This experiment was conducted to investigate the effect of lead, a heavy metal, on the growth of Vigna radiata seedlings. The seeds were treated with lead solution (as lead (II) nitrate) of various concentrations: 0, 20, 50, 100, 150, 200 and 300 ppm and later sown on petri dishes containing cotton wool moistened with the solution they were treated with. After 96 hours of incubation, the length of their roots and shoots (epicotyl) were measured and their total growth (seedling length) was determined. Results showed that lead has an inhibitory effect on the growth of Vigna radiata as the length of the seedlings decreased with the increase in lead concentration. A statistical analysis using the Pearson product-moment correlation (PMCC) test supports the experimental hypothesis; the higher the lead concentration, the more stunted the growth of Vigna radiata seedlings, as the value of correlation coefficient, r was calculated to be greater than the critical value at 5% significant level.
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Excessive and poorly managed human activities such as fossil fuel burning, logging and mining have caused much damage to the environment. The consequences are widespread, affecting many including the flora and fauna. Among them, heavy metal contamination would probably be the most detrimental.
The term 'heavy metal' has many definitions, but according to the International Union of Pure and Applied Chemistry (IUPAC), in terms of its toxicity, a heavy metal is 'an element commonly used in industry and generally toxic to animals and to aerobic and anaerobic processes, but not every one of it is neither dense nor entirely metallic' . Some heavy metals play a dual purpose in the metabolism of plants. At low levels of concentration, they serve as essential micronutrients but when they reach a certain critical amount, these elements become toxic . An example of such an element is chromium, Cr. Other heavy metals, however, are harmful to plants by nature. This includes lead, Pb.
Lead, or also known as plumbum is a highly toxic pollutant. It is one of the most common heavy metals and its exposure is of utmost concern due to its toxic nature, extensive occurrence and long life in biological system. Lead occurs naturally in substantial amounts in the earth's surface, present in all soils, lakes, rivers and sea water. However, throughout the years, the quantity of this element seems to be on the rise due to the discharge of man-made activities, notably from automobile exhaust and through industrial effluents .
In humans, prolonged exposure to lead may cause poisoning which affects the health severely. In plants, on the other hand, lead uptake from soil and water can cause significant negative effects on their growth and germination such as retardation and poor seed viability. In addition, the production of seeds and fruits will also be reduced. This happens as lead induces changes in many of the structure and biochemical processes in their system .
There has been much work in regard to the effect of heavy metal on the growth of plants, mostly focusing on elements such as nickel, Ni and zinc, Zn (Durby and Dwivedi, 1987; Peralta, Tiemann, Gomez, Rascon, Gornez, Arteaga and Parsons, 2001; Munzuroglu and Geckil, 2002; Ahmad, Hussain and Saddiq, 2007) using mainly soybean and alfalfa as subjects.
In this experiment, Vigna radiata seeds were used to study the toxicity of lead. Also known as mung beans, Vigna radiata is a very common plant famous for its use in cooking. Its fast growth and high adaptability makes it a perfect subject for this experiment. Little information is available on the effects of lead on the growth of Vigna radiata. Hence, this investigation was carried out to study its effect on one of the parameters used to determine plant growth, which is the seedling length.
Research on the effect of heavy metal, especially lead on plants by bioassay has benefited many, especially those involved in agriculture. Knowing the dangers that it poses to potential food crops, steps have been taken to treat and remediate contaminated soils, usually through immobilization and by removing them physically. A much more recent method is phytoremediation which is the use of hyperaccumulator plants to remove the heavy metals through uptake and accumulation .
The higher the lead concentration, the more stunted the growth of Vigna radiata seedlings.
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There is no correlation between different lead concentrations and the growth of Vigna radiata seedlings.
A trial experiment was conducted to find the best range of lead concentration and the most suitable time required to soak the seeds for imbibition.
The concentrations required were 0 (distilled water), 50, 100, 250, 500 and 1000 ppm. The stock solution of 1000 ppm was prepared by dissolving 1 g of lead (as 1.60 g lead (II) nitrate) in 1000 ml of distilled water. Other concentrations were later prepared using the stock solution as the basis by serial dilution method. To make lead solution of 500 ppm, equal amounts of 1000 ppm lead solution and distilled water were added. The same step was followed when making 250 ppm lead solution, only substituting the 1000 ppm lead solution with that of 500 ppm.
At the beginning of the trials, the Vigna radiata seeds were separately soaked in different concentrations of lead as prepared before, for imbibition (to hasten seed germination). As part of the trials, these seeds were soaked for different periods of time; 6, 12 and 24 hours before sowing. In the meantime, petri dishes containing cotton wool moistened with appropriate volumes of the said concentrations were prepared. Once soaked, the solutions were disposed of and 20 seeds of comparable sizes were each sown on the petri dishes according to the solution they were priorly immersed in. Observation was made 72 hours after incubation. The number of seeds which germinated (when there is a 1-mm radicle emergence) was counted and the germination rate was calculated. In addition, the seedling length (sum of root and shoot length) of each growing seed was measured. The mean was obtained by calculation involving only the three longest seedlings.
Germination rate (%)
Mean seedling length (cm)
Table 1: The germination rate (%) and seedling length (in cm) of Vigna radiata seedlings soaked in different concentrations of lead at varying periods, after sowing for 72 hours
From the trials, it could be seen that the germination rate was generally highest when seeds were soaked for a period of 12 hours while the length of the seedlings showed significant decrease. Germination rate of Vigna radiata seeds was lowest at 1000 ppm of lead solution, dropping to an average of 30%. Hence, subsequent experiments were carried out at a smaller range of concentrations, ranging from 0-300 ppm since it is also known that the concentration of 300 ppm is the safe lead concentration commonly followed for agriculture purpose . Additionally, the period of incubation was also extended to 96 hours to make the observation more pronounced.
Concentration of lead solution
Different concentrations (0, 20, 50, 100, 150, 200 and 300 ppm) of lead solution were used to soak Vigna radiata seeds for imbibition and for growth.
Seedling growth (length)
The root and shoot of the Vigna radiata seedlings were measured using a metre rule with the help of a string.
Type and number of seedlings, soaking period, volume of solution used for soaking, volume of solution used to moisten the cotton wool and environmental factors (temperature, humidity and light intensity)
Apparatus and Materials
50 ml beaker, 50 ml measuring cylinder, glass rod, petri dishes, spatula, 1 L volumetric flask, dropper, metre rule, distilled water, lead (II) nitrate (Pb(NO3)2) salt, Vigna radiata seeds, cotton wool, and string.
The effect of each concentration of lead was studied on 20 seeds of Vigna radiata. Vigna radiata seeds were firstly surface sterilized by immersion with a dilute solution of sodium hypochlorite (1.2%) for 5 minutes and later washed with deionized water. The seeds were then soaked separately in 50 ml lead solution of varying concentrations (0 (distilled water), 20, 50, 100, 150, 200, and 300 ppm), applied as lead (II) nitrate, Pb(NO3)2 in labelled petri dishes for 12 hours. Swollen seeds were later sown on petri dishes containing cotton wool wetted with 20 ml of lead solution in which they were immersed in. Each treatment was replicated three times. The seeds were provided with 160-watt light and kept at room temperature (28Â±1 Â°C). The root and shoot length of the five tallest seedlings were measured at 96 hours of incubation using a string and metre rule. Data obtained were analysed statistically by Pearson product-moment correlation (PMCC) test.
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Gloves were worn while preparing the lead solution as the lead (II) nitrate salt used is toxic and may cause lead poisoning. Close contact with the salt and its solution was strictly avoided. Besides that, the apparatus used were handled with care as they are fragile and may cause injury if broken. In the experiment, to prevent damage, the soaked Vigna radiata seeds and the seedlings were transferred and handled cautiously.
Root length (cm)
Shoot length (cm)
Mean seedling length (cm)
Table 2: The root, shoot and mean seedling length (in cm) of Vigna radiata seedlings after sowing for 96 hours at varying lead concentrations
Graph 1: Graph of seedling length against lead concentration
The data obtained from this experiment were analysed statistically by using the Pearson product-moment correlation coefficient (PMCC) test. This test was conducted to determine the degree of correlation (linear dependence) between lead concentration, x and seedling length, y by calculating the correlation coefficient, r.
The coefficient ranges between -1 and 1. A value of 1 means that the relationship between x and y is perfectly linear (y increases with x) whereas the negative sign implies an inverse relationship between both variables. If r = 0 however, it means that x and y shows no correlation at all .
The calculations and steps involved are shown below.
âˆ‘x = 820
âˆ‘y = 36.82
âˆ‘x2 = 165,400
âˆ‘y2 = 218.11
âˆ‘xy = 3,135.2
Table 3: Calculations involved in finding the correlation coefficient, r
Table 4: Critical values for Pearson correlation
r = -0.905 < -0.754 (critical value for 5% confidence level)
A value of r = -0.905 shows that there exists a strong significant negative correlation between the concentration of lead and the length of the Vigna radiata seedlings. Since the calculated value of r is greater than the critical value (at 5% significant level), it is accepted that the null hypothesis can be rejected.
Table 2 shows the root, shoot, and mean seedling length of Vigna radiata seedlings treated with different concentrations of lead solution after 96 hours of incubation. From the table, it could be seen that an increase in lead concentration has caused a decrease in the length of the seedlings. Seedlings in the control group (0 ppm, treated with distilled water) seem to grow the tallest, at an average of 8.67 cm. However, as the concentration of lead slowly increased, the length of seedlings became shorter, down to a minimum length of 3.00 cm in 300 ppm of lead solution. Evidently, both the growth of roots and shoots of the seedlings became inhibited as lead becomes more concentrated since both parts show a reduction in length. This relationship is further validated by the statistical analysis using the PMCC test.
Graph 1, on the other hand, illustrates clearly the general trend and correlation between both variables. For the most part, the seedling length decreased with an increase in lead concentration. A line of best fit drawn almost perfectly fit the decreasing trend. There is however, a noted anomaly. The seedling length seems to level off at concentrations 150-200 ppm. From the results obtained, the difference in length between seedlings in respective concentrations was slight, only by 0.06 cm (1.6%). This anomaly may have arisen due to certain experimental errors and limitation. These will be further discussed in the following section.
Generally, Vigna radiata seedlings treated with distilled water showed the tallest growth while those in 300 ppm of lead were the most stunted. At a concentration of 20 ppm (normal lead concentration in most soils), the seedling length showed a 19.7% decrease while that in 300 ppm decreased by almost 65.4%. Even a small application of lead is shown to greatly reduce the growth of the seedlings. In agriculture, such an effect will give a huge impact on the yield produced and this will definitely have an influence on the industry and economy.
Germination initiates the beginning of life for a plant. Regarded as the first step in the growth of embryo, seeds first take up water (imbibition) in order for this to happen. Water uptake is a critical step since seeds are practically dry, containing only about 5-10% water. After imbibition, enzymes in the seeds become active and will be rapidly released to digest the stored food into smaller molecules required for metabolism. These will later be transported and converted into energy for the growth of the seedlings . The radicle will start to swell as germination takes place.
Since Vigna radiata is a dicotyledonous species, the part of the plant that first emerges from the seed is the radicle (embryonic root). The radicle enables the seedlings to anchor themselves firmly on the ground as they absorb water for further metabolism. Over time, the plumule (shoot) will start to develop. When the reserve food supply in the endosperm gets depleted, cotyledons will emerge from the seed coat, enlarge, undergo photosynthesis for a brief period, and will eventually wither and die .
The growth of seedlings continues as roots and shoots elongate. This happens around the meristematic region of the plants. The meristems (sites in the plant body where cell divides and where differentiation into specialized cells and tissues is initiated) are mostly held responsible for the growth of newly formed plants (seedling). Under favourable conditions, the unspecialized cells in these sites elongate, divide and mature as they grow, lengthening the roots and shoots . However, inhibitory responses may occur when changes takes place in these processes.
Lead uptake by seedlings has been found to affect the cell cycle of the cells in the meristematic region of the plant. A previous study has shown that lead deposits accumulated in the walls and vacuoles of root tip cells extends the period of S and G2 phase of mitosis (by 216% and 55% respectively), ultimately lengthening the whole cell cycle. It was discovered that protein synthesis in late G2 phase becomes inhibited, causing a deficit of tubulin. Consequently, cells lack properly formed mitotic spindles, hence disturbing the normal course of mitosis . The reduction in the mitotic rate of meristematic cells explains the poor growth in lead treated seedlings as there will be less cells responsible for division and elongation, and hence growth.
It is also suggested that growth inhibition by heavy metals, such as lead can be reasoned out by the rapid effects on the loss of cell turgor which happens as plasma membranes get damaged by the metals, leading to leakage of soluble constituents of the cells. Also, in a previous report, zinc has been found to impair water uptake capacity, promote metabolite leakage and depress chlorophyll development, all of which are important for normal growth under normal circumstances . This may be able to justify the effects of lead on the seedlings length.
There are other possibilities as well. It is reported that lead deposition induces stomata closure, resulting in reduced photosynthetic rates among plants. Hence, less food will be available for seedlings to grow, which will later affect their length. Lead also causes inhibition of enzyme activities which is important in the breakdown of food into a more soluble form . This is required by plants for proper and normal growth.
The procedures were designed to eliminate as much experimental errors as possible. In practice, however, there is always a possibility for such errors to occur. In this experiment, these may have arisen due to mistakes made during the preparation of the solutions. Hence, the exact concentrations used may have been inaccurate. In addition, within the four days of incubation, it was also possible that the growth of seedlings were affected by the lack of water since the damp cotton wool became dry due to evaporation over time. Furthermore, there were tendencies for the seedlings to get damaged by snapping, especially during transfer and the process of measuring their length. The measured seedling length may have not portrayed its actual length.
There were also some noticeable limitations to this experiment. For instance, although the seedlings were exposed to the same environmental factors, the humidity and temperature of the surroundings in which they were incubated in were not constant and fluctuated over time. This may have affected their potential growth. In addition, the embryo of some seeds may have already been damaged prior to the start of the experiment. The seedlings also differed in their growth rate and some may have a greater tolerance towards lead than the others. As a result, some seedlings may have been able to grow well even at high concentrations of lead. It should be noted that the seeds were chosen solely based on their physical appearances: the size, colour, shape and the condition of the seed coat. Nevertheless, the use of a fairly big amount of samples and several replicates should provide reliable results.
Certain modifications can be made to improve this experiment and further validate the hypothesis. There are many other parameters that can be used in determining plant growth. Other than seedling length, measurements on the seedling's dry/fresh weight or root/shoot ratio are a good indication of its growth. Seeds can also be sown in treated soils as an alternative to see how they respond to lead toxicity in relation to other abiotic stress under normal circumstances. Furthermore, apart from Vigna radiata, other variety of seeds can be used to test the variables since lead tolerance differs among different species of plants.
Based on the results obtained, it can be concluded that lead does have an inhibitory effect on the growth of Vigna radiata seedlings. The roots and shoots of the seedlings showed a marked decrease in length as the concentration of lead increases. The null hypothesis can therefore be rejected.
Information on the growth of plant seedlings were mostly obtained from the book Handbook of Plant Science Volume 1 & 2 (2007) and Plant Biology 2nd Edition (2006). Since these books were published and written by professionals in their respective fields, the facts provided should be highly reliable. In addition, the contents can be considered up-to-date due to their recent publish. Facts on lead as a heavy metal were acquired from an extension research from a university website (University of Minnesota - http://www.extension.umn.edu/distribution/horticulture/DG2543.html). This source is well-grounded as much of the information provided were referred to from reputable sources. The details on the experiment were obtained from peer-reviewed journals (Sources 9, 10, 11, 12 and 13) written by expert scholars. These articles can be obtained from scientific websites such as ScienceDirect (www.sciencedirect.com) and SpringerLink (www.springerlink.com) which are regarded by many of the scientific community as very trustworthy sources.
Figure 1: The initial stages of plant growth
Figure 2: Vigna radiata seeds soaked in lead solution