ENVIRONMENTAL PARAMETERS AS LIMITING FACTORS

In Fulfillment of BIO 2202L General Ecology Laboratory Requirement

Presented to the Biology Department
University of San Carlos

Presented by:
Abato, Liezyl Jane

Presented to:
Val B. Salares

February 2024
 TABLE OF CONTENTS

Cover Page …………………………………………………………………………….…………… i

Table of Contents ……………………………………………………………………………….………… 1

List of Tables ……………………………………………………………………………….………… 2

List of Figures ………………………………………………………………………….……………… 2

Introduction ………………………………………………………….…………………………….. 3

Materials and Methods ……………………….……………………………………………………………… 3

Results ………………………………………………….……………………………………… 8

Discussion ………………………………………………………………………………………… 16

References ………………………………………………………………………………………… 22

Documentation ………………………………………………………………………………………… 23

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 List of Tables
Table 1. Table of x² values. Chi-Square Analysis
Table 2. Chi-Square Analysis

List of Figures
Figure 1. Graph on the Effects of Temperature on Tadpoles, measured in Mortality
Figure 2. Pie Charts of the Number of Antlion Distribution in Different Soil Moisture Gradients (A)
Trial 1; (B) Trial 2; (C) Trial 3
Figure 3. Pie Chart of the Average Number of Antlion Distribution in Different Soil Moisture
Gradients
Figure 4. The Number of Germinated Seeds in Different pH Conditions
Figure 5. The Number of Live Shrimps in Different pH Conditions
Figure 6. Average Number of Live Shrimps in Different pH Conditions
Figure 7. Hatching success of Artemia franciscana over a pH range
Figure 8. Effects of Salinity in Zea mays Germination in Percentage
Figure 9. Effects of Salinity in Mung Bean Germination
Figure 10. Effects of Salinity in Seed Germination in Percentage
Figure 11. Effects of Salinity in Seed Germination in Percentage
Figure 12. Effects of Salinity in Snail Mortality
Figure 13. Effects of Salinity in Snail Mortality
Figure 14. Taken from Moreira et al. (2021) about the different body temperatures of ectotherms
(629) and endotherms (876).
Figure 15. Abstract figure from the Study of Zheng et al. (2015).

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INTRODUCTION

Nature regulates population growth in a variety of ways. All organisms function within a

set of conditions that become critical to their growth, development, and survival. The principle

of limiting factors refers to any factors in an environment capable of limiting these processes of

growth, abundance, or distribution of a population of organisms in an ecosystem. These factors

can be categorized into two (1) density-dependent factors; and (2) density-independent factors

(Edwards & Edwards, 2011). In addition, the Law of Tolerance in the success of an organism

depends on a complex set of environmental conditions (environmental factors). And that

organism would have the definite minimum, maximum, and optimum environmental factors that

determine success (Shelford, 1931). The principle of limiting factors is an ecological

generalization that suggests that productivity is constrained by a single, metabolically essential

factor that is present in the least supply relative to the potential biological demand. The limiting

factor represents a type of ecological stress that if alleviated will result in greater productivity

and development of the ecosystem. In this exercise, the students will investigate the responses

of certain plant and animal species to varying levels of environmental factors such as

temperature, soil moisture, pH , and salinity to determine the limiting effects of such factors.

Moreover, this experiments aims to identify the range at which the factor becomes limiting to

the particular experimental species.

MATERIALS AND METHODS

This section provides a comprehensive account of the experimental methods employed

in our experiments. Different procedures were performed on plants and animals to test and

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determine various limiting factors of the organisms. The experiments were performed in three

(3) laboratory sessions starting on February 8, 13, 15, 2024, 9:00 AM - 12:00 noon at the Botany

Laboratory, Room ES231 of Arnoldus Science Building, University of San Carlos - Talamban

Campus.

I. Temperature Effects

This experiment aims to obtain data about temperature effects as the limiting factor for

tadpoles. Thirty (30) tadpoles were utilized as samples for this experiment. There were three (3)

set-ups prepared with each beaker halfway filled with tap water and labeled as 1, 2, and 3. Beaker

1 served as the control set-up with room temperature recorded at 27˚ C. In Beaker 2, we lowered

the temperature by adding ice water, maintaining 10-15˚ C. For Beaker 3, we increased the

temperature by adding freshly boiled water, maintaining 40 - 45˚ C. Ten (10) tadpoles were gently

added to each beaker and were observed within 10-20 minutes. After twenty minutes of

exposure to different temperatures, we counted the number of dead tadpoles and determined

the percent (%) mortality for each temperature regime.

II. Soil Moisture Gradient

Instead of sowbugs, twenty (20) antlions were utilized as the samples for this experiment.

Four (4) soil samples weighed 500g each and were oven-dried at 105˚ C. The first soil sample was

set aside without the addition of water while the remaining 3 samples were sprinkled with tap

water at different ratios. We sprinkled the second soil sample with 10 mL water, the third sample

with 30 mL, and the fourth sample with 60 mL, respectively. After this, we divided the container

into four quadrants and placed each of the four soil samples in separate sections. The twenty

(20) antlions were then placed at the center of the quadrant and observed the antlions move

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around and disappear into the soil layers. After 10-15 minutes, we observed and counted the

number of antlions found in each quadrant. The antlions were given a few minutes to rest to

recover from the stress before doing the next trial. This experiment had three (3) trials.

Moreover, the data were analyzed using a Chi-Square Analysis. Chi-square analysis was

used because this test measures the degree of correspondence of observed values with the

expected ones. Values expected based on no differential response may be calculated by dividing

the total number of individuals counted during the run by the number of sections in the gradient

and results from simple gradient preference tests with individual species could be tested using

this analysis. The expected and observed numbers of each section of the gradient may be

compared by calculating a chi-square value (x2) by the following formula:

Chi-Square (x2) = Σ (Observed – Expected)²

Expected
For a valid chi-square test, expected values should be at least 5 for all gradient sections.

The probability that calculated chi-square values represent a significant deviation of observed

values from those expected may be determined by comparing the computed value to the values

in a chi-square table (Table 1) for a number of degrees of freedom, i.e. one less than the number

of gradient sections. An observed value exceeding the tabulated value for the 95% Or 995 levels

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indicates a significant deviation observed from expected values at those confidence levels.

Table 1. Table of x² values. (analystprep.com)

III. pH Effects

This experiment has two parts, Part A for plants and Part B for animals. Both experiments

are to test for the pH effects on Zea mays for Part A and Artemia eggs for Part B.

A. On Plants

Test solutions were prepared using Hydrogen Chloride (HCl) and distilled water to obtain

different pH levels. Using a pH meter, we determined the pH levels 2, 4, 7, and 9 and also used

buffers (1 N NaOH) to set the desired pH constant during the entire experiment. 12 petri dishes

were also prepared with each lined with filter paper at the bottom. Monggo seeds were utilized

as the samples for this test. Ten (10) Monggo seeds were then placed into each petri dish with

different pH: 1st set of dishes – pH 2, 2nd set of dishes – pH 4; 3rd set of dishes – pH 7 (neutral);

4th set of dishes – pH 9. The set-ups were observed for five (5) days for seed germination and

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the number of seeds that germinated in each dish. Moisture within the dishes was also

maintained and checked for the entire duration of the experiment. The following formula was

also used to calculate the percentage germination in each dish.

% Germination = Number of germinated seeds x 100

Total number of seeds
B. On Animals

In this experiment, brine shrimp (Artemia) eggs were the samples utilized to test for the

effect of pH. Twelve (12) culture tubes were filled with different pH level solutions - 1st set of

tubes – pH 2; 2nd set of tubes – pH 4; 3rd set of tubes – pH 7 (neutral); 4th set of tubes – pH 9.

Brine shrimps were inoculated approximately with the same amount into each culture tube and

were given five (5) days for observation. After five days, we counted the number of living shrimps

using a dissecting microscope in five randomly selected fields of view of each culture and the

results were recorded.

IV. Salinity

Effect salinity was also tested both on plants and animals. The experiment was divided

into two (2) parts (Part A. Plants and Part B. Animals). Both experiments are to test for the effect

of salinity on Monggo seeds and Zea mays for plants and snails for animals.

A. On Plants

Solutions were prepared by dissolving 0 ppt, 2.5 ppt, 5 ppt, 10 ppt, and 20 ppt salts in

1000 mL of distilled water to obtain the desired salt concentrations. One hundred (100) Zea mays

(corn seeds) and one hundred (100) monggo seeds were utilized as samples for this experiment.

These seeds were soaked overnight before performing salinity tests. There were five (5) sets of

Petri dishes with each set having three replicates and ten (10) soaked but ungerminated corn and

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monggo seeds into each dish with the corresponding salt solution. After five (5) days of

observation, we measured the root length and shoot length with a ruler (in cm) of the germinated

seeds in each dish. Root-shoot ratio was calculated and results were recorded.

B. On Animals

In this experiment, snails were utilized to test for the effect of salinity on animals. Similar

set-up from the test of salinity on plants, five (5) sets of candy jars contained a corresponding salt

concentration with each set having three replicates. In five (5) candy jars, a salt with 0 ppt, 2.5

ppt, 5 ppt, 10 ppt, and 20 ppt were dissolved in 1000 mL of distilled water, respectively. The level

of the solution in the jar was just enough to immerse the snail and covered the glass jars with a

mesh net to keep the snails from escaping. It was observed for five (5) days and recorded the

total number of dead snails in each set-up.

RESULTS

I. Temperature Effects

Figure 1. Graph on the Effects of Temperature on Tadpoles, measured in Mortality

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 The figure above shows the effect of temperature on the tadpole, measured in the

mortality of the specimens. The graph shows that the tap and distilled water at 40-45° Celsius

had the highest mortality for the tadpoles, with all trials having 100% mortality in both

temperatures. The distilled water at 10-15° Celsius took about 60% of the total tadpoles.

II. Soil Moisture Gradient

Figure 2. Pie Charts of the Number of Antlion Distribution in Different Soil Moisture Gradients
(A) Trial 1; (B) Trial 2; (C) Trial 3

Figure 3. Pie Chart of the Average Number of Antlion Distribution in Different Soil Moisture
Gradients

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 Based on the pie chart on the average number of observed antlions in different soil

moisture gradients, it can be inferred that the limiting factor is the soil moisture itself. The inverse

relationship between soil moisture and the number of antlions can be attributed to the fact that

modes of foraging for antlion larval groups require dry soil with loose structure and fine grains,

making natural dry grasslands-covered dunes their excellent habitats (Szentkirályi et al., 2008).

Critical Value
Number of Degrees of Chi-Square
N (95% Significance P-Value
Antlions Freedom Value
Level

Observed 20
3 1.356 0.35 0.718
Expected 20

Table 2. Chi-Square Analysis

There was a significant difference between the number of Antlions in different soil

moisture gradients, Χ2 (3, N = 20) = 1.356, p = .7108. Tolerance range: 0mL-40mL ( 0%-8.7%

moisture content) up to 17% (Fukada & Nisimura, 2021).

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 III. pH Effects
A. On Plants

Figure 4. The Number of Germinated Seeds in Different pH Conditions

Water acidity can influence seed germinability. Acidity has a detrimental effect on seed

germination because of the inhibition of enzyme synthesis needed for germination. Lower or

higher pH in water also affects seed germination negatively (Muttaqin et al., 2019).

IV. On Animals

Figure 5. The Number of Live Shrimps in Different pH Conditions

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 Figure 6. Average Number of Live Shrimps in Different pH Conditions

Figure 7. Hatching success of Artemia franciscana over a pH range

Seawater at pH 7.3 was found to be optimum for hatching cysts of Artemia franciscana.

At pH 4.5, there was a significant decrease in overall hatching success as compared with the pH

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1.3 group. It was also found that the percentage of cysts arrested at the E-2 stage (late

prenauplius) decreased significantly (Doyle & McMahon, 1995).

V. Salinity
A. On Plants

Figure 8. Effects of Salinity in Zea mays Germination in Percentage

The graph above shows the trend of the germination percentage in different salinity

levels. It can be seen that as salinity increases, there is a decrease in the germination of the maize

seeds. Salt stress influences seed germination primarily by lowering the osmotic potential of the

soil solution to retard water absorption by seeds, by causing sodium and/or chloride toxicity.

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 Figure 9. Effects of Salinity in Mung Bean Germination

Figure 10. Effects of Salinity in Seed Germination in Percentage

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 Figure 11. Effects of Salinity in Seed Germination in Percentage

B. On Animals

Figure 12. Effects of Salinity in Snail Mortality

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 Figure 13. Effects of Salinity in Snail Mortality

Salt essentially draws the water out of their skin -– an osmosis effect – and they die within

minutes of dehydration.

DISCUSSION

I. Temperature Effects

In contrast to the initially assumed and expected results, there was a 100% mortality rate

in the tadpoles placed inside a beaker with tap water at room temperature, as presented above.

Within the three trials performed, all ten tadpoles eventually died after 20 minutes of being

transferred from the stagnant rainwater to the tap water despite the observed relatively normal

swimming movement in the early stages of the experiment. This result was unexpected, given

that 27° Celsius provides favorable temperature conditions for tadpoles to thrive and survive

(Happy Pets Now, 2023). In fact, in the study by Goldstein et al. (2017), tadpoles placed under

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25° Celsius conditions (technically close to the temperature in the experiment) showed a 94%

survival rate. This leads to the inference that temperature was not the mortality factor specific

to the setup, thus bringing up other pOne possible cause of this may be the water used itself,

given that tadpoles were directly transferred from their original stagnant rainwater to tap water,

and death may have been caused by a possible immediate change in the chemistry of the water

itself. Tap water is not a recommended water source for tadpoles, as it may often contain

chlorine or chloramines harmful to amphibians (Wellington, 2021). These chemicals can be

harmful to amphibians, including tadpoles, as they can affect their skin and gill functions.

Additionally, tap water might have other substances like heavy metals or impurities that could

be detrimental to tadpoles. The heightened sensitivity brought by the much younger stage of the

tadpole subjects could also be the driving factor for higher mortality rates (Vasconcelos et al.,

2016).

Surprisingly, it can be observed in the results that within the three trials performed, only

a 60% mortality rate was recorded on tadpoles in distilled water at 10-15° Celsius. Swimming

movement immediately stopped as the tadpoles were transferred from the stagnant rainwater

to the cold distilled water. However, some of the subjects began to swim again as they were

reintroduced back to the original stagnant rainwater after the 20-minute experiment duration.

Thus, the idea of tadpoles surviving in cold waters is supported again by the findings of Goldstein

et al. (2017), since at 15° Celsius, about a 64% survival rate was recorded. However, the difference

in the survival rates may be attributed to the range in temperature, variation in tadpole species,

and relative age of tadpole subjects. Even some species, especially those in tropical areas, may

lack or may have less ability to acclimate to low temperatures, given the lack of selection pressure

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for acclimation in the tropics (Vo & Gridi-Papp, 2017). Nevertheless, again, this does not disregard

the fact that some tadpoles did survive in the cold water, with the low temperatures possibly

slowing down their metabolism, explaining the stoppage in the movement/swimming observed.

Graph 1 shows a 100% mortality of tadpoles in distilled water at 40-45 ° Celsius. In the

three trials, all ten tadpoles died when transferred from the stagnant rainwater to the boiled

water. Tadpoles are sensitive creatures, and their environment can significantly impact their

development. The ideal water temperature for tadpoles to survive is between 21 and 27° Celsius

(Martin, 2021). Any drastic change in their water temperature will significantly affect their

growth and health. The lethal temperature for young tadpoles is 35° Celsius and above, while

older tadpoles are more resilient and can stay longer before perishing (Biggs, 2010). However,

the specimens utilized in this experiment are relatively young and, as such, quickly died after

being slowly submerged in the hot water. In addition, the tadpoles are inferred to have

experienced heat stress due to the sudden temperature change in their environment.

Temperature plays an important role in the functioning of metabolic activities of living

organisms (Raju, 2004). Ectotherms, such as tadpoles, are particularly susceptible to changes in

the environment’s temperature due to their natural dependence on it (Deutsch et al., 2008;

Kingsolver, 2009; Bonino et al., 2011, Bonino et al., 2015a, Bonino et al., 2015b). Amphibians, in

particular, are more susceptible to these changes because of their highly permeable skin (Winter

et al., 2016). Tadpoles cannot thermoregulate, and the external temperature could be a good

indicator of its internal temperature. As such, these small organisms are optimal for studying

their thermal biology relative to the immediate environment (Bonino et al., 2020). On the other

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hand, endotherms with their ability to thermoregulate their internal temperature have much

lower tolerance ranges as seen in Figure 14.

Figure 14. Taken from Moreira et al. (2021) about the different body temperatures of
ectotherms (629) and endotherms (876).

II. Soil Moisture Gradient

Soil-dwelling organisms that inhabit sandy soils are strongly affected by the physical

characteristics of their substrate (Büsse et al., 2021). Among these soil-dwelling organisms are

the antlions. Antlions are typical ambush predators: they rely on the soil to capture prey, with or

without the traps these organisms have set up (Scharf & Ovadia, 2006; Scharf et al., 2011; Badano

et al., 2017). When it comes to deciding their “pits”, wet soils are generally avoided (Simberloff

et al., 1978) and sandy moisture of 9-17% is preferred (Fukada & Nisimura, 2021). Some degree

of moisture is preferred due to their relatively higher respiratory rate and may lead to water loss.

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 A myriad of benefits are associated with soil-dwelling organisms, like nutrient cycling and

aerating the soil, all of which are important for a plant’s growth and development. These depend

on conditions for their survival such as moisture, air, and pore size of the particular soil.

Earthworm casts and tunnels, termite mounds, and ant heaps help aerate the soil and increase

its porosity (European Union, 2010).

III. pH Effects

Microorganism activity, and nutrient solubility and availability, are key factors for the

development of plants depend on pH (Gentili et al., 2018). Reductions in seawater pH levels have

detrimental effects on the development and reproductive processes of many marine organisms

(Pörtner et al., 2004, 2005; Raven et al., 2005).

Figure 15. Abstract figure from the Study of Zheng et al. (2015).

The change in pH can affect land and water in different ways. Macronutrients like calcium,

nitrogen, potassium, and magnesium in soil are readily available at pH 6.0-6.5, and

micronutrients become less available at higher, alkaline pH (Ferrarezi et al., 2022). Furthermore,

most rhizobacteria were identified at pH 9 (Ferrarezi et al., 2022). Ocean acidification is a result

of the formation of carbonic acid. A study by Zheng et al. (2015) showed that the hatching rate,

growth at the late stage of acidification, and animal survival rate were reduced at lower pH than

20
in the control. Additionally, changes in gene expression were observed with lower pH, with genes

such as MyD88 and Notch, and several key enzyme activities like catalase and peroxidase were

also affected.

IV. Salinity

Salinity directly affects the metabolic processes of the body, most especially

osmoregulation (Yuliani et al., 2018). Osmoregulators regulate their body’s osmolarity by

developing mechanisms and osmoconformers match their body’s osmolarity to the environment

through the concentration of urea for example Also overfertilization.

Mongo seeds were determined to be moderately salt-sensitive relative to other species.

Two studies suggest that corn is most sensitive to salinity during the vegetative growth stage.

Piruzyan (1959) observed that corn grown in naturally saline soils was most sensitive during

emergence and seedling growth but became more tolerant by the flowering stage of growth.

Some crustaceans are vertically and horizontally affected by salinity (Imamsyah et al., 2020).

Fluctuations in salinity can increase or decrease the osmoregulatory ability of the hemolymph,

which causes shrinkage and swelling of cells through regulation of body fluids that can affect their

growth, development, and survival (Amin et al 2016).

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DOCUMENTATION

I. Temperature Effects

Figure 15. Preparation and Experimental Set-Up

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II. Soil Moisture Gradient

Figure 15. Preparation and Experimental Set-Up

III. pH Effects

Figure 16. Preparation of Materials

24
 Figure 17. Experimental Set-Up

IV. Salinity

Figure 18. Preparation of Materials

Figure 19. Experimental Set-up on Plants

25
Figure 20. Experimental on Animals

26