2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

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:

16 Feb 2016 to 15 Mar 2016

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:

1992

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Fresh samples of activated sludge were withdrawn on 16th February 2016 (day of test start) from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany, which is mainly fed with municipal wastewater.
- Preparation of inoculum for exposure: Since the sludge had not been taken from a high rate treatment plant and was not thought to contain inhibitors, the samples were not washed with mineral medium. After the arrival at the laboratory, they were kept aerobic until use. The sludge was left for settlement for ca. one hour. Subsequently the supernatant was discarded and the concentration of suspended solids was measured in the remaining sludge. The concentration was adjusted to 4.1 g/L and verified by dry mass measurement. The concentration used in the test was 29.6 mg dry mass/litre (7.40 mg dry mass/250 mL).

Duration of test (contact time):

28 d

Initial conc.:

50 mg/L

Based on:

test mat.

Remarks:

corresponding to 115.5 mg ThOD considering total nitrification and 51.3 mg ThOD considering the total lack of nitrification.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST WATER
The mineral medium prepared for the test contained 10 mL/L of the mineral stock solution (a) and 1 mL/L of the mineral stock solutions (b)–(d), diluted with deionized water free from inhibitory concentrations of toxic substances (e.g. Cu2+ ions). See 'Any other information on materials and methods' for abbrevations. The organic carbon content was checked at regular intervals (monthly) by dissolved organic carbon (DOC) analysis. The maximum DOC content was verified in the last sampling before and the first sampling after medium preparation and was found to be 0.45 mg/L, corresponding to 1.7 % of the organic carbon content introduced by the test material (26.08 mg C per liter). According to the OECD 301 guideline, a value of 10 % should not be surpassed. An overview of the reagents used for the preparation of the stock solution is tabulated in 'Any other information on materials and methods incl. tables'.

PREPARATION OF TEST VESSELS
- Reference substance:The concentration in the test vessels with reference item (procedural control, toxicity control) was 100 mg per litre mineral test medium (25 mg/250 mL). The solution of the reference substance in the mineral medium equivalent to a concentration of 100 mg/L was prepared using an aqueous stock solution (10 g/L). The aqueous stock solution was prepared by dissolving 500 mg of the reference substance in 50 mL deionised water. 2.5 mL of the aqueous stock solution were then filled up to 250 mL with inoculated mineral medium.
- Test item: The concentration in the test assays were 50 mg per litre mineral test medium (12.5 mg/250 mL). 50 mg test item correspond to 115.5 mg theoretical oxygen demand (ThOD) considering total nitrification and 51.3 mg ThOD considering the total lack of nitrification. No emulsifiers or solvents were used. The required amount to ensure a final concentration of 50 mg/L (12.5 mg per 250 mL) was added directly on a weight basis via Teflon discs. Subsequently, the mineral medium was added to the vessels. A further solution containing both test and reference item at the same concentrations as in the individual solutions (test item 50 mg/L, reference item 100 mg/L) was prepared to determine the possible toxicity of the test item against the inoculum. For this, the required amount of test item was added directly on a weight basis via Teflon discs. Subsequently, the required volume of aqueous reference stock solution and mineral medium were added to the vessels. Further flasks with mineral medium only were prepared for inoculum controls. Abiotic controls were also prepared to measure any possible abiotic degradation; a solution of the test item at about 50 mg/L was sterilized by the addition of 1 mL/L HgCl2 (10 g/L). In all test assays except of the abiotic controls, 1.82 mL of the inoculum stock solution were transferred for inoculation and the pH was measured. Before test start the pH values of all solutions were determined. At the end of the test the pH values of all solutions were determined again.

TEST CONDITIONS
- Apparatus: The test flasks (500 mL reaction vessels, labeled with the necessary information to ensure unique identification) were incubated under continuous stirring in SAPROMAT respirometer (VOITH Inc.). Oxygen consumption was measured and recorded continuously throughout the duration of the test.
- Light conditions: Darkness
- Test temperature: 22 °C, maintained with a built-in thermostat and checked twice per week.
- pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum. At the end of incubation, the pH was measured again in each test flask.

CALCULATIONS
The percentage biodegradation of the test item was calculated using the BOD and ThOD values. The ThOD was calculated with and without nitrification effects. The ThOD values for the reference substance, the test item and the toxicity control were determined as follows:
- ThOD(NH3) test item: 1.03 mg O2/mg test item
- ThOD(NO3) test item: 2.31 mg O2/mg test item
- ThOD(Na) Benzoate: 1.67 mg O2/mg reference item
- ThOD(NH3) toxicity control: 1.45 mg O2/mg substance mixture
- ThOD(NO3) toxicity control: 1.88 mg O2/mg substance mixture

Biodegradation was calculated based on both ThOD(NH3) and ThOD(NO3).

Reference substance:

benzoic acid, sodium salt

Test performance:

The Manometric Respirometry Test fulfills the validity criteria of the guideline:
- With a maximum of 4 %, the difference of extremes of replicate values of the removal of the test item at the end of the test was less than 20 %.
- The percentage degradation of the reference item has exceeded the pass level of 60 % by day 14.
- The oxygen uptake of the inoculum blank is <60 mg/L in 28 days and the pH value was inside the range of 6.0 - 8.5.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

based on ThOD(NH3)

Value:

1.9

St. dev.:

2.8

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

based on ThOD(NO3)

Value:

0.9

St. dev.:

1.2

Sampling time:

28 d

Details on results:

The percent biodegradation of the test item was calculated based on the theoretical oxygen demand of 1.03 mg O2/mg test item ThOD(NH3) and of 2.31 mg O2/mg test item ThOD(NO3). The average percent biodegradation of the test item in the test media was found to be 1.9 % (SD = 2.8 %) based on ThODNH3 and 0.9 % (SD = 1.2 %) based on ThODNO3, respectively, after 28 days of incubation. No biodegradation within the 10-day-window could be calculated since the start criterium of the window (10 % degradation rate) was not reached within the 28 days of incubation. Details on results on cumulated oxygen consumption per day and percentage of degradation considering the total nitrification (ThOD NH3 and NO3) are tabulated in 'Any other information on results incl. tables' for details on results.
- Toxicity control: The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the ThOD of the test item and the reference item. Within 14 days of exposure, biodegradation amounted to 67 % based on the ThOD(NH3) and to 51 % based on the ThOD(NO3). Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 50 mg/L because biodegradation in the toxicity control was >25 % within 14 days.
- Procedural control: In the procedural controls, the reference item was degraded by an average of 87 % by Exposure Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 94 %.

Results with reference substance:

The reference item was degraded by an average of 87 % by Exposure Day 14, thus confirming suitability of the activated sludge. At the end of the test (Day 28), the reference item was degraded by an average of 94 %.

Table: Cumulated Oxygen Consumption (mg O2/L) in the Test Flasks

Time	Inoculum blank		Test suspension		Abiotic control		Procedural control		Toxicity control
days	1	2	1	2	1	2	1	2	1	2
0	0	0	0	0	0	0	0	0	0	0
1	5	6	5	4	0	0	56	54	61	60
2	8	9	7	6	0	0	83	85	92	89
3	9	10	9	8	0	0	102	102	114	113
4	11	12	11	9	0	0	118	119	133	130
5	12	13	12	11	0	0	129	140	144	137
6	13	14	13	12	0	0	136	147	150	142
7	13	14	13	12	0	0	142	151	153	144
8	13	15	14	13	0	0	146	154	156	147
9	14	16	14	14	0	0	148	157	158	149
10	14	16	15	14	0	0	150	159	160	150
11	14	16	15	14	0	0	150	159	160	150
12	14	16	15	14	0	0	150	159	160	150
13	18	17	16	15	1	0	159	164	168	157
14	19	18	17	16	2	0	160	166	169	158
15	19	18	17	16	2	0	161	167	171	158
16	19	18	18	16	2	0	162	168	172	159
17	19	18	18	16	2	0	163	170	173	160
18	19	18	19	18	2	0	164	171	175	160
19	20	18	19	18	2	0	165	171	176	161
20	20	18	19	18	2	0	166	172	177	162
21	20	18	20	18	2	0	167	173	178	163
22	20	18	20	18	2	0	168	174	179	164
23	20	18	20	18	2	0	170	174	180	165
24	21	18	21	18	2	0	171	175	181	166
25	21	18	21	18	2	0	172	176	181	167
26	21	18	21	18	2	0	173	177	182	167
27	21	18	22	20	2	0	174	178	183	168
28	22	18	22	20	2	0	175	179	184	169

Oxygen consumption. Cumulated consumption (mg O2/L) at each day in the 28 day incubation period.

Single values of the parallel test vessels.

Test suspension: 50 mg/L;

procedural control: 100 mg/L;

Toxicity control: 150 mg/L.

 

Table: Percent Degradation in the Test Flasks considering the total lack of nitrification ThOD(NH3) 

	Test suspension		Abiotic control		Procedural control		Toxicity control
days	1	2	1	2	1	2	1	2
0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
1	-1.0	-2.9	0.0	0.0	30.3	29.1	25.5	25.0
2	-2.9	-4.9	0.0	0.0	44.7	45.9	38.3	36.9
3	-1.0	-2.9	0.0	0.0	55.5	55.5	48.0	47.5
4	-1.0	-4.9	0.0	0.0	63.9	64.5	55.8	54.4
5	-1.0	-2.9	0.0	0.0	70.0	76.6	60.4	57.1
6	-1.0	-2.9	0.0	0.0	73.6	80.2	62.7	59.0
7	-1.0	-2.9	0.0	0.0	77.2	82.6	64.0	59.9
8	0.0	-1.9	0.0	0.0	79.3	84.1	65.2	61.0
9	-1.9	-1.9	0.0	0.0	79.9	85.3	65.6	61.5
10	0.0	-1.9	0.0	0.0	81.1	86.5	66.6	62.0
11	0.0	-1.9	0.0	0.0	81.1	86.5	66.6	62.0
12	0.0	-1.9	0.0	0.0	81.1	86.5	66.6	62.0
13	-2.9	-4.9	2.2	0.0	85.0	88.0	69.1	64.0
14	-2.9	-4.9	4.4	0.0	85.0	88.6	69.1	64.0
15	-2.9	-4.9	4.4	0.0	85.6	89.2	70.0	64.0
16	-1.0	-4.9	4.4	0.0	86.2	89.8	70.5	64.5
17	-1.0	-4.9	4.4	0.0	86.8	91.0	70.9	64.9
18	1.0	-1.0	4.4	0.0	87.4	91.6	71.8	64.9
19	0.0	-1.9	4.4	0.0	87.7	91.3	72.1	65.2
20	0.0	-1.9	4.4	0.0	88.3	91.9	72.5	65.6
21	1.9	-1.9	4.4	0.0	88.9	92.5	73.0	66.1
22	1.9	-1.9	4.4	0.0	89.5	93.1	73.4	66.6
23	1.9	-1.9	4.4	0.0	90.7	93.1	73.9	67.0
24	2.9	-2.9	4.4	0.0	91.0	93.4	74.1	67.2
25	2.9	-2.9	4.4	0.0	91.6	94.0	74.1	67.7
26	2.9	-2.9	4.4	0.0	92.2	94.6	74.6	67.7
27	4.9	1.0	4.4	0.0	92.8	95.2	75.0	68.2
28	3.9	0.0	4.4	0.0	93.1	95.5	75.3	68.4

Percent degradation considering the total lack of nitrification. Degradation (%) at each day in the 28 day incubation period.

Single values of the parallel test vessels.

 

 

Table: Percent Degradation in the Test Flasks considering total nitrification ThOD(NO3) 

	Test suspension		Abiotic control		Procedural control		Toxicity control
days	1	2	1	2	1	2	1	2
0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
1	-0.4	-1.3	0.0	0.0	30.3	29.1	19.7	19.3
2	-1.3	-2.2	0.0	0.0	44.7	45.9	29.6	28.5
3	-0.4	-1.3	0.0	0.0	55.5	55.5	37.1	36.7
4	-0.4	-2.2	0.0	0.0	63.9	64.5	43.1	42.0
5	-0.4	-1.3	0.0	0.0	70.0	76.6	46.6	44.1
6	-0.4	-1.3	0.0	0.0	73.6	80.2	48.4	45.6
7	-0.4	-1.3	0.0	0.0	77.2	82.6	49.5	46.3
8	0.0	-0.9	0.0	0.0	79.3	84.1	50.3	47.2
9	-0.9	-0.9	0.0	0.0	79.9	85.3	50.7	47.5
10	0.0	-0.9	0.0	0.0	81.1	86.5	51.4	47.9
11	0.0	-0.9	0.0	0.0	81.1	86.5	51.4	47.9
12	0.0	-0.9	0.0	0.0	81.1	86.5	51.4	47.9
13	-1.3	-2.2	1.0	0.0	85.0	88.0	53.4	49.5
14	-1.3	-2.2	2.0	0.0	85.0	88.6	53.4	49.5
15	-1.3	-2.2	2.0	0.0	85.6	89.2	54.1	49.5
16	-0.4	-2.2	2.0	0.0	86.2	89.8	54.4	49.8
17	-0.4	-2.2	2.0	0.0	86.8	91.0	54.8	50.2
18	0.4	-0.4	2.0	0.0	87.4	91.6	55.5	50.2
19	0.0	-0.9	2.0	0.0	87.7	91.3	55.7	50.3
20	0.0	-0.9	2.0	0.0	88.3	91.9	56.0	50.7
21	0.9	-0.9	2.0	0.0	88.9	92.5	56.4	51.1
22	0.9	-0.9	2.0	0.0	89.5	93.1	56.7	51.4
23	0.9	-0.9	2.0	0.0	90.7	93.1	57.1	51.8
24	1.3	-1.3	2.0	0.0	91.0	93.4	57.3	51.9
25	1.3	-1.3	2.0	0.0	91.6	94.0	57.3	52.3
26	1.3	-1.3	2.0	0.0	92.2	94.6	57.6	52.3
27	2.2	0.4	2.0	0.0	92.8	95.2	58.0	52.7
28	1.7	0.0	2.0	0.0	93.1	95.5	58.2	52.8

Percent degradation considering total nitrification. Degradation (%) at each day in the 28 day incubation period.

Single values of the parallel test vessels.

Validity criteria fulfilled:

yes

Remarks:

See 'Test performance'

Interpretation of results:

not readily biodegradable

Conclusions:

The test item was not readily biodegradable under the conditions of the test according to the OECD TG 301F.

Executive summary:

STUDY DESIGN

At the Fraunhofer Institute for Molecular Biology and Applied Ecology the ready biodegradability of the test item was investigated at a concentration of 50 mg/L in a manometric respirometry test using domestic aerobic activated sewage sludge incubated at 22 °C in the dark over 28 days under continuous stirring. The manometric respirometry test was carried out according to the OECD guideline for Testing of Chemicals No. 301 F (1992) and the Council Regulation (EC) No 440/2008. The biochemical oxygen demand (BOD) was measured daily and the percentage degradation was calculated from the theoretical oxygen demand (ThOD) after correcting for the BOD of inoculum controls.

RESULTS

The average percent biodegradation of the test item in the test media was found to be 1.9 % (SD = 2.8 %) based on ThODNH3 and 0.9 % (SD = 1.2 %) based on ThODNO3, respectively, after 28 days of incubation. No biodegradation within the 10-day-window could be calculated since the start criterium of the window (10 % degradation rate) was not reached within the 28 days of incubation. In the toxicity control, containing both the test item and the reference item sodium benzoate, biodegradation was greater than 25 %. According to the guidelines this indicates that the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration. In the procedural controls, the reference substance sodium benzoate was degraded by an average of 87 % by exposure day 14, and reached an average biodegradation of 94 % by the end of the test (day 28), thus confirming suitability of the activated sludge. The difference of extremes of replicate values of the removal of the test item at the end of the test is less than 20 %. Therefore, the test can be considered as valid.

CONCLUSION

The test item was not readily biodegradable under the conditions of the test according to the guidelines listed above in 'Study design'.

Description of key information

Not readily biodegradable, OECD 301F, Manometric Respirometry Test, Simon 2016

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test item was investigated at a concentration of 50 mg/L in a manometric respirometry test using domestic aerobic activated sewage sludge incubated at 22 °C in the dark over 28 days under continuous stirring. The manometric respirometry test was carried out according to the OECD guideline for Testing of Chemicals No. 301 F (1992) and the Council Regulation (EC) No 440/2008. The biochemical oxygen demand (BOD) was measured daily and the percentage degradation was calculated from the theoretical oxygen demand (ThOD) after correcting for the BOD of inoculum controls. The average percent biodegradation of the test item in the test media was found to be 1.9% (SD = 2.8%) based on ThODNH3 and 0.9% (SD = 1.2%) based on ThODNO3, respectively, after 28 days of incubation. No biodegradation within the 10-day-window could be calculated since the start criterion of the window (10 % degradation rate) was not reached within the 28 days of incubation. In the toxicity control, containing both the test item and the reference item sodium benzoate, biodegradation was greater than 25%. According to the guidelines this indicates that the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration. In the procedural controls, the reference substance sodium benzoate was degraded by an average of 87% by exposure day 14, and reached an average biodegradation of 94% by the end of the test (day 28), thus confirming suitability of the activated sludge. The difference of extremes of replicate values of the removal of the test item at the end of the test is less than 20%. Therefore, the test can be considered as valid. The test item was not readily biodegradable under the conditions of the test according to the guidelines.

Furthermore, a study is available in which the stability of 5 and 50 ppm solution of the test substance in biologically active river water under aerated conditions at room temperature in subdued lighting was investigated. Experiments were also conducted in the presence of ‘Gramoxone U.K.’ and with filtered river water. The following solutions were tested:1: Thames + sediment, aerated 5 ppm solution labelled test substance, 2: Thames + sediment, aerated 5 ppm solution of labelled test substance + Gramoxone U.K., 3: Thames + sediment, aerated 50 ppm solution of labelled test substance, 4: Blackwater + sediment, aerated, 5 ppm solution of labelled test substance, 5: Filtered Thames water, aerated, 5 ppm solution of labelled test substance, 6: Thames + sediment, aerated, control for ATP analysis at termination of experiment, and 7: Blackwater + sediment, aerated, control for ATP analysis at termination of experiment. The trapping solutions were designed to monitor any evolution of volatile radioactivity. However, scintillation counting of the samples taken after 7 and 40 days revealed that the traps contained insignificant amounts of radioactivity (<0.1% (one value 0.4%)). The radioactivity present in the aqueous part of the samples as percentage of the calculated radioactivity were 2.34E+05 dpm/mL for the approximately 5 ppm solutions and 4.68E+05 dpm/mL for the 50 ppm solutions. To avoid disturbing the sediment in the samples the test substance was added to solutions 1-4 without agitation. This was probably the cause of the high values obtained for the 2 hour samples since complete mixing would not have been attained. Nevertheless when the day 1 and day 28 samples are compared there is a decrease of the order of 10% of the radioactivity in solutions 1-4 whereas solution 5 showed no decrease. This suggests that there was a small degree of physical absorption onto the sediment. This ‘absorbed’ activity has not been investigated further. The autoradiograms of chromatograms showed that all of the radioactivity which was chromatographed was due to parent material, even after 28 days. Identical results were obtained with elution by either solvent system. At day 4 an average of 89% of the applied activity was recovered from the chromatogram; of this an average of 96% was the test substance. At day 28 an average of 85% of the applied activity was recovered from the chromatogram and of this an average of 92% was the test substance. These results were typical of recoveries from the application of aqueous solutions of the labelled test substance. The results suggest that the test substance is resistant to microbial attack by the types of organisms present in two British rivers in the summer time. There may be a limited adsorption of activity onto the sediment.