Hexanoic acid

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

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

Adequacy of study:

key study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented publication.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 E (Bioaccumulation: Flow-through Fish Test)

Deviations:

yes

Remarks:

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

GLP compliance:

no

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 after 16 days in the top two exposure concentrations. Furthermore in the higher exposure concentrations dead fish were removed after 4 to 8 days and also sampling of tissues was performed.

- 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 liquid scintillation counting method (LSC).

- Sample storage conditions before analysis: Fish were stored at - 20 °C for one week until tissues were analyzed.

- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
1) Concentration of lauric acid in test media: Lauric acid was extracted (n =16, one sample from each concentration with surviving fish on four occasions) from the water samples (preserved with 3% formalin [v/v]; 40% formaldehyde). Five mL of sample was put in a 50- mL separating flask, and 50 mL of 1 M sulphuric acid was added. The acidified sample was then extracted by shaking three times with 5 mL of diethyl ether (5 min each time), after which the combined extracts were back-extracted with 10 ml of deionized water. The water was removed from the ether extract with anhydrous sodium sulfate, and the extract was then evaporated to dryness in a sample concentrator under nitrogen and redissolved in 500 mL of acetonitrile before analysis.
2) Total radioactivity in fish surviving was determined by dissolving the tissue in Soluene-350 tissue solubilizer (Packard). Two milliliters of Soluene-350 was added to each scintillation vial, which contained one fish. Vials were incubated at 50°C for 3 h or left at room temperature overnight. After cooling, sample coloration was reduced by addition of 50 or 300 µL of H2O2. Lauric acid was extracted from whole fish by matrix solidphase dispersion extraction.

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 35–40°C) 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.
- Controls: yes, only vehicle control
- Chemical name of vehicle: dimethyl sulfoxide (DMSO) was used for the radiolabeled lauric acid
- Concentration of vehicle in test medium: final concentration 0.5 µL/L
- Evidence of undissolved material: No undissolved material observed in the stocks

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Age at study initiation: approx. 2 months old
- Length at study initiation: not reported
- Weight at study initiation: 68.9 mg ± 9.8 mg
- Weight at termination: 111.3 ± 7.8 mg
- Method of breeding: feeding based on body weight, before feeding, surface scum (precipitated calcium laurate) was removed to minimize uptake of laurate via dietary sources
- Health status: healthy
- Feeding during test: yes, Fish were fed during the week and 4% of their body wet weight on weekends. Feeding levels were adjusted for mortalities that occurred during the test. To recalculate the food ratio for each exposure vessel, fish were not fed on day 13, as well not on day 27 (a day before the test termination).
- Food type: Tetramint and Artemia
- Amount: 2% of wet weight during the week, 4% of wet weight in weekends
- Frequency: during the week the fish was feed once daily with each type of food, respectively, at weekend once only with Tetramint.

ACCLIMATION
- Acclimation period: not stated

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

0 d

Hardness:

96.5 ± 4.5 mg/L CaCO3

Test temperature:

21.5 ± 0.5 ℃

pH:

7.6 ± 0.2

Dissolved oxygen:

8 ± 0.7

TOC:

not specified

Salinity:

not specified

Details on test conditions:

TEST SYSTEM
- Test vessel: 5 Liter
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: not reported
- 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
- Biomass loading rate: no data

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
- Light intensity: no data

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: no details
- Results used to determine the conditions for the definitive study: acute toxicity to fish for sodiun laurate was 11 - 63 mg/L, published data

Nominal and measured concentrations:

nominal laurate concentrations: 0, 2.0, 3.6, 6.4, 11.2 and 20.0 mg/L
measured laurate 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 was derived based on the measured laurate concentration in media (mg/L) and the body burden of nonmetabolised lauric acid (mg/kg wet weight).

Type:

BCF

Value:

234 - 249 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:2.0 mg/L

Type:

BCF

Value:

236 - 282 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:3.6 mg/L

Type:

BCF

Value:

238 - 288 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Conc.in environment / dose:6.4 mg/L

Type:

BCF

Value:

255 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: average

Metabolites:

less polar compounds, possibly triacylglycerols

Details on results:

- Mortality of test organisms: yes, 6% at 3.6 mg/L, 14% at 6.4 mg/L and 75% at total concentrations of 11 and 20 mg/L. The estimated 4-, 8-, 15-,
and 28-d median lethal concentrations (LC50s) were 20, 12, 9.9 and 9.8 mg/L, respectively.
- Behavioural abnormalities: no reported
- Observations on body length and weight: see table 3 showed below
- Organ specific bioaccumulation: no determined
- Mortality and/or behavioural abnormalities of control: No mortality and behavioural abnormalities observed.
- Loss of test substance during test period: no loss

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. For the pseudospecific growth rate, 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.

1) Exposure concentrations and the estimated water solubility of laurate in the test media   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).

 

No shorter-chain carboxylic acids (<12 carbons) were observed by GC–FID, indicating that the test substance in aqueous media was present as intact sodium laurate throughout the exposure period.

2) Tissue analyses

 

Table 1. 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.

 

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. Authors suggested that fatty acid having also a hydrophobic alkyl chain and a hydrophilic head group, the metabolic process involving v- and b-oxidation that leads to progressive shortening of the alkyl chain with subsequent excretion of a short-chain derivative, similar to anionic surfactants, such as sodium dodecyl sulphate (SDS). Although it is unclear whether the enzymes involved in fatty acid esterification are indicative, although in rainbow trout it was found that the rate of palmitic acid esterification by adipose tissue increased with increasing dietary lipid. In the present study, a small percentage of radioactivity could not be eluted with even the most polar solvent, indicating the label may also have become incorporated into water-soluble structures such as proteins or carbohydrates. In addition, radiolabeled TLC analysis indicated that the bulk of the radioactivity was associated with substances other than lauric acid, possibly triacylglycerols. This suggests that lauric acid could have been used as an energy source via b-oxidation but also could have entered an anabolic pathway, leading to their incorporation into larger lipids.

3) Bioconcentration factor

Table 2. Estimated lauric acid bioconcentration factors in zebrafish

Nominal laurate concn. [mg/L]	Mean measured water conc. [mg/L]	Lauric acid body burden [mg/kg]^1 (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
Average			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.

4) Effects on survival and growth

Exposure to sodium laurate caused mortalities at total concentrations of 3.6 (6%), 6.4 (14%), 11 (75%), and 20 mg/L

(75%). The estimated 4-, 8-, 15-, and 28-d median lethal concentrations (LC50s) were 20, 12, 9.9, and 9.8 mg/L, respectively. Based on soluble concentrations of laurate, LC50 values for the above time intervals were 10, 7.6, 7.6, and 7.6 mg/L. The 28-d NOEC for mortality was 2 mg/L.

 

Control fish increased in weight by 18 and 47% after 14 and 28 d, respectively. Fish exposed to the lower concentrations (2–6.4 mg/L) all increased in weight, and this increase was greater than that seen in control animals. It is known that fatty acids formed de novo and those originating from dietary lipids are easily esterified into neutral lipids and phospholipids. Once absorbed, laurate could perhaps be used as an additional food source by these fish, resulting in slightly enhanced growth compared to that of the control fish. The 28-d NOEC for growth was 6.4 mg/L.

Key value for chemical safety assessment

BCF (aquatic species):

255 L/kg ww

Additional information

A fish bioaccumulation study is available for the analogue substance C12 fatty acid-sodium laurate which showed negligible evidence of bioaccumulation potential in fish tissues with an estimated BCF of 255 L/kg after 28 days exposure.

As fatty acids are naturally stored in the form of triacylglycerols primarily within fat tissue until they are used for energy production (fat storage tactic), it is therefore considered that there will be no risk to aquatic organisms from potential bioconcentration/biomagnification of fatty acids.