6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Oct - 17 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

Modified Sturm Test, Jul 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany (29 Sep 2016)

Test material

Test material information

Constituent 1

Reference substance name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

EC Number:

254-074-6

EC Name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Cas Number:

38668-46-1

Molecular formula:

C9H13N7

IUPAC Name:

6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine-2,4-diamine

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Municipal wastewater treatment plant AZV Staufener Bucht, Germany (17 Oct 2017)
- Pretreatment: The activated sludge was washed twice with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.
- Concentration of sludge: 4.0 g/L dry solid content
- Storage length: 2 d
- Preparation of inoculum for exposure: 11.3 mL activated sludge was filled up to 1500 mL with 1488.7 mL mineral medium, corresponding to 30 mg dry solids/L.
- Initial cell/biomass concentration (test vessel): 30 mg dry solids/L

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to guideline
- Test temperature: 20.6 - 24.0 °C
- Suspended solids concentration: 30 mg dry solids/L
- Continuous darkness: No, diffuse light for 28 d

TEST SYSTEM
- Culturing apparatus: 2000 mL gas wash bottles with lateral connecting pieces for butyl rubber septa, filled with 1500 mL test solution and 2 cm stir bars, were used as reactors.
- Number of culture flasks/concentration: 3 flasks. The test vessels were prepared by directly adding 60.7 - 61.1 mg test item to the test vessels, corresponding to 20.0 - 20.1 mg/L organic carbon.
- Method used to create aerobic conditions: Prior to test start, the system was sealed and aerated with CO2-free air overnight under constant mixing (magnetic stirrers).
- Measuring equipment: Total carbon analyser TOC-L/ TOC-5050A with autosampler ASI 5000A (Shimadzu, Duisburg, Deutschland). Inorganic carbon was purged with H3PO4 (25%) using a non-dispersive infrared detector.
- Details of trap for CO2: The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series, each filled with 200 mL 0.2 M NaOH, containing 5.26 mg/L inorganic carbon.
- Procedure: The reactors were aerated with CO2-free air overnight. On the next day, the absorber wash bottles were filled with 0.2 M NaOH and the test item/reference substance were added into the test vessels, as required. The aeration rate was kept at 30 - 100 mL/min.
- Other: The CO2-free air production system consisted of an air compressor, three 1000 mL gas wash bottles filled with dry soda lime in series followed by one bottle filled with 0.1 M NaOH. At the end of the system 1 gas wash bottle was filled with demineralised water, followed by an empty one to catch condensation water. A colour change of the soda lime from white to blue indicates that the CO2 absorption capacity is depleted. The CO2-free air is passed on to an air distributor with 2 input and 22 output channels through PE tubes.

SAMPLING
- Sampling frequency: After 0, 4, 7, 11, 14, 21, and 28 d
- Sampling method: 4 mL NaOH from the first two CO2-absorber flasks connected in line was sampled and the inorganic carbon content was determined. The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air. On Day 28 2 mL of 4 M hydrochloric acid was added into each reactor to release the CO2 dissolved in water. On Day 29 inorganic carbon was determined in both CO2-absorber flasks in line.
- Other: Sampling was performed through a lateral connecting piece through a butyl rubber septum using 5 mL PE syringes.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, 3 replicates
- Reference compound: Yes, 3 replicates (5.15 mL of a 10.0 g/L sodium benzoate stock solution, corresponding to 20.0 mg/L organic carbon)
- Toxicity control: Yes, 1 replicate (61.0 mg test item and 5.15 mL reference stock solution (10 g/L), corresponding to 40.0 mg/L organic carbon)

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

6.5

Sampling time:

28 d

Remarks on result:

other: after acidification

Remarks:

ThCO2

Details on results:

In the toxicity control, degradation was 46.1% after 14 d. Therefore, the test item had no inhibitory effect on the inoculum, according to the guideline.
The mean CO2 evolution of the blank flasks was 31.9 mg/L on Day 28 after acidification.

BOD5 / COD results

Results with reference substance:

The reference compound sodium benzoate reached the pass level of 60% for ready biodegradability within 4 d.

Any other information on results incl. tables

VALIDITY CRITERIA

The test is valid according to the OECD test guideline 301 B (Table 1).

Table 1: Validity criteria for OECD 301.

Criterion from the guideline	Outcome	Validity criterion fulfilled
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	The difference of extremes of replicate values of the test item at the end of the test was < 20%.	Yes
Percentage degradation of the reference compound has reached the pass levels by day 14.	The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 4.	Yes
The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.	Degradation in the toxicity control was 46.1% after 14 days.	Yes
The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC, and the total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.	The IC content in the test vessel was 0.87 mg/L and the CO2 evolution in the inoculum blank at the end of the test was 31.9 mg/L.	Yes

 

RESULTS

Results are summarized in Table 2.

Table 2. Ultimate biodegradation after x day [% of ThCO2]

	0 d	4 d	7 d	11 d	14 d	21 d	28 d	29 d
test flask	0	-0.1	-0.2	-0.5	-1.1	-4.1	-0.7	-0.9
	0	5.3	5.4	6.0	7.1	9.6	12.8	11.9
	0	3.1	3.4	4.1	4.6	6.9	9.3	8.5
reference flask	0	68.7	84.1	86.3	87.3	101.4	89.5	89.9
	0	80.9	89.9	92.2	92.2	106.6	97.8	99.3
	0	73.9	86.4	86.3	88.9	107.2	96.3	98.3
toxicity control	0	37.3	44.2	45.0	46.1	53.6	49.4	49.5

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Interpretation of results:

under test conditions no biodegradation observed