Growth and photosynthetic responses to copper in wild grapevine

The present study evaluates the tolerance and accumulation potential of Vitis vinifera ssp. sylvestris under moderate and high external Cu levels. A greenhouse experiment was conducted in order to investigate the effects of a range of external Cu concentrations (0–23 mmol L⁻¹) on growth and photosynthetic performance by measuring gas exchange, chlorophyll fluorescence parameters and photosynthetic pigments. We also measured the total copper, nitrogen, phosphorus, sulphur, calcium, magnesium, iron, potassium and sodium concentrations in the plant tissues. All the experimental plants survived even with external Cu concentrations as high as 23 mmol L⁻¹ (1500 mg Cu L⁻¹), although the excess of metal resulted in a biomass reduction of 35%. The effects of Cu on growth were linked to a reduction in net photosynthesis, which may be related to the effect of the high concentration of the metal on photosynthetic electron transport. V. vinifera ssp. sylvestris survived with leaf Cu concentrations as high as 80 mg kg⁻¹ DW and growth parameters were unaffected by leaf tissue concentrations of 35 mg Cu kg⁻¹ DW. The results of our study indicate that plants of V. vinifera ssp. sylvestris from the studied population are more tolerant to Cu than the commercial varieties of grapevine that have been studied in the literature, and could constitute a basis for the genetic improvement of Cu tolerance in grapevine.     

Growth, photosynthesis and antioxidant responses of two microalgal species, Chlorella vulgaris and Selenastrum capricornutum, to nonylphenol stress

The effect of nonylphenol (NP) on growth, photochemistry and biochemistry of two green microalgae, Chlorella vulgaris and Selenanstrum capricornutum, and their ability to degrade NP were compared. The 96 h EC₅₀ of C. vulgaris and S. capricornutum were greater than 4.0 and 1.0 mg L⁻¹ NP, respectively, suggesting that the former species was more tolerant to NP. Both microalgae acclimated to NP stress through down-regulating their photosynthetic activities, including antenna size (chlorophyll a content), maximal photochemistry (Fv/Fm) and the light absorbed by PSII (ABS/CS₀), but the dissipation of energy from reaction centres (DI₀/RC) increased with the increase of NP concentrations. In C. vulgaris, the changes of these parameters were more significant than in S. capricornutum and recovered completely after a 96 h exposure. The antioxidant responses, such as GSH content, CAT and POD activities in C. vulgaris increased with the increase of NP concentrations after a 24 h exposure, but these changes disappeared with exposure time and recovered to the control levels after 96 h. In S. capricornutum, although GSH content, CAT and POD activities also increased when exposed to low- to moderate-NP concentrations, these values were significantly reduced at a high concentration (4 mg L⁻¹) even after a 96 h exposure, indicating its antioxidant responses were significantly delayed. It is clear that the more NP-tolerant species, C. vulgaris, acclimated better with a faster recovery of its photosynthetic activity from the NP-induced damage, and exhibited more efficient and rapid responses to NP-induced oxidative stress. C. vulgaris also had a higher NP degradation ability than S. capricornutum.     

Salicylic acid involved in the regulation of nutrient elements uptake and oxidative stress in Vallisneria natans (Lour.) Hara under Pb stress

In this study, the alterations in nutrient elements content, reactive oxygen species level and antioxidant response were studied in leaves of Vallisneria natans (Lour.) Hara exposed to salicylic acid (SA, 10 or 100 μM), or Pb (50 μM) or their combinations for 4 d. No significant alterations in Mn and Ca content were observed but content of Cu, Zn, Fe and P decreased in plants exposed to SA alone. SA application inhibited the uptake of Pb and partially reversed Pb-induced the alterations in Mn, Ca and Fe content in leaves of V. natans exposed to 50 μM Pb. The decreased chlorophyll (a + b) and increased malondialdehyde and O²⁻ and H₂O₂ content were detected in plants exposed to 100 μM SA, 50 μM Pb, 10 μM SA + 50 μM Pb or 100 μM SA + 50 μM Pb. Application SA partially inhibited Pb-induced the increase of malondialdehyde, O²⁻ and H₂O₂ content. 100 μM SA decreased the activity of NADH oxidase and the content of non-protein thiols, carotenoids and ascorbic acid and increased the content of dehydroascorbate in plants treated with or without Pb. SA alone decreased the ascorbate peroxidase activity and increased the catalase and peroxidase activity, while SA application increased catalase activity but had no significant effect on ascorbate peroxidase and peroxidase activity in V. natans exposed to Pb. The results indicate that SA involves in the regulation of Pb uptake, nutrient balance and oxidative stress.     

Calcium protects roots of Sedum alfredii H. against cadmium-induced oxidative stress

Sedum alfredii is a well-known Cd (cadmium) hyperaccumulator native to China. The impacts of exogenous Ca on Cd-induced oxidative stress and antioxidant systems in roots of S. alfredii were investigated by using cellular and biochemical approaches. Supplementation of the medium with higher Ca levels resulted in alleviated growth inhibition and decreased Cd concentration, as well as increased Ca concentration in roots. Cadmium induced lipid peroxidation and loss of plasma membrane integrity, reactive oxygen species overproduction, as well as ultrastructural changes of root cells were largely reversed by Ca supplementation in the medium. Calcium application significantly altered the Cd effects on antioxidant enzymes and non-enzyme antioxidants (non-protein thiols), and significantly increased glutathione (GSH) biosynthesis. The results suggest that Ca is able to protect the roots of S. alfredii against Cd toxicity by restoration of Cd-displaced Ca, alleviation of the metal induced oxidative stress, as well as promotion of GSH biosynthesis.     

Application of phosphate-solubilizing bacteria for enhancing bioavailability and phytoextraction of cadmium (Cd) from polluted soil

In this study, phosphate-solubilizing bacteria (PSB), Bacillus megaterium, were used to enhance Cd bioavailability and phytoextractability of Cd from contaminated soils. This strain showed a potential for directly solubilizing phosphorous from soils more than 10 folds greater than the control without inoculation. The results of pot experiments revealed that inoculation with B. megaterium significantly increased the extent of Cd accumulation in Brassica juncea and Abutilon theophrasti by two folds relative to the uninoculated control. The maximum Cd concentrations due to inoculation were 1.6 and 1.8 mg Cd g⁻¹ plant for B. juncea and A. theophrasti after 10 wk, respectively. The total biomass of A. theophrasti was not significantly promoted by the inoculation treatment, yet the total biomass of B. juncea increased from 0.087 to 0.448 g. It is also worth to mention that B. juncea predominantly accumulates Cd in its stems (39%) whereas A. theophrasti accumulates it in its leaves (68%) after 10 wk. The change of the Cd speciation indicated that inoculation of B. megaterium as PSB increased the bioavailabilty of Cd and consequently enhanced its uptake by plants. The present study may provide a new insight for improving phytoremediation using PSB in the Cd-contaminated soils.     

Cytogenetic status of human lymphocytes after exposure to low concentrations of p,p'-DDT, and its metabolites (p,p'-DDE, and p,p'-DDD) in vitro

Despite that the use of DDT has been restricted for more than 40 years to malaria affected areas, low doses of this pesticide and its metabolites DDE and DDD can be found in the environment around the world. Although it has been shown that these pollutants induce cell and DNA damage, the mechanisms of their cytogenotoxic activity remains largely unknown. This study looks into their possible genotoxic effects, at doses that can be found in body fluids, on human lymphocytes using the cytokinesis-block micronucleus assay and the comet assay. After exposure for 1, 6, and 24 h compounds p,p′-DDT (0.1 μg mL⁻¹), p,p′-DDE (4.1 μg mL⁻¹), and p,p′-DDD (3.9 μg mL⁻¹) showed increase in DNA damage. The most significant results were observed at exposure period of 24 h where number of micronucleated cells increased from control 2.5 ± 0.71 to 23.5 ± 3.54, 13.5 ± 0.71, and 16.5 ± 6.36 for DDT, DDE, and DDD, respectively. Similar effect was observed using comet test where the percentage of DNA in comets tail increased from control 1.81 ± 0.16 to 17.24 ± 0.55, 11.21 ± 0.56 and 9.28 ± 0.50 for each compound, respectively. At the same time Fpg-comet assay failed to report induction of oxidative DNA damage of these pollutants. Additionally, the type of cell death was determined using diffusion assay and necrosis dominated. Our findings suggest that even at low concentrations, these pesticides could induce cytogenetic damage to human peripheral blood lymphocytes and in that manner have the impact on human health as well.     

Histopathological and biochemical alternations of the heart induced by acute cadmium exposure in the freshwater crab Sinopotamon yangtsekiense

Cadmium (Cd) is a highly toxic element in water. Its toxicity has been attributed to oxidative stress mediated by free radicals. Here we investigated the effects of Cd on the histopathology, antioxidant enzymes and lipid peroxidation of crustacean heart. The freshwater crabs Sinopotamon yangtsekiense were exposed to different concentrations of Cd for 1, 3, 5 and 7 d. After exposure, histological abnormalities were discovered, including myocardial edema, vacuolar and vitreous degeneration, and infiltration of inflammatory cells. Additionally, alterations in nuclei, mitochondria, rough endoplasmic reticulum as well as myofibrils were observed. Meanwhile, superoxide dismutase (SOD) activity was significantly increased after Cd exposure. Catalase (CAT) activity was only increased in the group exposed to 14.50 mg L⁻¹ Cd on day 5 and decreased with increasing Cd concentration and exposure time. Glutathione peroxidase (GPx) activity was increased in groups treated with 29.00, 58.00 and 116.00 mg L⁻¹ on days 1 and 3, and decreased thereafter. Besides, malondialdehyde (MDA) levels were significantly increased after 3 d of Cd exposure at all the indicated concentrations. These results showed that acute Cd exposure led to harmful effects on the histology of crab heart, which are most likely linked to Cd-induced oxidative stress.     

Freshwater Ulva (Chlorophyta) as a bioaccumulator of selected heavy metals (Cd, Ni and Pb) and alkaline earth metals (Ca and Mg)

We analyzed the ability of freshwater taxa of the genus Ulva (Ulvaceae, Chlorophyta) to serve as bioindicators of metal in lakes and rivers. Changes in heavy metal (Ni, Cd and Pb) and alkaline earth metal (Ca and Mg) concentrations in freshwater Ulva thalli were investigated during the period from June to August 2010. The study was conducted in two ecosystems in Western Poland, the Malta lake (10 sites) and the Nielba river (six sites). Three components were collected for each sample, including water, sediment and Ulva thalli. The average concentrations of metals in the water sample and in the macroalgae decreased in the following order: Ca > Mg > Ni > Pb > Cd. The sediment revealed a slightly altered order: Ca > Mg > Pb > Ni > Cd. Ca and Mg were found at the highest concentrations in thalli due to the presence of carbonate on its surface. Among the examined heavy metals in thalli, Ni was in the highest concentration, and Cd found in the lowest concentration. There were statistically significant correlations between the levels of metals in macroalgae, water and sediment. Freshwater populations of Ulva exhibited a greater efficiency to bioaccumulate nickel as compared to species derived from marine ecosystems.     

Effect of octylphenol on physiologic features during growth in Arabidopsis thaliana

Alkylphenol ethoxylates are widely used as detergents, emulsifiers, solubilizers, wetting agents and dispersants. Octylphenol (OP) ethoxylates, one of alkylphenol ethoxylates, represent 15–20% of the market, and their metabolic residues may be discharged to surface waters, sediments and soils as a persistent and ubiquitous pollutant. We tested the response of Arabidopsis thaliana to different concentrations of OP. OP affected the germination percentage and mean germination period. 10 d treatment with OP, especially high concentration (10 and 50 mg L⁻¹), decreased shoot and root biomass and root length of 30 d-old A. thaliana. Content of chlorophyll was decreased but that of proline was increased in leaves with OP treatment. OP caused oxidative stress in leaves; malondialdehyde content was increased, and the activities of ascorbate peroxidase, catalase and superoxide dismutase were induced. OP affects the physiologic and morphologic features of A. thaliana during growth. Because plants might be exposed to OP for a long time in the surroundings, more attention needs to be paid to the effect of OP on plants.     

Differential physiological,ultramorphological and metabolic responses of cotton cultivars under cadmium stress

Cadmium (Cd) stress may cause serious physiological, ultramorphological and biochemical anomalies in plants. Cd-induced physiological, subcellular and metabolic alterations in two transgenic cotton cultivars (BR001, GK30) and their parent line (Coker 312) were evaluated using 10, 100 and 1000 μM Cd. Germination, fresh biomass of roots, stems and leaves were significantly inhibited at 1000 μM Cd. Root volume tolerance index significantly increased (124.16%) in Coker 312 at 1000 μM Cd. In non-Cd stressed conditions, electron micrographs showed well-configured root meristem and leaf mesophyll cells. At 1000 μM Cd, greater ultramorphological alterations were observed in BR001 followed by GK30 and Coker 312. These changes were observed in nucleus, vacuoles, mitochondria and chloroplast. Dense precipitates, probably Cd, were seen in vacuoles, which were also attached to the cell walls. A considerable increase in number of nuclei, vacuoles, starch granules and plastoglobuli was observed in the electron micrographs of both roots and leaves at 1000 μM Cd. MDA contents were higher in roots of BR001 at 1000 μM Cd. Mean values of SOD activity in leaves of both BR001 and GK30 at 1000 μM Cd significantly increased as compared to the controls. POD activity in roots of BR001 and Coker 312 was greater at all Cd (10, 100, 1000 μM) levels over the control. Regarding APX, highest percent increase (71.64%) in roots of GK30 at 1000 μM Cd was found. Non-significant differences in CAT activity were observed at all levels of Cd stress in leaves of BR001 and GK30. Both transgenic cotton cultivars and their parental line invariably responded towards Cd stress. However, Coker 312 showed Cd-resistant behavior as compared to its progeny lines (BR001 and GK30).     

Response of Holm oak (Quercus ilex subsp. ballota) and mastic shrub (Pistacia lentiscus L.) seedlings to high concentrations of Cd and Tl in the rhizosphere

The impairment of root growth and photosynthetical functioning are the main impacts of trace elements on woody plant seedlings. In this work, we assessed the response of Holm oak (Quercusilex subsp. ballota) and mastic shrub (Pistacia lentiscus) seedlings to high concentrations of Cd and Tl in the rhizosphere. These are non-essential trace elements, with a potential high mobility in the soil-plant system. Seedlings of these species are frequently used in the afforestation of degraded soils in mining areas. Plants were exposed to different levels of Cd (20, 80 and 200 mg L⁻¹) and Tl (2, 10 and 20 mg L⁻¹) in a sand culture. Biomass allocation, growth rates, chlorophyll fluorescence and gas exchange were studied. Both metals affected root biomass. Cadmium produced an increase in the root mass ratio and a decrease in the specific leaf area of the plants in oak seedlings, while Tl did not provoke such response. Mastic plants were more sensitive to Tl and Cd than oak plants. Between elements, Tl provoked more severe toxic effects in the plants, affecting the antennae complexes and reaction centers of the photosystem II. Both elements decreased net assimilation rates (down to a 20% of the control plants) and stomatal conductance (5-10% of the values for the control plants). Cadmium was highly retained in the roots of both species, while Tl was highly translocated into the leaves. In general, Holm oak showed a higher tolerance for Cd than for Tl, and a higher resistance to both metals than mastic shrub, due to a high capacity for Cd retention at the root level. However, such accumulation in roots may induce water stress in the seedling exposed to Cd.     

Arbuscular mycorrhizal fungi enhance both absorption and stabilization of Cd by Alfred stonecrop (Sedum alfredii Hance) and perennial ryegrass (Lolium perenne L.) in a Cd-contaminated acidic soil

A greenhouse pot experiment was conducted to compare the phytoextraction efficiencies of Cd by hyper-accumulating Alfred stonecrop (Sedum alfredii Hance) and fast-growing perennial ryegrass (Lolium perenne L.) from a Cd-contaminated (1.6 mg kg⁻¹) acidic soil, and their responses to the inoculations of two arbuscular mycorrhizal (AM) fungal strains, Glomus caledonium 90036 (Gc) and Glomus mosseae M47V (Gm). Ryegrass and stonecrop were harvested after growing for 9 and 27 wk, respectively. Without AM fungal inoculation, the weekly Cd extraction by stonecrop (8.0 μg pot⁻¹) was 4.3 times higher than that by ryegrass (1.5 μg pot⁻¹). Both Gc and Gm significantly increased (P < 0.05) root mycorrhizal colonization rates, soil acid phosphatase activities, and available P concentrations, and thereby plant P absorptions (except for Gm-inoculated ryegrass), shoot biomasses, and Cd absorptions (except for Gm-inoculated stonecrop), while only Gc-inoculated stonecrop significantly accelerated (P < 0.05) the phytoextraction efficiency of Cd by 78%. In addition, both Gc and Gm significantly decreased (P < 0.05) phytoavailable Cd concentrations by 21–38% via elevating soil pH. The results suggested the potential application of hyper-accumulating Alfred stonecrop associated with AM fungi (notably Gc) for both extraction and stabilization of Cd in the in situ treatment of Cd-contaminated acidic soil.     

Impact of nano-CuO stress on rice (Oryza sativa L.) seedlings

Indiscriminate release of metal oxide nanoparticles (NPs) into the environment due to anthropogenic activities has become a serious threat to the ecological system including plants. The present study assesses the toxicity of nano-CuO on rice (Oryza sativa cv. Swarna) seedlings. Three different levels of stress (0.5 mM, 1.0 mM and 1.5 mM suspensions of copper II oxide, <50 nm particle size) were imposed and seedling growth performance was studied along control at 7 and 14 d of experiment. Modulation of ascorbate–glutathione cycle, membrane damage, in vivo ROS detection, foliar H₂O₂ and proline accumulation under nano-CuO stress were investigated in detail to get an overview of nano-stress response of rice. Seed germination percentage was significantly reduced under stress. Higher uptake of Evans blue by nano-CuO stressed roots over control indicates loss of root cells viability. Presence of dark blue and deep brown spots on leaves evident after histochemical staining with NBT and DAB respectively indicate severe oxidative burst under nano-copper stress. APX activity was found to be significantly increased in 1.0 and 1.5 mM CuO treatments. Nevertheless, elevated APX activity might be insufficient to scavenge all H₂O₂ produced in excess under nano-CuO stress. That may be the reason why stressed leaves accumulated significantly higher H₂O₂ instead of having enhanced APX activity. In addition, increased GR activity coupled with isolated increase in GSH/GSSG ratio does not seem to prevent cells from oxidative damages, as evident from higher MDA level in leaves of nano-CuO stressed seedlings over control. Enhanced proline accumulation also does not give much protection against nano-CuO stress. Decline in carotenoids level might be another determining factor of meager performance of rice seedlings in combating nano-CuO stress induced oxidative damages.     

Phytoextraction of heavy metal polluted soils using Sedum plumbizincicola inoculated with metal mobilizing Phyllobacterium myrsinacearum RC6b

The aim of this study was to investigate the effects of metal mobilizing plant-growth beneficial bacterium Phyllobacterium myrsinacearum RC6b on plant growth and Cd, Zn and Pb uptake by Sedum plumbizincicola under laboratory conditions. Among a collection of metal-resistant bacteria, P. myrsinacearum RC6b was specifically chosen as a most favorable metal mobilizer based on its capability of mobilizing high concentrations of Cd, Zn and Pb in soils. P. myrsinacearum RC6b exhibited a high degree of resistance to Cd (350 mg L⁻¹), Zn (1000 mg L⁻¹) and Pb (1200 mg L⁻¹). Furthermore, P. myrsinacearum RC6b showed multiple plant growth beneficial features including the production of 1-aminocyclopropane-1-carboxylic acid deaminase, indole-3-acetic acid, siderophore and solubilization of insoluble phosphate. Inoculation of P. myrsinacearum RC6b significantly increased S. plumbizincicola growth and organ metal concentrations except Pb, which concentration was lower in root and stem of inoculated plants. The results suggest that the metal mobilizing P. myrsinacearum RC6b could be used as an effective inoculant for the improvement of phytoremediation in multi-metal polluted soils.     

Distribution of Se and its species in Myriophyllum spicatum and Ceratophyllum demersum growing in water containing Se (VI)

The uptake of Se (VI) by two aquatic plants, Myriophyllum spicatum L. and Ceratophyllum demersum L., and its effects on their physiological characteristics have been studied. Plants were cultivated outdoors under semi-controlled conditions and in two concentrations of Na selenate solution (20 μg Se L⁻¹ and 10 mg Se L⁻¹). The higher dose of Se reduced the photochemical efficiency of PSII in both species, while the lower dose had no effect on PSII. Addition of Se had no effect on the amounts of chlorophyll a and b. The concentration of Se in plants grown in 10 mg Se L⁻¹, averaged 212 ± 12 μg Se g⁻¹ DM in M. spicatum (grown from 8-13 d), and 492 ± 85 μg Se g⁻¹ DM in C. demersum (grown for 31 d). Both species could take up a large amount of Se. The amount of soluble Se compounds in enzyme extracts ranged from 16% to 26% in control, and in high Se solution from 48% to 36% in M. spicatum and C. demersum, respectively. Se-species were determined using HPLC-ICP-MS. The main soluble species in both plants was selenate (∼37%), while SeMet and SeMeSeCys were detected at trace levels.     

Zinc tolerance and accumulation in the salt-marsh shrub Halimione portulacoides

The halophytic shrub Halimione portulacoides is known to be capable of growth in soils containing extremely high concentrations of Zn. This study evaluated in detail the tolerance and accumulation potential of H. portulacoides under moderate and high external Zn levels. A greenhouse experiment was conducted in order to investigate the effects of a range of Zn concentrations (0-130 mmol L⁻¹) on growth and photosynthetic performance by measuring relative growth rate, total leaf area, specific leaf area, gas exchange, chlorophyll fluorescence parameters and photosynthetic pigment concentrations. We also determined the total zinc, nitrogen, phosphorus, calcium, magnesium, sodium, potassium, iron and copper concentrations in the plant tissues. H. portulacoides demonstrated hypertolerance to Zn stress, since it survived with leaf concentrations of up to 2300 mg Zn kg⁻¹ dry mass, when treated with 130 mmol Zn L⁻¹. Zinc concentrations greater than 70 mmol L⁻¹ in the nutrient solution negatively affected plant growth, in all probability due to the recorded decline in net photosynthesis rate. Our results indicate that the Zn-induced decline in the photosynthetic function of H. portulacoides may be attributed to the adverse effect of the high concentration of the metal on photosynthetic electron transport. Growth parameters were virtually unaffected by leaf tissue concentrations as high as 1500 mg Zn kg⁻¹ dry mass, demonstrating the strong capability of H. portulacoides to protect itself against toxic Zn concentrations. The results of our study indicate that this salt-marsh shrub may represent a valuable tool in the restoration of Zn-polluted areas.     

Differential toxicity of silver and titanium dioxide nanoparticles on Drosophila melanogaster development, reproductive effort, and viability: size, coatings and antioxidants matter

Silver and titanium dioxide nanoparticles are known to induce oxidative stress in vitro and in vivo. Here we test if they impact development, mating success, and survivorship in Drosophila melanogaster, and if so, if these effects are reversible by antioxidants. Ingestion of nanotitanium dioxide during the larval stage of the life cycle showed no effects on development or survivorship, up to doses of 200 μg mL⁻¹. Conversely, ingestion of nanosilver had major dose, size, and coating-dependent effects on each of these aspects of life history. Each of these effects was partially or fully reversible by vitamin C. Larvae growing on nanosilver supplemented with vitamin C showed a greater than twofold increase in survivorship compared to flies reared on nanosilver alone, and a threefold increase in mating success. Vitamin C also rescued cuticular and pigmentation defects in nanosilver fed flies. Biochemical assays of superoxide dismutase and glutathione show these markers respond to nanotitanium dioxide and nanosilver induced oxidative stress, and this response is reduced by vitamin C. These results indicate that life history effects of nanosilver ingestion result from oxidative stress, and suggest antioxidants as a potential remediation for nanosilver toxicity. Conversely, the lack of nanotitanium dioxide life history toxicity shows that oxidative stress does not necessarily result in whole organism effects, and argues that nanoparticle toxicity needs to be examined at different levels of biological organization.     

Heavy metal exposure in large game from a lead mining area: Effects on oxidative stress and fatty acid composition in liver

The Pb mining area of the valley of Alcudia and the Sierra Madrona mountains (S. Spain) has been exploited intermittently for over 2100 years, since Roman occupation and up until the late 1900s. Red deer (n = 168) and wild boar (n = 58) liver and bone (metacarpus) were analyzed for Pb, Zn, Cu, Cd, As and Se. Lipid peroxidation, total and oxidized glutathione (GSH), superoxide dismutase (SOD), GSH peroxidase (GPX) and fatty acid composition were studied in liver of red deer. The concentrations of Pb in liver and bone of red deer and wild boar were higher in the mining area than in the control area, and higher in the wild boar than red deer, but well below the level associated with clinical signs of Pb poisoning. Liver levels of Cu, Cd and Se were also higher in red deer from the mining area. Red deer from the mining area had 39% less total GSH than in the control area. The percentage of docosahexaenoic acid in liver of red deer from the mining area was 16% lower than in the control area.