1,3-diisopropylbenzene

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:

10 DEC 1985 to 19 MAY 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

A radiorespirometric method was used to determine the compatibility of the test substance with secondary waste treatment microorganisms . The procedure used microorganisms from a laboratory-maintained activated sludge unit. These microorganisms were exposed to [14C] glucose and the test substance. The extent of conversion of the [14C] glucose to [14C] carbon dioxide in the presence of the test article was compared to glucose conversion without test article.

GLP compliance:

not specified

Remarks:

pre-GLP

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.87 ± 0.04 mg/L
- Sampling method: Three 200 mL samples of the test article solution and three 200 mL samples of distilled water were added to 250 mL separatory funnels and extracted three times with 50 mL portions of methylene chloride. After each extraction the organic layer was decanted into a 250 mL Erlenmeyer flask and the volume reduced to approximately 5 mL with air flow at room temperature. Upon completion of the third extraction the volume was reduced to less than 2 mL and the samples were transferred to graduated test tubes and brought to 2 mL final volume with methylene chloride. Each sample was then analyzed by gas chromatography.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock test solutions were prepared in distilled water as follows: 100 mg of the test article was mixed with 1000 mL of distilled water by mechanical stirring for 24 hours at 22 °C. The mixture was centrifuged at 10,000xG for 10 minutes. After centrifugation, the top several mLs of liquid was discarded and the remainder saved for use as the stock test article solution. The pH was not measured and no pH adjustment was made. This stock solution subsequently was diluted by a factor of four in the test vessels to achieve the final nominal test article concentrations of (1/4)C, where C denotes the concentration of the test substance in the stock solution.

Reagents and solutions
- Potassium phosphate buffer (0.02M pH 6.9)
This buffer solution was prepared by dissolving 1.36 g of potassium dihydrogen phosphate and 1.74 g of dipotassium hydrogen phosphate in a total of 1.0 L of distilled water. The resulting solution was adjusted to pH 6.9 with 2N hydrochloric acid
- Nutrient broth stock solution
A nutrient broth stock solution was prepared from 24 g of Bacto nutrient broth (Difco laboratories) and 1.5 g of urea dissolved in a total of 3.0 L of 0.02 M phosphate buffer. The solution was steam sterilized at 121 °C for 15 minutes, then refrigerated.
- D-[U-14C] Glucose broth solution
This solution was prepared just before use. It consisted of 95 mL of distilled water, 10.0 mL of the nutrient broth stock solution, 0.10 mL of a 500 mg/mL non radiolabelled glucose solution, and 1.2 mL (approximately 12 µCi) of a D-[U-14C] glucose solution. The broth solution had an activity of 0.11 µCi/mL.

Test organisms (species):

activated sludge

Details on inoculum:

- Laboratory culture: Microorganisms were cultured in sludge tanks containing two chambers: a 300 mL aeration/mixing chamber and a 75 mL clarifying chamber. The contents of the sludge tanks were mixed and aerated by water-saturated air which entered at the bottom of the aeration/mixing chamber. The microorganisms were maintained on a glucose-peptone-nutrient mixture. This solution was pumped dropwise into the sludge tank aeration/mixing chamber. The sludge unit had a retention time of 6 hours. Cellular debris, waste products, and excess fluid were drained off at the overflow sidearm located on the settling chamber. Two sludge units were used. Air and nutrients were introduced simultaneously into both sludge tanks.
- Preparation of inoculum for exposure: Sludge units were seeded periodically with microorganisms obtained from an industrial waste treatment plant. Seeding occurred once per week, with about 10 % of each unit being replaced with new mixed liquor suspended solids (MLSS).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

5 h

Test temperature:

27 °C

Nominal and measured concentrations:

0.22 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: modified 50 mL Erlenmeyer flasks, fitted with downward curving 3/4 inch diameter sidearms, were used for the radiorespirometric assay. Scintillation vials containing a CO2 trapping solution composed of 0.2 mL distilled water and 1.0 mL diethanolamine were attached to the sidearms.
- Selection of microorganisms for testing: Between 350 and 400 mL of mixed liquor from the laboratory maintained domestic activated sludge units were centrifuged in tared 50 mL plastic centrifuge tube at 7000xG for 5-15 minutes at 4 °C. The supernatant liquid was decanted and the precipitate was washed in 300 mL of oxygenated 0.02 M potassium phosphate buffer, pH 6.9. The tube contents were then centrifuged as before, the supernatant liquid was decanted, and the tube and contents were weighed. The contents of the tube were resuspended in sufficient oxygenated potassium phosphate buffer to give a final activated sludge concentration of 10-20 mg/mL.
- Exposure: To each of 6 respirometer flasks were added to 5.0 mL of activated sludge solution that contained 20 mg of sludge solids per mL. Three of these flasks then received 2.5 mL of a stock test article solution. The remaining three flasks then received 2.5 mL of 0.02 M phosphate buffer(negative control). Each of the 6 flasks then received 2.5 mL of D-[U-14C] glucose broth solution (approx. 0.3 µCi). The flasks were sealed with clean serum stoppers and incubated in the dark at 27 °C in a reciprocating incubator-shaker at 70 cycles per minute for 5 hours. At the end of the incubation period the flasks were removed from the shaker and 5 mL of 2N HCl was added through the serum stopper of each flask using a syringe. The flasks were stored overnight at room temperature prior to analysis.

Reference substance (positive control):

no

Duration:

5 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.22 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: inhibition of conversion of radiolabelled glucose

Details on results:

Representative secondary waste treatment microorganism were not effected by test item at 0.22 mg/L. This is 1/4 the maximum concentration achieved after vigorously stirring a mixture of the test article and phosphate buffer for 24 hrs. The test item is not expected to affect secondary waste treatment adversely even at its limiting solubility.

Validity criteria fulfilled:

yes

Conclusions:

Exposure to 0. 22 mg/L of test item, the highest concentration tested, had no observed inhibitory effects on the microorganisms.

Executive summary:

A radiorespirometric method was used to determine the compatibility of test item with secondary waste treatment microorganisms. The procedure used microorganisms from a laboratory-maintained activated sludge unit. These microorganisms were exposed to (14C) glucose and 1,3-Diisopropylbenzene. The extent of conversion of the (14C) glucose to (14C) carbon dioxide in the presence of the test article was compared to glucose conversion without test article. Exposure to 0.22 mg/L of test item, the highest concentration tested, had no observed inhibitory effects on the microorganisms.

Description of key information

- Robilard, 1986:  non-guideline study with m-diisopropylbenzene, exposure to 0.22 mg/L of test item, the highest concentration tested, had no observed inhibitory effects on the microorganisms. Thus, the NOAEC is 0.22 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

0.22 mg/L

Additional information

A radiorespirometric method was used to determine the compatibility of 1.3-diisopropylbenzene with secondary waste treatment microorganisms (Robilard, 1986). The procedure used microorganisms from a laboratory-maintained activated sludge unit. These microorganisms were exposed to (14C) glucose and 1.3-diisopropylbenzene. The extent of conversion of the (14C) glucose to (14C) carbon dioxide in the presence of the test article was compared to glucose conversion without test article. Exposure to 0.22 mg/L of test item, the highest concentration tested, had no observed inhibitory effects on the microorganisms.