Heavy metals in reindeer and their forage plants

A n attempt was made to assess the level of heavy metal transfer from forage plants to reindeer (Rangifer tarandus L.) in an area in northern Lapland affected from dust from an open pit copper mine. Botanical analyses of rumen contents from reindeer provided information about the main plant species in the diet. Representative plant material was collected from sample plots within an 8 km radius from the central part of the mine and from a reference area situated about 200 km upwind of the mining site. The following plant species were analysed: Bryoria jremontii, Br. juscescens, Cladina rangiferina, Equisetum fluviatile, Descbampsiaflexuosa, Eriopborum vaginatum, Salix glauca, Salix pbylicifolia, Betula nana, and Vaccinium myrtillus. The greatest difference between metal concentrations in the plants collected from dust contaminated area and from the reference area was found in lichens. Copper is the main metallic component of the ore and was found in higher concentrations in lichens coming from the area around the mine than in lichens from the reference area. Smaller differences were found in vascular plants. Dust particles, remaining on outer surfaces after snow smelt contributed to a limited extent to the metal contents. Species—specific accumulation of metals was observed in some plants. The uptake of lead and cadmium in some vascular plants was somewhat higher in the reference area compared with plants growing in the perifery of the mining center, probably due to the metal concentrations in the bedrock. Organ material (liver and kidney) was collected from reindeer in both areas. N o noticable effect on metal concentrations in the liver of the reindeer were found. Although the lead, cadmium and copper concentrations were higher in the organs collected from animals in the reference area than in those from the mining area, the levels were still below the concentrations regarded as harmful for the animals from toxicological point of view. The material collected during this study would also lend itself to research into the trace element nutrition of the reindeer.


Introduction
Copper is extracted through open pit mining at Aitik, county of Norbotten, Sweden (Fig. 1.).
Applying for permission to increase production Boliden Mining Co. Ltd. was instructed by the National Franchise Board for Environment Protection to examine whether content of certain metals in and on reindeer forage plants and reindeer organ tissues exceeded current toxicological standards as prerequisites for a good state of health.This clam was caused by the fact that the mining area is situated within the summer range of Gallivare Forest Saami Community and that fears had been uttered by the Saami Community that the reindeer (and consequently the meat market) might be affected by heavy metals deposited with the dust.

The contents of copper and zinc in the moss
Pleurozium schreberi and reindeer lichen (Cladi-na spp.) growing around the mine were analysed by Ruhling (1978Ruhling ( 5C 1979)).High copper concentrations (1600 mg/kg) were observed in the moss growing in the immediate vicinity of the mine, and concentrations over 100 mg/kg were found as far as at a three kilometre distance.
The regional background level of 5 -10 mg/kg was not reached until some ten kilometres from the mine, and elevated zinc contents were observed only in the immediate neighbourhood of the mine centre.Similar conditions but with lower metal concentrations (not more than 370 mg Cu/kg) were observed in reindeer lichen (Cladina rangiferina).
The Aitik area is used by the Ratukka Group of the Gallivare Saami Community (SOU 1966).Direct movements between seasonal grazing areas are made only to a limited extent.
The movements of the reindeer herds are described as "roving" or migration grazing (Planverket, 1974).Grazing of the area occurs mainly when there is no snow on the ground (July through September) but reindeer herds may be present in the area even at other times of the year (Erik Lindstrom, the County Government; Ake Nordvall, the Ratukka Group).

Aims
This study aims at -clarifying the reindeer's choice of forage plants in the Aitik area during summer time, -studying the content of metals in important reindeer forage plants, and -studying the metal content in reindeer organs.
Winds from W -NW and from SE are dominating throughout the year (Fig. 4).
The reference area was considered free of deposition and man-made effects, but it is situated in the periphery of an ore province (Ores, industrial minerals, and rocks in Sweden.Map, SGU, ser. Ba No. 29, 1979).

Materials
Collecting has been carried out around Aitik and within a reference area (cf.In mid-May, mid-June, mid-July, and mid-August 1982, plant samples were collected within all sampling plots.As shown in Fig. 3 and Table 1, stress was laid on plants of importance to reindeer during the sampling period.
In July 1985 Bryoria fuscescens and Br.fremontii were once more sampled within both areas.
In August 1982, plant material equivalent to that of Aitik was collected at five locations within the reference area (Fig. 1).
Reindeer lichen from the June sampling at Aitik was used for determining wheter a metal accumulation in reindeer lichen occurred after the snow-melting period.
The plant material was classified, air dried, and packed for subsequent chemical analysis.
b) Sampling of rumen content for quantitative estimation of botanical composition.
During two periods (June 11-14 and August 12 -19) five grazing reindeer, 10 animals totally, were shot in the Aitik area, and on one occasion (July 21) five reindeer were sampled during an ear-tagging session (Fig. 2).
From each animal about 2 litres of stirred rumen content was collected and preserved for quantitative botanical analysis.
Figure 2. The sampling sites of reindeer around Aitik.

c) Sampling of organ material
Organ tissue samples were collected from the 15 reindeer which had also contributed rumen samples (Aitik I).
Organ tissue samples were also collected from 29 reindeer (Aitik II) at routine slaughter in the autumn.Of these, 9 were slaughtered at Sjungberget, north of the mine, and 20 at Kartijarvi south of the mining area (cf.Fig. 2).
It should be pointed out that the degree of contact with the vicinity of the mine of the Aitik II reindeer has escaped determination.However, according to the local herdsmen most of the Ratukka-reindeer regularly utilize summer pastures round Aitik.
Reference samples from 20 reindeer were collected in connection with regular slaughter at Aberget (Fig. 1) in mid-December.A herd that had previously grazed within the reference area was then rounded up during its migration to the winter pastures.
The organ tissue samples consisted of liver and kidneys.The left metacarpal bone (for estimation of nutritional state) and the left mandibular bone (for age determination) were sampled as well.The Aitik material was examined at the SVA (the National Veterinary Institute), whereas corresponding investigations of the reference material was carried out at the slaughter site by members of the sampling staff.
The organ material was transferred to the SVA, where it was stored at -20° C until chemical analysis was performed.Sweden) according to a standard ashing program (Frank 1976(Frank , 1983)).
The air dried plant samples were cut to small pieces.One gram of each sample was wet digested according to the standard procedure.
Analysis.-The residue after wet ashing was evaporated to dryness and the dry residue dissolved in an ionic buffer solution for analysis using a direct current plasma-tomic emission spectrometer (SpectraSpan IIIA, Applied Research Laboratories Inc., Sunland, USA).The analysis comprised simultaneous determination of the concentration of i.a.lead, cadmium, copper, and zinc (Frank &C Petersson, 1983).
Analytical results for plant material are given as mg/kg air dry weight, which means about 7 -9 per cent water content; organ material results are given as mg/kg wet weight.
Mercury in the livers from the Aitik I and Aitik II materials was determined using neutron activation analyses (IVL, Institutet for vattenoch luftvardsforskning, Stockholm, Sweden).The area frequency of plant groups was converted to frequency by weight using weight constants specific for plant groups and particle sizes (Eriksson et al., 1981).

Results and discussion
The summer diet of reindeer in the Aitik area

Plant material
Figure 5 shows the copper concentrations in Cladina rangiferina from the mining area.
The concentrations for copper, zinc, lead and cadmium are shown in Tables 2 and 3.
They show the lowest and highest metal concentrations found in reindeer forage plants in the Aitik and the reference areas, respectively, as well as the date of sampling.
Considering the presence of gradients in the material from the Aitik area, which may be Metals are transferred to and accumulated in lichens by airborn dust or dissolved in rain water (Monitor, 1987).According to Tuominen and Jaakola (1973), lichens also are able to take up and accumulate metals from the substratum via living or dead basal parts of thalli.
Metals are usually accumulated in lichens as ions adsorbed to cell walls (Monitor, 1987).The mining company, within its own measuring program, is continuosly monitoring the dust downfall and its copper concentration at 9 measuring stations around Aitik (Goransson, 1983). of the reference and the Aitik area are not as great.

The differences in copper concentration in
Betula nana between the reference area (2.3 -3.8 mg/kg) and Aitik are conspicuous.The highest concentration of 52 (June) and 25 (July) mg/kg were found near the centre of the mining area.
Of the grass species examined, Descbampsia flexuosa from the reference area has low copper concentrations (1.1 -1.9 mg/kg).The highest value from the mining area is 9 mg/kg.
Larger variations in concentration are shown by Eriopborum vaginatutn (cf.For Bryoria spp.higher lead concentrations can be recorded for Bryoria fuscescens than for Br.fremontii.In the latter species the lead concentrations exceeded the highest value of 5.9 mg/kg from the reference area at 7 measuring plots only, whereby the highest value round the mine was 15 mg/kg.In Br. fuscescens all values were higher (8.2 -30 mg/kg) than the highest ones of the reference area (cf.Table 3).
There is, however, a different picture to be seen when studying the lead uptake by vascular In similarity to lead it can be noted as a remarkable observation, that the lowest cadmium concentrations in Salix glauca and S.phylicifolia were higher in plants from the reference area (0.89 and 0.80 mg/kg, respectively) than in those from Aitik (0.39, 0.28, and 0.18 mg/kg).
This phenomenon can be interpreted, partly as an indication of species specificity, partly as an expression for the higher degree of mineralization of the soils of the reference area.
The cadmium uptake of the other vascular plants was largely of the same magnitude in the two areas examined, possibly somewhat larger in the reference area.
Even for cadmium the same phenomenon as for lead can be observed, namely that the downfall at the mine implies a certain cadmium burden, but the greater mineralization in the soils of the reference area seems to bring about an increased cadmium uptake by vascular plants.
A constant characteristic of the metal concentrations (copper, zinc, lead, and cadmium) in sampled Bryoria juscescens and Br.fremontii from Aitik was that the former had 138-282 per cent higher metal concentrations than the latter.Br. juscescens was collected on Norway spruce (Picea abies) whereas Br. fremontii was collected on Scots pine (Pinus sylvestris).One Table 5. Regional means (X), medians (X) and ranges (min -max) of lead, copper, zink and mercury in liver and kidney of reindeer (mg per kg wet weight) Rangifer, Special Issue No. 3, 1990.
Cu in Cladina rangiferina mg/kg 1401 0- Copper: Mean values (medians within brackets) for copper concentrations from Aitik I, Aitik II, and the reference area are 21 (16), 26 (19), and 29 (21) mg Cu/kg, respectively.Range was considerable and largely of the same magnitude in all groups, excepting the highest copper concentration in liver of 131 mg/kg, which was found in the group from Aitik II.The value is considered moderately elevated.In a material from Norway higher concentrations were observed, 3.8 per cent of the reindeer examined having copper levels of more than 150 mg/kg liver (Fraslie et al. 1987).
The copper concentrations in liver varied considerably, an observation which coincides with earlier data on copper in the livers of wild ruminants (Bonniwell 1986).The concentrations found in the present material were in several instances critically low, seen from the nutri-tional point of view.As upper limit 10 mg Cu/kg wet weight is regarded for copper deficiency in domestic ruminants, especially in sheep.Serious deficiency in considered at levels lower than 3 -5 mg/kg (Blood et al. 1989, Fraslie et al. 1987, Schwan et al. 1987).
If the same criteria are applied to reindeer, copper levels are inadequate in 27 percent of the Aitik I, in 31 per cent of the Aitik II, but in 5 per cent only of the reference material.No serious deficiency was found in our material, whereas in the Norwegian investigation 43 per cent of the material was reported to be deficient (Freslie et al. 1987).The copper supply of the reindeer in the present investigation seems to be better than that in the Norwegian one.
The renal copper levels are within normal values in all groups.The mean concentrations (medians within brackets) were 5.4 (5.3), 5.0 (4.8), and 4.0 (4.0) mg Cu/kg for Aitik I, Aitik II, and the reference area, respectively.It was interesting to note that the copper concentration in the livers was lowest in Aitik I and highest in the reference reindeer, where as in the kidneys the case was reversed.This fact is true both for mean values and medians (cf.Fig. 7).
The following thoughts may contribute to a   The constants A and B calculated for age dependent cadmium accumulation in kidney and liver for the three groups are shown in Table 7.  Cd mg/kg KIDNEY w.w.
Fig. 1 and Fig. 5).The test material consists of reindeer forage plants and reindeer organs.a) Sampling of plants To describe the distribution pattern of heavy metals within the Aitik area metal concentrations of some important reindeer forage plants were used.For chemical analysis the plants were collected at about the same time in both Aitik and the reference area.At Aitik sampling plots were located at a distance of 1000, 2000, 4000, and 8000 m from the centre of the mine along NW -SE and NE -SW transects.The transects coincide with dominant wind directions (Fig. 4).In addition plots were chosen in all points of the compass, right outside the fence surrounding the mine.
Pretreatment of samples.-Combustion of organ material (5 g liver and kidney, respectively) was performed by automatic wet digestion.An oxidizing mixture (15 ml) of nitric/perchloric acids = 7:3 (vol.:vol.) was added to the samples in boro-silicate glass tubes, in an electrically heated block of aluminium.During the automatic wet digestion time and temperatures are controlled by a microprocessor (Autostep, Digestor: Model 40, Tecator AB, Hoganas, Statistical analysis were performed by using standard programs (Statgraphics) for i.a.calculation of linear regression.b) Quantitative botanical analysis of rumen content One litre of stirred rumen content was washed through a set of six sieves (mesh size ranging HERBS DWARF SHRUBS between 4000 -125 n m).Particles smaller than 125 /j.m were discarded.The plant fragments were separated by aid of a grid and examined in a microscope.

Figure 3 Figure 3 .
Figure 3 illustrates that dwarf shrubs contribute as much as 21 -76 per cent to the summer diet of reindeer.In June Vaccinium myrtillus dominates, in July and August no species dominance was observed.The dwarf shrubs compartment then contains mainly Vaccinium spp., Calluna vulgaris, Empetrum nigrum, Andromeda polifolia and others.Leaves from Betula spp.and Salix spp.were found in the rumens during the entire summer.In August there was a noticeable increase from 15 per cent by weight to 53 per cent.In June Salix leaves dominated, in July Betula nana and in August Betula spp.Herbs occurred only in minor quantities (less than 1 per cent by weight).Graminoids quantitatively played a major role in June only, when 12 per cent by weight Equisetum fluviatile was frequent in June only, when it amounted to 13 per cent by weight.The high concentration of sodium (500 -600 mg Na/kg D.M. and values even as high as 2530 mg/kg D.M. was determined in the reference area) should be of great importance to reindeer as a major sodium source.The sodium concentration in other vascular plants as well as in lichens were mainly between 100 and 200 mg/kg D.M. Lichens occurred in the diet on all three sampling occasions.A marked increase was noted from 6 per cent in July to 17 per cent in August.The plant species preference shown by the Aitik reindeer corresponded fairly well to the observations of Skuncke (1968) and Waren¬ berg (1977) on domestic reindeer in Sweden, and those of Helle (1981) on wild Finnish forest reindeer (Rangifer tarandus fennicus).

For these 9 Figure 4 .
Fig.6).How much of the downfall from the time before measuring that had contributed to the copper content of CI. rangiferina is, however, not known.
plants.In the latter the lowest lead values of the plants from the reference area are often higher than those from, e.g.Beula nana, Salix glauca, 5. phylicifolia, Deschampsia flexuosa, Eriophorum vaginatum, and Equisetum fluviatile from Aitik.Thus, there are indications that the soils of the reference area are richer in minerals than those of Aitik, the latter mainly carrying cupreous minerals.The higher lead concentration of the Aitiklichens compared to those of the reference areas, may be explained both by anthropogenic sources in the ares, such as automobile transports etc. and by the lead concentration of the dust, the latter explanation finding support in the efficiency of Bryoria juscescens in dust enrichment.The results indicate that a certain deposition of lead occurs in the neighbourhood of the mine, but the mineralization and the lead uptake is larger in the vascular plants of the reference area than in those of Aitik.Cadmium: In Cladina rangiferina of the reference area the highest cadmium concentration was 0.18 mg/kg.In Aitik 0.28 and 0.30 mg/kg were found to be the highest values.Differences are greater in the efficiently dust filtering Bryoria spp.In Br. fremontii and Br.juscescens 0.41 and 0.58 mg Cd mg/kg, respectively, were observed as the highest concentrations in Aitik, whereas the cadmium concentration of the reference area did not exceed 0.27 mg/kg.Thus, cadmium levels increased towards the mining centre, probably as a result of increased dust downfall.

Figure 6 .
Figure 6.Dry deposition of copper at nine monitoring stations compared to copper concentrations in Cladina rangiferina (weighted mean values).might suggest that the canopy of Norway spruce funnels wet downfall towards the parts of the tree where arboreal lichens grow more efficiently than does the canopy of Scots pine.Organ material Results of analysis of reindeer organs from Aitik I and II and from the reference area are shown in Table 5 and are further illustrated by Figures 7 & 8.There was a wide range in several elements, and therefore both mean and median values are given.

Figure 7 .
Figure 7. Lead, copper, and zinc concentrations in the liver and kidney of the reindeer from Aitik I, Aitik II and reference areas.
burden of an animal species without considering the age of the individual.The longer an animal has lived in a certain environment, the higher the cadmium levels in the organs be-come.Cadmium accumulates in the liver, but mainly in the kidneys.The correlation between cadmium concentration and age can be approximately define in the form of a linear regression and be expressed by the equation: Y = A • X + B, where Y is the cadmium concentration of the organ and X is the age of the animal.A and B are constants, A (slope of the regression line) is characteristic for a certain region and/or population, and should be used together with B, the intercept of the regression line.

Figure 8
Figure 8 illustrates the relation between age and cadmium accumulation in kidney in the groups examined from Aitik I, Aitik II, and the reference area.Where the slope is the largest the same goes for the cadmium burden.The latter is largest in the reference area, as is the uptake of copper and lead, the explanation being the high degree of soil mineralization of the reference area.Similar calculations were reported in an investigation of Norwegian wild as well as domestic reindeer (Froslie et al. 1986).The regression coefficients (slopes) represent three regions in Norway with values of 1.8 in the South, 1.5 in the middle, and 0.25 in the

Figure 8 .
Figure 8. Age related cadmium accumulation in the kidney of reindeer from Aitik I, Aitik II and reference areas.The intercepts, shown in Fig. 8 and the values given in Table 7, but not given in the Norwegian investigation, show a great variation from positive to negative values depending on individual variations in the investigated groups.As the regression line in total expresses the relationship between age and cadmium concentration of the kidney, neither A nor B must be neglected.Consequently, only the slopes do not give the true values, on the other hand taken together with the intercept result in unrealistic values.The material has been treated also according to another statistical model, which is less prone to objection and allows comparison of the cad- visual estimation and not by closer examination of cut sections of the teeth means a somewhat uncertain procedure.Medians are preferable means and show the average yearly cadmium uptake in the respective organ in the actual population and the respective region in question.The calculation illustrate the same feature, namely, that the cadmium burdens like the uptake of other trace elements by animals and vascular plants were heaviest in the reference area.Summary Activities at the open pit copper mine at Aitik, county of Norrbotten, causes fallout of con-siderable amounts of metal-containing dust in the neighbourhood of the mining area.Mapping of metal contamination was made round the mine and within a reference area by analysing important reindeer forage plants as well as liver and kidneys from reindeer.The highest metal contents were demonstrated in lichens, which accumulate i.a.metals by airborne deposition and with precipitation.Elevated concentrations of copper could be registered within an area with the mine as its centre and with the radius of 8 kilometers.Outside that range background levels were generally obtained.For zinc, lead, and cadmium higher values than normal background levels were not found other than in close vicinity of the mine.Dominating winds contributed to an increased contamination NE -SE of the mine.Near the centre of the mining area copper concentration even of the vascular plants was higher, probably due to the metal-containing dust.A clear trend towards elevated metal concentration in the vascular plants of the reference area could also be registered, even though no notable presence of airborne dust could be observed.This phenomenon is thought to be due to the fact that the reference area is situated on the border to an ore province.The finding even of a species specific metal accumulation has been made during the study.Analysis of reindeer organs revealed largely the same offering of metals as did the analysis of the forage plant material.Copper, lead, and cadmium levels were higher in the reindeer of the reference area than in those of the mining area.In comparison with the animals from the reference area the differences in metal concentrations were smaller between reindeer from Aitik I and Aitik II.The latter, though, presented signs of the stronger influence of metals, which in turn, may be due to the possibility that they may have been grazing in other -unknown -areas with a heavy metal burden, or to the fact that they were slaughtered in September and had consequently the time to consume considerable amount of lichens after slaughtering of the Aitik I reindeer had occured.Reindeer migrate and utilize widely spread pastures.A local source of metal emission, consisting in the present investigation of copper mainly, does not seem to imply any health hazard worth mentioning.

Table 1 .
Sampled and analyzed forage plants

Table 2
. Minimum and maximum values of copper and zinc concentrations in forage plants of reindeer in Aitik (n= 18) and the reference area (n= 5) in June, July, August 1982, and in July 1985.Values in mg per kg DM. cens were registered.Among grasses Deschampsia flexuosa was common.

Table 3 .
Minimum and maximum (lowest and highest)values of lead and cadmium concentrations in forage plants of reindeer in Aitik (n= 18) and the reference region (n= 5) in June, July,August 1982,  and July 1985.Values in mg per kg DM.

Table 2
Zinc: Zinc concentrations in Cladina rangiferina from Aitik (13 -30 mg/kg) do not differ remarkably from those of the reference area (15 -22 mg/kg, cf.Table 2) but they are about 2 -3 times higher in Bryoria spp.from Aitik (61 and 86 mg/kg, respectively, as the maximum values), than from the reference area with zinc concentrations of 32 mg/kg as the highest values.In Cladina rangiferina from Aitik concentrations of 25 -56 mg/kg were found by Riihl-Differences in Vaccinium myrtillus, Betula nana, Graminidae, and Equisetum fluviatile are negligible as shown in Table 2. Lead: A study of the highest lead values found does not give any indication for a general tendency for lead accumulation.If, however, separate lead concentrations are studied in detail a less uniform picture emerges.

Table 8 .
No weighting for age distribution has been made, as the size of the population is limit-