Octasodium 2-(6-(4-chloro-6-(3-(N-methyl-N-(4-chloro-6-(3,5-disulfonato-2-naphthylazo)-1-hydroxy-6-naphthylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,5-disulfonato-1-hydroxy-2-naphthylazo)naphthalene-1,5-disulfonate

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:

From September 16, 1993 to October 14, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch number: NBY 3507/15
Source: Central notification unit, Zeneca Limited
Purity: 66.9%
Colour: orange/red
Physical state: solid

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Buckland Sewage Treatment Works, Newton Abbot, Devon.
- At the laboratory the activated sludge was kept aerated at 20 and pH maintianed at approximately 7.
- Preparation of inoculum: One week prior to the activated sludge was centrifuged resuspended in the test medium and the solids concentration determined. This sludge was then aerated and stirred for one week prior to use.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The test medium contained the following nutrients per litre of deionised water: KH,1304 85 mg; K,HPO4 217.5 mg; Na2HPO4.21120 334 mg; NH.4C1 5 mg; MgSO4,7r120 22.5 mg; CaC12.21120 36.4 mg; FeC11.611,0 0.25 mg.
- Test temperature: 20 ± 2°C
- pH: 7
- Preperation of inoculum: Following the week of pre-conditioning, nine units were set up for the test. The units containing test substance were dosed from a primary stock containing 1000 mg/L. Oxygen uptake was recorded daily during the experimental period. The readings were corrected for the blank and the volume of test solution. The biodegradation was calculated as a percentage of the chemical oxygen demand for the substance under test

TEST SYSTEM
- Culturing apparatus: Closed respirometer flask (Hach manometric biochemical oxygen demand (BOD) apparatus)
- Number of culture flasks/concentration: Three for inoculum blank and test substance; two each for a biotic control, reference control and toxicity control.
- Details of trap for CO2 and volatile organics if used: CO2 produced was trapped in potassium hydroxide.

SAMPLING
- Sampling frequency: every day

CONTROL AND BLANK SYSTEM
- Inoculum blank: Containing only inoculum but without test substance
- Abiotic sterile control: Yes, 200 mg/L sodium acetate in inoculated mineral medium.
- Toxicity control: Yes, a solution of 100 mg/L test substance and 200 mg/L sodium acetate in inoculated mineral medium.

Reference substance:

acetic acid, sodium salt

Remarks:

(tested at 200 mg/L concentration)

Key result

Parameter:

other: % degradation (DNPOC)

Remarks:

Dissolved non purgeable organic carbon

Value:

7

Sampling time:

28 d

Details on results:

1) Biochemical oxygen demand (BOD):

Test substance BOD value after 28 d indicated that no measurable biodegredation has occurred. See the table 1 in below results section.

2) Spectophotometric and chromatographic analysis:

A comparison between the chromatograms obtained from freshly prepared standard and the contents of the bottle indicates that test substance does not undetgone any structural changes during the course of the study. Refere to table number 2 and 3 in below results section.

3) Chemical oxygen demand (COD):

The measured COD values of test substance and sodium acetate were 0.71 and 0.69 g of oxygen per gram respectively. The COD obtained for sodium acetate compares favourably with this theoretical oxygen demand of 0.68 g of O2/g thus demonstrating the validity of the analytical technique.

4) Dissolved non purgeable organic carbon (DNPOC):

Analysis of 1000 mg/L stock solution used to dose test substance test bottles gave a value of 29% carbon. Elemental analysis carried out on the test material gave a carbon value of 30.1%. Analysis of the 2000 mg/L stock solution of sodium acetate gave a value of 29% carbon which matches with the THOD for sodium acetate. The 28 d DNPOC results are summarised in table 4 of below results section. Sodium acetate values were low which correspond to high level of biodegradation as expected. The residual DNPOC values for test substance indicate that greater than 90% nominal amount of test substance was still present at the end of the test.

Results with reference substance:

Sodium acetate achieved BOD:COD ratio in excess of 60% expected for biodegradable substance

Table 1: Test substance biochemical oxygen demand results

	Bottle no	BOD (mg 02/L)
		Test day number
		5	10	15	20	28
Blank control	1-3	L7	4.2	5.7	6	7
Sodium acetate (200 mg/L)	4-6	93	98	103	107	108
Sodium acetate - blank	-	91	94	97	101	101
BOD sodium acetate (g 02/g)	-	0.46	0.47	0.49	0.51	0.51
% Biodegradability ([BOD/COD] x 100%)^b	-	67	68	71	74	74
test substance (100 mg/L)	7	0	0	0	0	0
	8	0	0	0	5	5
	9	0	5	5	5	5
Mean	7-9	0	1.7	1.7	3.3	3.3
X595 (100mg/1) mean - blank		0	0	0	0	0
BOD test substance (g 02/g)		0	0	0	0	0
% Biodegradability ([BOD/COD] x 100%)^c		0	0	0	0	0

Table 2: Concentration of test substance after 28 d spectrophotometric analysis:

Bottle number	Sample	Concentration of test susbtance determined (mg/L)	% Of nominal concentration	Mean %
1	control	< 0.35	-	-
2	control	< 0.35	-
3	control	< 0.35	-
4	100 mg/L test substance	100	100	99
5	100 mg/L test substance	97	97
6	100 mg/L test substance	100	100

Table 3: Concentration of test substance after 28 d chromatographic analysis:

Bottle number	Sample	Concentration of test substance determined (mg/L)	% Of nominal concentration	Mean
1	Control	<0.044
2	Control	<0.044	-	-
3	Control	<0.044
4	100 mg/1 test substance	110	110	110
5	100 mg/1 test substance	110	110
6	100 mg/1 test substance	110	110

Table 4: Dissolved carbon results after 28 d:

	Unit no	Mean DNPOC (mg/L)
Blank control	1-3	1.9
Sodium acetate (200 mg/L)	4-6	2.1
Sodium acetate blank	-	0.2
% degradation of sodium acetate	-	> 99%
Test substance (100 mg/L)	7-9	28.8
Test substance blank	-	26.9
% degradation of test substance	-	7%

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the study conditions, the substance was not considered to be readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the substance according to OECD Guideline 301 F and EU Method C.4 -D, in compliance with GLP. Activated sludge was inoculated with the test (100 mg/L) or reference (acetic acid, sodium salt; 200 mg/L) substance, kept in darkened, enclosed respirometer flasks (20±2°C) and observed for degradation by measurement of the dissolved oxygen concentration over a 28 d period. Biodegradation of the test substance was determined to be 7%, based on BOD and DNPOC (dissolved non purgeable organic carbon), after 28 d. Spectrophotometric and chromatographic analysis of test substance at the end of 28 d test period showed mean test substance concentration of 99 and 110% respectively of the nominal starting concentration without any structural change. Therefore, under the study conditions, the substance was not considered to be readily biodegradable (Comber, 1993).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

A study was conducted to determine the ready biodegradability of the substance according to OECD Guideline 301 F and EU Method C.4 -D, in compliance with GLP. Activated sludge was inoculated with the test (100 mg/L) or reference (acetic acid, sodium salt; 200 mg/L) substance, kept in darkened, enclosed respirometer flasks (20±2°C) and observed for degradation by measurement of the dissolved oxygen concentration over a 28 d period. Biodegradation of the test substance was determined to be 7%, based on BOD and DNPOC (dissolved non purgeable organic carbon), after 28 d. Spectrophotometric and chromatographic analysis of test substance at the end of 28 d test period showed mean test substance concentration of 99 and 110% respectively of the nominal starting concentration without any structural change. Therefore, under the study conditions, the substance was not considered to be readily biodegradable (Comber, 1993).