Introducing Environmental Toxicology in Instructional Labs: The Use of a Modified Amphibian Developmental Toxicity Assay to Support Inquiry-Based Student Projects

Students will:

1. (1) Learn the scientific method, the process of science, by formulating hypothesizes about the effects of chemicals or samples on vertebrate development.

2. (2) Design and perform experiments using the developmental toxicology assay.

3. (3) Collect data, organize it into tables and graphs, analyze it with simple statistics, and interpret the meaning of their results.

4. (4) Present their results to the class, science fairs, or other venues, learning how to communicate scientific data.

Instructors must follow all relevant institutional and government safety and disposal guidelines. Students must get the instructor’s prior permission to bring in samples so that appropriate safety measures are taken. Animal care should be performed within the institution’s regulations for animals in the classroom. Further information can be found at http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-Laboratory-animals.pdf (National Research Council, 2011).

Because frog embryos are aquatic, high-ionic-strength solutions may adversely affect development. Insoluble chemicals require carrier solvents that must be included in the controls. If highly acidic or basic chemicals are used, pH must be adjusted to 6.5–8.5 daily. As with any animal breeding studies, there can be variation in the number and quality of embryos.

Male and female X. laevis frogs (Xenopus One, Dexter, MI) are kept in separate aquaria (5 frogs per 10 gallons) to prevent spontaneous mating. The aquaria are kept at 22 ± 3°C with a 12:12 light:dark photoperiod and are best equipped with aerators and filters. Frogs are fed daily high-protein fishmeal pellets (Purina Aquamax Fish Pellets, ¼ inch). These standardized conditions (Bantle & Sabourin, 1991) for frog rearing ensure consistent and reproducible results for the assay.

The FETAX solution below (pH 7.6–7.9) optimally supports embryonic growth and should be used for controls and all dilutions of toxicants (Dawson & Bantle, 1987).

Deionized/distilled water 10 L

NaCl 6.25 g

NaHCO₃ 0.96 g

KCl 0.30 g

CaCl₂ 0.15 g

CaSO₄ × 2 H₂O 0.60 g

MgSO₄ 0.75 g

Instructors induce breeding and amplexus in Xenopus by subcutaneous injections of human chorionic gonadotropin (HCG; Sigma CG-5 dissolved in sterile saline at 1000 IU/mL) into the dorsal lymph sac (Figure 1A) using a 1-mL tuberculin syringe using either a 1-day or 2-day procedure (Table 1). The 2-day procedure gives higher breeding success at certain times of the year.

Use a covered false-bottom breeding chamber (e.g., plastic sifting cat litter boxes) that allows the eggs to fall away from the frogs (Figure 1B) (McCallum & Rayburn, 2006). Fill the bottom chamber 3–4 inches deep with FETAX solution with aeration. Keep the breeding chamber in a dark, quiet location overnight. Generally, fertilized eggs (1000–3000) are visible the next morning (Bantle et al., 1998).

Egg dejellying removes the sticky egg jelly (Figure 2A) coat to allow easy sorting of eggs. Place embryos in fresh dejellying solution (100 mL, 2% L-cysteine [Sigma C-7352] in FETAX solution, pH to 8) and swirl until the jelly coat is removed (∼2 minutes; Figure 2B). Once the jelly has been removed, pour out the dejellying solution and wash the eggs 5 times with fresh FETAX solution (Bantle et al., 1998). Excess dejellying causes egg and embryo damage.

Assisting in egg sorting (1–3 hours) educates students on the procedures. Sort the eggs in a two-stage procedure by placing them in 100-mm glass Petri dishes with FETAX solution (Figure 3A). Remove unfertilized eggs (bloated and creamy white), and eggs or embryos with splotches, abnormal shape, or yolk leakage (Figure 3B, C), using plastic transfer pipettes. Perform a second sorting under a dissecting microscope using dissecting probes or pipettes to select normal blastulas and early gastrulas, which have numerous small cells, even dorsal pigmentation, and a cream-colored ventral region (Figure 3D).

Each replicate should contain 20 embryos placed (with pipettes) into 60-mm plastic Petri dishes with 8-mL test solutions (Bantle & Sabourin, 1991). Each experiment has a negative control (FETAX solution) and a positive control with a known toxicity value. We recommend acetone (LC50 = 2.28% v/v; EC50 = 1.3% v/v; minimum concentration to inhibit growth [MCIG]) = 1.4% v/v) (Rayburn et al., 1991). For statistical analysis, use 3 or 4 replicates for each treatment (including controls). For chemicals, use a “rangefinder” of 3–6 concentrations over a 100- to 1000-fold range or 3 serial dilutions of environmental samples. Keep test solutions between pH 6.5 and 8.5 or abnormal development will occur, adjusting with dilute HCl or KOH as needed. Incubate embryos at 24°C or in a room at 20–25°C. Temperatures >25°C can disrupt development.

Students daily observe, count live embryos, and remove dead embryos, slowing bacterial growth (∼1 hour). Students change solutions and monitor/adjust the pH daily. Basic organogenesis is completed by the end of the assay (96 hours). Instructors can also demonstrate embryonic development (Nieuwkoop & Faber, 1975).

Figure 4A shows embryos from cleavage to blastula. The 24-hour embryos (Figure 4B) respond to stimuli. By 48 hours, embryos swim if disturbed. The 72-hour embryos actively swim (Figure 4B). At 96 hours, tadpoles are free-living (Figure 4C).

Although the standard FETAX assay runs for 96 hours, the larvae or tadpoles can be transferred to larger containers to grow through metamorphosis. Feed tadpoles (10 µL/tadpole of a 10% slurry of strained baby peas) daily after day 4.

The embryos are anesthetized with a few drops of 20 mg/mL MS–222 (Sigma A – 5040) per dish. The embryos are examined under a dissecting microscope, scored for malformations, photographed (with ruler underneath), and their lengths measured using image analysis software (∼1–4 hours). Alternatively, using a piece of string (calibrated using the image of the ruler), students measure the head to tip-of-tail length, using a printed image. At the end of the analysis, the embryos are euthanized.

Sample data sheets (modified from Bantle et al., 1998) are illustrated in Tables 2, 3, and 4; controls and two experimental conditions (EC) are shown for brevity. Instructors can make data sheets for classroom use with spreadsheet programs. Controls are filled out as an example.

Figure 5A shows a normal larva. Common malformations include distorted facial structure (Figure 5B), eye abnormalities (missing eyes, differential eye sizes, or rifts in the eyes; Figure 5C), abnormal gut coiling or shape (Figure 5D), fluid-filled swellings (edemas; Figure 5E, F), or axial malformations (sharp tail or body bends; Figure 5G). Embryos with multiple malformations, often accompanied by severe axial malformations and edemas, are classed as “severe” (Figure 5H).

Analysis can be performed either by hand (Steel & Torrie, 1980) or by using spreadsheet programs, teaching students how to enter scientific data (Kamin, 2010). Students calculate means and standard deviations of percent dead, percent malformed, and embryo lengths of the replicates for each condition (Sokal & Rohlf, 2012). T-tests can determine whether two groups are significantly different from one another, demonstrating the importance of variation in experimental data (Thompson et al., 2011). Students plot graphs showing linear regressions, which allows them to demonstrate a concentration response. See the EPA website at http://www.epa.gov/eerd/stat2.htm.

Instructors should explain basic amphibian development, sensitivity of developing embryos, the relevance of vertebrate models to humans, and the use of basic statistics to analyze data and draw conclusions. Instructors must also explain and enforce lab safety procedures prior to starting experiments. Student teams of 4 or more, with instructor assistance, form hypotheses/questions/predictions and design experiments (Figure 6). Each experiment requires 180–400 embryos, depending on design.

Some ideas for possible student projects and sample hypotheses are described below.

• Household chemicals: Students can investigate the toxicity of common household chemicals (e.g., detergents or cleaning agents) to determine which are more toxic. These experiments promote understanding of the environmental effects of improper chemical disposal. Example: Are phosphate-free laundry detergents more or less toxic than phosphate-containing detergents?

• Food additives: Food additives such as preservatives, flavoring ingredients, artificial colors, anticaking agents, and emulsifiers can be investigated (Rayburn & Friedman, 2010). Example: Is sucralose more toxic than sucrose or aspartame?

• Garden chemicals and fertilizers: Environmental toxicity from agricultural chemicals is well established (Hecnar, 1995). Students can investigate fertilizers, plant foods, herbicides, and pesticides. Example: Are glyphosate-based herbicides less toxic than diquat-based herbicides?

• Pharmaceuticals: Pharmaceutical contamination of watersheds is an increasing problem due to the wide use of pharmaceuticals (Conners et al., 2009). Students can investigate the effects of common over-the-counter medicines, singly (Wolfe & Rayburn, 2001) or in combination (Moser & Rayburn, 2007). Example: Are naproxen-containing analgesics more or less toxic than aspirin?

• Petroleum products: In many recreational watersheds, contamination by oil and gasoline is a serious problem. Students can investigate oil, gasoline, additives, or mixtures (Hatch & Burton, 1998). Example: Is used motor oil more toxic than fresh oil?

• Environmental samples: Students can test local waters, especially near sites of pollution, to determine whether these waters are safe for frog embryo development. Be sure to get permission if collecting from private property.

• Metals: Metal ions from mining and manufacturing are a major watershed contaminant. Students can investigate common metal pollutants, including iron, copper, and aluminum. Warning: Nickel, chromium, lead, and mercury are very toxic (Rayburn et al., 1991).

• Plant extracts: Many plants contain toxins in their leaves, stems, and roots that discourage predation. Extract plants in FETAX solution and test serial dilutions (Friedman et al., 1991). Example: Are tomato leaves more toxic than maple leaves?

1. (1) Evaluation of questions and hypothesizes about environmental effects of chemicals.

2. (2) Evaluation of the experimental design and completion of the experiment. Students should demonstrate knowledge of embryonic development.

3. (3) Evaluation of data and laboratory notebooks containing the scoring of embryos for mortality, malformations, and length. Students should demonstrate skills in generating tables and figures to communicate relationships. Drawing of conclusions by the use of statistical analysis demonstrates student understanding of scientific processes.

4. (4) Evaluation of presentations of the students’ research to the class or to science fairs. A mini-symposium, where students present their data and conclusions, simulates the process of scientific data presentation. Extramural student presentations generate excitement and pride, as well as recognition for the school itself, and can stimulate further investigations and collaborations.

Assessment of goals 3 and 4 will need to be flexible because experiments may not work (embryos may all die or all live), and student evaluation of the failure of the experiment could be substituted for the original hypotheses.

A simplified version of a widely used environmental toxicity assay adapted to classroom use can support student-initiated, guided mini-research projects that allow student teams to investigate potential environmental toxins and samples. Students, with guidance, formulate hypotheses, design and perform experiments to investigate their hypotheses, statistically analyze their data, and present their results. The opportunity for students to perform research projects can generate student interest and experience in the scientific process and appreciation for the environmental effects of industry, agriculture, and improper chemical disposal.