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Determination of Phytotoxicity of Several Volatile Organic Compounds by Investigating the Germination Pattern of Tobacco Pollen
J Plant Physiol. Vol. 145. pp. 514-518 (1995)
Determination of Phytotoxicity of Several Volatile Organic Compounds by Investigating the Germination Pattern of Tobacco Pollen* UTE SCHUBERT, LINDA WISANOWSKY,
and ULRICH KULL
Biologisches Institut der Universitat Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany Received February 1, 1994 . Accepted May 30, 1994
Summary
Hydrated pollen of Nicotiana tabacum L. (var. xanthi nc) was exposed to different concentrations of several volatile organic compounds. The germination rate of the pollen was determined by applying a modified test procedure originally developed by Kappler and Kristen (1990). A stimulation of pollen germination resulting through a rise in pC02 was avoided by using a short exposure period. In the series of chlorinated ethanes, the ED50 (and ED25) values decrease with increasing number of chlorine atoms, which are correlated to a rise of the octanol-water partition coefficient. For the chlorinated ethenes a similar effect is probable, but less obvious. The toxicity of compounds with the same number of chlorine atoms is higher for ethane derivatives than for derivatives of ethene. The ED50 and ED25 values are compared with threshold values of physiological effects obtained after the application of 1,1, I-trichloroethane for 24h to tobacco leaves. We presume that chlorinated hydrocarbons may cause stress even at lower concentrations than those suggested by the pollen test. Therefore the listed ED values (Table 1) should be considered as maximum tolerable values regarding phytotoxic effects in experimental investigations. For the important solvent 1,1, I-trichloroethane the ED 25 value of phytotoxicity is lower than 4 mg/L; according to literature the concentration supposedly having no effeet on man is 1.9 - 5.2 mg/L.
Key words: Nicotiana tabacum L., air pollutants, aromatic hydrocarbons, chlorinated hydrocarbons, pollen germination, 1,1, I-trichloroethane. Abbreviations: EDso = 50 % effective dose; ED25 bon dioxide. Introduction
Pollen germination and pollen tube growth are affected by air pollutants in vivo and in vitro, however, the response is more striking in vitro (Wolters and Martens 1987, Pfahler 1992). From their data these authors concluded that pollen under standardized conditions may be used as sensitive biological indicators of pollution. Simple pollen test systems were developed by Kappler and Kristen (1987, 1990) and " Dedicated to Prof. Dr. H. K. Lichtenthaler on the occasion of his 60th birthday. © 1995 by Gustav Fischer Verlag, Stuttgart
= 25 % effective dose; pC02 =
partial pressure of car-
for pollutants contained in water - by Hoffmann et al. (1990) respectively. In both tests the toxic substance is dissolved in the germination medium. These test methods alIowa comparison of non volatile as well as volatile substances, although being rather unphysiological for volatile substances. Many volatile organic compounds (VOCs) are - at least locally - important air pollutants. As considered already by Lichtenthaler and Buschmann (1984), organic halogen compounds may be secondary effectors of tree damage. However, few data concerning their phytotoxicity is available (Schulze and Stix, 1990). Some substances were investigated
Phytotoxicity of volatile organic compounds by Masaru et al. (1976), Meyberg et al. (1987) and Kappler and Kristen (1990). Since volatile compounds under natural conditions reach the pollen as gases and many of them are rather insoluble in water we have established a modified test procedure measuring the germination rate of tobacco pollen. The pollen, suspended in a very thin film of germination medium, is exposed to the gaseous pollutant. The method was tested using several chlorinated hydrocarbons and some aromatic hydrocarbons. A preliminary report containing some of the results was published as an abstract (Schubert et al.
1992). Materials and Methods Plants
Plants of Nicotiana tabacum L. (var. xanthi nc) were raised under greenhouse conditions, such that enough pollen was available throughout the year. Immediately after anthesis, the pollen was directly dusted into Eppendorf-tubes and stored at -20°C until use. Only pollen from plants showing pollen viability of more than 70 % was used in the experiments. Substances tested
The chlorinated hydrocarbons tested are listed in Table 1. For comparison the toxicity of the aromatic hydrocarbons benzene, toluene and xylene (mixture of the dimethyl-benzenes) and of acetone were also investigated. All chemicals were bought from MerckDarmstadt.
515
bars in the figures indicate the standard deviations of (at least three) germination rates. The EDso value (50% effective dose) is the concentration which causes a decrease of pollen germination to 50 % of the total amount. The EDso values are calculated by linear interpolation between the two nearest concentrations. In the same wayan ED2S value can be calculated. This value is commonly used in ecotoxicology as a threshold value separating «sublethal» and «lethal» effects of a toxic substance (Padar and Angerhofer 1991).
Results The results obtained for the chlorinated hydrocarbons are shown as dose-response curves (Figs. 1- 3). The EDso and ED 2S values are given in Table 1, which lists all compounds investigated so far. Also included in Table 1 are the octanolwater partition coefficients of the compounds according to Koch (1989). All compounds show high vapour-pressure values (e.g. 1.38 x 104 Pa for trichloroethane; 6.7 x 102 Pa for tetrachloroethane). In the series of chlorinated ethanes the EDso and ED 2S values decrease with an increasing number of chlorine atoms, which is correlated with an increase of the octanol/water partition coefficient. Although less clear, a similar relationship for the chlorinated ethenes may be presumed. The toxicity of compounds with the same number of chlorine atoms is higher for saturated hydrocarbons than for derivatives of ethene. The EDso and ED 2S values for the investigated aromatic hydrocarbons are rather high and decrease with a rising substitution of the benzene ring, which in return is correlated with a rise of the octanollwater partition coefficient.
Germination test
An amount of 0.5 mg pollen is placed into 3.5 cm Petri dishes and held in a water saturated atmosphere for one hour. This imbibition procedure is advantageous for a high germination rate because it effects the hydration and thereby the reorganization of biomembranes (Heslop-Harrison et al. 1984; Hoekstra and van der WaI1988). Then, a 1 mL germination medium (as described by Kappler and Kristen 1987) is added and the pollen uniformly suspended by cautios shaking of the dishes. No tenside is applied. The concentration of pollen is about 0.5-1 x 106 mL -1. The germination medium must have room-temperature to avoid cold injuries of pollen (Hoekstra and van der Wal 1988). The Petri dishes are then put into small glasstroughs (8.5 x 5.7 x 6.7 cm) which are sealed tightly. The volatile compound is injected by a GC-syringe into the trough where it evaporates at once. As a control the same amount of water is injected into control troughs. Since drought stress would reduce the germination rate, all incubation troughs contain water saturated air. The pollen is exposed to different concentrations of the gaseous compounds at 22°C and in darkness. Final concentrations in the troughs are calculated using the applied amount and the (corrected) volumes of the glass troughs. Simultaneous controls for all concentrations of investigated pollutants are conducted. After an incubation period of 2 h the percentage of germinated pollen is counted by microscopy. The pollen is considered germinated if the pollen tube is unequivocally longer than the diameter of the pollen grain. From each Petri-dish six samples of pollen are transferred to a cell-counting chamber (Schilling-Kreuznetz), where the mean value is determined. For each concentration of a compound three separate tests are investigated. The mean values of these three tests are used to calculate a dose-response curve as described by Kappler and Kristen (1987). The inhibition rate is calculated by setting the germination rate of the control pollen (70 - 85 % of total amount) at 100 %. The
Discussion
Method Literature reports several different media for pollen germination (d. Stanley and Linskens 1985). Preliminary experiments using different media (Brewbaker and Kwak 1963, Portnoi and Horovitz 1977, Hoffmann et al. 1990) showed that the medium of Kappler and Kristen (1987) was most suitable to get high germination rates. Our method avoids a disadvantage of the test-system of Kappler and Kristen (1987, Table 1: EDso and ED 2S values based on the tobacco pollen germination test for chlorinated hydrocarbons, aromatics, and acetone. compound
EDso mg/L
1,2-dichloroethane 1, 1, I-trichloroethane 1,1,2,2-tetrachloroethane
17.1 18.1 0.055
trans-l,2-dichloroethene trichloroethene tetrachloroethene
64.0 31.7 34.3
ppm 4,170 3,270 5
mg/L
ED zs
9.2 <4 0.014
15,890 5,800 4,980
41.7 <4 12.3
19 p*
ppm 2,210 < 720 1
1.7 2.8 3.0
10,350 < 730 1,780
2.4 2.95
chloroform - trichloromethane
33
6,770
24.0
4,920
2.02
benzene toluene xylene
36.5 28.9 17.1
11,420 7,560 3,870
18.2 9.8 7.2
5,710 2,560 1,620
1.8 2.39 2.9
acetone
30.9
13,040
12.2
5,170
* Octanol-water partition coefficient according to Koch (1989).
516
UTE SCHUBERT, LINDA WISANOWSKY,
Inhibition of germination
Inhibition of germination
(% control)
100 90 80 70 60 50 40
0
(% control)
1,1,1 - trichloroethane
l
,---!/.l
30 20 10
/1
1
2
4 6 810
20
4060
100 90 1,2 - dichloroethane 80
trichloroethene
/
(
rl
*
I----f
4 6 810
2
20
~
4060
Inhibition of germination
,
i--
10 10
20
20
4060
tetrachloroethene
90 80 70 60 50 40 30 20 10 0
/r
x
*
t/ 1
)
Fig. I: Dose-response curves for several chlorinated hydrocarbons in the pollen germination test. Bars: standard deviations. The calculated ED50 values are marked by stars.
concentration (mg / dm 3)
Fig. 2: Dose-response curves for trans-I,2dichloroethane and I, 1,2,2-trichloroethane in the pollen germination test (different concentration scales!). Bars: standard deviations. The calculated ED50 values are marked by stars.
2
4 6 810
20
4060
concentration (mg / dm
3
Inhibition of germination
(% control)
trans-l,2 -dichloroethene
20
4 6 810
concentration (mg / dm 3)
100
concentration (mg / dm 3)
(% control)
100 90 80 70 60 50 40 30
2
Inhibition of germination
rI'
30 20 10
1
(% control)
1/
70 60 50 40
1
0
concentration (mg / dm 3)
Inhibition of germination
0
100 90 80 70 60 50 40 30 20 10
(% control)
0
and ULRICH KULL
4060 100 200
concentration (mg / dm 3)
100 90 80 70 60 50 40 30
20 10 0 0.001
l,l,2,2-tetrachloroethane
I-I
I
I)
0.01
1990) for volatile and especially for water-insoluble volatile compounds, for which these authors had to use dimethylsulfoxide as a solvent. Compared to the method used by Kappler and Kristen (1987, 1990) the use of a short exposure
0.1
1
period avoided a rise of the pC02 in the closed incubation system, which otherwise may cause a stimulation of pollen germination (Sfakiotakis et al. 1972). However, because we applied the VOCs as gases they have to dissolve in a thin
Phytotoxicity of volatile organic compounds
Inhibition of germination
Inhibition of germination
(% control)
100-r--------------, 90
xylene
80
Fig. 3: Dose-response curves for xylene and toluene in the pollen germination test. Bars: standard deviations. The calculated ED50 values are marked by stars.
I
0
1
2
*l
4 6 810
20
4060
concentration (mg / dm 3)
layer of the medium to reach the pollen. For the purpose of comparison, we tested some compounds already investigated by Kappler and Kristen (1990). Although the values are in good agreement for toluene (7560/7888 ppm), we found a significantly lower ED50 value for acetone (ED50 value from our test: 13,040 ppm; ED50 value of Kappler and Kristen: 20,500 ppm). The test-method described above may also be combined with the automatisable procedures of the measurement of pollen tube growth of Kappler and Kristen (1990). As found by Kappler and Kristen (1987) for their tested compounds, the dose-response curves showed steep inclines in the ED50 range. This is not true for all compounds regarding ED 25 ; therefore, in two cases no equivocal ED 25 value could be obtained. Because of the deviations of the individual measurements with very low concentrations of the VOC, it was not possible to calculate satisfactory EDlO values, which are somtimes used as threshold values of toxicity (Parlar and Angerhofer 1991).
Phytotoxicity of chlorinated hydrocarbons Some of the chlorinated hydrocarbons investigated are used as solvents and cleansers; however, there are no data on their phytotoxicity. Owing to their broad application, the concentration of such compounds could locally rise to values (Debus et al. 1989, Baumbach 1990) which according to our data (Table 1) are phytotoxic. The sensitivity of plant cells is rather high compared with the LD (lethal dose) values for mammals and man as collected by Koch (1989). To establish whether the phytotoxicity values obtained by the pollen test are well-founded, threshold doses causing effects on tobacco plant leaves are now under investigation. It is well known that photosynthesis is affected by most air pollutants in a direct or indirect manner (summarized in Schulte-Hostede et al. 1987, Lichtenthaler and Rinderle 1988, Treshow and Anderson 1989) which is perhaps a main cause of large scale forest damage (Lichtenthaler et al. 1985). For 1,1, 1-trichloroethane concentrations higher than 0.09 mg/ dm 3 applied for 24 h effects were found concerning the photosynthetic gas exchange of tobacco leaves (Schubert, unpublished). It is well known that changes in polyamine metabo-
toluene
70
1/
10
100-r--------------,
80
If
60 50 40 30 20
(% control) 90
70
517
60 50 40 30 20 10 0
1
2
J 1/1 */ 4 6 810
20
4060
concentration (mg / dm 3)
lism are caused by stress (Flores 1992). With 1,1, 1-trichloroethane, tobacco leaves show significant changes in the ratio putrescine/spermidine + spermine at concentrations higher than 0.1 mg/dm 3 applied for 24h (Haas 1992). Through concentrations effective near places of emission chlorinated hydrocarbons may therefore cause severe stress. We presume that the ED values obtained by the 2 h pollen test (Table 1) can be considered as threshold values of phytotoxic concentrations of the compounds. For 1,1, 1-trichloroethane, the concentration which is supposed to have no effects on man is 350-950ppm (1.9-5.2mg/dm 3) (Koch 1989) and in Germany the MAK value (maximum acceptable concentration at the place of work) is 1.08mg/dm3. Comparing the concentrations effecting plants and regarding the safety-factor of MAK values one may presume that the phytotoxicity is higher than toxicity for man. The same may be true for trichloroethene. The octanol-water partition coefficient is a key parameter for uptake of lipophilic organic compounds by plant cells and tissues (Paterson et al. 1990). We find a rise of phytotoxicity in each series of compounds tested correlated with an increase of the partition coefficient. From this correlation an uptake preferably through the membrane lipid bilayer may be concluded. This is also obvious for the aromatic hydrocarbons. For animals and man the toxicity of benzene is much higher because of its cancerogenicity. Regarding phytotoxicity, benzene is less toxic than toluene, probably due to its lower lipophily. Combinations of air pollutants (inorganic gases and formaldehyde) are known to lead to more than additive toxic effects (Lichtenthaler and Buschmann 1984). This was proven by Masaru et al. (1976) with a germination test using lily pollen. Investigations are being carried out to determine how far such synergistic effects can be detected by our pollen test system in a mixture of volatile organic compounds occurring in industrial emissions. Acknowledgements
Thanks are due to Mrs. Solis-Schreiter and Mr. Carsten Hoffmann for improvement of the English.
518
UTE SCHUBERT, LINDA WISANOWSKY, and ULRICH KULL
References BAUMBACH, G.: Luftreinhaltung. Springer, Berlin (1990). BREWBAKER, J. L. and B. H. KWACK: The essential role of calcium ion in pollen germination and pollen tube growth. Amer. J. Bot. 50, 859-865 (1963). DEBus, R., B. DITTRICH, P. SCHR6DER, and J. VOLMER: Biomonitoring organischer Luftschadstoffe. Aufnahme und Wirkung in Pflanzen. Ecomed, Landsberg (1989). FLORES, H. E.: Changes in polyamine metabolism in response to abiotic stress. In: SLOCUM, R. D. and H. E. FLORES: Biochemistry and Physiology of Polyamines in Plants. CRC-Press, Boca Raton, 213-228 (1992). HAAs, H.: EinfluB von 1,1,1-Trichlorethan auf Gehalt und Zusammensetzung freier Polyamine in Nicotiana tabacum Samsun EN. Diplomarbeit, Biologisches Institut, Universitat Stuttgart (1992). HESLOP-HARRiSON, J., Y. HESLOP-HARRiSON, and K. R. SHIVANNA: The evaluation of pollen quality and a further appraisal of the fluorochromatic (FCR) test procedure. Theor. App!. Genetics 67, 367 -375 (1984). HOEKSTRA, F. A. and E. W. VAN DER WAL: Initial moisture content and temperature of imbibition determine extent of inbibitional injury in pollen. J. Plant Physio!. 133, 257 -262 (1988). HOFFMANN, F., B. A. MARTIN, R. B. SmLEY, and S.-S. TSAY: Pollen germination is impeded by tap water. Environ. Pollut. 63, 179187 (1990). KAPPLER, R. and U. KRISTEN: Photometric quantification of in vitro pollen tube growth: a new method suited to determine the cytotoxicity of various environmental substances. Environ. Exp. Bot. 27, 305-309 (1987). - - Pollen test system. The toxicity of chemicals is determined by measuring inhibition of pollen tube growth. Invittox Protocol55, 1-7 (1990). KOCH, R.: Umweltchemikalien. VCH-Verlagsgesellsch. Weinheim (1989). LICHTENTHALER, H. K. and C. BUSCHMANN: Das Waldsterben aus botanischer Sicht. Verlauf, Ursachen und MaBnahmen. G. Braun, Karlsruhe (1984). LICHTENTHALER, H. K., G. SCHMUCK, and M. D6LL: Photosyntheseaktivitat bei Nadeln gesunder und geschadigter Koniferen. LISBerichte 57, 87 -105 (1985).
LICHTENTHALER, H. K. and U. RINDERLE: The role of chlorophyll fluorescence in the detection of stress conditions in plants. Critical Reviews Analyt. Chern. 19, Suppl. I, 29-85 (1988). MASARU, N., F. SYOZO, and K. SABURO: Effects of exposure to various injurious gases on germination of lily pollen. Environ. Pollut. 11, 181-187 (1976). MEYBERG, M., R. KApPLER, and U. KRISTEN: Bestimmung der Cytotoxizitat organischer Luftschadstoffe. VDI-Berichte 609, 507512 (1987). PAOLETTI, E. and L. M. BELLANI: The in·vitro response of pollen germination and tube length to different types of acidity. Environ. Pollut. 67, 279-286 (1990). PARLAR, H. and D. ANGERH6FER: Chemische Okotoxikologie. Springer Verlag, Berlin (1991). PATERSON, S., D. MACKAY, D. TAM, and W. Y. SHIV: Uptake of organic chemicals by plants: a review of processes, correlations and models. Chemosphere 21, 297 -331 (1990). PFAHLER, P. L.: Analysis of ecotoxic agents using pollen tests. In: LINSKENS, H. F. and J. F. JACKSON (ed.): Plant Toxin Analysis, Modern Methods of Plant Analysis, N.S., Vol. 13, 317-331 (1992). PORTNOI, L. and A. HOROVITZ: Sugars in natural and artificial pollen germination substrates. Ann. Bot. 41, 21- 27 (1977). SCHUBERT, U., L. WISANOWSKY, and U. KULL: Determination of the phytotoxicity of volatile organic compounds by measuring the germination rate of tobacco pollen. Plant Physiol. 99, Supplement, Abstr. 877 (1992). SCHULTE-HoSTEDE, S., N. M. DARRAL, L. W. BLANK, and A. R. WELLBURN: Air pollution and plant metabolism. Elsevier, London (1987). SCHULZE, E. and E. ST!X: Beurteilung phytotoxischer Immissionen, fur die noch keine Luftqualitatskriterien festgelegt sind. Angew. Bot. 64, 225-235 (1990). SFAKIOTAKIS, E. M., D. H. SIMONS, and D. R. DILLEY: Pollen germination and tube growth: dependent on carbon dioxide and independent of ethylene. Plant Physiol. 49, 963 -967 (1972). STANLEY, R. G. and H. F. LINSKENS: Pollen. Biologie, Biochemie, Gewinnung und Verwendung. Freund, Greifenberg (1985). TRESHOW, H. and K. ANDERSON (eds.): Plant stress from air pollution. Wiley and Sons, Chichester (1989). WOLTERS, J. H. B. and M. J. M. MARTENS: Effects of air pollutants on pollen. Bot. Rev. 53, 372-414 (1987).
Determination of Phytotoxicity of Several Volatile Organic Compounds by Investigating the Germination Pattern of Tobacco Pollen* UTE SCHUBERT, LINDA WISANOWSKY,
and ULRICH KULL
Biologisches Institut der Universitat Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany Received February 1, 1994 . Accepted May 30, 1994
Summary
Hydrated pollen of Nicotiana tabacum L. (var. xanthi nc) was exposed to different concentrations of several volatile organic compounds. The germination rate of the pollen was determined by applying a modified test procedure originally developed by Kappler and Kristen (1990). A stimulation of pollen germination resulting through a rise in pC02 was avoided by using a short exposure period. In the series of chlorinated ethanes, the ED50 (and ED25) values decrease with increasing number of chlorine atoms, which are correlated to a rise of the octanol-water partition coefficient. For the chlorinated ethenes a similar effect is probable, but less obvious. The toxicity of compounds with the same number of chlorine atoms is higher for ethane derivatives than for derivatives of ethene. The ED50 and ED25 values are compared with threshold values of physiological effects obtained after the application of 1,1, I-trichloroethane for 24h to tobacco leaves. We presume that chlorinated hydrocarbons may cause stress even at lower concentrations than those suggested by the pollen test. Therefore the listed ED values (Table 1) should be considered as maximum tolerable values regarding phytotoxic effects in experimental investigations. For the important solvent 1,1, I-trichloroethane the ED 25 value of phytotoxicity is lower than 4 mg/L; according to literature the concentration supposedly having no effeet on man is 1.9 - 5.2 mg/L.
Key words: Nicotiana tabacum L., air pollutants, aromatic hydrocarbons, chlorinated hydrocarbons, pollen germination, 1,1, I-trichloroethane. Abbreviations: EDso = 50 % effective dose; ED25 bon dioxide. Introduction
Pollen germination and pollen tube growth are affected by air pollutants in vivo and in vitro, however, the response is more striking in vitro (Wolters and Martens 1987, Pfahler 1992). From their data these authors concluded that pollen under standardized conditions may be used as sensitive biological indicators of pollution. Simple pollen test systems were developed by Kappler and Kristen (1987, 1990) and " Dedicated to Prof. Dr. H. K. Lichtenthaler on the occasion of his 60th birthday. © 1995 by Gustav Fischer Verlag, Stuttgart
= 25 % effective dose; pC02 =
partial pressure of car-
for pollutants contained in water - by Hoffmann et al. (1990) respectively. In both tests the toxic substance is dissolved in the germination medium. These test methods alIowa comparison of non volatile as well as volatile substances, although being rather unphysiological for volatile substances. Many volatile organic compounds (VOCs) are - at least locally - important air pollutants. As considered already by Lichtenthaler and Buschmann (1984), organic halogen compounds may be secondary effectors of tree damage. However, few data concerning their phytotoxicity is available (Schulze and Stix, 1990). Some substances were investigated
Phytotoxicity of volatile organic compounds by Masaru et al. (1976), Meyberg et al. (1987) and Kappler and Kristen (1990). Since volatile compounds under natural conditions reach the pollen as gases and many of them are rather insoluble in water we have established a modified test procedure measuring the germination rate of tobacco pollen. The pollen, suspended in a very thin film of germination medium, is exposed to the gaseous pollutant. The method was tested using several chlorinated hydrocarbons and some aromatic hydrocarbons. A preliminary report containing some of the results was published as an abstract (Schubert et al.
1992). Materials and Methods Plants
Plants of Nicotiana tabacum L. (var. xanthi nc) were raised under greenhouse conditions, such that enough pollen was available throughout the year. Immediately after anthesis, the pollen was directly dusted into Eppendorf-tubes and stored at -20°C until use. Only pollen from plants showing pollen viability of more than 70 % was used in the experiments. Substances tested
The chlorinated hydrocarbons tested are listed in Table 1. For comparison the toxicity of the aromatic hydrocarbons benzene, toluene and xylene (mixture of the dimethyl-benzenes) and of acetone were also investigated. All chemicals were bought from MerckDarmstadt.
515
bars in the figures indicate the standard deviations of (at least three) germination rates. The EDso value (50% effective dose) is the concentration which causes a decrease of pollen germination to 50 % of the total amount. The EDso values are calculated by linear interpolation between the two nearest concentrations. In the same wayan ED2S value can be calculated. This value is commonly used in ecotoxicology as a threshold value separating «sublethal» and «lethal» effects of a toxic substance (Padar and Angerhofer 1991).
Results The results obtained for the chlorinated hydrocarbons are shown as dose-response curves (Figs. 1- 3). The EDso and ED 2S values are given in Table 1, which lists all compounds investigated so far. Also included in Table 1 are the octanolwater partition coefficients of the compounds according to Koch (1989). All compounds show high vapour-pressure values (e.g. 1.38 x 104 Pa for trichloroethane; 6.7 x 102 Pa for tetrachloroethane). In the series of chlorinated ethanes the EDso and ED 2S values decrease with an increasing number of chlorine atoms, which is correlated with an increase of the octanol/water partition coefficient. Although less clear, a similar relationship for the chlorinated ethenes may be presumed. The toxicity of compounds with the same number of chlorine atoms is higher for saturated hydrocarbons than for derivatives of ethene. The EDso and ED 2S values for the investigated aromatic hydrocarbons are rather high and decrease with a rising substitution of the benzene ring, which in return is correlated with a rise of the octanollwater partition coefficient.
Germination test
An amount of 0.5 mg pollen is placed into 3.5 cm Petri dishes and held in a water saturated atmosphere for one hour. This imbibition procedure is advantageous for a high germination rate because it effects the hydration and thereby the reorganization of biomembranes (Heslop-Harrison et al. 1984; Hoekstra and van der WaI1988). Then, a 1 mL germination medium (as described by Kappler and Kristen 1987) is added and the pollen uniformly suspended by cautios shaking of the dishes. No tenside is applied. The concentration of pollen is about 0.5-1 x 106 mL -1. The germination medium must have room-temperature to avoid cold injuries of pollen (Hoekstra and van der Wal 1988). The Petri dishes are then put into small glasstroughs (8.5 x 5.7 x 6.7 cm) which are sealed tightly. The volatile compound is injected by a GC-syringe into the trough where it evaporates at once. As a control the same amount of water is injected into control troughs. Since drought stress would reduce the germination rate, all incubation troughs contain water saturated air. The pollen is exposed to different concentrations of the gaseous compounds at 22°C and in darkness. Final concentrations in the troughs are calculated using the applied amount and the (corrected) volumes of the glass troughs. Simultaneous controls for all concentrations of investigated pollutants are conducted. After an incubation period of 2 h the percentage of germinated pollen is counted by microscopy. The pollen is considered germinated if the pollen tube is unequivocally longer than the diameter of the pollen grain. From each Petri-dish six samples of pollen are transferred to a cell-counting chamber (Schilling-Kreuznetz), where the mean value is determined. For each concentration of a compound three separate tests are investigated. The mean values of these three tests are used to calculate a dose-response curve as described by Kappler and Kristen (1987). The inhibition rate is calculated by setting the germination rate of the control pollen (70 - 85 % of total amount) at 100 %. The
Discussion
Method Literature reports several different media for pollen germination (d. Stanley and Linskens 1985). Preliminary experiments using different media (Brewbaker and Kwak 1963, Portnoi and Horovitz 1977, Hoffmann et al. 1990) showed that the medium of Kappler and Kristen (1987) was most suitable to get high germination rates. Our method avoids a disadvantage of the test-system of Kappler and Kristen (1987, Table 1: EDso and ED 2S values based on the tobacco pollen germination test for chlorinated hydrocarbons, aromatics, and acetone. compound
EDso mg/L
1,2-dichloroethane 1, 1, I-trichloroethane 1,1,2,2-tetrachloroethane
17.1 18.1 0.055
trans-l,2-dichloroethene trichloroethene tetrachloroethene
64.0 31.7 34.3
ppm 4,170 3,270 5
mg/L
ED zs
9.2 <4 0.014
15,890 5,800 4,980
41.7 <4 12.3
19 p*
ppm 2,210 < 720 1
1.7 2.8 3.0
10,350 < 730 1,780
2.4 2.95
chloroform - trichloromethane
33
6,770
24.0
4,920
2.02
benzene toluene xylene
36.5 28.9 17.1
11,420 7,560 3,870
18.2 9.8 7.2
5,710 2,560 1,620
1.8 2.39 2.9
acetone
30.9
13,040
12.2
5,170
* Octanol-water partition coefficient according to Koch (1989).
516
UTE SCHUBERT, LINDA WISANOWSKY,
Inhibition of germination
Inhibition of germination
(% control)
100 90 80 70 60 50 40
0
(% control)
1,1,1 - trichloroethane
l
,---!/.l
30 20 10
/1
1
2
4 6 810
20
4060
100 90 1,2 - dichloroethane 80
trichloroethene
/
(
rl
*
I----f
4 6 810
2
20
~
4060
Inhibition of germination
,
i--
10 10
20
20
4060
tetrachloroethene
90 80 70 60 50 40 30 20 10 0
/r
x
*
t/ 1
)
Fig. I: Dose-response curves for several chlorinated hydrocarbons in the pollen germination test. Bars: standard deviations. The calculated ED50 values are marked by stars.
concentration (mg / dm 3)
Fig. 2: Dose-response curves for trans-I,2dichloroethane and I, 1,2,2-trichloroethane in the pollen germination test (different concentration scales!). Bars: standard deviations. The calculated ED50 values are marked by stars.
2
4 6 810
20
4060
concentration (mg / dm
3
Inhibition of germination
(% control)
trans-l,2 -dichloroethene
20
4 6 810
concentration (mg / dm 3)
100
concentration (mg / dm 3)
(% control)
100 90 80 70 60 50 40 30
2
Inhibition of germination
rI'
30 20 10
1
(% control)
1/
70 60 50 40
1
0
concentration (mg / dm 3)
Inhibition of germination
0
100 90 80 70 60 50 40 30 20 10
(% control)
0
and ULRICH KULL
4060 100 200
concentration (mg / dm 3)
100 90 80 70 60 50 40 30
20 10 0 0.001
l,l,2,2-tetrachloroethane
I-I
I
I)
0.01
1990) for volatile and especially for water-insoluble volatile compounds, for which these authors had to use dimethylsulfoxide as a solvent. Compared to the method used by Kappler and Kristen (1987, 1990) the use of a short exposure
0.1
1
period avoided a rise of the pC02 in the closed incubation system, which otherwise may cause a stimulation of pollen germination (Sfakiotakis et al. 1972). However, because we applied the VOCs as gases they have to dissolve in a thin
Phytotoxicity of volatile organic compounds
Inhibition of germination
Inhibition of germination
(% control)
100-r--------------, 90
xylene
80
Fig. 3: Dose-response curves for xylene and toluene in the pollen germination test. Bars: standard deviations. The calculated ED50 values are marked by stars.
I
0
1
2
*l
4 6 810
20
4060
concentration (mg / dm 3)
layer of the medium to reach the pollen. For the purpose of comparison, we tested some compounds already investigated by Kappler and Kristen (1990). Although the values are in good agreement for toluene (7560/7888 ppm), we found a significantly lower ED50 value for acetone (ED50 value from our test: 13,040 ppm; ED50 value of Kappler and Kristen: 20,500 ppm). The test-method described above may also be combined with the automatisable procedures of the measurement of pollen tube growth of Kappler and Kristen (1990). As found by Kappler and Kristen (1987) for their tested compounds, the dose-response curves showed steep inclines in the ED50 range. This is not true for all compounds regarding ED 25 ; therefore, in two cases no equivocal ED 25 value could be obtained. Because of the deviations of the individual measurements with very low concentrations of the VOC, it was not possible to calculate satisfactory EDlO values, which are somtimes used as threshold values of toxicity (Parlar and Angerhofer 1991).
Phytotoxicity of chlorinated hydrocarbons Some of the chlorinated hydrocarbons investigated are used as solvents and cleansers; however, there are no data on their phytotoxicity. Owing to their broad application, the concentration of such compounds could locally rise to values (Debus et al. 1989, Baumbach 1990) which according to our data (Table 1) are phytotoxic. The sensitivity of plant cells is rather high compared with the LD (lethal dose) values for mammals and man as collected by Koch (1989). To establish whether the phytotoxicity values obtained by the pollen test are well-founded, threshold doses causing effects on tobacco plant leaves are now under investigation. It is well known that photosynthesis is affected by most air pollutants in a direct or indirect manner (summarized in Schulte-Hostede et al. 1987, Lichtenthaler and Rinderle 1988, Treshow and Anderson 1989) which is perhaps a main cause of large scale forest damage (Lichtenthaler et al. 1985). For 1,1, 1-trichloroethane concentrations higher than 0.09 mg/ dm 3 applied for 24 h effects were found concerning the photosynthetic gas exchange of tobacco leaves (Schubert, unpublished). It is well known that changes in polyamine metabo-
toluene
70
1/
10
100-r--------------,
80
If
60 50 40 30 20
(% control) 90
70
517
60 50 40 30 20 10 0
1
2
J 1/1 */ 4 6 810
20
4060
concentration (mg / dm 3)
lism are caused by stress (Flores 1992). With 1,1, 1-trichloroethane, tobacco leaves show significant changes in the ratio putrescine/spermidine + spermine at concentrations higher than 0.1 mg/dm 3 applied for 24h (Haas 1992). Through concentrations effective near places of emission chlorinated hydrocarbons may therefore cause severe stress. We presume that the ED values obtained by the 2 h pollen test (Table 1) can be considered as threshold values of phytotoxic concentrations of the compounds. For 1,1, 1-trichloroethane, the concentration which is supposed to have no effects on man is 350-950ppm (1.9-5.2mg/dm 3) (Koch 1989) and in Germany the MAK value (maximum acceptable concentration at the place of work) is 1.08mg/dm3. Comparing the concentrations effecting plants and regarding the safety-factor of MAK values one may presume that the phytotoxicity is higher than toxicity for man. The same may be true for trichloroethene. The octanol-water partition coefficient is a key parameter for uptake of lipophilic organic compounds by plant cells and tissues (Paterson et al. 1990). We find a rise of phytotoxicity in each series of compounds tested correlated with an increase of the partition coefficient. From this correlation an uptake preferably through the membrane lipid bilayer may be concluded. This is also obvious for the aromatic hydrocarbons. For animals and man the toxicity of benzene is much higher because of its cancerogenicity. Regarding phytotoxicity, benzene is less toxic than toluene, probably due to its lower lipophily. Combinations of air pollutants (inorganic gases and formaldehyde) are known to lead to more than additive toxic effects (Lichtenthaler and Buschmann 1984). This was proven by Masaru et al. (1976) with a germination test using lily pollen. Investigations are being carried out to determine how far such synergistic effects can be detected by our pollen test system in a mixture of volatile organic compounds occurring in industrial emissions. Acknowledgements
Thanks are due to Mrs. Solis-Schreiter and Mr. Carsten Hoffmann for improvement of the English.
518
UTE SCHUBERT, LINDA WISANOWSKY, and ULRICH KULL
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