Fate of diuron and terbuthylazine in soils amended with two-phase olive oil mill waste

The addition of organic amendments to soil increases soil organic matter content and stimulates soil microbial activity. Thus, processes affecting herbicide fate in the soil should be affected. The objective of this work was to investigate the effect of olive oil production industry organic waste (alperujo) on soil sorption-desorption, degradation, and leaching of diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and terbuthylazine [N2-tert-butyl-6-chloro-N4-ethyl-1,3,5-triazine-2,4-diamine], two herbicides widely used in olive crops. The soils used in this study were a sandy soil and a silty clay soil from two different olive groves. The sandy soil was amended in the laboratory with fresh (uncomposted) alperujo at the rate of 10% w/w, and the silty clay soil was amended in the field with fresh alperujo at the rate of 256 kg per tree during 4 years and in the laboratory with fresh or composted alperujo. Sorption of both herbicides increased in laboratory-amended soils as compared to unamended or field-amended soils, and this process was less reversible in laboratory-amended soils, except for diuron in amended sandy soil. Addition of alperujo to soils increased half-lives of the herbicides in most of the soils. Diuron and terbuthylazine leached through unamended sandy soil, but no herbicide was detected in laboratory-amended soil. Diuron did not leach through amended or unamended silty clay soil, whereas small amounts of terbuthylazine were detected in leachates from unamended soil. Despite their higher sorption capacity, greater amounts of terbuthylazine were found in the leachates from amended silty clay soils. The amounts of dissolved organic matter from alperujo and the degree of humification can affect sorption, degradation, and leaching of these two classes of herbicides in soils. It appears that adding alperujo to soil would not have adverse impacts on the behavior of herbicides in olive production.     

Comparative sorption and leaching study of the herbicides fluometuron and 4-chloro-2-methylphenoxyacetic acid (MCPA) in a soil amended with biochars and other sorbents

Biochar, the solid residual remaining after the thermochemical transformation of biomass for carbon sequestration, has been proposed to be used as a soil amendment, because of its agronomic benefits. The effect of amending soil with six biochars made from different feedstocks on the sorption and leaching of fluometuron and 4-chloro-2-methylphenoxyacetic acid (MCPA) was compared to the effect of other sorbents: an activated carbon, a Ca-rich Arizona montmorillonite modified with hexadecyltrimethylammonium organic cation (SA-HDTMA), and an agricultural organic residue from olive oil production (OOW). Soil was amended at 2% (w/w), and studies were performed following a batch equilibration procedure. Sorption of both herbicides increased in all amended soils, but decreased in soil amended with a biochar produced from macadamia nut shells made with fast pyrolysis. Lower leaching of the herbicides was observed in the soils amended with the biochars with higher surface areas BC5 and BC6 and the organoclay (OCl). Despite the increase in herbicide sorption in soils amended with two hardwood biochars (BC1 and BC3) and OOW, leaching of fluometuron and MCPA was enhanced with the addition of these amendments as compared to the unamended soil. The increased leaching is due to some amendments' soluble organic compounds, which compete or associate with herbicide molecules, enhancing their soil mobility. Thus, the results indicate that not all biochar amendments will increase sorption and decrease leaching of fluometuron and MCPA. Furthermore, the amount and composition of the organic carbon (OC) content of the amendment, especially the soluble part (DOC), can play an important role in the sorption and leaching of these herbicides.     

Influence of organic amendment on the adsorption and leaching of ethametsulfuron-methyl in acidic soils in China

The sorption and leaching of ethametsulfuron-methyl by an acidic soil, after organic amendment with humic acid (HA) and a commercial peat, were studied in batch and soil column experiments. Adsorption capacity (K_f) values, obtained by fitting the experimental data to the Freundlich equation, ranged from 4.39 for the original soil containing 1.02% OC to 10.56 for the organic amended soils containing 2.61% OC. The increase in herbicide adsorption by organic amendment addition to soil was attributed to the high adsorptive capacity of the insoluble organic matter added to the soil. Evidence provided by FT-IR analysis suggested multifunctional hydrogen bonds were involved in the adsorption of ethametsulfuron-methyl on organic matter. The distribution of ethametsulfuron-methyl along the soil profile, obtained from soil column experiments, indicated that the amount of ethametsulfuron-methyl retained ranged from 68.4% for the column filled with the original soil to 92.4% for that filled with the organic amended soil. Amounts of ethametsulfuron-methyl recovered in the leachates, which ranged from 7.7% (organic amended soil) to 23.7% (unamended soil) of that applied, depended upon the loading rate and the source of organic amendment. Organic amendments significantly reduced the leaching of ethametsulfuron-methyl, and humic acid showed the higher potential than peat. This research suggests that organic amendment may be an effective management practice for controlling pesticide leaching.     

Availability of triazine herbicides in aged soils amended with olive oil mill waste

Amendments are frequently added to agricultural soils to increase organic matter content. In this study, we examined the influence of alperujo, an olive oil mill waste, on the availability of two triazine herbicides, terbuthylazine and atrazine, in two different sandy soils, one from Sevilla, Spain, and the other from Minnesota. The effect of aging on herbicide sorption and bioavailability was also studied. Soils were amended with alperujo at a rate of 3-5% (w:w) in laboratory studies. Apparent sorption coefficients for the triazine herbicides were calculated as the ratio of the concentrations of each herbicide sequentially extracted with water, followed by aqueous methanol, at each sampling time. These data showed greater sorption of terbuthylazine and atrazine in amended soils as compared to nonamended soils, and an increase in the amount of herbicide sorbed with increasing aging time in nonamended soils. The triazine-mineralizing bacterium Pseudomonas sp. strain ADP was used to characterize triazine bioavailability. Less mineralization of the herbicides by Pseudomonas sp. strain ADP was observed in soils amended with alperujo, as compared to the unamended soils, and, despite the increase in sorption with aging in unamended soils, herbicide mineralization also increased in this case. This has been attributed to Pseudomonas sp. strain ADP first using alperujo as a more readily available source of N as compared to the parent triazines. In summary, addition of alperujo to the soils studied was shown to increase triazine herbicides sorption and hence to reduce its availability and potential to leach.     

Influence of soil moisture on sorption and degradation of hexazinone and simazine in soil.

Sorption and degradation rates of hexazinone and simazine on soil were determined in a sandy loam soil incubated, during 44 days, at 25 degrees C with moisture contents ranging from 4% to 18%. Herbicide levels in soil solution were also measured, after extraction of this solution by a centrifugation method. All experiments were conducted with treated soil in plastic columns, and the results showed that this method is suitable for the simultaneous study of pesticide sorption and degradation in soil at different environmental conditions. In general, sorption of both herbicides was higher for aged herbicide residues compared to recently applied herbicides, and soil subjected to drying and rewetting cycles had the highest sorption values. K(f) values ranged from 0.5 to 1.2 for simazine and from 0.2 to 0.4 for hexazinone. Degradation rates increased with soil moisture content for both herbicides, and drying-rewetting of soil yielded degradation rates slower than that obtained at 10% soil moisture content. Hexazinone concentration in soil solution decreased with incubation time faster than simazine.     

Effects of Wood Amendments on the Degradation of Terbuthylazine and on Soil Microbial Community Activity in a Clay Loam Soil

The herbicide terbuthylazine is widely used within the EU; however, its frequent detection in surface and groundwater, together with its intrinsic toxicological properties, may pose a risk both for human and environmental health. Organic amendments have recently been proposed as a possible herbicide sorbent in soil, in order to limit herbicide movement from soil to water. The environmental fate of terbuthylazine depends not only in its mobility but also in its persistence. The latter is directly dependent on microbial degradation. For this reason, the effects of pine and oak residues on terbuthylazine soil microbial community functioning and on the potential of this community for terbuthylazine degradation were studied. For this purpose, degradation kinetics, soil dehydrogenase activity and the number of live bacteria were assessed in a clay loam soil treated with terbuthylazine and either amended with pine or oak wood or unamended (sterilised and non-sterilised). At day 65, 85?% of the herbicide applied still persisted in the sterile soil, 73?% in the pine-amended one and 63?% in the oak-amended and unamended ones. Pine residues increased the sorption of terbuthylazine to soil and hampered microbial degradation owing to its high terbuthylazine sorption capacity and a decrease in the bioavailability of the herbicide. On the contrary, in the presence of oak residues, the herbicide sorption did not increase significantly. The overall results confirm the active role of the soil microbial community in terbuthylazine degradation in amended and unamended soils and in a liquid enrichment culture performed using an aliquot of the same soil as the inoculum. In this clay loam soil, in the absence of amendments, the herbicide was found to be quite persistent (t _1/2?>?95?days), while in the enrichment culture, the same natural soil bacterial community was able to halve terbuthylazine in 24?days. The high terbuthylazine persistence in this soil was presumably ascribable to its texture and in particular to the mineralogy of the clay fraction.     

Biochar mediated alterations in herbicide breakdown and leaching in soil

Biochar application to soil has been proposed as a mechanism for improving soil quality and the long term sequestration of carbon. The implications of biochar on pesticide behavior, particularly in the longer term, however, remains poorly understood. Here we evaluated the influence of biochar type, time after incorporation into soil, dose rate and particle size on the sorption, biodegradation and leaching of the herbicide simazine. We show that typical agronomic application rates of biochar (10-100 t ha⁻¹) led to alterations in soil water herbicide concentrations, availability, transport and spatial heterogeneity. Overall, biochar suppressed simazine biodegradation and reduced simazine leaching. These responses were induced by a rapid and strong sorption of simazine to the biochar which limits its availability to microbial communities. Spatial imaging of ¹⁴C-labeled simazine revealed concentrated hotpsots of herbicide co-localized with biochar in the soil profile. The rate of simazine mineralization, amount of sorption and leaching was inversely correlated with biochar particle size. Biochar aged in the field for 2 years had the same effect as fresh biochar on the sorption and mineralization of simazine, suggesting that the effects of biochar on herbicide behavior may be long lasting. We conclude that biochar application to soil will reduce the dissipation of foliar applied pesticides decreasing the risk of environmental contamination and human exposure via transfer in the food chain, but may affect the efficacy of soil-applied herbicides.     

Valorization of Organic Wastes to Reduce the Movement of Priority Substances Through a Semiarid Soil

In this work, we examined the effect of two different organic wastes, composted sheep manure and coir, on the sorption, persistence, and mobility of three pesticides (alachlor, chlorfenvinphos, and chlorpyrifos) included as priority substances in European Directive 2013/39/EU. With this aim, leaching studies were conducted using disturbed soil columns filled with a typical agricultural soil (hipercalcic calcisol) from a semiarid area (southeastern Spain) to determine their potential for groundwater pollution. The three compounds were found in leachates of unamended soil although in different proportions: 53% (alachlor), 9% (chlorfenvinphos), and 6% (chlorpiryfos). The addition of organic wastes significantly increased the sorption of the studied pesticides. As a consequence, the half-lives of the studied pesticides were higher in amended than in unamended soils. A marked reduction of the amount recovered in leachates was observed in the amended soils, except for chlorpiryfos, whose recoveries barely changed. According to their potential groundwater pollution calculated as the groundwater ubiquity score (GUS) index, alachlor and chlorfenvinphos show medium leachability while chlorpiryfos is unlikely to leach.     

Biochar concomitantly increases simazine sorption in sandy loam soil and lowers its dissipation

Biochar application has been receiving much attention as pesticide pollution mitigator because it reduces harmful chemicals. However, direct comparisons between the effect of biochar and straw on the simazine fate in soils remain poorly understood. We explored the impact of biochars and straw on the simazine behavior in a soil using a ¹⁴C labeling approach. Biochar was produced by the thermal treatment of wheat straw at four contrasting temperatures (250, 350, 450 and 550°C) and was incorporated into a sandy loam soil. The sorption of simazine in the biochar soil from 83.9% to 87.5% was significantly higher than 43.0% in the unamended soil and 35.7% in the soil amended with unprocessed straw, thus resulting in low samizine leaching from 21.8% to 42.6% in the biochar soil. However, biochar application suppressed the simazine decomposition, which is contrast in the straw soil. Furthermore, the biogeochemical behavior of simazine varied with the pyrolysis temperature. These results indicate biochar application can significantly increase simazine adsorption and reduce leaching, which is benefit to the environmental pollution. In conclusion, the simazine behaviors in the soil are strongly influenced by the biochar properties. In comparison to straw, biochar has potential to mitigate simazine pollution.     

Changes in the microbial activity in a soil amended with oak and pine residues and treated with linuron herbicide

This work studies the effects of wood amendments on soil microbial community functioning and on the potential of this community for linuron degradation. For this purpose, soil dehydrogenase activity and the number of live bacteria, which represent broad scale measurements of the activity and viability of soil organisms, were assessed in soil treated with linuron and either amended with pine or oak wood or unamended (sterilized and non-sterilized). The overall results show that the microbial community had a significant role in linuron degradation. The linuron half-life values indicated a slower degradation rate in pine and oak amended soils than in unamended ones. This is attributed both to the higher sorption of linuron by these soils compared to the unamended ones and a consequent lower bioavailability of the herbicide for microbial degradation, and to the use of the pine and oak as an alternative carbon source by degrading microorganisms. Linuron did not affect the microbial community in terms of dehydrogenase activity and number of live bacteria, presumably because it had adapted to the herbicide. However, the dehydrogenase activity was significantly higher in the soils amended with pine or oak than in the non-amended ones, indicating that the presence of a carbon source favoured the overall bacterial community.     

Sorption-desorption isotherms and biodegradation of glyphosate in two tropical soils aged with eucalyptus biochar

ABSTRACT

The objective of this study was to evaluate the sorption-desorption process and biodegradation of glyphosate in two tropical soils aged with biochar derived from eucalyptus. The biochar aging period was 30 d. There was little difference between the amounts of sorbed glyphosate in Ultisol (96.8, 96.8 and 96.4%) and Alfisol (97.1, 97.5 and 97.4%) soils that were unamended or amended with biochar aged for 0 or 30 d, respectively. Similar amounts of desorbed herbicide occurred in Ultisol (3.3, 3.3 and 3.4%) and Alfisol (4.1, 4.2 and 3.9%) soils, respectively. The degradation time half-life (DT50) of glyphosate in Ultisol unamended and initial amended were higher (38 and 36 d, respectively) than DT50 in the amended soil with 30 d of biochar aging (27 d); and in the Alfisol DT50 was higher in unamended soil (38 d), and similar in soil unamended at 0 and 30 d of biochar aging (21 and 26 d, respectively). The addition of biochar to two tropical soils or its aging did not have any effect on the sorption and desorption of glyphosate and its biodegradation in relation to the unamended soils, and it can did not affect the transport and persistence of this herbicide in soil.     

Raw or incubated olive-mill wastes and its biotransformed products as agricultural soil amendments-effect on sorption-desorption of triazine herbicides

Raw olive-mill waste and soil amendments obtained from their traditional composting or vermicomposting were added, at rates equivalent to 200 Mg ha-1, to a calcareous silty clay loam soil in a laboratory test, in order to improve its fertility and physicochemical characteristics. In particular, the effects on the sorption-desorption processes of four triazine herbicides have been examined. We found that comparatively hydrophobic herbicides terbuthylazine and prometryn increased their retention on amended soil whereas the more polar herbicides simazine and cyanazine were less affected. Soil application of olive cake, without transformation, resulted in the highest herbicide retention. Its relatively high content in aliphatic fractions and lipids could explain the increased herbicide retention through hydrophobic bonding and herbicide diffusion favored by poorly condensed macromolecular structures. On the other hand, the condensed aromatic structure of the compost and vermicompost from olive cake could hinder diffusion processes, resulting in lower herbicide sorption. In fact, the progressive humification in soil of olive-mill solid waste led to a decrease of sorption capacity, which suggested important changes in organic matter quality and interactions during the mineralization process. When soil amended with vermicompost was incubated for different periods of time, the enhanced herbicide sorption capacity persisted for 2 months. Pesticide desorption was reduced by the addition of fresh amendments but was enhanced during the transformation process of amendments in soil. Our results indicate the potential of soil amendments based on olive-mill wastes in the controlled, selective release of triazine herbicides, which varies depending on the maturity achieved by their biological transformation.     

Natural soil colloids To retard simazine and 2,4-D leaching in soil

Natural or synthetic sorbents for pesticides can be used to reduce contamination of soils and natural waters. The sorption of simazine and 2,4-D on montmorillonite minerals has been studied and their potential use to retard pesticide leaching in soil evaluated. Simazine and 2,4-D did not sorb on high-layer charge montmorillonite, whereas sorption on the lower layer charge montmorillonite SWy varied depending on the saturating cation. Simazine sorption increased in the order Ca(2+)SWy < K(+)SWy < Fe(3+)SWy. Simazine molecules sorb on hydrophobic microsites of the montmorillonite. Once protonated, further sorption through cation exchange takes place in the interlamellar space of the montmorillonite, as corroborated by X-ray diffraction and FT-IR studies. 2,4-D does not sorb on K(+)SWy or Ca(2+)SWy, but does sorb on Fe(3+)SWy, because the acidic character of this sorbent allows the molecular form of 2, 4-D to sorb by hydrogen bonding and/or by hydrophobic interactions. Leaching experiments in hand-packed soil columns indicate that simazine and 2,4-D application as a complex with FeSWy renders later breakthrough and lower maximum concentration peaks, and the total herbicide leached is lower than when applied as the pure analytical grade compound. These results suggest the possible use of natural soil colloids as sorbents for herbicides such as simazine and 2,4-D to retard pesticide leaching in soil, thus reducing their ground water contamination potential.     

Persistence of simazine and terbuthylazine in a semiarid soil after organic amendment with urban sewage sludge

The persistence of two herbicides, simazine and terbuthylazine, and appearance of their principal dealkylated chloro-s-triazine metabolites have been studied in agricultural soil after the addition of urban sewage sludge as organic amendment. Both herbicides and metabolites were monitored during long-term laboratory incubation (140 days) and analyzed by gas chromatography with a nitrogen-phosphorus detector (GC-NPD). Residues were confirmed by gas chromatography with a mass selective detector (GC-MSD). A sonication microextraction method was used to extract the compounds. The organic amendments used were urban sewage sludge and the humic fraction of this sludge, to increase the organic matter content of the soil from 1% to 2%. For both compounds, simazine and terbuthylazine, the degradation began earlier in the amended soils. Simazine showed a higher dissipation rate than terbuthylazine, the percentage of the former at the end of the experiment being lower than 2% in all cases, while for terbuthylazine the corresponding percentage ranged from 5% to 46%. Organic amendment, mainly its humic fraction, caused a certain stabilization of terbuthylazine in the soil, but did not greatly influence the residual amount of simazine at the end of the experiment. The periodic aeration of the soil caused a greater degradation in the case of terbuthylazine. Only mono-deethylsimazine and deethylterbuthylazine were isolated from the soil during the time the experiment lasted, while the di-deethylated metabolite of simazine was not found.     

生物质炭施用及老化对石灰性紫色土中磺胺类抗生素迁移特征的影响

以长江上游低山丘陵区广泛分布的石灰性紫色土旱地的耕作层土壤为对象,采用室内批量平衡吸附试验和填装土柱穿透试验,研究了施用1%生物质炭及3年老化作用对2种磺胺类抗生素(磺胺嘧啶和磺胺二甲基嘧啶)吸附和迁移特征的影响。结果表明,Freundlich方程能更好地拟合抗生素在土壤中的等温吸附曲线,施用生物质炭提高了土壤对抗生素的吸附能力,吸附常数KF值依次为:老化1%施炭土新鲜1%施炭土对照土;土柱出流液的磺胺嘧啶和磺胺二甲基嘧啶相对浓度峰值均表现为:老化1%施炭土新鲜1%施炭土对照土,说明生物质炭的添加能有效减少控制紫色土中抗生素的淋失迁移,以对磺胺二甲基嘧啶的阻控效果较好;生物质炭老化3 a后土壤对抗生素的吸附作用与阻控效果均有所提高,主要归因于土壤pH的提高。     

Influence of organic by-products and nitrogen source on chemical and microbiological status of an agricultural soil

We assessed the influence of the addition of four municipal or agricultural by-products (cotton gin waste, ground newsprint, woodchips, or yard trimmings), combined with two sources of nitrogen (N), [ammonium nitrate (NH₄NO₃) or poultry litter] as carbon (C) sources on active bacterial, active fungal and total microbial biomass, cellulose decomposition, potential net mineralization of soil C and N and soil nutrient status in agricultural soils. Cotton gin waste as a C source promoted the highest potential net N mineralization and N turnover. Municipal or agricultural by-products as C sources had no affect on active bacterial, active fungal or total microbial biomass, C turnover, or the ratio of net C:N mineralized. Organic by-products and N additions to soil did not consistently affect C turnover rates, active bacterial, active fungal or total microbial biomass. After 3, 6 or 9 weeks of laboratory incubation, soil amended with organic by-products plus poultry litter resulted in higher cellulose degradation rates than soil amended with organic by-products plus NH₄NO₃. Cellulose degradation was highest when soil was amended with newsprint plus poultry litter. When soil was amended with organic by-products plus NH₄NO₃, cellulose degradation did not differ from soil amended with only poultry litter or unamended soil. Soil amended with organic by-products had higher concentrations of soil C than soil amended with only poultry litter or unamended soil. Soil amended with organic by-products plus N as poultry litter generally, but not always, had higher extractable P, K, Ca, and Mg concentrations than soil amended with poultry litter or unamende soil. Agricultural soil amended with organic by-products and N had higher extractable N, P, K, Ca and Mg than unamended soil. Since cotton gin waste plus poultry litter resulted in higher cellulose degradation and net N mineralization, its use may result in faster increase in soil nutrient status than the other organic by-products and N sources that were tested. Received: 15 May 1996     

加入不同量生物质炭盐渍化土壤盐分淋洗的差异与特征

生物质炭作为土壤调理剂,能够显著地改良培肥土壤,但对盐渍化土壤盐分淋洗的影响缺乏研究和了解。本研究采用土柱模拟试验,将蘑菇棒生物质炭按照不同的质量比(0%、2%、5%、10%),添加到内蒙古河套地区硫酸盐盐渍化土壤0～20 cm的土层中,并进行淋洗,测定淋出液和土壤盐分及主要盐分离子含量,以期了解生物质炭对土壤盐分和主要盐分离子洗脱的影响。结果表明:加入生物质炭的土柱,淋洗液出现的时间提前了5～36 d,电导率降低至5 mS cm-1缩短了41～100 d;生物质炭加入量越大,淋洗液出现的时间越早,电导率降低至5 mS cm-1所需的时间也越少。其中,生物质炭用量2%的处理,淋洗结束表层脱盐效果较好,含盐量与对照相比降低了34.25%。显然,向盐渍化土壤加入生物质炭,不仅能够缩短盐分洗脱时间,而且提高洗盐效率,但对盐分离子洗脱先后顺序及其速率,并没有表现出明显的影响。     

Nitrate leaching in an Atlantic Coastal Plain soil amended with poultry manure or urea-ammonium nitrate: Influence of thiosulfate

Nitrate leaching, which can lead to groundwater contamination, is a common occurrence, especially in sandy, well drained soils. Nitrogen from poultry manure (PM) and ammonium fertilizers has been shown to undergo rapid nitrification upon addition to soils, making it highly susceptible to nitrate leaching. Any management technique that could delay nitrification and thereby reduce nitrate leaching would be desirable. Ammonium thiosulfate has been shown to be an effective nitrification inhibitor in laboratory studies and may be useful in reducing nitrate leaching. Soil columns, 75 cm long and inner diameter 19.6 cm, were packed with a reconstituted profile of a Rumford loamy sand and amended with urea-ammonium nitrate (UAN) or PM. Corn was grown in the columns to create a dynamic soil/plant system. Columns were placed in a greenhouse and were leached periodically for a period of 10 weeks with deionized water in amounts intended to simulate early spring and summer rainfall patterns in the Atlantic Coastal Plain. Column leachates, as well as plant and soil samples were collected and analyzed for NO₃-N and NH₄-N. Nitrate-N leaching was largely dependent upon the amount of water moving through the system. Ammonium thiosulfate did not significantly decrease NO₃-N leaching or increase plant N uptake when used in combination with UAN or PM. Comparable amounts of NO₃-N leaching were observed for the UAN and the PM treated column. Additionally, large amounts of NO₃-N leaching were observed with the control columns, suggesting that residual soil N from previous crops can contribute significantly to NO₃-N leaching and may deserve further investigation.     

Identification of in situ 2,4-dichlorophenoxyacetic acid-degrading soil microorganisms using DNA-stable isotope probing

Stable isotope probing (SIP) was used to investigate the microorganisms responsible for degradation of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) in soil samples. Soils were unamended or amended with either unlabeled 2,4-D or UL(ring) ¹³C-2,4-D. Degradation of 2,4-D was complete after 17 days, whereas little removal (11±3%) was observed in the sterile controls. Terminal restriction fragment length polymorphism (TRFLP) on soil DNA after 17 days indicated a consistent increase in the relative abundance of one fragment (217 bp in Hae III digests) in soils spiked with 2,4-D (both unlabeled and labeled samples) compared to the unamended soils. DNA extracts from labeled and unlabeled 2,4-D amended soils were subject to ultracentrifugation, fractionation of centrifuged samples, followed by TRFLP on each fraction. TRFLP profiles from ultracentrifugation fractions illustrated that the same fragment experienced an increase in buoyant density (BD) in samples spiked with ¹³C-labeled 2,4-D. This increase in DNA BD indicates the organisms represented by this fragment were responsible for uptake and degradation of the herbicide. 16S rRNA sequencing of the heavy, ¹³C-enriched fraction suggests the organisms belong to the β subdivision of Proteobacteria. Herein, SIP facilitated the identification of unique organisms degrading 2,4-D in soil without the need for isolation and provided more direct evidence for a functional role of these organisms than would have been possible with the molecular-based methods alone.