity
Details on results:: Tissue concentration data is reported in Table 2. BAFs were calculated only for lettuce in three soils at the 10 µg/L water concentration (Table 3). Concentrations in lettuce leaf and irrigation water followed a generally linear relationship in the 0.4% OC soil (R² = 0.94, see Illustration), and BAF values are expected for other concentrations would be in a similar range. Plant tissue BAFs were calculated based on estimated soil concentration. Soil concentrations were estimated using measured concentration in irrigation water and experimental Koc values.
Reported statistics:: Statistical significance was determined by Analysis of Variance (ANOVA) with Tukey’s Test (α = 0.05); homogeneity of variance was assessed by
Levene’s Test (α = 0.05). Statistical analyses were calculated using OriginPro 9.0.
Validity criteria fulfilled:: not applicable
Conclusions:: The Bioaccumulation Factor of PFBS in soils irrigated with reclaimed wastewater amended with pure materials is 58.3 - 316 for lettuce (Lactuca sativa var. Multy) leaves grown to maturity. Actual concentrations in soil were not measured and buildup of PFBS during the experiment cannot be excluded.
Executive summary:: PFBS bioaccumulation potential was assessed in a test conducted on lettuce (Lactuca sativa var. Multy) and strawberry (Fragaria ananassa var. Albion). Plants were grown in soils irrigated with reclaimed wastewater. Wastewater did not contain detectable levels of PFBS and was amended with pure PFBS and other perfluoroalkyl acid substances at a range of concentrations. Plants were grown to maturity. Edible tissues (lettuce leaves and strawberry fruits) were collected for analysis. In addition, root and above-ground stem & leaf fruit tissues were analyzed for strawberry plants. Soil concentrations were not measured. Instead, soil-water distribution ratios were determined and soil concentrations were estimated from the irrigation water concentrations. Strawberry fruit concentrations were generally very low or non-detectable, but were higher in non-edible strawberry tissues as well as lettuce leaf samples. BAFs were reported only for lettuce leaves. However, the calculation method was based on estimated soil concentrations. Given the experimental method, stable concentrations of PFBS in soils cannot be assured, and the possibility of increasing test substance concentration over time cannot be excluded. The study addresses the specific case of continuous input of PFBS to agricultural systems only. It is deemed not relevant to the general case of bioaccumulation.

Reference 14

Endpoint:: bioaccumulation: terrestrial
Type of information:: experimental study
Adequacy of study:: other information
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 free acid PFBS in a laboratory setting using hydroponic growth medium. As PFBS is a strong acid that will exist predominantly in ionic form in the environment and in hydrophonic solution, plants would be exposed to PFBS- anions regardless of the original material (PFBS acid v. potassium salt). Therefore, this study is relevant to the examination of bioaccumulation in terrestrial plants.
Qualifier:: no guideline available
Principles of method if other than guideline:: - Principle of test: Uptake by plant tissues
- Short description of test conditions: Hydroponic plant growth
- Parameters analysed / observed: Concentrations in plant tissues
GLP compliance:: no
Remarks:: Academic research publication
Specific details on test material used for the study:: Perfluorobutane sulfonic acid, and other acid substances, was obtained from Wellington Laboratories (ON, Canada).

Five other perfluoroalkyl acid substances were tested simultaneously
Perfluorobutanoic acid (PFBA)
perfluorooctanoic acid (PFOA)
perfluorodecanoic acid (PFDA)

perfluorohexane sulfonic acid (PFHxS)
perfluorooctane sulfonic acid (PFOS)
Radiolabelling:: no
Details on sampling:: - Sampling intervals/frequency for test organisms: Aerial plant tissues were sampled on days 1, 2, 6, 13, and 20 and fresh weight measured
- Sampling intervals/frequency for test medium samples: Test medium was sampled on days 1, 2, 6, 13, and 20 for abiotic test chambers only
- Sample storage conditions before analysis: -20 °C

- Details on sampling and analysis of test organisms and test media samples:
Plant samples: Florisil 60–100 mesh (Acros Organics, New Jersey, USA) was washed with 50:50 v/v acetonitrile:methanol and air dried. Plant samples (0.5 g) were ground with 1.5 g florisil in a glass mortar and pestle. The homogenized material was then transferred to a polypropylene column with a cellulose frit at bottom, capped with another cellulose frit. Columns were then eluted with 7 mL 50:50 v/v acetonitrile:methanol, which was reduced to 5 mL. One-mL aliquots of the extracts were transferred to a new polypropylene tube containing 30 mg ENVI-Carb 120-140 mesh (Supelco, Bellefonte, USA), vortex-mixed for one minute, and centrifuged 10 minutes at 4000 RPM. Supernatant were filtered through 0.22 μm polypropylene syringe filters into polypropylene vials and analyzed by LC-MS/MS.

Water samples: Aqueous extracts were analyzed directly after fitration through 0.22 μm polypropylene syringe filters into polypropylene vials.
Vehicle:: no
Test organisms (species):: other: Bromus diandrus
Details on test organisms:: Grass (Bromus diandrus Roth.) seeds were germinated for one week in Petri dishes containing two layers of sterile filter paper moistened with water. Five selected seedlings of uniform size were selected for each test container.
Total exposure / uptake duration:: 20 d
Test temperature:: 22 ± 1 °C (illuminated), 16 ± 1 °C (darkness)
Details on test conditions:: TEST SYSTEM
- Test container: PVC container with plastic grid
- Amount of soil or substrate: 170 mL Hewitt's nutrient solution maintained at level of grid by adding more nutrient solution
- No. of organisms per container (treatment): five
- No. of replicates per treatment group: six (one container per day)
- No. of replicates per control / vehicle control: six (one container per day)

OTHER TEST CONDITIONS
- Photoperiod: 16 h:8h light:dark
- Light intensity: 250 - 300 µE/m²∙s PAR
- Relative humidity: 60%
- Five abiotic test vessels were held in the growth chambers alongside the plants to assess abiotic losses over time.
Nominal and measured concentrations:: Nominal: Blank, 0.5 µg/mL, 1 µg/mL
: Key result
Type:: BCF
Value:: 3.071 other: mL/g
Basis:: other: aerial tissue concentration
Time of plateau:: 20 d
Calculation basis:: other: concentration at end of experiment
Remarks on result:: other: BCF for 1 µg/mL nominal medium concentration
Type:: BCF
Value:: 3.332 other: mL/g
Basis:: other: aerial tissue concentration
Time of plateau:: 20 d
Calculation basis:: other: concentration at end of experiment
Remarks on result:: other: BCF for 0.5 µg/mL nominal medium concentration
Details on results:: Tissue concentrations in the 1 µg/mL exposure group reached an apparent plateau within 20 days, whereas concentrations in the 0.5 µg/mL group did not (see Illustration).

Concentrations of PFAAs in the abiotic test containers showed statistically significant declines in concentration, but the rate of decline was low (between 0.004/d and 0.007/d for all PFAAs). PFBS concentrations were 87% to 91% of initial by day 20 of the test. Loss of test material was therefore not taken into account.
Reported statistics:: Analysis of variance (ANOVA) with the least-significant-difference procedure (LSD) for a multiple statistical comparison of groups of means and p = 0.05 to evaluate the statistical significance of differences between samples subjected to the same experimental conditions.
Validity criteria fulfilled:: not applicable
Conclusions:: The Bioaccumulation Factors of PFBS in aerial portions of grass (Bromus diandrus Roth) grown hydroponically for 20 days were 3.1 mL/g at a 1.0 µg/L and 3.3 mL/g at a 0.5 µg/L loading rate. Tissue concentrations at the higher loading rate appeared to plateau by 20 days. Actual concentrations in active growth medium were not measured and buildup of PFBS during the experiment cannot be excluded.
Executive summary:: PFBS bioaccumulation potential was assessed in a test conducted on grass (Bromus diandrus Roth). Plants were grown hydroponically in Hewitt's nutrient solution with or without a mixture of pure PFBS and other perfluoroalkyl acid substances at two concentrations, 0.5 µg/L (each substance) and 1 µg/L. After 20-days' exposure, aerial tissues were collected for analysis. Media samples from actively growing test vessels were not analyzed. Instead, parallel abiotic growth vessels were set up and the nutrient solution analyzed during the test to account for abiotic losses of test material. Loss of material in the abiotic vessels was minimal during the exposure period, and nominal concentrations were used to calculate bioaccumulation factor. Given the experimental method, stable concentrations of PFBS in nutrient solution cannot be guaranteed, and in other studies examining hydroponic exposure PFBS concentrations were shown to increase throughout the test owing to preferential uptake of water. While total accumulation was low, the numerical accuracy of the result cannot be guaranteed. The result is reliable with restrictions and suitable for use in a Weight of Evidence approach.

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Three laboratory studies evaluating bioaccumulation in earthworms from soil are available. Only the Zhao et al. study had a lengthy (28-day) depuration period with enough data collected to calculate the kinetic, organic carbon and lipid-corrected BSAF (BSAFkinetic = 0.048 soil wet wt.). In the second study, organisms were only allowed 24 hours for depuration and a depuration rate was not calculated. The third study measured PFBS in the soil but none was detected in the worms after exposure.

Although several studies have shown that PFBS is capable of uptake by green plants, there is uncertainty concerning mechanisms that may be involved and influences of other parameters such as soil characteristics and concentrations as well as differences in plant tissues. Additionally, it is unclear how to use plant bioaccumulation data in a general risk assessment. It is not surprising that a highly water soluble, non-volatile, non-sorbing substance would be taken up into plant tissues. The REACH guidance does not offer information on how uptake into plants should be evaluated in a PBT assessment. Data are included here for completeness. Authors reported the accumulation factors using a variety of descriptors including BCF, BAF, Transfer Factor (TF), Root Concentration Factor (RCF) and Leaf Soil Accumulation Factor (LSAF). Plants were exposed via soil or in hydroponic growth solution. The reported tissue-specific accumulation factors ranged from 0.1 to 70. An additional study on plant bioaccumulation was not considered reliable and is not discussed further.

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Nominal water concentration	Lettuce leaf concentration¹	Strawberry fruit concentration²	Strawberry root concentration¹	Strawberry shoot concentration¹
Tap water	<0.071	<0.714	─	─
Reclaimed water	<0.071	<0.714	─	─
0.2	10.3 ± 3.56	<0.714	─	─
0.4	44.4 ± 10.2	<0.714	41.6 ± 10.8	23.1 ± 5.03
1	89.9 ± 22.3	<0.714	─	─
2	133 ± 17.4	<0.714	─	─
4	474 ± 85.9	<0.714	373 ± 127	124 ± 29.9
10	1150 ± 259	18.2 ± 6.00	─	─
20	2080 ± 175	27.0 ± 2.70	─	─
40	3670 ± 678	55.1 ± 7.18	2830 ± 203	1470 ± 192

Soil	PFBS concentration	PFBS BAF
0.4% OC	1150 ± 259	102 ± 22.9
2% OC	4870 ± 679	316 ± 44.1
6% OC	1190 ± 359	58.3 ± 17.6

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

Bioaccumulation: terrestrial

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