Quaternary ammonium compounds, benzyl-C16-18-alkyldimethyl, chlorides

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

EPA OPP 165-4 (Laboratory Studies of Pesticide Accumulation in Fish)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Details on sampling:

- A stock solution of 2.4 mg/mL was formulated by mixing radiolabeled test substance with unlabeled test substance and diluting with water.
- Following dilution with unlabeled test substance, the percent radiolabeled test substance in the mixture was 0.337 or 0.00337 as a decimal. On a combined test substance basis (both labeled and unlabeled) the calculated specific activity was 227013 dpmug x 0.00337 = 765.03 dpm/ug

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

Test organism:
- Common name: Bluegill
- Strain: SLS Lot 88A9
- Source: Froma culture maintained at Springborn Life Sciences, Inc.
- Length at study initiation (lenght definition, mean, range and SD): 65 mm
- Weight at study initiation (mean and range, SD): 3.6g
- Health status: Normal and healthy
- Frequency: Daily

Acclimation:
- Acclimation period: 14d prior to test initiation
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Dry pelleted food, ad libitum, daily throughout the 14d period, except 24h before testing.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

ca. 35 d

Total depuration duration:

ca. 21 d

Hardness:

22-32 mg/L CaCO3

Test temperature:

16-21°C

pH:

6.7-7.6

Dissolved oxygen:

6.2 -10.1 mg/L (64-94% of saturation)

Details on test conditions:

Test system
- Test vessel: 76 x 40 x 30 cm aquaria
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: 75L
- Renewal rate of test solution (frequency/flow rate): 4-4.7 tank volume replacements/d; 2L water to each aquaria at an average rate of 375 times/d
- No. of organisms per vessel: 190
- Biomass loading rate: 0.91 g/L/day

Test medium / Water parameters:
- Source/preparation of dilution water: Dilution water used for this test was from the same source as the water which flowed into the fish holding tank and was characterised weekly.
- Alkalinity: 21-24 mg/L CaCO3
- Conductance: 100-130 umhos/cm

Other test conditions:
- Photoperiod: 16h light/ 8h dark

Sampling period:
- To monitor initial concentration of 14C residues in water: 5 mL of samples were collected on Days 4, 3, 2, and 1 before introduction of fish
- To monitor concentration of 14C residues in water during the test: 5 mL of samples were collected on Days 1, 3, 7, 8, 9, 10, 14, 21, 23, 28 and 35 of exposure
- To quantify the accumulation and elimination of 14C residues in the edible, non-edible tissue of fish: 5 fish were collected, eviscerated and filleted on Day 1, 3, 7, 10, 14, 21, 28 and 35 of exposure.
- To quantify background 14C residues of test substance: 5 control fish were also collected, eviscerated and filleted on Day 35 of exposure and Day 21 of depuration.
- To estimate the half-life of the accumulated 14C residue present in the fish during depuration period: Water and tissue samples were collected from the tankon Day 1, 3, 7, 10, 14, 16 and 21 of depuration. Five fish were collected at each interval for analysis.

Others:
- Steady state was determined by measuring 14C residue conc. for 3 consecutive sampling intervals.
- After 35d of exposure, 35 of remaining fish in the treatment aquarium were transferred to a clean aquarium into which untreated dilution water was introduced at a rate equal to flow rate during exposure.
- Triplicate edible fish tissue samples from Day 35 of exposure were prepared for a hexane and methanol extraction procedure.
- To estimate the amount of 14C residues bound to the external surface of the skin of fish: 10 fish were removed after 35 d of exposure, eviscerated, filleted and the muscle portions were scraped off the skin. The skin portions were subsequently combustible to determine the residue conc. of the test substance.

Nominal and measured concentrations:

Nominal concentration: 0.05 mg/L
Measured concentration: 0.076 mg/L

Details on estimation of bioconcentration:

BCF factors for edible, non-edible and whole body fish tissue were determined by dividing the mean measured equilibrium 14C tissue concentration for each tissue type by the mean measured water concentration for the entire exposure period. For comparison, an additonal method of calculating BCF factors wher the ratio of the uptake constant (Ku) to the depuration constant (Kd) was utilized. i.e.,
BCF = Ku/Kd

Key result

Type:

BCF

Value:

79 dimensionless

Basis:

whole body w.w.

Calculation basis:

other: mean

Remarks on result:

other: predicted BCF (calculated using uptake and depuration constants) = 110

Remarks:

conc. in environment / dose: 0.076 mg/L

Key result

Type:

BCF

Value:

160 dimensionless

Basis:

non-edible fraction

Calculation basis:

other: mean

Remarks on result:

other: predicted BCF = 190

Remarks:

conc. in environment / dose: 0.076 mg/L

Key result

Type:

BCF

Value:

33 dimensionless

Basis:

edible fraction

Calculation basis:

other: mean

Remarks on result:

other: predicted BCF = 50

Remarks:

conc. in environment / dose: 0.076 mg/L

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

21 d

Details on results:

- Mortality of test organisms: two treated fish (in a population of 190) died
- Behavioural abnormalities: None

Exposure phase:
- The mean 14C residues measured in the edible tissue reached steady state on Day 14 and remained relatively constant throughout the remainder of the exposure (mean range 2.10-3.36 mg/kg).
- Mean steady state conc. in edible tissue and mean conc. in the water during the period 0-35d: 2.54±0.67 mg/kg and 0.076±0.024 mg/L. The mean steady state BCF factor in the edible tissue during the 35d of exposure was determined to be 33X.
- The mean 14C residues measured in the non-edible tissue reached steady state on Day 14 and ranged from 11-13 mg/kg during the remainder of the 35-d exposure.
- Mean steady state in non-edible tissue and mean conc. in the water during the period 0-35 d: 12±2.3 mg/kg and 0.076±0.024 mg/L. The mean steady state BCF factor in the non-edible tissue during the 35d of exposure was determined to be 160X.
- The mean steady state conc. of 14C residue conc. in whole fish and mean conc. in the water during the period 0.35 d: 6±1.5 mg/kg and 0.076±0.024 mg/L.. The mean steady state BCF factor in the whole body of fish during the 35d of exposure was determined to be 79X.
- The residues in control were below the detectable level; small residues seen were due to contamination.
- Analytical results of the polar (methanol) solvent and non-polar (hexane) solvent extractions of edible tissue samples revealed 11% of the 14C residues accumulated in the exposure were extractable with methanol, 2.4% with hexane and 75% were not extractable with either solvent.

Depuration phase:
- Analyses of the tissues during the depuration phase indicated that in each case there was a continuous elimination of 14C residues from the respective tissues over the course of the 21d depuration period.
- Concentration of 14C residues present in the water of the depuration aquarium remained <= 0.014 mg/L, the limit of radiometric detection throughout the 21d depuration period.
- Half-life for non-edible tissue was: Between Days 14 and 21. By Day 21, the fish had eliminated 29%, 60% and 44% of the 14C residues that had been present on the last day of exposure on each of the tissues (i.e., edible, non-edible and whole body tissues respectively).
- Half-life based on model calculations (using 1st order kinetics) were: 16, 10 and 12d for edible, non-edible and whole body tissues respectively.
- The conc. of the residual test substance on the skin was observed to be higher than those found in the total edible tissue (skin and muscle combined) indicating significant binding of the test substance to skin and scales of fish.

Given the known strong affinity for the cationic compounds like the test substance to bind to essentially all surfaces, a logical explanation for the observed slow depuration is that the 14C residues are strongly bound to surfaces exposed to the treated water, e.g., gills, skin and intestine. The phenomenon has been clearly demonstrated with other quarternary ammonium compounds.

Validity criteria fulfilled:

not specified

Conclusions:

Under the study conditions, the whole body BCF of the read across substance was determined to be 79, indicating low potential to bioaccumulate.

Executive summary:

A study was conducted to determine the aquatic bioaccumulation of the read across substance, C12 -16 ADBAC (30.64% active; 98.9% radiolabeled purity) in Lepomis macrochirus (bluegill fish) under flow-through conditions, according to EPA OPP 165-4, in compliance with GLP. The blue gill fish were continuously exposed to a nominal concentration of 0.050 mg/L of the read across substance (equivalent to a measured concentration of 0.076 mg/L) in well water for 35 days, followed by transfer of 35 fish into flowing uncontaminated water for a 21-d depuration period. Sampling was carried out on Days 0, 1, 3, 7, 9, 10, 14, 21, 23, 28 and 35 for the exposure period and Days 1, 3, 7, 10, 14 and 21 for the depuration period. Water samples were collected on Day 8 of the exposure period and Day 16 of the depuration for analytic determination of the read across substance concentration. Radiometric analyses of the water and selected fish tissues revealed that the mean steady state bioconcentration factor (BCF) in the edible, non-edible and whole-body fish tissue during the 35 days of exposure to be 33, 160 and 79 L/kg. The half-life for non-edible tissue was attained between Days 14 and 21, while it could not be reached for the edible and whole-body fish tissues by the end of 21-d depuration period. By Day 21 of the depuration period, the 14C residues present on the last day of exposure in the edible, non-edible and whole-body fish tissues had been eliminated by 29, 60 and 44% respectively. Analysis of skin tissue after 35 d of exposure showed residue levels somewhat higher than those observed for edible tissue at the same sampling period, indicating that there is likely significant binding of 14C-ADBAC to the skins and scales of exposed bluegill, as expected behaviour of cationic surfactants. Under the conditions of the study, the whole body BCF of the read across substance was determined to be 79, indicating low potential to bioaccumulate (Fackler, 1989). Based on the results of the read across study, a similar low bioaccumulation potential is expected for the test substance.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

13 Jan 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

QSAR prediction from a well-known and acknowledged tool. See below under "attached background material section" for methodology and QPRF.

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals

Principles of method if other than guideline:

The Bioconcentration factor (BCF) value was predicted for the test substance using BCFBAF v3.02 program of EPI Suite v 4.11. Since the test substance is an UVCB containing ionic and non-ionic constituents, the BCF values were predicted using regression-based and Arnot-Gobas BAF-BCF models respectively and using SMILES codes as the input parameter.

Justification for method:

other:

Details on estimation of bioconcentration:

The Bioconcentration factor (BCF) value was calculated for the test substance using BCFBAF v3.02 program of EPI Suite v 4.11. Since the test substance is an UVCB containing ionic and non-ionic constituents, the BCF values were predicted using regression-based and Arnot-Gobas BAF-BCF models respectively and using SMILES codes as the input parameter.

Key result

Type:

BCF

Value:

ca. 1.55 - ca. 162.4 other: L/Kg ww

Basis:

whole body w.w.

Calculation basis:

other: regression-based method and Arnot-gobas based method for ionic and non-ionic respectively

Remarks on result:

other: low bioaccumulation potential

Key result

Type:

BCF

Value:

70.77 other: L/Kg ww

Basis:

whole body w.w.

Remarks:

(weighted average value)

Calculation basis:

other: regression-based method and Arnot-gobas based method for ionic and non-ionic respectively

Remarks on result:

other: low bioaccumulation potential

Results:

The predicted BCF values for the different constituents using the regression-based equation and Arnot-Gobas methods were as follows:

SMILES	Constituents/Carbon chain length	Mole fraction Xi = (mi/Mi)/∑ (mi/Mi)	BCF (L/kg wet-wt) BCFBAF (regression based)	BCF (L/kg wet-wt) BCFBAF (Arnot-Gobas methodology based)	BCF x Xi (L/kg wet-wt) BCFBAF	Domain evaluation
CCCCCCCC[N+](C)(C)CC1=CC=CC=C1	C8	1.44E-03	3.16	-	4.56E-03	MW, Log KOW (ID)
CCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1	C10	1.30E-03	3.16	-	4.10E-03	MW, Log KOW (ID)
CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1	C12	1.18E-03	70.8	-	8.35E-02	MW, Log KOW (ID)
CCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1	C14	4.32E-02	70.8	-	3.06E+00	MW, Log KOW (ID)
CCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1	C16	4.98E-01	70.8	-	3.53E+01	MW, Log KOW (ID)
CCCCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1	C18 and longer	3.93E-01	70.8	-	2.78E+01	MW, Log KOW (ID)
CCCCCCCC\C=C\CCCCCCCC[N+](C)(C)CC1=CC=CC=C1	C18'	1.86E-03	70.8	-	1.32E-01	MW, Log KOW (ID)
CCCCCCCCCCCCCCCCN(C)C	N,N-dimethylhexadecan-1-amine	2.67E-02	-	162.4	4.34E+00	MW, Log KOW, Corr. Fac (ID)
C1=CC=C(C=C1)CCl	Benzyl chloride	0.00E+00	15.3	-	0.00E+00	MW, Log KOW (ID)
C1=CC=C(C=C1)C=O	Benzaldehyde	1.70E-02	-	3.566	6.05E-02	MW, Log KOW, no Corr. Fac
C1=CC=C(C=C1)CO	Benzyl alcohol	1.67E-02	-	1.549	2.58E-02	MW, Log KOW, Corr. Fac (ID)
		1.00E+00	BCF=		70.77
			Log BCF		1.85

Details on results
C8 ADBAC	SMILES : CCCCCCCCN(H)(C)(C)Cc1ccccc1	Domain evaluation
	CHEM  :
	MOL FOR: C17 H31 N1	MW	Max
	MOL WT : 249.44	ID	991.8
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 0.50 (BCF = 3.16 L/kg wet-wt)
	Biotransformation Half-Life (days) : 0.434 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 1.80 (BAF = 62.7 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 2.96	ID	11.26
	Log Kow (experimental): not available from database
	Log Kow used by BCF estimates: 2.96
	Equation Used to Make BCF estimate:
	Log BCF = 0.50 (Ionic; Log Kow dependent)
	Estimated Log BCF = 0.500 (BCF = 3.162 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Linear C4 terminal chain [CCC-CH3]      | 0.0341 | 0.0341
	Frag | 1 | Unsubstituted phenyl group (C6H5-)       | -0.6032 | -0.6032
	Frag | 1 | Aromatic-CH2                             | -0.3365 | -0.3365
	Frag | 5 | Aromatic-H                               | 0.2664 | 1.3319
	Frag | 3 | Methyl [-CH3]                           | 0.2451 | 0.7353
	Frag | 7 | -CH2- [linear]                          | 0.0242 | 0.1693
	Frag | 1 | Quaternary amine                         | 0.0000 | 0.0000
	Frag | 1 | Benzene                                  | -0.4277 | -0.4277
	L Kow| * | Log Kow =  2.96 (KowWin estimate)       | 0.3073 | 0.9109
	MolWt| * | Molecular Weight Parameter               |        | -0.6397
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | -0.3626
	RESULT  |           Bio Half-Life (days)           |        | 0.4339
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 1.598 /day (10 gram fish)
	kM (Rate Constant): 0.8984 /day (100 gram fish)
	kM (Rate Constant): 0.5052 /day (1 kg fish)
	kM (Rate Constant): 0.2841 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):
	Estimated Log BCF (upper trophic) = 1.798 (BCF = 62.74 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 1.798 (BAF = 62.74 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 1.702 (BCF = 50.34 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 1.702 (BAF = 50.35 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 1.664 (BCF = 46.1 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 1.665 (BAF = 46.19 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 1.995 (BCF = 98.96 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 2.048 (BAF = 111.7 L/kg wet-wt)
C10 ADBAC	SMILES : CCCCCCCCCCN(H)(C)(C)Cc1ccccc1
	CHEM  :
	MOL FOR: C19 H35 N1	MW	Max
	MOL WT : 277.50	ID	991.8
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 0.50 (BCF = 3.16 L/kg wet-wt)
	Biotransformation Half-Life (days) : 0.823 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 2.40 (BAF = 253 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 3.95	ID	11.26
	Log Kow (experimental): not available from database
	Log Kow used by BCF estimates: 3.95
	Equation Used to Make BCF estimate:
	Log BCF = 0.50 (Ionic; Log Kow dependent)
	Estimated Log BCF = 0.500 (BCF = 3.162 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Linear C4 terminal chain [CCC-CH3]      | 0.0341 | 0.0341
	Frag | 1 | Unsubstituted phenyl group (C6H5-)       | -0.6032 | -0.6032
	Frag | 1 | Aromatic-CH2                             | -0.3365 | -0.3365
	Frag | 5 | Aromatic-H                               | 0.2664 | 1.3319
	Frag | 3 | Methyl [-CH3]                           | 0.2451 | 0.7353
	Frag | 9 | -CH2- [linear]                          | 0.0242 | 0.2177
	Frag | 1 | Quaternary amine                         | 0.0000 | 0.0000
	Frag | 1 | Benzene                                  | -0.4277 | -0.4277
	L Kow| * | Log Kow =  3.95 (KowWin estimate)       | 0.3073 | 1.2127
	MolWt| * | Molecular Weight Parameter               |        | -0.7116
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | -0.0843
	RESULT  |           Bio Half-Life (days)           |        | 0.8235
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 0.8417 /day (10 gram fish)
	kM (Rate Constant): 0.4733 /day (100 gram fish)
	kM (Rate Constant): 0.2662 /day (1 kg fish)
	kM (Rate Constant): 0.1497 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):
	Estimated Log BCF (upper trophic) = 2.403 (BCF = 253 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 2.403 (BAF = 253.1 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 2.425 (BCF = 266.4 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 2.428 (BAF = 267.7 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 2.420 (BCF = 263.3 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 2.431 (BAF = 269.5 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 2.962 (BCF = 916.8 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 3.291 (BAF = 1956 L/kg wet-wt)
C12 ADBAC	SMILES : CCCCCCCCCCCCN(H)(C)(C)Cc1ccccc1
	CHEM  :
	MOL FOR: C21 H39 N1	MW	Max
	MOL WT : 305.55	ID	991.8
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 1.85 (BCF = 70.8 L/kg wet-wt)
	Biotransformation Half-Life (days) : 1.56 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 2.78 (BAF = 607 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 4.93	ID	11.26
	Log Kow (experimental): not available from database
	Log Kow used by BCF estimates: 4.93
	Equation Used to Make BCF estimate:
	Log BCF = 1.85 (Ionic; 11 or more -CH2- groups)
	Estimated Log BCF = 1.850 (BCF = 70.79 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Linear C4 terminal chain [CCC-CH3]      | 0.0341 | 0.0341
	Frag | 1 | Unsubstituted phenyl group (C6H5-)       | -0.6032 | -0.6032
	Frag | 1 | Aromatic-CH2                             | -0.3365 | -0.3365
	Frag | 5 | Aromatic-H                               | 0.2664 | 1.3319
	Frag | 3 | Methyl [-CH3]                           | 0.2451 | 0.7353
	Frag | 11 | -CH2- [linear]                          | 0.0242 | 0.2661
	Frag | 1 | Quaternary amine                         | 0.0000 | 0.0000
	Frag | 1 | Benzene                                  | -0.4277 | -0.4277
	L Kow| * | Log Kow =  4.93 (KowWin estimate)       | 0.3073 | 1.5146
	MolWt| * | Molecular Weight Parameter               |        | -0.7835
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | 0.1940
	RESULT  |           Bio Half-Life (days)           |        |  1.563
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 0.4435 /day (10 gram fish)
	kM (Rate Constant): 0.2494 /day (100 gram fish)
	kM (Rate Constant): 0.1402 /day (1 kg fish)
	kM (Rate Constant): 0.07886 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):
	Estimated Log BCF (upper trophic) = 2.779 (BCF = 600.6 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 2.783 (BAF = 607.1 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 2.885 (BCF = 768.2 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 2.922 (BAF = 835.5 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 2.913 (BCF = 818.6 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 3.028 (BAF = 1067 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 3.843 (BCF = 6963 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 4.913 (BAF = 8.186e+004 L/kg wet-wt)
C14 ADBAC	SMILES : CCCCCCCCCCCCCCN(H)(C)(C)Cc1ccccc1
	CHEM  :
	MOL FOR: C23 H43 N1	MW	Max
	MOL WT : 333.61	ID	991.8
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 1.85 (BCF = 70.8 L/kg wet-wt)
	Biotransformation Half-Life (days) : 2.97 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 3.14 (BAF = 1.38e+003 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 5.91	ID	11.26
	Log Kow (experimental): not available from database
	Log Kow used by BCF estimates: 5.91
	Equation Used to Make BCF estimate:
	Log BCF = 1.85 (Ionic; 11 or more -CH2- groups)
	Estimated Log BCF = 1.850 (BCF = 70.79 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Linear C4 terminal chain [CCC-CH3]      | 0.0341 | 0.0341
	Frag | 1 | Unsubstituted phenyl group (C6H5-)       | -0.6032 | -0.6032
	Frag | 1 | Aromatic-CH2                             | -0.3365 | -0.3365
	Frag | 5 | Aromatic-H                               | 0.2664 | 1.3319
	Frag | 3 | Methyl [-CH3]                           | 0.2451 | 0.7353
	Frag | 13 | -CH2- [linear]                          | 0.0242 | 0.3144
	Frag | 1 | Quaternary amine                         | 0.0000 | 0.0000
	Frag | 1 | Benzene                                  | -0.4277 | -0.4277
	L Kow| * | Log Kow =  5.91 (KowWin estimate)       | 0.3073 | 1.8165
	MolWt| * | Molecular Weight Parameter               |        | -0.8555
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | 0.4723
	RESULT  |           Bio Half-Life (days)           |        |  2.967
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 0.2336 /day (10 gram fish)
	kM (Rate Constant): 0.1314 /day (100 gram fish)
	kM (Rate Constant): 0.07389 /day (1 kg fish)
	kM (Rate Constant): 0.04155 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):
	Estimated Log BCF (upper trophic) = 3.012 (BCF = 1027 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 3.141 (BAF = 1384 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 3.145 (BCF = 1398 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 3.548 (BAF = 3531 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 3.185 (BCF = 1532 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 3.871 (BAF = 7437 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 4.309 (BCF = 2.037e+004 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 6.319 (BAF = 2.084e+006 L/kg wet-wt)
C16 ADBAC	SMILES : CCCCCCCCCCCCCCCCN(H)(C)(C)Cc1ccccc1
	CHEM  :
	MOL FOR: C25 H47 N1	MW	Max
	MOL WT : 361.66	ID	991.8
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 1.85 (BCF = 70.8 L/kg wet-wt)
	Biotransformation Half-Life (days) : 5.63 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 3.94 (BAF = 8.65e+003 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 6.89	ID	11.26
	Log Kow (experimental): not available from database
	Log Kow used by BCF estimates: 6.89
	Equation Used to Make BCF estimate:
	Log BCF = 1.85 (Ionic; 11 or more -CH2- groups)
	Estimated Log BCF = 1.850 (BCF = 70.79 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Linear C4 terminal chain [CCC-CH3]      | 0.0341 | 0.0341
	Frag | 1 | Unsubstituted phenyl group (C6H5-)       | -0.6032 | -0.6032
	Frag | 1 | Aromatic-CH2                             | -0.3365 | -0.3365
	Frag | 5 | Aromatic-H                               | 0.2664 | 1.3319
	Frag | 3 | Methyl [-CH3]                           | 0.2451 | 0.7353
	Frag | 15 | -CH2- [linear]                          | 0.0242 | 0.3628
	Frag | 1 | Quaternary amine                         | 0.0000 | 0.0000
	Frag | 1 | Benzene                                  | -0.4277 | -0.4277
	L Kow| * | Log Kow =  6.89 (KowWin estimate)       | 0.3073 | 2.1184
	MolWt| * | Molecular Weight Parameter               |        | -0.9274
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | 0.7506
	RESULT  |           Bio Half-Life (days)           |        |  5.631
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 0.1231 /day (10 gram fish)
	kM (Rate Constant): 0.06922 /day (100 gram fish)
	kM (Rate Constant): 0.03893 /day (1 kg fish)
	kM (Rate Constant): 0.02189 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):
	Estimated Log BCF (upper trophic) = 2.926 (BCF = 844.1 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 3.937 (BAF = 8649 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 3.067 (BCF = 1168 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 4.441 (BAF = 2.762e+004 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 3.110 (BCF = 1289 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 4.743 (BAF = 5.53e+004 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 4.127 (BCF = 1.34e+004 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 7.047 (BAF = 1.114e+007 L/kg wet-wt)
C18 and longer ADBAC	SMILES : CCCCCCCCCCCCCCCCCCN(H)(C)(C)Cc1ccccc1
	CHEM  :
	MOL FOR: C27 H51 N1	MW	Max
	MOL WT : 389.71	ID	991.8
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 1.85 (BCF = 70.8 L/kg wet-wt)
	Biotransformation Half-Life (days) : 10.7 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 4.45 (BAF = 2.81e+004 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 7.87	ID	11.26
	Log Kow (experimental): not available from database
	Log Kow used by BCF estimates: 7.87
	Equation Used to Make BCF estimate:
	Log BCF = 1.85 (Ionic; 11 or more -CH2- groups)
	Estimated Log BCF = 1.850 (BCF = 70.79 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Linear C4 terminal chain [CCC-CH3]      | 0.0341 | 0.0341
	Frag | 1 | Unsubstituted phenyl group (C6H5-)       | -0.6032 | -0.6032
	Frag | 1 | Aromatic-CH2                             | -0.3365 | -0.3365
	Frag | 5 | Aromatic-H                               | 0.2664 | 1.3319
	Frag | 3 | Methyl [-CH3]                           | 0.2451 | 0.7353
	Frag | 17 | -CH2- [linear]                          | 0.0242 | 0.4112
	Frag | 1 | Quaternary amine                         | 0.0000 | 0.0000
	Frag | 1 | Benzene                                  | -0.4277 | -0.4277
	L Kow| * | Log Kow =  7.87 (KowWin estimate)       | 0.3073 | 2.4202
	MolWt| * | Molecular Weight Parameter               |        | -0.9994
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | 1.0289
	RESULT  |           Bio Half-Life (days)           |        |  10.69
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 0.06486 /day (10 gram fish)
	kM (Rate Constant): 0.03647 /day (100 gram fish)
	kM (Rate Constant): 0.02051 /day (1 kg fish)
	kM (Rate Constant): 0.01153 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):
	Estimated Log BCF (upper trophic) = 2.401 (BCF = 251.7 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 4.449 (BAF = 2.811e+004 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 2.542 (BCF = 348.7 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 4.743 (BAF = 5.53e+004 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 2.586 (BCF = 385.2 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 4.921 (BAF = 8.343e+004 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 3.608 (BCF = 4058 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 7.129 (BAF = 1.345e+007 L/kg wet-wt)
C18' ADBAC	SMILES : CCCCCCCCC=CCCCCCCCCN(H)(C)(C)Cc1ccccc1
	CHEM  :
	MOL FOR: C27 H49 N1	MW	Max
	MOL WT : 387.70	ID	991.8
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 1.85 (BCF = 70.8 L/kg wet-wt)
	Biotransformation Half-Life (days) : 13.1 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 4.78 (BAF = 5.99e+004 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 7.66	ID	11.26
	Log Kow (experimental): not available from database
	Log Kow used by BCF estimates: 7.66
	Equation Used to Make BCF estimate:
	Log BCF = 1.85 (Ionic; 11 or more -CH2- groups)
	Estimated Log BCF = 1.850 (BCF = 70.79 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Linear C4 terminal chain [CCC-CH3]      | 0.0341 | 0.0341
	Frag | 1 | Unsubstituted phenyl group (C6H5-)       | -0.6032 | -0.6032
	Frag | 1 | Aromatic-CH2                             | -0.3365 | -0.3365
	Frag | 5 | Aromatic-H                               | 0.2664 | 1.3319
	Frag | 3 | Methyl [-CH3]                           | 0.2451 | 0.7353
	Frag | 15 | -CH2- [linear]                          | 0.0242 | 0.3628
	Frag | 2 | -C=CH [alkenyl hydrogen]                | 0.0988 | 0.1977
	Frag | 1 | Quaternary amine                         | 0.0000 | 0.0000
	Frag | 2 | -C=CH [alkenyl hydrogen]                | 0.0000 | 0.0000
	Frag | 1 | Benzene                                  | -0.4277 | -0.4277
	L Kow| * | Log Kow =  7.66 (KowWin estimate)       | 0.3073 | 2.3541
	MolWt| * | Molecular Weight Parameter               |        | -0.9942
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | 1.1173
	RESULT  |           Bio Half-Life (days)           |        |   13.1
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 0.05291 /day (10 gram fish)
	kM (Rate Constant): 0.02975 /day (100 gram fish)
	kM (Rate Constant): 0.01673 /day (1 kg fish)
	kM (Rate Constant): 0.009409 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):
	Estimated Log BCF (upper trophic) = 2.675 (BCF = 472.8 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 4.777 (BAF = 5.986e+004 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 2.818 (BCF = 658 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 5.004 (BAF = 1.009e+005 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 2.862 (BCF = 727.7 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 5.143 (BAF = 1.39e+005 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 3.723 (BCF = 5283 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 7.156 (BAF = 1.432e+007 L/kg wet-wt)
Benzyl chloride	SMILES : c1ccc(cc1)CCL
	CHEM  :
	MOL FOR: C7 H7 CL1	MW	Max
	MOL WT : 126.59	ID	991.8
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 1.18 (BCF = 15.3 L/kg wet-wt)
	Biotransformation Half-Life (days) : 0.148 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 1.18 (BAF = 15.1 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 2.79	ID	11.26
	Log Kow (experimental): 2.30
	Log Kow used by BCF estimates: 2.30
	Equation Used to Make BCF estimate:
	Log BCF = 0.6598 log Kow - 0.333 + Correction
	Correction(s):                   Value
	No Applicable Correction Factors
	Estimated Log BCF = 1.185 (BCF = 15.29 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Aliphatic chloride  [-CL]               | 0.3608 | 0.3608
	Frag | 1 | Unsubstituted phenyl group (C6H5-)       | -0.6032 | -0.6032
	Frag | 1 | Aromatic-CH2                             | -0.3365 | -0.3365
	Frag | 5 | Aromatic-H                               | 0.2664 | 1.3319
	Frag | 1 | Benzene                                  | -0.4277 | -0.4277
	L Kow| * | Log Kow =  2.30 (experimental  )       | 0.3073 | 0.7069
	MolWt| * | Molecular Weight Parameter               |        | -0.3246
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | -0.8296
	RESULT  |           Bio Half-Life (days)           |        | 0.1481
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 4.682 /day (10 gram fish)
	kM (Rate Constant): 2.633 /day (100 gram fish)
	kM (Rate Constant): 1.48 /day (1 kg fish)
	kM (Rate Constant): 0.8325 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):
	Estimated Log BCF (upper trophic) = 1.178 (BCF = 15.06 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 1.178 (BAF = 15.06 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 1.079 (BCF = 11.99 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 1.079 (BAF = 11.99 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 1.041 (BCF = 10.99 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 1.041 (BAF = 11 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 1.347 (BCF = 22.22 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 1.362 (BAF = 23.03 L/kg wet-wt)
Benzaldehyde	SMILES : c1ccc(cc1)C=O
	CHEM  :
	MOL FOR: C7 H6 O1	MW	Max
	MOL WT : 106.13	ID	959.17
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 0.64 (BCF = 4.4 L/kg wet-wt)
	Biotransformation Half-Life (days) : 0.156 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 0.55 (BAF = 3.57 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 1.71	ID	11.26
	Log Kow (experimental): 1.48
	Log Kow used by BCF estimates: 1.48
	Equation Used to Make BCF estimate:
	Log BCF = 0.6598 log Kow - 0.333 + Correction
	Correction(s):                   Value
	No Applicable Correction Factors
	Estimated Log BCF = 0.644 (BCF = 4.401 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Aldehyde  [-CHO]                        | 0.2465 | 0.2465
	Frag | 1 | Unsubstituted phenyl group (C6H5-)       | -0.6032 | -0.6032
	Frag | 5 | Aromatic-H                               | 0.2664 | 1.3319
	Frag | 1 | Benzene                                  | -0.4277 | -0.4277
	L Kow| * | Log Kow =  1.48 (experimental  )       | 0.3073 | 0.4549
	MolWt| * | Molecular Weight Parameter               |        | -0.2721
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | -0.8069
	RESULT  |           Bio Half-Life (days)           |        |  0.156
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 4.443 /day (10 gram fish)
	kM (Rate Constant): 2.499 /day (100 gram fish)
	kM (Rate Constant): 1.405 /day (1 kg fish)
	kM (Rate Constant): 0.7902 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):	log Kow	Max
	Estimated Log BCF (upper trophic) = 0.552 (BCF = 3.566 L/kg wet-wt)	ID	11.26
	Estimated Log BAF (upper trophic) = 0.552 (BAF = 3.566 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 0.450 (BCF = 2.817 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 0.450 (BAF = 2.817 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 0.418 (BCF = 2.617 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 0.418 (BAF = 2.617 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 0.615 (BCF = 4.123 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 0.621 (BAF = 4.176 L/kg wet-wt)
Benzyl alcohol	SMILES : c1ccc(cc1)CO
	CHEM  :
	MOL FOR: C7 H8 O1	MW	Max
	MOL WT : 108.14	ID	959.17
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 0.14 (BCF = 1.37 L/kg wet-wt)
	Biotransformation Half-Life (days) : 0.0267 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 0.19 (BAF = 1.55 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 1.08	ID	11.26
	Log Kow (experimental): 1.10
	Log Kow used by BCF estimates: 1.10
	Equation Used to Make BCF estimate:
	Log BCF = 0.6598 log Kow - 0.333 + Correction
	Correction(s):                   Value	Corr.	Instance
	Aromatic ring-CH-OH            -0.256	ID	1
	Estimated Log BCF = 0.137 (BCF = 1.371 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Aliphatic alcohol [-OH]                 | -0.0616 | -0.0616
	Frag | 1 | Unsubstituted phenyl group (C6H5-)       | -0.6032 | -0.6032
	Frag | 1 | Aromatic-CH2                             | -0.3365 | -0.3365
	Frag | 5 | Aromatic-H                               | 0.2664 | 1.3319
	Frag | 1 | Benzene                                  | -0.4277 | -0.4277
	L Kow| * | Log Kow =  1.10 (experimental  )       | 0.3073 | 0.3381
	MolWt| * | Molecular Weight Parameter               |        | -0.2773
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | -1.5734
	RESULT  |           Bio Half-Life (days)           |        | 0.02671
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 25.96 /day (10 gram fish)
	kM (Rate Constant): 14.6 /day (100 gram fish)
	kM (Rate Constant): 8.208 /day (1 kg fish)
	kM (Rate Constant): 4.616 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):
	Estimated Log BCF (upper trophic) = 0.190 (BCF = 1.549 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 0.190 (BAF = 1.549 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 0.179 (BCF = 1.51 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 0.179 (BAF = 1.51 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 0.171 (BCF = 1.482 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 0.171 (BAF = 1.482 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 0.350 (BCF = 2.24 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 0.354 (BAF = 2.259 L/kg wet-wt)
N,N-dimethylhexadecan-1-amine	SMILES : CCCCCCCCCCCCCCCCN(C)C
	CHEM  :
	MOL FOR: C18 H39 N1	MW	Max
	MOL WT : 269.52	ID	959.17
	--------------------------------- BCFBAF v3.01 --------------------------------
	Summary Results:
	Log BCF (regression-based estimate): 3.33 (BCF = 2.13e+003 L/kg wet-wt)
	Biotransformation Half-Life (days) : 2.49 (normalized to 10 g fish)
	Log BAF (Arnot-Gobas upper trophic): 2.98 (BAF = 950 L/kg wet-wt)
	=============================
	BCF (Bioconcentration Factor):
	=============================	log Kow	Max
	Log Kow (estimated) : 7.41	ID	11.26
	Log Kow (experimental): not available from database
	Log Kow used by BCF estimates: 7.41
	Equation Used to Make BCF estimate:
	Log BCF = -0.49 log Kow + 7.554 + Correction
	Correction(s):                   Value	Corr.	Instance
	Alkyl chains (8+ -CH2- groups) -0.596	ID	1
	Estimated Log BCF = 3.329 (BCF = 2133 L/kg wet-wt)
	===========================================================
	Whole Body Primary Biotransformation Rate Estimate for Fish:
	===========================================================
	------+-----+--------------------------------------------+---------+---------
	TYPE | NUM | LOG BIOTRANSFORMATION FRAGMENT DESCRIPTION | COEFF | VALUE
	------+-----+--------------------------------------------+---------+---------
	Frag | 1 | Linear C4 terminal chain [CCC-CH3]      | 0.0341 | 0.0341
	Frag | 1 | Tertiary amine                           | -0.7829 | -0.7829
	Frag | 3 | Methyl [-CH3]                           | 0.2451 | 0.7353
	Frag | 15 | -CH2- [linear]                          | 0.0242 | 0.3628
	L Kow| * | Log Kow =  7.41 (KowWin estimate)       | 0.3073 | 2.2759
	MolWt| * | Molecular Weight Parameter               |        | -0.6911
	Const| * | Equation Constant                        |        | -1.5371
	============+============================================+=========+=========
	RESULT  |       LOG Bio Half-Life (days)           |        | 0.3970
	RESULT  |           Bio Half-Life (days)           |        |  2.495
	NOTE    | Bio Half-Life Normalized to 10 g fish at 15 deg C  |
	============+============================================+=========+=========
	Biotransformation Rate Constant:
	kM (Rate Constant): 0.2778 /day (10 gram fish)
	kM (Rate Constant): 0.1562 /day (100 gram fish)
	kM (Rate Constant): 0.08786 /day (1 kg fish)
	kM (Rate Constant): 0.04941 /day (10 kg fish)
	Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):
	Estimated Log BCF (upper trophic) = 2.211 (BCF = 162.4 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 2.978 (BAF = 950.5 L/kg wet-wt)
	Estimated Log BCF (mid trophic)  = 2.349 (BCF = 223.6 L/kg wet-wt)
	Estimated Log BAF (mid trophic)  = 3.767 (BAF = 5844 L/kg wet-wt)
	Estimated Log BCF (lower trophic) = 2.392 (BCF = 246.6 L/kg wet-wt)
	Estimated Log BAF (lower trophic) = 4.238 (BAF = 1.731e+004 L/kg wet-wt)
	Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):
	Estimated Log BCF (upper trophic) = 3.861 (BCF = 7256 L/kg wet-wt)
	Estimated Log BAF (upper trophic) = 7.156 (BAF = 1.433e+007 L/kg wet-wt)

Validity criteria fulfilled:

not applicable

Conclusions:

Using the regression-based and Arnot-gobas based methods of the BCFBAF v.3.02 program (EPI SuiteTM v4.11) for ionic and non-ionic constituents respectively, the BCF values were predicted to range from 1.55 to 162.40 L/kg ww (weighted average: 70.77 L/Kg ww).

Executive summary:

The Bioconcentration factor (BCF) value of test substance, C16 -18 ADBAC was predicted using regression-based and Arnot-Gobas BAF-BCF models of BCFBAF v3.02 program (EPI SuiteTMv4.11). The Arnot-Gobas method, takes into account mitigating factors, like growth dilution and metabolic biotransformations, therefore the BCF values using this method is considered to be more realistic or accurate. Therefore, except for ionic, pigments and dyes, perfluorinated substances, for which it is not recommended (as of now), the Arnot-Gobas method is used preferentially used for BCF predictions. Considering that the test substance is an UVCB containing majorly ionic (e.g., (e.g., the quaternary ammonium salts) and few non-ionic constituents (e.g., amines), the BCF values were predicted using regression-based and Arnot-Gobas BAF-BCF models respectively and using SMILES codes as the input parameter. The BCF values for the constituents ranged from 1.55 to 162.40 L/kg ww (log BCF: 0.19 to 2.21), indicating a low bioaccumulation potential. On comparing with domain descriptors, all constituents were found to meet the MW, log Kow and/or maximum number of correction factor instances domain criteria as defined in the BCFBAF user guide of EPISuite. Further, given that the major constituents are structurally very similar and vary only in the carbon chain length, a weighted average value, which takes into account the percentage of the constituent in the substance, has been considered to dampen the errors in predictions (if any). Therefore, the weighted average BCF value was calculated as 70.77 L/Kg ww (Log BCF = 1.85). Overall, considering either the individual BCF predictions for the constituents or the weighted average values, the test substance is expected to have a low bioaccumulation potential. However, taking into consideration the model’s training set and validation set statistics and the fact that the training set only contains 61 ionic compounds, the BCF predictions for the individual constituents are considered to be reliable with moderate confidence.

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test

Deviations:

not specified

Principles of method if other than guideline:

The biomagnification study was carried out following the principles of OECD TG 305 and were performed in accordance with the German animal welfare act under the Landesamt für Naturschutz, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany (permit 81-02.04.2018.A023).

GLP compliance:

not specified

Radiolabelling:

no

Details on sampling:

In the main study five animals of each group were sampled randomized on day 7 and day 14 of the uptake phase and after 10 h, 24 h, 2 days, 3 days, 7 days and 14 days of depuration. Samplings were done before feeding of the animals. After each sampling the remaining biomass of each group was determined by weighing the complete group of fishes to adjust the daily feed ration. The sampled animals were anesthetized in a water bath containing 150 mg/L MS 222 (Sigma Aldrich) and euthanized by a deep cut through the neck. Animals were weighed, blotted and the compartments (liver, gastrointestinal tract (GIT) and carcass) were dissected. In addition, the GIT was rinsed with ultra-pure water to remove remaining feed or feces presumably containing the test items. After weighing all compartments, the samples were shock frozen and stored at -20 °C until further sample processing for chemical analysis. The experimental diet was analysed before and after the exposure phase to verify the stability of the test items during the uptake phase.

Vehicle:

yes

Remarks:

methanol

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

Preparation of the experimental diets for pre and main study
For preparation of enriched test feed for the preliminary study commercially available fish feed pellets (Inicio Plus® BioMar, Denmark) with a size of 2 mm were spiked according to the method described by Goeritz et al. (2013). Feed batches prepared for the pre-study were spiked with one test substance each. The spiking procedure applied for the cationic test substance is described below. Specific informations related to the preparation of the test diets are summarized in below table.

An application solution of test substance was prepared taking the purity and the reduced molecular weight of the ion into account. Therefore, 23.5 mg test substance were dissolved in 20 mL methanol, corresponding to a concentration of 1 mg/mL. For spiking, 100 g fish feed were placed in a 2 L pear-shaped flask connected to a rotary evaporator equipped with a stainless steel capillary to apply the test solution via a solvent-inlet tube to the pellets under vacuum. 3.44 mL application solution were applied to the feed particles by spray application, while the feed was thoroughly mixed by rotation to ensure a homogenous distribution of the test item on the pellets. During the spiking procedure, a low pressure of approximately 700 mbar was applied. After administration of the application solution 0.5 – 1.0 mL pure solvent was utilized to rinse the beaker of the application solution which was also applied via the spray-apparatus to ensure transfer of the whole amount of test item. Afterwards, a vacuum of 350 mbar was set to evaporate the solvent in the flask. In order to remove potential solvent residues, spiked pellets were dispersed in an aluminium tray and left in the fume hood overnight. Subsequently, spiked feed pellets were coated with sodium alginate and calcium chloride to avoid test item loss through leaching into test water. For this, a 2 % sodium alginate solution (w/w) was prepared by dissolving 3 g of sodium alginate in 150 g distilled ultra-pure water (UHQ). The suspension was heated to 100 °C and stirred until a homogenous viscous solution was obtained. 3.90 g of this solution were applied in a heated 1 L glass bottle and after even distribution of the sodium alginate solution on the inner walls of the bottle by gentle shaking, 100 g of the spiked feed pellets were added. The bottle was shaken thoroughly until no feed pellets were sticking to the wall anymore. In a last step, 0.89 g calcium chloride per 100 g feed were added and the bottle was mixed until no visual inhomogeneity could be observed.

During the main study, test substance was applied in pairs. The co-exposure of two test items reduced the number of separate studies and thus the required number of fish. The experimental diets were prepared and treated the same way as described above. Here, 250 g feed with a pellet size of 1.1 mm were prepared by solvent spiking. The application solution volumes of the ion pairs (Tetrabutylphosphonium bromide, TBP and test substance) were mixed beforehand and applied concurrently. For alginate coating, two batches of 100 g and one batch of 50 g feed were handled. The amount of sodium alginate and calcium chloride dihydrate applied to each batch were adjusted according to the protocol. The control diet used during the uptake phase for feeding control fish, was prepared in exactly the same way, but without the test substance in the spiking solvent. Homogeneity and content test substance in the spiked test diets were analysed directly after preparation in five replicates.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Juvenile rainbow trout were purchased from Fischzucht Störk (Bad Saulgau, Germany) and maintained in a flow-through system in 200 to 250 L tanks filled with copper ion reduced tap water. Animals were kept under constant aeration at 14 ± 2 °C and under a 16:8 light-dark cycle until the start of the study for acclimatization purposes. The fish were fed with a commercially available food for fish breeding (Inicio Plus®, BioMar, Denmark).

Route of exposure:

feed

Justification for method:

dietary exposure method used because stable, measurable water concentrations cannot be maintained

Test type:

flow-through

Water / sediment media type:

other: copper reduced tap water

Total exposure / uptake duration:

ca. 14 d

Total depuration duration:

ca. 14 d

Details on test conditions:

Design of biomagnification studies with ionic test substance
A preliminary test was carried out before the main study to test the palatability and potential toxic effects of each experimental diet spiked with test substances. Treatment and control group, each consisting of 8 fishes, were tested during an uptake phase lasting 14 days. The feeding rate was increased from 2.0 to 2.5 % in the second week of the uptake phase. Experimental conditions during the pre-test were similar to the main study as described below. A single sampling was carried out at the end of the uptake phase. Two of the eight animals were shock frozen in whole and stored at -20 °C until chemical analysis. The other six animals were dissected and liver, GIT, filet and carcass of three animals were pooled to one replicate. The two pooled replicates per compartment were shock frozen and stored at -20 °C until analysis.

For the main study rainbow trout with an average weight of 5.42 ± 1.14 g (n = 160) were fed per treatment test diets enriched with two different ionic organic compounds, each. The first experimental diet was enriched with two cations (TBP and test substance). The resulting treatments and one control group (each 40 animals) were tested simultaneously. 80 L glass tanks were used as experimental tanks filled with 75 L of copper reduced tap water with a flow rate of 15.6 L/h resulting in a 5-fold water exchange per day. The condition of the test animals was examined with the aid of score sheets at least once per day. The water temperature, oxygen content (saturation in % and mg/L) and pH were checked every second day. Concentrations of nitrite, nitrate and ammonia were determined on day one, day seven and at the end of the tests.
The uptake phase of 14 days was followed by a depuration phase lasting 14 days. All animals were fed the non-spiked feed during the depuration phase. A feeding rate equivalent to 2 % of the body wet weight of the test animals per day was applied in compliance with the recommendation given by the feed manufacturer. Feces which may result in uptake via aqueous exposure, feces were removed at least three times per day to reduce the risk of a secondary exposure pathway via the water due to the release of freely dissolved test substance from feed residues and fecal matter.

Determination of content, homogeneity and stability of test substances in experimental diets
For the determination of substance concentrations in fish feed approximately 1 g feed was weighed into a 15 mL PP-vial. 4 mL MeOH were added, homogenized for 30 seconds and then treated in an ultrasonic bath for 10 min. The samples were centrifuged for 5 min at 5 000 rpm and the supernatants transferred into a volumetric flask. After homogenization, the dispersing tool was washed with 3 mL MeOH. This solution was then used for the second extraction step which is similar to the first one. A third extraction step (using the methanol for washing the dispersing tool) was performed and the combined supernatants were filled up with methanol to a volume of 10 mL. The extracts of the feed samples were diluted by a factor of 1000 in two steps yielding a concentration within the calibration range. For external calibration, calibration ranges of 0.1 - 20 μg/L for the cations were used. Here, at least 6 different calibration points containing blank matrix, test substance and MeOH were mixed. The amount of matrix in the calibration samples was equal to the amount of matrix in the samples. The specific concentrations measured in each sample were calculated based on the recorded weight.
The homogeneity of spiked feed was determined by measuring substance concentrations in five replicates. The stability of spiked feed was measured in triplicates after specific time points. For the preliminary study, the substance concentrations in the experimental diets were presented as the mean concentration measured at test start and test end. For the main study, the average feed concentration during the study was calculated by taking the mean of the concentrations measured at test start and at the end of the uptake phase.

Determination of test substance content in fish samples
The concentrations of the test substances in fish samples were determined by chemical analysis and all tissue concentrations were calculated based on a wet weight basis. For analysis, all tissue samples, except for liver and GIT, were homogenized first. Afterwards, approximately 1 g of homogenate was weighed into a 15 mL PP-vial and after addition of 4 mL MeOH mixed with a dispersing tool for 30 seconds. The whole liver and GIT samples were homogenized using a dispersing tool following addition of 4 mL MeOH. After mixing, samples were treated in an ultrasonic bath for 10 min, centrifuged for 5 min at 5,000 rpm and the supernatants were transferred into a volumetric flask. The dispersing tool was rinsed with 3 mL MeOH, which was then used to repeat the extraction step followed by a further rinse. Supernatants obtained from the three extraction steps were combined and filled up to a total volume of 10 mL. The extraction efficiency of the used extraction protocols was assessed. Therefore, blank matrices (GIT, liver and carcass) were spiked with a known amount of test substance:

- GIT: 1 μg/L for cations
- Liver: 2 μg/L for cations
- Carcass: 5 μg/L for cations

The extraction was performed as described above. For assessment, recovery rates were calculated. The matrix-matched calibrations with at least 6 points ranged between 0.05 - 20 μg/L for the cations. The extracts of the different matrices were diluted depending on the substance concentrations to yield a concentration within the calibration range. For the main study, TBP GIT samples from the uptake phase and 10 h and 24 h depuration phase were measured with a 1:5 dilution. Time points 48 h, 72 h, 7 d and 14 d of depuration of TBP GIT samples and all TBP liver and carcass samples were diluted 1:1. All test substance GIT samples were diluted 1:80 and all liver and all carcass samples 1:1. The organic content in each measured solution was between 50 - 55 %. For the calibration solutions, depending on the end volume (200 -1000 μL), 10 to 50 μL of the substance stock solutions were diluted with blank matrix, UHQ and MeOH leading to the approximate ratio of 1:1 (v:v) of organic solvent and UHQ. Importantly, depending on the dilution of the samples the amount of the matrix in the calibration solutions was adjusted.

Nominal and measured concentrations:

Mean content in feed samples [mg/kg]: 22.8 ± 0.27 (preliminary study), 23.6 ± 1.28 (main study)

Reference substance (positive control):

not specified

Details on estimation of bioconcentration:

Biomagnification and distribution factors were calculated based on the tissue concentrations measured at the end of the uptake phase. For calculation of steady-state biomagnification factors (BMFss) the substance concentration in the whole fish measured at the end of the uptake phase was divided by the concentration of the enriched feed. In case of the main study, the sum of the compartment concentrations was used to derive 'whole fish' concentrations. Individual tissue distribution factors of the different substances were calculated from the compartment-specific tissue concentration divided by the concentration in the whole fish.

Calculation of kinetic biomagnification factors of different matrices and of the whole fish (main study)
- The assimilation efficiency (α, absorption of the test item across the gut) was calculated.
- The feeding rate (I) used in the calculation was adjusted for fish growth to give an accurate assimilation efficiency (α). The growth-corrected feeding rate Ig was calculated.
- The kinetic BMF (BMFk) was calculated by multiplying the assimilation efficiency (α) with the feeding rate constant (I), divided by the product of the overall depuration rate constant (k2).
- The depuration rate constant (k2) was calculated by performing a linear regression of ln(concentration in fish) versus time [day].
- The growth-corrected biomagnification factor was calculated using the growth corrected depuration rate constant.

For more details on the calculation methods, kindly refer to the attached background section of the IUCLID.

Key result

Conc. / dose:

ca. 23.6 other: mg/kg in feed

Type:

BMF

Value:

ca. 0.027 other: g/g

Basis:

whole body w.w.

Calculation basis:

steady state

Key result

Conc. / dose:

ca. 23.6 other: mg/kg in feed

Type:

BMF

Value:

ca. 0.04 other: g/g

Basis:

whole body w.w.

Calculation basis:

kinetic

Key result

Conc. / dose:

ca. 23.6 other: mg/kg in feed

Type:

BMF

Value:

ca. 0.046 other: g/g

Basis:

whole body w.w.

Calculation basis:

kinetic, corrected for growth

Details on results:

Determination of homogeneity, content and stability of test substance on diet
For all test substances a linear relationship between the peak area of the quantifier mass trace and the concentration in the matrix-matched calibrations was found. The homogenous distribution of the test substance on feed was confirmed by the analysis of the test substance concentrations in five individually processed replicates. These measurements also represented the starting point of the stability measurement. The results of the homogeneity test of the two feed batches spiked with the two cations TBP and test substance. The relative standard deviations (RSD) were <10 % for both ions. Thus, a homogenous distribution of the test substances in the feed can be assumed.

All feed batches showed a homogenous distribution of the test substances. The stability measurements were performed to confirm stable concentrations during the whole uptake phase. Stability was defined as the recovery of a test substance in the spiked diet in comparison to the feed concentration measured immediately after feed preparation. In the preliminary test, recoveries of 96.1 – 136 % in comparison to the day of feed preparation were determined. During the main test the stability of the dietary concentrations of the test substances were assessed twice. First, 14 days after diet preparation (96.1 – 123% recovery) and second, at the end of the uptake phase, where recoveries of 86.2 – 109 % relative to the original concentrations were ascertained.

Based on the results of the stability measurements the dietary concentrations were calculated. The test diets had mean concentrations of 22.8 ± 0.27 mg/kg for the preliminary study and 23.6 ± 1.28 mg/kg during the main study for the test substance.

Biological Observation
No mortality or abnormal behaviour of the test animals were observed during the main study. The experimental diets were accepted by the test animals and showed a decent digestibility as confirmed by the texture and appearance of the feces. One fish in the “cation” was euthanized at day 25 due to injuries. The specific growth rates of the animals ranged from 1.95 to 2.71 %/d over the entire experiment. During the study, the feed conversion ratio (FCR) was 0.69 to 0.95.

Growth correction
Fish were measured and weighed at the beginning of the experiment as well as at respective sampling time points to monitor growth and associated growth-dilution effects during the feeding study. Growth of the test animals is also an important measure to detect potential adverse effects that may occur following dietary exposure. Growth rate constants were determined separately for the uptake and depuration phases, for the treatments and the control group, using the ln-transformed weights of the fish. A subsequent parallel line analysis (PLA, as suggested by the OECD Guideline) resulted in no statistical differences between the uptake and the depuration phase among the treated groups with P = 0.7784 for TBP/TMOA. No statistically significant difference was detected with regard to the growth of the treated groups (P = 0.7162). Hence it was deduced that neither adverse nor toxic effects were caused by the enriched diets. Weight data of the treatment groups could be pooled deriving the overall fish growth rate constant kg. The value determined for kg of 0.021 1/d, based on ln-transformed weight data of all fish, was then used for growth correction of the depuration constants.

Analysis of test substance content in fish samples
During the main study the extraction efficiency of the used extraction protocol was assessed by spiking samples with a known concentration in five replicates. Subsequently, tissue samples were extracted with the same protocol and recovery rates regarding the applied amount of test substance were determined. The estimated recovery rates were in a range of 80 - 120 % confirming the suitability of the applied extraction procedure. Note, standard deviations ranged from 5.0 - 28.0 %.

Calculation of substance specific tissue distribution and biomagnification factors in preliminary study
Based on the tisuue concentrations measured in fish collected on day 14 of the uptake phase (preliminary study), tissue distribution and magnification factors were calculated. The biomagnification factors was determined to be 0.028 for test substance.

Calculation of biomagnification factors (main study)
As steady state seemed to be reached after 14 days of exposure, steady state biomagnification factors (BMFss) could be calculated. Tissue concentrations measured during the uptake and depuration phase were used to calculate BMFk and BMFkg values for the different compartments and for the 'whole fish'. All derived kinetic BMF values are based on at least three time points measured during depuration, starting at day 0 of the depuration phase (day 14 of uptake phase). The calculated BMF values show that test substance in this study did not seem to biomagnify after dietary exposure. The BMFk and BMFkg were calculated for the test substance to be 0.040 and 0.046, respectively. The BMFk and BMFkg were 0.489 and 0.542 for test substance in the GIT. In general, the GIT and the liver showed the highest values for the BMFk and BMFkg. Growth corrected depuration rate constants k2g, were used to calculate BMF values. The absolute tissue concentrations during the uptake and depuration phase and all parameters that were used for the calculation of the BMF values.

Evaluation
The spiking procedure used for the enrichment of the experimental diets was suitable and led to stable and homogenous concentrations of the IOCs. The BMF values were determined as BMFss, BMFk and BMFkg. For the cations (TBP and Test substance) the values of the BMFkg were 1.7 times higher than the values of the BMFss. indicating that the steady-state conditions were not reached for the cation tissue concentrations at the end of the uptake phase. Neither the BMF values determined for the whole fish nor for any compartment indicate a significant biomagnification potential of the tested compounds in fish. Nevertheless, differences in the biomagnification potential of the different compounds were observed. Test substance led to BMF values that were 5 to 10 times higher than those of the other ions. In addition, a clear pattern of compartment or tissue specific magnification was observed. For all compounds the highest BMF values were determined for the GIT followed by the liver and finally the carcass. This might be explained by the location of the GIT and the transport of the compounds by blood via the vena portae from the GIT to the liver.

Interpretation of results
Overall, the measured BMF-values are low. The BMF for test substance was 0.0463 g/g. Overall, the study report authors concluded that the experimental BMF are low due to slow uptake and rapid depuration.

Conclusions for the assessment of ionic substances
The bioaccumulation of test substance was tested in an OECD 305 dietary uptake study. Test substance did not show distinct biomagnification (BMFkg < 0.1 g/g) and the highest tissue concentrations of test substance was found in the GIT. The results of this study showed that the uptake and elimination kinetics are decisive for the dietary BMF of the investigated test substance. The tissue analysis indicated that the low BMF of the investigated organic cations was due to a slow uptake from the GIT into the blood. This was also seen for some of the organic anions, but for those, the main reason for the low BMF was a rapid elimination, i.e. a high depuration rate. Hence, screening parameters primarily predicting adsorption, like Kfish/water, log Kow, log Dow, or the KHSA, may not be well suited to indicate high biomagnification following dietary uptake. Overall, it was concluded from the screening that ionization lowers the tendency of a chemical to bioaccumulate, compared to non-ionized chemicals. Aside of the well-known lipophobicity of ionized groups, fast depuration seems to be a major reason for the observed low biomagnification of ionic compounds, in particular anions. Fast depuration may happen due to rapid metabolism or conjugation of charged compounds, and future studies should test this hypothesis.

Results

Determination of homogeneity of cationic substances in fish feed used in pre and main study

	Preliminary study			Main study
	Content in feed samples [mg/kg]	Mean [mg/kg]	RSD [%]	Content in feed samples [mg/kg]	Mean [mg/kg]	RSD [%]
Test substance	20.96	22.5	4.3	25.16	25.9	4.4
	23.62			26.98
	23.46			27.35
	22.04			24.26
	22.36			25.81

 

Results of stability investigations of feed batches for pre and main study

		Preliminary study			Main study
Test substance		tstart	t14 d	tend	tstart	t14 d	tend
	mean conc. [mg/kg]	22.5		23.0	25.9	24.9	22.3
	SD [mg/kg]	0.976		1.71	1.14	0.63	1.07
	RSD [%]	4.34		7.43	4.41	2.54	4.81
	mean recovery [%]			102		96.1	86.2

With tstart = day of preparation (here n = 5), t14 d = after 14 days (here n = 3), tend = end of the uptake phase (here n = 3), SD = standard deviation, RSD = relative standard deviations.

Feed conversion ratio (FCR) and specific growth rate (SGR) of experimental animals during the feeding study.

		Uptake phase (days 1 - 14)	Depuration phase (days 15 - 28)	Total experiment (days 1 - 28)
Test substance	Average body weight gain (g/fish)	2.39	2.37	4.76
	Average feed intake (g/fish)	1.66	2.25	3.90
	FCR	0.69	0.95	0.82
	Wt1 (g)	5.18	7.57	5.18
	Wt2 (g)	7.57	9.94	9.94
	t2-t1 (d)a	14	14	28
	SGR(%/d)	2.71	1.95	2.33

For accumulation: t1 = day 0 and t2 = day 14; for the depuration, t1 = day 14 and t2 = day 28, for the total experiment, t1 = day 0 and t2 = day 14. Wt1 = average body weight at t1; Wt2 = average body weight at t2.

Overview of extraction efficiencies of test substance

	Compartment	Recovery
Test substance	GIT	83.2 ± 7.67 %
	liver	102 ± 20.5 %
	carcass	115 ± 8.61 %

 

Test substance concentrations in fish samples collected at end of uptake (pre and main study)

		Preliminary study: Conc. ± SD [mg/kg]	Main study: Conc. ± SD [mg/kg]
Test substance	(calc.) whole fish	0.633 ± 0.042	0.6399
	filet	0.206 ± 0.081	---
	GIT	2.324 ± 0.799	8.2331 ± 1.661
	liver	1.632 ± 0.389	1.0178 ± 0.190
	carcass	1.218 ± 0.094	0.0862 ± 0.020

 

Overview of determined tissue specific BMFk and BMFkg

	GIT		Liver		Carcass
Test substance	BMFk	BMFkg	BMFk	BMFkg	BMFk	BMFkg
	0.489	0.542	0.0621	0.0833	0.00450	0.00742

 

Summary of 'whole fish' BMFk, BMFk and BMFkg

BMFk	BMFk	BMFkg
0.02709	0.04044	0.04629

 

Parameter for BMF calculation of test substance in compartments and 'whole fish'

	Unit		GIT	Liver	Carcass	Fish
C0,d	mg/kg	conc. at start of dep.	12.104	1.129	0.0632	0.955
k2	1/day	dep. rate constant	0.222	0.0862	0.0557	0.173
t	day	duration uptake	14	14	14	14
I	gfeed/gfish * day	feed ingestion rate	0.02	0.02	0.02	0.02
Cfeed	mg/kgfeed	conc. in feed	25.9	25.9	25.9	25.9
α		assimilation efficiency	5.43	0.268	0.0125	0.350
kg	1/d	growth rate constant	0.0219	0.0219	0.0219	0.0219
k2g	1/d	growth rate corrected k2	0.200	0.0643	0.0338	0.151
t1/2 (uncorrected)	days	substance-specific half life (based on uncorr. k2)	3.12	8.04	12.4	4.00
t1/2 (corrected)	days	substance-specific half life (based on growth-corr. k2)	3.46	10.8	20.5	4.58
BMFk		kinetic BMF	0.489	0.0621	0.00450	0.0404
BMFkg		growth-corrected BMF	0.542	0.0833	0.00742	0.0463

For more details on the results, kindly refer to the attached background section of the IUCLID.

Validity criteria fulfilled:

not specified

Conclusions:

Under the study conditions, the read across substance BMFss, BMFk and BMFkg values on whole body wet weight basis in rainbow trout were determined to be 0.02709, 0.0404 and 0.0463 g/g, respectively.

Executive summary:

A study was conducted to determine the biomagnification of the read across substance, C18 TMAC (purity 95%), following the principles of OECD TG 305. For the main study rainbow trout with an average weight of 5.42 g were fed per treatment test diets enriched with read across substance (23.6 mg/kg read across substance in feed). The resulting treatment and one control group (each 40 animals) were tested simultaneously. The uptake phase of 14 days was followed by a depuration phase lasting 14 days. All animals were fed the non-spiked feed during the depuration phase. The concentrations of the read across substances in fish samples were determined by chemical analysis and all tissue concentrations were calculated based on a wet weight basis. Chemical analysis of the read across substances was performed by liquid chromatography with coupled mass spectrometry (LC-MS/MS). In the main study five animals of each group were sampled randomized on day 7 and day 14 of the uptake phase and after 10 h, 24 h, 2 days, 3 days, 7 days and 14 days of depuration. Biomagnification factor (BMF) and distribution factor were calculated based on the tissue concentrations measured at the end of the uptake phase. No mortality or abnormal behaviour of the test animals were observed during the main study. The experimental diets were accepted by the test animals and showed a decent digestibility as confirmed by the texture and appearance of the feces. One fish was euthanized at day 25 due to injuries. The specific growth rates of the animals ranged from 1.95 to 2.71 %/d over the entire experiment. During the study, the feed conversion ratio (FCR) was 0.69 to 0.95. Fish were measured and weighed at the beginning of the experiment as well as at respective sampling time points to monitor growth and associated growth-dilution effects during the feeding study. Growth rate constants were determined separately for the uptake and depuration phases, for the treatments and the control group, using the ln-transformed weights of the fish. A subsequent parallel line analysis (PLA, as suggested by the OECD Guideline) resulted in no statistical differences between the uptake and the depuration phase among the treated groups with read across substance. No statistically significant difference was detected with regard to the growth of the treated groups. Hence it was deduced that neither adverse nor toxic effects were caused by the enriched diets. As steady state seemed to be reached after 14 days of exposure, steady state biomagnification factors (BMFss) could be calculated as 0.02709 g/g, which showed that read across substance did not biomagnify after dietary exposure. In general, the GIT and the liver showed the highest values for the BMFk and BMFkg. The kinetic BMF (BMFk) and growth-corrected biomagnification factor (BMFkg) were calculated for the read across substance to be 0.0404 and 0.0463, respectively. Overall, it was concluded from the screening that ionization lowers the tendency of a chemical to bioaccumulate, compared to non-ionized chemicals. Aside of the well-known lipophobicity of ionized groups, fast depuration seems to be a major reason for the observed low biomagnification of ionic compounds, in particular anions. Fast depuration may happen due to rapid metabolism or conjugation of charged compounds, and future studies should test this hypothesis. Under the study conditions, the read across substance BMFss, BMFk and BMFkg values on whole body wet weight basis in rainbow trout were determined to be 0.02709, 0.0404 and 0.0463 g/g, respectively (Schlechtriem, 2021). Based on the results of the read across study, a similar low bioaccumulation potential is expected for the test substance.

Description of key information

The results of the read across study, supported with the estimated BCF value for the test substance together with its ionic nature indicates a low bioaccumulation potential. The experimental BCF value of 79 L/kg ww from the read across study and the growth corrected kinetic biomagnification factor (BMFkg) value of 0.0463 based on read across to C18 TMAC, has been considered further for hazard/risk assessment. 

Key value for chemical safety assessment

BCF (aquatic species):

79 L/kg ww

BMF in fish (dimensionless):

0.046

Additional information

Study 1:A study was conducted to determine the aquatic bioaccumulation of the read across substance, C12 -16 ADBAC (30.64% active; 98.9% radiolabeled purity) in Lepomis macrochirus (bluegill fish) under flow-through conditions, according to EPA OPP 165-4, in compliance with GLP. The blue gill fish were continuously exposed to a nominal concentration of 0.050 mg/L of the read across substance (equivalent to a measured concentration of 0.076 mg/L) in well water for 35 days, followed by transfer of 35 fish into flowing uncontaminated water for a 21-d depuration period. Sampling was carried out on Days 0, 1, 3, 7, 9, 10, 14, 21, 23, 28 and 35 for the exposure period and Days 1, 3, 7, 10, 14 and 21 for the depuration period. Water samples were collected on Day 8 of the exposure period and Day 16 of the depuration for analytic determination of the read across substance concentration. Radiometric analyses of the water and selected fish tissues revealed that the mean steady state bioconcentration factor (BCF) in the edible, non-edible and whole-body fish tissue during the 35 days of exposure to be 33, 160 and 79 L/kg. The half-life for non-edible tissue was attained between Days 14 and 21, while it could not be reached for the edible and whole-body fish tissues by the end of 21-d depuration period. By Day 21 of the depuration period, the 14C residues present on the last day of exposure in the edible, non-edible and whole-body fish tissues had been eliminated by 29, 60 and 44% respectively. Analysis of skin tissue after 35 d of exposure showed residue levels somewhat higher than those observed for edible tissue at the same sampling period, indicating that there is likely significant binding of 14C-ADBAC to the skins and scales of exposed bluegill, as expected behaviour of cationic surfactants. Under the conditions of the study, the whole body BCF of the read across substance was determined to be 79, indicating low potential to bioaccumulate (Fackler, 1989).  

Study 2:The Bioconcentration factor (BCF) value of test substance, C16 -18 ADBAC was predicted using regression-based and Arnot-Gobas BAF-BCF models of BCFBAF v3.02 program (EPI SuiteTMv4.11). The Arnot-Gobas method, takes into account mitigating factors, like growth dilution and metabolic biotransformations, therefore the BCF values using this method is considered to be more realistic or accurate. Therefore, except for ionic, pigments and dyes, perfluorinated substances, for which it is not recommended (as of now), the Arnot-Gobas method is used preferentially used for BCF predictions. Considering that the test substance is an UVCB containing majorly ionic (e.g., (e.g., the quaternary ammonium salts) and few non-ionic constituents (e.g., amines), the BCF values were predicted using regression-based and Arnot-Gobas BAF-BCF models respectively and using SMILES codes as the input parameter. The BCF values for the constituents ranged from 1.55 to 162.40 L/kg ww (log BCF: 0.19 to 2.21), indicating a low bioaccumulation potential. On comparing with domain descriptors, all constituents were found to meet the MW, log Kow and/or maximum number of correction factor instances domain criteria as defined in the BCFBAF user guide of EPISuite. Further, given that the major constituents are structurally very similar and vary only in the carbon chain length, a weighted average value, which takes into account the percentage of the constituent in the substance, has been considered to dampen the errors in predictions (if any). Therefore, the weighted average BCF value was calculated as 70.79 L/Kg ww (Log BCF = 1.85). Overall, considering either the individual BCF predictions for the constituents or the weighted average values, the test substance is expected to have a low bioaccumulation potential. However, taking into consideration the model’s training set and validation set statistics and the fact that the training set only contains 61 ionic compounds, the BCF predictions for the individual constituents are considered to be reliable with moderate confidence. 

Study 3: A study was conducted to determine the biomagnification (BMF) potential of the read across substance, C18 TMAC (purity 95%), following the principles of OECD TG 305. For the main study rainbow trout (Oncorhynchus mykiss) with an average weight of 5.42 g were fed test diets enriched with read across substance (23.6 mg/kg read across the substance in feed. The resulting treatment and one control group (each 40 animals) were tested simultaneously. The uptake phase of 14 days was followed by a depuration phase lasting 14 days. All animals were fed the non-spiked feed during the depuration phase. The concentrations of the read across substance in fish samples were determined by chemical analysis and all tissue concentrations were calculated based on a wet weight basis. Chemical analysis of the read across substance was performed by liquid chromatography with coupled mass spectrometry (LC-MS/MS). In the main study five animals of each group were sampled randomized on Day 7 and Day 14 of the uptake phase and after 10 h, 24 h, 2 days, 3 days, 7 days and 14 days of depuration. Biomagnification factor (BMF) and distribution factor were calculated based on the tissue concentrations measured at the end of the uptake phase. No mortality or abnormal behaviour of the test animals was observed during the main study. The experimental diets were accepted by the test animals and showed a decent digestibility as confirmed by the texture and appearance of the feces. One fish was euthanized at Day 25 due to injuries. The specific growth rates of the animals ranged from 1.95 to 2.71 %/d over the entire experiment. During the study, the feed conversion ratio (FCR) was 0.69 to 0.95. Fish were measured and weighed at the beginning of the experiment as well as at respective sampling time points to monitor growth and associated growth-dilution effects during the feeding study. Growth rate constants were determined separately for the uptake and depuration phases, for the treatments and the control group, using the ln-transformed weights of the fish. A subsequent parallel line analysis (PLA, as suggested by the OECD Guideline) resulted in no statistical differences between the uptake and the depuration phase among the treated groups with the read across substance. No statistically significant difference was detected with regard to the growth of the treated groups. Hence it was deduced that neither adverse nor toxic effects were caused by the enriched diets. As steady state seemed to be reached after 14 days of exposure, steady state biomagnification factors (BMFss) could be calculated as 0.02709 g/g, which showed that read across substance did not biomagnify after dietary exposure. In general, the GIT and the liver showed the highest values for the BMFk and BMFkg. The kinetic BMF (BMFk) and growth-corrected biomagnification factor (BMFkg) were calculated for the read across substance to be 0.0404 and 0.0463, respectively. Overall, it was concluded from the screening that ionization lowers the tendency of a chemical to bioaccumulate, compared to non-ionized chemicals. Aside from the well-known lipophobicity of ionized groups, fast depuration seems to be a major reason for the observed low biomagnification of ionic compounds, in particular anions. Fast depuration may happen due to rapid metabolism or conjugation of charged compounds, and future studies should test this hypothesis. Under the study conditions, the read across substance BMFss, BMFk and BMFkg values on whole body wet weight basis in rainbow trout were determined to be 0.02709, 0.0404 and 0.0463 g/g, respectively, suggesting low biomagnification potential (Schlechtriem, 2021). Based on the results of the read across study, a similar low biomagnification potential is expected for the test substance. 

This is further supported by the no bioaccumulation potential evidence observed in in the two toxicokinetic studies in mammals with the read across substance, C12-16 ADBAC (Selim, 1987 and Appelqvist, 2006). Also, the biocides assessment reports available from RMS Italy on C12-16 ADBAC, concluded the substances to show low potential for bioaccumulation, based on the results from the above study (Fackler, 1989). They further stated this finding to be in line with the mode of action of the c12-16 ADBAC, which mainly possesses irritant/corrosive properties (ECHA biocides assessment report, 2015).

Overall, the results of the read across study, supported with the estimated BCF value for the test substance together with its ionic nature indicates a low bioaccumulation potential. The experimental BCF value of 79 L/kg ww from the read across study and the growth corrected kinetic biomagnification factor (BMFkg) value of 0.0463 based on read across to C18 TMAC, has been considered further for hazard/risk assessment.