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Administrative data

toxicity to microorganisms
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
No data
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline study used but comparable to national guidelines/standards No data on actual exposure concentrations given as dilutions at different volume ratios with the factor 2 were prepared.

Data source

Reference Type:
Comparison of the toxicity thresholds of water pollutants to bacteria, algae, and protozoa in the cell multiplication inhibition test
Bringmann G & Kuhn R
Bibliographic source:
Water Research 14: 231-241

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
The toxic effect of a substance is tested in the cell multiplication inhibition test during 16 h. Under this method, the onset of the inhibition of cell multiplication under the influence of hazardous water pollutants is determined.
GLP compliance:

Test material

Constituent 1
Reference substance name:
Oxalic acid
EC Number:
EC Name:
Oxalic acid
Cas Number:
oxalic acid
Details on test material:
No data
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data

Sampling and analysis

Analytical monitoring:
Details on sampling:
No data

Test solutions

Details on test solutions:
No data

Test organisms

Test organisms (species):
Pseudomonas putida
Details on inoculum:
No data

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
16 h
Post exposure observation period:

Test conditions

No data
Test temperature:
The pH was not adjusted as the effect of the pH of the hazardous water pollutant solution to be studied was part of the test.
Dissolved oxygen:
No data
No data
Nominal and measured concentrations:
No data on actual exposure concentrations given as dilutions at different volume ratios with the factor 2 were prepared.
Details on test conditions:
Leave both inoculated and non-inoculated dilution series at 25°C for 16 h.

- Test vessel: flask
- Type (delete if not applicable): closed by cotton-lined plastic caps
- Material, size, headspace, fill volume: 100 ml

- Source/preparation of dilution water: double distilled water
-Culture medium different from test medium:
Nutrient medium (for stock and preliminary cultures)

Dissolve in 1000 ml double-distilled water:
1.060 g sodium nitrate, NaNO3, A.R.;
0.600 g dipotassium hydrogen phosphate.
K2HPO4, anhydrous, high purity;
0.300 g potassium dihydrogen phosphate,
KH2PO4, A.R.;
0.200 g magnesium sulphate, MgSO, - 7 H 20,
10.000 g D(+) glucose (for biochemical and
microbiological purposes);
18.000 g Difco Bacto agar;
0.010 g ferrous sulphate, FeSO4 • 7 H2O, A.R.;
1.5 ml trace elements solution.

Sterilize the solution in a steam sterilizer for 1.5 h, after which add 3 ml of vitamin solution.

Trace elements solution (in grams per liter of double-distilled water)

0.055 A12 (S04)3.18 H2O;
0.028 KJ, A.R.;
0.028 KBr, A.R.;
0.055 TiO2 LAB;
0.028 SnCl2.2 H 20, A.R.;
0.028 LiCI, A.R.;
0.389 MnC12.4 H2O, A.R.;
0.614 H3B03, A.R.;
0.055 ZnSO4.7 H2O, A.R.;
0.055 CuSO4.5 H2O, A.R.;
0.059 NiSO,•6 H2O, A.R.;
0.055 Co(N03)2.6 H2O, A.R.

Vitamin solution

0.2 mg biotin (as D + biotin);
2.0 mg nicotinic acid
1.0 mg thiamine (as thiamine HCI);
I.0 mg p-aminobenzoic acid;
0.5 mg panthothenic acid (as D-panthothenic acid,
5 mg Pyndoxamine (as pyridoxamine dihydro-
2.0 mg cyanocobalamin (vitamin B 12);
100 ml double-distilled water.

Fill 6 ml each of the nutrient medium into culture tubes, sterilize the latter in a steam sterilizer by fractionated sterilization (three times) for 30 min. Let solidify in slant position.

Stock solution I

20.000 g D(+) glucose (for biochemical and mic-
robiological purposes);
4.240 g sodium nitrate, NaNO3, A.R.;
2.400 g dipotassium hydrogen phosphate,
K2HPO4 anhydrous, high Purity;
1.200 g potassium dihydrogen
KH2PO4, A.R.;
30 ml trace elements solution.

Dissolve glucose and nutrient salts separately in 500 ml double-distilled water each, sterilize in a steam sterilizer for 30 min and unite solutions when cooled

Stock solution II
0.200 g ferrous sulphate, FeSO4.7H20, A.R.;
4.000 g magnesium sulphate, MgSO4.7H2O,
in 1000 ml sterile double-distilled water.

0.500 g sodium chloride, NaCl, A.R.

in 1000 ml double-distilled water. Sterilize solution in a steam sterilizer for 30 min.

- Adjustment of pH: yes
- Photoperiod: 16 h

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
extinction of the monochromatic radiation at 436 nm in a 10-mm layer in the inoculated dilution series.

- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
not specified

Results and discussion

Effect concentrations
16 h
Dose descriptor:
other: Toxicity Threshold
Effect conc.:
1 550 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
No data
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
For evaluation of the toxicological findings at the end of the test period, the mean value (A) of the extinction is calculated for all test cultures that are free from both toxic influence and stimulation of growth except for those having extinction values outside a standard deviation of < 3% and also, the mean value (B) of the extinction for those test cultures having the lowest toxic pollutant concentration within the dilution series.
For mathematical evaluation by means of a suitable electronic calculator (a) (highest non-toxic pollutant concentration is plotted against (A) and (b) (lowest toxic pollutant concentration) against (B) as coordinates. After entering (A-3%), the pollutant concentration at which the inhibitory action (c) begins may be obtained from the regression line between (a;A) and (b;B) if a negative deviation of the mean extinction by a 3% difference against the mean extinction value for all test cultures having a non-toxic and non-stimulating pollutant concentration is used as an indicator of the beginning of inhibitory action.

Applicant's summary and conclusion

Validity criteria fulfilled:
No data on actual exposure concentrations given as dilutions at different volume ratios with the factor 2 were prepared.
The 16 h toxicity threshold was 1550 mg/l
Executive summary:
The bacteria Pseudomonas putida was exposed to oxalic acid according to the cell multiplication inhibition test. A 16 h toxicity threshold of 1550 mg/l was determined.