Introducing students to the process of scientific inquiry is a major goal of high school and college labs. Environmental toxins are of great concern and public interest. Modifications of a vertebrate developmental toxicity assay using the frog Xenopus laevis can support student-initiated toxicology experiments that are relevant to humans. Teams of students formulate hypotheses, perform experiments, analyze data, and present their results. By performing experiments to investigate the toxicity of household chemicals, pharmaceuticals, or agricultural chemicals, students will gain an appreciation of the environmental effects of improper disposal of common chemicals and industrial or agricultural run-off.

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. (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.

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 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.

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

NaHCO3 0.96 g

KCl 0.30 g

CaCl2 0.15 g

CaSO4 × 2 H2O 0.60 g

MgSO4 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.

Figure 1.

(A) Xenopus laevis with dorsal lymph sac highlighted. (B) Frogs in breeding chamber in amplexus. Embryos are seen attached to grate.

Figure 1.

(A) Xenopus laevis with dorsal lymph sac highlighted. (B) Frogs in breeding chamber in amplexus. Embryos are seen attached to grate.

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 DaysDaySexUnits
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 
Number of DaysDaySexUnits
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 

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.

Figure 2.

(A) An early-cleavage embryo with jelly coat. (B) Embryos being dejellied in 2% L-cysteine.

Figure 2.

(A) An early-cleavage embryo with jelly coat. (B) Embryos being dejellied in 2% L-cysteine.

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).

Figure 3.

(A) Xenopus embryos in Petri dish, immediately after dejellying. Unfertilized eggs are creamy white and bloating (dorsal view). (B) Representative bad eggs/embryos after sorting (dorsal view). (C) Embryos showing yolk leakage (dorsal view). (D) Good embryo (side view).

Figure 3.

(A) Xenopus embryos in Petri dish, immediately after dejellying. Unfertilized eggs are creamy white and bloating (dorsal view). (B) Representative bad eggs/embryos after sorting (dorsal view). (C) Embryos showing yolk leakage (dorsal view). (D) Good embryo (side view).

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°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).

Figure 4.

(A) Normal embryos from 2 cells (left) to late blastula stage (right). (B) Normal embryos/larvae from top to bottom at 24, 48, 72, and 96 hours. (C) Detailed view of normal 96-hour larvae.

Figure 4.

(A) Normal embryos from 2 cells (left) to late blastula stage (right). (B) Normal embryos/larvae from top to bottom at 24, 48, 72, and 96 hours. (C) Detailed view of normal 96-hour larvae.

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.

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.

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 hr48 hr72 hr96 hrTotal DeadPercent Dead (Total Dead/Total Exposed)
Sample       
Control A 4/20 = 20% 
Control B 2/20 = 10% 
Control C 0/20 = 0% 
Mean      6/60 = 10% 
       
EC 1A       
EC 1B       
EC 1C       
Mean       
       
EC 2A       
EC 2B       
EC 2C       
Mean       
       
Frog Embryo Mortality: Number of Dead
24 hr48 hr72 hr96 hrTotal DeadPercent Dead (Total Dead/Total Exposed)
Sample       
Control A 4/20 = 20% 
Control B 2/20 = 10% 
Control C 0/20 = 0% 
Mean      6/60 = 10% 
       
EC 1A       
EC 1B       
EC 1C       
Mean       
       
EC 2A       
EC 2B       
EC 2C       
Mean       
       
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 MalformationsPercent Malformed
Malformation typeFacialEyeGutEdemasTail/Body BendHeartStuntedSevereTotal Malformed/Total Surviving
Control A 3/16* = 18.8% 
Control B 1/18* = 5.6% 
Control C 2/20* = 10.0 % 
Mean         6/54 = 11.1% 
          
EC 1A          
EC 1B          
EC 1C          
Mean          
          
EC 2A          
EC 2B          
EC 2C          
Mean          
          
          
Frog Embryo MalformationsPercent Malformed
Malformation typeFacialEyeGutEdemasTail/Body BendHeartStuntedSevereTotal Malformed/Total Surviving
Control A 3/16* = 18.8% 
Control B 1/18* = 5.6% 
Control C 2/20* = 10.0 % 
Mean         6/54 = 11.1% 
          
EC 1A          
EC 1B          
EC 1C          
Mean          
          
EC 2A          
EC 2B          
EC 2C          
Mean          
          
          

*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
ControlControlControl
Length (cm)ABCEC 1AEC 1BEC 1CEC 2AEC 2BEC 2C
0.96 0.75 0.8       
0.54 0.92 0.83       
1.1 1.09 1.01       
0.75 1.06 1.03       
0.97 0.84       
0.69 0.89 1.05       
1.05 0.66 1.09       
1.06 1.02 0.94       
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
ControlControlControl
Length (cm)ABCEC 1AEC 1BEC 1CEC 2AEC 2BEC 2C
0.96 0.75 0.8       
0.54 0.92 0.83       
1.1 1.09 1.01       
0.75 1.06 1.03       
0.97 0.84       
0.69 0.89 1.05       
1.05 0.66 1.09       
1.06 1.02 0.94       
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       

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).

Figure 5.

(A) Normal 96-hour larvae. (B) Facial abnormalities with edema, heart, and gut abnormalities. (C) Eye abnormalities showing eye pigmentation abnormalities. (D) Abnormal gut with edema. (E) Severe edema with gut abnormalities and axial malformations. (F) Severe edema and gut abnormalities. (G) Axial abnormalities with two tails. (H) Multiple severe abnormalities.

Figure 5.

(A) Normal 96-hour larvae. (B) Facial abnormalities with edema, heart, and gut abnormalities. (C) Eye abnormalities showing eye pigmentation abnormalities. (D) Abnormal gut with edema. (E) Severe edema with gut abnormalities and axial malformations. (F) Severe edema and gut abnormalities. (G) Axial abnormalities with two tails. (H) Multiple severe abnormalities.

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 http://www.epa.gov/eerd/stat2.htm.

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.

Figure 6.

Flow chart explaining hypothesis formation and testing.

Figure 6.

Flow chart explaining hypothesis formation and testing.

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. (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.

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.

References

References
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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 (Xenopus laevis) exposed to selective serotonin reuptake inhibitors, fluoxetine and sertraline, throughout metamorphosis. Environmental Toxicology and Chemistry, 28, 2671–2676.
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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.
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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.
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Rayburn, J.R. & Friedman, M. (2010). L-cysteine, N-acetyl-L-cysteine and glutathione protect Xenopus laevis embryos against acrylamide-indcued malformations and mortality in the frog embryo teratogenesis assay. Journal of Agricultural and Food Chemistry, 58, 11172–11178.
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