Article pubs.acs.org/jchemeduc
Project-Based Learning in Undergraduate Environmental Chemistry Laboratory: Using EPA Methods To Guide Student Method Development for Pesticide Quantitation Eric J. Davis,*,† Steve Pauls,‡ and Jonathan Dick‡ †
Department of Biology and Chemistry, Azusa Pacific University, 901 East Alosta Avenue, Azusa, California 91702, United States Department of Chemistry, Fresno Pacific University, 1717 South Chestnut Avenue, Fresno, California 93702, United States
‡
S Supporting Information *
ABSTRACT: Presented is a project-based learning (PBL) laboratory approach for an upper-division environmental chemistry or quantitative analysis course. In this work, a combined laboratory class of 11 environmental chemistry students developed a method based on published EPA methods for the extraction of dichlorodiphenyltrichloroethane (DDT) and its environmental degradation products (dichlorodiphenyldichloroethane [DDD] and dichlorodiphenyldichloroethylene [DDE]) through Soxhlet extraction and quantitation using gas chromatography−mass spectrometry (GC−MS). From the derived procedures, the students spent the remainder of the semester performing extractions and analyses to detect pesticide drift from the San Joaquin Valley of California into the Sierra Nevada Mountain Range. The EPA methods used allow for a multitude of research-level questions which could be answered within a similar course with a specific question appropriate to student interest and regional environmental issues. This pedagogical approach provided numerous teaching moments regarding the pitfalls of research and low-level quantitation in environmental samples, as well as development opportunities for student teamwork skills as each individual focused on a specific aspect of the project throughout the semester. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Environmental Chemistry, Problem Solving/Decision Making, Inquiry-Based/Discovery Learning, Quantitative Analysis, Chromatography, Laboratory Instruction
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DDT Analysis in the Environment
INTRODUCTION
Dichlorodiphenyltrichloroethane (DDT) is a persistent, synthetic, organochlorine compound used extensively from the 1940s into the 1970s as a broad-spectrum pesticide.13−19 Its persistent nature made it an ideal long-term pesticide, especially for the control of malaria-carrying mosquitos. However, several studies in the 1960s culminated in the publication of Silent Spring by Rachel Carson, in which the environmental impacts of DDT were detailed, especially the effect on the eggshells of nesting birds due to environmental and biological accumulation of DDT. This, and other publications, resulted in a ban on the use of DDT in the United States in 1972. However, the extensive use of DDT and its highly persistent nature20 led to its continued detection in the environment to this day. Significant research has been performed indicating the continued existence of DDT and its environmental degradation products dichlorodiphenyldichloroethane (DDD) and dichlorodiphenyldichloroethylene (DDE).9,11,14−18,20−23 In these studies, substantial concentrations can be found in most matrices tested, including water, soil, plant, animal, and even human samples. Modern instrumental techniques allow the detection of DDT and its analogues down to parts per billion (ppb) or parts per trillion (ppt) levels using gas chromatography with electron capture detection (GC-ECD).9,11,15 GC− MS has also be utilized in DDT detection. While this technique
Project-Based Learning
Project-Based Learning (PBL) in undergraduate chemistry typically involves the assignment of a short-term project wherein students are given the tools needed to solve a problem and are allowed to solve it using their intuition and applied course skills.1−3 PBL projects have measured key components in groundwater and simulated hazardous waste,3,4 and include a multitude of general or analytical chemistry projects.1,2,5−8 These types of projects have the capacity to alter a student’s perception of science from canned, “formulaic”-based laboratories to a more realistic understanding of research or industry-standard analyses. The prevalence and availability of standard methods produced by the Environmental Protection Agency (EPA) provide a wide range of topics for PBL laboratories. The work described herein utilized several EPA methods9−12 for the determination of DDT and its analogues in environmental samples and is intended to be performed within a single semester by an 8−24 student laboratory section. This approach may be modified for less time consumed during a semester or for smaller class sizes through the selection of the specific research question. The procedure described below is intended as an outline of what was achieved with a group of 11 students of mixed chemistry backgrounds in a single semester laboratory section as a primer for using this concept in similar courses. © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: May 13, 2016 Revised: December 28, 2016
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DOI: 10.1021/acs.jchemed.6b00352 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
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Figure 1. Map of approximate selected sampling locations for DDT drift analysis. San Joaquin Valley locations (A, B, and C correspond to FPU Campus, Selma, and Madera, respectively) were selected to encompass a breadth of the Valley, while Sierra Nevada Mountain locations (D, Shaver Lake; E, Dune Creek) were selected on the basis of a westerly prevailing wind direction from each Valley site. Used with permission. Copyright 2016 Google.
experiment that could be conducted within the time frame allotted for the course (1 semester), personnel (11 students), and resources (GC−MS, Soxhlet extractors, soil sampling probes, water sampling probes, and aquatic biological samples) available. The students of FPU chose to pursue DDT in soil in the SJV and nearby SNM range to elucidate possible drift of these pesticides during use. Following this discussion, each student was assigned to write a short document describing their proposed methods for analyzing DDT in soil (student assignment templates and rubrics available in Supporting Information). While the results provided herein focus on soil samples, the EPA protocols utilized also allow for the determination of DDT in biological (fish or plants) samples and numerous other matrices. In the following week, a new discussion was held to determine the exact experimental protocols to be followed as standard operating procedure (SOP). A Google Doc was displayed to the class with one student assigned as the recorder so all could see the procedure as it was determined and outlined. This document was made available to the class as a whole once completed and remained a live document for SOP modifications when problems arose. Due to time constraints, the SOP determined during this discussion was tested through application to the problem. Thus, mistakes in the procedure were common and led to several errors within the experiment. These provided numerous teaching opportunities in how to ethically deal with poor data and when it may still be utilized or when samples must be reobtained or reanalyzed. Once the protocol was established, a calendar was arranged organize students’ schedules. Due to the long extraction steps involved in the EPA procedures, several students were involved with setup while others came the next day to stop the extraction and concentrate the samples. With varied student schedules,
provides slightly higher detection limits over GC-ECD, the mass spectrometer provides qualitative confirmation of the analyte identity that is unavailable in GC-ECD-based techniques. The ubiquitous research that has been performed in the area of environmental DDT detection and quantification makes it an ideal candidate for a PBL-based laboratory. Through the EPA, methods are freely available describing best practices in the quantification of DDT and its analogues from nearly any sample available to the researcher.9−12 In addition, Fresno Pacific University’s (FPU) unique location in the heart of the San Joaquin Valley (SJV) of California lends itself toward the study of environmental trends and agricultural impacts. As this is a highly agricultural region, DDT was used heavily in the SJV prior to its ban in 1972, with much of it it delivered through crop-dusting,13 making airborne particulate drift a likely scenario. Prevailing winds travel from the SJV in a southwesterly direction into the Sierra Nevada Mountain range (SNM) (see map in Figure 1).23 Thus, it was hypothesized by the students that detectable levels of DDT contamination exist within portions of the SNM.
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PEDAGOGICAL APPROACH This work describes a PBL-based laboratory that was utilized in an upper-division environmental chemistry course with 11 students enrolled (instructor notes available in Supporting Information). On the first day of laboratory, students were provided with a series of EPA methods9−12 and a description of an experiment where these methods had been utilized.19 In the following week, the students were expected to come to class prepared with ideas for what they would like to test as a combined class. The instructor’s role in this portion of the course was to guide and focus this discussion to a simple B
DOI: 10.1021/acs.jchemed.6b00352 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Article
many of them “specialized” within one aspect of the experiment (sample preparation and extraction setup, postextraction concentration, GC−MS analysis of the resultant samples, etc.). This was encouraged in the students in order to form a team where each could utilize his/her strengths to the benefit of the group as a whole and to simulate an industrial analysis laboratory environment. However, each student was trained in every aspect of the project to ensure a broad understanding of environmental sample preparation and analysis by the students of this Environmental Chemistry course. Each student was expected to maintain an active laboratory notebook detailing their work on the project throughout the semester. Students were required to submit brief (1−2 page) outlines of the work that had been performed and data obtained twice throughout the semester to assess their progress and encourage continuous data analysis habits (assignment descriptions and rubrics available in Supporting Information). In the final weeks of the laboratory course, students were encouraged to thoroughly analyze the data as a group. Once completed, the students were expected to submit individual journal article style papers with a full literature review and all expected sections (in the format for the journal Analytical Chemistry). As a group, the students also prepared a research poster for presentation in an on-campus research event. Course assignments and workload are summarized in Table 1.
through a batch-analysis process using the software available with the GC−MS used.
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MATERIALS AND METHODS As part of the pedagogical approach to the course, the procedures outlined below were generated by the students after a review of the appropriate EPA methods and a guided discussion with the entire course covering the protocols in question as well as sampling and relevant statistics. However, due to the lack of experience on the part of the students, the GC column, injection (using an autosampler), temperature programming, MS parameters, and quantitation method were developed by the instructor (as per EPA protocols), and students were trained on the insertion of their samples to the autosampler and the initiation of the GC−MS experiment. Reagents
Anhydrous sodium sulfate (reagent grade, VWR, Radnor, PA) was used for removal of water from extraction reagents through precontact. A 1:1 (v/v) mixture of acetone and hexanes (HPLC grade, VWR, Radnor, PA) was used for extractions. (Note deviation from EPA Method 3540 which called for pesticide grade reagents. These were avoided due to cost.) HPLC grade methanol (VWR, Radnor, PA) was used for sample dilution prior to GC−MS analysis. ACS grade naphthalene and 1,4dichlorobenzene (VWR, Radnor, PA) were diluted to a concentration of 1000 ppm in the methanol for internal standard spikes. EPA 8081 pesticide standards mixture (SigmaAldrich, St. Louis, MO) provided calibration standards of known concentration.
Table 1. Outline of Laboratory Course Work and Assessment Weeksa 1
a
Activity
2 3
Introduction; lecture on sampling statistics; assignment of EPA method reading Procedure design week 1 Procedure design week 2
4−12 13−15
Data collection Data collection
Assessment Discussion of EPA procedures for DDT analysis
Safety
Business proposal including outline of proposed procedure and experiments to be performed Data summaries (2×) Formal journal article; Research poster
Acetone, methanol, hexanes, and 1,2-dichlorobenzene are flammable liquids and contact irritants. Methanol is highly toxic in large exposure. Naphthalene is a flammable solid and a contact irritant, and is toxic with ingestion. Standard laboratory personal protective equipment (gloves, goggles, and lab coat) should be maintained at all times. All extractions were performed in a fume hood using controlled heating jackets to avoid ignition sources. GC−MS analyses were performed under direct instructor supervision, and all extraction setups were inspected by the instructor prior to use (specific hazards for all reagents are provided in Supporting Information).
This lab was performed in a single, 15-week semester.
In the semester this lab was offered, the students opted to test five separate sites. Each site provided four samples brought to the laboratory, and three portions of each sample were extracted (detailed descriptions of the sampling and extraction procedures are outlined in Materials and Methods). Thus, a total of 60 extractions were performed throughout the semester. Each of the resultant extracts were analyzed 3 times via GC−MS following internal standard spiking, resulting in a total of 180 chromatograms with statistically relevant results. This was a labor-intensive process and would not have been possible with fewer students. However, the experimental goals are flexible to account for differing course sizes, and three sites could have been studied by as few as eight students, while additional sites could be studied for larger groups assuming additional equipment is available for use. It is not recommended to attempt the procedure outlined herein if an autosampler is not available due to the large number of samples analyzed. In addition, a method for batch processing of data is highly advised. In the procedure outlined below, one student specialized in the use of the GC−MS software and provided summarized data (peak times and areas) to the rest of the class
Equipment
For all experiments outlined below, an HP-6890 gas chromatograph with attached HP-5973 mass spectrometer and HP7673C autosampler was utilized for GC−MS analyses. An Agilent HP-1, 15 m × 0.250 mm column with a 1.00 μm film was used with a temperature program initially held for 2 min at 150 °C, followed by a ramp rate of 20 °C/min, and finally held at 280 °C for 1 min. An injection volume of 2.0 μL was utilized for all injections with 6 solvent (methanol) and 2 sample washings between injections. Each sample was analyzed through three consecutive GC−MS analyses. The mass spectrometer was set in SIM mode with preset m/z values of 128 (naphthalene, internal standard), 146 (1,4-dichlorobenzene, internal standard), 317 (DDE), 318 (DDD), and 353 (DDT) daltons. Soxhlet extraction followed the procedure outlined in EPA method 3540C10 with 10−15 cycles per hour, 24 h extraction period, and 300 mL of solvent. A rotary evaporator was utilized in place of a Kuderna−Danish apparatus for sample concentration due to availability (note deviation from EPA methods). C
DOI: 10.1021/acs.jchemed.6b00352 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Figure 2. Internal standard calibration curves for (A) naphthalene and (B) 1,4-dichlorobenzene internal standards vs concentration of DDT and DDT analogues DDD and DDE. 20 ppm calibration points were eliminated from the 1,4-dichlorobenzene curve due to the limit of linearity. These curves provided internal standard calibration factors for subsequent quantification. All standards were diluted from EPA 8081 certified mixtures.
Experimental Procedure
sample was placed into a Soxhlet extractor in between glasswool plugs that had been precontacted with the 50/50 (v/v) acetone/hexane extraction solution. A 300 mL portion of extraction solvent was utilized with boiling chips to avoid bumping in each extraction. Extraction was performed at a rate of 10−15 cycles per hour for 24 h.10,11 After extraction was completed, each flask was rotaryevaporated to dryness (note change from EPA method), after which the boiling chips were removed and rinsed with fresh acetone/hexanes put into the flask. A small amount (
