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

Endpoint summary

Description of key information

Abiotic degradation; Hydrolysis

The estimated half-life at 25 °C of the test item was determined to be > 1 year at pH 4, 117 h at pH 7 and 96.9 h at pH 9. Rate constants were reported as 5.93 x 10E-03 h-1 at pH 7 and 7.15 x 10E-03 h-1 at pH 9 (OECD 111 and EU Method C.7).

 

Ready biodegradation

The test item attained 0 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable (OECD 301 B and EU Method C.4-C).

Biodegradation in water and sediment

[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.

 

Bioaccumulation

Dietary exposure of fish

Bluegill were exposed to a control and treatment feed for 14 days. Mean measured concentration of the treatment diet was 497 μg/g test item. The growth and lipid corrected BMF value was 0.0032 in analytically determined whole fish tissues in the treatment group. The measured time zero concentration (tissue concentration at Day 14 uptake) in whole fish tissue was 3.10 μg/g, while the derived time zero concentration in whole fish tissue was 0.268 μg/g suggesting the presence of undigested food in the gut tract. When whole fish tissues were analyzed without the gut tract, the mean measured concentration in fish tissue was 1.15 μg/g (OECD 305)

Exposure of sediment organisms

The Day 28 bioaccumulation factor (BAF) for oligochaetes (Lumbriculus variegatus) exposed to sediment spiked with the test item at a nominal concentration of 100 mg test substance/kg dry sediment (mean measured concentration during the uptake phase of 92.1 mg the test item equivalents/kg dry sediment) was 6.40. The accumulation in tissue was swift and the elimination was quick but variable. The non-eliminated residue content on Day 10 of the elimination phase was 34.9%. The uptake rate was 0.670 and the elimination rate was 0.105. The BAFK was 6.35 mg/kg. The lipid content from the organisms found in the four negative control replicates on Day 28 of the uptake phase was determined to be 2.17%. The percent organic carbon in the sediment was 1.5%. The normalized Day 28 BAF, based on the lipid content and percent organic carbon in the sediment was 4.42 (OECD 315).

 

Adsorption coefficient

The adsorption coefficient of the test item was determined to be greater than 1.63 x 10E+04 and log10 Koc was reported as >4.21 (OECD 121 and EU Method C.19).

Additional information

Abiotic degradation; Hydrolysis

Assessment of hydrolytic stability was carried out using a procedure designed to be compatible with Method C7 Abiotic Degradation, Hydrolysis as a Function of pH of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 111 of the OECD Guidelines for Testing of Chemicals, 13 April 2004.

The test system consisted of buffer solutions at pH 4, 7 and 9. The buffer solutions were subjected to ultrasonication and degassing with nitrogen to minimise the dissolved oxygen content.

Sample solutions at pH 4, 7 and 9 were maintained at 50.0 ± 0.5 °C for a period of 192 hours for pH 4 and 120 hours for pH 7 and 9 (preliminary test / Tier 1). Results from the Preliminary test/Tier 1 showed it was necessary to undertake further testing at pH 7 and pH9, with solutions being maintained at 40.0 ± 0.5 °C, 50.0 ± 0.5 °C, 60.0 ± 0.5 °C and 70.0 ± 0.5 °C for various periods of time (Tier 2). Identification of hydrolysis products was then assessed (Tier 3).

Sample solutions were taken from the water bath at various times and the pH of each solution recorded. The concentration of test item in the stock solutions and sample solutions was determined by high performance liquid chromatography – mass spectrometry (HPLC-MS).

The estimated half-life at 25 °C of the test item was determined to be > 1 year at pH 4, 117 h at pH 7 and 96.9 h at pH 9. Rate constants were reported as 5.93 x 10E-03 h-1 at pH 7 and 7.15 x 10E-03 h-1 at pH 9 (OECD 111 and EU Method C.7).

Ready biodegradation

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method was designed to be compatible with OECD Guidelines for Testing of Chemicals (1992) No 301B "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4 -C of Commission Regulation (EC) No 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph m).

An initial experiment was conducted at a concentration of 10 mg carbon/L. The toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25 % biodegradation after 14 days. These results indicated that, under the strict terms and conditions of the OECD guidelines, the test item would be classed as exhibiting inhibitory effects.

Therefore, following the recommendations of the test guidelines, in the definitive test, the test item, at a reduced concentration of 5 mg carbon/L was exposed to activated sewage sludge microorganisms with mineral medium in sealed culture vessels in the dark at temperatures of between 19 and 22 °C for 28 days.

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

The test item attained 0 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B. It is also clear from the test item results that some inhibition of the activated sewage sludge microorganisms had occurred as the CO2 evolution rates in the test item vessels were lower than those in the control vessel after 28 days. Care should therefore be taken in the interpretation of the results due to the inhibitory nature of the test item to the activated sewage sludge.

The test item attained 0 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.

Biodegradation in water and sediment

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.

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.

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.

[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.

Bioaccumulation

Dietary exposure of fish

The objective of this study was to obtain laboratory data characterizing the bioaccumulation potential of test item in the bluegill,Lepomis macrochirus. The protocol was based on procedures outlined in OECD Guidelines for Testing of Chemicals, Guideline 305:Bioaccumulation in Fish: Aqueous and Dietary Exposure.As the BMF is a comparison of the concentration of a substance in an organism with that in the organism’s food, lipid is taken into account by correcting for the contents of lipid in the organism and in the food.

 

The test was divided into two phases: uptake and depuration. During the uptake phase, bluegill were exposed an isotopic mixture of test item in diet at a sub-lethal concentration. The bluegill in the control group were exposed to an untreated diet. The nominal concentration of500 μg/g test item was selected in consultation with the Sponsor. Each group consisted of one test chamber with 70 fish in each chamber. During the depuration phase, fish were exposed to an untreated diet only. The duration of the uptake phase was 14 days and the depuration phase was 12 days. During both phases of the test, test organisms and water samples were collected and analyzed for 14C radioactivity. These values were used to determine the growth-corrected substance-specific half-life (t1/2g, from the growth-corrected elimination rate constant, k2g), the assimilation efficiency (absorption across the gut; α), the kinetic biomagnification factor (BMFK) and the lipid-corrected kinetic biomagnification factor (BMFKL) for whole fish tissues.

 

Bluegill were exposed to a control and treatment feed for 14 days. Mean measured concentration of the treatment diet was 497 μg/g test item. The growth and lipid corrected BMF value was 0.0032 in analytically determined whole fish tissues in the treatment group. The measured time zero concentration (tissue concentration at Day 14 uptake) in whole fish tissue was 3.10 μg/g, while the derived time zero concentration in whole fish tissue was 0.268 μg/g suggesting the presence of undigested food in the gut tract. When whole fish tissues were analyzed without the gut tract, the mean measured concentration in fish tissue was 1.15 μg/g.

Exposure of sediment organisms

The objective of this study was to determine the bioaccumulation potential of the test item in the oligochaete, Lumbriculus variegatus, through sediment exposure. The protocol was based upon the OECD Guidelines for Testing of Chemicals, Guideline 315: Bioaccumulation in Sediment-dwelling Benthic Oligochaetes

 

An initial trial was conducted but was terminated early and repeated due to high mortality in the treatment group. The test concentration was lowered for the definitive test.

 

The test was divided into two phases: the uptake (exposure) phase and the elimination (post-exposure) phase. During the uptake phase, oligochaetes were exposed to one sub-lethal test concentration and a negative and solvent control. The nominal test concentration selected in consultation with the sponsor was 100 mg the test item mg/kg of sediment based on the dry weight of the sediment. Oligochaetes in the solvent control group were exposed under identical conditions without test substance, but with the same amount of solvent used in the treatment group. Oligochaetes in the negative control group were exposed without test substance or solvent. Each test chamber contained the same quantities of sediment and overlying water. Thirty-six replicate test chambers were prepared for both the treatment group and the solvent control group for exposure of the test organisms (i.e. 18 replicates for sampling during the uptake phase plus 18 replicates for sampling during the elimination phase). An additional three replicates were prepared without organisms for the treatment group and the solvent control group for analytical sampling on Day 0. Four replicate test chambers were prepared for the negative control group for sampling at the end of the uptake phase.

 

The results of the study are based on the mean measured test concentrations in the sediment during the uptake phase. The duration of the uptake phase typically will vary according to the time required to reach steady-state, but was not to exceed 28 days. Sediment and overlying water samples were collected and analyzed from three sacrificed replicates of the treatment group and solvent control group on Days 0, 1, 3, 7, 14, 21 and 28 of the uptake phase. Results of the analyses in the sediment were used to verify the exposure over time. Additionally, sediment and overlying water samples were collected on Days 0, 1, 3, 5, 7 and 10 of the elimination phase. The duration of the elimination phase typically will vary according to the time required to reach 10% of the concentration measured in tissues at the end of the uptake phase, but was not to exceed 10 days. Worm tissue samples were collected from the culture on Day 0 and from three replicates from the treatment group and solvent control group on days 1, 3, 7, 14, 21 and 28 of the uptake phase and on days 0, 1, 3, 5, 7 and 10 of the elimination phase. An additional four replicates from the negative control group were sacrificed at the end of the uptake phase for the determination of lipid content in the tissue. The tissue concentrations were used to calculate the uptake rate constant (ks), the elimination rate constant (ke), and the kinetic bioaccumulation factor (BAFK). The bioaccumulation factor (BAF) was calculated, based on the concentration of the test item in the oligochaetes compared to the concentration of the test item in the sediment. Additionally, the residue level in the oligochaetes at the end of the elimination phase (non-eliminated residue; NER) was also determined.

 

The Day 28 bioaccumulation factor (BAF) for oligochaetes (Lumbriculus variegatus) exposed to sediment spiked with the test item at a nominal concentration of 100 mg test substance/kg dry sediment (mean measured concentration during the uptake phase of 92.1 mg the test item equivalents/kg dry sediment) was 6.40. The accumulation in tissue was swift and the elimination was quick but variable. The non-eliminated residue content on Day 10 of the elimination phase was 34.9%. The uptake rate was 0.670 and the elimination rate was 0.105. The BAFK was 6.35 mg/kg. The lipid content from the organisms found in the four negative control replicates on Day 28 of the uptake phase was determined to be 2.17%. The percent organic carbon in the sediment was 1.5%. The normalized Day 28 BAF, based on the lipid content and percent organic carbon in the sediment was 4.42.

Adsorption coefficient

The determination was carried out using the HPLC screening method, designed to be compatible with Method C.19 Adsorption Coefficient of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 121 of the OECD Guidelines for Testing of Chemicals, 22 January 2001.

 

The test utilised a High Performance Liquid Chromatograph and a commercially available cyanopropyl reverse phase HPLC column containing lipophilic and polar moieties was used. The dead time was determined by measuring the retention time of formamide (purity quoted by supplier: 99.94%) at 612 mg/L in methanol:water (55:45 v/v). Solutions of reference standards were prepared in methanol and test item (0.1039 g) was diluted to 100 mL with methanol.

 

The adsorption coefficient of the test item was determined to be > 1.63 x 10E04 with Log10 Koc > 4.21.