Characterization of alkylphenol metabolites in fish bile by enzymatic treatment and HPLC-fluorescence analysis

Alkylphenol (AP) metabolites were characterized in the bile of Atlantic cod (Gadus morhua L.) after exposure to nine individual compounds (10mg/kg fish), 2-methylphenol (2-MP), 4-methylphenol (4-MP), 3,5-dimethylphenol (3,5-DMP), 2,4,6-trimethylphenol (2,4,6-TMP), 4-tert-butylphenol (4-t-BP), 4-tert-butyl-2-methylphenol (4-t-B-2-MP), 4-n-pentylphenol (4-n-PP), 4-n-hexylphenol (4-n-HexP) and 4-n-heptylphenol (4-n-HepP), and a mixture (total dose; 13.5 mg/kg fish) of the nine APs by inter-muscular injection. The degree of alkylation ranged from methyl (C1) to heptyl (C7) and represents the types of APs present in produced water. Fish bile was collected on day 4 and 16 (exposure groups 2-MP, 3,5-DMP, 2,4,6-TMP and 4-t-B-2-MP) following exposure. Characterization of major metabolites was accomplished by enzymatic de-conjugation and analysis by high performance liquid chromatography connected to a fluorescence detector (HPLC-F) acquiring at ex/em 222/306 nm. Two solid phase extraction (SPE) columns were evaluated for clean-up of samples prior to analysis. Independent of alkyl homologue, the glucuronide conjugated APs were the most abundant metabolites (73-100%), whereas sulfates, glucosides and unchanged compounds were excreted in amounts of 0-21%, 0-6.1% and 0-6.3%, respectively. The total concentration of measured metabolites in the bile, determined as their respective APs after de-conjugation, increased with increasing degree of alkylation (3.2+/-2.6 microg/g bile for 2-MP and 571+/-81 microg/g bile for 4-n-HepP) after exposure to an equal dose of AP. Comparison of metabolite concentrations in bile sampled 4 and 16 days after exposure, showed that the levels of 2-MP, 2,4,6-TMP and 4-t-B-2-MP were reduced by 55%, 30% and 45%, respectively whereas 3,5-DMP increased by 25% (not significant). This study suggests that analysis of de-conjugated metabolites in fish bile can be used to monitor AP exposure to fish, due to the relatively high and persistent level of these compounds. However, although HPLC-F is suitable for laboratory exposures, it might not be sufficient selective for field studies.     

13C NMR spectroscopy in the analysis of conjugate metabolites in the bile of fish exposed to petroleum

The first natural abundance ¹³C NMR investigation of a complex mixture of conjugate metabolites obtained from the gall bladder bile of fish exposed to hydrocarbons is presented. Cunners were exposed to water accommodated No. 2 fuel oil containing about 68% saturates and 22% aromatics. Spectral analysis indicated that the hydrocarbon derivatives were present predominantly as β-glucuronides, with the oxygen at carbon-1 of glucuronic acid preferentially attached to an aliphatic carbon. The conjugate metabolites were enriched in aromatic-type carbons when compared to the fuel oil or the aromatic fraction of oil.     

Some factors influencing EROD activity in winter flounder (Pleuronectes americanus) exposed to effluent from a pulp and paper mill

EROD (ethoxyresorufin-O-deethylase) activity was determined in winter flounder, a sediment-inhabiting and non-migratory fish species, living near a pulp and paper mill in Newfoundland in relation to temperature, gender, sexual maturity, and lesions in the liver. Samples of liver were taken from fish captured by SCUBA divers at 0 degrees C, 5 degrees C and 16 degrees C. Enzymic activity was detected in fish living only above 0 degrees C. Adult males and juvenile fish had higher levels of EROD activity than prespawning females at 5 degrees C. Macrophage aggregates only or occurring simultaneously with bile ductule hyperplasia and clear cell foci in the liver, did not impair EROD activity but necrosis had a negative effect. Results from this study indicate the importance of water temperature, gender, sexual maturity and liver pathology in assessing EROD activity of fish in biomonitoring programs.     

N-Methyl-beta-carboline-3-carboxamide (FG 7142): An anxiogenic agent in cigarette smoke condensate and its mechanism of formation

beta-Carboline-3-carboxylic acid methylamide (FG 7142), an anxiogenic agent has been found in cigarette smoke condensate, but not in the cigarette itself. When a cigarette, except its filter portion, was immersed in 20 ml of potassium phosphate buffer, pH 7.4, then heated at 60 degrees C for 2 days with or without presence of methylamine, FG 7142 was detected only in the mixture containing methylamine. Furthermore, when the mixtures of beta-carboline derivatives and various amounts of methylamine hydrochloride were heated at 60 degrees C for 5 days, FG 7142 was formed only in the mixtures containing methylamine and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-caroxylic acid (MTCA) or 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (TCCA). FG 7142 was also produced in the mixture of glucose, l-tryptophan and methylamine when heated at 200 degrees C in a dry condition. These observations suggest that FG 7142 is formed through the smoking process and that methylamine in cigarette smoke may play an important role in the formation of FG 7142.     

Metabolism of crufomate (4-tert-butyl-2-chlorophenyl methyl methylphosphoramidate) administered topically to a sheep.

A sheep dosed topically with 14C-crufomate (4-tert-butyl-2-chlorophenyl methyl methylphosphoramidate) excreted 45.5% of the 14C dose in the urine within 9 days. The feces contained 1.2% and the carcass 40.4% (this included the 37.7% of the dose remaining on the skin in the dosing area) of the dose. At sacrifice, the fat, liver, kidney, lung, and skin (where the dose was applied) contained the highest concentrations of 14C. Fourteen urinary metabolites were isolated and characterized by mass spectrometry. The metabolic reactions involved were oxidations of the t-butyl moiety, O-demethylation, replacement of the H-N-CH3 moiety with a hydroxyl group, oxidation of the N-methyl group to yield N-formyl phosphoramidates, hydrolysis of the phosphoramidate moiety to yield phenols, conjugation with glucuronic acid and combinations of these reactions.     

Extended acid digestion for inactivation of fecal coliforms.

The objective of this research was to establish a correlation between inactivation of fecal coliforms caused by organic acids in their unionized form in batch acid digesters and semicontinuously fed acid digesters at both mesophilic (38 degrees C) and low-mesophilic (24 degrees C) temperatures. Batch acid digesters achieved a U.S. Environmental Protection Agency Class A level of fecal coliforms within 6 to 7 days of digestion at both temperatures. Semicontinuously fed, staged, acid-digestion systems achieved Class A standards on average only at mesophilic temperature at a solids retention time of 11 days. Systems operated at low-mesophilic temperatures did not achieve Class A standards.     

Metabolism of crufomate [(4-tert-butyl-2-chlorophenyl methyl methylphosphormidate)] by the rat.

Fifteen metabolites of crufomate (4-tert-butyl-2-chlorophenyl methyl methylphosphoramidate, I) were identified in the excreta from rats given single oral doses of I. Compound I was not detected in either the urine or the feces. The metabolic reactions observed were N-and O-demethylation, oxidations of the t-butyl moiety, replacement of the H-N-CH3 with an OH moiety, hydrolysis of the phosphoramidate moiety to yield the phenol, conjugation with glucuronic acid, and combinations of these reactions. No ring dehalogenation or ring substitution was observed.     

Comparative study of different exposure routes on the biotransformation and genotoxicity of PAHs in the flatfish species, Scophthalmus maximus

In this study, laboratory experiments were carried out in order to come to a better understanding of the fate of polycyclic aromatic hydrocarbons (PAHs) in the marine environment and especially on their bioaccumulation, biotransformation and genotoxic effects in fish. Juveniles of turbot (Scophthalmus maximus) were exposed to PAHs through different routes via (1) a mixture of dissolved PAHs, (2) a PAH-polluted sediment and (3) an oil fuel elutriate. Fish were exposed 4 days followed by a 6-day depuration period. In each experiment, PAH concentrations in the seawater of the tanks were analysed regularly by gas chromatography coupled with mass spectrometry. Muscle and liver samples were also analysed for parent PAH levels and PAH bioconcentration factors were calculated. Biotransformation was evaluated by measuring the levels of PAH metabolites in fish bile. Genotoxicity was assessed by the alkaline comet assay. Regardless of exposure route, the parent PAH concentrations in the liver and muscle showed a peak level 1 day after the beginning of the exposure, followed by a decrease up to the background level towards the end of the experiment, except for the exposure to dissolved PAHs for which levels were relatively low throughout the study. As a consequence, no bioaccumulation was observed in fish tissues at the end of the experiment. In contrast, regardless of exposure routes, a rapid production of biliary metabolites was observed throughout the whole exposure experiment. This was especially true for 1-hydroxypyrene, the major metabolite of pyrene. After 6 days of recovery in clean water, a significant decrease in the total metabolite concentrations occurred in bile. Fish exposed through either route displayed a significant increase in DNA strand breaks after 4 days of exposure, and significant correlations were observed between the level of biliary PAH metabolites and the level of DNA lesions in fish erythrocytes. Overall results indicate that exposure to either a mixture of dissolved PAHs, a PAH-contaminated sediment or a dispersed oil fuel elutriate leads to biotransformation and increase in DNA damage in fish. The quantification of PAH metabolites in bile and DNA damage in erythrocytes appear to be suitable for environmental monitoring of marine pollution either in the case of accidental oil spills or sediment contamination.     

Use of blood parameters in fish to assess acidic stress and chloride pollution in French running waters

The study investigated whether plasmatic parameters in fish such as Cl- content could serve as physiological indicators to evaluate water quality. The variations of plasma Cl- content in two fish species caught in a wide range of rivers representative of the hydrographic system of Lorraine (N-E France) were investigated. First, we studied autochthonous populations of chub (Leuciscus cephalus) which is a widespread species in the rivers of Lorraine. Organisms living in highly mineralised rivers (>1500 microScm(-1))--either naturally or due to salt mine contamination--showed plasma Cl- content significantly greater than organisms living in river with a lower mineralisation. Second, we investigated plasma Cl- content of brown trout (Salmo trutta fario) in poorly mineralised streams (<80 microS cm(-1)) with different degrees of acidification, both on autochthonous and transferred organisms. While indigenous trout maintained their Cl- content even in the acidic streams, transferred trout exhibited an important decrease of Cl- content after 48 h of exposure under acidic conditions.     

Free proline content and susceptibility of poplar (Populus) cuttings to the action of SO2, NaCl and PEG at different temperatures

A study has been made of the influence of separate and simultaneous action of SO(2), NaCl and polyethylene glycol 8000 (PEG) on leaf injuries and free proline content of two poplar cuttings, Populus 'Robusta' and P. 'Hybrida 275', at an optimum (+15 degrees C), a lowered (+3 degrees C) and an increased (+35 degrees C) temperature. Proline accumulation was greatest in the most damaged cuttings of both cultivars for all three treatments. Simultaneous action of SO(2) and NaCl, as well as of SO(2) and PEG, caused significantly greater accumulation of this imino acid compared with the action of each factor separately, and the influence was more than additive. An increase in free proline content under the influence of the treatment combinations investigated here was proportional to the temperature during exposure. Greatest accumulation of proline under the experimental conditions was obtained by the simultaneous action of SO(2) and NaCl, as well as of SO(2) and PEG, at the temperature of 35 degrees C.     

Effect of anions and cations on cadmium sorption kinetics from aqueous solutions by chitin: experimental studies and modeling

The effect of ions, including Na(+), Mg(2+), Ca(2+), Cl(-), SO(4)(2-) and CO(3)(2-), at various initial concentrations, on the kinetics of cadmium sorption by chitin was studied at 25 degrees C and free initial pH solution in batch conditions. The presence of these ions in solution was found to inhibit the uptake of cadmium by chitin to different degrees: sodium and chloride ions have no significant effect. For Mg(2+), Ca(2+), SO(4)(2-) and CO(3)(2-) ions, the effects ranged from a large inhibition of cadmium by Ca(2+) and CO(3)(2-) to a weak inhibition by Mg(2+) and SO(4)(2-). These results indicate that the uptake sites of these ions are the same. No ion was found to enhance cadmium uptake. The results also showed that the kinetics of sorption are best described by a pseudo second-order expression than a first or second-order model.     

The impact of constituent ions of acid mist on assimilation and stomatal conductance of Norway spruce prior and post mid-winter freezing

Norway spruce seedlings were sprayed twice weekly with one of a range of artificial mists at either pH 2.5, 3.0 or 5.6, for three months. The mists consisted of either (NH4)2SO4 (pH 5.6), NH4NO3 (pH 5.6), water (pH 5.6), HNO3 (pH 2.5), H2SO4 (pH 2.5). In late December 1988 and early January 1989 the light response of assimilation and stomatal conductance were assessed in the laboratory following a 4-day equilibration period at 12 degrees C. The intact trees were then subjected to a mild (-10 degrees C), brief (3 h) frost in the dark and the recovery of light saturated assimilation (Amax) was followed during the subsequent light period. The same trees were then subjected to a second 3 h (-18 degrees C) frost. The recovery of Amax during the next day was followed. All ion-containing mists stimulated Amax and apparent quantum yield relative to control trees, irrespective of pH. The mists containing SO4 made stomatal conductance unresponsive to light flux density and caused the stomata to lock open. Frosts of -10 degrees C and -18 degrees C did not inhibit the Amax of control trees for longer than 200 min into the light period. In contrast, the ion-containing mists exerted a significant inhibitory effect upon the recovery of Amax. Nitric acid inhibited Amax to 35% of the pre-frost value, whilst the remaining treatments inhibited Amax between 15% and 40% of the pre-frost value. It is concluded that SO4 causes increased mid-winter frost sensitivity and NO3 ameliortes this effect. The results are discussed in relation to forest decline.     

Morphological effects of Bisphenol-A on the early life stages of medaka (Oryzias latipes).

Bisphenol-A (BPA), a widely used polycarbonate plasticizer, has been of concern because it has been shown to leach out of plastics and other epoxy products. Primary sources of environmental releases are expected to be from BPA and epoxy manufacturing facilities. Although environmental concentrations may be limited, little is known about the effects of this compound on fish, particularly during their most sensitive early life stages. A pilot study exposing newly hatched medaka to 200 microg BPA/l for 96 h showed to be non-lethal. In addition, medaka embryos were exposed beginning at 5 h post-fertilization, for 9 days at 25 degrees C, to concentrations of 20 or 200 microg BPA/l (24 h static renewal). Embryos were monitored daily for development and gross abnormalities. Embryos exposed to 200 microg BPA/l did not exhibit abnormalities until after day 4. However, between days 4 and 8 the severity index (SI) score of embryos was significantly greater than those exposed to lesser concentrations. By day 9, SI scores were not statistically different among treatments. BPA caused transient embryonic deformities in medaka at environmentally relevant concentrations.     

Bioavailability of PAHs in the Biobio river (Chile): MFO activity and biliary fluorescence in juvenile Oncorhynchus mykiss.

Cytochrome P450-dependent monooxygenase (MFO) activity and levels of bile PAH metabolites were measured in fish (Oncorhynchus mykiss) exposed to Biobio river (Chile) water. Experiments were performed simultaneously in both the field and laboratory to investigate whether the river water contained notable bioavailable PAH fractions. The field experiment was conducted using fish caged for 41 days at La Mochita (Biobio river mouth) whereas the laboratory experiment involved a 21-day exposure treatment with water collected in the same area. Induction of hepatic MFO, assayed by benzo(a)pyrene monooxygenase activity (BaPMO), and levels of PAH metabolites in the bile were measured in groups of eight (field experiment) and four specimens (laboratory experiment) sampled after 5, 10, 21 and 41 days of exposure. BaPMO was induced by a factor of 23 and 25 in fish caged for 21 and 41 days, respectively. Likewise, a significant BaPMO induction was found in the fish group experimentally exposed to river water for 21 days. Biliary fluorescence measurements, recorded by fixed wavelength fluorescence (FF) and synchronous fluorescence spectroscopy (SFS), revealed significantly high levels of PAH metabolites in bile samples of fish caged for 21 and 41 days. A positive relationship was found between BaPMO activity and biliary PAH metabolites. This study provides evidences that (i) Biobio river water contains a significant bioavailable PAH fraction in terms of marked effects on fish and (ii) the biological indicators MFO activity and bile PAH metabolites represent an excellent screening methodology to assess PAH exposure.     

The effect of winter stress on Ilex aquifolium L.previously fumigated with ozone

European Holly (Ilex aquifolium) received either charcoal-filtered air (CFA) or CFA with 70 nl l(-1) ozone added for 7 h day(-1) over a 28 day period. Plants were then transferred into cooling incubators for hardening (4 degrees C day/2 degrees C night; day length 12 h) for 7 days and then to the frosting stage (2 degrees C day and -5, -10 or -15 degrees C night) for 4 days. The plants were then placed in ambient conditions. Treatment produced significant differences in chlorophyll fluorescence data. Stomatal conductance was significantly higher for the ozone treatments though both showed a general decline over all temperature regimes. Ozone also significantly increased electrolyte leakage and reduced winter survival. These results show that ambient concentrations of ozone can reduce the tolerance of I. aquifolium to freezing stress, which may have serious implications for its establishment and survival.     

Treatment of wastewater from red and tropical fruit wine production by zeolite anaerobic fluidized bed reactor

A study of the anaerobic treatment of wastewaters derived from red (RWWW) and tropical fruit wine (TFWWW) production was carried out in four laboratory-scale fluidized bed reactors with natural zeolite as bacterial support. These reactors operated at mesophilic temperature (35 degrees C). Reactors R1 and R2 contained Chilean natural zeolite, while reactors R3 and R4 used Cuban natural zeolite as microorganism support. In addition, reactors R1 and R3 processed RWWW, while reactors R2 and R4 used TFWWW as substrate. The biomass concentration attached to zeolites in the four reactors studied was found to be in the range of 44-46 g volatile solids (VS)/L after 90 days of operation time. Both types of zeolites can be used indistinctly in the fluidized bed reactors achieving more than 80%-86% chemical oxygen demand (COD) removals for organic loading rates (OLR) of up to at least 20 g COD/L d. pH values remained within the optimal range for anaerobic microorganisms for OLR values of up to 20 and 22 g COD/L d for RWWW and TFWWW, respectively. Toxicity and inhibition levels were observed at an OLR of 20 g COD/L d in reactors R1 and R3 while processing RWWW, whereas the aforementioned inhibitory phenomena were not observed at an OLR of 24 g COD/L d in R2 and R4, treating TFWWW as a consequence of the lower phenolic compound content present in this substrate. The volatile fatty acid (VFA) levels were always lower in reactors processing TFWWW (R2 and R4) and these values (< 400 mg/L, as acetic acid) were lower than the suggested limits for digester failure. The specific methanogenic activity (SMA) was twice as high in reactors R2 and R4 than in R1 and R3 after 120 days of operation when all reactors operated at an OLR of 20 g COD/L d.     

Adsorption of microcystin-LR from water with iron oxide nanoparticles

Adsorption of microcystin-LR (MC-LR) from water using iron oxide (alpha-Fe2O3) nanoparticles was investigated in this study. Adsorption of MC-LR adsorption was well-described by a pseudo second order kinetics model and Freundlich and Langmuir isotherm equations at 15 to 35 degrees C. Thermodynamic analysis showed that the Gibbs free energy was negative, whereas standard enthalpy and entropy changes were positive at this temperature range. These findings suggest that the adsorption of MC-LR on iron oxide nanoparticles was spontaneous and endothermic. The effects of initial pH, inorganic cations, and competing compounds with carboxyl groups on absorption of MC-LR were also evaluated. Typically, adsorption efficiency decreased with increasing pH from 2 to 11. Sodium ions did not appear to significantly affect MC-LR adsorption, whereas calcium ions slightly enhanced the MC-LR adsorption capacity of the iron oxide nanoparticles. Moreover, the inhibiting effect of competing organic compounds was increased with increasing numbers of carboxyl groups, as follows: citric acid (3)>oxalic acid (2)>benzoic acid (1).     

Regrowth of fecal coliforms in class A biosolids.

Eight types of Class A biosolids were tested for fecal coliform (FC) reactivation and/or regrowth at 20, 35, and 50 degrees C for 21 days. Growth of FC did not occur at 20 or 50 degrees C, but it was observed in two samples incubated at 35 degrees C after a lag period of 48 hours. In undigested biosolids, final FC concentration exceeded 10(4) MPN/g, whereas in thermophilically digested biosolids, the final FC concentration remained below 10(3) MPN/g, as FC regrowth may have been affected by the presence of the anaerobic bacterial consortium responsible for the digestion process. Fecal-coliform reactivation and regrowth within treatment plant operations seem unlikely but can occur in land application of biosolids.     

Metabolic rate and uptake and loss of cadmium from food by the fish Noemacheilus barbatulus L. (stone loach)

Fish, Noemacheilus barbatulus (stone loach), of different body weights were used to study rates of uptake and loss of cadmium during and after dietary exposure. Fish were kept singly in a continuous-flow system, and fed tubificid worms. The worms had a range of cadmium levels, but all levels were below that needed to cause acute lethal toxicity in the fish. Body weight affected both the maintenance ration and the amount of food consumed ad libitum, but the exponent for body weight (0.78+/-0.04), relating body weight to food consumption, was unaffected by either temperature or the size of feeding ration. The cadmium content of the worms did not affect the size of the maintenance ration. Metal burden in fish changed rapidly both during and after exposure. After exposure, the cadmium burden of starved fish usually declined more rapidly than in fed fish. A 58-fold increase in cadmium content in the food produced a 28% increase of body burden in the fish, and there was no evidence for biomagnification. Maintenance ration and ration ad libitum and rates of uptake and loss of cadmium increased with temperature within the range 8-18 degrees C, but exposure to cadmium at 16 degrees C yielded a higher asymptotic body burden than either 8 degrees C or 18 degrees C. Rate constants for loss of cadmium after exposure appear to be lower than for loss during exposure. Rates of uptake and loss of cadmium vary with metabolic rate. A maximum in the rate of oxygen consumption was measured at 16 degrees C, above which the rate dropped, presumably due to stress. The exponent for body weight was unaffected by activity or temperature. Body weight of fish appeared to affect both the rates of uptake and loss of cadmium, and feeding rations and respiration to the same extent: body weight exponents were not dissimilar.     

A protocol for the evaluation of genotoxicity in bile of carp (Cyprinus carpio) exposed to lake water treated with different disinfectants

A sensitive and rapid method to evaluate toxic and genotoxic properties of drinking water supplied from Lake Trasimeno (Umbria, Central Italy) was worked out analysing bile in Cyprinus carpio exposed for 20 d to lake water treated with 3 different disinfectants, sodium hypochlorite (NaClO), chlorine dioxide (ClO₂) and peracetic acid (PAA). Fish were sacrificed at 0, 10 and 20 d in order to investigate the time course of these endpoints. An aliquot of bile samples was fractionated by adsorption on C₁₈ silica cartridges and the genotoxic potential of whole bile and of bile fractions was evaluated by the single-cell microgel-electrophoresis (comet) assay on human colonic adenocarcinoma cells (Caco-2). Bile (both whole and fractionated) from specimens exposed to the three disinfectants always showed a genotoxic activity as compared to the control group. The results of this study provide evidence that all three disinfectants cause an increase in bile genotoxicity of chronically exposed fish.