Various species of Basidiomycota fungi reveal different abilities to degrade pharmaceuticals and also different pathways of degradation.

The presence of pharmaceuticals (PhACs) in the aquatic environment is an emerging problem worldwide. PhACs reach surface water via the effluents of wastewater treatment plants (WWTPs). WWTPs, although able to remove organic pollutants, do not always remove PhACs. Currently, in the treatment of sewage with the activated sludge method, numerous microorganisms are used, mostly bacteria. Nevertheless, these microorganisms are not resistant to many drug contaminants, and some may also pose a risk to human health. White-rot fungi (WRF), which degrade a wide spectrum of environmental pollutants, may be used as an alternative to microorganisms. However, little data exists comparing the removal of various PhACs by different WRF. In this study, we aimed to determine the ability of three WRF Basidiomycota species, Armillaria mellea, Phanerochaete chrysosporium, and Pleurotus ostreatus, to remove PhACs from various therapeutic groups over the course of 1 h-4 days. Additionally, we identified the fungal metabolites of PhACs, proposed the degradation pathways, and assessed the toxicity of the post-culture media. All selected WRF removed PhACs, but the degree of removal depended on WRF species and PhACs type. Antidepressants and immunosuppressants were removed most efficiently by P. ostreatus, cardiovascular drugs and sulfamethoxazole by A. mellea, and erythromycin by P. chrysosporium. The vast differences observed highlight the need for more intensive testing of different WRF species to select the best species for removing pharmaceuticals of interest. The structure of metabolites generated during degradation strongly depended on WRF species, but the most frequent xenobiotic transformations were oxidation and dealkylation. The obtained results gave insight into the substrate specificity of selected WRF while also providing a broad extension of the knowledge of pharmaceutical degradation by A. mellea.

Multi- and unilamellar liposomal encapsulation of ciprofloxacin as ways to modify its phototoxicity and photodegradation.

Liposomes are vesicular preparations that improve bioavailability of many pharmaceuticals, used even in ocular therapy. In addition, it is well documented that vesicular carriers could affect the photodegradation of molecules encapsulated inside, which is especially important for drugs that may exhibit phototoxicity when they are applied topically on sensitive light-exposed tissues. In this study, we investigated the effect of ciprofloxacin encapsulation into liposomes on its photodegradation, phototoxicity and photogenotoxicity in vitro at the concentration ranges applied in ophthalmology. We tested two variants of liposomes: large unilamellar vesicles (LUV) and multilamellar vesicles (MLV) in comparison to antibiotic solutions without phospholipids (CPX). On the basis of our research, the kinetics of ciprofloxacin photolysis was the fastest in formulations with vesicles with low drug-to-lipid ratio. Depending on vesicles type (drug-to-lipid ratio, MLV or LUV) and time of irradiation different degradants were produced. We proposed structures of the novel ciprofloxacin photolysis products characteristic for vesicles. We did not notice any photoprotective effect of application of ciprofloxacin encapsulation into liposomes, but it significantly affected the photodegradation product profile of the drug and the Photo-Irritation-Factor of the vesicular preparations. In the MTT and micronucleus assays impact of encapsulation was not as clearly visible.

Analytical, toxicological and kinetic investigation of decomposition of the drug diclofenac in waters and wastes using gamma radiation.

The radiolytic decomposition of the drug diclofenac (DCF), and in limited extent, also two other widely used drugs, ibuprofen and carbamazepine, was examined using liquid chromatography (LC) methods. The efficiency of DCF decomposition was examined in function of the absorbed dose of gamma radiation, and also in the presence of selected scavengers of radicals, which are commonly present in natural waters and wastes. Three different tests were employed for the monitoring of toxicity changes in the irradiated DCF solutions. The LC/mass spectrometry (MS) was used for the determination of products of DCF radiolysis. Using pulse-radiolysis method with the spectrophotometric detection, the rate constant values were determined for reactions of DCF with the main products of water radiolysis: hydroxyl radicals (1.24 ± 0.02) × 10(10) M(-1) s(-1) and hydrated electrons (3.1 ± 0.2) × 10(9) M(-1) s(-1). Their values indicate that both oxidative and reductive processes in radiolytic decomposition of DCF can take place in irradiated diluted aqueous solutions of DCF. The possibility of decomposition of all examined analytes was investigated in samples of river water and hospital waste. Compared to the previous studies, the conducted measurements in real samples were carried out at the concentration levels, which are close to those reported earlier in environmental samples. Graphical abstract ᅟ.

An assessment of the genotoxic effects of landfill leachates using bacterial and plant tests.

Two bacterial tests (the Ames test and the umu-test) and the Allium test were used to assess the genotoxicity of aqueous leachates from municipal solid waste landfill sites in Southern Poland. A comparison of the sensitivity of the applied tests was performed. None of the tested samples revealed genotoxic activity in the umu-test and thus did not appear to be sensitive enough for evaluations of leachates. Two out of 22 leachates were described as genotoxic in the Ames test and 3 out of 6 leachates in the Allium test. All of the analyzed leachates samples affected cell divisions. Two of the tests applied, the Allium and Ames test, revealed the high genotoxicity of leachate 4. Among the bioassays used in these studies, the Allium test proved to be more sensitive than bacterial tests for the investigation of leachate toxicity. The results suggest that the Allium cytogenetic bioassay is efficient and simple for genotoxicity studies of leachates. The potential correlations between the chemical characteristics and genotoxic effects is discussed. The biological effects of selected appeared to be related to chemical parameters. Leachates have a genotoxic potential and pose a risk to human health and the environment. A combination of biotests and chemical analyses is the best approach for the assessment of the risk or impact of leachates.

Radiolytic decomposition of pesticide carbendazim in waters and wastes for environmental protection.

The radiolytic degradation of widely used fungicide, carbendazim, in synthetic aqueous solutions and industrial wastewater was investigated employing γ-irradiation. The effect of the absorbed dose, initial concentration and pH of irradiated solution on the effectiveness of carbendazim decomposition were investigated. Decomposition of carbendazim in 100 µM concentration in synthetic aqueous solutions required irradiation with 600 Gy dose. The aqueous solutions of carbendazim have been irradiated in different conditions, where particular active radical species from water radiolysis predominate. The obtained data have been compared with the kinetic modeling. The reversed-phase high-performance liquid chromatography was used for the determination of carbendazim and its radiolytic decomposition products in irradiated solutions. The changes of toxicity of irradiated solutions were examined with different test organisms and human leukemia cells.

Application of microbial assay for risk assessment biotest in evaluation of toxicity of human and veterinary antibiotics.

The microbial assay for risk assessment (MARA) is a multispecies, growth inhibition microplate toxicity test with 11 microorganisms individually lyophilized in microplate wells. The microbial species representing wide diversity, generated 11 microbial toxic concentration (MTC) values were obtained giving a unique "toxic fingerprint" profile of the test sample. The toxicity of 14 antibiotics was evaluated with the MARA test. The fingerprints for each group of antibiotics differ, indicating a particular toxic profile. Tetracyclines were the most toxic antibacterials with the minimum MTC value of 3.6 µg L(-1). In the group of tetracyclines the order of the three most sensitive microbial strains was the same 2 > 6 > 7. Quinolones affected the most sensitive strain(s) at concentrations of 12-75 µg L(-1). The MARA bioassay exhibited different spectra of toxic responses to microbial strains for the first and second generation quinolones. However, for first generation quinolones strain 6 was substantially more sensitive than the other microorganisms, while second generation quinolones were most toxic to strain 3, followed by 6 then 4. In this instance, the three strains belong to two different phylogenetic groups-strain 3 is γ-proteotype and strains 4 and 6 are ß-proteotype.

Application of a microbiotests battery for complete toxicity assessment of rivers.

Acute hazard classification based on selected microbiotests was proposed to assess and compare the toxicity of rivers including surface-water, sediment and soil from floodplains. No direct relationship between the classification of pollution for surface-water based on physical-chemical parameters and proposed acute hazard classification based on organisms' sensitivity was observed. The quality of water according to hazard classification was better than in the mandatory classification of pollution, with the domination of Class II (slight acute hazard). The samples of sediment and soil were more toxic and represented Class II or Class III (acute hazard). The results indicated a need to complete the mandatory monitoring of surface-water in rivers with biological monitoring with toxicity assessment of rivers including water, sediment and soil from floodplains based on acute hazard classification. This integral approach enables a complete evaluation of the toxicity of aquatic life together with an estimation of negative changes in river systems.

Radiolytic degradation of the herbicide dicamba for environmental protection.

The radiolytic degradation of the widely used herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid), employing gamma irradiation in laboratory batch conditions and with a beam of accelerated electrons in flow-through installation, was investigated. The effects of dose magnitude, ozone or hydrogen peroxide in irradiated solution, and scavengers such as nitrate and hydrogen carbonate on the effectiveness of dicamba decomposition and the products formed were investigated. Changes in the toxicity of irradiated solutions were measured with the Microtox and Spirotox toxicity tests. The application of radiolytic degradation was also examined for decomposition of herbicides in commercial agrochemical preparations.

A comparison of sensitivity of spirotox biotest with standard toxicity tests.

The Spirotox test utilizes a large ciliate protozoan, Spirostomum ambiguum, as a bioindicator. Two kinds of test responses were observed after 24 and 48 h of incubation: different deformations and lethal response. The toxicity of 78 organic compounds evaluated by the Spirotox-volatile procedure varied over six orders of a magnitude from -log (24-h LC(50)) = -3.0 (methanol) to 3.0 (pentachlorophenol). Deformations of the S. ambiguum were two to three times more sensitive toxic response than lethality. The sensitivity of the Spirotox test was compared to four bioassay systems used worldwide: Tetrahymena pyriformis, Microtox, Daphnia magna, and Pimephales promelas. The toxicants were sorted according to the mechanism of action. The toxicity data for both protozoa. T. pyriformis and S. ambiguum were almost identical. Microtox, D. magna, and P. promelas were in general six- to eightfold more sensitive than the 24-h LC(50) Spirotox. The best agreement of toxicity data appeared in the group of nonpolar narcotics. In contrast the toxicity data for all tests only slightly correlated for polar narcotics. Very low slope for polar narcotics indicates that the toxicity range was much broader for the Spirotox test comparing to the others. The most toxic polar narcotics were even more toxic to Spirotox than to Microtox, Daphnia, and fish.

Tests for the toxicity assessment of cyanobacterial bloom samples.

Cyanobacterial (blue-green algal) blooms are one of the common consequences of the increasing eutrophication of surface waters. The production of cyanobacterial toxins and their presence in drinking and recreational waters represents a growing danger to human and animal health. Due to a lack of toxin standards and to resource limitations on the wide-scale use of analytical methods (e.g., high-performance liquid chromatography, enzyme-linked immunosorbent assay (ELISA)) in cyanobacterial toxin monitoring, it is necessary to assess and to develop additional methods for their detection and estimation. Microbiotests using invertebrates offer a possible approach for the inexpensive and straightforward detection and assessment of cyanobacterial bloom toxicity. Three microbiotests with: Thamnocephalus platyurus, Daphnia magna, and Spirostomum ambiguum were examined with bloom samples containing hepatotoxic microcystin-LR and up to five additional microcystin variants. Two kinds of cyanobacterial bloom sample preparations were tested: crude extracts (CE) and purified extracts (PE). The highest toxicity was found when CE was used for microbiotests. The sensitivity of microorganisms decreased from S. ambiguum to T. platyurus and to D. magna. A statistically significant correlation was found between microcystin concentration and T. platyurus biotest, and between mouse bioassay and S. ambiguum results. Addition of Me2SO (1%, v/v) is a possible method to increase the sensitivity of the microorganisms for microcystin-LR.

Spirotox--a new tool for testing the toxicity of volatile compounds.

A new method for estimating the toxicity of volatile compounds was developed. The test was carried out in the disposable polystyrene multiwells. After the organisms, protozoa Spirostomum ambiguum, were added to the wells, microplate was tightly closed using silicone grease and polyethylene film. The toxicities of 21 organic compounds were estimated. No control mortality was observed in all cases. Transparent PE film enabled good observation of test response. The toxicity of tested compounds varied over 4 orders of magnitude. Deformations were 2-4 more sensitive toxic response then lethality. The toxicity of tested compounds in Spirotox test correlates well with the log Kow and toxicity results from other bioassays: Microtox, D. magna and T. pyriformis.

Toxicity of inorganic compounds in the Spirotox test: a miniaturized version of the Spirostomum ambiguum test.

The Spirostomum ambiguum toxicity test has been intensively studied in the Department of Environmental Health Sciences, Warsaw University of Medicine for the last 5 years. The purpose of the present work was to develop and evaluate a miniaturized microplate version of the test, called the Spirotox test, and to estimate the toxicity of selected inorganic compounds to the Spirostomum ambiguum. The test was carried out in conventional 24-well (6 x 4) polystyrene multiwell plate. Preliminary test was one control and 11 toxicant concentrations with two duplicates. Definitive test was one control and five toxicant concentrations with three duplicates per concentration. Dilution of the sample was made directly in the plate. Toxicity series of heavy metals based on 24-h LC50 may be established as follows: Cu > Ag > Hg > Cr > Cd > Zn > Ni > Pb > Co > Mn. The series may be divided into four classes: extremely toxic: below 0.1 ppm (Cu, Ag, Hg); very toxic: 0.1-1.0 ppm (Cr, Cd, Zn, Ni); toxic: 1.0-10 ppm (Pb, Co); and low toxic: above 10 ppm (Mn). Anions were much less toxic to S. ambiguum than cations. Using the same classification, only cyanide (CN) was toxic, other anions were low toxic. Toxicity series based on 24-h LC50 may be established as follows: CN > SeO3 > Cr2O7 > NO2 > S2O3 >WO4 > BO3.

Evaluation of toxicity of medical devices using Spirotox and Microtox tests: I. Toxicity of selected toxicants in various diluents.

Significant effort has been directed toward developing in vitro alternatives, which can be the first step of toxicity analysis. Tissue culture assays are currently the most popular in vitro tests for evaluating acute toxicity. The possibility of applying two bioassays using microorganisms in assessing the toxicity of extracts of medical devices was investigated. The Microtox test system--a luminescent bacteria toxicity test--assesses changes in light output from a luminescent bacteria, Vibrio fischeri. Spirotox used a large ciliate protozoan: Spirostomum ambiguum. The most widely used extraction solvent, 0.9% NaCl, must be concentrated up to 2% for Microtox and diluted nine times for the Spirotox test. The organic solvents ethanol, DMSO, and polyethylene glycol 400 were not toxic in either test in concentrations of 1-2%. The toxicity of reference compounds Hg, Cd, Zn, Pb, and SDS was examined in various diluents. The sequence of toxicity of the tested compounds in Spirotox and Microtox was: Hg > Cd > Zn > Pb > SDS, and Hg > Pb = Zn > SDS > Cd, respectively. Addition of organic solvents changed the toxicity of compounds tested in 60% of Spirotox tests and only in 25% of Microtox tests. Changes were low, not exceeding 100% in almost all cases. No correlation was observed between diluent and toxicant in either bioassay.