Rapid determination of silver in cultivated Japanese and South Korean oysters and Japanese rock oysters using the 24.6-s neutron activation product 110Ag and estimation of its average daily intake
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- Fukushima, M. & Chatt, A. J Radioanal Nucl Chem (2013) 296: 563. doi:10.1007/s10967-012-2122-2
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Soft tissues of cultivated Japanese (Miyagi Prefecture) and South Korean (Koje-do and Kosong) oysters and Japanese rock oysters (Honshu Island) were analyzed to measure silver levels. The soft tissues, namely hepatopancreas, gill, muscle, and mantle were separated, freeze-dried, pulverized, and analyzed by an instrumental neutron activation analysis method in conjunction with compton suppression spectrometry (INAA-CSS). The method consisted of the irradiation of samples in a neutron flux of 5 × 1011 cm−2 s−1 using the rapid transfer system in an inner pneumatic irradiation site of the Dalhousie University SLOWPOKE-2 reactor (DUSR) facility for 12–15 s, decay for 20 s, and counting for 60 s. The 657.8-keV gamma-ray of the 24.6-s nuclide 110Ag was used for assaying silver. The method was validated using NIST, NRC and NIES certified reference materials. An absolute detection limit of 0.05 μg silver using NIST SRM 1566b Oyster Tissue was achieved. About 10–50 times higher levels of silver were found in cultivated Japanese oysters compared to the South Korean ones. The silver concentrations in cultivated oysters in Miyagi Prefecture showed the following trend: gill > mantle > hepatopancreas > muscle as well as on the age. Rock oysters generally had higher silver content compared to cultivated oysters. A very preliminary value of about 0.466 μg silver average intake per person per day was estimated from the consumption of oysters by the people living in the Sendai city of Miyagi Prefecture.
KeywordsNeutron activationShort-lived 110AgOystersJapanese daily intake
Oysters are considered a delicacy by some Japanese people. In 2009, about 128 million Japanese consumed a total of 48,700 tons soft tissues of oysters. People living in Sendai city of Miyagi Prefecture ate around 1,035 g of soft tissues of oyster per family per year compared to an average of 635 g per family in entire Japan . In 2009, the total yield of soft tissues of Japanese cultivated oysters was about 33,830 tons, of which about 4,918 tons (14.5 %) were from Miyagi Prefecture . It is obvious that the Japanese production of oyster was not sufficient to meet domestic needs. The shortfall was made up by imports of oysters from South Korea.
Silver is known to exist since ancient times. Its crustal and oceanic abundances are about 7.5 × 10−2 and 4 × 10−5 mg kg−1, respectively. Silver can be found as pure deposits, in its ores such as argentite and along with ores of lead, gold or copper. It has been used to make currency coins, jewelry, utensils, etc. Pure silver is applied to make electrical contacts, conductors, printed circuit boards, etc. Silver with zinc or cadmium is employed to make high capacity batteries. Sliver nitrate is used to manufacture photographic films and papers. Silver iodide is employed to seed clouds to produce rain in drought-stricken areas. The potential for the discharge of silver ions and compounds to bays, lakes, rivers and other water bodies from the sewage of mining and industrial operations mentioned above exists.
Silver ions and certain silver compounds are known to exert toxic effects on some bacteria and viruses. Some people claim that silver has healing properties and germicidal effect. In Japan, the internal walls of some mineral water bottles are coated with silver compounds to prevent growth of microorganisms. European Union has approved the use of silver in food coloring (E174 designation). It is a common practice to cover certain Indian sweets with a very thin decorative silver foil. However, the safety of silver usage in food is not free from dispute as the element is not known to play any essential biological role in humans. Moreover, certain silver salts may be carcinogenic. The pharmacological and toxicological effects of silver have recently been reviewed . Silver content of food is not readily available. Silver is one of the trace elements whose levels are generally low in most biological materials. However, rather high levels of silver can be found in marine bivalves. Oysters are known to accumulate silver in their soft tissues. From the toxicological point of view, it is important to measure silver levels in oysters and estimate its daily or annual intake.
Silver, in particular at low levels, is a rather difficult element to determine by almost all analytical techniques. Oysters have high levels of sodium which interfere with their analysis for several trace elements including silver by most instrumental analytical techniques. Oysters also contain rather high amounts of polysaccharides which are difficult to decompose by acids for techniques which require samples in a liquid state. Fortunately, instrumental neutron activation analysis (INAA) can be employed without the physical destruction, such as dissolution in acids, of the sample compared to other techniques. Most INAA methods reported in the literature for the determination of trace elements in biological materials generally employ minutes-to-hours-to-days of irradiation, decay and counting times. The stable isotopes of at least 38 elements, however, produce both short- and long-lived radioactive isotopes on thermal neutron irradiations . Many of these short-lived nuclides can be used for the sensitive determination of several elements.
Naturally occurring silver consists of two stable isotopes 107Ag (51.839 %) and 109Ag (48.161 %) . Twenty-eight radioisotopes of Ag have been characterized so far. The analytically useful isotope is 109Ag. It produces the metastable state of 110mAg (half-life = 249.79 days) with a cross section of 3.2 b under thermal neutron bombardment. The 109Ag isotope also produces 110Ag (half-life = 24.6 s) with a cross section of 89 b under similar conditions. It is evident that the short-lived 110Ag will be much more sensitive than the long-lived 110mAg for the same short irradiation, decay and counting times . Many more samples can also be analyzed within a short time using 110Ag.
Conventional INAA methods consisting of one-shot irradiation-decay-counting scheme have generally been employed for assaying short-lived nuclides [4, 7]. Sensitivity, detection limit, precision, and accuracy of measurements can be significantly improved by using methods based on the principles of cyclic activation analysis. In cyclic INAA (CINAA) the irradiation-decay-counting process of a sample is repeated for a suitable number of cycles. Although Chatt and coworkers [8–14] have extensively used pseudo-cyclic INAA (PC-INAA) and CINAA since 1979 for the determination of several trace elements including silver in a variety of matrices, we could not use it advantageously in oysters because of the high activities produced by major elements such as sodium, chlorine, bromine, magnesium, etc. present in the samples. We then developed a conventional INAA method in conjunction with compton suppression spectrometry (INAA-CSS) for detecting silver in oysters. We have applied this method to cultivated and rock oysters from Japan and cultivated oysters from South Korea to understand the distribution of silver in their organs and to estimate the intake of silver from their consumption by Japanese population groups.
Mass of soft tissues of cultivated oysters (g, ww, n = 10, avg ± sd) from Miyagi Prefecture, Japan
Mass of soft tissue
8.2 ± 3.4
9.0 ± 1.3
8.4 ± 2.6
15.0 ± 4.3
15.2 ± 2.9
12.1 ± 3.3
17.7 ± 2.5
8.2 ± 2.4
11.4 ± 2.2
Mass of soft tissues of rock oysters (g, ww, avg ± sd) from Japan
Mass of soft Tissue
26.6 ± 1.7 (n = 4)
17.8 ± 3.6 (n = 5)
26.6 ± 5.1 (n = 5)
20.9 ± 4.8 (n = 5)
Mass of soft tissues of cultivated oysters (g, ww, n = 5, avg ± sd) from South Korea
Mass of soft tissue
13.6 ± 2.4
8.0 ± 1.8
10.1 ± 1.4
5.6 ± 1.7
12.5 ± 3.1
7.0 ± 1.6
8.4 ± 1.8
Irradiation and counting
We placed between 200 and 400 mg of the freeze-dried pulverized oyster tissue samples in 1.2-mL precleaned polyethylene sample vials, capped and heat-sealed. We then placed these vials in 7-mL precleaned polyethylene irradiation vials, capped and heat-sealed.
We prepared silver comparator standards from the plasma emission spectroscopy standard solution with a certified purity of >99.999 % supplied by SCP Canada Ltd. We pipetted about 1 mL of the standard solution into 1.2-mL polyethylene vial, capped and heat-sealed. We calibrated Eppendorf pipettes used for dilutions and transfers. The comparator standards were of identical geometry and contained approximately similar amounts of silver as the samples. We used distilled deionized water (DDW) for making and diluting solutions and washing all apparatus. We analyzed all materials and reagents used in this work for “blanks”.
We validated the INAA-CSS method using the following NIST standard reference materials (SRM) and NRC and NIES certified reference material (CRM): NIST Oyster Tissue (SRM 1566b), NRC Dogfish Liver (CRM DOLT-3), NRC Lobster Hepatopancreas (CRM TORT-3), and NIES CRM No. 9 Sargasso. We used between 100 and 500 mg of these materials, depending on the minimum mass recommended by the issuing agency.
We irradiated all samples, comparator standards, and various SRM and CRMs at a thermal neutron flux of 5 × 1011 cm−2 s−1 using the rapid sample transfer system in an inner pneumatic irradiation site of the DUSR facility. The stability, homogeneity, and reproducibility of the DUSR neutron flux have previously been described [15–17]. The irradiation time (ti), decay time (td), and counting time (tc) depended mainly on the major elements in the sample. In general, these parameters were: ti of 12–15 s, td of 20 s, and tc of 60 s.
We irradiated a sample, namely the soluble fraction of an oyster after in vitro enzymolysis, for 1 h at a thermal neutron flux of 2.75 × 1013 cm−2 s−1 in the Kyoto University Reactor (KUR), Japan, and counted it for 20 min after a decay of 30 days to assay silver content through its long-lived nuclide 110mAg. We used a Canberra GC3020-7600SLS detector with a relative efficiency of 30 %, and a resolution of 1.87 keV at the 1332 keV photopeak of 60Co. The peak-to-Compton plateau ratio of this system was 54.3:1 at the 662-keV γ-ray of 137Cs.
Results and discussion
Method and its validation
We were aware that the soft tissues of oysters were rather difficult to dissolve completely due to their high polysaccharide content and that the levels of silver were particularly low in certain species of oyster. So we started out with the objective of using a non-destructive (instrumental) NAA method with high sensitivity, precision, accuracy and rapidity. In the past Chatt and coworkers [7, 9, 11, 13] have been quite successful in developing several PC-INAA and CINAA methods for the measurement of low levels of silver in different biological materials using the 24.6 s nuclide 110Ag. When we attempted to apply some of these methods to oysters, we observed that high activities 24Na, 38Cl, 80Br and 27Mg interfered with the assay of silver via 110Ag. These interfering activities kept on increasing from cycle to cycle leading to high dead times and a small 657.8-keV photopeak of 110Ag sitting on a very high background giving results with high uncertainties. In similar situations Chatt and coworkers [11, 16, 18] used epithermal INAA. We then attempted to analyze the oyster samples for silver by irradiating them in the epi-cadmium neutron flux of 2 × 1011 cm−2 s−1 at the DUSR facility. Since this flux was 2.5 times lower than the total reactor neutron flux of 5 × 1011 cm−2 s−1at 8 kW operating power, we could not reliably measure low levels of silver in oysters by either conventional one-shot epithermal INAA (EINAA), pseudo-cyclic EINAA (PC-EINAA) or cyclic EINAA (C-EINAA) methods. Recently, Pun and Landsberger  reported the successful usage of C-EINAA for the determination of silver in ores. In 2005 when we were carrying out this project on silver in oysters, we decided to develop a conventional INAA method in conjunction with compton suppression spectrometry for measuring low levels of silver in oysters.
Zhang  from our laboratory reported a value of 0.96 ± 0.04 as the experimentally determined peak efficiency reduction factor (PERF) ± 1σ for the 657.8-keV gamma-ray of 110Ag. Since this PERF is close to 1, anticoincidence counting will not reduce the efficiency of its measurement beyond the normal experimental errors. In other words, the INAA-CSS method can reduce background activities from interfering nuclides such as 24Na, 38Cl, and 27Mg without sacrificing the sensitivity for 110Ag.
The 657.8-keV gamma-ray (intensity = 94.64 %) of 110Ag (half-life = 24.6 s) could be potentially interfered with by the 657.0-keV gamma-ray (6.17 %) of 76As (26.32 h). In view of the irradiation times of 12–15 s used in these experiments as well as the respective half-lives, cross sections, and intensity of gamma-rays involved, we concluded that no such interference occurred for measuring silver levels in oysters. We believe that even samples containing percentage quantities of arsenic and silver at mg kg−1 level, the INAA-CSS method can still produce reliable silver values since the PERF for the 657.0-keV gamma-ray of 76As is 0.24 ± 0.03 leading to negligible interference .
Silver content of several reference materials by INAA-CSS (mg kg−1)
SRM and CRMs
0.639 ± 0.047 (n = 3)
0.666 ± 0.009
1.28 ± 0.04 (n = 3)
1.20 ± 0.07
7.83 ± 0.80 (n = 4)
NIES No. 9
0.36 ± 0.04 (n = 4)
0.31 ± 0.02
Silver levels in oysters
We found that the mass of soft tissues of cultivated oysters, both collected in 2005 Oct, from South Korea (Table 3) were generally smaller than the ones from Miyagi Prefecture (Table 1). Moreover, we noticed that the hepatopancreas in South Korean oysters did not grow as much as that of Miyagi oysters. It could mean that the growth stage of South Korean oysters was shorter than those of Miyagi particularly when both were the same species of oyster. The slower growth could also be due to lower levels of oxygen, plankton, and chlorophyll in seawater caused mainly by a higher density of oysters cultivated in those areas. The most important factor influencing growth in bivalves is food supply which, in turn, is controlled by factors such as temperature, aerial exposure, water depth, and population density .
We originally planned to study bioaccessibility of silver in oyster using a method similar to that prescribed by AOAC  and used by us . We managed to process only one sample for a preliminary study. We could not analyze that sample by the INAA-CSS method at the DUSR facility because it was being decommissioned at that time. So we decided to analyze the sample at the Kyoto University Reactor facility in Japan although it did not have a convenient fast transfer facility. So we used the long-lived 110mAg nuclide which gave a poorer detection limit. We did not detect the silver photopeak in the gamma spectra of the residue which is thought to be non-bioaccessible. It was then inferred that most of the silver in oyster was bioaccessible.
It is difficult to calculate the average daily dietary intake of silver primarily because of the lack of data. We made an attempt here to obtain a rough estimate of the intake from the consumption of oysters. It was mentioned earlier that the intake of oyster tissues in 2009 was 1,035 g per family in Miyagi Prefecture. We selected from our data the mass of silver in oyster soft tissues at 1 m depth in Higashimatsushima in 2005 Oct as 0.34 mg/kg on wet weight (ww) basis. In 2009 Dec, there were 1,012,882 people in 451,318 families in the Sendai city. They consumed a total of 467,114,130 g (ww) oysters containing a total of 172,430,845 μg (dw) of silver. Then the average intake of silver was about 0.466 μg per person per day assuming that (i) the silver content of oysters was constant during 2005–2009, (ii) the silver level did not change within the “oyster season” (Sep-Mar), (iii) the Miyagi Prefecture family consisted of only 4 members; (iv) the family ate only the oysters cultivated at 1 m depth in the Higashimatsushima area; (v) oyster is the only source of silver for this family; and (vi) silver in oysters is fully bioaccessible. It should be noted here that this intake value was obtained using a lot of assumptions which are prone to errors and therefore should be used with caution. No such estimate of intake of silver has previously been reported to the best of author’s knowledge.
We developed a conventional instrumental neutron activation analysis (INAA) method in conjunction with compton suppression spectrometry (CSS) for the detection of low levels of silver in oysters. We used the 657.8-keV gamma-ray of the 24.6-s nuclide 110Ag for rapid assay of silver. We validated the method using NIST, NRC and NIES certified reference materials and found it to be quite reliable. We achieved an absolute detection limit of 0.05 μg silver using NIST SRM 1566b Oyster Tissue. We applied this method to cultivated and rock oysters from Japan and cultivated oysters from South Korea to understand the distribution of silver in various organs and to obtain a rough estimate of the average daily intake of about 0.466 μg per person of silver from oyster consumption by the residents of the Sendai city in Miyagi Prefecture.
The authors would like to thank (i) Dr. J. Holzbecher and Mr. Blain Zwicker of the DUSR facility for their assistance in irradiation and counting; (ii) Dr. T. Matsutani of Ishinomaki Senshu University, Dr. H. Tamate of Yamagata University, Mr. Shigeru Watanabe in Higashimatsushima City, Miyagi Prefecture and MPFTI for collecting oysters and discussions; (iii) KUR for irradiation and counting; (iv) the Mitsubishi Foundation for financial assistance to M. Fukushima; and (v) the Natural Sciences and Engineering Research Council of Canada for a Discovery Grant to A. Chatt.