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Toxicity to microorganisms

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Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
13 Oct - 17 Nov 1978
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Inconsistent results
Qualifier:
no guideline followed
GLP compliance:
no
Analytical monitoring:
not specified
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Sample was weighed in clean, dried and tared one milliliter beakers and then introduced into the CAD flask or BOD dilution bottle.
Test organisms (species):
other: "biological seed"
Test type:
not specified
Water media type:
not specified
Limit test:
no
Remarks on exposure duration:
no data
Reference substance (positive control):
no
Details on results:
No results are given in the study report. It was considered to be invalid, since the measured oxygen demand was higher than the theoretical oxygen demand.
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
other information
Study period:
not reported
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
no guideline followed
Principles of method if other than guideline:
Bacillus subtilis was tested with different substances and the optical densitiy measured after 1 h to calculate the inhibition.
GLP compliance:
no
Analytical monitoring:
not specified
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Inoculum was placed into plastic tubes or Erlenmeyer flasks which already contained the test substance
- Controls: 2 with solvent, 2 with distilled water
- Chemical name of vehicle: Ethanol, dimethylsulfoxide, sodium hydroxide or distilled water
Test organisms (species):
Bacillus subtilis
Details on inoculum:
- Pretreatment: Plated with tryptose blood agar base (33 g/L) were inoculated from single colonies of Bacillus subtilis and incubated for 7 h. Afterwards the cells were supended and diluted at different low titers into three flasks containing the MCV medium and shaken at room temperature overnight. If the cultures had not grown overnight beyond an optical density at 600 nm (OD600) of 1.0 it was not used. The cultures reaching 1.0 were utilised for the test.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
60 min
pH:
7.2, adjusted with KOH
Details on test conditions:
TEST SYSTEM
- Test vessel: Plastic tubes or Erlenmeyer flasks
- Aeration: Tubes were aerated.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: MCV medium contains 1.4% K2HPO4 , 0.6% KH2PO4, 0.2% MgSO4 * 7 H2O, 0.1% sodium citrate, 25 µg/mL L-tryptophan, 10 µg/mL L-methionine and 1 % vitamin-free casein hydrolysate

OTHER TEST CONDITIONS
- Adjustment of pH: with KOH to 7.2

EFFECT PARAMETERS MEASURED: optical density at 600 nm after 60 min in the plastic tubes and after 15 or 20 min in the Erlenmeyer flasks

Reference substance (positive control):
no
Duration:
60 min
Dose descriptor:
EC50
Effect conc.:
1.8 mmol/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Endpoint:
toxicity to microorganisms, other
Remarks:
Pseudomonas putida, respiration inhibition, 18 h
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Mar - 16 Mar 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given (comparable to guideline)
Qualifier:
equivalent or similar to guideline
Guideline:
other: ISO 10712
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution containing 680 mg prod./L and an oversaturated solution containing 1360 mg/L were prepared using sterile Milli-Q water which was mixed with an magnetic stirrer for 10 min. The pH was adjusted to 6.8 - 7.2 with NaOH.
- Controls: 10 with inoculum without test substance (series B) to determine turbidity and for each test concentration 1 control without inoculum to determine occuring colouration
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: obtained from RIVM, Bilthoven, Netherlands
- Method of cultivation: kept in agar plant tubes with a nutrient stock solution, incubated at 25 °C
- Preparation of inoculum for exposure: 7 days old stock cultures were inoculated in a fluid nutrient medium in Erlenmeyer flasks for 16 - 20 h at 25 °C. Subsequently the extinction of the monochromatic radiation was measured at 436 nm in a 10 mm layer of the bacterial suspension by photoelectric measurement. On basis of the measured values the turbidity was adjusted with sterile saline to the value of the Formazin standard suspension (TU/F/436 nm = 10) with which the spectrophotometer was calibrated.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
18 h
pH:
6.8 - 7.2, adjusted with NaOH
Nominal and measured concentrations:
nominal: 0.266, 0.531, 1.063, 2.125, 4.25, 8.50, 17.00, 34.00, 68.00, 136.00, 272.00, 544.00, 1088.00 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks stoppered with aluminum caps containing 100 mL test medium
- No. of vessels per concentration (replicates): 3 + 1 control (please refer to "details of test solution" above)
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile Milli-Q water (Milipore Corp., Bedford, USA)

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to 6.8 - 7.2 with NaOH solution

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : turbidity after 16 -20 h
Reference substance (positive control):
yes
Remarks:
Methanol
Duration:
18 h
Dose descriptor:
EC10
Effect conc.:
912 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mean extinction value
Results with reference substance (positive control):
- Results with reference substance valid? yes
Reported statistics and error estimates:
The toxicity threshold (TT) is determined graphically. The mean extinction values of each dilution step were plotted against the logarithm of the mean values of the test substance concentration.

Table 1: Extinction of the test substances solutions after 16 - 20 h

Concentration

[mg prod./L]

Extinction at 436 nm

Replicate I

Replicate II

Replicate III

Arithm. mean of replicates

Replicate without inoculum

0.226

0.490

0.500

0.470

0.487

0.000

0.531

0.471

0.480

0.469

0.473

0.000

1.063

0.463

0.460

0.456

0.460

0.000

2.125

0.452

0.450

0.460

0.454

0.000

4.250

0.458

0.430

0.466

0.451

0.000

8.500

0.447

0.450

0.459

0.452

0.000

17.000

0.469

0.434

0.463

0.455

0.000

34.000

0.458

0.450

0.470

0.459

0.000

68.000

0.525

0.512

0.497

0.511

0.000

136.000

0.588

0.580

0.570

0.579

0.000

272.000

0.710

0.740

0.745

0.732

0.000

544.000

0.985

0.979

0.990

0.985

0.000

1088.000

0.198

0.212

0.220

0.210

0.000

Table 2: Extinction after 16 - 20 h in the reference substance and the blank control

Concentration

[mg prod./L]

Extinction at 436 nm

Reference substance

Blank controls

 4937.500

0.488

0.449

 9875.000

0.440

0.440

 19750.00

0.415

0.452

 39500.00

0.270

0.459

 79000.00

0.155

0.458

 

 

 

 

0.449

0.433

0.439

0.440

0.438

Arithmetic mean

0.446

With respect to the historical test with the reference substance methanol, it can be concluded that the test conditions were optimal and the results are valid.

Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
other information
Study period:
not reported
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only short abstract available
Qualifier:
no guideline followed
Principles of method if other than guideline:
Six different bacteria were anaerobically incubated and the turbitidy measured to determine inhibition on growth.
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution was prepared by dissolving measured quantities of the test substance in absolute ethanol. First a 250 mM stock solution was prepared and then further diluted with ethanol to 200, 175, 150, 125, 100, 75, 50, 25, 10, 5 and 2.5 mM.
- Controls: Since the test compund produces culture turbidity, a dilution series with medium and test compound without inoculum were prepared.
Test organisms (species):
other: Bifidobacterium bifidum type a, Bifidobacterium bifidum type b, Bifidobacterium infantis sp infantis type a, Bifidobacterium infantis sp infantis type b, Escherichia coli, Salmonella typhimurium
Details on inoculum:
- Preparation of inoculum for exposure: The PYG medium was dispensed into a 500 mL screw-capped bottle and sterilised at 121 °C for 15 min. While cooling, 3.0 mL were delivered into metal capped culture tubes which were inoculated with 0.03 mL of the substance stock solutions (2.5 - 20 mM). The culture tubes were placed in an anaerobic cabinet and maintained at 37°C over night. Oxygen free gas (20% nitrogen in hydrogen) was supplied.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
29 h
Test temperature:
37 °C
pH:
4.5 - 6.6, the pH was measured in the highest and lowest substance concentrations.
Nominal and measured concentrations:
Nominal: 200, 175, 150, 125, 100, 75, 50, 25, 10, 5 and 2.5 mM
Details on test conditions:
TEST SYSTEM
- Test vessel: culture tubes
- No. of vessels per control (replicates): one series (2.5 - 200 mM test substance) with test substance without inoculum to determine turbidity

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The PYG media (Petone-yeast extract glucose) with 1% glucose, was prepared and dispersed anaerobically and sterilised. Anaerobic culture counts were performed on PYG-agar roll tubes.

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: turbidity after 29 h
Reference substance (positive control):
no
Duration:
29 h
Dose descriptor:
EC50
Effect conc.:
80 other: nmol
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: B. bifidum type a
Duration:
29 h
Dose descriptor:
EC50
Effect conc.:
15 other: nmol
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: B. infantis sp infantis type a
Duration:
29 h
Dose descriptor:
EC50
Effect conc.:
2.5 other: nmol
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: B. infantis sp infantis type b
Duration:
29 h
Dose descriptor:
EC50
Effect conc.:
0 other: nmol
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: S. typhimurium
Details on results:
No 50 % inhibition on bacterial growth was achieved on E. coli and B. bifidum type b.

Description of key information

EC10 (18 h) = 912 mg/L (ISO 10712)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
912 mg/L

Additional information

One reliable study, investigating the toxicity of the substance to Pseudomonas putida, is available for octanoic acid. This cell multiplication inhibition test (1988) was conducted similarly to ISO 10712. The test organism was exposed to a wide range of nominal concentrations (0.266 - 1088.00 mg/L). The study determined an EC10 (18h) of 912 mg/L (nominal).

A further study report (1978) is available with biological seed as the test organisms, but was rated unreliable (RL3) due to inconsistent results and poor reporting.

Supportive information is withdrawn from short abstracts of two publications. In the first study (Freese, 1979) conducted with Bacillus subtilis, a EC50 of 0.25 mmol/L was obtained after 60 min. In the second study (Powell and May, 1981) with six different bacterial strains incubated anaerobically, different sensitivity of the various organisms to decanoic acid were observed. However, due to limited documentation and consequently limited information, these two literature studies were rated as not assignable.