4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

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:

supporting study

Study period:

2016-02-29 to 2016-04-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test) and Guideline for testinf of chemical, Degradation and accumulation. 301 F Ready biodegradability Manometric respirometry test. China Environmenal Press 2013, Second edition. The substance is adequately identified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Guideline for testing of chemicals, Degradation and accumulation. 301 F Ready biodegradability Manometric respirometry test. China Environmenal Press 2013, Second edition.

Deviations:

not specified

GLP compliance:

yes

Remarks:

GLP compliance statement done on April 20th, 2016.

Specific details on test material used for the study:

- Storage condition of test material: between 3-5 °C, in full and tightly closed containers in a dark cool place.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The activated sludge derived from sewage treatment plant in the east of Suzhou city (a conventional treatment plant for domestic waste) was selected as inoculum.

- Pretreatment: The freshly sampled activated sludge was collected, kept aerobic during transport, and filtered through fine filter sieve to remove the coarse particles. And then washed using the test medium. After settling for about 10 minutes, the supernatant was discarded. The sludge was resuspended in test medium and repeat the above operation for 3 times. An amount of 10 mL washed activated sludge was sampled for four replicates and dried under 105 °C for 2 hours to determine the concentration of suspended solid. Through measuring, the concentration of the sludge was determined to be 7.2 g suspended solid (SS)/L. The sludge was prepared with test medium to yield a concentration of 4.0 g SS/L and then aerated for 5 days under 22 ± 2 °C. Before test, the activated sludge suspended solid was determined to be 3.9 g SS/L. From this result, the added amount of activated sludge was calculated.

Duration of test (contact time):

28 d

Initial conc.:

28.6 mg/L

Based on:

test mat.

Initial conc.:

28.8 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 250 mL of test medium containing inoculum was firstly added to BOD flasks (test supensions) TS-1 and TS-2, and then 7.19 mg and 7.15 mg of test item were added to the two BOD flasks containing the test suspensions TS-1 and TS-2 respectively.

TEST CONDITIONS
- Composition of medium: Stock solution A : KH2PO4, K2HPO4-3H2O, Na2HPO4-12H2O, NH4CL; Stock solution B : CaCl2; Stock solution C : MgSO4-7H2O; Stock solution D : FeCl3-6H2O
- Test temperature: 20 +/- 1 °C
- pH: 7.5
- Suspended solids concentration: Before test, the activated sludge suspended solid was determined to be 3.9g SS/L.
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2 test concentrations : 28.8 mg/Land 28.6 mg/L; toxicity control: 29.8 mg of test item /L
- Test performed in closed flasks
- Measurement of the consumption of oxygen : The consumption of oxygen was determined from the change in pressure in the bottles. The amount of oxygen taken up by the microbial population during biodegradation of the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of ThOD.
-BOD analyzer : OxiTop Control, WTW, Internal No. TTE20141540

SAMPLING
- Sampling frequency: The BOD value of the test solution was checked 2-3 times a week during the test.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 250 mL of test medium containing inoculum , concentration of 30 mg SS / L
- Procedure control: 72mL stock solution of reference item was added to a volumetric flask and diluted with test medium (not containing inoculum), and then 11.5mL of inoculum was added and made up to 1500mL with test medium to obtain the test solution of procedure control (the concentration of reference item in the test solution was 48.0mg/L and the concentration of inoculum was 30mg SS/L). And 250mL test solution of procedure control was added to BOD flask PC (procedure control)
- No abiotic sterile control

Reference substance:

other: sodium benzoate

Preliminary study:

None

Test performance:

No data

Key result

Parameter:

% degradation (O2 consumption)

Value:

-1.8

Sampling time:

28 d

Remarks on result:

other: Mean of replicat 1 (-2.7% biodegradation) and replicat 2 (-0.9% biodegradation)

Details on results:

At the end of the test, the percentage biodegradation of the two test suspensions were -2.7 % and -0.9 %, giving an average of -1.8 % biodegradation after 28 days, based on ThOD.

Results with reference substance:

In the procedure control, the biodegradation percentage of reference item on day 14 was 80.8%.

Table 1 : Results of BOD value

Time (Day)	BOD Values (mg/L)
	Inoculum Blank		Test Suspension		Procedure Control	Toxicity Control
	IC-1	IC-2	TS-1	TS-2	PC	TC-1
1	2.8	1.4	4.2	1.4	40.9	39.5
2	5.6	5.6	7.0	4.2	50.7	50.7
3	7.0	7.0	8.5	5.6	59.2	59.2
4	8.5	8.5	9.9	7.0	64.8	64.8
5	8.5	8.5	9.9	7.0	69.0	67.6
6	9.9	9.9	11.3	8.5	70.5	70.5
7	9.9	9.9	11.3	9.9	71.9	71.9
8	11.3	11.3	12.7	9.9	73.3	73.3
9	12.7	11.3	12.7	11.3	74.7	74.7
10	12.7	12.7	12.7	11.3	76.1	76.1
11	12.7	12.7	12.7	12.7	77.5	77.5
12	14.1	12.7	14.1	12.7	77.5	78.9
13	14.1	12.7	14.1	12.7	78.9	78.9
14	14.1	14.1	14.1	14.1	78.9	80.3
15	14.1	14.1	14.1	14.1	80.3	81.7
16	14.1	14.1	15.5	14.1	80.3	81.7
17	14.1	14.1	15.5	14.1	81.7	81.7
18	14.1	14.1	15.5	14.1	81.7	83.1
19	14.1	15.5	15.5	14.1	81.7	83.1
20	15.5	15.5	15.5	15.5	81.7	83.1
21	15.5	15.5	15.5	15.5	83.1	84.5
22	18.3	18.3	18.3	16.9	86.0	86.0
23	18.3	18.3	18.3	18.3	86.0	87.4
24	18.3	19.7	18.3	18.3	86.0	87.4
25	15.5	14.1	14.1	15.5	83.1	84.5
26	15.5	15.5	14.1	15.5	83.1	84.5
27	15.5	15.5	14.1	15.5	83.1	86.0
28	16.9	15.5	14.1	15.5	84.5	84.5

Table 2 : Result of Biodegradation Percentage

Time (Day)	Biodegradation Percentage (%)
	Test Suspension			Procedure Control	Toxicity Control
	TS-1	TS-2	Average	PC	TC-1
1	2.7	-0.9	0.9	48.4	23.5
2	1.8	-1.8	0.0	56.3	28.3
3	2.0	-1.8	0.1	65.1	32.7
4	1.8	-2.0	-0.1	70.2	35.3
5	1.8	-2.0	-0.1	75.5	37.1
6	1.8	-1.8	0.0	75.6	38.0
7	1.8	0.0	0.9	77.3	38.9
8	1.8	-1.8	0.0	77.3	38.9
9	0.9	-0.9	0.0	78.2	39.3
10	0.0	-1.8	-0.9	79.1	39.8
11	0.0	0.0	0.0	80.8	40.7
12	0.9	-0.9	0.0	80.0	41.1
13	0.9	-0.9	0.0	81.7	41.1
14	0.0	0.0	0.0	80.8	41.5
15	0.0	0.0	0.0	82.6	42.4
16	1.8	0.0	0.9	82.6	42.4
17	1.8	0.0	0.9	84.3	42.4
18	1.8	0.0	0.9	84.3	43.3
19	0.9	-0.9	0.0	83.5	42.8
20	0.0	0.0	0.0	82.6	42.4
21	0.0	0.0	0.0	84.3	43.3
22	0.0	-1.8	-0.9	84.5	42.5
23	0.0	0.0	0.0	84.5	43.4
24	-0.9	-0.9	-0.9	83.6	42.9
25	-0.9	0.9	0.0	85.2	43.7
26	-1.8	0.0	-0.9	84.3	43.3
27	-1.8	0.0	-0.9	84.3	44.2
28	-2.7	-0.9	-1.8	85.2	42.8

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

At the end of the test, the percentage biodegradation of the two test suspensions were -2.7 % and -0.9 %, giving an average of -1.8 % biodegradation after 28 days. The pass-level was not reached. Consequently, the test item is considered as not readily biodegradable.

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 according to the Guideline for testing of chemicals, Degradation and accumulation, 301 F Ready biodegradability Manometric respirometry test, China Environmenal Press 2013, Second edition.

The concentration of test item present in the two test flasks was 28.8 mg/L and 28.6 mg/L respectively.

The percentage biodegradation of the two test suspensions, based on ThOD, were -2.7 % and -0.9 %, giving an average of -1.8 % biodegradation after 28 days. In the procedure control, the biodegradation percentage of reference item on day 14 was 80.8%.

The test material is consequently considered as not readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15-07-2009 to 12-08-2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed in 2009 according to OECD Guideline 310 and GLP principles. No deviations were noted. The substance is adequately identified but some data on composition is missing. Therefore, validity applies with restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO 1493:2005 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels, CO2 headspace test)

Version / remarks:

Adopted in 2005

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was stored in a closed vessel at room temperature

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt ), biologic sewage treatment plant treating mostly household sewage
- Date of collection : 15 July 2009
- Preparation of inoculum for exposure: The inoculum was taken from its source, washed, aerated and the dry matter was determined.
- Pretreatment: The sludge was filtrated through a cloth, then washed with tap water twice, with test medium once and resuspended in test medium. It was then aerated. The dry matter was determined with 4260 mg suspended solids/L.

Duration of test (contact time):

28 d

Initial conc.:

32.1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

inorg. C analysis

Remarks:

Analyses of the emitted CO2 were made by IC measurement

Details on study design:

TEST CONDITIONS
- Composition of medium: composed of 4 solutions : solution a : KH2PO4, K2HPO4, NA2HPO4-2H2O, NH4Cl ; solution b : CaCl2-2H2O ; solution c : MgSO4-7H2O ; solution d : FeCl3-6H2O

- Test temperature: 22 +/- 2°C
- pH: adjusted to 7.4 +/- 0.1
- pH adjusted: yes
- Suspended solids concentration: Inoculum was added to all flasks except abiotic control to give a concentration of suspended solids of 4 +/- 1 mg/L.
- Continuous darkness: No, test performed without direct lightning

TEST SYSTEM
- Number of culture flasks/concentration: one flask for the blank, 2 flasks for the controls, 2 flasks for the positive controls, 2 test flasks with one concentration tested of 32.1 mg/L, one flask for the abiotic control, one flask for the toxicity control.
- Method used to create aerobic conditions: the inoculum was aerated

- Measuring equipment: Carbon analyser TOC multi N/C 2100 S
- Test performed in closed vessels due to significant volatility of test substance: The vessels were closed with silicon septa and lids.
- Details of trap for CO2 : The emitted C02 in the headspace and test solution was absorbed with NaOH.

SAMPLING
- Sampling frequency: Days 0, 2, 12, 21, 23, 26, and 28. During the exponential degradation phase, samples were taken every two or three days, else every five or six days. At the end of the test (day 28), six vessels (test series control, positive control, test), three vessels (test series blank, abiotic control, toxicity control) were measured.
- Sampling method: To each vessel to be measured, 1.5 mL of NaOH 7-m was added by syrringe and needle and the vessel was shaken for one hour again. After settling down, a sample of 2 mL was pipetted into toc-Vials which were closed.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1500 mL of test medium
- Abiotic sterile control: 1492.5 mL of test medium + 7.5 mL of HgCl2-solution
- Toxicity control: 1485 mL of test medium + 15 mLof stock solution positive control + 1,41 mL of Inoculum
- Positive control : 2970 mL of test medium + 30 mL of stock solution positive control + 2.82 mL of inoculum

Reference substance:

aniline

Remarks:

Aniline was chosen as positive control.

Preliminary study:

None

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

8.5

Sampling time:

28 d

Remarks on result:

other: Mean of replicat 1 (12.8% biodegradation) and replicat 2 (4.2% biodegradation)

Details on results:

Biodegradability was calculated from IC net Inoganic Carbon values (calculated from the measured IC values).
The degrees of degradation of the test suspensions ( test substance + sludge, n=2), calculated from the net Inorganic Carbon values were 12.8 and 4.2 %, giving a mean of 8.5 % degradation.

Results with reference substance:

Degradation of the positive control (aniline) was 63 % within 14 days (63 % reached after 2 days).
The mean net IC content of controls at the end of the test was < 3 mg/L (2.15 mg/L).
Results with reference substance are valid as more than 60% of biodegradation were reached on day 2 (before Day 14).

Table 1 : Inorganic carbon values in ppm

Day	Blank	Control 1	Control 2	Positive control 1	Positive control 2	Test 1	Test 2	Abiotic control	Toxicity control
0	3.15	3.00	3.97	2.80	2.43	2.53	3.36	1.97	5.42
2	0.45	1.93	3.50	13.81	16.84	2.26	2.54	3.29	2.65
12	0.47	1.52	2.27	19.82	20.54	3.90	2.37	1.73	19.45
21	0.52	2.14	2.06	19.75	19.57	2.24	2.01	1.34	19.81
23	1.08	2.2	2.53	19.63	19.16	2.64	4.51	1.33	20.18
26	2.26	3.06	7.02	21.91	22.34	5.03	3.97	2.29	21.41
28	1.05	2.75	2.71	25.03	22.99	4.50	4.79	2.92	25.35
28	1.10	3.61	3.99	22.87	23.20	7.64	4.37	2.90	22.75
28	0.88	2.75	3.12	23.77	21.93	19.14	3.18	2.27	23.04

The value which was measured on day 28 (in bold italics) was attributed to a contamination and not included in evaluation.

Table 2 : Net Inorganic carbon values in ppm

 

The net inorganic carbon was calculated from the measured IC values by substraction of the blank (medium only without inoculum).

Day	Control 1	Control 2	Positive control 1	Positive control 2	Test 1	Test 2	Abiotic control	Toxicity control
0	-0.15	0.82	-0.35	-0.72	-0.62	0.21	-1.18	2.27
2	1.48	3.05	13.36	16.39	1.81	2.09	2.84	2.20
12	1.05	1.80	19.35	20.07	3.43	1.90	1.26	18.98
21	1.62	1.54	19.23	19.05	1.72	1.49	0.82	19.29
23	1.12	1.45	18.55	18.08	1.56	3.43	0.25	19.10
26	0.80	4.76	19.65	20.08	2.77	1.71	0.03	19.15
28	1.74	1.70	24.02	21.98	3.49	3.78	1.91	24.34
28	2.60	2.98	21.86	22.19	6.63	3.36	1.89	21.74
28	1.74	2.11	22.76	20.92	--	2.17	1.26	22.03

Negative values are attributed to measurement uncertainties and variation within the test flasks. In these low concentrations, this is considered as typical.

Table 3 : Degradation values in %

Day	Positive control 1	Positive control 2	Positive control mean	Test 1	Test 2	Test mean	Abiotic control	Toxicity control
2	55.9	71.2	63.5	-2.0	-0.8	-1.4	2.5	-0.2
12	90.3	93.9	92.1	8.8	2.1	5.4	-0.7	41.0
21	88.9	88.0	88.5	0.6	-0.4	0.1	-3.3	41.3
23	87.0	84.6	85.8	1.2	9.4	5.3	-4.5	41.6
26	85.0	87.1	86.1	0.0	-4.7	-2.4	-12.1	38.2
28	104.5	98.5	101.5	12.8	4.2	8.5	-2.0	48.0

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The degrees of degradation of the test suspensions (test substance + sludge, n=2), calculated from the measured IC values (corrected into net IC) were 12.8 % and 4.2 %. The mean biodegradability of the test substance was 8.5 %. The test item should be considered as not readily biodegradable according to this test, following ISO 14593:2005 and OECD Guideline 310, since the pass level of 60% biodegradation was missed at the end of the 10-day window and at the end of the test.

Executive summary:

The aerobic ready biodegradability of the test material was determined using a CO2-Headspace test following ISO 14593:2005 / OECD Guideline 310, with GLP compliance.

A nominal concentration of test material of 32.1 mg/L was introduced into the system.

The degrees of degradation of the test suspensions (test substance + sludge, n=2), calculated from the measured IC values (corrected into net IC) were 12.8 % and 4.2 %. The mean biodegradability of the test substance was 8.5 %. The test item showed virtually no biological degradation. No lag-phase and no 10-day window could be detected.

Degradation behaviour of positive control and toxicity control was normal: the positive control showed ready biodegradation; the replicates showed very good correspondence.

Degradation in the toxicity control correlated well with degradation in the test and the positive control series.

As the degradation in the toxicity control flask was 48% at the end of the test, the test item can be stated as "not toxic towards the inoculumn in a concentration of 32.1 mg/L. Abiotic degradation was observed.

The result of the test can be considered as valid.

The test item should be considered as not readily biodegradable according to this test, following ISO 14593:2005 and OECD Guideline 310, since the pass level of 60% biodegradation was missed at the end of the 10-day window and at the end of the test.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 May 2019 to 14 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to an internationally recognised method, and under GLP. The substance is adequately identified.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Specific details on test material used for the study:

- Molecular Formula: C11H22O2
- Molecular Weight: 186.30 g/mol
- Water Solubility: 6.89 g/L at 25 ºC (iSafeRat ® v1.8)
- Vapour Pressure: 2.27 Pa at 25 ºC (iSafeRat ® v1.3)
- Log Kow: 2.20 at 20 °C (EEC A8, Shake-Flask Method, Phytosafe 2009)

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): A mixed population of sewage treatment micro-organisms was obtained on 13 May 2019 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at temperatures of between 19 and 21 °C prior to use.

Duration of test (contact time):

60 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

226 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

The BOD values for the inoculum control, test item, procedure control and the toxicity control were measured daily.

Details on study design:

TEST CONDITIONS
- Composition of 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).
The pH of the mineral media was measured to be pH 7.7 and was adjusted to pH 7.4 using a diluted hydrochloric acid solution.

TEST SYSTEM
The following test preparations were prepared and inoculated in 500 mL amber glass bottles:
a) Five replicate bottles containing inoculated mineral medium to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L to act as the procedure control.
c) Five replicate bottles containing inoculated mineral medium and the test item at a concentration of 100 mg/L.
d) Two replicate bottles containing the test item at a concentration of 100 mg/L in inoculated mineral medium plus the reference item, aniline, at a concentration of 100 mg/L to act as toxicity control vessels.
All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.
On Day 0, the test and reference items were added to the mineral medium and the pH of all vessels was measured using a Hach HQ40d Flexi handheld meter prior to the addition of inoculum and adjusting to the final volume of 500 mL with mineral medium.
On Day 0, two inoculum control and test item vessels were sampled for compound specific analysis. In order to confirm that the aniline stock solution was prepared correctly, a diluted 100 mg/L stock solution (in reverse osmosis water) was also sampled for Total Organic Carbon (TOC) analysis.
All remaining inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.
The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.
As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask (see Figure 1). The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated computer.
The test was conducted in diffuse light at temperatures of between 20 and 22 °C.
On Day 60, two inoculum controls, one procedure control, two test item and one toxicity control vessel that were considered to have given the most consistent BOD values over the study period were sampled for compound specific analysis and/or pH analysis.
The remaining inoculum control and test item vessels which were not sampled were stored frozen for further analysis if required.
The remaining vessels which were not sampled were discarded and are not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

TEST ITEM PREPARATION
A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) with the aid of ultrasonication for 15 minutes prior to the pH values being measured using a Hach HQ40d Flexi handheld meter and the addition of inoculum (5 mL). The volume was adjusted to 500 mL with mineral medium to give the test concentration of 100 mg/L.
The inoculum control vessels were prepared by inoculating mineral medium (495 mL) with inoculum (5 mL). The pH values were measured using a Hach HQ40d Flexi handheld meter prior to the addition of inoculum.
A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guidelines.

REFERENCE ITEM PREPARATION
A reference item, aniline (C6H5NH2), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving a nominal amount of freshly distilled aniline (500 mg) directly in mineral medium (500 mL) with the aid of ultrasonication for approximately 20 minutes. An aliquot (50 mL) of this stock solution was diluted with mineral medium (350 mL) prior to measurement of the pH value and then addition of inoculum (5 mL) and adjusting to a final volume of 500 mL with mineral medium, to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.
The pH of the reference item stock solution was 7.4. The pH value was measured using a Hach HQ40d Flexi handheld meter.

TOXICITY CONTROL PREPARATION
A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) with the aid of ultrasonication for 15 minutes prior to the addition of an aliquot (50 mL) of the 1000 mg/L aniline stock solution and measurement of the pH value using a Hach HQ40d Flexi handheld meter. The inoculum (5 mL) was then added prior to adjusting to a final volume of 500 mL with mineral medium to give the test concentration of 100 mg test item/L and 100 mg aniline/L.

Reference substance:

aniline

Remarks:

Purity: 99+%; Batch A0358342

Preliminary study:

Information provided by the Sponsor indicated that the water solubility of the test item was 6.89 mg/L at 25 ºC (iSafeRat ® v1.8). Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation. From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, 1995) it was concluded that the best testable dispersions were found to be obtained when using the ultrasonication, silica gel with high shear mixing and solvent with filter paper and high shear mixing methods of preparation. As there was no difference in the observations of the test item dispersions it was considered appropriate to use ultrasonication to disperse the test item into the test system.

Key result

Parameter:

% degradation (O2 consumption)

Value:

7

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Value:

20

Sampling time:

60 d

Details on results:

The test item attained 7% biodegradation after 28 days and 20% biodegradation after 60 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours showed measured concentrations of 105 and 101% of nominal. After 60 days, measured test concentrations of 101 and 98% of nominal were obtained which indicated no significant losses of test item occurred over the exposure period.
Whilst there was no loss of test item shown from chemical analysis, a mean biodegradation value of 20% (19% for Test Item Replicate 1 and 20% for Test Item Replicate 2) was shown after 60 days. This difference was not considered to be critical given that the BOD values were within the acceptable limit of variation and there could have possibly been potential variability within the analytical method. Therefore priority is given to the use of the BOD values. Additionally, information supplied by the Sponsor indicated that the test item was a mixture of cis- and trans-isomers whiler the method for chemical analysis did not differentiate between these isomers. The peaks shown on the chromatography for chemical analysis of the Day 0 and Day 60 test samples containing inoculum were similar with the same ratio and intensity indicating no degradation of the test item occurred. Therefore, it is unlikely that the two peaks on each chromatogram on Day 0 and Day 60 for the test item represented the whole test substance (cis- and trans- isomers). The chemical analysis may only detect some isomers with the other isomer(s) being degraded, which is shown as 20% biodegradation after 60 days but undetected by chemical analysis.

Results with reference substance:

Total Organic Carbon of the diluted aniline stock solution confirmed that it had been prepared correctly.
Aniline (procedure control) attained 65% biodegradation after 14 days in a 10-Day Window, 67% biodegradation after 28 days and 81% degradation after 60 days thereby confirming the suitability of the inoculum and test conditions.

Table 5.2.1/1: Biological Oxygen Demand Values

Day	BOD (mg O2/L)
	Inoculum Control		Procedure Control	Test Item		Toxicity Control
	R1	R2		R1	R2
0	0.00	0.00	0.00	0.00	0.00	0.00
1	0.58	0.00	0.50	0.92	1.00	0.16
2	3.04	0.00	7.78	4.12	3.96	3.62
3	4.62	0.00	73.80	6.74	6.12	53.82
4	6.50	1.50	129.90	9.82	8.54	108.66
5	7.92	3.12	174.48	12.54	10.74	160.36
6	8.88	4.46	185.22	14.78	12.42	174.60
7	10.12	5.92	193.00	16.82	14.28	183.84
8	11.20	6.96	199.50	18.66	15.74	190.34
9	12.08	8.04	203.96	20.50	17.12	194.92
10	12.66	8.88	206.58	21.82	18.28	198.38
11	13.24	9.50	208.46	23.16	19.24	200.72
12	14.74	11.24	210.96	25.62	21.28	203.62
13	18.20	14.74	215.62	29.66	24.90	208.08
14	18.82	15.78	217.42	31.36	26.04	209.96
15	19.04	16.24	218.54	32.28	26.78	210.66
16	19.62	16.90	219.88	33.52	27.74	211.84
17	20.28	18.04	221.46	35.06	28.90	213.38
18	20.62	18.54	222.50	36.20	29.70	214.62
19	21.82	20.04	224.54	38.40	31.48	217.12
20	22.50	20.82	226.04	40.10	32.58	219.50
21	23.08	21.62	227.16	41.48	33.56	221.50
22	23.74	22.58	228.58	43.14	34.56	223.92
23	24.40	23.74	229.94	44.78	35.52	226.28
24	24.70	24.44	230.58	45.74	36.28	228.08
25	25.58	25.66	231.90	47.44	37.56	230.50
26	25.98	26.36	232.74	48.60	38.40	231.82
27	26.62	27.48	233.78	50.06	39.56	233.04
28	27.24	28.44	234.74	51.44	40.74	234.12
29	27.66	29.20	235.36	52.52	41.74	234.74
30	28.28	30.36	236.32	53.94	42.94	235.74

31	28.90	31.36	237.28	55.26	44.20	236.66
32	29.52	32.36	238.16	56.60	45.52	237.66
33	29.94	33.02	238.70	57.68	46.48	238.12
34	30.28	33.66	239.08	58.76	47.40	238.70
35	30.82	34.48	239.62	59.90	48.56	239.16
36	31.44	35.28	240.24	61.14	49.86	240.12
37	32.20	36.36	241.24	62.64	51.32	241.28
38	32.66	37.16	241.74	63.64	52.72	241.82
39	33.32	38.06	242.20	64.60	54.44	242.28
40	33.98	39.06	242.40	65.34	56.40	242.48
41	34.48	39.94	242.56	65.98	58.40	242.62
42	36.12	42.10	243.94	67.98	61.98	244.16
43	37.24	43.70	245.12	69.60	65.68	245.28
44	38.10	44.82	245.74	70.72	73.26	245.78
45	38.82	46.10	246.10	73.52	75.90	245.98
46	39.74	47.44	246.48	76.56	77.92	246.28
47	42.98	48.92	248.16	80.80	80.70	247.74
48	44.30	50.32	249.48	84.66	83.80	248.82
49	45.88	51.44	250.60	87.38	87.56	249.60
50	47.54	52.88	251.32	89.76	91.36	249.98
51	49.18	54.84	251.94	92.44	94.02	250.28
52	50.64	56.22	252.90	95.10	96.50	250.90
53	52.48	57.76	254.36	97.32	99.12	252.02
54	53.98	59.24	255.52	100.46	102.22	253.32
55	55.76	61.24	259.36	103.80	105.18	260.48
56	56.88	62.66	272.72	106.70	107.08	276.06
57	58.32	63.86	280.18	109.70	109.96	284.56
58	59.36	65.24	292.72	112.38	112.20	293.92
59	60.72	66.32	308.54	114.46	115.32	318.24
60	62.46	67.30	314.50	115.84	117.36	324.58

R= Replicate

Table 5.2.1/2: Percentage Biodegradation values

Day	Biodegradation (%)
	Procedure Control	Test Item			Toxicity Control
		R1	R2	Mean
0	0	0	0	0	0
1	0	0	0	0	0
2	2	1	1	1	0
3	23	2	1	2	9
4	41	2	2	2	18
5	55	3	2	3	27
6	58	3	2	3	29
7	60	3	2	3	31
8	62	4	3	4	32
9	63	4	3	4	32
10	63	4	3	4	33
11	64	4	3	4	33
12	64	5	3	4	33
13	64	5	3	4	33
14	65	5	3	4	34
15	65	6	3	5	34
16	65	6	4	5	34
17	65	6	4	5	34
18	66	6	4	5	34
19	66	7	4	6	34
20	66	7	4	6	34
21	66	7	4	6	35
22	66	8	4	6	35
23	67	8	4	6	35
24	67	8	4	6	35
25	67	8	4	6	36
26	67	8	5	7	36
27	67	9	5	7	36
28	67	9	5	7	36
29	67	9	5	7	36
30	67	9	5	7	36

31	67	9	5	7	36
32	67	10	5	7	36
33	67	10	6	7	36
34	67	10	6	8	36
35	67	10	6	8	36
36	67	10	6	8	36
37	67	11	6	8	36
38	67	11	7	8	36
39	67	11	7	9	36
40	67	11	7	9	36
41	66	11	8	9	36
42	66	11	9	9	36
43	66	11	9	10	36
44	66	11	12	10	36
45	66	12	13	10	35
46	66	12	13	12	35
47	65	13	13	13	35
48	65	14	14	13	35
49	65	15	15	13	35
50	65	15	15	14	35
51	65	15	16	15	34
52	65	16	16	15	34
53	64	16	17	16	34
54	64	16	17	16	34
55	65	17	18	17	35
56	69	18	18	17	38
57	71	18	18	18	39
58	75	19	19	19	40
59	79	19	19	19	44
60	81	19	20	20	45

R= Replicate

Table 5.2.1/3: pH values of the test preparations on days 0 and 60

Test Vessel	pH
	Day 0	Day 60
Inoculum ControlR1	7.4	8.5
Inoculum Control R2	7.4	8.5
Procedure Control	7.4	8.8
Test Item R1	7.4	8.4
Test Item R2	7.4	8.5
Toxicity Control	7.4	8.5

 R  =  Replicate

Table 5.2.1/4: Analytical measurements

Time Point (day)	Nominal concentration of test item in test sample (mg/L)	Sample dilution factor (F)	Determined concentration of test item in test sample	Percentage of nominal concentration (%)
0	Control – R1 Control – R2 100 – R1 100 – R2 P. Rec 1 – 98.9 P. Rec 2 – 98.9	0.1 0.1 0.1 0.1 0.1 0.1	<LOQ <LOQ 105 101 96.1 94.7	- - 105 101 97 96
60	Control – R1 Control – R2 100 – R1 100 – R2 P. Rec 1 – 102 P. Rec 2 – 102	0.1 0.1 0.1 0.1 0.1 0.1	<LOQ <LOQ 101 98.3 99.0 94.8	- - 101 98 97 93

R = Replicate

LOQ = Liquid of Quantification (0.72 mg/L)

- = Not applicable

P. Rec = Procedural recovery

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 7% biodegradation after 28 days and 20% biodegradation after 60 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Executive summary:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guideline 301F, EU Method C.4 -D and EPA OPPTS 835.3110, with GLP statement.

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 20 and 22 ºC for 60 days. At the request of the Sponsor the study was extended from 28 to 60 days in order to assess if any further degradation would occur. The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values andcompound specific analyses on Days 0 and 60. Control solutions with inoculum and the reference item, aniline, and a toxicity control were used for validation purposes.

The test item attained 7% biodegradation after 28 days and 20% biodegradation after 60 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Aniline (procedure control) attained 65% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 67% biodegradation was attained with 81% biodegradation after 60 days.

Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours showed measured concentrations of 105 and 101% of nominal. After 60 days, measured test concentrations of 101 and 98% of nominal were obtained which indicated no significant losses of test item occurred over the exposure period.

Whilst there was no loss of test item shown from chemical analysis, a mean biodegradation value of 20% (19% for Test Item Replicate 1 and 20% for Test Item Replicate 2) was shown after 60 days. This difference was not considered to be significant given that the BOD values were within the acceptable limit of variation and there could have possibly been potential variability within the analytical method. Additionally, information supplied by the Sponsor indicated that the test item was a mixture of cis- and trans-isomers. However the method for chemical analysis did not differentiate between these isomers. The peaks shown on the chromatography for chemical analysis of the Day 0 and Day 60 test samples containing inoculum were similar with the same ratio and intensity indicating no degradation of the test item occurred. Therefore, it is unlikely that the two peaks on each chromatogram on Day 0 and Day 60 for the test item represented the whole test substance (cis- and trans- isomers). The chemical analysis may only detect some isomers with the other isomer(s) being degraded, which is shown as 20% biodegradation after 60 days but undetected by chemical analysis.

Description of key information

OECD Guideline 301F, EU Method C.4 -D, EPA OPPTS 835.3110, GLP, Key study, validity 1:

7% biodegradation after 28 days and 20% biodegradation after 60 days

Not readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

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

The first study (COVANCE, 2019), assessed as the key study, was performed on the registered substance according to OECD Guideline 301F, EU Method C.4 -D and EPA OPPTS 835.3110, with GLP compliance. The test substance, at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 20 and 22 ºC for 60 days. The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values and compound specific analyses on Days 0 and 60. The test substance attained 7% biodegradation after 28 days and 20% biodegradation after 60 days and therefore cannot be considered to be readily biodegradable.

The second study (LAUS, 2009), assessed as a supporting study, was performed on the registered substance according to OECD Guideline 310 and ISO 14593:2005, with GLP compliance. The test substance, at a concentration of 32.1 mg/L was exposed to activated sludge for 28 days, in aerobic conditions. The mean biodegradability of the test substance was measured at 8.5 %. Therefore, the test substance should be regarded as not readily biodegradable according to this test.

The third study (CTI-CSERC Co. Ltd., 2016), assessed also as a supporting study, was performed on the registered substance according to the OECD Guideline No. 301 F and the corresponding China Guideline (the guideline for testing of chemicals, Degradation and accumulation, 301 F Ready biodegradability manometric respirometry test, China Environmenal Press 2013, Second edition), with GLP compliance. The test substance, at a concentration of 28.6-28.8 mg/L, was exposed to activated sludge for 28 days, in aerobic conditions. The mean biodegradability of the test substance was measured at -1.8% after 28 days. Therefore, the test substance is not readily biodegradable according to this test.

In conclusion, according to the key biodegradation study (and supported by two other studies), the registered substance is not readily biodegradable.