Accessing Planktothrix species diversity and associated toxins using quantitative real-time PCR in natural waters

Tese de Doutoramento em Biologia apresentada à Faculdade de Ciências da Universidade do Porto, 2015.

The coevolution of two phytoplankton species on a single resource: allelopathy as a pseudo-mixotrophy

Without the top-down effects and the external/physical forcing, a stable coexistence of two phytoplankton species under a single resource is impossible — a result well known from the principle of competitive exclusion. Here I demonstrate by analysis of a mathematical model that such a stable coexistence in a homogeneous media without any external factor would be possible, at least theoretically, provided (i) one of the two species is toxin producing thereby has an allelopathic effect on the other, and (ii) the allelopathic effect exceeds a critical level. The threshold level of allelopathy required for the coexistence has been derived analytically in terms of the parameters associated with the resource competition and the nutrient recycling. That the extra mortality of a competitor driven by allelopathy of a toxic species gives a positive feed back to the algal growth process through the recycling is explained. And that this positive feed back plays a pivotal role in reducing competition pressures and helping species succession in the two-species model is demonstrated. Based on these specific coexistence results, I introduce and explain theoretically the allelopathic effect of a toxic species as a ‘pseudo-mixotrophy’—a mechanism of ‘if you cannot beat them or eat them, just kill them by chemical weapons’. The impact of this mechanism of species succession by pseudo-mixotrophy in the form of alleopathy is discussed in the context of current understanding on straight mixotrophy and resource-species relationship among phytoplankton species.

Copper-mediated amyloid-beta toxicity is associated with an intermolecular histidine bridge

Amyloid-beta peptide (A beta) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic A beta-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/ peptide ratios of > 0.6:1 by EPR spectroscopy. The toxicity of the A beta-Cu2+ complex to cultured primary cortical neurons was attenuated when either the pi- or tau-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl- 1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. P-31 magic angle spinning solid-state NMR showed that A beta and A beta-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the A beta toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge.

Plantas ornamentais tóxicas: contributo para a sua caracterização e perceção do risco de exposição ocupacional

Mestrado em Segurança e Higiene do Trabalho.

Intervención en salud pública relacionada con la proliferación de microalgas tóxicas en una playa de Mijas (Málaga; España)

This paper provide an overview of the surveillance environmental efforts and risk management for the public health linked to the register of a disease outbreak related to an episode of toxic potentially and harmful microalgae, identified during the summer of 2010 in a recreational beach of a touristic municipality in the coast of Málaga (Spain). Phytoplankton analyses showed the presence of different species producers of marine biotoxins, dominating Ostreopsis cf. ovata in the followed immediately days to the communication of 39 clinical cases of people that required health cares. The risks of the toxins produced by microalgae must be taken into account in the health networks surveillance for recreational waters. This study suggests the possibility to review the actions of the public health services from Public Administration, to the light of the increasing information on episodic harmful algal blooms.

Spatial and temporal variation of natural toxicity in cnidarians, bryozoans and tunicates in Mediterranean caves

The natural toxicity of cnidarians, bryozoans and tunicates in two caves was assessed using the Microtox® technique in spring and autumn. One cave was located in the Cabrera Archipelago (Balearic Islands) and the other in the Medes Islands (Catalan littoral). The organisms analysed were good representatives of the coverage of each Phylum in the communities; however, these Phyla are less abundant than sponges which are the dominant group in these caves. Seventy-one percent of the species of cnidarians and bryozoans analysed were toxic in one of the caves, communities or seasons, which indicates the relevance of bioactive species in these groups. The tunicate Lissoclinum perforatum was the most toxic species. Although all three Phyla had some highly toxic species, a common pattern that related the caves, communities and seasons was not found. Seasonal variation of toxicity in cnidarians and bryozoans was higher in the Cabrera than in the Medes cave. Moreover, variation in toxicity either between communities or between seasons was a common trait for most cnidarians and bryozoans, whereas tunicates remained toxic throughout communities and seasons.

Amphibians in the diet of European Barn Owls

We present a review of the propensity to eat amphibians in the Barn Owl Tyto alba in Europe. Based on the analysis of 596 published studies reporting 3.32 million prey items identified in pellets, 17 869 amphibians (0.54%) were found. An analysis of 9036 amphibians identified to the species level showed that Barn Owls avoid consuming toxic species, and they are able to capture tree frogs (Hylidae) only rarely. The true frogs (Ranidae) are by far the most frequently captured amphibians followed by spadefoot toads (Pelobatidae) and Parsley frogs (Pelodytidae).

Method development for the simultaneous determination of methylmercury and inorganic mercury in seafood

This paper reports the method development for the simultaneous determination of methylmercury MeHgþ) and inorganic mercury (iHg) species in seafood samples. The study focused on the extraction and quantification of MeHgþ (the most toxic species) by liquid chromatography coupled to on-line UV irradiation and cold vapour atomic fluorescence spectroscopy (LC-UV-CV-AFS), using HCl 4 mol/L as the extractant agent. Accuracy of the method has been verified by analysing three certified reference materials and different spiked samples. The values found for total Hg and MeHgþ for the CRMs did not differ significantly from certified values at a 95% confidence level, and recoveries between 85% and 97% for MeHgþ, based on spikes, were achieved. The detection limits (LODs) obtained were 0.001 mg Hg/kg for total mercury, 0.0003 mg Hg/kg for MeHgþ and 0.0004 mg Hg/kg for iHg. The quantification limits (LOQs) established were 0.003 mg Hg/kg for total mercury, 0.0010 mg Hg/kg for MeHgþ and 0.0012 mg Hg/kg for iHg. Precision for each mercury species was established, being 12% in terms of RSD in all cases. Finally, the developed method was applied to 24 seafood samples from different origins and total mercury contents. The concentrations for Total Hg, MeHg and iHg ranged from 0.07 to 2.33, 0.003-2.23 and 0.006-0.085 mg Hg/kg, respectively. The established analytical method allows to obtain results for mercury speciation in less than 1 one hour including both, sample pretreatment and measuring step.

Especiação de Arsênio - uma revisão

This paper provides a review on separation methods and analytical techniques for the determination of several species of organic and inorganic arsenic in different matrices. Arsenic is an element whose speciation is of particular interest due to the great variation of toxicity levels exhibited for its different chemical forms. Arsenic (III) and As (V) are the most toxic species while organic compounds such as arsenobetaine (AsB), produced by methylation of inorganics species (carcinogenics) are relatively less toxic, hence the great importance of arsenic speciation in the determination of the degree of contamination of an environmental or biological system.

Diagnose of the ingestion of Asclepias mellodora St. Hil. by sheep through microhistological analysis of their digestive contents

Asclepias mellodora St. Hil. is a native acute toxic species frequent in the grasslands of the Buenos Aires province, Argentina, whose toxicity had not been assessed until now. This study evaluates the minimal lethal dose of this species for sheep, and the possibility of microscopically recognizing its fragments in gastrointestinal contents as a complementary diagnostic tool in necropsies. Three Frisona sheep (average LW=55±4.5 kg) were dosed via an esophageal tube with each one of the following doses of asclepias: 8.0, 5.0, 2.0 and 0.8 g DM.kg LW-1. Sheep poisoned with the three higher doses died between 10 and 85 h after intoxication, but those receiving the lower dose did not. During necropsies we: 1) determined the dry weight of the contents of rumen+reticulum, omasum+abomasum, and large intestine, 2) estimated the percentages of asclepias fragments by microanalysis correcting for digestion effects on fragment recognition, and 3) calculated the total mass of asclepias in the digestive tract of each animal. For the three higher doses, the mass of asclepias identified in the total ingesta was 12.3±3.4% of the amount supplied, possibly because of the strong diarrhea its ingestion produced. The percentages of asclepias in rumen+reticulum did not differ from the average quantified for the entire tract. The results of this study indicate that the minimal lethal doses of asclepias for sheep is between 2.0 and 0.8g DM·kg LW-1, and that the microhistological analysis of the rumen+reticulum, the easiest region to sample, can be used to confirm the ingestion of this toxic species, although the estimated percentage will be not a good estimator of the ingested percentage.

Planktic Cyanobacteria from upper Tietê basin reservoirs, SP, Brazil

Considering the great ecological and sanitary importance of the cyanobacteria and the need of detailed information about these organisms in Brazilian water bodies, the present study aims at contributing towards the knowledge of the cyanobacterial flora of five reservoirs belonging to the upper Tietê Basin, São Paulo: Billings, Guarapiranga, Jundiaí, Pirapora, Ponte Nova and Taiaçupeba. In the past several years, these reservoirs have been submitted to severe environmental deterioration and have repeatedly presented cyanobacterial blooms, including those of toxic species. The samples were collected between 1997 and 2003 either with plankton net (20 µm mesh) or van Dorn's bottle, and preserved with lugol solution or formaldehyde. Some species were isolated and maintained in culture. Forty-eight species of cyanobacteria were identified, with predominance of the order Chroococcales (58%), followed by the orders Oscillatoriales (21%) and Nostocales (21%). Among the 48 studied species, 17 (35%) were considered potentially toxic. The occurrence and biodiversity of the cyanobacteria in each reservoir depend on the environmental conditions. Among the five water bodies, Billings Reservoir presented the most adequate situation for the development of a greater number of species (34), probably due to its high pH values (around 8). Pirapora Reservoir on the other hand, with highest conductivity (445.0 µS cm-1) and lowest Secchi depth values (0.2 m), presented the lowest cyanobacterial biodiversity (14 species).

Avaluació de la toxicitat de metalls pesants i arsènic en diferents models biològics

En aquest estudi, la toxicitat de diversos metalls pesants i l'arsènic va ser analitzada utilitzant diferents models biològics. En la primera part d'aquest treball, el bioassaig de toxicitat Microtox, el qual està basat en la variació de l'emissió lumínica del bacteri luminiscent Vibrio fischeri, va ser utilitzat per establir les corbes dosi-resposta de diferents elements tòxics com el Zn(II), Pb(II), Cu(II), Hg(II), Ag(I), Co(II), Cd(II), Cr(VI), As(V) i As(III) en solucions aquoses. Els experiments es varen portar a terme a pH 6.0 i 7.0 per tal de mostrar que el pH pot influir en la toxicitat final mesurada d'alguns metalls degut als canvis relacionats amb la seva especiació química. Es varen trobar diferents tipus de corbes dosi-resposta depenent del metall analitzat i el pH del medi. En el cas de l'arsènic, l'efecte del pH en la toxicitat de l'arsenat i l'arsenit es va investigar utilitzant l'assaig Microtox en un rang de pHs comprès entre pH 5.0 i 9.0. Els valors d'EC50 determinats per l'As(V) disminueixen, reflectint un augment de la toxicitat, a mesura que el pH de la solució augmenta mentre que, en el cas de l'As(III), els valors d'EC50 quasi bé no varien entre pH 6.0 i 8.0 i només disminueixen a pH 9.0. HAsO42- i H2AsO3- es varen definir com les espècies més tòxiques. Així mateix, una anàlisi estadística va revelar un efecte antagònic entre les espècies químiques d'arsenat que es troben conjuntament a pH 6.0 i 7.0. D'altra banda, els resultats de dos mètodes estadístics per predir la toxicitat i les possibles interaccions entre el Co(II), Cd(II), Cu(II), Zn(II) i Pb(II) en mescles binàries equitòxiques es varen comparar amb la toxicitat observada sobre el bacteri Vibrio fischeri. L'efecte combinat d'aquests metalls va resultar ser antagònic per les mescles de Co(II)-Cd(II), Cd(II)-Zn(II), Cd(II)-Pb(II) i Cu(II)-Pb(II), sinèrgic per Co(II)-Cu(II) i Zn(II)-Pb(II) i additiu en els altres casos, revelant un patró complex de possibles interaccions. L'efecte sinèrgic de la combinació Co(II)-Cu(II) i la forta disminució de la toxicitat del Pb(II) quan es troba en presència de Cd(II) hauria de merèixer més atenció quan s'estableixen les normatives de seguretat ambiental. La sensibilitat de l'assaig Microtox també va ser determinada. Els valors d'EC20, els quals representen la toxicitat llindar mesurable, varen ser determinats per cada element individualment i es va veure que augmenten de la següent manera: Pb(II) < Ag(I) < Hg(II)  Cu(II) < Zn(II) < As(V) < Cd(II)  Co(II) < As(III) < Cr(VI). Aquests valors es varen comparar amb les concentracions permeses en aigues residuals industrials establertes per la normativa oficial de Catalunya (Espanya). L'assaig Microtox va resultar ser suficientment sensible per detectar els elements assajats respecte a les normes oficials referents al control de la contaminació, excepte en el cas del cadmi, mercuri, arsenat, arsenit i cromat. En la segona part d'aquest treball, com a resultats complementaris dels resultats previs obtinguts utilitzant l'assaig de toxicitat aguda Microtox, els efectes crònics del Cd(II), Cr(VI) i As(V) es varen analitzar sobre la taxa de creixement i la viabilitat en el mateix model biològic. Sorprenentment, aquests productes químics nocius varen resultar ser poc tòxics per aquest bacteri quan es mesura el seu efecte després de temps d'exposició llargs. Tot i això, en el cas del Cr(VI), l'assaig d'inhibició de la viabilitat va resultar ser més sensible que l'assaig de toxicitat aguda Microtox. Així mateix, també va ser possible observar un clar fenomen d'hormesis, especialment en el cas del Cd(II), quan s'utilitza l'assaig d'inhibició de la viabilitat. A més a més, diversos experiments es varen portar a terme per intentar explicar la manca de toxicitat de Cr(VI) mostrada pel bacteri Vibrio fischeri. La resistència mostrada per aquest bacteri podria ser atribuïda a la capacitat d'aquest bacteri de convertir el Cr(VI) a la forma menys tòxica de Cr(III). Es va trobar que aquesta capacitat de reducció depèn de la composició del medi de cultiu, de la concentració inicial de Cr(VI), del temps d'incubació i de la presència d'una font de carboni. En la tercera part d'aquest treball, la línia cel·lular humana HT29 i cultius primaris de cèl·lules sanguínies de Sparus sarba es varen utilitzar in vitro per detectar la toxicitat llindar de metalls mesurant la sobreexpressió de proteines d'estrès. Extractes de fangs precedents de diverses plantes de tractament d'aigues residuals i diferents metalls, individualment o en combinació, es varen analitzar sobre cultius cel·lulars humans per avaluar el seu efecte sobre la taxa de creixement i la capacitat d'induir la síntesi de les proteïnes Hsp72 relacionades amb l'estrès cel·lular. No es varen trobar efectes adversos significatius quan els components s'analitzen individualment. Nogensmenys, quan es troben conjuntament, es produeix un afecte advers sobre tan la taxa de creixement com en l'expressió de proteins d'estrès. D'altra banda, cèl·lules sanguínies procedents de Sparus sarba es varen exposar in vitro a diferents concentracions de cadmi, plom i crom. La proteïna d'estrès HSP70 es va sobreexpressar significativament després de l'exposició a concentracions tan febles com 0.1 M. Sota les nostres condicions de treball, no es va evidenciar una sobreexpressió de metal·lotioneïnes. Nogensmenys, les cèl·lules sanguínies de peix varen resultar ser un model biològic interessant per a ser utilitzat en anàlisis de toxicitat. Ambdós models biològics varen resultar ser molt adequats per a detectar acuradament la toxicitat produïda per metalls. En general, l'avaluació de la toxicitat basada en l'anàlisi de la sobreexpressió de proteïnes d'estrès és més sensible que l'avaluació de la toxicitat realitzada a nivell d'organisme. A partir dels resultats obtinguts, podem concloure que una bateria de bioassaigs és realment necessària per avaluar acuradament la toxicitat de metalls ja que existeixen grans variacions entre els valors de toxicitat obtinguts emprant diferents organismes i molts factors ambientals poden influir i modificar els resultats obtinguts.

Do phytoplankton communities evolve through a self-regulatory abundance-diversity relationship?

A small group of phytoplankton species that produce toxic or allelopathic chemicals has a significant effect on plankton dynamics in marine ecosystems. The species of non-toxic phytoplankton, which are large in number, are affected by the toxin-allelopathy of those species. By analysis of the abundance data of marine phytoplankton collected from the North-West coast of the Bay of Bengal, an empirical relationship between the abundance of the potential toxin-producing species and the species diversity of the non-toxic phytoplankton is formulated. A change-point analysis demonstrates that the diversity of non-toxic phytoplankton increases with the increase of toxic species up to a certain level. However, for a massive increase of the toxin-producing species the diversity of phytoplankton at species level reduces gradually. Following the results, a deterministic relationship between the abundance of toxic phytoplankton and the diversity of non-toxic phytoplankton is developed. The abundance–diversity relationship develops a unimodal pathway through which the abundance of toxic species regulates the diversity of phytoplankton. These results contribute to the current understanding of the coexistence and biodiversity of phytoplankton, the top-down vs. bottom-up debate, and to that of abundance–diversity relationship in marine ecosystems.

Determination of fluoroacetate and fluoride in blood serum by capillary zone electrophoresis using capacitively coupled contactless conductivity detection

Fluoroacetate is a highly toxic species naturally found in plants and in commercial products (compound 1080) for population control of several undesirable animal species. However, it is non-selective and toxic to many other animals including humans, and thus its detection is very important for forensic purposes. This paper presents a sensitive and fast method for the determination of fluoroacetate in blood serum using capillary electrophoresis with capacitively coupled contactless conductivity detection. Serum blood samples were treated with ethanol to remove proteins. The samples were analyzed in BGE containing 15 mmol/L histidine and 30 mmol/L gluconic acid (pH 3.85). The calibration curve was linear up to 75 mu mol/L (R(2) = 0.9995 for N = 12). The detection limit in the blood serum was 0.15 mg/kg, which is smaller than the lethal dose for humans and other animals. Fluoride, a metabolite of the fluoroacetate defluorination, could also be detected for levels greater than 20 mu mol/L, when polybrene was used for reversion of the EOF. CTAB and didecyldimethylammonium bromide are not useful for this task because of the severe reduction of the fluoride level. However, no interference was observed for fluoroacetate.

Caracterização limnológica e determinação da capacidade suporte do reservatório mendubim, (Rio Grande do Norte) para o cultivo de peixes em tanques-rede

The aims of this study were: i) assessing the trophic state of the Mendubim reservoir (semi-arid, Rio Grande do Norte, Brazil; 05° 38 99,0 S 36°55 98,0 W) based on chlorophyll-a, total phosphorus and nitrogen concentrations and water transparency; ii) relating the patterns of temporal variation of zooplankton and phytoplankton to the trophic state of the reservoir and iii) investigating the carrying capacity of the reservoir for cage fish farming. The samplingwas done monthly from July 2006 to July 2007 in three stations at the reservoir: next to the dam (barrage), in the central region and in the mouth of the main tributary. The abiotic and biotic variables analyzed were: Secchi depth, volatiles and fixed suspended solids, chlorophyll-a, total phosphorus and nitrogen, TN:TP ratio and mesozooplankton and phytoplankton composition and biomass. The results showed that the reservoir can be considered as mesotrophic with mean concentrations of total nitrogen, phosphorus and chlorophyll-a equal to 1711, 1 μg.L-1, 30,8 μg.L-1 and 5,62 μg.L-1 respectively. The Cyanophyceae class was the most representative in terms of density, with the presence of potentially toxic species such as Microcystis aeruginosa, Planktothrix planctonica, Cylindrospermopsis raciborskii, Aphanizomenon sp. ,Aphanocapsa delicatissima and Pseudanabaena acicularis. Among the zooplankton, the genus Notodiaptomus presented the largest biomass values. Overall, our results show that the light limitation should explain the weak relationship between chlorophyll-a and total phosphorus and nitrogen concentrations. We concluded that the water of Mendubim reservoir is suitable for intensive fish cage aquaculture. Based on the carrying capacity calculations for this reservoir, we found that the maximum sustainable yield of tilapias in cages in the reservoir is 126 ton per year assuming a factor of food conversion of 1.5: 1.0 and a phosphorus content in the fish food of 1%