Abstract: The uptake, accumulation and effects of environmentally relevant platinum group elements (PGE; Rh, Pd and Pt) on Lactuca sativa were investigated. Seedlings were exposed to nutrient solution amended with mixed solutions of PGE (0, 0.3, 0.5, 0.7 and 1.0 ppm each) of PGE (Pt, Pd and Rh as a mixture) for a period of 4 weeks. The results showed a significant (p<0.05) reduction in plant dry weight, nitrate reductase activity, protein content and photosynthesis efficiency when treated with concentrations greater than 0.5 ppm. The treatments also induced oxidative stress and membrane damage, as indicated by catalase activity and lipid peroxidation assays, respectively. Metal analysis in plant tissues by ICP-MS showed that accumulation followed the order Rh ~ Pt >> Pd. Seedlings exposed to 1.0 ppm of PGE for a period of 4 weeks accumulated about 50 mg kg-1 of Rh and Pt and about 30 mg kg-1 of Pd. The results of this study have exemplified the potential effects of these elements, albeit at concentrations in excess of typical environmental levels.
INTRODUCTION
In recent years, there has been a requirement in many countries that all new motor vehicles must be equipped with catalytic converters in an effort to reduce air pollution. The modern three-way catalysts consist of a ceramic monolith in the form of honeycomb. On the monolith, a wash-coat of γ-alumina is applied so as to increase the active surface and PGE (platinum, palladium and rhodium) are then dispersed on the wash-coat. The catalytic converters can remove about 90% of CO, unburnt HC and NOx from the exhaust gasses. Although the benefits of this technology to the environment are obvious, studies have indicated that due to thermal, mechanical and chemical impacts, particles containing PGE are emitted into the environment (Verstraete et al., 1998). As a result, a considerable increase in the concentrations of Pt, Pd and Rh in vegetation, soils and surface waters, especially at sites adjacent to motorways with high traffic, have been observed over the past few decades (Locatelli et al., 2005).
Despite growing concerns over the potential environmental and biological impacts of PGE, there is limited quantitative and mechanistic information about their uptake and behaviour in natural systems. In particular, their potential to enter the human food chain deserves attention because it has been reported that compounds of Pt (IV) and Pd (II) are nephrotoxic and gastrointestinal irritants, while Pt (II) compounds may have mutagenic, genotoxic or carcinogenic properties (Vlasankova, 2001). The practice of cultivating leafy vegetables for both human and livestock consumption in gardens adjacent to roads carrying heavy traffic therefore represents a significant vector for the exposure of PGE to humans. To this end, the study reported here aimed to investigate the uptake, accumulation and effects of aqueous PGE on hydroponically cultivated Lactuca sativa (lettuce), a leafy vegetable of economic importance.
MATERIALS AND METHODS
Germination Study
Individual plasma emission standards of Rh (III), Pd (II) and Pt (IV) in
1.2 M HCl were obtained from BDH/Merck. Lettuce seeds were purchased from a
local seed store in the city of Plymouth, UK in a single batch and enough for
the study. The nutrient solution was a quarter-strength Hoagland’s solution
(pH 6.5) as modified by Punshon and Dickinson (1997) containing (mM): 2.5 NH4
(NO3)2, 1.25 Ca (NO3)2, 1.5 KNO3,
0.5 KH2PO4, 1.5 MgSO4 and 0.25 NaCl and (μM):
10.0 H3BO3, 2.3 MnCl2 and 0.025 H2M0O4.
Each Petri dish contained 10 seeds and received 2 mL of solution every 2 days
for 10 days; all dishes were placed in a temperature controlled growth cabinet
at 23°C.
Seedling Growth and Treatments
Lettuce seedlings of similar height (10 cm) were selected for experimental
use. Nutrient solution devoid of the PGE served as control. Seedlings were grown
in 15 mL glass test-tube, one per tube and exposed to one of five concentrations
of PGE (0, 0.3, 0.5, 0.7 and 1.0 ppm) dissolved in the nutrient solution described
above. Both the control and treated solutions were adjusted to pH 6.5 using
potassium phosphate buffer. The experiment was carried out in a growth chamber
maintained at 23°C, a photoperiod of 16:8 h (L:D), relative humidity of
61.3% and irradiance of 70 μmol m-2 s-1 for 6 weeks.
Solutions were replaced every 5 days to avoid depletion of nutrient and metals.
There were 20 replicates per treatment.
Growth Measurements and Chemical Analyses
Lipid Peroxidation
Lipid peroxidation in the leaves was estimated by measuring the content
of thiobarbituric acid reactive compounds (TBAR), mainly malondialdehyde according
to a modified procedure of Wang and Jin (2005). Fresh leaves (0.2 g) were ground
in liquid N2 and extracted in 1 mL 20% trichloroacetic acid. The
extract was centrifuged (10000 g) for 5 min at 4°C. Supernatant (0.5 mL)
was mixed with 0.5 mL of 0.6% (w/v) thiobarbituric acid solution comprising
10% TCA. The mixture was incubated for 30 min in a boiling water bath and allowed
to cool; measurements were made at 450, 532 and 600 nm. Malondialdehyde (μM/g
f. wt) was calculated as 6.45 (A532-A600)-0.56 A450.
Nitrate Reductase Activity
The extraction and assay of nitrate reductase was estimated by a modified
method of Fan et al. (2002). Fresh leaves (0.2 g) was ground and extracted
in 2 mL of distilled water, centrifuged (10000 g) for 5 min at 4°C. One
milliliter of the crude extract was incubated at 25°C in the dark for 1
h in 3 mL of the substrate assay solution in a test tube. The substrate assay
solution contained 1 mL each of 0.1 M KNO3, 15 mL L-1
propan-1-ol and 0.1 M potassium phosphate buffer (pH 7.5). One milliliter of
the solution was transferred using a pipette after the incubation period into
a clean test tube. One milliliter each of 1% sulphanilic acid and 0.02% naphthylenediamine
(NED) was added, the mixture was thoroughly shaken and left to stand for 1 h
to allow full colour development. A blank was prepared by mixing 1 mL of substrate
assay solution, 1 mL of 1% sulphanilic acid and 1 mL of 0.02% naphthylenediamine
in a test tube. The mixture was allowed to stand for 1 h for colour development.
Colour development due to nitrite was then measured spectrophotometrically at
540 nm. The values were compared to a standard curve generated using solutions
of NaNO2.
Protein Estimation
This was achieved according to the method outlined by Bradford (1976). Thus,
100 mg of Coomassie Brilliant Blue G-250 were dissolved in 50 mL of 95% ethanol.
To this, 100 mL of 85% potassium phosphate buffer (pH 7.5) were added. The mixture
was made to 1000 mL with distilled water and this is the protein reagent. Five
mL of the reagent were mixed with 0.1 mL of extract and read after 2 min at
595 nm. Bovine serum albumin (BSA) standard solutions (10-100 μg mL-1)
protein in 0.1 mL was mixed with 5 mL of reagent and absorbance read at 595
nm. The blank was 0.1 mL of buffer and 5 mL of reagent. Protein content was
determined from a standard curve generated from solutions of BSA.
Chlorophyll Fluorescence
Chlorophyll fluorescence of photosystem II of the plant was measured with
a PAM fluorometer (FMS 1, Hansatech Instruments Ltd., Norfolk, UK). The measurements
were performed at room temperature (24°C). The time of measuring was 5 sec
and irradiance was set at 75% of maximum. The efficiency of photosystem II photochemistry
(ΦPSII), maximum quantum yield (Fv/Fm), the efficiency
of water splitting apparatus (Fo/Fv), the plastoquinone
pool (Fv/2), initial fluorescence (F0) and the variable fluorescence
(Fv) were recorded.
Metal Analysis in Plant Tissues
About 0.5 g of oven dried tissue samples were weighed individually in to
quartz digestion vessels. Ten milliliter of concentrated HNO3 and
2 mL of 30% HCl were added and the contents heated to near-dryness. Ten milliliter
of 20% HNO3 were then added and the contents refluxed. After cooling,
the digests were filtered through Whatman No. 1 filter papers into individual
25 mL volumetric flasks and adjusted to volume with 2% HNO3. Metal
concentrations were determined in the digests inductively coupled plasma-mass
spectrometry (ICP-MS) using a Thermo elemental Plasma Quad 2+ fitted with a
Meinhard dissolved solids nebuliser. 115-In and 193-Iridium were added to all
samples and mixed standards (prepared over the working range in 10% HNO3)
at a concentration of 100 ppb each to act as internal standards over the mass/charge
range studied.
Data Analysis
All measurements in triplicates were analysed using one way analysis of
variance and differences in means were determined by Tukey’s pairwise comparisons.
RESULTS
Effect of PGM on Germination of Lactuca Sativa Seeds
PGE concentrations of < 0.7 ppm had no effect on the germination efficiency
of the seeds, but above this concentration, there was a significant (p = 0.05)
reduction in the number of germinated seeds with corresponding increase in PGE
concentration (Table 1).
| Table 1: | Effects of PGE on the germination of Lactuca sativa |
| Different letter(s) indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison | |
| Table 2: | The dry weight (g) of Lactuca sativa after 4 weeks of treatment with PGE |
| Different letter(s) indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison | |
| Table 3: | Leaf area (cm2) of Lactuca sativa treated with various concentrations (ppm) of PGE |
| Different letter(s) indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison | |
Plants’ Dry Weight
There was no significant (p = 0.05) difference in the dry weight of the
control and those exposed to 0.3 ppm of PGE. Plants treated with 7.0 and 1.0
ppm had dry weights of 0.0399±0.0007 and 0.0336±0.0005 g, respectively.
These values were significantly (p = 0.05) lower than 0.0494±0.0004 g
observed for the control plants (Table 2).
Effect on Leaf Area
The leaf area of the plant decreased with increase in PGM concentrations.
The control plants had a mean value of 3.91±0.16 cm2, while
plants treated with 0.5, 0.7 and 1.0 ppm had 3.46±0.12, 2.71±0.08
and 2.33±0.02 cm2, respectively (Table 3).
Effect of PGM on Catalase Activity
We examined the effect of PGM on catalase activity of the plant. Catalase
activity at 25°C for the control plants was 0.283±0.006 mmol mg-1
protein min-1. There was a significant (p = 0.05) increase
in the enzyme activity of plants treated with PGE (Fig. 1). The increase in
the enzyme activity was proportional to the concentration of the PGE to which
they were exposed.
Effect on Lipid Peroxidation
The malondialdehyde content measured spectrometrically was found to be significantly
(p = 0.05) more in PGE treated plants than the control (Fig. 2). A mean value
of 2.827±0.199 μM g-1 f.wt was recorded for the control
plant while 3.787±0.163, 5.217±0.199 and 8.850±0.060 μM
g-1 f.wt were the mean values for plants treated with 0.3, 0.7 and
1.0 ppm of PGE, respectively.
Nitrate Reductase Activity
The effect of PGE on nitrate reductase activity (NRA) in the crude extract
of L. sativa was examined. The result showed a reduction in NRA when
exposed to PGE. The control plants had a mean NRA value of 0.209±0.012
μmol h-1 g-1 f.wt. The value was significantly (p
= 0.05) lower than 0.117±0.003 μmol h-1 g-1
f.wt observed for plants treated with 1.0 ppm of PGE (Fig. 3).
Effect of PGM on Total Protein Content
A decrease in total protein content was observed in all the PGE-treated
plants (Fig. 4). The lowest value (0.389±0.003 mg g-1 f. wt)
was observed in plants treated with 1.0 ppm of PGE.
| Fig. 1: | Catalase activity of Lactuca sativa treated with PGE. Different letter(s) indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison |
| Fig. 2: | Lipid peroxidation in leaves of Lactuca sativa treated with PGE. Different letter(s) on columns indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison |
| Fig. 3: | Nitrate reducatase activity of Lactuca sativa exposed to various concentrations of PGE. Different letter(s) indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison |
| Fig. 4: | Effect of various concentrations of PGE on the protein content of Lactuca sativa. Different letter(s) indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison |
Chlorophyll Fluorescence Parameters
Chlorophyll fluorescence parameters of leaves are presented in Fig.
5-10. The efficiency of photosystem II photochemistry
(ΦPSII) in leaves of PGE-treated plants was lower than those of the control.
The effect was significant (p = 0.05) when PGE concentration was 1.0 ppm. Similar
trend was found in Fv/Fm, a parameter that measures the
intrinsic (or maximum) efficiency of photosystem II.
| Fig. 5: | Toxicity of PGE on the photosytem II of Lactuca sativa. Different letter(s) indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison |
| Fig. 6: | PGE-induced reduction in maximum quantum yield of Lactuca sativa. Different letter(s) indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison |
| Fig. 7: | Effects of PGE on the efficiency of the water-splitting apparatus (Fo/Fv) of Lactuca sativa. Different letter(s) indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison |
| Fig. 8: | Effects of PGE on the plastquinone pool of Lactuca sativa. The same letter(s) indicate no significant difference at p = 0.05 according to Tukey’s pairwise comparison |
| Fig. 9: | Initial fluorescence (Fo) of Lactuca sativa as affected by different concentrations of PGE. The same letter(s) indicate no significant difference at p = 0.05 according to Tukey’s pairwise comparison |
| Fig. 10: | Effect of PGE on the variable fluorescence (Fv) of Lactuca sativa. The same letter(s) indicate no significant difference at p = 0.05 according to Tukey’s pairwise comparison |
The PGE reduced this parameter in a concentration-dependent manner, which was more pronounced in plants treated with 1.0 ppm of PGE. The PGE-induced changes in chlorophyll fluorescence kinetics also included a progressive rise in values of F0/Fv which was significant (p = 0.05) when treatment oncentration was 1.0 ppm (Fig. 7). Figure 8 presents the impact of the PGE on plastoquinone pool (Fv/2) of L. sativa.
| Fig. 11: | Metal concentrations (mg kg-1 d.wt) in Lactuca sativa following exposure to concentrations (ppm) of PGE. Different letter(s) on identical columns indicate significant difference at p = 0.05 according to Tukey’s pairwise comparison |
The result showed that plastoquinone pool values were lowered by PGE in a concentration-dependent manner. Data showing the effects of PGE on initial fluorescence (F0) and variable fluorescence (Fv) of L. sativa are as shown in Fig. 9 and 10. The treatment increased the F0 fluorescence level. In contrast, the variable fluorescence yield (Fv) was decreased by the treatment. The severity of the effects as indicated by both parameters was concentration dependent.
Metal Accumulation in Plants
Data showing the accumulation of Pt, Pd and Rh by the experimental plant
is as shown in Fig. 11. The accumulation was concentration dependent. Accumulation
of Pd was the least while Pt and Rh were at pa (Fig. 11). Plants exposed to
1.0 ppm of PGE for the experimental period had mean values of 52.65±2.46,
27.01±3.29 and 53.51±5.24 of Pt, Pd and Rh, respectively.
DISCUSSION
The changes in parameters measured due to PGE treatments revealed that these metals are toxic to plants. The inhibition of growth as indicated by dry weight and leaf area could be a result of direct interference with enzymes of physiological importance. Similar effects have been reported from studies involving metals like Cd, Cu, Pb, Hg and Ni (Clijsters et al., 1999; Chatterjee and Chatterjee, 2000; Odjegba and Fasidi, 2004). Catalase is one of the scavenging enzymes for reactive oxygen species (ROS) found in plants and it is primarily involved in the decomposition of H2O2 to water and molecular oxygen. ROS are produced as a consequence of electron transport processes in photosynthesis and aerobic respiration (Foyer et al., 1994; Mano et al., 2002). ROS are reactive and potentially harmful to cells, causing oxidation of lipids, proteins and nucleic acids. Its generation by the chloroplast can be increased by factors such as excess light, drought, salt and heavy metal stress and in some cases conditions of limiting carbon dioxide by activation of the photorespiratory pathway resulting in H2O2 production in the peroxisomes. The result from this study indicated increased activity of catalase which was concentration dependent. The corresponding increase in catalase activity was necessary to meet up with the detoxification process in order to keep the cells alive.
The result of our study indicated higher contents of malondiadehyde in PGE-treated plants, indicating that exposure to PGE induced cell membrane damage through lipid peroxidation. The membrane damage may be related to the excessive generation of ROS as a result of PGE treatments. Similar results have been reported that a high level of copper induced lipid peroxidation in Silene cucubalus roots (De Vos et al., 1991) and Triticum aestivum leaves (Luna et al., 1997). A significant decrease in nitrate reductase activity was observed at PGE level as low as 0.5 ppm. The interaction of the PGE with the enzyme could be as a result of the metals binding to functional ligands of the enzyme involved in the catalytic action. Nitrate reductase contains essential-SH Cys residues (Campbell, 1996) which could be modified by PGE. The low protein content in PGE-treated plants could be related to disruption of nitrogen metabolism in the affected plants (Solomonson and Barber, 1990).
Chlorophyll fluorescence analysis has become one of the most widely used techniques available to plant physiologists and ecophysiologists. Light energy absorbed by chlorophyll molecules in a leaf undergo one of three processes: (I) It can be used in photosynthesis, (ii) excess energy can be dissipated as heat or (iii) It can be re-emitted as light-chlorophyll fluorescence. These three processes occur in competition and an increase in the efficiency of one results in a decrease in the yield of other two (Maxwell and Johnson, 2000). Therefore, by measuring the yield of chlorophyll fluorescence, information about changes in the efficiency of photosynthesis and heat dissipation can be gained. Our study showed that PGE reduced the efficiency of photosystem II photochemistry (ΦPSII) as well as the maximum quantum yield of PSII (Fv/Fm) indicating the phenomenon of photoinhibition which may be due to partial blocking of electron transport from PSII to PSI by the stress factor. The results agreed with the findings of Bjorkman and Demmig (1987) and Johnson et al. (1993). There was an increase in the values of F0/Fv at high PGE concentrations, indicating that the test metals had effect on the water-splitting apparatus. The reduction in the plastoquinone pool as represented by the Fv/2 ratio (Fig. 8) could be one of the possible causes for the reduced quantum yield under metal stress. The result from this study showed that minimal fluorescence (F0) increased under PGE stress. The F0 is an emission from the excited chlorophylls in PSII antenna in competition to excitation energy transfer to the PSII reaction center, which takes place before the excitons reach the reaction centre (Mallick and Mohn, 2003). Since F0 originates from chlorophyll a antennae associated with the PSII light-harvesting complex (Karukstis, 1991), it is evident from this study that the efficiency of energy transfer from this complex to the PSII reaction center was reduced under PGE stress.
The study showed that Lactuca sativa accumulated substantial amounts of the metals capable of altering the physiological status and inhibiting the normal growth of the plant. In conclusion, the study reported here established the status of PGE as potent environmental pollutants based on the results presented. Plants could accumulate these metals to significant concentrations enough to cause harmful effects to the plant itself, livestock and humans. Therefore, the release of these metals into the environment requires serious attention.
ACKNOWLEDGMENTS
We are grateful to The Royal Society of London for granting a Fellowship to Victor Odjegba. We also thank Andrew Fisher and Angela Watson for their assistance and contributions.