Chemosphere 62 (2006) 545–558

www.elsevier.com/locate/chemosphere

Phytotoxicity of nitroaromatic energetic compoundsfreshly amended or weathered and aged in sandy loam soil

Sylvie Rocheleau a, Roman G. Kuperman b, Majorie Martel a,Louise Paquet a, Ghalib Bardai a, Stephen Wong a,Manon Sarrazin a, Sabine Dodard a, Ping Gong a,1,

Jalal Hawari a, Ronald T. Checkai b, Geoffrey I. Sunahara a,*

a Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue,

Montreal, Que., Canada H4P 2R2b US Army Edgewood Chemical Biological Center, AMSRD-ECB-RT-TE, 5183 Blackhawk Road,

Aberdeen Proving Ground, MD 21010-5424, USA

Received 5 February 2005; received in revised form 30 May 2005; accepted 14 June 2005Available online 19 August 2005

Abstract

The toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), 2,4-dinitrotoluene (2,4-DNT), and 2,6-dinitrotoluene (2,6-DNT) to terrestrial plants alfalfa (Medicago sativa L.), Japanese millet (Echinochloa crusgalli L.),and perennial ryegrass (Lolium perenne L.) were determined in Sassafras sandy loam soil using seedling emergence, freshshoot, and dry mass measurement endpoints. A 13-week weathering and aging of energetic materials in soils, whichincluded wetting and drying cycles, and exposure to sunlight of individual soil treatments, was incorporated into thestudy design to better reflect the soil exposure conditions in the field than toxicity determinations in freshly amendedsoils. Definitive toxicity tests showed that dinitrotoluenes were more phytotoxic for all plant species in freshly amendedtreatments based on EC20 values for dry shoot ranging from 3 to 24 mg kg�1 compared with values for TNB or TNTranging from 43 to 62 mg kg�1. Weathering and aging of energetic materials (EMs) in soil significantly decreased thetoxicity of TNT, TNB or 2,6-DNT to Japanese millet or ryegrass based on seedling emergence, but significantlyincreased the toxicity of all four EMs to all three plant species based on shoot growth. Exposure of the three plant spe-cies to relatively low concentrations of the four compounds initially stimulated plant growth before the onset of inhi-bition at greater concentrations (hormesis).� 2005 Elsevier Ltd. All rights reserved.

Keywords: TNT; TNB; Dinitrotoluene; Plant toxicity; Weathering and aging; Hormesis; Natural soil

0045-6535/$ - see front matter � 2005 Elsevier Ltd. All rights reservdoi:10.1016/j.chemosphere.2005.06.057

* Corresponding author. Tel.: +1 514 496 8030; fax: +1 514496 6265.

E-mail addresses: sylvie.rocheleau@cnrc-nrc.gc.ca (S. Ro-cheleau), geoffrey.sunahara@cnrc-nrc.gc.ca (G.I. Sunahara).1 Present address: Analytical Services, Inc., 3909 Halls Ferry

Road, Vicksburg, MS 39180, USA.

1. Introduction

Elevated levels of explosives and related materials areoften found in soil at military installations that involvemunitions manufacturing, disposal, testing, and train-ing. Concentrations of 2,4,6-trinitrotoluene (TNT) in

ed.

546 S. Rocheleau et al. / Chemosphere 62 (2006) 545–558

soil have been reported to exceed 87000 mg kg�1 (Siminiet al., 1995). By-products of TNT production such asdinitrotoluenes (DNTs) and 1,3,5-trinitrobenzene (TNB)are present worldwide at munitions manufacturingand post-production sites. Aerobic metabolites ofmicrobial degradation of TNT, including 2,4-DNT and2,6-DNT are also associated with soil TNT contamina-tion (Gorontzy et al., 1994; Spain et al., 2000). Exceptfor TNT, studies of soil-based phytotoxicity of nitroaro-matic energetic materials (EM) are scant (Sunaharaet al., 2001). Phytotoxicity of TNT has been evaluatedusing yellow nutsedge (Cyperus esculentus) (Palazzoand Leggett, 1986), poplar (Populus sp. deltoides x nigra,DN34) (Thompson et al., 1998), lettuce (Lactuva sativa)(Toussaint et al., 1995), and tall fescue (Festuca arundin-

acea) (Peterson et al., 1996). Scheidemann et al. (1998)showed that alfalfa (Medicago sativa) could not growin soil contaminated with 100 mg TNT kg�1, whereaswheat (Triticum aestivum) and bush bean (Phaseolus vul-garis) could develop at 500 mg kg�1 TNT in soil. Siminiet al. (1995) evaluated the toxicity to cucumber (Cucumis

sativus) and radish (Raphamus sativus) of soil from theJoliet Army Ammunition Plant containing a mixture ofTNT, TNB and heavy metals. They determined that toxi-city was mostly related to TNT and TNB, with lowestobservable effect concentrations (LOEC) of 7 to 19 mgTNT kg�1. Reddy et al. (1994) assessed the toxicity ofTNB in sand using lettuce (L. sativa) and oat (Avenasativa) and reported 5-d median effect concentration(EC50) values for seedling emergence of 19 mg kg�1 and>375 mg kg�1, respectively. Toussaint et al. (1995) re-ported an EC50 value of 2.34 mg l�1 for the effect ofTNT on lettuce root elongation. Gong et al. (1999b)compared the toxicity of TNT to cress (Lepidium sati-

vum), turnip (Brassica rapa), oat (A. sativa) and wheat(T. aestivum), and determined a lowest observable ad-verse effect concentration (LOAEC) of 50 mg kg�1, andstimulation of seedling growth at concentrations ofTNT from 5 to 50 mg kg�1. Robidoux et al. (2003) esti-mated median inhibition concentration (IC50) and IC20

values for lettuce and barley (Hordeum vulgare) seedlingemergence, fresh shoot mass, dry shoot mass, and rootmass in forest soil and silica. Picka and Friedl (2004) re-ported NOAEC and EC50 of TNT, 2,4-DNT, 2,6-DNTand seven reduction products for wheat (T. aestivum

L.), mustard (Sinapis alba L.), lettuce (Lactuva sativa

L.) and lentil (Lens culinaris Med.). Phytotoxicity datafor EMs in the majority of these studies were determinedusing nominal chemical concentrations and only a fewwere based on standard toxicity tests. Ecotoxicity databased on nominal concentrations can be incomplete be-cause it does not address chemical transformations, sorp-tion onto soil constituents and other fate processesaffecting EM bioavailability to plants. This issue presentsa challenge for site managers who wish to distinguishthose sites that pose significant environmental risks from

those that do not, prioritize contaminated sites by thelevel of risk posed, quantify the risks at each site, and de-velop appropriate remedial actions and cleanup goals.Scientifically based ecological soil screening levels (Eco-SSLs) are needed to identify contaminant explosive levelsin soil that present an acceptable ecological risk (USE-PA, 2003). To our knowledge, the present study is thefirst to report ecotoxicological benchmarks for TNB ordinitrotoluenes on the basis of measured chemical con-centrations and standardized terrestrial plant toxicitytests (Talmage et al., 1999), which are required for thedevelopment of Eco-SSLs (USEPA, 2003).

Bioavailability and toxicity of organic chemicals insoil can change over time (Alexander, 2000). Assessmentof the effects of weathering and aging of contaminantEMs in soil on the exposed terrestrial plants is criticalfor developing toxicity benchmarks that adequately re-flect potential ecological risks. Weathering and agingof organic contaminants in soil, i.e., subjecting contam-inated soils to wetting and drying cycles, and to sunlightexposure over a certain period of time, may alter theexposure environment of soil biota due to photodecom-position, hydrolysis, reaction with organic matter, sorp-tion/fixation, precipitation, immobilization, microbialtransformation and other fate processes that commonlyoccur at contaminated sites. Alexander (2000) reportedthat binding of organic compounds to soil particles maydecrease the toxicity to ecological receptors. Certain fateprocesses, including microbial or photochemical trans-formations of nitroaromatic EMs and other organiccompounds, including polycyclic aromatic hydrocar-bons (PAHs), can also produce chemicals that are morebioavailable or more toxic to soil organisms comparedwith parent compounds freshly introduced into soil.Weathering and aging of TNT in soil significantlydecreased adult survival and juvenile production bythe potworm Enchytraeus crypticus (Kuperman et al.,2005). Anthracene, benzo[a]pyrene, and fluoranthene ex-posed to simulated solar radiation induced greater inhi-bition of seedling emergence, root and shoot growth incanola (B. napus), compared to unexposed controls(Ren et al., 1996). Therefore, we designed our studiesto obtain phytotoxicological data for TNT, TNB, 2,4-DNT, and 2,6-DNT. Weathering and aging of theseEMs in soil prior to exposing plant species was also in-cluded in order to test the hypothesis that such a processcan affect the EM toxicity to plants, while allowing us tomore closely simulate the exposure effects in the field.

2. Materials and methods

2.1. Chemicals and reagents

Energetic materials TNT (CAS: 118-96-7; Purity:99%), and TNB (CAS: 99-35-4; Purity: 99.7%) were

S. Rocheleau et al. / Chemosphere 62 (2006) 545–558 547

obtained from the Defense Research and Developmentof Canada (Val Belair, QC, Canada). 2,4-DNT (CAS:121-14-2; Purity: 97%), and 2,6-DNT (CAS: 606-20-2;Purity: 98%) were obtained from Sigma–Aldrich Can-ada (Oakville, ON, Canada). Certified standards of theenergetic materials (AccuStandard, Inc., New Haven,CT) were used for HPLC determinations. Boric acid(H3BO3; CAS: 10043-35-3; Purity: 99.9%) was used asthe positive control. ASTM type I water (AmericanSociety for Testing and Materials, 2004) was obtainedusing the Millipore� Super Q water purification sys-tem (Millipore�, Nepean, ON, Canada) and was usedthroughout the studies. All other chemicals were eitheranalytical or certified grade. Glassware was washed withphosphate-free detergent, followed by rinses withtap water, ASTM type I water, acetone, analytical re-agent grade nitric acid 1% (v/v), then with ASTM typeI water.

2.2. Test soil

Sassafras sandy loam [Fine-loamy, siliceous, mesicTypic Hapludult] (USDA/ARS, 1999; SSL) was usedin this study to assess the phytotoxicity of EMs. This soilwas selected for developing ecotoxicological values pro-tective of soil biota because it has physical and chemicalcharacteristics supporting relatively high bioavailabilityof EMs (USEPA, 2003), including low organic matterand clay contents (69% sand, 13% silt, 17% clay, 1.2%organic matter, 5.5 cmol kg�1 cation-exchange capacity,and pH 5.2). The SSL soil was collected from an opengrassland field on the property of the US Army Aber-deen Proving Ground (Edgewood, MD). Vegetationand the organic horizon were removed to just belowthe root zone, and the top 15 cm of the A horizon wasthen collected. Soil was sieved through a 5-mm2 meshscreen, air-dried at room temperature for at least 72 hand mixed periodically to ensure uniform drying, thenstored at room temperature before use in testing. Soilanalyses showed that no EM compounds were presentabove analytical detection limits. Total concentrationsof metals and nutrients were within regional backgroundranges for SSL soil, and were reported previously byRobidoux et al. (2004).

2.3. Soil amendment and weathering

and aging procedures

The SSL soil was separately and independentlyamended with TNT, TNB, 2,4-DNT, or 2,6-DNT todetermine toxicity benchmarks for each EM. IndividualEMs were dissolved in acetone and transferred evenlyacross the soil surface, ensuring that the volume of solu-tion added at any one time did not exceed 15% (vol-ume mass�1) of the dry mass soil. The acetone wasallowed to volatilize (minimum of 18 h) in a darkened

chemical hood, and then mixed overnight (18 ± 2 h)using a three-dimensional rotary mixer. ASTM type Iwater was added to adjust the soil moisture to a levelequivalent to 75% of the water holding capacity(WHC). The WHC of SSL soil was 18% of the soildry weight. Soils prepared for the EM weathering andaging procedure were treated in the same manner asthe freshly amended soil. These EMs in soil were sub-jected to wetting and drying cycles, and to sunlight expo-sure in a greenhouse for a period of 13 weeks. All soiltreatments were brought to 75% of the WHC at 24 hprior to the initiation of toxicity tests. Each week,ASTM type I water was added to adjust the soil mois-ture to its initial value and then allowed to dry untilthe next addition of water.

2.4. Plant toxicity tests

The plant toxicity tests were performed followingASTM (1998) and USEPA (1982) standard protocols.Range-finding tests were performed with Zea mays L.‘‘Kandy corn Canada no. 1’’ (Williams Dam SeedsLtd., Dundas, Ontario, Canada), lettuce L. sativa L.‘‘Buttercrunch’’ (Stokes Seeds Ltd., Thorold, Ontario,Canada), alfalfa M. sativa L. ‘‘Canada no. 1’’ (WilliamsDam Seeds Ltd., Dundas, Ontario, Canada), perennialryegrass Lolium perenne L. ‘‘Express’’ (Pickseed CanadaInc., St-Hyacinthe, Quebec, Canada) and Japanese mil-let Echinochloa crusgalli L. Beauv. ‘‘Common no. 1’’(Labon Inc. Boucherville, Quebec, Canada) to selectthree plant species for definitive toxicity testing. Nomi-nal treatment concentrations for each EM in range-find-ing tests included 1, 10, 100, 1000, and 10000 mg kg�1.Control treatments included negative (ASTM type Iwater) and a carrier (acetone). All treatments were rep-licated (n = 3). Based on the results of range-findingtests alfalfa, Japanese millet, and perennial ryegrass wereselected for the subsequent definitive tests.

Definitive tests were performed using four replicatesand 6–9 concentrations (up to 1600 mg kg�1) of eachEM, in addition to negative (ASTM type I water) andacetone controls. Boric acid was used as the positivecontrol (Aquaterra Environmental, 1998; ASTM, 1998).Twenty seeds of each plant species were sown per10-cm pot containing 200 g dry soil. Alfalfa seeds wereinoculated with nitrogen-fixing bacteria prior to sowing.ASTM type I water was added to the soil to obtain 75%of the WHC. Plant pots were placed in polyethylenebags to minimize water loss. Plant toxicity tests wereperformed in a temperature and light controlled growthchamber. Plants were incubated in the dark for the first2 d and then exposed to a diurnal cycle afterwards. Thegrowth chamber conditions were set as follows: lightcycle at 5000 ± 500 lx at 25 �C for 16 h, dark cycle at20 �C for 8 h. Luminosity level was measured weeklyusing a LX-102 light meter (Lutron, Coopersburg, PA)

548 S. Rocheleau et al. / Chemosphere 62 (2006) 545–558

and the light intensity was adjusted when needed. Thenumbers of emerged seedlings were counted after 5 dfor alfalfa, and Japanese millet, and after 7 d for rye-grass. The number of shoots, shoot fresh mass, andshoot dry mass were measured after 16 d for alfalfa,and Japanese millet, and after 19 d for ryegrass. Shootdry mass was obtained after drying at 70 �C for24 ± 2 h. Results of the definitive toxicity tests were ac-cepted when seedling emergence in the negative controlswas P85% for ryegrass or Japanese millet, or P70% foralfalfa, and when EC50 values for boric acid werewithin ±2 times standard deviation of the establishedlaboratory baseline.

2.5. Chemical extractions and analyses

Acetonitrile extractions of soils were performed atthe beginning of each definitive test. Samples for chem-ical analysis were taken after the 24-h soil rehydration.For each treatment, triplicate 2-g soil aliquots wereweighed into 50-ml polypropylene centrifuge tubes,10 ml acetonitrile containing 1,3-DNB recovery stan-dard was added and the samples were vortexed for1 min, then sonicated in the dark for 18 ± 2 h at 20 �C(modified EPA Method 8330A; USEPA, 1998). Fivemilliliters of supernatant was transferred to a glass tube,to which 5 ml of CaCl2 solution (5 g l�1) was added. Forsoil samples amended with TNB, NaHSO4 was added tothe CaCl2 solution to prevent TNB degradation. Super-natants were filtered through 0.45 lmMillex-HV syringecartridges. Extraction was repeated if 1,3-DNB internalstandard recovery was lower than 90%.

Soil extracts were analyzed using a Thermo Separa-tion Products HPLC system composed of model P4000pump, a model AS1000 injector, a temperature controlmodule, and a model UV6000LP photodiode-arraydetector. A Supelcosil C8 column (25 cm · 4.6 mm ID,5 lm particles) and an 18% 2-propanol/82% watermobile phase were used. The flow rate was 1 ml min�1

and the run time was 40 min. The injection volumewas 50 ll. The detector was set to scan from 200 to350 nm and chromatograms were examined at 254 nm.The instrument limit of quantification was 0.05 mg l�1

per chemical, corresponding to 0.5 mg kg�1 soil.

2.6. Data analysis

Measurement endpoint data were analyzed usingnonlinear regression models described in Stephensonet al. (2000). Regression models were evaluated to iden-tify the best fit for the individual data set generated ineach toxicity test. The selection was based on severalparameters, including: examination of histograms ofthe residuals and stem-and-leaf graphs to ensure that

normality assumptions were met; examination of theregression line generated by each model to select onewith the closest fit to the data points; comparison ofthe variances of residuals, asymptotic standard errors,coefficients of determination, and the range of 95% con-fidence intervals (CI) associated with the point estimates,in order to identify the model that generated the smallestvalues/range for each parameter. Hormesis, a stimula-tory effect caused by exposure to low levels of potentiallytoxic chemicals followed by inhibitory effects at higherconcentrations (Stebbing, 1982; Calabrese et al., 1987),was also considered. The following nonlinear regressionmodels were used:

Logistic Gompertz model:

Y ¼ a� eð½logð1�pÞ��½C=ECp�bÞ

Exponential model:

Y ¼ a� eðð½logð1�pÞ�=ECpÞ�CÞþb

Logistic Hormetic model:

Y ¼ ðt � ½1þ hC�=f1þ ½ðp þ hECpÞ=ð1� pÞ�� ½C=ECp�bgÞ

where Y = value for a measurement endpoint (e.g., dryshoot mass); a = control response; t = control responsein the hormetic model; e = base of the natural loga-rithm; p = percent inhibition/100 (e.g., 0.50 for EC50);C = exposure concentration in test soil; ECp = estimateof effect concentration for a specified percent effect;h = hormetic effect parameter; and b = scale parameter.The ECp parameters used in this study included theEM concentration producing a 20% (EC20) or 50%(EC50) reduction in the measurement endpoint. TheEC20 parameter based on growth endpoint is the pre-ferred parameter for deriving Eco-SSL values for plants(USEPA, 2003). The EC50, a commonly reportedvalue, was included to provide additional toxicologi-cal information and enable comparisons of the resultsproduced in this study with results reported by otherresearchers.

Analysis of variance (ANOVA) was used to deter-mine the bounded (when applicable) LOEC or LOAECvalues for seedling emergence or growth data. LOAECvalues were determined in tests with hormetic response.Means separations were done using Fisher�s least signif-icant difference (LSD) pairwise comparison tests. A sig-nificance level of p 6 0.05 was accepted for determiningthe LO(A)EC values. Student�s t-test (two-tailed) withsignificance level of p 6 0.05 was used in comparisonsof analytical data for EMs freshly amended, and weath-ered and aged in soil. Statistical analyses were performed

S. Rocheleau et al. / Chemosphere 62 (2006) 545–558 549

using SYSTAT 7.01 (SPSS, 1997) or Microsoft EXCEL97, and were based on measured EM concentrations.

3. Results and discussion

3.1. Analytical determinations of energetic materials

in soil

Recovery of EMs in freshly amended soils wereabove 80% (Fig. 1A–D, white bars), except for somelow concentrations of TNT or 2,6-DNT (Fig. 1A andD). Weathering and aging of EMs in amended soils sig-nificantly (p 6 0.05) decreased concentrations of TNT,TNB, 2,4-DNT, and 2,6-DNT, compared with initialconcentrations in soils (Fig. 1A–D, black bars). Thegreatest percentage decrease occurred in treatmentsbelow 100 mg kg�1, with recoveries ranging from 2%to 10% for TNT, from 0% to 28% for TNB, from 25%to 37% for 2,4-DNT, and from 12% to 20% for 2,6-DNT. Percentage decrease in EM concentrations above100 mg kg�1 was not as pronounced, with recoveriesranging from 32% to 90% for TNT, from 67% to 98%for TNB, from 44% to 73% for 2,4-DNT, and from21% to 45% for 2,6-DNT. Recovery of 1,3-DNB stan-dard was above 90%.

The primary efforts of our studies centered on assess-ing the net phytotoxicological effects of weathering andaging of nitroaromatic EMs in contaminated soil. How-ever, transformation products of these nitroaromaticEMs were qualitatively detected in soil treatments usedin our study. Results of analytical determinations indi-cated that a portion of each EM was strongly sorbedonto soil or was transformed into reduced products, likeaminodinitrotoluenes, during the 13-week weatheringand aging period in soil. Comparable results were foundin studies with TNT (Kuperman et al., 2005), TNB, 2,4-DNT, and 2,6-DNT freshly amended or weathered andaged in SSL soil under similar conditions (Kupermanet al., 2004). In contrast, Dodard et al. (2003) reportedan average of 99% recovery of TNT in the study withOECD standard artificial soil immediately extracted fol-lowing the amendment with nominal concentrationsranging from 50 to 1000 mg kg�1. Such high recoveryof TNT can be attributed to both the properties of thecomponents of that synthetic soil, the absence or insuffi-cient biotic transformation/degradation of TNT in dryartificial soil compared with hydrated natural SSL soil,and the absence of physical sorption processes drivenby wetting/drying cycles of weathering. Lower extractedquantities of TNT compared with nominal TNT concen-trations in studies with standard Lufa 2.2 soil were ob-served by Schafer (2002). The author reported thatonly 29% and 54% of the freshly amended TNT couldbe recovered in 150 and 300 mg kg�1 nominal treatments

and considerably greater percentage decrease from theinitial TNT concentrations due to aging in soil, with nodetectable TNT in the 150 mg kg�1 nominal treatmentincubated at 20 �C for two months.

Abiotic factors that can affect fate of EMs in soil,including photolysis, wetting/drying cycles, and temper-ature, were controlled in our study and were similar inall treatments. Possible explanations of the observed in-verse relationship between percent recovery of EMsand the initial concentration in freshly amended soilcould be the suppression of microbial activity due totoxicity at higher concentrations (Dodard et al., 1999)and increasing saturation of binding sites of the soilconstituents. In contrast, lower recovery of 2,6-DNT(below 45% at all tested concentrations) may indi-cate that 2,6-DNT was readily transformed or that aportion of 2,6-DNT was sorbed onto soil. Traces of2-aminodinitrotoluene (2-ADNT) and 4-aminodinitrotol-uene (4-ADNT), two transformation products of TNT,and of 3,5-dinitroaniline (3,5-DNA), a transformationproduct of TNB, were detected in SSL soils that werefreshly amended with TNT or TNB, respectively, priorto initiation of the phytotoxicity test (identification con-firmed by UV spectrum; data not shown). Concentra-tions of 2-ADNT, 4-ADNT and 3,5-DNA increasedby the end of the phytotoxicity tests, suggesting that(bio)transformation of TNT and TNB occurred duringthese tests. None of these transformation products weredetected either at the beginning or at the end of the phy-totoxicity tests with 2,4-DNT or 2,6-DNT in freshlyamended soil. In tests with TNT, TNB, or 2,4-DNTweathered and aged in soils, these transformation prod-ucts were detected in greater amounts. Presence of2-ADNT and 4-ADNT was detected in all concentra-tions of TNT weathered and aged in soil, but in greateramount at concentrations between 50 and 200 mg kg�1.Dinitroaniline was detected in all concentrations of TNBweathered and aged in soil, but in greater amount atconcentrations between 40 and 80 mg kg�1. Measurableamounts of 2-amino-4-nitrotoluene (2A-NT), and 4-amino-2-nitrotoluene (4A-NT), two transformationproducts of 2,4-DNT, were detected in weathered andaged 2,4-DNT treatments at concentrations between25 and 200 mg kg�1. No nitroaromatic transformationproducts were detected in weathered and aged 2,6-DNT treatments. Previous studies showed that amino-dinitrotoluenes 4-ADNT and 2-ADNT were formed inTNT-amended OECD standard artificial soil (Dodardet al., 2003, 2004). The amino-nitrotoluene intermediatesare the most commonly detected products of TNT deg-radation and can be formed by soil bacteria in either aer-obic or anaerobic conditions (Hawari et al., 1998). Theinclusion of weathering and aging component in theEM phytotoxicity assessments allowed us to incorporatepotential alterations in chemical bioavailability and

0

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2 5 10 20 40 60 80 120 160 250 320 600 800 1200 1600

TNB nominal concentration (mg kg-1)

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2,4-DNT nominal concentration (mg kg-1)

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2,6-DNT nominal concentration (mg kg-1)

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TNT nominal concentration (mg kg-1)

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A

B

C

D

Fig. 1. Percent recovery of TNT (A), TNB (B), 2,4-DNT (C), and 2,6-DNT (D) freshly amended or weathered and aged in Sassafrassandy loam soil using acetonitrile extraction (USEPA method 8330A). Symbols used: (h) freshly amended treatment; (j) weatheredand aged treatment.

550 S. Rocheleau et al. / Chemosphere 62 (2006) 545–558

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resulting toxicity at contaminated sites, in the devel-opment of toxicological benchmarks for terrestrialplants.

3.2. Phytotoxicity of EMs in freshly amended soil

Both dinitrotoluenes (2,4-DNT and 2,6-DNT) weremore toxic to the plant species tested as compared toTNB or TNT (Table 1). Plant growth was a more sensi-tive endpoint compared with seedling emergence, and

Table 1Summary of toxicological benchmarks for TNT, TNB, 2,4-DNT, andsandy loam soil (SSL) determined for alfalfa, Japanese millet, and ry

Seedling emergence Shoot fresh m

LOEC(mg kg�1)

EC20

(mg kg�1)EC50

(mg kg�1)LOEC(mg kg�1)

EC(m

Alfalfa

Freshly amended treatmentTNT 150 95 (87–104) 142 (134–151) 41 41TNB 171a 145 (69–221) 172 (156–188) 39 382,4-DNT 99 >47 >47 5b 112,6-DNT 14 11 (6–16) 19 (14–24) 4 1.3

Weathered/aged treatmentTNT 215 38 (16–60) 119 (50–188) 10 3 (TNB 113a 109 (107–112) 114 114 202,4-DNT 121a 104 (91–117) 115 (109–121) 10 7 (2,6-DNT 15a 26 (0–128) 55 (9–100) 5 1.6

Japanese millet

Freshly amended treatmentTNT 194 40 (0–87) 210 (152–268) 0.3b 33TNB 125 109 (74–144) 204 (168–239) 22a 162,4-DNT 22 55 (46–63) 70 (63–78) 5 3.52,6-DNT 89 40 (28–52) 57 (46–68) 4a 13

Weathered/aged treatmentTNT 0.1a 105 (23–187) 673 (398–948) 10 6 (TNB 197 139 (0–294) 163 (64–262) 0.3b 0.3

2,4-DNT 90 86 >90 4 3.52,6-DNT 140a >15 >15 3 4.8

Ryegrass

Freshly amended treatmentTNT 95 90 (73–107) 137 (110–165) 0.3a 94TNB 125 28 (1–55) 95 (77–114) 125a 452,4-DNT 17a 8 (7–9) 16 (15–17) 4a 112,6-DNT 4b 29 (26–32) 38 (33–44) 89a 18

Weathered/aged treatmentTNT 0.2a 34 (22–46) 106 (69–143) 0.1a 15

TNB 197 107 (81–133) 150 (131–168) 0.3b 462,4-DNT 8 >8 >8 8 5 (

2,6-DNT 37 42 (38–45) 54 (52–56) 20 24

Confidence intervals (95%) are presented in brackets.ND: Not determined.a Lowest observed adverse effect concentration (LOAEC). Bold fonb Unbounded lowest observed adverse effect concentration (LOAEC

shoot dry mass was a more robust measure of growththan shoot fresh mass for assessing the effects of EMson alfalfa, Japanese millet, and ryegrass (Table 1). TheEC20 values (mg kg�1) for shoot dry mass ranged from2.8 to 26 for 2,6-DNT, from 11 to 34 for 2,4-DNT, from43 to 61 for TNT, and from 43 to 62 for TNB. The EC50

values (mg kg�1) for shoot dry biomass ranged from 9.5to 39 for 2,6-DNT, from 13 to 56 for 2,4-DNT, from 86to 173 for TNT, and from 89 to 129 for TNB. Resultsof our studies are comparable with those reported by

2,6-DNT freshly amended, or weathered and aged in Sassafrasegrass

ass Shoot dry mass

20

g kg�1)EC50

(mg kg�1)LOEC(mg kg�1)

EC20

(mg kg�1)EC50

(mg kg�1)

(22–60) 77 (43–112) 0.3a 43 (20–67) 93 (68–119)(10–66) 107 (72–141) 88 62 (28–96) 129 (97–161)(0–24) 38 (17–58) 5b 34 (10–59) 56 (33–79)(0–2.9) 5 (2–8) 4 2.8 (0–6) 9.5 (4–15)

1–4) 8 (4–12) 0.1a 1.4 (0.5–2.3) 4 (2–7)

(0–49) 63 (19–107) 114 46 (2–89) 92 (59–125)2–11) 30 (20–40) 10a 15 (9–21) 42 (29–56)(0.1–3.2) 7 (4–11) 5 0.4 (0–1.4) 5 (0–11)

(0–69) 99 (73–124) 0.7 56 (0–156) 173 (0–486)(12–21) 36 (27–45) 64 43 (27–59) 89 (73–104)(1.6–5.4) 10 (8–13) 9 25 (18–33) 34 (28–40)(12–14) 16 (15–18) 14a 11 (9–13) 18 (16–20)

3–10) 33 (22–43) 10 11 (1–21) 52 (30–74)(0.1–0.4) 0.9 (0.4–1.4) 0.3b 0.7 (0.4–0.9) 2.0 (1.2–2.8)

(2.3–4.6) 6.5 (5.4–7.5) 8 6 (5–8) 10 (9–12)

(3.9–5.8) 9 (8–10) 5 6 (3–9) 11 (8–13)

(82–107) 129 (106–152) 0.3b 61 (44–78) 86 (70–103)(35–56) 75 (59–91) 125a 56 (43–67) 89 (70–109)(10–12) 13 (12–15) 17a 11 (10–12) 13 (12–15)(4–32) 39 (19–59) 30a 26 (21–32) 39 (31–46)

(6–23) 48 (34–61) 64 13 (1–24) 48 (27–69)

(13–78) 83 (61–104) 81 51 (30–72) 86 (74–99)4–7) 7 (6–8) 8a 2 (0–4) 8 ND(21–27) 39 (36–41) 20 21 (18–23) 34 (32–36)

t denotes significant effect of weathering and aging.).

552 S. Rocheleau et al. / Chemosphere 62 (2006) 545–558

Reddy et al. (1994). Robidoux et al. (2003) deter-mined slightly higher EC20 values for barley exposedto TNT in freshly amended forest soil, with EC20 valuesfor shoot growth (fresh and dry mass) of 139 and272 mg kg�1. Lower EC20 values for TNT determinedin our study may be partially attributed to contrastingsoil properties of SSL that support greater bioavailabil-ity of TNT than the forest soil used by Robidoux et al.(2003), which contained more organic matter content.

Best fitted nonlinear regression models varied amongspecific compounds and plant species (Figs. 2–5). Hor-

Japanese m

Alfalfa

Ryegras

0 100 200 300 400 5000.0

0.01

0.02

0.03

0.04

0.05

0.06

Pla

ntdr

y m

ass

(g)

0 100 200 300 400 5000.0

0.01

0.02

0.03

0.04

0.05

0.06

100 200 300 400 500

TNT concentration (mg kg-1)

0.02

0.03

0.04

0.05

100 200 300 400 500

0.01

0.02

0.03

0.04

0.05

F/AR2 = 0.969

F/AR2 = 0.971

F/AR2 = 0.911

Pla

nt d

ry m

ass

(g)

00.0

0 100 200 300 4000.0

0.01

0.02

0.03

0.04

0.05

0.06

0 100 200 300 4000.0

0.01

0.02

0.03

0.04

0.05

0.06

Pla

nt d

ry m

ass

(g)

TNT concentration (mg kg-1)

TNT concentration (mg kg-1)

Fig. 2. Effect of TNT freshly amended (F/A) or weathered and aged (Wryegrass growth based on acetonitrile extractable concentrations.

metic effects, i.e., growth stimulation at low concentra-tions followed by inhibition at greater concentrations,were observed with all four energetic compounds testedin our study. We observed hormetic effects with all plantspecies exposed to TNT, with ryegrass exposed to TNB,with ryegrass exposed to 2,4-DNT, and with Japanesemillet and ryegrass exposed to 2,6-DNT (Figs. 2–5).Hormesis has been reported in plants exposed to heavymetals and aromatic hydrocarbons (Stebbing, 1982;Calabrese et al., 1987). Our results are consistent withother studies in which hormetic effects of EM were ob-

illet

s

0 200 400 600 8000.

0.01

0.02

0.03

0.04

0.05

0.06

Pla

nt d

ry m

ass

(g)

0 200 400 600 8000.0

0.01

0.02

0.03

0.04

0.05

0.06

0 200 400 600 8000.0

0.02

0.04

0.06

0.08

0.10

Pla

ntdr

y m

ass

(g)

0 200 400 600 8000.0

0.02

0.04

0.06

0.08

0.10

0 200 400 600 800

TNT concentration (mg kg-1)

0.0

0.01

0.02

0.03

0.04

0.05

0.06

Pla

nt d

ry m

ass

(g)

0 200 400 600 8000.0

0.01

0.02

0.03

0.04

0.05

0.06

W/AR2 = 0.967

W/AR2 = 0.974

W/AR2 = 0.979

TNT concentration (mg kg-1)

TNT concentration (mg kg-1)

/A) in Sassafras sandy loam soil on alfalfa, Japanese millet and

Alfalfa

Japanese millet

Ryegrass

0 50 100 150 2000.0

0.01

0.02

0.03

0.04

0.05

Pla

nt d

ry m

ass

(g)

0 50 100 150 2000.0

0.01

0.02

0.03

0.04

0.05

0 100 200 3000.0

0.01

0.02

0.03

0.04

0.05

0.06

Pla

nt d

ry m

ass

(g)

0 100 200 3000.0

0.01

0.02

0.03

0.04

0.05

0.06

0 50 100 150 2000.0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Pla

nt d

ry m

ass

(g)

0 50 100 150 2000.0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 200 400 6000.0

0.01

0.02

0.03

0.04

Pla

nt d

ry m

ass

(g)

0 200 400 6000.0

0.01

0.02

0.03

0.04

BF/A R2 = 0.972

W/A R2 = 0.966

Pla

nt d

ry m

ass

(g)

0.00.05

0.0

W/A R2 = 0.972

F/A R2 = 0.985

W/AR2 = 0.979

F/A R2 = 0.980

0 200 400 600 800

TNB concentration (mg kg-1)

0.01

0.02

0.03

0.04

0 200 400 600 800

0.01

0.02

0.03

0.04

TNB concentration (mg kg-1)

TNB concentration (mg kg-1)TNB concentration (mg kg-1)

TNB concentration (mg kg-1)TNB concentration (mg kg-1)

00.0

0.01

0.02

0.03

0.04

0.05

Pla

nt d

ry m

ass

(g)

00.0

0.01

0.02

0.03

0.04

0.05

200 400 600200 400 600

Fig. 3. Effect of TNB freshly amended (F/A) or weathered and aged (W/A) in Sassafras sandy loam soil on alfalfa, Japanese millet andryegrass growth based on acetonitrile extractable concentrations.

S. Rocheleau et al. / Chemosphere 62 (2006) 545–558 553

served for offspring production by Daphnia magna andsoil microbial populations exposed to EMs (Baileyet al., 1985; Gong et al., 1999a). Hormetic effects onjuvenile production by E. crypticus were reported forTNT in both freshly amended and in weathered andaged treatments (Kuperman et al., 2005) and for TNBfreshly amended into SSL soil (Kuperman et al., 2004).Hormetic effects have been measured in several plantsand animals following the exposure to pesticides, poly-cyclic aromatic hydrocarbons, heavy metals and other

organic chemicals (Calabrese et al., 1987). The mecha-nisms responsible for hormetic effects of explosives atspecific concentrations are not yet understood. Stebbing(1982) suggested that hormesis is the cumulative conse-quence of transient and sustained over-corrections ofbiosynthesis, i.e., a rate-controlled process controlledby end-product inhibition. Steevens et al. (2002) sug-gested that these mechanisms could include a direct ef-fect on test organisms through the release of metabolicproducts of explosives that may have a specific effect

Fig. 4. Effect of 2,4-DNT freshly amended (F/A) or weathered and aged (W/A) in Sassafras sandy loam soil on alfalfa, Japanese milletand ryegrass growth based on acetonitrile extractable concentrations.

554 S. Rocheleau et al. / Chemosphere 62 (2006) 545–558

on growth and reproduction, or indirect effects throughincreased supply of nitrogen from mineralization ofexplosives.

3.3. Phytotoxicity of weathered and aged EMs in soil

Very few studies have investigated the effects ofweathering and aging of energetic contaminants in soilon the exposure of terrestrial plants and resulting soiltoxicity. The net phytotoxicological effects determinedin our studies encompassed plant exposures to boththe residual parent EMs and to transformation products

of these EMs formed during weathering and aging inSSL soil. Weathering and aging of EMs in soil signifi-cantly decreased the toxicity of TNT treatments to Jap-anese millet, and of TNB or 2,6-DNT treatments toryegrass based on 95% CI for EC50 values for seedlingemergence (Table 1). Toxicity for shoot growth was sig-nificantly (95% CI basis) increased in weathered andaged treatments of TNT for alfalfa, Japanese milletand ryegrass, of TNB for Japanese millet, of 2,4-DNTfor Japanese millet and ryegrass, and of 2,6-DNT forJapanese millet. These differential effects of weatheringand aging of EMs in soil on toxicity endpoints for plants

Fig. 5. Effect of 2,6-DNT freshly amended (F/A) or weathered and aged (W/A) in Sassafras sandy loam soil on alfalfa, Japanese milletand ryegrass growth based on acetonitrile extractable concentrations.

S. Rocheleau et al. / Chemosphere 62 (2006) 545–558 555

may be attributed to a protective effect of the seed coatof dormant seeds, which limits transport of water solu-ble chemicals, including nutrients and contaminants.Once seed germination has occurred, chemicals can betaken up by plant roots and affect shoot growth.

Specific mechanisms of differential phytotoxicity ofnitroaromatic EMs in freshly amended soil comparedto phytotoxicity of EMs weathered and aged in soiltreatments are not yet fully understood. Weatheringand aging of EMs in soil may reduce exposure of terres-trial plants to the parent material due to fate processesthat commonly occur at contaminated sites. These fate

processes can decrease the amount of chemical that isbioavailable compared to freshly contaminated soils,or may reveal increased toxicity due to the presence ofmore toxic transformation products than parent com-pound freshly introduced into soil. Different intermedi-ates and condensation products like azoxy compoundscan be formed under aerobic soil conditions, some ofwhich are potentially more bioavailable and toxic thantheir precursors (Rieger and Knackmuss, 1995). In astudy investigating the toxicities of selected TNT reduc-tion products, Lachance et al. (2004) determined LC50

values for adult earthworm Eisenia andrei exposed to

556 S. Rocheleau et al. / Chemosphere 62 (2006) 545–558

TNT, 4-ADNT, or 2-ADNT of 132, 105, and215 mg kg�1, respectively, and gave the following orderof toxicity: 4-ADNT > TNT > 2-ADNT. Several of theseproducts can be found in TNT-contaminated soil (Daunet al., 1998; Frische, 2002), and a few, including 2-ADNT,4-ADNT, 2,4-diamino-6-nitrotoluene (2,4-DANT), and2,6-diamino-4-nitrotoluene (2,6-DANT), have been de-tected in earthworms E. andrei and Lumbricus terrestris

exposed to TNT contaminated soils (Johnson et al.,2000; Renoux et al., 2000; Robidoux et al., 2000).

4. Conclusions

Toxicities of TNT, TNB, 2,4-DNT, and 2,6-DNT,freshly amended or weathered and aged in SSL soil var-ied among plant species tested. Dinitrotoluenes weremore phytotoxic compared with TNB or TNT for allplant species in freshly amended treatments. Plantgrowth was a more sensitive endpoint compared withseedling emergence for assessing the effects of nitroaro-matic EMs on alfalfa, Japanese millet, and ryegrass.Exposure of the three plant species to relatively low con-centrations of each of the four energetic materials testedhad an hormetic effect on plant growth. Overall resultsof our study showed that weathering and aging ofTNT, TNB, 2,4-DNT, and 2,6-DNT in SSL soil signifi-cantly affected toxicity to the plant species tested.Weathering and aging of EMs in soil significantly de-creased the toxicity of TNT treatments to Japanese mil-let, and of TNB or 2,6-DNT treatments to ryegrassbased on seedling emergence, but significantly increasedthe toxicity in weathered and aged treatments of TNTfor alfalfa or ryegrass, of TNB for Japanese millet, of2,4-DNT for Japanese millet or ryegrass, and of 2,6-DNT for Japanese millet based on shoot growth. Theseresults show that consideration given to the effects ofweathering and aging of these nitroaromatic EMs in soilfor assessing phytotoxicity was well justified. Toxicitybenchmark values generated from this research will con-tribute to development of Ecological Soil Screening Lev-els that better represent the exposure conditions ofterrestrial plants at contaminated sites. Our findings ofincreased toxicity to plant growth in weathered and agedsoil treatments clearly show that additional studies arerequired to further investigate and understand the toxic-ity of the transformation products of nitroaromaticenergetic soil contaminants.

Acknowledgements

This research project was supported by the USDepartment of Defense, through the Strategic Environ-mental Research and Development Program (SERDP

Project CU-1221). This paper has been assigned NRCpublication number 47241.

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