Adenine

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:

2018-08-09 to 2018-09-06

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)

Version / remarks:

adopted July 17, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of activated sludge: Municipal sewage treatment plant, 31137 Hildesheim, Germany; receiving predominantly municipal sewage and hardly any industrial chemical waste
- Pretreatment: The activated sludge was washed twice with tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration with CO2 free air until test start (2 days).
- Concentration of sludge: The amount of inoculum used to initiate inoculation was 3.84 mL/L (25.1 mg/L dw).
- Initial cell/biomass concentration: Colony forming units (CFU) of the inoculum were determined prior to test start by standard dilution plate count: 1.30 x 109 CFU/L. The CFU concentration of the inoculum corresponds to approx. 0.50 x 107 CFU/L in the final test solution.

Duration of test (contact time):

28 d

Initial conc.:

88 mg/L

Based on:

ThOD

Remarks:

corresponding to an oxygen demand of 51.9 mg O2/L

Initial conc.:

32 mg/L

Based on:

ThOD

Remarks:

corresponding to an oxygen demand of 94.4 mg O2/L with nitrification

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 301 F
- Additional substrate: No
- Test temperature: Nominal: 20 - 24, ± 1 °C; actual: 21.0 – 21.3 °C
- pH: 7.71 - 7.77 (start), 5.31 - 7.29 (end); replicates outside of the range of 6.0 – 8.5 at test end were not evaluated
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: incubator
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: shaker
- Measuring equipment: OxiTop measuring system

SAMPLING
- Sampling frequency: 360 measuring points (every 112 minutes) during the 28 d incubation period
- Sampling method: OxiTop measuring system

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Functional control: 1
- Toxicity control: 1

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Not performed

Test performance:

The percentage degradation of the functional control reached the pass level of 60 % within 3 days (validity criterion: 60 % after 14 days).

The mean oxygen depletion in the inoculum control was 36.1 mg oxygen/L on day 28 (validity criterion: < 60 mg oxygen/L).

The pH-value of all test replicates was in the range of 6.0 - 8.5 at the end of the test, except for the test item replicates with a test item concentration of 88 mg/L. But since the BOD of these replicates was outside of the measurement range of the respirometer, these replicates were not evaluated anyway.

The difference of extremes of replicate values of removal of the test item at the end of the test was less than 20 % (1% difference on day 28).

The percentage degradation of the toxicity control reached the pass level of 25 % within 2 days (validity criterion: 25 % after 14 days).

Key result

Parameter:

% degradation (O2 consumption)

Value:

99

Sampling time:

28 d

Details on results:

At test end, the concentration of nitrite and nitrate was determined by photometric measurement from aliquots of the test item replicates. The corresponding oxygen consumption was calculated with the factors 3.43 for nitrite and 4.57 for nitrate, as given in OECD 301F. In both test item replicates the formation of oxidized nitrogen was determined. Accordingly, the biodegradation was calculated with the ThODNO3. Both test item replicates reached the 10% level (beginning of biodegradation) on day 5 and the 60% pass level on day 10. The mean biodegradation on day 28 was 99%. ThODNO3 of 94.4 mg O2/L (i.e. with nitrification) was considered, since the concentration of nitrate increased during the test.

Results with reference substance:

The pass level for ready biodegradation of 60% degradation was reached on day 3. The validity criterion that the degradation should be ≥ 60% after 14 d was fulfilled. The adaptation phase changed to the degradation phase within 2 days (degradation > 10%), with a maximum of 89% on day 27.

Table 1: Biological Oxygen Demand (BOD) of Inoculum Control, Functional Control, Test Item (88 mg/L) and Toxicity Control

Date	[d]	BOD [mg O2/L]
		Inoculum control	Functional Control		Test item (88 mg/L)				Toxicity Control Test Item (88 mg/L) + Reference Item
			R1		P1		P2		T1
		mean	gross	net	gross	net	gross	net	gross	net
10.08.2018	1	1.3	4.3	3.0	1.5	0.2	2.7	1.4	3.8	2.5
11.08.2018	2	4.1	31.9	27.8	3.3	-0.8	5.9	1.8	35.8	31.7
12.08.2018	3	6.9	37.2	30.2	6.5	-0.4	9.7	2.8	52.5	45.6
13.08.2018	4	10.1	42.0	31.9	11.0	0.9	15.0	5.0	73.9	63.8
14.08.2018	5	12.6	45.9	33.2	20.3	7.6	26.7	14.0	94.5	81.8
15.08.2018	6	14.9	50.2	35.3	35.3	20.5	43.7	28.8	107.3	92.5
16.08.2018	7	16.8	54.6	37.8	51.7	34.9	61.6	44.8	118.9	102.1
17.08.2018	8	18.2	57.7	39.4	64.8	46.6	73.9	55.7	135.7	117.4
18.08.2018	9	20.5	60.9	40.4	75.2	54.7	87.9	67.4	162.9	142.4
19.08.2018	10	22.5	64.2	41.6	89.0	66.5	108.3	85.8	199.8	177.3
20.08.2018	11	24.1	66.2	42.1	108.2	84.1	136.3	112.2	232.8	208.6
21.08.2018	12	25.5	68.2	42.7	137.0	111.5	173.1	147.6	254.6	229.1
22.08.2018	13	26.7	69.9	43.2	175.4	148.7	212.3	185.6	269.3	242.6
23.08.2018	14	27.7	71.4	43.7	211.8	184.1	238.0	210.3	277.9	250.3
24.08.2018	15	28.4	72.1	43.8	231.2	202.9	251.9	223.6	282.7	254.3
25.08.2018	16	29.8	73.3	43.5	243.3	213.5	261.5	231.7	285.9	256.2
26.08.2018	17	30.8	74.5	43.6	251.4	220.5	268.3	237.5	287.8	256.9
27.08.2018	18	31.9	75.3	43.4	257.0	225.1	273.1	241.2	289.0	257.1
28.08.2018	19	32.5	76.1	43.6	261.5	229.0	276.6	244.2	289.1	256.6
29.08.2018	20	32.8	76.1	43.3	264.8	232.0	278.6	245.8	289.0	256.2
30.08.2018	21	33.0	76.4	43.4	267.3	234.3	279.8	246.8	289.0	256.0
31.08.2018	22	33.5	77.4	43.9	269.9	236.4	282.2	248.7	289.0	255.5
01.09.2018	23	34.4	78.4	43.9	271.8	237.4	283.8	249.3	289.0	254.6
02.09.2018	24	34.8	78.9	44.1	272.8	237.9	285.1	250.3	289.0	254.2
03.09.2018	25	35.2	78.9	43.7	274.8	239.6	285.8	250.6	289.0	253.8
04.09.2018	26	35.5	79.5	44.0	276.0	240.5	286.2	250.6	288.4	252.9
05.09.2018	27	35.8	80.3	44.5	277.4	241.5	286.7	250.8	288.1	252.2
06.09.2018	28	36.1	80.5	44.4	278.2	242.0	287.9	251.8	287.6	251.5

Table 2: Biological Oxygen Demand (BOD) of Inoculum Control, Functional Control and Test Item (32 mg/L)

Date	[d]	BOD [mg O2/L]
		Inoculum control	Functional Control		Test item (32 mg/L)
			R1		P1		P2
		mean	gross	net	gross	net	gross	net
10.08.2018	1	1.3	4.3	3.0	2.3	1.0	2.2	0.9
11.08.2018	2	4.1	31.9	27.8	4.7	0.6	4.6	0.5
12.08.2018	3	6.9	37.2	30.2	8.6	1.7	8.5	1.6
13.08.2018	4	10.1	42.0	31.9	15.5	5.4	15.0	5.0
14.08.2018	5	12.6	45.9	33.2	26.0	13.4	25.0	12.4
15.08.2018	6	14.9	50.2	35.3	38.4	23.5	37.5	22.7
16.08.2018	7	16.8	54.6	37.8	47.7	30.9	46.7	29.9
17.08.2018	8	18.2	57.7	39.4	56.9	38.7	55.9	37.7
18.08.2018	9	20.5	60.9	40.4	68.8	48.3	68.1	47.6
19.08.2018	10	22.5	64.2	41.6	85.1	62.6	84.7	62.1
20.08.2018	11	24.1	66.2	42.1	104.0	79.9	103.0	78.9
21.08.2018	12	25.5	68.2	42.7	112.9	87.4	111.4	85.9
22.08.2018	13	26.7	69.9	43.2	116.5	89.8	114.8	88.1
23.08.2018	14	27.7	71.4	43.7	117.6	90.0	116.9	89.3
24.08.2018	15	28.4	72.1	43.8	118.8	90.5	117.8	89.5
25.08.2018	16	29.8	73.3	43.5	120.8	91.0	120.3	90.5
26.08.2018	17	30.8	74.5	43.6	122.2	91.4	121.3	90.5
27.08.2018	18	31.9	75.3	43.4	123.5	91.6	122.5	90.6
28.08.2018	19	32.5	76.1	43.6	124.0	91.5	123.5	91.0
29.08.2018	20	32.8	76.1	43.3	124.2	91.4	124.3	91.5
30.08.2018	21	33.0	76.4	43.4	124.5	91.5	124.6	91.6
31.08.2018	22	33.5	77.4	43.9	125.2	91.7	125.3	91.8
01.09.2018	23	34.4	78.4	43.9	126.4	91.9	126.8	92.3
02.09.2018	24	34.8	78.9	44.1	127.1	92.3	127.2	92.3
03.09.2018	25	35.2	78.9	43.7	127.9	92.7	127.9	92.7
04.09.2018	26	35.5	79.5	44.0	128.0	92.5	128.3	92.8
05.09.2018	27	35.8	80.3	44.5	128.0	92.2	129.5	93.7
06.09.2018	28	36.1	80.5	44.4	128.3	92.2	129.9	93.8

Table 3: Biodegradation [%] of the Test Item (32 mg/L) in Comparison to the Functional Control and Toxicity Control

Date	[d]	Biodegradation [%]
		Functional Control	Test item (32 mg/L)		Toxicity Control* Test Item (88 mg/L) + Reference Item
		R1	P1	P2	T
10.08.2018	1	6	1	1	1
11.08.2018	2	55	1	1	10
12.08.2018	3	60	2	2	15
13.08.2018	4	64	6	5	21
14.08.2018	5	66	14	13	26
15.08.2018	6	70	25	24	30
16.08.2018	7	76	33	32	33
17.08.2018	8	79	41	40	38
18.08.2018	9	81	51	50	46
19.08.2018	10	83	66	66	57
20.08.2018	11	84	85	84	67
21.08.2018	12	85	93	91	74
22.08.2018	13	86	95	93	78
23.08.2018	14	87	95	95	81
24.08.2018	15	87	96	95	82
25.08.2018	16	87	96	96	83
26.08.2018	17	87	97	96	83
27.08.2018	18	87	97	96	83
28.08.2018	19	87	97	96	83
29.08.2018	20	86	97	97	83
30.08.2018	21	87	97	97	83
31.08.2018	22	88	97	97	83
01.09.2018	23	88	97	98	82
02.09.2018	24	88	98	98	82
03.09.2018	25	87	98	98	82
04.09.2018	26	88	98	98	82
05.09.2018	27	89	98	99	81
06.09.2018	28	89	98	99	81

Table 4: pH-Values at Test Start and Test End

	pH-Value
	Start	End
Inoculum Control	7.75	1) 7.29
		2) 7.29
Functional Control	7.77	7.46
Test Item *	7.7	1) 5.53
(88 mg/L)		2) 5.31
Toxicity Control	7.72	6.46
Test Item	7.77	1) 6.93
(32 mg/L)		2) 6.92

1), 2) = First, second replicate

* The pH was outside of the range of 6.0 – 8.5 at test end. These replicates were not evaluated, see above

Table 5: Nitrite and Nitrate Concentrations at Test End

	Nitrite-N	Nitrate-N	corresponding Oxygen consumption	Biodegradation calculated with nitrification after 28 days
	[mg/L]	[mg/L]	[mg/L]	[%]
Test item repl. P1*	0.007	43.6	199	-*
Test item repl. P2*	0	43.8	200	-*
Test item repl. P1N	0.004	18	82.3	98
Test item repl. P2N	0.005	17.7	80.9	99
Toxicity control T*	0.267	19.6	180	81*

*) manometric data out of range

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In a valid and GLP-compliant Manometric Respirometry Test according to OECD TG 301 F the test item was found to be readily biodegradable by OECD criteria (99% biodegradation after 28 days).

Executive summary:

The ready biodegradability of the test item was determined with non-adapted activated sludge in a GLP-compliant Manometric Respirometry Test according to OECD TG 301 F for a period of 28 days. The test item concentration selected as appropriate was 88 mg/L, corresponding to a ThOD of 51.9 mg O₂/L and 32 mg/L test item with 94.4mg O₂/L (in case of nitrification) per test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day. In result, the mean oxygen depletion in the inoculum control was 36.1 mg O₂/L on day 28. In order to check the activity of the test system sodium benzoate was used as functional control. The pass level of 60% was reached on day 3. The biodegradation reached a maximum of 89% degradation on day 27. In the toxicity control containing both test and reference item, 81% degradation occurred within 14 days, with 81% after 28 days. The degradation of the reference item was not inhibited by the test item. Both test item replicates reached the 10% level (beginning of biodegradation) on day 5 and the 60% pass level on day 10. The mean biodegradation on day 28 was 99%. ThOD_NO3 of 94.4 mg O₂/L (i.e. with nitrification) was considered, since the concentration of nitrate increased during the test. Since the test item replicates with a concentration of 88 mg/L were out of the measurement range of the respirometer and the pH was out of the range, these replicates were not considered for evaluation. The validity criteria of the guideline are fulfilled.

Description of key information

In a Manometric Respirometry Test according to OECD TG 301 F the test item was found to be readily biodegradable by OECD criteria (99% biodegradation after 28 days) (reference 5.2.1-1).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of the test item was determined with non-adapted activated sludge in a GLP-compliant Manometric Respirometry Test according to OECD TG 301 F for a period of 28 days. The test item concentration selected as appropriate was 88 mg/L, corresponding to a ThOD of 51.9 mg O₂/L and 32 mg/L test item with 94.4mg O₂/L (in case of nitrification) per test vessel. In the toxicity control containing both test and reference item, 81% degradation occurred within 14 days, with 81% after 28 days. The degradation of the reference item was not inhibited by the test item. Both test item replicates reached the10% level (beginning of biodegradation) on day 5 and the 60% pass level on day 10. In result, the mean biodegradation on day 28 was 99%. ThOD_NO3 of 94.4 mg O₂/L (i.e. with nitrification) was considered, since the concentration of nitrate increased during the test. The validity criteria of the guideline are fulfilled (reference 5.2.1-1).