10 research outputs found
Toxic phytoplankton response to warming in two Mediterranean bays of the Ebro Delta
The analysis of the phytoplankton and environmental parameters of the time series in Alfacs and Fangar bays (north western Mediterranean) from 1990 to 2009 shows some trends. There is an increase in the average water column temperature, 0.11, 0.01, 0.80 and 0.23 ÂşC for spring, summer, fall and winter respectively in Alfacs Bay and 1.76, 0.71, 1.33, 0.89 ÂşC for spring, summer, fall and winter in Fangar Bay. The trends in phytoplankton populations show a shift in the timing of occurrence of Karlodinium spp. blooms and an increase of the Pseudo-nitzschia spp. abundances. There is a lack of correlation between the average seasonal temperatures and the toxic phytoplankton abundances
Prevalence and persistence of gymnodimines in clams from the Gulf of Gabes (Tunisia) studied by mouse bioassay and LC-MS/MS
In this work we studied the toxicity in clams from the Gulf of Gabes, Tunisia (Southern
Mediterranean). Samples from two stations (M2 and S6) were collected monthly from
January 2009 to September 2010, and analyzed by the official control method of
mousse bioassay (MBA) for lipophilic toxins. All samples were also analyzed with the
LC-MS/MS method for the determination of lipophilic toxins, namely: okadaic acid
group, pectenotoxins, yessotoxins and azaspiracids, spirolides and gymnodimines
(GYMs). The results showed prevalence of GYMs since it was the only toxin group
identified in these samples with a maximum of 2,136 ÎĽg GYM -A kg-1 (February 2009
at M2). Furthermore, GYMs showed persistence in the area, with only one blank sample
below the limit of detection. Interestingly, this blank sample was found in June 2009
after an important toxic episode which supports the recent findings regarding the high
detoxification capability of clams, much faster than that reported for oysters. In
comparison, good agreement was found among MBA, the LD50 value of 80-100 ÎĽg kg-1
reported for GYM- A, and quantitative results provided by LC-MS/MS. On the contrary
to that previously reported for Tunisian clams, we unambiguously identified and
quantified by LC-MS/MS the isomers GYM- B/C in most samples. Phytoplankton
identification and enumeration of Karenia selliformis usually showed higher densities at
site M2 than S6 as expected bearing in mind toxin results, although additional results
would be required to improve the correlation between K. selliformis densities and
quantitative results of toxins. The prevalence and persistence of GYMs in this area at
high levels strongly encourages the evaluation of the chronic toxic effects of GYMs.
This is especially important taking into account that relatively large quantities of GYMs
can be released into the market due to the replacement of the official control method
from mouse bioassay to the LC-MS/MS for lipophilic toxins (Regulation (EU) No
15/2011), and the lack of Regulation for this group of toxins
The implementation of liquid chromatography tandem mass spectrometry for the official control of lipophilic toxins in seafood: Single-laboratory validation under four chromatographic conditions
We performed a comprehensive study to assess the fit for purpose of four chromatographic conditions for the determination of six groups of marine lipophilic toxins (okadaic acid and dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins, gymnodimine and spirolides) by LC-MS/MS to select the most suitable conditions as stated by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). For every case, the elution gradient has been optimized to achieve a total run-time cycle of 12 min. We performed a single-laboratory validation for the analysis of three relevant matrices for the seafood aquaculture industry (mussels, pacific oysters and clams), and for sea urchins for which no data about lipophilic toxins have been reported before. Moreover, we have compared the method performance under alkaline conditions using two quantification strategies: the external standard calibration (EXS) and the matrix-matched standard calibration (MMS). Alkaline conditions were the only scenario that allowed detection windows with polarity switching in a 3200 QTrap mass spectrometer, thus the analysis of all toxins can be accomplished in a single run, increasing sample throughput. The limits of quantification under alkaline conditions met the validation requirements established by the EURLMB for all toxins and matrices, while the remaining conditions failed in some cases. The accuracy of the method and the matrix effects where generally dependent on the mobile phases and the seafood species. The MMS had a moderate positive impact on method accuracy for crude extracts, but it showed poor trueness for seafood species other than mussels when analyzing hydrolyzed extracts. Alkaline conditions with EXS and recovery correction for OA were selected as the most proper conditions in the context of our laboratory. This comparative study can help other laboratories to choose the best conditions for the implementation of LC-MS/MS according to their own necessities
An integrated approach for the assessment of HAB dynamics in two NW Mediterranean bays from a GEOHAB perspective
Alfacs and Fangar Bay in the Ebro Delta, NW Mediterranean are the major sites in Catalonia for
shellfish cultivation. These bays are subject to occasional closures in shellfish harvesting due to the
presence of phycotoxins. Fish kills have also been associated with harmful algal blooms. The
comparison of phytoplankton dynamics in both bays offers the opportunity to reveal differences in
bloom patterns of species known to be harmful for the ecosystem and aquaculture activities. Field
research is underway under the GEOHAB framework within the Core Research Project on HABs in
Fjords and Coastal Embayments. The overall objective of this study is to improve our understanding
of HAB biogeographical patterns, and key elements driving bloom dynamics in time and space within
these semi-constrained embayments. Via the comparative approach we aim to improve the prediction
for monitoring purposes, with a focus on Karlodinium spp. associated with massive kills of
aquaculture species. This objective is addressed by incorporating long-term time series of
phytoplankton identification and enumeration with the first results of recent field work in both bays.
The latter includes the application of optical sensors, to yield a complementary view with enhanced
spatial and temporal resolution of bloom phenomena
Marine biotoxins in the Catalan littoral: could biosensors be integrated into monitoring programmes?
Aquest article descriu els sensors enzimĂ tics i immunosensors
electroquĂmics que s’han desenvolupat als nostres grups per a
la detecciĂł de la biotoxina marina Ă cid okadaic (OA), i discuteix
la possibilitat d’integrar-los en programes de seguiment. Els
sensors enzimĂ tics per a OA que es presenten es basen en la
inhibiciĂł de la proteĂŻna fosfatasa (PP2A) per aquesta toxina i la
mesura electroquĂmica de l’activitat enzimĂ tica mitjançant l’ús
de substrats enzimĂ tics apropiats, electroquĂmicament actius
després de la seva desfosforació per l’enzim. Els immunosensors
electroquĂmics descrits en aquest article es basen en un
enzimoimmunoassaig sobre fase sòlida competitiu indirecte
(ciELISA), amb fosfatasa alcalina (ALP) o peroxidasa (HRP)
com a marcatges, i un sistema de reciclatge enzimĂ tic amb diaforasa
(DI). Els biosensors presentats aquà s’han aplicat a
l’anà lisi de dinoflagel·lats, musclos i ostres. Les validacions
preliminars amb assaigs colorimètrics i LC-MS/MS han demostrat
la possibilitat d’utilitzar les bioeines desenvolupades
per al cribratge preliminar de biotoxines marines en mostres de
camp o de cultiu, que ofereixen informaciĂł complementĂ ria a
la cromatografia. En conclusiĂł, tot i que encara cal optimitzar
alguns parĂ metres experimentals, la integraciĂł dels biosensors
a programes de seguiment Ă©s viable i podria proporcionar
avantatges respecte a altres tècniques analĂtiques pel que fa al
temps d’anà lisi, la simplicitat, la selectivitat, la sensibilitat, el fet
de poder ser d’un sol ús i l’efectivitat de cost.
This article describes the electrochemical enzyme sensors and
immunosensors that have been developed by our groups for
the detection of marine biotoxin okadaic acid (OA), and discusses
the possibility of integrating them into monitoring programmes.
The enzyme sensors for OA reported herein are
based on the inhibition of immobilised protein phosphatase 2A
(PP2A) by this toxin and the electrochemical measurement of
the enzyme activity through the use of appropriate enzyme substrates,
which are electrochemically active after dephosphorylation
by the enzyme. The electrochemical immunosensors described
in this article are based on a competitive indirect Enzyme-
Linked ImmunoSorbent Assay (ciELISA), using alkaline
phosphatase (ALP) or horseradish peroxidase (HRP) as labels,
and an enzymatic recycling system with diaphorase (DI). The
biosensors presented herein have been applied to the analysis
of dinoflagellates, mussels and oysters. Preliminary validations
with colorimetric assays and LC-MS/MS have demonstrated
the possibility of using the developed biotools for the preliminary
screening of marine biotoxins in field or cultured samples, offering
complementary information to chromatography. In conclusion,
although optimisation of some experimental parameters is
still required, the integration of biosensors into monitoring programmes
is viable and may provide advantages over other analytical
techniques in terms of analysis time, simplicity, selectivity,
sensitivity, disposability of electrodes and cost effectiveness
Conjugation of genetically-engineered protein phosphatases to magnetic particles for okadaic acid detection
This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3ÎĽg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2ÎĽg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1ÎĽg/L)
Levels of PSP toxins in bivalves exposed to natural blooms of Alexandrium minutum in Catalan harbours
The development, validation, comparison and evaluation of analytical methods for marine
toxins rely on the availability of toxic material. Within the project JACUMAR PSP, our
interest is mainly focused on autochthonous bivalve species with the toxic profile of
Alexandrium minutum, since this is the principal species involved regionally in PSP
outbreaks. Mussels and oysters were exposed during few days in the harbor of Vilanova i la
GeltrĂş, to blooms reaching a maximum A. minutum concentration of 200,000 cells L-1 in
2008, and 40,000 and 800,000 cells L-1, in 2009. Mussels, oysters and clams were exposed to
one bloom of 22,000 cells L-1 in the harbor of Cambrils in 2009. In all situations higher toxic
levels analyzed by HPLC-FD with postcolumn oxidation were observed in mussels (i.e.
1,200-2,500 ÎĽg eq. STX kg-1) than in oysters (i.e. 60-800 ÎĽg eq. STX kg-1) exposed to the
same bloom. Blooms with higher concentrations of A. minutum did not correspond to higher
levels of PSP toxins in bivalves. These differences may be explained by differences in A.
minutum population dynamics, toxin production or in the physiological state or behaviour of
shellfish. These results confirm that mussels concentrate more PSP toxins from A. minutum
than oysters and clams
Protein phosphatase inhibition assays for okadaic acid detection in shellfish: matrix effects, applicability and comparison with LC-MS/MS analysis
The applicability of the protein phosphatase inhibition assay (PPIA) to the determination of okadaic acid
(OA) and its acyl derivatives in shellfish samples has been investigated, using a recombinant PP2A and a
commercial one. Mediterranean mussel, wedge clam, Pacific oyster and flat oyster have been chosen as
model species. Shellfish matrix loading limits for the PPIA have been established, according to the
shellfish species and the enzyme source. A synergistic inhibitory effect has been observed in the presence
of OA and shellfish matrix, which has been overcome by the application of a correction factor (0.48).
Finally, Mediterranean mussel samples obtained from Rı´a de Arousa during a DSP closure associated to
Dinophysis acuminata, determined as positive by the mouse bioassay, have been analysed with the PPIAs.
The OA equivalent contents provided by the PPIAs correlate satisfactorily with those obtained by liquid
chromatography–tandem mass spectrometry (LC–MS/MS)
Adaptive Pathways : Possible Next Steps for Payers in Preparation for Their Potential Implementation
Altres ajuts: There was no funding body. However, the writeup work was in part supported by grants from the Karolinska Institutet, Sweden.Medicines receiving a conditional marketing authorization through Medicines Adaptive Pathways to Patients (MAPPs) will be a challenge for payers. The "introduction" of MAPPs is already seen by the European Medicines Agency (EMA) as a fait accompli, with payers not consulted or involved. However, once medicines are approved through MAPPs, they will be evaluated for funding by payers through different activities. These include Health Technology Assessment (HTA) with often immature clinical data and high uncertainty, financial considerations, and negotiations through different types of agreements, which can require monitoring post launch. Payers have experience with new medicines approved through conditional approval, and the fact that MAPPs present additional challenges is a concern from their perspective. There may be some activities where payers can collaborate. The final decisions on whether to reimburse a new medicine via MAPPs will have more variation than for medicines licensed via conventional processes. This is due not only to increasing uncertainty associated with medicines authorized through MAPPs but also differences in legal frameworks between member states. Moreover, if the financial and side-effect burden from the period of conditional approval until granting full marketing authorization is shifted to the post-authorization phase, payers may have to bear such burdens. Collection of robust data during routine clinical use is challenging along with high prices for new medicines during data collection. This paper presents the concept of MAPPs and possible challenges. Concerns and potential ways forward are discussed and a number of recommendations are presented from the perspective of payers