4-(α,α-dimethylbenzyl)phenol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted per OECD Test Guideline 201 following GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-(α,α-dimethylbenzyl)phenol

EC Number:

209-968-0

EC Name:

4-(α,α-dimethylbenzyl)phenol

Cas Number:

599-64-4

Molecular formula:

C15H16O

IUPAC Name:

4-(2-phenylpropan-2-yl)phenol

Constituent 2

Reference substance name:

290-968-0

IUPAC Name:

290-968-0

Details on test material:

p-Cumylphenol (PCP, CAS #599-64-4); Purity: 98-100%.

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Details on test solutions:

The test solutions were prepared from a 20 mg/L stock solution of p-Cumylphenol. A 100-mL volume was dispensed to a 250-mL plastic container for the highest test concentration (20 mg/L nominal test concentration). A second 100-mL volume of the stock solution was dispensed to another 250-mL container and serially diluted with 100-mL volumes of dilution water to obtain the remaining test concentrations (0.3, 0.6, 1.3, 2.5, 5.0, and 10 mg/L nominal test concentrations). The excess 100-mL volume was discarded.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23 ± 2 degrees C

pH:

Measured in one control and on highest concentration replicate at test initiation and termination, but not adjusted.

Nominal and measured concentrations:

Nominal concentrations: 0.3, 0.6, 1.3, 2.5, 5.0, 10 and 20 mg/L
Measured concentrations: 0.3, 0.5, 0.9, 2.0, 4.6, 8.2 and 17.5 mg/L

Details on test conditions:

The test was initiated with exponentially growing cells from in-house cultures maintained at 23 ± 2 degrees C under continuous light (3,870 lux). The culture had a weekly cell yield within 10% of historical levels. The cultures were grown under axenic conditions in 2-L flasks containing 1 L of artificial media, aerated with filtered sterile air.

Growth inhibition was assessed as the decrease in cell numbers relative to controls. Cell numbers were obtained from absorbance measurements at 430 nm calibrated against particle and cell counts at test termination.

The test solutions, prepared as described above, were spiked with 1 mL of a concentrated nutrient solution and then inoculated (1 mL) to give an initial cell density of ~10,800 cells/mL. The R. subcapitata inoculum was taken from an exponentially growing culture, washed twice with a sodium bicarbonate solution, and the cell number adjusted to give the desired initial cell density in the 100-mL test volume. Cell counts were done with a Coulter Counter Model ZBI® particle counter equipped with a 100-µm aperture. The solutions were osmotically adjusted prior to counting. At test termination, 0.5 mL was removed from six wells (equal volumes from each well) of the control, 2.0 mg/L, and 17.5 mg/L concentrations from each replicate plate. Each solution was counted until successive counts were within 10% of each other.
The test was conducted in 96-well microplates (Costar®, Corning Incorporated). The well volume was 300 µL. The test volume was 150 µL. There were three sets of controls on each plate. The pH was measured in one of the control wells and one well of the highest concentration at test initiation and termination. The test was conducted in a controlled environment chamber at 23 ± 2 degrees C under continuous light with an intensity at the plate surface of 3,870 lux provided by cool white fluorescent lights. The plates were read and rotated to a different position under the light bank each day. Absorbance measurements at 430 nm were taken at test initiation and at 24, 48, and 72 hours.
Particle counts were made on the controls, the 2.0 mg/L, and 17.5 mg/L test solutions at test termination. The counts were converted to cell densities with the factor 93.6 (dilution factor of 80 times 1.17, an empirical constant relating instrument counts to cell numbers). The cell densities were then regressed against the absorbance readings (correlation coefficient (r2) of 0.96).
The absorbance readings of the six replicate wells per concentration per plate were averaged and the averages converted into cell densities. The three columns of six control wells on each plate were averaged into a single value for derivation of the toxicity values. The toxicity values for the inhibiting effects of p-Cumylphenol on growth of R. subcapitata were derived from the areas under the growth curves.
The percent inhibition of cell growth at each test substance concentration (IA) was calculated as the difference between the area under the control curve (Ac) and the area under the growth curve of each test substance concentration (At; IA = ( Ac - At ) / Ac * 100). The results from each replicate plate were treated separately for derivation of the toxicity values (three replicates). In cases where the average absorbance value of a concentration resulted in a calculated cell density less than the inoculum, the value was set equal to the inoculum. This resulted when the differences in absorbance measurements amongst the controls and concentrations were within the expected variation of the microplate reader (±0.005 absorbance units).

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.9 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Two sets of samples were collected for analysis of p-Cumylphenol. The samples of the test solutions and controls were collected and incubated in 50-mL polypropylene centrifuge tubes. They were frozen and stored in darkness at –19 +/- 2 degrees C and shipped frozen, on ice to Reimer Analytical & Associates Inc. for p-Cumylphenol analysis. Measured initial concentrations of p-Cumylphenol were, on average, 14% lower than nominal concentrations. There was only one measured concentration that was greater than the nominal concentration (0.4 mg/L compared to the nominal value of 0.3 mg/L). Final measured concentrations of p-Cumylphenol were, on average, 28% lower than nominal concentrations. The final measured concentrations were lower than initial measured concentrations. The test concentrations inhibiting growth by 50% relative to controls at 48 and 72 hours were 1.0 and 1.4 mg/L respectively. The NOEC’s at 48 and 72 hours were 0.5 and 0.9 mg/L respectively. The NOEC’s were approximated from the concentration-effect relationship for p-Cumylphenol with the assumption that a 20% change was considered significant from a biological perspective. Negative values for growth inhibition can result if the cell density of the concentration exceeds that of the control. For example, negative values were obtained for the 0.3 mg/L concentration at 48 and 72 hours. An increase in cell density relative to controls was not considered an adverse effect.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the 48 and 72 hour EC50, in respect to growth rate, of p-Cumylphenol exposure on algae were 1.0 and 1.4 mg/L, respectively. The 48 and 72 hour NOEC were 0.5 and 0.9 mg/L.