DTIC ADA567433: Remote Detection of Plant Physiological Responses to TNT Soil Contamination

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Remote detection of plant physiological responsesto TNT soil W911NF-06-1-0074
contamination
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JC_Naumann, JE_Anderson, DR_Young
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14. ABSTRACT
Our study was aimed at understanding physiological responses to trinitrotoluene (TNT) soil
contamination, and using optical methods to detect TNT-induced stress in a woody plant prior to visible
changes. Myrica cerifera plants were potted in soil concentrations of TNT ranging from 30–500 mg kg?1.
Physiological measurements were significantly affected by TNT exposure at all treatment levels, and
photosynthetic decline likely resulted from metabolic impairment rather than stomatal closure as the
15. SUBJECT TERMS
Hyperspectral reflectance . Chlorophyll fluorescence . Photosynthesis
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a. REPORT b. ABSTRACT c. THIS PAGE ABSTRACT OF PAGES Donald Young
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Report Title
Remote detection of plant physiological responsesto TNT soil contamination
ABSTRACT
Our study was aimed at understanding physiological responses to trinitrotoluene (TNT) soil
contamination, and using optical methods to detect TNT-induced stress in a woody plant prior to visible
changes. Myrica cerifera plants were potted in soil concentrations of TNT ranging from 30–500 mg kg?1.
Physiological measurements were significantly affected by TNT exposure at all treatment levels, and
photosynthetic decline likely resulted from metabolic impairment rather than stomatal closure as the
experiment progressed. Several reflectance indices were able to detect TNT-induced stress before any
changes in chlorophyll concentrations occurred. The most sensitive index was the simple ratio R761/R757 which is
linked to fluorescence in-filling of the 02 atmospheric absorption. Changes at R740/R850 and R735/R850 may be
attributed to both fluorescence and structural characteristics of leaf anatomy in the near infrared region. This could
have been influenced by transformation and conjugation of TNT metabolites with other compounds. chlorophyll
index (CHL) or in the water band index (WBI970), which are indices typically associated with drought stress, and
may provide a means of separating stress due to explosives. Further studies need to be conducted with a combination
of stressors (TNT and natural) to
determine if responses are in fact generalized or if any of these changes are separable from natural stress.
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Remote detection of plant physiological respons ...
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© 2010 . Published in Plant and Soil, Vol. Ed. 0 329, (3) (2010), (, (3). DoD Components reserve a royalty-free, nonexclusive
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Approved for public release; distribution is unlimited.
PlantSoil(2010)329:239–248
DOI10.1007/s11104-009-0148-1
REGULAR ARTICLE
Remote detection of plant physiological responses
to TNT soil contamination
Julie C. Naumann&John E. Anderson&
Donald R. Young
Received:19June2009/Accepted:17August2009/Publishedonline:29August2009
#SpringerScience+BusinessMediaB.V.2009
Abstract Our study was aimed at understanding withothercompounds.chlorophyllindex(CHL)orin
physiological responses to trinitrotoluene (TNT) soil the water band index (WBI ), which are indices
970
contamination, and using optical methods to detect typically associated with drought stress, and may
TNT-induced stress in a woody plant prior to visible provide a means of separating stress due to explo-
changes. Myrica cerifera plants were potted in soil sives. Further studies need to be conducted with a
concentrationsofTNTrangingfrom30–500mgkg−1. combination of stressors (TNT and natural) to
Physiologicalmeasurementsweresignificantlyaffect- determine if responses are in fact generalized or if
ed by TNT exposure at all treatment levels, and anyofthesechangesareseparablefromnaturalstress.
photosynthetic decline likely resulted from metabolic
impairment rather than stomatal closure as the Keywords Trinitrotoluene.
experiment progressed. Several reflectance indices Hyperspectralreflectance.Chlorophyllfluorescence.
were able to detect TNT-induced stress before any Photosynthesis
changes in chlorophyll concentrations occurred. The
most sensitive index was the simple ratio R /R Abbreviations
761 757
which is linked to fluorescence in-filling of the 0 TNT Trinitrotoluene
2
atmospheric absorption. Changes at R /R and PPFD photosynthetic photon flux density
740 850
R /R may be attributed to both fluorescence and g stomatal conductance
735 850 wv
structural characteristics of leaf anatomy in the near A net photosynthetic rate
Net
infrared region. This could have been influenced by F minimalfluorescenceindark-adaptedleaves
o
transformation and conjugation of TNT metabolites F maximalfluorescenceindark-adaptedleaves
m
F /F maximum quantum use efficiency of PSII
v m
in the dark-adapted state
ResponsibleEditor:LizShaw.
F′ minimalfluorescenceinlight-adaptedleaves
: o
J.C.Naumann(*) J.E.Anderson F′ maximalfluorescenceinlight-adaptedleaves
m
USArmyERDC,FluorescenceSpectroscopyLab,
F steady-state fluorescence
7701TelegraphRoad, s
ΔF/F′ fraction of absorbed photons that are used
Alexandria,VA22315,USA m,
e-mail:[email protected] for photochemistry in a light-adapted leaf
F′ /F′ effectivequantumuseefficiencyofPSIIin
v m
D.R.Young
the light-adapted state
DepartmentofBiology,
PSII photosystem II
VirginiaCommonwealthUniversity,
Richmond,VA23284,USA WBI water band index
240 PlantSoil(2010)329:239–248
CHL chlorophyll index examining the physiological responses of these plants
PRI physiological reflectance index are limited, particularly to studies of plants grown in
cultureorhydroponicsolutions,whichdonottranslate
asreadilytofieldapplication.Byinvestigatingtheeffect
ofTNTonavarietyofspeciesatdifferentphenological
Introduction stages, we may be able to find methods for exploiting
plantsassentinelstodetectstresscausedbylandmines
The concept of using vegetation as sentinels to thatarelikelyleachingchemicalsintothesoil.
indicate presence or absence of toxic contaminants While numerous studies have looked at the uptake
is not new and could potentially provide an ideal and biotransformation of TNT in plants (Medina and
mechanismforlarge-scaledetection.Vegetationindices McCutcheon1996;Sensetal.1999;Bestetal.2008),
have been developed based on variations in plant little is known about how these compounds and
reflectance,whichsensechangesinbiomass,chlorophyll associatedmetabolitesdirectlyimpactthephotosystem,
content and leaf water status; however, differences in resultingindecreasedbiomassandchlorosis.Ithasbeen
manyoftheseindicesoftentakeplaceaftervisiblesigns proposedthatxenobioticsaretakenupandcarriedtothe
of stress have occurred. It has also been difficult to leavesthroughtranspiration,transformedandconjugated
positively attribute spectral phenomena to a specific with other compounds and compartmentalized in the
plant-contaminantinteraction(CarterandKnapp2001); vacuole,cellwallorlignin(TrappandMcFarlane1995).
and until recently there was no link between spectral Reflectance changes in both the cell wall structure and
indices and photosynthetic function. photosynthetic functioning of plants exposed to TNT
Advances in fluorescence spectroscopy and could be used for potential remote detection of
reflectance-derived fluorescence have made possible contaminated soils.
earlierdetectionofstressinplants(Zarco-Tejadaetal. ResearchinvolvingTNTeffectsonplantsislargely
2003, 2009; Evain et al. 2004), especially before focused on herbaceous species or woody plants
changes in chlorophyll content are visible. This grown in hydroponic media, while examination of
research approach has been applied to many areas of effects on woody plants growing in soil is lacking.
environmental stress, most notably drought, salinity, Thereisaneedtounderstandhowothergrowthforms
nitrogenconcentrationsandothernutrientdeficiencies respond to TNTcontamination for ease of scaling up
(Moralesetal.2000; Dobrowskietal. 2005; Campbell fromlaboratorystudiestolandscapelevelapplications.
etal.2008;Naumannetal.2008a,2009).Despitethese Weusedwoodyshrubsaplingsforourinvestigationto
advances in remote sensing technology, understanding aid in future field studies where mature plants may be
and application to anthropogenic stressors, particularly growingonTNTcontaminatedsoils.Theobjectivesof
explosives, remains limited. As our understanding of our study were to (1) understand the physiological
plant responses to various contaminants improves, it responsesofawoodyshrubgrowninsoilcontaminated
maybefeasibletousenaturallyoccurringvegetationto with various concentrations of TNT and (2) to use
monitor the environment, particularly in regard to hyperspectral remote sensing and chlorophyll fluores-
explosive compounds, which could have tremendous cence to possibly detect uptake in plant leaves in a
military and environmental impacts. laboratory setting. Investigating the use of remote
Research in the field of TNT detection and sensing methods to identify TNT contamination in
remediation has been driven by the need to clean up laboratorystudiesmayfacilitatedetectioninlargerscale
contaminated sites due to munitions production and fieldstudies.
processingfacilitiesaswellasmilitaryactivities(Best
et al. 2008). Many studies have focused on seedling
germination and early growth in TNT contaminated Materials and methods
soils (Gong et al. 1999; Robidoux et al. 2003; Ali et
al. 2006), yet few studies investigate the effect of Soil contamination
TNTonmatureplants.Althoughitiswelldocumented
thatreductionsinbiomassarearesultofTNTexposure For TNT treatments, 200 ml acetone containing
(Krishnanetal.2000; Robidoux et al. 2003), research different concentrations of TNT was added and
PlantSoil(2010)329:239–248 241
homogenized with 4.2 kg of a low nitrogen soil to relative humidity, and 28°C air temperature (LI- 6400,
obtaininitialconcentrationsof30mg,100mg,250mg LI-CORBiosciences,Inc.,Lincoln,NE).
and500mgofTNTkg−1drysoil.Forcontrolplantswe Light-adapted and dark-adapted measurements of
used soil treated only with 200 ml of acetone. Control chlorophyll fluorescence were conducted on the
and TNT treated soils samples were kept 72 h in the fourth or fifth fully expanded leaf of each plant using
dark to avoid photodegradation of TNT and to apulseamplitudemodulatedleafchamberfluorometer
evaporate the acetone from the soil (Ali et al. 2006). (LI- 6400, LI-COR Biosciences, Inc., Lincoln, NE).
Minimal fluorescence values in the dark-adapted state
Plant materials (F ) were obtained by application of a low intensity
o
far-red measuring light source (740 nm), while
Myrica cerifera L., Myricaceae, (wax myrtle) was maximalfluorescencevalues(F )weremeasuredafter
m
chosen as the study species because the physiology applyingasaturatinglightpulseof8,000μmolm−2s−1
and natural stress response has been well quantified. (630 nm). Minimum (F′ ) and maximum (F′ ) values
o m
Myrica cerifera is an evergreen shrub that forms of fluorescence in the light-adapted state at
extensive, dense thickets and is the dominant woody 700 μmol m−2 s−1 were also obtained in this manner.
species along the Atlantic Coast (Ehrenfeld 1990; Using these parameters, the following ratios were
Young et al. 1994). Coastal military facilities are calculated: ΔF/ F′ =(F′ −F)/ F′ , quantum yield of
m m s m
frequently inhabited with M. cerifera, and this could xanthophyll-regulated thermal energy dissipation and
provide future field opportunities in contaminated maximum quantum use efficiency of photosystem II
soils. Fruits of M. cerifera were collected from Hog (PSII) in the dark-adapted state, F /F = (F −F )/F .
v m m o m
Island (37° 40′N; 75° 40′W), a barrier island located Leaves were dark-adapted for 30 min using dark-
on the Eastern Shore of Virginia, and crushed with a adaptingleafclips(LI-CORBiosciences,Inc.,Lincoln,
mortar and pestle to break the waxy coating and NE) for F /F measurements.
v m
scarify seeds. Seeds were sown in transparent plastic
trays filled with one inch of Perlite growth medium, Chlorophyll concentrations
and watered as necessary with water. Plants with at
least three sets of secondary leaves were transplanted Leafsampleswerecollectedbypunchingforty0.32cm2
into 2 L plastic pots and grown for at least 5 months disks from each plant at the end of the experiment.
prior to experimentation. Plants were grown in a Chlorophyll concentrations were determined based on
Conviron environmental chamber (CMP 3244, methods recommendedbyŠesták (1971) byextracting
Controlled Environments Limited, Asheville, NC) chlorophyll using a 100% acetone solution. Samples
under a photosynthetic photon flux density (PPFD) werethengroundwithamortarandpestle,filtered,and
of approximately 700 μmol m−2 s−1, 48% relative analyzed using a Spectronic 21 spectrophotometer.
humidity, a photoperiod of 14 h, and a day/night Chlorophyll concentrations were calculated using
temperature of 30/25°C. Saplings were 50 cm at the equations described by Holm (1954).
beginning of the experiment and kept continually
moist, but well-drained throughout the experiment. Reflectance measurements
Measurements of gas exchange and fluorescence Measurements of leaf reflectance were taken concur-
rently with physiological and fluorescence measure-
PlantresponsestoTNTtreatmentswerequantifiedby ments. An ASD FieldSpec Pro reflectance radiometer
measuringstomatalconductancetowatervapor(g ), (Analytical Spectral Devices, Inc., Boulder, CO) was
wv
leaf net photosynthesis (A ), leaf fluorescence and used to measure the spectral reflectance of leaves
Net
leaf reflectance (n=5 per treatment). Measurements between 350 nm–2,500 nm. The ASD spectral
were conducted mid-day (1,000–1,400 h) once a resolution is approximately 1–3 nm from the visible
week for 9 weeks. Rates of stomatal conductance totheshort-waveinfrared.Laboratoryleafmeasurements
and leaf net photosynthesis were measured using a were collected perpendicular to the fore-optic using a
portable infrared gas analyzer at a photosynthetic 3,200°Klampasan illuminationsource. Thefore-optic
photon flux density of 700 μmol m−2 s−1, 48% oftheradiometerwasheldnadiratanominaldistanceof
242 PlantSoil(2010)329:239–248
0.25 m using an 8° field-of-view. To acquire a significantly lowered stomatal opening relative to
representative value, multiple spectra were collected control plants(F=6.77, P=0.0013); however, stomatal
and averaged for each leaf. Data were reduced from conductancevaluesdidnotremainlowoverthecourse
binary using the manufacturer’s software. A NIST of the experiment for plants treated with 30 mg TNT
spectralon reflectance standard was used as a white kg−1. Values remained lower than controls throughout
reference to optimize instrument gains prior to each the course of the experiment for all other treatments,
canopy measurement. This standard provides a near with the exception of week 4, where no significant
100% lambertian reflectance surface for calibration. differences were observed (F=1.90, P=0.1501).
Usingtheresultingreflectancevalues,severalreflectance Measurements of photosynthesis revealed signifi-
indices examining potential changes in pigments, cant reductions in all treatments by week 1 (F=12.68,
reflectance-derivedfluorescenceandwatercontentwere P<0.0001) and remained lower than controls through-
calculated(Table1). out the experiment (Fig. 1; Table 2), despite weeks
where stomatal conductance values did not differ
Statistical analysis from controls. Stomatal control did not appear to be
the main mechanism for lowering photosynthesis in
Variations in photosynthetic characteristics, stomatal TNT treated plants (F=81.78, P<0.0001, r2=0.33;
conductance, fluorescence, reflectance indices and Fig. 2).
chlorophyll concentrations among TNT treated plants Both light- and dark-adapted measurements were
relative to control plantswere analyzedwith one-way quantified to assess stress due to TNT uptake. An
analysis of variance for each stress experiment (Zar initial reduction in ΔF/F′ occurred in plants treated
m
1999). Dunnett’s multiple comparisons (α=0.05) with250mgkg−1and500mgkg−1byweek2(F=7.50,
identified significant differences in treatment plants P=0.0007;Fig.3;Table2)andremainedthroughweek
relative to controls. 3. Recovery occurred for both treatments by week 4
(F=2.84, P=0.0517). By week 6, plants treated with
100 mg kg−1, 250 mg kg−1 and 500 mg kg−1 had
Results significantly lower ΔF/F′ than controls (F=5.77,
m
P=0.0030) and remained lower throughout the exper-
Gas exchange and fluorescence measurements iment. Plants treated with 30 mg TNT kg−1 exhibited
lower ΔF/F′ values during week 7 only (F=19.68, P
m
Physiological stress in M. cerifera was induced by <0.0001).Dark-adapted fluorescence values (F /F )
v m
TNTas seen in stomatal conductance, photosynthesis decreased in plants treated with 250 mg and 500 mg
andfluorescencemeasurements.Adecreaseinstomatal TNTkg−1 dry soil by week 5 (F=13.02,P<0.0001).
conductance was seen in plants treated with 100 mg, Plants at 250 mg kg−1 recovered by week 6,
250mgand500mgTNTkg−1byweek2(F=9.79,P< however, F /F remained significantly lower than
v m
0.0001;Fig.1;Table2).Byweek3,alltreatmentshad controls for the remainder of the experiment in
Table1 Selectedvegetation
indicesusedinstatistical Reflectanceindex Formula Reference
analyses
Waterbandindex(WBI ) R /R (Peñuelasetal.1993)
970 970 900
Chlorophyllindex(CHL) (R –R )/(R +R ) (GitelsonandMerzlyak1996)
750 705 750 705
Structuralinsensitivepigment (R –R )/(R +R ) (Peñuelasetal.1995)
800 445 800 680
Index(SIPI)
Physiologicalreflectance (R –R )/(R +R ) (Gamonetal.1992)
531 570 531 570
Index(PRI)
Greennormalizeddifference (R –R )/(R +R ) (Gitelsonetal.1996)
801 550 801 550
Vegetationindex(GreenNDVI)
Curvatureindex(CI) R 2/(R *R ) (Zarco-Tejadaetal.2000)
683 675 691
PlantSoil(2010)329:239–248 243
Fig.1 EffectsofTNT
contaminatedsoilon
160
stomatalconductance(g )
wv
andnetphotosynthesis
)
(A )forMyricacerifera. 1
Net -
s
Symbolsrepresentmeans 2 120
forcontrolandtreatment -m
plants 0
2
H
ol 80
m
m
(
v
w
g 40
)
1
-
s
2 8
-
m
2
O
C
ol
m 4
µ
(
et control
N
A 30 mg/kg
100 mg/kg
250 mg/kg
0
500 mg/kg
0 10 20 30 40 50 60
Time (days)
plantstreatedat500mgkg−1(Fig.3;Table2).These Reflectance
significant differences occurred before any visible
signs of stress. TNT induced stress was apparent in multiple indices
during weeks 6, 7 and 8 (Table 4). The water band
Chlorophyll content index (WBI) and chlorophyll index (CHL) did not
change throughout the experiment. The physiological
Chlorophyllaconcentrationsdecreasedwithincreasing reflectance index (PRI), which is linked to photosyn-
stress, ranging from 316±16 to 270±8 mg m−2, but thetic functioning, was successful at detecting stress
therewerenosignificantdifferencesamongtreatments in plants at 500 mg kg −1 from week 6 and through
(F=2.26, P=0.100; Table 3). Chlorophyll b concen- the remainder of the study (Table 4). By the end of
trations ranged from 193±41 to 244±16 mg m−2 the experiment, PRI was significantly lower in
without significant differences among treatments plants at 250 mg kg−1 and 500 mg kg−1. Several
(F=1.72, P=0.186). There were no significant differ- indices that are considered to be reflectance-derived
ences among TNT treatments for total chlorophyll fluorescence showed significant changes in the
(F=2.12, P=0.118) or the chlorophyll a:b ratio (F= 500 mg kg−1 treatment. Most notable is the simple
0.84, P=0.516; Table 3). ratio R /R , which remained significantly lower
761 757
244 PlantSoil(2010)329:239–248
Table2 Summaryof
P-valuesfromDunnett’s Week Treatmentlevel Stomatalconductance Netphotosynthesis ΔF/F′m Fv/Fm
multiplecomparisons, (mgkg−1)
whichidentifiedsignificant
differencesintreatment 1 30 0.99 0.02 0.87 1.00
plantsrelativetocontrols. 100 1.00 0.02 0.97 0.02
Boldnumbersindicate
250 0.88 0.01 1.00 0.74
significanceatα=0.05
500 1.00 0.04 0.94 0.00
2 30 0.21 0.04 0.99 0.98
100 0.04 0.01 0.98 0.92
250 0.00 0.00 0.03 0.61
500 0.00 0.00 0.00 1.00
3 30 0.03 0.01 0.99 0.99
100 0.09 0.00 0.99 0.09
250 0.00 0.00 0.02 0.10
500 0.00 0.00 0.00 0.73
4 30 0.31 0.03 0.07 0.98
100 0.72 0.01 0.90 0.41
250 0.14 0.00 0.99 0.08
500 0.07 0.00 0.97 0.26
5 30 0.29 0.00 0.07 0.99
100 0.03 0.00 0.18 0.98
250 0.04 0.00 0.00 0.00
500 0.03 0.00 0.51 0.00
6 30 0.11 0.00 0.95 0.53
100 0.00 0.00 0.01 0.98
250 0.02 0.00 0.03 0.16
500 0.01 0.00 0.01 0.00
7 30 0.00 0.00 0.03 0.98
100 0.00 0.00 0.00 0.97
250 0.00 0.00 0.00 1.00
500 0.00 0.00 0.00 0.00
8 30 0.37 0.00 0.98 0.98
100 0.00 0.00 0.00 0.09
250 0.00 0.00 0.06 0.99
500 0.00 0.00 0.00 0.00
in multiple treatments from weeks 6–8, and was metabolites in the leaves, stems and roots (Sens et
significantly lower in all treatment levels during al. 1999). Very few studies have examined the effect
week 7 (Table 4). ofTNTonphysiologicalresponsesofplants(Thompson
etal. 1998; Alietal. 2006),thusthe exact mechanism
withwhichTNTcausesperturbationsinthephotosystem
Discussion remains unknown. By furthering our understanding of
how TNT affects plants, we may be able to develop
TNT affects photosynthesis and the light reactions, methodsfordetectingexplosivesuptakeinplantswhich
which has been documented as changes in biomass would have enormous benefits in both military and
and degradation of chlorophyll in leaves (Thompson environmental applications. Our study was aimed at
et al. 1998; Best et al. 2008). It is well known that understanding both physiological responses to TNT
plants take up TNT and convert it to various contamination,aswellasusingopticalmethodstodetect
PlantSoil(2010)329:239–248 245
16 TNT-induced stress in a woody plant prior to visible
changes.
1)12 ResponseandgrowthofplantinTNTcontaminated
2- s soils appearstobespecies dependent (Scheidemannet
-m
2 8 al.1998).OurresultsindicatethatM.ceriferawasable
CO to grow in concentrations up to 500 mg kg−1 with no
mol 4 obvious signs of visible damage to leaves. Other
µ
(et studies have reported growth of plants in soils up to
AN 0 750 mg kg−1 with no obvious effects (Mueller et al.
1995). Reductions in biomass, although not measured,
were apparent in plants treated with higher TNT
0 50 100 150 200 250
gwv (mmol H20 m-2 s-1) concentrations relative to controls by the end of the
experiment.Despitelowphotosyntheticrates,plantsat
Fig. 2 Relationship between stomatal conductance (g ) and all concentrations continued to grow.
wv
net photosynthesis (ANet) for Myrica cerifera under TNT Partial stomatal closure was observed in response
treatment
to TNT exposure in all treatments, which may
decreasetheinfluxofCO to maintain photosynthesis.
2
After a few weeks of exposure, some plants increased
Fig.3 Changesinthe 0.68
fluorescencemeasurements
ΔF/F′ andF /F for
m, v m
MyricaceriferaunderTNT 0.64
treatmentsovertime.
Symbolsrepresentmeansfor
controlandtreatmentplants 0.60
m
F/F' 0.56
∆
0.52
0.48
0.81
0.80
0.79
m
F
F/v
0.78
0.77
control
30 mg/kg
0.76 100 mg/kg
250 mg/kg
500 mg/kg
0.75
0 10 20 30 40 50 60
Time (days)