Engaging students in the process of science to increase learning and critical thinking has become a key emphasis in undergraduate education. Introducing environmental topics, such as the effects of endocrine-disrupting chemicals, into undergraduate courses offers a new means to increase student engagement. Daphnia magna can serve as a model organism for endocrine disruption, and its ease of handling, rapid reproduction rate, and clearly defined endpoints make it useful in short-term, student research projects. The concept of endocrine disruption can be tested through a 21-day reproductive study of D. magna exposed to varying concentrations of the pesticide fenoxycarb. Students will observe an altered reproduction rate and increased production of males under conditions that would typically result only in the production of female offspring. This research system allows students to formulate hypotheses, set up experiments, analyze data, and present results, leading to a greater appreciation of and interest in science.

Why Study Endocrine Disruption?

Endocrine-disrupting chemicals (EDCs) can inhibit or promote the expression of hormonal actions by interacting with hormone receptors, binding to the hormone itself, or altering other portions of hormone-signaling pathways. Several studies had identified the potential for endocrine disruption from naturally occurring compounds in the environment, but the idea that anthropogenic compounds could act as EDCs gained national attention from a position statement, known as the “Wingspread Statement,” produced by a group of concerned scientists (Bern et al., 1992). Since then, EDCs have been well documented and shown to affect a wide range of organisms – such as aquatic invertebrates, insects, fish, amphibians, and humans – in concentrations much lower than lethally toxic levels (Rodríguez et al., 2007). These chemicals are released into the water supply primarily by wastewater discharge and by direct deposition from recreation, agricultural runoff, and other sources. The majority of studies pertaining to potential EDCs have been focused on vertebrate models, presumably because of their higher degree of correlation with humans. However, recent understanding of crustacean endocrinology and the realization that these organisms may serve as model organisms to identify EDCs have prompted an increase in using crustacean models such as Daphnia magna (Tatarazako et al., 2003; deFur, 2004; Lampert, 2006). The potential for environmental contaminants to cause endocrine disruption frequently inspires widespread public interest (e.g., bisphenol a in water bottles), suggesting that the topic will be exciting to students.

Why Use Daphnia?

Daphnia magna is an aquatic crustacean species of critical importance to aquatic ecosystems because it serves as a primary consumer of algae, thus affecting water quality, and is a major constituent in aquatic food chains. Daphnia reproduce primarily by cyclic parthenogenesis, an asexual process whereby the females of the species produce varying numbers of female offspring under normal environmental conditions. These parthenogenic females are diploid clones of the mother. Environmental stress – such as colder temperatures, decreased photoperiods, food scarcity, and crowding – can stimulate the production of males capable of sexual reproduction (Hobaek & Larsson, 1990). Males are able to fertilize special haploid eggs to produce specialized structures called epiphia (singular epiphium). Epiphia, or “resting eggs,” are encased in a protective layer that enables them to survive in the environment, presumably to allow the species to survive in winter or drying conditions. When environmental conditions return to normal, a female emerges from the epiphium. Males and females can be distinguished by a variety of phenotypic traits. Specifically, female Daphnia tend to be larger, with a short first antenna and a pointed rostrum (Figure 1).

Figure 1.

Sex determination by length of first antenna in Daphnia magna (Tatarazako et al., 2004).

Figure 1.

Sex determination by length of first antenna in Daphnia magna (Tatarazako et al., 2004).

The onset of poor environmental conditions stimulates the release of a hormone that brings about the production of males. Since the discovery of methyl farnesoate as the hormone responsible for the production of males, it has been identified in over 30 crustacean groups, including Daphnia, although the specific receptor responsible for its binding has not been determined (LeBlanc, 2007). Methyl farnesoate is a terpenoid chemically related to juvenile hormone III (JHIII), an insect hormone (Laufer et al., 1992). Studies have identified several anthropogenic chemical analogues of methyl farnesoate – such as the insecticides kinoprene, fenoxycarb, and pyriproxyfen – that can induce the production of males (Haeba et al., 2008). Fenoxycarb in particular has demonstrated the potential to produce males in minute concentration (i.e., in the low µg/L range) (Oda et al., 2007).

Daphia magna has been adopted by several international organizations, including the Organisation for Economic Co-operation and Development (OECD), as a model organism for aquatic toxicity testing. Its high reproduction rate, inexpensiveness, ease of handling, sensitivity, and multiple clearly defined endpoints make it a good model organism. As part of this project, a Daphnia culture was maintained in a college teaching laboratory with low input of supplies or time.

Endpoints used to assess endocrine disruption have typically included reproductive rate, larval development, size, age of first reproduction, and molting abnormalities. One more recent endpoint that has gained interest in the determination of a chemical's ability to mimic methyl farnesoate is sex ratio (Tatarazako & Oda, 2007). This simple endpoint can determine methyl farnesoate agonists that mimic the natural hormonal effects, leading to increased male production under environmental conditions favorable to female production. One proposed mechanism of action is that the agonist, along with the natural hormone, binds to the cellular receptors that lead to signal transduction and to the eventual cellular effects that result in male development (Laufer & Biggers, 1992).

Goals & Objectives

EDCs have become a current and substantial environmental concern that should be included in undergraduate laboratory studies in biology, environmental science, physiology, and research courses. At the same time, there has been a call to include research in undergraduate courses to provide students authentic experiences, increase critical thinking, and promote lasting learning (AAAS, 2011). Therefore, we suggest a laboratory exercise to demonstrate and learn the concept of endocrine disruption that alleviates the majority of inherent concerns by using the invertebrate model organism D. magna. The objective of the experiment the students conduct is to determine whether endocrine disruption is occurring after exposure to a pesticide. Through the proposed activities, the goals for the instructor are to

  1. engage students in the process of science, which includes hypothesis formation, data collection, and analysis of results;

  2. expose students to endocrinology and illustrate the complex changes that can occur if the endocrine system is disrupted; and

  3. aid students in interpreting the meaning of data using simple forms of statistical analysis such as mean, standard deviation, and t-tests.

Teacher Laboratory Preparation

Chemicals & Solutions

Fenoxycarb (CAS no. 72490-01-8) powder, purchased from VWR (Sugar Land, TX), was used to produce stock solutions dissolved in acetone. Neat material was used to produce a primary stock of 1 million µg/L, followed by a secondary stock diluted to 25,000 µg/L, a concentration suitable for the production of test waters. All stock solutions were stored in the freezer to help conserve the reported concentration.

Daphnia

Daphnia, purchased from Aquatic Biosystems (Fort Collins, CO), were reared in glass-covered 1 L glass jars containing dechlorinated tap water produced by filtering tap water through a charcoal filter, followed by 24 hours of bubbling using a common fish-tank pump. Charcoal filters sold for drinking water are adequate, or bottled spring water could be used for ease. Environmental conditions favorable for the production of females included temperatures 20–24°C; pH between 6 and 9; 16:8 hour light:dark cycle; and low population density, between 40 and 50 Daphnia per jar. Cultures were fed daily with the 2 mL of the algae Selenastrum capricornutum (3.7 × 107 cells/mL) and 1 mL of yeast–trout chow (YTC) three times a week (both purchased from Aquatic BioSystems, Fort Collins, CO). Alternatively, both Daphnia and appropriate food sources such as Spirulina can be purchased individually or as a kit from common biological supply companies such as Carolina (Burlington, NC). Cultures were refreshed weekly by pouring off three-quarters of the culture and refilling to appropriate levels with fresh culture water. Cultures were reared for several weeks to ensure proper acclimation to the environmental parameters. Prior to the beginning of the experiment, several adult Daphnia were isolated in a separate jar to obtain enough neonates (<24 hours old) to initiate the experiment. Daphnia were transferred between jars using a large-bore pipette (10 mL) connected to a fast-release pump to minimize the risk of physical damage.

Student Introductory Activities

Prior to the beginning of the investigation, students were provided with the necessary background activities to increase the effectiveness of the investigation; these activities included a review of the scientific process, required readings in primary peer-reviewed literature, and research ethics. To begin the investigation, students were given an introduction to fenoxycarb and the “Daphnia magna Reproduction Test” (OECD test 211) and asked to brainstorm the background information needed to conduct an appropriate study concerning the potential for fenoxycarb to produce toxicological effects on Daphnia populations. The ensuing discussion and instructor guidance resulted in the following student research background topics: Daphnia life cycle and ecology, test parameters, previous studies of fenoxycarb, chemical nature of fenoxycarb, endocrine disruption, and Daphnia as a model organism. Small groups of students were assigned to research each component, which was followed by an informal presentation of that research to the class.

Daphnia from reproducing jars were used to practice transferring Daphnia via pipette to Petri dishes for observation of the length of the female's first antenna using a stereomicroscope. A second period of sex determination was conducted on ethanol-euthanized Daphnia that were previously exposed to fenoxycarb, producing a mix of males and females to observe.

Student Laboratory Methods

Initial Setup

Experiments were adapted from the OECD (2004). The secondary stock was used to produce test waters in 1 L volumetric flasks in concentrations of 2 µg/L, 1 µg/L, 0.5 µg/L, and 0.25 µg/L fenoxycarb (Table 1). Modifications of transfer amounts may need to be adjusted according to primary stock concentrations. Previous studies have shown these concentrations to produce a range of effects on reproductive rates, day of first reproduction, and male:female sex ratios in D. magna (Oda et al., 2007; S. Layton, unpublished data). For each concentration, 100 mL (±5 mL) of test solution was dispensed into 10 separate glass containers, followed by 0.2 mL of S. capricornutum (3.7 × 107 cells/mL) and 0.1 mL of YTC.

Table 1.
Instructions for producing fenoxycarb test-water concentrations.
Secondary Stock Concentration (c1)Transfer Volume (v1)Final Volume (v2)Final Water Concentration (c2)
25,000 µg/L 80 µL 1L (1,000,000 µL) 2 µg/L 
25,000 µg/L 40 µL 1L (1,000,000 µL) 1 µg/L 
25,000 µg/L 20 µL 1L (1,000,000 µL) 0.5 µg/L 
25,000 µg/L 10 µL 1L (1,000,000 µL) 0.25 µg/L 
Secondary Stock Concentration (c1)Transfer Volume (v1)Final Volume (v2)Final Water Concentration (c2)
25,000 µg/L 80 µL 1L (1,000,000 µL) 2 µg/L 
25,000 µg/L 40 µL 1L (1,000,000 µL) 1 µg/L 
25,000 µg/L 20 µL 1L (1,000,000 µL) 0.5 µg/L 
25,000 µg/L 10 µL 1L (1,000,000 µL) 0.25 µg/L 

One D. magna (<24 hours old) was transferred to each container via pipetting. An acetone solvent control was set up under the same conditions; afterward, all jars were covered with glass.

Data Collection & Experimental Maintenance

Daily observations of mortality, irregularities in behavior, and number of offspring were documented on record sheets modified from the OECD (Table 2). Each test container was fed S. capricornutum five times a week and YTC three times a week, corresponding to water changes when appropriate. Water changes are meant to maintain the target concentration of fenoxycarb at reported levels and should be conducted at least two or three times a week. For quality control, the parameters of dissolved oxygen concentrations (≥3 mg/L), pH (6–9), and temperature (20–24°C) were checked weekly in one test jar just before and one jar just after a water change.

Table 2.
Daphnia reproduction and parent mortality data sheet.
Experiment Number:_____ Date Started:__________ Medium:__________
Type of Food:__________ Test Chemical:________________ Concentration:__________ 
Day 10 11 12 13 14 15 16 17 18 19 20 21    
Medium renewal (check)                          
Food provided (check)                          
pH*                       New   
                      Old  
O2 (mg/L)*                       New   
                      Old  
Temp (°C)*                       New   
                      Old  
Number of live offspring**                         Total 
Jar 1                          
Jar 2                          
Jar 3                          
Jar 4                          
Jar 5                          
Jar 6                          
Jar 7                          
Jar 8                          
Jar 9                          
Jar 10                          
                       Total   
Experiment Number:_____ Date Started:__________ Medium:__________
Type of Food:__________ Test Chemical:________________ Concentration:__________ 
Day 10 11 12 13 14 15 16 17 18 19 20 21    
Medium renewal (check)                          
Food provided (check)                          
pH*                       New   
                      Old  
O2 (mg/L)*                       New   
                      Old  
Temp (°C)*                       New   
                      Old  
Number of live offspring**                         Total 
Jar 1                          
Jar 2                          
Jar 3                          
Jar 4                          
Jar 5                          
Jar 6                          
Jar 7                          
Jar 8                          
Jar 9                          
Jar 10                          
                       Total   
*

Pick one new and one old jar a week to check pH, oxygen, and temperature. Record the reading in the appropriate cell.

**

Record any adult mortality with an “M” in the appropriate cell.

Offspring were counted and euthanized for later sex determination by pipetting the Daphnia into vials containing methanol. Euthanized Daphnia sex determination was made by stereomicroscope observation. Daphnia were pipetted into drops on a Petri dish and manipulated with dissection probes to maneuver them onto their sides. Males were identified by the presence of a prominent first antenna located just beneath the beak-like projection or rostrum (Figure 1).

Endpoint & Statistical Analysis

The mean and standard deviation of the number of offspring and the number of days until the first reproduction were calculated for each control and treatment concentration; t-tests were used to determine whether the number of offspring in each fenoxycarb concentration differed significantly from the control. Percentages of the number of males and females were calculated per concentration and compared to the control; the appearance of any males under the test conditions indicates potential endocrine disruption. The upper fenoxycarb concentrations in this lab produced ~90% males, the lower fenoxycarb concentration ~10% males, and the control 0% males. Several forms of graphical data can be generated from the endpoint statistics, such as histograms or dose response curves.

Outcomes of Instructional Activity

This research project has been successfully implemented in an introductory college course and has shown substantial promise as a learning system for the scientific process, endocrine disruption, and statistical data interpretation. A group of 10 freshmen at a Kansas community college, with no formal research training or prior knowledge of Daphnia life cycles or male-to-female determination, were enrolled in a research methods class as part of a scholarship requirement or on a volunteer basis. The self-reported student majors included nursing, chemistry, dental hygiene, elementary education, biology, and neuroscience. Students were evaluated throughout the process for the quality of initial research, introductory activities, completion of methods, Daphnia sex determination, selection of appropriate statistical measures, and making relevant conclusions. The final project required students to produce a written paper in the form of a peer-reviewed journal article and give a group oral presentation. Students were assessed using the rubric in Table 3. After the conclusion of the lab procedures, 9 of the 10 students were adept at transferring, experimental setup, and sex determination of Daphnia (the one student's inadequate lab skills were most likely related to poor attendance and not to the difficulty of the procedures). Students demonstrated the ability to research acceptable sources, formulate hypotheses, reach valid conclusions, and present their findings in both written and oral forms; 90% of the students earned an A (60%) or B (30%) for the project as a whole. The successful implementation and completion of the project, given the students' lack of background knowledge and diverse majors, indicate that the lab can serve as a meaningful exercise to teach research-based skills and principles of endocrine disruption to a diverse set of students. Informal discussions with students and high levels of participation and commitment outside of class hours suggest that the project promoted student interest.

Table 3.
Daphnia–fenoxycarb endocrine-disruption rubric.
Project ComponentIncomplete (0 points)Poor (4 points)Marginal (6 points)Good (8 points)Excellent (10 points)Grade
Research No research, or research without references Research not complete and not thorough, or inaccurate Research mostly complete but not thorough, or some inaccuracies All research complete but not thorough, or few inaccuracies All research complete, thorough, and accurate  
Background activities/
readings 
No background activities or preparation
 
Few background activities complete and preparation for activities not evident Most background activities complete but preparation for activities not always evident All background activities complete but preparation for activities not always evident All background activities complete on time with significant evidence of preparation  
Project goals, including hypotheses No input provided for project goals or hypotheses Little significant input provided for project goals or hypotheses Some input in setting project goals and hypotheses; improper hypotheses based on research Major input in setting project goals and hypotheses; improper hypotheses based on research Major input in setting project goals and hypotheses; hypothesis proper based on research  
Procedures No procedures completed Few procedures completed Several procedures completed Most procedures completed effectively and responsibly and/or no substitutes were found to cover absences All procedures completed effectively and responsibly or substitutes were found to cover rare absences  
Daphnia sex determination Did not complete sex determination Mistakes regularly observed in sex determination Some mistakes observed in sex determination Mistakes rarely observed in sex determination No observed mistakes in sex determination  
Graphical and statistical data No statistical or graphical data provided Neither statistics nor graphs appropriate for findings Either statistics or graphs not appropriate for data Statistics and graphs appropriate for data; few reporting errors Statistics and graphs appropriate for data; no reporting errors  
Conclusions No conclusion of results provided Conclusions not appropriate based on the data Conclusions appropriate based on the data but overstated, and data not compared to findings from prior studies Conclusions appropriate based on the data but overstated, or data not compared to findings from prior studies Conclusions appropriate based on the data and not overstated; data compared to prior studies  
Written presentation of project No paper submitted, or paper submitted without references or plagiarized Paper not written clearly or concisely; has significant errors, or not all sections of the paper included Paper not written clearly, has some errors; includes Abstract, Introduction, Methods, Results, Discussion, and References sections Paper written clearly, concisely, with some errors; includes Abstract, Introduction, Methods, Results, Discussion, and References sections Paper written clearly, concisely, with no significant errors; includes Abstract, Introduction, Methods, Results, Discussion, and References sections  
Oral presentation of project No participation in the oral presentation of the project Presentation not well practiced; little evidence of knowledge about the project Presentation not well practiced; some knowledge of the project evident, but not complete; lacks the ability to answer audience questions Presentation well practiced; knowledge of the project evident; inability to accurately answer audience questions Presentation well practiced; knowledge of the project evident; accurately answered audience questions  
Final Grade ____/100 
Project ComponentIncomplete (0 points)Poor (4 points)Marginal (6 points)Good (8 points)Excellent (10 points)Grade
Research No research, or research without references Research not complete and not thorough, or inaccurate Research mostly complete but not thorough, or some inaccuracies All research complete but not thorough, or few inaccuracies All research complete, thorough, and accurate  
Background activities/
readings 
No background activities or preparation
 
Few background activities complete and preparation for activities not evident Most background activities complete but preparation for activities not always evident All background activities complete but preparation for activities not always evident All background activities complete on time with significant evidence of preparation  
Project goals, including hypotheses No input provided for project goals or hypotheses Little significant input provided for project goals or hypotheses Some input in setting project goals and hypotheses; improper hypotheses based on research Major input in setting project goals and hypotheses; improper hypotheses based on research Major input in setting project goals and hypotheses; hypothesis proper based on research  
Procedures No procedures completed Few procedures completed Several procedures completed Most procedures completed effectively and responsibly and/or no substitutes were found to cover absences All procedures completed effectively and responsibly or substitutes were found to cover rare absences  
Daphnia sex determination Did not complete sex determination Mistakes regularly observed in sex determination Some mistakes observed in sex determination Mistakes rarely observed in sex determination No observed mistakes in sex determination  
Graphical and statistical data No statistical or graphical data provided Neither statistics nor graphs appropriate for findings Either statistics or graphs not appropriate for data Statistics and graphs appropriate for data; few reporting errors Statistics and graphs appropriate for data; no reporting errors  
Conclusions No conclusion of results provided Conclusions not appropriate based on the data Conclusions appropriate based on the data but overstated, and data not compared to findings from prior studies Conclusions appropriate based on the data but overstated, or data not compared to findings from prior studies Conclusions appropriate based on the data and not overstated; data compared to prior studies  
Written presentation of project No paper submitted, or paper submitted without references or plagiarized Paper not written clearly or concisely; has significant errors, or not all sections of the paper included Paper not written clearly, has some errors; includes Abstract, Introduction, Methods, Results, Discussion, and References sections Paper written clearly, concisely, with some errors; includes Abstract, Introduction, Methods, Results, Discussion, and References sections Paper written clearly, concisely, with no significant errors; includes Abstract, Introduction, Methods, Results, Discussion, and References sections  
Oral presentation of project No participation in the oral presentation of the project Presentation not well practiced; little evidence of knowledge about the project Presentation not well practiced; some knowledge of the project evident, but not complete; lacks the ability to answer audience questions Presentation well practiced; knowledge of the project evident; inability to accurately answer audience questions Presentation well practiced; knowledge of the project evident; accurately answered audience questions  
Final Grade ____/100 

Students demonstrated various levels of comprehension in calculating and understanding the meaning of the statistics, most likely due to their individual math backgrounds and time constraints at the end of the course. Oral presentations were another challenging area for students, including their ability to answer open questions. Most likely, the lower performance on the presentation was due to nervousness, since most students showed good to excellent progress in the presentation practice sessions. Additional statistical and presentation exercises may help in future projects.

The suggested lab provides an easy-to-learn, student-guided research project that allows students to engage in the process of science and observe the unintentional endocrine effects of chemicals on nontarget populations. Experiencing the different aspects of the scientific process can lead to a greater appreciation of science and generate further interest in science disciplines.

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