1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters

Toxicity to microorganisms

Link to relevant study record(s)

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.

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.