Potassium sodium amino-hydroxy-[(3-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]-[(2-sulfonato-4-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-disulfonate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 29 October 2014 and 05 December 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

None

GLP compliance:

yes

Specific details on test material used for the study:

None

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 04 November 2014 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.:

418 mg/L

Based on:

test mat.

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.

An amount of test item (352.8 mg) was dissolved in mineral medium (1000 mL) to give a 352.8 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 amount of test item (352.8 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.

A mixed population of activated sewage sludge micro-organisms was obtained on
29 October 2014 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 and the volume adjusted to 2 liters to give a 2000 mg/L stock solution. An aliquot (418 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 418 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 (418 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 418 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.


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

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.

Test performance:

No data

Key result

Parameter:

% degradation (DOC removal)

Value:

0

Sampling time:

28 d

Remarks on result:

other: St. dev. not specified

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 51% biodegradation after 14 days and 51% biodegradation after 28 days, therefore confirming that the test item was not toxic to the sewage treatment micro-organisms used in the study.

The pH values did not fall below 6.8 in any test vessel during the study and were readjusted to pH 7 to 8 where necessary on sampling days. Aerobic conditions in the test vessels were maintained throughout the study as dissolved oxygen concentrations remained at or above 4.1 mg O2/L in all culture vessels.

Results with reference substance:

The procedure control, diethylene glycol attained 100% biodegradation after 14 days and 101% biodegradation after 28 days, thereby confirming the suitability of the inoculum and culture conditions. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation.

Dissolved Organic Carbon (DOC) Values on Each Sampling Occasion

Vessel		DOC (mg C/L)
		Day
		0 Hours	3 Hours	1	7	14	21	27	28
Inoculum Control	R1	1.01	1.36	1.93	3.03	3.25	3.18	3.70	4.18
	R2	1.04	1.33	1.83	3.17	3.15	3.28	3.58	3.56
	Mean	1.03	1.35	1.88	3.10	3.20	3.23	3.64	3.87
Procedure Control	R1	105.40	105.00	103.20	2.96	3.21	3.12	3.09	3.29
	R2	104.70	105.30	102.40	2.92	3.16	3.16	3.20	3.22
	Mean	105.05	105.15	102.80	2.94	3.19	3.14	3.15	3.26
Test Item	R1	107.80	108.20	105.70	110.10	109.00	109.70	107.10	111.50
	R2	106.30	106.90	104.80	107.60	107.40	112.00	109.40	112.70
	Mean	107.05	107.55	105.25	108.85	108.20	110.85	108.25	112.10
Toxicity Control		205.90	205.80	201.70	110.80	102.40	104.00	100.60	103.80

R₁- R₂= Replicates 1 and 2

Percentage Biodegradation / DOC Loss Values

Day	% Biodegradation / DOC Loss
	Procedure Control	Test Item	Toxicity Control
1	3	3	2
7	100	0	47
14	100	1	51
21	100	0	51
27	100	1	53
28	101	0	51

 

Dissolved Organic Carbon (DOC) Values from the Preliminary Investigational Work

In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge the following samples were analyzed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-V_CPHTOC analyzer.

 

Sample	DOC Concentration		% of Nominal Carbon Content
	mg C/L	mg C/L corrected for appropriate control
Mineral Medium	<LOQ	-	-
Control, inoculated at 300 mg ss/L, Filtered	1.16	-	-
Control, inoculated at 300 mg ss/L, Centrifuged	1.18	-	-
353 mg/L Untreated	83.26	83.26	99
353 mg/L Filtered	83.29	83.29	99
353 mg/L, inoculated at 300 mg ss/L, Filtered	84.60	83.44	99
353 mg/L, inoculated at 300 mg ss/L, Centrifuged	85.31	84.13	100

These results indicated that the test item did not adsorb to filter matrices or activated sewage sludge. Therefore, for the purpose of the test, the samples taken for DOC analysis were filtered to remove the suspended solids present without causing a loss of any test item.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

No biodegradation observed under the conditions employed, hence the test item was considered to be not inherently biodegradable.

Executive summary:

A study was performed to assess the inherent biodegradability of the test item in an aerobic aqueous media according to OECD Guideline 302B, EU Method C.9 and US EPA Test Guideline OCSPP 835.3200. In this study, 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 temperatures of between 20 and 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 and a toxicity control were used for validation purposes. The test item attained 0% biodegradation after 28 days. The test item cannot therefore be considered as inherently biodegradable under the experimental conditions employed in this study.

Description of key information

The test item was considered as not inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

A study was performed to assess the inherent biodegradability of the test item in an aerobic aqueous media according to OECD Guideline 302B, EU Method C.9 and US EPA Test Guideline OCSPP 835.3200. In this study, 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 temperatures of between 20 and 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 and a toxicity control were used for validation purposes. The test item attained 0% biodegradation after 28 days. The test item cannot therefore be considered as inherently biodegradable under the experimental conditions employed in this study. The test item attained 0% biodegradation after 28 days and cannot therefore be considered as inherently biodegradable under the experimental conditions employed in this study.