tert-butyl peroxypivalate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-04-15 to 1991-06-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Robra Test

Principles of method if other than guideline:

The toxicity towards bacteria was determined according to the Robra test. Substances that are toxic towards bacteria inhibit the enzyme activity resulting in a decrease of their oxygen consumption. The reduction in oxygen consumption refects the toxicity of a substance towards the test organisms.

GLP compliance:

not specified

Test organisms (species):

Pseudomonas putida

Details on inoculum:

Culture medium for test culture:
- Pepton from caseine (tryptic): 15.6 g
- Yeast extract: 2.8 g
- Natrium chloride: 5.6 g
- D(+) glucose: 1.0 g
- Agar: 12.0 g
- pH at 37 °C: 7.5 +/-0.1

These ingredients were dissolved in a 1 L boiling, deionised water. This culture medium was filled in flasks (each 6 - 8 mL). The flasks were sterilized in the autoclave for 15 minutes at 121 °C. Afterwards this culture medium was solidified.

Liquid media used for the cultivation of the test organisms:
- Pepton from caseine (tryptic): 15.6 g
- Yeast extract: 2.8 g
- Natrium chloride: 5.6 g
- D(+) glucose: 1.0 g

These ingredients were dissolved in a 1 L deionised water. The pH of this solution was adjusted to 7.2 +/-0.1 at 25 °C. For each liquid medium 200 mL were filled in a 500 mL Erlenmeyer flask. These flasks were closed with cotton and was autoclaved for 20 minutes at 121 °C.

Physiological NaCl solution:
9.0 g NaCl, p.a. was dissolved in 1 L distilled water. 10 mL of this physiological NaCl solution was filled in each flask. Afterwards the flasks were atuoclaved for 15 minutes at 121 °C.

Phosphate buffer (0.05 M):
- KH2PO4, p.a.: 27.2 g/L = 0.2 M
- NaOH, p.a.: 8.0g/L = 0.2 M
- pH at 20 °C: 7.2 +/-0.1

For the adjustment of the pH = 7.2 g 250 mL 0.2 M KH2PO4 solution was added to 175 mL NaOH solution. Distilled water was added up to 100 mL. 0.05 M phosphate buffer was stored in the refrigerator at 4 °C.

Synthetic BSB water:
stock solution 1:
- KH2PO4, p.a.: 8.5 g
- K2HPO4, p.a.: 21.75 g
- Na2HPO4 * H2O, p.a.: 33.4 g
- NH4Cl, p.a.: 1.7 g
- pH at 20 °C: 7.2 +/-0.1

stock solution 2: MgSO4 * 7 H2O, p.a.: 22.5 g

stock solution 3: CaCl2, p.a.: 27.5 g

stock solution 4: FeCl3 * 6 H2O, p.a.: 0.25 g

The components of each stock solution was dissolved in distilled or deionised water (total volume: 1 L). These solutions were autoclaved for 30 minutes at 121 °C. Under sterile conditions 1 mL was taken from each stock solution and was added to 1 L deionised or distilled water.

D(+) glucose stock solution (1 M): D(+) glucose: 198.1 g/L
Glucose was dissolved in distilled water and was sterilized in an autoclave for 30 minutes. The amount needed was filled into flask under sterile conditions each day.

Cultivation of test organisms:
For inoculation of the liquid media bacteria were slurrying in 10 mL 0.9% NaCl solution. Under sterile conditions 200 mL of this cultivation media was inoculated with 0.1 mL cell suspension. Further cultivation of the test organisms were performed for 16 +/- 2 h on a vibration machine at 22 +/- 1 °C.

Test type:

flow-through

Limit test:

no

Total exposure duration:

30 min

Remarks on exposure duration:

incubation time according to "Bewertung toxischer Wasserinhaltsstoffe aus ihrer Inhibitorwirkung auf Substratoxydation von Pseudomonas Stamm Berlin mit Hilfe polarographischer Sauerstoffmessungen" Abwasser, vol. 117, 1976, p. 80-86.

Test temperature:

37 °C

pH:

Culture medium for test culture: 7.5 +/-0.1

Nominal and measured concentrations:

1, 10, 100, 1 000 and 10 000 mg/L

Details on test conditions:

Cell suspension for oxygen consumption measurements:
After the exponential growth period the cells were centrifugated for 10 minutes ( 5000 - 7000 rpm). Afterwards the cells were washed twice with 0.05 M KH2PO4-NaOH buffer (pH 7.2) and were suspenden in buffer solution. This homogenised cell suspension was stored in the refrigerator at 4 °C.
For the oxygen consumption measurements the cell suspension was adjusted to a cell density of 0.3 absorption units at the wavelength 436 nm. Absorption measurements of the cell suspension were performed with an aliquote (diluted test medium 1:100) in a 1 cm cuvette (reference: buffer).

Test performance:
- Dilution series: According to the dilution ratios the liquid media were pipetted in a 100 mL volumetric flask and 5 mL of 1 M D(+) glucose solution with synthetic BSB water was added to fill up to 100 mL. Afterwards these solutions were transferred into 300 mL Erlenmeyer flasks and 2.5 mL adjusted cell suspension was added. In a water bath the temperature was adjusted at 20 °C +/- 1 °C.

As blind sample a sample was used without any toxic substances. As reference sample for toxicity a sample with a toxic substance was used.

Test assembly:
The measurements of the oxygen consumption was performed in a flow-through cell, which is equipped with an gold-amalgam electrode (Clark system). The cell is made of Plexiglas and has a total volume of 15 cm³. The oxygen saturated cell suspension was pumped with out air bubbles through the measuring cell. After pumping twice the volume of the cell the flow through the cell was stopped and the oxygen decrease was measured. Between the measurements the cell was purged with distilled water.

Reference substance (positive control):

yes

Remarks:

unknown toxic substance (not reported)

Key result

Duration:

30 min

Dose descriptor:

EC10

Effect conc.:

> 10 000 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

other: oxygen consumption rate

Details on results:

Test item concentration (mg/L) / % inhibition:
- 1 / 0
- 10 / 0
- 100 / 0
- 1 000 / 0
- 10 000 / 0

Conclusions:

Under the test conditions tert-butyl peroxypivalate does not show inhibition of the bacterial oxygen consumption up to 10 000 mg/L. Therefore, the EC10 of tert-butyl peroxypivalate was determined to be higher than 10 000 mg/L.

Executive summary:

The toxicity of tert-butyl peroxypivalate to microogranisms was assessed according to the Robra test. Under the test conditions tert-butyl peroxypivalate does not show any inhibition of the bacterial oxygen consumption up to 10 000 mg/L. Therefore, the EC10 of tert-butyl peroxypivalate was determined to be higher than 10 000 mg/L.

Description of key information

Under the test conditions tert-butyl peroxypivalate does not show inhibition of the bacterial oxygen consumption up to 10 000 mg/L. Therefore, the EC10 of tert-butyl peroxypivalate was determined to be higher than 10 000 mg/L.

Additional information

The toxicity of tert-butyl peroxypivalate to microogranisms was assessed according to the Robra test. Under the test conditions tert-butyl peroxypivalate does not show any inhibition of the bacterial oxygen consumption up to 10 000 mg/L. Therefore, the EC10 of tert-butyl peroxypivalate was determined to be higher than 10 000 mg/L.