Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

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:

6 November 1991 - 4 December 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to recent EU test guidance in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Principles of method if other than guideline:

The respirometric test was a modified version of the OECD Test Guideline 301C in which activated sludge obtained from a local sewage treatment works was used as the inoculum. The biodegradation was measured over a 28-day period in a manometric respirometer and at the end of the test period the concentration of Dissolved Organic Carbon (DOC) and test substance remaining were determined.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

EC Number:

413-550-5

EC Name:

Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

Cas Number:

142068-96-0

Molecular formula:

Hill formula: C53H28Cl2N14Na8O26S8

IUPAC Name:

octasodium 5-[(4-chloro-6-{[4-({[4-chloro-6-({7-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-8-hydroxy-3,6-disulfonatonaphthalen-1-yl}amino)-1,3,5-triazin-2-yl]amino}methyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]-3-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate

Details on test material:

The test sample Substance HI 12339 (reference NBY 405/74) was supplied by the Central Notification Unit, Imperial Chemical Industries PLC.

The sample was received on 29 September 1991 and was assigned the code V723.

Information supplied with the test sample V723 indicated that it contained 10.5% of water and test concentrations were adjusted accordingly.

The test sample was a dark brown powder. It was stored in the dark at ambient temperature in the container in which it was received until required for testing when, an appropriate subsample was provided for the test operator.

Specific details on test material used for the study:

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

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Activated sludge was obtained from Newton Abbot Sewage Treatment Works (Newton Abbot, Devon) which treats sewage of primarily domestic origin. At the laboratory the activated sludge was kept aerated at 20°C and the pH maintained at 7 ± 0.2.

One week prior to the test 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.

The use of activated sludge (A/S) from a sewage treatment works is a deviation from the original OECD Guideline 301C. This alternative procedure was proposed by the UK and is included in Draft Method DG XI/283/82 considered by the EEC. The one week pre-conditioning of the A/S is a procedure recommended by ECETOC.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

112 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Reference substance

The reference substance employed was sodium acetate as recommended in OECD Guideline Method 301C.

Test medium

The test medium was made up according to OECD Guideline 301C and contained the following nutrients per litre of deionised water KH2P04 85 mg; K2HPO4 217.5 mg; Na2HPO4.2H20 334 mg; NH4Cl 25 mg; MgSO4.7H2O 22.5 mg; CaCl2.2H2O 36.4 mg; FeCl3.6H2O 0.25 mg.

Test apparatus

The measurement of oxygen uptake was conducted in the Hach manometric biochemical oxygen demand (BOD) apparatus (Hach Chemical Co, Ames, Iowa, USA). Each individual unit consisted of a dark glass bottle (nominal capacity 500 ml) connected to a closed end mercury manometer. Carbon dioxide produced during respiration was absorbed by potassium hydroxide (KOH) placed in a cup in the neck of each bottle; the oxygen taken up was measured as a decrease in pressure by reading directly the BOD (mg 02/l) on a calibrated scale.

Sets of units were assembled together and stirred by means of motor driven magnets which rotated PTFE coated stirrer bars in each bottle. The whole apparatus was maintained in a temperature controlled room at 20 ± 2°C.

Test organisms

Activated sludge was obtained from Newton Abbot Sewage Treatment Works (Newton Abbot, Devon) which treats sewage of primarily domestic origin. At the laboratory the activated sludge was kept aerated at 20°C and the pH maintained at 7 ± 0.2.

One week prior to the test 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.

Experimental design

Following the week of pre-conditioning described above, nine units were set up for the test.

Oxygen uptake was recorded daily during the experimental period. The readings were corrected for the blank and the volume of test solution.

At the end of the experiment (28 days) the contents of the bottles were centrifuged and the supernatant analysed for dissolved organic carbon (DOC). The supernatants of the bottles containing Substance HI 12339 were also analysed for the substance itself using the method outlined above.

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

Preliminary study:

Not applicable

Test performance:

Biochemical Oxygen Demand (BOD)

The results which are summarised in Table 2 below show that sodium acetate (the positive control) has the BOD:TOD (Theoretical Oxygen Demand) ratio in excess of 60% expected for a biodegradable substance.

The results for Substance HI 12339 show a zero BOD over the 28-day period of the test

Dissolved Organic Carbon (DOC) analysis

At the end of the test, the contents of the units were centrifuged and analysed for their DOC content and the results are summarised in Table 3 below. The residual DOC values for the sodium acetate positive control were low, corresponding to a high level of biodegradation as expected for this substance. The residual DOC values for Substance HI 12339 corresponded to zero biodegradation.

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

The Biochemical Oxygen Demand (BOD) of Substance HI 12339 was measured in a respirometric test over 28 days at a nominal test concentration of 100 mg/1. Only a negligible Biochemical Oxygen Demand (BOD) was found during the 28 days of the test and the BOD5 and BOD28 of the test substance are essentially zero.

The Dissolved Organic Carbon (DOC) level as determined at the end of the test corresponded to zero biodegradation as essentially did specific analysis. The Chemical Oxygen Demand (COD) was measured as 0.88 g O2/g Substance HI 12339.

BOD5 / COD results

BOD5 / COD

Parameter:

BOD5

Value:

0 g O2/g test mat.

Results with reference substance:

The results show that sodium acetate (the positive control) has the BOD:TOD (Theoretical Oxygen Demand) ratio in excess of 60% expected for a biodegradable substance.

At the end of the test, the contents of the units were centrifuged and analysed for their DOC content. The residual DOC values for the sodium acetate positive control were low, corresponding to a high level of biodegradation as expected for this substance

Any other information on results incl. tables

TABLE 2

 

STUDY V723/C: BIOCHEMICAL OXYGEN DEMAND RESULTS

 

	Unit no	BOD (mgO2/l)
		Test day number
		5	10	15	20	28
Blank control	1-3	3.2	5.3	6.3	6.3	6.7
Sodium acetate (200 mg/l)	4-6	83.3	96.7	96.7	96.7	96.7
Sodium acetate - blank	-	80.1	91.4	90.4	90.4	90.0
BOD sodium acetate (g 02/g sodium acetate)	-	0.40	0.46	0.45	0.45	0.45
BOD sodium acetate (% TOD^1)	-	59	68	66	66	66
V723 (112 mg/l)	13 - 15	0	0	0	0	0
V723 (112 mg/l) - blank	-	0	0	0	0	0
BOD V723 (g O2/g V723)		0	0	0	0	0

The theoretical oxygen demand (TOD) of sodium acetate is calculated as: 0.68 g O₂/g sodium aceta

TABLE 3

STUDY V723/C: DISSOLVED ORGANIC CARBON (DOC) RESULTS AFTER 28 DAYS

 

	Unit no	Mean DOC(mg/1)
Blank control	1 - 3	1.6
Sodium acetate (200 mg/1)	4-6	2.5
Sodium acetate - blank	-	0.9
% Biodegradation^1	-	98%
V723 (112 mg/1)	13 - 15	34.0
V723 - blank	-	32.4
% Biodegradation^2	-	0%

¹    The theoretical DOC of 200 mg/1 of sodium acetate is 59 mg/1; this figure was used to calculate the % biodegradation.

 

²    The measured DOC of 1120 mg/1 of V723 is 312 mg/1; this figure was used to calculate the % biodegradation.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The Biochemical Oxygen Demand (BOD) of Substance HI 12339 was measured in a respirometric test over 28 days at a nominal test concentration of 100 mg/l. Only a negligible Biochemical Oxygen Demand (BOD) was found during the 28 days of the test and the BOD5 and BOD28 of the test substance are essentially zero.

The Dissolved Organic Carbon (DOC) level as determined at the end of the test corresponded to zero biodegradation as essentially did specific analysis. The Chemical Oxygen Demand (COD) was measured as 0.88 g O2/g Substance HI 12339.

Executive summary:

The substance cannot be considered to be biodegradable on the basis of the results of the study.