N-tert-butylbenzothiazole-2-sulphenamide

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:

August - November 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):

(1) Place: The sludge was collected from the following 10 locations throughout Japan:
　Fushikogawa Treatment Plant (Sapporo City, Hokkaido)
　Nakahama Treatment Plant (Osaka City, Osaka Prefecture)
　Kitakami River (Ishinomaki City, Miyagi Prefecture)
　Yoshino River (Tokushima City, Tokushima Prefecture)
　Hiroshima Bay (Hiroshima City, Hiroshima Prefecture)
Fukashiba Treatment Plant (Kashima-gun, Ibaraki Pref.)
Ochiai Treatment Plant (Shinjuku-ku, Tokyo)
Shinano River (Nishikanbara-gun, Niigata Prefecture)
Lake Biwa (Otsu City, Shiga Prefecture)
Lake Biwa (Otsu city, Shiga pref.) Dokai bay (Kitakyushu city, Fukuoka pref.)

(2) Time: June 1995

(3) Collection method:
- City sewage returned: sludge from sewage treatment plant
　 - Rivers, lakes and oceans: Topsoil at the edge of wave action in contact with surface water and the atmosphere

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

other: analysis of the degradation/hydrolysis products

Parameter followed for biodegradation estimation:

test mat. analysis

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Synthetic sewage: Glucose, peptone, and potassium dihydrogen
phosphate were dissolved in purified water to 5 (w/v) % each and pH was adjusted to 7.0±1.0
with sodium hydroxide
- Test temperature: 25±2℃
- pH: The pH was adjusted to 7.0±1.0 and exposed to air in a culture tank.
- pH adjusted: yes
- Suspended solids concentration: 5400 mg/kg
- Aeration: Outdoor air was passed through a pre-filter and used for aeration.

PREPARATION OF ACTIVATED SLUDGE AND CULTIVATION
The sludge filtrate (500 mL) was collected from 10 different locations throughout Japan in June 1995. The filtrate was made up to 10 liters by mixing 500 mL of each filtrate with 5 liters of the old activated sludge filtrate that had been used for the test. The pH was adjusted to 7.0±1.0 and exposed to air in a culture tank.

After stopping the aeration in the culture tank for about 30 minutes, about 1/3 of the total volume of supernatant liquid was removed. An equal amount of purified water was added to the supernatant and the tank was aerated again.
Synthetic sewage was added to make the concentration of the supernatant exchange liquid 0.1%. This operation was repeated once a day, and the sludge was incubated and made into activated sludge. The incubation temperature was set at 25±2℃.

During incubation, the appearance of the supernatant liquid and the condition of activated sludge formation were observed, and the settling property, pH, temperature, and dissolved oxygen concentration of the activated sludge were measured and recorded. The biota of the activated sludge was observed using an optical microscope as appropriate, and no abnormality was confirmed before the test.

TEST SYSTEM
- Culturing apparatus: Closed system oxygen consumption measurement device (Okura Electric
Co.)
- Number of culture flasks/concentration: Six test containers (300 mL culture bottle, activated
sludge concentration 30 mg/L)
- Stirring method: rotating stirring by magnetic stirrer
- Preparation of basic culture medium: The following test solutions a)-d) were prepared. The test
solutions b), c), and d) were inoculated with activated sludge to achieve a suspended solid
concentration of 30 mg/L by diluting with purified water and adjusting the pH to 7.0.
a) (water + test substance) system (1 vessel): 300 mL of purified water was added to the test
container, and the concentration of the test substance was set to 100 mg/L (accurately weighed
using an electronic analytical balance).
b) (sludge + test substance) system (3 test vessels): The test solution containing 100 mg/L test
substance (accurately weighed using an electronic analytical balance) and activated sludge
was placed in a test vessel (300 mL total volume)
c) (sludge + aniline) (1 vessel): The test solution containing 100 mg/L aniline (29.5 μL (30mg,
density= 1.022 g/cm3) of aniline are added by a micro syringe) and activated sludge was placed
in a test vessel (300 mL total volume).
d) The sludge blank system (1 vessel) contained activated sludge without test substance in a
test vessel (300 mL total volume)

- Measuring equipment: Data processing equipment manufactured by Asahi Keiki Kogyo
- Details of trap for CO2 and volatile organics if used: Carbon dioxide absorber Soda lime, sea 1 (For carbon dioxide absorption, manufactured by Wako Pure Chemical Industries)

SAMPLING
(1) Measurement of biochemical oxygen demand (BOD) using a closed system oxygen consumption analyzer (sampling after 7, 14, 21 and 28 days)
(2) Analysis of the test substance and hydroysis products (MBT, MBTS and tert-butyl amine) by high-performance liquid chromatography (HPLC) after 28 days

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1 replicate, test solution d)

STABILITY OF THE TEST SUBSTANCE
Stability check: The infrared absorption spectrum of the test substance was measured before and after the incubation of the test solution. The results showed that the test substance was stable under the storage conditions (refrigerated storage). As a result, it was confirmed that the test substance was stable under the storage conditions.

IDENTITY OF THE TEST SUBSTANCE
The structure of the test substance was confirmed by infrared (IR) spectroscopy, mass spectrometry (MS) and nuclear magnetic resonance (NMR).

Reference substance:

aniline

Key result

Parameter:

other: Degree of decomposition by HPLC

Remarks:

Degradability (%) = ((Sw- Ss)/ Sw) x 100 // Ss : Residual amount of test substance in the system (sludge + test substance) (measured value) (mg) // Sw : Residual amount of test substance in (water + test substance) system 　　　

Value:

87

Sampling time:

28 d

Key result

Parameter:

other: Degree of degradation by BOD　　　　　　

Remarks:

Degradability (%)= ((BOD - B)/ TOD) x 100 BOD: Biochemical oxygen demand of the system (sludge + test substance) (mg) B: Biochemical oxygen demand of sludge blank system (mg) TOD: Theoretical oxygen demand (calculated value assumuning 100% purity) (mg)

Value:

0

Sampling time:

28 d

Key result

Parameter:

BOD5

Remarks on result:

other: BOD

Results with reference substance:

The degradation of aniline (test concentration 100 mg/L) after 7 and 14 days was 63% and 74%, respectively, as determined from the BOD, confirming that the test conditions of this study were effective.

The degradation degree after 28 days was as follows, indicating that TBBS underwent abiotic degradation (hydrolysis), whereas no biodegradation was observed: 

	Degradation degree [%]
	vessel 2 (cond. b))	vessel 3 (cond. b))	vessel 4 (cond. b))	mean [%]
BOD result	0	0	0	0
HPLC result	80	81	100	87

The analysis result after 28 days was as follows:

The theoretical value refers to the amount of the substance present in the extract according to the corresponding extraction procedure. 

(a) Water + test substance; (b) (Sludge + test substance) system

		(a)	(b)			theoretical value
		vessel 1	vessel 2	vessel 3	vessel 4
BOD*	mg	0.0	0.0	0.0	0.0	74.4
test substance (HPLC)	mg	0.0	5.9	5.6	0.0	30
	%**	0	20	19	0	-
MBT (HPLC)	mg	7.9	0.5	0.3	0.2	21
	%**	38	2	2	1	-
MTBS (HPLC)	mg	2.9	14.1	12.0	13.6	20.9
	%**	14	67	57	65	-
tert-Butylamine (HPLC)	mg	8.9	7.7	7.7	9.5	9.2
	%**	97	84	83	103	-

*The (sludge + test substance) system was displayed by subtracting the value of the sludge blank system.
**The residual rate (%) and the generation rate (%) were calculated based on the following formulas, and the digits after the decimal point were rounded and displayed as integers.
Residual rate (%) ＝ (Residual amount (mg)/ Theoretical amount (mg)) x 100　

Summary of the BOD results 　

Vessel no.	sample description	BOD [mg]
		7th day	14th day	21st day	28th day
1	Water + test substance	0.0	0.0	0.0	0.0
2	Sludge + test substance	0.8	2.8	3.0	3.9
3	Sludge + test substance	0.9	2.4	2.4	3.3
4	Sludge + test substance	0.7	2.2	2.2	2.3
5	Control blank (B)	0.2	3.0	3.2	4.1
6	Sludge + Aniline	57.5	69.8	70.2	71.6

　　　

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The study was conducted in accordance with OECD guideline 301 C. The biodegradation of N-tert-butylbenzothiazole-2-sulphenamide was determined at 0 % degradation after 4 weeks. N-tert-butylbenzothiazole-2-sulphenamide hydrolyzed in water to form 2-mercaptobenzothiazole, 2,2'-dithiobis(benzothiazole), tert-butylamine, 2-sulfo(sulfino)benzothiazole and benzothiazole.

Executive summary:

The biodegradation of N-tert-butylbenzothiazole-2-sulphenamide was determined according at the OECD guideline method 301 C and showed a degradation of 0 % after 4 weeks.

N-tert-butylbenzothiazole-2-sulphenamide hydrolyzed in water to form 2-mercaptobenzothiazole, 2,2'-dithiobis(benzothiazole), tert-butylamine, 2-sulfo(sulfino)benzothiazole and benzothiazole.

Description of key information

Under the test conditions 0 % degradation of TBBS was observed within 28 days; and hence the substance has to be classified as not readily biodegradable. The hydrolysis products of TBBS, such as MBT and tert-butylamine, are also reported as not biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed