Workshop on Environmental Nanosafety: Biological Interactions of

Activity Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

pubs.acs.org/jchemeduc

Workshop on Environmental Nanosafety: Biological Interactions of Plastic Nanoparticles Tommy Cedervall,†,‡ Mikael T. Ekvall,†,‡ Karin Mattsson,†,‡,§ and Martin Lundqvist*,†,‡ †

Division of Biochemistry and Structural Biology, Lund University, Box 124, 22100 Lund, Sweden NanoLund, Lund University, Box 118, 22100 Lund, Sweden

‡

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S Supporting Information *

ABSTRACT: The one-hour workshop, containing both a demonstration and hands-on experiments on the topic of nanosafety, is based on current science on a topic of general interest. The workshop aims to provide a deeper knowledge and understanding of nanoparticles. The participants get an introduction to what nanoparticles are, why nanosized materials are interesting, how nanomaterials interact with biological molecules, and potential risks associated with nanoparticles. Furthermore, by participating in the workshop the audience gains insights into how research about nanoparticles is conducted. The participants carry out experiments to demonstrate that daily-used plastic products can be disintegrated into particles in the nanometer size range, which may have important implications for the environment. KEYWORDS: General Public, High School/Introductory Chemistry, First-Year Undergraduate/General, Interdisciplinary/Multidisciplinary, Public Understanding/Outreach, Hands-On Learning/Manipulatives, Inquiry-Based/Discovery Learning, Nanotechnology, Proteins/Peptides, Surface Science

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Currently there is neither consensus in the research field nor clear guidelines on how nanomaterials should be risk-assessed. Excellent activities concerning different aspects of nanomaterials, including optical properties of gold,8−10 solar cell mechanism,11 and toxicity,12 have previously been published in this Journal. The aim with this workshop is to provide a broader perspective and exercises that give direct hands-on experience to drive home concepts of commonly used terms in today’s news media such as nano, nanoparticles, and nanotoxicity. The idea of this workshop originated from a collaboration between us and producers at a local theater in Lund, Sweden. The workshop was designed as a complement to Daniel Pennac’s play entitled “The Sixth Continent”. This play illustrates the pathway leading us to the problem we are facing today, where large areas (“continents”) of plastics are floating around in the oceans. Plastic waste has during the past decade, and particularly more recently, attracted great interest in the news media all around the world.13−16 The effects of ocean

BACKGROUND TO THE WORKSHOP The new and rapidly developing field of nanotechnology first emerged during the 1990s, and many new applications based on nanomaterials have since been developed. Today, commercial products that contain nanotechnology are pervasive.1,2 There are two main factors that make nanoparticles attractive for industrial applications. First, a major advantage of bringing materials down to the nanosize range is the significantly larger surface area per unit mass provided by smaller particles compared with larger ones, and hence, less material can do more. An important example is provided by catalytic converters, in which rare metals are used.3 Second, the material may obtain different properties when it reaches the nanosize range compared with the bulk material. For example, aluminum, which is relatively benign and inert in bulk, becomes explosive,4 and the melting point of gold changes from 1064 °C for sizes above 10 nm to under 500 °C for a 1.5 nm gold particle.5 Risk assessments of nanomaterials present a challenge since risk assessments for a material in the macrosize range may not apply when the material is produced in the nanosize range.6 Moreover, the material may also have different effects within the nanosize range, i.e., different size populations of a material will give rise to different responses, for example acute toxicity.7 © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: December 21, 2018 Revised: June 11, 2019

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DOI: 10.1021/acs.jchemed.8b01032 J. Chem. Educ. XXXX, XXX, XXX−XXX

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polystyrene foam board soaked in the water for 5 min, as described in more detail in the Supporting Information (SI) and in previously published results.25 The participants do not need to watch the whole process. After ca. 15 s of mixing, when they have seen the process of how the material is broken up, the participants are divided into three groups and continue with one of three hands-on experiments.

plastics on wildlife have been widely documented, for example, birds eating plastics,17 birds and fish getting trapped in plastics,18 and plastic bags choking whales.19 The total production of different types of plastics increases annually,20 and plastics can nowadays be found virtually everywhere. During the last decades the trend has been to find ways to improve the recycling of plastics; however, we still have a long way to go, and the majority of the produced plastics is not being recycled.21 Plastics are often burned to produce energy, discarded in landfills, or simply left in the environment. In the environment, plastics may be exposed to different chemical environments, such as changes in pH or salt concentration, or to a variety of physical factors causing them to break down, including exposure to UV radiation and mechanical friction (e.g., by scraping against stones and sand at a beach with the help of waves and currents). These factors lead to the degradation of the large macrosized plastics into smaller and smaller plastic pieces that eventually may be taken up by aquatic living organisms. We have previously shown that polystyrene nanoparticles can travel up in a food chain and significantly affect organisms at higher trophic levels.22−24 The described workshop is based on our own scientific explorations and experiences gained in the nanoparticle field. This has allowed us to design a workshop that introduces nano as a concept as well as illustrates the potential hazard posed by degradation of larger macrosized plastics into smaller plastic objects. The described workshop has been appreciated by participants aged 13 and up.

Hands-On Sessions 1−3

Each of the three hands-on experiments targets a particular research question (see the SI): 1. What happens to the total surface area if a block is divided into many smaller blocks? 2. Do natural biomolecules interact with plastic nanoparticles? 3. Can zooplankton take up nanoparticles from their surrounding environment? To answer the first question, the students calculate the surface area of an assembled block of LEGOs with the help of a ruler and a calculator. Thereafter, they divide the block into the individual LEGO pieces and calculate their combined surface area. This illustrates one of the important reasons why nanosized objects are of interest for a wide range of applications: the surface area per unit mass of a material is much larger for nanosized objects than for macrosized objects. To explore the second question, polystyrene nanoparticles and the proteins immunoglobulin G (IgG) and bovine serum albumin (BSA) are used to illustrate that proteins may adsorb on nanoparticles. The experiment demonstrates that different proteins interact differently with a nanoparticle and that the outcome of the interaction between proteins and nanoparticles can be concentration-dependent, as described in more detail in our previously published results.26,27 The third experiment involves the planktonic crustacean zooplankton, Daphnia magna, which is called a filter feeder because of the large amounts of water it filters while feeding. In this experiment, the students investigate whether water treated with fluorescent nanoparticles (quantum dots) can be found inside Daphnia magna. The students are supplied with a sheet that contains zooplankton that have been fed with water containing quantum dots or regular water. In other words, some of the Daphnia magna have filtered clean water while others have filtered water containing quantum dots. The students then use a USB microscope and a laser pen that emits blue light. This pen is used to excite the quantum dot particles in order to visualize them and to observe that they have accumulated inside the Daphnia magna. This experiment builds directly on published scientific investigations.28

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THE WORKSHOP The workshop has three main aims. The first aim is to illustrate what nano-objects are, how they interact with their surroundings, and why they are interesting from an industrial perspective. The second aim is to show that daily-used plastic materials can be disintegrated into plastic nano-objects. The third aim is to illustrate how research focused on environmental nanosafety is conducted. The workshop contains one practical demonstration, three hands-on experiments, and one extra task, if time permits, for further learning. Demonstration: Can We Manufacture Plastic Nanoparticles from Bulk Plastics? Part 1

The workshop starts with part 1 of the demonstration (Figure 1) by asking the general question, “Can macro plastics be broken down into nano plastics?”. To answer this question, a piece of expanded polystyrene foam board is divided into smaller (1−5 cm3) pieces and put into a bowl containing Milli-Q water. A household immersion blender is then used to break up the

Extra Task

In the extra task, the participants have the opportunity to learn more about how the sizes of nanoparticles can be measured on the basis of how light interacts with its surroundings. They also see that the size of a material may affect the properties of the material. More information on this activity is provided in the SI. After the participants have finished with the hands-on experiments it is time for the part 2 of the demonstration. Demonstration: Can We Manufacture Plastic Nanoparticles from Bulk Plastics? Part 2

Figure 1. Outline of the workshop. The estimated time per segment can be adjusted to the total time planned for the workshop (40−60 min). The students are divided into three groups, and the groups do the three different hands-on sessions in parallel, changing sessions after 10−15 min until all three groups have done all three sessions.

The liquid portion of the plastic/water mixture generated in part 1 is recovered using a 20 mL syringe. The liquid is then passed through a 0.8 μm syringe filter and collected into a small vial (preferably rectangular in shape) with transparent walls. B

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Thereafter, a green laser beam is first shone through the control vial, containing pure water, demonstrating that there is nothing in the liquid to scatter the laser light (Figure 2). The same

schools (students studying programs focusing on various subjects from art to natural science), and for university students and teachers (from all levels and subjects). More than 300 persons have completed the workshop. In order to assess the participants’ understanding of the workshop, two different surveys were given out in immediate connection to the workshop (see the SI). Is There a Need for a Workshop about Nano?

The first survey, consisting of Statements 1−4, is aimed at investigating the participants’ own perceptions of the terms nano, nanotechnology, and nanotoxicity and was handed out before the participants performed the workshop (see the SI for Statements 1−4 and the results, which are presented in Figure S1). The data are limited, as 74 participants have answered so far, but the trend shows that a small majority consider themselves informed on what nanotechnology is. A majority of them did not know whether they have purchased items containing nanotechnology, and they were generally not informed about any potential risks regarding nanotechnology. The most surprising result was that a clear majority of the participants were indifferent to the word nano, which contradicts the preconception among researchers in the nanofield that the concept nano has a bad connotation among the general public. The conclusion from the answers to Statements 1−4 is that there is a need to increase the knowledge about nano, nanotechnology, and nanotoxicity. Participants’ Evaluation of the Workshop

A second survey, consisting of Statements 5−8, was handed out to the participants after they had finished the workshop to provide them with the opportunity to evaluate their experience of the workshop. Among the 114 participants who have answered this survey to date, the statements can be found in the SI, and the results are shown in Figure S2. A clear majority of the participants have found that the workshop is useful, informative, and at a reasonable level of difficulty. This may be counterintuitive since the participants come from diverse backgrounds, including students from elementary school to upper secondary school and natural science teachers; however, the participants’ questions at each individual workshop govern the depth of the explanations to their different observations, i.e., the level is adapted to the current audience.

Figure 2. (a) Scattering of green laser light in the control, Milli-Q water only (to the right), and the filtered water/plastic sample (to the left). (b) Size distribution data for the produced particles, detected with the nanoparticle tracking analysis instrument. Each line in the graph corresponds to an expanded polystyrene foam board sample that has been mixed and then filtered through a 0.8 μm filter. In total, the results from five individual samples are shown.

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procedure is then performed with the vial containing the freshly filtered sample. The small plastic particles (smaller than 0.8 μm since larger objects have been filtered out) scatter the green laser light (Figure 2). The size distribution of the produced plastic particles is reproducible, and measurements conducted with a nanoparticle tracking analysis (NTA) instrument reveal that the filtered sample contains particles with sizes of 160 ± 60 nm (Figure 2). The size can vary depending on the plastic starting material and the syringe filter used (see Ekvall et al.25 for more information).

CONCLUSION A one-hour workshop that introduces the participants to the nano field has been presented. The response to the workshop is positive regardless of the participants’ educational background. The workshop works for participants from seventh grade and higher.

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ASSOCIATED CONTENT

S Supporting Information *

HAZARDS Lasers may harm the eyes and hence should be handled with care and not pointed directly toward the eyes. Nanoparticles may be harmful and should be collected and handed in to the instructor for destruction. Suitable eye protection and lab coats should be worn during all lab procedures.

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b01032. Surveys and a detailed description of each segment of the workshop (PDF, DOCX) NTA movie of nanoparticles made in the workshop (AVI)

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IMPACT OF THE WORKSHOP During the last three years we have held this workshop at different venues, including elementary schools, upper secondary

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. C

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ORCID

(16) McCarthy, N. The World’s Oceans Are Infested With Plastic (March 22, 2017). https://www.statista.com/chart/8616/the-worldsoceans-are-infested-with-plastic/ (accessed April 2019). (17) Wilcox, C.; Van Sebille, E.; Hardesty, B. D. Threat of plastic pollution to seabirds is global, pervasive, and increasing. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (38), 11899−11904. (18) Blue Planet 2: How plastic is slowly killing our sea creatures, fish and birds. http://www.bbc.co.uk/newsbeat/article/42030979/blueplanet-2-how-plastic-is-slowly-killing-our-sea-creatures-fish-and-birds (accessed April 2019). (19) Kaladharan, P.; Asokan, P. K.; Koya, K. M.; Bhint, H. M. Plastic debris in the stomach of a Longman’s Beaked Whale, Indopacetus pacif icus (Longman, 1926) stranded off Sutrapada, Veraval, Saurashtra coast, India. J. Mar. Biol. Assoc. India 2014, 56 (2), 92−94. (20) Global plastic production from 1950 to 2016 (in million metric tons). https://www.statista.com/statistics/282732/global-productionof-plastics-since-1950/ (accessed April 2019). (21) Geyer, R.; Jambeck, J. R.; Law, K. L. Production, use, and fate of all plastics ever made. Science Advances 2017, 3 (7), e1700782. (22) Cedervall, T.; Hansson, L.-A.; Lard, M.; Frohm, B.; Linse, S. Food Chain Transport of Nanoparticles Affects Behaviour and Fat Metabolism in Fish. PLoS One 2012, 7 (2), e32254. (23) Mattsson, K.; Ekvall, M. T.; Hansson, L. A.; Linse, S.; Malmendal, A.; Cedervall, T. Altered Behavior, Physiology, and Metabolism in Fish Exposed to Polystyrene Nanoparticles. Environ. Sci. Technol. 2015, 49 (1), 553−561. (24) Mattsson, K.; Johnson, E. V.; Malmendal, A.; Linse, S.; Hansson, L.-A.; Cedervall, T. Brain damage and behavioural disorders in fish induced by plastic nanoparticles delivered through the food chain. Sci. Rep. 2017, 7, 11452. (25) Ekvall, M. T.; Lundqvist, M.; Kelpsiene, E.; Š ileikis, E.; Gunnarsson, S. B.; Cedervall, T. Nanoplastics formed during mechanical breakdown of daily used polystyrene products. Nanoscale Adv. 2019, 1, 1055−1061. (26) Cukalevski, R.; Ferreira, S. A.; Dunning, C. J.; Berggård, T.; Cedervall, T. IgG and fibrinogen driven nanoparticle aggregation. Nano Res. 2015, 8 (8), 2733−2743. (27) Cukalevski, R.; Lundqvist, M.; Oslakovic, C.; Dahlback, B.; Linse, S.; Cedervall, T. Structural Changes in Apolipoproteins Bound to Nanoparticles. Langmuir 2011, 27 (23), 14360−14369. (28) Feswick, A.; Griffitt, R. J.; Siebein, K.; Barber, D. S. Uptake, retention and internalization of quantum dots in Daphnia is influenced by particle surface functionalization. Aquat. Toxicol. 2013, 130−131, 210−218.

Mikael T. Ekvall: 0000-0002-4452-0580 Martin Lundqvist: 0000-0003-1175-9103 Present Address §

K.M.: University of Gothenburg, Department of Marine Sciences, Kristineberg Marine Research Station, 45178 Fiskebäckskil, Sweden.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Teater Sagohuset for initiating the collaboration that resulted in this workshop and all of the students and teachers that participated in the workshop, especially those that filled in different questionnaires. We also thank the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) for funding to T.C. (Grants 2015-01222 and 2018-02501) and Prof. Karin Åkerfeldt and Dr. Celia Cabaleiro Lago for valuable comments.

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REFERENCES

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DOI: 10.1021/acs.jchemed.8b01032 J. Chem. Educ. XXXX, XXX, XXX−XXX