Contamination status of harbour porpoises in the southern North Sea

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 Carcasses collection

The national stranding network in France (RNE; Réseau National d’échouage) established in 1972 consists of local correspondents available to intervene when a stranding of marine mammal occurs along the French coast. The network is coordinated by the « Centre de Recherche sur les Mammiferes Marins » (CRMM) under the supervision of the Ministry of the Environment. Once a stranding of marine mammal is reported, the CRMM prevent the corresponding of the area to intervene and examine the animal. In our study area, the « Observatoire pour la Conservation et l’Etude des Animaux et Milieux Marins » (OCEAMM) and the « Ligue Protectrice des Animaux du Nord » (LPA) Calais were the correspondents in case of a stranding along northern France coast. For the strandings along the Bay of Biscay coast, different correspondents may intervene (such as CRMM, Ligue pour la Protection des Oiseaux (LPO) marais Breton, LPO marais de Mullembourg, etc). Whereas for the marine mammals stranded along the Belgian coast, the Belgian Marine Mammals Network intervenes in case of stranding. On the coast where the stranding occurred, basic information such as the species in question, sex, size, status, date and place were systematically recorded. Other parameters as well as tissues and organs collection were obtained after dissection.

Protocol of dissection, collection and preservation of samples

Freshly dead or slightly decomposed carcasses of porpoises stranded along the southern North Sea (Figure 2.1) were transported to the Veterinary Medicine, University of Liege as soon as possible to be preserved in freezing rooms for further necropsies and investigations (Figure 2.2). All individuals provided for necropsies were labeled. Animals were measured, weighed and the sex and the overall length were determined. For the harbour porpoises stranded along the Bay of Biscay, necropsies were held at the CRMM, La Rochelle – France. Stranding locations are presented in figure 2.3.

Metallic analyses

Livers and kidneys were collected for metal analysis and stored at -20°C in plastic bags until analysis were carried out. In order to avoid contamination for trace metal analyses, two acidified bath wash (molar mixture HCl: HNO3 for analysis, Merck) were used to decontaminate all the used equipments (vials, beakers, syringes, etc). After decontamination, all materials were rinsed with Milli-Q ultrapure water (18.2 MΩ.cm, Millipore Corporation). The drying of equipments as well as all the handling (dissection, grinding, preparation of solutions, etc) were carried out under a laminar flow hood « Federal Standard » (n°209a, class 100). Tissues of livers and kidneys were freeze-dried for 48 hours then homogenized with an agate mortar and pestle. In Teflon vials (Savillex PFA), an aliquot of ~150 mg from each material was digested in a concentrated solution of nitric acid (65 %, Suprapur, Merck) at room temperature for 24 hours and then at 100°C for 4 hours. After digestion, all samples were diluted to ~15% then filtered out using polyethylene syringes equipped with cellulose acetate filter (0.2μm). Finally, before spectroscopic analysis, all samples were diluted to ~2%. This dilution helps to reach a concentration below 2g.L-1 in total dissolved salt for best instrument performance and stability. For each analysis series, reagent blanks were treated and analyzed in the same conditions as samples. The signal of all blanks remains below 1% compared to the signal of all samples and for all elements reported in this study. Therefore, no corrections have been made.
Besides total mercury analysis which were conducted on an Advanced Mercury Analyzer Spectrophotometer (AMA-254, Altec), other metal concentrations were determined by Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES, Thermo Scientific iCAP 6500) and Inductively Coupled Plasma Mass Spectrometer (ICP-MS, Varian 820-MS) depending on concentration levels of each metal. Metal concentrations are reported in μg.g-1 dry weight (dw). ICP-AES and ICP-MS are analytical techniques used for multi-elemental measurements available at Centre Commun de Mesure (CCM, Université du Littoral Côte d’Opale, Dunkerque). The inductively coupled plasma principle is common for the two spectroscopic techniques but the detection mode is different (figure 2.4).

Analysis on ICP-MS

Via the interface cone, the elements in the sample converted into ions are bought into the mass spectrometer. An intermediate vacuum region created by two intermediate cones: the sampler and the skimmer (~1mm) transmit the ions coming from the ICP torch with an atmospheric pressure into a low pressure region of the mass spectrometer. The mass spectrometer used is a quadrupole mass filter. It consists on the fact that electrostatic lenses with a positive charge serve to collimate the ion beam (also positively charged) and focus it into the entrance slit of the mass spectrometer. The ions are therefore separated by their mass-to-charge ratio (m/e). This mass filter only transmits the ions with an m/e ratio according to the frequency previously applied to the quadrupole. For a given isotope, the signal is the number of pulses which is converted into concentration after calibration. ICP-MS is a fast, multi-elemental technique and has much lower detection capabilities compared to ICP-AES. Elements such as As, Cr, Cd, Pb and V were determined in livers and kidneys (depending on concentration levels in the sample) using ICP-MS. Limits of quantification are presented in table 2.3.

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Table of contents :

PREFACE
RESUME DETAILLE DE LA THESE
LIST OF FIGURES
LIST OF TABLES
CHAPTER 1 INTRODUCTION
The harbour porpoise (Phocoena phocoena)
Distribution and description
Distribution on a local scale
Protection status of harbour porpoises in the European waters
Diet of harbour porpoises
Threats
Assessment of chemical contamination
Studying the feeding ecology of harbour porpoises
Objectives of the study
Outline of the study
CHAPTER 2 STRATEGIES, PROTOCOLS AND METHODOLOGIES
1. Sample collection and necropsies
1.1. Carcasses collection
1.2. Protocol of dissection, collection and preservation of samples
2. Chemical analyses
2.1. Metallic analyses
2.1.1. Analysis on ICP-AES
2.1.2. Analysis on ICP-MS
2.1.3. Analysis on AMA-254
2.1.4. Quality control procedures
2.2. Persistent Organic Pollutants analyses
2.3. Data treatment
3. Feeding ecology
3.1. Stomach content analysis
3.1.1. Otoliths
3.1.2. Diet composition
3.1.3. Feeding strategy
3.2. Stable isotopes analysis
3.3. Fatty acids analysis
3.4. Compound-Specific Stable Isotope Analysis (CSIA)
3.5. Data treatment
3.5.1. Mixing model: Stable isotope analysis in R (SIAR)
3.5.2. Fatty acids data treatment
3.6. Environmental data
CHAPTER 3 HARBOUR PORPOISES (PHOCOENA PHOCOENA) STRANDED ALONG THE SOUTHERN NORTH SEA: AN ASSESSEMENT THROUGH METALLIC CONTAMINATION
Abstract
Introduction
1. Materials and methods
1.1. Sampling and data collection
1.2. Metal analysis
1.3. Data treatment
2. Results
2.1. Metal contaminants and maturity status
2.2. Metal contaminants and causes of death
3. Discussion
3.1. Non essential elements
3.2. Essential elements
3.3. Temporal trends
Conclusion
Acknowledgements
CHAPTER 4 ORGANOCHLORINES IN HARBOUR PORPOISES (PHOCOENA PHOCOENA) STRANDED ALONG THE SOUTHERN NORTH SEA BETWEEN 2010-2013
Abstract
Introduction
1. Materials and methods
1.1. Sampling and data collection
1.2. POP analysis
1.3. Data treatment
2. POP results
2.1. POPs and maturity status
2.2. POPs and causes of death
3. Discussion
3.1. PCB levels
3.2. DDX levels
Conclusion
Acknowledgements
CHAPTER 5 THE DIET OF HARBOUR PORPOISES (PHOCOENA PHOCOENA) IN THE SOUTHERN NORTH SEA: A RELATIONSHIP WITH PREY AVAILABILITY
Abstract
Introduction
1. Materials and methods
1.1. Sampling and data collection
1.2. Stomach content analyses
1.3. Stable isotopes analyses
1.4. Fatty acids composition
1.5. Compound-Specific Stable Isotope Analysis (CSIA)
1.5. Data treatment
2. Results
2.1. Stomach contents
2.2. Stable isotopes and SIAR
2.3. Fatty acids composition and CSIA
3. Discussion
3.1. Diet of harbour porpoises stranded along the southern North Sea
3.2. General diet composition
Conclusion
Acknowledgements
CHAPTER 6 FEEDING HABITS OF HARBOUR PORPOISES (PHOCOENA PHOCOENA) FROM THE SOUTHERN NORTH SEA AND THE BAY OF BISCAY INFERRED FROM A MULTI APPROACH DIETARY ANALYSES
Abstract
Introduction
1. Materials and methods
1.1. Sampling and data collection
1.2. Stomach content analysis
1.3. Stable isotopes analysis
1.4. Fatty acids analysis
1.5. Data treatment
2. Results
2.1. Stomach contents
2.2. Stable isotope analyses of δ13C and δ15N
2.3. Lipid composition
3. Discussion
3.1. Diet composition
3.2. Comparison with previous studies
Conclusion
Acknowledgements
CHAPTER 7 GENERAL DISCUSSION
Contamination status of harbour porpoises in the southern North Sea
Changes in the distribution of harbour porpoises in the North Sea
Value of a multi-approach dietary analysis
Conclusions and perspectives
REFERENCES .

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