uptake of nitroaromatic compounds in plants

Research Articles Nitroaromatic Compounds in Plants

Research Articles

Uptake of Nitroaromatic Compounds in Plants

Implications for Risk Assessment of Ammunition Sites

1Klaus Schneider, 1jan Oltmanns, 2Thomas Radenberg, 2Thomas Schneider, 3Dagmar Pauly-Mundegar

I Forschungs- und Beratungsinstitut Gefahrstoffe GmbH (FoBiG), Werderring 16, D-79098 Freiburg, Germany e Abfallentsorgungs- und Altlastensanierungsverband Nordrhein-Westfalen, Werkstralge 15, D-45527 Hattingen, Germany

Umweltamt - Chemisches Untersuchungsinstitut Leverkusen, Di]sseldorferstraRe 153, D-51379 Leverkusen, Germany

Corresponding author: Dr. Klaus Schneider

Abstract The uptake of nitroaromatic compounds by plants from the soil was studied at an ammunition site. After the development of analytical methods for 2,4,6-trinitrotoluene, aminodinitrotoluenes and dinitrotoluenes in plant material, we could show that these substances ac- cumulated in the roots of plants and are found to a lesser extent in- leaves and stems. We observed only moderate differences between various plant species. It is likely that a metabolic transformation in plants leads to the formation of dinitrotoluenes which are consid- ered to be potent carcinogens. Results from soils with a wide range of explosive concentrations show a good correlation between the plant and soil concentrations. The relative accumulation in plant material is higher at lower soil concentrations. At low soil concentrations of about I mg trinitrotoluene/kg soil, an accumulation factor of about 0.5 can be derived. These data are an important in- put for the risk assessment of ammunition sites.

Keywords: Risk assessment of ammunition sites; plant uptake and metabolism of nitroaromatic compounds; 2,4,6-trinitrotoluene; aminodinitrotoluenes; dinitrotoluenes

1 Introduction

Soils f rom ammuni t ion sites, where explosives such as 2,4,6-trinitrotoluene (TNT) had formerly been produced, even after decades contain large amounts of T N T and related nitroaromatic compounds. In the case of inhabited sites these substances represent a risk for exposed people due to their toxic and carcinogenic properties [1, 2, 3]. The uptake of T N T in edible plants is therefore very important for the assessment of possible health risks on ammunition sites.

Various authors were able to show that T N T and other nitroaromatics are taken up by plants, either under hydro- culture conditions [4, 5, 6, 7] or after the addition of T N T to soil [8, 9]. 2-amino-4,6-dinitrotoluene (2-ADNT) and 4- amino-2,6-dini t ro to luene (4-ADNT) consti tute the pri- mary metabolites of T NT in plants. In all of these experiments, the largest amounts were found in the plant roots.

The properties of the soil determine the accumulation, e.g. the availability and the uptake of T N T in the plants was higher in soils with a low content of organic carbon [8]. In this study [8] with 14C-labelled TNT, a large propor t ion of the detected radioactivity could not be related to TNT, 2- A D N T or 4-ADNT, but existed ra ther in the form of unidentified metabolites.

As far as we are aware, only one study on the plant uptake of T N T from contaminated soil at an ammunit ion site has been performed to date [10]. In this study, soil from an ammunition site in Hessen, Germany was dug out, homogenized and used for plant cultivation. The authors used two soils with either low (3 mg/kg, sum of TNT, 2-ADNT, and 4-ADNT, median) or high (499 mg/kg) contaminant concentrations and eight different plant species. The availability of T N T to plants could be shown, although its entry into the soil had occurred decades ago. Severe phytotoxic effects were obvious at the highly contaminated area. Only three of eight plant species were able to grow on this soil. Bush bean roots demonstrated the highest concentration of nitroaromatic compounds (2-ADNT, 4 - A D N T and TNT). In the area with a lower contamination, the plant uptake of T N T could be shown only for beans and alfalfa. Interest- ingly, the authors found dinitrotoluenes (2,4-DNT, 2,6- DNT) in the roots of bush beans from the highly contaminated area, although these compounds could not be detected in the corresponding soil samples. DNTs in particu- lar have to be regarded as critical from the toxicological point of view since 2,4- and 2 ,6-DNT have proved to be potent animal carcinogens (for review of toxic effects, see e.g. [11]). Furthermore, epidemiologic studies point to a carcinogenic activity of these compounds in humans [12].

In order to assess the exposure via plants, a systematic plant analysis program was carried out at an ammunit ion site in Nor th Rhine-Westphalia, Germany. Due to the pro- duction of explosives until 1926, the soil of this site con- tains a number of nitroaromatic compounds, with the main contaminant being TNT. The site has been used as a resi- dential area with gardens and children's playgrounds for

ESPR - Environ. Sci. & Pollut. Res. 3 (3) 135-138 (1996) �9 ecomed publishers, D-86899 Landsbera, Germany

135

Nitroaromatic Compounds in Plants Research Articles

some decades and the population amounts to several hun- dreds of people. The program aimed at

- examining whether the existing knowledge on TNT-uptake by plants is transferable to the scenario of typical garden utilization

- investigating the question as to whether or not TNT is metabolized to DNTs and

- elucidating the dependence of plant transfer on the concentration in soil.

2 Mater ia l s and M e t h o d s

2.1 Hydroponic experiments

The absorption and enrichment of TNT in plants was analyzed using hydroponic experiments with bush beans (Phaseolus vulgaris). Bean seeds were placed on moist cot- ton in glass beakers covered with perforated parafilm. 3-5 days after germination, each seedling was transferred to a 500 ml Erlenmeyer flask containing 300 ml of a commer- cially available culture medium (Substral) which was titrated to pH 5.8 using acetate buffer. The culture medium was aerated and renewed three times weekly. Absorption experiments were initiated with 3-week-old plants which were transferred to 300 ml culture medium containing 10 mg/l TNT (99 % purity, Prochem, Wessel). After 3, 5, and 7 days, plants were removed from this culture medium and the roots were rinsed briefly in methanol/H20 (80/20; v/v) to remove TNT adhering to the roots. The roots were care- fully wiped dry and the roots, stem and leaves were separated and analyzed individually. Beans grown as described above, but with the omission of TNT, served as controls.

2.4 Analysis of nitroaromatics in plants

Because of the complex matrix of plants, a comprehensive extraction procedure was required. Several samples of each vegetable from a plot were homogenized in a moulinette mixer, 50 ml of 1M HC1 was added to 2 g of this ho- mogenate and cell structures were disrupted with an Ultra- Turrax. An internal standard (1,4-dinitrobenzene) was added and hydrolysis was carried out for 1 h in a water bath (90 ~ The solution was then extracted with 30 ml of toluene under acid and subsequently under alkaline conditions (addition of 15 ml 4M NHs) in a separatory funnel with 40 ml methanol to prevent emulsification. The com- bined organic extracts were concentrated to 0.5 ml and made up to 5 ml with cyclohexane/ethyl acetate (50/50; v/v) and used in the first purification step with gel perme- ation chromatography on Bio-Beads | S-X3 with cyclohexane/ethyl acetate (50/50; v/v) as an eluting solvent. Fractions containing nitroaromatics were concentrated to 0.5 ml and diluted in cyclohexane/ethyl acetate (80/50; v/v) and then applied to a florisil | column. The first fraction was again concentrated to 0.5 ml and made up to a defined volume with ethyl acetate. The nitroaromatics in ethyl acetate solutions were separated and quantified by GC-ECD on two columns with different polarity (DB-5/OV-1701- CB). With the method described, recovery rates of more than 70 % and - depending on the plant matrix - detection limits between 5 and 15 lag/kg fresh weight were achieved. A total of 120 samples were analyzed using this method.

Statistical tests were carried out using C-Stat for Windows (Cherwell Scientific Publishing Ltd, Oxford).

2.2 Outdoor experiments

Outdoor experiments were carried out in the spring of 1993. Two vegetable beds of approx. 5-10 m 2 per plot were laid in eight different plots of land in an area known to be contaminated with TNT. The two beds (bed A and B) of the same plot were used for cultivation of different plant species. An homogenization of the earth was not performed prior to plant cultivation. Carrots (Daucus carota), radishes (Raphanus sativus), kale (Brassica oIeracea) (Bed A), lettuce (Lactuca sativa), lamb lettuce (Valerianella Io- custa) and bush beans (Bed B) were planted with seeds of uniform quality. After harvesting, vegetables were thor- oughly cleaned and deep frozen until analysis. The edible parts, roots and leaves of the vegetables were analyzed; carrots and radishes were pared and the parings and underly- ing tissue were analyzed seperately. Before analysis, the water content of the samples was determined by drying at 70 ~ for 48 h.

2.3 Analysis of nitroaromatics in soil samples

Soil samples were removed from each bed from a depth of 0-0.3 m, 0.3-0.6 m, and 0.6-1 m using a Piirckhauer probe [15]. The samples were sieved (< 8 mm), homogenized, dried with sodium sulphate and sonicated after the addition of toluene. Toluene extracts were directly analyzed by GC-MS (single determinations).

3 Results and D i s c u s s i o n

For this investigation, plots with high and low degrees of contamination were deliberately chosen and areas with an extremely high contamination (> 200 mg/kg) were ex- cluded. Correspondingly, the soil analysis of the beds des- ignated for the growing of the plants yielded a wide range of contamination between below 1 mg/kg and up to 200 mg/kg nitroaromatics (-~ Table 1). The main contaminant in soil was TNT, followed by ADNTs, whereas dinitrotoluenes (2,4-DNT and 2,6-DNT) in most plots were present only in trace amounts (--) Table 1). The experiments show, as was to be expected, that plants take up nitroaromatic compounds from the soil. The analytical method applied in this study reduced the disturbing effects of matrix components without extreme demands on manpower and apparatus and with high recovery rates even in the presence of critical matrix conditions (lamb lettuce, radish).

The concentration of the substances in the plants corre- lated with the concentration in soil (--9 Fig. 1) while pro- nounced species-specific differences were not seen.

In agreement with earlier studies [8, 5, 10], the highest concentration was found in the roots of plants. The concentrations in leaves and stems amounted to approximately 20 % of the corresponding concentration in the roots, the carrot being the exception where approximately 80 % was found (data not shown).

136 ESPR- Environ. Sci. & Pollut. Res. 3 (3) 1996

R e s e a r c h A r t i c l e s N i t r o a r o m a t i c C o m p o u n d s in P l a n t s

Table 1: Concentrations of nitroaromatic compounds in soil. From each plot, two beds (A and B) were probed separately. Three other plots showed no detectable amounts of contaminants

Substance

TNT

2-ADNT

4-ADNT

2,4-DNT

2,6-DNT

nitroaromatic compounds

n. d.: not detectable

Sample No. and concentrations (mg/kg soil DW)

1A 1B 2 A 2 B 3 A 3B 4 A 4 B 5 A 5B

1.29 n.d. 1.40 0.85 0.13 0.13 55.50 162.00 0.34 0.42

0.04 n.d. 0.16 n.d. n.d. n.d. 2.80 5.25 n.d. n.d.

0.04 n.d. 0.18 0.08 n.d. n.d. 3.45 4.1 n.d. n.d.

n.d. n.d. 0.10 1.50 n.d. n.d. 0.03 0.7 n.d. 0.04

n.d. n.d. n.d. 0.39 n.d. n.d. n.d. n.d. n.d. n.d.

1.42 n.d. 1.83 2.91 0.13 0.13 61.78 172.35 0.34 0.45

Fig. 1: Concentrations of nitroaromatic compounds (l~'qT, ADNTs, DNTs) in the roots of several plant species in relation to soil contamination (open fields: plant species not investigated in this bed)

Although TNT was the main contaminant in soil, ADNTs dominated in the plants. After the TNT-uptake from soil in gardens, an extensive metabolism to ADNTs obviously takes place. Similar observations were made in other inves- tigations under hydroponic conditions [5] or after the uptake of TNT in plants from homogenated soil [10]. The re- lationship between TNT and ADNTs as observed in our study is shown in Figure 2 for bush beans. It is seen to be within the same order of magnitude for the other plant species as well.

In some of the root samples, small amounts of 2,4-DNT and 2,6-DNT could be detected, although these contaminants were not found in the corresponding soil samples. Previously, GORGE et al. [10] found small amounts of DNTs in plants which had been grown on TNT- contaminated soil containing no DNTs. Our hydroponic experiments in which seedlings of bush beans were grown in culture medium with the addition of pure TNT confirm these results since we also found traces of 2,4-DNT and 2,6- DNT in some roots ( ~ Table 2). It seems likely that DNTs can be formed in plants as a result of TNT-metabolism.

Fig. 2: Relative quantities of TNT, 2-ADNT and 4-ADNT in the soil and roots of bush beans (mean values for all soil concentrations investigated)

Table 2: Concentrations of TNT, 2-ADNT, 4-ADNT, 2,4-DNT, and 2,6-DNT in the roots of bush beans after growing the plants in hydroponic solutions containing TNT (10 rng/l)

Substance

3d

TNT 0.1

2-ADNT 0.71

4-ADNT 4.13

2,4-DNT 0.13

2,6-DNT 0.77

n. d.: not detectable

Time with TNT-medium contact (concentrations in pg/g fresh weight)

5 d 7 d 15 min no contact

1.32 0.09 0.15 n.d.

3.6 0.91 0.47 n.d.

28 6.28 1.13 n.d.

0.09 0.17 n.d. n.d.

1.42 3.86 0.12 n.d.

Since the soil was not homogenized prior to plant cultivation, the measured concentration in the soil can only be regarded as an approximation of the actual contamination near the rhizosphere. Nevertheless the dependence of the concentration in the plant on the measured concentration in the soil is obvious. The concentrations of nitroaromatics in plant roots (all species) demonstrate a statistically sig- nificant correlation with the logarithm of the measured concentrations in soil (-~ Fig. 3). This means that there is

ESPR- Environ. Sci. & Pollut. Res. 3 (3) 1996 137

N i t r o a r o m a t i c C o m p o u n d s in P lant s R e s e a r c h Ar t i c l e s

a more effective uptake via the plant root at lower concentrations in the soil. At low soil concentrations of about 1 mg/kg, an accumulation factor for plant roots (concentration of nitroaromatics in the root divided by the concentration in the soil, each on a dry weight basis) of about 0.5 can be derived from the regression analysis. For leaves and stems there is a much higher variation and the evaluation yields an accumulation factor of about 0.1. These accumulation factors enable us to assess the order of magnitude of a possible exposure through the consumption of edible plants and, thus, represent a valuable aid in the site-specific risk assessment of ammunition sites 113]. The experiments therefore confirm the importance of the plant as a pathway of exposure in the case of a garden being used to cultivate edible plants.

Sum of nitroaromatic compounds in the roots of plants (pglkg DW)

2800J . . . . . . I

2400 ' |

- - - [ I J

2000 : . . . . - ! EY = 453.37x ~- 537.54r = 4.71

I

1600 . . . . ~ - - - I

, oo.oo . . . . . . . . . . . .

4 0 0 �9 .~_~-- ! l - -

0 ' - - - " . ' ;1 ~ d

tl i

4 . . . . . /

- - l - { II __m

t 0 2 3

log sum of nitroaromatlc compounds In soil (mglkg DW)

Regression analysis of the dependence of plant root concentrations upon soil concentrations (correlation statistically signifi- cant, spearman rank coefficient s=0.768, p<0.001)

Fig. 3:

The assessment of TNT-metabolites in plants remains problematic. Apart from the finding that DNTs may be formed in the plant, the fact that a large amount of the TNT taken up by plants is presumably present in the form of unidentified metabolites [8] is of major concern. FELLOWS et al. [14] were able to show that acidqabile conjugates of ADNTs are formed in plants and that these conjugates are the chemical species responsible for the translocation from the roots to the leaves where further metabolites are formed. The toxicological relevance of these latter compounds is not known.

4 C o n c l u s i o n s

The uptake of TNT in plants takes place on a large scale and can lead to an intake of nitroaromatic compounds into the human body if these plants are consumed. This exposure pathway is therefore of critical importance for the risk assessment of ammunition sites, in which TNT is a com- mon contaminant. Meanwhile, from this and former inves- tigations, there are improvements in knowledge regarding

the accumulation of TN T and ADNTs, However, the unidentified metabolites formed in plants still have to be characterized, both in respect of their chemical analysis and their potential toxic effects, in order to allow a more comprehensive assessment of this exposure pathway.

Acknowledgements The authors gratefully acknowledge the financial support from the Ab- fallentsorgungs- und Ahlastensanierungsverband Nordrhein-Westfalen (AAV).

5 References

[1] G. Koss; A. LOMMEL; I. OLLROGE; L TESSERAUX; R. HAAS; A. D. KAPPOS: Zur Toxikologie der Nitrotoluole und weiterer Nitroaro- maten aus riistungsbedingten Altlasten. Bundesgesundheitsblatt 32, 527 (1989)

i2] B. S. LEVINE; J. H. RUST; J. J. BARKLEY; E. M. FUREDI; P. M. LISH: Six month oral toxicity study of trinitrotoluene in beagle dogs. Toxicology 63,233 (1990)

[3] R. H. ROSS; W. R. HARTLEY: Comparison of water quality criteria and health advisories for 2,4,6-trinitrotoluene. Regul. Toxicol. Pbarmacol. 11, 114 (1990)

[4] S. D. HARVEY; R. J. FELLOWS; D. A. CATALDO; R. M. BEAN: Analy- sis of 2,4,6-trinitrotoluene and its transformation products in soils and plant tissues by high-performance liquid chromatography. J. Chromatogr. 518, 361 (1990)

[5] E. GORGE; S. BRANDT; D. WERNER: Uptake and metabolism of 2,4,6-trinitrotoluene in higher plants. ESPR - Environ. Sci. Pollut. Res. 1,229 (1994)

[6] A.J. PALAZZO; D. C. LEGGEVr: Effect and disposition of TNT in a terrestrial plant. J. Environ. Qual. 15, 49 (1986)

[7] C. MCFARLANE; O. NOLT; C. WICKLIFF; T. PFLEEGER; R. SHIMABUKU: Uptake, distribution and metabolism of four organic chemicals by soybean plants and barley roots. J. Environ. Tox. Chem. 6, 847 (1987)

[8] D. A. CATALDO; S. D. HARVEY; R. J. FELLOWS; R. M. BEAM; B. D. MCVEETY: An Evaluation of the Environmental Fate and Behav- ior of Munitions Material (TNT, RDX) in Soil and Plant Systems. Environmental Fate and Behavior of TNT. Battelle Memorial In- stitute, Richland, WA, USA, 1989

[9] J. C. PENN1NGTON: Soil Sorption and Plant Uptake of 2,4,6-Trini- trotoluene. Army Engineer Waterways Experiment Station, Vicks- burg, MS, Environmental Lab., USA, 1988

[10] E. GORGE; S. BRANDT; D. WERNER: Aufnahme von 2,4,6-Trinitro- toluol in Pflanzen - Freilandversuche auf dem Gel~inde der ehema- ligen Sprengstoffabrik in Stadtallendorf. UWSF - Z. Umwelt- chem. Okotox. 7, 139 (1995)

[11] D. E. R1CKERT; B. E. BUTTERWORTH; J. A. Popp: Dinitrotoluol: Acute toxicity, oncogenicity, genotoxicity, and metabolism. CRC Crit. Rev. Toxicol. 13, 217 (1984)

[12] L.T. STAYNER; A. L. DANNENBERG; T. BLOOM; M. THUN: Excess he- patobiliary cancer mortality among munitions workers exposed to dinitrotoluene. Journal of Occupational Medicine 35,291 (1993)

[13] K. SCHNEIDER; M. HASSAUER; E KALBERLAH: Toxikologische Be- wertung von Ri~stungsaltlasten. 1. Expositionsanalyse als erster Schritt zur Bewertung von Gesundheitsgef~ihrdungen und zur Ableitung yon standortspezifischen Bodenbeurteilungskriterien. UWSF - Z. Umweltchem. Okotox. 6, 271 (1994)

[14] R.J. FELLOWS; S. D. HARVEY; D. A. CATALDO: An evaluation of the environmental fate and behavior of munitions material (tetryl and polar metabolites of TNT) in soil and plant systems. Preliminary evaluation of TNT-polar metabolites in plants. Battelle Pacific Northwest Labs., Richland, WA, USA, 1992

[15] LOLF, Landesanstah fi~r Okologie, Landschaftsentwicklung und Forstplanung Nordrhein-Westfalen: Mindestuntersuchungspro- gramm Kuhurboden. Recklinghausen, 1988

Received: May 23, 1996 Accepted: September 24, 1996

138 ESPR - Environ. Sci. & Pollut. Res. 3 (3) 1996

uptake of nitroaromatic compounds in plants

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