1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

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 July 2021 to 13 August 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, seven days before the test.

- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution by shaking and was again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed (5.555 g wet weight), dried and the ratio of wet sludge to dry weight (0.5115 g dry weight) was determined. Based on this ratio, calculated amount of wet sludge (5 g dry weight that was equivalent to 54.3 g wet sludge) was suspended in mineral medium to yield a concentration equivalent to about 5 g per litre (on dry weight basis).
The pH of the activated sludge inoculum after preparation was 7.52, just before use the pH was: 6.74. A pH adjustment of activated sludge inoculum was not performed.

- Pretreatment: Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium) for 7 days at test temperature (the actual temperature: 20.0 – 20.9 °C).

- Concentration of sludge: 5 g per litre (on dry weight basis)

- Initial cell/biomass concentration: ~ 10^8 /L

- Water filtered: yes

- Type and size of filter used, if any: cotton wool

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

The biodegradation of the test material was followed by the oxygen uptake of the microorganisms during exposure to the test material.

Details on study design:

The test material (at a concentration of 3.0 mg/L) was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at a controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item, sodium benzoate (at a concentration of 3.0 mg/L) was tested simultaneously under the same conditions as the test material, and functioned as a procedure control (reference control). Additionally, inoculum (containing the filtered inoculum, only) and toxicity (containing both the test item and reference item) controls were examined.
The chosen test material concentration was based on the performed preliminary experiments: on solubility properties, behaviour, and toxicity of the test material; furthermore, on the calculated ThODNH3 value of the test material, based on the provided molecular structure (ThODNH3: 1.96 mg O2/mg).
The suitability of the used activated sludge inoculum was judged based on the percentage biodegradation of the reference item. A toxicity control was used to examine whether the test material at the applied concentration level had an inhibitory effect on the activated sludge microorganisms.

Reference substance:

benzoic acid, sodium salt

Remarks:

Sodium benzoate at a concentration of 3 mg/L

Test performance:

The study was considered as valid since the oxygen depletion in the inoculum control was 0.8 mg O2/L on average, and did not exceed the validity criteria of 1.5 mg O2/L after 28 days.
The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any time. The lowest value was 3.00 mg O2/L, which was measured on the 28th day in the toxicity control.
The difference of duplicate values for the degradation at the plateau or at the end of the test was not greater than 20 %.
The differences between the duplicate biodegradability values in the procedure control and toxicity control groups were 1-19 % throughout the experiment.
In the test material group higher difference values were obtained during the incubation period: 378 % on the 7th day, 179 % on the 14th day, 64 % on the 21st day but the difference between the duplicate biodegradability values was 16 % on the 28th day (at the end of the test).
The high differences were considered not contradictive with the validity of the study since the test material biodegradability was minimal, a biodegradation curve cannot be established. The difference between the corresponding measured dissolved oxygen concentration values was minimal, about 0-3 %; therefore, these results do not affect the validity of the study.
The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. The percentage degradation of the reference item was 72.6 % on the 14th day.
All validity criteria were met as required by the test guideline OECD 301.

Key result

Parameter:

% degradation (O2 consumption)

Value:

2.1

Sampling time:

28 d

Remarks on result:

other: Mean % biodegradation of test material

Parameter:

% degradation (O2 consumption)

Value:

78.3

Sampling time:

28 d

Remarks on result:

other: Mean % biodegradation of reference item (sodium benzoate)

Details on results:

- Correction for Oxygen Uptake for Interference with Nitrification
Errors due to not considering nitrification in the assessment by oxygen uptake of the biodegradability of test substances not containing N are marginal (not greater than 5 %), even if oxidation of the ammonium N in the medium occurs erratically as between test and blank vessels. However, for test substances containing N, serious errors can arise if the observed oxygen uptake is not corrected for the amount of oxygen used in oxidizing ammonium to nitrite and nitrate. For that reason, at this N-containing test material, the oxidized nitrogen (nitrate and nitrite) concentrations were determined following each oxygen measurement with photometric method using nitrite and nitrate cell tests. The LOQ (Limit Of Quantification) of the measurements was 0.03 mg NO2−/L and 0.4 mg NO3−/L, respectively.
The nitrate and nitrite concentrations in the start, 7-, 14-, 21-, and 28-day samples (test material, inoculum control and toxicity control) were below the corresponding LOQ in all measurement occasions, throughout the study.
The oxygen uptake resulting from a possible ammonium oxidation did not influence the amount of oxygen taken up by microbial population. Any correction of the measured dissolved oxygen concentrations was not performed.


- Biodegradation of the Test Material
Under the test conditions, the percentage biodegradation of the test material reached a minimal biodegradation, a mean of 2.1 % after 28 days based on its ThODNH3. (The highest biodegradability value of 3.7 % was noticed on the 21st day of the test.)
The pass level for ready biodegradability is the removal of 60 % ThODNH3 in a 10-day window. The obtained biodegradation value remained far below the pass level; therefore, the test material was considered to be not readily biodegradable.


- Biodegradation of the Toxicity Control
In the toxicity control containing both the test material and the reference item, a mean of 31.6 % biodegradation was noted within 14 days and the calculated biodegradation was 35.0 % after 28 days of incubation (the biodegradation values reached a plateau on about the 7th day of the experiment and from this day on, the slight subsequent changes were considered as being within the biological variability range of the applied test system).
Thus, the test material can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).

Results with reference substance:

The reference item, sodium benzoate, was sufficiently degraded to a mean of 72.6 % after 14 days, and to a mean of 78.3 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum. The biodegradation reached its plateau on about the 14th day and from this day the slight changes were considered as being within the biological variability range of the applied test system.

Percentage biodegradation over time during the study

Treatment	Conc. [mg/L]	Bottle Number	Percentage biodegradation after n days
			7	14	21	28
Test Material	3	1a	-0.7	0.2	4.9	2.3
		1b	2.2	3.1	2.6	2.0
		mean	0.8	1.6	3.7	2.1
Ref. Item	3	2a	62.0	71.4	74.8	78.5
		2b	55.0	73.8	75.6	78.1
		mean	58.5	72.6	75.2	78.3
Toxicity Control	Test item: 3 Ref. Item: 3	4a	28.6	32.2	28.2	34.4
		4b	33.1	31.0	34.0	35.6
		mean	30.8	31.6	31.1	35.0

Biodegradation % = (BOD (mg O2/mg T.m. or R.i.)/ThODNH3 (mg O2/mg T.m. or R.i.))*100

Where:

T.m. = test material

R.i. = reference item

BOD = Biochemical Oxygen Demand

ThODNH3 = Theoretical Oxygen Demand

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Remarks:

The test material is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % theoretical oxygen demand (ThODNH3) in a 10-day window. The percentage biodegradation of the test material reached a mean of 2.1 % on the 28th day of the test based on its ThODNH3.

Conclusions:

Under the conditions of this study the test material is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % theoretical oxygen demand (ThODNH3) in a 10-day window. The percentage biodegradation of the test material reached a mean of 2.1 % on the 28th day of the test based on its ThODNH3.

Executive summary:

The ready biodegradability of the test material was investigated according to OECD Guideline for Testing of Chemicals No. 301 D, EU Method C.4-E and EPA Guideline 712-C-98-076: OPPTS 835.3110 in accordance with GLP via the closed bottle test. Using this method, the test material was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The biodegradation of the test material reached a mean of 2.1% after 28 days. Therefore, under the conditions of this study the test material can be considered to be not readily biodegradable.

Description of key information

Under the conditions of thE study the test material can be considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Additional information

The ready biodegradability of the test item was investigated according to OECD Guideline for Testing of Chemicals No. 301 D, EU Method C.4-E and EPA Guideline 712-C-98-076: OPPTS 835.3110 in accordance with GLP via the closed bottle test. The study was assigned a reliability score of 1 in accordance with the criteria set forth by Klimisch (1997).

Using this method, the test material was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The biodegradation of the test material reached a mean of 2.1% after 28 days. Therefore, under the conditions of this study the test material can be considered to be not readily biodegradable.