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Endpoint:
toxicity to microorganisms, other
Remarks:
cell multiplication inhibition test with P. putida
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
Propan-2-ol (Isopropyl alcohol) is both reagents used in the manufacture of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol. Therefore, Propan-2-ol (Isopropyl alcohol) need to be considered in the assessment of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol .
Qualifier:
equivalent or similar to guideline
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
Deviations:
not specified
Principles of method if other than guideline:
The concentration of the bacterial suspension is measured turbidimetrically; it is expressed by the extinction of the primary light of the monochromatic radiation at 436 nm for a layer of 10 mm thickness. The concentration at which the inhibitory action of a pollutant starts will be present in that step of a dilution series of the pollutant having an extinction value at the end of the test period that is ≥ 3% below the mean value of extinction for non-toxic dilutions of the test cultures.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Before preparing the test cultures neutralize the pollutant solution having a known content in sterile double-distilled water to be tested by using the minimum volume of acid or alkaline solution. The initial concentration of the pollutant solution was not reported.

From this pollutant solution, prepare four parallel dilution series in 300 ml Erlenmeyer flasks, stoppered with cotton-lined plastic caps. Each of the dilutions contains 1 part v/v of polutant solution in 2E0 to 2E14 parts v/v mixture. Prepare the dilution series as follows: the first flask of each series contains 160 ml of pollutant solution at the start. Starting from this flask prepare the subsequent dilution steps at a constant dilution ratio by consistently mixing 80 ml of preliminary pollutant dilution and 80 ml double distilled water. Consequently, each flask contains 80 ml of culture liquid at the start. Make up each flask of the three dilution series to be inoculated to 100 ml by adding 5 ml each of stock solution I, 5 ml of stock solution II and 10 ml each of the prepared bacterial suspension from the preliminary culture having a known adjusted extinction value.
Test organisms (species):
Pseudomonas putida
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Hardness:
not reported
Test temperature:
25ºC
pH:
not reported
Dissolved oxygen:
not reported
Salinity:
freshwater
Nominal and measured concentrations:
2E0 to 2E14 parts v/v mixture
Details on test conditions:
Following inoculation, the extinction value of the monochromatic radiation at 436 nm for a 10-mm layer of the bacterial suspension of the test cultures will correspond to the extinction value of the Formazin standard suspension TE/F/436 nm = 10.

Leave both inoculated and non-inoculated dilution series at 25ºC for 16 hours. After termination of the test period measure the extinction of the monochromatic radiation at 436 nm in a 10-mm layer in the inoculated dilution series.

Nutrient medium (for stock and preliminary cultures)
Dissolve in 1000 ml double -distilled water:
1.060 g sodium nitrate, NaNO3
0.600 g dipotassium hydrogen phosphate, K2HPO4, anhydrous
0.300 g potassium dihydrogen phosphate, KH2PO4
0.200 g magnesium sulphate, MgSO4.7 H2O
10.000 g D(+) glucose
18.00 g Difco Bacto agar
0.010 g ferrous sulphate, FeSO4.7 H2O
1.5 ml trace elements solution.

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

Trace elements solution (in grams per liter of double-distilled water)
0.055 Al2(SO4)3.18 H2O
0.028 KI
0.028 KBr
0.055 TiO2
0.028 SnCl2.2 H2O
0.028 LiCl
0.389 MnCl2.4 H2O
0.614 H3BO3
0.055 ZnSO4.7 H2O
0.055 CuSO4.5 H2O
0.059 NiSO4.6 H2O
0.055 Co(NO3)2.6 H2O

Vitamin solution
0.2 mg biotin (as D+ biotin)
2.0 mg nicotinic acid
1.0 mg thiamine (as thiamine HCl)
1.0 mg p-aminobenzoic acid
0.5 mg panthothenic acid (as D-panthothenic acid, Na-salt)
5 mg pyridoxamine (as pyridoxamine dihydrochloride)
2.0 mg cyanocobalamin (vitamin B12)
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
4.240 g sodium nitrate, NaNO3
2.400 g dipotassium hydrogen phosphate, K2HPO4 anhydrous
1.200 g potassium dihydrogen phosphate, KH2PO4
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, FeSO4.7 H2O
4.000 g magnesium sulphate MgSO4.7 H2O

in 1000 ml sterile double distilled water.

Saline
Dissolve:
0.500 g sodium chloride, NaCl

in 1000 ml double-distilled water. Sterilize solution in a steam sterilizer for 30 min.
Reference substance (positive control):
no
Duration:
16 h
Dose descriptor:
other: Toxicity threshold
Effect conc.:
1 050 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
For evalution 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, (a) (highest non-toxic pollutant concentration) is plotted against (A) and (b) (lowest toxic pollutant concentration) against (B) as coordinates. After having entered (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 of 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:
not applicable
Conclusions:
A 16h Toxicity threshold concentration of 1050 mg/l has been determined for growth inhibition effects to Pseudomonas putida of the test substance. Propan-2-ol (Isopropyl alcohol) is both reagents used in the manufacture of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol. Therefore, Propan-2-ol (Isopropyl alcohol) need to be considered in the assessment of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol .
Executive summary:

According to the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.7b: Endpoint specific guidance section R.7.8.17.1 Laboratory data on toxicity on STP microorganisms, results of the cell multiplication inhibition test with P. putida (Bringmann and Kühn 1980) can be used for calculation of the PNECmicro-organisms.

Endpoint:
activated sludge nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
Propan-2-ol (Isopropyl alcohol) is both reagents used in the manufacture of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol. Therefore, Propan-2-ol (Isopropyl alcohol) need to be considered in the assessment of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol .
Qualifier:
no guideline followed
Principles of method if other than guideline:
The saprozoic flagellate protozoon Chilomonas paramaecium served as model organism in a standardized medium. The test period required for determination of the TGK was 48h. An electronic cell counter (Coulter) was used for quantitative determination of the protozoa inoculated and their multiplication within the test cultures.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: TS was dissolved in sterile ddH2O. Whenever necessary, pH was adjusted to 6.9.
Test organisms (species):
Chilomonas paramaecium
Details on inoculum:
- Laboratory culture: yes
- Method of cultivation: C. paramaecium cultures (20 mL organic mineral medium) were incubated in 300 mL Erlenmeyer flasks, closed with metal caps, at 20 °C with the same medium as the test culture
- Preparation of inoculum for exposure: after 72 or 96 h at 20 °C, the pre-cultures were examined over an inverted microscope
- Pretreatment: none
- Initial biomass concentration: 4 mL of appr. 15000 cell/mL
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
npne
Hardness:
not reported
Test temperature:
20 °C
pH:
6.9
Dissolved oxygen:
not reported
Salinity:
freshwater
Nominal and measured concentrations:
1 part TS solution (concentration not specified) in 2E0 - 2E14 parts mixture (i.e. 1:1, 1:2, 1:4, 1:8... 1:16384) (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask covered with metal caps
- Type: open
- Material: glass; size: 300 mL; fill volume: 20 mL
- Aeration: shaken
- No. of organisms per vessel: appr. 60000 cell
- No. of vessels per concentration: 2
- No. of vessels per control: 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: double distilled water
- Medium (20 mL) prepared with 8 mL Stammlösung 1 + 8 mL TS + 4 mL C. paramecium culture

Stammlösung 1
8 g glycine
290 mg Ca(NO3)2*4(H2O)
70 mg Mg(NO3)2*7(H2O)
40 mg KNO3
26 mg K2HPO4
dissolved in 1 L ddH2O, pH 6.9. To the sterile Stammlösung 1 were then added 7.5 mL pasteurized Stammlösung 2.

Stammlösung 2
0.2 mg D-biotine
2.0 mg nicotinic acid
1.0 mg thiamin HCl
1.0 mg p-aminobenzoic acid
0.5 mg sodium D-pantothenate
5 mg pyridoxamine dihydrochloride
2.0 mg cyanocobalamine
dissolved in 100 mL ddH2O. Pasteurized 30 min at 60 °C

- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: none

EFFECT PARAMETERS MEASURED: cell number (Coulter counter)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
other: Toxicity Threshold Concentration
Effect conc.:
104 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
A 48h Toxicity threshold concentration of 104 mg/l has been determined for growth inhibition effects to Chilomonas paramaecium of the test substance.
Validity criteria fulfilled:
not applicable
Remarks:
Dilution series long enough (15 concentrations) to determine a dose effect relationship
Conclusions:
A 48h Toxicity threshold concentration of 104 mg/l has been determined for growth inhibition effects to Chilomonas paramaecium of the test substance. Propan-2-ol (Isopropyl alcohol) is both reagents used in the manufacture of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol. Therefore, Propan-2-ol (Isopropyl alcohol) need to be considered in the assessment of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol .
Executive summary:

A 48h Toxicity threshold concentration of 104 mg/l has been determined for growth inhibition effects to Chilomonas paramaecium of the test substance.Propan-2-ol (Isopropyl alcohol) is both reagents used in the manufacture of Reaction mass of O-isopropyl ethylthiocarbamateand n-butanol and propan-2-ol.Therefore, Propan-2-ol (Isopropyl alcohol) need to be considered in the assessment of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol  and propan-2-ol .

Description of key information

A 16h Toxicity threshold concentration of 1050 mg/l has been determined for growth inhibition effects to Pseudomonas putida of the test substance. Propan-2-ol (Isopropyl alcohol) is both reagents used in the manufacture of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol. Therefore, Propan-2-ol (Isopropyl alcohol) need to be considered in the assessment of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol  and propan-2-ol .
 
A 48h Toxicity threshold concentration of 104 mg/l has been determined for growth inhibition effects to Chilomonas paramaecium of the test substance. Propan-2-ol (Isopropyl alcohol) is both reagents used in the manufacture of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol. Therefore, Propan-2-ol (Isopropyl alcohol) need to be considered in the assessment of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol  and propan-2-ol .

Key value for chemical safety assessment

EC50 for microorganisms:
520 mg/L
EC10 or NOEC for microorganisms:
104 mg/L

Additional information

A 16h Toxicity threshold concentration of 1050 mg/l has been determined for growth inhibition effects to Pseudomonas putida of the test substance. Propan-2-ol (Isopropyl alcohol) is both reagents used in the manufacture of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol. Therefore, Propan-2-ol (Isopropyl alcohol) need to be considered in the assessment of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol  and propan-2-ol .

 

A 48h Toxicity threshold concentration of 104 mg/l has been determined for growth inhibition effects to Chilomonas paramaecium of the test substance. Propan-2-ol (Isopropyl alcohol) is both reagents used in the manufacture of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol and propan-2-ol. Therefore, Propan-2-ol (Isopropyl alcohol) need to be considered in the assessment of Reaction mass of O-isopropyl ethylthiocarbamate and n-butanol  and propan-2-ol .