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Survey of bioavailable selenium in Sweden with the moose (Alces alces L.) as monitoring animal
the Science of the Total Embnment The Science
of the Total
Environment
172 (1995)
37-45
Survey of bioavailable selenium in Sweden with the moose ( Alces alces L.) as monitoring animal Vera Galgan* a, Adrian Frankb aDepartment of Chemistry, National Veterinary Institute, P.O. Box 7073, S-750 07 Uppsala, Sweden bThe Centre for Metal Biology, University of Uppsala, P.O. Box 535, S-751 21 Uppsala, Sweden Received
25 December
1994; accepted
10 January
1995
Abstract
Liver tissueswere collected from moose(Alces &es L.) from the regular hunting seasonsof 1981and 1982.The material (from about 4300 animals)was stored at -20°C at the National Veterinary Institute (SVA). From this material, 2080 specimensfrom 12 counties representing 14 regions were analyzed for selenium.The counties included were Norrbotten, Vastemorrland (northern region), Uppsala, Kopparberg, Gavleborg, Jtimtland (central region), north and south Kalmar (easternregion), Halland, north and southAlvsborg (westernregion), and Blekinge, Kristianstad and Malmohus (southern region). The analysiswas performed by a combination of continuoushydride generationusinga flow-injection techniqueand atomic absorptionspectrometry.The medianseleniumconcentration in the liver in the entire material was0.15 and the mean (-t S.D.) 0.25 f 0.29(range: 0.03-3.11mg/kg wet wt. The highest medianvalues obtained were 0.26, 0.28 and 0.29 mg/kg, in the countiesof Gavleborg (central region) and north and south Alvsborg (western region), respectively. The lowest medians,0.09-0.10 mg/kg, were found in the counties of Uppsala (central region), north and south Kalmar (eastern region), and Jamtland (central region). Intermediate median values were obtained in the other counties: 0.13 mg/kg in the county of Wsternorrland (northern region), 0.15-0.16 mg/kg in the counties of Blekinge and Malmohus (southern region) and Norrbotten (northern region), 0.18 mg/kg in the county of Halland (western region), and 0.23 mg/kg in the counties of Kristianstad and Kopparberg (southern and central regions,respectively). Seleniumconcentrations < 0.1 mg/kg in the liver of cattle are consideredto be deficient. In someof the regions,50-60% of the mooselivers showedvalues below this level. The results indicate that the mooseis useful for monitoring the amount of seleniumavailable for wild-grazing animalsand confirm that the Swedishenvironment is poor in selenium. Keywords: Moose; Liver; Selenium;Monitoring
necrosis in rats deficient in vitamin E. The biochemical role of the element was elucidated in the 70s by the discovery that glutathione peroxidase (GSH-Px) is a Se-containing enzyme [24]. Together with other defensive factors, e.g. superoxide dismutase (SOD), catalase, and vitamins E and C, this enzyme protects the organism against
1. Introduction
Selenium (Se) is an essential element. Schwarz and Foltz [25] showed that Se could prevent liver
* Corresponding
author.
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A. Frank
/ The Science of the Total Environment
oxidative stress caused by free radicals [17]. Several other mammalian selenoproteins have now been identified, including another Se-dependent enzyme, type l-iodothyronine deiodinase [l]. Selenium is also important for immunodefense and for the synthesis of certain antibodies [18]. Sweden is a selenium deficient country. The poor natural occurrence of this element in Sweden has led to nutritional Se deficiency in domestic animals in the past and presumably a suboptima1 nutritional Se status in the population. Nutritional muscular dystrophy has been observed in pigs, cattle, sheep and horses fed with home-produced fodder [21]. Among these affected animals, sudden heart failure caused death, particularly in fast-growing lambs and calves. Diffuse subclinical signs may also appear, such as loss of appetite, retarded growth, impaired fertility and disturbances of reproduction 1231. Since the beginning of the SOS,Se supplementation of fodder has been allowed in Sweden to improve the Se status in domestic animals, and has resulted in better production and economy for the farmer. Little is known about the Se status of terrestrial wild animals in Sweden. Wild animals are dependent on the occurrence of this element in nature. Its bioavailability may be influenced by several factors such as the geochemical background, aerial deposition from natural [19] and anthropogenic sources [6], acidification, the buffer capacity of the soil, and the chemical form of the Se. All these factors together are decisive for the uptake of Se by plants eaten by herbivores. The factors mentioned above may cause regional differences in the bioavailability, and these might be monitored by measuring the Se concentration in the organs of herbivorous animals, preferably in the liver. It has been shown earlier that the moose, a wild ruminant, is a suitable animal for monitoring the bioavailability of some elements occurring in the natural environment [9]. The moose is widespread in Sweden, and is hunted according to an organized system, which facilitates collection of organ tissues. The moose is relatively stationary and its migration is limited [26]. Age determination of the animals is feasible and is also neces-
172 (1995) 37-45
sary, as the concentration of some elements is age-dependent. The aim of this investigation was to obtain time-related basic data for the bioavailability of Se in Sweden using the moose as a monitoring animal. This was done by determining the Se concentration in the liver of moose from different regions of the country. It was considered that such reference material could be used for comparison with future information on environmental changes with possible consequences for the wild Swedish fauna. 2. Materials
and methods
2.1. Liver tissue samples
Liver and kidney tissues were collected from about 4300 moose in 25 regions of Sweden during the hunting seasons of 1981 and 1982 for determination of the bioavailability of cadmium [93. This material was stored at -20°C. Compared with that in fresh tissue, the water content decreased by about 5% during storage. The age of the animals was determined by cutting the first molar tooth of the left mandible and examining it under a microscope. Of the liver tissue material, 2080 samples from 14 regions (represented by 12 counties) were investigated for Se. Two of the counties, Kalmar and Alvsborg, were divided into two hunting districts, north and south. The samples analyzed were collected from northern, southern, central, eastern and western regions of Sweden as shown in Table 1 and Fig. 1. 2.2. Analytical
methods
The desiccated outer layer of the liver specimens was cut off and samples were taken from the inner part. The samples were prepared by automated wet digestion according to a standardized procedure [7,8]. A mixture of 65% nitric acid and 70% perchloric acid (7:3 by vol.) was used as oxidizing agent. The digestion was performed in borosilicate glass tubes using an electrically heated aluminium block connected to a microprocessor for control of time and temperature. The digestion was stopped at 225°C.
39
K Galgan, A. Frank / The Science of the Total Environment I72 (I 99.5) 37-45 Table 1 The counties from which the moose tissue samples were collected, representing different regions of Sweden Regions Northern
Central
Southern
Eastern
Western
Norrbotten VSstemorrland
JSrntland Givleborg Kopparberg Uppsala
Blekinge Kristianstad MahnGhus
Kahnar
Halland
A combination of continuous hydride generation by flow injection and atomic absorption spectrometry [12,13] was used for Se determination. Atomization was carried out in an electrically heated quartz cuvette. The analytical system and the collection and calculation of analytical data were controlled by computer. The analytical results are presented on a wet weight basis (mg/kg wet wt.). The analytical method was checked by analyzing standard reference materials (Bovine Liver NIST 1577 and 1577a). 2.3. Statistical methods For calculations of the analytical results, the programme Statgraphics (Version: 5.1, STSC, Inc., Rockville, MD) was used. As the analytical data were not normally distributed (Kolmogorov-Smirnov test), nonparametric methods were used in the calculations. The age dependence of the Se concentrations was checked by testing calves (0.5 year) against 1.5year-old animals using the Kruskal-Wallis test. In addition, all age groups were tested against Se concentrations with Spearman-rank correlation. The distribution pattern of the Se concentrations in livers from the 12 counties is presented in the form of a notched box-and-whisker plot. 3. Results
The selenium concentrations in the ers from the 12 counties are presented as mean and standard deviation (Z f dian, and range. High Se concentrations with median 0.25 mg Se/kg wet wt. were found in
moose livin Table 2 S.D.), mevalues > the north-
ern and southern parts of the county of Alvsborg (western region), as well as in the county of Gavleborg (central region). The highest single concentration (3.05 mg/kg) was noted in a liver from the county of Gavleborg. Low Se concentrations with median values of about 0.09-0.1 mg/kg were obtained in the northern and southern parts of the county of Kalmar (eastern region) and in the counties of Uppsala and Jlmtland (both central regions). The lowest Se values, 0.027 and 0.029 mg/kg, were found in the counties of Vasternorrland and Jamtland (central region), respectively. The total material is presented graphically in the form of a notched box-and-whisker plot in Fig. 2. For better resolution in the figure, 1 mg Se/kg liver was chosen as the upper level. Wide ranges with high concentrations (some of them over 2 mg Se/kg), resulting in long boxes and many outliers (+ sign) are seen in Fig. 2 for the counties of Blekinge, Kristianstad, Alvsborg, Kopparberg and Gavleborg (K, L, P, W and Y, respectively). A narrow distribution pattern, short boxes and few outliers are shown by the counties of Uppsala (C) and northern Kalmar (Hn). The narrow box for Malmijhus (M) is proportional to the few tissue samples investigated (N = 47). The largest number of samples analyzed came from Norrbotten (BD) (N = 281), resulting in a broad box. The median values for the county of Alvsborg (Pn and Ps) on the west coast of Sweden are significantly higher than those for the counties of Uppsala (C) and Kalmar (Hn and Hs) on the east coast (Kruskal-Wallis test, P < 0.001) (Fig. 2). The ages of the animals investigated were between 0.5 and 14.5 years. The group of animals
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/ The Science
of the
1.5 years old represented 59% of the whole material, and calves only 15%. However, the age dis-
mg
Se/kg
> 0.1
liver
W.W.
- 5 0.2 > 0.2
C
not
analyzed
Total Environment
I72 (1995)
37-45
tribution of the moose populations in the different counties varied. Using Spearman-rank correlation, a positive correlation was found between Se concentration in the liver and increasing age in three out of 12 counties, namely northern Kalmar (Y = 0.27, P < O.OOl>, Kristianstad (r = 0.25, P < 0.011, and Halland (r = 0.23, P < 0.01). A significantly higher Se concentration was noted in the 1.5 year-old animals than in the calves in the regions of north Kalmar (P < O.OOl), south Kalmar (P < 0.05) and Kristianstad (P < O.Ol), as well as in the whole material (P < 0.01). The material was divided into four Se concentration ranges in each of the 12 counties and the percentage number of samples in each range is shown in Fig. 3. In the counties of Kristianstad (L), north and south Alvsborg (Pn and Ps), Kopparberg (W), and GGleborg (X), Se concentrations of over 0.2 mg Se/kg were found in more than 50% of the material. The corresponding figures (> 0.2 mg Se/kg) for the other counties were: north Kalmar (Hn) l%, south Kalmar (Hs) 14%, Uppsala (C) 2%, and Jlmtland (Z) 15%. Animals with concentrations below 0.1 mg Se/kg were found in the following counties in the given proportions: Uppsala (C) 47%, north Kalmar (Hn) 63%, south Kalmlar (Hs) 59%, Vasternorrland (Y) 36%, and Jamtland (Z> 46%. 4. Discussion
Fig. 1. Selenium in livers collected in 1982 from moose in 12 counties representing 14 regions of Sweden. Median values were calculated for each respective region. The values are classified into three ranges and depicted by shaded areas. The darker the shading, the higher the hepatic selenium concentration. The values for the unshaded areas have not yet been analyzed. The letters given in the figure are the letters used officially in Sweden to represent the different counties. Those included in this investigation are: BD, county of Norrbotten; C, Uppsala; H, Kalmar; K, Blekinge; L, Kristianstad; M, Malmlihus; N, Halland; P, hvsborg; W, Kopparberg; X, Gvleborg; Y, Vastemorrland; Z, Jlmtland;
Reports on Se concentrations in organs from terrestrial wild animals are rare. Ohlendorf 1221 found Se concentrations of < 2 mg/kg dry weight (dry wt.) in livers from wild herbivorous mammals, corresponding to about < 0.4-0.5 mg Se/kg wet wt. Selenium concentrations in moose liver have been reported from Norway by Froeslie et al. [lO,ll]. Their tissue material was collected from six regions. The mean Se concentrations (* S.D.) in their whole material (N = 542) was 0.62 f 0.95 (range 0.07-5.6) mg/kg wet wt., compared with that in the present study (N = 2080) of 0.25 + 0.29 (range 0.03-3.1) mg/kg wet wt. In the Norwegian study, median values were also presented for each region. The highest median value, 1.0 mg/kg, was found in Finnmark in
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the northern part of Norway, and the lowest, 0.18 mg/kg, in Hedmark and Oppland, in the central part of the country. The corresponding values in the present study were 0.29 mg/kg in the southern part of the county of Avsborg on the west coast of Sweden and 0.09 mg/kg in the county of Kalmar on the east coast.
Determinations of the Se concentrations in biogeochemical samples (plant roots from the banks of small streams) by the Geological Survey of Sweden (SGU) revealed a similar pattern with high concentrations on the west and south coast and low on the east coast. In the present investigation, high median Se concentrations were found in moose liver samples from the western regions of Sweden, such as the northern and western areas of the county of hvsborg, and from the county of Kristianstad in the south. Low liver Se concentrations were found in the counties of Uppsala (central region) and north and south Kalmar (eastern region). These findings are in good accordance with the observations on biogeochemical samples. The Se concentration is low in the Scandinavian Precambrian rock and also in the soil. By weathering and microbial activity, insoluble Se compounds are converted into soluble forms available to plants. These compounds can also be leached or volatilized 13,141. Younger sedimentary rocks, such as limestone in the province of Sk&e in the south of Sweden, contain higher Se concentrations, as shown in the investigation of biogeochemical samples [4]. The total Se content of the soil is not well
4.1. Regional diRerewes
Increased Se concentrations were found in the livers from the moose in the south of Norway, especially in areas near the coast, an observation which could be explained by atmospheric deposition [lo]. This finding correlated well with the results of investigations in forest moss samples, which showed higher Se concentrations in the southern and south-western parts of Norway. Norwegian 1191 and Swedish studies 1161 have shown similarities in Se concentrations in coastal forest soils. Increased concentrations were found on the Norwegian south and south-east coast, as well as on the Swedish south-west coast compared with the east coast. As plausible explanations for these findings, high mean annual precipitation, deposition from natural marine sources, and anthropogenic contributions from industrial areas were suggested. Table 2 Selenium
concentrations
in moose
County
liver
N
from
12 counties,
41
172 (199.5) 37-45
representing
14 regions
of Sweden
%
&S.D.
Median
Range
0.197 0.102 0.099 0.128 0.311 0.332 0.198 0.292
0.146 0.033 0.043 0.110 0.413 0.252 0.114 0.321
0.155 0.101 0.094 0.093 0.158 0.227 0.152 0.182
0.048-1.633 0.042-0.257 0.053-0.585 0.046-0.633 0.050-2.754 0.066-1.572 0.090-0.593 0.071-2.158
Norrbotten (BD) Uppsala (C) Kalmar, north (HII) Kalmar, south (Hs) Blekinge (K) Kristianstad (L) Malmohus (M> Halland (N)
281 150 183 80 120 149 47 128
Avsborg,
(Pn)
1.59
0.373
0.375
0.277
0.07.5-2.672
hvsborg, south (Ps) Kopparberg (W) Gkleborg (X) Vastemonland (Y) Jamtland (Z)
167 169 154 122 171
0.462 0.309 0.383 0.146 0.132
0.437 0.272 0.398 0.099 0.105
0.290 0.226 0.258 0.124 0.098
0.065-2.061 0.057-2.320 0.050-3.054 0.027-0.555 0.029-0.695
north
Total N, number weight.
2080 of liver
samples;
means
0.250 and standard
deviations
0.288 (ji. +- SD.),
0.151 median
values
0.027-3.054 and ranges
are expressed
in mg Se/kg
wet
42
V. Galgan, A. Frank
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C
/The
Hn
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Hs
K
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L
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M
N
Pn
172 (1995) 37-45
Ps
W
X
Y
2
Fig. 2. Notched box-and-whisker plots showing selenium concentrations in moose liver specimens from 12 Swedish counties, expressed in mg/kg liver wet weight. The concentration of 1 mg Se/kg liver was chosen as the upper limit. For explanation of letters representing counties, see Fig. 1. Hn and Hs, north and south Kalmar; Pn and Ps, north and south kvsborg.
correlated to the Se content of plants in general. Its availability to plants is influenced by several factors, including the pH and the microflora of the soil and waters, climatological factors, and interaction with compounds of different elements, as well as by fertilization [14,15]. In acid soils (pH 4.5-6.5) and under high moisture conditions, Se in the form of selenite is bound to colloids, as
iron hydroxide selenium complexes [5]. These are insoluble and less available to plants. In basic soils (pH 7.5~8.5), Se is present as soluble selenate, which is taken up by plants especially when precipitation and leaching are low, thus contributing to the Se nutrition in herbivorous animals. Other elements, such as sulfur, may interact and influence the uptake of Se by plants, which is a
T/. Galgan,
A. Frank/The
Science of the Total Environment
mg
K
L M
N
Pn Ps W
X
Y
Z
Se/kg
liver
I
5 0.05
I
5 0.05
- I 0.1
5 0.01
- s 0.2
B
ED C Hn Hs
43
172 (1995) 37-45
W.W.
a- 0.2
Tot
Fig. 3. Selenium in moose liver. Percentage number of samples in different Se concentration ranges in the 12 counties investigated and in the entire material (Tot). For explanation of letters representing counties, see Fig. 1. Hn and Hs, north and south Kalmar; Pn and Ps, north and south Avsborg.
passive procedure [6]. Sulfate in high concentrations in soils competes with Se compounds, causing the Se uptake by plants to decrease. The Swedish west coast is strongly affected by acidification and is one of the areas that suffers most from atmospheric pollution by sulfuric compounds and from environmental pollution from the European continent and Great Britain. The effect of acidification has been demonstrated in an investigation of the Cd burden of moose from
different geographic regions of Sweden [9]. A heavy Cd burden was found in the strongly aciditied southern and western parts of the country, and declined to the east and north. Despite heavy acidification, high Se concentrations were observed on the west coast of Sweden in the present material. It would seem that atmospheric deposition from marine and anthropogenic sources is an important factor in Se nutrition.
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4.2. Age dependency
In humans as well as in animals, the Se concentration decreases in the liver after birth 120,281. As the concentration of Se in milk is low, the liver and probably also some other organs serve as a selenium depot during the first period of life. Thus, lower Se concentrations are expected in the liver from moose calves than from adult animals. Higher concentrations in yearlings than in calves were observed for the entire material (2080 animals), as well as for the material from the counties of Kalmar and Kristianstad. However, a statistically significant correlation between the age of the animals and their hepatic Se concentrations was only found for three regions (north Kalmar, Kristianstad and Halland). In the present investigation, the group of yearlings was the predominant age group, representing 59% of the animals, whereas the group of calves consisted of only 15%. In addition, the age distribution varies between the regions. The growth rate of calves is highest during the first months of life, i.e. between July and September [26]. The calves begin to graze very young, some days after birth. However, the suckling period lasts until November-December. At the time of the hunting season, the calves seem to have almost reached the nutritional Se status of that of the adult animals. Thus, it is difficult to recognize concentration differences within the material. Another reason for the lack of a statistically significant difference in the hepatic Se concentration between calves and adults in some regions could be the relatively small number of calves in the groups. Obviously, the hepatic Se concentrations in calves and adults become equalized at the time of hunting, and in general no difference was found. Further, even within a homogeneous age group of moose, great variations in Se concentrations were observed. Consequently, the entire material was presented without age grouping. No correlation between age and hepatic Se concentration was found in moose in the Norwegian investigation 1111. 4.3. Nuttitional
Available
data for evaluation
Se status in wild animals are limited. Values obtained in domestic ruminants may be used for this purpose [6]. Concentrations in the liver of above 0.3 mg Se/kg wet wt. are considered sufficient [2], and between 0.15 and 0.3 marginal. The selenium status in cattle is evaluated according to similar principles at the National Veterinary Institute. Concentrations 5 0.05 mg Se/kg liver are regarded as severe Se deficiency, > 0.05-10.1 as deficiency, and > 0.1-s 0.2 as marginal/insufficient. Liver Se concentrations above this latter value are regarded as adequate. According to these criteria, 50-60% of the animals in the counties of Uppsala (central region) and Kalmar (eastern region) have an insufficient or marginal Se status. These latter counties were found to be the most Se poor regions in the present investigation (Fig. 3). Selenium-deficiency diseases in wild animals are difficult to diagnose. Clinical signs are seldom apparent. Subclinical deficiency may exist, but is difficult to recognize. The most plausible way of ascertaining a subclinical deficiency status is by supplementation, which will result in improved reproduction, as reported in deer [6]. In addition, as mentioned previously, Se together with other compounds such as vitamin E are involved in the protection of the organism against oxidative damage [27]. References [II
El
[31 [41
[Sl
Se status
of the optimum
172 (1995) 37-45
D. Behne, C. Weiss-Nowak, M. Kalckhisch, C. Westphal, H. Gessner and A. Kyriakopoulos, Studies on new mammalian selenoproteins, in M. Anke, D. Meissner and C.F. Mills (Eds.), Trace Elements in Man and Animals, TEMA 8, Proceedings of an International Symposium, 1993, pp. 516-524. D.C. Blood and O.M. Radostits, Veterinary Medicine. A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats, and Horse, 7th edn., Ball&e Tindall, London, 1989, pp. 1187-1202. G.F. Combs, Jr., S.B. Combs, 2. Selenium in the environment, in The Role of Selenium in Nutrition, Academic Press, New York, 1986, pp. l-40. J. Ek, S.O. Ohlsson and 0. Selinus, Bly, kadmium, selen - hela Sverige kartllggs. (Lead, Cadmium, selenium the whole of Sweden is mapped.) Forskning och Framsteg 2/1988, in Swedish. L. Fishbein, Selenium, in E. Merian (Ed.), Metals and their Compounds in the Environment, VCH, Weinheim, 1991, pp. 1153-1190.
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W.T. Flueck, Moglicher Einfluss von Immissionen auf die Spuren-elementversorgung wildlebender Wiederkauer: Selen als Beispiel. Z. Jagdwiss., 36 (1990) 179-185. Automated wet ashing and multi-metal de171 A. Frank, termination in biological materials by atomic absorption spectrometry. Z. Anal. Chem., 279 (1976) 101-102. accessory to an automated wet t81 A. Frank, Semi-micro digestion system for ashing small sample amounts, in P. Bratter and P. Schramel (Eds.), Trace Element Analysis in Medicine and Biology, Vol. 5, Walter de Gruyter, Berlin, 1988, pp. 78-83. and L.R. Petersson, Assessment of bioavail[91 A. Frank ability of cadmium in the Swedish environment using the moose (Alces alces) as indicator. Z. Anal. Chem., 317 (1984) 652-6.53. DOI A. Froeslie, G. Norheim, J.P. Rambaek and E. Steinnes, Levels of trace elements in liver from Norwegian moose, reindeer and red deer in relation to atmospheric deposition. Acta Vet. Stand., 25 (1984) 333-345. 1111 A. Froeslie, G. Holt, R. Hoeie and A. Haugen, Levels of copper, selenium and zinc in liver of Norwegian moose ( Alces alces), reindeer (Rangifer tarandus), roedeer (Capreolus capreolus) and hare (Lepus timidus). Norsk Landbruksforskning, 1 (1987) 243-249. 1121 V. Galgan and A. Frank, Automated system for determination of selenium in biological materials, in P. Bratter and P. Schramel (Eds.), Trace Element Analysis in Medicine and Biology, Vol. 5, Walter de Gruyter, Berlin, 1988, pp. 84-89. Notes and comments on the [131 V. Galgan and A. Frank, determination of selenium in biological materials. Norwegian J. Agric. Sci. Suppl., 11 (1993) 57-74. U.C. Gupta, M. Lamand and T. [141 G. Gissel-Nielsen, Westermarck, Selenium in soils and plants and its importance in livestock and human nutrition. Adv. Agronomy, 37 (1984) 397-460. General aspects of selenium fertilizaWI G. Gissel-Nielsen, tion. Norwegian J. Agric. Sci. Suppl., 11 (1993) 135-140. [I61 L. Johnsson, Se-levels in the Mor layer of Swedish forest soils. Swed. J. Agric. Res., 19 (1987) 21-28. Relation of selenium to other antioxidants, 1171 B. Karlmark, with special reference to free radicals. Norwegian J. Agric. Sci. Suppl., 11 (1993) 11-20.
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H.-J.S. Larsen, Relations between selenium and immunity. Norwegian J. Agric. Sci. Suppl., 11 (1993) 105-119. of sele1191 J. L%g and E. Steilmes, Regional distribution nium and arsenic in humus layers of Norwegian forest soils. Geoderma, 20 (1978) 3-14. DO1 D.C. Mahan, A.L. Moxon and M. Hubbard, Efficacy of inorganic selenium supplementation to sow diets on resulting carry-over to their progeny. J. Anim. Sci., 46 (1977) 738-746. E. Norrman, Selen ett livsniidvandigt splramne. [Xl (Selenium - an essential trace element.1 Aktuellt f&n lantbruksuniversitetet. nr. 324, Husdjur, Uppsala, 1983, in Swedish. Bioaccumulation and effects of seleD-4 H.M. Ohlendorf, nium in wildlife, in Selenium in Agriculture and the Environment, SSSA Special Publication no. 23, 1989, Madison, WI, USA, pp. 133-177. [231 B. Pehrson, Diseases and diffuse disorders related to selenium deficiencies in ruminants. Norwegian J. Agric. Sci. Suppl., 11 (1993) 79-93. 1241 J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, D.G. Hafeman and W.G. Hoekstra, Selenium: biochemical role as a component of glutathione peroxidase. Science, 179 (1973) 588-590. I251 K. Schwarz and C.M. Foltz, Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Sot., 79 (1957) 3292-3293. (The moose - a D.61 F. S&felt, Algen - en presentation. presentation), in H. Ekman, N. Hermansson, J.O. Pettersson, J. Riilcker, M. Steen and F. Stilfelt (Eds.), Algen - djuret, skijtseln och jakten, Svenska JPgarefiirbundet, Spinga, Sweden, 1992, pp. 46-49, in Swedish. I271 D.E. Ullrey, S.M. Schmitt, T.M. Cooley, P.K. Ku and P.A. Whetter, Selenium, vitamin E and capture myopathy in white-tailed deer, in C.F. Mills, I. Bremner and J.K. Chesters (Eds.), Trace Elements in Man and Animals TEMA 5, Proceedings of the International Symposium, 1984, pp. 113-115. Selenium content of tissues in Finnish PSI T. Westermarck, infants and adults with various diseases, and studies on the effects of selenium supplementation in neuronal ceroid lipofuscinosis patients. Acta Pharmacol. Toxicol., 41 (1977) 121-128.
of the Total
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Survey of bioavailable selenium in Sweden with the moose ( Alces alces L.) as monitoring animal Vera Galgan* a, Adrian Frankb aDepartment of Chemistry, National Veterinary Institute, P.O. Box 7073, S-750 07 Uppsala, Sweden bThe Centre for Metal Biology, University of Uppsala, P.O. Box 535, S-751 21 Uppsala, Sweden Received
25 December
1994; accepted
10 January
1995
Abstract
Liver tissueswere collected from moose(Alces &es L.) from the regular hunting seasonsof 1981and 1982.The material (from about 4300 animals)was stored at -20°C at the National Veterinary Institute (SVA). From this material, 2080 specimensfrom 12 counties representing 14 regions were analyzed for selenium.The counties included were Norrbotten, Vastemorrland (northern region), Uppsala, Kopparberg, Gavleborg, Jtimtland (central region), north and south Kalmar (easternregion), Halland, north and southAlvsborg (westernregion), and Blekinge, Kristianstad and Malmohus (southern region). The analysiswas performed by a combination of continuoushydride generationusinga flow-injection techniqueand atomic absorptionspectrometry.The medianseleniumconcentration in the liver in the entire material was0.15 and the mean (-t S.D.) 0.25 f 0.29(range: 0.03-3.11mg/kg wet wt. The highest medianvalues obtained were 0.26, 0.28 and 0.29 mg/kg, in the countiesof Gavleborg (central region) and north and south Alvsborg (western region), respectively. The lowest medians,0.09-0.10 mg/kg, were found in the counties of Uppsala (central region), north and south Kalmar (eastern region), and Jamtland (central region). Intermediate median values were obtained in the other counties: 0.13 mg/kg in the county of Wsternorrland (northern region), 0.15-0.16 mg/kg in the counties of Blekinge and Malmohus (southern region) and Norrbotten (northern region), 0.18 mg/kg in the county of Halland (western region), and 0.23 mg/kg in the counties of Kristianstad and Kopparberg (southern and central regions,respectively). Seleniumconcentrations < 0.1 mg/kg in the liver of cattle are consideredto be deficient. In someof the regions,50-60% of the mooselivers showedvalues below this level. The results indicate that the mooseis useful for monitoring the amount of seleniumavailable for wild-grazing animalsand confirm that the Swedishenvironment is poor in selenium. Keywords: Moose; Liver; Selenium;Monitoring
necrosis in rats deficient in vitamin E. The biochemical role of the element was elucidated in the 70s by the discovery that glutathione peroxidase (GSH-Px) is a Se-containing enzyme [24]. Together with other defensive factors, e.g. superoxide dismutase (SOD), catalase, and vitamins E and C, this enzyme protects the organism against
1. Introduction
Selenium (Se) is an essential element. Schwarz and Foltz [25] showed that Se could prevent liver
* Corresponding
author.
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/ The Science of the Total Environment
oxidative stress caused by free radicals [17]. Several other mammalian selenoproteins have now been identified, including another Se-dependent enzyme, type l-iodothyronine deiodinase [l]. Selenium is also important for immunodefense and for the synthesis of certain antibodies [18]. Sweden is a selenium deficient country. The poor natural occurrence of this element in Sweden has led to nutritional Se deficiency in domestic animals in the past and presumably a suboptima1 nutritional Se status in the population. Nutritional muscular dystrophy has been observed in pigs, cattle, sheep and horses fed with home-produced fodder [21]. Among these affected animals, sudden heart failure caused death, particularly in fast-growing lambs and calves. Diffuse subclinical signs may also appear, such as loss of appetite, retarded growth, impaired fertility and disturbances of reproduction 1231. Since the beginning of the SOS,Se supplementation of fodder has been allowed in Sweden to improve the Se status in domestic animals, and has resulted in better production and economy for the farmer. Little is known about the Se status of terrestrial wild animals in Sweden. Wild animals are dependent on the occurrence of this element in nature. Its bioavailability may be influenced by several factors such as the geochemical background, aerial deposition from natural [19] and anthropogenic sources [6], acidification, the buffer capacity of the soil, and the chemical form of the Se. All these factors together are decisive for the uptake of Se by plants eaten by herbivores. The factors mentioned above may cause regional differences in the bioavailability, and these might be monitored by measuring the Se concentration in the organs of herbivorous animals, preferably in the liver. It has been shown earlier that the moose, a wild ruminant, is a suitable animal for monitoring the bioavailability of some elements occurring in the natural environment [9]. The moose is widespread in Sweden, and is hunted according to an organized system, which facilitates collection of organ tissues. The moose is relatively stationary and its migration is limited [26]. Age determination of the animals is feasible and is also neces-
172 (1995) 37-45
sary, as the concentration of some elements is age-dependent. The aim of this investigation was to obtain time-related basic data for the bioavailability of Se in Sweden using the moose as a monitoring animal. This was done by determining the Se concentration in the liver of moose from different regions of the country. It was considered that such reference material could be used for comparison with future information on environmental changes with possible consequences for the wild Swedish fauna. 2. Materials
and methods
2.1. Liver tissue samples
Liver and kidney tissues were collected from about 4300 moose in 25 regions of Sweden during the hunting seasons of 1981 and 1982 for determination of the bioavailability of cadmium [93. This material was stored at -20°C. Compared with that in fresh tissue, the water content decreased by about 5% during storage. The age of the animals was determined by cutting the first molar tooth of the left mandible and examining it under a microscope. Of the liver tissue material, 2080 samples from 14 regions (represented by 12 counties) were investigated for Se. Two of the counties, Kalmar and Alvsborg, were divided into two hunting districts, north and south. The samples analyzed were collected from northern, southern, central, eastern and western regions of Sweden as shown in Table 1 and Fig. 1. 2.2. Analytical
methods
The desiccated outer layer of the liver specimens was cut off and samples were taken from the inner part. The samples were prepared by automated wet digestion according to a standardized procedure [7,8]. A mixture of 65% nitric acid and 70% perchloric acid (7:3 by vol.) was used as oxidizing agent. The digestion was performed in borosilicate glass tubes using an electrically heated aluminium block connected to a microprocessor for control of time and temperature. The digestion was stopped at 225°C.
39
K Galgan, A. Frank / The Science of the Total Environment I72 (I 99.5) 37-45 Table 1 The counties from which the moose tissue samples were collected, representing different regions of Sweden Regions Northern
Central
Southern
Eastern
Western
Norrbotten VSstemorrland
JSrntland Givleborg Kopparberg Uppsala
Blekinge Kristianstad MahnGhus
Kahnar
Halland
A combination of continuous hydride generation by flow injection and atomic absorption spectrometry [12,13] was used for Se determination. Atomization was carried out in an electrically heated quartz cuvette. The analytical system and the collection and calculation of analytical data were controlled by computer. The analytical results are presented on a wet weight basis (mg/kg wet wt.). The analytical method was checked by analyzing standard reference materials (Bovine Liver NIST 1577 and 1577a). 2.3. Statistical methods For calculations of the analytical results, the programme Statgraphics (Version: 5.1, STSC, Inc., Rockville, MD) was used. As the analytical data were not normally distributed (Kolmogorov-Smirnov test), nonparametric methods were used in the calculations. The age dependence of the Se concentrations was checked by testing calves (0.5 year) against 1.5year-old animals using the Kruskal-Wallis test. In addition, all age groups were tested against Se concentrations with Spearman-rank correlation. The distribution pattern of the Se concentrations in livers from the 12 counties is presented in the form of a notched box-and-whisker plot. 3. Results
The selenium concentrations in the ers from the 12 counties are presented as mean and standard deviation (Z f dian, and range. High Se concentrations with median 0.25 mg Se/kg wet wt. were found in
moose livin Table 2 S.D.), mevalues > the north-
ern and southern parts of the county of Alvsborg (western region), as well as in the county of Gavleborg (central region). The highest single concentration (3.05 mg/kg) was noted in a liver from the county of Gavleborg. Low Se concentrations with median values of about 0.09-0.1 mg/kg were obtained in the northern and southern parts of the county of Kalmar (eastern region) and in the counties of Uppsala and Jlmtland (both central regions). The lowest Se values, 0.027 and 0.029 mg/kg, were found in the counties of Vasternorrland and Jamtland (central region), respectively. The total material is presented graphically in the form of a notched box-and-whisker plot in Fig. 2. For better resolution in the figure, 1 mg Se/kg liver was chosen as the upper level. Wide ranges with high concentrations (some of them over 2 mg Se/kg), resulting in long boxes and many outliers (+ sign) are seen in Fig. 2 for the counties of Blekinge, Kristianstad, Alvsborg, Kopparberg and Gavleborg (K, L, P, W and Y, respectively). A narrow distribution pattern, short boxes and few outliers are shown by the counties of Uppsala (C) and northern Kalmar (Hn). The narrow box for Malmijhus (M) is proportional to the few tissue samples investigated (N = 47). The largest number of samples analyzed came from Norrbotten (BD) (N = 281), resulting in a broad box. The median values for the county of Alvsborg (Pn and Ps) on the west coast of Sweden are significantly higher than those for the counties of Uppsala (C) and Kalmar (Hn and Hs) on the east coast (Kruskal-Wallis test, P < 0.001) (Fig. 2). The ages of the animals investigated were between 0.5 and 14.5 years. The group of animals
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1.5 years old represented 59% of the whole material, and calves only 15%. However, the age dis-
mg
Se/kg
> 0.1
liver
W.W.
- 5 0.2 > 0.2
C
not
analyzed
Total Environment
I72 (1995)
37-45
tribution of the moose populations in the different counties varied. Using Spearman-rank correlation, a positive correlation was found between Se concentration in the liver and increasing age in three out of 12 counties, namely northern Kalmar (Y = 0.27, P < O.OOl>, Kristianstad (r = 0.25, P < 0.011, and Halland (r = 0.23, P < 0.01). A significantly higher Se concentration was noted in the 1.5 year-old animals than in the calves in the regions of north Kalmar (P < O.OOl), south Kalmar (P < 0.05) and Kristianstad (P < O.Ol), as well as in the whole material (P < 0.01). The material was divided into four Se concentration ranges in each of the 12 counties and the percentage number of samples in each range is shown in Fig. 3. In the counties of Kristianstad (L), north and south Alvsborg (Pn and Ps), Kopparberg (W), and GGleborg (X), Se concentrations of over 0.2 mg Se/kg were found in more than 50% of the material. The corresponding figures (> 0.2 mg Se/kg) for the other counties were: north Kalmar (Hn) l%, south Kalmar (Hs) 14%, Uppsala (C) 2%, and Jlmtland (Z) 15%. Animals with concentrations below 0.1 mg Se/kg were found in the following counties in the given proportions: Uppsala (C) 47%, north Kalmar (Hn) 63%, south Kalmlar (Hs) 59%, Vasternorrland (Y) 36%, and Jamtland (Z> 46%. 4. Discussion
Fig. 1. Selenium in livers collected in 1982 from moose in 12 counties representing 14 regions of Sweden. Median values were calculated for each respective region. The values are classified into three ranges and depicted by shaded areas. The darker the shading, the higher the hepatic selenium concentration. The values for the unshaded areas have not yet been analyzed. The letters given in the figure are the letters used officially in Sweden to represent the different counties. Those included in this investigation are: BD, county of Norrbotten; C, Uppsala; H, Kalmar; K, Blekinge; L, Kristianstad; M, Malmlihus; N, Halland; P, hvsborg; W, Kopparberg; X, Gvleborg; Y, Vastemorrland; Z, Jlmtland;
Reports on Se concentrations in organs from terrestrial wild animals are rare. Ohlendorf 1221 found Se concentrations of < 2 mg/kg dry weight (dry wt.) in livers from wild herbivorous mammals, corresponding to about < 0.4-0.5 mg Se/kg wet wt. Selenium concentrations in moose liver have been reported from Norway by Froeslie et al. [lO,ll]. Their tissue material was collected from six regions. The mean Se concentrations (* S.D.) in their whole material (N = 542) was 0.62 f 0.95 (range 0.07-5.6) mg/kg wet wt., compared with that in the present study (N = 2080) of 0.25 + 0.29 (range 0.03-3.1) mg/kg wet wt. In the Norwegian study, median values were also presented for each region. The highest median value, 1.0 mg/kg, was found in Finnmark in
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the northern part of Norway, and the lowest, 0.18 mg/kg, in Hedmark and Oppland, in the central part of the country. The corresponding values in the present study were 0.29 mg/kg in the southern part of the county of Avsborg on the west coast of Sweden and 0.09 mg/kg in the county of Kalmar on the east coast.
Determinations of the Se concentrations in biogeochemical samples (plant roots from the banks of small streams) by the Geological Survey of Sweden (SGU) revealed a similar pattern with high concentrations on the west and south coast and low on the east coast. In the present investigation, high median Se concentrations were found in moose liver samples from the western regions of Sweden, such as the northern and western areas of the county of hvsborg, and from the county of Kristianstad in the south. Low liver Se concentrations were found in the counties of Uppsala (central region) and north and south Kalmar (eastern region). These findings are in good accordance with the observations on biogeochemical samples. The Se concentration is low in the Scandinavian Precambrian rock and also in the soil. By weathering and microbial activity, insoluble Se compounds are converted into soluble forms available to plants. These compounds can also be leached or volatilized 13,141. Younger sedimentary rocks, such as limestone in the province of Sk&e in the south of Sweden, contain higher Se concentrations, as shown in the investigation of biogeochemical samples [4]. The total Se content of the soil is not well
4.1. Regional diRerewes
Increased Se concentrations were found in the livers from the moose in the south of Norway, especially in areas near the coast, an observation which could be explained by atmospheric deposition [lo]. This finding correlated well with the results of investigations in forest moss samples, which showed higher Se concentrations in the southern and south-western parts of Norway. Norwegian 1191 and Swedish studies 1161 have shown similarities in Se concentrations in coastal forest soils. Increased concentrations were found on the Norwegian south and south-east coast, as well as on the Swedish south-west coast compared with the east coast. As plausible explanations for these findings, high mean annual precipitation, deposition from natural marine sources, and anthropogenic contributions from industrial areas were suggested. Table 2 Selenium
concentrations
in moose
County
liver
N
from
12 counties,
41
172 (199.5) 37-45
representing
14 regions
of Sweden
%
&S.D.
Median
Range
0.197 0.102 0.099 0.128 0.311 0.332 0.198 0.292
0.146 0.033 0.043 0.110 0.413 0.252 0.114 0.321
0.155 0.101 0.094 0.093 0.158 0.227 0.152 0.182
0.048-1.633 0.042-0.257 0.053-0.585 0.046-0.633 0.050-2.754 0.066-1.572 0.090-0.593 0.071-2.158
Norrbotten (BD) Uppsala (C) Kalmar, north (HII) Kalmar, south (Hs) Blekinge (K) Kristianstad (L) Malmohus (M> Halland (N)
281 150 183 80 120 149 47 128
Avsborg,
(Pn)
1.59
0.373
0.375
0.277
0.07.5-2.672
hvsborg, south (Ps) Kopparberg (W) Gkleborg (X) Vastemonland (Y) Jamtland (Z)
167 169 154 122 171
0.462 0.309 0.383 0.146 0.132
0.437 0.272 0.398 0.099 0.105
0.290 0.226 0.258 0.124 0.098
0.065-2.061 0.057-2.320 0.050-3.054 0.027-0.555 0.029-0.695
north
Total N, number weight.
2080 of liver
samples;
means
0.250 and standard
deviations
0.288 (ji. +- SD.),
0.151 median
values
0.027-3.054 and ranges
are expressed
in mg Se/kg
wet
42
V. Galgan, A. Frank
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C
/The
Hn
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Hs
K
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L
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M
N
Pn
172 (1995) 37-45
Ps
W
X
Y
2
Fig. 2. Notched box-and-whisker plots showing selenium concentrations in moose liver specimens from 12 Swedish counties, expressed in mg/kg liver wet weight. The concentration of 1 mg Se/kg liver was chosen as the upper limit. For explanation of letters representing counties, see Fig. 1. Hn and Hs, north and south Kalmar; Pn and Ps, north and south kvsborg.
correlated to the Se content of plants in general. Its availability to plants is influenced by several factors, including the pH and the microflora of the soil and waters, climatological factors, and interaction with compounds of different elements, as well as by fertilization [14,15]. In acid soils (pH 4.5-6.5) and under high moisture conditions, Se in the form of selenite is bound to colloids, as
iron hydroxide selenium complexes [5]. These are insoluble and less available to plants. In basic soils (pH 7.5~8.5), Se is present as soluble selenate, which is taken up by plants especially when precipitation and leaching are low, thus contributing to the Se nutrition in herbivorous animals. Other elements, such as sulfur, may interact and influence the uptake of Se by plants, which is a
T/. Galgan,
A. Frank/The
Science of the Total Environment
mg
K
L M
N
Pn Ps W
X
Y
Z
Se/kg
liver
I
5 0.05
I
5 0.05
- I 0.1
5 0.01
- s 0.2
B
ED C Hn Hs
43
172 (1995) 37-45
W.W.
a- 0.2
Tot
Fig. 3. Selenium in moose liver. Percentage number of samples in different Se concentration ranges in the 12 counties investigated and in the entire material (Tot). For explanation of letters representing counties, see Fig. 1. Hn and Hs, north and south Kalmar; Pn and Ps, north and south Avsborg.
passive procedure [6]. Sulfate in high concentrations in soils competes with Se compounds, causing the Se uptake by plants to decrease. The Swedish west coast is strongly affected by acidification and is one of the areas that suffers most from atmospheric pollution by sulfuric compounds and from environmental pollution from the European continent and Great Britain. The effect of acidification has been demonstrated in an investigation of the Cd burden of moose from
different geographic regions of Sweden [9]. A heavy Cd burden was found in the strongly aciditied southern and western parts of the country, and declined to the east and north. Despite heavy acidification, high Se concentrations were observed on the west coast of Sweden in the present material. It would seem that atmospheric deposition from marine and anthropogenic sources is an important factor in Se nutrition.
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4.2. Age dependency
In humans as well as in animals, the Se concentration decreases in the liver after birth 120,281. As the concentration of Se in milk is low, the liver and probably also some other organs serve as a selenium depot during the first period of life. Thus, lower Se concentrations are expected in the liver from moose calves than from adult animals. Higher concentrations in yearlings than in calves were observed for the entire material (2080 animals), as well as for the material from the counties of Kalmar and Kristianstad. However, a statistically significant correlation between the age of the animals and their hepatic Se concentrations was only found for three regions (north Kalmar, Kristianstad and Halland). In the present investigation, the group of yearlings was the predominant age group, representing 59% of the animals, whereas the group of calves consisted of only 15%. In addition, the age distribution varies between the regions. The growth rate of calves is highest during the first months of life, i.e. between July and September [26]. The calves begin to graze very young, some days after birth. However, the suckling period lasts until November-December. At the time of the hunting season, the calves seem to have almost reached the nutritional Se status of that of the adult animals. Thus, it is difficult to recognize concentration differences within the material. Another reason for the lack of a statistically significant difference in the hepatic Se concentration between calves and adults in some regions could be the relatively small number of calves in the groups. Obviously, the hepatic Se concentrations in calves and adults become equalized at the time of hunting, and in general no difference was found. Further, even within a homogeneous age group of moose, great variations in Se concentrations were observed. Consequently, the entire material was presented without age grouping. No correlation between age and hepatic Se concentration was found in moose in the Norwegian investigation 1111. 4.3. Nuttitional
Available
data for evaluation
Se status in wild animals are limited. Values obtained in domestic ruminants may be used for this purpose [6]. Concentrations in the liver of above 0.3 mg Se/kg wet wt. are considered sufficient [2], and between 0.15 and 0.3 marginal. The selenium status in cattle is evaluated according to similar principles at the National Veterinary Institute. Concentrations 5 0.05 mg Se/kg liver are regarded as severe Se deficiency, > 0.05-10.1 as deficiency, and > 0.1-s 0.2 as marginal/insufficient. Liver Se concentrations above this latter value are regarded as adequate. According to these criteria, 50-60% of the animals in the counties of Uppsala (central region) and Kalmar (eastern region) have an insufficient or marginal Se status. These latter counties were found to be the most Se poor regions in the present investigation (Fig. 3). Selenium-deficiency diseases in wild animals are difficult to diagnose. Clinical signs are seldom apparent. Subclinical deficiency may exist, but is difficult to recognize. The most plausible way of ascertaining a subclinical deficiency status is by supplementation, which will result in improved reproduction, as reported in deer [6]. In addition, as mentioned previously, Se together with other compounds such as vitamin E are involved in the protection of the organism against oxidative damage [27]. References [II
El
[31 [41
[Sl
Se status
of the optimum
172 (1995) 37-45
D. Behne, C. Weiss-Nowak, M. Kalckhisch, C. Westphal, H. Gessner and A. Kyriakopoulos, Studies on new mammalian selenoproteins, in M. Anke, D. Meissner and C.F. Mills (Eds.), Trace Elements in Man and Animals, TEMA 8, Proceedings of an International Symposium, 1993, pp. 516-524. D.C. Blood and O.M. Radostits, Veterinary Medicine. A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats, and Horse, 7th edn., Ball&e Tindall, London, 1989, pp. 1187-1202. G.F. Combs, Jr., S.B. Combs, 2. Selenium in the environment, in The Role of Selenium in Nutrition, Academic Press, New York, 1986, pp. l-40. J. Ek, S.O. Ohlsson and 0. Selinus, Bly, kadmium, selen - hela Sverige kartllggs. (Lead, Cadmium, selenium the whole of Sweden is mapped.) Forskning och Framsteg 2/1988, in Swedish. L. Fishbein, Selenium, in E. Merian (Ed.), Metals and their Compounds in the Environment, VCH, Weinheim, 1991, pp. 1153-1190.
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W.T. Flueck, Moglicher Einfluss von Immissionen auf die Spuren-elementversorgung wildlebender Wiederkauer: Selen als Beispiel. Z. Jagdwiss., 36 (1990) 179-185. Automated wet ashing and multi-metal de171 A. Frank, termination in biological materials by atomic absorption spectrometry. Z. Anal. Chem., 279 (1976) 101-102. accessory to an automated wet t81 A. Frank, Semi-micro digestion system for ashing small sample amounts, in P. Bratter and P. Schramel (Eds.), Trace Element Analysis in Medicine and Biology, Vol. 5, Walter de Gruyter, Berlin, 1988, pp. 78-83. and L.R. Petersson, Assessment of bioavail[91 A. Frank ability of cadmium in the Swedish environment using the moose (Alces alces) as indicator. Z. Anal. Chem., 317 (1984) 652-6.53. DOI A. Froeslie, G. Norheim, J.P. Rambaek and E. Steinnes, Levels of trace elements in liver from Norwegian moose, reindeer and red deer in relation to atmospheric deposition. Acta Vet. Stand., 25 (1984) 333-345. 1111 A. Froeslie, G. Holt, R. Hoeie and A. Haugen, Levels of copper, selenium and zinc in liver of Norwegian moose ( Alces alces), reindeer (Rangifer tarandus), roedeer (Capreolus capreolus) and hare (Lepus timidus). Norsk Landbruksforskning, 1 (1987) 243-249. 1121 V. Galgan and A. Frank, Automated system for determination of selenium in biological materials, in P. Bratter and P. Schramel (Eds.), Trace Element Analysis in Medicine and Biology, Vol. 5, Walter de Gruyter, Berlin, 1988, pp. 84-89. Notes and comments on the [131 V. Galgan and A. Frank, determination of selenium in biological materials. Norwegian J. Agric. Sci. Suppl., 11 (1993) 57-74. U.C. Gupta, M. Lamand and T. [141 G. Gissel-Nielsen, Westermarck, Selenium in soils and plants and its importance in livestock and human nutrition. Adv. Agronomy, 37 (1984) 397-460. General aspects of selenium fertilizaWI G. Gissel-Nielsen, tion. Norwegian J. Agric. Sci. Suppl., 11 (1993) 135-140. [I61 L. Johnsson, Se-levels in the Mor layer of Swedish forest soils. Swed. J. Agric. Res., 19 (1987) 21-28. Relation of selenium to other antioxidants, 1171 B. Karlmark, with special reference to free radicals. Norwegian J. Agric. Sci. Suppl., 11 (1993) 11-20.
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H.-J.S. Larsen, Relations between selenium and immunity. Norwegian J. Agric. Sci. Suppl., 11 (1993) 105-119. of sele1191 J. L%g and E. Steilmes, Regional distribution nium and arsenic in humus layers of Norwegian forest soils. Geoderma, 20 (1978) 3-14. DO1 D.C. Mahan, A.L. Moxon and M. Hubbard, Efficacy of inorganic selenium supplementation to sow diets on resulting carry-over to their progeny. J. Anim. Sci., 46 (1977) 738-746. E. Norrman, Selen ett livsniidvandigt splramne. [Xl (Selenium - an essential trace element.1 Aktuellt f&n lantbruksuniversitetet. nr. 324, Husdjur, Uppsala, 1983, in Swedish. Bioaccumulation and effects of seleD-4 H.M. Ohlendorf, nium in wildlife, in Selenium in Agriculture and the Environment, SSSA Special Publication no. 23, 1989, Madison, WI, USA, pp. 133-177. [231 B. Pehrson, Diseases and diffuse disorders related to selenium deficiencies in ruminants. Norwegian J. Agric. Sci. Suppl., 11 (1993) 79-93. 1241 J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, D.G. Hafeman and W.G. Hoekstra, Selenium: biochemical role as a component of glutathione peroxidase. Science, 179 (1973) 588-590. I251 K. Schwarz and C.M. Foltz, Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Sot., 79 (1957) 3292-3293. (The moose - a D.61 F. S&felt, Algen - en presentation. presentation), in H. Ekman, N. Hermansson, J.O. Pettersson, J. Riilcker, M. Steen and F. Stilfelt (Eds.), Algen - djuret, skijtseln och jakten, Svenska JPgarefiirbundet, Spinga, Sweden, 1992, pp. 46-49, in Swedish. I271 D.E. Ullrey, S.M. Schmitt, T.M. Cooley, P.K. Ku and P.A. Whetter, Selenium, vitamin E and capture myopathy in white-tailed deer, in C.F. Mills, I. Bremner and J.K. Chesters (Eds.), Trace Elements in Man and Animals TEMA 5, Proceedings of the International Symposium, 1984, pp. 113-115. Selenium content of tissues in Finnish PSI T. Westermarck, infants and adults with various diseases, and studies on the effects of selenium supplementation in neuronal ceroid lipofuscinosis patients. Acta Pharmacol. Toxicol., 41 (1977) 121-128.