Fatty acids, montan-wax, stearyl esters

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Remarks:

Toxicity control of ready biodegradability test.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Mar - 27 Apr 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 301 F

Version / remarks:

17 Jul 1992

GLP compliance:

yes (incl. QA statement)

Remarks:

Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, Japan (22 Dec 2016)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Silica gel 60 (0.5 g per test vessel filled with 300 mL test solution)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The toxicity control was prepared by accurately weighing and adding 9 mg test item to the test vessel, which was then heated at 100 °C for 20 min in an oven. Then, 0.5 g Silica gel 60 was added to the test vessel and stirred for 30 min at room temperature. Subsequently, 30 mg of the reference compound sodium benzoate and 3.20 mL activated sludge were added to the test vessels, which was then filled to 300 mL with mineral medium. The final concentration of test item was 30 mg/L and the final concentration of sodium benzoate was 100 mg/L.
- Chemical name of vehicle Silica gel 60
- Concentration of vehicle in test medium: 0.5 g in 300 mL (= 500 mg/0.3 L = 1666.66 mg/L)
- Evidence of undissolved material: No, the state of the test item could not be confirmed because of the presence of silica gel. The test solution was colorless.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Kurume central sewage treatment center (Kurume, Fukuoka, Japan), 30 Mar 2018
- Method of cultivation: After collection, the activated sludge was aerated at about 22 °C for about 3.5 h until use in the test.
- Initial suspended solids concentration: 30 mg/L

Test type:

static

Water media type:

freshwater

Total exposure duration:

28 d

Test temperature:

22 ± 1 °C

pH:

7.4 (mineral medium)

Nominal and measured concentrations:

30 mg/L test item and 100 mg/L sodium benzoate (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel with closed system oxygen consumption measuring apparatus
- No. of vessels per concentration (replicates): 2
- No. of vessels per control blank (replicates): 2
- No. of vessels per vehicle control (replicates): 0
- Sludge concentration at test start: 30 mg/L suspended solids

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Mineral medium prepared with purified water (Japanese Pharmacopoeia, Takasugi Pharmaceutical)

OTHER TEST CONDITIONS
- Adjustment of pH: The mineral medium was adjusted to 7.4
- Photoperiod: Incubation in the dark
- Other: Each test solution was stirred with a stirrer and placed in a temperature controlled bath.

EFFECT PARAMETERS MEASURED:
- Biological oxygen demand: Continuous measurement

Reference substance (positive control):

yes

Remarks:

sodium benzoate

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biological oxygen demand

Remarks on result:

other:

Remarks:

The percentage biodegradation was 58% after 14 d.

Details on results:

- Any observations that might cause a difference between measured and nominal values: No, the state of the test item could not be confirmed because of the presence of silica gel. The test solution was colorless.
- Effect concentrations exceeding solubility of substance in test medium: Yes
- Adsorption (e.g. of test material to the walls of the test container): No
- Blank controls oxygen uptake rate: 18 mg/L
- Coefficient of variation of oxygen uptake rate in control replicates: 0

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 84% biodegradation of sodium benzoate after 14 d

In the toxicity control, the percentage biodegradation by BOD was 58% after 14 days. According to the test guideline, if the percentage biodegradation is less than 25% within 14 days, the test item is toxic. Therefore, the test item can be assumed not to be inhibitory to activated sludge microorganisms at the applied concentration level of 30 mg/L.

According to the ECHA guidance R.7b (2017) the information content of a ready biodegradability test can be used to derive a NOEC when a toxicity control has been included that shows good degradation of a positive control substance (e.g. sodium benzoate) in the presence of the test substance.

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

The average biodegradation of the reference item sodium benzoate in the toxicity control was 58% by BOD after 14 d. Therefore, the test item is not inhibitory to activated sludge microorganisms at the applied concentration level of 30 mg/L.

Description of key information

NOEC ≥ 30 mg/L (nominal, OECD 301 F)

Additional information

The microbial toxicity of Fatty acids, montan-wax, stearyl esters (EC No. 269-642-9, CAS 68308-30-5) was inferred from the toxicity control of a standard ready biodegradability test conducted according to OECD testing guideline 301 F and GLP, as recommended by the Guidance document “Chapter R.7b Endpoint specific Guidance on Information Requirements and Chemical Safety Assessment (ECHA, 2018) in absence of a microbial toxicity test according to OECD 209.

In the manometric respirometry test, the toxicity control was prepared by inoculating 30 mg/L test item with (0.5 g/300 mL) silica gel 60, 30 mg/L reference item (sodium benzoate) and 30 mg/L activated sludge (suspended solids) in a final volume of 300 mL mineral medium for 28 d in the dark. Biodegradation was followed by continuously measuring the biochemical oxygen demand with a closed system oxygen consumption measuring apparatus.

After 14 d, degradation of the reference item in the toxicity control was 58%and thus above the criterion for inhibition effects to the inoculum (< 25% degradation in 14 d). Based on this result, the test item had no toxic effect on the inoculum and a NOEC (14 d) of ≥ 30.0 mg/L was derived according to the Guidance document “Chapter R.7b Endpoint specific Guidance on Information Requirements and Chemical Safety Assessment” (ECHA, 2017).