Comparative evaluation of a bioluminescent bacterial assay in terrestrial ecotoxicity testing

Despite the widespread and successful use of luminescence-based bioassays in water testing, their applications to soils and sediments is less proven. In part this is because such bioassays have mainly been carried out in an aqueous-based medium and, as such, favour contaminants that are readily water-soluble. In this study, aqueous solutions and soils contaminated with heavy metals (HM), polar organic contaminants and hydrophobic organic contaminants (HOCs) were tested using a range of luminescence-based bioassays (Vibrio fischeri, Escherichia coli HB101 pUCD607 and Pseudomonas fluorescens 10586r pUCD607). For the first two chemical groups, the assays were highly reproducible when optimised extraction procedures were employed but for HOCs the bioassay response was poor. Quantitative structure-activity relationships (QSARs) obtained from aqueous solutions had a linear response although correlation for the chemicals tested using bacterial bioassays was significantly less sensitive than that of sublethal tests for Tetrahymena pyriformis. Bacterial and Dendrobaena veneta bioassay responses to extracts from HM amended soils showed that a clear relationship between trophic levels could be obtained. There is no doubt that the wide range of bioluminescent-based bioassays offers complementary applications to traditional testing techniques but there is a significant need to justify and optimise the extraction protocol prior to application.     

Comparison of response of six different luminescent bacterial bioassays to bioremediation of five contrasting oils

The performance of six different bioluminescent bacteria for the assessment of oil bioremediation was compared. Three contained lux genes linked to promoters from hydrocarbon degradation pathways: Pseudomonas fluorescens HK44 (pUTK21), Escherichia coli HMS174 (pOS25) and E. coli DH5 alpha (pGEc74, pJAMA7), responding to naphthalene, isopropylbenzene and octane, respectively. The other three expressed lux constitutively: E. coli HB101 (pUCD607) and P. putida F1 (pUCD607) are genetically engineered, while Vibrio fischeri is naturally bioluminescent and was included to facilitate comparison with previous work. Five different oils (four crude oils plus diesel) were spiked into soil, and the progress of remediation was followed over a period of 119 d by monitoring both hydrocarbon disappearance and changes in the microbial response to soil extracts. The octane bioassay was the only one of the hydrocarbon-responsive bacterial assays to show any appreciable response, with up to 20-fold induction by light crude oils. Heavy crude oil and diesel elicited a much weaker response. The metabolic (lux constitutively expressed) bioassays showed that there was a general increase in toxicity over the course of the experiment, although toxicity to E. coli HB101 (pUCD607) appeared to be decreasing by the final sampling point. The metabolic bioassay response was much less variable between the different oils than for the first three, catabolic, strains.     

Whole-cell bacterial biosensors for rapid and effective monitoring of heavy metals and inorganic pollutants in wastewater

The increasing number of potentially harmful pollutants in the wastewater effluent discharge necessitates the need for the development of fast and cost effective analytical techniques for extensive monitoring programmes to assess the effectiveness of the treatment process. This study compared the use of bacterial biosensors to the conventional Daphnia magna assay, Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) tests as well as chemical analysis, for monitoring the toxicity of wastewater. The bacterial biosensors constructed in this study, using S. sonnei and E. coli, were found to be sensitive to the toxicity of the wastewater effluents. A linear increase in bioluminescence with increasing concentration of heavy metals and inorganic pollutants in water was observed, with a correlation coefficient (r(2)) as high as 0.995 and 0.997, respectively. No notable correlation between biosensor toxicity and BOD and COD test results was observed. These bacterial biosensors could provide appropriate alternatives for a rapid, sensitive and cost effective detection of wastewater quality. However, the differences in sensitivity obtained for the different systems suggest that the use of a battery of toxicity assays may be required to provide a real ecotoxicological assessment of wastewater samples.     

Soil solution extraction techniques for microbial ecotoxicity testing: a comparative evaluation

The suitability of two different techniques (centrifugation and Rhizon sampler) for obtaining the interstitial pore water of soil (soil solution), integral to the ecotoxicity assessment of metal contaminated soil, were investigated by combining chemical analyses and a luminescence-based microbial biosensor. Two different techniques, centrifugation and Rhizon sampler, were used to extract the soil solution from Insch (a loamy sand) and Boyndie (a sandy loam) soils, which had been amended with different concentrations of Zn and Cd. The concentrations of dissolved organic carbon (DOC), major anions (F- , CI-, NO3, SO4(2-)) and major cations (K+, Mg2+, Ca2+) in the soil solutions varied depending on the extraction technique used. Overall, the concentrations of Zn and Cd were significantly higher in the soil solution extracted using the centrifugation technique compared with that extracted using the Rhizon sampler technique. Furthermore, the differences observed between the two extraction techniques depended on the type of soil from which the solution was being extracted. The luminescence-based biosensor Escherichia coli HB101 pUCD607 was shown to respond to the free metal concentrations in the soil solutions and showed that different toxicities were associated with each soil, depending on the technique used to extract the soil solution. This study highlights the need to characterise the type of extraction technique used to obtain the soil solution for ecotoxicity testing in order that a representative ecotoxicity assessment can be carried out.     

Combined toxicity of three chlorophenols 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol to Daphnia magna

The toxicity of single and combined mixtures of 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP) to Daphnia magna was studied. The toxicity ranking of these three single chlorophenols (CPs) to Daphnia magna was PCP > 2,4-DCP > 2,4,6-TCP. The toxic units (TU) approach was used to estimate the combined effects in experiments, the median effective concentration (EC(50)) values were 0.87-1.21 and 0.46-0.59 for binary and ternary mixtures, respectively. Response surface models of General Linear Models (R(2) > 0.90, residual deviation < 3.25) were established for all three binary mixtures. The toxicity for ternary mixtures based on the EC(50)-value and 10% effective concentration (EC(10))-value fixed mixture ratio presented a synergism. The risk based on the single CP's toxicity test may be underestimated. In addition, four approaches (concentration addition, toxicity equivalency factors, effect summation, and independent action) were used for the calculation of combined effects of the mixture. The experimental results showed that concentration addition and toxicity equivalency factor approaches were effective methods for calculation of additive effects of mixtures from binary systems of CPs; while independent action and effect summation (low simulated tail) predicted lower toxicity than experimental results. Limitations of the traditional focus on the effects of single agents were highlighted; hazard assessments ignoring the possibility of joint action of CPs will almost certainly lead to significant underestimations of risk.     

中国沿海三省主要饮用水源有机物监测

采用固相萃取、吹扫捕集和气相色谱-质谱联用仪(GC-MS)对江苏、浙江、山东省的21个主要地表饮用水源、126份水样中的25种VOCs、38种SVOCs进行定量检测。结果有19种VOCs、10种SVOCs至少在一个水源地多次被检出,其中挥发性氯代烃、苯系物、氯代苯类、酚类、硝基苯类、酞酸酯类、农药为中国或美国环境保护局提出的"水中优先控制污染物",最高检出量为27.79μg/L。三省主要地表水受到有机物轻度污染,水体中VOCs、SVOCs含量尚未超过《地表水环境质量标准》(GB 3838-2002)和《生活饮用水卫生标准》(GB 5749-2006)限值,工业废水排放及农田雨水径流是地表水有机物的重要来源。     

Ecotoxicological and microbiological characterization of soils from heavy-metal- and hydrocarbon-contaminated sites

The aims of this study were to characterize soils from industrial sites by combining physicochemical, microbiological, and ecotoxicological parameters and to assess the suitability of these assays for evaluation of contaminated sites and ecological risk assessment. The soil samples were taken from long-term contaminated sites containing high amounts of heavy metals (sites 1 and 2) or petroleum hydrocarbons (site 3) located in the upper Silesia Industrial Region in southern Poland. Due to soil heterogeneity, large differences between all investigated parameters were measured. Microbiological properties revealed the presence of high numbers of viable hetrotrophic microorganisms. Soil enzyme activities were considerably reduced or could not be detected in contaminated soils. Activities involved in N turnover (N mineralization and nitrification) were significantly (P?<?0.05) higher in samples from the metal-contaminated sites than in samples from the hydrocarbon-contaminated site, whereas the opposite was observed for phosphatase activity. The Microtox test system appeared to be the most appropriate to detect toxicity and significant differences in toxicity between the three sites. The Ostracodtoxkit test was the most appropriate test system to detect toxicity in the hydrocarbon-contaminated soil samples. Correlation analysis between principal components (obtained from factor analysis) determined for physicochemical, microbiological, and ecotoxicological soil properties demonstrated the impact of total and water-extractable contents of heavy metals on toxicity.     

Heat shock protein 47 stress responses in Chinese hamster ovary cells exposed to raw and reclaimed wastewater

As wastewater reclamation and reuse becomes more widespread, risks of exposure to treated wastewater increase. Moreover, an unlimited number of pollutants can be identified in wastewater. Therefore, comprehensive toxicity assessment of treated wastewater is imperative. The objective of this study was to perform a comprehensive toxicity assessment of wastewater treatment systems using stress response bioassays. This powerful tool can comprehensively assess the toxicity of contaminants. In this study, samples from conventional activated sludge treatment, membrane bioreactors (MBRs) with different pore sizes and sludge retention times (SRTs), rapid sand filtration, coagulation, nano-filtration (NF) and reverse osmosis (RO) were investigated. The results of stress response bioassays confirmed that the secondary effluent showed higher stress response than influent indicating that biological treatment generates toxic compounds. The results obtained from molecular weight fractionation of water samples demonstrated that organic matter with a higher molecular weight fraction (>0.1 μm) causes toxicity in secondary effluent. Furthermore, supernatant from MBR reactors showed toxicity regardless of SRT. On the other hand, stress response was not detected in MBR permeates except for an MBR equipped with a larger pore size membrane (0.4 μm) and with a short SRT (12 days). While rapid sand filtration could not remove the toxic compounds found in secondary effluent, coagulation tests, operated at an appropriate pH, were effective for reducing stress response in the secondary effluent. Experimental findings also showed that stress response was not detected in cases of NF and RO permeate subsequent to MBR treatment.     

Validation of plant based bioassays for the toxicity testing of Indian waters

Plant-based bioassays have recently gained remarkable popularity among the toxicological/eco-toxicological assessment procedures. The reasons for their wide use are comparative simplicity, sensitivity, and cost-effectiveness as well as a good correlation with other toxicity tests. The present study describes the use of two plant bioassays, Allium cepa test and seed germination test in the evaluation of the toxicity/genotoxicity of industrial waste water and river water and standardization with the commonly occurring pollutants in Indian waters namely heavy metals and phenolics. Both tests were standardized to suit the Indian conditions, and the local varieties were used. Both bioassays responded significantly with the test range of heavy metals and phenolics. The toxicity of heavy metals was in the order of Cu > Ni > Cd in both the tests whereas 2,4-dinitrophenol was the most toxic among the phenolic compounds. Cabbage, millet, and cucumber, respectively, were found to be the most sensitive in the seed germination test for the test heavy metals and phenols. Significant amounts of chromosomal abnormalities including bridges, stickiness, and fragmentations were recorded with both the industrial waste water and the XAD concentrated river water samples by A. cepa test.     

Toxicity assessment for petroleum-contaminated soil using terrestrial invertebrates and plant bioassays

The assessment of soil quality after a chemical or oil spill and/or remediation effort may be measured by evaluating the toxicity of soil organisms. To enhance our understanding of the soil quality resulting from laboratory and oil field spill remediation, we assessed toxicity levels by using earthworms and springtails testing and plant growth experiments. Total petroleum hydrocarbons (TPH)-contaminated soil samples were collected from an oilfield in Sfax, Tunisia. Two types of bioassays were performed. The first assessed the toxicity of spiked crude oil (API gravity 32) in Organization for Economic Co-operation and Development artificial soil. The second evaluated the habitat function through the avoidance responses of earthworms and springtails and the ability of Avena sativa to grow in TPH-contaminated soils diluted with farmland soil. The EC₅₀ of petroleum-contaminated soil for earthworms was 644 mg of TPH/kg of soil at 14 days, with 67 % of the earthworms dying after 14 days when the TPH content reached 1,000 mg/kg. The average germination rate, calculated 8 days after sowing, varied between 64 and 74 % in low contaminated soils and less than 50 % in highly contaminated soils.     

Synergistic effects of metal nanoparticles and a phenolic uncoupler using microdroplet-based two-dimensional approach

A droplet-based microfluidic technique for testing multiple reagent concentrations is presented. We used this experimental approach to study combined effects of gold (AuNP) and silver nanoparticles (AgNP) with the phenolic uncoupler 2,4-dinitrophenol (DNP) with respect to the growth of Escherichia coli. In order to evaluate the toxicity of binary mixtures, we first encapsulated the E. coli cells and particle mixtures inside the microdroplets using PEEK (polyetherketone) modules. Two-dimensional concentration spaces with about 500 well separated droplets were addressed. We subsequently analyzed the cell growth, the viability and the autofluorescence intensity (metabolic activity) of the bacteria with a micro-flow-through fluorometer and photometer. Dose-dependent synergistic effects were found for the binary mixture of AgNPs and DNP, which indicated a stronger interaction in the mixture than it was expected from effect summation. For the binary mixture of DNP and AuNPs in non-toxic concentrations, we found only weak synergistic effects at low DNP concentrations. Furthermore, the non-toxic tested AuNPs causes effect summation in the binary mixture with the phenolic uncoupler. In general, we demonstrated the efficiency of a droplet-based microfluidic system for fast high-throughput screenings of binary and multiple mixtures. This work also confirmed the relevance of highly resolved droplet-based assays for the miniaturization of ecotoxicological aquatic test systems.     

2,4,6-Trinitrotoluene detection using recombinant antibodies

The ability to detect low levels of 2,4,6-trinitrotoluene (TNT) in aqueous samples is important due to the toxicity of both TNT and its breakdown products. We have been characterizing recombinant anti-TNT antibodies isolated from the Griffin library of phage displayed scFvs by selection for binders to the TNT-surrogate 2,4,6-trinitrobenzene (TNB) coupled to the protein bovine serum albumin. Two candidate antibody fragments, TNB1 and TNB2, were isolated and evaluated by ELISA for their ability to bind to TNB coupled to the protein ovalbumin. Competition ELISA was then used to demonstrate antibody fragment binding to TNT in solution and to examine cross-reactivity towards several TNT-related compounds and other explosives. Both recombinant antibody fragments were incorporated into a continuous flow assay for the detection of TNT. TNB2, the best single chain antibody, showed a limit of detection of 1 ng ml(-1), comparable to a commercially available anti-TNT antibody in the same assay format.     

Bacterial biosensors for rapid and effective monitoring of biodegradation of organic pollutants in wastewater effluents

Significant amounts of toxic substances which are hazardous to animals, plants, microorganisms, and other living organisms including humans are released annually into aquatic and terrestrial environments, mostly from improper wastewater discharges. Early detection of such pollutants in wastewater effluents and proper monitoring before their final release into the environment is therefore necessary. In this study, two whole-cell bacterial biosensors were constructed by transforming competent cells of Shigella flexneri and Shigella sonnei with pLUX plasmids and evaluated for their potential to monitor wastewater samples undergoing degradation by measuring bioluminescence response using a microplate luminometer. Both bacterial biosensors were found to be extremely sensitive to the wastewater samples, with different patterns, concomitant with those of the COD removals demonstrated at the different days of the degradation. Generally higher bioluminescence values were obtained at the later days of the degradation period compared to the initial values, with up to 571.76% increase in bioluminescence value obtained at day 5 for 0.1% (v/v) effluent concentration. Also, a steady decrease in bioluminescence was observed for the bacterial biosensors with increasing time of exposure to the wastewater effluent for all the sampling days. These biosensor constructs could therefore be applicable to indicate the bioavailability of pollutants in a way that chemical analysis cannot, and for in situ monitoring of biodegradation. This has great potential to offer a risk assessment strategy in predicting the level of bioremediation required during municipal wastewater treatment before their final discharge into the aquatic milieu.     

Risk assessment of soils contaminated by mercury mining, Northern Spain

Analytical results of soil samples taken in three different mercury mining sites in Northern Spain are studied to assess the potential adverse health effects of the exposure to trace elements associated with the mining process. Doses contacted through ingestion and inhalation and the dose absorbed through the skin were calculated using USEPA's exposure parameters and the US Department of Energy's toxicity values. The results of the risk assessment indicate that the highest risk is associated with ingestion of soil particles and that the trace element of major concern is arsenic, the exposure to which results in a high cancer risk value for all the sites ranging from 3.3 × 10(-5) to 3.6 × 10(-3), well above the 1 × 10(-5) probability level deemed unacceptable by most regulatory agencies. Regarding non-cancer effects, exposure to polluted soils yields an aggregate hazard index above the threshold value of 1 for all three sites, with As and Hg as the main contributors. Risk assessment has proven to be a very useful tool to identify the contaminants and exposure pathways of most concern in the soils from metal mining sites, as well as to categorize them in terms of action priority to ensure fitness for use.     

In vitro bioassay for reactive toxicity towards proteins implemented for water quality monitoring

Reactive organic chemicals comprise a large number of compounds with a variety of reactive moieties. While most assays for reactive toxicity focus on DNA damage, reactivity towards proteins can also lead to irreparable damage, but reactivity towards proteins is typically not included in any test battery for water quality assessment. Glutathione (GSH) is a small tripeptide whose cysteine moiety can serve as a model for nucleophilic sites on proteins. GSH is also an important indicator of detoxification processes and the redox status of cells and due to its protective role, depletion of GSH ultimately leads to adverse effects. A bioassay based on genetically modified Escherichia coli strains was used to quantify the specific reactivity towards the protein-like biological nucelophile GSH. The significance of GSH for detoxification was assessed by comparing the growth inhibition induced by reference chemicals or water samples in a GSH-deficient strain to its fully functional parent strain. The GSH deficient strain showed the same sensitivity as the GSH proficient strain to non-reactive and DNA damaging chemicals, but was more sensitive to chemicals that attack cysteine in proteins. The difference in effect concentrations for 50% inhibition of growth assessed as biomass increase (EC(50)) between the two strains indicates the relevance of GSH conjugation as a detoxification step as well as direct reactivity with cysteine-containing proteins. Seven reference compounds serving as positive and negative controls were investigated. The E. coli strain that lacks GSH was four times more sensitive towards the positive control Sea-Nine, while negative controls benzo[a]pyrene, 2-aminoanthracene, phenol, t-butylhydroquinone, methyl methane sulfonate and 4-nitroquinoline oxide showed equal effect concentrations in both strains. Water samples collected across an indirect potable reuse scheme representing the complete water cycle from sewage to drinking water in South East Queensland, Australia were used to evaluate the applicability of the E. coli assay for reactive toxicity in water samples. While the EC(50) values of the GSH+ strain showed similar trends as in other biological endpoints over the various treatment chains, the specific response indicative of protein damage was only observed in samples that had undergone chlorination as a disinfection process. High natural organic matter or other matrix components disturbed the bioassay so much that we recommend it for future routine testing only in tertiary treated water or drinking water.     

TNT红水污染土壤中二硝基甲苯磺酸盐的分析方法

火炸药厂红水污染土壤中的主要污染物为二硝基甲苯磺酸盐[DNTS,包括2,4-二硝基甲苯-3-磺酸盐(2,4-DNT-3-SO_3~-)和2,4-二硝基甲苯-5-磺酸盐(2,4-DNT-5-SO_3~-)2种异构体]。研究建立了振荡提取-高效液相色谱法测定土壤中DNTS的方法。4种土壤(黄土、棕土、红壤和黑土)和5个浓度水平(50、100、250、500、1 000 mg/kg)时,DNTS的回收率为69.4%~111%,变异系数为0.28%~6.62%(n=3)。应用该方法测定甘肃某火炸药厂红水污染土壤样品,2,4-DNT-3-SO_3~-和2,4-DNT-5-SO_3~-浓度实测值(平均值分别为505、25.2 mg/kg)重复性好,变异系数为1.29%和1.53%(n=3)。     

Ecotoxicity monitoring of hydrocarbon-contaminated soil using earthworm (Eisenia foetida)

In order to assess the applicability of an earthworm bioassayas a technique for monitoring the soil flushing process, short-term and long-term toxicity tests were conducted on remediatedsoil using various pore volumes of surfactant solution. Resultsobtained on short-term toxicity testing indicated that biomassincreased as the soil flushing proceeded, and on diesel-contaminated soils this testing showed that the effect of dieselis lethal and that 25 pore volumes of soil surfactant were notsufficient to abrogate the toxic effect of diesel. These short-term tests also showed strong sublethal relationships between the development of biomass, and the concentrations of toxic chemicals in the soil. Although relationships between contaminants and the various bioassay parameters examined werenot significant in long-term testing, an increase in the numberof juveniles was observed over time, which may have been a consequence of a reduction in toxicity associated with the flushing process.     

烷基酚类化合物对斑马鱼胚胎的毒性效应

采用斑马鱼胚胎发育技术,观察邻甲酚、对甲酚、间甲酚和2,4-二叔丁基酚联合作用对斑马鱼胚胎的影响,以研究烷基酚类化合物的毒性.结果表明,4种受试物的联合对斑马鱼胚胎发育均有明显的抑制作用及不同程度的致死等效应.Ohpf染毒主要表现为:24 h后卵凝结和尾部无延展,眼点发育受抑制及无主动活动;48 h卵凝结和心率异常,无...     

Heavy metal contamination from mining sites in South Morocco: Monitoring metal content and toxicity of soil runoff and groundwater

The aim of the present work is the assessment of metal toxicity in runoff, in their contaminated soils and in the groundwater sampled from two mining areas in the region of Marrakech using a microbial bioassay MetPLATE™. This bioassay is based on the specific inhibition of the β-galactosidase enzyme of a mutant strain of Escherichia coli, by the metallic pollutants. The stream waters from all sampling stations in the two mines were all very toxic and displayed percent enzyme inhibition exceeding 87% except SWA₄ and SWB₁ stations in mine C. Their high concentrations of copper (Cu) and zinc (Zn) confirm the acute toxicity shown by MetPLATE. The pH of stream waters from mine B and C varied between 2.1 and 6.2 and was probably responsible for metal mobilization, suggesting a problem of acid mine drainage in these mining areas. The bioassay MetPLATE™ was also applied to mine tailings and to soils contaminated by the acidic waters. The results show that the high toxicity of these soils and tailings was mainly due to the relatively concentration of soluble Zn and Cu. The use of MetPLATE™ in groundwater toxicity testing shows that, most of the samples exhibited low metal toxicity (2.7–45.5% inhibition) except GW3 of the mine B (95.3% inhibition during the wet season and 82.9% inhibition during the dry season). This high toxicity is attributed to the higher than usual concentrations of Cu (189 μg Cu l⁻¹) and Zn (1505 μg Zn l⁻¹). These results show the potential risk of the contamination of different ecosystems situated to the vicinity of these two metalliferous sites. The general trend observed was an increase in metal toxicity measured by the MetPLATE with increasing total and mobile metal concentrations in the studied matrices. Therefore, the MetPLATE bioassay is a reliable and fast bioassay to estimate the metals toxicity in the aquatic and solids samples.