SEASONAL PATTERN OF HEAVY METAL LEVELS IN KIDNEYS OF ROE DEER (Capreolus capreolus), SHOT IN SLOVENIA (CENTRAL EUROPE)

Bostjan Pokorny, Cvetka Ribaric-Lasnik

ERICo Velenje, Ecological Research & Industrial Cooperation, Koroska 58, 3320 Velenje, Slovenia

e-mail: bostjan.pokorny@erico.si

ABSTRACT

Kidneys of 164 roe deer were collected in three areas of Slovenia in 1998. Levels of Cd, Pb, Zn and Hg were measured and some factors influencing those levels were estimated in order to assure reliability of the species for the bioindicative purposes. We found strong seasonal influence on Hg and Pb levels, being significantly the highest in August and September. Cd and Zn levels showed similar seasonal pattern, although it was not as much pronounced. Seasonal variation in metal levels in roe deer tissues originate in both quantitative as well as qualitative (availability of different forage) differences in nutrition of the species. Since the seasonal peaks for majority of metals appear in late summer and early autumn, it seems that some plant taxa, such as fungi, might represent important source of heavy metal (especially Hg) intake in mammalian organism.

INTRODUCTION

Since it fulfils numerous criteria (e.g. widely geographic distribution, relatively small home range, territorial living and browsing nutrition strategy, relatively long life-span, huge availability of basic data, relatively simple sampling procedure) roe deer (Capreolus capreolus) has been often mentioned in the literature (e.g. Müller 1985, Wren 1986, Tataruch 1991, Holm 1993, Chyla et al. 1996) as a good or even excellent accumulative bioindicator of heavy metal burdens in the environment. The importance of the species for passive bioindication has been clearly confirmed also in Slovenia (a small country in the southern part of the Central Europe), where heavy metal levels (HML) in roe deer viscera were intensively studied in last decade (Doganoc / Sinigoj-Gacnik 1995, Gnamus et al. 1995, Pokorny 2000a, Pokorny / Ribaric-Lasnik 2000). However, some internal and external factors, such as age, sex and season of sample collection, might significantly affect HML in animal tissues (Wren 1986). Therefore, the influence of those factors should be estimated and eliminated to assure sufficient reliability of spatial and temporal comparisons, which are essential for bioindicative purposes.

Unlike age- and sex-related influences on HML in tissues of different deer species, which have been ubiquitously studied world-wide (e.g. Kocan et al. 1980, Müller 1985, Froslie et al. 1986, Eriksson et al. 1990, Tataruch 1991), ascendance of the season has been of minor interest so far. Nevertheless, some studies have revealed significantly higher autumnal HML in deer tissues in comparison with spring (Crete et al. 1989, Michalska / Zmudzki 1992, Holm 1993). On the contrary, Gamberg and Scheuhammer (1994) found the highest Cd levels in tissues of white-tailed deer in spring, while Blottner et al. (1999) did not reveal seasonal influence on Cd levels in roe deer kidneys. Since previous results about the seasonal influence on HML in deer tissues were both scarce and contradictive, we tried to ascertain the seasonal pattern of Cd, Pb, Hg and Zn levels in roe deer kidneys in some polluted as well as pristine areas of Slovenia.

METHODOLOGY

Kidneys of 164 roe deer were collected in the period from 16 May to 31 December 1998 by local hunter authorities in three areas of Slovenia: in the Salek Valley (a prealpine region in a central Slovenia, where the major Slovene power plant of Sostanj is located; n = 114), at the Pokljuka high plateau (an alpine region in northern Slovenia, situated in the Triglav National Park; n = 29) and in the Kocevsko region (a karst woodland in southern Slovenia with many close-to-nature and even primeval forests; n = 21). The last two regions were included in the study as control areas without any local emission sources, thus only limited number of samples were collected there. All animals were shot with a legal permission (see Pokorny 2000a for details).

After dissection tissues were packed separately, frozen and sent to the laboratory at ERICo Velenje, where they were stored at -18 °C till the analyses. Tissues were homogenised with Büchi-Mixer B-400 with a ceramic knife. A CEM MSP 1000 microwave digestion system was used for a wet digestion of samples. Tissue samples (1.4 to 1.5 g, wet weight) were weighed to ± 0.1 mg into microwave digestion vessels to which 7 ml of conc. HNO₃ and 1 ml of H₂O₂ were added. The inductively coupled plasma mass spectrometry (Hewlett Packard 4500 plus ICP-MS) was used for the determination of Cd, Pb and Zn, while hydride technique (atomic absorption spectrometer Perkih Elmer SIMAA 6000) was used for the determination of Hg. Standard reference material (Bovine liver BCR 185) was used for analytical quality control.

Log-transformation of elementary data was performed to assure normality and homogeneity of variances. One-way ANOVA with posterior calculation of lowest significant differences (LSD) was used for comparison among three periods: May–July (period I), August–September (II) and October–December (III), respectively. Periods were defined considering both social behaviour of the species (I: a formation of territories and a rut period; II: living in territories after the rut; III: a disintegration of territories and a formation of family groups) as well as availability of food sources (I: availability of young herbs and fresh browse; II: a nutrition with mature herbs, availability of fungi; III: browsing of woody plants, availability of maize). Data for both sexes were subsequently pooled since one previous study (Pokorny 2000b) had not revealed differences in levels of any single metal between males and females. However, dissimilar age-related accumulation of heavy metals was ascertained in the same study. On the contrary to Pb and Hg levels, which were proved to be independent of animal’s age, Cd and Zn levels significantly increased with the age of animal analysed (ibid.). Therefore, we pooled data for all age classes before statistical processing of Pb and Hg, while for Cd and Zn only adults were taken into a consideration since this age class was seasonally the most equal distributed in the annual bag. Because of a sample size limitation, the seasonal influence on HML was fully studied in the Salek Valley only, while nothing but Hg was examined in both control areas. In the case that HML was below the detection limit of analytic method (<0.01 for Cd and Hg, <0.05 for Pb, <1.0 for Zn, respectively), half value of the detection limit was applied in data processing. In the following sections, all results are given as mg/kg on a wet weight basis.

RESULTS

The highest burdens of all metals of interest were measured in the period August–September (Table 1). The peak in punctually distribution of daily HML very obviously appeared between 220^th (in mid-August) and 280^th (in mid-October) consecutive day of the year (Graph 1), which strongly coincided with the second study period. However, the seasonal differences are significant only for Hg (p<0.001) and Pb (p<0.01). In the case of Cd and Zn, differences among periods are evident but not significant (Table 2) due to relatively small sample size as well as age-related influences, which predominate over seasonal behaviour of both metals.

Graph 1: Seasonal influence on Hg (A) and Pb (B) levels in kidneys of roe deer, shot in the Salek Valley in 1998. Each point represents metal content in kidney of every single animal with the corresponding day of culling. Beginning of the first period: 135^th consecutive day of the year; beginning of the second period: 213^th day; beginning of the third period: 273^th day.

Table 1: Levels of Hg, Pb, Cd and Zn (mg/kg wet weight) in kidneys of roe deer, shot in the Salek Valley in three seasons of 1998. The arithmetic means (ā) with the 95 % confidence intervals and range (Min, Max) are given.

	May–July n = 29 (Hg, Pb) or 15 (Cd, Zn)			August–September n = 43 (Hg, Pb) or 22 (Cd, Zn)			October–December n = 35 (Hg, Pb) or 12 (Cd, Zn)
	ā ± t0.05*SE	Min	Max	ā ± t0.05*SE	Min	Max	ā ± t0.05*SE	Min	Max
Hg	0.03 ± 0.01	0.01	0.14	0.27 ± 0.20	0.01	3.20	0.07 ± 0.03	0.01	0.60
Pb	0.12 ± 0.05	0.04	0.76	0.15 ± 0.04	0.01	0.54	0.08 ± 0.02	0.01	0.27
Cd	4.00 ± 1.26	1.29	11.4	7.23 ± 2.79	0.73	32.6	4.00 ± 1.18	0.88	7.40
Zn	51.5 ± 19.0	12.7	138	67.9 ± 22.7	12.8	239	34.9 ± 12.6	9.40	91.3

Table 2: Parameters of statistical comparison (one-way ANOVA) of HML (log-transformed data) in kidneys of roe deer, shot in the Salek Valley in three seasons of 1998 (for n see Table 1).

	Hg		Pb		Cd		Zn
	H(2) = 19.69; p<0.001^a		F(2,101) = 5.66; p<0.01		F(2,46) = 2.55; p>0.05		F(2,46) = 2.65; p>0.05
Season	Aug–Sep	Nov–Dec	Aug–Sep	Nov–Dec	Aug–Sep	Nov–Dec	Aug–Sep	Nov–Dec
May–Jul	p<0.001	p<0.01	NS	p<0.01	NS	NS	NS	NS
Aug–Sep	/	NS	/	p<0.05	/	NS	/	NS

NOTES:

a: Since log-transformation had not assured the homogeneity of variances, Mann-Whitney U-test was applied instead of ANOVA.

NS: Differences are not statistically significant.

DISCUSSION

Our results revealed that the season of shooting has a strong impact on heavy metal (especially Hg and Pb) levels in roe deer kidneys, being the highest in late summer and early autumn. Differences in availability of food sources (e. g. Tataruch 1991) as well as in physiological processes (e. g. Crete et al. 1989) have been employed as two major explanations for seasonal differences in HML in deer tissues. Inside physiological characteristics, three groups of processes should be discussed: a) processes which are directly connected with heavy metal intake rate (seasonally different energetic needs influence the quantity of food intake; e. g. Holm 1993); b) processes which are directly connected with elimination of heavy metals from soft tissues, such as antler growth (Sawicka-Kapusta 1981) or formation of foetus and lactation (Krynski et al. 1982); c) process of catabolism, which results in decreasing weight of viscera and consequently in increasing HML, expressed on organ weight basis in winter period (Crete et al. 1989). However, processes connected with elimination of heavy metals occur in close season of bucks hunting (antler growth generally begins in winter and ceases before the end of May) and does hunting (the hunting season begins in Slovenia on 1 September, whiles lactation ceases in early summer), therefore they have definitely no influences on results, obtained in our study. Moreover, if the process of catabolism provoked seasonal influences in HML, metal burdens in roe deer kidneys would be the highest in the last study period (see above), what is just the opposite to our results. Accordingly, our findings suggest that the seasonal variation of HML in roe deer tissues originate in differences in nutrition of the species.

In continental Europe, the intensity of food intake by roe deer is the highest in August and September, when an adult animal consumes at average 0.55 and 0.60 kg of dry plant matter per day, respectively (Drodz / Osiecki 1973, cit. Avila et al. 1999). Although differences in the ingestion rates between second and first study period are unimportant (an average consumption rate of 0.55 kg per day in period May-July), the differences between second and third period are much more pronounced (an average consumption rate of 0.55, 0.45 and 0.35 kg per day in October, November and December, respectively). As it is shown in Table 1, relative seasonal differences in levels of Pb, Cd and Zn in roe deer kidneys get along fairly well with differences in consumption rate among seasons. Accordingly, the quantity of ingested food, which depends on momentary energetic needs as well as availability of food sources, has doubtless strong influence on HML in roe deer tissues. However, differences in Hg levels among seasons are much higher as they would be expected considering seasonal differences in food intake only. Since seasonal peak of Hg (but also Pb) levels appears in very short period of time (Graph 1), it should be concluded that HML in roe deer tissues reflect the impact of some food sources, which accumulate high amounts of heavy metals, in summer–autumnal nutrition of the species.

Mushrooms are known to be an important food source for roe deer in late summer and early autumn (Adamic 1990, Tataruch 1991, Strandberg / Knudsen 1994). Because of specific anatomical and physiological characteristics (e. g. unprotected surface of their vegetative cells, the large ratio of surface to volume of the mycelium, mycelia are in contact with a large volume of the upper soil), higher fungi are very good bioaccumulators of heavy metals, especially Hg and Cd (Wodratschek / Röder 1993). In Slovenia, HML are much higher in fungi in comparison with some green plants (as much as one and two orders of magnitude for Cd and Hg, respectively; Byrne et al. 1976). To our best knowledge, fungi have not been interpreted as an important factor influencing HML in roe deer tissues so far. However, also radiocaesium burdens in roe deer tissues show similar seasonal pattern as heavy metals, with very evident peak between July and October (Kiefer et al. 1996 for Germany; Avila et al. 1999 for Sweden). Since Cs is markedly accumulated in fungi, which in Sweden represent about 76 % of total food intake by roe deer in summer-autumnal period (Strandberg / Knudsen 1994), it was concluded that mushrooms should be considered as very important source of Cs in this period (Avila et al. 1999). We believe that the same conclusion can be made also for heavy metals, especially Hg.

The significance of fungi ingestion for elevated levels of Hg in roe deer kidneys was confirmed also in the Kocevsko region and at the Pokljuka plateau, which are both known as important mushrooms gathering areas of Slovenia. Although they were included in the study as control areas without any local emission sources, the average Hg levels in roe deer tissues from both areas are approximately twofold higher in comparison with the Salek Valley (Pokorny 2000b). Elevated Hg levels in tissues of roe deer, shot at the Pokljuka plateau, were also found in year 1997 (Pokorny / Ribaric-Lasnik 2000). For understanding seasonal mechanisms it should be noted that the peak in Hg levels in the Kocevsko region coincided with the peak in the Salek Valley (between 220^th and 260^th consecutive day of the year), while at the Pokljuka plateau it appeared much later (between 250^th and 300^th day of the year). In comparison with the Salek Valley (altitude 300–900 m above sea level) and the Kocevsko region (400–1000 m), a vegetation period at the Pokljuka plateau (1200–1900 m) is much shorter, what influences HML in roe deer kidneys in two-way. Firstly, the winter begins earlier in the last area, thus the highest food intake rate of roe deer appears earlier at high plateau (for explanation about winter digestive strategy of the species, see Holand 1992). If only quantity of ingested food influenced HML in deer tissues, the peak would certainly appear sooner at the Pokljuka plateau. Secondly, vegetation period begins later in higher altitudes, therefore the start of growth period is delayed there. If the availability of certain plant species (e. g. fungi) exerted influence upon HML in deer tissues, the peak of HML would be delayed as well. Since our findings are in the opposition to the first thesis and in close agreement with the second one, it seems that the seasonal availability of some plant species is the most important factor, influencing Hg levels in roe deer kidneys. Considering well-known accumulative abilities of fungi together with their seasonal importance in the nutrition of roe deer, it appears that mushrooms can certainly represent an important source of heavy metal intake in organism of this species. The conclusion can be easily applied also for some other mammalians, including humans. However, a monitoring of metal levels in higher fungi and precise knowledge about feeding habits of consumers should be of great importance for drawing clear conclusions about their significance for heavy metal transfer along food chains.

ACKNOWLEDGEMENT

The research was financially supported by the Sostanj Thermal Power Plant and The Ministry of Science and Technology of the Republic of Slovenia. We are grateful to Simona Diklic, who helped us with the translation. Finally, we owe a deep dept of gratitude to all those who helped with the collection of samples.

REFERENCES

Adamic M (1990), Food Habits as an Element of the Management of Ungulate Populations with the Emphasis on the Red Deer (Cervus elaphus L.). Ljubljana, Institut za gozdno in lesno gospodarstvo (In Slovene).

Avila R, Johanson KJ, Bergström R (1999), J. Environ. Radioact. 46:99-112.

Blottner S, Frölich K, Roelants H, Streich J, Tataruch F (1999), Reproduc. Toxicol. 13:261-267.

Byrne AR, Ravnik V, Kosta L (1976), Sci. Total Environ. 6:65-78.

Chyla A, Lorenz K, Gaggi C, Renzoni A (1996), Environ. Protect. Engin. 22:65-70.

Crete M, Nault R, Walsh P, Benedetti J, Lefebvre MA, Weber J (1989), Sci. Total Environ. 80:103-112.

Doganoc D, Sinigoj-Gacnik K (1995), Bull. Environ. Contam. Toxicol. 54:166-170.

Eriksson O, Frank A, Nordkvist M, Petersson LR (1990), Rangifer 3:315-331.

Froslie A, Haugen A, Holt G, Norheim G (1986), Bull. Environ. Contam. Toxicol. 37:453-460.

Gamberg M, Scheuhammer AM (1994), Sci. Total Environ. 143:221-234.

Gnamus A, Horvat M, Stegnar P (1995), Z. Jagdwiss. 41:198-208

Holand O (1992), Can. J. Zool. 70:1331-1335.

Holm J (1993), Sci. Total Environ. 139/140:237-249.

Kiefer P, Pröhl G, Müller H, Lindner G, Drissner J, Zibold G (1996), Sci. Total. Environ. 192:49-61.

Kocan AA, Shaw MG, Edwards WC, Eve JH (1980), J. Wildl. Diseas. 16:593-596.

Krynski A, Kaluzinski J, Wlazelko M, Adamowski A (1982), Acta Theriol. 35:499-507.

Michalska K, Zmudzki J (1992), Medycyna Wet. 48:160-162.

Müller P (1985), Z. Jagdwiss. 31:146-153.

Pokorny B (2000a), Web Ecology (In Press).

Pokorny B (2000b), Levels of Heavy Metals in Roe Deer Tissues from Polluted Areas of Slovenia (the Salek Valley, the Zasavje Region and the Meza Valley). Velenje, ERICo (In Slovene).

Pokorny B, Ribaric-Lasnik C (2000), Bull. Environ. Contam. Toxicol. 64:20-26.

Sawicka-Kapusta K, Perzanowski K, Bobek B (1981), Ekologia Polska 29:353-359.

Strandberg M, Knudsen H (1994), J. Environ. Radioact. 23:189-203.

Tataruch F (1991), VDI Berichte 901:925-936.

Wondratschek I, Röder U (1993), In: Plants as Biomonitors: Indicators for Heavy Metals in the Terrestrial Environment. (B Markert, Editor), Weinheim, VCH Verlagsgesellschaft, pp. 345-363.

Wren CD (1986), Environ. Monit. Assess. 6:127-144.