p-tert-butylstyrene

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

Study period:

From May 09, 2017 to May 12, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

see ''Principles of method if other than guideline''

Principles of method if other than guideline:

1. The protocol states that the test solution temperature will be maintained at 24 ± 2 ºC by controlling the air temperature within the environmental chamber. At 24 hours of exposure, the temperature was out of range by -1 ºC on one of the test shelves used for the exposure. The temperature was only slightly out of range for only one day of the exposure and all validity criteria were met for this study; therefore, this deviation did not have an adverse impact on the results or interpretation of the study.

2. Protocol Amendment 1 states that two samples will be taken from each vial, and two counts made on each sample. During the exposure, additional cell counts were made for some replicates when a cell count was unusually high or low relative to others, and the counts were averaged. The additional cell counts were averaged with the initial counts, thereby provided a more accurate cell density determination for the above replicates. Therefore, this deviation did not have an adverse impact on the results or interpretation of the study.

GLP compliance:

yes

Specific details on test material used for the study:

Lot No.: 122016TBSSV
Purity: 95.9% (analyzed on 20 December 2016); 96.0% (reanalyzed on 10 May 2017)

Analytical monitoring:

yes

Details on sampling:

ALGAL DENSITY

At each 24-hour interval, algal density was determined in three replicate vials (two for each treatment level and one for the negative control) and in six for the solvent control using a Beckman Coulter Multisizer 4e Particle Analyzer. Two samples were removed from each test vessel, and two counts were performed on each sample. The two counts determined for each sample were averaged, and the two average cell counts determined for each replicate were averaged. The reported cell density was the value determined from the final average of the four cell counts for each replicate.

Replication:
Fourteen replicates per treatment level (5 treatmet levels) and the negative control and twenty-three replicates for the solvent control

Additional Replication:
Replicate vials of the 0.25 mg/L (nominal) treatment level, not inoculated with algae were analyzed at 24 and 72 hours of exposure, respectively.
Results of the solutions without algae were compared with the results for the corresponding 0.25 mg/L solutions containing algae to assess the impact of algal biomass on test substance concentration.


CHEMICAL ANALYSIS

Sampling Intervals and Solution Composition:
0 Hour: 1 replicate of the solvent control and 1 replicate of the negative control and treatment levels
24 Hour: 1 replicate of the solvent control and 1 replicate of the negative control and treatment levels; additional replicate for solution without algae
72 Hour: 1 replicate of the solvent control and 1 replicate of the negative control and treatment levels; additional replicate for no algae solution

Number of Samples Taken from Each Solution: One sample at each interval
Sampling Location: Approximate midpoint from the surface, bottom, and sides of the vessel
Quality Control (QC) Samples: 3 samples per sampling interval, prepared in dilution water at nominal concentrations approximating the test concentration range

Vehicle:

yes

Remarks:

DMF

Details on test solutions:

Primary Stock Solution Preparation
Primary Stock Solution Concentration: 10 mg/mL
Stock Solution Vessel and Volume: 25-mL volumetric flask
Amount of Test Substance Added: 0.2608 g (0.2501 g as active ingredient)
Diluent: DMF

The primary and secondary stock solutions were mixed by shaking and inversion of the volumetric flask and were observed to be clear and colorless with no visible undissolved test substance following preparation.

Exposure solutions were individually prepared in the replicate test vessels for each concentration. The exposure solutions were observed to be clear and colorless with no visible undissolved test substance following preparation. Negative control and solvent control vessels were maintained under the same conditions as the treatment level solutions. Observations of the exposure solutions with respect to precipitation, clarity, and material adhered to the sides of the vessels were recorded daily during the exposure period.

Test organisms (species):

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

Details on test organisms:

Strain: 1648
Source: UTEX The Culture Collection of Algae at the University of Texas, Austin, Texas (maintained in stock culture at Smithers Viscient)
Age (Inoculum): Four days since previous transfer

Culture Conditions Prior to Testing
Growth Medium: Algal Assay Procedure (AAP) medium, prepared with sterile, deionized water
pH: 7.5 ± 0.1, initial pH adjusted with dilute hydrochloric acid or sodium hydroxide prior to use if necessary
Vessels: 250-mL glass flasks covered with stainless steel caps which permitted gas exchange (volume = 100 mL per vessel)
Photoperiod: None (continuous illumination)
Light Intensity: Photosynthetically-active radiation (PAR) of 60 to 80 μE/m2/S
Temperature: 23 ± 1 °C, controlled using an environmental chamber
Agitation: Continuous at a rate of 100 ± 10 rpm on an orbital shaker

Required Starting Cell Density: Approximately 10,000 cells/mL
Inoculum Culture Density: 1,788,000 cells/mL
Inoculum Volume Added: 0.375 mL per vessel, added aseptically
Initial (0-Hour) Cell Density: Approximately 10,000 cells/mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 to 22 °C

pH:

7.5 ± 0.1, initial pH adjusted with dilute hydrochloric acid (HCl) or sodium hydroxide (NaOH) prior to use if necessary

Conductivity:

Measured at exposure initiation using a YSI Model No. 3100 - 115 V conductivity meter

Nominal and measured concentrations:

Nominal Concentrations: negative control, solvent control, 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L
Geometric Mean Measured Concentrations: negative control, solvent control, 0.049, 0.10, 0.21, 0.44, and 0.55 mg/L
Co-Solvent Load: 0.10 mL/L

Details on test conditions:

Exposure System: closed system, static conditions
Temperature Control: environmental chamber set to maintain temperature at 24 ± 2 °C
Photoperiod: none (continuous)
Light Intensity: 60 to 80 μE/m2/S; Approximately 4440 to 6010 lux, measured at exposure initiation using a Fisher Scientific light meter
Agitation: continuous at a rate of 100 rpm
Control of Bias: random placement of exposure vessels and position reassignment daily, based on computer-generated random numbers

Water Quality
pH: measured at exposure initiation and termination using a Yellow Springs Instrument (YSI) pH100A pH meter and combination electrode
Conductivity: measured at exposure initiation using a YSI Model No. 3100 - 115 V conductivity meter
Temperature: continuously monitored with a Fisher Scientific minimum-maximum thermometer in a flask of water adjacent to the test flasks in the environmental chamber

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL:

Remarks:

0.38-0.50

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL:

Remarks:

0.48-0.54

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL:

Remarks:

0.45-0.56

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

CHEMICAL ANALYSIS

The measured concentrations at exposure initiation closely approximated the nominal concentrations. Measured concentrations were generally consistent throughout the exposure and maintained the expected concentration gradient with the exception of the 1.0 mg/L treatment level, which had a recovery of 30% of nominal concentration at 24 hours of exposure. The 72-hour measured concentration for this treatment level was similar to the concentration determined at 0-hour. The variability in measured concentrations at this treatment level is attributed to solubility since this exposure level closely approximated the functional water solubility limit of the p-tert butylstyrene under the testing conditions.

The analytical results of the 24- and 72-hour samples from the 0.25 mg/L nominal treatment with algae present were 0.23 and 0.19 mg/L, respectively. Analysis of the corresponding test solutions without algae present resulted in recoveries of 0.24 and 0.23 mg/L, respectively, and demonstrated that the presence of algae had no impact on the concentration of test substance in the exposure solution.

Analysis of eight of the nine QC samples resulted in measured concentrations which were consistent with the predetermined recovery range

BIOLOGICAL RESULTS

- During the 72-hour exposure, cells observed in the control and all the treatment levels tested were observed to be healthy and normal in appearance.
- No statistically significant difference was detected between the 72-hour negative control and solvent control data for growth rate. Treatment data for 72-hour growth rate were compared to the solvent control.
- No statistically significant difference was detected between the 72-hour negative control and solvent control data for yield. Treatment data for 72-hour yield were compared to the solvent control.

Results with reference substance (positive control):

Reference Substance: Zinc chloride (ZnCl2)
Duration: 96 h
EC50: 0.082 mg Zn/L, with 95% confidence intervals of 0.065 to 0.092 mg Zn/L (historical mean = 0.089 mg/L, March 2005 to present)
The result is within the expected range for Pseudokirchneriella subcapitata reported by Greene et. al. (1991);

Reported statistics and error estimates:

Prior to NOEC and LOEC determinations, the data were first checked for normality using Shapiro-Wilk’s Test (U.S. EPA, 2002) and for homogeneity of variance using Bartlett's Test (U.S. EPA, 2002). If the data passed the tests for homogeneity and normality, then a parametric statistical test, e.g., Dunnett’s Multiple Comparison Test (U.S. EPA, 2002) was used to determine the NOEC and LOEC. If the data did not pass the tests for homogeneity and normality, then the NOEC and LOEC were determined using an appropriate non-parametric statistical test, e.g., Dunn’s Test with Bonferroni-Holm’s Adjustment (U.S. EPA, 2002). All statistical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilk’s and Bartlett's Tests, where the 99% level of certainty was applied.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions growth rate and biomass growth were determined as follows, growth rate: NOEC: 0.44 mg/L; EC50: > 0.55 mg/L (meas.); biomass: NOEC: 0.44 mg/L; EC50: 0.50 mg/L (meas.).

Executive summary:

A study was conducted to determine the toxicity of the test substance to freshwater alga Pseudokirchneriella subcapitata according to OECD Guideline 201, in compliance with GLP. The study was conducted using 5 nominal concentrations of the test substance ranging from 0.063 to 1 mg/L, a co-solvent (dimethylformamide) and negative controls with an exposure period of 72 h. The definitive exposure of this study was conducted under static conditions in a closed exposure system to minimize the volatilization of the test substance. Due to the nature of the test substance, volatile organic analysis (VOA) vials with Teflon-lined screw caps were used. A study performed with the reference substance zinc chloride confirmed the sensitivity of the system. The measured concentrations at exposure initiation closely approximated the nominal concentrations. Measured concentrations were generally consistent throughout the exposure and maintained the expected concentration gradient. The results of this study were based on geometric mean measured concentrations of the test substance. Under the study conditions, the NOEC and EC50 of the test substance based on growth rate were determined to be 0.44 mg/L and >0.55 mg/L (Softcheck, 2019).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.44 mg/L

EC10 or NOEC for freshwater algae:

0.55 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance to freshwater alga Pseudokirchneriella subcapitata according to OECD Guideline 201, in compliance with GLP. The study was conducted using 5 nominal concentrations of the test substance ranging from 0.063 to 1 mg/L, a co-solvent (dimethylformamide) and negative controls with an exposure period of 72 h. The definitive exposure of this study was conducted under static conditions in a closed exposure system to minimize the volatilization of the test substance. Due to the nature of the test substance, volatile organic analysis (VOA) vials with Teflon-lined screw caps were used. A study performed with the reference substance zinc chloride confirmed the sensitivity of the system. The measured concentrations at exposure initiation closely approximated the nominal concentrations. Measured concentrations were generally consistent throughout the exposure and maintained the expected concentration gradient. The results of this study were based on geometric mean measured concentrations of the test substance. Under the study conditions, the NOEC and EC50 of the test substance based on growth rate were determined to be 0.44 mg/L and >0.55 mg/L (Softcheck, 2019).