Seabird tissues as efficient biomonitoring tools for Hg isotopic

Blood and feathers are the two most targeted avian tissues for environmental biomonitoring. 19 studies, with mercury (Hg) concentration in blood and b...
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Seabird tissues as efficient biomonitoring tools for Hg isotopic investigations: implications of using blood and feathers from chicks and adults Marina Renedo, David Amouroux, Bastien Duval, Alice Carravieri, Emmanuel Tessier, Julien Pierre, Gilbert Barre, Sylvain Berail, Zoyne Pedrero, Yves Cherel, and Paco Bustamante Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00422 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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Seabird tissues as efficient biomonitoring tools for Hg isotopic

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investigations: implications of using blood and feathers from

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chicks and adults

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Marina Renedo1,2*, David Amouroux2, Bastien Duval2, Alice Carravieri3, Emmanuel Tessier2,

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Julien Barre2, Sylvain Bérail2, Zoyne Pedrero2, Yves Cherel3, Paco Bustamante1*.

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1

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2 rue Olympe de Gouges, 17000 La Rochelle, France

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2

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chimie pour l'Environnement et les Matériaux, UMR 5254, 64000, Pau, France

Littoral Environnement et Sociétés (LIENSs), UMR 7266 CNRS-Université de la Rochelle,

CNRS/ UNIV PAU & PAYS ADOUR, Institut des Sciences Analytiques et de Physico-

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3

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Rochelle, 79360 Villiers-en-Bois, France

Centre d'Etudes Biologiques de Chizé (CEBC), UMR 7372 CNRS-Université de La

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*Corresponding authors: [email protected], [email protected]

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Keywords: skua, penguins, Hg isotopes, bioaccumulation, marine ecosystems, bioindicators

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Abstract

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Blood and feathers are the two most targeted avian tissues for environmental biomonitoring

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studies, with mercury (Hg) concentration in blood and body feathers reflecting short and long-

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term Hg exposure, respectively. In this work, we investigated how Hg isotopic composition

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(e.g. δ202Hg and ∆199Hg) of blood and feathers from either seabird chicks (skuas, n=40) or

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adults (penguins, n=62) can accurately provide information on exposure to Hg in marine

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ecosystems. Our results indicate a strong correlation between blood and feather Hg isotopic

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values for skua chicks, with similar δ202Hg and ∆199Hg values in the two tissues (mean

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difference: -0.01±0.25 ‰ and -0.05±0.12 ‰, respectively). Since blood and body feathers of

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chicks integrate the same temporal window of Hg exposure, this suggests that δ202Hg and

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∆199Hg values can be directly compared without any correction factors within and between

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avian groups. Conversely, penguin adults show higher δ202Hg and ∆199Hg values in feathers

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than in blood (mean differences: 0.28±0.19 ‰ and 0.25±0.13 ‰), most likely due to tissue-

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specific Hg temporal integration. Since feathers integrate long-term (i.e. the inter-moult

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period) Hg accumulation, whereas blood exhibits short-term (i.e. seasonal) Hg exposure in

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adult birds, the two tissues provide complementary information of trophic ecology at different

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time scales.

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Introduction

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Mercury (Hg) and more specifically methylmercury (MeHg) is a highly toxic

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environmental pollutant with severe risks for animal and human health 1. The amount of Hg

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released into the environment has increased since pre-industrial times as a consequence of

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human activities

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levels of Hg have been reported in high trophic level predators

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present elevated Hg concentrations in their tissues and have been reported as efficient

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bioindicators of the environmental pollution 6. Since seabirds display contrasted foraging

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strategies and feed at different trophic levels, they are considered appropriate models to assess

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Hg contamination of the marine environment.

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2,3

. Due to its persistence and biomagnification in marine food webs, high 4,5

. For example, seabirds

Hg exposure in birds is essentially attributed to dietary uptake (especially MeHg), which 7–9

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is then distributed by the blood stream to internal organs and tissues

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excrete Hg in feathers during plumage synthesis. Therefore, moulting is considered as the

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main detoxification route in most avian species 10. Hg is sequestered in feathers by binding to

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keratin molecules 11, which impedes its reincorporation into internal tissues. Between 70 and

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90% of the whole Hg body burden has been shown to be remobilised from internal tissues and

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excreted into the growing feathers 9, mainly under its organic form (more than 90% of THg as

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MeHg)

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for biomonitoring studies because they do not involve lethal-sampling. Each tissue presents a

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specific Hg turnover rate, thus potentially providing complementary information at different

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temporal scales of Hg exposure. In adult birds, feathers, a metabolically inactive tissue, are

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representative of Hg incorporation during the inter-moult period (one whole year in most

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seabirds). In contrast, blood is a metabolically active tissue representative of a shorter-term

12–14

. Birds efficiently

. In birds, blood and feathers (and also eggs) are the most frequently used tissues

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Hg intake than feathers, meaning a few weeks/months before sampling 15. Therefore, tissue-

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specific integration times must be considered for the selection of the most appropriate avian

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tissue depending on the scientific purposes

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simultaneously growing feathers of large chicks reflect recent Hg intake over a similar time-

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period, because chicks synthesise their first adult-like feathers at the end of the chick-rearing

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period 16.

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. In contrast, both sampled blood and

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In the last decades, the measurement of Hg isotopic mass-dependent (MDF, δ202Hg) and

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mass-independent fractionation (MIF, ∆199Hg) has become a documented tool for identifying

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sources of Hg and biogeochemical processes within the different compartments of the

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environment

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mixing of isotopically distinct sources. Hg MDF can occur during all Hg specific

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transformation processes such as volatilization

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demethylation reactions

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combination of these processes in the environment can induce similar or opposite MDF in

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different degrees of magnitude, so that processes and Hg reservoirs cannot so distinctly be

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recognized, especially when using complex bioindicators. In contrast, significant Hg MIF is

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induced exclusively during photochemical reactions and mainly concerns the two odd

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isotopes (199 and 201)

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signature is thus assumed to be preservedthroughout the food web

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MIF signatures in tissues of top predators allow investigating photochemical processes in the

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marine ecosystem prior to uptake in the food web

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provide complementary information and they are used as tracers for both Hg potential sources

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and transformation pathways in aquatic ecosystems

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been successfully used in ecotoxicological studies for a better understanding of detoxification

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and excretion processes and Hg metabolic responses, such as hepatic demethylation in marine

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. Hg isotopes can vary as a result of fractionation during reactions and by

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, reduction

20–23

, photochemical reactions

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, bacterial methylation or

and metabolic processes

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. Hg MIF is not affected by biological processes

19,21,23

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. A

and its

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. Consequently, Hg

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. Therefore, both Hg MDF and MIF

29–33

. Hg isotopic signatures have also

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or human exposure by excretion biomarkers such as urine

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and hairs

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mammals

.

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Concerning avian samples, two studies have revealed the efficiency of Hg isotopic analyses in

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seabird eggs for investigating factors controlling Hg cycling in aquatic ecosystems 37,38.

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Here, we present the first data on Hg isotopic composition (δ202Hg and ∆199Hg) of blood

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and body feathers of the same individual seabirds with the double objective of (i)

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investigating Hg isotopic relationships between the two tissues and (ii) evaluating their

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suitability as tracers of the fate of Hg in marine ecosystems. This study was performed on

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different seabird species from the Southern Ocean exhibiting contrasted ecological

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characteristics (i.e. food and feeding ecology) and breeding in distant sites (i.e. over a

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latitudinal gradient) in order to cover a wide range of tissue Hg concentrations

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focused on two seabird models considered as representative of the local contamination, hence

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chicks (which were fed only with food from around the colony and reflect a relatively short

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period of exposure, 2-3 months) and penguins (which are more restricted to the colony area

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than migratory seabirds and reflect a longer period of exposure than chicks, one year). Since

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Hg integrated in chick blood and feathers correspond to a similar time frame, we first focused

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on skua chicks, from four different populations, to explore inter-tissue isotopic relationships

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and the allocation of Hg following transport and potential fractionation from blood to

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feathers. Potential biological processes or transport among the two tissues were hypothesized

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to induce Hg MDF, leading to differences in δ202Hg values between blood and feathers. In

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contrast, ∆199Hg values in blood and feathers of chicks were predicted to be similar, because

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Hg MIF is not induced by in vivo processes. In a second step, we investigated δ202Hg and

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∆199Hg values in blood and feathers from adult penguins to evaluate the influence of specific

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Hg exposure temporal windows on Hg isotopic compositions of both tissues. We expect that

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seasonal changes in penguin food and feeding ecology can produce significant Hg isotopic

39,40

. We

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variations among tissues. Penguins from six different species that breed from high-Antarctica

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to the subtropics were selected to confirm this hypothesis.

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Material and methods

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Sites and fieldwork

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Sample collection was conducted during the austral summer 2011-2012 (from October

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to February) in four sites of the Terres Australes et Antarctiques Françaises, depending on

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seabird species: Pointe Géologie, Adélie Land (Antarctic Zone, 66°40’S, 140°10’E),

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Amsterdam Island (Subtropical Zone, 37°50’S, 77°31’E), Crozet Islands (Subantarctic Zone,

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46°26’S, 51°45’E) and Kerguelen Islands (Subantarctic Zone, 49°21′S, 70°18′E) (Figure S1

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and Table S1). Ten to eleven birds were randomly chosen in each group. Only one chick per

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skua nest was sampled to avoid pseudo-replication. Penguins were all adult breeding birds at

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the end of the reproductive cycle. Blood samples were collected from a wing vein,

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centrifuged, and red blood cells were kept frozen at -20°C until analysis. A few body feathers

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were collected at random from the lower back of skuas and from the belly of penguins.

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Seabird chicks and adult penguins renew their entire plumage within a short time-period (i.e.,

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2-4 weeks), meaning that all body feathers grow almost simultaneously, thus showing very

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limited inter-feather variations in their Hg concentrations 11, 12.

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Reference materials, sample preparation, total Hg and Hg species concentrations analysis

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Due to the absence of feather and bird blood certified reference materials (CRM) for

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Hg and MeHg concentrations, two internal reference samples were prepared with pooled

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samples collected from different individuals of king penguin (KP) from Crozet Islands: F-KP

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(feathers) and RBC-KP (red blood cells). The two reference samples were analyzed at each

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analytical session. For the validation of the results, human hair CRM (IAEA-086) was

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additionally analyzed due to its similar chemical composition to feathers (i.e. keratin).

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Feathers samples were cleaned, oven dried and homogenized as detailed in a previous study ACS Paragon Plus Environment

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Altec) thus allowing at intercomparing with Hg total concentrations obtained by Hg

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speciation analyses, i.e. the sum of inorganic and organic Hg. For feather analyses, a matrix

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dependent calibration was performed with human hair reference material (NIES-13) as

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described elsewhere

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study 40. For Hg speciation analyses, feathers were prepared following a previously developed

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method

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digestion with 5 mL of tetramethylammonium hydroxide (25% TMAH in H2O, Sigma

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Aldrich)

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detailed elsewhere 14.

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Total Hg isotopic composition analysis

. Total Hg concentration was also quantified by using an advanced Hg analyzer (AMA-254,

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. Blood samples analyses were performed as described in a previous

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. Hg was extracted from blood samples (0.10-0.15 g) by alkaline microwave

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. Details of the extraction method, analysis and quantification of Hg species are

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Samples (0.05-0.10 g) were digested with 3 or 5 mL of HNO3 acid (65%, INSTRA

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quality) after a predigestion step overnight at room temperature. Two different mineralization

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systems were successfully tested and used: High Pressure Asher (HPA) and Hotblock. HPA

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mineralization was performed at high conditions of pressure (130 bar) and temperature

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(temperature ramp: 80ºC-120ºC (2ºC/min) - 300ºC (2.5 h) - 80ºC (1 h)). Hotblock

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mineralization was carried out in Savillex vessels at 75ºC during 8 h (6 h in HNO3 and 2 h

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more after addition of 1/3 of the total volume of H2O2 (30%, ULTREX quality)). Hg isotopic

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composition

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Instruments), as detailed previously

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calculated relative to the bracketing standard NIST SRM-3133 reference material to allow

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inter-laboratory comparisons, as described in SI. NIST SRM-997 thallium standard solution

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was used for the instrumental mass-bias correction using the exponential law. Secondary

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standard NIST RM-8160 (previously UM-Almadén standard) was used for validation of the

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analytical session (Table S2).

was determined

using cold-vapor generator (CVG)-MC-ICPMS (Nu 30

. Hg isotopic values were reported as delta notation,

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Recoveries of extraction were verified for all samples by checking the signal intensity

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obtained on the MC-ICPMS for diluted extracts relative to NIST 3133 standard (with an

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approximate uncertainty of ±15%). Total Hg concentrations in the extract solution were

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compared to the concentrations found by AMA-254 analyses to assess method recovery.

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Average recoveries obtained were 98±14% for feathers (n=104) and 100±2% for blood

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samples (n=102). Accuracy of Hg isotopic analyses for keratin matrixes was evaluated with

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human hair material NIES-13, whose reference values are validated 43. Hg isotopic results for

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soft-tissues (blood samples) were evaluated with validated reference values of Lake Michigan

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fish tissue NIST SRM 1947. Previously published Hg isotopic values for human hair IAEA-

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086 and tuna fish ERM-CE-464 materials were used for intercomparison

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reference samples of feathers (F-KP) and avian blood (RBC-KP) were also measured.

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Repeatability was estimated by analyzing the same extract of each reference material over the

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long term (during 2 weeks of analysis). Uncertainty for delta values was calculated using 2SD

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typical errors for each internal reference material (Table S2). Internal reproducibility was also

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assessed for numerous measurements of the two internal reference samples of feathers (F-KP)

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and avian blood (RBC-KP) within 2-10 months of interval (Table S3).

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Statistical analyses

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Statistical tests were performed using R 3.3.2 (RStudio)

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. Internal

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. Hg isotopic variations

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between blood and feathers from the same individuals of the different skua and penguin

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populations were assessed using paired t-tests. Statistical differences between Hg MIF ratios

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for each seabird species were tested by non-parametrical test (Kruskal–Wallis with Conover-

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Iman test).

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homogeneity of variances using Shapiro–Wilk and Breusch-Pagan tests, respectively.

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Statistically significant results were set at α=0.05. Relationships between blood and feather

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Hg isotopic composition were tested using Pearson correlation rank tests.

Before these analyses, data were checked for normality of distribution and

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Results and discussion

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Information on Hg speciation in each tissue is essential for the interpretation of total Hg

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isotopic signatures. As expected, blood and feather samples analyzed in this study present a

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large proportion of MeHg (n = 102, 92±6% and 92±3%, respectively). This is in agreement

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with previous reported results

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within the organism, including growing feathers

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widely amongst skua chick populations for both blood (0.51 to 3.78 µg g-1) and feathers (1.82

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to 11.78 µg g-1) and between adult penguin blood (0.43 to 1.95 µg g-1) and feathers (0.35 to

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3.90 µg g-1) (Figures S2 and S3). Hg species concentrations for each seabird are included in

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Table S1.

14,46

and highlights the role of blood as a Hg transport tissue 15

. Mean MeHg concentrations differed

Blood and feather Hg MDF (δ202Hg) values followed the predicted theoretical MDF line

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(calculation details in SI). Hg odd-MIF were reported as ∆199Hg values, i.e. the difference

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between measured δ199Hg values and δ199Hg values predicted by the theoretical MDF line. All

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blood and feather samples showed significant MIF of the odd isotopes (199Hg and

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Significant to very small MIF of even isotopes (∆200Hg) - a potential tracer of atmospherically

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sourced Hg

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differences were found between blood and feather samples.

49–51

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Hg).

- was also observed (ranging from -0.14 to 0.17 ‰). No significant ∆200Hg

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Skua chicks: identical Hg isotopic composition in blood and feathers

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Chick blood δ202Hg values range from 0.23±0.13 to 1.56±0.08 ‰. Likewise, feather

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δ202Hg values vary widely, from 0.00±0.36 to 1.51±0.20 ‰ (Table 1). Low variability is

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found in ∆199Hg between the four sites, with blood values ranging from 1.51±0.08 to

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1.70±0.05 ‰ and feather values varying from 1.46±0.07 to 1.76±0.05 ‰ (Table 1). The range

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of δ202Hg and ∆199Hg values obtained for the different skua populations are within the range

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of previous measurement performed on marine and pelagic organisms such as seabird eggs

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37,38

and pelagic fish 33,52. Skua chick blood samples display an overall ∆199Hg/∆201Hg slope of

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1.14±0.07 (Pearson correlation, R2=0.86, p