Continuous Flow-through System Research Articles

Nanofluidic preconcentration and detection of nanoparticles

The fast detection and characterization of nanoparticles, such as viruses or environmental pollutants, are important in fields ranging from biosensing to quality control. However, most existing techniques have practical throughput limitations, which significantly limit their applicability to low analyte concentrations. Here, we present an integrated nanofluidic scheme for preconcentration and subsequent detection of nanoparticle samples within a continuous flow-through system. Using a Brownian ratchet mechanism, we increase the nanoparticle concentration ∼27-fold. Single nanoparticles are subsequently detected and characterized by optical heterodyne interferometry. A wide range of potential applications can be foreseen, including real-time analysis of clinically relevant virus samples and contamination control of processing fluids used in the semiconductor industry.

Hexavalent chromium‐induced multiple biomarker responses in liver and kidney of goldfish, Carassius auratus

Hexavalent chromium [Cr (VI)] is a constituent of chromite ore. Although it is known to have several industrial and technological applications, its release into the aquatic environment as a result of chemical spill or inadequate waste discharge may hamper the health of aquatic organisms. In this study, we have investigated the effects of Cr (VI) on multiple biomarkers responses in goldfish under subchronic exposure conditions. Laboratory-acclimatized fish were exposed to 4.25 ppm and 8.57 ppm Cr (VI) for four weeks using a continuous flow-through system. During exposure, fish samples were collected on a weekly basis and analyzed for multiple biomarkers including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), metallothionein (MT), and total protein in liver and kidney. Study results indicated that the CAT activity and total protein levels in Cr (VI) - treated goldfish did not significantly differ (P > 0.05) from their respective controls during experimentation. However, highly significant up-regulations (P < 0.05) of SOD, GPx, and MT expression in Cr (VI) - treated goldfish were recorded at different exposure times depending on Cr (VI) concentration, test organ, and/or biomarker of interest. For example, significantly higher liver GPx levels were found at weeks 2 and 3 in the 4.25 ppm concentration, and at weeks 3 and 4 in the 8.57 ppm, while kidney GPx levels were significantly higher at weeks 1, 2 and 3 in the 4.25 ppm concentration, and at weeks 2, 3 and 4 in the 8.57 ppm concentration. In summary, Cr (VI)-induced oxidative stress was characterized by statistically significant increases in SOD, GPx, and MT expression in goldfish tissues; with the kidney showing a relatively higher sensitivity to Cr (VI) toxicity compared with the liver.

An in vitro urinary tract catheter system to investigate biofilm development in catheter-associated urinary tract infections

Biofilm development in urinary tract catheters is an often underestimated problem. However, this form of infection leads to high mortality rates and causes significant costs in health care. Therefore, it is important to analyze these biofilms and establish avoiding strategies. In this study a continuous flow-through system for the cultivation of biofilms under catheter-associated urinary tract infection conditions was established and validated. The in vitro urinary tract catheter system implies the composition of urine (artificial urine medium), the mean volume of urine of adults (1mLmin–1), the frequently used silicone catheter (foley silicon catheter) as well as the infection with uropathogenic microorganisms like Pseudomonas aeruginosa. Three clinical isolates from urine of catheterized patients were chosen due to their ability to form biofilms, their mobility and their cell surface hydrophobicity. As reference strain P. aeruginosa PA14 has been used. Characteristic parameters as biofilm thickness, specific biofilm growth rate and substrate consumption were observed. Biofilm thicknesses varied from 105±16 μm up to 246±67 μm for the different isolates. The specific biofilm growth rate could be determined with a non invasive optical biomass sensor. This sensor allows online monitoring of the biofilm growth in the progress of the cultivation.

TiO2/AC Composites for Synergistic Adsorption-Photocatalysis Processes: Present Challenges and Further Developments for Water Treatment and Reclamation

Titanium dioxide supported on activated carbon, or TiO2/AC composite, exhibits bifunctionality of adsorption and photocatalysis in synergism. The authors review the TiO2/AC synthesis techniques, characteristics, and performances in removing organic pollutants in water. Practical issues pertinent to applications of the TiO2/AC composite in water treatment and reclamation are discussed. These include dispersing the particles and recovering from the product water, UV introduction and attenuation in the photoreactor, long-term photostability and mechanical stability of the composite, potential TiO2 deactivation by the organic and inorganic matrices, assessment of intermediates and byproducts, and regeneration techniques for the exhausted or fouled TiO2/AC. Coupling with a membrane separation process to recover and regenerate TiO2/AC in various continuous flow-through system configurations is proposed. There are also possible integrations of TiO2/AC treatment systems with other treatment processes that may result in effective pollutant removal with reduced energy and chemical cost. Future developments including incorporation of solar energy are proposed. A modified TiO2/AC that can be photoexcited by solar light has been developed by the authors, and its performance in adsorbing and photocatalytic degradation (PCD) of bisphenol-A is presented.

Effects of Produced Water on Reproductive Parameters in Prespawning Atlantic Cod (Gadus morhua)

Produced water (PW) discharged from offshore oil industry activities contains substances that are known to contribute to a range of mechanisms of toxicity. In the present study selected reproductive biomarkers were studied in prespawning Atlantic cod (Gadus morhua) exposed to PW. The fish were exposed for 12 wk within a continuous flow-through system at realistic environmental near-field concentrations. Concentrations of polyaromatic hydrocarbon (PAH) and alkylphenol (AP) compounds were analyzed by gas chromatography with mass spectrometric detection measurement, as were PAH and AP metabolites in fish bile for verification of exposure conditions and presence of compounds in PW. A suite of reproductive biomarkers (vitellogenin, zona radiata protein, and plasma steroid concentrations) and histological alterations of the gonads were determined. Results showed that exposure to sufficiently high levels of PW produced an increase in vitellogenin levels in female fish compared to the control. Impaired oocyte development and reduced estrogen levels were also observed in PW-exposed female fish. In male fish testicular development was altered, showing a rise in amount of spermatogonia and primary spermatocytes and a reduction in quantity of mature sperm in the PW-exposed fish compared to control. Data indicate that sufficiently high levels of PW have the potential to adversely affect the reproductive fitness of cod.

Effects of 4- tert-octylphenol on Xenopus tropicalis in a long term exposure

Endocrine disrupting chemicals that activate the estrogen receptor are routinely detected in the environment and are a concern for the health of both exposed humans and indigenous wildlife. We exposed the western clawed frog ( Xenopus tropicalis) to the weak estrogen octylphenol from Nieuwkoop–Faber (NF) stage 46 tadpoles through adulthood in order to document the effects of a weak estrogen on the life history of an amphibian species. Frogs were exposed to 1, 3.3, 11 and 36 μg/L octylphenol in a continuous flow-through water system. Just prior to completion of metamorphosis (NF 65), a random subsample of froglets was collected and assessed, while the remaining frogs received continued exposure through 31 weeks of exposure when the remaining animals were sampled. Significant induction of the female egg yolk protein precursor vitellogenin was observed in the high treatment at the larval subsampling for both males and females, but not at the final sampling for either sex. No significant deviation from the control sex ratio was observed for either sampling period, suggesting minimal to no effect of octylphenol exposure on gonad differentiation. No effects in the adult frogs were observed for mortality, body mass and size, liver somatic index, estradiol and testosterone serum levels, sperm counts, or oocyte counts. The development and growth of oviducts, a female-specific secondary sex characteristic, was observed in males exposed to octylphenol. These results indicate that octylphenol exposure can induce vitellogenin in immature froglets and the development of oviducts in male adult frogs. The lack of effect observed on the developing gonads suggests that in amphibians, secondary sex characteristics are more susceptible to impact from estrogenic compounds than the developing gonads.

Application of a Burkholderia cepacia lipase-immobilized silica monolith to batch and continuous biodiesel production with a stoichiometric mixture of methanol and crude Jatropha oil

BackgroundThe enzymatic production of biodiesel through alcoholysis of triglycerides has become more attractive because it shows potential in overcoming the drawbacks of chemical processes. In this study, we investigate the production of biodiesel from crude, non-edible Jatropha oil and methanol to characterize Burkholderia cepacia lipase immobilized in an n-butyl-substituted hydrophobic silica monolith. We also evaluate the performance of a lipase-immobilized silica monolith bioreactor in the continuous production of biodiesel.ResultsThe Jatropha oil used contained 18% free fatty acids, which is problematic in a base-catalyzed process. In the lipase-catalyzed reaction, the presence of free fatty acids made the reaction mixture homogeneous and allowed bioconversion to proceed to 90% biodiesel yield after a 12 hour reaction time. The optimal molar ratio of methanol to oil was 3.3 to 3.5 parts methanol to one part oil, with water content of 0.6% (w/w). Further experiments revealed that B. cepacia lipase immobilized in hydrophobic silicates was sufficiently tolerant to methanol, and glycerol adsorbed on the support disturbed the reaction to some extent in the present reaction system. The continuous production of biodiesel was performed at steady state using a lipase-immobilized silica monolith bioreactor loaded with 1.67 g of lipase. The yield of 95% was reached at a flow rate of 0.6 mL/h, although the performance of the continuous bioreactor was somewhat below that predicted from the batch reactor. The bioreactor was operated successfully for almost 50 days with 80% retention of the initial yield.ConclusionsThe presence of free fatty acids originally contained in Jatropha oil improved the reaction efficiency of the biodiesel production. A combination of B. cepacia lipase and its immobilization support, n-butyl-substituted silica monolith, was effective in the production of biodiesel. This procedure is easily applicable to the design of a continuous flow-through bioreactor system.

Growth, Protein Retention, and Body Composition of Fingerling Indian Major Carp, Rohu, Labeo rohita (Hamilton), Fed Diets with Various Levels of Lysine

Optimum level of dietary lysine for fingerling rohu, Labeo rohita (4.42 ± 0.1 cm; 0.66 ± 0.08 g), was determined by feeding isonitrogenous (40% crude protein) and isocaloric (4.28 kcal/g) diets with graded concentrations of lysine (1.20, 1.40, 1.60, 1.80, 2.0, and 2.20% dry diet) to triplicate groups of fish in 80-L circular polyvinyl troughs (water volume 55 L) fitted with a continuous flow-through system (1-1.5 L/min) for 12 wk. Growth performance was evaluated on the basis of absolute weight gain (AWG g/fish), feed conversion ratio (FCR), protein efficiency ratio (PER), and protein retention efficiency (PRE%). Adequacy of dietary lysine was estimated using broken-line and nonlinear regression models. When assessed using broken-line model, these parameters were best at 1.82, 1.81, 1.80, and 1.81%, respectively, dietary lysine, corresponding to 4.55, 4.52, 4.50, and 4.52%, respectively, of the dietary protein, whereas when subjected to second-degree polynomial regression analysis optimum dietary lysine was found at 1.91, 1.90, 1.91, and 1.91% of dry diet, corresponding to 4.80, 4.75, 4.80, and 4.75%, respectively, of the dietary protein. Based on the above regression analyses, optimum dietary lysine requirement for fingerling L. rohita was found to range between 1.80 and 1.91% of the diet, corresponding to 4.50-4.80% of dietary protein.

Uptake and elimination, and effect of estrogen-like contaminants in estuarine copepods: an experimental study

In recent years, anthropogenic chemicals which can disrupt the hormonal systems of both humans and wildlife have been raised to a major cause of concern. The aim of the present work was to determine the bioconcentration factors of the two major alkylphenols (AP) of the Seine Estuary [4-nonylphenol (4 NP) and nonylphenol acetic acid (NP1EC)] and of the synthetic estrogen, estrogen ethinylestradiol (EE2), in Eurytemora affinis after exposure in a continuous flow-through system under environmental realistic conditions. Moreover, the elimination of these compounds in copepods from the Seine Estuary has been investigated by measuring concentrations after 1 week in clean water in comparison to background levels. In this study, the dominant copepod species of the Seine Estuary, E. affinis, was exposed at environmental relevant concentrations under laboratory-controlled conditions to selected waterborn contaminants, a mixture of 4 NP/NP1EC, and a synthetic EE2. The uptake and the elimination of these contaminants by E. affinis have been studied. The results show that, at the end of the uptake period, both 4 NP and NP1EC, and also EE2 were accumulated in exposed copepods with respective concentration factors of 324, 3,020, and 5,383. A rapid elimination of these compounds was also observed in copepods placed in clean water since 54% of total NP1EC and 100% of EE2 amounts have been lost after 3 days. Pregnenolone was synthesized after exposure to EE2 and AP mixture. These results demonstrate that E. affinis has the potency to accumulate but also to eliminate endocrine disrupters which suggests a non-negligible role of this copepod species in the biogeochemical cycles of these contaminants in estuarine ecosystems. Hence, these results also suggest that a transfer of 4 NP, NP1EC, and EE2 to copepod predators and subsequently that secondary poisoning of these organisms might be possible. Estrogen-like contaminants can induce pregnenolone synthesis and affect the reproduction of E. affinis. These results suggest the important role of this copepod species in biogeochemical cycles of non-ionic surfactants as well as synthetic steroids in estuarine ecosystems. E. affinis could be a non-negligible route of exposure for juvenile fish and underline the potential for deleterious effects on copepod predators.

Various biomarker and bioindicator responses in muddy loach (Misgurnus anguillicadatus) exposed to endosulfan for 21 days

To evaluate the effects for endocrine disruption and the toxic effects on the biomarkers and bioindicators the muddy loach (Misgurnus anguillicadatus) was exposed to endosulfan using a continuous flow-through system for a period of 21 days. This study evaluated the effect of endosulfan using the biomarkers (VTG, AChE, EROD, Comet) and bioindicators (CF, GSI, LSI) should be deleted because it is logically redundant. Male muddy loach exposed to the 2.0 μg/L of endosulfan showed a steep increase of VTG rate compare to the control groups (p 0.05). In conclusion, endosuflan caused vitellogenesis, cytochrome p450 induction, AChE damage and DNA damage in the muddy loach (Misgurnus anguillicaudatus), indicating that those effects should be considered in the case of the EDCs risk aβeβment of the endosulfan especially in benthic freshwater fish. In addition, those biomarkers can be used for the monitoring of endosulfan contamination in aquatic ecosystems.

Subacute toxicity of Karate to Sarotherodon galilieus (Linne, 1758)

The study investigated subacute toxic effects of Lambda-cyhalothrin (Karate) on Sarotherodon galilieus in a continuous flow-through system which was conducted by Fisheries Department, F. U.T, Minna. Sarotherodon galilieus was exposed to the following concentration of Karate: 0.00 ug/l, 0.5 ug/l and 1 ug/l. Histopathological studies were carried out on the Karate-exposed experimental fish. Severe gill disruptions and mutilations was observed which lead to curving and calcification of lamellae, lipidosis in liver were observed, kidney cells were congested when compared with the control. The effects of this toxicant on the liver, gills, and kidney cells of this fish increase with an increased concentration. The physicochemical parameters were within the tolerable limit recommended for the normal physiological processes in fish, however, they were affected by the toxicity of Karate. Temperature was negatively correlated with toxicity of karate and affect the conductivity significantly. The main behavioral changes observed in the fish were agitated swimming, air gulping, erratic swimming, lateral bending of the body, loss of equilibrium, quiescence, and death.

In situ investigation and visualisation of microbial attachment and colonisation in a heap bioleach environment: The novel biofilm reactor

In this paper, the development of a novel means of investigating the attachment and subsequent biofilm formation of mineral bioleaching micro-organisms to mineral surfaces in situ is described. The protocol was developed to investigate the interactions of micro-organisms with sulfide minerals and low-grade chalcopyrite ore under conditions resemblant of a bioheap environment. The method makes use of a biofilm reactor in which thin sections of mineral ore are mounted. The reactor is operated as a continuous flow-through system. Attachment of pure and mixed cultures of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum is assessed. The technique allows for the investigation of microbial ecology with special regard to microbe–mineral attachment, site and mineral specific associations of micro-organisms and spatial organisation of microbial communities present through the use of fluorescent microscopy techniques. Preliminary fluorescent in situ hybridisation (FISH) analysis of the attachment of L. ferriphilum and A. ferrooxidans to massive chalcopyrite sections, as well as to low-grade chalcopyrite containing ore sections is presented. In the case of both low-grade and massive sulfide mineral samples, attachment of mixed micro-colonies was observed in regions where surface defects were prevalent. In low-grade samples, preferential attachment was observed in regions where sulfide minerals were present. The density of the attached micro-colonies increased with an increase in contacting time (from 20, 72 and 96 h) and was indicative of an actively growing mono-layered biofilm.

Reduction of Alkali-Labile Phosphates in Mussels Exposed to Primary-Treated Wastewaters Undergoing Ozone and Ultraviolet Disinfection: A Pilot Study

Abstract The purpose of this study was to examine the estrogenic potential of a primary municipal effluent, undergoing ozone or ultraviolet disinfection, in Elliptio complanata mussels. Mussels were exposed for seven weeks, using a continuous flowthrough system, to a primary effluent from a major city before and after disinfection. Results showed that the effluents, regardless of the disinfection procedure, readily affected gametogenesis in mussels as determined by gonado-somatic index, and by DNA synthesis as determined by the activity of aspartate transcarbamoylase activity, a rate-limiting enzyme for pyrimidine synthesis. The estrogenic potential of the effluents was observed by the increased levels of alkali-labile phosphate, a generic measure for vitellogenin-like proteins, where the disinfection procedures did not completely remove the estrogenic effects of the primary effluent. Thus, the introduction of an ozone or ultraviolet disinfection step to a primary-treated effluent will likely diminish the estrogenic potential, but not entirely.

Population heterogeneity in Methylobacterium extorquens AM1

Heterogeneity of cells within exponentially growing populations was addressed in a bacterium, the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methanol-inducible promoter (P(mxaF)) and gfp(uv) was used with flow cytometry to analyse the distribution of gene expression in populations grown on either succinate or methanol, correlated with forward scatter as a measure of cell size. These cell populations were found to consist of three major subpopulations defined by cells that were actively growing and dividing, newly divided, and non-dividing. Through the use of flow cytometry, it was demonstrated that a significant percentage of the total population did not respond to carbon shift. In addition, these experiments demonstrated that a small subset of the total population was significantly brighter than the rest of the population and dominated fluorimetry data. These results were corroborated with a continuous flow-through system and laser scanning microscopy, confirming that subpopulations, not discernible in the population average, dominate population response. These results demonstrate that the combination of flow cytometry and microscopic single-cell analysis can be effectively used to determine the dynamics of subpopulations in population response. In addition, they support the concept that physiological diversity in isogenic populations can poise some proportion of the population to respond appropriately to changing conditions.

Impact of PEF treatment inhomogeneity such as electric field distribution, flow characteristics and temperature effects on the inactivation of E. coli and milk alkaline phosphatase

Abstract High intensity pulsed electric field (PEF) treatment was investigated focusing on the alteration of electric field distribution, flow characteristics and temperature distribution due to the modification of the treatment chamber. The aim was the improvement of the effectiveness of microbial inactivation of E. coli and to reduce the PEF impact on alkaline phosphatase (ALP) activity in raw milk. Mathematical simulation of the PEF process conditions considering different treatment chamber setups was performed prior to experimental verification. Finally the impact of the treatment chamber modifications on microbial inactivation and enzyme activity was determined experimentally. Using a continuous flow-through PEF system and a co-linear treatment chamber configuration the insertion of stainless steel and polypropylene grids was performed to alter the field strength distribution, increase the turbulence kinetic energy and improve the temperature homogeneity. The Finite Element Method (FEM) analysis showed an improved electric field strength distribution with increased average electric field strength and a reduced standard deviation along the center line of the treatment zone indicating a more homogenous electric field. The velocity profile was improved resulting in an increase of turbulence kinetic energy due to the insertion of the grids. As revealed by mathematical modeling, the temperature of the liquid was decreased, and formation of temperature peaks was avoided. Measured inactivation of heat sensitive alkaline phosphatase (ALP) was reduced from 78% residual activity to 92% after PEF treatment and it could be shown that thermal effects and temperature peaks have been the main reason for enzyme inactivation due to PEF. At the same time, an increase of microbial inactivation of 0.6 log–cycles could be determined experimentally due to the modification of the treatment chamber design. Industrial relevance The application of pulsed electric field as a non-thermal pasteurization technology requires an accurately defined treatment intensity followed by a predictable microbial inactivation. Unavoidable thermal effects occurring during PEF treatment due to ohmic heating have to be minimized to assure the retention of heat-sensitive nutrients and bioactive compounds. The presented investigations contribute to the fulfilment of these requirements for further successful industrial implementation of the PEF technology such as the selective inactivation or retention of enzyme activity in liquid food systems.

Uptake and elimination of hydrophobic organic contaminants in estuarine copepods: An experimental study

Polycyclic aromatic hydrocarbons (PAHs) are considered to be rapidly biotransformed by organisms, whereas polychlorinated biphenyls (PCBs) are strongly bioaccumulated. In the present study, the estuarine copepod Eurytemora affinis was exposed in a continuous flow-through system to dissolved PAH (500 ng/L) and PCB (300 ng/L) mixtures for 86 h, whereas control groups were placed in a continuous flow-through system with clean water. Both PCB and PAH body residues were measured and compared in exposed and in nonexposed copepods to assess the uptake and the elimination of these two contaminant classes in this copepod species. After the exposure, exposed copepods exhibited concentration factors, based on a dry-weight basis, of 25, 750, and 1,200, respectively, for total PCBs and PAHs. The lower concentrations of PAHs in the nonexposed versus exposed copepods in contrast to small differences for PCBs suggest a higher rate of metabolism of PAHs compared with PCBs and could explain the differences observed in the accumulation. Furthermore, uptake as well as elimination of both PCBs and PAHs were compound selective in E. affinis. Therefore, higher-molecular-weight PCBs and PAHs were preferentially accumulated, while lower-molecular-weight compounds were preferentially eliminated. These results suggest the importance of copepods in the biogeochemical cycles of hydrophobic organic contaminants in estuarine ecosystems.

Serum Inhibits P. aeruginosa Biofilm Formation on Plastic Surfaces and Intravenous Catheters

Biofilm formation on medical devices such as intravenous catheters is a serious manifestation of Pseudomonas aeruginosa infections. Serum has bactericidal activity, a function of multiple serum components. In this study, we determined the effect of serum and serum components on the formation of P. aeruginosa biofilm. We examined the effect of adult bovine serum (ABS) or bovine serum albumin (BSA) on biofilm development on plastic coverslips. This was done using both static and continuous flow-through culture systems and P. aeruginosa strain PAO1. Biofilms were quantified using crystal violet assays and visualized using confocal scanning laser microscopy and scanning electron microscopy. We examined the effect of ABS on PAO1 swimming and twitching motilities (both contribute to P. aeruginosa biofilm development). We also analyzed the inhibitory effect of adult human serum (AHS) and plasma (AHP) on PAO1 biofilm development on plastic coverslips and intravenous catheters. Compared with M9 minimal medium (M9), 10% ABS-supplemented medium (M9/ABS-10) caused a significant decrease in biofilm development. Coverslips precoated with M9/ABS-10 failed to develop biofilm when placed in M9. In addition to reduced biofilm formation, adding ABS to M9 reduced an already-developed PAO1 biofilm. Compared with M9, M9/ABS-10 enhanced PAO1 twitching motility considerably, but did not affect swimming motility. Similar to ABS, BSA blocked biofilm formation but did not affect PAO1 twitching motility. Both AHS and AHP blocked PAO1 biofilm formation on plastic coverslips and intravenous catheters. These results suggest that as part of the host innate resistance, serum inhibits P. aeruginosa biofilm formation on plastic surfaces, including intravenous catheters. Two possible scenarios for this inhibition include blocking the direct interaction between P. aeruginosa and the substrates, and the enhancing P. aeruginosa twitching motility.

In vitro Skin Permeability of Different Terbinafine Hydrochloride Formulations

The objectives of the study are to determine the in vitro permeability of different terbinafine hydrochloride formulations through human skin and to measure the respective concentrations of each formulation within the exposed skin tissue. The permeation of three commercially available 1% terbinafine hydrochloride formulations and two terbinafine hydrochloride solutions of 10 and 20 mg/ml through human skin was investigated using an in vitro continuous flow-through perfusion system. The terbinafine hydrochloride retained in the skin was extracted and analysed. The terbinafine hydrochloride from the different formulations readily diffused into the skin tissue. However, no flux values for any of the terbinafine hydrochloride formulations through the skin into the receptor fluid were found. The mean terbinafine hydrochloride concentrations in the skin after 24 h exposure to the three commercial formulations were 3.589, 1.590 and 4.219 μg/ml respectively. The mean terbinafine hydrochloride concentrations in the skin after 24 h of exposure to the terbinafine hydrochloride solutions (PBS/Methanol 1:1) of 10 and 20 mg/ml were 85.280 and 154.680 μg/ml respectively. The mean terbinafine hydrochloride concentration in the skin exposed to the 10 mg/ml PBS/Methanol solution was higher than those from the three commercial formulations. Terbinafine seems to accumulate in skin/bind to the skin, rather than to diffuse through the skin into the receptor compartment. This unique pharmacokinetic property of terbinafine hydrochloride may enhance its efficacy as topical antifungal and reduce systemic side effects.

Whole-cell luminescence-based flow-through biodetector for toxicity testing

A new type of biodetector was designed based on a bioluminescence test with the bacterium Vibrio fischeri performed in a liquid continuous flow-through system. Here we describe the modification of a commercial tube luminescence detector to work in the flow mode by building a new flow cell holder and a new case including "top cover" to connect the flow cell with the waste and the incubation capillary in a light-proof manner. As different samples were injected successively it was necessary to keep the individual peaks separated. This was done using an air-segmented flow in the reaction coil. To afford fast screening, the incubation time of the sample and the Vibrio fischeri, which equaled the dead time of the detection system, was set at 5.6 min. Rapid monitoring of toxic substances is achieved by using 20 microL of sample and flow-rates of 110-150 microL min(-1). As a proof-of-principle, we show results for the detection of five selected di-, tri- and tetrachlorophenols at different concentrations varying from 1 to 200 mg L(-1). Calculation of inhibition rates and EC50 values were performed and compared with corresponding values from the DIN EN ISO 11348-2 microplate format. Compared with the latter, the inhibition rates obtained with our flow-through biodetector for the compounds tested were generally about twofold lower, but importantly, a much faster detection is possible.

Effects of the wood extractives dehydroabietic acid and betulinol on reproductive physiology of zebrafish ( Danio rerio)—A two-generation study

Two wood extractives, dehydroabietic acid (DHAA) and betulinol (BET), present in wood industry effluents were evaluated for their potential effects on the reproductive physiology of zebrafish. Adult zebrafish (F0) were exposed in a continuous flow-through system to 50 μg/l DHAA, 5 μg/l BET and 0.27 μg/l (1 nM) 17β-estradiol (E2) for 3 months. Eggs were collected from F0 fish and the following F1 generation was exposed for 6 months. Biomarkers analyzed in both F0 and F1 fish were plasma vitellogenin (Vtg), testosterone (T), E2 (only females) and gonadal histology. DHAA and BET affected growth in terms of increased condition factor, and spawning was stimulated in BET-exposed fish of the F0 generation. F0 males exposed to DHAA and F0 females exposed to BET showed lower plasma Vtg concentration, but F1 males exposed to BET showed an increase in Vtg. In fish exposed to E2, the positive control for estrogenic effects, a pronounced increase in Vtg concentration was observed. Plasma sex steroids were not significantly affected by the wood extractives. However, although not statistically significant, the T concentration tended to be lower in fish of all BET treatments. The histological study revealed alterations in spermatogenic stages of F0 males exposed to DHAA and BET, which were different from those caused by E2. In F1 females, the percentage of vitellogenic oocytes was decreased in DHAA, BET and E2 exposures. This study shows that DHAA and BET may contribute to growth alterations and reproductive disturbances reported in fish exposed to pulp and paper mill effluents. Further, these wood extractives may have different effects in F0 and F1 generation fish, which highlights the value of two-generation studies in investigations regarding endocrine disrupting compounds.