Growth and domoic acid content of Pseudo-nitzschia australis isolated from northwestern Baja California,Mexico, cultured under batch conditions at different temperatures and two Si:NO3 ratios

Domoic acid (DA) poisoning in the southern part of the California Current System has been associated typically with blooms of Pseudo-nitzschia australis. The environmental variables that promote growth and DA production in the Mexican part of this system have not been identified. The present study investigated the effect of temperature and two nutrient ratios on the growth characteristics and DA content of two (BTS-1, BTS-2) P. australis strains isolated from the Pacific coast of northern Baja California peninsula, México. Of the different temperatures assayed (10, 12, 14, 15, 18 and 20 °C), the maximum cell abundance was detected at 12 °C for BTS-2 and 14 °C for BTS-1. The highest maximum specific growth rate (1.69 day⁻¹) was measured at 15 °C for BTS-2. With the exception of cells maintained at 15 °C, growth characteristics were similar in P. australis cultured in a high Si:NO₃ (2.5) or low Si:NO₃ (0.5) ratio at each temperature. Dissolved (dDA) and cellular (cDA) DA content measured at the stationary phase of growth was similar in cells cultivated at the different temperatures. No difference in cDA (between 0.11 and 1.87 pg DA cell⁻¹) was observed in cells cultivated at the two nutrient ratios. To evaluate if P. australis accumulates DA (cDA + dDA) at different stages of the culture and not only during the stationary phase of growth, the BTS-1 strain was cultivated at 14 °C and the content of this toxin was measured during culture development. The cultures were maintained at high (HL; 200 μmol quanta m⁻² s⁻¹) and low light (LL; 30 μmol quanta m⁻² s⁻¹) and in the two nutrient ratios to evaluate the effect of these variables on DA content. The photosynthetic performance and pigment concentration were measured as indicators of the physiological condition of the cells. cDA was detected in all culture conditions and during the different stages of growth. The highest DA content was measured during the lag phase of growth and it was present mainly in the medium (dDA = 70.83 pg DA cell⁻¹). Cells cultivated at HL produced more DA than LL cultured cells. P. australis cultured in HL presented lower photosynthetic rates than LL cells and had similar concentrations of photoprotective pigments and the highest maximum photosynthetic rates were detected during the lag phase of growth in all culture conditions. The results demonstrate that P. australis from northern Baja California peninsula presents a narrow temperature range for optimal growth under batch culture conditions. P. australis produce DA at different stages of growth, and DA content was related to the light intensity at which the cells were cultivated.     

Nutrient ratios influence variability in Pseudo-nitzschia species diversity and particulate domoic acid production in the Bay of Seine (France)

The population dynamics of different Pseudo-nitzschia species, along with particulate domoic acid (pDA) concentrations, were studied from May 2012 to December 2013 in the Bay of Seine (English Channel, Normandy). While Pseudo-nitzschia spp. blooms occurred during the two years of study, Pseudo-nitzschia species diversity and particulate domoic acid concentrations varied greatly. In 2012, three different species were identified during the spring bloom (P. australis, P. pungens and P. fraudulenta) with high pDA concentrations (∼1400 ng l⁻¹) resulting in shellfish harvesting closures. In contrast, the 2013 spring was characterised by a P. delicatissima bloom without any toxic event. Above all, the results show that high pDA concentrations coincided with the presence of P. australis and with potential silicate limitation (Si:N < 1), while nitrate concentrations were still replete. The contrasting environmental conditions between 2012 and 2013 highlight different environmental controls that might favour the development of either P. delicatissima or P. australis. This study points to the key role of Pseudo-nitzschia diversity and cellular toxicity in the control of particulate domoic acid variations and highlights the fact that diversity and toxicity are influenced by nutrients, especially nutrient ratios.     

Domoic acid in a marine pelagic food web: Exposure of southern right whales Eubalaena australis to domoic acid on the Península Valdés calving ground,Argentina

The gulfs that surround Península Valdés (PV), Golfo Nuevo and Golfo San José in Argentina, are important calving grounds for the southern right whale Eubalaena australis. However, high calf mortality events in recent years could be associated with phycotoxin exposure. The present study evaluated the transfer of domoic acid (DA) from Pseudo-nitzschia spp., potential producers of DA, to living and dead right whales via zooplanktonic vectors, while the whales are on their calving ground at PV. Phytoplankton and mesozooplankton (primary prey of the right whales at PV and potential grazers of Pseudo-nitzschia cells) were collected during the 2015 whale season and analyzed for species composition and abundance. DA was measured in plankton and fecal whale samples (collected during whale seasons 2013, 2014 and 2015) using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The genus Pseudo-nitzschia was present in both gulfs with abundances ranging from 4.4 × 10² and 4.56 × 10⁵ cell l⁻¹. Pseudo-nitzschia australis had the highest abundance with up to 4.56 × 10⁵ cell l⁻¹. DA in phytoplankton was generally low, with the exception of samples collected during a P. australis bloom. No clear correlation was found between DA in phytoplankton and mesozooplankton samples. The predominance of copepods in mesozooplankton samples indicates that they were the primary vector for the transfer of DA from Pseudo-nitzschia spp. to higher trophic levels. High levels of DA were detected in four whale fecal samples (ranging from 0.30 to 710 μg g⁻¹ dry weight of fecal sample or from 0.05 and 113.6 μg g⁻¹ wet weight assuming a mean water content of 84%). The maximum level of DA detected in fecal samples (710 μg DA g⁻¹ dry weight of fecal sample) is the highest reported in southern right whales to date. The current findings demonstrate for the first time that southern right whales, E. australis, are exposed to DA via copepods as vectors during their calving season in the gulfs of PV.     

Determination of selenium and its compounds in marine organisms

In this study, we investigate the type and quantity of selenium compounds in fish and marine organisms, using ion-pair reversed phase LC–ICP-MS, developed and applied for the analysis of Atlantic cod, Atlantic salmon, Greenland halibut, Atlantic herring, blue mussel, common crab, scallop, calanus, and Euphasia super. Of the samples examined, the lowest level of selenium was found in farmed Atlantic salmon (0.17 mg Se kg⁻¹ dm). The total selenium extraction efficiency by phosphate buffer was 2.5 times higher in sea plankton and shellfish samples than in fish samples. Analysis of Se species in each hydrolysate obtained by proteolysis showed the presence of selenomethionine, which constituted 41.5% of the selenium compounds detected in hydrolysates of Atlantic herring and 98.4% of those in extracts of Atlantic salmon. Inorganic compounds, such as selenates and selenites, were detected mainly in sea plankton and shellfish samples (<0.13 mg Se kg⁻¹ wm), although no correlation was found between the presence of inorganic compounds and total selenium concentration. The accuracy of the total selenium determination was validated using a certified reference material (oyster tissue (NIST 1566b)). A lyophilised powder of cod (Gadus morhua) was used to validate speciation analysis, enzymatic hydrolysis of lyophilised powder of cod recovered 54 ± 6% of total selenium, and SeMet constituted 83.5 ± 5.28% of selenium detected in hydrolysates. The chromatographic detection limits were, respectively, 0.30 ng mL⁻¹, 0.43 ng mL⁻¹, 0.54 ng mL⁻¹, 0.55 ng mL⁻¹, 0.57 ng mL⁻¹ and 0.72 ng mL⁻¹ for selenate, selenomethionine, selenite, Se-methyl-selenocysteine, selenocystine and selenomethionine selenoxide.The data on selenium concentrations and speciation presented here could be useful in estimating levels of selenium intake by seafood consumption.     

A toxic Pseudo-nitzschia bloom in Todos Santos Bay,northwestern Baja California,Mexico

A toxic Pseudo-nitzschia spp. bloom in the Todos Santos Bay area (31.8°N), Mexico, is described. This is the southernmost report of the presence of domoic acid (DA) in the California Current System and it is also the first report of the distribution of toxic Pseudo-nitzschia species and DA on the Baja California west coast. The maximum cell abundance of Pseudo-nitzschia was 3.02 × 10⁵ cells L^?1 and the maximum concentration of DA in particulate matter (pDA) was 0.86 μg L^?1. P. australis constituted the major proportion of cells identified as Pseudo-nitzschia. The environmental conditions associated with wind-driven upwelling were the cause for the accumulation of toxic cells. Maximum pDA and cell concentration were detected around 14 °C. The ratio of the concentration of macronutrients seemed to be the important factor for the accumulation of P. australis. The highest cell abundance was detected in areas with a high Si(OH)₄ to N ratio in the entire water column. Therefore, the relative increase of silicate concentration related to upwelling conditions was the probable cause for the accumulation of P. australis. Maximum photosystem II (PSII) quantum efficiency of charge separation (F_v/F_m) was negatively correlated to the pDA to fucoxanthin ratio. This ratio was used in this work as an index of cellular DA content. Therefore, the photosynthetic competence of the cells might be an important factor that affected their DA cellular content.     

Heterotrimeric G protein α-subunit is localized in the plasma membrane of Pinus bungeana pollen tubes

Heterotrimeric G proteins are involved in a variety of cellular responses, but relatively little is known about their function and biochemistry in plant pollen. In this paper, we establish the presence of a G protein associated with the plasma membranes of Pinus bungeana pollen tube. A 40 kDa polypeptide is detected and immunolocalized predominantly in pollen tube plasma membranes by polyclonal antisera directed against conserved peptides of mammalian Gα-subunit during pollen tube development. Cholera and pertussis toxins exhibited biphasic actions on tube growth, that is to say, inhibited pollen tube growth and result in rupture of tubes at concentrations less than 400 ng mL⁻¹, whereas stimulated pollen tube growth at concentration over 500 ng mL⁻¹. Fourier transform-infrared (FT-IR) spectra showed that the two toxins at concentrations of 400 ng mL⁻¹ resulted in enhanced synthesis of phenolics and reduced synthesis of cellulose, hemicellulose, and xylan of pollen tube wall, which may account for incidental rupture of pollen tubes at the concentration. These results suggest that the two toxins possibly affect pollen tube growth via downstream pertussis or cholera toxin-sensitive functional proteins, which regulate tube wall biosynthesis than at the Gα-subunit in P. bungeana tube growth.     

Mixotrophy in the newly described dinoflagellate Yihiella yeosuensis: A small,fast dinoflagellate predator that grows mixotrophically,but not autotrophically

To investigate tropical roles of the newly described Yihiella yeosuensis (ca. 8 μm in cell size), one of the smallest phototrophic dinoflagellates in marine ecosystems, its trophic mode and the types of prey species that Y. yeosuensis can feed upon were explored. Growth and ingestion rates of Y. yeosuensis on its optimal prey, Pyramimonas sp. (Prasinophyceae), as a function of prey concentration were measured. Additionally, growth and ingestion rates of Y. yeosuensis on the other edible prey, Teleaulax sp. (Cryptophyceae), were also determined for a single prey concentration at which both these rates of Y. yeosuensis on Pyramimonas sp. were saturated. Among bacteria and diverse algal prey tested, Y. yeosuensis fed only on small Pyramimonas sp. and Teleaulax sp. (both cell sizes = 5.6 μm). With increasing mean prey concentrations, both specific growth and ingestion rates of Y. yeosuensis increased rapidly before saturating at a mean Pyramimonas concentration of 109 ng C mL⁻¹ (2725 cells mL⁻¹). The maximum growth rate (mixotrophic growth) of Y. yeosuensis fed with Pyramimonas sp. at 20 °C under a 14:10-h light-dark cycle of 20 μE m⁻² s⁻¹ was 1.32 d⁻¹, whereas the growth rate of Y. yeosuensis without added prey was 0.026 d⁻¹. The maximum ingestion rate of Y. yeosuensis fed with Pyramimonas sp. was 0.37 ng C predator⁻¹ d⁻¹ (9.3 cells predator⁻¹ d⁻¹). At a Teleaulax concentration of 1130 ng C mL⁻¹ (66,240 cells mL⁻¹), growth and ingestion rates of Y. yeosuensis fed with Teleaulax sp. were 1.285 d⁻¹ and 0.38 ng C predator⁻¹ d⁻¹ (22.4 cells predator⁻¹ d⁻¹), respectively. Thus, Y. yeosuensis rarely grows without mixotrophy, and mixotrophy supports high growth rates in Y. yeosuensis. Y. yeosuensis has the highest maximum mixotrophic growth rate with the exception of Ansanella graniferaamong engulfment feeding mixotrophic dinoflagellates. However, the high swimming speed of Y. yeosuensis (1572 μm s⁻¹), almost the highest among phototrophic dinoflagellates, may prevent autotrophic growth. This evidence suggests that Y. yeosuensis may be an effective mixotrophic dinoflagellate predator on Pyramimonas and Teleaulax, and occurs abundantly during or after blooms of these two prey species.     

An o-aminobenzoic acid film-based immunoelectrode for detection of the cardiac troponin T in human serum

An amperometric immunosensor for cardiac troponin T detection in human serum troponin T, a marker considered as “gold standard” for acute myocardial infarction diagnosis, is described. A stable carboxylic film to covalently bind antibodies against cTnT onto electrode surface was achieved with electropolymerization of the o-aminobenzoic acid. A fractional factorial study was performed to optimize the electropolymerization parameters. Cyclic voltammetry assays were carried out for characterize steps of the modified electrode surface. The obtained calibration curve at −0.05 V by amperometry presented a good linear response range from 0.05 to 5.0 ng mL⁻¹ cTnT with a correlation coefficient of 0.992 (n = 6) and 0.016 ng mL⁻¹ detection limit. The electrodes showed a good stability upon the analytical responses retaining 91.6% of its initial response after 18 days. This sensor showed outgoing results regarding sensitivity allowing reliable measurements of the cTnT at levels of clinical significance for acute myocardial infarctions diagnosis.     

Simultaneous determination of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam in human serum using gas chromatography–mass spectrometry

A method for the quantitation of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam from human serum capable of monitoring concentrations achieved under therapeutic conditions is presented. The substances were extracted under basic conditions with toluene and the hydroxy metabolites transformed to their tert-butyldimethylsilyl derivatives with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. The samples were measured by gas chromatography–mass spectrometry. The limits of detection are 0.2 ng ml⁻¹ for midazolam and 0.1 ng ml⁻¹ for 1-hydroxy- and 4-hydroxymidazolam. The coefficients of variation are 3.9% at 5 ng ml⁻¹ for midazolam, 6.7% at 2 ng ml⁻¹ for 1-hydroxymidazolam and 8.8% (22.2%) at 0.5 (0.2) ng ml⁻¹ for 4-hydroxymidazolam.     

Development and validation of a rapid method for the determination and confirmation of 10 nitroimidazoles in animal plasma using liquid chromatography tandem mass spectrometry

A rapid LC–MS/MS method has been developed and validated for the simultaneous identification, confirmation and quantitation of 10 nitroimidazoles in plasma. The method validated in accordance with Commission Decision (CD) 2002/657/EC is capable of analysing for metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), ipronidazole (IPZ) and their hydroxy metabolites MNZ-OH, HMMNI (hydroxymethyl, methyl nitroimidazole), IPZ-OH. The method is also capable of analysing carnidazole (CRZ), ornidazole (ORZ) and ternidazole (TRZ) which are rarely analysed by modern methods. MNZ, DMZ and RNZ have a recommended level (RL) of 3 ng mL^?1 which this method is easily able to detect for all the nitroimidazole compounds. Plasma samples are extracted with acetonitrile, and NaCl is added to help remove matrix contaminants. The acetonitrile extract undergoes a liquid–liquid wash step with hexane; it is then evaporated and reconstituted in mobile phase. The reconstituted samples are analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS). The decision limits (CCα) range from 0.5 to 1.6 ng mL^?1 and the detection capabilities (CCβ), range from 0.8 to 2.6 ng mL^?1. The results of the inter-assay study, which was performed by fortifying bovine plasma samples (n = 18) on three separate days, show the accuracy calculated for the various analytes range between 101% and 108%. The precision of the method, expressed as CV% values for the inter-assay variation of each analyte at the three levels of fortification (3, 4.5 and 6.0 ng mL^?1), ranged between 4.9% and 15.2%. A day 4 analysis was carried out to examine species variances in animals such as avian, ovine, porcine and equine.     

Trace-level sensing of dopamine in real samples using molecularly imprinted polymer-sensor

Induced-fit responsive dopamine (DA) imprinted polymer, poly (melamine-co-chloranil), was used as a suitable coating material for the modification of a hanging mercury drop electrode. The zwitterionic conformation of the imprinted polymer responded differential pulse, cathodic stripping voltammetric current, without any false-positive or false-negative contributions of non-specific sorptions, in aqueous environment of complex matrices. The limit of detection (3σ) of dopamine was found to be as low as 0.148 ng mL⁻¹, by the proposed sensor that could be considered a sensitive marker of dopamine depletion in Parkinson's disease (PD).     

Morphology and molecular phylogeny of Nitzschia bizertensis sp. nov.—A new domoic acid-producer

A new toxin-producing marine diatom, Nitzschia bizertensis sp. nov., isolated from the Bizerte Lagoon (Tunisia, Southwest Mediterranean Sea) is, based on studies on eight different strains, characterized morphologically by light microscopy, transmission and scanning electron microscopy, and phylogenetically using the nuclear rDNA regions: SSU, ITS1, 5.8S, ITS2 and D1–D3 of the LSU. The species belongs to the sections Lanceolatae or Lineares as defined by Cleve and Grunow (1880). These sections are characterized by species having linear-lanceolate valves with an eccentric raphe where the fibulae does not extend into the valve, and are otherwise famous for the lack of characters useful for delineation of species. Nitzschia bizertensis differs from most other species in these sections by having a high density of interstriae. The morphological and phylogenetic studies and comparisons with previously described Nitzschia species showed Nitzschia bizertensis sp. nov. to be a new species. Batch culture experiments were conducted for estimations of maximum growth rate and production of domoic acid (DA). Maximum cellular DA content of the examined strains ranged from 2 × 10⁻⁴ to 3.6 × 10⁻² pg cells⁻¹. The total DA concentration (pg mL⁻¹) was high already in exponential growth phase maybe due to reinoculation of “old” stationary phase cells, and increased into stationary growth phase where it reached a stationary level varying among the strains from ca. 4500 to 9500 pg mL⁻¹. Nitzschia bizertensis represents a new domoic acid-producing diatom and is the second toxin producing Nitzschia species. The resolution of Nitzschia bizertensis and Nitzschia navis-varingica in different parts of the LSU phylogenetic tree, and the recovery of the Pseudo-nitzschia species phylogenetically distant from those two species suggests that the ability to produce DA either evolved multiple times independently or was lost multiple times.     

The relationship between Pseudo-nitzschia (Peragallo) and domoic acid in Scottish shellfish

The diatom genus Pseudo-nitzschia (Peragallo) associated with the production of domoic acid (DA), the toxin reposnsible for amnesic shellfish poisoning, is abundant in Scottish waters. A two year study examined the relationship between Pseudo-nitzschia cells in the water column and DA concentration in blue mussels (Mytilus edulis) at two sites, and king scallops (Pecten maximus) at one site. The rate of DA uptake and depuration differed greatly between the two species with M. edulis whole tissue accumulating and depurating 7 μg g⁻¹ (now expressed as mg kg⁻¹) per week. In contrast, it took 12 weeks for DA to depurate from P. maximus gonad tissue from a concentration of 68 μg g⁻¹ (now mg kg⁻¹) to <20 μg g⁻¹ (now mg kg^‐1). The DA depuration rate from P. maximus whole tissue was <5% per week during both years of the study. Correlations between the Pseudo-nitzschia cell densities and toxin concentrations were weak to moderate for M. edulis and weak for P. maximus. Seasonal diversity on a species level was observed within the Pseudo-nitzschia genus at both sites with more DA toxicity associated with summer/autumn Pseudo-nitzschia blooms when P. australis was observed in phytoplankton samples. This study reveals the marked difference in DA uptake and depuration in two shellfish species of commercial importance in Scotland. The use of these shellfish species to act as a proxy for DA in the environment still requires investigation.     

Determination of metoclopramide in human plasma by LC–ESI-MS and its application to bioequivalance studies

An LC–MS method for the determination of metoclopramide in human plasma was developed and validated. Sample preparation involved extraction with ethyl acetate. Chromatographic separation was performed on a Thermo Hypersil-Hypurity C₁₈ (150 mm × 2.1 mm, 5 μm) with the mobile phase consisting of 40 mM ammonium acetate–methanol–acetonitrile. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H]⁺ ions at m/z 300 for metoclopramide and at m/z 384 for the internal standard (prazosin). The method was validated over 0.78–50.00 ng mL^?1 for metoclopramide. The recovery was 67.8–83.1%, and the limit of quantitation (LOQ) detection was 0.78 ng mL^?1 for metoclopramide. The intra- and inter-day precision of the method at three concentrations was 5.0–13.6% with accuracy of 99.2–104.0%. Stability of compounds was established in a battery of stability studies. The method was successfully applied to bioequivalence studies of metoclopramide hydrochloride tablets to obtain the pharmacokinetic parameters.     

Expansion of harmful brown tides caused by the pelagophyte,Aureoumbra lagunensis DeYoe et Stockwell,to the US east coast

Brown tides caused by the pelagophyte Aureoumbra lagunensis DeYoe et Stockwell have formed ecosystem disruptive algal blooms in shallow lagoons of Texas (TX), USA, for more than two decades but have never been reported elsewhere. During the summer of 2012, a dense brown tide occurred in the Mosquito Lagoon and northern Indian River Lagoon along the east coast of Florida (FL), USA. While chlorophyll a levels in this system have averaged 5 μg L⁻¹ during the past two decades, concentrations during this brown tide reached ∼200 μg L⁻¹. Concurrently, levels of nitrate were significantly lower than average and levels of dissolved organic nitrogen were significantly higher than average (p < 0.001 for both). Sequences of the 18S rRNA gene of the bloom community and of single cell isolates were identical to those of Aureoumbra lagunensis DeYoe et Stockwell from TX. The A. lagunensis brown tide in FL bloomed to densities exceeding 10⁶ cells mL⁻¹ (quantified with a species-specific immuno-label) from July through September, began to dissipate in October, but maintained densities exceeding 10⁵ cells mL⁻¹ in some regions through December of 2012. The decline of the bloom was associated with near-hypoxic conditions and more than 30 fish kills reported within the Mosquito Lagoon in September 2012, a number far exceeding all prior monthly reports in this system dating to 1996. Wild northern quahog populations (a.k.a. hard clam, Mercenaria mercenaria) suffered mass die offs during the brown tide and eastern oysters (Crassostrea virginica) that settled during 2012 were significantly smaller than prior years. Clearance rates of hard clams and eastern oyster were significantly reduced in the presence of Mosquito Lagoon bloom water and A. lagunensis monocultures isolated from the Mosquito Lagoon at densities of ∼10⁶ cells L⁻¹. The expansion of harmful brown tides caused by A. lagunensis to these estuaries represents a new threat to the US southeast coast.     

The measurement of ecstasy in human hair by triple phase directly suspended droplet microextraction prior to HPLC-DAD analysis

New pre-concentration technique, triple phase suspended droplet microextraction (SD-LPME) and liquid chromatography-photodiode array detection was applied to determine ecstasy, MDMA (3,4-methylendioxy-N-methylamphetamine) in hair samples. In this research MDMA in hair was digested and after treatment extracted. The effective parameters were investigated and method was evaluated. Under the optimal conditions, the MDMA was enriched by factor 98.11. Linearity (r = 0.9921), was obtained in the range of 10–15,000 ng mL^?1 and detection limit was 0.1 ng mL^?1.     

Algicidal and growth-inhibiting bacteria associated with seagrass and macroalgae beds in Puget Sound,WA, USA

The algicidal and growth-inhibiting bacteria associated with seagrasses and macroalgae were characterized during the summer of 2012 and 2013 throughout Puget Sound, WA, USA. In 2012, Heterosigma akashiwo-killing bacteria were observed in concentrations of 2.8 × 10⁶ CFU g⁻¹ wet in the outer organic layer (biofilm) on the common eelgrass (Zostera marina) in north Padilla Bay. Bacteria that inhibited the growth of Alexandrium tamarense were detected within the biofilm formed on the eelgrass canopy at Dumas Bay and North Bay at densities of ∼10⁸ CFU g⁻¹ wet weight. Additionally, up to 4100 CFU mL⁻¹ of algicidal and growth-inhibiting bacteria affecting both A. tamarense and H. akashiwo were detected in seawater adjacent to seven different eelgrass beds. In 2013, H. akashiwo-killing bacteria were found on Z. marina and Ulva lactuca with the highest densities of ∼10⁸ CFU g⁻¹ wet weight at Shallow Bay, Sucia Island. Bacteria that inhibited the growth of H. akashiwo and A. tamarense were also detected on Z. marina and Z. japonica at central Padilla Bay. Heterosigma akashiwo cysts were detected at a concentration of 3400 cysts g⁻¹ wet weight in the sediment from Westcott Bay (northern San Juan Island), a location where eelgrass disappeared in 2002. These findings provide new insights on the ecology of algicidal and growth-inhibiting bacteria, and suggest that seagrass and macroalgae provide an environment that may influence the abundance of harmful algae in this region. This work highlights the importance of protection and restoration of native seagrasses and macroalgae in nearshore environments, in particular those regions where shellfish restoration initiatives are in place to satisfy a growing demand for seafood.     

Determination of phenothiazine derivatives in human urine by using ionic liquid-based dynamic liquid-phase microextraction coupled with liquid chromatography

A simple and rapid method for the determination of seven phenothiazines derivatives (chlorpromazine, promethazine, levomepromazine, prochlorperazine, trifluoperazine, fluphenazine and thioridazine) in human urine samples is presented. The analytes are extracted from the sample in 50 μL of the ionic liquid 1-butyl-3-methyl-imidazolium hexafluorophosphate working in an automatic flow system under dynamic conditions. The chemical affinity between the extractant and the analytes allows a good isolation of the drugs from the sample matrix achieving at the same time their preconcentration. The separation and detection of the extracted compounds is accomplished by liquid chromatography and UV detection. The proposed method is a valuable alternative for the analysis of these drugs in urine within the concentration range 0.07–10 μg mL^?1. Limits of detection were in the range from 21 ng mL^?1 (thioridazine) to 60 ng mL^?1 (levomepromazine). The repeatability of the proposed method expressed as RSD (n = 5) varied between 2.2% (levomepromazine) and 3.9% (chlorpromazine).     

Analysis of aminoglycoside residues in bovine milk by liquid chromatography electrospray ion trap mass spectrometry after derivatization with phenyl isocyanate

A derivatization procedure using phenyl isocyanate was adapted to liquid chromatography ion trap mass spectrometry (LC–MSⁿ) for confirmation and quantification of aminoglycoside residues in milk. Aminoglycoside residues were extracted from milk with acid and isolated from the matrix with a weak cation exchange solid-phase extraction cartridge. After isolating the compounds from the milk, derivatives of gentamicin, neomycin, and tobramycin were formed by reacting the drugs with phenyl isocyanate in the presence of triethylamine. The analytes were separated using a dilute formic acid/acetonitrile gradient on a reversed-phase LC column. The derivatized compounds were analyzed using positive ion electrospray LC–MSⁿ with ion trap detection. Product ion spectra were generated from the derivatized protonated molecules. Specific ion transitions were evaluated for quantitative determination and qualitative confirmation of residues in milk. Using this procedure, residues were qualitatively confirmed in milk samples fortified with gentamicin and neomycin at levels ranging from 15 to 300 ng mL^?1. Gentamicin has four major components that were successfully separated and confirmed independently; for quantitative determination the peak areas from the four analogs were summed. Tobramycin was added as an internal standard for quantitation to mitigate the effects of matrix ion suppression and variable recoveries. Overall recoveries for this method ranged from 80% to 120% with relative standard deviations of less than 25%. The method detection limits are 9.8 ng mL^?1 for NEO and 12.8 ng mL^?1 for total GEN residues.     

Interactive effects of salinity and water depth on the growth of Melaleuca ericifolia Sm. (Swamp paperbark) seedlings

Melaleuca ericifolia Sm. (Swamp paperbark) is a common tree species in freshwater and brackish wetlands in southern and eastern Australia. The survival of this species in many wetlands is now threatened by increased salinity and inappropriate water regimes. We examined the response of 5-month-old M. ericifolia seedlings to three water depths (exposed, waterlogged and submerged) at three salinities (2, 49 and 60 dS m⁻¹). Increasing water depth at the lowest salinity did not affect survival, but strongly inhibited seedling growth. Total biomass, leaf area and maximum root length were highest in exposed plants, intermediate in waterlogged plants and lowest in submerged plants. Although completely submerged plants survived for 10 weeks at the lowest salinity, they demonstrated negative growth rates and were unable to extend their shoots above the water surface. At the higher salinities, M. ericifolia seedlings were intolerant of waterlogging and submergence: all plants died after 9 weeks at 60 dS m⁻¹. Soil salinities increased over time, and by Week 10, exceeded external water column salinities in both the exposed and waterlogged treatments. In exposed sediment, ∼90% of plants survived for 10 weeks at 60 dS m⁻¹ even though soil salinities reached ∼76 dS m⁻¹. No mortality occurred in the exposed plants at 49 dS m⁻¹, and small but positive relative growth rates were recorded at Week 10. We conclude that at low salinities M. ericifolia seedlings are highly tolerant of sediment waterlogging, but are unlikely to tolerate prolonged submergence. However, at the higher salinities, M. ericifolia seedlings are intolerant of waterlogging and submergence and died rapidly after 5 weeks exposure to this combination of environmental stressors. This research demonstrates that salinity may restrict the range of water regimes tolerated by aquatic plants.