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

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

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 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test Material Name: Methacryloxyisopropyl acid phthalate
Chemical Name: 1,2-Benzenedicarboxylic acid, mono(1-methyl-2-((2-methyl-1-oxo-2-propen-1-yl)oxy)ethyl)ester
Synonyms: Reaction mass of 2-[({1-[(2-methylprop-2-enoyl)oxy]propan-2-yl}oxy)carbonyl]benzoic acid and 2-({2-[(2-methylprop-2-enoyl)oxy]propoxy}carbonyl)benzoic acid
Lot/Reference/Batch Number: YY00GCV000
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the purity of the test material as 62.8 wt% (The Dow Chemical Company, 2016).
Test Material Stability Under Storage Conditions: The certificate of analysis lists the test material as having an expiration date of 8 months after date of manufacture (The Dow Chemical Company, 2016).

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Inoculum:
The microbial inoculum consisted of activated sludge mixed liquor, collected from the oxidation ditch bioreactor at the Saginaw Charter Township Wastewater Treatment Plant (Saginaw, Michigan) on March 8, 2017. This facility treats an excess of 24 million liters of wastewater per day, of which > 80% is from domestic sources. The activated sludge was collected one day prior to initiation of the test, and was continuously aerated until
used. Prior to use, the activated sludge was screened through 500 μm nylon mesh, and briefly homogenized in a Waring blender (Waring Products Inc., Torrington, Connecticut). The mixed liquor suspended solids (MLSS) content of the homogenized sludge was determined gravimetrically to be 3,097 mg/L. Based on this determination, an appropriate volume of the homogenized activated sludge will be added to the sterilized mineral medium to yield a final MLSS concentration of 29.7 mg/L.

Duration of test (contact time):

28 d

Initial conc.:

43 mg/L

Based on:

test mat.

Initial conc.:

50.3 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Oxygen consumption is used as the primary indicator of biodegradation.

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

CO2 evolution supplemental measure of biodegradation.

Parameter followed for biodegradation estimation:

DOC removal

Remarks:

DOC removal supplemental measure of biodegradation.

Details on study design:

Route of Administration:
The test system and route of administration was selected based on the OECD Guideline 301 (OECD, 1992), and in consideration of the physical/chemical properties of the test material. The test material occurs as a liquid that is poorly soluble in water (64.9 mg/L, estimated) and therefore evaluation of biodegradability required direct addition to the aqueous reaction mixtures on a weight or volume basis. The dispersion of such materials in the aqueous reaction mixtures is recommended for evaluating ready biodegradability, using one of several techniques described in Annex III to OECD Guideline No. 301 (OECD 1992). For this study, dispersion of the test material within the test medium was facilitated by coating the test material onto a silica gel powder. Coating of the test material onto silica gel helps to evenly disperse the test material within the test medium and maintain contact between it and the microbial inoculum. Prior to use, the silica gel (35-60 mesh) was fired in a muffle furnace at 550°C to remove any trace organic and water contaminants. The test material was mixed with silica gel at a nominal gravimetric loading of 20.4% wt. The mixture was vortexed for ~10 seconds and then placed on a hematology mixer inside an incubator set to 35°C. After 2 hours, the mixture was a freeflowing, homogenous mixture. The vial was removed and stored at room temperature until use. Weighed portions of the fired silica gel, and test material amended silica gel were analyzed for total organic carbon (by combustion) to determine the homogeneity with which the test material was incorporated onto the silica gel carrier. A weighed portion of the test material-amended silica gel was added directly to the reaction mixtures to yield the required test material concentrations.

Solubility and Stability Assessment:
A prior determination of stability in the test medium was not relevant to biodegradation studies, as the test conditions were intended to promote degradation by biodegradation, hydrolysis, and oxidation/reduction reactions.
The test material is poorly soluble in water. The extent to which the test material was dissolved and dispersed in the test medium was assessed by analyzing dissolved organic carbon (DOC) in the biodegradation reaction mixtures at test initiation.

Chemicals and Reagents:
De-ionized water used to prepare the mineral medium and test chemical/reference material stock solutions was purified through a PURELAB Ultra water treatment system (ELGA LabWater, High Wycombe, United Kingdom) producing ultrapure water. All other chemicals used were purchased from commercial sources and have appropriate documentation of identity and purity.

Mineral Medium:
A defined mineral medium was prepared as specified in OECD Guideline 301F, by dissolving appropriate volumes of concentrated mineral stock solutions (Table 1) in ultrapure water. The pH of the finished mineral medium was recorded, and adjusted (if necessary) within the range of 7.2 – 7.6. The finished mineral medium was filtersterilized with a Corning 0.22 μm membrane sterilization unit prior to addition of the inoculum.

Test Procedure:
The biodegradation reaction mixtures were prepared in specially designed 0.5-liter glass reaction vessels, each containing a 400 mL portion of the inoculated mineral medium. The reaction vessels are designed with flat glass bottoms to accommodate stirring with large PTFE-coated magnetic stir bars. The reaction vessels, stir bars, and media carboys were autoclaved one day prior to the start of the study. The various reaction mixtures
which comprised the experimental design for this study were labeled appropriately directly on the bottle for proper identification of the vessels. Inoculum Blanks, containing the inoculated mineral medium and without added test or reference material, were prepared in duplicate. These Inoculum Blanks were used to determine mean values for cumulative O2 consumption, CO2 evolution, and DOC concentration in the absence of added test or reference material. These mixtures also contained an amount of unamended silica gel which is approximately equal to that added in the Test Mixtures. Biodegradation of a reference material, sodium benzoate (99.5 mg/L), was determined in duplicate Positive Control mixtures to verify the viability of the inoculum. Biodegradation of the test material in the Test Mixtures was determined by adding the test material (as weighed portions of test material-coated silica gel) to the inoculated mineral medium (400 mL) at a concentration yielding 50.3 mg/L chemical oxygen demand (COD). A single Abiotic Control was prepared by adding mercuric chloride (248 mg/L) to inoculated mineral media containing the test material at a concentration yielding 51.3 mg/L COD. This Abiotic Control is used to determine the amount of O2 consumption, CO2 evolution, and changes in DOC concentrations recorded in the Test Mixtures which are attributed to abiotic reactions. A single Toxicity Control was prepared by adding test material (as weighed portions of test material-coated silica gel) at a concentration yielding 51.3 mg/L COD and 99.5 mg/L of sodium benzoate. This Toxicity Control was used to determine whether the concentrations of the test material used in the test mixtures are inhibitory to the microbial inoculum.
After addition of test material, sodium benzoate, and chemical sterilant to the appropriate vessels, the reaction mixtures were sealed and stirred for at least 30 minutes to homogenize contents prior to initiation of the test. The initial pH of each mixture was determined, and adjusted (if necessary) within the range of 7.2 – 7.6. Samples (30 mL) of the Inoculum Blanks, Positive Controls, Test Mixtures, Abiotic Control, and Toxicity Control were collected for initial DOC analyses. The biodegradation reaction mixtures were incubated in the darkness at a temperature in the range of 20-24°C, and maintained within ± 1°C of the average incubation temperature. The reaction mixtures were continuously stirred by a PTFE-coated magnetic stir bar rotating at approximately 150 revolutions per minute (r.p.m.).

Statistics and Calculations:
Descriptive statistics (mean, standard deviation) were used where applicable.

Reference substance:

other: sodium benzoate

Key result

Parameter:

% degradation (O2 consumption)

Value:

150

St. dev.:

5

Sampling time:

28 d

Parameter:

% degradation (CO2 evolution)

Value:

151

St. dev.:

5

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Value:

99.4

St. dev.:

0

Sampling time:

28 d

Details on results:

Biological Oxygen Demand (BOD):
Biological oxygen demand (BOD) is used as the primary indicator of biodegradation in the OECD 301F: Manometric Respirometry test. These measurements of BOD showed the extent of biodegradation of the test material under the conditions of this test. The time required for average biodegradation to exceed 10% DO2 (i.e., the lag period) was 2.8 days and the 60% DO2 level was exceeded after 4.3 days (See Table 2). By the end of the 28-day test, % biodegradation of the test material reached 150 ± 5.0% DO2 (mean ± 1 SD, n = 2). Additional cellular respiration and decay from new cells produced during growth can result in apparent extents of degradation which exceed 100% of COD. Such results are by no means considered to indicate an invalid test condition or measurement method.

Results with reference substance:

Biodegradation of the reference material (sodium benzoate) exceeded 60% by 3.5 days, verifying the viability of the activated sludge inoculum.

Test Material Coating:

Triplicate weighed portions of the test material-amended silica gel were analyzed to verify the concentration and homogeneity with which the test material was coated onto the silica gel carrier. These results showed an average carbon concentration of 149 ± 10.3 mg/g (n = 3, 1 SD), which equates to a loading of 24.2% test material by weight (based on theoretical carbon content).

Biological Oxygen Demand (BOD):

The time required for average biodegradation to exceed 10% DO2 (i.e., the lag period) was 2.8 days and the 60% DO2 level was exceeded after 4.3 days.

TABLE 2. Summary of Biodegradation Based on Oxygen Consumption (DO2)

Reaction Mixtures	Time (Days) to Achieve		DO2 (%)* at
	10% DO2	60% DO2	10-d Window	Day 28
Positive Controls	0.8	3.5	84.9 ± 2.6	97.3 ± 0.6
Test Mixtures	2.8	4.3	123 ± 1	150 ± 5
Toxicity Control	1.0	NA**	38**	31.7**

*Mean ± 1 SD, n = 2

**DO2 result after 7 days. Instrument malfunctioned on Day 7, and no further data was recorded.

CO2 Evolution:

Two of the other OECD tests for ready biodegradability utilize measurements of CO2 evolution to indicate the extent of test material mineralization. The pass criterion for these tests is 60% of theoretical carbon dioxide evolution. While measurement of CO2 evolution is not a requirement of OECD Guideline No. 301F, these supplemental measurements of CO2 evolution confirmed the extent of test material biodegradation and ready biodegradability conclusion derived from oxygen consumption. Biodegradation of the material exceeded 10% DCO2 after 3.0 days, and after 28 days

reached 151 ± 5.0% DCO2 (mean ± 1 SD, n = 2). Therefore, the rates and extents of biodegradation determined from CO2 evolution closely reflected those determined from BOD.

DOC Analyses:

Analyses of DOC were performed on all vessels to determine the percent degradation of the test and reference materials. The analyses of the Test Mixtures at test initiation indicated a mean blank-corrected concentration of 26.4 ± 0.9 mg/L (mean ± 1 SD, n = 2), which based on a theoretical carbon content of 61.6%, equates to 42.8 mg/L test material. Therefore, the test material was considered as fully soluble at the nominal concentration tested (43 mg/L). The mean blank-corrected DOC concentrations in the Test Mixtures at day 28 were 0.15 mg/L. The extent of DOC removal in the Test Mixtures was 99.4 ± 0.0 % (mean ± 1 SD., n=2) at day 28. These results confirm the ultimate biodegradation of the test material and suggest that little or no persistent and water soluble degradation products were formed as a result of its biodegradation.

Test Validation:

Several criteria are specified by the OECD for validating the results of its tests for ready biodegradability (OECD, 1992). These criteria are based on parameters such as inoculum viability, precision among replicate reaction mixtures, maintenance of temperature and pH of the reaction mixtures.

The inoculum used in this test consumed an average of 31 mg/L oxygen over 28 days, where the OECD guideline indicates that this background oxygen consumption should not exceed 60 mg/L. The inoculum produced > 60% biodegradation of the reference material, sodium benzoate, within the required 10-day window prior to day 14 of the test. The 60% DO2 pass level was exceeded after 3.5 days, and biodegradation based on O2 consumption, CO2 production and DOC removal reached 97.3 ± 0.6%, 80.6 ± 7.4%, and 100.1 ± 0.1% (mean ± 1 SD, n = 2) respectively, at the end of the test, which verified the viability of the activated sludge inoculum.

For the Test Mixtures and Positive Controls, the extent of biodegradation recorded for replicate reaction mixtures must not differ by more than 20% DO2 at the end of the 10-day window, plateau of degradation, or the end of the test (OECD, 1992). In this test, the percentage of test material biodegradation in the replicate Test Mixtures differed by < 7.5% DO2 over all sample intervals of the 28-day test. The maximum difference in percentage of sodium benzoate biodegradation in replicate reaction mixtures was 7.1% DO2 over the course of the study. The results indicate that the procedures used to prepare, incubate, and analyze the biodegradation reaction mixtures resulted in sufficient precision in the test results.

Temperature of the incubator which contained the biodegradation reaction mixtures was recorded periodically throughout the study using a calibrated min/max digital thermometer. The recorded minimum temperatures averaged 22.2 ± 0.1°C (mean ± 1 SD., n = 28) and the maximum temperatures averaged 22.3 ± 0.1°C (mean ± 1 SD., n = 28), over the entire duration of this test. Therefore, the incubation temperature fell within the required range of 20-24°C, and was maintained within the required precision of ± 1°C.

The pH of the biodegradation reaction mixtures remained within the required range of 6.0 to 8.5 over the duration of this test. The pH of the Test Mixtures decreased by no more than 0.13 pH units from their initial values over 28 days, and showed only a 0.19 pH unit (maximum) difference relative to the Inoculum Blanks at the end of the test. This minimal variation in pH indicates that the mineral medium contained adequate buffering capacity for the inoculum and test materials evaluated in this test.

Toxicity Controls:

The Toxicity Control mixtures containing 99.5 mg/L sodium benzoate and 43.6 mg/L of the test material showed no evidence for inhibition of the microbial inoculum by the test material. The OECD guideline specifies that toxicity or inhibition of the inoculum is indicated when net oxygen consumption remains less than 25% of the total applied ThOD over the first 14 days of the test (OECD, 1992). The extent of biodegradation exceeded 25% by Day 2; however, the instrument malfunctioned around Day 7 and thus no oxygen consumption data was recorded after Day 7. The results of the CO2 evolution and DOC analysis of the Toxicity Control provide further evidence that no inhibition of the inoculum occurred.

Abiotic Controls:

A single Abiotic Control mixture was included in the experimental design to determine the extent to which abiotic processes may result in degradation of the test material. The mixture contained 43.8 mg/L of test material in the inoculated mineral medium, which was chemically sterilized by addition of 248 mg/L HgCl2. The Abiotic Control mixture exhibited no O2 consumption or CO2 production over the duration of the 28-day test. Therefore, the O2 consumption and CO2 production in the Test Mixtures was solely attributed to biodegradation of the test material.

Validity criteria fulfilled:

yes

Remarks:

Results of this test met or exceeded each of the OECD-specified criteria for validation of the ready biodegradability test.

Interpretation of results:

readily biodegradable

Conclusions:

The methacryloxyisopropyl acid phthalate exceeded 60% biodegradation within 4.3 days of the start of the test and within the 10-day window detailed in OECD 301F guideline. The results of this test demonstrated that methacryloxyisopropyl acid phthalate can be classified as “readily biodegradable”, according to the OECD 301F: Manometric Respirometry Test (OECD, 1992).

Executive summary:

The ready biodegradability of methacryloxyisopropyl acid phthalate was determined using the OECD Guideline No. 301F: Manometric Respirometry Test. Biodegradation of methacryloxyisopropyl acid phthalate exceeded 60% biodegradation within 4.3 days of the start of the test and within the 10-day window detailed in OECD 301F guideline. The results of this test demonstrated that methacryloxyisopropyl acid phthalate can be classified as “readily biodegradable”, according to the OECD 301F: Manometric Respirometry Test. This study employed a series of biodegradation reaction mixtures containing activated sludge inoculum collected from the Saginaw Charter Township Wastewater Treatment Plant (Saginaw, Michigan), which was suspended in a defined mineral medium at a concentration of 29.7 mg/L (dry solids). Biodegradation of the test material was evaluated in reaction mixtures at a concentration of 43.0 mg/L, which was equivalent to 50.3 mg/L chemical oxygen demand (COD). Reaction mixtures were incubated in the darkness at a constant temperature between 20 to 24°C, and maintained within ± 1°C. Oxygen consumption in the biodegradation reaction mixtures was

continuously recorded at 6 hr intervals, using an automated respirometer system. The onset of methacryloxyisopropyl acid phthalate biodegradation (i.e. oxygen consumption ≥ 10% of COD) occurred after 2.8 days in the Test Mixtures, and biodegradation exceeded the pass level of 60% COD consumption after 4.3 days. At the end of the 28 day test, the extent of biodegradation based on BOD, CO2 evolution and DOC removal reached 150 ± 5.0%, 151 ± 5.0%, and 99.4 ± 0.0% (mean ± 1 SD, n = 2), respectively. A Toxicity Control mixture, containing both aniline (99.5 mg/L) and the test material (43.6 mg/L), showed no evidence for inhibition of the microbial inoculum. Oxygen consumption and CO2 evolution observed in the reaction mixtures could be attributed solely to biological activity, as no net O2 consumption or CO2 evolution was measured in an Abiotic Control mixture containing the test material and a chemical sterilant (HgCl2). Thus, methacryloxyisopropyl acid phthalate is not inhibitory of the

inoculum and can be classified as “readily biodegradable”.

Other results of this test met or exceeded each of the OECD-specified criteria for validation of the ready biodegradability test. These include parameters such as viability of the inoculum, control of pH and temperature, and precision in percentage biodegradation recorded among replicate test mixtures containing a biodegradable reference material. Biodegradation of the reference material (sodium benzoate) exceeded 60% by 3.5 days, verifying the viability of the activated sludge inoculum. Therefore, the results of this study are considered fully valid, and indicate that methacryloxyisopropyl acid phthalate exhibits potential for rapid and ultimate degradability in various environments.

Description of key information

In the key study, the ready biodegradability of methacryloxyisopropyl acid phthalate was determined using the OECD Guideline No. 301F: Manometric Respirometry Test. Biodegradation of methacryloxyisopropyl acid phthalate exceeded 60% biodegradation within 4.3 days of the start of the test and within the 10-day window detailed in OECD 301F guideline. The results of this test demonstrated that methacryloxyisopropyl acid phthalate can be classified as “readily biodegradable”, according to the OECD 301F: Manometric Respirometry Test. This study employed a series of biodegradation reaction mixtures containing activated sludge inoculum collected from the Saginaw Charter Township Wastewater Treatment Plant (Saginaw, Michigan), which was suspended in a defined mineral medium at a concentration of 29.7 mg/L (dry solids). Biodegradation of the test material was evaluated in reaction mixtures at a concentration of 43.0 mg/L, which was equivalent to 50.3 mg/L chemical oxygen demand (COD). Reaction mixtures were incubated in the darkness at a constant temperature between 20 to 24°C, and maintained within ± 1°C. Oxygen consumption in the biodegradation reaction mixtures was continuously recorded at 6 hr intervals, using an automated respirometer system. The onset of methacryloxyisopropyl acid phthalate biodegradation (i.e. oxygen consumption ≥ 10% of COD) occurred after 2.8 days in the Test Mixtures, and biodegradation exceeded the pass level of 60% COD consumption after 4.3 days. At the end of the 28 day test, the extent of biodegradation based on BOD reached 150 ± 5.0% (mean ± 1 SD, n = 2). A Toxicity Control mixture, containing both aniline (99.5 mg/L) and the test material (43.6 mg/L), showed no evidence for inhibition of the microbial inoculum. Oxygen consumption and CO2 evolution observed in the reaction mixtures could be attributed solely to biological activity, as no net O2 consumption or CO2 evolution was measured in an Abiotic Control mixture containing the test material and a chemical sterilant (HgCl2). Thus, methacryloxyisopropyl acid phthalate is not inhibitory of the inoculum and can be classified as “readily biodegradable”.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable