Reaction products of diphenylamine with nonene, branched

Biodegradation in water and sediment: simulation tests

Description of key information

No significant mineralisation was observed in a surface water simulation degradation study according to OECD 309 at 12°C.

Additional information

Aerobic mineralization of the 14C radiolabelled UVCB substance in surface water amended with small amounts of suspended sediment was investigated under defined laboratory conditions at 12°C for 60 days. Two test item concentrations were tested (2 and 10μg/L).14C aniline was used as reference substance. Radioactivity in the water phase, the suspended sediments and solvent extractions from test vessels was measured by Liquid scintillation counter. Mineralization was determined by measuring 14CO₂in absorption liquids.

Analytical investigations of the test substance and potential degradation products was performed with radio-HPLC and LC-MS/MS.

The total mean recoveries were 65-86% of the total applied radioactivity (TAR) for the high dose and 84-95% TAR for the low dose experiments.

Formation of radioactive carbon dioxide did not exceed 1%TAR in any of the doses.

 

Results of the control substance

About 25 %TAR was measured as 14CO₂from test assays with the control substance 14C aniline. 14CO₂production in the test assays with control substance and solvent (ethanol) was doubled compared to the control assays without solvent, indicating co-metabolism or enhancement of aniline degraders. However, biodegradation of 14C-aniline as control substance in control assays as well as solvent control assays confirmed the biological activity in the test system. 86 x 10³colony forming units (cfu) per mL was measured at the begin of exposure from the test system and 69 x 10³cfu/mL at the end of exposure.

 

In the water phase the radioactivity ranged from 35 to 59% TAR in the high dose and from 22 to 69%TAR in the low dose.

The radioactivity of the suspended solids measured in pellets after centrifugation of the water phase was 0.1 – 11% TAR for the high dose and 0.2 – 16% TAR for the low dose.

Since the uvcb substance and its constituents are known to have a high adsorption potential to various surfaces, it was assumed that the missing radioactivity was still present in the test vessel and tightly adsorbed to the glass wall. Several extraction steps were performed to achieve the final recovery.

The radioactivity of washing fractions of the test vessels was ca. 5 – 24% TAR for the high dose and 5 – 50% TAR for the low dose.

 

The uvcb substance consists mainly of two branched isomer groups either monoalkylated or dialkylated. During analytical investigations of the water phase it could be observed that the two main isomer groups decreased during the study, the monoalkylated faster than the dialkylated isomer group. Further peaks were observed from day 0 to day 60 (high dose) and day 55 (low dose), respectively. In the high dose the radioactivity in the water phase extracts for analytical investigation decreased over 60 days. The concentrations of both isomer groups decreased but the increase of further peaks was lower than the decrease of parent substances. Isomer group 1 could not be identified as distinguished peak from day 55 to day 60.

 

In the low dose the radioactivity in the water phase extracts for analytical investigation decreased over 60 days. The concentrations of both isomer groups decreased but the increase of further peaks was lower than the decrease of parent substances. Isomer group 1 could not be identified as distinguished peak from day 7 to day 60.

 

Decrease of the parent isomer groups and further peaks were also observed in the sterile samples with a high test concentration after 60d. The presence of further peaks (7-8% TAR) in sterile test assays indicate additionally abiotic degradation processes.

Peak detection of these radioactive fractions was at the edge of the analytical limit of quantification, and radioactive fractions of further peaks could not be separated further into individual peaks and identified.

 

The DT50s in the water phase were 0.1-5.1d for isomer group 1 and 4.2-130d for isomer group 2.  

 

As the test item is an uvcb substance which consists of isomer groups of different size and solubility in water, decrease of the concentration of the constituents due to dissolution, degradation and adsorption is difficult to differentiate. The dissolution behaviour of the single constituents can affect the solubility of the other constituents in the uvcb substance. The solubility of the single constituents in the current surface water test system is not known. Based on QSAR data, isomer group 1 with the lower molecular weight has a higher solubility and therefore bioavailability for degradation than isomer group 2 with a higher molecular weight. The ratio of the different constituent groups in the uvcb substance can change in aquatic media because of this effect, which makes it difficult to interpret results on half-life in degradation tests with uvcb substances. However, based on the available data it can be assumed that the isomer group 1 will disappear faster in aquatic media than isomer group 2. This disappearance is probably due to degradation processes but other processes like adsorption are overlaying these effects.

  

The constituents of the uvcb substance have high sorption capacities. Although the test concentrations of 2 and 10μg/L are very low, the long incubation in an aqueous medium obviously lead to a slow but continuous sorption at the inner surface of the test vessel which resulted in a slow decrease of material balance.

However, the distribution of radioactivity in the test systems clearly show that the applied test conditions as required by the Guideline OECD 309 are not suitable to investigate the biodegradation of strong sorbing substances in aqueous environments. It can be concluded that the portion of substance sorbed to the test vessel surface was not further available for microbial degradation processes.

The interpretation of the data was very complex or even impossible as the parent substance is an uvcb substance and degradation pathways of constituents as well as adsorption processes of the different degradation products and the parent constituents itself cannot be distinguished when testing a uvcb substance.

Furthermore, the results of the control substance indicate worst case conditions for biodegradation in the test which can be explained with the low test temperature and the low concentration of suspended particulate matter of 15 mg/L in the test.