Universidade de Lisboa
Faculdade de Ciências
Departamento de Biologia Animal
Impact of metal contamination on early life history stages of
common cuttlefish (Sepia officinalis): a biochemical approach
Rui Filipe Vieira Cereja
Dissertação de Mestrado
Mestrado em Ecologia Marinha
Universidade de Lisboa
Faculdade de Ciências
Departamento de Biologia Animal
Impact of metal contamination on early life history stages of
common cuttlefish (Sepia officinalis): a biochemical approach
Rui Filipe Vieira Cereja
Dissertação de Mestrado orientada pelo Professor Doutor Rui
Rosa e co-orientada pela Doutora Joana Raimundo
Mestrado em Ecologia Marinha
i
Agradecimentos:
À Doutora Joana Raimundo do IPMA (antigo IPIMAR) e Doutor Rui Rosa do Laboratório Marítimo da Guia, meus orientadores que entre o imenso trabalho que tiveram me ajudaram a concluir esta tese, assim como a todo o pessoal do Departamento de Ambiente Aquático do IPMA e do Laboratório Marítimo da Guia que sempre me receberam bem e me ajudaram sempre que precisei.
À Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, em especial ao Professor Doutor Mário Diniz da secção de Bioquímica e Biofísica ao Professor Doutor Pedro Costa do Departamento de Ciências e Engenharias do Ambiente desta instituição, que me ajudaram na grande parte deste trabalho realizada na FCT-UNL. Obrigado pela vossa disponibilidade e opiniões e desculpas por alguns mal entendidos. À Tatiana Teixeira e Ana Rita Lopes que me ajudaram com muito do trabalho de laboratório na FCT-UNL.
Aos meus pais e a todos os meus familiares que me ajudam e continuam a ajudar, e sem os quais não teria conseguido aqui chegar.
Aos meus colegas e amigos da FCUL, Ana Vicente, Audrey Lopes, Ana João, Joana Castro, Vanessa Madeira, Joana Coelho, Sara Serra, Maria João Sebastião, Raquel Redol, Vanessas Pires (que são duas), Miguel Guerreiro, Miguel Landum, Vitor Pereira, Andreia Teixeira, Mónica Vidal, Catarina Rodrigues e Irina Silva que tornaram estes últimos anos excelentes e muitos acompanharam desde a minha chegada cá até agora. Muito obrigado pelos bons momentos, opiniões, desabafos, aventuras e histórias para contar não só deste ano mas dos últimos cinco.
À Mafalda que sempre teve paciência e disponibilidade para mim e acompanhou toda a evolução e problemas que tive ao longo deste percurso. Obrigado pelo teu apoio e presença constante.
E finalmente à Estrutura de Missão para a Extensão da Plataforma Continental (EMEPC) pelas oportunidades que me deu nos últimos três meses e a toda a sua equipa, com quem passei bons momentos e sempre me ajudou em tudo o que foi necessário.
A todos, muito obrigado!
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Index
1. Resumo 1 2. Summary 6 3. General Introduction 7 3.1. References 104. Trace elements partitioning between eggshell and embryo of common cuttlefish (Sepia officinalis) during embryonic development exposed to different
environmental contamination (Sado estuary, Portugal) 14
4.1. Resumo 14
4.2. Abstract 15
4.3. Introduction 16
4.4. Material and Methods 18
4.4.1. Sampling 18
4.4.2. Analytic methodology 19
4.4.3. Statistical Analysis 21
4.5. Results 21
4.5.1. General Considerations 26
4.5.2. Variation between stages for each area 26 4.5.3. . Variation between areas for each embryonic stage 28
4.6. Discussion 29
4.6.1. . Elemental partitioning between eggshells and embryos 30 4.6.2. Elemental differences between embryonic stages in the three capture
areas 31
4.6.3. Parental transference (Eurominas) 33
4.7. Final considerations 34
4.8. References 35
5.
Changes in heat shock response and oxidative stress during
embryogenesis of the common cuttlefish (Sepia officinalis) in distinct
estuarine zones
39 5.1. Resumo 39 5.2. Abstract 40 5.3. Introduction 41 5.4. Methods 44 5.4.1. Sampling 44 5.4.2. Analytical Methodology 445.4.2.1. Preparation of tissue extracts 44
5.4.2.2. Glutathione S-Transferase (GST) 45
5.4.2.3. Catalase (CAT) 46
5.4.2.4. Superoxide dismutase (SOD) 47
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5.4.2.6. Heat shock proteins 48
5.4.3. Statistical Analysis 49
5.5. Results 49
5.5.1. CAT 49
5.5.2. SOD 50
5.5.3. GST 50
5.5.4. Malondialdehyde concentration (indicator of lipid peroxidation) 51
5.5.5. HSP 52
5.6. Discussion 53
5.6.1. Antioxidant enzyme concentration 53
5.6.2. Variations of antioxidants between areas and stages 54
5.6.3. Damage caused 55
5.7. Final considerations 56
5.8. References 57
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List of Figures:
Figure 1 - Cuttlefish egg tissues, adapted from wolf et al. (1985). From up to down
there is eggshell (s), germinal disc (gd), yolk (y), viteline membrane (vm), periviteline space (ps), chorion (ch) and finally basal ring (br). The used tissues in this work was yolk and germinal disc (or the embryo in later stages) and eggshell. 8
Figure 2 - Medians, 25 and 75% percentiles, minimum and maximum ranges of V, Cr,
Mn, Co, Ni, Cu, Zn, As, Se and Pb concentrations (µg g-1, dw) for eggshells and embryos in three embryonic stages (S0, S1, S2 and S3) collected in Caldeira (white boxes), Eurominas (grey boxes) and Setnave (black boxes). 22 - 25
Figure 3 - Changes in superoxide dismutase activity (U mg-1, ww) during embryogenesis (S1 initial stage , S2 – intermediate stage, S3 – final stage) of the common cuttlefish (Sepia officinalis) in three distinct zones of the Sado Estuary (Caldeira de Troia – White boxes, Eurominas – grey boxes, and Setnave – black boxes).. Values represent
median 25% and 75% distributions. 50
Figure 4 - Changes in Glutathione S-Transferase (pmol min-1 mg-1, ww) during embryogenesis (S1 initial stage , S2 – intermediate stage, S3 –final stage) of the common cuttlefish (Sepia officinalis) in three distinct zones of the Sado Estuary (Caldeira de Troia – White boxes, Eurominas – grey boxes, and Setnave – black boxes.). Values represent median 25% and 75% distributions. 51
Figure 5 - Changes in malondialdehyde concentration (pmol mg-1, ww) during embryogenesis (S1 initial stage , S2 – intermediate stage, S3 – final stage) of the common cuttlefish (Sepia officinalis) in three distinct zones of the Sado Estuary (Caldeira de Troia – White boxes, Eurominas – grey boxes, and Setnave – black boxes). Values represent median 25% and 75% distributions. 52
Figure 6 - Changes in HSP70/HSC70 (ng hsp70/hsc70 mg−1, ww) during embryogenesis (S1 initial stage , S2 – intermediate stage, S3 – final stage) of the common cuttlefish (Sepia officinalis) in three distinct zones of the Sado Estuary (Caldeira de Troia – White boxes, Eurominas – grey boxes, and Setnave – black boxes). Values represent
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List of Tables:
Table 1 - Vanadium, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Mo, Cd and Pb (µg g-1, dw) concentrations of fish protein (DORM-3), fish liver (DOLT-4) and lobster hepatopancreas (TORT-2) obtained in the present study and certified values. 20
Table 2.1 - Median (and ranges) of V, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Mo, Cd and Pb
concentrations (µg g-1, dw) in embryos and eggshells of Sepia officinalis in embryonic stages 1 (S1), 2 (S2) and 3 (S3) collected in Caldeira. Annex1
Table 2.2 - Median (and ranges) of V, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Mo, Cd and Pb
concentrations (µg g-1, dw) in embryos and eggshells of Sepia officinalis in embryonic stages 1 (S1), 2 (S2) and 3 (S3) collected in Eurominas. Annex 1
Table 2.3 – Median (and ranges) of V, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Mo, Cd and Pb
concentrations (µg g-1, dw) in embryos and eggshells of Sepia officinalis in embryonic stages 1 (S1), 2 (S2) and 3 (S3) collected in Setnave. Annex 1
Table 3 – Kurskall-Wallis test values (H) and p-values for the concentrations of V, Cr,
Mn, Co, Ni, Cu, Zn, As, Se, Cd, Mo and Pb for the samples collected in Caldeira, Eurominas and Setnave, at stage 1 (S1), 2 (S2) and 3, (S3). p<0.05 are marked with a
grey shade Annex 1
Table 4 – Kurskall-Wallis test values (H) and p-values of differences between
concentrations of V, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Cd, Mo and Pb for the samples collected in Eurominas between stage0 (S0), 1 (S1), 2 (S2) and 3 (S3). p<0.05 are marked with a grey shade. For Cd, due to only S0 and S3 have values above detection limit, the values are from Man-Whitney U test Annex 1
Table 5 - Table 4: Kurskall-Wallis test values (H) and p-values for antioxidant enzymes
SOD and GST. MDA and HSP70/HSC70 concentrations in the different areas and stages. p<0.05 are marked with a grey shade Annex 2
Table 6 – Median and ranges for SOD (U mg-1), GST (pmol min-1 mg-1), MDA (pmol mg−1) and HSP70/HSC70 (ng hsp70/hsc70 mg−1) concentrations for embryonic stages 1 (S1), 2 (S2) and 3 (S3) in Caldeira, Eurominas and Setnave Annex 2
1
1. Resumo:
O aumento exponencial da população mundial e o desenvolvimento industrial têm vindo a provocar um aumento das descargas de poluentes e degradação do meio aquático. Os estuários e as zonas costeiras são os principais locais de descargas e de retenção de poluentes, nomeadamente metais (Herut e Kress, 1997). Sendo considerados locais com elevada biodiversidade e importância ecológica, a protecção destas zonas tem, nos últimos anos, surgido como de extrema importância.
Os efluentes domésticos e industriais, a contaminação pelo tráfego marinho e a poluição atmosférica são algumas da fontes antropogénicas de metais (Bryan et al., 1971). Os níveis de metais no meio podem aumentar também devido à erosão, aos detritos dos organismos e aos produtos de decomposição da matéria orgânica (Niencheski et al., 1994). Alguns metais, por exemplo o Fe, o Cu e o Zn, em concentrações naturais têm funções essenciais e são indispensáveis para a realização de funções metabólicas dos organismos, como parte integrante de enzimas, regulação de ADN e fluídos sanguíneos (Bryan, 1971; Joyce and Steitz, 1994; Ferraro, 2004). Outros metais não possuem funções metabólicas conhecidas, por exemplo o Cd e o Pb (Bustamante, 1998). A exposição dos organismos a elevadas concentrações de metais pode provocar efeitos nocivos ao nível bioquímico ou celular (Depledge et al., 1995; Goksyr et al., 1996), causando danos de formas muito variadas, como por exemplo danos ao nível do ADN, inactivação ou estimulação de actividade enzimática e produção de radicais livres de oxigénio (Lindquist and Crayg., 1988; Ushmar and Halliwell, 1996; Raimundo et al., 2010; Pereira et al., 2010).Radicais livres de oxigénio são radicais resultantes de moléculas de oxigénio como o radical superoxido (O2-*), radical hidroxila (OH-*),
radical peroxilo (RO2*), acilo radical (RO*), radical hidroperoxila (HO-2*) e peroxido de
hidrogénio (H2O2) e ácido hipocloroso (HOCl) (Halliwell and Gutterridge, 1999). Muitas destas
moléculas são produzidas durante o normal metabolismo das células, havendo mecanismos de defesa que previnem que estas causem danos nos componentes celulares. Os metais (assim
2 como vários outros factores de stresse) aumentam a quantidade destes radicais ao alterarem o metabolismo por indução de stresse. Para além disso, alguns metais também criam radicais directamente através das reacções de Feton e diminuem a quantidade de enzimas antioxidades por inibição das mesmas. Todos estes efeitos vão alterar o equilíbrio entre enzimas antioxidantes e os radicais de oxigénio levando a que estes comecem a reagir com outras moléculas presentes como proteínas, lípidos e ADN causando danos nestes (Winston and Di Giulio, 1991; Singh et al., 2004). Ao reagirem com os radicais, os lípidos passam a peróxidos lipídicos (LPO) como hidroperoxidos lipídicos (lipid-ooh) que por sua vez formam radicais hidroperoxila (ROO*·) e acilo (RO*·) que por sua vez reagem com os lípidos vizinhos propagando a oxidação em cadeia (Ushmar and Halliwell, 1996). Esta oxidação cria vários problemas na célula, desde oxidação de proteínas, perda de Ca2+, perda de fluidez da membrana e colapso da membrana levando à morte da célula (Ushmar and Halliwell, 1996; Storey, 1996).
As “Heat Shock Proteins” (HSP) são proteínas essências para o normal funcionamento da célula actuando como chaperonas permitindo a normal enrolamento (“folding”) das proteínas (Fink, 1999) e são o sistema mais semelhante em todos os grupos de seres vivos, sendo comum entre arquias, bactérias, plantas e animais (Lindquist and Craig, 1988). Estas proteínas são conhecidas como proteínas de stresse, pois a sua expressão é principalmente associada a diversos eventos, respondendo a temperatura (função pela qual foram nomeadas HSP), stress oxidativo, metais, campos magnéticos e etanol, sendo que todos estes factores causam danos na estrutura terciária das proteínas. A sua função durante eventos de stresse é evitar o enrolamento errado de proteínas, e caso já tenha ocorrido corrigi-lo ou eliminar proteínas que tenham perdido a sua função (Parsell and Lindquist, 1993). As famílias de HSP que são normalmente associadas à resposta a stresse causado por metais, são as famílias HSP60 e HSP70. As proteínas “Heat Shock Congnate 70KDa” (HSC70), são proteínas traduzidas de forma constitutiva que estão encarregues da normal agregação das novas proteínas formadas no
3 retículo endoplasmático e as que vão ser transferidas para as mitocôndrias (Turner et al., 1999). Quando em stresse as células aumentam a produção destas proteínas acumulando-as nos agregados de proteínas com o objectivo de promover o correcto enrolamento, ou corrigir ou eliminar o enrolamento mas efectuados (Dastoor and Dreyer, 2000).
Os cefalópodes são exclusivamente marinhos, com uma elevada distribuição, encontrando-se desde a superfície do oceano até às zonas abissais. O choco comum, Sepia officinalis, vive em alto mar durante o inverno vindo depois para as zonas costeiras e estuários para se reproduzir. Nestas zonas, as fêmeas depositam os ovos em substrato duro como troncos de árvores, algas e artes de pesca, ficando estes ovos expostos à poluição destas zonas (Boucaud-Camou and Boismery, 1991). De forma a impedir a entrada de contaminantes provenientes da água a membrana glico-lipo-proteica dos ovos possui uma camada glicoproteica que, com o avançar do desenvolvimento e o crescimento do embrião, começa a tornar-se mais fina permitindo a passagem de alguns contaminantes e a acumulação no embrião (Bustamante et al., 2004; Lacoue-Labarthe et al., 2008, 2010). Vários estudos foram realizados analisando a variação de concentrações de metais durante a embriogénese mas quase todos em condições laboratoriais controladas sendo importante explorar o comportamento desta variação em meios naturais. O objectivo deste trabalho foi estudar a partição de metais na membrana glico-lipo-proteica e embrião de choco ao longo da ontogenia expostos a diferentes níveis de contaminação ambiental. Foram selecionadas três áreas de estudo no Estuário do Sado com conhecidas diferenças na contaminação (Caeiro et al., 2005). Foi também estudado o stresse induzido nos embriões devido ao aumento dos teores de metais através da análise da concentração de enzimas de antioxidantes (CAT, SOD e GST), “Heat Shock Proteins” e peroxidação lipídica. O estuário do Sado junta áreas altamente urbanizadas e industrializadas com áreas de reservas naturais e quase pristinas, sendo um bom sistema para comparar as respostas em áreas pristinas com áreas contaminadas (Catarino et al., 1987).
4 Segundo Caeiro et al. (2005) podemos encontrar vários níveis de contaminação ao longo do Estuário do Sado. Desses locais usou-se três diferentes locais sendo por ordem crescente de contaminação: Caldeira (pristino), Eurominas (contaminação intermédia) e Setnave (muito contaminado). Para analisar a evolução da acumulação dos metais ao longo da embriogénese os embriões foram separados em três diferentes estados sendo o estado 1 (S1) equivalente aos estados I-VI o estado 2 (S2) aos estados XI-XV e o estado 3 do estado XIX (estados retirados de Naef’s, 1928).
Os embriões foram colhidos à mão sendo de imediato separada a membrana glico-lipo-proteica do embrião deixando apenas o embrião, há excepção de estado 1 onde foi deixada também a membrana vitelina, pois caso contrário estes embriões tornar-se-iam de difícil manuseio. Assim que separadas, ambas as partes foram congeladas a -80º C. De seguida, as amostras foram liofilizadas e homogeneizadas e submetidas a uma digestão ácida a várias temperaturas de acordo com Ferreira et al. (1990). Posteriormente foram determinados os níveis de V, Cr, Mn, Ni, Co, Cu, Zn, As, Se, Mo, Cd e Pb na membrana glico-lipo-proteica e no embrião por Espectrometria de Massa com Plasma Induzido Acoplado (ICP-MS).
Para quantificar a concentração das proteinas antioxidantes, HSP e peroxidação lipídica as amostras foram homogeneizadas em tampão de homogeneização e congeladas a -20º C. As concentrações de superóxido dismutase, catálase e Glutationa S-Transferase (GST) foram medidas através do protocolo descrito em Correia et al. (2003), a peroxidação lipídica pelo protocolo descrito em Diniz et al. (dados não publicados) e as HSP70/HSC70 através do protocolo adaptado por Njemini et al. (2005).
Depois de testados os prossupostos de normalidade e homocedasticidade foi escolhido o teste estatístico não paramétrico Kurskal-Wallis.
5 De um modo geral, os elementos analisados apresentaram níveis detectáveis na membrana glico-lipo-proteica e no embrião. O Zn foi o elemento que registou os teores mais elevados seguido do As e do Cu. Os três elementos são considerados essenciais para o desenvolvimento do ovo, sugerindo uma regulação metabólica pelo mesmo. No entanto, comparando as duas matrizes, a membrana apresentou as concentrações mais elevadas comparativamente ao embrião. Este resultado sugere que esta membrana consegue proteger eficazmente o embrião, formando uma barreira para a entrada de elementos vindos do ambiente. Contudo, com o crescimento do ovo a membrana glico-lipo-proteica vai ficando mais fina permitindo uma entrada de contaminantes. Este efeito foi mais evidente no estado mais avançado de desenvolvimento, S3, na área mais poluída, Setnave. Associado ao crescimento do ovo, era também esperado um efeito de diluição nas concentrações dos elementos metais, mas tal efeito só pode ser visto para as concentrações de As, sugerindo assim uma maior importância da entrada de elementos via ambiente que do efeito de diluição.
Nos ovos retirados da fêmea, observou-se uma elevada concentração de metais, principalmente de elementos essenciais. Este padrão sugere um importante efeito de transferência parental. Esta transferência por parte dos progenitores pode explicar grande parte da contaminação encontrada por exemplo na Caldeira (local menos contaminado).
Com a subida da concentração dos vários elementos era expectável um aumento de stresse oxidativo e danos no embrião. De acordo com os dados recolhidos nesta experiência nenhum destes cenários se verificou não havendo em geral qualquer alteração significativa. Por outro lado, parece haver um outro factor de stresse com maior importância que os elementos analisados, para estas condições ambientais, que não tenha sido controlado neste estudo. Não tendo sido controlado este fator, as variações encontradas nas enzimas antioxidantes, HSP e MDA neste estudo são aparentemente naturais. Uma prova de tal factor é a variação da GST que, embora sem diferenças significativas, apresentou um padrão de variação, aumentando
6 sempre do S1 para o S2 e tendo uma pequena redução para o S3. A mesma variação foi verificada nas concentrações de SOD e MDA mas menos evidentes, podendo ser apenas consequência da variabilidade natural da amostra e não resultantes de um factor de stresse. A variação destes biomarcadores com a contaminação ambiental que deverá ser analisado em estudos futuros.
2. Summary
The coastal and estuarine areas are impacted systems due to their proximity to populated and industrial areas. Estuaries receive contamination via river in addition to the direct input and are considered especially sensitive. These activities increase the concentrations of elements that otherwise would be in harmless to life. The present work aims to search the influence of environmental contamination in the eggshell and embryos of cuttlefishes during the embryonic development. To achieve this objective eggs were captured in areas with different contamination levels and separate into eggshell and embryo in three/four embryonic stages. Moreover, stress induced by contaminants in the embryo was evaluated by measuring antioxidant enzymes, such as lipid peroxide and heat shock proteins 70Kda/heat shock cognate 70Kda (HSP70/HSC70) concentrations. The eggs presented high values of essential elements even in the ones taken from the female. Almost all elements were able to accumulate in the eggshell during embryonic development and in much lower levels in the embryo. These results suggested that the eggshell is a good protective shield against waterborne metals. The antioxidant enzymes have shown no relation to trace elements in the studied environment concentrations, having no significant differences for any capture area. On other hand, a pattern of GST variation was seen (although
7 without significant differences) with increasing concentrations from S1 to S2 and a small decrease to S3. The same pattern was verified in SOD and MDA concentrations but not so evident, suggesting a biological variation and not as result of environmental influence.
3. General Introduction
The coastal and estuarine areas are impacted systems because their proximity to populated and industrial areas. Estuaries receive contamination via river in addition to the direct input and are considered especially sensitive. These systems are also very important both biologically and as resources, which made them targets of growing attention and legislation as the water framework directive (Muxica et al., 2007).
Trace elements enter the water either by land erosion or pollution. When in normal concentrations such elements are important to the development and growth of all life beings since they are needed to many cell processes and components, such as enzymes, DNA and blood fluids (Bryan, 1971; Joyce and Steitz, 1994; Ferraro, 2004). Contamination makes trace elements concentration rise to values higher than normal becoming harmful to the organisms, damaging DNA, proteins and lipids, by inhibiting enzymes or creating reactive oxygen species (ROS) (Lindquist and Crayg, 1988; Ushmar and Halliwell, 1996; Pereira et al., 2010, Raimundo et al., 2010)
Reactive Oxygen Species (ROS), as superoxide and hydrogen peroxide, are radicals formed from oxygen, usually produced by the normal metabolism of organisms which are in balance with antioxidant enzymes (cells defence mechanism, which transform ROS in harmless molecules or less harmful ROS) being harmless to the cells (Singh et al., 2004, Bayir, 2005). Contaminants, such as metals, disturb this balance by causing
8 stress, creating more ROS or inhibiting antioxidant enzymes causing oxidative damage over lipids creating lipid peroxides which also act as oxidants, propagating the damage (Winston and Di Giulio, 1991). As mentioned above, oxidative stress can also damage proteins and DNA being the Heat Shock Proteins (HSP) an important defence against stress, mainly HSP70 family.
Common cuttlefish (Sepia officinalis) is a cephalopod which lives offshore in adult life stages coming to shallow waters only to reproduce (Boucaud-Camou and Boismery, 1991). In shallow waters it lays eggs in clusters attached to hard surfaces like wood sticks, algae, and fishing nets. Their eggs are composed by many different layers as presented in Figure 1.
Fig. 1 – Cuttlefish egg tissues, adapted from wolf et al. (1985). From up to down there is eggshell (s), germinal disc (gd), yolk (y), viteline membrane (vm), periviteline space (ps), chorion (ch) and finally basal ring (br). The used tissues in this work was yolk and germinal disc (or the embryo in later stages) and eggshell.
The eggshell prevents many waterborne contaminants, such as metals, from reaching the embryo and harming it. With one month and the growth of the embryo, the eggshells starts getting thinner allowing some elements to pass through and
9 accumulating in the embryo causing damaging is cells (Bustamante et al., 2004; Lacoue-Labarthe et al., 2008, 2010).
The Sado estuary, located on the west coast of Portugal near the city of Setúbal is the second largest in the country. This area have plenty industrial activity near the city of Setúbal such as shipyards, mining activities, pulp and paper, pesticides, fertilizers, yeast, food and harbour activities (Catarino et al., 1987) and traditional rice farms, salt pans and intensive fish farms also occupy a large land area around the estuary. On the other hand, the major part of the estuary is a natural reserve with salt marshes areas, a small dolphin’s family and many important ecological components. This blend of industrial areas with natural reserves and some pristine areas turns this estuary a good place to compare areas with different contamination levels. According to Caeiro et al. (2005) the area of Caldeira, a semi enclosed area in Tróia peninsula, has always showed the lower contamination levels. On the other hand, the areas near the Eurominas mines and the Setnave shipyards have more contaminated waters, being Setnave the area with highest contamination levels in all parameters.
This thesis aims to evaluate the eggshell permeability to waterborne contaminants, such as metals, and how different environmental contamination levels and embryogenesis influence such permeability. Moreover, it is also measured the stress induced and damage suffered by the cells exposed to different environmental contamination levels at different embryonic stages.
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3.1. Referências
Bayir, H., 2005. Reactive Oxygen Species. Crit Care Med. 33, S498-S501
Boucaud-Camou, E., Boismery, J., 1991. The migrations of the cuttlefish (Sepia officinalis L.) in the English Channel. In: Boucaud-Camou, E. (Ed.), Actes du 1 er Symposium international sur la Seiche. Centre de Publications de L'Université de Caen. Caen, 1–3 juin 1989, 179–189
Bryan, W. G., 1971. The Effects of Heavy Metals (other than Mercury) on Marine and Estuarine Organisms. P Roy Soc Lond B Bio. 177, 389-410
Bustamante, P. 1998. Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs. Thesis, University of La Rochelle.
Bustamante, P., Teyssié, J., Danis, B., Fowler, S., Miramand S., Cotret, O and Warnau, A., 2004. Uptake, transfer and distribution of silver and cobalt in tissues of the common cuttlefish Sepia officinalis at different stages of its life cycle. Mar Ecol Prog Ser. 269, 185-195
Caeiro, S., Costa, M.H., Ramos, T.B., Fernandes, F., Silveira, N., Coimbra, A., Medeiros, G., Painho, M., 2005. Assessing heavy metal contamination in Sado Estuary sediment: An index analysis approach. Ecol Indic. 5, 151-169
Catarino, J., Peneda, M.C., Santana, F., 1987. Estudo do Impacto da Indústria no Estuário do Rio Sado. Estimativas da Poluição Afluente. DEII, INETI - Instituto Nacional de Engenharia e Tecnologia Industrial, Lisbon, Portugal
Correia, A. D., Costa, M.H., Luis, O. J., Livingstone, D.R., 2003. Age-related changes in antioxidante enzyme activities, fatty acid composition and lipid peroxidation in
11 whole body Gammarus locusta (Crustacea: Amphipoda). J Exp Mar Biol Ecol. 289, 83-101.
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13 Parsell, D. and Lindquist, S., 1993. The Function of Heat-Shock Proteins In Stress Tolerance: Degradatin and Reactivation of Damaged Proteins. Annu. Rev. Genet. 27, 437-496
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14
4. Trace elements partitioning between eggshell and embryo of common
cuttlefish (Sepia officinalis) during embryonic development exposed to
different environmental contamination (Sado estuary, Portugal)
4.1. Resumo:
As concentrações de doze elementos: V, Cr, Mn, Ni, Co, Cu, Zn, As, Se,Mo, Cd and Pb foram medidas na membrana glico-lipo-proteica e embriões de choco, Sepia officinalis, capturados em três diferentes áreas do estuário do Sado que possuem diferentes níveis de contaminação. Também se procuraram diferenças entre diferentes estados de desenvolvimento embrionário de forma a permitir observar como é feita a acumulação dos diferentes elementos entre a membrana e o embrião ao longo da embriogénese em condições naturais. Foram também analisados ovos no estado 0 (antes da postura), de uma fêmea apanhada na Eurominas, de forma a avaliar a importância da transferência parental na contaminação. A membrana mostrou-se um bom escudo contra contaminantes provenientes da água mantendo a concentração de metais no embrião reduzida. Por outro lado os elementos essenciais Cu e As encontravam-se em concentrações superiores no embrião em comparação com a membrana e o Zn em concentrações semelhantes na maior parte dos estados, o que aparenta uma regulação destes elementos. Para a maioria dos elementos a membrana apresentou valores mais elevados para o S3 e na zona mais poluída, a Setnave. Neste trabalho, para as condições ambientais encontradas, a entrada de elementos do meio exterior mostrou ter maior importância na concentração dos elementos no embrião do
15 que a diluição por crescimento. Também a transferência parental mostrou ter uma grande importância na concentração de metais presente no embrião.
4.2. Abstract:
Concentrations of V, Cr, Mn, Ni, Co, Cu, Zn, As, Se, Mo, Cd and Pb were determined in eggshells and embryos of cuttlefish, Sepia officinalis, captured in three areas of the
Sado estuary with different contamination degree. Differences between embryonic stages were searched to evaluate elemental partitioning between eggshells and embryos through the embryogenesis in natural conditions. Eggs in stage 0 (before posture) capture in Eurominas, were also analysed in order to evaluate elemental parental transference. Eggshell was found to be a good shield against waterborne contaminants keeping metal concentration in the embryo low. The exception was the essential metals such as Cu and As which were higher in the embryo and Zn which were similar at most stages, suggesting a regulation of these elements. For the majority of the elements, eggshell in stage 3 showed higher concentrations in the most contaminated area, probably associated with the diminishing thickness of the eggshell. In the present study, the input of contaminants via environment/water may be more important than the elemental dilution associated with growth. Parental transference was also considered as an important source of elements to the egg.
16
4.3. Introduction
The coastal and estuarine areas are impacted systems because their proximity to populated and industrial areas. Estuaries receive contamination via river in addition to the direct input and are considered especially sensitive. These systems are also very important both biologically and as resources. Common cuttlefish, Sepia officinalis, live offshore during winter migrating to coastal waters and estuaries to mate and reproduce (Boucaud-Camou and Boismery, 1991). In these waters they are exposed to more contaminated environments, favouring higher elemental concentrations in tissues. Once the eggs are laid they will be exposed to contaminants available in the environment (Miramand et al., 2006; Lacoue-Labarthe et al., 2009, 2010a). For cuttlefish embryo to accumulate waterborne metals, such elements have to be able to pass through eggshell that acts as an effective shield against metal penetration. In addition to the direct contamination from the environment, the cuttlefish egg could also possibly suffer from a second contamination pathway, viz. the transfer of metals from the female during the prespawning period (Lacoue-Labarthe et al., 2008).
Laboratory studies with cuttlefish eggs showed that the association with the embryo or eggshell depends on the element. Silver, Zn, Cu, Co, Cs, Mn and Hg were showed to be preferentially accumulated in the embryo (Bustamante et al., 2002, 2004; Miramand et al., 2006; Lacoue-Labarthe et al. 2009, 2010) while Am, Cd, Pb, and V were mainly associated to the glicoproteinic eggshell passing only a very small amount to the embryo (Bustamante et al., 2004, 2006; Miramand et al., 2006; Lacoue-Labarthe et al., 2010a). Many of the metals which accumulate in embryos only pass through the
17 eggshell after the first month of life, when the eggshell begins getting thinner (Wolf et al., 1985; Miramand et al., 2006; Lacoue-Labarthe et al., 2008, 2009, 2010a;).
Some metal effects on cuttlefish embryos are already known. For instance, Cu at elevated concentrations is known to make embryos hatch earlier and weaker, even when malformations don’t occur (Paulij et al., 1990). Cadmium is known to inhibit Acid Phosphatase and Cathepsin activity, the enzymes responsible for the oviparous embryos feeding process from the yolk sack (Lacoue-Labarthe et al., 2010b). In vitro, cathepsin activity is also inhibited by Cu and Zn in the digestive gland cells of adult Sepia officinalis, and Ag stimulates enzyme activity (Le Bihan et al., 2004). Cadmium is also known for possibly replacing metals as Zn and Cu in the proteins due to their similar sizes (Zauke and Petri, 1993) .However, little is known about the temporal changes in metal concentrations during embryogenesis in native S. officinalis.
This study reposts the partitioning of V, Cr, Mn Co, Ni, Cu, Zn, As, Se, Mo, Cd and Pb in eggshells and embryos of feral cuttlefish eggs from three spawning habitats with different contamination. Elements in not spawned eggs were also quantified to identify elements coming from mother transference.
18
4.4. Material and Methods 4.4.1. Sampling
Cuttlefish, Sepia officinalis, eggs were collected during low tide at the spawning season, during March and May of 2011, in three areas with different contamination levels (Caeiro et al., 2005) in the Sado estuary. In ascending order of contamination, the sampling areas were: Caldeira, Eurominas and Setnave. The eggs were obtained by hand fishing and with the help of local fishermen. A mature female was also captured, and eggs were taken directly.
Eggs were separated according to the stage of maturation into three different classes S1, S2 and S3 (based on Naef’s 1928 descriptions), namely: Stage 1 (initial) – with a clear cleavage and differentiation of the germinal disc (Naef’s stages I-VI); Stage 2 (intermediate) –eye balls with a delicate circular ridge that forms the iris fold rudiment; the gill rudiments are differentiated, there is also a clearly differentiation of the arms and the fin appears (Naef’s stages XI-XV); Stage 3 (final) – almost newly-hattached juveniles (Naef’s stages XIX). Additionally, eggs from the female ovary were also separated and classified as stage 0 (S0). This class of maturation was only analysed in Eurominas, where female was captured. The tissues from these eggs were not divided as the spawned eggs were.
Eggshells, basal ring and vitelline membrane (washed with Milli-Q water in order to remove sediments) were separated from embryos and yolk and stored in criovials according to collection area and stage, at -80 ºC. However, the separation was not performed in all stages. In S1, the vitelline membrane was kept because once it is removed the yolk may scatter, and in S0 no tissues were feasible to separate.
19
4.4.2. Analytical methodology
Trace elements were analysed in freeze-dried, grinded and homogenised samples after an acid digestion according to the method described in Raimundo et al. (2010a) and Ferreira et al. (1990). All lab ware was cleaned with HNO3 (20 %) for two days and
rinsed with Milli-Q water to avoid contamination. Procedural blanks were prepared using the same analytical procedure and reagents, and included within each batch of 10 samples. Concentrations of V, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Mo, Cd and Pb were determined by a quadrupole ICP-MS (Thermo Elemental, X-Series). The accuracy of analytical methods was assessed by the analysis of international certificate reference materials (DORM-3 – fish protein; DOLT-2– Fish liver and TORT-2 – lobster hepatopancreas, values presented in tables 1 and 2). The results obtained were in good agreement with the certified values (p>0.05). Procedural blanks always accounted for less than 1 % of the total element in the samples. All the results are given as medians and ranges in microgram per gram of tissue dry weight (µg g-1, dw). Detection limits were: 0.0030 µg g-1 for V, 0.0019 µg g-1 for Cr, 0.0016 µg g-1 for Mn,
0.0020 µg g-1 for Co, 0.00088 µg g-1 for Ni, 0.0015 µg g-1 for Cu, 0.0032 µg g-1 for Zn,
0.0029 µg g-1 for Se, 0.74 µg g-1 As, 0.0004 µg g-1 for Mo, 0.0030 µg g-1 for Cd and
20 Table 1 – Vanadium, Cr, Mn, Co, Ni, Cu, Zn, As, Se, Mo, Cd and Pb (µg g-1, dw) concentrations of fish protein (DORM-3), fish liver (DOLT-4) and lobster
hepatopancreas (TORT-2) obtained in the present study and certified values.
Standard V Cr Mn Co Ni Cu Zn As Se Mo Cd Pb (µg g-1) DORM-3 Obtained 1.50±0.10 1.7±0.020 2.28±0.078 0.24±0.010 1.16±0.023 15.3±0.64 47.9±1.2 6.45±0,070 3.46±0.13 0.29±0.055 0.27±0.010 0.25±0.021 Certified - 1.89±0.17 4.6* 1.89±0.19 1.28±0.24 15.5±0.63 51.3±3.1 6.88±0.30 3.3* - 0.290±0.020 0.395±0.050 DOLT-4 Obtained 0.55±0.072 1.34±0.020 7.83±0.63 0.22±0.010 0.71±0.15 29.5±1.1 109.3±1.8 7.7±0,28 7.82±0.26 0.96±0.22 21.0±1.2 0.12±0.0058 Certified 0.60* 1.4* - 0.25* 0.97±0.11 31.2±1.1 116±6 9.66±0.62 8.3±1.3 1.0* 24.3±0.8 0.16±0.04 TORT-2 Obtained 1.87±0.067 0.75±0.028 10.19±0.73 0.51±0.024 2.72±0.69 93.8±8.3 166.1±15 19.7±2.2 5.77±0.20 0.91±0.27 25.4±0.88 0.34±0.029 Certified 1.64 ± 0.19 0.77 ± 0.15 13.6 ± 1.2 0.51 ± 0.09 2.50 ± 0.19 106±10 180±6 21.6±1.8 5.63 ± 0.67 0.95 ± 0.10 - - * Reference values
21
4.4.3. Statistical analysis
Prior to statistical tests application, element concentrations were tested for normality and equality of variances. Since variables were not parametric, the nonparametric test Kurskall-Wallis was used to verify differences between sampled stages and differences between areas either for embryos and eggshells and the Man-Whitney test to compare eggshells with embryos. In Eurominas, differences between S0 and the other three stages were also searched.
4.5. Results:
Figure 2 presents the median, 25 and 75% percentiles, maximum and minimum of. V, Cr, Mn, Co, Ni, Cu, Zn, As, Se and Pb concentrations (µg g-1, dw) in eggshells and embryos in stages 0 (S0), 1 (S1), 2 (S2) and 3 (S3) collected in Caldeira, Eurominas and Setnave. To simplify interpretation figure scales were reduced to better adjust the distribution, hiding outliers as consequence in some cases. The median and ranges are presented in annex 1 table 2.1 (Caldeira), 2.2 (Eurominas) and 2.3 (Setnave), respectively. The p-values resulting from Kurskall-Wallis and Mann-Whitney tests are showed in annex 1, Table 3
22
Eggshells Embryos
Figure 2 – Medians, 25 and 75% percentiles, minimum and maximum ranges of V, Cr, Mn, Co, Ni, Cu, Zn, As, Se and Pb concentrations (µg g-1, dw) for eggshells (left) and embryos(right) in three embryonic stages (S0, S1, S2 and S3) collected in Caldeira (white boxes), Eurominas (grey boxes) and Setnave (black boxes).
S1 S2 S3 0 2 4 6 8 [V ] (µ g g -1) S0 S1 S2 S3 0,0 0,2 0,4 0,6 0,8 [V ] (µ g g -1 d w ) S1 S2 S3 0 2 4 6 [C r] ( µ g g -1) S0 S1 S2 S3 0,0 0,5 1,0 1,5 2,0 [C r] ( µ g g -1)
23
Eggshells Embryos
Figure 2(Cont.) - Medians, 25 and 75% percentiles, minimum and maximum ranges of V, Cr, Mn, Co, Ni, Cu, Zn, As, Se and Pb concentrations (µg g-1, dw) for eggshells and embryos in three embryonic stages (S0, S1, S2 and S3) collected in Caldeira (white boxes), Eurominas (grey boxes) and Setnave (black boxes).
S1 S2 S3 0 40 80 120 160 200 [M n ] (µ g g -1 d w ) S0 S1 S2 S3 0 2 4 6 [M n ] (µ g g -1) S1 S2 S3 0 2 4 6 8 10 [C o ] (µ g g -1) S0 S1 S2 S3 0,00 0,03 0,06 0,09 0,12 [C o ] (µ g g -1) S1 S2 S3 0 10 20 30 40 [N i] ( µ g g -1) S0 S1 S2 S3 0,0 0,5 1,0 [N i] ( µ g g -1)
24
Eggshells Embryos
Figure 2(Cont.) - Medians, 25 and 75% percentiles, minimum and maximum ranges of V, Cr, Mn, Co, Ni, Cu, Zn, As, Se and Pb concentrations (µg g-1, dw) for eggshells and embryos in three embryonic stages (S0, S1, S2 and S3) collected in Caldeira (white boxes), Eurominas (grey boxes) and Setnave (black boxes).
S1 S2 S3 0 20 40 60 80 [C u ] (µ g g -1) S0 S1 S2 S3 0 20 40 60 80 100 [C u ] (µ g .g -1) S1 S2 S3 0 40 80 120 160 200 240 [Z n ] (µ g g -1) S0 S1 S2 S3 0 20 40 60 80 100 [Z n ] (µ g g -1) S1 S2 S3 0 2 4 6 8 10 [A s ] (µ g g -1) S0 S1 S2 S3 0 40 80 120 160 [A s ] (µ g g -1)
25
Eggshells Embryos
Figure 2(Cont.) - Medians, 25 and 75% percentiles, minimum and maximum ranges of V, Cr, Mn, Co, Ni, Cu, Zn, As, Se and Pb concentrations (µg g-1, dw) for eggshells and embryos in three embryonic stages (S0, S1, S2 and S3) collected in Caldeira (white boxes), Eurominas (grey boxes) and Setnave (black boxes).
S1 S2 S3 0 1 2 3 [S e ] (µ g g -1) S0 S1 S2 S3 0 1 2 3 4 [S e ] (µ g g -1) S1 S2 S3 0 2 4 6 8 [P b ] (µ g g -1) S0 S1 S2 S3 0,0 0,2 0,4 0,6 0,8 [P b ] (µ g g -1)
26
4.5.1. General Concentrations:
Zinc was the most abundant element reaching one to four orders of magnitude above the remaining elements, followed by Cu and As. Median values varied from 28 µg g-1 to 160 µg g-1 for Zn, 16 µg g-1 to 82 µg g-1for Cu and 3.6 µg g-1 to 106 µg g-1 for As. The medians of the remaining elements decreased from 91 to 0.00092 µg g-1 in the following order: Mn>Ni>V>Co>Se>Cr>Pb>Cd>Mo.
The concentrations of As and Cu, were significantly (p <0.05) higher in embryos (medians: 73 µg g-1 for As and 53 µg g-1 for Cu) in comparison to eggshells (medians: 4.3 µg g-1 for As and 29 µg g-1 for Cu). For the other analysed elements, eggshells showed, in general, higher values except for Se that presented similar concentrations in eggshells and embryos.
4.5.2. Variation between stages for each area
Elemental concentrations in the different embryonic stages varied according to the capture area:
Caldeira – The higher values registered were 84 µg g-1 iat eggshells in S3 for Zn and 106 µg g-1 embryos at S1 for As. Selenium levels decreased from S1 to S3. Chromium, Co, Ni, Cu, Zn, Se and Cd concentrations were significantly different in eggshells (p <0.05) with an increasing concentration from S1 to S2 and/or S3. For the embryos, only V concentrations increased significantly from S1 to S3 (p <0.05). Manganese also presented significant differences but with lower levels in S2 in comparison to the other two stages. Most of the embryonic stages showed significant differences between eggshells and embryos, except S1 for Cr and Se, and S2 and S3 for Zn.
27
Eurominas – The higher values found were 126 µg g-1 in S3 for eggshells and 85 µg g-1 in S2 for embryos, both for Zn. Stage 0 had significant differences (p<0.05) for V increasing from S0 (0.21 µg g-1) to S3 (0.52 µg g-1), Co (82 µg g-1) and Se (3.3 µg g-1) decreased after S1 (52 and 1.8 µg g-1 to Co and Se) while Mn (S0, 3.5 µg g-1) and Pb (S0, 0.37 µg g-1) only have significant differences after S2 (2.6 and 0.095 µg g-1 of Mn and Pb for).
Enhanced concentrations of Mn, Co, Ni, Cu, Zn, Cd and Pb were registered in eggshells from S1 to S3 (p<0.05). Vanadium and Cr showed significant differences (p<0.05), mainly because S2 (medians of 3.3 µg g-1 for V and 1.2 µg g-1 for Cr) presented lower concentrations than the other two stages (medians S1: 4.4 µg g-1 for V and 2.5 µg g-1 for Cr; median S3: 4.96 µg g-1 for V and 3.13 µg g-1 for Cr), although V values in S3 were slightly higher than S1. For the embryos, significantly differences were registered for V (medians: 0.29 µg g-1 in S1 0.28 µg g-1 in S2 and 0.52 µg g-1 in S3) and Co (medians: 0.035 µg g-1 in S1, 0.029 µg g-1 in S2 and 0.083 µg g-1 in S3) concentrations, with enhanced concentrations in S3.
Setnave – In general, elements presented significant increasing concentrations from S1
to S3 in eggshells. Exceptions were Se and Pb with no significant differences (p<0.05) between embryonic stages, medians of 1.1 – 1.2 µg g-1 for Se and 1.7 – 2.2 µg g-1 for Pb. On the other hand, concentrations in embryos only varied for V, Mn, Co and Zn, with higher levels in S3 (0.6, 2.3, 0.060 and 86 µg g-1, respectively) comparing to S1 (0.3, 1.9, 0.016 and 72 µg g-1, respectively). Arsenium levels also showed significant differences (p<0.05), decreasing from S1 (median of 69 µg g-1) to S3 (median of 48 µg g
-1
). For all stages significant differences (p <0.05) were observed between eggshells and embryos with exception of: S3 for Cu, S1 for Zn and S1, S2 and S3 for Se.
28
4.5.3. Variation between areas for each embryonic stage
Stage 1 – Concentrations of V, Co, Ni, Cu, Zn, As, Se, Cd and Pb in eggshells and V, Cr,
Mn, Ni, Cu, Zn and Pb in embryos presented significant differences (p<0.05) between sampling areas. For the eggshells: Caldeira (pristine area) was the area with higher concentrations of V, As, Se and Pb (4.6 µg g-1, 7.3 µg g-1, 1.6 µg g-1 and 3.5 µg g-1, respectively); Eurominas presented significantly (p<0.05) higher concentrations of Co (3.3 µg g-1), Ni (7.5 µg g-1), Zn (70 µg g-1) and Cd (0.52 µg g-1); and in Setnave, Cu concentrations were significantly (p<0.05) higher (25µg g-1) in comparison to the other sampling areas (28 µg g-1 in Caldeira and 25 µg g-1 in Eurominas). In the embryos variation in element concentrations between areas was the follow: Caldeira showed significantly (p<0.05) higher concentrations of Cr, Mn and Pb with medians of 1.7 µg g
-1, 27 µg g-1 and 3.5 µg g-1, respectively; Eurominas showed higher median
concentrations (p<0.05) of Ni (0.18 µg g-1) and Cu (52 µg g-1) and, together with Caldeira, the highest concentrations of Zn (81 and 82 µg g-1, respectively). Setnave and Eurominas displayed higher V concentrations (medians of 0.30 µg g-1 and 0.29 µg g-1, respectively).
Stage 2 – Significant differences (p<0.05) between areas were found for various
elements in both embryos and eggshells at S2. For V and Cr levels in embryos and eggshells, and for Se values in embryos no variations were observed between sampling areas. Eggshells collected in Caldeira presented enhanced levels of As, Se and Cd (medians of 6.7, 2.2 and 0.75 µg g-1, respectively). Manganese, Co and Ni had similar values between Eurominas and Setnave (p<0.05). Vanadium, Cr, Cu and Zn (medians of: 6.0 µg g-1 for V, 2.5 µg g-1 for Cr, 32 µg g-1 for Cu and 116 µg g-1 for Zn) showed higher concentrations in eggshells from Setnave. In embryos: Mn (4.2 µg g-1), As (81 µg
29 g-1) and Pb (0.21 µg g-1) were higher in Caldeira (p<0.05); Cu (54 µg g-1), Zn (85µg g-1) and Cd (0.0045 µg g-1) were higher in Eurominas and Mn (2.3 µg g-1 and 1.9 µg g-1) and Ni (0.15 µg g-1 and 0.16 µg g-1) in Eurominas and Setnave.
Stage 3 – Significant differences (p<0.05) were registered for concentrations of V, Mn,
Co, Ni, Cu, Zn, As and Cd in eggshells and V, Mn, Co, Ni, As, Cd and Pb in embryos. Eggshells from the eggs collected in Setnave displayed, for the majority of the analysed elements the highest concentrations. For embryos: Caldeira displayed the higher concentrations of Mn, As and Pb, median levels of 4.5 µg g-1, 84 µg g-1 and 0.25µg g-1, respectively; Eurominas registered higher Co (0.083 µg g-1), Ni (0.24 µg g-1) and Cd (0.0045 µg g-1) concentrations than the ones from other areas; and Setnave had the higher concentrations of V (0.60 µg g-1). Cu and Zn concentrations had no significant differences among areas in the embryos.
4.6. Discussion:
The examination of elemental compartmentalization among eggshells and embryos, of feral eggs, is highly important to better interpret defence mechanisms and tolerance during embryonic development of cuttlefish. Moreover, in this species, egg and embryos growth is not continuous throughout the duration of development (Lacoue-Labarthe et al., 2008a) being important to evaluate differences during the embryonic development.
Zinc was one of the elements that presented higher concentrations followed by As and Cu. These elements, at certain concentration intervals, are considered essential, being necessary for cuttlefish growth and development (Villanueva and Bustamante, 2006). On the other hand, Co, Ni, Cd, Mo and Pb presented the lowest concentrations. Co and
30 Ni are also essential metals when in small amounts (Villanueva and Bustamante, 2006) but Cd, Mo and Pb are harmfull elements, being Cd and Pb effects in cephalopods already studied (Raimundo et al., 2010a, 2010b; Labarthe et al., 2010b)
Comparing present data to levels found in Sepia officinallis and Loligo vulgaris hatchlings and Octopus vulgaris eggs (Villanueva and Bustamante, 2006), Cr, Mn, Co, Ni, Cu, Zn, As, Cd and Pb concentrations fall within the interval levels registered for octopus eggs. On the other hand, concentrations in cuttlefish eggs from Sado estuary were in general higher than values registered in Miramand et al. (2005) (only S3). Conversely, Co and Pb in embryos and Cd in eggshells showed lower concentrations in the present work. These differences may result from a higher contamination found in the three capture areas in comparison to the studied areas in Miramand et al. (2005).
4.6.1. Elemental partitioning between eggshells and embryos
For most of the analysed elements a clear partition was observed with enhanced levels associated to the eggshells in comparison to embryos. These findings are in line with laboratory exposure studies with cuttlefish (Bustamante et al., 2002; Lacoue-Labarthe et al., 2008a, 2010a), evidencing that eggshells acts as an efficient shield protecting the embryos against contaminant exposure. Selenium concentrations were, in general, similar in eggshells and embryos. Different association was observed in cuttlefish eggs from laboratory postures from exposed females, where Se is mainly found in the vitellus (75% of the whole egg burden) (Lacoue-Labarthe et al., 2008b). Selenium is an essential element known to be necessary to the proper synthesis and functioning of the glutathione peroxidase, which is a major cellular antioxidant enzyme (Bell et al., 1986). The similar levels found in the eggs collected in Sado, may suggest a protecting
31 mechanisms from the high values found in the environment. In contrast, higher concentrations of As were found to be accumulated in the embryos. This association was also observed in the pristine area, Caldeira (Caeiro et al., 2005), suggesting a strong affinity of As to the embryos. In general, higher levels of Cu and, in a less extend, of Zn were also registered in the embryos in the three capture areas. The essentiality of these elements and the presence in several cellular structures may favour the permeability of the eggshells to these elements. However, a different pattern was observed by Miramand et al. (2006), which observed an enhancement of Cu concentrations in the eggshells. The enhancement of Zn concentrations observed in the embryos is in agreement with finding made by Bustamante et al. (2002). Furthermore, Cu and Zn were the elements that presented the lowest coefficients of variation in eggshells and more clearly in embryos. This low variability may result from the metabolic control of these two essential elements as proposed by Villanueva and Bustamante (2006).
4.6.2. Elemental differences between embryonic stages in the three capture areas
Once the eggs are laid they are potentially subject to environmental contamination. Caldeira was used as the pristine area, however eggs from this area presented enhanced concentrations of As and Se in the eggshells and Mn, As and Pb in the embryos. These elevated levels may result from parental transfer (elements with high concentration in S0 from Eurominas, discussed further on) justifying the high concentrations in the eggs collected in an area with low environmental contamination (Caeiro et al., 2005). The most contaminated area, Setnave, only presented the highest
32 values of the majority of the analysed elements once the eggshells get thinner (S3) and subsequently the elements may be easily incorporated.
The differences on the elemental composition during the ontogenetic development have already been observed in cephalopods and it depends on the considered element and on the species (Bustamante, 1998). In the eggshells of cuttlefish, all elements presented increased concentrations with ontogenic development in the most polluted area, Setnave. Among the analysed elements, Mn, Co, Ni, Cu, Zn, Se and Cd also increased in Eurominas and Pb only increased in the last area. Such increases probably result from a continuous environmental exposure and subsequent accumulation in the eggshells during the embryosgenesis. This association of metals with the eggshells was also described in early studies (Miramand et al., 2006, Labarthe et al., 2008a 2010a). Concentrations of V, Co, Ni, Cu and Cd presented an increase tendency from S1 to S3 in embryos captured in Eurominas and Setnave. Manganese and Zn presented the same pattern but only in embryos from the most contaminated site (Setnave). This increase tendency observed in Setnave, may result from the lower Mn and Zn levels in embryos in S1, in comparison to Eurominas, leading to a higher accumulation capacity. Arsenic concentrations had a different variation during the embryonic development decreasing from S1 to S3. Even though the comparison among embryonic stages was not made in terms of content to eliminate potential elemental dilution with somatic growth (Boletzky, 2003), the decrease observed for As concentrations in embryos may result from dilution/growth. On other hand, the above mentioned elements showed an increase tendency both in eggshells and embryos, suggesting that the input of contaminants via environment/water may be more important than the elemental dilution associated with growth. The enrichment observed in the embryos in S3 may
33 also be associated with the thickness of the eggshells that diminishes with the development (Miramand et al., 2006) being more permeable to waterborne elements near the hatching.
4.6.3. Parental transference (Eurominas)
In addition to the direct contamination from the environment, the cuttlefish egg could also be contaminated via parental transfer. The somatic tissues of the cuttlefish female are partly used to produce the eggs (Guerra and Castro, 1994) and a fraction of their contaminant burden may therefore be transferred to the eggs and then to offspring. In order to evaluate elemental parental transfer, concentrations in embryos from S0 were compared to S1, only for eggs collected in Eurominas. No comparison was made with S2 and S3 since the contamination via the environment was probably more important than parental transference. In the present study, only V and Cr concentrations were lower in S0 in comparison to S1, Co, Ni and Zn were, generally, similar between stages and Mn, Cu, As, Mo, Cd and Pb were superior in S0. In a laboratory study, among eight radiotracers present in the prey of the female cuttlefish, only two essential elements (i.e. Zn and Se) and one non-essential metal (i.e. Ag) were transferred efficiently to the vitellus of the eggs (Lacoue-Labarthe et al., 2008b).
4.7. Final considerations
In sum, eggshells function as a protection for embryos for the majority of the analysed elements except Se. With the embryonic development higher element concentrations were registered in the most contaminated area, Setnave, probably associated with the diminishing thickness of the eggshell. In the present study, the input of contaminants
34 via environment/water may be more important than the elemental dilution associated with growth. There is also a very important mother transference effect for Mn, Cu, As, Mo, Cd and Pb. Yet this importance must be re-evaluated since some of the metals on S0 may also belong to the eggshells which have a considerable concentration of metals placed in order to form a shield against waterborne elements.
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