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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail. For read-across justification see Section 13.
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 aerated.
- Initial biomass concentration: approx. 10 x 10E5 bacteria/mL, adjusted with sterile NaCl solution by photometrical measurement at 436 nm (extinction 0.306)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
4.75 h
Remarks on exposure duration:
The incubation time is slightly below the scheduled value, but this had no influence on the oxygen consumption (4.5 mg O2/L)
Post exposure observation period:
none
Hardness:
not mentioned
Test temperature:
Preculture: 24.8 - 24.9 °C
Incubation: 25.4 - 26.5 °C
pH:
not mentioned
Dissolved oxygen:
see "Remarks on results"
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 aerated.
- Cell concentration of the inoculum: approx. 10 x 10E5 bacteria/mL, adjusted with sterile NaCl solution by photometrical measurement at 436 nm (extinction 0.306)
TEST SYSTEM
- Number of culture flasks:
a) 4 flasks per concentration with test substance, 2 of this flasks were used for determination of auto-oxidation 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: 4.75 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 (sterile 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:
4.75 h
Dose descriptor:
EC10
Effect conc.:
> 0.98 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 > 0.98 g act. ingr./L
EC50 > 0.98 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 statistics performed

Oxygen concentration

   control  1 mg/L  10 mg/L  100 mg/L  1000 mg/L
 Initial average  8.24  8.32  8.08  8.24  8.30
 Average after incubation  3.74  3.71  3.77  3.66  3.53
 O2 consumption during incubation  4.50  4.61  4.31  4.58  4.78
 Absolute difference    -0.11  0.19  -0.08  -0.28
 Relative difference [%]    -2.4  4.3  -1.7  -6.2

All oxygen concentrations are given in mg O2 / L

Validity criteria fulfilled:
yes
Conclusions:
The test substance did not affect the bacterial respiration in the tested concentration range.
Executive summary:

The study of the bacteriotoxicity of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters was carried out by a method developed by the testing laboratory. Principle of the method: 100 mL BOD bottles closable by means of ground stoppers are to be charged with the culture solution, the suspension of bacteria (Pseudomonas putida Migula) and the test substance in stepped concentrations, closed so as to leave no air bubbles and incubated for 5 -6 hours at 25 ± 2 °C in a shaking incubator. Batches free of test substance serve as control. The difference between the oxygen content of the solutions in the individual containers at time zero and after the incubation time gives the bacterial oxygen consumption. Comparison of the amounts of oxygen consumed in the control and test batches gives information about the concentration-dependent influence of the test substance on the oxygen consumption.

For the test 2.0 g of the test substance was weighed in 20 mL neutralised emulsifier (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, corresponding to 980 mg a.i./L). For further test concentrations the test solution was adequately diluted with emulsifying solution (dilution 1:10 each).

The test substance did not affect the bacterial respiration in the tested concentration range (1, 10, 100 and 1000 mg/L; corresponding to 1, 9.8, 98 and 980 mg a.i./L),:

EC10 > 0.98 g a.i./L, EC50 > 0.98 g a.i./L.

Description of key information

NOEC/EC10: >932 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
932 mg/L

Additional information

Data on the substance itself are not available. Data are available on a structural analogue of the substance, a trimellitate ester with mixed C8 -C10 side chains. This was investigated using a method developed by the testing laboratory. Principle of the method: 100 mL BOD bottles closable by means of ground stoppers are to be charged with the culture solution, the suspension of bacteria (Pseudomonas putida Migula) and the test substance in stepped concentrations, closed so as to leave no air bubbles and incubated for 5 -6 hours at 25 ± 2 °C in a shaking incubator. Batches free of test substance serve as control. The difference between the oxygen content of the solutions in the individual containers at time zero and after the incubation time gives the bacterial oxygen consumption. Comparison of the amounts of oxygen consumed in the control and test batches gives information about the concentration-dependent influence of the test substance on the oxygen consumption. For the test 2.0 g of the test substance was weighed in 20 mL neutralised emulsifier (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, corresponding to 980 mg a.i./L). For further test concentrations the test solution was adequately diluted with emulsifying solution (dilution 1:10 each). The tested substance did not affect the bacterial respiration in the tested concentration range (1, 10, 100 and 1000 mg/L; corresponding to 1, 9.8, 98 and 980 mg a.i./L),: EC10 > 0.98 g a.i./L, EC50 > 0.98 g a.i./L. This corresponds to an EC10 > 932 mg/L for the registered substance when differences in molecular weight are taken into account.