Reaction mass of 2-(((1-(methacryloyloxy)propan-2-yl)oxy)carbonyl)benzoic acid and 2-((2-(methacryloyloxy)propoxy)carbonyl)benzoic acid

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:

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:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: YY00GCV000
- Expiration date of the lot/batch: 2017-08-09
- Purity test date: 2017-01-03

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient
- Stability under test conditions: Elevated temperature aging studies (non-GLP) for ZM-120M (unknown lot number) indicate the active ingredient remains within production specification at 25°C and 37°C for 200 days (~28 weeks) and at 50°C for 150 days (~21 weeks).
- Solubility and stability of the test substance in the solvent/vehicle: Results of the non-GLP range-finding test and preliminary methodology work indicated that the test material was expected to be soluble in AAP medium at the selected test concentrations for the study. The test solutions were utilized on the same day as preparation; thus, assessment of stability of the test solutions was not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
An aliquot containing 318 mg methacryloxyisopropyl acid phthalate (adjusted for 62.8% purity) was added to a 2-L volumetric flask and filled to volume with AAP medium, creating a 100 mg/L bulk test solution. Following direct addition, the bulk test solution was clear and colorless with undissolved clear droplets on the bottom and throughout the solution. The 100 mg/L bulk test solution was then stirred on a magnetic stir plate utilizing a Teflon lined stir bar at approximately 300 revolutions per minute (RPM) for approximately 24 hours. The bulk test solution was then pH adjusted (3.8 to 7.3) with NaOH prior to use. Following stirring and pH adjustment, the bulk test solution was clear and colorless with white precipitate visible on the surface.

Analytical monitoring:

yes

Details on sampling:

Samples were collected from the bulk test solutions at test initiation and from pooled replicate samples at exposure termination. Blank replicates were sampled separately at exposure termination. All test solutions were analyzed within 24 hours of preparation; therefore, no stability assessment was required.

Vehicle:

no

Details on test solutions:

An aliquot containing 318 mg methacryloxyisopropyl acid phthalate (adjusted for 62.8% purity) was added to a 2-L volumetric flask and filled to volume with AAP medium, creating a 100 mg/L bulk test solution. Following direct addition, the bulk test solution was clear and colorless with undissolved clear droplets on the bottom and throughout the solution. The 100 mg/L bulk test solution was then stirred on a magnetic stir plate utilizing a Teflon lined stir bar at approximately 300 revolutions per minute (RPM) for approximately 24 hours. The bulk test solution was then pH adjusted (3.8 to 7.3) with NaOH prior to use. Following stirring and pH adjustment, the bulk test solution was clear and colorless with white precipitate visible on the surface.

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata from in-house cultures was initially obtained from the University of Texas at Austin Culture Collection (UTEX1648; lot # 060215) in June 2015. This species is widely accepted and recommended for toxicity testing by the test guidelines. Stock cultures of this organism were maintained axenically by periodic transfer into sterile medium. Typical culture conditions are listed in Table 1. Algae were cultured under continuous illumination of approximately 5,200 ± 520 lux at a temperature of 23 ± 2ºC. 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 0.839 mL aliquot of the culture was required to inoculate each test vessel at an initial cell density of approximately 10,000 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure period

Test temperature:

22 – 23°C

pH:

7.1 -7.9

Nominal and measured concentrations:

Nominal: 0 (AAP medium control), 0.300, 1.00, 3.10, 9.80, 31.3 and 100 mg methacryloxyisopropyl acid phthalate/L
Measured:

Details on test conditions:

TEST SYSTEM
Test vessels were sterilized 250-mL Erlenmeyer flasks with foam stoppers each containing 50 mL test medium. Each flask was uniquely labeled (i.e., study, replicate, test level) for identification purposes.
Definitive Test
The definitive test was conducted under static conditions for approximately 72 hours from 10 to 13 July 2017. Three replicate test vessels were prepared per test level. Six replicates were prepared for the AAP medium control. Each replicate contained 50 mL of the appropriate test solution and was inoculated with approximately 10,000 cells/mL. An additional replicate at each test level and AAP medium control 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 ± 2ºC. 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 medium 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 medium control. Temperature was continuously monitored with a minimum/maximum thermometer placed in a representative containing deionized water located in the test area. At test initiation, light intensity was measured at each position where replicate vessels were placed during the exposure.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
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 Lake Huron water supplied to The Dow Chemical Company by the City of Midland Water Treatment Plant. The base water (laboratory dilution water (LDW)) 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. 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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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 (average of two cell counts per replicate test vessel) were determined at approximately 24, 48 and 72 hours (± 1 hour from test initiation). 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. The cell count values for the blank replicates were used to correct for background in daily calculations. In addition, morphological observations were done at exposure termination on a composited sample of the inoculated replicates at each test level. 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
A non-GLP 72-hour range-finding test was conducted from 20 to 23 June 2017. Two replicate vessels per test level (6 for control) were inoculated with a predetermined aliquot of algal inoculum to achieve 10,000 cells/mL and exposed to nominal test concentrations of 0 (AAP medium control), 1, 10 and 100 mg methacryloxyisopropyl acid phthalate/L (adjusted for 62.8% purity), over the course of a 72-hour static exposure period. An uninoculated replicate (counting blank) was prepared at each test level and control. An aliquot containing 159 mg methacryloxyisopropyl acid phthalate was added to a 1-L volumetric flask and filled to volume with AAP medium, creating a 100 mg/L bulk test solution. Following direct addition, the bulk test solution was clear and colorless with undissolved clear droplets on the bottom and throughout the solution. The 100 mg/L bulk test solution was then stirred on a magnetic stir plate, utilizing a Teflon lined stir
bar, at approximately 300 revolutions per minute (RPM) for approximately 24 hours. The bulk test solution was then pH adjusted (4.2 to 7.5) with NaOH prior to use. Following stirring and pH adjustment, the bulk test solution was clear and colorless with no apparent undissolved test material visible. The remaining bulk test solutions were prepared as dilutions of the 100 mg/L bulk test solution. The control consisted of AAP medium without the addition of the test material. The prepared bulk test solutions were apportioned into individual test vessels. All bulk test solutions, including the AAP medium control, were clear and colorless at test initiation. Blank replicate test vessels for the AAP medium control, 1 and 10 mg/L test levels remained clear and colorless over the course of the exposure period. The blank replicate test vessel for the 100 mg/L test level was clear and colorless, with a small amount of white precipitate present at the surface,
throughout the exposure period. Cell counts were taken after approximately 72 hours of exposure. Following 72 hours of exposure, mean yield inhibition values were 20, 14 and 54% for the 1, 10 and 100 mg methacryloxyisopropyl acid phthalate/L test levels, respectively. Based on information from the range-finding test, the following nominal test concentrations were used in the definitive study: 0 (AAP medium control), 0.300, 1.00, 3.10, 9.80, 31.3, and 100 mg methacryloxyisopropyl acid phthalate/L (adjusted for 62.8% purity).

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

All biological results are expressed in terms of nominal concentrations of methacryloxyisopropyl acid phthalate.

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 316.2, 301.7, 317.8, 250.5, 263.4, 257.1 and 156.1 (x104) cells/ml for the AAP medium control, 0.300, 1.00, 3.10, 9.80, 31.3 and 100 mg methacryloxyisopropyl acid phthalate/L test levels, respectively. At 72 hours, the mean inhibition response relative to the AAP medium control ranged from -1 to 51% inhibition of yield. Based on statistical analysis of the results (Dunnett’s test, α = 0.05), cell yield at the 100 mg methacryloxyisopropyl acid phthalate/L test level was significantly different from the AAP medium control. Thus, the 72-hour NOEC for yield is reported as 31.3 mg methacryloxyisopropyl acid phthalate/L. The statistically calculated EyC50 is >100 mg methacryloxyisopropyl acid phthalate/L (highest concentration tested).

Growth Rate
All growth rate data were normally distributed and homogeneous (Shapiro-Wilk Test, p > 0.01 and Levene’s Test, p > 0.01). Mean specific growth rates from 0 and 72 hours were 1.918, 1.901, 1.921, 1.831, 1.855, 1.845 and 1.681 (day-1) for the AAP medium control, 0.300, 1.00, 3.10, 9.80, 31.3 and 100 mg methacryloxyisopropyl acid phthalate/L test levels, respectively. From 0 to 72 hours, mean inhibition response relative to the AAP medium control ranged from 0 to 12% of the mean specific growth rate. Based on statistical analysis (Dunnett’s test, α = 0.05) of the results, mean specific growth rate at the 100 mg methacryloxyisopropyl acid phthalate/L test level was significantly different from the AAP medium control. Thus, the 72 hour NOEC for growth rate is reported as 31.3 mg methacryloxyisopropyl acid phthalate/L. The statistically calculated ErC50 is >100 mg methacryloxyisopropyl acid phthalate/L (highest concentration tested).

Morphological Observations
At test termination, microscopic evaluation of algal cells at each test level, including the AAP medium control, revealed no abnormal observations.

Results with reference substance (positive control):

Not applicable

Analytical Results

At test initiation (0 hours), measured methacryloxyisopropyl acid phthalate concentrations of the bulk test solutions (without algae) ranged from 90.0 to 97.3% of nominal concentrations. At exposure termination (72 hours), the analysis of pooled replicate test solutions (with algae) yielded methacryloxyisopropyl acid phthalate concentrations ranging from 94.6 to 103% of nominal concentrations. Analysis of blank replicate solutions (without algae) conducted at 72 hours revealed methacryloxyisopropyl acid phthalate concentrations ranging from 94.2 to 104% of nominal concentrations. Mean measured concentrations of the bulk test solutions at 0 hours and pooled replicates solutions (with algae) at 72 hours were 0.301, 0.973, 2.88, 9.26, 29.4 and 95.2 mg methacryloxyisopropyl acid phthalate/L. Similarly, the mean measured concentrations of the bulk test solutions (without algae) at 0 hours and the blank replicate solutions (without algae) at 72 hours were 0.302, 0.962, 2.91, 9.30, 29.4 and 94.6

methacryloxyisopropyl acid phthalate/L. None of the analyses of the medium control exhibited a concentration exceeding the lower limit of quantitation (LLQ) equivalent to 0.250 mg methacryloxyisopropyl acid phthalate/L.

Acceptance Criteria

The following acceptance criteria set forth in the OECD 201 guideline (2006) were met:

1) The cell density in the control cultures should increase exponentially by a factor of at least 16 within 72 hours. The 72-hour mean cell density increased by approximately 317-fold, thereby exceeding the minimal criteria.

2) The coefficient of variation average for specific growth rates throughout the exposure (days 0 – 3) in AAP medium control replicates was 2.1%, 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 AAP medium 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 AAP medium control replicates was 8.6%, which was below the maximum criterion.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity values for Pseudokirchneriella subcapitata exposed to methacryloxyisopropyl acid phthalate over a 72-hour static exposure period and based on nominal concentrations were as follows:
o Cell yield
72-hour EyC50: >100 mg/L (highest concentration tested)
72-hour NOEC: 31.3 mg/L
o Growth rate
72-hour ErC50: >100 mg/L (highest concentration tested)
72-hour NOEC: 31.3 mg/L

Executive summary:

The purpose of this study was to assess the potential effects of methacryloxyisopropyl acid phthalate to the freshwater green alga, Pseudokirchneriella subcapitata. The study was performed for 72 hours with target nominal concentrations of 0 (AAP control), 0.300, 1.00, 3.10, 9.80, 31.3 and 100 mg methacryloxyisopropyl acid phthalate/L. Cell density was determined at approximately 24, 48 and 72 hours (±1 hour from test initiation). Temperatures during the exposure period ranged from 22-23°C. The pH ranged from 7.1-7.9 and the light intensity ranged from 4490-5320 lux. Test solutions were analyzed at test initiation and exposure termination by high performance liquid chromatography/mass spectrometry (HPLC/MS-MS). None of the analyses of the AAP medium control exhibited a concentration exceeding the lower limit of quantitation (LLQ) equivalent to 0.250 mg methacryloxyisopropyl acid phthalate/L. Measured concentrations of methacryloxyisopropyl acid phthalate ranged from 90.0 to 104% of nominal concentrations over the course of the exposure period. The resulting mean measured test concentrations were 0.301, 0.973, 2.88, 9.26, 29.4 and 95.2 mg methacryloxyisopropyl acid phthalate/L. Because the mean measured concentration were within ± 20% of the target nominal concentrations, study endpoints were based on nominal concentrations.

The acute toxicity values for Pseudokirchneriella subcapitata exposed to methacryloxyisopropyl acid phthalate over a 72-hour static exposure period and based on nominal concentrations were as follows:

o Cell yield

72-hour EyC50: >100 mg/L (highest concentration tested)

72-hour NOEC: 31.3 mg/L

o Growth rate

72-hour ErC50: >100 mg/L (highest concentration tested)

72-hour NOEC: 31.3 mg/L

Description of key information

In a growth inhibition test with Pseudokirchneriella subcapitata,the 72-hour E_rC50 (based on growth rate) for methacryloxyisopropyl acid phthalate was >100 mg/L. The 72-hour NOEC was determined to be 31.3 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

31.3 mg/L

Additional information

One static toxicity study with the green algae,Pseudokirchneriella subcapitata,was available for review and was found to be of good quality and reliable for use in the risk assessment process (Klimisch score = 1). This study measured both growth rate (r) and biomass as indicators of growth inhibition over 72 hours of exposure to methacryloxyisopropyl acid phthalate.  The study was performed as per OECD TG 201 and in accordance with the Principles of Good Laboratory Practice (GLP). The test was performed at 22-23 °C under static conditions with a control (0 mg/l), and 6 nominal concentrations of methacryloxyisopropyl acid phthalate (0.300, 1.00, 3.10, 9.80, 31.3 and 100 mg/l). The dilution water was sterile enriched media adjusted to a pH of 7.5 ± 0.4. No insoluble material was noted during the test. Under the conditions of the study, the 72-hour no observed effect concentration was 31.3 mg/l methacryloxyisopropyl acid phthalate. The 72-hour EC50 was >100 mg/l when calculated using the both number of cells/ml and using the average specific growth rate.