Reaction mass of pentyl ester butanoic acid and 2-methylbutyl ester butanoic acid

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 October 2019 - 05 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

adopted 17th July, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the
Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Reaction mass of 2-methylbutyl butyrate and pentyl butyrate
EC number: 908-712-1
Description: Clear colorless to pale yellow liquid
Purity: 99.8%
Storage conditions: Controlled room temperature (15-25 °C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials will be applied to ensure personnel health and safety.

Oxygen conditions:

not specified

Inoculum or test system:

sewage, domestic (adaptation not specified)

Details on inoculum:

The inoculum: Secondary effluent, microorganisms from a domestic waste water treatment plant.

Origin: The secondary effluent will be supplied from one of the sewage plant for domestic sewage in Veszprém county, Hungary. Details of the source of inoculum to be used in the study will be specified in the appendix of the study plan and documented in the data.

Conditioning: After collection of a fresh sample of secondary effluent will be allowed to settle for approximately one hour or filtrated through a coarse filter paper. Thereafter the decanted effluent or filtrate will be aerated until use.

Duration of test (contact time):

28 d

Initial conc.:

2.4 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

APPARATUS
Type and Size: BOD bottles (300 ml) with special neck and glass stoppers.
Identification: Each test flasks was uniquely identified with at least study code, test group, days of measurement and replicate number.

TEST CONDITIONS
- The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) which was maintained at a temperature of 22 ± 2°C in accordance with the guideline. The test flasks were placed into an incubator and kept at 21.6 - 23.8 °C, in the dark. Temperature was measured at least each day of oxygen consumption measurement, but on each working day normally.
- The oxygen concentration of test water was 8.7 mg/L at the start of the test.
- The pH value of the test water was checked prior to study start. The pH of the test water was 7.24.

The test conditions were measured with suitable instruments and documented in the raw data.

STOCK SOLUTIONS
Stock solutions were prepared using analytical grade salts* and deionised water in the following ratios:
a) Solution: KH2PO4 2.125 g / 250 mL water
K2HPO4 5.4375 g / 250 mL water
Na2HPO4 x 12H2O 16.795 g / 250 mL water
NH4Cl 0.125 g / 250 mL water
b) Solution: MgSO4 x 7 H2O 5.625 g / 250 mL water
c) Solution: CaCl2 x 2 H2O 9.10 g / 250 mL water
d) Solution: FeCl3 x 6 H2O 0.25 g / 1L water


RATIO OF INGREDIENTS
A 1 mL aliquot of each of the stock solutions a) - d) were combined and made up to a final volume of 1000 mL with deionised water. The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature.The dissolved oxygen concentration was 8.7 mg/L at about 22 °C.

PREPARATION OF THE TEST SOLUTIONS

Before the preparation of the respective test solutions with Test Item an aqueous stock solution of the test item with a concentration of 24.0 mg/L was prepared and diluted accordingly (using ultrasonic bath for 10 minutes). The test item concentration in the test solutions was 2.4 mg/L.
The chosen test item concentration was based on theoretical oxygen demand (ThOD) of 2.53 mg O2/mg test item (calculated according to equation given in the guidelines) and on the performed 14-d preliminary test.

The components were applied in the amounts/volumes in the test flasks:

1.) Test Item (flasks 1a and 1b)
Based on the theoretical oxygen demand (ThOD) of 2.53 mg O2/mg test item, test item stock solution* (corresponding to 8.16 mg of Test Item) was thoroughly mixed into 3.40 litres of aqueous test medium (corresponding to 2.4 mg/L test item, with a ThOD of about 6.072 mg O2/L).
* The concentration of the stock solution was: 24.0 mg/L.

2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)
Based on the theoretical oxygen demand (ThOD) of Sodium benzoate (1.67 mg O2 per mg; details on calculation are given in the guidelines), stock solution* corresponding to 12.168 mg of Sodium benzoate was mixed into 3.38 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThOD of about 6.012 mg O2/L).
* The concentration of the stock solution was: 360 mg/L.

3.) Inoculum Control (flasks 3a and 3b)
Only filtered inoculum was added to 3.38 litres of aqueous test medium.

4.) Toxicity Control (flasks 4a and 4b)
The Test Item* (340.0 mL) and the reference item stock solutions* (34.0 mL) were mixed into 3.40 litres of aqueous test medium corresponding to 2.4 mg/L test item (ThOD of 6.072 mg O2/L) and 3.6 mg/L reference item (ThOD of 6.012 mg O2/L).
* The concentration of the stock solution was: 24.0 mg/L.
** The concentration of the reference item stock solution was: 360 mg/L.

Microbial inoculum (0.5 mL per litre) was added to each preparation bottle.

PREPARATION OF TEST FLASKS
Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stoppers.

THE TEST BOTTLES
The number of test bottles were as follows:
- at least 10 bottles containing the test item and inoculum
- at least 10 bottles containing the reference item and inoculum (procedure control)
- at least 10 bottles containing only inoculum (inoculum control)
- at least 10 bottles containing the test item, reference item and inoculum (toxicity control)

Reference substance:

benzoic acid, sodium salt

Preliminary study:

A 14-day preliminary test was conducted to determine the approximate biodegradation of the test item. The chosen test item concentration was based on the theoretical oxygen demand (ThOD) of 2.53 mg O2/mg test item (calculated according to equation given in the guidelines).

Test performance:

The study met the validity criteria.

Parameter:

% degradation (O2 consumption)

Remarks:

Test 1a

Value:

26.4

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

Test 1b

Value:

26.4

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Remarks:

Mean

Value:

26.4

Sampling time:

28 d

Parameter:

ThOD

Value:

2.53 mg O2/g test mat.

Results with reference substance:

14-day preliminary test with reference item:
Test 2a - 73.3 % degradation at 28 d
Test 2b - 75.0 % degradation at 28 d
Mean - 74.2 % degradation at 28 d

14-day preliminary test with toxicity control:
Test 4a - 38.9 % degradation at 28 d
Test 4b - 41.3 % degradation at 28 d
Mean - 40.1 % degradation at 28 d

 The % biodegradation results are summarised in the following table:

Treatment	Concentration [mg/L]	Flask no.	Percent of biodegradation after n days of exposure
			7.0	14.0	21.0	28.0
Test item	2.4	1a	-1.6	6.6	29.7	26.4
		1b	0.0	9.9	29.7	26.4
		mean	-0.8	8.2	29.7	26.4
Reference item	3.6	2a	63.3	68.3	75.0	73.3
		2b	65.0	70.0	73.3	75.0
		mean	64.2	69.2	74.2	74.2
Toxicity control	Test item: 2.4 Reference item: 3.6	4a	31.8	34.2	39.7	38.9
		4b	31.8	34.2	42.1	41.3
		mean	31.8	34.2	40.9	40.1

The dissolved oxygen concentration, oxygen depletion and BOD results are appended in the 'background material' section

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item (Reaction mass of 2-methylbutyl butyrate and pentyl butyrate) was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of Reaction mass of 2-methylbutyl butyrate and pentyl butyrate reached a mean of 26.4 % after 28 days based on the ThOD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore, the test item is considered not readily biodegradable.

The reference item Sodium benzoate was sufficiently degraded to a mean of 69.2 % after 14 days, and to a mean of 74.2 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 34.2 % biodegradation was noted within 14 days and 40.1 % biodegradation after 28 days of incubation.

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 2.4 mg/L on the secondary effluent
microorganisms because degradation was >25 % within 14 days.

The study met the validity criteria.

The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.

Based on the results of this study, the test item is considered not readily biodegradable.

Executive summary:

The test item (Reaction mass of 2-methylbutyl butyrate and pentyl butyrate) was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of Reaction mass of 2 -methylbutyl butyrate and pentyl butyrate reached a mean of 26.4 % after 28 days based on the ThOD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore, the test item is considered not readily biodegradable.

The reference item Sodium benzoate was sufficiently degraded to a mean of 69.2 % after 14 days, and to a mean of 74.2 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 34.2 % biodegradation was noted within 14 days and 40.1 % biodegradation after 28 days of incubation.

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 2.4 mg/L on the secondary effluent microorganisms because degradation was >25 % within 14 days.

The study met the validity criteria.

The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.

Based on the results of this study, the test item is considered not readily biodegradable.

Description of key information

Study conducted to recognised testing guidelines with GLP certification

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

Based on the results of this study, the test item is considered not readily biodegradable.

The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.