Journal of Chromatography A, 1419 (2015) 109–115

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Determination of emerging contaminants in wastewater utilizing

comprehensive two-dimensional gas-chromatography coupled with
time-of-flight mass spectrometry
Sarah Prebihalo a , Adrienne Brockman a , Jack Cochran a,b , Frank L. Dorman a,c,∗
a
Pennsylvania State University, Forensic Science Program, University Park, PA 16802, United States
b
Restek Corporation, Bellefonte, PA 16823, United States
c
Pennsylvania State University, Department of Biochemistry and Molecular Biology, University Park, PA 16802, United States

a r t i c l e i n f o a b s t r a c t

Article history: An analytical method for identification of emerging contaminants of concern, such as pesticides and
Received 15 May 2015 organohalogens has been developed and utilized for true discovery-based analysis. In order to achieve
Received in revised form the level of sensitivity and selectivity necessary for detecting compounds in complex samples, com-
23 September 2015
prehensive gas chromatography coupled with time-of-flight mass spectrometry (GC × GC–TOFMS) was
Accepted 24 September 2015
Available online 28 September 2015
utilized to analyze wastewater samples obtained from the Pennsylvania State University wastewater
treatment facility (WWTF). Determination of emerging contaminants through a process of combining
samples which represent “normal background” and comparing this to new samples was developed.
Keywords:
GC × GC–TOFMS Results show the presence of halogenated benzotriazoles in wastewater samples as well as soil sam-
Comprehensive two-dimensional gas ples from Pennsylvania State University agricultural fields. The trace levels of chlorinated benzotriazoles
chromatography observed in the monitoring wells present on the property indicate likely environmental degradation of
Time-of-flight mass spectrometry the chlorinated benzotriazoles. Preliminary investigation of environmental fate of the substituted ben-
Environmental forensics zotriazoles indicates their likely degradation into phenol; an Environmental Protection Agency (USEPA)
priority pollutant.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
The toxicological effects of unrecognized emerging contam-
inants in the environment have long been a point of interest
among environmental scientists [1]. One well-known example of
the dangers of environmental pollutants is the “Love Canal” inci-
dent, where residents were forced from their homes due to the
presence of 19,000 metric tons of chemical waste once dumped
below the surface [2]. More recently, hundreds of thousands of
West Virginia residents were without water for days due to the
negligent storing of 4-methylcyclohexanemethanol (used as a coal
cleaning agent) which caused a massive chemical spill in to the
Elk River, Charleston’s main water source. The company responsi-
ble, Freedom Industries, ultimately declared bankruptcy in January
2014 [3]. After such instances, investigations into the cause and
clean-up are often lead by government agencies such as the U.S.
Environmental Protection Agency (USEPA), the Centers for Disease
∗ Corresponding author at: Pennsylvania State University, Department of Bio-
chemistry and Molecular Biology, University Park, PA 16802, United States.
E-mail address: frank@peak-diagnostics.com (F.L. Dorman).
Control and Prevention (CDC) and The Chemical Safety Board. The
approach can be categorized as “reactive”, in that the source of
contamination is determined after a problem is known to exist.
The importance of determining the presence of emerging con-
taminants is outlined as one of the top five goals of the Strategic
Plan (2000) for the U.S. Environmental Protection Agency’s (USEPA)
Office of Research and Development. The focus of environmental
forensics has primarily been on acutely toxic/carcinogenic pesti-
cides and industrial intermediates considered “priority” pollutants
that demonstrate environmental persistence. Unfortunately, the
compounds that fall under this characterization represent only a
small fraction of the chemicals present in the environment [4].
Additionally, present analytical methodologies mainly focus on
individual compounds, rather than the mixtures that contain the
compounds. As a result, environmental scientists have a limited
understanding of the complete contaminant profile in a particular
event [5].
Due to the multitude of tests required to not only identify the
contaminant, but also determine its source, environmental foren-
sics has traditionally been extremely time consuming. For the
approach to change from reactive to proactive, a representative
matrix is needed for the identification of emerging contaminants

http://dx.doi.org/10.1016/j.chroma.2015.09.080
0021-9673/© 2015 Elsevier B.V. All rights reserved.
110 S. Prebihalo et al. / J. Chromatogr. A 1419 (2015) 109–115

in the environment. Wastewater contains compounds and their has been established, this same procedure could be applied to other
respective metabolites which are continuously introduced into the samples of interest.
aquatic environment as complex mixtures. As a result, wastewater
is a commonly used matrix for analyzing environmental samples.
2. Materials and methods
In the past, environmental scientists have analyzed wastewater for
several contaminant classes, including illicit drugs, pharmaceut-
2.1. Water sample preparation
icals and personal care products (PPCPs) and endocrine disruptors
[6–8]. Through these studies, the relevance of utilizing wastewater
The Pennsylvania State University Wastewater Treatment Facil-
to determine contaminants in environmental samples is evident.
ity (WWTF) (Office of the Physical Plant, State College, PA) was
Discovery analysis utilizing wastewater and other complex
used as a control facility to refine analytical methodology. Over
matrices requires a separation technique capable of distinguishing
the course of a year several water samples were collected through-
the thousands of potential compounds present in a sample. When
out the various steps in the treatment process into clean 4 L amber
the source, transport and fate of contaminants are of concern, tra-
glass bottles. At the PSU WWTF, influent sewage is divided into
ditional methodologies have utilized gas-chromatography–mass
two separate flow streams, with one serving the Pennsylvania State
spectrometry (GC–MS) as the dominant instrumentation [9–11].
University Campus and one serving the borough of State College.
GC–MS methods have been developed for the identification and
Wastewater samples were also collected from the pre-treatment
quantification of “priority contaminants” in water such as poly-
settling tanks as well as post-treatment water tanks. Settling tanks
chlorinated biphenyls (PCBs), pesticides, estrogenic compounds
are used as a first treatment step to separate the solid material
and polycyclic aromatic hydrocarbons (PAHs) [12–15]. Despite
inherent to wastewater. The Pennsylvania State University WWTF
the ability of these methods to determine the presence of spe-
participates in a unique open loop system in which treated water is
cific compounds, the complexity of wastewater or soil matrices
discharged onto over 600 acres of farmland and forest area that act
often does not allow for sufficient separation using traditional
as a “living filter”. The living filter system was proposed in the 1960s
gas chromatography. When analyzing samples containing more
after it was determined Pennsylvania State University chemical
than 150–250 relevant compounds, the separation power of tra-
dumping was killing fish in Spring Creek [23]. This filter acts as an
ditional (single column) gas chromatography decreases and has
intermediate step to further treat the water before it is introduced
limited peak capacity [5]. Comprehensive two-dimensional gas
into Spring Creek, and represents an additional “purification” step
chromatography (GC × GC) has been demonstrated as a technique
that most WWTF systems would not employ. This system works by
capable of enhanced separation of compounds within a complex
removing treated effluent from the facility and transferring it to the
matrix [16–19]. The two orthogonally aligned columns are com-
farmland located off campus. The water then enters the sprinkler
prised of two different stationary phase chemistry which allows
system and is distributed to the various areas in the field. As a result,
for the increased peak capacity. In order to produce narrow bands
soil samples, spray water and monitoring well water were also able
in the second dimension column, a quad-jet cryogenic modulator
to be collected into 500 mL amber glass jars for additional analy-
(LECO, St. Joseph, MI) is utilized in order to trap and refocus analyte
sis. Soil samples were collected from several areas; two from lines
bands as they elute from the first dimension column. The decrease
actively spraying, and two from a line sprayed in the last few days.
in band width is primarily responsible for the increase in sensitivity.
Both surface soils and soils 6 inches from the surface were collected.
It is due to these features that two-dimensional gas chromatogra-
Additionally, water directly from sprinkler head was collected.
phy has been demonstrated as having the capability to resolve an
Samples were extracted and concentrated to a final volume
order of magnitude more compounds from a complex matrix than
of 2 mL from a nominal starting volume of 500 mL, which was
traditional methods [9]. One example is the application of two-
removed from the homogenized 4 L collection sample, following
dimensional gas chromatography to the petrochemical industry,
a modified USEPA 3510c extraction technique [24]. Three recovery
specifically the determination of oil spill sources [20]. Additionally,
standards were spiked into the samples in final extract concen-
Focant et al. [21] and Megson et al. [22] have developed a method to
tration of 200 pg/␮L: QuEChERS internal standard mix for GC–MS
improve the separation of 209 polychlorinated biphenyl congeners.
analysis (cat# 33267), acid surrogate mix (cat# 31025) and revised
With the increased peak capacity of two-dimensional gas
B/N surrogate mix (cat#31024) (Restek Corp., Bellefonte, PA, USA).
chromatography comes a need for a detector capable of rapidly
These standards were chosen as they contain environmentally rel-
acquiring the large packets of data generated during the separa-
evant compounds such as polychlorinated biphenyls (PCBs) and
tion. Additionally, the narrow second dimension peaks produced
cover a wide range of both acidic and basic compounds. These com-
during cryogenic modulation require a mass spectrometer capa-
pounds are listed in Table 1. Each sample was serially extracted
ble of fast scanning rates for improved compound identification in
three times under acidic conditions (pH = 2) and three times under
complex matrices. With the addition of automatic spectral decon-
basic conditions (pH = 11) using dichloromethane as the extrac-
volution, co-eluting compounds can be distinguished based on
tion solvent (Avantor, Center Valley, PA, 18304 cat# 9264-03).
mass spectral data. Coupling fast-scanning time-of-flight mass
If an emulsion occurred, the sample was centrifuged for 3 min
spectrometry (TOFMS) with comprehensive two-dimensional gas
at 3000 rpm (IEC Centra-8 Centrifuge, Geneva 20-Switzerland) in
chromatography provides the capabilities necessary for identifica-
order to achieve necessary separation of phases. Kuderna–Danish
tion of compounds within a complex matrix.
evaporation was then used to concentrate samples to a final vol-
Traditionally, discovery based analysis of complex samples uti-
ume of 2 mL. Internal standards, SV internal standard mix (Restek
lizing the technique described above requires a sophisticated and
Corp., Bellefonte, PA, USA cat# 31885), were added to each sample
knowledgeable analyst. The large number of peaks produced neces-
to result in a final concentration listed in Table 1.
sitates the analyst be capable of rapidly determining peak identity
and isolating compounds of interest. This process is time con-
suming as most environmental samples are complex and contain 2.2. Soil sample preparation
background analytes introduced via normal biological processes. In
order to reduce data processing time, a “normal” background can The soil samples were prepared using a modified USEPA 3550C
be defined in order to subtract previously recognized compounds ultrasonic extraction technique [25]. 30 g of each sample and 60 g
from subsequent samples to then identify emerging contaminants of anhydrous sodium sulfate (nominal weights) were weighed
of interest. Once the “normal” background of a particular matrix (Metler Toledo-MS204S, Columbus, OH) and mixed together in a
 S. Prebihalo et al. / J. Chromatogr. A 1419 (2015) 109–115 111

Table 1 oven was temperature programmed at 40 ◦ C for 1.5 min, 4 ◦ C/min

Compounds utilized as recovery standards and final concentrations (assuming 100%
to 315 ◦ C with a final hold time of 15 min. The secondary oven
recovery).
offset was 5 ◦ C and followed the same oven program ramp as in
Standard name Components Final extract the primary oven. The modulator offset was 15 ◦ C to the primary
concentration
oven temperature program. The MS transfer line temperature was
(pg/␮L)
330 ◦ C. Ionization was with electron ionization (EI) energy of 70 eV
QuEChERS internal 2,2 ,5- 200 and an ion source temperature of 250 ◦ C. The collected mass range
standard mix for Trichlorobiphenyl
was 50–550 amu with an acquisition rate of 200 scans per s after
GC–MS analysis
2,4,4 - 200 a solvent delay of 450 s. The detector voltage was set as the volt-
Trichlorobiphenyl age needed to yield a S/N of approximately 1000 for the 284 ion of
2,2 ,5,5 - 200 hexachlorobenzene (Restek Corp., Bellefonte, PA, USA cat# 32231)
Tetrachlorobiphenyl
injected on column, under standard (non-modulated 1D) condi-
Triphenylmethane 40
Tris(1,3- 200 tions at 200 pg/␮L prior to the analysis of any samples. Data was
dichloroisopropyl) acquired using a mass defect of 20 mu/100 u.
phosphate
Triphenylphosphate 80 2.4. Data processing
Acid surrogate mix 2-Fluorophenol 200
Phenol-d6 200
2,4,6-Tribromophenol 200 All data were processed using ChromaTOF software (LECO Corp.,
Revised B/N Nitrobenzene-d5 200 St. Joseph, MI, USA), version 4.51. Signal-to-noise minima were set
surrogate mix to 100, and the full chromatogram was processed over the complete
2-Fluorobiphenyl 200
mass range following the solvent delay. Positive identification of all
Pyrene-d10 200
p-Terphenyl-d14 200
compounds was based on the analysis of reference materials prior
to the analysis of sample extracts. Mass spectral identification was
also used for confirmation, and was based on both the spectra of the
400 mL beaker until a free flowing powder was achieved. As with reference materials and also on the National Institute of Standards
the water samples, QuEChERS internal standard mix for GC–MS and Technology (NIST, Gaithersburg, MD) mass spectral library, ver-
analysis, acid surrogate mix and revised B/N surrogate mix (Restek sion 2011. A minimum similarity match of 700 was required before
Corp., Bellefonte, PA, USA) were used as recovery standards and peak identification was tentatively assigned. Due to the modulation
added to a final concentration in extract of 200 pg/␮L (refer to process in which peaks are fractionated before entering the second
Table 1 for compound list). Each sample was serially extracted dimension column (2D), the 2D chromatographic peaks are repre-
three times using a 1:1 (vol/vol) acetone:dichloromethane solution sented in slices which are recombined to form a single 2D peak if
(Avantor, Center Valley, PA). A Branson 450 Sonifier equipped with second-dimension retention times of each slice were in agreement
a 3/4 inch solid horn and output control setting of 10, and 50% duty and mass spectral matches of 500 or more were met.
cycle, was used for each extraction. Kuderna–Danish evaporation Quantification was based on internal standards, using aver-
was used to concentrate samples to a final volume of 2 mL. age relative response factors pre-determined from the calibration
Additionally, laboratory control blanks were also extracted and curves for each of the target compounds. The internal standards
analyzed during each extraction process. For the water sample lab- used were deuterium-labeled polycyclic aromatic hydrocarbon
oratory control, 500 mL of Milli-Q Water (EMD Millipore Catalog compounds (Restek Corp., Bellefonte, PA, USA, cat# 31885), added
number QGARD00D2) was extracted. 30 g of anhydrous sodium sul- to all samples at a final extract concentration of 200 pg/uL. A
fate was extracted as the soil sample laboratory control (method standard curve was analyzed prior to the extracts using 8270
blank). MegaMix, a calibration standard containing 76 of the most com-
monly analyzed components (Restek Corp., Bellefonte, PA, USA
2.3. Instrumentation cat# 31850). Calibration standards were analyzed over range of
2–1000 pg/uL for each target compound.
All sample extracts were analyzed using a Pegasus 4D
GC × GC–TOFMS instrument (LECO Corp., St. Joseph, MI, USA). This 2.5. Reference method
system utilizes a non-moving quad-jet modulator consisting of
two cold nitrogen jets and two pulsed hot-air jets to trap and The “Reference Method” option within ChromaTOF software
refocus compounds eluting from the first dimension (1D) column. was utilized in order to prepare a data processing method in which
The gas chromatograph was a 7890A GC System with a 7683B future samples could be analyzed to determine the presence of out-
Series Injector (Agilent Technologies, DE, USA). The column ensem- liers. This feature allows the analyst to make pair-wise comparisons
ble consisted of an Rxi-5 Sil MS 60 m × 0.25 mm ID × 0.25 ␮m df between two samples in order to determine differences in sample
(Restek Corp., Bellefonte, PA, USA cat# 13626) coupled to an Rtx- composition. In this case the method blank for each sample matrix
200 1.3 m × 0.25 mm ID × 0.25 ␮m df (Restek Corp., Bellefonte, PA, and the samples themselves were compared in order to determine
USA cat# 15124) which was located inside the second dimension possible “outliers”. The reference sample to be compared to all soil
oven. The two columns were connected using a CPM Union (Agi- samples was 30 g of sodium sulfate taken through the soil sample
lent Technologies, DE, USA, cat# G3182-60581) and UltiMetal Plus preparation. The components identified in the reference sample
Flexible Metal Ferrules (FMF) (Agilent Technologies, DE, USA cat# were to be considered “normal”, and compared to the addition of
G3188-27501). The modulation period was 2.5 s. The hot pulse any new samples. The main parameters set to compare the refer-
duration was 0.60 s. Helium was used as the carrier gas at a con- ence analytes to those in the new samples were; retention time
stant flow rate of 1 mL/min. The injection volume was 1 ␮L of the deviation in the first and second dimension (in seconds), tolerance
final extract in dichloromethane. percent and the match similarity. Tolerance percent defines how
The sample was injected using a standard split/splitless injector closely the area of the analyte in the sample must match the ref-
held at 250 ◦ C in splitless mode for a 1.5 min hold time, and was erence sample. Table 2 outlines the reference method parameters.
equipped with a 4 mm single taper liner with deactivated quartz Each analyte in the unknown samples were labeled as one of the
wool (Restek Corp., Bellefonte, PA, USA cat# 23303.5). The primary following: “Match” (compound was found in both the reference and
112 S. Prebihalo et al. / J. Chromatogr. A 1419 (2015) 109–115

Table 2 survive the wastewater environment or treatment due to likely

ChromaTOF reference method parameters.
Parameter
1st dimension retention deviation (s)
Tolerance (%)
S/N threshold
Match threshold
unknown sample), “Not found” (compound is only present in the
reference), “Out of Tolerance” (compound is present in both the
reference and unknown, but is outside method parameters), and
“Unknown” (compound is only present in the unknown sample).
3. Results and discussion
3.1. Identification of contaminants
When using the reference method procedure, multiple com-
pounds were found in the water obtained from the Pennsylvania
State University WWTF that is considered “outliers”. Initially, dur-
ing manual data reduction, these compounds were tentatively
identified by a NIST library search as chlorine-substituted iso-
cyanates. Fig. 1 shows NIST library spectra which demonstrate the
high-quality, though inaccurate, match.
A total ion chromatogram (TIC) highlighting the identified
compounds is illustrated in Fig. 2. Isocyanates are unlikely to
rapid hydrolysis of the cyanate group [26]. To prove the misidenti-
Set point fication, six chlorine substituted methyl phenyl isocyanate isomer
2.5 standards were analyzed: 4-chloro-3-nitrophenyl isocyanate,
20 3-chloro-2-methylphenyl isocyanate, 4-chloro-2-methylphenyl
20 isocyanate, 2-chloro-6-methylphenyl isocyanate, 4-chloro-3-
700 methylphenyl isocyanate and 5-chloro-2-methylphenyl isocyanate
(Sigma–Aldrich, St. Louis, MO). While the mass spectral data
observed between the standards and samples is similar, the
large difference in first and second dimension retention times
supports the theory that these compounds were not chlorinated
isocyanates.
The mass spectral data of the NIST identified isocyanates clearly
indicates the presence of a single chlorine atom, due to the iso-
tope ratio pattern demonstrated in Fig. 3. In order to determine
the potential identity of these compounds, the mass of chlo-
rine was subtracted from a “suspected candidate” molecular ion
(m/z = 167), leaving a [M–Cl]+ mass of m/z = 133. When plotting an
extracted ion chromatogram at m/z = 133, unhalogenated benzotri-
azole compounds were identified with high mass spectral similarity
in the chromatogram at a lesser first-dimension retention time.
These compounds also exhibited several isomers similar to the
un-identified chloro-derivatives, suggesting that these compounds
may belong to the same chemical family. Additionally, their simi-
lar (later) second-dimension retention times allowed for improved
detection by resolving them away from the more prevalent com-
pounds in the sample matrix (Figs. 4–6).

Fig. 1. Peak true mass spectra vs. library isocyanate identification for influent collected at the WWTF.

Fig. 2. Total ion chromatogram displaying highlighted compounds of interest in Penn State’s influent.
 S. Prebihalo et al. / J. Chromatogr. A 1419 (2015) 109–115 113

Fig. 3. Chlorine isotope pattern observed in the mass spectrum of the NIST identified Fig. 6. Example mass spectrum of halogenated benzotriazoles observed in effluent
chloro-isocyanate compounds in influent collected from the WWTF. collected from the WWTF.

Further investigation of the halogenated benzotriazoles indi-
cated they occurred at roughly 100 times higher concentrations
in the “Penn State Campus” influent as compared to the “State
College Borough” influent, and these compounds seem to be
present continuously. This indicates that a significant source is
likely located on the Penn State campus. Chlorinated benzotria-
zoles are often used as corrosion inhibitors and in heating and
cooling systems. The Office of the Physical Plant (OPP) at Pennsyl-
vania State University confirmed that open-loop systems are used
for some of the cooling towers on campus. MSDS information for
the chemical process confirms the use of halogenated benzotria-
zoles in the open-loop system used by the University. This could
explain the frequency and concentration of these compounds
observed in the samples, as they were observed in the WWTF efflu-
ent consistently, which suggests probable deposition onto the Penn
State spray fields.
To confirm the identity of halogenated benzotriazoles in Penn
State’s waste water, several triazole standards were analyzed
(Sigma–Aldrich, St. Louis, MO). Although the availability of stan-
dards is limited and isomer position is difficult to determine
in the samples, the standards were used to confirm com-
pound class. Two standards, 1(chloromethyl)-1H-benzotriazole
and 5-chloro-7-methyl[1,2,4]triazolo[1,5-A]pyridine were con-
firmed to elute in the same area of the chromatogram as the
compounds of interest, indicating similar chemical class. The
high similarity between the mass spectral fragmentation and
retention times between the 1(choromethyl)-1H-benzotriazole
and a NIST identified chlorinated isocyanate indicates that
the compounds of interest are in fact halogenated benzotria-
zoles.
As the chlorinated benzotriazoles are observed in both the
influent and effluent at Penn State’s WWTF at comparative lev-
els, indicating they pass relatively unchanged through the normal
treatment process, their environmental transport and fate is of con-
cern. Additionally, halogenated benzotriazoles are a part of the
organohalogen class, which have been shown to exhibit environ-
mental persistence and bioaccumulation [27]. The Pennsylvania
State University utilizes a living filter system to further treat water
before it enters Spring Creek, meaning that the chlorinated benzo-
triazoles are being deposited onto the agricultural fields, potentially
causing consequences to human or animal health. It is important to
note that at wastewater treatment plants that do not implement a
“living filter” system, contaminants would be discharged into other
water sources without a further treatment step, so the relatively
unique situation with the PSU WWTF allows for simpler transport
and fate determination.

Fig. 4. TIC showing compounds identified in effluent collected from the WWTF.

Fig. 5. Extracted ion chromatogram (m/z 167) highlighting halogenated benzotriazole compounds observed in effluent collected from the WWTF.
114 S. Prebihalo et al. / J. Chromatogr. A 1419 (2015) 109–115
3.2. Environmental transport and fate
Soil samples from the Penn State agricultural fields at the surface
and a depth of six inches show an approximate twenty fold decrease
in the peak area of chlorinated benzotriazoles as compared to
treated effluent; indicating a drastic reduction at shallow depth
from what would be expected given the continuous emission of the
compounds, as long as these compounds are not bio-transformed
or efficiently transported through the soil depths. This is a qual-
itative comparison, but was deemed significant as the triazoles
were not detected at significant concentration at lower depths or
in the monitoring wells. This indicates an environmental factor
may be contributing to the significant decrease in concentration.
Biological/environmental degradation may be responsible for the
disappearance of the compounds observed in the monitoring well
samples, as the compounds move through the “living filter” process.
Photodegradation was the first process investigated, but laboratory
studies completed in November 2013 by Brockman and Dorman
[28] show halogenated benzotriazoles remaining relatively consis-
tent over time when exposed to sunlight, indicating this is not likely
the cause of disappearance in the spray fields. Additional studies
have shown that halogenated and non-halogenated benzotriazoles
degrade into a variety of compounds, including phenol, when they
are introduced to a soil environment [29]. Phenol has been classified
as a “priority pollutant” according to the United States Environmen-
tal Protection Agency (USEPA) and its presence in the environment
is a human health concern [30].
As described above, halogenated benzotriazoles have been
demonstrated to degrade to phenol, suggesting that the drastic
decrease in concentration of halogenated benzotriazoles in soil
samples may be due to environmental factors potentially causing
breakdown to phenol. Phenol was detected in each soil sample at
low concentrations, as well as the monitoring wells, which could
potentially be of concern due to the well water ultimately enter-
ing Spring Creek. However, the initial levels prove to be below the
limit set by the USEPA of 6 ppm in drinking water [31]. The soil
samples show the transport of phenol through the soil “filter” that
ultimately is deposited into monitoring wells. Spray field gradient
maps provided by the Office of the Physical Plant, show Well W2
being located at the lowest elevation and closest to “Toftrees Pond”.
A summary of results is shown in Table 3.
3.3. Reference method results
As most current methods are designed to detect certain “tar-
get” compounds such as PCBs or pesticides, contaminants that fall
outside these chemical classes have the potential to be overlooked
when utilizing these targeted methodologies. Traditionally, in the
case of true discovery work, the analyst would be required to man-
ually evaluate the significance of each peak, and make inferences
as to whether the compound is “significant” or a part of the nor-
mal matrix. However, when the analyst is able to separate “normal”
compounds from those considered to be “significant” the amount
Table 3
Summary of phenol concentrations observed in soil and well water samples from
the Penn State agricultural fields.
Sample ID Phenol concentration (ppm)
A14 spray water 0.0085
A14 surface soil 0.032
A14 6 deep soil 0.014
F122 surface soil 0.0064
F122 6 deep soil 0.016
Well W2 0.0061
Well W6 0.075
Well W7 0.023
of time required to evaluate potential contaminants is drastically
decreased. In order to create a reference method, the analyst first
defines what is considered “normal” in the matrix of interest. In this
case, the compounds identified in the method blanks were consid-
ered “background”. Method parameters such as match threshold
and retention time deviation were set in order to prevent the soft-
ware from inappropriately assigning library matches to the various
peaks. A data processing method referencing this “normal” sam-
ple was then used to process the new sample, similar to the data
processing methods used to calculate concentration of target com-
pounds. However, it is important to note that due to the nature of
this technique, all samples need to be analyzed under the same gas
chromatography and mass spectrometry methods.
Each soil sample was analyzed using the reference method pre-
pared from the soil method blank, which utilized sodium sulfate as
the extracted material. Compounds identified in the method blank
can be considered “background” and thus considered insignificant
in the new sample. At least fifty such compounds can be effec-
tively removed from the hit table of the new sample, a quantity
which could be increased if a “normal” sample was to be used for
comparison. When the reference method was applied to soil sam-
ples from Penn State’s agricultural fields, phenol was identified as
an “unknown” compound when compared to the method blank,
indicating phenol could potentially be considered “significant” and
require further investigation.
In addition to identifying phenol as an “unknown” compound in
spray field soil samples, the reference method technique was uti-
lized to compare “State College Borough” influent to “Penn State
Campus” influent. While chlorinated benzotriazoles were easily
observed at high levels in the “Penn State Campus” influent, ana-
lyzing with the reference method allowed the compounds to be
labeled as “out of tolerance”, indicating the necessity for further
investigation. The identification of phenol and chlorinated ben-
zotriazoles by the reference method illustrates the ability of the
program to quickly identify compounds that differ from what is
considered “normal”.
4. Conclusion
This work allowed for the development of an analytical
method capable of rapidly identifying emerging contaminants
in wastewater. The targeted-discovery method, which utilized
GC × GC–TOFMS combined with the reference method function
within ChromaTOF, permitted the identification of halogenated
benzotriazoles in Pennsylvania State University’s wastewater and
the subsequent identification of phenol in soil samples. Traditional
targeted analytical testing at the WWTF would likely not identify
low level compounds, potentially causing them to enter the envi-
ronment unrecognized. Due to the identification of halogenated
benzotriazoles at Penn State’s WWTF, which utilizes an open-loop
system sending treated effluent to water agricultural fields, the
environmental transport and fate of these organohalogens are of
concern. Reduced presence of these compounds suggests environ-
mental degradation as a potential factor of concern. Studies show
halogenated benzotriazoles have the potential to degrade to phe-
nol, a USEPA regulated compound.
As observed in the detection of chlorinated benzotriazoles in
wastewater, determining emerging contaminants and their envi-
ronmental transport and fate is a complex process. The technique
described above proved to be a valuable tool in resolving complex
matrices, as it allows the analyst to separate “normal” compounds
from those considered “significant”. Traditional data processing
requires the analyst to spend a significant quantity of time exam-
ining the chromatogram for compounds of interest. With the
implementation of the ChromaTOF reference method tool, the data
 S. Prebihalo et al. / J. Chromatogr. A 1419 (2015) 109–115
interpretation time is decreased by 50%, as halogenated benzotri-
azoles and other emerging contaminants are separated from those
to be considered “background”. As the amount of processing time
decreases, more time is available for analysts to investigate envi-
ronmental transport and fate, potentially leading to the discovery
of USEPA targeted compounds such as phenol in locations not typ-
ically monitored. Ultimately, the use of this method in monitoring
levels of contaminants after an environmental disaster could prove
extremely useful as the post processing time needed to analyze the
dense data sets is decreased.
Acknowledgements
The authors would like to acknowledge the Pennsylvania
State University Waste Water Treatment Facility for their contin-
ued support. Additionally, Restek Corporation, Leco and Aglient
Technologies all provided support in terms of consumables and
hardware which made this work possible.
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