Abstract:
Interactions of polycyclic aromatic hydrocarbons (PAHs) in crude oil with leaf plant tissues from Spartina alterniflora and Avicennia germinans were measured in laboratory experiments. Experiments were performed with both PAHs in air and water in contact with the leaf tissues. For air contact, PAH concentrations were measured over time using gas chromatography-mass spectrometry (GC-MS) and using 2-laser confocal microscopy which can visually image phenanthrenes in leaf tissue. For PAHs in the water phase, concentrations in the water and plant tissues were measured simultaneously using GC-MS.
Suggested Citation:
Pardue, John H.. 2021. Interaction of alkylated phenanthrenes and naphthalenes with leaf tissue of Spartina alterniflora and Avicennia germinans. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/YV6ZHYWY
Purpose:
This data was collected to better understand how polycyclic aromatic hydrocarbons (PAHs) behave when volatilized PAHs interact with plant tissues. The chamber experiment used 2 wetland plants, Spartina alterniflora (smooth cordgrass) and Avicennia germinans (Black mangrove), and phenanthrene as a model PAH. The experiment compared confocal microscopic visualization techniques for phenanthrene and chemical analysis of phenanthrene for the same plant tissues.
Methods:
Phenanthrene exposure to A. germinans and S. alterniflora leaves
Avicennia germinans and S. alterniflora plants were harvested with permission from a marsh just north of Fourchon Beach in Port Fourchon, LA and potted in a greenhouse at LSU using soil from the site. Plants were sampled in 2017 and 2018 and watered with a synthetic brackish water mixture and a micronutrient mixture every other watering. The longitude and latitude coordinates of the location of the marsh where samples of A. germinans and S. alterniflora plants were harvested (Fourchon Beach in Port Fourchon, LA) are 29 9.993’ N, and 90 5.564’ W. Plants were located on a segment of Fourchon Beach impacted by the Deepwater Horizon oil spill. Cleanup activities on this segment ceased in 2014 and some surface oil still exists on the beach surface.
To expose plants to phenanthrene, a chamber setup was constructed similar to a previous study in accordance with laboratory best practices (Wild et al. 2006). A glass thirty-gallon aquarium with a lid was first cleaned with soap and water and a 50:50 solution of hexane and acetone. Aluminum-faced duct tape was used to seal the edges where caulking was present. Phenanthrene (30 mg of 99.9%, Sigma-Aldrich) was dissolved in 10 mL of isooctane in an amber vial to decrease UV light exposure (Wild et al. 2006). To ensure the least amount of damage to the plant cuticles, isooctane was used instead of acetone because in a preliminary cuticle test, isooctane removed the least amount of cuticle. All 10 mL of the dissolved phenanthrene was applied to sections of glass paneling with a glass micro-syringe and the solvent was allowed to evaporate. Potted plants (either A. germinans or S. alterniflora) were placed in the chambers in either large glass jars or in a plastic container covered in aluminum foil. At the initiation of the study, initial leaf samples (day 0) were collected from A. germinans or S. alterniflora (two leaves for microscopy and two leaves for gas chromatography for three replicates each day) in Teflon centrifuge tubes. At the initiation of the exposure period, plants were placed in the center of the tank and the phenanthrene-contaminated glass panels were placed in the tank, leaning on the tank sides with the contaminated side of the glass facing the inside of the tank. The lid was placed on the tank and a small fan inserted into a hole in the tank lid to circulate the air through the chamber. Separate experiments were set up for A. germinans and S. alterniflora. At 24 hours and 48 hours, the lid was opened and leaves were sampled for microscopy and chemical analysis as described below.
Chemical analysis of PAHs
To measure the PAH concentrations in the cuticle of the leaf, the leaf was swirled in a glass jar containing dichloromethane (DCM) until the leaf is just covered (100 mL). The solvent, DCM, was then exchanged with 50:50 hexane acetone in a RapidVap Vacuum, N2 Evaporation System Model 7910000 (LABCONCO Corporation, Kansas City, MO), first by evaporating the DCM from cuticle sample to 1.5 mL at 30 °C, then adding 50 mL of 50:50 hexane acetone which was concentrated down to 10 mL at 70 °C. One milliliter aliquot of this 10 mL sample was used for analysis by GC-MS. PAH analysis was performed using a Hewlett Packard 6890N gas chromatograph equipped with a 5973N mass selective detector. The GC conditions were: 1 µl of the sample; DB 5 capillary column (30 m x 0.25 mm x 0.25 µm film), carrier gas (helium) at a rate of 5.7 mL/min, temperature program: injector 300°C, detector 280°C, oven temperature: 45°C for 3 min then increased at 6°C/min to 315°C and held for 15 min. For each set of samples, the QA/QC included blanks, internal standards for each sample, and a calibration check sample for each run. Once the GC-MS has run on the current PAH method, the data was reintegrated in Agilent Chemstation for data quality control and accuracy. The value in ng/uL was then multiplied by 1000 to get ng/uL and then multiplied by the reciprocal of the aliquot fraction, in this case, involved multiplying the final result by 10.
Imaging using 2-laser confocal microscopy
Both intact leaves, as well as epidermal peels, were used in the imaging process. To prepare the epidermal peels, leaves were placed in a beaker containing a mixture of 30% hydrogen peroxide and acetic acid (50:50) in a water bath between 80 °C - 100 °C for 12-20 hours until the leaves turned white (Jain 1976). The leaves were then rinsed with deionized (DI) water and the mesophyll tissue was softly brushed out. It was important to note which were the adaxial and abaxial sides of the leaves as well as the inside and outside of the epidermal peel for analysis later.
For whole leaves, a sterile six-millimeter diameter hole puncher was used to punch coupons out of the leaves. The circular coupon disks were places in an uncontaminated 50 mm diameter cell culture dish with a glass bottom. A few drops of Phosphate Buffer Saline (PBS) were pipetted on the leaf coupon and a small glass piece that was specially cut to fit in the 50 mm cell culture dish was placed on top to hold the leaf coupon down as flat as possible.
Imaging was conducted using the Leica TCS SP5 MP at Pennington Biomedical Research Institute (Baton Rouge, LA) equipped with an SP680 short-pass filter and a BS442 beam splitter for BP 320-430 (375/110) to PMT NDD2 and BP 486-506 (496/20) to PMT NDD1. The two-photon confocal settings to view phenanthrene are known to be at 375 nm (Wild et al. 2006). In order to fluoresce alkylated and non-alkylated phenanthrenes, the wavelength emission range was set between 350 and 390 nm with the laser power at 700 nm (Karlitschek et al. 1998). The high voltage photomultiplier tubes non-descanned detector number one (PMT NDD1) and two (PMT NDD2) were both set for 800. The lens was a 25.0 x 0.95 water objective (Leica HCX IRAPO L). The leaves were observed with MP microscopy as well as chemical extraction to compare grey values with the actual concentrations. At first, we examined the whole leaf for three days and then repeated the study with cuticle/epidermal peel for six days.
Image processing
For all of the images, the smart gain was set to 703. The average mean grey value (MGV) was taken as the intensity of phenanthrene fluorescence. The leaves were not flat enough to get a satisfactory image on one plane so z-stacks were created for the whole section of interest to be analyzed. The z-stack is a number of slices or images that are stacked the same distances apart in the z-direction so that when stacked can create an almost 3D image when interpolated. To analyze the z-stacks, the maximum intensity projection (max intensity) was chosen as the standard of comparison because, for each pixel in the z-stack, the highest intensity value is chosen regardless of where it is located in the stack and the image it created was with the pixels that are most “in focus” throughout the whole stack. The issue with max intensity could however arise if the image was photo-bleached for a number of reasons like the laser was on the spot for too long or if the sample was highly contaminated. On day zero of the experiment, it was paramount that the MP laser settings were set correctly to account for a larger range of intensity on the higher end so that photo-bleaching can be avoided.
Fiji by way of ImageJ was the software used for image analysis. Using Fiji, the image analysis method used on the Z-stacks was initiated by channel separation. Since the focus was on the phenanthrene channel (channel two), Image> Color > Split Channels was used and channel one was not used for this analysis. For the channel two window, the maximum intensity projection was created by Image > Stacks > Z project. The start and stop were filled in automatically and ‘Max Intensity’ was selected. Once the Max Intensity Projection was created, a “random points macro” generated ten points on the image. Since we had previously established that phenanthrene moved only through the interlamellar tissue in Avicennia, we adjusted random points to the nearest interlamellar space if it wasn’t assigned there randomly. Points were added to the Region of Interest (ROI) manager and the ROI was saved. Point intensities were measured and results were saved from the results window. Images were saved as a TIFF or PNG to preserve data integrity. The Look Up Table (LUT) was modified for qualitative analysis. The yellow and magenta pseudo-color LUT or green and blue LUT is a good choice for color blindness (Ferreira and Rasband 2012).
Provenance and Historical References:
Ferreira, T., and W. Rasband. 2012. ImageJ User Guide. 198.
Jain, K. K. 1976. Hydrogen Peroxide and Acetic Acid for Preparing Epidermal Peels from Conifer Leaves. Stain Technology 51 (3): 202–4. DOI: 10.3109/10520297609116701.
Karlitschek, P., F. Lewitzka, U. Bünting, M. Niederkrüger, and G. Marowsky. 1998. Detection of Aromatic Pollutants in the Environment by Using UV-Laser-Induced Fluorescence. Applied Physics B: Lasers and Optics 67 (4): 497–504. DOI: 10.1007/s003400050535.
Wild, Edward, John Dent, Gareth O. Thomas, and Kevin C. Jones. 2006. Visualizing the Air-To-Leaf Transfer and Within-Leaf Movement and Distribution of Phenanthrene: Further Studies Utilizing Two-Photon Excitation Microscopy. Environmental Science & Technology 40 (3): 907–16. DOI: 10.1021/es0515046.
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