Transfer of metals to plants and red deer in an old lead mining area in Spain

S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 6 ( 2 00 8 ) 2 8 7–2 97

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v

Transfer of metals to plants and red deer in an old lead mining area in Spain M.M. Reglero, L. Monsalve-González, M.A. Taggart, R. Mateo ⁎ Instituto de Investigación en Recursos Cinegéticos, IREC (CSIC, UCLM, JCCM), Ronda de Toledo s/n, 13071 Ciudad Real, Spain

AR TIC LE I N FO

ABS TR ACT

Article history:

Lead mining in the Sierra Madrona mountains and the valley of Alcudia in Southern Spain

Received 18 March 2008

began in the 1st millennium B.C., and the area was intermittently exploited up until the end

Received in revised form 3 June 2008

of the 20th century. The degree of contamination by Pb, Zn, Cd, Cu, As and Se of soil, water

Accepted 3 June 2008

and sediment, and the transfer to 13 species of plants, and then to red deer (Cervus elaphus)

Available online 14 July 2008

have been studied. Mined areas had higher concentrations in stream sediments than control areas. The highest concentrations were observed for Pb (1481 µg g− 1 d.w.) and As

Keywords:

(1880 µg g− 1) in the sediment of a stream flowing beside the spoil dump at Mina de Horcajo.

Cervus elaphus

Plants from mining sites contained consistently higher levels of Pb and As, and their

Large game

concentrations in plants were correlated. The highest concentrations of Pb were in

Food chain

Gramineae (Pb: 97.5, As: 2.4 µg g− 1 d.w.), and the lowest in elmleaf blackberry (Rubus

Acid drainage

ulmifolius). The highest mean liver concentrations were found in red deer from the mining

Plant tissue

sites for Pb (0.805 µg g− 1 d. w.), Cd (0.554 µg g− 1), Se (0.327 µg g− 1), and As (0.061 µg g− 1), although these were well below the levels associated with clinical poisoning. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Heavy metal contamination from mining and smelting activities has been studied worldwide in different environmental compartments, including water (Younger, 2001), soil (Álvarez et al., 2003) and biota (Brotheridge et al., 1998; Mateo et al., 2006; Wilson and Pyatt, 2007), and reviewed in several extensive reports (Dudka and Adriano, 1997; Johansen and Asmund, 2001; Johnson and Hallberg, 2005). As is the case in this study, several investigations have looked at the impact of Pb mines (Chukwuma, 1995; Pugh et al., 2002; Beyer et al., 2005; Deng et al., 2006; Li et al., 2006). Metal mining generates large volumes of crushed waste rock (spoil) and tailings with high concentrations of residual heavy metals. In former days such waste was left untreated after mines close, and then continues as a locally important long term, point source of environmental metal contamination (Jacob and Otte, 2004). As spoil weathers, metal sulphides become oxidised and sulphuric acid is generated which can cause acidification of nearby

waterbodies. Likewise, this oxidation and weathering process commonly increases both the solubility and bioavailability of many metals for plants inhabiting such areas. Some plants are accumulators or even hyperaccumulators of metals and such species may be used to decontaminate soils by “phytoextraction” (Yanqun et al., 2004; Freitas et al., 2004a; Sun et al., 2005). However, the accumulation of toxic heavy metals in plants may increase the risk of transfer to herbivorous wild mammals, or to livestock and game animals (Chaney, 1989; Madejón et al., 2002; Taggart et al., 2005). Moreover, aerial borne contamination of vegetation by wind blown particulates may occur within the vicinity of the spoil dump. This may occur to a greater degree if the spoil is of a fine particle size (more readily entrained) and if it remains without vegetation and therefore physically unstable. Aerial borne particles deposited on above ground plant surfaces may pose an important route of exposure for herbivores, as may be the direct inhalation of metal rich dust (Ma, 1996). Further, Beyer et al. (2007) observed extremely high Pb exposure in deer due

⁎ Corresponding author. Tel.: +34 926295450; fax: +34 926295451. E-mail address: [email protected] (R. Mateo). 0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2008.06.001

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to the ingestion of polluted soils near a mine, because soil can constitute a significant dietary component for certain vertebrate species, including mammals. Mining in the Sierra Madrona mountains and the valley of Alcudia in Southern Spain started at the beginning of the 1st millennium B.C. At the end of the 2nd century B.C., during the Roman expansion through the Iberian peninsula, there was an increase in the extraction and smelting of metals in this area, the focus being on the extraction of argentiferous galena. During the Middle Ages mining activities shifted towards the mercury deposits in Almadén (Valley of Alcudia), and then at the end of the 15th and during the 16th century, extraction of argentiferous galena re-emerged. The 17th and 18th centuries were relatively inactive but extensive mining operations recommenced during the 19th–20th centuries as technical improvements to the extraction process made the area more viable again (Hevia, 2003). This province became the major Pb producing area in Spain during the second half of the 19th century, but from the 1930s onwards, production fell and in 1988 the last mine, San Quintín, closed. Around 484 old mines and prospects are located in an area spanning approximately 2500 km2, the majority of which targeted Pb–Zn veins. Other metals were also produced in the area though, i.e., Ag, Cu, Sb, Sn, W, As and Bi. Although many of the worked deposits were quite small in size, San Quintín (500,000 Mt of Pb), Minas de Horcajo (300,000 Mt of Pb), and Diógenes (200,000 Mt of Pb) were all important producers within Spain (Palero-Fernández and Martín-Izard, 2005). The land affected by these mines has never been remediated. Currently, the Sierra Madrona area is largely devoted to and occupied by hunting estates, where several hundred red deer (Cervus elaphus) are bagged every year, and are destined for human consumption. The key aim of our research was therefore to establish the degree of contamination in this mining area of the soil, sediment, water and plants, and to consider, using red deer tissues, the degree of food-chain transfer of metals that may be occurring.

2.

Materials and methods

2.1.

Study area

Fig. 1 – Study area in the Province of Ciudad Real. Two sampling sites (Minas de Horcajo and Navalmartina) are in the mining area, and two are control sites (Torneros and Hornias Bajas).

Four sampling sites were selected within the region, two of them in the mining area of Sierra Madrona, and two in control areas in the Montes de Ciudad Real. One mine sampling site was at Minas de Horcajo-La Garganta in Almodóvar del Campo. The soil and plants sampled at this site were from the vegetated hillside adjacent to the spoil dump for the mine, and the water and sediment were collected from a creek flowing between the vegetated hillside and the dump. The dump itself was not sampled because no vegetation occurred there. The second sampling site within the mined zone was at Navalmartina in Fuencaliente. Although this site has no mines within the hunting estate, it is around 10 km downstream of a Roman smelter located in the Valderreprisa pass (Hevia, 2003). As such, this site is perhaps intermediate between the first, actively mined area, and the 2 control areas. Sampling sites in the control areas were in the hunting estates of Torneros in Saceruela and Hornias Bajas in Piedrabuena. All these sampling sites were in the Province of Ciudad Real.

2.2. The Sierra Madrona and Alcudia Valley region is in the northern part of the Eastern Sierra Morena in the Province of Ciudad Real, Southern Spain (Fig. 1). The elevation here ranges between 500 and 1300 m. The climate is Mediterranean sub-humid with marked seasons, and Mediterranean shrubland is dominant. The most important geological features in this area are a series of major Hercynian WNW–ESE trending anticlines and synclines. The lithostratigraphic sequence is composed of siliciclastic rocks with some interlayered volcanics and rare carbonate levels (Palero-Fernández and Martín-Izard, 2005). The Montes de Ciudad Real region (where the control sites were located) has altitudes between 500 and 1000 m, has similar geomorphological, climatic and biogeographic characteristics to Sierra Madrona, and is considered to be part of the same natural macroregion (García-Rayego, 1994). However, this area has not been widely mined historically and is not known to be rich in mineralised zones with high metal contents.

Sampling

Samples of 0–5 cm soil (100 g) were collected from each sampling site (n = 3 or 6 per site). Water (50 ml) and sediment (100 g) were collected from the nearest stream to the point of soil sampling when it was possible (n = 3 or 4 per site). The water was preserved with 1% nitric acid (Analytical grade, Panreac). The plants present at these points were sampled (50 g of leaves) if they were known to be components of the diet of red deer (Carranza, 2004). Three of the species sampled were collected at all four sampling sites: holm oak (Quercus ilex), gum rockrose (Cistus ladanifer), elmleaf blackberry (Rubus ulmifolius). Plus, various grasses (Gramineae) were also collected at all 4 sites. Grasses were sampled at early stages of growth and the species confirmed at the sampling site after total seed head development. The species analyzed were bristly dogstail (Cynosurus echinatus), medusahead (Taeniatherum caput-medusae), Holcus sp., slender false brome (Brachypodium sylvaticum),

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Bromus sp., tall oat-grass (Arrhenatherum album) and big quaking grass (Briza maxima). Nine further species were sampled but were not present at all sampling sites, these were Algerian oak (Quercus canariensis), large grey willow (Salix atrocinerea), oneseed hawthorn (Crataegus monogyna), sageleaf rockrose (Cistus salvifolius), strawberry tree (Arbutus unedo), dog rose (Rosa canina), narrow leafed ash (Fraxinus angustifolia), false olive (Phillyrea angustifolia) and heather (Erica sp.). In addition to leaves, acorns of Q. ilex and flowers of C. ladanifer were also sampled. Liver and bone (metacarpus) samples of 72 adult red deer (≥3 years old) were obtained from hunters during the autumn and winter of 2004–05 and 2005–06. The deer were from the hunting estates at La Garganta (surrounding Minas de Horcajo; n = 13), Navalmartina (n = 21), Hornias Bajas (n = 18) and Torneros (n = 20). Sex was recorded at the time of sampling and 45 males and 27 females were utilised.

2.3.

Laboratory analysis

Soil samples were passed through a 0.5 mm stainless-steel sieve to obtain a homogeneous sample and oven-dried at 50 °C to constant weight. Plant remains or small stones were removed from sediments prior to freeze-drying (Christ Alpha 1–2, Braun Biotech). Dry sediment was sieved as for soils. Leaves were thoroughly cleaned with distilled water, dried at 50 °C and homogenized using a cutting mill prior to digestion. Acorns, liver and bone were freeze-dried. Dry samples of liver, bone and leaves (0.5 g) were digested with 3 ml of HNO3 (69% Analytical Grade, Panreac), 1 ml of H2O2 (30% v/v Suprapur, Merck) and 4 ml of H2O (Milli-Q grade) with a microwave oven (Ethos E, Milestone). The program for the digestion began at a potency of 750 W and ramped for

15 min up to 180 °C, after which, samples were held for 10 min at 800 W and at a temperature of 180 °C. Digested samples were diluted to a final volume of 50 ml with Milli-Q H2O. The digestion of dry samples of soil and sediment (0.3 g) was also achieved with the microwave oven, but using 6 ml of HNO3, 2 ml of HF and 2 ml of HCl. Water samples (9 ml) were digested with 1 ml of HNO3 in the microwave oven and then diluted to a final volume of 10 ml. The analysis of As, Se, Cd and Pb was achieved using a graphite furnace-atomic absorption spectroscopy system (AAnalyst 800, Perkin Elmer) equipped with an autosampler (AS 800, Perkin Elmer), using 50 µg NH4H2PO4 and 3 µg Mg (NO3)2 as matrix modifiers in each atomization for Pb and Cd, and 5 µg Pd and 3 µg Mg(NO3)2 for As and Se. The analyses of Cu and Zn were achieved using a flame atomic absorption spectrometer system (AAnalyst 800 equipped with an autosampler AS 90 plus, Perkin Elmer). Solutions used for calibration were prepared from commercial stock standards with 1 g/l of each element (Panreac). The limits of detection (LODs, in µg g− 1 dry weight, back-calculated to concentrations in samples) were, for As: 0.022, Se: 0.124, Cd: 0.004, Zn: 1.06, Cu: 1.48 and Pb: 0.065. Blanks were processed in each batch of digestions. A certified reference sample of bush branches and leaves (NCS DC 73349A, China National Analysis Center for Iron and Steel) was analyzed (n = 5) and the recovery (mean % recovery ± SE) was 105.9 ± 1.4% for Pb, 97.8 ± 1.0% for Zn, 97.5 ± 1.5% for Cu and 121± 2.2% for As. A sample spiked with 0.1 µg g− 1 Cd was analyzed (n = 6) and the recovery was 96.2 ± 2%. A reference sample of bovine liver (BCR 185R, Community Bureau of Reference) was analyzed (n = 8) and the recovery was 94.4 ± 5.8% for Pb, 95.8 ± 0.7% for Zn, 100.4 ± 0.6% for Cu, 99.0 ± 4.0% for Cd, and 74.3 ± 2.2% for Se. A reference sample of bone ash (SRM 1400, National Institute of Standards and Technology, USA) with certified levels of Pb and Zn was

Table 1 – Concentrations (mean ± SE) of heavy metals and metalloids in water, sediment and soil from four sampling sites in the Province of Ciudad Real Sample (units)

Mining sites

−1 a

Water (ng ml )

Pb Cd As Se Sediment (μg g− 1) Pb Zn Cu Cd As Se Soil (μg g− 1) Pb Zn Cu Cd As Se

Control sites

Minas de Horcajo

Navalmartina

Hornias Bajas

Torneros

n=3 19.6 ± 6.5 0.020 ± 0.015 50.7 ± 14.8A nd n=2 1481 ± 1136A 72.8 ± 30.6AB 37.1 ± 29.7A 0.284 ± 0.192A 1880 ± 350A 0.994 ± 0.935 n=3 7.78 ± 1.39 31.2 ± 1.9AB 66.4 ± 6.1A 0.079 ± 0.010A 43.1 ± 8.8A 0.769 ± 0.031A

n=4 17.6 ± 10.3 0.007 ± 0.002 1.07 ± 0.24C nd n=3 44 ± 34B 79.3 ± 6.3A 25.6 ± 3.2A 0.072 ± 0.017AB 4.01 ± 0.77B 0.487 ± 0.076 n=6 16.9 ± 6.9 56.9 ± 17.4A 14.2 ± 7.0AB 0.093 ± 0.029A 4.53±0.39B 0.317 ± 0.062B

n=3 36.2 ± 17.3 nd 6.04 ± 2.54B 1.00 ± 0.35 n=3 16 ± 6B 19.4 ± 7.0BC 5.4 ± 1.6A 0.014 ± 0.010BC 1.51 ± 0.33C 0.111 ± 0.052 n=3 9.5 ± 3.5 12.8± 2.0B 4.0± 0.4B 0.018 ± 0.006B 1.94 ± 0.83C 0.282 ± 0.119B

n=3 3.0 ± 2.2 nd ndC 1.06 ± 0.41 n=3 10 ± 1B 8.0 ± 0.8C 0.394 ± 0.094B 0.001 ± 0.001C 2.55 ± 0.38BC 0.326 ± 0.185 n=3 19.4 ± 1.6 14.7 ± 3.2AB 4.3 ± 0.6B 0.036 ± 0.005AB 2.41 ± 0.32BC 0.346 ± 0.031B

Those means sharing the same capital letter did not differ among locations (Tukey test, p b 0.05). a Zn and Cu were below the detection limit by FAAS in water.

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analyzed (n= 12) and the recovery was 94.5 ±1.8% for Pb and 92.6 ± 1.3% for Zn. All concentrations are given in dry weight (d.w.).

2.4.

Statistical analysis

Data below the detection limit were assigned values of half of the respective LOD for each element. The concentrations of the elements were log-transformed to attain or approach a normal distribution of the data. Differences in the concentrations of each element among the four plant species (Q. ilex, C. ladanifer, R. ulmifolius and Gramineae) sampled in all locations were studied with a general linear model (GLM), with the factors species and location as independent variables. Differences among species at each location or among locations for each species, including incidences where species were not sampled at every location, were studied with one-way ANOVAs. The differences among locations in soil, water and sediment concentrations were also studied with one-way ANOVA tests. Post-hoc differences were established with Tukey tests. The relationship between the substrate (soil, sediment and water) concentration of the different elements and the plant concentrations from each sampling site was studied by linear correlations at a sampling point scale (n = 5–14). The differences among locations in concentrations of the different elements in red deer tissues were studied with a GLM, including sex as a factor. Linear correlations between the concentrations of the different elements were studied within each type of sample (soil, sediment, water, plants and deer tissues). Statistical significance was set at pb 0.05.

3.

Results

3.1. water

Metal levels and relationships in soil, sediment and

The concentration of Pb was significantly higher in the sediment of the stream flowing through Minas de Horcajo that in the other sampling sites (p = 0.008; Table 1; Fig, 2). However, this difference was not significant for Pb in terms of water or soil. The concentrations of As in water, sediment and soil were higher in Minas de Horcajo than in the other sites (p b 0.001), where mean levels in water exceeded 50 ng ml− 1 and in sediment were over 1800 µg g− 1. Selenium in soil (not logtransformed) was significantly higher in Minas de Horcajo than in the other areas (p = 0.003). Zinc, Cu and Cd tended to be higher in sediment and soil from Minas de Horcajo and Navalmartina than in the other sites (all with p b 0.02; Table 1). Cadmium was detectable in water from the mining area, but not in the control sites, while the opposite was true for Se. Correlations among sediment concentrations of Zn, Cd and Cu were highly significant (all with r N 0.9, p b 0.001), but Pb was only correlated with Cd (r = 0.77, p = 0.12) and As (r = 0.88, p b 0.001). Concentrations of sediment Cd and As were significantly correlated (r = 0.632, p = 0.37). Correlations in soil were significant between Zn–Cd, Zn–Cu, Se–Cu, Se–As, As–Cu, and As–Cd (all with r values between 0.55 and 0.74, p b 0.05). Element concentrations were not correlated in water.

Fig. 2 – Pb concentrations (mean ± SE) in stream sediments, plants and red deer tissues from mining and control areas. Those means sharing a capital letter did not differ among locations (Tukey test, p b 0.05).

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Table 2 – Concentration of Pb (μg g− 1 d.w.) in plants from four sampling sites in the Province of Ciudad Real Species

Quercus ilex Quercus canariensis Cistus ladanifer Cistus salvifolius Salix atrocinerea Arbutus unedo Erica sp. Crataegus monogyna Rosa canina Rubus ulmifolius Fraxinus angustifolia Phillyrea angustifolia Gramineae

Sample

Leaf Acorn Leaf Leaf Flower Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf

Minas de Horcajo n

Mean ± SE

3 3 3 3 1 – 3 – 1 1 1 3 – 2 4

Aa

21.1 ± 14.8 0.226 ± 0.184b 2.99 ± 0.622a 13.6 ± 2.65Aa 2.60 – 2.26 ± 0.482a – 4.41 7.80 41.3 7.60 ± 3.16Aa – 3.56 ± 0.589Aa 97.5 ± 37.5Aa

Navalmartina n

Mean ± SE

6 6 – 5 – 1 – 4 – 3 3 3 3 – 4

Ba

1.18 ± 0.391 ndb – 0.949 ± 0.147Ba – 0.499 – 0.255 ± 0.015ab – 0.213 ± 0.105ab 0.080 ± 0.063ab 0.860 ± 0.148Aa 0.508 ± 0.444ab – 0.792 ± 0.241Ba

Hornias Bajas n

Torneros

Mean ± SE C

4 4 – 3 3 – – 3 – 1 2 4 3 3 4

0.043 ± 0.013 0.092 ± 0.039 – 0.137 ± 0.042C 0.065 ± 0.021 – – 0.140 ± 0.070 – 0.403 0.110 ± 0.025 ndB 0.063 ± 0.062 0.140 ± 0.004B 0.416 ± 0.236B

n

Mean ± SE

3 3 – 2 3 – – 3 – – – 3 3 3 3

0.143 ± 0.082C 0.235 ± 0.191 – 0.065 ± 0.025C 0.131 ± 0.023 – – 0.105 ± 0.013 – – – ndB 0.084 ± 0.017 0.566 ± 0.425AB 0.248 ± 0.044B

Those means sharing a lower case letter did not differ among species and those sharing a capital letter did not differ among locations (Tukey test, p b 0.05).

3.2.

Metal levels and relationships in plants

The concentrations of Pb were higher in the plants from Minas de Horcajo and Navalmartina, both in the mining area, than in the control sites (p b 0.001; Table 2; Fig 2). There were significant differences among plant species, with the Gramineae from Minas de Horcajo having the highest concentration of Pb (p b 0.001; Table 2; Fig 2). The concentration of Zn differed among species (p b 0.001; Table 3), but only R. ulmifolius had a significantly lower concentration in Minas de Horcajo than in the control sites (Table 3). The concentration of Cu differed among areas (p = 0.032) and species (p = 0.017; Table 4). In particular, Cu levels were higher in C. ladanifer, P. angustifolia and Gramineae from the mining areas, than in the control sites. The concentration of Cd differed among locations (p = 0.004) and species (p b 0.001), in particular Q. ilex from

Minas de Horcajo had higher Cd levels than those from Hornias Bajas (Table 5). The concentration of As differed among locations (p b 0.001) and species (p = 0.001), being at its highest in the Gramineae from Minas de Horcajo (Table 6). The concentration of Se in plants did not differ among locations or species, with the exception of the acorn of Q. ilex from Minas de Horcajo which had a higher level than in Torneros (Table 7). Several differences among species were consistently found at different locations. The lowest concentrations of Zn and Cu were observed in the acorns of Q. ilex (Tables 3 and 4) at all sites, and Pb in the mined area. The leaves of C. ladanifer had the highest concentrations of Cd (as did one sample of C. salvifolius), whereas P. angustifolia and F. angustifolia had the lowest (Table 5). When results for all four areas were combined, a significant correlation was observed between Pb and As concentrations in

Table 3 – Concentration of Zn (μg g− 1 d.w.) in plants from four sampling sites in the Province of Ciudad Real Species

Quercus ilex Quercus canariensis Cistus ladanifer Cistus salvifolius Salix atrocinerea Arbutus unedo Erica sp. Crataegus monogyna Rosa canina Rubus ulmifolius Fraxinus angustifolia Phillyrea angustifolia Gramineae

Sample

Leaf Acorn Leaf Leaf Flower Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf

Minas de Horcajo

Navalmartina

Hornias Bajas

Torneros

n

Mean ± SE

n

Mean ± SE

n

Mean ± SE

n

Mean ± SE

3 3 3 3 1 – 3 – 1 1 1 3 – 2 4

19.6 ± 1.69a 6.41 ± 0.95d 30.1 ± 6.48bc 98.5 ± 1.0a 36.9 – 113.3 ± 7.25a – 11.9 39.4 34.7 25.4 ± 1.10Cbc – 43.9 ± 5.23b 35.2 ± 7.37bc

6 6 – 5 – 1 – 4 – 3 3 3 3 – 4

19.1± 2.23c 7.15± 0.63d – 64.1 ± 17.3a – 70.1 – 26.9 ± 2.61bc – 44.4 ± 3.66ab 25.7 ± 1.17bc 32.8 ± 2.94BCabc 55.8 ± 5.83Aa – 32.6 ± 4.08abc

4 4 – 3 3 – – 3 – 1 2 4 3 3 4

23.9 ± 0.89ab 9.94 ± 1.51c – 40.4 ± 3.72ab 32.6 ± 1.97ab – – 25.9 ± 2.55ab – 19.8 23.9 ± 12.6b 56.7 ± 3.17Aa 26.2 ± 4.17Bab 28.1 ± 5.60ab 35.2 ± 6.64ab

3 3 – 2 3 – – 3 – – – 3 3 3 3

29.1 ± 4.38a 9.23 ± 1.87b – 40.4 ± 2.04a 26.9 ± 1.36a – – 32.6 ± 1.82a – – – 43.2 ± 2.95ABa 35.5 ± 5.21ABa 33.7 ± 2.33a 36.5 ± 7.63a

Those means sharing a lower case letter did not differ among species and those sharing a capital letter did not differ among locations (Tukey test, p b 0.05).

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Table 4 – Concentration of Cu (μg g− 1 d.w.) in plants from four sampling sites in the Province of Ciudad Real Species

Quercus ilex Quercus canariensis Cistus ladanifer Cistus salvifolius Salix atrocinerea Arbutus unedo Erica sp. Crataegus monogyna Rosa canina Rubus ulmifolius Fraxinus angustifolia Phillyrea angustifolia Gramineae

Sample

Minas de Horcajo

Leaf Acorn Leaf Leaf Flower Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf

Navalmartina

Hornias Bajas

Torneros

n

Mean ± SE

n

Mean ± SE

n

Mean ± SE

n

Mean ± SE

3 3 3 3 1 – 3 – 1 1 1 3 – 2 4

11.2 ± 0.78a 2.2 ± 0.31b 16.7 ± 1.94a 12.5 ± 0.45Aa 10.8 – 16.9 ± 1.37a – 11.6 14.5 27.2 14.6 ± 1.04a – 11.9 ± 0.72Aa 15.6 ± 1.19Aa

6 6 – 5 – 1 – 4 – 3 3 3 3 – 4

7.86 ± 1.64ab 3.56 ± 0.57c – 10.6 ± 1.11ABab – 5.35 – 8.58 ± 0.16Ab – 14.1 ± 2.04ab 14.4 ± 3.45ab 12.1 ± 4.96ab 22.3 ± 0.77a – 12.1 ± 1.75ABab

4 4 – 3 3 – – 3 – 1 2 4 3 3 4

11.3 ± 1.39ab 2.89 ± 0.89c – 6.37 ± 0.91Babc 5.07 ± 0.57abc – – 3.44± 0.54Bbc – 8.42 7.81 ± 0.89abc 17.6 ± 2.25a 9.96 ± 3.96abc 5.05 ± 0.47Babc 5.75 ± 1.57Babc

3 3 – 2 3 – – 3 – – – 3 3 3 3

12.7 ± 1.67a 2.90 ± 0.46b – 8.74 ± 1.75ABab 4.16 ± 2.12b – – 6.25± 1.26ABab – – – 15.1 ± 1.19a 11.8 ± 2.49ab 6.04 ± 0.76Bab 9.47 ± 3.29Abab

Those means sharing a lower case letter did not differ among species and those sharing a capital letter did not differ among locations (Tukey test, p b 0.05).

the leaves of the four species sampled in all areas (r = 0.699, p b 0.001; Fig. 3). However, R. ulmifolius showed lower levels of As with respect to Pb when compared to the other species. Other, less significant correlations were found between Pb and Cd (r = 0.36, p = 0.006), Cd and As (r = 0.378, p = 0.004) and Cd and Zn (r = 0.31, p = 0.019).

3.3.

in deer from the mining areas (p = 0.001). Correlations among liver concentrations were positive for Zn–Cd (r = 0.635, p b 0.001), Cu–Cd ( r = 0.421, p b 0.001), Se–Cd ( r = 0.242, p b 0.048) and Se–As (r = 0.301, p = 0.013), and negative for Zn–As (r = − 0.26, p = 0.033). Bone Pb concentrations were negatively correlated with As (r = − 0.458, p = 0.001) and Se (r = − 0.417, p = 0.002).

Metal levels and relationships in red deer tissues

Red deer from the mining area, especially from Navalmartina, had a higher concentration of Pb in liver (p b 0.001; Table 8) than those from the control sites. The concentration of Se in liver and bone was also higher in deer from the mining area than in those from control sites (p ≤ 0.001), as was the case for liver Cd and As (p b 0.05). In contrast, bone Zn levels were lower

3.4. Relationship between metal concentrations in substrate, plants and deer tissues A clear pattern was observed in the means of the Pb concentrations found at the four sampling sites for sediment, plants and tissues of red deer (Fig. 2). At the sampling point level, the Pb concentrations were correlated between

Table 5 – Concentration of Cd (μg g− 1 d.w.) in plants from four sampling sites in the Province of Ciudad Real Species

Sample

Minas de Horcajo n

Quercus ilex Quercus canariensis Cistus ladanifer Cistus salvifolius Salix atrocinerea Arbutus unedo Erica sp. Crataegus monogyna Rosa canina Rubus ulmifolius Fraxinus angustifolia Phillyrea angustifolia Gramineae

Leaf Acorn Leaf Leaf Flower Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf

3 3 3 3 1 – 3 – 1 1 1 3 – 2 4

Mean ± SE Acd

0.050 ± 0.010 0.010 ± 0.002dc 0.024 ± 0.007cde 0.486 ± 0.214a 0.163 – 0.315 ± 0.025ab – 0.004 nd 0.008 0.006 ± 0.001e – 0.004 ± 0.002e 0.076 ± 0.022bc

Navalmartina n 6 6 – 5 – 1 – 4 – 3 3 3 3 – 4

Mean ± SE ABb

0.026 ± 0.005 0.019 ± 0.003b – 0.376 ± 0.111a – 0.481 – 0.048 ± 0.006b – 0.015 ± 0.001b ndc ndc ndc – 0.011 ± 0.005b

Hornias Bajas n 4 4 – 3 3 – – 3 1 2 4 3 3 4

Torneros

Mean ± SE Bab

0.010 ± 0.004 0.017 ± 0.007ab – 0.100 ± 0.018a 0.046 ± 0.007ab – – 0.033 ± 0.008ab – 0.005 ndc 0.010 ± 0.006bc ndc ndbc 0.017 ± 0.013ab

n

Mean ± SE

3 3 – 2 3 – – 3 – – – 3 3 3 3

0.023 ± 0.010ABab 0.017 ± 0.005abc – 0.180 ± 0.005a 0.073 ± 0.015ab – – 0.076 ± 0.023ab – – – ndcd ndd 0.005 ±0.001bcd 0.030 ± 0.019ab

Those means sharing a lower case letter did not differ among species and those sharing a capital letter did not differ among locations (Tukey test, p b 0.05).

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Table 6 – Concentration of As (μg g− 1 d.w.) in plants from four sampling sites in the Province of Ciudad Real Species

Quercus ilex Quercus canariensis Cistus ladanifer Cistus salvifolius Salix atrocinerea Arbutus unedo Erica sp. Crataegus monogyna Rosa canina Rubus ulmifolius Fraxinus angustifolia Phillyrea angustifolia Gramineae

Sample

Leaf Acorn Leaf Leaf Flower Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf

Minas de Horcajo

Navalmartina

Hornias Bajas

Torneros

n

Mean ± SE

n

Mean ± SE

n

Mean ± SE

n

Mean ± SE

3 3 3 3 1 – 3 – 1 1 1 3 – 2 4

b

6 6 – 5 – 1 – 4 – 3 3 3 3 – 4

0.418 ± 0.302 0.147 ± 0.051 – 0.131 ± 0.012AB – 0.117 – 0.048 ± 0.015 – nd nd 0.052 ± 0.019 nd – 0.100 ± 0.025B

4 4 – 3 3 – – 3 – 1 2 4 3 3 4

0.040 ± 0.011 nd – 0.059 ± 0.006BC nd – – nd – nd nd nd nd 0.052 ± 0.019 0.124 ± 0.061B

3 3 – 2 3 – – 3 – – – 3 3 3 3

0.025 ± 0.004 nd – 0.044 ± 0.023C 0.059 ± 0.015 – – 0.043 ± 0.016 – – – 0.029 ± 0.004 nd 0.025 ± 0.004 0.025 ± 0.004B

0.078 ± 0.045 0.432 ± 0.233b ndb 0.248 ± 0.104Aab nd – 0.168±0.135b – 0.190 nd nd 0.054 ± 0.021b – 0.069 ± 0.036b 2.406 ± 1.371Aa

Those means sharing a lower case letter did not differ among species and those sharing a capital letter did not differ among locations (Tukey test, p b 0.05).

sediment and Gramineae (r = 0.858, p = 0.013), but no other correlations were observed between the substrate (soil, sediment or water) and the other plant species. However, significant correlations were found for Pb levels between the four species of plants sampled at all the four locations (all with r N 0.75). As for the other elements, significant correlations were observed for As between soil and Gramineae and C. ladanifer (r N 0.74), for Se between soil and Gramineae (r = 0.61), and for Cd and Zn between soil and C. ladanifer (r N 0.69). Correlations between plant species were significant for Cd between Gramineae and Q. ilex (r = 0.65), for Cu between C. ladanifer and Gramineae (r = 0.83), for Zn between R. ulmifolius and Q. ilex, for Se between R. ulmifolius and Gramineae (r = 0.83) and for Cu and As between C. ladanifer and Gramineae (r N 0.76).

4.

Discussion

The mining area of the Valle de Alcudia in the Sierra Madrona has been exploited intermittently for over 2100 years, since Roman occupation and up until the late 1900s (Hevia, 2003). The concentrations of several metals studied herein are currently significantly higher in stream sediments, plants and herbivores within this Pb mining area than in control sites located within the same province (Ciudad Real).

4.1.

Metal levels in substrates and plants

The highest concentration of Pb was found in the streams flowing through the two mining sites. The concentrations of

Table 7 – Concentration of Se (μg g− 1 d.w.) in plants from four sampling sites in the Province of Ciudad Real Species

Quercus ilex Quercus canariensis Cistus ladanifer Cistus salvifolius Salix atrocinerea Arbutus unedo Erica sp. Crataegus monogyna Rosa canina Rubus ulmifolius Fraxinus angustifolia Phillyrea angustifolia Gramineae

Sample

Leaf Acorn Leaf Leaf Flower Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf

Minas de Horcajo

Navalmartina

Hornias Bajas

Torneros

n

Mean ± SE

n

Mean ± SE

n

Mean ± SE

n

Mean ± SE

3 3 3 3 1 – 3 – 1 1 1 3 – 2 4

0.164 ± 0.013b 0.288 ± 0.025Aab 0.278 ± 0.038ab 0.369 ± 0.053ab 0.169 – 0.473 ± 0.160a – 0.135 0.278 0.471 0.265 ± 0.047ab – 0.261 ± 0.039ab 0.244 ± 0.029ab

6 6 – 5 – 1 – 4 – 3 3 3 3 – 4

0.209 ±0.016ab 0.235 ± 0.008AB – 0.197 ± 0.017b – 0.258 – 0.229 ± 0.020ab – 0.299 ± 0.015a 0.294 ± 0.015a 0.249 ± 0.018ab 0.223 ± 0.012ab – 0.196 ± 0.010b

4 4 – 3 3 – – 3 – 1 2 4 3 3 4

0.189 ± 0.067 0.286 ± 0.023AB – 0.353 ± 0.014 0.361 ± 0.005 – – 0.368 ± 0.153 – 0.331 0.294 ± 0.047 0.341 ± 0.092 – 0.210 ± 0.066 0.216 ± 0.070

3 3 – 2 3 – – 3 – – – 3 3 3 3

0.248 ± 0.058 0.229 ± 0.023B – 0.241 ± 0.176 0.231 ± 0.069 – – 0.206 ± 0.079 – – – 0.295 ± 0.087 0.184 ± 0.016 0.258 ± 0.099 0.256 ± 0.057

Those means sharing a lower case letter did not differ among species and those sharing a capital letter did not differ among locations (Tukey test, p b 0.05).

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Fig. 3 – Relationship between As and Pb concentrations, expressed as logarithms, in plants collected in four sampling sites in the Province of Ciudad Real, Spain.

Pb in the sediment at Minas de Horcajo (1481 µg g− 1 d.w.) was comparable to that found in soil and sediment from other Pb mining areas (752–12,139 µg g− 1; Chukwuma, 1995; Yanqun et al., 2004; Brown et al., 2005; Liu et al., 2005; Deng et al., 2006; Li et al., 2006; Conesa Alcaraz et al., 2007). The soil Pb concentration in Minas de Horcajo was much lower than this because the samples were collected from a vegetated hillside near the spoil dump, where the plants eaten by red deer were growing. The unvegetated soil from the spoil dump probably had much higher Pb concentrations, but this was not sampled. The sediment in the stream flowing through the mining area may give a better reflection of the overall metal pollution in the catchment. Plants also reflected the difference between the mining and control areas, in terms of Pb concentrations, and Pb concentrations in plants were higher than in soils in Minas de Horcajo. This may be because there is often a significant amount of aerial deposition on plants of dust containing Pb around mines (Madejón et al., 2004; Hasselbach et al., 2005; Wilson and Pyatt, 2007). The significant correlation between Pb and As in plants (Fig. 3) may reflect this aerial deposition, since the uptake by and translocation into plants of these two elements are probably under quite differing control (Meharg et al., 1994; Huang and Cunningham, 1996). In terms of food-chain transfer to herbivores, the presence of adsorbed metals to the surface of plants may be more important than the amounts absorbed by roots and translocated to the aerial parts of the plant (Madejón et al., 2002). The species that accumulated the most Pb were Gramineae, Q. ilex and C. ladanifer. Gramineae from Minas de Horcajo had a concentration (97 µg g− 1) within the range for expected phytotoxicity (30–300 µg g− 1) according to Pugh et al. (2002), and at this level, grasses would also be potentially toxic to livestock (30 µg g− 1; Chaney, 1989). The levels observed in Gramineae are not dissimilar to the concentrations found in Cynodon dactilon and Sorghum halepense growing in soils affected by the Aznacóllar mine spill in Southern Spain (148 µg g− 1 d.w.; Madejón et al., 2002). The species with the

lowest Pb concentration in Minas de Horcajo was R. ulmifolius (7.6 µg g− 1). Other studies have also found relatively low Pb concentrations in Rubus sp. growing in heavy metal polluted sites, i.e., 1.8 µg g− 1 in a study by Freitas et al. (2004b) and 22 µg g− 1 in work by Yoon et al. (2006). Finally, the concentration of Pb in acorns of Q. ilex or in the flowers of C. ladanifer was much lower than in their respective leaves. As a general rule, the translocation of toxic heavy metals from the roots to aerial plant parts, and especially to the reproductive structures of the plant, is restricted by various mechanisms (Chukwuma, 1995; Deng et al., 2006; An, 2006; Li et al., 2006). The level of Cu in the soil from Minas de Horcajo (66 µg g− 1) is within the range of potential toxicity to plants (60–125 µg g− 1) according to Kabata-Pendias and Pendias (1992), and is within the range of soil Cu levels observed in similar mining areas in NE Portugal (31–225 µg g− 1; Freitas et al., 2004b). Selenium levels in soil are normally around 0.2 µg g− 1 (Rosenfeld and Beath, 1964) but they can be highly geographically variable. Soils and sediments analyzed here, when compared to our regionally relevant control area, are however elevated in Minas de Horcajo. Selenium levels in water are just 1 μg l− 1 which is an order of magnitude lower than that considered safe in drinking water for humans (WHO, 2006). The As levels in sediments at Minas de Horcajo (1880 µg g− 1) were over twice that of the most polluted sediment after the Aznalcóllar mine spill, and within the range of concentrations determined for the mine waste sludge associated with that disaster (Taggart et al., 2005). The water in the stream at Minas de Horcajo contains a mean of just over 50 μg l− 1, which is 5 times higher than the generally accepted drinking water standard for As for humans (WHO, 2006). Concentrations of As in plants were below phytotoxic levels (3–10 µg g− 1) and the maximum level tolerated by livestock (50 µg g− 1; Chaney, 1989).

Table 8 – Concentrations (µg g− 1 d.w.) of heavy metals and metalloids in tissues of adult red deer (Cervus elaphus) from four sampling sites in the Province of Ciudad Real Minas de Horcajo

Navalmartina

Liver n = 12 Pb 0.766 ± 0.280AB

n = 17 0.805 ± 0.155A

Cd 0.554 ± 0.090A

0.463 ± 0.069AB

Cu 50.8 ± 7.8 Zn 89.9 ± 3.9 As 0.061 ± 0.003A

51.7 ± 9.1 97.6 ± 7.8 0.046 ± 0.006AB

Se Bone Pb Cd Cu Zn As Se

0.327 ± 0.033A

0.224 ± 0.026B

n = 13 3.53 ± 0.92 nd nd 62.5 ± 3.0C 0.193 ± 0.007

n = 20 2.29 ± 0.98 nd nd 67.4 ± 2.3BC 0.261 ± 0.031

0.475 ± 0.044A

0.647 ± 0.046A

Hornias Bajas n = 18 0.357 ± 0.086BC 0.249 ± 0.022AB 47.3 ± 6.4 89.0 ± 2.7 0.044 ± 0.006B 0.193 ± 0.024B n=8 1.24 ± 0.41 nd nd 74.2±3.9AB 0.218 ± 0.026 0.280 ± 0.065B

Torneros n = 20 0.199 ± 0.081C 0.226 ± 0.031B 55.9 ± 6.9 95.4 ± 2.6 0.047 ± 0.007AB 0.200 ± 0.005B n = 10 0.93 ± 0.10 Nd Nd 81.0 ± 3.5A 0.231 ± 0.011 0.242 ± 0.027B

Those means sharing a capital letter did not differ among locations (Tukey test, p b 0.05).

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The detected Zn concentrations in plants were below the levels associated with phytotoxicity (500–1500 µg g− 1) and toxicity towards livestock (300–1000 µg g− 1; Chaney, 1989). The plant species with the highest Zn level in this study was S. atrocinerea, and other Salix species have been shown to have elevated Zn levels in other mining areas (Pugh et al., 2002). Another species with high Zn levels in this study was C. ladanifer at the Minas de Horcajo site, and the levels were comparable to those found in mining areas in Portugal (Freitas et al., 2004a, 2004b). R. canina from Minas de Horcajo was the only plant with Cu levels (27.2 µg g− 1) which can be associated with phytotoxicity (25–40 µg g− 1), and with levels above that tolerated by sheep (25 µg g− 1; Chaney, 1989). Phytotoxicity of Se in plants is highly variable but toxic effects are not considered to normally occur below 5 µg/g in plant tissues (Reilly, 1996). Selenium levels in analyzed plants were at least 10 times below this value. All the Cd concentrations found in plants in the present study are well below the level at which we would expect phytotoxicity (N5–700 µg g− 1), but in certain cases, i.e., for C. ladanifer in Minas de Horcajo (0.49 µg g− 1), the concentrations seen were close to the levels which would be of concern for livestock (N0.5 µg g− 1; Chaney, 1989).

4.2.

Metal levels in deer tissues

The concentration of Pb in deer liver was higher in the mining sites than in the control sites. The mean concentrations of Pb in bone of deer showed a similar trend to the levels observed in sediments and plants (Fig. 2), but significant differences between locations were not noted. Liver tends to reflect short term acute exposure, while bone accumulates more than 90% of the total body burden of Pb over time, and therefore reflects long term exposure (Ma, 1996). Hence, the higher Pb level in liver (but not in bone) in red deer from the mining sites compared to the control sites, may indicate that there is a temporal aspect to the Pb exposure here. However, red deer from the mining area showed a liver concentration well below the liver concentration associated with clinical signs of Pb poisoning (30 µg g− 1 d.w. or 10 µg g− 1 w.w.; Ma, 1996; Frøslie et al., 2001). Frøslie et al. (2001) reviewed liver Pb levels in red deer from different parts of Europe and the mean values ranged from 0.03 to 2.5 µg g− 1 w.w. Several studies with cervids from sites polluted by mining and smelting activities have described higher tissue Pb concentrations than in the present study. Red deer from near a Cu–Ni–Fe ore smelter in Ontario, Canada, had liver and bone Pb levels around 1.47 and 6.78 µg g− 1 d.w., respectively (Parker and Hamr, 2001). Liver Pb concentrations in roe deer (Capreolus capreolus) near a Pb smelter in Slovenia were also higher (0.71 µg g− 1 w.w; Pokorny and Ribarič-Lasnik, 2000) than those reported here. Red deer from the Province of Córdoba (Spain), on the southern side of Sierra Morena, had mean liver Pb concentrations of 0.57 µg g− 1 w.w., although some 4.3% of the deer had N2 µg g− 1 w.w. (Santiago et al., 1998). Much lower concentrations (0.030– 0.045 µg g− 1 w.w.) have been observed in cattle from Northern Spain (Miranda et al., 2001). The diet of red deer in Spain is based on grass between autumn and spring and on leaves of bushes and trees during the summer with some variation depending on the geographic area. In Sierra Morena, grasses represent 75% of the diet in spring and only 20% in summer. The species of bushes and

295

trees more commonly consumed by red deer in Spain are P. angustifolia, A. unedo, Quercus sp., Cistus sp., Erica sp., Olea europaea, Mirtus communis and Pistacia lentiscus (Carranza, 2004). In the present study, several of these species contained lower Pb levels than grasses (Gramineae) in the polluted site of Minas de Horcajo, and consequently some seasonal variations in the exposure to Pb in the deer may be expected. However, other sources of exposure to Pb should be evaluated in the future, because the Pb concentrations in liver and bone of the deer from Minas de Horcajo and Navalmartina were very similar despite the fact that the plants from the first site contained Pb levels more than one order of magnitude higher than they were in the second site. This may be because deer from the La Garganta hunting estate around Minas de Horcajo do not have full access to the most polluted areas, or, it may be that direct soil ingestion is a more important source of Pb exposure than plants are, as suggested by Beyer et al. (2007). Levels of Cu and Zn in liver were no different in mined areas than in control sites. Liver Cu levels were within the range observed in roe and red deer (4–30 µg g− 1 w.w.) and well below the levels associated with Cu poisoning (N150 µg g− 1 w.w.; Frøslie et al., 2001). Selenium was higher at Minas de Horcajo that at the other 3 sites, while mean levels of Cd in liver were approximately twice as high in Minas de Horcajo than in one of the control sites. Liver As levels were comparable to those reported in European wild ungulates (0.02 µg g− 1 w.w.; Frøslie et al., 2001), and Cd liver levels were similar to those found in red deer from Sierra Morena (0.21 µg g− 1 w.w.; Santiago et al., 1998) and cattle from Spain and elsewhere (0.03–0.31 µg g− 1 w. w.; Miranda et al., 2001). The highest correlation between elements in red deer tissues was observed in the liver for Zn and Cd. Both elements can induce the cellular production of metallothionein, a low-molecular-weight metal-binding protein, and consequently the correlation between them may be expected (Gamberg and Scheuhammer, 1994; López-Alonso et al., 2002).

5.

Conclusions

The concentrations of Pb and other elements in the sediments, plants and red deer from mining areas, especially from Mina de Horcajo, were higher than in the control sites. The high concentration of Pb and As detected in the sediment of a stream flowing beside the spoil dump at Mina de Horcajo, and the elevated levels of As observed in water indicate the need for remedial action at the site. Phytostabilization of the dump would reduce the wind dispersion of metals and the volume of drainage to the stream. However, the possibility of an increased transfer to herbivores must be considered, especially if plants with the potential to accumulate are used. Some species of plants growing at Minas de Horcajo, like Gramineae and R. canina may exceed the levels of Pb and Cu associated with toxicity to livestock. On the contrary, P. angustifolia and R. ulmifolius had some of the lowest values for several metals. Similar scenarios to Mina de Horcajo are scattered throughout the historically mined area of the Sierra Madrona and the Valle de Alcudia, so further work should be done to identify autochthonous plant species that could be used in phytostabilization. Species combining both high

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tolerance and very low translocation would perhaps make suitable candidates.

Acknowledgments We thank E. Arias, P. Talavera and D. Vidal for their help with sample collection, P. Camarero, and S. Sánchez-Maroto, for their help with laboratory work, P. Ferrandis for plant identification and W.N. Beyer for his comments on the manuscript. This project was funded by a grant from the Department of Education and Science of the Autonomous Government of Castilla-La Mancha (PCC-05-004-2) and a grant from the University of Castilla-La Mancha.

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