4-(α,α-dimethylbenzyl)phenol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

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 °C and shipped frozen, on ice to Reimer Analytical & Associates Inc. for p-Cumylphenol analysis.

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 (species):

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

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 ± 2 °C

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 °C under continuous light (3870 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 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 but was not adjusted. The test was conducted in a controlled environment chamber at 23 ± 2 °C under continuous light with an intensity at the plate surface of 3870 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 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).

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

Key result

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

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

Key result

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:

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 NOECs at 48 and 72 hours were 0.5 and 0.9 mg/L respectively. The NOECs 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.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72 hour EC50, with respect to growth rate, was 1.4 mg/L. The NOEC was 0.9 mg/L.

Executive summary:

A reliable algal growth inhibition test to OECD Guideline 201 was performed wth R. subcapitata at concentrations of p-Cumylphenol ranging from 0.3 to 17.5 mg/L for 72 hours under static conditions with continuous illumination.

Under the conditions of this study, the 48 and 72 hour EC50 values, with respect to algal growth rate, were 1.0 and 1.4 mg/L, respectively. The 48 and 72 hour NOEC values were determined to be 0.5 and 0.9 mg/L.

Description of key information

In a freshwater green algae study, the 72 hour EC50 and NOEC of p-Cumylphenol were 1.4 and 0.9 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.4 mg/L

EC10 or NOEC for freshwater algae:

0.9 mg/L