Reaction products of 1-(substitutedphenyl)urea coupled with diazotated potassium sodium substituted-5-{[2-(substituted)ethyl]sulfonyl}benzenesulfonate, further condensed with 2,4,6-trichloro-1,3,5-triazine, further converted with disubstituted benzene-1,4-disulfonic acid in aq. sodium hydroxide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: screening test, other

Remarks:

Inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 20 October 2015 and 24 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.9 (Biodegradation: Zahn-Wellens Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3200 (Zahn-Wellens / EMPA Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Test System
A mixed population of activated sewage sludge micro-organisms was obtained on 26 October 2015 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.


Preparation of Inoculum
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of DOC that may have been present. A sub-sample of the washed sewage sludge was then removed and the suspended solids concentration determined.

This inoculum was used for preparation of the replicate inoculum control, procedure control and test item vessels and the single toxicity control vessel.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

other: carbon

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

Mineral Medium
The mineral medium used in this study was that recommended in the OECD Guidelines.

Procedure
Preliminary Investigational Work
During the test, samples are taken for DOC analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge solids. Thus the following work was conducted and samples analyzed for DOC using a Shimadzu TOC-VCPH TOC analyzer.

A nominal amount of test item (329 mg) was dissolved in mineral medium (1000 mL) to give a 329 mg/L stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter). A further nominal amount of test item (329 mg) was dissolved in mineral medium and inoculated at a concentration of 300 mg suspended solids (ss)/L prior to adjusting to a final volume of 1000 mL. Two samples were taken for DOC analysis; one after filtration through a Gelman 0.45 µm AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and the other after centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 300 mg ss/L and then filtering or centrifuging as per the test item samples.

For the inoculum, a mixed population of activated sewage sludge micro-organisms was obtained on 20 October 2015 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.


Experimental Preparation
Test Item
The test item was dissolved directly in reverse osmosis purified deionized water prior to dispersal in inoculated mineral medium to give the required test concentration.

A nominal amount of test item (4000 mg) was dissolved in reverse osmosis purified deionized water with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 2 liters to give a 2000 mg/L stock solution. An aliquot (421 mL) of the 2000 mg/L stock solution was dispersed in inoculated mineral medium and the volume adjusted to 2 liters to give a final test concentration of 421 mg/L, equivalent to 100 mg carbon/L.

The volumetric flask containing the stock solution was inverted several times to ensure homogeneity of the solution.
A test concentration of 100 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Toxicity Control
A toxicity control, containing the test item and diethylene glycol was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.

An aliquot (421 mL) of the 2000 mg/L test item stock solution was dispersed in inoculated mineral medium along with an aliquot (220 mL) of the 2000 mg/L reference item stock solution. The volume was adjusted to 2 liters to give a final concentration of 421 mg test item/L, plus
220 mg diethylene glycol/L, equivalent to a final concentration of 200 mg carbon/L.


Preparation of Test System
The following test solutions were prepared and inoculated in 3 liter glass test vessels each containing 2 liters of solution:

a) An inoculum control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (diethylene glycol), in duplicate, in inoculated mineral medium to give a final concentration of 100 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 100 mg carbon/L.
d) The test item plus diethylene glycol, one replicate, in inoculated mineral medium to give a final concentration of 200 mg carbon/L to act as a toxicity control.

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.

Each test vessel was inoculated with the prepared inoculum at a final concentration of 300 mg suspended solids/liter in order to give an inoculum to carbon ratio of 3:1.

The test vessels were covered with polypropylene lids to reduce evaporation and incubated at temperatures of between 20 to 25 °C in darkness for 28 days. The test vessels were constantly aerated with compressed air via glass tubes and stirred constantly by a magnetic stirrer.


Evaluations
DOC Analysis
Samples (approximately 30 mL) were filtered through 0.45 µm Gelman AcroCap disposable filters. The first approximate 5 mL of filtrate was discarded.

Samples were taken for analysis at 0 and 3 hours and on Days 1, 7, 14, 21, 27 and 28.

Prior to sampling for analysis any losses due to evaporation were corrected by the addition of deionized water and any material adhering to the culture vessel resuspended.

The samples were analyzed for DOC using a Shimadzu TOC-VCPH TOC analyzer. Samples (50 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyzer. Total Carbon analysis is carried out at 680 ºC using a platinum catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involved conversion by 25% orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in triplicate.

Water Quality Criteria
Immediately prior to sampling the temperature and dissolved oxygen concentration (by means of a Hanna Instruments HI 93510 digital thermometer and a Hach HQ40d Flexi handheld meter) of each vessel were recorded. The pH of each vessel was also determined using a Hach HQ40d Flexi handheld meter and adjusted to pH 7 to 8, where necessary, with sodium hydroxide or hydrochloric acid solutions.


Data Evaluation
Determination of Carbon Content
From analysis of three replicate 329 mg/L stock solutions, the percentage carbon in the test item was determined to be 23.74%.

The theoretical amount of carbon present in the reference item, diethylene glycol (C4H10O3) was calculated as follows:

((no of C atoms x mol wt of C) / mol wt of diethylene glycol) x 100

= ((4 x 12.011) / 106.12) x 100 = 45.27 %

Thus for a test concentration of 220 mg/L the theoretical carbon concentration was approximately 100 mg C/L.


Percentage Biodegradation or Loss of DOC
The percentage biodegradation or loss of DOC for each sample was calculated using the following equation:

Dt (%) = [1 – (Ct – Cbt) / (Co – Cbo)] x 100

Where:
Dt = Biodegradation or loss of DOC (%) at time t
Ct = mean mg DOC/L of sample at time t
Cbt = mean mg DOC/L of control (blank) at time t
Co = mean mg DOC/L of sample after 3 hours ± 30 minutes
Cbo = mean mg DOC/L of control (blank) after 3 hours ± 30 minutes

Reference substance:

diethylene glycol

Preliminary study:

Preliminary Investigational Work
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.

Parameter:

% degradation (DOC removal)

Value:

0

Sampling time:

28 d

Details on results:

Definitive Test
From DOC analysis the test item attained 0% biodegradation after 28 days.

OECD Guideline No. 302B does not give any definitive pass levels for test items, however the “Summary of Considerations in the Report from the OECD Expert Group on Degradation and Accumulation” suggests that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralisation may be regarded as evidence for ultimate biodegradation.

The toxicity control attained 48% biodegradation after 14 days and 44% biodegradation after 28 days, therefore confirming that the test item was not toxic to the sewage treatment micro-organisms used in the study. The slight decrease in biodegradation between days 14 and 28 was considered to be due to sampling/analytical variation.

The pH values did not fall below 7.0 in any test vessel during the study. Aerobic conditions in the test vessels were maintained throughout the study as dissolved oxygen concentrations remained at or above 6.5 mg O2/L in all culture vessels.

Results with reference substance:

The procedure control, diethylene glycol attained 100% biodegradation after 14 and 28 days, thereby confirming the suitability of the inoculum and culture conditions.

Dissolved Organic Carbon (DOC) Values on Each Sampling Occasion

Vessel		DOC (mg Carbon/L)
		Day
		0 Hours	3 Hours	1	7	14	21	27	28
Inoculum Control	R1	1.06	2.39	2.15	3.42	3.34	3.74	3.24	3.72
	R2	0.94	1.40	1.75	3.32	3.42	3.41	3.97	4.82
	Mean	1.00	1.90	1.95	3.37	3.38	3.58	3.61	4.27
Procedure Control	R1	98.41	92.16	94.51	3.90	3.67	3.50	3.46	4.20
	R2	98.17	92.81	95.86	5.19	3.15	3.15	2.91	3.45
	Mean	98.29	92.49	95.19	4.55	3.41	3.33	3.19	3.83
Test Item	R1	109.70	102.80	103.90	115.70	103.60	112.60	105.40	114.90
	R2	110.60	103.60	110.00	116.40	102.80	110.20	99.78	108.30
	Mean	110.15	103.20	106.95	116.05	103.20	111.40	102.59	111.60
Toxicity Control		213.00	201.80	201.40	119.50	106.40	113.60	105.40	115.40

R₁- R₂= Replicates 1 and 2

Percentage Biodegradation

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
1	0	0	0
7	99	0	42
14	100	1	48
21	100	0	45
27	100	2	49
28	100	0	44

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item attained 0% biodegradation after 28 days.

OECD Guideline No. 302B does not give any definitive pass levels for test items, however the “Summary of Considerations in the Report from the OECD Expert Group on Degradation and Accumulation” suggests that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralization may be regarded as evidence for ultimate biodegradation.

The test item can therefore be considered not to have exhibited evidence of inherent, primary biodegradation under the experimental conditions employed in this study.

Executive summary:

Introduction

The study was performed to assess the inherent biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 302B, “Inherent Biodegradability; Modified Zahn-Wellens/EMPA Test”, Method C.9 of Commission Regulation (EC) No. 440/2008, and the US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3200.

 

Methods…….

The test item was exposed to activated sewage sludge micro-organisms at a concentration of 100 mg carbon/L with mineral medium in the dark at a temperatures of between 20 to 25 ºC for 28 days. The biodegradation of the test item was assessed by the determination of dissolved organic carbon removal. Control cultures with inoculum and the reference item, diethylene glycol, together with a toxicity control were used for validation purposes.

 

Results…….

The test item attained 0% biodegradation after 28 days.

 

OECD Guideline No 302B does not give any definitive pass levels for test items, however the “Summary of Considerations in the Report from the OECD Expert Group on Degradation and Accumulation” suggests that a figure of more than 20% biodegradation may be regarded as evidence for inherent, primary biodegradability. A figure of more than 70% mineralisation may be regarded as evidence for ultimate biodegradation.

 

The test item can therefore be considered not to have exhibited evidence of inherent, primary biodegradation under the experimental conditions employed in this study.

Description of key information

The study was performed to assess the inherent biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 302B, “Inherent Biodegradability; Modified Zahn-Wellens/EMPA Test”.

The test item was exposed to activated sewage sludge micro-organisms at a concentration of 100 mg carbon/L for 28 days. 

The biodegradation of the test item was assessed by the determination of dissolved organic carbon removal. Control cultures with inoculum and the reference item, diethylene glycol, together with a toxicity control were used for validation purposes.

 

The test item attained 0% biodegradation after 28 days.

The test item can therefore be considered not to have exhibited evidence of inherent, primary biodegradation under the experimental conditions employed in this study.

 

 

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed