Copper Ion Effects On Mung Beans Germination Environmental Sciences Essay

This experiment was planned to investigate the effect of different concentrations on copper ion on seed germination of mung beans. The experiment uses seed germination as a parameter in the presence of varying concentration of copper ion solution (Cu2+) in copper (ii) sulphate (CuSO4). Solutions used were 300 ppm, 200 ppm, 100 ppm, 75 ppm, 50 ppm, 25 ppm, and 0 ppm and the seeds were all soaked for 6 hours, and then sown on Petri dishes with a wetted cotton layer. The seeds were watered with the same volume of Cu2+ solution of respective concentrations. The numbers of seeds germinated were recorded after 20 hours, and the germination rate was calculated. Results showed that germination rate increases as concentration decreases. An analysis was done using the Pearson product-moment correlation coefficient (PMCC), and it showed a statistically significant negative linear relationship between concentration of Cu and germination rate as the calculated r-value was greater than critical value (Cr) at 5% significant level.

Research and Rationale

Plants as micronutrients require a number of heavy metals, which are found naturally in soil.13 However, the global buildup of metals in the environment is increasingly becoming a problem.1 Toxic metals continue polluting the biosphere by volcanoes, natural weathering of rocks, and by industrial activities such as combustion of fossil fuels and mining.2 Heavy metal pollution has accelerated since the beginning of the industrial revolution. Copper (Cu) is one of the main metal pollutants, and usually results from human activities such as mining and the use of fertilizers.4 Cu is an essential element needed in trace amounts in plants, about 4-30 ppm of the approximate dry weight in plants (Raven and Johnson 1999), associated with enzyme activity which catalyses the oxidative reactions in various metabolic pathways.(4)

An excess causes a reduce in germination, growth, respiration, photosynthesis and also causes severe membrane decomposition.4 It becomes toxic as it interferes with the enzyme activity, acting as a non-competitive inhibitor, destructing the tertiary bonds in some enzymes, thus altering and inhibiting enzyme activity.4 Human life becomes at risk once these plants develop tolerance mechanisms against Cu, and when these plants are incorporated into our food chain.(2) Mung bean is part of the human food chain as it is a favourite ingredient in Asian food.5 It is commercially grown in many regions of Asia. An ability of it to become Cu tolerant would pose a high risk on human health due to Cu accumulation in the body.2

This investigation also serves to show the type of soils suitable for mung bean growth in agriculture. Soils contaminated by copper or near industries are deemed unsuitable. The seed- Vigna radiata, commonly known as mung beans was chosen as it has a short life cycle and is small, thus large numbers of the seeds can be used as not much space is taken.

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Germination rate, which is usually expressed in percentage, shows the number of seeds that is likely to germinate; based on a particular plant species.6 Germination is one of the most critical stages of development in a plant’s life cycle. It is at this stage where the plants are more susceptible to injuries, water stress or diseases. 7

Experimental Hypothesis

The lower the concentration of Cu2+ in the solution, the greater the germination rate of mung beans.

Null Hypothesis

There is no correlation between the different concentrations of Cu2+ in the solution and the germination rate of mung beans.

Variables:

Manipulated variable : Different concentrations of Cu2+ from Cuso4. (ppm)

Responding variable : Germination rate of mung beans (%)

Fixed variables : Mass of Cuso4 (g)

Volume of solution used for soaking (30 ml/beaker) and for moistening the cotton (50 ml/petri dish)

Temperature

Light Intensity

Humidity

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Apparatus: Petri dishes

Label stickers

Weighing balance

Dropper

Beakers (600 ml)

Volumetric flask (1000 ml)

Measuring cylinders (100 ml, and 500 ml)

Pipette

Schott bottles

Materials: 3.937g of Cuso4

Distilled water

Mung bean seeds (A)

Cotton

Planning

Number of seeds used: 15

The first trial was to determine the condition needed for germination. Three different conditions were identified- in the dark (inside a cupboard), normal laboratory conditions, and outside the laboratory (under normal environment). The seeds were soaked for an hour in distilled water and then sown on wetted cotton. For each condition, three replicates were prepared. The results were observed after 20 hours.

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Conditions

Number of seeds Germinated

Mean Number of Seeds Germinated

Replicate 1

Replicate 2

Replicate 3

Laboratory

In the Cupboard

Garden

Table 1: Germination rate of seeds in different conditions

Germination rate= Number of seeds germinated/Total number of seeds – 100

The results showed that the seeds grown in the dark had the highest germination rate. This is in line with what has been written about the germination of mung beans in websites stating that mung beans germinate in darkness.5 Therefore, it was decided that further germination of the seeds would be carried out in the dark under room temperature of 25°C in the mornings and 22°C during night.

The second trial was a combination to find out the most suitable duration to soak the seeds and the best concentrations needed to carry out the experiment. For starters, the concentrations decided were a range from 1000 parts per million (ppm), 750 ppm, 50 ppm, 250 ppm, 100 ppm and 0 ppm (distilled water). The stock solution of 1000 ppm was prepared by the following method.

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To make 1000 ppm of Cu using CuSo4.5H2o

Molar Mass of CuSo4.5H2o= 249.5g

Atomic weight of Cu=63.5g

1g of Cu in relation to molar mass of salt= 249.5/63.5

= 3.931g

Hence, 3.931g is weighed out from CuSo4.5H2o and dissolved in 1000ml of distilled water to make a standard solution of 1000ppm of Cu.8

From the stock solution, the serial dilution method was applied to make a concentration of 750 ppm. From 1000 ml of the stock solution, 750 ml of the solution was diluted in 250 ml of distilled water in a volumetric flask, to make up 1000 ml of 750 ppm solution of Cu. To make up 500 ppm solution, 500 ml of stock solution was diluted in 500 ml of distilled water and so on.

The duration to soak was set to one hour, three hours, six hours and 12 hours. The seeds were soaked in 6 beakers containing the different concentrations, and sown to germinate on Petri dishes. Wetted cotton was used, and the respective concentration of the solution the seeds were soaked in was used to wet the cotton. 15 soaked seeds of similar size were chosen to germinate in two replicates.

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Concentration (ppm)

Duration of Soaking (Hours)

1

3

6 A

B

Mean

Germination Rate (%)

73.3

60.0

73.3

100 A

B

Mean

Germination Rate (%)

26.7

86.7

250 A

B

Mean

Germination Rate (%)

33.3

26.7

500 A

B

Mean

Germination Rate (%)

33.3

750 A

B

Mean

Germination Rate (%)

0.0 1000

A

B

Mean

Germination Rate (%)

0.0

Table 2: Germination rate of seeds in different conditions soaked for different durations

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Observations were made 20 hours later, and in concentrations of 1000 ppm and 750 ppm, no seeds germinated. 500 ppm was the highest concentration whereby seeds germinated while for 0%, the germination rate was 80%. The seeds that was soaked for six hours showed the highest seed germination rate for the concentrations whereby seeds did germinate. Therefore, it was decided that the best range of concentrations to be used included 0 ppm, and also a value slightly lower than 500 ppm, and the duration of soaking was 6 hours.

For seeds soaking in 500 ppm solutions ad above, the seeds appeared slightly purplish, and some even turned black. The seeds germinated at concentration of 500 ppm had its roots stunted with necrotic tips.

To ensure the results were more reliable statistically, the number of seeds to be used in the actual experiment was increased to 50 seeds per concentration instead of 15. The actual experiment also included two replicates, using similar methods to obtain more reliable results. The Pearson product-moment correlation coefficient (PMCC) was chosen to analyse the data obtained.

Experimental Procedures

A stock solution of 1000ppm of Cu2+ was prepared from 3.937g of Cuso4 (as indicated in the trial).

The serial dilution method is used to prepare different concentrations of Cu2+ in Cuso4 solution. For example, 10 ml of 1000ppm solution was pipetted out and mixed with 990ml of distilled water in a 1000 ml volumetric flask to obtain a 10ppm solution, and so on. Besides 10 ppm, 25 ppm, 50 ppm, 75 ppm, 100 ppm, 200 ppm, and 300 ppm were prepared and stored in labeled Schott bottles.

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At least a 150 seeds were chosen randomly and placed in a beaker to be soaked with a 300 ppm solution (50 ml) for six hours.

Two Petri dishes were prepared and labeled A and B. Two layers of white cotton were placed in each Petri dish, spread evenly. The cottons were moistened with 300 ppm solution of the same volume (30 ml).

50 seeds of similar size were chosen and sown in each labeled Petri dish and placed inside the cupboard to germinate.

Observation was made 20 hours after sowing. Germination was said to have occurred once the radicle was visibly extended from the surface of the seed for about a minimum of 5mm.

The number of seeds germinated was determined, and the mean and the germination rate were calculated.

Steps 2 to 8 were repeated for different concentrations- 200 ppm, 100 ppm, 75 ppm, 50 ppm, 25 ppm, and 0 ppm, and all results were recorded in Table 3, a graph was plotted and a PMCC test was used to analyse the data.

Risk Assessment

All glasswares, such as measuring cylinders and volumetric flask were handled with extra care as they can break easily and may then cause injuries. The soaked seeds were handled with gently as they are very fragile. While using the measuring cylinder, volumetric flask and pipette to measure the amount of solution needed, the eye-level was ensured to be perpendicular with the scale on those apparatus as to prevent parallax error. This was crucial while mixing the stock solution for a little difference may actually affect the percentages of all the other solutions.

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Results

Concentration (ppm)

Number of Seeds Germinated

Mean Number of Seeds Germinated

Germination Rate (%)

A

B

100

25

50

75

100

200

300

Table 3: Germination of seeds in different concentrations

Calculating germination rate:

=

= 78%

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Statistical Analysis

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The PMCC test was chosen to measure the strength of linear dependence between two variables-concentrations and germination rate.

The correlation coefficient, r ranges from +1 to -1. A value of zero indicates no linear correlation between the said variables, while +1 indicates a linear equation explains the relationship between concentration and germination rate perfectly, with all the points from the data lying on a line for which germination rate increases as concentration increases. A value of -1 indicates that all points from the data lie on a line whereby germination rate decreases as concentration increases.9

x

100

200

300

∑X=750

y

100

∑Y=424

x2

625

2500

5625

10000

40000

90000

∑x2= 148750

y2

10000

8836

8100

6084

784

324

256

∑y2=34384

yx

2350

4500

5850

2800

3600

4800

∑xy= 23900

SS (x) =

=

=68392.857

SS (y) =

=

=8701.714

SS (xy) =

=

= -21528.571

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The correlation coefficient, r, is found using the formula:

= -0.8825 (negative indicating a negative correlation between variables)

r = 0.8825 > 0.754 Cr for 5% confidence level.

The analysis using PMCC proved a statistically significant negative linear relationship between the concentrations of Cu2+ in CuSO4 and germination rate, as the calculated r value was larger than Cr at 5% confidence level.

Hence, null hypothesis is rejected and experimental hypothesis is accepted. A higher concentration of Cu2+ leads to a lower germination rate of mung beans.

Data Analysis

The statistical analysis using PMCC proved that low concentration of Cu did result in a greater germination rate of mung beans, as shown in Table 3 too which shows the number of seeds germinated after treatment with respective concentrations of Cu2+ in CuSO4 solutions for 20 hours. The table shows that only a very low concentration of Cu allows germination.

Graph 1 illustrates the trend and relationship between the variables. It can be seen clearly that as the concentration of Cu2+ in CuSO4 increases, the rate of germination decreases. Distilled water recorded the highest germination at 100%, while the lowest germination rate was noted when the concentration used was 300 ppm.

There is a great difference in germination rate from 75 ppm to 100 ppm, about 50%.

Increase the concentration from 25 ppm to 50 ppm and 200 ppm to 300 ppm only showed a decrease in 4% and 2% respectively. This may one of the inconsistencies of the experiment.

Distilled water, or 0 ppm acted as a control in this experiment. From the results obtained, all the seeds germinated when soaked in distilled water. From the trials, seeds soaked in distilled water did not germinate more than 80%, however in the actual experiment it was 100%. After much research, I concluded that the results obtained from the actual experiment was valid as it supports the results obtained from various other research published.1, 10

The inconsistencies that occurred may have been due to:

Some of the seeds may have been already damaged (trials) and so germination was impossible

The cotton layers used in 0 ppm during trials were too thick, and became dry fast, thus made it impossible for the seeds to absorb the water and enable germination in 20 hours.

Mixing errors could have occurred causing the drop between 75 ppm and 100 ppm, as the concentrations of the solutions could have been higher than it was suppose to be.

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Evaluation

The degree of seed germination in the presence of Cu2+ is to some extent a measure of resistance of mung beans to this toxic metal.11 Germination, is a process said to occur when a dormant seed begins sprouting into a seedling, under the right growing conditions.14 This process is highly dependent on external and internal conditions. Light or darkness, temperature, water and oxygen and considered to be the important external conditions that may affect germination.

Imbibition, the process by which water is taken up by the seeds, causes the soaked mung bean seeds to swell and start softening, thus the breaking of the seed coat.11, 6 This makes germination easier. Hydrolytic enzymes are activated (due to water), and these enzymes digest the food source in the seeds into chemicals, that are useful metabolically. 6, 7

Excess Cu is detrimental as it becomes toxic. It interferes with the enzyme activity, acting as a non-competitive inhibitor, destructing the tertiary bonds in some enzymes, thus altering and inhibiting enzyme activity.4 It combines with the thiol groups, breaking the hydrogen bonds and disulphide bridges holding the 3-D shape together (of an enzyme). In order to germinate, amylose is needed, and is metabolized by amylase. The interference of toxic level of Cu causes the inhibition of amylases, thus preventing the food store in the cotyledon to be broken down, depriving the embryo from carbohydrate needed for respiration and production of energy for germination.4

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There were some limitations in this experiment. The temperature of the surroundings is one of it. It was assumed that the temperature in the cupboard was to be 22 and 25 at night and morning respectively. However, along the day, the temperature could have fluctuated. This could have affected the germination process, as the time taken for germination to occur could have been longer.

Besides, the humidity of the surroundings was also not measured and thought to be constant all the time. However, due to changes in weather (rain at night, hot sun in the mornings) during the time of experiment, there could have been changes in relative humidity and temperature too. Some of the seeds may have already been damaged but appeared normal. These seeds could have been sown for germination, thus it can be certain that the seeds that did not germinate were purely due to high toxic Cu levels or not.

The use of a large sample had given enough replicates to support the conclusion for this experiment. The results are reliable as the experiment can be controlled and also repeated. This investigation only stressed on the effects of Cu on the germination of mung beans, without subjecting the seeds to different abiotic or biotic pressures.

Modification could be made by comparing the, effects of other heavy metals such as Nickel, Cadmium and Lead on the germination of mung beans. Besides investigating the rate of germination only, the length of the radicle can measured (plant growth) to see the effects on growth of mung beans. The growth of plants may show a more visible change due to the toxic metals. Plus, other varieties of seeds such as sunflower, orca or cabbage can be used to test the levels of metal toxicity tolerance on other plants species.

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Conclusion

With reference to the results obtained and statistical analysis made, it can be concluded that the lower the concentration of Cu in the solution, the greater the germination rate of mung beans, Vigna radiata. This is because Cu is an enzyme, non-competitive inhibitor which alters and inhibits enzyme activity. Thus experimental hypothesis is accepted, while null hypothesis is rejected.

Evaluation of Sources

Source 7 and 13 are published books written by well-known authors in the scientific community. The source is reliable as it has been revised, recognized and undergone a series of editing before being published.

Sources 1,2,3, 10 and 11 are scientific journals. Scholars wrote it and the journals have been peer-reviewed, and have a high level of scientific creditability. Therefore, these sources should contain valid information.

Sources 4,5,6,8,9,12 and 14 are websites. These are sites that have been referral site to by many people and the information in it are also cited, thus it is reliable.

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Appendix

Figure The apparatus used to make the stock solution-500ml Measuring Cylinder, 100ml Measuring cylinder, 1000ml volumetric flask

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