Simple and sensitive determination of o-phenylphenol in citrus fruits using gas chromatography with atomic emission or mass spectrometric detection

In this work, a simple and sensitive method for the analysis of the pesticide o-phenylphenol (OPP) on citrus fruits was developed. OPP is extracted with dichloromethane by ultrasonication and derivatized with ferrocenecarboxylic acid chloride. Using ferrocene as a label, residues of OPP are determined by gas chromatography with atomic emission detection in the iron selective mode or with mass spectrometric detection. Sample cleanup is simple and rapid and merely involves a removal of excess reagent on an alumina minicolumn. The method detection limit is 2 ng of OPP/g of fruit, and recoveries from lemon samples fortified at levels of 35 and 140 ng/g are 101 and 106%, respectively. The citrus fruits analyzed (oranges, grapefruits, lemons) contained between 60 ng/g and 0.37 microg/g OPP (RSD = 8-13%), and the results were in good agreement with results obtained when OPP was analyzed using an established HPLC-FLD method. Several alcohols could also be identified in the fruit peel.     

Residue levels and effectiveness of pyrimethanil vs imazalil when using heated postharvest dip treatments for control of Penicillium decay on citrus fruit

The influence of fungicide concentration and treatment temperature on residue levels of pyrimethanil (PYR) in comparison with the commonly used fungicide imazalil (IMZ) was investigated in orange fruits following postharvest dip treatments. The dissipation rate of PYR residues was recorded as a function of storage conditions. The fungicide efficacy against green and blue molds caused by Penicillium digitatum and Penicillium italicum, respectively, was evaluated on different citrus varieties following the fungicide application at 20 or 50 degrees C. Residue levels of PYR in Salustiana oranges were significantly correlated with the fungicide dosage, but residue concentrations were notably higher (ca. 13-19-fold) after treatment at 50 degrees C as compared to treatments at 20 degrees C. After treatment at temperatures ranging from 20 to 60 degrees C, PYR and IMZ residues in Salustiana oranges were significantly correlated with dip temperatures. Dissipation rates of PYR during storage were negligible in both Salustiana and Tarocco oranges. Results obtained on wounded, noninoculated Miho satsumas revealed that when treatments were performed at 50 degrees C, PYR or IMZ concentrations needed to achieve the complete control of decay were 8- and 16-fold less than by treatment at 20 degrees C. When fruits were inoculated with either P. digitatum or P. italicum, the application of 400 mg L(-1) PYR at 20 degrees C or 100 mg L(-1) PYR at 50 degrees C similarly reduced green and blue mold development. These results were corroborated by storage trials on Marsh grapefruits and Tarocco oranges. The lowest concentration of PYR required to achieve almost total protection of the fruit against decay accounted for 100 mg L(-1) at 50 degrees C and 400 mg L(-1) at 20 degrees C, respectively. Treatments did not affect fruit external appearance, flavor, and taste. It is concluded that postharvest PYR treatment represents an effective option to control green and blue mold in citrus fruit and that integration of fungicide applications and hot water dips may reduce the possibility of selecting fungicide-resistant populations of the pathogen, as a consequence of increased effectiveness of the treatment.     

Determination of oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with tandem mass spectrometry

A new methodology is described for rapidly determining the herbicide oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with negative ion electrospray ionization tandem mass spectrometry (LC/MS/MS). Oryzalin is extracted from water using a polymeric sorbent solid phase extraction (SPE) column and from fruit using methanol. The water samples require no further purification, but an aliquot of the fruit sample extracts is diluted with water and purified using a polymeric 96 well SPE plate. Purified extracts are concentrated prior to determination by LC/MS/MS at m/z 345 (Q1) and m/z 281 (Q3) using an external standard for calibration. The validated limits of quantitation were 0.05 microg/L in water (drinking water, surface water, and groundwater) and 0.01 microg/g in citrus fruits (oranges and lemons) and stone fruits (peaches and cherries). Recoveries averaged 102% for water samples and 85-89% for the various types of fruit samples. For all fortification levels combined, the relative standard deviations ranged from 4 to 6% for water and from 2 to 4% for fruit.     

Orthophenylphenol and phenylhydroquinone residues in citrus fruit and processed citrus products after postharvest fungicidal treatments with sodium orthophenylphenate in California and Florida

Sodium orthophenylphenate (SOPP) has been used extensively for >40 years to control postharvest diseases of citrus fruits. Studies of the metabolism of [(14)C]SOPP have identified orthophenylphenol (OPP) as the major metabolite with phenylhydroquinone (PHQ) as a minor metabolite. The whole-fruit tolerance in the United States for OPP is 10 ppm. This study was conducted to quantify terminal OPP and PHQ residues in whole Navel oranges, grapefruit, and lemons following SOPP applications at maximum application rates and following commercial application and fruit storage practices. OPP and PHQ residues also were determined in products processed from treated Navel oranges. OPP residues in lemons, Navel oranges, and grapefruit treated with SOPP using foamer wash and shipping wax applications remained below the 10 ppm tolerance, and PHQ residues were all < or =0.439 ppm. PHQ residues in whole fruit increased with time in commercial storage. OPP residues in all Navel orange matrices except oil remained relatively stable with time in commercial storage; residues in oil declined substantially while in storage.     

New <Emphasis Type="Italic">Citrus</Emphasis> chloroplast haplotypes revealed by molecular markers using Algerian and Spanish accessions

Thirty-seven chloroplast molecular markers were used to evaluate the genetic diversity and infer the phylogenetic relationship of 24 Algerian Citrus accessions from the Institut Technique de l′Arboriculture Fruitière et de la Vigne germplasm bank. The reliability and consistence of the clustering distribution was further asserted including 5 Spanish accessions from the Instituto Valenciano de Investigaciones Agrarias. The accessions were positioned on a phylogenetic tree of the genus Citrus based on previous analyses of the whole sequence of citrus chloroplast. Algerian accessions clustered into two main clades mostly differentiated by the occurrence of either mandarin or pummelo chloroplast types. All 7 mandarins analyzed were grouped in the same clade while the other cluster subdivided in 4 groups, included 1 lumia, 3 lemons, 2 grapefruits and 11 sweet oranges. Algerian grapefruit accessions were grouped together with the pummelos in a single cluster while all sweet oranges formed an independent and homogenous clade. Interestingly, the lemons studied were clustered in 3 different subclusters while Citrus lumia genotype was isolated in a different group. These results suggest that in contrast to the studied Algerian mandarins or sweet oranges, that share all the same mandarin or sweet orange chloroplast haplotype, the high diversity of current lemon accessions is at least partially correlated with the identity of different pummelo progenitors which evolved from a common ancestor. In addition, the data indicate that Citrus lumia is a new type of citrus chloroplast that appears to be phylogenetically related to the chloroplasts of the pummelo and micrantha group.     

Monoclonal enzyme immunoassay for the analysis of carbaryl in fruits and vegetables without sample cleanup

The N-methylcarbamate pesticide carbaryl is one of the most important insecticides used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) for the determination of this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 101.0 +/- 26.9 ng/L and with a dynamic range between 31.6 and 364.0 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbaryl at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on both extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop and the fortification level, recoveries in the 59.0--120.0% range were obtained for purified samples and in the 70.0--137.7% range for crude extracts. The carbaryl immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbaryl at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was excellent (y = 1.04x + 0.71, r(2) = 0.992, n = 33), with HPLC being more precise than ELISA (mean coefficients of variation of 5.2 and 12.0%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.28x - 0.59, r(2) = 0.987, n = 33) while maintaining similar precision. Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbaryl in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases sample throughput.     

Validation of a monoclonal enzyme immunoassay for the determination of carbofuran in fruits and vegetables

The N-methylcarbamate pesticide carbofuran is a very important insecticide used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) to determine this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 740 ng/L and with a dynamic range between 200 and 3100 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbofuran at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop, mean recoveries in the 43.9--90.7% range were obtained for purified samples and in the 90.1--121.6% range for crude extracts. The carbofuran immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbofuran at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was very good (y = 0.90x + 2.66, r(2)() = 0.958, n = 25), with HPLC being more precise than ELISA (mean coefficients of variation of 4.1 and 11.5%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.02x + 10.44, r(2)() = 0.933, n = 29). Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbofuran in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases the sample throughput.     

Complexation of imazalil with beta-cyclodextrin,residue uptake,persistence, and activity against penicillium decay in citrus fruit following postharvest dip treatments

A method for the inclusion of imazalil (IMZ) in the beta-cyclodextrin (betaCD), structural characterization of the inclusion complex and its antifungal activity against Penicillium digitatum and P. italicum assessed by in vitro and in vivo tests are reported. According to the starting stoichiometry of betaCD with respect to IMZ, an equimolar ratio beta-cyclodextrin-IMZ (betaCD-IMZ) was detected by (1)H NMR. In vitro assays showed that the freshly prepared betaCD-IMZ was as effective as IMZ, although 1- and 4-day-old betaCD-IMZ mixtures were more effective. Studies on Star Ruby grapefruit showed no significant differences in residue uptake between treatments with an IMZ commercially available fungicide (Deccozil) or betaCD-IMZ when equal active ingredient (a.i.) concentrations (250 mg/L) and dip temperatures (20 or 50 degrees C) were used. By contrast, treatments of Tarocco oranges and Di Massa lemons with 250 mg/L betaCD-IMZ at 50 degrees C produced significant differences in residue uptake in comparison with 250 mg/L Deccozil treatments at 50 degrees C. The a.i. degradation rate in grapefruit during postquarantine and simulated marketing period (SMP) at 20 degrees C was not affected by the type of formulation used, whether at 20 or 50 degrees C. Conversely, IMZ in oranges and lemons had greater persistence when applied at 50 degrees C. All fungicide treatments showed a comparable efficacy against decay in grapefruit and oranges, whereas treatment in lemons at 250 mg/L a.i. of heated fungicides had higher suppressive effects against decay than unheated chemicals having equal a.i. concentrations and comparable activity at 1200 mg/L IMZ at 20 degrees C.     

Enantioseparation of imazalil residue in orange by capillary electrophoresis with 2-hydroxypropyl-beta-cyclodextrin as a chiral selector

Chiral resolution of imazalil, a fungicide, was performed by capillary electrophoresis (CE) using 2-hydroxypropyl-beta-cyclodextrin as a chiral selector. Factors affecting the chiral resolution and migration time of imazalil were studied. The optimum running conditions were found to be 5 mM ammonium dihydrogenphosphate-50 mM phosphate buffer (pH 3.0) containing 4 mM 2-hydroxypropyl-beta-cyclodextrin with an effective voltage of +25 kV at 20 degrees C using direct detection at 200 nm. Under these conditions, the resolution (Rs) of racemic imazalil was approximately 6. The extraction of imazalil from orange samples was done with acetonitrile under basic conditions. The extract was purified with a solid-phase extraction cartridge (Sep-Pak plus PS-2) and was analyzed by the above CE method. Eight orange samples were analyzed, and imazalil was detected in seven samples. In four of these seven oranges, the level of (-)-imazalil was the same as that of (+)-imazalil, but in the other three oranges, the level of (-)-imazalil was found to be lower than that of (+)-imazalil, suggesting that (-)-imazalil was degraded more quickly than (+)-imazalil in oranges.     

Gas chromatographic determination of fenpropimorph residues in citrus fruit

A rapid gas chromatographic method for determining fenpropimorph residues in citrus fruit is reported. The fungicide is extracted with hexane after pH adjustment of the fruit homogenate. A short liquid-liquid partitioning process is performed before gas chromatography on an OV-17 column with nitrogen-phosphorus specific detection. The limit of detection of the method was 0.01 mg/kg, based on a 25 g sample. Recovery was always higher than 70%. Fenpropimorph residues in "Washington Navel" oranges and "Hernandina" clementine fruits dipped in a 1500 mg/L fungicide solution were determined. The fungicide remains mainly in the peel, with levels less than 0.1 mg/kg in the pulp. Fungicide residues in the peel decrease during storage, mainly in Washington Navel peel, where values decreased from 5.2 to 2.8 mg/kg.     

Measurement of food flavonoids by high-performance liquid chromatography: A review

The flavonoids are plant polyphenols found frequently in fruits, vegetables, and grains. Divided into several subclasses, they include the anthocyanidins, pigments chiefly responsible for the red and blue colors in fruits, fruit juices, wines, and flowers; the catechins, concentrated in tea; the flavanones and flavanone glycosides, found in citrus and honey; and the flavones, flavonols, and flavonol glycosides, found in tea, fruits, vegetables, and honey. Known for their hydrogen-donating antioxidant activity as well as their ability to complex divalent transition metal cations, flavonoids are propitious to human health. Computer-controlled high-performance liquid chromatography (HPLC) has become the analytical method of choice. Many systems have been developed for the detection and quantification of flavonoids across one, two, or three subclasses. A summary of the various HPLC and sample preparation methods that have been employed to quantify individual flavonoids within a subclass or across several subclasses are tabulated in this review.     

Citrus Growth and Rhizosphere Properties

Fifteen citrus varieties (four varieties of limes/lemons, three varieties of mandarins, and eight varieties of sweet oranges) were tested in a row-to-row multireplicate field experiment on Typic Rhodustalf. Pre-bearing growth behavior of different citrus varieties showed a significant difference (P ≤ 0.05) with respect to canopy volume (1.221 m³ with Bearss lemon and 0.220 m³ with Cara Cara Navel) governed by changes in different rhizospheric properties (soil-available nutrients, soil microbial population, and soil microbial biomass nutrients). Response in canopy volume was more governed by soil microbial biomass nutrients [carbon (C_mic), nitrogen (N_mic), and phosphorus (P_mic)] followed soil microbial population and soil available nutrients in decreasing order. Indices developed through diagnosis and recommendation integrated system further helped in partitioning interrhizosphere nutrient deficiencies. These studies suggested that (i) biological properties of rhizosphere soils of limes and lemons were of much superior quality and (ii) rhizospheric biological properties are transformed according to plant species and variety.     

便携式双档位柑橘多品质无损检测装置设计与试验

为了解决普通检测装置难以覆盖不同果径（25～95mm）柑橘的检测需求问题,研发了覆盖多果径柑橘的便携式双档位多品质无损检测装置。以砂糖橘（果径25.35～48.61mm）和武鸣沃柑（果径53.24～94.71mm）为研究对象,基于研发的双档位探头,在赤道部位每隔120°采集一次光谱,平均光谱作为该柑橘的原始光谱。经标准正态变量变换（standard normal variable,SNV）、多元散射校正（multiplicative scatter correction,MSC）预处理,再利用竞争性自适应加权抽样算法（competitive adaptive reweighted sampling, CARS）筛选特征波长,分别建立了沃柑和砂糖橘的可溶性固形物含量（soluble solids content,SSC）和水分的偏最小二乘预测模型。沃柑的SSC和水分预测模型验证集相关系数分别为0.937、0.951,均方根误差分别为0.382°Brix、0.491%;砂糖橘的SSC和水分预测模型验证集相关系数分别为0.921、0.935,均方根误差分别为0.460°Brix、0.673%...     

Determination of acequinocyl and hydroxyacequinocyl on fruits and vegetables by HPLC-DAD

A method for determining residues of the new reduced-risk pesticide acequinocyl and its deacetylated derivative hydroxyacequinocyl on fruits and vegetables (grapes, lemons, pears, and tomatoes) by HPLC is described. The pesticides were extracted from the fruits and vegetables with hexane and ethyl acetate solution (1:1, v/v), determined by HPLC-DAD at 250 nm and confirmed by LC/MS. No cleanup was necessary. This method is characterized by recoveries (0.01-4 mg/kg) > 77%, while the coefficient of variation was determined to be less than 11%. The limit of quantitation for both acequinocyl and hydroxyacequinocyl was 0.01 mg/kg for all matrixes.     

Differences in the carotenoid content of ordinary citrus and lycopene-accumulating mutants

High-performance liquid chromatography, coupled with photodiode array detection, was used to analyze the carotenoid composition of peel and juice vesicle tissues of ordinary and lycopene-accumulating mutants (referred to as red mutants in this article) of orange, pummelo, and grapefruit. Thirty-six major carotenoids, including some cis-trans isomers, were separated on a C30 reversed phase column, and 23 of them were identified on the basis of retention times and spectral characteristics with authentic standards. Carotenoid profiles varied with tissue types, citrus species, and mutations. beta-Citraurin occurred in the peel of oranges but not in juice vesicles, whereas the reverse was found for violaxanthin, 9-cis-violaxanthin, and luteoxanthin. The diversity of carotenoids in peel and juice vesicle tissues and the fact that there was over 250 times higher content of total carotenoids in peels of Yuhuan pummelo than juice vesicles suggested that the biosynthesis of carotenoids in these two tissues was independent and exchange of carotenoids between the tissues was not likely. Lutein was observed in peels of pummelos and grapefruits and juice vesicles of ordinary pummelo but not in orange tissues. Accumulation of lycopene and beta-carotene was observed in red mutant citrus, except for the peel of Cara Cara red orange. Additionally, phytoene accumulated in all tissues except for the peel of Chuzhou Early Red pummelo. No obvious change in the total content of xanthophylls was observed in the Cara Cara red orange. Ordinary grapefruit (Marsh) tissues and pummelo (Yuhuan) juice vesicles were almost devoid of carotenoids, and in red mutants, the content of total carotenoids increased dramatically up to 790-fold. The different changes in carotenoid content and profiles in mutant(s) of different citrus species suggest that the underlying mechanisms for the mutations might be different.     

Effect of the iron supply on the nutrition of different citrus variety/rootstock combinations using dris 1

The Diagnosis and Recommendation Integrated System (DRIS) has been found reliable in diagnosing nutrient requirements, despite changes in the tissue sampling or the time sampling. Relatively few researchers use the results obtained by hydroponic culture to make a DRIS data bank. In this work, results obtained in this way have been utilized as DRIS reference values to evaluate the iron (Fe) supply influence on the nutrition of different citrus combinations under normal and deficient Fe conditions. The DRIS indices have been compared with standard methods to evaluate the effectiveness of this system of interpretation. The Citrus macrophylla rootstock for lemons is more resistant to Fe chlorosis than Sour orange. Based on the diagnosis of visual symptoms, the Citrus volkameriana is more resistant to this nutritional deficiency for oranges than the Cleopatra mandarin.     

基于改进YOLOv7模型的柑橘表面缺陷在线检测

柑橘表面缺陷是水果检测分级的重要依据,针对传统柑橘表面缺陷检测方法效率低、精度低等问题,该研究提出一种柑橘表面缺陷的实时检测方法。该方法首先对柑橘图像进行图像增强,然后利用提出的YOLOv7-CACT模型对柑橘表面缺陷进行检测,该模型在YOLOv7模型骨干网络中引入坐标注意力模块(coordinate attention, CA),从而提高模型对缺陷部分的关注度。在网络头部引入CT(contextual transformer,CT)模块,融合静态和动态上下文表征特征,从而增强缺陷部分特征表达能力。通过试验确定CA模块和CT模块的最佳位置。改进后的YOLOv7-CACT模型检测结果平均精度均值（mean average precision,mAP）相较于原始模型增加了4.1个百分点,达到91.1%,满足了实际生产中对柑橘缺陷检测精度的要求。最后将基于YOLOv7-CACT的柑橘检测模型通过TensorRT进行部署,试验结果表明模型的推理时间满足柑橘生产线10个/s的实时分选要求,总体的检测精度达到94.4%,为柑橘表面缺陷在线检测提供了一种精准的实时检测方法。     

基于实例分割的柑橘花朵识别及花量统计

柑橘隔年结果现象严重,花量统计有助于果园的规划管理,并对产量预测有重要意义,但是柑橘单一植株花量巨大,花朵紧凑密集,花期树叶遮挡覆盖,对花量计算造成很大的阻碍。对此该研究提出基于实例分割的柑橘花朵识别与花量统计方法,以花期的柑橘树冠图像为样本进行花朵实例的识别及分割,通过对Mask R-CNN主体卷积部分和掩膜分支部分的优化,实现对复杂结构图像中密集小尺度柑橘花朵目标的高效检测、获取图像中可见花数量。结果显示,该方法花量识别神经网络的平均精度为36.3,花量计算误差为11.9%,对比未优化Mask R-CNN网络在训练和识别的时间效率上均有显著提升。该研究解决了柑橘花量统计难度高的问题,有助于柑橘早期测产和落花监测,并为花量控制提供决策依据。     

Molecular and immunochemical characteristics of monoclonal and recombinant antibodies selective for the triazine herbicide simetryn and application to environmental analysis

A monoclonal antibody (mab) selective for the thiomethyl-s-triazine herbicide simetryn was obtained and characterized in enzyme-linked immunosorbent assay (ELISA). An IC(50) value for simetryn was 8.5 ng/mL, and the detection range extended from 1.1 to 70 ng/mL in ELISA. The cDNAs encoding variable heavy chain (VH) and variable light chain (VL) of the mab were cloned to produce various recombinant antibodies. Single-chain variable fragment (scFv) antibodies derived from the mab were characterized in ELISA and showed similar reactivities and specificities to the parent mab. A urea denaturation test revealed that the scFv antibodies bound to simetryn were more stable than those in the absence of antigen. A sandwich ELISA based on VH and VL fragments of the mab was successfully developed and showed similar sensitivity to those based on the mab and scFv antibodies in ELISA. In the recovery experiments using spiked environmental samples, the results obtained in ELISA based on the mab were favorably correlated with those by HPLC.     

New approach to immunochemical determinations for triclopyr and 3,5,6-trichloro-2-pyridinol by using a bifunctional hapten,and evaluation of polyclonal antiserum

The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed ELISA turned out to be a powerful tool for monitoring of residual triclopyr or 3,5,6-trichloro-2-pyridinol in water samples at trace level.