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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 26, 2020 to January 20, 2021

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:

equivalent or similar to guideline

Guideline:

other: US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Method C.4-D of Commission Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: A mixed population of sewage treatment micro-organisms

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage. 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 21 ±1°C prior to use. 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).

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Preliminary Solubility Work: the test substance was partly soluble in water. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation

Test Substance Preparation
A nominal amount of test substance (50 mg) was dispersed in mineral medium (350 mL) and subjected to high shear mixing (at approximately 7500 rpm for 15 minutes) prior to the addition of inoculum (5 mL) and made to a volume of 500 mL with mineral media to give the test concentration of 100 mg/L. The inoculum control vessels were prepared in a similar manner without the addition of test substance.

Reference Substance Preparation:
A reference substance, 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 reference substance, (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 using a Hach HQ40d Flexi handheld meter. The addition of inoculum (5 mL) was then added and adjusted 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.

Toxicity control:
A toxicity control, containing the test substance and aniline, was prepared in order to assess any toxic effects of the test substance on the sewage treatment micro-organisms used in the test. A nominal amount of test substance (50 mg) was dispersed in mineral medium (350 mL) and subjected to high shear mixing (at approximately 7500 rpm 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. 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.

Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL glass bottles:
a) Three replicate bottles containing inoculated mineral medium to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference substance, aniline, at a concentration of 100 mg/L to act as the procedure control.
c) Three replicate bottles containing the test substance and inoculated mineral medium at a concentration of 100 mg/L.
d) Two replicate bottles containing the test substance at a concentration of 100 mg/L in inoculated mineral medium and the reference substance, 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 substance were added to the mineral medium. The pH of all vessels was measured using a Hach HQ40d Flexi handheld meter. All inoculum control, test substance, 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 four times a day on the hard disk drive of a non-dedicated computer. The test was conducted in diffuse light at a temperature of 20 to 21ºC. On Day 28, two inoculum controls, one procedure control, two test substance and one toxicity control vessel were sampled for pH. The remaining vessels which were not sampled were 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.

Assessments
(a) Oxygen Consumption Measurements: The Biological Oxygen Demand (BOD) values for the inoculum control, test item, procedure control and the toxicity control were measured daily.
(b) Temperature Measurements: The temperature of the water bath was recorded daily.
(c) pH Measurements: In order to confirm whether the pH of the test preparations changed, the pH was measured using a Hach HQ40d Flexi handheld meter on Days 0 and 28.

Data evaluation
The Theoretical Oxygen Demand (ThOD) and percentage biodegradation in terms of oxygen consumption were calculated. For more details kindly refer to attached background material section of the IUCLID.

Validation Criteria and Biodegradation
The BOD of the inoculated mineral medium (control) is normally 20 to 30 mg O2/L and should not exceed 60 mg O2/L after 28 days. Values higher than 60 mg O2/L require critical examination of the data and the experimental technique. If the pH of the inoculated test vessels after 28 days is outside the range 6.0 to 8.5 and the biodegradation rate of the test substance is less than 60%, the test should be repeated at a lower test concentration. The test is considered valid if the difference between extremes of replicate BOD values at the time the plateau is reached, at the end of the test or at the end of the 10 Day window, as appropriate, is less than 20%. The toxicity control (test substance and aniline) should attain greater >=25% biodegradation by Day 14 for the test substance to be considered as non-inhibitory. The percentage biodegradation of aniline calculated from oxygen consumption values must be >=60% (in a 10 Day window) after 14 days. Test substances giving a measured BOD value which is >=60% of the ThOD value (within 28 days) are regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation rate exceeding 10%. However, according to the revised introduction to the OECD Guidelines for Testing of Chemicals, Section 3 (OECD, 2006), if a test on a mixture is performed and it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results.

Reference substance:

aniline

Remarks:

(C6H5NH2)

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 68

Sampling time:

28 d

Details on results:

The test item attained 68% biodegradation after 28 d. As the test item was UVCB, the 10 d window criteria does not apply and therefore the test item can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301

Results with reference substance:

Aniline (procedure control) attained 72% biodegradation after 14 days with greater than 60% degradation being attained in a 10 day window. After 28 days 78% biodegradation was attained thereby confirming the suitability of the inoculum and test conditions.

Results

Definitive Test

Daily BOD values for the test substance, procedure control, toxicity control and inoculum control vessels are given in Table 1. Percentage biodegradation values of the test substance and procedure control and the toxicity control are given in Table 2. The pH values of each individual vessel on 0 and 28 d are given in Table 3. For Figures, kindly refer to the picture/graph section of the IUCLID. The calculated Theoretical Oxygen Demand values for the test and procedure control were 293 and 309 mg O2/L, respectively.

Table 1: Biological Oxygen Demand Values

Day	BOD (mg O2/L)
	Inoculum Control		Procedure Control	Test substance		Toxicity Control
	R1	R2		R1	R2
0	0.00	0.00	0.00	0.00	0.00	0.00
1	1.58	1.66	1.38	2.42	1.58	2.66
2	3.34	3.46	2.66	14.74	3.16	9.46
3	3.62	3.62	2.88	43.44	34.44	43.82
4	3.62	3.62	3.24	58.40	50.98	107.46
5	4.92	5.58	8.74	74.80	65.94	182.26
6	6.66	7.54	47.98	94.96	79.22	253.28
7	7.28	8.34	132.98	112.50	100.00	301.26
8	7.28	8.34	180.76	129.98	121.12	343.86
9	9.88	10.82	208.70	151.98	145.06	376.94
10	10.54	11.50	217.88	159.64	154.72	388.88
11	11.78	12.62	225.20	166.56	161.68	397.34
12	12.50	13.16	230.70	171.84	167.02	403.50
13	13.28	13.86	234.74	176.60	171.76	408.54
14	14.32	14.92	238.16	181.18	176.42	413.08
15	15.00	15.78	240.78	184.64	180.38	417.08
16	15.00	15.78	242.98	188.00	184.26	421.88
17	15.74	16.36	245.56	191.64	188.18	427.16
18	16.62	17.20	247.90	195.26	191.76	431.08
19	16.66	17.20	248.74	197.68	194.00	433.46
20	16.66	17.20	250.56	201.12	197.38	436.86
21	18.86	19.32	253.60	205.84	201.76	442.20
22	19.32	19.66	255.10	209.04	204.50	446.86
23	20.04	20.04	256.74	212.84	207.16	450.86
24	20.66	20.66	258.32	216.62	209.54	455.02
25	20.74	20.70	259.28	219.46	211.04	457.98
26	21.20	20.90	260.94	222.54	213.12	461.20
27	21.58	21.40	262.40	224.96	214.84	463.48
28	22.24	22.08	264.26	227.70	217.00	465.86

R = Replicate

Table 2: Percentage Biodegradation Values

Day	Biodegradation (%)
	Procedure Control	Test substance			Toxicity Control
		R1	R2	Mean
0	0	0	0	0	0
1	0	0	0	0	0
2	0	4	0	2	1
3	0	14	11	13	7
4	0	19	16	18	17
5	1	24	21	23	29
6	13	30	25	28	41
7	41	36	31	34	49
8	56	42	39	41	56
9	64	48	46	47	61
10	67	51	49	50	63
11	69	53	51	52	64
12	71	54	53	54	65
13	72	56	54	55	66
14	72	57	55	56	66
15	73	58	56	57	67
16	74	59	58	59	68
17	74	60	59	60	68
18	75	61	60	61	69
19	75	62	60	61	69
20	76	63	62	63	70
21	76	64	62	63	70
22	76	65	63	64	71
23	77	66	64	65	72
24	77	67	64	66	72
25	77	68	65	67	73
26	78	69	66	68	73
27	78	69	66	68	73
28	78	70	66	68	74

R= Replicate

Table 3: pH Values of the Test Preparations on Days 0 and 28

Test Vessel	pH
	Day 0	Day 28
Inoculum Control R1	7.4	7.3
Inoculum Control R2	7.3	7.3
Procedure Control	7.3	7.9
Test Item R1	7.4	7.3
Test Item R2	7.4	7.3
Toxicity Control	7.4	7.7

R  =   Replicate

Validation Criteria and Biodegradation:

The mean BOD of the inoculated mineral medium (control) was 22.16 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines. The pH of the inoculated test substance vessels on Day 28 was 7.3 and hence satisfied the validation criterion given in the OECD Test Guidelines. The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines. The test substance attained 68% biodegradation after 28 days. As the test substance was UVCB, the 10-day window criteria does not apply and therefore the test substance can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301 The toxicity control attained 66% biodegradation after 14 days and 74% biodegradation after 28 days thereby confirming that the test substance was not toxic to the sewage treatment micro-organisms used in the test. Aniline (procedure control) attained 72% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. After 28 days 78% biodegradation was attained thereby confirming the suitability of the inoculum and test conditions.

Total Organic Carbon Confirmation

Total Organic Carbon of the diluted aniline stock solution confirmed that it had been prepared correctly.

Conclusion

The test substance attained 68% biodegradation after 28 days. As the test substance was UVCB, the 10-day window criteria does not apply and therefore the test substance can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under study conditions, the test substance was considered to be readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the test substance using manometric respirometry method, according to OECD Guideline 301F, in compliance with GLP. Three replicate bottles of the test substance at a concentration of 100 mg/L were exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at 20 to 21°C for 28 days. The biodegradation of the test substance was assessed by the measurement of daily oxygen consumption values for 28 days. Three replicate bottles of control solutions with inoculum and two replicate bottles of the reference substance, aniline (100 mg/L), and a toxicity control (100 mg/L test + 100 mg/L aniline) were used for validation purposes. The test substance attained 68% biodegradation after 28 days. As the test substance was UVCB, the 10-day window criteria does not apply and therefore the test substance can be considered to be readily biodegradable. The toxicity control attained 66% biodegradation after 14 days and 74% biodegradation after 28 days thereby confirming that the test substance was not toxic to the sewage treatment micro organisms used in the test. Aniline (procedure control) attained 72% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window.  After 28 days 78% biodegradation was attained thereby confirming the suitability of the inoculum and test conditions. The pH of the inoculated test substance vessels on Day 28 was 7.3. The mean BOD of the inoculated mineral medium (control) was 22.16 mg O2/L after 28 days. The difference between extremes of replicate BOD values at the end of the test was less than 20%. The test met all the validity criteria. Under the study conditions, the test substance was considered to be readily biodegradable (Labcorp, 2021).

Description of key information

Based on the study results, the test subsatance is considered to be readily biodegradable (68% after 28 days).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

A study was conducted to determine the ready biodegradability of the test substance using manometric respirometry method, according to OECD Guideline 301F, in compliance with GLP. Three replicate bottles of the test substance at a concentration of 100 mg/L were exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at 20 to 21°C for 28 days. The biodegradation of the test substance was assessed by the measurement of daily oxygen consumption values for 28 days. Three replicate bottles of control solutions with inoculum and two replicate bottles of the reference substance, aniline (100 mg/L), and a toxicity control (100 mg/L test + 100 mg/L aniline) were used for validation purposes. The test substance attained 68% biodegradation after 28 days. As the test substance was UVCB, the 10-day window criteria does not apply and therefore the test substance can be considered to be readily biodegradable. The toxicity control attained 66% biodegradation after 14 days and 74% biodegradation after 28 days thereby confirming that the test substance was not toxic to the sewage treatment micro organisms used in the test. Aniline (procedure control) attained 72% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window.  After 28 days 78% biodegradation was attained thereby confirming the suitability of the inoculum and test conditions. The pH of the inoculated test substance vessels on Day 28 was 7.3. The mean BOD of the inoculated mineral medium (control) was 22.16 mg O2/L after 28 days. The difference between extremes of replicate BOD values at the end of the test was less than 20%. The test met all the validity criteria. Under the study conditions, the test substance was considered to be readily biodegradable (Labcorp, 2021).