NOTES
Use of Indicator Plants to Evaluate Atmospheric Levels of Nitrogen Dioxide in the Vicinity of a Chemical Plant Alexander E. Donagi and Ayana 1. Goren* Research Institute for Environmental Health, Sackler School of Medicine, Tel-Aviv University, Tel Aviv, Israel
A combination of engineering design, stack sampling, environmental monitoring, and biological indicators was used to solve a serious public health problem resulting from a NO2-emitting chemical plant. Indicator plants (Phaseolus uulgaris) were placed a t distances of about 600 m from the source in 8 different directions. After 4 days of exposure, necrotic lesions appeared on the mature leaves of the Phaseolus vulgaris plants at all stations. Good correlation between the duration of wind blowing from the source toward the plant site and the extent of injury (percent of injured leaves and percent of leaf area injured) observed after 2 to 3 weeks exposure a t this site could be found. Studies concerning the effects of NO2 on plants are quite rare; most have dealt with acute damage caused as a result of chamber fumigations with as high as 50 ppm of NO2 ( I ) . Other investigators reported about injury as a result of chamber exposures to NO2 concentrations lower than the earlier studies by one order of magnitude ( 2 4 ) .Four successive fumigations with 0.16 ppm of NO2 for 6 h each caused typical necrotic lesions on alfalfa leaves ( 4 ) . Long exposures to NO2 concentrations of 0.3-0.4 ppm caused significant growth suppression to tomatoes and pinto beans ( 3 ) and reduced fruit yield of orange trees ( 5 ) . Tingey et al. (6) and Bennett et al. ( 7 ) reported synergistic effects of the phytotoxic pollutants SO? and NO2 (in concentrations less than 0.5 ppm each) on plants exposed in growth chambers. Nitrogen dioxide damage to plants in the field has been observed around acid-producing plants in Germany (8),in the United States (9),and in Italy (10).Ambient levels of NO, up to 0.585 ppm and SO2 up to 0.690 ppm measured in the vicinity of a polluting source caused acute injury symptoms to Eastern White Pine seedlings (11). Studies reported in the literature (12) indicate that the common NO2 concentrations in the ambient air are unlikely to cause visible plant injury in the field, excluding cases of accidental releases from industries, where this gas is involved in certain processes. However, the fact that cumulative effects are caused by successive fumigations with low NO2 concentrations and the synergistic interaction between low NO2 and SO2 concentrations indicates that NO2 may be a more important phytotoxicant than was previously thought. Darley (13) described, for example, up to 30% growth reduction in tomatoes exposed to ambient concentrations of NO2 in Riverside, Calif. Close proximity of residential zones to a fairly big NOn emitting industrial plant in the sea shore region of Israel has been the reason for numerous citizens’ complaints regarding odor nuisances, headaches, and breathlessness. Until July 1977 only two small absorption columns using Raschig rings filling and water as a scrubbing liquid were used for elimination of nitrogen oxides from the stack gases of the plant. In August 1977, two additional columns were installed and connected in series to the old ones, and the filling of all the columns was changed to stainless steel Pallrings, thus obtaining improved absorption capability. Only after changing the absorbing liquid in the last column to Has04 was the 986
Environmental Science & Technology
problem of air pollution from this plant practically solved. This fact was confirmed by environmental monitoring and stack sampling, which were carried out by the health authorities throughout the different stages of the development of engineering solutions for elimination of the NO? emission from this plant. The ambient air monitoring was carried out a t that direction where there were the most complaints. Because of the relative scarcity of continuous NO? monitors and their high cost, it was proposed to expose sensitive indicator plants around the polluting source, in order to have a cumulative indicator for injurious NO2 concentrations a t different parts of the community. Due to the fact that the injury threshold of plants to NO2 is relatively high, the injury signs per se may also serve as an indication for eventual hazardous effects to the surrounding population. Experimental Stack sampling was done according to the EPA recommended method for determination of nitrogen oxide emission from stationary sources (14). Environmental monitoring was carried out using a Thermo-Electron-Corporation chemiluminescent NO, analyzer, which was placed a t the vicinity of site 4 (Figure 1).Continuous monitoring was performed during three time periods (6124-7/31/77; 8115-9/22/77; 1211-121 31/77), which represented the three subsequent stages of the development of engineering solutions. The exposure of the indicator plants was carried out during June and July 1977, while only two absorption columns were operating and stack levels of NO2 were extremely high. I
I
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/
/
Residential I
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4\ \ /
Industrial Zone
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Figure 1. Location of
the sampling stations around the plant
0013-936X/79/0913-0986$01.00/0 @ 1979 American
Chemical Society
Table 1. Stack Sampling and Environmental Monitoring NO2 Data for the Various Stages of the Pollution Abatement System in the Plant 2 absorption columnsa
slack sampling ___ concn.
dale
LSI. peak
112-h
ppm
22000 34000 23 000
6/26/77 8/27/77 8/28/77 6I 29177 7/6/77 7/9/77 7/10/77 7. /. 1.1./.7.7. 7/11/77 7/13/77 7/23/77
1.6 2.2 2.2 2.8 1.6 2.2 1.2 4.4 1.5 1.2
1.1 2.0 2.0 1.0 0.5 1.0 0.6 1.0 1.0 0.9
dale
Feb 1976
envlron monitoring EOIIEII, ppm
~
June 32000 1976 28000 30 000
Water as a Scrubbing liquid.
-
4 absorption c o l ~ m n s ~ environ monitoring slack sampling concn, ppm COOEll. inst. 112-h dale peak peak dale ppm
_____
peak
Oct 1977
16 000 14 000 10 500 15 000
8/24/77 8/25/77 8126 I 77 8128I 77 8130I 77 9/1/77 9/2/77 9/4/77 9/5/77 9/6/77 9/7/77 9/10/77 9/12/77 9/16/77 9/17/77 9/18/77 9/19/77
1.2 0.6 0.6 1.4 1.8 0.6 1.0
0.6 0.5
4 absorption soiumnsb ~
slack sampling concn. dale ppm
Nov 1977
2000 2500 5000 4000 3000
envlron monitoring concn, ppm ihrl. 112-h dale peak peak
12/6/77 12/7/77 12/6/77 12/10/77
0.7 0.6 0.5 0.7
0.3 0.4 0.5
0.5 0.5
0.8 1.9 0.8 1.8
1.2 0.6 0.8
Scrubbing liquids: first three columns. water: fowth co
Phaseolus vulgaris was chosen as the indicator plant for NO?, because of its relatively low injury threshold as well as its rapid response to high NO2 concentrations (15).Seeds of Phaseolus vulgaris were swelled in a dark chamber and transferred to pots (30 X 40 X 10 cm) containing vermiculite, after 5 days. Ten plants per pot were grown in a growth chamber, a t a day temperature of 27 f 1 "C and a night temperature of 17 f 1 "C. The day length was 12 hand light intensity 600 ft-c. Plants were irrigated throughout the experiment with half-strength Hoagland's nutrient solution. At the age of 3.5 weeks, the plants were placed a t distances of about 600 m from the source in eight different directions (Figure 1).Only stations 5 and 8 were located a t a distance of 500 and 700 m, respectively, from the source. For control purposes an identical pot with 10 plants, which were grown "IIUC'
"_--_
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university campus, was used. The plants were irrigated every 2 dlays, and app,earance of injury symptoms was recorded throiighout the ex periment. P1 leaves, and indexes Measurements of plant height, number 01 of injury were taken weekly. The following indexes of injury were determined percentage of injured plants, percentage of injured leaves, and percentage of leaf area injured (estimation of necrotic area, as a percentage of total leaf area). At the end of the experiment, fresh and dry weights of leaves, stems, and roots were determined, and the area of the leaves of every plant was measured. mnmlnoiral Wind direction data were obtained from a metc...-.va.v... station in the vicinity of the factory. Possible correlations between the cumulative duration of wind blowing toward the different stations (f12.5" from every station) and the extent of injury on the plants exposed a t them were investigated. For statistical evaluation of the results. t tests. one-wav WIare
used.
Rt
ination oFNO2 emissions. Stack samplingdata and extremely
Figure 2. Injury symptoms on mature following 4 days of exposure, at site 2
leaves of
Phaseolus vulgaris
.-'
high instantaneous and half-honrl) _.. ...V.....-..ll .--~" are given in Table I. The environmental monitoring that was carried out while only two columns were used-the stage through which the field exposure of the indicator plants was carried o u t s h o w e d very high peak values (Table I). The half-hourly Israeli standard (relevant for 99%of the time) of 0.5 ppm of NO? and even the half-hourly standard for 100% of the time (1.0 ppm of NO?) were quite often exceeded. After 4 days of exposure, necrotic lesions (Figure 2) of various extents appeared along the margins of the mature leaves and between the veins, in all of the stations. t tests that were conducted showed that in all 8 exposure stations and during all of the observations the injury was highly significant ( a 6 0.005). No injury symptoms appeared in the control plants throughout the whole experiment. A one-way analysis of variance for evaluating the injury on the leaves a t the end of the experiment showed that there were significant differences in the extent of the injury among the eight exposure stations. The level of significance that was ~
Volume 13, Number 8, August 1979
987
50
i
6o
I
2o
0
1I
y: 4.71 r0.10x 20
I
I
,
9
I
I
I
I
l0
20
30
40
50
60
70
80
,
90
0
i I o
,
100
Duration of wind blowing towards stations,hr.
1 0
10
20
30
40
50
60
70
80
90
100
PERCENTAGE OF INJURED LEAVES
0 PERCENTAGE
OF LEAVES’AREA INJURED
Figure 3. Correlation between cumulative duration of wind blowing in a certain direction and plant injury indexes, after 2 weeks of exposure
found for the percentage of injured leaves was 0.002, and for the percentage of leaves’ area injured it was 0.01. A multiple range test showed that there were no differences in the percentage of injured leaves and in the percentage of leaves’ area injured, in the plants exposed a t sites 1, 2,3,4, and 8. Investigation of the variance of most of the various growth parameters (plants’ height during the exposure, and fresh and dry weights a t the end of the experiment) showed that there were no significant differences among the stations. However, a highly significant difference among the exposure stations could be found, a t the end of the experiment, only for the total area of the leaves (a: 6 0.000 1).A multiple range test that was done in regard to the area of the leaves a t the end of the experiment showed that in sites 1, 2, and 4 there were no differences in the area of the leaves. At these stations, the area of the leaves was considerably smaller than a t the other ones. This stems from the high injury extent and, as a result of it, the severe leaf drop (mainly at the end of the experiment) a t these stations. As can be seen from Figures 3 and 4, a good correlation (T = 0.7; T = 0.8) exists between the cumulative duration of wind blowing in a certain direction and various indexes of plant injury (percentage of injured leaves and percentage of leaves’ area injured), as calculated for all the stations. Appropriate correlation coefficients for 2 and 3 weeks of exposure were calculated and regression lines were calculated and drawn.
Discussion Effects of NO2 on growth parameters are not specific and, as can be seen from the results, most of them also do not reflect significant differences among the stations. Out of the various growth parameters investigated, only the leaf area-which is directly influenced by the pollutant-exhibits significant differences in the different exposure sites. Total leaf area was measured only a t the end of the experiment and therefore gave but restricted information about the effects of NO*. On the other hand, injury symptoms, which are specific to NOz, vary significantly in their extent in different exposure sites. The extent of injury was estimated throughout the whole experiment and gave a continuous cumulative measurement of NOz. Of all the parameters of plant response measured, only the percent of leaves injured and the percent of area injured cor988
i
Environmental Science & Technology
Duration of wind blowing towards stations, hr. 0
PERCENTAGE OF INJURED LEAVES
0 PERCENTAGE OF IEAVES’ AREA INJURED
Figure 4. Correlation between cumulative duration of wind blowing in a certain direction and plant injury indexes, after 3 weeks of exposure
related significantly with the duration of wind and presumably with NO2 dosage. One can divide the eight exposure stations in this experiment into two groups, according to the injury extent observed on the plants exposed in them: (a) highly polluted sites, 1,2,3,4,and 8 (station 8 was somewhat farther from the source), and (b) polluted sites, 5,6, and 7 (station 5 was somewhat nearer to the source). As a matter of fact, sites 1 , 2 , 3 , 4 ,and 8 are the sites of the most frequent wind directions, 1, 2, and 8 during daytime and 3 and 4 during nighttime. Since the factory is located in a rural region, it constitutes the single source for air pollution in the experimental area. Actual field monitoring a t the vicinity of the factory showed that no other air pollutant other than NO2 reached a level beyond the usual background. Synergistic effects with other pollutants can therefore be excluded, and all the injury symptoms should be attributed to NO2 alone. This experiment demonstrates the success of using Phaseolus vulgaris as an indicator plant for cumulative monitoring of NO2 in the vicinity of polluting sources. In the present study, a successful combination of engineering design, stack sampling, and environmental monitoring, as well as biological monitoring, assisted in solving a serious public health problem.
Acknowledgment The authors express their appreciation to Mrs. Ida Kukliansky for her assistance in the data processing. Literature Cited (1) Benedict, H. M., Breen, W. H., Proc. N a t l . Air Pollut. Symp., 3rd
(1955). ( 2 ) Middleton, J. T., Darley, E. F., Brewer, R. F., J . Air Pollut. Control Assoc., 8, 9-15 (1958). ( 3 ) Taylor, 0. C., Eaton, E. M., Plant Physiol., 41,132-5 (1966). (4) Tingey, D. T., M.A. Thesis, Department of Botany, University of Utah, Salt Lake City, 1968. (5) Thompson, C. R., Hensel, E. G., Kats, G., Taylor, 0. C., Atmos. Enuiron., 4, 349-55 (1970). 16) Tingev. D. T.. Reinert, R. A,, Dunning, J., Heck, W. W., Phytop a t h c h g y , 61,1506 (1971). (7) Bennett, J. H., Hill, A. C., Soleimani, A,, Edwards, W. H., Enuircn. Pollut., 9,127-32 (1975).
(8) Berge, H., in “Phytotoxische Immissionen”, Paul Parey Verlag,
Berlin. 1963. (9) Thomas, M. D., Annu. Reu. Plant Physiol., 2,293-322 (1951). (10) Janone, G., Humus, 10,17-9 (1954). (11) Skelly, J. M., Moore, L. D., Stone, L. L., Plant Dis. Rep., 56,3-6 (1972). (12) Taylor. 0. C., Adu. Chem. Ser., No. 122 (1973). (13) Darley, E. F., Air Pollut., Proc. Eur. Congr., lst, 137-42 (1968).
(14) Code of Federal Regulations 40, Protection of Environment,
131-2, US. Government Printing Office, Washington, D.C., 1972. (15) Taylor, 0. C., Maclean, D. C., in “Recogqition of Air Pollution Injury to Vegetation: A Pictorial Atlas”, Jacobson, J. S., Hill, A. C., Eds., Air Pollution Control Association, Pittsburg, Pa., 1970.
Received for review July 28,1978. Accepted February 21,1979.
Microbial Degradation of Organic Compounds at Trace Levels Robert S. Boethling‘ and Martin Alexander* Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, N.Y. 14853
A sensitive method was developed for measuring the biodegradation of organic chemicals based on the formation of “TO2 from I4C-labeled compounds. It was shown that glucose at an initial concentration of 18 ng/L was degraded by microorganisms in culture a t rates well below those predicted by Michaelis-Menten kinetics from the degradation rates at higher glucose levels. The rate of glucose degradation a t 18 ng/L was affected by the density of the bacterial population. T h e maximum rate of biodegradation of dimethylamine, diethylamine, and diethanolamine added t o stream water was proportional to the initial amine concentration over a range of concentrations from several nanograms t o several milligrams per liter. In recent years, the production and use of synthetic organic chemicals have risen dramatically, and concomitantly the discharge of these chemicals into diverse ecosystems, both aquatic and terrestrial, has increased. The number and the identities of many of the substrates introduced into natural waters and soils are unknown, but knowledge of the activities o f society and environmental monitoring ( I ) suggest that the number and diversity of organic pollutants are large. Unfortunately, few generalizations exist to explain why some of these chemicals are biodegradable and disappear rapidly, whereas others are not subject to rapid microbial degradation or are not attacked at all. Concentration is one factor t h a t may govern the susceptibility of organic chemicals to microbial destruction in nature. Surprisingly, only a few studies on the effect of a wide range of concentrations on the persistence of organic chemicals have been reported ( 2 , 3). One reason for this scarcity of experimental data may be the lack of sensitivity of many techniques commonly used for the detection and quantification of organic compounds a t trace levels. The present study was designed t o assess the effect of a wide range of concentrations of biodegradable chemicals on the rate of their mineralization, Le., complete conversion to inorganic products. A sensitive method involving lT-labeled compounds was used for this purpose.
Materials and Methods Pseudomonas sp. was obtained from a n enrichment containing glucose, glycerol, and succinate as carbon sources ( 4 ) . Surface water samples were taken aseptically from Fall Creek in central New York and processed within 2 h of collection. All samples were analyzed for p H and total alkalinity by standard methods ( 5 ) .The water was amended with a n inorPres;;
address, Biocentrics, 480 Democrat Rd.. Gibbstown. N J .
08027. 0013-936X/79/0913-0989$01.00/0
ganic nutrient supplement (6)modified as follows: NaNO3 and NaHC0:j were omitted, Zn was added a t the same molarity as ZnSOCH20 rather than ZnClp, and NH4Cl was added a t 8.0 mg/L. Microbial populations were enumerated by plating appropriate dilutions in triplicate on nutrient agar containing 20 m M D-glUCOSe. T h e stream water contained 8-100 X lo3 bacterial cells/mL at the time of sampling. Microbial activity was monitored by measurement of I4CO2 produced from l4C-1abeled organic chemicals. AxeniC cultures of bacteria and stream water were incubated in 500-mL flasks that were modified to permit flushing with Nz subsequent to t h e incubation period. After specified periods of time, the contents of the flasks were acidified by adding 12 N HzS04 t o a final concentration of 0.12 N, the flasks were connected to a source of N2,and the COz in t h e flasks was flushed out and trapped in ethanolamine. If necessary, NaHC03 was added immediately before acidification to raise the concentration of bicarbonate plus carbonate t o at least 1.0 mM. The experimental flasks contained 100 or 200 mL of medium and were incubated a t 29 “C in the dark t o minimize photodecomposition of added organic chemicals and fixation by photosynthetic microorganisms of the I4CO2released from the degradation of the added 14C-labeledcompound. Axenic cultures were incubated on a rotary shaker operating a t 140 rpm, but flasks containing stream water were incubated without shaking. Measurements of dissolved oxygen ( 5 ) showed t h a t the medium remained aerobic. Samples (1.0 g) of ethanolamine containing trapped 14C02were added to 9 m L of scintillation fluid ( 7 ) ,and the radioactivity was measured in a Beckman LS-1OOC liquid scintillation spectrometer (Beckman Instruments, Fullerton, Calif.). The data were corrected for quench by the external standard/channels ratio method and expressed as disintegrations per minute (dpm). T h e I4C-labeled chemicals used were not sufficiently volatile to be trapped in ethanolamine after acidification of the medium, they were not detectably sorbed at microgramhiter levels t o particulate matter in the media or to the incubation vessels, and they were not converted to 14C02or other volatile products in autoclaved media. Pseudomonas sp. was grown in a mineral salts medium amended with 5 mM D-glucose to prepare inocula for biodegradation experiments or with the desired initial level of glucose in experiments assessing the biodegradability of low concentrations of this substrate. Growth of the cultures used as inocula was monitored spectrophotometrically a t 540 nm, and the cells were collected on 0.45-pm membrane filters when the cultures reached the late logarithmic phase of growth. The cells were washed with basal medium, resuspended in fresh medium without added glucose, and transferred to flasks
@ 1979 American Chemical Society
Volume 13, Number 8, August 1979
989
