Soil Extract Solution Research Articles

Quantitative detection of chloroethene-reductive bacteria Dehalococcoides spp. using alternately binding probe competitive polymerase chain reaction

Dehalococcoides spp. are responsible for the reductive dehalogenation of environmental contaminants and are candidates for engineered bioremediation. The development of a sensitive, reliable, and rapid method for the quantification of Dehalococcoides spp. is required for the effective use of the organisms in bioremediation sites. Here, we describe the quantification of the 16S rRNA gene of Dehalococcoides spp. using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The primers and probe sets that were newly designed for ABC-PCR were found to have a high specificity for Dehalococcoides spp. The standard curve of ABC-PCR had a good fitting (R = 0.999), and the lower detection limit was 10 copies/μl of template DNA. We also investigated the effects of inherent PCR-inhibiting compounds in an environmental sample on the quantification using ABC-PCR or real-time PCR by adding the soil extraction solution to PCR mixtures. ABC-PCR was more robust against the PCR amplification inhibitors than real-time PCR. The copy number of the 16S rRNA gene of Dehalococcoides spp. in soil and groundwater samples was successfully quantified using ABC-PCR. In conclusion, ABC-PCR is useful for the quantification of Dehalococcoides spp. populations and dynamics at bioremediation sites.

First Report of Phytophthora tentaculata Causing Root and Stem Rot of Oregano in Italy.

Oregano (Origanum vulgare L.; Lamiaceae) is cultivated for culinary and medicinal purposes and as an ornamental. In October of 2007, 1- to 2-year-old potted plants of oregano showed symptoms of decline associated with root and basal stem rot in a nursery in Liguria (northern Italy) that produces 1 million to 1.5 million potted aromatic plants per year. Aboveground symptoms included leaf russeting and chlorosis, wilt, defoliation and dieback of twigs, browning of the basal stem, and subsequent collapse of the entire plant. Approximately 80% of the plants died within 30 days after the appearance of the first symptoms on the canopy. Approximately 20% of a stock of 30,000 oregano plants was affected. Stocks of other aromatic species, such as mint, lavender, rosemary, and sage, appeared healthy. A Phytophthora species was consistently isolated from symptomatic stems and roots of oregano plants on BNPRAH selective medium (2). Ten pure cultures were obtained by single-hypha transfers, and the species was identified as Phytophthora tentaculata Kröber & Marwitz by morphological criteria and sequencing of the internal transcribed spacer (ITS) region of rDNA using the ITS 4 and ITS 6 universal primers for DNA amplification. Isolates from oregano formed stoloniferous colonies with arachnoid mycelium on potato dextrose agar and had a growth rate of 2 to 3 mm per day at 24°C with optimum, minimum, and maximum temperatures of 24, 8, and 34°C, respectively. Sporangia formed in soil extract solution and were papillate and spherical or ovoid to obpyriform with a length/breadth ratio of 1.3:1. Few sporangia were caducous and all had a short pedicel (<5 μm). Hyphal swellings and chlamydospores were produced in sterile distilled water and corn meal agar, respectively. All isolates were homothallic and produced globose terminal oogonia (mean diameter of 34 μm) with one or occasionally two paragynous, monoclinous, or diclinous antheridia. Amphigynous antheridia were also observed. The sequence of the ITS region of the rDNA (GenBank No. FJ872545) of an isolate from oregano (IMI 395782) showed 99% similarity with sequences of two reference isolates of P. tentaculata (Accession Nos. AF266775 and AY881001). To test for pathogenicity, the exposed root crowns of 10 6-month-old potted plants of oregano were drench inoculated with 10 ml of a suspension of 2 × 104 zoospores/ml of isolate IMI 395782. Sterile water was pipetted onto the roots of 10 control plants. All plants were maintained in 100% humidity at 22 to 24°C in a greenhouse under natural light and watered once a week. Within 3 weeks after inoculation, all inoculated plants developed symptoms identical to those observed in the nursery and died within 30 to 40 days after the appearance of the first symptoms. Control plants remained healthy. P. tentaculata was reisolated solely from symptomatic plants. P. tentaculata has been reported previously on several herbaceous ornamental plants (1,3). However, to our knowledge, this is the first report of this species on O. vulgare. Root and basal stem rot caused by P. tentaculata is the most serious soilborne disease of oregano reported in Italy so far.

First Report of Collar and Root Rot Caused by Phytophthora nicotianae on Daphne odora in Italy

Daphne odora is becoming popular in gardens because of its variegated foliage and fragrant flowers in late winter and early spring. During October of 2008 in a commercial nursery near Maggiore Lake (Verbano-Cusio-Ossola Province) in northwestern Italy, plants of D. odora showed extensive chlorosis and root rot. Diseased plants eventually wilted and died, dropping leaves in some cases. Most frequently, wilted leaves persisted on stems. At the soil level, dark brown-to-black water-soaked lesions that coalesced often girdled the stem. All of the crown and root system was affected. Disease was widespread and severe with 70% of 2,500 potted plants being affected. A Phytophthora-like organism was isolated consistently on a medium selective for oomycetes (4) after disinfestation of lower stem and root pieces of D. odora for 1 min in a solution containing 1% NaOCl. Tissue fragments of 1 mm2 were excised from the margins of the lesions and plated. The pathogen was identified based on morphological and physiological features as Phytophthora nicotianae (= P. parasitica) (2). Sporangia were produced for identification by growing a pure culture in sterilized soil extract solution at neutral pH (obtained by shaking and then centrifuging 300 g of soil in 1 liter of distilled water). They were spherical to ovoid, papillate, and measured 39.2 to 54.5 × 31.7 to 41.7 μm (average 44.8 × 34.5 μm). Papillae measured 2.4 to 4.9 μm (average 3.7 μm). Chlamydospores were spherical with a diameter ranging from 15.8 to 36.1 μm (average 25.4 μm). The internal transcribed spacer (ITS) region of rDNA of a single isolate was amplified using primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 804-bp segment showed a 100% homology with the sequence of P. nicotianae EF140988. The nucleotide sequence has been assigned GenBank No. FJ843100. Pathogenicity of two isolates obtained from infected plants was confirmed by inoculating 12-month-old plants of D. odora. Both isolates were grown for 15 days on a mixture of 70:30 wheat/hemp kernels and then 80 g/liter of the inoculum was mixed into a substrate containing sphagnum peat moss/pumice/pine bark/clay (50:20:20:10 vol/vol). One plant per 3-liter pot was transplanted into the substrate and constituted the experimental unit. Three replicates were used for each isolate and noninoculated control treatment; the trial was repeated once. All plants were kept in a greenhouse at temperatures from 20 to 25°C. Plants inoculated with isolate no. 1 developed symptoms of chlorosis and root rot within 14 days and then a wilt rapidly followed. Isolate no. 2 was less aggressive causing the same symptoms within 20 days. Control plants remained symptomless. P. nicotianae consistently was reisolated from inoculated plants. Previously, P. nicotianae (= P. parasitica) has been reported in several states of the United States on D. odora (3). To our knowledge, this is the first report of P. nicotianae on D. odora in Italy. The economic importance of the disease is low because of the limited number of farms that grow this crop in Italy, although spread could increase as the popularity of plantings expand.

Collar Rot on Italian Alder Trees in California Caused by Phytophthora siskiyouensis

In November 2006, trees of Italian alder (Alnus cordata) were observed declining in association with bleeding trunk cankers in a commercial landscape in Foster City, CA. A species of Phytophthora was isolated on PARP selective medium from the leading edge of the cankers. The Phytophthora species was homothallic with primarily paragynous antheridia and had oospores that were mostly globose and aplerotic. Sporangia were produced from mycelia on plugs of carrot piece agar in soil extraction solution and were semi-papillate and ovoid to ellipsoid in shape. The intergenic transcribed spacer region of rDNA from an alder isolate matched with 100% identity to isolates in GenBank of Phytophthora siskiyouensis, a recently described species associated with tanoak and found in the soil and waterways of coastal Oregon. Pathogenicity was tested on young alder trees growing in pots. Pathogenicity was confirmed on Italian alder trees and potential pathogenicity was demonstrated on red and white alder trees. Accepted for publication 11 March 2009. Published 13 April 2009.

First Report of Phytophthora cryptogea on Osteospermum spp. in South Africa.

Osteospermum is the largest genus of the tribe Calendulea of the Compositae (Asteraceae) and has a center of diversity in South Africa with approximately 40 known species in the Cape Province (3). This indigenous plant genus is also a popular floricultural crop grown in South Africa because of drought and high temperature tolerance. Two diseased Osteospermum sp. samples were submitted by commercial nurseries to the Stellenbosch University Plant Disease Clinic. Both samples showed similar symptoms including black lesions on the lower stem, petioles, and the base of lower leaves. Isolations were made from roots, stems, and petioles following surface disinfestation of the lesions and plating on potato dextrose agar (PDA), water agar, PARP, and PARPH (1). The only microorganism that was consistently isolated from all lesions was a species of Phytophthora, from which single hyphal tip cultures were prepared. The cultures were identified morphologically as Phytophthora cryptogea Pethybridge & Lafferty. Morphological characteristics and cardinal growth temperatures followed Lévesque and de Cock (2). Distinctive morphological characteristics included abundant hyphal swellings in sterile soil extract and hemp seed water cultures, nonpapil-lated, persistent, internally proliferating, obpyriform sporangia having mean dimensions of 36 × 26 μm and mean length/breadth ratios that ranged from 1.30 to 1.53 (1). Cultures had a petaloid pattern on PDA and no growth at 35°C that distinguished it from Phytophthora drechsleri (1). Analyses and comparison of the internal transcribed spacer (ITS) regions of isolate STE-U 6133 (GenBank Accession No. GI DQ479410) supported the P. cryptogea identification. STE-U 6133 has been deposited in the Stellenbosch University culture collection. Pathogenicity of isolate STE-U 6133 was tested on six plants of Osteospermum jucundum (Phill.) T. Norl. and O. ecklonis (DC.) Norl. in individual containers. Inoculum was grown on V8 agar for 7 days, and 10-mm culture disks were incubated in a filter (0.2 μm) sterilized 1% soil extract solution (1). The disks were vortexed vigorously and filtered through miracloth (Calbiochem Corp., La Jolla, CA) to remove agar disks and excess mycelium. The inoculum concentration was adjusted to 104 sporangia/ml and incubated at 4°C for 4 h. Six plants were inoculated with a 10-ml suspension of sporangia and zoospores by pipetting the suspension at the collar of each plant. Another six plants were treated with water as controls. The inoculated plants were kept at temperatures ranging from 22 to 25°C and were watered until saturation every second day. Initial symptoms were black lesions on the lower stem and petiole bases developing 14 to 18 days after inoculation, followed by wilting of some plants. Control plants remained healthy. Isolations from symptomatic plants yielded only P. cryptogea of which pure cultures were established, completing Koch's postulate. The experiment was repeated with similar results. To our knowledge, this is the first report of P. cryptogea as a pathogen of O. jucundum and O. ecklonis in South Africa. P. cryptogea has been reported as a pathogen on Osteospermum spp. in other countries (4).

Rapid quantification of polycyclic aromatic hydrocarbons in hydroxypropyl- β-cyclodextrin (HPCD) soil extracts by synchronous fluorescence spectroscopy (SFS)

Synchronous fluorescence spectroscopy (SFS) was directly applied to rapidly quantify selected polycyclic aromatic hydrocarbons (PAHs: benzo[a]pyrene and pyrene) in aqueous hydroxypropyl- β-cyclodextrin (HPCD) soil extract solutions from a variety of aged contaminated soils containing four different PAHs. The method was optimized and validated. The results show that SFS can be used to analyse benzo[a]pyrene and pyrene in HPCD based soil extracts with high sensitivity and selectivity. The linear calibration ranges were 4.0 × 10 −6–1.0 × 10 −3 mM for benzo[a]pyrene and 6.0 × 10 −6–1.2 × 10 −3 mM for pyrene in 10 mM HPCD aqueous solution alone. The detection limits according to the error propagation theory for benzo[a]pyrene and pyrene were 3.9 × 10 −6 and 5.4 × 10 −6 mM, respectively. A good agreement between SFS and HPLC was reached for both determinations of PAHs in HPCD alone and in soil HPCD extracts. Hence, SFS is a potential means to simplify the present non-exhaustive hydroxypropyl- β-cyclodextrin (HPCD)-based extraction technique for the evaluation of PAH bioavailability in soil.

Communities of P-solubilizing bacteria, fungi and arbuscular mycorrhizal fungi in grass pasture and secondary forest of Paraty, RJ - Brazil

Communities of P-solubilizing bacteria, fungi and arbuscular mycorrhizal fungi, were evaluated in two different ecosystems. Samplings taken from two areas of Atlantic forest, in Paraty-RJ, Brazil, one with a secondary forest and the other with a grass pasture were studied. Four growth media: GL (glucose and yeast extract), GES (glucose, soil extract, KNO3, CaCl2, MgSO4, NaCl, FeEDTA and micronutrients solution), GAGES (glucose, soil extract, arabinose, glycerol, CaCl2, MgSO4 and NaCl) and GELP (glucose, soil extract, yeast extract, peptone, CaCl2, MgSO4 and NaCl) were evaluated for the isolation of P-solubilizing microorganisms. The identification of P-solubilizing bacteria was based on 16 S rDNA sequence analysis, while the identification of P-solubilizing fungi and arbuscular mycorrhizal fungi was based on morphology. The greatest number of P-solubilizing bacteria was isolated using GL and GELP growth media. The greatest number of P-solubilizing fungi was isolated using GAGES and GES. The bacteria were identified as Enterobacteriaceae and Bacillus sp., while the P-solubilizing fungi were identified as Aspergillus sp. Glomus macrocarpum and Glomus etunicatum were the dominant mycorrhizal fungi in the secondary forest and grass pasture area, respectively.

First Report of Brown Rot and Wilt of Fennel Caused by Phytophthora megasperma in Italy.

Fennel (Foeniculum vulgare Mill. var. azoricum (Mill.) Thell.) in the Apiaceae family is native to southern Europe and southwestern Asia. It is an economically important crop in Italy that produces approximately 85% of all fennel worldwide. The main producing regions are Apulia, Campania, Latium, and Calabria. During the late winter of 2004 in the Crotone Province of the Calabria Region, following heavy rains, patches of fennel plants with symptoms of brown, soft rot of the bulb-like structure formed by the thickened leaf bases, development of yellow leaves, stunting, and wilting of the entire plant were observed in fields. A homothallic Phytophthora sp. was isolated consistently from the brownish tissues of the stout stems and leaf bases of symptomatic plants using a selective medium (3). Pure cultures were obtained by single hyphal tip transfers. On potato dextrose agar (PDA), the diameter of oospores varied from 28 to 42 μm (mean = 36.3 ± 0.4). Antheridia were primarily paragynous. Sporangia were not produced on solid media but were formed in sterile soil extract solution. They were nonpapillate, noncaducous, ovoid and obpyriform (25 to 45 × 35 to 60 μm), and internally proliferating. Optimum and maximum temperatures for radial growth of the colonies on PDA were 25 and 30°C, respectively. At 25°C, radial growth rate was approximately 6 mm per day. On the basis of morphological and cultural characteristics, the isolates were identified as Phytophthora megasperma Drechsler. Electrophoretic patterns of mycelial proteins and four isozymes (acid and alkaline phosphatase, esterase, and malate dehydrogenase) on polyacrylamide gels of the fennel isolates were identical to those of reference isolates of P. megasperma of the BHR (broad host range) group included in P. gonapodyides-P. megasperma Clade 6 (1,3), but distinct from those of the isolates of other nonpapillate species included in Waterhouse's taxonomic group VI. Internal transcribed spacer (ITS) regions of rDNA sequences (2) confirmed that fennel isolates belonged to P. megasperma BHR group. Pathogenicity of a fennel isolate from Calabria (IMI 391711) was confirmed by pouring a zoospore suspension at 2 × 104 zoospores per ml on the soil of 10 3-month-old potted fennel plants. The soil of the inoculated and 10 control seedlings was flooded for 24 h. After 10 days, stems and leaf bases of the seedlings showed a brown rot. Chlorosis and wilting of all seedlings developed after 20 days. Controls inoculated with water did not develop any symptoms. The pathogen was reisolated from typical brown rot and tests were repeated with similar results. To our knowledge, this is the first report of P. megasperma causing disease on fennel.

Photo–Fenton treatment of TNT contaminated soil extract solutions obtained by soil flushing with cyclodextrin

The technical feasibility and performances of coupling flushing abilities of cyclodextrin solutions for 2,4,6-trinitrotoluene (TNT) removal from contaminated soil and the ability of Photo–Fenton treatment for final disposal of soil extract solutions containing high TNT loads have been investigated at laboratory scale. Methylated-β-cyclodextrin (MCD) has shown better ability than hydroxypropyl-β-cyclodextrin (HPCD) to complex TNT. The MCD solution increased the aqueous concentration of TNT in soil extract effluents as much as 2.1 times the concentrations obtained during the water flush of the soil. TNT in soil extract solution has been treated by Photo–Fenton. Our results indicate that MCD has a beneficial effect on the degradation rates of TNT. This relative improvement of TNT degradation rate (1.3 time) in presence of high amounts of hydroxyl radical scavengers can be ascribed to the formation of a ternary complex (TNT-cyclodextrin-iron) which can direct hydroxyl radical reaction toward TNT. Complete mineralization of soil extraction solutions was not achieved and TNT degradation pathway has been elucidated in order to ensure that no potential toxic intermediate is left at the end of the treatment time. After successive TNT hydroxylations, oxidative opening of the TNT aromatic ring quickly occurred, leading to the accumulation of short chain carboxylic acids such as oxalic acid and formic acid.

Coupling enhanced water solubilization with cyclodextrin to indirect electrochemical treatment for pentachlorophenol contaminated soil remediation

This study undertakes to examine, at laboratory scale, the technical feasibility, mechanisms and performances provided by coupling the enhanced flushing abilities of cyclodextrin solutions for pentachlorophenol (PCP) removal from contaminated soil with indirect electrochemical treatment for the final disposal of soil extract solutions containing high PCP loads (0.77 mmol L −1). The hydroxypropyl-β-cyclodextrin (HPCD) solution increased the aqueous concentration of PCP in soil extract effluents to as much as 3.5 times the concentrations obtained during the water flush of the soil. PCP was treated with electrochemically generated Fenton's reagent in an aqueous medium. The increase in PCP water solubility in the presence of HPCD is balanced by the corresponding decrease in PCP degradation rate under indirect electrochemical treatment. This is due to the high carbon content (HPCD and dissolved natural organic matter) in the soil extract solutions, which compete for the non-selective hydroxyl radical reaction to PCP. However, our results indicate that HPCD has a beneficial effect on the degradation rates of PCP. This relative improvement in PCP degradation could be explained by the formation of the ternary complex (PCP-cyclodextrin-iron) which may direct hydroxyl reaction to PCP and which would, in any case, justify the use of a Fenton -like process for the final treatment of soil extract solutions. Total disappearance of PCP and 90% abatement of the chemical oxygen demand were achieved within an 11 h electrolysis treatment time. Elucidation of the PCP degradation pathway indicates that after successive PCP hydroxylations, oxidative opening of the PCP aromatic ring quickly occurred, leading to small unstable non-chlorinated or partially chlorinated short chain carboxylic acids, such as monochloroacetic and dichloromaleic acid. Determination of the concentration of these acids shows that indirect electrochemical treatment leads to oxalic acid accumulation in aqueous solutions treated. A decrease in toxicity was observed at the end of the treatment time.

Effect of humic acid on the underpotential deposition-stripping voltammetry of copper in acetic acid soil extract solutions at mercaptoacetic acid-modified gold electrodes

Electrochemical measurements were undertaken for the investigation of the underpotential deposition-stripping process of copper at bare and modified gold electrodes in 0.11 M acetic acid, the first fraction of the European Union’s Bureau Communautaire de Références (BCR) sequential extraction procedure for fractionating metals within soils and sediments. Gold electrodes modified with mercaptoacetic acid showed higher sensitivity for the detection of copper than bare gold electrodes, both in the absence and in the presence of humic acid in acetic acid solutions, using the underpotential deposition-stripping voltammetry (UPD-SV) method. In the presence of 50 mg l −1 of humic acid, the mercaptoacetic acid modified electrode proved to be 1.5 times more sensitive than the bare gold electrode. The mercaptoacetic acid monolayer formed on the gold surface provided efficient protection against the adsorption of humic acid onto the gold electrode surface. Variation of the humic acid concentration in the solution showed little effect on the copper stripping signal at the modified electrode. UPD-SV at the modified electrode was applied to the analysis of soil extract samples. Linear correlation of the electrochemical results with atomic spectroscopic results yielded the straight-line equation y (μg l −1) = 1.10 x − 44 (ppb) ( R=0.992, n=6), indicating good agreement between the two methods.

Re‐evaluation of Factors Affecting Total Nitrogen Measurements in Soil Extract Solutions Using Microwave‐Assisted Persulfate Oxidation

Interest and the use of persulfate oxidation for the measurement of total nitrogen (N) in soil extracts and related solutions has been increasing. Persulfate has a number of advantages relative to Kjeldahl digestion including greater sensitivity, ease of use, opportunities for automation of the procedure or portion of it and reduced requirement for waste disposal. A wide variety of persulfate solution formulations and digestion conditions have been proposed and their effectiveness have been compared to other methods such as Kjeldahl, but not with each other. A study was conducted to re‐examine various components of the persulfate method. The components were the composition of the oxidation solution, digestion conditions and compatibility of the digestion with quantification of the product (nitrate) of the oxidation. Tests suggested that the oxidation step was quite tolerant to the composition of the oxidant (persulfate solution) if there was sufficient to excess quantity of the oxidant to decompose organic compounds present and oxidize all resulting N compounds to nitrate, and if sufficient energy (heat) was applied. It was determined that microwaving the sample with 4% potassium persulfate oxidant in a sealed vessel for 10 minutes at 120°C was adequate. We also found that including boric acid in the oxidant as a pH buffer resulted in the solution being alkaline (pH 9) for a relatively wide range of sample matrices after the digestion was complete. Alkalinity was necessary for quantifying the resulting nitrate using flow injection analysis based on the commonly used colorimetric determination of N as nitrite after cadmium column reduction. Since the apparent recovery of organic N in solutions used for soil extractions was largely due to the effect of the cations and anions on nitrate quantification, it is essential to standardize and calibrate with an appropriate matrix of the solution containing the sample and an organic N standard through both the oxidation and quantification steps of the procedure. With these three precautions, persulfate oxidation can provide effective and efficient measurement of total N in a variety of soil extracts and related solutions.

Fenton's Reagent Degradation of 2,4-Dinitrotoluene in Water–Acetone Mixtures

This article presents laboratory studies using Fenton's reagent (Fe+2 + H2O2) to degrade 2,4-dinitrotoluene (DNT) in water-acetone mixtures representative of contaminated soil extract solutions. Specifically, DNT degradation and kinetics as a function of Fenton's reagent dosage and variable acetone concentration (0-15.5 M) were examined. DNT degradation was successful (>98%) in water only, but was significantly impacted by acetone. At acetone concentrations greater than 3 M, the maximum DNT degradation was 20%. Greater than 65% DNT degradation, however, was measured at acetone concentrations less than or equal to 1.38 M. The presence of acetone also reduced DNT degradation kinetics. DNT degradation behavior in the presence of acetone suggests acetone is a significant hydroxyl radical scavenger; however, it may also contribute to DNT removal, possibly through secondary reactions involving acetone degradation products and oxygen. Comparison of model and experimental results show that DNT degradation is as much as 38% greater than predicted by direct reaction with hydroxyl radical alone.

First Report of Phytophthora cinnamomi on Quercus laurifolia.

In May 2001, bleeding cankers were observed on several laurel oak (Quercus laurifolia) trees in central Florida. Affected trees had chlorotic leaves, sparse canopies, and little new growth. Multiple cankers were present on the trunk and extended from the soil line up to approximately 5 m. Each canker had a reddish to dark brown or black exudate. From two of the infected trees, tissue samples were taken from beneath the bark around the edge of an actively growing lesion and transferred directly to Phytophthora-selective medium (1), and three soil cores (2 cm in diameter, 20 cm deep) were collected from the base of each tree. A baiting bioassay (with camellia leaf disks and shore juniper and eastern hemlock needles as baits) was used to assay fresh composite soil samples for Phytophthora species (1). P. cinnamomi was recovered from both tissue and soil samples (2). Mycelia were coralloid with abundant hyphal swellings. Sporangia were produced in 1.5% nonsterilized soil extract solution. Sporangia were ovoid to ellipsoid in shape and nonpapillate. Average sporangium size was 72 × 45 μm (length × width). To our knowledge, this is the first report of P. cinnamomi on laurel oak trees.

Development of a kinetic basis for bioavailability of sorbed naphthalene in soil slurries

The degradation of naphthalene in soil–slurry systems was studied using four different organisms and two soils. Organisms with zero-order, first-order, and Michaelis–Menten rates were selected. The soils had substantially different sorption distribution coefficients. Sorption and desorption was evaluated in abiotic soil–slurry systems. The desorption process was described by a model that accounts for equilibrium, rate-limited and non-desorbing sites. Biodegradation parameters were measured in soil-extract solutions. Bioavailability assays, inoculated soil slurries, were conducted and both liquid- and sorbed-phase naphthalene concentrations were measured over time. For the less sorptive soil, the results could be explained by sequential desorption and degradation processes. For the other soil, enhanced degradation was clearly observed for the organisms with first-order and Michaelis–Menten rates. Several explanations are explored for these observations including direct sorbed-phase degradation and the development of elevated substrate concentrations at the organism/sorbent interface. No enhancement was found for the organism with zero-order kinetics.

Biodegradation of non-desorbable naphthalene in soils.

The degradation of naphthalene was studied in soil-slurry systems, and a quantitative model was developed to evaluate the bioavailability of sorbed-phase contaminant. Four soils with different organic matter contents were used as sorbents. Two naphthalene-degrading organisms, Pseudomonas putida G7 and NCIB 9816-4, were also selected. Sorption isotherms and single and series dilution desorption studies were conducted to evaluate distribution coefficients, desorption parameters, and the amount of non-desorbable naphthalene. Biodegradation kinetics were measured in soil extract solutions and rate parameters estimated. Bioavailability assays involved establishing sorption equilibrium, inoculating the systems with organisms, and measuring naphthalene concentrations in both sorbed and dissolved phases over time. For all four soils, the sorption isotherms were linear, and desorption could be described by a model involving three types of sites: equilibrium, nonequilibrium, and non-desorption. Enhanced bioavailability, as evidenced by faster than expected degradation rates based on liquid-phase concentrations, were observed in soils with the higher sorption distribution coefficients. These observations could be described using model formulations that included solid-phase degradation. In all soils studied, degradation of non-desorbable naphthalene was observed.

PREDICTING ALACHLOR MOBILITY USING BATCH SORPTION KINETIC DATA

The ability to accurately predict the mobility of a soil-applied herbicide is important to product registration and recommendations for environmentally safe use. Commonly, mobility is affected by time-dependent sorption processes. Parameters for such nonequilibrium sorption may be conveniently determined from batch kinetic studies; however, applicability of these parameters for subsequent use in transport modeling must be verified. The objective of this study, therefore, was to determine whether batch kinetic data were appropriate for predicting nonequilibrium sorption during alachlor [2-chIoro-N-(2,6-diethylphenyl)-N-(methoxymethyl)-acetamide] transport in Dundee silt loam soil. Batch (solution and extraction) data previously generated in this laboratory were used to determine sorption parameters for two- or three-site equilibrium/kinetic sorption models. Predictions of alachlor mobility generated from the corresponding transport models were then compared with miscible displacement data. Use of sorption parameters determined from batch solution data alone resulted in only qualitative predictions of alachlor movement. However, more accurate predictions were obtained if sorption parameters were based on both soil extraction and soil solution data.

The determination of glyphosate in soils with moderate to high clay content

AbstractAn improved method for the quantitative analysis of the herbicide glyphosate [N‐(phosphonomethyl)glycine] in soils containing moderate to high clay content is described. Critical evaluations of previously published methods have indicated that recoveries of glyphosate from soils with high clay content are often low. Where acceptable recovery estimates have been reported, these methods also report increased interferences and rarely include soils with clay content exceeding 30%.The proposed method was developed and characterized using six soils of different clay content (25–87% clay), with other physical and chemical properties as described. Recoveries of glyphosate from the soils were determined after duplicate extractions with 0.1 M potassium hydroxide. Clean‐up of soil extract solutions was by cation‐exchange column chromatography. Subsequent quantitation was by HPLC with post‐column oxidation, followed by derivatization using OPA‐MERC with fluorometric detection. No interferences were detected.Recovery estimates for each fortified sample were determined over a concentration range (0.56–11.25 mg glyphosate kg −1 ) with all recoveries being greater than 80%. Detection limit for glyphosate in soil was 0.04 mg kg −1, and instrument response was linear for solutions up to 50.0μg glyphosate ml −1. Reproducibility relative confidence interval, for a single sample analysis, was determined as.

Reusable fiber-optic-based immunosensor for rapid detection of imazethapyr herbicide

Imazethapyr is a herbicide belonging to the imidazolinone class of compounds. It is the active ingredient of a commercial herbicide PURSUIT®. Polyclonal antibodies have been prepared in rabbits and sheep which specifically recognize this class of imidazolinone compounds. Using the immune rabbit serum, an enzyme-linked immunosorbent assay (ELISA) has been developed for the entire class of commercial imidazolinone herbicides including imazaquin, imazapyr, imazethapyr and imazamethabenz methyl. The quantitation of imidazolinones is soil requires a certain amount of sample pretreatment, thus the through-put is not ideal. A simpler immunoassay method for screening large amount of soil samples economically would be useful. Taking the sheep polyclonal immune serum, we used a fluorescent immunoassay employing optical fiber and fluorescence (US Patent 4,582,809, 1986), to assay for imazethapyr. Purified sheep antibody was immobilized on quartz fibers. A mixture of fluorescein-labelled imazethapyr analog and free imazethapyr was presented to the fiber for direct competition of the antibody binding sites or displacement of a previously fluorescein labelled fiber. The response time for the detection of imazethapyr ranged from seconds to minutes. The sensitivity of the assay was 1 nM. This binding of the fluorochrome to the fiber was reversible by washing with a phosphate buffered saline. Multiple measurements were easily processed with a single fiber over the course of several hours. Analysis of imazethapyr residue in soil can be accomplished by subjecting a clarified soil extract solution directly for analysis without further treatment. The crossreactivity data indicates that the assay is apparently specific for the imidazolinone class of compounds.

The dynamics of Rhizobium leguminosarum biovar trifolii introduced into soil as determined by immunofluorescence and selective plating techniques

After the introduction of Rhizobium leguminosarum biovar trifolii into a loamy sand and a silt loam, high recovery percentages were determined using quantitative immunofluorescence. Soil type, but not inoculum density between 10 4 and 10 8 cells per gram of soil, significantly influenced the recovery percentage of the immunofluorescence technique. Recovery percentages determined using selective plating were independent of either soil type or inoculum density and exceeded those determined by immunofluorescence. The serological and genetic markers used for detection were stable during 55 days of incubation in phosphate-buffered saline and soil extract solution. After the introduction of R. leguminosarum biovar trifolii into both sterilized soil types, the population increased to 0.5–1×10 9 cells per gram of soil, but a decline was demonstrated in non-sterile loamy sand and silt loam during incubation of 90 days at 15°C. Starvation of rhizobial cells in the phosphate-buffered saline and soil extract solution, as well as incubation in both soil types, resulted in a significant decrease in mean cell size.