Investigating the effect of varying concentrations of nitric acid on the stem growth of Vigna radiata seeds

How does the different concentrations (0.0125, 0.5000, 0.0750, 0.1000, 0.1250 moles) of nitric acid (HNO₃) affect the length of stem growth of Vigna radiata (green gram) seeds grown in the nitric acid solution for 7 days?

In recent times there has been more awareness raised on pollution and its effects on the world, in my different IB HL classes I have learnt more about the different aspects of pollution from the political side in Global politics as well as the chemical side in Chemistry. But in my IB biology class, I learnt about global warming and the greenhouse effect and how the chemicals released into the atmosphere not only increase warming globally but also result in acidification in ecosystems and its effect on life which to me was the most interesting. Acidification can be defined as the increased concentration of acids (hydrogen ions) in soil or water which decreases the overall pH ("Acidification - Definition, Meaning & Synonyms", 2022). These conditions are not conducive to ecosystems with a large array of plants that require neutral conditions for optimal growth.

Acidification is primarily as a result of acid rain which is rainfall or any form of precipitation that is made acidic as a result of atmospheric pollution. This is primarily caused by the industrial burning of coal and other fossil fuels where the waste gasses produced as a by-product containing sulfur and nitrogen oxides gather and in the atmosphere and react with atmospheric water to form liquid acids which are precipitated on the earth's surface ("What is Acid Rain? | US EPA", 2022).

As seen in figure 1, due to the sulfur and nitrogen oxides that are released into the atmosphere the resulting precipitation in various ecosystems affects biotic factors such as heterotrophic consumers and autotrophs both on land and in the ocean. This is due to the corrosive nature of acid rain when precipitated towards the ground it dissolves nutrients such as magnesium and calcium found within the soil, such nutrients are regularly absorbed by plants as these nutrients are essential and cannot be synthesized outside the plant they must be readily consumed, the lack of these nutrients in soil negatively affects plant growth (EPA, 2023). Further, when there is a high level of acid being precipitated it not only causes rain but acid fog in affected areas which strips nutrients from the foliage of a tree and results in the death of the leaves due to the lack of photosynthesis for nutrient production the entire tree ("Effects of Acid Rain | US EPA", 2022). In addition, similar studies were done on the effect of pH conditions on Vigna radiata growth as well as other flowering plants have shown that natural soils are slightly acidic ranging from pH 5-7 and an increase in alkalinity or acidity has an inverse relationship with stem growth (Gentili, Ambrosini, Montagnani, Caronni, & Citterio, 2018). Studies have also shown that increased nitric acid reacts with magnesium and chloride ions to provide salts which are insoluble for plants resulting in limited uptake of essential minerals. Vigna radiata when isolated from soil can provide nutrition necessary for stem growth, however, when in acidic conditions caused by chemical substances such as nitric acid nutrients obtained through photosynthesis are easily corroded which causes a lack of nutrients within the plant to provide energy (in for on adenosine triphosphate) causing a lack of biological processes necessary for life.

Further, additional factors that limit plant growth include light intensity, carbon dioxide concentration and temperature. As acid rain is a by-product of pollution, temperature is also invariably affected, due to pollution causing global warming. Therefore areas generally affected by acid rain are additionally affected by increased atmospheric temperatures which can cause denaturation of various cell organelles essential to photosynthesis to denature and be incapable of generating the necessary nutrients for plant life.

Because of the adverse effects of acid rain on various plant life, my investigation is focused on Vigna radiata commonly known as green grams. I choose this specific plant because of its significance to my home region Kenya which is the main exporter of this food. Where my home is located there are many farms that export Vigna radiata, which is a livelihood for farmers in my region with the demand for the product being high. However, because of the increasing effects of global warming on regions within Kenya in the seasonal shift where the dry season lasts longer as well as the rainy season, both of which are not favorable for farming. Because of this, I wanted to focus my biology IA on acidification conditions and the reaction that the Vigna radiata have to these conditions as research shows that growth and growth are best at pH levels of 6.0-7.5 ("Green Gram Production", 2022). As stated above. Therefore measuring the effect of different concentrations of nitric acid by increasing the amount in every trial will help me gauge how negatively Vigna radiata crops in my region would react in the largely acidic conditions, this can provide significant information to farmers in my region as it enables them to forecast crop growth in the event of acid rain.

If the concentration of nitric acid is increased the growth of a Vigna radiata the length of the stem grown within 7 days will decrease as the increased acidic conditions will corrode essential minerals and nutrients found in the surrounding environment as well as the cotyledon of the seedling, this restricts the mass of minerals and nutrients that are accessible for stem growth hindering stem growth of a Vigna radiata seedling.

Independent variable: the increasing concentrations of nitric acid (0.0125, 0.5000, 0.0750, 0.1000, 0.1250 moles). The concentration of it will be manipulated by increasing the concentration of nitric acid that will be added to each petri dish, therefore, increasing the surrounding conditions of the Vigna radiata seedling. Dependent variable: length of the Vigna radiata stem after 7 day. The length of the Vigna radiata stem (Measured using a centimeter ruler) will vary at each trial in the varying concentrations of nitric acid within a set amount of time displaying a visible effect on the growth and survival of the Vigna radiata seedling.

One controlled variable includes the volume of nitric acid solution. Each dilute solution with the fixed volume of moles will be 100ml in volume. 1 ml of each nitric acid solution will be placed in each petri dish for the respective trial. The change in volume causes an increase or decrease in the moles of each nitric acid solution which would invariably affect the stem growth of each seedling.

Another controlled variable includes the duration of each trial as I will conduct all trials over a period of 7 days. I choose this amount of time because it generally takes Vigna radiata seedlings to require 3 days to grow under normal conditions (Green Gram Seed Germination, Time Period, Process | Agri Farming, 2020). I, therefore, added an extra 4 days to the duration as the seedlings would be growing in unfavorable conditions, further in order to record a visible difference in stem length I increased the duration of time to improve the data gathered.

Another controlled variable includes light intensity exposure as I will place all the trial Petri dishes in the same area so that they have equal light exposure. As light intensity is one of the main factors that affect plant growth affecting the rate of photosynthesis, I decided to place all dishes in the same area, therefore, making the atmospheric conditions similar, limiting the major limiting factors to affect plant growth and processes.

Another controlled experiment includes room temperature as I will place all the trial Petri dishes in the same area so that the room temperature (30°C) is the same throughout the trials. I've done this as substantial increase or decrease in temperature would result in the rate at which the necessary processes are performed such as photolysis and photosynthesis would be increasingly affected. Further substantial increases in temperature cause the denaturation of cell organelles (enzymes). This limiting the effectiveness of the trial is necessary to make sure that all Vigna radiata seedlings are exposed to the same conditions.

My last controlled variable is the number of seedlings placed in each petri dish, each petri dish will have 5 seedlings totalling 25 for each trial. I've done this as the use of multiple seedlings in each petri dish is to decrease the random or systematic error for each trial. The stem of each seed will be measured and averaged between the five values for each trial, increasing the reliability of each data point.

Control experiment -

• Place a cotton pad into the bottom of 5 Petri dishes and wet it with distilled water.
• Using a stirring rod, press down on the cotton pads so that 40ml of the water is entirely saturated.
• Place 5 Vigna radiata seeds in each petri dish.
• Monitor uncontrolled growth over a period of 7 days

Preliminary experiment -

• Place a cotton pad into the bottom of 5 Petri dishes and wet it with distilled water.
• Using a stirring rod, press down on the cotton pads so that 40ml of the water is entirely saturated.
• Place 5 Vigna radiata seeds in each petri dish.
• Add 1.0cm³ of concentrations 0.200, 0.300, 0.400, 0.500 and 0.600 moles of nitric acid solution to each corresponding petri dish
• After 7 days have passed measure the length of the stem grown (using the average growth of the five seeds in each petri dish as 1 trial) using a centimeter ruler for each seedling at different concentrations.

Nitric acid sample preparations -

• Prepare 0.0125, 0.0500, 0.0750, 0.100, and 0.1250 moles of nitric acid solution
• Using the lab-provided 0.5 moles of nitric acid calculate the volume of dilute solution using known concentration.
• Measure 10ml of nitric acid (0.5 moles) using a measuring cylinder and place in the respective volumetric flask.
• Measure the calculated volume of distilled water in the respective volumetric flask to create 100ml solutions of nitric acid (each 0.0125, 0.0500, 0.0750, 0.100, 0.1250 moles respectively).

Procedure -

• Place a cotton pad into the bottom of each petri dish and wet it with distilled water.
• Label the petri dish from A to E, corresponding to all 5 concentrations.
• Using a stirring rod, press down on the cotton pads so that 40ml of the water is entirely saturated.
• Surface sterilized seeds in 70% ethanol and transfers the seeds aseptically to the Petri dishes under aseptic conditions in order to ensure a sterile environment and prevent cross-contamination of seeds that could hinder stem growth.
• Place 5 seeds in each corresponding petri dish.
• Place the Petri dishes next to a window that provides the necessary light.
• Leave seeds to germinate under the same temperature, light intensity and carbon dioxide concentration to create optimum conditions for 24 hours for seed germination.
• Add 1.0 cm^3 of the allocated concentration of nitric acid to each corresponding petri dish.
• After 7 days have passed measure the length of the stem grown (using the average growth of the five seeds in each petri dish as 1 trial) using a centimeter ruler for each seedling at different concentrations.
• Repeat steps 2-10 five times with each concentration (0.0125, 0.0500, 0.0750, 0.100, and 0.1250 moles) to improve the reliability of the raw data.

Safety- nitric acid is highly corrosive therefore gloves and a lab coat was worn throughout the experiment process when handling the materials (fragile glassware). Goggles were also worn as nitric acid can cause eye irritation. Ethical- as Vigna radiata are a source of food and nutrition the use of many Vigna radiata in this experiment is an ethical concern as there are people suffering with no food, because of this the number of Vigna radiata used was limited to what was highly necessary to avoid wastage. Environmental- as the pH was highly acidic (3.01) the cotton pads needed to be disposed of in the organic waste bin so as to prevent cross-contamination with other students. Furthermore, the formation of nitric acid produces nitrogen gas which is a greenhouse gas and therefore contributes negatively to the environment.

Sample calculations - Please note that all calculations are only given for 0.1000 concentration as an example for interpretation.

Average volume of water needed for a dilute solution of nitric acid with desired concentration. initial concentration × initial volume = final concentration × final volume 0. 5 × x = 0. 1 × 100 = 20cm³ of nitric acid added to 100cm 3 solution Therefore 80cm of water was added to create of 0.1 moles dilute solution.3

\(x=\frac{0.1x100}{0.5}\)

Average stem growth of Vigna radiata at different concentrations between the five trials.

\(\frac{9.0+8.5+8.0+9.5+8.0}{5}=806\,cm\)

Using the TI- 84 graphing calculator I derived the standard deviation in order to quantify how much the concentration of nitric acid and the average growth of the Vigna radiata stem differs. As seen in the image displayed the standard deviation for the data displayed in table 2 is σ = 0. 0387 This value indicates that the data points are closer together, therefore are closely correlated.

points is between 0.05 moles dm³ and 0.075 moles dm³, as the value changed from 16.6cm to 10.0cm which is a significant difference of 6.6cm when compared to the difference between the first and second data points, 19.7cm and 16.6cm and the difference is 3.1cm. From this, it can be assimilated that 0.075 moles dm³ was a critical concentration for Vigna radiata as the growth of the stem rapidly deteriorated between the concentration 0.0500 and 0.0750 indicating that the critical pH value that Vigna radiata seedlings can withstand is between these 2 data points.

Further, I used statistical analysis through Pearson's product correlation coefficient formula (PMCC) which measures the strength of a linear correlation between a set of data points. The null hypothesis indicates that the correlation coefficient is not significantly different to zero therefore, in this experiment it would indicate that there is no correlation between the increased concentration of nitric acid and the stem growth of the Vigna radiata seedling. The alternative hypothesis indicates that the correlation coefficient is significantly different from zero and the closer it is to a value of +1 or -1 the more significant the relationship between variables, therefore, in this experiment it would indicate that there is a correlation between the increased concentration of nitric acid and the stem growth of the Vigna radiata seedling. As shown, using the TI-84 graphing calculator I derived the PMCC value using the average stem growth value for each concentration of nitric acid (as displayed in table 2), the value derived is -0.968 (to 3 s.g.f). This PMCC value suggests that there is a strong negative relationship between the x variable (concentration of nitric acid) and the y variable (growth of Vigna radiata stem). Therefore as the concentration of nitric acid increases the growth of the stem will decrease.

From this it can be assimilated that because the Vigna radiata was isolated in the minerals and nutrients that were available to it during growth because of the use of cotton wool as compared to soil as the varying mineral and nutrient concentration in soil cannot be controlled and can indirectly impact the data of this experiment. This process includes the primary and secondary plant growth (apical and lateral meristems affected), photosynthesis (photosynthetic ability) and metabolic changes in order to attempt adaptation to those that are conducive of acidic environments Debnath & Ahammed, 2020). Because the minerals available were limited to those provided by the distilled water, the data is more reliable and it can be seen that other than acid rain affecting the supply of nutrients and mineral available plant growth in soil. When the plant is isolated the effect of the acidic conditions are more visible.

Qualitatively, i was able to observe this when collecting the data after 7 days as each petri dish had 5 seeds which i was going to average to provide a value for each trial, i noticed that some of the plants that did not proceed to stem growth were visibly shriveled as the seeds were leached of all nutrients and minerals available which caused the initial green color to change from green to brown as no green light was absorbed and reflected due to low photosynthetic ability which was all observed qualitatively. During the preliminary experiment when i used higher concentrations of nitric acid there was little to no stem growth as a majority of the seeds shriveled and were

unable to grow, which prompted me to create more dilute solutions of nitric acid in order to observe a discernable pattern between nitric acid concentration and stem growth. To a greater extent, the critical value of 0.075 suggests that the Vigna radiata seeds acid tolerance is relatively low as it is unable to withstand a high concentration of Nitric acid (low potential of hydrogen) when isolated from excess sources of minerals and nutrients. Further the error bars derived from the standard deviation value displays that there is lower margin of error within this experimental design and data, reducing the uncertainty and risk of random or systematic error that could have impacted the data.