Catalase activity in human erythrocytes: effect of phenoxyherbicides and their metabolites

The effects of exposure to different concentrations of phenoxyherbicides and their metabolites were studied in human erythrocytes, with particular attention to catalase (CAT-EC. 1.11.1. 6- hydrogen peroxide: hydrogen peroxide oxidoreductase). 4-chloro-2-methylphenoxyacetic acid (MCPA), 2,4-dimethylphenol (2, 4-DMP) and 2,4-dichlorophenoxyacetic acid (2,4-D) did not affect CAT activity, but 2,4-dichlorophenol (2,4-DCP) and 2,4,5-trichlorophenol (2,4,5-TCP) decrease its activity, the latter being the more inhibitory.     

Reductive dechlorination of chlorophenols in slurries of low-organic-carbon marine sediments and subsurface soils

The reductive dechlorination of 2,4- and 3,4-dichlorophenol (DCP) was studied in slurries of marine sediments and subsurface soils with dissolved organic carbon concentrations less than 1 ppm. Dechlorination was markedly greater in marine sediment slurries than in subsoil slurries, although similar products were observed in each case. From 25% to 98% of the 2,4- and 3,4-DCP (6.5 μm/l) added to most marine slurries was converted to 4- and 3-chlorophenol (CP) respectively, within 30 weeks. In contrast 2,4-DCP was dechlorinated to 4-CP (>90%) in only 1 of 24 replicate subsoil slurries after 32 weeks of incubation. Dechlorination was observed within 2 weeks when yeast extract was added to subsoil slurries; yeast extract additions also stimulated dechlorination in marine sediments but to a lesser extent. The intermediate monochlorophenol products did not persist in marine slurries but did persist in the subsoil slurries. It was concluded that the total organic carbon at a site is not always a good predictor of the site's ability to support dechlorination activity. Received: 3 December 1996 / Received revision: 28 February 1997 / Accepted: 7 March 1997     

Effects of 2,4-dichlorophenol on activated sludge

The effects of 2,4-dichlorophenol (2,4-DCP) on both acclimated and unacclimated activated sludge were investigated in batch reactors. The IC(50) values on the basis of maximum specific growth rate ( micro(m)), percent chemical oxygen demand (COD) removal efficiency and sludge activity were found to be 72, 60 and 47 mg l(-1), respectively, for unacclimated culture. The percent COD removal efficiencies of unacclimated culture were affected adversely, even at low concentrations, whereas culture acclimated to 75 mg 2,4-DCP l(-1) could tolerate about 200 mg 2,4-DCP l(-1)on the basis of COD removal efficiency. Although yield coefficient values of unacclimated culture increased surprisingly to very high values with the addition of 2,4-DCP, a linear decrease with respect to 2,4-DCP concentrations was observed for acclimated culture. Although no removal was observed with unacclimated culture, almost complete removal of 2,4-DCP up to a concentration of 148.7 mg l(-1) was observed with acclimated culture. It was showed that the culture could use 2,4-DCP as sole organic carbon source, although higher removal efficiencies in the presence of a readily degradable substrate were observed. Culture acclimated to 4-chlorophenol used 2,4-DCP as sole organic carbon source better than those acclimated to 2,4-DCP.     

Effects of 2,4-D and its metabolite 2,4-dichlorophenol on antioxidant enzymes and level of glutathione in human erythrocytes

The effects of in vitro exposure of human erythrocytes to different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) were studied. The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the level of reduced glutathione (GSH) were determined. The activity of erythrocyte superoxide dismutase SOD decreased with increasing dose of 2,4-D and 2,4-DCP, while glutathione peroxidase activity increased. 2,4-D (500 ppm) decreased the level of reduced glutathione in erythrocytes by 18% and 2,4-DCP (250 ppm) by 32%, respectively, in comparison with the controls. These results lead to the conclusion that in vitro administration of herbicide-2,4-D and its metabolite 2,4-DCP causes a decrease in the level of reduced glutathione in erythrocytes and significant changes in antioxidant enzyme activities. Comparison of the toxicity of 2,4-D and 2,4-DCP revealed that the most prominent changes occurred in human erythrocytes incubated with 2,4-DCP.     

Malonylated flavonol glycosides from the petals of Clitoria ternatea

Three flavonol glycosides, kaempferol 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside, quercetin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside, and myricetin 3-O-(2",6"-di-O-alpha-rhamnosyl)-beta-glucoside were isolated from the petals of Clitoria ternatea cv. Double Blue, together with eleven known flavonol glycosides. Their structures were identified using UV, MS, and NMR spectroscopy. They were characterized as kaempferol and quercetin 3-(2(G)- rhamnosylrutinoside)s, kaempferol, quercetin, and myricetin 3-neohesperidosides, 3-rutinosides, and 3-glucosides in the same tissue. In addition, the presence of myricetin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside was inferred from LC/MS/MS data for crude petal extracts. The flavonol compounds identified in the petals of C. ternatea differed from those reported in previous studies.     

Biodegradation kinetics of 2,4-dichlorophenol by acclimated mixed cultures

The biodegradation kinetics of 2,4-dichlorophenol (2,4-DCP) by culture (Culture M) acclimated to mixture of 4-chlorophenol (4-CP) and 2,4-DCP and the culture (Culture 4) acclimated to 4-CP only were investigated in aerobic batch reactors. Also, pure strains isolated from mixed cultures were searched for their ability towards the biodegradation of 2,4-DCP. Culture 4 was able to completely degrade 2,4-DCP up to 80 mg/L within 30 h and removal efficiency dropped to 21% upon increasing initial concentration to 108.8 mg/L. When the Culture M was used, complete degradation of 2,4-DCP in the range of 12.5-104.4 mg/L was attained. A linear relationship between time required for complete degradation and initial 2,4-DCP concentrations was observed for both mixed cultures. It was observed that the Haldane equation can be used to predict specific degradation rate (SDR) (R(2)>0.99) as a function of initial 2,4-DCP concentrations and it adequately describes 2,4-DCP concentration profiles. Both of the mixed cultures settled well, which is important to maintain good removal efficiency for longer periods of time for real full-scale applications. Although the pure strains isolated from mixed cultures were found to have higher SDR of 2,4-DCP compared to mixed cultures, they did not settle well under quiescent conditions.     

Simultaneous and Repetitious Removal of 2,4-Dichlorophenol and Copper from Soils Using an Aqueous Solution of Ethyl-Lactate-Amended EDDS

Heavy metals and the transformation products of herbicides, such as 2,4-dichlorophenol (2,4-DCP), are toxic soil pollutants. We assessed the ability of an aqueous solution of the “green solvent” ethyl lactate alone and combined with [S,S]-ethylenediaminedisuccinic acid (EDDS) to remove 2,4-DCP and copper simultaneously from soils. Ethyl lactate extracted 2,4-DCP from contaminated soil comparable to Triton X-100. Ethyl lactate/EDDS extracted more 2,4-DCP and Cu from contaminated soils than ethyl lactate alone. The enhanced extraction of Cu increased slightly with an increase in the EDDS/Cu molar ratio; the maximum Cu extraction efficiency was about 32.3% at an EDDS/Cu ratio of 5. An increase in the ionic strength (NaCl) of the ethyl lactate/EDDS solution decreased the amount of 2,4-DCP extracted by maximally 12% but increased the amount of Cu extracted by >500%. We tested the recycling of the ethyl lactate/EDDS solution with the cation-exchange resin 001×7 and the hyper-cross-linked polymer resin NDA-150. Fresh ethyl lactate/EDDS solution and two sequentially recycled solutions removed 31.4, 28.3, and 26.7% of the Cu in Cu-contaminated soil and 77.7, 62.9, and 56.8% of the 2,4-DCP in 2,4-DCP-contaminated soils, respectively. The ethyl lactate/EDDS solution removed 31.8% of the Cu and 73.0% of the 2,4-DCP in Cu- and 2,4-DCP-contaminated soils, and the solution remained effective even after two recyclings. The aqueous solution of ethyl lactate/EDDS can be used to effectively remove Cu and 2,4-DCP from complex contaminated soils and can be reactivated.     

Growth Rates of a Pseudomonad on 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol

The growth of a pseudomonad on 2,4-D (2,4-dichlorophenoxyacetic acid) and 2,4-DCP (2,4-dichlorophenol) was studied in batch and continuous culture. The optimum growth rate using 2,4-D was 0.14/h at 25 C in a pH range from 6.2 to 6.9. Highest specific growth rate using 2,4-DCP was 0.12/h at 25 C in a pH range from 7.1 to 7.8. Growth was strongly inhibited by 2,4-DCP above a concentration of 25 mg/liter whereas no appreciable inhibition was observed with 2,4-D at concentrations up to 2,000 mg per liter. Growth on 2,4-DCP was described by Monod kinetics at subinhibitory concentrations but the inhibition by 2,4-DCP exhibited an unusual linear response to substrate concentration, and did not fit a model based on noncompetitive inhibition. The lag phase of batch cultures was found to depend on both 2,4-DCP concentration and prior adaptation of the inoculum. A study such as this on the kinetics of growth on related substrates may be useful as a method of finding the rate-limiting step in a metabolic sequence.     

Modeling chlorophenols degradation in sequencing batch reactors with instantaneous feed-effect of 2,4-DCP presence on 4-CP degradation kinetics

Two instantaneously fed sequencing batch reactors (SBRs), one receiving 4-chlorophenol (4-CP) (SBR4) only and one receiving mixture of 4-CP and 2,4-dichlorophenol (2,4-DCP) (SBRM), were operated with increasing chlorophenols concentrations in the feed. Complete degradation of chlorophenols and high-Chemical oxygen demand (COD) removal efficiencies were observed throughout the reactors operation. Only a fraction of biomass (competent biomass) was thought to be responsible for the degradation of chlorophenols due to required unique metabolic pathways. Haldane model developed based on competent biomass concentration fitted reasonably well to the experimental data at different feed chlorophenols concentrations. The presence of 2,4-DCP competitively inhibited 4-CP degradation and its degradation began only after complete removal of 2,4-DCP. Based on the experimental results, the 4-CP degrader’s fraction in SBRM was estimated to be higher than that in SBR4 since 2,4-DCP degraders were also capable of degrading 4-CP due to similarity in the degradation pathways of both compounds.     

Effect of carbon sources and shock loading on the removal of chlorophenols in sequential anaerobic-aerobic reactors

The effect of carbon sources and shock loadings have been studied using two sets of sequential upflow anaerobic sludge blanket (UASB) and rotating biological contactor (RBC) reactors viz., UASB-I followed by RBC-I and UASB-II followed by RBC-II for the removal of two different priority pollutants, 2-CP and 2,4-DCP present in simulated wastewaters. Sodium formate, sodium propionate, glucose and methanol were used separately as four different carbon sources in the feed as co-substrate. Methanol was found to be the best carbon source for UASB reactors showing 95% 2-CP and 81.1% 2,4-DCP removals. The carbon sources formate and propionate were not found suitable in UASB reactors as only 22.6-46.8% 2-CP and 41.9-42.8% 2,4-DCP removals were observed. With glucose as carbon source 93.7% 2-CP and 79.6% 2,4-DCP removals were observed in UASB reactors. For all the four carbon sources more than 97.6% 2-CP and 99.7% 2,4-DCP removals were observed in sequential reactors. Although all the four carbon sources could not serve as good carbon source for UASB reactor alone but could be successfully used by the sequential reactors for the removal of chlorophenols. The Performance of sequential reactors was also evaluated at five different chlorophenolic shock loadings. During shock loading study the concentration of chlorophenols in the wastewaters was increased to 45, 60, 75, 90 and 105 mg/l as compared to the normal feed containing 30 mg/l 2-CP or 2,4-DCP. During shock loading study complete removal of 2-CP and more than 99.6% removal of 2,4-DCP was observed in sequential reactors. Sequential reactors successfully withstood all the shock loadings and produced high quality effluents.     

Purification and characterization of two extracellular peroxidases from Streptomyces sp. strain AM2, a decolorizing actinomycetes responsible for the biodegradation of natural humic acids

Two extracellular humic acids peroxidases called HaP1 and HaP2 were isolated from the Streptomyces sp. strain AM2 and, based on MALDI-TOF MS analysis. The purified enzymes were determined as monomers with molecular masses of 40,351.11 and 25,175.19 Da, respectively. The N-terminal amino acid sequences of HaP1 and HaP2 were identified, and their optimum pH values were determined as 6 and 7.5, respectively. Standard 2,4-dichlorophenol (2,4-DCP) assays showed that both enzymes had maximal activity at 55 °C. HaP2 was stable at 55 °C for more than 24 h and had a half-life of 90 min at 65 °C. Although the catalytic properties of HaP1 and HaP2 were nearly identical, their stabilities and Reinheitzahl (RZ) values were substantially different. Both peroxidases were found to be heme proteins that catalyzed the oxidation of a wide range of substrates in the presence of hydrogen peroxide (H₂O₂), with HaP2 exhibiting a broader range of substrate specificity. The characterization of peroxidase activity revealed activity against humic acids, guiacol, 2,4-DCP, l-3,4-dihydroxyphenylalanine, and 2,4,5-trichlorophenol as well as other chlorophenols in the presence of H₂O₂. However, the inhibition of peroxidase activity by the addition of potassium cyanide and sodium azide also indicated the presence of heme components in the tertiary structure of these enzymes.     

Degradation of 2,4-dichlorophenol by Bacillus sp. isolated from an aeration pond in the Baikalsk pulp and paper mill (Russia)

A pure culture of 2,4-dichlorophenol (2,4-DCP)-degrading bacteria was isolated from a natural enrichment that had been adapted to chlorophenols in the aeration pond of the Baikalsk pulp and paper mill (Russia). The bacteria were identified by 16S rDNA intergenic region analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (1545 bp) in the GenBank database revealed that these bacteria are related to Bacillus cereus GN1. Degradation of 2,4-DCP was studied using this culture in liquid medium under aerobic conditions, at initial concentrations of 20–560 μM 2,4-DCP. The 2,4-DCP degradation rates by B. cereus GN1 could be determined at concentrations up to 400 μM. However, higher concentrations of 2,4-DCP (560 μM) were inhibitory to cell growth.     

Flavones and flavone glycosides from Halophila johnsonii

Halophila johnsonii Eiseman is a shallow-water marine angiosperm which contains UV-absorbing metabolites. Studies on methanol extracts of H. johnsonii by means of HPLC-UV, NMR, HPLC-MS resulted in isolation and identification of seven previously unknown flavone glycosides: 5,6,7,3′,4′,5′-hexahydroxyflavone-7-O-β-glucopyranoside (1), 5,6,7,3′,4′,5′-hexahydroxyflavone-7-O-(6″-O-acetyl)-β-glucopyranoside (2), 6-hydroxyluteolin-7-O-(6″-O-acetyl)-β-glucopyranoside (3), 6-hydroxyapigenin-7-O-(6″-O-acetyl)-β-glucopyranoside (4), 6-hydroxyapigenin-7-O-(6″-O-[E]-coumaroyl)-β-glucopyranoside (5), 6-hydroxyapigenin-7-O-(6″-O-[E]-caffeoyl)-β-glucopyranoside (6) and 6-hydroxyluteolin-7-O-(6″-O-[E]-coumaroyl)-β-glucopyranoside (7). Also isolated were three known flavone glycosides, 6-hydroxyluteolin 7-O-β-glucopyranoside (8), scutellarein-7-O-β-glucopyranoside (9), and spicoside (10), and five known flavones, pedalitin (11), ladanetin (12), luteolin (13), apegenin (14) and myricetin (15). Qualitative comparison of the flavonoid distribution in the leaf and rhizome-root portions of the plant was also investigated, with the aim of establishing the UV-protecting roles that flavonoids played in the sea grass.     

Biodegradation of 2,4-Dichlorophenol through a Distal meta-Fission Pathway

Alcaligenes eutrophus JMP222, a derivative of A. eutrophus JMP134 which has lost plasmid pJP4 (encoding the tfd genes for the ortho fission pathway), was induced for the meta fission pathway when grown on o-cresol. Resting cell suspensions, grown on o-cresol, oxidized 2,4-dichlorophenol (2,4-DCP), a degradation product of 2,4-dichlorophenoxyacetic acid, to 3,5-dichlorocatechol. Further degradation of 3,5-dichlorocatechol was observed by the production of a yellow ring fission product with liberation of chloride. Oxidation of 2,4-DCP (305 (mu)M) in 47 hs resulted in 69% dehalogenation through this pathway. The ring fission product was characterized as 2-hydroxy-3,5-dichloro-6-oxo-hexa-2,4-dienoic acid by gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared spectroscopy. These data indicate that 2,4-DCP is degraded through a distal meta ring fission pathway, in contrast to either a suicidal proximal fission or the standard ortho fission pathway.     

Kinetic and isothermal studies on liquid-phase adsorption of 2,4-dichlorophenol by palm pith carbon

Adsorption studies were conducted to study the removal of 2,4-dichlorophenol (2,4-DCP) from aqueous solution on palm pith carbon under varying experimental conditions such as agitation time, adsorbent dose, pH and temperature. Higher 2,4-DCP was removed with decrease in the initial concentration of 2,4-DCP and increase in amount of adsorbent used. Kinetic study showed that the adsorption of 2,4-DCP on palm pith carbon was a gradual process. Adsorption capacities were 19.16 mg/g for the particle size of 250-500 microm. The equilibrium time was 60 and 80 min for 10 and 20 mg/L and 100 min for both 30 and 40 mg/L phenol concentrations, respectively. Acidic pH was favourable for the adsorption of 2,4-DCP. Studies on pH effect and desorption showed that chemisorption seemed to play a major role in the adsorption process. Thermodynamic study showed that adsorption of 2,4-DCP on palm pith carbon was more favoured. The change in entropy (DeltaS0) and heat of adsorption (DeltaH0) of palm pith carbon was estimated as 30.72 J/mol/k and 7.16 kJ/mol, respectively. The high positive value of change in Gibbs free energy indicated the feasible and spontaneous adsorption of 2,4-DCP on palm pith carbon. The results indicated that palm pith carbon was an attractive candidate for removing phenols from wastewater.     

Degradation of 2,4-dichlorophenol by Bacillus sp. isolated from an aeration pond in the Baikalsk pulp and paper mill (Russia)

A pure culture of 2,4-dichlorophenol (2,4-DCP)-degrading bacteria was isolated from a natural enrichment that had been adapted to chlorophenols in the aeration pond of the Baikalsk pulp and paper mill (Russia). The bacteria were identified by 16S rDNA intergenic region analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (1545 bp) in the GenBank database revealed that these bacteria are related to Bacillus cereus GN1. Degradation of 2,4-DCP was studied using this culture in liquid medium under aerobic conditions, at initial concentrations of 20–560 μM 2,4-DCP. The 2,4-DCP degradation rates by B. cereus GN1 could be determined at concentrations up to 400 μM. However, higher concentrations of 2,4-DCP (560 μM) were inhibitory to cell growth.     

Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.     

Degradation of dissolved and sorbed 2,4-dichlorophenol in soil columns by suspended and sorbed bacteria

The influence of sorption of bacteria, as well as 2,4-dichlorophenol (2,4-DCP), on themineralization of 100 g l^-1 of the organic compound was examined in an aquifer material under advective flow conditions (column displacement technique). The study was designed to distinguish the rates and extent of biodegradation of the sorbed and the dissolved trace organic and the contribution of sorbed and suspended bacteria to the degradation. The degradation of dissolved 2,4-DCP was significantly faster thanthe degradation of the same compound sorbed to the solids, and suspended bacteriadegraded the dissolved compound at a higher rate than sorbed bacteria, also on a percell basis. The suspended bacteria degraded 8–12% of the added dissolved 2,4-DCP, while sorbed bacteria made a smaller contribution by degrading about 5% of sorbed 2,4-DCP. No degradation was seen with sorbed 2,4-DCP and suspended bacteria, and a marginal contribution was made by sorbed bacteria on the degradation of dissolved 2,4-DCP (<0.4%).     

Genotoxic effect of 2,4-dichlorophenoxy acetic acid and its metabolite 2,4-dichlorophenol in mouse.

The cytogenetic effect of 2,4-dichlorophenoxy acetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) was studied in bone-marrow, germ cells and sperm head abnormalities in the treated mice. Swiss mice were treated orally by gavage with 2,4-D at 1.7, 3.3 and 33 mg kg(-1)BW (1/200, 1/100 and 1/10 of LD(50)). 2,4-DCP was intraperitoneally (i.p.) injected at 36, 72 and 180 mg kg(-1)BW (1/10, 1/5, 1/2 of LD(50)). A significant increase in the percentage of chromosome aberrations in bone-marrow and spermatocyte cells was observed after oral administration of 2,4-D at 3.3 mg kg(-1)BW for three and five consecutive days. This percentage increased and reached 10.8+/-0.87 (P<0.01) in bone-marrow and 9.8+/-0.45 (P<0.01) in spermatocyte cells after oral administration of 2,4-D at 33 mg kg(-1)BW for 24 h. This percentage was, however, lower than that induced in bone-marrow and spermatocyte cells by mitomycin C (positive control). 2,4-D induced a dose-dependent increase in the percentage of sperm head abnormalities. The genotoxic effect of 2,4-DCP is weaker than that of 2,4-D, as indicated by the lower percentage of the induced chromosome aberrations (in bone-marrow and spermatocyte cells) and sperm head abnormalities. Only the highest tested concentration of 2,4-DCP (180 mg kg(-1)BW, 1/2 LD(50)) induced a significant percentage of chromosome aberrations and sperm head abnormalities after i.p. injection. The obtained results indicate that 2,4-D is genotoxic in mice in vivo under the conditions tested. Hence, more care should be given to the application of 2,4-D on edible crops since repeated uses may underlie a health hazard.     

Bioregeneration of granular activated carbon in simultaneous adsorption and biodegradation of chlorophenols

The objectives of this study are to obtain the time courses of the amount of chlorophenol adsorbed onto granular activated carbon (GAC) in the simultaneous adsorption and biodegradation processes involving 4-chlorophenol (4-CP) and 2,4-dichlorophenol (2,4-DCP), respectively, and to quantify the bioregeneration efficiency of GAC loaded with 4-CP and 2,4-DCP by direct measurement of the amount of chlorophenol adsorbed onto GAC. Under abiotic and biotic conditions, the time courses of the amount of chlorophenol adsorbed onto GAC at various GAC dosages for the initial 4-CP and 2,4-DCP concentrations below and above the biomass acclimated concentrations of 300 and 150 mg/L, respectively, were determined. The results show that the highest bioregeneration efficiency was achieved provided that the initial adsorbate concentration was lower than the acclimated concentration. When the initial adsorbate concentration was higher than the acclimated concentration, the highest bioregeneration efficiency was achieved if excess adsorbent was used.