Ammonium undecafluorohexanoate

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Data from the different publications are compiled in this target entry.

Adequacy of study:

weight of evidence

Justification for type of information:

The study was performed for the corresponding acid (perfluorohexanoic acid) of the target substance ammonium undecafluorohexanoate. Due to the very low pKa value of 3.29 the anionic form of the acid is the predominant form in the environment. Thus, it is considered justified to use experimental data from the corresponding acid of the ammonium salt in order to assess the bioaccumulation potential of the target substance.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Remarks on result:

not measured/tested

Remarks:

The primary route of potential accumulation of perfluorinated compounds is not considerd to be the fat tissue. Thus, lipid normalization was not considered to be relevant for this study.; Data from publication Martin et al. 2003a

Remarks on result:

not measured/tested

Remarks:

Data from publication Martin et al. 2003b

Remarks on result:

not determinable

Remarks:

A BCF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples. / Data from publication Yeung & Mabury, 2013

Temp.:

12 °C

Type:

BCF

Value:

4 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Carcass analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Temp.:

12 °C

Type:

BCF

Value:

27 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Blood analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Temp.:

12 °C

Type:

BCF

Value:

8 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Liver analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Remarks on result:

not determinable

Remarks:

A BAF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples. / Data from publication Martin et al. 2003b

Remarks on result:

not determinable

Remarks:

A BCF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples. / Data from publication Yeung & Mabury, 2013

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

5.2 d

Remarks on result:

other: Carcass analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

4.5 d

Remarks on result:

other: Blood analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

3.9 d

Remarks on result:

other: Liver analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Remarks on result:

not determinable

Remarks:

A BAF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples. / Data from publication Martin et al. 2003b

Remarks on result:

not determinable

Remarks:

A BCF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples. / Data from publication Yeung & Mabury, 2013

Rate constant:

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

Value:

0.53

Remarks on result:

other: Carcass analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Rate constant:

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

Value:

4.1

Remarks on result:

other: Blood analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Rate constant:

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

Value:

1.4

Remarks on result:

other: Liver analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Rate constant:

overall depuration rate constant (d-1)

Value:

130

Remarks on result:

other: Carcass analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Rate constant:

overall depuration rate constant (d-1)

Value:

150

Remarks on result:

other: Blood analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Rate constant:

overall depuration rate constant (d-1)

Value:

180

Remarks on result:

other: Liver analysis; Result for perfluorooctanoic acid (PFOA)

Remarks:

Data from publication Martin et al. 2003a

Remarks on result:

not determinable

Remarks:

A BAF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples. / Data from publication Martin et al. 2003b

Remarks on result:

not determinable

Remarks:

A BCF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples. / Data from publication Yeung & Mabury, 2013

Metabolites:

Metabolites were not analysed since perfluorinated acids are considered to be stable. No metabolism is expected.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

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 followed

Principles of method if other than guideline:

- Principle of test: Tissue distribution and bioconcentration study with rainbow trout (Oncorhynchus mykiss) exposed simultaneously to a suite of perfluoroalkyl carboxylates and sulfonates of varying fluorinated chain lengths in a flow-through aqueous exposure. The compound-specific bioaccumulation parameters were determined by liquid chromatography–tandem mass spectrometry (LC-MS-MS).
- Short description of test conditions: Juvenile rainbow trout were exposed to a 1,000-fold dilution of the stock perfluorinated acids (PFAs: C5 - C14) solution in a flow-through exposure design for 12 d, followed by 33 d of depuration in clean water. Fish growth was monitored by weighing the total biomass. Three fish from the exposure tank and one from the control tank were sampled at each predetermined interval during the uptake phase of the experiment (4.5, 9, 18, 36, 72, 144, and 288 h). At 288 h, the remaining fish were transferred to new aquaria receiving clean water. During the depuration phase, three fish from the treatment tank and one from the control tank were sampled at each time interval (4.5, 9, 18, 36, 72, 144, 288, 456, and 792 h). During the uptake phase, water samples were collected below the surface at 0.25, 4.5, 12, 18, 36, 72, 144, 197, 244, and 288 h. Water samples were also collected at 48 and 96 h of the depuration phase from both tanks to check for contamination. Sampled fish were anesthetized. The gut, consisting of esophagus, stomach, pyloric ceca, spleen, and intestines, was removed but not analyzed. The blood, liver, and carcass samples were analyzed separately for PFAs at each sampling time to determine the kinetics of uptake and depuration.

GLP compliance:

no

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: 4.5, 9, 18, 36, 72, 144, and 288 h (uptake phase), 4.5, 9, 18, 36, 72, 144, 288, 456, and 792 h (depuration phase)
- Sampling intervals/frequency for test medium samples: 0.25, 4.5, 12, 18, 36, 72, 144, 197, 244, and 288 h (water samples, uptake phase), 48 and 96 h (depuration phase)
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): Sampled fish were anesthetized. The gut, consisting of esophagus, stomach, pyloric ceca, spleen, and intestines, was removed but not analyzed. The blood, liver, and carcass samples were analyzed separately for PFAs at each sampling time to determine the kinetics of uptake and depuration. Liver and blood samples were homogenized in 15-mL plastic (polypropylene copolymer) centrifuge tubes containing 3 mL of Na2CO3 (0.25 M), 1 mL of water, 1 mL of the ion-pairing agent tetrabutylammonium hydrogensulfate (0.5 M adjusted to pH 10), and 100 mL (25 ng) of the internal standard, perfluorononanoic acid (PFNA, C9). Carcass samples were first reduced to a fine powder by using a mortar and pestle with liquid nitrogen, and subsequently were homogenized in 50-mL plastic centrifuge tubes containing 10 to 20 mL of Na2CO3. An exact quantity (2–4 g) of the homogenate was then transferred to a separate centrifuge tube containing 1 mL of water, 1 mL of tetrabutylammonium hydrogensulfate, and 100 mL of PFNA.

Vehicle:

yes

Remarks:

methanol

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing all the test compounds was produced by first dissolving PFAs in a small amount of methanol, which subsequently was dissolved in 30 L of reverse osmosis laboratory water. The resulting solution was contained in a polypropylene container and was stirred for 3 d before the beginning of the experiment. After allowing the solution to settle for several hours, some of the test compounds had not completely dissolved, based on the appearance of a white solid at the surface of the solution. This material was removed by filtering the entire solution through glass (GF/C) microfiber filters (Whatman, Kent, UK). The resulting solution was used for exposure and was constantly stirred while being delivered to exposure tanks.
- Controls: yes, test medium control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): methanol

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Rainbow springs (Thamesford, Ontario, Canada)
- Age at study initiation (mean and range, SD): juvenile fish
- Weight at study initiation (mean and range, SD): 5-10 g (mean: 7.3 g for exposure and 7.9 g for control)
- Health status: Only healthy fish were used.
- Description of housing/holding area:
- Feeding during test
- Food type: Trout feed was purchased from Martin Mills (Thamesford, Ontario, Canada)
- Amount&Frequency: Fish were fed daily at a rate of 1.5% body weight per day, corrected for growth throughout the experiment.

ACCLIMATION
- Acclimation period: 2 weeks
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: same as test

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: The test item is well soluble in water and has a low potential to adsorb to sediment or food particles.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

12 d

Total depuration duration:

33 d

Test temperature:

12 °C

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria lined with polypropylene bags (Volume: 45 L)
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): 500 mL dilution water/min; 0.5 mL test substance/min
- No. of organisms per vessel: no information given; three fish were sampled at each timepoint
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: 8 g/L (exposure), 2.5 g/L (control)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Carbon-filtered and dechlorinated water (with Na2SO3)
- Holding medium different from test medium: no, same as test

OTHER TEST CONDITIONS
- Photoperiod: 12 h light / 8 h darkness

Nominal and measured concentrations:

Measured: 0.014 - 1.7 µg/L (for individual components; mixture of perfluorinated acids was tested); perfulorohexanoic acid was present in the test vessels at concentrations of 1.7 µg/L (RSD: 10%).

Reference substance (positive control):

no

Remarks on result:

not measured/tested

Remarks:

The primary route of potential accumulation of perfluorinated compounds is not considerd to be the fat tissue. Thus, lipid normalization was not considered to be relevant for this study.

Temp.:

12 °C

Type:

BCF

Value:

4 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Carcass analysis; Result for perfluorooctanoic acid (PFOA)

Temp.:

12 °C

Type:

BCF

Value:

27 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Blood analysis; Result for perfluorooctanoic acid (PFOA)

Temp.:

12 °C

Type:

BCF

Value:

8 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Liver analysis; Result for perfluorooctanoic acid (PFOA)

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

5.2 d

Remarks on result:

other: Carcass analysis; Result for perfluorooctanoic acid (PFOA)

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

4.5 d

Remarks on result:

other: Blood analysis; Result for perfluorooctanoic acid (PFOA)

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

3.9 d

Remarks on result:

other: Liver analysis; Result for perfluorooctanoic acid (PFOA)

Rate constant:

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

Value:

0.53

Remarks on result:

other: Carcass analysis; Result for perfluorooctanoic acid (PFOA)

Rate constant:

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

Value:

4.1

Remarks on result:

other: Blood analysis; Result for perfluorooctanoic acid (PFOA)

Rate constant:

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

Value:

1.4

Remarks on result:

other: Liver analysis; Result for perfluorooctanoic acid (PFOA)

Rate constant:

overall depuration rate constant (d-1)

Value:

130

Remarks on result:

other: Carcass analysis; Result for perfluorooctanoic acid (PFOA)

Rate constant:

overall depuration rate constant (d-1)

Value:

150

Remarks on result:

other: Blood analysis; Result for perfluorooctanoic acid (PFOA)

Rate constant:

overall depuration rate constant (d-1)

Value:

180

Remarks on result:

other: Liver analysis; Result for perfluorooctanoic acid (PFOA)

Metabolites:

Metabolites were not analysed since perfluorinated acids are considered to be stable. No metabolism is expected.

Details on results:

- Mortality of test organisms: 2% in exposure tank, 0% in control
- Observations on body length and weight: A growth rate of 4.9E-03 g/d was determined in the exposure tank and 5.1E-03 g/d in the control.
- Organ specific bioaccumulation: In general, PFA concentrations were greatest in the blood, kidney, liver, gall bladder, and lowest in the gonads, adipose and muscle tissue. Within the blood, the plasma contained between 94 and 99% of total PFAs, with only a minor fraction detectable in the cellular fraction. Perfluorinated acids were also detectable in the gills.

All tissue concentrations were corrected for growth dilution by determining the percent increase in freshweight at each sampling interval, relative to t = 0, by using the predicted exponential growth rate equation.

Perfluorohexanoic acid (C6) could not be detected in the analysed tissues. Thus, the results for the perfluorinated acids >C6 are presented only. It can be concluded that perfuloroheaxanoic acid does not bioaccumulate in the analysed tissues.

Table 1: Bioconcentration factors in carcass for all perfluorinated acids found

Compound	BCF [L/kg]
Perfluorooctanoic acid (PFOA:C8)	4.0 ± 0.6
Perfluorodecanoic acid (PFDA:C10)	450 ± 62
Perfluoroundecanoic acid (PFUnA:C11)	2700 ± 400
Perfluorododecanoic acid (PFDoA:C12)	18000 ± 2700
Perfluorotetradecanoic acid (PFTA:C13)	23000 ± 5300

Table 2: Bioconcentration factors in blood for all perfluorinated acids found

Compound	BCF [L/kg]
Perfluorooctanoic acid (PFOA:C8)	27 ± 9.7
Perfluorodecanoic acid (PFDA:C10)	2700 ± 350
Perfluoroundecanoic acid (PFUnA:C11)	11000 ± 1400
Perfluorododecanoic acid (PFDoA:C12)	40000 ± 4500
Perfluorotetradecanoic acid (PFTA:C13)	30000 ± 4200

Table 3: Bioconcentration factors in liver for all perfluorinated acids found

Compound	BCF [L/kg]
Perfluorooctanoic acid (PFOA:C8)	8.0 ± 0.59
Perfluorodecanoic acid (PFDA:C10)	1100 ± 180
Perfluoroundecanoic acid (PFUnA:C11)	4900 ± 770
Perfluorododecanoic acid (PFDoA:C12)	18000 ± 2900
Perfluorotetradecanoic acid (PFTA:C13)	30000 ± 6000

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

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 followed

Principles of method if other than guideline:

- Principle of test: Juvenile rainbow trout (Oncorhynchus mykiss) were simultaneously exposed to a homologous series of perfluoroalkyl carboxylates and sulfonates for 34 d in the diet, followed by a 41 d depuration period. Carcass and liver concentrations were determined by using liquid chromatography–tandem mass spectrometry, and kinetic rates were calculated to determine compound specific bioaccumulation parameters.
- Short description of test conditions: Fish were fed with treated food during uptake phase for 34 d and untreated food during depuration phase for 41 d. For analysis, fish were sampled on days 4, 7, 14, 21, 28, and 34 (uptake) and 7, 14, 21, 28, 34, and 41 (depuration) and concentrations were analysed in carcass and liver.
- Parameters analysed / observed: Concentration of perfluoroalkyl carboxylates and sulfonates was analysed in different compartments (carcass, liver) of fish during uptake and depuration phase. Growth rate, mortality, bioaccumulation factor (BAF), feeding rate, assimilation efficiency and depuration half life were determined.

GLP compliance:

no

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: Fish were sampled on days 4, 7, 14, 21, 28, and 34 during uptake phase and 7, 14, 21, 28, 34, and 41 during depuration phase.
- Sampling intervals/frequency for test medium samples: same as for test organisms
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): Fish were always sampled before feeding and approximately 24 h after the previous feeding. Sampled fish were anesthetized with MS-222 and euthanized by a blow to the head and cervical dislocation. An incision was made along the ventral surface from the anus to the gills, and the entire liver was removed. The gut, consisting of esophagus, stomach, pyloric ceca, spleen, and intestines, was removed to avoid contamination of the carcass sample with feces and unabsorbed food. The resulting liver and carcass samples were analyzed separately throughout the experiment to determine the kinetics of uptake and depuration.

Vehicle:

yes

Remarks:

methanol (evaporated before addition of spiked food)

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

PREPARATION OF SPIKED FISH FOOD
- Spiked food was prepared by adding starter food (Martin Mills, Tavistock, ON, Canada) to a round-bottom flask containing a solution of all the test compounds in methanol. The flask was placed on a rotary evaporator and the mixture was slowly dried under vacuum. The resulting spiked food was further dried in an oven at 60 °C for 3 h to remove excess methanol. Control food was prepared in the same manner; minus test compounds.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: purchased from Rainbow Springs (Thamesford, ON, Canada)
- Age at study initiation (mean and range, SD): juvenile fish
- Weight at study initiation (mean and range, SD): 2-5 g
- Health status: only healthy fish were used
- Description of housing/holding conditions: Carbon-filtered and dechlorinated water (with Na2SO3) flowed through each tank at 2 L/min, the temperature was maintained at 12 °C, and a 12-h photoperiod.
- Feeding during test
- Food type: spiked food
- Amount: at a rate of 1.5% body weight
- Frequency: daily

ACCLIMATION
- Acclimation period: two weeks
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: commercial trout food (Martin Mills, Tavistock, Ontario, Canada)

Route of exposure:

feed

Justification for method:

dietary exposure method used for following reason: Bioaccumulation via the dietary route was performed in addition to the aqueous route.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

34 d

Total depuration duration:

41 d

Test temperature:

12 °C

Details on test conditions:

TEST SYSTEM
- Test vessel: not specified
- Renewal rate of test solution (frequency/flow rate): 2 L/min
- No. of organisms per vessel: not specified
- No. of vessels per concentration (replicates): 2 vessels
- No. of vessels per control / vehicle control (replicates): 1 vessel
- Biomass loading rate: 2.0 - 2.2 g/L (control: 1.1 g/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Carbon-filtered and dechlorinated water (with Na2SO3)
- Holding medium different from test medium: same as test

OTHER TEST CONDITIONS
- Photoperiod: 12 h light / 12 h darkness
- For dietary exposure fish bioaccumulation, overall daily feeding rate used in the study: 1.5% d
- For dietary exposure fish bioaccumulation, number of feeds per day (number of feeds daily ration split between): Fish were fed daily

Nominal and measured concentrations:

Concentration of perfluorohexanoic acid in food: 0.52 µg/g

Reference substance (positive control):

no

Remarks on result:

not measured/tested

Remarks on result:

not determinable

Remarks:

A BAF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples.

Remarks on result:

not determinable

Remarks:

A BAF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples.

Remarks on result:

not determinable

Remarks:

A BAF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples.

Metabolites:

Not analysed since perfluorinated compounds are known to be stable to transformation and degradation.

Details on results:

- Mortality of test organisms: 0% in contol and treatments
- Observations on feeding behavior: Feeding rate was 1.5% of body weight fed daily (g food/g fish/d)
- Observations on body length and weight: No statistically significant difference was found in the rate of growth for exposed fish relative to controls.

Water samples collected before and after feeding revealed no trace of PFAs (≤ 5 ng/L), indicating negligible transfer of contaminants to the water column during the feeding process. Only carboxylates with more than six perfluoroalkyl carbons and sulfonates with more than four perfluoroalkyl carbons were detectable in fish tissues at all given sampling times. At any time during the experiment, perfluoropentanoic acid, perfluorohexanoic acid (PFHxA, C10), and perfluoroheptanoic acid (PFHpA, C7) were not detected, thus, their bioaccumulation potential was expected to be negligible (i.e., BAF < 0.1).

Reference 4

Endpoint:

bioaccumulation in aquatic species: fish

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 followed

Principles of method if other than guideline:

- Principle of test: The bioconcentration of perfluoroalkylates in juvenile rainbow trout was studied. Fish were exposed to two aqueous film-forming foams (AFFF) under flow-through conditions. During uptake phase three fish were samples on day 1, 3, 6 and 11. During depuration phase fish were sampled on day 3, 7, 13 and 25. Liver and carcass homogenates were analyzed by LC-MS/MS for twenty perfluoroalkylates including perfluorohexanoic acid.

GLP compliance:

no

Remarks:

Publication

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: During uptake phase three fish were samples on day 1, 3, 6 and 11. During depuration phase three fish were sampled on day 3, 7, 13 and 25.
- Sampling intervals/frequency for test medium samples: Water samples were taken at the same time as fish samples.
- Sample storage: The fish samples were subsequently kept frozen, and the water samples were stored at 4 °C until analysis.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): Fish were euthanized using a lethal dose of 100 mg/mL of tricaine methane sulfonate and were briefly washed with Milli-Q water, dried with paper towel, and weighed. The liver was removed, and then it and the rest of the carcass were homogenized separately using a blender.

Vehicle:

no

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with commercially available foam solutions was prepared and administered to the exposure tanks at 1 mL/min to give a final concentration of 1000 µg/L.
- Controls: yes, test medium control

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Humber Springs Trout Hatchery (Orangeville, Ontario, Canada)
- Age at study initiation (mean and range, SD): juvenile
- Weight at study initiation (mean and range, SD): 5−7 g
- Feeding during test
- Food type: commercial fish food
- Amount: 1.5% of body weight
- Frequency: daily

ACCLIMATION
- Acclimation period: 2 weeks prior to exposure
- Acclimation conditions (same as test or not): same as test

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: The test item is well soluble in water and has a low potential to adsorb to sediment or food particles.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

11 d

Total depuration duration:

36 d

Test temperature:

approx. 18 °C

Details on test conditions:

TEST SYSTEM
- Test vessel: 475 L fiberglass tanks
- Type of flow-through (e.g. peristaltic or proportional diluter): not specified
- Renewal rate of test solution (frequency/flow rate): approx. 10 L/min
- No. of organisms per vessel: not specified (three fish were sampled at each sampling point)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: carbon filtered and dechlorinated tap water
- Holding medium different from test medium: same as test

OTHER TEST CONDITIONS
- Photoperiod: 12 h light/12 h dark

Nominal and measured concentrations:

Measured: 44 ± 16 ng/L (perfluorohexanoic acid)

Reference substance (positive control):

no

Remarks on result:

not measured/tested

Remarks on result:

not determinable

Remarks:

A BCF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples.

Remarks on result:

not determinable

Remarks:

A BCF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples.

Remarks on result:

not determinable

Remarks:

A BCF could not be determined since perfluorohexanoic acid could not be detected in carcass and liver samples.

Metabolites:

Not analysed since perfluorinated compounds are known to be stable to transformation and degradation.

Details on results:

- Mortality of test organisms: 0% mortality
- Other biological observations: No increase in hepatosomatic index was recorded indicating that fish were not under metabolic stress from chemical exposure.
- Mortality and/or behavioural abnormalities of control: 0% mortality
- Observations on fish weight: Fish weight increased at the end of exposure: Control 9.5 ± 0.2 g (test start), 13.8 ± 0.9 g (test end); 3M foam 8.5 ± 0.3 g (test start), 14.4 ± 0.5 g (test end); Angus fire 8.7±0.5 g (test start), 14.8 ± 0.8 g (test end)

Reported statistics:

A nonparametric Mann−Whitney U test was used to evaluate any significant difference in the growth rate or the hepatosomatic index between the treatment and the control groups. The significance level was α = 0.05.

Perfluorohexanoic acid could not be detected in fish samples.

Reference 5

Endpoint:

bioaccumulation in aquatic species: fish

Remarks:

Uptake of the substance in fish was measured.

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 Sep - 03 Oct 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted in referece to: Nawaji, T., Mizoguchi, N., Ono, M., Matuura, T., Seki, M., Teraoka, H. (2018): Comparing time-series of chemical concentrations in zebrafish (Danio rerio) embryos/larvae exposed to teratogens with different hydrophobicity; caffeine, sodium valproate, and diethylstilbestrol. J. Toxicol. Sci., 43, 267-273.

GLP compliance:

no

Remarks:

This study was not initially performed for REACh purposes but for other regulatory programs where the studies are not needed to be in compliance with GLP.

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: All test levels were measured at test start and test end.
- Sampling intervals/frequency for test medium samples: Every 24 h fish were analyzed for concentration of test item.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): The test samples were diluted with an appropriate amount of test water in advance. The larvae and embryos were first washed with clean dechlorinated tap water (4 times) and transferred to 1.5 mL sampling tubes. Afterwards 100 µL methanol/ultrapure water (1/1 v/v) was added and the sample was homogenized with an electric homogenizer for about 1 min. 300 µL methanol/ultrapure water (1/1 v/v) was added followed by centrifugation (20000xg for 10 min). The supernatant was processed and filled up to 1 mL with methanol/ultrapure water (1/1 v/v) and filtered with a membrane filer (Millex-LG, pore size 0.20 µm). 80 µL of the filtrate was taken, 80 µL methanol/ultrapure water (1/1 v/v) was added and vortexed for 10 sec. This sample was processed by analytical methods.

Vehicle:

no

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The weighed test sample (0.50 g) and 1 L dilution water were mixed, stirred and dissolved to prepare a stock solution of 500 mg/L. The stock solution was used to prepare the respective test solutions.
- Controls: yes, dilution water

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Strain: NIES-R
- Source: Originally supplied by National Institute for Environmental Studies, Japan
- Health status: only healthy fish were used
- Feeding during test : No feeding

ACCLIMATION FOR SPAWNING
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: every day fed with appropriate amount of food (Artemia nauplii)

PAIRING AND COLLECTION OF FERTILIZED EGGS
- Two males and on female per container (approx. 3 L); no feeding during pairing. Fertilized eggs were collected from three breeding groups. Eggs were within 5 h after fertilization.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: Substance is sufficiently stable in water and the potential for adsorption is low. Thus, concentrations can be sufficiently maintained.

Test type:

static

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

6 d

Test temperature:

28.6 - 28.7 °C

pH:

7.7

Dissolved oxygen:

7.3 - 7.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL glass tanks (for exposure), 24-well plates for observation of symptoms
- Aeration: no
- No. of organisms per vessel: 24 (morphological observations); 45 per replicate/test level (180 in total)
- No. of vessels per concentration (replicates): 4 per test level (for measuring concentration in embryos/larvae); 24 per test level (for observation of symptoms)
- No. of vessels per control / vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (ISO 6341-1982)
- Holding medium different from test medium: same as test

OBSERVATIONS DURING TEST
- Observation of test organisms (mortaliy, malformation and oter visible abnormalities were observed every 24 h.
- Body weight was measured every 24 h
- Appearance of test solutions was checked at test start and end.
- Test item concentrations in embryo/larvae + test medium was measured.

OTHER TEST CONDITIONS
- Adjustment of pH: not adapted
- Photoperiod: 16 h light/8 h darkness

Nominal and measured concentrations:

Nominal: 100, 250, 500 mg/L
Measured: 101, 255, 511 mg/L (geometric mean)

Reference substance (positive control):

no

Remarks on result:

not measured/tested

Remarks on result:

not measured/tested

Remarks on result:

other:

Remarks:

See "Any other information on results incl. tables"

Remarks on result:

not measured/tested

Mortality

No mortality was recorded.

Morphology

No morphological abnormalities were recorded.

Table 1: Concentrations in test medium

Nominal concentration [mg/L]	Measured concentration [mg/L]
	Test start	Test end	Geometric mean
Control	Not detected	Not detected	-
100	101	101	101
250	254	256	255
500	501	522	511

Table 2: Concentrations in embryos/larvae (amount per embryo/larvae)

Nominal concentration [mg/L]	Replicate	24 h			48 h			72 h			96 h			120 h			144 h
		Conc. [ng/embryo or larvae]	Mean	S.D.	Conc. [ng/embryo or larvae]	Mean	S.D.	Conc. [ng/embryo or larvae]	Mean	S.D.	Conc. [ng/embryo or larvae]	Mean	S.D.	Conc. [ng/embryo or larvae]	Mean	S.D.	Conc. [ng/embryo or larvae]	Mean	S.D.
Control	1	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-
	2	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-
100	1	2.42	2.43	0.19	17.7	24.4	5.6	22.9	20.4	2.9	26.7	31.5	5.2	23.1	19.2	3.4	12.0	12.7	2.2
	2	2.43			21.8			21.2			27.4			21.0			11.0
	3	2.20			28.2			21.3			35.0			15.6			15.9
	4	2.67			29.8			16.2			36.8			17.3			12.0
250	1	4.31	4.34	0.14	19.5	29.8	9.3	28.9	33.5	7.4	39.4	44.1	6.0	16.6	16.5	2.2	9.85	11.6	1.2
	2	4.20			28.8			44.0			49.2			14.8			12.3
	3	4.32			42.2			27.9			49.3			14.9			12.0
	4	4.53			28.9			33.2			38.4			19.6			12.5
500	1	5.74	5.77	0.09	38.0	36.8	3.3	41.7	37.0	6.7	58.8	50.5	6.3	42.0	43.4	1.6	20.3	24.0	8.2
	2	5.66			37.3			43.3			51.8			44.9			22.9
	3	5.86			32.0			33.9			44.5			44.8			35.7
	4	5.82			39.8			29.0			46.9			42.1			17.0

Table 3: Concentrations of test item (amount per body weight)

Nominal concentration [mg/L]	Replicate	24 h			48 h			72 h			96 h			120 h			144 h
		Conc. [mg/kg]	Mean	S.D.	Conc. [mg/kg]	Mean	S.D.	Conc. [mg/kg]	Mean	S.D.	Conc. [mg/kg]	Mean	S.D.	Conc. [mg/kg]	Mean	S.D.	Conc. [mg/kg]	Mean	S.D.
Control	1	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-
	2	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-	n.d.	-	-
100	1	9.36	9.39	0.74	63.2	87.0	20.1	74.9	66.7	9.5	86.2	102	17	70.1	58.5	10.3	37.0	39.3	6.7
	2	9.39			78.0			69.4			88.4			63.8			34.0
	3	8.51			101			69.6			113			47.5			49.1
	4	10.3			106			52.9			119			52.5			36.9
250	1	16.6	16.8	0.5	69.7	107	33	94.5	109	24	127	142	19	50.6	50.1	6.8	30.4	36.0	3.8
	2	16.2			103			144			159			44.9			38.0
	3	16.7			151			91.1			159			45.4			37.0
	4	17.5			103			108			124			59.6			38.5
500	1	22.2	22.3	0.3	136	131	12	136	121	22	190	163	20	128	132	5	62.7	74.0	25.3
	2	21.9			133			142			167			136			70.8
	3	22.6			114			111			144			136			110
	4	22.5			142			94.6			151			128			52.4

The concentration of test item in all exposure levels reached maximum values at 96 hpf (hours post fertilization) and then gradually decreased to less than half the corresponding maximum concentrations.

The temporal behavior was similar to data of diethylstilbestrol (DES) previously reported (Nawaji et al., 2018). According to the report, DES is highly soluble in fat and it was considered that and the gradual decrease of concentrations of DES in embryos/larvae was due to a decline of total lipid concentration in whole embryos/larvae because of the energetic costs of development and growth. Although the test item is an ammonium salt and has a high water solubility the amphipathic property of the test item may lead to the result of temporal behaviors similar to DES.

Moreover, organogenesis of the zebrafish liver begins between 60-72 hpf and the liver becomes visible at 96 hpf (Chu and Sadler, 2009). Consequently, it was considered that much of metabolic function was supposed to be obtained at approximately 96 hpf and the activity in the liver may have been mainly responsible for the decline of concentration of test item in embryos/larvae. Additionally, the activity of drug-metabolizing enzymes in the liver and/or in sites other than the liver might have partially contributed to the rapid decrease at 120 hpf and 144 hpf. No peaks other than test item were observed on the chromatogram obtained under the analytical conditions in this study.

Validity criteria fulfilled:

not applicable

Description of key information

Ammonium undecafluorohexanoate (CAS 21615-47-4) is expected to have a negligible potential to bioaccumulate based on the available data.

Additional information

The potential of a substance for bioaccumulation is normally described by its potential to assimilate in the lipid tissue of aquatic organisms. The affinity of a substance to partition to the fat tissue can be screened using the log Pow. A substance with a log Pow ≥ 4.5 is considered to have a potential for bioaccumulation in fat tissue and the bioaccumulation needs to be assessed further (ECHA, 2017). For perfluorinated substances the log Pow is not considered to be an adequate parameter to screen the bioaccumulation potential (ECHA, 2017).
Ammonium undecafluorohexanoate (CAS 21615-47-4) is the ammonium salt of perfluorohexanoic acid. Due to the very low pKa value of 3.29 the anionic form of the acid is the predominant form in the environment. In aqueous medium NH3 generally exists in aqueous media as ammonium hydroxide. The anionic functional group and the dipolar nature of the carbon/fluorine backbone comprise to the hydrophobic as well as lipophobic properties of the substance. Thus, the log Pow is considered to be inappropriate to describe the accumulation potential of perfluorinated compounds. Instead several studies have indicated that perfluorinated substances tend to bind to proteins (proteinophilic) instead of assimilating in lipid (ECHA, 2017; Conder et al., 2007). Due to these very specific properties of the substance it is considered necessary to investigate the bioaccumulation potential of perfluorohexanoic acid in specific tissues of aquatic species and not derive a bioconcentration factor based on whole body wet weight. This might underestimate the bioaccumulation potential. Martin et al. (2003a) investigated the potential bioaccumulation of several perflurocarboxylates in fish during aqueous exposure under flow-through conditions. The study was not performed according to an accepted guideline but the study design is well described and comparable to OECD Guideline 305. Juvenile rainbow trout were exposed to a mixture of perflurocarboxylates with C-chains of C5 (perfluoropentanoic acid) to C14 (perfluorotetradecanoic acid). Perfluorohexanoic acid was detected in the test medium at a mean concentration of 1.7 µg/L (relative standard deviation: 10%) throughout exposure. The exposure was split into an uptake phase of 12 d and a depuration phase of 33 d. To investigate the accumulation the fish were anesthetized. The gut, consisting of esophagus, stomach, pyloric ceca, spleen, and intestines, was removed but not analyzed. The blood, liver, and carcass samples were analyzed separately for perflurocarboxylates at each sampling time to determine the kinetics of uptake and depuration. Perfluorohexanoic acid could not be detected in any of the analysed samples of carcass, blood and liver assuming that the substance does not bioaccumulate. The results from this study clearly show that bioaccumulation of perflurocarboxylates is dependent on the chain length of the carbon-fluorine backbone and the analysed tissue. Blood was found to be the tissue with the highest bioconcentration factors (BCF). The BCF in blood increased from 4.0 L/kg (C8) up to 40,000 L/kg (C12) and started to decrease to 30,000 for perfluorododecanoic acid (C14). All BCF values were corrected for growth to exclude any impact of growth dilution during exposure. In conclusion this study clearly shows that the perflorohexanoic acid (C6) does not bioaccumulate in fish via aqueous exposure. This result is supported by a study performed by Yeung & Mabury (2013). In this study juvenile rainbow trout (Oncorhynchus mykiss) were exposed to two commercial products containing several perfluorinated compounds (including perfluorohexanoic acid). The concentration of perfluorohexanoic acid in the test medium during the uptake phase was 44 ± 16 ng/L. Analysis of carcass and liver samples did not reveal any uptake/accumulation from the aqueous phase in the different tissues which is consistent with the study from Martin et al. (2003a). Martin et al. (2003b) further investigated a potential accumulation of perfluorohexanoic acid via dietary exposure. Juvenile fish (Oncorhynchus mykiss) were exposed to spiked trout food during an uptake phase of 24 d followed by a depuration phase of 41 d. A mixture of the same perfluorocarboxylates as already used for the aqueous exposure were mixed into trout food. Perfluorohexanoic acid was measured in the food at a concentration of 0.52 µg/g. The analysed fish samples (liver and carcass) did not contain perfluorohexanoic acid. Thus, it can be assumed that the substance is not taken up by fish via dietary exposure and biomagnification based on the diet is not expected. These findings of limited bioaccumulation in fish can be supported further by monitoring data from a marine environment. Thompson et al. (2011) analysed water, sediment and biota samples for the occurrence of different perfluorinated alkyl compounds. Perfluorohexanoic acid was found in 100% of the analysed water samples in concentrations of 2.9 ± 0.4 ng/L (n=20) whereas the substance was not found in sediment samples (n=10). Moreover, perfluorohexanoic acid could not be detected in all biota samples (oysters, white ibis eggs, gull eggs, fish muscle and fish liver). Although the substance was found in water it could not be measured in any biota sample indicating that accumulation is negligible. Moreover, Conder et al. (2008) concluded that perfluorinated substances with a C-chain ≤ 7 do not bioaccumulate and do not biomagnify within the food chain. BCF and BAF values from laboratory and field measurements were analysed for C7, C8, C9, C10 and C11 perfluorinated acids and none of the studies for C7 resulted in a potential for bioaccumulation.

In addition to the presented studies on the bioaccumulation of PFHxA an additional study investigated the uptake of the substance in fish larvae/embryo of Danio rerio (Nawaji, 2018). The study did not follow a standard guideline but a method described in a peer-reviewed journal. Fertilized eggs were exposed to concentrations of PFHxA of 100, 250 and 500 mg/L under static conditions for 6 d. The concentrations were maintained as confirmed by analytical measurements (101, 255 and 511 mg/L (geometric mean)). An increase of concentrations in organisms was obvious starting on day 2 in all three concentrations. After day 4 the concentrations in fish embryos/larvae decreased to approximately the level on day 1. This decrease of concentrations in tissue was explained by the beginning of the metabolic function in fish which normally starts after 60-72 h after fertilization. Thus, it is likely that metabolizing enzymes in the developing liver and/or other sites of the body may have partially contributed to the rapid decrease of concentrations on day 5 and 6. This study result supports the above findings that in most of the environmental tissue samples analysed no perfluorohexanoic acid was found. It further supports the hypothesis that organisms (e.g. fish) are capable to metabolize/excrete the substance efficiently. 

Taking all information from laboratory and environmental monitoring into account it can be assumed that perfluorohexanoic acid has a low potential for bioaccumulation and biomagnification in the food chain.