Environ. Sci. Technol. 2001, 35, 3103-3108
Multielemental Analysis of Purpleback Flying Squid Using High Resolution Inductively Coupled Plasma-Mass Spectrometry (HR ICP-MS) H I D E K I I C H I H A S H I , * ,† HIROYOSHI KOHNO,‡ KURUNTHACHALAM KANNAN,§ AKITO TSUMURA,| AND SHIN-ICHI YAMASAKI¶ National Research Institute of Fisheries and Environment of Inland Sea, Maruishi 2-17-5, Ohno-cho, Hiroshima, 739-0452, Japan, Okinawa Regional Research Center, Tokai University, Sakiyama 165, Taketomi 907-15, Japan, 213 National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824, National Institute of Agro-Environmental Sciences, Kan-non-dai 3-1-1, Tsukuba 305-0856, Japan, and Faculty of Agriculture, Tohoku University, Tsutsumi-Dori-Amamiya-cho 1-1, Sendai 981-0914, Japan
Forty-four elements were analyzed in 21 tissues of purpleback flying squid, Sthenoteuthis oualaniensis, by high resolution inductively coupled plasma-mass spectrometry (HR ICP-MS) and inductively coupled plasma atomic emission spectrophotometry (ICP-AES). Greater concentrations of V, Fe, Co, Ni, Cu, Ag, Cd, Pb, and Bi were found in liver, pancreas, and ink sac than in other tissues. Ink sac concentrated remarkable levels of Ca and Sr in addition to the above-mentioned elements. Several alkalis, alkaline earth, and rare earth elements preferentially accumulated in muscle. Among the hard tissues, accumulation of V and U in beak, Ni, Zn, and Cd in gladius and Cr in skin was prominent. K, Rb, Cs, Pb, Bi and some transition elements (V, Co, Cu, Zn, Ag, Cd) were significantly (p < 0.05) higher in the livers of adult than in juvenile squids. Sodium, alkaline earth, and rare earth elements were higher in the livers of juveniles than in adult squids.
Introduction The squids, which feed upon nectonic fishes and squids themselves, occupy a high trophic level in the marine food web (1). Most small and medium size squids grow rapidly with a lifespan of less than 1 year (2). Owing to their rapid growth rate, squids are expected to be a potential food resource in the future (2). Furthermore, squids are important prey organisms for marine mammals (3) and seabirds (4, 5). * Corresponding author phone: +81-829-55-3756; fax: +81-82954-1216; e-mail:
[email protected];
[email protected]. † National Research Institute of Fisheries and Environment of Inland Sea. ‡ Tokai University. § Michigan State University. | National Institute of Agro-Environmental Sciences. ¶ Tohoku University. 10.1021/es010653v CCC: $20.00 Published on Web 07/03/2001
2001 American Chemical Society
Squids have been reported to be useful as bioindicators to monitor oceanic pollution (6, 7). However, differences in the concentrations of elements at various life stages and distribution among different body tissues are not well characterized. Furthermore, earlier studies of elemental analysis in marine biota have focused only on a few heavy metals. In this study, 44 elements were analyzed in 21 tissues of purpleback flying squid, Sthenoteuthis oualaniensis, a pelagic squid, using a high resolution inductively coupled plasma mass spectrometer (HR-ICP-MS) and a inductively coupled plasma atomic emission spectrophotometer (ICPAES). Elemental concentrations, distribution in tissues and organs, and size related changes in the accumulation were examined.
Materials and Methods Squid Samples. Purpleback flying squid is widely distributed in the Indo-Pacific Ocean. Squids were collected from the waters off Iriomote Island in Ryukyu Islands, Japan, during Aug-Sep 1992 and 1993. Available biometric measurements for individual squids are shown in Table 5 (Supporting Information). The dorsal mantle length (DML) of the squids ranged from 78.9 to 303 mm (Figure 5; Supporting Information showing the relation between dorsal mantle length and body weight of squid), which are considered to be medium sized individuals. Adult and subadults of greater than 100 mm DML were jigged off the waters. Juveniles (78.9-97.1 mm in DML) were found stranded alive along the coast of Iriomote Islands (24′19′′N, 123′41′′E). All the adults analyzed had a photophore (luminescent organ). The body weight of the juvenile squids ranged from 11 to 26 g, whereas those of the adult ranged from 90 to 1300 g. After rinsing with seawater, samples were sealed in polyethylene bags and kept frozen at -20 °C until analyses. Prior to analysis, samples were dissected on a clean polypropylene board using fine ceramic scissors and Teflon coated tweezers. Eye, ganglion, liver (digestive gland), pancreas (digestive duct appendage), ink sac, stomach, caecum, gill, branchial heart, spermatophore, Needham’s sac, prostate gland, seminiferous duct, testis, Nidamental gland, matured ova, mantle, fin, arm, funnel, mouth, skin, gladius, cartilage, beak, and stomach contents were separated for the analysis of elements in individual tissues and organs. Trace Element Analyses. Samples were digested in Teflon containers using a microwave digestor (MDS-81D, CEM Corporation, U.S.A.) following the method described elsewhere (8). Tissues were homogenized, and 100 mg of wet tissues was weighed into 120 mL Teflon vials and digested overnight with 10 mL of pure nitric acid (EL grade, specific gravity: 1.38, Kanto Chemical Co. Ltd., Japan). The microwave parameters were 300 W power for 30 min, held with no power for 30 min and then 600 W power for 60 min. The digested contents were transferred to acid washed polypropylene bottles and made up to 100 mL with double distilled water and subjected to AES analyses. For MS analysis, internal standard (200 pg/mL Rh) was spiked and diluted further with double distilled water. External standards (Plasma Standards; SPEX Industries Inc., USA) were diluted to appropriate concentrations for instrumental calibration. S, P, Mg, Ca, Sr, Na, and K were analyzed by an axially mounted inductively coupled plasma atomic emission spectrophotometer (axial ICP-AES: Maxim III/Applied Research Laboratory, Switzerland). The flow rates of argon to coolant, plasma, and carrier were kept at 13, 1.2, and 0.45 L/min, respectively. Sample flow rate was set at 3 mL/min. The VOL. 35, NO. 15, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Reported Concentrations of Elements in Livers of Ommastrephid Squids (µg/g) species
location
wt basis
n
statistics
Mn
Fe
Co
8 0.52 100 8 0.54 120 7 320
1.3 1.3
Cu
Zn
Ag
Cd
Cs
Pb
refs
Sthenoteuthis oualaniensis Ogasawara, Japan wet Central Pacific dry Ogasawara, Japan wet
median (mature) mean (mature) mean composite
Central Pacific Pacific, Japan North Pacific
dry wet wet
mean composite composite
Sea of Japan Pacific, Japan Sea of Japan Sea of Japan Sea of Japan Sea of Japan
wet wet dry dry wet wet
mean composite mean (winter) mean (summer) composite mean
Off Victoria New Zealand
wet wet
composite mean
South Pacific Australia
dry dry
mean mean
26 4.2 6
Argentina Argentina
wet wet
mean mean
10 0.73 9 1.1
120 35 4.8 82 0.005 0.33 this study 220 50 5.6 79 0.005 0.44 1700 510 24 780 (13) 0.48 (27)
Ommastrephes bartrami 14 0.46
199 81
200 160 12 540 78
(13) (28) (29)
290
0.42
Todarodes pacificus 3 0.73 150 60 1.6 120 22 3.1 320
0.48
110
55
38
(30) (28) (10) (10) (27) (31)
75
74
170
(29) (31)
24 96 43 390 92 510 120
46 0.003 2.7
71 150
0.28
a
130
a
150
Nototodarus sloani 0.38
Nototodarus gouldi 750
250 700 360 830
3.3
50 33
11 32
0.6 0.3
36 38
0.24 12 0.6 12
Illex argentinus
a
8.1
24 54
33 47
Not reported.
TABLE 2. Reported Concentrations of Elements in Ink Sac and Gladius of Teuthoidea Dibranchiates Squids (µg/g, Mean) species
location
S. oualaniensis
Okinawa, Japan
T. pacificus I. argentinus L. opalescens
Sea of Japan Argentina California, USA
S. oualaniensis
Okinawa, Japan
unidentified squid I. argentinus L. patagonica L. opalescens
Texas, USA Argentina Argentina California, USA
a
wt basis
na
Mn
Fe
wet wet wet wet wet
Ink Sac with Ink 1 2.9 17 1 1.4 18 32 15 3 6.0 4(16) 1.8 7.1
wet wet dry wet wet wet
Gladius (Pen, Arrow) 1 0.23 3.3 1 0.69 5.5 1 2.1 160 1(10) 1.4 3.5 2(20) 0.81 5.9 4(16) 1.9 3.2
Cu
Zn
Ag
Cd
Pb
refs
110 360 54 36 130
120 110 25 150 40
0.97 2.2
25 120 5.7 2.2 6.3
0.16 0.32
this study this study (15) (12) (17)
110 160 65 4.8 82 66
340 310 260 85 110 99
1.3 0.49
3.6 0.39
0.50 0.44
68 62 1.3 0.97 11 6.3
34 3.2 0.08 0.15 2.3 0.67 4.3 1.6
this study this study (33) (12) (16) (17)
Values in parentheses indicate the number of individuals pooled.
wavelengths for S, P, Mg, Ca, Sr, Na, and K were 180.733, 214.916, 279.552, 393.365, 407.771, 589.595, and 766.488 nm, respectively. V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Rb, Y, Ag, Cd, Sn, Sb, Cs, La, Ce, Pr, Nd, Sm, Eu, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hg, Tl, Pb, Bi, Th, and U were analyzed by a high resolution inductively coupled plasma mass spectrometer (HR-ICP-MS: PlasmaTrace/VG Elemental, U.K.) with an ultrasonic nebulizer (USN: Applied Research Laboratory, Switzerland). The flow rates of argon to coolant, plasma, and carrier were kept at 14, 0.7, and 1 L/min, respectively. Sample flow rate was set at 1.8 mL/min. The abundance and m/z values selected for various elements are shown (Table 6, Supporting Information). The resolution for MS analyses was selected from 300 to 7000. Further details of the analytical conditions have been reported elsewhere (9). Analyses were performed in a class 100 clean room or in a class 1000 clean booth. All containers were placed in 1% pure nitric acid for over a month and thoroughly rinsed with double distilled water prior to use. 3104
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Statistical Analyses. Concentrations of some elements did not follow a normal distribution pattern. Therefore, nonparametric Mann-Whitney U-test was applied to test the significance at 95 or 99% confidence interval. The application software used for statistical analysis was SPSS/ PC (Version 5.0) (SPSS Inc., USA).
Results and Discussions Tissue/Organ Distribution of Concentrations. Elemental concentrations and distribution in various organs and tissues of a female squid (303 mm in DML) are presented in Table 7 (Supporting Information). The liver (also known as digestive gland, hepatopancreas, or mid-gut gland) contained high concentrations of heavy metals as reported earlier (10-12). Concentrations of 3d-transition metals (except Cr and Mn) and Ag, Cd, Hg, Pb, Bi, and U were also high in the liver. Concentrations of alkalis, rare earth elements, and nonmetals (S, P, and As) did not differ greatly among organs and tissues.
FIGURE 3. Concentration pattern of rare earth elements in the liver of S. oualaniensis in relation their atomic numbers.
FIGURE 1. Distribution of body burden of elements in a mature female purpleback flying squid. FIGURE 4. Seawater normalized concentrations (SNC) of elements in the liver of S. oualaniensis in relation to their atomic numbers.
FIGURE 2. Elemental concentrations (minimum, median, and maximum) in the liver of S. oualaniensis in relation to their atomic numbers. Reported mean and median concentrations of selected elements in livers of adult Ommastrephid squids from various locations are shown in Table 1. Observed concentrations of Mn and Fe in the present study were comparable to those reported elsewhere. However, concentrations of Co were much higher than those reported in other studies. Concentrations of Cu (1720 µg/g dry wt), Zn (513 µg/g), Ag (24.1 µg/g), and Cd (782 µg/g) in the liver of S. oualaniensis caught off the central Pacific (13) were evidently higher than those found in the present study even after converting the values to wet weight basis. High concentrations of Ag have been commonly encountered in livers of Ommastrephid squids. The reason for the high accumulation of Ag in squids is still unclear (10-12, 14). Pancreas (digestive duct or pancreatic appendage) contained relatively high concentrations of V, Mn, Fe, Co, Ni, Cu, Zn, and Cd. Renal appendages (kidney) in two species of cephalopods, Eledon cirrhosa and Sepia officinalis, contained high concentrations of Mn, Ni, Cu, and Pb (6). Cadmium concentrations in kidney were much lower than those in pancreas. The ink sac of S. oualaniensis contained high concentrations of Ca and Sr. Noticeable concentrations of V, Fe, Co, Ni, Cu, Ag, Cd, Pb, and Bi were also found in the ink sac. Reported concentrations of trace elements in the ink sac and gladius of squids are listed in Table 2. Cd concentrations in the ink sac of S. oualaniensis were much higher than the mean value of 5.7 µg/g (dry weight) recorded for Todarodes pacificus collected from the Japan Sea in 1980 (15). However, it should be noted that jigging may result in the release of ink from the sac (as a defense mechanism), which may affect the reported concentrations. Similar problems have been
encountered in the analyses of gall bladder, which contains bile in mammals. Presence of considerable concentrations of elements in ink suggests excretion of heavy metals and alkaline earth elements. Furthermore, high concentration of some elements in ink is noteworthy because of its use in foodstuffs in some Mediterranean countries and in Japan. In hard tissues, especially in the gladius, concentrations of Cr, Ni, Zn, Cd, and U were high. The elemental concentrations in gladius were comparable to those reported for Illex argentinus and Loligo spp. from Argentina and the U.S. coastal waters (12, 16, 17). On the other hand, concentrations of alkalis, alkaline earth, and rare earth elements were low in partially calcified gladius (Table 7, Supporting Information). Gladius is a suitable tissue for the analysis of trace elements in squids because it is prominent and easy to separate from other tissues. Isomorphous substitution of rare earth elements to Ca was known in mineralogy (18). Even though high concentrations of Ca were observed in the ink sac of squids, concentrations of rare earth elements were low. Individual tissues and organs of a male squid was also analyzed to examine the distribution of elements. Distribution of elements in tissues of male squid was similar to that in female. Although specific enrichment of heavy metals in gonads of filter feeding bivalve mollusks has been shown, such a feature was not found in S. oualaniensis indicating that the influence of spawning on elemental concentrations in this species might be low. Body Burden of Elements in Squids. The distribution and body burden of elements in S. oualaniensis were calculated as the product of concentrations and weight of individual tissues (Figure 1). Liver accounted for only about 3% of the body weight in purpleback flying squid. This is small compared to liver to body weight ratios of 9.5% (8.610.3) and 11.6% (9.3-13.2) in Todarodes pacificus and Watasenia scintillans, which are oegopsid teuthoid squids. The burdens of V, Fe, Co, Ni, Ag, Cd, Hg, Bi, and U were high in the liver of S. oualaniensis despite its relatively smaller proportion to body mass. Most of the burden of elements were distributed (>50%) in muscle since this tissue occupied >80% of the body weight. However, none of the elements showed elevated concentrations in muscle. Distributions of Ca and Sr in the ink sac, Cr VOL. 35, NO. 15, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Comparison of Element Concentrations in Livers of Purpleback Flying Squid Juveniles and Adults (µg/g, Wet Weight) juvenile (n ) 10)
DML (mm) weight (g) Na Mg P K Ca V Cr Mn Fe Co Ni Cu Zn Ga As Rb Sr Y Ag Cd Sn Sb Cs La Ce Pr Nd Sm Eu Gd Tb Dy Ho Er Tm Yb Lu Hg Tl Pb Bi Th U a
adult (n ) 8)
median
mean
min.
max.
median
mean
min.
max.
U-test (p < 0.05)
86.9 18.8 5150 800 2150 1850 1380 0.341 0.126 0.719 151 0.209 0.400 12.0 8.36 0.003 6.96 1.04 9.40 0.0048 1.49 20.3 0.235 0.0042 0.0037 0.012 0.017 0.0020 0.0074 0.0017 0.00045 0.020 0.00037 0.0013 0.00032 0.0010 0.00013 0.00063 0.00002 0.06 0.00086 0.189 0.0020 0.013 0.051
87.6 19.0 5170 825 2110 1940 3260 0.418 0.128 0.953 148 0.208 0.364 13.1 9.16 0.004 7.54 1.01 31.7 0.014 1.63 20.8 0.234 0.0046 0.0041 0.029 0.059 0.0076 0.026 0.0051 0.00094 0.066 0.0023 0.0044 0.00079 0.0037 0.0003 0.0018 0.0003 0.06 0.0010 0.207 0.0018 0.025 0.049
78.8 11.0 3800 490 1000 1500 710 0.20 0.027 0.33 75.6 0.11 0.013 4.0 6.7
