N-butyl phenyl ether

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:

May 16, 2016 to September 15, 2016

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:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test Material: Butyl phenyl ether
Chemical Name: Butoxybenzene
Synonyms: None
Lot/Reference/Batch Number: 06027KH
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the purity as 99.5% by gas liquid chromatography with structure confirmation by infrared spectroscopy (Rajzer, 2007).
Stability: Butyl phenyl ether, lot 22466, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under U.S. EPA OPPTS Guideline 830.6317 (Ferruzzi, 2016).

Analytical monitoring:

yes

Details on sampling:

Samples were collected from the bulk test solutions at test initiation and from pooled replicate samples at termination. Blank replicates were analyzed separately. To assess analytical method precision and solution homogeneity, three additional samples were collected at test initiation from the highest and lowest concentration bulk test solutions. To confirm butyl phenyl ether concentrations, the collected samples were analyzed using high performance liquid chromatography with diode array UV absorbance detection (HPLC/DAD). All test solutions were used within 24 hours of preparation; therefore, no stability assessment was required.

The bulk exposure solutions (AAP medium control, 0.1, 0.3, 1.0, 3.1 and 10 mg/L solutions) were sampled for analytical confirmation of test material. At test initiation (0 hours), single aliquots (~ 5 mL) of AAP medium control, 0.3, 1, and 3.1 mg/L bulk exposure solutions were collected using a glass pipette and transferred to 20-mL vials. At test initiation, four aliquots (~ 5 mL) of 0.1 and 10 mg/L bulk exposure solution were collected from the top, top middle, bottom middle, and bottom of its container to evaluate homogeneity.

The contents of algal test vessels were pooled at exposure termination (72 hours) to provide one composite sample of the control and of each test material exposure for analytical confirmation. Aliquots (0.3 - 0.5 mL) were collected using a Hamilton syringe and transferred to 2 mL glass vials. Pooled samples were vortex-mixed. Single aliquots (~1 mL) were collected from the single counting blank test vessels (containing test material but not inoculated with algae) using a Hamilton syringe and transferred to 2 mL glass vials.

Aliquots (0.500 mL) of sample were transferred to centrifuge tubes containing 0.500 mL of acetonitrile and centrifuged at 16,100 rcf for 4.0 minutes. Afterwards, ~ 0.300 mL aliquots were transferred to ultra high recovery autosampler vials for analysis by HPLC/DAD. The concentrations of butyl phenyl ether were quantified using external standard calibration, and reported concentrations were not corrected for purity of the test material.

Vehicle:

no

Details on test solutions:

Preparation of Test Solutions:
Due to the potential volatility of the test material, discovered during preliminary non-GLP work, the study was conducted in sealed test vessels. Test vessels were 125-mL glass serum bottles which were crimp-sealed with an aluminum seal containing a 20 mm PTFE silicone septum. Each test vessel contained 100 mL of the appropriate test solution. The test material was expected to be soluble in AAP at the concentrations selected for the study.
A primary stock (bulk solution; equal to the highest test concentration) was prepared via direct addition of the test material to AAP. Butyl phenyl ether (actual weight: 0.0200 g; no adjustment for purity) was added to 2 L of AAP to achieve a 10 mg butyl phenyl ether/L bulk solution. The solution was stirred on a magnetic stir plate utilizing a Teflon lined magnetic stir bar for 30 minutes. Following stirring, the primary stock appeared clear and colorless. The remaining bulk solutions were prepared as dilutions of the primary stock (bulk solution).

Remaining bulk solutions were clear and colorless. Portions of the prepared bulk solutions were poured into individual test vessels. Test solutions were utilized on the same day as preparation; thus, assessment of stability of the test solutions was not required. The dispersal of the test material in the surrounding medium (AAP) was considered to represent the most probable route of exposure in the environment.

Test organisms (species):

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

Details on test organisms:

Test Organism:
Pseudokirchneriella subcapitata from in-house cultures was initially obtained from the University of Texas at Austin Culture Collection (UTEX1648; lot # 060215A) in June 2015. This species is widely accepted and recommended for toxicity testing by the test guideline. Stock cultures of this organism were maintained axenically by periodic transfer into sterile medium. Typical culture conditions are listed below. Algae were cultured under continuous illumination of approximately 5,200 ± 520 lux at a temperature of 23 ± 2 ºC.
The algal inoculum for the test was prepared from a 3-day old stock culture. A Coulter Multisizer 3 (Beckman Coulter, Brea, California) was used to determine the cell density of the stock culture. This evaluation determined that a 0.550 mL aliquot of the culture was required to inoculate each test vessel at an initial cell density of approximately 7,500 cells/mL.

Organism: Pseudokirchneriella subcapitata
Temperature: 23 ± 2°C
Light (lux): 5200 ± 520 (test subculture)
Photoperiod: Continuous (24 hours light/day)
Medium: Algal assay procedure medium (AAP)
pH: Range: approximately 7.0–7.5
Culture Conditions: Axenic
Culture Volume: 50 mL
Culture Vessel: 250-mL Erlenmeyer flask
Culture Vessel Cap: Foam cap

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not measured

Test temperature:

Temperature Range: 22 – 23°C

pH:

pH Range: 7.7 -10.1

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Nominal and measured concentrations:

NOMINAL TEST CONCENTRATIONS: 0 (AAP medium control), 0.10, 0.30, 1.0 and 10 mg butyl phenyl ether/L
MEASURED TEST CONCENTRATIONS: < LLQ (AAP medium control), 0.0600, 0.183, 0.580, 1.65 and 5.55 mg butyl phenyl ether/L

Details on test conditions:

TEST SYSTEM
Test Vessels:
Test vessels were 125-mL glass serum bottles which were crimp sealed with an aluminum seal containing a 20 mm PTFE silicone septum. Each vessel contained 100 mL of test solution. Each vessel was uniquely labeled for identification purposes.

GROWTH MEDIUM
- Standard medium used: yes, Algal assay procedure medium (AAP)

TEST MEDIUM / WATER PARAMETERS
Culture and Test Medium:
The algae was cultured in freshwater algal nutrient medium (i.e., AAP medium), prepared with sterile deionized water and reagent grade chemicals. The water source for the deionized water system was municipal water produced by the City of Midland Water Treatment Plant. The base water used to prepare the medium was passed through a series of activated carbon, (two) deionization polymer (US Filter Mixed Bed, Type 1), and a final filtration unit, prior to collection and autoclaving in clean glass containers. Prior to treatment, the base water used to prepare the medium is analyzed periodically to verify that no contaminants are present at levels that may interfere with the test results.
Due to the exposure design (i.e., sealed test vessels), the AAP medium used in the study was prepared with 20X the standard amount of sodium bicarbonate (NaHCO3). The additional NaHCO3 provided a carbon source for the algae in lieu of atmospheric CO2 which was unavailable during the study.

OTHER TEST CONDITIONS
Exposure Scenario: 72-hour static
Temperature Range: 22 – 23°C
Light Intensity Range: 4470-5550 lux
pH Range: 7.7 -10.1
Shaking Rate: 100 rpm
Photoperiod: 24-hour light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Cell Density Determination and Morphological Observations:
Algal cell densities of the initial inoculum and test solutions were determined by electronic particle counting using a Coulter Multisizer 3 (Beckman Coulter, Brea, California) fitted with a 100-μm aperture tube. Total cell counts were determined at approximately 24, 48 and 72 hours. Cells were cumulatively counted at a lower threshold equivalent spherical diameter of approximately 2.6 μm to a higher threshold equivalent spherical diameter of approximately 8.7 μm. Two cell count readings were made per replicate and averaged. The readings for the blank replicates were used to correct for background in daily calculations. The adjusted cell counts were converted to cells x 10,000/mL (cell density) for statistical analysis and reporting.
In addition, morphological observations were done at test termination on a composited sample of the inoculated replicates at each test concentration. The cells were observed under a microscope (Olympus BH Microscope (Olympus Corporation, Tokyo, Japan); 20x or 40x objective lens; WF10x eyepiece; 1.25x Dual Observation Deck).

TEST CONCENTRATIONS
Selection of Test Concentrations:
A non-GLP 72-hour range-finding test was conducted from 02 to 05 May 2016. Test solutions were prepared as described below. Two replicate vessels per test level (6 for control) were inoculated with a predetermined aliquot of algal inoculum to achieve 7,500 cells/mL and exposed to nominal concentrations of 0 (AAP control), 0.5, 5.0, and 50 mg butyl phenyl ether/L. An uninoculated replicate (counting blank) was prepared at each test level and control.
Cell counts were taken after approximately 72 hours of exposure. Cell density was approximately 99 % inhibited at the 5.0 and 50 mg butyl phenyl ether/L test levels following 72 hours of exposure. No inhibition was observed at the 0.5 mg butyl phenyl ether/L test level. Based on information from the range-finding test, the following nominal test concentrations were set for the definitive study: 0 (AAP control), 0.10, 0.30, 1.0, 3.1, and 10 mg butyl phenyl ether/L.

Definitive Test:
The definitive test was conducted under static conditions for 72 hours from 23 to 26 May 2016. Three replicate test vessels were prepared per test level. Six replicates were prepared for the control AAP media. Each replicate contained 100 mL of the appropriate test solution and was inoculated with approximately 7,500 cells/mL. An additional replicate at each test and control level was prepared but not inoculated with algae to serve as a counting blank. These blanks were used to correct the daily counts for the potential interference of the test material and to monitor pH without the algal biomass. At test initiation and following sampling for cell densities at 24 and 48 hours, the replicate test vessels were placed in a walk-in environmental chamber (Lab-Line Environmental Chamber, Lab-Line Inc., Melrose, Illinois) on a shaker table (set at approximately 100 rpm) according to a computer-generated randomization. The target test temperature was 23 ± 2 ºC. The photoperiod was set at 24 hours of continuous light with a target light intensity of 5,200 ± 780 lux.
The pH was measured from bulk test solutions at 0 hours and from blanks and pooled replicate samples of each test level and AAP control at 72 hours. Pooled samples were prepared by withdrawing and combining approximately 2.5-mL volumes from each inoculated replicate at each test level and AAP control. Temperature was continuously monitored with a minimum/maximum thermometer placed in a representative vessel which was incubated adjacent to the test material exposures. At test initiation, light intensity was measured at each position where inoculated replicate vessels were placed during the exposure.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.67 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(Yield)

Remarks on result:

other: 95% CI = 1.56-1.79 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.183 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(Yield)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.84 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI = 2.26-3.56 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.183 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analytical Results:
At test initiation, measured butyl phenyl ether concentrations of the bulk test solutions ranged from 58.7 to 66.7% of the nominal test concentrations. At 72 hours, the analysis of pooled replicate test solutions (with algae) yielded butyl phenyl ether concentrations ranging from 44.5 to 63.0% of nominal concentrations. Analysis of blank solutions (without algae) conducted at 72 hours revealed butyl phenyl ether concentrations ranging from 51.0 to 79% of nominal concentrations. Mean measured concentrations of the bulk test solutions at 0 hours and pooled replicates solutions (with algae) at 72 hours were 0.0600, 0.183, 0.580, 1.65 and 5.55 mg butyl phenyl ether/L.
Similarly, the mean measured concentrations of the bulk test solutions at 0 hours and the blank solutions (without algae) at 72 hours were 0.0646, 0.207, 0.625, 1.75 and 5.90 butyl phenyl ether/L. These results indicate that the presence of algae had no substantial affect on test material exposure concentrations in the test vessels. None of the analyses of the media control exhibited a concentration exceeding the lower limit of quantitation (LLQ) equivalent to 0.05 mg butyl phenyl ether/L.
Mean measured concentrations, calculated from measured concentrations of the bulk test solutions (0 hrs.) and pooled replicate test solutions containing algae (72 hrs.), were used in reporting the study endpoint values.

Test Conditions:
Temperature during the exposure period ranged from 22 to 23ºC. Light intensity ranged from 4470 to 5550 lux. The pH was 7.7 at test initiation. At test termination, the pH ranged from 8.2 to 10.1 in replicates with algae. The pH of replicates without algae was 7.9. The pH increase in replicates containing algae is most likely due to atmospheric CO2 being unavailable to the algal cells during the exposure (i.e., sealed test vessels).

Biological Results:
All biological results are expressed in terms of mean measured concentrations of butyl phenyl ether.

Yield:
All cell yield data were normally distributed and homogeneous (Shapiro-Wilk Test, p > 0.01 and Levene’s Test, p > 0.01).
Mean yields at 72 hours were 145.9, 135.7, 141.2, 125.4, 72.17 and 0.4927 (x10^4) cells/mL for the AAP control, 0.0600, 0.183, 0.580, 1.65 and 5.55 mg butyl phenyl either//L test levels, respectively. At 72 hours, the mean inhibition response
relative to the control ranged from 3 to 100 % inhibition of yield. At 72 hours, yields at the 0.580, 1.65 and 5.55 mg butyl phenyl ether/L test levels were significantly different from the AAP control. Thus, the 72-hour NOEC for yield is
reported as 0.183 mg butyl phenyl ether/L. The calculated EyC50 (95% confidence intervals) is 1.67 (1.56-1.79) mg butyl phenyl ether/L.

Growth Rate:
Mean specific growth rate data were normally distributed (Shapiro-Wilk Test, p > 0.01) but were not homogeneous (Levene’s Test, p < 0.01).
Mean specific growth rates at 72 hours were 1.758, 1.734, 1.747, 1.708, 1.525 and 0.1328 (dayE-1) for the control, 0.0600, 0.183, 0.580, 1.65 and 5.55 mg butyl phenyl ether/L test levels, respectively. At 72 hours, mean inhibition response relative to the control ranged from 1 to 92% of the mean specific growth rate. Mean specific growth rates for 0.0600, 0.580, 1.65 and 5.55 mg butyl phenyl ether/L at 72 hours were statistically different from the control. The 0.183 mg butyl phenyl ether/L test level was not statistically different from the control at 72 hours and based on biological interpretation will be reported as the 72-hour NOEC for specific growth rate. The 0.0600 and 0.183 mg butyl phenyl ether/L test levels both exhibited one percent inhibition relative to the control. Thus, the growth rate reduction associated with the 0.0600 mg/L test level was considered as biologically irrelevant. The calculated ErC50 is 2.84 (2.26-3.56) mg butyl phenyl ether/L.

Morphological Observations:
At test termination, microscopic evaluation of algal cells at the 0.0600, 0.183, 0.580 and 1.65 mg butyl phenyl ether/L test levels and AAP control revealed no abnormal appearances. No algal cells were present at the 5.55 mg butyl phenyl ether/L test level.

Acceptance Criteria:
The following acceptance criteria set forth in the OECD Guideline 201 (2006) were met:
1) The cell density in the control cultures increased exponentially by a factor of at least 16 within 72 hours. In this study, the 72-hour mean cell density increased by approximately 196-fold, thereby exceeding the minimal criterion.
2) The coefficient of variation average for specific growth rates throughout the exposure (days 0 – 3) in replicate control cultures was 0.965%, which is below the limit of 7% set by the guideline.
3) The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) must be 35% or less. The growth rate (per day) for each control replicate was calculated for each time period. A mean, standard deviation and coefficient of variation was calculated for each replicate based on each daily growth rate. The mean coefficient of variation for the control replicates was 11.8%, which was below the maximum criterion.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

For each endpoint, mean values were calculated for each test level and the control. Percent inhibition values were then determined based on differences in mean test level values as compared to control values.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity values for Pseudokirchneriella subcapitata exposed to butyl phenyl ether over a 72-hour static exposure period and based on mean measured concentrations were as follows:
Cell yield:
72-hour EyC50: 1.67 mg/L (95% CI = 1.56 - 1.79 mg/L)
72-hour NOEC: 0.183 mg/L

Growth rate:
72-hour ErC50: 2.84 mg/L (95% CI = 2.26 - 3.56 mg/L)
72-hour NOEC: 0.183 mg/L

Executive summary:

The purpose of this study was to assess the potential effects of butyl phenyl ether to the freshwater green alga, Pseudokirchneriella subcapitata. The study was performed over a 72-hour static exposure period with target nominal concentrations of 0 (AAP control), 0.10, 0.30, 1.0, 3.1 and 10 mg butyl phenyl ether/L. Cell counts were determined at approximately 24, 48 and 72 hours (± 1 hour from exposure initiation) of exposure. Temperatures during the exposure period ranged from 22-23 °C. The pH ranged from 7.7-10.1 and the light intensity ranged from 4470-5550 lux.

Test solutions were analyzed at test initiation and termination by high performance liquid chromatography with diode array UV absorbance detection (HPLC/DAD). None of the analyses of the media control exhibited a concentration exceeding the lower limit of quantitation (LLQ) equivalent to 0.05 mg butyl phenyl ether/L. Measured concentrations ranged from 44.5 to 79% of nominal concentrations over the course of the exposure period. The resulting mean measured concentrations were < LLQ, 0.0600, 0.183, 0.580, 1.65 and 5.55 mg butyl phenyl ether/L.

The acute toxicity values for Pseudokirchneriella subcapitata exposed to butyl phenyl ether over a 72-hour static exposure period and based on mean measured concentrations were as follows:

Cell yield:

72-hour EyC50: 1.67 mg/L (95% CI = 1.56-1.79 mg/L)

72-hour NOEC: 0.183 mg/L

Growth rate:

72-hour ErC50: 2.84 mg/L (95% CI = 2.26-3.56 mg/L)

72-hour NOEC: 0.183 mg/L

Description of key information

The 72 -hr EC50 (growth rate) of butyl phenyl ether to the freshwater green alga, Pseudokirchneriella subcapitata, was determined to be 2.84 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.84 mg/L

EC10 or NOEC for freshwater algae:

0.183 mg/L