6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

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:

02 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

OECD Guidelines for Testing of Chemicals, No.: 209 Adopted: 22 July 2010.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part C, C.11

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Version / remarks:

Product Properties Test Guideline OCSPP 850.3300 (Public Draft OPPTS 850.6800, April 1996) of the United States Environment Protection Agency (EPA) "Modified Activated Sludge, Respiration Inhibition Test", June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

not specified

Details on sampling:

Not specified

Vehicle:

no

Details on test solutions:

Because of the low solubility, the test item was added by directly weighing the test item into the bottles (and using ultrasonic bath for 20 minutes). The test formulation was freshly prepared at the beginning of the experiment, in the testing laboratory.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
Source: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The activated sludge was supplied by the sewage plant for domestic sewage two days before the start of the experiment. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage per litre and kept aerated at 20 ± 2°C until use.
The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined.
Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.60. The activated sludge was used directly after conditioning.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

No post exposure observation period specified.

Hardness:

Not specified

Test temperature:

20.1 – 21.4 °C

pH:

7.21 - 8.02

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

The test was performed at three concentrations of the test item: 10 (TI11, TI12, TI13, TI14, TI15), 100 (TI21, TI22, TI23, TI24, TI25) and 1000 mg/L (TI31, TI32, TI33, TI34, TI35) were used.

Details on test conditions:

Test units
Type and size: Appropriate glass beakers for 500 mL volume and BOD bottles with 300 mL volume.
Identification: Each test flask was uniquely identified with study code, treatment and replicate codes.

Test conditions
Surrounding type: Temperature controlled laboratory
Temperature: 20.1 – 21.4 °C
Aeration: With compressed air (about 1 L/min)
Recording: Test conditions were measured with suitable instruments and documented in the raw data.

Preparations of the test flasks
One test solution with a final volume of 400 mL (ratio of composition of each test mixture referring to 500 mL according to the guideline) was prepared per treatment in a glass flask.
A volume of 12.8 mL synthetic sewage and an adequate amount of the test item were placed into the test flask by direct addition (see the Table 1) and filled up with deionised water to 200 mL before the start of the incubation. At the start of the test, 200 mL activated sludge inoculum with a sludge concentration of 3 g per litre of suspended solids was added to all flasks, with the exception of the abiotic control flasks.
Abiotic controls in 5 parallels (A1, A2, A3, A4 and A5), and one untreated blank (B1) control was started as the first step of the test. Then at appropriate time intervals of approximately 30 minutes the further test groups were started. The second series were 4 different concentrations (without replicates) of the reference item (REF1, REF2, REF3, REF4) and to the second blank control (B2). The third series was the 5 replicates of the test item concentration of 10 mg/L nominal concentration (TI11, TI12, TI13, TI14 and TI15) and the third blank control (B3). The fourth series was the five replicates of test item concentration 100 mg/L nominal concentration (TI21, TI22, TI23, TI24 and TI25) and the fourth blank control (B4). The final series was the five replicates of test item concentration 1000 mg/L nominal concentration (TI31, TI32, TI33, TI34 and TI35) and the fifth blank control (B5).
In summary, 3 test item concentration series and one abiotic control series were prepared in 5 parallels with one blank control series by series. In parallel with the above test mixtures four reference item concentrations without replicates were also prepared with one blank control.

Measurement of Respiration Rate (Dissolved Oxygen)
For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after about 3 hours incubation time, and was not further aerated. The oxygen concentration was measured for about fifteen minutes, with an O2 electrode and was recorded in every half minute. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.

Measurement of pH and temperature
The pH of the test mixtures was determined at the start and at the end of the incubation period in all test vessels.
The temperature was measured in the laboratory with a min/max thermometer during the experiment.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

In comparison to the inoculum controls the inhibition of the respiration rate in the case of the activated sludge was between -10.41 % and -6.32 % in the examined range of 10 – 1000 mg/L of test item.
The average specific respiration rate (RS) of the untreated (Blank) controls (B1-B5) was 30.61 mg O2/L/h/g solid.
The variation coefficient of oxygen uptake rate in above untreated replicates was 1.64 %.

Results with reference substance (positive control):

The following nominal concentrations of the reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as in the case of the test item: 1.0, 3.2, 10.0 and 32.0 mg/L.
In comparison to the controls the inhibition of the respiration rate of the activated sludge was 77.59 % at the highest nominal concentration of 32.0 mg/L.
At the nominal concentrations of 1.0, 3.2 and 10.0 mg/L 12.63 %, 25.97% and 42.02 % inhibition of the respiration rate was calculated respectively.
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 10.22 mg/L with 95% confidence limits of 7.83 – 13.35 mg/L.

Reported statistics and error estimates:

Not specified

Influence of test item on oxygen consumption of activated sludge

Test group		Conc. of test item in test mix (mg/L)	Total oxygen consumption rate (RT) (mg O2/L/h)	Specific respiration rate (Rs) (mg O2/L/h/g solid)	Inhib. (%)	pH values^#		Temperature (°C) (measured in the Laboratory)^*	Variation coefficient of O2consumption rate (%)
ID	Name					Start	End
A1	Abiotic	1000	-1.03	-	-	7.46	8.00	20.1/21.4	-
A2		1000	-1.12	-	-	7.56	8.00		-
A3		1000	0.08	-	-	7.55	8.00		-
A4		1000	-0.08	-	-	7.53	8.02		-
A5		1000	0.21	-	-	7.49	8.01		-
B1	Blank	0.00	44.91	29.94	-	7.21	7.81	20.1/21.4	1.64
B2		0.00	45.73	30.49	-	7.31	7.82
B3		0.00	46.50	31.00	-	7.40	7.84
B4		0.00	45.60	30.40	-	7.52	7.71
B5		0.00	46.80	31.20	-	7.35	7.77
REF1	Ref. item	1.00	39.72	26.48	12.63	7.34	7.78	20.1/21.4	-
REF2		3.20	33.60	22.40	25.97	7.35	7.77		-
REF3		10.00	26.23	17.49	42.02	7.33	7.82		-
REF4		32.00	9.90	6.60	77.59	7.31	7.86		-
TI1	Test item	10	48.42	32.28	-6.32	7.46	7.85	20.1/21.4	-
TI2		100	50.30	33.53	-10.41	7.60	7.68		-
TI3		1000	49.41	32.94	-8.47	7.33	7.76		-

^#: Individual pH values of the BOD flasks, except TI1-3, where the average values are reported.

^*: Minimum/Maximum temperature (°C) measured in the Laboratory, during the experiment.

Validity criteria fulfilled:

yes

Conclusions:

Activated sludge respiration inhibition test (carbon and ammonium oxidation) was carried out with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol to evaluate the influence of the test item on the activity of activated sludge microorganisms by measuring the respiration rate under defined conditions.
Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was the following:
The 3-hour EC50: > 1000 mg/L
Based on the results of this study, the test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary.

Executive summary:

A laboratory test was carried out with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

The test concentrations (10, 100 and 1000 mg/L) were chosen to permit the determination of the approximate EC50.

 

The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate.

 

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was the following:

The 3-hour EC50: > 1000 mg/L

 

Based on the results of this study, the test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary.

Description of key information

The 3-hour EC50: > 1000 mg/L nominal loading rate

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol was the following:

The 3-hour EC50: > 1000 mg/L

Based on the results of this study, the test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary.