A method for quantitative analysis of flavor-tainting alkylphenols and aromatic thiols in fish

Simultaneous steam distillation-extraction (SDE) of fortified rainbow trout tissue resulted in greater than 95% recovery of 2-isopropylphenol, 3-isopropylphenol, 4-isopropylphenol, 2,4-diisopropylphenol, 2,5-diisopropylphenol, 3,5-diisopropylphenol, carvacrol and thymol. Lower recoveries were obtained for 2,6-diisopropylphenol (81%), thiophenol (55%), and thiocresol (85%). Analysis of concentrated extracts by gas chromatography/mass spectrometry operated in the selected ion monitoring mode allowed quantitative detection of these compounds down to 0.5 ppb based on 20 g of initial sample.     

Coupling of 2,4,6-trinitrotoluene (TNT) metabolites onto humic monomers by a new laccase from Trametes modesta

During degradation of trinitrotoluene (TNT) by Trametes modesta, addition of humic monomers prevented the accumulation of all major stable TNT metabolites (aminodinitrotoluenes [AMDNT]) by at least 92% in the presence of 200 mM ferulic acid and guaiacol. Acute toxicity tests with individual TNT metabolites and in T. modesta cultures supplemented with 200 microM TNT demonstrated that the TNT biodegradation process lead to less toxic metabolites. Toxicity decreased in the order TNT>4-HADNT (4-hydroxylaminodinitrotoluene)>2-HADNT>2,6-DNT (2,6-dinitrotoluene)>2',2',6,6-azoxytetranitrotoluene>4-AMDNT>2-AMDNT>2,4-diamninonitrotoluene (2,4-DAMNT) while 2,4-DNT and 2,6-DAMNT were the least toxic. Ferulic acid is the best candidate for immobilization TNT biodegradation metabolites since it prevented the accumulation of AMDNTs in cultures during TNT biodegradation and its products were less toxic. All humic monomers were very effective in immobilizing 2-HADNT [100%], 4-HADNT [100%] and 2,2,6,6-azoxytetranitrotoluene [100%]. Two distinct laccase isoenzymes (LTM1 and LTM2) potentially involved in immobilization of TNT degradation products were purified to electrophoretic homogeneity. LTM1 and LTM2 have molecular weights of 77.6 and 52.5 kDa, are 18% and 24% glycosylated, have pI values of 3.6 and 4.2, respectively. Both enzymes oxidized all the typical laccase substrates tested. LTM1 showed highest kinetic constants (K(m)=0.03 microM; K(cat)=8.8 4x 10(7)s(-1)) with syringaldazine as substrate.     

Oxidation of MCPA and 2,4-D by UV radiation, ozone, and the combinations UV/H2O2 and O3/H2O2

The phenoxyalkyl acid derivative herbicides MCPA (4-chloro 2-methylphenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) were oxidized in ultrapure water by means of a monochromatic UV irradiation and by ozone, as well as by the combinations UV/H2O2 and O3/H2O2. In the direct photolysis of MCPA, the quantum yield at 20 degrees C was directly evaluated and a value of 0.150 mol Eins(-1) was obtained in the pH range 5-9, while a lower value of 0.41 x 10(-2) mol Eins(-1) was determined at pH=3. Similarly, for 2,4-D a value of 0.81 x 10(-2) mol Eins(-1) was deduced, independent of the pH of work. The influence of the additional presence of hydrogen peroxide was established in the combined process UV/H2O2, and the specific contribution of the radical pathway to the global photo-degradation was evaluated. The oxidation by ozone and by the combination O3/H2O2 was also studied, with the determination of the rate constants for the reactions of both herbicides with ozone and hydroxyl radicals at 20 degrees C. These rate constants for the direct reactions with ozone were 47.7 and 21.9 M(-1) s(-1) for MCPA and 2,4-D respectively, while the found values for the rate constants corresponding to the radical reactions were 6.6 x 10(9) and 5.1 x 10(9) M(-1) s(-1).     

Modeling the quantum yields of herbicide 2,4-D decay in UV/H2O2 process

The photodecay of herbicide 2,4-D in a hydrogen peroxide-aided photolysis process was studied and modeled. The decay rate of 2,4-D was known to be low in the natural environment, but rate improvement was achieved in an H2O2/UV system. The 2,4-D decay quantum yields under ultraviolet (UV) light at 253.7 nm increased from 4.86 x 10(-6) to 1.30 x 10(-4) as the ratio of [H2O2]/[2,4-D] increased from 0.05 to 12.5. Apparently, in the presence of UV light, the decay rate of 2,4-D could be greatly improved as the concentration of hydrogen peroxide increased. However, the efficiency of 2,4-D photodecay was retarded if the concentration of H2O2 was overdosed, because the excess hydrogen peroxide consumes the hydroxyl radicals (HO*) in the solution, resulting in a much weaker oxidant HO2*. The decay of 2,4-D was also pH dependent. A ranking of acid (highest), base (middle) and neutral (lowest) was observed owing to the property change of reactants and the shifting of dominant mechanisms among photolysis, photohydrolysis and chemical oxidation. Two mathematical models were proposed to predict the quantum yield for various [H2O2]/[2,4-D] ratios and initial pH levels, in which very good correlation was found for the ranges of regular application.     

Biodegradation of haloacetic acids by bacterial enrichment cultures

Haloacetic acids (HAAs) are toxic organic chemicals that are frequently detected in surface waters and in drinking water distribution systems. The aerobic biodegradation of HAAs was investigated in serum bottles containing a single HAA and inoculated with washed microorganisms obtained from enrichment cultures maintained on either monochloroacetic acid (MCAA) or trichloroacetic acid (TCAA) as the sole carbon and energy source. Biodegradation was observed for each of the HAAs tested at concentrations similar to those found in surface waters and in drinking water distribution systems. The MCAA culture was able to degrade both MCAA and monobromoacetic acid (MBAA) with pseudo-first order rate constants of 1.06 x 10(-2) and 1.13 x 10(-2) l(mg protein)(-1) d(-1), respectively, for concentrations ranging from 10(-5) to 2 mM. The pseudo-first order rate constant for TCAA degradation by the TCAA culture was 6.52 x 10(-3) l(mg protein)(-1) d(-1) for concentrations ranging from 5.33 x 10(-5) to 0.72 mM. The TCAA culture was also able to degrade MCAA with the rate accelerating as incubation time increased. Experiments with radiolabeled HAAs indicated that the 14C was primarily converted to 14CO2 with minor incorporation into cell biomass. The community structure of the enrichment cultures was analyzed by both cultivation-dependent and cultivation-independent approaches. Denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene fragments showed that each of the two enrichment cultures had multiple bacterial populations, none of which corresponded to HAA-degrading bacteria cultivated on HAA-supplemented agar plates. This research indicates that biodegradation is a potential loss mechanism for HAAs in surface waters and in drinking water distribution systems.     

Biochemical characterization of a multiple heavy metal, pesticides and phenol resistant Pseudomonas fluorescens strain

Pseudomonas fluorescens SM1 isolate was found to be resistant to some major water pollutants namely Cd2+, Cr6+, Cu2+, Ni2+, Pb2+, BHC, 2,4-D, mancozeb and phenols up to a concentration four times to the normal levels occurring in the highly pollulated regions. Curing experiment brought about the loss of one or more resistance markers indicating the plasmid born resistance. Plasmid profile of SM1 strain showed the presence of one DNA band of 43.6 kb. This Plasmid was isolated from SM1 strain and introduced into Escherichia coli DH5 alpha with a transformation frequency of 6.7 x 10(-4)transformants/recipient cell. The test SM1 strain was also capable of biotransforming Cr(VI) to Cr(III) which is less toxic compounds. Present studies further indicated that the test SM1 strain was not only resistant to 2,4-D, phenols and catechol but also capable of bioremediating these toxicants quite efficiently. Moreover, studies with inhibitors like sodium azide, 2,4-DNP and chloramphenicol suggested that the major mechanism for the bioremediation of the heavy metals other than Cr6+ would be the biosorption process.     

Ecotoxicity characterization of dinitrotoluenes and some of their reduced metabolites

In the present study, the toxic effects of 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT) and a selection of their respective metabolites were examined and compared to 2,4,6-trinitrotoluene (TNT) using the 15-min Microtox (Vibrio fischen) and 96-h freshwater green alga (Selenastrum capricomutum) growth inhibition tests. All of the compounds tested were less toxic than TNT. Using the Microtox assay, 2,6-DNT was more toxic than 2,4-DNT and the order of toxicity for 2,6-DNT and its metabolites was: 2,6-DNT > or = 2A-6NT > 2,6-DAT; whereas that for 2,4-DNT was: 4A-2NT > 2A-4NT > 2,4-DNT > 2,4-DAT. For the algal test, 2,4-DNT was more toxic than 2,6-DNT and the order of toxicity for 2,4-DNT and its metabolites was: 2,4-DNT > 2,4-DAT approximately equal to 4A-2NT = 2A-4NT. The order of toxicity for 2,6-DNT and its reduced metabolites using the algal test was very similar to the Microtox bioassay. These results demonstrate that the reduced metabolites of 2,6-DNT tested in this study were less toxic than that of the parent compound, but certain partially reduced metabolites of 2,4-DNT can be more toxic than the parent molecule. These data put into question the general hypothesis that reductive metabolism of nitro-aromatics is associated with a sequential detoxification process.     

Enhanced chemical oxidation of aromatic hydrocarbons in soil systems

Fenton's destruction of benzene, toluene, ethylbenzene, and xylene (BTEX) was investigated in soil slurry batch reactors. The purpose of the investigation was to quantify the enhancement of oxidation rates and efficiency by varying process conditions such as iron catalyst (Fe(II) or Fe(III); 2, 5, and 10mM), hydrogen peroxide (H2O2; 30, 150, 300 mM), and metal chelating agents (l-ascorbic acid, gallic acid, or N-(2-hydroxyethyl)iminodiacetic acid). Rapid contaminant mass destruction (97% after 3h) occurred in the presence of 300 mM H2O2 and 10 mM Fe(III). An enhanced removal rate (>90% removal after 15 min and 95% removal after 3h) was also observed by combining Fe(III), N-(2-hydroxyethyl)iminodiacetic acid and 300 mM H2O2. The observed BTEX mass removal rate constants (3.6-7.8 x 10(-4)s(-1)) were compared to the estimated rate constants (4.1-10.1 x 10(-3)s(-1)). The influence of non-specific oxidants loss (by reaction with iron hydroxides and soil organic matter) was also explored.     

Bioconcentration factor of relatively low concentrations of chlorophenols in Japanese medaka

Bioconcentration factors (BCF) for pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP) in Japanese medaka (Oryzias latipes) were determined at five different concentrations of the chemicals, between 0.1 and 10 microg/l (PCP), 0.3 and 30 microg/l (2,4-DCP), in the ambient water. Medaka were exposed to each chemicals in a continuous-flow system during the embryonic development period and 60 days after hatching from eggs collected in the laboratory. Both the exposure time and the aqueous concentrations are much more realistic and closer to natural aquatic environments than those used in conventional BCF studies. The BCF values of PCP were from (4.9+/-2.8)x10(3) at the aqueous concentration of 0.074+/-0.028 microg/l to (2.1+/-1.4)x10(3) at 9.70+/-0.56 microg/l. The BCF value of 2,4-DCP were from (3.4+/-3.0)x10(2) at 0.235+/-0.060 microg/l to 92+/-27 at 27.3+/-1.6 microg/l. Generally, BCF values increased as the aqueous concentrations of PCP or 2,4-DCP decreased. This finding suggests that a relatively low and realistic aqueous concentration of these compounds is necessary to more accurately determine their BCF values in natural aquatic environments. Conventional BCF experiments at higher aqueous concentrations may underestimate the BCF values.     

Enhancing the performance of sequencing batch reactors by adding crushed date seeds to remove high concentrations of 2,4-dinitrophenol

Wastewater treatment systems using simultaneous adsorption and biodegradation processes have been successful in treating toxic pollutants present in industrial wastewater. The goal of this investigation was to assess the effectiveness of date seeds in reducing the toxic effects of 2,4-dinitrophenol (DNP) on activated sludge microorganisms. Two identical sequencing batch reactors (SBRs) (4-L glass vessel), each with a 3.5-L working volume, were used. The initial DNP concentrations in the reactor were 50, 75, 100, 250, and 500 mg/L. The reactor amended with date seeds was capable of degrading DNP at significantly greater rates (11 +/- 2.5 mg/L x h) than the control SBR (4 +/- 1.2 mg/L x h) at a 95% confidence level. Date seeds can be added to the mixed liquor of activated sludge treatment plants to remove high concentrations of DNP from wastewater, to protect the treatment plant against toxic components in the influent and enhance the settling characteristics of the mixed liquor.     

Toxicity and bioaccumulation of reduced TNT metabolites in the earthworm Eisenia andrei exposed to amended forest soil

Soils contaminated with 2,4,6-trinitrotoluene (TNT) and TNT primary reduction products have been found to be toxic to certain soil invertebrates, such as earthworms. The mechanism of toxicity of TNT and of its by-products is still not known. To ascertain if one of the TNT reduction products underlies TNT toxicity, we tested the toxicity and bioaccumulation of TNT reduction products. 2-Amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6-nitrotoluene (2,4-DANT) and 2,6-diamino-4-nitrotoluene (2,6-DANT) were tested separately in adult earthworms (Eisenia andrei) following a 14-d exposure to amended sandy loam forest soil. TNT, 4-ADNT, and 2-ADNT were lethal to earthworms (14-d LC(50) were: 580, 531 and 1088 micromol kg(-1), or 132, 105 and 215 mgkg(-1) dry soil, respectively) and gave the following order of toxicity: 4-ADNT>TNT>2-ADNT. Exposure to 2,4-DANT and to 2,6-DANT caused no mortality at 600 micromol kg(-1) or 100 mgkg(-1) dry soil. We found that all four TNT reduction products accumulated in earthworm tissues and 2-ADNT reached the highest levels at 3.0+/-0.3 micromol g(-1) tissue. The 14-d bioaccumulation factors were 5.1, 6.4, 5.1 and 3.2 for 2-ADNT, 4-ADNT, 2,4-DANT and 2,6-DANT, respectively. Results also suggest that some TNT metabolites are at least as toxic as TNT and should be considered when evaluating the overall toxicity of TNT-contaminated soil to earthworms.     

Rapid quantification of polycyclic aromatic hydrocarbons in hydroxypropyl-beta-cyclodextrin (HPCD) soil extracts by synchronous fluorescence spectroscopy (SFS)

Synchronous fluorescence spectroscopy (SFS) was directly applied to rapidly quantify selected polycyclic aromatic hydrocarbons (PAHs: benzo[a]pyrene and pyrene) in aqueous hydroxypropyl-beta-cyclodextrin (HPCD) soil extract solutions from a variety of aged contaminated soils containing four different PAHs. The method was optimized and validated. The results show that SFS can be used to analyse benzo[a]pyrene and pyrene in HPCD based soil extracts with high sensitivity and selectivity. The linear calibration ranges were 4.0x10(-6)-1.0x10(-3)mM for benzo[a]pyrene and 6.0x10(-6)-1.2x10(-3)mM for pyrene in 10mM HPCD aqueous solution alone. The detection limits according to the error propagation theory for benzo[a]pyrene and pyrene were 3.9x10(-6) and 5.4x10(-6)mM, respectively. A good agreement between SFS and HPLC was reached for both determinations of PAHs in HPCD alone and in soil HPCD extracts. Hence, SFS is a potential means to simplify the present non-exhaustive hydroxypropyl-beta-cyclodextrin (HPCD)-based extraction technique for the evaluation of PAH bioavailability in soil.     

Hydrogen peroxide-assisted UV photodegradation of Lindane

Aqueous solutions of gamma-hexachlorocyclohexane (Lindane) were photolyzed (lambda=254 nm) under a variety of solution conditions. The initial concentrations of hydrogen peroxide (H(2)O(2)) and Lindane varied from 0 to 20 mM and 0.21 to 0.22 microM, respectively, the pH ranged from 3 to 11, and several concentration ratios of Suwannee River humic acid and fulvic acid were dissolved in the irradiated solutions. Lindane rapidly reacted, and the maximum reaction rate constant (9.7 x 10(-3) s(-1)) was observed at pH 7 and initial [H(2)O(2)]=1 mM. Thus, 90% of the Lindane is destroyed in approximately 4 min under these conditions. In addition, within 15 min, all chlorine atoms were converted to chloride ion, indicating that chlorinated organic by-products do not accumulate. The reactor was characterized by measuring the photon flux (7.04 x 10(-6) E s(-1)) and the cumulative production of *OH during irradiation. The cumulative *OH production during irradiation was fastest at an initial [H(2)O(2)]=5 mM (k=0.77 micro M s(-1)).     

Assessing the effects of the three herbicides acetochlor, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid on the compound action potential of the sciatic nerve of the frog (Rana ridibunda)

To assess the relative toxicity of the herbicides acetochlor and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on the nervous system, the sciatic nerve of the frog (Rana ridibunda) nerve was incubated in saline inside a specially designed recording chamber. This chamber permits monitoring of the evoked compound action potential (CAP) of the nerve, a parameter that could be used to quantify the vitality of the nerve in normal conditions as well as when the nerve was exposed to the compounds under investigation. Thus, when the nerve was exposed to acetochlor, the EC(50) was estimated to be 0.22mM, while for 2,4,5-T the EC(50) was 0.90mM. Using the identical nerve preparation, the EC(50) of 2,4-D was estimated to be 3.80mM [Kouri, G., Theophilidis, G., 2002. The action of the herbicide 2,4-dichlorophenoxyacetic acid on the isolated sciatic nerve of the frog (Rana ridibunda). Neurotoxicol. Res. 4, 25-32]. The ratio of the relative toxicity for acetochlor, 2,4,5-T and 2,4-D was found to be 1:4:17.2. However, because it is well-known that the action of 2,4-D is dependent on the pH, the relative toxicity of the three compounds was tested at pH 3.3, since it has been found that the sciatic nerve of the frog is tolerant of such a low pH. Under these conditions, the EC(50) was 0.77mM (from 0.22mM at pH 7.2) for acetochlor, 0.20mM (from 0.90mM) for 2,4,5-T and 0.24mM (from 3.80mM at pH 7.2) for 2,4-D. Thus, the relative toxicity of the three compounds changed drastically to 1:0.25:0.31. This change in the relative toxicity is due not only to the increase in the toxicity of 2,4,5-T and 2,4-D at low pH levels, but also to the decrease in the toxicity of acetochlor at pH 3.3.     

Photodegradation mechanism for bisphenol A at the TiO2/H2O interfaces

Bisphenol A (BPA) can be decomposed photocatalytically under UV-illumination in aqueous TiO2 dispersion. The two methyl groups in BPA were initially attacked with .OH and/or .OOH radicals having strong oxidizing power, followed by the cleavage of the two phenyl moieties. Finally, the photomineralization to CO2 gas occurred via oxidative processes involving carboxylic acids and aldehydes. The decomposition of structurally similar substances of 4,4'-ethylidenebisphenol (EBP) and 4,4'-methylenebisphenol (MBP) was compared. The decomposition of BPA gave more kinds of intermediates such as 4-isopropylphenol, 4-ethylphenol, etc. On the other hand, that of EBP gave mainly 4-isopropylphenol and that of MBP gave a predominant product of 4-hydroxybenzaldehyde. These photooxidative pathways were proposed on the base of the evidence of oxidative intermediate formation.     

2,4-D mineralization in unsaturated and near-saturated surface soils of an undulating, cultivated Canadian prairie landscape

The herbicide 2,4-D [2,4-(dichlorophenoxy) acetic acid] is one of the most widely used pesticides in the Canadian prairies and is frequently detected as a ground and surface water contaminant. The objective of this paper was to determine the magnitude and extent of variation of 2,4-D mineralization in a cultivated undulating prairie landscape. Microcosm incubation experiments, using a 4 x 3 x 2 factorial experimental design (soil moisture, 4 levels: 60, 85, 110, 135% of field capacity; slope position, 3 levels: upper-, mid- and lower-slopes; soil depth, 2 levels: 0-5 and 5-15 cm), were used to assess 2,4-D mineralization. The first-order mineralization rate constant (k(1)) varied from 0.03 to 0.22 day(- 1), while total 2,4-D mineralization varied from 31 to 52%. At near-saturated conditions (110 and 135% of field capacity), the onset of 2,4-D degradation was delayed in soil obtained from the upper- and mid-slopes but not in soils obtained from the lower-slope position. The k(1) and total 2,4-D mineralization was significantly influenced by all three factors and their interactions. The Freundlich sorption coefficient of 2,4-D ranged from 0.83 to 2.46 microg (1-1/n)g(- 1) mL(1/n) and was significantly influenced by variations in soil organic carbon content across slope positions. The infield variability of 2,4-D sorption and mineralization observed across slope positions in this undulating field was comparable in magnitude and extent to the regional variability of 2,4-D sorption and mineralization observed in surface soils across Manitoba. The large variability of 2,4-D mineralization and sorption at different slope positions in this cultivated undulating field suggests that landform segmentation models, which are used to delineate slope positions, are important considerations in pesticide fate studies.     

Bioherbicidal activity of terpenes and phenylpropenes against Echinochloa crus-galli

Abstract

This study was undertaken to evaluate the herbicidal activity of twelve natural compounds belonging to monoterpenes, phenylpropenes, and sesquiterpenes against Echinochloa crus-galli under laboratory and glasshouse conditions. Experiments were conducted to determine the impact of different concentrations (0.5, 1, 2, 4 and 8?mM) of these compounds on the seed germination and root and shoot growth of barnyard grass. trans-Cinnamaldehyde, eugenol, and thymol caused the highest impact on barnyard grass reducing its seed germination and shoot growth. p-Cymene (EC₅₀ = 0.22?mM) and trans-cinnamaldehyde (EC₅₀ = 0.34?mM) were the most potent compounds in limiting the root growth of the E. crus-galli. In a post-emergent experiment, thymol, trans-cinnamaldehyde, eugenol, farnesol, and nerolidol significantly reduced the shoot growth, fresh and dry weight of two-leaf stage barnyard grass after 2 days of the foliar treatment with the concentrations of 0.5, 1.0 and 2.0%. These compounds induced severe visible injury symptoms where trans-cinnamaldehyde, eugenol, farnesol and nerolidol showed a complete weed control at 1.0 and 2.0%. These compounds were successfully formulated as emulsifiable concentrates and showed higher herbicidal activities against barnyard grass. Altogether, our data showed that trans-cinnamaldehyde, eugenol, thymol, farnesol, and nerolidol can be developed as novel bioherbicides for managing E. crus-galli.     

Immobilization of polyphenol oxidase onto mesoporous activated carbons -- isotherm and kinetic studies

Investigations were carried out in batch modes for studying the immobilization behavior of polyphenol oxidase (PPO) on two different mesoporous activated carbon matrices, MAC400 and MAC200. The PPO was immobilized onto MAC400 and MAC200 at various enzyme activities 5 x 10(4), 10 x 10(4), 20 x 10(4), 30 x 10(4)Ul(-1), at pH 5-8, and at temperature ranging from 10 to 40 degrees C. The intensity of immobilization of PPO increased with increase in temperature and initial activities, while it decreased with increase in pH. Immobilization onto MAC400 followed the Langmuir model while Langmuir and Freundlich models could fit MAC200 data. Non-linear pseudo first order, pseudo second order and intraparticle diffusion models were evaluated to understand the mechanism of immobilization. The free and immobilized enzyme kinetic parameters (K(m) and V(max)) were determined by Michaelis-Menten enzyme kinetics. The K(m) values for free enzyme, PPO immobilized in MAC400 and in MAC200 were 0.49, 0.41 and 0.65 mM, respectively. The immobilization of PPO in carbon matrices was confirmed using FT-IR spectroscopy and scanning electron microscopy.     

Kinetics of 2,4-dichlorophenol and 4-Cl-m-cresol degradation by Pseudomonas sp. cultures in the presence of glucose

Comparison of the ability of Pseudomonas sp. to degrade 2,4-dichlorophenol and 4-Cl-m-cresol in separate cultures in the presence of glucose, as a conventional carbon source, is reported. The specific growth rates at 0.1 mM 2,4-dichlorophenol and 4-Cl-m-cresol were estimated to be 0.181 and 0.154 h(-1), respectively, showing that Pseudomonas sp. is mainly inhibited by 4-Cl-m-cresol. The percentage of consumption ranges between 65% and 11% for 2,4-dichlorophenol and between 37% and 8% for 4-Cl-m-cresol, respectively, depending on its initial concentration. The dechlorination of the two compounds was investigated in the growth media and it was found that chloride liberation in the case of 2,4-dichlorophenol took place during the exponential phase of growth, followed by pH decrease from 6.1 to 5.8 at 0.1 mM. In contrast, in the case of 4-Cl-m-cresol chloride ion release was observed to a lesser extent, indicating the different metabolic pathway of 4-Cl-m-cresol. 2,4-Dichlorophenol and 4-Cl-m-cresol degradation followed a first-order kinetics model, whereas glucose consumption fitted well a zero-order kinetics model.     

Toxicity of chlorinated phenoxyacetic acid herbicides in the experimental eukaryotic model Saccharomyces cerevisiae: role of pH and of growth phase and size of the yeast cell population

The inhibitory effect of the herbicides 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in Saccharomyces cerevisiae growth is strongly dependent on medium pH (range 2.5-6.5). Consistent with the concept that the toxic form is the liposoluble undissociated form, at values close to their pK(a) (3.07 and 2.73, respectively) the toxicity is high, decreasing with the increase of external pH. In addition, the toxicity of identical concentrations of the undissociated acid form is pH independent, as observed with 2,4-dichlorophenol (2,4-DCP), an intermediate of 2,4-D degradation. Consequently, at pH values above 3.5 (approximately one unit higher than 2,4-D pK(a)), 2,4-DCP becomes more toxic than the original herbicide. A dose-dependent inhibition of growth kinetics and increased duration of growth latency is observed following sudden exposure of an unadapted yeast cell population to the presence of the herbicides. This contrasts with the effect of 2,4-DCP, which essentially affects growth kinetics. Experimental evidences suggest that the acid herbicides toxicity is not exclusively dependent on the liposolubility of the toxic form, as may essentially be the case of 2,4-DCP. An unadapted yeast cell population at the early stationary-phase of growth under nutrient limitation is significantly more resistant to short-term herbicide induced death than an exponential-phase population. Consequently, the duration of growth latency is reduced, as observed with the increase of the size of the herbicide stressed population. However, these physiological parameters have no significant effect either on growth kinetics, following growth resumption under herbicide stress, or on the growth curve of yeast cells previously adapted to the herbicides, indicating that their role is exerted at the level of cell adaptation.