Chemistry and Analysis of Radionuclides: Laboratory Techniques and Methodology
On the adequate phenological stage for each species, susceptible and resistant plants must be adequately identified by treatment. The leaves that have been predetermined to receive the radiolabeled herbicide must be covered with plastic film, aluminum paper or small paper envelopes. The radiolabeled herbicide solution must be prepared on a solution containing its commercial formulation at the recommended dose for the considered phonologic stage.
It is important for the radiolabeled herbicide to be applied with at least Bq of specific activity, in the case of studies with most of the annual weeds Nandula and Vencil, The choice for the leaf on which the application will occur depends on the studied species. Each plant or part of the plant must be collected according to the pre-established times for each situation. However, it is suggested that at least six collection times are used, in addition to time zero immediately after the application , and that the untreated plants are included as control.
For each collection, the treated leaf from each plant must be rinsed with the adequate solvent. The concentration v v -1 of the solvent must be established on preliminary tests with the studied molecule. Then, the radioactivity during the rinsing must be quantified by LSS in order to determine the non-absorbed radioactivity. The leaf absorption is calculated by the difference between the applied and the non-absorbed radioactivity. The plants must be dried with an absorbing paper, pressed and dried on an air circulation oved at 70 o C for 48 hours.
Then, the steps to evaluate the translocation are conducted. Usually, the translocation studies are conducted right after the absorption studies, although they demand more work and time. Differently from the absorption, which occurs within hours after the treatment, the translocation of herbicides may take up to days after the treatment. Due to this reason, in order to evaluate the translocation, the previous knowledge must be considered in order to determine the times after the treatment in which this variable should be evaluated. The biological combustion is the most used procedure to quantify the translocation of herbicides on plants.
However, care must be taken when stating that the detection of the radioactivity on other parts of the plant, outside the treated leaf, means that the herbicide is on its parental form. It might have been converted into a non-phytotoxic metabolite. In order to state this, one must investigate the potential for the herbicide to have been metabolized by the studied weed, through the information available in the literature.
To study the movement of herbicides on plants, the qualitative techniques involving autoradiography or phosphorus blade images have been used for over 50 years Nandula and Vencil, While the biological combustion offers a quantitative estimation of the herbicide on the treated plant, autoradiography Figure 4 or the phosphorus blade image provide a qualitative measurement of the movement of the herbicide on the plant, in addition to the location where it occurs. For the exposition of the treated and untreated plants, the use of phosphorus blade images is safer in comparison to the use of autoradiography, since it does not require handling chemical compounds that are harmful to the health.
Despite more expensive, the technique is also quicker. A single day of exposition of a plant on a phosphorus blade resulted on images with superior quality than the exposition for three weeks with the X-ray film Wehtje et al. Therefore, in order to study the translocation, the plants treated as on the absorption study must be exposed on phosphorus blade for 72 hours, in order to scan the image for qualitative analysis. The usual procedure to quantify the translocation of herbicides on plants is the biological combustion, in which dry samples of each part of the plant both the treated leaf and the part above and below it, as well as the roots are oxidized by the presence of O 2 , and the resulting CO 2 is captured on a special solvent.
Then, the radioactivity must be measured on the scintillation counter. The quantitative analysis of the translocation may also be conducted through the volume analysis, offered by the software provided together with the image scanner, as of its purchase.
The volume is the total signal intensity of the radioactivity within defined limits of the image. The translocation is then expressed as the rate between the percentage of signal intensity on the applied zone, as well as above and below it, and the total signal intensity on a defined image containing 14 C Ahmad-Hamdani et al. Source: Reis et al. The use of radiolabeled herbicides to investigate whether the herbicide is being metabolized on the plant is an efficient method, and it is the most indicated method to diagnose the resistance related to other phenomena that are not related to the change on the action site of the herbicide Tranel, The analytical method aiming at studying the metabolism of herbicides in plants comprehends three fundamental steps: preparation of the plants and application of treatments; extraction and separation; and identification of the herbicide and its metabolites, if any.
The steps to conduct the study on the metabolism of herbicides in plants are described as follows. The preparation of plants and application of the treatments must be conducted as described for the absorption and translocation study. In case the fresh samples of plants are not adequate for processing after the collection, the ideal is to store them at o C to assure the stability of the active substances and metabolites.
For the extraction, the adequate system of solvents for the studies herbicide must be known. The treated leaf must be rinsed with non-polar solvent usually ethanol or methanol. Then, the plant must be dried with an absorbing paper, immediately frozen in liquid nitrogen and stored at o C up to its use. A stainless steel homogenizer may also be used. The supernatants must be mixed, and the radioactivity must be determined by LSS, in order to know the mass balance, which is expressed as the rate between the radioactivity applied at the beginning of the experiment and the total radioactivity measured originated from rinsing all parts of the plant.
The mass balance may be also referred to as the radioactivity recovery percentage. The study of biodegradation with the use of radiometric techniques is based on measuring the mineralization and degradation rates of a test-substance according to the microorganisms of the soil. The method consists in treating soil samples with the test-substance pesticide and incubating them in biometric flasks, under controlled temperature and soil humidity conditions.
The molecule biodegradation process is quantified at previously established intervals, in which soil samples are extracted and analyzed in order to determine the concentrations of the original substance or metabolites. The mineralization is evaluated by the evolution of the 14 CO 2 captured in the solution, as well as by the formation of bound residue through the oxidation of the soil samples Dias, The soil samples must be collected from a layer with at most 20 cm in depth, having previously cleaned the vegetable layer that covers the soil.
This is important because it is on the superficial layers that most of the microorganisms on the soil are. After sifting, the water content of the soil and the field capacity of the soils that will be used must be determined. This determination may be conducted as described below. The amount of 10 g of soil must be placed on Petri dishes, previously weighted.
After this period, the dishes must be removed from the oven and placed on a desiccator until cooled. Then, they must be weighted again. Under that technique, the airdried soil, sifted on a sieve with a 2 mm mesh, must be placed on a mL beaker until it is filled. Then, an amount of 2 mL of distilled water must be dripped for a period of 10 seconds; thus, a moist clod will be created, which must be separated from the rest of the soil.
It is noteworthy that other techniques may be used in order to determine the field capacity, such as by calculating the density of the water flow, for its determination in the field, and by the water content corresponding to a certain tension on the retention curve, determined in the field and at the laboratory, as described by Brito et al. According to the standards published by OECD a , for biodegradation studies, the highest recommended dose for the culture must be tested for the herbicide that will be studied.
However, depending on the objective of the study, other doses may be used. In order to calculate the equivalent of the dose to be used for the preparation of the work solution, the soil density equal to 1. The volume of the work solution to be applied on each flask depends on the soil mass that will be added to the soil. It is important to point out that, during the study, periodic verifications of the water content of the soils on the Bartha flasks must be conducted. The application of the work solution on the soil must be made with a micropipette, taking care for it to be distributed across the entire soil surface.
After the application, the soil must be homogenized, and the flask, closed. For the mineralization study, collections of the NaOH 0,2 mol L -1 solution must be conducted 7, 14, 21, 28, 35, 42, 56, 63 and 70 days after the application of the test-substance. On each evaluation period, samples of the solution must be collected and transferred into liquid scintillation flasks, containing the scintillating solution, in order for the 14 C on the solution to be determined using the Liquid Scintillation Spectrometer LSS.
The rest of the solution must be removed, and a new solution must be added to the side arm. The determination of the radioactivity on the soil using the extraction method must be conducted with the incubated soil collections, on the same incubation periods. The extraction is conducted to determine the extractable radioactivity from the soil and, then, with other available techniques, the concentration of the original product and its possible metabolites. In some cases, this extraction period may be extended, in order to reach a higher evolved 14 CO 2 percentage.
The extraction must be conducted according to the methodology available in the literature; in some cases, pre-tests are recommended in order to verify whether the solvents and extraction methods are adequate. Source: Adapted from Dias The study of the degradation of herbicides with the use of thin layer chromatography is conducted after the reduction of the extracts on a rotaevaporator. Shares of the reduced extract and the standard solution of the radiolabeled herbicide must be applied on silica plates. The elution must be conducted with adequate solvents for each test-substance. The products and possible metabolites may be observed with the help of ultraviolet light, X-ray film or radioscanner.
After the extraction, the remaining 14 CO 2 non-extractable residue from the soil samples must be determined. For such, soil samples must be oxidized on a biological oxidizer, and the resulting 14 CO 2 from the combustion must be collected on a scintillating solution and quantified on a LSS.
At the end of the study, the sum of percentages of the test-substance found on the NaOH solution 0. The soil samples are collected on the superficial layer at most 0 to 20 cm in depth , with the previous cleaning of the vegetable layer, on locations with cultivated areas with agricultural crops of an agronomic interest.
After drying, the samples go through a sieve with a 2. The physical-chemical properties must be analyzed at least, the cation exchange capacity, pH, organic carbon or organic matter, clay content, mineralogy, density and porosity of the samples in reference laboratories, and the classification of the studied soils must be described according to the Brazilian Soil Classification System Embrapa, Glass columns with glass wool with 0.
At least two columns per soil must be prepared two repetitions for each herbicide treatment. Source: Dias The soil columns are prepared closing their end with quartz wool, filling the conic part with rinsed quartz sand dried on an oven at o C and adding soil samples in the column up to 0. Remove g of soil from the upper part and set apart for application of the herbicide, if applied on dry soil; if not, do not remove the g portion from the surface. Avoid excessive vibrations in order not to separate the soil particles.
The soil samples stored on the columns must be weighted, in order to control the reproducibility of the packaging process of the columns. The soil columns are placed inside a 2 L test tube, and they are slowly moistened with the ascending flow of a CaCl 2 0. The soil sample must be flooded for approximately 30 min. After the flooding process of the columns, when the CaCl 2 solution reached the top of the columns due to the ascending flow, they are removed from the test tube and installed on the holder for one or two hours in order for the CaCl 2 solution to be drained.
The amount of g initially removed where the herbicide was applied if that is the case must be placed inside the column, or the herbicide must be directly applied on the humid soil. The herbicide must be applied at the highest recommended dose for the studied culture in the field, and radioactivity around After the application, the surface of the soil sample must be covered by a quartz wool disk Figure 7 B , with an inverted funnel fitted in, where a tube will be connected, through which the CaCl 2 0.
A flow of approximately 8 mL h -1 must be simulated for 48 hours, using the CaCl 2 0. The study must be conducted on an acclimatized environment, with little light and controlled temperature between 18 and 25 o C. At every 12 hours preferably , three 10 mL portions of the leachate are collected, having 10 mL of insta-gel solution added for measurement on the Liquid Scintillation Spectrometer LSS. After 48 hours of the application of radiolabeled herbicides, corresponding to the end of the drainage of the CaCl 2 0. Whenever this is not possible, the glass column with the soil must be frozen right after the end of the leaching and draining of the soil since the excess of water on the soil may break the glass column , in order for the soil to be removed from the glass column afterwards.
The soil samples are air-dried, weighted, macerated and homogenized Figure 8 D. Three sub-samples 0. The leaching study on soil columns does not quantify the leaching potential of the herbicide in the field. The results are expressed as a percentage of the radioactivity found on the leachate and on each segment of the column, in relation to the initially applied radioactivity.
These results help in the decision-making in order to request in-field tests for herbicides that show a high leaching potential in the laboratory. In order to verify the repeatability and analytical sensitivity of the method, the oxidized soil samples and leachate samples are in duplicates. The soil samples are collected on the superficial layer at most 0 to 20 cm of depth , with the previous cleaning of the vegetable layer, on locations with cultivated areas with agricultural crops of an agronomic interest.
The analyses of the physical-chemical properties must be conducted at least, the cation Exchange capacity, pH, organic carbon or organic matter, clay content, mineralogy, density and porosity of the samples at a reference laboratory, and the classification of the studied soils must be conducted according to the Brazilian Soil Classification System Embrapa, If necessary, the soil samples may be stored in a laboratory, on adequately identified plastic bags, with the location, date and name of the collector.
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The experimental design is completely randomized, with at least two replications. Each experimental unit is constituted by a Teflon flask with capacity for 50 mL and screw cap. Portions of each soil are weighted in duplicates on the flasks, and the volume of the calcium chloride solution CaCl 2 0. The pre-test must be conducted in order to determine the adequate soil:solution relationship, as well as the equilibrium time and the amount of the herbicide sorbed on the soil and on the walls of the Teflon flasks.
At least 45 mL of CaCl 2 0. In order to conduct this study, samples in duplicates from two soils with contrasting texture must be used. It is suggested that the soil:solution relationship is 2 g of soil and 50 mL of solution , 5 g of soil and 50 mL of solution or 25 g of soil and 50 mL of solution , with at least 50 mL of volume used in all relationships.
METHODOLOGIES TO STUDY THE BEHAVIOR OF HERBICIDES ON PLANTS AND THE SOIL USING RADIOISOTOPES
The initial solution must be prepared with the herbicide at the highest dose to be used on the sorption study on CaCl 2 0. Then, this solution is applied on the Teflon flasks, and it is mechanically stirred on a horizontal table for 48 hours. This allows the selection of the most adequate soil:solution relationship in order to conduct the sorption study. The analytical standards non-radiolabeled and amounts of the radiolabeled herbicide are added in order to obtain a final concentration corresponding to the recommended dose of the active ingredient g ha -1 for the culture in which the herbicide is recorded.
Whenever possible, one of the concentrations of the herbicide used must correspond to the maximal in-field dose recommended and contain at least 80 Bq mL -1 of radioactivity. The calculations of the dilutions to prepare the stock-solutions depend on the solubility, polarity, hydrophobicity K ow and the recommended dose of the herbicide.
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The scintillating solutions are prepared by mixing 0. Then, add 4 g of PPO 2. Complete with toluene up to 1 L. On sorption studies, shares of the radiolabeled solutions are transferred in duplicates to independent flasks containing 10 mL of scintillating solution insta-gel plus, and the amount of the initial concentration of 14 C-herbicide is determined within five minutes according to the counting on a Liquid Scintillating Spectrometer ECL. In duplicate, 10 mL of the radiolabeled solutions of all concentrations are added to the Teflon flasks containing the soils with the exact amount of the soil:solution concentration found on the preliminary studies, which is usually The flasks are stirred on an horizontal shaker table, in a dark room 20 to 25 o C , at approximately rpm 4 g to reach the equilibrium concentration during the time found on the preliminary studies, which is usually 24 hours.
The sorbed amount of the herbicide is calculated by the difference between the initial concentration and the concentration on the supernatant after the equilibrium. The desorption studies are conducted immediately after the sorption one, under the same conditions. On the Teflon flasks containing soil and the sorbed radiolabeled herbicide from the sorption study, 10 mL of the CaCl 2 0. After the stirring period, the flasks are centrifuged, and 1 mL shares of the supernatant are pipetted in duplicate to a scintillation flask containing 10 mL of scintillating solution and analyzed by LSS.
The desorped amount is calculated by the difference between the sorbed radioactivity on the soil and the one remaining on the supernatant. The studies must contain two controls: flasks with the CaCl 2 0. The soils from the Teflon flasks are dried on an oven at 40 o C for 48 hours. After drying, they are grinded and stored on plastic packages. Sub-samples from each soil are weighted in triplicate 0. From that, the mass balance is conducted by the total amount of herbicide applied, represented by the sum of the sorption, desorption and soil combustion sum.
The Kd value of the desorption is also calculated in a similar manner than the Kd values of the sorption. The data must follow the normal distribution, and the variances must be homogeneous in order to provide an appropriate interpretation of tests and, also, the additivity of the effects of variation factors. According to the information adapted from Nandula and Vencill and Kniss et al. In case of comparison between behavioral studies on herbicides between two or more soils or plants, within the same collection time, the data may be analyzed using separation procedures for common means.
Autoradiography is purely a qualitative measurement and, therefore, it does not involve any statistical application. The peaks obtained by chromatography may be separated and identified according to the Rf values available in the scientific literature or by the use of the metabolite standards. Regarding the data related to biodegradation, in case of significant effects, tests must be applied for multiple comparisons of the means of these significant effects. In order to interpret the results, the leaching data are compared to the ones from the treatment without the herbicide dose zero ; however, when there is no interaction between the variation sources, the means of the treatments on the curves are used.
The data are subjected to analysis of variance according to the F test, using a statistical program and, then, regression equations and the curves are plotted, when the data are quantitative, and a comparison test for the means is conducted, when the data are qualitative. Every radioactive installation or radioactive waste deposit must have a radioactive waste management plan, within the context of the licensing and control processes CNEN, All and every material that gets directly in contact with the radioactive solution are considered as radioactive waste.
All radioactive waste from the laboratory shall be stored in packages identified with the international symbol for the presence of radiation, fixated on a clear and visible manner, and stored on an adequate deposit room for such purposes, which shall be sufficiently far away from the work area. This location shall be rigorously controlled by a responsible technician, designated to receive, store and control all the radioactive material, as well as for the several protection measurements; the access must be limited to the staff that is authorized to handle the radioactive material.
After stored on the radioactive waste deposit, the waste is sent to IPEN Institute for Energetic and Nuclear Researches , in order to be stored or eliminated. The segregation of waste must be conducted on the same location where the waste was generated or on an adequate environment, taking into consideration the characteristics of the radioactive material, such as: physical state; half-life; compactable or non-compactable; organic or inorganic; biological putrescible and pathogenic ; in addition to hazardous characteristics regarding explosivity, combustibility, inflammability, corrosivity and chemical toxicity CNEN, The solid waste is placed on plastic containers with a screw cap and adequately labeled.
The materials soils and plants , after conducting the studies on radiolabeled herbicides, must be dehydrated at room temperature, on stainless steel trays. In case their value is below the value specified as an activity concentration limit for disposal, the materials must be disposed as regular waste. The liquid waste aqueous solution with low activity lower than 5. In the case of scintillating and organic solutions, they must be carefully transferred from the scintillating flasks to the polypropylene tanks, with a sealing screw cap, adequately identified.
All scintillating solutions with radioactive activity above the allowed limit for disposal on the sanitarian swage network must be stored on polypropylene tanks with a sealing screw cap, adequately identified. When the volume of the solution on the flask reaches the limit, the responsible technician must be immediately warned in order to measure the final specific activity Bq m The technician must adequately fill the waste record book of the laboratory and send the flask to the deposit of radiolabeled materials.
Any leftover of the radiolabeled work solution must be transferred to the glass flasks, with a spigot, screw cap and capacity for 10 mL or more. After identification product, radioisotope, total activity, specific activity, responsible person and date , these flasks must be stored on the freezer designated for such purposes. Use the radiolabeled waste collector from the laboratory. Otherwise, this material might be disposed as regular waste. At the end of each month, the responsible technician must send the radioactive waste to the deposit, filling out the waste records.
There is the need of further disclosure within the scientific community connected to the study of weeds regarding the use of radiolabeled herbicides on absorption, translocation and metabolism studies on plants and the biodegradation, mineralization, leaching and sorptiondesorption on the soil according to the Brazilian conditions.
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On this review, a step-by-step was suggested for the methodologies in order to meet this need, including the management of resulting radioactive waste from the studies conducted in the laboratory. Techniques that use radioisotopes such as tracers are extremely useful to study the dynamics of herbicides on the environment, since the radiometric techniques offer the possibility of accurately determining very small amounts in a relatively short time.
Ahmad-Hamdani M. Herbicide resistance endowed by enhanced rates of herbicide metabolism in wild oat Avena spp.
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