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Introducing environmental toxicology in instructional labs: the use of a modified amphibian developmental toxicity assay to support inquiry-based student projects.

A current focus of instructional biology labs is to transition from traditional labs to inquiry-based formats that model the scientific process in which students formulate hypotheses, perform experiments, and analyze data. Environmental toxins and contamination generate concern and media coverage. Using a vertebrate developmental bioassay, students perform experiments investigating the environmental effects of toxins and contamination, encouraging interest in science. Experiments involving household chemicals such as cleaning agents, fertilizers, and pesticides give appreciation for their proper disposal.

The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) assay is a widely used, validated vertebrate toxicity assay that uses the South African clawed frog Xenopus laevis (Bantle & Sabourin, 1991). Late-blastula embryos are incubated in control or test solutions under standardized conditions for 96 hours; then the 1-cm tadpoles are assessed for growth, malformations, and mortality (Nieuwkoop & Faber, 1975). Because fundamental developmental mechanisms are conserved, this model illustrates vertebrate embryonic development and is more relevant to humans than invertebrate models. Unlike most amphibians, Xenopus embryos are transparent, allowing easy observation of internal organs. The assay can be simplified to meet institutional constraints, provide valuable experiences in "hands on" science, and promote understanding of human environmental impacts. This assay is well suited for introductory college courses, AP Biology high school labs, and science fair projects.

* Learning Goals

Students will:

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

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

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

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

* Materials & Methods

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 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 [+ or -] 3[degrees]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,1/4 inch). These standardized conditions (Bantle & Sabourin, 1991) for frog rearing ensure consistent and reproducible results for the assay.

* FETAX Solution

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
NaHC[O.sub.3]                  0.96 g
KCl                            0.30 g
Ca[Cl.sub.2]                   0.15 g
CaS[O.sub.4] x 2 [H.sub.2]O    0.60 g
MgS[O.sub.4]                   0.75 g

* Xenopus Breeding

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 & Sorting

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).

* Experimental Procedures

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[degrees]C or in a room at 20-25[degrees]C. Temperatures >25[degrees]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 [micro]L/ tadpole of a 10% slurry of strained baby peas) daily after day 4.

* Endpoint Analysis

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.

* Examples of Malformations

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).

* Statistical Analysis

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

* Preparation

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?

* Student Assessment

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

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

(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) 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.

* Conclusion

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.

DOI: 10.1525/abt.2012.74.7.12


Bantle, J.A., Dumont, J.N., Finch, R.A., Linder, G. & Fort, D.J. (1998). Atlas of Abnormalities: A Guide for the Performance of FETAX, 2nd Ed. Stillwater, OK: Oklahoma State University Press.

Bantle, J.A. & Sabourin, T.D. (1991). Standard guide for conducting the frog embryo teratogenesis assay-Xenopus (FETAX). American Society for Testing and Materials E1439-91.7p0

Conners, D.E., Rogers, E.D., Armbrust, K.L., Kwon, J.-W. & Black, M.C. (2009). Growth and development of tadpoles (Xenopuslaevis) exposed to selective serotonin reuptake inhibitors, fluoxetine and sertraline, throughout metamorphosis. Environmental Toxicology and Chemistry, 28, 2671-2676.

Dawson, D.A. & Bantle, J.A. (1987). Development of a reconstituted water medium and preliminary validation of the frog embyro teratogenesis assay-Xenopus (FETAX). Journal of Applied Toxicology, 7, 237-244.

Friedman, M., Rayburn, J.R. & Bantle, J.A. (1991). Developmental toxicology of potato alkaloids in the frog embyro teratogenesis assay-Xenopus (FETAX). Food and Chemical Toxicology, 29, 537-547.

Hatch, A.C. & Burton, G.A., Jr. (1998). Effects of photoinduced toxicity of fluoranthene on amphibian embryos and larvae. Environmental Toxicology and Chemistry, 17, 1777-1785.

Hecnar, S.J. (1995). Acute and chronic toxicity of ammonium nitrate fertilizer to amphibians from southern Ontario. Environmental Toxicology and Chemistry, 14, 2131-2137.

Kamin, L.F. (2010). Using a five-step procedure for inferential statistical analyses. American Biology Teacher, 72, 186-188.

McCallum, M.L. & Rayburn, J. (2006). A simple method for housing Xenopus during oviposition and obtaining eggs for use in FETAX. Herpetological Review, 37, 331-332.

Moser, B. & Rayburn, J. (2007). Evaluation of developmental toxicity of interaction between caffeine and pseudoephedrine using frog embryo teratogenesis assay-Xenopus (FETAX). Bios, 78, 1-9.

National Research Council. (2011). Guide for the Care and Use of Laboratory Animals, 8th Ed. Washington, D.C.: National Academies Press.

Nieuwkoop, P.D. & Faber, J. (1975). Normal Table of Xenopus laevis (Daudin), 2nd Ed. Amsterdam, The Netherlands: North-Holland.

Rayburn, J.R., DeYoung, D.J., Bantle, J.A., Fort, D.J. & McNew, R. (1991). Altered developmental toxicity caused by three carrier solvents. Journal of Applied Toxicology, 11, 253-260.

Rayburn, J.R., Fort, D.J., McNew, R. & Bantle, J.A. (1991). Synergism and antagonism induced by three carrier solvents with t-retinoic acid and 6-aminonicotinamide using FETAX. Bulletin of Environmental Contamination and Toxicology, 46, 625-632.

Rayburn, J.R. & Friedman, M. (2010). L-cysteine, N-acetyl-L-cysteine and glutathione protect Xenopus laevis embryos against acrylamide-induced malformations and mortality in the frog embryo teratogenesis assay. Journal of Agricultural and Food Chemistry, 58, 11172-11178.

Sokal, R.R. & Rohlf, F.J. (2012). Biometry, 4th Ed. New York, NY: W.H. Freeman. Steel, R.G.D. & Torrie, J.H. (1980). Principles and Procedures of Statistics: A Biometrical Approach, 2nd Ed. New York, NY: McGraw-Hill.

Thompson, E.D., Bowling, B.V., Whitson, M. & Naczi, R.F.C. (2011). Engaging students in natural variation in the introductory biology laboratory via a statistics-based inquiry approach. American Biology Teacher, 73, 100-104.

Wolfe, A. & Rayburn, J. (2001). The developmental toxicity of meclizine using the frog embryo teratogenesis assay-Xenopus (FETAX). Alabama Academy of Science, 72, 27-38.

ROGER SAUTERER and JAMES R. RAYBURN are Professors of Biology at Jacksonville State University, 700 Pelham Road North, Jacksonville, AL 36265. E-mail:

Table 1. Protocol for 1-day and 2-day subcutaneous
injections of human chorionic gonadotropin into
the dorsal lymph sac of Xenopus using a 1-mL
tuberculin syringe.

Number of
Days         Day      Sex       Units

1 Day                 Male      200-500 IU
                      Female    300-800 IU

2 Days       Day 1    Male      150 IU
             Day 1    Female    150 IU
             Day 2    Male      250 IU
             Day 2    Female    350 IU

Table 2. Sample mortality data sheet. Instructions: Score the number
of dead embryos in each dish daily in the appropriate column. Because
dead embryos quickly disintegrate, calculate the number dead by:
[Daily Dead Embryos = Original embryo number - (previous dead + current
surviving embryos)]. Score the total dead at the 96-hour endpoint and
calculate the percentage dead.

                  Frog Embryo Mortality:
                     Number of Dead

             24 hr   48 hr   72 hr   96 hr   Total      Percent Dead
                                              Dead      (Total Dead/
                                                       Total Exposed)

Control A      1       0       1       2       4         4/20 = 20%
Control B      0       0       1       1       2         2/20 = 10%
Control C      0       0       0       0       0         0/20 = 0%
Mean                                                     6/60 = 10%



Table 3. Sample malformations data sheet. Instructions: At the 96-
hour endpoint, each individual malformation is scored in the
appropriate category, even if a single embryo has more than one
malformation. Enter the total percent and number of malformed embryos
in the "% malformed" column. Embryos with multiple malformations are
scored as one malformed embryo here.

               Frog Embryo Malformations

Malformation                                 Tail/Body
type           Facial   Eye   Gut   Edemas     Bend

Control A        1       0     2      3          0
Control B        1       1     1      1          1
Control C        0       0     2      0          0



               Frog Embryo Malformations   Percent Malformed

Malformation                               Total Malformed/
type           Heart    Stunted   Severe    Total Surviving

Control A        0         1        1      3/16 * = 18.8%
Control B        1         1        0      1/18 * = 5.6%
Control C        2         0        0      2/20 * = 10.0%
Mean                                       6/54 = 11.1%



* Number of living embryos (see mortality table).

Table 4. Sample embryo-length data sheet. Instructions: Record length
of each surviving embryo in the appropriate column, then calculate
mean, variance, and standard deviation (SD) for each column.

            Frog Embryo Length Data

            Control   Control   Control

Length         A         B         C      EC 1A   EC 1B

1            0.96      0.75       0.8
2            0.54      0.92      0.83
3             1.1      1.09      1.01
4            0.75      1.06      1.03
5            0.97        1       0.84
6            0.69      0.89      1.05
7            1.05      0.66      1.09
8            1.06      1.02      0.94
9            0.52       1.1      0.72
10           0.68       0.8      0.77
11           0.52      0.85      0.69
12           0.71      0.55      0.63
13           0.67      0.61      1.02
14           0.74      0.58      0.92
15           1.06      0.67      1.05
16           1.01      0.82      1.08
17                     0.59      0.83
18                      1.1      0.56
19                               0.97
20                               0.79

Mean         0.814     0.837     0.881
Variance     0.044     0.038     0.025
SD           0.211     0.196     0.158

            Frog Embryo Length Data

Length      EC 1C   EC 2A   EC 2B   EC 2C


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Author:Sauterer, Roger; Rayburn, James R.
Publication:The American Biology Teacher
Article Type:Report
Geographic Code:1USA
Date:Sep 1, 2012
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