hexasodium 5-[(4-{3-[(8-benzamido-1-hydroxy-3,6-disulfonato-2-naphthyl)diazenyl]-4-sulfonatoanilino}-6-[(2-sulfonatoethyl)amino]-1,3,5-triazin-2-yl)amino]isophthalate

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:

The study was conducted between 6 August 2007 and 12 October 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances" (November 21, 2003; No. 1121002)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Magenta T-43

IUPAC Name:

Magenta T-43

Test material form:

solid: particulate/powder

Details on test material:

Chemical name:
Mixture of hexasodiun 5-{4-[3-(8-benzoylamino-1-hydroxy-3,6-disulfonatonaphthalene-2-yldiazenyl)-4-sulfonatoanilino]-6-(2-sulfonatoethylamino)-1,3,5-triazine-2-ylamino} benzene-1 ,3-dicarboxylate (main product) and heptasodiun 5,5'-{6-[3-(8-benzoylamino-1-hydroxy-3,6-disulfonatonaphthalene-2-yldiazenyl)-4-sulfonatoanilino]-2,4-(1,3,5-triazine)diyldiimino} bis(benzene-1 ,3-dicarboxylate) (byproduct)

Molecular formula:
Main Product: C36H23N9Na6O18S4
By Product: C42H22N9Na7O19S3

Appearance: Dark red powder
Storage conditions: Dark storage place at room temperature
Lot Number: H186-5
Purity of Test Item: 91.9 % (main component 90.6 %, byproduct 1.3 %)
Impurity: Water= 5.5%
Inorganic component (Na2SO4) = 0.1%
Total of unknown organic component = 2.5% (each component <1%)
The test item was treated with correction by the purity

Stability: Stable (The test item was stable under the storage conditions, as shown by the finding that IR spectra of the test item before the experimental start and after the experimental completion were identical).

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

Activated sludge
Preparation of activated sludge:
Activated sludge used in the present test was prepared.

Sampling sites:
On-site sludge sampling was carried out at the following 10 locations in Japan:
Fushikogawa city sewage plant (Sapporo-shi, Hokkaido)
Fukashiba industrial sewage plant (Kamisu-shi, Ibaraki)
Nakahama sewage treatment plant (Osaka-shi, Osaka)
Ochiai treatment plant (Shinjuku-ku, Tokyo)
Kitakami River (lshinomaki-shi, Miyagi)
Shinano River (Niigata-shi, Niigata)
Yoshino River (Tokushima-shi, Tokushima)
Lake Biwa (Otsu-shi, Shiga)
Hiroshima Bay (Hiroshima-shi, Hiroshima)
Dokai Bay (Kitakyushu-shi, Fukuoka)

Sampling method
Sewage plant:
Return sludge was collected.

Rivers, lake and sea:
Surface water and surface soil which was in contact with the atmosphere were collected.
Sampling date: June, 2007

Preparation method of activated sludge:
Activated sludge was prepared as follows to maintain its uniformity. The filtrate (5 L) of the supernatant of the activated sludge* cultivated about for 3 months was mixed with the mixed filtrate (5 L) of the supernatant of a sludge collected newly at each location. The mixed filtrate (10 L) was aerated** after the pH value of the mixture was adjusted to 7.0 ± 1.0.

(*The activated sludge cultivated the mixed filtrate (10 L) of the supernatant of sludge collected at the 10 locations according to the cultivation methods described later.
**Pre-filtered open air was used).

Cultivation
Approximately 30 minutes after ceasing aeration of the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed. Dechlorinated water was added to the remaining portion so that the total volume reached 10 L. This mixture was aerated for 30 minutes or more, and then a pre-determined amount of synthetic sewage* was added to the mixture so that the concentration of the synthetic sewage was 0.1% in the volume of dechlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 25 ± 2 °C.

(*Synthetic sewage:
Glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water to obtain 50 g/L of the solution for each component. The pH of the solution was adjusted to 7.0±1.0 with sodium hydroxide).

Control and use:
During cultivation, the appearance of the supernatant, sedimentation of the sludge, formation of flock, pH, dissolved oxygen concentration in the solution and temperature were checked to maintain a normal state of sludge. It was confirmed that these were within the scope of the control standard stipulated in the "Testing Methods for New Chemical Substances", and these results were stored as raw data. Microflora in the activated sludge was microscopically observed and sludge with no abnormal symptoms was used for the test. The activated sludge, which was cultivated for 19 hours after it had been added the synthetic sewage, was used.

Inspection of activity and date of initiation of use of activated sludge:
Inspection of activity: Activity of the sludge was assessed using standard items before initiation of use.
Date of initiation of use: July 17, 2007

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 1

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Measurement of Biochemical Oxygen Demand (BOD)

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

DOC removal

Remarks:

Measurement of Total Organic Carbon

Details on study design:

Performance of biodegradation test
Preparations for test:
Measurement of concentration of suspended solid in activated sludge. The concentration of suspended solid was measured to determine the amount of activated sludge to add.

Method: In accordance with Japanese Industrial Standards (JIS) K 0I02-1998 Section 14.1
Date: August 6, 2007
Result: Concentration of suspended solid in the activated sludge was 3870 mg/L.

Preparation of basal culture medium:
Each 3 mL of solutions A, B, C and D, which are prescribed in JIS K 0102-1998 Section 21, were made up to 1000 mL with purified water (Japanese Pharmacopeia, Takasugi Pharmaceutical Co., Ltd.) and then the pH of this solution was adjusted to 7.0.

Reference item:
Aniline (reagent grade, Wako Pure Chemical Industries, Ltd. Lot No. TSR3919) was used as a reference item to confirm that the sludge was sufficiently active.

Instruments and conditions of cultivation:

Instruments for cultivation:
Closed system oxygen consumption measuring apparatus:
Temperature controlled bath and measuring unit: Asahi Techneion Co., Ltd.
Data sampler: Asahi Techneion Co., Ltd.
Vessel 300 mL in volume (improved type)
Absorbent for carbon dioxide: Soda lime No.1 (for absorption of carbon dioxide, Wako Pure Chemical Industries, Ltd.)

Conditions of cultivation:
Cultivation temperature: 25 ± 1°C
Cultivation duration: 28 days (under conditions of darkness)
Stirring method: Each test solution was stirred by a magnetic stirrer.

Observation and measurement of test conditions:
Observation of test solution
During the cultivation, the appearance of the test solution was observed once a day and conditions of the instruments were checked properly.

Measurement of biochemical oxygen demand (BOD)
During the cultivation period, the change in BOD of the test solutions was measured by autorecording using a data sampler. Cultivation temperature was measured and recorded once a day.

Preparation of test solutions:
The following test solutions were prepared and cultivated under the conditions described.

Addition of test item or aniline:
(a) Test solution (water + test item) (n= 1, Vessel No.1)
In one test vessel, 3 mL of 10.0 g/L of the test item in water was added to 297 mL of purified water, so that the concentration of the test item reached 100 mg/L and then the pH of the solution was measured. 10.0 g/L of the test item in water was pre-treated as follows. The test sample was accurately weighed and dissolved in purified water to obtain it.

(b) Test solution (sludge + test item) (n=3, Vessel No. 2, 3 and 4)
In each test vessel, 3 mL of 10.0 g/L of the test item in water was added to the basal culture medium [the volume was less than 297 mL by the volume (2.33 mL) of activated sludge inoculated], so that the concentration of the test item reached 100 mg/L and then the pH of the solution was measured. 10.0 g/L of the test item in water was pretreated as follows. The test sample was accurately weighed and dissolved in purified water to obtain it.

(c) Test solution (sludge + aniline) (n=1, Vessel No.5)
In one test vessel, 29.5 µL [30 mg = 29.5 µL x 1.022 g/cm3 (density)] of aniline was taken out by microsyringe and added to [the volume was less than 300 mL by the volume (2.33 mL) of activated sludge inoculated], so that the concentration of aniline reached 100 mg/L.

(d) Test solution (control blank) (n=1, Vessel No.6)
In one test vessel, nothing was added to the basal culture medium [the volume was less than 300 mL by the volume (2.33 mL) of activated sludge inoculated].

Inoculation of activated sludge:
The activated sludge cultivated under the conditions described earlier was added to each test vessel, (b), (c) and (d), so that the concentration of the suspended solid reached 30 mg/L.

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Remarks:

Biochemical Oxygen Demand

Value:

0

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (DOC removal)

Value:

1

Sampling time:

28 d

% Degradation 3

Parameter:

% degradation (test mat. analysis)

Remarks:

Percentage biodegradation of test item by HPLC

Value:

1

Sampling time:

28 d

Details on results:

Percentage Degradation:
Data for percentage biodegradations after 28 days were recorded.

Overall Results:

Percentage biodegradation by BOD = average 0%
Percentage biodegradation by DOC = average 1%
Percentage biodegradation of test item (HPLC) = average 1%

BOD5 / COD results

Results with reference substance:

Points of degradation plot (reference substance):
56 % degradation after 7 d
71 % degradation after 14 d
76 % degradation after 21 d
78 % degradation after 28 d

Any other information on results incl. tables

Analytical results of test solutions

Analytical results of the test solutions after 28 days were as follows.

In the test solution (water + test item) and the test solutions (sludge + test item), approximately theoretical amount of the test item remained and no peak except the test item was detected on the HPLC chromatogram. Therefore, any converted products were judged not to be produced and then not analyzed.

Appearance of test solutions

Appearances of test media in cultivation vessels were as follows.

 

	Test Solution	Appearance	pH
At the start of cultivation	Water + test item	The test item was dissolved. The test solution was red	Vessel-1 6.8
	Sludge + test item	The test item was dissolved. The test solution was red	Vessel-2 6.9 Vessel-3 6.9 Vessel-4 6.9
At the end of cultivation	Water +test item	Insoluble compound was not observed. The test solution was red.	Vessel-1 7.0
	Sludge + test item	Insoluble compound except the sludge was not observed. Growth of the sludge was not observed. The test solution was red.	Vessel-2 7.2 Vessel-3 7.2 Vessel-4 7.2

 

Analytical results of test solutions

Analytical results of the test solutions after 28 days were as follows.

In the test solution (water + test item) and the test solutions (sludge + test item), approximately theoretical amount of the test item remained and no peak except the test item was detected on the HPLC chromatogram. Therefore, any converted products were judged not to be produced and then not analyzed.

 

		Water + test item	Sludge + test item			Theoretical Amount
		Vessel-1	Vessel-2	Vessel-3	Vessel-4
BOD*	mg	0.7	-0.4	-0.4	0.4	40.0*
Residual amount and percentage residue of DOC**	mgC	12.1	12.1	12.1	11.9	11.5*
	%	105	106	105	104	-
Residual amount and percentage residue of test item (HPLC)***	mg	30.3	30.1	30.0	29.9	30.1
	%	101	100	100	99	-

*Calculated from the molecular formula of main component

**The value of control blank was subtracted from the values of the test solutions (sludge + test item).

*** These were calculated based on the total peak area on the chromatograms.

 

Percentage degradation after 28 days

 

Percentage biodegradations after 28 days were as follows:

 

		Sludge + test item
		Vessel-2	Vessel-3	Vessel-4	Average
Percentage biodegradation by BOD	%	-1	-1	1	0
Percentage biodegradation by DOC	%	0	0	2	1
Percentage biodegradation of test item (HPLC)	%	1	1	1	1

 

Discussion

The percentage biodegradation of each peak of the test item by HPLC was as follows.

 

Percentage biodegradation of each peak of test item

		Sludge + test item
		Vessel-2	Vessel-3	Vessel-4	Average
Peak 1 (Main product)	%	1	1	1	1
Peak 2 (by product)	%	-2	-3	-3	0 (-2)*

*The average percentage biodegradation was regarded as 0 % because the calculated average value shown in parentheses was negative.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The substance is not considered to be readily biodegradable.

Executive summary:

Summary:

Biodegradation study of T-43 by microorganisms

 

Conditions of cultivation

(1)Concentration of test item (100 mg/L)

(2)Concentration of activated sludge (30 mg/L, as the concentration of suspended solid)

(3)Volume of test solution (300 mL)

(4)Cultivation temperature 25 ±1 °C)

(5)Cultivation duration (under conditions of darkness, 28 days)

 

 

Measurement and analysis for calculation of percentage biodegradation

 

(1)Measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus.

(2)Determination of dissolved organic carbon (DOC) by a total organic carbon analysis (TOC)

(3) Determination of test item by high-performance liquid chromatography (HPLC)

 

Results

(1) Percentage biodegradation by BOD -1%, -1%, 1%; (average 0%)

(2) Percentage biodegradation by DOC 0%, 0%, 2%; (average 1%)

(3) Percentage biodegradation of test item (HPLC) 1%, 1%, 1%; (average 1%)

 

 

Conclusion:

The test item was not biodegraded by microorganisms under the test conditions of this study.