German_phosphate&phytoplankton

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Florida Atlantic University *

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293

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Chemistry

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Dec 6, 2023

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Phosphate and Phytoplankton Lab Elizabeth German Lab Partners: Tehya Fulcher, Otto Martinez, & Brandon Sheetz Lab preformed: 03/06/2023 & 03/13/2023 1 | P a g e

I. Abstract: In this 2-part experiment, the absorbance levels of samples varying in phosphate and chlorophyll concentrations were examined. The absorbance level of a substance is the amount of light the solution absorbs. The samples in this lab that contained a higher phosphate or chlorophyll concentration on average had a higher absorbance rate as well. The highest absorbance rates that were found were from the pond water and groundwater samples. The pond and groundwater samples also had the highest concentration of phosphate and absorbance level of the water sample set, which included groundwater, pond water, canal water, boat basin water, and marine salt water. II. Introduction: Phosphate is a key nutrient to algae. Phosphates typically follow a seasonal cycle that can range from high and low phosphate levels in many water sources due to water salinity and temperature. This phosphate cycle correlates directly with the growth rates of algae, as they tend to follow the same seasonal cycle. The amount of phosphate in a water sample can help to determine how much chlorophyll and algae are in the water. Chlorophyll is a major photosynthetic and can be found in almost all plants, for this experiment the plant that was focused on was algae. The mass of algae in a sample can be correlated directly with the overall chlorophyll concentration within a said sample, therefore the higher the mass of algae the higher the chlorophyll concentration will be. The chlorophyll concentration in water samples can be measured using a spectrometer because chlorophyll will absorb the light. As it is much easier for researchers to measure the amount of chlorophyll in a sample of water and then make an approximation on algae content 2 | P a g e

because of the direct correlation. The lab procedure that was done was a small-scale example of how researchers would perform this experiment in the field. III. Methods and Materials: In this laboratory experiment, three different sample sets were made. First deionized water, phosphate "stock" solution (60 μ moles/liter), and phosphate reagent were used to make different samples of varying phosphate concentrations ranging from 3 micromole/L to 12micromol/L. Next, four 100 ml volumetric flasks, 13 100 ml plastic bottles, disposable pipettes, a pipette bulb, a graduated cylinder, and water samples were collected from different environments (groundwater, pond water, canal water, boat basin water, and marine salt water.) The last set of samples was made using 25 mm 0.45 μm membrane filters, filter holders and flasks, forceps, centrifuge tubes with caps, test tube racks, acetone, and then the previously stated water samples that were collected from outside. All these different samples were then pipetted into cuvettes and examined in a spectrophotometer. Ultimately, this experiment was conducted in three sections, and in the first section samples of various concentrations were tested. First, the four standards at various concentrations were made by placing 5, 10, 15, and then 20mL of the stock solution into each of the respective 100mL volumetric flasks. After the stock solution was added to each volumetric flask, they were diluted using deionized water and were filled to the 100mL mark creating the concentrations of 3, 6, 9, and 12μm/L respectively. 10 mL of each diluted solution was then added to the plastic bottles. Three of each bottle were made. An extra bottle was filled with only deionized water to be used as the control. 1mL of the mixed reagent was then added to each bottle including the bottle of 3 | P a g e

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only deionized water using a transfer pipette. All samples were left then in the dark for 5 minutes after the reagent was added to allow the samples to react. Once the samples were allowed to react, each sample was transferred into a cuvette and ran in the spectrophotometer at 885 nm. In between each trial, a blank of the deionized water with the reagent was run in the spectrophotometer to keep the results accurate. The absorbance levels of all the samples were then recorded in a lab notebook. For the second part of the experiment 10mL of each water, sample was then placed into 3 separate plastic bottles allowing 3 trials to be tested for each sample, 13 bottles in total were used. In the 13th bottle, there was no sample just 10mL of deionized water was added, and this sample acted as the control. 1mL of the mixed reagent was then added to each of the bottles using a transfer pipette. All samples were then left to react in the darkroom for approximately 5 minutes. The samples were then placed in cuvettes and ran in the spectrophotometer at 885 nm with a blank sample being ran in between, all data was then recorded in a lab notebook. In the last part of this experiment 5mL of each water sample was obtained and then filtered using a 25 mm membrane filter. Once filtered, the filter paper was placed in a centrifuge tube. 8 ml of 90% acetone was added to each tube before being capped and shaken vigorously. All samples were then allowed to react in the dark for 30 minutes. The absorbance of each sample was then run at 665, 645, and 630 nm three different times to ensure accuracy with a blank being ran between each test. The concentration of chlorophyll in each sample was then calculated using the following equation, Chl a = 15.6 x A665 - 2.0 x A645 - 0.8A630. 4 | P a g e

IV. Results: During the first section of this experiment, it was found that the highest absorbance came from the sample with the highest concentration of phosphate (12 micromol/L) and it was 0.269±0.0058nm. As stated previously, the relationship between the concentration of phosphate in a sample is directly related to the absorbance level so the rate of absorbance from highest to lowest was 12 micromol/L, 9micromol/L, 6 micromol/L, and the 3 micromol/L. The absorbance levels were 0.269±0.0058nm, 0.195±0.0082nm, 0.137±0.0022 nm, and 0.077±0.0024 nm, respectively. The direct relationship between the absorbance levels and the phosphate concentration is relative to each sample’s absorbance level. During the test of the second set of samples which examined the phosphate concentrations in collected water samples. The pond water sample at 0.087±0.0042 nm and the groundwater sample at 0.072±0.0016nm had the two highest absorbance rates. While the samples that had the lowest absorbance rates were the boat basin sample at 0.026±0.0028 nm and the marine salt water at 0.023±0.0028nm. The absorbance levels between the different water samples are relative to the water conditions of each area. The last part of the experiment was calculating the concentration of chlorophyll in the different collected water samples. It was found that the pond water sample had the highest levels of chlorophyll with 3.72 micromol/L followed by the groundwater sample with a concentration being 2.49 micromol/L. These results further support the previous section of the lab as the water samples with the highest absorbance dues to phosphate concentration were also the pond water and groundwater samples. As the concentration of chlorophyll for the boat basin was 1.68 micromol/L, marine saltwater was 1.83 micromol/L, and the canal water sample was 1.71 micromol/L. 5 | P a g e

V. Discussion: In this experiment, we were able to see how concentrations of certain substances in a sample of water can affect its absorbance rates. Overall, it was found the higher the concentration of either phosphate or chlorophyll the higher the absorbance of that specific sample will be. Throughout the different times of the year, these concentrations can fluctuate though because of the changing environment. Chlorophyll is specifically found in plants such as algae in the water and phosphate is a key nutrient to these algae. Algae will have specific times in the year that it will have a larger growing season due to the cycle of phosphate concentration. The phosphate levels in the second part were predicted to be what they are because of where the sample was taken from. The freshwater retention pond and the koi pond both had higher concentrations of phosphate and this was to be expected because these ponds are enclosed environments where it is easier to control the level of phosphate and overall nutrients. Overall, the data that was collected was what was predicted to occur. The only potential error was in part three the acetone being used caused etching on the cuvettes that could have caused the absorbance levels to be skewed. 6 | P a g e

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VI. Figures and tables: Figure 1: Absorbance verses concentration Note 1: Absorbance in nm verses the concentration of phosphate in micromol/L in made water samples. Data is shown depicting a direct relationship between the two variables which cause the capability of inferences to be easier. Figure 2: absorbance vs. concentration Note 2: Absorbance in nm verses the concentration of phosphate in micromol/L in collected water samples. The data shows that the samples are no longer directly related 7 | P a g e

Figure 3: Chlorophyll concentration Marien saltwater Pond water Canal water Ground water Boat Basian 0 0.5 1 1.5 2 2.5 3 3.5 Chlorophyll Concentration Water sample Phosphate concentration (micromoles/liter) Note 3: Chlorophyll concentration bar graph. The freshwater retention pond has a significantly higher concentration compared to the saltwater and boat basin samples. The pond sample is higher than the saltwater and boat basin samples but not as higher as the freshwater retention pond. 8 | P a g e