Student Activity: Detecting Heavy Metal Ion Concentrations in Shellfish

by Larry Jerdal

PURPOSE

This student experiment allows extraction of the lead ion (Pb²⁺) in shellfish with determination of the lead concentration compared with a known standard.

DESCRIPTION

Designed for first year general chemistry courses, this experiment may be completed when presenting topics such as solution chemistry or oxidation-reduction behavior of metal ions. The learning objectives for this activity include: students will learn the theory and practice of spectrophotometry, students will perform an instrumental calibration using known standard and control solutions, and students will conduct a spectrophotometric measurement by absorbance in determining the lead ion concentration in an environmental sample.

TIME

45 minutes using fully prepared reagents and sample extract. More time may be allowed depending on student participation in preparation of materials.

MATERIALS

Chemicals

• 0.0005% by weight solution, dithizone in methylene chloride
• (0.50g dithizone (Diphenyl thiocarbazone) in 1L of methylene chloride)
• 2L methylene chloride (CH₂Cl₂)
  SAFETY CONCERN: Use only in hood, students should not be exposed to this chemical without adequate ventilation!

Equipment

• 1 shellfish knife
• 1 spectrophotometer with cuvettes (appropriate for single wavelength determination)
• 2 250mL beakers
• 1 stirring rod
• 3 pipetes (Beral or 2mL glass)
• 1 Bunsen burner
• 1 ring stand with screen
• 1 pr forceps
• 2 large test tubes with stoppers
• 1 clam, oyster, mussel, or other shellfish

SAFETY CONCERN

Use only shellfish of known collection history or those sold for human consumption. Wash hands and surfaces with which shellfish may have come in contact.

SAFETY INSTRUCTIONS

Shellfish should be prepared prior to student use. Consult local regulations concerning the use of live animals for student instruction. Bivalve shellfish are especially difficult (particularly oysters) for inexperienced persons. Carefully cut the adductor muscle and use extreme care in handling the knife used for shellfish preparation!!!

Students should not prepare the dithizone solvent solution. Instructors should use caution in preparing this solution assuring that adequate fume hood exhaust ventilation is available in both preparation and experimental use by students. Dithizone will stain skin and clothing. Methylene chloride is highly toxic if ingested and should not contact the skin. Methylene chloride is flammable and should not be used in a room with exposed flames. Consult appropriate safety references and a complete MSDS for handling procedures for methylene chloride. Make sure adequate hood exhaust and ventilation is maintained for the duration of the laboratory experiment.

PROCEDURE

Shellfish Extraction

Weigh the shellfish viscera to the nearest 0.1g after removal from the shell. Place the shellfish viscera, cut into four or five pieces, and all shell liquor in a 250mL beaker. Add 100mL of water. Place the beaker on a ringstand with screen and heat to a low boil. Continue carefully heating the beaker and contents for 20 minutes, making sure that the contents do not splatter or boil over. Remove the beaker from the heat and allow to cool until the beaker and contents may be handled. Decant the shellfish liquor from the beaker. Discard the solid shellfish residue. Pipet 10mL of the cool extract into a large test tube. Perform solvent transfer operations and venting in a hood. Add 10mL of the dithizone prepared solution and 15mL of methylene chloride. Stopper the tube. Shake well by repeated inversions, relieve any vapor pressure periodically by carefully opening the stopper in a hood. If the lead ion has been extracted from the shellfish, a distinct color will develop in the clear layer within a few minutes. After extraction is complete, pipet 2mL of the colored layer into a test tube or spectrometer cuvette. Set the tube aside until the control and standard tubes are ready for measurement.

Control and Standard Preparation

Prepare the control solution for measurement using the same procedure for extraction as completed with the shellfish extract. Standard solution series test tubes of known concentration will be available for use near the spectrophotometer or data will be provided to you for analysis.

Spectrophotometric Measurement

Set the spectrophotometer at 510nm when the machine is ready for use. Set the meter "zero" on the machine with no cuvette inserted. Place the control test tube in the test tube slot and, when the machine is stabilized, set the meter at 100% transmittance (0% absorbance). Place the shellfish extract tube into the spectrophotometer and record the absorbance reading.

CALCULATIONS

The following data is provided as a sample of results which could be used for student calculations.

LEAD ION CONCENTRATION AT 510NM
(1 Liter of Sediment)

mg Pb2+	Absorbance
200mg	0.215
150mg	0.193
100mg	0.170
75mg	0.155
50mg	0.140
25mg	0.124

This data may be treated graphically to demonstrate a calibration curve. The data may also be used to determine unknown values for shellfish lead concentrations by graphic or regression methods.

DISCUSSION

Many public beaches and shorelines, freshwater and marine, have been declared dangerous for public use and closed in recent years. Closure results from many reasons, chief among them being pollution from human or animal waste with bacterial or viral, metal, or medical waste/foreign object contamination. With the waters contaminated with foreign material, the organisms which live in aquatic edge ecosystems may carry burdens of contaminants which could be harmful if ingested. Metal ions, particularly, are subtle toxins as they are not visbile and do not usually cause acute reactions as do bacterial and viral agents. Such metals as lead, nickel, and chromium may be concentrated by shellfish and, if ingested in high dosages, cause various symptoms and complaints, even resulting in death.

Sources of pollution in the aquatic environment are many including discharges of untreated or undertreated sewage waste, industrial wastewater, urban and agricultural runoff, and simple litter. All of these discrete or diffuse sources of pollution undergo complex chemical reactions in the environment as well as biochemical reactions in various organisms.

LABORATORY DISCUSSION QUESTIONS

1. What is the color of the lead extraction layer (the dithizone complex) in the liquid-liquid/solid extraction?

2. What is the process of machine adjustment and calibration used to determine the shellfish lead concentration? Outline the major steps.

3. What is the relationship between the shellfish extract color and the measurement wavelength used on the spectrophotometer?

4. What are legal and practical levels of exposure to toxic metals (mercury, nickel, lead, chromium, arsenic, selenium) in food according to Environmental Protection Agency (EPA) guidelines? Food and Drug Administration (FDA) guidelines? Your state or local regulatory agency?

5. What are corresponding levels of exposure for drinking water?

6. How would you design a major sampling project to determine the safety of shellfish sold for human consumption in a particular area?

LABORATORY DISCUSSION QUESTION RESPONSES

1. Red/pink.

2. The zero is adjusted to indicate no light transmission from the light source. The "blank" is adjusted for solution variation at 100% transmission, which accounts for all chemical and physical interactions except for the material determined, known concentration levels are added and taken through the entire analytical preparation, and, from the response data, a calibration curve or regression is run to determine the slope of the curve of the analytical determinations. The concentration of the unknown is either interpolated on a graph or solved by equation of the line. The data set should always be graphed to identify outlying points indicating errors or sample problems.
   3.510 nm is close to the color pink visible in the sample.

3. Various sources may be used to identify these values including APHA Standard Methods and EPA document #440-5-86-008. These values may also be obtained by writing the state and federal regulatory agencies.

4. These values may change frequently depending on new research findings.

5. Responses will vary but should pay attention to environmental sampling and sampling density, methods of completing the analyses, cost of program, and what to do in the case of identified contamination.

REFERENCES

Staff and participants, 1989 Dreyfus Institute on Environmental Chemistry, Princeton University, Princeton, NJ.

Vogel, Arthur I., 1987. Vogel's Textbook of Quantitative Inorganic Analysis, 4th Ed.

American Public Health Association. Standard Methods for the Examination of Water and Wastewater.

Woodrow Wilson Leadership Program in Chemistry lpt@www.woodrow.org
The Woodrow Wilson National Fellowship Foundation webmaster@woodrow.org
CN 5281, Princeton NJ 08543-5281 Tel:(609)452-7007 Fax:(609)452-0066