Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

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:

June 06, 2016 to August 24, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

EU, OECD method, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

EC Number:

276-423-1

EC Name:

Disodium [6-hydroxy-5-[(2-hydroxy-5-sulphophenyl)azo]naphthalene-2-sulphonato(4-)]cuprate(2-)

Cas Number:

72152-71-7

Molecular formula:

C16H8CuN2O8S2.2Na

IUPAC Name:

Cuprate(2-), [6-(hydroxy-kO)-5-[2-[2-(hydroxy-kO)-5-sulfophenyl]diazenyl-kN1]-2-naphthalenesulfonato(4-)]-, sodium

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Species/Origin: Aerobic activated sludge, micro organisms from a domestic waste water treatment plant was supplied by the sewage plant Rossdorf, Germany.
Conditioning: The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The purpose of this study was to determine the ready biodegradability of the test item Sanodal-Rot B3LW. The test item was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.
This type of study is recognized by the international test guidelines and should provide a rational basis to assess the ready biodegradation properties of the test item when incubated with aerobic activated sludge.
Test Units
Type and Size: Manometric Test System with test flasks containing a volume of approximately 500 mL.
Apparatus: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany
Principle: The test flasks were incubated at 22°C * 1°C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

Preparation of Test Flasks:The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used.

Incubation: The closed test flasks were incubated in a climatised room under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.
Test Duration: 28 days
Measurement of Oxygen: The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany) each day.
Temperature: Temperature was measured each working day in the climatised room.
pH-Value: pH-values were measured in control, procedure control and a separately prepared test flask with test item at test start (to prevent loss of test item in the test flasks) and in all flasks at the end of the test using a pH-electrode WTW pH 340i.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guide-line. The reference item sodium benzoate was degraded to more than 60% after 4 days of incubation.

Results and discussion

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

Biodegradation of Test Item
Percentage Biodegradation:
The test item contains nitrogen; therefore the evaluation of biodegradation has to be based ThODNH4 and ThODNO3. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days.
The mean biodegradation at test end after 28 days was 0% (ThODNH4, ThODNO3).
Conclusion: Degradation of the test item was 0% after 28 days.
Therefore, Sanodal-Rot B3LW is considered to be not readily biodegradable.

Biodegradation of Reference Item Sodium Benzoate
Percentage Biodegradation: The reference item sodium benzoate was sufficiently degraded to 83% after 14 days and to 89% after 28 days of incubation.
Conclusion: The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Biodegradation in the Toxicity Control
Percentage Biodegradation: In the toxicity control containing both, the test item and the reference item sodium benzoate, 55% (ThODNH4) biodegradation was noted within 14 days and 56% (ThODNH4) biodegradation after 28 days of incubation (50% and 52% based on ThODNO3). According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.
Conclusion: According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

Abiotic Control
Oxygen Demand: The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no use to correct the degradation of the test item and toxicity control.

BOD5 / COD results

Results with reference substance:

Biodegradation of Reference Item Sodium Benzoate
Percentage Biodegradation: The reference item sodium benzoate was sufficiently degraded to 83% after 14 days and to 89% after 28 days of incubation.
Conclusion: The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Any other information on results incl. tables

Table2. Cumulative Biochemical Oxygen Demand (mg O₂/L) in Test Flasks during the Test Period of 28 Days

Time	Flask No.
(days)	1	2	3	4	5	6	7
1	5	5	0	0	55	0	15
2	5	5	5	5	85	0	75
3	10	5	5	10	100	0	95
4	10	10	5	10	125	0	110
5	10	10	10	10	130	0	120
6	10	10	10	10	140	0	135
7	15	10	10	15	140	0	140
8	15	10	10	15	145	0	150
9	15	15	10	15	150	0	155
10	15	15	15	15	150	0	155
11	15	15	15	15	155	0	160
12	20	15	15	20	155	0	160
13	20	15	15	20	160	0	165
14	20	15	15	20	160	0	165
15	20	15	15	20	165	0	165
16	20	15	15	20	165	0	165
17	20	15	15	20	165	0	165
18	20	15	15	20	165	0	170
19	20	15	20	20	170	0	170
20	20	15	20	20	170	0	170
21	20	15	20	25	170	0	170
22	20	15	20	25	170	0	170
23	20	15	20	25	170	0	170
24	20	15	20	25	170	0	170
25	20	15	20	25	175	0	170
26	20	15	20	25	175	0	170
27	20	15	20	25	175	0	170
28	20	15	20	25	175	0	175

            Flasks 1 and 2: Sanodal-Rot B3LW

            Flasks 3 and 4: inoculum control

Flask 5:reference(procedure control)

            Flask 6: abiotic control

            Flask 7: toxicity control

Table3. Percentage Biodegradation of the Test Itemand of the Toxicity Control based on
ThOD_NO3and of Sodium Benzoatebased on ThOD_NH4

Time		Percentage Biodegradation^1
(Days)		Sanodal-Rot B3LW^1			Sodium Benzoate^2		Toxicity control^1, 2
		Flask 1 [%]		Flask 2 [%]		Flask 5 [%]		Flask 7 [%]
1		4		4		32		5
2		0		0		46		24
3		2		0		54		30
4		2		2		68		35
5		0		0		70		37
6		0		0		76		42
7		2		0		74		43
8		2		0		77		47
9		2		2		80		48
10		0		0		78		47
11		0		0		81		49
12		2		0		80		48
13		2		0		83		50
14		2		0		83		50
15		2		0		86		50
16		2		0		86		50
17		2		0		86		50
18		2		0		86		52
19		0		0		87		51
20		0		0		87		51
21		0		0		86		50
22		0		0		86		50
23		0		0		86		50
24		0		0		86		50
25		0		0		89		50
26		0		0		89		50
27		0		0		89		50
28		0		0		89		52

            ¹ThOD_NH3of Sanodal-Rot B3LW: 1.208 mg O₂/mg test item
            ²ThOD_NH4of sodium benzoate: 1.666 mg O₂/mg reference item

Table4.pH-Values at the End of the Test

Flask No.	Treatment	pH-value
1	Sanodal-Rot B3LW	7.6
2	Sanodal-Rot B3LW	7.6
3	Inoculum control	7.6
4	Inoculum control	7.6
5	Reference item (procedure control)	7.8

 

 

 

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Degradation of the test item was 0% after 28 days.
Therefore, the test item is considered to be not readily biodegradable.

Executive summary:

Title:	Ready Biodegradability in a Manometric Respirometry Test
Guidelines:	-     Commission Regulation 440/2008/EC, Method C.4-D of: Manometric Respirometry Test (EEC Publication No. L 142/496, May 2008) -     OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry Test", adopted
Material and Methods:
Test Item:	Batch No.:Lot 101115
Test Species:	Aerobic activated sludge (microorganisms from a domestic waste­water treatment plant) was supplied by the sewage treatment plant of Rossdorf, Germany.
Test Design:	The test item was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.
Endpoints:	Degradation rate of test item calculated by the oxygen consumption of the aerobic activated sludge microorganisms after 28 days of incubation.
Test Item Loading Rate (initial concentration in medium C0):	103.1 mg/L corresponding to an oxygen demand of about 99.6 mg/L (ThODNH4) and 124.5 mg/L (ThODN03)
Reference Item:	Sodium Benzoate
Reference Item Loading Rate:	103.3 mg/L corresponding to an oxygen demand of about 172.1 mg/L (ThODNH4)
Test Conditions:	22°C ± 1°C, darkness
Results:
Biodegradation of Sanodal-Rot B3LW:	The test item contains nitrogen; therefore the evaluation of biodegradation has to be based ThODNH4and ThODNO3. The criterion for ready biodegradability under the conditions of a manometric respirometry test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days. The mean biodegradation at test end after 28 days was 0% (ThODNH4, ThODNO3). Therefore, the test item is considered to be not readily biodegradable based on ThODNH4/ ThODNO3.
Biodegradation of Sodium Benzoate:	The reference item sodium benzoate was sufficiently degraded to 83% after 14 days and to 89% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.
Biodegradation of the Toxicity Control:	In the toxicity control containing both, the test item and the reference item sodium benzoate, 55% (ThODNH4) biodegradation was noted within 14 days and 56% (ThODNH4) biodegradation after 28 days of incubation (50% and 52% based on ThODNO3). According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

This study is classified acceptable and satisfies the guideline requirements for ready biodegradability studies.