Vibrio Fischeri Test Research Articles

Matrix effect in the hydroxyl radical induced degradation of β-lactam and tetracycline type antibiotics

Due to the spread of antibiotic resistant bacteria, elimination of antibiotics from purified wastewater is a highly important task. Purified wastewater contains large variety of organic/inorganic compounds that strongly influence the efficiency of advanced oxidation processes (AOP). In this work, we investigate the radiation-induced degradation of selected antibiotic contaminants (oxacillin and cloxacillin from the β-lactams; tetracycline and chlortetracycline from the tetracyclines) in four matrices: pure water, tap water, synthetic wastewater and purified wastewater received from a wastewater treatment plant. Changes in technically important parameters, such as COD, TOC, BOD, OUR, acute toxicity and antibacterial activity, were investigated at 0.1 mmol dm−3 (40–48 mg dm−3) antibiotic concentration. None of the four antibiotics were biodegradable in any of the four matrices, however, after irradiation with relatively low doses the obtained products were biodegradable. Oxacillin and cloxacillin were not toxic in Vibrio fischeri test, while tetracycline and chlortetracycline showed toxicity that was strongly reduced by the irradiation treatment. Both COD and TOC decreased after irradiation, their ratio shifted towards TOC indicating an increase in the average oxidation state (AOS). The increase in AOS was lower in purified wastewater matrices: this matrix may contain various small, highly oxidized molecules in high concentration, which degrade very slowly in AOP. The antibacterial activity in most of matrices was greatly reduced or completely disappeared at around 2–4 kGy. However, in purified wastewater matrices some antibiotic activity remained even at 4 kGy. Here the degradation of antibiotic is slow (small ΔCOD/dose value) because a large fraction of the reactive radicals is scavenged presumably by small, highly oxidized molecules in the solution. Although the water radiolysis product H2O2 affected some of the bioassays, this phenomenon was absent in purified wastewater. The purified wastewater after ionizing radiation treatment can be safely released into the receiving lakes or rivers.

Bacterial Toxicity Testing: Modification and Evaluation of the Luminescent Bacteria Test and the Respiration Inhibition Test

The activated sludge respiration inhibition test and the luminescent bacteria test with Vibrio fischeri are important bacterial test systems for evaluation of the toxicity of chemical compounds. These test systems were further optimized to result in better handling, reliability and sensitivity. Concerning the Vibrio fischeri test, media components such as yeast extract and bivalent cation concentrations like Ca2+ and Mg2+ were optimized. The cultivation, storage conditions and reactivation process of the stored bacteria were also improved, which enabled simpler handling and led to good reproducibility. Additionally, the respiration inhibition test with a prolonged incubation time was further analyzed using different chlorinated phenols as reference compounds. It could be stated that a longer incubation period significantly improved the sensitivity of the test system.

Toxic response of the bacterium Vibrio fischeri to sodium lauryl ether sulphate residues in excavated soils.

Sodium lauryl ether sulphate (SLES) is the main chemical component in several lubricant products used for soil conditioning in the mechanized excavation industry using Earth Pressure Balance-Tunnel Boring Machines. During the tunnelling process, huge amounts of excavated soil are produced and the SLES presence can affect the subsequent re-use of this material as a by-product. Currently, there is still no regulatory indication of reliable and sensitive bioassays for monitoring soil quality during the excavation process. The main objective of this work was to verify if the Vibrio fischeri screening test was suitable as a consistent and precautionary tool for this specific purpose. Firstly, the ecotoxicity (EC20 and EC50) of the SLES standard solution and three commercial products (SLES content from 10 to 50%) were evaluated to select the most environmental friendly product. Subsequently, soil samples from about 2 years of tunnelling in a real construction site, conditioned with the selected product, were evaluated for their environmental compatibility with the prescriptions of an Italian site-specific protocol. The latter established 2 mg/L as a threshold value for SLES concentration in soil water extracts and a no toxic response (≤20%) for the Vibrio fischeri test. The comparison of the bacterium bioluminescence inhibition values (%) with analytical determinations showed an ecotoxicity when SLES was >2 mg/L. The toxicity was directly related to SLES concentration, indicating that the V. fischeri test and the SLES analyses are suitable tools for assessing excavated soil as a by-product, ensuring its safe reuse in accordance with a green production process (circular economy).

Class Regular–Increased– and Class Iso–Toxicity, the two clearly defined and recognisable forms of the phenomenon. A developed Köln–Model (Hydractinia echinata Test–System. V.) study

According to the currently developed Köln–Model based on Hydractinia echinata Test–System (HeTS), the methodical toxicity investigations conducted with amphiphilic and non–amphiphilic singular derivatives with homo– and heteronomous segmentation from 20 different classes, reveals that the effectiveness noted M (mol/L) are characteristic for the Aromatic Basis of Toxicity (ABT) model, for the Class Regular–Increased Toxicity (CRIT) and for Class Iso–Toxicity (CIT), respectively. The phenomenon of toxicity is the result of monovalent interaction between the larval cellular receptor (R) of the marine colonial hydroid H. echinata organism and the xenobiotic substrate (SbX) with the molecular aggregate transitional–complex (RSbX) formation and the information–signal generation (SI), according to the Dual Causality Principle that biological phenomena are simultaneously determined by genetic programs and physics laws. The SI consists of chemical (SIch) and electromagnetic (SIem) components, and is directly responsible for the simultaneous onset of the metamorphosis quantified by the M toxicity values and the metabolizing process of the SbX, with the formation of the reaction products (P). The CRIT, respectively CIT approach was also successfully applied to published data obtained with the ciliate protozoan Tetrahymena pyriformis (T. pyriformis), the freshwater fish Pimephales promelas (P. promelas), the freshwater crustacean Daphnia magna (D. magna) and the marine bacterium Vibrio fischeri (name Aliivibrio fischeri) test–systems (TS’s).

Diclofenac biodegradation by newly isolated Klebsiella sp. KSC: Microbial intermediates and ecotoxicological assessment

The presence of diclofenac, a frequently used analgesic drug in the environment, can be harmful to a variety of organisms like algae, crustaceans and fish. In this study, a bacterial strain of Klebsiella sp. KSC (Gen Bank, accession number KX500307) able to biodegrade high concentration of diclofenac was isolated from livestock soil and identified. The exposure of Klebsiella sp. KSC to 70 mg/L of diclofenac resulted in diclofenac mineralization after 72 h. This is the first study that points out substantial biodegradation of high concentration of diclofenac (70 mg/L) in less than 72 h; and this shows the potential of this strain to be bioaugmented in contaminated sites or to bioreactors. The chemical structure of twelve biotransformation products of diclofenac are proposed. Its transformation pathway may involve hydroxylation, dehydroxylation, decarboxylation and dechlorination of the central ring of diclofenac. Acute ecotoxicity assay with Vibrio fischeri test showed that the main biotransformation product (TP298) of diclofenac by Klebsiella sp. KSC was less toxic than the parent compound.

3,4-Dimethylpyrazole phosphate and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture nitrification inhibitors: Quantification in plant tissues and toxicity assays

Nitrification inhibitors are used to maintain ammonium available in the soil for longer periods while reducing nitrate leaching and N2O emission. In this work we evaluated the potential toxicity effects of 3,4-dimethylpyrazole phosphate (DMPP) and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) nitrification inhibitors. In order to determine the potential plant capacity to take up and translocate these inhibitors, we grew clover plants in hydroponic conditions and we developed a novel methodology for extracting DMPP and DMPSA that we quantified by HPLC. In addition, we also did toxicity bioassays: seed germination and Vibrio fischeri test. When clover was exposed to high amounts of nitrification inhibitors, plants accumulated DMPP, predominantly in leaves, and also DMPSA that preferentially accumulated in roots. These inhibitors did not provoke phytotoxicity at the equivalent of the maximum amount estimated in agriculture (0.5mg/kg soil). DMPP only provoked detrimental effects in plants at very high dose (100mg/kg soil). Interestingly, DMPSA was innocuous.

Transformation of the Β-Triketone Pesticides Tembotrione and Sulcotrione by Reactions with Ozone: Kinetic Study, Transformation Products, Toxicity and Biodegradability

ABSTRACTRate constants between ozone and triketone herbicides, tembotrione and sulcotrione, were determined between pH 2 and 12. Both compounds are completely transformed within seconds by ozone under real water treatment conditions. A reaction pathway was proposed based on the identification of transformation products. Toxicity evaluation using Vibrio fischeri test showed that a residual toxicity remained even after degradation of the major by-products, but biodegradation significantly increased from 50% to 70% for molar ozone to pesticide ratios from 0 to 2. Thus, ozonation coupled with biological treatment would drastically reduce the risk associated to the presence of triketone herbicides.

Reactions of clofibric acid with oxidative and reductive radicals—Products, mechanisms, efficiency and toxic effects

The degradation of clofibric acid induced by hydroxyl radical, hydrated electron and O2−∙/HO2∙ reactive species was studied in aqueous solutions. Clofibric acid was decomposed more effectively by hydroxyl radical than by hydrated electron or O2−∙/HO2∙. Various hydroxylated, dechlorinated and fragmentation products have been identified and quantified. A new LC–MS method was developed based on 18O isotope labeling to follow the formation of hydroxylated derivatives of clofibric acid. Possible degradation pathways have been proposed. The overall degradation was monitored by determination of sum parameters like COD, TOC and AOX. It was found that the organic chlorine degrades very effectively prior to complete mineralization. After the treatment no toxic effect was found according to Vibrio fischeri tests. However, at early stages some of the reaction products were more harmful than clofibric acid.

Air pollution impact assessment in the area surrounding the industrial settlement of Priolo/Augusta (Italy): A multidisciplinary approach.

Background. During the past years air pollution impact on environment and health in areas surrounding industrial sites has received an increasing attention in order to prevent or minimize adverse effects or clean-up compromised ecosystems. The measurement of pollutants emissions into the environment by chemical analysis provide little information on the toxicological activity of the contaminants. Biological monitoring assesses the effects of air mixtures on reactive organisms, highlighting the interactions among individual compounds. Exposure to air pollutants causes adverse effects both on environment, agriculture and human health. Aim. In order to evaluate the impact of atmospheric pollutants emitted by the industrial settlement of Priolo/Augusta (Italy) heavy metals (Pb, Cd, As, V, Hg, Rh, Pd, Pt, Ni) levels in air were monitored in the year 2010 and Vibrio fischeri test was used to appraise airborne pollutant ecotoxicity. Method. Sampling and determination of heavy metals was performed according to UNI EN 14902. From filtres it was made an aqueous suspensions and measured the ecotoxicity (EC50 values) of samples. Statistical analysis is made by R software. The inhibition levels and effective toxicity concentrations of these samples and solutions were determined. Interactive toxicity effects among the metals were investigated. Results. Pb, Cd, As and Ni were found in trace amounts but always below law limits 155/2010. Hg, Rh, Pd and Pt were always below the detection limit. The impacts of the single heavy metal solutions were compared with each other, a toxicity ranking of Cd>Pb>As>V>Ni was obtained in order of decreasing severity. The total effective concentrations of these five metals were in the ranges of 0.094–0.543 µg/mc. The interactive toxicity effects of the heavy metals in samples were classified as antagonistic. Conclusion. High levels of heavy metals may constitute an important input in the biochemical cycle and may have significant impacts, but our findings are not considered concern for human health.

Organophosphorus insecticides: Toxic effects and bioanalytical tests for evaluating toxicity during degradation processes

Organophosphorus insecticides have been the most applied group of insecticides for the last two decades. Their main toxic effects are related to irreversible inactivation of acetylcholinesterase (AChE). Actually, they covalently bind to serine OH group in the enzyme active site forming phosphorylated enzyme that cannot hydrolyze acetylcholine. Organophosphorus insecticides in the environment undergo the natural degradation pathway including mainly homogeneous and heterogeneous hydrolysis (especially at high pH) generating non-inhibiting products. Additionally, thio organophosphates are easily oxidized by naturally present oxidants and UV light, forming more toxic and stable oxons. Thus, oxidative degradation procedures, generally referred as advanced oxidation processes (AOP), have been applied for their efficient removal from contaminated waters. The most applied bioassays to monitor the organophosphate toxicity i.e. the detoxification degree during AOP are Vibrio fischeri and AChE bioassays. Vibrio fischeri toxicity test exploits bioluminescence as the measure of luciferase activity of this marine bacterium, whereas AChE bioassay is based on AChE activity inhibition. Both bioanalytical techniques are rapid (several minutes), simple, sensitive and reproducible. Vibrio fischeri test seems to be a versatile indicator of toxic compounds generated in AOP for organophosphorus insecticides degradation. However, detection of neurotoxic AChE inhibitors, which can be formed in AOP of some organophosphates, requires AChE bioassays. Therefore, AChE toxicity test is more appropriate for monitoring the degradation processes of thio organophosphates, because more toxic oxo organophosphates might be formed and overlooked by Vibrio fischeri bioluminescence inhibition. In addition, during organophosphates removal by AOP, compounds with strong genotoxic potential may be formed, which cannot be detected by standard toxicity tests. For this reason, determination of incidence of micronuclei and cell proliferation index in cultivated human lymphocytes and fibroblasts is suitable for evaluation of organophosphorus insecticides and their break down products inducing cytogenetic damage.

Removal of dexamethasone from aqueous solution and hospital wastewater by electrocoagulation

This study is concerned with the removal of the anti-inflammatory dexamethasone from aqueous solution and hospital wastewater by electrocoagulation. The variation of the toxicity during the electrocoagulation was also studied through experiments that were designed and optimized by means of response surface methodology. The coagulation efficiency was evaluated by measuring the dexamethasone concentration by high performance liquid chromatography coupled to a diode array detector. In addition, variation was evaluated through a Vibrio fischeri test. The results showed an increase in the removal of dexamethasone (up to 38.1%) with a rise of the current applied and a decrease of the inter-electrode distance, in aqueous solutions. The application to hospital effluent showed similar results for the removal of dexamethasone. The main effect of the electrocoagulation was that it removed colloids and reduced the organic load of the hospital wastewater. Regarding the current applied, the calculated energy efficiency was 100%. Without pH adjustment of the aqueous solution or hospital wastewater, the residual aluminum concentration always remained lower than 10mgL−1, and, with adjustment (to pH6.5), lower than 0.30mgL−1, at the final stage. No toxicity variation was observed during the electrocoagulation process in aqueous solution, either in the presence or absence of dexamethasone.

HPLC Determination of Antihistaminic Drugs in Surface Waters and Toxicological Evaluation by Vibrio Fischeri Test

A HPLC method for the simultaneous determination of three widely used antihistaminic drugs, diphenhydramine, oxatomide and promethazine, in surface water samples and a toxicological assay using the bioluminescent bacterium Vibrio fischeri to assess a comprehensive contamination were developed. The analytical procedure was extended to the by-products from promethazine photodegradation. The method involved pre-concentration and clean-up by solid phase extraction and HPLC analysis with diode array detection. Validation of the method was performed on synthetic mixtures and surface water samples spiked with the drugs, showing mean recovery ranging from 93 and 107%, with RSD below 5.5%. Limits of quantitation in surface waters, calculated on 1.0 l of sample, were in the range 1.9 – 2.5 μg/l. The toxicological tests demonstrated toxicity due to promethazine from a concentration of 3.94 μg/ml while the other antihistamines showed no significant toxicity up to 50.0 μg/ml. However, the toxicity of drug mixtures was found greater than the sum of the values from samples containing single drugs. The presence of the studied drugs was monitored in waters sampled from two rivers in Calabria (Italy), collected along a period of seven months. Diphenhydramine and oxatomide were not detected in any samples. On the contrary, promethazine was found in two samples taken in July at a concentration of 2.0 and 2.3 μg/l, significantly below the values causing toxicity. Keywords: Acute toxicity, Bioluminescence inhibition, Diphenhydramine, Drug photodegradation, HPLC, Oxatomide, Promethazine, SPE, Surface waters, Vibrio fischeri test

Evaluation of heavy metal distribution and biological toxicity in agglomeration bed material during artificial waste incineration in fluidized bed

This study discusses the impact of different operating parameters on the bed material particle size and heavy metal distribution, and evaluates the impact of bed material heavy metal on the environment through TCLP and Vibrio fischeri test. The experimental results show that the bed material particle size distribution inclines to smaller particle sizes as the operating temperature increases. When there is Na, the particle size increases due to the agglomeration of eutectic. As for the heavy metal distribution, the combination of the fine particle sizes ( 0.84 mm) and fine (<0.59 mm) particle sizes have the maximum leaching concentration. As for the biological toxicity, when the temperature is 700 °C or Na concentration is 0.3%, the biological toxicity is at its maximum, which may due to a high accumulation of heavy metals.

Sunlight‐Assisted Fenton Reaction Catalyzed by Gold Supported on Diamond Nanoparticles as Pretreatment for Biological Degradation of Aqueous Phenol Solutions

Gold nanoparticles supported on Fenton-treated diamond nanoparticles (Au/DNPs) have been reported as one of the most efficient solid catalysts effecting the Fenton reaction, achieving a turnover number (TON) as high as 321,000. However, at room temperature the main limitation for the catalytic activity of Au/DNPs is the pH of the solution, which should be less than 5. In this paper, we report that exposure of Au/DNPs to sunlight enhances the catalytic activity of Au/DNPs up to the point that it can promote the Fenton reaction at room temperature even at slightly basic pH values. Also, in addition to performing a deep Fenton treatment and considering that the excess of H(2)O(2) used in the process should be minimized, we have achieved in our study, using a mild Fenton reaction promoted by Au/DNPs under sunlight irradiation, an optimum in the biodegradability, a minimum in the ecotoxicity, and no toxicity for the Vibrio fischeri test. The results have shown that, by using an H(2)O(2) -to-phenol molar ratio of 5.5 or higher, it is possible to achieve a high biodegradability as well as a complete lack of ecotoxicity and of Vibrio fischeri toxicity. The stability of Au/DNPs was confirmed by analyzing the gold leached to the solution and by performing four consecutive reuses of the catalyst with initial pH values ranging from 4 to 8. It was observed that, after finishing the reaction and exhaustive washings with basic aqueous solutions, the initial reaction rate of the used catalyst is recovered to the value exhibited by the fresh solid. Overall, our study shows that the synergism between catalysis and photocatalysis can overcome the limitations found for dark catalytic reactions and that the reaction parameters can be optimized to effect mild Fenton reactions aimed at increasing biodegradability in biorecalcitrant waste waters.

Computerized sperm motility analysis in toxicity bioassays: A new approach to pore water quality assessment

The aim of this study was to test the sensitivity of computerized sperm motility analysis in the sea urchin Paracentrotus lividus as the endpoint in toxicity bioassays. The tested matrices were pore water samples collected in an agriculture-impacted Mediterranean lagoon, Lake Varano (Italy). Two standardized bioassays were also conducted as controls, the P. lividus spermiotoxicity test and the Vibrio fischeri (Microtox ®) test. VCL (curvilinear velocity), VSL (straight line velocity), VAP (average path velocity), and the percentage of rapid spermatozoa recorded by the Sperm Class Analyzer ® system showed high sensitivity and discrimination ability, to a degree comparable with the larval development endpoint of the spermiotoxicity test. The test evaluated in this study requires small volumes of matrices, involves minimal sample manipulation, and can easily be extended to many other bioindicator species. It may therefore be considered a promising “quick response tool” following hazardous events that may adversely affect an aquatic ecosystem.

Sun light degradation of 4-chloroaniline in waters and its effect on toxicity. A high performance liquid chromatography – Diode array – Tandem mass spectrometry study

This paper studies the degradation reactions that 4-chloroaniline can naturally undergo in waters for the action of sun light. 10.00 mg L −1 4-chloroaniline aqueous solution, without any addition of organic solvent, are undergone to photoirradiation under conditions that simulate sun light. The degradation pathway, followed by HPLC-DAD–MS/MS methods, is complex since the pollutant gives rise to many photoproducts: the predominant species are characterized by m/ z values of 217 (P5) and 218 (P6) and are compatible with dimeric structures of 4-chloroaniline. Vibrio fischeri tests indicate that the photoproducts of 4-chloroaniline are characterized by a toxicity level significantly greater than the precursor.

Air pollution impact assessment on agroecosystem and human health characterisation in the area surrounding the industrial settlement of Milazzo (Italy): a multidisciplinary approach

In order to evaluate the impact of atmospheric pollutants emitted by the industrial settlement of Milazzo (Italy) on agriculture, sulphur dioxide and ozone levels in air were monitored and the data were used to estimate yield losses of the most widespread cultures. Trace element concentrations in crops and soils were also detected and metabolic profiles of soil microbial communities were considered. Vibrio fischeri test was used to appraise airborne pollutant ecotoxicity and epidemiological studies on causes of death distribution were carried out to characterize health state of people living in the area. All the sampling points were selected in farms on the basis of a theoretical meteo-diffusive model of industrial air pollutants. Experimental SO2 and O3 values mainly exceeded the threshold established by Italian and EU regulations to protect vegetation and they correspond to estimated significant crop losses. Conversely toxic element residues in soils and in agroalimentary products were generally lower than the fixed values. SO2 and O3 concentrations, toxic element contents and ecotoxicity levels of airborne pollutants were not related only to industrial site emissions, while the fluctuations on metabolic profiles of soil microbial communities seem to agree with the predicted deposition of xenobiotic compounds from the industrial plants. The epidemiological study evidenced a better health state of populations living in the investigated area than in the Messina province and the Sicily region but, inside the area, males living in the municipalities closest to the industrial settlement exhibited a worst health state than those in the very far ones.

Toxicity evaluation with Vibrio fischeri test of organic chemicals used in aquaculture

The evaluation of acute toxicity by Vibrio fischeri test for different organic chemicals (antibiotics, pesticides, therapeutants, herbicides) commonly applied in aquaculture and a degradation product of surfactants, 4-nonylphenol, is presented in this work. Simazine, atrazine, emamectin benzoate and leucomalachite green have no toxic effects on V. fischeri at the concentration tested (up to 6mgl(-1)) which correspond to the maximum water solubility. Ciprofloxacin, terbutryn and deltamethrin, caused inhibition effects of 28%, 22% and 30% at concentrations up to 5mgl(-1). Toxic effects were not observed in the case of flumequine and oxolinic acid at the maximum concentration tested (0.189mgl(-1)). According to the toxicity categories established in the EU legislation, ciprofloxacin, terbutryn and deltamethrin could be considered non-harmful for V. fischeri. Malachite green and 4-nonylphenol are "very toxic to aquatic organisms" (EC(50,30min)=0.031mgl(-1) and 0.48mgl(-1), respectively). Carbaryl is "toxic to aquatic organisms" (2.4mgl(-1)). and glyphosate is harmful to V. fischeri (EC(50,30min)=44.2mgl(-1)). The matrix effect was evaluated comparing the toxicity measurements of the target compounds solubilized in seawater and distilled water. Malachite green, 4-nonylphenol and glyphosate, showed higher toxicity in distilled water than in seawater. Carbaryl was more toxic in seawater. All the compounds tested in seawater were not harmful at concentrations of ngl(-1) (10 and 50). However, 4-nonlylphenol and malachite green may act as toxic compounds in the environment at a low ppb level, since both may be detected in water at this concentration level.

Pretreatment of Highly Polluted Pharmaceutical Waste Broth by Wet Air Oxidation

The preoxidation of a highly polluted waste pharmaceutical fermentation broth using wet air oxidation (WAO) has been studied as a possible method for the effective removal of organics. The applied (pre)treatment method should enhance the biotreatability of the pharmaceutical fermentation broth in terms of reduced initial toxicity and higher biodegradability. Preliminary experiments in the pilot biological treatment plant were successful only at low organic loads, whereas the system collapses at higher ones. The characterization of the fermentation broth was started by common physicochemical analysis, whereas several bioassays were used to determine its impact on biological treatment plants and the environment. Toxicity prior to and after WAO was determined using the acute Vibrio fischeri test, measurement of inhibition of O2 consumption, and the Daphnia magna acute test. Ready biodegradability of the treated and untreated broth has also been assessed. WAO experiments were accomplished in the 2 L batch rea...

Microbial activities in soils of a former sawmill area

To find out microbial metabolic functioning and toxicity in a former sawmill area, carbon dioxide evolution, methane oxidation potential, 10 hydrolytic enzyme activities, Vibrio fischeri test, fluorescein diacetate hydrolysis activity (FDA), soil pH, carbon, nitrogen and pentachlorophenol (PCP) content were measured at four sites. The area is contaminated with aged chlorophenols. Chlorophenol content of soil was analyzed with a novel HPLC-MS technique, which allowed to measure chlorophenols without derivatization. The sites had a pollution gradient from 0.5 to 15 microg PCP g dw of soil(-1). Endogenous carbon dioxide evolution, methane oxidation potential, butyrate-esterase, acetate-esterase, sulphatase and aminopeptidase activities were lower at the site 2 than 3, although the site 2 and 3 had similar content of carbon and nitrogen. The soil was toxic in V. fischeri test at the site 2, which had high content of PCP (3.93+/-1.00 microg PCP g dw of soil(-1)). The results indicated that endogenous carbon dioxide evolution, methane oxidation potential, butyrate-esterase, acetate-esterase, sulphatase and aminopeptidase activities were sensitive to PCP in the soil. The results indicated that alpha-glucosidase, beta-glucosidase, beta-xylosidase, beta-cellobiosidase, phosphomonoesterase, N-acetyl-glucosaminidase activity and FDA hydrolysis activity were not sensitive to PCP in the soil. Soil processes involved in the cycling of carbon, nitrogen, sulphur and phosphorus were only slightly vulnerable in the former sawmill area and most sensitive microbial species were probably replaced with more tolerant ones to maintain and recover functioning of the former sawmill soils.