Fatty acids, C16-18 (even numbered), ammonium salts

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

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

Remarks:

Internationally accepted method, EPI Suite, EPA (USA)

Justification for type of information:

See attached the QMRF and QPRF for the QSAR model

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

Meylan, W.M., Howard, P.H, Aronson, D., Printup, H. and S. Gouchie. 1997. "Improved Method for Estimating Bioconcentration Factor (BCF) from Octanol-Water Partition Coefficient", SRC TR-97-006 (2nd Update), July 22, 1997; prepared for: Robert S. Boethling, EPA-OPPT, Washington, DC; Contract No. 68-D5-0012; prepared by: ; Syracuse Research Corp., Environmental Science Center, 6225 Running Ridge Road, North Syracuse, NY 13212.
Meylan, W.M., Howard, P.H., Boethling, R.S. et al. 1999. Improved Method for Estimating Bioconcentration / Bioaccumulation Factor from Octanol/Water Partition Coefficient. Environ. Toxicol. Chem. 18(4): 664-672 (1999).

Specific details on test material used for the study:

SMILES: N(H)(H)(H)(H)OC(=O)CCCCCCCCCCCCCCCCC

Key result

Type:

BCF

Value:

70.79 L/kg

Remarks on result:

other: (Log BCF from regression-based method = 1.85; log Kow used: 5.07)

The BCF of ammonium stearate was 70.79 L/kg wet-wt (log BCF from regression-based method = 1.850).

Validity criteria fulfilled:

not applicable

Conclusions:

The BCF of ammonium stearate was 70.79 L/kg wet-wt (log BCF from regression-based method = 1.850).

Executive summary:

Using the BCFBAF v3.01 model of EPI Suite v4.11, the BCF of ammonium stearate was 70.79 L/kg wet-wt (log BCF from regression-based method = 1.850).

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

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

Remarks:

Internationally accepted method, EPI Suite, EPA (USA)

Justification for type of information:

See attached the QMRF and QPRF for the QSAR model.

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

Meylan, W.M., Howard, P.H, Aronson, D., Printup, H. and S. Gouchie. 1997. "Improved Method for Estimating Bioconcentration Factor (BCF) from Octanol-Water Partition Coefficient", SRC TR-97-006 (2nd Update), July 22, 1997; prepared for: Robert S. Boethling, EPA-OPPT, Washington, DC; Contract No. 68-D5-0012; prepared by: ; Syracuse Research Corp., Environmental Science Center, 6225 Running Ridge Road, North Syracuse, NY 13212.
Meylan, W.M., Howard, P.H., Boethling, R.S. et al. 1999. Improved Method for Estimating Bioconcentration / Bioaccumulation Factor from Octanol/Water Partition Coefficient. Environ. Toxicol. Chem. 18(4): 664-672 (1999).

Specific details on test material used for the study:

SMILES: N(H)(H)(H)(H)OC(=O)CCCCCCCCCCCCCCC

Key result

Type:

BCF

Value:

70.79 L/kg

Remarks on result:

other: (Log BCF from regression-based method = 1.850. log Kow used: 4.09)

The BCF of ammonium palmitate was 70.79 L/kg wet-wt (log BCF from regression-based method = 1.850).

Validity criteria fulfilled:

not applicable

Conclusions:

The BCF of ammonium palmitate was 70.79 L/kg wet-wt (log BCF from regression-based method = 1.850).

Executive summary:

Using the BCFBAF v3.01 model of EPI Suite v4.11, the BCF of ammonium palmitate was 70.79 L/kg wet-wt (log BCF from regression-based method = 1.850).

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Deviations:

yes

Remarks:

(depuration phase was not determined; fish were only sampled at the end of exposure)

GLP compliance:

not specified

Radiolabelling:

yes

Details on sampling:

- Sampling intervals/frequency for test organisms:
Sampling of tissue was perfomed at the termination of exposure in surviving fish, which was after 28 days in the low exposure concentrations and 16 days in top two exposure concentrations. Also after 4 to 8 days in dead fish at the higher exposure concentrations sampling was conducted.
Total radioactivity in fish surviving to the end of the test (0, 2, 3.6, and 6.4 mg/L) or to day 16 (11.2 and 20 mg/L) was determined in about five fish at each concentration by dissolving the tissue in Soluene-350 tissue solubilizer (Packard).
Lauric acid was extracted from whole fish by matrix solidphase dispersion extraction and extraction efficiency was determined by extraction from
spiked fish tissue (unexposed fish, 0.3–0.6 g wet weight). Fractions were analyzed by GC–mass spectrometry (MS) on a Fisons Instruments MD800 bench-top GC–MS. Additionally, radioactivity recovered in each fraction was determined for some samples.

- Sampling intervals/frequency for test medium samples:
Concentrations of laurate in all the test media were determined frequently throughout the test (n=21; one sample per test concentration on each sampling occasion) using LSC. A limited number of samples (three to six per concentration) were also centrifuged to estimate crudely the water-soluble fraction. Lauric acid was extracted (n=16, one sample from each concentration with surviving fish on four occasions) from the water samples to determine if biodegradation products significantly contributed to total radioactivity. Samples were analyzed by GC–FID using a Perkin Elmer GC 8700 chromatograph.

- Sample storage conditions before analysis: At test termination (day 28), fish were anesthesied, frozen and stored at - 20 °C for one week until tissues were analyzed.

Vehicle:

yes

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

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stocks of sodium laurate were prepared daily by warming (above the Kraft point of 35–40ºC) equimolar quantities of sodium hydroxide and lauric acid in distilled water in a water bath to keep the soap in solution (stock solutions of 5.7, 3.2, 1.82, 1.03, and 0.57 g/L). Radiolabeled lauric acid in dimethyl sulfoxide (DMSO) was added to each stock (final concentration, 0.5 ml DMSO/L) to give specific activities of 0.57, 1.02, 1.79, 3.16, and 5.71 mCi/g, respectively. The concentrations of sodium laurate were measured in the stocks on six occasions during the experiment (n=25; concentrations 99 ± 2% of nominal) by liquid scintillation counting (LSC).
- Controls: Yes, carbon-filtered tap water containing the same amount of DMSO of the stock solutions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethyl sulfoxide (DMSO)

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Age at study initiation (mean and range, SD): approx. 2 months old
- Weight at study initiation (mean and range, SD): 68.9 mg ± 9.8 mg
- Weight at termination (mean and range, SD): 111.3±7.8 mg
- Feeding during test: Yes. Feeding levels were adjusted for mortalities that occurred during the test. Fish were not fed on day 13 and were reweighted on day 14 to recalculate the food ratio. Moreover, fish were not fed on day 27 and reweight on day 28 (test termination).
Before feeding, surface scum (precipitated calcium laurate) was removed to minimize uptake of laurate via dietary sources.
- Food type: Fish food (Tetramin) and Artemia (each one daily)
- Amount: 2% of wet weight during the week, 4% of wet weight in weekends
- Frequency: During the week: fish food (Tetramint) and Artemia, each one daily. During the weekend: Fish food (Tetramint) once.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Hardness:

96.5 ± 4.5 mg/L CaCO3

Test temperature:

21.4 ± 0.5 ºC

pH:

7.6 ± 0.2

Dissolved oxygen:

8 ± 0.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 5 Liter
- Type (delete if not applicable): open
- Aeration: flow -though
- Type of flow-through: proportional diluter
- Renewal rate of test solution (frequency/flow rate): 170 -180 mL/min.
- No. of organisms per vessel: 16
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Holding medium different from test medium: no
- Intervals of water quality measurement: yes
- Intervals of test medium replacement: every 28 min.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: no details
- Results used to determine the conditions for the definitive study: The concentration range was selected after a review of the published acute toxicity values to fish for sodium laurate (11 and 63 mg/L [1]; .27 mg/L, Unilever Research, unpublished results).

Nominal and measured concentrations:

Nominal concentrations: 0, 2.0, 3.6, 6.4, 11.2 and 20.0 mg/L
Measured concentrations: 0, 2.2, 3.7, 6.6, 12.9 and 20.1 mg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Bioconcentration derived from the body burdens and average test media concentrations.

Key result

Conc. / dose:

2 mg/L

Type:

BCF

Value:

242 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: (BCF mean value of 243, 234 and 249 L/kg)

Key result

Conc. / dose:

3.6 mg/L

Type:

BCF

Value:

263 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: (BCF mean value of 282, 270 and 236 L/kg)

Key result

Conc. / dose:

6.4 mg/L

Type:

BCF

Value:

263 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: (BCF mean value of 238 and 288 L/kg)

Metabolites:

Analysis of fish tissue indicated that laurate was extensively metabolized. Metabolites were more hydrophobic than the parent material, which could have been incorporated into larger lipids.

Details on results:

- Mortality of test organisms: Exposure to sodium laurate caused mortalities at total concentrations of 3.6 (6%), 6.4 (14%), 11 (75%), and 20 mg/L (75%).
- Observations on body length and weight: Differences existed between the mean weights at day 0 (p 5 0.03, ANOVA), which prevented comparison of the mean weights of control fish with exposed fish after 14 and 28 d. Growth rates were higher in laurate-exposed fish than in control animals, although only at 3.6 mg/L (days 0–14) and at 2 and 3.6 mg/L (days 0–28) were these significantly different (p , 0.05). Between days 14 and 28, no significant differences existed in growth rate of control and exposed fish.
- Bioconcentration:
Increasing exposure concentrations of laurate led to increasing concentrations of lauric acid in fish tissue. After 28 d, fish exposed to 2, 3.6, and 6.4 mg/L laurate had accumulated 535, 970, and 1,735 mg/kg lauric acid (wet weight). This equates to 2.7, 4.8, and 8.7 mmol/kg.
The mean 28-d bioconcentration factor (BCF) was 255 L/kg with an SD of 22 based on intact lauric acid. Growth dilution is unlikely to have affected the BCF given the relatively slow growth rate over the 28-d study period and the rapid time for lauric acid to reach a steady state concentration.
Extraction of fish tissue with various solvents clearly showed that the radioactivity recovered from fish was not associated with intact lauric acid. In fish that died shortly after the start of the test, the bulk of the intact lauric acid eluted in the toluene fraction, but the majority of the radioactivity was found in the hexane and toluene fractions. In survivors at the end of the exposure period, a much larger proportion of the radioactivity (>70%) eluted in the hexane fraction. These findings indicate that lauric acid was rapidly metabolized to more hydrophobic molecules, especially in fish
that survived to day 28.
- Other results: Recovery of laurate concentration in media: 100 to 115% of nominal concentrations. At concentrations of 2, 3.6, and 6.4 mg/L, there was little difference between total and soluble laurate, but approximate 8.6 mg/L and 8.0 mg/L of soluble laurate were measured in the 11.2 and 20 mg/L test media, corresponding to 80 and 40% of the total laurate, respectively. Therefore, an approximate solubility limit for laurate in the test medium was 8 to 9 mg/L. During the test, precipitates were visible, particularly at the higher concentrations (11.2 and 20.0 mg/L).

Reported statistics:

Mean weights and pseudospecific growth rates were compared using nonparametric analysis of variance (Kruskal-Wallis one-way ANOVA) followed by Dunn’s test (two-tailed test, comparison against a control group), as a test for normality (Shapiro-Wilk) indicated some of the data were not normally distributed. The pseudospecific growth rate expresses the individual growth rate compared to the mean initial weight of the tank population. Statistical tests were carried out using Unistat (version 4). Toxicity data were analyzed by nonlinear interpolation or, if the data allowed, by the probit method.

Extraction of lauric acid from fish tissue:

Nominal laurate concn. (mg/L)	No.fish	Tissue weight^1(mg)	Hexane extract (µg)	Toluene extract (µg)	Ethyl acetate extract (µg)	Methanol extract (µg)	Total lauric acid (µg)	Lauric acid in fish (mg/kg)^1	Intact lauric acid^2(%^14C)	Duration of exposure (days)
0	6	557	0.2	0.7	1.9	0.1	2.9	5	-	28
	5	480	0.2	0.6	3.1	0.3	4.2	9	-	28
2.0	3	405	14.8	164	38.6	0.2	218	538	3.0	28
	4	489	17.3	174	61.5	0.3	253	517	-	28
	4	471	15.5	190	53.3	0.3	259	551	3.7	28
3.6	3	382	21.3	289	86.7	0.2	397	1040	3.4	28
	3	417	30.2	288	97.0	0.3	416	996	3.9	28
	4	458	26.7	291	80.0	0.7	399	871	-	28
6.4	3	391	42.7	493	78.0	0.4	614	1570	3.1	28
	3	336	39.9	505	91.8	0.7	638	1900	4.3	28
11.2^3	6	486	24.3	264	71.8	7.0	367	754	6.8	4 - 6
20.0^3	5	396	9.1	151	23.4	6.3	198	500	6.5	4 - 5
	7	577	33.7	521	188	14.8	749	1300	15.1	5 - 8

1) wet weight

2) percentage of measured intact lauric acid of total14C in tissues at the end of exposure

3) nonmetabolised lauric acid measured in fish died 5 to 8 days after the start of the exposure.

After 28 days exposure, the body burden of nonmotabolised lauric acid increased with increasing of exposure concentrations. Only a small fraction of the radioactivity (3–15%) recovered from fish was associated with nonmetabolized lauric acid.

Estimated lauric acid bioconcentration factors in zebrafish:

Nominal laurate concn. [mg/L]	Mean measured water conc. [mg/L]	Lauric acid body burden [mg/kg] (no. fish)	Bioconcentration factor (BCF) [L/kg]
2.0	2.21	538 (3), 517 (4), 551 (4)	243, 234, 249
3.6	3.69	1040 (3), 996 (3), 871 (4)	282, 270, 236
6.4	6.60	1570 (3), 1900 (3)	238, 288
Mean			255 ± 22

1) wet weight

Although this study was not designed to measure the bioconcentration of lauric acid, according to OECD guidance, the estimated time to reach 95% of steady state is 78 h (3.3 days, based on logKow = 4.6), is well within the duration of the test. Hence, the estimated bioconcentration factors derived from the measured body burdens of nonmetabolised lauric acid and average test media concentrations of 255 ± 22 can be applied to justify the bioconcentration of lauric acid as well.

Validity criteria fulfilled:

yes

Remarks:

(temperature variation <±2ºC, dissolved oxygen did not fall bellow 60% of saturation, concentration of test item manteined within 20%, no adverse effects on control fish)

Conclusions:

The bioconcentration factor of sodium laurate was estimated to be 255 L/kg.

Executive summary:

Bioaccumulation to zebrafish (Danio rerio) of sodium laurate was analyzed in a 28 day growth rate study. Groups of 16 juvenile fish (~2 months old) were exposed to the test item concentration of 0 (control), 2.0, 3.6, 6.4, 11.2 and 20 mg/L for 28 days under flow-through conditions (1 replicate). At test termination (day 28), fish were anesthesied, frozen and stored at - 20 °C for one week until tissues were analyzed. Bioconcentration derived from the body burdens and average test media concentrations in the tissues (lauric acid). Laurate was extensively biotransformed to metabolites, including less polar compounds, possibly triacylglycerols. At the end of the exposure period, which was considered long enough for steady state to be achieved, the bioconcentration factor of laurate was estimated to be 255 L/kg.

Description of key information

Weight of evidence: Based on the estimation of BCF by BCFBAF v3.01 model of EPI Suite v4.11 for ammonium stearate and ammonium palmitate (BCF = 70.79), and the experimental results obtained on sodium laurate (BCF = 255 L/Kg), the weight of evidence approach was applied and the BCF for the substance Fatty acids, C16-18 (even numbered), ammonium salts was determined to be 255 L/Kg based on the worst case assumption.

Key value for chemical safety assessment

BCF (aquatic species):

255 L/kg ww

Additional information

Weight of Evidence Approach (see rationale attached in IUCLID5 Section 13):

The bioaccumulation factor of ammonium stearate and ammonium palmitate, constituents of the substance Fatty acids, C16 -18 (even numbered), ammonium salts, were calculated by BCFBAF v3.01model of EPI Suite v4.11. For both constituents the BCF was estimated to be 70.79 L/kg wet-wt (log BCF from regression-based method = 1.850). A a study reported by Van Egmond R et al. (1999), the bioaccumulation to zebrafish (Danio rerio) of sodium laurate was analyzed in a 28 day growth rate study. Fish were exposed to the test item up to 20 mg/L for 28 days under flow-through conditions. At test termination, the tissues were analyzed. Laurate was extensively biotransformed to metabolites, including less polar compounds, possibly triacylglycerols. The bioconcentration factor of laurate was estimated to be 255 L/kg.

 

Based on the available data, the weight of evidence approach was applied and the bioaccumulation factor of the substance Fatty acids, C16 -18 (even numbered), ammonium salts was determined to be 255 L/kg (based on the worst case assumption).