Arch. Environ. Contam. Toxicol. 50, 240–248 (2006)
DOI: 10.1007/s00244-005-7058-x
Alkaline Digestion and Solid Phase Extraction Method for Perfluorinated
Compounds in Mussels and Oysters from South China and Japan
M. K. So,1 S. Taniyasu,2 P. K. S. Lam,1* G. J. Zheng,1 J. P. Giesy,1,3 N. Yamashita2
1
Centre for Coastal Pollution and Conservation, Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon,
Hong Kong SAR, Peoples Republic of China
2
National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki 305-859, Japan
3
Department of Zoology, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, East Lansing,
Michigan 48824, USA
Received: 10 March 2005 /Accepted: 8 May 2005
Abstract. Perfluorinated compounds (PFCs), such as perfluorooctane sulfonate (PFOS), have been identified in the
coastal waters of China and Japan. An alkaline digestion
method, coupled with solid-phase extraction (SPE), and highperformance liquid chromatography interfaced with high-resolution electrospray tandem mass spectrometry was developed
to determine PFCs in mussel and oyster samples from coastal
waters of south China and Japan. These techniques produced
adequate recoveries and reporting limits with small quantities
of PFCs. Concentrations of individual PFCs in mussels and
oysters from south China and Japan ranged from 113.6 to
586.0 pg/g, wet weight (ww) for PFOS, 63.1 to 511.6 pg/g, ww
for perfluorohexane sulfonate, 9.3 to 30.1 pg/g, ww for perfluorobutane sulfonate and 37.8 to 2957.0 pg/g, ww for perfluorooctane sulfonamide. The quantification of perfluorinated
carboxylates was compromised by interferences from carboxylates in the procedural blanks. Perfluoroundecanoate and
perfluorononanoate had relatively great blank interferences,
which resulted in relatively poor limits of quantification for
these compounds. Some PFCs were only identified in a limited
number of samples: perfluorododecanoate in samples from
Tokyo Bay, Japan (195.9 pg/g, ww); and perfluorodecanoate in
Fuzhou, China (131.7 pg/g, ww) and Tokyo Bay (118.6 pg/g,
ww). The greatest concentrations of perfluorooctanoate, perfluoroheptanoate, and perfluorohexanoate were observed in
samples from Tokyo Bay and Bei Hai, south China.
The extensive application of perfluorinated compounds (PFCs)
in many commercial products has led to the widespread
occurrence of PFCs in the environment, and PFCs are found
globally in tissues of many wildlife species, and in blood
serum of both occupationally and non-occupationally exposed
people (Hoff 2003; Kannan et al. 2001; Kuklenyik et al. 2004;
*Correspondence to: P. K. S. Lam; email: bhpksl@cityu.edu.hk
Olsen et al. 1999, 2003; Van de Vijver et al. 2003, 2004). The
widespread occurrence and potential toxicities of some PFCs,
have led to their production being voluntarily phased out in
2001 (Martin et al. 2004a).
Among the PFCs, perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorooctane sulfonamide (PFOSA), perfluorohexane sulfonate (PFHS), and perfluorobutane
sulfonate (PFBS) have received the most attention (Tomy et al.
2004). PFOS has been found in almost every wildlife species
in which it has been measured. Although PFOS and PFOA
have been measured most frequently, long-chained perfluorocarboxylates (PFCAs), with carbon numbers ranging from 9 to
15, have been found in ng/g, wet weight (ww) concentrations
in liver samples of various species from the Canadian Arctic.
In mink liver, the mean perfluorononanoate (PFNA) (16 ng/g,
ww) concentration was greater than that of PFOS (8.7 ng/g,
ww) (Martin et al. 2004b). Longer-chained PFCAs [the series
from perfluorohexanoate (PFHxA) to 14-carbon version of
PFOA (perfluorotetradecanoate, PFTA)] have also been observed in the fish liver tissues collected from Etobicoke Creek,
Toronto, Canada, where an accidental release of fire-fighting
foam occurred (Moody et al. 2002). The possibility of bioaccumulation and bioconcentration of long-chained perfluoroalkyl carboxylates and sulfonates (Martin et al. 2003a,
2003b) emphasizes the necessity of monitoring and evaluating
the toxicological effects of these compounds in the marine
environment.
In order to monitor these PFCs, a reliable method with
acceptable precision and accuracy is needed. Many previous
studies utilized ion-pairing methods for extracting PFCs in
blood, liver, and tissue samples. Those methods, however,
gave poor recoveries for some of the analytes (Martin et al.
2004a). A new automated solid-phase extraction and measurement method for PFCs in serum and milk achieved
acceptable recoveries for most analytes, except perfluoropentanoate (PFPeA) and perfluorododecanoate (PFDoDA), of
which the recoveries were poor (22% and 33% in serum,
respectively) (Kuklenyik et al. 2004). Most of the published
sample preparation methods do not involve any special
241
Perfluorinated Compounds in Mussels, Oysters from China and Japan
cleanup procedures, which results in interferences during
instrumental analysis (Martin et al. 2004a). Solid-phase
extraction (SPE) shows promise as a reliable and relatively
simple cleanup technique, especially for biological samples
(Kuklenyik et al. 2004; Martin et al. 2004a). The aim of this
study was to develop a practical method for measuring a broad
range of PFCs in biological matrices.
With rapid industrialization and development in the past
decade, the coastal waters of south China and Hong Kong have
been increasingly influenced by pollutant exposures from a
wide range of industrial and domestic sources (So et al. 2004).
Previous studies of these coastal waters have primarily focused
on traditional persistent organic pollutants including PCBs,
DDTs, PAHs, and other OC pesticides (Fu et al. 2003; Mai et
al. 2002; Yang et al. 1997). A recent study revealed that
concentrations of PFCs, mainly PFOS and PFOA, in the
coastal waters of south China and Hong Kong were similar to
those found in the coastal marine environment of Japan and
south Korea (So et al. 2004). Ranges of concentrations of
PFOS in Hong Kong, and the Pearl River Delta, China, were
0.09–3.1 and 0.02–12 ng/L, respectively, whereas those for
PFOA were 0.73–5.5 and 0.24–16 ng/L, respectively (So et al.
2004). Although all the concentrations were less than those
that would be expected to cause adverse effects to aquatic
organisms or their predators, the presence of PFCs in coastal
waters suggests their widespread distribution and potential
releases from adjacent industrialized areas in Hong Kong and
south China. Further monitoring and identification of sources
of PFCs are necessary for a more comprehensive risk assessment.
Fig. 1. The sampling locations along the East Coast of China and in
Japan: Qinzhou (QZ), Fang Cheng (FC), Bei Hai (BH), Xiamen (XI),
Fuzhou (FZ), SengSi Dao (SS), Tokyo Bay (TB)
methanol before use. Disposable PP copolymer laboratory ware was
used to minimize potential analyte loss. Teflon and glass containers
were avoided; the former cause analytical interferences, and the latter
may bind the surfactants in an aqueous solution.
Sample Preparation
Materials and Methods
Chemicals and Standards
External standards, a mixture of perfluorinated compounds (PFCmix)
containing PFOS, PFHS, PFBS, PFOSA, PFDoDA, perfluoroundecanoate (PFUnDA), perfluorodecanoate (PFDA), PFNA, PFOA, perfluoroheptanoate (PFHpA) and PFHxA, at 100 ng/mL were supplied
by the National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan. Methanol (high-performance liquid
chromatography (HPLC) grade), distilled water (HPLC grade),
potassium hydroxide (KOH, 85%), and ammonium acetate were
purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan).
Nylon mesh filters (12 lm, 13 mm i.d.) were purchased from Iwaki
(Tokyo, Japan). All solvents and reagents were used as received.
Frozen tissue samples from each location were thawed and homogenized by a mechanical homogenizer in a PP bottle. After homogenizing duplicate samples from one location, the homogenizer probe
and laboratory ware were thoroughly washed in the sequence of tap
water, distilled water, and methanol before homogenizing the next set
of samples to avoid cross contamination. Approximately 1 g of
homogenized tissue was transferred to a 50-mL PP centrifuge tube,
and 150 lL of 100 ng/mL PFCmix and 30 mL of 0.01 N KOH/
methanol were added to the PP tube. The mixture was shaken at 250
rpm at room temperature (25 oC) for 16 h. After digestion, 1 mL of
the tissue solution mixture was added to a 1-L PP bottle containing
100-mL distilled water and was shaken thoroughly. This tissue–water
mixture was then extracted for subsequent analysis.
Sample Extraction by Solid Phase Extraction Cartridge
Sample Collection
Green-lipped mussels, Perna viridis, were collected from six locations,
namely, Qinzhou (QZ), Fang Cheng (FC), Bei Hai (BH), Xiamen (XI),
Fuzhou (FZ) and Seng Si Dao (SS) along the east coast of China in
April 2004. Oysters (Crassostrea gigas) were also collected from Tokyo Bay (TB) in Japan in July 2004 (Figure 1). All the samples were
stored in polyethylene bags, kept in ice, and transported to the laboratory immediately. Mussel and oyster soft tissues were removed from
shell and placed in polypropylene (PP) tubes. The samples were stored
at –20C until analyzed. All equipment, storage containers, and laboratory ware were cleaned by rinsing with distilled water followed by
The tissue–water homogenate was extracted by using an Oasis HLB
cartridge (Oasis HLB: 0.2 g, 6 cm3; Waters Corp. Milford, Massachusetts). The procedures are a slight modification of a previously
described method (So et al. 2004). Each sample was extracted with a
separate cartridge. Prior to loading the cartridge with a sample, the
cartridge was preconditioned by eluting with 5 mL of methanol followed by 5 mL of distilled water at a rate of 2 drops/sec. The tissue–
water mixture was then loaded onto the cartridge, eluting at a rate of 1
drop/sec, and the eluate was discarded. The cartridge was prevented
from becoming dry at all times during the loading of the samples.
As part of method development, a separate and preliminary
washing step was evaluated: each HLB cartridge was washed with 5
M. K. So et al.
242
Table 1. Multiple-reaction-monitoring analysis of PFCs indicating precursor and product negative ion masses and MSMS conditions
Analyte
Abbreviation
Perfluorooctanesulfonate
Perfluorobutanesulfonate
Perfluorohexanesulfonate
Perfluorooctanesulfonamide
Perfluorohexanoate
Perfluoroheptanoate
Perfluorooctanoate
Perfluorononanoate
Perfluorodecanoate
Perfluoroundecanoate
Perfluorododecanoate
PFOS
PFBS
PFHS
PFOSA
PFHxA
PFHpA
PFOA
PFNA
PFDA
PFUnDA
PFDoDA
Precursor ion
Product ion
Collision energies (eV)
Cone voltages (V)
498.6
298.7
398.7
497.7
312.8
362.8
413
462.7
512.8
563
612.7
79.7
79.7
79.7
77.7
268.8
318.8
368.7
418.8
468.8
519
568.8
35
25
30
25
7
8
10
10
10
10
10
90
35
70
55
35
35
35
35
35
35
35
mL of 40% methanol in water at a rate of 1 drop/sec, and the eluate
was collected in a 15-mL PP centrifuge tube (Fraction 1). Following
this step, the cartridge was allowed to run dry and the remaining water
was completely removed from the cartridge by using a PP syringe. A
second fraction (Fraction 2) was eluted with 10 mL of 100% methanol
at a rate of 1 drop/sec. The eluates in Fraction 1 and Fraction 2 were
then reduced to 3 mL and 1 mL, respectively, under a gentle stream of
high-purity nitrogen gas. After volume reduction, the eluates were
passed through separate 0.1 lm 13 mm nylon mesh filters (which
were pre-washed with 2 · 0.5 mL methanol), and then transferred into
the corresponding 15-mL PP centrifuge tube. Any eluates that remained in the nylon filter were washed through the filter with 0.5 mL
of 100% methanol. The final volume of the eluate was reduced by
high-purity nitrogen gas to 3 mL for Fraction 1 and 0.5 mL for
Fraction 2. One hundred microliters of eluate was transferred to a PP
vial for instrumental analysis.
Instrumental Analysis
Concentrations of PFCs in tissue samples were analyzed using HPLC
with high-resolution, electrospray tandem mass spectrometry (HPLCMS/MS). Separation of analytes was performed by an Agilent HP1100
liquid chromatograph (Agilent, Palo Alto, California) interfaced with
a Micromass Quattro II mass spectrometer (Waters Corp., Milford,
Massachusetts) operated in electrospray negative mode. A 10-lL
aliquot of extract was injected onto a Keystone Betasil C18 column
(2.1 mm i.d. · 50-mm length, 5 lm) with 2 mM ammonium acetate
and methanol as the mobile phases starting at 10% methanol at a flow
rate of 300 lL/min. The gradient was increased to 100% methanol at
10 min before reverting to the original conditions at 12 min. The
desolvation gas flow and temperature were kept at 750 L/h and 400C.
The collision energies, cone voltages, and MS/MS parameters for the
instrument were optimized individually for each analyte and are
summarized (Table 1).
Data Analysis
Acquisition analysis for all of the samples, blanks, and QC materials
were performed using MassLynx software of the Micromass Quattro II
system (Waters Corp., Milford, MA). Each ion of interest in the chromatogram was automatically selected and integrated. Concentrations of
target analytes were quantified by using calibration curves constructed
using external standards of six different concentrations (2, 10, 50, 200,
1000, and 20,000 ng/L). Apart from a few outliers, the standard calibration curve showed strong linearity (correlation coefficients >0.99).
Prior to sample quantification, the instrument was tuned to obtain
the maximum peak resolution and the greatest mass accuracy. Mul-
tiple reaction monitoring (MRM) mode, having a higher sensitivity,
was used in the present study for quantification. In MRM mode, a
daughter ion, usually the one with the greatest intensity, produced
from a selected parent ion was monitored and quantified.
Results and Discussion
Optimization of Alkaline Digestion
Due of the specific protein-binding properties of PFCs,
biological matrix interference reduces the sensitivity of the
analytical technique. Therefore, it is necessary to remove the
proteins to release PFCs for increased sensitivity. Alkaline
digestion of lipids and proteins before extraction was suggested to achieve an accurate and reliable measurement of
PFCs in biological samples. Current sample preparation
methods do not include a cleanup step to remove these
matrix interferences (Martin et al. 2004a). To evaluate the
effect of alkaline digestion on target analytes, 10 lL of 100
ng/mL PFCmix was added to 1 mL of different concentrations of KOH in water (0.01, 0.1, 0.3, 0.5, 1, and 2 N) or
methanol (0.01, 0.1, and 0.3 N). The percentage recoveries
for all the target analytes under different KOH concentrations are summarized in Table 2. In general, all alkaline/
water mixtures gave poor recoveries for PFDoDA and
PFUnA and other PFCs, and were judged unacceptable for
the alkaline digestion protocol. The alkaline/methanol solution (0.01 N KOH/methanol) was found to have the most
consistent recoveries for all PFCs (>70%). For many PFCs,
greater KOH concentrations reduced recovery rates. The
duration for the alkaline digestion process was also tested by
shaking approximately 1 g of homogenized mussel tissue
sample with 30 mL 0.01 N KOH/methanol solution. Small
particles were still observed after shaking for 2 and 6 h.
Consequently, the samples were digested overnight (10 h) to
ensure complete digestion of the fat and proteins in the
homogenized tissue.
Quality Assurance and Control
Blank Analysis
The occurrence of relatively great concentrations of some
PFCs in blanks is a persistent problem in the analyses of these
243
Perfluorinated Compounds in Mussels, Oysters from China and Japan
Table 2. The effect of concentration of KOH/methanol on the recoveries of PFCs
Concentration (ng/L)
Conc. Of KOH in methanol
(KOH/M) or water (KOH/W) (N)
PFOS
PFHS
PFBS
PFOSA
PFDoDA
PFUnDA
PFDA
PFNA
PFOA
PFHpA
PFHxA
0.01NKOH/W
0.1NKOH/W
0.3NKOH/W
0.5NKOH/W
1NKOH/W
2NKOH/W
0.01NKOH/M
0.1NKOH/M
0.3NKOH/M
106.6
108.7
72.9
46.6
37.8
25.1
108.9
125.2
116.8
89.5
97.4
88.7
79.1
68.4
60.9
80.9
93.5
82.8
92.1
91.7
84.9
77.4
72.7
64.8
73.6
81.6
66.9
56.0
95.5
70.5
46.6
34.4
20.6
91.3
100.0
88.6
24.8
32.6
6.5
2.1
2.7
2.6
86.9
99.6
64.7
53.7
62.1
24.6
9.2
8.5
3.5
91.5
98.8
66.9
109.6
98.6
72.2
80.3
26.6
12.5
107.0
112.3
82.3
90.6
91.0
73.4
50.8
40.5
28.4
81.7
84.5
58.2
193.7
186.6
223.0
233.6
195.7
140.4
244.8
250.7
187.5
87.2
100.6
94.6
90.7
73.5
62.7
81.6
86.0
61.1
106.2
101.2
98.6
90.6
77.2
66.4
85.4
82.1
51.8
Table 3. Background level of perfluorinated compounds (pg) in HPLC grade methanol, nylon mesh filter, and the whole procedures
Concentration (pg)
PFOS
PFHS
PFBS
PFOSA
PFDoDA
PFUnDA
PFDA
PFNA
PFOA
PFHpA
PFHxA
<1.6
<6.7
<1.6
<1.6
<1.6
<1.6
<1.6
<1.6
<1.6
<1.6
<1.6
27.6
<10
<2
<2
<2
2.7
3.3
<2
<2
<2
<2
<2
2.8
2.8
34.6
<10
<50
<50
<10
<10
<10
<10
2.6
<2.6
2.6
<2.6
5.1
<5.1
<8.2
8.2
<61.8
61.8
36.8
<36.8
<50
<50
31.3
<31.3
a
HPLC grade methanol
Nylon filtera
Without washing
With washing
Procedural blank
Fraction 1b
Fraction 2c
a
b
c
<43.6
43.6
<10
<10
<11.8
11.8
Amount in the final 1 mL solution and 10 lL was injected into the LC/MS/MS for quantification.
Amount in the final 3 mL solution and 10 lL was injected into the LC/MS/MS for quantification.
Amount in the final 0.5 mL solution and 10 lL was injected into the LC/MS/MS for quantification.
compounds. The sources of contamination can be laboratory
products containing fluoropolymers such as polytetrafluoroethylene (Martin et al. 2004a). The difficulties presented by
background contamination of PFCs in ‘‘blank’’ test matrix was
evaluated and minimized by the following procedures: (1)
elimination of Teflon-containing equipment, (2) use of precleaned and methanol-rinsed glassware, and (3) use of PP
storage vials. Contamination of equipment and solvent were
tested for presence of ‘‘background’’ concentrations of target
analytes. The methanol, nylon mesh filter, as well as the entire
procedure, were evaluated as sources of background levels of
target analytes before samples were analyzed. Evaluation of
methanol contamination was performed by concentrating 30
mL of methanol to 1 mL by gentle nitrogen blown-down and
then injected into the LC/MS/MS for analysis. Concentrations
for all of the compounds of interest were less than the limit of
quantification (LOQ), indicating that HPLC-grade methanol
would not contribute much interference for PFC analysis
(Table 3).
Contamination from the nylon mesh filter was evaluated by
comparing prewashed versus nonwashed filters using standard
cleaning procedures. Pre- and nonwashed filters were rinsed
with 1 mL of 100% methanol; the eluate was collected and
analyzed. Trace amounts of PFOS (27.6 pg) and PFOA (34.6
pg) were found in the nontreated nylon filter. The levels of
these compounds in the background were greatly reduced by
washing the filter with methanol prior to sample loading
(Table 3).
In previous analyses of water (So et al. 2004), direct SPE
extraction of PFCs without digestion was utilized to evaluate
coastal waters of Hong Kong, south China, and Korea. However, in the presence of a biological matrix, the technique had
to be modified with alkaline digestion to attempt to reduce
matrix interferences. Procedural blank, following the same
extraction procedure, was carried out for each set of samples to
test for possible contamination. Considerable amounts of
PFDoDA (8.2 pg), PFUnDA (43.6 pg), PFDA (11.8 pg), and
PFNA (61.8 pg) were detected in the target fraction (F2), while
concentrations of all the other chemicals were less than the
LOQ. Trace amounts of PFOA (36.8 pg) and PFHxA (31.3 pg)
were also found in Fraction 1 (Table 3). The LOQ for each
target analyte was determined as the smallest mass of standard
injected that resulted in a reproducible measurement of peak
areas consistent with the calibration curve. The LOQs (amount
in the final 0.5 mL solution and 10 lL was injected for
quantification) were 10 pg for PFOS, 2.6 pg for PFHS and
PFBS, 5.1 pg for PFOSA, 8.2 pg for PFDoDA, 43.6 pg for
PFUnDA, 11.8 pg for PFDA, 61.8 pg for PFNA, 36.8 pg for
PFOA, 50 pg for PFHpA, and 31.3 pg for PFHxA.
Spike Recovery Analysis
Recoveries of PFCs were performed to determine the precision
and accuracy of the extraction and analytical procedures. Two
M. K. So et al.
244
Table 4. Recoveries (%) for individual PFCs with and without alkaline digestion
Recovery (%)
PFOS
PFHS
PFBS
PFOSA
PFDoDA
PFUnDA
PFDA
PFNA
PFOA
PFHpA
PFHxA
Without alkaline digestion (n = 2)
With alkaline digestion (n = 2)
Fraction 1
Fraction 2
Fraction 1
Fraction 2
Fraction 1+2
0
0.1
0.1
0.1
0
0.1
0.1
0.2
0.5
0
0.2
115.7
104.8
102.2
117.9
73.5
93.7
108.2
107.4
109.6
111.6
100.4
0.9
66.5
93.6
0.6
0.4
2.6
2.3
17.5
104.8
120.7
120.6
149.1
33.2
1.3
104.0
91.6
110.5
114.7
101.1
24.1
1.7
1.6
150.0
99.7
94.8
104.6
92.0
113.0
116.9
118.6
128.9
122.4
122.2
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15.4
8.0
0.8
2.7
1.8
1.7
1.9
3.5
1.4
5.6
5.0
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1.2
31.0
27.5
0.3
0.3
1.2
1.5
2.9
5.8
8.9
2.0
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11.5
10.5
0.3
8.9
6.4
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1.6
3.6
0.3
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15.7
41.5
27.8
8.7
6.7
3.1
1.4
1.3
9.3
8.7
2.4
Note: The values are mean € standard deviations.
separate spike recovery evaluations are reported, which evaluate (1) the alkaline digestion effect and (2) the biological
matrix effect.
Alkaline Digestion. To determine the recoveries of PFCs
with alkaline digestion, 150 lL of 100 ng/mL PFCmix was
spiked into 30 mL KOH/methanol, digested for 16 h, and
then extracted following the same procedures used for the
biological samples. The recoveries of PFCs without alkaline
digestion were studied following a similar procedure, except
that the 150 lL PFCmix was added directly to 100 mL distilled water. The recoveries for all the target analytes are
summarized (Table 4). Without alkaline digestion, most of
the chemicals were detected in Fraction 2 with recoveries of
greater than 70%. The recoveries in Fraction 1, usually regarded as a washing step, were less than 0.5% for most of the
compounds.
Recoveries of various PFCs after alkaline digestion are
given (Table 4). The recoveries in Fraction 2 were 149.1%
for PFOS, 33.2% for PFHS, 104% for PFOSA, 91.6% for
PFDoDA, 110.5% for PFUnDA, 114.7% for PFDA, and
101.1% for PFNA. Relatively great recoveries were also
detected in Fraction 1 for perfluorinated sulfonic acids (PFHS
and PFBS) and carboxylic acids (PFOA, PFHpA and PFHxA)
with carbon chain lengths of less than 6 and 8, respectively.
The detection of target analytes in Fraction 1 may be
attributed to (1) alterations in the structure of these compounds after alkaline digestion; and/or (2) the effect of KOH/
methanol on the adhesion capacity of the analytes to the
cartridge sorbent. By combining Fractions 1 and 2, most of
the compounds achieved recoveries of more than 92%. On
the basis of the above observations, the washing step (Fraction 1) was eliminated from final analytical scheme, and all
target PFCs were eluted directly with 15 mL of 100%
methanol.
Matrix Effects. The effect of biological matrix on the
recoveries of PFCs was investigated with a matrix spike
recovery test. Approximately 1 g of homogenized oyster
tissue collected from Tokyo Bay was spiked with 150 lL of
100 ng/mL PFCmix and digested by 30 mL KOH/methanol
for 16 h, and then extracted following the same procedures as
described above (n = 2). A second series of duplicate samples, without the addition of oyster homogenate, was processed in the same manner. The recoveries of various PFCs
from this spike exercise are presented (Table 5). In general,
recoveries of PFCs were comparable between samples with
or without oyster tissue present; recoveries of all PFCs exceeded 76% and were judged acceptable as quantitative
estimates of PFC concentrations in tissues, except for PFOA,
which had a high recovery in samples without oyster tissue
(197%) compared with those with oyster tissue (93%). This
high recovery was probably the result of PFOA contamination during the extraction, and further analysis is required to
ascertain this point. Notwithstanding, results of the present
study indicated that the sample matrix does not cause a
significant reduction in concentrations spiked onto the oyster
tissue matrix. Further study should examine the matrix effects of other tissue types.!?A3B2 tpb=3.8mm?>
Linearity
The accuracy of the above method was further assessed by
performing linearity tests using four different amounts of
oyster tissues (0.8, 1.1, 1.7, 2.0 g) for extraction. The correlation between increased amounts of analyte with proportional increases of tissue weight is evaluated by linear
regression (Table 6). Good linearity was found between
amounts of oyster tissues and several PFCs including PFOS,
PFBS, PFUnDA, and PFOA. The r2 values were 0.73 for
PFOS, 0.94 for PFBS, 0.70 for PFUnDA, and 0.89 for
PFOA. The r2 values for PFHS, PFDA, and PFNA ranged
from 0.56 to 0.58, and those for PFOSA, PFDoDA, PFHpA,
and PFHxA were less than 0.50. The apparent lack of
strong linear relationship for these latter compounds is
probably related to the small quantities of oyster tissues
used, and the relatively small concentrations of these PFCs
in the tissues.
245
Perfluorinated Compounds in Mussels, Oysters from China and Japan
Table 5. Recoveries (%) for individual PFCs with and without oyster samples
Recovery (%)
Without oyster samples (n = 2)
PFOS
PFHS
PFBS
PFOSA
PFDoDA
PFUnDA
PFDA
PFNA
PFOA
PFHpA
PFHxA
Fraction 1
Fraction 2
0
46.0
88.0
0.5
0
1.0
2.0
5.5
83.0
124.0
119.5
109.5
58.5
1.0
102.0
121.0
123.5
127.5
113.5
114.0
3.0
2.5
€ 5.7
€ 1.4
€ 0.7
€
€
€
€
€
€
0
0
2.1
7.1
8.5
2.1
€
€
€
€
€
€
€
€
€
€
€
9.2
3.5
0
5.7
12.7
10.6
2.1
0.7
8.5
0
0.7
With oyster samples (n = 2)
Fraction 1 + 2
109.5
104.5
89.0
102.5
121.0
124.5
129.5
119.0
197.0
127.0
122.0
€
€
€
€
€
€
€
€
€
€
€
9.2
2.1
1.4
6.4
12.7
10.6
2.1
1.4
1.4
8.5
2.8
Fraction 1
Fraction 2
0
58.0
74.5
2.5
0
1.5
1.0
12.5
68.0
115.5
110.5
158.5
41.5
1.0
79.5
89.5
97.5
122.5
99.0
24.5
1.5
2.0
€ 2.8
€ 3.5
€ 0.7
€
€
€
€
€
€
0.7
0
4.9
9.9
0.7
10.6
€
€
€
€
€
€
€
€
€
€
€
Fraction 1 + 2
0.7
0.7
0
0.7
3.5
2.1
3.5
4.2
3.5
0.7
0
158.5
99.5
75.5
82.0
89.5
99.0
123.5
111.5
92.5
117.0
112.5
€
€
€
€
€
€
€
€
€
€
€
0.7
2.1
3.5
1.4
3.5
2.8
3.5
0.7
13.4
0
10.6
The values are mean € standard deviations.
Table 6. Linearity test between amounts of tissue weights and
concentration of PFCs
Target analyte
Correlation coefficient (r2); n = 4
PFOS
PFHS
PFBS
PFOSA
PFDoDA
PFUnDA
PFDA
PFNA
PFOA
PFHpA
PFHxA
0.73
0.56
0.94
0.43
0.50
0.70
0.58
0.56
0.89
0.06
0.22
Table 7. Recoveries (%) and level of blank (pg) for individual PFCs.
PFOS
PFHS
PFBS
PFOSA
PFDoDA
PFUnDA
PFDA
PFNA
PFOA
PFHpA
PFHxA
Level of blank (pg)*
Recovery (%)
Mean
S.D.
Mean
S.D.
<10
<2
<2
<2
3.4
42.0
<10
53.2
<10
<50
<10
—
—
—
—
3.3
15.4
—
2.0
—
—
—
119.7
93.4
84.7
75.1
57.4
82.5
84.6
92.8
100.5
97.2
94.5
6.4
7.0
0.3
2.8
6.9
7.1
3.3
7.2
11.2
11.9
0.1
Amount in 0.2 mL final extract solution and 10 lL was injected into
the LC/MS/MS for quantification.
Modification and Finalization of Solid Phase Extraction
Method
As a result of the presence of certain target analytes in Fraction
1, the preliminary washing step was eliminated. The cartridge
was directly eluted with 15 mL (instead of 10 mL) 100%
methanol after sample loading, and the final volume was reduced to 0.2 mL instead of 0.5 mL. All the other procedures
were not altered. With the change in solvent volume and
washing process, solution spike recovery after alkaline digestion was performed to determine the accuracy and precision of
the modified method. A matrix spike recovery test was not
carried out at this time because the results of the solution spike
recovery evaluation were consistent with the earlier evaluation. Procedural blanks were carried out for each set of
extraction to test for possible sources of contamination. The
recovery (%) and the level of blank (pg) in 0.2 mL final extract
solution are summarized (Table 7). Except for a trace amount
of perfluorinated carboxylic acid (PFDoDA, PFUnDA, and
PFNA) found in the procedural blank, all the other analytes
were less than the LOQ. The recoveries for the target analytes
were more than 75%, except for PFDoDA, which had a
recovery of 57.4%.
Most of the previous studies utilized an ion-pairing method
for the quantitation of a limited number of PFCs from biological samples such as liver and serum. It was not until 2003
that a study measured the concentrations of a much wider
range of PFCs from biological tissue samples (fish carcasses)
(Martin et al. 2003a). The recoveries were approximately 95%
for PFHpA, 100% for PFOA, 80% for PFDA, PFUnA, PFHxS
and PFOS, 65% for PFDoDA as well as 55% for PFTA and
PFBS (Martin et al. 2003a). In the present study, except for
PFDoDA, all the other PFCs had recoveries at least comparable to, if not higher than, that of previous study. In particular,
the mean recovery for PFBS (84.7%) was much higher in the
present study. In addition, the method described here also allowed the analysis of PFHxA with a good recovery (94.5%).
Application of the Technique to Mussel and Oyster
Samples from China and Japan
The modified method was used to analyze mussel samples
along the east coast of China, and oyster samples collected
from Tokyo Bay in Japan. Concentrations of PFCs, in terms of
M. K. So et al.
1028.9 € 39.8 <12,468.8 <1593.8
<2343.8
<1429.7
<863.6
<12,090.0 <1545.5
<2272.7
<1386.4
<651.4
<9120.0 <1165.7
<1714.3
<1045.7
<863.6
<12,090.0 <1545.5
<2272.7
<1386.4
<897.6
<12,566.9 2184.3 € 51.2 2884.3 € 487.4 2442.5 € 526.8
<616.2
<8627.0 1774.1 € 73.5 2741.1 € 303.8 1875.1 € 239.5
770.1
<10,363.6 4289.0
<1948.1
1517.5
2247.7
1778.0
€ 52.0
305.7
€ 55.3
342.4
€ 43.3
297.6
€ 4.3
336.2
19201.3
€
€
€
€
€
€
23.4
179.5
134.9
69.7
101.6
58.9
<796.9
<772.7
<582.9
<772.7
<803.1
<551.4
1272.1
<9843.8
<9545.5
<7200.0
<9545.5
<9921.3
<6810.8
<8181.8
Fig. 2. Composition of perfluorinated sulfonic acid and sulfonamide
in mussel and oyster samples from different sampling locations
1544.5
1263.6
2012.0
1042.4
1014.2
614.1
3805.2
87.2
86.8
82.5
86.8
87.3
81.5
84.6
The values are mean € standard deviations.
<93.8
<90.9
172.0
222.0
148.8
103.2
166.2
358.6 3996.9 € 82.0
282.6 478.0 € 167.4
144.0 677.7 € 64.6
98.5 1012.1 € 31.8
139.4 771.7 € 121.3
27.6
468.6 € 1.1
1309.7
9.1
7.3
5.5
0.8
€
€
€
€
<12.0
<12.0
30.1
29.3
18.9
19.1
25.6
10.5
22.1
11.3
4.2
15.4
0.2
€
€
€
€
€
€
511.6
63.1
118.6
133.6
98.0
86.7
201.7
45.9
37.3
25.2
13.0
17.7
5.1
€
€
€
€
€
€
197.7
166.8
352.1
137.6
128.8
113.6
586.0
Wet weight basis
FZ
XI
QZ
FC
SS
BH
TB
Dry weight basis
FZ
XI
QZ
FC
SS
BH
TB
87.2
86.8
82.5
86.8
87.3
81.5
84.6
€
€
€
€
€
€
<183.0
<183.0
<183.0
<183.0
310.2 € 66.9
346.9 € 44.3
233.7
<300.0
<300.0
<300.0
<300.0
366.3 € 61.9
507.1 € 56.2
<300.0
<204.0
<204.0
<204.0
<204.0
277.4 € 6.5
328.2 € 13.6
660.5
<1596.0
<1596.0
<1596.0
<1596.0
<1596.0
<1596.0
<1596.0
287.7
234.7
53.5
45.2
37.8
62.2
2957.0
€
€
€
€
€
€
3.0
23.7
23.6
9.2
12.9
10.9
<102.0
<102.0
<102.0
<102.0
<102.0
<102.0
195.9
<1260.0
<1260.0
<1260.0
<1260.0
<1260.0
<1260.0
<1260.0
131.7 € 5.1
<114.0
<114.0
<114.0
<114.0
<114.0
118.6
PFHpA
PFOA
PFNA
PFDoDA PFUnDA PFDA
PFOSA
PFBS
Location
Concentration (pg/g)
Moisture
content (%) PFOS
PFHS
Table 8. Concentrations of PFCs (pg/g, wet weight and dry weight) in mussel and oyster samples collected from East Chinese Coast and Tokyo Bay in Japan
PFHxA
246
pg/g, in mussel and oyster samples are summarized in Table 8.
Unless otherwise stated, all concentrations of PFCs in mussel
or oyster tissues measured in this study are expressed on a wet
weight basis. Most of the mussel and oyster samples contained
detectable concentrations of PFOS, PFHS, PFBS, and PFOSA.
Concentrations ranged from 113.6 to 586.0 pg/g for PFOS,
63.1 to 511.6 pg/g for PFHS, <12.0 to 30.1 pg/g for PFBS, and
37.8 to 2957.0 pg/g for PFOSA. Oyster samples from Tokyo
Bay had the greatest concentrations of PFOS (586.0 pg/g) and
PFOSA (2957.0 pg/g). Nevertheless, the level of PFOS
[3805.2 pg/g (recalculated on a dry weight basis)] was much
less than the greatest PFOS concentration (1,106,000 pg/g dry
weight) measured in oysters from Hog Point, Chesapeake Bay
(Kannan et al. 2002). The comparatively small concentrations
of perfluorinated sulfonic acid and sulfonamide in mussel
samples from China indicated that the east coast of China is
probably not severely polluted by PFCs. In general, concentrations of perfluorinated sulfonic acids (expressed on a dry
weight basis) were in a similar range as PCBs (1330–13,000
pg/g), chlordanes (170–9910 pg/g), and hexachlorocyclohexanes (130–1860 pg/g), but less than that of DDTs (14,450–
640,000 pg/g), in mussels collected along the east coast of
China (Monirith et al. 2003). The composition of perfluorinated sulfonic acids and sulfonamide were compared among
different sampling locations (Figure 2). Similar compositions
were found in mussels from Bei Hai, Seng Si Dao, Fang
Cheng, and Qingzhou, with PFOS as the major component. A
completely different composition profile was observed in
oysters from Tokyo Bay, where PFOSA accounted for
approximately 80% of the PFCs. Assuming that the uptake of
different PFCs by mussels and oysters followed a similar
mechanism, these findings suggest that the sources of PFC
contamination in Japan and China could be quite different. In
Japan, the high PFOSA concentrations could probably be due
to the extensive use of insecticide to control termites and ants.
In Fuzhou, PFHS showed the greatest concentration among all
the PFCs, indicating that a source of PFHS contamination
might exist in this area.
The analysis of PFCAs in the present study had two major
technical difficulties: (1) trace amounts of carboxylic acids,
especially PFUnDA (210 pg/mL) and PFNA (266 pg/mL),
were found in the procedural blank; and (2) the concentrations
of several PFCs in the samples were comparable to or only
slightly greater than those found in the procedural blank.
247
Perfluorinated Compounds in Mussels, Oysters from China and Japan
Fig. 3. The comparison of total concentration of perfluorinated carboxylic and sulfonic compounds in mussel and oyster
samples from individual sampling location
Concentrations of PFUnDA and PFNA in all the samples were
less than the LOQ. PFDoDA was only detected in oysters from
Tokyo Bay (195.9 pg/g), whereas PFDA was detected in
samples from Fuzhou (131.7 pg/g) and Tokyo Bay (118.6 pg/
g). The greatest concentrations of PFOA were found in samples from Tokyo Bay, whereas the greatest concentrations of
PFHpA and PFHxA were found in samples from Bei Hai. The
total concentrations of perfluorinated carboxylic and sulfonic
compounds in mussels and oysters from individual sampling
locations were compared (Figure 3). Total concentrations of
sulfonic compounds were higher than carboxylic compounds
in samples from Fuzhou and Tokyo Bay and vice versa for
those from Seng Si Dao and Bei Hai. This pattern indicates
that sulfonyl-containing compounds were or are commonly
used in areas near Fuzhou and Tokyo Bay.
Although a bioconcentration study of PFCs on rainbow
trout demonstrated that the bioconcentration potential of
PFCs increased with increasing perfluoroalkyl chain length
(Martin et al. 2003a), such a pattern was not observed for
perfluorinated carboxylates in the present study. Except for
the Fuzhou mussels, samples from other locations showed
greater concentrations of longer-chained perfluorinated sulfonates. The reason for the absence of such a pattern for
carboxylates is not known, but could be due to different
organisms and different environments that were investigated
in the respective studies.
Summary
Alkaline digestion, coupled with Oasis HLB solid-phase
extraction, were developed and evaluated for a range of
long-chained and short-chained PFCs from biota samples.
This rapid and relatively simple extraction method performed as well as other published methods for tissue analysis of PFCs. Acceptable recoveries were achieved for most
of the target analytes, but trace amounts of certain PFCs in
the procedural blank restricted the applicability of this
method to samples containing relatively high concentrations
with respect to those compounds. Further work will be required to minimize the blank level by improving the solid
phase extraction procedure.
Acknowledgment. This study was supported by the Area of Excellence Scheme under the University Grants Committee of the Hong
Kong Special Administration Region, China (Project No. AoE/P-04/
2004), and a RGC-CERG (cityU1401/05M).
References
Fu J, Mai B, Sheng G, Zhang G, Wang X, Peng P, Xiao X, Ran R,
Cheng F, Peng X, Wang Z, Tang UW (2003) Persistent organic
pollutants in environment of the Pearl River Delta, China: An
overview. Chemosphere 52:1411–1422
Hoff PT, Van de Vijver K, Van Dongen W, Esmans EL, Blust R, De
Coen WM (2003) Perfluorooctane sulfonic acid in bib (Trisopterus luscus) and plaice (Pleuronectes platessa) from the
Western Scheldt and the Belgian North Sea: Distribution and
biochemical effects. Environ Toxicol Chem 22:608–614
Kannan K, Koistinen J, Beckmen K, Evans T, Gorzelany JF, Hansen
KJ, Jones PD, Helle E, Nyman M, Giesy JP (2001) Accumulation
of perfluorooctane sulfonate in marine mammals. Environ Sci
Technol 35:1593–1598
Kannan K, Hansen KJ, Wade TL, Giesy JP (2002) Perfluorooctane
sulfonate in oysters, Crassostrea virginica, from the Gulf of
Mexico and the Chesapeake Bay, USA. Arch Environ Contam
Toxicol 42:313–318
Kuklenyik Z, Reich JA, Tully JS, Needham LL, Calafat AM (2004)
Automated solid-phase extraction and measurement of perfluorinated organic acids and amides in human serum and milk.
Environ Sci Technol 38:3698–3704
Mai BX, Fu JM, Sheng GY, Kang YH, Lin Z, Zhang G, Min YS, Zeng
EY (2002) Chlorinated and polycyclic aromatic hydrocarbons in
riverine and estuarine sediments from Pearl River Delta, China.
Environ Pollut 117:457–474
Martin JW, Mabury SA, Solomon KR, Muir DCG (2003a) Dietary
accumulation of perfluorinated acids in juvenile rainbow trout
(Oncorhynchus mykiss). Environ Toxicol Chem 22:189–195
Martin JW, Mabury SA, Solomon KR, Muir DCG (2003b) Bioconcentration and tissue distribution of perfluorinated acids in rainbow
trout (Oncorhynchus mykiss). Environ Toxicol Chem 22:196–204
Martin JW, Kannan K, Berger U, Voogt PD, Field J, Franklin J, Giesy
JP, Harner T, Muir DCG, Scott B, Kaiser M, JTrnberg U, Jones
KC, Mabury SA, Schroeder H, Simcik M, Sottani C, Bavel BV,
KTrrman A, Lindstrçm G, Leeuwen SV (2004a) Analytical
challenges hamper perfluoroalkyl research. Environ Sci Technol
248A–255A
Martin JW, Smithwick MM, Braune BM, Hoekstra PF, Muir DCG,
Mabury SA (2004b) Identification of long-chain perfluorinated
acids in biota from the Canadian Arctic. Environ Sci Technol
38:373–380
Monirith I, Ueno D, Takahashi S, Nakata H, Sudaryanto A, Subramanian A, Karuppiah S, Ismail A, Muchtar M, Zheng J, Richardson BJ, Prudente M, Hue ND, Tana TS, Tkalin AV, Tanabe S
248
(2003) Asia-Pacific mussel watch: monitoring contamination of
persistent organochlorine compounds in coastal waters of Asian
countries. Mar Pollut Bull 46:281–300
Moody CA, Martin JW, Kwan WC, Muir DCG, Mabury SA (2002)
Monitoring perfluorinated surfactants in biota and surface water
samples following an accidental release of fire-fighting foam into
Etobicoke Creek. Environ Sci Technol 36:545–551
Olsen GW, Burris JM, Mandel JH, Zobel LR (1999) Serum perfluorooctane sulfonate and hepatic and lipid clinical chemistry tests in
fluorochemical production employees. J Occup Environ Med
41:799–806
Olsen GW, Hansen KJ, Stevenson LA, Burris JM, Mandel JH (2003)
Human donor liver and serum concentrations of perfluorooctanesulfonate and other perfluorochemicals. Environ Sci Technol
37:888–891
So MK, Taniyasu S, Yamashita N, Giesy JP, Zheng J, Fang Z, Im SH,
Lam PKS (2004) Perfluorinated compounds in coastal waters of
Hong Kong, South China and Korea. Environ Sci Technol
38:4056–4063
M. K. So et al.
Tomy GT, Tittlemier SA, Palace VP, Budakowski WR, Braekevelt
E, Brinkworth L, Friesen K (2004) Biotransformation of Nethyl perfluorooctanesulfonamide by rainbow trout (Onchorhynchus mykiss) liver microsomes. Environ Sci Technol 38:
758–762
Van de Vijver KI, Hoff PT, Van Dongen W, Esmans EL, Blust R,
De Coen WM (2003) Exposure patterns of perfluorooctane
sulfonate in aquatic invertebrates from the Western Scheldt
estuary and the Southern North Sea. Environ Toxicol Chem 22:
2037–2041
Van de Vijver KI, Hoff PT, Das K, Van Dongen W, Esmans EL,
Siebert U, Bouquegneau JM, Blust R, De Coen WM (2004)
Baseline study of perfluorochemicals in harbour porpoises
(Phocoena phocoena) from Northern Europe. Mar Pollut Bull
48:992–997
Yang YH, Sheng GY, Fu JM, Min YS (1997) Organochlorinated
compounds in waters of the Pearl River Delta region. Environ
Monit Assess 44:569–575