5-propyloxan-2-one

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:

disregarded due to major methodological deficiencies

Study period:

2007-11-02 to 2008-01-31

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The substance identification is incomplete. The study was performed according to the EU Method C.4-E : Determination of the ready biodegradability - Closed bottle test with significant methodological modifications. The test substance was dissolved in a partially biodegradable solvent, chloroform was used to prepare a "stock solution" prior to adding an aliquot of this solution to the mineral medium. The biodegradation of the solvent in the test substance solutions was determined by substracting the degradation in a solvent control. However, it cannot be ascertained directly to what extent the chloroform degraded in the solutions containing test substance, the results of the study are not interpretable and the study is considered technically invalid.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection on March 15th and16th 2007

Specific details on test material used for the study:

- Storage condition of test material: stored at room temparature and protected from direct sunlight
- Treatment of test material prior to testing: Chloroform was used as solvent for the preparation of the test solution : a stock solution was prepared in chloroform.

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Remarks:

The inoculum was derived from surface waters.

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The inoculum was derived from surface waters collected in the near area of Pau, France. The surface waters receive predominantly domestic sewage.
- Preparation of inoculum for exposure: The freshly collected sample of surface water was previously pre-conditionned to the experimental conditions under aerobic conditions for 6 days in the mineral medium at the test temperature : 400 mL of surface water were added with 0.5 mL of stock solutions (a mixture of chloroform and test substance).
- Storage conditions: The resulting solution was maintained under strong aeration and agitation at 20 +/- 1 °C for 6 days.
- Concentration of inoculum : The pre-conditionned inoculum wa further used at a rate of 2 mL/L of medium (for Test 1) or 0.7 mL/L of medium (Test 2).
- Suspended solids : were measured to represent 4.4 µg/L (Test 1) or 0.9 µg/L (Test 2).

Duration of test (contact time):

>= 28 - < 36 d

Initial conc.:

5.02 mg/L

Based on:

test mat.

Initial conc.:

2.04 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: 8.5 g/L of KH2PO4, 21.75 g/L K2HPO4, 33.40 g/L Na2HPO4 - 2H2O, 0.50 g/L NH4Cl, 27.50 g/L CaCl2, 22.50 g/L MgSO4 - 7H2O, 2.5 g/L FeCl3 - 6H2O
- Test temperature: 20 +/- 1 °C
- pH : Test 1 : 7.50 ; Test 2 : 7.40
- Suspended solids concentration: 4.4 µg/L (Test1), 0.9 µg/L (Test 2).
- bottles were incubated in dark

TEST SYSTEM
- Culturing apparatus: vessels
- Number of culture flasks/concentration: test 1 : 2 blank replicates, 2 test replicates, 1 solvent replicate, 1 toxic replicate, 2 aniline replicates ; test 2 : 2 blank replicates, 2 test replicates, 1 solvent replicate, 1 toxic replicate, 2 aniline replicates
- Measuring equipment: Dissolved oxygen was assessed by the electrode method : Oxi38 equipped with StirrOx G probe, WTW

SAMPLING
- Sampling frequency: blank series, test series and reference series were withdrawn every 3-4 days


CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, 300 mL mineral medium + 50 mL pre-conditionned inoculum
- Abiotic sterile control: Not performed
- Toxicity control: Yes, 300 mL mineral medium + 100 µL aniline + 20 µL Figolide + 50 mL pre-conditionned inoculum

Reference substance:

aniline

Preliminary study:

None

Test performance:

No data

Key result

Parameter:

% degradation (O2 consumption)

Value:

50

Sampling time:

36 d

Remarks on result:

other:

Remarks:

The 10-day window criteria was not fulfilled.

Details on results:

50.0 % biodegradation occurred after 36 days of incubation, in Test 2. The pass-level was almost reached but it did not occur within the 10-day window and the tested substance was not considered as easily biodegradable.

Results with reference substance:

In the toxic control series more than 25 % biodegradation of aniline had occurred on day 14.

The validity criteria were fulfilled for Test 2, at 2.04 mg/L :

-Mean oxygen uptake in the blank vessels < 1.5 mg/L at the end of the test,

-the residual concentration of oxygen in the test vessels did not fall below 0.5 mg/L at any time,

-differencies of extremes of replicate values of the removal of the test item was less than 20 % of mean value at the end of test,

percentage biodegradation of the reference compound (aniline) had reached the pass-level by day 14,

-in the toxic control series more than 25 % biodegradation of aniline had occurred on day 14.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

50 % biodegradation occurred within 36 days, the pass-level was almost reached but this did not occur within the 10-day window. Consequently, the test substance was not considered as easily biodegradable.

Executive summary:

This study was performed according to the EU Method C.4-E : Determination of the ready biodegradability - Closed bottle test.

The test material was exposed to inoculum from surface waters, at a concentration of 2.04 mg/L in Test 2, in culture medium, in closed bottles, in the dark, at 20 +/- 1 °C, for 36 days.

The degradation of the test material was assessed by the measurement of oxygen consumption.

50 % biodegradation occurred within 36 days and the 10-day window criteria was not fulfilled, even if the pass-level was almost reached.

However, in both tests, the test substance was dissolved in a partially biodegradable solvent, chloroform was used to prepare a "stock solution" prior to adding an aliquot of this solution to the mineral medium. The biodegradation of the solvent in the test substance solutions was determined by substracting the degradation in a solvent control. However, it cannot be ascertained directly to what extent the chloroform degraded in the solutions containing test substance, the results of the study are not interpretable and the study is considered technically invalid.

Consequently, the test material should be regarded as not readily biodegradable according to this test.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2011-09-02 to 2012-08-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

It was evaluated that this study has a reliability of 2 (Klimisch rating) as : - the Cerficate of Analysis of the test substance was not provided in the study report and the substance identification is incomplete. -no sterile abiotic control was performed -the concentration of tested substance was 21,4 mg/L instead of 100 mg/L like the guideline demands -more measurements could have been made during the 10-day window, allowing a better evaluation of the 10-day window criteria -the unknown volatility of the substance was not discussed in the results, whereas it could have helped in the assessment of the amount of remaining substance.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Guideline for testing of chemicals, 301F Ready Biodegradability: Manometric Respirometry test. First edition. Beijing:China Environmental Science Press. 2004: 363-369.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP Compliance Statement performed by China National Accreditation Service for Conformity Assessment, datinf from 2010-11-24, valid until 2013-11-23

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test substance was weighted and added directly to the bottles. After adding 100 mL of test medium, an ultrasonic treatment of 15 min was performed.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

No data

Duration of test (contact time):

28 d

Initial conc.:

21.4 mg/L

Based on:

not specified

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: 8.5 g KH2PO4, 28.5 g K2HPO4-3H2O, 67.15 g Na2HPO4-12H2O, 0.5 g NH4Cl ; 13.75 g CaCl2 ; 11.25 g MgSO4-7H2O ; 0.125 g FeCl3 6H2O

- Test temperature: 22 +/- 11°C
- pH: 7.4 +/- 0.2
- pH adjusted: yes
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
3 bottles used as "test suspensions containing test substance inoculum and test medium : initial concentration of 21.4 mg/L
3 bottles used as "inoculum blank" containing inoculum and test medium : do not contain test substance
1 bottle used as "procedure control" containing reference substance, inoculum and test medium : initial concentration of 30.1 mg/L
1 bottle used as "toxicity control" containing test substance, reference substance, inoculum and test medium : initial concentration of 30.4 mg/L.


- Method used to create aerobic conditions: The test bottles were aerated on OxiTop Control BOD at 22°C +/- 1°C
- Measuring equipment: The consumption of BOD of each test bottle was determined and recorded automatically by the respirometer OxiTop Control BOD
- Test performed in closed vessels as volatility of the test substance was unknown.
- Details of trap for CO2 and volatile organics if used: No details


SAMPLING
- Sampling frequency: At the start of the test and at the end of the test.
- Sampling method: The consumption of BOD of each test bottle was determined and recorded automatically by the respirometer OxiTop Control BOD. pH values of 2 specific bottles were determined and the pH adjusted in each test bottle at the start of the test. pH values of each test bottle were also determined at the end of the test.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 bottles containing inoculum and test medium
- Abiotic sterile control: No abiotic sterile control performed
- Toxicity control: 1 bottle containing test substance, reference substance, inoculum and test medium

Reference substance:

other: sodium benzoate

Preliminary study:

None

Test performance:

No data

Key result

Parameter:

% degradation (O2 consumption)

Value:

67.6

Sampling time:

28 d

Remarks on result:

other: At the end of the test, the percentage biodegradation of the 2 test suspensions were 62.5% and 72.7 %. The average was 67.6%. During the 10-day window the percentage biodegradation of the test substance did not reach 60% under the test conditions.

Details on results:

The consumption of BOD of each test bottle was determined and recordred automatically by the respirometer OxiTop Control BOD.
At the end of the test , the percentage biodegradation of the two test supsensions were 62.5 % and 72.7 %. The average was 67.6 %. During the 10-day window, the percentage biodegradation of the test substance did not reach 60 % under the conditions of the test.

Results with reference substance:

On the 14th day of the test, the percentage biodegradation (based on ThOD) of the reference substance was 83.8%. It passed the level of 60 %, thereby passing the validity criteria.

Table 1 : Biochemical oxygen demend (BOD) of each test bottle during the test

Time (d)	Test suspensions		Inoculum blank			Procedure control	Toxicity control
	bottle 1 (mg/L)	bottle 2 (mg/L)	bottle 3 (mg/L)	bottle 4 (mg/L)	Mean (mg/L)	bottle 5 (mg/L)	bottle 6 (mg/L)
1	0,0	0,0	0,0	0,0	0,0	0,0	0,0
4	6,2	7,9	9,0	8,4	8,7	42,4	39,5
7	13,0	17,0	10,9	10,1	10,5	49,2	53,5
10	27,7	31,7	12,6	12,1	12,4	53,7	66,2
14	36,2	40,1	14,3	14,3	14,3	56,5	77,5
18	43,0	47,5	15,4	16,0	16,0	59,4	86,0
20	43,0	47,5	16,3	16,3	16,3	59,9	84,5
22	43,5	48,0	16,6	16,5	16,5	61,0	86,0
25	46,9	51,4	17,1	17,3	17,3	62,2	78,9
28	49,2	54,3	17,7	18,0	18,0	62,7	94,4

Table 2 : The percentage degradation calculated by BOD

Group	7d		14d		21d		28d
	BOD (mg)	degradation (%)	BOD (mg)	degradation (%)	BOD (mg)	degradation (%)	BOD (mg)	degradation (%)
test suspension 1	13,0	5,0	36,2	43,8	43,0	53,4	49,2	62,5
test suspension 2	17,0	13,0	40,1	51,6	47,5	62,4	54,3	72,7
Procedure control	49,2	76,9	56,5	83,8	59,9	86,6	62,7	88,9
Toxicity control	53,5	43,2	77,5	63,0	84,5	67,9	94,4	75,9

Table 3: Biodegradation percentage at each day (not present in the study report but calculated by the assessor)

Days	% biodegradation for Rep1	% biodegradation for Rep2
1 4 7 10 14 18 20 22 25 28	0 -5 5 31 44 55 53 54 59 62	0 -2 13 39 52 64 63 63 69 73

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

At the end of the test , the percentage biodegradation of the two test supsensions were 62.5 % and 72.7 %. The average was 67.6 %. The 10-day window criterion was not fulfilled.

Executive summary:

The ready biodegradability of the test material has been determined by the Manometric Respirometry Test according to the OECD Guideline No. 301 F and the Chinese guideline for the testing of chemicals : 301 F ready biodegradability : Manometric Respirometry Test.

A nominal concentration of test material of 21.4 mg/L was introduced in the system.

The percentage biodegradation of the two test suspensions, based on ThOD, were 62.5 % and 72.7 %. The average was 67.6 %. The 10-day window criteria was not fulfilled.

On the 14th day of the test, the percentage biodegradation (based on ThOD) of the reference substance was 83.8% surpassing the level of 60 % and thereby passing the validity criteria.

The test material can consequently be considered as ready biodegradable failing the 10-day window.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 March 2019 to 10 April 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The substance is adequately characterised but purity is out of specifications. Therefore validation applies with restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21/08/2018; Date of issue: 19/11/2018

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Inoculum: A mixed population of activated sewage sludge micro-organisms, obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum: The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1-Hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.1 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

other: carbon content

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

- Medium: The mineral medium used in this study was that recommended in the OECD Guidelines. The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

- Test item preparation: From the preliminary solubility work it was concluded that the test item was insoluble in water at a concentration of 100 mg/L using the conventional methods of sonication and high shear mixing. Therefore following the recommendations of the International Standards Organisation (ISO 10634, 1995) and Handley et al (2002) it was concluded that the best testable dispersions were found to be obtained when using the high shear mixing method of preparation and the solvent with high shear mixing method of preparation. As there was no difference between the observations of the two preparations it was considered acceptable to use high shear mixing alone to disperse the test item into the test system. The test item was dispersed directly in mineral medium. An amount of test item (44.4 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 14.8 mg/L, equivalent to 10 mg carbon/L. A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

- Reference item preparation: A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium prior to the volume being adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

- Toxicity control: A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An amount of test item (44.4 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 14.8 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

PREPARATION OF TEST SYSTEM
The following were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of preparation:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 21 and 24 °C for 28 days, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 42.9 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter and adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all of the vessels being adjusted to 3 liters by the addition of mineral medium, which had been purged overnight with CO2-free air. The inoculum control vessels were prepared in a similar manner without the addition of test item or reference item.
In order to confirm that the sodium benzoate solution was prepared correctly, a diluted 100 mg/L stock solution (in reverse osmosis water) was sampled for Total Organic Carbon (TOC) analysis.
The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using deionsed reverse osmosis water.

Reference substance:

benzoic acid, sodium salt

Remarks:

Batch SLBT3039; Purity > 99.5%

Key result

Parameter:

% degradation (CO2 evolution)

Value:

88

Sampling time:

28 d

Remarks on result:

other: within the 10d window

Details on results:

The total CO2 evolution in the inoculum control vessels on Day 28 was 33.52 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.
Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels.
The IC analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
The test item attained 88% biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
The toxicity control attained 56% biodegradation after 14 days and 73% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Results with reference substance:

Sodium benzoate attained 84% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines. After 28 days 90% biodegradation was attained.
TOC of the diluted sodium benzoate stock solution confirmed that it had been prepared correctly.

Table 5.2.1/1: Percentage Biodegradation values

Day	Biodegradation (%)
	Procedure Control	Test item	Toxicity Control
0 2 6 8 10 14 21 28 29*	0 50 77 75 89 84 82 80 90	0 4 23 41 55 69 81 87 88	0 10 23 24 27 56 73 73 73

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Table 5.2.1/2: Total and Inorganic Carbon Values in the Culture Vessels on day 0

Test vessel	Total Carbon* (mg/L)	Inorganic Carbon* (mg/L)	IC Content (% of TC)
Test item 10 mg C/L R1	10.12**	0.14	1
Test item 10 mg C/L R2	9.95**	-0.09	0

* Corrected for control values. Negative values are due to measured concentrations being less than control values

** TC value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

Table 5.2.1/3: pH values of the test preparations on days 0 and 28

Test vessel	pH
	Day 0 Pre-adjustment	Day 0 Post-adjustment	Day 28
Inoculum Control R1 Inoculum Control R2 Procedure Control R1 Procedure Control R2 Test item R1 Test item R2 Toxicity control	7.7 7.7 7.7 7.7 7.7 7.7 7.7	7.4 7.4 7.4 7.4 7.4 7.4 7.4	7.5 7.4 7.5 7.5 7.4 7.5 7.5

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 88% biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport and Transformation Test Guidelines OCSPP 835.3110.

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 21 and 24 °C for 28 days. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

The test item attained 88% biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Sodium benzoate attained 84% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Description of key information

OECD Guideline 301B, GLP, Key study, validity 1:

88% biodegradation after 28 days, within the 10-day window.

The substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

To assess the ready biodegradability of the registered substance, three studies are available.

The first study, from Covance, 2019, assessed as the key study (despite restrictions on test item composition), was performed on the registered substance according to OECD Guideline 301B and EU Method C.4-C with GLP compliance. The substance, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 21 and 24 °C for 28 days. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes. The test substance attained 88% biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The substance can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The second study, from Guangdong Detection Center of Microbiology, 2012, assessed as the supporting study, was performed on the registered susbstance according to OECD Guideline 301F with GLP compliance, and according to the Chinese Guideline 301F Ready Biodegradability: Manometric Respirometry test. Activated sludge was exposed to the test substance at a concentration of 21.4 mg/L, with culture medium, for 28 days, in aerobic conditions. According to this test, the degree of degradation, calculated from the O2 consumption of two replicates was 62.5 % and 72.7 % respectively, mean of 67.6 %, after 28 days, but without the 10 -day window. Thus, the test substance should be regarded as readily biodegradable but failling the 10-day window criteria. More measurements could have been made during the 10 -day window, allowing a better evaluation of the 10 -day window criteria.

The third study, from Phytosafe, 2008, assessed as a disregarded study, was performed on the registered substance according to the EU Method C.4-E: Determination of the ready biodegradability - Closed bottle test. The test material was exposed to inoculum from surface waters, at a concentration of 2.04 mg/L in Test 2, in culture medium, in closed bottles, in the dark, at 20 +/- 1 °C, for 36 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. The validity criteria were fulfilled for Test 2, for a concentration of test substance of 2.04 mg/L. 50 % biodegradation occurred within 36 days and the 10-day window criteria was not fulfilled, even if the pass-level was almost reached. However, in both tests, the test substance was dissolved in a partially biodegradable solvent, chloroform was used to prepare a "stock solution" prior to adding an aliquot of this solution to the mineral medium. The biodegradation of the solvent in the test substance solutions was determined by substracting the degradation in a solvent control. However, it cannot be ascertained directly to what extent the chloroform degraded in the solutions containing test substance, the results of the study are not interpretable and the study is considered technically invalid. Consequently, the test cannot be considered valid.

In conclusion, based on the key study, the registered substance is readily biodegradable.