Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

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:

2017-08-10 to 2017-10-26

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:

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

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

January 1998

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 (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 09 August 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 (5.832 g wet weight), dried and the ratio of wet sludge to dry weight (0.5033 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (5 g dry weight that was equivalent to 57.94 g wet sludge) was suspended in mineral medium (Section 5.4; ad. 1000 mL) to yield a concentration equivalent to about 5 g per litre (on dry weight basis). The prepared activated sludge inoculum was aerated under test conditions (for 6 days) until use. The pH of the activated sludge inoculum after preparation was 7.38, just before use the pH was: 7.41. 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 August 09 to August 15, 2017) at test temperature (the actual temperature: 20.0 – 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.

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

other: based on the results of the preliminary solubility and toxicity tests

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- Method: 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 (75 mg) of Leuco Sulfur Blue 20P was suspended (using ultrasonic bath for about 10-15 min.) in the respective volume (500 mL) of aqueous test medium to prepare a 150 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.

- 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 21.70 g
Na2HPO4 x 12H2O 67.16 g
NH4Cl 0.50 g
Water ad 1000 mL
B) Solution: CaCl2 x 2 H2O 36.40 g
Water ad 1000 mL
C) Solution: MgSO4 x 7 H2O 22.50 g
Water ad 1000 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).

Preparation of Mineral Medium (Ratio of Ingredients)

The mineral medium was prepared in the following ratio: 1 mL of the stock solutions A - D) (Section: 5.4.1) 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 8.61 mg/L. The pH of the mineral medium was 7.36.
Environmental Conditions

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 20.0 to 21.4 °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 20.0-20.2 °C. During the pre-conditioning of activated sludge inoculum the temperature was 20.0 - 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 8.61 mg/L. The pH was checked prior study start and found to be 7.36; 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: 5 L),
- 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 °C with a temperature deviation of ±1 °C) environment room (and/or incubator) with thermometer with exclusion of light,
- Balance, Centrifuge, Ultrasonic bath.

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.
- 10 (+2 reserve) bottles containing the test item and inoculum (source: bottle 1a and 1b)
- 10 (+2 reserve) bottles containing the sodium benzoate and inoculum (source: bottle 2a and 2b)
- 10 (+2 reserve) bottlescontaining only inoculum (inoculum control)(source: bottle 3a and 3b)
-10 (+2 reserve) bottlescontaining the test item, sodium benzoate and inoculum (toxicity control)(source: bottle 4a and 4b)

PRELIMINARY EXPERIMENTS
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 4.0 mg/L. Unequivocal toxic effect of the test item was not found at this investigated concentration (in the toxicity control group 27.1 % (higher than 25 %) degradation occurred within 14 days). 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).

Reference substance:

benzoic acid, sodium salt

Preliminary 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 4.0 mg/L. Unequivocal toxic effect of the test item was not found at this investigated concentration (in the toxicity control group 27.1 % (higher than 25 %) degradation occurred within 14 days). In the toxicity control containing both, the test item and the reference item, a mean of 35.6 % biodegradation was noted within 14 days .

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 1.753 mg O2/ mg test item of Leuco Sulfur Blue 20P 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 Leuco Sulfur Blue 20P reached a mean of 6.1 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 14th day of the experiment. From this day the sight changes were considered as being within the biological variability range of the applied test system. The concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred. 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 65.5 % after 14 days, and to a mean of 69.9 % 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 35.6 % biodegradation was noted within 14 days and 36.0 % 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)

Value:

>= 3.1 - <= 6.1

Sampling time:

28 d

Details on results:

VALIDITY OF THE TEST
The study was considered as valid since oxygen depletion in the inoculum control was 1.25 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 3.36 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 (~19 %) between duplicate values for degradation was calculated in the test item group on the 14th 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 65.5 % on the 14th day. Thus, all validity criteria were met as required by the test guideline OECD 301.

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 item, 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 measured quantities of nitrite in the inoculum control, test item and toxicity control samples were below the LOQ at the measurement occasions.
The nitrate concentration of the samples was less than 0.4 mg/L in all measurement occasions, throughout the study.

BIODEGRADATION OF THE TEST ITEM
Under the test conditions the percentage biodegradation of Leuco Sulfur Blue 20P reached a mean of 6.1 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 14th day of the experiment. From this day on, 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 SUBSTANCE
The reference item Sodium benzoate was sufficiently degraded to a mean of 65.5 % after 14 days, and to a mean of 69.9 % 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 35.6 % biodegradation was noted within 14 days and 36.0 % 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:

1.753 other: mg O2/ mg test item

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 65.5 % after 14 days, and to a mean of 69.9 % 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	8.38	7.40	6.98	6.91	6.80
	3.0	1b	8.46	7.32	7.00	6.98	6.90
		mean	8.42	7.36	6.99	6.95	6.85
Reference item		2a	8.27	4.54	3.92	3.89	3.76
	3.0	2b	8.44	4.40	4.02	3.83	3.46
		mean	8.36	4.47	3.97	3.86	3.61
Inoculum control	–	3a	8.47	7.63	7.32	7.29	7.32
		3b	8.49	7.54	7.42	7.32	7.14
		mean	8.48	7.59	7.37	7.31	7.23
Toxicity control	Test item: 3.0 Reference item: 3.0	4a	8.39	4.56	3.76	3.72	3.61
		4b	8.46	4.08	3.57	3.45	3.36
		mean	8.43	4.32	3.67	3.59	3.49

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	0.09	0.29	0.30	0.33
		1b	0.25	0.35	0.31	0.31
Reference item	3.0	2a	2.84	3.24	3.21	3.26
		2b	3.15	3.31	3.44	3.73
Toxicity control	Test item: 3.0 Reference item: 3.0	4a	2.94	3.52	3.50	3.53
		4b	3.49	3.78	3.84	3.85

 

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)

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	0.03	0.10	0.10	0.11
		1b	0.08	0.12	0.10	0.10
Reference item	3.0	2a	0.95	1.08	1.07	1.09
		2b	1.05	1.10	1.15	1.24
Toxicity control	Test item: 3.0 Reference item: 3.0	4a	0.49	0.59	0.58	0.59
		4b	0.58	0.63	0.64	0.64

 

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	1.6	5.5	5.6	6.3
	3.0	1b	4.7	6.7	5.8	5.9
		mean	3.1	6.1	5.7	6.1
Reference item		2a	56.7	64.8	64.1	65.2
	3.0	2b	62.9	66.2	68.7	74.6
		mean	59.8	65.5	66.4	69.9
Toxicity control	Test item: 3.0 Reference item: 3.0	4a	28.6	34.3	34.1	34.4
		4b	34.0	36.8	37.4	37.5
		mean	31.3	35.6	35.7	36.0

Biodegradation % =

where:

T.i.        =test item

R.i.       =reference item

CODof the test item = 1.753 mg O₂/mg test item

ThOD_NH3of reference item = 1.67 mg O₂/mg reference item

Nitrate Concentrations
Analytical samples	Measured nitrate concentration (mg/L) in the test bottles
	1a	1b	3a	3b	4a	4b
0 day	---	---	---	---	---	---
7^thday	---	---	---	---	---	---
14^thday	---	---	---	---	---	---
21^stday	---	---	---	---	---	<0.4
28^thday	---	---	---	---	---	---

Remarks:            LOQ of nitrate determination: 0.4 mg NO₃/L

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

                              ---: nitrate concentrations were not detectable.

 

 

Nitrite Concentrations
Analytical samples	Measured nitrite concentration (mg/L) in the test bottles
	1a	1b	3a	3b	4a	4b
0 day	<0.03	---	<0.03	---	---	---
7^thday	---	---	<0.03	---	---	---
14^thday	---	---	---	---	---	<0.03
21^stday	---	---	---	---	---	---
28^thday	---	---	---	---	---	---
Remarks:            LOQ of nitrite determination: 0.03 mg NO2/L                               1a, 1b, 3a, 3b, 4a and 4b mean the bottle numbers,                               ---: nitrite concentrations were not detectable.

 

Quality Control Samples
Analytical occasions	Nitrite			Nitrate
	Nominal concentration			Nominal concentration
	0.05 mg/L	0.5 mg/L	2 mg/L	0.5 mg/L	2 mg/L
	(Measured concentrations mg/L)
7^thday	0.05	0.50	2.00	0.5	2.0
21^stday	0.05	0.50	2.01	0.5	2.0
28^thday	0.05	0.50	2.00	0.5	2.0

 Measurement of the QC samples was performed for providing information about the method applicability.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item was considered to be not readily biodegradable (6.1 % biodegradation on day 28). According to the test guidelines the pass level for ready biodegradability is 60 % of COD.

Executive summary:

The ready biodegradability of the test item was assessed in a valid and GLP-compliant study according to OECD Guideline 301 D, EU Method C.4 and US EPA Guideline 712-C-98-076: OPPTS 835.3110. The test item 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. 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. The chemicaloxygen demand (COD) of 1.753 mg O2/ mg test item 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 the test item reached a mean of 6.1 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 14thday of the experiment. From this day the slight changes were considered as being within the biological variability range of the applied test system. The concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred. 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 65.5 % after 14 days, and to a mean of 69.9 % 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 35.6 % biodegradation was noted within 14 days and 36.0 % 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 (6.1 % 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

The ready biodegradability of the test item was assessed in a valid and GLP-compliant study according to OECD Guideline 301 D, EU Method C.4 and US EPA Guideline 712-C-98-076: OPPTS 835.3110. The test item 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. 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. The chemicaloxygen demand (COD) of 1.753 mg O2/ mg test item 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 the test item reached a mean of 6.1 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 14th 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 concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred. 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 65.5 % after 14 days, and to a mean of 69.9 % 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 35.6 % biodegradation was noted within 14 days and 36.0 % 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).