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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-02-14 to 1995-02-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
other: O2 consumption test (Huels method)
Principles of method if other than guideline:
The "Oxygen Consumption Test" offers the possibility of measuring chronic toxicity effects in bacteria with difficult substances e.g. those which are sparingly soluble, coloured, emulsifying or dispersive, even slightly volatile products can be tested.
Principle of the test: Glass vessels (e.g. 100 mL BOD-flasks) with known volume will be completely filled with culture solution, bacterial suspension and test substance in different concentrations (emulsified if insoluble in water), bubble-free sealed with glass stoppers and incubated for 5 to 6 hours at 25 ± 2°C on a shaking machine. Test vessels without bacterial suspension and vessels without test substance serve as controls. The differences between the oxygen content in the vessels at the beginning and the end of the incubation time is a measurement for the bacterial oxygen consumption.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 2.0 g of the test substance was weighed in 20 mL neutralised emulsifier and emulsified in a shaking incubator at 25 °C for 19 h at 200 rpm. 1 mL of this test solution was given into the test vessel (100 mL) for the highest test concentration (1000 mg/L). For further test concentrations the test solution was adequately diluted with emulsifying solution (dilution 1:10 each).
- Controls: yes
- Chemical name of vehicle (emulsifier): Nonylphenol plus 10 EO plus 5 PO
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 1 mL/100 mL
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not mentioned
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: yes
- Method of cultivation: not described
- Preparation of inoculum for exposure: approx. 19 h incubation of liquid culture of test organism
- Pretreatment: 200 mL stock solution I and II each were filled in an sterile 5 L-flask, containing 3520 mL deionized water and a magnetic stirrer bar, the solution were stirred for 30 min. and aerated. Thereafter 80 mL pre-culture were added and additionally 5 min stirred and areated.
- Initial biomass concentration: approx. 10 x 10E5 bacteria/mL, adjusted with sterile NaCl solution by photometrical measurement at 436 nm (extinction 0.293)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
5 h
Post exposure observation period:
none
Hardness:
not mentioned
Test temperature:
Preculture: 24.0 °C
Incubation: 25.0 - 26.1 °C
pH:
not mentioned
Dissolved oxygen:
not mentioned
Salinity:
see "Details on test conditions"
Nominal and measured concentrations:
1, 10, 100 and 1000 mg/L (nominal concentrations)
Details on test conditions:
INOCULUM/TEST ORGANISM
- Species/strain: Pseudomonas putida migula
- Source: Dr. Reinhard Kanne, Bayer AG, Leverkusen, Germany
- Method of cultivation: not described
- Preparation of inoculum: approx. 19 h incubation of liquid culture of test organism
- Pretreatment: 200 mL stock solution I and II each were filled in an sterile 5 L-flask, containing 3520 mL deionized water and a magnetic stirrer bar, the solution were stirred for 30 min. and aerated. Thereafter 80 mL pre-culture were added and additionally 5 min stirred and areated.
- Cell concentration of the inoculum: approx. 10 x 10E5 bacteria/mL, adjusted with sterile NaCl solution by photometrical measurement at 436 nm (extinction 0.293)
TEST SYSTEM
- Number of culture flasks:
a) 4 flasks per concentration with test substance, 2 of this flasks were used for determination of autooxidation of the test substance by adding of 1 mL HgCl2 solution, final concentration in the flasks approx. 3 mg/L
b) 4 flasks without test substance but with HgCl2 addition for determination of the oxygen content at the beginning of the test
c) 5 flasks without test substance, without HgCl2 for determination of the uninfluenced bacterial oxygen consumption. 1 flask of these was used to measure the oxygen contents after 4.5 h to determine the end of incubation. This value was not used for evaluation.
- Measuring equipment: oxygen electrode
- Termination of test: 1 mL HgCl2 solution (final concentration approx. 3 mg HgCl2/L) were added to each test vessel to stop the reaction.
TEST SUBSTANCE CONCENTRATIONS: 1, 10, 100 and 1000 mg/L
- Preparation of test substance solutions: 2.0 g of the test substance was weighed in 20 mL neutralised emulsifying solution (Nonylphenol, 10 EO, 5 PO) and emulsified in a shaking incubator at 25 °C for 19 h at 200 rpm. 1 mL of this test solution was given into the test vessel (100 mL) for the highest test concentration (1000 mg/L). For further test concentrations the test solution was adequately diluted with emulsifying solution (dilution 1:10 each).
DURATION OF THE TEST: 5.0 h
ANALYTICAL PARAMETER: O2 consumption
TEST CONDITIONS
- Composition of solutions:
a) Trace element solution (autoclaved), per 500 mL distilled water:
0.0275 g Al2(SO4)3 x n H2O
0.014 g KJ
0.014 g KBr
0.0275 g TiO2
0.014 g SnCl2 x 2 H2O
0.014 g LiCl
0.1945 g MnCl2 x 4 H2O
0.307 g H3BO3
0.0275 g ZnSO4 x 7 H2O
0.0275 g CuSO4 x 5 H2O
0.0295 g NiSO4 x 6 H2O
0.0275 g Co(NO3)2 x 6 H2O
b) Stock solution I (sterilized):
20 g D(+)Glucose
4.24 g Na NO3
2.40 g K2HPO4
1.20 g KH2PO4
30 mL Trace element solution (a)
1000 mL distilled water
c) Stock solution II (steril filtrated):
0.200 g FeSO3 x 7 H2O
3.71 g MgSO4 x 6 H2O
made up to 1 L with distilled water
d) NaCl solution (autoclaved):
0.500 g NaCl made up to 1 L distilled water


Reference substance (positive control):
no
Duration:
5 h
Dose descriptor:
EC10
Effect conc.:
> 0.99 g/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The test substance did not affect the bacterial respiration in the tested concentration range:
EC10 > 0.99 g act. ingr./L
EC50 > 0.99 g act. ingr./L

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: not mentioned
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
No statitistics performed
Validity criteria fulfilled:
yes
Conclusions:
Toxicity of the test item was evaluated against Pseudomonas putida. No respiration inhibition was observed up to the highest tested concentration (990 mg/L).
Executive summary:

Toxicity of the test item was evaluated against Pseudomonas putida based on the Hüls method. The "Oxygen Consumption Test" offers the possibility of measuring chronic toxicity effects in bacteria with difficult substances e.g. those which are sparingly soluble, coloured, emulsifying or dispersive, even slightly volatile products can be tested. Glass vessels were  completely filled with culture solution, bacterial suspension and test substance in different concentrations (emulsified), bubble-free sealed with glass stoppers and incubated for 5 to 6 hours at  25 ± 2°C on a shaking machine. Test vessels without bacterial suspension and vessels without test substance served as controls. The differences between the oxygen content in the vessels at the beginning and the end of the incubation time is a measurement for the bacterial oxygen consumption. No respiration inhibition was observed up to the highest tested concentration (990 mg/L).

Description of key information

No inhibition of respiration of Pseudomonas putida up to 0.99 g/L.

Key value for chemical safety assessment

Additional information

Two studies are available on benzyltoluene toxicity towards microorganisms. Both are based on inhibition of respiration. The Diefenbach study (1995) has been made according to a test procedure in accordance with generally accepted scientific standards and described in sufficient details. Benzyltoluene was dispersed with a non ionic surfactant. At none of tested concentrations, between 1 and 1000 mg/L, any inhibition of respiration of Pseudomonas putida has been observed. Too few information is available from Bouraly (1981) at the only concentration tested 1g/L where there is an inhibition of about 60%. Test conditions were not described and the only testing concentration did not allow to derive precise effect concentration values.