Effect of Copper, Zinc and Lead and Their Combinations on the Germination Capacity of Two Cereals

The majority of researchers have studied the following group of microelements: B, Zn, Mn, Cu, Na, Co, Mo, I, Sn, Cl, Al, V, F, Cr, Hg, Cs, Li, Cd, As, Th, Rb, Cr, W, Ti, Sn, Se, Ba, Br. Sporadically, the following elements have been mentioned too: Au, Ra, Hg and Pb. In this study, the effects of copper treatments and their combination with zinc and lead microelements on the germination of maize and barley were investigated using different concentrations of these microelements. Six treatments were used: 1. Copper-sulphate (CuSO4) applied alone, 2. Zinc-sulphate (ZnSO4) applied alone, 3. Copper applied with zinc, 4. Leadnitrate (Pb(NO3)2) applied alone, 5. Copper applied with lead and 6. Untreated control. Maize (Kiskun SC 297) and barley caryopsis were treated with copper and zinc solutions in the following concentrations: 0.03%, 0.003% and 0.0003%. Maize and barley caryopsis were treated with these solutions for 12 and 24 hours. Maize and barley caryopsis were also treated with lead solutions Pb(NO3)2 with different concentrations: 0.0005%, 0.005% and 0.05%. Maize and barley were treated with these solutions for 12 and 24 hours. In the combined treatments (3 and 5), the same concentration was used for each microelement as in treatments 1, 2 and 4. Control treatments were treated with water for both plant species. Our results showed that copper microelements significantly inhibit germination compared to the untreated control. The toxicity of copper is higher if concentration increases. Zinc microelements also inhibit germination, however its effect highly depends on the microelement concentration. Treatments of copper + zinc also inhibit germination. The two microelements applied together cause more phytotoxicity than they do alone. Lead is highly toxic to plants even in low concentrations. The toxic effect on germination dramatically increased when lead was applied with copper.

Copper content in plants varies over a large scale.Cereals, wheat, barley and oat are more vulnerable to copper deficiency than maize and rye (Caramete et al., 1973;Ross, 1994;Salisbury and Ross, 1992;Taiz and Zeiger, 1991;Trifu and Bărbat, 1997).In maize, the content of copper in normal leaves is more than 8 ppm, and in unhealthy plants, the copper content decreases to below 6 ppm.The symptoms of copper deficiency are dark green leaves with necrosed spots (Mohr and Schopfer, 1995;Salisbury and Ross, 1992).Leaves present malformations and those which are young are permanently whithered.Vegetative development is not as affected as germinative.Development of roots decreases, and they are long, thin and white (Trifu, 1976;Trifu and Bărbat, 1997).The critical level of copper toxicity in plants is around 20-30 mg/kg dry tissue (Vangrosveldt and Clijsters, 1994).The most important symptoms of copper toxicity are chlorosis, modifications in root morphology and development, decrease of stem development and eventual, perturbation in the plant development (Bathory et al., 1997;Mehra and Farago, 1994;Ouzounidou, 1994Ouzounidou, , 1995aOuzounidou, , 1995b;;Punz and Siegardt, 1993).It can also be mentioned that copper, as a fungicide, can also cause phytotoxicity (rusetting) in several plant species (Holb and Heijne, 2001;Merrington et al., 2002;Holb et al., 2003;Stover et al., 2004).
The content of zinc in faulty plants varies by species (Caramete et al., 1973;Salisbury and Ross, 1992).The symptoms of zinc deficiency vary from species to species and are manifested by a decrease in plant development, malformations of position and morphology of leaves, and also observed were yellow colored leaves (sometimes with yellow lines).Maize is one of the plants which responds very quickly to zinc deficiency.The symptoms in maize are: chlorosis, thin lines on leaves and whithered appearance (Cakmak, 2000;Mehra and Farago, 1994;Salisbury and Ross, 1992;Trifu and Bărbat, 1997).Excess of zinc in plants is harmful, because it blocks the assimilation of iron and the plant becomes chlorotic.Zinc toxicity shows generally a deficiency in development, chlorosis, and red-brown pigmentation in the chlorotic area (Caramete et al., 1973;Davis and Parker, 1993;Mehra and Farago, 1994).
The factors which determine lead content in plants are: mining activities, traffic density, distance from road and climatic conditions.Roots accumulate and fix large amounts of lead.The inhibitory effects of lead on enzyme activities were studied in maize leaves.These include perturbated photosynthesis, respiration, the content of photo assimilation pigments and some important enzymes.
The aim of our study was to compare the effect of copper and its combination with zinc and lead on the germination of maize and barley.

MATERIAL AND METHODS
The effects of copper microelements and their combinations with zinc and lead on the germination of maize and barley were investigated using different concentrations of microelements.Six treatments were used: 1. Copper-sulphate (CuSO 4 ) applied alone, 2. Zinc-sulphate (ZnSO 4 ) applied alone, 3. Copper applied with zinc, 4. Lead-nitrate (Pb(NO 3 ) 2 ) applied alone, 5. Copper applied with lead, 6. Untreated control.
Maize (Kiskun SC 297) and barley caryopsis were treated with copper and zinc solutions in the following concentrations: 0.03%, 0.003% and 0.0003%.Maize and barley caryopsis were treated with these solutions for 12 and 24 hours.Maize and barley caryopsis were also treated with lead solutions Pb(NO 3 ) 2 at different concentrations: 0.0005%, 0.005% and 0.05%.Maize and barley were treated with these solutions for 12 and 24 hours.In the combined treatments (3 and 5), the same concentration was used for each microelement as in treatments 1, 2 and 4. Control treatments were treated with water for both plant species.After the treatment application, the capacity of germination was determined in percentages.All treatments were made in three replications.Significant effects of the treatments were analysed by analysis of variance.

RESULTS AND DISCUSSIONS
The capacity for germination after 12 hours of treatments decreased with increasing copper concentrations (Figure 1).This decrease was significant for copper concentrations of 0.3 and 0.03%, compared to the control treatment.After 24 hour treatments, the germination capacity also decreased with an increasing copper concentration.The decrease of 0.3, 0.03 and 0.003% concentrations of copper was significant compared to the control treatment.
The capacity for germination after 12 hours of treatments decreased with increasing zinc concentrations (Figure 2).This decrease was significant for zinc concentrations of 0.0003 and 0.03%, compared to the control treatments.After 24 hour treatments, germination capacity also decreased with increasing copper concentration.The decrease of 0.03 and 0.003% concentrations of zinc was significant compared to the control treatment.
The capacity for germination after 12 hours of treatments decreased with increasing copper + zinc concentrations (Figure 3).This decrease was significant at all concentrations compared to the control treatments.However, after 24 hours treatments, the decrease of germination capacity was not clear and there were no significant differences among treatments.Some authors on wheat plants observed that plants grow on copper treated plots; however plant growth significantly decreased when it was compared the untreated control plots (Lanaras et al., 1993).The plants height was 25% lower on copper soils than in soils without copper treatments.These results are in agreement with other published data (Breckel, 1991;Kahle, 1993;Arduini et al., 1994;Ouzounidou, 1994Ouzounidou, , 1995aOuzounidou, , 1995b)).In contrary, zinc is very important in the vegetative organs of plants (Barman et al., 1999(Barman et al., , 2000;;Tomulescu et al., 2000b).
The capacity for germination after 12 and 24 hours of treatment decreased with an increase in lead concentrations (Figure 4).This decrease was significant for all concentrations, compared to the control treatments.
After 12 and 24 hours of treatment, the capacity for germination dramatically decreased with an increase in copper + lead concentrations (Figure 5).The highest concentration of copper and lead mixture completely inhibited germination.This decrease was significant at all concentrations compared to the control treatments.Some authors (Mehra and Farago, 1994) stated that effects determined by lead toxicity are diverse and often contradictory.It seems that lead ions penetrating the plant obstruct the enzymes and disturb the electo sion and negatively influence plant germination and development.Our results are in agreement with previous research (Băliban et al., 1999;Tomulescu andBurcă, 2003a, 2003b;Tomulescu et al., 2003aTomulescu et al., , 2003b)).In barley, lead may be more toxic to germination than copper.Our results on lead phytotoxicity are omulescu et al., 2000a, 2000b).

CONCLUSIONS
The following conclusions were drawn from the comparative study of the effects of copper microelements and combined zinc and lead on the germination of corn and barley.1. Copper microelements inhibit germination significantly compared to the untreated control.The toxicity of copper is higher if concentration increases.2.

Figure 1 :
Figure 1: Capacity for germination after 12 and 24 hour treatments of different concentrations of copper microelement

Figure 4 :Figure 5 :
Figure 4: Capacity for germination after 12 and 24 hour treatments of different concentrations of lead microelement . J.-Jong, de P. F.-Heijne, B. (2003): Efficacy and Kah f trees to heavy metals.Lan and some photosynthetic measurements on field-culti wheat growing on ore bodies enriched in Cu.Physiol.Plant.