bis(branched-alkyl) 2-{[(1-phenylethyl)amino]methyl}alkanedioate

Biodegradation in water and sediment: simulation tests

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Administrative data

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 March 2017 to 26 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4400 (Anaerobic Aquatic Metabolism)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- The test substance was received neat at EAG Laboratories - Hercules. Upon receipt, 6 mL of acetonitrile was added, and the test substance was stored frozen (~ - 80°C) when not in use.
- Radiochemical purity checks of the test substance (performed before and after application) were conducted.
- A copy of the certificate of analysis of the reference standard was provided.
- The reference standard solution was prepared by adding acetonitrile to a weighed amount of the standard into an amber glass vial, to obtain approximately 25 or 50 mg/mL final concentration.
- The reference standard was stored frozen when not in use.

Radiolabelling:

yes

Study design

Oxygen conditions:

anaerobic

Inoculum or test system:

natural water / sediment: freshwater

Details on source and properties of surface water:

SOURCE OF TEST SYSTEMS
- Sediment and water systems were collected from two sites in the United States. The first was collected from Brandywine Creek, Pennsylvania on 25 January 2017 by Wildlife International. This system was received at EAG Laboratories - Hercules on 27 January 2017. The second test system was collected from Choptank River, Maryland on 25 January 2017 by Wildlife International and received at EAG Laboratories - Hercules on 27 January 2017. Sediments were collected from the top 15 cm of the sediment layer while in flooded conditions. Water samples were collected from the same sites at the same time as the sediment collections.
- On arrival at EAG Laboratories - Hercules, the systems were stored refrigerated in the dark until use in the study. The water layers were sieved through a 0.2 mm sieve and sediment layers were sieved through a 2 mm sieve prior to use in the study. The test systems were referred to as “Pennsylvania” or “Brandywine Creek,” and “Maryland” or “Choptank River” during use in the study.
- Locations of the sites were: Pennsylvania (PA) GPS coordinates 39.8909, -75.6128; Maryland (MD) GPS coordinates 38.7749, -75.9284.

Details on source and properties of sediment:

CHARACTERISATION OF THE TEST SYSTEMS
- The moisture content of the sediments was determined by using a Metter Toledo HB43-S moisture analyzer with five determinations for each sediment. Physicochemical characterization of the water and sediment was performed by Agvise Laboratories, Northwood, ND.
- A summary of the water and sediment characteristics is presented in Table 3 (attached).
- Results of the pH, O2, and ORP measurements (taken during sampling) are presented in Table 4 (attached).

MICROBIOLOGICAL VIABILITY OF TEST SYSTEM
- The sediments used in this study were analyzed for microbial viability (microbial biomass) prior to use (shortly following receipt), at the beginning of the incubation (experimental start) and at the end of the study.
- Microbial biomass results are presented in Table 3 (attached).
- Sediment viability was evaluated using the substrate induced respiration rate (SIR) method (production rate of CO2 after glucose addition).
- Additionally, the test systems used in this study were analyzed for anaerobic microbial viability at the beginning of the study and at the end of the study for both test systems. Viability was determined by enumerating the total colony-forming units (CFU) of anaerobic bacteria on anaerobic agar. Water/sediment samples were diluted in sterile microbial buffer solution and plated in duplicate. The culture plates were incubated at 35 °C for 3 days.

Duration of test (contact time):

<= 102 d

Initial test substance concentration

Initial conc.:

10 other: μCi per 100 μL

Based on:

other: radiolabelled test item

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

SOLVENTS AND REAGENTS
- All solvents and reagents (reagent grade or better) were obtained from Fisher Scientific, or EMD.
- All water used was HPLC grade or purified with a Barnstead E-Pure system (ASTM Type I).

EQUIPMENT
- Laboratory Balances
- Barnstead Four Module E-Pure
- Liquid Scintillation Counters (LSC) (Beckman LS6000IC and LS6500 Series)
- Wrist-Action Shaker
- Buchi Roto-evaporators
- Nitrogen Evaporators
- Microwave Accelerated Reaction System (MARS 6)
- Thermo Scientific Savant Speed-Vac
- Vortex mixers
- Sonicators
- Shimadzu TOC-5050A Total Organic Carbon Analyzer
- Mettler Toledo HB43-S Moisture Analyzer
- Laboratory Centrifuges (Sorvall RC-2B, Sorvall RC-5B, Sorvall RT7)
- Orion Model 501 pH/O2/ORP Meter
- Harvey Biological Oxidizer OX500 and OX600
- Storm 820 Optical Scanner
- TC Pal Automated Fraction Collector
- Gilson FC 204
- PerkinElmer MicroBeta2 LumiJET Microplate Counters 2450
- High Performance Liquid Chromatography: Agilent 1200 series HPLC equipped with degasser, quaternary pump, column compartment with heater and variable wavelength detector (VWD) or diode array detector (DAD). β-RAM flow through monitor system with Laura software (LabLogic) for detection of radiocarbon on HPLC.

TEST SYSTEM IDENTIFICATION
- All test systems were identified by a EAG project number, study type, test system, test substance, date/initials, storage/incubation temperature, laboratory notebook/page number, and a unique sample designation (i.e. “Sample #1”).

Results and discussion

Half-life of parent compound / 50% disappearance time (DT50)

Half-life of parent compound / 50% disappearance time (DT50) 1

Compartment:

entire system

DT50:

56 d

Type:

other: single first order (SFO) kinetic model

Temp.:

20 °C

Remarks on result:

other: Pennsylvania test system

Half-life of parent compound / 50% disappearance time (DT50) 2

Compartment:

entire system

DT50:

27 d

Type:

other: single first order (SFO) kinetic model

Temp.:

20 °C

Remarks on result:

other: Maryland test system

Transformation products:

yes

Identity of transformation products

Identity of transformation products 1

No.:

#1

Identity of transformation products 2

No.:

#2

Details on transformation products:

DEGRADATION OF [14C] TEST ITEM IN WATER/SEDIMENT SYSTEMS UNDER ANAEROBIC CONDITIONS
- The percent of test item in water layers and sediment extracts was determined by HPLC. Results are summarized in Table 7 and Table 8 (attached) for the PA and MD test systems, respectively, and representative radiochromatograms with concurrent injection of the test item reference standard were shown in Figure 7 of the study report for PA test system and Figure 8 of the study report for the MD test system. Graphical representations of the decline of test item, and formation/decline of major residues, and formation of 14CO2 in each system are shown in Figure 9 (attached). The data for all HPLC analyses and calculation relating HPLC results with the percent of applied radiocarbon were summarized in Appendix 7 of the study report. The degradation pathway for test item in water/sediment systems under anaerobic conditions is presented in Figure 11 (attached).

PENNSYLVANIA (PA) TEST SYSTEM
- [14C]Test Item in the PA test system degraded from 99.2% AR at time 0 to an average of 63.8% AR at 29 DAT. Test item declined further to 41.1% AR after 102 days of anaerobic incubation. Amounts of test item in water layers alone declined from an average of 72.3% AR at time 0 to 2.6% AR by 102 DAT. Test Item in sediment layers alone increased from 26.9% AR at 0 DAT to a maximum of 59.5% AR at 29 DAT, then declined to 38.5% AR at the end of the incubation period.
- A major unknown degradate (designated as U-1) was detected at a retention time of approximately 9.5 minutes by HPLC in both water layers and sediment extracts for all time points after 0 DAT. U-1 reached a maximum average of 27.0% AR at 21 DAT and then declined to 14.2% AR by the end of the study (102 DAT). U-1 was later identified by LC/MS to be a hydrolysis product of test item from cleavage of one of the ester bonds.
- A second major unknown degradate (designated as U-2) was detected a retention time of 3.5 minutes by HPLC and it was observed in more significant amounts in the water layers. U-2 increased throughout the study to represent a maximum average of 18.2% AR by the end of the study (102 DAT). Structural elucidation by LC/MS confirmed that the remaining ester bond in U-1 hydrolyzed to form U-2 (details were given in Appendix 8 of the study report).
- Unknown degradates U-3 (Rt = ~8.7 min) and U-4 (Rt = ~9 min) were detected later in the study (60+ DAT), but they were minor, representing maximum averages of 1.9% AR and 5.9% AR at 60 DAT. Both degradates declined by the end of the study (102 DAT) to ≤ 1.2% AR.
- Other uncharacterized chromatographic peaks were detected throughout the study, but all individual peaks represented at most 3.5% AR in any single sample (details were given in Appendix 7 of the study report).

MARYLAND (MD) TEST SYTEM
- [14C]Test Item degraded in the MD test system degraded from 98.3% AR at time 0 to an average of 42.4% AR at 29 DAT. Test item declined further to 21.1% AR after 102 days of anaerobic incubation. Amounts of test item in water layers alone declined at a similar rate as the PA water layer, decreasing from 77.5% AR at time 0 to 2.7% AR by 102 DAT. Test item in sediment layers alone increased from 20.9% AR at 0 DAT to a maximum of 38.7% AR at 14 DAT, then declined to 18.4% AR at the end of the incubation period (102 DAT).
- A major unknown degradate (designated as U-1) was detected at a retention time of approximately 9.5 minutes by HPLC (Method 1, Section 3.12) in both water layers and sediment extracts for all time points after 0 DAT. U-1 reached a maximum average of 40.9% AR at 21 DAT and then declined to 15.9% AR by the end of the study (102 DAT). U-1 was later identified by LC/MS to be a hydrolysis product of test item from cleavage of one of the ester bonds.
- A second major unknown degradate (designated as U-2) was detected a retention time of 3.5 minutes by HPLC and it was observed in more significant amounts in the water layers. U-2 increased throughout the study to represent a maximum average of 40.7% AR by the end of the study (102 DAT). Structural elucidation by LC/MS confirmed that the remaining ester bond in U-1 hydrolyzed to form U-2 (details were given in Appendix 8 of the study report).
- Unknown degradates U-3 (Rt = ~8.7 min) and U-4 (Rt = ~9 min) were detected later in the study (60+ DAT), but they were minor, representing maximum averages of 8.4% AR and 7.5% AR at 60 DAT. Both degradates declined by the end of the study (102 DAT) to 3.2% (U-3) and 5.9% AR (U-4). These degradates were considered minor (≤ 10% AR at any interval and declining by the end of the study) and therefore, characterization was not needed.
- Other uncharacterized chromatographic peaks were detected throughout the study, but all individual peaks represented at most 3.9% AR in any single sample (details were given in Appendix 7 of the study report).

CONFIRMATION OF [14C] TEST ITEM AND IDENTIFICATIONS OF MAJOR TRANSFORMATION PRODUCTS BY LC/MS
- The confirmation of test item and identifications of its major degradates U-1 and U-2 were assessed by LC/MS of a selected sample and the detailed results were presented in Appendix 8 of the study report.
- The proposed structures for the major degradates U-1 (2 possible structures) and U-2 are attached.

DEGRADATION RATE OF [14C] TEST ITEM IN ANAEROBIC WATER/SEDIMENT SYSTEMS
- The DT50, and DT90 results in the total test system and water layers were shown in Figure 10 of the study report and a summary table (attached) was provided.
- The SFO model was an acceptable fit to the data due to the low chi-squared error (≤ 14.2%) and the high R2 values (≥ 0.8138).

PROPOSED DEGRADATION PATHWAYS OF TEST ITEM IN ANAEROBIC AQUATIC WATER/SEDIMENT SYSTEMS
- In both MD and PA test systems, hydrolysis of the parent esters was the predominant route of degradation, yielding U-1 (hydrolysis of 1 ester). U-1 subsequently underwent further hydrolysis of the second ester to form the major degradate U-2. Subsequent degradation finally resulted in mineralization to carbon dioxide and formation of bound residues. A proposed degradation pathway was given in Figure 11 (attached).

Details on results:

RADIOCHEMICAL PURITY AND STABILITY OF [14C] TEST ITEM
- The radiopurity of the test substance was determined by HPLC to be 100% prior to use in the study. The stability of the test substance under conditions of administration was confirmed by HPLC analysis of the dose solution following the application. Analysis of the test substance following dosing indicated radiopurity greater than 99%.

APPLICATION RATE
- The target dose rate was 10 μCi per vessel. Aliquots of the application solution taken throughout the dosing process were used to confirm the treatment rate of 9.9 μCi/vessel for both test systems. The aliquots of the dose solution were within a relative standard deviation of 1.2% (n = 8), confirming homogeneity of the dosing solution during application (see Appendix 5, attached).

CHARACTERISTICS OF THE TEST SYSTEMS
- The anaerobic microbiological activity of the sediment system of a slurry of the sediment and corresponding water layer was determined by plate count (colony forming units per gram) at the experimental start and at the end of the incubation period. The results are shown in Table 3 (attached). The total organic content (TOC) of the water and sediment was also measured at the start and end of the incubation period. The results revealed that the test systems were anaerobically viable at the end of the incubation (see Table 3, attached).
- Throughout the study, the pH in the water layers averaged 6.12 for the PA test system and 7.36 for the MD test system. Similarly, the pH in the sediment layers averaged 5.86 for the PA test system and 7.09 for MD test system, throughout the study. The dissolved oxygen averaged 0.52 ppm for the PA test system (individually ranging from 0.16 to 0.89 ppm) and 0.27 ppm for the MD test system (ranging from 0.09 to 0.52 ppm), indicating anoxic conditions (O2 < 1 ppm) for all samples. The ORP in the water layers averaged -167 mV for the PA test system (ranging from -392 to +3 mV) and -192 mV for the MD test system (ranging from -403 to +77 mV). The ORP in the sediment layers averaged -254 mV for the PA test system (ranging from -491 to -121 mV) and -251 mV for the MD test system (ranging from -424 to -116 mV) (see Table 4, attached).

RADIOCARBON DISTRIBUTION AND MASS BALANCE
- The mass balance was based on the sum of radiocarbon in water layers, sediment extracts, volatile traps and residual sediment radiocarbon (post-extracted sediment), and is expressed as percent of applied radiocarbon (AR). Mass balance for the study is presented in Table 5 (attached) and Table 6 (attached) for the two test systems. Graphical radiocarbon distribution for the study is presented in Figure 6 (attached). Transcriptions of the raw data were presented in Appendix 6 of the study report.

PENNSYLVANIA (PA) TEST SYSTEM
- The mass balance for [14C]Test Item in the PA test system averaged 99.5 ± 2.0% AR (individual sample range 95.5 to 103.0% AR). At time 0, average radiocarbon recovered in the water layers and the sediment extracts was 72.3 and 26.9% AR, respectively. Recovery in water layers decreased to an average of 23.7% AR by the end of the incubation period (102 DAT). Extractable radiocarbon (from sediment) increased to a maximum average of 72.0% AR at 29 DAT, and then decreased for the remainder of the study to average 55.6% AR by 102 DAT (see Table 5, attached).
- Additional extractions performed on the representative 102 DAT post-extracted sediment samples recovered averages of 0.8% AR in the toluene extract and 2.3% AR in the tetrahydrofuran (THF) extract.
- Post-extracted sediment residues represented 0.8% AR at time 0, increased to a maximum of 12.6% AR at 60 DAT, and then decreased (due to the addition of toluene and THF extractions) to 11.4% AR at 102 DAT.
- Minor amounts of mineralization were detected in the NaOH traps (as 14CO2), increasing to a maximum average of 3.0% AR by 102 DAT. Formation of organic volatiles was minor during the study, as recoveries in foam plug traps represented at most 0.2% AR for all samples (see Table 5, attached).

MARYLAND (MD) TEST SYSTEM
- The mass balance for [14C]Test Item in the MD test system averaged 99.2 ± 1.6% AR (individual sample range 96.5 to 101.9% AR). At time 0, average radiocarbon recovered in the water layers and the sediment extracts was 77.5% and 20.9% AR, respectively. Recovery in water layers decreased to an average of 53.2% AR by the end of the incubation period (102 days). Extractable radiocarbon (from sediment) increased to a maximum average of 52.8% AR at 29 DAT, and then decreased for the remainder of the study to average 37.6% AR by 102 DAT (see Table 6, attached).
- Post-extracted sediment residues represented an average of 0.2% AR at time 0, increased to a maximum of 3.5% AR at the end of the study (102 DAT).
- Minor amounts of mineralization were detected in the NaOH traps (as 14CO2), increasing to a maximum average of 2.4% AR by 102 DAT. Formation of organic volatiles was minor during the study, as recoveries in foam plug traps represented at most 0.4% AR for all samples (see Table 6, attached).

Any other information on results incl. tables

DISCUSSION

- An anaerobic aquatic metabolism study using [14C]Test Item was conducted on two water/sediment test systems (Pennsylvania and Maryland) at a dose of approximately 10 μCi per vessel. The samples were incubated for up to 102 days under anaerobic conditions at 20 °C.

- Mass balance for the study, defined as the sum of radiocarbon in the water layers, sediment extracts, post-extracted sediment, sodium hydroxide traps, and foam plug traps for organic volatiles, was at least 95.5% AR (applied radiocarbon) for all samples. Average mass balance for both test systems was at least 99.2% AR. The suitability of the experimental setup and sampling methods are shown by the high recoveries.

- Test item represented at least 97.6% AR at Time 0 in both test systems. Test item then represented 41.1% AR at the end of the study in the Pennsylvania test system and 21.1% AR at the end of the study in the Maryland test system.

- Disappearance times (DTs) were generated for the total test item in the total water/sediment system and water layers only using single first order (SFO) kinetics, following the FOCUS approach and KinGUI software. The DT50 of test item in the total system (sediment and water layer) was 56 days for the Pennsylvania test system and 27 days for the Maryland test system. Test item degraded in the water layers for both test systems at approximately the same rate (DT50 of 9 days).

- U-1 (retention time of 9.5 minutes) was a major degradate detected in both test systems, reaching maximum averages of 27.0 and 40.9% AR in the Pennsylvania and Maryland test systems, respectively. U-2 (retention time of 3.5 minutes) was another major degradate that reached maximum averages of 18.2 and 40.7% AR by the end of the study (102 DAT) in the Pennsylvania and Maryland test systems, respectively. U-1 and U-2 were later identified by LC/MS to be the hydrolysis products of test item.

- U-3 (retention time of 8.7 minutes) was a minor degradate that reached maximum averages of 1.9 and 8.4% AR in the Pennsylvania and Maryland test systems, respectively. U-4 (retention time of 9 minutes) was another minor degradate that reached maximum averages of 5.9 and 7.5% AR in the Pennsylvania and Maryland test systems, respectively. Both U-3 and U-4 degradates declined by the end of the study.

- Other uncharacterized chromatographic peaks were observed in the Pennsylvania test system, but none exceeded 3.5% AR in any single sample. Uncharacterized chromatographic peaks in the Maryland test system represented at most 3.9% AR in any single sample.

- Bound residues in sediment increased to significant levels only in the Pennsylvania test system, reaching 14.4% AR by 102 DAT. The residual sediments of the final time point (102 DAT) for the Pennsylvania test system were subjected to additional extractions with toluene and tetrahydrofuran solvents, recovering 0.8 and 2.3% AR, respectively. In contrast, the bound residues in the Maryland sediment were minor, increasing up to 3.5% AR by the end of the study (102 DAT).

- Generation of 14CO2 was minor during the study, as recoveries in NaOH traps reached 3.0% AR in the Pennsylvania test system and 2.4% AR in the Maryland test system by the end of the study. No further characterization was performed on the NaOH traps. Negligible amounts of organic volatiles were detected in foam plug trap extracts during the study, representing at most 0.4% AR in any single sample.

Applicant's summary and conclusion

Conclusions:

[14C]Test Item was not persistent in both the Pennsylvania and Maryland test systems under anaerobic conditions, as indicated by the DT50 values representing 56 days and 27 days, respectively. Test item dissipated primarily by formation of U-1, U-2, followed by formation of bound residues and/or ultimately mineralization to carbon dioxide.

Executive summary:

GUIDELINE

The objective of this study was to evaluate the rate and route of degradation of [14C]Test Item in water/sediment systems under anaerobic conditions in accordance with OCSPP guideline 830.4400: Anaerobic Aquatic Metabolism and OECD 308: Aerobic and Anaerobic Transformation in Aquatic Sediment Systems.

 

METHODS 

Two water/sediment systems were freshly collected from sites in Brandywine Creek, Pennsylvania, USA and Choptank River, Maryland, USA. The water/sediment samples were treated with [14C]Test Item and incubated in the dark at 20 °C for periods of up to 102 days. The phenyl ring of the test item was uniformly labelled and is designated as UL-[14C] or [phenyl-U-14C]Test Item. The samples were prepared in biometer-type flasks and periodically flushed with nitrogen to exclude oxygen, then sealed during the incubation period. Sample flasks contained a 10 % aqueous NaOH trap for CO2 and a foam plug for organic volatiles. Radioassay was performed on samples at designated intervals by liquid scintillation counting (LSC).

 

[14C]Test Item and degradates were identified and quantified by high performance liquid chromatography (HPLC) of water layers and sediment extracts with co-injection of test item analytical reference standard. The HPLC assignments of [14C]Test Item and its major transformation products (defined as ≥ 10 % of the dose at any interval) were confirmed/identified by high resolution accurate mass liquid chromatography / mass spectrometry (HR-AM-LC/MS).

 

Mass balance for the study was defined as the sum of the radiocarbon in the water layers, sediment extracts, post-extracted sediment combustions, and volatile traps. At 7 days after treatment (DAT), mass balance recoveries were low, ranging from 58.6 – 89.6% of applied radiocarbon (AR) in all samples. The radiocarbon loss was investigated in the 14 DAT sampling, where anaerobicity measurements were only taken on one of the replicates for each test system (rep B). It was then discovered that the parent compound adhered to the characterization probes (used pH, dissolved oxygen, redox potential readings) due to its hydrophobicity. Therefore, the 7 DAT and 14 DAT rep B samples were unused for kinetics and mass balance calculations, and an additional time point (21 DAT) was added to the study.

 

RESULTS

For the Pennsylvania test system (designated as PA), the average mass balance of radiocarbon was 99.5 ± 2.0% AR (applied radiocarbon) (individual samples ranging from 95.5 to 103.0% AR). For the Maryland test system (designated as MD), the study average for mass balance radiocarbon recoveries was 99.2 ± 1.6% AR (sample range 96.5 – 101.9% AR).

 

Radiocarbon in the water layers decreased in both test systems from averages of 72.3 – 77.5% AR at Time 0 to averages of 23.7% (PA) and 53.2% AR (MD) by the end of the study (102 DAT). Radiocarbon in the sediment extracts increased to maximum averages of 72.0% (PA) and 52.8% AR (MD) at 29 DAT and then decreased to 55.6% (PA) and 37.6% AR (MD) by 102 DAT.

 

Minor amounts of14CO2were detected in the sodium hydroxide traps, increasing up to averages of 3.0 and 2.4% AR by the end of the study (102 DAT) in the PA and MD test systems, respectively. Minor amounts of organic volatiles (≤ 0.4% AR) were detected in foam plug traps during the study for both test systems.

 

Bound residues in sediments represented ≤ 1.0% AR at time 0 across both test systems. Bound residues increased slowly in the MD sediment to average 3.5% AR by the end of the study (102 DAT). In contrast,14C-residues bound quickly to the PA sediment to average 14.4% AR by the end of the study. Due to the high levels of radiocarbon in the PA sediment, both replicates of the residual sediments from the final time point were subjected to additional extractions with anon-polar solvent (toluene) and a polar solvent with a low dielectric constant [tetrahydrofuran (THF)]. The additional extractions only released 0.8% AR in toluene and 2.3% AR in THF.

 

[14C]Test Item was detected at 97.6-100.2% AR (for the total water/sediment system) at time 0 for both test systems. [14C]Test Item declined in both test systems to averages of 41.1% in the PA total system and 21.1% AR in the MD total system at the end of the anaerobic incubation (102 DAT).

 

Disappearance times (DT50, and DT90) for the degradation of [14C]Test Item in the two test systems were calculated using the KinGUI program (Model 2.2012.320.1629) following the FOCUS approach. The calculated disappearance times of [14C]Test Item in the total system, and water layer, were as follows using single first-order (SFO) kinetics: Pennsylvania (PA) total system DT50 = 56 days; Pennsylvania (PA) total system DT90 = 186 days; Pennsylvania (PA) water layer DT50 = 9 days; Pennsylvania (PA) water layer DT90 = 30 days; Maryland (MD) total system DT50 = 27 days; Maryland (MD) total system DT90 = 88 days; Maryland (MD) water layer DT50 = 9 days; Maryland (MD) water layer DT90 = 29 days.

 

[14C]Test Item degraded primarily by hydrolysis of one or both ester bonds as elucidated by LC/MS analysis. The major degradate eluting at approximately 9.5 minutes by HPLC (designated as U-1) was the product of a single ester cleavage of test item, and it increased to maximum averages of 27% (PA) and 40.9% AR (MD) at 21 DAT, and then declined during the remainder of the study to average 14.2% (PA) and 15.9% AR (MD) by the end of the study (102 DAT). The major degradate eluting at approximately 3.5 minutes by HPLC (designated as U-2) was formed from hydrolysis of both ester bonds. U-2 increased throughout the entire study to represent 18.2% AR (PA) and 40.7% AR (MD) by the end of the study. Other minor degradates were observed, but they did not exceed an average of 8.4% AR, and they decreased by the end of the study.

 

CONCLUSION

[14C]Test Item was not persistent in both the Pennsylvania and Maryland test systems under anaerobic conditions, as indicated by the DT50 values representing 56 days and 27 days, respectively. Test item dissipated primarily by formation of U-1, U-2, followed by formation of bound residues and/or ultimately mineralization to carbon dioxide.