Contamination and bioaccumulation of benzotriazole ultraviolet stabilizers

in sh from Manila Bay, the Philippines using an ultra-fast liquid

chromatographytandem mass spectrometry
Joon-Woo Kim
a,b
, Tomohiko Isobe
a,b,
, Babu Rajendran Ramaswamy
a,c
, Kwang-Hyeon Chang
d
,
Atsuko Amano
e
, Todd M. Miller
a
, Fernando P. Siringan
f
, Shinsuke Tanabe
a
a
Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan
b
Senior Research Fellow Center, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan
c
Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamilnadu, India
d
Department of Environmental Science and Engineering, Kyung Hee University, Seochen-dong 1, Giheung-gu, Yongin-Si, Gyeonggi-Do 446-701, Republic of Korea
e
National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8567, Japan
f
Marine Science Institute, University of Philippines Diliman, Quezon City 1101, Philippines
a r t i c l e i n f o
Article history:
Received 26 April 2011
Received in revised form 7 June 2011
Accepted 12 June 2011
Available online 7 July 2011
Keywords:
BUVSs
Bioaccumulation
Stable isotope
Fish
Manila Bay
a b s t r a c t
Benzotriazole ultraviolet stabilizers (BUVSs) used in plastic products, building materials and personal
hygiene products were analyzed in shes collected from Manila Bay, the Philippines. BUVSs were
detected at ng g
1
level in all the sh samples, indicating their ubiquitous contamination in coastal
waters. Among the targeted eight BUVSs, UV-328 was predominantly found with a mean concentration
of 34.2 ng g
1
lipid weight, implying large scale production and use of this compound in the Philippines.
High concentrations of
P
BUVSs were found in bumpnose trevally (Carangoides hedlandensis), bluetail
mullet (adult) (Valamugil buchanani), common ponysh (Leiognathus equulus) and coral grouper (adult)
(Epinephelus corallicola) indicating their active uptake and/or lower metabolic capacity to eliminate
BUVSs. Among BUVSs, UV-P showed signicant positive relationship (p < 0.05) between concentration
and sh length (r = 0.29) and sh weight (r = 0.31). Levels of UV-P in demersal species had positive cor-
relation with d
15
N, indicating that possibile sink of UV-P is bottom sediment in the bay, and ultimately
accumulate through benthic food web rather than pelagic food web. To our knowledge, this is the rst
study on BUVSs distribution in sh from developing countries.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
Benzotriazole ultraviolet stabilizers (BUVSs) are used as absorb-
ers and stabilizers in textiles, household products, fabrics, plastics,
optical products, agricultural chemicals and many other material
from 0.05% to 2% by weight on or into the product to protect
against UV-irradiation (Fent et al., 2008), and also used in poly-
meric material and paints to improve their stability, and prevent
light-induced degradation and yellowing. In addition, they are also
used in personal-hygiene products such as body lotions, creams,
shampoos, fragrances, and sunscreen cosmetics (METIJ, 2006).
The concentration of a specic UV stabilizer in sunscreens varies
between 0.5% and 10%, but may even up to 25% (Hauri et al.,
2003). In terms of production and use, the 2-hydroxyphenyl deriv-
atives of benzotriazole constitute one of the most important fami-
lies of UV stabilizers (Crawford, 1999). Currently, 27 UV stabilizers
are listed in Annex VII of the Cosmetics Directive, and in addition a
further 43 chemicals are listed as UV stabilizer ingredients in cos-
metic products in the EU (Wahie et al., 2007).
The consumption volume of benzotriazole derivatives for both
plastic and to a lesser extent non-plastic uses increased during
the past few decades all over the world; however, their environ-
mental occurrence was sparsely documented (Crawford, 1999).
Carpinteiro et al. (2010a) analyzed BUVSs in water samples using
headspace solid-phase microextraction (SPME) followed by gas
chromatography tandem mass spectrometry. The same authors
also reported the levels of BUVSs in indoor dust (Carpinteiro et
al., 2010b). Even though the occurrence of BUVSs (UV-320, UV-
327 and UV-328) in the environment (river water, wastewater,
and sediment has been reported in the late 1970s (Jungclaus
et al., 1978). Recently, Zhang et al. (2011) reported the detection
of benzophenone and benzotriazole compounds from sediment
and sludge samples in China and USA. However, the studies on
their fate in biological samples, and possible harmful effects on
wildlife and human health are still scarce. Pruell et al. (1984)
0045-6535/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2011.06.054

Corresponding author at: Center for Marine Environmental Studies (CMES),

Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan. Tel./fax: +81 89
927 8162.
E-mail address: t.isobe@agr.ehime-u.ac.jp (T. Isobe).
Chemosphere 85 (2011) 751758
Contents lists available at ScienceDirect
Chemosphere
j our nal homepage: www. el sevi er. com/ l ocat e/ chemospher e
detected UV-327 and UV-328 in hard shell clam (Mercenaria
mercenaria) purchased from a seafood store in Rhode Island, USA.
Presence of lipophilic UV stabilizers (4-MBC, EHMC, Octocrylene
(OC), BP-3, homosalate (HMS)) up to 3800 ng g
1
lw (lipid weight)
in lake sh from Germany was reported (Nagtegaal et al., 1997).
Buser et al. (2006) and Fent et al. (2010) also reported UV stabiliz-
ers in sh from the rivers in Switzerland. Furthermore, a couple of
studies reported the occurrence and fate of BUVSs in marine food
chain and in marine mammals from the coastal waters of western
Japan (Nakata et al., 2009; Nakata and Shinohara, 2010).
Apart from many benecial properties, some UV stabilizers are
suspected to disrupt the endocrine system, and adversely affect
the fecundity, reproduction and development in organisms (Sch-
lumpf et al., 2001, 2004). Danovaro et al. (2008) found that some
sunscreen commercial products caused abrupt and complete
bleaching of hardcorals at low(10 lL L
1
) concentrations. Weisbrod
et al. (2007) reported the signicant estrogenic effects of the com-
monUVlter, BP-2, onvitellogenininduction, secondarysexcharac-
teristics, gonadal development andreproductioninfatheadminnow
(Pimephales promelas).
Generally, UV stabilizers can enter the aquatic environment
either directly via wash-off from the human body and cloth or
via wastewater from the catchment area. Domestic wastewater is
considered as a signicant source that brings large amount of
BUVSs. BUVSs are lipophilic (log K
ow
: 4.318.28) and persistent,
so it can be expected that once BUVSs enter the aquatic environ-
ment, they can be accumulated in biological tissues of various
organisms, especially in sh. Fish are suitable organisms that can
be used to monitor the presence of persistent lipophilic contami-
nants in aquatic systems. Moreover, humans may be exposed to
such chemicals including UV stabilizers not only by use of personal
care products but also through the consumption of sh.
Manila Bay is one of the pollution hot spots in the seas of East
Asia and about one third of population (26 million) in the Philip-
pines inhabit around the bay. The bay supports sheries and aqua-
culture activities signicantly, and serves as a sink and transit area
for the domestic and industrial wastes from Metro Manila and the
surrounding provinces (Velasquez et al., 2002). Since many people
in Metro Manila depend on sh from the bay for food, contamina-
tion of sh in the bay can be a threat to human health in this area.
The environmental contamination by polychlorinated biphenyls,
organochlorine pesticides and trace elements has already been re-
ported in Manila Bay (Prudente et al., 1994, 1997; Carvalho et al.,
2009; Villeneuve et al., 2010). However, no study has been carried
out on the contamination status of emerging BUVSs in commercial
species of sh from Manila Bay, till date. Based on this background,
we collected sh from the local sh market around the bay, and
analyzed eight BUVSs to evaluate their fate in the present aquatic
ecosystem, coastal marine area. We have also assessed the dietary
intake of BUVSs through sh to evaluate the possible threats, if any,
to human health in this area.
2. Material and methods
2.1. Chemicals
All standards were obtained in the highest available purity.
UV-P (CAS 2440-22-4), UV-326 (CAS 3896-11-5), UV-328 (CAS
25973-55-1), UV-329 (CAS 3147-75-9) were purchased from Wako
Pure Chemical (Osaka, Japan). UV-9 (CAS 2170-39-0), UV-234 (CAS
70321-86-7), UV-320 (CAS 3846-71-7), and UV-327 (CAS 3864-99-
1) were purchased from SigmaAldrich (St. Louis, MO, USA). Iso-
tope-labeled standards (ISs), TnBP-d
27
and TPhP-d
15
were supplied
by Tokyo Chemical Industry (Tokyo, Japan) and SigmaAldrich (St.
Louis, MO, USA), respectively. Acetone, hexane, dichloromethane,
methanol and 5% water-impregnated silica gel were purchased
from Wako Pure Chemical (Osaka, Japan).
2.2. Sample collection
During January and June 2008, 58 specimens belonging to 20
species were collected from the local sh markets around Manila
Bay (in Metro Manila and Cavite City), taking into consideration
the most popular species available at the time of collection. We se-
lected only shes that were collected from Manila Bay, and select
the species including juveniles and adults at the same sh market
considering sh collection time and place. Fishes were packed in
clean polyethylene bags, frozen, and transported on dry ice to
the Environmental Specimen Bank (es-BANK) of Ehime University
(Tanabe, 2006), Japan and stored at 25 C until analysis. The de-
tails of the species, biometric data (length and weight), feeding
habits and habitat are given in Table 1.
2.3. Chemical analysis
Details of the analytical method were described elsewhere (Kim
et al., 2011b). Briey, 5 g of sh muscle tissues were freeze-dried at
80 C for at least 72 h and homogenized with anhydrous sodium
sulfate (Na
2
SO
4
) and 10 ng of TnBP-d
27
was added to each sample
as an internal standard, then extracted with a mixture of hexane
and acetone (1:1, v/v) using a High Speed Solvent Extractor
(SE-100, Mitsubishi Chemical Analytechs, Japan) at 30 C at a
10 mL min
1
ow rate for 30 min. Extracts were concentrated
using a rotary evaporator (EYELA, Japan) and made up to 10 mL
with n-hexane. A portion of the extract (2 mL) was used for lipid
measurement gravimetrically.
One milliliter of the extract was subjected to further clean up as
follows. Four grams of 5% H
2
O deactivated silica gel was stirred
with hexane and the slurry was transferred to a glass column
(200 10 mm i.d) having a glass wool plug, then 1 g of Na
2
SO
4
was layered above the silica gel and conditioned with 25 mL of
hexane. The extract was loaded onto the column and eluted with
100 mL of dichloromethane. Then the dichloromethane fraction
was evaporated using a rotary evaporator until about 1 mL and
transferred into a 2 mL glass vial and dried under a gentle stream
of nitrogen gas. After complete solvent evaporation the analytes
were reconstituted in 1 mL of methanol and 1 ng of TPhP-d
15
was
added as syringe standard.
BUVSs including UV-P, UV-9, UV-234, UV-320, UV-326, UV-327,
UV-328, and UV-329, were analyzed by an ultra-fast liquid chro-
matography (UFLC)-XR system (Shimadzu, Japan) coupled with
MS/MS (AB Sciex, Tokyo, Japan). Chromatographic separation was
performed by an Asentis express C
18
column (2.7 lm,
100 mm 2.1 mm; Supelco, Bellefonte, USA) at a ow rate of
0.2 mL min
1
using (A) 0.1% (v/v) formic acid in Milli-Q water
and (B) 10 mM ammonium acetate in methanol. The gradient con-
ditions for positive mode were: 02 min, 20% B; 3.0 min, 95% B;
7.0 min, 95% B; 8.014 min, 100% B. For optimal stability, the tem-
perature of sample tray was set at 10 C. Peak identication and
quantication were carried out with Analyst 1.5.1 software and de-
tails of MS parameters were given in our previous paper Kim et al.
(2011b).
2.4. Stable isotope measurement
Levels of the stable isotopes (d
13
C and d
15
N) were measured in
sh muscle tissues. Homogenized tissue samples were dried for
24 h at 60 C and ground into ne powder with a mortar and pes-
tle. The solvent-extractable lipid was removed by extracting the
sample with a mixture of chloroform:methanol (2:1). The residues
were obtained by centrifugation using microtubes, and dried
752 J.-W. Kim et al. / Chemosphere 85 (2011) 751758
initially at room temperature and later at 60 C for 24 h. Stable car-
bon and nitrogen isotopes were measured using Integra CN d
13
C
and d
15
N analyzer (Sercon Co., UK). All isotopic data are reported
in the conventional d notation.
d
13
C or d
15
N R
sample
=R
standard
1 1000
where R is
13
C/
12
C or
15
N/
14
N ratio for d
13
C or d
15
N, respectively. Pee
Dee Belemnite and N
2
in air were used as standards for d
13
C or d
15
N,
respectively.
2.5. Quality assurance and quality control
Working standards (from 0.01 to 10 ng mL
1
) of BUVSs were
prepared and stored at 4 C in dark. Standard calibration curves
were obtained by plotting nominal analyte concentrations versus
internal standard. Excellent linearity with a correlation coefcient
value of R
2
> 0.998 was achieved for all the eight analytes. The
method detection limits (MDLs) and method quantication limits
(MQLs) were determined as the concentrations which would give
three and ten times, respectively the standard deviation of the
peak area for seven replicates of the blank sample. MDL and MQL
were 0.29 pg g
1
lipid wt. and 127 pg g
1
lipid wt., respectively.
Recoveries of the analytes were assessed by subtraction concentra-
tion observed in non-spiked samples from those in spiked (1 ng of
each standard) samples. The recoveries of the BUVSs ranged from
70.9% to 112% with the relative standard deviations (% RSD) of
0.711.7%. TnBP-d
27
was also spiked as an internal standard to
check the performance of the method. The recovery of TnBP-d
27
in the samples ranged between 80.5% and 90.7% and the nal con-
centrations were not corrected with recoveries. The concentrations
of analytes were calculated by comparing the peak area of each
compound in sample extract relative to those in standard and
the nal concentrations were not corrected for recovery. A proce-
dural blank (Na
2
SO
4
) was analyzed with every batch of seven sam-
ples to check for interference and contamination, and blank value
was subtracted from sample concentration. In this study, the aver-
age concentrations of target compounds in blank samples were
ranged between ND (UV-320) and 0.05 ng mL
1
(UV-326).
The data analysis was performed using the SPSS software (SPSS
for Windows: SPSS Inc., 2001). The correlation hypothesis between
the measured sh variables (length, weight, and stable isotopes) of
each species and their respective BUVSs concentrations were
tested by the Spearmans rank correlation. A probabilistic value
<0.05 was considered as statistically signicant.
3. Results and discussion
3.1. Extent of contaminant detection and concentration
BUVSs targeted in the present study were detected in almost all
sh samples from Manila Bay, the Philippines and their concentra-
tions were given in Table 2. Among the eight BUVSs, UV-328, UV-P,
UV-320 and UV-234 were frequently detected in 88%, 86%, 79% and
55% of analyzed specimens (n = 58), respectively. These detection
frequency shows the ubiquitous contamination UV-328, UV-P
and UV-320 in the ecosystem of Manila Bay, and the usage of these
compounds in the Philippines may be extensive compared to the
other BUVSs. On the other hand, UV-327, UV-329, UV-326 and
UV-9 were detected in less than half of the samples (Table 2).
UV-9 (log K
ow
4.31) was detected in only one sh out of 58 samples
(<2%), suggesting its smaller amount of use or lower bioavailabil-
ity. Nakata et al. (2009) reported that higher concentrations of
UV-328 compared with the other BUVSs (UV-320, UV-326, and
UV-327) in marine organisms from the Ariake Sea, Japan. Our re-
sults were consistent with their ndings, implying that the produc-
tion and usage of UV-328 may be prevalent in the AsiaPacic
region. Generally, the concentrations of UV-320, UV-326, UV-327
and UV-328 in sh samples from the Philippines were higher than
those reported in marine shes (athead, solesh, sea bass, mullet,
etc.) from shallow waters of Japan (Nakata et al., 2009). This sug-
gests that large scale usage of BUVSs and/or the release of un-
treated wastewater containing BUVSs to the coastal waters
resulted in elevated levels of contamination in Manila Bay.
Although UV-320 was frequently detected, the concentrations
were lower than that of UV-234 and UV-328. Our results are con-
sistent with Nakata et al. (2009) who found low concentrations
(<0.057 ng g
1
wet wt.) of UV-320 than UV-328 in the organisms
Table 1
Biometric data (mean SD) of the analyzed specimens and few ecological notes about them.
Family Common name Scientic name N Mean SD Major food items
a
Habitat
Length (cm) Weight (g)
Carangidae Bumpnose trevally Carangoides hedlandensis 3 13.3 0.2 38.4 1.7 Shrimps, small crabs, and shes Demersal
Purse-eye scad Selar crumenthalmops 3 20.2 1.6 132 27.8 Shrimps and small invertebrates Pelagic
Yellowtail scad Atule mate 3 11.0 0.5 14.2 1.7 Crustaceans and cephalopods Pelagic
Yellowstripe scad Selaroides leptolepis 3 11.2 0.8 16.7 4.4 Ostracods, gastropods, and euphausiids Demersal
Redtail scad Decapterus kurroides 2 19.5 0.7 68.2 2 Zooplankton and invertebrates Pelagic
Mugilidae Bluetail mullet (adult) Valamugil buchanani 1 31.0 531 Phytoplankton and crustaceans Demersal
Bluetail mullet (juvenile) Valamugil buchanani 2 24 0.7 157 18 Phytoplankton and crustaceans Demersal
Flathead grey mullet Mugil cephalus 3 14.7 0.4 33.4 2.1 Zooplankton, detritus, and benthic organisms Demersal
Clupeidae Chacunda gizzard shad Anodontostoma chacuda 3 12.4 0.1 20.9 2.3 Phytoplankton, zooplankton, and crustaceans Pelagic
Toli shad Tenualosa toli 7 12.4 0.6 20.5 3.0 Zooplankton Pelagic
Gerreidae Deep-bodied mojarra Gerres erythrourus 3 13.5 0.9 43.6 13.3 Zooplankton, bivalves, crustaceans, and shes Demersal
Longn mojarra Pentaprion longimanus 3 12.7 0.7 3.4 4.5 Benthic animals Demersal
Teraponidae Fourlined terapon Pelates quadrilineatus 3 11.5 1.3 21.0 5.6 Invertebrates Pelagic
Engraulidae Indian anchovy Stolephorus indicus 3 8.50 0.5 7.3 1.9 Zooplankton Pelagic
Scombridae Indian mackerel Rastreliliger kanagurta 1 20.0 93.4 Phytoplankton and zooplankton Pelagic
Polynemidae Common ponysh Leiognathus equulus 3 8.67 0.6 11.4 1.2 Polychaetes, crustaceans, shes, and worms Demersal
Sillaginidae Silver sillago Sillago sihama 2 14.3 0.4 23.9 1.4 Polychaete worms and crustaceans Demersal
Mullidae Yellowstriped goatsh Upeneus vittatus 2 13.5 0.7 32.4 5.2 Small crustaceans Demersal
Scatophagidae Spotted scat Scatophagus argus 3 15.5 1.1 119 15.3 Worms, crustaceans, insects, and plant matter Pelagic
Labridae Tripletail wrasse Cheilinus trilobatus 1 20.0 156 Benthic invertebrate and crustaceans Demersal
Serranidae Coral grouper (adult) Epinephelus corallicola 1 27.0 369 Crustaceans and shes Demersal
Coral grouper (juvenile) Epinephelus corallicola 3 14.3 1.1 47.4 10.6 Crustaceans and shes Demersal
a
Source: FishBase (2010).
J.-W. Kim et al. / Chemosphere 85 (2011) 751758 753
from the Ariake Sea, Japan. This may indicate the differences in
accumulation and biodegradability of this compound. According
to some papers summarizing the toxicities of UV-320, acute toxic-
ity of UV-320 on Daphnia pulex was not high (Kim et al., 2011a).
However, Hirata-Koizumi et al. (2008) reported increase in liver
weight, centrilobular hepatocyte hypertrophy and focal necrosis
of hepatocytes in rats chronically exposed for UV-320 by oral
administration for a period of 52 weeks. Due to its persistent, bio-
accumulative, and toxic properties, UV-320 is categorized as a
Class I Specied Chemical Substance by Japanese government since
2007 (METIJ, 2006). Therefore, extensive monitoring on the distri-
bution of this compound in the environment and biota are
necessary.
Based on Spearmans correlation coefcients (at 0.05 level), sig-
nicant positive correlations were found between UV-234 and UV-
328 (r = 0.69), UV-234 and UV-329 (r = 0.63), UV-320 and UV-327
(r = 0.62), and UV-320 and UV-328 (r = 0.72) (Table 3). Similarly,
Nakata et al. (2009) also found a positive correlation between
UV-326 and UV-327, UV-326 and UV-328, and UV-327 and UV-
328 in sediment from the Ariake Sea and suggested that these
compunds might have been originated from same sources such
as plastic materials. The signicant correlations observed in the
present study suggest that sh in Manila Bay are exposed to BUVSs
originating from the same sources which are distributed homoge-
neously in the bay.
Further research is needed to understand the distribution and
behavior of these compounds in aquatic organisms. Information
on production and consumption of these chemicals are also
necessary.
3.2. Composition of BUVSs in shes
Relative contributions of each BUVSs to total concentrations of
analytes of the present study in the sh samples are shown in
Fig. 1. The percentage contributions of UV-P and UV-328 were
found predominant. Interestingly, compositions of BUVSs were dif-
ferent even in shes belonging to the same family. On the other
hand, same composition pattern was observed in shes belonging
to different families. For example, the composition of BUVSs in
fourlined terapon belonging to teraponidae was similar to yellow-
stripe scad belonging to carangidae with pecentage compositions
of 9697% for UV-P and 34% for UV-320. In addition, composition
of BUVSs in sliver sillago belonging to sillaginidae and in indian
mackerel belonging to scombridae were uniform (Fig. 1). This
may be due to lower metabolic capacity or selective uptake of
UV-P and UV-328 in these shes. Higher contribution of UV-328
in indian anchovy (54%), common ponysh (68%), bumpnose
trevally (66%), and athead grey mullet (62%) belonging to differ-
ent families may be due to the greater avalability of this compound
and active uptake by these sh species.
Table 2
BUVSs concentrations (mean SD, range) (ng g
1
lipid wt.) in marine species from Manila Bay, the Philippines.
Lipid content
(%)
UV-P UV-9 UV-234 UV-320 UV-326 UV-327 UV-328 UV-329
P
BUVSs
Detection frequency (%) 86 2.0 55 79 19 43 88 41
Bumpnose trevally 0.40 0.17 41.2 53.2 ND
a
5.91 10.2 6.25 9.18 22.2 38.5 ND 207 308 33.3 54.9 316 460
(ND101) (ND17.7) (0.7916.9) (ND66.7) (ND563) (1.1496.7)
Purse-eye scad 2.61 0.26 14.7 16.7 ND 2.23 3.21 2.47 4.01 ND 1.07 1.85 3.32 4.44 1.86 3.22 33.2 22.7
(2.9733.8) (ND5.91) (ND7.10) (ND3.21) (ND8.37) (ND5.57)
Yellowtail scad 2.43 0.40 17.0 21.8 5.40 9.36 9.24 15.5 5.98 9.61 3.78 6.55 8.42 12.0 8.55 11.8 7.12 12.3 65.5 98.6
(1.6141.9) (ND16.2) (ND27.1) (ND17.1) (ND11.3) (1.1922.3) (ND22.0) (ND21.4)
Yellowstripe scad 2.49 0.44 6.20 10.7 ND ND 0.28 0.44 ND ND ND ND 6.48 11.1
(ND18.5) (ND0.79)
Redtail scad 1.18 0.28 15.8 3.77 ND ND 0.99 0.18 ND 6.94 5.02 10.6 2.18 1.46 2.07 35.9 12.9
(13.218.5) (0.871.12) (3.3910.5) (9.0912.2) (ND2.93)
Bluetail mullet (adult) 0.23 57.4 ND ND 9.60 211 2.57 18.4 ND 299
Bluetail mullet (juvenile) 1.32 0.59 21.2 7.20 ND 1.52 0.71 0.44 0.26 ND ND 3.71 1.38 0.47 0.66 27.3 4.71
(16.126.3) (1.022.02) (0.250.62) (2.734.69) (ND0.94)
Flathead grey mullet 0.72 0.37 3.02 5.24 ND 34.6 12.6 6.88 2.56 6.32 10.9 9.70 9.28 105 74.2 4.86 4.25 171 110
(ND9.07) (22.047.1) (4.119.15) (ND19.0) (ND18.5) (30.2179) (ND7.89)
Chacunda gizzard shad 1.60 0.58 7.29 2.98 ND 7.36 10.3 0.02 0.03 ND 0.46 0.80 4.18 2.78 2.54 3.17 21.8 18.5
(4.9410.7) (0.8519.2) (ND0.06) (ND1.38) (1.567.09) (ND6.09)
Toli shad 1.23 0.23 8.70 9.80 ND 4.23 7.39 1.00 1.40 ND 0.37 0.97 5.81 7.81 ND 20.1 23.8
(ND26.4) (ND17.6) (ND3.97) (ND2.57) (ND19.7)
Deep-bodied mojarra 0.84 0.27 6.60 2.05 ND 2.35 1.58 0.70 0.68 ND 5.30 3.86 5.88 6.93 1.97 2.19 22.8 15.6
(4.278.09) (0.653.76) (ND1.36) (2.219.63) (ND13.5) (ND4.33)
Longn mojarra 0.49 0.23 81.5 122 ND 8.44 7.84 3.64 4.15 23.8 41.2 109 122 9.83 11.4 12.0 10.6 248 311
(7.13222) (1.1416.7) (0.128.21) (ND71.3) (ND221) (ND22.3) (ND19.8)
Fourlined terapon 2.50 0.95 6.87 7.10 ND ND 0.19 0.21 ND ND ND ND 7.06 7.27
(1.5314.9) (ND0.41)
Indian anchovy 1.57 0.28 6.59 5.12 ND 6.96 12.1 0.37 0.64 ND ND 22.2 27.9 4.72 8.18 40.9 49.1
(1.6111.8) (ND20.9) (ND1.10) (1.5253.9) (ND14.2)
Indian mackerel 1.18 10.8 ND ND 0.39 ND ND 4.98 ND 16.2
Common ponysh 1.11 0.15 19.5 23.2 ND 8.36 6.02 22.5 7.59 ND 38.6 20.9 193 68.7 4.08 3.81 286 110
(2.5146.0) (2.8214.8) (14.028.7) (20.861.5) (119255) (ND7.55)
Silver sillago 0.23 0.06 26.6 13.2 ND ND 0.71 0.59 ND ND 11.0 3.21 ND 38.3 17.0
(17.235.9) (0.291.13) (8.7213.3)
Yellowstriped goatsh 0.18 0.09 18.9 26.7 ND 62.9 88.9 5.88 6.35 ND 18.8 26.5 36.9 52.2 ND 143 147
(ND37.8) (ND126) (1.3910.4) (ND37.5) (ND73.9)
Spotted scat 0.43 0.04 40.6 19.2 ND 9.50 9.07 4.64 0.93 40.7 28.5 23.4 26.9 26.3 20.0 11.4 11.6 157 98.6
(29.362.8) (3.2319.9) (3.785.63) (9.9666.2) ND (12.449.2) (4.6324.9)
Tripletail wrasse 0.27 17.9 ND 4.64 5.67 31.4 7.50 ND 4.77 71.9
Coral grouper (adult) 0.13 160 ND 14.3 0.78 ND 18.5 21.1 39.4 254
Coral grouper (juvenile) 0.39 0.29 35.0 38.2 ND 3.57 6.19 3.96 3.44 32.9 30.0 5.45 9.43 4.40 7.61 1.39 2.41 86.6 60.2
(ND75.7) (ND10.7) (ND6.23) (ND58.8) (ND16.3) (ND13.2) (ND4.17)
a
ND indicates the values below the method detection limit (MDL).
754 J.-W. Kim et al. / Chemosphere 85 (2011) 751758
3.3. Species-specic accumulation of BUVSs
Signicant positive correlation between body weight and length
were found in all sh species (r = 0.94; p < 0.001) (data not shown).
Among the eight BUVSs analyzed in this study, size-dependent
accumulation was observed only for UV-P. Signicant positive cor-
relations between UV-P concentration and both length (r = 0.29;
p < 0.05) and weight (r = 0.32; p < 0.05) were observed. This accu-
mulation pattern is possibly because of efcient uptake, and low
elimination rate of UV-P. However, concentrations of the rest of
the BUVSs did not show any relation with sh length and weight.
Therefore, differences in accumulation/exposure pattern indicate
the species specicity in sh samples.
Concentrations of BUVSs measured in 20 sh species varied
greatly depending on the species within one to two orders of mag-
nitude (Table 1). Bumpnose trevally (Carangoides hedlandensis) had
the highest concentrations (316 460 ng g
1
lipid wt.) of
P
BUVSs
than the other species, whereas yellowstripe scad (Selaroides
leptolepis) had the lowest concentrations (6.48 11.1 ng g
1
li-
pid wt.) of
P
BUVSs. This wide variation in concentrations indi-
cates species-specic accumulation and elimination of BUVSs.
Concentrations of total BUVSs in adult bluetail mullet (Valamugil
buchanani) (299 ng g
1
lipid wt.) and coral grouper (Epinephelus
corallicola) (254 ng g
1
lipid wt.) were found one order of magni-
tude higher than those in their juveniles (27.3 and 86.6 ng g
1
li-
pid wt., respectively) (Table 1), suggesting the possibility of
greater food consumption and growth-dependent accumulation
in adults. It is interesting to note that UV-326 was not detected
in the juveniles of blue mullet (V. buchanani) but detected at higher
concentration (211 ng g
1
lipid wt.) in adult. However, this accu-
mulation pattern of UV-326 was not observed in coral grouper
(E. corallicola), where, higher (mean) concentration (32.9 ng g
1
-
lipid wt.) was found in juveniles, similarly, UV-320 was also higher
in juvenile (3.96 ng g
1
lipid wt.) than adult grouper (0.78 ng g
1
lipid wt.). In addition, UV-234 and UV-329 were not found in adult
but detected in juvenile bluetail mullet (V. buchanani).
The accumulation of BUVSs was highly species specic, among
carangids; total BUVSs in bumpnose trevally was about 550 times
higher than in the other four species of that family (Table 1). Fish
belonging to clupidae (Anodontostoma chacuda and Tenualosa toli)
and teraponidae families (Pelates quadrilineatus) accumulated less
amount of BUVSs. Further, UV-9 was found only in yellowtail scad
(Atule mate) (ND16.2 ng g
1
lipid wt.). Relatively large variation in
concentrations observed in the reported data can be related to
feeding habit and/or metabolic capacity of each sh species.
3.4. Relationship between concentrations of BUVSs and stable isotopes
Analysis of stable isotopes (d
13
C and d
15
N) in organisms is an
useful indicator of the feeding relationship in food web (Peterson
and Fry, 1987). In general, d
13
C values of organisms vary around
1 in accrodance with single trophic level increase when organ-
isms feed on the same carbon sources (Rau et al., 1983; Mills
et al., 1984). d
15
N can be used as an index of trophic level, because
Table 3
Spearmans rank correlations among the eight BUVSs.
UV-P UV-9 UV-234 UV-320 UV-326 UV-327 UV-328 UV-329
UV-P 1 0.17 0.22 0.25 0.44
**
0.16 0.24 0.43
**
UV-9 1 0.21 0.21 0.22 0.20 0.13 0.23
UV-234 1 0.53
**
0.33
*
0.44
**
0.69
**
0.63
**
UV-320 1 0.47
**
0.62
**
0.72
**
0.47
**
UV-326 1 0.35
**
0.29
*
0.45
**
UV-327 1 0.49
**
0.52
**
UV-328 1 0.55
**
UV-329 1
*
p < 0.05.
**
p < 0.01.
0% 20% 40% 60% 80% 100%
Chacunda gizzard shad
Toli shad
Purse-eye scad
Redtail scad
Yellowtail scad
Yellowstripe scad
Fourlined terapon
Deep-bodied mojarra
Longfin mojarra
Yellowstriped goatfish
Spotted scat
Silver sillago
Indian mackerel
Indian anchovy
Common ponyfish
Bumpnose trevally
Flathead grey mullet
Tripletail wrasse
Bluetail mullet (adult)
Bluetail mullet (juvenile)
Coral grouper (adult)
Coral grouper (juvenile)
UV-P
UV-9
UV-234
UV-320
UV-326
UV-327
UV-328
UV-329
Fig. 1. Composition of BUVSs in shes collected from Manila Bay, the Philippines.
J.-W. Kim et al. / Chemosphere 85 (2011) 751758 755
the d
15
N for predators is 3.4 greater than that of prey owing to
isotopic fractionation on assimilation of nitrogen (Allen, 1991;
Hobson and Welch, 1992).
We found a considerable variation in both d
13
C and d
15
N values
of all 58 specimens from Manila Bay (Fig. 2). Most species except
Indian anchovy and bluetail mullet (juvenile), showed the d
13
C val-
ues ranging from13to 19with a corresponding increase of
d
15
N, indicating that they belonged to the same food web in Manila
Bay (r = 0.48, p < 0.0005). d
13
C and d
15
N of indian anchovy and
bluetail mullet (juvenile) were out of ranges, suggesting that they
had different feeding habits and/or different food sources rather
than bay-origin food. Different from its adults which occupy coast-
al habitat, the young bluetail mullet stays in estuaries and also of-
ten ascend rivers, and dwells in benthic habitat (Allen, 1991). For
the trophic level of other sh species, d
15
N varied from 7.58 for
spotted scat (Scatophagus argus) a detritivore to 14.3 for coral
grouper (adult). Hobson and Welch (1992) reported that d
15
N val-
ues of the same food web samples do not obviously correlate with
d
13
C values after the rst trophic shift from primary producers to
lter feeders. From the narrow range of d
13
C values and wide var-
iation of d
15
N values, the sh species of the present study includes
at least 23 different trophic level species. When we examined the
relationship between BUVSs concentrations and d
15
N values, no
biomagnication pattern was obeserved for most of the analytes.
However, we observed a signicant accumulation of UV-P in
demersal sh (p < 0.01) (Fig. 3). It has been suggested that BUVSs
such as UV-320 and UV-327 are often adsorbed on soil (46.9%
and 43.3%) and sediment (51.2% and 55.4%) with high concentra-
tions than in water (1.9% and 1.3%) and air (0.00002% and
0.00004%) (METIJ, 2007). Therefore, bioaccumulation of UV-P in
demersal sh observed in the present study suggests that it mainly
settles down to bottom sediment, and ultimately accumulats
through benthic food chain rather than pelagic food chain. Benthic
food web could be the main pathway of bioaccumulation of UV-P.
On the other hand, there is no signicant correlation between con-
centrations of several BUVSs and d
15
N values in both pelagic and
demersal sh. Our results suggest that each BUVS has different
accumulation behavior in food web depending on its physico-
chemical properties and bioavailability in the ambient environ-
ment. The comprehensive and continuous investigations on the
distribution and accumulation of BUVSs in food web are necessary
to clarify their species-specic distribution and risk assessment for
the betterment of sher resources in the bay.
3.5. Daily intake of BUVSs through shes
Number of emerging contaminants raises a considerable toxico-
logical and public concern, especially when human health based
guideline values are unavailable. Human may be exposed to UV
stabilizers directly by use of cosmetics, or indirectly through food.
Intake of BUVSs through seafood may be one of the exposure
routes for human, since it is one of the most important food and
protein sources. Daily intake of BUVSs by human in the Philippines,
through consumption of sh was estimated based on their concen-
trations (wet wt. basis) in sh and per diem sh consumption. Ta-
ble 4 shows the estimated daily intake of UV-320. Annual sh
consumption of 31.8 kg year
1
for the Philippines (Food and Agri-
culture Organization of the United Nations) and the average body
weight of 60 kg were used for this estimation in the present study.
No observed adverse effect level (NOAEL) value of UV-320 was
0.1 mg kg
1
d
1
for male rat (Hirata-Koizumi et al., 2008). Based
on Schriks et al. (2010), Tolerable Daily Intake (TDI) of UV-320
6
8
10
12
14
16
18
-22 -20 -18 -16 -14 - 12 - 10
1
5
N

(

)
13
C ()
Chacunda gizzard shad
Purse-eye scad
Bumpnose trevally
Bluetail mullet (adult)
Bluetail mullet (juvenile)
Longfin mojarra
Coral grouper (adult)
Coral grouper (juvenile)
Tripletail wrasse
Spotted scat
Flathead grey mullet
Yellowstriped goatfish
Silver sillago
Indian mackerel
Redtail scad
Toli shad
Yellowstripe scad
Common ponyfish
Indian anchovy
Fourlined terapon
Yellowtail scad
Deep-bodied mojarra
r = 0.48, p < 0.001
Indian anchovy
Bluetail mullet (juvenile)
Fig. 2. Relationship between d
13
C and d
15
N values in marine sh from Manila Bay.
0
50
100
150
200
250
8 10 12 14 16
C
o
n
c
e
n
t
r
a
t
i
o
n

(
n
g
/
g
l
w
)
15
N
r = 0.55, p < 0.01
Fig. 3. Relationships between d
15
N and UV-P in demersal shes from Manila Bay.
Table 4
Dietary intake (range and mean) of BUVSs through sh consumption for the
population in the Philippines.
Fish UV-320 (lg d
1
)
Min Max Mean
Bumpnose trevally 0.0004 0.003 0.001
Purse-eye scad ND
a
0.006 0.002
Yellowtail scad ND 0.05 0.02
Yellowstripe scad ND 0.0008 0.0003
Redtail scad 0.0007 0.001 0.001
Bluetail mullet (adult) 0.002
Bluetail mullet (juvenile) 0.0004 0.0005 0.0004
Flathead grey mullet 0.002 0.006 0.004
Chacunda gizzard shad ND 0.0001 0.00004
Toli shad ND 0.004 0.001
Deep-bodied mojarra ND 0.0006 0.0004
Longn mojarra 0.0001 0.002 0.0009
Fourlined terapon ND 0.0006 0.0004
Indian anchovy ND 0.0006 0.0002
Indian mackerel 0.0004
Common ponysh 0.01 0.03 0.02
Silver sillago 0.0001 0.0002 0.0001
Yellowstriped goatsh 0.0003 0.001 0.0007
Spotted scat 0.002 0.002 0.002
Tripletail wrasse 0.001
Coral grouper (adult) 0.0001
Coral grouper (juvenile) ND 0.001 0.0008
Calculated guideline value 6.0
Average sh consumption in the Philippines: 87 g d
1
.
a
ND: <LOD and/or <0.05.
756 J.-W. Kim et al. / Chemosphere 85 (2011) 751758
was calculated from the rat NOAEL value subtracted by using an
uncertainty factor of 1000 and found as 0.1 lg kg
1
d
1
. NOAEL
or LOAEL is divided by an appropriate uncertainty factor to correct
for extrapolation from animal to human data, and in adequacy of
the database (Van Leeuwen, 2000). The guideline value for UV-
320 (6 lg d
1
) was calculated from the TDI value. As a conse-
quence, daily intake of UV-320 in all shes is two to ve orders
of magnitude lower than the calculated guideline value (6 lg d
1
).
Therefore, adverse health effects by UV-320 may not occur through
consumption of sh by human in the Philippines, at present. How-
ever, there is a need to consider additional effects, because little is
known about the toxicological effect of BUVSs on aquatic and ter-
restrial organisms and wildlife. Although acute toxicities of BUVSs
were not high, long-term and multi-effect studies of these com-
pounds are required. Moreover, further studies should be con-
ducted on the potential human health effects of contamination
by BUVSs as these compounds are used in many personal care
products to protect human skin from direct exposure to the harm-
ful UV radiation from sunlight.
4. Conclusions
To our knowledge, this is the rst study on the contamination of
BUVSs in shes from developing countries and also the rst report
on the quantication of UV-P, UV-9, UV-234 and UV-329 in sh.
The BUVSs were detected in almost all the shes analyzed, sug-
gesting that the production and usage of these compounds are
widespread in the Philippines. High concentrations of BUVSs found
in bumpnose trevally, bluetail mullet (adult), common ponysh
and coral grouper (adult) at hundreds of ng g
1
lipid wt., indicate
that these compounds are preferably accumulated in these species
and/or these species may have low metabolic capacity to elimate
these compounds. The body weight and body length were the best
predictors of within-species differences in UV-P concentrations.
UV-P concentration in demersal sh species was positively corre-
lated with trophic level, indicating its accumulation through ben-
thic food web rather than pelagic. Human intake of BUVSs by
consuming sh from Manila Bay is far lower when compared to
TDI (6 lg d
1
), and will not pose any threat to consumers at this
moment. Since information on ecotoxicological aspects of BUVSs
are meager, extensive studies are needed to assess their ecotoxico-
logical risks associated with their distribution and fate in different
trophic level organisms in the aquatic environment.
Acknowledgements
We would like to thank Dr. A. Subramanian, CMES, Ehime
University, Japan for critical reading of the manuscript. Financial
support was provided by Grants-in-Aid for Scientic Research
(S: 20221003), for Young Scientist (B:23710077) and Global COE
Program of the Japanese Ministry of Education, Science, Sports,
Culture and Technology (MEXT) and Japan Society for the Promo-
tion of Science (JSPS). This research was partly supported by MEXT
program Promotion of Environmental Improvement for Indepen-
dence of Young Researchers under the Special Coordination Funds
for Promoting Science and Technology.
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