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Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 23, 2020 to March 26, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
Version / remarks:
While OECD test guideline 305 recommends that the concentration of test substance in the test chambers should be maintained within ± 20% of the mean of measured values during the uptake phase, in this study the concentration of test substance was not maintained within this limit due to the rapid biodegradability of the test substance. The Coefficient of Variation for the time-weighted mean measured concentration was approximately 14% in both treatment groups and sampling occurred every 3-4 days through the 28 d exposure period to characterize exposure concentrations to compensate for the expected loss due to biodegradation.
Deviations:
no
Remarks:
(see details in remarks section)
GLP compliance:
yes (incl. certificate)
Remarks:
[U.S. Environmental Protection Agency (40 CFR Part 792)]
Radiolabelling:
no
Details on sampling:
- Water samples: Water samples were collected from the negative control and both treatment groups (0.050 and 0.100 mg/L) on 4, 2 and 1 days prior to the start of the exposure period to confirm the concentrations after conditioning the diluter system. In the negative control and both treatment groups, water samples were collected on days 0 (hours 0 and 4), 1, 3, 6, 7, 10, 14, 17, 21, 24 and 28 of the uptake phase, and days 1, 4, 7 and 10 of the depuration phase for the analysis of tridecyl (C13) - LA S. On Days 6 and 7 of the uptake phase, additional samples were collected due to a delivery malfunction and the syringe pump was replaced due to a second malfunction. On each sampling day, two water samples were analyzed from the negative control test chamber and each treatment group test chambers. All water samples were collected into 20-mL glass scintillation vials that were prefilled with 2000 µL of 50.0 µg a.i./L internal standard in methanol. Samples were collected from mid-depth in the test chamber using a graduated glass pipette. Water samples were processed immediately for analysis. Additional water samples were collected at each interval as backup during both the uptake and depuration phases.

- Tissue samples: Tissue samples were collected from the negative control and both treatment groups (0.050 and 0.100 mg/L) on uptake days 0 (hour 4), 1, 3, 7, 10, 14, 17, 21, 24 and 28 of the uptake phase, and days 1, 4, 7 and 10 of the depuration phase. At each tissue sampling interval, a sufficient number of fish were collected to provide at least four replicate samples of negative control fish and four replicate samples from each tridecyl (C13) - LAS treatment group. Fish were impartially removed from the test chambers, rinsed with dilution water, blotted dry and euthanized by making an incision from just posterior to the base of the pectoral fin dorsally through the spinal cord. The fish were measured for total length and wet weight within approximately 15 minutes of collection. All tissue samples were processed immediately or stored frozen.

- Lipid sampling: Twenty-seven additional fish were collected from the control and each treatment group to determine lipid content (nine from the control and each treatment group). Of these fish sampled, the control samples collected on Day 0 of uptake, after confirmation of steady state (Day 28 uptake) and at the end of depuration (Day 11 depuration) were analyzed for lipid content. All fish collected for lipid content were stored frozen until analysis.
Vehicle:
no
Details on preparation of test solutions, spiked fish food or sediment:
A primary stock solution was prepared 6 times during the uptake phase test by mixing a calculated amount of test substance into reverse osmosis (RO) water (at a nominal concentration of 200 mg/L). It was sonicated for approximately 30 minutes after bringing to a partial volume. The stock solution was then brought to a final volume of 2 L and stirred on a magnetic stir plate for approximately 15 minutes. The stock solution was clear and colorless with foam on the surface. The stock was held refrigerated in glass amber bottles. Fresh aliquots of each stock were placed on the delivery system pump daily during the test.

Test solution concentrations were based on test substance as received and not adjusted for the active ingredient of test substance.
Test organisms (species):
Lepomis macrochirus
Details on test organisms:
TEST ORGANISM
- Common name: Bluegill
- Source: Osage Catfisheries, Inc., Osage Beach, Missouri
- Age at study initiation: Approximately 8 months old at test initiation
- Length at study initiation: 6.0 ± 0.33 cm (range: 5.3 – 6.5 cm)
- Weight at study initiation: 2.86 ± 0.488 g (range: 1.94 – 3.58 g)
- Mean measured lipid content at test initiation (in whole fish tissue of negative control): 5.31%
- Food type: Commercially prepared diet of Sera Vipan supplied by Sera, North America of Montgomeryville, Pennsylvania - Amount: 2% of fish body weight
- Frequency: At least once daily

ACCLIMATION
- Acclimation period: 2 weeks (14 days)
- Acclimation conditions: Same as test
- Type of food: Commercially prepared diet of Sera Vipan supplied by Sera, North America of Montgomeryville, Pennsylvania
- Amount of food: 2% of fish body weight
- Feeding frequency: At least once daily
- Health during acclimation: All fish in the culture during this time appeared normal
Route of exposure:
aqueous
Water / sediment media type:
other: well water (filtered, sterilized)
Total exposure / uptake duration:
28 d
Total depuration duration:
11 d
Hardness:
139 - 142 mg/L (as CaCO3)
Test temperature:
21.9 - 22.3 °C
pH:
8.1 - 8.3
Dissolved oxygen:
≥ 7.6 mg/L (88% of saturation)
TOC:
i) Uptake: 1.27 - 1.35 mg C/L
ii) Depuration: 1.19 - 1.79 mg C/L
Conductivity:
345 - 349 µS/cm
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Uptake phase: 127-L Teflon-lined stainless steel aquaria filled with 80 L of test solution having 19.2 cm (approx.) depth of the test water.
- Depuration phase: 54-L Teflon-lined stainless steel aquaria filled with 45 L of dilution water (well water) having 24.6 cm (approx.) depth of the test water
- Renewal rate of test solution: 87.5 µL stock solution/minute (low treatment group) and 175 µL stock solution/minute (high treatment group)
- Type of flow-through: Continuous flow diluter
- Delivery rate of mixed solution (stock solution and dilution water) from mixing chamber: 350 mL/minute for achievement of desired nominal concentration
- No. of organisms per vessel: 80
- No. of vessels per concentration: 1
- No. of vessels per control / vehicle control (replicates): 1
- Loading rate: 0.45 g fish/L/day
- Instantaneous loading rate: 2.9 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Freshwater obtained from a well approximately 40 meters deep located on the Eurofins-Easton site. The water was filtered to 0.45 µm and passed through a ultraviolet sterilizer prior to use.
- Pesticides: < 0.011 - <22 µg/L
- Metals: <0.0050 - 34.2 mg/L
- Chloride: 3.9 mg/L
- Alkalinity: 172 (164 – 180) mg/L as CaCO3
- Specific conductance: 327 (301 - 348) µS/cm

- Intervals of water quality measurement:
- Temperature: Measured in each test chamber at the beginning and end of the test and at weekly intervals (approx.) during the test. Temperature was also monitored continuously in the negative control test chamber.
- pH: Measured in each test chamber at the beginning and end of the test and at weekly intervals (approx.) during the test
- Dissolved oxygen: Measured in each test chamber on a daily basis.
- Hardness, alkalinity and conductivity: Measured in the dilution water and high treatment level at the beginning and end of each phase of the test.
- TOC: Total organic carbon (TOC) was measured in the dilution water, low treatment level and high treatment level at the beginning and end of each phase, and approximately weekly during the test.
- Intervals of test medium replacement: 6 exchange additions of test solution in each test chamber per day

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of dark
- Light intensity at surface of water:
- Starting of uptake phase: 904 lux
- Starting of depuration phase: 881 lux
- Transition period: 30 minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: 0.100 mg/L (nominal concentration)
- Results used to determine the conditions for the definitive study:
- Time to steady state: 10.5 days
- Time to reach 50% clearance: 2.5 days
- BCF (kinetic): 893 L/kg
- BCF (steady state): 862 L/kg
Nominal and measured concentrations:
Nominal concentrations (uptake phase): 0, 0.050 and 0.100 mg/L
Time weighted mean measured concentrations (uptake phase): 0, 0.019 (38% of nominal concentration) and 0.035 (35% of nominal concentration) mg/L
Details on estimation of bioconcentration:
BASIS FOR CALCULATION OF BCF
The steady-state bioconcentration factor (BCF) values were determined from the mean tissue concentrations at apparent steady-state divided by the time weighted average water concentration. Tissue concentrations were considered to be at apparent steady-state if three or more consecutive sets of tissue concentrations were within ± 20% of each other, and there was no significantly increasing trend in residue concentration in time between the first and last successive analyses (p > 0.05). All statistical tests were performed using a personal computer with the SAS System for Windows software. Nonlinear regression was used to sequentially solve for k1 (uptake rate constant) and k2 (depuration rate constant) using fish tissue data from the uptake phase and depuration phase. These rate constants were used to calculate a kinetic bioconcentration factor (BCFK = k1/k2) and also to calculate half-life for clearance in tissue (t½) and, time to reach 95% of steady state (t95). The kinetic uptake and depuration rate constants were determined using nonlinear regression described by Newman. In the sequential method, data from the depuration (elimination) phase was used to first estimate k2, and then using both the k2 estimate and fish tissue from the uptake phase to estimate k1. In addition, lipid-normalized and growth-corrected kinetic and steady state bioconcentration factors (BCFSSL, BCFKL, BCFKg and BCFKLG) as well as growth corrected half-life (t½g) were determined.

Details on equations used for calculations of various parameters are in PDF file attached in "attached background material section" due to limitations of IUCLID in handling equations.
Lipid content:
5.31 %
Time point:
start of exposure
Remarks on result:
other: Determined in whole tissue from the negative control fish
Lipid content:
6.95 %
Time point:
end of exposure
Remarks on result:
other: Determined in whole tissue from the negative control fish
Lipid content:
6.28 %
Time point:
other: Day 11 of depuration phase
Remarks on result:
other: Determined in whole tissue from the negative control fish
Key result
Conc. / dose:
0.019 mg/L
Type:
BCF
Value:
476 L/kg
Basis:
whole body w.w.
Calculation basis:
steady state
Key result
Conc. / dose:
0.035 mg/L
Type:
BCF
Value:
495 L/kg
Basis:
whole body w.w.
Calculation basis:
steady state
Key result
Conc. / dose:
0.019 mg/L
Type:
BCF
Value:
393 L/kg
Basis:
whole body w.w.
Time of plateau:
14 d
Calculation basis:
kinetic
Key result
Conc. / dose:
0.035 mg/L
Type:
BCF
Value:
399 L/kg
Basis:
whole body w.w.
Time of plateau:
12 d
Calculation basis:
kinetic
Key result
Conc. / dose:
0.019 mg/L
Type:
BCF
Value:
415 L/kg
Basis:
whole body w.w.
Calculation basis:
kinetic, corrected for growth
Key result
Conc. / dose:
0.035 mg/L
Type:
BAF
Value:
420 L/kg
Basis:
whole body w.w.
Calculation basis:
kinetic, corrected for growth
Key result
Conc. / dose:
0.019 mg/L
Type:
BCF
Value:
283 L/kg
Basis:
normalised lipid fraction
Calculation basis:
kinetic
Key result
Conc. / dose:
0.035 mg/L
Type:
BCF
Value:
287 L/kg
Basis:
normalised lipid fraction
Calculation basis:
kinetic
Key result
Conc. / dose:
0.019 mg/L
Type:
BCF
Value:
298 L/kg
Basis:
normalised lipid fraction
Calculation basis:
kinetic, corrected for growth
Key result
Conc. / dose:
0.035 mg/L
Type:
BCF
Value:
302 L/kg
Basis:
normalised lipid fraction
Calculation basis:
kinetic, corrected for growth
Key result
Conc. / dose:
0.019 mg/L
Type:
BCF
Value:
342 L/kg
Basis:
normalised lipid fraction
Calculation basis:
steady state
Key result
Conc. / dose:
0.035 mg/L
Type:
BCF
Value:
356 L/kg
Basis:
normalised lipid fraction
Calculation basis:
steady state
Key result
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
3.4 d
Remarks on result:
other: half-life value based on time weighted mean measured test concentration of 0.019 mg/L.
Key result
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
2.8 d
Remarks on result:
other: half-life value based on time weighted mean measured test concentration of 0.035 mg/L.
Key result
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
3.4 d
Remarks on result:
other: growth corrected half-life value based on time weighted mean measured test concentration of 0.019 mg/L.
Key result
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
2.9 d
Remarks on result:
other: growth corrected half-life value based on time weighted mean measured test concentration of 0.035 mg/L.
Key result
Rate constant:
overall uptake rate constant (L kg-1 d-1)
Value:
84.4
Remarks on result:
other: based on time weighted mean measured test concentration of 0.019 mg/L
Key result
Rate constant:
overall depuration rate constant (d-1)
Value:
0.215
Remarks on result:
other: based on time weighted mean measured test concentration of 0.019 mg/L.
Key result
Rate constant:
overall uptake rate constant (L kg-1 d-1)
Value:
99.8
Remarks on result:
other: based on time weighted mean measured test concentration of 0.035 mg/L.
Key result
Rate constant:
overall depuration rate constant (d-1)
Value:
0.25
Remarks on result:
other: based on time weighted mean measured test concentration of 0.035 mg/L
Key result
Rate constant:
growth rate constant (d-1)
Value:
0.019
Remarks on result:
other: control group (uptake phase).
Key result
Rate constant:
growth rate constant (d-1)
Value:
-0.015
Remarks on result:
other: control group (depuration phase).
Key result
Rate constant:
growth rate constant (d-1)
Value:
0.013
Remarks on result:
other: based on time weighted mean measured test concentration of 0.019 mg/L (uptake phase).
Key result
Rate constant:
growth rate constant (d-1)
Value:
-0.016
Remarks on result:
other: based on time weighted mean measured test concentration of 0.019 mg/L (depuration phase).
Key result
Rate constant:
growth rate constant (d-1)
Value:
0.015
Remarks on result:
other: based on time weighted mean measured test concentration of 0.035 mg/L (uptake phase).
Key result
Rate constant:
growth rate constant (d-1)
Value:
0.008
Remarks on result:
other: based on time weighted mean measured test concentration of 0.035 mg/L (depuration phase).
Key result
Rate constant:
growth rate constant (d-1)
Value:
0.012
Remarks on result:
other: pooled and based on time weighted mean measured test concentration of 0.019 mg/L.
Key result
Rate constant:
growth rate constant (d-1)
Value:
0.013
Remarks on result:
other: pooled and based on time weighted mean measured test concentration of 0.035 mg/L
Key result
Rate constant:
growth-corrected depuration rate constant (d-1)
Value:
0.203
Remarks on result:
other: based on time weighted mean measured test concentration of 0.019 mg/L.
Key result
Rate constant:
growth-corrected depuration rate constant (d-1)
Value:
0.238
Remarks on result:
other: based on time weighted mean measured test concentration of 0.035 mg/L.
Details on kinetic parameters:
- Estimated time to reach 95% steady state at nominal exposure of 0.050 mg/L: 14 days
- Estimated time to reach 95% steady state at nominal exposure of 0.100 mg/L: 12 days
Details on results:
- Mortality of test organisms: There was one mortality in 0.050 mg/L (0.019 mg/L time weighted mean measured concentration) treatment groups during the study, however these were considered to be incidental and not treatment related
- Clinical signs of toxicity: Fish appeared normal throughout the test duration
- Observations on body length and weight: No statistical differences were observed between the growth data in the control group and the treatment groups
- Depuration phase: Tridecyl (C13) - LAS quickly depurated from all fish tissue and by Day 7 and 10 of depuration in low and high treatment groups, respectively tissue concentrations were - Determination of steady-state: It was determined the study had reached steady state on Days 21, 24 and 28 in both treatment groups. However the residue concentrations of these successive analyses were not within ± 20% of each other, and was attributed to the decreasing tissue concentrations on Day 24. To confirm steady-state was achieved, the Day 24 interval was excluded from analysis to evaluate steady-state. When the measured concentrations from Days 17, 21 and 28 were evaluated, the residue concentrations were within ± 20% of each other and it was statistically confirmed that that the study had reached steady state by Day 28.
Reported statistics:
- All statistical tests were performed using a personal computer with SAS System for Windows software and student's t-test (p=0.05) was used for statistical comparison of slopes of the linear regression.

Table 1: Measured concentrations of tridecyl (C13) - LAS in pre-test diluter verification samples

Nominal Test Concentration (mg/L) Sample Number (106A-140) Sampling Time (Day) Measured Concentration (mg/L)1,2 Percent of Nominal
Negative control PT-1 -4 <LOQ --
Negative control PT-2 -4 <LOQ --
Negative control   PT-134 -2 <LOQ --
Negative control   PT-144 -2 <LOQ --
Negative control PT-19 -1 <LOQ --
Negative control PT-20 -1 <LOQ --
0.050 PT-3 -4 0.0324 64.7
0.050 PT-4 -4 0.0321 64.1
0.050  PT-153 -2 0.028 56
0.050  PT-163 -2 0.0274 54.8
0.050 PT-21 -1 0.0139 27.9
0.050 PT-22 -1 0.013 25.9
0.100 PT-5 -4 0.051 51
0.100 PT-6 -4 0.0563 56.3
0.100  PT-173 -2 0.0412 41.2
0.100  PT-183 -2 0.0441 44.1
0.100 PT-23 -1 0.0364 36.4
0.100 PT-24 -1 0.0356 35.6

1The method limit of quantitation (LOQ) for the analyses of Tridecyl (C-13)-LAS in freshwater is defined as 0.0100 mg/L, the lowest nominal concentration in a fortified sample at which a mean recovery of 70-110% has been obtained.

2Results were generated using Excel 2010. Manual calculations may differ slightly.

3Aeration was added after sampling.

 

Table 2: Measured concentrations of tridecyl (C13) - LAS in water samples from a bluegill bioconcentration test during the uptake phase

Nominal Test Concentration (mg/L) Sample Number (106A-140) Sampling Time (Day) Measured Concentration (mg/L)1,2 Percent of Nominal2 Mean Measured Concentration (mg/L)3 Mean Measured Percent of Nominal3
Negative control 1 0 <LOQ -- -- --
Negative control 2 0 <LOQ --
Negative control 7 0.17 <LOQ --
Negative control 8 0.17 <LOQ --
Negative control 13 1 <LOQ --
Negative control 14 1 <LOQ --
Negative control 19 3 <LOQ --
Negative control 20 3 <LOQ --
Negative control 25 6 <LOQ --
Negative control 26 6 <LOQ --
Negative control 31 7 <LOQ --
Negative control 32 7 <LOQ --
Negative control  374 7 <LOQ --
Negative control  384 7 <LOQ --
Negative control 43 10 <LOQ --
Negative control 44 10 <LOQ --
Negative control 49 14 <LOQ --
Negative control 50 14 <LOQ --
Negative control 55 17 <LOQ --
Negative control 56 17 <LOQ --
Negative control 61 21 <LOQ --
Negative control 62 21 <LOQ --
Negative control 67 24 <LOQ --
Negative control 68 24 <LOQ --
Negative control 73 28 <LOQ --
Negative control 74 28 <LOQ --
0.050 3 0 0.0134 26.8 0.019 ± 0.0027 (CV – 14.3%) 38
0.050 4 0 0.0128 25.7
0.050 9 0.17 0.0128 25.6
0.050 10 0.17 0.0123 24.6
0.050 15 1 0.00924 18.5
0.050 16 1 0.00983 19.7
0.050 21 3 0.0101 20.3
0.050 22 3 0.00904 18.1
0.050  274 6  < LOQ5 --
0.050  284 6  < LOQ5 --
0.050  336 7  < LOQ5 --
0.050  346 7  < LOQ5 --
0.050  397 7 0.0143 28.5
0.050  407 7 0.0174 34.8
0.050  45-RE8 10 0.0129 25.8
0.050 46 10 0.0145 29
0.050 51 14 0.0194 38.8
0.050 52 14 0.0225 45.1
0.050 57 17 0.029 58.1
0.050 58 17 0.0244 48.7
0.050 63 21 0.0307 61.3
0.050 64 21 0.0237 47.4
0.050 69 24 0.0259 51.8
0.050 70 24 0.0309 61.7
0.050 75 28 0.026 52
0.050 76 28 0.0219 43.7
0.100 5 0 0.0227 22.7 0.035 ± 0.0050 (CV – 14.1%) 35
0.100 6 0 0.0224 22.4
0.100 11 0.17 0.0241 24.1
0.100 12 0.17 0.0237 23.7
0.100 17 1 0.0243 24.3
0.100 18 1 0.0223 22.3
0.100 23 3 0.0213 21.3
0.100 24 3 0.0232 23.2
0.100  294 6  < LOQ5 --
0.100  304 6  < LOQ5 --
0.100  356 7  < LOQ5 --
0.100  366 7  < LOQ5 --
0.100  417 7 0.0392 39.2
0.100  427 7 0.0375 37.5
0.100 47 10 0.0325 32.5
0.100 48 10 0.0308 30.8
0.100 53 14 0.0395 39.5
0.100 54 14 0.0331 33.1
0.100 59 17 0.0331 33.1
0.100 60 17 0.0304 30.4
0.100 65 21 0.0496 49.6
0.100 66-RE8 21 0.0699 69.9
0.100 71 24 0.0583 58.3
0.100 72 24 0.0596 59.6
0.100 77 28 0.0272 27.2
0.100 78 28 0.0255 25.5

1The method limit of quantitation (LOQ) for the analyses of Tridecyl (C-13)-LAS in freshwater is defined as 0.0100 mg/L, the lowest nominal concentration in a fortified sample at which a mean recovery of 70-110% has been obtained.

2Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly.

3Results were generated using Excel 2010. Manual calculations may differ slightly.

4At first checklist, syringe pump was found with run light flashing indicating no delivery. All stopcocks were open, and syringes were seated properly in the syringe. Samples were collected when the delivery malfunction was found, and flows were restored. Syringe pump was confirmed to be delivering at the end of the day.

5When equivalents were <LOQ, ½ LOQ (0.0050 mg/L) was used for calculations.

6Syringe pump was found with run light flashing once again indicating no delivery. Syringes were checked for alignment on pump. After failing to find a cause for syringe pump failure, the pump was replaced, and flows were restored. Analytical samples were taken at the time the malfunction was found.

7A second set of samples were taken at the end of the day to confirm test concentrations had returned to expected levels. 

8The initial result was extrapolated above the range of the calibration curve, and the reanalysis diluted within the range of the calibration curve is presented here.

 

Table 3: Measured concentrations of tridecyl (C13) - LAS in water samples from a bluegill bioconcentration test during the depuration phase

Nominal Test Concentration (mg/L) Sample Number (106A-140) Sampling Time (Day) Measured Concentration (mg/L)1,2 Percent of Nominal2
Negative control 79 1 <LOQ --
Negative control 80 1 <LOQ --
Negative control 85 4 <LOQ --
Negative control 86 4 <LOQ --
Negative control 91 7 <LOQ --
Negative control 92 7 <LOQ --
Negative control 97 10 <LOQ --
Negative control 98 10 <LOQ --
0.050 81 1 <LOQ --
0.050 82 1 <LOQ --
0.050 87 4 <LOQ --
0.050 88 4 <LOQ --
0.050 93 7 <LOQ --
0.050 94 7 <LOQ --
0.050 99 10 <LOQ --
0.050 100 10 <LOQ --
0.100 83 1 <LOQ --
0.100 84 1 <LOQ --
0.100 89 4 <LOQ --
0.100 90 4 <LOQ --
0.100 95 7 <LOQ --
0.100 96 7 <LOQ --
0.100 101 10 <LOQ --
0.100 102 10 <LOQ --

1The method limit of quantitation (LOQ) for the analyses of Tridecyl (C-13)-LAS in freshwater is defined as 0.0100 mg/L, the lowest nominal concentration in a fortified sample at which a mean recovery of 70-110% has been obtained.

2Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly

 

Table 4a: Mean measured concentrations of tridecyl (C13) - LAS in water samples by day during the uptake phases for negative control and  0.05 mg/L treatment group

Nominal Test Concentration (mg/L) Study Phase Sampling Interval (Day) Number of Samples Measured Concentration (mg/L)1 Percent of nominal1
0 Uptake 0 2 <LOQ --
0 Uptake 0 (Hour 4) 2 <LOQ --
0 Uptake 1 2 <LOQ --
0 Uptake 3 2 <LOQ --
0 Uptake 6 2 <LOQ --
0 Uptake 7 2 <LOQ --
0 Uptake 7 2 <LOQ --
0 Uptake 10 2 <LOQ --
0 Uptake 14 2 <LOQ --
0 Uptake 17 2 <LOQ --
0 Uptake 21 2 <LOQ --
0 Uptake 24 2 <LOQ --
0 Uptake 28 2 <LOQ --
0.050 Uptake 0 2 0.0131 26.2
0.050 Uptake 0 (Hour 4) 2 0.0126 25.1
0.050 Uptake 1 2 0.0095 19.1
0.050 Uptake 3 2 0.0096 19.1
0.050 Uptake 6 2 <LOQ --
0.050 Uptake 7 2 <LOQ --
0.050 Uptake 7 2 0.0159 31.7
0.050 Uptake 10 2 0.0137 27.4
0.050 Uptake 14 2 0.021 41.9
0.050 Uptake 17 2 0.0267 53.4
0.050 Uptake 21 2 0.0272 54.4
0.050 Uptake 24 2 0.0284 56.8
0.050 Uptake 28 2 0.024 47.9
Mean1 0.015 ± 0.0076 (CV 52.8%) 28.90%
Time‑weighted Mean1 0.019 ± 0.0027 38.40%

1Results generated using Excel 2010 in full precision mode. Manual calculations may differ slightly. Non-weighted and time-weighted means calculated using the average concentration of each sampling interval.

 

Table 4b: Mean measured concentrations of tridecyl (C13) - LAS in water samples by day during the uptake phases for 0.100 mg/L treatment group

Nominal Test Concentration (mg/L) Study Phase Sampling Interval (Day) Number of Samples Measured Concentration (mg/L)1 Percent of nominal1
0.100 Uptake 0 2 0.0226 22.6
0.100 Uptake 0 (Hour 4) 2 0.0239 23.9
0.100 Uptake 1 2 0.0233 23.3
0.100 Uptake 3 2 0.0223 22.3
0.100 Uptake 6 2 <LOQ --
0.100 Uptake 7 2 <LOQ --
0.100 Uptake 7 2 0.0384 38.4
0.100 Uptake 10 2 0.0317 31.7
0.100 Uptake 14 2 0.0363 36.3
0.100 Uptake 17 2 0.0318 31.8
0.100 Uptake 21 2 0.0598 59.8
0.100 Uptake 24 2 0.059 59
0.100 Uptake 28 2 0.0264 26.4
Mean1 0.027 ± 0.0154 (CV 56.4%) 27.30%
Time‑weighted Mean1 0.035 ± 0.0050 (CV = 14.1%) 35.20%

1Results generated using Excel 2010 in full precision mode. Manual calculations may differ slightly. Non-weighted and time-weighted means calculated using the average concentration of each sampling interval.

 

Table 4c: Mean measured concentrations of tridecyl (C13) - LAS in water samples by day during depuration phases

Nominal Test Concentration (mg/L) Study Phase Sampling Interval (Day) Number of Samples Measured Concentration (mg/L)1 Percent of nominal1
0 Dep 1 2 <LOQ --
0 Dep 4 2 <LOQ --
0 Dep 7 2 <LOQ --
0 Dep 10 2 <LOQ --
0.050 Dep 1 2 <LOQ --
0.050 Dep 4 2 <LOQ --
0.050 Dep 7 2 <LOQ --
0.050 Dep 10 2 <LOQ --
0.100 Dep 1 2 <LOQ --
0.100 Dep 4 2 <LOQ --
0.100 Dep 7 2 <LOQ --
0.100 Dep 10 2 <LOQ --

1Results generated using Excel 2010 in full precision mode. Manual calculations may differ slightly. Non-weighted and time-weighted means calculated using the average concentration of each sampling interval.

 

Table 5: Measured concentrations of tridecyl (C13) - LAS in whole fish tissues in the negative control group from a bluegill bioconcentration test during the uptake phase

Sample ID (106A-140- ) Uptake Day Whole Fish Concentration (mg/kg)1,2 Mean Measured Concentration (mg/kg)3
WF-1 0.17 <LOQ --
WF-2 0.17 <LOQ
WF-3 0.17 <LOQ
WF-4 0.17 <LOQ
WF-16 1 <LOQ --
WF-17 1 <LOQ
WF-18 1 <LOQ
WF-19 1 <LOQ
WF-31 3 <LOQ --
WF-32 3 <LOQ
WF-33 3 <LOQ
WF-34 3 <LOQ
WF-46 7 <LOQ --
WF-47 7 <LOQ
WF-48 7 <LOQ
WF-49 7 <LOQ
WF-61 10 <LOQ --
WF-62 10 <LOQ
WF-63 10 <LOQ
WF-64 10 <LOQ
WF-76 14 <LOQ --
WF-77 14 <LOQ
WF-78 14 <LOQ
WF-79 14 <LOQ
WF-91 17 <LOQ --
WF-92 17 <LOQ
WF-93 17 <LOQ
WF-94 17 <LOQ
WF-106 21 <LOQ --
WF-107 21 <LOQ
WF-108 21 <LOQ
WF-109 21 <LOQ
WF-121 24 <LOQ --
WF-122 24 <LOQ
WF-123 24 <LOQ
WF-124 24 <LOQ
WF-136 28 <LOQ --
WF-137 28 <LOQ
WF-138 28 <LOQ
WF-139 28 <LOQ

1The method limit of quantitation (LOQ) for the analyses of Tridecyl (C-13)-LAS in bluegill tissue is defined as 2.50 mg/kg, the lowest nominal concentration in a fortified sample at which a mean recovery of 70-110% has been obtained.

2Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly.

3Results were generated using Excel 2010. Manual calculations may differ slightly.

 

Table 6: Measured concentrations of tridecyl (C13) - LAS in whole fish tissues in the low exposure group (nominal 0.050 mg/L) from a bluegill bioconcentration test during the uptake phase

Sample ID (106A-140- ) Uptake Day Whole Fish Concentration (mg/kg)1,2 Mean Measured Concentration (mg/kg)3
WF-6 0.17 <LOQ <LOQ
WF-7 0.17 <LOQ
WF-8 0.17 <LOQ
WF-9 0.17 <LOQ
 WF-21-RE4 1  <LOQ5 <LOQ
 WF-22-RE4 1  <LOQ5
 WF-23-RE4 1 2.41
 WF-24-RE4 1  <LOQ5
WF-36 3 2.6 2.69
WF-37 3 3.08
WF-38 3 2.64
WF-39 3 2.43
 WF-51-RE6 7 3.57 3.83
 WF-52-RE6 7  <LOQ5
 WF-53-RE6 7 3.3
WF-54 7 7.18
WF-66-RE6 10 3.2 3.91
WF-67-RE6 10 2.66
WF-68-RE6 10 2.94
WF-69 10 6.82
WF-81 14 2.8 6.88
WF-82 14 6.57
WF-83 14 7.53
WF-84 14 10.6
WF-96 17 8.42 9.75
WF-97 17 11.5
WF-98 17 8.98
WF-99 17 10.1
WF-111 21 13.5 8.91
WF-112 21 6.85
WF-113 21 5.19
WF-114 21 10.1
 WF-126-RE4 24 5.09 6.36
WF-127 24 6.78
WF-128 24 6.14
WF-129 24 7.41
WF-141 28 10.8 9.04
WF-142 28 6.86
WF-143 28 10.6
WF-144 28 7.88

1The method limit of quantitation (LOQ) for the analyses of Tridecyl (C-13)-LAS in bluegill tissue is defined as 2.50 mg/kg, the lowest nominal concentration in a fortified sample at which a mean recovery of 70-110% has been obtained.

2Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly.

3Results were generated using Excel 2010. Manual calculations may differ slightly.

4Samples were reanalyzed at a reduced dilution factor as the original results were extrapolated below the range of the calibration curve. The results of the reanalysis are presented here.

5When equivalents were <LOQ, ½ LOQ (1.25 mg/kg) was used for calculations.

 

Table 7: Measured concentrations of tridecyl (C13) - LAS in whole fish tissues in the high exposure group (nominal 0.100 mg/L) from a bluegill bioconcentration test during the uptake phase

Sample ID (106A-140- ) Uptake Day Whole Fish Concentration (mg/kg)1,2 Mean Measured Concentration (mg/kg)3
WF-11 0.17 <LOQ <LOQ
WF-12 0.17 <LOQ
WF-13 0.17 <LOQ
WF-14 0.17 <LOQ
 WF-26-RE4 1 3.73 3.28
 WF-27-RE4 1 2.5
 WF-28-RE4 1 3.13
 WF-29-RE4 1 3.76
WF-41 3 5.55 5.34
 WF-42-RE5 3 4.27
WF-43 3 5.33
WF-44 3 6.19
 WF-56-RE5 7 8.53 8.81
 WF-57-RE5 7 9.02
 WF-58-RE5 7 8.14
 WF-59-RE5 7 9.55
WF-71 10 10.8 10.2
WF-72 10 12
WF-73 10 11
WF-74-RE5 10 6.9
WF-86 14 12.5 10.6
WF-87 14 9.25
WF-88 14 14.1
WF-89 14 6.37
WF-101 17 22.3 16.7
WF-102 17 15.5
WF-103 17 14
WF-104 17 15.1
WF-116 21 19.3 20.5
WF-117 21 21.4
WF-118 21 22.6
WF-119 21 18.5
 WF-131-RE4 24 7.33 9.63
 WF-132-RE4 24 10.1
WF-133 24 11.3
 WF-134-RE4 24 9.8
WF-146 28 11 17.3
WF-147-RE4 28 27.4
WF-148 28 20.6
WF-149 28 10.3

1The method limit of quantitation (LOQ for the analyses of Tridecyl (C-13)-LAS in bluegill tissue is defined as 2.50 mg/kg, the lowest nominal concentration in a fortified sample at which a mean recovery of 70-110% has been obtained.

2Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly.

3Results were generated using Excel 2010. Manual calculations may differ slightly.

4Samples were reanalyzed at a reduced dilution factor as the original results were extrapolated outside the range of the calibration curve. The results of the reanalysis are presented here.

5Original results of these samples were outside of the range of the calibration curve and had to be extrapolated. Samples were reanalyzed, and the results of the reanalysis are presented here.

 

Table 8: Measured concentrations of tridecyl (C13) - LAS in whole fish tissues in the negative control group from a bluegill bioconcentration test during the depuration phase

Sample ID (106A-140- ) Depuration Day Whole Fish Concentration (mg/kg)1,2 Mean Measured Concentration (mg/kg)3
WF-151 1 <LOQ --
WF-152 1 <LOQ
WF-153 1 <LOQ
WF-154 1 <LOQ
WF-163 4 <LOQ --
WF-164 4 <LOQ
WF-165 4 <LOQ
WF-166 4 <LOQ
WF-175 7 <LOQ --
WF-176 7 <LOQ
WF-177 7 <LOQ
WF-178 7 <LOQ
WF-187 10 <LOQ --
WF-188 10 <LOQ
WF-189 10 <LOQ
WF-190 10 <LOQ

1The method limit of quantitation (LOQ for the analyses of Tridecyl (C-13)-LAS in bluegill tissue is defined as 2.50 mg/kg, the lowest nominal concentration in a fortified sample at which a mean recovery of 70-110% has been obtained.

2Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly.

3Results were generated using Excel 2010. Manual calculations may differ slightly.

 

Table 9: Measured concentrations of tridecyl (C13) - LAS in whole fish tissues in the low exposure group (nominal 0.050 mg/L) from a bluegill bioconcentration test during the depuration phase

Sample ID (106A-140- ) Depuration Day Whole Fish Concentration (mg/kg)1,2 Mean Measured Concentration (mg/kg)3
WF-155 1 6.63 7.56
WF-156 1 6.41
WF-157 1 11.3
WF-158 1 5.91
WF-167 4  1.844 2.54
WF-168 4  1.844
WF-169 4 2.91
WF-170 4 3.57
WF-179 7 <LOQ <LOQ
WF-180 7 <LOQ
WF-181 7 <LOQ
WF-182 7 <LOQ
WF-191 10 <LOQ <LOQ
WF-192 10 <LOQ
WF-193 10 <LOQ
WF-194 10 <LOQ

1The method limit of quantitation (LOQ for the analyses of Tridecyl (C-13)-LAS in bluegill tissue is defined as 2.50 mg/kg, the lowest nominal concentration in a fortified sample at which a mean recovery of 70-110% has been obtained.

2Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly.

3Results were generated using Excel 2010. Manual calculations may differ slightly.

4Below LOQ but within the acceptable recovery of the LOQ.

 

Table 10: Measured concentrations of tridecyl (C13) - LAS in whole fish tissues in the high exposure group (nominal 0.100 mg/L) from a bluegill bioconcentration test during the depuration phase

Sample ID (106A-140- ) Depuration Day Whole Fish Concentration (mg/kg)1,2 Mean Measured Concentration (mg/kg)3
WF-159 1 10.1 9.44
WF-160 1 7.09
WF-161 1 9.46
WF-162 1 11.1
WF-171 4  < LOQ4 <LOQ
WF-172 4  1.835
WF-173 4 3.58
WF-174 4 2.67
WF-183 7  < LOQ4 <LOQ
WF-184 7 2.58
WF-185 7 2.51
WF-186 7  < LOQ4
WF-195 10 <LOQ <LOQ
WF-196 10 <LOQ
WF-197 10 <LOQ
WF-198 10

<LOQ

1The method limit of quantitation (LOQ for the analyses of Tridecyl (C-13)-LAS in bluegill tissue is defined as 2.50 mg/kg, the lowest nominal concentration in a fortified sample at which a mean recovery of 70-110% has been obtained.

2Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly.

3Results were generated using Excel 2010. Manual calculations may differ slightly.

4When equivalents were <LOQ, ½ LOQ (1.25 mg/kg) was used for calculations.

5Below LOQ but within the acceptable recovery of the LOQ.

 

Table 11: Mean measured concentrations of tridecyl (C13) - LAS in whole fish tissues in the low and high exposure groups by day during the uptake and depuration phases

Nominal Concentration Tridecyl (C13) - LAS (mg/L) Study Phase Sampling Interval (Day) Mean Whole Fish Conc.1,2(mg/kg)
0.050 Uptake 0.17 < LOQ
0.050 Uptake 1 < LOQ
0.050 Uptake 3 2.69
0.050 Uptake 7 3.83
0.050 Uptake 10 4.52
0.050 Uptake 14 6.88
0.050 Uptake 17 9.75
0.050 Uptake 21 8.91
0.050 Uptake 24 6.36
0.050 Uptake 28 9.04
0.050 Depuration 1 7.56
0.050 Depuration 4 2.54
0.050 Depuration 7 < LOQ
0.050 Depuration 10 < LOQ
0.100 Uptake 0.17 < LOQ
0.100 Uptake 1 3.28
0.100 Uptake 3 5.34
0.100 Uptake 7 8.81
0.100 Uptake 10 10.7
0.100 Uptake 14 10.6
0.100 Uptake 17 16.7
0.100 Uptake 21 20.5
0.100 Uptake 24 9.63
0.100 Uptake 28 17.3
0.100 Depuration 1 9.44
0.100 Depuration 4 < LOQ
0.100 Depuration 7 < LOQ
0.100 Depuration 10 < LOQ

1The method limit of quantitation (LOQ for the analyses of Tridecyl (C-13)-LAS in bluegill tissue is defined as 2.50 mg/kg, the lowest nominal concentration in a fortified sample at which a mean recovery of 70-110% has been obtained.

2Results were generated using Excel 2010. Manual calculations may differ slightly.

 

Table 12a: Confirmation of Steady State (Day 21, 24 and 28)

Tissue Type Nominal Water Concentration (mg/L) Mean Measured Tissue Concentration (mg/kg) For Last Three Sampling Intervals1, 2 Mean SD p value3
Day 21 Day 24 Day 28 
Whole Fish 0.050 8.91 6.36 9.04 8.10 1.51 0.2321
Whole Fish 0.100 20.5 9.63 17.3 15.80 5.57 0.2791

1Mean measured tissue concnetration at each interval were not within ± 20% of the mean tissue concentration.

2Although measurements from Days 21, 24 and 28 were not within ± 20% of the mean tissue concentration, additional analysis of previous interval confirms steady-state was achieved by Day 28 of uptake.

3p = 0.05

 

Table 12b: Confirmation of Steady State (Day 17, 21 and 28)

Tissue Type Nominal Water Concentration (mg/L) Mean Measured Tissue Concentration (mg/kg) For Last Three Sampling Intervals1, 2 Mean SD p value3
Day 17 Day 21 Day 28 
Whole Fish 0.050 9.75 8.91 9.04 9.23 0.45 0.8259

Whole Fish

0.100 16.7 20.5 17.3 18.20 2.04 0.7697

1Mean measured tissue concnetration at each interval were not within ± 20% of the mean tissue concentration.

2Additional analysis was conducted using the intervals of Day 17, 21 and 28 to confirm steady state was achieved.

3p = 0.05

 

Table 13: Steady-State BCF Values for Bluegill

Tissue Type Time - Weighted Mean Measured Test Concentration (mg/L) Uptake Days at Steady State Mean Measured Steady-State Tissue Concentration (mg/kg) Steady-State BCF1 BCFSS95% Confidence Interval2
Whole Fish 0.019 21-24-28  9.04 476 285 – 705
Whole Fish 0.035 21-24-28  17.3 495 113 – 951

1Steady-State BCF = (mean tissue concentration at last interval of steady-state)/(time-weighted mean measured water concentration). Although apparent steady-state is confirmed over three intervals, only the tissue concentrations for the last steady-state interval are used for the BCFSS calculation.

2Calulated using Excel 2010.

 

Table 14: Kinetic Estimates of the BCFK for Bluegill

Tissue Type Nominal Exposure (mg/L) Uptake Rate Constant (k1, L/kg/d)1 Depuration Rate Constant (k2, Day-1)1 Kinetic Bioconcentration Factor (BCFK)2 BCFK95% Confidence Interval3 Estimated Time to Reach 95% Steady State (Days)2 Estimated Time to Reach 50% Clearance (Days)2
Whole Fish 0.050 84.4 0.2151 393 324 – 475 14 3.4
Whole Fish 0.100 99.8 0.2502 399 301 – 528 12 2.8

1Calculated using SAS.

2Calculated in Excel 2010 using methods outlined in OPPTS guideline 850.1730.

3Calculated using Excel 2010 according to methods described by Bailer et al. 2000. Estimated and Testing Bioconcentration Factors. Environmental Toxicology and Chemistry, Vol. 19 No. 9, pg. 2338-2340.

 

Table 15: Growth Rate Constants

 Growth rate constant kgvalues
Control1 0.050 mg/L2,3,4 0.100 mg/L2,3,4
Uptake 0.01906 0.01274 0.01507
Depuration -0.01456 -0.01577 0.0082
Pooled -- 0.01165 0.01261

1Statistical difference between slopes of the uptake and depuration phases (p< 0.05).

2No statistical difference between slopes of uptake and depuration phases (p>0.05).

3Growth measurements from both the uptake and depuration phases of the treatment group were pooled for the overall growth rate constant. 

4No statistical difference between the uptake and depuration phases in the control and treatment groups (p>0.05). Therefore, the control group was used for lipid analysis and lipid correction.

 

Table 16: Growth Corrected Values in Whole Fish Tissues

Parameters 0.019 mg/L 0.035 mg/L
k2 0.2151 0.2502
kg 0.01165 0.01261
k2g 0.2035 0.2376
BCFKg 415 420
BCFKL 283 287
BCFKgL 298 302
t1/2g 3.4 2.9
BCFSSL 342 356

k2: depuration rate constant.

kg: growth rate constant.

k2g: growth corrected depuration rate constant.

t1/2g: growth corrected half- life (days).

BCFSSL: lipid corrected steady state BCF value

BCFKg: growth corrected kinetic BCF value.

BCFKL: lipid corrected kinetic BCF value.

BCFKgL: growth and lipid corrected kinetic BCF value.

 

Table 17: Whole Fish Lipid Determination

Sample Number (106A-140-) Treatment Group Sampling Interval (Day) Tissue Weight (grams) Lipid Weight (grams)  Percent Lipids1(%) Mean Percent Lipids1(%)
L1 Negative Control Day 0, Hour 0 Uptake 2.6678 0.0958 3.59 5.31
L2 Negative Control Day 0, Hour 0 Uptake 2.7294 0.1766 6.47
L3 Negative Control Day 0, Hour 0 Uptake 3.9529 0.2326 5.88
L10 Negative Control Day 28 Uptake 3.0389 0.2231 7.34 6.95
 Ln = 0.72
L11 Negative Control Day 28 Uptake 3.2567 0.2131 6.54
L12 Negative Control Day 28 Uptake 3.8569 0.2691 6.98
L19 Negative Control Day 11 Depuration 2.2648 0.1436 6.34 6.28
L20 Negative Control Day 11 Depuration 3.624 0.2458 6.78
L21 Negative Control Day 11 Depuration 5.0063 0.2866 5.72

1Results were generated using Excel 2010. Manual calculations may differ slightly.

2Ln = lipid normalization factor based on lipid content of the fish at the end of uptake normalized to 5%.

 

Table 18: Means and Ranges of Water Quality Parameters

Mean Measured Test Concentration (mg/L) Temperature1(°C) DO2(mg/L) pH Conductivity (µS/cm) Alkalinity (mg/L as CaCO3) Hardness (mg/L as CaCO3) TOC Uptake3(mg C/L) TOC Depuration3(mg C/L)
Negative Control 22.1 (21.9 – 22.3) 8.1 (6.8 – 8.8) 8.1 (7.9 – 8.2) 345 (310 – 358) 183 (180 – 188) 139 (136 – 144) 1.27 (<LOQ – 2.77) 1.79 (<LOQ – 3.40)
0.019 22.1 (21.9 – 22.3) 7.8 (6.8 – 8.7) 8.2 (8.0 – 8.3) -- -- -- 1.35 (<LOQ – 2.52) 1.57 (<LOQ – 2.73)
0.035 22.1 (21.9 – 22.3) 7.7 (6.7 – 8.7) 8.2 (7.9 – 8.3) 349 (310 – 364) 182 (178 – 188) 142 (140 – 148) 1.35 (<LOQ – 2.73) 1.19 (<LOQ – 1.62)

1Continuous measurements of temperature in the Solvent Control test chamber ranged from 21.90 to 22.23°C measured continuously with PointView Central Monitoring System to the nearest 0.01°C

2A dissolved oxygen concentration of 5.2 mg/L is 60% saturation at 22°C in freshwater.

3All extrapolated values below the lowest calibration standard are reported as < 1 mg C/L.

 

Table 19: Cumulative mortality and treatment-related effects in negative control during uptake phase

Day Observations1 Cumulative Number Dead Number Remaining Number Sampled
0 AN 0 75 5
1 AN 0 70 5
2 AN 0 70 0
3 AN 0 65 5
4 AN 0 65 0
5 AN 0 65 0
6 AN 0 65 0
7 AN 0 60 5
8 AN 0 60 0
9 AN 0 60 0
10 AN 0 55 5
11 AN 0 55 0
12 AN 0 55 0
13 AN 0 55 0
14 AN 0 50 5
15 AN 0 50 0
16 AN 0 50 0
17 AN 0 45 5
18 AN 0 45 0
19 AN 0 45 0
20 AN 0 45 0
21 AN 0 40 5
22 AN 0 40 0
23 AN 0 40 0
24 AN 0 35 5
25 AN 0 35 0
26 AN 0 35 0
27 AN 0 35 0
282 AN 0 27 8

1Observed Effects:  AN = Appears Normal.

2All fish were transferred to a clean diluter containing only clean dilution water. Fish were enumerated during the transfer.

 

Table 20: Cumulative mortality and treatment-related effects in negative control during depuration phase

Day Observations1 Cumulative Number Dead Number Remaining Number Sampled
0 AN 0 27 0
1 AN 0 23 4
2 AN 0 23 0
3 AN 0 23 0
4 AN 0 19 4
5 AN 0 19 0
6 AN 0 19 0
7 AN 0 15 4
8 AN 0 15 0
9 AN 0 15 0
10 AN 0 11 4
11 AN 0 82 3

1Observed Effects:  AN = Appears Normal.

2Fish remaining after lipid sampling were euthanized and stored frozen for possible future analysis.

 

Table 21: Cumulative mortality and treatment-related effects in 0.050 mg/L treatment group during uptake phase

Day Observations1 Cumulative Number Dead Number Remaining Number Sampled
0 AN 0 75 5
1 AN 0 70 5
2 AN 0 70 0
3 AN 0 65 5
4 AN 0 65 0
5 AN 0 65 0
6 AN 0 65 0
7 AN 0 60 5
8 AN 0 60 0
9 AN 0 60 0
10 AN 0 55 5
11 AN 0 55 0
12 AN 0 55 0
13 AN 0 55 0
14 AN 0 50 5
15 AN 0 50 0
16 AN 0 50 0
17 AN 0 45 5
18 AN 0 45 0
19 AN 0 45 0
20 AN 0 45 0
21 AN 0 40 5
22 1 X; Rest AN 1 39 0
23 AN 0 39 0
24 AN 0 34 5
25 AN 0 34 0
26 AN 0 34 0
27 AN 0 34 0
282 AN 0 26

8

1Observed Effects:  AN = Appears Normal; X = Dead.

2All fish were transferred to a clean diluter containing only clean dilution water. Fish were counted during the transfer.

 

Table 22: Cumulative mortality and treatment-related effects in 0.050 mg/L treatment group during depuration phase

Day Observations1 Cumulative Number Dead Number Remaining Number Sampled
0 AN 0 26 0
1 AN 0 22 4
2 AN 0 22 0
3 AN 0 22 0
4 AN 0 18 4
5 AN 0 18 0
6 AN 0 18 0
7 AN 0 14 4
8 AN 0 14 0
9 AN 0 14 0
10 AN 0 10 4
11 AN 0 72 3

1Observed Effects:  AN = Appears Normal.

2Fish remaining after lipid sampling were euthanized and stored frozen for possible future analysis

 

Table 23: Cumulative mortality and treatment-related effects in 0.100 mg/L treatment group during uptake phase

Day Observations1 Cumulative Number Dead Number Remaining Number Sampled
0 AN 0 75 5
1 AN 0 70 5
2 AN 0 70 0
3 AN 0 65 5
4 AN 0 65 0
5 AN 0 65 0
6 AN 0 65 0
7 AN 0 60 5
8 AN 0 60 0
9 AN 0 60 0
10 AN 0 55 5
11 AN 0 55 0
12 AN 0 55 0
13 AN 0 55 0
14 AN 0 50 5
15 AN 0 50 0
16 AN 0 50 0
17 AN 0 45 5
18 AN 0 45 0
19 AN 0 45 0
20 AN 0 45 0
21 AN 0 40 5
22 AN 0 40 0
23 AN 0 40 0
24 AN 0 35 5
25 AN 0 35 0
26 AN 0 35 0
27 AN 0 35 0
282 AN 0 27 8

1Observed Effects:  AN = Appears Normal.

2All fish were transferred to a clean diluter containing only clean dilution water. Fish were enumerated during the transfer.

 

Table 24: Cumulative mortality and treatment-related effects in 0.100 mg/L treatment group during depuration phase

Day Observations1 Cumulative Number Dead Number Remaining Number Sampled
0 AN 0 27 0
1 AN 0 23 4
2 AN 0 23 0
3 AN 0 23 0
4 AN 0 19 4
5 AN 0 19 0
6 AN 0 19 0
7 AN 0 15 4
8 AN 0 15 0
9 AN 0 15 0
10 AN 0 11 4
11 AN 0 82 3

1Observed Effects:  AN = Appears Normal.

2Fish remaining after lipid sampling were euthanized and stored frozen for possible future analysis.

 

Table 25: Growth measurements of Bluegill during uptake phase

Sample Number Depuration Interval (Day) Nominal Tridecyl (C13) - LAS Concentration (mg/L) Total Length1(mm) Wet Weight2(g)
1 0.17 0 56 2.4659
2 0.17 0 59 2.6783
3 0.17 0 52 1.8806
4 0.17 0 51 1.7697
5 0.17 0 54 2.1752
6 0.17 0.050 52 1.989
7 0.17 0.050 59 2.8051
8 0.17 0.050 64 3.0502
9 0.17 0.050 54 2.0831
10 0.17 0.050 52 1.7611
11 0.17 0.100 62 3.2439
12 0.17 0.100 57 1.9675
13 0.17 0.100 56 2.244
14 0.17 0.100 57 2.2523
15 0.17 0.100 58 2.5287
16 1 0 59 2.9191
17 1 0 52 1.5262
18 1 0 58 2.4141
19 1 0 52 1.9469
20 1 0 51 1.5979
21 1 0.050 63 3.4624
22 1 0.050 61 2.9245
23 1 0.050 57 2.3271
24 1 0.050 59 2.9532
25 1 0.050 57 2.5974
26 1 0.100 60 2.8748
27 1 0.100 59 2.6513
28 1 0.100 52 1.8667
29 1 0.100 49 1.4487
30 1 0.100 55 2.2917
31 3 0 63 3.1318
32 3 0 63 2.9272
33 3 0 55 2.0096
34 3 0 62 3.1774
35 3 0 54 1.9622
36 3 0.050 55 2.0088
37 3 0.050 59 2.4732
38 3 0.050 52 1.7067
39 3 0.050 59 2.4087
40 3 0.050 54 2.1408
41 3 0.100 53 1.9449
42 3 0.100 60 2.9033
43 3 0.100 52 1.7915
44 3 0.100 57 2.3605
45 3 0.100 50 1.5912
46 7 0 62 2.8109
47 7 0 57 2.2499
48 7 0 63 3.1317
49 7 0 58 2.6162
50 7 0 62 3.2993
51 7 0.050 54 2.0809
52 7 0.050 65 3.3642
53 7 0.050 71 4.7045
54 7 0.050 59 2.3966
55 7 0.050 55 2.0977
56 7 0.100 53 1.859
57 7 0.100 65 3.3044
58 7 0.100 62 3.2859
59 7 0.100 53 1.8035
60 7 0.100 56 2.3039
61 10 0 56 2.5331
62 10 0 60 3.1385
63 10 0 55 2.155
64 10 0 63 3.1257
65 10 0 60 2.5374
66 10 0.050 58 2.6133
67 10 0.050 59 2.9288
68 10 0.050 60 2.6565
69 10 0.050 54 1.8105
70 10 0.050 51 1.6792
71 10 0.100 67 4.0929
72 10 0.100 55 2.2207
73 10 0.100 61 2.7928
74 10 0.100 58 2.5559
75 10 0.100 53 2.1306
76 14 0 63 3.1139
77 14 0 57 2.3793
78 14 0 61 3.1525
79 14 0 61 3.0455
80 14 0 60 2.7412
81 14 0.050 67 4.5463
82 14 0.050 55 2.1202
83 14 0.050 52 1.9083
84 14 0.050 51 1.6216
85 14 0.050 56 2.3265
86 14 0.100 62 3.4888
87 14 0.100 63 3.8913
88 14 0.100 65 3.9072
89 14 0.100 64 3.4083
90 14 0.100 57 2.3509
91 17 0 62 3.2584
92 17 0 63 3.3154
93 17 0 63 3.43
94 17 0 50 1.5646
95 17 0 69 4.6031
96 17 0.050 55 2.36
97 17 0.050 67 3.8312
98 17 0.050 57 2.4527
99 17 0.050 60 2.6174
100 17 0.050 71 4.6604
101 17 0.100 68 4.045
102 17 0.100 56 2.1309
103 17 0.100 66 3.7218
104 17 0.100 55 2.1051
105 17 0.100 63 3.2787
106 21 0 56 2.3797
107 21 0 55 2.3044
108 21 0 68 4.3035
109 21 0 70 4.567
110 21 0 62 2.77
111 21 0.050 66 3.7245
112 21 0.050 64 3.4572
113 21 0.050 64 3.263
114 21 0.050 62 3.1923
115 21 0.050 64 3.7344
116 21 0.100 62 3.1797
117 21 0.100 61 2.9517
118 21 0.100 55 2.1711
119 21 0.100 57 2.4807
120 21 0.100 61 2.9259
121 24 0 59 2.8646
122 24 0 69 5.0251
123 24 0 62 3.5325
124 24 0 63 3.4407
125 24 0 59 3.0178
126 24 0.050 60 2.8143
127 24 0.050 66 3.6445
128 24 0.050 58 3.0136
129 24 0.050 65 3.9138
130 24 0.050 58 2.7746
131 24 0.100 60 3.1378
132 24 0.100 57 2.6665
133 24 0.100 53 2.3391
134 24 0.100 70 4.3935
135 24 0.100 61 3.5019
136 28 0 70 4.0735
137 28 0 77 5.9312
138 28 0 61 2.9173
139 28 0 63 3.149
140 28 0 68 3.907
141 28 0.050 63 3.4989
142 28 0.050 63 3.1534
143 28 0.050 61 3.0775
144 28 0.050 60 2.6799
145 28 0.050 67 3.8144
146 28 0.100 65 3.4944
147 28 0.100 68 3.7235
148 28 0.100 65 3.6637
149 28 0.100 61 3.1919
150 28 0.100 62 3.1381
151 1 0 58 2.8887
152 1 0 66 4.0265
153 1 0 63 3.5907
154 1 0 64 3.6164
155 1 0.050 69 4.7511
156 1 0.050 69 5.1185
157 1 0.050 62 2.976
158 1 0.050 64 3.9663
159 1 0.100 57 2.5662
160 1 0.100 56 2.4717
161 1 0.100 64 4.1565
162 1 0.100 62 3.2494
163 4 0 69 4.0811
164 4 0 72 4.6562
165 4 0 62 2.973
166 4 0 67 3.6445
167 4 0.050 63 2.8565
168 4 0.050 61 2.7402
169 4 0.050 62 2.9037
170 4 0.050 62 2.8996
171 4 0.100 74 5.3306
172 4 0.100 72 5.2499
173 4 0.100 60 2.5289
174 4 0.100 60 2.5515
175 7 0 67 4.0262
176 7 0 66 4.0622
177 7 0 73 4.7703
178 7 0 57 2.3293
179 7 0.050 63 3.2007
180 7 0.050 78 6.5547
181 7 0.050 69 4.1051
182 7 0.050 62 2.8649
183 7 0.100 69 4.2511
184 7 0.100 62 2.9106
185 7 0.100

67

3.7383
186 7 0.100 63 3.0928

1Total length measured from the tip of the snout to the end of the tail with a metric ruler.

2Wet weight measured after rinsing each fish with dilution water (well water) and blotting dry.

 

Table 26: Growth measurements of bluegill during depuration phase

Sample Number Depuration Interval (Day) Nominal Tridecyl (C13) - LAS Concentration (mg/L) Total Length1(mm) Wet Weight2(g)
151 1 0 58 2.8887
152 1 0 66 4.0265
153 1 0 63 3.5907
154 1 0 64 3.6164
155 1 0.050 69 4.7511
156 1 0.050 69 5.1185
157 1 0.050 62 2.976
158 1 0.050 64 3.9663
159 1 0.100 57 2.5662
160 1 0.100 56 2.4717
161 1 0.100 64 4.1565
162 1 0.100 62 3.2494
163 4 0 69 4.0811
164 4 0 72 4.6562
165 4 0 62 2.973
166 4 0 67 3.6445
167 4 0.050 63 2.8565
168 4 0.050 61 2.7402
169 4 0.050 62 2.9037
170 4 0.050 62 2.8996
171 4 0.100 74 5.3306
172 4 0.100 72 5.2499
173 4 0.100 60 2.5289
174 4 0.100 60 2.5515
175 7 0 67 4.0262
176 7 0 66 4.0622
177 7 0 73 4.7703
178 7 0 57 2.3293
179 7 0.050 63 3.2007
180 7 0.050 78 6.5547
181 7 0.050 69 4.1051
182 7 0.050 62 2.8649
183 7 0.100 69 4.2511
184 7 0.100 62 2.9106
185 7 0.100 67 3.7383
186 7 0.100 63 3.0928
187 10 0 74 3.5956
188 10 0 60 2.7954
189 10 0 61 3.1548
190 10 0 61 2.7738
191 10 0.050 67 4.4358
192 10 0.050 58 2.2567
193 10 0.050 56 2.2199
194 10 0.050 69 4.4267
195 10 0.100 60 3.0998
196 10 0.100 61 2.9425
197 10 0.100 60 3.1964
198 10 0.100 65 4.4083

1Total length measured from the tip of the snout to the end of the tail with a metric ruler.

2Wet weight measured after rinsing each fish with dilution water (well water) and blotting dry.

 

Table 28: Statistical evaluation of slopes for growth data

  Negative Control 0.019 mg/L 0.035 mg/L
p value2,3,4(Uptake and Depuration Comparison1) 0.0338 0.2343 0.7306
p value2,5(Uptake Comparison) -- 0.2239 0.4253
p value2,5(Depuration Comparison) -- 0.9656 0.3647

1Slopes (kg) of growth data of the treatment groups were compared to the control using the student t-test.

2p = 0.05

3Significant difference between the slopes of the uptake and depuration phase growth measurements in the negative control (p < 0.05).

4No significant difference between the slopes of the uptake and depuration phase growth measurements in the treatment groups (p > 0.05).

5No significant difference between the slopes of the treatment groups in comparison to the negative control.

Validity criteria fulfilled:
yes
Conclusions:
A BCF study using the bluegill sunfish (Lepomis macrochirus) was conducted with a pure C13 LAS 2,3-phenyl compound to determine the bioconcentration behaviour in a flow through experiment. Two exposure levels were used 0.019 and 0.035 mg/L (time-weighted mean measured concentrations). The uptake (exposure) phase was 28 d in duration after which the fish were transferred to clean water for a depuration phase lasting 11 d. The test was performed GLP using OECD Guideline 305-I (2012), Aqueous Exposure Bioconcentration Fish Test. The study was performed using a moderately hard, well water characterized with respect to temperature, pH, water hardness, dissolved oxygen, conductivity, and Total organic carbon (TOC). All validity criteria identified in the Guideline were met. Steady state and kinetic determinations of the BCF were estimated and were given as uncorrected values, lipid normalized, and growth corrected values. Analytical measurements of water and tissue samples was performed by HPLC with tandem mass spectrometric detection (LC/MS/MS) including the use of a internal pure deuterated dodecyl (C12 LAS) standard. BCFs were expressed as steady state (SS) and kinetic (K) values with lipid normalization and growth corrections. Uncorrected BCFss were 476 and 495 L/kg for 0.019 and 0.035 mg/L exposures, respectively and BCFk were 393 and 399 L/kg for 0.019 and 0.035 mg/L. Lipid normalized, growth-corrected BCFk values were 298 and 302 L/kg for 0.019 and 0.035 mg/L, respectively. Mean % lipid of bluegill ranged from 5.31-6.95% over the duration of the study. Estimated time to reach 95% of steady state was 12-14 d and to reach 50% clearance was 2.8-3.2 d. The recommended final bioconcentration value based on kinetic modelling, lipid and growth correction is 302 L/kg.
Executive summary:

This study was conducted for determining the bioconcentration potential of tridecyl (C13) - LAS in Lepomis macrochirusbluegill, through aqueous exposure.

 

Experimental setup consisted of a negative control group, low dose treatment group (0.05 mg/L) and high dose treatment group (0.10 mg/L). Measured concentrations for the treatment groups expressed as time-weighted mean measured water concentrations were 0.019 and 0.035 mg/L, respectively. Each group consisted of one test chamber with 80 fish in the negative control and each treatment level test chamber. Fish were exposed to the two test concentrations during a 28 day uptake phase followed by an 11 day depuration phase following removal to clean dilution water. During both phases of the experiment, test organisms and water samples were collected and analyzed for tridecyl (C13) – LAS using high performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) including the use of a internal pure deuterated dodecyl (C12 LAS) standard. These values were used to determine substancespecific half-lives, kinetic and steady state bioconcentration factors for the test substance in fish, as well as lipid-normalized and growthcorrected kinetic and steady state bioconcentration factors.

 

Steady-state BCF values (uncorrected) for the low and high treatment groups were 476 and 495 L/kg in whole fish tissue, respectively. The lipid-normalized whole fish BCFss (steady state) value in the low and high treatment groups were 342 and 356 L/kg, respectively. BCFK (kinetic) values derived by nonlinear regression for the low and high treatment groups were 393 and 399 in whole fish tissue, respectively. The time to reach 95% steady state based on kinetics was 14 and 12 days and time to reach 50% clearance was 3.4 and 2.8 days for whole fish, respectively. The lipid normalized growth corrected kinetic BCF in whole fish tissue was 298 in the low treatment group and 302 in the high treatment group. The growth corrected half-life was 3.4 and 2.9 days in the low and high treatment groups, respectively.

The recommended value for subsequent characterization of the bioconcentration behavior of C13 LAS is the lipid normalized, growth corrected BCF value measured in the 0.035 mg/L exposure of 302 L/kg.

This BCF study is classified as acceptable and satishfies the OECD Guideline 350-I.

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:
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:
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).

 

A recent log Kow measurement for C13 LAS provided a value of 3.67, therefore, a new study on the bioaccumulation of C13 LAS was commissioned to confirm estimates from previous studies.A pure C13 LAS was exposed to bluegill sunfish (Lepomismacrochirus) for 28 d at time-weighted mean measured concentrations of 0.019 and 0.035 mg/L followed by an 11 d depuration period in clean water (Schneider et al., 2020). BCFs were expressed as steady state (SS) and kinetic (K) values with lipid normalization and growth corrections. Uncorrected BCFss were 476 and 495 L/kg for 0.019 and 0.035 mg/L exposures, respectively and BCFk were 392 and 399 L/kg for 0.019 and 0.035 mg/L. Lipid normalized, growth-corrected BCFk values were 298 and 302 L/kg for 0.019 and 0.035 mg/L, respectively. Mean % lipid of bluegill ranged from 5.31-6.95% over the duration of the study. The BCF values were consistent with previously determined values for C13 LAS from Tolls et al. (1997, 1998, 2000).


Study 1 (C13 LAS):

 

A BCF study using the bluegill sunfish (Lepomis macrochirus) was conducted with a pure C13 LAS 2,3-phenyl compound to determine the bioconcentration behaviour in a flow through experiment. Two exposure levels were used 0.019 and 0.035 mg/L (time-weighted mean measured concentrations). The uptake (exposure) phase was 28 d in duration after which the fish were transferred to clean water for a depuration phase lasting 11 d. The test was performed GLP using OECD Guideline 305-I (2012), Aqueous Exposure Bioconcentration Fish Test. The study was performed using a moderately hard, well water characterized with respect to temperature, pH, water hardness, dissolved oxygen, conductivity, and Total organic carbon (TOC). All validity criteria identified in the Guideline were met. Steady state and kinetic determinations of the BCF were estimated and were given as uncorrected values, lipid normalized, and growth corrected values. Analytical measurements of water and tissue samples was performed by HPLC with tandem mass spectrometric detection (LC/MS/MS) including the use of a deuterated internal pure deuterated dodecyl (C12 LAS) standard. BCFs were expressed as steady state (SS) and kinetic (K) values with lipid normalization and growth corrections. Uncorrected BCFss were 476 and 495 L/kg for 0.019 and 0.035 mg/L exposures, respectively and BCFk were 392 and 399 L/kg for 0.019 and 0.035 mg/L. Lipid normalized, growth-corrected BCFk values were 298 and 302 L/kg for 0.019 and 0.035 mg/L, respectively. Mean % lipid of bluegill ranged from 5.31-6.95% over the duration of the study. Estimated time to reach 95% of steady state was 12-14 d and to reach 50% clearance was 2.8-3.2 d. The recommended final bioconcentration value based on kinetic modelling and lipid and growth correction is 302 L/kg. (Schneider et al, 2020). This value is very similar tothe results of the Tolls studies, and supports the use of the Tolls data as a basis to calculate a weighted BCF for the UVCB mixture. 

   

Study 2 (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 3 (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 4 (C12-2, C11-5, C12-5 and C13-5 LAS mixture):  

 

 A BCF study withPimephales promelaswas 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 5 (C12-2, C11-5, C12-6 and C12-3 LAS mixture): 

 

 A BCF study withPimephales promelaswas 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 6 (C12-2 LAS): 

 

 A study was conducted to investigate the potential use of severalin vitrosystems 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 7 (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