Abstract Map Ta Phut Industrial Estate is a major
industrial area in Thailand for both petrochemical and
heavy industries. The release of hazardous wastes and other
pollutants from these industries increases the potential for
contamination in foods in the surrounding area, especially
farmed shellfish. This study determined the arsenic (As),
cadmium (Cd), and manganese (Mn) concentrations in the
edible flesh of farmed shellfish, including Perna viridis,
Meretrix meretrix, and Scapharca inaequivalvis, around
the Map Ta Phut area using inductively coupled plasma
mass spectrometry. The results showed that shellfish samples
contained high levels of total As [1.84–6.42 mg kg-1
wet weight (ww)]. High Mn concentrations were found in
P. viridis and M. meretrix, whereas S. inaequivalis contained
the highest Cd. Arsenobetaine (AsB) was found to
be the major As species in shellfish ([45 % of total As).
The in vitro cytotoxicity of these elements was evaluated
using human cancer cells (T47D, A549, and Jurkat cells).
An observed decrease in cell viability in T47D and Jurkat
cells was mainly caused by exposure to inorganic As (iAs)
or Mn but not to AsB or Cd. The combined elements
(AsB?Mn?Cd) at concentrations predicted to result from
the estimated daily intake of shellfish flesh by the local
people showed significant cytotoxicity in T47D and Jurkat
cells.
Map Ta Phut Industrial Estate, located in Rayong province,
eastern Thailand, is a major industrial estate hosting both
petrochemical and heavy industries. It was established in
1989 as part of the government’s policy to develop the
eastern seaboard. It consists of 66 companies that include 32
petrochemical companies, 2 oil refineries, 8 steel companies,
7 chemical and fertilizer manufacturers, and 17 public
utilities including the power, steam, and gas sector (Langkulsen
et al. 2011). The rapid development of this industrial
estate has impacts on environmental and occupational
health, as well as the health of residents in the surrounding
area, through effects on the quality of the local air, soil, and
water sources. During its operation, there have been many
reports indicating that the release of toxic chemicals and
other pollutants has caused increased health risks for people
living in this area (Jadsri et al. 2006; Peluso et al. 2008;
Aungudornpukdee and Vichit-Vadakan 2009; Aungudornpukdee
et al. 2010; Peluso et al. 2010). Toxic pollutants are
released from this industrial estate into the sea and accumulated
in aquatic organisms. This potentially places the
health of people living in Map Ta Phut at risk through the
consumption of contaminated seafood. The Pollution Control
Department of the Thai Ministry of Natural Resources
and Environment collected water samples around Map Ta
Phut industrial estate in 2008 (groundwater n = 40; shallow
water n = 30) and 2009 (groundwater n = 45; shallow
water n = 47), and determined the element concentrations
(Pollution Control Department 2010). The results from
shallow water showed that although the concentrations of
arsenic (As), manganese (Mn), iron (Fe), and lead (Pb)
decreased from 2008 to 2009, Mn and Fe concentrations
still exceeded the standard limit values of groundwater for
consumption set by the Department of Mineral Resources,
Thailand ([0.5 and 1.0 mg L-1, respectively). By contrast,
the cadmium (Cd) concentration in shallow water increased
from not detectable in 2008 to 0.0354 mg L-1 in 2009,
which was greater than the standard limit value
([0.01 mg L-1). The results for groundwater were also
similar to those of shallow water in that the concentrations
of Fe and Mn were greater than the standard limit values. In
addition, the concentrations of Fe and Mn in groundwater
(18.12 and 1.71 mg L-1 in 2009, respectively) were also
greater than those found in shallow water (2.73 and
0.45 mg L-1 in 2009, respectively).
In recent years, farming of shellfish around the bay of
Map Ta Phut industrial estate has increased. Perna viridis,
Meretrix meretrix, and Scapharca inaequivalvis are commercially
grown in the sea at the Ban Takuan area, which
receives wastewater released from the industrial estate.
Therefore, bioaccumulation of some toxic elements, especially
As and Cd, in farmed shellfish from this area is
possible. The results of the present study aimed to determine
the concentrations of some elements, including As,
Cd, and Mn, in the edible flesh of farmed shellfish samples
of P. viridis, M. meretrix, and S. inaequivalvis from the
Ban Takuan area. As and Cd are known as metalloestrogens
(Stoica et al. 2000; Bodwell et al. 2006; Davey et al.
2007), which mimic the biological effects of estradiol in
breast cancer cells, and have also been reported to be
associated with increased risk of cancer development (Huff
et al. 2007; Tokar et al. 2012; Aballay et al. 2012; Julin
et al. 2012). Therefore, an evaluation of the in vitro cytotoxic
effects of selected individual and combined elements
(As, Cd, and Mn) was also performed in different human
cancer cells: human hormone-dependent breast cancer cells
(T47D), human lung cancer cells (A549), and human acute
lymphoblastic leukemic T cells (Jurkat clone E6-1). The
selection of the concentrations of these elements to be
tested was based on the estimated daily intake of contaminated
shellfish.
Materials and Methods
Sample Collection
Shellfish samples, P. viridis (n = 50), M. meretrix
(n = 20), and S. inaequivalvis (n = 20) were purchased
from local shops along the sea (shellfish farming area) at
Ban Takuan during 2009–2011 (July and December 2009,
May 2010, and October 2011). The collected samples were
packed in ice and transported to the laboratory.
Sample Preparation
The soft tissues of shellfish were excised with a stainless
steel scalpel and thoroughly washed with distilled and
deionized water (Ddw) three times and freeze-dried. The
samples were pulverized in a stainless steel blender and
stored at -20 C before microwave-assisted acid digestion.
Suprapur nitric acid (65 %) (Merk, Germany) was used
for digestion of samples, and all glassware and digestion
vessels were soaked in Analar nitric acid (10 %). Highpurity
water (Milli-Q purification system; Millipore, Bedford,
Massachusetts, USA) was used to prepare all solutions
for digestion and for rinsing equipment. Standard reference
material (SRM), DORM2 dogfish muscle certified by the
National Research Council of Canada, was used for method
validation. For inductively coupled plasma-mass spectrometry
(ICP-MS) analysis, standard solutions of As, Cd,
and Mn were obtained from Fisher Chemical (UK).
Microwave-Assisted Acid Digestion
A Mars 5 Microwave Accelerated Reaction System (CEM,
USA) was used for microwave digestion of the samples.
Dried powders (0.2500 g) of shellfish tissues and the SRM
were placed into polytetrafluoroethylene vessels. Six milliliters
of concentrated nitric acid (65 % HNO3) (Suprapur,
Merk, Germany) and 2.0 mL of Ddw were added to the
vessels. A negative control was included that contained only
deionized water and 65 % HNO3. The vessels were sealed,
placed on a rotating on a platform, and subjected to microwave
digestion with agitation using a power setting of
1,200 W, a ramp time of 30 min at 190 C, and a pressure of
180 psi. The holding time was 30 min. Two replicates were
made for each sample. The digested sample volume was then
adjusted to 50 mL with deionized water, and the samples
were stored in polyethylene bottles at 4 C until analysis
Abstract Map Ta Phut Industrial Estate is a major
industrial area in Thailand for both petrochemical and
heavy industries. The release of hazardous wastes and other
pollutants from these industries increases the potential for
contamination in foods in the surrounding area, especially
farmed shellfish. This study determined the arsenic (As),
cadmium (Cd), and manganese (Mn) concentrations in the
edible flesh of farmed shellfish, including Perna viridis,
Meretrix meretrix, and Scapharca inaequivalvis, around
the Map Ta Phut area using inductively coupled plasma
mass spectrometry. The results showed that shellfish samples
contained high levels of total As [1.84–6.42 mg kg-1
wet weight (ww)]. High Mn concentrations were found in
P. viridis and M. meretrix, whereas S. inaequivalis contained
the highest Cd. Arsenobetaine (AsB) was found to
be the major As species in shellfish ([45 % of total As).
The in vitro cytotoxicity of these elements was evaluated
using human cancer cells (T47D, A549, and Jurkat cells).
An observed decrease in cell viability in T47D and Jurkat
cells was mainly caused by exposure to inorganic As (iAs)
or Mn but not to AsB or Cd. The combined elements
(AsB?Mn?Cd) at concentrations predicted to result from
the estimated daily intake of shellfish flesh by the local
people showed significant cytotoxicity in T47D and Jurkat
cells.
Map Ta Phut Industrial Estate, located in Rayong province,
eastern Thailand, is a major industrial estate hosting both
petrochemical and heavy industries. It was established in
1989 as part of the government’s policy to develop the
eastern seaboard. It consists of 66 companies that include 32
petrochemical companies, 2 oil refineries, 8 steel companies,
7 chemical and fertilizer manufacturers, and 17 public
utilities including the power, steam, and gas sector (Langkulsen
et al. 2011). The rapid development of this industrial
estate has impacts on environmental and occupational
health, as well as the health of residents in the surrounding
area, through effects on the quality of the local air, soil, and
water sources. During its operation, there have been many
reports indicating that the release of toxic chemicals and
other pollutants has caused increased health risks for people
living in this area (Jadsri et al. 2006; Peluso et al. 2008;
Aungudornpukdee and Vichit-Vadakan 2009; Aungudornpukdee
et al. 2010; Peluso et al. 2010). Toxic pollutants are
released from this industrial estate into the sea and accumulated
in aquatic organisms. This potentially places the
health of people living in Map Ta Phut at risk through the
consumption of contaminated seafood. The Pollution Control
Department of the Thai Ministry of Natural Resources
and Environment collected water samples around Map Ta
Phut industrial estate in 2008 (groundwater n = 40; shallow
water n = 30) and 2009 (groundwater n = 45; shallow
water n = 47), and determined the element concentrations
(Pollution Control Department 2010). The results from
shallow water showed that although the concentrations of
arsenic (As), manganese (Mn), iron (Fe), and lead (Pb)
decreased from 2008 to 2009, Mn and Fe concentrations
still exceeded the standard limit values of groundwater for
consumption set by the Department of Mineral Resources,
Thailand ([0.5 and 1.0 mg L-1, respectively). By contrast,
the cadmium (Cd) concentration in shallow water increased
from not detectable in 2008 to 0.0354 mg L-1 in 2009,
which was greater than the standard limit value
([0.01 mg L-1). The results for groundwater were also
similar to those of shallow water in that the concentrations
of Fe and Mn were greater than the standard limit values. In
addition, the concentrations of Fe and Mn in groundwater
(18.12 and 1.71 mg L-1 in 2009, respectively) were also
greater than those found in shallow water (2.73 and
0.45 mg L-1 in 2009, respectively).
In recent years, farming of shellfish around the bay of
Map Ta Phut industrial estate has increased. Perna viridis,
Meretrix meretrix, and Scapharca inaequivalvis are commercially
grown in the sea at the Ban Takuan area, which
receives wastewater released from the industrial estate.
Therefore, bioaccumulation of some toxic elements, especially
As and Cd, in farmed shellfish from this area is
possible. The results of the present study aimed to determine
the concentrations of some elements, including As,
Cd, and Mn, in the edible flesh of farmed shellfish samples
of P. viridis, M. meretrix, and S. inaequivalvis from the
Ban Takuan area. As and Cd are known as metalloestrogens
(Stoica et al. 2000; Bodwell et al. 2006; Davey et al.
2007), which mimic the biological effects of estradiol in
breast cancer cells, and have also been reported to be
associated with increased risk of cancer development (Huff
et al. 2007; Tokar et al. 2012; Aballay et al. 2012; Julin
et al. 2012). Therefore, an evaluation of the in vitro cytotoxic
effects of selected individual and combined elements
(As, Cd, and Mn) was also performed in different human
cancer cells: human hormone-dependent breast cancer cells
(T47D), human lung cancer cells (A549), and human acute
lymphoblastic leukemic T cells (Jurkat clone E6-1). The
selection of the concentrations of these elements to be
tested was based on the estimated daily intake of contaminated
shellfish.
Materials and Methods
Sample Collection
Shellfish samples, P. viridis (n = 50), M. meretrix
(n = 20), and S. inaequivalvis (n = 20) were purchased
from local shops along the sea (shellfish farming area) at
Ban Takuan during 2009–2011 (July and December 2009,
May 2010, and October 2011). The collected samples were
packed in ice and transported to the laboratory.
Sample Preparation
The soft tissues of shellfish were excised with a stainless
steel scalpel and thoroughly washed with distilled and
deionized water (Ddw) three times and freeze-dried. The
samples were pulverized in a stainless steel blender and
stored at -20 C before microwave-assisted acid digestion.
Suprapur nitric acid (65 %) (Merk, Germany) was used
for digestion of samples, and all glassware and digestion
vessels were soaked in Analar nitric acid (10 %). Highpurity
water (Milli-Q purification system; Millipore, Bedford,
Massachusetts, USA) was used to prepare all solutions
for digestion and for rinsing equipment. Standard reference
material (SRM), DORM2 dogfish muscle certified by the
National Research Council of Canada, was used for method
validation. For inductively coupled plasma-mass spectrometry
(ICP-MS) analysis, standard solutions of As, Cd,
and Mn were obtained from Fisher Chemical (UK).
Microwave-Assisted Acid Digestion
A Mars 5 Microwave Accelerated Reaction System (CEM,
USA) was used for microwave digestion of the samples.
Dried powders (0.2500 g) of shellfish tissues and the SRM
were placed into polytetrafluoroethylene vessels. Six milliliters
of concentrated nitric acid (65 % HNO3) (Suprapur,
Merk, Germany) and 2.0 mL of Ddw were added to the
vessels. A negative control was included that contained only
deionized water and 65 % HNO3. The vessels were sealed,
placed on a rotating on a platform, and subjected to microwave
digestion with agitation using a power setting of
1,200 W, a ramp time of 30 min at 190 C, and a pressure of
180 psi. The holding time was 30 min. Two replicates were
made for each sample. The digested sample volume was then
adjusted to 50 mL with deionized water, and the samples
were stored in polyethylene bottles at 4 C until analysis
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