Laboratory Experiment pubs.acs.org/jchemeduc
Demonstrating the Effect of Surfactant on Water Retention of Waxy Leaf Surfaces Yu-Chun Chiu,† Matthew A. Jenks,† Michelle Richards-Babb,‡ Betsy B. Ratcliff,‡ John A. Juvik,§ and Kang-Mo Ku*,† †
Division of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia 26505, United States C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506, United States § Department of Crop Sciences, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States ‡
S Supporting Information *
ABSTRACT: We report here the development of an inexpensive and engaging laboratory-based activity that can help students learn about the scientific method and the role of plant epicuticular waxes and surfactant function on waxy plant leaves as real life example in the agricultural industry. Three each of nontreated collard leaves (Brassica oleraceae L. Acephala group) and brushed-leaves were sprayed with water to demonstrate hydrophobicity of epicuticular waxes. Another set of three nontreated collard leaves was sprayed with Tween 20 containing water to demonstrate function of surfactant. Then, water retention rate was calculated by subtracting between before and after spray treatments. Water contact angles were measured by ImageJ software from nontreated and brushed-leaves from pictures taken by students’ smartphone. This lab activity provides a foundation for instruction on the importance of the plant epicuticular waxes that is the outermost hydrophobic barrier in agrochemical applications and the function of surfactant on waxy leaf surface. KEYWORDS: High School/Introductory Chemistry, First-Year Undergraduate/General, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Agricultural Chemistry, Plant Chemistry, Surface Science
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INTRODUCTION Epicuticular waxes form the outermost barrier of the plant surface, serving as a hydrophobic coating over most aboveground organs that protect plants from many forms of environmental stress.1−3 Epicuticular waxes consist of long chain (20−40 carbons) fatty acids, alcohols, aldehydes, alkanes, and esters, along with various other lipids. The hydrophobic nature of epicuticular waxes contributes to plant drought tolerance by decreasing transpiration, and provides pathogen resistance by shedding surface water needed for spore germination.1,3 The epicuticular wax layer also creates a physical barrier that (i) protects plant organs from mechanical damage, (ii) prevents pathogen infection, and (iii) deters insect feeding.3 Further, the hydrophobic properties of the surface wax layer have been identified as a major barrier to effective application of chemicals (e.g., herbicides, pesticides, fungicides, and growth regulators) in agricultural sprays.3,4 For instance, the retention of pesticide spray droplets on leaf surfaces is reduced significantly by waxy or hairy leaf surfaces resulting in low chemical application performance, overuse of pesticide, and the potential for environmental pollution. However, surfactant chemicals, added as an adjuvant to pesticide agricultural sprays, enhance spray deposition, droplet adhesion and coverage, and overall retention of pesticide droplets on leaf surfaces. © XXXX American Chemical Society and Division of Chemical Education, Inc.
Surfactants also enhance the movement of herbicide from leaf surfaces through the wax layer and into plant tissues promoting the destruction of unwanted plants (i.e., weeds). Thus, it is important for students in many different disciplines (e.g., biology, chemistry, plant science, horticulture, agronomy, forestry, and entomology) to understand the chemical and physical properties of plant epicuticular waxes and how the hydrophobicity of leaf surfaces can be modified by surfactant. As scientists in every discipline employ some form of the scientific method, particularly during research at the early stages,5 it is important for students to understand the scientific method and its role in fundamental research. Thus, scientists have fashioned activities6−9 and experiments10−12 to introduce students to the scientific method with a focus on learning how to do science instead of doing science to learn.13 Students familiar with the research process from start (e.g., hypothesis formulation and experimental testing) to finish (e.g., use of inferential statistics to validate or reject a hypothesis) are better able to evaluate the significance of scientific information Received: July 22, 2016 Revised: October 14, 2016
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DOI: 10.1021/acs.jchemed.6b00546 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
and 14 h/10 h day/night temperature regimen. At 20 days after germination, seedlings were transferred to 4 L pots. These seedlings were grown for 5−6 weeks before being used as plant material for the experiment described herein. Three leaves were provided to each students’ group (mature collard plant can provide 5−8 flat leaves). Alternatively, collard leaf samples purchased in grocery stores can be used. However, the waxy surfaces of store-bought produce are often damaged during harvest, transportation, and in-store handling.
communicated by diverse sources (i.e., from popular news media to peer-reviewed publications). In the experiment described herein, students are introduced to and use the scientific method to study the physical properties (e.g., contact angles and wettability or water retention) of collard green leaves (Brassica oleraceae L. Acephala group) with and without epicuticular wax and in the presence and absence of surfactant. The predominant wax components on collard greens, C29 alkane (n-nonacosane) and C29 ketone (15nonacosanone), play a major role in the waxy crystal structure.14 Surface hydrophobicity is assessed by measuring the contact angle of liquid water droplets on leaf surfaces. Small contact angles (≤90°) correspond to high wettability and low surface hydrophobicity, whereas large contact angles (>90°) correspond to low wettability and high surface hydrophobicity. The crystal structure of epicuticular wax on the leaf surface has a significant impact on plant surface hydrophobicity.15,16 Previous studies explain how hydrophobicity alone can lead to water contact angles on leaf surfaces of up to 120°, whereas a highly crystalline surface wax is necessary to create higher contact angles [e.g., Sacred Lotus (Nelumbo nucifera Gaertn.)].17 Wettability is the ability of a solid surface to reduce the surface tension of a contacting liquid droplet, and cause it to spread and wet the surface. On plant surfaces, the shape, density, and chemical composition of epicuticular wax crystals is the primary determinant of surface wettability,3,18 and thus the ability of the surface to retain water. Whereas educational materials for the teaching laboratory have been published demonstrating the surface physical properties (e.g., wettability and droplet contact angles) of synthetic materials,19−21 no educational materials demonstrating the effects of surfactant on the physical properties of natural materials (i.e., plant leaf surfaces) have been reported. Smartphones and laptops have been used in teaching laboratories to expand student capacity to conduct a wide variety of experiments in the biological and physical sciences.22,23 Smartphones, along with free image analysis software, have substituted for expensive, inaccessible instrumentation for measurement of contact angles and colorimetry in hands-on laboratory exercises.21−24 Likewise, we have developed a cost-effective, hands-on laboratory experiment in which students use (i) their smartphone devices to take photographic images and (ii) the open source, free image analysis software (ImageJ) available from the National Institutes of Health (NIH)25 to learn about the scientific method, plant epicuticular waxes, and how surfactant modifies the waxy surface. Considering the experiment’s (i) simplicity, (ii) limited equipment needed, and (iii) focus on the scientific method, this experiment can be used by students enrolled in general chemistry coursework in both secondary or postsecondary school settings.
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Determining Leaf Surface Wettability
The experiment consists of two parts: (i) analysis of wettability (or water retention) on plant leaves with and without epicuticular wax and in the presence and absence of surfactant and (ii) measurement of contact angles on leaves with and without epicuticular wax. Students work in pairs on all parts of the experiment with one smartphone device per pair of students. In addition, students must avoid touching leaf surfaces during handling as the epicuticular wax layer is easily disturbed. A total of nine collard leaf sections (3 × 3 cm2 each and cut from 1−2 collard leaves) per pair of students is needed for the wettability experiment. Students remove epicuticular wax from the surfaces of three experimental leaf sections using a 11/2 in. wide bristle paintbrush (Craftsmart, Blackburn North, VIC, Australia) and gently swiping across the leaf surface. After epicuticular wax removal, the leaf surface should be glossy, indicating a reduction in light reflectance from wax crystals or reduced wax crystal surface density (see Figures 1 and 2) as
Figure 1. Representative image of water retention experiment. Waxy leaf (left), brushed leaf (middle), and Tween 20 surfactant sprayed on waxy leaf (right).
demonstrated by Grant et al.26 Several other methods including use of gum arabic, surface wiping with dry glass fabric, and peeling of waxes with cryo-adhesives have been developed to remove epicuticular waxes.27 However, the brushing method is simple, inexpensive, and reproducible enough for the purposes of this experiment. Six other leaf sections, three control and three leaf sections for the surfactant study, are left undisturbed. Students determine the weights of the three control and three brushed experimental replicate leaf sections and then place the sections on a flat cardboard surface inclined by 40°. Students spray the leaves with distilled water infused with red food coloring (0.1% Red 40, Neon Food Color, McCormick, Sparks, MD) using a spray bottle. The red color allows enhances visualization of the water droplets retained on leaf surfaces and improves photographic image quality (see Figure 1).
EXPERIMENTAL DETAILS
Preparation of Plants
Seeds of Brassica oleracea L. Acephala group cultivars “Top Bunch” (collard greens) were purchased from Johnny’s Selected Seeds (Winslow, ME). Seeds of each cultivar were germinated in 32-cell plant plug trays filled with Sunshine LC1 (Sun Gro Horticulture, Vancouver, British Columbia, Canada) professional soil mix. Seedlings were grown in the Evansdale greenhouse at West Virginia University under a 25 °C/17 °C B
DOI: 10.1021/acs.jchemed.6b00546 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Figure 2. Representative image of contact angles before (left) and after epicuticular wax removal (right) from collard leaf.
Figure 3. Surface water retention per area (mg/cm2) from triplicate analysis of collard leaves.
Afterward, students lower the cardboard to horizontal and, one-by-one and without disturbing the surface water, transfer the leaf sections to the balance and reweigh. Students repeat this process with the three unbrushed experimental leaf sections, but spray the surfaces with a mixture containing distilled water infused with red food coloring and a surfactant adjuvant (Tween 20, P1379, Sigma). After measuring each leaf’s area, students calculate water retention per area (mg/cm2) for each leaf section. Step-by-step student instructions and instructor tips for the wettability experiment are provided in Supporting Information.
contact angle means and standard deviations for leaf sections (N = 3) before and after brushing (wax removed) and paired ttest statistic and corresponding p-value between leaves with and without epicuticular wax (see Figure 4).
Determining Contact Angles
Students cut three leaf sections (3 × 3 cm2; avoiding veins and curled areas) from 1−2 collard leaves for this part of the experiment. Using double sided adhesive tape, students secure the leaf sections to the laboratory bench, near the edge. Students use the paintbrush to remove epicuticular wax from the left side of each leaf section, use a 1 mL syringe to pipet two drops of 0.1% Red 40 aqueous solution onto both left (unbrushed) and right sides (brushed) of each leaf section, and take separate smartphone photos of the droplets on each leaf section (see Figure 2). Contact angles of water droplets on the three leaf sections before and after brushing and removing epicuticular waxes are obtained by analysis of the photos using the ImageJ software. Step-by-step student instructions and instructor tips for the contact angle experiment are provided in Supporting Information.
Figure 4. Contact angle for applied water droplets both before and after epicuticular wax removal from collard greens leaves (N = 5).
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HAZARDS Although this experiment has few hazards, students should use care when cutting leaves with scissors. Syringes should be discarded according to institutional policy. In addition, students should wear chemical splash goggles, protective apron or laboratory coat, and gloves when spraying aqueous solutions containing Red 40 artificial coloring and Tween 20 surfactant.
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RESULTS AND DISCUSSION This experiment was (i) piloted by students in a nonmajors Principles of Plant Science course (PLSC 206; N = 94) during the fall of 2015 and (ii) implemented in a nonscience majors Survey of Chemistry II course (CHEM 112; N = 14) during the summer of 2016. The time required to perform both activities (wettability and contact angle) is about 90 min for paired student groups. Our results show that normal waxy leaf surfaces retain much less sprayed-on water than leaves having their waxes removed mechanically (by brushing) or than leaves sprayed with water containing Tween 20 surfactant adjuvant. Whole untreated waxy leaves of collard greens retained only 1.53 ± 1.00 mg/cm2 of sprayed-on water, whereas leaf surfaces with epicuticular waxes removed retained 8.07 ± 0.93 mg/cm2 of water (p
