Propan-2-ol

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

Remarks:

Cell proliferation inhibition

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without detailed documentation.

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:

other: mean extinction value

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

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.

Description of key information

The 16 hour Toxicity Threshold concentration of 1050 mg/L for Pseudomonas putida (Bringmann & Kühn, 1980) is selected as the key value.

Toxicity Threshold = 1050 mg/L; 16 hours, Pseudomonas putida (Bringmann & Kühn, 1980)

Toxicity Threshold = 1050 mg/L; 16 hours, Pseudomonas putida (Bringmann & Kühn, 1977)

Toxicity Threshold = 4930 mg/L; 72 hours, Entosiphon sulcatum (Bringmann, 1978)

Toxicity Threshold = 3425 mg/L; 20 hours Uronema parduzci (Bringmann, 1980)

Toxicity Threshold = 104 mg/L; 48 hours Chilomonas paramaecium (Bringmann et. al., 1980)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 050 mg/L

Additional information

The toxicity of iso-propyl alcohol (IPA) to Pseudomonas putida was assessed in a published non-guideline study that predates GLP requirements for excotoxicity studies (Bringmann & Kühn 1980). The study used a static freshwater system. Cell culture was maintained on nutrient agar slants and examined for purity periodically. The inoculum was prepared by growing the microorganism on nutrient agar plates for 24 hours. The cells were washed off from the medium and resuspended in a nutrient medium. By determining the extinction of the monochromatic radiation at 436 nm for a 10 mm layer of the bacterial suspension, the final turbidity value of the bacterial suspension was adjusted, by means of sterile saline, such that it corresponded to the extinction value of a Formazin standard suspension TE/F/436 nm = 10.

 

Four parallel dilution series in 300 mL Erlenmeyer flasks were prepared of test solution. Each dilution contains 1 part v/v of test solution in 2⁰ to 2¹⁴parts v/v mixture. The first flask in the series contained 160 mL of test solution at the start. Starting from this flask, the subsequent dilution steps were prepared at a constant dilution ratio by consistently mixing 80 mL of preliminary test dilution and 80 mL double distilled water. Consequently, each flask contained 80 mL of culture liquid at the start. Each flask of the three dilution series were 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. Both inoculated and non-inoculated flasks were incubated at 25ºC for 16 hours. After 16 hours, the extinction of the monochromatic radiation at 436 nm for a 10 mm layer of the bacterial suspension was determined.

 

The results were analyzed on a semi-logarithmic chart according to the following scheme. The calculated values (A), (A-3%), and (B) were located on the Y-axis (linear): the average (A) of all the absorbance of the non-inhibited and non-stimulated cultures (as long as those values were below a standard deviation of less than 3 %); and the average (B) of all the absorbance of the cultures showing the lowest toxic effect. The highest non-toxic concentration (a) and the lowest toxic concentration (b) were located on the X-axis.

 

The placing of (A-3 %) on the line between the coordinates (a, A) and (b, B) allowed the value (C) to be determined on the abscissa of the initial concentration at which growth inhibition was determined or Toxicity Threshold. The Bringmann & Kühn (1980) results indicate that Pseudomonas putida had a 16 hour Toxicity Threshold concentration of 1050 mg/L.

 

None of the five published studies presented documenting the toxicity of IPA to microorganisms were conducted with a mixed inoculum that would asses the functioning of the entire microbial community in a sewage treatment plant, rather the tests were based on single species systems. 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 sewage treatment plant microorganisms, results of the cell multiplication inhibition test with P. putida (Bringmann and Kühn 1980) can be used as the key value for chemical safety assessment.

 

The supporting data report higher Toxicity Thresholds for Entosiphon sulcatum (flagellated protozoa) (Bringmann, 1978) and Uronema parduzci (ciliated protozoa) (Bringmann, 1980) and a lower Toxicity Threshold for Chilomonas paramaecium (flagellated protozoa) (Bringmann et. al., 1980). According to the ECHA Guidance on information requirements and chemical safety assessment, Chapter R.7b: Endpoint specific guidance section R.7.8.16.1 Laboratory data on toxicity to STP microorganisms and its sources, no correlation exists between activated sludge and ciliated protozoa test results. The lower Toxicity Threshold is for flagellated protozoa and a contact time of 48 hours.  In general in accordance with the hydraulic retention time in a sewage treatment plant, short-term toxicity measurements in the order of hours are preferred. As these three protozoan results are equal to or greater than 20 hours contact time, these results should not be used as the key value for chemical safety assessment.