In the Laboratory
Undergraduate Introductory Quantitative Chemistry Laboratory Course: Interdisciplinary Group Projects in Phytoremediation
W
Debra L. Van Engelen* Department of Chemistry, University of Redlands, Redlands, CA 92373; *
[email protected] Steven W. Suljak Department of Chemistry, Santa Clara University, Santa Clara, CA 95053 J. Patrick Hall and Bert E. Holmes Department of Chemistry, University of North Carolina at Asheville, Asheville, NC 28804
A semester-long quantitative laboratory course taught concurrently with the second-semester general chemistry course allows students to devise mini-research projects around the general area of phytoremediation. Within the context of this course, students learn to plan a project with a specific hypothesis while designing experiments to test and control variables in a complex system. While there are many courses that use sampling and analysis methods to survey pollution in the environment, this course specifically is intended to model a research experience where students actually design experiments to test a definite hypothesis. In addition to teaching beginning students particular laboratory techniques, analytical methods, and principles of environmental chemistry, the course is also designed to purposely facilitate the type of critical thinking, group process, creativity, and methods of scientific discovery needed for later successful undergraduate research experiences in any discipline or project. Perfecting a method of investigation is of limited use until a person applies it to unresolved questions. By working to solve open-ended problems that are of interest to them and are interdisciplinary in nature, students learn to think critically, becoming better problem solvers, and the learning process is more exciting and relevant (1–7). Early exposure to interdisciplinary problem solving encourages students to enthusiastically remain in science and actively seek undergraduate research experiences. A number of research-active undergraduate institutions have developed courses, which are interdisciplinary in nature, that address open-ended questions related to an area of current research focus in science (8–12).
Overview of the Course and Experimental Procedures
Background The study of plant uptake of metals is an area of considerable research interest and publication in current scientific literature. Certain plants known as hyperaccumulators have adapted biological mechanisms to tolerate and uptake metal cations, accumulating from 1% to 25% of their dry mass as metal ions. The plant species are usually found in limited areas and frequently develop a resistance to certain toxic metals
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only. A number of review articles and books describe phytoremediation, or the process of using plants to remediate pollution (13–17). Phytoremediation may be a viable pollution remediation option that involves growing plants that hyperaccumulate target substances from contaminated soil or water sites to remove or neutralize the pollutants. While there has been much recent interest in phytoremediation, the identification of plants that might be useful, the mechanism(s) of plant uptake, and the effects of soil composition and chemistry are very complex and still largely being discovered. Contamination of soils with heavy metals is of local concern in many communities and, therefore, research in remediation of soils by plants can be linked to environmental concerns that are relevant to the local region. Students will typically find these projects important and motivating wherever the laboratory course is offered. For example, in western North Carolina where the course described herein was first offered, there is a fairly high level of mining and effluent discharge from industry, exposed acidic ores in some locations, and areas with serious acidic deposition problems. These factors lead to a number of local sites that are contaminated by metal cations. Other sources of contaminated soils exist in virtually every community, for instance, near old houses with lead-based paint or near roadways contaminated by vehicles burning leaded fuels. In general, it is not difficult to find local areas with metal-contaminated soils in need of remediation that will be useful and interesting for students to study in their projects.
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The majority of students take the course during their second or third academic year although prepared students may take the course during the second semester of their first year. The course meets once per week for four hours although the laboratory is open at other times so that students may care for plants. Each section is limited to fifteen students with an advanced chemistry major as a teaching assistant in addition to the instructor.
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In the Laboratory Table 1. General Schedule for the Course Week
Individual Activities
Group Activities
1
Safety review, lab notebooks; use of volumetric pipets, flasks, and burets; making solutions and dilutions
Description of phytoremediation and group project format
2
Continue individual technique practice
Assignment of groups; literature searches and citing references; groups meet outside class for planning and reviewing literature
3
Excel spreadsheets for data analysis; pH measurement of water and soils; vacuum filtration
Preliminary group project description due; collect samples, assign duties for group members, plant seeds (if planting)
4
H2O2/HNO3 digestion of soil samples; periodic notebook checks
Sample collection, identification of plants, planting seeds, care of plants
Practice sample preparation techniques; experiment: Fe in Soil by AAS; experiment: Potentiometric Titration of carbonates; experiment: Cation Exchange Capacity of Soils with Ammonia Electrode; write two formal laboratory reports
Prepare digested soil and water run-off samples; analyze run-off samples by IC; prepare standard solutions for AAS or IC
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Harvest plants, digest, and prepare all project samples and appropriate standards for analysis; Use AAS or IC to analyze project samples; interpret and present data, draw conclusions; formal group project report and presentation to class
5–10
11–15
The general outline of the experiments and schedule for the course are shown in Table 1. The course includes topics which are rarely taught in a beginning chemistry laboratory course: • Soils • Natural waters • Plant uptake mechanisms • Pollution sources
In addition to the “mini-research experience”, this course is planned to develop proficiency in many laboratory methods and techniques. A structured series of short experiments is completed by students so that they become more proficient with general chemical analysis methods while concurrently working on the semester group project. These techniquebuilding experiments are taught in a traditional format but are adapted to this particular environmental context. For instance, there are experiments that teach students to measure the pH of soils, titrate carbonate potentiometrically (as is found in limestone rock), and determine the cation-exchange capacity of soils using an ammonia-sensing electrode. Ion chromatography (IC) is used to study the extractable anions that are normally present in surface drainage water. Under very controlled conditions (see Hazards below), students follow a standard procedure to digest either soil or plants with concentrated nitric acid and hydrogen peroxide. They determine the naturally occurring iron content in their soil sample by atomic absorption spectrometry (AAS). The preliminary material and experiments are described in detail in the Supplemental Material.W While completing short experiments to learn laboratory techniques, project groups are formed and the semester-long
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projects are designed in consultation with the instructor. The project experiments are initiated within the first few weeks of the course. The students are allowed flexibility in selecting the area and question that they would like to investigate. Some groups focus on variables of soil chemistry or natural waters such as pH, nitrate content, or cation-exchange capacity. Other students are more interested in different species of plants or conditions of plant growth such as nutrients, temperature, or light levels. Often, students decide to study one environmental pollutant, specifically. Most projects include laboratory growth container experiments to study specific heavy-metal accumulation in plants, although some groups choose to collect soil and plant samples from the field. Students learn to use the Chemical Abstracts Service SciFinder Scholar database to locate background information for their project design. Because phytoremediation is a topic of current research interest with hundreds of related articles that are published annually, students quickly realize the wide scope of research interests in this interdisciplinary field. With careful planning and frequent discussions, the students are guided by the instructor to focus their project hypothesis or question to match the scope of the course and design appropriate experiments that they will be able to complete. After a number of relevant analytical methods have been practiced by the students early in the semester, they work closely with the instructor to decide the exact analytical methods that will be used for determination of particular analytes at the completion of their projects. Most transition metals are determined by IC with post-column derivatization and detection with a photodiode array detector. The chromatograms of a 1.0 ppm standard of transitionmetal cations (A) and of a digested grass sample from a stu-
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In the Laboratory List 1. Titles of Representative Group Projects in Phytoremediation • Effects of Selected Species of the Family Brassicaceae on Lead Contaminated Soil • Uptake of Heavy Metals from Soils Surrounding the Municipal Sewer District's Sludge Disposal Lagoon • Hyperaccumulatory Effects of Datura stramioninum, Spinacia oleracea, and Lupinus texensis on Arsenic-Contaminated Soil near Pressure-Treated Lumber • EDTA-Enhancement of Cadmium Adsorption in Mustard Plants at Various Plant Life Stages • Phytoextraction of Transition Metals by Barley Grass and Soy • Effect of pH on the Uptake of Lead by Collard Greens • Hyperaccumulation of Heavy Metals in C3 Grasses • Uptake of Heavy Metals at Different Stages of Plant Growth by Indian Mustard (Brassica juncea) • Trifolium pratense L with Symbiotic Microbial Amendments for the Remediation of Lead, Copper, and Zinc in Roadside Soil
Hazards
Figure 1. Transition-metal cation chromatogram using a Dionex IonPac CS5A column with mobile phase of 8.0 mM oxalic acid, 50 mM KOH , and 100 mM tetramethylammonium hydroxide, and flow rate of 0.30 mL/min. Post-column derivatization with PAR postcolumn reagent (either 0.060 g/L or 0.12 g/L 4-(2-pyridylazo)resorcinol in PAR postcolumn diluent, 1.0 M 2-dimethylaminoethanol, 0.50 M ammonium hydroxide, and 0.30 M sodium bicarbonate) at flow rate of 0.15 mL/min. Photodiode array detection with single-wavelength chromatogram shown at 530 nm. (A) 1.0 ppm standard with retention times (in minutes): lead ion, 2.37; copper ion, 2.77; cadmium ion, 3.67, cobalt, 5.39; zinc, 7.13; nickel, 8.66. (B) Digested plant sample of grass collected near creek with industrial effluent discharge upstream with 0.018 ppm copper, 0.227 ppm cadmium, and 0.100 ppm zinc.
dent project (B) are shown in Figure 1. The titles of some of the projects completed in 2001–2002 are shown in List 1. Students have the option of also using AAS for the analysis of their project samples. In some cases, this is recommended for analysis of particularly toxic analytes, such as lead, that have a lower sensitivity on IC. Some groups may choose to use both IC and AAS for completion of their projects. At the culmination of the group project, students prepare and analyze project samples, collect data, statistically analyze the results, and communicate their findings to their peers in a seminar format. The “need-to-know” specific chemical concepts and techniques for their research project and in writing the summary report motivates students and leads them to pursue a deeper understanding of numerous chemical principles.
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Extreme care must be taken to avoid spilling the concentrated nitric acid or the 30% hydrogen peroxide during the digestion procedure as both cause severe burns. Addition of these chemicals and hotblock digestion must be done in the fume hood; toxic gas is evolved as nitrogen oxides. Use of acid-resistant gloves, aprons, and safety goggles is essential. The reaction of dried, ground leaf material with hydrogen peroxide may become vigorous and froth. Care must be taken so that no closed containers are used where a buildup of gas might occur. For beginning students, we recommend that the digestion procedure be closely supervised by an advanced teaching assistant student or the instructor who may actually perform some of the steps for the students in the class. Any spilled acid must be immediately neutralized with either NaHCO3 or a commercially available spill kit. Care must also be taken to avoid contact with samples or standards, such as lead nitrate, that contain toxic metals. Conclusion Because students have much latitude in the design of the project, the question to be studied, and the final methods of analysis, this course closely resembles a research experience for students. The laboratory course is designed to develop both individual skills and promote cooperative learning while beginning students work on projects in a specific area of environmental chemistry and analysis. The course is dynamic and open-ended but has a strong and clear structure that makes the experience cohesive and comprehensible to students. In general, student achievement has been high and the projects are of good quality. Most students readily report that they enjoyed the format and structure of the course, had a rewarding and positive educational experience, and had greater interest in continuing scientific studies as a result of this course. Surveys are conducted at the end of each semes-
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In the Laboratory
ter with both structured ranking-format and open-ended questions. While surveys are subjective evaluation tools, it is clear that most of the survey responses are positive, consistent with student comments made directly to the instructors. The overwhelming majority of responses from students indicate that they felt they learned more working as part of a group, found the projects to be interesting, and felt more confident of their ability to succeed in science after taking this laboratory course. A summary of some of the survey questions given and frequency of responses is presented in more detail in the Supplemental Material.W The responses also indicate that this type of course boosts confidence and interest in beginning undergraduate research soon after completion of the course. Acknowledgments The authors thank Kevin Moorhead of the UNCA Environmental Studies Department for his helpful suggestions. This project was supported by a National Science Foundation, Course, Curriculum and Laboratory Improvement Program (NSF-CCLI) grant, No. 9952797. W
Supplemental Material
Notes for the instructor, including the survey questions and responses, detailed instructions for the students, and background information on the techniques are available in this issue of JCE Online.
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