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Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
Deviations:
yes
Remarks:
(Growth was not measured and a kinetic BCF value was not calculated)
GLP compliance:
no
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: For exposure phase: After 3, 8, 78 and 120 hours For depuration phase: After 3 hours

- Sampling intervals/frequency for test medium samples: Duplicate water samples were collected daily

- Details on sampling and analysis of test organisms and test media samples: Fish (n=6) were sampled after 3, 8, 78 and 120 hours exposure. Six remaining fish were transferred to clean water, allowed to depurate for 3 hours and then sampled. Fish were euthanized by cervical dislocation, and skin, gills, head, fillet, liver, and internal organs (IO: Intestine, kidney, spleen, stomach, pyloric caeca, heart, gall bladder, and gonads) were dissected. Samples were stored at -20°C until analysis. The livers of all six fish sampled at one time point were pooled. Likewise, the IO, skin, gills, and heads of a pair of two specimens were pooled into one sample. Muscles of individual fish were analyzed.
Vehicle:
not specified
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Vijge Hatchery (Vaassen, Netherlands)
- Age at study initiation: 5 months old
- Length at study initiation: No data
- Weight at study initiation: 2.25 ± 0.50 g
- Lipid content at test initiation: No data
- Health status: No data
- Description of housing/holding area: Fish were kept at 14°C
- Feeding during test
- Food type: Trouvit dryfeed,
- Amount: 1% of their body weight
- Frequency: daily

ACCLIMATION
- Acclimation period: Minimum of one week
- Acclimation conditions: Fish were kept at 14°C
- Type and amount of food: Trouvit dryfeed, 1% of their body weight
- Feeding frequency: Daily
- Health during acclimation: No data
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
other: reconstituted water – freshwater
Total exposure / uptake duration:
120 h
Total depuration duration:
3 h
Hardness:
1.21 mM
pH:
7.2
Details on test conditions:
- No of organisms: 30; 24 fish (uptake exposure) and 6 fish (depuration exposure)
- No. of vessels per concentration (replicates): No data
- No. of vessels per control / vehicle control (replicates): No data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted from distilled water with 0.75 mM CaCl2.2H2O, 0.46 mM MgSO4.7H2O, 1.51 mM NaHCO3, 0.04 mM KH2PO4, 1.01 mM NaNO3, and 0.10 mM Na2SiO3 salts
Nominal and measured concentrations:
C10-2-LAS: 1.32 ± 0.11 µM (equivalent to 0.42 ± 0.04 mg/L)
C11-2-LAS: 0.62 ± 0.12 µM (equivalent to 0.21 ± 0.04 mg/L)
C12-2-LAS: 0.20 ± 0.05 µM (equivalent to 0.07 ± 0.02 mg/L)
C13-2-LAS: 0.08 ± 0.03 µM (equivalent to 0.03 ± 0.01 mg/L)
Reference substance (positive control):
not specified
Details on estimation of bioconcentration:
BASIS FOR CALCULATION OF BCF

BCFss: ratio of Cf/Cw at steady state (dCf/dt=0). Steady state was achieved at 78 hours.

BCFss,mix: Cf,tot/Cw,tot = Sum Cf,i/Sum Cw,i.

where, BCFss, refers to steady state bioconcentration factor for LAS
BCFss,mix refers to bioconcentration of mixture at steady state
Key result
Type:
BCF
Value:
>= 1.4 - <= 372 L/kg
Time of plateau:
78 h
Calculation basis:
steady state
Remarks on result:
other: Range of BCFss values for individual constituents in the LAS mixture (C10-2, C11-2, C12-2, C13-2)
Key result
Type:
BCF
Value:
23.3 L/kg
Time of plateau:
78 h
Calculation basis:
steady state
Remarks on result:
other: BCFss,mix for LAS mixture (C10-2, C11-2, C12-2, C13-2)
Details on results:
Tissue and whole body BCFs were determined. The tissue specific concentrations were referred to as Ctis. The average test compound concentration in two pooled fish samples (Cf) was calculated by summing up the amounts of the test compound in the individual tissues and dividing by the sum of the body weights of two paired fish pooled.

The time course of Ctis was evaluated after normalizing Ctis to the concentration in the water (Cw) because the concentration of the test compound in the exposure solution decreased by about a factor of 2 during the 120 hour exposure period.

The ranking of the average of the steady-state Ctis/Cw values was skin > gills > liver > head > IO > muscle for C11-2-, C12-2, and C13-2-LAS while C10-2-LAS deviated slightly from this pattern. Notwithstanding the high metabolic activity in the liver, Cliver/Cw was high relative to IO, head, and muscle implying that LAS loss by biotransformation was compensated for by rapid uptake from the water and transport to the liver.

The steady-state Ctis/Cw ranged from 1.7-684 L/kg for gills and 4.0-717 L/kg for skin.

Whole body BCFss values for the individual components of the mixture ranged between 1.4 and 372 L/kg in rainbow trout. BCF values increased with increasing length of the alkyl chain. The whole body BCF at steady-state for the mixture (BCFss,mix) was 23.3 L/kg.

Table 1: Bioconcentration factors for individual components in the LAS mixture and for the mixture for Rainbow trout (Tolls et al., 2000)

Component LAS (µM) LAS (mg/L) BCFss (L/kg) BCFss,mix (L/kg)
C10-2 1.32 ± 0.11 0.42 ± 0.04 1.4 23.3
C11-2 0.62 ± 0.12 0.21 ± 0.04 6
C12-2 0.20 ± 0.05 0.07 ± 0.02 82
C13-2 0.08 ± 0.03 0.03 ± 0.01 372
Validity criteria fulfilled:
yes
Remarks:
(OECD 305-I was followed)
Conclusions:
A bioconcentration factor study with Oncorhynchus mykiss was conducted with a C10-13 LAS mixture consisting of C10-2, C11-2, C12-2 and C13-2 LAS (average carbon chain length: 10.6). The ratio of LAS in tissues to water was higher in gills and skin than other tissues and ranged from 1.7-684 and 4.0-717 L/kg, respectively. Whole body BCFss values for the individual components of the mixture ranged from 1.4-372 L/kg. The whole body BCFss for the mixture (BCFss,mix) was 23.3 L/kg.
Executive summary:

A bioconcentration factor study with Oncorhynchus mykiss was conducted with a C10-C13 LAS mixture consisting of C10-2, C11-2, C12-2 and C13-2 LAS (average chain length:10.6).

Fish (n=6) were sampled after 3, 8, 78 and 120 hours exposure.  Six remaining fish were transferred to clean water, allowed to depurate for 3 hours and then sampled. Fish were euthanized by cervical dislocation, and skin, gills, head, fillet, liver, and internal organs (IO: Intestine, kidney, spleen, stomach, pyloric caeca, heart, gall bladder, and gonads) were dissected. The livers of all six fish sampled at one time point were pooled. Likewise, the IO, skin, gills, and heads of a pair of two specimens were pooled into one sample. Muscles of individual fish were analyzed. Water samples and fish were extracted by solid-phase extraction and matrix solid-phase dispersion extraction, respectively.

LAS concentrations in fish reached steady state after approximately 78 hours. The ratio of Ctis/Cw was higher in gills and skin than other tissues and ranged from 1.7-684 and 4.0-717 L/kg, respectively.  Whole body BCFss values ranged from 1.4-372 L/kg for individual components of the LAS mixture. BCFs increased with increasing alkyl chain length.  The whole body BCFss for the mixture (BCFss,mix) was 23.3 L/kg.

This BCF study is classified as acceptable and satisfies the guideline requirement for the current OECD Guideline 305-I (Aqueous Exposure Bioconcentration Fish Test).

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
Deviations:
yes
Remarks:
Growth was not measured
GLP compliance:
no
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: Replicates of four fish were sampled several times during the intial portion of the uptake phase and sampled five times during the steady state portion of the uptake phase.
- Sampling intervals/frequency for test medium samples: Duplicate water samples (ca. 40 mL) were collected daily.
- Sample storage conditions before analysis: Water samples were either extracted immmediately or preserved by addition of 10% volume of MeOH and stored in the refrigerator under N2 for no longer than 2 days. Fish were collected, blotted dry and killed by immersion into liquid N2 and stored at -20 degree C until analysis.

Vehicle:
not specified
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION
Stock solutions were prepared by dissolving the test compounds in reconstituted water and kept under N2 to prevent aerobic biodegradation. The exposure solution was continuously prepared by dilution of the stock solution of the test compounds in reconstituted water with O2-saturated reconstituted water. Two peristaltic pumps were employed to deliver the desired volumes of the stock solution as well as reconstituted water to a mixing vessel from where the exposure solution entered the exposure aquarium.

Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead Minnow
- Source: Reared in Utrecht University hatchery
- Weight: 0.5-1 g
- Lipid content: 5.4 ± 1.7% (n=20)
- Health status: free of observable diseases and abnormalities
- Feeding during test: To minimize the concentration of suspended solids in the exposure solution, the fish were allowed to feed for 30 minutes in a separate aquarium receiving the effluent of the exposure aquarium and were transferred back from the feeding aquarium with a net. In the elimination phase, fish were fed in the elimination aquarium.
- Amount: 1% of their body weight
- Frequency: Daily

ACCLIMATION
- Acclimation period: minimum of 1 week
- Acclimation conditions (same as test or not): not specified
- Amount of food: 1% of their body weight
- Feeding frequency: Daily
- Health during acclimation: free of observable diseases and abnormalities
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
other: reconstituted water - freshwater
Total exposure / uptake duration:
>= 168 - <= 192 h
Hardness:
1.21 mM
Test temperature:
20.7-22.5°C
TOC:
1.6 mg/L
Details on test conditions:
TEST SYSTEM
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Biomass loading rate: 1 L/g/d

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted from distilled water with CaCl2-2H2O 0.75 mM, MgSO4-7H2O 0.46 mM, NaHCO3 1.51 mM, KH2PO4 0.04 mM, NaNO3 1.01mM, Na2SiO3 0.10mM salts.

RANGE-FINDING / PRELIMINARY STUDY
Expt A, as noted in the publication, was conducted as a preliminary study and was a mixture of the following:
C10-2 1.77 µM (0.57 mg/L)
C11-2 0.98 µM (0.33 mg/L)
C12-2 0.47 µM (0.16 mg/L)
C13-2 0.32 µM (0.12 mg/L)
- Results used to determine the conditions for the definitive study: Organisms did not achieve steady-state during the 48 h uptake phase in the preliminary study so exposure was extended for remaining experiments.
Nominal and measured concentrations:
Measured concentrations:
C12-2 0.02 µM (0.01 mg/L)
C10-2 0.26 µM (0.08 mg/L)
C11-2 0.07 µM (0.02 mg/L)
C13-2 0.03 µM (0.01 mg/L)
C10-in 2.49 µM (0.80 mg/L)
C11-in 0.66 µM (0.22 mg/L)
C12-in 0.19 µM (0.07 mg/L)
C13-in 0.28 µM (0.10 mg/L)
Details on estimation of bioconcentration:
BASIS FOR CALCULATION OF BCF

1. Steady-state bioconcentration factor (BCFss): It is defined as ratio of Cf/Cw at steady state (dCf/dt=0). Steady state was assumed to be reached if the increase of Cf/Cw during the last 24 hours of the experiment was less than 15%.

2. Kinetic bioconcentration factor (BCFk) : It is defined as ratio of k1/k2. k1 was determined from the initial linear part of the uptake curve by linear regression of the ratio Cf/Cw (t) against time. Values of k2 were determined by a non- linear curve fit of (Cf(t)/ Cf(t=0)) = e(-k2t).

3. BCFss and the percent in water for the individual components in the mixture were used to calculate a mixture BCF

BCF values were lipid normalized to 5% lipid: BCFxx,l = 5%/% lipid * BCFxx.
xx refers to steady-state (ss) or kinetic (k)
Lipid content:
>= 3.7 - <= 7.1 %
Remarks on result:
other: Mean 5.4%
Key result
Type:
BCF
Value:
>= 3 - <= 987.2 L/kg
Time of plateau:
74 h
Calculation basis:
steady state
Remarks on result:
other: Range of BCFss values for individual constituents in the LAS mixture (C10-2, C11-2, C12-2, C13-2, C10-in, C11-in, C12-in and C13-in)
Key result
Type:
BCF
Value:
>= 2.8 - <= 914.1 L/kg
Basis:
normalised lipid fraction
Time of plateau:
74 h
Calculation basis:
steady state
Remarks on result:
other: Range of BCFss,l values for individual constituents in the LAS mixture (C10-2, C11-2, C12-2, C13-2, C10-in, C11-in, C12-in and C13-in)
Key result
Type:
BCF
Value:
>= 7.1 - <= 1 070.3 L/kg
Time of plateau:
74 h
Calculation basis:
kinetic
Remarks on result:
other: Range of BCFk values for individual constituents in the LAS mixture (C10-2, C11-2, C12-2, C13-2, C10-in, C11-in, C12-in and C13-in)
Key result
Type:
BCF
Value:
>= 6.6 - <= 991 L/kg
Basis:
normalised lipid fraction
Time of plateau:
74 h
Calculation basis:
kinetic
Remarks on result:
other: Range of BCFk,l values for individual constituents in the LAS mixture (C10-2, C11-2, C12-2, C13-2, C10-in, C11-in, C12-in and C13-in)
Key result
Type:
BCF
Value:
22.6 L/kg
Time of plateau:
74 h
Calculation basis:
steady state
Remarks on result:
other: BCFss,mix for LAS mixture (C10-2, C11-2, C12-2, C13-2, C10-in, C11-in, C12-in and C13-in)
Key result
Type:
BCF
Value:
20.9 L/kg
Basis:
normalised lipid fraction
Time of plateau:
74 h
Calculation basis:
steady state
Remarks on result:
other: BCFss,mix,l for LAS mixture (C10-2, C11-2, C12-2, C13-2, C10-in, C11-in, C12-in and C13-in)
Details on kinetic parameters:
- Uptake rate constant (k1) values ranged from 6.4-642.2 L/kg/d for individual constituents in the LAS mixture (C10-2, C11-2, C12-2, C13-2, C10-in, C11-in, C12-in and C13-in)
- Depuration rate constant (k2) values ranged from 0.6-1.4/day for individual constituents in the LAS mixture (C10-2, C11-2, C12-2, C13-2, C10-in, C11-in, C12-in and C13-in)
Details on results:
The uptake curves indicated that steady state was reached between 74 and 96 h.

BCF values for the individual components of the mixture ranged from 3-987 L/kg and 7 -1070 L/kg, for BCFss and BCFk, respectively. Lipid normalized BCF values, using a mean lipid value of 5.4%, ranged from 3-914 L/kg and 7-991 L/kg for BCFss,l and BCFk,l, respectively.

BCFss and BCFk are not significantly different from each other (p<0.05).

BCFs increased with increasing alkyl chain length for a given isomer and were higher when the p-sulfophenyl moiety was positioned closer to the terminal carbon of the alkyl chain. 

BCFss and the percent in water for the individual components in the mixture were used to calculate a steady-state mixture BCF (BCFss,mix) of 22.6 L/kg. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 20.9 L/kg.

Table 1: BCF values from Expriment D, expressed for the individual components in the mixture as steady-state (ss) and kinetic (k). The steady-state mixture BCF (BCFss,mix) is based on the individual component BCFss values and the % in water. BCF values were lipid normalized (l) to 5% using a mean lipid value of 5.4% measured during the experiment D (Toll et al., 1997).

Experiment Component % in mixture LAS (mg/L) k1 (L/kg/d) k2 (d-1) BCFss (L/kg) BCFss,l (5.4%) (L/kg) BCFk (L/kg) BCFk,l (5.4%) (L/kg) BCFss, mix (L/kg) BCFss, mix,l (5.4%) (L/kg)
D C10-2 0.065 0.08 10.9 0.9 6.0 5.6 12.1 11.2 22.6 20.9
C11-2 0.017 0.02 61.7 0.8 31.9 29.5 77.1 71.4
C12-2 0.005 0.01 260.1 0.7 211.5 195.8 371.6 344.1
C13-2 0.008 0.01 642.2 0.6 987.2 914.1 1070.3 991.0
C10-in 0.623 0.80 6.4 0.9 3.0 2.8 7.1 6.6
C11-in 0.165 0.22 26.8 1.4 9.1 8.4 19.1 17.7
C12-in 0.047 0.07 98.9 1.1 29.9 27.7 89.9 83.2
C13-in 0.071 0.10 187.8 0.6 112.4 104.1 313.0 289.8

Where, k1 refers to uptake rate constant

k2, refers to elimination rate constant

BCFss, refers to steady state bioconcentration factor for LAS

BCFss,l, refers to steady-state bioconcentration factor for LAS, lipid normalized to 5%

BCFk, refers to kinetic bioconcentration factor for LAS

BCFk,l refers to kinetic bioconcentration factor for LAS, lipid normalized to 5%

BCFss,mix, refers to bioconcentration of mixture at steady state

BCFss,mix,l, refers to bioconcentration of mixture at steady state, lipid normalized to 5%

Of the information in above table, LAS concentration, BCFss,l, BCFk, BCFk,l, BCFss,mix and BCFss,mix,l  were calculated by the assessor using the molecular weight of the constituents and details present in the references.

Validity criteria fulfilled:
yes
Remarks:
OECD 305-I was followed
Conclusions:
A bioconcentration factor study with Pimephales promelas was conducted with a C10-C13 LAS mixture with an average chain length of 10.6. BCF values were lipid normalized to 5% using a mean lipid value of 5.4%. Lipid normalized BCFss and BCFk values for the individual components of the mixture ranged from 3-914 and 7-991 L/kg, respectively. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 20.9 L/kg.
Executive summary:

A BCF study using the fathead minnow was conducted with a C10-C13 LAS mixture ((C10-2, C11-2, C12-2, C13-2, C10-in, C11-in, C12-in and C13-in) with an average chain-length of 10.6 to understand the bioconcentration behavior of individual n-(p-sulfophenyl) alkanes in flow-through bioconcentration experiments. The method was comparable to OECD 305-I (2012).

Fish were exposed to the LAS mixture between 168 and 192 hours and after exposure they were allowed to depurate in clean water. Temperature, water hardness and TOC were also measured during the test.  Analytical measurement was performed by HPLC after extraction of the test compounds from fish and water.

BCFss and BCFk values for the individual components of the mixture ranged from 3-987 and 7-1070 L/kg, respectively. The BCFss value for the mixture was 22.6 L/kg. These values were lipid normalized to 5% using a mean lipid value of 5.4%. Lipid normalized BCFss and BCFk values for the individual components of the mixture ranged from 3-914 L/kg and 7-991 L/ kg, respectively. BCFss and BCFk values for an individual component of the mixture were not significantly different from each other. BCFs increased with increasing alkyl chain length for a given isomer and were higher when the p-sulfophenyl moiety was positioned closer to the terminal carbon of the alkyl chain. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 20.9 L/kg.

This BCF study is comparable to current OECD Guideline 305-I (Aqueous Exposure Bioconcentration Fish Test).

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
Deviations:
yes
Remarks:
(Growth was not measured and the maximum acceptable value for total organic carbon exceeded the recommended limit of 2 mg/L)
GLP compliance:
no
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: Replicates of four fish were sampled five times during the steady state portion of the uptake phase.
- Sampling intervals/frequency for test medium samples: Duplicate water samples (ca. 40 mL) were collected daily.
- Sample storage conditions before analysis: Water samples were either extracted immediately or preserved by addition of 10% volume of MeOH and stored in the refrigerator under N2 atmosphere for no longer than 2 days. Fish were collected, blotted dry and killed by immersion into liquid N2 and stored at -20°C until analysis.
Vehicle:
not specified
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION

Stock solutions were prepared by dissolving the test compounds in reconstituted water and kept under N2 to prevent aerobic biodegradation. The exposure solution was continuously prepared by dilution of the stock solution of the test compounds in reconstituted water with O2-saturated reconstituted water.

Two peristaltic pumps were employed to deliver the desired volumes of the stock solution as well as reconstituted water to a mixing vessel from where the exposure solution entered the exposure aquarium.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead Minnow
- Source: Reared in Utrecht University hatchery
- Weight: 0.5-1 g
- Lipid content: 5.4 ± 1.7% (n=20). No lipid determinations were done for experiment B but since the experiments were conducted in the same laboratory under the same conditions, mean lipid data from Experiment D of 5.4 (± 1.7) % was used to calculate lipid normalized data for Experiment B.
- Health status: free of observable diseases and abnormalities
- Feeding during test. To minimize the concentration of suspended solids in the exposure solution, the fish were allowed to feed for 30 minutes in a separate aquarium receiving the effluent of the exposure aquarium and were transferred back from the feeding aquarium with a net. In the elimination phase, fish were fed in the elimination aquarium.
- Amount: 1% of their body weight.
- Frequency: Daily

ACCLIMATION
- Acclimation period: minimum of 1 week
- Acclimation conditions (same as test or not): not specified
- Amount of food: 1% of their body weight
- Feeding frequency: Daily
- Health during acclimation: free of observable diseases and abnormalities
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
other: reconstituted water – freshwater
Total exposure / uptake duration:
>= 168 - <= 192 h
Hardness:
1.21 mM
Test temperature:
20.7-22.5°C

TOC:
3.1 mg/L
Details on test conditions:
TEST SYSTEM
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Biomass loading rate: 1 L/g/d
- Renewal rate of test solution (frequency/flowrate):
For exposure phase: water renewal rate was 1L/d/g fish
For elimination phase: water flow rate was 0.5-1 L/d/g fish

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted from distilled water with 0.75 mM CaCl2-2H2O, 0.46 mM MgSO4-7H2O, 1.51 mM NaHCO3, 0.04 mM KH2PO4, 1.01mM NaNO3 and 0.10 mM Na2SiO3 salts.

RANGE-FINDING / PRELIMINARY STUDY
Experiment A, as noted in the publication, was conducted as a preliminary study and was a mixture of the following:

C10-2 1.77 μM (0.57 mg/L)
C11-2 0.98 μM (0.33 mg/L)
C12-2 0.47 μM (0.16 mg/L)
C13-2 0.32 μM (0.12 mg/L)

- Results used to determine the conditions for the definitive study: Organisms did not achieve steady state during the 48 h uptake phase in the preliminary study so exposure was extended for remaining experiments.
Nominal and measured concentrations:
Measured concentrations:
C12-2: 0.24 mg/L
C11-5: 0.59 mg/L
C12-5: 0.36 mg/L
C13-5: 0.26 mg/L
Details on estimation of bioconcentration:
BASIS FOR CALCULATION OF BCF

1. Steady-state bioconcentration factor (BCFss): It is defined as ratio of Cf/Cw at steady state (dCf/dt=0). Steady state was assumed to be reached if the increase of Cf/Cw during the last 24 hours of the experiment was less than 15%.

2. Kinetic bioconcentration factor (BCFk) : It is defined as ratio of k1/k2. k1 was determined from the initial linear part of the uptake curve by linear regression of the ratio Cf/Cw(t) against time. Values of k2 were determined by a non- linear curve fit of (Cf(t)/ Cf(t=0)) = e(-k2t).

3. BCFss and the percent in water for the individual components in the mixture were used to calculate a mixture BCF

BCF values were lipid normalized to 5% lipid: BCFxx,l = 5%/% lipid * BCFxx.
xx refers to steady-state (ss) or kinetic (k)
Lipid content:
>= 3.7 - <= 7.1 %
Remarks on result:
other: No lipid determinations were done for experiment B but since the experiments were conducted in the same laboratory under the same conditions, mean lipid data from Experiment D of 5.4 (± 1.7)% was used to calculate lipid normalized data for Experiment B
Key result
Type:
BCF
Value:
>= 6.1 - <= 99.1 L/kg
Time of plateau:
74 h
Calculation basis:
steady state
Remarks on result:
other: Range of BCFss values for individual constituents in the LAS mixture (C12-2, C11-5, C12-5 and C13-5)
Key result
Type:
BCF
Value:
>= 5.6 - <= 91.8 L/kg
Basis:
normalised lipid fraction
Calculation basis:
steady state
Remarks on result:
other: Range of BCFss,l values for individual constituents in the LAS mixture (C12-2, C11-5, C12-5 and C13-5)
Key result
Type:
BCF
Value:
>= 3.6 - <= 89.6 L/kg
Calculation basis:
kinetic
Remarks on result:
other: range of BCFk values, for individual constituents in the LAS mixture (C12-2, C11-5, C12-5 and C13-5)
Key result
Type:
BCF
Value:
>= 3.3 - <= 83 L/kg
Basis:
normalised lipid fraction
Calculation basis:
kinetic
Remarks on result:
other: Range of BCFk,l values for individual constituents in the LAS mixture (C12-2, C11-5, C12-5 and C13-5)
Key result
Type:
BCF
Value:
27 L/kg
Calculation basis:
steady state
Remarks on result:
other: BCFss,mix for LAS mixture (C12-2, C11-5, C12-5 and C13-5)
Key result
Type:
BCF
Value:
25 L/kg
Basis:
normalised lipid fraction
Calculation basis:
steady state
Remarks on result:
other: BCFss mix,l for LAS mixture (C12-2, C11-5, C12-5 and C13-5)
Details on kinetic parameters:
- Uptake rate constant (k1) values ranged from 4.3-134.4 L/kg/d for individual constituents in the LAS mixture (C12-2, C11-5, C12-5 and C13-5)
- Depuration rate constant (k2) values ranged from 1.2-1.5/day for individual constituents in the LAS mixture (C12-2, C11-5, C12-5 and C13-5)
Details on results:
The uptake curves indicated that steady state was reached between 74 and 96 h.

BCF values for the individual components of the mixture ranged from 6.1-99.1 L/kg and 3.6-89.6 L/kg, for BCFss and BCFk, respectively. Lipid normalized BCF values, using a mean lipid value of 5.4%, ranged from 5.6-91.8 L/kg and 3.3-83.0 L/kg for BCFss,l and BCFk,l, respectively.

BCFss and BCFk are not significantly different from each other (p<0.05).

BCFs increased with increasing alkyl chain length for a given isomer and were higher when the p-sulfophenyl moiety was positioned closer to the terminal carbon of the alkyl chain.

BCFss and the percent in water for the individual components in the mixture were used to calculate a mixture BCF of 27.0 L/kg. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 25.0 L/kg.

Table 1: BCF values from Experiment B, expressed for the individual components in the mixture as steady-state (ss) and kinetic (k). The mixture BCF (BCFmix) is based on the individual component BCFss values and the % in water. BCF values were lipid normalized (l) to 5% using a mean lipid value of 5.4% measured during Experiment B (Tolls et al., 1997)

Experiment Component % in mixture LAS (mg/L) k1(L/kg/d) k2(d-1) BCFss (L/kg) BCFss,l (5.4%) (L/kg) BCFk (L/kg) BCFk,l (5.4%) (L/kg) BCFss, mix (L/kg) BCFss, mix,l (5.4%) (L/kg)
B C12-2 0.163 0.24 134.4 1.5 99.1 91.8 89.6 83.0 27.0 25.0
C11-5 0.42 0.59 4.3 1.2 6.1 5.6 3.6 3.3
C12-5 0.247 0.36 11.1 1.2 10.0 9.3 9.3 8.6
C13-5 0.17 0.26 45.5 1.2 34 31.5 37.9 35.1

Where, k1 refers to uptake rate constant

k2, refers to elimination rate constant

BCFss, refers to steady state bioconcentration factor for LAS

BCFss,l, refers to steady-state bioconcentration factor for LAS, lipid normalized to 5%

BCFk, refers to kinetic bioconcentration factor for LAS

BCFk,l refers to kinetic bioconcentration factor for LAS, lipid normalized to 5%

BCFss,mix, refers to bioconcentration of mixture at steady state

BCFss,mix,l, refers to bioconcentration of mixture at steady state, lipid normalized to 5%

Of the information in above table, LAS concentration, BCFss,l, BCFk, BCFk,l, BCFss,mix and BCFss,mix,l  were calculated by the assessor using the molecular weight of the constituents and details present in the references.

Validity criteria fulfilled:
yes
Remarks:
OECD 305-I was followed
Conclusions:
A bioconcentration factor study with Pimephales promelas was conducted with a LAS mixture (C12-2, C11-5, C12-5 and C13-5) with an average chain length of 11.7. BCF values were lipid normalized to 5% using a mean lipid value of 5.4%. Lipid normalized BCFss and BCFk values for the individual components of the mixture ranged from 5.6-91.8 and 3.3-83.0 L/kg, respectively. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 25.0 L/kg.
Executive summary:

A BCF study with Pimephales promelas was conducted with an LAS mixture (C12-2, C11-5, C12-5 and C13-5) with an average chain-length of 11.7 to understand the bioconcentration behavior of individual n-(p-sulfophenyl) alkanes in flow-through bioconcentration experiments. The method was comparable to OECD 305-I (2012).

Fish were exposed to the LAS mixture between 168 and 192 hours and after exposure they were allowed to depurate in clean water. Temperature, water hardness and TOC were also measured during the test. Analytical measurement was performed by HPLC after extraction of the test compounds from fish and water.

BCFss and BCFk values for the individual components of the mixture ranged from 6.1-99.1 and 3.6-89.6 L/kg, respectively. The BCFss value for the mixture was 27.0 L/kg. These values were lipid normalized to 5% using a mean lipid value of 5.4%. Lipid normalized BCFss and BCFk values ranged from 5.6-91.8 and 3.3-83.0 L/kg, respectively. BCFss and BCFk values for an individual component of the mixture were not significantly different from each other. BCFs increased with increasing alkyl chain length for a given isomer and were higher when the p-sulfophenyl moiety was positioned closer to the terminal carbon of the alkyl chain. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 25.0 L/kg.

This BCF study is comparable to current OECD Guideline 305-I (Aqueous Exposure Bioconcentration Fish Test)

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
Deviations:
yes
Remarks:
(Growth was not measured and the maximum acceptable value for total organic carbon exceeded the recommended limit of 2 mg/L)
GLP compliance:
no
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: Three replicates of four fish were sampled five times during the steady state portion of the uptake phase.
- Sampling intervals/frequency for test medium samples: Duplicate water samples (ca. 40 mL) were collected daily.
- Sample storage conditions before analysis: Water samples were either extracted immediately or preserved by addition of 10% volume of MeOH and stored in the refrigerator under N2 atmosphere for no longer than 2 days. Fish were collected, blotted dry and killed by immersion into liquid N2 and stored at -20°C until analysis.
Vehicle:
not specified
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION

Stock solutions were prepared by dissolving the test compounds in reconstituted water and kept under N2 to prevent aerobic biodegradation. The exposure solution was continuously prepared by dilution of the stock solution of the test compounds in reconstituted water with O2-saturated reconstituted water.

Two peristaltic pumps were employed to deliver the desired volumes of the stock solution as well as reconstituted water to a mixing vessel from where the exposure solution entered the exposure aquarium.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead Minnow
- Source: Reared in Utrecht University hatchery
- Weight: 0.5-1 g
- Lipid content: 5.4 ± 1.7% (n=20). No lipid determinations were done for experiment C but since the experiments were conducted in the same laboratory under the same conditions, mean lipid data from Experiment D of 5.4 (± 1.7)% was used to calculate lipid normalized data for Experiment C.
- Health status: free of observable diseases and abnormalities
- Feeding during test. To minimize the concentration of suspended solids in the exposure solution, the fish were allowed to feed for 30 minutes in a separate aquarium receiving the effluent of the exposure aquarium and were transferred back from the feeding aquarium with a net. In the elimination phase, fish were fed in the elimination aquarium.
- Amount: 1% of their body weight.
- Frequency: Daily

ACCLIMATION
- Acclimation period: minimum of 1 week
- Acclimation conditions (same as test or not): not specified
- Amount of food: 1% of their body weight
- Feeding frequency: Daily
- Health during acclimation: free of observable diseases and abnormalities
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
other: reconstituted water – freshwater
Total exposure / uptake duration:
>= 168 - <= 192 h
Hardness:
1.21 mM
Test temperature:
20.7-22.5°C

TOC:
3.7 mg/L
Details on test conditions:
TEST SYSTEM
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Biomass loading rate: 1 L/g/d
- Renewal rate of test solution (frequency/flowrate):
For exposure phase: water renewal rate was 1L/d/g fish
For elimination phase: water flow rate was 0.5-1 L/d/g fish

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted from distilled water with 0.75 mM CaCl2-2H2O, 0.46 mM MgSO4-7H2O, 1.51 mM NaHCO3, 0.04 mM KH2PO4, 1.01mM NaNO3 and 0.10 mM Na2SiO3 salts.

RANGE-FINDING / PRELIMINARY STUDY
Experiment A, as noted in the publication, was conducted as a preliminary study and was a mixture of the following:

C10-2 1.77 μM (0.57 mg/L)
C11-2 0.98 μM (0.33 mg/L)
C12-2 0.47 μM (0.16 mg/L)
C13-2 0.32 μM (0.12 mg/L)

- Results used to determine the conditions for the definitive study: Organisms did not achieve steady state during the 48 h uptake phase in the preliminary study so exposure was extended for remaining experiments.
Nominal and measured concentrations:
Measured concentrations
C12-2: 0.07 mg/L
C11-5: 0.56 mg/L
C12-6: 0.21 mg/L
C12-3: 0.11 mg/L
Details on estimation of bioconcentration:
BASIS FOR CALCULATION OF BCF

1. Steady-state bioconcentration factor (BCFss): It is defined as ratio of Cf/Cw at steady state (dCf/dt=0). Steady state was assumed to be reached if the increase of Cf/Cw during the last 24 hours of the experiment was less than 15%.

2. Kinetic bioconcentration factor (BCFk) : It is defined as ratio of k1/k2. k1 was determined from the initial linear part of the uptake curve by linear regression of the ratio Cf/Cw(t) against time. Values of k2 were determined by a non- linear curve fit of (Cf(t)/ Cf(t=0)) = e(-k2t).

3. BCFss and the percent in water for the individual components in the mixture were used to calculate a mixture BCF

BCF values were lipid normalized to 5% lipid: BCFxx,l = 5%/% lipid * BCFxx.
xx refers to steady-state (ss) or kinetic (k)
Lipid content:
>= 3.7 - <= 7.1 %
Remarks on result:
other: No lipid determinations were done for experiment C but since the experiments were conducted in the same laboratory under the same conditions, mean lipid data from Experiment D of 5.4 (± 1.7)% was used to calculate lipid normalized data for Experiment C
Key result
Type:
BCF
Value:
>= 9.8 - <= 168.4 L/kg
Time of plateau:
74 h
Calculation basis:
steady state
Remarks on result:
other: Range of BCFss values for individual constituents in the LAS mixture (C12-2, C11-5, C12-6 and C12-3)
Key result
Type:
BCF
Value:
>= 9.1 - <= 155.9 L/kg
Basis:
normalised lipid fraction
Calculation basis:
steady state
Remarks on result:
other: Range of BCFss,l values for individual constituents in the LAS mixture (C12-2, C11-5, C12-6 and C12-3)
Key result
Type:
BCF
Value:
>= 15.5 - <= 359.1 L/kg
Calculation basis:
kinetic
Remarks on result:
other: range of BCFk values, for individual constituents in the LAS mixture (C12-2, C11-5, C12-6 and C12-3)
Key result
Type:
BCF
Value:
>= 14.4 - <= 332.5 L/kg
Basis:
normalised lipid fraction
Calculation basis:
kinetic
Remarks on result:
other: Range of BCFk,l values for individual constituents in the LAS mixture (C12-2, C11-5, C12-6 and C12-3)
Key result
Type:
BCF
Value:
30.2 L/kg
Calculation basis:
steady state
Remarks on result:
other: BCFss,mix for LAS mixture (C12-2, C11-5, C12-6 and C12-3)
Key result
Type:
BCF
Value:
28 L/kg
Basis:
normalised lipid fraction
Calculation basis:
steady state
Remarks on result:
other: BCFss mix,l for LAS mixture (C12-2, C11-5, C12-6 and C12-3)
Details on kinetic parameters:
-Uptake rate constant (k1) values ranged from 12.4-251.4 L/kg/d for individual constituents in the LAS mixture (C12-2, C11-5, C12-6 and C12-3)
- Depuration rate constant (k2) values ranged from 0.5-1.5/day for individual constituents in the LAS mixture (C12-2, C11-5, C12-6 and C12-3)
Details on results:
The uptake curves indicated that steady state was reached between 74 and 96 h.

BCF values for the individual components of the mixture ranged from 9.8-168.4 L/kg and 15.5-359.1 L/kg, for BCFss and BCFk, respectively. Lipid normalized BCF values, using a mean lipid value of 5.4%, ranged from 9.1-155.9 L/kg and 14.4 -332.5 L/kg for BCFss,l and BCFk,l, respectively.

BCFss and BCFk are not significantly different from each other (p<0.05).

BCFs increased with increasing alkyl chain length for a given isomer and were higher when the p-sulfophenyl moiety was positioned closer to the terminal carbon of the alkyl chain.

BCFss and the percent in water for the individual components in the mixture were used to calculate a mixture BCF of 30.2 L/kg. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 28.0 L/kg.

Table 1: BCF values from Experiment C, expressed for the individual components in the mixture as steady-state (ss) and kinetic (k). The mixture BCF (BCFmix) is based on the individual component BCFss values and the % in water. BCF values were lipid normalized (l) to 5% using a mean lipid value of 5.4% measured during Experiment C (Tolls et al., 1997)

Experiment Component % in mixture LAS (mg/L) k1(L/kg/d) k2(d-1) BCFss (L/kg) BCFss,l (5.4%) (L/kg) BCFk (L/kg) BCFk,l (5.4%) (L/kg) BCFss, mix (L/kg) BCFss, mix,l (5.4%) (L/kg)
C C12-2 0.075 0.07 251.4 0.7 168.4 155.9 359.1 332.5 30.2 28.0
C11-5 0.592 0.56 12.4 0.8 9.8 9.1 15.5 14.4
C12-6 0.214 0.21 47.0 0.5 31.9 29.5 94 87
C12-3 0.118 0.11 128.4 1.5 42.1 39 85.6 79.3

Where, k1 refers to uptake rate constant

k2, refers to elimination rate constant

BCFss, refers to steady state bioconcentration factor for LAS

BCFss,l, refers to steady-state bioconcentration factor for LAS, lipid normalized to 5%

BCFk, refers to kinetic bioconcentration factor for LAS

BCFk,l refers to kinetic bioconcentration factor for LAS, lipid normalized to 5%

BCFss,mix, refers to bioconcentration of mixture at steady state

BCFss,mix,l, refers to bioconcentration of mixture at steady state, lipid normalized to 5%

Of the information in above table, LAS concentration, BCFss,l, BCFk, BCFk,l, BCFss,mix and BCFss,mix,l  were calculated by the assessor using the molecular weight of the constituents and details present in the references.

Validity criteria fulfilled:
yes
Remarks:
OECD 305-I was followed
Conclusions:
A bioconcentration factor study with Pimephales promelas was conducted with a LAS mixture (C12-2, C11-5, C12-6 and C12-3) with an average chain length of 11.4. BCF values were lipid normalized to 5% using a mean lipid value of 5.4%. Lipid normalized BCFss and BCFk values for the individual components of the mixture ranged from 9.1-155.9 L/kg and 14.4-332.5 L/kg, respectively. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 28.0 L/kg.
Executive summary:

A BCF study with Pimephales promelas was conducted with an LAS mixture (C12-2, C11-5, C12-6 and C12-3) with an average chain length of 11.4 to understand the bioconcentration behavior of individual n-(p-sulfophenyl) alkanes in flow-through bioconcentration experiments. The method was comparable to OECD 305-I (2012).

Fish were exposed to the LAS mixture between 168 and 192 hours and after exposure they were allowed to depurate in clean water. Temperature, water hardness and TOC were also measured during the test. Analytical measurement was performed by HPLC after extraction of the test compounds from fish and water.

BCFss and BCFk values for the individual components of the mixture ranged from 9.8-168.4 L/kg and 15.5-359.1 L/kg, respectively. The BCFss value for the mixture was 30.2 L/kg. These values were lipid normalized to 5% using a mean lipid value of 5.4%. Lipid normalized BCFss and BCFk values ranged from 9.1-155.9 L/kg and 14.4-332.5 L/kg, respectively. BCFss and BCFk values for an individual component of the mixture were not significantly different from each other. BCFs increased with increasing alkyl chain length for a given isomer and were higher when the p-sulfophenyl moiety was positioned closer to the terminal carbon of the alkyl chain. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 28.0 L/kg.

This BCF study is comparable to current OECD Guideline 305 -I (Aqueous Exposure Bioconcentration Fish Test)

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Refer to the section 13 of the dataset for details on the read across justification.
Qualifier:
equivalent or similar to
Guideline:
other: OECD 319A (for rainbow trout hepatocytes) and OECD 319B (rainbow trout S9 fractions)
Deviations:
yes
Remarks:
In the given study, rainbow trout and common carp microsomal fractions were used instead of combination of cytosol and microsomal fractions as per OECD 319 B Guideline.
GLP compliance:
not specified
Radiolabelling:
yes
Details on sampling:
- Sampling intervals/frequency: Cellular systems for biotransformation were sampled at 0.5, 2, 5, 10, 24 and 48 hours. Subcellular fractions for biotransformation were sampled at the end of the incubation period i.e. 2 hours.
- Sample storage conditions before analysis: For cellular systems, media and cells were immediately frozen in liquid nitrogen, and then stored at -80°C. For subcellular system, samples were centrifuged and the supernatant was evaporated to dryness until TLC analysis.
- Details on analysis (e.g. sample preparation, analytical methods): Liquid scintillation counting (LSC) and thin layer chromatography (TLC) were used for analysis. Further details are provided in “Details on analytical methods” section.

Vehicle:
no
Details on preparation of test solutions, spiked fish food or sediment:
For cellular systems, test substance solutions were prepared in Leibovitz-15 media and verified via liquid scintillation counting.

For subcellular system, 14C-Labeled test substance (4167 Bq) was directly incubated in a buffered system and a NADPH-generating system. No information is available for test solution preparation.
Test organisms (species):
other: Cellular fractions from Common carp (Cyprinus carpio) and an immortalized cell line (PLCH-1) and subcellular fractions from Common carp and Rainbow trout (Oncorhynchus mykiss)
Details on test organisms:
Culture I: Common carp (Cyprinus carpio)
Source: Aquatic Research Organisms (ARO, Hampton, NH, USA)
Acclimation period: at least 8 days
Feed type: Hikari Koi Staple Diet (Hayward, California, USA)
Frequency: twice daily
Organisms were not fed 18 h prior to test initiation
Water quality: The culture was maintained in 890 L flow through aquaria supplied with blended well water with the following controlled conditions:
Water temperature: 22-26°C
Hardness: Approximately 180 mg CaCO3/L
pH: ~8
Photoperiod conditions: 16 h light/ 8 h dark
Light intensities: Ranging from 50-90 lux

Culture II: Common carp (Cyprinus carpio) and Rainbow trout (Oncorhynchus mykiss)
a) Common carp:
Source: Station Experimentale Piscicole de l’Indre (SEPIB, Le Blanc, France)
Feed type: carp intensive diet (SARB, Tours, France)
Frequency: twice daily
Weight at study initiation: approximately 250 g
Water quality: water temperature was 24 ± 2°C, hardness was between 150 and 220 mg CaCO3/L and pH was 7.2.

b) Rainbow trout (Oncorhynchus mykiss)
Source: INRA pisciculture at Donzacq, France
Feed type: pelleted commercial feed (Trouvit, Fontaine Les Vervins, France).
Frequency: once daily
Weight at study initiation: approximately 220 g
Water quality: water temperature was 17 ± 1°C, hardness was approximately 120 mg CaCO3/L and pH was 7.4.

PLHC-1 cells
Source: Dr. L. Hightower, University of Connecticut, Storrs, CT, USA.
Cryogenically preserved in liquid nitrogen

CELLULAR SYSTEM
Primary hepatocytes: Primary hepatocytes prepared from excised liver tissue from Common carp (Cyprinus carpio) were processed with various reagents (Hank’s balanced salt solution (HBSS), collagenase, calcium chloride, trypsin, EDTA and Leibovitz-15 media. Cells in L-15 media were centrifuged (100g for 7 minutes). The resulting pellet was remixed with media closing parantheses and filtered. The primary cells (approximately 400000 cells per well) were seeded in 96-well microtiter plates.

PLHC-1 cell culture: Cultures were used within 20 passages of being removed from cryogenic storage. Cells were grown to confluency and then-sub-cultured .Approximately 100000 PLHC-1 cells were seeded per well in 96-well microtiter plates.

SUB-CELLULAR SYSTEM:
Hepatic homogenates and microsomal fractions: Subcellular systems were prepared from thawed carp and trout livers. Livers were washed twice with cold homogenizing buffer (HEPES, EDTA, DTT and glycerol adjusted to pH 7.4), homogenized and filtered. Samples were centrifuged at 600g for 10 minutes to obtain liver homogenate fractions. The supernatant was centrifuged at 10000g for 20 minutes and for microsomal fractions, the supernatant was centrifuged again at 105000g for 60 minutes. Subcellular fractions were characterized for total protein, esterase and ECOD (ethoxycoumarin O-deethylase) activities.

Complete details on preparation of these in vitro systems are available in the publication (Dyer SD et al., 2008).
Route of exposure:
other: in vitro exposure
Justification for method:
other: As per the current regulatory standards (EU REACH), there is an increasing pressure to reduce, refine or replace animal testing, hence, there is an urgent need to evaluate the feasibility of in vitro systems useful for bioaccumulation estimation.
Test type:
static
Details on test conditions:
The subcellular systems used were hepatic microsomal fraction and liver homogenates from the Common carp (Cyprinus carpio) and hepatic microsomal fraction from Rainbow trout (Oncorhynchus mykiss). Cellular systems were comprised of primary hepatocytes from the Common carp (Cyprinus carpio) and PLHC-1 cell culture derived from Poeciliopsis lucida.

CELLULAR SYSTEM

1 Primary hepatocytes cells and PLHC-1 cells were placed approximately 24 hours prior to dosing with surfactant (25 µM) in the microtiter plates at cell density of 400000 and 100000 cells per well, respectively,
2. Each test well was dosed with 200 µL of the test substance solutions (prepared in Leibovitz-15 media). Control wells were filled with 200 µL of L-15 media only. Additionally, wells without cells (blanks) were dosed for determination of surfactant absorption to plate well surfaces.
3. Microtiter plates were sampled (in triplicate) at several time points i.e. 0.5, 2, 5, 10, 24 and 48 hours to determine the proportion of radiolabel bio-transformed. Media overlying cells was used for total counts (LSC) and to determine the proportion of radiolabel transformed. Media and cells were stored at 80°C..

SUBCELLULAR SYSTEMS
1. 14C-Labeled test substance (4167 Bq) was incubated at various concentrations (10–250 µM) in 1 mL PO4 Na/K buffer 0.1 M, 5 mM MgCl2 buffer (pH 7.4) containing the subcellular fraction (homogenate: 10 mg protein, or microsomes: 2 mg protein) and a NADPH-generating system (NADP 1.27 mM, glucose 6-phosphate 5 mM and glucose 6-phosphate dehydrogenase 2 IU) in a water bath at 25 C for 2 hours while shaking.
2. Blanks (inactivated subcellular fraction by immersion in boiling water for about 3 minutes) were also conducted in parallel.
3. After 2 hours incubation, samples were exposed to methanol to stop the reaction process. It was followed by two centrifugations at 8000 rpm for 10 minutes. Finally, an aliquot of the supernatant (500 µg/L, corresponding to ca. 500 Bq) was evaporated to dryness. The evaporated samples were resuspended in methanol before TLC analysis.

Further details on the test methodology are available in the publication (Dyer SD et al., 2008).
Nominal and measured concentrations:
Cellular Systems – 25 µM
Subcellular Systems - 10–250 µM
Reference substance (positive control):
not specified
Details on estimation of bioconcentration:
Intrinsic clearance rates (Vmax/Km) calculated from the subcellular system (i.e. microsomal fraction) and parent chemical loss (due to biotransformation) calculated from cellular systems were used as inputs to an in vitro to in vivo extrapolation model for bioconcentration (Cowan Ellsberry et al., 2008).

This extrapolation model takes the in vitro loss rates and scales them to in vivo liver loss rates and then combines the total in vivo hepatic clearance with information on the amount of blood flowing through the liver and the effect of chemical binding to proteins and lipids in blood, relative to that in the test system.

Next, the whole body transformation rate constant, KMET, was estimated. KMET takes volume of distribution of chemical (from the blood to other tissues) into account.

Finally, bioconcentration factors (BCFs) of the test substance were predicted by the mass-balance model (Arnot and Gobas; 2003, 2004).
Key result
Type:
BCF
Value:
88 dimensionless
Calculation basis:
other: Predicted using an in vitro to in vivo metabolic rate extrapolation model (Cowan-Ellsberry et al., 2008) linked to a mass-balance model (Arnot and Gobas; 2003, 2004)
Remarks on result:
other: Estimated for primary hepatocytes of Common carp
Key result
Type:
BCF
Value:
98 dimensionless
Calculation basis:
other: Predicted using an in vitro to in vivo metabolic rate extrapolation model (Cowan-Ellsberry et al., 2008) linked to a mass-balance model (Arnot and Gobas; 2003, 2004)
Remarks on result:
other: Estimated for hepatic microsomal fraction of Rainbow trout
Key result
Type:
BCF
Value:
90 dimensionless
Calculation basis:
other: Predicted using an in vitro to in vivo metabolic rate extrapolation model (Cowan-Ellsbery et al., 2008) linked to a mass-balance model (Arnot and Gobas; 2003, 2004)
Remarks on result:
other: Estimated for hepatic microsomal fraction of Common carp
Details on results:
CELLULAR SYSTEMS
1. Primary hepatocytes
- Thin layer chromatograms indicated that the test substance was rapidly taken up by the primary hepatocyte cell fraction and biotransformed to more polar metabolites.
- The proportion of metabolites increased throughout the exposure period in both cellular and media fractions.
- The resulting metabolites were found in both media and cellular fractions, evidence of metabolite efflux from the cellular fraction as well as partitioning within the cell.
- Considering these findings and assuming a completely mixed model, the parent loss rate (In vitro clearance rate) from the entire test system (media and cell fractions) was determined to be 0.01 nmol/cell/h. Considering the test volume and concentration of test material, the in vitro intrinsic clearance rate was 8E-08 mL/cell/h. This resulted in predicted fish kMET of 0.122 per day and predicted BCF of 88.

2. PLHC-1 cells
- The proportion of radiolabeled material converted to metabolites was much less, compared to primary cells, suggesting that efflux pumps may play a greater role in C12-2-LAS loss from PLHC-1 cells compared to primary hepatocytes.
- Normalized on a per protein basis, the rates of uptake and loss were greater for the PLHC-1 cells than the primary hepatocytes.

SUBCELLULAR SYSTEMS
- TLC analysis of carp liver homogenates and carp and trout microsomal fractions indicated biotransformation to more polar metabolites.
- Michaelis–Menten parameters (Vmax and Km) for the production of metabolites were calculated according to the nonlinear regression equation from microsome incubations.
- Mean Vmax ± (SD), pmol/min/mg prot, and Km ± (SD) µM, parameters for carp microsomes incubated with C12LAS were 278.14 ± 28.90 and 59.12 ± 19.91, respectively, whereas they were 173.40 ± 35.80 and 144.10 ± 344.30 from trout microsomal incubations.
- The time course of formation of metabolites by carp liver microsomes showed a rapid decrease of the parent C12-2LAS. Within 2 hours of incubation, 52% of C12-2LAS was consumed.
- The ratio of the Vmax and Km provides the in vitro intrinsic clearance rate. In vitro clearance rates were 282 and 72.2 for carp and trout, respectively. Predicted in vivo metabolism rates (KMET) for the subcellular systems were 0.094/day and 0.017/day for carp and trout and the resulting predicted BCF were 90 and 98, respectively.

Based on these values, the predicted BCF values were all less than 99 (BCF value based on Log Kow of 3). The predicted BCF values corresponded closely to measured values in several fish species, verifying the utility of in vitro systems in refining Kow-based BCFs via the inclusion of biotransformation rates
Reported statistics:
Values were compared using one-way analysis of variance (ANOVA). When there is significant (p < 0.05) changes, differences were further tested using a Tukey multiple comparison test.

Table 1: First-order kinetics of uptake and loss of C12-2-LAS in primary hepatocytes from the common carp and PLHC-1 cells from Poeciliopsis lucida (Dyer et al., 2008)

Test system k1uptake (µmol/g/h) k2loss (1/h) R2
Primary hepatocytes, test 1 0.323 0.024 0.946
Primary hepatocytes, test 2 0.286 0.009 0.933
PLHC-1 cells, test 1 0.754 0.062 0.924
PLHC-1 cells, test 2 0.768 0.762 0.946

where, k1 represents uptake of parent molecules into cells

k2 represents first order rate loss over time.

Table 2: First-order in vitro clearance rates based on exposure of C12-2-LAS to Rainbow trout and Common carp microsomes, and primary hepatocytes from Common carp (Dyer et al., 2008)

Test system In vitro clearance rate Predicted fish Predicted BCF
Subcellular  (mL/h/g protein) Cellular (mL/cell/h) KMET(per day)
Microsomal fraction of Rainbow trout 72.2 - 0.017 98
Microsomal fraction of Common carp 282 - 0.094 90
Primary hepatocytes of Common carp - 8 x 10-8 0.122 88

Clearance rates were extrapolated to predict in vivo metabolism (KMET) rates and BCF using the approach discussed in Cowan-Ellsberry et al. (2008). Assuming KMET= 0, the default BCF for the surfactants (log Kow: 3) is 99.

Validity criteria fulfilled:
yes
Remarks:
(similar to OECD Guideline 319 A and 319 B)
Conclusions:
The predicted C12-2-LAS bioconcentration factors (BCF) were 88 (in primary hepatocytes of common carp), 98 (in microsomal fraction of rainbow trout) and 90 (in microsomal fraction of common carp). BCF values were estimated using an in vitro to in vivo metabolic rate extrapolation model (Cowan-Ellsberry et al., 2008) linked to a mass-balance model (Arnot and Gobas; 2003, 2004).

These predicted BCF values corresponded closely to measured values in several fish species, verifying the utility of these in vitro systems in refining Kow-based BCFs via the inclusion of biotransformation rates.
Executive summary:

This study investigates the potential use of several in vitro systems to assess the bioconcentration of C12-2-LAS.

The subcellular systems used were microsomes and homogenates from the common carp (Cyprinus carpio) and microsomes from rainbow trout (Oncorhynchus mykiss). Cellular systems included primary hepatocytes from the Common carp (Cyprinus carpio) and PLHC-1 cell culture derived from Poeciliopsis lucida. These in vitro models were used to calculate intrinsic clearance rates (Vmax/Km) from the subcellular systems (i.e. microsomal fraction) and parent chemical loss (due to biotransformation) from primary hepatocytes, which were used as inputs for an vitro to in vivo metabolic rate extrapolation model linked to a mass-balance model for bioconcentration.

Cellular systems indicated that test substance was rapidly taken up by the primary hepatocyte cell fraction and biotransformed to more polar metabolites. Results with the subcellular systems also showed a rapid decrease of the parent C12-2LAS within 2 hours of incubation.

The predicted C12-2-LAS bioconcentration factors (BCF) were 88 (in primary hepatocytes of common carp), 98 (in microsomal fraction of Rainbow trout) and 90 (in microsomal fraction of Common carp).

These predicted BCF values corresponded closely to measured values in several fish species, verifying the utility of these in vitro systems in refining Kow-based BCFs via the inclusion of biotransformation rates.

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Refer to the section 13 of the dataset for details on the read across justification.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
Deviations:
yes
Remarks:
no depuration phase was used in study
GLP compliance:
not specified
Radiolabelling:
no
Details on sampling:
Fish were sampled in triplicates after 1 and 2 hour during the initial part of experiment.
Also, for experiment 1, approx. 143, 145 and 165 hours were the sampling points. Whereas, for experiment 2 it was approx. 95, 115, 140 and 165 hours
Vehicle:
no
Details on preparation of test solutions, spiked fish food or sediment:
Peristaltic pumps were used to prepare exposure solution, i.e. reconstituted water at 22°C + aqueous stock solution of test substance, in a mixing vessel.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: fathead minnow
- Source: Six to eight months old fish were obtained from the hatchery of Utrecht, The Netherlands.
- Weight: 0.74 ± 0.11 g
- Lipid – This was not provided in the study, but organisms are from the same laboratory and are a similar size to organisms in Tolls 1998 (Bioconcentration of Surfactants – PhD thesis), summarized in Bioaccumulation: aquatic/sediment: 002 under CAS RN 68411-30-3 IUCLID dataset. The lipid content in Fathead minnow was 5.4 ± 1.7%.
- Feeding during test: Once per day

ACCLIMATION
- Acclimation period: 1 week minimum
Route of exposure:
aqueous
Test type:
other: measured flow-through
Water / sediment media type:
other: Reconstituted freshwater
Total exposure / uptake duration:
> 165 - < 168 h
Hardness:
Not reported
Test temperature:
22°C
pH:
Not reported
Dissolved oxygen:
Not reported
TOC:
Experiment 1: 3.8 mg/L
Experiment 2: 2.0 mg/L
Salinity:
Not reported
Conductivity:
Not reported
Details on test conditions:
The fish were exposed to Na C12-2-LAS in two separate experiments, i.e. 1 and 2, using a flow-through system. Peristaltic pumps were used to prepare exposure solution (i.e. reconstituted water at 22°C + aqueous stock solution of test substance) in a mixing vessel. From the vessel, the exposure solution entered the aquarium (volume: 2 L; Renewal: 8 and 9 times/day in experiment 1 and 2) that was homogenized by continuous stirring. For feeding purpose, the fish were transferred to a separate vessel (once/day) that receives the effluent from aquarium. Fish displayed normal swimming behavior and did not show any signs of food avoidance post their transfer. Feeding was performed at same time every day in order to synchronize it with fecal excretion. The exposure lasted for 168 hours (experiment 1) and 165 hours (experiment 2). The concentrations of Na C12-2-LAS and C4-3-SPC in the aquarium and fish were determined after the steady-state was attained. For this purpose, the sampling of fish was started after 95 hours.

Temperature, pH, oxygen concentration and water flow rates were measured three times a day. In both experiments, less than 5% variation was observed in temperature, pH and oxygen concentration Total Organic Carbon was determined in acidified water samples (pH = 1) by OI TOC analyzer at the end of experiment. TOC concentration was 3.8 and 2 mg/L in experiment 1 and 2, respectively. The mass of fish and water volume were determined gravimetrically. Compound specific chemical analysis of Na C12-2-LAS and C4-3-SPC in fish and water samples was performed using Matrix solid-phase dispersion (MSPD) extraction with subsequent ion-pair liquid-liquid (IP-LL) partitioning of the extract method where C12-1-LAS and C4-3-SPC were used as internal standards. The method was sufficiently sensitive (LOQ of LAS in fish, 0.2 µg‚g-1) and reliable to investigate bioaccumulation of LAS in laboratory experiments.

The steady state bioconcentration factor (BCFss-LAS) of Na C12-2-LAS was determined using the ratio of the concentration in fish (Cf) to the concentration in water (Cw).
In experiment 1, the rate constant of uptake (k1-LAS), the steady state bioconcentration factor of Na C12-2-LAS (BCFss-LAS), and the steady-state ratio (R) of the concentration of C4-3-SPC and Na C12-2-LAS were determined. Experiment 2 was carried out to confirm the steady-state findings of experiment 1, thus, only BCFss-LAS and R were determined.
Nominal and measured concentrations:
The average concentrations of Na C12-2-LAS during the steady-state part of the exposure phase were 0.104 and 0.194 µM in experiments 1 and 2, respectively
Reference substance (positive control):
not specified
Details on estimation of bioconcentration:
The bioconcentration factor was determined by as the ratio of the concentration of Na C12-2-LAS in fish to the concentration in water at steady-state.

Key result
Temp.:
22 °C
Type:
BCF
Value:
106 L/kg
Basis:
not specified
Calculation basis:
steady state
Remarks on result:
other: BCFss-LAS for experiment 1
Key result
Temp.:
22 °C
Type:
BCF
Value:
98 L/kg
Basis:
normalised lipid fraction
Remarks on result:
other: BCFss-LAS,l for experiment 1
Key result
Temp.:
22 °C
Type:
BCF
Value:
128 L/kg
Basis:
not specified
Calculation basis:
steady state
Remarks on result:
other: BCFss-LAS for Experiment 2.
Key result
Temp.:
22 °C
Type:
BCF
Value:
119 L/kg
Basis:
normalised lipid fraction
Remarks on result:
other: BCFss-LAS,l for Experiment 2.
Key result
Rate constant:
overall uptake rate constant (L kg-1 d-1)
Value:
159
Remarks on result:
other: Experiment 1.
Details on kinetic parameters:
Uptake rate constant (k1): Experiment 1: 159 L kg-1 d-1
Experiment 2: An uptake rate constant was not determined.
Experiments 1 and 2: No depuration phase.
Metabolites:
C4-3-SPC [3-n-(p-sulfophenyl)-butyric acid]
- C4-3-SPC was present in the exposure water and it increased during both exposure experiments and the parent compound decreased during the study. The average concentration during steady state were 0.138 and 0.055 mM in experiments 1 and 2, respectively.
- Mass flux calculations demonstrated that the formation of C4-3-SPC per unit of time was smaller than the uptake of Na C12-2-LAS by fish. It is thus likely that microbial degradation of Na C12-2-LAS rather than biotransformation by fish was the prevalent process leading to formation of C4-3-SPC in the water.
- C4-3-SPC was present in fish samples at 8.6 and 19.4 µmol/kg. The ratio of Cf,SPC to Cf,LAS appeared to be time dependent indicating that the biotransformation process in the fish had attained steady state.
- Biotransformation intermediates/metabolites other than C4-3-SPC were not detected in water, while small peaks (in chromatogram) eluting between C4-3-SPC and Na C12-2-LAS were indicative of low concentrations of unknown metabolites in the fish.
- Due to microbial degradation of Na C12-2-LAS in water phase, presence of C4-3-SPC can be either due to uptake from water or biotransformation of Na C12-2-LAS. Investigations revealed that the ratio of Cf,SPC to Cf,LAS was not dependent on the Cw,SPC which indicates uptake of C4-3-SPC from water does not contribute perceivably to the concentration of this compound in the fish. Biotransformation was the only significant source of C4-3-SPC in fish.
Details on results:
BCF values were lipid normalized to 5% lipid: BCFxx,l = 5%/% lipid * BCFxx.
Here, xx represents ss for BCFss.

Table 1: Summary of bioconcentration parameters from Experiments 1 and 2 with Na C12-2-LAS (Tolls et al., 2000).

Parameter Experiment 1 Experiment 2
Cw-LAS 0.104 ± 0.062 µM 0.194 ± 0.061 µM
Cw-SPC 0.138 ± 0.053 µM 0.055 ± 0.019 µM
Kdec 0.48 d(-1) 0.22 d(-1)
Cf-LAS 11 ± 2.6 µmol/kg (n=9) 24.9 ± 10.4 µmol/kg (n=20)
Cf-SPC 8.6 ± 2 µmol/kg (n=9) 19.4 ± 11.5 µmol/kg (n=20)
BCFss-LAS 106 (n=9) 128 (n=20)
BCFss-LAS,l 98 119
k1-LAS 159 ± 16 L/kg ND
R 0.81 ± 0.22 (n=9)  0.68 ± 0.37 (n=20)

where,

Cw-LAS = Concentration of Na C12-2-LAS flowing out of the aquarium

Cw-SPC = Concentration of C4-3-SPC flowing out of the aquarium

Kdec = First-order rate constant of the decrease of the concentration of Na C12-2-LAS in the water in the exposure aquarium

Cf-LAS = Concentration of Na C12-2-LAS in fish

Cf-SPC = Concentration of C4-3-SPC in fish

BCFss-LAS = Steady state bioconcentration factor for LAS (Cf-LAS/Cw-LAS)

BCFss-LAS,l= Steady-state bioconcentration factor for LAS, lipid normalized to 5%

k1-LAS = Uptake rate constant for LAS

R = Ratio of the concentrations of C4-3-SPC and Na C12-2-LAS in fish at steady state

ND=not determined

Validity criteria fulfilled:
yes
Remarks:
(Comparable to OECD Guideline 305-I)
Conclusions:
The steady-state bioconcentration factors (BCFss-LAS) for Na C12-2-LAS in fathead minnow (Pimephales promelas) were 106 and 128 L/kg in experiments 1 and 2, respectively. Lipid normalized BCF values were 96 and 119 L/kg in experiments 1 and 2, respectively.
Executive summary:

The study was conducted to determine the bioconcentration factor of Na C12-2-LAS in Fathead minnow (Pimephales promelas).

The fish were exposed to Na C12-2-LAS in two separate experiments.The steady-state bioconcentration factor (BCFss-LAS) of Na C12-2-LAS was determined as the ratio of the concentration in fish to the concentration in water at steady-state.

The metabolite C4-3-SPC was present in fish samples at 8.6 and 19.4 µmol/kg in experiments 1 and 2, respectively.  Biotransformation intermediates other than C4-3-SPC were not detected in water, while small peaks eluting between C4-3-SPC and Na C12-2-LAS were indicative of low concentrations of metabolites in the fish.

The steady-state bioconcentration factors (BCFss-LAS) for Na C12-2-LAS in fathead minnow (Pimephales promelas) were 106 and 128 L/kg in experiments 1 and 2, respectively. Lipid normalized BCF values were 96 and 119 L/kg in experiments 1 and 2, respectively.

Description of key information

The registered test substance, MEA-LAS, dissociates into MEA and LAS in aqueous media. MEA is not bioaccumulative (ECHA, 2020). Bioaccumulation of the registered substance focuses on the more hazardous constituent, LAS. LAS is not bioaccumulative (Section 4.3.1 Aquatic Bioaccumulation) based on several bioaccumulation and metabolism studies. A series of octanol:water partition coefficient studies were completed that provide log Kow estimates of 1.8, 2.42, 3.04, and 3.67 for LAS chain lengths of C10, C11, C12 and C13, respectively (Fraunhofer IME, 2019). Tolls et al. (1997, 1998, 2000) determined steady state BCFs ranged from 1.4-5.6, 6-29.5, 82-195.8 and 372-914.1 L/kg for C10, C11, C12, and C13, respectively. Dyer et al. (2008) confirmed a high rate of cellular metabolism in fish for C12 LAS which was used as input to a physiological model of fish bioaccumulation by Cowen-Ellsberry et al. (2008) to estimate a C12 LAS BCF of 98 L/kg, within modelling expectations of the authors. The registered UVCB substance, whose substance identity profile for the commercially relevant distribution of chain lengths is given in section 1.2 of the dataset, has an estimated BCF of 103 L/kg for rainbow trout to 250 L/kg for fathead minnow based on measured BCFs versus chain length regressions established from Tolls et al. (1997, 1998, 2000).

 

Because of the recent log Kow measurement for C13 LAS of 3.67, a new study on the bioaccumulation of C13 LAS was commissioned to confirm estimates from previous studies. A previous communication to ECHA delivered on 5 December 2019 indicated a delay to remedy a data request from ECHA (CCH-D-2114391910-46-01/F) for the follow-on study, Bioaccumulation in aquatic species (Annex IX, Section 9.3.2.; test method: Bioaccumulation in fish: aqueous and dietary exposure, OECD TG 305) with the registered substance. At this time, the in-life portion of the new bioaccumulation study is completed, and the preliminary results are consistent with the expected BCF value showing C13 LAS does not bioaccumulate. Following data review and quality control, we anticipate having the final GLP report in May 2020 and the data will then be integrated into the dossier without undue delay.

Study 1 (C10-13 LAS):

A BCF study using the fathead minnow (Pimephales promelas) was conducted with a C10-13 LAS mixture ((C10-2, C11-2, C12-2, C13-2, C10-in, C11-in, C12-in and C13-in) with an average chain-length of 10.6 to understand the bioconcentration behavior of individual n-(p-sulfophenyl) alkanes in flow-through bioconcentration experiments. The method was comparable to OECD Guideline 305-I (2012). Fish were exposed to the LAS mixture between 168 and 192 h and after exposure they were allowed to depurate in clean water. Temperature, water hardness and TOC were also measured during the test.  Analytical measurement was performed by HPLC after extraction of the test compounds from fish and water. BCFss and BCFk values for the individual components of the mixture ranged from 3-987 and 7-1070 L/kg, respectively. The BCFss value for the mixture was 22.6 L/kg. These values were lipid normalized to 5% using a mean lipid value of 5.4%. Lipid normalized BCFss and BCFk values for the individual components of the mixture ranged from 3-914 L/kg and 7-991 L/ kg, respectively. BCFss and BCFk values for an individual component of the mixture were not significantly different from each other. BCFs increased with increasing alkyl chain length for a given isomer and were higher when the p-sulfophenyl moiety was positioned closer to the terminal carbon of the alkyl chain. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 20.9 L/kg (Tolls, 1997 and 1998).

Study 2 (C10-13 LAS):

A bioconcentration study with rainbow trout (Oncorhynchus mykiss) was conducted with a C10-13 LAS mixture consisting of C10-2, C11-2, C12-2 and C13-2 LAS (average chain length: 10.6). The method was comparable to OECD Guideline 305-I (2012). Fish (n=6) were sampled after 3, 8, 78 and 120 h of exposure.  Six remaining fish were transferred to clean water, allowed to depurate for 3 h and then sampled. Fish were euthanized by cervical dislocation, and skin, gills, head, fillet, liver and internal organs (IO: Intestine, kidney, spleen, stomach, pyloric caeca, heart, gall bladder and gonads) were dissected. The livers of all six fish sampled at one time point were pooled. Likewise, the IO, skin, gills and heads of a pair of two specimens were pooled into one sample. Muscles of individual fish were analyzed. Water samples and fish were extracted by solid-phase extraction and matrix solid-phase dispersion extraction, respectively. LAS concentrations in fish reached steady state after approximately 78 h. The ratio of Ctis/Cw was higher in gills and skin than other tissues and ranged from 1.7-684 and 4.0-717 L/kg, respectively.  Whole body BCFss values ranged from 1.4-372 L/kg for individual components of the LAS mixture. BCFs increased with increasing alkyl chain length.  The whole body BCFss for the mixture (BCFss,mix) was 23.3 L/kg (Tolls, 2000).

Study 3 (C12-2, C11-5, C12-5 and C13-5 LAS mixture):

A BCF study with Pimephales promelas was conducted with an LAS mixture (C12-2, C11-5, C12-5 and C13-5) with an average chain-length of 11.7 to understand the bioconcentration behavior of individual n-(p-sulfophenyl) alkanes in flow-through bioconcentration experiments. The method was comparable to OECD Guideline 305-I (2012). Fish were exposed to the LAS mixture between 168 and 192 h and after exposure they were allowed to depurate in clean water. Temperature, water hardness and TOC were also measured during the test. Analytical measurement was performed by HPLC after extraction of the test compounds from fish and water. BCFss and BCFk values for the individual components of the mixture ranged from 6.1-99.1 and 3.6-89.6 L/kg, respectively. The BCFss value for the mixture was 27.0 L/kg. These values were lipid normalized to 5% using a mean lipid value of 5.4%. Lipid normalized BCFss and BCFk values ranged from 5.6-91.8 and 3.3-83.0 L/kg, respectively. BCFss and BCFk values for an individual component of the mixture were not significantly different from each other. BCFs increased with increasing alkyl chain length for a given isomer and were higher when the p-sulfophenyl moiety was positioned closer to the terminal carbon of the alkyl chain. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 25.0 L/kg (Tolls, 1997 and 1998).

Study 4 (C12-2, C11-5, C12-6 and C12-3 LAS mixture):

A BCF study with Pimephales promelas was conducted with an LAS mixture (C12-2, C11-5, C12-6 and C12-3) with an average chain length of 11.4 to understand the bioconcentration behavior of individual n-(p-sulfophenyl) alkanes in flow-through bioconcentration experiments. The method was comparable to OECD Guideline 305-I (2012). Fish were exposed to the LAS mixture between 168 and 192 h and after exposure they were allowed to depurate in clean water. Temperature, water hardness and TOC were also measured during the test. Analytical measurement was performed by HPLC after extraction of the test compounds from fish and water. BCFss and BCFk values for the individual components of the mixture ranged from 9.8-168.4 L/kg and 15.5-359.1 L/kg, respectively. The BCFss value for the mixture was 30.2 L/kg. These values were lipid normalized to 5% using a mean lipid value of 5.4%. Lipid normalized BCFss and BCFk values ranged from 9.1-155.9 L/kg and 14.4-332.5 L/kg, respectively. BCFss and BCFk values for an individual component of the mixture were not significantly different from each other. BCFs increased with increasing alkyl chain length for a given isomer and were higher when the p-sulfophenyl moiety was positioned closer to the terminal carbon of the alkyl chain. The lipid normalized BCFss for the mixture (BCFss,mix,l) was 28.0 L/kg (Tolls, 1997 and 1998).

Study 5 (C12-2 LAS):

A study was conducted to investigate the potential use of several in vitro systems to assess the bioconcentration of C12-2 LAS. The subcellular systems used were microsomes and homogenates from the common carp (Cyprinus carpio) and microsomes from rainbow trout (Oncorhynchus mykiss). Cellular systems included primary hepatocytes from the Common carp (Cyprinus carpio) and PLHC-1 cell culture derived from Poeciliopsis lucida. These in vitro models were used to calculate intrinsic clearance rates (Vmax/Km) from the subcellular systems (i.e. microsomal fraction) and parent chemical loss (due to biotransformation) from primary hepatocytes, which were used as inputs for an vitro to in vivo metabolic rate extrapolation model linked to a mass-balance model for bioconcentration. Cellular systems indicated that test substance was rapidly taken up by the primary hepatocyte cell fraction and biotransformed to more polar metabolites. Results with the subcellular systems also showed a rapid decrease of the parent C12-2-LAS within 2 h of incubation. The predicted C12-2-LAS bioconcentration factors (BCF) were 88 (in primary hepatocytes of common carp), 98 (in microsomal fraction of Rainbow trout) and 90 (in microsomal fraction of Common carp). These predicted BCF values corresponded closely to measured values in several fish species, verifying the utility of these in vitro systems in refining Kow-based BCFs via the inclusion of biotransformation rates (Dyer, 2008; Cowan-Ellberry, 2008; Arnot 2003 and 2004).

Study 6 (C12-2 LAS):

A study was conducted to determine the bioconcentration factor of C12-2-LAS, sodium salt in Fathead minnow (Pimephales promelas). The fish were exposed to the test substance in two separate experiments. The steady-state bioconcentration factor (BCFss-LAS) was determined as the ratio of the concentration in fish to the concentration in water at steady-state. The metabolite C4-3-SPC was present in fish samples at 8.6 and 19.4 µmol/kg in Experiments 1 and 2, respectively.  Biotransformation intermediates other than C4-3-SPC were not detected in water, while small peaks eluting between C4-3-SPC and C12-2-LAS, sodium salt were indicative of low concentrations of metabolites in the fish. The steady-state bioconcentration factors (BCFss-LAS) for the test substance in Fathead minnow (Pimephales promelas) were 106 and 128 L/kg in Experiments 1 and 2, respectively. Lipid normalized BCF values were 96 and 119 L/kg in Experiments 1 and 2, respectively (Tolls, 1998 and 2000).

Key value for chemical safety assessment

BCF (aquatic species):
20.9 L/kg ww

Additional information