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Placental transfer of arsenic to fetus of Dall’s porpoises (Phocoenoides dalli)
Marine Pollution Bulletin 51 (2005) 845–849 www.elsevier.com/locate/marpolbul
Placental transfer of arsenic to fetus of DallÕs porpoises (Phocoenoides dalli) Reiji Kubota
a,1
, Takashi Kunito b, Junko Fujihara a,2, Shinsuke Tanabe Jian Yang c, Nobuyuki Miyazaki c
a,*
,
a Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan Department of Environmental Sciences, Faculty of Science, Shinshu University, Asahi 3-1-1, Matsumoto, Nagano 390-8621, Japan Center for International Cooperation, Ocean Research Institute, The University of Tokyo, Minamidai 1-15-1, Nakano-ku, Tokyo 164-8639, Japan b
c
Abstract Concentrations of total arsenic and individual arsenic compounds were determined in liver, muscle, kidney and blubber of mother and fetus of DallÕs porpoises collected from off Sanriku, Japan, in the year 2000 to characterize the placental transfer of arsenic to fetus in cetaceans. Arsenic was detected in all the tissues of DallÕs porpoises. Total arsenic concentrations in liver, kidney, muscle and blubber were 0.76, 0.69, 0.35 and 0.55 lg/g wet wt, respectively, for mother and 0.28, 0.23, 0.26 and 0.07 lg/g wet wt, respectively, for fetus. In all the tissues, concentrations of total arsenic in mother DallÕs porpoise were higher than in fetus. Arsenic speciation revealed that arsenobetaine was the major arsenic compound in liver, kidney and muscle of both mother and fetus. The percentage of arsenobetaine to total arsenic ranged from 76.0 to 91.0% in the tissues. Dimethylarsinic acid, arsenocholine, methylarsonic acid and an unidentified arsenic compound were also detected in tissues of both mother and fetus as minor constituents, whereas tetramethylarsonium ion was not detected in tissues of the fetus. These results suggest that arsenobetaine, dimethylarsinic acid, arsenocholine and methylarsonic acid are transferable from mother to fetus in DallÕs porpoises. To our knowledge, this is the first report on placental transfer of arsenic compounds to fetus in marine mammals. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Arsenic; Arsenobetaine; Cetacean; Marine mammal; Placental transfer
1. Introduction The behavior and toxicity of arsenic depend on the chemical forms in the environment and in the organisms (Cullen and Reimer, 1989; Goering et al., 1999; Goyer, 1991; Styblo et al., 2000). Therefore, chemical speciation of arsenic is a key factor to understand the metabolism
*
Corresponding author. Tel./fax: +81 89 927 8171. E-mail address: [email protected] (S. Tanabe). 1 Present address: Division of Environmental Chemistry, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan. 2 Present address: Department of Legal Medicine, Shimane University School of Medicine, Enya 89-1, Izumo, Shimane 693-8501, Japan. 0025-326X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2005.01.011
and toxic effects of arsenic. Until now, various investigations have been conducted on arsenic accumulation in lower trophic marine animals, such as fish, crustacean, cephalopod, shellfish and marine algae (Cullen and Reimer, 1989). In contrast, information about arsenic accumulation in higher trophic marine animals is still limited. Our previous study reported that arsenobetaine and dimethylarsinic acid are transferable to eggs of black-tailed gull (Kubota et al., 2002a). However, as far as we know, there is no information on the placental transfer of arsenic compounds to fetus in marine mammals. In the present study, concentrations of total arsenic and individual arsenic compounds were determined in several tissues of mother and fetus DallÕs porpoises
846
R. Kubota et al. / Marine Pollution Bulletin 51 (2005) 845–849
(Phocoenoides dalli) to characterize placental transfer of arsenic compounds to fetus.
Chemical speciation of arsenic was conduced following the method described previously (Kubota et al., 2003a). Arsenic compounds were extracted from freeze-dried tissues with a mixture of methanol/MilliQ water (9:1 v/v). Arsenic compounds were identified and quantified with a high performance liquid chromatograph (HPLC; Shimadzu, LC10A Series) combined with inductively coupled plasmamass spectrometer (ICP-MS; HewlettPackard HP4500) as an arsenic-specific detector. Supelcosil LC-SCX cation-exchange column (pyridine buffer) and Hamilton PRP-X100 anion-exchange column (NH4H2PO4 buffer) were used for separation of different arsenic compounds. Methylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AB), arsenocholine (AC), trimethylarsine oxide (TMAO), tetramethylarsonium iodide (TeMA), arsenite (As(III)) and arsenate (As(V)) were used as standard substances. The ion intensities at m/z 75 (75As), 77 (40Ar37Cl, 77Se) and 87 (87Rb: internal standard) were monitored. In this study, the concentration of arsenic compounds are expressed as lg As/g wet weight.
2. Materials and methods 2.1. Samples Mother and fetus DallÕs porpoises used in the present study (Table 1) were caught for commercial purpose from off Sanriku, Japan, in 2000 under appropriate permit. Liver, kidney, muscle and blubber were dissected out and each tissue was weighed. Some portions of these tissues were provided by fishermen and were stored in deep-freezer at 20 °C before being freeze-dried. The liver, kidney and muscle were freeze-dried as soon as possible after collecting. 2.2. Chemical analyses Total arsenic was determined as described previously (Kubota et al., 2001). Wet tissue (blubber) and freezedried tissues (liver, kidney and muscle) were digested by heating with mixture of acid (HNO3:HClO4: H2SO4 = 1:2:1) to over 300 °C. Total arsenic concentrations in tissues were determined using a hydride generator (Shimadzu HVG-1) coupled to an atomic absorption spectrometer (Shimadzu AA680). In this study, total arsenic concentration was expressed on a wet weight basis (lg As/g wet wt). Moisture content of tissues ranged from 63.7 to 77.0% (Table 2).
3. Results and discussion 3.1. Arsenic accumulation in tissues Concentrations of total arsenic and several arsenic compounds in tissues of mother and fetus DallÕs porpoises are shown in Table 2. Arsenic was detected in all the tissues of mother and fetus, ranging from 0.07
Table 1 Biometric data of DallÕs porpoises
Mother Fetus a
Location
Date
Age
Sex
Type
Body length (cm)
Body weight (kg)
Off Sanriku, Japan Off Sanriku, Japan
January 28, 2000 January 28, 2000
6 years 6 monthsa
Female Female
Truei-type Truei-type
181 55
103 2.6
After conception.
Table 2 Concentrations of arsenic compounds in tissues of DallÕs porpoises Tissues
n
Moisture content (%)
Concentration (lg As/g wet wt) MMA
DMA
AB
AC
TeMA
Total
Mother
Liver Kidney Muscle Blubber
1 1 1 1
63.7 66.7 66.0 N.A.
0.01 <0.01 <0.01 N.A.
0.04 0.04 0.02 N.A.
0.58 0.56 0.32 N.A.
0.08 0.06 <0.01 N.A.
0.04 0.02 0.01 N.A.
0.76 0.69 0.35 0.55
Fetus
Liver Kidney Muscle Blubber
1 1 1 1
70.3 77.0 74.9 N.A.
<0.01 <0.01 <0.01 N.A.
0.01 0.01 0.02 N.A.
0.25 0.21 0.23 N.A.
0.01 0.01 <0.01 N.A.
N.D. N.D. N.D. N.A.
0.28 0.23 0.26 0.07
MMA: methylarsonic acid; DMA: dimethylarsinic acid; AB: arsenobetaine; AC: arsenocholine; TeMA: tetramethylarsonium ion; N.A.: not analyzed.; N.D.: not detected.
R. Kubota et al. / Marine Pollution Bulletin 51 (2005) 845–849
847
Fig. 1. Arsenic distribution in tissues of mother and fetus of DallÕs porpoises.
3.2. Chemical speciation of arsenic Concentrations of arsenic compounds in liver, kidney and muscle of mother and fetus DallÕs porpoises are shown in Table 2. Arsenobetaine and dimethylarsinic acid were observed in all the three tissues of both mother and fetus (Table 2). In addition, methylarsonic acid and arsenocholine were also detected in the three tissues of mother and fetus, but the levels were below the limit
16000
Mother AB
12000 DMA
8000 4000
Ion intensity
(blubber of fetus) to 0.76 (liver of mother) lg/g wet wt. The highest arsenic concentration was observed in the liver, followed by kidney, blubber and muscle in mother. In contrast, the concentration sequence of the fetus was liver > kidney > muscle > blubber. Interestingly, total arsenic concentration in blubber of mother was relatively high, which was consistent with the results of ringed seals and pilot whales (Ebisuda et al., 2002; Julshamn et al., 1987), whereas the level in fetus was extremely low (Table 2). The difference between mother and fetus might be due to the chemical characteristics of arsenic compounds in blubber which will be discussed later in this paper. Distribution of arsenic burden among the four tissues of mother and fetus DallÕs porpoises was calculated using the total arsenic concentration and the weight of each tissue (Fig. 1). In the mother, arsenic burden was highest in blubber (59.6%), followed by muscle (33.5%), kidney (3.7%) and liver (2.6%). In contrast, arsenic burden of muscle (59.0%) was highest in fetus, followed by blubber (25.2%), liver (12.8%) and kidney (3.1%). The low content of arsenic in blubber of fetus, compared to that of mother, is due to its low arsenic concentration as mentioned above (Table 2).
MMA
AC TeMA
Unknown
As(V)?
0 0
200
400
600
800
1000
1200
1400
6000 AB
Fetus
5000 4000 3000
DMA MMA As(V)?
2000
Unknown
1000
AC
0 0
200
400
600
800
1000
1200
1400
Retention time (seconds) Fig. 2. HPLC/ICP-MS chromatograms (column, Supelcosil LC-SCX) of arsenic compounds in liver of mother and fetus DallÕs porpoises.
of detection in some tissues (Table 2 and Fig. 2). In contrast, tetramethylarsonium ion was detected only in the tissues of mother (Table 2). An unidentified arsenic compound was observed in all the tissues of both mother and fetus (Fig. 2). An unidentified compound with the similar retention time was also observed in tissues of short-finned pilot whale, harp seal, ringed seal, loggerhead turtle, green turtle and black-tailed gull (Kubota et al., 2002a,b, 2003a; Ebisuda et al., 2002). This compound may be trimethyl(2-carboxyethyl)arsonium ion
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R. Kubota et al. / Marine Pollution Bulletin 51 (2005) 845–849
Fig. 3. Composition of arsenic compounds in tissues of mother and fetus DallÕs porpoises.
because of the similarity in the retention time of the unknown compound in standard reference material DORM2 of National Research Council Canada (probably trimethyl(2-carboxyethyl)arsonium ion; Francesconi et al., 2000) and the unknown compound in the present study. Composition of arsenic compounds in liver, kidney and muscle of mother and fetus DallÕs porpoises is shown in Fig. 3. The composition of arsenic compounds was similar between mother and fetus. Arsenobetaine was the dominant arsenic compound in all the tissues of mother and fetus, and the percentage of arsenobetaine to total arsenic ranged from 76.0% (liver of mother) to 91.0% (muscle of mother). In contrast, percentages of dimethylarsinic acid, arsenocholine, methylarsonic acid and tetramethylarsonium ion were low, ranging from 1.8% (methylarsonic acid in liver of mother) to 11.2% (methylarsonic acid in liver of mother). The predominance of arsenobetaine in tissues of DallÕs porpoise was consistent with the results of other higher trophic marine animals, such as marine mammals, seabirds and sea turtles (e.g., Fujihara et al., 2003, 2004;Goessler et al., 1998;Kubota et al., 2003b). 3.3. Placental transfer of arsenic to fetus Total arsenic concentrations in tissues of fetus were lower than those of mother (Table 2). Yang et al. (2004) reported that concentrations of toxic elements, such as Hg, Cd and Ag, in the liver of fetus were apparently lower than those of mother DallÕs porpoises. Mea-
dor et al. (1993) also found that concentrations of As, Cd, Hg and Se in liver and kidney of fetus were lower than those of mother of long-finned pilot whales. Hence, placental transfer of toxic elements, including arsenic, seems to be limited to some extent by some physiological system such as placental barrier in marine mammals. In the tissues of fetus, arsenobetaine, dimethylarsinic acid, arsenocholine and methylarsonic acid were detected, although only arsenobetaine, dimethylarsinic acid and arsenocholine could be quantified (Table 2). These results indicate that arsenobetaine, dimethylarsinic acid, arsenocholine and methylarsonic acid are transferable to fetus in DallÕs porpoises. Our previous study showed that arsenobetaine and dimethylarsinic acid can be transferred from mother bird to eggs of the seabird (black-tailed gull, Larus crassirostris) (Kubota et al., 2002a). Percentages of arsenobetaine and dimethylarsinic acid were similar between mother and fetus (or eggs) for both DallÕs porpoises (Fig. 3) and black-tailed gulls (Kubota et al., 2002a). Hence, arsenobetaine and dimethylarsinic acid might be generally transferable to fetus and egg of wildlife, and the composition of arsenic compounds in fetus and eggs reflect those of mother. In the mother DallÕs porpoise, arsenic burden of fetus was only 1.8% because of its low mass (Fig. 1) and low arsenic concentration (Table 2). The arsenic burden in fetus (1.8%) was lower than that of body weight (0.6%), suggesting that transfer of arsenic to fetus is limited to some extent. Interestingly, arsenic burden of blubber was extremely different between mother and fetus as mentioned above (Fig. 1). We assume that this
R. Kubota et al. / Marine Pollution Bulletin 51 (2005) 845–849
difference might be due to the low placental transferability of lipid-soluble arsenic compounds present in the blubber of mother DallÕs porpoises. Recently, Ebisuda et al. (2002, 2003) reported that distribution of lipidsoluble and water-soluble arsenic compounds in several tissues (blubber, liver, kidney, muscle and gonad) of ringed seals (Pusa hispida): high percentage of dimethylarsinic acid-containing lipid-soluble arsenic compounds were observed in blubber (about 90% of total arsenic), whereas the lipid-soluble arsenic compounds were minor constituents in liver, kidney, muscle and gonad. According to Tanabe et al. (1982), hydrophobic highchlorinated chemicals are less transferable from mother to fetus of striped dolphins (Stenella coeruleoalba). Collectively, lipid-soluble arsenic compounds are less transferable than water-soluble arsenic compounds such as arsenobetaine, dimethylarsinic acid and arsenocholine, leading to the low concentration and low burden of arsenic in blubber of fetus DallÕs porpoises. Additional characterization of these lipid-soluble arsenic compounds would help in understanding the placental transfer and accumulation of arsenic in fetus of marine mammals.
Acknowledgments This study was supported by Grants-in-Aid for Scientific Research (A) (no. 12308030) from Japan Society for the Promotion of Science and for Scientific Research on Priority Areas (A) (no. 13027101) and ‘‘21st Century COE Program’’ from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
References Cullen, W.R., Reimer, K.J., 1989. Arsenic speciation in the environment. Chemical Reviews 89, 713–764. Ebisuda, K., Kunito, T., Kubota, R., Tanabe, S., 2002. Arsenic concentrations and speciation in the tissues of ringed seals (Phoca hispida) from Pangnirtung, Canada. Applied Organometallic Chemistry 16, 451–457. Ebisuda, K., Kunito, T., Fujihara, J., Kubota, R., Shibata, Y., Tanabe, S., 2003. Lipid-soluble and water-soluble arsenic compounds in blubber of ringed seal (Pusa hispida). Talanta 61, 779– 787. Francesconi, K.A., Khokiattiwong, S., Goessler, W., Pedersen, S.N., Pavkov, M., 2000. A new arsenobetaine from marine organisms identified by liquid chromatography–mass spectrometry. Chemical Communications 12, 1083–1084.
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Fujihara, J., Kunito, T., Kubota, R., Tanabe, S., 2003. Arsenic accumulation in livers of pinnipeds, seabirds, and sea turtles: Subcellular distribution and interaction between arsenobetaine and glycine betaine. Comparative Biochemistry and Physiology Part C 136, 287–296. Fujihara, J., Kunito, T., Kubota, R., Tanaka, H., Tanabe, S., 2004. Arsenic accumulation and distribution in tissues of black-footed albatrosses. Marine Pollution Bulletin 48, 1153–1160. Goering, P.L., Aposhian, H.V., Mass, M.J., Cebria´n, M., Beck, B.D., Waalkes, M.P., 1999. The enigma of arsenic carcinogenesis: Role of metabolism. Toxicological Sciences 49, 5–14. Goessler, W., Rudorfer, A., Mackey, E.A., Becker, P.R., Irgolic, K.J., 1998. Determination of arsenic compounds in marine mammals with high-performance liquid chromatography and an inductively coupled plasma mass spectrometer as element-specific detector. Applied Organometallic Chemistry 12, 91–501. Goyer, R.A., 1991. Toxic effects of metals. In: Amdur, M.O., Doull, J., Klaassen, C.D. (Eds.), Casarett and DoullÕs Toxicology: The Basic Science of Poisons. Pergamon, New York, pp. 623–680. Julshamn, K., Andersen, A., Ringdal, O., Mørkøre, J., 1987. Trace elements intake in the Faroe Islands I. Element levels in edible parts of pilot whales (Globicephalus meleanus). The Science of the Total Environment 65, 53–62. Kubota, R., Kunito, T., Tanabe, S., 2001. Arsenic accumulation in the liver tissue of marine mammals. Environmental Pollution 115, 303–312. Kubota, R., Kunito, T., Tanabe, S., Ogi, H., Shibata, Y., 2002a. Maternal transfer of arsenic to eggs of blacktailed gull (Larus crassirostris) from Rishiri Island, Japan. Applied Organometallic Chemistry 16, 463–468. Kubota, R., Kunito, T., Tanabe, S., 2002b. Chemical speciation of arsenic in the livers of higher trophic marine animals. Marine Pollution Bulletin 45, 218–223. Kubota, R., Kunito, T., Tanabe, S., 2003a. Occurrence of several arsenic compounds in the liver of birds, cetaceans, pinnipeds, and sea turtles. Environmental Toxicology and Chemistry 22, 1200– 1207. Kubota, R., Kunito, T., Tanabe, S., 2003b. Is arsenobetaine the major arsenic compound in the liver of birds, marine mammals, and sea turtles?. Journal de Physique IV 107, 707–710. Meador, J.P, Varanasi, U., Robish, P.A., Chan, S., 1993. Toxic metals in pilot whale (Globicephala melaena) from strandings in 1986 and 1990 on Cape Cod, Massachusetts. Canadian Journal of Fisheries and Aquatic Sciences 50, 2698–2706. Styblo, M., Del Razo, L.M., Vega, L., Germolec, D.R., LeCluyse, E.L., Hamilton, G.A., Reed, W., Wang, C., Cullen, W.R., Thomas, D.J., 2000. Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Archives of Toxicology 74, 289–299. Tanabe, S., Tatsukawa, R., Maruyama, K., Miyazaki, N., 1982. Transplacental transfer of PCBs and chlorinated hydrocarbon pesticides from the pregnant striped dolphin (Stenella coeruleoalba) to her fetus. Agricultural and Biological Chemistry 46, 1249–1254. Yang, J., Kunito, T., Anan, Y., Tanabe, S., Miyazaki, N., 2004. Total and subcellular distribution of trace elements in the liver of a mother-fetus pair of DallÕs porpoises (Phocoenoides dalli). Marine Pollution Bulletin 48, 1122–1129.
Placental transfer of arsenic to fetus of DallÕs porpoises (Phocoenoides dalli) Reiji Kubota
a,1
, Takashi Kunito b, Junko Fujihara a,2, Shinsuke Tanabe Jian Yang c, Nobuyuki Miyazaki c
a,*
,
a Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan Department of Environmental Sciences, Faculty of Science, Shinshu University, Asahi 3-1-1, Matsumoto, Nagano 390-8621, Japan Center for International Cooperation, Ocean Research Institute, The University of Tokyo, Minamidai 1-15-1, Nakano-ku, Tokyo 164-8639, Japan b
c
Abstract Concentrations of total arsenic and individual arsenic compounds were determined in liver, muscle, kidney and blubber of mother and fetus of DallÕs porpoises collected from off Sanriku, Japan, in the year 2000 to characterize the placental transfer of arsenic to fetus in cetaceans. Arsenic was detected in all the tissues of DallÕs porpoises. Total arsenic concentrations in liver, kidney, muscle and blubber were 0.76, 0.69, 0.35 and 0.55 lg/g wet wt, respectively, for mother and 0.28, 0.23, 0.26 and 0.07 lg/g wet wt, respectively, for fetus. In all the tissues, concentrations of total arsenic in mother DallÕs porpoise were higher than in fetus. Arsenic speciation revealed that arsenobetaine was the major arsenic compound in liver, kidney and muscle of both mother and fetus. The percentage of arsenobetaine to total arsenic ranged from 76.0 to 91.0% in the tissues. Dimethylarsinic acid, arsenocholine, methylarsonic acid and an unidentified arsenic compound were also detected in tissues of both mother and fetus as minor constituents, whereas tetramethylarsonium ion was not detected in tissues of the fetus. These results suggest that arsenobetaine, dimethylarsinic acid, arsenocholine and methylarsonic acid are transferable from mother to fetus in DallÕs porpoises. To our knowledge, this is the first report on placental transfer of arsenic compounds to fetus in marine mammals. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Arsenic; Arsenobetaine; Cetacean; Marine mammal; Placental transfer
1. Introduction The behavior and toxicity of arsenic depend on the chemical forms in the environment and in the organisms (Cullen and Reimer, 1989; Goering et al., 1999; Goyer, 1991; Styblo et al., 2000). Therefore, chemical speciation of arsenic is a key factor to understand the metabolism
*
Corresponding author. Tel./fax: +81 89 927 8171. E-mail address: [email protected] (S. Tanabe). 1 Present address: Division of Environmental Chemistry, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan. 2 Present address: Department of Legal Medicine, Shimane University School of Medicine, Enya 89-1, Izumo, Shimane 693-8501, Japan. 0025-326X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2005.01.011
and toxic effects of arsenic. Until now, various investigations have been conducted on arsenic accumulation in lower trophic marine animals, such as fish, crustacean, cephalopod, shellfish and marine algae (Cullen and Reimer, 1989). In contrast, information about arsenic accumulation in higher trophic marine animals is still limited. Our previous study reported that arsenobetaine and dimethylarsinic acid are transferable to eggs of black-tailed gull (Kubota et al., 2002a). However, as far as we know, there is no information on the placental transfer of arsenic compounds to fetus in marine mammals. In the present study, concentrations of total arsenic and individual arsenic compounds were determined in several tissues of mother and fetus DallÕs porpoises
846
R. Kubota et al. / Marine Pollution Bulletin 51 (2005) 845–849
(Phocoenoides dalli) to characterize placental transfer of arsenic compounds to fetus.
Chemical speciation of arsenic was conduced following the method described previously (Kubota et al., 2003a). Arsenic compounds were extracted from freeze-dried tissues with a mixture of methanol/MilliQ water (9:1 v/v). Arsenic compounds were identified and quantified with a high performance liquid chromatograph (HPLC; Shimadzu, LC10A Series) combined with inductively coupled plasmamass spectrometer (ICP-MS; HewlettPackard HP4500) as an arsenic-specific detector. Supelcosil LC-SCX cation-exchange column (pyridine buffer) and Hamilton PRP-X100 anion-exchange column (NH4H2PO4 buffer) were used for separation of different arsenic compounds. Methylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AB), arsenocholine (AC), trimethylarsine oxide (TMAO), tetramethylarsonium iodide (TeMA), arsenite (As(III)) and arsenate (As(V)) were used as standard substances. The ion intensities at m/z 75 (75As), 77 (40Ar37Cl, 77Se) and 87 (87Rb: internal standard) were monitored. In this study, the concentration of arsenic compounds are expressed as lg As/g wet weight.
2. Materials and methods 2.1. Samples Mother and fetus DallÕs porpoises used in the present study (Table 1) were caught for commercial purpose from off Sanriku, Japan, in 2000 under appropriate permit. Liver, kidney, muscle and blubber were dissected out and each tissue was weighed. Some portions of these tissues were provided by fishermen and were stored in deep-freezer at 20 °C before being freeze-dried. The liver, kidney and muscle were freeze-dried as soon as possible after collecting. 2.2. Chemical analyses Total arsenic was determined as described previously (Kubota et al., 2001). Wet tissue (blubber) and freezedried tissues (liver, kidney and muscle) were digested by heating with mixture of acid (HNO3:HClO4: H2SO4 = 1:2:1) to over 300 °C. Total arsenic concentrations in tissues were determined using a hydride generator (Shimadzu HVG-1) coupled to an atomic absorption spectrometer (Shimadzu AA680). In this study, total arsenic concentration was expressed on a wet weight basis (lg As/g wet wt). Moisture content of tissues ranged from 63.7 to 77.0% (Table 2).
3. Results and discussion 3.1. Arsenic accumulation in tissues Concentrations of total arsenic and several arsenic compounds in tissues of mother and fetus DallÕs porpoises are shown in Table 2. Arsenic was detected in all the tissues of mother and fetus, ranging from 0.07
Table 1 Biometric data of DallÕs porpoises
Mother Fetus a
Location
Date
Age
Sex
Type
Body length (cm)
Body weight (kg)
Off Sanriku, Japan Off Sanriku, Japan
January 28, 2000 January 28, 2000
6 years 6 monthsa
Female Female
Truei-type Truei-type
181 55
103 2.6
After conception.
Table 2 Concentrations of arsenic compounds in tissues of DallÕs porpoises Tissues
n
Moisture content (%)
Concentration (lg As/g wet wt) MMA
DMA
AB
AC
TeMA
Total
Mother
Liver Kidney Muscle Blubber
1 1 1 1
63.7 66.7 66.0 N.A.
0.01 <0.01 <0.01 N.A.
0.04 0.04 0.02 N.A.
0.58 0.56 0.32 N.A.
0.08 0.06 <0.01 N.A.
0.04 0.02 0.01 N.A.
0.76 0.69 0.35 0.55
Fetus
Liver Kidney Muscle Blubber
1 1 1 1
70.3 77.0 74.9 N.A.
<0.01 <0.01 <0.01 N.A.
0.01 0.01 0.02 N.A.
0.25 0.21 0.23 N.A.
0.01 0.01 <0.01 N.A.
N.D. N.D. N.D. N.A.
0.28 0.23 0.26 0.07
MMA: methylarsonic acid; DMA: dimethylarsinic acid; AB: arsenobetaine; AC: arsenocholine; TeMA: tetramethylarsonium ion; N.A.: not analyzed.; N.D.: not detected.
R. Kubota et al. / Marine Pollution Bulletin 51 (2005) 845–849
847
Fig. 1. Arsenic distribution in tissues of mother and fetus of DallÕs porpoises.
3.2. Chemical speciation of arsenic Concentrations of arsenic compounds in liver, kidney and muscle of mother and fetus DallÕs porpoises are shown in Table 2. Arsenobetaine and dimethylarsinic acid were observed in all the three tissues of both mother and fetus (Table 2). In addition, methylarsonic acid and arsenocholine were also detected in the three tissues of mother and fetus, but the levels were below the limit
16000
Mother AB
12000 DMA
8000 4000
Ion intensity
(blubber of fetus) to 0.76 (liver of mother) lg/g wet wt. The highest arsenic concentration was observed in the liver, followed by kidney, blubber and muscle in mother. In contrast, the concentration sequence of the fetus was liver > kidney > muscle > blubber. Interestingly, total arsenic concentration in blubber of mother was relatively high, which was consistent with the results of ringed seals and pilot whales (Ebisuda et al., 2002; Julshamn et al., 1987), whereas the level in fetus was extremely low (Table 2). The difference between mother and fetus might be due to the chemical characteristics of arsenic compounds in blubber which will be discussed later in this paper. Distribution of arsenic burden among the four tissues of mother and fetus DallÕs porpoises was calculated using the total arsenic concentration and the weight of each tissue (Fig. 1). In the mother, arsenic burden was highest in blubber (59.6%), followed by muscle (33.5%), kidney (3.7%) and liver (2.6%). In contrast, arsenic burden of muscle (59.0%) was highest in fetus, followed by blubber (25.2%), liver (12.8%) and kidney (3.1%). The low content of arsenic in blubber of fetus, compared to that of mother, is due to its low arsenic concentration as mentioned above (Table 2).
MMA
AC TeMA
Unknown
As(V)?
0 0
200
400
600
800
1000
1200
1400
6000 AB
Fetus
5000 4000 3000
DMA MMA As(V)?
2000
Unknown
1000
AC
0 0
200
400
600
800
1000
1200
1400
Retention time (seconds) Fig. 2. HPLC/ICP-MS chromatograms (column, Supelcosil LC-SCX) of arsenic compounds in liver of mother and fetus DallÕs porpoises.
of detection in some tissues (Table 2 and Fig. 2). In contrast, tetramethylarsonium ion was detected only in the tissues of mother (Table 2). An unidentified arsenic compound was observed in all the tissues of both mother and fetus (Fig. 2). An unidentified compound with the similar retention time was also observed in tissues of short-finned pilot whale, harp seal, ringed seal, loggerhead turtle, green turtle and black-tailed gull (Kubota et al., 2002a,b, 2003a; Ebisuda et al., 2002). This compound may be trimethyl(2-carboxyethyl)arsonium ion
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Fig. 3. Composition of arsenic compounds in tissues of mother and fetus DallÕs porpoises.
because of the similarity in the retention time of the unknown compound in standard reference material DORM2 of National Research Council Canada (probably trimethyl(2-carboxyethyl)arsonium ion; Francesconi et al., 2000) and the unknown compound in the present study. Composition of arsenic compounds in liver, kidney and muscle of mother and fetus DallÕs porpoises is shown in Fig. 3. The composition of arsenic compounds was similar between mother and fetus. Arsenobetaine was the dominant arsenic compound in all the tissues of mother and fetus, and the percentage of arsenobetaine to total arsenic ranged from 76.0% (liver of mother) to 91.0% (muscle of mother). In contrast, percentages of dimethylarsinic acid, arsenocholine, methylarsonic acid and tetramethylarsonium ion were low, ranging from 1.8% (methylarsonic acid in liver of mother) to 11.2% (methylarsonic acid in liver of mother). The predominance of arsenobetaine in tissues of DallÕs porpoise was consistent with the results of other higher trophic marine animals, such as marine mammals, seabirds and sea turtles (e.g., Fujihara et al., 2003, 2004;Goessler et al., 1998;Kubota et al., 2003b). 3.3. Placental transfer of arsenic to fetus Total arsenic concentrations in tissues of fetus were lower than those of mother (Table 2). Yang et al. (2004) reported that concentrations of toxic elements, such as Hg, Cd and Ag, in the liver of fetus were apparently lower than those of mother DallÕs porpoises. Mea-
dor et al. (1993) also found that concentrations of As, Cd, Hg and Se in liver and kidney of fetus were lower than those of mother of long-finned pilot whales. Hence, placental transfer of toxic elements, including arsenic, seems to be limited to some extent by some physiological system such as placental barrier in marine mammals. In the tissues of fetus, arsenobetaine, dimethylarsinic acid, arsenocholine and methylarsonic acid were detected, although only arsenobetaine, dimethylarsinic acid and arsenocholine could be quantified (Table 2). These results indicate that arsenobetaine, dimethylarsinic acid, arsenocholine and methylarsonic acid are transferable to fetus in DallÕs porpoises. Our previous study showed that arsenobetaine and dimethylarsinic acid can be transferred from mother bird to eggs of the seabird (black-tailed gull, Larus crassirostris) (Kubota et al., 2002a). Percentages of arsenobetaine and dimethylarsinic acid were similar between mother and fetus (or eggs) for both DallÕs porpoises (Fig. 3) and black-tailed gulls (Kubota et al., 2002a). Hence, arsenobetaine and dimethylarsinic acid might be generally transferable to fetus and egg of wildlife, and the composition of arsenic compounds in fetus and eggs reflect those of mother. In the mother DallÕs porpoise, arsenic burden of fetus was only 1.8% because of its low mass (Fig. 1) and low arsenic concentration (Table 2). The arsenic burden in fetus (1.8%) was lower than that of body weight (0.6%), suggesting that transfer of arsenic to fetus is limited to some extent. Interestingly, arsenic burden of blubber was extremely different between mother and fetus as mentioned above (Fig. 1). We assume that this
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difference might be due to the low placental transferability of lipid-soluble arsenic compounds present in the blubber of mother DallÕs porpoises. Recently, Ebisuda et al. (2002, 2003) reported that distribution of lipidsoluble and water-soluble arsenic compounds in several tissues (blubber, liver, kidney, muscle and gonad) of ringed seals (Pusa hispida): high percentage of dimethylarsinic acid-containing lipid-soluble arsenic compounds were observed in blubber (about 90% of total arsenic), whereas the lipid-soluble arsenic compounds were minor constituents in liver, kidney, muscle and gonad. According to Tanabe et al. (1982), hydrophobic highchlorinated chemicals are less transferable from mother to fetus of striped dolphins (Stenella coeruleoalba). Collectively, lipid-soluble arsenic compounds are less transferable than water-soluble arsenic compounds such as arsenobetaine, dimethylarsinic acid and arsenocholine, leading to the low concentration and low burden of arsenic in blubber of fetus DallÕs porpoises. Additional characterization of these lipid-soluble arsenic compounds would help in understanding the placental transfer and accumulation of arsenic in fetus of marine mammals.
Acknowledgments This study was supported by Grants-in-Aid for Scientific Research (A) (no. 12308030) from Japan Society for the Promotion of Science and for Scientific Research on Priority Areas (A) (no. 13027101) and ‘‘21st Century COE Program’’ from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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