Water Research 212 (2022) 118122

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Abiotic oxidative transformation of 6-PPD and 6-PPD quinone from tires

and occurrence of their products in snow from urban roads and in
municipal wastewater
Bettina Seiwert a, Maolida Nihemaiti a, Mareva Troussier b, Steffen Weyrauch a,
Thorsten Reemtsma a, c, *
a
Helmholtz-Centre for Environmental Research – UFZ, Department of Analytical Chemistry, Permoserstrasse 15, 04318 Leipzig, Germany
b
Sigma Clermont, Department of Chemistry, 23 Rue Roche Genès, 63170 Aubière, France
c
University of Leipzig, Institute for Analytical Chemistry, Linnéstrasse 3, 04103 Leipzig, Germany

A R T I C L E I N F O A B S T R A C T

Key words: The antiozonant N-phenyl-N’-(1,3-dimethylbutyl)-p-phenylenediamine (6-PPD) is added to tires to increase their
Snowmelt lifetime and is emitted with tire and road wear particles into the environment. Recently, one of its transformation
Road runoff products (TPs), 6-PPD quinone (6-PPDQ), has gained attention due to its toxicity towards coho salmon. In this
Urban runoff
study, the abiotic oxidative transformation of 6-PPD is investigated by a series of ozonation experiments in the
6PPD quinone
lab followed by analysis of TPs using liquid chromatography-high resolution-mass spectrometry (LC-HRMS). A
6PPDQ
TRWPs total of 38 TPs were detected and tentatively identified, which were formed either directly from 6-PPD or via 6-
PPDQ as intermediate. A suspect screening by LC-HRMS showed 32 of these TPs to occur in snow collected from
urban roads as surrogate of road-runoff, where 6-PPDQ, 4-aminodiphenylamine (4-ADPA), TP 213, and TP 249
were the most prominent besides 6-PPD. More than 90% of the total load of 6-PPD and its TPs was found in the
particulate fraction of snow. Thus, retaining the particulate fraction of road runoff before its discharge into
surface water would substantially reduce the emission of 6-PPD and many of its TPs. Some TPs prevailed in the
water phase of the snow due to their higher polarity. A total of 13 TPs were detected by suspect screening in the
dissolved phase of a wastewater treatment plant (WWTP) influent. Their total load was markedly enhanced
during a day of snowmelt (approx. 1100 g/d) and rainfall (approx. 2000 g/d) compared to dry weather (approx.
190 g/d). 6-PPD and 6-PPDQ contributed to less than 1% to this total load in the water phase (estimated con­
centrations of max 0.1 µg/L). The elimination of the estimated total loads of 6-PPD related TPs from the water
phase in WWTP ranged from 22 to 67% depending on weather conditions. Eventually TP 249, 4-ADPA and TP
259_2 dominated in WWTP effluent (estimated concentration from 0.5 up to 2 µg/L). Thus TP 249 and TP 259_2
are, likely, the most specific and stable TPs of 6-PPD to be determined in the environment.

1. Introduction
Tire and road wear particles (TRWPs) are generated on roads as an
inevitable consequence of tire use in automotive traffic. There is
increasing scientific interest into both, the occurrence and fate of TRWPs
in the environment and of tire related compounds, which were added to
tires and may be released from TRWPs while they reside in the
environment.
One prominent class of compounds are N, N’-disubstituted phenyl­
enediamines, which are added to tires to prevent the oxidative
degradation of the rubber and, thus, to maintain its structure and tire
function. They protect rubber because they are highly reactive towards
oxygen or ozone (Layer and Lattimer, 1990). This reactivity inevitably
results in various transformation products (TPs) of antiozonants in tires,
and later on, also in TRWPs. One of the most frequently used members of
this class of compounds is N-phenyl-N’-(1,3-dimethylbutyl)-p-­
phenylenediamine (6-PPD), because it is less reactive towards molecular
oxygen but reacts well with ozone. The reaction mechanism of 6-PPD
with ozone and some of its ozonation products have been studied from
the perspective of material protection (Lattimer et al., 1983). Five TPs

* Corresponding author.
E-mail address: thorsten.reemtsma@ufz.de (T. Reemtsma).

https://doi.org/10.1016/j.watres.2022.118122
Received 24 November 2021; Received in revised form 21 January 2022; Accepted 24 January 2022
Available online 25 January 2022
0043-1354/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
B. Seiwert et al.
with lower molecular weight and another 5 TPs with higher molecular
weight than 6-PPD were reported. The structure of one of those TPs was
erroneously assumed to be a nitrone (Lattimer et al., 1983), which was
later confirmed to be 6-PPD quinone (6-PPDQ) (Tian et al., 2021) .
6-PPDQ was recently proven to be toxic to coho salmon in the US
(Tian et al., 2021); other fish (chum salmon), however, appear to be less
affected by it (Hiki et al., 2021; McIntyre et al., 2021). Recent screening
studies reported the occurrence of 6-PPDQ in road runoff and in urban
streams affected by road runoff, but quantitative data are scarce and
await confirmation (Challis et al., 2021; Haddad et al., 2018; Johan­
nessen et al., 2021).
Some studies reported organic chemicals that leach from tire parti­
cles (Müller et al., 2021; Peter et al., 2018; Seiwert et al., 2020). How­
ever, such chemicals may also be transformed either in tires during their
production and use, in TRWPs during aging in the environment, or in
aqueous environment after their release from TRWPs (Wagner et al.,
2021). Such transformation processes significantly enhance the number
of compounds of potential environmental relevance. This is also true for
6-PPD, as it is obvious that 6-PPDQ is only one of its several TPs (Lat­
timer et al., 1983; Unice et al., 2015).
This study revisits the abiotic oxidative transformation of 6-PPD, this
time from an environmental perspective. Specifically, it addresses the
following questions: Which TPs are formed from 6-PPD? This question is
answered by laboratory ozonation experiments and analysis by liquid
chromatography-high resolution mass spectrometry (LC-HRMS). Which
of these 6-PPD related TPs can be found in road runoff? This is studied by
a suspect screening of snow collected from roads as surrogate of road
runoff for the TPs found in lab experiment. How do these compounds
partition between the particulate and the aqueous fraction of road
runoff? This is considered by analyzing the water and particle phase of
snow separately. How much of 6-PPD and its TPs arrives at and leaves
from municipal wastewater treatment plants (WWTPs) receiving road
runoff? For this purpose, suspect screenings of the influent and effluent
of a WWTP during dry weather, snowmelt and a rain event are
performed.
2. Materials and methods
2.1. Chemicals
All chemicals were of analytical grade and used as received without
further purification. Methanol, acetonitrile, and formic acid for LC-MS
analysis were provided by Biosolve (Valkenswaard, Netherlands).
Disodium hydrogen phosphate (≥99%) and potassium dihydrogen
phosphate (≥99%) were supplied from Merck (Darmstadt, Germany).
The analytical standard of 6-PPDQ was purchased from HPC Standards
GmbH (Borsdorf, Germany). Nitroso-diphenylamine (NO-DPA, 95%)
and N-(1,3-Dimethylbutyl)-N’-phenyl-1,4-phenylenediamine (6-PPD,
98%) were purchased from abcr GmbH (Karlsruhe, Germany). 4-hydrox­
ydiphenylamine (4-HDPA, 98%) were supplied by Alfa Aesar (Kandel,
Germany). 4-aminodiphenylamine (4-ADPA), and N,N’-diphenyl-p-
phenylene diamine (DPPD) are from Sigma-Aldrich. Ultrapure water
was obtained from a Merck MilliQ Integral 5 system (Merck, Darmstadt,
Germany).
2.2. Sampling and sample preparation
Snow from urban streets was collected after a week of cold weather
(< 0 ◦ C) following a snow fall event in February 2021. The snow samples
were collected from main and side roads in the City of Leipzig, Germany
into glass bottles of 250 mL size filled up to 2/3. The samples were kept
frozen until further treatment and analysed on the same day. An aliquot
(20 mg) was taken and transferred to a centrifugation tube and thawed
at room temperature. After centrifugation the supernatant were trans­
ferred to a vial and analyzed immediately. The remaining particles were
extracted by adding 2 mL of methanol and applying ultrasonic bath for
2
Water Research 212 (2022) 118122
two hours. The extracts were injected into UPLC-HRMS after diluting 1:1
with ultrapure water.
Sampling of influent and effluent of a WWTP connected to a com­
bined sewer system was performed on mid of the week: 17.02.2021
(snow melting event, 6 samples), 17.03.2021 (rain event, 6 samples),
and 16.06.2021 (dry weather condition, 10 samples). The WWTP size is
550,000 population equivalents (PE) and performs enhanced nutrient
removal with tertiary treatment. Briefly, 1-hour composite samples of
the influent to the biological stage (effluent of the grit channel) and the
effluent of the settling basin were collected over the day, stored at 4 ◦ C in
15 mL glass bottles and analyzed one day after sampling as one sample
set. The average pH was 8.5 (snow), 8.4 (rain) and 7.6 (dry conditions)
for the influent and pH 7.3, 7.4 and 7.8 for the effluent.
Two mL of wastewater were filtered using syringe filters (0.45 µm,
RC membrane, Minisart RC4, Sartorius) and filled into 2 mL glass vials
and used for direct injection to UPLC–HRMS and UPLC-MS/MS.
2.3. Lab-scale ozonation experiments
The stock solution of ozone was prepared by introducing ozone
containing oxygen into ice-cooled ultrapure water; the ozone was pro­
duced by an ozone generator (CMG, 10–5, INNOVATEC Ger­
aetetechnik). The ozone stock solution was standardized by its UV
absorption at 258 nm (ε= 3000 M− 1 cm− 1) (Elovitz and von Gunten,
1999). Experiments were performed in amber glass vials at room tem­
perature (23±2 ◦ C). Ozonation in solution was initiated by spiking the
pre-determined volume of ozone stock solution into 10 mM phosphate
buffer solution (pH 7) containing 1 mg/L of 6-PPD. Initial doses of ozone
were 1, 2, 5 and 10 mg/L. For ozonation in solid phase, the solid 6-PPD
was prepared by adding 15 µL of its methanol stock solution in a glass
vial and evaporate until dryness under a gentle stream of argon. The
glass vial was then placed in a flask saturated with continuous flow of
ozone containing oxygen. After being exposed to gaseous ozone, the
glass vials were taken out and 1 mL of methanol/water (1:1) added. The
samples were directly analyzed using RPLC-MS without further
enrichment.
The ozonation of 6-PPD TPs, namely 4-ADPA, 4-HDPA, and 6-PPDQ,
was individually investigated under similar experimental conditions,
aiming to achieve the full picture of the 6-PPD ozonation pathways. The
gas phase and aqueous phase ozonation of DPPD, another N,N’-disub­
stituted phenylenediamine, was also studied in comparison to 6-PPD.
2.4. Analysis with UPLC-TOF-MS
Aqueous samples and extracts of particles were analyzed by ultra-
performance liquid chromatography time-of-flight mass-spectrometry
(UPLC-TOF-MS). The instrument consisted of an ACQUITY UPLC-System
(HSS T3 column; 100 × 2.1 mm, 1.7 μm) coupled to a XEVO XS Q-TOF-
MS (Waters GmbH, Eschborn, Deutschland) as described elsewhere
(Klöckner et al., 2021). The following parameters were applied: flow
rate: 0.45 μL/min, column temperature: 45 ◦ C, capillary voltage: 0.7 kV
(positive ionization mode), source temperature: 140 ◦ C, desolvation
temperature: 550 ◦ C, sampling cone voltage: 20 V, source offset: 50 V,
cone gas: nitrogen, collision gas: argon, desolvation gas flow: 950 L/h,
scan time: 0.15 s, collision energy: 4 eV (molecular ions) and 15 – 35 eV
(fragment analysis). The mobile phase consisted of (A) water with 0.1%
formic acid (v/v) and (B) methanol with 0.1% formic acid (v/v). The
solvent gradient was: 0 min 2% B, 12.25 min 99% B, 15.00 min 99% B;
15.10 min 2% B, 17.00 min 2% B.
Quantitative analysis of a few compounds available as reference
compounds (6-PPD, 4-HDPA, and 6-PPDQ) was performed by HPLC
(Agilent 1290) connected to a Qtrap5500 (Sciex) triple Quad MS by
multiple reaction monitoring (transitions and HPLC conditions given in
Table S5 and S6) and external calibration. The reference compounds
were dissolved in acetonitrile and diluted in MeOH/water (1:1) directly
before analysis to avoid their transformation. The LOQ was 15 ng/L for
B. Seiwert et al. Water Research 212 (2022) 118122

4-HDPA, 25 ng/L for 6-PPDQ, and 100 ng/L for 6-PPD. 3. Results & discussion

2.5. Identification of TPs from lab-scale experiments 3.1. Lab-scale ozonation of 6-PPD and related compounds

Identification of TPs was done by evaluating the RPLC-MS data in a
retention time window of 1 to 10 min and a mass range of m/z 50 to
1200. MarkerLynx was used to perform the peak picking for the mo­
lecular ion trace (function 1) with 0.1 min deviation in retention time
and 0.01 Da deviation in exact mass. The marker table with relative
intensities (to the total marker intensity) was exported to Excel and all
further evaluations were performed there. The peaks with a relative
intensity above 1 and newly appeared in the ozonated samples
compared to controls were selected. Sum formulas were assigned by
using a mass tolerance of 5 ppm and an elemental composition of C
(0–100), H (0–100), N (0–20), O (0–20), and Na (0–2), and by taking
into account the fragment ions. The fragment ions were used, if avail­
able, to propose a structure based on possible transformations that may
happen to the parent compound 6-PPD.
The confidence levels were reported according to Schymanski
(Schymanski et al., 2014). TPs that do not have a structure that could be
proven by standard compounds were named according to their observed
nominal mass ([M + H]+, [M+Na]+ or [M-H]− ). If two TPs exhibited the
same nominal mass “_2′′ was added to the second one. JChem for Office
(Excel) was used for chemical database access, structure based property
calculation (log D values at pH 7.4), search and reporting (JChem for
Office 20.14.0.688, 2020, ChemAxon (http://www.chemaxon.com)
Lab-scale ozonation was performed to study the TPs that originate
from oxidation processes of 6-PPD, either during tire use or after its
abrasion and transformation into TRWPs. The reaction of solid 6-PPD
with ozone gas may simulate the transformation of 6-PPD in tires and
TRWPs, while the reaction with ozone in aqueous solution including
hydrolytic reactions may simulate reactions after the release of 6-PPD
from tires or from TRWPs into the surrounding water.
In total, 38 TPs of 6-PPD were detected in these two sets of ozonation
experiments by LC-HRMS and identified to various levels by their exact
mass (formula) and product ions (structure) or by reference compounds
(Tab. S1). A total of 26 of these TPs have not been reported before in
literature. The various TPs of 6-PPD were arranged along six pathways
(A to F, Fig. 1), in extension of the scheme of Lattimer et al. (Lattimer
et al., 1983), who investigated the gas phase ozonation of 6-PPD to study
its effectiveness as an antiozonant, and by incorporating the work of
Tian et al. (Tian et al., 2021). Separate ozonation experiments with two
commercially available TPs (4-ADPA and 4-HDPA) supported the
pathway (Fig. 1), which is further extended by 6-PPDQ ozonation
(pathway G and H, Fig. 2).
The reactions of 6-PPD with ozone gas and with ozone in water form
similar TPs, but with different relative yields (Fig. 1, Fig. S1). Such
differences may be due to the different reaction mechanisms and ki­
netics of a homogenous (solution) and a heterogenous (solid/gas) re­
action, and the presence or absence of water for hydrolytic reactions.

Fig. 1. Tentatively identified TPs formed from 6-PPD by ozone (solid reacted with ozone gas and aqueous solution reacted with dissolved ozone) arranged along
different (partially hypothetic) pathways: A) Quinone pathway, B) Nitroso aryl pathway, C) HDPA pathway, D) Aryl hydrolysis pathway, E) Formaldehyde addition
pathway, F) Side chain oxidation pathway. TP number in bold: detected in ozonation experiment, in snow sample (aqueous phase) and in the WWTP, standard:
detected in ozonation experiment and in the snow sample (aqueous phase), italic and gray: detected only in the ozonation experiment. For more information on the
TPs please refer to Tab. S1.

3
B. Seiwert et al.
However, both reaction conditions are of environmental relevance.
One initial step under both conditions is the oxidation/dehydroge­
nation of 6-PPD to the more conjugated benzoquinone imine, N-(4-(1,3-
dimethylbutyl)imino)− 2,5-cyclohexadiene-1-ylidene (6-PPDQI) (m/z
267, ESI pos) (Duchacek and Kuta, 1990; Huntink, 2003). A mix of
6-PPD and 6-PPDQI was reported to be used in tire manufacturing
(Datta et al., 2004). In aqueous solution, an equilibrium likely exists
between both substances depending on the redox conditions; immedi­
ately after preparing the aqueous solution of 6-PPD in contact with air,
6-PPDQI is also detectable (Fig. S 2). This is one reason why 6-PPD
quantification in aqueous solution is difficult. Likely, the further
oxidation of 6-PPDQI by two subsequent oxygen additions via TP283_1
leads to 6-PPDQ (m/z 299).
The TPs that were detected and tentatively identified in this study
can be arranged along different pathways (Fig. 1). Given that many of
these reactions are competitive rather than consecutive and do not only
involve oxidation reactions, experiments at different ozone-dosages
were not instrumental in verifying these pathways. Therefore, much of
Fig. 1 awaits experimental verification. Of the many TPs, the following
appeared to be most relevant in terms of their peak areas (Figure S1):
Along the quinone pathway (A), TP283_1, the direct product of 6-
PPDQI, as well as its two oxidation products, 6-PPDQ (formed by oxy­
gen addition) and TP 281 (formed by dehydrogenation) were the major
products. However, several other products are formed along this path
(Fig. 1).
Along the nitroso aryl pathway (B), TP285_3, formed by the oxidation
of one of the amino groups of 6-PPD, and NO-DPA, product of further
oxidation and oxidative cleavage of the aliphatic side chain were
dominating. TP285_3 has been found to be relatively stable (Lattimer
et al., 1983), which could explain its comparatively high signal intensity
in our experiment (Fig. S1). The next two pathways (C, D in Fig. 1) were
more relevant for ozonation of 6-PPD in water than for ozonation of dry
6-PPD by ozone in air; both these pathways involve hydrolysis of
amino-bonds of 6-PPD. The HDPA-pathway (C) leads to 4-HDPA. Hy­
drolysis of 6-PPD has been reported to be quite fast in water (half-life of
8 h at pH 7) (ECHA, 2021a). 4-HDPA may, however, also be formed from
6-PPDQI, the hydrolysis of which is even faster (half-life of 4.7 h at pH 7
(ECHA, 2021b)). Along the Aryl hydrolysis pathway (D) TP 194 is the
major product (Fig. S1). Oxidation of TP 194 may lead to TP 138, the TP
with the lowest molecular weight detected in these experiments. From
the Side chain oxidation pathway (F), TP 285_4 was detected as an initial
product. This can break down to form 4-ADPA, a comparably large peak
4
Water Research 212 (2022) 118122
Fig. 2. Tentatively identified TPs formed from
6-PPDQ by ozone (solid reacted with ozone gas
and aqueous solution reacted with dissolved
ozone) arranged along different (partially
hypothetic) pathways: G) Dealkylation pathway
H) Rearrangement pathway. TP number in bold:
detected in ozonation experiment, in snow
sample (aqueous phase) and in the WWTP,
standard: detected in ozonation experiment and
in the snow sample (aqueous phase), italic and
gray: detected only in the ozonation experi­
ment. For more information on the TPs please
refer to Tab. S2.
(Fig. S1), or leads to TP 269 and TP 227. The side chain oxidation of
6-PPDQI leads to TP 283_2. An overview of all TPs and their identifi­
cation level is given in Tab. S1.
6-PPD is not the only N,N’-disubstituted phenylenediamine that may
be added as antioxidant or, specifically, as antiozonant to tires. DPPD is
also of importance and its ozonation in aqueous phase and gas phase
were additionally investigated in this study. Five TPs known from 6-PPD
were also found as DPPD products, belonging to the nitroso aryl pathway,
the aryl hydrolysis pathway or the HDPA pathway (Tab. S3). DPPD has not
played a role in the most recent discussion on chemicals released from
tires or TRWPs. Although its leaching potential is lower than that of 6-
PPD (Müller et al., 2021), its TPs could contribute to tire derived
leachables.
Analogous to 6-PPD, the oxidative transformation of 6-PPDQ was
studied and 12 further TPs were detected (Fig. 2). All these TPs were also
found during 6-PPD ozonation, with TP 249 and TP 265 showing the
highest intensity (Fig. S1). The products detected from 6-PPDQ ozona­
tion confirm that its quinone system was primarily attacked by ozone
(pathway H). This is in line with previous studies on ozonation of qui­
nones (Ramseier and Gunten, 2009). All the TPs within pathway H result
from an attack on the quinone ring followed by ring opening and rear­
rangement (one exception: TP 351, only oxidation of the quinone ring
and alkyl chain). Other TPs are explainable by the loss of the aniline
moiety or the alkyl chain (pathway G (Fig. 2, Tab. S2). These results show
that 6-PPDQ is subjected to further degradation in the presence of oxi­
dants. In agreement to this, the half-life of 6-PPDQ in dechlorinated
drinking water was recently reported to be 33 h at room temperature
(Hiki et al., 2021).
Above discussion on the relative importance of different TPs in lab-
scale ozonation experiments is based on their peak intensities
(Fig. S1), because reference chemicals were only available for 6-PPD and
three TPs, preventing a full quantification of all the TPs detected.
However, the response factors of these four compounds in LC-HRMS
were comparatively similar and ranged from 0.44 (4-HDPA) to 1.2 (6-
PPD) (Tab. S4). On this basis, it appears justified to evaluate the rele­
vance of different TPs of 6-PPD based on their peak intensities. However,
this cannot substitute a later full quantification of those TPs that are or
will become available as reference compounds.
3.2. Occurrence of 6-PPD and its TPs in snow from urban roads
In urban environment, snow is expected to accumulate particles
B. Seiwert et al. Water Research 212 (2022) 118122

generated on roads for the period from snowfall to snow melt. Therefore,
three snow samples collected in Leipzig, Germany in February 2021
were analyzed by LC-HRMS and a suspect screening was performed for
the TPs found from lab-scale experiments (Fig. 1, Fig. 2, Tab. S1, Tab.
S2). The water and the particulate fraction of the snow samples were
analyzed separately.
A total of 32 TPs of 6-PPD, including 6-PPDQ, were detected in the
water or the particulate phase of all three snow samples (Fig. 3b,
Fig. S4b). Analytically, this confirms the previous finding that lab ex­
periments can be very useful to generate suspects for the screening of
environmental samples (Seiwert et al., 2016). More importantly, this
result outlines that the diversity of TPs formed by ozonation of 6-PPD in
the lab is of relevance also in the environment. Indeed, 6-PPDQ is only
one of those TPs and the detection of 9 of its TPs proves that 6-PPDQ is
not a dead-end product. The TPs, which are known from the lab ex­
periments, but not detected in snow, are primarily intermediates such as
TP283_1, 6-PPDQI, and TP285_2 (Fig. 1).
The load in the snow (particles and water) of 6-PPD and the 32 TPs
detected was clearly dominated by 6-PPD, with 40 – 50% of the total
peak area (TPA) (Fig. 3a). This shows that 6-PPD is much stabilized by
its embedding into the rubber matrix of TRWPs. The TPs with the
highest relative contribution (based on peak areas) are 4-ADPA and 6-
PPDQ (both around 8% of TPA), followed by TP 213, TP 297_3, TP
249, TP 205, and TP 257 (all ≥ 2% TPA on average). 4-ADPA may,
however, also originate from other disubstituted phenylenediamines in
tires, like DPPD, as demonstrated in this study (Tab. S3). The compo­
sitions of all three snow samples are comparatively similar, even though
they were collected from different streets.
With ongoing aging of TRWPs and ongoing transformation of 6-PPD
and its TPs, the pattern of 6-PPD related compounds in the environment
may differ from the ones observed from snow samples. Likely, TPs
occurring later in the transformation pathways (Fig. 1) are, then, gaining

Fig. 3. a) Relative importance (relative peak area of their LC-HRMS signals) of 6-PPD and its TPs in three snow samples (water + particulate phase) from roads. Error
bars represent standard deviation of the three samples. b) Concentration (signal intensity) ratio of the water to the particulate phase for each of the compounds in the
three snow samples. The dotted 1:1 line represents equal signal intensities for the water and the particulate phase.

5
B. Seiwert et al.
relative importance.
Recent studies have shown 6-PPD and 6-PPDQ to occur in surface
runoff and in urban streams affected by surface runoff (Challis et al.,
2021; Johannessen et al., 2021; Tian et al., 2021) as well as in road dust
(Huang et al., 2021). However, other TPs except 6-PPDQ have not yet
been considered, nor has the particulate phase been considered in such
studies.
The overwhelming majority of 6-PPD in the snow is observed in the
particulate phase containing TRWPs, rather than in water (distribution
ratio water/particle 0.001, Fig. 3b). Similarly, the particulate phase
dominates the load in snow for most of the TPs of 6-PPD as well, with a
proportion of 90% (ratio 0.1) to 99% (ratio 0.01) for individual com­
pounds (Fig. 3b). For 6-PPDQ, the share of the aqueous phase is only
little higher than for 6-PPD. These findings agree to the suggestion of 6-
PPDQ being a potential chemical marker for TRWPs in the environment
(Klöckner et al., 2021).
For several TPs formed from 6-PPDQ (namely TP 197, TP 299 and TP
302, right side of Fig. 3b), the proportion in the water phase equals or
even exceeds the proportion in the particle phase of the snow (ratio > 1).
This may reflect the higher polarity of these TPs, expressed by their
lower predicted logD values compared to most TPs directly formed from
6-PPD (Fig. S5).
Summing the peak areas of 6-PPD and all its TPs, 90 - 93% of TPA are
found in the particulate phase and only 6 - 10% in the water phase of all
three snow samples. This is primarily due to the large contribution of 6-
PPD (40 - 50% of TPA), which is almost exclusively found in the par­
ticulate phase.
In a recent leaching study, 3% of 6-PPD was determined in the
leachate (Müller et al., 2021), a value much higher than the < 0.1%
found here. This may indicate that equilibration between the particulate
and water phase had not taken place in all snow samples in this study,
despite the melting and sample processing in the lab at room tempera­
ture for approx. 2 h. Also, the contact time of particles in surface runoff
before its entry into a sewer system is limited and may not be sufficient
to reach the equilibrium concentrations in the two phases.
Non-equilibrium is even more likely because chemicals transport within
the TRWP/rubber phase is highly hindered (Huntink, 2003). Therefore,
it is reasonable to assume that the strong dominance of the particle
phase for the load of 6-PPD and many of its TPs in the snow samples is
also true for surface runoff in times of rainfall.
The dominance of the particulate phase for the load of 6-PPD and its
TPs outlines the option to effectively control their emission into envi­
ronment by retaining the particles of road runoff before its discharge. In
the case of combined sewer systems, this can be effectively done in
WWTPs (see Section 3.3). The situation may, however, be different for
separate sewer systems, even when settling basins are in place to retain
the particles of surface runoff; 6-PPD and its TPs may slowly be released
into the water phase during extended residence times of TRWPs in such
basins.
Moreover, the above data suggest that analyzing 6-PPD and 6-PPDQ
only in the water phase of discharges rich in particles, such as combined
sewer overflow or untreated surface runoff, but not in the particulate
phase being discharged together with the water would drastically un­
derestimate the amounts transferred to surface waters.
3.3. Occurrence of 6-PPD and its TPs in a wastewater treatment plant
In combined sewer systems, the surface runoff is collected together
with wastewater from other sources (households, industry, etc.) and
treated in WWTPs. Therefore, the influent and effluent of a WWTP
treating wastewater from a combined sewer system was analyzed by
suspect screening for 6-PPD and its TPs over one day during snowmelt,
rainfall, and dry weather (6 – 10 samples per day, 22 samples of each,
influent and effluent, in total). Only the dissolved phase was analyzed as
particle removal in WWTPs is known to be very high (Cheng et al.,
2021).
6
Water Research 212 (2022) 118122
Of the 38 TPs found from lab-scale experiments, 13 TPs (including 3
TPs of 6-PPDQ) were detected in the water phase of the WWTP influent
(Fig. 4, Fig. S6). 6-PPD could only be determined during snowmelt,
while 6-PPDQ was determined during snowmelt and rainfall. Neither of
the two was detected during dry weather (LOQ approx. 25 ng/L). Their
contribution to the sum of the 6-PPD related compounds in the influent
was low (approx. 1% of TPA). The major signals in the influent, how­
ever, correspond to TP257, TP259_1 and TP259_2 (pathway A), 4-ADPA
and NO-DPA (pathway B), all originating from 6-PPD (Fig. 1), as well as
TP249 from 6-PPDQ (Fig. 2). The load of all these compounds was
clearly enhanced during snowmelt and rainfall (Fig. 4). This supports
the assumption that these compounds do originate from surface runoff
and from TRWPs, rather than from the other wastewater sources
collected in combined sewer systems (households, industry, etc.).
4-ADPA, NO-DPA, 4-HDPA, TP194, and TP138 were detected in the
water phase of WWTP influent also during dry weather, although at
much lower concentration than during snowmelt and rainfall. 4-ADPA is
known to be applied in pharmaceuticals, photography, and in dyestuff
industries (Khanna et al., 1987), so that surface runoff indeed may not
be the only source of 4-ADPA in municipal wastewater. However, the
peak area of all 6-PPD related compounds during dry weather was one
order of magnitude lower than during snow melt or rainfall (Fig. 4).
4-ADPA, NO-DPA, 4-HDPA and TP138 could originate from other
diphenylamine antioxidants (Tab. S3), which were recently detected to
be present in treatment plant influents as well (Zhang et al., 2021).
In WWTP influent, 4-HDPA and 6-PPDQ were quantified based on
available reference compounds. The mean concentration of 4-HDPA
ranged from 0.07 µg/L for the day with snowmelt to 0.02 µg/L at dry
weather (Table 1). Concentrations of 6-PPDQ were even higher espe­
cially during snowmelt, reaching 0.1 µg/L. Based on the mean flow of
water at the sampling days, the loads were around 5 g/d for 4-HPDA and
< 5 g/d for 6-PPDQ at dry weather, and increased to 17 g/d and 26 g/d,
respectively, for the day with snowmelt (Table 1). The estimated total
load of 6-PPD and its TPs in the influent was 190 g/d at dry weather and
increased to 1100 and 2000 g/d at the day of snowmelt and rainfall,
respectively (Table 1).
In WWTP effluent, 14 TPs of 6-PPD could still be observed. Their
estimated total load was reduced by 22% (rainfall) and 45% (snow melt)
to approx. 1600 g/d and 600 g/d. Removal increased to 65% for the day
with regular dry weather conditions (Table 1). No clear indication of
removal in WWTP was found for TP 259_2, 4-ADPA, TP 213, and TP 235.
The compounds dominating the effluent pattern were TP 249, TP 259_2,
and 4-ADPA. These TPs appear stable enough to withstand biological
wastewater treatment, at least partially. Especially TP 249 and TP 259_2
are, thus, promising candidates to search for in the aquatic environment.
Their identification was not successful in this study, but would be
desirable.
While 6-PPD was detected in the effluent only during the snowmelt
event, 6-PPDQ was found during snow melt and rainfall, but contributed
to 1% or less to the 6-PPD related compounds in WWTP effluent.
However, it should be noted that the biological treatment process in
WWTPs may form additional TPs of 6-PPD or one of its TPs. This study
confines itself to the 38 TPs that were detected in lab experiments on
abiotic oxidative transformation (Fig. 1, Fig. 2).
For WWTP effluents, a median concentration of 0.018 µg/L of 6-PPD
from 5 samples was reported (ECHA, 2021c). Data on the concentration
of 6-PPD in influents and effluents of municipal WWTPs, and the extent
of 6-PPD removal are not available yet. No reports appear to exist on
concentration of any of 6-PPD TPs in municipal wastewater.
For stormwater, a median value of 0.11 µg/L for 6-PPD was docu­
mented for four samples (ECHA, 2021c). For 6-PPDQ recent studies
report stormwater concentrations in the range of 0.2 – 1.2 µg/L (Challis
et al., 2021; Tian et al., 2022). The concentrations of 6-PPDQ found here
in the influent of the WWTP during snowmelt and rainfall are even lower
than the reported data on road runoff, which may in part be due to the
dilution of road-runoff with wastewater from households and industry in
B. Seiwert et al. Water Research 212 (2022) 118122

Fig. 4. a) Estimated daily load of 6-PPD and its TPs in the influent of a WWTP connected to a combined sewer system during one day of snow melt (6 samples/day),
rainfall (6 samples/day) and dry weather (10 samples/day).

Table 1
Mean concentrations (µg/L) and loads (g/d) of dissolved 4-HDPA and 6-PPDQ and estimated total concentration and load of 6-PPD and its TPs in the influent and
effluent of a municipal WWTP treating wastewater from a combined sewer system during one day of snowmelt, rainfall and dry weather.
a)
4-HDPA 6-PPDQ 6-PPD and TPs

Concentration (µg/L) Load (g/d) Concentration (µg/L) Load (g/d) Estimated total Estimated total load
concentration (µg/L) (g/d)

Day Influent effluent influent effluent influent effluent influent effluent influent effluent influent effluent

Snowmelt 0.070 ± 0.013 0.018 ± 0.014 17.2 4.5 0.105 ± 0.037 n.d.b) 25.8 <6.2 4.4 2.4 1082 601
Rainfall 0.061 ± 0.020 0.018 ± 0.025 8.5 2.5 0.052 ± 0.022 n.d.b) 7.3 <3.5 14.3 11.2 2001 1565
Dry weather 0.023 ± 0.025 n.d.b) 4.9 <3.3 n.d.b) n.d.b) <5.4 <5.4 0.9 0.3 188 66

a) Estimated from their total peak area and the calibration curve of 6-PPD.
b) n.d.: not determined. LOQ approx. 15 ng/L for 4-HDPA and 25 ng/L for 6-PPDQ.

the combined sewer systems.
4. Conclusions
• A total 38 TPs were identified to various confidence levels in this
study as products of the abiotic oxidative transformation of 6-PPD.
While 6-PPDQ may be of specific concern due to its biological ef­
fects, understanding the environmental fate of 6-PPD requires a
much broader view on its TPs.
• 34 TPs of 6-PPD are detected in the snow from urban roads.
• The major load of 6-PPD and its TPs in snow from urban roads is in
the particulate phase, namely from 6-PPD and 6-PPDQ (90 – 99%).
With increasing polarity of the TPs, the aqueous phase is gaining
importance. This, likely, also applies for surface runoff after rainfall.
• Retaining the particulate fraction of road runoff before its discharge
into surface water would substantially reduce the emission of 6-PPD
and many of its TPs.
• Up to 13 TPs of 6-PPD were detected in the influent to a WWTP
treating combined sewer water. The proportion of 6-PPD and 6-
PPDQ to the total signal area of all 6-PPD related compounds in
the dissolved phase was < 1%, outlining that in this phase other TPs
are becoming increasingly important.
• The estimated load of 6-PPD and its TPs in municipal wastewater was
elevated by about one order of magnitude during snowmelt and rain
compared to dry weather conditions.
• Their elimination in WWTP was moderate with 22 – 65% depending
on the weather conditions. Total 14 TPs of 6-PPD were still detect­
able in the effluent of WWTP, with TP 249, TP 259_2, and 4-ADPA as
the dominant compounds.
• The reactivity of 6-PPD and the large number of TPs complicates
studying the environmental fate of 6-PPD and assessing its environ­
mental risk.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This study was funded in part by The Helmholtz Association of
German Research Centers through its research program “POF IV” and
the research unit “Chemicals in the Environment” (CITE). Technical
support by Petra Keil is gratefully acknowledged. Staff of the WWTP are
acknowledged for providing wastewater samples and discussions of
results.

7
B. Seiwert et al.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.watres.2022.118122.
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