2,7-Naphthalenedisulfonic acid, 5-(acetylamino)-3-[2-[5-[[4-chloro-6-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-4-hydroxy-, sodium salt (1:4)

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 January 4, 2019 to March 14, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Details on inoculum:

The surface water was considered as source of inoculum. The surface water was sourced from Shameerpet Lake in Ranga Reddy District of Telangana State, India. The inoculum was aerated for 5 days followed by microbial colony forming unit count was evaluated after 5 days of aeration. The domestic waste was not considered as source of inoculum because it was contaminated by chlorinating agent.

Duration of test (contact time):

28 d

Initial conc.:

18 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

The experimental set up included, the Zero air cylinder which was connected to manifold from which the air devoid of oxides of carbon and nitrogen were transferred through silicon tubing at one atmospheric pressure. The air was passed to five, 5.0 litre narrow mouthed glass flasks containing mineral medium. The inlet air which comes from the manifold of Zero air cylinder was connected to the glass tube extended to the bottom of the 5.0 litre glass flasks, while the outlet at the top of 5.0 litre narrow mouth glass flask were used to remove excess air and carbon dioxide.
The inoculum, surface water 50.0mL taken in a measuring cylinder (50mL capacity) was added to the mineral medium. This was followed by rinsing of measuring cylinder with additional 50mL of distilled water and the same was transferred to respective flasks. The mineral medium along with inoculum was aerated to purge out carbon dioxide at the rate of 30 mL/min for a period of 5 days, without connecting the barium hydroxide traps.
After 24 hours of aeration with the inoculum, the test item and reference item were added in calculated amounts as mentioned below.
The experiment was conducted with the following number of flasks and labeled appropriately.
Flasks 1 & 2 - Contains both test item and inoculum in each flask (test suspension)
Flasks 3 & 4 - Contains only inoculum in each flask (inoculum blank)
Flask 5 - Contains both reference item and inoculum (reference suspension)

To flask 1 & 2 calculated volumes of around 18 mg equivalent carbon per litre (Dissolved organic carbon) of the test item was added. The test item was insoluble in aqueous medium, there forced continuous stirring was provided for homogenization of test item. To flasks 3 & 4 only inoculum was added. To the 5th flask calculated reference item, around 18 mg per litre with respect to (DOC) carbon content was added. All the flasks were covered with black color poly ethylene bag to avoid light transparency. To each flask, three 0.0125M Barium hydroxide (100mL) traps were connected in series. The outlet tubing was connected to the inlet of the first barium hydroxide trap extended to bottom of the trap, while the outlet from the first barium hydroxide trap was connected to the inlet of second trap using silicon tubing. The same was followed for third trap.
The test was initiated by bubbling CO2 free air through the suspensions at a rate of around 30 ml/min. The study was conducted for 28 days at a temperature 22±2℃. The CO2 was quantified on the 0 to 13, 17, 20, 24, 26 to 28 day. The closest barium hydroxide absorber to the test vessel was subjected to titration against 0.05M HCl using phenolphthalein indicator. The next absorber in series was connected immediate to test vessel while fresh 100mL barium hydroxide absorber was connected to last in the series. This pattern of replacement was followed during the course of study. The volumn of 0.05M Hydrochloric acid consumed for each trap was recorded.
On the end of 28th day analysis, 1.0mL of concentrated Hydrochloric acid was added to each test vessel and aerated them overnight to drive off the carbon dioxide present in the test suspensions.
On day 29the the last analysis of evolved carbon dioxide trapped in remaining barium hydroxide absorber and same was evaluated.
The test was found to be valid as the data generated for positive control yielded greater than 60% biodegradation within a period of 14 days window.

Reference substance:

other: Sodium Benzoate

Remarks:

C7H5O2.Na

Parameter:

% degradation (CO2 evolution)

Value:

< 60

Sampling time:

29 d

Details on results:

The microbial count evaluated for the surface water on the day of exposure to experiment bottles was found to be around 10^7 units of CFU.
The test item after exposure to aqua matrix with inoculum showed a negative effect on the growth of microorganisms. This was augmented by the low release of carbon dioxide from the test suspension. The short lag phase to achieve 10% degradation calculated in terms of evolved carbon dioxide was between 13th and 16th day. Moreover at the end of 28th day the test item bio-degraded to a level of 20.46% in terms of carbon dioxide evolved.
The details regarding test item added, theoretical carbon dioxide, and carbon dioxide produced and % biodegradation in test suspension was given in Table 1, 3 and 4.
The bio degradation was effective for reference suspension, as the pass level of 60% degradation was attained within the 14 day window period. This substantiates the validity of the study. The details on titer value and calculations were provided in Table 5-7. The inoculum blank titer value and carbon dioxide produced was presented in Table 2.

Results with reference substance:

At Day 14 during the study, the biodegradation was >60 %.

Table 1. Theoretical Carbon Dioxide - Test Suspension

Test item	Carbon	Theoretical CO2
Weight (mg)	Content (mg)	CO2(mg)	per gram test item
*134.52	45.00	164.99	1.227
** 134.63	45.03	165.13	1.227

*quantity added to flask-1, test item suspension

**quantity added to flask-2, test item suspension

Table 2. Carbon Dioxide Produced in Inoculum Blank

Inoculum Blank
Flask 3				Flask 4
Time (day)	HCl Normality	Titre value (mL)	CO2 produced (mg)	Time (day)	HCl Normality	Titre value (mL)	CO2 produced (mg)
0	0.05	50.00	0.00	0	0.05	50.00	0.00
1	0.05	49.10	0.99	1	0.05	49.60	0.44
2	0.05	48.30	1.87	2	0.05	48.70	1.43
3	0.05	48.00	2.20	3	0.05	48.40	1.76
4	0.05	48.40	1.76	4	0.05	48.30	1.87
5	0.05	48.80	1.32	5	0.05	48.60	1.54
6	0.05	48.60	1.54	6	0.05	48.50	1.65
7	0.05	48.70	1.43	7	0.05	48.50	1.65
8	0.05	48.90	1.21	8	0.05	48.60	1.54
9	0.05	48.50	1.65	9	0.05	48.50	1.65
10	0.05	48.40	1.76	10	0.05	48.30	1.87
11	0.05	48.90	1.21	11	0.05	48.20	1.98
12	0.05	48.10	2.09	12	0.05	48.40	1.76
13	0.05	48.20	1.98	13	0.05	48.30	1.87
17	0.05	48.60	1.54	17	0.05	48.70	1.43
20	0.05	47.90	2.31	20	0.05	47.50	2.75
24	0.05	47.50	2.75	24	0.05	48.10	2.09
26	0.05	48.60	1.54	26	0.05	48.30	1.87
27	0.05	49.10	0.99	27	0.05	49.00	1.10
28	0.05	49.30	0.77	28	0.05	49.20	0.88

Table 3. Carbon Dioxide Produced in Test Suspension

Inoculum Blank
Flask 1				Flask 2
Time (day)	HCl Normality	Titre value (mL)	CO2 produced (mg)	Time (day)	HCl Normality	Titre value (mL)	CO2 produced (mg)
0	0.05	50.00	0.00	0	0.05	50.00	0.00
1	0.05	48.90	1.21	1	0.05	48.70	1.43
2	0.05	48.70	1.43	2	0.05	48.60	1.54
3	0.05	48.80	1.32	3	0.05	48.70	1.43
4	0.05	48.50	1.65	4	0.05	48.90	1.21
5	0.05	48.60	1.54	5	0.05	48.80	1.32
6	0.05	48.50	1.65	6	0.05	48.70	1.43
7	0.05	47.00	3.30	7	0.05	47.20	3.08
8	0.05	47.20	3.08	8	0.05	47.10	3.19
9	0.05	47.10	3.19	9	0.05	47.30	2.97
10	0.05	46.50	3.85	10	0.05	46.40	3.96
11	0.05	46.20	4.18	11	0.05	46.30	4.07
12	0.05	45.80	4.62	12	0.05	45.70	4.73
13	0.05	45.60	4.84	13	0.05	45.50	4.95
17	0.05	44.20	6.38	17	0.05	44.10	6.49
20	0.05	43.10	7.59	20	0.05	43.20	7.48
24	0.05	44.30	6.27	24	0.05	44.20	6.38
26	0.05	47.10	3.19	26	0.05	47.30	2.97
27	0.05	47.30	2.97	27	0.05	47.60	2.64
28	0.05	47.40	2.86	28	0.05	47.10	3.19

 

Table 4. Percentage Bio Degradation

Time (Day)	CO2 produced (mg)					Cumulative CO2 Produced (mg), A-B			% Biodegradation
	Test item (A)		Blank (B)
	Flask 1	Flask 2	Flask 3	Flask 4	Mean	Flask 1		Flask 2	Flask 3	Flask 4	Mean
0	0.00	0.00	0.00	0.00	0.00	0.00		0.00	0	0	0
1	1.21	1.43	0.99	0.44	0.71	0.50		0.71	0.30	0.43	0.37
2	1.43	1.54	1.87	1.43	1.65	0.28		0.60	0.17	0.37	0.27
3	1.32	1.43	2.20	1.76	1.98	-0.38		0.05	-0.23	0.03	-0.10
4	1.65	1.21	1.76	1.87	1.82	-0.55		-0.55	-0.33	-0.33	-0.33
5	1.54	1.32	1.32	1.54	1.43	-0.44		-0.66	-0.27	-0.40	-0.33
6	1.65	1.43	1.54	1.65	1.60	-0.39		-0.83	-0.23	-0.50	-0.37
7	3.30	3.08	1.43	1.65	1.54	1.38		0.71	0.83	0.43	0.63
8	3.08	3.19	1.21	1.54	1.38	3.08		2.53	1.87	1.53	1.70
9	3.19	2.97	1.65	1.65	1.65	4.62		3.85	2.80	2.33	2.57
10	3.85	3.96	1.76	1.87	1.82	6.65		5.99	4.03	3.63	3.83
11	4.18	4.07	1.21	1.98	1.60	9.24		8.47	5.60	5.13	5.36
12	4.62	4.73	2.09	1.76	1.93	11.94		11.28	7.23	6.38	7.03
13	4.84	4.95	1.98	1.87	1.93	14.85		14.30	9.00	8.66	8.83
17	6.38	6.49	1.54	1.43	1.49	19.75		19.31	11.97	11.69	11.83
20	7.59	7.48	2.31	2.75	2.53	24.81		24.26	15.03	14.69	14.86
24	6.27	6.38	2.75	2.09	2.42	28.66		28.22	17.37	17.09	17.23
26	3.19	2.97	1.54	1.87	1.71	30.14		29.48	18.27	17.85	18.06
27	2.97	2.64	0.99	1.10	1.05	32.07		31.08	19.43	18.82	19.13
28	2.86	3.19	0.77	0.88	0.83	34.10		33.44	20.67	20.25	20.46

Table 5. Theoretical Carbon Dioxide - Reference Suspension

Ref item	Carbon	Theoretical CO2
Weight (mg)	Content (mg)	CO2(mg)	per gram Ref item
*77.13	45.00	165.00	2.139

*Quantity of reference item added to test flask 5

Table 6. Carbon Dioxide Produced in Reference Suspension

Flask 5
Time (day)	HCl Normality	Titre value (ml)	CO2 Produced (mg)
0	0.05	50.00	0.00
1	0.05	49.20	0.88
2	0.05	45.90	4.51
3	0.05	44.80	5.72
4	0.05	43.60	7.04
5	0.05	42.70	8.03
6	0.05	39.30	11.77
7	0.05	38.80	12.32
8	0.05	36.40	14.96
9	0.05	35.50	15.95
10	0.05	32.10	19.69
11	0.05	33.70	17.93
12	0.05	35.90	15.51
13	0.05	39.40	11.66
17	0.05	40.30	10.67
20	0.05	40.40	10.56
24	0.05	42.90	7.81
26	0.05	44.70	5.83
27	0.05	46.00	4.40
28	0.05	46.20	4.18

Table 7. Percentage Bio Degradation in reference Suspension

Time (Day)	CO2 produced (mg)					Cumulative CO2 Produced (mg), A-B			% Biodegradation
	Reference item (A)		Blank (B)
	Flask 5		Flask 3	Flask 4	Mean	Flask 5			Flask 5
0	0.00		0.00	0.00	0.00	0.00			0.00
1	0.88		0.99	0.44	0.71	0.16			0.10
2	4.51		1.87	1.43	1.65	3.03			1.83
3	5.72		2.20	1.76	1.98	6.77			4.10
4	7.04		1.76	1.87	1.82	11.99			7.27
5	8.03		1.32	1.54	1.43	18.59			11.27
6	11.77		1.54	1.65	1.60	28.77			17.43
7	12.32		1.43	1.65	1.54	39.55			23.97
8	14.96		1.21	1.54	1.38	53.13			32.20
9	15.95		1.65	1.65	1.65	67.43			40.87
10	19.69		1.76	1.87	1.82	85.31			51.70
11	17.93		1.21	1.98	1.60	101.64			61.60
12	15.51		2.09	1.76	1.93	115.23			69.83
13	11.66		1.98	1.87	1.93	124.96			75.73
17	10.67		1.54	1.43	1.49	134.15			81.30
20	10.56		2.31	2.75	2.53	142.18			86.17
24	7.81		2.75	2.09	2.42	147.57			89.43
26	5.83		1.54	1.87	1.71	151.69			91.93
27	4.40		0.99	1.10	1.05	155.05			93.97
28	4.18		0.77	0.88	0.83	158.40			96.00

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The solid test item CR SR94 does not biodegrade under stringent laboratory conditions. However, this does not mean that test item does not degrade rather research with different matrix with different conditions requires much attention.

Executive summary:

The study was conducted to evaluate the ready biodegradability of CR SR94 under stringent laboratory conditions. The temperature during the study period was maintained at 22±2℃ for 28 days.

 

The microbial interaction with the test item in test suspension was almost identical with respect to blank control (mineral medium and inoculum). The microbial count evaluated for the surface water on the day of exposure to experiment bottles was found to be 10^7 units of CFU.

 

The validity of the study was supported by the reference suspension behavior in which the biodegradation was achieved greater than 60% within 14 days window period. The test item was found to be non-biodegradable. The reason is, during the whole study duration only 20.46% was found to be undergone degradable. Hence, the study was concluded that the test item does not readily biodegradable.

 

However, this does not mean that the test item will not undergo degradation. The result of 20.46% degradation evince that it may degrade slowly and much work on favorable conditions has to be done for its degradation.

Description of key information

Not readily biodegradable (OECD TG 301B).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed