Thresholds for Nitrogen Oxide Emissions for -Gas Turbines on

Australian Native Plants

by

. Dr Frank Murray, Dr Colin Walker and Mr Roger Monk.

A report to the Energy Research and Development Corporation

December 1992

FINAL REPORT TO THE ENERGY RESEARCH AND DEVELOPMENT

CORPORATION

PROJECT TITLE:

Thresholds ·of nitrogen . oxide

turbines on Australian

AUTHORS:

emissions from gas

native plants .

DRFRANKMURRAY,DRCOLINWALKERANDMRROGERMONK

ENVIRONMENTAL SCIENCE MURDOCH UNIVERSITY MURDOCH W A 6150

TELEPHONE NUMBER (09) 360 6000

FAX NUMBER (09) 310 4997

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TABLE OF CONTENTS

ABSTRACT .................... : ...... : .................................................................................... : .... 4

· SUMMARY ............................... , ......................................... , ......... ~ ......... ·........................ 5

1 INTRODUCTION ......................... ~ .......................................................................... lO _ 1.1 PROJECT OBJECTIVES ........................................... _; .................................. , ... 10 1.2 NITROGEN OXIDES IN THE ENVIRONMENT: A NEW VIEW ....... ll 1.3 SOURCES OF NITROGEN OXIDES AND ASSOCIATED ISSUES ...... 14

1.3.1 Sources 1.3.2 Reactions Controlling Atmospheric Nitrogen Oxides

. · 1.4 EFFECTS OF NITROGEN OXIDES ON PLANTS ........................ -: .......... 17 1.4.1 Symptoms 1.4.2 Yield Effects 1.4.3 Deposition, Assimilation and Physiological Effects

1.5 EFFECTS OF NITROGEN OXIDES ON AUSTRALIAN NATIVE .... ;.24 AND OVERSEAS SPECIES

2 GENERAL METHODOLOGY ................. : ................................................. ' ............. 27 2.1 SPECIES SELECTION .......................... -.......................... ; ..................................... .-.. 27

2.1.1 Banksia attenuata (Banksia) 2.1.2 Eucalyptus calophylla (Marri) 2.1.3 Eucalyptusglobulus (Tasmanian Blue Gum) 2.1.4 Eucalyptusmaculata (Spotted Gum) 2.1.5 Eucalyptus marginata · . _ (Jarrah) . 2.1.6 Eucalyptus microcorys (Tallowwood) 2.1.7 Eucalyptus pilularis (Blackbutt) 2.1.8 Pinus radiata · (Radiata Pine)

2.2 FUMIGATION CHAMBERS .......... ; ......................................................... 29 2.3 POLLUTANT DISPENSING AND MONITORING ............................ 30 ' ' . . 2.4 EXPERIMENTAL D.ESIGN ........................................................................ 30 2.5. ENVIRONMENTAL CONDITIONS ...................................................... 32 2.6 RESPONSE PARAMETERS ................... ~ .................................................. 33 2.7 STATISTICAL ANALYSIS ......................................... · ........................ -: ....... 33

3 THE SYMPTOMS OF FUMIGATION WITH HIGH LEVELS OF ................ 35 NITROGEN OXIDES ON NATIVE PLANTS.

3.1 INTRODUCTION ................................................................................... _.-........ 35 3.2 METHODOLOGY ...... , ................ ~ .................. : .................................. : ............. -36

3.2.1 Plant Species . 3.2.2 Fumigation

32.3 Observations and Photographic Documentation 3.204 Biochemical Tests on Leaf Tissue

3.3 RESULTS ................... ~ .............................................................. _ ...................... 37 3.3.1 Observations and Photographs 3.3.2 Biochemical Tests· on Lea£ Tissue

3.4 DISCUSSION ................................................................................................. 39

4 THE EFFECTS OF NITROGEN OXIDES ON BLUEGUM, JARRAH, ....... .40 TALLOWWOOD AND BLACKBUTT . . 4.1 INTRODUCTION .......................................................... .-................................ 40 4.2 MATERIALS AND METHODS ................................................................. 40

4.2.1 Plant cultivation . 4.2.2 Fumigation regime

4.2.3 Harvest procedure 4.3 RESULTS ........................................................................................................ 41 ·

4.3.1 Eucalyptus microcorys (Tallowwood) 4.3.2 Eucalyptus marginata (Jarrah) 4.3.3 Eucalyptus globulus ·(Tasmanian Blue Gum) 4.3.4 Eucalyptus pilularis (Blackbutt)

4.4 DISCUSSION ........... · ................ : ..................................... i ............................... 47

5 THE EFFECTS OF NITROGEN OXIDES ON JARRAH, MARRI, ............. 64 . TALLOWWOOD AND SPOTTED GUM.

5.1 INTRODUCTION .................. ; ...... ; ............................................... , ................ 64 5.2 MATERIALS AND METHODS .................................................................. 65

5.2.1 Plant cultivation 5.2.2 Fumigation Conditions 5.2.3 Porometry 5.2.4 Harvest Procedure

5.3 RESULTS ......................... .-................................................................................. 67 5.3.1 Eucaiyptus calophylla (Marri) 5.3.2 Eucalyptus maculata (Spotted gum) 5.3.3 Eucalyptus marginata (Jarrah)

5.3.3.1 Biomass. 5.3.3.2 Nitrogen 5.3.3.3 Nutrient Analysis 5.3.3.4 Amino Acids 5.3.3.5 Stomatat Conductance

5.3.4 Eucalyptus microcorys (Tallowwood) 5.4 DISCUSSION ....................... : ........................................................................... 94

5.4.1 Growth and Total Nitrogen Concentration 5.4.2 Effects on Amino Acid Physiology of E. marginata. 5.4.3 Correlations between Amino Acids and Tissue Nitrogen

of E. marginata. 5.4.4 General Discussion

6 THE EFFECTS OF NITROGEN OXIDES AND SULPHUR DIOXIDE ON .. 98 JARRAH, MARRI, BANKSIA ATTENUATA, AND PINUS RADIATA.

6.1 INTRODUCTION .......................... : ................................................ -.............. 98 6.2 MATERIALS AND METHODS ............................................................. , ... 98

6.2.1 Plant cultivation 6.2.2 Fumigation Conditions· 6.2.3 Harvest Procedure 6.2.4 Analyses

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6.3 RESULTS ...................................................................................................... 101 6.3.1 Pinus radiata 6.3.2 Banksia attenuata 6.3.3 Eucalyptus marginata (Jarrah)

6.3.3.1 Biomass. 6.3.3.2 Nitrogen 6.3.3.3 Sulphur

6.3.4 Eucalyptus microcorys (Tallowwood) 6.3.4; 1 Biomass 6..3.4.2 Nitrogen 6.3.4.3 Sulphur

6.4 DISCUSSION .... · ........... -............................................. _ .................................... 113

7 DOSE RESPONSES OF MAJOR AUSTRALIAN FOREST SPECIES TO.l16 NITROGEN OXIDES.

7.1 INTRODUCTION ....................................................................................... 116 7.2 NITROGEN 0XIDES ........................ ; ......................................................... 117

7.2.1 Plant Species 7 .2.2 ·Seasonal Effects

7.3 INFREQUENT·FUMIGATIONS ...... :··· .. , ............................................. ; .. 120 7.4 PLANT GROWTE ~'ARAMETERS ......... , ............................................. 123

. .

TECHNOLOGY TRANSFER ... : ............................................................................... 125

REFERENCES ............................................................................................................. 127

APPENDIX .................. : .................... ~ ........................................................................... 137

ACKNOWLEDGEMENTS .............. ~ ............................... -..... .' ................................ : ... 139

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ABSTRACT

Natural vegetation in Europe and North America has been damaged by emissions of nitrogen oxides, but there are no published studies of effects of nitrogen oxides on Australian native vegetation. To determine the potential for emissions of nitrogen oxides from gas-fired power stations to affect Australian native vegetation, eight economically or ecologically important tree species were exposed to nitrogen oxides at various concentrations and frequencies of exposure. The experiments were conducted in open-top chambers under field climatic conditions, and under realistic exposure conditions.

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The results showed that although there were some species differences, concentrations of up to 200 nL L:·1 for 2 hour/day, 3 days/week, stimulated height and weight by about 10:..20%. However, a decrease in height and weight of up to 15% resulted. from exposure to 100-500 nL L-1 for 2 hour/day, 3 days/week, depending on the species. There was no visible injury. Changes in the frequency of exposure had little effect on plant response, but there were interactions with sulphur dioxide, cold and possibly drought, which can accentuate the effects of nitrogen oxides.

The symptoms of nitrogen dioxide injury to leaves of Australian native plants have not been previously described, but are described in this report. They are unlikely to be found in the field as they only occur at very high concentrations, such as would only be found around the sites of large accidental releases.

SUMMARY

A program to assess the impact of gaseous emissions from the energy industry on .the vegetation in Australia has continued with a study on the threshold for nitrogen oxide emissions from gas-fired power stations to modify plant growth of important tree species. This study follows from the deficiencies in management criteria, noted in a previous study (Murray et al., 1991), in that:

"Australia has no national secondary ambient air quality standards for the protection of the environment from damage caused by emissions of sulphur dioxide and nitrogen oxides."

Such standards are a commonplace regulatory benchmark within the environmental protection mechanisms put in place by legislation and inspection and monitoring protocols in many nations (United Nations, 1987).

The study was conducted as:

· • there is now widespread and increasing damage to native vegetation in nutrient poor environments (such as heathland nature reserves) in Western Europe caused by nitrogen oxides.

• the important role of nitrogen oxides in forest decline in Europe and· North America is being increasingly recognised.

• little is known about the effects of nitrogen oxides on native Australian plants, although there is evidence to suggest that Australian native plants, many of which have evolved in ~md adapted to low nitrogen environments, are sensitive to nitrogen deposition.

• there is increasing emphasis on natural gas as a fuel for electricity generation in Australia, and for more general use as a clean and relatively pollution-free fuel. However combustion of natural gas is a ~ignificant source of nitrogen oxide emissions.

The objectives of this ERDC funded project are:-

• to determine the range of tolerance to nitrogen oxides of the most economically and ecologically important groups of Australian native plants under field conditions.

• to quantify .the maximum concentrations of nitrogen oxides that Australian native species can tolerate. This will facilitate better planning and siting of gas turbines to minimise adverse effects while avoiding unnecessary expenditure.

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• to characterise the symptoms of nitrogen oxides injury to native vegetation: This will assist with monitoring and to respond to public . . concern. ·

Some 8 major Australian forest species, mostly Eucalyptus species, have been exposed to nitrogen oxides under field climatic conditions. These experiments used an open-top chamber fumigation system within which the plants were propagated for several months, a procedure commonly used overseas to establish the effects of gas pollutants in field situations. During this period they were exposed to nitrogen oxides on up to eighty occasions in two hour fumigation sessions.

As plants may respond both to the concentration of a pollutant and the frequency of exposure, two types pf exposure regime were conducted. One type examined the response to varying concentrations (ambient to 500 nL L-1) at frequencies of two hours per day, three days per week for about five months. A second type of exposure· regime examined responses to constant concentration (100 or 500 nL Vl) at varying frequency.

In one experiment plants were grown in the soil, simulating natural growth conditions, and in two experiments plants were grown in pots which enabled effects on root growth to be measured, as European studies have shown that air pollutants can change the distribution of assimilate, favouring shoot growth over root growth, and exposing. trees to the effects of drought.

As it is rare that only one air pollutant is present in the atmosphere, an experiment was conducted to determine if the other major pollutant from electricity generation, sulphur dioxide, influenced. the response of plants to nitrogen oxides. The dose-response data drawn from these studies will help establish a satisfactory background on which to consider the possible impact of

·nitrogen oxides on native plant communities.

The following program of works was undertaken:

• A review of the literature on the effects of nitrogen oxides on plants and reactions of nitrogen oxides in the environment, focussing on the physiological effects of the nitrogen oxides, their sources, the atmospheric chemistry of nitrogen oxides, and the assimilation of nitrogen oxides in plants.

• Selection of the most economically- and ecologically-important Australian tree species growing in forest regions near energy-intensive facilities.

• Operation of an open-top chamber fumigation system to the experimental protocol devised to study the development of dose response relationships for these species. ·

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• Assessment of the symptoms, physiological and growth effects of nitrogen oxide fumigations on these species.

The findings of the review were that a reasonable body of literature now exists describing. the effects of very high concentrations of nitrogen oxides in plants, particularly examining physiological responses, however almost all of the species studied are from Europe, North America, or Japan. · Similar situations apply to reports of the sensitivity of particular species and symptoms of nitrogen oxides damage to foliage. Little of the information available can be used to characterise the concentrations of nitrogen oxides, alone or in combination with other pollutants, which cause damage to vegetation under field conditions.

Several issues in the literature should be noted as they reflect the limitations of any experimental protocols to correctly register the effects of nitrogen oxides pollutants on vegetation in the field. Firstly, long term effects of nitrogen oxides on heath communities favour the replacement of these with grasslands over periods of twenty years or so. Other long term effects such as forest decline have been known for some time. Yet it is difficult and expensive to conduct long-term studies. Secondly, the chemical reactions of atmospheric nitrogen oxides are well understood in principle, but poorly understood in terms of their kinetics. Thus~ while the conversion of nitric oxide to nitrogen dioxide may have some significance in subsequent effects on vegetation as nitrogen dioxide is considered to be more toxic than nitric oxide, the rate of conversion may be among the least certain component of a model of these effects. Thirdly, indirect effects may be at least as important as direct effects. There is considerable evidence and mounting concern that the entry of nitrogen oxides into native vegetation may diminish frost or drought resistance or improve the foliage as a substrate fm: insects. The metabolic components of the leaf tissue that might be ,associated with the last of these effects are not dear, but soluble amino acids are a candidate.

·The results of the project show that, over some eight tree species, there exists consistent beneficial effects of nitrogen oxides on growth of 10-20% at low concentrations, and at concentrations up to nearly 200 nL L-1 in some species. Above this concentration, all species decline in growth, returning to, or below the growth of non-fumigated plants at between 100-500 nL L-1. The declines in height and yield at high nitrogen oxide concentrations were as much as 15% less than control plants.

When certain species were fumigated with nitrogen ~oxides they tended to increase the proportion of leaves to the rest of the plant. This may render plants exposed to nitrogen oxides more susceptible to drought and other root derived stresses. Some variation of the magnitude and nature of responses to nitrogen oxides has been observed in different species studied. Pinus radiata is among the least affected by nitrogen oxide fumigations, whereas Eucalyptus calophylla is clearly affected, among several other Eucalyptus species.

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Experiments to determine the influence of sulphur dioxide on plant response to nitrogen oxides showed that increasing nitrogen oxides concentrations generally caused decreases or had little effect on weight or height. When plants were simultaneously exposed to both pollutants, weight or height decreases caused by nitrogen oxides were accentuated by sulphur dioxide.

Some variations in the response of plants to nitrogen oxides in seasonally different climatic. conditions were observed, suggesting an interaction between nitrogen oxides and climate, so that under cold conditions, the effects of nitrogen oxides may alter plant habit. These effects may relate to variations in growth patterns of the different species, with greater capacity to detoxify and utilise the nitrogen of the absorbed nitrogen oxides when the plant had a higher relative growth rate. Infrequent fumigations, at once a week or less, did not result in identifiable trends in the growth of the group of 8 species studied overall. Little evidence was found of increase in plant response to increasing frequency of exposure, suggesting that the plants were able to recover from a fumigation event before the next fumigation.

Symptoms of nitrogen ~xide injury were observed in acute fumigation experiments, and are reproduced in chapter 3. Death of leaf tissue in a pattern of coalesced patches, each of a millimetre or so in diameter were characteristic symptoms. The necrosis appeared within 24 hours of fumigation at high · concentrations (10,000 nL L-1) for appearance. In the range of fumigants used

· in other experiments (ambient to 500 nL L-1), the plants showed no visible symptoms of injury.

Discussion within the document considers some of the issues. of importance in applying secondary criteria to pollutant emissions. Assessment of native plant community health has in the past been largely undertaken by visual inspection, noting the crown structure and density, the presence or absence of dead limbs, and the chlorosis of foliage. However, as the effects of nitrogen oxides are so visually asymptomatic, an alternative is to consider a range of morphological, physiological and biochemical parameters as indicative of nitrogen oxides exposure. Tissue amino acids and ammonia levels were examined as potential indicators, however very little response of amino. acids · to nitrogen oxide fumigation was observed in E. marginata, the species chosen for this exploratory work. Levels of free ammonia in leaf tissue increased about two-fold immediately after intense nitrogen oxide fumigation and it is of interest to examine whether such a response persists over time and at lower concentrations ..

In conclusion, the Australian native tree species showed:

• a stimulation by 10-20% of height and weight at nitrogen oxides concentrations of up to 200 nL L-1 for two hours per day, three days per week, depending on the species. ·

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"'/i.

• a decrease by up to 15% in height and weight when exposed to 100-500 nL L -1 of nitrogen oxides for two hours per day on three days per week, depending on the species. · ·

• an inte~action between nitrogen oxides and cold weather, which alters plant habit

• little evidence of variation in plant response with increasing frequency of exposure.

• plants fumigated with nitrogen oxides have a smaller root weight in comparison with foliage weight, which may render them more susceptible to drought and other root-derived stresses.

• sulphur dioxide accentuates the effects of nitrogen dioxides.

• symptoms of acute nitrogen oxide injury on leaves consist of small patches of dead tissue, one millimetre or so in diameter which coalesce. However this only occurs at very high concentrations and is unlikely to be seen in the field under realistic ambient concentrations.

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CHAPTER 1: INTRODUCTION

1.1 PROJECT OBJECTIVES

. Nitrogen oxides are predominantly generated by reaction of atmospheric dinitrogen with atmospheric oxygen in the combustion flame, with a smaller proportion generated from fuel nitrogen (de Soete, 1992). In a developed nation like the United States, fuel combustion in stationary sources is responsible for 56% of nitrogen oxides emissions, while transportation

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. contributes around 40% of nitrogen oxide emissions (US EPA, 1991). In the LaTrobe Valley industry emits 97% of the nitrogen oxides,but in Melbourne it only emits only 15%, as 81% is emitted by vehicles (Streeton, 1990). In the electricity generation industry, nitrogen oxides are generated as the sole major pollutant from gas turbine power stations, although other pollutants such as 'sulphur dioxide are also emitted from coal- and oil-fired power stations. These types of power sources are the main-stay of the Australian electricity generation industry. Substantial increases in power generated in Australia has been through increased generation by gas turbine power stations in recent years (ESAA, 1992). With annual increases in consumption of electricity of 5% per year since 1987 (ESAA, 1992), and increasing numbers of vehicles in · Australia, nitrogen oxides are anticipated to be an increasingly important component of the atmosphere in Australia.

It is well established that responses of plants to air pollutant exposures vary · between different spec'ies, and that native yegetation is a sensitive receptor of

air pollutants (Legge and Crowther, 1987; Wolfenden and Mansfield, 1991). Further, it is considered the economic and ecological values of native trees are beststudied by measurement of growth (and related plant parameters) in relatively long term experimental designs, to emulate field exposures. It is clear that short term studies, examining effects of pollutants through · exposures under constant environmental conditions, for example in growth chambers, tend to characterise effects irt plants with high growth rates and under optimum conditions. Further, the results of these experiments do not correlate with observations and measurement of the impact of air pollution in the field. Most studies of pollutant gas effects have been under short term studies, but these overlook the modifying effect of sporadic exposures and stresses of field conditions (Krupa and Kickert, 1987). Finally, a characterisation of the visible effects of acute exposure is necessary to assist monitoring and respond to public inquiry. This study addresses these needs.

The objectives of this ERDC funded project are:-

1. to determine the range of tolerance to nitrogen oxides of the most economically and ecologically important groups of Australian native plants under field conditions~

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2. to quantify the maximum concentrations of nitrogen oxides that Australian native species can tolerate. This will facilitate better planning and siting of gas turbines to minimise adverse effects while avoiding unnecessary expenditure. ·

3. to characterise the symptoms of nitrogen oxides injury to native vegetation. This will assist with monitoring and to respond to public concern.

1.2 NITROGEN OXIDES IN THE ENVIRONMENT: A NEW VIEW

Understanding of the environmental impact of nitrogen oxides is changing rapidly. In the past decade, studies on nitrogen oxides have identified damage to the environment caused by, or involving nitrogen oxides. Nitrogen oxides have been associated with the production of photochemical

· smog and acid rain for decades. Nitrogen oxides are linked with industry, vehicles and agricultural activity, and tend to be involved with most major atmospheric problems. Concern about the role of nitrogen oxides in damage to the environment has led to unprecedented levels of international cooperation and discussion, with many nations embracing restrictive measures to curb emissions of these pollutants (Clarke and Williams, 1991).

Currently there is intense interest in Europe in the effects of atmospheric nitrogen oxides due to the damage which is occurring to natural vegetation. A series of studies have established that the rate of reversion of heathland nature reserves to grasslands has increased in association with regional increases in gaseous nitrogen pollutants from human activities (See section 1.5). Decline of forest trees in both Europe and the eastern US is also linked to nitrogen oxides. In the forest trees, a nitrogen fertilization effect leads to imbalances in nutrition and sensitivity to cold and drought conditions (Section 1.5). ·

There is action in several European countries, and proposals for international conventions, to mandate decreases in nitrogen oxides emissions. These are similar in approach to the conventions on sulphur dioxide emissions. The draft of the fifth EEC Environmental Action Programme has proposed a stabilisation of nitrogen oxides emissions at 1990 levels by 1994, followed by a 30% reduction by 2000. These targets already exist for large combustion plants as a directive, adopted in 1988, requires that nitrogen oxides emissions from these sources be reduced by 30% from 1980 levels by 1998. Natural gas is a very clean fuel; normally resulting in negligible emissions of sulphur dioxide, carbon monoxide, particulates and hydrocarbons, and much smaller emissions ofcarbon dioxide than coal or oil. However, combustion of natural

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gas is a major source of rutrogen oxide emissions. Nitrogen oxide emissions from natural gas combustion depend upon many factors including flame temperature and geometry, and the surrounding medium (Section 1.3)~ Typical gas-fired plant have been estimated to emit about 150 ng J-1 (Mandyczewsky et al. 1992). While there is no suggestion of moves in Australia along the lines of the emission regulations cited above at this stage, this project was established to investigate the potential for fertilisation/toxicity responses of native trees to nitrogen oxide emissions. ·

Perhaps even closer to the immediate circumstances of the urban.inhabitant is evidence of the role of nitric oxide in physiological functions in the body. Nitrogen oxides are known to irritate lungs, lower resistance to respiratory infection, and can cause the accumulation of fluid in the lung (US EPA, 1991). Over the last year or so, nitric oxide has been the focus of intense study due to its role in areas of neurophysiology, reproduction, and immunology. These have been high profile studies reporting in prominent journals such as Science (Snyder, 1992; Burnett et al:, 1992; Finkel et al., 1992; Izumi et al., 1992). Further, descriptive review papers explaining these studies are rapidly appearing in the popular science journals (Lancaster, 1992; Snyder and Bredt, 1992). Thus, nitric oxide has roles in the death of neurons during illness and injury, as a signal ~olecule in synaptic function, in preventing aggregation of · blood platelets, as a cellular toxin in the killing of infected cells and in the effective destructionof can..:erous cells by macrophages, or white blood cells, of the bodies defence system. Certain of these functions have been known to man for some time, in the form of the preservative effects of nitrite salts, but understanding of the effects and metabolic production of nitric oxide by living cells has advanced rapidly in recent years.

Considerable effort to minimize emissions of nitrogen oxides hasbeen made. This· has been done through monitoring emissions and setting safe levels, and by studying economical procedures to minimize the emissions. Monitoring emissions and setting levels has largelybeen undertaken by regulatory authorities in various nations, setting standards, objectives or guidelines, depending on their power to administer these. Primary standards are intended to protect human health, whereas secondary standards are intended to protect the wide range of activities making up human welfare .. one uniform level of 53 nL L-1 as an annual arithmetic mean has. been set for· nitrogen dioxide for primary and secondary standards (US EPA, 1991). The World Health Organisation (1987) recommends nitrogen dioxide levels of 210 nL L-1 and 80 nL L-1 as primary or human health guidelines for one-hour and twenty four-hour averaging levels respectively. The Victorian EPA (Australia) has established objectives for nitrogen dioxide at three levels of safety for one hour and twenty four~ hour averaging levels (Streeton, 1990). These are 150 nL L-1 and 60 nL L-1 as acceptable levels and 250 nL L-1 and 150 nL L-1 as detrimental levels, for one-hour and twenty four-hour averaging levels respectively, and 500 nL L-1 as an alert level at or above which "most members of the exposed population are likely to be adversely affected. a

Some guidelines .for vegetation exposure levels has also been published (World Health Organisation, 1987). These guidelines suggest that:

" In the presence of levels of sulphur dioxide and ozone not higher than 30 IJ,g m-3 (11.3nL L-1) and 60 IJ,g m-3 (30 nLL-1) respectively, the atmospheric concentrations of nitrogen dioxide should be no higher than 30 IJ,g m-3 (15.7 nL L-1) as a yearly average of twenty four-hour means and no higher than 95 IJ,g m-3 (49.6 nL L-1) as a four-hour average.

·In order to protect sensitive ecosystems, the total nitrogen deposition should not exceed 3 g m-2 per year. "

Overall, the ambient standard levels have been chosen on the basis of being lower than the ·lowest-observed-effect level in man or plant. Most nations have developed national strategies for air pollution control that are similar across boundaries and in control levels (United Nations, 1987). These use a range of legislative measures providing. licensing authorities, zoning of activities, impact assessments, investment incentives and disincentives, and the authorities formed specify control technology that should be employed and maintain an inspection regime over this.

Studies have been undertaken to optimize energy production at minimum nitrogen oxide production levels (Kido et al., 1992; Burd, 1992); to capture stack gases in useful forms (Lin, 1992); to modify forms of rutrogen in stack emissions, for example by urea injection (de Soete, 1992); and to integrate and identify further remediation procedures (de Soete, 1992).

Also, the interaction of soils and vegetation with atmospheric nitrogen oxides has become better understood, as a result of understanding of possible mechanisms and circumstances in which nitrogen oxides are emitted by plants. Earlier workers had focussed on uptake alone, expecting to characterise an assimilation driven by metabolic energy captured through photosynthesis (Wellburn, 1990). However, a number of studies have demonstrated that agricultural systems can be significant sources of nitrogen oxide emission, particularly where high levels of nitrogenous fertilizers have been applied, as in much European agriculture, and horticulture worldwide (Mosier .and Schimel, 1991; Skiba et al., 1992).

The atmospheric inputs of nitrogen oxides place a nitrogen burderi on native plant communities. Many Australian soils are characteristically poor in nitrogen and other nutrients, and many non-leguminous native plants are adapted to low levels of nitrogen in the environment. Effective utilisation of may be limited by low relative growth rates, unlike agricultural plants. Uptake of nitrogen oxides by forest systems can be considered remediation, and appropriate siting of industry can facilitate this minimal cost approach. Studies to quantify the long-term capacity of forests to remove nitrogen oxides from air are few. Researchers have attempted to quantify more passive fluxes, that is, wet and dry deposition, of nitrogen compounds. Plant death or

increased susceptibility to frost or insect damage were serious consequences in the Northern hemisphere forests to nitrogen oxides deposition (Wolfenden and Mansfield, 1991; Alstad et al., 1982).

However, plants have some capacity to actively regulate nitrogen fluxes in their environment. Hargreaves et al. (1992) describe a nett uptake of nitrogen dioxide by a pasture, while simultaneously emitting nitric oxide, with regulation of fluxes through stomatal conductance. Complex interactions of nitrogen oxides with trace volatile organic compounds led to a daily accumulation of nitrogen oxides in the canopy of pine forest (Enders et al., 1992). Native plant communities can tolerate or benefit from a modest levels of nitrogen oxides in the atm_osphere (Adams and Attiwill, 1991, Murray et al., 1991). ·Adams and Attiwill (1991) observe that eucalypt forest actively absorb nitrogen from rain in the crown canopy, Some modelling of the mechanism of entry of nitrogen oxides with plants has been undertaken (Enders et al., 1992; Hargreaves et al.,1992), however these only consider the micro-meteorology of the atmosphere-plant interaction. This is unsatisfactory, as the development of appropriate models in this· area may lead to improved siting of industries, and possibly better production from forests and agriculture.

In this context, this review attempts to identify the potential mechanisms that plant communities, particularly natives, may have for uptake of nitrogen oxides from the atmosphere. The issues at large revolve around the following questions. Firstly, what levels of nitrog~n oxide assimilation are sustainable in the long term~ without leading to detrimental effects. Secondly, what capacity is there to adapt plant communities to more efficiently assimilate nitrogen oxides, for example, with improvements in nutrient status. Thirdly, what indicators, tests, monitoring and management procedures may be appropriate for plant communities providing such assimilation functions. In this context, the review will briefly examine the known physiological processes associated with plant uptake of nitrogen oxides, the intercoiwersions of oxides and plant metabolites, and some of the issues already noted above.

1.3 SOURCES OF NITROGEN OXIDES AND AS SOCIA TED ISSUES

1.3.1 Sources

Nitric oxide is largely derived from vehicles, power plants, domestic and general industrial sauces, and nitrification in soils (Dasch,1992'; Clarke and Williams, 1991; Skiba et al. , 1992). Nitric oxide production from combustion results from several mechanisms which each progress best under slightly different combustion conditions such as temperature, fuel:oxygen ratio, or fuel qualities(de Soete, 1992). These mechanisms proceed in response to any of combustion temperature, fuel nitrogen, or the formation of CX radicals. As a consequence, studies to estimate (or understand the chemistry of) nitric oxide production with different fuels are common (e.g., Kido and Hirasawa, 1991). Prediction of nitrogen oxide formation in stationary combustion has

progressed to reliable levels with well known factors including plant layout, control of gas circulation, and staged combustion (Clarke and Williams, 1991; Burd, 1992}. However the same can not be said for vehicle combustion where catalytic convertor efficiency may be a determinant (Dasch, 1992), or soil nitrification where anaerobic conditions may be important (Arah, 1992). One possible source of nitric oxide yet to be examined in terms of potential to contribute to total input to the atmosphere is from plants. This occurs where there has been adequate nitrogen supply (Farquhar et al., 1983), or where herbicide-treatments have been applied (Klepper, 1979); both increasingly common circumstances in agriculture. Other sources of nitric oxide include the oxidation of atmospheric nitrous oxide (Arah, 1992), and natural sources such as volcanic activity and wildfires.

Nitrogen dioxide production mostly comes from similar sources to nitric oxide, that is, from vehicle emissions, stationary combusticm plants, and also possibly from plants. Oxidation to nitrogen dioxide provides C). removal mechanism for stratospheric nitric oxide, and the nitrogen dioxide formed at low elevations may return to soils and plants and be assimilated as nitrate (Arah, 1992). This step is assisted by the higher solubility of nitrogen dioxide . than nitric oxide in rainwater (Garsed, 1984). Further oxidation to nitric acid ·may also occur. These conversions lead to cycling of nitrogen oxides in the atmosphere.

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Nitrogen oxides in the atmosphere commonly have a daily cycle in urban regions with peaks of high concentration in the morning and evening associated with high levels of vehicle traffic (WHO, 1987). These peaks are of a few ·hours duration, superimposed over a background level contributed from other sources. Typical urban levels throughout the world are 10 to 50 nL L-1.

·In Melbourne, vehicles contribute around 81% of atmospheric nitrogen oxides while industry contributes 15% and domestic sources the remaining 4% (Streeton, 1990). The frequency of high levels in any given urban region depends on time of the day, on prevailing meteorological conditions and seasons, on the levels of various human activities (WHO, 1987), and in certain cases such as Los Angeles, the effect of the surrounding topography on the urban air mass (Solomon et al., 1992). In Melbourne, the one-hour average acceptable level of 150 nL L-1 of nitrogen dioxide has been exceeded one or two times per year {Streeton, 1990). Domestic exposure mainly results from gas heating and cooking devices (Mandyczewsky et al. , 1992) and cigarette smoking. These sources can result in substantially higher atmospheric concentrations of nitrogen oxides than external sources (WHO, 1987).

1.3.2· Reactions Controlling Atmospheric Nitrogen Oxides

Several workers have referred to complexities in the gas-phase reactions which nitric oxide and nitrogen dioxide undergo. These influence the levels of nitrogen dioxide present .in equilibrium with nitric oxide through the reactions:

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arid

NO + 03 ~ N02 + h v ~ 0+02+M~

N02 NO

03

+ + +

02 0 M

where M is a particulate surface absorbing the energy ~f the reaction. These reactions work against each other to achieve a steady-state in this area of trace gas chemistry, under given conditions of light, temperature, particulates and

16

other sinks for ozone such as hydrocarbons (Wellburn, 1988; Hargreaves et .al., 1992; Enders et al., 1992). Ozone in thelroposphere is mostly generated by the second two of these reactions, that is, the photolysis of nitrogen dioxide and . subsequent reaction of atomic oxygen.

Oxidationofnitric oxide to nitrogen dioxide in the atmosphere allow . concentrations is a well studied process. Initially it was thought that either ozone or dimolecular oxygen could be involved in such reactions. · Oxidation of nitric oxide by reaction with ozone is an exothermic reaction, releasing 201.4 J g-mol-1 at 298 K (Samsonov, 1973). Altshuller (1956) has calculated that 50% conversion of atmospheric nitric oxide proceeds in around a minute when both nitricoxide and ozone are at concentrations of 100 nL L-1. Oxidation of nitric oxide by reaction with bimolecular oxygen is less exothermic, releasing 114 J g-mol-1 of 02 at298 K, .and occurs as a two-step reaction via an intermediate complex oxide (N0.02) which is unstable, usually decomposing to the initial nitric oxide and molecular oxygen (Samsonov, 1973; Mellor/ 1962; op. citl The oxidation reaction with ozone is considered to be the most · important (WHO, 1987), .however it also consumes ozone. During the Latrobe Valley Airshed study (Manins, 1986), the proportion of nitrogen dioxide to total nitrogen oxides increased by 15 to 40 % per hour in power station plumes

. · containing around 10 nL L~1 of total nitrogen oxides. ·

The spatial and temporal distribution of nitrogen oxides in urban environments is also in dynamic equilibrium with other gas emissions such as ammonia (Solomon et al. , 1992). It has been observed in the Los Angeles basin that nitric acid concentrations in the atmosphere are up to 20 11g m-3. Nitric acid is formed in air by the oxidation of nitrogen oxides mostly from vehicle emissions. Nitric acid concentrations diminish when it intercepts NH3 evolved from animal wastes in dairy livestock areas, forming NH4N03 at concentrations up to 130 ~-tg m-3. The nett result is a fallout of NH4N03 aerosol. The atmospheric reaction with NH3 is similar to the flue gas treatment with urea, ammonia or cyanuric acid (de Soete, 1992). However the latter yields N2 and H20, the end-produ~ts of the reaction at temperatures around 900°C. Reactions between NH3 and S02 at flue gas temperatures of 60-65°C are found to yield ammonium sulphite products (Hjuler and Dam-Johansen, 1992), which may be more analogous to atmospheric NH4N03 formation. Temperature has been found to be an important controlling factor in these processes, as well as the lime- flue gas reactions used by Lin (1992).

17

Nitrogen oxides also r~act with each other to form a range of polymeric nitrogen oxides (N204, N20s), with water vapour forming nitric and nitrous acids, with carbon monoxide to form carbon dioxide and dinitrogen, with ClO forming chlorine nitrate (ClON02, Folkins and Brasseur, 1992), and with . peroxides of volatile organic hydrocarbons to form peroxyacyl nitrates (known as PANs, Wellburn, 1988).

1.4 EFFECTS OF NITROGEN OXIDES ON PLANTS

1.4.1 Symptoms

Visible symptoms of nitrogen oxides damage on plants are generally clearly delineated patches of necrosis on leaves .. These patches are often on the tips of longer, narrow leaves or on both the leaf margin and interveinal regions of broad leaves. Photographic atlases of air pollution injury in plants have been published (Jacobson and Hill, 1970; Taylor et al., 1987; Lacasse and Treshow, 1978) .. The acute symptoms generally appear after fumigation with nitrogen oxides at concentrations of several parts per million for an hour or more. Such high concentrations would not be commonly experienced by vegetation. Considerable variations in sensitivity to nitrogen oxides of different species and interactions of nitrogen oxides damage with other environmental effects have been reported (Table 1.1).

Symptoms alone are of little use in diagnosing exposure to nitrogen oxide pollution. Several reports depict the foliar symptoms of certain nutrient . disorders, plant diseases, and other pollutants (for example, sulphur dioxide), which can be sufficiently similar to symptoms of nitrogen oxide damage to require further situation information and analysis to allow an effective · diagnosis (Taylor et al., 1987; Grundon, 1987; Snowball and Robson, 1983). Certain typical patterns of damage on foliage, geographic relationships of damaged plants to sources, and patterns of distribution of injury may be considered in a micrometeorological approach to help identify sources of damage.

Unfortunately, further analysis to uniquely identify nitrogen oxide pollution is quite. difficult, since effects on plant tissue composition may be small. Under less acute conditions, nitrogen oxides can initially cause plants to be a deeper green colour (Wellburn; 1990). Chlorophyll levels in leaf may be shown to increase. The researcher is restricted to eliminating other possibilities by analysis for nutrient imbalances, observing the absence of pathogens under the microscope, and eliminating other sources of stress which could result in the observed effects. Leaf tissue of plants suffering nitrogen oxide damage should usually have adequate levels of tissue nitrogen. · No correlations between symptoms and yieldshas been shown in studies reviewed by Legge and Crowther (1987).

17 18

Table 1.1. Relative sensitivity to atmospheric nitrogen oxides (from Taylor et al., 1987).

Sensitive Intermediate Tolerant

Trees and Shrubs Apple Japanese maple Austrian pine Larch Jasmine Beech Pear Lawson cypress Copper beech Pyracantha Lime Elder Silver Birch Norway maple Ginkgo*

Privet Hornbeam Rhododendron Locust tree Silver fir Oak* Spruce Pine*

Yew

Agricultural and Barley Beet Kale horticultural crops Bean* Buckwheat Kohlrabi

Broccoli Com Onion Carrot Cucumber Potato Clover Rape Red cabbage Leek* Rye White cabbage Lettuce Ryegrass Lucerne Strawberry. Oats Swiss chard Parsley Sprouts Pea* Tomato Radish Wheat Rhubarb Spinach

Ornamentals Aster Fuschia Gladiolus Azalea Petunia Heather* Begonia Lily of the Valley Chrysanthemum Hibiscus. Lupin Rose Snapdragon Sunflower Sweet pea

Wild species Brome grass Annual poa Chickweed* Clover Cocksfoot Fat hen Common vetch* Dandelion Ox-eye daisy Plantain Italian ryegrass Rose Red fescue

Ryegrass Smooth stalked meadow grass Timothy

* = very sensitive or very tolerant

1

1.4.2 Yield Effects

Where plants are nitrogen deficient, increases in biomass from low concentrations of nitrogen oxide pollution can occur through nutritive mechanisms. This effect is not observed in all studies, and generally the view is that air pollution with nitrogen oxides will lower plant growth. Wellburn (1990) has explored this situation comprehensively. Responses appear to vary with the sensitivity of species, the available sunlight, and through interactions

. with other pollutants and nutrients. Spierings (1971) and Taylor et al. (1975) provide examples where sensitive species have shown small yield decreases with low levels of nitrogen dioxide. Several other studies have indicated that fumigation with nitrogen oxides alone do not result in major changes in _yields, posing little threat to crop productivity (Amundson and Madean, Some reviews of the field (Rajagopal and Saxe, 1988; Rowland et al., 1985) conclude that there usually is a decline in growth while Legge and Crowther (1987) conclude that most studies with nitrogen dioxide below 1000 nL L-1. have shown inconclusive effects.· A recent report of this group (Murray et al. , 1991) examines previous studies on the effects of ·nitrogen oxides on plant growth and points out the use of unrealistically high concentrations of fumigants in some European and US studies. However high concentrations may be reached in enclosed atmospheres such as in heated glass houses, and a recent study has examined the suitability of a wide range of species for cultivation in such atmospheres (Saxe, 1992). ·

One area of emphasis is on interactive effects of nitrogen oxides with sulphur dioxide rather than direct single pollutant studies. In cereals, nitrogen oxides ameliorate the damaging effects of sulphur dioxide on plant growth, whereas in legumes and other plants the opposite occurs. In Australia, recent studies of nitrogen oxide effects on plant species have focussed on its interaction with sulphur dioxide (Murray et al., 1991) and effects on grain yield of crop species and growth of pasture species (Murray and Wilson, 1992a, b). One study on the forest species hoop pine has also been undertaken (Murray et al. , in press). Attempting to summarise the diverse responses in order to develop secondary ambient air quality criteria for sulphur dioxide and nitrogen oxides,· it was concluded that the information base on nitrogen oxides was uncertain. The report (Murray et al., 1991) suggested that both nitric oxide and nitrogen dioxide were toxic to plants and offered a recommendation that:

"in the presence of levels of sulphur dioxide not higher than 25 nL L-1, the atmospheric concentration of nitrogen oxides (the sum of the nitric oxide and nitrogen dioxide concentrations) should be no higher than 25· nL L-1 as a four hour arithmetic mean."

. This was intended to protect plants against visible injury as well as any decline in yield. The criterion was based on several experiments carried out during the project.

Generally, supply of nitrogenous fertilisers has well known effects of increased yields, greater efficiency of water usage, increased harvest index (that is, the

19

t

a

L____

20

yield of grain as a proportion of the whole top), and higher grain nitrogen concentration after exceeding a low threshold where it declines (Beevers, 1976; Broadbent and Carlton, 1978; Embleton and Jones, 1978). Nitrogen fertilisation also tends to impart a delay in flowering of annuals, increased leaf area and photosynthetic light harvesting, and a susceptibility to fungal diseases (Graham, 1983). How these aspects of plant growth are affected by atmospheric nitrogen oxides entering through the foliage as distinct from soil-applied nitrogen has not been studied. In principle, foliar fertilisation which is very commonly undertaken with urea as a nitrogen source (Smith et al, 1979; Alexander, 1986; Alexander and Schroeder, 1987) should have quite similar effects to nitrogen oxides.

Both nitrogen and sulphur from acid deposition can act as nutrients to plants through leaf absorption (Irving and Miller, 1981; Troiano et al., 1983). In . discussing foliar nutrient uptake, Franke (1986) draws attention to channels termed ectotechoides in the external walls of the epidermis, below the leaf cuticle, which may allow entry of nutrients. Much more needs to be learnt of leaf structures and how these interact with surface applied compounds. There is ample evidence that processes such as acid deposition of pollutants will modify the forms of soluble, titratable chemicals on the leave surface and in this way regulate their movement into leaves (Smith et al, 1979). Use of alkaline products (oxides, carbonates) in deliberate foliar applications of other nutrients to plants experiencing acid deposition would seem very appropriate.

1.4.3 Deposition, Assimilation and Physiological Effects

Cape and Unsworth(1988) have provided a detailed discussion ofthe deposition processes of pollutants into plants. The dominant process is usually direct uptake of gaseous pollutants from the atmosphere, but deposition of particulate aerosols and wet deposition, particularly of acidic mists at high altitudes, can occur.

Atmospheric gases diffuse into leaves under well understood flux relationships guided by the conductances of canopy and stomata. The fluxes may involve turbulent. flow, laminar flow and molecular diffusion processes (Finnigan and Raupach, 1987). In the discrete plant canopies, such as trees in an open forest, dispersal of atmospheric pollutants into the tree canopies often occur as cool downdrafts from the bulk atmosphere within the canopies (Finnigan and Raupach, 1987). Around the trees, warmer, more buoyant air rises from near the warmer substratum and soil generating a pattern of eddy transfer. Thus, the occasionally downdraft air movement feeding assimilation in forest communities differs from the occasionally updraft air· movement experienced by plants grown and fumigated in open-top chambers. Whether this has any significant effect on the plant growth and habit responses to pollutants in unclear. Other differences in environmental circumstances and procedural aspects of experimentation with open-top chambers and pollutant have been discussed by Unsworth (1991).

2

When nitrogen oxides have entered the substomatal cavity of the leaf they have sever().l possible fates. Initially, they may dissolve in the solution in the apoplastic cell wall space. From there they may react with amine functions in organic constituents to form adducts such as nitrosamines. Nitric oxides may coordinate with iron in sites in the cell wall and in soluble low-molecular weight complexes involved in vascular transport of iron in plants, such as nicotianamine (Lancaster, 1992; Walker and Welch, 1986). In aqueous solution, nitrogen oxides are reversibly hydrated to form nitric and nitrous acids which in turn will be in dissociation equilibrium with their respective anions. As the unhydrated oxides, these small neutral forms are quite permeable through membranes, and can easily enter the internal space of the leaf cells. The extent to which this occurs will depend on the equilibria mentioned above, and these will effectively be regulated by the overall concentrations of nitrogen oxides and the pH of the solution in the apoplastic cell wall space. Acid conditions will favour the free oxides and greater entry of these into the cell.

Once in the cell, the nitrogen oxides may react with amine and iron-containing species, including many proteins. The nitrate and nitrite salts may be actively accumulated into the vacuole. ·The nitrogen may enter the organic nitrogen pool through reduction by nitrate and nitrite reductase (Fig. 1.1). The distribution of entering nitrogen oxides into these processes will be governed by the activities of several enzymes. These include the activities of nitrate and nitrite reductase, the vacuolar membrane transport complexes, and the many other energy-consuming enzyme reactions that will compete with these for available energy. Accumulation of nitrogen oxides is reported to lower the pH of vacuolar fluid, as distinct from cytoplasmic or apoplastic fluids (Freer-Smith, 1987), so it may be anticipated that a substantial proportion of nitrogen oxides does in fact enter the cell. ·

Nitrate reductase and nitrite reductase assimilation of·atmospheric nitrogen oxides is dependent on the provision of energy to drive the reaction (Fig. L1). As these enzymes are located in the chloroplast, their energy supply is usually derived from photosynthesis, arld competes in particular with the fixation of carbon dioxide. Chloroplast energy is usually quite low when available light declines (Bidwell, 1979). Nitric oxide may well also inhibit the reduction of its anion form since electron transport to nitrite reductase passes through the iron protein ferredoxin sirohaeme, as well as several cytochromes at earlier steps of photosynthetic electron transport.

Once reduced to ammonia, nitrogen then enters amino acid metabolism (Fig. 1.1). The best known of the ammonia assimilating reactions is the glutamate dehydrogenase: glutamine-oxaloglutarate 'aminotransferase (Beevers, 1976; Bidwell, 1979). Another reaction that is often overlooked is the glycolate-glycine pathway (Fig. 1.2). This reaction may well respond rapidly to short-term variations in nitrogen oxides in the following way. Nitrogen oxides entering the substomatal cavity will lower the rate of photosynthesis in stomatal guard cells, causmg guard cell conductance to increase through less

21

of

photosynthesis -'~ carbohydrates

~ reducing power

J NADH or NADPH

l

l

/ . n

respirat1o ~

""'' ~ ferredoxin sirohaeme

i nitrate nitrite red.uctase reductase

N03- -. ---7- N02- ---7- NH.3

\ / 2N02(aq) + H20 ~ HN02 + HN03

I 2N02(g)

22

carbon skeletons

amino 7- acids

Fig. 1.1. A general scheme of the metabolism of nitrogen dioxide via nitrate reduction. - Modified from Bidwell, R.G.S. (1979) Plant Physiology 2nd Ed. Macmillan, New York.

----'

CHLOROPLASTS

PEROXISOMES

glycolysis gluconeogenisis?

MITOCHONDRIA

· sugars, starch

bRuBP C02

'Te-p ~ PGA

I glycerate

glycerate

OH-pyruvate

serine

P-glycolate

glycolate

glyoxylate NH3 from

N H3 ~=----+-----+- nitrate ~---....~ reduction

glycine

Fig. 1.2. ·Reactions of the glycolate pathway in the assimilation of ammonia. Note that two molecules of glycine are required to synthesize one molecule of serine. -From Bidwell, R.G.S. (1979) Plant Physiology 2nd Ed. Macmillan, New York.

23 (c 24

energy for solute transport to maintain their turgor. As a result, lower internal C02 concentrations are found within the leaf and a competing reaction with 02 proceeds at a greater rate. The 02 reaction forms glycolate and PGA (phosphoglyceric acid), rather than a usual two molecules of PGA when reacting with C02 (Fig. 1.2). The rest of the reactions which result in a net assimilation of ammonia are also shown in Fig. 1.2.

The synthesis of amino acids is organised into several branching networks of reactions with the incorporation of amino nitrogen functions at a early steps (Fig. 1.3}. These first amino acids formed are termed head group amino acids (Beevers, 1976; Bidwell, 1979). These groupings have been quite valuable in understanding many ways in which physiological processes responds to

. modifying factors.

1.5 EFFECTS OF NITROGEN OXIDES ON AUSTRALIAN NATIVE AND OVERSEAS SPECIES

As we have seen above, nitrogen oxides may provide nitrogen nutrition to plants. However, a substantial proportion of Australian natives have adapted to soil conditions providing relatively little available nitrogen. The . exceptions to this are to befound among native legumes generally, the natural vegetation community of the Briga.low where the dominant tree species is a legume and cohabiting species also benefit, and those regions where water or other nutrients such as phosphorus are more severe limitations. Although it is difficult to demonstrate; there have been many suggestions that adaption to low levels of ambient supply of a given nutrient leads to less tolerance for a higher supplyof that nutrient. If this is the case for nitrogen, examples of nitrogen oxides sensitive species may be found among Australian natives.

Studies in Europe have shown that emissions of ammonia from intensive agriculture, and nitrogen oxides from vehicle, domestic and industrial emissions, are readily taken up by plants through the stomata and leaf cuticle, and metabolised to amino acids and proteins (reviewed by Wellburn, 1988 and 1990). The increase in the plant nitrogen concentration, and consequent changes· in plant metabolism,· including increased concentatioris in some amino acids and proteins, results in succulent growth in heath plants, rendering plants more attractive to insects (Dohmen et al. 1984; Bolsinger and Fliickiger, 1989) and more sensitive to frost damage and water stress (Dueck et al. 1990). As a consequence there is, now considerable concern in North-West Europe about the rate at which the heathlands nature reserves are reverting to grasslands (Liljelund an:d Torstensson, 1988). The rate of reversion of Calluna and Erica-dominated heathlands to grassland dominated by Deschampsia, Molinia and other grasses has increased dramatically in the last two decades in these areas, associated with increases in ammonia emissions from intensive agriculture, and nitrogen oxide emissions from urban, vehicle and industrial

PHENYLALANINE I

TYROSINE I pentose ?-

26

sources (Van Dijk and Roelofs, 1988; Uren and Ashmore, 1991). Nitrogen fertilization experiments confirm that this change is a .consequence of nitrogen enrichment (Boxman et al., 1988).

In Europe and Eastern North America, atmospheric nitrogen compounds, especially nitrogen oxides are thought to contribute to the widespread problem of forest decline by promoting growth at the cost of nutrient imbalances (Lange et al.1989; Norby et al. 1989) or by disturbing the development of winter hardiness {Nihlgard, 1985; Waring, 1987) and drought tolerance (Dueck et al. 1990)~ This results in canopy thinning, apical shoot death and a general

. decline or death of trees (US EPA and German Ministry of Research and Technology, 1988).

There is little published information on the effects of atmospheric nitrogen oxides on Australian native species. It is well established that some species of Eucalyptus are sensitive to soil nitrogen status in the field. Tree decline in rural areas of Australia is a complex problem with many, poorly-understood factors involved (Land~berg and Wylie, 1990). Some forms of tree decline in rural areas are associated with a high nitrogen status of the soils and in the foliage of Eucalyptus trees, although the expression of the tree decline may also be associated with secondary factors, such as insect infestation, and may be accentuated by climatic and hydrological factors (reviewed by Landsberg and Wylie, 1990). ·

CHAPTER 2: GENERAL METHODOLOGY

The basis of the choice of species selection and the methodology common to all three experiments is discussed below. The fumigation regimes, growth conditions, cultivation and harvest procedures varied slightly for each experiment and therefore will be discussed under the relevant chapter for each experiment.

2.1 SPECIES SELECTION

The following species were selected for study. This selection was based on information received from agriculture departments, forestry commissions and electrical generation and supply authorities in each state of Australia as to which native plant species they considered to be of the greatest ecological, economic and aesthetic importance.

Banksia attenuata Eucalyptus calophylla Eucalyptus globulus Eucalyptus maculata Eucalyptus marginata Eucalyptus microcorys Eucalyptus pilularis Pinus radiata

(Banksia) (Marri) (Tasmanian Blue Gum) (Spotted Gum) (Jarrah) (Tallowwood) (Blackbutt) (Radiata Pine)

To ensure that the data collected would be relevant to future resource management and planning decisions, emphasis was placed on selecting tree species that commonly grow close to major electricity generating facilities and are of ecological significance in native forests or of economic importance to the forestry industry. Pinus radiata, although not native to Australia, was selected due to its prominent economic importance to the forestry industry.

2.1.1 Banksia attenuata ·(Banksia)

Banksias occur in all states of Australia. Their characteristically large, persistent inflorescences are easily recognisable in Australian forests and form an important part of the wildflower industry. They are very hardy and can grow on poor soils. They are also particularly important to apiarists as they produce copious quantities of nectar and pollen. This property also renders them indispensible to many species of native. fauna (Boland et al, 1984). Banksia attenuata is a common Western Australian banksia that usually grows to between five to eight metres tall.

27

2.1.2 Eucalyptus calophylla (Marri)

Marri is a conspicuous forest tree indigenous to Western Australia. It has a wide distribution over the southwest of Western Australia and commonly grows in association with jarrah and karri. It is a major component of the forests in the Pinjar area where electricity generating plants are operating. It grows to40m and is the primary source of wood chip material in Western Australia (Boland et al, 1984).

2.1.3 Eucalyptus globulus (Tasmanian Blue Gum)

Tasmanian blue gum occurs naturally in Tasmania, Victoria and New South Wales growing to between 15 and 20m tall (Boland et al, 1984). It is a hardy species, capable of strong growth under a wide range of climatic and edaphic conditions (Hillis and Brown, 1984). For this reason it is used in many countries in the world for reforestation work. Under ideal conditions it is one of the fastest growing eucalypts and is an important plantation forestry species in Victoria and New South Wales.

2.1.4 Eucalyptus maculata (Spotted Gum)

Spotted gum is widely distnbuted on the east coast of Australia, growing from central Queensland down to eastern Victoria at sites up to 400km inland (Boland et al, 1984). It is a conspicuous species growing to between 20 and 35:n tall and is a major component of forests in the Hunter Valley and Central Coast of New South Wales. It commonly grows in pure stands although is often associated with ironbark, blackbutt, and tallowwood (Boland et al, 1984). It is one of the ten most important saw log species in both Queensland and New South Wales (Queensland Forest Service pers com; Forestry Commissionof New South Wales, 1986). It is the primary winter nectar source for bees in New South Wales and commercial apiarists depend on the flowers of the spotted gum to support their industry.

2.1.5 Eucalyptus marginata (Jarrah)

Jarrah, a native ofWestern Australia, is considered to be one of the most important hardwoods in Australia, being particularly sought after for the . construction of high quality furniture. It is by far the most important sawlog in Western Australia. It occurs in the southwest of Western Australia mainly between Perth and Albany. It often grows in pure stands but is also found in association with marri and karri. It is a tall tree, growing to between 30 and 40m tall.

2.1.6 Eucalyptus microcorys (Tallowwod)

Tallowwood is widely distributed in southeastern Queensland and northern New South Wales. Individuals grow to between 35 and 60m heigh and is

. commonly found on rainforest fringes associated with other eucalypts such as

th

th as

blackbutt, flooded gum and white mahogany (Boland et al., 1984). This species comprises approximately 10% of the total volume of log timber in New South Wales (Forestry Commission of New South Wales, 1986), and is one of the ten most important logged species in Queensland (Queensland Forest Service pers com). It is considered to be one of the best native hardwood timbers (Boland et al., 1984).

2.1.7 Eucalyptus pilularis (Black britt)

Blackbutt has a similar distribution to that of tallowwood except that it extends_ further southwards, almost to Victoria (Boland et al., 1984). It sometimes grows in pure stands, but is also commonly associated with several other eucalypts such as tallowwood and spotted gum. It is a very tall tree growing to 70 m. This species is one of the most important hardwoods in Australia, and is the principal species sawn in coastal New South Wales and south-eastern Queensland (Boland et al., 1984). In New South Wales it comprises 15% of the total volume of log timber (Forestry Commission of New South Wales, 1986).

2.1.8 Pinus radiata (Radiata Pine)

This species is the most important plantation species nationally, on both privately and publicly owned land. In total, P. radiata plantations cover' over half a million hectares, comprising 68%' of the total area of plantations in Australia. It is the major plantati_on species in New South Wales, Victoria, Western Australia, South Australia and Tasmania (Department of Primary Industry,1985).

2.2 FUMIGATION CHAMBERS

To derive dose-response relations for Australian flora, the approach used by the United States Environmental Protection Agency (USEPA) and the European Economic Community (EEC) to assess air pollution injury to vegetation was adopted. Both of these bodies independently selected open top chambers for their crop loss assessment networks established to determine dose-response information for major air pollutants. Alter-native approaches such as mechanistic and physiological models have virtually been abandoned in both the United States of America (USA) and Europe since the mid 1970s. This was due to both the complexity of the model characteristics, which meant that it would have been decades before the models could have been used predictively, and the impracticality of growing most crops to full maturity in controlled environment chambers. The USA National Crop Loss Assessment Network (NCLAN), which used open top chambers, yielded useful data on field grown crops exposed to pollutants withinfour years (Adams et al., 1985).

29

Open-top chambers of the same design as those used by the USEP A and the EEC were used in this study in a series of three experiments. Sixteen cylindrical open-top chambers (Heagle et al., 1973) were used for this group of fumigation experiments. Each chamber was 3m in diameter and 2.4m high, consisting of a rigid aluminium frame covered by UV -stabilised PVC plastic. The upper half of the frame was covered by a single layer of plastic and the lower half was covered by a double layer forming an envelope having the inner layer perforated by holes 25mm in diameter. Air was drawn by a fan through a dust filter and then forced along a duct, into the chamber through the holes in the lower envelope and then out tlvough the open top. The output of the fan was 1 m3s-1 enabling about 3.5 air changes per minute.

2.3 POLLUTANT DISPENSING AND MONITORING

The dispensing system and the monitoring equipment for the pollutant gases were housed in seperate temperature controlled buildings to prevent any leakage from the dispensing system registering on the monitoring equipment and to assist in maintaining stable fumigation conditions in the chambers. Anhydrous nitric oxide gas was mixed with N2 and dry air and then introduced to the inlet ducts of the chambers. Flowmeters were used to control the relative volumes of N and air, so controlling the nitric oxide to nitrogen dioxide ratio in the chambers. The nitric oxide and nitrogen dioxide concentrations in the chambers were also controlled by flowmeters. In the third experiment, sulphur dioxide was introduced to some of the chambers in addition to nitric oxide and nitrogen dioxide.

The concentrations of nitrogen dioxide and nitric oxide were measured using a Monitor Labs Model 8840 Chemiluminescent Nitrogen Oxides Analyzer calibrated by the gas phase titration technique. The standard reference nitric . oxide gas was obtained from CIG, certified by EPA Victoria, and diluted with a Monitor Labs Model 8500 calibrator. A Thermo Electron Model 143 calibrator was used with certified permeation tubes to check the functioning of the catalytic converter (that reduces nitrogen dioxide to nitric oxide) in the analyser.

The concentration of sulphur dioxide was measured using a Monitor Labs Model 8850 Pulsed Fluorescent sulphur dioxide Analyser, calibrated with the Model 143 calibrator, with NBS traceable certified permeation tubes.

2.4 EXPERIMENTAL DESlGN

Three experiments are described. These have been named; Trees1, Trees 2 & Trees 3. Each consisted of 8 duplicated treatments (Table 2.1). In Trees 1 and Trees 2, four different concentrations of nitrogen oxides and four different

c.

the

I

L

frequencies of fumigation were used. The eight treatments were divided into two. regimes each consisting of five treatments (two treatments were common to both regimes):

Regime 1 - Increasing frequency, constant concentration.

Regime 2 - Increasing concentration, constant frequency.

In Trees 3, four different concentrations of nitrogen oxides, two different concentrations of sulphur dioxide and one fumigation frequency was used.

Table 2:1 The experimental design use·d in t'le three major fumigation experiments.

a Trees 1 Treatment Regime Nominal Fumigation frequency

conc.(nL L-1) , 1 1&2 0 none (control) 2 1 100 2hrs/5 months

.3 1 100 · 2hrs I month 4 1 100 2hrs/week 5 2 25 2hrs 3 times I week· 6 2 50 2hrs 3 times/ week 7 1&2 100 2hrs 3 times/week 8 2 200 2hrs 3 times I week

b Trees 2 Treatment Regime Nominal NOx Fumigation frequency

conc.{nL L-1) 1 1&2 0 none (control) . 2 1 500 2hrs I 5 months 3 1 500 2hrs I month 4 1 500 2hrs/week 5 2· 50 2hrs 3 times I week 6 2 100 2hrs 3 times I week 7 2 300 2hrs 3 times/week. 8 1&2 500 2hrs 3 times/week

I

31

c T rees 3 ( 11 f f 2hr 3 . ti a uffilga ons are or s trmes /week) Treatment Nominal NOx Nominal S02

conc.(nL L-1) conc.(nL L-1) 1 0 0 2 100 0 3 •c 200 0 4 400 0 5 0 250 6 100 250 7 200 250 8 400 250

Chronic effects on plants of air pollutants are more likely to be a result of cumulative stress of periodic fumigation episodes rather than of a continuous fumigation (Krupa and Kickert, 1987). As plants may respond both to the concentration of a pollutant and to the frequency of exposure to that pollutant it was necessary to subject them to varying concentrations and frequencies of fumigation. ·

The concentrations and frequencies of fumigation '"Nere chosen to span the ambient values likely to be found in the vicinity of power generating facilities. One hour averages in excess of 500 nL L~1 of nitrogen oxides have been recorded periodically in the Latrobe Valley (Latrobe Valley Airshed Study, 1986). Hence a concentration range of 0-500nL Vl was used in this study. The frequency range of between once in five months to three times per week was chosen to cover the range of fumigation events likely to be experienced in an· airshed due to varying atmospheric conditions.

Trees 1 and Trees 2 were designed to show the effects of concentration and frequency of nitrogen oxides fumigations. Trees 3 was designed to establish the interactive effects of sulphur dioxide, and nitrogen oxides. The pollutant concentrations for all three experiments were chosen to simulate worst case scenarios under conditions of inversion break-up and plume grounding. Thus, the decision was made to fumigate for only two hours per day between 1100 and 1300, three days per week to simulate break-up of the atmospheric inversion layer which typically begins mid-morning.

2.5 ENVIRONMENTAL CONDITIONS

Meteorological recordings were obtained from the Perth Bureau of Meteorology from their recording station located in WeUington Street, East Perth. This is 15km from the experimental site and there is no significant difference between meteorological conditions at the two places. Mean daily maximum and minimum temperatures were calculated for the fumigation period of each experiment, in addition to mean daily relative humidities for

for

the recording times used by the Bureau of 0900 and 1500. From previous experiments it has been established that mean temperatures are about 1 oc higher inside the chambers than outside and relative humidity is about 1.5% lower inside the chambers than outside.

2.6 E.ESPONSE PARAMETERS

At and after harvest a range of parameters was recorded. These included growth parameters such as weight, height and trunk diameter and physiological parameters such as tissue concentrations of nitrogen, sulphur and amino acids. Growth parameters indicated the effects of fumigations on tree structure and growth whilst physiological parameters gave indications as to the mechanisms of these effects.

2.7 STATISTICAL ANALYSIS

Fumigation monitoring data were analyzed using Statview 512+ and SuperANOVA (Abacus Concepts Inc, 1986). Analysis of Variance (ANOVA) was followed by a Scheffe's F-test of the means to indicate duplication of

. fumigation regimes.

The intention of the experimental designs of the three major fumigation experiments was to utilise sixteen cha'mbers, attempting to provide eight treatments in duplicate. Analysis of the means of fumigation gas concentrations indicate that this effort was successful. However, analysis of individual plant parameters does indicate thatoccasional cases of significant

·effects of chamber nested within the treatments occur. These occurences can be attributed to other environmental differences between the individual chambers, small differences in fumigation gas concentrations between the chamber pairs in a given treatment that are compensated for over the course of the plant growth during the experiment, and small differences in the gas concentrations within a single fumigation event.

Because of the intention to use the chambers as duplicates, plant data from duplicate chambers were pooled for the descriptive statistics. The presented st

Where an ANOVA demonstrated a significantly low probability for a null hypothesis, this probability has been annotated in the data tables (Chapters 4-6) with asterisks in the following common convention:

* the probability of obtaining the data distribution without there existing an effect of the modifying treatments (in this study, fumigants) on the measured plant parameter

.Jies between one in twenty (P== 0.0500) and one in a hundred (P==O.OlOO),

** the same probability lies between one in a hundred (P==O.OlOO) and one in a thousand (P==O.OOlO), and

*** the same probability lies below one in a thousand (P==O.OOlO).

CHAPTER 3: THE SYMPTOMS OF FUMIGATION WITH HIGH LEVELS OF NITROGEN OXIDES ON NATIVE PLANTS.

3.1 INTRODUCTION

This experiment was carried out to characterise the symptoms of acute exposure to nitrogen oxides in the native vegetation species used in this . project. These observations will assist in monitoring and in responding to the concern of the public. The species chosen for the study take in a range of native plants occuring in all states of Australia. See section 2.1 for an outline of these.

Symptoms of nitrogen oxide exposure rarely occur in the field. The occurences reported in recent years tend to be in areas subject to intermittent very high levels (fertiliser production plants, ammunition plants: Krause, 1988). The appearance of acute nitrogen oxides symptoms iri. the field should be regarded as possible evidence of serious and abnormal releases of nitrogen oxides, perhaps of an accidental or systems failure nature. However, it is also likely that similar field symptoms are derived. from other sources, as has been: · discussed in section 1.4.1, or through interaction of such sources with nitrogen oxides. Chronic injury, as exploreq in later chapters (4-6), tends not to be manifest as symptoms, but rather as modified growth.

The opportunity was taken to examine the physiological responses of native · plants to a high level of nitrogen oxides. Physiological effects of chronic exposure to nitrogen oxides on several plant species have been describes earlier (Section 1.4.3). The visual symptoms do not indicate specific metabolic processes that have been damaged or overloaded, rather there is a nonspecific discolouration and death of leaf tissue. It is widely accepted that nitrogen oxides dissolve in the apoplastic solution of the cell wall (WHO, 1987), but itis not clear whether the nitrogen oxides that enter the cell (and may or may not be involved in metabolism), or the nitrogen oxides remaining in the cell wall are responsible for the damage manifest as visible symptoms. Jn fact; there is little information on the proportions of nitrogen oxides in these locations. Much of this may depend on the relative proportions of nitric oxide and nitrogen dioxide, since there is some debate about the relative solubility and, therefore, relative toxicity of these two nitrogen oxides.

As a result, it is appropriate to identify some more distinctive physiological symptoms that might occur in the field, to more positively identify nitrogen oxides damage. In this context, we examined the levels of ammonia and urea in leaf tissue immediately after exposure. These simple nitrogen metabolites were chosen because of their close metabolic association with nitrate and nitrite, the soluble anions from the nitrogen oxides, and the quick and convenient assay techniques available for them.

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3.2 ME1HODOLOGY

3.2.1 Plant Species

Five plant species were used in this experiment, these were: Eucalyptus calophylla (Marri) Eucalyptus globulus (Tasmanian Blue Gum) Eucalyptus marginata (Jarrah) Eucalyptus microcorys (Tallowwood) Banksia attenuata (Banksia)

Plants were obtained at a five to ten leaf stage in small nursery pots, growing in potting mixes, and used as such.

3.2.2 Fumigation

Fumigation was carried out in glass chambers of 80 L volume, stirred with small instrument fans. Plants were placed in the chambers, and nitric oxide was released into one chamber, the concentration measured, and the chamber sealed up for two hours. An identical chamber held a_ set of control plants of similar age, appearance, and in identical pots and soils. This was also sealed at the same time, enclosing ambient airfor two 1\ours. Nitrogen oxides in the ambient air were below 5 nL L-1 at this time, whereas the fumigated plants were in an atmosphere of 10 000 nL L-1 of nitrogen oxides, this being over 99% nitrogen dioxide. Air temperature in the chambers increased from 24°C to 27°C over the two hour period. The chambers were situated.outside, and conditions were overcast with occasional sunny spells.

3.2.3 Observations and Photographic Documentation

Plants were observed during fumigation and immediately after removal from the glass chambers. Photographic records were taken 24 to 48 hours after

. fumigation. Typical leaves of control and fumigated plants of each species were excised and photographed in pairs under a sheet of glass.

3.2.4 Biochemical Tests on Leaf Tissue

Immediately after removal from the fumigation chambers, individual plants were cut at soil level, and the shoot sealed in plastic bags and frozen at -25°C. For estimation of free ammonia and urea in these plants, the technique of Walker et al. (1985) was modified as follows. Samples were chopped upon thawing, subsampled and extracted with deionised water in centrifuge tubes for 30 minutes in a hot water bath at 80°C. The obtained solutions were assayed for ammonia by a direct microdiffusion in small vials as described by Burris (1972). A preliminary step using urease. to hydrolyse urea to ammonia was used to estimate the combined urea and ammonia (Walker et al. , 1985)

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t

and urea obtained by difference. Tissue concentrations were expressed on a fresh weight basis (Table 3.1)~ Insufficient material was obtained to allow total nitrogen measurements.

3.3 RESULTS

3.3.1 Observations and Photographs

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Immediately on removal of the plants from the chambers, it was observed that the Eucalyptus globulus plants had a somewhat more blue-green coloration. Leaves of some other fumigated plant species tended to have a red-brOwn tinge to their margins. The significance of these colour changes is not clear, but may be partly attributed to formation of ammonia within the tissue (Taylor et al., 1987). After 24 to 48 hours distinct portions of the leaves of fumigated plants had died. Leaf necrosis was most pronounced on E. marginata and least apparent on Banksia attenuata plants (see Fig. 3.1).

3.3.2 Biochemical Tests on Leaf Tissue

Free ammonia in the fresh leaf tissue was assayed almost immediately after removal from the fumigation chamber. The ammonia in fumigated plants was significantly higher than in control plants (Table 3.1 ). Increases in ammonia were observed in both the tips (terminal 1 em portion) and the

Table 3.1. Ammonia concentrations (jlmol g-1 fresh weight) in fumigated and control plants after exposure to 10 000 nL L -1 of nitrogen dioxide. See text for conditions of exposure,

Control Plants Fumigated Plants

Species· Leaf Tip Rest of Leaf Leaf Tip Rest of Leaf

E. · microcorys 0.70 0.40 1.73 1.70 E. marginata 0.46 0.21 2.27 1.53 E. glogulus 0.41 0.23 1.85 1.37

·E. maculata 0.53 0.31 1.14 0.54 B. attenuata 0.31 0.20 . 0.59 0.56

Average Increase 320% 438% over Control

remaining blade of the leaves. The tips of the leaves were somewhat higher in ammonia than the rest of the. leaf in both control and fumigated plants (Table 3.1). Urea concentrations were also estimated in the tissues, but concentrations were low in both treatments and have not been tabulated.

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3.4 DISCUSSION

The symptom development experiment found symptoms resembling those reported elsewhere with nitrogen oxides (Taylor et al., 1987, see also Section 1.4.1). Small necrotic patches developed on the foliage of the native species following exposure to nitrogen oxides (10 000 nL L-1) for two hours (see Fig. 3.1). Responses varied among the species, the Banksia attenuata being the least sensitive.

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Increases in tissue ammonia have not been commonly reported in response to nitrogen oxides fumigation. Rowland et al., (1988) reviews the area of amino acid metabolism in plants, describing the frequent positive responses to nitrogen oxides reported for total and individual amino acids. Ammonia may be rapidly formed on excising fresh plant tissue, largely through the enzymatic breakdown of free amino acids. Therefore researchers have largely overlooked ammonia levels in tissue of nitrogen oxides affected plants. However, with adequate controls, that is, equivalent sampling of control plants, tissue ammonia levels may be useful in understanding mechanisms of the effects of nitrogen dioxide on the physiology of plants.

The increases in ammonia reported in this experiment place a limit on the extent to which free amino acids might have varied quantitatively in vivo from that analysed. For example, in the experiment reported in Chaptei 5, the measured total free amino acid levels in E. marginata were maintained at around 10 J..lmol·g-1(dry weight) quite uniformly with increasing concentrations of nitrogen oxides (Table 5.8). In the current study, fumigation increased ammonia by around 1.5 J..lmol g-1 (fresh weight) (Table 3.1) or around 3.9 J..lmol g-1(dry weight) (FW:DW ratio= 2.6, Table 5.6). If formation of ammonia is similar at the lower nitrogen oxides fumigation concentrations, measurement of the tissue ammonia pool could be quite significant in quantifying effects. However, very rapid harvesting (in the manner carried out above) of large experiments such as that described in chapter 5 is not possible in practise. Since it is possible that the ammonia accumulation is a temporary phenomenon in plants during nitrogen oxides fumigations, more. elaborate sampling procedures may be necessary for mass balance studies.

It is important to note that the fumigant involved in this study was chiefly nitrogen dioxide. Although, some workers attribute a greater phytotoxicity to nitrogen dioxide (eg., Krause, 1988), recent studies have characterised metabolic roles for nitricoxide, and that nitric oxide is rapidly coordinated to iron . contained in many proteins (Lancaster, 1992). The affinity of nitric oxide for iron complexes was first described by Priestley last century (Mellor, 1962) .

. . There is now considerable speculation as to the consequences of this association in many areas of biology, as discussed earlier (See 1.2, 1.4).

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CHAPTER 4: THE EFFECTS OF NITROGEN OXIDES ON BLUEGUM, JARRAH, t ALLOWWOOO AND BLACKBUTT .

. 4.1 INTRODUCTION

This experiment wa's carried out to test the hypothesis that the growth of four species of eucalypts would be diminished by exposure to nitrogen oxides at concentrations likely to be experienced adjacent to fossil fuel powered electricity generating stations under various climatic conditions. The experiment was designed to determine the range of nitrogen oxide concentrations that these species could tolerate under field conditions.

4.2 MATERIALS AND METHODS

4.2.1 Plant cultivation

. /

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Three-month old nursery-grovT seedlings of four tree species were planted in the soil within the fumigation chambers. Ten seedlings of each species were randomly assigned to each of 16 chambers. The organic content of the sandy soil had· previously been augmented with a potting mix (2 sand, 3 sawdust, 3 woodfibre and 4 pinebark). At planting each seedling was fertilised with lOg ot

·. slo