Distribution and Mineralization of <sup>14</sup>C-Hexazinone in Soil Microcosm with the Riparian Forest Specie <em>Cecropia Hololeuca</em> | OMICS International
ISSN: 2155-6199
Journal of Bioremediation & Biodegradation

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Distribution and Mineralization of 14C-Hexazinone in Soil Microcosm with the Riparian Forest Specie Cecropia Hololeuca

Marinho DA1*, Bicalho ST2, Ferreira EM3 and Langenbach T3
1Postgraduation Program in Plant Biotechnology, CCS, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
2FATEC-ID, Induiutaba, S.P. Brazil
3Institute of Microbiology Professor Paulo de Góes, CCS, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Corresponding Author : Marinho DA
Universidade Federal do Rio de Janeiro
CCS, Bloco I, Instituto de Microbiologia
sala 29 Cidade Universitária
CEP 21491-590, Rio de Janeiro, Rio de Janeiro, Brazil
E-mail: [email protected]
Received: Spetember 07, 2011; Accepted: November 29, 2011; Published: December 02, 2011
Citation: Marinho DA, Bicalho ST, Ferreira EM, Langenbach T (2011) Distribution and Mineralization of 14C-Hexazinone in Soil-Plant Microcosm with the Riparian Forest Specie Cecropia Hololeuca. J Bioremed Biodegrad S1:002. doi:10.4172/2155-6199.S1-002
Copyright: © 2011 Marinho DA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Although Brazil is one of the world’s largest consumers of the herbicide hexazinone there are few studies, in the country, on the fate of this compound in the environment. This work studied the distribution and mineralization of hexazinone in a microcosm, with and without plants. The herbicide 14C-hexazinone was applied to microcosms with soil and C. hololeuca seedlings and mineralization and volatilization was monitored over a two month period. After this incubation period, radioactivity was measured in different soil layers, the rhizosphere, as well as in parts of the plant: fine and thick roots, stems, and leaves. The results showed that evaporation promoted an upward movement of hexazinone to higher soil layers which was enhanced in the presence of plants, due to evapotranspiration. The mass balance showed an uptake of 12.2 % in plants, and the leaves had the highest bioaccumulation value (9.1%). Mineralization in the soil microcosm was low, about 0.6 % but with the presence of plants it increased to 1.15 % without increasing the CFU of hexazinone and lignin biodegrader fungi. Bioremediation of hexazinone by biodegradation and accumulation in plant biomass reduced its concentration in the soil slightly, showing that this molecule can have hazardous consequences for the environment.

Soil pollution; Herbicides; Biodegradation; Microcosms; Tropical riparian forest
Hexazinone is an herbicide in the triazine chemical family used in Brazil in pre-and post-emergence for weed control in sugarcane plantations [1]. This substance is highly soluble in water (33g/L) and therefore highly mobile in the environment.
Brazil is among the largest consumers of hexazinone in the world [2]. This herbicide is used in the sugarcane crops for ethanol production that will probably double in the next decade [3]. Although there are only a few studies about the behavior of this herbicide in this country some researchers have noted its leaching in soils [4-6] also there is more scientific data available for other triazines. Although hexazinone is more soluble and more mobile in the environment than atrazine, the latter is considered to be the most polluting groundwater pesticide in Europe and United States [7,8].
Atrazine and other triazines have shown hazardous impacts on flora and fauna. Atrazine causes feminization of amphibians due to their endocrine disruption effects which contribute to the current decline of these animals in ecosystems [9], and it is very toxic for Atlantic salmon, an endangered specie [10]. Hexazinone is considered low to moderate toxicity, which is maybe why there are more ecotoxicological studies with atrazine; however some studies have shown that hexazinone can cause environmental changes whose effects have yet to be analyzed. The presence of hexazinone can affect non target species thereby introducing of changes in the natural terrestrial ecosystems affecting the food chain by decreasing the food supply for primary consumers [11]. Primary productivity is a key factor for the maintenance of life in aquatic ecosystems, hexazinone can decrease the periphyton photosynthetic activity [12], and atrazine can decrease the marine phytoplankton [13] affecting the primary productivity of this ecosystems. The mechanism is not known, but some studies indicate that atrazine and hexazinone can interfere with nutrient cycling processes in both marine [13] and terrestrial [14] ecosystems respectively, increasing the release of phosphorus and nitrogen by organisms that inhabit these places.
Hexazinone degradation by abiotic factors, like moisture, light and temperature, occurs very slowly. Among these factors, moisture and light are the ones that contribute most to its degradation. However, most hexazinone biodegradation is carried out by microorganisms [2,15]. In one of the few available studies on the mineralization of hexazinone, Rhodes obtained between 45 and 75% of biodegradation in eighty days, and concluded that degradation in soil is primarily due to demethylation and hydroxylation of the cyclohexyl ring [16]. Some microorganisms that have the ability to degrade hexazinone have been isolated. Among these bacteria we can cite the genera Methylobacterium, Paenibacillus, Microbacterium, Rhodococcus and Methylobacterium, in tropical climates [17], and also those of the genera Pseudomonas and Enterobacter [18], all grown under aerobic conditions. Hexazinone degradation under anaerobic conditions has also been observed [19]. Although fungi are known to degrade a large number of pollutants [20], there are no studies that demonstrate their ability to degrade hexazinone.
Strong tropical rains increase the problem of using highly soluble, mobile and persistent pesticides in the environment. In tropical regions pesticides are easily leached or seep into the soil, reaching surface and subsurface waters in significant concentrations. Many Brazilian crops are grown close to streams and rivers. Data of herbicide efficiency are available but information about their hazardous effects and the retaining capacity of riparian forests, are few. Studies in our laboratory with riparian forests species have shown enhanced atrazine mineralization as well as high uptake in biomass (31 to 45%). These processes can remove this substance from the soil and/or groundwater partially [21] reducing its bioavailability and hazardous effects.
Soil management influences mineralization, which is higher in soils under no-tillage (NT) than in soils with conventional tillage (CT) as observed with atrazine in our laboratory [22]. Others have shown that NT soil management increases b-glucosidase, acid phosphatase and arylsulfatase enzymatic activities [23]. The straw that remains in the soil has a lignin content of about (10-20%) and the ligninase enzyme is important in the processes for cellulose and hemi-cellulose degradation, as well as being efficient in the biodegradation of many persistent molecules [24, 20].
Materials and Methods
Experimental design
Experiments were performed with 14C-hexazinone to trace the molecule distribution in the soil layers as well as in the different parts of the plant and mineralization. Another similar experiment was carried out with rhizosphere and soil samples to obtain the number of lignolytic and hexazinone degrader fungi using the colony forming units (CFU) method. Experiments were performed with the following treatments in triplicate: microcosms with no plants as controls and microcosms with plants. The experiments allowed us to compare the behavior of hexazinone in the presence and absence of plants on a laboratory scale.
Experiment 1
1. Microcosms: The microcosms were PVC tubes that were 35cm high with a 15cm diameter. A nylon screen was placed at the bottom to prevent any soil loss but it allowed the entry of water from the dishes that were under them. At the bottom of the microcosm there was a layer of sand before the column of soil.
2. Soil: The soil was classified as Red-Yellow Latosol (Oxisol), with sandy-clay texture, taken from the secondary forest of the Pontifical Catholic University of Rio de Janeiro (PUC-Rio), located at a latitude 22° 97’ and longitude of 43° 23’ W. After litter removal the soil was collected from the upper 20cm layer, dried at room temperature and sieved through a 2mm mesh. The soil was fertilized with 30g NPK 4-14-8 in each microcosm and limed to pH 5 to 5.5. Soil characteristics are summarized in Table 1.
3. Plants: Six month old Cecropia hololeuca species were kindly donated from the nursery garden of Guaratiba Environment Department of RJ. The seedlings were removed from the plastic bags and the roots were water-soaked for a few minutes and shaken gently to remove all the substrate. The seedlings were put into microcosms individually and soil was added carefully. Plants were acclimatized for one month. The plants were watered daily during acclimatization and afterwards three times a week with the amount of water equivalent to 200 mm/month during the experiment. The water was added to a plastic plate, commonly used in gardening, placed underneath the microcosm.
4. Hexazinone: In this experiment a non radiolabeled hexazinone solution, with a tenth of the recommended application dose of 132g a.i. ha-1, corresponding to 1.32μg/cm was used. To this solution was added 14C- hexazinone (97% purity) uniformly ring labeled in the amount of 78,000 Bq for each microcosm diluted in methanol. After 1 min of ultrasonication the solution was applied and carefully distributed at the bottom of the microcosms.
A tenth of the recommended application dose was used for two reasons: preliminary tests carried out to verify the tolerance of C. hololeuca to the hexazinone showed that a tenth was the highest tolerated dose before toxic symptoms, like leaf chlorosis, appeared. In addition the herbicide was applied at the bottom of the microcosms, simulating the groundwater pollution by this molecule; in this case the amount of herbicide would be lower than the field recommended dose applied on the surface.
5. Microcosms assembly: Twenty-four hours before applying the solution of 14C-labeled hexazinone watering was stopped. The hexazinone was applied at the bottom of the microcosm, after which the top of the microcosm was sealed with a polyurethane sponge that had a hole in the center for the stem to pass through. Then the microcosm was closed with a sheet of aluminum foil, and finally a top layer of plastic, which was fixed with a tape.
The tube for 14C-CO2 capture was positioned in the space between the soil surface and the polyurethane sponge and connected two chemical traps in sequence, containing 35mL of ethylene glycol monobutyl ether and ethanolamine (1:1) each. The airflow in the system with the traps was maintained using a vacuum pump during the experiment with a rotameter that maintained the flow values between 10 to 15L/h.
6. Volatilization: At the end of the experiment, the microcosm was opened, the sponges were removed and the radioactive material was extracted by maceration with 50mL of acetone in a beaker glass with a pestle for 1 min and followed by 3 min. ultrasonication. The acetone was poured into a concentration flask. This procedure was repeated twice and the acetone was again added to the flask that was then concentrated to dryness. This material was resuspended in 20mL of Aquasolv liquid scintillation cocktail (4g PPO, 0.25g POPOPO, 333 ml of Triton X100 and 667mL of toluene). Aliquots of 5ml were added to 5ml of scintillation solution and radioactivity was measured in triplicate in a Packard liquid scintillator counter.
7. Mineralization: The solution from the chemical traps was collected three times a week in the first fifteen days and from then on twice a week until the end of the experiment. From each collected sample a radioactivity aliquot was measured in a Packard liquid scintillation counter.
8. Hexazinone distribution in plant-soil system: During the experiment the leaves that fell were collected and their radioactivity measured. At the end of the experiment the microcosms were opened, the bottom nylon screen removed and the soil was sliced into 5 cm layers and then dried at room temperature. Thin (≤1mm) and thick (≥1mm) roots were separated from each soil fraction manually. Rhizosphere was carefully removed from the roots with paintbrushes. The soil and rhizosphere samples were ground in a blender and the total weight recorded. The aerial part of the plant was divided into leaves and stems which were divided into: region 1 closest to the root collar region; region 2in the middle part; and region 3 the upper part of the stem. All samples were ground in a blender for homogenization and total weight was determined.
From the total amounts of each sample aliquots of 0.5 to 1g in triplicate were weighed and burned in an Oxidizer (Zinsser - Oximat 500). 14C-CO2 was absorbed by another liquid scintillation cocktail (1L Aquasolv + 666 mL methanol + 416 mL ethanolamine).
The nylon screens and plates were stored separately and radioactivity was measured.
Experiment 2
The aim of this experiment was to follow hexazinone biodegrading fungi as well as the lignolytic fungi population in the soil and rhizosphere under normal plant growing conditions.
1. Rhizosphere separation: Disassembly was obtained by removing the plant with the soil from the microcosm. Immediately after this procedure the plants were shaken carefully to avoid rupturing the roots and to separating the bulk soil. The remaining soil retained on the roots was rhizosphere and was manually removed with glove protected hands. The rhizosphere and bulk soil were immediately stored in separated plastic bags at 4°C and processed within two weeks. The soil moisture was measured by gravimetry.
2. Culture medium and CFU determination: Chloramphenicol (400 mg/L) plus amoxicillin (500 mg/L) was added to the modified Katayama culture medium [25] and the pH was adjusted to 5.0 and then the medium was autoclaved. The culture medium was divided into two parts. Filtered hexazinone (Millipore 0.22mM) was added to one of them until reaching final concentration of 1 mg / L, and to the other part of the medium a filtered solution with the Remazol Brilliant Blue R (RBBR) dye was added in order to reach a final concentration of 0.04%.
All medium reagents used were from Vetec Fine Chemistry. Statistical analysis was performed with Microsoft Excel software.
The determination of CFU was carried out according to the standard methods; sterility tests were carried out with all the Petri dishes, and incubation was at 28°C for 72h.
Microcosms radioactivity distribution
Mass balance: The leaves of Cecropia seedlings had some chlorosis and fell during the experiment, but all seedlings survived the herbicide dose. The total recovery of radioactivity was about 110±12% in microcosms without plants and 89±6% in microcosms with intact plants. The radioactivity distribution in the mass balance (Figure 1) showed that 73.4 ± 4% remained in the soil, up to 12.2±1.4% was incorporated in the plant and mineralization was below 1.15 %. The increasing values in the different compartments of the microcosm with plants can be placed in the following sequence: thick roots 0.8±0.1%, stem 0.9±0.5%, fine roots 1.3±0.4%, rhizosphere 2.0±0.3%, leaves 9.1±1.1%. Radioactivity retained in the nylon screen and in the bottom plate was less than 0.01 %.
Distribution in soil: The microcosms without plant showed that evaporation induces an upward movement of hexazinone. Plant evapotranspiration increased this movement as shown in Figure 2. The amount of rhizosphere radioactivity followed the distribution of the hexazinone concentrations in the soil with the highest values in the two layers from 12 to 22cm deep in the microcosm with plants (Figure 2).
Distribution in plants: In the fine roots the highest radioactivity value was found in the layers between 12 and 17cm, this tendency was also observed in the thick roots, with the highest radioactivity values in the same layers. Nevertheless the amount of radioactivity in the thick roots of upper layers was higher than in the soil of the same layers (Figure 3). Also, in these layers the roots weighed more than in the other layers (data not shown).
Stems divided in 3 equal parts showed an increase of radioactivity from the bottom to the upper segment (data not shown). Leaves had the major accumulation of hexazinone in the plant (Figure 1).
Specific Radioactivity: The relation between radioactivity and the weight of each part of the microcosm showed the accumulation capacity with values in Bq.g-1 (Table 2). In the stems increasing bioaccumulation capacity occurred in the upper portion (70 Bq.g-1). During the incubation period some leaves fell and were collected but specific activity did not show major differences between the leaves collected at the end of the experiment. Soil specific radioactivity was higher in middle microcosm layers (12-22cm) with 30.7 ± 2.5 Bq.g-1.
Volatilization: In microcosms without a plant the volatilization was about 0.02% of the total radiation applied, and with plants 0.03%. These values had no statistically significant differences.
Mineralization: Total mineralization showed significant differences (α=0.05, p<0.009) between microcosm with only soil 0.6%, and with C. hololeuca 1.15%. The slope of mineralization with plants is a little higher than soil without plants (Figure 4).
Hexazinone fungi and lignin biodegraders: Hexazinone bio-degraders as well as the lignolytic fungi population showed minimum and maximum values of log106 to log 1.5x107.g-1 soil CFUs, demonstrating no significant differences between the distinct treatments measured one and two months after application.
Enhanced plant uptake and mineralization in the mass balance shows that bioremediation is influenced by plants. Plant transpiration is the main suction force that moves the soil water solution upward. It has a greater intensity than soil evaporation. As a consequence the hexazinone distributed in the soil layers moved toward the surface and a part (12.2%) was accumulated in the C. hololeuca tissues. However this value is much smaller than the bioaccumulation of atrazine observed in a similar experiment with the same species, C. hololeuca (45%) and another riparian forest species T. micrantha (31%) [11]. In this case the surface soil, with higher organic matter content than the subsurface soil used for atrazine, can sorb [26] the residue more intensively and a lower amount of hexazinone was captured by the plant. Nevertheless other factors can interfere such as the difference in chemical nature between hexazinone and atrazine. Another possible factor can be the differences in uptake by the plant due to barriers against substances such as hexazinone with very low Kow (octanol-water coefficient) compared to more lipophilic substances like atrazine [26]. To be absorbed and translocated the pesticide needs to go through the cells of the endoderm to get to the root xylem. The spaces between the endodermal cells have the Casparian stretch barrier, a very hydrophobic structure. Hydrophilic pesticides such as hexazinone pass through this layer of cells slowly [27,28]. It is possible that in this work this barrier prevented this herbicide from accumulating in the C. hololeuca tissues, particularly in roots. Concentrations of approximately 7.9% (Vaccinum sp.) and 10.1% (Solidago fistulosa) of 14C-hexazinone were found in a study with one-year-old plants, in agreement with our results [29]. Due to its hydrophilic character hexazinone translocates easily in fine (1.3%) and thick roots (0.8%) without major accumulation and concentrates bioaccumulation in the leaves (9%). In the study with atrazine [26] the highest concentration was also in the leaves (22%), but the roots had a significantly higher concentration (3% fine roots and 15% thick roots) than in our study with hexazinone. It is probable that the more hydrophobic character of atrazine increases its adsorption in roots.
In the plants the highest amount of total and specific radioactivity (bioaccumulation) was in the leaves probably due to the mode of action of hexazinone. The hexazinone mode of action is to replace plastoquinone QB from its binding pocket located in D1 protein in photosystem II, blocking the electron transport chain with the inhibition of photosynthesis. A chain reaction is triggered culminating in the oxidation of membrane lipids, destroying cell membranes, tissues and killing the plant [30,31].
This work showed a small mineralization of hexazinone under humid soil conditions, a process that can be enhanced by Cecropia hololeuca seedlings. Rhodes got very high mineralization of hexazinone, between 45 and 75% [16], but their experiments were performed in biometer flasks with a capacity of only 250 mL, very different conditions of our experiments. Hexazinone and lignin fungi degraders in the rhizosphere and bulk soil did not change significantly during the two months of the experiment indicating that their participation in the degradation of this herbicide under the conditions of this study was small. Also there was a low increase in mineralization of hexazinone from 0.6 % to 1.1 % when the microcosms contained plants. Plant exudates induce soil changes increasing microbial population as well as being able to decrease the hydrophobic index in the soil [32]. This would make more hexazinone, which has a low hydrophobicity, available for mineralization. Further studies are needed to identify the main factors involved in this process.
High amounts of residues in the leaves are returned to the soil by senescence and the distribution and mineralization begins again. This pesticide cycling prevents it from reaching the ground and surface water and keeps this molecule in the upper layers of soil where a higher population of biodegrading microorganisms is present. In this work the phytostabilization by absorption and translocation of hexazinone in the plant arrived at 12.2% plus 1.5% mineralization, reducing the bioavailable molecules in the soil and thus reducing the hazardous effects on the environment.
We thank the financial support of the “National Counsel of Technological and Scientific Development” (Cnpq), “Fundação Carlos Chagas Filho de Amparo à pesquisa do estado do Rio de Janeiro” (FAPERJ), Programa de apoio a Núcleos de Excelência (PRONEX), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and to the nursery garden of Guaratiba Environment Department of RJ for the C. hololeuca seedl Xenopus laevis ings.
We thank Dupont in Brazil for the donation of hexazinone.

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