Lorenox™

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:

From May 10, 2012 to June 7, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study is reliable with restrictions: - No date of the final report was reported (no signature, just stated "June 2012"); No Certificate of Analysis was provided in the report; The substance is adequately identified, but some data on composition is missing; The temperature variation range exceed the normal range. However, the test results revealed this experiment met the quality requirements and the deviation did not affect the validity of the test results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The temperature variation range exceeded the normal range. Time points when temperature was in the range of 23.0°C-23.5°C was 10. The results revealed this experiment met the quality requirements; the deviation did not affect the validity of the results.

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 301F Ready Biodegradability: Manomettric Respiremetry Test. First edition. Beijing: China Environmental Science Press. 2004: 363-369.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: GB/T 21801－2008 Chemicals—Ready Biodegradability: Manomettric Respiremetry Test.

Deviations:

not specified

GLP compliance:

yes

Remarks:

Statement of GLP compliance; no signature date.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source: The secondary of Liede Sewage Treatment Plant of Guangzhou
- Treatment: The sludge was washed by centrifugation, the supernatant liquid phase was decanted and the solid material resuspended in a mineral medium. A homogenized aliquot of the final sludge suspension was weighted, thereafter dried and the ratio of wet to dry weight was calculated as 9.8 g/L. Based on this ration, calculated amount of wet sludge was suspended in mineral medium to obtain a concentration equivalent to 2.4 g dry material per liter. Appropriate volume of inoculum was inoculated in each bottle to give a final concentration of 28 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

16.4 mg/L

Based on:

test mat.

Remarks:

Bottles 1, 2, and A, corresponding to 50 mg ThODNH4 /L

Initial conc.:

16.5 mg/L

Based on:

test mat.

Remarks:

Bottles 6, corresponding to 50mg ThODNH4 /L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST SYSTEM
- Test water: Deionized water
- Test medium: The test medium was prepared with the deionized water.
- Test grouping:
Bottles 1, 2 & A (test suspension): Containing test substance, inoculum and test medium
Bottles 3, 4 & B (inoculum blank): Containing inoculum and test medium
Bottle 5 (procedure control): Containing reference substance, inoculum and test medium
Bottle 6 (toxicity control): Containing test substance, reference substance, inoculum and test medium
Bottles A & B for determination of pH value of the test suspensions and inoculum blanks at the start of the test
- Addition of the test substance: 6.1 mg test substance was weighted and added directly to the bottles 1, 2 and A respectively, with ultrasonic treatment for 15min after 100mL test medium were added, and the suspension were obtained. 4.2 mg test substance was weighted and added directly to the bottle 6, with ultrasonic treatment for 15min after 100mL deionized water were added, and the suspension were obtained.
- Addition of inoculum: 4.3mL inoculum was added to bottles 1, 2 and A; 5.0mL inoculum was added to bottles 3, 4 and B, 2.9mL inoculum was added to bottle 6.
- Addition of the test medium: Test medium was added to bottles 1, 2, 5 and A to make the final volume reached 365mL; Test medium was added to bottles 3, 4 and B to make the final volume reached 432mL; Test medium was added to bottle 6 to make the final volume reached 250mL.

TEST CONDITIONS
The test bottles were agitated and aerated on OxiTop® Control BOD at 22°C±1°C in the dark over 28days period.

ANALYSIS AND DETERMINATION
The consumption of BOD of each test bottle was determined and recorded automatically by the respirometer OxiTop® Control BOD. pH value of bottles A and B were determined, and adjusted pH value of the solution in each test bottle to 7.4±0.2 at the start of the test. And pH value of each test bottle were determined at the end of the test.

Reference substance:

benzoic acid, sodium salt

Remarks:

30.1mg/L (procedure control, corresponding to 50mg ThOD NH4/L); 30.4mg/L (toxicity control, corresponding to 51mg ThOD NH4/L)

Preliminary study:

ThODNH4 of the test substance is 3.05mg/mg, and ThODNH4 of the reference substance is 1.67mg/mg.

Test performance:

At the start of the test, pH values of test suspension and blank group were 7.70 and 7.48, so the test suspensions were adjusted to 7.34.
Temperature during the test period was from 21.9°C-23.5°C.

Key result

Parameter:

% degradation (O2 consumption)

Value:

-4.2

Sampling time:

28 d

Remarks on result:

other: Test suspension 1

Key result

Parameter:

% degradation (O2 consumption)

Value:

-2

Sampling time:

28 d

Remarks on result:

other: Test suspension 2

Details on results:

At the end of the test, pH values of test suspensions were both 7.26 those of inoculum blanks were 7.54 and 7.55 (2 replicates), and those of the procedure control and toxicity control were 7.73 and 7.23 respectively.
At the end of the test, the mean oxygen uptake of two inoculum blanks was 23.0mg/L.
At the end of the test, the percentage biodegradation of two test suspensions were both 0%.

Results with reference substance:

On the 14th day of the test, the percentage biodegradation of the reference substance and toxicity control was 80.0% and 38.9% respectively, which showed that the inoculum activity could meet the requirement of the test and the test material was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study.

Table 5.2.1/1: Biochemical oxygen demand (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)
4 7 11 14 18 21 25 28	15.3 15.8 17.0 17.5 18.7 19.2 20.3 20.9	15.3 16.4 17.0 18.1 19.2 19.8 21.5 22.0	12.9 16.0 18.0 19.4 21.1 22.5 23.3 24.7	13.2 14.9 17.3 17.7 18.8 19.7 20.8 21.3	13.1 15.5 17.7 18.6 20.0 21.1 22.1 23.0	46.3 53.1 57.1 58.8 60.5 61.6 62.7 62.7	45.1 50.7 55.0 57.8 60.6 62.0 63.4 66.2

Table 5.2.1/2: The percentage degradation calculated by BOD

Group	14d		28d
	BOD (mg/mg)	Degradation (%)	BOD (mg/mg)	Degradation (%)
Test suspension 1 Test suspension 2 Procedure control Toxicity control	-0.064 -0.027 1.336 0.839	-2.1 -0.9 80.0 38.9	-0.128 -0.061 1.317 0.923	-4.2 -2.0 78.9 42.9

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the test conditions, the percentage biodegradation of the test substance on the 28 days was 0%.

Executive summary:

This study was performed according to the test guideline 301F of the OECD (adopted 17 July 1992) and China Environmental Science Press (2004: 363 -369) with GLP statement.

A measured volume of inoculated mineral medium with activated sludge (28 mg dry material/L), containing a known concentration of test substance (16.4 -16.5 mg test substance/L, corresponding to 50 mg ThOD/L) as the nominal sole source of organic carbon, is stirred in a closed bottle at a constant temperature for up to 28 days. The microorganisms draw oxygen to degrade organic substances from the amount of air remaining in the closed system. The carbon dioxide formed by this process is absorbed. Due to the reduction in the amount of oxygen, the pressure in the bottle sinks. This change is detected and stored by the measuring head. After the data transfer to the controller, it is used to determine the BOD value. The amount of BOD taken up by the microbial population during biodegradation of the test substance (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD.

The consumption of oxygen of each test bottle was determined and recorded automatically by OxiTop® Control BOD Test System.

At the start of the test, pH values of test suspension and blank group were 7.70 and 7.48, so the test suspensions were adjusted to 7.34.

Temperature during the test period was from 21.9°C-23.5°C.

At the end of the test, pH values of test suspensions were both 7.26 those of inoculum blanks were 7.54 and 7.55 (2 replicates), and those of the procedure control and toxicity control were 7.73 and 7.23 respectively. At the end of the test, the mean oxygen uptake of the two inoculum blanks was 23.0 mg/L. On the 14th day of the test, the percentage biodegradation of the reference substance and toxicity control was 80.0% and 38.9% respectively, which showed that the inoculum activity could meet the requirement of the test and the test material was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study. At the end of the test, the percentage biodegradation of two test suspensions were both 0%. More precisely, -4.2% and -2.0% biodegradation were reported for the test suspensions 1 and 2, respectively.

This study is reliable with restrictions, for the following reasons:

- no date of the final report was reported (no signature, just stated "June 2012");

- no Certificate of Analysis was provided in the report;

- the substance is adequately identified, but some data on composition is missing;

- the temperature variation range exceed the normal range. However, the test results revealed this experiment met the quality requirements and the deviation did not affect the validity of the test results.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 May 2019 to 08 July 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The substance is adequately identified, but some data on composition is missing, so validation applies with restrictions.

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: C16H28O
- Molecular Weight: 236.4 g/mol
- Water Solubility: 2.09 mg/L at 25 ºC (iSafeRat ® v1.8)
- Vapour Pressure: 0.138 Pa at 20 ºC (OECD 104/EEC A4, effusion method, Laus 2011)
- Log Kow: 5.13 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 07 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 20 °C prior to use.

Duration of test (contact time):

60 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

305 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 7.8 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. 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 or frozen 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) and subjected to high shear mixing (approximately 7500 rpm, 15 minutes) prior to allowing to cool to temperatures of approximately 21 °C and measurement of 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 and allowed to cool to a temperature of approximately 21 °C prior to use. 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) and subjected to high shear mixing (approximately 7500 rpm, 15 minutes) prior to allowing to cool to temperatures of approximately 21 °C and then the addition of an aliquot (50 mL) of the 1000 mg/L aniline stock solution (see Section 3.6.3) 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 2.09 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 dispersion was found to be obtained when using the high shear mixing, high shear mixing with silica gel and high shear mixing with solvent method of preparations. As there was no difference between the observations of these dispersions, it was considered appropriate to use only high shear mixing in order to disperse the test item into the test system.

Key result

Parameter:

% degradation (O2 consumption)

Value:

3

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Value:

5

Sampling time:

60 d

Details on results:

The test item attained 3% biodegradation after 28 days and 5% 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 93 and 101% of nominal were obtained. A decline in measured test concentration was observed on Day 60 to 24 and 20% of nominal, (calculated to be a 76 and 80% loss over the test duration assuming 100% recovery on Day 0).
The losses observed by chemical analysis were higher than those observed by oxygen consumption. This was considered to be due to the presence of degradation and/or hydrophobic peaks in the chromatography (see chromatograms in "Attached background material") as a result of chemical degradation, biotransformation or hydrolysis of the test item. Additionally although the test item did not adsorb to the inoculum initially, which was confirmed by the validation work and the results from the test item vessels on Day 0, the test item may have adsorbed to the inoculum over the 60 day study period although it is beyond the scope of this study to confirm this.

Results with reference substance:

Total Organic Carbon of the diluted aniline stock solution gave a result of 101% of nominal and therefore confirmed that it had been prepared correctly.
Aniline (procedure control) attained 70% biodegradation after 14 days in a 10-Day Window, 73% biodegradation after 28 days and 76% degradation after 60 days thereby confirming the suitability of the inoculum and test conditions (> 60%, in a 10 -day window, after 14 days).

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	1.54	1.16	1.50	3.20	3.74	3.84
2	2.34	2.00	2.84	4.54	5.12	4.88
3	2.96	2.66	24.36	5.50	6.16	6.20
4	3.62	3.42	88.72	6.46	7.08	16.74
5	4.20	4.00	168.22	7.24	7.92	111.50
6	4.78	4.66	192.26	8.20	8.82	160.72
7	5.42	5.38	205.04	9.20	9.78	166.94
8	6.00	6.04	211.20	9.82	10.54	170.02
9	6.00	6.04	214.42	9.92	10.66	171.84
10	6.00	6.24	217.28	10.42	11.00	174.44
11	6.62	6.96	219.82	11.42	12.00	177.26
12	7.12	7.58	221.74	12.20	12.74	179.64
13	7.66	8.16	223.32	13.04	13.54	181.84
14	8.24	8.82	224.96	13.96	14.32	184.18
15	8.78	9.46	226.58	14.86	15.16	186.26
16	9.32	10.04	228.20	15.82	15.96	187.96
17	9.96	10.70	229.62	16.82	16.82	189.50
18	12.20	12.92	232.78	19.20	19.24	192.42
19	12.70	13.36	234.04	19.86	19.90	193.54
20	13.20	13.86	235.24	20.36	20.66	194.72
21	13.28	13.86	235.82	20.46	20.82	195.38
22	13.42	14.20	237.08	21.08	21.70	196.54
23	14.20	14.92	238.28	22.00	22.82	197.88
24	14.24	14.92	238.70	22.08	23.04	198.46
25	15.74	16.42	240.78	23.86	25.00	200.66
26	16.96	17.50	242.16	24.94	26.16	202.16
27	17.40	17.90	242.98	25.48	26.82	203.16
28	18.24	18.70	243.98	26.32	27.78	204.46
29	18.78	19.20	244.78	26.94	28.48	205.42
30	18.90	19.36	245.40	27.44	28.90	206.34

31	20.04	20.40	246.74	28.66	30.08	207.88
32	20.54	20.90	247.60	29.32	30.74	209.12
33	20.90	21.28	248.36	29.86	31.32	210.16
34	21.32	21.70	249.16	30.58	31.90	211.20
35	21.90	22.24	250.10	31.24	32.58	212.16
36	22.20	22.58	250.90	31.90	33.16	213.16
37	23.04	23.32	252.02	32.66	34.02	214.20
38	23.66	24.04	253.06	33.48	34.94	215.34
39	23.66	24.04	253.40	33.62	34.98	215.66
40	24.16	24.44	254.24	34.12	35.62	216.50
41	24.66	24.94	254.78	34.52	36.06	217.08
42	25.32	25.78	256.24	35.56	37.28	218.58
43	25.98	26.48	257.18	36.48	38.16	219.82
44	26.94	27.28	258.02	37.20	39.02	220.66
45	27.54	27.70	258.44	37.48	39.36	221.16
46	27.54	27.70	258.60	37.52	39.36	221.28
47	27.54	27.70	258.68	37.52	39.40	221.50
48	28.24	28.40	260.22	38.90	40.78	223.32
49	29.28	29.44	261.40	40.02	41.94	224.62
50	29.58	29.70	261.86	40.44	42.28	225.12
51	30.02	30.08	262.40	41.06	42.90	225.90
52	30.02	30.08	262.40	41.10	42.90	226.04
53	31.12	31.16	264.02	42.90	44.36	227.78
54	32.02	32.02	265.06	44.02	45.32	228.86
55	32.48	32.48	265.64	44.74	45.86	229.66
56	32.98	32.86	266.14	45.40	46.36	230.32
57	32.98	32.86	266.18	45.52	46.36	230.54
58	32.98	32.86	266.56	46.28	46.78	231.40
59	33.98	33.94	267.94	47.78	48.06	232.74
60	34.82	34.72	268.82	48.64	48.84	233.62

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	1	1	1	0
2	0	1	1	1	0
3	7	1	1	1	1
4	28	1	1	1	2
5	53	1	1	1	17
6	61	1	1	1	25
7	65	1	1	1	26
8	66	1	1	1	27
9	67	1	2	2	27
10	68	1	2	2	27
11	69	2	2	2	28
12	69	2	2	2	28
13	70	2	2	2	28
14	70	2	2	2	29
15	70	2	2	2	29
16	71	2	2	2	29
17	71	2	2	2	29
18	71	2	2	2	29
19	72	2	2	2	29
20	72	2	2	2	30
21	72	2	2	2	30
22	72	2	3	3	30
23	72	2	3	3	30
24	73	2	3	3	30
25	73	3	3	3	30
26	73	3	3	3	30
27	73	3	3	3	30
28	73	3	3	3	30
29	73	3	3	3	30
30	73	3	3	3	30

31	73	3	3	3	31
32	73	3	3	3	31
33	74	3	3	3	31
34	74	3	3	3	31
35	74	3	3	3	31
36	74	3	4	4	31
37	74	3	4	4	31
38	74	3	4	4	31
39	74	3	4	4	31
40	74	3	4	4	31
41	74	3	4	4	31
42	75	3	4	4	31
43	75	3	4	4	32
44	75	3	4	4	32
45	75	3	4	4	32
46	75	3	4	4	32
47	75	3	4	4	32
48	75	3	4	4	32
49	75	3	4	4	32
50	75	4	4	4	32
51	75	4	4	4	32
52	75	4	4	4	32
53	75	4	4	4	32
54	75	4	4	4	32
55	75	4	4	4	32
56	75	4	4	4	32
57	75	4	4	4	32
58	76	4	5	5	32
59	76	5	5	5	32
60	76	5	5	5	32

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	7.6
Inoculum Control R2	7.4	7.6
Procedure Control	7.4	8.5
Test Item + Inoculum R1	7.4	7.6
Test Item + Inoculum R2	7.4	7.6
Toxicity Control	7.4	8.3

 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 (mg/L)	Percentage of nominal concentration (%)
0	Control – R1 Control – R2 100 – R1 100 – R2 P. Rec 1 – 103 P. Rec 2 – 103	0.1 0.1 0.1 0.1 0.1 0.1	<LOQ <LOQ 93.0 101 102 108	- - 93 101 98 104
60	Control – R1 Control – R2 100 – R1 100 – R2 P. Rec 1 – 103 P. Rec 2 – 103	0.1 0.1 0.1 0.1 0.1 0.1	<LOQ <LOQ 23.8 20.1 100 104	- - 24 20 97 101

R = Replicate

- = Not applicable

P. Rec = Procedural recovery

LOQ = Limit of Quantification (1.0 mg/L)

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 3% biodegradation after 28 days, and 5% 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 medium. 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 and compound 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 3% biodegradation after 28 days, and 5% 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 70% biodegradation after 14 days with greater than 60% degradation being attained within the 10-Day window. After 28 days 73% biodegradation was attained with 76% biodegradation after 60 days.

Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours resulted in measured concentrations of 93 and 101% of nominals. A decline in measured test concentration was observed on Day 60 to 24 and 20% of nominal, (equating to 76 and 80% loss over the test duration assuming 100% recovery on Day 0). 

The losses observed by chemical analysis were higher than those observed by oxygen consumption. This was considered to be due to the presence of degradation and/or hydrophobic peaks in the chromatogram as a result of chemical degradation, biotransformation or hydrolysis of the test item which nevertheless did not lead to mineralisation. Additionally although the test item did not adsorb to the inoculum initially, which was confirmed by the validation work and the results from the test item vessels on Day 0, the test item may have adsorbed to the inoculum over the 60 day study period although it was beyond the scope of this study to confirm this. 

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study is only a non-GLP screening test. In addition, the substance is adequately identified but some data on composition is missing. Therefore, validation applies with restrictions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Ammonium chloride was omitted from the medium to prevent nitrification.

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Remarks:

AND river water

Details on inoculum:

- Activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to 2.0 mg DW/in the biological oxygen demand (BOD) bottles (van Ginkel and Stroo, 1992).
- River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating. The river water spiked with mineral salts of the nutrient medium was used undiluted.

Duration of test (contact time):

56 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- Test procedures of Closed Bottle test:
The Closed Bottle test was performed according to Test Guidelines (OECD 1992). The nutrient medium of the Closed Bottle test contained per liter of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrifi-cation.
The test substance was dosed on silica gel in a 50-mL serum flask to obtain a concentration of 3.0 mg / g silica gel. Only the upper layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw cap and mixed vigorously. Subsequently, 0.20 g of silica gel was administrated to each test bottle. Administering of the water-insoluble test substance was also accomplished by dissolving 1.0 g/L of test substance in dichloromethane (DCM). The test substance in DCM (0.6 mL) was directly added to bottles. The solvent was allowed to evaporate by placing the bottles on a roller bank in a ventilated hood for 4 hours to obtain an even distribution of the test substance on the walls of the bottles.
The tests were performed in 0.3 L BOD bottles with glass stoppers. Use was made of 3 bottles containing only respective inoculum and silica gel and 3 bottles with test substance and the respective inoculum. The final concentration in the bottles was 2.0 mg/L.
Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium. Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

- Analyses:
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW, Germany). The pH was measured using a calibrated pH meter (EUTECH instruments, Canada). The temperature was measured and recorded with a thermo couple connected to a data logger (ThermoScientific, USA). The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a pre-weighed 12 m cellulose nitrate filter. This filter was dried for 1.5 hours at 104°C and weighed after cooling. The dry weight was calculated by subtracting the weighed filters and by dividing this difference by the filtered volume.

Key result

Parameter:

% degradation (O2 consumption)

Value:

1

Sampling time:

28 d

Remarks on result:

other: Inoculum = sludge / Administering method: DCM

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Inoculum = sludge / Administering method: silica gel

Key result

Parameter:

% degradation (O2 consumption)

Value:

-2

Sampling time:

28 d

Remarks on result:

other: Inoculum = river water / Administering method: DCM

Key result

Parameter:

% degradation (O2 consumption)

Value:

-4

Sampling time:

28 d

Remarks on result:

other: Inoculum = river water / Administering method: silica gel

Parameter:

% degradation (O2 consumption)

Value:

1

Sampling time:

56 d

Remarks on result:

other: Inoculum = sludge / Administering method: DCM

Parameter:

% degradation (O2 consumption)

Value:

-1

Sampling time:

56 d

Remarks on result:

other: Inoculum = sludge / Administering method: silica gel

Parameter:

% degradation (O2 consumption)

Value:

-1

Sampling time:

56 d

Remarks on result:

other: Inoculum = river water / Administering method: DCM

Parameter:

% degradation (O2 consumption)

Value:

-2

Sampling time:

56 d

Remarks on result:

other: Inoculum = river water / Administering method: silica gel

Details on results:

- Test conditions:
The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of the media was 7.3 (activated sludge) and 8.0 (river water) at the start of the test. The pH was 7.1±0.1 (activated sludge) and 8.0±0.1 (river water) at day 28. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption. Slight inhibition of the endogenous respiration of the inoculum was detected with the test substance. The inhibition the test item administered with DCM was noted during the first two weeks. Biodegradable substances having the low inhibition as found with the test substance do usually biodegrade in the prolonged Closed Bottle tests.

- The Closed Bottle test results:
Ring opening of the test item requires three to four enzymes. The first enzyme hydroxylates a carbon in the ring. The alcohol formed due to this hydroxylation must then be oxidized to a ketone (second enzyme). Next an ester bond has to be formed through a Baeyer Villiger reaction which requires a third enzyme and finally the ester has to be hydrolysed. This sequence of reactions has been described for other simple cyclic hydrocarbons (mono-constituents). However, the test item is a mixture of 16 enantiomers with four substituents on the ring. This means that there is a high chance that the substituents on ring of the parent constituents might not be positioned correctly for enzymes responsible for ring cleavage. The test item is therefore thought to be persistent.
Two valid readily biodegradation studies are available and only ≤ 3% and 5% biodegradation was found after 28 and 60 days, respectively (Covance, 2019 and Guangdong Detection Center of Microbiology, 2012). The ThOD used to calculate the biodegradation percentages is 3.1 g/g. In this study, biodegradation of the test substance was not found within 8 weeks with both activated sludge and river water. This finding is in line with the other tests carried out. The test
substance should therefore not be classified as readily biodegradable and in the absence of other studies, is considered persistent in the environment. A final GLP test will not be carried out.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Biodegradation of the test substance was not found within 8 weeks with both activated sludge and river water. The test substance should therefore not be classified as readily biodegradable and in the absence of other studies, is considered persistent in the environment.

Executive summary:

A non-GLP screening test was performed with two inoculums (activated sludge and river water) to assess the inoculum giving the most advantageous classification of the registered substance in the Closed Bottle test. The Closed Bottle tests are performed according to slightly modified OECD Test Guidelines (OECD, 1992). Ammonium chloride was omitted from the medium to prevent nitrification. The test substance was dosed on silica gel in a 50-mL serum flask to obtain a concentration of 3.0 mg / g silica gel. Only the upper layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw cap and mixed vigorously. Subsequently, 0.20 g of silica gel was administrated to each test bottle. Administering of the water-insoluble test substance was also accomplished by dissolving 1.0 g/L of test substance in dichloromethane (DCM). The test substance in DCM (0.6 mL) was directly added to bottles. The solvent was allowed to evaporate by placing the bottles on a roller bank in a ventilated hood for 4 hours to obtain an even distribution of the test substance on the walls of the bottles. The tests were performed in 0.3 L BOD bottles with glass stoppers. Use was made of 3 bottles containing only respective inoculum and silica gel and 3 bottles with test substance and the respective inoculum. The final concentration in the bottles was 2.0 mg/L. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium. Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992). The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW, Germany). The pH was measured using a calibrated pH meter (EUTECH instruments, Canada). The temperature was measured and recorded with a thermocouple connected to a data logger (ThermoScientific, USA). The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a pre-weighed 12 µm cellulose nitrate filter. This filter was dried for 1.5 hours at 104°C and weighed after cooling. The dry weight was calculated by subtracting the weighed filters and by dividing this difference by the filtered volume.

The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of the media was 7.3 (activated sludge) and 8.0 (river water) at the start of the test. The pH was 7.1±0.1 (activated sludge) and 8.0±0.1 (river water) at day 28. Temperatures ranged from 22 to 24°C. The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption. Slight inhibition of the endogenous respiration of the inoculum was detected with the test substance. The inhibition of the test item administered with DCM was noted during the first two weeks. Biodegradable substances with relatively low inhibition, as found in the case of this test substance usually biodegrade in prolonged Closed Bottle tests.

Ring opening of the test item requires three to four enzymes. The first enzyme hydroxylates a carbon in the ring. The alcohol formed due to this hydroxylation must then be oxidized to a ketone (second enzyme). Next an ester bond has to be formed through a Baeyer Villiger reaction which requires a third enzyme and finally the ester has to be hydrolysed. This sequence of reactions has been described for other simple cyclic hydrocarbons (mono-constituents). However, the test item is a mixture of 16 enantiomers with four substituents on the ring. This means that there is a high chance that the substituents of the parent constituents might not be positioned correctly for enzymes responsible for ring cleavage to be able to break the ring open. The test item is therefore thought to be potentially persistent, or at least not mineralisable under the conditions of a ready test.

Two other valid readily biodegradation studies are available and in these only ≤ 3% and 5% biodegradation was found after 28 and 60 days, respectively (Covance, 2019 and Guangdong Detection Center of Microbiology, 2012). The ThOD used to calculate the biodegradation percentages is 3.1 g/g. In this study, biodegradation of the test substance was not found within 8 weeks with both activated sludge and river water. This finding is in line with the other tests carried out. The test substance should therefore not be classified as readily biodegradable and in the absence of other studies, is considered persistent in the environment. A final GLP test will not be carried out.

Description of key information

OECD Guideline 301F, EU Method C4-D, EPA OPPTS 835.3110, GLP, Key study, validity 2:

3% biodegradation after 28 days and 5% biodegradation after 60 days, with activated sludge

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 biodegradation potential of the registered substance, three experimental studies are available.

The biodegradation study, performed by Covance in 2019 and assessed as the key study, was performed on the registered substance to assess the ready biodegradability of the test item in an aerobic aqueous media, according to 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. 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 only 3% biodegradation after 28 days, and 5% biodegradation after 60 days 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 was carried out resulting in measured concentrations of 93 and 101% of nominals. A decline in measured test concentration was observed on Day 60 to 24 and 20% of nominal, (calculated as 76 and 80% loss over the test duration assuming 100% recovery on Day 0). The losses observed by chemical analysis were higher than those observed by oxygen consumption. This was considered to be due to the presence of degradation and/or hydrophobic peaks in the chromatogram as a result of chemical degradation, biotransformation or hydrolysis of the test item but not to the extent of increasing mineralisation. Additionally, although the test substance did not adsorb to the inoculum initially, which was confirmed by the validation work and the results from the test item vessels on Day 0, the test substance, with a log Kow at 5.13, may have adsorbed to the inoculum over the 60 day study period although it was beyond the scope of the study to confirm this. 

Two other experimental studies, assessed as supporting studies, were performed on the registered substance.

The biodegradation study, performed by Guangdong Detection Center of Microbiology in 2012, was performed according to the OECD Guideline 301F and China Environmental Science Press (2004: 363 -369) with GLP statement. The test substance attained no biodegradation after 28 days and therefore the substance is not considered readily biodegradable.

Finally, the biodegradation study, performed by Nouryon in 2020, was performed as a screening study according to a slightly modified OECD Guideline 301D and without GLP statement. This study was conducted to assess if the use of another inoculum (river water instead of activated sludge) can improve biodegradation of the registered substance. In this study, biodegradation of the test substance was not found within 8 weeks with both activated sludge and river water. This finding is in line with the other tests carried out. The test substance should therefore not be classified as readily biodegradable and in the absence of other studies, is considered persistent in the environment.