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Acid Rain Effects on the Production and Composition of Epicuticular Waxes of Turf Grass, Poa-pratensis-lolium-festuca

Natalie Buch Syracuse University, Syracuse NY

Mentors: Maureen Conte and JC Weber

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Abstract An experiment was performed for 1 week to determine the physiological and morphological response of Poa-pratensis–lolium-festuca to simulated acid rain. Five pH treatments (5.6,4.4,3.5,2.4 and 1.6) were selected to examine if any differences in wax production and composition resulted from this stress. The five treatments were chosen to resemble real pH rainwater values -unpolluted rainwater pH or control, the current northeastern US pH, the current Hong Kong pH, the lowest pH recorded in Europe and the lowest recorded in the US, respectively. The waxes were quantified and identified with a GM/MS after being oven dried, ultrasonicated with an organic solvent, centrifuged, folch’s extracted, transesterified, and finally derivatized the samples. There was a slight discrepancy between the control and the rest of the treatments in the total new growth, however no trend in growth or biomass with increasing acidity. pH 1.6 had a lower C:N molar ratio, this treatment also had a higher total wax concentration –confirmed with the SEM qualitative analysis- and an increase in the longer chain distributions of alcohols and acids. The vegetation was characterized by high abundance of C 26 n-alcohol. The results of relative distribution of class and chain length of compounds were very similar and reproducible, but the results suggest there was subtle change as a physiological response. No trends of the isotopic fractionation appeared with increasing acidity, indicating the stomatal mechanism was not altered. Keywords Acid rain, leaf waxes, wax class composition, chain length distribution

Introduction

As anthropogenic pollution, mainly from fossil-fuel combustion, continues to be released

into the atmosphere acid deposition comes along as a product of Sulfur dioxide and Nitric oxides

after being oxidized in the atmosphere. This term is applied to precipitation with a pH below 5. In

the United States and more specifically the northeast, acidification values have decreased since

the Clean Air Act (1990), however the global emissions have increased. Asia, Africa, Canada and

South America are more likely to continue increasing the discharge given the reality of

population growth and their potential for industrial expansion (Galloway, 1989). High levels of

sulfuric, but also nitric acid can affect the growth of vegetation, leaf and species composition, soil

buffering capacity among other components of terrestrial ecosystems (Larssen et al, 2006).

Plants protect their aerial surfaces with an external hydrophobic cuticle layer, coated with

epicuticular wax, which acts as an obstacle to drought, low temperature, UV-B radiation and

pathogens and herbivores (Maffei, 1996). Since waxes are at the interphase with the atmosphere,

they also play an important role in gas exchange by reducing transpiration.

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The morphology of the waxes is mainly influenced by growth conditions (Baker and Hunt,

1986). The crystalline deposits that overlay the cuticle vary in forms, such as plates, ribbons,

tubes and rods (Baker, 1982). Variations in wax morphology may be caused by differences in the

chemical composition of the material or different arrangement of the same compounds. The

composition of the leaf wax is subject to continuous change as leaves expand and constantly

ablate by natural exfoliation, wind and dust abrasion and regenerate the waxes (Hadley and Smith

1989; Rogge et al. 1993). They also vary with species, genus and parts within a plant according to

their ecological function in the environment (Eigenbrode, 1995).

These waxes are formed when the epidermal cells secrete fatty acids that polymerize from

exposure to oxygen. They are a complex heterogeneous mixture of long-chain (C20-C34) fatty

acids and their derivatives that during biosynthesis are further modified into major aliphatic

components (Rhee et al, 1998), mainly hydrocarbons, esters, primary alcohols, triacylglycerols,

fatty acids, ketones and aldehydes, (Barthlott et al, 1998) that reflect the episodic nature of the

transported atmospheric material.

Manipulation of the acidity of the water source could be used as a projected response of

plants to the possible future advection of weathering (Baker and Hunt, 1986) and pollutants, for

waxes are indicators of air pollution effects (Turunen, et al 1997). Growth will also be monitored

to estimate biomass differences among treatments. The SEM will allow me to compare and

understand the morphological changes of the wax structure and distribution on the leaf surface.

Imaging the conglomerations that form in the cells that surround the stomata can tell more about

wax production and crystals of the grass. A qualitative examination of the stomatal openings

should reflect changes in stomatal conductance and therefore determine carbon fractionation,

even though terrestrial photosynthesis discriminates against 13CO2 (Conte and Weber, 2002), if

plants are stressed they tend to utilize less 12CO2 and have a heavier carbon signal (Igamberdiev,

2003).

Waxes are not only important in an individual level because they define the surface

properties of the leaf, but can also affect the secondary production of its ecosystem. The effects of

their composition can affect grazers and upper trophic levels that obtain energy from this plant

source. An example of this is how cuticle thickness interferes with bacterial colonization of the

plants (Lindow et al, 2003). A lower relative abundance of waxes can lead to modifications due

to more microbial activity. The extent at which the cuticle is being affected should be assessed

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because leaves become less hydrophobic following exposure to gaseous air pollutants and

become more susceptible to nutrient leaching (Percy and Baker, 1987). Air pollutants also alter

the structure and arrangement of the waxes, and this can result in wax erosion (Riding and Percy,

1985).

Waxes are biologically important, but their role protecting plants is of economic interest

because they have a commercial value (Seigler, 1998), they are important for the pharmaceutical,

cosmetic and food industries (Olubunmi, 2010). Understanding the changes in wax production

and composition also has major implications and use in paleo studies. Poaceae are a source of

biomass in the geological record of soils, as well as lake and marine sediments (Rommerskirchen,

2006). Grasses are useful for this experiment, for they have a rapid growth rate. The main wax

component of grasses are alcohols or esters, in rainforests long chain alkanes are more abundant

(Tulloch and Hoffman, 1977; Vogts et al, 2009). In addition they are a major food source for both

humans, grazing and domestic animals.

Objectives

My objectives include studying how different pH levels of wet acid deposition affect

epicuticular waxes of leaves by measuring total wax biomass, wax concentrations, component

class composition of n-alkanes, n-alcohols and n-acids, and carbon and nitrogen abundance and

isotopic fractionation. The leaf phyllosphere will also be examined to correlate it to the

physiological results.

Materials and methods

Sod grass (Poa pratensis- lolium- festuca) was purchased from Mahoney’s Garden Center

in East Falmouth MA, put into pots, and allowed to acclimate in a growth chamber for one week

before the experimental simulated precipitation treatment began. At the start of the experimental

phase, the grass was cut to the lowest extent possible to be able to measure the new growth. The

grass was under 200 C, and received approximately 1,550 µE m−2 s−1 (medium-high setting in

the chamber) for a 16-h light period per day. As for simulated precipitation, five treatments with

pH values of 5.5, 4.4, 3.5, 2.5, and 1.5 were prepared by dilution of reagent grade sulfuric acid

and nitric acid (70% and 30%, respectively) with deionized water. Rainfall was simulated

applying the acid solutions from hand-held atomizers once a day between 20:30 and 21:00. I

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selected this period of time because the light of the growth chamber turned off at 21:00; this way

I was reducing evapotranspiration. 75ml of water was calculated to be 2x the average rainfall (0.1

inches) per area (6.5 cm) in the northeast, and it was supplemented with spritzers that contained

the acid treatments. These treatments were chosen for, pH 5.6 is the natural pH of unpolluted

rainwater, pH 4.4 is the average pH of northeastern US, pH 3.5 is the average rainfall pH in

Tseung Kwan O, Hong Kong and 2.4 is the lowest pH recorded in Europe (Scotland), and pH 1.5

is the lowest recorded in the US (West Virginia) . Soil pH was also measured, the first and the

final day of the experiment. The soil was collected and diluted with water at a 5:1 ratio and

measured by a Hannah pH probe.

After 7 days of growth, the grass was clipped and weighed to obtain the total wet biomass

of each treatment. I isolated 3 blades from each treatment for the SEM analysis before drying the

samples to obtain the dry mass. The isotopic and epicuticular composition of the grass species

was then to be examined. A total of 10 (three duplicates, two single samples, and a procedural

blank) samples were tested for extraction of waxes. Subsequently, they were all oven-dried at 55-

60°C overnight. The blades were grounded into a powder using a MeOH- rinsed mortar-pestle

and 50 mg of each sample were transferred into solvent rinsed 16 mm Pyrex tubes for

epicuticular wax extraction. At this phase, a subsample was taken (≈2 mg) for carbon and

nitrogen bulk abundance and stable isotope analysis using a Europa ANCA-SL elemental

analyzer preparation unit interfaced with a Europa 20-20 Continous-Flow Isotope Ratio Mass

Spectrometer in the Ecosystem Center’s Stable Isotope Laboratory.

An internal standard mixture containing compounds from each wax component class was

added to all the pyrex tubes, to be able to quantify the wax compounds. The concentrations of the

standard were 10.20 µg of 21 fatty alcohol, 10.28 µg of 5 alpha cholestane, 11.26 µg  of 23 fatty

acid and 11.31 µg of 36 alkane. The ultrasonic extraction consists in immersing the dry material

in an organic solvent, dichloromethane (DCM) and ultra-sonicating twice with a Misonix

Ultrasonic Processor for ten minutes to extract the lipids. All samples were then centrifuged

around 3000 rpm and the DCM organic extract vacummed filtered through fritted funnels into

separatory funnels to eliminate transfer of leaf particulates. A Folch’s extraction was done using,

10 ml of 0.88% aqueous KCl to separate water-soluble proteins and carbohydrates from the

organic extract. The extracts were then passed though anhydrous sodium sulfate columns to

remove excess water that would interfere in the reactions of transesterification, when the complex

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molecules are broken down to component classes and free fatty acids are derivatized into methyl

esters.

The transesterification process occurs after resuspending the extracts in 0.5 ml toluene and

adding 2 ml transesterification reagent (10% methanolic HCL),(made with 30 ml of anhydrous

methanol, 3.0 ml of acetyl chloride). The result will be the phase separation of the glycerol from

the extract products. Esterification occurs after the reaction mixture is capped under nitrogen and

heated at 50°C overnight. After this step the fatty acids will convert to fatty acid methyl esters.

To remove the non-lipid contaminants from the extracts a hexane extraction was done, it consists

in shaking the mixture with an aqueous 5% NaCl solution (Christie, 1993) and 2ml of hexane.

The transesterified extracts were hexane extracted and evaporated to just dry using a Svant

SpeedVac and resuspended in DCM.

The next step will render highly polar materials to be sufficiently volatile without thermal

decomposition or molecular re-arrangement. During the derivatization process, the dry samples

were resuspended in 50µl pyridine and 50µl of the catalyst 1% TMCS/ trimethylchlorosilane and

the silylating reagent BSTFA/ N,O-bis(trimethylsilyl) trifluoroacetamide were added , capped

under nitrogen and heated at 55ºC for 1 hr. Derivatives react with active hydrogen atoms of the

sample and a trimethyl group is attached to the hydroxyl groups. Derivatization is required, for

fatty acids and fatty alcohols are not volatile, otherwise the GC could not monitor these (Orata,

2012). By derivatizing, the adsorption of the analyte in the GC is reduced and it will increase

detectability.

In a gas chromatography (a GC Agilent technologies 7890A model with a CPSil SCB

60m x 0.25mm x 0.25µm film column and a temperature program of 50(2) 150 320 (30) (for

a total of 85 min) volatile organic compounds are separated as a result of equilibria between the

solutes and GC column identifies and quantifies individual classes of waxes and molecular

species. Afterwards, a Mass Spectrometer (MS Agilent technologies 5975C with triple-Axis

detector) analyzed the components of the samples by charging the specimen molecules,

accelerating them through a magnetic field, breaking the molecules into charged fragments and

detecting the different charges (Christie, 2003). Chromperfect software (Justice Laboratories) was

later used to retrieve the peak areas of the FID readout that were used for quantification.

Before the Scanning Electron Microscopy (Zeiss Supra40VP) could be utilized to

examine the samples, a blade tip from the pH 1.6, 35 and 5.6 treatments were cut in small pieces

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and affixed to aluminum stubs by double-sided adhesive tape and later air-dried (Barthlott, 1998).

The stubs were then coated with a platinum layer. Both sides of a blade of the three treatments

will be observed, for a total of 6 samples.

Results  

The growth of Poa pratensis-lolium-festuca was slightly affected by the acid treatments,

the control had about 24% taller than the average of the rest of the treatments. There was not a

declining trend in the total growth (Fig.1). On average, the difference in biomass between the

least and most acidic treatment was 0.04 g per dry weight (Fig. 2). However, there was a notable

morphological difference between the pots of the pH 1.6 treatment and the rest; foliar damage

was evident in all the blades of this treatment.

As to elemental analysis, the percent C by dry weight decreased with increasing acidity

(Fig. 3). Nitrogen content increased with increasing acidity (Fig.4). For this, the lowest pH

treatment had a much lower carbon to nitrogen molar ratio and the ratios increased

logarithmically with increasing pH (Fig.5). As to isotopic fractionation, the variations in the δ 13

C and δ 15 N values did not correlate to expected results (Fig. 6, 7). The pH 3.5 and pH 5.6

treatments δ 15 N had high variability between replicates (>0.5 mils).

In all treatments n-alcohols were the dominant class component, the relative distribution

percentage of n-alcohols in the treatments ranged from 62 to 72% (Fig. 8). n-acids formed ~20%

of the waxes, whereas n-alkanes conformed ~10%. Treatment pH 1.6 had higher total wax

concentration to organic carbon ratio (Fig. 9). It had an average of 111.11 µg/g C; the treatments

pH 2.4, 3.5, 4.4 and 5.6 had 80.25, 86.49, 83.54, 80.73 µg/g C respectively after adding the FAL

24-32, FAME 20-34 and ALK 29-33.

The molecular distribution of longer chain alcohol and acid components increased with

acidity. 26 fatty alcohol in the wax on the leaves in the pH 1.6 treatment is ~21 µg/g C or 30%

higher (Fig. 10a) than the average of the rest of the treatments (Fig. 10b-10e). The long chained

C28 and C30 fatty alcohols had higher concentrations in the most acidic treatment (Fig. 11a).

Although there is variation between replicates, this treatment also had higher concentrations in

the longer fatty acid chain lengths, (Fig. 12) A higher abundance of longer chain compounds in

the C30-C34 n-acids correlates positively with the longer chain abundance of C30-C32 n-alcohols

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(Fig. 13). A peculiar outcome of the 1.6 pH treatment is seen in the alkane distribution of C29 and

C31 (Fig. 14). This treatment’s average resulted in a higher C29 in relation to C31 pH (Fig. 15), I

should note that there was greater variability in the concentration of alkanes in the lowest acidity

treatment. Another interesting result is the higher concentration of sterols in the pH 1.6 treatment

(Fig. 16).

The images retrieved from the SEM support the quantitative results of total wax; a more

uneven distribution was observed in the control treatment (Fig. 17b vs Fig. 19b). Less wax

conglomerates were seen near the midrib of the leafs (Fig. 17c ). No differences were noted in the

stomata among treatments (Fig. 17e vs Fig. 19c ). Crystalline structures were only present in the

most acidic treatment (Fig. 18e,f), these ranged between ~10 and ~30 µm. As to the intermediate

treatment, the wax distribution looked similar to the 5.6 pH treatment (Fig. 18b).

Discussion

It has been proven that plant species vary in their susceptibility to the effects of acid rain,

and Poa- pratensis-lolium-festuca had a physiological response after the period of one week. The

minor differences in growth and biomass are not surprising because this outcome has been

observed in many other studies, for growth is highly dependent on the soil’s buffering capacity

(Amthor, 1984). And, the soil utilized for the experiment was very alkaline (Table 1).

The trend of lower carbon percentage in relation to dry weight with increasing acidity has

been observed in an earlier study. Ferenbaugh’s data of Phaseolus plants in 1976, indicated that

the acidified plants sustained a loss of capacity to produce carbohydrates. A theory this author

gives is that this might due to a slight increase in the respiration rate that would not allow the

plant to catabolize the additional carbohydrate. For the pH 1.6, the decrease in carbon shows that

even though there was a higher wax concentration the acid rain is still affecting carbon abundance.

An explanation for the higher nitrogen abundance of the most acidic treatment could be explained

by the plant incorporating N from the nitric acid irrigation. However, a study done in the

Howland forest in Maine states that by incorporating N plants also increase carbon sequestration

and this was not the case for the C of my experiment. Nonetheless, a possibility is that since N

and dark respiration rates are related, they increase linearly (Osaki, 2001).

The relative distributions of the class components are very similar and reproducible, this

was expected, for the reason that they are characteristic of their genus and the molecular

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composition determines the three dimensional structure of the wax which can vary greatly among

different types of plants. The SEM images support the results of total wax concentrations, higher

wax concentrations in the most acidic treatment as a defensive response. Looking into the

molecular distribution of alcohols and acids, the trend of higher concentration in the longer chain

compounds suggests that the plant is subtly responding by producing longer and therefore, more

resistant and refractory compounds in order to protect itself from degradation. This effect has also

been observed after plants have been under water stressed conditions (Bondada, 1996). The

increase of total wax concentration of treatment pH 1.6 is also noteworthy; it’s another defensive

response after such a short period of time.

Yet, after qualitatively examining the stomata of treatments pH 1.6, 3.5 and 5.6 there were

no notable differences in the morphology. Which leads to the isotopic response; we would have

expected a higher fractionation in treatment pH 1.6. Perhaps, this indicates that the wax protected

the stomata enough that it did not affect the gas exchange process. I would speculate that if the

treatments were applied for a longer period of time, there would be a change in both the openings

and the 13CO2 signal. Perchance in a future study a more quantitative analysis could allow to

make more accurate comparisons between the acid treatments. Another factor that might interfere

with the material observed in the SEM is the lag time between the harvest time and the time when

the plants were affixed to the stubs. The technique to fix the samples might have also altered the

observed results.

It would be interesting to determine the implications this has for paleoenvironmental

reconstruction studies. Also, to examine if there is an equivalent response after being acidified by

dry deposition. Longer chain distributions could tell whether plants were under stressful

atmospheric conditions. Looking at class components shouldn’t be enough to determine

differences, for the plant will preserve its signature associated with its genus. But, the molecular

composition should also be studied. In conclusion, subtle changes in chain length can infer

changes in the precipitation’s water quality.

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Figure 1.

Figure 1.

0  

1  

2  

3  

4  

5  

6  

7  

0   1   2   3   4   5   6  

blade  grow

th  (cm)  

pH  treatment  

Total  growth  

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Figure 2.

Figure 2.

0  

0.05  

0.1  

0.15  

0.2  

0.25  

0.3  

0.35  

0.4  

0.45  

0   1   2   3   4   5   6  

new  biomass  (g/dw)  

pH  treatment  

Total  Biomass  

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Figure 3.

40  

41  

41  

42  

42  

43  

43  

44  

44  

45  

45  

46  

0   1   2   3   4   5   6  

%  C  

pH  treatment  

%  Carbon  by  dry  weight  

13

Figure 4.

3.5  

4.0  

4.5  

5.0  

5.5  

6.0  

0   1   2   3   4   5   6  

%  N  

pH  treatment  

%  Nitrogen  by  dry  weight  

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Figure 5.

y  =  2.6463ln(x)  +  8.2179  R²  =  0.91388  

8  

9  

10  

11  

12  

13  

14  

0   1   2   3   4   5   6  

Mole  C:N  ratio  

pH  treatment  

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Figure 6.

Figure 6.

-­‐31.0  

-­‐30.5  

-­‐30.0  

-­‐29.5  

-­‐29.0  

-­‐28.5  0   1   2   3   4   5   6  

d  13  C  

pH  

d13C  (o/oo  vs.  PDB)  

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Figure 7.

-­‐1.2  

-­‐1.0  

-­‐0.8  

-­‐0.6  

-­‐0.4  

-­‐0.2  

0.0  

0.2  

0   1   2   3   4   5   6  

d  15  N  

pH    

d15N    (o/oo  vs.  AIR)  

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Figure 8.

0  

10  

20  

30  

40  

50  

60  

70  

80  

90  

100  

ph  1.6   ph  2.4   ph  3.5   ph  4.4   ph  5.6  

%  Wax  

Percent  wax  per  class  

FAL   FAME   ALK  

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Figure 9.

0  

20  

40  

60  

80  

100  

120  

140  

0   1   2   3   4   5   6  

Concen

tra5

on  µg/  g  C  

pH  treatment  

Total  wax  concentration  FAL+FAME+ALK  

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Figure 10.

a) b)

c) d)

e)

20

Figure 11.

c) d)

a) b)

e)

21

Figure 12.

a) b)

c) d)

e)

22

Figure 13.

R²  =  0.55013  

25.0  

27.0  

29.0  

31.0  

33.0  

35.0  

37.0  

4.0   4.5   5.0   5.5   6.0   6.5   7.0   7.5   8.0  

%  FAM

E  

%  FAL  

Long  Chained  (30-­‐32)  FAME/FAL  

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Figure 14

a) b)

c) d)

e)

24

Figure 15

0.0  

0.2  

0.4  

0.6  

0.8  

1.0  

1.2  

1.4  

1.6  

1.8  

0   1   2   3   4   5   6  

µg/gdw

 

pH  treatment  

C29/31  Alkane  ratio  

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Figure 16.

400  

450  

500  

550  

600  

650  

700  

750  

800  

0   1   2   3   4   5   6  

µg/gdw

 

pH  treatment  

Total  Sterols  

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Figure 17.

a)

c) d)

b)

e)

27

Figure 18.

a) b)

c)

28

Figure 16.

Figure 19.

a) b)

c) d)

e) f)

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Table 1. Treatment initial soil pH 11/12/13 final soil pH 11/19/13 1.6 (1) 6.6 6 1.6 (2) 6.5 6.2 2.4 (1) 7.1 7.6 2.4 (2) 7.2 7.6 3.5 (1) 7.2 7.6 3.5 (2) 7.1 7.6 4.4 (1) 7.3 7.4 4.4 (2) 7.3 7.3 5.6 (1) 7.3 7.4 5.6 (2) 7.4 7.3

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Acknowledgements Special thanks go to my advisors Maureen Conte and JC Weber for guiding me through the course of these 5 weeks. Fiona Jevon, Alice Carter, Sarah Nalven and Rich McHorney for their support, Louis Kerr for sharing your knowledge of SEM, and Marshall Otter for his work done in the Ecosystem Center’s Stable Isotope Laboratory in the MBL. To everyone who was a part of the SES program, you are all wonderful. Literature Cited: Adams C. et al. 1990. Crystal Occurrence and Wax Disruption on Leaf Surfaces of Cabbage Treated with Simulated Acid Rain. New Phytologist, 114(1):147-158 Amthor J. 1984. Does acid rain directly influence plant growth? Some comments and observations. Environmental Pollution Series A, Ecological and Biological 36(1):1-6 Baker and Hunt. 1981. Developmental changes in leaf epicuticular waxes in relation to foliar penetration. The New Phytologist, 88:731-747 Baker EA. 1982. Chemistry and morphology of plant epicuticular waxes. The Plant Cuticle. Academic Press, NY. 139-165 Baker EA and Hunt G. 1986. Erosion of waxes from leaf surfaces by simulated rain. New Phytologist 102:161-173. Barthlott W, et al. 1998. Classification and terminology of plant epicuticular waxes. Botanical Journal of the Linnean Society 126: 237-260. Bondada, et al. 1996. Effects of water stress on the epicuticular wax composition and ultrastucture of cotton. Environmental and Experimental Botany, 36(1): 61-69 Christie W. 2003. Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids, third edition. The Oily Press, UK. pp.129 Christie. 1993. Advances in Lipid Methodology. The Oily Press, Dundee. Two, pp. 195-213 Conte M, Weber JC. 2002. Plant biomarkers in aerosols record isotopic discrimination of terrestrial photosynthesis. Nature Publishing group 417: 639-641. Eigenbrode S, et al. 1995. Effects of plant epicuticular lipids on insect herbivores. Ann. Rev. Entomol 40:171-94. Ferenbaugh R. 1976. Effects of simulated acid rain on phaseolus vulgaris L. (Fabaceae).American Journal of Botany, 63 (3) 283-288 Folch J. et al. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497-509. Galloway J. 1989. Atmospheric Acidification: Projections for the Future. Springer on behalf of

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surfaces of urban plants. Environmental Science & Technology, 27 (13): 2700-2711 Rommerskirchen F. et al. 2006. Chemotaxonomic significance of distribution and stable carbon isotopic composition of long-chain alkanes and alkan-1-ols in C< sub> 4</sub> grass waxes. Organic Geochemistry, 37(10): 1303-1332. Seigler D. 1998. Plant waxes. Plant Secondary Metabolism, Springer. 51-55 Shepherd T. et al. 2006. The Effects of Stress on Plant Cuticular Waxes. New Phytologist , 171(3):469-499 Tulloch, A. P., & Hoffman, L. L. 1977. Composition of epicuticular waxes of some grasses. Canadian Journal of Botany, 55(8): 853-857 Turunen M, et al. 1997. A review of the response of epicuticular wax of conifer needles to air pollution. J. Environ. Qual. 19: 35-45 Vogts, A. et al. 2009. Distribution patterns and stable carbon isotopic composition of alkanes and alkan-1-ols from plant waxes of African rain forest and savanna C< sub> 3</sub> species. Organic geochemistry, 40(10): 1037-1054. Figure 1. Total new growth after 1 week of treatment Figure 2. Total new biomass after 1 week of treatment Figure 3. Percent carbon by dry weight Figure 4. Percent nitrogen by dry weight Figure 5. Molar CN ratio Figure 6. del 13C Figure 7. del 15N Figure 8. Relative Distribution of Classes Figure 9. Total wax concentrations Figure 10. FAL molecular distribution Figure 11. FAL molecular distribution at a smaller scale Figure 12. FAME molecular distribution Figure 13. Covariance between long chain FAME and FAL Figure 14. Alkane molecular distribution Figure 15. Relationship between alkane C29 and C31 Figure 16. Total sterols Figure 17. SEM images of the 5.6 pH treatment. Figure 18. SEM images of the 3.5 pH treatment. Figure 19. SEM images of the 1.6 pH treatment Table 1. Measured soil pH at the initial and final day of the treatments