Tetrabutylammonium bromide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Experimental result using OECD guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Remarks:

The actived sludge was collected from STP and which was maintained at test conditions for the period ranging from 5 -7 days

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP Bhandewadi, Nagpur, India, there is not history of contamination of test item, sampling depth 1-2 feet
- Laboratory culture and - Method of cultivation: : Preparation of inoculum for exposure and pre-treatment: Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º C test temperature
- Concentration of sludge: Aliquot of the final sludge suspension weighed, dried and the ratio of wet sludge to its dry weight determined and concentration of sludge was 0.4 g dry material per litre final test medium.
- Water filtered: yes
-Biomass concentration: A concentration of 32 mL/400 mL of activated sludge solution was added to every flask. This concentration gave suspended solid of 1.0 g/L.
- Method of cultivation: Aeration of incoming domestic sludge with micro-organisms by means of diffuser
- Preparation of inoculum for exposure and pre-treatment: Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º C test temperature.

Duration of test (contact time):

42 d

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

BOD

Details on study design:

Stock Solution
Test Chemical: 0.2 g of the test chemical was dissolved in 200 mL mineral media. And the mixture was stirred for 24 h. The actual value obtained after analytical determination was 1057.22 mg/L used in the test.
Procedure Control (Sodium Benzoate): 0.1 g of the procedure control was dissolved in 100 mL water. And the mixture was stirred for 24 h. The actual value obtained after analytical determination was 858.57 mg/L used in the test.
N-ATU (N-Allylthio urea): 0.1 g of the procedure control was dissolved in 100 mL water. And the mixture was stirred for 24 h. The actual value obtained after analytical determination was 1087.63 mg/L used in the test.

TEST CONDITIONS
- Composition of medium: OECD medium is used in the study details are given in any other infromation of material and methods
- Additional substrate: Not applicable
- Solubilising agent (type and concentration if used): Not applicable
- Test temperature: 20°C
- pH:
0 day blank control : 7.4 and 7.0 on day 42
- pH adjusted: no
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: BOD bottles
- Number of culture flasks/concentration: 2 culture flasks per concentrations
- Measuring equipment: DO meter is used to measure the dissolved oxygen.
- Test performed in open system: BOD bottles were closed with LID


SAMPLING
- Sampling frequency: 0 day, 7 day, 14 day, 21 day, 28 day, 35 day, and 42 day
- Sample storage before analysis: Bottles were immediately analysed for BOD


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes included in the test, it contains only test inoculum
- Toxicity control: yes included in the test, reference compound and inoculum

STATISTICAL METHODS:

Reference substance:

benzoic acid, sodium salt

Test performance:

Test was conducted for the period 42 days to understand the biodegradibilty nature of test chemical, based on the oxygen consumption. which was measured at regular intervals of 7 days.

Parameter:

% degradation (O2 consumption)

Value:

42.7

Sampling time:

42 d

Remarks on result:

other: degradation after 28 days was 36.51

Details on results:

The oxygen consumed by the test systems was corrected for oxygen consumption occurring in the blank test systems. The BOD Values (mgO2/mg) and percent biodegradation results for each test system are reported in tabes in additional information. The BOD42 value of test chemical was observed to be 1.03 mgO2/mg. ThOD was determined by calculation as 2.41 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test item and was determined to be 42.7 % at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 89.82 %. Degradation of Sodium Benzoate exceeds 64.67 % on 7 days & 88.62 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The toxicity control was > 25% after 14 days of exposure.

Results with reference substance:

The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 89.82%. Degradation of Sodium Benzoate exceeds 64.67 % on 7 days & 88.62 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid.

 Table 1

D.O Values(mg/L)

No. of Days	Inoculum Blank (Control)	Test Suspension	Procedure Control (Reference Item)	Toxicity Control
0	8.2	8.0	8.2	8.2
7	8.0	6.5	3.7	6.3
14	7.8	5.5	1.9	5.2
21	7.6	4.2	1.0	3.9
28	7.2	3.5	0.8	3.4
35	6.8	2.6	0.6	1.9
42	6.5	2.2	0.5	1.3

 

TABLE 2

BOD values (mgO2/mg)

No. of Days	Test Suspension	Procedure Control (Reference Item)	Toxicity control
0	0	0	0
7	0.33	1.08	0.43
14	0.53	1.48	0.65
21	0.80	1.65	0.93
28	0.88	1.60	0.95
35	1.0	1.55	1.23
42	1.03	1.50	1.30

 

TABLE 3

PERCENT BIODEGRDATION RESULTS

No. of Days	Test Suspension (%)	Procedure Control (Reference Item) (%)	Toxicity control (%)
0	0%	0%	0%
7	13.69	64.67	17.84
14	21.99	88.62	26.97
21	33.19	98.80	38.58
28	36.51	95.81	39.41
35	41.49	92.81	51.03
42	42.73	89.82	53.94

 

Table 4

BOD42, THOD AND % BIODEGRADATION VALUES

Method details	BOD42 (mgO2/mg)	ThOD (mgO2/mg)	% Biodegradation
Test Item	1.03	2.41	42.73
Reference Item	1.50	1.67	89.82
Toxicity control	1.30	2.41	53.94

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The test chemical undergoes 42.73 % biodegradation after 42 days in the test condition. Thus, the test chemical was considered to be inherently biodegradable.

Executive summary:

42-days Closed Bottle test following the OECD guideline 301 D was performed to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C. The test system included control, test item and reference item and toxicity control. the test was conducted using activated sludge, . The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. This was pre-conditioned at the test temperature. 1 mL supernatant was diluted to 100 mL with mineral medium and from this solution 0.125 mL was added to 125 mL test bottles. This gave the bacterial count as 10e4 to 10e6 CFU/L. .The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The oxygen consumed by the test systems was corrected for oxygen consumption occurring in the blank test systems. The BOD Values (mgO2/mg) and percent biodegradation results for each test system are reported in tabes in additional information. The BOD42 value of test chemical was observed to be 1.03 mgO2/mg. ThOD was determined by calculation as 2.41 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test item and was determined to be  42.7 % at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 89.82 %. Degradation of Sodium Benzoate exceeds 64.67 % on 7 days & 88.62 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The toxicity control was > 25% after 14 days of exposure. Based on the results, the test chemical, under the test conditions, was considered to be inherently biodegradable in nature.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP Bhandewadi, Nagpur, India, there is not history of contamination of test item, sampling depth 1-2 feet
- Laboratory culture and - Method of cultivation: : Preparation of inoculum for exposure and pre-treatment: Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º C test temperature
- Activated sludge details: Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport.
- Water filtered: yes
-Biomass concentration: Aliquot of the final sludge suspension weighed, dried and the ratio of wet sludge to its dry weight determined and concentration of sludge was 0.96 g dry material per litre final test medium.
- Method of cultivation: Aeration of incoming domestic sludge with micro-organisms by means of diffuser
- Preparation of inoculum for exposure and pre-treatment: Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º C test temperature.

Duration of test (contact time):

28 d

Initial conc.:

250 mg/L

Based on:

test mat.

Initial conc.:

659 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

STODK SOLUTION:
Test Chemical:1 g of the test item was dissolved in 1000 mL mineral media as test item is soluble in water no solubilizing agent was used during stock preparation. from which 500 ml was further dissolved in 2000 ml of medium which contain 659 mg/L of COD
Procedure Control (Aniline): 1 g of the procedure control was dissolved in 1000 mL mineral media. The final test vessel has 661 mg/L of COD

TEST CONDITIONS
- Composition of medium: OECD medium is used in the study details are given in any other infromation of material and methods
- Additional substrate: Not applicable
- Solubilising agent (type and concentration if used): Not applicable
- Test temperature: 20 °C
- pH adjusted: no
- Suspended solids concentration: 0.96 g dry material per litre final test medium.
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Glass vessels
- Number of culture flasks/concentration: 2 vessels/ concentrations
- Method used to create aerobic conditions: continues stirring
- Measuring equipment: Hanna COD vials (Medium Range Reagents) in which all test samples were added and later refluxed in HI 839800 COD reactor for 2h at 150 ± 10°C. After reflux all the test samples were estimated on HI 83099 COD and multiparameter bench photometer for COD parameter.


SAMPLING
- Sampling frequency: Sampling of all test vessels from each test group concentration were collected for analysis at zero-time (immediately after set-up), 3h, 7th, 14th, 21st, 27th and 28th day during the incubation period.
- Sampling method: Samples were removed at regular intervals by pipetting 30 mL of sample, measured COD for all the test vessels.
- Sample storage before analysis: samples were analysed immediately after sampling


CONTROL AND BLANK SYSTEM
- Inoculum blank: Containing inoculum and mineral media;
- Test Suspension: Containing test item, inoculum and mineral media;
- Procedure Control: Containing procedure control (aniline), inoculum and mineral media.
- Toxicity Control: Containing test item, procedure control (aniline), inoculum and mineral media.


STATISTICAL METHODS:

Reference substance:

aniline

Test performance:

A mixture containing the test substance, mineral nutrients and a relatively large amount of activated sludge in aqueous medium is agitated and aerated at 20± 2° C in the dark or in diffuse light for up to 28 days. Blank controls, containing activated sludge and mineral nutrients but no test substance, are run in parallel. The biodegradation process is monitored by determination of COD in filtered samples taken at daily or other time intervals. The ratio of eliminated COD, corrected for the blank, after each time interval, to the initial COD value is expressed as the percentage biodegradation at the sampling time. The percentage biodegradation is plotted against time to give the biodegradation curve.

Parameter:

% degradation (DOC removal)

Value:

80.74

Sampling time:

28 d

Remarks on result:

other: Based on the COD value

Details on results:

The percent degradation of test chemical was found to be 80.74% based on the COD.

Results with reference substance:

The reference compound degraded upto 90.01 % after 28 days of exposure.

COD VALUES (mg/L)

No. of Days	Blank Control			Test item			Procedure Control			Toxicity Control
	R1	R2	Mean	R1	R2	Mean	R1	R2	Mean	R1	R2	Mean
0 h (before Inoculum added)	52	59	56	678	640	659	664	658	661	972	981	977
0 h (after 3 h)	57	53	55	629	665	647	648	670	659	959	978	969
7	40	49	45	470	512	491	195	225	210	475	525	500
14	30	50	40	147	244	197	117	161	139	232	268	250
21	40	30	35	149	190	170	111	77	94	162	198	180
27	35	22	29	168	122	145	40	46	43	88	92	90
28	21	31	26	133	146	140	44	31	38	61	78	70

 

TABLE 3

% BIODEGRADATION (Mean)

No. of Days	Test Item (%)	Procedure Control (%)	Toxicity Control (%)
0	-	-	-
7	24.66	72.68	50.22
14	73.65	83.61	76.81
21	77.20	90.23	84.14
27	80.41	97.68	93.33
28	80.74	98.01	95.19

 

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The percent degradation of test chemical was found to be 80.74% based on the COD, After exposure period of 28 days. thus it can be classified as inherently biodegrabale in water.

Executive summary:

The test item Tetrabutylammonium bromide was investigated for its inherent biodegradability in a Zahn-Wellens/EMPA Test ( OECD 302B). The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38ºC) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. Preparation of inoculum for exposure and pre-treatment: Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º Ctest temperature. Concentration of Sludge: Aliquot of the final sludge suspension weighed, dried and the ratio of wet sludge to its dry weight determined and concentration of sludge was 0.96 g dry material per litre final test medium. The biodegradation was determined by following the COD (Chemical Oxygen Demand) of the test item in the incubation vessels during exposure. Aniline was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. 5L glass vessels (beakers) were used in the study. The test system includes an inoculum blank control group, a procedure control group, and a test item group a toxicitycontrol group, each maintained in replicates (total 8 vessels). All the test vessels were kept on magnetic stirrer and aerated by means of air blower for 28^thdays and later all test vessels were incubated at BOD incubator at a constant temperature of 20±2°C. Sampling of all test vessels from each test group concentration were collected for analysis at zero-time (immediately after set-up), 3h, 7^th, 14^th, 21^st, 27^thand 28^thday during the incubation period. Samples were removed at regular intervals by pipetting 30 mL of sample, measured COD for all the test vessels. Hanna COD vials (Medium Range Reagents) in which all test samples were added and later refluxed in HI 839800 COD reactor for 2h at 150±10°C. After reflux all the test samples were estimated on HI 83099 COD and multiparameter bench photometer for COD parameter. The procedure control Aniline was sufficiently degraded to 90.23 % after 13 days, and to 98.01 % after 28 days of incubation, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the procedure control aniline, 76.81% biodegradation was noted within 14 days and 95.19% biodegradation was determined after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the activated sludge microorganisms.  Under the test conditions the mean percentage biodegradation of Tetrabutylammonium bromide reached 80.74% after 28 days of incubation based on COD elimination. As the final biodegradation is above 70% in this test and all validity criteria were met, Tetrabutylammonium bromide can be considered to be potentially inherently biodegradable.

Description of key information

Biodegradation in water:

42-days Closed Bottle test following the OECD guideline 301 D was performed to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C. The test system included control, test item and reference item and toxicity control. the test was conducted using activated sludge, . The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. This was pre-conditioned at the test temperature. 1 mL supernatant was diluted to 100 mL with mineral medium and from this solution 0.125 mL was added to 125 mL test bottles. This gave the bacterial count as 10e4 to 10e6 CFU/L. .The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The oxygen consumed by the test systems was corrected for oxygen consumption occurring in the blank test systems. The BOD Values (mgO2/mg) and percent biodegradation results for each test system are reported in tabes in additional information. The BOD42 value of test chemical was observed to be 1.03 mgO2/mg. ThOD was determined by calculation as 2.41 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test item and was determined to be  42.7 % at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 89.82 %. Degradation of Sodium Benzoate exceeds 64.67 % on 7 days & 88.62 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The toxicity control was > 25% after 14 days of exposure. Based on the results, the test chemical, under the test conditions, was considered to be inherently biodegradable in nature.

Inherently biodegradable in water:

The test item Tetrabutylammonium bromide was investigated for its inherent biodegradability in a Zahn-Wellens/EMPA Test ( OECD 302B). The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days.The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38ºC) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. Preparation of inoculum for exposure and pre-treatment: Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º Ctest temperature. Concentration of Sludge: Aliquot of the final sludge suspension weighed, dried and the ratio of wet sludge to its dry weight determined and concentration of sludge was 0.96 g dry material per litre final test medium.The biodegradation was determined by following the COD (Chemical Oxygen Demand) of the test item in the incubation vessels during exposure. Aniline was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.5L glass vessels (beakers) were used in the study. The test system includes an inoculum blank control group, a procedure control group, and a test item groupatoxicitycontrol group, each maintained in replicates (total 8 vessels).All the test vessels were kept on magnetic stirrer and aerated by means of air blower for 28^thdays and later alltest vessels wereincubatedatBOD incubatorat aconstant temperatureof20±2°C.Sampling of all test vessels from each test group concentration were collected for analysis at zero-time (immediately after set-up), 3h, 7^th, 14^th, 21^st, 27^thand 28^thday during the incubation period.Samples were removed at regular intervals by pipetting 30 mL of sample, measured COD for all the test vessels.Hanna COD vials (Medium Range Reagents) in which all test samples were added and later refluxed in HI 839800 COD reactor for 2h at 150±10°C.After reflux all the test samples were estimated on HI 83099 COD and multiparameter bench photometer for COD parameter.The procedure control Aniline was sufficiently degraded to 90.23 % after 13 days, and to 98.01 % after 28 days of incubation, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the procedure control aniline, 76.81% biodegradation was noted within 14 days and 95.19% biodegradation was determined after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the activated sludge microorganisms.  Under the test conditions the mean percentage biodegradation of Tetrabutylammonium bromide reached 80.74% after 28 days of incubation based on COD elimination. As the final biodegradation is above 70% in this test and all validity criteria were met, Tetrabutylammonium bromide can be considered to be potentially inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater

Additional information

Experimental studies have been reviewed biodegradation in water endpoint and their results are summarized below.

42-days Closed Bottle test following the OECD guideline 301 D was performed to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C. The test system included control, test item and reference item and toxicity control. the test was conducted using activated sludge, . The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. This was pre-conditioned at the test temperature. 1 mL supernatant was diluted to 100 mL with mineral medium and from this solution 0.125 mL was added to 125 mL test bottles. This gave the bacterial count as 10e4 to 10e6 CFU/L. .The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The oxygen consumed by the test systems was corrected for oxygen consumption occurring in the blank test systems. The BOD Values (mgO2/mg) and percent biodegradation results for each test system are reported in tabes in additional information. The BOD42 value of test chemical was observed to be 1.03 mgO2/mg. ThOD was determined by calculation as 2.41 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test item and was determined to be  42.7 % at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 89.82 %. Degradation of Sodium Benzoate exceeds 64.67 % on 7 days & 88.62 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The toxicity control was > 25% after 14 days of exposure. Based on the results, the test chemical, under the test conditions, was considered to be inherently biodegradable in nature.

Next study was reviewed from authoritative database (J check, 2018) in this Biodegradation experiment was conducted for 28 days for evaluating the percentage biodegradability of test chemical in this experiment activated sludge was used as a test inoculums. The initial test substance conc. used in the study was 7.2 mg/l. The percentage degradation of test substance was determined to be 0% by O2 consumption, DOC removal and HPLC parameter in 28 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

Inherently biodegradable in water:

The test item Tetrabutylammonium bromide was investigated for its inherent biodegradability in a Zahn-Wellens/EMPA Test ( OECD 302B). The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days.The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38ºC) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. Preparation of inoculum for exposure and pre-treatment: Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º Ctest temperature. Concentration of Sludge: Aliquot of the final sludge suspension weighed, dried and the ratio of wet sludge to its dry weight determined and concentration of sludge was 0.96 g dry material per litre final test medium.The biodegradation was determined by following the COD (Chemical Oxygen Demand) of the test item in the incubation vessels during exposure. Aniline was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.5L glass vessels (beakers) were used in the study. The test system includes an inoculum blank control group, a procedure control group, and a test item groupatoxicitycontrol group, each maintained in replicates (total 8 vessels).All the test vessels were kept on magnetic stirrer and aerated by means of air blower for 28^thdays and later alltest vessels wereincubatedatBOD incubatorat aconstant temperatureof20±2°C.Sampling of all test vessels from each test group concentration were collected for analysis at zero-time (immediately after set-up), 3h, 7^th, 14^th, 21^st, 27^thand 28^thday during the incubation period.Samples were removed at regular intervals by pipetting 30 mL of sample, measured COD for all the test vessels.Hanna COD vials (Medium Range Reagents) in which all test samples were added and later refluxed in HI 839800 COD reactor for 2h at 150±10°C.After reflux all the test samples were estimated on HI 83099 COD and multiparameter bench photometer for COD parameter.The procedure control Aniline was sufficiently degraded to 90.23 % after 13 days, and to 98.01 % after 28 days of incubation, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the procedure control aniline, 76.81% biodegradation was noted within 14 days and 95.19% biodegradation was determined after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the activated sludge microorganisms.  Under the test conditions the mean percentage biodegradation of Tetrabutylammonium bromide reached 80.74% after 28 days of incubation based on COD elimination. As the final biodegradation is above 70% in this test and all validity criteria were met, Tetrabutylammonium bromide can be considered to be potentially inherently biodegradable.

By considering the result of first study which was performed according to OECD guideline and of Klimisch rating 1 the test chemical is considered to be ultimate inherently biodegradable in nature.