butanedioic acid, 2-octenyl

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: Octenylsuccinic acid
Chemical Name: 2-(Octen-1-yl) butanedioic acid
Synonyms: DF-20 Acid, OSAC
Lot/Reference/Batch Number: 8515025
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.2% area by high performance liquid chromatography with identification by liquid chromatography mass spectrometry and nuclear magnetic resonance (Megregian, 2016).
Test Material Stability Under Storage Conditions: Octenylsuccinic acid, lot 8515025, 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 830.6313 (Megregian and Crispin, 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 Midland Municipal Wastewater Treatment Plant (Midland, Michigan) on May 12, 2016. This facility treats an excess of 11 million liters of wastewater per day, of which > 90% 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 1,527 mg/L. Based on this determination, homogenized activated sludge was added to the sterilized mineral medium to yield a final MLSS concentration of 29.6 mg/L (dry wt).

Duration of test (contact time):

28 d

Initial conc.:

53.4 mg/L

Based on:

ThOD

Initial conc.:

25.4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

primary indicator of biodegradation

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

supplemental indicator of biodegradation

Parameter followed for biodegradation estimation:

DOC removal

Remarks:

supplemental indicator of biodegradation

Details on study design:

Test System Justification and 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. For this study, dispersion of the test material within the test medium was facilitated by coating the material onto silica gel. Since the test material is a long-chain fatty acid, it has potential to precipitate out as Ca or Mg salts in the reaction mixtures, which would occur as a floating “scum” on the surface of the biodegradation reaction mixtures. Coating on silica gel helped to evenly disperse the test material within the test medium and maintained contact between it and the microbial inoculum. Prior to use, the silica gel was fired in a muffle furnace at 550 °C to remove any trace organic contaminants. The test material was then coated onto the silica gel (35-60 mesh) at a gravimetric loading of 19.9% wt. Coated silica gel was mixed to give a free-flowing homogenous powder. Prior to study initiation, both blank silica and portions of the test material-coated silica were analyzed for total organic carbon (TOC) to verify loading and homogeneity of the test material. The amount of coated silica gel added to the biodegradation reaction mixtures was determined based on the test material theoretical oxygen demand (ThOD). Weighed portions of the test material-coated silica gel were added to the reaction mixtures to yield the required concentrations of the test material.

Solubility and Stability Assessment:
A prior determination of stability in the test medium is not relevant to biodegradation studies, as the test conditions are intended to promote degradation by biodegradation, hydrolysis, and oxidation/reduction reactions.
The test material was expected to be 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 reference material stock solutions was purified though 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 had 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 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 filter-sterilized with a Corning 0.2 μm membrane sterilization unit prior to addition of the inoculum. See Table 1 in "Other Methods" section for Mineral Medium Composition.

Test Procedure:
The biodegradation reaction mixtures were prepared in specially designed 1-liter glass reaction vessels, each containing an approximately 500 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. These vessels are also fitted with 20 x 105 mm glass side baffles to facilitate complete mixing/aeration of the stirred reaction mixtures. All reaction vessels were labeled using a numbering system for vessel identification.
Inoculum Blanks, containing the inoculated mineral medium with unamended silica gel 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 dissolved organic carbon (DOC) concentration in the absence of added test material. Biodegradation of a reference material, aniline, was determined in duplicate Positive Control mixtures to verify the viability of the inoculum. These reaction mixtures contained 100.5 mg/L aniline, which was added to the inoculated mineral medium as a concentrated aqueous solution. Biodegradation of the test material in the Test Mixtures was determined by adding test material-amended silica gel to the inoculated mineral medium (500 mL) at a concentration of approximately 25.4 mg/L, yielding approximately 53.4 mg/L theoretical oxygen demand (ThOD). A single Abiotic Control mixture was prepared by adding mercuric chloride (248 mg/L) to inoculated mineral medium containing the test material-amended silica gel. This Abiotic Control was used to determine the amount of O2 consumption, CO2 evolution, and changes in DOC concentration measured in the Test Mixtures which is attributed to abiotic reactions.
After addition of test material, aniline, and chemical sterilant to the appropriate vessels, the pH of the reaction mixtures were measured and adjusted as necessary to 7.4 ± 0.2, then stirred for 30 minutes to homogenize their contents prior to initiation of the test. Samples (30 mL) of the Inoculum Blanks, Positive Controls, Test Mixtures and Abiotic Control were collected for initial analyses of DOC. Samples (30 mL) of the Positive Controls were also analyzed for dissolved nitrate and nitrite. Prior to measurement of initial oxygen and CO2 concentrations, the headspace volume of each individual reaction vessel was determined by the automated respirometer system. Other specific operating
parameters for the respirometer system are described in detail in the Study File. The biodegradation reaction mixtures were incubated in the darkness at a constant temperature between 20 to 24 °C, and maintained within ± 1°C. The reaction mixtures were continuously stirred by a PTFE-coated magnetic stir bar rotating at a setting of 150 rpm.

Frequency of Sampling:
Concentrations of oxygen and CO2 in the headspace of each reaction vessel were recorded at six-hour intervals over the entire 28-day test period. Upon completion of these measurements on day 28, the pH and DOC concentrations in Inoculum Blanks, Positive Controls, Test Mixtures, and Abiotic Control were determined. Additional analyses of dissolved nitrate and nitrite were performed for the Positive Controls.

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

Reference substance:

aniline

Key result

Parameter:

% degradation (O2 consumption)

Value:

132

St. dev.:

6

Sampling time:

28 d

Remarks on result:

other: By the end of the 28-day test, % biodegradation of the test material reached 132 ± 6% DO2 (mean ± 1 SD, n =2).

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 3.6 days and the 60% DO2 level was exceeded after 4.6 days (See Table 2 in "Any other information on results" section). By the end of the 28-day test, % biodegradation of the test material reached 132 ± 6% DO2 (mean ± 1 SD, n =2). Based on these results, octenylsuccinic acid can be classified as “ready biodegradable”. Note that the measured extent of test or reference material biodegradation in the OECD 301F test can sometimes reach or exceed 100%. This is typically observed when a tested substance is very rapidly and completely degraded within the first few days of the test, and is thought to be the result of rapid growth and then decomposition (and associated oxygen consumption) of the microbial population associated with test material degradation.

Results with reference substance:

Biodegradation of the reference material (aniline) exceeded 60% by 4.6 days.

Biological Oxygen Demand (BOD):

By the end of the 28-day test, % biodegradation of the test material reached 132 ± 6% DO2 (mean ± 1 SD, n =2).

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	3.8	4.6	88.5 ± 1.2	99.2 ± 0.8
Test Mixtures	3.6	4.6	121 ± 5	132 ± 6

*mean ± standard deviation, n = 2

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 183 ± 1.50 mg/g (1 SD), which equates to a loading of 29.1% test material by weight.

CO2 Production:

Two 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 within 28 days. 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 substance biodegradation and ready biodegradability conclusion derived from measurement of oxygen consumption. Biodegradation of the substance exceeded 10% DCO2 after 4.1 days, and after 28 days reached 85.1 ± 4.8% DCO2 (mean ± 1 SD, n = 2). Therefore, the rates and extents of biodegradation determined from CO2 evolution closely reflected those determined from BOD, and confirm the ready- and ultimate biodegradability of the test material under the conditions of this test.

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 16.6 mg/L, which based on a theoretical carbon content of 63.1%, equates to 26.3 mg/L test material. Therefore, the test material was considered as fully soluble at the nominal concentration tested (25.4 mg/L). The extent of DOC removal for the Test Mixtures was 96.2 ± 0.0 mg/L (mean ± 1 SD., n = 2) at day 28. The mean blank-corrected DOC concentration in the Test Mixtures at day 28 was 0.64 mg/L. These results confirm the biodegradability of the test material and suggest that little or no persistent and water soluble degradation products were formed as a result of test substance 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, and maintenance of temperature and pH of the reaction mixtures.

The inoculum used in this test consumed an average of 38 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, aniline, within the required 10-day window prior to day 14 of the test. The 60% DO2 pass level was exceeded after 4.6 days, and biodegradation based on O2 consumption, CO2 production and DOC removal reached 99.2%, 62.9% and 92.4%, respectively, at the end of the test.

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 < 8.6%

DO2 over all sample intervals of the 28-day test. The maximum difference in percentage of aniline biodegradation in replicate reaction mixtures differed by < 1.94% DO2 at the end of the 10-day window. 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 daily minimum temperatures averaged 21.6 ± 0.3°C (± 1 SD, n = 15) and the maximum temperatures averaged 22.0 ± 0.3°C, 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 ranged from 7.27 to 7.33 and remained within the required range of 6.0 to 8.5 over the duration of this test. The pH of the Test Mixtures changed by no more than 0.05 pH units from their initial values over 28 days, and showed only a 0.15 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 substances evaluated in this test.

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 substance. The mixture contained approximately 24 mg/L of test material (equivalent to approximately 50 mg/L ThOD) 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 (data not shown). The initial blank-corrected DOC concentration in this reaction mixture was 15.8 mg/L and the final concentration was 17.4 mg/L, indicating no net removal of DOC under the abiotic incubation conditions. Therefore, the O2 consumption, CO2 production, and DOC removal measured in the Test Mixtures were solely attributed to biodegradation of the test material.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Biodegradation of the octenylsuccinic acid exceeded the 60% pass criterion for demonstrating “ready biodegradability” in the manometric respirometry test. This result was achieved within the required 10-day window falling within the 28 day test period. The results of this test therefore demonstrate that octenylsuccinic acid can be classified as “readily biodegradable”, according to the OECD 301F: Manometric Respirometry Test (OECD, 1992). By virtue of this definition and the stringent nature of this biodegradation screening test, octenylsuccinic acid can be expected to rapidly and ultimately biodegrade in a variety of aerobic environments.

Executive summary:

The ready biodegradability of octenylsuccinic acid was determined using the OECD Guideline No. 301F: Manometric Respirometry Test. This study employed a series of biodegradation reaction mixtures containing activated sludge inoculum collected from the City of Midland Wastewater Treatment Plant (Midland, Michigan), which was suspended in a defined mineral medium at a concentration of 29.6 mg/L (dry solids). Due to the potential for the test material to precipitate out as Ca or Mg salts, the test material was coated onto silica gel to facilitate its dispersion in the biodegradation reaction mixtures. Biodegradation of the test material was evaluated in reaction mixtures at a concentration of 25.4 mg/L, which was equivalent to 53.4 mg/L theoretical oxygen demand (ThOD). 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 hour intervals, using an automated respirometer system. The onset of octenylsuccinic acid biodegradation (i.e. oxygen consumption > 10% of ThOD) occurred after 3.6 days in the Test Mixtures, and biodegradation exceeded the pass level of 60% ThOD consumption after 4.6 days. At the end of the 28 day test, the extent of biodegradation based on BOD, CO2 production, and DOC removal reached 132 ± 6 %, 85.1 ± 4.8%, and 96.2 ± 0.0% (mean ± 1 SD), respectively. 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, octenylsuccinic acid 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 (aniline) exceeded 60% by 4.6 days, verifying the viability of the activated sludge inoculum. Therefore, the results of this study are considered fully valid, and indicate that octenylsuccinic acid exhibits

potential for rapid and ultimate degradability in various aerobic environments.

Description of key information

The biodegradability of octenylsuccinic acid in water was assessed in an OECD 301F manometric respirometry test, conducted in compliance with GLP standards (Reliability 1). All validity criteria specified in the testing guideline were satisfied. At the end of the 28 day test, the extent of degradation based on oxygen consumption was 132%, with 60% degradation achieved after 4.6 days. Therefore, octenylsuccinic acid was classified as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

In the manometric respirometry test with octenylsuccinic acid, the time to reach 10% degradation based on oxygen consumption was 3.6 days, and the 60% threshold was exceeded after 4.6 days. At the end of the 10-day window, the extent of degradation was 121%, with a final value of 132% at the conclusion of the 28-day test. The extent of biodegradation after 28 days based on supplemental measurements of carbon dioxide evolution and dissolved organic carbon (DOC) removal was 85% and 96%, respectively, which supported the conclusion that octenylsuccinic acid can be classified as readily biodegradable.