2,5-di-tert-butylhydroquinone

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13th January 2019 - 3rd February

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes

Specific details on test material used for the study:

Test Item Name
(As per TIDS) : YAPOX2245
Chemical Name (IUPAC) : 2, 5-Di-tertiary butyl hydroquinone
CAS Number : 88-58-4
Physical Appearance : White crystalline powder
Batch No. : 20015011219
Purity : 99.37%
Date of Manufacture : June 2019
Best Before Use : May 2021
Storage Condition : Ambient (21°C to 29°C)
Batch Produced by
(Name and Address) : Yasho Industries Limited
Plot No. 2514/2515, Phase IV, G.I.D.C.,
Vapi-396195, Gujarat, India.
Test Item Code by Test Facility : D819-002

Analytical monitoring:

no

Details on sampling:

- Concentrations: 3 concentrations ranging from 1000 to 10 mg/L nominal concentrations.

Vehicle:

yes

Remarks:

deionised water

Details on test solutions:

The test was performed using three concentrations of the test item, i.e., 10 mg/L, 100 mg/L and 1000 mg/L with three blank controls and three abiotic controls with the highest concentrations of the test item at test start and test end, respectively. Since the test item was considered to be insoluble in water, the test item was weighed directly into the test vessel to achieve the appropriate concentration.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The sludge was collected and coarse particles were removed by settling for a short period, for about 15 minutes, and decanting the upper layer of finer solids. Alternatively, the sludge was homogenized in a blender for about 15 seconds. The suspended solids concentrations of the activated sludge were prepared for about 2 g/L to 4 g/L in a bulk quantity. The sludge was collected, the coarse particles were removed by settling for a short period for about 15 minutes, and decanting the upper layer of finer solids. The sludge was washed off and centrifuged for a period of 10 minutes at 10,000 m/s2 to produce a clear supernatant and pellet of sewage solids. The supernatant liquid was discarded and the sludge was re-suspended in deionised water with shaking and the wash-water was removed by re-centrifuging and discarding again. The washing and centrifuging process was repeated twice. The dry mass of a known volume of the stock suspended solids concentration was determined and the sludge was diluted further in deionised water. Prepared sludge solid concentration was 3.2 g/L. The activated sludge was continuously aerated at the test temperature (20 ± 2°C) until usage.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 ± 2°C.

Nominal and measured concentrations:

Nominal concentrations used were of 10 mg/l, 100 mg/l and 1000 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL glass beakers
- Aeration: aerated upto the exposure period of 3 hours with the compressed air at a flow rate of approximately 0.6 L per minute at test temperature.
- No. of vessels per concentration (replicates): 3 replicates at 1000mg/L; 1 replicate at 10 and 100 mg/L
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3
- No. of vessels per abiotic control (replicates): 3

Activated sludge was used as the microbial inoculum. Activated sludge was collected from the exit of the aeration tank of a well-operated wastewater treatment plant of Siddaganaga Institute of Technology, Tumkur-572103 receiving predominantly domestic sewage.
The sludge was collected and coarse particles were removed by settling for a short period, for about 15 minutes, and decanting the upper layer of finer solids. Alternatively, the sludge was homogenized in a blender for about 15 seconds.
The suspended solids concentrations of the activated sludge were prepared for about 2 g/L to 4 g/L in a bulk quantity. The sludge was collected, the coarse particles were removed by settling for a short period for about 15 minutes, and decanting the upper layer of finer solids. The sludge was washed off and centrifuged for a period of 10 minutes at 10,000 m/s2 to produce a clear supernatant and pellet of sewage solids. The supernatant liquid was discarded and the sludge was re-suspended in deionised water with shaking and the wash-water was removed by re-centrifuging and discarding again.
The washing and centrifuging process was repeated twice. The dry mass of a known volume of the stock suspended solids concentration was determined and the sludge was diluted further in deionised water. Prepared sludge solid concentration was
3.2 g/L.
The activated sludge was continuously aerated at the test temperature (20 ± 2°C) until usage.

Range finding test
The test mixtures, reference mixtures and blank solutions were aerated upto the exposure period of 3 hours with the compressed air at a flow rate of approximately
0.6 L per minute at test temperature. The test was performed at a temperature within the range 20 ± 2°C. In each test vessel (1 L glass beaker) the respective amount of test item, reference item stock solution, dilution water, and synthetic sewage were applied. The total volume in the vessels was 500 mL, considered the volume of inoculum to be added to start the test. Before use, the pH was checked.
The test was initiated with the first blank controls by adding the microbial inoculum and aeration started. The procedure was repeated at approximately 13 minute intervals with the reference substance and then the test item to give a series of vessels containing different concentrations of the reference and test substance. The final flasks were blank controls, prepared exactly as the first. The test was conducted in the following order so that the reference substance, test item and abiotic control were bracketed by the controls:

a. Blank control FB1, FB2 and FB3
b. Reference substance FR1, FR2 and FR3
c. Test item FT1 , FT2, FT3, FT4 and FT5
d. Abiotic control FA1, FA2 and FA3
e. Blank control FB4, FB5 and FB6

Three Abiotic control FA (synthetic sewage feed and test item at the highest concentration (i.e., 1000 mg/L) without microbial inoculum) test was evaluated.
The test was performed using three concentrations of the test item such as 10 mg/L, 100 mg/L and 1000 mg/L with a blank control and abiotic control with the highest concentrations of the test item.
The test was performed using three concentrations of the test item, i.e., 10 mg/L,
100 mg/L and 1000 mg/L with three blank controls and three abiotic controls with the highest concentrations of the test item at test start and test end, respectively. Since the test item was considered to be insoluble in water, the test item was weighed directly into the test vessel to achieve the appropriate concentration.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (Purity 99.1%)

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

A known inhibitor reference substance 3, 5-dichlorophenol was used to check the sensitivity of the sludge at 2, 6, and 36 mg/L nominal concentrations. The EC50-value for the reference substance was found to be between the 2 to 40 mg/L of acceptance criteria.

 RANGE FINDING TEST - PERCENTAGE OF INHIBITION AFTER 3 HOURS EXPOSURE PERIOD

Sample Code	Oxygen Consumption Rate (R)	Specific Respiration Rate (Rs)		Mean Specific Respiration Rate (Rs)	Inhibition Rate IT(%)	Mean Inhibition Rate IT(%)	SD (±)	RSD (%)
FB1- Blank Control	32.64	RTB1	21.76	21.79	-	-	-	-
FB2- Blank Control	32.34	RTB2	21.56		-	-	-	-
FB3- Blank Control	33.78	RTB3	22.52		-	-	-	-
FB4- Blank Control	32.94	RTB4	21.96		-	-	-	-
FB5- Blank Control	33.24	RTB5	22.16		-	-	-	-
FB6- Blank Control	31.2	RTB6	20.80		-	-	-	-
FR1- Ref Sub. 2 mg/L	30.6	RTR1	20.40	-	6.39	-	-	-
FR2- Ref Sub. 6 mg/L	17.82	RTR2	11.88	-	45.49	-	-	-
FR3- Ref Sub. 36 mg/L	1.80	RTR3	1.20	-	94.49	-	-	-
FT1- Test Sub. 10 mg/L	32.82	RTS1	21.88	-	-0.40	-	-	-
FT2-Test Sub. 100 mg/L	32.40	RTS2	21.60	-	0.89	-	-	-
FT3-Test Sub. 1000 mg/L	32.58	RTS3	21.72	-	0.34	0.40	0.11	26.65
FT4-Test Sub. 1000 mg/L	32.52	RTS4	21.68	-	0.52
FT5- Test Sub. 1000 mg/L	32.58	RTS5	21.72	-	0.34
FA1- Abiotic Control 1000 mg/L	0.00	RTA	0.00	-	-	-	-	-
FA2-Abiotic Control 1000 mg/L	0.00	RTA	0.00
FA3-Abiotic Control 1000 mg/L	0.00	RTA	0.00

Validity criteria fulfilled:

yes

Conclusions:

Test item YAPOX 2245 is non-toxic 3-h NOEC as ≥ 1000 mg/L to the activated sludge.

Executive summary:

Activated Sludge Respiration Inhibition Test for the test item YAPOX 2245 obtained from Yasho Industries Limited was evaluated according to OECD Guideline No. 209. Activated sludge obtained from waste sewage treatment plant was used as test system. The test was carried out under the test conditions with the test item concentrations 10 mg/L, 100 mg/L, 1000 mg/L. The blank controls (without the test item or reference substance) mean oxygen uptake rate was greater than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. The respiration rates of the blank control, reference substance and test item replicates were measured after the exposure period of three hours, and the inhibitory effects of the test item and reference substance were determined in comparison to the blank control respiration rates. A known inhibitor reference substance 3, 5-dichlorophenol was used to check the sensitivity of the sludge at 2, 6, and 36 mg/L nominal concentrations. The EC50-value for the reference substance was found to be between the 2 to 40 mg/L of acceptance criteria. Total respiration inhibition for the test item YAPOX 2245 with at the nominal concentrations of 10 mg/L, 100 mg/L and 1000 mg/L have no significant effect on activated sludge respiration inhibition.

Description of key information

3-h NOEC ≥ 1000 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

Activated Sludge Respiration Inhibition Test for the test item YAPOX 2245 obtained from Yasho Industries Limited was evaluated according to OECD Guideline No. 209. Activated sludge obtained from waste sewage treatment plant was used as test system. The test was carried out under the test conditions with the test item concentrations 10 mg/L, 100 mg/L, 1000 mg/L. The blank controls (without the test item or reference substance) mean oxygen uptake rate was greater than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. The respiration rates of the blank control, reference substance and test item replicates were measured after the exposure period of three hours, and the inhibitory effects of the test item and reference substance were determined in comparison to the blank control respiration rates. A known inhibitor reference substance 3, 5-dichlorophenol was used to check the sensitivity of the sludge at 2, 6, and 36 mg/L nominal concentrations. The EC50-value for the reference substance was found to be between the 2 to 40 mg/L of acceptance criteria. Total respiration inhibition for the test item YAPOX 2245 with at the nominal concentrations of 10 mg/L, 100 mg/L and 1000 mg/L have no significant effect on activated sludge respiration inhibition.