Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives

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:

03 November - 01 December, 2017

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 17, 1992

Deviations:

no

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

January 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives

EC Number:

288-896-1

EC Name:

Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives

Cas Number:

85940-08-5

Molecular formula:

not applicable

IUPAC Name:

Reaction product of N-(2,4-dinitrophenyl)acetamide with 1-methyl-2,4-dinitrobenzene and polysulfide, leuco derivatives

Test material form:

solid: particulate/powder

Details on test material:

Test item: Leuco Sulphur Brown 46
Appearance: Brown powder
CAS No: 85940-08-5

Specific details on test material used for the study:

Date of production: 04.08.2016
Expiration date: 04.08.2021

Study design

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 27 October 2017
(seven 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 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 (5.370 g wet weight), dried and the ratio of wet sludge to dry weight (0.4224 g dry weight)
determined. Based on this ratio, calculated amount of wet sludge (5 g dry weight that was equivalent to 63.56 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 7 days) until use.
The pH of the activated sludge inoculum after preparation was 7.53, 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 ) for 7 days (from October 27 to November 03, 2017) at test
temperature (the actual temperature: 20.1 – 21.7 oC). Before use 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 these plating experiments by manual colony counting.
The approximately cell count of aerated inoculum was ~108/L; therefore, before the test the inoculum was further diluted 50 000x with mineral medium to
reach the necessary ~104 – 106 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.
Nutrient agar:Supplier: MERCK; Batch Number: VM756750, Expiry date: 11 October 2021

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

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 (100 mg) of Leuco Sulphur Brown 46 was suspended (using ultrasonic bath for about 10-15 min.) in the respective volume (500 mL) of aqueous test medium (mineral medium, see Section: 5.4) to prepare a 200 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.

Mineral Medium

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 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).

The details of the used chemicals are summarized in the Table 1.

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 8.30 mg/L. The pH of the mineral medium was 7.43.


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 20.1 - 21.0 °C. The test bottles were incubated in an incubator at 22 ± 2 ºC, 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 20.1 – 21.7 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 8.30 mg/L.
The pH was checked prior study start and found to be 7.43; 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, Freezer,
Spectrophotometer (for nitrate, nitrite and COD determination).

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, and 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 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 4.0 mg/L. Unequivocal toxic effect of the test item was not found at this investigated concentration (in the toxicity control group 30.6 % (higher than 25 %) degradation occurred within 14 days).

COD Determination

The COD (chemical oxygen demand) of the test item (3.23 mg O2/mg test item) was determined in the test facility using COD Cell Test (MERCK®) as described in Section: 6.5.4.

The chosen test item concentration of 4.0 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

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

The test item solubility, behavior, and toxicity were tested in a 14-day preliminary 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 30.6 % (higher than 25 %) degradation occurred within 14 days).
In the toxicity control containing both, the test item and the reference item, a mean of 25.8 % biodegradation was noted within 14 days.

Test performance:

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. The chemical oxygen demand (COD) of 3.23 mg O2/ mg test item of Leuco Sulphur Brown 46 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 Sulphur Brown 46 reached a mean of 3.8 % 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 concurrently conducted analytical determination of possible nitrite and nitrate development showed slight changes in nitrite concentrations in the 28-day samples; however the measured dissolved oxygen concentrations in the inoculum control and toxicity control bottles did not correspond to the consumed oxygen of ammonium oxidation processes. Likely technical effects (turbidity and/or discoloration) also influenced the nitrite concentration determinations. 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 79.7 % after 14 days, and to a mean of 82.8 % 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 25.8 % biodegradation was noted within 14 days and 30.3 % 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).

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

3.8

Sampling time:

28 d

Details on results:

Validity of the Study

The study was considered as valid since oxygen depletion in the inoculum control was 1.4 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 1.59 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 (~15 %) between duplicate values for degradation was calculated in the procedure control group on the 21st 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 79.7 % on the 14th day.

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

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 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 test item samples were below the LOQ throughout the study. The measured quantities of nitrite in the inoculum
control and toxicity control samples were below the LOQ in the 0-day, 7-day, 14-day and 21-day samples. In the 28-day inoculum control samples in a
verage 0.11 mg/L, (see Appendix II, Table 7). In the 28-day toxicity control samples in average 0.12 mg/L nitrite was noticed .
The nitrate concentration of the samples was less than 0.4 mg/L in all measurement occasions, throughout the study
According to the referred OECD 301 guideline (Annex V) the oxygen consumed in nitrite formation is 3.43 x increase of nitrite-N concentration.
Accordingly in the inoculum control the oxygen consumed by ammonium oxidation processes was 0.11 x 3.43 = 0.38 mg/L. The measured average oxygen
depletion from 21st day to 28th day of the test was 0.28 mg/L .
In contrast in the toxicity control the oxygen consumed by ammonium oxidation processes was 0.12 x 3.43 = 0.41 mg/L. The measured average oxygen
depletion from 21st day to 28th day of the test was 0.89 mg/L .
In this study the change of the measured dissolved oxygen concentrations in the inoculum control and toxicity control bottles did not correspond to the
consumed oxygen of ammonium oxidation processes.
Because of the relationship between oxygen uptake resulting from a possible ammonium oxidation and oxygen uptake of applied microbial population was
equivocal, any correction of the measured dissolved oxygen concentrations was considered as not possible. In the case of inoculum control the calculated
oxygen consumed by ammonium oxidation processes was higher (0.38 mg/L) than the measured oxygen depletion (0.28 mg/L). The measured higher nitrite
concentration values were caused likely by a technical effect (possible discoloration of the solutions and/or turbidity). Similar effect could cause higher
nitrite concentration values in the toxicity control as well.

Biodegradation of the Test Item

Under the test conditions the percentage biodegradation of Leuco Sulphur Brown 46 reached a mean of 3.8 % 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 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 Item

The reference item Sodium benzoate was sufficiently degraded to a mean of 79.7 % after 14 days, and to a mean of 82.8 % after 28 days of incubation,
based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum. 1.

Biodegradation of the Toxicity Control

In the toxicity control containing both, the test item and the reference item, a mean of 25.8 % biodegradation was noted within 14 days and 30.3 %
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).

BOD5 / COD results

BOD5 / COD

Key result

Parameter:

COD

Value:

3.23 other: mg O2/ mg test item

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 79.7 % after 14 days, and to a mean of 82.8 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.
In the toxicity control , a mean of 25.8 % biodegradation was noted within 14 days and 30.3 % biodegradation after 28 days of incubation.

Any other information on results incl. tables

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	8.25	7.17	6.78	6.64	6.34
	4.0	1b	8.27	7.20	6.73	6.60	6.41
		mean	8.26	7.19	6.76	6.62	6.38
Reference item		2a	8.26	4.16	3.28	3.34	2.82
	3.0	2b	8.27	4.12	3.34	2.73	2.63
		mean	8.27	4.14	3.31	3.04	2.73
Inoculum control	–	3a	8.15	7.64	7.20	7.16	6.82
		3b	8.20	7.50	7.21	6.96	6.73
		mean	8.18	7.57	7.21	7.06	6.78
Toxicity control	Test item: 4.0 Reference item: 3.0	4a	8.27	3.82	2.94	2.80	1.59
		4b	8.28	3.49	2.81	2.33	1.77
		mean	8.28	3.66	2.88	2.57	1.68

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	0.48	0.50	0.50	0.51
		1b	0.46	0.57	0.56	0.46
Reference item	3.0	2a	3.50	4.01	3.81	4.04
		2b	3.55	3.96	4.43	4.24
Toxicity control	Test item: 4.0 Reference item: 3.0	4a	3.85	4.36	4.36	5.28
		4b	4.19	4.50	4.84	5.11

 

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	4.0	1a	0.12	0.13	0.12	0.13
		1b	0.12	0.14	0.14	0.12
Reference item	3.0	2a	1.17	1.34	1.27	1.35
		2b	1.18	1.32	1.48	1.41
Toxicity control	Test item: 4.0 Reference item: 3.0	4a	0.55	0.62	0.62	0.75
		4b	0.60	0.64	0.69	0.73

 

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	3.7	3.9	3.8	3.9
	4.0	1b	3.6	4.4	4.3	3.6
		mean	3.6	4.1	4.1	3.8
Reference item		2a	69.9	80.2	76.1	80.8
	3.0	2b	70.9	79.2	88.5	84.8
		mean	70.4	79.7	82.3	82.8
Toxicity control	Test item: 4.0 Reference item: 3.0	4a	22.4	25.4	25.4	30.8
		4b	24.4	26.3	28.2	29.8
		mean	23.4	25.8	26.8	30.3

Biodegradation % =where:T.i.        =test itemR.i.       =reference item

Nitrate Concentrations
Analytical samples	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
7^thday	< 0.4	< 0.4	---	< 0.4	---	< 0.4
14^thday	---	---	< 0.4	---	---	---
21^stday	---	---	---	---	---	---
28^thday	< 0.4	< 0.4	< 0.4	< 0.4	< 0.4	< 0.4

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	---	---	---	---	---	---
7^thday	---	---	---	---	---	---
14^thday	---	---	---	< 0.03	---	---
21^stday	< 0.03	< 0.03	< 0.03	< 0.03	---	---
28^thday	< 0.03	< 0.03	0.13	0.09	0.13	0.10
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.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item was considered to be not readily biodegradable (3.8 % 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. This study is recognized by following test guidelines: OECD Guideline for Testing of Chemicals No. 301 D: “Ready Biodegradability: Closed Bottle Test”, adopted July 17, 1992,Commission Regulation (EC) No 440/2008, Annex Part C, C.4., Part VI: “Closed Bottle Test (Method C.4-E)”. Dated May 30, 2008, EPA Guideline 712-C-98-076: OPPTS 835.3110, “Ready Biodegradability”, January 1998.

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 4.0 mg/L investigated in the main test was based on the results of the preliminary solubility and toxicity tests. The chemicaloxygen demand (COD) of 3.23 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 3.8 % after 28 days based on its COD. Based on the dissolved oxygen depletion, the resulting biodegradation values reached a plateau on about the 7^thday 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 showed slight changes in nitrite concentrations in the 28-day samples; however the measured dissolved oxygen concentrations in the inoculum control and toxicity control bottles did not correspond to the consumed oxygen of ammonium oxidation processes. Likely technical effects (turbidity and/or discoloration) also influenced the nitrite concentration determinations. 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 79.7 % after 14 days, and to a mean of 82.8 % 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 25.8 % biodegradation was noted within 14 days and 30.3 % 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).