Phenol, 4-methyl-, reaction products with dicyclopentadiene and isobutylene

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 February - 22 March 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed according to DIN guideline and according to GLP principles. Due to the water insolubility of the test substance, the guideline method was modified. Although the DIN method is not the preferred method under REACH, the results can be used for the calculation of the PNEC for microorganisms when no other test results are available (ECHA Guidance on information requirements and chemical safety assessment, chapter R.7b, section R.7.8.17.1)

Qualifier:

according to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

Deviations:

yes

Remarks:

Due to the water insolubility of the test substance, the test was modified. For details see below.

GLP compliance:

yes

Analytical monitoring:

not required

Vehicle:

yes

Details on test solutions:

Test 1: aqueous test suspension:
Test substance and Tween 80 (0.1 g/L) were mixed in ultrapure water for 15 min in an ultrasonic bath. Then the suspension was mixed overnight at 50°C using a magnetic stirrer, after which the test solution was filtered and the filtrate was used in the test and diluted.

Test 2: ethanol-based test suspension:
A suspension of test substance and ethanol was prepared and mixed in ultrapure water for 15 min in an ultrasonic bath. Then the suspension was mixed overnight at 50°C using a magnetic stirrer, after which it was used in the test and diluted. The highest test concentration contained some undissolved test substance.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

Stockculture: DSM-No. 50026, lyophilisate from 29 Jan 1991.

Stock cultures were inoculated weekly in stock agar. These cultures (max. 7 days old) were used in the test. To adapt the cultures to fluid media, three 250 mL Erlenmeyer flasks were inoculated with bacteria in 100 mL of nutritional preculture solution. After 18+/-2 hours at 21°C, opacity was determined and set at FNU 100 using preculture medium. This suspension was used as the inoculum for the test-preculture.
Then 90 mL of preculture medium was inoculated with 10 mL of bacterial suspension (FNU 100). This bacterial suspension (FNU 10) was incubated for 7h at 21°C and subsequently the opacity was set at 50 FNU. This suspension was used as the inoculum in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

17 h

Post exposure observation period:

not applicable

Hardness:

no data

Test temperature:

21 +/- 1°C

pH:

Test 1: pH of undiluted filtrate was 5.79
Test 2: pH of highest test concentration solution was 6.52

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Test 1: 10000, 6250, 3125, 1562.5 and 781.3 mg/L
Test 2: 150.9, 94.3, 47.2, 23.6 and 11.8 mg/L.

Details on test conditions:

For each test concentration, 3 test cultures and one test substance control (i.e. no inoculum) were used. In 250 mL erlenmeyer flasks, 100 mL of test solution + bacterial suspension (FNU 50; 10 ml) were added. Controls (i.e. without test substance but with Tween 80 or ethanol) were also tested.
All test cultures were incubated for 17h at 21°C at a rotation shaker.

Reference substance (positive control):

no

Duration:

17 h

Dose descriptor:

NOEC

Effect conc.:

>= 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

filtrate of aqueous suspension

Basis for effect:

growth inhibition

Duration:

17 h

Dose descriptor:

NOEC

Effect conc.:

>= 150.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

solved in ethanol and diluted in water

Basis for effect:

growth inhibition

Details on results:

Test 1: Growth inhibition at 10000, 6250, 3125, 152.5 and 781.3 mg/L: 4.4%, -6.8%, -4.6%, -3.4% and -5.9%, respectively.
Test 2: Growth inhibition at 150.9, 54.3, 7.2, 23.6 and 11.8 mg/L: -30.8, -8.5, -6.9, -6.9 and -4.3%, respectively.

Validity criteria fulfilled:

yes

Remarks:

The opacity of the control cultures at the start of the test (i.e. 5 FNU) increased at least 100 times during the test.

Conclusions:

The test substance does not induce growth inhibition of Pneudomonas putida when tested as filtrated test solutions up to a concentration of 10000 mg/L or when tested after being dissolved in ethanol and then diluted in water up to a concentration of 150.9 mg/L. The measured pH values of the highest test concentrations, pH 5.79 and pH 6.52, respectively, did not affect the cell multiplication.

Description of key information

One GLP study is presented.

The IBR study was conducted according to DIN 38412 (part 8) and exposed Pseudomonas putida for 17h in a static test to nominal concentrations up to 10000 mg/L of Lowinox CPL (filtrate of an aqueous suspension) and up to 150.9 mg/L (first dissolved in ethanol and then diluted in water). The EC50 was >150.9 mg/L and the NOEC was 150.9 mg/L, based on the lowest nominal concentrations.

Key value for chemical safety assessment

EC50 for microorganisms:

150.9 mg/L

EC10 or NOEC for microorganisms:

150.9 mg/L

Additional information

Due to the low water solubility, the test substance solutions in the IBR study were prepared in two ways. The first stock solution was prepared by dissolving the test substance in water in the presence of Tween 80, ultrasonicating for 15 min and stirring at 50°C overnight. The filtrate of this suspension was used as the highest test concentration (10000 mg/L) and for further dilutions.

The second stock solution was prepared by dissolving the test substance in ethanol and subsequent mixing with water, ultrasonicating for 15 min and overnight stirring at 50°C. This suspension with visible undissolved material was used as the highest test concentration (150.9 mg/L) and for further dilutions.

The bacteria did not show any effects on growth. It can be concluded that the substance does not cause growth effects in bacteria at concentrations which are higher than its water solubility.