Laboratory Experiment pubs.acs.org/jchemeduc
Determination of Mercury in Fish: A Low-Cost Implementation of Cold-Vapor Atomic Absorbance for the Undergraduate Environmental Chemistry Laboratory Brian K. Niece* and James F. Hauri Department of Chemistry, Assumption College, Worcester, Massachusetts 01609-1296, United States S Supporting Information *
ABSTRACT: Mercury is a known neurotoxin that is particularly harmful to children and unborn fetuses. Consumption of contaminated fish is one major route of mercury exposure. This laboratory experiment gives students an opportunity to measure mercury concentrations in store-bought seafood and compare the results to suggested exposure limits. The U.S. Environmental Protection Agency (EPA) recommended method for determination of mercury concentrations is cold-vapor atomic spectroscopy. We propose a method of adapting an existing flame atomic absorbance spectrometer for this technique with little additional cost, thus allowing students to learn about this important technique. Students measured mercury concentrations in swordfish and tuna purchased at a local supermarket. Mercury levels in both fish were within the range found by the U.S. Food and Drug Administration (FDA). Students gained experience with sample digestion, cold-vapor analysis, and data analysis. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Environmental Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Applications of Chemistry, Atomic Spectroscopy, Food Science
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determine mercury concentrations. Murcia et al. have reported a solution-phase spectrophotometric determination of mercury.8 Armenta et al.,9 Cizdziel,10 and Jenkins et al.11 all describe laboratory exercises measuring mercury using commercial mercury analysis systems. Rice et al. suggest using a commercial direct mercury analyzer to study concentrations of mercury in locally caught fish.12 The U.S. Environmental Protection Agency (EPA) recommended method for determination of mercury concentration in fish is by cold-vapor atomic absorbance (CV-AAS) or coldvapor atomic fluorescence spectroscopy.13,14 Given the importance of this technique to measuring such a widespread environmental contaminant, we wanted to give the chemistry and environmental science students hands-on experience with the method. We also wanted them to acquire some understanding of how the cold-vapor method differs from flame and furnace atomic spectroscopies.
ercury has long been known to be harmful to humans, particularly children and developing fetuses. The neurotoxic effects of the metal are worse for young children, but will adversely affect all people. The primary route of exposure to mercury tends to be fish consumption. Both freshand saltwater seafood have been found to have significant concentrations of mercury. Much of the mercury in the environment is released as a byproduct of burning coal and can disperse through the air to contaminate otherwise relatively unimpacted water bodies. Fish from these “unpolluted” ponds have also been shown to have measurable concentrations of mercury in their tissue.1 As a result, local residents who eat fish caught in such ponds may be unknowingly exposed to this mercury. Because of the biomagnifying properties of organic mercury, top of the food chain saltwater fish such as mackerel, shark, and swordfish often have the highest body burden of mercury. These fish also tend to be the most attractive for human consumption.2 The U.S. Food and Drug Administration (FDA) monitored mercury levels in commercial seafood from 1990− 2010 and found concentrations exceeding 1 ppm in some samples of swordfish and tuna.3 The importance of educating the students about mercury contamination and analysis was raised as long ago as 1972.4 More recently, Judd pointed out the relevance of mercury in the environment and suggested that contemporary reports, particularly concerning contaminated fish, could be used to raise student interest in this important topic.5 Ramsay6 and Romero et al.7 have described experiments using titration to © 2013 American Chemical Society and Division of Chemical Education, Inc.
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LOW-COST INSTRUMENT The environmental course in which this experiment is used has a relatively small enrollment and is only offered once every other year. The chemistry department currently has no other need for mercury analysis. Commercial mercury analysis systems cost $25,00015 or more, a cost that is prohibitively expensive given the modest needs for the instrument. Therefore, we set out to find a way to expose students to the CV-AAS technique using the existing atomic absorption Published: February 19, 2013 487
dx.doi.org/10.1021/ed300471w | J. Chem. Educ. 2013, 90, 487−489
Journal of Chemical Education
Laboratory Experiment
resulting solution was transferred to a volumetric flask and diluted to volume. Droplets of fat remained in the mixture after digestion. They were avoided when removing aliquots of the sample for analysis.
spectrometer and other hardware found in the chemistry laboratories. Thistlethwaite and Trease described the construction of a complete cold-vapor analysis system without the use of a commercial spectrometer,16 but the effort was unreasonable given our modest needs. Lieu, et al. described the conversion of an ordinary condenser into a cold-vapor cell;17 however, the work involved was beyond the author’s (B.K.N.) glassblowing skills. The crucial breakthrough that allowed assembly of this system from existing lab components was the realization that sodium chloride is transparent to UV radiation at 254 nm. Therefore, it is possible to use a 10 cm gas IR cell with NaCl windows borrowed from the physical chemistry course in place of the quartz cell ordinarily used for CV-AAS. Consequently, the only equipment purchase was a mercury hollow cathode lamp (Perkin-Elmer Lumina, $432). It may even be possible to adapt some spectrometers to use a low-cost mercury lamp in place of the hollow cathode lamp,18 although that option has not been tested with this equipment.
Analysis
Portions of 1 μg/mL mercury standard or digested fish sample were diluted to 100 mL with a solution of nitric and sulfuric acids, and 20 mL of a reducing solution containing sulfuric acid, sodium chloride, hydroxylamine sulfate, and tin(II) chloride was added. House air at a flow rate of 2 L/min was bubbled through the resulting mixture, sweeping the reduced mercury into the analytical cell mounted in the AA spectrometer. The absorbance was recorded for 60 s at a wavelength of 253.7 nm, and the peak height recorded. After passing through the cell, the air was bubbled through a scrubbing solution of iodine and potassium iodide before being vented to a fume hood. See the Supporting Information for a detailed description of the apparatus.
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HAZARDS The nitric and sulfuric acids used in the diluting solution and for preparing the mercury standard are corrosive and students should wear appropriate protective clothing. The boiling mixture of concentrated nitric and sulfuric acids used during the digestion is extremely hazardous. This portion of the experiment should be performed in a fume hood and the students closely supervised. Mercury is toxic and the standard solution is prepared in diluted sulfuric acid. It is therefore caustic and should be handled with gloves. Iodine and hydroxylamine sulfate are corrosive and irritants. Vanadium(V) oxide is extremely toxic, and hydrogen peroxide is a strong oxidizer. They should be handled with gloves. All waste material potentially contains traces of mercury and should be disposed of according to local, state, and federal environmental regulations.
EXPERIMENTAL OBJECTIVES This laboratory experiment is intended for an upper-level undergraduate instrumental or environmental chemistry course. It is also appropriate for an analytical course. The students undertake a complicated analyte extraction in digesting the fish samples. The experiment is performed after a flame atomic absorbance experiment, allowing students to gain experience with the less common cold-vapor technique. Using a system assembled in-house rather than a commercial instrument gives the students a better understanding of the differences between the cold-vapor technique and standard atomic absorption methods. Finally, the students are exposed to a current environmental concern, the biomagnification of mercury in seafood. The use of store-bought fish samples piques student interest in the outcome of the experiment.
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EXPERIMENTAL OVERVIEW The analytical samples for this experiment were skinless, boneless tuna and swordfish steaks purchased frozen at a local supermarket. The origin of the two types of fish was indicated as Indonesia and Singapore, respectively. The students were divided into groups of 2−3 and each group analyzed both types of fish. In addition, the entire class worked together to complete the standard analyses for the calibration curve. Each group set up the digestion19,20 at the beginning of the laboratory period. While the digestion mixtures were boiling, the students began measuring the absorbance of calibration solutions. All sample and standard measurements were completed in a single three-hour lab session. Mercury concentrations were determined19−21 using a Perkin-Elmer 3100 atomic absorbance (AA) spectrometer fitted with a cold-vapor system assembled from parts already available in the chemistry department. Solutions were prepared using 18 MΩ deionized water. All glassware was washed with 1 M nitric acid and rinsed with deionized water prior to use. Chemicals were reagent grade purchased from Sigma Aldrich unless specified otherwise.
RESULTS AND DISCUSSION The results measured by students using this procedure show more scatter than would be expected from a dedicated mercury analyzer, but are acceptable for an introduction to the technique. A calibration curve produced from duplicate measurements of five mercury standard quantities is shown in Figure 1. The detection limit (three times standard deviation) calculated from this graph using triplicate blank measurements
Digestion
Homogenized fish samples were boiled for 15 min in a 1:1 mixture of nitric and sulfuric acids (both VWR, Aristar Plus trace metal grade) containing vanadium(V) oxide. After rinsing the reflux condenser with water and hydrogen peroxide, the
Figure 1. Calibration curve for cold-vapor analysis showing absorbance at 254 nm vs μg of mercury added to the analysis flask. R2 = 0.90. 488
dx.doi.org/10.1021/ed300471w | J. Chem. Educ. 2013, 90, 487−489
Journal of Chemical Education
Laboratory Experiment
is 0.5 μg of mercury. This detection limit corresponds to a concentration of 5 ppb mercury in the analysis solution and is slightly higher than the detection limit of 1 ppb reported in the literature for CV-AAS without preconcentration or improved aeration techniques.21 We point out to the students that, although this detection limit could be improved, it is sufficient for our purposes and other factors such as cost must also be considered in determining how to perform an analysis. Using 10 g fish samples, the detection limit for the method corresponds to a mercury level of 0.21 ppm in the fish. The mercury content measured in two fish samples purchased at a local supermarket are shown in Table 1. Each
(2) Zahir, F.; Rizwi, S. J.; Haq, S. K.; Khan, R. H. Low Dose Mercury Toxicity and Human Health. Environ. Toxicol. Pharmacol. 2005, 20, 351−360. (3) U.S. Food and Drug Administration. Mercury Levels in Commercial Fish and Shellfish (1990−2010). http://www.fda.gov/ food/foodsafety/product-specificinformation/seafood/foodborne pathogenscontaminants/methylmercury/ucm115644.htm (accessed Jan 2013). (4) Klein, D. H. Some General and Analytical Aspects of Environmental Mercury Contamination. J. Chem. Educ. 1972, 49, 7− 10. (5) Judd, C. S. News from Online: Mercury and Our Environment. J. Chem. Educ. 2001, 78, 570−571. (6) Ramsay, C. G. EDTA Titration of Cadmium and Mercury: An Exercise in Logic. J. Chem. Educ. 1977, 54, 714−717. (7) Romero, M.; Guidi, V.; Ibarrolaza, A.; Castells, C. Complexometric Determination of Mercury Based on a Selective Masking Reaction. J. Chem. Educ. 2009, 86, 1091−1093. (8) Murcia, N. S.; Lundquist, E. G.; Russo, S. O.; Peters, D. G. Quincy Meets Perry Mason: An Experience in Chemistry and Law A Simple Spectrophotometric Method for the Determination of Traces of Mercury(II) and Lead(II). J. Chem. Educ. 1990, 67, 608−611. (9) Armenta, S.; de la Guardia, M. Determination of Mercury in Milk by Cold Vapor Atomic Fluorescence: A Green Analytical Chemistry Laboratory Experiment. J. Chem. Educ. 2011, 88, 488−491. (10) Cizdziel, J. V. Mercury in Environmental and Biological Samples Using Online Combustion with Sequential Atomic Absorption and Fluorescence Measurements: A Direct Comparison of Two Fundamental Techniques in Spectrometry. J. Chem. Educ. 2011, 88, 209−215. (11) Jenkins, J. D.; Orvis, J. N.; Smith, C. J.; Manley, C.; Rice, J. K. Including Non-Traditional Instrumentation in Undergraduate Environmental Chemistry Courses. J. Chem. Educ. 2004, 81, 22−23. (12) Rice, J. K.; Jenkins, J. D.; Manley, A. C.; Sorel, E.; Smith, C. J. Rapid Determination of Mercury in Seafood in an Introductory Environmental Science Class. J. Chem. Educ. 2005, 82, 265−268. (13) Method 1631, Revision E; Mercury in Water by Oxidation, Purge and Trap, and Cold Vapor Atomic Fluorescence Spectrometry; EPA: Washington, DC, 2002. (14) Method 245.7, Revision 2.0; Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry; EPA: Washington, DC, 2005. (15) Dwyer, M. CETAC Technologies, Omaha, NE. Personal communication, 2012. (16) Thistlethwaite, P. J.; Trease, M. Determination of Mercury by a Simple Atomic Absorption Method. J. Chem. Educ. 1974, 51, 687−688. (17) Lieu, V. T.; Cannon, A.; Huddleston, W. E. A Non-Flame Atomic Adsorption Attachment for Trace Mercury Determination. J. Chem. Educ. 1974, 51, 752−753. (18) Sands, R. D. An Inexpensive Mercury Vapor Lamp. J. Chem. Educ. 1985, 62, 526. (19) Method 977.15: Mercury in Fish: Alternative Flameless Atomic Absorption Spectrophotometric Method. In Official Methods of Analysis of AOAC International, 16th ed.; Cunniff, P., Ed.; AOAC International: Arlington, VA, 1995; pp 21−22. (20) Method 977.21: Mercury in Food: Flameless Atomic Absorption Spectrophotometric Method. In Official Methods of Analysis of AOAC International, 16th ed.; Cunniff, P., Ed.; AOAC International: Arlington, VA, 1995; pp 20−21. (21) Clevenger, W. L.; Smith, B. W.; Winefordner, J. D. Trace Determination of Mercury: A Review. Crit. Rev. Anal. Chem. 1997, 27, 1−26.
Table 1. Mercury Content in Store-Bought Fish Samples
a
Fish
Origin
Hg Concentration (ppm)a
Swordfish Tuna
Singapore Indonesia
0.34 0.29
95% Confidence Interval (ppm)a
Hg per 3 oz Serving/μg
0.16 0.19
29 25
n = 9.
sample was analyzed in triplicate by three groups of students for a total of nine measurements per sample. Both varieties of fish contained mercury at a concentration above the method detection limit and in the range observed by the FDA (0−3.2 ppm for swordfish and 0−1.8 ppm for tuna3). In both types of fish, the quantity of mercury in a 3 oz serving is higher than the EPA suggested daily intake of 20 μg for a 150 lb adult.
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SUMMARY AND CONCLUSIONS This experiment has been used in an environmental chemistry course, allowing the students to gain hands-on experience with the cold-vapor technique for analysis of mercury. At the same time, they learned about the widespread problem of mercury contamination in seafood and biomagnification of mercury in species higher in the food chain. The use of store-bought fish samples piqued student interest in this analysis.
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ASSOCIATED CONTENT
S Supporting Information *
Student instructions, instructor notes, and instructions for assembling the cold vapor apparatus are available. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the assistance of Darlene Thornton, Claudia Restrepo, Soo Hwang, and Jen Niece. Michael Mattera spent considerable time conducting preliminary tests on the cold-vapor system.
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REFERENCES
(1) Rose, J.; Hutcheson, M. S.; West, C. R.; Pancorbo, O.; Hulme, K.; Cooperman, A.; DeCesare, G.; Isaac, R.; Screpetis, A. Fish Mercury Distribution in Massachusetts, USA Lakes. Environ. Toxicol. Chem. 1999, 18, 1370−1379. 489
dx.doi.org/10.1021/ed300471w | J. Chem. Educ. 2013, 90, 487−489
