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EC number: 200-661-7
CAS number: 67-63-0
According to the ECHA Guidance on information requirements and chemical
safety assessment, Chapter R.7b: Endpoint specific guidance section
R.220.127.116.11 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.
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
Toxicity Threshold = 3425 mg/L; 20 hours Uronema parduzci (Bringmann,
Toxicity Threshold = 104 mg/L; 48 hours Chilomonas paramaecium (Bringmann
et. al., 1980)
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.18.104.22.168
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
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.22.214.171.124 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.
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