Octanoic acid

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

13 Oct - 17 Nov 1978

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Inconsistent results

Qualifier:

no guideline followed

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Sample was weighed in clean, dried and tared one milliliter beakers and then introduced into the CAD flask or BOD dilution bottle.

Test organisms (species):

other: "biological seed"

Test type:

not specified

Water media type:

not specified

Limit test:

no

Remarks on exposure duration:

no data

Reference substance (positive control):

no

Details on results:

No results are given in the study report. It was considered to be invalid, since the measured oxygen demand was higher than the theoretical oxygen demand.

Reference 2

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

other information

Study period:

not reported

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

Bacillus subtilis was tested with different substances and the optical densitiy measured after 1 h to calculate the inhibition.

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Inoculum was placed into plastic tubes or Erlenmeyer flasks which already contained the test substance
- Controls: 2 with solvent, 2 with distilled water
- Chemical name of vehicle: Ethanol, dimethylsulfoxide, sodium hydroxide or distilled water

Test organisms (species):

Bacillus subtilis

Details on inoculum:

- Pretreatment: Plated with tryptose blood agar base (33 g/L) were inoculated from single colonies of Bacillus subtilis and incubated for 7 h. Afterwards the cells were supended and diluted at different low titers into three flasks containing the MCV medium and shaken at room temperature overnight. If the cultures had not grown overnight beyond an optical density at 600 nm (OD600) of 1.0 it was not used. The cultures reaching 1.0 were utilised for the test.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

60 min

pH:

7.2, adjusted with KOH

Details on test conditions:

TEST SYSTEM
- Test vessel: Plastic tubes or Erlenmeyer flasks
- Aeration: Tubes were aerated.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: MCV medium contains 1.4% K2HPO4 , 0.6% KH2PO4, 0.2% MgSO4 * 7 H2O, 0.1% sodium citrate, 25 µg/mL L-tryptophan, 10 µg/mL L-methionine and 1 % vitamin-free casein hydrolysate

OTHER TEST CONDITIONS
- Adjustment of pH: with KOH to 7.2

EFFECT PARAMETERS MEASURED: optical density at 600 nm after 60 min in the plastic tubes and after 15 or 20 min in the Erlenmeyer flasks

Reference substance (positive control):

no

Duration:

60 min

Dose descriptor:

EC50

Effect conc.:

1.8 mmol/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Reference 3

Endpoint:

toxicity to microorganisms, other

Remarks:

Pseudomonas putida, respiration inhibition, 18 h

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Mar - 16 Mar 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given (comparable to guideline)

Qualifier:

equivalent or similar to guideline

Guideline:

other: ISO 10712

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution containing 680 mg prod./L and an oversaturated solution containing 1360 mg/L were prepared using sterile Milli-Q water which was mixed with an magnetic stirrer for 10 min. The pH was adjusted to 6.8 - 7.2 with NaOH.
- Controls: 10 with inoculum without test substance (series B) to determine turbidity and for each test concentration 1 control without inoculum to determine occuring colouration

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: obtained from RIVM, Bilthoven, Netherlands
- Method of cultivation: kept in agar plant tubes with a nutrient stock solution, incubated at 25 °C
- Preparation of inoculum for exposure: 7 days old stock cultures were inoculated in a fluid nutrient medium in Erlenmeyer flasks for 16 - 20 h at 25 °C. Subsequently the extinction of the monochromatic radiation was measured at 436 nm in a 10 mm layer of the bacterial suspension by photoelectric measurement. On basis of the measured values the turbidity was adjusted with sterile saline to the value of the Formazin standard suspension (TU/F/436 nm = 10) with which the spectrophotometer was calibrated.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

18 h

pH:

6.8 - 7.2, adjusted with NaOH

Nominal and measured concentrations:

nominal: 0.266, 0.531, 1.063, 2.125, 4.25, 8.50, 17.00, 34.00, 68.00, 136.00, 272.00, 544.00, 1088.00 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks stoppered with aluminum caps containing 100 mL test medium
- No. of vessels per concentration (replicates): 3 + 1 control (please refer to "details of test solution" above)
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile Milli-Q water (Milipore Corp., Bedford, USA)

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to 6.8 - 7.2 with NaOH solution

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : turbidity after 16 -20 h

Reference substance (positive control):

yes

Remarks:

Methanol

Duration:

18 h

Dose descriptor:

EC10

Effect conc.:

912 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mean extinction value

Results with reference substance (positive control):

- Results with reference substance valid? yes

Reported statistics and error estimates:

The toxicity threshold (TT) is determined graphically. The mean extinction values of each dilution step were plotted against the logarithm of the mean values of the test substance concentration.

Table 1: Extinction of the test substances solutions after 16 - 20 h

Concentration [mg prod./L]	Extinction at 436 nm
	Replicate I	Replicate II	Replicate III	Arithm. mean of replicates	Replicate without inoculum
0.226	0.490	0.500	0.470	0.487	0.000
0.531	0.471	0.480	0.469	0.473	0.000
1.063	0.463	0.460	0.456	0.460	0.000
2.125	0.452	0.450	0.460	0.454	0.000
4.250	0.458	0.430	0.466	0.451	0.000
8.500	0.447	0.450	0.459	0.452	0.000
17.000	0.469	0.434	0.463	0.455	0.000
34.000	0.458	0.450	0.470	0.459	0.000
68.000	0.525	0.512	0.497	0.511	0.000
136.000	0.588	0.580	0.570	0.579	0.000
272.000	0.710	0.740	0.745	0.732	0.000
544.000	0.985	0.979	0.990	0.985	0.000
1088.000	0.198	0.212	0.220	0.210	0.000

Table 2: Extinction after 16 - 20 h in the reference substance and the blank control

Concentration [mg prod./L]	Extinction at 436 nm
	Reference substance	Blank controls
4937.500	0.488	0.449
9875.000	0.440	0.440
19750.00	0.415	0.452
39500.00	0.270	0.459
79000.00	0.155	0.458
		0.449
		0.433
		0.439
		0.440
		0.438
Arithmetic mean		0.446

With respect to the historical test with the reference substance methanol, it can be concluded that the test conditions were optimal and the results are valid.

Reference 4

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

other information

Study period:

not reported

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Only short abstract available

Qualifier:

no guideline followed

Principles of method if other than guideline:

Six different bacteria were anaerobically incubated and the turbitidy measured to determine inhibition on growth.

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution was prepared by dissolving measured quantities of the test substance in absolute ethanol. First a 250 mM stock solution was prepared and then further diluted with ethanol to 200, 175, 150, 125, 100, 75, 50, 25, 10, 5 and 2.5 mM.
- Controls: Since the test compund produces culture turbidity, a dilution series with medium and test compound without inoculum were prepared.

Test organisms (species):

other: Bifidobacterium bifidum type a, Bifidobacterium bifidum type b, Bifidobacterium infantis sp infantis type a, Bifidobacterium infantis sp infantis type b, Escherichia coli, Salmonella typhimurium

Details on inoculum:

- Preparation of inoculum for exposure: The PYG medium was dispensed into a 500 mL screw-capped bottle and sterilised at 121 °C for 15 min. While cooling, 3.0 mL were delivered into metal capped culture tubes which were inoculated with 0.03 mL of the substance stock solutions (2.5 - 20 mM). The culture tubes were placed in an anaerobic cabinet and maintained at 37°C over night. Oxygen free gas (20% nitrogen in hydrogen) was supplied.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

29 h

Test temperature:

37 °C

pH:

4.5 - 6.6, the pH was measured in the highest and lowest substance concentrations.

Nominal and measured concentrations:

Nominal: 200, 175, 150, 125, 100, 75, 50, 25, 10, 5 and 2.5 mM

Details on test conditions:

TEST SYSTEM
- Test vessel: culture tubes
- No. of vessels per control (replicates): one series (2.5 - 200 mM test substance) with test substance without inoculum to determine turbidity

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The PYG media (Petone-yeast extract glucose) with 1% glucose, was prepared and dispersed anaerobically and sterilised. Anaerobic culture counts were performed on PYG-agar roll tubes.

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: turbidity after 29 h

Reference substance (positive control):

no

Duration:

29 h

Dose descriptor:

EC50

Effect conc.:

80 other: nmol

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: B. bifidum type a

Duration:

29 h

Dose descriptor:

EC50

Effect conc.:

15 other: nmol

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: B. infantis sp infantis type a

Duration:

29 h

Dose descriptor:

EC50

Effect conc.:

2.5 other: nmol

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: B. infantis sp infantis type b

Duration:

29 h

Dose descriptor:

EC50

Effect conc.:

0 other: nmol

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: S. typhimurium

Details on results:

No 50 % inhibition on bacterial growth was achieved on E. coli and B. bifidum type b.

Description of key information

EC10 (18 h) = 912 mg/L (ISO 10712)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

912 mg/L

Additional information

One reliable study, investigating the toxicity of the substance to Pseudomonas putida, is available for octanoic acid. This cell multiplication inhibition test (1988) was conducted similarly to ISO 10712. The test organism was exposed to a wide range of nominal concentrations (0.266 - 1088.00 mg/L). The study determined an EC10 (18h) of 912 mg/L (nominal).

A further study report (1978) is available with biological seed as the test organisms, but was rated unreliable (RL3) due to inconsistent results and poor reporting.

Supportive information is withdrawn from short abstracts of two publications. In the first study (Freese, 1979) conducted with Bacillus subtilis, a EC50 of 0.25 mmol/L was obtained after 60 min. In the second study (Powell and May, 1981) with six different bacterial strains incubated anaerobically, different sensitivity of the various organisms to decanoic acid were observed. However, due to limited documentation and consequently limited information, these two literature studies were rated as not assignable.