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EC number: 273-227-8
CAS number: 68953-84-4
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
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.
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
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.
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
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.
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
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
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.
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