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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:

2021-11-25 to 2022-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The pH measurement in the reactors was not performed at the end of the test (day 28), but 12 days later (at day 40). No impact on the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot number: RR1286
- Purity: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep containers tightly closed in a dry, cool and well-ventilated place. Recommended storage temperature: 0 - 40 °C
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable under recommended storage conditions
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis:
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: soluble in water
- Reactivity of the test material with the incubation material used (e.g. plastic ware): n.a.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge from the municipal wastewater treatment plant AZV Breisgauer Bucht was used. The treatment plant
clarifies predominantly domestic wastewater and has a capacity of 600,000 inhabitant
equivalents. Sampling date of activated sludge was 30 November 2021.
- Preparation of inoculum for exposure: The activated sludge was washed twice
with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.
- Concentration of sludge: 30 mg dry solids per litre
- Dry solid content of the activated sludge: 4.7 g/L

Duration of test (contact time):

28 d

Initial conc.:

35.4 mg/L

Based on:

test mat.

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium consists of mineral components, namely, potassium and sodium phosphates plus ammonium chloride, calcium chloride, magnesium sulphate and iron (III) chloride
- Test temperature: 19.9 – 21.9°C
- pH: The pH measurement in the reactors was not performed at the end of the test (day 28), but 12 days later (at day 40) and the pH value was 7.6
- pH adjusted: no
- Aeration of dilution water: The system was sealed and aerated with CO2-free air. The aeration rate was kept at a rate of 30 - 100 mL / min (1.6 - 5.5 bubbles / second) and determined visually daily on working days.
- Suspended solids concentration: 30 mg dry solids per litre
- Continuous darkness: no

TEST SYSTEM
- Culturing apparatus: Gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septa were used as reactors.
- Number of culture flasks/concentration: In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound and one reactor containing test item and reference compound (toxicity control) were set up.
- Method used to create aerobic conditions: The CO2-free air production system consists 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 (sodium hydroxide).
- Measuring equipment: Total carbon analyzer TOC-L with autosampler ASI-L, Shimadzu Deutschland, Duisburg
- Details of trap for CO2 and volatile organics if used: 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.

SAMPLING
- Sampling frequency: On the 4th, 7th, 11th, 14th, 21st and 28th day, 8 mL NaOH were sampled
- Sampling method: Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 10 mL PE syringes.


CONTROL AND BLANK SYSTEM
- Inoculum blank: three reactors containing only inoculum was used as blank
- Toxicity control: one reactor containing test item and reference compound was used as toxicity control
- Other: three reactors containing the reference compound was used as positive control

Reference substance:

benzoic acid, sodium salt

Test performance:

The IC content of the test item reactor at the beginning of the test was 1.3% of the TC and
therefore less than 5%.
● The CO2 evolution in the inoculum blank at the end of the test was 22.3 mg/L and therefore
below 40 mg/L.
● The difference of extremes of replicate values of the test item at the end of the test was less
than 20%.
● The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 4.
● The degradation extent in the toxicity control after 14 days was above 25% of ThCO2.
The test is valid according to OECD Test Guideline 301 B (July 1992).

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

% of ThCO2

Value:

-1.8

Sampling time:

28 d

Remarks on result:

other: with IC reactor; values were -1.6, -1.8 and -1.9 % of ThCO2

Results with reference substance:

The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 4.

Table 1: Ultimate biodegradation after x days [% of ThCO2]

Replicate	day 0	day 4	day 7	day 11	day 14	day 21	day 28	day 28 with IC reactor
Test flasks
1	0	-0.6	-0.7	-0.6	-0.8	0.0	-1.1	-1.6
2	0	-5.0	-0.1	0.1	-1.0	-1.2	-1.6	-1.8
3	0	-2.1	-0.9	-0.7	-0.6	-1.3	-2.0	-1.9
mean	0	-2.6	-0.6	-0.4	-0.8	-0.8	-1.6	-1.8
Reference flasks
1	0	69.7	76.2	81.6	86.3	89.8	91.5	91.7
2	0	72.1	78.0	85.7	87.4	89.7	91.1	91.2
3	0	68.6	82.5	87.4	89.0	90.1	92.9	93.0
mean	0	70.1	78.9	84.9	87.6	89.9	91.8	92.0
Toxicity control flask (Test item and Reference item)
1	0	35.5	39.1	42.7	44.7	43.5	45.0	45.0

 

 

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

No degradation of the test item could be observed within the test duration of 28 days. The degradation of the test item at the end of the test was -1.8%.

Executive summary:

The ready biodegradability of 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea was assessed on basis of the OECD Guideline 301B (CO2 evolution method). 
The activated sludge inoculum was derived from a German municipal wastewater treatment plant. Degradation was followed by determining the carbon dioxide produced and absorbed to sodium hydroxide via IC-measurement (IC = inorganic carbon).
The mean percentage of biodegradation of 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea was -1.8% of ThCO2 after 28 days. Thus, no degradation of the 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea was observed within the test duration.

Description of key information

On basis of a OECD 301 B test 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea is not biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

The Biodegradability of 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea was investigated using the CO2 evolution method according to OECD 301B. No degradation of the test item could be observed within the test duration of 28 days. The degradation of the test item at the end of the test was -1.8%