v2i3-murphy

Research Paper

Sentinels and What They Tell Us: Monitoring Metal Content as a Probe of the Health, Nutrition, and Environment of Deer, Duck, Monarch Butterflies, and Asian Lady Beetles

Jason Christopher Reynolds, Bailey Burns, Caroline Cooper, Ebony Echols, Aubrey Hill, Casey Kropp, Alexandra Marchese, Benjamin Marler, Shelbee Mathews, Victoria Parks, Seth Warren, Aubree Archie, Emily K. Fike, Logan Gildea, Sidney Ison, Mary Stanley, and Maureen Kendrick Murphy*

Department of Chemistry & Biochemistry, Huntingdon College, Montgomery, Alabama 36106, USA.

*Corresponding author, Maureen Kendrick Murphy, email:maureenm@hawks.huntingdon.edu

Received 16 April 2018, revised 2 August 2018, accepted 3 August 2018

Publication Date (Web): August 3, 2018

© Frontiers in Science, Technology, Engineering and Mathematics

Abstract

The health, nutrition, and environment of several sentinel species were monitored by ICP-OES to reflect normal and abnormal levels of trace elements. Levels of trace elements in these species can be correlated with trace element concentrations in the surrounding environment. We present extensive studies of the metal content, and in one case, boron content in: (a) deer hooves from ten counties in Alabama, (b) Mallard duck feet from Arkansas counties, (c) Asian lady beetles from Alabama, and (d) Monarch butterflies obtained during 2014-2016 from overwintering locations adjacent to the Monarch Butterfly Biosphere Reserve in Mexico. Our results demonstrate that metal and boron content in these selected species can be used as sentinels of health, nutrition, and the environment. The usefulness of ICP-OES for bio-monitoring programs and for engaging undergraduates in broad research projects with a common goal is demonstrated.

Keywords

Sentinel species, ICP-OES, Optical emission spectroscopy, Plasma, Calibration, Digestion, Metals, Bio-monitoring, Bio-accumulation

Introduction

Interest in the chemistry and biochemistry of sentinel species that can serve as bio-sensors of the environment as well as report on the species’ health and nutrition received heightened awareness when Chemical & Engineering News published a cover story on the topic (Ritter, 2017). In order for a species to serve as a sentinel, the species should be readily available, easily handled, and have consistent and regularly measurable responses to health, nutrition, and changes in the air, water, and soil in the surrounding environment.

Various sentinel species have been used to monitor the environment.  These include salmon, honeybees, liches, seagulls, mussels, and caribou (Ritter, 2017).  Lead levels in salmon were monitored concomitant with deteriorating water quality and high lead levels in the waters where the salmon were collected.  Honeybees have been analyzed for neonicotinoids via GC-MS to determine the source of these compounds, which have been implicated in the decline in the U.S. honeybee population by 30-40% in the last decade. Table 1 summarizes the results from the determination of organic and inorganic compounds/elements in research involving sentinel species.

The purpose of this research was three-fold: (a) to examine the metal and boron content in sentinel species in order to provide baseline levels and examine the possible effects of environment on metal and boron content related to the health and nutrition of each species, (b) to engage undergraduate research teams in broad projects with a common goal, and (c) to demonstrate the usefulness of ICP-OES in bio-monitoring research programs.

Our Sentinel Species

a) Deer     

The white-tailed deer (Odocoileus virginianus), also known as the whitetail or Virginia deer, is a medium-sized deer native to the United States and common in Alabama. During the 2017-2018 hunting season, state game wardens in Alabama reported a total of 76,007 deer harvested in the state (https://game.dcnr.alabama.gov/Report/County/Deer). Deer were selected as sentinel species in Alabama due to its prevalence in the state, its presence on private and public land, its connection to air, soil, vegetation, and its ease of analysis.  Deer hooves were selected as reporters of health, nutrition, and the environment because they are composed of keratin (Wang, 2016), and like human fingernails, concentrate metals and trace elements from diet and the environment into their hooves.

b) Mallard Duck

The Mallard duck (Anas platyrhynchos) is a dabbling duck that breeds throughout the temperate and subtropical Americas and in various locations around the world. Mallards live in wetlands, eat water plants and small animals, and are social animals preferring to congregate in groups or flocks of varying sizes. These ducks were selected as sentinels because of their commonness in the south, especially around lakes in Arkansas, their connection to water, plants, and air, and the ease with which duck feet can be analyzed.

c) Multicolored Asian Lady Beetle  

The multicolored Asian lady beetle (Harmonia axyridis) is native to Asia but occurs in many areas of the United States. This insect was imported and released as early as 1916 in attempts to naturally control certain insect pests. Over the years, federal, state and private entomologists released the insect at a number of locations. Since 1998, Asian lady beetles have been reported in every county in Alabama. Outside its native area of eastern Asia, these beetles primarily show only one of three basic color types: red or orange with black spots, black with four red spots, and black with two red spots. The form with anywhere from zero to 19 spots is known as the succinea form.  Figure 1 depicts the sixteen known multicolored Asian lady beetles. Three of these (#3, #10, and # 13) were analyzed in this study; all were located in Alabama, and a total of 398 samples were collected from Number Six Mine Lake in Franklin County, Alabama. This lake was formed from the iron mining spoils when the mine was operated from 1848-1972. Iron spoils lakes form when open-cut mining operations cease and the remaining pit fills with ground, surface, and rainwater. Number Six Mine Lake is 63 acres and has a maximum depth of 25 feet. 

d) Monarch Butterfly

The Monarch butterfly (Danaus plexippus) is a milkweed butterfly in the family Nymphalidae. It is a familiar North American butterfly, and is considered an iconic pollinator species. Its wings feature an easily recognizable black, orange, and white pattern. The eastern North American monarch population is notable for its annual southward late-summer/autumn migration from the northern and central United States and southern Canada to Florida and Mexico. Because of its common presence and mobility, it was selected as a sentinel species in our studies.

Table 1. Sentinel species analyzed (1980-2018), adapted from Ritter (2017).

1Technique abbreviations for methods: AAS (atomic absorption spectroscopy), AES (atomic emission spectroscopy), GC-MS (gas chromatography/mass spectrometry), ICP-MS (inductively coupled plasma/mass spectrometry), ICP-OES (inductively coupled plasma optical emission spectroscopy), MS-mass spectrometry.

Materials and Methods

All chemicals were obtained from Sigma-Aldrich Chemical Co. and all ICP-OES standard samples were used as received from Inorganic Ventures, Inc. Mineral free nitric acid (HNO3) was purchased from Sigma-Aldrich Chemical Co. Ultra-high purity (UHP) argon gas used for the plasma generation and auto-sampler was obtained from Air Gas, Inc.

Elemental Analysis of Samples

Determination of Al, B, Cu, Zn, Co, Mg, Mn, Fe, Cr, Cd, Ni, Pb, and Zn content was made directly on each final solution using an inductively coupled plasma optical emission spectrometer (Varian VISTA ICP-OES) and associated auto-sampler. Standard solutions of each sample were prepared according to Varian manufacturer procedures for ICP-OES. Known standards (IV-26) containing 26 elements/metal ions were purchased from Inorganic Ventures, Inc. at various concentrations, including 1000 mg/L and 5000 mg/L. Standards were volumetrically diluted to 500 ppb, 1000 ppb, and 5000 ppb for use as calibration standards. All metals/elements standard solutions selected for analysis passed the calibration test of the ICP-OEP software within +/- 5.0%.

A calibration blank was composed of the same solvent as was used in the analysis of samples and standards (matrix-matched).  All samples and standards were analyzed in triplicate in the same run and under the exact same instrumental settings. Prior to loading each measured sample into the auto-sampler of the ICP-OES, each sample was filtered through a syringe filter into a polycarbonate sample tube. The analysis of each sample via ICP-OES was completed without error signals. All calibration curves exhibited r2 values greater than 0.9990. The sample preparation and analysis methods used in this research duplicate those of other researchers (Hidirogou and Williams 1986; Abdulrahman et al 2012) in which percent recovery tests were conducted to certify the ICP-OES protocol used in this paper.

Deer Hoof Sample Preparation

The deer hooves were removed from each male deer just after killing and were immediately frozen at 4_C after assigning a sample number to each hoof. For analysis, each hoof was cut 1.0 cm above the keratin hoof to remove non-hoof material, dried, and weighed. The mean mass of each deer sample (2 hooves per sample) was 65.03 g, with samples ranging from 57.05-71.43 g. When ready for analysis, the trimmed hooves were allowed to defrost, and were dissolved completely in concentrated nitric acid and kept covered in a hood overnight to prevent excessive foaming, while digesting. After filtering each sample solution through Whatman-Grade 4 qualitative filter paper, exactly 1.0 mL of each sample was removed from the hooves-nitric acid mixture and was diluted volumetrically to 100 mL with 7% nitric acid. A total of 34 deer hooves from ten counties in Alabama were analyzed.

Mallard Duck Feet Sample Preparation

The Mallard duck feet were removed from each duck just after killing the duck and were immediately frozen at 4_C after assigning a sample number to each pair of duck feet, and weighing each pair. When ready for analysis, the duck feet were allowed to defrost, and were dissolved completely in concentrated nitric acid and kept covered in a hood overnight to prevent excessive foaming, while digesting. After filtering each sample solution through Whatman-Grade 4 qualitative filter paper, exactly 1.0 mL of each sample was removed from the golden colored nitric acid mixture and was diluted volumetrically to 100 mL with 7% nitric acid.

Figure 1. Sixteen known variants of the multicolored Asian lady beetle. [Photo credit: http://www. entomart.be/INS-0038.html]

Asian Lady Beetle Sample Preparation

Three hundred and ninety eight (398) Asian lady beetles were collected at Number Six Mine Lake in Franklin County, Alabama.  The beetles were sorted into three variants, labeled as Beetle #3 (red with black spots), Beetle #10 (orange with black spots) and Beetle #13 (orange with no spots). See Figure 1 for a depiction of each variant.  Each sample was subdivided into groups of 16-25 lady beetles to make a mass of 1.25 grams.  Each of these samples was dissolved and digested in 25.0 mL of concentrated nitric acid, then filtered through a syringe filter.  Following this, exactly 1.0 mL of each sample was diluted to the mark with 7% HNO3 in a 100.0 mL volumetric flask. Each sample was analyzed and the mean of each variant of beetle samples was reported. 

Table 4. Metal Analysis in plants Salvinia, Eichhornia, Hydrilla and metal analysis of water samples determined by Atomic Absorption Spectrophotometry (Perkin Elmer; Analyst 300 [concentration of metals in ppm]. Reference: Sabale SR, Tamhankar BV, Dongare MM, Mohite BS (2012) Extraction, Determination and Bioremediation of Heavy Metal Ions and Pesticide Residues from Lake Water. J Bioremed Biodegrad,  3, 143.

Asian Lady Beetles

Results from the ICP-OES analysis of three variants of multicolored Asian lady beetles collected from an iron mining spoils lake in Franklin County, Alabama showed comparative mean metal concentrations of 398 beetle samples to be Ni>Fe>Cu>Zn>Al>Mo>Pb>Co>Cr>Cd. However, as seen in Table 5, these results are affected by the extremely high Ni content detected in one variant (Beetle #13, See Figure 1), the multicolored Asian lady beetle that was orange with no spots. This lady beetle was more difficult to find, as it was always present in lower in numbers in groups of lady beetles discovered. For five separate samples of 23, 23, 24, 23 and 16 beetles of this kind (orange, no spots), the Ni concentration detected was over 100 ppm for each sample, with a mean of 150 ppm for all Beetle #13 samples (See Table 5). A research study on the identification and quantification of the odorants produced by the multicolored Asian lady beetles (Cai et al, 2007) reported that the orange lady beetle (Beetle #13 in our study) produced and contained more methoxypyrazine, the compound responsible for the foul smell of the lady beetles, than the other variants. In addition, the enzyme that catalyzes the reaction that places a methyl and/or a methoxy group on the pyrazine molecule is a nickel-containing enzyme (Ragsdale, 2009). Detection of comparatively high levels of Ni in this variant of multicolored Asian lady beetles may be an indicator of the different role of this variant in the multicolored Asian lady beetle population.

Table 5. Mean metal content of three multicolored Asian lady beetle variants: #3:red with black spots, #10 orange with black spots, and #13 orange with no spots determined by ICP-OES. See Figure 1 for detail of each variant.

Figure 3. Mean metal concentration in ppm in Monarch butterfly wings (n=54 butterflies) from Mexico, collected during 2014-2017. Error bars reflect +/- 5% of measured value, based upon 5% error in each metal concentration calibration curve (ICP-OES).

Monarch Butterflies                                                                                                                                    

Results from metal content in 54 Monarch butterflies (27 sets of 2 wings per sample) showed high concentrations of Fe and Cu, some 12 times higher than other metal concentrations detected. Every sample analyzed showed high Fe and Cu concentrations compared to the other metals detected (See Figure 3). The order of concentration levels detected in our Monarch butterfly wings was found to be: Fe>Cu>Al>Mn>Zn>Pb>Ni>Cd>Co>Cr>Mo. Monarch butterflies can obtain these metals from drinking water which may be contaminated with mining spill-tainted water in Mexico. A toxic spill in August 2014 at the Grupo Mexico copper mine in Sonora, Mexico spewed 10 million gallons of mining waste, including copper sulfate, into the Sonora and Bacanuchi rivers. Hundreds of miles of waterways in Mexico were contaminated with metals from the mining waste. The Monarch butterfly population has decreased by over 80 percent throughout the past few decades and estimates placed 145 million butterflies at the wintering grounds in 2017 compared to 1 billion in 1990 (Skoiec et al, 2017). Our results show high levels of metals in Monarch butterflies collected in Mexico from 2014-2017, which may indicate risks to the health of the species.

Conclusions

Our research demonstrates that ICP-OES analyses of fresh deer hooves, Mallard duck feet, multicolored Asian lady beetles, and Monarch butterflies reflect comparative levels of metals and boron, indicating similar nutrition and health of these sentinel species and in some cases, indicating elevated levels of elements produced by environmental effects.  These sentinel species in our study reflect comparatively high Fe levels in deer hooves from iron mining lake areas, healthy Zn:Cu ratios in deer hooves from 70% of counties studied, increased B content in ducks killed near rice fields adjacent to lakes where B is bio-accumulated in the lake, and high Fe and Cu content in Monarch butterflies due to mining spills in Mexico affecting wintering sites of the butterflies. In addition, Ni-containing enzymes that catalyze the O-methylation of pyrazine (Ragsdale, 2009) may be responsible for increased Ni content in the orange, non-spotted multicolored Asian lady beetle that has been found to secrete the most methoxypyrazine as an odorant when compared to other variants of these Asian lady beetles (Cai et al., 2009). The results from this research can be used to gauge the health, nutrition, and environment of these sentinel species. Finally, the use of ICP-OES coupled with standard analytical methods, provides an engaging method to involve undergraduate research teams in broad bio-monitoring projects with a common goal of understanding the chemistry of sentinel species.

Acknowledgements

We are grateful to Steve Rodopoulos and the Montgomery Water Works (MWW) for the donation of the Varian VISTA ICP-OES instrument, supplies, and training. We also acknowledge Karen Blake of MWW, for training faculty and students in the use and maintenance of the instrument, and Chad Commander of Agilent Technologies, Inc. for getting the instrument operational and software optimized. We acknowledge two researchers whose helpful peer-reviews contributed to the improvement of this manuscript.

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Citation:

Jason Christopher Reynolds, Bailey Burns, Caroline Cooper, Ebony Echols, Aubrey Hill, Casey Kropp, Alexandra Marchese, Benjamin Marler, Shelbee Mathews, Victoria Parks, Seth Warren, Aubree Archie, Emily K. Fike, Logan Gildea, Sidney Ison, Mary Stanley, and Maureen Kendrick Murphy* (2018) Sentinels and What They Tell Us: Monitoring Metal Content as a Probe of the Health, Nutrition, and Environment of Deer, Duck, Monarch Butterflies, and Asian Lady Beetles, Frontiers in Science, Technology, Engineering and Mathematics, Volume 2, Issue 3, 148-158