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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
Study period:
1994-11-21 to 1994-11-22
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
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
Specific details on test material used for the study:
- Name of test material (as cited in study report): WITAMOL 118
- Substance type: product
- Physical state: liquid
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 2.0 g test substance was added to 20 ml of vehicle and emulsified at about 200 rpm in the shaking incubator for at least 19 h at 25°C. 1 ml of this solution was added to the test flasks. Additionally this solution was further diluted with emulsifier to obtain the 1 ml solution for the next concentration step.
- Controls: yes, vehicle control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): neutralized nonylphenol, ethoxylated (10 EO), propoxylated (5 PO)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 1 % (v/v)
- 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: 0.292)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
4.92 h
Post exposure observation period:
none
Hardness:
not mentioned
Test temperature:
24.8 - 26.9 °C
pH:
not mentioned
Dissolved oxygen:
not mentioned
Salinity:
see "Details on test conditions"
Nominal and measured concentrations:
Nominal concentrations: 1, 10, 100, 1000 mg/L (=1, 9.9, 99, 990 mg/L active ingredients)
Details on test conditions:
INOCULUM/TEST ORGANISM
- Species/strain: Pseudomonas putida, strain Berlin
- Source: Institut für Wasser-, Boden- und Lufthygiene des BGA, Berlin, 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: 0.292)
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, 1000 mg/L
- Preparation of test substance solutions: The test substance was directly pipetted into the test flasks.
DURATION OF THE TEST: 4.92 h
ANALYTICAL PARAMETER: O2 consumption
TEST CONDITIONS
- Composition of solutions:
a) Trace element solution (autoclaved):
0.055 g/L Al2(SO4)3 x n H2O
0.028 g/L KJ
0.028 g/L KBr
0.055 g/L TiO2
0.028 g/L SnCl2 x 2 H2O
0.028 g/L LiCl
0.389 g/L MnCl2 x 4 H2O
0.614 g/L H3BO3
0.055 g/L ZnSO4 x 7 H2O
0.055 g/L CuSO4 x 5 H2O
0.059 g/L NiSO4 x 6 H2O
0.055 g/L 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 deionized water
c) Stock solution II (steril filtrated):
0.200 g FeSO3 x 7 H2O
4.00 g MgSO4 x 7 H2O, resp. 3.70 g MgSO4 x 6 H2O
made up to 1 L with deionized water
Reference substance (positive control):
no
Duration:
4.92 h
Dose descriptor:
EC10
Effect conc.:
> 990 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: O2 consumption
Details on results:
Inhibition of the bacterial O2 consumption (mean values):
1 mg/L: 0 %
10 mg/L: 0.8 %
100 mg/L: 3.2 %
1000 mg/L: -0.3 %
The EC10 could not be established.
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
No statitistics performed
Validity criteria fulfilled:
yes
Conclusions:
The test substance did not inhibit bacterial respiration in the tested concentration range.
Executive summary:

1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esterst did not inhibit bacterial respiration in the tested concentration range. The EC50 was in excess of 990 mg/L.

Description of key information

1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esterst did not inhibit bacterial respiration in the tested concentration range. The EC50 was in excess of 990 mg/L.

Key value for chemical safety assessment

Additional information

The available study with 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters shows that the substance does not inhibit bacterial respiration in the tested concentration range. The EC50 was in excess of 990 mg/L.