Embed Size (px)
Citation preview
This article was downloaded by: [Pennsylvania State University]On: 23 April 2013, At: 07:56Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,UK
Journal of Plant NutritionPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/lpla20
Fate of DTPA, EDTA, andEDDS in Hydroponic Mediaand Effects on Plant MineralNutritionTimothy M. Vadas a , Xinning Zhang a b , Ashley M.Curran a & Beth A. Ahner aa Department of Biological and EnvironmentalEngineering, Cornell University, Ithaca, New York,USAb Department of Environmental Science andEngineering, California Institute of Technology,Pasadena, California, USAVersion of record first published: 23 Aug 2007.
To cite this article: Timothy M. Vadas , Xinning Zhang , Ashley M. Curran & Beth A.Ahner (2007): Fate of DTPA, EDTA, and EDDS in Hydroponic Media and Effects on PlantMineral Nutrition, Journal of Plant Nutrition, 30:8, 1229-1246
To link to this article: http://dx.doi.org/10.1080/01904160701555119
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes.Any substantial or systematic reproduction, redistribution, reselling, loan,sub-licensing, systematic supply, or distribution in any form to anyone isexpressly forbidden.
The publisher does not give any warranty express or implied or make anyrepresentation that the contents will be complete or accurate or up to
date. The accuracy of any instructions, formulae, and drug doses should beindependently verified with primary sources. The publisher shall not be liablefor any loss, actions, claims, proceedings, demand, or costs or damageswhatsoever or howsoever caused arising directly or indirectly in connectionwith or arising out of the use of this material.
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
Journal of Plant Nutrition, 30: 1229–1246, 2007
Copyright © Taylor & Francis Group, LLC
ISSN: 0190-4167 print / 1532-4087 online
DOI: 10.1080/01904160701555119
Fate of DTPA, EDTA, and EDDS in Hydroponic
Media and Effects on Plant Mineral Nutrition
Timothy M. Vadas,1 Xinning Zhang,1,2 Ashley M. Curran,1
and Beth A. Ahner1
1Department of Biological and Environmental Engineering, Cornell University, Ithaca,New York, USA
2Current address: Department of Environmental Science and Engineering, CaliforniaInstitute of Technology, Pasadena, California, USA
ABSTRACT
Synthetic chelators are commonly used in hydroponic media to solubilize iron (Fe);however, the fate of these chelators is unknown. This study examined the persistenceof three synthetic chelators, ethylenediaminetetraacetate (EDTA), diethylenetriamine-pentaacetate (DTPA), and ethylenediaminedisuccinate (EDDS) in a bench-scale lettuceproduction system. The EDDS concentration decreased rapidly within 7d, most likelydue to biodegradation. The EDTA and DTPA concentrations stayed steady throughoutthe experiments despite additions to maintain a constant volume and loss of chelator mayhave been due to either plant uptake or photodegradation of the chelator. Despite largedifferences in solution chemistry, the final shoot concentrations of iron (Fe), manganese(Mn), copper (Cu), and zinc (Zn) were similar among chelator treatments, whereasroot concentrations of these same elements were highly variable. The concentration ofDTPA in a commercial lettuce production system was measured and highly variableconcentrations were found.
Keywords: DTPA, EDDS, EDTA, hydroponics, chelator, nutrient solutions, lettuce
INTRODUCTION
Metal chelators are widely used in algal and plant growth solutions to maintainiron (Fe) solubility in hydroponic solutions, demonstrate limitation effects orexamine metal transport mechanisms (Webb et al., 1993; Parker and Norvell,1999). Synthetic chelators such as ethylenediaminetetraacetic acid (EDTA) are
Received 12 May 2006; accepted 4 October 2006.Address correspondence to Cornell University, 320 Riley-Robb Hall, Ithaca, NY
14853. E-mail: [email protected]
1229
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
1230 T. M. Vadas et al.
assumed to be relatively stable, but may be degraded chemically or biologically,or taken up by the plant.
It is well known that Fe-organic complexes are photoreactive(Svenson et al., 1989) and degradation rates are dependent on irradiance, tem-perature (Albano and Miller, 2001) and pH of the solution (Metsarinneet al., 2004). Half lives of Fe-EDTA and Fe-diethylenetriaminepentaacetic acid(DTPA) were measured under high sunlight conditions to be 11 min and 8 min,respectively (Svenson et al., 1989). Hangarter and Stasinopoulos (1991) foundthat under cool white fluorescent lamps in the laboratory, EDTA bound to Fein an agar growth solution was broken down into glyoxylic acid and formalde-hyde, which could inhibit plant growth. Several other studies have establishedthat EDTA in natural waters and EDTA, DTPA, or ethylenediaminedisucci-nate (EDDS) in laboratory-prepared solutions containing Fe were degradedsignificantly by sunlight and more specifically by UV light (Nowack andBaumann, 1998; Metsarinne et al., 2001). Photolytic degradation of the Fe-chelate complex in nutrient solutions for plant growth in the presence of blueand UV light was confirmed by Albano and Miller (2001).
It is less certain whether microbial degradation of synthetic chelators issignificant in hydroponic solutions. At low concentrations in natural surfacewaters, EDTA is not biologically degraded (Nowack and Baumann, 1998),but in wastewater treatment plants, depending on the pH, concentration, andbacterial species present, EDTA can be degraded (Nortemann, 1999). Morespecialized wastewater treatment processes involving coupled electrochemicaland biological treatment have been designed to remove up to 90% of DTPAfrom waste streams (van Ginkel et al., 2002), but DTPA is generally resistantto microbial degradation (Hinck et al., 1997).
Another potential loss of chelator from the hydroponic solutions is trans-port into the plant tissue. A study with 14C-labeled EDTA (Bell et al., 2003)showed accumulation of 14C in the shoots of swiss chard after 21 h; the levelsof EDTA accumulated were comparable to the amount potentially taken up bytranspirational flow. Other studies on chelate-assisted lead (Pb) uptake haveshown that at EDTA concentrations higher than 0.25 mM in solution, Pb accu-mulates in plant tissues along with EDTA and can be measured in the xylemsap after 48 h (Vassil et al., 1998; Epstein et al., 1999). At high chelate concen-trations, calcium (Ca2+) may be removed from the membrane through ligandcomplexation, compromising the root membrane integrity (Vassil et al., 1998),while at lower concentrations the EDTA complex could enter the xylem acrossan underdeveloped casparian band at the root tips (Bell et al., 1991).
Most studies involving chelators in hydroponic nutrient solutions have fo-cused on the mineral nutrition of the plant. Typically, the elemental compositionof both solution and plant are analyzed to determine the influence of the chelatoron metal uptake or availability (Handreck, 1992; Laurie et al., 1991a, 1991b).The primary focus of this study is on the temporal changes in the concentrationof chelator in the hydroponic nutrient solutions, which in laboratory studies are
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
Chelator Concentrations in Hydroponic Medium 1231
replaced frequently to maintain constant conditions, but in a commercial facil-ity, such as that run by the Cornell controlled environment agriculture (CEA)program, where volumes greater than 1000 gallons are not unusual, solutions arerecycled for economic and environmental reasons. DTPA and EDTA are com-monly used to solubilize Fe, but little is known about the fate of these chelatorsin solution. They might accumulate and therefore decrease the bioavailabilityof other essential metals [copper (Cu), zinc (Zn)], or may degrade or otherwisebe removed leading to the precipitation of Fe.
In this study, laboratory experiments have been conducted to compare thepersistence of three different chelators, EDTA, DTPA, and ethylenediaminedis-uccinic acid (EDDS) during a month-long experiment to evaluate the resultingeffects on plant nutrition. Both EDTA and DTPA are commonly used in hydro-ponic medium, whereas EDDS is a relatively new, more biodegradeable chelator(Vandevivere et al., 2001) and, to our knowledge, this is the first time it has beenused in hydroponic solutions. In each experiment, the chelator concentrationwas monitored during the crop cycle and the final elemental composition of thesolution and plant tissue was analyzed.
MATERIALS AND METHODS
A bench-scale lettuce production system was designed to emulate the systemat the Cornell CEA greenhouse, having similar volume to surface area ratios,plant spacing (4.5 plants per sq ft; 48 plants/m2) and greenhouse control oflight (a daily integral of 16 moles/m2) and temperature (day/night of 24/18◦C).An opaque 68 L Rubbermaid
©R container, dimensions 13′′ × 20′′ × 16.5′′ (0.33m × 0.5 m × 0.42 m), was used as the nutrient basin and a 1/2
′′ (1.3 cm)styrofoam sheet was used for the crop support and solution cover. The tub wasinitially filled with 47 L of nutrient solution (described below) and continuouslyaerated. Black plastic was used to cover the seam between the styrofoam andthe tub. Lettuce seedlings, Latuca sativa cv, (obtained from Cornell CEA) weregerminated with an ebb and flood system in rockwool cubes. At age 11 d,the seedlings were rinsed in distilled water for several minutes, and 6 plantswere inserted in a styrofoam sheet for each experiment. Throughout the growthcycle, the pH of the solution was maintained between 5.6 and 6.3. The ECwas monitored and maintained between 1200 and 1400 μS/cm through dailyadditions of nutrient solution or water to sustain a constant volume. Individualexperiments were conducted over the course of two years.
Nutrient Solution Composition
Two types of stock solutions (200×) were prepared from laboratory gradechemicals. Stock A (one for each chelator), once diluted, resulted in nutrient
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
1232 T. M. Vadas et al.
solution concentrations of 1.0 mM potassium nitrate (KNO3) (EMD Chemicals,Inc.), 2.1 mM calcium nitrate [Ca(NO3)2] (EM Science), 0.18 mM ammoniumnitrate (NH4NO3) (EM Science), 17 μM iron nitrate [Fe(NO3)3·9H2O] (FisherScientific), and 17 μM H5DTPA (Aldrich) or 17 μM Na2H2EDTA·2H2O(Sigma Aldrich) or 40 μM EDDS (Courtesy of P&G). Stock B, once di-luted, resulted in nutrient solution concentrations of 3.4 mM KNO3, 1.0 mMmonopotassium phosphate (KH2PO4) (Mallinckrodt), 0.5 mM magnesium sul-fate (MgSO4·7H2O) (Fisher Scientific), 63 μM potassium sulfate (K2SO4)(EM Science), 15 μM boric acid (H3BO3) (J.T. Baker), 3.5 μM zinc sulfate(ZnSO4·7H2O) (Fisher Scientific), 2.5 μM manganese sulfate (MnSO4·H2O)(Mallinckrodt), 0.37 μM copper sulfate (CuSO4·5H2O) (Fisher Scientific), and0.25 μM sodium molybdate (Na2MoO4·2H2O) (Mallinckrodt). Stock A solu-tions were wrapped in foil and stored in the dark when not in use. Followingdilution of stock solutions, the pH of the resulting nutrient solution was adjustedto 5.9.
The chemical speciation was calculated with the chemical equilibriumprogram CHEAQS (Verweij, 2005). Stability constants were obtained from theNIST critical stability constant database (Martell and Smith, 2003). As a resultof adding equimolar Fe and EDTA or DTPA, nearly 100% of the Fe is boundto chelator in solution, as is greater than 80% of the added Cu and 60% of theadded Zn. More than 2-fold additional EDDS than Fe was added because itrapidly degraded in a preliminary experiment and it is not as strong a chelatoras EDTA or DTPA; as a result, equilibrium calculations predicted nearly 100%chelation of Cu and Zn by EDDS in addition to the Fe. The free metal ionconcentrations are nearly the same in the DTPA and EDTA solutions, but thefree ion concentrations of Fe, Cu, and Zn are initially much lower in the EDDSsolution because of the excess chelator. About 90% of the Mn is present as freeMn2+ in both EDTA and DTPA solutions and 85% in the EDDS solution.
Sampling Protocol
For each of the bench-scale greenhouse experiments, solution samples for chela-tor analyses were taken on a regular basis, typically daily. In most experiments,chelator samples were collected into clear polypropylene tubes, but amber bot-tles were used for the final experiment, and then stored in the dark at 4◦C untilanalysis if the sample was to be analyzed that day or frozen at −10◦C for longerperiods. An additional solution sample was taken for elemental analysis. Uponharvest, triplicate lettuce heads were sampled by taking one old leaf (dark greenouter leaves), one new leaf (light green leaves near the center), and root tissue fortotal elemental analysis. Samples were placed in either pre-weighed polypropy-lene tubes or acid-washed glass screw-top tubes and dried to a constant weightat 70◦C prior to digestion. In the first two experiments, which included the oneEDDS experiment, harvested roots were rinsed with distilled water for 1 m. In
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
Chelator Concentrations in Hydroponic Medium 1233
the last two experiments, harvested roots were rinsed for 10 m, 3 successivetimes, with 1 mM EDTA adjusted to pH 6. However, the change in the rinsingmethod did not result in a consistent difference in measured metal concentra-tions and therefore values from different experiments were averaged together.The remaining lettuce heads and root tissues were placed in paper bags anddried at 70◦C until a constant weight was reached for dry weight measurement.Rockwool was also sampled for elemental composition.
In addition, solution samples were collected from the Cornell CEA hydro-ponic greenhouse from July 16, 2003 to October 28, 2004, intermittently for thefirst year and then more regularly during the last two months. The samples in thelast two months were consistently collected at about 6 PM in clear polypropy-lene tubes, and all samples were filtered through a 0.2 μm HT Tuffryn
©R syringefilter (Pall Corporation) and stored in the freezer prior to analysis.
HPLC Analysis of Chelators
For HPLC standard preparation, stock solutions of 0.01 M EDDS, and 0.01M EDTA, and 0.01 M DTPA were prepared using a 40% EDDS solution,Na2H2EDTA·2H2O, and H5DTPA, respectively. Standard concentrations rang-ing from 1 to 30 μM were prepared from these stocks. A salt solution, consist-ing of the major cations and anions from the nutrient solution [4.4 mM KNO3,2.1 mM Ca(NO3)2·4H2O, and 0.18 mM NH4NO3], was added to EDDS andEDTA standards. DTPA standards were prepared with all components of thenutrient solution except for the Fe and DTPA present in Stock A. The stocksand standards were wrapped in aluminum foil and stored in the refrigerator.
The following EDTA and EDDS sample preparation is based largely on amethod published by Nowack et al. (1996). An Fe(III) solution was prepared bydissolving 0.01 M iron nitrate [Fe(NO3)3·9H2O] in 0.01 M nitric acid (HNO3).Formate buffer was prepared with 0.02 M formic acid adjusted to pH 3.3 withHNO3. Tetrabutylammonium bromide (TBA-Br) solution was prepared by dis-solving 0.05 M TBA-Br in formate buffer. Prior to analysis of EDTA and EDDS,samples were thawed in a covered water bath at 60◦C and 0.5 mL aliquots weredried in 2 mL centrifuge tubes using a vacuum centrifuge. Formate buffer (1 mL)and Fe(III) solution (20 μL) were then added to each tube and the samples weresealed and heated at 90◦C for 3 h in a dry heat block. After the samples cooledto room temperature, 40 μL of TBA-Br solution was added and the sampleswere transferred to amber autosampler vials for HPLC analysis.
A new sample preparation protocol based on Nowack et al. (1996) wasdeveloped for DTPA since experimentation with various methods revealed thatthe presence of phosphate greatly improved DTPA recovery (Vadas, 2006). Toeach 1 mL of DTPA sample or standard, 100 μL of a 20 mM KH2PO4 solutionand 20 μL of the Fe(III) solution was added and subsequently heated at 90◦Cfor 3 h. Samples were then transferred to amber vials for HPLC analysis.
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
1234 T. M. Vadas et al.
The following isocratic HPLC methods for analyzing EDDS, EDTA, andDTPA are based on methods developed for EDTA by Nowack et al. (1996) andDTPA by Richardson et al. (1994). Analyses were performed with a WatersNova-Pak C18 column (3.9 × 300 mm) on a Beckman HPLC system equippedwith a 125 Solvent module and a Shimadzu SIL-10ADvp autoinjector with asample injection volume of 200 μL. The analyte was detected with a ShimadzuSDP-10ADvp UV-vis detector set at 258 nm. The flow rate was fixed at 1mL/min. The EDDS/EDTA mobile phase consisted of 0.02 M formate buffer,8% acetonitrile (ACN), and 0.001 M TBA-Br adjusted to pH 3.3 with 0.1 MHNO3. The DTPA mobile phase included a mixture of two solutions: 91%solution A containing 4 mM octylamine and 8% ACN, adjusted to pH 6 withH2SO4, and 9% solution B containing 100% ACN. The Fe-EDDS complexeluted at about 5 m, the Fe-EDTA complex at about 5.5 m, and the Fe-DTPAcomplex at about 9.2 m.
Elemental Analysis
Dry tissue samples from the first two experiments were digested with 5 mL ofHNO3 heated at 65◦C for 24 h in a drying oven, diluted with water and analyzedon an inductively coupled plasma optical emission spectrometer (ICP-OES) atthe Analytical Laboratory in the Department of Horticulture at Cornell Univer-sity. Tissue samples for the last two experiments were sequentially digested,first with the addition of 5 mL concentrated HNO3 (heated at 110◦C for 15 mand allowed to react overnight) and then with 3 mL of H2O2 (heated 15 mat 110◦C). Samples were diluted and then analyzed by ICP-OES at the USDAFederal Nutrition Laboratory on the Cornell campus.
Photodegradation of EDTA and DTPA Experiments
Photodegradation of EDTA and DTPA was examined under typical greenhouseconditions in July of 2005. Polypropylene sample vials with DTPA- or EDTA-containing nutrient solutions (as described above) were exposed to natural sun-light in the greenhouse. Subsamples were collected over time into amber tubesand were analyzed for chelator concentration using the HPLC methods de-scribed above. Half-lives were calculated assuming first order kinetics.
Extraction of EDTA and DTPA from Plant Tissue
Leaf and root tissue samples collected for chelator extraction were rinsed bysubmersion in DI water for 10 m, 3 successive times. Samples were placed inplastic bags and frozen at −80◦C until analysis. An extraction protocol based
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
Chelator Concentrations in Hydroponic Medium 1235
on methods described in Vassil et al. (1998) was used to extract chelators fromplant tissue. Approximately 0.5 g of tissue was placed in 1.5 mL of a 50:50ethanol:water solution. Triplicate tissue samples were homogenized using aTissue TearorTM (Biospec Products, Inc.) and subsequently heated at 80◦C for10 m, then centrifuged at 2000 rpm for 20 m. The supernatant was then dividedinto two samples and the resulting replicate samples were dried using a vacuumcentrifuge. An internal standard was added to one replicate. Subsequent stepswere the same as described above. Samples were analyzed on the HPLC usingthe isocratic elutions described above followed by 20 m of 100% ACN and20 m of re-equilibration with the mobile phase to rinse the column betweentissue samples.
RESULTS
Chelator Concentrations in Hydroponic Medium During
the Growth of Lettuce
The EDDS concentration in the nutrient solution decreased rapidly within thefirst week from 40 μM to below 3 μM and remained between 0 and 4 μMfor the duration of the experiment (Figure 1a). This was most likely due tobiodegradation and/or photodegradation of the EDDS in solution. This wasamong the first experiments performed and was not replicated. In hydroponicmedium containing EDTA and DTPA, concentrations during the first two weeksgradually increased as expected on the basis of how much was added and thenleveled off at approximately17 μM for the remainder of the experiment. Onlydata from the final EDTA and DTPA experiments is shown (Figure 1b,c). At theend of the experiment, concentrations of EDTA and DTPA could theoreticallyhave reached 25 and 27 μM, respectively (as shown by the dotted line in Fig-ure 1b,c), but 34% and 37% of the EDTA and DTPA were lost, respectively.To maintain a constant concentration, degradation or plant uptake must havebalanced chelator addition to the nutrient solution. Replicate experiments con-ducted for both EDTA and DTPA were comparable, revealing similar trends(Vadas, 2006).
Photochemical Degradation of EDTA and DTPA
Photodegradation experiments were conducted to determine potential loss ratesof EDTA and DTPA from medium exposed to natural sunlight in the greenhouse.Photodegradation of both DTPA and EDTA was rapid; initial concentrationsdecreased by about 80% over 1 h (Figure 2). Half-lives for each compoundwere calculated assuming first order kinetics (ln [C/C0] = −kpt); t1/2 for EDTAwas 28.3 m and 22.7 m for DTPA (R2 = .978 for EDTA and .998 for DTPA).These values are within the range of published values (Metsarinne et al., 2001;
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
1236 T. M. Vadas et al.
Figure 1. EDDS, EDTA, and DTPA concentrations (�) in the nutrient medium duringthe course of one experiment. The cumulative expected concentration (dotted line) wascalculated from the volume of nutrient solution replenished over the course of eachexperiment. Values are means ± SD (n = 3). Where not shown, error bars are withinthe height of the symbol.
Metsarinne et al., 2004; Svenson et al., 1989). Chelator concentration in nu-trient solutions exposed to fluorescent light in the laboratory did not decreasesignificantly over one hour (data not shown); for this reason, we assumed thatlight exposure during sample preparation in the laboratory would not affect our
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
Chelator Concentrations in Hydroponic Medium 1237
Figure 2. Fraction of initial added DTPA or EDTA (C/C0) remaining in solution fol-lowing incubation under natural light in the greenhouse at 24◦C, pH 6. Values are means± SD (n = 3).
measurements. Photodegradation of EDDS was not examined because it wasclear from plant growth experiments (Figure 1a) that it would not be an effectivechelator in recirculating hydroponic systems.
Extraction of EDTA and DTPA from Plant Tissues
Both EDTA and DTPA were measured in roots and shoot tissue extracts. Inroots, DTPA accumulated to greater levels than EDTA, whereas in shoots, av-erage EDTA concentrations were higher than those of DTPA (Table 1). The
Table 1Average EDTA and DTPA concentration (μmoles/g DW) measured in lettuce tissueextracts and % recovery of missing chelator from the final experiment. New shootand old shoot tissue were assumed to account for 25% and 75%, respectively, of thetotal lettuce head biomass. Values followed by the same letter within a column are notsignificantly different from each other (P > 0.05). Below detection values were averagedas zero. n = 3.
Total in 6Roots Old shoots New shoots plants %
(μmoles/g) (μmoles/g) (μmoles/g) (μmoles) recovery
EDTA 0.029 ± 0.007 a 0.015 ± 0.005 a 0.048 ± 0.004 a∗ 43.9 10.9DTPA 0.045 ± 0.004 b 0.005 ± 0.009 a 0.009 ± 0.015 a∗ 13.3 2.77
∗Significantly different with P < 0.06.
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
1238 T. M. Vadas et al.
higher concentration of EDTA in shoots might be due to the smaller size andcharge of EDTA compared to DTPA. Based on these analyses, an estimate ofthe total amount of EDTA and DTPA in lettuce tissue at the end of the finalexperiment is 43.9 and 13.3 μmoles respectively, which represents 11% and3%, respectively, of the total chelator missing from solution. Photodegradationor biological transformation of chelators within the plant tissue during the longgrowing period likely has an impact on the concentration of chelators measuredwithin tissues. Also, the tissue measurements represent a minimum concentra-tion because of poor peak separation and a high background absorbance inextraction samples. The fact that intact chelators were found in tissues indi-cates that they are taken up by the plant and suggests that this loss mechanismmay be important for loss of chelator from the nutrient solution. In addition,the chelator concentration remains fairly constant throughout the experiments;since there is less added early on, the removal rate must be lower. The increasedloss of chelator towards the end of the experiments could be related to highertranspiration rates of older plants.
Plant Yields and Mineral Nutrition
Within individual experiments, crops grown in medium containing DTPA didnot have significantly different shoot or root dry weights from crops grownwith EDDS or EDTA (e.g., 7.59 ± 0.75 g vs. 7.71 ± 0.56, for EDTA andDTPA shoots, respectively, from the third experiment). Biomass yields betweenexperiments cannot be compared because growth periods were different.
The shoot concentrations of Fe, Cu, and Zn were similar and not influ-enced by the chelator used (Table 2), reflecting the plant’s ability to efficientlyregulate uptake of these elements regardless of the solution chemistry. AverageFe and Zn shoot concentrations were within the range reported for greenhouseproduction of the same lettuce species by Cornell CEA (LaDue, 2005) usingthe same nutrient solution composition with DTPA as the chelator (75–94 re-ported here vs. 68–198 ppm and 85–120 reported here vs. 42–134 ppm forFe and Zn, respectively). Shoot copper concentrations were slightly elevated(11–31 reported here vs. 5–12 ppm Cu) most likely due to solution contamina-tion. Average shoot Mn concentrations in young leaf tissues were lower thanreported values (20–35 reported here vs. 36–126 ppm Mn), but old leaf tissueMn concentrations were within the range (data not shown).
The concentration of Fe measured in solution at the end of the experimentswas nearly zero in the EDDS experiment and an average of 13 and 14 μM forthe EDTA and DTPA experiments, respectively. The solution Fe concentrationin most cases was equal to or less than the measured chelator concentration,except in the third DTPA experiment where the total dissolved Fe concentrationwas 18 μM, yet the chelator concentration was only 15 μM, suggesting othercompounds, possibly breakdown products, are also chelating Fe. Whereas no
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
Tabl
e2
Ave
rage
plan
tFe,
Mn,
Cu,
Zn
conc
entr
atio
nsin
shoo
ts,r
oots
,and
nutr
ient
solu
tions
.Roo
tsfr
omth
efir
sttw
oex
peri
men
tsw
ere
rins
edin
dist
illed
wat
erfo
ron
em
inut
e.R
oots
from
the
final
two
expe
rim
ents
wer
eri
nsed
in1
mM
ED
TAfo
r10
m,3
succ
essi
vetim
es.H
owev
er,t
hese
diff
eren
tri
nsin
gte
chni
ques
did
notr
esul
tin
cons
iste
ntdi
ffer
ence
sin
mea
sure
dm
etal
conc
entr
atio
ns,t
here
fore
valu
esfr
omal
lexp
erim
ents
wer
eav
erag
ed.
Roo
tcon
cent
ratio
nssh
ould
bein
terp
rete
das
tota
lint
race
llula
ran
dsu
rfac
ebo
und
met
als.
Shoo
ttis
sues
conc
entr
atio
nspr
esen
ted
repr
esen
tyou
ngle
aftis
sue
mea
sure
men
tsas
abe
tteri
ndic
ator
ofnu
triti
onal
stat
us.V
alue
sfo
llow
edby
the
sam
ele
tterw
ithin
aco
lum
nar
eno
tsig
nific
antly
diff
eren
tfr
omea
chot
her
(p>
0.05
).V
alue
sar
e±
SD.
Plan
ttis
sues
(μg/
gD
W)
FeM
nC
uZ
n
Roo
tsSh
oots
Roo
tsSh
oots
Roo
tsSh
oots
Roo
tsSh
oots
ED
DS1
(n=
3)71
00±
1800
a94
±70
a50
0±
100
a20
±3
a11
00±
39a
31±
14a
1300
±17
a12
0±
31a
ED
TA2
(n=
12)
2400
±16
00b
75±
17a
140
±80
b35
±17
b50
±29
∗b
11±
8a
130
±40
∗b
88±
23a
DT
PA3
(n=
15)
2000
±10
00a
78±
32a
340
±22
0a
24±
8ab
59±
46b
14±
10a
240
±12
0c
85±
20a
Nut
rien
tmed
ium
(μM
)In
itial
6Fi
nal
Initi
al6
Fina
lIn
itial
6Fi
nal
Initi
al6
Fina
l
ED
DS1
(n=
1)17
n.d.
2.5
0.27
0.37
1.7
3.6
1.6
ED
TA2,
4(n
=3)
1713
±3.
52.
51.
1±
1.3
0.37
4.5
±4.
33.
65.
8±
3.6
DT
PA3,
5(n
=4)
1714
±3.
42.
50.
37±
0.37
0.37
4.3
±5.
23.
64.
7±
1.8
1A
vera
gefr
om1
expe
rim
ent.
2A
vera
gefr
om3
expe
rim
ents
.3A
vera
gefr
om4
expe
rim
ents
.4M
easu
red
initi
also
lutio
nco
ncen
trat
ions
(μM
)fr
omth
efin
altw
oE
DTA
expe
rim
ents
rang
edfr
om15
.5to
15.9
for
Fe,1
.49
to2.
24fo
rM
n,1.
99to
2.69
for
Cu,
and
3.67
to6.
71fo
rZ
n.5M
easu
red
initi
also
lutio
nco
ncen
trat
ions
(μM
)fr
omth
efin
altw
oD
TPA
expe
rim
ents
rang
edfr
om15
.7to
17.1
for
Fe,2
.13
to2.
26fo
rM
n,0.
66to
3.98
for
Cu,
and
4.11
to4.
33fo
rZ
n.6In
itial
expe
cted
conc
entr
atio
nba
sed
onnu
trie
ntso
lutio
nco
mpo
sitio
n.∗ n
=6,
ED
TAre
sults
from
the
thir
dex
peri
men
twer
ere
mov
eddu
eto
high
solu
tion
cont
amin
atio
nw
ithZ
nan
dC
u;if
thes
eva
lues
wer
ein
clud
ed,
the
aver
age
root
conc
entr
atio
nsar
e25
0±
230
ppm
and
960
±90
0fo
rC
uan
dZ
n,re
spec
tivel
y.
1239
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
1240 T. M. Vadas et al.
Figure 3. Mn concentrations in hydroponic media with EDTA (�) and DTPA (� ) as afunction of time during the final experiment. * contamination suspected in these samples.
significant differences in shoot concentrations of Fe were observed, root con-centrations of Fe were significantly higher in the EDDS experiment. Since lessthan 1 μM EDDS remained in solution at the end of the experiment (Figure 1a)and Fe is extremely insoluble at the pH of our nutrient solution, Fe likely pre-cipitated on the root surface. The Fe-oxide precipitates on the roots also likelycontributed to the high levels of root-associated Cu, Zn, and manganese (Mn)in the EDDS-grown plants.
In contrast to steady Fe concentrations measured in solution during thefinal experiment (data not shown), the Mn concentration decreased steadily(Figure 3). This was likely typical of all experiments since the solution concen-tration of Mn measured at the end of all but one experiment was low (Table 2).A mass balance was conducted on Mn during the final experiment, revealingthat most of the Mn was sorbed to the rockwool. The sorption to rockwoolaccounts for about 108 ± 20% and 97 ± 9% of the Mn lost from the mediumin the EDTA and DTPA systems, respectively, whereas only 18 ± 5% of theMn in the EDTA system, and 12 ± 4% in the DTPA system ended up in planttissue. Total recovery from the nutrient solution, plant tissue and rockwool [1Mhydrochloric acid (HCl) extracted] was 129 ± 24% for the EDTA system and114 ± 14% for the DTPA system.
The shoot concentrations of Mn in the EDTA experiments were signifi-cantly higher than the EDDS experiment, but not significantly different fromthe DTPA experiments. During the final experiment, Mn remained in solutionfor a longer period in the EDTA system than the DTPA system (Figure 3). Man-ganese added in these experiments is not thermodynamically stable in the pres-ence of oxygen and oxidation of Mn, though slow, can be kinetically enhancedby microorganisms. Manganese-oxidizing organisms are known to require Cu
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
Chelator Concentrations in Hydroponic Medium 1241
in order to accomplish this, but at high concentrations (>0.15 μM), Cu canbecome toxic (Zhang et al., 2002). The DTPA solution may have provided amore favorable environment for biological Mn oxidation due to greater bindingof Cu by DTPA and its photodegradation byproducts resulting in lower solu-tion free Cu2+ concentrations. The lower Mn concentrations associated withthe roots from the EDTA experiments therefore may reflect less Mn oxidationon the root surface. With less oxidation, Mn remains in solution longer and istherefore more available for uptake and translocation to the shoots.
In all but one experiment, the Cu concentrations in the hydroponic mediumat the end of the experiment were 4- to 32-fold higher than the intended nutrientsolution concentration (Table 2). It appears that the Cu concentration was highfrom the onset of the experiments (data only available for the final two exper-iments), ranging from 1.8 to 11 times the intended concentration and steadilyincreased over the course of the final experiment (data not shown). This con-tamination was likely from the greenhouse DI water used to dilute the stocksolutions, dust, or contact with the plastics used in the study. This could have ledto the elevated Cu concentrations seen in the shoots. The root concentrations ofCu from EDTA and DTPA experiments were significantly lower than the EDDSexperiment. The high Cu values measured for EDDS-treated roots is likely dueto sorption of Cu to Fe-oxide coated roots in the absence of any chelator.
In general, contamination of Zn was less of a problem, since solution Znconcentrations at the end of the experiments were only 0.4 to 2.7 times the ini-tial solution concentrations. The root concentrations of Zn were significantlydifferent between all experiments, with much higher values in the EDDS exper-iment likely due to sorption to the roots in the absence of chelator as with Cu.The higher Zn root concentration observed for the DTPA experiment comparedto the EDTA experiment may have been related to the postulated greater Mnoxidation on the roots in the DTPA system and subsequent sorption of Zn tothe Mn oxides.
Cornell CEA Chelator Concentrations
The concentration of DTPA at the Cornell CEA greenhouse was tracked formore than a year. Tank 1 contained lettuce at the seedling stage (12–22 d) ofthe crop cycle and tank 2 contained the mature lettuce heads (23–35 d) forthe last portion of the crop cycle. Samples over the entire time course showedconcentrations in the range of about 1 to 22 μM (Table 3). There was a largeincrease in DTPA concentration in both tanks between November 2003 andJanuary 2004 (from about 4 μM to 21 μM in tank 1 and from 6 μM to 18 μMin tank 2, Table 3) which we tentatively attribute to less photodegradationduring the cloudy winter months. In order to characterize variation that maybe happening on a finer scale, more frequent samples were taken starting onSeptember 20, 2004. These samples also showed large changes in concentration
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
1242 T. M. Vadas et al.
Table 3DTPA concentrations (μM) in a recirculating hydroponic solution from a commercialscale system growing lettuce from 7/16/2003 to 10/8/2004. Tank 1 contained plantsage 12–22 d while tank 2 contained plants age 23–35 d old. The system is maintainedyear-round by Cornell CEA.
Seasonal sampling Intensive sampling
Date Tank 1 Tank 2 Date Tank 1 Tank 2
7/16/2003 0.8 2.5 ± 0.4 9/20/2004 3.1 ± 0.2 9.9 ± 0.111/6/2003 3.8 ± 0.6 5.9 ± 0.3 9/24/2004 4.4 ± 0.3 8.8 ± 0.31/12/2004 22 ± 0.1 18 ± 0.2 9/27/2004 5.6 ± 0.3 9.5 ± 0.42/11/2004 2.6 ± 0.2 9.9 ± 0.1 9/29/2004 11 ± 0.2 9.2 ± 0.24/5/2004 3.5 ± 0.2 na 10/4/2004 6.4 ± 0.2 6.2 ± 0.26/16/2004 5.7 ± 0.2 2.9 ± 0.1 10/8/2004 5.7 ± 0.0 4.6 ± 0.0Averagea 6.3 ± 7.6 7.9 ± 6.5 Averagea 6.0 ± 2.7 8.0 ± 2.1
aAverage values are not significantly different (P > 0.05).
over only a few days. The DTPA concentration in tank 1 increased from 3 μMto 11 μM over nine days and decreased to 6 μM over the next nine days, whilethe DTPA concentration in tank 2 decreased from about 10 μM to 5 μM over18 days (Table 3).
Operation of the greenhouse for commercial production adds additionalvariables to consider. During some harvests, large sections of the ponds areleft uncovered for variable periods of time allowing light to reach the watersurface. Also, the solution in tank 2 likely receives less light exposure duringnon-harvest times since it contains mature crops and the plants form a canopythat covers most of the styrofoam and edges. However, that did not seem to havea large impact because the concentrations in both tanks on average are similar.Each tank was independently replenished with nutrient stock as needed, whichwould explain the different concentrations measured in each tank during theintensive sampling period.
DISCUSSION
This study examined the fate of the chelators EDDS, EDTA, and DTPA, inhydroponic solutions over the course of one crop cycle. The concentration ofEDDS decreased rapidly, most likely due to biological degradation since it isknown to be readily degraded (Vandevivere et al., 2001). Concentrations ofEDTA and DTPA appear to level off at near initial concentrations, and lossescould have been due to plant uptake and photodegradation of the chelator eitherin the solution or following uptake into the plant material. The plant nutrition
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
Chelator Concentrations in Hydroponic Medium 1243
did not appear to be affected over one crop cycle in these experiments, buthigh root concentrations of some metals and nearly complete loss of Mn fromsolution were observed, which could potentially lead to nutrient deficiencies ina recirculating hydroponic system.
It is important for greenhouse managers to realize that chelator concen-trations can be highly variable. One of our original hypotheses was that thechelator concentration would increase over time decreasing the bioavailabilityof micronutrients Cu and Zn, although there was no evidence for this in ourstudy. During periods of low chelator concentration, some metals may be re-moved from the system disproportionally to other nutrients due to adsorption orprecipitation on the roots causing mineral deficiencies over time or the bioavail-ability of some metals may be elevated (e.g., high Cu levels in shoots from theEDDS system). However, we found that loss processes balanced addition inour experiments and that at the CEA facility, levels were never greater than130% of the target chelator concentration and sometimes dipped to very lowlevels. For year-round recirculating systems, seasonal variations in light maylead to different rates of photodegradation. For optimal management of chelatorconcentrations, exposure of the nutrient solutions to light must be minimizedand plant transpiration rates controlled for a more predictable loss of chelatorthrough plant uptake.
One of the more striking results was that the dissolved Mn concentrationwas very low at the end of almost all of the experiments. This is consistent withproblems at the CEA greenhouse where plant failures have sometimes beencorrelated with Mn deficiencies. In some instances, Mn may be removed bybiological manganese oxidation, decreasing its availability to the plants. Mnwas also found to associate with the rockwool, but whether this is adsorption ofMn (II) or oxide formation on the fibers of the rockwool is unknown. High Mnconcentrations already present in rockwool may autocatalyze the oxidation ofMn2+ from the nutrient solution. Regardless of mechanism, lower than averageMn values were found in our experimental plant shoots after only one cropcycle. It may be useful to either find a substitute support material or treat therockwool in some way to minimize Mn adsorption or oxidation.
In one DTPA experiment, the dissolved Fe concentration at the end ofthe experiment exceeded the chelator concentration (18 vs 15 μM), suggestingthat there were likely other compounds present which are also able to chelateFe. Metsarinne et al. (2004) identified several degradation products of DTPAusing GC-MS such as diethylenetriaminetriacetic acid and ethylenediamine-triacetic acid which could potentially chelate Fe, but little is known about thebinding constants and half-lives of these chemicals. The HPLC chromatogramsfrom several DTPA solution samples, most notably those from the photodegra-dation experiment, did show an additional peak increasing in size over the courseof the experiment eluting at about 4 m (Vadas, 2006). When EDTA-containingsamples were analyzed using this method, EDTA eluted close to 4 m, whichsuggests that one degradation product of DTPA is similar in size and charge to
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
1244 T. M. Vadas et al.
EDTA. This compound could also be chelating Fe in the hydroponic solution. Inaddition, some photodegradation byproducts may be harmful to the plants (e.g.formaldehyde; Hangarter and Stasinopoulos, 1991). In order to understand thepotential problems associated with these byproducts, they must be identifiedand their metal binding ability characterized.
From these experiments we have shown that EDDS is not an effectivechelator for hydroponic systems, but both EDTA and DTPA can be used, thoughsubtle differences in plant mineral nutrition may occur due to different ratesof removal from solution. The effective use of chelators to solubilize metals inrecirculating hydroponic nutrient solutions can be enhanced when care is takento decrease chelator losses and a better understanding of trace metal lossescan be used to maintain optimal nutrient concentrations.
REFERENCES
Albano, J. P., and W. B. Miller. 2001. Photodegradation of FeDTPA in nu-trient solutions. I. Effects of irradiance, wavelength, and temperature.Hortscience 36: 313–316.
Bell, P. F., R. L. Chaney, and J. S. Angle. 1991. Free metal activity and total metalconcentrations as indices of micronutrient availability to barley [Hordeumvulgare (L.) ‘Klages’]. Plant and Soil 130: 51–62.
Bell, P. F., M. J. McLaughlin, G. Cozens, D. P. Stevens, G. Owens, and H.South. 2003. Plant uptake of C-14-EDTA, C-14-citrate, and C-14-histidinefrom chelator-buffered and conventional hydroponic solutions. Plant andSoil 253: 311–319.
Epstein, A. L., C. D. Gussman, M. J. Blaylock, U. Yermiyahu, J. W. Huang,Y. Kapulnik, and C. S. Orser. 1999. EDTA and Pb-EDTA accumulation inBrassica juncea grown in Pb-amended soil. Plant and Soil 208: 87–94.
Handreck, K. A. 1992. Relative effectiveness of iron sources for an iron-inefficient species growing in a soilless medium. Journal of Plant Nutrition15: 179–189.
Hangarter, R. P., and T. C. Stasinopoulos. 1991. Effect of Fe-catalyzed photoox-idation of EDTA on root-growth in plant culture media. Plant Physiology96: 843–847.
Hinck, M. L., J. Ferguson, and J. Puhaakka. 1997. Resistance of EDTA andDTPA to aerobic biodegradation. Water Science and Technology 35: 25–31.
LaDue, R. 2005. Lettuce tissue concentration ranges of Fe, Mn, Zn, Cu grownat the Cornell CEA facility. Ithaca, NY. Personal communication.
Laurie, S. H., N. P. Tancock, S. P. McGrath, and J. R. Sanders. 1991a. Influenceof complexation on the uptake by plants of iron, manganese, copper andzinc. 1. Effect of EDTA in a multi-metal and computer-simulation study.Journal of Experimental Botany 42: 509–513.
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
Chelator Concentrations in Hydroponic Medium 1245
Laurie, S. H., N. P. Tancock, S. P. McGrath, and J. R. Sanders. 1991b. Influenceof complexation on the uptake by plants of iron, manganese, copper andzinc. 2. Effect of DTPA in a multi-metal and computer-simulation study.Journal of Experimental Botany 42: 515–519.
Martell, A. E., and R. M. Smith. (2003) NIST Standard Reference Database 46Version 7.0: NIST Critically selected stability constants of metal complexes7.0. NIST, Gaithersburg, MD.
Metsarinne, S., P. Rantanen, R. Aksela, and T. Tuhkanen. 2004. Biologicaland photochemical degradation rates of diethylenetriaminepentaacetic acid(DTPA) in the presence and absence of Fe(III). Chemosphere 55: 379–388.
Metsarinne, S., T. Tuhkanen, and R. Aksela. 2001. Photodegradation ofethylenediaminetetraacetic acid (EDTA) and ethylenediamine disuccinicacid (EDDS) within natural UV radiation range. Chemosphere 45: 949–955.
Nortemann, B. 1999. Biodegradation of EDTA. Applied Microbiology andBiotechnology 51: 751–759.
Nowack, B., and U. Baumann. 1998. Biodegradation of the photolysis productsof Fe(III)EDTA. Acta Hydrochimica et Hydrobiologica 26: 104–108.
Nowack, B., F. G. Kari, S. U. Hilger, and L. Sigg. 1996. Determination ofdissolved and adsorbed EDTA species in water and sediments by HPLC.Analytical Chemistry 68: 561–566.
Parker, D. R., and W. A. Norvell. 1999. Advances in solution culture methodsfor plant mineral nutrition research. In Advances in Agronomy, Vol 65. SanDiego: Academic Press Inc. pp. 151–215.
Richardson, D. E., G. H. Ash, and P. E. Harden. 1994. Determination of di-ethylenetriaminepentaacetic acid in pulp-mill effluent by ion-interactionreversed-phase liquid-chromatography. Journal of Chromatography A 688:47–53.
Svenson, A., L. Kaj, and H. Bjorndal. 1989. Aqueous photolysis of the iron(III) complexes of NTA, EDTA and DTPA. Chemosphere 18: 1805–1808.
Vadas, Timothy M. 2006. Fate of DTPA, EDTA, and EDDS in hydroponicmedia and effects on plant mineral nutrition. M.S. Thesis, Biological andEnvironmental Engineering, Cornell University, Ithaca, NY.
van Ginkel, C. G., B. J. W. Tuin, V. G. Aurich, and W. Maassen. 2002. Couplingof electrochemical and biological treatment to remove diethylenetriamine-pentaacetic acid (DTPA) from pulp and paper effluents. Acta Hydrochimicaet Hydrobiologica 30(2–3): 94–100.
Vandevivere, P., F. Hammes, W. Verstraete, T. Feijtel, and D. Schowanek.2001. Metal decontamination of soil, sediment, and sewage sludge bymeans of transition metal chelant [S,S]-EDDS. Journal of Environmen-tal Engineering-ASCE 127(9): 802–811.
Vassil, A. D., Y. Kapulnik, I. Raskin, and D. E. Salt. 1998. The role of EDTAin lead transport and accumulation by Indian Mustard. Plant Physiology117: 447–453.
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
1246 T. M. Vadas et al.
Verweij, W. 2005. CHEAQ: A program for calculating Chemical Equilibria inAquatic Systems, Ver L06. National Institute for Public Health and theEnvironment, Bilthoven, The Netherlands.
Webb, M. J., W. A. Norvell, R. M. Welch, and R. D. Graham. 1993. Using achelate-buffered nutrient solution to establish the critical solution activityof Mn2+ required by barley (Hordeum-Vulgare L). Plant and Soil 153(2):195–205.
Zhang, J., Lion, L. W., Nelson, Y. M., Shuler, M. L., and Ghiorse, W. C. 2002.Kinetics of Mn(II) oxidation by Leptothrix discophora SS1. Geochimicaet Cosmochimica Acta 65(5): 773–781.
Dow
nloa
ded
by [
Penn
sylv
ania
Sta
te U
nive
rsity
] at
07:
56 2
3 A
pril
2013
