Phytoremediation

CHY 116 Phytoremediation of Copper-Polluted Water

by Eichornia crassipes1

Learning Goals

Water hyacinths will be used to remove trace quantities of copper from polluted water samples, introducing the concept of phytoremediation. Low concentrations of copper will be detected and quantified spectrophotometrically by forming Cu2+-cuprizone complexes.

Lecture Connections

Experimental Background

Mining and other industrial operations can sometimes deposit heavy metals in the local environment. Some of these metals (e.g. As, Pb, Hg, and Cd) are exceptionally toxic, causing health problems for humans when ingested.2,3Compounding the problem, heavy metals are neither destroyed nor excreted by the plants and animals that ingest them. Tissue concentrations of heavy metals therefore increase moving up the food chain, a process called bioaccumulation. Heavy metal poisoning in humans can result from ingesting an organism (particularly a predator) that has lived in a contaminated environment. This phenomenon is responsible for the warnings about mercury poisoning from eating too much tuna (a predatory fish).

Remediation (clean up) of areas polluted with heavy metals is a daunting task. How does one selectively remove a heavy-metal pollutant from a lake (or an acre of soil) when its concentration is only at the  parts per million (ppm) level? (1 part per million = 1 milligram per liter)

Phytoremediation is one approach to solving this problem.3 Some plants absorb heavy metals selectively and irreversibly from soil or water with minimal harm to themselves. These plants effectively scrub their growing media of toxic metals. Once the plants have absorbed the pollutant, they can be uprooted and the metals recovered in a safe manner. For more information on phytoremediation, read  the provided article Promises & Prospects of Phytoremediation (pdf).

This lab will test the ability of an aquatic plant, the water hyacinth (Eichornia crassipes) to remove copper ions from water. Water hyacinths are free-floating plants, with an extensive root system, commonly found in lakes and ponds. Copper is less toxic than some heavy metals (making it safe for us to use in lab), but it can still cause health problems for some people.4

The quantity of dissolved copper in polluted” water samples will be measured spectrophotometrically with a Baush and Lomb Spectronic 20 spectrophotometer. The molar absorptivity of CuSO4, the Cu2+ source, is relatively low. This means that solutions of Cu2+ must be quite concentrated to give absorbances the spectrophotometers can detect. (Equation 1)

Beer’s Law:                  A=ebc (Equation 1)

A = absorbance; e = molar absorptivity (M-1*cm-1); b = path length (cm); c = concentration (M) (refer to the Measuring an Equilibrium Constant experiment to review absorbance)

The contaminated wetland water samples contain aqueous Cu2+ at very low concentrations (1 - 10 ppm). To detect Cu2+ at these concentrations, Cu2+ will be reacted with the complexing agent cuprizone (oxalic acid bis-(cyclohexylidenehydrazide), C14H22N4O2). The resulting Cu2+-cuprizone coordination complex has a high enough molar absorptivity that it can be detected at ppm-level concentrations.

To ensure that the Cu2+-cuprizone coordination complex is formed quantitatively, we will use excess cuprizone and control the solution pH with a phosphate buffer to a pH of 7.9. The Cu2+-cuprizone coordination complex forms slowly so we must allow time for the reaction to reach equilibrium (30 minutes).

Before measuring the polluted water samples, standards with known Cu2+-cuprizone concentrations will be prepared and a Beer’s Law analysis will be performed, to generate a calibration curve.

Once the standard data is collected, the contaminated wetland water samples will be evaluated. Using the cuprizone complexation technique, the Cu2+ concentration in the samples will be measured before adding water hyacinths (time 0). The hyacinths will then be added, and the Cu2+ concentration will be monitored over time. This will provide information about how much Cu2+ a single water hyacinth can remediate and how quickly this processoccurs.

Note:

-   the cuprizone and buffer reagents are not added until the contaminated wetland water solution has been exposed to the plant and a sample has been removed at each specific time. This ensures that the buffer and cuprizone are not affecting the plant or the process of remediation.

-   some buffer systems might interfere with the analysis – although the Cu2+-cuprizone seems to be stable over a range of pH, the effect of pH on dissolved Cu2+ needs to be considered. If too basic, Cu2+ might precipitate as the oxide or hydroxide. The phosphate buffer, at fairly neutral pH, is a good choice for this experiment.

You will make your absorbance measurements with a Vernier Spectrophotometer. 

References

1This experiment is adapted from Kelley, C.; Gaither, K.; Baca-Spry, A.; Cruickshank, B.Incorporation of Phytoremediation Strategies into the Introductory Chemistry Laboratory. Chem. Educator 2000, 5, 140-143, and from Bowdoin College Department of Chemistry Remediation of Copper Polluted Water, Spring 2011/Fall 2011.

2Toxicological profiles for thousands of substances can be found at the Agency for Toxic Substances and Disease Registry: http://www.atsdr.cdc.gov/toxprofiles/index.asp.  This organization also maintains a priority list for hazardous substances (http://www.atsdr.cdc.gov/cercla/); the heavy metals mentioned in the text top this list.

3Jadia, C. D.; Fulekar, M. H. Phytoremediation of heavy metals: Recent Techniques. African Journal of Biotechnology 2009, 8, 921-928.

4"Wilson's Disease". Mayo Clinic. http://www.mayoclinic.com/health/wilsons-disease/DS00411. 3/15/11

Experimental Procedure

You will work in groups of 2 in this experiment. This is a 2 week lab. During the first week, standard Cu2+ solutions will be prepared at varying concentrations. Using Excel, absorbance vs. concentration will be graphed in order to obtain a calibration curve. During the second week, a contaminated wetland water sample (polluted with Cu2+) will be exposed to a water hyacinth plant, which over time will  absorb Cu2+ present in solution. At specific time intervals, samples will be removed from the contaminated water in order to evaluate the Cu2+ concentration remaining, and determine the efficiency of the water hyacinth in remediation. Using Excel, concentraton vs. time will be graphed.

Note about units: parts per million (ppm) = milligrams per liter (mg/L)

Week 1:

a.) Preparation of standards

Properly rinse and fill a buret with the Cu2+ stock solution (3.5 ppm CuSO4). Dispense exactly 5, 10, 15, and 20 mL of this solution into four clean, labeled 25 mL volumetric flasks.* Add 1 mL of 0.5% Cuprizone solution and 3 mL of pH 7.9 phosphate buffer to each flask. Carefully fill to the mark with deionized water, stopper and mix. In a beaker, prepare a blank standard of 1 mL 0.5% cuprizone, 3 mL of pH 7.9 buffer, and 21 mL deionized water (carefully measure with a graduated cylinder). Before measuring absorbance, allow the solutions to stand for 30 minutes or longer for the color to fully develop. Calculate the exact Cu2+ concentrations of the standards (M1V1 =M2V2).

*  If any of the solutions become colored at this stage, there is copper contamination present. Clean the flasks by rinsing once with 1M HCl, once with tap water, and finally three rinses with distilled water.

While you are waiting for the color to develop, practice with a pipet pump / bulb, a 5 mL volumetric pipet, and water, so that next week you will be able to rapidly remove samples at the proper time intervals.

b.) Measuring the absorbance of standards

You will be evaluating the samples at a wavelength of 600 nm (the maximum wavelength of absorption for Cu2+-cuprizone). Obtain a sample cuvette and make sure it is clean inside and out. Avoid touching the the bottom third of the cuvette to keep its light path clear. Rinse the cuvette in with a small amount (~1 mL) of distilled water (the blank), then fill the cuvette about 3/4 full. Wipe off the outside of the cuvette and insert it into the sample compartment with the orientation mark aligned with the mark on the spectrophotometer. Calibrate the spectrophotometer. 

NOTE: this blank standard will also be your first data point (zero concentration of Cu2+ should give you zero absorbance).

Return the blank standard to its beaker. rinse in the cuvette with the solution prepared with the 5 mL of the Cu2+ stock solution, wipe off the outside, insert it into the instrument, and read the absorbance. Repeat for the remaining calibration solutions (least to most concentrated), recording the absorbance measurements to three decimal places in an appropriately labeled  data table in your lab notebook.

Finally, check to be sure that the instrument is still properly blanked and zeroed, as follows: rinse in and fill the cuvette again with the blank standard. Read its absorbance. It should still be very close to 0.000. Remove the cuvette and read transmittance. It should still be close to 0.00. If either reading has changed by more than 5 in the last decimal place, you should repeat the absorbance measurements for the standard solutions. More specific directions for instrument operation will be provided in the laboratory.

Week 2:

a.) Remediation with Water Hyacinths

In a plastic bucket, add 500 mL of the Cu2+ solution (10 ppm) – this is the contaminated wetland water sample. Properly rinse a 5 mL volumetric pipet and pipet 5 mL of this solution into a clean, labeled 25 mL volumetric flask. This is the time zero sample.

Obtain a water hyacinth. Let the water drain from its root mass, and note its relative size. Start your stopwatch, and place the water hyacinth into the bucket of Cu2+ solution.

With the volumetric pipet, remove 5 mL samples at 5, 10, 20, 30, 45, and 60 minutes, placing each sample into clean, labeled 25 mL volumetric flasks. Prior to sampling, slosh the plant around to homogenize the solution. Record the actual elapsed time of each sample.

As time permits, add 1 mL 0.5% cuprizone and 3 mL pH 7.9 phosphate buffer to the samples, dilute and mix as for the standards. You will need to prepare a new blank standard: in a beaker, add 1 mL 0.5% cuprizone, 3 mL of pH 7.9 buffer, and 21 mL deionized water (carefully measure with a graduated cylinder).

In between sampling, set up the spectrophotometer (600 nm), and calibrate with the blank standard as done last week.

b.) Measuring the absorbance of phytoremediated samples

Before reading the absorbance values, make sure you have waited 30 minutes to allow the color to fully develop.

Read absorbance values for the timed phytoremediation solutions as follows. Rinse in the cuvette  with the 60-minute phytoremediation solution, wipe off the outside, insert it into the instrument, and read the absorbance. Repeat for the remaining samples of decreasing time (least concentrated to most concentrated), recording absorbances to three decimal places in an appropriately labeled data table in your lab notebook.

All solutions may be disposed of down the drain. In the waste hood, rinse the volumetric flasks and  caps with 1M HCl, in the sink rinse thoroughly with tap water, followed by deionized H2O. Rinse the burets and volumetric pipets with deionized H2O. Return all cleaned items.

Pre-lab Assignment

Pre-Lab Library excercise:

Download and submit as a pdf document one primary research article that describes an aspect of phytoremediation. Sarah Lucchesi, the USM library chemistry liaison prepared an instructional video on how to search the libraries various holdings and databases.  The mp4 movie is available here (mp4). The file is 55 MB.

Notebook Preparation:

In your lab notebook, prepare the following information:

·      A brief (2-3 sentences) objective of the lab.

·    A table of glassware, equipment and chemicals to be used. Include relevant properties and safety information for each chemical. Be sure to include the hazards associated with each chemical.

·      Several “bullet points” summarizing the tasks involved in the procedure.

Be sure to read and study the Experimental Background material, along with the procedures, before coming to lab. Keep full, legible records of your work, data, and observations in your Laboratory Notebook.

If your watch or cell phone has a stopwatch function, bring it to lab the second week for timing your sampling.

Experimental Videos

        (1) Preparation of calibration solutions 1, 42.5 min

        (2) Preparation of calibration solutions 2, 6.5 min

        (3) Absorbance measurements, 15.3 min

        (4) Examination of water hyacinth, 8.0 min

        (5) 5 minute aliquot, 0.8 min

        (6) 10 minute aliquot, 1.0 min

Experimental Data 

       Calibration standards data &

      Phytoremediation data (pdf)

After lab

1.) Use Excel to plot your standard data as absorbance (vertical axis) vs. concentration (horizontal axis). There are 5 data points to be graphed. Don’t overlook the blank sample. Fit a straight line to your data, and obtain the equation of the line and the regression coefficient (R2). Label the axes of the graph and give it an informative title from which the reader can readily understand the relationship represented on the graph. If your data points have a high degree of accuracy, the value for b should have an absolute value of less than 0.010 and the correlation coefficient should be ≥ 0.99. Make two copies of the graph. Hand in one with your notebook pages and report form, and tape the other one on the corresponding pages of your lab notebook.

 You may want to set up a data table similar to this in your notebook:

*In your notebook, be sure to show how you calculated the diluted concentrations from the stock solution.

2.) In your notebook, calculate the concentration of Cu2+ for your phytoremediation samples using the equation of the line determined for the standards. This gives you the concentration of the solutions in  the 25.00 mL volumetricflasks.

3.) In your notebook, calculate the concentration of Cu2+ in the bucket for each timed sample. This is a M1V1 = M2V2 calculation (M is concentration) but the tricky part is that you know the concentration of the dilute solution (in the 25.00 mL volumetric flask; M2 which you determined from experimental absorbance and the standard curve in Question 2) and need to calculate the concentration of the more concentrated solution (5.00 mL samples taken at various times; M1). This gives you the concentration of Cu2+ in the bucket containing the water hyacinths at the various times.

 For 2 and 3, you may want to set up a data table similar to this in your notebook. 

4.) Use Excel to plot [Cu2+] (remaining in the bucket) vs. time in minutes using a column graph, with the columns vertically oriented. Give the graph a title that explains the experiment and incorporates the two variables being graphed (y & x) and some mention of the plant. DO NOT include a trendline for this graph.

Grading Rubric (pdf)

Post Experimental Analysis complete the following REPORT FORM and answer the after lab questions above in your notebook.

Report Form Name                                                        

CHY 116: Phytoremediation of Copper-Polluted Water by Eichornia crassipes

1.) Standards Data & Results: Report the equation of the line and the correlation coefficient for the copper standards evaluated. Comment on the quality of this data using the absolute value of the y- intercept and the correlation coefficient. Your discussion must include a frame of reference for what constitutes “good” data and the source for your frame of reference.

2.) Phytoremediation Data & Results:  Show a complete calculation  for determining the concentration of Cu2+(ppm) in the remediated sample at time = 60 minutes. Include units, unit cancellation, and correct significant figures for full credit.

3.) Comment on the graph from the phytoremediation results. Does concentration decrease linearly over time? If not, provide and explanation.

4.) Go to https://www.epa.gov/ground-water-and-drinking-water/national-primary-drinking-water-regulations. Record the mg/L level allowed for copper in drinking water. Record the health effects of short and long term exposure to copper in drinking water.

5.) Did your water hyacinth render your contaminated wetland water solution drinkable as far as copper levels are concerned? Refer to your answer from Question 4 remembering that mg/L = ppm. Include your experimental results to help support your answer.

6.) Look up Wilson’s disease (cite your source) and briefly describe what it is and the primary treatment. (Be sure to address the term chelation - you may have to refer to some other sources.)

7.) The drug used to treat Wilson’s disease is similar in chemical function to what chemical that we are using to analyze for copper? Explain.

8.) In addition to the sources of copper pollution listed in the EPA table, find three other sources of copper pollution in the environment.

9.) Type a brief summary of this experiment. In the summary include a topic sentence describing the goal of the experiment. In the body of the summary, focus on the results and outcomes (not procedural details, not intermediate calculations, but final results) and a final conclusion sentence. Your summary should consist of three paragraphs with each paragraph addressing the following information:

Paragraph 1: What was the purpose of the lab that you performed and what did you do?

Paragraph 2: What were your results and what was the error associated with the results. Include numerical results and how do your results compare to literature values (if appropriate include percent error from literature values)?

Paragraph 3: Provide an error analysis. Address the precision of the instrument(s) and method(s) that you used. Analyze the %RSD of the method(s) used if appropriate.

Turn in as one pdf document:

1.) all pages from your laboratory notebook for this experiment, 

2.) your Excel spreadsheet including the standard curve, 

3.) all pages of this Report Form, and 

4.) a typed Summary.

Staple them into a single package.