Introduction restoring eventual acute contaminations events is necessary. In this context, phytoremediation emerge
1. Introduction restoring eventual acute contaminations events is necessary. In this context, phytoremediation emerge
In the Humid Pampa, the main agricultural region as an effective, environmental friendly and in Argentina, the application of herbicides associated solar-energy driven technology with strong to the intensive agronomic practices implies the use of application potential and public acceptance [5]. about 7000 tons of atrazine (ATZ) every season [1]. However, despite phytoremediation is the best As it was demonstrated in countries with intensive technology to be applied on these wide extensions of ATZ application, the herbicide input frequently moderately polluted soils, the application context overcomes the ecosystems natural attenuation capacity have characteristics that necessary involve an and its residues pollute soils and groundwater, integral approach and strategies specially designed increasing the risk on human health and wildlife [2, case by case [6]. As an example, weeds which are 3]. robust, low requirements, fast growing and naturally Considering that Argentina is one of the mayor selected herbicide resistant vegetal species, are worldwide ATZ consumers [4], development of logically excluded from the agronomic context. In specific technologies designed for minimizing the the same way, the intensive agronomic practices environmental impact of the agronomic practices and model narrows the technology application to a
parallel or integrative approach, with minimal Corresponding author: Luciano J. Merini, Ph.D., teaching
assistant; research fields: biotechnology, bio/rhizoremediation interference with standard agronomic procedures or of organic pollutants. E-mail: [email protected].
Biotechnological Strategies for Phytoremediating Triazinic
Herbicides in the Humid Pampa (Argentine)
impact on crops yield. Therefore, these additional implementation strategies compatibles with the variables plus the intrinsic complexity of conventional agronomical practices. Considering these phytoremediation make the implementation strategy
observations and the particular soil and climate one of the most relevant aspects to take into account.
characteristics of the region, Chenopodium quinoa, Besides, considering that intensive agronomic
Lotus glaber and Lulium multiflorum come up as practices are applied over about 2 million hectares of
candidates to be employed in the Argentinean Humid the Humid Pampa, it is highly relevant to design
Pampa.
strategies which allow overcoming the obstacles of
2.2 Strategies Design for Selected Plant Species field scale implementation and tackling its
associated pollution. In addition, integral Combining the special particularities of the remediation strategies could have a deep influence
intensive agronomic practices and the soil, weather in a context where the narrowing of the urban/rural
and topogeographical characteristics of the Humid gap has transformed the once environmental Pampa, implementation strategies were designed for problem in a public health issue.
the three agronomic relevant vegetal species proposed. The objective of this work is designing integral
C. quinoa : regarding to herbicide tolerance, this phytoremediation strategies in order to minimize the
specie share the same Genus and is closely related to environmental impact of ATZ pollution in the Humid
C. album , a well known multi-resistant weed, Pampa agronomical context. Under the integral
frequently used as experimental model for ATZ approach, intensive agriculture is considered as a
tolerance assessing.
continuous, open field industrial process which needs Agronomically, C. quinoa is “pseudocereal” which
a coupled biotechnological solution to its running off grains have acquired a rising interest as high protean or leaching wastewaters.
value food since, unlike others; they contain all In this framework and considering the soil and
essential amino acids. It is currently considered as a topogeographical characteristics of the Humid Pampa
“new old crop” and it has an added value that makes [7], the ATZ tolerance of three agronomical relevant
of its production a profitable business. Those vegetal species and their capacity to stimulate
characteristics allow implementing it as a rizospheric degradation were assessed and their
phytoremediation tool by fully replacing the application strategies proposed.
traditional crop until complete soil decontamination.
2. Material and Methods
The replacement phytoremediation strategies planned for C. quinoa was specially designed for Austral
2.1 Plant Selection Pampa soils type (Fig. 1). Despite the cultivable soils
During the selection of suitable vegetal species it is of the region represent a comparatively small portion necessary to consider, besides herbicide tolerance, its
of Humid Pampa; they participate in the intensive implementation feasibility in a sustainable farming
agronomical exploitation with extensive use of practice context. Plants selected as candidates have to
herbicides.
be tolerant to agronomical doses of herbicide, able to Lotus glaber : these legumes possess excellent settle fast with minimal water and nutritional nutritional properties as a pasture and a special resources and capable to develop an abundant root
tolerance to salinity and flooding which allow system and biomass [8]. In addition, considering the
developing strategies specially adapted to the unique application context, candidates species should be
topogeographical characteristics of the Salado River agronomical profitable and allow the design of
basin [9]. The Salado River basin covers vast zones of
Biotechnological Strategies for Phytoremediating Triazinic
Herbicides in the Humid Pampa (Argentine)
the Humid Pampa and its topohydrography shapes the the pastures quality. In the current agronomical agricultural practices. Concerning that, the wavy
management, corn is cropped in the knoll and half profiles of the Depressed Pampa (Fig. 1) (region
knoll for feeding cattle while the bottoms (fresh and comprehending one third of the Humid Pampa) with
alkaline salty) are used as pasture and water source floodplains and high salinity bottoms (Fig. 2) cover
[10]. It is important to remark the strong association the 17 million hectares of the Salado River basin all
between agricultural and livestock activities in this across the central part of the region.
zone, where most ATZ dependant corn crops are The designed strategy for Lotus sp. would be the
meant to be used for feeding cattle. implantation of several furrows in the half knoll and
L. multiflorum (annual ryegrass): is a graminea with the fresh bottoms in order to prevent herbicide runoff
fast and broad implantation which has excellent from the cultured knoll and half knoll to the fresh and
agronomical characteristics for the Humid Pampa salty alkaline flooded bottoms (Fig. 2). In this strategy,
region [11] and a background of tolerance to besides interrupting ATZ run-off and reduce herbicide
herbicides like glyphosate and simazine [12, 13]. In spreading, a secondary benefit is the improvement of
this case, the designed strategy is based on a modern
Fig. 1 Map of the Humid Pampa indicating the: I) Rolling Pampa, II) Depressed Pampa and III) Austral Pampa sub regions.
Fig. 2 Scheme of the wavy topogeography of the Depressed Pampa presenting the zones dedicated to agriculture and livestock activity and herbicide movement (arrow size is proportional to movement chances).
Biotechnological Strategies for Phytoremediating Triazinic
Herbicides in the Humid Pampa (Argentine)
agronomical practice like the late intercropping of Once sterilized by any of the mentioned method, ryegrass in normally ATZ dependant crops (i.e.: seeds were soaked for 12 h in sterile distilled water at corn,sorghum) which is currently employed in the
4 °C in order to break dormancy and synchronize region [14, 15]. The intercropping strategy reduces the
germination.
reusing time of the soils by stabilizing them and Parallel, 40 mL of 0.8% agar, sugar free increasing the rizospheric effect on herbicide removal,
Murashige-Skoog (MS) medium [16] sumplemented which diminish the dispersion of ATZ and its
with vitamins RT [17] were poured into 360 mL glass metabolites.
flasks and spiked with increasing volumes (in the µL
Finally, it is important to remark that independently -1 range) of a 2,000 mg·L ATZ ethanol solution to from the strategy, the herbicide dissipation capacity or
reach concentration equivalent to field application rizospheric removal enhancement, the sole presence of -1 rates of 1-50 mg·kg . The range was meant to hold
the plant in the polluted soil is an advantage since it ATZ contents from that corresponding to a heavy reduces herbicide spreading, restores soil structure and
agronomical application rates (1 mg·kg -1 ) to those improves its agronomical properties.
found in heavily polluted “hot spot” around herbicide container disposal or sprayer washing sites (50
2.3 ATZ Tolerance Assay mg·kg -1 ). Flasks were screw capped and autoclaved at
In order to ensure maximum herbicide 121 °C and 1 atm for 20 minutes. Although ethanol is bioavailability and avoid microorganism interference,
less toxic than methanol (usual ATZ vehicle) and ATZ tolerance of the candidate plant species was
represents less than 1% of the medium, a vehicle assessed in sterile semisolid agar medium. For this
control group was included to assess its improbable experiment, seeds of the different species were treated
but possible interference on seeds germination or plant separately, according to the special requirements of
development.
their integument characteristics. Then, 10 seeds were sterile sown in each of the 0, 1,
C. quinoa seeds, the most fragile ones, were surface -1 5, 10, 50 mg·kg ATZ containing flask, sealed with sterilized by treatment with 96° ethanol for 1 minute
plastic film and incubated in a growing chamber and 20% bleach (55 g·L -1 of active Cl
2 ) solution for 30
settled at 24 ± 1 °C, 40% relative humidity and 16/8
minutes, rinsing carefully with sterile distilled water -1 hours of photoperiod (400 µmol·cm ·seg ). between treatments. This procedure also helps to
2.4 Soil Sampling
remove the surface saponins present on C. quinoa seed.
Representative soil samples were collected in the On the other hand, L. glaber seeds have a hard
proximities of Colón city, in the Humid Pampa region, integument which requires a previous step of sand
in the north of Buenos Aires province, Argentine paper scarification to facilitate the germination before
(33°52 ′23.33″ S and 61°80′10.61″ W). The average being surface sterilized by ethanol and bleach solution
temperatures in the region are 8-10 °C in winter and treatment as described above.
22-23 °C in summer and the rainfall about 1,024
Finally, L. multiflorum seeds were surface sterilized -1 mm·yr . This soil is representative of the dominant
order in the Humid Pampa, the Molisoles and was minutes and 96° ethanol for 1 minute since, in
by sequential treatment with 0.1% HgCl 2 for 30
classified as a Sandy Loam Argiudoll, not salty and previous studies, the presence of bleach treatment
well drained. Soils sampling was carried out in resistant endophytic fungi were detected during
pristine wild pasture plots with no record of herbicide sterilization method tuning.
application in the last 15 years.
Biotechnological Strategies for Phytoremediating Triazinic
Herbicides in the Humid Pampa (Argentine)
2.5 ATZ Remediation Assay in Soil Microcosms was performed on sterile condition. According to the L. multiflorum
sacrificial experimental design, triplicates were assisted ATZ remediation
established for every treatment and sampling time (Fig. experiment was designed to assess the effects of the
3). Samples were collected at 0 (to), 7, 14 and 21 days plant and the natural attenuation independently (Fig.
after ATZ addition. In every sample time, a set of 3). In this regard, 360 mL screw capped glass flasks
triplicates was disposed and soil samples were were loaded with 200 g of soil without any record of
collected for ATZ and metabolites measuring. ATZ application. For setting the “with plant”
microcosms, L. multiflorum seeds were surface
2.6 Soil Extraction
sterilized as described before, soaked at 4 °C in sterile water for 12 h. Next day, 10 seeds were sawn in each
For ATZ content measuring, soil was carefully microcosm and then incubated in a growing chamber
mixed and 15 g weighted and extracted with as it was previously described. Plantlets were let to
methanol:water 80:20, taking into account the special grow until the 3rd leave stage (21 days), when the
requirements of high humic matter content soils [18]. experiment was started by adding 20 mL of a 10
Extracts were separated and methanol was evaporated mg·L -1 ATZ aqueous solution in order to reach a final
at 25 °C under gentle N 2 stream until constant volume. concentration of 1 mg·kg -1 of soil. The microcosms
Then, aqueous remain was freeze dried, resuspended without herbicide were watered with 20 mL of sterile
in 1 mL of acetonitrile, filtered and analyzed by distilled water. Herbicide addition as well as watering
HPLC.
Fig. 3 Experimental design for assessing plant assisted remediation and natural attenuation of atrazine in soil microcosms.
Biotechnological Strategies for Phytoremediating Triazinic
Herbicides in the Humid Pampa (Argentine)
2.7 HPLC Analysis containing an ATZ concentration which doubles a heavy field application level. The capacity of
High performance liquid chromatography was germinate in a ATZ containing environment is carried out by using a hybrid particles XBridge RP significant from the technological point of view since C18 column (250 × 4.6 mm i.d., 5 µm), suited in a the field application strategies were designed based on Jasco HPLC modular device equipped with a the use of standard seeding machinery and LG-980-02 ternary gradient unit, PU-980 intelligent transplantation is not an economically viable option. pump, AS-950 autosampler and UV-975 UV detector. Despite some plant survived in presence of 5 and 10 Then, system was conditioned with a binary mobile
mg·kg -1 of ATZ (Fig. 4), the appropriated plant
phase of acetonitrile (A) and KH 2 PO 4 0.01 M buffer
develop and the absence of macroscopic toxicity
(pH = 4.7) (B) at a flow rate of 1 mL·min -1
effects at the 1 mg·kg -1 level, led to the selection of solvent program started with 1% A for 6 min, then
. The
this concentration for further soil microcosms 28% A for 4 min, increased to 70% A for the next 4
dissipation experiments (Fig. 5).
min and finally decreased to 1% A for 3 min in order to restore initial conditions. Chromatograms obtained
3.2 ATZ Dissipation in Soil Microcosms at 220 nm were compared with ATZ and metabolites
During the dissipation assay, L. multiflorum curves in the µg/mL level, prepared from 1 mg/mL
plantlets were able to tolerate the addition 1 mg·kg -1 of stock standard solutions of ATZ, desethyl-ATZ,
ATZ (more than 4 times the standard application rate). desisopropyl-ATZ, hydroxyl-ATZ and This high tolerance capacity is convenient in the desethyl-desisopropyl-ATZ. design of a phytoremediation strategy since agronomic
3. Results and Discussion
integral approaches for the Depressed Pampa sub-region could imply the L. multiflorum
3.1 ATZ Tolerance Assays implantation at bottom sites, where the herbicide
Both Q. quinoa and L. glaber were ATZ-sensitive naturally tends to accumulate as a consequence of the and although the seed were able to germinate under
run-off [20]. In addition, considering that Carruthers experimental conditions, plantlets died in the first 72
et al . (2000) demonstrated that interseeding had no hours. These results emphasize the complexity of
effect on yields of the intercropped species and its finding an appropriated plant species for high performance on ATZ tolerance, it is reasonable phytoremediating chemical compounds specially to suggest an early implantation of L. multiflorum in designed as a “plant killer” under the already
corn cultured fields would minimize herbicide discussed restrictions of the agronomical framework.
spreading, improving the ecological and agronomical On the other hand, L. multiflorum was able to
benefits of the phytoremediation process. germinate and grow in presence of 1 mg·kg -1 of ATZ
After 21 days of incubation, ATZ degradation rate and some plantlets survived even in the 5 and 10
was significantly higher in the L. multiflorum implanted mg·kg -1 ATZ containing media (Fig. 4). It is important
microcosms, improving the soil natural ATZ to point out that, in order to avoid sugar induced ATZ
attenuation capacity in a 20% (Fig. 6). This capability tolerance genes expression [19], MS media was
support the proposed intercropping strategy in typical prepared sucrose free which tightens the experimental
corn cultures since L. multiflorum implantation conditions. It is also remarkable that tolerance
significantly improves ATZ degradation plus the assessing started from seeds, which germinated and
benefits of stabilize the soil, reduce the herbicide developed in a sugar free maximum availability media
run-off and restore soil structure and agronomic
Biotechnological Strategies for Phytoremediating Triazinic
Herbicides in the Humid Pampa (Argentine)
Fig. 4 L. multiflorum tolerance to ATZ in MS sugar free medium. (n = 3, error bars represent SD).
Control 1 mg ·kg -1
Fig. 5 Photography of the L. multiflorum tolerance to ATZ in MS sugar free medium showing the absence of macroscopic effects at the 1 mg ·kg -1 ATZ level.
properties via root exudates [21]. Furthermore, after microcosms, where the soil natural attenuation performing the appropriated studies to guarantee it can
capacity was overcome by a 30% in the first 7 days.
be safely grazed, an additional profit can be obtained That fast degradation capacity can be exploited in an from the use of ryegrass as a pasture [22].
anti-runoff barrier strategy specially designed for the In addition, a statistically significant difference was
undulated profile of the Depressed Pampa. In observed in the dissipation rate of the implanted
accordance with the observed by Delin and London
Biotechnological Strategies for Phytoremediating Triazinic
Herbicides in the Humid Pampa (Argentine)
Fig. 6 Atrazine dissipation assay in soil microcosms with and without L. multiflorum plants. (n = 3, error bars represent SD).
(2002) and considering that slope associated herbicide [4] K.A. Wulfeck-Kleier, M.D. Ybarra, T.F. Speth, M.L. run-off represent the main variable in ATZ spreading, Magnuson, Factors affecting atrazine concentration and quantitative determination in chlorinated water, Journal
ryegrass implantation in a “anti-run off barrier” way of Chromatography A 1217 (2010) 676-682. could be an excellent alternative for the undulated
[5] K.E. Gerhardt, X-D Huang, B.R. Glick, B.M. Greenberg, profile of this Humid Pampa sub-region.
Phytoremediation and rhizoremediation of organic soil
Finally, the exceptional performance of L. contaminants: Potential and challenges, Plant Science 176 (2009) 20-30.
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systems in agrochemicals-contaminated soils allow the designing strategies either for the Rolling
phytoremediation research, in: I.A. Goluber (Ed.), Handbook of Phytoremediation, Nova Science Publishers,
Pampa (undersowing) or the Depressed Pampa Inc. Hauppauge NY, ISBN 978-1-61728-753-4, 2011, pp.
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667-690.
E.F. Viglizzo, F. Lértora, A.J. Pordomingo, J.N.
Bernardos, Z.E. Roberto, H. Del Valle, Ecological [1] CASAFE-Mercado de productos fitosanitarios 2009 vs.
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