Large Numbers Of Environmental Samples Research Articles

A Simple and Effective Visual Fluorescent Sensing Paper-Based Chip for the Ultrasensitive Detection of Mercury Ions in Environmental Water.

Traces of mercury ions in environmental water can harm humans and animals. Paper-based visual detection methods have been widely developed for the rapid detection of mercury ions; however, existing methods are not sensitive enough to be used in real environments. Here, we developed a novel, simple and effective visual fluorescent sensing paper-based chip for the ultrasensitive detection of mercury ions in environmental water. CdTe-quantum-dots-modified silica nanospheres were firmly absorbed by and anchored to the fiber interspaces on the paper's surface to effectively avoid the unevenness caused by liquid evaporation. The fluorescence of quantum dots emitted at 525 nm can be selectively and efficiently quenched with mercury ions, and the ultrasensitive visual fluorescence sensing results attained using this principle can be captured using a smartphone camera. This method has a detection limit of 2.83 µg/L and a fast response time (90 s). We successfully achieved the trace spiking detection of seawater (from three regions), lake water, river water and tap water with recoveries in the range of 96.8-105.4% using this method. This method is effective, low-cost, user-friendly and has good prospects for commercial application. Additionally, the work is expected to be utilized in the automated big data collection of large numbers of environmental samples.

Determinants of associations between codon and amino acid usage patterns of microbial communities and the environment inferred based on a cross-biome metagenomic analysis

Codon and amino acid usage were associated with almost every aspect of microbial life. However, how the environment may impact the codon and amino acid choice of microbial communities at the habitat level is not clearly understood. Therefore, in this study, we analyzed codon and amino acid usage patterns of a large number of environmental samples collected from diverse ecological niches. Our results suggested that samples derived from similar environmental niches, in general, show overall similar codon and amino acid distribution as compared to samples from other habitats. To substantiate the relative impact of the environment, we considered several factors, such as their similarity in GC content, or in functional or taxonomic abundance. Our analysis demonstrated that none of these factors can fully explain the trends that we observed at the codon or amino acid level implying a direct environmental influence on them. Further, our analysis demonstrated different levels of selection on codon bias in different microbial communities with the highest bias in host-associated environments such as the digestive system or oral samples and the lowest level of selection in soil and water samples. Considering a large number of metagenomic samples here we showed that microorganisms collected from similar environmental backgrounds exhibit similar patterns of codon and amino acid usage irrespective of the location or time from where the samples were collected. Thus our study suggested a direct impact of the environment on codon and amino usage of microorganisms that cannot be explained considering the influence of other factors.

Strontium isotope evidence for Neanderthal and modern human mobility at the upper and middle palaeolithic site of Fumane Cave (Italy)

To investigate the mobility patterns of Neanderthals and modern humans in Europe during the Middle-to-Upper Palaeolithic transition period, we applied strontium isotope analysis to Neanderthal (n = 3) and modern human (n = 2) teeth recovered from the site of Fumane Cave in the Monti Lessini region of Northern Italy. We also measured a large number of environmental samples from the region, to establish a strontium ‘baseline’, and also micromammals (vole teeth) from the levels associated with the hominin teeth. We found that the modern humans and Neanderthals had similar strontium isotope values, and these values match the local baseline values we obtained for the site and the surrounding region. We conclude that both groups were utilizing the local mountainous region where Fumane Cave is situated, and likely the nearby Lessini highlands and Adige plains, and therefore the strontium evidence does not show differening mobility patterns between Neanderthals and modern humans at the Fumane site.

Data-independent acquisition with ion mobility mass spectrometry for suspect screening of per- and polyfluoroalkyl substances in environmental water samples

Per- and polyfluoroalkyl substances (PFASs) have environmentally persistent, and the various types of PFASs have been detected in water environments. Many previous studies have performed data-dependent acquisition (DDA) of mass spectra from an environmental sample by high-resolution mass spectrometry to identify PFAS suspects individually. In comparison, the data-independent acquisition (DIA) of comprehensive mass spectra of the sample is a technology which enables to know the occurrences of suspects and non-targets simultaneously. However, it is difficult to associate the fragment ions of targeted precursor ions by retention time only, because of the existence of co-eluting ions derived from environmental samples. Since the separation of ions is not enough with only the conventional DIA method, here, we attempted to support it using ion mobility mass spectrometry (IMS) to distinguish the relevant ions from co-eluting ions by drift time. Firstly, suspect screening of PFASs with eternal database resulted in determining 32–96 PFAS suspects in firefighting foam impacted groundwater samples (n = 8) by suspect screening. Among all the pairs of respective precursor ions and fragment ions of the PFAS suspects, 5%–19% (4–9 PFASs) of them were associated without considering the drift time of IMS, while 37%–49% (15–43 PFASs) of them were associated with considering the drift time. The consideration of the drift time increased the association ratios in all samples. In a sample, most precursor ions could be associated with their fragment ions (41 of 43 PFASs) because at least one probable fragment ion was observed among three of maximum intensity fragment ions. Thus, the method improved the identification by excluding the unrelated co-eluting ions by IMS. Moreover, the method can acquire a certain reliable MS/MS spectra of suspects in environmental samples in one analysis. It is not essential to conduct instrumental analyses again for samples stored for a long time even when the data sets and/or methodologies of data analyses are modified (e.g., the original database, screening list, or statistical filtering/data cleaning approach). It will be particularly useful for studies that must analyze a large number of environmental samples.

Determination of glyphosate residues in Egyptian soil samples

A sensitive linker-assisted enzyme-linked immunosorbent assay (L'ELISA) was developed for the analysis of glyphosate in Egyptian soil samples. Polyclonal glyphosate antibodies were produced from rabbits immunized with glyphosate protein conjugate. The conjugate was prepared by activating the carboxylic groups of proteins; thyroglobulin or bovine serum albumin with 1-ethyl-3- (3-diaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide followed by directly coupled to the amino group of glyphosate. The L'ELISA used succinic anhydride to derivatize glyphosate, which mimics the epitopic attachment of glyphosate to thyroglobulin. L'ELISA recognized the derivatized glyphosate with a limit of detection (LOD) of 0.8 ng g−1 and sensitivity (IC50 value) of 0.018 μg g−1. The recovery values of the spiked soil samples with different concentrations of glyphosate were in the range of 87.4–97.2%. Good correlation was achieved between L'ELISA and conventional high-pressure liquid chromatography (HPLC) with fluorescence detection. This study demonstrated the utility and convenience of the sensitive, simple, practical and cost-effective L'ELISA method for glyphosate analysis in soil samples. Also, it is ideal for rapid screening of a large number of environmental samples.

A novel approach for rapidly and cost-effectively assessing toxicity of toxic metals in acidic water using an acidophilic iron-oxidizing biosensor

Contamination by heavy metals and metalloids is a serious environmental and health concern. Acidic wastewaters are often associated with toxic metals which may enter and spread into agricultural soils. Several biological assays have been developed to detect toxic metals; however, most of them can only detect toxic metals in a neutral pH, not in an acidic environment. In this study, an acidophilic iron-oxidizing bacterium (IOB) Strain Y10 was isolated, characterized, and used to detect toxic metals toxicity in acidic water at pH 2.5. The colorimetric acidophilic IOB biosensor was based on the inhibition of the iron oxidizing ability of Strain Y10, an acidophilic iron-oxidizing bacterium, by metals toxicity. Our results showed that Strain Y10 is acidophilic iron-oxidizing bacterium. Thiobacillus caldus medium (TCM) (pH 2.5) supplied with both S4O62− and glucose was the optimum growth medium for Strain Y10. The optimum temperature and pH for the growth of Strain Y10 was 45 °C and pH 2.5, respectively. Our study demonstrates that the color-based acidophilic IOB biosensor can be semi-quantitatively observed by eye or quantitatively measured by spectrometer to detect toxicity from multiple toxic metals at pH 2.5 within 45 min. Our study shows that monitoring toxic metals in acidic water is possible by using the acidophilic IOB biosensor. Our study thus provides a novel approach for rapid and cost-effective detection of toxic metals in acidic conditions that can otherwise compromise current methods of chemical analysis. This method also allows for increased efficiency when screening large numbers of environmental samples.

Donor/acceptor nanoparticle pair-based singlet oxygen channeling homogenous chemiluminescence immunoassay for quantitative determination of bisphenol A.

Bisphenol A (BPA) is widely used in consumer products such as plastic bottles and food containers. It has become a ubiquitous environmental contaminant and poses a serious risk to human health. A rapid, sensitive, and high-throughput method for detecting BPA is therefore desirable. Herein, a donor/acceptor nanoparticle pair-based singlet oxygen channeling chemiluminescence homogenous immunoassay is developed for the determination of BPA. The donor nanoparticles were modified with phthalocyanine as a photosensitizer and were then coated with streptavidin. The acceptor nanoparticles were doped with thioxene derivatives and Eu(III) as a chemiluminescence emitter and then coated with anti-BPA antibody. Under light irradiation, oxygen near the donor surface transforms to singlet oxygen (1O2), which migrates to the acceptor and reacts with it, generating luminescence. Because 1O2 has a very short lifetime, luminescence is generated only when the donor and acceptor are in close proximity. This occurs when they are brought together by the antigen/antibody and streptavidin/biotin reaction. Based on this singlet oxygen channeling mechanism, a competitive homogenous chemiluminescence immunoassay for BPA was developed on 384 microplates. The assay exhibited linear detection over the range 10-1000ng/mL and a limit of detection of 2.9ng/mL. The intra- and inter-assay precisions were both below 5.1%. The average recoveries of three spiked samples in tap and river water samples were in the range 95.5-121.0%, in agreement with values obtained using high-performance liquid chromatography. The homogeneous assay is rapid, low cost, sensitive, and allows high-throughput, so is well suited for screening large numbers of environmental samples. Graphical abstract Principle of the singlet oxygen channeling homogenous chemiluminescence competitive immunoassay based on nanoparticle pairs for determination of BPA.

Trace-Level Mercury Ion (Hg2+) Analysis in Aqueous Sample Based on Solid-Phase Extraction Followed by Microfluidic Immunoassay

Mercury is considered the most important heavy-metal pollutant, because of the likelihood of bioaccumulation and toxicity. Monitoring widespread ionic mercury (Hg(2+)) contamination requires high-throughput and cost-effective methods to screen large numbers of environmental samples. In this study, we developed a simple and sensitive analysis for Hg(2+) in environmental aqueous samples by combining a microfluidic immunoassay and solid-phase extraction (SPE). Using a microfluidic platform, an ultrasensitive Hg(2+) immunoassay, which yields results within only 10 min and with a lower detection limit (LOD) of 0.13 μg/L, was developed. To allow application of the developed immunoassay to actual environmental aqueous samples, we developed an ion-exchange resin (IER)-based SPE for selective Hg(2+) extraction from an ion mixture. When using optimized SPE conditions, followed by the microfluidic immunoassay, the LOD of the assay was 0.83 μg/L, which satisfied the guideline values for drinking water suggested by the United States Environmental Protection Agency (USEPA) (2 μg/L; total mercury), and the World Health Organisation (WHO) (6 μg/L; inorganic mercury). Actual water samples, including tap water, mineral water, and river water, which had been spiked with trace levels of Hg(2+), were well-analyzed by SPE, followed by microfluidic Hg(2+) immunoassay, and the results agreed with those obtained from reduction vaporizing-atomic adsorption spectroscopy.

Environmental assessment of estrogenic pollutants in Nam River of Korea using indirect competitive ELISA and E-screen assay

Endocrine disrupting chemicals (EDCs) in ecosystem have raised a serious public health concern due to their potential adverse effects of interfering endogenous endocrine systems of organisms, including human being. Nam River is a main stream in southern part of Korea and has been used as a source of drinking water and for a variety of water-based activities of the people in this region. For the present study, water samples were collected from 11 different locations of Nam River, including two wastewater treatment plants (WWTPs). The collected water samples were analyzed with indirect competitive enzymelinked immunosorbent assay (IC-ELISA) and E-screen assay for the quantitative measurement of estrogens and the evaluation of their estrogenic activities, respectively. IC-ELISA showed that significant levels of estrogens (as estrogen equivalent concentration (EEC)) were present in WWTPs influents (5.537 ng-EEC/L for Jinju WWTP and 16.817 ng-EEC/L for Gyeongsang national university WWTP, respectively), but not detected in other river water samples. E-screen assay also showed similar results of the high estrogenic activities in WWTP influents. Unlike IC-ELISA, however, the results of E-screen revealed the presence of some weak estrogenic activities in all of the environmental water samples other than WWTP. One of the possible reasons of the difference is the presence of some estrogenic pollutants which are structurally unrelated to estrogens. From this, it is considered that the IC-ELISA method is a quick and reliable assay for the 1st-trial screening of large number of environmental samples with a relatively low cost. The present study could be helpful for the assessment of estrogen pollutants in environmental samples.

Measures of phylogenetic differentiation provide robust and complementary insights into microbial communities

High-throughput sequencing techniques have made large-scale spatial and temporal surveys of microbial communities routine. Gaining insight into microbial diversity requires methods for effectively analyzing and visualizing these extensive data sets. Phylogenetic β-diversity measures address this challenge by allowing the relationship between large numbers of environmental samples to be explored using standard multivariate analysis techniques. Despite the success and widespread use of phylogenetic β-diversity measures, an extensive comparative analysis of these measures has not been performed. Here, we compare 39 measures of phylogenetic β diversity in order to establish the relative similarity of these measures along with key properties and performance characteristics. While many measures are highly correlated, those commonly used within microbial ecology were found to be distinct from those popular within classical ecology, and from the recently recommended Gower and Canberra measures. Many of the measures are surprisingly robust to different rootings of the gene tree, the choice of similarity threshold used to define operational taxonomic units, and the presence of outlying basal lineages. Measures differ considerably in their sensitivity to rare organisms, and the effectiveness of measures can vary substantially under alternative models of differentiation. Consequently, the depth of sequencing required to reveal underlying patterns of relationships between environmental samples depends on the selected measure. Our results demonstrate that using complementary measures of phylogenetic β diversity can further our understanding of how communities are phylogenetically differentiated. Open-source software implementing the phylogenetic β-diversity measures evaluated in this manuscript is available at http://kiwi.cs.dal.ca/Software/ExpressBetaDiversity.

Development of ERE/DRE-dual CALUX bioassays system for monitoring estrogen- and dioxin-like persistent organic pollutants

Estrogen- and dioxin-like responsive element mediated Chemically Activated LUciferine gene eXpression (ERE/DRE-dual CALUX) was utilized to develop dualbioassays for simultaneously determining estrogen and dioxin levels. The devised ERE/DRE-dual CALUX bioassay system, was applied to the analysis of 103 estrogen and dioxin-related isomers, PAHs, PCBs, and pesticides. The nonylphenol highly induced estrogen-like activity (9.95 times) while benzo(k)fluoranthene, chrysene (zonerefined), and 3,3′,4,4′,5-pentachlorobiphenyl, highly induced dioxin-like activity (32.23, 42.39, and 34.47 times) respectively as compared with the untreated control. To confirm the validity of the devised ERE/DRE-dual CALUX bioassay system, we compared this bioassay with high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) to assess five air samples taken in Korea. The correlation coefficient between the ERE/DRE-dual CALUX bioassay system and HRGC/HRMS method showed a good relationship (r2 = 0.92). This study indicates that ERE/DRE-dual CALUX is a simple, high-throughput, valid, and inexpensive bioassay method for screening large numbers of environmental samples.

Quantification of uranium-238 in environmental samples using gamma-ray spectrometry

A large number of environmental samples are routinely measured world-wide using gamma-ray spectrometry some of its assets being easy sample preparation and comprehensive data for many radionu-clides in one analysis. Although other techniques can be considered more suitable for analysing 238U in environmental samples, it is also routinely done by gamma-ray spectrometry. One mainly uses γ-ray emissions following the decay of the first daughter, 234Th, for determining the 238U activity. However, the low-energy gamma-rays at 63 keV and 92.5 keV are very difficult to quantify in a robust way due to high attenuation and interferences. This paper quantifies parameters affecting the possibility of making robust quantification of 238U via 234Th using gamma-ray spectrometry. It addresses the use of correct decay data, suitable detectors, optimised sample size, enhanced spectral amplification, correction for peak interferences and control of background.

Prediction of Soil Organic Properties in a Diverse Population of Ohio Soils Using Near Infrared Reflectance Spectroscopy Analysis

AbstractA Near Infrared Spectroscopy (NIRS) method was used to predict soil organic carbon and amino sugar concentrations, and β-Glucosidase and β-Glucosaminidase activity in 184 diverse soils of Ohio. The measured variable values were calibrated to NIR spectral data with partial least squares regression analysis. The multivariate models developed were validated using the full cross validation method and the test set method with a test set size of 50 samples. Statistical analysis of the spectral data was done using the multivariate analysis software Unscrambler 8.0 (CAMO Inc). The first differential transformation of the NIR region (1100-2498 nm) gave good results. The NIRS method predicted well the organic carbon (OC) and amino sugar concentrations in soil (R2 was 0.91 for OC and 0.90 for amino sugar). The enzyme activity values were also well predicted (R2 was 0.82 for soil β-glucosidase and β-glucosaminidase enzyme activity). Thus, the NIRS method has the potential to rapidly and accurately predict values of carbon-related variables, including the carbon enzymes β-Glucosidase and β-Glucosaminidase, in soils. The equipment needed is not expensive and the NIRS method can be used where very large numbers of environmental samples need to be rapidly analyzed. Indeed, the prediction equations can be constantly improved as more data points are entered into the correlations between laboratory measured values and NIRS values.

Molecular detection of Pseudomonas aeruginosa in recreational water

The aim of this study was the development of a new molecular assay for Pseudomonas aeruginosa identification in recreational water. The method includes bacterial cell concentration through membrane filtration, a short (6 h) culture-enrichment step, DNA extraction and its amplification through a Real-Time PCR assay. The performance of the molecular approach was evaluated on 44 samples of swimming pool water and compared with the reference method UNI EN ISO 16266:2008. Positivity rates of 6% and 74% in pool and inlet water, respectively, with the standard culture method, and of 23% and 74% with the molecular method were found. Statistical analysis indicated “substantial agreement” (Cohen's Kappa index: 0.6831) between the two approaches. RAPD typing of P. aeruginosa isolates showed identical fingerprint profiles, indicating their epidemiological correlation. The developed protocol showed very high specificity and a detection limit of 10 genomic units. This technique has the potential to screen large numbers of environmental samples, and could be proposed as part of a self-monitoring plan for recreational facilities, improving surveillance and early warning systems.

BIPES, a cost-effective high-throughput method for assessing microbial diversity

Pyrosequencing of 16S rRNA (16S) variable tags has become the most popular method for assessing microbial diversity, but the method remains costly for the evaluation of large numbers of environmental samples with high sequencing depths. We developed a barcoded Illumina paired-end (PE) sequencing (BIPES) method that sequences each 16S V6 tag from both ends on the Illumina HiSeq 2000, and the PE reads are then overlapped to obtain the V6 tag. The average accuracy of Illumina single-end (SE) reads was only 97.9%, which decreased from ∼99.9% at the start of the read to less than 85% at the end of the read; nevertheless, overlapping of the PE reads significantly increased the sequencing accuracy to 99.65% by verifying the 3' end of each SE in which the sequencing quality was degraded. After the removal of tags with two or more mismatches within the medial 40-70 bases of the reads and of tags with any primer errors, the overall base sequencing accuracy of the BIPES reads was further increased to 99.93%. The BIPES reads reflected the amounts of the various tags in the initial template, but long tags and high GC tags were underestimated. The BIPES method yields 20-50 times more 16S V6 tags than does pyrosequencing in a single-flow cell run, and each of the BIPES reads costs less than 1/40 of a pyrosequencing read. As a laborsaving and cost-effective method, BIPES can be routinely used to analyze the microbial ecology of both environmental and human microbiomes.

NESTED AUTOMATED RIBOSOMAL INTERGENIC SPACER ANALYSIS: A RAPID AND ACCURATE METHOD FOR COMPARISON OF BACTERIAL COMMUNITY COMPOSITION

ABSTRACT Nested automated ribosomal intergenic spacer analysis (ARISA) was used to examine the community structure of epilithic biofilms in freshwater streams experiencing different levels of human impact. This molecular fingerprinting technique generated reproducible profiles of bacterial community structure that varied significantly between stream sites. Nested ARISA was determined to be a cost‐effective, high‐throughput approach to assess bacterial community composition from very small sample volumes, requiring little sampling effort and without the need for taxonomic identification of individual organisms. In combination with multidimensional scaling, nested ARISA provides a rapid and sensitive method to carry out complex analyses of bacterial community structure. PRACTICAL APPLICATIONSNested automated ribosomal intergenic spacer analysis (ARISA) provides a high‐throughput molecular method with which to screen large numbers of environmental samples for differences in microbial community structure. This sensitive approach benefits assessments from small sample volumes or environments exhibiting reduced microbial biomass (both aquatic and terrestrial). Differences in bacterial community structure (obtained from ARISA profiles) could be used to characterize the impact of anthropogenic disturbance on freshwater systems, analogous to the current use of macroinvertebrate indicators of freshwater ecological health.

Development of a Genotyping Method for Potato Scab Pathogens Based on Multiplex PCR

Scab disease significantly damages potato and other root crops. Streptomyces scabiei, S. acidiscabiei, and S. turgidiscabiei are the best-known causal agents of this disease. We have developed a novel genotyping method for these potato scab pathogens using multiplex PCR, whose benefits include rapid and easy detection of multiple species. We designed a species-specific primer set (6 primers, 3 pairs) for the 16S rRNA genes and 16S-23S ITS regions of these potato scab pathogens. The specificity of the primer set was confirmed by testing 18 strains containing potato scab pathogens, other Streptomyces species, and strains of other genera. The application of the developed method to potato field soil and potato tissue samples resulted in the clear detection and identification of pathogens. Since this method is applicable to a large number of environmental samples, it is expected to be useful for a high-throughput analysis of soil and plant tissues of scab disease.

Estrogenicity profile and estrogenic compounds determined in river sediments by chemical analysis, ELISA and yeast assays

An effects-directed strategy was applied to bed sediments of a polluted tributary in order to isolate and identify the major estrogenic chemicals it discharges into the River Po, the principal Italian watercourse. Sediment extract was concentrated by solid phase extraction and then fractioned into 10 fractions by reversed phase high performance liquid chromatography (RP-HPLC). Estrogenic activity of whole extract and fractions were determined using a recombinant yeast assay containing the human estrogen receptor (YES). The 10 fractions and whole extract were analysed for target compounds, e.g. estrone (E1), 17β-estradiol (E2), estriol (E3), 4-nonylphenol (NP), 4- tert-octylphenol ( t-OP), bisphenol A (BPA), using both liquid chromatography–tandem mass spectrometry (LC–MS/MS) and non-competitive enzyme-linked immunosorbent assays (ELISA). The YES assay determined high estrogenic activity in whole sediment (15.6 ng/g EE2 equivalents), and positive results for fractions nr 1, 2, 6, 7 and 8. E1, E3 and NP were the main estrogenic chemicals, however, other unidentified compounds contributed to sediment estrogenicity, particularly for polar fractions nr 1 and 2. A GC–MS screening performed in scan mode identified other potential contributors such as phthalates (DBP, BBP), and OP isomers. A next sampling campaign extended to other tributaries and receiving stretches of the River Po confirmed E1, E3 and NP as major estrogenic chemicals potentially threatening other sites of the main river. In general, target compound ELISAs have been shown to be suitable tools for a rapid screening of wide areas or large numbers of environmental samples for estrogenic risk. The potential for interferences suggests however to use cautiously the concentration values obtained from some of the immunoassays.

Completely automated short-term genotoxicity testing for the assessment of chemicals and characterisation of contaminated soils and waste waters

The umu-test was developed for the detection of effects of chemical mutagens and carcinogens in environmental samples. It is performed according to ISO 13829 with Salmonella choleraesius subsp. chol. (strain TA1535/pSK1002). By automating the entire test, large numbers of toxicants and environmental samples as well as more treatments and parallels can be tested and, additionally, only low sample volumes are needed. In this work, an automated umu-test has been set up by installing a robotic XYZ-platform and a microplate reader inside a cabin. The use of established technical equipment for the automation in combination with a performance according to ISO standards was the essential aim of the approach. After initial preparation, the test is conducted software-controlled, follows the standard and fulfils the validity criteria of the standard procedure. For the optimization of the automated test umu-tests with one concentration of methyl methanesulfonate (MMS) of 166.7 mg/L were carried out. After optimization of incubation and pipetting conditions in the automated test, dose-response curves of various chemicals and environmental samples were assessed. The results of the automated umu-test have been compared with those of the standard manual test. The aim of the study was to show the applicability of an automated test system for the assessment of the genotoxic effects of various chemicals and environmental samples. During optimization, tests with 166.7 mg/L of MMS in every well of the microplate are carried out. Chemicals with different physical, chemical and toxicological properties are applied in both test systems. Water samples from different waste water treatment plants, and water extracts of contaminated and uncontaminated soils are assessed in the umu-test. The test is performed in parallel manually according to the standard and automatically using the robotic platform. Dose-response relationships and DLI-values are recorded and compared. The umu-test is applied on a RoboSeq 4204 SE pipetting station (MWG AG, Ebersberg, Germany). The robot is equipped with four holders for disposable tips to avoid undesired mixing of liquids while testing. With this system, it is possible to pipette all liquids. Photometric measurements are performed using a microplate reader. The pipetting station and the photometer are placed in an incubation cabin. According to the standards, exposure and growth of the bacteria are performed at 37 degrees C and the enzyme activity is assessed at 28 degrees C. Since both temperatures can't be adjusted simultaneously in the cabin, the test is performed in deviation from the standard at 33 degrees C. The results show that both the testing of non-volatile substances with strong or moderate genotoxic effects and the testing of water and soil samples in the automatic system work very well. Nevertheless, it is still difficult to characterize volatile chemicals automatically. This is illustrated, e.g. by testing 2- +/- -hydroxyquinoline. In this case, the chemical would not be assessed genotoxically after automatic performance of the umu-test. Sealing of the microplates, for example, avoids the loss of volatile substances, but this step of the procedure can not be performed automatically. Discussion. Only very few studies deal with the automation of bioassays. Eisentraeger et al. (2004) showed the suitability of a miniaturized and automated algae test for the testing of large numbers of environmental samples. Genotoxicity with an automated liquid handling are introduced by White et al. (1996) and Janz et al. (1989). A complete automation including liquid handling, incubation and photometric measurement is a new approach and leads to satisfying results. Optimization and suitability of the automated test are demonstrated in this study. Induction rates and growth factors do not differ significantly if the incubation temperature and the pipetting mode are optimized. Due to flexible scripting, the newly developed automated test system can also be used to perform other genotoxicity tests. Most results clearly show that genotoxicity tests can be automated completely allowing rapid testing that can be performed over night, for instance. Nevertheless, the test performance has to be optimized step by step depending on the technical characteristics of the automat. In order to overcome these technical problems, detailed knowledge of the hardware and the software of the automat and of the respective genotoxicity test system are needed. During this process of automation, it is very useful to test one genotoxic substance with an identical concentration in every well. The automated test system based on the RoboSeq 4204 SE pipetting station (MWG AG, Ebersberg, Germany) still has to be optimized with respect to the testing of volatile compounds. There is a need for removable, gas-tight microplate covers. For non-volatile chemicals and environmental samples, it can be used routinely. Nevertheless, the experiences made during this study can only partly be transferred to other robotic platforms and other bioassays, and automation of bioassays still can be a time-consuming matter.

DRE-CALUX bioassay in comparison with HRGC/MS for measurement of toxic equivalence in environmental samples

CALUX, Chemically Activated LUciferase gene eXpression bioassay, has proven valuable for screening for and assessing toxic equivalents of dioxin-like compounds, because it detects all AhR (arylhydrocarbon receptor) ligands in a variety of sample matrices. In this study, we tried to validate DRE (dioxin-response elements)-CALUX bioassay, which has been developed by cloning mouse cyp1a1 gene in front of luciferase reporter gene. We compared DRE-CALUX bioassay with high resolution gas chromatography/mass spectrometry (HRGC/MS) for assessing environmental samples from Korea. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dose response study showed high correlation (r2=0.89) between DRE-CALUX bioassay and EROD (ethoxyresorufin O-deethylase) bioassay, a commonly used bioassay method. The mean TEQ value of water samples was 0.57 pg-TEQ(HRGC/MS)/L and 4.97 pg-TEQ(CALUX)/L. For soil samples, HRGC/MS-TEQ values ranged from 0 to 47.18 pg-TEQ/g (dry) and correlated well (r2=0.98) with values obtained by CALUX-TEQ which ranged from 0.92 to 649.97 pg-TEQ/g (dry). The difference between the absolute TEQ values might be due to the presence of dioxin-like compounds without WHO-TEQ values rather than the difference between CALUX-REP and WHO-TEQ. Based on this study, we suggest that DRE-CALUX bioassay can serve as an alternative bioassay method for high-throughput analysis of large number of environmental samples.