Marine Environmental Research 20 (1986) 143-- 160

Age-Related Accumulation of Heavy Metals in Bone of the Striped Dolphin, Stenella coeruleoalba

K. Honda, Y. Fujise,* R. Tatsukawa

Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan

K. Itano

Department of Food Chemistry, Osaka City Institute of Public Health and Environmental Sciences, Tojo-cho 8-34, Tennouji-ku,

Osaka 543, Japan

&

N. Miyazaki

National Science Museum, Hyakunin-cho 3-23-1, Shinjuku-ku, Tokyo 160, Japan

(Received: 28 February, 1986)

A BSTRA CT

Concentration levels of metals (Fe, Mn, Zn, Cu, Pb, Ni, total Hg, methyl Hg and Se) in whole bone of the striped dolphin, Stenella coeruleoalba, and their variations with growth and sex were investigated. The metal concentrations characteristically changed with bone growth. Accumula- tion of Fe, Mn, Zn and Cu depended upon the metabolic turnover connected with ossification whilst, for the accumulation of Pb, Ni, Cd and Hg, the age or exposure time was a dominant factor. However, Pb and Ni were markedly accumulated during the suckling period and probably transferred from mother to calf via milk. The Cd concentration

* Present address: Research Institute of North Pacific Fisheries, Faculty of Fisheries, Hokkaido University, 1-1, Minato-cho 3-chome, Hakodate 041, Japan.

143

Marine Environ. Res. 0141-1136/86/503"50 © Elsevier Applied Science Publishers Ltd, England, 1986. Printed in Great Britain

144 K. Honda et al.

rapidly increased during the late period of the suckling stage, indicating higher absorption efficiency and accumulation of Cd through the digestive tract of the calf than those of mature dolphin. The transfer of total Hg to calf via milk was negligible, and its concentration increased with age until about 20 years, while methyl Hg reached a plateau at about 10 )'ears. A significant positive correlation (r = O" 798, p < 0"01) was found between the concentrations of total Hg and Se in the bone, and the molar ratio depended upon that of the blood. Furthermore, the concentrations of Fe and Cd in the bone were higher in the female than in the male, while Pb and Ni were lower in the female; the), are probabl)' due to parturition and lactation.

INTRODUCTION

The concentration levels and compositions of some heavy metals and major inorganic and organic components in the bones of terrestrial animals like cattle, pig, rat and human and their changes during bone- loss, protein-calorie malnutrition, ageing and various dietary deficiencies of minerals have been extensively studied (Stewart, 1975; Jowsey, 1976; Brown et aL, 1978; Driessens, 1980; Indritz & Hegarty, 1980). However, the heavy metal concentrations in bones of marine mammals and their growth-related changes largely remain to be studied, although the accumulation of heavy metals, Hg in particular, in soft organs and tissues of small cetaceans has been extensively studied because of their toxicities and pollution in the marine environment (Gaskin et al., 1972; Koeman et aL, 1973; Harms et al., 1978; Arima & Nagakura, 1979; Honda & Tatsukawa, 1983; Honda et al., 1983; Itano et aL, 1984a, b, c). While examining distribution characteristics of nine heavy metals and some major chemical components in various organs and tissues of striped dolphin, S. coeruleoalba, Honda et al. (1982; 1984a, b) reported the higher accumulation of Zn, Mn and Pb in the bones compared with those in the soft tissues. Concentrations of the metals and major chemical components also varied widely between parts of the bone structure. Further, the concentrations of major chemical components in the whole bone of striped dolphin changed with age, during foetal and calf periods at times of rapid bone growth (Honda et aL, 1984b). Accumulated load of certain heavy metals in the bone cannot, therefore, be disregarded in terms of accumulation rate, and certain heavy metal accumulations in the bone may also vary with different ages and life stages. Up to now,

Age-related heavy metals m bone of the striped dolphin 145

however, the growth-related accumulations of heavy metals in the bone of the marine mammals are unreported.

This paper describes the concentration levels of heavy metals (Fe, Mn, Zn, Cu, Pb, Ni, Cd, total Hg, methyl Hg and Se) in whole bone of the striped dolphin, Stenella coeruleoalba, at different ages, including the foetus, and also investigates their variations with growth and sex.

MATERIALS AND METHODS

Forty striped dolphins of different ages, including thirteen pregnant females, were captured alive at Taiji on the southern end of the Kii peninsula during the winter-catching seasons between 1978 and 1980. All specimens were in good health with no macroscopic pathological symptoms. The animals were frozen at -20°C as soon as field conditions allowed (1-8 h). Body weight, length, sex, basic morphometric data and weight of various organs and tissues were recorded for all specimens. The mandibular teeth were taken from all the immature and mature animals. Animals were aged following the method of Kasuya (1976). The age/body length regression equation was applied to deter- mine the age of the foetus and those calves younger than 0-5 year, as described by Miyazaki (1977). The relevant biometric details have already been reported (Miyazaki et al., 1981).

The entire bone of foetus and the 10th dorsal vertebra of the immature and mature animals were selected for metal determinations. Our preliminary investigations of the metal distribution in various bones of the striped dolphin have reported that the dorsal vertebrae, which consist of fifteen bones, contained high loads of heavy metals relative to the whole bone burdens, and also that the metal concentrations in the 10th dorsal vertebra, in particular, closely correlated with those in the entire bone (Honda et al., 1984a, b). Adhering muscle and ligament were carefully removed from the bone samples. The surfaces of the bone samples were gently washed with distilled water, excess water was dried by filter paper and the samples were weighed before analysis. For moisture determination, the bone samples were dried at 80°C for 12 h. Volatile lipid loss at this temperature was very low ( < 1%), so analytical error of the moisture content should be negligible. All the dry samples were pulverized and stored in polyethylene bags until analysis.

One to two grams of each dry bone sample were digested with a

146 K. Honda et al.

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Age-related hear)' metals in bone of the striped dolphin 147

mixture of nitric and perchloric acids. The resulting solution was analyzed by atomic absorption spectrophotometry (AAS) for Fe, Mn, Zn, Cu, Pb, Ni and Cd, and flameless AAS, cold vapor technique, for total Hg (Honda et al., 1984a). Se was determined by fluorometric spectrophotometry after 2,3-diaminonaphthalene chelation (Itano et al., 1984b). Determinations of methyl Hg was performed by gas chromatography, as described by West66 (1967). Contents of lipid, protein and Ca were measured following the methods of Honda et al. (1984b). Metal concentrations in the 10th dorsal vertebra of calf, immature and mature animals were converted to equivalent con- centrations of the whole bone using regression equations of metal concentrations given by Honda et al., (1984a, b).

RESULTS AND DISCUSSION

Metal concentration levels in the whole bone

Table 1 shows the average concentrations of heavy metals, Ca and major biochemical components in whole bone of striped dolphin. Based on measurements of body length and age and using data on weaning and sexual maturity of this species from Miyazaki (1977), each individual was classified into four stages of growth; namely, foetus, calf, immature adult and mature adult. Data for male and female dolphins are shown separately. For the four growth classes, one-way analysis of variance (ANOVA) of the concentration for each component was made.

The results shown in Table 1 indicate that the difference by growth stage was statistically significant for all metals and major components and highly significant for Cd, total Hg, Pb and lipid (p <0.01). The difference was generally proved to occur between two groups, the foetus and the others, the former being higher in concentration of Mn, Cu and moisture than the latter. The concentration of Mn, however, was highest in calves and the concentrations of Fe, total Hg and methyl Hg were considerably lower in calves. For some metals, sexual differences were also significant; the concentrations of Fe and Cd were higher in females than in males, whilst the opposite was observed for Pb and Ni.

Reports concerning the hard tissues of marine mammals, such as bone and teeth, are very scarce. Roberts et al. (1976) reported the concentrations of Pb (3-5 _+ 2.5/~g/wet g), Cd (0.41 + 0.33 pg/wet g) and

148 K. Honda et al.

Hg (0" 17 _+ 0-22/~g/wet g) in the rib of ten common seals, Phoca vitulma, of different ages from East Anglia and West Scotland. Braham (1973), examining Pb concentrations in nineteen organs and tissues of six immature California sea lions, Zalophus californianus, from the coast of San Luis Obispo County, California, reported that the mean concentrations of Pb were 34.2 for the humerus, 20.6 for the femur and 8-7/~g/dry g for the rib, and also that their concentrations were significantly higher than those of the soft tissues. The striped dolphin does not have a femur and the metal concentrations also vary among part of the bone structure (Honda et al., 1984a).

Age-related accumulation of metals in the whole bone

Significant differences between the metal concentrations were observed with growth, and their concentrations varied widely during the foetal and calf stages (Table 1). The period between foetus and calf had a rapid increase in bone weight, and a maximum of relative growth rate (bone weight/body weight) was also found in the foetus just before birth (Fig. 1). When the metal concentrations were plotted against age,

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Age trends of the bone weight (x) and bone/body weight (O) of striped do lph in , Stenella coeruleoalba. T h e foetal age is s h o w n for the pe r iods o f - 1 to 0 year

on the abscissa .

Age-related heavy metals in bone of the striped dolphin 149

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Fig. 2. Age trends of the heavy metal concentrations in whole bone of the striped dolphin, Stenella coeruleoalba. The foetal age is shown for the periods of - 1 to 0 year

on the abscissa. O: Female. 0 : Male. x : Methyl Hg.

150 K. Honda et al.

marked metal specific variations were found in the periods of rapid bone growth (Fig. 2).

iron The concentration of Fe in bone increased with gestational age and, after birth rapidly decreased through the calf stage, after which, the Fe concentration increased with age until approximately 8 years. The concentration in the matured male dolphin then remained constant. This age trend of bone Fe was similar to that of the liver- and blood- Fe of striped dolphin previously reported by Honda et al. (1983). While the hemoglobin-Fe in the blood increased with age up to maturity (Fig. 3), the serum-Fe concentration was relatively high in the foetal period, especially just before and after birth; its concentration markedly de- creased through the calf period. After that period, the serum-Fe concentration increased with age up to maturity. The age trend of the serum-Fe concentration agreed well with that of the bone Fe concentration, which means that the serum-Fe concentration indicates the bone Fe accumulation. The serum-Fe concentration varies with changes of Fe requirements in organs and tissues, so the accumulated Fe in the bone during the foetal period is largely used up during the rapid growth of organs and tissues of the calf. For the maturing stage,

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Fig. 3. Age trends of the total-Fe ( x ) and hemoglobin-Fe (O) concentrations in blood of the male striped dolphin, Stenella coeruleoalba. The shaded area shows approximately the serum-Fe concentrations, and the foetal age is shown for the period of -1 to 0

year on the abscissa.

Age-related heavy metals in bone of the striped dolphin 151

the bone Fe is accumulated as storage Fe via food and thereafter remains unchanged. The Fe reserve in the matured female dolphin, however, varies widely, probably reflecting the reproductive activities of parturi- tion and lactation.

Z&c The bone Zn (Fig. 2) showed relatively low concentration in the foetus and after birth it sharply increased during the calf stage. After this, Zn concentration roughly increased with age until approximately 8 years, thereafter remaining stable. A similar trend was observed for bone Ca (Fig. 4). The relationship between both metal concentrations (Zn and Ca) showed a significant positive correlation (r=0"859, p < 0"005). This suggests that Zn accumulates in bone with calcification. A close observation, however, showed that the highly accumulated Zn in the bone after birth decreased slightly up to about 3 years; such a trend was not found with the bone Ca. The concentration ratios of

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Fig. 4. Age trends of the contents of Ca (©) and moisture ( 0 ) in whole bone of the striped dolphin, Stenella coeruleoalba. The foetal age is shown for the period of - l to

0 year on the abscissa,

152 K. Honda et al.

Zn/Ca for three groups, foetus, calf, and immature plus mature adults, were calculated to see if any difference occurs in the accumulation of bone Zn with calcification between the growth stages. Mean ratios were as follows: foetus, 1.14 × 10-3; calf, 3.37 × 10-3: and immature plus mature adults, 2.41 × 10 -3. This indicates that calves have the highest accumulation rate of Zn with calcification, followed by the immature and mature adults and the foetus. Calves have a rapid growth of bone (Fig. 1) and also the highest activity of alkaline phosphatase (ALP) in the bone for the whole life span of striped dolphin (Saito et al., 1986). It is known that the role of ALP in ossification is its removal of the inhibiting pyrophosphate(s) (Chan et al., 1972). Some investigators have also speculated that Zn may participate in the synthesis of ALP (Westmorland & Hoekstra, 1969; Iqbal, 1969) and the amount of ALP is lower in Zn-deficient rat (Iqbal, 1971). The fact that, in the present study, the Zn concentration in bone sharply increased soon after birth and was followed by a slight decrease until about 3 years of age suggested that usually a large amount of Zn in the bone was incorporated with calcium apatite, but a minor portion in the calves was used for the synthesis of ALP.

Manganese The Mn concentration in bone of the foetus (Fig. 2) was about twice that of immature and mature dolphins; it sharply increased from about the tenth month of gestation until a maximum level at birth. This trend was similar to the accumulation rates of bone Zn and Ca, i.e. the high accumulations of bone Mn in foetus and newborn corresponded with marked increases of Zn and Ca concentrations. It is known that Mn, as well as Zn, is essential for bone formation, and also that the activity of ALP is lower in Mn-deficient rat and chick (Underwood, 1971). We assume that the high concentration of bone Mn in foetus and newborn is connected with an increase of ALP activity and that the chemical form of bone Mn at these stages may be different from the stable form of the bone Mn combined with calcium apatite at the other stages. Furthermore, after birth, the bone Mn concentration rapidly decreased during suckling when the overall Mn burden in bone decreased. However, the total body burden of Mn increased throughout the whole life span (Fig. 5). This means that the bone Mn, which is a major source of available Mn, was redistributed throughout other organs and tissues, rather than the excess being excreted.

Age-related heavy metals in bone of the striped dolphin 153

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Fig. 5. Age trends of Mn burdens in whole bone (0) and whole body (x) of the striped dolphin, Stenella coeruleoalba. The foetal age is shown for the period of - 1 to 0 year on the abscissa. The whole body burden of Mn was calculated from the weight of organs and tissues and their Mn concentrations, previously reported by Honda et

al. (1983) and Miyazaki et al. (1981).

Copper The bone Cu concentration (Fig. 2) was highest in the early foetal stage and decreased linearly with age during the foetal and suckling periods (0-1.5 year). After 1-5 year, the concentration of Cu remained constant. A similar trend was found for the moisture content in the bone, and the relationship between the concentrations of Cu and moisture showed a significant positive correlation (r = 0.923, p < 0-005). Cu in b o n e is associated with the formation of collagen in cartilage and its presence is concentrated in the blood in bone. The rapid decrease of Cu concentration in bone at this time can be explained by a dilution effect of the increased Ca concentration with bone formation.

Lead and nickel

The Pb concentration in bone of the foetus was relatively low and, after birth, its concentration increased with age (Fig. 2). A similar trend was also found for Ni. The concentrations of these two metals, however,

154 K. Honda et al.

were considerably higher in the early foetal period than in the late one, which may suggest transplacental transfer of Pb and Ni in the early embryonic stage. These metal concentrations might also be diluted by increased weight of the foetal bone. After birth, the Pb and Ni concentrations rapidly increased during the suckling period, and their accumulation rates were faster than those of the immature and mature dolphins. This indicates that absorption efficiency and accumulation of Pb and Ni via milk is higher than those via food.

Furthermore, the concentrations of Pb and Ni were lower (about two times, see Table 1) in the female than in the male, which may be due to an excretion of Pb and Ni through parturition and lactation. A similar effect by lactation was observed for liver, kidney and muscle of this species (Honda et al., 1983) and this 'cleansing' by the female, i.e. the lactating female using the milk as a sink for pollutants, has great implications for pollutant transfer. Detailed effects of lactation on the accumulation of these metals will be published elsewhere.

C a d m i u m

The Cd concentrations of bone of the foetus and young calf (0-0.33 year) were very low (Table 1 and Fig. 2). The same tendency was found with the Cd concentrations in the other organs and tissues during the same periods, and the Cd concentration in milk was also very low (<0-03pg/wet g) (Honda & Tatsukawa, 1983). These observations indicate that Cd is not preferentially transferred to the foetus and calf via the placenta and milk from the mother. After 0.33 year, the Cd concentration markedly increased up to about 1-5 year; afterward, its concentration in male dolphin slightly decreased with age until approximately 20 years. For the male dolphin, the decrease of Cd concentration during 5 to 20 years is explained by a dilution effect of the bone growth. The rapid increase of Cd concentration in the bone between 0-33 and 1"5 years of age implies higher absorption efficiency and accumulation of Cd through the digestive tract of calf dolphins compared with those of adults. According to Miyazaki et al. (1977), the mean weaning age of the striped dolphin is 1.5 year, although some dolphins continue to take milk up to 2 to 3 years of age. They also reported that the dolphin started feeding on solid food such as fish, squid and shrimp at 0.25 years of age. Therefore, further studies are needed to estimate the relative uptake rates of Cd from milk and solid food during the weaning period.

Age-related heavy metals in bone of the striped dolphin 155

A sexual difference in the bone Cd concentration was observed. The concentration in mature female dolphins was higher than that in the mature males and varied widely. This trend agreed well with the high concentration of bone Fe in mature female dolphins seen in Fig. 2; the relationship between the concentrations of Fe and Cd showed a significant positive correlation (r = 0.950, p < 0"001). Both the bone Cd and Fe are present in the blood and bone marrow and the contents of blood and red marrow varied widely with sexual status such as pregnancy, parturition, lactation and anoestrus (Honda & Tatsukawa, 1983; Honda et al., 1983, 1984a, b). The wide variation of bone Cd concentration in the mature female dolphin therefore appears to be due to changes of the blood and red marrow connected with changes in sexual status.

Total and methy l mercury The concentrations of total and methyl Hg in the bone (Fig. 2) were relatively low in the foetus; from birth, the total Hg concentration increased with age until about 20 years and later remained constant, while methyl Hg concentration reached a plateau at about 10 years of age. The calf dolphin, however, had a very similar concentration of total and methyl Hg to the foetus, which means limited transfer of Hg via milk because of the very low concentration of Hg in milk (0.002_ 0.002/~g/wet g) (Itano et al., 1984c).

Although the methylated Hg comprised about 90% of the total Hg in the foetus and calf, the percentage decreased with age thereafter until about 20 years and then remained constant. A similar trend was reported also for the soft tissues such as muscle, kidney, blubber, etc. (Itano et al., 1984b). In other words, these observations indicate that Hg in the body from age 1.5 year is accumulated mainly as non-methyl Hg, although Hg in the food items, such as squid and myctophid, was present mainly as methyl Hg (Itano et al., 1984b). This suggests a mechanism of demethylation, the biological significance of which remains to be studied.

Selenium The Se concentration in the bone of foetus was very low, and after birth its concentration increased during the suckling period (0-1.5 year). After 1.5 years of age, the concentration gradually increased up to about 20 years and later appeared to remain unchanged. The rapid increase of

156 K. Honda et al.

Se concentrat ion during the suckling period is probably due to relatively high transfer of Se via milk, as with Pb and Ni concentrations.

Relationship between total Hg and Se in the bone

Se is essential for the growth and reproduction of animals (Underwood, 1971) and has the capacity to detoxify metals such as Hg, Cd, As and Cu by mutual interaction (Venugopal & Luckey, 1978). A significant positive correlation (r = 0-798, p < 0"01) between the concentrations of total Hg and Se in the bone was found for immature and mature dolphins and the result is shown in Fig. 6. The regression equation was about 2.5 for the molar slope constant and 0-58 + 0.21 (mean + standard deviation) for the molar ratio of total Hg/Se. This molar slope constant was considerably higher and the molar ratio lower than those of the other organs and tissues of striped dolphin, being approximately unity for the molar slope constant and above unity for the molar ratio (Itano et al., 1984b). However, the molar ratio of total Hg/Se for the blood reported by Honda et al. (1984b) was below unity (0.61 + 0.25, mean + standard deviation), and its value was similar to that of bone in this study. Both Hg and Se are present in the blood and the bone

o oo o 110 0.6 0.8 ~g/we~ g ~ ' ,

10 nmo~e/wet g 12 5e

Fig. 6. Relationship between the concentrations of total Hg and Se in whole bone of the striped dolphin, Stenella coeruleoalba, immature plus mature (O), calf (O). The regression equation was calculated for the immature and mature dolphins. Total Hg

(mole/wet g) = 2.48 x Se (mole/wet g) - 1.38 (r = 0'798, p < 0"01).

Age-related heavy metals m bone of the striped dolphin 157

marrow (Honda et al., 1984b), and hence the molar ratios of total Hg/Se in the bone are linked with that in the blood, although no plausible explanation of the relatively high slope constant and low ratio in the bone compared with that in the other organs and tissues can be advanced.

CONCLUSIONS

Plankton and fish show a relatively fast uptake and accumulation of heavy metals and their accumulation processes are approximated by a simple mathematical model. As for mammals and birds, their metal accumulations are varied with species-specific processes (Honda & Tatsukawa, 1985). The results in this study also showed that the concentrations of heavy metals in bone varied widely with metal species, and with age. An accumulation of essential metals such as Fe, Mn, Zn and Cu depended upon the metabolic turnover connected with ossification. While the concentrations of Ni, Pb, Hg and especially Cd, were very low in foetal bone and after birth, their concentrations increased with age, indicating that age or exposure time is a dominant factor for their accumulation. However, the concentrations of Ni, Pb, Cd and Hg in the bone were influenced by the biological factors such as pregnancy, parturition and growth rates of bone. The concentrations of Ni and Pb rapidly increased by metal uptake via milk, but not significantly for Cd and Hg. The concentrations of Ni, Pb and Cd remained constant through 1-20 years as a result of rapid increase of bone weight. Such a dilution effect was not significant for Hg and the bone concentration of Hg increased with age until about 20 years. Additionally, a close correlation between Hg and Se concentrations in the bone indicated a detoxification effect by Se because of it having the capacity to detoxify Hg.

The very wide variations of the metal concentrations found indicate that consideration of age and biological processes in the dolphin is necessary for understanding bioaccumulation phenomena for heavy metals and also their effects on this species. In particular, the rapid and wide variation of the metal concentrations during the foetal and calf periods warrants a detailed study on bioaccumulation processes in these stages.

158 K. Honda et al.

A C K N O W L E D G E M E N T S

Specimens were collected through the co-operation of the fishermen's unions at Taiji. This work was supported in part by Grant-in-Aid for Scientific Research (Project Nos. 58030020 and 59030053) and by the Toyota Foundat ion (80-I-123).

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Age-related beau' metals in bone of the striped dolphin 159

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