Toxicity evaluation of single and mixed antifouling biocides measured with acute toxicity bioassays

Antifouling biocides used in boat paints were analyzed with a battery of toxicity bioassays to evaluate the toxic effects of these compounds on Vibrio fischeri, Daphnia magna and Selenastrum capricornotum. The antifoulants tested were Irgarol 1051, Kathon 5287, chlorothalonil, diuron, dichlofluanid, 2-thiocyanomethylthiobenzothiazole (TCMTB) and tributyltin (TBT). In most cases, the sensitivity of the organisms towards the toxicants followed the order: S. capricornotum > D. magna > V. fischeri. Toxicity by concentration level had the following order: TBT=Kathon 5287>chlorothalonil>Irgarol 1051>diuron>dichlofluanid>TCMTB for S. capricornotum. For D. magna (48 h test), the toxicity order of compounds was TBT>Kathon 5287>chlorothalonil>TCMTB>dichlofluanid>Irgarol 1051>diuron. For V. fischeri (30 min test), the compound toxicity had the following order: Kathon 5287>TBT>TCMTB>dichlofluanid>Irgarol 1051>chlorothalonil.Degradation products of Irgarol 1051 and diuron were also tested. Degradation product of Irgarol 1051 was found to be less toxic to the crustacean and the microalga but more toxic to the bacterium. Degradation products of diuron were less toxic to the microalga in comparison with the bacterium. For mixtures of compound, toxicities were additive in only 33% of the cases and 21% of mixtures were less toxic than expected based on the sum of concentrations of toxicants (antagonistic effect). Synergistic enhancements of toxicity were observed for a majority (46%) of the mixtures.The average reproducibility of the EC₅₀ and LOEC measurements was 27, 24 and 28%, respectively, in the V. fischeri, S. capricornotum and D. magna bioassays. For single compound, the reproducibility of EC₅₀ was better than ±20% for a vast majority of the measurements with the V. fischeri system, thus agreeing closely with the reported reproducibility values for this relatively well-known assay.     

Comparative evaluation of the effects of pesticides in acute toxicity luminescence bioassays

Acute toxicity of pesticides in water was assessed singly and in mixtures using the responses of the luminescent bacterium Vibrio fischeri (BioTox™), the aquatic invertebrate Daphnia magna (Daphtoxkit™), and the MitoScan™ assay. The latter utilized fragmented mitochondria to enzymatically convert β-nicotinamide adenine dinucleotide (NADH) to its oxidized form, NAD⁺. The rate of the conversion being sensitive to type and concentration of toxicants. The pesticides tested were Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate), Cyromazine (N-cyclopropyl-1,3,5-triazine-2,4,6-triamine), Fenamiphos (ethyl 4-methylthio-m-tolyl isopropylphosphoramidate), and Formetanate (3-dimethylaminomethyleneiminophenyl methylcarbamate). The toxicity bioassays were characterized in terms of relative sensitivity, reproducibility, range of the linear response, and the ability to reveal synergistic/antagonistic interactions among toxicants. The D. magna assay was the most sensitive and best able to detect toxic interactions of mixtures. Also, unlike the other assays used, the response of the daphnid system was linear over a 10-fold change in pesticide concentration. Relative to the BioTox™, the MitoScan™ was 2- to 11-fold more sensitive for the compounds and mixtures tested. The EC₅₀ reproducibility of all tests was within ±20% coefficient of variation; however, the lowest observable effect concentration (LOEC) were only reproducible to ±35% on average. Cyromazine was the least toxic of the pesticides tested. To test the predictive value of the concept of concentration addition, toxicities of binary and quaternary mixtures of four different pesticides were analyzed. Synergistic/antagonistic responses were most frequently observed in testing with D. magna. Synergistic/antagonistic effects were seen only in 25 and 50% of the cases with the BioTox™ and the MitoScan™ assays, respectively.     

Application of ring study: water toxicity determinations by bioluminescence assay with Vibrio fischeri

Acceptance of toxicity bioassays as effective analytical tools in environmental areas needs guarantees of standardization but also validation. Ten European laboratories took part in an inter-laboratory study using different commercial devices based on bioluminescence inhibition of bacteria Vibrio fischeri. Reproducibility and stability by short toxicity endpoints, effective concentration that gives 10%, 50% and 80% of inhibition (EC₁₀, EC₅₀ and EC₈₀) is evaluated. Parametric and non-parametric statistic is applied and performance of participant laboratories is addressed by z-scores calculated by non-parametric statistic. z-Score classification was based on harmonised protocol for proficiency testing of analytical laboratories (satisfactory |z| ≤ 2; questionable 2 < |z| ≤ 3; unsatisfactory |z| > 3). Tested samples were phenol, 3,5-dichlorophenol and influent wastewater. Based on z-score classification, more than 70% of the laboratories showed a satisfactory performance for phenol, 3,5-dichlorophenol and influent wastewater (86%, 90% and 70%, respectively). Reproducibility and stability was observed in toxicant references and in wastewater samples. EC₈₀ determination appears to be more robust that EC₁₀ and EC₅₀. EC determinations can be considered favorable at 5 and 15 min of exposition, in particular for EC₈₀. The use of different commercial devices can not be considered an additional source of variation.     

European ring exercise on water toxicity using different bioluminescence inhibition tests based on Vibrio fischeri, in support to the implementation of the water framework directive

An inter-laboratory comparison exercise was conducted under the European Union funded project entitled: Screening Methods for Water Data Information in Support of the Implementation of the Water Framework Directive (SWIFT-WFD) and coordinated by the Consejo Superior de Investigaciones Científicas (CSIC), in order to evaluate the reproducibility of different toxicity tests based on the bioluminescence inhibition of Vibrio fischeri, for the rapid water toxicity assessment.For the first time, this type of exercise has been organized in Europe, and using different tests based on the same principle. In this exercise, 10 laboratories from 8 countries (Austria, Cyprus, Germany, Greece, Italy, Portugal, Romania, and Spain) took place, and a total number of 360 samples were distributed.During the exercise, six series of six samples were analyzed along 5 months. Every batch of samples was composed by three real samples and three standard solutions. The real samples were: a raw influent and the effluent of a wastewater treatment plant (WWTP), and a sample from a first settlement of the WWTP spiked with a mixture of toxicant standards.A final number of 330 (91.7%) samples was analyzed, 3300 values in duplicate were collected, and the results for each sample were expressed as the 50% effective concentration (EC₅₀) values calculated through five points of dilution inhibition curves, after 5 and 15 min of incubation times.A statistical study was initiated using 660 results. The mean values, standard deviations (σ), variances (σ²), and upper and lower warning limits (UWL and LWL) were obtained, using the EC₅₀ values calculated with the result from the participating laboratories.The main objectives of this toxicity ring study were to evaluate the repeatability (r) and reproducibility (R) when different laboratories conduct the test, the influence of complex matrix samples, the variability between different tests based on the same principle, and to determine the rate at which participating laboratories successfully completed tests initiated.In this exercise, the 3.93% toxicity values were outliers according with the Z-score values and the Dixon test. The samples with the greater number of outliers were those with the smallest variability coefficient, corresponding to the greater and the smaller toxicity level.No relation was found through the cluster analysis, between the final results and the different commercial devices involved. Testing by multiple commercial devices did not appear to reduce the precision of the results, and the variability coefficient for the exercise was nearby to the average value for past editions carried out at national level, where the different participants used the same commercial device.Stability of samples was also followed during the exercise. While statistical significance differences were not found for the greater part of samples, for the sample from the WWTP influent, a significant decrease of the toxicity value was found along this study. Nevertheless, this was a type of sample with a high toxicity level during all the exercise.On the other hand, in order to obtain the chemical characterization of real samples, those were analyzed by chromatographic techniques, using different sequential solid phase extraction (SSPE) procedures, followed by liquid chromatography coupled with mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). Good agreement was found between the chemical analysis results and the toxicity level of the samples.     

Selective immobilization of Acinetobacter junii on the natural zeolitized tuff in municipal wastewater

The immobilization of desired bacteria onto material was usually performed in synthetic media. The aim of this study was to test the immobilization of phosphate (P)-accumulating bacteria Acinetobacter junii onto natural zeolitized tuff (NZ) in the raw or sterilized municipal wastewater containing the common bacteria Escherichia coli and Enterococcus faecalis and the performance of immobilized A. junii in the same type of wastewater. In the sterilized wastewater which contained the mixture of A. junii, E. coli and E. faecalis, the A. junii was selectively immobilized onto NZ in significantly higher numbers than E. coli and E. faecalis. The A. junii added in the form of bioparticles to the wastewater containing E. coli and E. faecalis, multiplied and removed P from wastewater. The P removal from wastewater was a function of biomass of P-accumulating bacteria and not the amount of NZ or bioparticles used. The performance of A. junii was significantly better in membrane filtered than in autoclaved wastewater. The experiments that were performed in raw non sterilized wastewater showed that A. junii can be successfully immobilized onto NZ in competition with natively present heterotrophic bacteria, retain its metabolic activity and successfully remove P from such water, which makes this technology feasible from biotechnological aspect.     

Assessment of the acute toxicity of triclosan and methyl triclosan in wastewater based on the bioluminescence inhibition of Vibrio fischeri

In this work, the contributions of triclosan and its metabolite methyl triclosan to the overall acute toxicity of wastewater were studied using Vibrio fischeri. The protocol used in this paper involved various steps. First, the aquatic toxicities of triclosan and methyl triclosan were determined for standard substances, and the 50% effective concentrations (EC₅₀) were determined for these compounds. Second, the toxic responses to different mixtures of triclosan, methyl triclosan, and surfactants were studied in different water matrices, i.e., Milli-Q water, groundwater and wastewater, in order to evaluate (i) the antagonistic or synergistic effects, and (ii) the influence of the water matrices. Finally, chemical analysis was used in conjunction with the toxicity results in order to assess the aquatic toxicities of triclosan and its derivative in wastewaters. In this study, the toxicities of 45 real samples corresponding to the influents and effluents from eight wastewater treatment works (WWTW) were analyzed. Thirty-one samples were from a wastewater treatment plant (WWTP) equipped with two pilot-scale membrane bioreactors (MBR), and the influent and the effluent samples after various treatments were characterized via different chromatographic approaches, including solid-phase extraction (SPE), liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), and SPE coupled to gas chromatography–mass spectrometry (GC–MS). The toxicity was determined by measuring the bioluminescence inhibition of Vibrio fischeri. In order to complete the study and to extrapolate the results to different WWTPs, the toxicity to V. fischeri of samples from seven more plants was analyzed, as were their triclosan and methyl triclosan concentrations. Good agreement was established between the overall toxicity values and concentrations of the biocides, indicating that triclosan is one of the major toxic organic pollutants currently found in domestic wastewaters.     

Ecotoxicity of, and remediation with, engineered inorganic nanoparticles in the environment

This article presents recent developments on the use of inorganic nanoparticles (NPs) for environmental remediation in polluted soil, water and gas. The number of publications on these topics has grown exponentially in recent years, especially those focused on wastewater treatment. Among these topics, removal of metals has become the most popular, although some works relate to the use of nanomaterials for the elimination of nutrients (e.g., nitrogen and some persistent organic pollutants). However, this growth has not been accompanied by knowledge about the behavior of NPs once used and released into the environment. In this article, we also comment upon the current situation with respect to NP toxicology (nanotoxicology). A remarkable number of different toxicology tests has been applied to NPs, often making it very difficult to interpret or to generalize the results. We analyze in detail the bioluminescence, Daphnia magna and other tests, and give some preliminary results obtained in our work.     

A sensitive, rapid and inexpensive way to assay pesticide toxicity based on electrochemical biosensor

We reported a rapid toxicity assay method using electrochemical biosensor for pesticides, Escherichia coli (E. coli) was taken as a model microorganism for test. In this method, we adopted ferricyanide instead of natural electron acceptor O₂, and then microbial oxidation was substantially accelerated. Toxicity assays measured the effect of toxic materials on the metabolic activity of microorganisms. The current signal of ferrocyanide produced from the metabolism was proven to be directly related to the toxicity, which could be amplified by ultramicroelectrode array (UMEA). The ratio of the electrochemical signals, recorded in the presence and absence of toxin, provided an index of inhibition. Accordingly, a direct toxicity assessment (DTA) based on chronoamperometry was proposed to detect the effect of toxic chemicals on microorganisms. 3,5-Dichlorophenol (DCP) was taken as the reference toxicant, its IC50 was estimated to be 8.0 mg/L. Three pesticides were examined using this method. IC50 values of 6.5 mg/L for Ametryn, 22 mg/L for Fenamiphos and 5.7 mg/L for Endosulfan were determined and in line with EC50 values reported in the literature. Atomic force microscopy (AFM) was also used for morphology characterization of E. coli induced by three pesticides. These results confirmed the present electrochemical method used is reliable. In addition, the electrochemical method is a sensitive, rapid and inexpensive way for toxicity assays of pesticides.     

One-pot synthesis of multivalent arrays of mannose mono- and disaccharides

The synthesis of a selection of multivalent arrays of mannose mono- and disaccharides, that are of potential use as anti-infective agents against enterobacteria infections, is described.     

Determination of microcystin-LR in surface water using high-performance liquid chromatography/tandem electrospray ionization mass detector

The need for a rapid, sensitive, and reliable analytical method for microcystin-LR has been emphasized by the awareness of toxic cyanobacteria as a human-health risk through drinking water. Microcystin-LR is the most commonly reported microcystin which is produced by cyanobacteria. The WHO guideline for microcystin-LR in drinking water is 1 μg/l. In this paper, an effective method has been developed by application of high-performance liquid chromatography/tandem electrospray ionization mass detector in the determination of microcystin-LR in surface water sample. At the LC-MS-MS CID-full scan mode, different relative collision energies have been tested with 30% being used for further microcystin-LR analysis. The possible mass dissociation path has been proposed. Based on 30% relative collision energy, present method has an excellent method detection limit (MDL), which is as low as 2.6 ng/l. To the best of our knowledge, this represents one of the most sensitive methods in existence for the microcystin-LR analysis. This method has also been validated by evaluation of the calibration linearity, precision, accuracy, recovery, and mass ratio stability.     

Aggregation and dispersion of silver nanoparticles in exposure media for aquatic toxicity tests

Silver nanoparticles (AgNPs) are currently being very widely used in industry, mainly because of their anti-bacterial properties, with applications in many areas. Once released into the environment, the mobility, bioavailability, and toxicity of AgNPs in any ecosystem are dominated by colloidal stability. There have been studies on the stability or the aggregation of various nanoparticles (NPs) under a range of environmental conditions, but there is little information on fully characterised AgNPs in media used in (eco)toxicity studies. In this study, monodisperse 7, 10 and 20 nm citrate-stabilised AgNPs were synthesised, characterised and then fractionated and sized by flow field-flow fractionation (FFF) and measured with dynamic light scattering (DLS) in different dilutions of the media recommended by OECD for Daphnia magna (water flea) toxicity testing. Stability of NPs was assessed over 24 h, and less so over 21 days, similar time periods to the OECD acute and chronic toxicity tests for D. magna. All particles aggregated quickly in the media with high ionic strength (media1), resulting in a loss of colour from the solution. The size of particles could be measured by DLS in most cases after 24h, although a fractogram by FFF could not be obtained due to aggregation and polydispersity of the sample. After diluting the media by a factor of 2, 5 or 10, aggregation was reduced, although the smallest NPs were unstable under all media conditions. Media diluted up to 10-fold in the absence of AgNPs did not induce any loss of mobility or fecundity in D. magna. These results confirm that standard OECD media causes aggregation of AgNPs, which result in changes in organism exposure levels and the nature of the exposed particles compared to exposure to fully dispersed particles. Setting aside questions of dose metrics, significant and substantial reduction in concentration over exposure period suggests that literature data are in the main improperly interpreted and nanoparticles are likely to have far greater biological effects than suggested thus far by poorly controlled exposures. We recommend that the standard OECD media is diluted by a factor of ca. 10 for use with these NPs and this test media, which reduces AgNP aggregation without affecting the viability of the text organism.     

Synthesis, structural and larvicidal studies of some triorganotin 2-(p-chlorophenyl)-3-methylbutyrates

A series of triorganotin 2-(p-chlorophenyl)-3-methylbutyrates, (R₃SnO₂CCH(CH(CH₃)₂)C₆H₄Cl-4), where R = methyl, ethyl, n-propyl, n-butyl, phenyl and cyclo-hexyl, have been synthesized. Elemental analyses, Mössbauer, Infrared and NMR spectroscopies have been used to characterize their structures. Based on the spectroscopic results, all the complexes with the exception of the tricyclohexyl compound were found to be five-coordinated in the solid state while the tricyclohexyltin derivative was determined to be four-coordinated. Structural assignments based on spectroscopic data are supported by the crystallographic results of four of the triorganotin butyrates (trimethyl-, tri-n-propyl-, tri-n-butyl- and tricyclohexyltin 2-(p-chlorophenyl)-3-methylbutyrate). Multinuclear NMR spectroscopy studies indicated that all the complexes were tetrahedral in solution. Larvicidal activities of the complexes were evaluated against the 2nd instar stage of the Anopheles stephensi, Aedes aegypti and Culex pipiens quinquefasciatus mosquitoes. The toxicity data indicate that there does not appear to be any significant differences of the compounds towards the different mosquito species based on their averaged toxicity values. In addition, the toxicity of the triorganotin compounds towards the mosquito larvae was concluded to be dependent on both the compound and the species of mosquito larvae.     

Advanced mass spectrometric methods applied to the study of fate and removal of pharmaceuticals in wastewater treatment

This overview is of analytical methodologies based on gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, applied in environmental monitoring of pharmaceutical residues and their known degradation products. We also consider the ability of time-of-flight (TOF) and quadrupole-TOF instruments to provide sufficiently accurate-mass measurements and full-scan spectra for unequivocal confirmation of target compounds and investigation of their degradation products, which are either known or unknown.

We focus attention on the fate and the behavior of pharmaceutical residues during conventional and advanced wastewater treatments. Wastewater-treatment plants are designed to remove conventional pollutants (e.g., suspended solids and biodegradable organic compounds), but not low concentrations of synthetic pollutants (e.g., pharmaceutically active compounds).

Membrane bioreactor systems represent a new generation of processes that have proved to outperform conventional activated sludge treatment in terms of sludge production and effluent quality. In the past few years, there has been much attention paid to their capability for removing trace organic contaminants from sewage. This review highlights their improved performance in removing pharmaceutical residues from wastewater compared to conventional treatment.     

Rolling--a new application technique for luminescent bacteria on high-performance thin-layer chromatography plates

High-performance thin-layer chromatography (HPTLC) coupled with bioluminescence detection using Vibrio fischeri bacteria can be used for screening for unknown substances. This is accomplished by dipping the HPTLC plate in an aqueous bacteria solution. Especially polar substances, however, can start to dissolve during this process, which leads to blurring and tailing of the zones on the plate. To overcome this disadvantage, we applied the bacteria solution by rolling. This method has been described for chemical derivatizations, but is very rarely used. The rolling device was made of commercially available household articles. Using octhilinone and methylparaben as test compounds, rolling was compared with dipping. Despite of performing the rolling process manually, the results were reproducible. Depending on the substance and its amount on the HPTLC plate, peaks were narrower, up to a factor of 4 higher and with a higher signal-to-noise ratio than after dipping.     

Microbially induced separation of quartz from calcite using Saccharomyces cerevisiae

Cells of Saccharomyces cerevisiae and their metabolites were successfully utilized to achieve selective separation of quartz and calcite through microbially induced flotation and flocculation. S. cerevisiae was adapted to calcite and quartz minerals. Adsorption studies and electrokinetic investigations were carried out to understand the changes in the surface chemistry of yeast cells and the minerals after mutual interaction. Possible mechanisms in microbially induced flotation and flocculation are outlined.     

New biologically active linear triterpenes from the bark of three new-caledonian Cupaniopsis species

In the course of automated screening for small-molecule agonists to peroxisome proliferator-activated receptor-γ (PPAR-γ), 10 new linear triterpenes 1-10 have been isolated from the bark of three neocaledonian Cupaniopsis species, SAPINDACEAE. The structures were elucidated by extensive mono- and bi-dimensional spectroscopy and mass spectrometry.     

Five novel norcembranoids from Sinularia leptoclados and S. parva

Three new norcembrane-based diterpenoids, leptocladolides A (1), B (4) and C (5), along with five known metabolites 6-10, have been isolated from the dichloromethane extract of a Taiwanese soft coral Sinularia leptoclados. Furthermore, a chemical investigation on the dichloromethane extract of S. parva has resulted in the isolation of two new related isomers, 1-epi-leptocladolide A (2) and 7E-leptocladolide A (3), in addition to 1 and 7. The structures of new metabolites 1-5 were elucidated on the basis of extensive spectroscopic analyses and their relative stereochemistries were determined by NOESY experiments. The new metabolites 1 and 3 have been shown to exhibit significant cytotoxic activity against KB and Hepa59T/VGH cancer cell lines.     

Development and validation of a high-performance liquid chromatographic method for the analysis of antioxidative phenolic compounds in fennel using a narrow bore reversed phase C18 column

A simple high-performance liquid chromatography (HPLC) method for the separation and quantitative determination of the main antioxidant phenolic compounds from bitter fennel, Foeniculum vulgare, was developed. The use of a narrow bore reversed phase (RP) C₁₈ column and an acidic mobile phase enabled ten compounds (caffeoylquinic acids, dicaffeoylquinic acids, flavonoids and rosmarinic acid) to be separated within a 40 min time analysis. The method was validated to demonstrate its selectivity, linearity, precision, accuracy and robustness. In addition, some parameters were studied to optimize the complete extraction of the phenolic compounds. The method was applied to the evaluation of three different fennel materials: distilled and non-distilled aerial parts, as well as defatted fruits. Distilled fennel was found to contain a higher proportion of antioxidant phenolic compounds than the non-distilled plan material.     

Inhibitory study of some novel Schiff base derivatives on Staphylococcus aureus by microcalorimetry

The effect of a series of novel Schiff base compounds on Staphylococcus aureus was studied by microcalorimetric method at 37 °C The results showed that all of the organic compounds had the capacity to inhibit the growth of S. aureus in different extent. And the extent and duration of the inhibitory effect on the growth of S. aureus, judged from the rate constant (k), varied with the different structure of the Schiff base compounds. According to the power-time curves, the multiplication rate constant and inhibition ratio were calculated. The growth rate constant of S. aureus (in log phase) in the presence of Schiff base compounds decreased with the increasing of the concentrations of these compounds regularly. The experimental results revealed that the hydrophilicity of Schiff bases had a great influence on their antibacterial activity. Of these Schiff bases, the greater their hydrophilicity, the higher their antibacterial activity. The antibacterial structure-activity relationship (SAR) of Schiff base derivatives was also briefly discussed.