Improvement of an efficient separation method for chemicals in diesel exhaust particles: analysis for nitrophenols

GOAL, SCOPE, AND BACKGROUND: Diesel exhaust is believed to consist of thousands of organic constituents and is a major cause of urban pollution. We recently reported that a systematic separation procedure involving successive solvent extractions, followed by repeated column chromatography, resulted in the isolation of vasodilatory active nitrophenols. These findings indicated that the estimation of the amount of nitrophenols in the environment is important to evaluate their effect on human health. The isolation procedure, however, involved successive solvent extractions followed by tedious, repeated chromatography, resulting in poor fractionation and in a significant loss of accuracy and reliability. Therefore, it was crucial to develop an alternative, efficient, and reliable analytical method. Here, we describe a facile and efficient acid-base extraction procedure for the analysis of nitrophenols. MATERIALS AND METHODS: Diesel exhaust particles (DEP) were collected from the exhaust of a 4JB1-type engine (ISUZU Automobile Co., Tokyo, Japan). Gas chromatography-mass spectrometry (GC-MS) analysis was performed with a GCMS-QP2010 instrument (Shimadzu, Kyoto, Japan). RESULTS: A solution of DEP in 1-butanol was extracted with aqueous NaOH to afford a nitrophenol-rich oily extract. The resulting oil was methylated with trimethylsilyldiazomethane and subsequently subjected to GC-MS analysis, revealing that 4-nitrophenol, 3-methyl-4-nitrophenol, 2-methyl-4-nitrophenol, and 4-nitro-3-phenylphenol were present in significantly higher concentrations than those reported previously. DISCUSSION: Simple acid-base extraction followed by the direct analysis of the resulting extract by GC-MS gave only broad peaks of nitrophenols with a poor detection limit, while the GC-MS analysis of the sample pretreated with (trimethylsilyl)diazomethane gave satisfactorily clear chromatograms with sharp peaks and with a significantly lowered detection limit (0.5 ng/ml, approximately 100 times). CONCLUSION: The present method involving an acid-base extraction, in situ derivatization, and GC-MS analysis has shown to be a simple, efficient, and reliable method for the isolation and identification of the chemical substances in DEP.     

Naphthalene metabolism in Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic microorganism

The thermophilic bacterium Nocardia otitidiscaviarum strain TSH1, originally isolated in our laboratory from a petroindustrial wastewater contaminated soil in Iran, grows at 50 degrees C on a broad range of hydrocarbons. Transformation of naphthalene by strain TSH1 which is able to use this two ring-polycyclic aromatic hydrocarbon (PAH) as a sole source of carbon and energy was investigated. The metabolic pathway was elucidated by identifying metabolites, biotransformation studies and monitoring enzyme activities in cell-free extracts. The identification of metabolites suggests that strain TSH1 initiates its attack on naphthalene by dioxygenation at its C-1 and C-2 positions to give 1,2-dihydro-1,2-dihydroxynaphthalene. The intermediate 2-hydroxycinnamic acid, characteristic of the meta-cleavage of the resulting diol was identified in the acidic extract. Apart from typical metabolites of naphthalene degradation known from mesophiles, benzoic acid was identified as an intermediate for the naphthalene pathway of this Nocardia strain. Neither phthalic acid nor salicylic acid metabolites were detected in culture extracts. Enzymatic experiments with cell extract showed the catechol 1,2-dioxygenase activity while transformation of phthalic acid and protocatechuic acid was not observed. The results of enzyme activity assays and identification of benzoic acid in culture extract provide strong indications that further degradation goes through benzoate and beta-ketoadipate pathway. Our results indicate that naphthalene degradation by thermophilic N. otitidiscaviarum strain TSH1 differs from the known pathways found for the thermophilic Bacillus thermoleovorans Hamburg 2 and mesophilic bacteria.     

Modulation of steroidogenesis by coastal waters and sewage effluents of Hong Kong, China, using the H295R assay

BACKGROUND, AIM, AND SCOPE: The presence of a variety of pollutants in the aquatic environment that can potentially interfere with the production of sex steroid hormones in wildlife and humans has been of increasing concern. The aim of the present study was to investigate the effects of extracts from Hong Kong marine waters, and influents and effluents from wastewater treatment plants on steroidogenesis using the H295R cell bioassay. After exposing H295R cells to extracts of water, the expression of four steroidogenic genes and the production of three steroid hormones were measured. MATERIALS AND METHODS: Water samples were collected during the summer of 2005 from 24 coastal marine areas and from the influents and effluents of two major waste water treatment plants (WWTPs) in Hong Kong, China. Samples were extracted by solid phase extraction (SPE). H295R cells were exposed for 48 h to dilutions of these extracts. Modulations of the expression of the steroidogenic genes CYP19, CYP17, 3betaHSD2, and CYP11beta2 were determined by measuring mRNA concentrations by real-time polymerase chain reaction (Q-RT-PCR). Production of the hormones progesterone (P), estradiol (E2), and testosterone (T) was quantified using enzyme linked immunosorbent assays (ELISA). RESULTS: Extracts from samples collected in two fish culture areas inhibited growth and proliferation of H295R cells at concentrations greater or equal to 10(5) L equivalents. The cells were exposed to the equivalent concentration of active substances in 10,000 L of water. Thus, to observe the same level of effect as observed in vitro on aquatic organisms would require a bioaccumulation factor of this same magnitude. None of the other 22 marine samples affected growth of the cells at any dilution tested. Twelve of the marine water samples completely inhibited the expression of CYP19 without affecting E2 production; inhibition of CYP17 expression was observed only in one of the samples while expression of CYP11beta2 was induced as much as five- and ninefold after exposure of cells to extracts from two locations. The expression of the progesterone gene 3betaHSD2 was not affected by any of the samples; only one sample induced approximately fourfold the production of E2. Although more than twofold inductions were observed for P and T production, none of these values were statistically significant to conclude effects on the production of these two hormones. While influents from WWTPs did not affect gene expression, an approximately 30% inhibition in the production of E2 and a 40% increase in P occurred for the exposure with influents from the Sha Tin and Stonecutters WWTPs, respectively. Effluents from WWTPs did not affect the production of any of the studied hormones, but a decrement in the expression of the aldosterone gene CYP11beta2 was observed for the Sha Tin WWTP exposure. No direct correlation could be established between gene expression and hormone production. DISCUSSION: Observed cytotoxicity in the two samples from fish culture areas suggest the presence of toxic compounds; chemical analysis is required for their full identification. Although effluents from WWTPs did not affect hormone production, other types of endocrine activity such as receptor-mediated effects cannot be ruled out. Interactions due to the complexity of the samples and alternative steroidogenic pathways might explain the lack of correlation between gene expression and hormone production results. CONCLUSIONS: Changes observed in gene expression and hormone production suggest the presence in Hong Kong coastal waters of pollutants with endocrine disruption potential and others of significant toxic effects. The aromatase and aldosterone genes seem to be the most affected by the exposures, while E2 and P are the hormones with more significant changes observed. Results also suggest effectiveness in the removing of compounds with endocrine activity by the WWTPs studied, as effluent samples did not significantly affect hormone production. The H295R cell showed to be a valuable toll in the battery required for the analysis of endocrine disrupting activities of complex environmental samples. RECOMMENDATIONS AND PERSPECTIVES: Due to the intrinsic complexity of environmental samples, a combination of analytical tools is required to realistically assess environmental conditions, especially in aquatic systems. In the evaluation of endocrine disrupting activities, the H295R cell bioassay should be used in combination with other genomic, biological, chemical, and hydrological tests to establish viable modes for endocrine disruption and identify compounds responsible for the observed effects.     

Effects of pig manure compost and nonionic-surfactant Tween 80 on phenanthrene and pyrene removal from soil vegetated with Agropyron elongatum

This paper evaluates the effects of pig manure compost (PMC) and Tween 80 on the removal of phenanthrene (PHE) and pyrene (PYR) from soil cultivated with Agropyron elongatum. Soils spiked with about 300mgkg(-1) of PHE and PYR were individually amended with 0%, 2.5%, 5% and 7.5% (dry wt) of PMC or 0, 20 and 100mgkg(-1) of Tween 80. Unplanted and sterile microcosms were prepared as the controls. PAH concentration, total organic matter (TOM), dissolved organic carbon (DOC), total heterotrophic and PAH degrading microbial populations in soil were quantified before and after 60d period. The results indicated that A. elongatum could significantly enhance PYR removal (from 46% to 61%) but had less impact on PHE removal (from 96% to 97%). Plant uptake of the PAHs was insignificant. Biodegradation was the key mechanism of PAH removals (<3% losses in the sterile control). Increase in PMC or Tween 80 levels increased the removal of PYR but not of PHE. Maximal PYR removal of 79% and 92% were observed in vegetated soil receiving 100mgkg(-1) Tween 80 and 7.5% PMC, respectively. Enhanced PYR removal in soil receiving PMC could be explained by the elevated levels of DOC, TOM and microbial populations as suggested by Pearson correlation test. While the positive effect of Tween 80 on PYR removal could probably due to its capacities to enhance PYR bioavailability in soil. This paper suggests that the addition of either PMC or nonionic-surfactant Tween 80 could facilitate phytoremediation of PAH contaminated soil.     

The zebrafish embryo model in environmental risk assessment—applications beyond acute toxicity testing

BACKGROUND, AIM, AND SCOPE: The use of fish embryos is not regulated by current legislations on animal welfare and is therefore considered as a refinement, if not replacement of animal experiments. Fish embryos represent an attractive model for environmental risk assessment of chemicals since they offer the possibility to perform small-scale, high-throughput analyses. MAIN FEATURES: Beyond their application for determining the acute toxicity, fish embryos are also excellent models for studies aimed at the understanding of toxic mechanisms and the indication of possible adverse and long-term effects. Therefore, we have reviewed the scientific literature in order to indicate alternative applications of the fish embryo model with focus on embryos of the zebrafish. RESULTS AND DISCUSSIONS: The analysis of the mode of action is important for the risk assessment of environmental chemicals and can assist in indicating adverse and long-term effects. Toxicogenomics present a promising approach to unravel the potential mechanisms. Therefore, we present examples of the use of zebrafish embryos to study the effect of chemicals on gene and protein patterns, and the potential implications of differential expression for toxicity. The possible application of other methods, such as kinase arrays or metabolomic profiling, is also highlighted. Furthermore, we show examples of toxicokinetic studies (bioconcentration, ABC transporters) and discuss limitations that might be caused by the potential barrier function of the chorion. Finally, we demonstrate that biomarkers of endocrine disruption, immune modulation, genotoxicity or chronic toxicity could be used as indicators or predictors of sub-acute and long-term effects. CONCLUSIONS: The zebrafish embryo represents a model with an impressive range of possible applications in environmental sciences. Particularly, the adaptation of molecular, system-wide approaches from biomedical research is likely to extend its use in ecotoxicology. RECOMMENDATIONS AND PERSPECTIVES: Challenges for future research are (1) the identification of further suitable molecular markers as indicators of the mode of action, (2) the establishment of strong links between (molecular) effects in short-term assays in embryos and long-term (toxic) effects on individuals, (3) the definition of limitations of the model and (4) the development of tests that can be used for regulatory purposes.     

The lanthanum precipitation method. Part 1: a new method for technetium(IV) speciation in humic rich natural groundwater

A new and quick method for direct speciation of Tc(IV) in humic rich solutions, based on the induced aggregation of humic substances in the presence of the trivalent cation La(3+), is presented. This method (the "La-precipitation method") allows flocculating all the humic substances and also the Tc(IV) associated with humic substances. The method is tested on solutions containing Tc(IV) and Gorleben humic substances. The influence of different parameters (humic substance concentration, Tc concentration, reaction time and pH) is investigated on the observed free Tc(IV) concentration after precipitation of all humic substances. None of these parameters had a (significant) influence on the observed Tc(IV) concentration in solution after addition of La(3+) to Tc(IV)-HS containing solutions. It is therefore proposed that the method can be used to separate the Tc(IV) bound to humic substances from the free inorganic Tc species in solution.     

Heavy metal accumulation in wheat plant grown in soil amended with industrial sludge

The concentrations of different forms of Zn, Cu, Mn, Ni, Cd, Cr, Pb and Fe metals were determined for the roadside sludge collected from pickling-rolling and electroplating industrial area. In sludge the relative abundance of total heavy metals were Fe>Mn>Cr>Ni>Cu>Pb>Zn>Cd and DTPA-extractable metals were in the order--Fe>Ni>Mn>Cr>Cu>Zn>Pb>Cd. Pot-culture experiment was conducted in soils amended with sludge (0%, 10%, 20%, 30%), pretreated with lime (0%, 0.5% and 1%). The soils were alkaline in nature (pH>8.3) with organic carbon contents were 0.34% and 0.72%. The most abundant total and bio-available metal was Fe. Two wheat seedlings were grown in each pot containing 3kg sludge-amended or control soil and the experiment was conducted till harvesting. Application of sludge increased both total and bio-available forms of metals in the soils, while lime application decreased the bioavailability of heavy metals in sludge-amended soils. The content of organic carbon showed positive correlation with all metals except Zn, Cr and Pb. CEC also showed a strong positive correlation (R2>0.7) with Fe, Mn, Cu, Ni and Cd. Though wheat plants are not accumulators, the translocation efficiency was appreciably high. The translocation factor from shoot to grain was found smaller than that of root to shoot of wheat plants. This makes an implication that the heavy metal accumulation was proportionally lesser in grain than in shoot. In, 10% sludge with 0.5% lime-amended soils; each of these toxic heavy metals was found to be within permissible range (USEPA). Hence, on the basis of present study, the best possible treatment may be recommended.     

Role of blue green algae biofertilizer in ameliorating the nitrogen demand and fly-ash stress to the growth and yield of rice (Oryza sativa L.) plants

Rice is a major food crop throughout the world; however, accumulation of toxic metals and metalloids in grains in contaminated environments is a matter of growing concern. Field experiments were conducted to analyze the growth performance, elemental composition (Fe, Si, Zn, Mn, Cu, Ni, Cd and As) and yield of the rice plants (Oryza sativa L. cv. Saryu-52) grown under different doses of fly-ash (FA; applied @ 10 and 100 tha(-1) denoted as FA(10) and FA(100), respectively) mixed with garden soil (GS) in combination with nitrogen fertilizer (NF; applied @ 90 and 120 kg ha(-1) denoted as NF(90) and NF(120), respectively) and blue green algae biofertilizer (BGA; applied @ 12.5 kg ha(-1) denoted as BGA(12.5)). Significant enhancement of growth was observed in the plants growing on amended soils as compared to GS and best response was obtained in amendment of FA(10)+NF(90)+BGA(12.5). Accumulation of Si, Fe, Zn and Mn was higher than Cu, Cd, Ni and As. Arsenic accumulation was detected only in FA(100) and its amendments. Inoculation of BGA(12.5) caused slight reduction in Cd, Ni and As content of plants as compared to NF(120) amendment. The high levels of stress inducible non-protein thiols (NP-SH) and cysteine in FA(100) were decreased by application of NF and BGA indicating stress amelioration. Study suggests integrated use of FA, BGA and NF for improved growth, yield and mineral composition of the rice plants besides reducing the high demand of nitrogen fertilizers.     

Identification of key parameters controlling dissolved oxygen migration and attenuation in fractured crystalline rocks

In the crystalline rocks of the Canadian Shield, geochemical conditions are currently reducing at depths of 500-1000 m. However, during future glacial periods, altered hydrologic conditions could potentially result in enhanced recharge of glacial melt water containing a relatively high concentration of dissolved oxygen (O2). It is therefore of interest to investigate the physical and geochemical processes, including naturally-occurring redox reactions, that may control O2 ingress. In this study, the reactive transport code MIN3P is used in combination with 2k factorial analyses to identify the most important parameters controlling oxygen migration and attenuation in fractured crystalline rocks. Scenarios considered are based on simplified conceptual models that include a single vertical fracture, or a fracture zone, contained within a rock matrix that extends from the ground surface to a depth of 500 m. Consistent with field observations, Fe(II)-bearing minerals are present in the fractures (i.e. chlorite) and the rock matrix (biotite and small quantities of pyrite). For the parameter ranges investigated, results indicate that for the single fracture case, the most influential factors controlling dissolved O2 ingress are flow velocity in the fracture, fracture aperture, and the biotite reaction rate in the rock matrix. The most important parameters for the fracture zone simulations are flow velocity in the individual fractures, pO2 in the recharge water, biotite reaction rate, and to a lesser degree the abundance and reactivity of chlorite in the fracture zone, and the fracture zone width. These parameters should therefore receive increased consideration during site characterization, and in the formulation of site-specific models intended to predict O2 behavior in crystalline rocks.     

Effect of vegetation type on throughfall deposition and seepage flux

This paper compares different vegetation types (coniferous and deciduous forest, grassed and pure heathland) in terms of input (throughfall deposition) and output (seepage flux) in a region with intermediate nitrogen load (+/-20kg Nha(-1)y(-1) via bulk precipitation) in comparable conditions in north Belgium. Coniferous forest (two plots Pinus sylvestris and two plots Pinus nigra) received significantly higher nitrogen and sulphur throughfall deposition than deciduous forest and heathland. Grassed and pure heathland had significantly highest throughfall quantities of Ca(2+) and Mg(2+), respectively. The observed differences in throughfall deposition between the different vegetation types were not univocally reflected in the ion seepage flux. Considerable seepage fluxes of NO(3)(-), SO(4)(2-), Ca(2+) and Al(III) were only found under the P. nigra plots. We discuss our hypothesis that the P. nigra forests already evolved to a situation of N saturation, while the other vegetation types did not.