Reaction product of 4-aminophenol with 2-ethyl-6-methylbenzenamine and sodium polysulfide

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:

2017-02-22 to 2017-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

January 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
- Origin: The activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 17 February 2017 (six days before the main test). The prepared activated sludge was continuously aerated (2 L/minute) at the test temperature of 22 ± 2 °C, for about 6 days.
- Preparation of Activated Sludge Inoculum: 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 with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with mineral medium and then aerated under test conditions (for 6 days) until use. The pH of the activated sludge inoculum after preparation was 7.69, just before use the pH was: 7.28. A pH adjustment of activated sludge inoculum was not performed.
- Pre-conditioning of Activated Sludge Inoculum: Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium ) for 6 days (from February 17 to February 23, 2017) at test temperature (the actual temperature: 20.3 – 21.4°C). During the aeration the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating 0.1 mL of the different, 10^-1, 10^-2, 10^-3 and 10^-4 dilutions of cultures on nutrient agar plates. Plates were incubated at 37 °C for 24 hours.

The viable cell number of the cultures was determined by manual colony counting. The approximately cell count of aerated inoculum was ~108/L; therefore on the day of the test this inoculum was diluted 1000x with mineral medium to reach the necessary 10^5-10^6 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning (see above) improves the precision of the method.
The inoculum was not pre-adapted to the test chemical.

The chosen test item concentration of 4.0 mg/L investigated in the main test was based on the results of the preliminary solubility and toxicity tests.

Duration of test (contact time):

28 d

Initial conc.:

4 mg/L

Based on:

test mat.

Remarks:

based on the results of the preliminary solubility and toxicity tests

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

PREPARATION OF TEST SOLUTIONS
The test solutions used in the test were prepared by mechanical dispersion freshly, at the beginning of the experiment, in the testing laboratory as follows:
For the preparation of test item test solutions, at first the suitable amount (80 mg) of Blue Sema was suspended (using ultrasonic bath for about 10-15 min.) in the respective volume (1000 mL) of aqueous test medium (mineral medium, see Section: 5.4) to prepare a 80 mg/L stock solution (suspension).
Before the preparation of the test item suspension the test item was ground with a pestle and mortar (as fine as possible).
The test item stock solution (suspension) was continuously stirred until use to ensure a good dispersion and homogeneity (extra care was taken to avoid air bubbles in the stirred solution). During the incubation period the test solutions were not stirred further.


TEST CONDITIONS
The test was carried out in a controlled environment room (during the preparation, aeration and incubation of the mineral medium, preparation of test bottles (units), during the formulation, oxygen and pH measuring) at a temperature of 22 +/- 2°C according to the guideline. The actual temperature range was 20.7 to 21.3 °C. The test bottles were incubated in an incubator at 22 +/- 2°C , in the dark. During the incubation (28 days) of the test units the temperature range was 20.0-20.1 °C. During the pre-conditioning of activated sludge inoculum the temperature was 20.3-21.4 °C. Temperature was measured continuously using min/max thermometer (in controlled environment room) or built-in thermometer (in incubator) and recorded at least once a day. The oxygen concentration of test water (mineral medium) was in the range of 8-9 mg/L. It was measured at the start of the test and found to be 8.97 mg/L. The pH was checked prior study start and found to be 7.32; further pH adjustment was considered as not necessary. The test conditions were measured with suitable instruments and documented in the raw data.

EQUIPMENT
- Large glass tank (volume:~30 L) and
- Large glass bottles (volume: 5L),
- Narrow necked, Winkler bottles with glass stoppers,
- Funnels and coarse filter papers,
- Oxygen and pH meter with appropriate O2 and pH electrode,
- Aeration system, Moisture analyzer,
- Temperature controlled (in the range of 22 ± 2 oC with a temperature deviation of ±1 °C) environment room (and/or incubator) with thermometer with exclusion of light,
- Balance, Centrifuge.

TEST UNITS
- Type and Size: Winkler bottles (300 mL, coded) with special neck and glass stoppers.
- Identification: Each test bottle was uniquely identified with study number, test group, days of measurement and replicate number.

PREPARATION OF TEST SOLUTIONS AND CONTROLS
 A sufficient number of Winkler bottles were cleaned with 5 – 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing standing for about 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions (Section 6.3) were filled into the bottles bubble-free until the bottles were completely filled. (The volume of a completely filled bottle is approximately 300 mL.) Then they were tightly closed with glass stoppers.

The number of test bottles was the following:
- Test solution: 10 (+2 reserve) bottles containing the test item and inoculum
- Procedure control: 10 (+2 reserve) bottles containing the sodium benzoate and inoculum
- Blank control: 10 (+2 reserve) bottles containing only inoculum (inoculum control)
- Toxicity control: 10 (+2 reserve) bottles containing the test item, sodium benzoate and inoculum
 

PRELIMINARY TESTS
The pre-experiments on solubility of the test item, and the 14-day toxicity test for the determination of the test concentration for the main test were conducted non-GLP, and these pre-experiments are excluded from the Statement of Compliance in the final report. The raw data of these tests will be archived under the study code of the present study.

The test item solubility, behavior, and toxicity were tested in a 14-day preliminary experiment. The test design was the same as described at the main experiment. In the preliminary experiment the test item was investigated at the concentration of 4 mg/L. No toxic effect of the test item was found at this investigated concentration.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

In the preliminary experiment the test item was investigated at the concentration of 4 mg/L. No toxic effect of the test item was found at this investigated concentration.

Test performance:

VALIDITY OF THE STUDY
The study was considered as valid since oxygen depletion in the inoculum control was 1.18 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 2.51 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 %.

At the biodegradation plateaus (test item, procedure control, and toxicity control groups) or at the end of the test the highest difference (~6 %) between duplicate values for degradation was calculated in the reference control group on the 14th day of the test; furthermore in the test item group on the 7th day of the test.

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 80.9 % on the 14th day.

All validity criteria were met as required by the test guideline OECD 301.

Key result

Parameter:

% degradation (O2 consumption)

Value:

11.8

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

12.4

Sampling time:

21 d

Parameter:

% degradation (O2 consumption)

Value:

11.5

Sampling time:

14 d

Details on results:

Under the test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of the test item reached a mean of 11.8 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 7th day of the experiment. From this day the slight changes were considered as being within the biological variability range of the applied test system. The highest biodegradability value of 12.4 % was noticed on the 21st day of the test.

The concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred (the slight changes in nitrite and nitrate concentrations in the 21-day and 28-day samples were caused likely by a technical effect: turbidity and/or discoloration). Therefore the biodegradability value of the test item was calculated based on its COD; any correction, based on the measured nitrite and/or nitrate content was not performed.

In the toxicity control containing both, the test item and the reference item, a mean of 33.5 % biodegradation was noted within 14 days and 34.7 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).

Key result

Parameter:

COD

Value:

2.31 mg O2/g test mat.

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 80.9 % after 14 days, and to a mean of 80.2 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

Table 1: Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Bottle	mg O2/L after n days of exposure
	[mg/L]	No.	0	7	14	21	28
Test item		1a	8.67	6.92	6.73	6.49	6.37
	4.0	1b	8.64	6.95	6.65	6.44	6.40
		mean	8.66	6.94	6.69	6.47	6.39
Reference item		2a	8.69	4.07	3.87	3.70	3.58
	3.0	2b	8.76	4.05	3.69	3.65	3.49
		mean	8.73	4.06	3.78	3.68	3.54
Inoculum control	–	3a	8.73	8.03	7.86	7.71	7.67
		3b	8.71	7.95	7.78	7.63	7.41
		mean	8.72	7.99	7.82	7.67	7.54
Toxicity control	Test item: 4.0 Reference item: 3.0	4a	8.63	3.42	3.12	2.87	2.69
		4b	8.59	3.35	2.99	2.80	2.51
		mean	8.61	3.39	3.06	2.84	2.60

 

  Table 2:  Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Bottle	mg O2/L after n days of exposure
	[mg/L]	No.	7	14	21	28
Test item	4.0	1a	1.02	1.04	1.13	1.12
		1b	0.96	1.09	1.15	1.06
Reference item	3.0	2a	3.89	3.92	3.94	3.93
		2b	3.98	4.17	4.06	4.09
Toxicity control	Test item: 4.0 Reference item: 3.0	4a	4.48	4.61	4.71	4.76
		4b	4.51	4.70	4.74	4.90

 

oxygen depletion : (m_t0- m_tx) - (m_bo- m_bx), where:

 

m_t0: oxygen concentration (mg/L) of test group on day 0 (1a, 2a, 4a and 1b, 2b, 4b from Table 2)

m_tx: oxygen concentration (mg/L) of test group on day x (1a, 2a, 4a and 1b, 2b, 4b from Table 2)

m_b0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b from Table 2)

m_bx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b from Table 2)

Table 3: BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Bottle	BOD after n days of exposure
	[mg/L]	No.	7	14	21	28
Test item	4.0	1a	0.26	0.26	0.28	0.28
		1b	0.24	0.27	0.29	0.27
Reference item	3.0	2a	1.30	1.31	1.31	1.31
		2b	1.33	1.39	1.35	1.36
Toxicity control	Test item: 4.0 Reference item: 3.0	4a	0.64	0.66	0.67	0.68
		4b	0.64	0.67	0.68	0.70

 

BOD = = mg O₂/mg T.i and/or R.i.

where:

T.i.        =test item

R.i.       =reference item

i.control=inoculum control

 

Table 4: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration	Bottle	Percent of biodegradation after n days of exposure
	[mg/L]	No.	7	14	21	28
Test item		1a	11.1	11.3	12.2	12.1
	4.0	1b	10.4	11.8	12.5	11.5
		mean	10.7	11.5	12.4	11.8
Reference item		2a	77.8	78.4	78.8	78.6
	3.0	2b	79.6	83.4	81.2	81.8
		mean	78.7	80.9	80.0	80.2
Toxicity control	Test item: 4.0 Reference item: 3.0	4a	32.2	33.1	33.9	34.2
		4b	32.4	33.8	34.1	35.2
		mean	32.3	33.5	34.0	34.7

Biodegradation % =

where:

T.i.        =test item

R.i.       =reference item

i.control=inoculum control

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item was determined to be not readily biodegradable (11.8 % biodegradation on day 28). According to the test guidelines the pass level for ready biodegradability is 60 % of COD.

Executive summary:

To assess the ready biodegradability of the test item, a Closed Bottle Test according to OECD Guideline 301 D, EU Method C.4 -E and EPA OPPTS 835.3110 was conducted in compliance with GLP principles. The test item (initial concentration: 4 mg/L; based on the results of a preliminary study) was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. The biodegradation was followed by oxygen uptake of the microorganisms during exposure. The chemicaloxygen demand (COD) of 2.31 mg O2/ mg test item was determined at the start of the main experiment. In parallel, a procedure control with the reference substance Sodium benzoate (at a concentration of 3.0 mg/L), a blank control (containing the filtered inoculum only) and a toxicity control (containing both the test item and reference item) were examined. The biodegradability value of the test item was calculated based on its COD; any correction, based on the measured nitrite and/or nitrate content was not performed as the concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred. In result, under the test conditions ready biodegradation of the test item was not noticed. The percentage biodegradation of the test item reached a mean of 11.8 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 7th day of the experiment. From this day the slight changes were considered as being within the biological variability range of the applied test system. The highest biodegradability value of 12.4 % was noticed on the 21st day of the test. The reference item Sodium benzoate was sufficiently degraded to a mean of 80.9 % after 14 days, and to a mean of 80.2 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 33.5 % biodegradation was noted within 14 days and 34.7 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days). All validity criteria of the guidelines were fulfilled.

Description of key information

The substance was considered to be not readily biodegradable (11.8 % biodegradation on day 28). According to the test guidelines the pass level for ready biodegradability is 60 % of COD.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

To assess the ready biodegradability of the test item, a Closed Bottle Test according to OECD Guideline 301 D, EU Method C.4 -E and EPA OPPTS 835.3110 was conducted in compliance with GLP principles.The test item (initial concentration: 4 mg/L; based on the results of a preliminary study) was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. The biodegradation was followed by oxygen uptake of the microorganisms during exposure.The chemicaloxygen demand (COD) of 2.31 mg O2/ mg test item was determined at the start of the main experiment.In parallel, a procedure control with the reference substance Sodium benzoate (at a concentration of 3.0 mg/L), a blank control (containing the filtered inoculum only) and a toxicity control (containing both the test item and reference item) were examined. The biodegradability value of the test item was calculated based on its COD; any correction, based on the measured nitrite and/or nitrate content was not performed as the concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred. In result, the reference item Sodium benzoate was sufficiently degraded to a mean of 80.2 % after 28 days, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control a mean of 34.7 % biodegradation after 28 days of incubation was noted. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days). For the test item 11.8 % biodegradation on day 28 was reached. Therefore, the test item is not regarded to be readily biodegradabale by OECD criteria. The test is regarded to be valid.