Goncalves et al. studied the absorption capacity of Pinus elliottii from water contaminated with cadmium, lead and chromium and found that its bark is a good absorbent of heavy metals between pH of 5.0 and 7.0. Pine needles with a thick epicuticular wax layer are best suited for biomonitors since metals absorbed are not easily leached out (Mingorance et al., 2007 as cited by Serbula et al., 2012). Serbula et al. (2012) assessed the metal uptake of Pinus ssp. and Tila ssp. (Linden) and concluded that pine needles are more sentitive to heavy metal pollution and can be used to biomonitor air pollution. The results were supported by the fact that the enrichment factors (EFs) of pine needles, for all elements analysed, were higher compared to linden leaves which had an EF value of less than two which is not indicative of pollution. This is due to differences in physical and physiological nature.
1.3.2 Araucaria columnaris
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Araucaria columnaris also known as Coral reef araucaria is a conifer in the Araucariaceae family. It usually grows in warm climate and can attain 60 m height and can be used as a lead indicator.
Ceburnis and Steinnes (2000) found that conifer needles can accumulate heavy metals from the atmosphere but they observed low retention of the metals and could not find a correlation between soil and root uptake. They thus concluded that conifer needles may only be used close to pollution sites. The same authors were able to show that cadmium and manganese in needles but not in moss came from soil.
1.3 Methods of digestion
Microwave digestion is used to dissolve heavy metals in the organic molecules prior to analysis by atomic absorption spectroscopy (AAS) or inductive coupled plasma (ICP). This is done dissolving a particular amount of sample in a strong acid in a closed vessel, and raising the pressure and temperature through microwave irradiation thus increasing the speed of thermal decomposition of the sample, and promoting solubility of heavy metals in solution. Finally the digested solutions containing heavy metals can be analysed through elemental techniques. Different microwave digestion techniques exist and each method is suitable different type of sample whether plants, soil or any other organic matter. This section reports on the different digestion techniques used to analyse plant parts.
Dahmani-Muller et al. (1999) washed and cleaned plant samples for 20 minutes ultrasonically to remove foreign particles. They were then oven-dried for 15 h at 85oC and grinded and passed through a 500 µm sieve. 0.250 g of this sample was gradually warmed with hydrofluoric acid and perchloric acid was added after cooling. The solution was further heated to dryness and the residue was then redissolved while heating with hydrochloric acid and finally diluted to 50 ml with distilled water.
Many other studies made use of nitric acid for digestion, for instance Lobersli and Steinnes (1987) dried previously washed plants at approximately 60oC before digesting with concentrated nitric acid. Prior to this the metal level were estimated by extracting with 1M ammonium acetate pH 7.00 and pH 4.80 depending on the metals to be analysed. Or there is the case for Burns and Parker (1988) who digested 1 g of plant sample in a solution of 20:20:1 HCLO4, HNO3 and H2SO4 after drying for 48 h at 45oC and grinding. Residues were redissolved in 5ml of 1% HNO3.
Vousta et al. (1996) studied trace metals in vegetables by drying at 60oC for 4 days after washing. The samples were then grounded and place in a desiccator prior to ultrasonic extraction with concentrated HNO3 and HCl. Vegetables can also be analysed by drying at 70oC for 72h and digesting 0.5 g of grounded plant tissue with 5 ml HNO3 in a sealed polyvinyl fluoride crucible and left to stand for 2h at room temperature before placing the crucible in a high pressure metal cylinder. The metal cylinder is then placed in an oven at 100oC for 1h and then 170oC for 5h. Finally excess acid is evaporated on a hot plate at 105oC and the residues are diluted to 50 ml and stored at 4oC until ready for analysis. This technique was employed in the research of Cui et al., 2004. Kachenco and Singh (2004) also analysed vegetable samples by washing with 0.1 % teepol for 15 seconds, 0.1% hydrochloric acid for 15 seconds and finally three times with deionised water. Once clean, the samples were dried at 70oC in a dehydrator for 48 to 72 h depending on sample size. They were then weighed and grinded for digestion in a mixture of nitric acid, perchloric acid.
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The study of metals in the rings of Pinus sylvestris were studied by Lukaszewski et al., 1988 and VanÄ›k et al. (2010). The former digested 0.5 g of the grounded tree rings with hydrogen peroxide and nitric acid (Douy et al., 2003) after drying for 24 h at 105oC. The latter also dried the sample for 24h but at 70oC and digesting overnight with concentrated nitric acid at 190oC in 60 ml PTFE beakers. The solution obtained was dissolved in deionised water.
In the case of Zheljazhov et al. (2008) medicinal plants were dried at 35oC until they attained a constant weigh and further dried at 70oC for 3 days and passed through a 1 mm sieve and stored in closed vials before digesting 1g subsample in 10 ml nitric acid in a digestion tube overnight at room temperature. The mixture obtained was then decomposed on aluminium digestion block at 150oC for eight hours, filtered and made up to 50 ml in a volumetric flask with doubled distilled water.
Aznar et al. studied metal contamination in lichen. Each sample was cleaned, dried and freeze-dried in a 0.1 g aliquot and transferred in Savillex Teflon bombs with 5 ml tridistillated nitric acid and 1 µl fluohydric acid. Digestion was carried out in a high pressure autoclave for three hours at 110oC.
Nicolaidou and Nott (1998) used a similar digestion method as used by Bryan et al.(1985) by drying the plants at 80oC and digesting with 'Aristar' HNO3 followed by evaporation before redissolving in 1N 'Aristar' HCl.
1.4 Analysis of metals
The determination of heavy metals in the plant samples are usually done by flame or graphite atomic absorption spectroscopy with duplicates, blanks and certified reference materials (Yang et al., 2011). Graphite furnace AAS is usually used to analysed metals falling below the standard deviation of blank samples (Zheljazkov et al., 2008). Inductively coupled plasma can also be used.
1.5 Results from previous studies
Most studies of impact of smelters on plants found elevated levels of heavy metals. Nearly all samples analysed exceeded maximum level allowed, for instance, cadmium and lead were above the Australian Food standards maximum level (Kachenko and Singh, 2005). Aznar et al. (2007) even found Cd, Cu and Pb contamination 20 km from the smelter to be two times higher than those at the reference sites (50km away).
Many studies showed that contamination occurred from atmospheric deposition of heavy metals on the plant surface. Aznar et al. (2007) found that wind intensity, elevation and distance from smelter affected the level of cadmium, copper and lead on plants. Airborne deposition was also observed by Vousta et al. (1996), Vanek et al. (2010) and Kachenko and Singh (2005). Douay et al. ( ) found that lead content decreased considerably after smelter closedown accounting once again for superficial deposits of heavy metals.
The areas around smelters are often subjected to acid rain resulting from pollutants such as SO2 in the atmosphere. Soil pH also affects the availability of metal uptake by plants. Kozlov et al. (1995) demonstrated that the level of manganese fell with increasing distance mostly due to high acidity of the soil caused by acid rain in the vicinity of the smelter, this leads to direct leaching of Mn. Lobersli and Steinnes (1987) observed that low pH lead to higher availability of iron in plant. Kachenko and Singh (2005) observed high uptake of cadmium due to acidity of soil.
Some studies found a relationship between the level of metal in soil and plants. The level of zinc, lead, cadmium and copper in plant tissues depend on their concentrations in soil (Kachenko and singh, 2005). Low nickel and copper deposits on plants suggested that these metals were mostly taken up from the soil (Kozlov et al., 1995). Douay et al., ( ) came to the conclusion that cadmium content in wheat grains depended on its level in soil since the smelter closedown did not lead to a noticeable decrease of the element concentration. Lukaszewski et al. (1988) also observed the same result in tree rings of Pinus sylvestris L.).
Kachenko and Singh (2005) found interactions between Cd and Zn in plants. For example, high concentrations of zinc in soil resulted in high cadmium uptake by leafy vegetables. Kozlov et al. (1995) noted that for high levels of copper in plants, the zinc uptake was low.
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Different plant species have different accumulation capacity (Arunachalam et al., ). Ostrich fern is considered as an accumulator since it can absorb ten times the concentrations observed in Interrupted fern (Burns and Parker, 1988). Genetic differences among species account for the transport heavy metals as well as accumulation in different plant parts (Zheljazkov et al., 2008). Moreover, the physical and chemical structure of the plant may change over time, thus the uptake of heavy metals will be different from year to year (Watmough and Hutchinson, 2002).
Table 1 shows the concentration of metals observed in different plant species grown nears smelters.