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1. Differential effects of two explosive compounds on seed germination and seedling morphology of a native shrub,. 1. Morella cerifera. 2. Stephen M. Via a, Julie ...
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Differential effects of two explosive compounds on seed germination and seedling morphology of a native shrub, Morella cerifera

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Stephen M. Via a, Julie C. Zinnert a, Donald R. Young a

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a

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Corresponding Author: Julie Zinnert, phone: 1-804-828-0083, e-mail: [email protected]

Department of Biology, Virginia Commonwealth University, VA, USA

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Abstract Soils contaminated with explosive compounds occur on a global scale. Research demolition explosive

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(RDX) (hexahydro-1,3,5-trinitro-1,3,5-triazine) and trinitrotoluene (TNT) (2-methyl-1,3,5-trinitrobenzene) are the

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most common explosive compounds in the environment. These compounds, by variably impacting plant health, can

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affect species establishment in contaminated areas. Our objective was to quantify comparative effects of RDX and

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TNT on a native shrub, Morella cerifera, at two life stages. Morella cerifera seeds and juvenile plants were exposed

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to soil amended with RDX up to 1500 ppm and TNT up to 900 ppm. Percent germination was recorded for three

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weeks; morphological metrics of necrotic, reduced, and curled leaves, in addition to shoot length and number were

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counted and measured at the end of the experiment (eight weeks) for juvenile plants. All concentrations of RDX

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inhibited seed germination while TNT did not have an effect at any concentration. As contaminant concentration

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increased, significant increases in seedling morphological damage occurred in the presence of RDX, whereas TNT

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did not affect seedling morphology at any concentration. Overall the plants were more sensitive to the presence of

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RDX. Species specific responses to explosive compounds in the soil have the potential to act as a physiological

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filter, altering plant recruitment and establishment. This filtering of species may have a number of large scale

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impacts including: altering species composition and ecological succession.

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Keywords: Explosives Soil Contamination; Germination; Life Stages; Plant Morphology; RDX; TNT

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Introduction Explosive compound contaminated soils are present on nearly every continent (Pilon-Smits 2005); 68

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nations have declared contamination by unexploded ordnances (UXOs; UNICEF; 1995). The danger posed by these

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munitions is not simply their explosive nature but the ecotoxicological impacts of compounds contained within

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(Pitchel 2012). The most common organic explosive compounds in the environment are RDX (hexahydro-1,3,5-

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trinitro-1,3,5-triazine) and TNT (2-methyl-1,3,5-trinitrobenzene; Rylott and Bruce 2008; Pichtel 2012). Explosive

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compounds are readily absorbed via water into the roots and once inside the plant can induce a variety of

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physiological and morphological responses. These impacts vary based on contaminant concentration (Best et al.

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2006), environmental conditions (Best et al. 2009), plant species (Winfield et al. 2004; Pilon-Smits 2005), and plant

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life stage (Winfield et al. 2004). Among these response factors, life stage is by far the least understood.

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Three life stages of plants are: seed, seedling (juvenile), and adult. In relation to explosive soil

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contamination the seed stage is more sensitive than other life stages as it is especially dependent on environmental

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conditions (Vila et al. 2007). Soil contaminated with RDX and TNT can inhibit seed germination but concentrations

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required for impairment vary with species (Khatisashvili et al. 2009; Krishnan et al. 2000). A number of agronomic

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species are particularly tolerant to explosive compounds and some can tolerate RDX soil concentrations up to

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10,000 ppm without any impacts on seed germination (Rocheleau et al. 2005). However successful germination does

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not ensure survival and reproduction.

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In general seedlings are more resilient to impacts of explosive compound exposure than seeds (Winfield et

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al. 2004). The primary method which explosive compounds enter plants is by bulk flow of water in the xylem,

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induced by evapotranspiration (Paterson et al. 1990). Before entering the xylem, compounds pass through the

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Caspian strip into the endodermis. Ability to pass through this barrier depends on characteristics of both the

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compound and plant (Paterson et al. 1990). Compound structure also influences where a compound is sequestered

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and accumulated (Pichtel 2012). Substances that are highly hydrophobic (e.g. TNT) tend to bioaccumulate in roots

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(Paterson et al. 1990, Scheidemann et al. 1998; Best et al. 2009; Bretner et al. 2010) and those that are highly mobile

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(e.g. RDX) accumulate in leaves (Best et al. 2009; Bretner et al. 2010). The partitioning of RDX and TNT coincides

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with morphological impacts on various plant species.

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RDX is transported rapidly to leaves where it is bound to lignin, cellulose, and in vacuoles of cells without being degraded (Vila et al. 2007). In leaf tissue, RDX may undergo photolysis and degrade into a variety of

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compounds (Pichtel 2012), both harmful (e.g. NO) and benign (e.g. CO2). Reported adverse effects of RDX

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exposure include curled or irregular leaf margin, fused leaves, bifurcated leaves, atypical pigmentation, reduced

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shoot length, decreased leaf expansion, delayed emergence, atypical bilateral symmetry, thin stems, yellow spots,

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leaf chlorosis, necrotic lesions, underdeveloped roots, reduced root length, curled root tips, and decreased root

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exudate (Winfield et al. 2004; Vila et al. 2007). The most common response is leaf necrosis (Winfield et al. 2004),

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particularly at leaf edges (Vila et al. 2007). In contrast, TNT suppresses root and shoot growth (Gong et al. 1999;

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Peterson et al. 1998; Khatisashvili et al. 2009) with degree of impairment increasing with concentration (Krishnan et

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al. 2000). TNT is rapidly degraded (Burken et al. 2000) but can be found throughout the plant (Bretner et al. 2010)

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and may cause damage to both leaves and roots (Sens et al. 1999). These impacts have been observed across a broad

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range of plant species (Gong et al. 1999; Peterson et al. 1998; Winfield et al. 2004; Vila et al. 2007; Khatisashvili et

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al. 2009).

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Very few studies have investigated the impacts of RDX and TNT across life stages. There are studies on

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germination which terminate with germination success and others which follow species several days into the life of

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the embryo; most of these include transgenic and agronomic species. Furthermore, these studies tend to focus on

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only one contaminant while in a field setting multiple contaminants will most likely be present (Pitchel 2012). Lack

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of uniformity, avoidance of field conditions, and low representation of species found on contaminated sites prevents

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laboratory results from providing insight into field situations. Acting as a physiological filter, soil contaminants may

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even cause shifts in species recruitment and colonization. As such more research is needed into the basic responses

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of naturally occurring species to explosive contamination and an understanding of the mechanisms involved with the

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response to such contamination is also in need of clarification. Gaining an accurate understating of individual

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responses to explosives soil contamination provides insight towards a better understanding of community scale

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impacts. Our objective was to monitor impacts of two explosive compounds in various concentrations across two

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life stages of a native evergreen shrub Morella cerifera (also known as Myrica cerifera).

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Methods

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Plant material

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Morella cerifera L. (Myricaceae) naturally colonizes bombing ranges and occurs near munitions factories along the United States Atlantic coast (Naumann et al., 2010). This evergreen shrub has been studied extensively;

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especially physiology and stress responses (Young, 1992; Sande and Young 1992; Naumann et al. 2010; Via et al.

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2014). Morella cerifera seeds were collected from Hog Island, Virginia. Juvenile plants were reared from seeds for

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two months prior to experimentation. All seeds used were cold stratified for four months and scarified to remove

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the waxy coating of the fruit (Young and Young 2009).

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Soil

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Soil was prepared in a single batch for both germination and juvenile experiments. A 3:1 mixture of topsoil and

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sand was used to mimic natural conditions for M. cerifera. Soil was thoroughly dried and then treated with 200 ml

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acetone and the appropriate amount of the explosive compound. The acetone mixtures were sprayed on soil and

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allowed to dry for 72 hrs to allow acetone to evaporate. Soil was dried in the dark to prevent photodegradation of

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compounds (Ait Ali et al. 2006). Concentrations ranged from 0 ppm (Reference) to 1500 ppm for RDX and 0 ppm

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(Reference) to 900 ppm for TNT. Ranges were chosen to correspond with concentrations used in previous studies

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and as a conservative estimate of those found in contaminated field sites (Schneider et al. 1996; Best et al. 2006; Via

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et al., 2014).

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Germination

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Contaminated soil was placed in Petri dishes (n=5) and misted with water until damp. A total of 50 seeds were

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placed in each dish and all dishes were sealed in plastic bags to maintain optimal humidity (60%), with 14 hour

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day/night cycles, and a 25-20O C temperature range. Seeds were kept in a climate controlled Conviron S10H growth

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chamber (Conviron, Pembina ND, U.S.A). Seed germination was monitored daily for 21 days. A seed was

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classified as germinated when the radical emerged from the seed coat. Germinated seeds were removed from dishes

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to avoid duplicate counting.

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Juvenile Morphology

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Plants of roughly 12 cm in height were grown two to three per pot (n=5) and placed in catch dishes to

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prevent leaching of contaminants. Plants were grown in a glasshouse, watered to prevent wilting, and kept at an

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ambient temperature of 24o C ± 7oC (relative humidity ≈ 60%). Leaf count, necrosis, curling, and reduced leaf area

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were measured for 8 weeks. Leaves were defined as necrotic if ≥ 30% was covered in lesions, as curled if the leaf

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deviated ≥45o from the plane of the mid-vein, and as reduced if at maturation was