Ecotoxicology and Environmental Safety 112 (2015) 54–59

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Biodegradation of 2,4-dinitrotoluene by different plant species Radka Podlipná a, Blanka Pospíšilová b, Tomáš Vaněk a,n a b

Laboratory of Plant Biotechnologies, Institute of Experimental Botany, Czech Academy of Sciences, Praha 6, Czech Republic Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic

art ic l e i nf o

a b s t r a c t

Article history: Received 22 May 2014 Received in revised form 23 July 2014 Accepted 24 July 2014

Over the past century, rapid growth of population, mining and industrialization significantly contributed to extensive soil, air and water contamination. The 2,4-dinitrotoluene (2,4-DNT), used mostly as explosive, belongs to the hazardous xenobiotics. Soils and waters contaminated with 2,4-DNT may be cleaned by phytoremediation using suitable plant species. The ability of crop plants (hemp, flax, sunflower and mustard) to germinate and grow on soils contaminated with 2,4-DNT was compared. Stimulation of their growth was found at 0.252 mg/g 2,4-DNT. The lethal concentration for the growth for these species was around 1 mg/g. In hydropony, the above mentioned species were able to survive 200 mg/l 2,4-DNT, the concentration close to maximal solubility of 2,4-DNT in water. Metabolism of 2,4-DNT was tested using suspension culture of soapwort and reed. The degradation products 2-aminonitrotoluene and 4-aminonitrotoluene were found both in the medium and in the acetone extract of plant cells. The test showed that the toxicity of these metabolites was higher than the toxicity of the parent compound, but 2,4-diaminotoluene, the product of next reduction step, was less toxic in the concentration range tested (0–200 mg/l). & 2014 Published by Elsevier Inc.

Keywords: 2,4-Dinitrotoluene Degradation Phytotoxicity Phytoremediation

1. Introduction The production, use, and disposal of explosives, agricultural chemicals, pharmaceuticals, dyes, and plastics have led to a wide scale contamination of the environment. Environmental contamination by nitrocompounds is associated principally with the explosive industry. Modern explosives are nitrogen containing organic compounds with the potential for self-oxidation to small gaseous molecules (N2, H2, O2, and CO2). Many are polynitroaromatic compounds including 2,4,6-trinitrotoluene (TNT; which for many years dominated the explosive industry), 1,3,5-trinitrobenzenene (TNB), dinitrotoluenes (2,4-DNT, 2,6-DNT), dinitrobenzene (DNB), N-methyl-N,2,4,6-tetranitroaniline (Tetryl), and 2,4,6-trinitrophenol (picric acid). Aromatic nitro compounds are resistant to chemical or biological oxidation and to hydrolysis because of the electron-withdrawing nitrogroups (Achtnich et al., 1999; Rieger and Knackmuss, 1995) Dinitrotoluenes (DNT) are the precursors of TNT, once of the most widely manufactured military explosive in the world. DNT

Abbreviations: 2,4-DNT, 2,4-dinitrotoluene; 2,6-DNT, 2,6-dinitrotoluene; 2A-NT, 2-amino,4-nitrotoluene; 4A-NT, 4-amino,2-nitrotoluene; TNT, 2,4,6-trinitrotoluene n Correspondence to: Laboratory of Plant Biotechnologies, Institute of Experimental Botany, Czech Academy of Sciences, Rozvojová 263, Praha 6, Czech Republic. Tel.: +420225106832. E-mail address: [email protected] (T. Vaněk). http://dx.doi.org/10.1016/j.ecoenv.2014.07.026 0147-6513/& 2014 Published by Elsevier Inc.

contamination at military sites dates back to the World War I. Due to the relatively wide distribution of TNT manufactures, many areas became contaminated with mono- and dinitrotoluenes. While TNT production was decreased, DNTs have remained an important industrial chemicals, used as the precursors of toluene diisocyanate and methylene diphenyldiisocyanate in the production of polyurethane foams (Nishino et al., 2000). For example in China, more than 500 plants have until now produced TNT. This process generates a high number of intermediates, such as mononitrotoluenes and dinitrotoluenes, which cause pollution of water resources (Xu and Jing, 2012). Commercial grade DNT consist mainly of 2,4-dinitrotoluene (2,4-DNT, 76 percent), and 2,6-dinitrotoluene (2,6-DNT, 20 percent). Both main isomers are priority pollutants in the US-EPA (United States Environmental Protection Agency) list. Toxicity of these compounds to humans and animals has been documented (Tchounwou et al., 2003; Karnjanapiboonwong et al., 2009; Xu and Jing, 2012). DNT isomers are known to cause cancer, to lower sperm number and to reduce fertility in laboratory animals (Rickert et al., 1984). Lent et al. (2012) compared the subacute toxicity of individual DNT isomers using male SpragueDawley rats. The exposure to 2,4-, 2,6- and 3,5-DNT resulted in decrease of their tested mass and degenerative histopathological changes. Toxicity of DNTs to terrestrial plants was described by Rocheleau et al. (2006). Phytotoxicity tests showed that dinitrotoluenes are more toxic for all tested plant species than TNT. Results

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from Tradescantia micronucleus (Trad-MCN) bioassay indicated that 2,4-DNT is genotoxic in minimum effective dose of 30 mg/l (Gong et al., 2003). Degradation of DNT was initially described in the mixed population of microorganisms enriched from DNT-contaminated surface water (McCormick et al., 1978). The degradation mechanism has been, however, elucidated later on. The DNT has been found biodegradable, via oxidative or reductive pathways (Kaplan, 1992). The oxygenase or peroxidase enzymes were reported to initiate ring cleavage (Christopher et al., 2000). The reductive pathway results in formation of metabolites such as 2-amino-4-nitrotoluene (2A-NT), 4-amino-2-nitrotoluene (4A-NT), 2,4-diaminotoluene (DAT), azoxytoluene isomers, and 4-acetamido-2-nitrotoluene (McCormick et al., 1978; Freedman et al., 1996; Hudcová et al., 2011). In anaerobic systems, 2,4-DNT was transformed to aminonitrotoluenes 2A-NT and 4A-NT via nitrosonitrotoluenes (2-NO-4NT and 4-NO-2NT). The aminonitrotoluenes were further transformed to some unknown products (McCormick et al., 1978; Liu et al., 1984). The second pathway results to dihydroxylamino intermediates (Hughes et al., 1999). Cheng et al. (1996) reported that 2,4-DNT was reduced to DAT with ethanol as the primary substrate. On that results followed up with their study Wang et al. (2011). For efficient degradation of 2,4-DNT they used an anaerobic filter combined with biological aerated filter and ethanol as the electron donor. The toxicity of DNT degradation products was determined by Sax and Lewis (1989). In rats, the oral LD50 values for 2A-NT, 4A-NT, and 2,4-DAT were 574, 6860 and 260 mg/kg, respectively (Sax and Lewis, 1989). Houpt et al. (2013) focused on the toxicity of 4A-NT for rats. The lowest observed adverse effect level was 27 mg/kg/d of 4A-NT in consumed doses for male and 32 mg/kg/d for female rats based upon decreased body weight gain. The toxic effects of 2,4-DNt and its metabolites were examined using the 15-min Microtox (Vibrio fischeri) assay and 96-h fresh water green alga test. The Microtox assay showed the order of toxicity for 2,4-DNT and metabolites: 4A-NT4 2A-NT42,4-DNT42,4-DAT, in case of the algae test the order was: 2,4-DNT42,4-DAT
4A-NT¼ 2A-NT (Dodard et al., 1998). In spite of the fact that 2A-NT, 4A-NT and 2,4-DAT are toxic, these compounds are not specifically screened in the environment. Different microorganisms have been used as biological systems for remediation of sites and waste waters contaminated by such environmentally toxic compounds (Spanggord et al., 1991; Bradley et al., 1994, 1997; Boopathy, 1994; Cheng et al., 1996; Haidour and Ramos, 1996; French et al., 1999; Zhang et al., 2000; Hudcová et al., 2011). In addition to microorganisms, also plants can be used to remove, transform, or accumulate toxic chemicals from the soils, sediments, ground waters, surface waters, and even the atmosphere. Currently, phytoremediation is used for elimination of many classes of contaminants, including petroleum hydrocarbons, chlorinated solvents, dyes, pesticides, explosives, heavy metals and radionuclides, and landfill leachates (Rylott and Bruce, 2009; Podlipná et al., 2010, Watharkar and Jadhav, 2014). According to Best et al. (1997), approximately 80 percent of the polluted ground waters occur within 20 m of the ground surface. This suggests that a significant number of sites are potentially suitable for low cost phytoremediation (Susarla et al., 2002). Phytoremediation of nitroaromatic pollutants is aimed to degrade them into less- or non-toxic compounds and to diminish their further movement by sequestration and accumulation in plants. The fate and metabolism of TNT as the most common explosive has been studied extensively and its degradation products were determined in many plant systems (Panz and Miksch, 2012; Rylott and Bruce, 2009; Vaněk et al., 2003; Hannink et al., 2002). The DNT isomers, however, still need to attract the attention. Su and Zhu (2007) described uptake of DNT by rice roots and demonstrated that DNT transport is achieved mainly via the symplastic pathway. Su and Liang (2011) gave direct evidence of

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transport of 2,4-DNT from roots to shoots via xylem. 2,4-DNT concentration in wheat and tomato seedlings were linearly correlated with their concentration in external solution and also dependent on the transpiration rate. In contrast to microorganism systems, little information is available on the transformation of 2,4-DNT by plants. Yoon et al. (2006) indicated monoaminonitrotoluenes as 2,4-DNT transformation products in Arabidopsis seedlings. We focused on the optimizing of phytoremediation as a progressive cleaning method for the waste waters and soils in the areas of present or former ammunition plants. At first we determined the toxic effects of 2,4-DNT on the plant species potentially suitable for remediation (several crop plants and wetland plants). In the next step we followed the metabolism of 2,4-DNT in the plant species surviving on contaminated area (soapwort and reed). Finally, the phytotoxicity of 2,4-DNT degradation products was determined.

2. Materials and methods 2.1. Plant material The seeds of Helianthus annuus L., Sinapis alba L (AROS-osiva, Prague, CZ), Linum usitatissimum cv. Viola and Cannabis sativa cv. Beniko (Agritec, Šumperk, CZ) were used for modified standard mustard germination test (CSN EN ISO 7346-2). Twenty five seeds were placed in Petri dishes (diameter¼100 mm) on the filter paper soaked with5 ml of nutrient medium (588 mg/l CaCl2.2H2O, 246 mg/l MgSO4.7H2O, 129 mg/l Na2HCO3 and 11.5 mg/l KCl) supplemented with 2,4-DNT (50, 100 and 200 mg/l) and germinated in growth chamber on 24 1C in dark. The control seed were growing on untreated nutrient medium on the same condition. The number of germinated seeds was calculated after 3 days. Each treatment had three replicates. In the area of the ammunition plant, the soil samples were taken from the contaminated place completely without plant cover. The concentration of 2,4-DNT in that sandy soil was about 9 mg/g. In the experiments the soil was mixed with fresh substrate with the aim to decrease 2,4-DNT concentration. The seeds (2  25) of crop species mentioned above were sowed into flowerpots with the modified soil (4.75 mg/g, 1.36 mg/g and 0.25 mg/g of 2,4-DNT) and regularly watered. The plants were grown in greenhouse. The clear substrate was used as the control soil. The number of germinated seeds was counted after 3 days. After 60 days the plant were harvested and the fresh mass was determined. The seeds of soapwort (Saponaria officinalis) and ragwort (Senecio jacobaea) were harvested from the plants growing in the area of ammunition plant contaminated by a wide spectrum of nitrocompounds. Sterilized seeds of ragwort were germinated and seedlings were grown aseptically in Magenta boxes on the Murashige and Skoog (1962) medium without hormones, at 25 1C, with a16-h photoperiod at 72 mmol/m2/s. Callus culture of soapwort was initiated from the surface sterilized seeds setting on the basal MS medium supplemented with 1.14 mg/l 2,4-D, 0.5 mg/l BAP and 0.43 mg/l kinetin and solidified with agar (0.7 percent). The callus culture was transferred into the liquid medium and cultivated in Erlenmeyer’s flasks on horizontal shaker in the dark at 25 1C to obtain suspension cultures. The suspension culture was subcultured at 2-week interval. Ten g of aseptically filtered cell mass was inoculated to 100 ml of fresh medium in every flask. After 5 days of growth, the medium was supplemented with 2,4-DNT (final concentration 50 mg/l, 274 mM stock in methanol). Callus culture of reed (Phragmites australis) was initiated from the surface sterilized seeds setting on the Hoagland (1920) medium supplemented with 0.225 mg/l 2,4-D, and 0.215 mg/l kinetin. The callus culture was transferred into the liquid medium and cultivated in Erlenmeyer’s flasks on horizontal shaker in the dark at 25 1C to obtain suspension cultures. The suspension culture was subcultured at 2-week interval. Ten g of aseptically filtered cell mass was inoculated to 100 ml of fresh medium in every flask. After 5 days of growth the medium was supplemented with 2,4-DNT (final concentration 50 mg/l, 274 mM stock in methanol).

2.2. Determination of content of 2,4-DNT and degradation products The uptake of DNT by cell suspension was determined in the medium and in acetone extract from the plant cells using HPLC. The medium from the suspension culture was filtered out using a Buchner funnel and 2 ml of the medium was centrifuged (13,000 g, 2 min) to separate the impurities and directly analyzed by HPLC. The separated cells (approximately 20–30 g fresh mass) were resuspended and extracted using orbital shaker (50 rpm) with 50 ml acetone for 24 h. The

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filtered acetone extract was evaporated and the solid residue was dissolved in 2 ml methanol. The samples were analyzed on a reverse phase SiC18Biospher packed stainless steel column (250  4 mm). A linear gradient of methanol (10–100 percent) was applied for 25 min at a flow rate 1 ml/min. Substances were detected comparing spectra and retention times of samples and standards using detector Finnigan Spectra System UV6000 (Thermo electron corporation, USA). The content of known compounds was calculated from calibration curve of corresponding standards at 230 nm. The HPLC detection limit for all compounds was 0.1 mg/l. All experiments were done in triplicate and standard deviations were determined. 2.3. Test of toxicity We examined phytotoxicity of 2,4-DNT and its degradation products using the standard mustard germination test (CSN EN ISO 7346-2). Seeds (25) of white mustard (S. alba L.) germinated in Petri dishes on filtration paper moistened with cultivation medium (588 mg/l CaCl2.2H2O, 246 mg/l MgSO4.7H2O, 129 mg/l Na2HCO3 and 11.5 mg/l KCl) supplemented with different concentrations (0, 5, 10, 20, 50, 100 mg/l) of 2,4 DNT; 4A-NT or 2A-NT. The seeds sprouted in cultivated room at dark and 24 1C. The length of roots was measured after 3 days. 2.4. Mathematical treatment of data Data were processed using STATISTICA.CZ Version 12.0 (StatSoft, Prague, Czech Republic). Results are expressed as mean 7 standard deviation. Statistical significances of the differences were determined using STATISTICA.CZ. Differences with p o0.05 were considered significant and were determined by using of one way ANOVA test (particularly the Tukey test), which was applied for means comparison.

3. Results and discussion 3.1. Phytotoxicity of 2,4-DNT Both 2,4- and 2,6-DNT are highly toxic to different organisms including rats (Lent et al., 2012), carp (Zheng and Xu, 2012; Shen et al., 2010), reptile (Suski et al., 2008), amphibian (Paden et al.,2011) and invertebrates (Karnjanapiboonwong et al., 2009),. There is, however, little information about the toxicity of 2,4- and 2,6-DNT to higher plants, especially to the crop plants. In the present study the toxic effects of 2,4 DNT on germination of seeds of H. annuus L., S. alba L, L. usitatissimum cv. Viola and C. sativa cv. Beniko was described. The germination of crop plants was tested using the hydroponics medium supplemented with 2,4-DNT in concentration 50, 100 and 200 mg/l. The number of germinated seeds was compared with the control conditions. All species showed the significant decrease in germination (by 20–30 percent) only at the highest 2,4-DNT concentration

120 100

(%)

80 60 40

(200 mg/l) (Fig. 1). These data demonstrate that the crop plants are able to germinate in the presence of DNT in concentration corresponding to the maximum of 2,4-DNT solubility in water. In the next experiment, natural soil taken from an ammunition plant contaminated with 2,4-DNT during the manufacturing process was used. The concentration of 2,4-DNT in the soil was about 9 mg/g. The seeds (2  25) of the above mentioned species were sowed into pots with the soil. No species was able to grow on this soil, due to strong contamination (about 9 mg/g). After mixing of the original soil with the fresh mould we obtained the soil with following concentrations of 2,4-DNT: 4.75 mg/g, 1.36 mg/g and 0.25 mg/g. The content of 2,4-DNT higher than 1 mg per 1 g of soil was lethal for seeds of all species except sunflower, which was able to germinate by 50 percent on the soil contaminated with 2,4-DNT in concentration 1.3 mg/g (Table 1). The seedlings were, however, very poor and died in 3 weeks. Germination and growth at low DNT concentration (0.25 g/kg) was not inhibited. In opposite, the biomass of plants growing on contaminated soil was after three months even twice higher in comparison with control plants (Table 2). The growth stimulation could be caused by the enrichment of the soil with the nitrogen originated from degradation of 2,4-DNT and its metabolites by soil microorganisms. Our results are in contrast to the results of Picka and Friedl (2004), who determined the lethal concentration (LC50) of 2,4-DNT for wheat 0.049 mg/g, mustard 0.12 mg/g, lettuce 0.08 mg/g and lentil 0.17 mg/g. The results of different studies may be affected by the soil properties (organic matter, clay content, pH), which can influence adsorption of xenobiotics to the soil particles and therefore their bioavailability (Rafiq et al., 2014). Rocheleau et al. (2010) found that the toxicity of 2,4-DNT to three terrestrial plant species (alfalfa, barnyard grass, ryegrass) inversely and significantly correlated with the soil organic matter content. Therefore the difference in our results and data of Picka and Friedl may be caused by different types of soil. 3.2. Phytotransformation of 2,4-DNT The fate of 2,4-DNT, both in medium and tissue extract, was followed using the plantlets of S. jacobaea cultured in vitro and cell suspension cultures of S. officinalis and P. australis. In the case of

Table 1 The germination of seeds (in percentage) on the contaminated soil. The soil obtained from the ammunition plant was mixed with the mould in ratio 1:1 (conc. 4.75 mg/g) subsequently 1:3 (conc. 1.385 mg/g) and 1:6 (conc. 0.252 mg/g), 100%¼ number of seeds germinated on the natural mould.  -seeds were not sowed. 2,4-DNT

4.750 (mg/g)

1.358 (mg/g)

0.252 (mg/g)

Helianthus annuus Zea mays Linum utissittatisimum Sinapis alba Cannabis sativa

0  0 0 0

507 4.5 0 0 0 0

103.8 7 2.8 75.0 7 1.6 105.3 7 3.2 0 78.9 7 2.8 87.5 7 1.9

20 50mg/l 100mg/l 200mg/l

0 s

pi

na

Si

a

b al

H

eli

an

u

nn

sa

u th

m nu

Li

us u

sit

um

sim

is at

n Ca

na

s bi

a

tiv

sa

ys

a Ze

Table 2 Fresh mass of plants growing (60 days) in the flowerpots on the soil contaminated with 2,4-DNT (0.252 mg/g) in comparison to the control.

ma

Fig. 1. The germination of crop plants on different concentration of 2,4-DNT. The seeds were germinated 3 days on Petri dishes (3  25 seeds for every species) on the filter paper soaked with nutrient medium supplemented with 2,4-DNT in concentration 50, 100, and 200 mg/l.

Helianthus annuus Zea mays Linum utissittatisimum Sinapis alba Cannabis sativa

Control (g)

0.252 mg/gDNT (g)

25.20 7 2.31 25.157 3.89 23.78 7 2.23 21.487 1.96 24.887 3.5

58.007 4.11 38.45 7 3.64 30.53 7 2.77 29.157 2.06 49.257 3.92

R. Podlipná et al. / Ecotoxicology and Environmental Safety 112 (2015) 54–59

45 40 35

mg/l

30 25 20 15 10 5 0 1

2

4

7

10

17

25

32

days

Fig. 2. Degradation of 2,4-DNT by yellowweed (Senecio jacobaea). Plants were cultivated in vitro in Magenta boxes on the basal MS medium supplemented by 2, 4-DNT (initial concentration 50 mg/l). The curve means the decrease of 2,4-DNT content in the control boxes without plants caused probably by photolysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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S. jacobaea, the initial concentration of 2,4-DNT in the growth medium (50 mg/l) decreased to 20 mg/l in 17 days (Fig. 2). The relatively high standard deviations were given by different growth rate of plants in the independent biological experiments. The speed of degradation was dependent on the plant biomass and development of the root system. The decrease of 2,4-DNT concentration in control medium (without plants, by 50 percent) was caused probably by photolysis of this compound as was described by O´Sullivan et al. (2010) or by the sorption on the wall of plastic boxes. According to the uptake and degradation pathway of 2,4-DNT by microorganisms (Cheng et al., 1996) and the previous study on plants (Yoon et al., 2006; Su and Zhu, 2007) we supposed that the first 2,4-DNT degradation step was the reduction of one nitrogroup resulting in formation of 2A-NT and 4A-NT. When

2,4-DNT

25.0 35.0 20.0

2,4-DNT

30.0 15.0

mg/l

mg/l

25.0 20.0

10.0

15.0 5.0 10.0 0.0

5.0

0

1

2

3

4

7

days

0.0 0

0.25

1

2

3

6

9

Fig. 5. Degradation of 2,4-DNT by plant cell culture of reed (Phragmites australis). Concentration of 2,4-DNT (mg/ml) in the medium during the experiment (7 days) slowly decreased. The initial concentration was 50 mg/l. No degradation products were found.

5.0 4A-NT

2A-NT

4.0

mg/l

3.0 0.6 2.0

2,4-DNT

0.5 1.0

0.0 0

0.25

1

2

3

6

9

days

mg/g DW

0.4 0.3 0.2

Fig. 3. Degradation of 2,4-DNT by plant cell culture of soapwort (Saponaria officinalis). The initial concentration of 2,4-DNT (50 mg/l) in the medium rapidly decreased in six days and the products of first biotransformation aminonitrotoluenes (2-ANT and 4-ANT) appeared.

0.1 0.0 0

0.6

2

4

7

2

4

7

9.0

4A-NT

0.5

1

8.0

2,4-DNT

2A-NT

4A-NT

7.0 6.0 µg/g DW

mg/g DW

0.4 0.3 0.2

5.0 4.0 3.0 2.0

0.1 1.0 0

0.0 0

0.25

0.5

1

3

8

15

days

Fig. 4. Degradation of 2,4-DNT by plant cell culture of soapwort (Saponaria officinalis). 2,4-DNT was uptaken by the cells and metabolized by the enzymatic system. The columns show concentration of 2,4-DNT and its degradation product (4-ANT) in the acetone extract of cells.

0

1 days

Fig. 6. Degradation of 2,4-DNT by plant cell culture of reed (Phragmites australis). 2,4-DNT was uptaken by the cells and metabolized by the enzymatic system. The columns show concentration of 2,4-DNT and degradation products (4-ANT and 2-ANT) in the acetone extract of cells during the experiment.

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Fig. 7. Phytotoxicity of 2,4-DNT and its degradation products according the standard mustard germination test. The concentration of tested compounds (2,4-DNT; 2, 4-DAT; 4-ANT; 2-ANT) was 0–200 mg/l. The length of roots of three days old mustard seedlings were measured and compare to the untreated control. 2,4-DAT caused only small inhibition in comparison to 2,4-DNT in all concentration, but the inhibition of growth caused by nitrotoulenes (2-ANT, 4-ANT) in low concentration (12.5, 25.0) was significantly higher (p o0.05).

suspension culture of soapwort was used, 2,4-DNT (initial concentration 50 mg/l) was taken up from the medium in 6 days. The first degradation products were detected in the growth medium after 6 h. These compounds were identified as 4A-NT and 2A-NT and their concentration reached transient maximum in 2 days. At the end of experiment (after 9 days) the concentration of 4A-NT was 2.5 mg/l, while the concentrations of 2A-NT and 2,4-DNT were under detection limits (Fig. 3). 2,4-DNT was uptaken very quickly into the cells, the maximum of content of 2,4-DNT was detected 6 h after application, but we cannot confirm, that the maximum level had not been achieved earlier. The acetone extract of the plant cells contained only 4-ANT as the degradation product, similarly as the medium (Fig. 4). The rate of 2,4-DNT metabolic conversion in suspension culture of reed was much slower. No reduction products were found in the medium of reed cultures (Fig. 5.). On the other hand, the acetone extract of reed cells contained 2,4-DNT and both 4A-NT and 2A-NT, but in very low concentrations (Fig. 6). The small content of metabolites found in acetone extract may indicate that 2,4-DNT and its degradation products were during the detoxication process bound to the cell wall, similarly as reported for TNT (Nepovím et al., 2005). Using 14C-TNT, the author monitored distribution of radioactivity after 2-week treatment of reed plants, and 30 percent of radioactivity was determined in medium and aceton-soluble compounds accounted 23 percent of applied radioactivity. Using the radio-labeled 2,4-DNT, Yoon et al. (2006) described that the radioactivity bound to the tissue increased from 49 percent in the third day after application to 72 percent in 14 days. 3.3. Phytotoxicity of degradation products Until now, only limited information about toxic effect of 2A-NT and 4A-NT or 2,4-DAT has been published. We examined phytotoxicity of 2,4-DNT and its degradation products using the standard mustard germination test (ISO 7346). We used the concentration 12.5; 25; 50; 100; 200 mg/l, the last one is the maximal concentration of 2,4-DNT in water. At this concentration (200 mg/l) are DNT, 2A-NT and 4A-NT very toxic, the observed inhibition was over 90 percent. In contrast, 2,4-DAT caused only 60 percent inhibition. In the low concentration range (0–75 mg/l), 2A-NT and 4A-NT caused stronger inhibition than 2,4-DNT (Fig. 7). Similarly, Picka and Friedl (2004) found that the toxicity of 2,4-

DAT is much lower than that of 2,4-DNT. They found significantly lower toxic effect of 2A-NT and 4A-NT only in case of wheat (approximately 8-times lower than 2,4-DNT) that indicated the species specificity of the effect.

4. Conclusions We brought new evidence that the crop plants hemp, flax, sunflower, mustard could grow on places contaminated with 2,4-DNT (up to 1 mg/l) and therefore they could be used for phytoremediation. In the laboratory experiments, no negative effect on germination of tested species was observed until the concentration of 2,4-DNT in nutrient solution reached 200 mg/l. The growth of plants in the pots with the contaminated soil was inhibited by 100 percent at the concentrations of 2,4-DNT 4.75 g/kg and 1.36 g/kg. In contrast, the low concentration (0.252 g/kg) had the stimulatory effect, the plant biomass on soil with low concentration of 2,4-DNT was after 30 days higher than the control. The ability of plants to metabolize 2,4-DNT was demonstrated using in vitro culture of S. jacobaea and suspension cultures of S. officinalis and P. australis. Both 2-amino-4-nitrocompounds and 4-amino-2-nitrocompounds were found as the products of 2,4-DNT metabolism. The suspension culture of soapwort metabolized 2,4-DNT much more readily than the reed cells. The concentration of 4A-NT in the cell acetone extract was 100-times higher in soapwort tissue than in reed one. We predict presence of specific enzymes in soapwort cells, which enable the fast detoxification of nitrocompounds and therefore a troublefree growth on contaminated site. Toxicity of 2A-NT, 4A-NT and 2,4DAT was compared with that of the parent compound (2,4-DNT) using standard mustard ecotoxicity test. The phytotoxicity of both 2A-NT and 4A-NT was in low concentration (0–25 mg/l) significantly higher than that of the parent compound, but the 2,4-DAT was less toxic in all concentration (0–200 mg/l).

Acknowledgments This work was supported by Czech-USA grant (LH11048) of Ministry of Education, Youth and Sports.

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