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Environmental fate & pathways

Biodegradation in water: screening tests

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Description of key information

Using standard ready biodegradation test conditions, the test chemical displays negligible degradation. As a consequence the biodegradation potential of DAPD was further assessed in a number of inherent biodegradation studies.
In these inherent biodegradation tests it was demonstrated that slow inherent biodegradation of DAPD does occur, yielding 23 to 37% of mineralisation after 56-63 days. Furthermore, primary degradation of DAPD into water soluble metabolites was found to occur much faster and lead to the complete removal of the parent compound from the test system within 28 days.

This was confirmed in a simulation study according to OECD 309, from which a worst-case DT50 of 11.1 (12°C, low concentration run) and 29.6 (12°C, high concentration run) was determined.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

Two studies are available addressing the ready biodegradability of 1,4 -benzenediamine, N,N’-mixed Ph and tolyl derivs. (DAPD) (Kung, 1995 and Hartmann, 1990). In both studies, the test chemical was subjected to aerobic degradation conditions in the presence of activated sludge for 28 days using the oxygen uptake monitoring. Under these conditions, no biodegradation of 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. was observed.

 

Both tests fulfil the validity criterion as the used positive control substance showed a high degree of biodegradation. In the study by Kung (1995) one of the test flasks contained test chemical, control substance and inoculum. From this test run it could be concluded that 1,4 -benzenediamine, N,N’-mixed Ph and tolyl derivs. did not show microbial toxicity, as the control substance was degraded without any problem. It should be noted however, that these tests require the use of high levels of test compound (100 mg/L) in incubation media, far in excess (> 100 x) the water solubility of the the constituents of DAPD. As biodegradation can only occur for chemicals in solution, this test might not be the most appropriate one to evaluate the chemicals (realistic) biodegradation potential.

 

As a consequence the biodegradation potential of DAPD was further assessed in a number of inherent biodegradation studies. In these studies a surfactant is added to the test bottles in order to enhance the water solubility of the test substance, a lower concentration of test substance is used and an elevated sludge concentration when compared to a ready biodegradation test.

 

In total 3 studies have been undertaken to assess the inherent biodegradability of1,4 -benzenediamine, N,N’-mixed Ph and tolyl derivs. (DAPD (Commander and Daniel, 2011a, Commander and Daniel 2011b, Commander, Daniel and Mc. Cormack 2011). In the first study (Commander and Daniel, 2011a), the oxygen uptake of the test substance was again chosen as the parameter to be monitored. In this test, no degradation was observed at a DAPD concentration of 20 mg/L. Additionally, the test substance appeared to have some inhibitory effect on the respiration rate of the inoculum. There was, however, sufficient viability in the inoculum to degrade sodium benzoate when this positive control substance was added to the test vessels after 11 days of incubation.

 

The second test (Commander and Daniel, 2011b) made use of radiolabelled test substance R898. The formation of 14C-CO2 and the distribution of the remaining 14C over the aqueous phase and the sludge solids were examined. Due to the higher sensitivity of the methods used for the quantitation of the test chemical and the degradation products (14C-CO2) a test concentration closer to the water solubility could be implemented. As a consequence, in this experiment test substance concentrations of 100, 10 and 1 µg/L were used. The test results showed about 23% of mineralisation (CO2-formation) after 56 days, which demonstrates the inherent biodegradation potential of DAPD. Furthermore, it was found that a large portion of the radioactivity remained associated with the sludge solids, whereas no parent compound could be detected in the aqueous phase of the test system after 28 days.

 

The third test (Commander, Daniel and Mc. Cormack 2011) used the same principle and test substance concentrations (100 and 10 µg/L) as the previous one, but additional effort was put into the analysis of breakdown products. In this study, the maximum mineralisation was observed in the10 µg/L experiment) and yielded up to an average of 37% in 63 days. Furthermore, incorporation of the test substance into intracellular components (DNA/RNA, lipids, proteins et cetera) of the biomass was observed. It was also confirmed that no test substance remained in the aqueous test phase, although 14C levels of up to 55% of the applied radioactivity were found in the aqueous fraction. This suggests that water soluble metabolites were formed. However, due to their multiplicity and/or difficulties with the chemical separations they could not be characterized.

 

A water simulation test has been conducted according to OECD 309 to assess the degradation potential of R898 in freshwater. For this test, the R898 constituent has been selected as the worst-case constituent from DAPD. The study has been conducted with radio-labelled R898 (label on the outer rings of the molecule). Samples were incubated at 12°C at a low (8 µg/L) and high (40 µg/L) concentration for the purpose of determination of the DT50 of the parent. Fitting the pseudo first-order kinetics to the parent concentrations (using CAKE model) results in following DT50 values: 11.1 days for the low concentration sample set and 29.6 days for the high concentration sample set. For both the conclusion is hence that the substance is not persistent in freshwater. During up to 60 days of incubation, the parent substance was degraded to several transformation products. An immediate oxidation to N,N´-Bis(1-methylphenyl)-1,4-cyclohex-(2,5)diene-diimine (diimine species) was observed already in the 0d samples. Further degradation products were observed in course of the incubation. One, o-toluidine, was characterised by co-chromatography with a reference standard. A part of the degradation products could not be chromatographed as distinct signals on HPLC, but was forming a background signal eluting within a retention time range of approx. 10 minutes. It is assumed that this signal is originating from different degradation products representing small amounts of the applied radioactivity. For quantification the background radioactivity signal was divided up into retention time intervals. Up to 16 metabolite signals or retention time intervals were detected. Using high resolution mass spectrometry (HR-MS/MS) at least a molecular formula was proposed for the most metabolite signals or retention time intervals. For 5 metabolites also a molecular structure was suggested. These metabolites represent degradation products formed by subsequent degradation of the diimine species, indicating that the primary oxidation is followed by further degradation steps.

The obtained data sets were analysed using the program CAKE version 3.3. As it could not be fully excluded that the formation of diimine species at least partially occurs during sample work-up; this fast first degradation step was not considered in the kinetic evaluation. Instead the detected amounts of R898 and diimine species were merged and both considered as parent test item within the kinetic evalutation. The kinetic models considered for the analysis were SFO (Single First Order), DFOP (Double First Order in Parallel), HS (Hockey Stick), and FOMC (First Order Multi Compartment). According to the results the best fitting results were obtained when considering SFO kinetics. The following DT50 values were obtained from the 12°C tests: 11.1 days for the low concentration sample set and 29.6 days for the high concentration sample set.

Overall, it can be concluded from the full set of experimental results that DAPD does not fulfil the criteria for ready biodegradation. Nevertheless, slow inherent biodegradation of DAPD does occur, yielding 23 to 37% of mineralisation after 56-63 days in an inherent biodegradation test. Furthermore, primary degradation of DAPD into water soluble metabolites was found to occur much faster and lead to the complete removal of the parent compound from the test system within 14 days. The DT50 values obtained in the water simulation test according to OECD 309 are below 40 days, and therefore provide additional evidence that the substance is not persistent in freshwater.