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Potential mechanisms of acid mist injury to red spruce
EnvironmentalandExpennkmtalBotany,Vol. 35, No.
2, pp, 125-137, 1995 Elsevier Science Ltd Printed in Great Britain
Pergamon 0098-8472(95)00006-2
POTENTIAL MECHANISMS OF ACID MIST INJURY TO RED SPRUCE IAN D. LEITH, LUCYJ. SHEPPARD and MAUREEN B. MURRAY Institute of Terrestrial Ecology, Bush Estate, Penicuik, Midlothian EH26 0QB, U.K.
(Received 18 August 1994; acceptedin revisedform 28 November 1994) Leith I. D., Sheppard L.J. and Murray M. B. Potential mechanisms of acid mist injury to red spruce. Environmental and Experimental Botany 35, 125-137, 1995.--One-year-old red spruce seedlings (Picea rubens Sarg.) were exposed to six simulated mist treatments in open-top chambers, between 25 May and 8 November 1988. Mists were applied twice weekly to foliage and soil, with each application equivalent to 2 mm precipitation, applied over 30 min. The treatments contained pairwise combinations of H ÷, NH4÷, 8042- and NO3- ions at concentrations of 1.6 or 3.2 mol m -3, with one treatment including all four ions. Effects of individual ions and ion combinations on visible injury, growth and nutrition were determined. After four applications ofH2SO 4 at 3.2 mol m -3 (pH 2.5), current year needles turned red-brown. Subsequent application of HzSO4 at a reduced concentration of 0.5 mol m -3 (pH 3.0) resulted in no further increase in foliar injury symptoms. No foliar injury symptoms developed on seedlings receiving HNO3 (pH 2.5), NH4NO3 or (NH4)2SO4 treatments. Twenty-five per cent of needles were damaged on seedlings receiving H2SO4+ NH4NO3 (pH 2.5), but only after 24 applications of mist, suggesting that either NH4+ or NO3- ions ameliorated the effect ofH2SO 4. There was a stimulation in shoot and root biomass in those treatments containing N, with or without H2SO4. Contrast analysis indicated a positive growth response to acidity. Significant uptake of S and N occurred in response to treatment. This experiment indicates that it is the simultaneous presence of H + and SO42- in occult precipitation that has the greatest potential for inducing foliar injury. The presence of NO3- was found to ameliorate the toxic effects. Potential mechanisms underpinning these observations are discussed.
Key words : Red spruce, visible injury, foliar nutrients, growth, sulphate, acidity, nitrate.
INTRODUCTION
must have a lower p H to cause similar effects. (t6/ Both SO42- and H + ions were present in the simuThe decline of red spruce (Picea rubens Sarg.) has lated acid mist used by LeithJ 22)However, acid rain been investigated under controlled environments and cloud contain other ions, including pollutante.g. open-top chambers ~6/and more recently in the derived N O 3 - and NH4 + and the contribution of field. Various 'threshold' values for injury have been these N-containing ions on the development of reported but these are strongly influenced by seed- injury needs to be evaluated. ling age, duration and frequency of exposure and While symptoms of visible injury induced under the chemical composition of the applied experimental exposures have shown remarkable treatments. (I5'22'31~Visible injury to red spruce can consistency, conflicting results have been obtained be caused by sulphuric acid at p H 3.5, but it is from experiments designed to test the effects of known that mixtures of sulphuric and nitric acid simulated acid precipitation on the growth of red 125
126
I.D. LEITH et al.
spruce seedlings. No significant effects of simulated acid rain on growth and photosynthesis were reported by Laurence et aL, 12~1or by Taylor eta/., (45) who measured extension growth on seedlings after exposure to artificial acid mist at pH 3.6 and pH 5.1 containing both SO42- and NO3- ions. However, Percy(31) found significant growth reductions and morphological changes in red spruce seedlings treated with acid rain (containing sulphate and nitrate, ratio 2 : 1) at pH 2.6. Jacobson et al. ~16~also found reductions in leader extension in red spruce exposed to H2SO~ mist at pH 2.6. Wood and Bormann I48~ showed increases in the growth of P/nus strobus seedtings exposed to simulated acid mist applied as nitric acid at pH 2.3 for 20 weeks. In an experiment preceding that described in this paper, exposure of red spruce seedlings, grown in open-top chambers (OTCs) to simulated acid mist, induced direct foliar injury/discoloration. Development of foliar injury was found to be strongly dependent on the ionic concentration of the simulated acid mist. A linear increase in foliar injury to red spruce seedlings was observed as the ion concentrations in the mist increased. As the four major ions were in constant ratio to each other, the contribution of individual ions could not be identified. In the present experiment, the specific influence of these individual ions on red spruce was tested using paired combinations of the four major ions (NO3-, SO42-, NH4 +, H ÷) in six acid mist treatments. This paper reports foliar injury, growth, biomass production and nutrient status resulting from the application of these mist treatments over a period of 23 weeks between May and November 1988. Effects on frost hardiness from this experiment were reported by Cape et al. 131 Earlier experiments/16/have shown that the presence of N increased the acidity strength at which visible injury was observed, i.e. reduced plant sensitivity to the H + ion concentration. The significance of the anion-proton interaction has not, however, been specifically addressed. This paper is based on five treatments plus a control (Table 1) which were designed to explore the relative contributions of S, N and acidity on both the induction of visible injury and growth response. The following hypotheses were tested, and the significance of these observations for mechanisms by which the perturbing ions perturb are discussed. 1. The combination of H + and SO42-, not H +
2.
3.
4.
5.
and NOa-, ions causes visible injury (compare response to H 2 S O 4 and HNO3 with control). Neither SO42- nor NO3- in combination with NH4 + ions induce visible injury in the absence of acidity [compare responses of(NH4)2 SO4 and NH4NO3 treatments with control]. The form of added N (NH4+ or NO3-) determines the nature of effect on red spruce [compare responses to (NH4)2SO 4 and HNO3 treatments with control]. The presence o f S O 4 2- ions perturbs red spruce in the absence of acidity [compare response to NH4NO3 and (NH4)2SO4 with control]. The response to all four ions in combination produces similar effects to those observed in the 1987 experiment./22) METHODS
A d d mist treatments
The design and physical properties of the octagonal glass-sided open-top chambers (floor area 7.0 1/12, height 2.3 m including frustrum), were described in detail by Fowler et aL 191All chambers received filtered air, with 03, SO2 and NO2 removed by activated charcoal filter units. Ambient concentrations of these gases over the season averaged: SO2 2.1 nl 1-l, NO 1.1 nl 1-1, NO2 4.7 nl 1-l and 03 26.7 nl 1- i. The flow rate of air was supplied via ducted fan units to the chambers at a flow rate of 40 m 3 min-l (two complete air changes min-1). The seedlings, in 4 1 pots, were irrigated daily via capillary matting (holding capacity 7 1 m-2). Rainfall was excluded from the chambers by U.V. stabilised polyethylene lids fitted within the frustrum. The treatment design used paired combinations of the four major ions and one treatment containing all four ions (Table 1). Each treatment was randomly assigned to two chambers. (231Mist treatments were made from concentrated stock solutions. Prior to application, each solution was checked for electrical conductivity, acidity and presence or absence of sulphate using CaC12 solution. Mist treatments were applied using a pressurized system./9~ Each chamber received the equivalent of 2 mm precipitation (14 1) at a rate of 3 mm hr -~ twice weekly over a 23-week period. 122~Air turbulence within the chamber ensured an even distribution of mist throughout the chamber. Mist was applied between 25 May and 8 November 1988.
POTENTIAL MECHANISMS OF ACID MIST INJURY
127
Table 1. Ion concentrationsand deposition ratesfor the mist treatments. The concentrationof H2SO4 was modredfrom (a) afterfour applications of mist to (b) (see text) Ion concentrations mol m Solutions H20 NH4NO3 (NH4)2SO4 H2SO4 (a) (b) HNO3 H~SO4 + NH~IO3
NO3 8042 NH4+ H ÷ 0 1.6
0 1.6
3.2 1.6
1.6 0.5 1.6
Plant material Experimental material was propagated at Bush Estate, (55°51'N, 3°12'W, 185 m altitude) from seed collected from mature stands of red spruce at Hardy Brook, New Hampshire, U.S.A. (43°56'N, 71°32'W, altitude 490 m). Stratified seed was sown on 14 March 1987 into a sphagnum peat based compost containing 200 g Vitax Q4 compound fertiliser (N 5.3%, P 7.5%, K 10%) per 100 1 of compost. Seedlings were transferred to 0.1 1 pots containing the same compost medium but without additional fertiliser in late June 1987. Seedlings were maintained in a filtered glasshouse without supplementary lighting but protected from frost throughout the winter. Prior to final repotting, foliar N concentrations were increased by approximately 0.5% by four applications of Ingestad B and C,/j4/ equivalent to 15 mg N per pot. In April 1988, 1year old seedlings of uniform height were selected and repotted into 4 1 pots containing a 60% peat: 40% quartz grit compost by volume with no additional fertiliser, and transferred to the O T C s at Bush Estate. Plant growth measurements A destructive harvest of 10 seedlings of similar size to those selected for the main O T C experiment was undertaken as plants were repotted in April 1988. Height and root collar diameter were measured. The seedlings were then separated into shoot and root systems. Individual root systems were washed, partitioned into fine ( < 1 m m diam-
-3
Deposition kg ha-x application- a
pH
S
N
H
0 1.6 3.2
0 -
5.6 5.6 5.6
0 1
0 0.9 0.9
0 -
2.5 3.0 2.5 2.5
2.0 0.3
1.6
3.2 1.0 3.2 3.2
0.9 0.9
0.06 0.04 0.06 0.06
1
eter) and coarse ( > 1 m m diameter) roots, dried at 80°C for 4 days then weighed. The above-ground parts were also dried at 80°C for 4 days and needle and stem dry weight recorded.
Height growth measurements Immediately prior to transfer to the O T C s (April 1988) total plant height and root collar diameter were recorded for 10 randomly selected seedlings per chamber. Thereafter, weekly measurements of shoot leader extension were recorded. Root collar diameter was remeasured at the final harvest (5 December 1988). Comparison of height growth cessation The date of leader extension cessation was determined as the first assessment date (described by its Julian day where 1 August = 213) on which individual seedlings failed to produce an increase in height growth. The date of height cessation was found for each treatment chamber by calculating the chamber mean from 10 plants. Biomass assessment The 10 randomly selected seedlings in each of the 12 chambers which had been used for height growth measurements were destructively harvested between 5 and 7 December 1988. The procedure was similar to that described for pre-treatment harvest; the root collar diameter was measured, then the shoot system was severed from the root system at the root collar. Components of the shoots were
128
I.D. LEITH et al.
divided into year classes, dried at 80°C for 4 days, then weighed. Root systems were washed out carefully under a fine jet of water over a coarse sieve. Two to three first order lateral roots (approximately 15% of the total biomass) were removed from the thickened root collar and divided into coarse and fine size classes. The remaining laterals were removed from the thickened root collar. All the roots were ovendried for 4 days then weighed. The ratio of fine to coarse roots in the subsample was used to estimate the ratio of fine to coarse roots in the bulk sample. The weight of the thickened root collar was determined separately.
Chemical analysis Samples of rinsed dried, current year needles from each of 10 plants per chamber were bulked by chamber. One well-mixed subsample was ground ( < 0.8 mm) for chemical analysis. An aliquot of this ground material was redried at 105°C for 3 hr, 350 mg was digested in a modified Kjehldal digest./3°/ N, P, K, Ca and Mg were determined as described by Allen et aL I~l Sulphur was determined on the ground sample by X-ray fluorescence spectrometryJ 29/The results are expressed on a percentage dry weight basis. Accuracy was checked against internal reference material and external certiffed standards. Assessment of visible foliar injury Seedlings in all 12 chambers were assessed for foliar injury prior to initial mist application and on nine occasions throughout the exposure period, May-November 1988. Foliar injury was determined using a system of 12 needle damage classes. (22/ Only current year needles on marked individual seedlings were visually scored for incidence of necrotic and/or chlorotic damage by the same operator. Visible foliar injury was expressed on a percentage damage basis. Estimates of percentage damage within each chamber and between treatments were analysed for treatment differences using A N O V A , (37)following angular transformation of the data. Experimental design-analysis The six treatments were randomly assigned to the 12 chambers to provide a complete randomized design. The A N O V A (37)was conducted on chamber
means, i.e. two per treatment. Differences between treatment means were tested using Tukey's studentized range test. Contrasts were also constructed in the analysis of variance to test the effects of individual ions. These contrasts are not independent because most treatment ions were applied in combinations of ions and the concentration of SO42- had to be changed (Table 1). RESULTS
Visiblefoliar injury Applications of simulated acid mist treatments began on 25 May 1988. Injury symptoms developed after four applications (2 weeks), but only on the seedlings treated with p H 2.5 H2SO4. Injury symptoms resembled those described by Leith et al. (221 with an initial red-brown discoloration of the current year needles leading to complete necrosis and needle loss within 4 weeks. Red-brown discoloration was principally found along the whole length of the needles and on both the adaxial and abaxial surfaces. Observations of only partially necrotic needles showed browning beginning at the distal end of the needles progressing towards the proximal end and resulting in complete needle necrosis. The numbers of needles showing light brown chlorosis were small compared with those showing red-brown discoloration. This chlorosis affected the whole needle. The extent of the initial foliar necrosis in the H2SO4 treatment on 13 June 1988 was about 7% per plant, with approximately 70% of individuals affected in both replicate chambers. The laterals on the upper whorls exhibited more damage than the less exposed lower whorls. No visible injury was observed in the other treatments on this date (Fig. 1). After the initial observation of foliar necrosis, misting was withheld from all treatments between 10June and 1July. Continued application of H2SO4 at a concentration of 1.6 mol m -~ would have resulted in severe needle necrosis and probable seedling death. The concentration of H + and SO42- ions in the H2SO treatment was therefore reduced by a factor of 3 to 0.5 tool m -3 (Table 1). On 28June 1988, damaged seedlings were removed from the H2SO4 treatment and replaced with untreated seedlings of a similar age and size which had been kept in a glasshouse in filtered air. Twenty of the original seedlings which had been exposed to the p H 2.5
POTENTIAL MECHANISMS OF ACID MIST INJURY 0
i
80
of the injury increased steadily over the following 11 weeks so that by the end of November, 90% of seedlings showed foliar injury on 25% of their current year needles. Treatment with HNO3 (pH 2.5) or de-ionised water caused virtually no visible damage (< 1%) over the experimental period. The control plants, which received de-ionised water, appeared pale green in colour by comparison with other treatments, especially the treatment with HNO3, but did not exhibit visible foliar injury. By the beginning of November, the NHaNO3 (pH 5.6) and (NH4)2SO4 (pH 5.6) treated seedlings showed very little damage (< 2%) on 10% of the plants. Six per cent of plants treated with HNO3 pH 2.5 were damaged. Visible injury to plants receiving NH4NO3 was in the form of light brown~reen needle discoloration which was undetectable from a distance. Plants in the (NH4)2SO4 treatment showed needle yellowing, especially at the needle tips. No significant differences in the degree of damage or number of damaged plants were observed between the HNO3, NHqNO3, H2SO4 (pH 3), (NH4)2 SO4 and H20 treatments.
111804+ NH,NO,
IMNO
IO0 .
o
June
July
2
IIHO
"
40
0
Aug
Sept Oct
Nov
1988 25 2O
"~ 15
/ 0
Heightgrowth cessation-budset
' ~ , w
June
129
w
July
Aug
.
w
Sept
'
Oct
i
i
Nov
1988
Fig. 1. (a) Development of visible injury; percentages of seedlingsinjured by the different acid mist treatments. (b) Development of needle injury; percentage of damaged needles per plant.
Treatment effects were marginally significant (P = 0.06). The H 2 S O 4 t r e a t m e n t was the last to set bud, while the NH4NO3 and water treatments set bud first (Fig. 2). Contrast analysis revealed significant (P = 0.01"*) effects of S and acidity. The presence o f S O 4 2- appeared to delay budset-height growth cessation. B/0mass
retained in the chambers; of these, 10 were used in the biomass assessment. Misting resumed on 1July with the modified HzSO4 treatment and continued until 8 November. There was no further damage to the original plants (pH 2.5, pH 3.0) and almost no visible injury on the replacement seedlings, which only received pH 3.0 mist. Foliar injury symptoms were observed in the H2SO4+ NH4NO3 (pH 2.5) treatment from the beginning of August 1988 following 18 mist applications. By 19 August, 20% of the seedlings were showing foliar injury with approximately 5% of needles damaged per plant (Fig. 1a,b). The extent H2SO 4 were
Treatment effects on the sum of needle and stem (shoot) weight produced in the growing season, encompassing the experimental period were significant (P < 0.05) (Fig. 3). New growth increased in response to increasing acidity. Neither the presence of N or S significantly (P > 0.05) affected shoot dry weights. Needle weights were greatest in the NHaNO~, NH4NO3, + H 2 S O 4 and HNO3 treatments. Root weights (data not shown) were not significantly affected by individual treatments but were significantly (P < 0.05) higher when NO3- was included in the mist. Root/shoot ratios (data not shown) ranged from 0.9 (H2SO4) to 1.1 (NH4NO3) and were likewise not significantly affected by treatment. Separation of the roots into fine, coarse and
130
I.D. LEITH et aL 225 220 215
~'
T
_.]___
210
"O §
2O5
190
ab
a
ab
H=SO4+ (HNOa)= H2S04 NH4NOs (NH4)=SO, H20 NH4NOa
Fig. 2. Effect of different acid mist treatments on height growth cessation. Values with the same letter are not significantly different [(P> 0.05) Tukey's studentized range test].
tap root revealed differential treatment effects but no significant differences. Weights of fine roots ranged from 3.2 g (H2SO4) to 4.6 g (NH4NOs+ H2SO4) and coarse roots from 1.6 g [I-I20 , (NH4)2SO4] to 3.3 g (HNOs). The structural root component, coarse roots increased in response to NOs-N and root weights were highest in those treatments containing NO3-N. Significant treatment effects (P < 0.05) on the fine to coarse root ratio were observed (Fig. 4). Ratios were highest in the H 2 0 and (NH4)2SO4 treatments, reflecting the low coarse root weights. Root collar diameter was not significantly affected by treatment mainly because of the huge difference (67%) between the two chambers in the (NH4)2SO4 treatment. The largest root collar diameters were from seedlings receiving all four ions and the smallest from those receiving water only. Contrast analysis was undertaken to determine the influence of specific ions (PROC GLM), (sT/ although ion effects were not mutually independent in this experiment. The inclusion of acidity was found to significantly (P < 0.001) influence all the growth parameters highlighted in Table 2, [new needles and stem, the tap and coarse root, total root weight, leader extension, root/shoot ratio, and root
3.5
b 3.0 o 2.5 4
|
®
~3
¢2
2.0
ab
1.5 o. O i-
~
1.o 0.5
0
H-SO.+ N~, N'Os
HNOa H=SO4 NH4NO3 (NH4)=SO4 H=O
Fig. 3. Effect of acid mist treatment on total weight of new stem and needles. Value for total weight with the same letter are not significantly different [(P > 0.05) Tukey's studentized range test]. Note the larger proportion of stem to needles in the treatments lacking additional N.
o.o H=SO4* HNOa H2SO4 NH, NO3 (NH4)2SO4 H=O NH4NOa
Fig. 4. Effect of acid mist treatment on the fine root to coarse root ratio. Columns with the same letter are not significantly different [(P> 0.05) Tukey's studentized range test].
POTENTIAL MECHANISMS OF ACID MIST INJURY
131
Table 2. Results of a contrast analysisfor differences between treatments with and without individual ions for growth parameters. These ions are not mutually independent in this experiment. Valuesshow theprobabilitv ofa nulleffect (P < 0.001+++, P < 0.01++, P < 0.05+). +/- Indicate direction of effect, increase or decrease
New needles (Ne) New stem (St) New total (St+Ne) Tap root (g) Coarse root (g) Fine root (g) Root wt (g) R/S ratio Root collar diameter Leader extension Date height cessation
H+
NH4+
+++ +++ +++ ++ +++ n.s. n.s. +++ +++ +++ ++
collar diameter (P < 0.05)]. Root collar diameter was greater in the presence of acidity. The presence of the NH4 + ion was not significant (P> 0.05) for any of the above-mentioned parameters, but increased root collar diameter. The inclusion of NO3- significantly affected biomass production and leader extension (P < 0.01) with the exception of the fine roots. The presence of NO3- had no significant affect on the date of height growth cessation, but the presence ofNH4 + shortened the growing season. The inclusion of $042- had no significant effect on any of the growth (weight, diameter) parameters, only influencing the time the plants were growing and like H ÷, inclusion delayed budset. The overall effect of N was attributable to NO3- induced effects rather than the presence of the NH4 + ion. Foliar nutrient status No significant treatment effects were observed for foliar N, P, K, Ca or Mg concentrations (Tables 3 and 4). Treatments lacking additional N, i.e. H2804 and H 2 0 had the lowest N concentration. The occasional large differences between replicate chambers contributed to the absence of statistically significant effects. The base cations K, Mg and Ca showed least variability with treatment. Although some treatments contained N, foliar N concentrations were not significantly (P > 0.05) influenced by treatment. By contrast, foliar S concentrations were treatment-dependent. Those treatments which contained S had significantly
NO3-
SO42-
n.s.
+++
n.s.
+++
n.s. n.s.
+++ +++
n.s. n.s.
+++ +++
n.s.
+++
n.s.
+++
n.s.
+++
n.s.
+++
n.s.
n.s.
n.s.
+++
n.s.
+++
n.s.
+++
n.s.
+++
n.s.
+++
+
+
n.s.
++
n.s.
+++
n.s.
++
++
n.s.
n.s.
N
(P < 0.05) more S than those which lacked S. The H2SO 4 treatment, where the S concentration was modified from 1.6 to 0.5 mol m -g, had intermediate S concentrations but these were also significantly higher than those from the control, H 2 0 plants. Contrast analysis (Table 5) showed that foliar N concentrations were significantly (P = 0.04) influenced by the presence of the NH4 + ion. Despite the absence of K from the acid mist treatments, fofiar K concentrations were significantly increased by the presence of N, acidity and SO49-. DISCUSSION
Within the first 3 weeks of acid mist exposure, the first hypothesis was tested; the absence of injury in the HNO3 treatment supported the view that a combination of H + and SO42- ions are required to induce visible injury. The absence of immediate visible injury followed by its much slower and reduced development in the NH4 NO3+ H2 SO4 treatment further showed that the presence of N mitigates harmful effects of S. Likewise, the low level of damage in the (NH4)2 SO4 treatment provided further confirmatory evidence that SO42- and H + are necessary for the induction of significant amounts of needle necrosis (hypothesis 2). Injury was greatest when 8042- was present but in the absence of acidity damage was negligible. As observed in Leith's experiment, (22) significant amounts of injury were observed in the presence of
132
I.D. LEITH et al. Table 3. Analysis of variance offoliar S concentrations9%m red spruce shoots. The main factor is poUutedmist treatmentwith two replicatechambermeans (bulkedsamples) with single degreeoffieedom contrast analysis Source
DF
Sum of squares
Treatment Treatment x chamber Total
5 6 11
0.092 0.003 0.095
Source
Tests of hypothesis Error Treatment x Chamber
Contrast Effect of H Effect of NH4 Effect of NO3 Effect of SO4 Effect of N
DF 1 1 1 1 1
SS 0.007 0.071 0.003 0.090 0.156
F Value 32.49
P> F 0.0003
FValue 13.2 124.8 4.9 158.3 27.4
P> F 0.0109 0.0001 0.0679 0.0001 0.0020
Table 4. Foliar N, S, P, K, Mg and Ca concentrations (% d~y weight) representinga bulked samplej~om 10 plants for individual replicatedchambers (1 and 2). The treatment mean is shownfor S, where valuesfollowed by the same letterare not signoqcantly different (P > 0.05) using Tukey's studentized range test. Treatment effects were not signoqcantfor N, P, K, Mg and Ca N H20 NH4NO3 (NH4)2SO4 (pH 2.5 and pH 3.0)H2SO4 HNO~ H2SO4 + NH4NO~
Rep 1 Rep 2 1 2 1 2 1 2 1 2 1 2
0.96 1.40 1.60 1.70 1.4 1.5 1.1 1.2 1.2 1.3 1.4 1.4
all four ions and the level of injury escalated with the onset of cold hardening which appears to be triggered by daylength in red spruce (Sheppard, L.J. unpublished results.) This experiment was designed to distinguish between individual phytotoxic effects of the four major ions present in cloudwater. Combining SO42-, NH4 +, N O 3 - and H + at the p H extremes
S 0.15 0.18 0.19 0.20 0.34 0.39 0.27 0.25 0.21 0.19 0.42 0.37
0.17c 0.19bc 0.36a 0.26b 0.20bc 0.40a
P
K
Mg
Ca
0.18 0.24 0.22 0.25 0.27 0.28 0.19 0.21 0.24 0.21 0.26 0.18
0.91 1.00 0.83 0.99 1.1 1.1 0.98 1.0 1.1 1.1 1.1 1.0
.014 0.13 0.15 0.14 0.15 0.18 0.11 0.14 0.15 0.13 0.14 0.12
.058 0.64 0.74 0.72 0.69 0.72 0.63 0.65 0.73 0.63 0.73 0.56
of natural precipitation, wet deposition (27)provided a unique opportunity to identify some important synergistic effects. In the ensuing discussion we briefly relate our findings with respect to the corroboration or otherwise of previous studies with red spruce (6~ and then concentrate on potential mechanisms by which the specific ions may be influencing plant growth and nutrition.
POTENTIAL MECHANISMS OF ACID MIST INJURY
133
Table 5. Results of a contrast, F-test, for difference between treatments with and without individual ionsfor nutrient concentrations. Ions are not mutually independent in this experiment. Values show the probability of a null effect and the direction of the effect (+ = increase, - =
decrease,P < 0.001+++, P < 0.01++, P < 0.05+)
E~ctof H NH~ NO~ SO4 N
N
P
K
Ca
Mg
S
n.s. + n.s. n.s. +
n.s. n.s. n.s. n.s. n.s.
+ n.s. n.s. + -
n.s. n.s. n.s. + n.s.
n.s. n.s. n.s. n.s. n.s.
++ ++ n.s. +++ ++
Wib injucv Exposure of red spruce seedlings to H2SO 4 approximately 4 weeks after bud burst produced almost immediate necrosis in current year foliage. This observation is consistent with those of Jacobson et al. 116~who observed maximum injury when concentrations of both H + and SO42- were high. Inclusion of NHaNO3 with H2SO4 significantly delayed the rate of injury development but did not eradicate it. After 6 months 25% of foliage on all plants had either turned red-brown or abscised corroborating observations by Leith./22/ Reducing the level of acidity, H + ion concentration, in the H 2 S O 4 t r e a t m e n t by a factor of 3 resulted in almost no damage and it was concluded that red spruce was tolerant of acidity above p H 3. The observation that reducing acidity/ion concentration can arrest visible injury, was also seen for Sitka spruce,/43/indicating the importance of ion concentration/acidity in the phytotoxic response. The reduction in SO42concentration in the mist was reflected in lower foliar S concentrations. The characteristic necrotic red-brown coloration induced in current year needles during the growing season resembled that described by Leith et alJ 221 and has been reported for a number of conifer species exposed to simulated acid mist in controlled conditions. (2'26'46)Such necrosis of needles in the field has not been widely reported although reddening caused by freezing injury brought about through rapid warming and solar radiation has been observed in spring. Ill/ Yellow flecking and red-brown discoloration appear to be more common throughout European and North American forests./~8~ Advanced stages of nutrient deficiency, induced by anthropogenic pollutants causing nutri-
ent imbalances, e.g. N : K, (2°)however, can also take the form of red-brown needle discoloration. The lowered base cation concentrations in the H~SO4 treatments show how acid inputs can exacerbate nutrient imbalances with respect to N. Attacks by insects such as Ips ~pographus (spruce bark beetle) and fungal pathogens such as Lophodermium picea (spruce needle cast fungus) also cause red discoloration of needles and needle loss. Because of the lack of specificity of symptoms of needle injury, their diagnostic value in the field is limited. However, the induction of needle injury under controlled experiments is important because it provides conclusive evidence of pollutant-induced perturbations in the plant. The (NHfl~O3+H2SO4) pH 2.5 treatment provided the same ions as used in the previous dose response experiment./9~ However, in that experiment the mixture was composed of an equimolar solution of (NH4)2 SO4+ HNO3 (pH 2.5) providing 3.0 mol m -3 H +, 6.0 m o l m -3 NH4 +, 3.0 mol m -3 NO3- and 3.0 mol m 3 SO42-. By contrast, in the present experiment there were 3.2 mol m -3 H + and 1.6 mol m -3 each of NH4 +, NO3- and SO42- . Thus in the present experiment the ratio of H + to SO42is twice that of the previous experiment and the ratio of N : S is 2 : 1 compared with 3 : 1. In both experiments, the induction of injury took about 910 weeks, but the S dose required to induce injury in this experiment was < 50% of that received by the 2-year-old plants in the previous dose experiment. Results of a study byJacobson et al. 115'~suggest that this increased sensitivity may have been caused by the lower N:S ratio in this experiment. It is also possible that the increased H+:NO3 - ratio in the present study (2 : 1 compared with 1 : 1 in the dose
134
I.D. LEITH et al.
response study) may have played a part, since the assimilation of NO3- consumes protons/33/ (see later). This direct comparison between the two experiments provides corroborating evidence of the ameliorating effect of NO3- on SO42-/H + induced phytotoxicity. Growth and foliar nutrients
In the absence of additional fertilizer N the positive response to N seen here reflects that observed in the field on N limited sites. (2°/The presence of NO3-N and H + ions generally stimulated growth, while the presence of NH4 + or SO4- had no effect. The combination of H2SO4+ NH4NO3 increased both stem leader and new needle growth significantly, whereas treatment with (NH4)2SO4 had no effect on growth. Treatment with HNO3, NHdNO3 and H2SO 4 also enhanced growth by comparison with the H20 treatment. The proliferation in fine root growth in response to N and coarse root branching in response to H + is consistent with the findings of Deans et al. (sl The positive effect of acidity on growth contrasts the results of Percy/3~/ but is attributed to enhanced N availability(42)which may have overridden any negative effects of acidity. Tamm and Wiklander (44/ concluded that H2SO 4 treatment of soil enhanced N mineralization, particularly in the absence of additional N. Our growth data corroborates Jacobson's study/15~ that the presence of SO42- ions does not influence growth per se but that growth is stimulated by additions of NO3-N and acidity. The N-stimulated increase in dry matter production is all the more impressive since those treatments containing NH4NO~ set bud, and stopped growing first. By contrast, the presence of SO42- extended the growing season. This contrasts with the effects of SO42found in the spring, when treatment with SO42was found to delay bud burst./41/These results contradict the view put forward by Nihlgard (2a/ and Friedland et al. (~°1 that additional N extends the growing season, making plants more frost sensitive. However, it may provide an explanation for the increased frost sensitivity associated with SO42-,/3~ since the onset of winter hardening in north-temperate conifers is strongly coupled to growth cessation.(2a/ These data suggest that where N availability is restricting growth the benefits of additional N can exceed the negative effects associated with S inputs.
Implications for underpinning mechanisms
The aforementioned observations highlight some outstanding questions regarding the respective phytotoxic roles of 8042-, H +, NO 3- and NH4 + ions in acid mist. How do the presence of (a) H ÷ and (b) NH4NO3 modify the phytotoxicity of SO42-? L'Hirondelle et a/. (24) observed that H2SO 4 induced foliar injury was reduced when foliar N status was high. In this study foliar N status was almost identical and more than adequate (1.4% N) in the (NH4)2SO4 and H2SO4+ NH4NO3 treatments yet levels of injury differed significantly between these treatments. Foliar N concentrations from both the control and H2SO 4 treatments were low < 1.1% but visible injury was only observed in the H2SO4 treatment. Our results do not uphold the view that high foliar N concentrations per se protect foliage from acid (H+)/SO42- induced damage. However the inclusion of NHaNO3 with H2SO4 significantly enhanced dry matter production, despite the damage to foliage (needle necrosis), and despite not lowering S concentrations. We suggest therefore that the ameliorating influence of N on SO42- toxicity(6'4°)is founded on increased growth which provides for utilization of the absorbed S and thus S detoxification. When N availability is high and foliar N status is sufficient to promote growth, red spruce can compensate for needle necrosis through enhanced photosynthetic capacity, C assimilation. (8/ Thus the large amounts of potentially toxic S are detoxified either through glutathione production via assimilatory SO42- reduction (4~ or via incorporation into amino acids./4'a4'39~ Foliar S concentrations (% S) in themselves do not need to be low as long as the form or location of S is not toxic. Treatment with pH 2.5 HNO3 or pH 5.6 (NH4)2SO4 caused no significant visible injury to red spruce confirming hypothesis 1 that the combination of acidity and sulphuric acid are necessary to induce visible injury in red spruce. The likelihood of N causing phytotoxic effects is much smaller than that for S because plant demand for N exceeds that for S by a factor of 16J391Jacobson et al. 115~ also observed injury with sulphuric acid mist but concluded that acidity was the main predisposing agent and that 8042- exacerbated while NO3- ameliorated the damage. Eamus and Murray/7/ were unable to detect deleterious effects of high proton concentrations on A~,~ in Norway spruce seedlings which received the same treatments as here, and
POTENTIAL MECHANISMS OF ACID MIST INJURY they concluded that 8042- toxicity with respect to visible injury and Amax was (concentration x pH) dependent. With respect to 8042- phytotoxicity, Eamus and Murray (7) suggested that the impact of 8042- upon assimilation may relate to SO4 ~induced modification of the ionic balance of the chloroplast. Thylakoid architecture and function is extremely sensitive to the ionic balance of the stroma. They/7/observed 'locking open' of the stomata which could have been due to a reduction in chloroplast generated ATP or competition between SO42- and C1- ions in the guard cells which would influence the movement o f K + ions and hence guard cell opening (Cleland, R. pers. communication). The absence of visible injury in the presence of HNOs contradicts the opinion/15~ that acidity is the main cause of visible injury. The reductive assimilation of both NO3- and SO42-, consume protons (32/ although the former is much more important. (33~The neutralizing effect of NO3- (0.78 mol H + per mol NO3-), together with the plants high demand for N for growth and the growth process may explain the absence of injury by HNO3. The synergistic effects of H ÷ and SO42- with respect to their phytotoxicity may be due to the mutualistic way in which two ions are absorbed via the foliage. It is widely believed that the cuticle presents a relatively impermeable barrier to SO4 zuptake./35/However, acid rain containing H ÷ and SO42- ions causes significant erosion and fusion of the epicuticular wax./2s'36/ Needle wettability is increased and with it ion retention. Repeated treatment of isolated cuticles with either H 2 8 0 4 or HNO3 increases SO42- but not NO3- uptake./~2/ Jeffree et al. 1~7)have observed significant movement of SO42- into leaves pretreated with acidified SO42- solutions. There is now increasing evidence that as ion concentrations build up on leaf surfaces through drying, significant amounts of SO42- and H + can be mutually absorbed in a way that doesn't happen for NO3-. (12'~3'E8'19'38/The combination of 8042- and H + ions is phytotoxic because when leaves are simultaneously exposed to both ions their mutual uptake is significantly enhanced. These results further substantiate the view that 8042- is phytotoxic in high concentrations particularly when acidity is also high implying a synergism between the two ions. Nitrate ions ameliorate the effects probably through enhanced
135
assimilation and growth dilution. Observations of direct acid/SO42- induced injury to red spruce are rare in the field suggesting phytotoxic concentrations are rarely experienced for long enough to cause visible injury in the field. However, nonlethal SO42- concentrations, which can perturb plant physiology and result in reductions in frost hardiness, leading to increased injury from frost, have been seen in the field./47) Acknowledgements~The analytical staffat ITE Merlewood are thanked for undertaking the chemical analysis and Neil Cape for providing invaluable comment on earlier drafts. This research was supported by funds provided by the Northeastern Forest Experiment Station's Spruce-fir Research Co-operative within the joint U.S. Environmental Protection Agency-USDA Forest Service Forest Response programme. The Forest Response programme is part of the National Acid Precipitation Assessment Programme. This paper has not been subject to EPA or Forest Service peer review and should not be construed to represent the policies of either Agency.
REFERENCES
1. Allen S. E., Grimshaw H. M., Parkinson J. A. and Quarmby, C. (1974) Pages 81-159 in S. E. Allan, ed. Chemical ana~sis of ecological materials. Blackwell Scientific Publications, Oxford. 2. Brown K. A. (1989) Effects of ozone and acid mist and soil factors on Norway spruce in relation to forest damage. Research Report, National Power, Leatherhead, Surrey, U.K. 3. CapeJ. N., Leith I. D., Fowler D., Murray M. B., Sheppard L. J., Eamus D. and Wilson R. (1991) Sulphate and ammonium in mist decrease frost hardiness of red spruce. New Phytol. 118, 119-126. 4. Chen Y. and Wellburn A. R. (1989) Enhanced ethylene emissions from red and Norway spruce exposed to acid mists. Pl. Physiol. 91,357-361. 5. DeansJ. D., Leith I. D., Sheppard L.J., CapeJ. N., Fowler D., Murray M. B. and Mason P. (1990) The influence of acid mist on growth, dry matter partitioning, nutrient concentrations and mycorrhizal fruiting-bodies in red spruce seedlings. New Phytol. 115, 459 164. 6. Eagar C. and Adams M. B. (eds) (1992) Theecologyand decline of red spruce in the Eastern United States. SpringerVerlag, New York. 7. Eamus D. and Murray M. B. (1993) The impact of constituent ions of acid mist on assimilation and
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stomatal conductance of Norway spruce prior and post mid-winter freezing. Env. PoUut. 79, 135-142. 8. Eamus D. and Fowler D. (1990) Photosynthetic and stomatal conductance responses to acid mist of red spruce seedlings. Pl. CeUEnvir. 13, 349-357. 9. Fowler D., Cape J. N., Deans J. D., Leith I. D., Murray M. B., Smith R. I., Sheppard L. J. and Unsworth M. H. (1989) Effects of acid mist on the frost hardiness of red spruce seedlings. New Phytol. 113~ 321-335. 10. Friedland A. J., Hawley G.J. and Gregory R. A. (1985) Investigations of nitrogen as possible contributor to Red spruce (Picea rubens Sarg.) decline. Proc. Air Pollutants Effects on ForestEcosystems. Co-ordinated by The Acid Rain Foundation, St PaulMinnesota, pp. 95-106. 11. Hadley J. L., Friedland A. J., Herrick G. T. and Amundson R. G. (1991) Winter desiccation and solar radiation in relation to red spruce decline in the northern Appalachians. Can. J. For. Res. 21, 269272. 12. Hauser H. D., Waiters K. D. and Berg V. S. (1993) Patterns of effective permeability of leaf cuticles to acids. Pl. PhysioL 101,251-257. 13. Henderson C. G. McK. (1990) The nature of high altitude precipitation and its effect on Scots pine (Pinus sylvestris L.) and Sitka spruce [Picea sitchensis (Bong.) (Carr.)]. Ph.D. Thesis, University of Edinburgh, 346 pp. 14. Ingestad I. (1979) Mineral nutrient requirements of Pinus sylvestris and tS"ceaabies seedlings. Physiol. Pl. 45, 373-380. 15. JacobsonJ. S., Bethard T., Heller L. I. and Lassoie J. P. (1990) Response of Picea rubensseedlings to intermittent mist varying in acidity, and in concentrations of sulphur, and nitrogen-containing pollutants. Physiol. Pl. 78~ 595-560. 16. JacobsonJ. s., Heller L. I., Yamada K., Osmelosld J., Bethard T. and LassoieJ. P. (1990) Foliar injury and growth response of red spruce to sulphate and nitrate acidic mist. Can. J. For. Res. 20, 58-65. 17. Jeffree C. E., GraceJ. and Hoad S. P. (1994) K. E. Percy,J. N. Cape, R.Jangels and C.J. Simpson, eds. Spatial disOibution of sulphate uptake by wind-damagedbeech leaves. A# pollutants and the leaf cutich. NATO ASI Ser. Cr. Vol. 36, Springer-Verlag, Berlin, pp. 183-195. 18. Johnson A. H. (1987) Deterioration of red spruce in the Northern Appalachian Mountains. Pages 83-89 in T. C. Hutchison and K. M. Meema, eds. Effects of atmosphoic pollutants on forests, wetlands and agricultural ecosystems. Springer-Verlag, Berlin. 19. Klemm O. et aL (1989). 3C: Leaching and uptake of ions through above-ground Norway spruce tree parts. Pages 210-238 in E. D. Schulze, O. L. Lange and R. Oren, eds. Forestdeclineand air poUution: a study
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of spruce (Picea abies) on acid soils. Ecological Studies 77~ Springer-Verlag, Berlin. Kreutzer, K. (1993) Changes in the role of nitrogen in Central European Forests. Pages 82-96 in Hhetd/Muller-Doubois, eds. Forestdeclinein theAtlantic and Pacific regions. Springer-Verlag, Berlin Laurence J. A., Kohut R.J. and Amundson R. G. (1989) Response of red spruce seedlings exposed to ozone and simulated acidic precipitation in the field. Arch. Envir. Contam. Toxic. 18, 285-290. Leith I. D., Murray M. B., Sheppard L.J., CapeJ. N., DeansJ. D., Smith R. I. and Fowler D. (1989) Visible foliar injury of red spruce seedlings subjected to simulated acid mist. New Phytol. 113~ 313-320. LevittJ. (1980) Responsesofplants to environmentalstresses, Vol. 1, Chillin~ freezing and h~h temperaturestresses. 2nd Edition, Academic Press, London, 471 pp. L'Hirondelle S., Jacobson J. S. and Lassioe J. P. (1992) Acidic mist and nitrogen fertilization effects on growth, nitrate reductase activity, gas exchange and frost hardiness of red spruce seedlings. New Phytol. 121,611-622. Mengel K., Hogrebe A. M. R. and Esch A. (1988a) Effect of acidic fog on cuticle formation and water status of Picea abies (L.) Karst. Pages 132-138 inJ. Bervae, P. Mathy and P. Evers, eds. Relationships between above and below ground influences of air pollutants onforest trees. EUR 11 738 EN, CEC Belgium. Mengel, K., Breininger, M. Th. and Lutz, H. J. (1990) Effect of simulated acidic fog on carbohydrate leaching. CO2 assimilation and development of damage symptoms in young spruce trees (Picea abies L. Karst) Env. Exp. Bot. 30~ 165-173. Mohnen V. A. (1988) Mountain cloudwater chemistry project, wet, d~y and cloudwater deposition. Mountain Cloud Chemistry Project Report to USEP. Nihlgard B. (1985) The ammonium hypothesis - an additional explanation to the forest dieback in Europe. Ambio 14, 2-8. Parkinson J. A. (1987) The determination of total sulphur in vegetation. Pages 12-15 in P. A. Rowland, ed. Chemicalanalysis in environmentalresearch.Institute of Terrestrial Ecology. Symposium No 18. Merlewood Research Station, Abbots Ripton, Cambs. U.K. Parkinson J. A. and Allen S. E. (1975) A wet oxidation procedure suitable for the determination of nitrogen and mineral nutrients in biological material. Commun. Soil Sci. Pl. Anal 6, 1-11. Percy K. (1986) The effects of simulated acid rain on germinative capacity, growth and morphology of forest tree seedlings. New Phytol. 104~ 473-484. Raven, J. A. (1986) Biochemical disposal of excess H + in growing plants? New Phytol. 104~ 175-206. Raven J. A. (1988) Nitrogen acquisition by shoots and roots: occurrence and implications for acid base
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34.
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regulation. Pages 187-195 inJ. Bervaes, P. Mathy and P. Evers, eds. Relationships betweenabove and below ground influences of air pollutants on forest trees. EUR 11738EN, CEC Belgium. Rennenberg H. (1989) Synthesis and emission of hydrogen sulphide by higher plants. In E. S. Saltzman and W.J. Cooper, eds. Biogenic sulphur in the environment. American Chem. Soc., Washington. Riederer M. (1989) The cuticle of conifers: structure, composition and transport properties. Pages 157188 in Forest decline and air pollution: a study of spruce (Picea abies) on soils. Ecological Studies 77, SpringerVerlag, Berlin. Rinallo C., Raddi P. and di Lonardo V. (1986) Effects of simulated acid deposition on the surface structure of Norway spruce and silver fir needles. Eur. J. For. Pathol. 16, 440-446. SAS (1985) SAS User's Guide: Statistics, Version 5, SAS Institute Inc. Cary, NC, U.S.A. Scherbatskoy T. and Tyree M. (1990) Kinetics of exchange of ions between artificial precipitation and maple leaf surfaces. New Phytol. 114, 703-712. SchiffJ. A., Stern A. I., Saidha T. and LiJ. (1993) Some molecular aspects of sulphate metabolism in photosynthetic organisms. Pages 21-35 in L.J. de Kok, I. Stulen, H. Rennenberg, C. Brunold and W. E. Ranser, eds. Sulphur, nutrition and assimilation in h~her plants. SPB Academic Publishing. Sheppard L.J. (1994) Causal mechanisms by which sulphate, nitrate and acidity influence frost hardiness in red spruce: Review and hypothesis. New Phytol. 127, 69 82.
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41. Sheppard L.J., CapeJ. N. and Leith I. D. (1993a) Acid mist affects dehardening, bud burst and shoot growth in red spruce. For. Sd. 39, 680-691. 42. Sheppard L.J., CapeJ. N. and Leith I. D. (1993b) Influence of acidic mist on frost hardiness and nutrient concentrations in red spruce seedlings, Part 1: Exposure of the fofiage and rooting environment. New ehytol. 124, 595-605. 43. Sheppard L. J., Leith I. D., Smith C. M. S. and Kennedy V. (1995) Effects of soil chemistry on the response of potted Sitka spruce to acid mist in opentop chambers. Water, Air Soil PoUut. (in press). 44. Tamm C. and Wiklander, G. (1980) Effects of artificial acidification with sulphuric acid on tree growth in Scots pine forest. Proc. Int. Conf. Ecol. Impact and Acid Predp. Norway SNSF prog, pp. 188-189. 45. Taylor G. E. Jr., Norby, R.J., McLaughlin, S. B., Johnson, A. H. and Turner, R. S. (1986) Carbon dioxide assimilation and growth of red spruce (Picea rubens Sarg.) seedlings in response to ozone, precipitation chemistry and soil type. Oceologia70, 163 171. 46. Temple P.J. (1988) Injury and growth ofJeffrey pine and Giant sequoia in response to ozone and acidic mist. Env. Exp. Bot. 28, 323 333. 47. Vann D. R., Strimbeck G. R. and Johnson A. H. (1992) Effects of ambient levels of airborne chemicals on the freezing resistance of red spruce foliage. For. Ecol. Manage. 51, 69-79. 48. Wood T. and Bormann F. H. (1977) Short term effects of a simulated acid rain upon the growth and nutrient relations of Pinus strobus L. Wat. Air Soil PoUut. 7, 479488.
2, pp, 125-137, 1995 Elsevier Science Ltd Printed in Great Britain
Pergamon 0098-8472(95)00006-2
POTENTIAL MECHANISMS OF ACID MIST INJURY TO RED SPRUCE IAN D. LEITH, LUCYJ. SHEPPARD and MAUREEN B. MURRAY Institute of Terrestrial Ecology, Bush Estate, Penicuik, Midlothian EH26 0QB, U.K.
(Received 18 August 1994; acceptedin revisedform 28 November 1994) Leith I. D., Sheppard L.J. and Murray M. B. Potential mechanisms of acid mist injury to red spruce. Environmental and Experimental Botany 35, 125-137, 1995.--One-year-old red spruce seedlings (Picea rubens Sarg.) were exposed to six simulated mist treatments in open-top chambers, between 25 May and 8 November 1988. Mists were applied twice weekly to foliage and soil, with each application equivalent to 2 mm precipitation, applied over 30 min. The treatments contained pairwise combinations of H ÷, NH4÷, 8042- and NO3- ions at concentrations of 1.6 or 3.2 mol m -3, with one treatment including all four ions. Effects of individual ions and ion combinations on visible injury, growth and nutrition were determined. After four applications ofH2SO 4 at 3.2 mol m -3 (pH 2.5), current year needles turned red-brown. Subsequent application of HzSO4 at a reduced concentration of 0.5 mol m -3 (pH 3.0) resulted in no further increase in foliar injury symptoms. No foliar injury symptoms developed on seedlings receiving HNO3 (pH 2.5), NH4NO3 or (NH4)2SO4 treatments. Twenty-five per cent of needles were damaged on seedlings receiving H2SO4+ NH4NO3 (pH 2.5), but only after 24 applications of mist, suggesting that either NH4+ or NO3- ions ameliorated the effect ofH2SO 4. There was a stimulation in shoot and root biomass in those treatments containing N, with or without H2SO4. Contrast analysis indicated a positive growth response to acidity. Significant uptake of S and N occurred in response to treatment. This experiment indicates that it is the simultaneous presence of H + and SO42- in occult precipitation that has the greatest potential for inducing foliar injury. The presence of NO3- was found to ameliorate the toxic effects. Potential mechanisms underpinning these observations are discussed.
Key words : Red spruce, visible injury, foliar nutrients, growth, sulphate, acidity, nitrate.
INTRODUCTION
must have a lower p H to cause similar effects. (t6/ Both SO42- and H + ions were present in the simuThe decline of red spruce (Picea rubens Sarg.) has lated acid mist used by LeithJ 22)However, acid rain been investigated under controlled environments and cloud contain other ions, including pollutante.g. open-top chambers ~6/and more recently in the derived N O 3 - and NH4 + and the contribution of field. Various 'threshold' values for injury have been these N-containing ions on the development of reported but these are strongly influenced by seed- injury needs to be evaluated. ling age, duration and frequency of exposure and While symptoms of visible injury induced under the chemical composition of the applied experimental exposures have shown remarkable treatments. (I5'22'31~Visible injury to red spruce can consistency, conflicting results have been obtained be caused by sulphuric acid at p H 3.5, but it is from experiments designed to test the effects of known that mixtures of sulphuric and nitric acid simulated acid precipitation on the growth of red 125
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I.D. LEITH et al.
spruce seedlings. No significant effects of simulated acid rain on growth and photosynthesis were reported by Laurence et aL, 12~1or by Taylor eta/., (45) who measured extension growth on seedlings after exposure to artificial acid mist at pH 3.6 and pH 5.1 containing both SO42- and NO3- ions. However, Percy(31) found significant growth reductions and morphological changes in red spruce seedlings treated with acid rain (containing sulphate and nitrate, ratio 2 : 1) at pH 2.6. Jacobson et al. ~16~also found reductions in leader extension in red spruce exposed to H2SO~ mist at pH 2.6. Wood and Bormann I48~ showed increases in the growth of P/nus strobus seedtings exposed to simulated acid mist applied as nitric acid at pH 2.3 for 20 weeks. In an experiment preceding that described in this paper, exposure of red spruce seedlings, grown in open-top chambers (OTCs) to simulated acid mist, induced direct foliar injury/discoloration. Development of foliar injury was found to be strongly dependent on the ionic concentration of the simulated acid mist. A linear increase in foliar injury to red spruce seedlings was observed as the ion concentrations in the mist increased. As the four major ions were in constant ratio to each other, the contribution of individual ions could not be identified. In the present experiment, the specific influence of these individual ions on red spruce was tested using paired combinations of the four major ions (NO3-, SO42-, NH4 +, H ÷) in six acid mist treatments. This paper reports foliar injury, growth, biomass production and nutrient status resulting from the application of these mist treatments over a period of 23 weeks between May and November 1988. Effects on frost hardiness from this experiment were reported by Cape et al. 131 Earlier experiments/16/have shown that the presence of N increased the acidity strength at which visible injury was observed, i.e. reduced plant sensitivity to the H + ion concentration. The significance of the anion-proton interaction has not, however, been specifically addressed. This paper is based on five treatments plus a control (Table 1) which were designed to explore the relative contributions of S, N and acidity on both the induction of visible injury and growth response. The following hypotheses were tested, and the significance of these observations for mechanisms by which the perturbing ions perturb are discussed. 1. The combination of H + and SO42-, not H +
2.
3.
4.
5.
and NOa-, ions causes visible injury (compare response to H 2 S O 4 and HNO3 with control). Neither SO42- nor NO3- in combination with NH4 + ions induce visible injury in the absence of acidity [compare responses of(NH4)2 SO4 and NH4NO3 treatments with control]. The form of added N (NH4+ or NO3-) determines the nature of effect on red spruce [compare responses to (NH4)2SO 4 and HNO3 treatments with control]. The presence o f S O 4 2- ions perturbs red spruce in the absence of acidity [compare response to NH4NO3 and (NH4)2SO4 with control]. The response to all four ions in combination produces similar effects to those observed in the 1987 experiment./22) METHODS
A d d mist treatments
The design and physical properties of the octagonal glass-sided open-top chambers (floor area 7.0 1/12, height 2.3 m including frustrum), were described in detail by Fowler et aL 191All chambers received filtered air, with 03, SO2 and NO2 removed by activated charcoal filter units. Ambient concentrations of these gases over the season averaged: SO2 2.1 nl 1-l, NO 1.1 nl 1-1, NO2 4.7 nl 1-l and 03 26.7 nl 1- i. The flow rate of air was supplied via ducted fan units to the chambers at a flow rate of 40 m 3 min-l (two complete air changes min-1). The seedlings, in 4 1 pots, were irrigated daily via capillary matting (holding capacity 7 1 m-2). Rainfall was excluded from the chambers by U.V. stabilised polyethylene lids fitted within the frustrum. The treatment design used paired combinations of the four major ions and one treatment containing all four ions (Table 1). Each treatment was randomly assigned to two chambers. (231Mist treatments were made from concentrated stock solutions. Prior to application, each solution was checked for electrical conductivity, acidity and presence or absence of sulphate using CaC12 solution. Mist treatments were applied using a pressurized system./9~ Each chamber received the equivalent of 2 mm precipitation (14 1) at a rate of 3 mm hr -~ twice weekly over a 23-week period. 122~Air turbulence within the chamber ensured an even distribution of mist throughout the chamber. Mist was applied between 25 May and 8 November 1988.
POTENTIAL MECHANISMS OF ACID MIST INJURY
127
Table 1. Ion concentrationsand deposition ratesfor the mist treatments. The concentrationof H2SO4 was modredfrom (a) afterfour applications of mist to (b) (see text) Ion concentrations mol m Solutions H20 NH4NO3 (NH4)2SO4 H2SO4 (a) (b) HNO3 H~SO4 + NH~IO3
NO3 8042 NH4+ H ÷ 0 1.6
0 1.6
3.2 1.6
1.6 0.5 1.6
Plant material Experimental material was propagated at Bush Estate, (55°51'N, 3°12'W, 185 m altitude) from seed collected from mature stands of red spruce at Hardy Brook, New Hampshire, U.S.A. (43°56'N, 71°32'W, altitude 490 m). Stratified seed was sown on 14 March 1987 into a sphagnum peat based compost containing 200 g Vitax Q4 compound fertiliser (N 5.3%, P 7.5%, K 10%) per 100 1 of compost. Seedlings were transferred to 0.1 1 pots containing the same compost medium but without additional fertiliser in late June 1987. Seedlings were maintained in a filtered glasshouse without supplementary lighting but protected from frost throughout the winter. Prior to final repotting, foliar N concentrations were increased by approximately 0.5% by four applications of Ingestad B and C,/j4/ equivalent to 15 mg N per pot. In April 1988, 1year old seedlings of uniform height were selected and repotted into 4 1 pots containing a 60% peat: 40% quartz grit compost by volume with no additional fertiliser, and transferred to the O T C s at Bush Estate. Plant growth measurements A destructive harvest of 10 seedlings of similar size to those selected for the main O T C experiment was undertaken as plants were repotted in April 1988. Height and root collar diameter were measured. The seedlings were then separated into shoot and root systems. Individual root systems were washed, partitioned into fine ( < 1 m m diam-
-3
Deposition kg ha-x application- a
pH
S
N
H
0 1.6 3.2
0 -
5.6 5.6 5.6
0 1
0 0.9 0.9
0 -
2.5 3.0 2.5 2.5
2.0 0.3
1.6
3.2 1.0 3.2 3.2
0.9 0.9
0.06 0.04 0.06 0.06
1
eter) and coarse ( > 1 m m diameter) roots, dried at 80°C for 4 days then weighed. The above-ground parts were also dried at 80°C for 4 days and needle and stem dry weight recorded.
Height growth measurements Immediately prior to transfer to the O T C s (April 1988) total plant height and root collar diameter were recorded for 10 randomly selected seedlings per chamber. Thereafter, weekly measurements of shoot leader extension were recorded. Root collar diameter was remeasured at the final harvest (5 December 1988). Comparison of height growth cessation The date of leader extension cessation was determined as the first assessment date (described by its Julian day where 1 August = 213) on which individual seedlings failed to produce an increase in height growth. The date of height cessation was found for each treatment chamber by calculating the chamber mean from 10 plants. Biomass assessment The 10 randomly selected seedlings in each of the 12 chambers which had been used for height growth measurements were destructively harvested between 5 and 7 December 1988. The procedure was similar to that described for pre-treatment harvest; the root collar diameter was measured, then the shoot system was severed from the root system at the root collar. Components of the shoots were
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divided into year classes, dried at 80°C for 4 days, then weighed. Root systems were washed out carefully under a fine jet of water over a coarse sieve. Two to three first order lateral roots (approximately 15% of the total biomass) were removed from the thickened root collar and divided into coarse and fine size classes. The remaining laterals were removed from the thickened root collar. All the roots were ovendried for 4 days then weighed. The ratio of fine to coarse roots in the subsample was used to estimate the ratio of fine to coarse roots in the bulk sample. The weight of the thickened root collar was determined separately.
Chemical analysis Samples of rinsed dried, current year needles from each of 10 plants per chamber were bulked by chamber. One well-mixed subsample was ground ( < 0.8 mm) for chemical analysis. An aliquot of this ground material was redried at 105°C for 3 hr, 350 mg was digested in a modified Kjehldal digest./3°/ N, P, K, Ca and Mg were determined as described by Allen et aL I~l Sulphur was determined on the ground sample by X-ray fluorescence spectrometryJ 29/The results are expressed on a percentage dry weight basis. Accuracy was checked against internal reference material and external certiffed standards. Assessment of visible foliar injury Seedlings in all 12 chambers were assessed for foliar injury prior to initial mist application and on nine occasions throughout the exposure period, May-November 1988. Foliar injury was determined using a system of 12 needle damage classes. (22/ Only current year needles on marked individual seedlings were visually scored for incidence of necrotic and/or chlorotic damage by the same operator. Visible foliar injury was expressed on a percentage damage basis. Estimates of percentage damage within each chamber and between treatments were analysed for treatment differences using A N O V A , (37)following angular transformation of the data. Experimental design-analysis The six treatments were randomly assigned to the 12 chambers to provide a complete randomized design. The A N O V A (37)was conducted on chamber
means, i.e. two per treatment. Differences between treatment means were tested using Tukey's studentized range test. Contrasts were also constructed in the analysis of variance to test the effects of individual ions. These contrasts are not independent because most treatment ions were applied in combinations of ions and the concentration of SO42- had to be changed (Table 1). RESULTS
Visiblefoliar injury Applications of simulated acid mist treatments began on 25 May 1988. Injury symptoms developed after four applications (2 weeks), but only on the seedlings treated with p H 2.5 H2SO4. Injury symptoms resembled those described by Leith et al. (221 with an initial red-brown discoloration of the current year needles leading to complete necrosis and needle loss within 4 weeks. Red-brown discoloration was principally found along the whole length of the needles and on both the adaxial and abaxial surfaces. Observations of only partially necrotic needles showed browning beginning at the distal end of the needles progressing towards the proximal end and resulting in complete needle necrosis. The numbers of needles showing light brown chlorosis were small compared with those showing red-brown discoloration. This chlorosis affected the whole needle. The extent of the initial foliar necrosis in the H2SO4 treatment on 13 June 1988 was about 7% per plant, with approximately 70% of individuals affected in both replicate chambers. The laterals on the upper whorls exhibited more damage than the less exposed lower whorls. No visible injury was observed in the other treatments on this date (Fig. 1). After the initial observation of foliar necrosis, misting was withheld from all treatments between 10June and 1July. Continued application of H2SO4 at a concentration of 1.6 mol m -~ would have resulted in severe needle necrosis and probable seedling death. The concentration of H + and SO42- ions in the H2SO treatment was therefore reduced by a factor of 3 to 0.5 tool m -3 (Table 1). On 28June 1988, damaged seedlings were removed from the H2SO4 treatment and replaced with untreated seedlings of a similar age and size which had been kept in a glasshouse in filtered air. Twenty of the original seedlings which had been exposed to the p H 2.5
POTENTIAL MECHANISMS OF ACID MIST INJURY 0
i
80
of the injury increased steadily over the following 11 weeks so that by the end of November, 90% of seedlings showed foliar injury on 25% of their current year needles. Treatment with HNO3 (pH 2.5) or de-ionised water caused virtually no visible damage (< 1%) over the experimental period. The control plants, which received de-ionised water, appeared pale green in colour by comparison with other treatments, especially the treatment with HNO3, but did not exhibit visible foliar injury. By the beginning of November, the NHaNO3 (pH 5.6) and (NH4)2SO4 (pH 5.6) treated seedlings showed very little damage (< 2%) on 10% of the plants. Six per cent of plants treated with HNO3 pH 2.5 were damaged. Visible injury to plants receiving NH4NO3 was in the form of light brown~reen needle discoloration which was undetectable from a distance. Plants in the (NH4)2SO4 treatment showed needle yellowing, especially at the needle tips. No significant differences in the degree of damage or number of damaged plants were observed between the HNO3, NHqNO3, H2SO4 (pH 3), (NH4)2 SO4 and H20 treatments.
111804+ NH,NO,
IMNO
IO0 .
o
June
July
2
IIHO
"
40
0
Aug
Sept Oct
Nov
1988 25 2O
"~ 15
/ 0
Heightgrowth cessation-budset
' ~ , w
June
129
w
July
Aug
.
w
Sept
'
Oct
i
i
Nov
1988
Fig. 1. (a) Development of visible injury; percentages of seedlingsinjured by the different acid mist treatments. (b) Development of needle injury; percentage of damaged needles per plant.
Treatment effects were marginally significant (P = 0.06). The H 2 S O 4 t r e a t m e n t was the last to set bud, while the NH4NO3 and water treatments set bud first (Fig. 2). Contrast analysis revealed significant (P = 0.01"*) effects of S and acidity. The presence o f S O 4 2- appeared to delay budset-height growth cessation. B/0mass
retained in the chambers; of these, 10 were used in the biomass assessment. Misting resumed on 1July with the modified HzSO4 treatment and continued until 8 November. There was no further damage to the original plants (pH 2.5, pH 3.0) and almost no visible injury on the replacement seedlings, which only received pH 3.0 mist. Foliar injury symptoms were observed in the H2SO4+ NH4NO3 (pH 2.5) treatment from the beginning of August 1988 following 18 mist applications. By 19 August, 20% of the seedlings were showing foliar injury with approximately 5% of needles damaged per plant (Fig. 1a,b). The extent H2SO 4 were
Treatment effects on the sum of needle and stem (shoot) weight produced in the growing season, encompassing the experimental period were significant (P < 0.05) (Fig. 3). New growth increased in response to increasing acidity. Neither the presence of N or S significantly (P > 0.05) affected shoot dry weights. Needle weights were greatest in the NHaNO~, NH4NO3, + H 2 S O 4 and HNO3 treatments. Root weights (data not shown) were not significantly affected by individual treatments but were significantly (P < 0.05) higher when NO3- was included in the mist. Root/shoot ratios (data not shown) ranged from 0.9 (H2SO4) to 1.1 (NH4NO3) and were likewise not significantly affected by treatment. Separation of the roots into fine, coarse and
130
I.D. LEITH et aL 225 220 215
~'
T
_.]___
210
"O §
2O5
190
ab
a
ab
H=SO4+ (HNOa)= H2S04 NH4NOs (NH4)=SO, H20 NH4NOa
Fig. 2. Effect of different acid mist treatments on height growth cessation. Values with the same letter are not significantly different [(P> 0.05) Tukey's studentized range test].
tap root revealed differential treatment effects but no significant differences. Weights of fine roots ranged from 3.2 g (H2SO4) to 4.6 g (NH4NOs+ H2SO4) and coarse roots from 1.6 g [I-I20 , (NH4)2SO4] to 3.3 g (HNOs). The structural root component, coarse roots increased in response to NOs-N and root weights were highest in those treatments containing NO3-N. Significant treatment effects (P < 0.05) on the fine to coarse root ratio were observed (Fig. 4). Ratios were highest in the H 2 0 and (NH4)2SO4 treatments, reflecting the low coarse root weights. Root collar diameter was not significantly affected by treatment mainly because of the huge difference (67%) between the two chambers in the (NH4)2SO4 treatment. The largest root collar diameters were from seedlings receiving all four ions and the smallest from those receiving water only. Contrast analysis was undertaken to determine the influence of specific ions (PROC GLM), (sT/ although ion effects were not mutually independent in this experiment. The inclusion of acidity was found to significantly (P < 0.001) influence all the growth parameters highlighted in Table 2, [new needles and stem, the tap and coarse root, total root weight, leader extension, root/shoot ratio, and root
3.5
b 3.0 o 2.5 4
|
®
~3
¢2
2.0
ab
1.5 o. O i-
~
1.o 0.5
0
H-SO.+ N~, N'Os
HNOa H=SO4 NH4NO3 (NH4)=SO4 H=O
Fig. 3. Effect of acid mist treatment on total weight of new stem and needles. Value for total weight with the same letter are not significantly different [(P > 0.05) Tukey's studentized range test]. Note the larger proportion of stem to needles in the treatments lacking additional N.
o.o H=SO4* HNOa H2SO4 NH, NO3 (NH4)2SO4 H=O NH4NOa
Fig. 4. Effect of acid mist treatment on the fine root to coarse root ratio. Columns with the same letter are not significantly different [(P> 0.05) Tukey's studentized range test].
POTENTIAL MECHANISMS OF ACID MIST INJURY
131
Table 2. Results of a contrast analysisfor differences between treatments with and without individual ions for growth parameters. These ions are not mutually independent in this experiment. Valuesshow theprobabilitv ofa nulleffect (P < 0.001+++, P < 0.01++, P < 0.05+). +/- Indicate direction of effect, increase or decrease
New needles (Ne) New stem (St) New total (St+Ne) Tap root (g) Coarse root (g) Fine root (g) Root wt (g) R/S ratio Root collar diameter Leader extension Date height cessation
H+
NH4+
+++ +++ +++ ++ +++ n.s. n.s. +++ +++ +++ ++
collar diameter (P < 0.05)]. Root collar diameter was greater in the presence of acidity. The presence of the NH4 + ion was not significant (P> 0.05) for any of the above-mentioned parameters, but increased root collar diameter. The inclusion of NO3- significantly affected biomass production and leader extension (P < 0.01) with the exception of the fine roots. The presence of NO3- had no significant affect on the date of height growth cessation, but the presence ofNH4 + shortened the growing season. The inclusion of $042- had no significant effect on any of the growth (weight, diameter) parameters, only influencing the time the plants were growing and like H ÷, inclusion delayed budset. The overall effect of N was attributable to NO3- induced effects rather than the presence of the NH4 + ion. Foliar nutrient status No significant treatment effects were observed for foliar N, P, K, Ca or Mg concentrations (Tables 3 and 4). Treatments lacking additional N, i.e. H2804 and H 2 0 had the lowest N concentration. The occasional large differences between replicate chambers contributed to the absence of statistically significant effects. The base cations K, Mg and Ca showed least variability with treatment. Although some treatments contained N, foliar N concentrations were not significantly (P > 0.05) influenced by treatment. By contrast, foliar S concentrations were treatment-dependent. Those treatments which contained S had significantly
NO3-
SO42-
n.s.
+++
n.s.
+++
n.s. n.s.
+++ +++
n.s. n.s.
+++ +++
n.s.
+++
n.s.
+++
n.s.
+++
n.s.
+++
n.s.
n.s.
n.s.
+++
n.s.
+++
n.s.
+++
n.s.
+++
n.s.
+++
+
+
n.s.
++
n.s.
+++
n.s.
++
++
n.s.
n.s.
N
(P < 0.05) more S than those which lacked S. The H2SO 4 treatment, where the S concentration was modified from 1.6 to 0.5 mol m -g, had intermediate S concentrations but these were also significantly higher than those from the control, H 2 0 plants. Contrast analysis (Table 5) showed that foliar N concentrations were significantly (P = 0.04) influenced by the presence of the NH4 + ion. Despite the absence of K from the acid mist treatments, fofiar K concentrations were significantly increased by the presence of N, acidity and SO49-. DISCUSSION
Within the first 3 weeks of acid mist exposure, the first hypothesis was tested; the absence of injury in the HNO3 treatment supported the view that a combination of H + and SO42- ions are required to induce visible injury. The absence of immediate visible injury followed by its much slower and reduced development in the NH4 NO3+ H2 SO4 treatment further showed that the presence of N mitigates harmful effects of S. Likewise, the low level of damage in the (NH4)2 SO4 treatment provided further confirmatory evidence that SO42- and H + are necessary for the induction of significant amounts of needle necrosis (hypothesis 2). Injury was greatest when 8042- was present but in the absence of acidity damage was negligible. As observed in Leith's experiment, (22) significant amounts of injury were observed in the presence of
132
I.D. LEITH et al. Table 3. Analysis of variance offoliar S concentrations9%m red spruce shoots. The main factor is poUutedmist treatmentwith two replicatechambermeans (bulkedsamples) with single degreeoffieedom contrast analysis Source
DF
Sum of squares
Treatment Treatment x chamber Total
5 6 11
0.092 0.003 0.095
Source
Tests of hypothesis Error Treatment x Chamber
Contrast Effect of H Effect of NH4 Effect of NO3 Effect of SO4 Effect of N
DF 1 1 1 1 1
SS 0.007 0.071 0.003 0.090 0.156
F Value 32.49
P> F 0.0003
FValue 13.2 124.8 4.9 158.3 27.4
P> F 0.0109 0.0001 0.0679 0.0001 0.0020
Table 4. Foliar N, S, P, K, Mg and Ca concentrations (% d~y weight) representinga bulked samplej~om 10 plants for individual replicatedchambers (1 and 2). The treatment mean is shownfor S, where valuesfollowed by the same letterare not signoqcantly different (P > 0.05) using Tukey's studentized range test. Treatment effects were not signoqcantfor N, P, K, Mg and Ca N H20 NH4NO3 (NH4)2SO4 (pH 2.5 and pH 3.0)H2SO4 HNO~ H2SO4 + NH4NO~
Rep 1 Rep 2 1 2 1 2 1 2 1 2 1 2
0.96 1.40 1.60 1.70 1.4 1.5 1.1 1.2 1.2 1.3 1.4 1.4
all four ions and the level of injury escalated with the onset of cold hardening which appears to be triggered by daylength in red spruce (Sheppard, L.J. unpublished results.) This experiment was designed to distinguish between individual phytotoxic effects of the four major ions present in cloudwater. Combining SO42-, NH4 +, N O 3 - and H + at the p H extremes
S 0.15 0.18 0.19 0.20 0.34 0.39 0.27 0.25 0.21 0.19 0.42 0.37
0.17c 0.19bc 0.36a 0.26b 0.20bc 0.40a
P
K
Mg
Ca
0.18 0.24 0.22 0.25 0.27 0.28 0.19 0.21 0.24 0.21 0.26 0.18
0.91 1.00 0.83 0.99 1.1 1.1 0.98 1.0 1.1 1.1 1.1 1.0
.014 0.13 0.15 0.14 0.15 0.18 0.11 0.14 0.15 0.13 0.14 0.12
.058 0.64 0.74 0.72 0.69 0.72 0.63 0.65 0.73 0.63 0.73 0.56
of natural precipitation, wet deposition (27)provided a unique opportunity to identify some important synergistic effects. In the ensuing discussion we briefly relate our findings with respect to the corroboration or otherwise of previous studies with red spruce (6~ and then concentrate on potential mechanisms by which the specific ions may be influencing plant growth and nutrition.
POTENTIAL MECHANISMS OF ACID MIST INJURY
133
Table 5. Results of a contrast, F-test, for difference between treatments with and without individual ionsfor nutrient concentrations. Ions are not mutually independent in this experiment. Values show the probability of a null effect and the direction of the effect (+ = increase, - =
decrease,P < 0.001+++, P < 0.01++, P < 0.05+)
E~ctof H NH~ NO~ SO4 N
N
P
K
Ca
Mg
S
n.s. + n.s. n.s. +
n.s. n.s. n.s. n.s. n.s.
+ n.s. n.s. + -
n.s. n.s. n.s. + n.s.
n.s. n.s. n.s. n.s. n.s.
++ ++ n.s. +++ ++
Wib injucv Exposure of red spruce seedlings to H2SO 4 approximately 4 weeks after bud burst produced almost immediate necrosis in current year foliage. This observation is consistent with those of Jacobson et al. 116~who observed maximum injury when concentrations of both H + and SO42- were high. Inclusion of NHaNO3 with H2SO4 significantly delayed the rate of injury development but did not eradicate it. After 6 months 25% of foliage on all plants had either turned red-brown or abscised corroborating observations by Leith./22/ Reducing the level of acidity, H + ion concentration, in the H 2 S O 4 t r e a t m e n t by a factor of 3 resulted in almost no damage and it was concluded that red spruce was tolerant of acidity above p H 3. The observation that reducing acidity/ion concentration can arrest visible injury, was also seen for Sitka spruce,/43/indicating the importance of ion concentration/acidity in the phytotoxic response. The reduction in SO42concentration in the mist was reflected in lower foliar S concentrations. The characteristic necrotic red-brown coloration induced in current year needles during the growing season resembled that described by Leith et alJ 221 and has been reported for a number of conifer species exposed to simulated acid mist in controlled conditions. (2'26'46)Such necrosis of needles in the field has not been widely reported although reddening caused by freezing injury brought about through rapid warming and solar radiation has been observed in spring. Ill/ Yellow flecking and red-brown discoloration appear to be more common throughout European and North American forests./~8~ Advanced stages of nutrient deficiency, induced by anthropogenic pollutants causing nutri-
ent imbalances, e.g. N : K, (2°)however, can also take the form of red-brown needle discoloration. The lowered base cation concentrations in the H~SO4 treatments show how acid inputs can exacerbate nutrient imbalances with respect to N. Attacks by insects such as Ips ~pographus (spruce bark beetle) and fungal pathogens such as Lophodermium picea (spruce needle cast fungus) also cause red discoloration of needles and needle loss. Because of the lack of specificity of symptoms of needle injury, their diagnostic value in the field is limited. However, the induction of needle injury under controlled experiments is important because it provides conclusive evidence of pollutant-induced perturbations in the plant. The (NHfl~O3+H2SO4) pH 2.5 treatment provided the same ions as used in the previous dose response experiment./9~ However, in that experiment the mixture was composed of an equimolar solution of (NH4)2 SO4+ HNO3 (pH 2.5) providing 3.0 mol m -3 H +, 6.0 m o l m -3 NH4 +, 3.0 mol m -3 NO3- and 3.0 mol m 3 SO42-. By contrast, in the present experiment there were 3.2 mol m -3 H + and 1.6 mol m -3 each of NH4 +, NO3- and SO42- . Thus in the present experiment the ratio of H + to SO42is twice that of the previous experiment and the ratio of N : S is 2 : 1 compared with 3 : 1. In both experiments, the induction of injury took about 910 weeks, but the S dose required to induce injury in this experiment was < 50% of that received by the 2-year-old plants in the previous dose experiment. Results of a study byJacobson et al. 115'~suggest that this increased sensitivity may have been caused by the lower N:S ratio in this experiment. It is also possible that the increased H+:NO3 - ratio in the present study (2 : 1 compared with 1 : 1 in the dose
134
I.D. LEITH et al.
response study) may have played a part, since the assimilation of NO3- consumes protons/33/ (see later). This direct comparison between the two experiments provides corroborating evidence of the ameliorating effect of NO3- on SO42-/H + induced phytotoxicity. Growth and foliar nutrients
In the absence of additional fertilizer N the positive response to N seen here reflects that observed in the field on N limited sites. (2°/The presence of NO3-N and H + ions generally stimulated growth, while the presence of NH4 + or SO4- had no effect. The combination of H2SO4+ NH4NO3 increased both stem leader and new needle growth significantly, whereas treatment with (NH4)2SO4 had no effect on growth. Treatment with HNO3, NHdNO3 and H2SO 4 also enhanced growth by comparison with the H20 treatment. The proliferation in fine root growth in response to N and coarse root branching in response to H + is consistent with the findings of Deans et al. (sl The positive effect of acidity on growth contrasts the results of Percy/3~/ but is attributed to enhanced N availability(42)which may have overridden any negative effects of acidity. Tamm and Wiklander (44/ concluded that H2SO 4 treatment of soil enhanced N mineralization, particularly in the absence of additional N. Our growth data corroborates Jacobson's study/15~ that the presence of SO42- ions does not influence growth per se but that growth is stimulated by additions of NO3-N and acidity. The N-stimulated increase in dry matter production is all the more impressive since those treatments containing NH4NO~ set bud, and stopped growing first. By contrast, the presence of SO42- extended the growing season. This contrasts with the effects of SO42found in the spring, when treatment with SO42was found to delay bud burst./41/These results contradict the view put forward by Nihlgard (2a/ and Friedland et al. (~°1 that additional N extends the growing season, making plants more frost sensitive. However, it may provide an explanation for the increased frost sensitivity associated with SO42-,/3~ since the onset of winter hardening in north-temperate conifers is strongly coupled to growth cessation.(2a/ These data suggest that where N availability is restricting growth the benefits of additional N can exceed the negative effects associated with S inputs.
Implications for underpinning mechanisms
The aforementioned observations highlight some outstanding questions regarding the respective phytotoxic roles of 8042-, H +, NO 3- and NH4 + ions in acid mist. How do the presence of (a) H ÷ and (b) NH4NO3 modify the phytotoxicity of SO42-? L'Hirondelle et a/. (24) observed that H2SO 4 induced foliar injury was reduced when foliar N status was high. In this study foliar N status was almost identical and more than adequate (1.4% N) in the (NH4)2SO4 and H2SO4+ NH4NO3 treatments yet levels of injury differed significantly between these treatments. Foliar N concentrations from both the control and H2SO 4 treatments were low < 1.1% but visible injury was only observed in the H2SO4 treatment. Our results do not uphold the view that high foliar N concentrations per se protect foliage from acid (H+)/SO42- induced damage. However the inclusion of NHaNO3 with H2SO4 significantly enhanced dry matter production, despite the damage to foliage (needle necrosis), and despite not lowering S concentrations. We suggest therefore that the ameliorating influence of N on SO42- toxicity(6'4°)is founded on increased growth which provides for utilization of the absorbed S and thus S detoxification. When N availability is high and foliar N status is sufficient to promote growth, red spruce can compensate for needle necrosis through enhanced photosynthetic capacity, C assimilation. (8/ Thus the large amounts of potentially toxic S are detoxified either through glutathione production via assimilatory SO42- reduction (4~ or via incorporation into amino acids./4'a4'39~ Foliar S concentrations (% S) in themselves do not need to be low as long as the form or location of S is not toxic. Treatment with pH 2.5 HNO3 or pH 5.6 (NH4)2SO4 caused no significant visible injury to red spruce confirming hypothesis 1 that the combination of acidity and sulphuric acid are necessary to induce visible injury in red spruce. The likelihood of N causing phytotoxic effects is much smaller than that for S because plant demand for N exceeds that for S by a factor of 16J391Jacobson et al. 115~ also observed injury with sulphuric acid mist but concluded that acidity was the main predisposing agent and that 8042- exacerbated while NO3- ameliorated the damage. Eamus and Murray/7/ were unable to detect deleterious effects of high proton concentrations on A~,~ in Norway spruce seedlings which received the same treatments as here, and
POTENTIAL MECHANISMS OF ACID MIST INJURY they concluded that 8042- toxicity with respect to visible injury and Amax was (concentration x pH) dependent. With respect to 8042- phytotoxicity, Eamus and Murray (7) suggested that the impact of 8042- upon assimilation may relate to SO4 ~induced modification of the ionic balance of the chloroplast. Thylakoid architecture and function is extremely sensitive to the ionic balance of the stroma. They/7/observed 'locking open' of the stomata which could have been due to a reduction in chloroplast generated ATP or competition between SO42- and C1- ions in the guard cells which would influence the movement o f K + ions and hence guard cell opening (Cleland, R. pers. communication). The absence of visible injury in the presence of HNOs contradicts the opinion/15~ that acidity is the main cause of visible injury. The reductive assimilation of both NO3- and SO42-, consume protons (32/ although the former is much more important. (33~The neutralizing effect of NO3- (0.78 mol H + per mol NO3-), together with the plants high demand for N for growth and the growth process may explain the absence of injury by HNO3. The synergistic effects of H ÷ and SO42- with respect to their phytotoxicity may be due to the mutualistic way in which two ions are absorbed via the foliage. It is widely believed that the cuticle presents a relatively impermeable barrier to SO4 zuptake./35/However, acid rain containing H ÷ and SO42- ions causes significant erosion and fusion of the epicuticular wax./2s'36/ Needle wettability is increased and with it ion retention. Repeated treatment of isolated cuticles with either H 2 8 0 4 or HNO3 increases SO42- but not NO3- uptake./~2/ Jeffree et al. 1~7)have observed significant movement of SO42- into leaves pretreated with acidified SO42- solutions. There is now increasing evidence that as ion concentrations build up on leaf surfaces through drying, significant amounts of SO42- and H + can be mutually absorbed in a way that doesn't happen for NO3-. (12'~3'E8'19'38/The combination of 8042- and H + ions is phytotoxic because when leaves are simultaneously exposed to both ions their mutual uptake is significantly enhanced. These results further substantiate the view that 8042- is phytotoxic in high concentrations particularly when acidity is also high implying a synergism between the two ions. Nitrate ions ameliorate the effects probably through enhanced
135
assimilation and growth dilution. Observations of direct acid/SO42- induced injury to red spruce are rare in the field suggesting phytotoxic concentrations are rarely experienced for long enough to cause visible injury in the field. However, nonlethal SO42- concentrations, which can perturb plant physiology and result in reductions in frost hardiness, leading to increased injury from frost, have been seen in the field./47) Acknowledgements~The analytical staffat ITE Merlewood are thanked for undertaking the chemical analysis and Neil Cape for providing invaluable comment on earlier drafts. This research was supported by funds provided by the Northeastern Forest Experiment Station's Spruce-fir Research Co-operative within the joint U.S. Environmental Protection Agency-USDA Forest Service Forest Response programme. The Forest Response programme is part of the National Acid Precipitation Assessment Programme. This paper has not been subject to EPA or Forest Service peer review and should not be construed to represent the policies of either Agency.
REFERENCES
1. Allen S. E., Grimshaw H. M., Parkinson J. A. and Quarmby, C. (1974) Pages 81-159 in S. E. Allan, ed. Chemical ana~sis of ecological materials. Blackwell Scientific Publications, Oxford. 2. Brown K. A. (1989) Effects of ozone and acid mist and soil factors on Norway spruce in relation to forest damage. Research Report, National Power, Leatherhead, Surrey, U.K. 3. CapeJ. N., Leith I. D., Fowler D., Murray M. B., Sheppard L. J., Eamus D. and Wilson R. (1991) Sulphate and ammonium in mist decrease frost hardiness of red spruce. New Phytol. 118, 119-126. 4. Chen Y. and Wellburn A. R. (1989) Enhanced ethylene emissions from red and Norway spruce exposed to acid mists. Pl. Physiol. 91,357-361. 5. DeansJ. D., Leith I. D., Sheppard L.J., CapeJ. N., Fowler D., Murray M. B. and Mason P. (1990) The influence of acid mist on growth, dry matter partitioning, nutrient concentrations and mycorrhizal fruiting-bodies in red spruce seedlings. New Phytol. 115, 459 164. 6. Eagar C. and Adams M. B. (eds) (1992) Theecologyand decline of red spruce in the Eastern United States. SpringerVerlag, New York. 7. Eamus D. and Murray M. B. (1993) The impact of constituent ions of acid mist on assimilation and
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