Phenol, 2,4-dinitro-, sulfurized, thiosulfonated

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:

10 August - 26 October, 2017

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)

Specific details on test material used for the study:

Date of production: 30.11.2016
Expiration date: 30.11.2021

Oxygen conditions:

anaerobic

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 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.

The viable cell number of the cultures was determined by manual colony counting. The approximately cell count of aerated inoculum was ~109/L; therefore on the day of the test this inoculum was diluted 200000 x with mineral medium to reach the necessary 104-105 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 conc.:

4 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:

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 Solubilised Sulphur Black 1 was dissolved, suspended in the respective volume (250 mL) of aqueous test medium (mineral medium, see Section: 5.4) to prepare a 400 mg/L stock solution.
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 21.70 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) 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

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 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.

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 7-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.

Preliminary Toxicity Test

The test item solubility, behavior, and toxicity were tested in a 7-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 (in the toxicity control group higher than 25 % degradation occurred within 7 days).
Test Item Concentration

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).

Course of the Test

Preparation of Test Bottles

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.

Sulphuric acid: Supplier: CARLO ERBA; Lot Number: V2B680142B, Expiry date: February 2018;
Potassium iodide: Supplier: REANAL (lach:ner); Batch Number: PP/2015/17202, Expiry date: 08 June 2018;
Iodine: Supplier: MERCK; Batch Number: B1344561; Expiry date: 31 July 2021.

The Number of Test Bottles

The number of test bottles was the following:
 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) bottles containing only inoculum (inoculum control) (source: bottle 3a and 3b)
 10 (+2 reserve) bottles containing the test item, sodium benzoate and inoculum (toxicity control) (source: bottle 4a and 4b)

Oxygen Measurements

The incubation period of the closed bottle test was 28 days.
The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method].
Oxygen measurements were performed in all duplicate bottles in all test groups on days 0, 7, 14, 21 and 28.

Temperature Measurements

Temperature was measured continuously (with a built-in thermometer) in the incubator (during the 28-day incubation period) and recorded at least once a day.

Measurement of Total Oxidized N (Nitrite and Nitrate)

Because of the nitrogen content of the test item, samples for nitrate and nitrite analysis were taken from all vessels (of test item, inoculum control and toxicity control group) and the oxidized nitrogen (nitrate and nitrite) concentrations were measured.
For technical reason the nitrate and nitrite analysis of the start (day 0) and 7-day samples was performed together on the 7th day of the test, the analysis of 14-day samples was performed one week later, together with the 21-day samples, on the 21st day of the test. The start (day 0) and 14-day samples were adequately stored in freezer (at -15 -30°C) until determination of nitrate and nitrite. The 7-, 21- and 28-day samples were analyzed directly after oxygen
measurements.

Measurement of Chemical Oxygen Demand (COD)

The COD (chemical oxygen demand) of the test item was determined in the test facility using COD Cell Test (MERCK®)

Reference substance:

benzoic acid, sodium salt

Preliminary study:

The test item solubility, behavior, and toxicity were tested in a 7-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.
In the toxicity control containing both, the test item and the reference item, a mean of 34.5 % biodegradation was noted within 14 days.
No toxic effect of the test item was found at this investigated concentration (in the toxicity control group higher than 25 % degradation occurred within 7 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 1.297 mg O2/ mg test item of Solubilised Sulphur Black 1 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 Solubilised Sulphur Black 1 reached a mean of 12.1 % 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 34.5 % biodegradation was noted within 14 days and 36.2 % 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)

Value:

>= 6.7 - <= 12.6

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 1.25 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 3.43 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 (~18 %) between
duplicate values for degradation was calculated in the toxicity control 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.

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 inoculum control samples were below the LOQ at the measurement occasions.
The measured quantities of nitrite in the test item and toxicity control samples were minimal; however in the measurable range throughout the test. In these samples all of the measured nitrite concentration values were in the 0.03-0.07 mg/L range, any increasing tendency was not noticed.
The nitrate concentration of the samples was less than 0.4 mg/L in all measurement occasions, throughout the study.
The measured nitrite concentration values were caused likely by a technical effect (possible discoloration of the solutions and/or turbidity).

Biodegradation of the Test Item

Under the test conditions the percentage biodegradation of Solubilised Sulphur Black 1 reached a mean of 12.1 % 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 Item

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 34.5 % biodegradation was noted within 14 days and 36.2 % 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:

1.297 other: mg O2/ mg

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	8.47	7.28	6.69	6.65	6.62
	4.0	1b	8.50	7.20	6.75	6.73	6.59
		mean	8.49	7.24	6.72	6.69	6.61
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: 4.0 Reference item: 3.0	4a	8.54	4.92	4.17	3.86	3.67
		4b	8.56	4.05	3.56	3.66	3.43
		mean	8.55	4.49	3.87	3.76	3.55

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	0.30	0.67	0.65	0.60
		1b	0.40	0.64	0.59	0.66
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: 4.0 Reference item: 3.0	4a	2.73	3.26	3.51	3.62
		4b	3.62	3.89	3.73	3.88

 

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	0.07	0.17	0.16	0.15
		1b	0.10	0.16	0.15	0.17
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: 4.0 Reference item: 3.0	4a	0.39	0.47	0.50	0.52
		4b	0.52	0.56	0.53	0.55

 

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	5.7	12.9	12.4	11.6
	4.0	1b	7.8	12.3	11.5	12.7
		mean	6.7	12.6	12.0	12.1
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: 4.0 Reference item: 3.0	4a	26.3	31.4	33.8	34.9
		4b	34.9	37.5	35.9	37.4
		mean	30.6	34.5	34.9	36.2

Biodegradation % =where:T.i. =test item R.i.  =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	<0.4	<0.4	---	---	<0.4	<0.4
14^thday	<0.4	<0.4	---	---	<0.4	<0.4
21^stday	<0.4	<0.4	---	---	<0.4	<0.4
28^thday	<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	<0.03	0.03	<0.03	---	<0.03	0.03
7^thday	0.05	0.05	<0.03	---	0.07	0.05
14^thday	<0.03	0.03	---	---	0.03	0.03
21^stday	0.05	0.05	---	---	0.04	0.04
28^thday	0.05	0.05	---	---	0.05	0.04
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, Solubilised Sulphur Black 1, was considered to be not readily biodegradable (12.1 % 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 Solubilised Sulphur Black 1. 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 1.297 mg O2/mg test item of Solubilised Sulphur Black 1was 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 Solubilised Sulphur Black 1 reached a mean of 12.1 % 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 34.5 % biodegradation was noted within 14 days and 36.2 % 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, Solubilised Sulphur Black 1, was considered to be not readily biodegradable (12.1 % 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