Fish Kill Experiment Lab Report

Piper Stevens

Unknown Sample #1

Lab Date: January 28, 2017

Purpose
 Identify the presence of IA and IIA metal ions in a simulated water sample

from the Clark Fork of the Columbia River using emission spectroscopy.

Determine the concentration of copper and iron ions in the simulated water

sample using absorption spectroscopy, and report if those levels are too toxic

to support life. Use the collected data to determine a possible cause of the

major fish kill in 1984 in the Clark Fork of the Columbia River.

Introduction

As urban sprawl continues to expand into agricultural and forested

land, the threat of water contamination has become a major concern in the

public eye. Runoff from fertilizers and highways tends to carry dangerous

metal ions into drinking water sources. As technology improves, it is

becoming easier and easier for scientists to detect small levels of

contamination in water. It is becoming clear that human waste has an

incredible impact on freshwater systems that it flows through. Not only is

contamination a concern for public safety, it also poses a large threat to

animal life and ecosystems worldwide. When toxic metals reach reservoirs,

ponds, and rivers, they have a dramatic effect on the wildlife that inhabits

those water sources. A dangerous level of toxic metals in water has shown to

kill massive populations of wildlife in and around a natural water source. This

was the case in Clark Fork of the Columbia River where a major fish-kill

occurred in 1984. Scientists have attributed this killing to water

contamination in the Montana section of the Columbia River, and are looking

into which contaminants in particular caused this ecologic disaster.

In this experiment, known samples of IA and IIA metals will be tested

for emission spectrums and compared with the emission spectrum of a

simulated water sample from the Clark Fork of the Columbia River. To do this,

aqueous samples of each metal will be burned in a Bunsen burner and a

Spectrophometer will detect the emission spectrum of each known metal.

This will allow a direct comparison of the emission spectrums of each IA and

IIA metals to the emission spectrum of the lake sample. This will ultimately

determine which metal ions are present in Clark Fork, and if the salinity of

the water was too high to support wildlife. The levels of copper and iron in

the water solution will also be tested with this experiment using data from an

absorption spectrum. If the concentration of copper is above 1.3ppm, and if

the concentration of iron is above 0.3ppm, the water is considered toxic. The

toxicity of the water will help in determining the cause of the fish-kill. Using

known concentrations of a solution containing both copper and iron, it is

possible to bracket the unknown solution in order to determine the

concentration of copper and iron in the sample. The water sample is highly

concentrated in Cl- molecules, therefore the copper and iron ions are present

as chloride complexes. These chloride complexes only weakly absorb light,

and do not give usable absorption spectrum data. Because of this, a

complexing agent must be added to form compounds that will absorb

sufficient light to record an absorption spectrum. In this case, thiocyanate

 2+
ions (SCN-) will be added to form brightly colored complexes with and
C u

3+ + 2+
. Solutions ranging from 400ppm of
Fe [ CuSCN ] and [ FeSCN ]
to 50ppm

+ 2+
[ CuSCN ] and [ FeSCN ]
will be created and tested using the

Spectrophometer. These absorption spectrums will then be compared with

that of the unknown water sample to determine the concentration of copper

and iron in Clark Fork of the Columbia River.

This experiment addressed the central question: Which IA and IIA

metal ions are present in a simulated water sample from Clark Fork? As well

as, are the iron and copper concentrations in that sample at a toxic level?

This lab utilizes skills that I have obtained through doing both emission and

absorption labs in the past. In fact, I performed a lab that required me to test

the emission spectrums of various known IA and IIA salts. The skills I learned

testing emission spectrums in the infrared flares lab will help me test the

unknown water sample in this lab. Also, the emission spectrum data I

gathered on IA and IIA metals in that lab can be used in this lab so I will not

have to retest each metal. Likewise, in the Determination of Chromium(VI)

Concentrations Via Absorption Spectroscopy lab, I was required to determine

the concentration of copper in an unknown sample using its absorption

spectrum. In that lab, I learned how to use one standard solution to create

other solutions of varying concentrations. I also learned how to graph and

 determine the equation of a linear regression. All of the skills described

above will assist me in performing the complex lab presented to me with this

problem.

The possible outcomes of this lab vary hugely based on the simulated

water sample I am given. There could be any range and combination of IA

and IIA metals in the water sample. However, I predict that there will be at

least two detectable metals that I will be able to find traces of in the sample.

Furthermore, the concentration of copper and iron in the sample could also

vary hugely. I imagine that, because there was a major fish-kill, the water has

some form of contamination and either has high levels of copper, iron, or

both. I do not have any solid idea of the results I will get from doing this lab;

however, by the end I hope to have an explanation of what toxins in the

water at Clark Fork of the Columbia River caused the fish-kill in 1984.

Experimental

Chemical Reagents:

- 5mL Standard 0.5 M Na Cl solution

- 5mL Standard 0.5 M Li Cl solution
- 5mL Standard 0.5 M K Cl solution
- 5mL Standard 0.5 M Ca C l 2 solution
- 5mL Standard 0.5 M Ba C l 2 solution
- 5mL Standard 0.5 M Sr C l 2 solution
*I decided to leave the IA and IIA metals solutions on the chemical

reagents list; however, I did not use these solutions in my procedure.

Instead, I decided to use data that I had collected from a previous lab

to give me the emission spectrums of the IA and IIA metals.

 2+ 3+
- Standard Fe/Cu solution containing 400ppm and 20ppm in
C u F e

SCN- solution.
-10 mL each of four diluted Fe/Cu solutions having copper

concentrations of 320ppm, 240ppm, 160ppm, and 80ppm. And

iron concentrations of 16ppm, 12ppm, 8ppm, and 4ppm.

- 5mL 20ppm iron(III) nitrate solution (already mixed with SCN- ions)
- 5mL 400ppm copper (II) nitrate solution (already mixed with SCN- ions)
- Simulated Clark Fork of the Columbia River Water sample

Equipment

- MeasureNet spectrophometer and MeasureNet workstation

- 8 cuvettes
- Coiled nichrome wire
- 10 mL volumetric flask
- Watch glass
- Bunsen Berner
- Pipettes
- Test tubes
- Kimwipes

Constants and Mathematical Equations

P
-Transmittance =
P0

Where P = power of light after passing through the sample

P0 = power of light after passing through first wall of cuvette

1
-Absorbance = log
Transmitance

-Moles to ppm:

moles solvent grams solvent 1000 mg

ppm =
L solution 1 mol solvent 1g

Example converting 0.1 M Cu into ppm:

 0.1mol Cu 63.55 g Cu 1000 mg
=6355 ppm Cu
1L 1mol Cu 1g

- R2 = linear correlation

Where the closer R2 is to 1, the more correlated the data is.

- absorbance concentration

Proved using Beer Lamberts law and the molar absorptivity coefficient

proves this constant.

- ( Concentration1 ) ( Volume1 ) =( Concentration2 ) ( Volume 2)

Example finding the volume of 400ppm solution needed to create

10mL 330ppm solution.

(400ppm)(x)=(330ppm)(10ML) x=8.75mL

Setup

Emission Spectrum Absorption Spectrum

Part 1- Solution Preparation

1. Label a watchglass and pour 1-2mL of the unknown solution into the

watchglass. Place a labeled nichrome wire into the watchglass with the

unknown solution.
2. Fill two cuvettes with the 0.1 M iron (III) sulfate solution and the 0.1 M

copper (II) sulfate solution. These will act as a control test to show the

shape of the absorption graph for each metal. Only perform the

emission spectrums for these solutions if extra time is available.

2+
3. Label 6 test tubes and fill one of them with 10mL of 400ppm
C u

3+
/20ppm solution.
F e
2+ 3+
4. Place 8 mL of the 400ppm /20ppm solution into the 10mL
Cu Fe

volumetric flask and fill the remainder of the volume with distilled

water until the meniscus of the solution lines up with the 10mL line on

2+ 3+
the volumetric flask. This will create the 320ppm 16ppm .
Cu F e
5. Turn the flask upside down a few times to combine the solution. Poor

the solution into a labeled test tube and wash the volumetric flask.
6. Repeat steps 4 and 5 with different amounts of standard Fe/Cu solution

to create the remaining concentrations of copper and iron:

240ppm/12ppm, 160ppm/8ppm, 80ppm/4ppm. The amounts of

standard Fe/Cu solution to add for each concentration are in the chart

below:

Desired Concentration: 2+/

Volume of 400ppm
Copper Iron Cu
3+
20ppm
Fe
320ppm 16ppm 8.0mL
240ppm 12ppm 6.0mL
160ppm 8ppm 4.0mL
80ppm 4ppm 2.0mL
 7. Fill the remaining cuvettes with the Fe/Cu solutions of varying

concentrations. Fill the cuvette full of solution, and then wipe each

cuvette with a Kimwipe being careful not to leave fingerprints on the

glass sides.
8. Fill an additional cuvette with distilled water. This will serve as the

blank solution for the Spectrophometer.

Part 2: Test Emission Spectrums

9. Turn on the workstation that Spetrophometer data will be sent to and

press F5 Spectroscopy, then F1 Emission.

10. Press Setup to enter the scan parameters and use the arrow keys to

change the min and max x and y values.

- Y represents intensity: set the y min to 0 and the y max to 1500
- X represents wavelength: leave x at its default setting.
11. Press Display to accept the values. The MeasureNet system is now

ready to record emission spectrum.

12. Go to the MeasureNet Spectrophotometer and connect the

MeasureNet fiber optic cable to the workstation. Set up a Bunsen

burner about 6inches away from the probe of the measuring cable.

(Reference the picture under setup for a visual representation)

13. Press Station Number on the MeasureNet spectrophometer and

enter the station number of the workstation the data will be sent to.

Press Enter and the screen will display ready to scan

14. Completely cover the fiber optic cable with a finger and press ZERO

until the workstation reads ready to scan

15. Press Intensity and heat a nichrome wire soaked in distilled water in

a Bunsen burner flame and adjust the position of the Bunsen burner

until the intensity reading is above 500. Press Intensity again and a

ready to scan message will appear on the screen.

 - Only perform this process the first spectrum test.
16. Lift the nichrome wire from the watch glass containing the unknown

sample solution. Place the wire in the Bunsen burner flame- on the tip

of the inner cone of blue fire.

17. When the Bunsen burner light changes color due to the burning

solution, press Sample on the workstation and continue burning the

salt solution until the spectrum graph appears on the individual

workstation.
18. Return to the MeasureNet workstation and examine the emission

spectrum. Adjust the intensity if the data exceeds the original y values.

If this happens, press Setup, then F2 and enter new min and max

values for y. Press display and the MeasureNet will re-plot the data.
- This step is optional only if the data exceeds the original

parameters.
19. Press File Options, then press F3 to save the scan. Enter a 3-digit

code and press Enter

20. Press Display on the workstation to clear the previous scan and ready

the workstation for another scan.

Emission Spectrum Data Analysis

- Save the emission spectrum file for the unknown sample on a

flash drive or through email and open it in excel.

- Copy and paste the emission data from the Infrared Flares lab

onto the excel sheet and delete the data for Magnesium and

Cesium.
- Using a smoothed line scatter plot, convert all the data into a

graph.
 - Determine the wavelength of the highest emission for each

sample including the unknown and record them in the data table

below.
- Compare the graphs of the known IA and IIA metals with the

unknown sample to determine which metals are present in the

unknown sample.

Part 3- Test Absorption Spectrums

21. Turn on the workstation that the absorption Spectrophometer data will

be sent to and press F5 Spectroscopy, then F2 Absorbance.

22. Press Setup to enter the scan parameters and use the arrow keys to

change the min and max x and y values.

- Y represents absorption: set the Y max to 2.0 and press Enter
- X represents wavelength: leave x set at its default values
23. Press Display to accept the values. The MeasureNet system is now

ready to record an absorbance spectrum.

24. Go to the MeasureNet Spectrophometer. Be sure the Visible or UV-

Visible source power switch is ON. Press Station Number and enter

the station number of the workstation that the data will be sent to.

Press Enter and the screen will display Ready to Scan

25. Insert the cuvette containing all black solution into the sample

compartment then press ZERO. When screen reads Ready to Scan

take out the black cuvette.

26. Insert the cuvette containing distilled water into the sample

compartment then press Reference. When the screen reads Ready to

Scan take out the cuvette.

 2+/
27. Insert the cuvette containing the standard 400ppm 20ppm
C u

3+
solution and press sample. Make sure the absorption graph
Fe

appears on the individual workstation before removing the sample.

28. Return to the MeasureNet workstation and examine the absorption

spectrum. To save the data, press File Options then press F3 to save

the scan. Enter a 3-digit code and press enter.

29. Press Display on the workstation to clear the previous scan and ready

the station for the next scan.

30. Repeat steps 25-30 with the remaining prepared cuvettes. Then test

the simulated river water sample for its absorption spectrum. Save

each absorption graph before moving on to the next sample.

31. If there is enough time, test the 0.1M copper solution and the 0.1M

iron solution for their individual absorption spectrums.

32. Absorption Spectrum Data Analysis
- Save all the absorption spectrum files on a flash drive or through

email. This includes all the Fe/Cu solution concentrations, the unknown

sample, the 0.1M iron nitrate solution, and the 0.1M copper nitrate

solutions (if there was time to test the last two solutions).
- Open the files in excel. Using the smooth line scatter plot option,

create individual graphs for the 0.1 M iron nitrate solution and the 0.1M

copper nitrate solution. Look at the overall shape of each graph to get

an idea of where each metal will peak for the solution containing both

metals.
- Create another smooth line scatter plot with the Fe/Cu solutions as well

as the unknown water sample.

 - Identify the curve that represents the copper in the solution, and

identify the curve that represents the iron in the solution. Determine

the wavelength at which the most separation occurs between samples

for both iron and copper ( max . Then record the absorbance for each

concentration at the max in the data table below.

- Perform a linear regression for both the copper and iron curves of the

graph by entering the concentration versus absorbance at the max

for each metal into separate excel cells.

- Select the data and create an XY scatter plot for each metal.
- Add a Trendline to both graphs. Press options and click Display

Equation and Display r-squared.

- Use the equation from each graph and the absorbance of the unknown

sample at max to determine the concentration of each metal in the

unknown water sample and record the concentration in the Lab Report.

Results

Data Tables and Graphs

Solution Wavelength at Peak Intensity (nm)

Na Cl 570.3nm
Li Cl 670.80nm
K Cl 766.40nm 767.03nm
Ca C l 2 587.5.0nm 770.3.0nm
Sr C l 2 703.00nm 677.00nm
Unknown #1 588.69nm 766.4nm

Metals in Unknown water sample Sodium and Calcium

 IA and IA Emission Spectrums
4500
4000
Sodium
3500
Lithium
3000
Potassium
2500 Strontium
Intensity 2000 Barium
1500 Calcium
1000 Unknown #1
Lithium
500
0
100 200 300 400 500 600 700 800 900 1000
-500

Wavelength (nm)

Absorption Spectrum: Cu/Fe Dilutions

1

0.8

400ppm
0.6
320ppm
240ppm
Absorption 0.4 160ppm
80ppm
Unknown #1
0.2

0
100 200 300 400 500 600 700 800 900 1000

-0.2

Wavelength
 *Data series are labeled based on the concentration of copper in each

solution.

Absorption Spectrum: Copper

Wavelength at peak absorption ( max : 335.68nm

Concentration (ppm) Absorption at max

400ppm 0.776

320ppm 0.552

240ppm 0.312

160ppm 0.121

80ppm 0.002

Unknown #1 0.126
2 Trendline equation: y = 0.0025x -
R = 0.98592
0.2411

Concentration of Copper in Unknown Sample 146.84 ppm

Copper: wavelength 335.68nm

0.9
0.8 0.78
0.7 f(x) = 0x - 0.24
0.6 R = 0.99
0.55
0.5
Absorption 0.4
0.3 0.31

0.2
0.1 0.12

0 0
50 100 150 200 250 300 350 400 450

Concentration (ppm)
 Absorption Spectrum: Iron

Wavelength at peak absorption ( max : 458.53nm

Concentration (ppm) Absorption at max

20ppm 0.667

16ppm 0.521

12ppm 0.286

8ppm 0.136

4ppm 0.018

Unknown #1 0.077
Trendline equation: y = 0.0421x -
R2 = 0.98738
0.1793

Concentration of Iron in Unknown Sample 6.09 ppm

 Iron: wavelength 458.53nm
0.8

0.7
0.67
0.6 f(x) = 0.04x - 0.18
R = 0.99
0.5 0.52

0.4
Absorption
0.3 0.29

0.2
0.14
0.1

0 0.02
2 4 6 8 10 12 14 16 18 20 22

Concentration (ppm)

Calculations

Concentration of copper and iron in unknown sample:

Copper: Iron:

0.126 = 0.0025x - 0.2411 0.077 = 0.0421x - 0.1793

0.3671 = 0.0025x 0.2563 = 0.0421x

146.84ppm = x 6.09ppm = x

Final Solution

The metals present in the simulated water sample are sodium and

calcium. This can clearly be seen in the emission graphs of these metals

compared with the graph of the unknown sample. The wavelengths at which

each metal peaks line up with the two peaks of the unknown sample. The

concentration of copper in the sample is 146.8ppm and the concentration of

 iron is 6.09 ppm. These concentrations were determined by running

2+ 3+
absorption tests on dilutions of a 400ppm /20ppm solutions.
Cu Fe

Then, a linear regressions was created for the copper and iron curves of the

graph to determine the concentrations of each metal in the solution.

Discussion

The results of this experiment are in many ways relevant to the major

fish-kill in the Clark Fork of the Columbia River. The results of the emission

test show that there were two metals present in the water. This suggests that

there was a high level of salinity in the water at Clark Fork, which could have

contributed to the fish-kill. Furthermore, the concentration of copper

(146.8ppm) and the concentration of iron (6.09ppm) in the water are both

considered toxic by the EPA standards for drinking water. This experiment

has identified several toxicities in the water sample. This narrows the

reasons that the fish-kill took place in 1984 and may allow researchers and

environmentalists to make changes to the water conditions in order to

prevent any danger to wildlife.

In doing this lab, every measure was taken to ensure accuracy of the

data. However, there is always room for error when conducting an

experiment. To begin, there are many instances where mistakes could have

been made while analyzing the data. In particular, when determining which

curve on the absorption graph was caused by copper and which was caused
 by iron. Furthermore, the dilutions were made from adding distilled water to

a solution with known concentration of copper and iron. There was a high

chance for error while creating these samples. In the future, standard

solutions of each concentration would allow for more precision. Lastly, the

water sample had IA and IIA metal ions bonded to chlorine, while, in the data

I used those metals were bonded to nitrates. This caused a slight shift in the

emission graph of the sample compared to the known metals. If time had

allowed, running new tests with each IA and IIA metal chlorate would have

provided more accurate data for analysis. If this experiment were continued,

more accuracy in the solutions and testing would produce data with higher

accuracy.

Conclusions

The concentration of copper in the unknown sample is 146.8ppm, and

the concentration of iron in the unknown sample is 6.09ppm. I came to this

conclusion by analyzing the emission spectrum of solutions containing

different concentrations of copper and iron. By creating solutions of different

concentrations, I was able to bracket the concentration of copper and iron in

the sample solution in order to find the exact concentration. I used the

graphs from the 0.1M copper and 0.1M iron solutions to identify the max of

the Cu(II) and Fe(III) curves. Then I identified which curve on the absorption

graph was the copper absorption and which curve was the iron absorption.

After identifying the curves, I chose a wavelength for both the copper and
iron that had the most separation between each concentration. For copper

this was 335.68nm and for iron it was 458.53nm. Then, using the absorptions

for each concentration at those wavelengths, I created a scatter plot with a

trend line. I was then able to use the equation of the trend line to calculate

the exact concentration of copper and iron in the unknown sample. Both of

these concentrations are over the level of toxicity allowed in drinking water,

and are dangerous to wildlife.

The emission spectrum of the unknown sample shows that there are

traces of sodium and calcium present. I came to this conclusion by testing

the emission spectrum of the unknown sample using a Spectrophometer,

and then comparing the graph it created with the graphs of known IA and IIA

metals. The sample has peak intensities at two wavelengths which each line

up with one of the unknown metals. The peak at 588.69nm lines up with the

sodium peak and calcium peak, and the peak at 769.54nm lines up with

calcium. I reached the conclusion that calcium is present in the sample,

because the sample emission graph lines up with some of the smaller peaks

of the calcium graph. There is room for error in these results, however I am

confident in the data I collected and the conclusions I reached.

Bibliography

- Stanton, B., Zhu, L., & Atwood, C. H. (2006). Experiments in general

chemistry featuring MeasureNet. Belmont, CA: Thomson Brooks/Cole.

Appendix E, Appendix B-2, and Appendix D provided me with detailed

explanations of how to set up and use a Spectrophometer for both

 emission and absorption spectroscopy, and how to perform a linear

regression on Excel.
- "Dangerous for the Environment Hazard Symbol." Dangerous For The

Environment Hazard Symbol. N.p., n.d. Web. 29 June 2016.

This was the website where I found the picture on the title page of the

proposal.
- "Drinking Water Contaminants Standards and Regulations." EPA.

Environmental Protection Agency, n.d. Web. 29 June 2016.

I used the EPA website to find the concentrations of copper and iron

that are considered toxic.

- "National Geographic Freshwater 101: Pollution." National Geographic.

N.p., n.d. Web. 29 June 2016.

I used this website to gain information on freshwater pollution and

contamination that I used while writing the introduction of this

procedure proposal.