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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 29 November 2012 to 07 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected date: 14 June 2012 / Signed date: 10 January 2013

Specific details on test material used for the study:

- Physical state: white crystalline powder
- Storage condition: Room temperature protected from direct sun light
- Hydrosolubility = 5,4 mg/L
- ThOD = 3,01 g/g

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): The inoculum was derived from surface waters collected in the near area of Pau, France and receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The freshly collected sample of surface water was previously pre-conditioned to the experimental conditions under aerobic conditions for 5 days at the test temperature in the mineral medium used for testing. The resulting solution was continuously stirred for 5 days at 20+/- 1°C. The pre-conditioned inoculum was further used at a rate of 2 mL/L.

Duration of test (contact time):

28 d

Initial conc.:

3.33 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to Guideline. The mineral medium was strongly aerated for at least 20 minutes, and then allowed to stand for approximately 20 h at the test temperature before use.
- Test temperature: 20 +/- 1 °C
- Continuous darkness: yes
- Other: each BOD bottles was fitted with a glass stopper

TEST SYSTEM
- Culturing apparatus: 250 mL BOD bottles
- Number of culture flasks/concentration: 2
- Preparation of the bottles: The 250 mL BOD bottles were thoroughly cleaned before use using 5-10 mL of a wash solution (2.5 g iodine plus 12.5 g potassium iodide per litre of 1% w/v sulphuric acid). The bottles were shaken to coat the bottle walls and left to stand for 15 min. The wash solution was poured off and the bottles were thoroughly rinsed with tap water and finally demineralised water.
- Dissolved oxygen: Dissolved oxygen was assessed by the electrode method. The zero-time for dissolved oxygen was measured within 1h after the setting of the test system. Duplicate vessels of each the blank series, the test series and the reference series were sacrificed every 3-4 days for dissolved oxygen analysis. The toxic control series was measured out from one single vessel.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Blank group = mineral medium alone with inoculum (duplicate determinations)
- Abiotic sterile control: no
- Toxicity control: Toxic control = solution containing 2-5 mg/L of each the test substance and aniline in mineral medium, with inoculum (single determinations).
- Other: Reference item = 2-5 mg/L aniline solution in mineral medium with inoculum (duplicate determinations)

PREPARATION OF THE EXPERIMENTAL SOLUTIONS
6 L of water were set at the saturation level using excessive amount of the test item. The volume was placed at 20 +/- 2°C and stirred for the night with a magnetic stirrer. The solution was then allowed to remain undisturbed for 2 hours so that excess of the item separated. The test item concentration was checked in the resulting solution before use. The data showed taht the test item concentration was 5.54 mg/L. The mineral medium was then reconstituted.
The test system was set as follows:
- Blank group: 300 mL of mineral medium + 0.6 mL of pre-conditioned inoculum
- Test group: 180 mL of the above test solution + 120 mL of mineral medium + 0.6 mL of pre-conditioned inoculum.
- Reference group: A stock solution was prepared using 0.1216 g Aniline for 20 mL of water. 300 mL of mineral medium were added with 100 µL of the stock solution and 0.6 mL of pre-conditioned inoculum.
- Toxic control group: 180 mL of the test solution + 100 µL of the Aniline stock solution + 120 mL of mineral medium + 0.6 mL of pre-conditioned inoculum.
The nominal value for test item concentration in the test group and in the toxic control group was taken as 5.54 * 180/300 = 3.33 mg/L

Reference substance:

aniline

Remarks:

Purity >99.0%; batch No. 034K0126

Test performance:

The initial value for dissolved oygen concentration was 8.87 mg/L for the control, 8.22 mg/L for the test item treatment and for the toxic control, and 8.85 mg/L for the reference item treatment.
The consumption of oxygen in the controls was less than 1.5 mg/L over the 28d test period, as required.
For the test item treatment, the measured concentrations for dissolved oxygen were approximately the same as for the controls up to day 28 of the test period.
For the reference item treatment and for the toxic control, the difference with the controls was more tan 1 mg/L up from day 10 of the test period.

Key result

Parameter:

% degradation (O2 consumption)

Value:

8.6

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Details on results:

Oygen updake and related biodegradation:
See tables 5.2.1/1 and 5.2.1/2 in "Any other information on results incl. tables"
- Test substance: The calculated values for biodegradation were less than 10% for the entire test period.
- Toxic control: Based on total ThOD, the biodegradation was more than 25% up from day 14 of the test period.

Active ingredient content:
Mean measured initial concentrations represented 106.9% the nominal value (3.333 mg/L). The treatment application was considered as valid. At the end of the 28-day test period, mean measured concentrations still represented more than 95% of the nominal value in the test item solutions and in the toxic control (see Table 5.2.1/3 in "Any other information on results incl. tables")

Results with reference substance:

Aniline: The beginning of the 10-day window was observed between day 6 and day 10 of the test period. The pass-level was reached on day 14: mean value for biodegradation was 65.9%. The biodegradation stabilized at approximately 84%.

Table 5.2.1/1: Measured oxygen concentrations in the BOD bottles, mg/L

Days	Blank		Test substance		Toxic	Aniline
	Rep.1	Rep.2	Rep.1	Rep.2		Rep.1	Rep.2
0 3 6 10 14 17 20 24 28	8.87 8.40 8.35 8.28 8.11 8.20 8.27 8.23 8.19	8.87 8.53 8.38 8.38 8.24 8.16 8.29 8.21 8.20	8.22 7.85 7.57 7.59 7.44 7.37 7.35 7.43 7.41	8.22 7.87 7.56 7.53 7.39 7.41 7.40 7.39 7.31	8.22 7.91 7.59 4.42 3.58 4.01 3.85 3.45 3.45	8.85 8.53 8.37 5.69 4.85 4.25 4.20 4.21 4.12	8.85 8.52 8.12 5.64 5.02 4.37 4.14 3.93 4.03

Table 5.2.1/2: Calculated biodegradation, %

Days	Test substance			Toxic	Aniline
	Rep.1	Rep.2	Mean		Rep.1	Rep.2	Mean
0 3 6 10 14 17 20 24 28	0 3.70 6.50 6.30 7.80 8.50 8.70 7.90 8.10	0 3.50 6.60 6.90 8.30 8.10 8.20 8.30 9.10	0 3.60 6.55 6.60 8.05 8.30 8.45 8.10 8.60	0 2.08 4.23 25.52 31.16 28.27 29.34 32.03 32.03	0 -1.74 -0.51 53.75 67.80 80.21 83.29 81.85 83.19	0 -1.54 4.62 54.77 64.31 77.75 84.52 87.60 85.03	0 -1.64 2.05 54.26 66.06 78.98 83.90 84.72 84.11

Table 5.2.1/3: Measured concentrations of the test substance

	Replicate unit	Replicate determination	Test item, mg/L	% recovery
Test initiation	1	1 2	4.14 4.32	124.3% 130.0%
	2	1 2	3.30 3.16	99.4% 95.1%
	3	1 2	3.17 3.24	95.3% 97.5%
	Mean		3.56	106.9%
End of test
Test units	1	1 2	3.29 3.39	99.0% 101.8%
	2	1 2	3.00 3.02	90.3% 90.8%
	3	1 2	3.22 3.29	96.8% 99.0%
	Mean		3.20	96.3%
Toxic controls	1	1 2	2.88 2.77	86.5% 83.2%
	2	1 2	3.73 3.58	112.1% 107.6%
	Mean		3.24	97.3%

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substancei s not easily biodegradable (removal within 28 days < 10%)

Executive summary:

The biodegradation of the test substance was assessed according to the closed-bottle test method, appropriate for low water soluble substances.

The solution of the test substance (3.33 mg/L) in mineral medium was inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely full, closed bottle in the dark at constant temperature (20 +/- 1 °C).

Degradation was followed by analysis of dissolved oxygen over a 28 -day period. The amount of oxygen taken up by the microbial population during biodegradation of the test substance, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of the theorical oxygen demand (ThOD).

Additional analysis assessments were performed at test initiation and at test completion in the test vessels for quantification of the test item, so as to judge upon the primary degradation over the test period.

In the test item treatment, the oxygen uptake was similar to that of the controls for the entire test period. At the end of test, analytical check of the test item in the test solutions and in the toxic control proved that the initial value still remained. Therefore, the test substance was considered as not easily biodegradable (removal within 28 days < 10%).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 May 2019 to 06 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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: C16H26O
- Molecular Weight: 234.4 g/mol
- Water Solubility: 5.4 mg/L at 20 ºC (Phytosafe 2012, micro-column elution method, EEC A6)
- Vapour Pressure: 0.0949 Pa at 20 ºC (Laus 2013, effusion method, EEC A4)
- Log Kow: 4.77 at 25°C (Covance 2019, slow-stirring method, EEC A23/OECD TG 123)

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 03 June 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 20 and 21 °C prior to use.

Duration of test (contact time):

60 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

300 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. THe pH of the inoculum control and procedure control vessels were measured using a Hach HQ40d Flexi handheld meter prior to the addition of the inoculum and the volume in all the vessels being adjusted to 500 mL by the addition of mineral medium. The pH of the contents of the test item and toxicity control vessels were not measured prior to the addition of the inoculum due to the risk of test item being lost from the test system by adherence to the pH probe.
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.
Two inoculum control and two test item vessels were sacrificed for compound specific analysis on Day 0 and 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 ultrasonic disruption for 15 minutes prior to allowing to cool to temperatures of between 21 and 22 ºC. The inoculum (5 mL) was then added and the volume adjusted to a final volume of 500 mL with mineral medium to give the test concentration of 100 mg/L.
The inoculum control vessels were prepared in a similar manner without the addition of test item and ultrasonication. Prior to the addition of the inoculum the pH of the mineral media in each inoculum control vessel was measured using a Hach HQ40d Flexi handheld meter.
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 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 using a Hach HQ40d Flexi handheld meter 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 ultrasonication for 15 minutes prior to allowing to cool to temperatures of approximately 21 ºC. An aliquot (50 mL) of the 1000 mg/L aniline stock solution and 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 5.4 mg/L at 20 °C (Phytosafe 2012, micro-column elution method, EEC A6). 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 ultrasonication method of preparation.

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

60 d

Details on results:

The test item attained no biodegradation after 28 and 60 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
There were significant statistical differences between the Day 60 inoculum control and test item biological oxygen demand values. This significant statistical difference was considered to be due to the test item not being biodegradable under the conditions of this test and not that the test item was exhibiting an inhibitory effect at the test concentration employed in the study. This was confirmed by the toxicity control vessel which attained greater than 25% biodegradation by Day 14 of the test, thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro organisms used in the test.
Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours showed measured concentrations of 108 and 106% of nominal were obtained. A decline in measured test concentration was observed on Day 60 to 78 and 83% of nominal, (calculated to be a 22 and 17% 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. Although the test item did not adsorb to the inoculum initially, which was confirmed by the validation work of the analytical method and the results from the test item vessels on Day 0, with a log kow of greater than 4, it is likely that the test item may have adsorbed to the inoculum and/or glassware over the 60 day study period although it is beyond the scope of this study to confirm this.

Results with reference substance:

TOC of the diluted aniline stock solution confirmed that it had been prepared correctly.
Aniline (procedure control) attained 72% biodegradation after 14 days in a 10-Day Window and 74% biodegradation after 28 and 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	0.34	0.08	0.24	0.46	0.66	0.84
2	1.88	1.66	1.92	1.92	2.16	2.46
3	2.12	2.00	3.46	1.96	2.38	2.92
4	3.66	3.50	8.66	3.34	3.66	5.20
5	4.58	4.20	12.82	3.88	4.20	18.08
6	5.66	5.08	35.36	4.66	5.08	92.42
7	6.70	5.96	77.26	5.46	5.96	163.60
8	7.58	6.50	141.78	6.00	6.42	174.06
9	8.66	7.66	202.72	6.92	7.46	177.98
10	9.82	8.66	225.40	7.88	8.34	181.42
11	11.04	9.70	231.24	8.82	9.20	184.60
12	11.62	10.00	232.62	9.16	9.46	186.84
13	12.20	10.28	233.78	9.74	9.88	189.00
14	12.70	10.58	234.48	10.08	10.24	190.72
15	14.70	12.38	236.82	11.78	12.04	193.92
16	15.82	13.82	238.48	12.86	13.28	196.34
17	16.50	14.50	239.52	13.36	13.86	198.08
18	17.50	14.96	240.86	13.74	14.28	199.62
19	17.78	14.96	241.40	13.82	14.28	200.54
20	18.28	14.96	241.90	13.82	14.28	201.58
21	20.50	16.54	244.98	15.12	15.54	204.58
22	22.16	18.12	247.16	16.42	16.86	207.04
23	22.82	18.62	248.20	16.82	17.20	208.58
24	23.58	18.86	248.86	17.00	17.36	209.92
25	24.50	19.28	249.70	17.24	17.62	211.46
26	26.24	21.08	252.14	18.90	19.08	214.28
27	27.48	22.32	253.94	20.00	20.08	216.88
28	28.54	23.28	255.56	20.78	20.82	219.12
29	28.94	23.44	256.40	20.90	20.90	220.38
30	29.98	24.00	257.56	21.20	21.28	222.00

31	30.82	24.54	258.68	21.66	21.66	223.58
32	32.48	26.04	260.68	23.04	22.82	225.86
33	33.44	26.94	261.98	23.82	23.58	227.70
34	33.82	27.08	262.28	23.86	23.66	228.90
35	34.16	27.20	262.40	23.90	23.70	229.58
36	35.40	27.66	263.56	24.20	24.08	231.62
37	36.16	28.04	264.32	24.54	24.32	233.08
38	37.56	29.44	266.10	25.78	25.66	235.44
39	38.24	29.82	266.80	26.20	26.04	237.20
40	39.70	31.16	268.56	27.44	27.12	239.66
41	40.32	31.58	269.18	27.74	27.40	241.20
42	40.94	31.78	269.56	27.82	27.48	242.52
43	41.86	40.28	270.44	28.32	27.98	244.28
44	43.48	43.98	272.26	29.58	29.32	246.74
45	43.48	43.98	272.26	29.62	29.32	247.24
46	45.32	45.94	273.80	30.44	30.16	249.82
47	45.40	46.18	273.98	30.62	30.20	250.48
48	46.56	47.40	274.98	30.74	30.36	252.32
49	46.74	47.60	275.18	30.78	30.36	257.78
50	48.40	49.28	276.84	31.62	31.20	260.40
51	48.74	49.64	277.22	31.90	31.40	261.40
52	50.18	50.78	278.60	32.66	32.28	263.76
53	51.68	52.44	280.34	33.94	33.62	266.06
54	52.78	53.56	281.68	34.82	34.44	267.86
55	53.52	54.44	282.52	35.32	35.02	269.26
56	54.32	54.82	282.80	35.40	35.20	270.30
57	54.90	55.64	283.56	35.82	35.66	271.44
58	55.82	56.60	284.26	36.12	36.02	272.72
59	56.86	58.02	285.34	36.78	36.70	274.30
60	57.52	59.06	286.02	37.20	37.20	275.22

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	0	0	0	0	0
3	0	0	0	0	0
4	2	0	0	0	0
5	3	0	0	0	2
6	10	0	0	0	14
7	23	0	0	0	26
8	44	0	0	0	27
9	63	0	0	0	28
10	70	0	0	0	28
11	71	0	0	0	29
12	72	0	0	0	29
13	72	0	0	0	29
14	72	0	0	0	29
15	72	0	0	0	30
16	72	0	0	0	30
17	72	0	0	0	30
18	73	0	0	0	30
19	73	0	0	0	30
20	73	0	0	0	30
21	73	0	0	0	31
22	73	0	0	0	31
23	74	0	0	0	31
24	74	0	0	0	31
25	74	0	0	0	31
26	74	0	0	0	31
27	74	0	0	0	32
28	74	0	0	0	32
29	75	0	0	0	32
30	75	0	0	0	32

31	75	0	0	0	32
32	75	0	0	0	32
33	75	0	0	0	32
34	75	0	0	0	33
35	75	0	0	0	33
36	75	0	0	0	33
37	75	0	0	0	33
38	75	0	0	0	33
39	75	0	0	0	33
40	75	0	0	0	34
41	75	0	0	0	34
42	75	0	0	0	34
43	74	0	0	0	33
44	74	0	0	0	33
45	74	0	0	0	33
46	74	0	0	0	34
47	74	0	0	0	34
48	74	0	0	0	34
49	74	0	0	0	35
50	74	0	0	0	35
51	74	0	0	0	35
52	74	0	0	0	35
53	74	0	0	0	35
54	74	0	0	0	35
55	74	0	0	0	35
56	74	0	0	0	35
57	74	0	0	0	35
58	74	0	0	0	36
59	74	0	0	0	36
60	74	0	0	0	36

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	7.3	7.7
Inoculum Control R2	7.3	7.7
Procedure Control	7.4	8.5
Test Item R1	-	7.8
Test Item R2	-	7.8
Toxicity Control	-	8.3

 R  =  Replicate

-      =  The pH of the contents of the test item and toxicity control vessels were not measured prior to the addition of the inoculum due to the risk of test item being lost from the test system by adherence to the pH probe.

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 – 100 P. Rec 2 – 100	0.1 0.1 0.1 0.1 0.1 0.1	Non detected Non detected 108 106 103 103	- - 108 106 102 103
60	Control – R1 Control – R2 100 – R1 100 – R2 P. Rec 1 – 100 P. Rec 2 – 100	0.1 0.1 0.1 0.1 0.1 0.1	<LOQ Non detected 78.0 83.3 98.1 99.2	- - 78 83 98 99

R = Replicate

- = Not applicable

P. Rec = Procedural recovery

LOQ = Limit of Quantification (0.94 mg/L)

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

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

There were significant statistical differences between the Day 60 inoculum control and test item biological oxygen (BOD) values. This significant statistical difference was considered to be due to the test item not being biodegradable under the conditions of this test and not that the test item was exhibiting an inhibitory effect at the test concentration employed in the study. This was confirmed by the toxicity control vessel which attained greater than 25% biodegradation by Day 14 of the test, thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro‑organisms used in the test.

Aniline (procedure control) attained 72% biodegradation after 14 dayswith greater than 60% degradation being attained in a 10-Day window.  After 28 and 60 days 74% biodegradationwas attained.

Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours showed measured concentrations of 108 and 106% of nominal were obtained. A decline in measured test concentration was observed on Day 60 to 78 and 83% of nominal, (calculated to be a 22 and 17% 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. Although the test item did not adsorb to the inoculum initially, which was

confirmed by the validation work of the analytical method and the results from the test item vessels on Day 0, with a log kow of greater than 4, it is likely that the test item may have adsorbed to the inoculum and/or glassware over the 60 day study period although it is beyond the scope of this study to confirm this.

Description of key information

OECD Guideline 301F, EU Method C4-D, EPA OPPTS 835.3110, GLP, Key study, validity 1:

0% biodegradation after 28 and 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, two experimental studies are available.

The most recent study (Covance, 2019), 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 item attained no biodegradation after 28 days and 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 showed measured concentrations of 108 and 106% of nominal were obtained. A decline in measured test concentration was observed on Day 60 to 78 and 83% of nominal, (calculated to be a 22 and 17% 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. Although the test item did not adsorb to the inoculum initially, which was confirmed by the validation work of the analytical method and the results from the test item vessels on Day 0, with a log kow of greater than 4, it is likely that the test item may have adsorbed to the inoculum and/or glassware over the 60 day study period although it is beyond the scope of this study to confirm this.

The other study (Phytosafe, 2013) was also performed on the registered substance but according to the closed-bottle test method. The solution of the test substance (3.33 mg/L) in mineral medium was inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely full, closed bottle in the dark at constant temperature (20 +/- 1 °C). Degradation was followed by analysis of dissolved oxygen over a 28 -day period. Additional analysis assessments were performed at test initiation and at test completion in the test vessels for quantification of the test item, so as to judge upon the primary degradation over the test period. In the test item treatment, the oxygen uptake was similar to that of the controls for the entire test period. At the end of test, analytical check of the test item in the test solutions and in the toxic control proved that the initial value still remained. Therefore, the test substance was considered as not easily biodegradable (removal within 28 days < 10%). This result supports the key study.