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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed according to protocol under GLP.
Qualifier:
according to guideline
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
Deviations:
yes
Remarks:
Meatpepton and vitamin B1 were not included in the pre-culture growth medium. The test solutions were made from a test substance stock solution.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
A test substance stock solution was prepared using an analytical balance (AE 163, Mettler-Toledo AG, Greifensee, Switzerland. For the respiration test 5.0092 g test substance and 5.0793 g Tween 80 was mixed in a total of 500 mL deionized water, the pH was adjusted to 7.7 with 1M NaOH. A white homogeneous suspension was obtained after mixing for approximately one hour. A stock solution of the emulsifier was made by dissolving 10.0035 g Tween 80 in 100 mL deionized water.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
The test was carried out with the gram-negative, aerobic bacterium Pseudomonas putida (DSM 50026), obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, BRD on 1995.05.10. The strain was maintained on micro bank tubes after incubation for 24 hours at
approximately 25°C, and stored in the freezer. For the test a pre-culture was prepared by inoculation of the pre-culture growth medium with Pseudomonas putida from an agar slope. This culture was incubated at 21°C ± 1°C for 72 hours in a shaking waterbath (Sanyo-Gallenkamp, Breda, The Netherlands) at 100 rev/min before the start of the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 min
Test temperature:
21.1 °C
Dissolved oxygen:
The amount of oxygen in the test vessels at the start of the test was sufficient because air saturated buffer solution was used as dilution water (> 90%).
Nominal and measured concentrations:
Nominal: 31.1, 62.1, 124.3, 248.5 and 497 mg/L test substance (active ingredient).
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyers
- Type: closed
- Aeration: no
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Nutrient stock solutions were prepared separately, and stored at approximately 4°C in the dark for approximately one week. All solutions were transparent and homogenous at time of use. The nutrients were dissolved in 1 liter deionized water.
Stock 1 : 8.48 g KH2P04, 21.80 g K2HP04, 33.34 g Na2HP04.2H20. 1.68 g NH4Cl
Stock 2 : 22.48 g MgS04.7H20
Stock 3 : 27.48 g CaCl2
Stock 4 : 0.25 g FeCl3.6H20
From each stock solution 1 ml was transferred to a 1 liter conical flask and filled up to 1000 mL with deionized water. The solution was aerated before use. Phosphate buffer: 6.80 g KH2P04 and 1.40 g NaOH was dissolved in a 1000 mL conical flask and filled up to 1000 mL with deionized water. The pH of this solution was 7.2. Glucose stock solution: 49.54 g glucose-monohydrate was dissolved in 100 mL deionized water.

- Culture medium different from test medium: The synthetic growth medium was made by dissolving the following amounts in one liter of deionized water: 15 g peptone (Casein), 3 g yeast extract, 6 g NaCI, 1 g D(+}glucose. This solution was divided into 100 mL portions in 300 mL Erlenmeyer flasks. These flasks were sterilized at 121 °C for 10 min.
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Duration:
30 min
Dose descriptor:
NOEC
Effect conc.:
>= 497 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 497 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
During the test the respiration of Pseudomonas putida was not inhibited at nominal test concentrations up to 497 mg/L Bis(4-methylbenzoyl)peroxide. A slight stimulation of the respiration was observed at the lower concentrations. The respiration of the control with Tween 80 was not inhibited, but slightly stimulated which indicates that Tween 80 was not toxic for Pseudomonas putida at the tested concentration.
Reported statistics and error estimates:
No statistics were performed because no inhibition was observed.
Validity criteria fulfilled:
yes
Conclusions:
EC20, 50, 80 values of Bis(4-methylbenzoyl} peroxide for Pseudomonas putida could not be calculated because of the low toxicity of Bis(4-methylbenzoyl) peroxide. On the basis of the results as presented it can be concluded that Bis(4-methylbenzoyl)peroxide was not toxic to the gram negative aerobic bacterium Pseudomonas putida when exposed to nominal concentration up to 497 mg/L.
Executive summary:

In order to predict effects of chemicals in an aquatic environment, the toxicity of Bis(4methylbenzoyl) peroxide to bacteria was assessed. The toxicity was determined in the respiration inhibition test with the Gram-negative aerobic bacterium Pseudomonas putida in accordance with slightly modified DIN Test Guidelines and in compliance with the OECD Principles of Good Laboratory Practice.

Respiration by Pseudomonas putida was determined in the presence of various concentrations of BiS(4methylbenzoyl) peroxide. The test was conducted in a nutrient medium at a temperature of 21.1 °C. The bacteria were exposed to Bis(4-methylbenzoyl)peroxide at the following nominal concentrations (active ingredient) 0, 31.1. 62.1, 124.3, 248.5 and 497 mg/L. EC20, 50, 80 values of Bis(4-methylbenzoyl} peroxide for Pseudomonas putida could not be calculated because of the low toxicity of Bis(4-methylbenzoyl) peroxide. On the basis of the results as presented it can be concluded that Bis(4-methylbenzoyl)peroxide was not toxic to the gram negative aerobic bacterium Pseudomonas putida when exposed to nominal concentration up to 497 mg/L.

The test was valid as shown by the inhibition of the respiration at the lowest concentration of 0%, the respiration rates of the controls 1.2 to 1.3 mg/L/min and a deviation of 8% from the average value of the controls.

Description of key information

EC20, 50, 80 values of Bis(4-methylbenzoyl} peroxide for Pseudomonas putida could not be calculated because of the low toxicity of Bis(4-methylbenzoyl) peroxide. On the basis of the results as presented it can be concluded that Bis(4-methylbenzoyl)peroxide was not toxic to the gram negative aerobic bacterium Pseudomonas putida when exposed to nominal concentration up to 497 mg/L.

Key value for chemical safety assessment

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