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### Kam.m.ez'bauer H, H Selinger, R Rommelt, A ZieglerJons, D Knoppik, BfioCk. 1987, Toxic coraponents of motor vehicle exaissions for the spruce Pcic;a abies. Environ. pollut. 48:235-243.

C pj (--,Environmen.tal Pollution 48 (1987) 235-243



## Toxic Components of Motor Vehicle Emissions for the

Spruce Picea ables
H. Kaln7nerbauer,° H. Selinger, b R. R6inmelt,° A. Ziegler-Jons, b D. Knoppik' & B. Hocka* °Department of Botany, 6 Department of Physics, Technical University of Munich,

D-8050 Freising 12, Federal Republic of Germany (Received 17 February 1987; accepted 15 June 1987)

ABSTRACTSix-year-old Nortivay spruce trees were exposed for 30 nzin under standardised conditions to the exhaust fi•om an Otto engine running on leadfree petrol. Gas-exchange measurements in an open system using an infr

- ar•ed gas analyser showed a sudden, sevel•e drop in CO z assimilation. an .d transpiration rates. By using filters which absor

- bed differen tfi

- actions of the exhaust it could be dernonstrated that the toxic effects can be attr

- ibuted to the NO X fraction.



### INTRODUCTION The toxic effects of motor vehicle emissions on Norway spruce Picea abies have been investigated by Kammerbauer et al. (1986). For this purpose, a standard set-up with a five-cylinder Otto engine running on lead-free petrol was used. It produced emission loads which occur at speeds of c. 120 km h -1 in the fifth gear. A sudden drop in photosynthetic capacity and an impairment of stomatal regulation in spruce was observed after a fumigation of only 15 min, followed by changes in colour and, finally, by needle dropping. The visible symptoms at the shoot depended upon the developmental stage of the trees. If fumigation was performed in the spring, before sprouting, or even during early stages of sprouting, the buds were not * To whom all correspondence should be addressed. 235236 H. Kammerbauer, H. Selinger, R. Romnlelt, A. Ziegler-Jons, D. Knoppilc, B. Hock 

### visibly impaired and continued to develop normally. The twigs which grew were green with brown inner needles. Treatment, later in the season, however, led to a browning of the young shoots. No such symptoms were observed when a catalytic converter was added, which reduced NO c , hydrocarbons and CO to a tenth of the original concentrations. It was speculated that the toxic effects could be attributed to the NO Y fraction. By using filters which absorb various fractions of exhaust we can show that it is most likely the NO Y fraction which is acutely toxic to Norway spruce.

MATERIALS AND METHODS The experimental arrangement for fiimigation has been described in detail ; elsewhere (Kammerbauer et al., 

### 1986). Essentially, a five-cylinder Otto

engine was run on lead-free petrol under the load of an eddy-current brake to simulate conditions which occur at speeds of c. 120 km h -1 . The emissions
; 

### were cooled to room temperature, passed through filters (cf. 

### below) and
; 

### finally piped into the exposure chamber with the trees (6-year-old clones of

No. 814, Provenienz 84019 of the Bayerische Landesanstalt fi.ir Forstliche ' Saat- und Pflanzenzucht, Teisendorf), where gas samples were also taken for I

. 

### NO

.

,, hydrocarbon and CO analyses. The gas exchange was measured in an open system using an infrared gas analyser one day before and 3 h after I fumigation, as described elsewhere (Kammerbauer et al., 

### 1986). The gas ; exchange parameters V

~ m ~ X 

### and JmaX were calculated as described by Selinger i et al. (1987). 

### ! Filters were used to reduce the hydrocarbon and NO x concentrations (Table 1) and were mounted between the cooling site and the flow meter. ; Figure 1 shows the construction of the hydrocarbon filter. The charcoal

,. 

### i collar was made of two nesting cylinders of perforated galvanized metal

sheets which were joined at the bottom by a plate to form a stable unit. The space between the two cylinders was filled with active charcoal (Fa. Camfil, i D-2067 Reinfeld) and packed down with a vibrator. The collar was mounted

TABLE 1 Reduction of Exhaust Emissions by the Use of Filter Devices Filter• type NO

. , Hyclrocarbons CO (Pprll) (ppnn) (vol. %)
Activated chai

- coal filter 650 120 2•2 Chemisorbant filter 50 170 1•0 Control (without filter) 650 230 2•0

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### , ; __.__

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### .........

~ 1,
•e

C~ s 

### y 

### s

:1

~

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### i 

### f

C 1

i Toxic conzponents of motor vehicle emissions for Picea abies 237

inlet

activated charcoal perforated sheet metal cylinder

—• outlet

outer wall

Fig. 1. Activated charcoal filter (longitudinal section) for adsorbing hydrocat

- bons from exhaust emissions.

outlet

prefilter Fflg. 2. Chemisorbant f lter - for oxidising NO x in exhaust emissions.238 H. Kammerbauer, H. Selinger, R. Rommelt, A. Ziegler-J6tis, D. Knoppik, B. Hock 

### inside a plastic cylinder with an inlet for the exhaust and an outlet for the filtered gases. The NO x concentrations were reduced with a chemisorbant filter (ATEXLabor-Aircleaner, type LAC 3 s, ATEX-Filter Co., D-5830 Schwelm). It consisted of the following parts (Fig. 2): one Macrofalt M 45-2-36 prefilter, two chelnisorbant filters with PM-12-DP modules and one Macrofalt M 45-2-36 after-filter. The filter modules were tightly packed with PurafilChemisorbant II medium (Purafil Inc., Atlanta, USA), which consisted of activated aluminium oxide impregnated with 4% (w/v) potassium permanganate. During the oxidation of NO x to nitrate, the permanganate was reduced to MnO z . The optimal reactions were obtained at temperatures up to 40°C, a relative humidity between 15 and 95% and a holding time of at least 0•2 s. Particulate components were removed froin the exhaust by the cellulose prefilter.

RESULTS Trees were exposed to inotor emissions before sprouting (April 1986) for 30 min in the presence and absence of filters reducing different fractions of the emissions. Figure 3 shows representative measurements for the CO Z assimilation and transpiration rates obtained from single plants. The results of calculations for several gas exchange parameters, derived from four independent measurements, are given as percentage changes in Table 2. The controls were kept under the same conditions as the fumigated plants, but not exposed to the exhaust gases. No significant changes in the transpiration rate could be noted over the observation period (Table 2, Fig. 3a,b). However, when the trees were exposed to unfiltered exhaust einissions, a sudden and dramatic reduction in the CO Z assimilation (A350, A,,,

~ 

### tX) and transpiration (T350) rates was observed (Table 2). The changes were in the same range as those seen in earlier experiments (Kammerbauer et al., 1986), even though a different clone of Norway spruce was used and the fumigations were carried out at an earlier point in the season. This also holds true for the parameters which were computed from the CO 2 response curve, V ': ,,,., v which depends upon the ribulose bisphosphate carboxylase (RuBP caroxylase) activity, and J

~ 

### ,,., X , which is limited by the rate of photosynthetic electron transport. Similar dramatic changes were. observed when the fumigation was performed in the presence of an activated charcoal filter, which reduced the proportion oChydrocarbons to 52% by adsorption, but left the NO,. and CO concentrations essentially unchanged (Table 1). The decrease in the transpiration rate, the rise in the compensation point, and the loss of~

Toxic components oJ'inotor uehicle emissiazs for Picea abies 239

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-50 1 i i i i 11 i i i i 1 0 0 500 1000 1500 2000 0 500 1000 1500 2000 Intercellular CO2 partial pressure (μbar) Fig. 3. Rate of CO, assimilation, A(a, c; e) and rate of transpiration, T(b,d,f) versus intercellular CO, partial pressure measured 1 day before treatment (- -Q- -Q-) and 3 h after treatment (—G—tm'—). a,b, Control; c,d, tunligation in the presence of a chenzisorbant filter; e,f, fumigation in the presence of an activated charcoal filter. Arrows indicate net photosynthesis rates under normal atmospheric conditions (330 hbar CO,). 

### stomatal regulatory ability should be noticed, although no visible symptoms could be observed in this clone, in contrast to earlier experiments with other, more sensitive clones. Nevertheless, even here there was practically no recovery with respect to photosynthesis and transpiration when single trees were measured again o'iae month later. Wheirn the proportion of NO Y was reduced to 8% by means of a chemisorbant filter, no major differences from the controls (without fuznigation) could be observed. The photosynthetic and transpirational240 H. Karntnerbattet•, H. Selinger, R. Rommelt, A. Ziegler-J6ns, D. Knoppik, B. Hock

TABLE 2 Effect of Fumigation in the Presence and Absence of Filters on the Gas Exchange of Norway

Spruce (% Change compared to the values before fumigation.) 

### I Coiztrol Funiigation Fcunigation in the vvithout fzlter presettce of an (%) activated

charconl flter (%) Fumigation iii the

preserice of a c1leinisorbant filter (%) Measured parameters A 350 —0+5 —100+7 —87±5 4+1 A max +4±3 —68±8 —70±11 4-I-2
T350 —0±11 —87±15 —73±3 4±2 I' —9+3 +255+55 +670±260 5±2 gn (200)/ga (> 2000) —13 ± 6 — 66 ± 18 — 50 ± 6 23 ± 5 Calculated parameters V °m°x 4+6 —86+21 —75±14 5±2 J m°x 6-I- 5 — 66 ± 10 — 61 -I- 13 3-I- 4 The data were obtained from four independent experiments and are given as ineans + standard errors. A350+ Am°x; net; CO,,assimilation rate at ambient CO, partial pressure (350 Febar) and at saturating CO, partial pressure (>2000 μbar), respectively. T350: transpiration rate at ambient CO Z partial pressure (350μbar); I': CO, compensation point: ga (200), g;, (>2000): stomatal conductance at 200μbar and at >2000μbar CO, partial pressure, respectively; g; (200)/g; (>2000): stomatal regulatory ability. V°m°x: maximal carboxylation capacity; J, n;
, x : maximal ribulose bisphosphate regeneration capacity. 

### parameters remained essentially unchanged. Only the stomatal regulatory ability was increased by this treatment. Although the hydrocarbon and CO concentrations were also reduced to 74% and 50%, respectively, by this procedure, there are compelling reasons for attributing the absence of toxic effects to the reduction of NO x . This was suspected as soon as a motor adjustment which reduced NO X concentrations (90 ppm (- μl litre -1 )), but left high concentrations of hydrocarbons (250ppm) and CO (1•5vol.%), produced less toxic effects than when NO x concentrations relnained high (Kammerbauer 

### et al., 1986). On the other hand, the CO concentration r.anges applied in our experiments do not have lasting toxic effects on plants. It is taken up, oxidised in darkness to CO 2 and released, whereas in the light, it is incorporated into serine glycine and subsequently into sucrose (Bidwell & Bebee, 1974). These authors observed a recovery time of 60 to 90 min in air, after a 15 to 20 min exposure to 99 % CO for a variety of plants.

DISCUSSION Decreasing individual components in motor emissions made it evident that the NO„ fraction was responsible for the bulk of the toxic effects on Norway spruce. Nitrogen oxides arise during the high-temperature combustionToxic cornponents of rnotor oehicle emissions for Picea abies 241 

### processes, for the 7nost part as a result of the combination of atmospheric Y I oxygen and nitrogen. This process produces mainly NO, which is then

; oxidised at decreasing temperatures to NO 2 . The latter accounted for

20-40% of the total nitrogen oxides under our experimental conditions in the exposition chambers. Nitric oxide and NO 2 react at high partial pressures with water to form nitrite and nitrate according to the equations

NO + NO 2 "—'O) 2 HONO 2 NO 2 '"

— ' 0 ,- 

### HONO +'H 3 0 + + NO3 (cf. 

### Finlayson-Pitts & Pitts, 1986). The rate drops rapidly as the concentrations approach ambient levels. ; However, surface-catalyzed reactions, such as those occurring in plants, can

increase the rates. The importance of NO x as a pollutant has often been overlooked (cf. 

### Bell, 1986). Although toxicological studies were usually performed in combin-

- ation with other pollutants, such asSO Z or 0 3 , investigations dealing z exclusively with NO, in plants were carried out as early as 20 years ago

(Taylor & Eaton, 1966).
t 

### Small doses usually stimulate growth, because NO 2 is then used as a

nitrogen source. The resulting nitrite activates leaf nitrite reductase, which detoxifies the compound by reducing it to ammoniuin. This in turn is

~ 

### incorporated into amino acids (Rogers & Campbell, 1979; Yoneyama &

~ 

### Sasakawa,1979; Wellburn et a1.,1980). 

### Whereas loads of 62 ppb (- nl litre -1 ) ; caused an increase in shoot dry weight in some broad-leaf trees (21-week

exposure in Tilia cordata, 60-week exposure in Alnus incana; Freer-Smith, 1984), the conifer Pinus sylvestris exhibited a decrease in relative growth t ; rate after an 86-week exposure with the same concentration (Freer-Smith,

1985, cited in Bell, 1986). The ftimigation of 2-year-old Pinus strobus with
1 

### ; 0 1 ppm NO 2 for 4 h day -1 , over a period of 35 days, diminished the dry ; ' matter of needles and produced necrotic changes, whereas no symptoms ; were observed below 0•05 ppm (Yang et al., 1982). In most plants, how- ; ever, the threshold to chronic injuries is estimated to be c. 0•17 ppm

(Lichtenthaler, 1984). Above 3-4 ppm NO Z , the range of acute toxicity is

~ 

### reached (Kato et al., 1974; Yoneyama et al., 1979).

Injury by NO x is not restricted to a single biochemical reaction. The impairment of the net assimilation rate is assumed to originate, in part, in its action as an acid gas, which destroys the proton gradient across the thylacoid tneinbranes in the chloroplasts (Lichtenthaler & Buschmaml, 1984). Ultrastructural studies by Wellburn et al. (1972) demonstrated the swelling of thylacoids by NO 2 , indicating a disturbance of the ionic balance and ATP synthesis. As a radical, NO z initiates lipid peroxidation and i therefore leads to membrane destruction (Heath, 1980; Elstner, 1984). i242 H. KammerGaater, H. Selinger, R. Ronzmelt, A. Ziegler-Jons, D. Knoppik, B. Hock 

### The closure of stomata in response to motor emissions causes a decrease in transpiration and CO 2uptake. This is most likely due to NO 2 . A similar effect was recognised back in the 1950s by Koritz & Went (1953) as a response to the oxidative components of smog. Consequently, it is also characteristic of the action of ozone (cf. 

### summary by Dugger & Ting, 1970) and S0 2 (Majernik & Mansfield,1972). Stomatal closure, which is due to the loss of turgor pressure in the guard cells, is interpreted as the consequence of an impairment of inembrane functions causing the leaching of ions across the plasmalemma. Our experiments with motor emissions and individual fractions were in the range of acute toxicity. Of course, it is not possible to extrapolate from these data to the influence of ambient concentrations on plants. Besides the lower concentrations, the cumulative effects of other pollutants, which often work synergistically (cf. 

### Landolt & Keller, 1985), inust be considered. The formation of secondary air polh.itants in the presence of ozone and light must also be taken into account, since these lead to the production of yet other strong oxidants (Fuhrer, 1985). Therefore, special attention should be given to ways of reversing the present rise in NO x emissions.

ACKNOWLEDGEMENTS This work was supported by a grant from the Bayerisches Staatsministerium fiir Unterricht und Kultus to Professor Hock. The fumigation experiments were carried out at the Institute fur Verbrennungsmaschinen of the Technical University of Munich. We are indebted to Dr K. Zeilinger and Dipl.-Ing. H. J. Riedl for their technical help with the experiments.


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