Toxicity of Nanoparticles to Brine Shrimp: An Introduction to

Laboratory Experiment pubs.acs.org/jchemeduc

Toxicity of Nanoparticles to Brine Shrimp: An Introduction to Nanotoxicity and Interdisciplinary Science Melissa A. Maurer-Jones,‡,# Sara A. Love,‡,# Sharon Meierhofer,‡ Bryce J. Marquis,† Zhen Liu,‡ and Christy L. Haynes*,‡ †

Department of Chemistry, University of Central Arkansas, Conway, Arkansas 72035, United States Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States

‡

S Supporting Information *

ABSTRACT: Nanotoxicity is an area of intense research, stimulated by increased use of nanoparticles in commercially available products. Herein, using nanotoxicity as a platform, we describe an experiment that emphasizes interdisciplinary science in a collaborative work setting while expanding the traditional realm of chemistry and chemistry research. Students synthesize two noble metal nanoparticle suspensions (Au and Ag) and expose brine shrimp to these nanoparticles, assessing the brine shrimp viability after 24 h exposure. By examining two different nanoparticles at several concentrations, with appropriate control conditions, students can work together to collect and analyze all the toxicity results necessary to draw conclusions about the toxicity of Au and Ag nanoparticles. This experiment provides introductory chemistry students hands-on experience with a cutting-edge area of science while showcasing the real-world application of chemistry to an important modern scientific problem, nanoparticle toxicity. KEYWORDS: High School/Introductory Chemistry, First-Year Undergraduate/General, Interdisciplinary/Multidisciplinary, Laboratory Instruction, Collaborative/Cooperative Learning, Hands-On Learning/Manipulatives, Nanotechnology, Toxicology

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toxicity effects. Here, students utilize a simple aquatic organism, brine shrimp, as the model of study because they are simple to hatch, grow, and maintain.2 Although there are many indicators of toxicity, viability is most commonly explored. Viability, the toxicity measure that students use in this experiment, can be defined as the ability of a cell or organism to survive exposure to a toxicant or the number or percentage of organisms living versus dead. Students are tasked to examine brine shrimp survival after exposure to Ag or Au nanoparticles (that they have synthesized) at a variety of concentrations. By varying the concentrations within this experiment, the students can be exposed to basic statistical analysis tools. Pooling of the class’ replicate data allows students to practically apply these tools, leading to an understanding of differences in toxicity and when scientific data reveals statistical significance. This experiment was tested with high school chemistry students. An analysis of student comprehension, based on student’s paired pre- and post-experiment responses, is included herein; these data were used to improve the experimental procedure.

n modern science, the traditional disciplinary boundaries are increasingly blurred, where chemists study toxicology or material scientists work on cancer research. Interdisciplinary research can propel innovation with different perspectives contributing to important scientific and technological problems. One new field of research that benefits from an interdisciplinary approach is the field of nanotoxicology, or the study of the toxicity of nanoparticles. Nanoparticles are frequently, but not strictly, defined as particles with at least one dimension less than 100 nm. Because of their unique physical and chemical properties, which differ from atoms or bulk of the same material, nanomaterials have been increasingly incorporated into commercially available products, including silver nanoparticles in clothing to prevent odor and titanium dioxide in cosmetics as a sunscreen. This increased use necessitates the understanding of the possible toxicity these nanoparticles pose for humans and the environment. As such, numerous scientists, including chemists, material scientists, biologists, and toxicologists, have undertaken the study of the impacts of nanoparticles in biological and environmental models to better understand and hopefully predict their potential toxicity.1 In the experiment detailed herein, nanotoxicology is used as a platform to introduce chemistry students to interdisciplinary, collaborative science, including aspects from chemistry, materials science, toxicology, and statistics while also exposing them to an area of cutting-edge research. As is often the case in toxicology, a model organism is used to examine potential © 2013 American Chemical Society and Division of Chemical Education, Inc.

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EXPERIMENTAL OVERVIEW In this experiment, the students synthesize nanoparticles, perform a serial dilution of nanoparticle suspension, do a live/dead brine shrimp assessment, and analyze the resulting data. In the interest of time and conservation of materials, the Published: January 17, 2013 475

dx.doi.org/10.1021/ed3005424 | J. Chem. Educ. 2013, 90, 475−478

Journal of Chemical Education

Laboratory Experiment

prepared solutions in vials or test tubes labeled a−f (see Table 1 in the Supporting Information). The solutions can either be stored for future use or used immediately.

experiment is best performed in groups of 2 or 4 students (2 pairs of lab partners) so that the work is divided. This experiment was designed with high school students in mind, but would also be applicable to an introductory level chemistry or environmental science class, providing that the lab met multiple days in a week, as students need 2 or 3 consecutive days with 45−60 min per day to complete all components. Herein, we briefly describe the components of the experiment, with more detail found in the teacher packet, including our vision for the division of tasks, and the student worksheet in Supporting Information.

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LIVE/DEAD BRINE SHRIMP ASSAY The brine shrimp assay determines brine shrimp viability after a 24 h exposure to the control and nanoparticle solutions described above. Viability of the brine shrimp was assessed based on brine shrimp movement (see Figure 2 for image of brine shrimp egg and live brine shrimp).

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NANOPARTICLE SYNTHESIS Au and Ag nanoparticle syntheses were performed by the students from stock solutions, which were prepared beforehand by the instructor. Transmission electron microscopy (TEM) images of prepared nanoparticles can be seen in Figure 1.

Figure 2. Image of brine shrimp eggs (inset) and live brine shrimp.

The assay was performed in a multiwell plate, or other containers that can hold at least 2.5 mL each. Students measured 0.5 mL brine shrimp suspension, using a Pasteur pipet premarked with a lines for measuring 0.5 and 1 mL, and counted the moving brine shrimp while they were still in the pipet, then transferred the brine shrimp to an empty well and recorded the value and the well in which it was placed. This part of the experiment has significant potential for error, and therefore, students should take care counting. Students performed the viability in quadruplicate so that each control or nanoparticle concentration had multiple data points. This means that if a student or lab pair within a larger group was testing the viability of 3 sample solutions, they filled a total of 12 wells. After counting and recording the number of shrimp per well, 1 mL of serum solution (25% bovine calf serum, 75% artificial seawater by volume, see the Supporting Information), to prevent nanoparticle aggregation, and 1 mL of the control or nanoparticle solution were added to each well. Upon exposure, brine shrimp solutions were placed under a lamp, such as a desk or utility lamp, approximately 9 in. from the bulb and incubated for 24 h, after which the brine shrimp were counted again, only counting the shrimp that were moving (i.e., alive).

Figure 1. TEM images of Au and Ag nanoparticles used by the students within this experiment.

Au Nanoparticles

Citrate-reduced Au nanoparticles were synthesized (as modified from a previously published method3) by heating 15 mL of 1.1 mM HAuCl4 stock solution in a glass vial, periodically stirring, and upon boiling, adding 1.5 mL of 50 mM sodium citrate stock solution. Within a couple minutes, the solution changed from clear to a deep red-purple, indicating nanoparticle formation; the solution was heated with periodic stirring for 10 min after the color change. Ag Nanoparticles

Ag nanoparticles were synthesized with simple modifications to a previously published method.4 In glass vial or beaker, 13 mL of deionized water was brought to a boil, ideally under constant stirring, and 1 mL of 7.5 mM AgNO3 and 1 mL of 7.5 mM sodium citrate stock solutions were added and stirred. Next, 20 drops of 10 mM NaBH4 stock solution were added, and the solution immediately turned from clear to yellow as the nanoparticles formed; the solution was heated with periodic stirring for 10 min after the color change.

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DATA ANALYSIS An important component of chemistry, and science in general, is data analysis and statistics. In this experiment, students used the viability data to perform simple statistics. First, viability was calculated per well by # live brine shimp after exposure viability (in %) = # live brine shrimp before exposure

Exposure Solutions and Nanoparticle Dilution

In this experiment, students compared the toxicity of different concentrations of nanoparticles, along with water and metal ion (i.e., Ag+ or Au+) controls, on brine shrimp. To achieve the varied concentrations, a serial dilution was performed from the stock, as-synthesized nanoparticles yielding the following concentrations: 100% nanoparticles, 50% nanoparticles, 25% nanoparticles, and 12.5% nanoparticles. Although the exact concentration of nanoparticles may differ in the syntheses, the concentrations of the as-synthesized and diluted nanoparticles were comparable for the toxicity assay. Table 1 in the teacher packet indicates the solutions that were prepared and subsequently incubated with the brine shrimp. Students

× 100%

(1)

Once the viability was calculated per well, data was combined for each exposure condition and students calculated the average, standard deviation, and other statistical transformations (e.g., t testing). Using the combined data, students then plotted the data to see a graphical representation of the class’ 476




and based on their pre- and post-testing answers, the students showed improvement in their understanding of nanotoxicology and interdisciplinary science. However, limited improvement was seen from the statistic question, where student responses were largely unchanged after performing the experiment. The described experiment, with high school students, informed the final version of the experiment described in the methods section. Students’ results revealed a significant decrease in viability upon exposure to the Ag+ control but showed no dose-dependent decrease in viability upon exposure to either Au or Ag nanoparticles as compared to the control, contrary to our conclusions found in the labs’ development. It was concluded that the students’ observations were greatly affected by error within the brine shrimp counting. Therefore, changes were made to the division of experiments with more time given for careful brine shrimp counting. The Ag nanoparticle synthesis was also modified to a preparation that requires less time. This beta-test illuminated the multicomponent nature of nanotoxicity studies. That is, this experiment has many components and depending on prelaboratory preparation and lecture, the emphasis could be placed preferentially on one area or another. Clearly, based on the pre- and post-test question results above, our emphasis was placed largely on nanoparticles, nanotoxicity, and the advantages of interdisciplinary science, which is the bias of our academic research. However, we envision this experiment to be useful for a lot of different learning outcomes, depending on where emphasis is placed by the instructor.

results, making conclusions about the relative toxicity of Au and Ag nanoparticles. Variations to the viability experiment can be performed, such as varying the time of exposure (e.g., 24 vs 48 or 72 h) and number of replicates, depending on the aim of this experiment.

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HAZARDS

Sodium borohydride is flammable. AgNO3 and HAuCl4 may cause irritation to skin, eyes, and respiratory tract and may be harmful if swallowed or inhaled. Sodium citrate may cause irritation to skin, eyes, and respiratory tract. Goggles should be worn during this experiment in addition to the optional use of gloves and lab aprons. All nanoparticle solutions, including reagents and exposed brine shrimp, should be collected and disposed in accordance with hazardous waste procedures.

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DISCUSSION Three classes of high school chemistry students, two standard and one advanced placement level, performed this experiment. The students were given a short (five) question test (see the Supporting Information), both prior to and following the experiment, to assess their level of comprehension of the core themes (interdisciplinary science, nanotoxicology, and statistics) presented. Prelaboratory discussions were led by the experiment developers, in lieu of the high school teacher introductory lecture. Direct comparisons were made between the pre- and post-tests of individual students in each class; differences in responses (i.e., changes between incorrect/ correct and qualitative/quantitative answer) to the five posed questions were assessed and tabulated for all paired (pre- and post-experimental testing) responses. For question one, students were asked to define the “nano” prefix: responses ranged from qualitative (i.e., very small, tiny) to quantitative (i.e., correct 10−9 and incorrect 109). The most common error was an inversion in scale for those giving quantitative responses, for both the pre- and post-test responses. Overall, student comprehension shifted to more quantitative responses when students responded differently on the post-test. For question two, students were asked about their exposure to nanoparticles. Students largely (>65%) responded that they had been exposed to nanoparticles in the pre-test: post-testing showed students who had answered negatively changed their answers to affirmative responses. Question three asked students to describe safety determinations (for a chemical or technology); typical responses included testing (involving either animals or people) and experimentation for both pre- and post-test responses. The students were asked to interpret data suggesting viability of 70% ± 5% in question four: responses ranged from “give or take”, error/% error/range of error, and specifically stated ranges. Students responses were largely unchanged from pre- and post-testing, while the roughly 5% that did respond differently provided more descriptive, appropriate responses (e.g., percent error became percent range of live brine shrimp). To assess the students understanding of interdisciplinary science, students were asked which fields of science ask questions related to toxicity. Student responses included various fields from physical and biological sciences, medicine, and a more general response of “all fields”. Upon post-testing, student responses included similar branches of science but typically included more disciplines in their answers (e.g., toxicology became toxicology, chemistry, biology). The experiment was successfully completed in the laboratory classroom,

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CLASSROOM SCIENCE STANDARDS The high school national science education standards (NSES) addressed by this activity include “Science as Inquiry”, “Physical Science”, “Science and Technology”, and “History and Nature of Science”. More details on specific national standards can be found in the teacher packet.

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CONCLUSION This experiment was designed to be flexible for a variety of instructional aims (e.g., emphasis on statistical analysis in chemistry) and can be used fully or only partially (e.g., only nanoparticle synthesis or only viability assay). Through this experiment, students are exposed to the fields of nanotechnology and nanotoxicology, with hands-on experience with basic chemical, material, toxicological, and statistical principles. Beyond scientific principles, this experiment encourages class-wide collaboration and allows all students to contribute to the group data.

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ASSOCIATED CONTENT

S Supporting Information *

Student worksheet and teacher packet, which includes a supply list, extended instructions, pre- and post-lab assessment and tables of science standards. This material is available via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions #

These authors contributed equally.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Amanda Gavin and the students at East Ridge High School in Woodbury, MN for helping improve this laboratory experiment and Audrey F. Meyer for help in implementing the lab. This work was supported by the National Science Foundation (CHE-1152931), a UMN doctoral dissertation fellowship awarded to M.A.M-.J, and a NNIN Research Experience for Teachers fellowship awarded to S.M. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRSEC program.

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REFERENCES

(1) Oberdö rster, G.; Oberdö rster, E.; Oberdö rster, J. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 2005, 113, 823−839. (2) Lieberman, M. A brine shrimp bioassay for measuring toxicity and remediation of chemicals. J. Chem. Educ. 1999, 76, 1689−1691. (3) Marquis, B. J.; McFarland, A. D.; Braun, K. L.; Haynes, C. L. Dynamic measurement of altered chemical messenger secretion after cellular uptake of nanoparticles using carbon-fiber microelectrode amperometry. Anal. Chem. 2008, 80, 3431−3437. (4) Chinnapongse, S. L.; MacCuspie, R. I.; Hackley, V. A. Persistence of singly dispersed silver nanoparticle in natural freshwaters, synthetic seawater, and simulated estuarine waters. Sci. Total Environ. 2011, 409, 2443−2450.

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Toxicity of Nanoparticles to Brine Shrimp: An Introduction to

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