Professional Documents
Culture Documents
GC-50 glass lter, which was rinsed with isotonic ammonium formate to
remove the salts. Surface sediments (1e2 cm, <63 mm, n 3) were collected from
Clear Water Bay.
For measurements of background THg and MeHg concentrations in the bivalves,
individuals of different sizes for each bivalve species were randomly selected from
the collection. Selected bivalves were depurated without feeding for two days.
Afterwards the bivalves were dissected, freeze-dried, and ground into a ne powder.
For small individuals (shell length <1 cm), 10e20 individuals of similar size were
pooled to make up a composite sample. For each site, totally 50 samples were made,
among which the number of composite samples was between 10 and 15. Dried
tissues or sediments were weighed and approximately 0.2 g of which were digested
at 190
C with aqua regia (2 mL HNO
3
:6 mL HCl) in a microwave digestion system.
MeHg in bivalves were extracted by digesting approximately 40 mg of homogenized
soft tissues with 25% KOH in methanol at 60
C for 3 h.
All containers for sampling and analysis of Hg were vigorously cleaned with hot
4 N HCl acid. THg was analyzed using the single gold trap amalgamation technique
by CVAFS (QuickTrace
g
g
d
)
-
1
-
1
0.01
0.1
1
10
100
1000
MeHg
0.01 0.1 1
0.01
0.1
1
10
100
1000
Scallop
Clam
Oyster
Green mussel
Black mussel
Fig. 2. Inux rates of Hg(II) and MeHg in ve species of bivalves at different ambient
concentrations. Data presented as mean SD (n 4). The relationships between metal
inux rate (I
w
, mg g
1
d
1
) and metal concentration in the dissolved phase (C
w
, mg L
1
)
in the bivalves are as follows (r
2
0.99, p < 0.001). For Hg(II), I
w
32.8C
w
1.02
for
scallops; I
w
8.9C
w
0.87
for clams; I
w
4.4C
w
1.05
for oyster; I
w
11.4C
w
1.06
for green
mussels; I
w
3.5C
w
1.29
for black mussels; For MeHg, I
w
595.8C
w
1.16
for scallops;
I
w
82.4C
w
1.13
for clams; I
w
62.5C
w
1.14
for oysters; I
w
132.7C
w
1.24
for green mussels;
I
w
71.4C
w
0.96
for black mussels.
MeHg-Sediment
Time (h)
0 10 20 30 40 50
1
10
100
Hg(II)-Algae
0 10 20 30 40 50
%
r
e
t
a
i
n
e
d
1
10
100
MeHg-Algae
0 10 20 30 40 50
1
10
100
Scallop
Clam
Oyster
Green mussel
Black mussel
Hg(II)-Sediment
Time (h)
0 10 20 30 40 50
%
r
e
t
a
i
n
e
d
1
10
100
Fig. 3. Retention of Hg(II) and MeHg in ve species of bivalves following a pulse
feeding of radiolabeled diatoms or sediments. Data presented as mean SD (n 10).
K. Pan, W.-X. Wang / Environmental Pollution 159 (2011) 2500e2506 2503
Domouhtsidou and Dimitriadis, 2000). The higher percentage of
Hg(II) distributed in the organelles fraction in the green mussels
implies that the green mussels transported the Hg(II) extracellu-
larly through organelles (possibly lysosomes) more actively,
leading to higher efux rates in this species.
3.4. Biodynamics in controlling Hg body concentrations and the
MeHg to THg ratio in bivalves
Metal bioaccumulation in aquatic invertebrates can be highly
variable, even for animals within closely related taxa down to
species in the same genus (Rainbow, 2002). In our study, the body
concentrations of THg and MeHg in the bivalves also varied in terms
of concentration as well as MeHg to THg ratio. The site-specic
dissolved Hg concentration in seawater did not control the inter-
specic difference. For example, relatively higher THg concentra-
tions (0.35 ng L
1
) were observed inTolo Harbor, compared to those
observed in Dapeng Bay and Clear Water Bay (0.21 and 0.20 ng L
1
,
respectively). The green mussels collected from Tolo Harbor
contrarily contained the lowest THg concentrations in the tissues
among the ve bivalve species. Moreover, the clams, oysters, and
black mussels, which were all collected from Clear Water Bay,
accumulated THg differently (black mussels > oysters > clams).
Thus the differentiation of Hg bioaccumulation may be more likely
due to the biologically driven species-specic biodynamics. It
should be noted, however, that the concentrations of Hg(II) both in
water and in food were much higher than those of MeHg, and also
accounted for 60e90% of THg in the tissues of the bivalves. It is
possible that the biodynamics of Hg(II) had larger effects on the
concentrations of THg in the bivalves than that of MeHg.
The bivalves had contrasting k
u
for both Hg(II) and MeHg, which
varied by an order of magnitude (Table 2). Food quality also had
large effects on the AEs of Hg(II) and MeHg, indicating that food
choice may contribute to the species-specic Hg bioaccumulation
(see below). No signicant relationship can be found between the
k
u
or AE and the concentrations of THg and MeHg in the bivalves.
Not surprisingly, none of the parameters alone can explain the
interspecic body concentration of MeHg and THg, as bio-
accumulation is a result of the interaction between uptake and loss.
Yet, the biodynamics still has signicant implications for under-
standing the variations in the concentrations of MeHg and THg in
the bivalves. For example, the scallops had the highest k
u
both for
Hg(II) and MeHg, which may be associated with its highest MeHg
concentration. However, the high loss rate of Hg(II) (0.04 day
1
)
prevented the THg in scallops from ranking the highest.
Metal excretion rate constant (k
e
) was important for deter-
mining the intraspecic and interspecic differences in metal bio-
accumulation (Pan and Wang, 2009a, b). Oysters had the lowest k
e
of Hg(II) and MeHg in the bivalves (Table 2, Fig. 4). The k
e
of Hg(II) in
green mussels (0.06 day
1
) was two times higher than that in
oysters (0.02 day
1
). Similarly, the rate of MeHg in the green
mussels (0.007 day
1
) also overwhelmed that in the oysters (0.003
day
1
). Despite the fact that both the dissolved THg in Tolo Harbor
and the k
u
in green mussels were high, the fast loss rates of Hg(II)
and MeHg were partially responsible for the lowest THg and MeHg
concentrations in this species. A previous study has shown that the
Cu body concentrations in different bivalves were inversely corre-
lated with the Cu k
e
s (Pan and Wang, 2009a). Such a relationship
was also observed between the k
e
s of Hg(II) and the THg concen-
trations in the oysters, clams, scallops, green mussels (data not
shown). One exception was the black mussels. The k
e
s of Hg(II) and
MeHg (0.035 and 0.006 day
1
, respectively) were signicantly
higher than those in the oysters collected from the same site.
Contrarily, the black mussels contained higher THg concentrations
(92 ng g
1
) than the oysters (70 ng g
1
).
Growth dilution may also play a signicant role in controlling
the interspecic variation. Rapid growth is the major factor
resulting in reduced Hg concentrations in sh (Ward et al., 2010).
A specic growth rate ranging from 0.01 to 0.10 day
1
is commonly
MeHg
0 5 10 15 20 25
10
100
Hg(II)
Time (days)
0 5 10 15 20 25
%
r
e
t
a
i
n
e
d
10
100
Scallop
Clam
Oyster
Green mussel
Black mussel
Time (days)
Fig. 4. Depuration of Hg(II) and MeHg in ve species of bivalves over a 24-day period
following a 6-day combined exposure to dissolved and dietary Hg (Hg(II) or MeHg,
Mean SD, n 10).
S
c
a
l
l
o
p
C
l
a
m
O
y
s
t
e
r
B
m
u
s
s
e
l
G
m
u
s
s
e
l
S
c
a
l
l
o
p
C
l
a
m
O
y
s
t
e
r
B
m
u
s
s
e
l
G
m
u
s
s
e
l
0
20
40
60
80
MRG
Cellular debris
Organelles
HSP
MTLP
Hg(II)
%
m
e
t
a
l
i
n
e
a
c
h
f
r
a
c
t
i
o
n
0
20
40
60
80
MeHg
Fig. 5. Subcellular distribution of Hg(II) and MeHg in ve species of bivalves collected on the sixth day during a 24-day depuration period. (Mean SD, n 3). G mussel: green
mussel; B mussel: black mussel.
Hg(II) in organelles fraction (%)
0 5 10 15 20 25
E
f
f
l
u
x
r
a
t
e
(
d
a
y
)
-
1
0.00
0.02
0.04
0.06
0.08
0.10
Hg(II) in MTLP fraction (%)
0 20 40 60 80 100
0.00
0.02
0.04
0.06
0.08
0.10
y =0.0041 x -0.013
p<0.001
r
2
=0.96
Fig. 6. Relationship between the efux rate constant of ve species of bivalves and the
percentage of Hg(II) in either the organelles or the MTLP fraction. (n 10 for efux rate
and n 3 for subcellular distribution).
K. Pan, W.-X. Wang / Environmental Pollution 159 (2011) 2500e2506 2504
observed for marine bivalves (Clausen and Riisgrd, 1996;
Jrgensen, 1996). Thus, growth dilution can contribute to 10e30%
of the overall efux for Hg(II) when the specic growth rate
constant (g) is 0.01 per day, but can be up to 60e80% when the g is
as high as 0.10 per day. It is possible that the ve bivalve species
have different g and thus have different degrees of dilution for THg
and MeHg. However, the effects of growth may be complicated by
the rate of weight change compared to the rate of uptake and loss
for Hg, which may be the reason that the effect of growth dilution
on Hg accumulation was only observed in scallops and green
mussels (Fig. 1).
Biodynamics not only controls the body concentrations of Hg(II)
and MeHg, but also has confounding effects on the MeHg to THg
ratios. Bivalves effectively absorbed Hg(II) and MeHg from both
dissolved and dietary phases, with MeHg being accumulated more
efciently. Consequently, the MeHg to THg ratios in bivalves were
signicantly elevated compared to those in water and sediments
(less than 0.5%, Ullrich et al., 2001). Although MeHg concentrations
in bivalves did not vary widely (a difference of w2 times), there was
a difference of nearly four times (11e37%) in the MeHg to THg ratio
(Table 1). On the one hand, the relatively low percentage of Hg in
the form of MeHg in bivalves was largely determined by the much
higher concentrations of Hg(II) in water and food as compared to
MeHg. On the other hand, the difference in the MeHg to THg ratio
was affected by the differences between the biodynamics of Hg(II)
and MeHg in each species. However, it appeared that the k
u
and AE
could not explain the species-dependent ratio. For example,
difference in k
u
between Hg(II) and MeHg was most pronounced in
the black mussel (a difference of w20 times), whereas the ratio was
the lowest in this species. The proportion of MeHg was positively
correlated with the ratio of the efux rate of Hg(II) to that of MeHg
(Fig. 7, p 0.059), indicating that the species-specic MeHg to THg
ratio was possibly controlled by the relative difference between the
efux rate of Hg(II) and MeHg in each species. The ratio of the efux
rate of Hg(II) to that of MeHg in bivalves was found to be 6e9.
A similar difference (3e7 times) has also been observed in sh
(Wang and Wong, 2003; Wang et al., 2010). However, the
predominance of dietary exposure and ingestion of food of a higher
MeHg to THg ratio made the proportion of MeHg in high-trophic-
level shes overwhelm that in bivalves.
3.5. Feeding niche of bivalves and its effects on Hg accumulation
While the species-specic accumulation of Hg was driven by the
detoxication strategies of bivalves, the impacts of ecological
factors cannot be ruled out. Field studies have shown an average
15
Nenrichment of 3.4&per trophic level, and a lower value of 2.5&
was observed for herbivores (Vander Zanden and Rasmussen,
2001). The d
15
N only varied within a small range of 1& between
the ve bivalve species (Table 3), implying a similar trophic level
between the bivalves. There were however signicant differences
in d
13
C signals among the ve species of bivalves (Table 3), indi-
cating that they may rely on different carbon sources. The d
13
C
signals varied largely between clams (17.1&) and black mussels
(19.5&) collected from Clear Water Bay, with an intermediate
value of 18.8& for oysters. Numerous studies have shown that
phytoplankton or SPM are more depleted in d
13
C than sedimentary
organic matter (SOM) or benthic microalgae (France, 1995;
Middelburg and Nieuwenhuize, 1998; Ishihi, 2003), resulting in
pelagic feeders that are more depleted in d
13
C than benthic
consumers (Hobson et al., 1995). For example, Currin et al. (1995)
suggested that an average d
13
C of 14.9& was found for benthic
microalgae and an average of 21.1&for marine phytoplankton. In
our study, the clams appeared to feed on resuspended sediments,
whereas the black mussels relied onphytoplankton or SPMinwater
column. The rock oysters possibly relied on both sediments and
SPM. The different food sources and habitats may lead to different
bioavailability of Hg for clams, oysters and black mussels. It was
found that the THg concentration in the SPM (w100 ng g
1
) was
higher than that in the sediments (w65 ng g
1
), while the AEs of
Hg(II) and MeHg were both lower for sediments than phyto-
plankton. The black mussels may have a higher potential for
accumulating Hg from food than the clams and oysters. As a result,
the concentration of THg in black mussels was much higher than
that in oysters even though the oysters had the lowest elimination
rates of both Hg(II) and MeHg.
4. Conclusions
Our study has clearly demonstrated that bivalves have
substantial capacity to accumulate Hg(II) and MeHg fromwater and
food. All ve bivalve species, scallops, clams, oysters, green mussels
and black mussels, signicantly accumulated Hg from the low Hg
environments. The bivalves showed distinct detoxication strate-
gies for Hg, which was reected in their subcellular partitioning
and biodynamics. Hg(II) distributing in the organelles fraction was
closely related to the species-dependent Hg(II) elimination rate,
suggesting an interaction between the subcellular partitioning and
biokinetics of Hg(II) in bivalves. The biodynamics parameters were
only able to partially explain the different body concentrations of
THg and MeHg in bivalves, and the variations in the MeHg to THg
ratios were largely controlled by the relative difference between
the elimination rates of Hg(II) and MeHg. The interspecic body
concentrations of MeHg and THg were complicated by the differ-
ences in the biodynamics of Hg(II) and MeHg, and the different
feeding niches of bivalves.
Acknowledgements
We thank Prof. Ling-Feng Huang and his student Mr. Xin-Qing
Zheng of Xiamen University for their help in analyzing stable
isotopes in this study. This study was supported by the General
Research Funds from the Hong Kong Research Grants Council
(663009 and 662610).
k
e(Hg(II))
/k
e(MeHg)
5 6 7 8 9 10
M
e
H
g
/
T
H
g
r
a
t
i
o
(
%
)
0
10
20
30
40
50
y=7.84x-32.4
p=0.059
r
2
=0.74
Fig. 7. Relationship between the ratio of MeHg to THg and the ratio of the elimination
rates of Hg(II) and MeHg.
Table 3
The values of d
13
C and d
15
N in ve species of bivalves (mean SD, n 3).
Species Collection site d
13
C (&) d
15
N (&)
Scallop C. nobilis Dapeng Bay 18.7 0.1 9.0 0.1
Green mussel P. viridis Tolo Harbor 20.7 0.0 8.6 0.2
Clam R. philippinarum Clear Water Bay 17.1 0.0 8.3 0.2
Oyster S. cucullata Clear Water Bay 18.8 0.0 8.5 0.1
Black mussel S. virgatus Clear Water Bay 19.5 0.0 8.0 0.2
K. Pan, W.-X. Wang / Environmental Pollution 159 (2011) 2500e2506 2505
References
Canesi, L., Viarengo, A., Leonzio, C., Filippelli, M., Gallo, G., 1999. Heavy metals and
glutathione metabolism in mussel tissues. Aquatic Toxicology 46, 67e76.
Chen, C.Y., Serrell, N., Evers, D.C., Fleishman, B.J., Lambert, K.F., Weiss, J., Mason, R.P.,
Bank, M.S., 2008. Meeting report: methylmercury in marine ecosystems-from
sources to seafood consumers. Environmental Health Perspectives 116,
1706e1712.
Chen, C.Y., Dionne, M., Mayes, B.M., Ward, D.M., Sturup, S., Jackson, B.P., 2009.
Mercury bioavailability and bioaccumulation in estuarine food webs in the gulf
of Maine. Environmental Science and Technology 43, 1804e1810.
Clausen, I., Riisgrd, H.U., 1996. Growth, ltration and respiration in the mussel
Mytilus edulis: no evidence for physiological regulation of the lter-pump to
nutritional needs. Marine Ecology Progress Series 141, 37e45.
Currin, C.A., Newell, S.Y., Paerl, H.W., 1995. The role of standing dead spartina-
alterniora and benthic microalgae in salt-marsh food webs e considerations
based on multiple stable-isotope analysis. Marine Ecology Progress Series 121,
99e116.
Dallinger, R., 1993. Strategies of metal detoxication in terrestrial invertebrates. In:
Dallinger, R., Rainbow, P.S. (Eds.), Ecotoxicology of Metals in Invertebrates.
Lewis Publisher, Boca Raton, USA, pp. 245e289.
Dimitriadis, V.K., Domouhtsidou, G.P., Raftopoulou, E., 2003. Localization of Hg and
Pb in the palps, the digestive gland and the gills in Mytilus galloprovincialis (L.)
using autometallography and X-ray microanalysis. Environmental Pollution
125, 345e353.
Ditri, F.M., 1991. Mercury contamination e what we have learned since Minamata.
Environmental Monitoring and Assessment 19, 165e182.
Domouhtsidou, G.P., Dimitriadis, V.K., 2000. Ultrastructural localization of heavy
metals (Hg, Ag, Pb, and Cu) in gills and digestive gland of mussels, Mytilus
galloprovincialis (L.). Archives of Environmental Contamination and Toxicology
38, 472e478.
Fialkowski, W., Rainbow, P.S., Smith, B.D., Zmudzinski, L., 2003. Seasonal variation in
trace metal concentrations in three talitrid amphipods from the gulf of Gdansk,
Poland. Journal of Experimental Marine Biology and Ecology 288, 81e93.
Fowler, B.A., 1978. General subcellular effects of lead, mercury, cadmium, and
arsenic. Environmental Health Perspectives 22, 37e41.
France, R.L., 1995. C-13 enrichment in benthic compared to planktonic algae e
foodweb implications. Marine Ecology Progress Series 124, 307e312.
Gagnon, C., Fisher, N.S., 1997. Bioavailability of sediment-bound methyl and inor-
ganic mercury to a marine bivalve. Environmental Science and Technology 31,
993e998.
Herman, P.M.J., Middelburg, J.J., Widdows, J., Lucas, C.H., Heip, C.H.R., 2000. Stable
isotopes as trophic tracers: combining eld sampling and manipulative label-
ling of food resources for macrobenthos. Marine Ecology Progress Series 204,
79e92.
Hobson, K.A., Ambrose, W.G., Renaud, P.E., 1995. Sources of primary production,
benthic-pelagic coupling, and trophic relationships within the Northeast water
Polynya: insights from delta C-13 and delta N-15 analysis. Marine Ecology
Progress Series 128, 1e10.
Inza, B., Ribeyre, F., Boudou, A., 1998. Dynamics of cadmium and mercury
compounds (inorganic mercury or methylmercury): uptake and depuration in
Corbicula uminea. Effects of temperature and pH. Aquatic Toxicology 43,
273e285.
Ishihi, Y., 2003. Feeding of the bivalve Theora lubrica on benthic microalgae: isotopic
evidence. Marine Ecology Progress Series 255, 303e309.
Jrgensen, C.B., 1996. Bivalve lter feeding revisited. Marine Ecology Progress Series
142, 287e302.
Langston, W.J., Bebianno, M.J., Burt, G.R., 1998. Metal handling strategies in
molluscs. In: Langston, W.J., Bebianno, M.J. (Eds.), Metal Metabolism in Aquatic
Environments. Chapman & Hall, London, pp. 219e283.
Marigmez, I., Soto, M., Cajaraville, M.P., Angulo, E., Giamberini, L., 2002. Cellular
and subcellular distribution of metals in molluscs. Microscopy Research and
Technique 56, 358e392.
Mason, R.P., Reinfelder, J.R., Morel, F.M.M., 1995. Bioaccumulation of mercury and
methylmercury. Water Air and Soil Pollution 80, 915e921.
Mason, R.P., Reinfelder, J.R., Morel, F.M.M., 1996. Uptake, toxicity, and trophic
transfer of mercury in a coastal diatom. Environmental Science and Technology
30, 1835e1845.
Middelburg, J.J., Nieuwenhuize, J., 1998. Carbon and nitrogen stable isotopes in
suspended matter and sediments from the Schelde estuary. Marine Chemistry
60, 217e225.
Morel, F.M.M., Kraepiel, A.M.L., Amyot, M., 1998. The chemical cycle and bio-
accumulation of mercury. Annual Review of Ecology and Systematics 29,
543e566.
Pan, J.F., Wang, W.-X., 2004. Uptake of Hg(II) and methylmercury by the green
mussel Perna viridis under different organic carbon conditions. Marine Ecology
Progress Series 276, 125e136.
Pan, K., Wang, W.-X., 2008. Validation of biokinetic model of metals in the scallop
Chlamys nobilis in complex eld environments. Environmental Science and
Technology 42, 6285e6290.
Pan, K., Wang, W.-X., 2009a. Biodynamics to explain the difference of copper body
concentrations in ve marine bivalve species. Environmental Science and
Technology 43, 2137e2143.
Pan, K., Wang, W.-X., 2009b. Inter-individual variations in cadmium and zinc
biodynamics in the scallop Chlamys nobilis. Marine Ecology Progress Series 383,
151e160.
Rainbow, P.S., 1995. Biomonitoring of heavy metal availability in the marine envi-
ronment. Marine Pollution Bulletin 31, 183e192.
Rainbow, P.S., 2002. Trace metal concentrations in aquatic invertebrates: why and
so what? Environmental Pollution 120, 497e507.
Ross, S.T., 1986. Resource partitioning in sh assemblages e a review of eld
studies. Copeia, 352e388.
Rouleau, C., Block, M., 1997. Working methods paper e fast and high-yield synthesis of
radioactive (CH
3
Hg)-Hg-203(II). Applied Organometallic Chemistry 11, 751e753.
Tsui, M.T.K., Wang, W.-X., 2004. Temperature inuences on the accumulation and
elimination of mercury in a freshwater cladoceran, Daphnia magna. Aquatic
Toxicology 70, 245e256.
Ullrich, S.M., Tanton, T.W., Abdrashitova, S.A., 2001. Mercury in the aquatic envi-
ronment: a review of factors affecting methylation. Critical Reviews in Envi-
ronmental Science and Technology 31, 241e293.
Vander Zanden, M.J., Rasmussen, J.B., 2001. Variation in delta N-15 and delta C-13
trophic fractionation: implications for aquatic food web studies. Limnology and
Oceanography 46, 2061e2066.
Viarengo, A., Canesi, L., Moore, M.N., Orunesu, M., 1994. Effects of Hg
2
and Cu
2
on
the cytosolic Ca
2
level in molluscan blood-cells evaluated by confocal
microscopy and spectrouorometry. Marine Biology 119, 557e564.
Wallace, W.G., Lee, B.G., Luoma, S.N., 2003. Subcellular compartmentalization of Cd
and Zn in two bivalves. I. Signicance of metal-sensitive fractions (MSF) and
biologically detoxied metal (BDM). Marine Ecology Progress Series 249,
183e197.
Wang, W.-X., Fisher, N.S., 1996. Assimilation of trace elements and carbon by the
mussel Mytilus edulis: effects of food composition. Limnology and Oceanog-
raphy 41, 197e207.
Wang, R., Wang, W.-X., 2010. Importance of speciation in understanding mercury
bioaccumulation in tilapia controlled by salinity and dissolved organic matter.
Environmental Science and Technology 44, 7964e7969.
Wang, W.-X., Wong, R.S.K., 2003. Bioaccumulation kinetics and exposure pathways
of inorganic mercury and methylmercury in a marine sh, the sweetlips Plec-
torhinchus gibbosus. Marine Ecology Progress Series 261, 257e268.
Wang, W.-X., Stupakoff, I., Gagnon, C., Fisher, N.S., 1998. Bioavailability of inorganic
and methylmercury to a marine deposit feeding polychaete. Environmental
Science and Technology 32, 2564e2571.
Wang, R., Wong, M.H., Wang, W.-X., 2010. Mercury exposure in the freshwater
tilapia Oreochromis niloticus. Environmental Pollution 158, 2694e2701.
Ward, D.M., Nislow, K.H., Chen, C.Y., Folt, C.L., 2010. Rapid, efcient growth reduces
mercury concentrations in stream-dwelling Atlantic salmon. Transactions of the
American Fisheries Society 139, 1e10.
Watras, C.J., Bloom, N.S., 1992. Mercury and methylmercury in individual
zooplankton e implications for bioaccumulation. Limnology and Oceanography
37, 1313e1318.
K. Pan, W.-X. Wang / Environmental Pollution 159 (2011) 2500e2506 2506