Research Articles
Uptake of TNT in Plants
Uptake and Metabolism of 2,4,6-Trinitrotoluene in Higher Plants Elisabeth G6rge, Sebastian Brandt, Dietrich Werner Fachbereich Biologie, Philipps Universit~it Marburg, D-35032 Marburg, Germany
Corresponding author: Dr. Elisabeth G6rge
Abstract The fate of the explosive 2,4,6-TNT in plants is of major interest. Therefore, a method was developed to analyse TNT and derivatives in plant tissue. The method was utilized to investigate the uptake and metabolism of T N T in Medicago sativa and Allium schoenoprasum grown in hydroponic cultures containing TNT levels of 0.1 to 10 rag/1. Detectable concentrations of nitrotoluenes were significantly higher in Allium schoenoprasum than in Medicago sativa. The uptake of TNT in plants was directly related to the initial TNT level. The principal nitroaromatic components in roots and shoots of both plant species were identified as 4-ADNT and 2-ADNT in equal amounts, with substantially less TNT.
is no report that T N T or aminodinitrotoluenes could be identified in upper plant parts grown in contaminated soils or contaminated wasted sites. In this study a method was designed to analyse the uptake and metabolism of T N T using Medicago sativa and Alliurn schoenoprasum as test plants. The intention was to develop a method that could also be used to investigate uptake of T N T under field conditions.
2 2.1
1
Introduction
2,4,6-Trinitrotoluene (TNT) is one of the most widely used explosives. It occurs as a pollutant of soil and ground water especially at ammunition sites. It has been shown that T N T is toxic to a large variety of organisms, including aquatic organisms such as green algae (SMOCK et al. 1976) and duckweed (SCHOTT & WOV,VHLEY 1974) as well as terrestrial plants (PALAZZO & LEGGETT 1986). Toxicity to animals or humans (munition workers) causing liver damage, anaemia (DJER_~SSI& VITANY 1975, Koss et al. 1989, HATHAWAY1977) or cataract of the eyes (GRANT 1986) is well documented. In Germany there are many former ammunition sites with soils still contaminated with large amounts of T N T and derivatives. The persistence of these compounds in natural environments results in major problems with land use. Some of these sites are now used as real estates or even gardens. Since T N T can enter the food chain via plants, the uptake of T N T in plants is of great interest. Several studies examining the fate of T N T in' plants have revealed uptake of T N T (PALAZZO & LEGGETT 1986, FOLSOM et al. 1988, PENNINGTON 1988, CATALDO et al. 1989, HARVEY et al. 1990). The major identified products were the aminodinitrotoluene isomers, 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT). Studies examining the uptake of 14C T N T in bush beans indicated that adsorbed T N T and derivatives underwent major transformation. Large quantities of 14C were either bound to the pelleted plant residues or were found to be water soluble (HARVEYet al. 1990). Up to now there ESPR-Environ. Sci. & Pollut. Res. 1 (4) 229-233 (1994) 9 ecomed publishers, D-86899 Landsberg, Germany
Material and methods Chemicals
2,4,6-Trinitrotoluene was obtained from Dr. E.v.L0w, Hygiene Institut, Philipps University Marburg. 2-ADNT and 4-ADNT were synthesized by Dr. K. STHNBACH,Fachbereich Chemie, Philipps University Marburg. 2.2
Plant cultivation
The uptake and metabolism of T N T of plants were evaluated using Medicago sativa cv. Du Puits and Allium schoeno-
prasum. Seeds were washed with 0 . 1 % Twen 20, rinsed several times and then surf~ice sterilized by incubation with 30 % H202 for 10 min and subsequently washed with sterile water. Seedlings were germinated for two days on NB (Nutrient broth, Difco laboratories) agar plates to visualize contamination with bacteria or fungi. Uncontaminated seedlings were carefully transferred to the hydroponic system and were grown for 28 days in a growth chamber with 16 h, 25 ~ light period and 8 h, 20 ~ dark period. The plants were grown in sterile 100 ml test tubes containing 20 ml of glass beads ( 4 - 7 mm) to support the plants. The test tubes were partly shielded with black plastic tubes to minimize photolysis of T N T and to protect the roots from light. To avoid bacterial contamination they were dosed with a sterile plug of cotton wool and an aluminium cap. The nutrient solution used in all test tubes was a mineral medium modified from BROUGHTON& DILWORTH(1971) supplied with 0.5 mM KNO 3. The initial pH was 6.8. Stock solutions of T N T were prepared by adding 100 mg ground T N T to one litre of autoclaved medium. The solution was treated in an ultrasonic bath for 5 min and then stirred until the T N T had completely dissolved. The stock solutions were diluted to final T N T concentrations by adding autoclaved medium. The different T N T concentrations
229
Uptake of TNT in Plants
Research Articles
selected for study were 0, 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 mg/1. Each experiment was conducted twice with at least 3 replicates. Solutions were analysed for pH, unpolar nitrotoluenes and watersoluble aromatic amines immediately after contamination with T N T and at the time of harvest. Sterility was proven by plating an aliquot on NB agar plates. For analysis of nitrotoluenes the hydroponic solutions were extracted twice with methylene chloride. The extract was dried over anhydrous sodium sulfate, evaporated to dryness, dissolved with a small amount of ethyl acetate and subjected to analysis by GC/ECD. The aqueous solution, after methylene chloride extraction, was used for analysis of aromatic amines using a p-nitroanilin standard as representative of the overall aromatic content as described by v. LOw & HAAs (1986). After 28 days of growth, plants Were removed from the hydroponic solutions and separated into roots and shoots. The roots were rinsed first with water followed by methanol. After measurement of length and weight, plants were freezedried, minced with a dismembrator (Braun, Melsungen, F R G ) and stored at - 2 0 ~ until analysis. A 100 mg aliquot of freeze-dried plant material was extracted for 30 min in an ultrasonic bath with 4 ml of methylene chloride twice. After centrifugation the supernatant was removed, evaporated to dryness, redissolved in ethyl acetate and analysed with GC/ECD. The pellet was subjected to acid hydrolysis with 25 % H2SO 4 by immersing the tube in a 95 ~ water bath for 90 min (P~AZZO & LGGETT 1986). After cooling to room temperature the hydrolyzed material was extracted twice with 2 ml methylene chloride. The phases were separated by centrifugation for 10 min at 3 000 g. After removal of the methylene chloride layer, the acid layer was made basic by the addition of 8 M N a O H and extracted again with methylene chloride. The phases were again separated by centrifugation and the methylene chloride layer was combined with the methylene chloride-extract of the acid hydrolysis. After drying over anhydrous sodium sulfate, evaporation to dryness and redissolving in ethyl acetate the sample was subjected to further clean up or directly analysed by GC/ECD. Methylene chloride extracts of green plant parts were purified either by solid phase extraction with Adsorbex SCX-columns
180 -6 ~- 1 6 0
A.schoenoprasum
(Merck, Darmstadt, FRG) to remove chlorophylls, or Florisil columns as described by HARVEYet al. (1990). The columns had been previously washed with methanol and conditioned with methylene chloride. An additional 2 ml of methylene chloride was used to rinse the remaining sample out of the sample vial to the column. The eluate was evaporated to dryness, dissolved in ethyl acetate and subjected to analysis with GC/ECD.
2.3
Residue analysis
For analysis of all samples a gas chromatograph (Varian model 3 400) with ~3Ni electron capture detector was used. Some of the samples were additionally analysed by means of GC/MS. A 1/al aliquot of the ethyl acetate extract was injected (split 1:20) onto a 30 m DB-5, 25/am I.D. column containing a 1.0/~m film of stationary phase (J&W Scientific, Folsom, CA, USA). The components were separated with helium carrier gas using a temperature program from 140 ~ to 240 ~ at 10 ~ and 240 ~ to 260 ~ at 5 ~ Mass spectrometer studies utilized a Finnigan ITD 800, full scan or single scan modus, 70 eV, interfaced to a Varian 3 400 gas chromatograph, equipped as described above. Spiked tissue extracts with 100 pg T N T , 4-ADNT and 2-ADNT per/al were used as external calibration standards.
Results and discussion
3
3.1
Plant growth
Initial concentrations of 50 mg/l or more restricted plant growth totally. Root growth was most influenced by the presence of T N T . Visible symptoms of injury to roots consisted of growth restriction and browning of the root tips. Root length of Medicago sativa was reduced at 10 mg/1 ( ~ Fig. 1). Higher concentrations affected the shoot length as well. Allium schoenoprasum displayed symptoms of root damage already at lower levels of TNT. The root length was significantly reduced at concentrations of 5 mg/1. PALAZZO& LEGGETT (1986) also reported inhibition of root growth of Cyperus esculentus at 5 mg/1 in hydroponic solution, whereas duckweed was sensitive to T N T at concentrations of 1 mg/1 (ScHoTT & WORTHLEY1974). Thus some plants seem to be
M.sativa
c
o u 140 ~- 1 2 0 O
lOO _c
80
c
60
2
40
Fig. 1:
20 0
0
0.1
0.5
1.0
initial
230
5.0
concentration
0
0.1
o f TNT
0.5
.0 5 . 0
[r~g/t]
10.0
Length of roots and shoots of Allium schoenoprasum and Medicago sativa in hydroponic solution containing different TNT levelscompared to a control after 28 days of growth ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Research Articles
Uptake of TNT in Plants
more tolerant to T N T than others. TUCKERet al. (1989) investigated tolerance of different plant species to T N T , but there was no indication, that monocots in general are more tolerant to T N T than dicots.
Table 1: Quantity of T N T , 4-ADNT and 2-ADNT at the beginning of the study (to) and at the time of harvest (28 day) in hydroponic solutions TNT-level 0.0
3.2
Analysis of hydroponic solutions
The hydroponic solutions were analysed immediatly after addition of TNT. The recovery of nitrotoluenes was more than 95 %. Although only T N T was added, 6.7 % • 1.7 % of the initially analysed nitrotoluenes were identified as the aminodinitrotoluenes (ADNTs). No increase in formation of ADNTs could be observed in control solutions even after 4 weeks, although the total amount of detectable nitrotoluenes decreased slightly (--' Table 1). This indicated transformation of T N T to compounds that can not be extracted and analysed with the described method. Analysis of aromatic amines showed a slight increase even for the control tubes ( ~ Fig. 2). Products were not subjected to further analysis.
C~
0.4-
E 61
~
control A.schoenoprusum ~M.sativa
0.5
1.0
5.0
hydroponic solution to TNT 0.000g1 0 . 0 6 8 f g 4-ADNT 0.000e 2-ADNT 0.000e
0.004e 0.001e
control solution (28 day) TNT 0.000g 0.031fg 4-ADNT 0.000e 2-ADNT 0,000e
10.0 mg/I
0,001e 0,001e
0.407e 0.747d O.019cde 0.033c 0.010de 0.015de
3.787a 0.192a 0.111b
0,347e 0.370e 3,532b O.015cde O.015cde 0.119b O.012de 0,009de 0.095bc
9.617a~ 0.529a 0.269a 6,120b 0.262b 0.177b
Allium schoenoprasum (28 day) TNT 0,000g 0.014g 0.057 fg 4-ADNT 0.000e 0,004e 0.011de 2-ADNT 0,000e 0.006e 0.036d
0.151f 0.029 cd 0.072c
1.299c 0.113b 0,202a
-
Medicago sativa (28 day) TNT 0.000g 0.000g 4=ADNT 0.000e 0,000e 2-ADNT 0,000e 0,000e
0.000g 0.001e 0.002e
0,007g 0.035c 0.013de
0,046c 0,082c 0.054c
0.000g 0.002e 0.000e
Means for each compound followed by the same letter are not significantly different at the 0.05 level by WELLER/DUNCANtest. 2 Statistical tests for TNT level 10 mg/l were done separately. - not included
0.3
E 0
0.1
mg/I
0.2
4-, o
E 0.1 o 0
0.0
0 0.1 0.5 initial concentration
1,0 5,0 o f TNT [ m g / I ]
Fig. 2: Concentration of aromatic amines using a p-nitroaniline stand a m as representative of overall aromatic content in hydroponic solution containing different T N T levels after 28 day of incubation. T h e solutions were analysed for aromatic amines after extraction with methylene chloride.
High concentrated T N T solutions ( > 10 mg TNT/1) that were exposed to growth chamber lights turned brown to pink, probably due to the formation of a photodecomposition product. These products have already been observed and characterized by SPANGGORD et al. (1980) and have also been found in hydroponic solutions by HARVEY et al. (1990). This might also be a reason for the observed decline of nitrotoluenes in control solutions. Analysis of solutions used to support plant growth showed a major decrease of nitrotoluenes, dependent on the plant species ( ~ Table 1). In the case of Allium scboenoprasum with initial concentrations of 0.1 to 5 mg TNT/1 only 25 % to 39 % could be analysed as nitrotoluenes after 28 days of growth. Only 63.9 % + 15 % of the extracted nitrotoluenes were identified as T N T , 1 0 . 1 % + 2 . 9 % or 26.2 % • 12.2 % were analysed as 4-ADNT or 2-ADNT, respectively (n = 9).
ESPR-Environ. ScL& Pollut. Res. 1 (4) 1994
In tests with Medicago sativa, the decline of T N T was much higher ( ~ Table 1). At initial concentrations of 5 and 10 mg TNT/1, only 1.3 % and 8.7 % of the initial concentration could be analysed as nitrotoluenes after 28 d of growth. Of the total detectable amount, 48 % • 14 % was identified as 4-ADNT and 38.5 % • 18 % as 2-ADNT. That means that the majority of the initial T N T was transformed to the aminodinitrotoluenes or other compounds. T N T represented less than 11.8 % • 10 % of the total nitrotoluene fraction. At the time of harvest we found a dose-dependent increase of aromatic amines in the hydroponic solution ( ~ Fig. 2), probably also due to metabolism of the added T N T . During growth of Medicago sativa the pH of the solution dropped from pH 8.5 (control solution) to 6.9, indicating metabolic activity of the roots. For Allium schoenoprasum the pH also declined to pH 7.6, except for the highest T N T level (5 mg/1, pH 8.4), indicating only minor activity. This is in accordance with the restricted root growth for that T N T level. It is not yet proven, whether the formation of aminodinitrotoluenes and unknown products is due to root catalyzing effects or is a consequence of the metabolic activity of bacteria. Control of sterility of all test tubes revealed minor contamination with fungi or bacteria in some of the tubes. However, there was no correlation of significant differences in T N T concentrations with any detectable growth of microorganisms. In additional experiments one isolate of bacteria was able to transform T N T to the aminodinitrotoluenes and to unknown substances if it was grown in T N T containing NB-medium (10 mg/1). Incubation in mineral medium without the additional supply of C-sources, revealed only minor growth and no significant decrease of the initial
231
Uptake of TNT in Plants
Research Articles
TNT-level (unpublished results), which is in accordance with results of other authors (AME~,HANOVA & NAUMOVA 1978, OSMON & KLAUSMEmV.1972, P~uss et al. 1993). They suggested that T N T cannot be used as the sole C-source and is metabolized only by cometabolism. In our experiments root exudates could have provided C-sources for bacteria (WHIPPS 1990). Therefore, it is not possible to confirm whether transformation of T N T was due to root catalyzing effects or mediated by bacterial activity. 3.3
Uptake of TNT in plants
Extraction of plant material was always performed in three steps. After extraction with methylene chloride, the remaining pellet was subjected to acid hydrolysis and extracted again with methylene chloride, followed by a basidification with 8 M N a O H and a second extraction with methylene chloride. The most effective extraction step for spiked plant tissue, as well as for plants grown in T N T containing hydroponic solution or soil, was the acid hydrolysis ( - Table 2). This is in accordance with results of HARVEY et al. (1990), who also found acid hydrolysis to be very efficient. Table 2: Proportion of nitroaromatics and standard deviation of different extraction steps of root samples (n = 10) of Medicago sativa grown in contaminated sand (100 m g / k g ) for two months. TNT
4-ADNT % proportion
2-ADNT
16,9_+7,0 80,0 _+9,7 3,0+2,7
2,9_+ 5,7 63,3 _+26,4 33,8_+24,0
4,3_+ 5,7 78,2 + 25,9 17,5_+16,8
99.9
100.0
100.0
extract methylene chloride H2SO 4 NaOH total
The detectable amount of nitroaromatics in plant tissue was correlated to the initial concentration of T N T in the hydroponic solution ( ~ Tables 3, 4). Thus the amount that can be taken up by plants in soil is dependent on the bioavailability of T N T , especially in the soil solution. Factors controlling the adsorption/desorption in soils are therefore also controlling the bioavailability of T N T and the amount that can be taken up by plants. Significant accumulation in roots was detected at an initial concentration of 0.5 mg TNT/1. At this concentration no visible symptoms of injury occurred.
Table 3: Detectable concentration of T N T , 4 - A D N T and 2-ADNT in shoots and roots of Allium schoeneprasumand Medicagosativa after growth in hydroponic solution with different TNT-levels shoots
0.0
0.1
0.5 1.0 5.0 nitrotoluenes in shoots #gtg dryweight
Allium schoenoprasum TNT < 0.110c~ < 0.110c 4-ADNT < 0.110d < 0.110o' 2-ADNT < 0.110c < 0.110c
< 0.110c 0.350cd 0,583c
0.460b 0.693bc 1,870b
0.442b 1.677a 5.659a
Medicago sativa TNT < 0.033c 9 .ADNT < 0.033d 2-ADNT < 0.033c
< 0.033c 0.045d 0.039c
0.078c 0.102d 0.102c
0.860a 1.026b 0.885c
< 0.033c 0.045d 0.039c
1 Means for each compound followed by the same letter are not significandy different at the 0.05 level by WALLER/DUNCANtest. Means for control are detection limits.
roots
0.0
0.1
0.5 1.0 5.0 10.0 nitrotoluenes in roots pg/g d r y w e i g h t - -
Allium schoenoprasum TNT < 0.110o1 3.187d 4-ADNT < 0.110d 6.220d 2-ADNT < 0.110d 4.319d
Clean up procedures using SCX as well as Florisil solid phase extraction columns failed to retain the nitrotoluenes. So SCXcolumns were only used to remove chlorophyll from the extract. To evaluate the efficiency of the analytical method 1 ppm of T N T , 4-ADNT and 2-ADNT were added to freeze-dried material of Medicago sativa and then extracted as described. The recovery of the extraction method with methylene chloride was about 100 % for both T N T and 2-ADNT and 75 % for 4-ADNT. Acid hydrolysis released additional absorbed and transformed nitrotoluenes from plant tissue grown in contaminated substrate. Spiked tissue extracts were used as external standards and also to evaluate the calibration standards of the GC/ECDsystem. The ECD signal was linear for the range of 10 to 500 pg T N T , 4-ADNT and 2-ADNT per/A and was not disturbed by dry matter contents of up to 250 mg/ml. After 28 days of growth, plant roots and shoots were analysed for T N T and the aminodinitrotoluenes 2-ADNT and 4-ADNT. The detection limits for each of these compounds are given in Table 3.
232
Medicago sativa TNT < 0.025d 4-ADNT < 0.033d 2-ADNT < 0.033d
0.022d 1.142d 0.559d
7.289bcd 5.705bcd22.783a 10.780cd 19.222c 76.077ab 11.151c 21.610c 69.900ab 1.344d 3.811cd 12.77"3bc 13.811ab 7.622cd 12.521c 40.050bc 94.731a 8.173cd 15.471c 34.820bc 82.790a
1 Means for each compound followed by the same letter are not significantly different at the 0.05 level by WALLER/DuNcANtest. Means for control are detection limits. - not included
Table 4: Correlation coefficient for the content of nitroaromadcs in different plant organs in relation to the initial concentration of T N T in hydroponic solution species
plant part
A. schoenoprasum
root 2 shoot 2 root 3 shoot 2
M. sativa
1 after PEARSON
20.1 tO 5 mg TNT/I
n
correlation coefficient 1
15 15 26 12
0.941 0.921 0.765 0.977
30.1 to 10 mg TNT/I
ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Research Articles T h e m o n o c o t Allium schoenoprasum g r o w n in h y d r o p o n i c solution with 1 m g or 5 mg T N T / I showed a larger quantity o f detectable a m m u n i t i o n residues in roots and shoots c o m p a r e d to the dicot Medicago sativa. This could reflect a p l a n t specific difference in u p t a k e , accumulation or m e t a b o l i s m . C A T , D O et al. (1989) found the u p t a k e o f T N T to be higher in the dicot Phaseolus vulgaris t h a n in the e x a m i n e d m o n o c o t s Bromus rnollis a n d Avena sativa, and suggested that there might be principal differences between dicots and monocots. However, our results showed that there was no such difference in b e h a v i o u r between dicots and monocots. It is clear from the d a t a o f b o t h investigated p l a n t species that the m a j o r i t y of detectable nitrotoluenes were extracted from the r o o t tissues. F o r Allium schoenoprasum at initial T N T levels o f 5 m g / l the concentration of nitrotoluenes in roots was m o r e than 20 times higher than in shoots. F o r Medicago sativa the detectable concentration in r o o t s was m o r e than 30 times higher. A similar distribution o f T N T metabolites and residues was also found by PALAZZO & LEGGETT (1986) a n d was c o n f i r m e d in 14C experiments by H a r v e y et al. (1990). By analysis of r o o t tissues o f Medicago sativa only 9 % + 8.9 % o f the total detectable a m o u n t could be identified as T N T . 4 - A D N T and 2 - A D N T were found at equal concentrations of about 40 % of the total amount each. That means that the p a t t e r n o f distribution of nitrotoluenes in h y d r o p o n i c solution and p l a n t tissues are different. W h e t h e r this is due to a difference in u p t a k e or due to m e t a b o l i s m in p l a n t tissue still has to be elucidated.
4
Conclusions
In this study a m e t h o d was described to investigate u p t a k e of T N T in plants. T h e u p t a k e a n d accumulation seems to be dependent on p l a n t species, p l a n t organs and the initial T N T concentration o f the substrate. Since T N T can enter the food chain via plants, there is a need to investigate u p t a k e o f T N T under field conditions.
5
Uptake of TNT in Plants
DJERASSI,L. S.; L. VIT,~r Haemolytic episode in G6-PD deficient workers exposed to TNT. Brit. J. Ind. Med. 32, 54 (1975) FOLSOM,B. L.; J. C. PENNINGTON;S. L. TEETER;M. R. BARTON;J. A. BRIGHT: Effects of soil pH and treatment level on persistence and plant uptake of 2,4,6-trinitrotoluene. Waterways Experiment Station, Corps of Engineers, Vicksburg, MS, Technical Report EL-88 - 22 (1988) GRANT,W. M.: Toxicology of the eye. Charles C. Thomas Publishers, Springfield Illinois 1986, p. 953 HaTHaWAY,J. A.: Trinitrotoluene - a review of reported dose-related effects providing documentation for a workplace standard. J. Occupa. Med. 19, 341 (1977) H.~VEY, S. D.; R. J. FELLOWS;D. A. CAT,a-DO;R. M. BEAN:Analysis of 2,4,6-trinitrotoluene and its transformation products in soils and plant tissues by high-performance liquid chromatography. J. Chromato. 518, 361 (1990) Koss, G.; A. LOMMEL;I. OLLROGE;I. TESSERAUX;R. HAAS; A. D. KAVPOS: Zur Toxikologie der Nitrotoluole und weiterer Nitroaromaten aus rfistungsbedingten Altlasten. Bundesgesundhbl. 527 (1989) OSMON, J. L.; R. E. KLAUSMEIER:The microbial degradation of explosives. Dev. Ind. Microbiol. 14, 247 (1972) PALAZZO,A. J.; D. C. LEGGETT:Effect and disposition of TNT in a terrestrial plant. J. Environ. Qual. 15, 49 (1986) PENNINGTON, J.C.: Plant uptake of 2,4,6-trinitrotoluene, 4-amino-2,6-dinitrotoluene, and 2-amino-4,6-dinitrotoluene using 14C-labeled and unlabeled compounds. US Army Engineer Waterways Experiment Station, Miss Vicksburg: Technical Report EL-88-20th (1988) PREUSS, A.; J. FIMPEL;G. DIEKERT: Anaerobic transformation of 2,4,6-trinitrotoluene (TNT). Arch. Microbiol. 159, 345 (1993) SAS Institute: SAS/STAT Guide for Personal Computers, Version 6.03 Edition 1988 SCHOTr, C. D; E. G. WORTHLEY:The toxicity of TNT and related wastes to an aquatic flowering plant, Lemnaperpusilla. Edgewood Arsenal, Aberdeen Prov. Ground, MD. Techn. report 74016. AD-778158 (1974) SMOCK,L. A.; D. L. STONEBURGER;J. R. CLARK:The toxic effects of trinitrotoluene (TNT) and its primary degradation products on two species of algae and the fathead minnow. Water Res 10,537 (1976) SPANGGORD,R. J.; T. MILL;T.-W. CHOU; W. R. MAYBE;J. H. SMITH; S. LEE: Environmental fate studies on certain munition wastewater constituents. Final report. Phase II, Laboratory studies, SRI International, Menlo Park. CA (1980) TUCKER, W. L.; W. L. BANWART;T. C. GRANATO;J.J. HASSETT: Screening for plant tolerance to TNT. Report for USATHAMA by CERL and the University of Illinois (1989) V. LOw, E.; L. KAMINSKI;W. NEUMEIER; R. HAAS;K. STEINBACH: Mikrobieller Abbau von Nitroaromaten aus einer ehemaligen Sprengstoffproduktion. Forum St~idte-Hygiene 40, 347 (1989) WHIPPS,J. M.: Carbon economy. In: J. M. LYNCH,The rhizosphere. John Wiley & Sons, Chichester 1990, pp. 59
Literature
A M E ~ O V A , N. N.; R. P. NAUMOVA:2,4,6-Trinitrotoluene as a source of nutrition for bacteria. Microbiology 47, 318 (1978) BROUGHTON, W. J.; M. J. DtLWORTH:Control of leghaemoglobin in snake beans. Biochem. J. 125, 1075 (1971) CATALDO,D. A.; S. D. HARVEY; R. J. FELLOWS;R. M. BEAN; B. D. McVEETY: An evaluation of the environmental fate and behavior of munitions material (TNT, RDX) in soil and plant systems. Pacific Northwest laboratories, Richland, Report AD-A223 546 (1989)
ESVR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Received:April 7, 1994 Accepted:June 6, 1994
233
Uptake of TNT in Plants
Uptake and Metabolism of 2,4,6-Trinitrotoluene in Higher Plants Elisabeth G6rge, Sebastian Brandt, Dietrich Werner Fachbereich Biologie, Philipps Universit~it Marburg, D-35032 Marburg, Germany
Corresponding author: Dr. Elisabeth G6rge
Abstract The fate of the explosive 2,4,6-TNT in plants is of major interest. Therefore, a method was developed to analyse TNT and derivatives in plant tissue. The method was utilized to investigate the uptake and metabolism of T N T in Medicago sativa and Allium schoenoprasum grown in hydroponic cultures containing TNT levels of 0.1 to 10 rag/1. Detectable concentrations of nitrotoluenes were significantly higher in Allium schoenoprasum than in Medicago sativa. The uptake of TNT in plants was directly related to the initial TNT level. The principal nitroaromatic components in roots and shoots of both plant species were identified as 4-ADNT and 2-ADNT in equal amounts, with substantially less TNT.
is no report that T N T or aminodinitrotoluenes could be identified in upper plant parts grown in contaminated soils or contaminated wasted sites. In this study a method was designed to analyse the uptake and metabolism of T N T using Medicago sativa and Alliurn schoenoprasum as test plants. The intention was to develop a method that could also be used to investigate uptake of T N T under field conditions.
2 2.1
1
Introduction
2,4,6-Trinitrotoluene (TNT) is one of the most widely used explosives. It occurs as a pollutant of soil and ground water especially at ammunition sites. It has been shown that T N T is toxic to a large variety of organisms, including aquatic organisms such as green algae (SMOCK et al. 1976) and duckweed (SCHOTT & WOV,VHLEY 1974) as well as terrestrial plants (PALAZZO & LEGGETT 1986). Toxicity to animals or humans (munition workers) causing liver damage, anaemia (DJER_~SSI& VITANY 1975, Koss et al. 1989, HATHAWAY1977) or cataract of the eyes (GRANT 1986) is well documented. In Germany there are many former ammunition sites with soils still contaminated with large amounts of T N T and derivatives. The persistence of these compounds in natural environments results in major problems with land use. Some of these sites are now used as real estates or even gardens. Since T N T can enter the food chain via plants, the uptake of T N T in plants is of great interest. Several studies examining the fate of T N T in' plants have revealed uptake of T N T (PALAZZO & LEGGETT 1986, FOLSOM et al. 1988, PENNINGTON 1988, CATALDO et al. 1989, HARVEY et al. 1990). The major identified products were the aminodinitrotoluene isomers, 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT). Studies examining the uptake of 14C T N T in bush beans indicated that adsorbed T N T and derivatives underwent major transformation. Large quantities of 14C were either bound to the pelleted plant residues or were found to be water soluble (HARVEYet al. 1990). Up to now there ESPR-Environ. Sci. & Pollut. Res. 1 (4) 229-233 (1994) 9 ecomed publishers, D-86899 Landsberg, Germany
Material and methods Chemicals
2,4,6-Trinitrotoluene was obtained from Dr. E.v.L0w, Hygiene Institut, Philipps University Marburg. 2-ADNT and 4-ADNT were synthesized by Dr. K. STHNBACH,Fachbereich Chemie, Philipps University Marburg. 2.2
Plant cultivation
The uptake and metabolism of T N T of plants were evaluated using Medicago sativa cv. Du Puits and Allium schoeno-
prasum. Seeds were washed with 0 . 1 % Twen 20, rinsed several times and then surf~ice sterilized by incubation with 30 % H202 for 10 min and subsequently washed with sterile water. Seedlings were germinated for two days on NB (Nutrient broth, Difco laboratories) agar plates to visualize contamination with bacteria or fungi. Uncontaminated seedlings were carefully transferred to the hydroponic system and were grown for 28 days in a growth chamber with 16 h, 25 ~ light period and 8 h, 20 ~ dark period. The plants were grown in sterile 100 ml test tubes containing 20 ml of glass beads ( 4 - 7 mm) to support the plants. The test tubes were partly shielded with black plastic tubes to minimize photolysis of T N T and to protect the roots from light. To avoid bacterial contamination they were dosed with a sterile plug of cotton wool and an aluminium cap. The nutrient solution used in all test tubes was a mineral medium modified from BROUGHTON& DILWORTH(1971) supplied with 0.5 mM KNO 3. The initial pH was 6.8. Stock solutions of T N T were prepared by adding 100 mg ground T N T to one litre of autoclaved medium. The solution was treated in an ultrasonic bath for 5 min and then stirred until the T N T had completely dissolved. The stock solutions were diluted to final T N T concentrations by adding autoclaved medium. The different T N T concentrations
229
Uptake of TNT in Plants
Research Articles
selected for study were 0, 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 mg/1. Each experiment was conducted twice with at least 3 replicates. Solutions were analysed for pH, unpolar nitrotoluenes and watersoluble aromatic amines immediately after contamination with T N T and at the time of harvest. Sterility was proven by plating an aliquot on NB agar plates. For analysis of nitrotoluenes the hydroponic solutions were extracted twice with methylene chloride. The extract was dried over anhydrous sodium sulfate, evaporated to dryness, dissolved with a small amount of ethyl acetate and subjected to analysis by GC/ECD. The aqueous solution, after methylene chloride extraction, was used for analysis of aromatic amines using a p-nitroanilin standard as representative of the overall aromatic content as described by v. LOw & HAAs (1986). After 28 days of growth, plants Were removed from the hydroponic solutions and separated into roots and shoots. The roots were rinsed first with water followed by methanol. After measurement of length and weight, plants were freezedried, minced with a dismembrator (Braun, Melsungen, F R G ) and stored at - 2 0 ~ until analysis. A 100 mg aliquot of freeze-dried plant material was extracted for 30 min in an ultrasonic bath with 4 ml of methylene chloride twice. After centrifugation the supernatant was removed, evaporated to dryness, redissolved in ethyl acetate and analysed with GC/ECD. The pellet was subjected to acid hydrolysis with 25 % H2SO 4 by immersing the tube in a 95 ~ water bath for 90 min (P~AZZO & LGGETT 1986). After cooling to room temperature the hydrolyzed material was extracted twice with 2 ml methylene chloride. The phases were separated by centrifugation for 10 min at 3 000 g. After removal of the methylene chloride layer, the acid layer was made basic by the addition of 8 M N a O H and extracted again with methylene chloride. The phases were again separated by centrifugation and the methylene chloride layer was combined with the methylene chloride-extract of the acid hydrolysis. After drying over anhydrous sodium sulfate, evaporation to dryness and redissolving in ethyl acetate the sample was subjected to further clean up or directly analysed by GC/ECD. Methylene chloride extracts of green plant parts were purified either by solid phase extraction with Adsorbex SCX-columns
180 -6 ~- 1 6 0
A.schoenoprasum
(Merck, Darmstadt, FRG) to remove chlorophylls, or Florisil columns as described by HARVEYet al. (1990). The columns had been previously washed with methanol and conditioned with methylene chloride. An additional 2 ml of methylene chloride was used to rinse the remaining sample out of the sample vial to the column. The eluate was evaporated to dryness, dissolved in ethyl acetate and subjected to analysis with GC/ECD.
2.3
Residue analysis
For analysis of all samples a gas chromatograph (Varian model 3 400) with ~3Ni electron capture detector was used. Some of the samples were additionally analysed by means of GC/MS. A 1/al aliquot of the ethyl acetate extract was injected (split 1:20) onto a 30 m DB-5, 25/am I.D. column containing a 1.0/~m film of stationary phase (J&W Scientific, Folsom, CA, USA). The components were separated with helium carrier gas using a temperature program from 140 ~ to 240 ~ at 10 ~ and 240 ~ to 260 ~ at 5 ~ Mass spectrometer studies utilized a Finnigan ITD 800, full scan or single scan modus, 70 eV, interfaced to a Varian 3 400 gas chromatograph, equipped as described above. Spiked tissue extracts with 100 pg T N T , 4-ADNT and 2-ADNT per/al were used as external calibration standards.
Results and discussion
3
3.1
Plant growth
Initial concentrations of 50 mg/l or more restricted plant growth totally. Root growth was most influenced by the presence of T N T . Visible symptoms of injury to roots consisted of growth restriction and browning of the root tips. Root length of Medicago sativa was reduced at 10 mg/1 ( ~ Fig. 1). Higher concentrations affected the shoot length as well. Allium schoenoprasum displayed symptoms of root damage already at lower levels of TNT. The root length was significantly reduced at concentrations of 5 mg/1. PALAZZO& LEGGETT (1986) also reported inhibition of root growth of Cyperus esculentus at 5 mg/1 in hydroponic solution, whereas duckweed was sensitive to T N T at concentrations of 1 mg/1 (ScHoTT & WORTHLEY1974). Thus some plants seem to be
M.sativa
c
o u 140 ~- 1 2 0 O
lOO _c
80
c
60
2
40
Fig. 1:
20 0
0
0.1
0.5
1.0
initial
230
5.0
concentration
0
0.1
o f TNT
0.5
.0 5 . 0
[r~g/t]
10.0
Length of roots and shoots of Allium schoenoprasum and Medicago sativa in hydroponic solution containing different TNT levelscompared to a control after 28 days of growth ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Research Articles
Uptake of TNT in Plants
more tolerant to T N T than others. TUCKERet al. (1989) investigated tolerance of different plant species to T N T , but there was no indication, that monocots in general are more tolerant to T N T than dicots.
Table 1: Quantity of T N T , 4-ADNT and 2-ADNT at the beginning of the study (to) and at the time of harvest (28 day) in hydroponic solutions TNT-level 0.0
3.2
Analysis of hydroponic solutions
The hydroponic solutions were analysed immediatly after addition of TNT. The recovery of nitrotoluenes was more than 95 %. Although only T N T was added, 6.7 % • 1.7 % of the initially analysed nitrotoluenes were identified as the aminodinitrotoluenes (ADNTs). No increase in formation of ADNTs could be observed in control solutions even after 4 weeks, although the total amount of detectable nitrotoluenes decreased slightly (--' Table 1). This indicated transformation of T N T to compounds that can not be extracted and analysed with the described method. Analysis of aromatic amines showed a slight increase even for the control tubes ( ~ Fig. 2). Products were not subjected to further analysis.
C~
0.4-
E 61
~
control A.schoenoprusum ~M.sativa
0.5
1.0
5.0
hydroponic solution to TNT 0.000g1 0 . 0 6 8 f g 4-ADNT 0.000e 2-ADNT 0.000e
0.004e 0.001e
control solution (28 day) TNT 0.000g 0.031fg 4-ADNT 0.000e 2-ADNT 0,000e
10.0 mg/I
0,001e 0,001e
0.407e 0.747d O.019cde 0.033c 0.010de 0.015de
3.787a 0.192a 0.111b
0,347e 0.370e 3,532b O.015cde O.015cde 0.119b O.012de 0,009de 0.095bc
9.617a~ 0.529a 0.269a 6,120b 0.262b 0.177b
Allium schoenoprasum (28 day) TNT 0,000g 0.014g 0.057 fg 4-ADNT 0.000e 0,004e 0.011de 2-ADNT 0,000e 0.006e 0.036d
0.151f 0.029 cd 0.072c
1.299c 0.113b 0,202a
-
Medicago sativa (28 day) TNT 0.000g 0.000g 4=ADNT 0.000e 0,000e 2-ADNT 0,000e 0,000e
0.000g 0.001e 0.002e
0,007g 0.035c 0.013de
0,046c 0,082c 0.054c
0.000g 0.002e 0.000e
Means for each compound followed by the same letter are not significantly different at the 0.05 level by WELLER/DUNCANtest. 2 Statistical tests for TNT level 10 mg/l were done separately. - not included
0.3
E 0
0.1
mg/I
0.2
4-, o
E 0.1 o 0
0.0
0 0.1 0.5 initial concentration
1,0 5,0 o f TNT [ m g / I ]
Fig. 2: Concentration of aromatic amines using a p-nitroaniline stand a m as representative of overall aromatic content in hydroponic solution containing different T N T levels after 28 day of incubation. T h e solutions were analysed for aromatic amines after extraction with methylene chloride.
High concentrated T N T solutions ( > 10 mg TNT/1) that were exposed to growth chamber lights turned brown to pink, probably due to the formation of a photodecomposition product. These products have already been observed and characterized by SPANGGORD et al. (1980) and have also been found in hydroponic solutions by HARVEY et al. (1990). This might also be a reason for the observed decline of nitrotoluenes in control solutions. Analysis of solutions used to support plant growth showed a major decrease of nitrotoluenes, dependent on the plant species ( ~ Table 1). In the case of Allium scboenoprasum with initial concentrations of 0.1 to 5 mg TNT/1 only 25 % to 39 % could be analysed as nitrotoluenes after 28 days of growth. Only 63.9 % + 15 % of the extracted nitrotoluenes were identified as T N T , 1 0 . 1 % + 2 . 9 % or 26.2 % • 12.2 % were analysed as 4-ADNT or 2-ADNT, respectively (n = 9).
ESPR-Environ. ScL& Pollut. Res. 1 (4) 1994
In tests with Medicago sativa, the decline of T N T was much higher ( ~ Table 1). At initial concentrations of 5 and 10 mg TNT/1, only 1.3 % and 8.7 % of the initial concentration could be analysed as nitrotoluenes after 28 d of growth. Of the total detectable amount, 48 % • 14 % was identified as 4-ADNT and 38.5 % • 18 % as 2-ADNT. That means that the majority of the initial T N T was transformed to the aminodinitrotoluenes or other compounds. T N T represented less than 11.8 % • 10 % of the total nitrotoluene fraction. At the time of harvest we found a dose-dependent increase of aromatic amines in the hydroponic solution ( ~ Fig. 2), probably also due to metabolism of the added T N T . During growth of Medicago sativa the pH of the solution dropped from pH 8.5 (control solution) to 6.9, indicating metabolic activity of the roots. For Allium schoenoprasum the pH also declined to pH 7.6, except for the highest T N T level (5 mg/1, pH 8.4), indicating only minor activity. This is in accordance with the restricted root growth for that T N T level. It is not yet proven, whether the formation of aminodinitrotoluenes and unknown products is due to root catalyzing effects or is a consequence of the metabolic activity of bacteria. Control of sterility of all test tubes revealed minor contamination with fungi or bacteria in some of the tubes. However, there was no correlation of significant differences in T N T concentrations with any detectable growth of microorganisms. In additional experiments one isolate of bacteria was able to transform T N T to the aminodinitrotoluenes and to unknown substances if it was grown in T N T containing NB-medium (10 mg/1). Incubation in mineral medium without the additional supply of C-sources, revealed only minor growth and no significant decrease of the initial
231
Uptake of TNT in Plants
Research Articles
TNT-level (unpublished results), which is in accordance with results of other authors (AME~,HANOVA & NAUMOVA 1978, OSMON & KLAUSMEmV.1972, P~uss et al. 1993). They suggested that T N T cannot be used as the sole C-source and is metabolized only by cometabolism. In our experiments root exudates could have provided C-sources for bacteria (WHIPPS 1990). Therefore, it is not possible to confirm whether transformation of T N T was due to root catalyzing effects or mediated by bacterial activity. 3.3
Uptake of TNT in plants
Extraction of plant material was always performed in three steps. After extraction with methylene chloride, the remaining pellet was subjected to acid hydrolysis and extracted again with methylene chloride, followed by a basidification with 8 M N a O H and a second extraction with methylene chloride. The most effective extraction step for spiked plant tissue, as well as for plants grown in T N T containing hydroponic solution or soil, was the acid hydrolysis ( - Table 2). This is in accordance with results of HARVEY et al. (1990), who also found acid hydrolysis to be very efficient. Table 2: Proportion of nitroaromatics and standard deviation of different extraction steps of root samples (n = 10) of Medicago sativa grown in contaminated sand (100 m g / k g ) for two months. TNT
4-ADNT % proportion
2-ADNT
16,9_+7,0 80,0 _+9,7 3,0+2,7
2,9_+ 5,7 63,3 _+26,4 33,8_+24,0
4,3_+ 5,7 78,2 + 25,9 17,5_+16,8
99.9
100.0
100.0
extract methylene chloride H2SO 4 NaOH total
The detectable amount of nitroaromatics in plant tissue was correlated to the initial concentration of T N T in the hydroponic solution ( ~ Tables 3, 4). Thus the amount that can be taken up by plants in soil is dependent on the bioavailability of T N T , especially in the soil solution. Factors controlling the adsorption/desorption in soils are therefore also controlling the bioavailability of T N T and the amount that can be taken up by plants. Significant accumulation in roots was detected at an initial concentration of 0.5 mg TNT/1. At this concentration no visible symptoms of injury occurred.
Table 3: Detectable concentration of T N T , 4 - A D N T and 2-ADNT in shoots and roots of Allium schoeneprasumand Medicagosativa after growth in hydroponic solution with different TNT-levels shoots
0.0
0.1
0.5 1.0 5.0 nitrotoluenes in shoots #gtg dryweight
Allium schoenoprasum TNT < 0.110c~ < 0.110c 4-ADNT < 0.110d < 0.110o' 2-ADNT < 0.110c < 0.110c
< 0.110c 0.350cd 0,583c
0.460b 0.693bc 1,870b
0.442b 1.677a 5.659a
Medicago sativa TNT < 0.033c 9 .ADNT < 0.033d 2-ADNT < 0.033c
< 0.033c 0.045d 0.039c
0.078c 0.102d 0.102c
0.860a 1.026b 0.885c
< 0.033c 0.045d 0.039c
1 Means for each compound followed by the same letter are not significandy different at the 0.05 level by WALLER/DUNCANtest. Means for control are detection limits.
roots
0.0
0.1
0.5 1.0 5.0 10.0 nitrotoluenes in roots pg/g d r y w e i g h t - -
Allium schoenoprasum TNT < 0.110o1 3.187d 4-ADNT < 0.110d 6.220d 2-ADNT < 0.110d 4.319d
Clean up procedures using SCX as well as Florisil solid phase extraction columns failed to retain the nitrotoluenes. So SCXcolumns were only used to remove chlorophyll from the extract. To evaluate the efficiency of the analytical method 1 ppm of T N T , 4-ADNT and 2-ADNT were added to freeze-dried material of Medicago sativa and then extracted as described. The recovery of the extraction method with methylene chloride was about 100 % for both T N T and 2-ADNT and 75 % for 4-ADNT. Acid hydrolysis released additional absorbed and transformed nitrotoluenes from plant tissue grown in contaminated substrate. Spiked tissue extracts were used as external standards and also to evaluate the calibration standards of the GC/ECDsystem. The ECD signal was linear for the range of 10 to 500 pg T N T , 4-ADNT and 2-ADNT per/A and was not disturbed by dry matter contents of up to 250 mg/ml. After 28 days of growth, plant roots and shoots were analysed for T N T and the aminodinitrotoluenes 2-ADNT and 4-ADNT. The detection limits for each of these compounds are given in Table 3.
232
Medicago sativa TNT < 0.025d 4-ADNT < 0.033d 2-ADNT < 0.033d
0.022d 1.142d 0.559d
7.289bcd 5.705bcd22.783a 10.780cd 19.222c 76.077ab 11.151c 21.610c 69.900ab 1.344d 3.811cd 12.77"3bc 13.811ab 7.622cd 12.521c 40.050bc 94.731a 8.173cd 15.471c 34.820bc 82.790a
1 Means for each compound followed by the same letter are not significantly different at the 0.05 level by WALLER/DuNcANtest. Means for control are detection limits. - not included
Table 4: Correlation coefficient for the content of nitroaromadcs in different plant organs in relation to the initial concentration of T N T in hydroponic solution species
plant part
A. schoenoprasum
root 2 shoot 2 root 3 shoot 2
M. sativa
1 after PEARSON
20.1 tO 5 mg TNT/I
n
correlation coefficient 1
15 15 26 12
0.941 0.921 0.765 0.977
30.1 to 10 mg TNT/I
ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Research Articles T h e m o n o c o t Allium schoenoprasum g r o w n in h y d r o p o n i c solution with 1 m g or 5 mg T N T / I showed a larger quantity o f detectable a m m u n i t i o n residues in roots and shoots c o m p a r e d to the dicot Medicago sativa. This could reflect a p l a n t specific difference in u p t a k e , accumulation or m e t a b o l i s m . C A T , D O et al. (1989) found the u p t a k e o f T N T to be higher in the dicot Phaseolus vulgaris t h a n in the e x a m i n e d m o n o c o t s Bromus rnollis a n d Avena sativa, and suggested that there might be principal differences between dicots and monocots. However, our results showed that there was no such difference in b e h a v i o u r between dicots and monocots. It is clear from the d a t a o f b o t h investigated p l a n t species that the m a j o r i t y of detectable nitrotoluenes were extracted from the r o o t tissues. F o r Allium schoenoprasum at initial T N T levels o f 5 m g / l the concentration of nitrotoluenes in roots was m o r e than 20 times higher than in shoots. F o r Medicago sativa the detectable concentration in r o o t s was m o r e than 30 times higher. A similar distribution o f T N T metabolites and residues was also found by PALAZZO & LEGGETT (1986) a n d was c o n f i r m e d in 14C experiments by H a r v e y et al. (1990). By analysis of r o o t tissues o f Medicago sativa only 9 % + 8.9 % o f the total detectable a m o u n t could be identified as T N T . 4 - A D N T and 2 - A D N T were found at equal concentrations of about 40 % of the total amount each. That means that the p a t t e r n o f distribution of nitrotoluenes in h y d r o p o n i c solution and p l a n t tissues are different. W h e t h e r this is due to a difference in u p t a k e or due to m e t a b o l i s m in p l a n t tissue still has to be elucidated.
4
Conclusions
In this study a m e t h o d was described to investigate u p t a k e of T N T in plants. T h e u p t a k e a n d accumulation seems to be dependent on p l a n t species, p l a n t organs and the initial T N T concentration o f the substrate. Since T N T can enter the food chain via plants, there is a need to investigate u p t a k e o f T N T under field conditions.
5
Uptake of TNT in Plants
DJERASSI,L. S.; L. VIT,~r Haemolytic episode in G6-PD deficient workers exposed to TNT. Brit. J. Ind. Med. 32, 54 (1975) FOLSOM,B. L.; J. C. PENNINGTON;S. L. TEETER;M. R. BARTON;J. A. BRIGHT: Effects of soil pH and treatment level on persistence and plant uptake of 2,4,6-trinitrotoluene. Waterways Experiment Station, Corps of Engineers, Vicksburg, MS, Technical Report EL-88 - 22 (1988) GRANT,W. M.: Toxicology of the eye. Charles C. Thomas Publishers, Springfield Illinois 1986, p. 953 HaTHaWAY,J. A.: Trinitrotoluene - a review of reported dose-related effects providing documentation for a workplace standard. J. Occupa. Med. 19, 341 (1977) H.~VEY, S. D.; R. J. FELLOWS;D. A. CAT,a-DO;R. M. BEAN:Analysis of 2,4,6-trinitrotoluene and its transformation products in soils and plant tissues by high-performance liquid chromatography. J. Chromato. 518, 361 (1990) Koss, G.; A. LOMMEL;I. OLLROGE;I. TESSERAUX;R. HAAS; A. D. KAVPOS: Zur Toxikologie der Nitrotoluole und weiterer Nitroaromaten aus rfistungsbedingten Altlasten. Bundesgesundhbl. 527 (1989) OSMON, J. L.; R. E. KLAUSMEIER:The microbial degradation of explosives. Dev. Ind. Microbiol. 14, 247 (1972) PALAZZO,A. J.; D. C. LEGGETT:Effect and disposition of TNT in a terrestrial plant. J. Environ. Qual. 15, 49 (1986) PENNINGTON, J.C.: Plant uptake of 2,4,6-trinitrotoluene, 4-amino-2,6-dinitrotoluene, and 2-amino-4,6-dinitrotoluene using 14C-labeled and unlabeled compounds. US Army Engineer Waterways Experiment Station, Miss Vicksburg: Technical Report EL-88-20th (1988) PREUSS, A.; J. FIMPEL;G. DIEKERT: Anaerobic transformation of 2,4,6-trinitrotoluene (TNT). Arch. Microbiol. 159, 345 (1993) SAS Institute: SAS/STAT Guide for Personal Computers, Version 6.03 Edition 1988 SCHOTr, C. D; E. G. WORTHLEY:The toxicity of TNT and related wastes to an aquatic flowering plant, Lemnaperpusilla. Edgewood Arsenal, Aberdeen Prov. Ground, MD. Techn. report 74016. AD-778158 (1974) SMOCK,L. A.; D. L. STONEBURGER;J. R. CLARK:The toxic effects of trinitrotoluene (TNT) and its primary degradation products on two species of algae and the fathead minnow. Water Res 10,537 (1976) SPANGGORD,R. J.; T. MILL;T.-W. CHOU; W. R. MAYBE;J. H. SMITH; S. LEE: Environmental fate studies on certain munition wastewater constituents. Final report. Phase II, Laboratory studies, SRI International, Menlo Park. CA (1980) TUCKER, W. L.; W. L. BANWART;T. C. GRANATO;J.J. HASSETT: Screening for plant tolerance to TNT. Report for USATHAMA by CERL and the University of Illinois (1989) V. LOw, E.; L. KAMINSKI;W. NEUMEIER; R. HAAS;K. STEINBACH: Mikrobieller Abbau von Nitroaromaten aus einer ehemaligen Sprengstoffproduktion. Forum St~idte-Hygiene 40, 347 (1989) WHIPPS,J. M.: Carbon economy. In: J. M. LYNCH,The rhizosphere. John Wiley & Sons, Chichester 1990, pp. 59
Literature
A M E ~ O V A , N. N.; R. P. NAUMOVA:2,4,6-Trinitrotoluene as a source of nutrition for bacteria. Microbiology 47, 318 (1978) BROUGHTON, W. J.; M. J. DtLWORTH:Control of leghaemoglobin in snake beans. Biochem. J. 125, 1075 (1971) CATALDO,D. A.; S. D. HARVEY; R. J. FELLOWS;R. M. BEAN; B. D. McVEETY: An evaluation of the environmental fate and behavior of munitions material (TNT, RDX) in soil and plant systems. Pacific Northwest laboratories, Richland, Report AD-A223 546 (1989)
ESVR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Received:April 7, 1994 Accepted:June 6, 1994
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