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

Endpoint summary

Description of key information

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.

Additional information

Ready biodegradation

GUIDELINE

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).

METHODS

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.

RESULTS

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 microorganisms used in the study.

CONCLUSION

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

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.