2-tert-butyl-1,4-dimethoxybenzene

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:

2018-12-14

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: 2-tert-butyl-1,4-dimethoxybenzene
Public name: Vetimoss
EC number: 244-216-5
CAS number: 21112-37-8
Batch/Lot Number: A180118C
Description: Clear, pale yellow liquid
Expiry Date: 06 June 2019
Purity: 99.78%
Storage conditions: Room temperature (15-25°C, ≤70 relative humidity%), under inert gas. Protected from humidity (tight closed container).

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

FORMULATION
Based on poor solubility of the Test Item in water, a given amount Test Item was weighed directly into each individual flask, then approximately 10 minutes ultra-sonication was applied just before the start of the experiment.
ADMINISTRATION OF THE TEST ITEM

Test Concentrations: The test was performed at 3 concentrations of the Test Item with 5 replicates per concentration: 7.5 mg/L (TI11, TI12, TI13, TI14, TI15), 15 mg/L (TI21, TI22, TI23, TI24, TI25) and 30 mg/L (TI31, TI32, TI33, TI34, TI35) were used.

PERFORMANCE OF THE TEST

Preparations of the test flasks: Each glass flask had a final volume of 400 mL (ratio of composition of each test mixture referring to 500 mL according to the guideline). 
A volume of 12.8 mL synthetic sewage, an adequate amount of the Test Item and deionised water were placed into the test flask before the start of the incubation (see the Table 1). At the start of the test, 200 mL activated sludge inoculum with a sludge concentration of 3 g/L of suspended solids was added to all flasks, except 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 7.5 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 15 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 30 mg/L nominal concentration (TI31, TI32, TI33, TI34 and TI35) and the fifth blank control (B5).

In summary, 3 Test Item concentration series and 1 abiotic control series were prepared in 5 parallels with one blank control series by series. In parallel with the above test mixtures 4 Reference Item concentrations without replicates were also prepared with 1 blank control.

CONTROLS
Abiotic Control (A): In parallel to the study, abiotic control flasks (A1, A2, A3, A4 and A5) were tested without inoculum, but with the highest concentration of the Test Item (30 mg/L nominal concentration), under identical test conditions.

Untreated Control (B): 1-1 member of five replicates (B1, B2, B3, B4 and B5) of the untreated control group (deionised water, synthetic sewage and inoculum, without addition of the Test Item) was tested in parallel with all of the test series.

Reference Control (REF): In parallel with the Test Item, the Reference Item 3,5-Dichlorophenol (REF1, REF2, REF3, REF4) was tested at nominal test concentrations of 1.0; 3.2; 10 and 32 mg/L under identical test conditions.A stock solution of 3,5-Dichlorophenol was prepared according to the OECD Guideline No. 209 (1.0 g of 3,5-Dichlorophenol dissolved in 1000 mL of water). Warm deionised water was used to help the dissolution of the Reference Item and the volume of the solution was made up to 1.0 L with deionised water and then cooled to room temperature. The pH of the solution was checked and adjusted with NaOH solution (2 mol/L) to pH 7.43.

Reference Item: 3,5-Dichlorophenol
Manufacturer: ACROS ORGANICS
Batch No.: A0376465
Expiry Date: 31 August 2021
Control preparation: A stock solution of 3,5-Dichlorophenol was prepared according to the OECD Guideline No. 209 (1.0 g of 3,5-Dichlorophenol dissolved in 1000 mL of water). Warm deionised water was used to help the dissolution of the Reference Item and the volume of the solution was made up to 1.0 L with deionised water and then cooled to room temperature. The pH of the solution was checked and adjusted with NaOH solution (2 mol/L) to pH 7.43.

Test organisms (species):

activated sludge

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 3 days before the start of the experiment. Prior to use, the sludge was fed daily from Day-2 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 was 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/L (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.28. The activated sludge was used directly after conditioning.

Synthetic Sewage Feed (ratio of composition of culture media referring to 1000 mL)
- Peptone: 16.0 g
- Meat extract: 11.0 g
- Urea: 3.0 g
- NaCl: 0.7 g
- CaCl2 x 2H2O: 0.4 g
- MgSO4 x 7H2O: 0.2 g
- K2HPO4: 2.8 g
- Deionised water added to 1000.0 mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not reported

Test temperature:

Temperature: 19.6–21.3°C

Measurement: The temperature was measured in the laboratory with a min/max thermometer during the experiment.

pH:

- Measurement: The pH of the test mixtures was determined at the start and at the end of the incubation period in all test vessels.
- Activated sludge: The pH of the activated sludge inoculum was determined to be pH 7.28.

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

- Nominal concentrations: 7.5 mg/L, 15 mg/L and 30 mg/L
- Measured concentrations: No analysis undertaken

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 : 19.6–21.3ºC
Aeration: With compressed air (about 1 L/min)
Recording: Test conditions were measured with suitable instruments and documented in the raw data.

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 approximately 3-hours incubation, and was not further aerated. The oxygen concentration was measured for approximately 15 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.:

> 30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Test Item:

In comparison to the inoculum controls, the inhibition of the respiration rate in the case of the activated sludge was between -1.21-1.66% in the examined concentration range of 7.5–30 mg/L (nominal).

Blank controls

The average specific respiration rate of the untreated controls was 26.03 mg O2/L/h/g solid sludge.

Coefficient of variation in controls: The variation coefficient of oxygen uptake rate in Control replicates was 4.54%.

Conclusion

Under the conditions of this study, testing up to the highest concentration of test item which did not damage the measuring equipment, the observed endpoint for the effect of the Test Item was the 3-hour EC50 value which was considered to be >30 mg/L (nominal). Based on the results of this study, the Test Item had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 30 mg/L (nominal). No further testing above the highest technically feasible concentration was necessary due to the fact that the Test Item had a physical destructive effect on the caps of the oxygen electrodes during the preliminary trial formulation.

Results with reference substance (positive control):

Positive control

In comparison to the controls, the inhibition of the respiration rate of the activated sludge was 84.93% at the highest nominal concentration of 32.0 mg/L. At the nominal concentrations of 1.0, 3.2 and 10.0 mg/L 14.21%, 24.94% and 40.76% inhibition of the respiration rate was calculated respectively. The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 9.02 mg/L with 95% confidence limits of 7.09–11.47 mg/L.

Reported statistics and error estimates:

The 3-hour EC50 value of the Reference Item and its 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software. In case of the Test Item, the EC50 was determined directly from the values of percentage of inhibition.

Influence of Test Item on oxygen consumption of activated sludge

Test group	Conc. of Test Item in test mix.	Total oxygen consumption rate (RT)	Specific respiration rate (RS)	Inhibition	pH values[1]
Name, ID	mg/L (nominal)	mg O2/L/h	mg O2/L/h/g solid	%	start	end
Abiotic Control
A1	30	1.24	---	---	7.21	7.74
A2		1.03	---	---	7.21	7.77
A3		1.24	---	---	7.24	7.78
A4		1.08	---	---	7.27	7.79
A5		1.20	---	---	7.28	7.79
Blank Control
B1	0.0	36.60	24.40	---	7.18	7.54
B2		39.20	26.13	---	7.22	7.98
B3		39.00	26.00	---	7.48	8.07
B4		38.80	25.87	---	7.54	8.08
B5		41.60	27.73	---	7.60	8.10
Reference Item
REF1	1.0	34.65	23.10	14.21	7.19	7.93
REF2	3.2	30.46	20.31	24.94	7.19	7.93
REF3	10.0	24.29	16.19	40.76	7.20	7.96
REF4	32.0	7.04	4.69	84.93	7.21	8.00
Test Item
TI1	7.5	39.55	26.37	1.66	7.49	8.07
TI2	15	39.83	26.56	0.93	7.51	8.07
TI3	30	40.67	27.11	-1.21	7.59	8.10

[1] Individual pH values of the BOD flasks, except TI1-2-3, where the average values are reported.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, testing up to the highest concentration of test item which did not damage the measuring equipment, the observed endpoint for the effect of the Test Item was the 3-hour EC50 value which was considered to be >30 mg/L (nominal). Based on the results of this study, the Test Item had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 30 mg/L (nominal).

Executive summary:

The toxicity of Vetimoss to activated sludge respiration was determined during an OECD 209 guideline study. Due to the nature of the test item, a concentration of >30 mg/L was not able to be tested as it destroyed the dissolved oxygen meter. Based on this study, an EC50 of >30 mg/L and a NOEC of 30 mg/L were reported for activated sludge inhibition by Vetimoss.

Description of key information

The toxicity of Vetimoss to activated sludge respiration was determined during an OECD 209 guideline study. Due to the nature of the test item, a concentration of >30 mg/L was not able to be tested as it destroyed the dissolved oxygen meter. Based on this study, an EC50 of >30 mg/L and a NOEC of 30 mg/L were reported for activated sludge inhibition by Vetimoss.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

30 mg/L