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Environmental fate & pathways

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:
12Sep2018-12Dec2018
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:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Batch 1075126
Specific details on test material used for the study:
Identification: 2-[(4-aminophenyl)azo]-1,3-dimethyl-1H-imidazolium chloride
Batch (Lot) Number: 1075126
Expiry date: 24 July 2022 (expiry date)
Physical Description: Brown powder

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Freshly obtained sludge was used immediately. Before use, sludge was coarsely sieved (1 mm). After treatment concentration of suspended solids (SS) was determined to be 3.1 g/L in concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 3 mL per litre of mineral medium, leading to a SS concentration of 9.2 mg/L.
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 23 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test duration
28 days for inoculum blank and test item (last CO2 measurement on day 29).
14 days for procedure and toxicity control (last CO2 measurement on day 15).
During the test period, test media were aerated and stirred continuously.

Test vessels
2 litre brown coloured glass bottles.

Milli- RO water
Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.

Stock solutions of mineral components
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.

Mineral medium
1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.

Barium hydroxide
0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from air.

Synthetic air (CO2 < 1 ppm)
A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. Synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

Illumination
Test media were excluded from light.

Preparation of Bottles
Pre-incubation medium The day before start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Type and number of bottles
Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Procedure control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).

Preparation
At the start of the test (day 0), test and reference item were added to bottles containing microbial organisms and mineral components.
Volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before.
Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
>= 1 - <= 2
Sampling time:
28 d
Remarks on result:
other: not readily biodegradable
Details on results:
Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of test item (1% and 2%, based on ThCO2).
In the toxicity control, the test item was found not to inhibit microbial activity.
Since all criteria for acceptability of the test were met, this study was considered to be valid.

BOD5 / COD results

Results with reference substance:
Reference item was biodegraded by at least 60% (actual result: 82%) within 14 days.

Any other information on results incl. tables

1.     Reference item was biodegraded by at least 60% (actual result: 82%) within 14 days.

2.     Difference between duplicate values for %-degradation of test item was always less than 20% (actual result: ≤ 1%).

3.     Total CO2release in the blank at the end of the test did not exceed 40 mg/L (59.6 mg CO2per 2 litres of medium, corresponding to 29.8 mg CO2/L).

4.     Inorganic Carbon content (IC) of test item (suspension) in mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since test medium was prepared in tap-water purified by reverse osmosis (Milli- RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In conclusion, 2-[(4-aminophenyl)azo]-1,3-dimethyl-1H-imidazolium chloride was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

Since the test item was easily soluble in water test media were prepared using a stock solution of 1 g/L in Milli- RO water. Aliquots of 46 mL of the clear, dark red stock solution were added to test item bottles A and B and to the toxicity control. These test bottles contained medium with microbial organisms. Volumes of suspensions were made up to 2 litres with Milli- RO water. Test solutions were continuously stirred during the test, to ensure optimal contact between test item and test organisms. Test duration was 28 days for inoculum blank and test item (last CO2 measurement on day 29) and 14 days for procedure and toxicity control (last CO2 measurement on day 15).

Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of test item (1% and 2%, based on ThCO2).

In the toxicity control, the test item was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, 2-[(4-aminophenyl)azo]-1,3-dimethyl-1H-imidazolium chloride was designated as not readily biodegradable.