A sensitive, rapid ferricyanide-mediated toxicity bioassay developed using Escherichia coli

A need for rapid toxicity techniques has seen recent research into developing new microbiological assays and characterising their toxicity responses using a range of substances. A microbiological bioassay that determines changes in ferricyanide-mediated respiration for toxicity measurement (FM-TOX) shows particular promise. The development and optimisation of an improved FM-TOX method, incorporating novel features, is described using Escherichia coli as the biocatalyst. Omission of an exogenous carbon source, used in previously described FM-TOX assays, substantially improves the assay sensitivity. In addition, the development of a two-step procedure (toxicant exposure followed by determination of microbial respiratory activity) was found to enhance the inhibition of E. coli by 3,5-dichlorophenol and four other toxicants, compared to single-step procedures. Other assay parameters, such as the ferricyanide concentration, exposure times and optical density of the biocatalyst were also optimised, sometimes based on practical aspects. Toxicity tests were carried out using the adopted technique on both organic and inorganic toxicants, with classic sigmoid-shaped dose-response curves observed, as well as some non-standard responses. IC₅₀ data is presented for a number of common toxicants. The optimised assay provides a good foundation for further toxicity testing using E. coli, as well as the potential for expanding the technique to utilise other bacteria with complementary toxicity responses, thereby allowing use of the assay in a range of applications.     

Toxicity assessment of chlorophenols using a mediated microbial toxicity assay

While direct toxicity assessment (DTA) is now widely recognised as a useful tool for environmental risk assessment, many existing tests fail to meet end-user needs. This article describes the significant progress made to the MICREDOX® DTA assay, developed at Lincoln Ventures Ltd, brought about by miniaturising this assay to a multi-well plate format combined with limiting current microelectrode transduction. The benefits have been reduced: preparation time, reduced assay time, lower material costs and a higher level of replication achieved. To validate the precision of the miniaturised format, the concentrations required to cause a 50% decrease in signal (EC₅₀) by an archetypal group of toxicants, the chlorophenols, were determined using two terrestrial bacterial strains, Escherichia coli K12 and Klebsiella oxytoca 13183. The assay time was then reduced by stepwise adjustment of the incubation time, from 60 down to 5 min, and the EC₅₀s reported by E. coli to each of the toxicants after 45, 30, 15 and 5 min incubations were determined. The results obtained match closely with those reported by the Activated Sludge Respiration Inhibition Test and confirm the miniaturised multi-well plate MICREDOX® DTA assay reliably reports representative EC₅₀ values for these toxicants. The previously described trends of increasing toxicity with increasing chlorine substitution and the observation that meta-substituted chlorophenols are more toxic than their ortho-substituted counterparts are also confirmed. The ability to monitor toxicity using terrestrial organisms, in volumes amenable to multi-well microtitre plates and incubations requiring only a few minutes, facilitates the rapid generation of highly reproducible, easy to operate and inexpensive DTA measurements.     

A baseline inhalation toxicity model for narcosis in mammals

This paper presents the results of an analysis of the rodent inhalation literature and the development of a quantitative structure–activity relationships (QSAR) model for 4-hour LC50 as baseline toxicity to complement the baseline toxicity model for aquatic animals. We used the same literature review criteria developed for the ECOTOX database which selects only primary references with explicit experimental methods to form a high-quality database. Our literature review focused on the primary references reporting a 4-hour exposure for a single species of rodent in which the chemical had been clearly tested as a vapour and for which the exposure concentrations were not ambiguous. An expert system was used to remove reactive chemicals, receptor-mediated toxicants, and any test that produced symptoms inconsistent with non-polar narcosis. The QSAR model derived for narcosis in rodents was log LC50 = 0.69 × log VP + 1.54 which had an r ² of 0.91, which is significantly better than the baseline toxicity model for aquatic animals. This simple model suggests that there is no intrinsic barrier to estimating baseline toxicity for in vivo endpoints in mammalian or terrestrial toxicology.     

A novel approach to generate robust classification models to predict developmental toxicity from imbalanced datasets

Computational models to predict the developmental toxicity of compounds are built on imbalanced datasets wherein the toxicants outnumber the non-toxicants. Consequently, the results are biased towards the majority class (toxicants). To overcome this problem and to obtain sensitive but also accurate classifiers, we followed an integrated approach wherein (i) Synthetic Minority Over Sampling (SMOTE) is used for re-sampling, (ii) genetic algorithm (GA) is used for variable selection and (iii) support vector machines (SVM) is used for model development. The best model, M3, has (i) sensitivity (SE) = 85.54% and specificity (SP) = 85.62% in leave-one-out validation, (ii) classification accuracy of the training set = 99.67%, (iii) classification accuracy of the test set = 92.59%; and (iv) sensitivity = 92.68, specificity = 92.31 on the test set. Consensus prediction based on models M3–M5 improved these percentages by 5% over M3. From the analysis of results we infer that data imbalance in toxicity studies can be effectively addressed by the application of re-sampling techniques.     

入境废物原料浸出液毒性试验方法探讨

在分析入境危险废物浸出毒性试验方法的基础上,针对入境废物原料浸出液毒性试验方法,按照我国对危险废物浸出毒性试验方法基本要求内容,对确定的14种浸出物质试验方法进行了探讨,并将该法有效运用于对入境可用作原料的工业品废物环控指标普查的工作之中。     

Development of a mediated whole cell-based electrochemical biosensor for joint toxicity assessment of multi-pollutants using a mixed microbial consortium

Since most risk assessment for toxicants is based on individual single-species test, the deduction of such results to ecosystem evaluation is afflicted with uncertainties. Herein, we successfully developed a p-benzoquinone mediated whole-cell electrochemical biosensor for multi-pollutants toxicological analysis by co-immobilizing mixed strains of microorganism, including Escherichia coli (gram-negative bacteria), Bacillus subtilis (gram-positive bacteria) and Saccharomyces cerevisiae (fungus). The individual and combined toxicities of heavy metal ions (Cu²⁺, Cd²⁺), phenol (3,5-dichlorophenol) and pesticides (Ametryn, Acephate) were examined. The experimental results showed that the order of toxicity for individual toxicant was ranked as Cu²⁺ > 3,5-dichlorophenol (DCP) > Ametryn > Cd²⁺ > Acephate. Then the toxic unit (TU) model was applied to determine the nature of toxicological interaction of the toxicants which can be classified as concentration additive (IC50_mix = 1TU), synergistic (IC50_mix < 1TU) and antagonistic (IC50_mix > 1TU) responses. The binary combination of Cu²⁺ + Cd²⁺, Cu²⁺ + DCP, Cu²⁺ + Acephate, DCP + Acephate, Acephate + Ametryn were analyzed and the three kind of joint toxicity effects (i.e. additive, synergistic and antagonistic) mentioned above were observed according to the dose-response relationship. The results indicate that the whole-cell electrochemical biosensor based on mixed microbial consortium is more reasonable to reflect the joint biotoxicity of multi-pollutants existing in real wastewater, and combined effects of toxicants is extremely necessary to be taken into account in ecological risk assessment. Thus, present study has provided a promising approach to the quality assessment of wastewater and a reliable way for early risk warning of acute biotoxicity.     

Fireflies in the coalmine: luciferase technologies in next-generation toxicity testing

Whole-animal studies have been the mainstay of toxicity testing for decades. These approaches are too expensive and laborious to effectively characterize all of the chemicals currently in commercial use. In addition, there are social and ethical pressures to reduce, refine and replace animal testing in toxicology. The National Research Council (NRC) has outlined a new strategy to transition from animal-based tests to high throughput, cell-based assays and computational modeling approaches to characterize chemical toxicants. Critical to this vision, assays that measure toxicity pathways associated with adverse health effects must be developed. Bioluminescent assays are particularly well suited to the demands of next-generation toxicity testing because they measure a wide range of biological activities in a quantitative and high throughput manner. This review describes the limitations of traditional, animal-based toxicity testing and discusses the current and developing uses of bioluminescent technologies in next-generation testing based on three general assay formats: luciferase-limited assays, ATP-limited assays and luciferin-limited assays.     

Comparative proteome profiling of bleomycin‐induced lung toxicity in rats by 2D–nano‐LC–MS/MS and spectral counting

The mechanisms involved in bleomycin‐induced lung toxicity have not been fully understood to date. This work aimed to compare the proteome profiling of bleomycin‐induced lung toxicity by using 2D–nano‐LC–MS/MS and spectral counting. By comparing the spectral counts of identified proteins between control and bleomycin‐treated groups, we noted that 102 proteins were upregulated and 28 proteins were downregulated in the bleomycin‐treated group. Among these differently expressed proteins, five proteins were chosen for validation by Western blot analysis. The levels of these five proteins were consistent with proteomic results. These potential mediators can facilitate the translation of the underlying mechanisms of bleomycin‐induced lung toxicity to molecular targets in the clinical arena.     

SMILES as an alternative to the graph in QSAR modelling of bee toxicity

Simplified Molecular Input Line Entry System (SMILES) nomenclature has been used as elucidating the molecular structure in construction of the quantitative structure-activity relationships (QSAR) for predicting bee toxicity. On the basis of the symbols used in the SMILES notation numerical parameters have been obtained, which are simple and fast to calculate. The method has been used to develop a QSAR model to predict toxicity of pesticides on bees. Results on a heterogeneous set of pesticides are good. Statistical characteristics of this model are: n=85, R2=0.68, s=0.82, F=180 (training set); n=20, R2=0.72, s=0.68, F=46 (test set).     

Discriminating toxicant classes by mode of action: 3. Substructure indicators§

Decision support for selecting suitable QSARs for predictive purposes is suggested by a stepwise procedure: The first tier pre-filters the compounds based on substructure indicators for baseline versus excess toxicity. This step, if sufficiently conservative, discriminates chemicals, whose toxicity can be reliably estimated from their log?K _OW from those, that require further classification by biological and chemical domain. A test set of 115 chemicals from 9 different MOA classes was used to compare the discriminatory power of several classification schemes based on substructure indicators. Performance, evaluated by contingency table statistics, is varied and no single scheme provides sufficient applicability and reliability for pre-filtering chemical inventories. Major improvements are feasible with combined use of three classification schemes: assignments of baseline toxicants are protective, recognition of excess toxicants is acceptable and applicability range increases favourably.     

Effects of gamma-ray treatment on wastewater toxicity from a rubber products factory

In order to reduce the toxicity of both raw wastewater and effluent from a rubber products factory, γ-ray treatment was applied at different dose levels. The γ-ray treatment did not completely removed the toxicity, suggesting that there were major toxicants other than destroyable organic compounds. Toxicity identification evaluation (TIE phase 1) was conducted to characterize major toxicants using Daphnia magna. The suspected toxicants in both raw wastewater and effluent were mostly filterable materials and EDTA chelatable metals and, to some degree, non-polar organic compounds. Anion-exchange removable compounds, most likely organics, were found only in raw wastewater. Metal analyses showed that zinc and copper concentrations were above levels causing toxicity to D. magna. After 20 kGy γ-ray treatment of raw wastewater, filtrations both at pH 3 and at the initial pH (pH 3.6) showed dramatic change (9 to 77% and 29 to 85%, respectively) in toxicity reduction, suggesting the formation of toxic filterable materials which are stable even at acidic conditions. Unlike raw wastewater, there was no significant change in TIE results after γ-ray treatment at 20 kGy for rubber effluent.     

Development and applications of whole cell biosensors for ecotoxicity testing

Whole cell biosensors are the focus of considerable and increasing interest worldwide as methods for detecting and quantifying environmental toxicity, including biochemical oxygen demand (BOD), heavy metals, antibiotics, pesticides and herbicides. This review follows the development of whole cell biosensors from attempts to utilise changes in cellular metabolism to determine BOD and general toxicity, through the exploitation of unique metabolic pathways to detect specific toxicants, to the increasingly widespread use of genetic engineering to build new, and modify existing, sensing pathways.     

Toxicity Assessment of Cyanide and Tetramethylene Disulfotetramine （Tetramine） Using Luminescent Bacteria Vibrio-qinghaiensis and PbO= Electrochemical Sensor

The toxicities of cyanide and tetramethylene disulfotetramine （tetramine） were evaluated by two methods of luminescent bacteria and PbO2 electrochemical sensor. Vibrio-qinghaiensis, a kind of luminescent bacteria, can produce bioluminescence and the bioluminescence was decreased with the addition of toxicants. The toxicities of cyanide and tetrarnine were expressed as 10 min-EC50 value, which was the concentration of chemical that reduces the light output by 50% after contact for 10 min. Nano PbO2 modified electrode, a rapid toxicity determination method was also described in this work. By the PbO2 modified electrode, the current responses of Escherichia coli （E. coli） were changed with the addition of toxicants. The value of 10 min-EC50 was also provided with the PbO2 electrochemical sensor. Compared with the 10 min-EC50 and detection limits （38.38 and 0.60 μg/mL for cyanide, 0.24 and 0.02 μg/mL for tetramine） with luminescent bacteria, the PbO2 sensor provided a simple and convenient method with lower 10 min-EC50 and detection limits （26.37 and 0.52 μg/mL for cyanide, 0.21 and 0.01 μg/mL for tetramine） and fast response time.     

Laboratory systems to evaluate the toxicity of polymers combustion products and hazard assessment

Several laboratory systems have been proposed to evaluate the toxicity of combustion products of materials. Generally, the “fire models” were poorly instrumented, excepted the analysis of the major toxicants. Better informations being needed in this respect, a furnace has been made, based on the 2 zones NBS model, and allowing the evaluation of flammability, heat release and weight loss. Moreover, opacity and toxic gases are monitored. The time to incapacitation is probably the more realistic toxicological evaluation. It is worth while to rank materials by an hazard assessment, separating the variables involved in the fire hazard and the variables corresponding to the toxicological hazards and to plot them in a two-dimensional graphic which looses less information than an index.     

Radiation processing of wastewater evaluated by toxicity assays

Biological assays have been applied to industrial effluents and sewage influents, from distinct sites, before and after being submitted to ionizing radiation treatment. The objective of this study was to evaluate the efficiency of radiation, mainly electron beam accelerator, for the acute toxicity removal. The selected sampling presented a very toxic level and the radiation process was efficient for toxicity removal for 87.7% of irradiated samples. The sewage influents required lower radiation doses to reduce toxicity when compared to raw industrial effluents.     

A new microbial assay for the toxicity detection of contaminated soils

A comparative study to detect toxicity prior to bioremediation treatment was set in order to investigate dehydrogenase activity inhibition of a common soil bacterium caused by soil contaminated with Cu, Pb, and As. A spectrophotometric test with Pseudomonas fluorescens strain ATCC 13525 utilising the 2,3,5-triphenyl tetrazolium chloride (TTC) reduction by microbial dehydrogenase has been adapted for this purpose. Soil samples are incubated for 48 hours at 30 +/- 1 degrees C in 18-ml tubes in the presence of TTC as an artificial electron-acceptor. The reduced TTC forms a reddish colour substance named triphenyl formazan (TPF), which can be extracted from the microbial cells and measured colorimetrically. The rapid response of biological activity in microorganisms and the reported sensitivity to the toxicants in the contaminated samples are reflected by the TTC reduction method, which is a sensitive tool for toxicity screening of contaminated sites, routine monitoring of bioremediation processes, as well as for feasibility studies of bioremediation treatments, in order to assess whether a specific pollutant or any other substance at a site location could inhibit the microbiological processes.     

被动采样技术在测定沉积物中有机污染物的生物可利用性和毒性中的研究进展

综述了被动采样技术作为仿生萃取方法在测定沉积物中有机污染物的生物可利用性和毒性中的应用。对比了半渗透膜装置、聚乙烯膜装置、聚甲醛树脂萃取和固相微萃取这些常见被动采样技术在使用过程中的优缺点,并针对被动采样技术应用中的问题,提出可能的解决手段和研究需求。     

Controlled‐Potential‐Based Electrochemical Sulfide Sensors: A Review

Due to their potential applications in industry and potent toxicity to the environment, sulfides and their detection have attracted the attention of researchers. To date, a large number of controlled‐potential techniques for electrochemical sulfide sensors have been developed, thanks to their simplicity, reasonable limit of detection (LOD), and good selectivity. Different researchers have applied different strategies for developing selective and sensitive sulfide sensors. However, there has been no systematic review on controlled‐potential techniques for sulfide sensing. In light of this absence, the main aim of this review article is to summarize various strategies for detecting sulfide in different media. The efficiencies of the developed sulfide sensors for detecting sulfide in its various forms are determined, and the essential parameters, including sensing strategies, working electrodes, detection media, pH, LOD, sensitivity, and linear detection range, are emphasized in particular. Future research in this area is also recommended. It is expected that this review will act as a basis for further research on the fabrication of sulfide sensors for practical applications.