Environ. Sci. Technol. 1905, 19, 432-437

Effect of Ethylene and Related Hydrocarbons on Carbon Assimilation and Transpiration in Herbaceous and Woody S p e c i d

Sheila A. Squler," George E. Taylor, Jr., Wllliam J. Selvldge, and Carla A. Gunderson

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831

Although the hormonal effects of ethylene (CzH4) on plant growth and development are well documented, recent evidence suggests that carbon assimilation in some her- baceous species is highly sensitive to ethylene. Since ethylene is a common trace gas in many airsheds influ- enced by urban and industrial pollutant sources, elevated levels of ethylene may be affecting the productivity of some terrestrial vegetation. The objectives of this study were to investigate the resporisiveness of carbon assimilation to ethylene and related low molecular weight hydrocarbons in plant species of dissimilar growth and physiological features, to address the physiological mechanism of pho- tosynthetic inhibition, and to estimate minimum Czy4 concentrations causing incipient effects on carbon assi- milation. Of the four hydrocarbons studied only ethylene influenced carbon assimilation in a variety of species. The level of ethylene needed to elicit a change in carbon as- similation differed markedly among species. Estimated 5-h concentrations of ethylene required to influence carbon assimilatiori in ethylene-sensitive species ranged from 0.60 to 19.5 pmol/m3. The ethylene-induced inhibition of photosynthesis was correlated with a decline in stomatal conductance to HzO vapor.

Introduction Low molecular weight hydrocarbons (i.e., carbon atom

numbers 5 8) play a significant role in a humber of en- virohmental issues relating to air quality, most notably the potential gldbal climate change and the formation of photochemical smog, aerosols, and acidic precipitation (1). The most common aliphatics include methane, ethane, ethylene, acetylene, propane, propylene, butanes, and pentanes (2). Of the reactive or non-methane hydro- carbons, ethylene (CZH4) is the most common, with con- centrations 50.05 pmol/m3 in pristine areas (3), but often 2 orders of magnitude higher in urban airsheds or near major point sources (4) . The major anthropogenic sources of many low molecular weight hydrocarbons, including CZH4, are vehicular exhaust and volatization from petro- chemical processes (5), while advanced energy technologies may be significant point sources in the future (6).

The significance to terrestrial vegetation of elevated levels of low molecular weight hydrocarbons is not certain. With the exception of CzH4, little information is available regarding plant response to either short-term or long-term expoeure regimes. The hormonal effects of CzH4 are well documented under controlled laboratory conditions, and threshold concentrations to induce many physiological/ biochemical characteristics of premature senescence lie between 0.4 and 4 hmol/m3 (7). Heck et al. (8) observed symptoms of hormonal CzH4 action (i.e., whole-plant senescence) in cotton (Gossypium hirsutum) after 30-day exposures to 25 pmol/m3, while Abeles and Heggestad (9) noted similar hormonal effects on a number of herbaceous species treated for 70 days to CzH4 concentrations 11.0 c t ~ ~ l / m 3 , In conjunction with air quality data and field

'Publication No. 2457, Environmental Sciences Division, ORNL. Address correspondence to this author at the Juniata College,

Huntingdon, PA 16652.

studies, Abeles and Heggestad (9) concluded that CzH4 was an environmental stress influencing prihcipally urban vegetation. Some of the other hydrocarbon gases can affect plant growth at concentrations well above ambient levels ( lo) , and at least one compound, propylene, is reported to be a C2H4 analogue (11).

Early reports of C2H4 action in terrestrial vegetation ihdicated that foliar gaseous exchange of carbon dioxide and water vapor were not affected by CzH4 exposure. For example, Pallaghy and Raschke (12) observed no change in stomatal conductance to HzO vapor in corn (Zea mays) and pea (Pisum satiuum) following exposure to CzH4 concentrations as high as 410 pmol/m3 (duration of ex- posure not specified). At a similar concentration Aharoni (13) reported no effect of CzH4 on the conductivity of the HzO diffusive path in 10 herbaceous species. However, a more recent study by Kays and Pallas (14) demonstrated pronounced and very rapid physiological effects from CzH4 exposure. Following only a 2-h exposure to 10 pmol/m3 C2H4, photosynthesis ( P N ) in peanut (Arachis hypogaea) was reduced as much as 33%. Further studies have dem- onstrated additional PN responses among several herba- ceous species (15, 16). The observation that C2H4 can demonstrably affect carbon assimilation in a direct and immediate fashion (14) strengthens the proposal of Abeles and Heggestad (9) and suggests that the physiological mode of action may be a direct inhibition of carbon as- similation rather than a long-term hormonal effect on plant growth processes. The determination of threshold levels of CzH4 causing incipient effects on PN relative to reported CzH4 concentrations in the atmosphere is important in establishing the likelihood under natural conditions of a direct C2H4 effect on carbon assimilation.

The objectives of this study were to (i) determine the responsiveness of PN in five plant species to a number of low molecular weight hydrocarbons at high exposure con- centrations (screening investigation), (ii) for those hydro- carbon gases causing effects, determine the responsiveness of P, and transpiration (TR) at near-ambient concentra- tions, including estimates of the minimum concentration causing effects on P N , and (iii) investigate the physiological basis of changes in PN and T R .

Materials and Methods Plant Material. Species were selected to include a

range of growth habits (annual/herbaceous vs. woody/ perennials) and dissimilar carbon metabolism (C, vs. C4 assimilatory pathway). Herbaceous C3 (Glycine max L., cv. Davis; Nicotiana tobaccum L., cv. Bel W-3; Arachis hypogaea L., cv. Jumbo Virginia) and C4 (Zea mays L., cv. F1 hybrid Early Golden Giant) species were grown in Promix BX (Premier Brands, Inc., Rochelle, NY) in 1.0-L pots. After germination, seedlings were thinned to one per pot. Fraxinus pennsylvanica L. seedlings (green ash) were obtained as cold-stored, 1-year-old plants (Forest Keeling Nursery, Elsberry, MO) and were potted in Promix BX in 2.5-L pots. Plants were watered daily and supplied weekly with a full complement of liquid fertilizer (Peters Fertilicter, W. R. Grace, Allentown, PA). Plants were gown

a glasshouhe with the following environmental condi-

432 Environ. Sci. Technol., Vol. 19, No. 5, 1985 0013-936X/85/0919-0432$01 . sO/O 0 1985 American Chemical Society

Table I. Summary of Environmental Conditions in the Gas-Exchange Chambers

condition

chamber volume, m3 air exchange, min air temperature, "C relative humidity, pmol/m3 photosynthetic photon flux density, pmol m-2 s-l photoperiod, h outlet COz concentration, pmol/m3 range of hydrocarbon concentrations, pmol/ms boundarv laver conductance to H,O vauor, cm/s

range

0.92 4-6 31-35 0.8-1.3 320-500 15 11.5-14.3 0-164 10

tions: mean day/night temperatures of 35/18 "C, 15-h photoperiod, photosynthetic photon flux density of 0-1600 pmol m-2 s-l, and relative humidity of 4540%. The natural photoperiod was extended to 15 h with HID so- dium vapor lamps which provided 325 pmol m-2 s-l a t bench height. Studies were conducted 5-7 weeks after germination for the herbaceous species and 4-5 months after bud break for green ash.

Gaseous Exchange System. Exposures were con- ducted in an open gas-exchange system (1 7, 18) utilizing four matching, continuously stirred tank reactors. Oper- ating conditions unique to this study are outlined in Table I.

Hydrocarbon gases were dispensed from cylinders (Certified Grade Purity, Union Carbide Corp.), and the flow of pollutant gases to each chamber was regulated with rotameters. Hydrocarbon concentrations were monitored with a flame ionization method (Model 400 hydrocarbon analyzer, Beckman Instruments, Inc., Fullerton, CA) in which the ionization current was proportional to the rate a t which carbon atoms of hydrocarbon origin entered the burner assembly. Calibration was achieved with methane and ethylene standards (Primary Standards, Union Car- bide Corp.). Hydrocarbon concentrations were monitored at the outlet ports to each chamber, and the sample lines were heated and continuously exhausted. Concentrations of COz and HzO were monitored at each chamber's inlet and outlet ports using infrared gas analyzers (Beckman Instruments, Inc., Fullerton, CA) calibrated with a range of known gas concentrations (i.e., certified grade purity COz in nitrogen, Matheson, Morrow, GA; dilution of HzO-saturated air with dry nitrogen). The COz analysis was done after the HzO was cryogenically trapped. Where necessary, estimates of whole-plant gas exchange rates of COz and H20 were calculated as described in ref 17 and expressed as mmol m-2 h-' (projected leaf area).

Experimental Protocol. Two types of exposures were conducted to address the first two objectives. In the first (objective l), representative C3 (G. max) and C4 (2. mays) herbaceous species were exposed for 6 h to 0,41, 103, or 164 pmol/m3 propylene (C3H6), butane (C4H10), ethane (CzH6), or ethylene (CzH4). These gases were selected on the basis of preliminary data (6) that indicated each hy- drocarbon had some unique combination of chemical/ physical properties suspected of governing toxicity. Failure of a compound to elicit a response in this screening exercise was a basis for not pursuing subsequent studies (only CzH4 elicited a response). In the second type of exposure (ob- jective 2), herbaceous (G. max, A. hypogaea, and N . to- bacuum) and woody (F. pennsylvanica) species were ex- posed to CzH4 concentrations of 4, 10, 21, 41, or 82 pmol/m3. Exposures began 4 h after photoperiod initiation and continued uninterrupted for 28 h. Each combination of species and concentration was replicated at least twice.

The logistics for each type of exposure were the same. Plants (8-16) were watered to drip point and transferred

to each of four chambers the afternoon prior to exposure. The top of the pot was covered with clear plastic to min- imize the exchange of COz, HzO, and hydrocarbon gas between the soil and atmosphere. The following morning (i.e,, 14-h acclimation) before hydrocarbon exposures, rates of COz and HzO exchange were recorded for the plants in each chamber, These chamber-specific preexposure values were used to evaluate subsequent changes in COz and HzO exchange in both control and treated plants. A single hydrocarbon gas was added at different rates to the inlet plenum of three chambers, and the fourth served as a control. The COP, HzO, and hydrocarbon concentrations at inlet and/or outlet ports were recorded at 1-2-h in- tervals.

For those situations in which PN was influenced by ex- posure to a hydrocarbon gas (i.e., CZH4), stomatal con- ductance to HzO vapor (g,) was measured to investigate the physiological basis of inhibition (objective 3). Studies were conducted in one of the chambers that was modified with hand-access ports to minimize disturbance of the chamber's environmental conditions during porometry measurements. Stomatal conductance of the abaxial surface (LI 1600 steady state porometer, Li Cor., Inc., Lincoln, NE) was measured before and during exposure in both control and treated plants of all five species except A. hypogaea, in which g, of the adaxial leaf surface was monitored (A. hypogaea is hyperstomatous). Two con- ductance measurements per leaf were taken on three leaves per plant. The growing and exposure conditions (timing of exposure relative to photoperiod) were comparable to that used for objective 1, except a single C2H4 concentra- tion of 21 pmol/m3 (exceeded estimated threshold con- centration to influence PN in most CzH4 sensitive species) was administered.

Data Analysis. From each replicated exposure (i.e., combinations of plant species and hydrocarbon concen- tration), mean rates of COP and HzO exchange (expressed as percentages of initial rates) were calculated at common time intervals. The exchange rates of COz and HzO in control and treated plants were subsequently graphed as a function of time. For studies relating to objective 1, the rate of change over time in COz or HzO exchange for treated plants was expressed as a percentage of that which occurred in control plants a t the same time interval. Statistical tests of hydrocarbon effects on PN and TR were performed only on those data recorded at the last time interval. One-way analysis of variance was conducted on each combination of species and hydrocarbon gas, and the least significant difference was used to evaluate statistical significance between concentrations of a single gas. Sto- matal conductance data were evaluated by using paired t tests. All statistical tests were conducted at a = 0.05.

Results Rates of Carbon Assimilation (PN) and Transpi-

ration (TR). BefoSe exposure and after acclimation, ab- solute PN rates ranged from 10.9 mmol of C02 m-2 h-l in F. pennsylvanica to 55.4 mmol of COz m-2 h-l in 2. mays (Table 11). In all five species, PN in control plants rose throughout the first day and, after 6-8 h, had risen 10-20%. On the second full day of exposure, PN rates continued to remain above the initial preexposure values. Transpiration rates ranged from 5.84 mol m-2 h-l in N. tobaccum to 9.18 mol m-2 h-' in G. max (Table 11).

Objective 1. In 2. mays none of the four gases influ- enced PN at concentrations as high as 164 pmol/m3 (Table 111). At this highest concentration for each hydrocarbon gas, all mean PN rates in 2. mays were within f10% of that recorded in control plants. Carbon assimilation in G. man

Environ. Sci. Technol., Vol. 19, No. 5, 1985 433

Table 11. Initial Preexposure Rates of Net Photosvnthesis

41 C2H4/N. tobaccum

and Transpiration (Mean f SD)

net . photosynthesis, transpiration, mmol m-2 h-' mol m-2 h-'

Glycine rnax 36.2 f 11.7 9.18 f 1.94 Zea mays 55.4 f 11.8 8.80 f 0.98 Nicotiana tobaccum 51.1 f 21.4 5.84 f 1.15 Arachis hypogaea 22.3 i 5.3 8.34 f 1.11 Fraxinus pennsylvanica 10.9 * 4.0 6.39 i 1.86

Table 111. Mean Photosynthetic Rates (mmol m-* h-I) After 3-6 h of Exposure to Hydrocarbon Gasesa

gas concentration, pmol/m3

species gas

Z . mays C4H10 C2H6 C2H4 C.3H6

G . max C4H10 C2H6 CZH4 C3H6

A. hypogaea CZH, F. pennsylvanica C2H4

0 41 102 164

62.6 60.9 60.9 63.2 51.5 52.6 53.7 52.7 52.6 54.3 54.5 56.7 82.5 79.8 75.9 77.0 42.7 39.5 43.8 39.8 40.5 33.3 44.9 39.8 36.2 25.7 18.8 13.8 35.8 42.0 36.2 34.8

19.4 13.6 8.0 6.2 8.3 8.0 8.0 4.9

LSD NS NS NS NS NS NS

10.9 3.6

6.5 1.2

OData for each combination of species and gas were analyzed via one-way ANOVA, and statistically significant F values are those with numeric LSD values.

was not responsive to either C4HI0 or C.&, but both C2H4 and c3H6 exhibited statistically significant effects on PN (Table 111). The lowest (41 pmol/m3) and highest C2H4 concentrations reduced P N by 29% and 62% in G. max, respectively. In the same species exposed to C3H6, the only PN rate that was statistically different from the others was that at 41 pmol/m3, in which P N was 17% above that in control plants. Thus, a consistent concentration-depend- ent response in G. max was evident only for C2H4. In the remaining two species, PN was responsive to C2H4 exposure. In A. hypogaea, all concentrations markedly inhibited PN, and rates ranged from 32% (164 pmol/m3) to 70% (41 pmol/m3) of that in control plants, while in F. pennsyl- uanica, a statistically significant reduction in PN was re- corded only at the highest concentration (Table 111).

Objective 2. The objective of this study was to deter- mine the responsiveness of P N and TR to near ambient concentrations of CzH4. In G. max exposed to CzH4, P N declined at all concentrations >4 pmol/m3 relative to that in control plants (Figure la). By 1600 h on day 1, P N was demonstrably inhibited only at the highest concentration (21 pmol/m3), but by 1200 h on the second day P N was depressed at all concentrations. While P N in control plants a t the end of exposure had risen nearly 20% above initial rates, PN in C2H4-exposed plants either remained un- changed (4 and 10 pmol/m3) or was depressed to a level 50% below the initial rate (21 pmol/m3).

Carbon assimilation was more rapidly affected by C2H4 exposure in A. hypogaea; within 3.5-5 h, P N was less than the initial rate a t concentrations 1 4 pmol/m3, while P N in control plants had risen 8-12% (Figure lb). This C2H4-induced inhibition of P N continued through day 1. By 1200 h of day 2, P N a t 4 pmol/m3 of C2H4 was com- parable to that in control plants while that in the two highest concentrations (10 and 21 pmol/m3) was only 40-50% of the initial rate.

In N . tobaccum, the effects of C2H4 on PN were apparent within 2-3 h although a higher range of concentrations

434 Environ. Sci. Technol., Vol. 19, No. 5, 1985

50 t C2H4/G. rnax

0 I I I I I

I 1 I I I

100 p

uu t o

0 c

I- z > VI 0 c 8 50 a

y 100

I I I I I

50 t C,H4 /F pennsylvanlco

0 I 1 I I I I 0800 \ZOO 1600 0800 I200 (600

TIME ( h )

Flgure 1. Response of photosynthesis (as a percentage of initial value) as a function of time and CzH, concentration in 0 . max (a), A . hy- pogaea (b), N. fobaccum (c), and F. pennsyhanica (d). Numbers after each response line are CzH, concentrations (pmoi/rn3).

than used for A. hypogaea was needed to induce effects (Figure IC). Within 3.5 h of exposure, P N in control plants rose 15% but declined to rates of 50-80% of the initial values at C2H4 levels of 41 and 82 pmol/m3, respectively. In both C2H4 exposures, P N remained depressed through- out day 1, and by 1600 h all PN rates were substantially below that of control plants. Carbon assimilation on day 2 rose 20% in control plants, while P N was 130% of the initial rate a t C2H4 concentrations 2 2 1 pmol/m3.

The effects of C2H4 on PN in F. pennsyluanica (Figure Id) were not as pronounced as those recorded in the herbaceous species. At the end of day 1, P N a t concen- trations 110 pmol/m3 was below that in control plants although the percentage of inhibition remained constant through day 2.

To estimate minimum C2H4 concentrations required to inhibit carbon assimilation, the change in P N in each species was expressed relative to its respective controls at the same time interval and plotted as a function of the natural log of the product of concentration and time (18). The data for each species were subsequently analyzed by using linear regression techniques (Figure 2). With the exception of F. pennsyluanica, the regression coefficients (i.e., slopes) were significantly different from zero, and the regression accounted for 280% of the variation in PN. Estimates of minimum C2H4 concentrations needed to just

I I I 1 1 1 1 1 1 1 I I 1 1 1 1 1 1 1 I I I I

1 W I I

N. taboccum -\ 0 I I I I I I l l 1 I I IiI11111 I I I IO' IO2 I 03

CONCENTRATION X TIME

Flgure 2. Regression of the change in photosynthesis (as a percentage of that in contrd plants) as a function of natural logarithm of the product of C&14 concentration (pmol/m3) and time (h). Symbols are as follows: G. max (O), N . tobaccum (A), A . hypcgaea (0), and F . pennsyhanica (A).

200, I I I 1

z ; + o a4 2 0 0 , , I I I I 1

0 I I 1 1 I

0800 1200 1600 0800 (200 TIME OF DAY (h)

Figure 3. Response of transpiration (as a percentage of initial value) as a function of time and CpH, concentration in A . hypogaea and N . fobaccum , Numbers after each response line are C2H4 concentrations (~.~mol/m~).

inhibit PN (incipient inhibition) following a 5-h exposure were 0.60 (F. pennsylvanica), 1.4 (A. hypogaea), 2.8 (G. max), and 19.5 (N. tobaccum) pmol/m3. For this time interval (5 h) among all species the mean concentration for incipient inhibition of PN was 6.1 pmol/mS, and 1.6 pmol/m3 if only the more C2H4 responsive species were considered.

Transpiration ( TR). The only species exhibiting a marked and consistent TR response as a function of C2H4 exposure were A. hypogaea and N. tobaccum (Figure 3a,b). In A. hypogaea, TR rates by 1200 h on day 2 were 70% (10 pmol/m3) and 90% (21 pmol/m3) of initial rates, while that in the controls and 4 pmol/m3 C2H4 rose nearly 40%. Transpiration in N . tobaccum a t the end of each day at C2H4 concentrations 1 2 1 pmol/m3 was less than that in control plants, and on the second day TR ranged from 50 to 100% of initial rates in C2H4-exposed plants but nearly 160% in control plants.

Stomatal Conductance to H 2 0 Vapor (gs). With the exception of A. hypogaea, g, in control plants (0.0 pmol/m3 C2H4) of all species either remained unchanged (G. max) or declined over time (Table IV) although the decline never exceeded 19% (N. tobaccum). In A. hypogaea, g, increased more than 50% over time in control plants. In all species exposed to C2H4, g, declined over time although the change was statistically significant in only three of the five species

Table IV. Stomatal Conductance to HzO Vapor (g,) in Control and CzH4-Treated Plants (Mean * SD)

leaf conductance, species/ treat- cm/s change,

ment, pmol/m3 initial final %

N . tobaccum 0

21

F. pennsylvanica 0

21

G. mal: 0

21

2. mays 0

21

A. hypogaea 0

21

0.42 (0.04) 0.43

(0.03)

0.19 (0.05) 0.19

(0.06)

0.21 (0.01) 0.21 (0.04)

0.20 (0.02) 0.20

(0.01)

0.18 (0.03) 0.28

(0.05)

0.34 -19 (0.07) 0.20 -54"

(0.03)

0.18 -5 (0.09) 0.11 -42"

(0.02)

0.21 0 (0.02) 0.19 -10

(0.03)

0.18 -10 (0.03) 0.19 -5

(0.04)

0.28 +58" (0.05)

(0.06) 0.17 -41"

" Indicates statistically significant difference between initial and final leaf conductance values (paired t test).

(N. tobaccum, F. pennsylvanica, and A. hypogaea).

Discussion The first objective of this study was to determine the

responsiveness of PN to four hydrocarbon gases. Two of the gases, C2H6 and C4H10, are reported to be innocuous (19,20) although the physicochemical properties of C4H10 suggest that the gas is readily taken up by vegetation (6). Propylene is thought to be a C2H4 analogue and at high concentrations produces symptoms indicative of hormonal action (19). Reported C2H4 effects on PN are variable, with both substantial inhibition (14) and no demonstrable change (12) observed. Our results confirm the innocuous nature of C2HG and C4H10 in 2. mays and G. max, and C3H6 exhibited no consistent effects on PN in the same two species at concentrations 1164 pnol/m3. These results for C3H6 agree with that of Kays and Pallas (14).

The responsiveness of PN to trace C2H4 levels has been confirmed in several species, and the ambivalent nature of different reports in the literature appears to be a con- sequence of interspecific variation in the responsiveness of PN to C2H4. Carbon assimilation in 2. mays exhibited no response to C2H4 concentrations <82 pmol/m3. Pre- vious studies (12) with the same and a related C4 species (Sorghum halpense) also noted no C2H4 effects on PN at substantially higher concentrations. The remaining species tested were all responsive to C2H4, and there was no ap- parent pattern as a function of growth habit. At the in- traspecific level Pallas and Kays (15) noted a positive correlation between photosynthetic efficiency and the PN response to C2H4. This pattern does not explain differ- ences among species since the most photosynthetically efficient species (2. mays) was least responsive to C2H4 (Table 11).

An obvious physiological site of C2H4-induced changes in carbon assimilation is the stomate. In both A. hypogaea and N. tobaccum, TR declined in concert with PN. In each

Environ. Sci. Technol., Vol. 19, No. 5, 1985 435

Table V. Reported CzHa Concentrations (pmol/m*) in the Atmosphere

concentration (meanlmaxi-

environment continent mum) reference

pristine antarctica urbanlindustrial Australia

Europe Asia North America North America North America

North America North America North America North America North America North America

nonurban Europe

0.041a 0.261a 0.21/0.42 0.731436 0.70/ 1.10

0.21/0.42 0.20/0.40

0.08/0.08 0.021a 0.031a 0.09/0.15 0.04/0.14

15.1128.6

0.01/a

Maximum concentration not reported.

herbaceous and woody species exhibiting PN susceptibility to C2H4, stomatal conductance to water vapor (g,) during C2H4 exposure declined more than that in control plants although the decline in G. max was not statistically sig- nificant. Stomatal conductance did not respond to C2H4 in 2. mays, the only species whose PN rate was not re- sponsive to C2H4. Kays and Pallas (14) reported concur- rent declines in PN and g, in A. hypogaea during exposure to 41 pmol/m3 C2H4, and the 81% decline in g, was more than sufficient to account for the 65% drop in PN. How- ever, the observation that the COz compensation point rose 25% (15) suggests that C2H4 effects on carbon assimilation may be mediated by changes other than at the stomate. In fact, C2H4-induced inhibition of carbon assimilation in potato (Solanum tuberosum) is reported to occur without any change in g, (16), thus implicating nonstomatal factors (e.g., carboxylation reaction) as being C2H4 responsive. It is possible that PN inhibition precedes and is the cause of declining g, if CzH4 increases the intercellular gas-phase C02 concentration.

The responsiveness of carbon assimilation to C2H4 is of practical importance only if C2H4 concentrations in the atmosphere commonly exceed the minimum concentration needed to affect PN. Although C2H4 is not routinely measured, there are sufficient data to offer some gener- alizations (Table V). In urban/industrial airsheds, all reported mean values exceed 0.21 pmol/m3 (Table V). Maxima in urban settings average 3 times higher than the mean and range from 0.42 to 28.6 ~ m o l / m ~ . In rural lo- cations, the average of reported mean C2H4 concentrations is 0.07 pmol/m3, and the maxima tend to exceed the mean by only a factor of 1.6. Many of these ambient air C2H4 concentrations approach or even exceed the estimated minimum concentration for 5 h needed to affect PN in C2H4 responsive species (Figure 2). Since vehicular exhaust is the major anthropogenic source of C2H4 (5), the pro- nounced diurnal pattern of vehicular usage results in a corresponding cycle in C2H4 concentrations, with highest concentrations occurring during daylight hours in both the morning and late afternoon (21). Thus, the dynamics of C2H4 concentration are coincident with those periods when carbon assimilation is most active.

The responsiveness of carbon assimilation to trace levels of C2H4 may be involved in the mechanism of physiological effects of other common pollutants. Following exposure to toxic gases such as chlorine, ozone, and sulfur dioxide, many vascular plants produce C2H4 endogenously and

436 Environ. Sci. Technol., Vol. 19, No. 5, 1985

subsequently emit the compound into the intercellular space of the leaf interior (22,23). Emission rates of C2H4 are positively correlated with the magnitude of stress. One of the first physiological responses to the same toxic gases is a change in PN (24), so that the change in PN may be a consequence of endogenous C2H4 production. Thus, C2H4 would be a chemical agent mediating pollutant-in- duced changes in PN or g,.

In summary, the responsiveness of carbon assimilation to trace concentrations of C2H4 and related hydrocarbons was investigated in a variety of plant species. Only C2H4 had an effect on carbon assimilation. Differences between species in their responsiveness to C2H4 were a notable feature and were manifest as both qualitative (C2H4 re- sponsive and nonresponsive) and quantitative (variable minimum concentrations to affect PN among C2H4 re- sponsive species) effects. The physiological site of C2H4-induced changes in carbon assimilation may be in both the gas (i.e., stomate) and aqueous (Le., carboxylation) phase of the COz diffusive path. The minimum concen- tration to inhibit carbon assimilation for several herbaceous and woody species approximates reported mean atmos- pheric C2H4 concentrations in many urban/industrial re- gions. These results indicate that reported C2H4 effects on plant productivity under either field or laboratory conditions may not be solely accountable to the gas’ hor- monal action on plant developmental processes. An equally plausible explanation is a direct and immediate effect of C2H4 on carbon assimilation.

C3H6, 74-98-6; carbon, 7440-44-0.

Literature Cited

Registry NO. CdH10, 106-97-8; C&3, 74-84-0; CzH4,74-85-1;

(1) Rodhe, H.; Eliassen, A.; Isaksen, I.; Smith, F. B.; Whelpdale, D. M. “Tropospheric Chemistry and Air Pollution”. Geneva, 1982, World Meteorological Organization Technical Note 176.

(2) National Research Council “Vapor-Phase Organic Pollu- tants. Volatile Hydrocarbons and Oxidation Products”; National Academy of Sciences: Washington, DC, 1976.

(3) Robinson, E.; Robbins, R. C. “Sources, Abundance, and Fate of Gaseous Atmosphere Pollutants”. Stanford Research Institute, 1968, Report SRI Project PR-6755.

(4) U.S. Environmental Protection Agency “Air Quality Criteria for Ozone and Other Photochemical Oxidants”; Environ- mental Criteria and Assessment Office: Research Triangle Park, NC, 1978; EPR-600/8-78-004.

(5) Graedel, T. E. “Chemical Compounds in the Atmosphere“; Academic Press: New York, 1978.

(6) Taylor, G. E., Jr. Am. J. Hortic. Sci. 1983, 18, 684-689. (7) Abeles, F. B. “Ethylene in Plant Biology”; Academic Press:

(8) Heck, W. W.; Pires, E. G.; Hall, W. C. J. Air Pollut. Control ASSOC. 1961, 11, 549-556.

(9) Abeles, F. B.; Heggestad, H. E. J. Air Pollut. Control Assoc. 1973, 23, 517-521.

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Received for review July 16,1984. Revised manuscript received November 15, 1984. Accepted December 3, 1984. Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05- 840R21400 with Martin Marietta Energy Systems, Znc.

Determination of Total Sulfur in Lichens and Plants by Combustion-Infrared Analysis

Larry L. Jackson,* Edythe E. Engleman, and Janet L. Peard

U.S. Geological Survey, MS 928, Denver Federal Center, Denver, Colorado 80225

rn Sulfur was determined in plants and lichens by com- bustion of the sample and infrared detection of evolved sulfur dioxide using an automated sulfur analyzer. Va- nadium pentaoxide was used as a combustion accelerator. Pelletization of the sample prior to combustion was not found to be advantageous. Washing studies showed that leaching of sulfur was not a major factor in the sample preparation. The combustion-IR analysis usually gave higher sulfur content than the turbidimetric analysis as well as shorter analysis time. Relative standard deviations of less than 7% were obtained by the combustion-IR technique when sulfur levels in plant material ranged from 0.05 to 0.70%. Determination of sulfur in National Bureau of Standards botanical reference materials showed good agreement between the combustion-IR technique and other instrumental procedures. Seven NBS botanical reference materials were analyzed.

The determination of sulfur in plants and lichens is an integral part of many environmental studies. In particular, lichens have proven to be viable air quality indicators for pollutants such as the heavy metals and sulfur. Numerous studies have shown correlations between the elemental content of lichens and that of anthropogenic sources (1-5). In environmental studies such as these the methodology used varies greatly both in the preparation and in the analysis of the lichens. Frequently the methodologic biases are not clearly understood, which makes broad interpre- tation of the data difficult, if not impossible.

The determination of sulfur in plants has generally been restricted to turbidimetric procedures (6-8) and X-ray fluorescence spectroscopy (1, 9, 10). Combustion tech- niques with either photometric or titrimetric quantitation of evolved sulfur have also been used to a limited extent

We have applied a combustion technique with infrared (IR) detection to the determination of sulfur in lichens and higher order plants and have examined the viability of using this procedure in long-range studies such as the detection of contaminants from emission sources over several years. The analytical methodology used in studies

(1 1-1 4).

of this nature must be both accurate and precise to detect small changes with time. Due to the increasing importance of lichens as air quality indicators and the heterogeneous nature of lichens, a symbiotic relationship of an algae and fungus, our experimental work emphasized the analysis of lichens. The effects on precision due to sample size, pelletization, and use of accelerator were examined. We also examined sample preparation techniques, washing and grinding, which may affect the determination of sulfur in lichens through leaching or inhomogeneity effects. Through detailed study of sample preparation and the analytical technique, we have attempted to define the methodologic biases and their effects on data interpreta- tion.

The following plant species (with common name where appropriate) are dealt with in this paper: Medicago sativa L. (alfalfa); Vitis labruscana Bailey (Concord grape); Festuca sp. (fescue grass); Fraxinus pennsylvanica Marsh. (green ash); Parmelia chlorochroa Tuck.; P. sulcata Tayl.; Juniperus scopulorum Sarg. (red juniper); Artemisia tridentata Nutt. (big sagebrush); Lycopersicum esculen- tum Mill. (tomato); Triticum compactum Host (soft-white wheatgrain); Agropyron smithii Rydb. (western wheat- grass); Salix pulchra Cham. (willow).

Experimental Section Instrumentation. A Leco combustion-IR sulfur ana-

lyzer, Model SC-132 (Leco Corp., St. Joseph, MI), was used in this work. The instrument is composed of a micro- processor control unit, an electronic balance, resistance furnace, and a solid-state infrared detector. The sample (0.25 g) was combusted in a stream of pure oxygen at 1370 OC for approximately 2 min. Thirty seconds after com- bustion was initiated oxygen was blown directly into the combustion crucible. The evolved SO2 was detected and measured in an IR cell after the removal of water.

The instrument was calibrated daily with National Bu- reau of Standards reference material 1572, citrus leaves (0.407% sulfur). A coal sample was combusted after every fourth sample to flush condensed organic matter through the combustion train. Also, several coal samples were

Not subject to U.S. Copyright. Published 1985 by the American Chemical Society Envlron. Sci. Technoi., Vol. 19, No. 5, 1985 437