p-tert-butylstyrene

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:

From May 17, 2017 to August 17, 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))

Deviations:

yes

Remarks:

see ''Principles of method if other than guideline''

Principles of method if other than guideline:

- The reference compound 3,5-dichlorophenol had an EC50 slightly above the acceptable range of 2 to 25 mg/L during the preliminary test. This means for the preliminary test the batch of sludge may have been less sensitive to chemicals. This deviation is not considered to have had adverse impact on the study since the EC50 for the reference compound was within the acceptable range during the definitive test.

- During the preliminary study, an additional sampling at 50 minutes was performed to confirm the results from the 30 minute sampling. This deviation did not have an adverse impact on the results or interpretation of the study because the same results were achieved at 50 minutes as were achieved at 30 minutes, thereby confirming a 30 minute exposure duration in the definitive test would be sufficient.

GLP compliance:

yes

Specific details on test material used for the study:

Lot No. 122016TBSSV;
Purity: 95.9% (analyzed on 20 December 2016); 96.0% (reanalyzed on 10 May 2017); 96.0% (reanalyzed on 9 August 2017)

Analytical monitoring:

yes

Details on sampling:

The test was conducted in 250-mL labeled glass bottles. During the contact time (i.e., the 30 minutes that the microorganisms were in contact with the test substance or 3,5-dichlorophenol prior to the measurement of oxygen consumption), the solutions were gently mixed using a shaker table set to 150 rpm to provide oxygen to the inoculum.

Due to known volatility of the test substance, 30-minute samplings were conducted during the preliminary test in addition to the 3-hour samplings. The preliminary test showed less inhibition at 3 hours versus 30 minutes, which was attributed to the volatilization of the test substance. Therefore, only the 30-minute exposure was considered appropriate for the definitive test. During the definitive test, the test vessels were covered with perforated parafilm to allow for air exchange during shaking of the test vessels while on the shaker table. This set-up minimized the losses of the test substance while still being able to measure meaningful respiration rates. Although omission of a 3-hour measurement does not follow the standard test guideline proposal, the preliminary test results suggested that an additional 3-hour contact time in the definitive test would not yield valid results.

Vehicle:

no

Details on test solutions:

Standard Reagents:

All solutions were prepared using purified reagent water meeting ASTM Type II requirements generated from a Millipore Milli-Q Direct 8 water purification system.

Test Substance Stock Solution Preparation:

A 1.78 mg/L stock solution was prepared by bringing 2.1 µL of the test substance (relative density = 0.884) to a final volume of 1000 mL with purified reagent water in a 1-L Erlenmeyer flask. The flask was covered with parafilm and the solution was stirred using a Teflon-coated stir bar for approximately 45 minutes before use to dissolve the oil-like droplets.

A 5.09 mg/L stock solution was prepared by bringing 6.0 µL of the test substance (relative density = 0.884) to a final volume of 1000 mL with purified reagent water in a 1-L Erlenmeyer flask. The flask was covered with parafilm and the solution was stirred using a Teflon-coated stir bar for 45 to 60 minutes before use to dissolve the oil-like droplets.

3,5-Dichlorophenol Stock Solution Preparation:

A 500 mg/L 3,5-dichlorophenol stock solution was prepared by first placing 0.2565 g of the reference substance (0.2503 g as active ingredient) in 5.0 mL of 1 N sodium hydroxide (NaOH). Then 10 mL of purified reagent water was added to the solution. While on a stir plate, approximately 1.5 mL of 1.0 N sulfuric acid (H2SO4) was slowly added to the solution until a precipitate was observed. The solution was then transferred to a 500-mL volumetric flask and brought to volume with purified reagent water. Finally, the solution was transferred to an amber glass bottle and the pH of the resultant 3,5-dichlorophenol stock solution was 11.28, which required further adjustment with 1 N sulfuric acid (H2SO4) to a final pH of 7.49.

At test initiation (0 hour), 3.2 mL of synthetic sewage feed was brought to a volume of 50 mL with laboratory well water. The microbial inoculum (50 mL) was added to the solution, resulting in a final volume of 100 mL. This mixture prepared in a 250-mL glass bottle, covered with perforated parafilm and aeration through shaking on a shaker table at 150 rpm was immediately initiated. This test beaker was identified as Control 1 (C1). An abiotic control was prepared by adding an appropriate volume of test substance stock solution (same as the stock used to prepare the biotic test solutions for that concentration as stated in the table below) to synthetic sewage feed, and bringing to volume (100 mL) with laboratory well water; no microbial inoculum was added. The abiotic control solution was prepared in a 250-mL glass bottles, covered with perforated parafilm and aeration through shaking on a shaker table at 150 rpm was immediately initiated. The reference substance test systems (1.0, 3.0, 10, and 30 mg 3,5-dichlorophenol/L) were then prepared by adding appropriate volumes of the 3,5 dichlorophenol stock solution to the sludge mixture containing synthetic sewage feed and bringing to a volume of 50 mL with laboratory well water. The microbial inoculum (50 mL) was added to the reference solutions, resulting in a final volume of 100 mL. These vessels were all part of the first run or analysis. Control 2 (C2) was prepared in the same manner as Control 1, 15 minutes after the last reference substance test system was prepared. Lastly, test substance systems were prepared at each test substance concentration by adding an appropriate volume of test substance stock solution (listed in the table below) to the sludge mixture. Five replicates were prepared at each test substance concentration. One additional control was initiated with each series of test substance concentrations following the same procedure as Control 1 (C1). Since there were six test substance concentrations, there were six test substance runs after the 3,5-dichlorophenol run.

Test organisms (species):

sewage, domestic

Details on inoculum:

Prior to test initiation, approximately 8 L of activated sludge was obtained from the New Bedford Waste Water Treatment Plant (WWTP), New Bedford, Massachusetts, which receives primarily domestic sewage. Before use, the sludge solution was passed through a 2-mm stainless steel sieve, concentrated by centrifuging for 10 minutes at 1000 rpm, and the resulting supernatant was poured off. The sludge was then washed with laboratory well water and centrifuged at 1000 rpm for 10 minutes, for a total of four times, with the supernatant being discarded each time. The percent moisture content of the concentrated sludge was obtained with a Sartorius MA-150 moisture balance. The percent moisture was used to calculate the quantity of sludge required for suspension in water to obtain an activated sludge inoculum containing a solids level of 3 grams per liter (± 10%). Based on the percent moisture content of 94.72%, 142.74 g of the sludge was added to 2356 mL of laboratory well water to create the inoculum. The inoculum received 1.25 mL of an undiluted synthetic sewage feed and was aerated overnight until use. The inoculum was prepared one day before test initiation and had a pH value of 7.32. The solids content was determined to be 3.1 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Remarks on exposure duration:

Due to the volatility of the test substance the inoculum contact time was reduced to 30 min in the final test.

Test temperature:

20 ± 2 °C

pH:

The measured pH value in the abiotic control was 7.51 and the remaining pH values ranged from 7.30 to 7.67.

Nominal and measured concentrations:

Nominal concentrations: 0.0 (control), 0.01, 0.03, 0.09, 0.28, 0.81, and 2.4 mg/L
Adjusted nominal concentrations: 0.0 (control), 0.01, 0.02, 0.07, 0.2, 0.5, and 1.7 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

30 min

Dose descriptor:

EC10

Effect conc.:

0.107 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Nominal concentrations adjusted based on measured concentrations of the stock solutions were determined to be 0.01, 0.02, 0.07, 0.2, 0.5, and 1.7 mg/L. These adjusted nominal concentrations were used to calculate the EC10 and EC50 values.

At test termination, the minimum and maximum respiration rates for the seven control vessels were 22.3 and 35.3 mg O2/L/hr, respectively, which met the acceptability criterion (i.e., within 30% of each other during the test with the coefficient of variance equaling 17.1%). The mean respiration rate in the control vessels was 30.0 mg/L.

The mean respiration rates in the test substance test vessels (0.01, 0.02, 0.07, 0.2, 0.5, and 1.7 mg/L, adjusted nominal) were 30.7, 34.9, 25.9, 26.5, 19.2, and 22.7 mg O2/L/hr, respectively. Compared to the mean of the seven controls, the respiratory inhibition for the test substance vessels was -2.3, -16.5, 13.7, 11.6, 36.1, and 24.2% respectively.

Results with reference substance (positive control):

The respiration rates in the 3,5-dichlorophenol test vessels (1.0, 3.0, 10, and 30 mg/L) were 32.4, 34.5, 22.5, and 10.0 mg O2/L/hr, respectively. Compared to the respiration rate in the control 1 (34.4 mg/L), the respiratory inhibition for the 3,5-dichlorophenol test vessels was 5.8, -0.3, 34.6, and 70.9%, respectively. Based on these results, the EC50 value for 3,5-dichlorophenol was determined by linear regression using an Excel computer program and was determined to be 16.4 mg/L, which was within the acceptable limits specified in the OECD 209 Guideline (i.e., 2 to 25 mg/L).

Reported statistics and error estimates:

The EC50 is the estimated concentration of the test substance or reference substance that inhibits the respiration of the microorganisms present in activated sludge by 50%, as compared to the respiration in the control vessels. The EC10 is the estimated concentration of the test substance or reference substance that inhibits respiration of the microorganisms present in activated sludge by 10%, as compared to the respiration in the control vessels.
Using an Excel computer program, the percent inhibition was plotted against the log of the test and reference substance concentration and the EC10 and EC50 values were derived by linear regression.

The nominal test concentrations were adjusted based on the measured stock concentrations and used to calculate the final EC10 and EC50 values. 

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions the following results for the test substance were determined to be EC10 and EC50 values were 0.107 mg/L and greater than 1.7 mg/L (the highest concentration tested), respectively.

Executive summary:

A study was conducted to determine the toxicity of the test substance on the respiration of activated sludge according to OECD Guideline 209, in compliance with GLP. The dry matter of the inoculum (activated sludge of predominantly domestic origin) was determined as 3.1 g suspended solids/L. Due to the volatility of the test substance the inoculum contact time was reduced to 30 min. In the final experiment the test substance was tested using 6 concentrations ranging from 0.01 to 2.4 mg/L (nominal). The nominal test concentrations were adjusted from measured stock concentrations resulting in the target concentrations: 0, 0.01, 0.02, 0.07, 0.2, 0.5 or 1.7 mg/L. The reference substance, 3,5 -dichlorophenol, was tested concurrently to evaluate the sensitivity of the microbial population. The minimum and maximum respiration rates for the control vessels were determined to be 22.3 and 35.3 mg O2/L/h, respectively, meeting the acceptability criterion. The mean respiration rate in all the control vessels was 30.0 mg/L. Under the study conditions the EC10 and EC50 of the test substance for respiration inhibition in activated sludge bacteria were determined to be 0.107 mg/L and greater than 1.7 mg/L (the highest tested concentration), respectively (McLaughlin, 2019).

Key value for chemical safety assessment

EC50 for microorganisms:

1.7 mg/L

EC10 or NOEC for microorganisms:

0.107 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance on the respiration of activated sludge according to OECD Guideline 209, in compliance with GLP. The dry matter of the inoculum (activated sludge of predominantly domestic origin) was determined as 3.1 g suspended solids/L. Due to the volatility of the test substance the inoculum contact time was reduced to 30 min. In the final experiment the test substance was tested using 6 concentrations ranging from 0.01 to 2.4 mg/L (nominal). The nominal test concentrations were adjusted from measured stock concentrations resulting in the target concentrations: 0, 0.01, 0.02, 0.07, 0.2, 0.5 or 1.7 mg/L. The reference substance, 3,5 -dichlorophenol, was tested concurrently to evaluate the sensitivity of the microbial population. The minimum and maximum respiration rates for the control vessels were determined to be 22.3 and 35.3 mg O2/L/h, respectively, meeting the acceptability criterion. The mean respiration rate in all the control vessels was 30.0 mg/L. Under the study conditions the EC10 and EC50 of the test substance for respiration inhibition in activated sludge bacteria were determined to be 0.107 mg/L and greater than 1.7 mg/L (the highest tested concentration), respectively (McLaughlin, 2019).