Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples

The development of easy-to-use and rapid-monitoring immunoassay methods for organic environmental pollutants in a class-selective manner is a topic of considerable environmental interest. In this work, a heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) based on a monoclonal antibody (MAb) with broad-specificity for organophosphorus pesticides (OPs) was applied to the detection of O,O-diethyl and O,O-dimethyl OPs in water samples. The ciELISA conditions were carefully optimized to obtain a three to five-fold improvement of sensitivity for most OPs, and thirteen OPs were determined at concentrations ranging from 0.017 to 30 ng mL(-1). The determination of spiked environmental water samples showed average recoveries from 81.5% to 115.1%, with the coefficient of variation (CV) ranging from 6.1% to 20.9%, which showed satisfactory reproducibility of the developed ciELISA. To overcome the negative aspect of broad-specificity immunoassays not providing qualitative and quantitative analysis of individual OPs in blind samples, we used "percent inhibition rate" to make the developed ciELISA a semi-quantitative method, which allows the monitoring of positive samples from hundreds of negative samples. The determination of OPs in blind water samples by the developed ELISA with confirmation by HPLC-MS/MS analysis demonstrated that the assay is ideally suited as a screening method for OP residues prior to chromatographic analysis.     

Seasonal dynamics of freshwater pathogens as measured by microarray at Lake Sapanca,a drinking water source in the north-eastern part of Turkey

Monitoring drinking water quality is an important public health issue. Two objectives from the 4 years, six nations, EU Project μAqua were to develop hierarchically specific probes to detect and quantify pathogens in drinking water using a PCR-free microarray platform and to design a standardised water sampling program from different sources in Europe to obtain sufficient material for downstream analysis. Our phylochip contains barcodes (probes) that specifically identify freshwater pathogens that are human health risks in a taxonomic hierarchical fashion such that if species is present, the entire taxonomic hierarchy (genus, family, order, phylum, kingdom) leading to it must also be present, which avoids false positives. Molecular tools are more rapid, accurate and reliable than traditional methods, which means faster mitigation strategies with less harm to humans and the community. We present microarray results for the presence of freshwater pathogens from a Turkish lake used drinking water and inferred cyanobacterial cell equivalents from samples concentrated from 40 into 1 L in 45 min using hollow fibre filters. In two companion studies from the same samples, cyanobacterial toxins were analysed using chemical methods and those dates with highest toxin values also had highest cell equivalents as inferred from this microarray study.     

Gene-TEQ--a standardized comparative assessment of effects in the comet assay using genotoxicity equivalents

Existing methods for the comparison of genotoxic effects in the comet assay bear considerable disadvantages such as the problem to link information about concentration dependence and severity of effects. Moreover, given the lack of standardized protocols and the use of various standards, it may be extremely difficult to compare different studies. In order to provide a method for standardized comparative assessment of genotoxic effects, the concept of genotoxicity equivalents (Gene-TEQ) was developed. As potential reference compounds for genotoxic effects, three directly acting (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl-methanesulfonate, and N-methyl-N-nitrosourea) and three indirectly acting (cyclophosphamide, dimethylnitrosamine, and 4-nitroquinoline-oxide) genotoxic substances were compared with respect to their cytotoxic (neutral red) and genotoxic (comet assay) concentration-response profiles in the permanent fish cell line RTL-W1. For further comparison, two sediment extracts from the upper Danube River were investigated as environmental samples. Based on the results of cytotoxicity and genotoxicity testing, MNNG was selected as the reference compound. At several exposure levels and durations, genotoxic effects of both the other pure substances and the environmental samples were calculated as percentages of the maximum MNNG effect and related to the absolute MNNG effect (EC values). Thus, genotoxicity equivalent factors (Gene-TEQs) relative to MNNG could be calculated. Gene-TEQs can easily be applied to pure substances, mixtures and field samples to provide information about their toxicity relative to the reference compound. Furthermore, the Gene-TEQ concept allows a direct comparison of environmental samples from different laboratories.     

Ultracentrifugation as a direct method to concentrate viruses in environmental waters: virus-like particle enumeration as a new approach to determine the efficiency of recovery

Some health important enteric viruses are considered to be emerging waterborne pathogens and so the improvement of detection of these viruses in the aquatic environment is one of the most important steps in dealing with these pathogens. Since these viruses may be present in low numbers in water, it is necessary to concentrate water samples before viral detection. Although there are several methods to concentrate viruses in environmental waters, all present some drawbacks and consequently the method should be chosen that, despite its limitations, is adequate to achieve the aim of each study. As the effectiveness of the concentration methods is evaluated by determining the efficiency of viral recovery after concentration, it is important to use a simple and effective approach to evaluate their recovery efficiency. In this work ultracentrifugation, usually used as a secondary step for virus concentration, was evaluated as the main method to concentrate directly viruses in environmental water samples, using the microscopic enumeration of virus-like particles (VLP) as a new approach to estimate the efficiency of recovery. As the flocculation method is currently employed to concentrate viruses in environmental waters, it was also used in this study to assess the efficiency of the ultracentrifugation as the main viral concentration method in environmental waters. The results of this study indicate that ultracentrifugation is an adequate approach to concentrate viruses directly from environmental waters (recovery percentages between 66 and 72% in wastewaters and between 66 and 76% in recreational waters) and that the determination of VLP by epifluorescence microscopy is a simple, fast and cheap alternative approach to determine the recovery efficiency of the viral concentration methods.     

The first monoclonal antibody-based,matrix-resistant immunoassay for the carbamate herbicide asulam,in water

To date, no ligand binding assay has been described for the carbamate herbicide asulam, although a variety of physical methods, dependent on pre-concentration of water samples, have been documented for its assessment. However, asulam is increasingly used in sensitive agricultural areas, and statutory regulations concerning its monitoring will undoubtedly become more stringent. Antibodies are optimal partners in ligand binding assays, but it is commonly understood by immunological researchers that where no antibody reactive with a particular antigen has yet been described, the immunogenicity of the antigen may be particularly restricted. By the expedient of employing a specialised approach to final immunisation with an asulam-protein conjugate, prior to the immortalisation of a specific anti-asulam antibody-producing cell, we have succeeded in generating a monoclonal antibody reactive specifically with asulam that can be configured in a convenient immunoassay. This antibody may be used flexibly in a number of ways: small sample volumes of 10 microl can be assessed to sensitivities of 4.35 x 10(-7) M (10 microg L(-1)) while avoiding discrepancies contributed by the assay matrix; this antibody-based assay can also be formatted to deliver sensitivities at levels stipulated by regulatory authorities (e.g., 4.35 x 10(-9) M or 0.1 microg L(-1)) directly from a water sample, without prior pre-concentration.     

Evaluation of performance of time-saving extraction devices in the BCR three-step sequential extraction procedure

Two alternative extraction methods--a routine ultrasonic bath and a microwave oven--were developed and optimized for their eventual exploitation in the three-stage sequential extraction procedure proposed by the European Standards, Measurements and Testing (S, M & T) Programme, formerly Bureau Communitaire de Reference (BCR), for the operationally defined speciation of Cd, Cr, Cu, Ni and Zn. The conventional BCR three-stage sequential extraction procedure was modified at each stage, applying ultrasonic or microwave devices in order to shorten the required 16 h of shaking in all three steps. The experimental tests and the optimization of the operating parameters were carried out on a highly homogenized estuarine sediment reference material (RM S7) prepared by the Joint Research Centre (JRC) of ISPRA. Extractable metal contents were measured by inductively coupled plasma-mass spectrometry (ICP-MS) and the results obtained in each fraction by all three methods were statistically compared for all the studied elements. The conventional and newly developed alternative extraction methods were finally compared by the analysis of BCR 601, which is certified for the three-step BCR sequential extraction procedure. According to the statistical evaluation of the results, the proposed accelerated sequential extraction methods are valid alternatives to conventional shaking, with much shorter extraction times.     

A field screening test for the assessment of concentrations and mobility of potentially toxic elements in soils: a case study on urban soils from Rome and Novi Sad

The increasing demand for environmental pollution control results in the development and use of new procedures for the determination of dangerous chemicals. Simple screening methods, which can be used directly in the field for a preliminary assessment of soil contamination, seem to be extremely advantageous. In our laboratory, we developed and optimized a rapid test for a preliminary evaluation of both the concentration and the mobility of some potentially toxic metals in soils. This screening test consists of a single extraction of the soil sample with a buffer solution, followed by the titration of the extracted solution with dithizone to determine the contents of bi-valent heavy metals (such as Pb, Cu, Zn, and Cd). This screening method was then directly applied in the field during the sampling campaign in the framework of an Italian–Serbian collaborative project, finalized in the study of metal availability in soils. The results obtained in the field with the rapid test were compared with those obtained in the laboratory following the conventional procedure commonly used to evaluate metal bioavailability (diethylenetriaminepentaacetic extraction). Moreover, selected samples were analyzed sequentially in the laboratory using the standardized BCR three-step sequential extraction procedure. The screening test gave results conceptually in good agreement with those obtained via the BCR procedure. These preliminary data show that the proposed screening test is a reliable method for the preliminary rapid evaluation of metal total concentrations and of potential metal mobility in soils, supporting sampling activities directly in the field.     

不同酸碱度土壤阳离子交换量的测定研究

为了能够快速准确测定不同酸碱度土壤中的阳离子交换量,弥补现行标准方法的不足,对三氯化六氨合钴浸提-分光光度法中的土壤称样量、pH、浸提时间、浸提温度和离心时间等条件进行了优化。优化后的方法检出限为0.43 cmol^+/kg,测定下限为1.72 cmol^+/kg,方法实验室内精密度为0.2%~1.8%,适用于大批量土壤样品中阳离子交换量的测定。     

A rapid lateral flow immunoassay for the detection of fungal alpha-amylase at the workplace

Fungal alpha-amylase is a flour supplement which is added to improve the quality of bakery products. Various studies have shown that exposure to this enzyme is an important risk factor for the development of bakers' allergy and this allergy is reported to be one of the most frequent causes of occupational asthma. A rapid assay was developed to monitor exposure to occupational allergens directly at the workplace. The sensitivity of the developed assay is 0.32 ng amylase mL(-1) in a buffer system with the commercially available alpha-amylase preparation Fungamyl 1600S as the standard. Initial validation tests (n = 33) were performed with airborne and settled dust from an industrial bakery. The new lateral flow immunoassay detected amylase in 22 of the 26 samples regarded as positive in an enzyme immunoassay, and was negative for all seven enzyme immunoassay-negative samples, while the four lateral flow immunoassay-negative/enzyme immunoassay-positive samples all had levels below 2 ng mL(-1). The sensitivity of 2 ng mL(-1) of the amylase lateral flow immunoassay is sufficient for first screening purposes and, therefore, this simple and rapid assay may allow direct on-site demonstration of work-related hazards of bio-allergen exposure. This would be particularly useful in occupational hygiene practice, especially in traditional or small-scale bakeries which lack the technological skills for testing the exposure to respiratory allergens.     

Improving Salmonella determination in Sinaloa rivers with ultrafiltration and most probable number methods

Monitoring of waterborne pathogens is improved by using concentration methods prior to detection; however, direct microbial enumeration is desired to study microbial ecology and human health risks. The aim of this work was to determine Salmonella presence in river water with an ultrafiltration system coupled with the ISO 6579:1993 isolation standard method (UFS-ISO). Most probable number (MPN) method was used directly in water samples to estimate Salmonella populations. Additionally, the effect between Salmonella determination and water turbidity was evaluated. Ten liters or three tenfold dilutions (1, 0.1, and 0.01 mL) of water were processed for Salmonella detection and estimation by the UFS-ISO and MPN methods, respectively. A total of 84 water samples were tested, and Salmonella was confirmed in 64/84 (76%) and 38/84 (44%) when UFS-ISO and MPN were used, respectively. Salmonella populations were less than 5 × 10(3) MPN/L in 73/84 of samples evaluated (87%), and only three (3.5%) showed contamination with numbers greater than 4.5 × 10(4) MPN/L. Water turbidity did not affect Salmonella determination regardless of the performed method. These findings suggest that Salmonella abundance in Sinaloa rivers is not a health risk for human infections in spite of its persistence. Thus, choosing the appropriate strategy to study Salmonella in river water samples is necessary to clarify its behavior and transport in the environment.     

Development of an improved methodology to detect infectious airborne influenza virus using the NIOSH bioaerosol sampler

A unique two-stage cyclone bioaerosol sampler has been developed at NIOSH that can separate aerosols into three size fractions. The ability of this sampler to collect infectious airborne viruses from a calm-air chamber loaded with influenza A virus was tested. The sampler's efficiency at collecting aerosolized viral particles from a calm-air chamber is essentially the same as that from the high performance SKC BioSampler that collects un-fractionated particles directly into a liquid media (2.4 × 10(4) total viral particles per liter of sampled air (TVP/L) versus 2.6 × 10(4) TVP/L, respectively, after 15 min) and the efficiency is relatively constant over collection times of 15, 30 and 60 min. Approximately 34% of the aerosolized infectious virus collected after 15 min with the NIOSH bioaerosol sampler remained infectious, and infectious virus was found in all three size fractions. After 60 min of sampling, the infectious virus/liter air found in the NIOSH bioaerosol sampler was 15% of that found in the SKC BioSampler. This preservation of infectivity by the NIOSH bioaerosol sampler was maintained even when the initial infectivity prior to aerosolization was as low as 0.06%. The utility of the NIOSH bioaerosol sampler was further extended by incorporating an enhanced infectivity detection methodology developed in our laboratory, the viral replication assay, which amplified the infectious virus making it more readily detectable.     

Water quality indices across Europe--a comparison of the good ecological status of five river basins

The European Water Framework Directive (WFD) requires the definition of near-natural reference conditions to determine the extent of water bodies' deviation from "good ecological status" caused by stress gradients. However, the classification of ecological quality depends on the assessment method applied and the stressor concerned. While assessment methods that are generally applicable would be favourable, many European countries employ the locally developed water quality metrics that assess the impact of organic pollution (including eutrophication) and the associated decrease in dissolved oxygen. These indices do not specifically address stress from organic toxicants, such as pesticides. The aim of this study was to examine the performance of presently used assessment methods to identify reference conditions of non-contaminated streams in five selected European river basins, covering the geographical region from Spain to Finland, as a crucial prerequisite to indicate toxic gradients. The analysis comprised the Belgium biotic index (BBI), the biological monitoring working party (BMWP) scoring system and the revised German saprobic index. For comparison, we included an adaptation of the recently developed SPEAR index. In two previous field studies, this metric highly correlated with measured pesticide gradients. In this study, SPEAR was the only indicator that was generally applicable to all monitoring data and capable of determining "high ecological status" of reference conditions in all basins. Thus, based upon previous and own results, the authors suggest the species at risk (SPEAR) index to be potentially useful as a European-wide index to address deviations from "good ecological status" due to organic toxicants and recommend it for consideration in integrated water-resource evaluations under the WFD.     

Comparison of three sampling and analytical methods for the determination of airborne hexavalent chromium

A field study was conducted with the goal of comparing the performance of three recently developed or modified sampling and analytical methods for the determination of airborne hexavalent chromium (Cr(VI)). The study was carried out in a hard chrome electroplating facility and in a jet engine manufacturing facility where airborne Cr(VI) was expected to be present. The analytical methods evaluated included two laboratory-based procedures (OSHA Method ID-215 and NIOSH Method 7605) and a field-portable method (NIOSH Method 7703). These three methods employ an identical sampling methodology: collection of Cr(VI)-containing aerosol on a polyvinyl chloride (PVC) filter housed in a sampling cassette, which is connected to a personal sampling pump calibrated at an appropriate flow rate. The basis of the analytical methods for all three methods involves extraction of the PVC filter in alkaline buffer solution, chemical isolation of the Cr(VI) ion, complexation of the Cr(VI) ion with 1,5-diphenylcarbazide, and spectrometric measurement of the violet chromium diphenylcarbazone complex at 540 nm. However, there are notable specific differences within the sample preparation procedures used in three methods. To assess the comparability of the three measurement protocols, a total of 20 side-by-side air samples were collected, equally divided between a chromic acid electroplating operation and a spray paint operation where water soluble forms of Cr(VI) were used. A range of Cr(VI) concentrations from 0.6 to 960 microg m(-3), with Cr(VI) mass loadings ranging from 0.4 to 32 microg, was measured at the two operations. The equivalence of the means of the log-transformed Cr(VI) concentrations obtained from the different analytical methods was compared. Based on analysis of variance (ANOVA) results, no statistically significant differences were observed between mean values measured using each of the three methods. Small but statistically significant differences were observed between results obtained from performance evaluation samples for the NIOSH field method and the OSHA laboratory method.     

利用MALDI-TOF MS技术简便、快速检测水华蓝藻的方法研究

以水华蓝藻为研究对象,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术对其进行测定分析并获得了蛋白质指纹图谱。对铜绿微囊藻标准株(NIES-843)样品通过3种前处理方法得到的质谱图进行对比,确定了质谱分析的样品前处理方法,建立了利用MALDI-TOF MS技术简便、快速检测水华蓝藻的方法。对在水华中出现的4种不同蓝藻进行MALDI-TOF MS分析,结果表明各种蓝藻具有其特征性波谱,可据此对水华蓝藻进行区分和鉴定。该方法快速、简便、精确、可程序化,具有广泛的应用前景。     

An Approach to Multi-criteria Environmental Evaluation with Multiple Weight Assignment

In the domain of environmental protection, multi-criteria evaluation is used in cases where multiple alternatives, on the bases of multiple parameters, need to be evaluated. Depending on the particular goal of investigation, various approaches have been developed and applied. This various multi-criteria approaches differ in parameter weighting method, in data normalization method as well as in the method for assessment of alternatives. According to the applied multi-criteria methodology, the result, i.e., the rank of alternatives may differ to some extent. In this context, parameter weighting bears special significance in multi-criteria evaluation, while the choice of method is crucial for final result. The specificity of parameter weighting process in environment protection is directly related to pronounced interdisciplinary character of this area, as well as the large number of influential parameters. With this in mind, this paper presents an approach to multi-criteria evaluation which—through integration of three specific methods for parameter weighting—allows more flexible and multi-purpose application. Based on the established concept, a software application was developed. Besides automated parameter weighting, it also provides graphical interpretation of results. The developed approach and software have been verified on the case study related to evaluation of environmental loadings at six locations in the city of Novi Sad.     

Real-time PCR for detection of the Aspergillus genus

Aspergillus is a genus of mold that has strong indoor sources, including several species capable of acting as opportunistic pathogens. Previous studies suggest that Aspergillus could serve as an indicator for abnormal mold growth or moisture, making it an important genus for environmental monitoring. Here, a quantitative polymerase chain reaction (qPCR, or real-time PCR) assay is presented for Aspergillus. The assay shows good specificity for the genus, detecting all Aspergillus species tested, although a few non-Aspergillus species are also amplified. Sensitivity testing demonstrates that DNA representing one conidium can be detected. A validation study compared qPCR results against direct microscopy counts using A. fumigatus conidia aerosolized into a laboratory chamber. The assay was then used to quantify Aspergillus in indoor air samples, demonstrating its utility for environmental monitoring. Analysis of a small number of clinical sputum samples showed complete agreement with culturing results.     

吹扫捕集-气相色谱质谱法分析土壤和沉积物中挥发性有机物

采用吹扫捕集-气相色谱质谱法对土壤和沉积物中挥发性有机物进行分析,优化了实验条件,所有物质的相对标准偏差小于5.0％,土壤样回收率在78.2％ ～99.8％之间,沉积物样的回收率在55.2％～95.2％之间.     

Addressing the challenges of tetracycline analysis in soil: extraction, clean-up, and matrix effects in LC-MS

An optimized extraction and clean-up method for the analysis of chlortetracycline, doxycycline, oxytetracycline, and tetracycline antibiotics in soil is presented in this work. Soil extraction using different solvents was performed, but the use of a 50 : 50 (v/v) methanol : acetate buffer (pH 8) solvent mixture in a pressurized liquid extraction (PLE) system proved to give the best extraction efficiency and reproducibility. The effect of soil composition on the PLE extraction efficiency was also examined, and results indicated that recovery data for one soil is not necessarily consistent with other soil types containing different compositions of clay and organic matter content. The percent recoveries of the optimized PLE method varied between the soils and ranged from 22-99%, depending on soil type, and more specifically clay content. In addition, the extent of ionization suppression caused by co-extracted humic acids was examined in an ion trap mass spectrometer (MS), and a single quadrupole MS. It was found that under positive electrospray ionization, the single quadrupole MS was less susceptible to ionization suppression than the ion trap MS. Therefore, various sample clean-up procedures were evaluated to selectively reduce the amount of co-extracted humic acids in the soil extracts. The most effective clean-up was obtained from the use of StrataX sorbent in combination with a strong anion exchange cartridge.