2-methylpentyl 2-hydroxybenzoate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10-08-2020 to 10-10-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for the Testing of Chemicals, Effects on Degradation and Accumulation, Version 2, 301F Manometric Respirometry Test, China Environmental Press, China (2013)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Fresh activated sludge used in the test was collected from Suzhou Drainage Company Ltd., domestic waste sewage treatment plant : Suzhou city, The People’s Republic of China. The batch number of the inoculum is included in the full study report. The activated sludge of waste water treatment plant is recommended by test guideline.
- Storage conditions: See pretreatment field.
- Storage length: ca. 1 week; i.e. maximum 7 days from sampling to test initiation.
- Preparation of inoculum for exposure: The freshly sampled activated sludge was collected from the aeration basin and kept aerobic during transport. See pretreatment field for further information.
- Pretreatment: The activated sludge was filtered through fine filter sieve to remove the coarse particles, washed using the test medium and settled for ca. 10 minutes, after which the supernatant was discarded. The sludge was resuspended in test medium, repeated 3 times. An amount of 10 mL washed activated sludge was sampled (4 replicates) and dried at 105 °C for 2 hours to determine the concentration of suspended solid (SS}, which was determined to be 6.6 g SS/L. The sludge was diluted with test medium to a concentration of 4.1 g SS/L and aerated for 2 days at 22 ± 2 °C until required. Before test, the activated sludge suspended solid was determined to be 4.1 g SS/L. From this result, the added amount of activated sludge was calculated.
- Concentration of sludge: The inoculum was added into the test suspensions, the procedure control, the inoculum blank and toxicity control to give a final concentration of 30.0 mg SS/L.
- Water filtered: Yes.
- Type and size of filter used, if any: Fine filter, not otherwise specified. The water for preparing medium and solution was made by the Water Purifier (to analytical grade water, i.e. organic carbon concentration < or = 2 ppb). For each series of tests, the same synthetic media was used (details reported in the full study report).

Duration of test (contact time):

28 d

Initial conc.:

ca. 13.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Solution A [KH2PO4: 8.5005 g; K2HPO4.3H2O: 21.7508 g; Na2HPO4.12H2O: 67.1502 g; NH4Cl: 0.5005 g – made up to 1000 mL in purified water The pH value was measured to be 7.67 then adjusted to 7.53 with HCI (aq)]; Solution B [MgSO4.7H2O: 5.551 g made up to 250 mL water] ; Solution C [CaCl2: 9.1076 g made up to 250 mL water] ; Solution D [FeCl3,6H2O: 0.0624 g made up to 250 mL water with one drop HCl (aq)].
8 Litres test medium was prepared : 80 mL of stock solution A was firstly mixed with purified water, and then 8.000 mL of stock solution B, C and D were added, mixed thoroughly and made up to 8 Litres with purified water. The pH value was measured to be 7.67. Then the pH adjusted to 7.57 with HCl(aq).
- Solubilising agent (type and concentration if used): None.
- Test temperature: 22 ±1 °C (actual: 21.8 – 22.2 °C)
- pH: 7.57 (medium) and pH 7.75-7.93 at the end of the test.
- pH adjusted: No (not adjusted at end of test, since the pH was not outside the range of 6.0 to 8.5).
- Aeration of dilution water: Not reported
- Suspended solids concentration: The inoculum was added into the test suspensions, the procedure control, the inoculum blank and toxicity control to give a final concentration of 30.0 mg SS/L.
- Continuous darkness: No. Test conducted under diffuse light

TEST SYSTEM
- Culturing apparatus: Flasks with continuous stirring (magnetic stirrer)
- Number of culture flasks/concentration: In duplicate (Inoculum blank); duplicate (test system); single flasks (procedure control and toxicity control)
- Method used to create aerobic conditions: Screw sealed flasks with sensor head/CO2 trap.
- Method used to create anaerobic conditions: Not applicable.
- Measuring equipment: The respirometer used during this study is : BOD Analyzer: Aerobic respirometer, England CES, No.: TTE20170918
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: Not applicable.
- Details of trap for CO2 and volatile organics if used: 5mL of 2 mol/L sodium hydroxide was added to CO2 absorption traps.
- Other: Not applicable.

SAMPLING
- Sampling frequency: Daily (BOD)
- Sampling method: Respirometry measuring the oxygen uptake of the test medium.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. See table 1.
- Abiotic sterile control: No.
- Toxicity control: Yes.
- Other: Positive reference control ('Procedure control' ; Sodium Benzoate).

Reference substance:

benzoic acid, sodium salt

Remarks:

ca. 15.7 mg/L

Test performance:

(1) The oxygen uptake of the inoculum blanks were 36 mg/L, which was less than 60 mg/L in 28-d.
(2) At the end of the test, the pH values of each test vessel were 7.75-7.93, which was within the range of 6.0-8.5.
(3) The difference of extremes of replicate values of the removal of the test substance at the plateau, at end of the test or at the end of the 10-d window was less than 20% (actual day 14: 61.6% and 69.2% and actual day-28: 74.8% and 87.3%).
(4) The percentage degradation of the procedure control and the toxicity control were 100% and 72.2%, which had reached the pass levels of 60% and 25% of ThOD on 14d.
All validity criteria were considered to be met.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

mean (n=2)

Value:

81.1

Sampling time:

28 d

Remarks on result:

other: n=2; 10-d window met

Details on results:

The test item was degraded based on BOD: actual mean = 81.1% (n=2 ; 74.8% and 87.3%) at day 28
At the end of the 10-day window the test item was degraded by 61.6% and 69.2%. The 10-day window criterion was met.

Results with reference substance:

Degradation of sodium benzoate exceeded 100% after 14 days: the activity of the inoculum was thus verified (validity criterion).

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item mean biodegradation in duplicate was 81.1 % at day 28 based on BOD (10-day window met).

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301F and China Guidelines for the Testing of Chemicals, Effects on Degradation and Accumulation, Version 2, 301F Manometric Respirometry Test (2013) under GLP. The test substance, at a concentration of 30 mg/L was exposed to activated sewage sludge micro-organisms obtained from the Suzhou Drainage Company Ltd., domestic waste sewage treatment plant of Suzhou city, China, with culture medium in sealed culture vessels in diffuse light at 22°C ± 1°C for 28 days. The sludge was diluted in flasks to 30.0 mg SS/L within the test system and in the negative and toxicity controls. The degradation of the test item was assessed by daily measurement of oxygen consumption from days 0 and 28 using an respirometer system. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The oxygen uptake of the inoculum blank was 36.0 mg/L, which was less than 60 mg/L in 28-days. At the end of the test, the pH values of each test vessel were within the range of 6.0-8.5 (actual pH: 7.75 – 7.93). The difference of extremes of replicate values of the removal of the test substance at the plateau, at end of the test or at the end of the 10-d window was less than 20% (actual day 14: 61.6% and 69.2% and actual day-28: 74.8% and 87.3%). The percentage degradation of the procedure control and the toxicity control were 100% and 72.2%, which had reached the pass levels of 60% and 25% of ThOD on 14d. The toxicity test attained 60 % degradation after 28 days thereby confirming that the test substance was not inhibitory to the sewage treatment micro-organisms used in the study. Sodium Benzoate attained 100% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The test system therefore met the validation criteria of the guideline. The mean biodegradation for duplicate test flasks at 3 days for the test item was 5.6%. The mean biodegradation for duplicate test flasks at 4 days and 14 days for the test flasks was 35.0% and 65.4%, respectively. The mean biodegradation at 28 days for the test item was 81.1% (the 10-day window was met). Under the conditions of the study, test item is considered as readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-05-2020 to 18-08-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan guidelines : "II: Method for Testing the Biodegradability of Chemical Substances by Microorganisms (301F)"

Version / remarks:

stipulated in the "Testing Methods for New Chemical Substances" of Japanese Chemical Substances Control Law (March 31, 2011, Yakushokuhatsu 0331 No. 7; Heisei 23.03.29 Seikyoku No. 5; Kampokihatsu No. 110331009; latest revision, July 1, 2019, Yakuseihatsu 0701 No. 1; 20190619 Seikyoku No. 2; Kampokihatsu No. 1907011) and other citations within the full study report.

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge was obtained : from an aeration tank of a sewage treatment plant treating predominantly domestic sewage in Kurume-shi, Fukuoka, Japan. The sample was collected the day before test item exposure. The activated sludge of a (domestic) waste water treatment plant is recommended by test guideline.
- Storage conditions: See pretreatment field.
- Storage length: ca. 1 day from sampling to test initiation. The activated sludge was aerated at approximately 22°C until use for the test.
- Preparation of inoculum for exposure: See above.
- Pretreatment: Additive amount of the activated sludge into the test vessel was 3.26 mL on the basis of the concentration of suspended solid in the activated sludge which was determined by the following method: Method In accordance with Japanese Industrial Standards (JIS) K 0102:2019 : Section 14.1 (day before test initiation) : suspended solid concentration of activated sludge : 2760 mg/L , then diluted in test vessel to final test vessel suspended solid concentration : 30.0 mg/L).
- Concentration of sludge: The inoculum was added into the test suspensions, the procedure control, the inoculum blank and toxicity control to give a final concentration of 30.0 mg/L (dry weight).
- Water filtered: Not reported.
- Type and size of filter used, if any: Not reported.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

i.e. Biological Oxygen Demand (BOD)

Parameter followed for biodegradation estimation:

DOC removal

Remarks:

Determination of dissolved organic carbon (DOC) was not conducted because water solubility of the test item was less than 100 mg/L.

Parameter followed for biodegradation estimation:

other: Percentage biodegradation of test item by HPLC

Details on study design:

TEST CONDITIONS
- Composition of medium: Activated sludge was obtained : from an aeration tank of a sewage treatment plant treating predominantly domestic sewage in Kurume-shi, Fukuoka, Japan. The sample was collected the day before test item exposure. The activated sludge of a (domestic) waste water treatment plant is recommended by test guideline. Additive amount of the activated sludge into the test vessel was 3.26 mL on the basis of the concentration of suspended solid in the activated sludge which was determined by the following method: Method In accordance with Japanese Industrial Standards (JIS) K 0102:2019 : Section 14.1 (day before test initiation) : suspended solid concentration of activated sludge : 2760 mg/L , then diluted in test vessel to final test vessel suspended solid concentration : 30.0 mg/L). The mineral medium (3 L) was prepared at the same proportion as the following method: 10 mL of solution A and each 1 mL of solutions B, C, and D prescribed in the test methods, were made up to 1 L with purified water (The Japanese Pharmacopoeia, Takasugi Pharmaceutical), and then the pH of this solution was adjusted to 7.4.
- Additional substrate: Not applicable.
- Solubilising agent (type and concentration if used): Not applicable.
- Test temperature: 22 ±1 °C
- pH: Blank (water + test item): not reported ; Sludge + test item: 7.4 (at end of study) ; control blank: 7.3 to 7.4
- pH adjusted: No.
- CEC (meq/100 g): Not reported.
- Aeration of dilution water: Not reported.
- Suspended solids concentration: 30 mg SS/L.
- Continuous darkness: Yes.
- Other: Not applicable.

TEST SYSTEM
- Culturing apparatus: Glass vessel (ca. 300 mL).
- Number of culture flasks/concentration: Duplicate.
- Method used to create aerobic conditions: Not applicable.
- Method used to create anaerobic conditions: Not applicable.
- Measuring equipment: See previous sections.
- Test performed in closed vessels due to significant volatility of test substance: Not reported (although suspected)
- Test performed in open system: Not applicable.
- Details of trap for CO2 and volatile organics if used: Not applicable.
- Other: Not applicable.

SAMPLING
- Sampling frequency: BOD (oxygen consumption) was monitored continuously ; minimum end of incubation for DOC, where applicable and/or HPLC for parent and transformation products analysis by HPLC (and/or further analysis, for degradants if necessary).
- Sampling method: See above.
- Sterility check if applicable: Not applicable.
- Sample storage before analysis: Not applicable.
- Other: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: Not applicable.
- Toxicity control: Yes.
- Other: Reference item control (Sodium benzoate)

STATISTICAL METHODS: Not applicable.

Reference substance:

benzoic acid, sodium salt

Remarks:

100 mg/L

Test performance:

1. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled. Therefore, the test is considered valid (Actual: 1% in BOD and 0% in HPLC)
2. The BOD of the inoculated blank control was 19 and 21 mgO2/L and < 60 mgO2/L after 28 days
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual 7.4 for test item vessels)
4. Sodium benzoate attained 93% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
5. The percentage degradation of the toxicity control (sludge + test item + sodium benzoate) was 44% which had reached the pass level 25% of ThOD on 14 days.
All validity criteria were considered to be met.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

BOD ; mean

Value:

93

Sampling time:

28 d

Remarks on result:

other: actual mean = 93% (n=2 ; 92% and 93%) at day 28

Remarks:

10-day window met

Parameter:

% degradation (DOC removal)

Remarks:

DOC ; mean

Value:

89

Sampling time:

28 d

Remarks on result:

other: actual mean = 89% (n=2 ; 88% and 91%) at day 28

Parameter:

% degradation (test mat. analysis)

Remarks:

GC ; mean

Value:

100

Sampling time:

28 d

Details on results:

The test item was degraded based on BOD: actual mean = 93% (n=2 ; 92% and 93%) at day 28
At the end of the 10-day window the test item was degraded by 73% and 78%. The 10-day window criterion was met.
The test item was degraded based on HPLC: 100% at day 28.
At the end of the incubation, 100% of the test item had disappeared in the HPLC chromatograms. No peaks corresponding to degradation products were detected. Full details are provided in the full study report.

Results with reference substance:

Sodium benzoate attained 93% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item mean biodegradation in duplicate was 93 % at day 28 based on BOD (10-day window met).

Executive summary:

The ready biodegradability test was carried out according to Japanese guidelines and OECD TG 301F guideline under GLP. The test item, at a nominal concentration of 100 mg/L was exposed to mixed activated sewage sludge micro-organisms obtained from the activated sludge from an aeration tank of a sewage treatment plant treating predominantly domestic sewage in Kurume-shi, Fukuoka, Japan. The sample was collected the day before test item exposure. The activated sludge, was prepared in laboratory according to Japan guidelines. The test item was exposed in culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to 30 mg SS/L at exposure volume of 300 mL. The degradation of the test item was assessed by the measurement of oxygen consumption (BOD) on days 0 through 28 using a closed system oxygen consumption apparatus. DOC would be determined quantitatively by TOC analyser, as applicable. Determination of dissolved organic carbon (DOC) was not conducted because water solubility of the test item was less than 100 mg/L. The test item degradation was determined directly quantitatively by HPLC-DAD. Transformation products, are identified qualitatively by HPLC-DAD and/or further analysis, as applicable. Control solutions with inoculum and the reference substance, sodium benzoate, together with blank controls were used for validation purposes. In the test inoculum blank the oxygen uptake was ca. 21 mg O2/L. The pH value at the end of the test period 28 days did not exceed 7.4 in the test item system and 7.4 in the blank control system. The repeatability validity criterion of not more than 20% difference between replicates was fulfilled. Therefore, the test is considered valid. The test system met the validation criteria of the guideline. Sodium benzoate attained 93% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions. At the end of the 10-day window the test item was degraded by 73% and 78%. The 10-day window criterion was met. The mean biodegradation for duplicate test flasks at 28 days by BOD for the test item was 93% (actual 92% and 93%). The mean biodegradation at day 28 by direct analysis via HPLC was 100% and no residual parent substance was detected. No transformation products were detected in the HPLC. Under the conditions of the study, test item is considered as readily biodegradable.

Description of key information

Weight of evidence: substance is readily biodegradable, 2021

1. Biodegradation: readily biodegradable ; mean biodegradation 93 % by BOD and 100% by HPLC at 28-days. 10-day window met., OECD TG 301F, 2020a

2. Biodegradation: readily biodegradable, mean biodegradation 81.1% at 28-days, 10-day window met, OECD TG 301F, 2020b

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Key study : OECD TG 301F, 2020a : The ready biodegradability test was carried out according to Japanese guidelines and OECD TG 301F guideline under GLP. The test item, at a nominal concentration of 100 mg/L was exposed to mixed activated sewage sludge micro-organisms obtained from the activated sludge from an aeration tank of a sewage treatment plant treating predominantly domestic sewage in Kurume-shi, Fukuoka, Japan. The sample was collected the day before test item exposure. The activated sludge, was prepared in laboratory according to Japan guidelines. The test item was exposed in culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to 30 mg SS/L at exposure volume of 300 mL. The degradation of the test item was assessed by the measurement of oxygen consumption (BOD) on days 0 through 28 using a closed system oxygen consumption apparatus. DOC would be determined quantitatively by TOC analyser, as applicable. Determination of dissolved organic carbon (DOC) was not conducted because water solubility of the test item was less than 100 mg/L. The test item degradation was determined directly quantitatively by HPLC-DAD. Transformation products, are identified qualitatively by HPLC-DAD and/or further analysis, as applicable. Control solutions with inoculum and the reference substance, sodium benzoate, together with blank controls were used for validation purposes. In the test inoculum blank the oxygen uptake was ca. 21 mg O2/L. The pH value at the end of the test period 28 days did not exceed 7.4 in the test item system and 7.4 in the blank control system. The repeatability validity criterion of not more than 20% difference between replicates was fulfilled. Therefore, the test is considered valid. The test system met the validation criteria of the guideline. Sodium benzoate attained 93% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions. At the end of the 10-day window the test item was degraded by 73% and 78%. The 10-day window criterion was met. The mean biodegradation for duplicate test flasks at 28 days by BOD for the test item was 93% (actual 92% and 93%). The mean biodegradation at day 28 by direct analysis via HPLC was 100% and no residual parent substance was detected. No transformation products were detected in the HPLC. Under the conditions of the study, test item is considered as readily biodegradable.

 

Key study : OECD TG 301F, 2020b : The ready biodegradability test was carried out according to OECD TG 301F and China Guidelines for the Testing of Chemicals, Effects on Degradation and Accumulation, Version 2, 301F Manometric Respirometry Test (2013) under GLP. The test substance, at a concentration of 30 mg/L was exposed to activated sewage sludge micro-organisms obtained from the Suzhou Drainage Company Ltd., domestic waste sewage treatment plant of Suzhou city, China, with culture medium in sealed culture vessels in diffuse light at 22°C ± 1°C for 28 days. The sludge was diluted in flasks to 30.0 mg SS/L within the test system and in the negative and toxicity controls. The degradation of the test item was assessed by daily measurement of oxygen consumption from days 0 and 28 using an respirometer system. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The oxygen uptake of the inoculum blank was 36.0 mg/L, which was less than 60 mg/L in 28-days. At the end of the test, the pH values of each test vessel were within the range of 6.0-8.5 (actual pH: 7.75 – 7.93). The difference of extremes of replicate values of the removal of the test substance at the plateau, at end of the test or at the end of the 10-d window was less than 20% (actual day 14: 61.6% and 69.2% and actual day-28: 74.8% and 87.3%). The percentage degradation of the procedure control and the toxicity control were 100% and 72.2%, which had reached the pass levels of 60% and 25% of ThOD on 14d. The toxicity test attained 60 % degradation after 28 days thereby confirming that the test substance was not inhibitory to the sewage treatment micro-organisms used in the study. Sodium Benzoate attained 100% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The test system therefore met the validation criteria of the guideline. The mean biodegradation for duplicate test flasks at 3 days for the test item was 5.6%. The mean biodegradation for duplicate test flasks at 4 days and 14 days for the test flasks was 35.0% and 65.4%, respectively. The mean biodegradation at 28 days for the test item was 81.1% (the 10-day window was met). Under the conditions of the study, test item is considered as readily biodegradable.