A fast and reliable LC-MS-MS method for the quantification of saxitoxin in blood plasma samples.

Saxitoxins (STXs) are potent neurotoxins produced by marine dinoflagellates or freshwater cyanobacteria known to cause acute and eventually fatal human intoxications, which are classified as paralytic shellfish poisonings (PSPs). Rapid analysis of STXs in blood plasma can be used for a timely diagnosis and confirmation of PSPs. We developed a fast and simple method of STX extraction based on plasma sample acidification and precipitation by acetonitrile, followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Our approach provides the results ≤30 min, with a limit of detection of 2.8 ng/mL and a lower limit of quantification of 5.0 ng/mL. Within-run and between-run precision experiments showed good reproducibility with ≤15% values. Standard curves for calibration were linear with correlation coefficients ≥0.98 across the assay calibration range (5-200 ng/mL). In an interlaboratory analytical exercise, the method was found to be 100% accurate in determining the presence or absence of STX in human plasma specimens, with recovery values of 86-99%. This simple method for STX determination in animal or human plasma can quickly and reliably diagnose STX exposures and confirm suspected PSP cases to facilitate patient treatment or expedite necessary public health or security actions.

Hydrodynamic cavitation-enhanced activation of sodium percarbonate for estrogen removal.

The present paper investigated the potential of hydrodynamic cavitation (HC) as an effective tool for activating sodium percarbonate (SPC). The method's efficiency was demonstrated by effectively removing estrogens, which are pollutants that have adverse impacts on aquatic ecosystems. The effects of the SPC concentration, temperature of solution, and cavitation time were evaluated. After SPC/HC treatment, the removal of estrogens was monitored by liquid chromatography-tandem mass spectrometry (LC -MS/MS). Already after 4 s of treatment and 24 h of reaction time, more than 97% of estrogens (initial concentration of 300 ng/L) were removed. The effect of post-treatment time is not considered in several papers, even though it seems to be crucial and is discussed here. The results were supported by the values of degradation rate constants, which fit the pseudo-first-order kinetic model. We also verified that HC alone was not effective for estrogen removal under the selected conditions. The sustainability of the SPC/HC system was evaluated based on electric energy per order calculation. The combination of SPC and HC is a promising approach for rapidly degrading micropollutants such as estrogenic compounds without the need for additional technological steps, such as pH or temperature adjustment.

Potential toxicity of Schisandra chinensis to water environment: acute toxicity tests with water crustaceans.

Fruits of Schisandra chinensis, an East Asian liana plant, are currently more and more used to produce nutrient supplements that positively affect human health due to the content of various secondary metabolites. On the other hand, these substances because of their bioactivity can cause possible allelopathic or toxic effects concerning other organisms (algae, plants, animals). But the ecotoxicological properties of S. chinensis outside its area of origin have yet to be sufficiently verified. Two crustaceans, Daphnia magna and Thamnocephalus platyurus, were selected as model aquatic organisms to test the potential impact of S. chinensis active compounds on the aquatic environment. Crude water extract from S. chinensis fruits, simulating the natural leakage of active substances in water, was tested in treatments from 0.0045 to 45 mg/L (according to the content of schisandrin as the dominating lignan). Effective concentration (EC50) causing 50% lethal effect for D. magna was established to 0.0448 mg/L after 24 h and 0.0152 mg/L after 48 h. EC50 for T. platyurus reached 0.4572 mg/L after 24 h, i.e. more than ten times higher than for D. magna. This study showed that the potential environmentally relevant concentrations of S. chinensis bioactive compounds could represent a severe risk to aquatic ecosystems.

Bioactive compounds from Schisandra chinensis - Risk for aquatic plants?

Schisandra chinensis is a potential plant for production of nutrient supplements due to adaptogens content. The dominant bioactive substance, lignan schisandrin, has positive effects on human health, but it can cause possible allelopathic effects in relation to other plants. S. chinensis is not native to European ecosystems, and its ecotoxicological properties have not been verified yet. Lemna minor was selected as a model aquatic plant to test its potential impact on the aquatic environment. Crude water extract from S. chinensis fruits, simulating the natural soaking of active substances in a surface water body, was used in treatments from 0.045 to 45 mg/L (according to the content of schisandrin as the dominating lignan). During seven days of cultivation, the growth (number of plants, leaf area, fresh weight) and photosynthetic activity of L. minor fronds were assessed. In low treatments (0.045 and 0.09 mg/L), the extract of S. chinensis did not cause any changes in duckweed growth parameters or photosynthetic performance. Higher treatments (0.45 and 0.9 mg/L) caused significant limitations in plants' number, total leaf area, and fresh weight. The photosynthetic parameters (basal chlorophyll fluorescence, quantum yields) were affected only by 0.9 mg/L. The highest treatment, 45 mg/L, exhibited extreme toxicity to duckweed plants causing their death during the first five days of cultivation. Schisandrin and other bioactive substances extractable from S. chinensis fruits can negatively impact water biota in the case of massive contamination of surface water.

Graphene oxide interaction with Lemna minor: Root barrier strong enough to prevent nanoblade-morphology-induced toxicity.

The production of graphene oxide (GO) along with its applications in various aquatic environments is vastly increasing thanks to its rapidly expanding range of new GO-based environmental technologies. Therefore, the fate of GO in aquatic environments is an important issue, as it could become an environmental challenge if its potential toxic mechanism is not addressed properly. Number of studies reporting the toxicity of GO to various aquatic organisms is still increasing. However, research data on the possible toxic mechanism of GO towards aquatic plants have yet to be collected, especially regarding GO's surface chemistry. Here, we studied the interaction of three differently oxidized GO systems with model aquatic plant Lemna minor. We found that although none of the three GOs caused lethal phytotoxicity to Lemna after 7 days, the mechanism of action was dependent on the GO's surface oxidation. Based on the amount of functional surface groups, the GO was able to directly interact with the Lemna's root through its edges. However, in this case in contrast to algae and crustaceans, the interaction did not lead to a mechanical damage. Therefore, our results showed that GO is not hazardous to Lemna minor even at very high concentrations (up to 25 mg/L), because the root barrier proved to be strong enough to prevent GO's penetration and its consequent toxicity.

Oxidation of antibiotics by ferrate(VI) in water: Evaluation of their removal efficiency and toxicity changes.

Antibiotics in water and wastewater have been determined extensively. The treatment of antibiotics in water needs evaluation of possible harmful effects on aquatic ecosystems and human health. This paper presents the toxicity evaluation of antibiotics after their treatment with ferrate (VI) (FeVIO42-, Fe(VI)) in water. The antibiotics (sulfamethoxazole (SMX), erythromycin (ERY), ofloxacin (OFL), ciprofloxacin (CIP), tetracycline (TET), oxytetracycline (OXY), and trimethoprim (TMP)) were treated at pH 8.0 by applying two concentrations of Fe(VI) to have molar ratios of 5:1 and 10:1 ([Fe(VI)]:[antibiotic]). Under the studied conditions, incomplete removal of antibiotics was observed, suggesting that the treated solutions contained parent antibiotics and their transformation products. The toxicity of antibiotics without Fe(VI) treatment was tested against freshwater green alga Raphidocelis subcapitata and cyanobacterium Synechococcus elongatus, which were determined to be generally sensitive to antibiotics, with EC50 < 1.0 mg/L. The toxicity of Fe(VI) treated solution was tested against R. subcapitata. Results found no toxicity for the treated solutions of OFL, CIP, and OXY. However, SMX, ERY, and TET remained toxic after Fe(VI) treatment (i.e., more than 75% growth inhibition of R. subcapitata). Results demonstrated that R. subcapitata may be applied to test the toxicity of antibiotics after oxidative treatments.

The environmental fate of graphene oxide in aquatic environment-Complete mitigation of its acute toxicity to planktonic and benthic crustaceans by algae.

Graphene oxide (GO) as the most studied hydrophilic graphene derivative can be deployed in a broad spectrum of environmental technologies opening the issue of its ecotoxicity. Nevertheless, the information about its behavior in complex aquatic environment is still not sufficient. Here, we studied the interaction of three differently oxidized GO systems with planktonic and benthic crustaceans. By standard toxicity tests, we observed the importance of feeding strategy as well as the surface oxidation of GO with respect to GO's ecotoxicity. However, to gain a clearer insight into GO's environmental fate, we introduced a pre-treatment with algae as the most common source of food for crustaceans. Such an adjustment mimicking the conditions in real aquatic ecosystems resulted in complete mitigation of acute toxicity of GOs to all organisms and, more importantly, to the eradication of oxidative stress caused by GOs. We argue, that the pre-exposition of food is a crucial factor in GO's overall environmental fate, even though this fact has been completely neglected in recent studies. These experiments proved that GO is not a hazardous material in complex aquatic environments because its acute toxicity can be successfully mitigated through the interaction with algae even at very high concentrations (25 mg/L).

Interspecies interactions can enhance Pseudomonas aeruginosa tolerance to surfaces functionalized with silver nanoparticles.

Development of anti-fouling surfaces is a major challenge in materials research. Microorganisms growing as biofilms have enhanced tolerance to antimicrobial strategies including antibiotics and antiseptics complicating the design of anti-fouling surfaces. Silver nanoparticles (AgNPs) are a promising antimicrobial technology with broad spectrum efficacy with a reduced likelihood of microorganisms developing resistance to the technology. This study tested the efficacy of new immobilized AgNP-modified surface technology against three common opportunistic pathogens grown either as monocultures or as cocultures. The presented study fills a gap in the literature by quantifying the efficacy of immobilized AgNP particles against multispecies biofilms. Polyethylene (PE) surfaces functionalized with the AgNPs were highly effective against Pseudomonas aeruginosa biofilms reducing viable cell counts by 99.8 % as compared to controls. However, the efficacy of the AgNP-modified PE surface was compromised when P. aeruginosa was cocultured with Candida albicans. Interspecies interactions can strongly influence the efficacy of anti-fouling AgNP coatings highlighting the need to test surfaces not only against biofilm phenotypes but under conditions representative of applications including the presence of multispecies consortia.

Occurrence and toxicity of antibiotics in the aquatic environment: A review.

In recent years, antibiotics have been used for human and animal disease treatment, growth promotion, and prophylaxis, and their consumption is rising worldwide. Antibiotics are often not fully metabolized by the body and are released into the aquatic environment, where they may have negative effects on the non-target species. This review examines the recent researches on eight representative antibiotics (erythromycin, trimethoprim, sulfamethoxazole, tetracycline, oxytetracycline, ofloxacin, ciprofloxacin, and amoxicillin). A detailed overview of their concentrations in surface waters, groundwater, and effluents is provided, supported by recent global human consumption and veterinary use data. Furthermore, we review the ecotoxicity of these antibiotics towards different groups of organisms, and assessment of the environmental risks to aquatic organisms. This review discusses and compares the suitability of currently used ecotoxicological bioassays, and identifies the knowledge gaps and future challenges. The risk data indicate that selected antibiotics may pose a threat to aquatic environments. Cyanobacteria were the most sensitive organisms when using standard ecotoxicological bioassays. Further studies on their chronic effects to aquatic organisms and the toxicity of antibiotic mixtures are necessary to fully understand the hazards these antibiotics present.

High-pressure jet-induced hydrodynamic cavitation as a pre-treatment step for avoiding cyanobacterial contamination during water purification.

Due to specific physical properties, hydrodynamic cavitation (HC) is assigned to the powerful technologies for treating the biotic contamination in water including cyanobacteria. Contaminated water stream (CWS) can be cavitated directly by passing through some HC device, or indirectly when high-pressure jet stream (HPJS) is directed against its flow. Relatively small HPJS stream can thus treat a big volume of CWS in a short time or even work in continuous mode. Cyanobacteria floating in the CWS are forced to flow through the mixing cavitation zone. Within 2 h after single HC treatment, cyanobacterial cell suspensions showed disintegration of larger colonies and enhanced biomass sedimentation. Additional pre-treatment of CWS with low amounts of hydrogen peroxide (H2O2; 33, 66 and 99 µmol/L) enhanced the effect of HC and led to further inhibition of cyanobacterial photosynthesis (maximum quantum yield of photosystem II decreased by up to 60%). The number of cyanobacterial cells in the treated CWS decreased continuously over 48 and 72 h, though some cells remained alive and were able to recover photosynthetic activity. The technique proposed (direction of a HPJS against a CWS and pre-treatment with low H2O2 concentrations) provides (i) effective removal of cells from the water column, and (ii) reduced contamination by organic compounds released from the cells (especially cyanotoxins) as the cell membranes are not destroyed and the cells remain alive. This process shows potential as an effective pre-treatment step in water purification processes related to cyanobacterial contamination.

Application of passive sampling for sensitive time-integrative monitoring of cyanobacterial toxins microcystins in drinking water treatment plants.

Calibrated adsorption-based passive samplers were used for time-integrative monitoring of microcystins (MCs) in three full-scale drinking water treatment plants (DWTPs) in the Czech Republic during two vegetation seasons (Jun-Nov), in parallel with traditional discrete sampling. MCs were detected in epilimnetic water samples at concentrations up to 14 µg/L, but their levels in raw water in DWTPs were below 1 µg/L WHO guideline value for drinking water. Conventional treatment technologies (coagulation/filtration) eliminated cyanobacteria and intracellular toxins but had a limited removal efficiency for extracellular toxins. MCs were regularly detected in final treated water, especially in DWTPs equipped only with the conventional treatment, but their concentrations were below the quantitation limit of discrete sampling (<25 ng/L). Passive samplers in combination with LC-MS/MS analysis provided excellent sensitivity allowing to detect time-weighted average (TWA) concentrations of MCs as low as 20-200 pg/L after 14-d deployment. Median MC TWA concentrations in the treated water from the individual DWTPs were 1-12 ng/L, and most likely did not present significant health risks. Passive samplers well reflected spatiotemporal variations of MCs, actual concentrations of extracellular toxins, MC removal efficiency in DWTPs, and toxin concentrations in the treated water. Passive sampling can be effectively used for assessment and management of MC health risks during DWTP operation.

The effect of silver nanoparticles and silver ions on zebrafish embryos (Danio rerio).

OBJECTIVES: The aim of this study was to establish and evaluate the mortality rate, hatching rate and observe the presence of sublethal changes in zebrafish embryos after exposure to silver ions and nanoparticles. METHODS: Tests were conducted on newly fertilized zebrafish embryos, according to the modified OECD guideline 236, using a semistatic method and 96 hour incubation time. Silver nitrate and two different silver nanoparticles, stabilized with 0.01% solution of maltose and gelatine in the first case, and stabilized with polyvinylpyrrolidone, in the latter, were tested. RESULTS: Significant differences in toxicity of tested substances were recorded. The value of 96hLC50 for silver nitrate was 58.44 µg/L. The value of 96hLC50, calculated for silver nanoparticles stabilized with 0.01% solution of maltose and gelatine, was nearly 100 times higher, 4.31 mg/L. The value 96hLC50 for silver nanoparticles stabilized with polyvinylpyrrolidone exceeded 100mg/L, occurrence of sublethal effects caused by silver nanoparticles stabilized with polyvinylpyrrolidone was insignificant in most of the exposition groups, but only in this substance caused decreased hatching rate. CONCLUSION: Properties of different silver nanoparticles play an important role in levels of their toxicity and predominant mechanisms of action. In general, silver nanoparticles are less toxic for Danio rerio embryos than silver ions.

Iron and Iron Oxide Nanoparticles Synthesized Using Green Tea Extract: Improved Ecotoxicological Profile and Ability to Degrade Malachite Green.

In recent years, iron-based nanoparticles (FeNPs) have been successfully used in environmental remediation and water treatment. This study examined ecotoxicity of two FeNPs produced by green tea extract (smGT, GTFe) and their ability to degrade malachite green (MG). Their physicochemical properties were assessed using transmission electron microscopy, X-ray powder diffraction, dynamic light scattering, and transmission Mössbauer spectroscopy. Using a battery of ecotoxicological bioassays, we determined toxicity for nine different organisms, including bacteria, cyanobacterium, algae, plants, and crustaceans. GTFe, amorphous complex of Fe(II, III) ions and polyphenols from green tea extract, proved low capacity to degrade MG and was toxic to all tested organisms. Superparamagnetic iron oxide NPs (smGT) derived from GTFe, showed no toxic effect on most of the tested organisms up to a concentration of 1g/L, except for algae and cyanobacterium and removed 93 % MG at concentration 125 mg Fe/L after 60 minutes. The procedure described in this paper generates new superparamagnetic iron oxide NPs from existing and toxic GTFe, which are nontoxic and has degradative potential for organic compounds. These findings suggest low ecotoxicological risks and suitability of this green-synthesized FeNPs for environmental remediation purposes.

High-valent iron (Fe(VI), Fe(V), and Fe(IV)) species in water: characterization and oxidative transformation of estrogenic hormones.

This paper presents solid state synthesis and characterization of tetra-oxy iron(iv) and iron(v) species in their salt forms (Na4FeO4-Fe(IV) and K3FeO4-Fe(V)). Stability of the synthesized salts, commonly called ferrates, in water was determined by applying the (57)Fe Mössbauer spectroscopy technique. Within 2 s in water, Fe(IV) converted into Fe(III) while Fe(V) transformed into Fe(VI) and Fe(III) at pH = 8.2. Comparatively, Fe(VI) (bought as K2FeO4) remained stable in aqueous solution during the short time period. The oxidative removal efficiency of the high-valent iron species was then tested against five environmentally important estrogenic hormones (estron (E1), 17-ß-estradiol (E2), estriol (E3), 17-α-ethinylestradiol (EE2), and diethylstibestrol (DES)) in effluent water of a wastewater treatment plant. Three dosages of iron species (1, 10, and 100 mg L(-1)) were applied to the effluent water. An increase in the concentration of dosages enhanced the removal of estrogens. Both Fe(V) and Fe(VI) were effective in degrading estrogens, but Fe(IV) showed limited oxidation capacity to transform estrogens. The oxidized products of the estrogens were analyzed using Raman spectroscopy and high-performance liquid chromatography-mass spectrometry (HPLC-MS) techniques. Results demonstrated the transformation of estrogens into low molecular weight oxygenated compounds such as quinone-like and opened-aromatic ring species. A detailed study on E1 by using excess Fe(VI) showed the mineralization of the parent compound. The results demonstrate great potential of high-valent iron species in the degradation of endocrine disruptor chemicals like estrogens with several superior aspects including fast reactions, complete degradation and/or formation of benign organic species, and environmentally-acceptable iron oxide by-products.

Oxidation of microcystin-LR by ferrate(VI): kinetics, degradation pathways, and toxicity assessments.

The presence of the potent cyanotoxin, microcystin-LR (MC-LR), in drinking water sources poses a serious risk to public health. The kinetics of the reactivity of ferrate(VI) (Fe(VI)O4(2-), Fe(VI)) with MC-LR and model compounds (sorbic acid, sorbic alcohol, and glycine anhydride) are reported over a range of solution pH. The degradation of MC-LR followed second-order kinetics with the bimolecular rate constant (kMCLR+Fe(VI)) decreasing from 1.3 ± 0.1 × 10(2) M(-1) s(-1) at pH 7.5 to 8.1 ± 0.08 M(-1) s(-1) at pH 10.0. The specific rate constants for the individual ferrate species were determined and compared with a number of common chemical oxidants employed for water treatment. Detailed product studies using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) indicated the oxidized products (OPs) were primarily the result of hydroxylation of the aromatic ring, double bond of the methyldehydroalanine (Mdha) amino acid residue, and diene functionality. Products studies also indicate fragmentation of the cyclic MC-LR structure occurs under the reaction conditions. The analysis of protein phosphatase (PP1) activity suggested that the degradation byproducts of MC-LR did not possess significant biological toxicity. Fe(VI) was effective for the degradation MC-LR in water containing carbonate ions and fulvic acid (FA) and in lake water samples, but higher Fe(VI) dosages would be needed to completely remove MC-LR in lake water compared to deionized water.

Evaluation of antibacterial properties of novel phthalocyanines against Escherichia coli--comparison of analytical methods.

We analyzed antibacterial effects of several novel phthalocyanines against Escherichia coli and evaluated the suitability of flow cytometry for the detection of antibacterial effects of phthalocyanines in comparison with routinely used cultivation. After 3h of exposure under cool white light eight cationic phthalocyanines showed very high antibacterial activity in the concentration of 2.00 mg L(-1) and four of them were even efficient in the concentration of 0.20 mg L(-1). Antibacterial activity of neutral and anionic compounds was considerably lower or even negligible. No antibacterial effect was detected when bacteria were exposed without illumination. Binding affinity to bacterial cells was found to represent an important parameter influencing phthalocyanine antibacterial activity that can be modified by total charge of peripheral substituents and by the presence of suitable functional groups inside them. Agglomeration of cells observed in suspensions treated with a higher concentration of certain cationic phthalocyanines (the strongest binders to bacterial membrane) affected cytometric measurements of total cell counts, thus without appropriate pretreatment of the sample before analysis this parameter seems not to be fully valid in the evaluation of phthalocyanine antibacterial activity. Cytometric measurement of cell membrane integrity appears to be a suitable and even more sensitive parameter than cultivation.

Survey of cyanobacterial toxins in Czech water reservoirs--the first observation of neurotoxic saxitoxins.

The environmental occurrence and concentrations of cyanobacterial toxins (cyanotoxins) were investigated in the Czech Republic. Concentrations of microcystins (MCs), cylindrospermopsin (CYN) or saxitoxins (STXs) were determined immunochemically by ELISA assays in 30 water samples collected from the surface layers of 19 reservoirs during the summer season of 2010. MCs were detected in 18 reservoirs and 83 % of samples, with median and maximal concentration being 1.5 and 18.6 µg/L, respectively. The high frequency of MC occurrence coincided with prevalence of cyanobacterium Microcystis sp., which was detected in 87 % samples, followed by Dolichospermum (Anabaena) sp. observed in 33 % samples. CYN was detected by ELISA only in one sample at a concentration of 1.2 µg/L. STXs presence was indicated for the first time in Czech water reservoirs when the toxins were found at low concentrations (0.03-0.04 µg/L) in two samples (7 %) collected from two different reservoirs, where STXs co-occurred with MCs and eventually also with CYN. In both STX-positive samples, the phytoplankton community was dominated by Microcystis sp., but Dolichospermum sp. and/or Aphanizomenon sp. were also present as putative producers of STX and/or CYN. Cyanotoxins commonly occurred in Czech water reservoirs, and MCs frequently at concentrations possibly associated with human health risks. MCs were the most prevalent and abundant cyanotoxins, but also other cyanotoxins were detected, though sporadically. Further research and regulatory monitoring of cyanotoxins other than MCs is therefore required.

Acute, chronic and reproductive toxicity of complex cyanobacterial blooms in Daphnia magna and the role of microcystins.

Toxic cyanobacterial blooms are a global threat to human health and aquatic biota. While the ecotoxicity of cyanobacterial toxins such as microcystins has been studied extensively, little is known about the risks they pose in the wild, i.e. within complex biomasses. In this work, crustaceans (Daphnia magna) were exposed to varying concentrations (0-405 mg d.w L(-1)) of eight complex cyanobacterial water bloom samples in a series of acute (48 h) and chronic (21 day) toxicity experiments. Further acute and chronic exposure assays were performed using aqueous extracts of the crude biomass samples and two fractions prepared by solid phase extraction (SPE) of the aqueous extracts. The cyanobacterial biomasses differed with respect to their dominant cyanobacterial species and microcystin contents. High acute toxicity was observed for 6 of the 8 crude biomass samples. Chronic exposure assays were performed using one complex biomass sample and its various subsamples/fractions. The complex biomass, the crude aqueous extract, and the microcystin-free SPE permeate all elicited similar and significant lethal effects, with LC50 values of around 35.6 mg biomass d.w L(-1) after 21 days. The cyanobacterial biomass samples also affected reproductive health, significantly increasing the time to the first brood (LOEC = 45 mg d.w L(-1) exposure) and inhibiting fecundity by 50% at 15 mg d.w L(-1). Conversely, the microcystin-containing C18-SPE eluate fraction had only weak effects in the chronic assay. These results indicate that cyanobacterial water blooms are highly toxic to zooplankton (both acutely and chronically) at environmentally relevant concentrations. However, the effects observed in the acute and chronic assays were independent of the samples' microcystin contents. Our results thus point out the importance of other cyanobacterial components such as lipopolysaccharides, various peptides and depsipeptides, polar alkaloid metabolites or other unidentified metabolites in the overall ecotoxicity of complex cyanobacterial blooms.

Photodynamic effects of 31 different phthalocyanines on a human keratinocyte cell line.

Phthalocyanines (Pcs, colored macromolecular compounds with the ability to generate singlet oxygen) represent a promising group of photosensitizers due to their intense absorption in the red and UV portion of the spectrum which leads to their excitation. In order to characterize possible toxic effects associated with eventual practical use and application of these chemicals, we employed an in vitro cell culture model to evaluate cytotoxic effects of 31 different phthalocyanines using neutral red uptake assay. An immortalized human keratinocyte cell line HaCaT was exposed to the tested chemicals for 2 or 24h, either with or without illumination in the last 60 min of the exposure period. After 2- or 24-h exposure without illumination, no cytotoxic effects or weak cytotoxic effects were induced by any Pc under the study and EC50 values could not be obtained within the tested concentration ranges (1.25-20 mg L(-1) or 0.625-10 mg L(-1)). On the other hand, exposure to phthalocyanines under illumination induced a significant cytotoxic effect. The most pronounced cytotoxicity was elicited by Pcs previously shown to have high positive charge densities at peripheral parts of substituent groups, which is most likely the factor responsible for the binding of Pc to negatively charged membranes on the cell surface and thus guaranteeing the tight connection necessary for the singlet oxygen attack on the cell surface.

The influence of hard substratum reflection and calibration profiles on in situ fluorescence measurements of benthic microalgal biomass.

Measuring chlorophyll-a fluorescence is a commonly used method to determine microphytobenthic biomass expressed as chlorophyll-a per square centimetre. However, this in situ method is affected by reflection from the substratum which triggers an additional fluorescence signal within the microphytobenthic biofilm. Depending on the colour and texture of the natural substratum, this effect can lead to a considerable overestimation of microphytobenthic biomass. The results cannot be corrected for this effect by performing an auto-zero measurement, since the overestimation is not caused by an offset of the fluorometer. This article describes a substratum-specific correction procedure using a 700 nm signal to eliminate this effect by quantifying the fluorescence signal as a result of the reflection. An empirical relationship between the 700 nm signal and the additional fluorescence is used to calculate a correction factor for the reflective properties of the substratum. The factor is determined and applied during each biomass measurement, thereby making an additional calibration step for each individual type of substratum superfluous. This new method improves the reliability of the results significantly without increasing the time necessary to perform the measurements and without complicating the measurement procedure.