1. Biodegradability of the antioxidant diaryl-p-phenylene diamine using a
modified inherent biodegradation method at an environmentally
relevant concentration
R. Dailey a
, M. Daniel b,⇑
, A.P. Leber c
a
The Goodyear Tire & Rubber Company, 1485E Archwood Avenue, Akron, OH 44306, USA
b
Brixham Environmental Laboratory, Freshwater Quarry, Brixham, Devon TQ5 8BA, UK
c
Contributing Consultant to The Goodyear Tire & Rubber Company, Diplomat-AM. Board of Toxicology, 1344 Jefferson Avenue, Akron, OH 44313, USA
h i g h l i g h t s
The antioxidant DAPD was degraded in a modified inherent biodegradability test.
No parent compound was measured after 28 d.
37% Mineralisation was measured after 63 d and 29% incorporation into biomass.
Silica gel and surfactant were used to increase bioavailability.
a r t i c l e i n f o
Article history:
Received 19 December 2012
Received in revised form 17 May 2013
Accepted 25 May 2013
Available online xxxx
Keywords:
Diaryl-p-phenylene diamine
Degradation
Environmental
Antioxidant
DAPD
Polystay
a b s t r a c t
The chemical product diaryl-p-phenylene diamine (DAPD), produced by The Goodyear Tire Rubber
Company as POLYSTAY 100Ò
(CAS 68953-84-4), is employed as an antidegradant in polymers used in
tires and industrial rubber products. Previous evaluations pertaining to the ecological fate of DAPD indi-
cated a lack of biodegradative activity in aquatic media. In order to further pursue the biodegradation
potential of DAPD, it was deemed necessary to enhance the sensitivity of the aquatic biodegradation
assay through (a) employment of a radiotracer of the test substance, and (b) optimisation of conditions
for achieving maximal solubilisation of test material in the aquatic media of the incubation vessels. Test
vessels were prepared according to the OECD ready biodegradability test guidelines, with DAPD added on
silica gel at concentrations of 10 or 100 lg LÀ1
, together with a surfactant to aid solubilisation. After 63 d
incubation up to 37% mineralisation was measured and up to 29% of the applied radioactivity was incor-
porated into cell biomass. Also, after 28 d no DAPD could be measured in solution by radio-TLC and
HPLC–MS. These three results demonstrate that the antioxidant DAPD undergoes microbiologically med-
iated biodegradation and is highly unlikely to persist in the environment.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The chemical product diaryl-p-phenylene diamine (DAPD), pro-
duced by The Goodyear Tire Rubber Company as POLYSTAY 100Ò
(CAS 68953-84-4), is employed as an antidegradant in polymers
used in tires and industrial rubber products. Historically, various
aromatic amines have served this purpose, but one substance used
previously in this application (2-naphthylamine) was considered
an occupational hazard due to its carcinogenic activity (NTP,
2011). In contrast, DAPD has been subjected to chronic toxicity
studies, and exhibited no evidence of carcinogenicity or other sig-
nificant long-term health effects (Iatropoulos et al., 1997).
More than eighty percent of the manufactured product consists
of three constituents with the structures below (Fig. 1). In contem-
porary terminology, it is considered to be a ‘‘multi constituent sub-
stance’’ (ECHA, 2008). In addition to these three components, the
product contains 20% of higher molecular weight compounds
and trace levels of starting reactants, e.g., aniline.
Previous evaluations assessing the ecological fate of DAPD in a
standard biodegradation test indicated a lack of biodegradative
activity in aquatic media (Ricerca, 1995). Important physical prop-
erties that plausibly influenced this inactivity in standard assays in-
clude the components’ low water solubilities (1 mg LÀ1
), high
logKow values (P3.3), and elevated logKoc values (4.3) (Chemex,
2010a). These chemical structures are not compatible with abiotic
hydrolysis. Known oxidation products of many aromatic amines
are phenolic and quinone substances, which have higher polarity
0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemosphere.2013.05.072
⇑ Corresponding author. Tel.: +44 1803 884315; fax: +44 1803 882974.
E-mail address: maggie.daniel@astrazeneca.com (M. Daniel).
Chemosphere xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
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Please cite this article in press as: Dailey, R., et al. Biodegradability of the antioxidant diaryl-p-phenylene diamine using a modified inherent biodegrada-
tion method at an environmentally relevant concentration. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.072

2. and lower partition coefficients than the parent compound, leading
to the possibility that these compounds are more biodegradable
(Kuczkowski, 2003). In a fish bioaccumulation test performed with
radiolabelled R898, rapid depuration of radioactivity was observed
(t½ 5 d), consistent with the formation of a polar metabolite(s)
(Brixham Environmental Laboratory, 2011). Additional work is
required to identify metabolic products occurring in the fish. Past at-
tempts to assess the microbial biodegradation of the chemical
according to standard testing guidelines, such as the OECD 301B car-
bon dioxide evolution test (OECD, 1992) have not shown activity in
sludge-inoculated incubation media from sewage treatment plants.
The method included exposure of 30 mg test chemical per liter in
biodegradation incubation flasks which greatly exceeded the water
solubility of the chemical’s constituents. The absence of measurable
CO2 release in this assay indicated negligible ultimate degradation
under these assay conditions (Ricerca, 1995). No assessments of pri-
mary degradation i.e. removal of DAPD were made.
Toxicity testing in aquatic and terrestrial tests has shown the
toxicity of DAPD is very different in the presence or absence of sed-
iment or soil. Previous testing in aquatic toxicity tests to algae,
daphnia and fish displayed high toxicities (EC50s 1 mg LÀ1
) (Che-
mex, 2010b; Springborn Laboratories, 1995a,b, 1996, 1997a). How-
ever, testing of the chemical in the presence of sediment and soil
totally eliminated toxic activities (NOECs = 1000 mg kgÀ1
soil and
sediment) in resident species (chironomids and earthworms) (Che-
mex, 2010c; Mambo-Tox Ltd, 2010a, 2010b). These toxicity data
suggest that the strong adsorption of DAPD (supported by logKoc
values P 4.39) to sediment in aquatic systems attenuates the
chemical’s presence in the water column, and the level of toxicity
of the chemical in standard laboratory aquatic testing in the ab-
sence of sediment lead to results that do not represent bona fide
ecological conditions. While chironomid and earthworm species
may be more resistant to DAPD exposures and toxicity than aqua-
tic species, the very marked potencies observed for the aquatic
species suggest other factors are important in the attenuation of ef-
fects observed in soil/sediment species.
In order to further pursue the biodegradation assessment of
DAPD, it was deemed necessary to enhance the sensitivity of aqua-
tic biodegradation testing through (a) employment of a radiotracer
of the test substance, and (b) optimisation of conditions for achiev-
ing maximal solubilisation of test material in the aquatic media of
incubation vessels. The former was done through the de novo syn-
thesis of carbon-14 labeled R-898 (di-o-tolyl-p-phenylene dia-
mine, CAS 15017-02-4) with the central ring being the site of
carbon 14 labeling. A combination of surfactant plus silica gel sub-
strate was employed to enhance solubilisation of the radiolabelled
test chemical in the aquatic media as permitted by the REACH
technical guidance for degradation and persistence assessments
(ECHA, 2008). The choice of R-898, which constitutes approxi-
mately 20% by mass of the antioxidant POLYSTAY 100Ò
, as the sen-
tinel chemical for DAPD for this assay was based upon the fact that
R-898 is the component with the highest partition coefficient and
lowest water solubility, and was projected to be the least likely to
biodegrade due to its higher degree of methyl substitution com-
pared to other product components. Previous laboratory assays
using standard ready biodegradability methods showed DAPD
was not readily biodegradable, raising the possibility that it may
be persistent in the environment. However, due to the known abi-
otic oxidative degradation of this substance in its role as a polymer
antioxidant, it was considered important to reassess degradation of
DAPD including modifications and enhancements described in the
REACH guidance (ECHA, 2008). The modifications chosen were (a)
testing at a low concentration using a radioisotope, (b) adding the
test substance on an inert support (silica to enhance surface area
exposure to incubation media), and (c) use of a surfactant to ele-
vate solubilisation. The test was also enhanced by increasing expo-
sure time to 63 d, plus use of one treatment (5 replicates) with a
higher inoculum concentration.
2. Materials and methods
2.1. Materials
Radiolabelled N,N0
-di-o-tolyl,-p-[U-14
C]phenylene diamine (R-
898; CAS 15017-02-4) was obtained from Selcia Limited, Ongar,
UK and had a specific activity of 18.7 lCi mgÀ1
. The radiochemical
purity of the R-898 was determined as 95.5%. Silica gel, Synperonic
PE105 surfactant, and mineral salts were obtained from Sigma–Al-
drich, Poole, UK. Reverse osmosis water with a conductivity of
15 lS mÀ1
was obtained from the in-house reverse osmosis
system.
Radiolabelled benzoic acid was obtained from American Radio-
labeled Chemicals, St. Louis, USA, and was used as a positive con-
trol substance, to demonstrate activity of the inoculum. This was
combined with non-radiolabelled sodium benzoate from Sigma–
Aldrich, Poole, UK before dosing, to adjust the amount of radioac-
tivity to the required test concentration.
Activated sludge was collected from the aeration basin of a
waste water treatment plant at Totnes, Devon, UK, a plant which
treats sewage of predominantly domestic origin.
The mineral medium was made up according to the OECD 302C
guideline (OECD, 1981) and contained the following nutrients per
litre of reverse osmosis water: 25.5 mg of KH2PO4, 65.25 mg of
Fig. 1. Components of POLYSTAY 100Ò
.
2 R. Dailey et al. / Chemosphere xxx (2013) xxx–xxx
Please cite this article in press as: Dailey, R., et al. Biodegradability of the antioxidant diaryl-p-phenylene diamine using a modified inherent biodegrada-
tion method at an environmentally relevant concentration. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.072

3. K2HPO4, 66.54 mg of Na2HPO4Á2H2O, 67.5 mg of MgSO4Á7H2O,
109.2 mg of CaCl2Á2H2O, 0.75 mg of FeCl3Á6H2O (all from Sigma–
Aldrich).
2.2. Experimental set up
One litre test vessels were used, each containing mineral med-
ium, activated sludge, and either the antioxidant [14
C]R-898 or the
reference substance, at total volumes of 800 mL and the concentra-
tions shown in Table 1. Control vessels without test or reference
substance were not required, as the fate of the chemicals were fol-
lowed by radioactivity. The vessels were stirred and purged contin-
uously with humidified air at a rate of 30–100 mL minÀ1
. Vent
gases passed through activated carbon traps and 2 M sodium
hydroxide traps to collect volatile organic carbon compounds,
and carbon dioxide, respectively. Vessels were incubated, in the
dark, at nominal 25 ± 2 °C. The experimental set up is shown in
Fig. 2. To minimise the potential of R-898 adsorbing to the glass
surfaces the incubation vessels, all vessels used for preparing test
chemical solutions, and other equipment which came into contact
with test chemical were silanised before use. This was done by
soaking the glass items for at least 1 h with a 5% solution of dichlo-
rodimethylsilane in toluene, rinsing with methanol, and air drying.
A surfactant, Synperonic PE105 at 15 mg LÀ1
, and silica gel were
used to enhance the bioavailability of [14
C]R-898 and improve dis-
persion within the test system, as permitted in the OECD 301 Test
Guideline (OECD, 1992), ISO 10634 Standard (ISO, 1995) and
REACH Guidance (ECHA, 2008).
[14
C]R-898 was introduced into the biodegradation test vessels
on silica gel. Solutions of [14
C]R-898 were prepared in acetone at
6230 and 663 mg LÀ1
for the 100 and 10 lg LÀ1
test concentrations,
respectively, and 12.5 lL aliquots applied to individually weighed
silica gel samples (264 mg) in glass weighing boats. Care was taken
to ensure the solution was applied to the gel only, and did not
come into contact with the glass boats. The acetone was evapo-
rated by (i) air-drying at room temperature for approximately
1 h, leaving defined quantities of [14
C]R-898 coating each silica
gel sample, and (ii) oven-drying overnight at nominal 35 ± 2 °C.
This procedure ensured the acetone was completely removed. On
day 0 of the biodegradation study the [14
C]R-898-coated silica
gel was mixed to ensure uniform dispersal of R-898 and added to
the solutions of mineral medium, microbial inoculum and surfac-
tant in the incubation vessels.
The exposure time was extended to approximately 60 d, an
enhancement recommended in the REACH guidance for persis-
tency assessment for poorly water soluble chemicals (ECHA,
2008). This extended test period allowed sufficient time for (a)
the enrichment of a small community of competent microbial
degraders, and (b) slower rates of degradation related to mass
transfer issues and solubilisation of the test compound.
In a separate study the antioxidant [14
C]R-898 was introduced
into empty incubation vessels as a 743 mg LÀ1
solution in acetone,
sufficient to give a test concentration of 100 lg LÀ1
. The acetone
was then evaporated by air-drying at room temperature for
approximately 1 h, leaving a coating of [14
C]R-898 on the interior
surfaces of the vessels. To ensure the acetone was completely re-
moved the vessels were dried overnight in an oven at nominal
35 ± 2 °C. The biodegradation test was commenced the following
day after the addition of the OECD 302C mineral medium, micro-
bial inoculum at 300 mg LÀ1
dry suspended solids, and surfactant.
The experimental set up was as described above. Further additions
of [14
C]R-898 were made to two of these vessels after 28 and 49 d
incubation, by adding [14
C]R-898 on silica gel, applied using the
method described above, each equivalent to 100 lg LÀ1
[14
C]R-
898. This approach was intended to show if R-898 exposure re-
Table 1
Experimental design.
Number
of
replicates
Dry sludge
solids
concentration
(mg/L)
[14
C]R-898
concentration
(lg/L)
Reference
substance
concentration
(lg/L)
Mineralisation
(%)
5 30 10 0 37
5 30 100 0 27
5 300 100 0 15
3 30 0 100 76
ORBOTM
tubes
Empty
trap
Empty
trap
2M
NaOH
Trap 1
2M
NaOH
Trap 2
Central
vacuum
Empty
trap
Water
Test vessel
(stirred)
Water: Reverse osmosis water to humidify the influent air
OrboTM tubes : Activated carbon traps to capture evolved volatile organic material (Orbo32 followed by Orbo 91)
Empty traps: Empty traps to avoid siphoning of sodium hydroxide into test vessel or being sucked into the vacuum pump
Traps 1 and 2:2M NaOH to trap evolved 14CO2
Fig. 2. Experimental set up.
R. Dailey et al. / Chemosphere xxx (2013) xxx–xxx 3
Please cite this article in press as: Dailey, R., et al. Biodegradability of the antioxidant diaryl-p-phenylene diamine using a modified inherent biodegrada-
tion method at an environmentally relevant concentration. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.072

4. sulted in either the enrichment of a competent microbial popula-
tion or inactivation of the inoculum within a relatively short time
period for an inoculum source derived from a domestic site.
2.3. Sampling
At intervals during the biodegradation study (Days 1, 3, 7, 14,
21, 28, 56 and 63) 14
CO2 captured in the sodium hydroxide traps
was sampled and quantified by Liquid Scintillation Counting
(LSC, using a Tri-Carb 2810TR counter). Subsamples of 50 mL of
test solution were taken from the test vessels after 1 and 6 h incu-
bation, and on Days 1, 3, 7, 14, 21 and 28.
The subsamples were filtered through Whatman No 4 filter pa-
pers and the aqueous filtrate collected. The suspended solids col-
lected on the filter papers were rinsed with 10 mL aliquots of
acetone, then dichloromethane (DCM), in order to remove free test
chemical and possible breakdown products, solvent rinses were
collected. The water filtrate and solvent rinses were analysed by
LSC, and residual R-898 in the water and acetone was quantified
by radio-TLC. Subsamples were applied to Si gel 60 RP-18F254
plates prior to elution with 90:10:1 (v/v) acetonitrile:RO water:for-
mic acid. Radioactivity on the plate was visualised and the radioac-
tive intensities quantified using a Fuji FLA7000 phosphorimager.
The filter papers were combusted on a Packard 307 sample oxidiser
for up to 15 min, until combustion was complete, to determine the
radioactivity associated with suspended solids.
At the end of the study the entire bottle contents were filtered
through Whatman No 4 filter papers, and subjected to the same
procedures as had been used on the subsamples taken previously.
The activated carbon tubes were extracted by back flushing with
acetone and analysed by LSC to determine if any volatile organic
material was purged from the test system during the test period.
The suspended solids from duplicate vessels from each treatment
were scraped off filter papers and the biomass fractionated, using
the method described by Federle and Itrich (1997) to determine
if the radioactivity had been incorporated into the biomass of the
microbial inoculum.
In the fractionation process the suspended solids were trans-
ferred to micro-centrifuge tubes, then extracted using the solvents
(1 mL aliquots) and conditions shown in Table 2. After each extrac-
tion the samples were centrifuged, the supernatants removed and
analysed by LSC. The subcellular fractions associated with each
extraction stage are also shown in Table 2.
3. Results
3.1. Mineralisation to CO2
The [14
C]benzoic acid reference substance showed extensive
carbon dioxide evolution, or mineralisation, 76% by Day 14 of the
study when these vessels were terminated, as they had shown
the activated sludge microorganisms were viable. Benzoic acid is
soluble in water (2.9 g LÀ1
at 20 °C), with a logKOW of approxi-
mately 1.9 (Wibbertmann et al., 2000), and hence is readily avail-
able to the activated sludge microorganisms.
Mineralisation of the antioxidant [14
C]R-898, was observed in
all the treatments (Table 1 and Fig. 3). The most extensive miner-
alisation, 37% on Day 63, was in the lowest [14
C]R-898 concentra-
tion (10 lg LÀ1
), which is below its water solubility limit
(110 lg LÀ1
), and much lower than the concentrations recom-
mended in standard biodegradation tests (10–100 mg LÀ1
). This
finding agrees with data from an earlier experiment (data not
shown), where enhanced mineralisation was observed by reducing
test substance concentration.
In the higher [14
C]R-898 concentration vessels (100 lg LÀ1
)
more mineralisation was measured in the incubation vessels with
the lower sludge concentration. Mineralisation of 27% and 15% was
measured for the 30 and 300 mg LÀ1
suspended solids vessels,
respectively, probably indicating that more [14
C]R-898 was incor-
porated into biomass in the higher suspended solids concentration
vessels than in the lower, and was therefore not evolved as carbon
dioxide.
The methods described in the OECD test guidelines (OECD,
1992) and REACH (ECHA, 2008) and ISO (1995) guidance for
improving the bioavailability of low solubility test substances were
used here, and the use of silica gel and surfactant to increase the
bioavailability of R-898 helped achieve higher levels of biodegra-
dation than in previous studies, which were performed to the stan-
dard OECD guideline without modifications. These results are
consistent with findings of other work (van Ginkel et al., 2008),
where compounds which had previously thought to be non-
degradable were degraded under modified conditions.
The extended incubation period described in REACH guidance
for compounds where the test conditions lead to mass transfer is-
sues (e.g. poorly water soluble chemicals) allowed time for biodeg-
radation to reach a plateau at a higher level than that observed at
28 d.
When sequential additions of [14
C]R-898 were used, a ‘‘stepped
approach’’, the amount of mineralisation observed increased after
each addition, increasing from 10% to 18% and finally to 37% on
the third addition, as shown in Fig. 4. This indicates that the com-
Table 2
Biomass fractionation of suspended solids (Federle and Itrich, 1997).
Step Solvent or treatment Temperature (°C) Time Biomass/subcellular fractions in supernatants
1 10% Trichloroacetic acid (TCA) 0 (ice bath) 20 min Low MW cellular components
2 Ethanol:ether 1:1 50–55 (water bath) 20 min Lipid
3 5% TCA 100 (oven) 30 min DNA and RNA
4 10 M NaOH 25 (Room temperature) 18 h Protein
5 Burn residue Approximately 1100 Until burn complete Cell wall
-20
0
20
40
60
80
100
0 10 20 30 40 50 60 70
Mineralisationorparentconcentration
Time (days)
10 µg/L R-898, 30 mg/L suspended solids, mineralisation
100 µg/L R-898, 30 mg/L suspended solids, mineralisation
100 µg/L R-898, 300 mg/L suspended solids, mineralisation
100 µg/L R-898, 30 mg/L suspended solids, parent
100 µg/L R-898, 300 mg/L suspended solids, parent
(%radioactivity)
Fig. 3. Mineralisation of R-898 at two test concentrations and two concentrations
of suspended solids and concentration of extractable R-898 (error bars show one
standard deviation).
4 R. Dailey et al. / Chemosphere xxx (2013) xxx–xxx
Please cite this article in press as: Dailey, R., et al. Biodegradability of the antioxidant diaryl-p-phenylene diamine using a modified inherent biodegrada-
tion method at an environmentally relevant concentration. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.072

5. petent bacteria in those vessels were becoming enriched (i.e.
increasing in cell numbers) after exposure to the antioxidant
[14
C]R-898, and able to metabolise test compound with a greatly
reduced lag period between each successive addition. It also shows
the antioxidant R-898 was not toxic to the inoculum at that
concentration.
3.2. Incorporation into biomass
The lower carbon dioxide evolution from incubation vessels
with the higher suspended solids concentration displayed higher
incorporation rates of radioactivity into the biomass.
The results from the biomass fractionation procedure are given
in Table 3. The radioactivity measured in the low molecular weight
cellular compounds, the lipids, the DNA and RNA, and the proteins,
were combined to give the total radioactivity incorporated into
biomass. Radioactivity associated with the cell walls has not been
included in the total incorporated, as this may represent both met-
abolic biomass incorporation and radioactive compounds adsorp-
tion onto the surface of cells. Therefore, not including the cell
walls in the total metabolised is a worst case assumption. The asso-
ciation of radioactivity with the cell walls may have occurred
either through covalent binding of a reactive metabolite of R-
898, or through intermediary metabolic conversion(s) to lower
molecular weight natural substances that were subsequently
incorporated into the biomolecules found in subcellular fractions.
In sludge solids taken from the incubation vessels with the
higher suspended solids concentration almost 30% of the radioac-
tivity added to the vessels was incorporated into biomass, whereas
only 13–16% of that added to the low suspended solids concentra-
tion vessels was incorporated. The detection of radioactivity in the
cell proteins is a clear indication that R-898 was metabolically
transformed, and had entered biochemical pathways. The treat-
ment with the highest percentage of applied radioactivity incorpo-
rated into biomass and associated with cell walls also showed the
least mineralisation, indicating R-898 had been incorporated into
cells, as opposed to being used in respiration and mineralised.
The amount and distribution of radioactivity measured in the cel-
lular fractions was as expected, in that it was broadly similar to
previously published results on the biodegradation of another
moderately degradable chemical (Federle and Itrich, 1997).
3.3. Radio-TLC results
Radio-TLC results showed that the proportion of extractable
radioactivity which could be identified as R-898 was very low ini-
tially, and increased to a maximum after 1 d, then gradually re-
duced to an undetectable level on Day 28 (Fig. 3). The low initial
concentration is thought to be due to its low rate of solubilisation.
R-898 was added to the test vessels on the surface of silica gel, and
as it dissolved in the aqueous phase it would have reacted imme-
diately with any free radicals in the test solution. After the reac-
tions with free radicals had been completed all further
dissolution of R-898 would result in an increase in measurable R-
898 in the aqueous and solvent extract phases. After this initial in-
crease the measured concentration of R-898 decreased, and most
of the radioactivity in the samples was measured at the origin of
the TLC plates, indicating the formation of non-polar breakdown
products.
3.4. Mass balance
Between 94% and 97% of the applied radioactivity in the
[14
C]benzoic acid test vessels was recovered at the end of the
study.
The percentage of the radioactivity applied to each [14
C]R-898
test vessel which was recovered in all fractions at the end of the
study was between 72% and 92%, with one vessel at 56%. The ves-
sels with the lowest recovery had less radioactivity measured in
the activated sludge solids than the other replicates. Biomass frac-
tionation was done on suspended solids from two of these vessels,
which could have resulted in the loss of some radioactivity during
the extractions.
4. Discussion
Results from this study clearly demonstrate for the first time
primary and ultimate biodegradation of this antioxidant in an
aquatic assay. Production of 14
CO2 following incubation of the
radiolabelled DAPD component R-898 with sludge inoculum pro-
vides unequivocal evidence of the capacity of microorganisms to
degrade DAPD in an aqueous environment. This follows previous
observations that R-898 (a component of DAPD) is biologically
transformed in fish to rapidly-eliminated substances, and that soil
testing indicated significant degradation of the chemical over the
period of 1 year (Springborn, 1997b).
At termination of the biodegradation assay, 14
C was found in the
biomass of the incubation media. This suspended solid phase was
further fractionated (according to the method of Federle and Itrich,
0
10
20
30
40
50
0 10 20 30 40 50 60 70 80 90
Mineralisation(%appliedradioactivity)
Time (days)
Additional R-898 added
on Days 28 and 49
Fig. 4. Mineralisation of subsequent additions of the antioxidant R-898, assuming
no further contribution from previous additions (error bars show one standard
deviation).
Table 3
Bio-distribution of radioactivity in cells contained in suspended solids at study termination, day 63.
Vessel description % Of total applied radioactivity in each fraction
Low MW cellular Lipids DNA and RNA Protein Cell walls Total metabo-liseda
10 lg/L [14
C]R-898, 30 mg/L sludge 2.3 3.4 1.0b
8.9 6.5 15.6
100 lg/L [14
C]R-898, 30 mg/L sludge 1.8 3.1 5.2 7.1 3.8 12.9
100 lg/L [14
C]R-898, 300 mg/L sludge 1.2 4.5 2.7 20.9 17.3 29.3
a
Total of all fractions except cell walls.
b
Part of sample lost from one replicate.
R. Dailey et al. / Chemosphere xxx (2013) xxx–xxx 5
Please cite this article in press as: Dailey, R., et al. Biodegradability of the antioxidant diaryl-p-phenylene diamine using a modified inherent biodegrada-
tion method at an environmentally relevant concentration. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.072

6. 1997). The largest fractions were found to be in the cell proteins
(7–21% of the applied radioactivity) and cell walls (4–17% of the
applied radioactivity). Overall, 13–30% of the applied radioactivity
had been metabolised into cell biomass. According to the Univer-
sity of Minnesota Pathway Prediction System metabolic model,
plausible metabolites derived from R-898 include the amino acids
aspartic acid and glycine, which could ultimately be incorporated
into a cellular fraction (University of Minnesota, accessed 2012).
Radio-TLC results showed no [14
C]R-898 was present in the
aqueous test solutions after 28 d incubation again providing fur-
ther evidence that R-898 is not persistent. Mass spectrometry anal-
ysis (HPLC–MS) of aqueous test solutions from the end of the study
did not detect R-898, or the initial breakdown products predicted
by the University of Minnesota prediction system. Later products
could not be evaluated, as they were not likely to be detected by
the analysis method.
5. Conclusions
The use of a radiolabelled test substance allowed the low solu-
bility antioxidant, R-898, a component of DAPD, to be subjected to
biodegradation testing at more environmentally relevant concen-
trations. The availability of radiolabelled R-898 allowed the fate
of the molecule to be determined unequivocally, and with greater
accuracy than if it were not labelled.
The evolution of radioactive carbon dioxide in these studies
demonstrated mineralisation of up to 37% of the applied [14
C]R-
898. Incorporation of radioactivity into biomass also shows metab-
olism of R-898 had occurred. Increased biodegradation of R-898
with successive additions also shows enrichment of competent
bacteria occurs over a short time period. It also demonstrated that
R-898 was not toxic to the inoculum.
In addition, after 28 d there was no [14
C]R-898 remaining in the
test solution, and it can therefore be concluded that primary deg-
radation, mineralisation and incorporation into biomass results in
the total degradation of R-898. In addition, the primary and sec-
ondary predicted products from the Minnesota pathway prediction
system were searched for, but were not detected, indicating that
after 63 d the expected intermediates were no longer present.
The combined evidence of carbon dioxide evolution, incorpora-
tion into biomass, adaptation of bacteria, and disappearance of par-
ent compound, shows that the antioxidant DAPD is highly unlikely
to persist in the environment.
The use of modifications (silica gel and surfactant) to the OECD
test method within the REACH (ECHA, 2008) and ISO (1995) guid-
ance improved the bioavailability of the tested compound in the
biodegradation assessment of DAPD.
Acknowledgements
The authors would like to thank Ruth Commander (Brixham
Environmental Laboratory) for her valuable contribution to the
experimental work presented here, and Dr. Jason Snape (Brixham
Environmental Laboratory) for his vital advice and guidance
throughout the work, and during preparation of this paper.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.chemosphere.
2013.05.072.
References
Brixham Environmental Laboratory, 2011. In vivo dietary bioaccumulation, fish
flow through test for POLYSTAY 100. BR0456/B (Summary available on the
Registered Substances pages of the ECHA website, www.echa.europa.eu).
Chemex, 2010a. The Estimation of the Adsorption Coefficient (Koc) of DAPD
(Polystay 100): Amended Final Report 1, #ENV 9051/011010 AFR1. (Summary
available on the Registered Substances pages of the ECHA website,
www.echa.europa.eu).
Chemex, 2010b. The toxicity to Daphnia magna of DAPD (Polystay 100) as
determined by the 21-d reproduction test. Report # ENV 9052. (Summary
available on the Registered Substances pages of the ECHA website,
www.echa.europa.eu).
Chemex, 2010c. The Acute Toxicity of DAPD (Polystay 100) to Chironomus riparius
over a 28-d exposure period: Amended Final Report 2. #ENV9053/011010 AFR2.
(Summary available on the Registered Substances pages of the ECHA website,
www.echa.europa.eu).
ECHA, 2008. Guidance on information requirements and chemical safety
assessment.
Federle, T.W., Itrich, N.R., 1997. Comprehensive approach for assessing the kinetics
of primary and ultimate biodegradation of chemicals in activated sludge:
application to linear alkylbenzene sulfonate. Environ. Sci. Technol. 31, 1178–
1184.
Iatropoulos, M.J., Williams, G.M., Wang, C.X., Brunnemann, K.D., Leber, A.P., 1997.
Assessment of chronic toxicity and carcinogenicity in rats of Wingstay 100, a
rubber antioxidant/antiozonant. Exp. Toxicol. Pathol. 49, 153–165.
ISO, 1995. ISO Standard 10634:1995 Water Quality – Guidance for the preparation
and treatment of poorly water-soluble organic compounds for the subsequent
evaluation of their biodegradability in an aqueous medium.
Kuczkowski, J.A., 2003. Things I Learned about PPDS at my Mentor’s Knee and Other
Places. Presented at Fall Meeting of the Rubber Division, American Chemical
Society, Cleveland, OH, Oct 14–17 2003, Paper #83.
Mambo-Tox Ltd, 2010a. Determination of the acute toxicity of Polystay 100 Pastille
to the earthworm Eisenia fetida in an artificial soil substrate. #NON-GLP-
GOOD1 Rev #1. (Summary available on the Registered Substances pages of the
ECHA website, www.echa.europa.eu).
Mambo-Tox Ltd, 2010b. Determination of chronic (sub-lethal) toxicity of DAPD
(Polystay 100) to the earthworm Eisenia fetida in an artificial soil substrate.
#GOOD-10-1 Rev #1. (Summary available on the Registered Substances pages
of the ECHA website, www.echa.europa.eu).
NTP, 2011. Report on Carcinogens, twelfth ed. U.S. Department of Health and
Human Services, Public Health Service, National Toxicology Program, Research
Triangle Park, NC.
OECD, 1981. OECD Guideline for the Testing of Chemicals, Test Guideline 302C,
Inherent Biodegradability, Modified MITI Test (II).
OECD, 1992. OECD Guideline for the Testing of Chemicals, Test Guideline 301, Ready
Biodegradability.
Ricerca, 1995. Biodegradation Study of a Rubber Antioxidant – Wingstay 100.
Report # 6011-94-0037-BC-001 (Summary available on the Registered
Substances pages of the ECHA website, www.echa.europa.eu).
Springborn Laboratories, 1995a. Wingstay 100 – Acute Toxicity to Daphnids
(Daphnia magna) Under Flow-Through Conditions Amended report #96-1-
6328. (Summary available on the Registered Substances pages of the ECHA
website, www.echa.europa.eu).
Springborn Laboratories, 1995b. Wingstay 100 – Prolonged (14-d) Acute Toxicity to
Common Carp (Cyprinus carpio) under Flow-Through Conditions. Amended
report # 96–2-6362 (Summary available on the Registered Substances pages of
the ECHA website, www.echa.europa.eu).
Springborn Laboratories, 1996. Wingstay 100 – Toxicity to the Freshwater Green
Alga, Selenastrum capricornutum. Report # 96-4-6454. (Summary available on
the Registered Substances pages of the ECHA website, www.echa.europa.eu).
Springborn Laboratories, 1997a. Wingstay 100 – Prolonged (14-d) Acute Toxicity to
Rainbow Trout (Oncorhynchus mykiss) Under Flow-Through Conditions. Report
#96-11-6770 (Summary available on the Registered Substances pages of the
ECHA website, www.echa.europa.eu).
Springborn Laboratories, 1997b. Soil dissipation study of radiolabelled R-898.
Report # 13537-6150 (Summary available on the Registered Substances pages
of the ECHA website, www.echa.europa.eu).
University of Minnesota Biocatalysis/Biodgradation Database Pathway Prediction
System http://umbbd.msi.umn.edu/predict/ (accessed 16.01.12).
Van Ginkel, G.C., Gancet, C., Hirschen, M., Galobardes, M., Lemaire, Ph., Rosenblom,
J., 2008. Improving ready biodegradability testing of fatty amine derivatives.
Chemosphere 73, 506–510.
Wibbertmann, A., Kielhorn, J., Koennecker, G., Mangelsdorf, I., Melber, C., 2000.
Benzoic acid and sodium benzoate. WHO Concise International Chemical
Assessment Document 26.
6 R. Dailey et al. / Chemosphere xxx (2013) xxx–xxx
Please cite this article in press as: Dailey, R., et al. Biodegradability of the antioxidant diaryl-p-phenylene diamine using a modified inherent biodegrada-
tion method at an environmentally relevant concentration. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.072