Effects of anti-androgens cyproterone acetate, linuron, vinclozolin, and p,p'-DDE on the reproductive organs of the copepod Acartia tonsa.

The study was performed to detect the effects of anti-androgenic compounds on the reproduction. In this paper alterations observed in the marine calanoid copepod Acartia tonsa exposed to environmental concentrations of cyproterone acetate (CPA), linuron (LIN), vinclozolin (VIN), and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) for 21 days covering a full life cycle are described. Histological alterations were studied with a focus on reproductive organs, gonad and accessory sexual glands. Exposure to ≥1.2 µg L(-1) CPA caused degeneration of spermatocytes and deformation of the spermatophore in males. In a single male exposed to 33 µg L(-1) CPA, an ovotestis was observed. In CPA exposed females, enhancement of oogenesis, increase in apoptosis and a decrease in proliferation occurred. Exposure of males to ≥12 µg L(-1) LIN caused degenerative effects in spermatogonia, spermatocytes and spermatids, and at 4.7 µg L(-1) LIN, the spermatophore wall displayed an irregular formation. In LIN exposed females, no such structural alterations were found; however, the proliferation index was reduced at 29 µg L(-1) LIN. At an exposure concentration of ≥100 µg L(-1) VIN, distinct areas in male gonad were stimulated, whereas others displayed a disturbed spermatogenesis and a deformed spermatophore wall. In VIN exposed female A. tonsa, no effects were observed. Male A. tonsa exposed to p,p'-DDE displayed an impairment of spermatogenesis in all stages with increased degrees of apoptosis. In p,p'-DDE-exposed females, a statistical significant increase of the proliferation index and an intensification of oogenesis were observed at 0.0088 µg L(-1).

Residues of selected polychlorinated biphenyls (PCB) and organochlorine pesticides (OCP) in postmortem lungs from Epirus, northwestern Greece.

Organochlorine (OC) pesticides and polychlorinated biphenyls (PCB) are compounds characterized as persistent organic pollutants (POP) in the environment. These compounds are monitored globally since they enter the human body and accumulate in tissues, resulting in consequent adverse effects. In this study concentrations of selected OC compounds were determined in human autopsy lungs from Epirus, a relatively restricted region in northwestern Greece. This is the first epidemiologic study from Greece reporting on monitoring of environmental pollutants in human autopsy material. Thirty lungs collected from that number of autopsy cases were analyzed: 19 males and 11 females. The age range was 14-91 yr (mean ± SD = 61.8 ± 22.5 yr). Twenty-two cases (73%) were positive for at least one pollutant and eight cases were negative (27%). PCB were the most abundant class of contaminants, detected in 15 out of the 30 cases (50%). Dichlorodiphenyltrichloroethane (DDT) and hexachlorocyclohexanes (HCH) were second and third in abundance with 9 (30%) and 8 (27%) positive cases, respectively. The frequency of detection showed a tendency to increase with age of individuals. The patterns of OC found in human autopsy lungs were similar to those reported previously for other human specimens. Our results demonstrated a similar trend in contamination sources and distribution has occurred in western Greece as noted globally.

Wax screen-printable ink for massive fabrication of negligible-to-nil cost fabric-based microfluidic (bio)sensing devices for colorimetric analysis of sweat.

Fabric-based microfluidic analytical devices (µADs) have emerged as a promising material for replacing paper µADs thanks to their superior properties in terms of stretchability, mechanical strength, and their wide scope of applicability in wearable devices or embedded in garments. The major obstacle in their widespread use is the lack of a technique enabling their massive fabrication at a negligible-to-nil cost. In response, we report the development of a wax ink with proper thixotropic and hydrophobic properties, fully compatible with automatic screen-printing that allows the one step massive fabrication of microfluidics on a cotton/elastane fabric, with a printing resolution 400 µm (hydrophilic channel) and 1000 µm (hydrophobic barrier), without being necessary any post curing. The cost of the ink (50 g) and of each microfluidic device is ca. 2.3 and 0.007 €, respectively. The active component of the ink was a refined beeswax in a matrix based on ethyl cellulose in 2-butoxy ethyl acetate. Screen-printed fabric µADs were used for the simultaneous colorimetric determination of pH and urea in untreated human sweat by using multivariate regression analysis. This method enabled the direct measurement of urea using urease, regardless of the sweat's pH, and shows strong agreement with a reference method.

Evaluation of matrix solid-phase dispersion extraction for the determination of polycyclic aromatic hydrocarbons in household dust with the aid of experimental design and response surface methodology.

A simple, fast, and inexpensive procedure for sample preparation based on matrix solid-phase dispersion was developed for the determination of Environmental Protection Agency 16 priority polycyclic aromatic hydrocarbons in indoor dust samples. Parameters that affect the extraction efficiency such as type of dispersant, elution solvent, and solvent volume were evaluated and optimized with the aid of experimental design and response surface methodology. Analysis was performed by HPLC coupled with UV-Vis diode array detector (UV-DAD). For verification, a GC coupled with a mass spectrometer in SIM mode was also applied. Recoveries obtained were from 53 to 120% for all target analytes with detection limits ranging from 0.2 to 10 ng/g and 0.2 to 2 ng/g for LC-UV-DAD and GC-MS, respectively. The optimized method was used for the analysis of 11 household dust samples collected from private houses.

Development and application of chemometric-assisted dispersive liquid-liquid microextraction for the determination of suspected fragrance allergens in water samples.

A simple and green method based on dispersive liquid-liquid microextraction, mated to chemometrics and followed by mass spectrometric detection for the determination of suspected fragrance allergens in water samples is developed and assessed in this work. Volume of extraction and disperser solvent, pH, ionic strength, extraction time, sample volume, as well as centrifugation time were initially optimized in a fractional factorial design. The obtained significant factors were optimized by using a central composite design and the quadratic model between the dependent and the independent variables was built. The obtained optimal conditions were: aqueous sample of 3.8 mL, 100 µL chloroform, 1.40 mL acetone, 4 min centrifugation time, natural pH containing 5% (w/v) NaCl, and centrifugation speed 4000 rpm. Method proved to be linear over a wide range of concentration for all analytes with R(2) between 0.9807 and 0.9959. The repeatability and reproducibility of the proposed method, expressed as relative standard deviation, varied between 3-13% and 4-16%, respectively. The limits of detection ranged from 0.007 to 1.0 µg L(-1) . The recommended method was applied to water samples including baby bath as well as swimming pool water samples and was compared with a previously reported method.

Determination of pesticides and toxic potency of rainwater samples in western Greece.

Rainwater samples from four municipalities located in Achaia Prefecture, Greece, were collected from March to September 2006. The toxic potency of pollutants present in 36 rainwater samples was tested using Daphnia pulex. The pesticide determination was conducted with GC-MS. Only phosphamidon was detected, which appeared in 52% and 13% of the rural and urban areas, respectively. The toxicity of rainwater was determined in 52% and 46.7% of the rural and urban area samples, respectively. Chemical analyses showed that in rural areas, the PO(4)(3-) ions had higher concentrations than in urban areas. On the other hand, the SO(4)(2-), NO(-)(3), and NO(-)(2) anions are more highly concentrated in urban areas. Correlation analysis proved that the toxicity of the rainwater samples is moderate, affected by the presence of the insecticide only in the rural areas. The results indicated that toxicity can be directly assessed via bioassays, even when unknown pollutants are present.

Sustainable production of a new generation biofuel by lipase-catalyzed esterification of fatty acids from liquid industrial waste biomass.

In this work we suggest a methodology comprising the design and use of cost-effective, sustainable, and environmentally friendly process for biofuel production compatible with the market demands. A new generation biofuel is produced using fatty acids, which were generated from acidogenesis of industrial wastes of bioethanol distilleries, and esterified with selected alcohols by immobilized Candida antarctica Lipase-B. Suitable reactors with significant parameters and conditions were studied through experimental design, and novel esterification processes were suggested; among others, the continuous removal of the produced water was provided. Finally, economically sustainable biofuel production was achieved providing high ester yield (<97%) along with augmented concentration (3.35M) in the reaction mixtures at relatively short esterification times, whereas the immobilized lipase maintained over 90% of its initial esterifying ability after reused for ten cycles.

Determination of UV-filter residues in bathing waters by liquid chromatography UV-diode array and gas chromatography-mass spectrometry after micelle mediated extraction-solvent back extraction.

A preconcentration methodology utilizing the cloud point phenomenon is described in this study for the determination of sunscreen agent residues in bathing waters by reversed phase liquid chromatography with UV detection and gas chromatography (GC) with mass spectrometric (MS) detection. The method employs the entrapment of the analytes in the micelles of the non-ionic surfactant TX-114, upon increase of the solution temperature to 60 degrees C. The analytes are either re-extracted or back extracted from the final micellar extract into appropriate organic solvents, a procedure that facilitates the direct application of the method not only with liquid chromatography but mostly importantly with gas chromatographic analysis. Ultrasonication was employed to assist the procedure and accelerate the extraction of the analytes into the solvent phase. Under the optimum experimental conditions, the method affords satisfactory recoveries in the range of 95-102% and relative standard deviation lower than 6% without interference from the presence of the surfactant. The method was successfully applied to the determination of UV filters in natural waters.

Metolachlor photodegradation study in aqueous media under natural and simulated solar irradiation.

To elucidate the photochemical behavior of pesticide metolachlor, degradation was carried out in aqueous media of different compositions such as sea, river, lake, and distilled water under natural and simulated solar irradiation. In addition, the effect of important constituents of natural water such as dissolved organic matter (DOM, isolated from Pamvotis Lake) and nitrate ions was also examined. It was found that photodegradation proceeds via a pseudo-first-order reaction in all cases. The presence of DOM inhibits the photolysis reaction with half-lives ranging from 87 to 693 h whereas the degradation rate was accelerated up to 11 times in the presence of NO(3)(-). In addition, the toxicity of the degradation products formed (generally through hydroxylation, dealkylation, and cyclization reactions) was also performed using the marine luminescent bacterium Vibrio fisheri. Our results indicated a toxicity increase of the irradiated solution showing that photoproducts of higher acute toxic effects were formed.

Aquatic phototransformation study of the antifouling agent Sea-Nine 211: identification of byproducts and the reaction pathway by gas chromatography-mass spectroscopy.

The photochemical behaviour of the biocide Sea-Nine 211 was carried out in order to investigate several transformation products formed in different environmental matrices and under different conditions. Sea-Nine 211 photodecomposition was performed under laboratory conditions using a xenon light source and under natural sunlight conditions in sea, river, lake as well as in distilled water. In order to examine the effect of dissolved organic matter (DOM), the phototransformation of the tested biocide was studied also in the presence of various concentrations of humic and fulvic acids. The phototransformation was shown to proceed via pseudo-first-order reaction in all cases and the presence of humic and fulvic acids enhanced the photolysis reaction. Kinetic experiments were monitored with GC-ECD and the half-lives (t(1/2)) varied between 6 and 433 h. Irradiation of the aqueous Sea-Nine 211 solutions gave rise to a great number of transformation products that were isolated by means of SPE using SDB extraction disks while six of them were tentatively identified using GC-MS techniques. Based on this byproduct identification a possible transformation pathway is proposed for the decomposition of Sea-Nine 211 in aqueous media.

Analysis of antifouling biocides Irgarol 1051 and Sea Nine 211 in environmental water samples using solid-phase microextraction and gas chromatography.

This study develops a method for the analysis of biocides Irgarol 1051 and Sea Nine 211 in environmental water samples, using solid-phase microextraction (SPME). Their determination was carried out using gas chromatography with flame thermionic (FTD), electron-capture (ECD) and mass spectrometric detection. The main parameters affecting the SPME process such as adsorption-time profile, salt additives and memory effect were studied for five polymeric coatings commercially available for solid-phase microextraction: poly(dimethylsiloxane) (100 and 30 microm), polyacrylate, poly(dimethylsiloxane)-divinylbenzene (PDMS-DVB 65 microm) and Carbowax-divinylbenzene (65 microm). The method was developed using spiked natural waters such as tap, river, sea and lake water in a concentration range of 0.5-50 microg/l. All the tested fiber coatings have been evaluated with regard to sensitivity, linear range, precision and limits of detection. Typical RSD values (triplicate analysis) in the range of 3-10% were obtained depending on the fiber coating and the compound investigated. The recoveries of biocides were in relatively high levels 60-118% and the calibration curves were reproducible and linear (R2>0.990) for both analytes. The SPME partition coefficients (Kf) of both compounds were also calculated experimentally in the proposed conditions for all fibers using direct sampling. Finally the influence of organic matter such as humic acids on extraction efficiency was studied, affecting mostly Sea Nine 211 uptake by the fiber. Optimum analytical SPME performance was achieved using the PDMS-DVB 65 microm fiber coating in ECD and FTD systems for Sea Nine 211 and Irgarol 1051, respectively.

Determination of antifouling compounds in marine sediments by solid-phase microextraction coupled to gas chromatography-mass spectrometry.

Solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry was applied to determine the antifouling biocides chlorothalonil, dichlofluanid, sea nine 211 and irgarol 1051 in marine sediments. Two experimental approaches were selected before the submission of the aqueous extracts to SPME prior to GC determination. The extraction of the biocides from the sediment samples was conducted using (a) water (containing 5%, v/v, acetone) and (b) acetone which was then diluted with water to give a 5% (v/v) content. The recommended procedures were found to be applicable for quantitative determination of the selected antifouling compounds in sediments with R.S.D.s below 17% and limits of detection ranging from 0.5 to 25 ng/g. The acetone/SPME procedure showed lower detection limits (0.5 to 6 ng/g) and R.S.D. values (< 11%) as well as better recoveries (73 to 92%), proving that it could be successfully performed for the determination of antifouling compounds in sediment analysis, even in samples with high organic matter content. Both optimized water/SPME and acetone/SPME procedures were applied to the analysis of antifouling compounds in marine sediments and compared with the conventional liquid-liquid extraction with subsequent clean up by solid-phase extraction.

Determination of residues of UV filters in natural waters by solid-phase extraction coupled to liquid chromatography-photodiode array detection and gas chromatography-mass spectrometry.

This study describes a procedure for the enrichment, separation and quantification of four major UV filters in natural waters. Solid-phase extraction combined with liquid chromatography and photodiode array detection (LC-UV-DAD), and gas chromatography with mass spectroscopy (GC-MS) were employed for the analyses. LC of the four compounds with surfactant-modified hydro-organic eluents gave satisfactory resolution of overlapping peaks. In GC, a significant improvement of the detection limits was attained, but only three compounds could be detected. The method was validated for, and applied to, various water samples prone to UV filter accumulation due to recreational activities. Recoveries from real samples were 86-99% with LOQs as low as 0.5 ng/l depending on the sample volume and the analytical procedure.

Application of solid-phase microextraction in the monitoring of priority pesticides in the Kalamas River (N.W. Greece).

A solid-phase microextraction (SPME) method was applied to an extended monitoring survey of priority pesticides for the European Union for a period of 12 months in water of the Kalamas River (Epirus region of northwestern Greece) in order to determine their concentrations and seasonal variations. Polydimethylsiloxane-coated fiber (100 microm) was used. The samples were screened using gas chromatography with flame thermionic detection. Detection was confirmed by gas chromatographymass spectroscopy. The most frequently detected pesticides were some of the more commonly used herbicides, such as S-ethyl-N,N-di-n-propylthiol carbamate (EPTC), trifluralin, atrazine, deethylatrazine, terbuthylazine and alachlor, and insecticides, such as carbofuran, diazinon, disulfoton, parathion methyl, parathion ethyl, fenthion and ethion. Concentrations of individual compounds ranged from 0.020 to 0.3 microg/L. Greater pesticide concentrations occurred during the seasons of application. A comparison with a well-established solid-phase extraction (C18 disks) procedure was performed for samples of high-season application (May-September) in order to confirm the effectiveness of the SPME technique. The results demonstrate the suitability of the SPME method for routine screening multiresidue analysis in natural waters.

Photocatalytic degradation of propachlor in aqueous TiO2 suspensions. Determination of the reaction pathway and identification of intermediate products by various analytical methods.

The light-induced degradation of propachlor (2-chloro-N-isopropylacetanilide) under simulated solar irradiation has been investigated in aqueous solutions containing TiO2 suspensions as photocatalysts. The study focuses on the identification of possible intermediate products and the determination of inorganic ions formed during the process, using several powerful analytical techniques such as gas chromatography mass-spectrometry (GC-MS) and ion chromatography. The primary degradation of propachlor has been a fast process with a half-live time of 2.3 min and followed pseudo-first-order kinetics according to the Langmuir-Hinshelwood model. The mineralization of the organic carbon to CO2 after 240 min of irradiation was found to be < 63%. The corresponding stoichiometric transformation of organic chlorine into chloride ion was reached in 30 min, whereas oxidation of nitrogen to nitrate ions took place at delayed irradiation times. Various aromatic intermediates have been identified by GC/MS techniques during the treatment, which were involved into three tentative degradation routes. One, was based on the dechlorination through positive holes or solvated electrons to form the hydroxy or aliphatic derivative. The second route consists of the rupture of amide bond or the N-C bond via nitrogen ionization by positive holes and through the ring hydroxylation, respectively. Finally, the third possible degradation route was based on hydrogen abstraction by the hydroxyl radicals or positive holes, followed either by the addition of oxygen to the alkyl radical or the decomposition of the peroxyl radical formed. From the analytical data, a possible multi-step degradation scheme was proposed. This work points to the necessity of extended knowledge of the successive steps in a solar-assisted detoxification process.

Study of chlorothalonil photodegradation in natural waters and in the presence of humic substances.

Photodegradation of chlorothalonil was studied in different natural waters (sea, river and lake) as well as in distilled water under natural and simulated solar irradiation. The effect of dissolved organic matter (DOM) such as humic and fulvic substances on the photodegradation rate of chlorothalonil was also studied in simulated sunlight. The presence of DOM enhanced the photodegradation of chlorothalonil with the exception of seawater. The kinetics were determined through gas chromatography electron capture detection (GC/ECD) and the photodegradation proceeds via pseudo-first-order reaction in all cases. Half-life ranged from 1 to 48 h. In natural and humic water chlorothalonil photodegradation gave rise to two different intermediates compared to distilled water demonstrating that the transformation of chlorothalonil depend on the constitution of the irradiated media and especially from DOM. The byproducts identified by GC/MS techniques were: chloro-1,3-dicyanobenzene, dichloro-1,3-dicyanobenzene, trichloro-1,3-dicyanobenzene and benzamide.

Survey for the occurrence of antifouling paint booster biocides in the aquatic environment of Greece.

Since the restriction imposed by European Union regulations on the use of TBT-based antifouling paints on boats below 25 m in length, new terms have been introduced in the 'small boat' market. Replacement products are generally based on copper metal oxides and organic biocides. Several studies have demonstrated the presence of these biocides in European ports and marinas of Spain, France, Germany and the United Kingdom. An extended survey of the antifouling biocides chlorothalonil, dichlofluanid, irgarol 1051 and sea-nine 211 was carried out in Greek ports and marinas of high boating activities from October 1999 to September 2000. The sampling sites were: Piraeus, Elefsina, Thessaloniki, Patras, Chalkida, Igoumenitsa, and Preveza (Aktio). The extraction of these compounds from the seawater samples was performed off-line with C18 solid phase extraction (SPE) disks while the determination was carried out with gas chromatography coupled to electron capture (ECD), thermionic (FTD) and mass spectroscopy (MS) detectors. The concentration levels of biocides were higher during the period from April to October. This seasonal impact depends on the application time of antifouling paints and mimic trends in the seasonal distribution of biocides in other European sites.

Chemometric optimization of dispersive suspended microextraction followed by gas chromatography-mass spectrometry for the determination of polycyclic aromatic hydrocarbons in natural waters.

A dispersive suspended microextraction (DSME) method coupled with gas chromatography-mass spectrometry (GC-MS) was developed and validated for the simultaneous determination of ten polycyclic aromatic hydrocarbons in real water samples. The optimization of the method was achieved with a 2(7-4) Plackett-Burman design, while the significant factors were optimized using a central composite design (CCD). The parameters that were studied included the sample volume, organic solvent volume, extraction time, restoration time and organic solvent. The optimum experimental conditions for the proposed method comprised 4.3mL of the water sample, 93µL of toluene as the extraction solvent, a 104-s extraction time and a 10-min restoration time. The recoveries varied from 70 to 111%. Chrysene was the least recovered compound, while anthracene displayed the highest extraction efficiency. The analytical method (DSME) was shown to be linear (R(2)>0.993) over the studied range of concentrations, exhibiting satisfactory precision (RSD%<10.6%) and reaching limits of detection between 8 and 46ngL(-1).

Evaluation of magnetic nanoparticles to serve as solid-phase extraction sorbents for the determination of endocrine disruptors in milk samples by gas chromatography mass spectrometry.

A rapid magnetic solid-phase extraction (MSPE) is proposed based on C18-functionalized magnetic silica nanoparticles as sorbents, for the determination of endocrine disruptors - 20 organochlorine pesticides and 6 polychlorinated biphenyls - in milk samples. Magnetic nanoparticles are characterized by several techniques, such as Scanning Electron Microscopy, X-Ray diffraction, Brunauer-Emmett-Teller and Fourier transform-infrared. The MSPE is performed by dispersion of the Fe3O4@SiO2@C18 nanoparticles in milk samples with sonication, after protein precipitation. Then, the sorbent is collected by applying an external magnetic field and the analytes are desorbed by n-hexane. Several parameters affecting the extraction efficiency of target analytes by the magnetic nanoparticles are investigated, including washing and elution solvents, amount of sorbents, time of extraction and elution, sample and elution solvent volume. The proposed method is optimized by means of experimental design and response surface methodology. When coupled with gas chromatography-mass spectrometry detection and under optimum extraction conditions, average recoveries of target analytes are found to be in the range of 79% to 116%. The proposed MSPE-GC-MS analytical method has a linear calibration curve for all target analytes with coefficients of determination to range from 0.9950 to 0.9999. The limits of quantification are found to be between 0.2 and 1µg/L ensuring compliance with the maximum residue limits established by European Commission and Codex Alimentarius, for OCPs and PCBs residues in milk. The proposed method is applied to the determination of target analytes in milk samples from local markets.

Effects of methyltestosterone, letrozole, triphenyltin and fenarimol on histology of reproductive organs of the copepod Acartia tonsa.

The marine calanoid copepod Acartia tonsa was exposed to methyltestosterone (MET, 1.6-126 µg L(-1)), letrozole (LET, 10-1000 µg L(-1)), triphenyltin chloride (TPT, 0.0014-0.0088 µg L(-1) TPT-Sn) and fenarimol (FEN, 2.8-105 µg L(-1)) for 21 d covering a full life-cycle. All four compounds investigated are known to act as androgens in vertebrates. The digestive tract, musculature, nervous system, reproductive organs, gonad and accessory sexual glands were examined by light microscopy after routine staining and immune-labelling for detection of apoptosis and determination of proliferation activities. MET induced an inhibition of oogenesis, oocyte maturation and yolk formation, respectively, which was most pronounced at the lowest concentrations tested. In LET exposed males, spermatogenesis was enhanced with very prominent gamete stages; in some stages apoptosis occurred. The spermatophore was hypertrophied and displayed deformations. In females, LET induced a disorder of oogenesis and disturbances in yolk synthesis. TPT stimulated the male reproductive system at 0.0014 and 0.0035 µg TPT-SnL(-1), whereas inhibiting effects were observed in the female gonad at 0.0088 µg TPT-SnL(-1). In FEN exposed females proliferation of gametes was reduced and yolk formation showed irregular features at 2.8-105 µgL(-1). In FEN exposed males an elevated proliferation activity was observed. No pathological alterations in other organ systems, e.g. the digestive tract including the hindgut acting as respiratory organ, the nervous system, or the musculature were seen. This indicates that the effects on gonads might be caused rather by disturbance of endocrine signalling or interference with hormone metabolism than by general toxicity.