Environ Sci Pollut Res (2015) 22:11387–11400
DOI 10.1007/s11356-015-4289-x
RESEARCH ARTICLE
GC/MS analysis of triclosan and its degradation by-products
in wastewater and sludge samples from different treatments
Fatemeh Tohidi 1 & Zongwei Cai 1
Received: 29 October 2014 / Accepted: 26 February 2015 / Published online: 27 March 2015
# Springer-Verlag Berlin Heidelberg 2015
Abstract A gas chromatography/mass spectrometry (GC/MS)-
based method was developed for simultaneous determination of
triclosan (TCS) and its degradation products including 2,4-di-
chlorophenol (2,4-DCP), 2,8-dichlorodibenzo-p-dioxin (2,8-
DCDD), and methyl triclosan (MTCS) in wastewater and sludge
samples. The method provides satisfactory detection limit, accu-
racy, precision and recovery especially for samples with compli-
cated matrix such as sewage sludge. Liquid-liquid extraction and
accelerated solvent extraction (ASE) methods were applied for
the extraction, and column chromatography was employed for
the sample cleanup. Analysis was performed by GC/MS in the
selected ion monitoring (SIM) mode. The method was success-
fully applied to wastewater and sludge samples from three dif-
ferent municipal wastewater treatment plants (WWTPs).
Satisfactory mean recoveries were obtained as 91(±4)–106(±7)
%, 82(±3)–87(±4) %, 86(±6)–87(±8) %, and 88(±4)–105(±3) %
in wastewater and 88(±5)–96(±8) %, 84(±2)–87(±3) %, 84(±7)–
89(±4) %, and 88(±3)–97(±5) % in sludge samples for TCS, 2,4-
DCP, 2,8-DCDD, and MTCS, respectively. TCS degradation
products were detected based on the type of the wastewater
and sludge treatment. 2,8-DCDD was detected in the plant
Responsible editor: Leif Kronberg
Electronic supplementary material The online version of this article
(doi:10.1007/s11356-015-4289-x) contains supplementary material,
which is available to authorized users.
* Zongwei Cai
zwcai@hkbu.edu.hk
Fatemeh Tohidi
rtowhidi55@gmail.com
1
State Key Laboratory of Environmental and Biological Analysis,
Department of Chemistry, Hong Kong Baptist University, Kowloon
Tong, Hong Kong SAR, China
utilizing UV disinfection at the mean level of 20.3(±4.8) ng/L.
2,4-DCP was identified in chemically enhanced primary treat-
ment (CEPT) applying chlorine disinfection at the mean level of
16.8(±4.5) ng/L). Besides, methyl triclosan (MTCS) was detect-
ed in the wastewater collected after biological treatment (10.7±
3.3 ng/L) as well as in sludge samples that have undergone
aerobic digestion at the mean level of 129.3(±17.2) ng/g dry
weight (dw).
Keywords Triclosan . Wastewater treatment .
2,8-Dichlorodibenzo-p-dioxin . 2,4-Dichlorophenol . Methyl
triclosan . Sludge
Introduction
Triclosan (2,4,4′-trichloro-2′-hydroxydiphenyl ether (TCS))
as a prevalent antibacterial, antifungal, and antiviral agent
(Jones et al. 2000) in a variety of products (Tan et al. 2002,
Kazama et al. 1974, Chow et al. 1977, Singer et al. 2002) has
been widely focused since its elevated concentration in differ-
ent media along with the evidences of transformation to more
toxic compounds such as 2,4-dichlorophenol (2,4-DCP) and
2,8-dichlorodibenzo-p-dioxin (2,8-DCDD) has been docu-
mented (Aranami and Readman 2007, Buth et al. 2009,
2010, Ferrer et al. 2004, Kanetoshi et al. 1987, 1988, Latch
et al. 2003, 2005). Ubiquitous occurrence of TCS in the envi-
ronment is the result of widespread use and consequent trans-
port to the wastewater. TCS is partially treated in a wastewater
treatment plant (WWTP) and eventually discharged to the
environment. This compound is vastly found in wastewater
(Bester 2005, Buth et al. 2009, Gatidou et al. 2007, Greyshock
and Vikesland 2006, McAvoy et al. 2002, Singer et al. 2002,
Mezcua et al. 2004) and receiving water bodies ( Bester 2005,
Singer et al. 2002). Depending on the treatment technology,
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TCS was found to be removed from wastewater between 58 %
in trickling filter to 96.2 % in activated sludge process
(McAvoy et al. 2002). Singer et al. (2002) showed that 79 %
of the TCS was biodegraded in the biological treatment step in
a typical secondary treatment plant while 15 % was adsorbed
to the sludge. The remaining 6 % was discharged into the
receiving water. Although not recognized as a toxic com-
pound, TCS may transform to other products with potent tox-
icity. It has been evidenced that TCS may undergo
photodegradation (Latch et al. 2005, Lores et al. 2005, Son
et al. 2007) when exposed to UV light, biodegradation
(Balmer et al. 2004, Bester 2005, Lindstorm et al. 2002) in
aerobic condition, and oxidation during chlorination (Canosa
et al. 2005, Greyshock and Vikesland 2006) in the chlorine
disinfection process. 2,4-DCP, a probable transformation
product, is listed as the priority toxic pollutant by US
Environmental Protection Agency (1994). Moreover,
International Agency for Research on Cancer has classified
this compound in group 2B, possibly carcinogenic to humans
(World Health Organization 1999). Another important trans-
formation product, 2,8-DCDD is a member of dioxin family
with a recently reported relative potency factor of 1×10−4
compared to 2,3,7,8-TCDD (Samara et al. 2009). This com-
pound is suspected to be transformed to higher chlorinated
dioxins when exposed to light or chlorine (Buth et al. 2010).
Furthermore, chlorinated triclosans are believed to be the pre-
cursors of polychlorodibenzo-p-dioxins (PCDDs) (Buth et al.
2009), the most toxic synthetic compounds known. Lastly,
methyl triclosan (MTCS) resulted from microbial methylation
of TCS in aerobic condition. It has received much attention
recently due to its hydrophobicity (log Kow =5.2) (Waria et al.
2011) and persistency in the environment (Lindstorm et al.
2002). MTCS has been vastly detected in the biota as well
as wastewater and surface water (Balmer et al. 2004, Leikera
et al. 2009).
There are a number of analytical methods that reported the
determination of TCS in environmental samples. For instance,
Okumura and Nishikawa (1996) published a gas
chromatography/mass spectrometry (GC/MS)-based method
for the determination of TCS and its 3-chlorinated derivative
via methylation with diazomethane in water, sediment, and
fish samples. Detection limits were 0.030–0.0059 ng/mL,
1.7–4.6 ng/g, and 0.89–2.5 ng/g for surface water, sediment,
and fish samples, respectively. Besides, recoveries ranged 74–
112 %, 91–119 %, and 83–117 % for the abovementioned
samples, respectively. Bester (2005) developed a method
based on liquid-liquid extraction and stir bar sorption for the
extraction and cleanup to determine TCS and MTCS in water
and wastewater. Butler et al. (2012) utilized accelerated sol-
vent extraction (ASE) and C18 cartridge for extraction and
cleanup of soil. Eventually, GC/MS was applied for instru-
mental analysis of TCS and MTCS. The mean recovery of
94.3 % and the method detection limit of 1.4 ng/g were
Environ Sci Pollut Res (2015) 22:11387–11400
obtained. Buth et al. (2010) sought the presence of CTDs
(chlorinated TCS derivatives) originated from wastewater-
based TCS in the sediment cores by using soxhlet extraction,
Oasis HLB cartridge, and silica column for sample prepara-
tion. A modified US EPA Method 1613B was applied sepa-
rately to investigate DCDDs (dichlorodibenzoparadioxins)
and DCDFs (dichlorodibenzofurans) in the samples. Mezcua
et al. (2004) studied the presence of 2,7/2,8-DCDD in waste-
water using Oasis HLB cartridge for extraction of samples.
The recoveries and the relative standard deviations were
80 % and average 9 %, respectively. Among the studies car-
ried out on TCS determination, few have considered the anal-
ysis of sewage sludge probably due to the complexity of
sludge matrix that makes the sample cleanup a challenging
step especially for trace analysis purposes. As an example,
Ying and Kookana (2007) studied the occurrence of TCS in
Australian wastewater and biosolid. Wastewater samples were
extracted with C18 cartridge while biosolid samples were son-
icated and finally cleaned up with the same cartridge. Samples
were then analyzed with GC/MS. The recoveries of 101–
104 %, 89–96 %, and 73–74 % were obtained for surface
water, wastewater, and biosolid, respectively. In addition, the
limit of quantification was 3 ng/L and 5 μg/kg for water and
biosolid, respectively. Another example is the method report-
ed by Samaras et al. (2011) for the determination of acidic
pharmaceuticals and phenolic endocrine disrupting chemicals
in wastewater and sewage sludge. C18 cartridge was found to
be suitable for extraction of target compounds from wastewa-
ter. After the extraction, derivatization was carried out with a
mixture of N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA), 1 % trimethylchlorosilane (TMCS), and 10 μL pyr-
idine. For sludge extraction, the samples were sonicated with a
mixture of MeOH/H2O. The cleanup and derivatization pro-
cedure were the same as wastewater. Finally, the sample was
analyzed with GC/MS. Absolute recoveries of 67±1 and 86±
4 % were obtained for TCS in wastewater and sludge samples,
respectively. Moreover, limit of detection was 3.6 ng/L and
15 ng/g for wastewater and sludge, respectively.
We hypothesized that TCS transformation may take place
during the wastewater and sludge treatment and it may be
affected by the treatment technology. Therefore, various
TCS degradation products were chosen based on treatment
type and potent generation. Attempt was done to develop a
single method for analysis of the target compounds except 2,
8-DCDD in sewage sludge that required a lower matrix back-
ground. A method was developed for simultaneous determi-
nation of TCS and its degradation products including 2,8-
DCDD, 2,4-DCP, and MTCS in wastewater samples. In addi-
tion, since the major part of TCS and potent by-products could
be adsorbed to the sewage sludge, a robust and reliable meth-
od was developed for determination of the aforementioned
compounds in sludge samples. The study compared the effect
of various experimental parameters on each step of the
Environ Sci Pollut Res (2015) 22:11387–11400
developed methods to obtain the optimum condition, and fi-
nally, the methods were well validated against wastewater and
sludge samples collected from three WWTPs with three dif-
ferent treatment processes in Hong Kong. Moreover, the effect
of the applied treatment on the degradation of TCS and the
occurrence of resulted by-products are discussed.
Experimental
Sample collection
Wastewater samples were collected from raw sewage (influ-
ent), middle stage if the plant had more treatment stages, and
treated wastewater (effluent) of three WWTPs. Sludge sam-
ples were grab samples of primary sludge (sludge originated
from the primary sedimentation), secondary sludge (sludge
originated from the activated sludge process), and dewatered
sludge (treated sludge). Sampling site scheme is given in
Table 1S (online resource). Moreover, wastewater quality pa-
rameters are presented in Tables 2S and 3S (online resource).
The frequency of sampling was once per 3 months.
Wastewater samples were collected in two 4-L amber bottles
as grab samples. The bottles were prewashed with distilled
water, methanol, ethyl acetate, methanol, and distilled water.
After collecting sample and in situ pH measurement, phospho-
ric acid was added to reach pH 3–4 for stabilization. The
samples were then threaded with caps and Teflon and stored
in an ice-filling cooler for immediate delivery to the analytical
laboratory. The samples were stored at 4 °C in a refrigerator
for immediate analysis. Primary and secondary sludge sam-
ples were collected in amber glass jars, and appropriate
amount of methanol was in situ added. The samples were then
stored at 4 °C in a refrigerator for immediate analysis.
Dewatered sludge was collected in aluminum foil and stored
at −18 °C until further analysis. All information about the
plants was provided by the personnel and staff.
Reagents and chemicals
TCS and 2,8-DCDD were purchased from Wellington
Laboratories (Ontario, Canada). TCS-d3 was provided by
Accustandard Inc., USA. 2,4-DCP and MTCS were provided
by Dr Ehrenstorfer GmbH. Dichloromethane (DCM) and ethyl
acetate (EA) were from ACS Chemical Inc. Nonane and n-
hexane were from Anaqua Chemicals Supply. Methanol and
acetonitrile (ACN) were obtained from TEDIA Company Inc.
Anhydrous sodium sulfate, sodium hydroxide, and formic acid
were of analytical grade. Silica (0.063–0.2 mm) and alumina
activated acidic (mesh~150) were from Merk and Brockmann
grade 1, respectively. N-(tert-Butyldimethylsilyl)-N-methyl-
11389
trifluoroacetamide (MTBSTFA) was provided by Sigma-
Aldrich.
Sample preparation for the analysis of TCS, MTCS,
2,8-DCDD, and 2,4-DCP in wastewater
Two hundred fifty milliliters of wastewater sample was spiked
with 100 ng of TCS-d3 and allowed to equilibrate for 1 h. It
was then acidified with 250 μL formic acid and extracted with
3×25 mL dichloromethane. For influent samples that showed
emulsion in the interface, temperature decrease and mechani-
cal agitation of the organic phase were effective to disturb the
emulsion. The extract was dried over anhydrous sodium sul-
fate (0.1 g) and concentrated by use of nitrogen evaporator.
For cleanup, the concentrate was reconstituted to 0.5 mL with
n-hexane and quantitatively loaded onto a 10 % acidified sil-
ica column conditioned with 6 mL of n-hexane. Silica column
was prepared by dry-packing a Pasteur pipette with a plug of
glass wool, 0.6 g of silica gel (40–75 μm) acidified with ap-
propriate amount of sulfuric acid, and 0.2 g of anhydrous
sodium sulfate on the upper layer. The column was then eluted
by gravity with 7 mL of n-hexane/DCM (6:4) (F1), 7 mL of
DCM (F2), and 6 mL of DCM/acetone (98:2) (F3) for 2,8-
DCDD and MTCS, TCS, and 2,4-DCP fractions, respectively.
F1 and F2 fractions were mixed, dried over sodium sulfate
(0.1 g), and concentrated under a gentle stream of nitrogen.
Finally, the concentrate was reconstituted to 400 μL with
DCM. One microliter of the solution was injected to GC/
MS. Since 2,4-DCP is a polar compound, peak shape shows
tailing in the chromatogram that causes further poor resolu-
tion, uncertainty in the retention time, and error in quantifica-
tion. Therefore, determination of 2,4-DCP was based on de-
rivatization with MTBSTFA. For this purpose, 70 μL of the
reagent and 100 μL of ACN were added to the extract follow-
ed by vigorous shaking with a vortex mixer for 10 s and
keeping in the oven at 60 °C for 30 min. After the reaction
was complete, the solution was brought to 400 μL with DCM.
A 6–8-point calibration curve was constructed for 2,4-DCP
derivative (1 to 500 μg/L), 2,8-DCDD (5 to 250 μg/L), and
MTCS (10 to 500 μg/L), respectively. For each sequence of
samples, a method blank and for each sample a spiked sample
was analyzed with the same protocol described to check for
any contamination.
Sample preparation for the analysis of TCS, MTCS,
2,8-DCDD, and 2,4-DCP in sludge
All sludge samples except dewatered sludge were centrifuged at
6,000 rpm for 8 min. The water was separated, and the solid
sludge was further lyophilized in the freeze-dryer. 0.5 g of the
sample was spiked with 100 ng TCS-d3 and left for 1 h so that
solvent will be evaporated. Then, it was placed in an ASE cell
filled with inert diatomaceous earth material and was extracted
11390
with DCM as the solvent extractor. The ASE condition was as
follows: preheat 1 min, heat 5 min, static 5 min, flush 60 %,
purge 60 s, cycle two times, pressure 1,200 psi, and temperature
100 °C. After extraction, the sample was dried over 0.2 g anhy-
drous sodium sulfate, concentrated over a gentle stream of nitro-
gen, and then reconstituted to 1 mL with n-hexane. A pre-
prepared gel permeation chromatography (GPC) column was
used for the first step of cleanup. For this purpose, the column
was first washed with proper amount of DCM/Hex (1:1) and
then the extractant was loaded onto the top of the column. The
same solvent was used for elution. The next 20 mL of eluate was
collected after discarding the first 20-mL portion. In order to
remove sulfur-containing compounds, metallic copper was acti-
vated by HCl and added to the extract. The sample was decanted
and concentrated under a gentle stream of nitrogen and then
reconstituted to 1 mL with n-hexane. A multilayer silica column
was used for further cleanup. For this purpose, a glass column
was wet packed with 6 g of 5 % w/w deactivated silica topped
with 6 g of 10 % w/w acidified silica and 2 g of anhydrous
sodium sulfate on the upper layer. The column was firstly washed
with 30 mL of n-hexane. The extract was then loaded onto the
column and eluted with 30 mL n-hexane, 50 mL of DCM, and
50 mL of DCM/Ac (98:2) for washing and eluting fraction 1
(TCS, MTCS) and fraction 2 (2,4-DCP), respectively. Then,
the fractions were dried over sodium sulfate (0.2 g) and concen-
trated by using gentle stream of nitrogen. Fraction 2 was further
derivatized with MTBSTFA according to the previous protocol.
The extracts were reconstituted to 400 μL for GC/MS analysis.
For 2,8-DCDD determination, 10 g dry weight (dw) sample was
placed in an ASE cell filled with inert diatomaceous earth mate-
rial and extracted with toluene with the same condition described.
The extract was washed with 3×7 mL sulfuric acid and distilled
water. Then, it was dried over 1 g sodium anhydrous and con-
centrated to a final volume of 1 mL of n-hexane (solvent change).
The sample was eluted by 60 mL n-hexane through a multilayer
silica column containing 2 g basic silica 2 % w/w, 1 g neutral
silica, and 4 g acidic silica 30 % w/w, respectively, topped with
1 g anhydrous sodium sulfate. The eluate was then introduced
onto a column containing 4 g of acidic alumina prewashed with
20 mL hexane. The column was further washed with 30 mL of n-
hexane followed by 20 mL of 60:40 DCM/n-hexane (v/v). The
eluate was then concentrated to the final volume of 200 μL to be
analyzed by GC/MS.
GC/MS analysis
Instrumental analysis was performed on a gas chromatograph/
mass spectrometer Agilent 6890 with 5975B MSD. A HP-
5MS GC column (30 m × 250 μm internal diameter ×
0.25 μm film thickness, (5 %-phenyl)-methylsiloxane) was
used for the separation. GC/MS condition was as follows:
the injector and the transfer line temperature were set to 280
and 300 °C, respectively. Temperature program was initiated
Environ Sci Pollut Res (2015) 22:11387–11400
with 75 °C for 1 min, increased to 230 °C at 10 °C, then to
280 °C at 20 °C/min, and held for 15 min. Injection volume
was 1 μL in splitless mode, and solvent delay was 5 min to
prevent filament damage. The carrier gas was helium at a flow
rate of 1.2 mL/min. Mass spectrophotometer was operated in
electron ionization mode at 70 eV and was calibrated against
perfluorotributylamine (PFTBA) on a monthly basis. Selected
ion monitoring was applied for quantitative analysis. To
achieve the highest sensitivity and response, ions were chosen
based on maximum abundance and minimum presence of any
interference. For each compound except TCS, the most abun-
dant ion was selected for quantitation along with the next two
abundant ions for identification confirmation. The related ions
are shown in Table 1. Identification was based on retention
time for each compound in GC chromatogram, examination of
mass profile peak, mass peak shift, and isotopic ratio of se-
lected ions. Instrumental blank was utilized every five samples
to check for the analyte carryover. For a standard check solu-
tion, a mid-level calibration standard was analyzed at the be-
ginning and end of each sequence of the sample analysis to
ensure about the instrument performance.
A calibration curve made of 7 points was constructed for
TCS, and the area ratio of TCS peak to that of TCS-d3 was used
for the calculation of relative response factor (RRF). The con-
centration was then calculated based on the comparison of peak
area of TCS and the internal standard as well as RRF. Three
calibration standards with different concentrations were ana-
lyzed to obtain daily RRF and compare to the mean RRF ob-
tained from the calibration curve. Ideal value for RRF is unity,
and any deviation from this value arises from error in standard
solution preparation or different purities of the standards.
Results and discussion
Optimization of wastewater sample preparation
Extraction of wastewater samples
For wastewater liquid-liquid extraction, different pH values
were examined to evaluate the effect of pH on the extraction
Table 1 Selected ions for quantitation and confirmation in SIM mode
and chromatographic retention time of the target compounds
Compound Quantitation Confirmation Retention
ion (m/z) ion (m/z) time (min)
TCS 290 288, 218 17.14
TCS-d3 293 291, 221 17.14
2,8-DCDD 252 254, 189 15.69
2,4-DCP (silyl derivative) 219 221, 276 11.99
MTCS 302 304, 306 17.23
Environ Sci Pollut Res (2015) 22:11387–11400
efficiency (3, 5, 7, and 9). The experiments were first per-
formed by spiking various amounts of standard solution of
target compound to reagent blank sample (Milli-Q water) to
assess the sample preparation. Then, the experiment was re-
peated with the same procedure except pre-determined waste-
water sample was utilized as a real sample. One hundred
nanograms of TCS-d3 was added to the sample prior to GC
analysis for the recovery study. Experiment result was an av-
erage from duplicate analysis of three sets of samples. It was
found that the recovery was higher at low pH values for TCS
and 2,4-DCP due to their acidic characteristic while no signif-
icant recovery change was observed for other compounds.
Therefore, acidic condition was preferred for the extraction
of compounds. Solvents including n-hexane, DCM, and tolu-
ene were also examined as solvent extraction, and DCM was
found to be the suitable solvent for extraction as its polarity
could cover the variety of polarities of target compounds.
Moreover, various volumes of solvent were considered for
extraction (10–50 mL) and it was observed that 25 mL of
DCM suffices for three consecutive extractions. For lower
volume of solvent, the residue of TCS could still be found in
the extracted sample. Moreover, for higher solvent volume, no
improvement in the recovery and no TCS residue in the ex-
tracted sample were observed.
Cleanup of wastewater samples
For wastewater sample cleanup, two commercial SPE cartridges,
C18 and Oasis HLB, and a homemade silica column were stud-
ied. Recovery for TCS, MTCS, and 2,4-DCP when utilizing
HLB showed a better result comparing to C18 column, though
the recovery for 2,8-DCDD was not satisfactory for both car-
tridges (37±5–53±7 %). Therefore, a homemade silica column
was also considered. The results showed an improvement in
recovery of 2,8-DCDD and in signal to noise ratio in chromato-
gram. Therefore, silica column was applied for cleanup. The
related recovery data are given in Table 4S (online resource).
Optimization of derivatization
For the quantitative analysis of 2,4-DCP, two different
silylating agents BSTFA and MTBSTFA were examined.
MTBSTFA derivative showed more stability during the anal-
ysis. Moreover, the derivative peak in the chromatogram lo-
cated in a longer retention time which was more favorable
comparing to BSTFA derivative. Therefore, experiments were
performed to study various reagent volumes (30–100 μL),
temperatures (25–70 °C), and reaction times (15–60 min) to
obtain a complete and reproducible derivatization. It was
found that reagent with volume lower than 70 μL resulted in
lower sensitivity. Furthermore, temperature showed to have a
direct effect on kinetic of the reaction. At room temperature,
almost half of 2,4-DCP remained intact and after 1 h all the
11391
derivative compound transformed back to 2,4-DCP. However,
the derivative was stable when the reaction temperature in-
creased to 60 °C. No change in sensitivity or peak area was
observed beyond 60 °C. In the same manner, reaction time
shorter than 30 min resulted in incomplete derivatization and
presence of 2,4-DCP peak in the chromatogram. Therefore,
the reaction time was set to 30 min.
Optimization of sludge sample preparation
Extraction of sludge samples
Due to high content of organic matter in the sludge and
probable stronger binding of analytes, ASE was selected
for extraction to ensure efficient and complete extraction of
sludge samples. The experiments were first performed by
spiking various amounts of standard solution of target
compounds to the reagent blank sample (diatomaceous
earth material) to assess the sample preparation. Then, the
experiment was repeated with the same procedure except
pre-determined sludge sample was utilized as a real sam-
ple. For this section, the sludge was first extracted with 1:1
solution of MeOH:H 2 O in a sonication bath for about
30 min to extract TCS present in the sludge. Moreover, a
blank sample was analyzed for every batch of samples to
determine the TCS residue in the sample. It was observed
that TCS concentration was below the limit of detection in
most of the samples. Various solvents including EA, DCM,
and toluene were applied for extraction recovery assess-
ment. TCS and MTCS were observed to have higher re-
coveries with DCM while that of 2,4-DCP was higher
when EA was used. Though, since the difference was not
considerable, DCM was selected for extraction of TCS,
MTCS, and 2,4-DCP. However, the extraction recovery
for 2,8-DCDD dropped dramatically when extracted with
DCM probably due to stronger binding of this compound
to organic matter of the sludge. Therefore, an extraction
procedure with toluene as extractant was developed with
the same ASE parameters. Data concerning recovery re-
sults are given in Table 5S (online resource). Other ASE
parameters such as solvent, temperature, number of cycles,
and pressure were investigated as well. Pressure change
did not show a significant effect on the extraction efficien-
cy of all the compounds though temperature below 100 °C
decreased the extraction recoveries of all compounds.
Cycle numbers from 1 to 3 were also applied. While 1 cycle
left target compounds unextracted in the sample, 3 cycles
showed a decrease in silica cleanup recoveries of all com-
pounds due to clogging in the silica column as well as high
background and limit of detection in mass spectrum. It was
observed that 2 cycles was good enough for a complete
extraction with no effect of clogging. Moreover, the mass
spectra background did not affect the limit of detection.
11392
Optimization of GPC procedure
GPC column was calibrated for efficient elution. For this pur-
pose, the column was eluted after the standard solutions were
introduced to the column and eluate was collected in every
10 mL. After analyzing each portion and detecting the target
compounds, attempt was done to collect the eluate in every
1-mL fraction. It was observed that all the target compounds
were completely eluted in the second 10-mL portion; thus, for
the sample cleanup, the first 10-mL portion was discarded and
the second 10 mL was collected for further cleanup.
Approximately, 40–50 % of impurities and interferents were
separated in this step.
Optimization of silica column procedure
Due to complicated matrix of sludge samples, the commercial
cartridges were unable to clean up the samples; therefore, a
multilayer silica column was considered for an efficient clean-
up procedure. It was observed that the recovery for 2,4-DCP
was not satisfactory when plain silica was used. Moreover,
activation of the silica led to a decrease in recovery of TCS
and 2,4-DCP. This may be due to strong binding of silica-free
OH sites after activation with the two polar compounds.
Therefore, silica was 5 % w/w deactivated with water to par-
tially deactivate the silica OH sites. The result showed im-
provement in recovery for both compounds while no signifi-
cant effect on the recoveries of other compounds (Table 6S,
online resource).
Quantitative analysis
In order to achieve reliable quantitation of TCS, isotope dilu-
tion mass spectrometry (IDMS) approach was applied for
sample analysis. This method has been widely used for envi-
ronmental sample analysis (Diemer and Heumann 2000,
Mechlińska et al. 2012, Dai et al. 2012, Wang et al. 2011,
Itoh et al. 2011, Milton and Wang 2002). The theory renders
the introduction of a compound as close in structure as possi-
ble to the target molecule to the sample (Li 2001). While ideal
internal standard requirement is hardly met, IDMS exhibits a
perfect approach to achieve this goal by applying a stable
isotope-labeled analog of the compound of interest, hence
dramatic decrease in variance and increase in the accuracy
of the analysis (Colby et al. 1981, Vogl and Pritzkow 2010).
To apply IDMS, TCS-d3 was used as an internal standard. The
molecular ion of TCS, m/z 288 was observed to be the most
abundant ion. The two fragment ions at m/z 252 and 218 may
have resulted from losing one and two chlorines from the
molecular ion, respectively. Furthermore, ether bond cleavage
of the TCS molecule may lead to produce the ion at m/z 162
and lastly ion m/z 146 may come from hydroxyl group loss
from ion m/z 162. The same fragmentation pattern was
Environ Sci Pollut Res (2015) 22:11387–11400
observed for TCS-d3 except the three added mass units from
deuterium label.
The selection of quantitative ions of TCS and TCS-d3 was
important. Although TCS and TCS-d3 exhibited the most
abundant ions at m/z 288 and 291, respectively, the ion at m/
z 288 was not selected as the quantitative ion for TCS because
it produced an isotope ion at m/z 291 that has the same mass as
its corresponding ion from TCS-d3 (with 12.67 % abundance
of the base peak). Thus, the TCS ion at m/z 291 originated
from 37Cl and 13C isotopes might interfere with the quantita-
tion of the internal standard and cause significant analytical
error, especially when the level of TCS in samples was signif-
icantly different from the spiked level of TCS-d3. In this study,
the ion at m/z 290 from TCS was selected as the quantitative
ion and the corresponding ion at m/z 293 was selected for
TCS-d3. Because this ion has a relative abundance of 97 %
compared to that of the ion at m/z 288, the analytical sensitiv-
ity was expected to be similar. Although there is still a small
contribution of TCS to ion at m/z 293 with 4.1 % abundance
of the base peak, the resulted analytical error was minimal
after correction.
Method limit of detection
The limit of detection (LOD) can be improved by increasing
the sample volume/amount used for the analysis. However,
the effect of matrix might be enhanced as well.
Consequently, higher interference due to matrix will be ob-
served. It was found that the analysis of 250-mL sample pro-
vided satisfied results. Method detection limit was obtained as
the lowest sample concentration giving a signal-to-noise
greater than 3. As can be seen from Table 2, for 250 mL of
sample, method detection limit in the real sample was calcu-
lated to be 10.0, 6.0, 4.0, and 9.0 ng/L for TCS, 2,8-DCDD, 2,
4-DCP, and MTCS, respectively. Moreover, for 0.5 g of
sludge samples, the method detection limit for the target com-
pounds except 2,8-DCDD were 10, 1.0, and 5.0 ng/g for TCS,
2,4-DCP, and MTCS, respectively. For 2,8-DCDD, the detec-
tion limit was found to be 3 ng/g for 10 g of the sludge sample.
Table 2 Limit of detection and quantification of the target compounds
for wastewater and sludge samples
Compound Wastewater Sludge
LOD (ng/L) LOQ (ng/L) LOD (ng/g) LOQ (ng/g)
TCS 10.0 30.0 10.0 30.0
2,4-DCP 4.0 12.0 1.0 3.0
2,8-DCDD 6.0 18.0 3.0 6.0
MTCS 9.0 27.0 5.0 15.0
Environ Sci Pollut Res (2015) 22:11387–11400 11393

Table 3 Mean recoveries (in percent, n=3), precisions, and accuracies of the developed method for the target compounds in wastewater and sludge
samples

Compound Wastewater Sludge

Mean recovery Mean rel. error Mean RSD Mean recovery Mean rel. error Mean RSD

TCS 91(±4)–106(±7) −1.19 6.3 88(±5)–96(±8) −3.15 6.4

2,4-DCP 82(±3)–87(±4) −14.3 4.2 84(±2)–87(±3) −14.7 4.3
2,8-DCDD 86(±6)–87(±8) −13.4 4.4 84(±7)–89(±4) −13.4 4.9
MTCS 88(±4)–105(±3) −3.2 6.2 88(±3)–97(±5) −7.4 6.0
TCS-d3 92(±6)–110(±7) −0.8 5.2 88(±6)–99(±9) −1.4 9.2

Method accuracy and precision Analysis of triclosan in wastewater samples

The IDMS application improved the method accuracy
and precision. However, it should be mentioned that
low extraction recovery of the target compound can in-
crease the detection limit. Therefore, recovery test for
wastewater was performed by fortifying 250 mL reagent
blank and predetermined wastewater sample with vari-
ous amounts of TCS (10, 50, 100 ng), 2,4-DCP (10, 50,
100 ng), 2,8-DCDD (5, 10, 50 ng), and MTCS (10, 50,
100 ng). The test for sludge was carried out on the
diatomaceous earth as well as the extracted sludge. For
recovery efficiency examination, 100 ng of TCS-d3 was
added prior to GC injection. Experiment result was an
average from duplicate analysis of three sets of samples.
Table 3 shows the recovery, precision, and accuracy of
the developed method for the analytes in wastewater
and sludge samples.
In order to validate the developed methods, three major
WWTPs with different treatment technologies were selected
and the wastewater and sludge samples were analyzed during
February 2011 to February 2012 in dry (November–March)
and wet (April–September) seasons. The collected samples
after each treatment stage were analyzed to assess the removal
efficiency. TCS was detected with various loading concentra-
tion in the influent and a decreasing trend toward effluent in
wastewater samples (Table 4). TCS loading concentration var-
ied significantly in three plants and showed different elimina-
tion based on the treatment type practiced in each plant.
Cheung Chau (C.C.) WWTP runs primary treatment with
the average daily capacity of 8,600 m3/day to serve a popula-
tion of nearly 23,000. The mean flow rate for two sampling
events in dry season as well as that in wet season were 8,219
and 9,136 m3/day, respectively. Since the flow rate for two

Table 4 Mean level of the target compounds in wastewater and sludge samples from three studied WWTPs in Hong Kong

Compound Influent After primary After biological Before Effluent Primary sludge Secondary Dewatered
(ng/L) sedimentation sedimentation disinfection (ng/L) (ng/g) sludge (ng/g) sludge (ng/g)
(ng/L) (ng/L) (ng/L)

S.C. WWTP
TCS 348.7(±49.3) 253.5(±50.1) NA 253.5(±42.2) 236.9(±27.5) 462.7(±84.7) NA 455.2(±83.2)
2,4-DCP ND ND NA ND 16.8(±4.5) ND NA ND
2,8-DCDD ND ND NA ND ND ND NA ND
MTCS ND ND NA ND ND ND NA ND
S.T. WWTP
TCS 327.4(±76.3) 314.9(±73.9) 148.3(±27.5) 148.3(±27.5) 133.2(±24.3) 206.6(±22.1) 162.2(±16.2) 308.6(±32.3)
2,4-DCP ND ND ND ND ND ND ND ND
2,8-DCDD ND ND ND ND 20.3(±4.8) ND ND ND
MTCS ND ND 10.7(±3.3) 10.7(±3.3) 10.5(±4.1) ND 51.0(±7.5) 53.7(±7.1)
C.C. WWTP
TCS 512.8(±52.1) NA NA NA 395.8(±48.6) 2288.3(±244.4) NA 2505.9(±253.4)
2,4-DCP ND NA NA NA ND ND NA ND
2,8-DCDD ND NA NA NA ND ND NA ND
MTCS ND NA NA NA ND ND NA 129.3(±17.2)

NA not available, ND not detected

11394
sampling events in the same season had negligible variation,
mean flow rate was used. Primary treatment enables removal
of around 70 % of the particulates (total suspended solid
(TSS)) and 30 % of the biochemical oxygen demand (BOD)
(Table 2S, online resource). Influent was subjected to filtration
of grits and sedimentation. The effluent was then discharged
to the surface water without further treatment. Generated
sludge was aerobically digested, dewatered, and disposed to
landfill. The highest average of TCS loading in the influent
among the studied WWTPs belonged to this plant (729.0±
31.1 ng/L). Certainly, water consumption pattern per capita
of every region governs the variation in TCS loading to the
plant. TCS concentration was measured from 350.9±32.8 to
729.0±31.1 ng/L and from 285.3±22.5 to 560.2±55.3 ng/L
for influent and effluent, respectively. The mean influent con-
centration in wet season was lower (527.7±20.3 ng/L) com-
pared to that in dry season (664.5±24.2 ng/L) that could be
due to attenuation of sewage with urban runoff in wet season.
This trend was found to be consistent for the other two plants.
TCS concentration varied from 1649.2±227.3 to 2853.5±
276.4 ng/g dw in primary sludge and from 1070.8±140.8 to
Fig. 1 Chromatogram (a) and
mass spectrum (b) of TCS
detected in the sludge samples
Environ Sci Pollut Res (2015) 22:11387–11400
3525.8±376.5 ng/g dw in dewatered sludge. TSS in the influ-
ent was dramatically different during the sampling period (av-
erage 177 mg/L in dry season vs. average 95 mg/L in wet
season). Elevated TSS level in dry season could cause en-
hanced sedimentation and higher TSS removal compared to
wet season (average 73.4 % in dry season vs. average 55.7 %
in wet season) (Table 2S, online resource). Settlement of
suspended solids (TSS) could directly influence the partition
of organic pollutants onto the sludge. It is noteworthy to men-
tion that adsorption and accumulation of organic compounds
to the settled matter is a well-known process in wastewater
treatment. Other removal processes such as biodegradation or
stripping are not likely due to short retention time in sedimen-
tation tank and negligible volatility of TCS (Henry’s law con-
stant 1.52×10−7 atm m3/mol at 25 °C) (O’Neil 2001).
Stonecutters (S.C.) WWTP is currently the largest chemi-
cally enhanced primary treatment (CEPT) plant in Hong Kong
with the average daily capacity of 1.4 mm3/day. During the
sampling period, the mean flow rates in the dry and wet sea-
sons were 1,366,000 and 1,384,000 m3/day, respectively.
Coagulants are added to the sewage for a more efficient
Environ Sci Pollut Res (2015) 22:11387–11400
Fig. 2 Chromatogram (a) and
mass spectrum (b) of silyl
derivative of 2,4-DCP detected in
the effluent sample of S.C.
WWTP
flocculation and sedimentation of particles. Not only
suspended solid but also soluble organic compounds are re-
moved by specific adsorption or co-precipitation with the floc.
The generated sludge is further dewatered and disposed to
landfill. BOD and TSS are removed by 60 and 80 %
(Table 2S, online resource), respectively, that indicates the
effectiveness of chemical treatment comparing to primary sed-
imentation. TCS was detected in wastewater from 263.7±38.5
to 549.0±72.1 ng/L in the influent and from 199.8±41.8 to
384.5±74.1 ng/L in the effluent. The mean influent concen-
tration in wet season and dry season were 291.1±17.0 and
509.2±22.7 ng/L, respectively. Besides, TCS was found in
the primary sludge in the range of 263.0±67.4 to 655.4±
121.6 ng/g dw and in the dewatered sludge in the range of
255.2 ± 44.3 to 643.5 ± 87.7 ng/g dw. As can be seen in
Table 3S (online resource), despite the considerable variation
11395
in BOD and TSS of the influent in dry and wet seasons, these
two parameters showed almost constant removal (69.5–70.2
and 75–78.5 % for BOD and TSS, respectively). During the
sampling period, chlorination was applied to the last step for
disinfection purpose by using sodium hypochlorite.
Concentration of TCS showed a small decrease (up to
9.9 %) after chlorination chamber in dry season samples that
might indicate the effect of chlorination on TCS transforma-
tion. Though, no significant change was observed for wet
season samples. This observation might be due to lower con-
centration of TCS in wet season and negligible transformation
during chlorination process.
Sha Tin (S.T.) WWTP with daily output capacity 0.2 mm3/
day is currently the largest secondary treatment plant in Hong
Kong. During the sampling period, mean flow rate in the dry
and wet seasons were 220,000 and 225,000 m 3 /day,
11396
Fig. 3 Chromatogram (a) and
mass spectrum (b) of 2,8-DCDD
detected in the effluent sample of
S.T. WWTP
respectively. Wastewater treatment comprises primary sedi-
mentation, activated sludge (biological treatment), and UV
disinfection. In addition, anaerobic digestion and further
dewatering are applied to the generated sludge for stabilization.
TCS concentration was found to range from 326.7±76.1 to
430.0±65.3 ng/L in the influent and from 85.4±15.5 to 198.4
±37.6 ng/L in the effluent. High BOD and TSS removal (85
and 90 %, respectively, Table 2S, online resource) implies the
efficiency of biological treatment for removing the biodegrad-
able constituents from wastewater. S.T. WWTP had the least
TCS loading in the influent at the average level of 372.3 ng/L.
The mean influent concentration in wet season and dry season
were 329.4±16.3 and 415.3±18 ng/L, respectively. A sharp
decrease in TCS concentration after biological treatment unit
might indicate the involvement of two probable processes: bio-
degradation of TCS and sorption to biomass. TCS has been
proved to biodegrade in biological condition with various deg-
radation rates depending on the experiment condition (>98 %,
Federle et al. 2002, 95 %, Puthiya Veetil et al. 2012, 75 %,
Environ Sci Pollut Res (2015) 22:11387–11400
Chen et. al 2011). Therefore, both mentioned processes could
be involved in TCS elimination in the biological treatment step.
Concentration of TCS in primary, secondary, and dewatered
sludge was in the range of 141.3±15.6–250.5±26.1, 129.6±
15.8–220.1±15.4, and 206.4±21.0–370.4±25.5 ng/g dw, re-
spectively. Since the lost TCS mass in biological treatment
was not consistent with the mass observed in the secondary
sludge, it could be concluded that the major part of TCS was
biodegraded during microbial treatment. Concentration of TCS
in the primary sludge samples from dry season were higher
than that from wet season that might be attributed to the en-
hanced partition of TCS to the sludge as a result of elevated
TCS concentration in the influent in dry season.
Analysis of degradation products in wastewater
and sludge samples
Chromatogram and mass spectrum of TCS obtained from the
sludge sample are shown in Fig. 1a, b. Since TCS was
Environ Sci Pollut Res (2015) 22:11387–11400
Fig. 4 Chromatogram (a) and
mass spectrum (b) of MTCS
detected after secondary treatment
in S.T. WWTP
proposed to be subjected to transformation, probable degrada-
tion products were also considered to be monitored.
– 2,4-DCP was detected only in the effluent samples of S.C.
WWTP after chlorination disinfection in dry season sam-
pling events (Fig. 2a, b) at the mean concentration of 16.8
±4.5 ng/L. Besides, the final effluent of these sampling
events showed a small drop in TCS concentration before
and after the chlorination disinfection. As the physical
elimination of TCS is not probable at this step, it is pro-
posed that other known loss processes such as chlorina-
tion or degradation to other products such as 2,4-DCP
might be involved (Buth et al. 2009, Canosa et al.
2005). Chlorination practice in S.C. WWTP is carried
out by contacting treated wastewater with sodium
11397
hypochlorite solution followed by dechlorination to neu-
tralize any residual chlorine before discharge. The contact
time was 15–30 min during the sampling period. The
chlorination practice is of importance since it raises the
concern about the generation of disinfection by-products
(DBPs) from wastewater constituents such as trihalo-
methanes (THMs), haloalkenes, haloacetic acid, etc.
(Glaze and Henderson 1975, Krasner et al. 2006). It is
likely that the exposure time of TCS to chlorine as a
strong oxidant was long enough to cause TCS degrada-
tion to other by-products. 2,4-DCP accounted for 93.9
and 94.8 % of the TCS loss in December and February
effluent samples, respectively, while the compound was
not detected in the samples from wet season sampling
events. Moreover, no significant TCS concentration drop
11398
was found before and after chlorination for wet season.
This observation might be due to lower concentration of
TCS in wet season and negligible transformation during
chlorination process. 2,4-DCP was neither observed in
S.C. sludge samples nor in wastewater and sludge sam-
ples from other WWTPs (Table 4). These observations
might be the confirmation for TCS transformation during
chlorination of wastewater.
– 2,8-DCDD was detected in the effluent of S.T. WWTP
after UV disinfection chamber at the mean concentration
of 20.3±4.8 ng/L (Fig. 3a, b). As it is reported previously
(Latch et al. 2003), one of the possibilities for the forma-
tion of 2,8-DCDD is the transformation of TCS when ex-
posed to UV light. Since this compound was not detected
in the wastewater samples from other steps of S.T. WWTP
or in the sludge samples, it might have been formed as a
result of TCS photodegradation in the UV disinfection
chamber in which the wastewater is exposed to UV lamps
radiation (254 nm). Decrease in TCS concentration ob-
served before and after UV disinfection chamber might
also support this hypothesis. Although some reports have
mentioned the formation of 2,8-DCDD during chlorina-
tion process (Buth et al. 2009), this compound was not
detected in the effluent samples from S.C. WWTP or in
the sludge samples. The concentration of 2,8-DCDD was
almost constant in two sampling events.
– MTCS was detected in most of the wastewater samples
collected after biological treatment in S.T. WWTP
(Fig. 4a, b), though not in the samples from the other plants
that did not run the biological treatment. The concentrations
were found to be close to LOD (mean level of 10.7±3.3 ng/
L). It was reported previously that TCS could undergo mi-
crobial transformation to produce methylated TCS in the
biological condition (Miyazaki et al. 1984). MTCS was
also detected in the secondary sludge samples at the mean
concentration of 51.0±7.5 ng/g dw and with no significant
change in dewatered sludge samples. Therefore, it is pro-
posed that the source of MTCS in the sludge could be
microbial transformation of TCS during aerobic biological
treatment rather than anaerobic digestion of sludge. MTCS
was also found in the dewatered sludge samples from C.C.
WWTP at the mean concentration of 129.3±17.2 ng/g dw.
The source of MTCS in this plant could only be attributed
to the generation from TCS during aerobic sludge digestion
as MTCS was not observed in the wastewater samples as
well as primary sludge samples.
Conclusion
The developed analytical methods proved to be efficient for
simultaneous determination of TCS and its degradation
Environ Sci Pollut Res (2015) 22:11387–11400
products including 2,4-DCP, 2,8-DCDD, and MTCS in waste-
water and sludge samples with satisfactory accuracy, preci-
sion, limit of detection and recovery. For wastewater samples,
liquid-liquid extraction and silica column chromatography
were used for extraction and sample cleanup. For sludge sam-
ples, ASE along with GPC and multilayer silica column were
applied for sample extraction and cleanup. High signal to
noise ratio indicated that the method could remove the inter-
ferences effectively. Several parameters were examined to ob-
tain the best condition for analysis. Finally, the samples were
analyzed by GC/MS. SIM mode was selected in order to ob-
tain higher sensitivity and selectivity. TCS concentration
dropped significantly after each treatment step, and the loss
in each plant was a factor of treatment technology. Biological
treatment was the most effective treatment due to biodegrad-
ability of TCS while CEPT and primary treatment were the
next effective treatments. In dry season, concentration of TCS
in primary and secondary sludge increased that could be due
to higher TCS loading in the influent and consequent en-
hanced adsorption to the sludge. However, for S.C. WWTP,
the concentration of TCS was almost the same in dry and wet
seasons that might show the influence of chemical treatment
over physical sedimentation.
Although wastewater treatment is to remove/reduce con-
taminants, the process may be a source of generating other by-
products which may be potentially more toxic. 2,8-DCDD
was detected as a photodegradation product of TCS in the
effluent of the plant practicing UV radiation disinfection.
This observation implied direct influence of UV radiation on
the formation of 2,8-DCDD in the real condition of wastewa-
ter treatment. 2,4-DCP was detected merely in the effluent of
the plant applying chlorination as the disinfection, likely due
to the transformation of TCS to this degradation product.
Since 2,4-DCP was not detected in the sludge samples, it
might be concluded that the main source of this compound
is the chlorination. A significant drop in the concentration of
TCS could support this hypothesis. The presence of trace
amount of MTCS was noticed in the activated sludge treat-
ment. Moreover, it could be detected in the dewatered sludge
treated by the aerobic digestion process. Therefore, it could be
concluded that MTCS was a by-product of TCS that has un-
dergone aerobic treatment in wastewater and sludge treatment.
Although the transformation degree of TCS was small
during the treatment, production of the by-products such
as 2,4-DCP or 2,8-DCDD with higher potent toxicity or
MTCS with higher persistency is still much of concern
when daily huge amount of wastewater and sludge are
discharged to the environment. Apart from production of
by-products during wastewater and sludge treatment, high
accumulation of TCS in the sludge raises the concern of
more probable transformations when the sludge is incin-
erated at high temperatures or disposed to the environ-
ment at the final destination.
Environ Sci Pollut Res (2015) 22:11387–11400
Funding This study was supported by the National Natural Science
Foundation of China (21175025) and Faculty Research Grant from Hong
Kong Baptist University (FRG2/12-13/032).
Conflict of interest The authors declare that they have no conflict
of interest.
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