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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms, other
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
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.
Qualifier:
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:
no
Analytical monitoring:
no
Details on sampling:
No data
Vehicle:
no
Details on test solutions:
No data
Test organisms (species):
Pseudomonas putida
Details on inoculum:
No data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Post exposure observation period:
No
Hardness:
No data
Test temperature:
25ºC
pH:
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
Salinity:
No data
Conductivity:
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 SYSTEM
- Test vessel: flask
- Type (delete if not applicable): closed by cotton-lined plastic caps
- Material, size, headspace, fill volume: 100 ml

TEST MEDIUM / WATER PARAMETERS
- 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,
A.R.;
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,
Na-salt);
5 mg Pyndoxamine (as pyridoxamine dihydro-
chloride);
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
Dissolve:
0.200 g ferrous sulphate, FeSO4.7H20, A.R.;
4.000 g magnesium sulphate, MgSO4.7H2O,
A.R.
in 1000 ml sterile double-distilled water.

Saline
Dissolve:
0.500 g sodium chloride, NaCl, A.R.

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

OTHER TEST CONDITIONS
- 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.

TEST CONCENTRATIONS
- 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
Key result
Duration:
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.
Validity criteria fulfilled:
yes
Remarks:
No data on actual exposure concentrations given as dilutions at different volume ratios with the factor 2 were prepared.
Conclusions:
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.

Description of key information

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.

Key value for chemical safety assessment

EC50 for microorganisms:
1 550 mg/L

Additional information