Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

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:

November 02, 2016 - February 08, 2017

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:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

anaerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Test System
Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 27 October 2016 (seven days before the main test). The prepared activated sludge was continuously aerated (2 L/minute) at the test temperature of 22 ± 2 oC, for about 7 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 (see Section 5.4) and then aerated under test conditions (for 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.61, just before use the pH was: 7.31. 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, mineral medium ) for 7 days (from October 27 to November 03, 2016) at the test temperature (the actual temperature: 20.3 – 21.9 °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, usually 10-2, 10-3 and 10-4 dilutions of cultures on nutrient agar plates.

The viable cell number of the cultures was determined by these plating experiments by manual colony counting. The approximately cell count of aerated inoculum fell in the range of ~109/L; therefore on the day of the test this inoculum was diluted 10000 x with mineral medium to reach the necessary 105-106 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improves the precision of the method.
The inoculum was not pre-adapted to the test chemical.

The chosen test item concentration of 3.0 mg/L to be investigated in the main test was based on the available information about the solubility and toxicity of the test item (based on the preliminary toxicity test results.

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

other: based on the preliminary toxicity test results

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Formulation
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:
At first the suitable amount (60 mg) of Yellow 22 was suspended (using ultrasonic bath for about 10 min.) in the respective volume (1000 mL) of aqueous test medium (mineral medium) and a stock solution with a concentration of 60 mg/L was prepared. The test item stock solution was continuously stirred until use to ensure a good dispersion and homogeneity (extra care was taken to avoid of air bubbles in the stirred solution). This stock solution was adequately diluted in the test item containing test groups. During the incubation period the test solutions were not stirred.

Stock Solutions for Mineral Medium
In purified, deionized water analytical grade salts were added to prepare the following stock solutions:
A) Solution: KH2PO4 8.50 g
K2HPO4 21.75 g
Na2HPO4 x 12H2O 67.16 g
NH4Cl 0.50 g
Water ad 1000 mL
B) Solution: CaCl2 x 2 H2O 18.20 g
Water ad 500 mL
C) Solution: MgSO4 x 7 H2O 11.25 g
Water ad 500 mL
D) Solution: FeCl3 x 6 H2O 0.125 g
Water ad 500 mL
(The “D” stock solution was prepared on the day of the mineral medium preparation and was not further stored).

The mineral medium was prepared in the following ratio: 1 mL of the stock solutions A - D) were combined per 1000 mL total volume, filled with water (purified deionized) . The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was checked and found 8.76 mg/L. The pH of the mineral medium was 7.23

Environmental 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 20.5 21.9 oC.
The test bottles were incubated in incubator at 22 +/- 2 °C, in the dark. During the incubation (28 days) of the test units the temperature range was 20.0-20.3 oC.
During the pre-conditioning of activated sludge inoculum the temperature was 20.3-21.9 oC.
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 8.76 mg/L.
The pH was checked prior study start and found to be 7.23; 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 oC) 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.

Preliminary Experiments
The pre-experiments on solubility of the test item, and the 14-day toxicity test were conducted non-GLP for the determination of the test concentration for the main test, 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.

Preliminary Toxicity Test
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 3 mg/L. No toxic effect of the test item was found at this investigated concentration.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

The test item solubility, behavior, and toxicity were tested in a 14-day preliminary toxicity 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 3 mg/L. No toxic effect of the test item was found at this investigated concentration.

Test performance:

The chosen test item concentration of 3.0 mg/L investigated in the main test was based on the results of the preliminary solubility and toxicity tests. The chemical oxygen demand (COD) of 1.577 mg O2/ mg test item of Yellow 22 was determined at the start of the main experiment.
Under the test conditions ready biodegradation of this test item was not noticed, the percentage biodegradation of Yellow 22 reached a mean of 15.6 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 21st day of the experiment (where the highest biodegradability value of about 16.2 % was noticed). From this day the variations slight changes were considered as being within the biological variability range of the applied test system.
The concurrently conducted analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred (the slight changes in nitrite and nitrate concentrations 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.
The reference item Sodium benzoate was sufficiently degraded to a mean of 79.0 % after 14 days, and to a mean of 88.7 % 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 46.5 % biodegradation was noted within 14 days and 54.2 % 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:

% degradation (O2 consumption)

Remarks:

mean

Value:

9.3

Sampling time:

28 d

Parameter:

% degradation (CO2 evolution)

Remarks:

mean

Value:

9.5

Sampling time:

21 d

Parameter:

% degradation (CO2 evolution)

Remarks:

mean

Value:

8.9

Sampling time:

14 d

Details on results:

Under the test conditions the percentage biodegradation of Yellow 22 reached a mean of 15.6 % after 28 days based on its COD. The highest biodegradability value of 16.2% was noticed on the 21st day of the experiment. From this day the slight subsequent changes were considered as being within the biological variability range of the applied test system. The test item can be considered to be not readily biodegradable.

Biodegradation of the Reference Item
The reference item Sodium benzoate was sufficiently degraded to a mean of 79.0 % after 14 days, and to a mean of 88.7 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

Biodegradation of the Toxicity Control
In the toxicity control containing both, the test item and the reference item, a mean of 46.5 % biodegradation was noted within 14 days and 54.2 % 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:

1.577 mg O2/g test mat.

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 79.0 % after 14 days, and to a mean of 88.7 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

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	8.74	7.24	7.00	6.63	6.67
	3.0	1b	8.73	7.21	6.90	6.61	6.57
		mean	8.74	7.23	6.95	6.62	6.62
Reference item		2a	8.79	4.44	3.68	3.21	3.14
	3.0	2b	8.78	4.12	3.51	2.99	2.81
		mean	8.79	4.28	3.60	3.10	2.98
Inoculum control	–	3a	8.71	7.95	7.61	7.41	7.42
		3b	8.76	7.72	7.38	7.36	7.30
		mean	8.74	7.84	7.50	7.39	7.36
Toxicity control	Test item: 3.0 Reference item: 3.0	4a	8.74	3.66	2.95	2.44	2.13
		4b	8.74	3.48	3.01	2.16	2.06
		mean	8.74	3.57	2.98	2.30	2.10

  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	3.0	1a	0.60	0.50	0.76	0.70
		1b	0.62	0.59	0.77	0.79
Reference item	3.0	2a	3.45	3.87	4.23	4.28
		2b	3.76	4.03	4.44	4.60
Toxicity control	Test item: 3.0 Reference item: 3.0	4a	4.18	4.55	4.95	5.24
		4b	4.36	4.49	5.23	5.31

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	3.0	1a	0.20	0.17	0.25	0.23
		1b	0.21	0.20	0.26	0.26
Reference item	3.0	2a	1.15	1.29	1.41	1.43
		2b	1.25	1.34	1.48	1.53
Toxicity control	Test item: 3.0 Reference item: 3.0	4a	0.70	0.76	0.83	0.87
		4b	0.73	0.75	0.87	0.88

 

BOD = = mg O₂/mg T.i and/or R.i.where:

T.i.        =test item

R.i.       =reference item

i.control=inoculum control

 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	12.7	10.6	16.1	14.7
	3.0	1b	13.1	12.5	16.3	16.6
		mean	12.9	11.5	16.2	15.6
Reference item		2a	69.0	77.4	84.6	85.5
	3.0	2b	75.2	80.6	88.8	91.9
		mean	72.1	79.0	86.7	88.7
Toxicity control	Test item: 3.0 Reference item: 3.0	4a	43.0	46.8	50.9	53.8
		4b	44.8	46.1	53.8	54.5
		mean	43.9	46.5	52.3	54.2

Biodegradation % =where:

T.i.        =test item

R.i.       =reference item

i.control=inoculum control

Nitrate Concentrations
Analytical occasions	Measured nitrate concentration (mg/L) in the test bottles
	1a	1b	3a	3b	4a	4b
0 day	<0.4	<0.4	<0.4	<0.4	<0.4	<0.4
7^thday	<0.4	<0.4	<0.4	<0.4	<0.4	<0.4
14^thday	<0.4	<0.4	<0.4	<0.4	<0.4	<0.4
21^stday	0.6	0.5	<0.4	<0.4	<0.4	<0.4
28^thday	0.9	0.7	0.6	0.6	0.8	0.7

Remarks:            LOQ of nitrite determination: 0.03 mg NO₂/L

                              1a, 1b, 3a, 3b, 4a and 4b mean the bottle numbers.

 

 

 

	Nitrite Concentrations
	Analytical occasions	Measured nitrite concentration (mg/L) in the test bottles
		1a	1b	3a	3b	4a	4b
	0 day	<0.03	<0.03	<0.03	<0.03	<0.03	<0.03
	7^thday	<0.03	<0.03	<0.03	<0.03	<0.03	<0.03
	14^thday	0.04	0.04	<0.03	<0.03	0.05	0.04
	21^stday	0.48	0.43	0.25	0.25	0.30	0.41
	28^thday	<0.03	<0.03	<0.03	<0.03	<0.03	<0.03
Remarks:   LOQ of nitrite determination: 0.03 mg NO2/L                     1a, 1b, 3a, 3b, 4a and 4b mean the bottle numbers.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item was considered to be not readily biodegradable (15.6 % biodegradation on day 28). According to the test guidelines the pass level for ready biodegradability is 60 % of COD.

Executive summary:

The purpose of this study was to determine the ready biodegradability of the test item. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. 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 item, 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 item concentration of 3.0 mg/L investigated in the main test was based on the results of the preliminary solubility and toxicity tests. Under the test conditions ready biodegradation of this test item was not noticed, the percentage biodegradation of the test item reached a mean of 15.6 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 21^stday of the experiment (where the highest biodegradability value of about 16.2 % was noticed). From this day the variations slight changes were considered as being within the biological variability range of the applied test system.

The concurrently conducted analytical determination of a possible nitrite and nitrate development demonstrated that no nitrification occurred. Therefore the biodegradability value of the test item was calculated based on its COD.

The reference item Sodium benzoate was sufficiently degraded to a mean of 79.0 % after 14 days, and to a mean of 88.7 % after 28 days of incubation, based on ThOD_NH3, 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 46.5 % biodegradation was noted within 14 days and 54.2 % 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).

Description of key information

The test item was considered to be not readily biodegradable (15.6 % 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