Bioconcentration of chlorinated anilines and benzene in fish: Preliminary results

• 1.1. Seven chlorinated anilines and one chlorinated benzene were tested for their ability to bioconcentrate in guppies (Poecilia reticulata) under different experimental conditions.
• 2.2. Interactions between compounds in a mixture influence the bioconcentration of some chlorinated anilines. These interactions result in either an increase or a decrease of bioconcentration, depending on the compound studied.
• 3.3. Exposure concentration can have an effect on the extent of bioconcentration of some chlorinated anilines.

     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway.

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

Degradation of aniline and monochloroanilines by Rhodococcus sp. An 117 and a pseudomonad: a comparative study

Two newly isolated aniline-degrading bacterial strains were characterized with regard to their enzyme systems responsible for aniline catabolism. One of them identified as a Rhodococcus sp. metabolized aniline exclusively via the beta-ketoadipate pathway by means of inducible enzymes. The aniline-degrading enzyme system of the second isolate, presumably a pseudomonad, was shown to consist of an inducible aniline-converting enzyme and constitutive meta-pathway enzymes. Both isolates failed to metabolize monochlorinated anilines in the absence of additional carbon sources. To explain this the ring-cleaving enzymes of both isolates were examined for their substrate specificities. Furthermore, the effect of 4-chlorocatechol on the enzymes catalyzing aniline conversion and catechol oxygenation was investigated.     

Bacterial bioconcentration of chlorinated hydrocarbon insecticides from aqueous systems

The prevalence of chlorinated hydrocarbon insecticide uptake by chemoorganotrophic bacteria has been investigated. Thirteen bacterial species were observed to sorb and concentrate (bioconcentratc)α-chlordane,β-chlordane, dieldrin, heptachlor epoxide, and lindane from aqueous systems. Bioconcentration, as expressed by the ratio of cellular insecticide in ng/mg (dry wt) to supernatant insecticide in ng/μl, ranged from 10 for lindane byEnterobacter aerogenes to a high of 55,900 forβ-chlordane byCaulobacter vibrioides var.limonus. Amounts of cellular chlorinated hydrocarbon insecticides (CHI) detected and the bioconcentration ratios were observed to have the following order in magnitude:α- orβ-chlordane > dieldrin > heptachlor epoxide > lindane. This decreasing order was the inverse of reported water solubilities for the CHI and the inverse relationship was mathematically defined. The CHI were not easily removed from cells by washing (desorbing) and desorption was directly proportional to insecticide water solubility. Uptake of the CHI was rapid, near-maximum amounts being sorbed within 15 min, and pH 7 appeared optimal for bioconcentration as examined over the range pH 6 to 8. Implications of this investigation are that bioconcentration of CHI by bacteria might serve as a means of introducing these toxic compounds into aquatic food chains and that the bioconcentration phenomenon might lend itself as a treatment procedure for the intentional removal of residual CHI from water supplies and wastewater.     

Construction and application of an Escherichia coli bioreporter for aniline and chloroaniline detection

Aniline and chlorinated anilines (CAs) are classified as priority pollutants; therefore, an effective method for detection and monitoring is required. In this study, a green-fluorescence protein-based bioreporter for the detection of aniline and CAs was constructed in Escherichia coli DH5α, characterized and tested with soil and wastewater. The sensing capability relied on the regulatory control between a two-component regulatory protein, TodS/TodT, and the P _todX promoter of Pseudomonas putida T-57 (PpT57), since the gene expression of todS, todT, and todC2 are positively induced with 4-chloroaniline. The bioreporter system (DH5α/pPXGFP–pTODST) is markedly unique with the two co-existing plasmids. The inducibility of the fluorescence response was culture-medium- and time-dependent. Cells grown in M9G medium exhibited a low background fluorescence level and were readily induced by 4CA after 3-h exposure, reaching the maximum induction level at 9?h. When tested with benzene, toluene, ethyl-benzene and xylene, aniline and CAs, the response data were best fit by a sigmoidal dose–response relationship, from which the K _1/2 value was determined for the positive effectors. 3CA and 4CA were relatively powerful inducers, while some poly-chlorinated anilines could also induce green fluorescence protein expression. The results indicated a broader recognition range of PpT57’sTodST than previously reported for P. putida. The test results with environmental samples were reliable, indicating the potential application of this bioreporter in the ecotoxicology assessment and bioremediation of areas contaminated with aniline- and/or CAs.     

Bioconcentration of atrazine,zinc and iron in the blood of Tilapia sparrmanii (cichlidae)

1. The bioconcentration of atrazine, zinc and iron in the blood of Tilapia sparrmanii has been determined separately in each of six exposure groups.2. An increased bioconcentration of atrazine in the blood occurred with an increased exposure concentration.3. With exposure to zinc, there was a gradual decrease in bioconcentration of zinc in the blood of T. sparrmanii with an increased concentration in the water.4. A similar tendency was observed during iron exposure, except that the decrease in bioconcentration was not significant.     

Interpretation of the reactivity of peroxidase compound II with phenols and anilines using the Marcus equation

The catalytic cycle of heme peroxidases involves three processes: the formation of compound I, its conversion to compound II and regeneration of the native enzyme. Each of the processes consists of a reversible binding stage followed by an irreversible transformation stage. Our group has proposed a continuous, sensitive and reliable chronometric method for measuring the steady-state rate of peroxidase activity. Furthermore, we have derived an analytical expression for the steady-state rate and simplified it, taking into consideration the experimental values of the rate constants of some stages previously determined by other authors in stopped-flow assays. We determined the value of the constant for the transformation of a series of phenols and anilines by compound II, and found that it involves a deprotonation step and an electron transfer step. Study of the solvent deuterium isotope effect on the oxidation of phenol revealed the non-rate-limiting character of the deprotonation step in a proton inventory study. Usage of the Marcus equation showed that the electronic transfer step is rate-limiting in both cases, while phenols and anilines were oxidised at different rates for the same potentials. This can be attributed to the shorter electron-tunnelling distance for electron transfer to the iron ion in the phenols than in the anilines.     

藻类对多环芳香烃(PAHs)的富集和代谢

概述了藻类对PAHs的富集和代谢的研究进展。环境中多环芳香烃(PAHs)的污染能导致严重的健康问题,利用生物特别是微生物去除污染环境中的PAHs是一项新的技术。藻类对PAHs的富集与有机污染物的类型、藻类的种类及藻类的生物量有关,活细胞和死细胞对PAHs均有富集能力。还阐述了PAHs在真菌、细菌和藻类体内代谢的途径以及代谢过程中起关键作用的酶,PAHs在藻类中的代谢途径和细菌及真菌都不同,谷胱甘肽转移酶(GST)在藻类代谢PAH过程中起重要作用,但细胞色素P450酶所起的作用则不详。     

Isolation of a glucosyltransferase from Arabidopsis thaliana active in the metabolism of the persistent pollutant 3,4-dichloroaniline

The pollutant 3,4-dichloroaniline (DCA) was rapidly detoxified by glucosylation in Arabidopsis thaliana root cultures, with the N-beta-d-glucopyranosyl-DCA exported into the medium. The N-glucosyltransferase (N-GT) responsible for this activity was purified from Arabidopsis suspension cultures and the resulting 50 kDa polypeptide analysed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) following tryptic digestion. The protein was identified as GT72B1. The GT was cloned and the purified recombinant enzyme shown to be highly active in conjugating DCA and 2,4,5-trichlorophenol, as well as several other chlorinated phenols and anilines, demonstrating both N-GT and O-GT activity. GT72B1 showed little activity towards natural products with the exception of the tyrosine catabolite 4-hydroxyphenylpyruvic acid. Both O-GT and N-GT activities were enhanced in both plants and cultures treated with herbicide safeners, demonstrating the chemical inducibility of this detoxification system in Arabidopsis.     

Conjugal transfer of plasmid pNB2 to activated sludge bacteria leads to 3-chloroaniline degradation in enrichment cultures

AIMS: The involvement of the aniline-degradative plasmid pNB2 in degradation of 3-chloroaniline (3-CA) was investigated. METHODS AND RESULTS: Plate matings of a Pseudomonas putida strain containing pNB2 with a mixed bacterial culture derived from activated sludge was carried out. After inoculation of the mating mixtures into batch cultures containing 3-CA, degradation of the compound was observed. A total of five different transconjugant strains could be isolated from one of the batch cultures and two of them were able to degrade 3-CA. These two isolates were identified as Comamonas testosteroni by partial 16S rDNA sequencing. CONCLUSIONS: It can be assumed that pNB2 carries a part of the genes involved in the catabolism of 3-CA, but that completion of the pathway must be provided by chromosomal genes in the host strain. SIGNIFICANCE AND IMPACT OF THE STUDY: pNB2 is a candidate plasmid which can be used in plasmid-mediated bioaugmentation of wastewater bacteria involved in degradation of chlorinated anilines.     

Biodegradability of chlorinated solvents and related chlorinated aliphatic compounds

The biodegradability of chlorinated methanes, chlorinated ethanes, chlorinated ethenes, chlorofluorocarbons (CFCs), chlorinated acetic acids, chlorinated propanoids and chlorinated butadienes was evaluated based on literature data. Evidence for the biodegradation of compounds in all of the compound categories evaluated has been reported. A broad range of chlorinated aliphatic structures are susceptible to biodegradation under a variety of physiological and redox conditions. Microbial biodegradation of a wide variety of chlorinated aliphatic compounds was shown to occur under five physiological conditions. However, any given physiological condition could only act upon a subset of the chlorinated compounds. Firstly, chlorinated compounds are used as an electron donor and carbon source under aerobic conditions. Secondly, chlorinated compounds are cometabolized under aerobic conditions while the microorganisms are growing (or otherwise already have grown) on another primary substrate. Thirdly, chlorinated compounds are also degraded under anaerobic conditions in which they are utilized as an electron donor and carbon source. Fourthly, chlorinated compounds can serve as an electron acceptor to support respiration of anaerobic microorganisms utilizing simple electron donating substrates. Lastly chlorinated compounds are subject to anaerobic cometabolism becoming biotransformed while the microorganisms grow on other primary substrate or electron acceptor. The literature survey demonstrates that, in many cases, chlorinated compounds are completely mineralised to benign end products. Additionally, biodegradation can occur rapidly. Growth rates exceeding 1 d^-1 were observed for many compounds. Most compound categories include chlorinated structures that are used to support microbial growth. Growth can be due to the use of the chlorinated compound as an electron donor or alternatively to the use of the chlorinated compound as an electron acceptor (halorespiration). Biodegradation linked to growth is important, since under such conditions, rates of degradation will increase as the microbial population (biocatalyst) increases. Combinations of redox conditions are favorable for the biodegradation of highly chlorinated structures that are recalcitrant to degradation under aerobic conditions. However, under anaerobic conditions, highly chlorinated structures are partially dehalogenated to lower chlorinated counterparts. The lower chlorinated compounds are subsequently more readily mineralized under aerobic conditions.     

Ames试验在水质检测方面的应用

近年来 ,水体污染日趋严重 ,各国学者从氯化后饮水中分离出多种致突变、致癌物质。为此我们采用Ames试验 ,对珠江流域主要取水点的水源水和对应自来水中的有机致突变物污染情况进行了研究。研究表明 ,部分取水点的有机致突变物污染较严重 ,并且氯化消毒后自来水的致突性大于水源水的致突性。因而 ,加强饮水中致突变物质的检测 ,改进净水消毒剂和净水流程很有必要。     

Toxicité aigüe et bioconcentration du lindane et de la deltaméthrine par les tetards de Rana temporaria et les gambusies (Gambusia affinis)

Acute toxicity and bioconcentration capacities of lindane and deltamethrin were studied in Rana temporaria tadpoles and in mosquitofish. These studies show that the toxicity of deltamethrin is about 100 to 1 000 times greater than that of lindane and bioconcentration factors are very different for these two insecticides. The bioconcentration factor of lindane, a stable chemical, low volatility, hydrophobic and a poorly metabolized molecule is considerable in either static or flow through contamination systems. For deltamethrin, an quickly metabolized molecule, this factor is weak or null. Moreover a comparison of various methods of contamination (static or flow through systems) showed that the experimental conditions of exposure to the insecticide strongly influence the concentration in the tested species.

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Toxicité aigüe et bioconcentration du lindane et de la deltaméthrine par les tetards de Rana temporaria et les gambusies (Gambusia affinis)

     

Reductive dechlorination pathways for substituted benzenes: a correlation with electronic properties

Electronic properties were correlated with observed reductive dechlorination pathways by unacclimated consortia for chlorinated phenols, dihydroxybenzenes, benzoic acids, and anilines. Molecular structures and properties were calculated using the semi-empirical Modified Neglect of Differential Overlap method at the Cornell Supercomputing Facility. Observed preferential positions for reductive dechlorination by unacclimated consortia correlate well with the largest negative value for the carbon-chlorine bond charge. Of 16 dechlorination pathways observed for unacclimated bacteria, the most negative carbon-chlorine bond charge correlated with 15 pathways.This correlation between the observed dechlorination position and the parent compound's electronic properties is consistent with the observed reductive dechlorination of chlorophenols and chlorinated dihydroxybenzenes at the ortho position, and the meta dechlorination of chlorobenzoic acids. Net carbonchlorine bond charges also correlate with the preferred dechlorination position for two of three known chloroaniline pathways, suggesting preferential removal of chlorines from the ortho position of chloroanilines.Abbreviations CA chloroaniline - CBz chlorobenzoic acid - CC chlorocatechol - CP chlorophenol - DCA dichloroaniline - DCBz dichlorobenzoic acid - DCC dichlorocatechol - DCH dichlorohydroquinone - DCP dichlorophenol - DCR dichlororesorcinol - PCP pentachlorophenol - TCA trichloroaniline - TCBz trichlorobenzoic acid - TCC trichlorocatechol - TCH trichlorohydroquinone - TCP trichlorophenol - TCR trichlororesorcinol - TeCA tetrachloroaniline - TeCBz tetrachlorobenzoic acid - TeCC tetrachlorocatechol - TeCH tetrachlorohydroquinone - TeCP tetrachlorophenol - TeCR tetrachlororesorcinol     

Kinetic and molecular orbital studies on the rate of oxidation of monosubstituted phenols and anilines by horseradish peroxidase compound II

The second-order rate constant (k4) for the oxidation of a series of aromatic donor molecules (monosubstituted phenols and anilines) by horseradish peroxidase (HRP) compound II was examined with a stopped-flow apparatus. The electronic states of these substrates were calculated by an ab initio molecular orbital method. It was found that in both phenols and anilines log k4 values correlate well with the highest occupied molecular orbital (HOMO) energy level and the lowest unoccupied molecular orbital (LUMO) energy level, but not with the net charge or frontier electron density on atoms of these molecules. The HOMO and LUMO energy levels of phenols and anilines further showed linear relationships with Hammett's sigma values with negative slopes. Similar results were obtained in the oxidation of substrates by HRP compound I, except that the rate of reaction was much higher than in the case of HRP compound II. In addition, the rates of oxidation of phenols by compound I or II were found to be about 1000 times higher than those of anilines with similar HOMO energy levels. On the basis of these results, the mechanism of electron transfer from the substrate to the heme iron of HRP compound II is discussed.     

Temperature effects on bioconcentration of DDE by Daphnia

1. Lake temperatures vary with season, latitude, elevation and as a result of thermal pollution. In addition, lake temperatures may increase with global warming. Radiotracer experiments were conducted to determine the effects of temperature on the bioaccumulation of lipophilic organic contaminants by zooplankton. Daphnia pulex were exposed to ¹⁴C-labelled DDE, a stable metabolite of the organochlorine pesticide DDT, in particle-free water for 24 h. An increase in temperature from 5 to 25 °C resulted in a 314% increase in bioconcentration factor (the ratio of contaminant concentration in the organism to contaminant concentration in the water).
2. To mimic the fluctuating temperatures experienced by zooplankton during diel vertical migration, we conducted experiments in which animals were exposed to 25 °C for 12 h in the light, then 15 °C for 12 h in the dark. Exposure to this fluctuating temperature regime for 48 h resulted in a 27–33% increase in bioconcentration factor relative to a constant 20 °C control.
3. Live animals accumulated more than twice the amount of DDE than freshly killed animals, indicating that the activity of the organism is important in bioconcentration. This finding sheds light on the possible mechanisms for the increase in bioconcentration at higher constant temperatures. Daphnia pump more water through their branchial chambers at higher temperatures. Thus, if the thoracic limbs are an important site of contaminant uptake, then animals are exposed to more contaminant molecules at higher temperatures. Other possible mechanisms include changes in the thickness of the diffusive boundary layer and changes in cell membrane permeability.     

Passive sampler for chlorinated pesticides in estuarine waters

ABSTRACT

Monitoring organic contaminants in the marine environment and identifying their bioconcentration pathways presents great difficulties, as the analytical techniques require preconcentration steps that are time-consuming and tedious. Samplers have been developed that can concentrate extremely low levels of hydrophobic compounds, such as organochlorine pesticides, from water bodies, for direct analysis with minimal sample preparation. The passive samplers consist of polyethylene tubes filled with iso-octane that are deployed within weighted wire cages in estuaries and rivers for a period of three weeks. Hydrophobic compounds such as chlorinated pesticides are continuously partitioned from the water column into the solvent within the tube, providing an integrated sample over the duration of exposure. Pesticides are concentrated 200–300 fold in three weeks. Upon retrieval, the contents can be analysed directly by gas chromatography—electron capture detector (GC—ECD) or concentrated for analysis by gas chromatography-mass spectrometry (GC—MS). Laboratory experiments to determine the uptake rate of the compounds into the sampler enable the calculation of average water concentration over the sampling period. The samplers are cheap and easy to prepare, enabling their deployment in numerous sites over long periods if required. They are envisaged as being a quick and easy monitor for background levels of these contaminants within our waterways.     

Temperature effects on bioconcentration of DDE by Daphnia

1. Lake temperatures vary with season, latitude, elevation and as a result of thermal pollution. In addition, lake temperatures may increase with global warming. Radiotracer experiments were conducted to determine the effects of temperature on the bioaccumulation of lipophilic organic contaminants by zooplankton. Daphnia pulex were exposed to ¹⁴C-labelled DDE, a stable metabolite of the organochlorine pesticide DDT, in particle-free water for 24 h. An increase in temperature from 5 to 25 °C resulted in a 314% increase in bioconcentration factor (the ratio of contaminant concentration in the organism to contaminant concentration in the water).
2. To mimic the fluctuating temperatures experienced by zooplankton during diel vertical migration, we conducted experiments in which animals were exposed to 25 °C for 12 h in the light, then 15 °C for 12 h in the dark. Exposure to this fluctuating temperature regime for 48 h resulted in a 27–33% increase in bioconcentration factor relative to a constant 20 °C control.
3. Live animals accumulated more than twice the amount of DDE than freshly killed animals, indicating that the activity of the organism is important in bioconcentration. This finding sheds light on the possible mechanisms for the increase in bioconcentration at higher constant temperatures. Daphnia pump more water through their branchial chambers at higher temperatures. Thus, if the thoracic limbs are an important site of contaminant uptake, then animals are exposed to more contaminant molecules at higher temperatures. Other possible mechanisms include changes in the thickness of the diffusive boundary layer and changes in cell membrane permeability.     

Growth conditions impact 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE) accumulation by Cucurbita pepo

Laboratory experiments were conducted to study the effects of soil moisture content, planting density, plant age, and the growth of multiple generations on the bioconcentration of weathered p,p'-DDE by the plant Cucurbita pepo. As soil moisture content increased from 7.4% to 29.9% (by weight), rates of contaminant accumulation by plant roots were increased by more than a factor of 2. Higher planting density also led to higher uptake, as the root bioconcentration factor (BCF, dry-weight ratio of contaminant concentration in the tissue to that in the soil) increased by 15-fold as the number of plants per pot was raised from 1 to 3. Concentrations of the compound in plant roots were inversely related to plant age, with root BCF declining by approximately a factor of 3 as plants aged from 14 to 28 d. Finally, no change in the bioavailability of the compound was observed in successive generations of plants grown in the same contaminated soil. The results suggest that phytoremediation is influenced by a number of factors and that the cleanup of contaminated soil can be enhanced by an understanding of environmental and other conditions affecting plant growth and bioconcentration.