monachus science poster
Vol. 5 (1): May 2002



ANALYSIS OF HEAVY METALS IN BLUBBER AND SKIN
OF MEDITERRANEAN MONK SEALS

A. Dosi, S. Adamantopoulou, P. Dendrinos,
S. Kotomatas, E. Tounta & E. Androukaki

MOm/Hellenic Society for the Study and Protection of the Monk Seal


Introduction

During the last few decades, seals and other fish-consuming marine mammals have received attention as indicators of environmental pollution (Holden, 1970; 1975; 1978; Olsson et al., 1992) as they can reflect contaminant concentrations in their prey species as well (Law et al., 1992; Marcovecchio, 1994).

Mediterranean monk seals, Monachus monachus, occupy a high trophic level in the food web. Therefore they may be considered useful as an indicator species for investigating the biomagnification of heavy metals in the Greek waters and in the East Mediterranean in general. The IUCN/UNEP (1988) Action Plan for the Mediterranean monk seal has considers that pollution from chemical compounds may be an important limiting factor for the species’ welfare but its potential impact has not been assessed up until now. Studies on the incidence of metals on the Eastern Mediterranean subpopulations of the species, as well as from the whole Mediterranean region in general, are confined to one study of mercury concentrations in body hair of Monachus monachus (Yediler et al., 1993).


Objectives of the study

Given the endangered status of Monk seals and the potential for heavy metals to have detrimental effects upon marine vertebrates (Rawson et al., 1993), there is a clear need to augment the almost inexistent amount of data regarding heavy metal burdens in marine mammals from the Mediterranean Sea. This study is an initial investigation of heavy metal concentrations in this species and its aim is to form a baseline reference for future work.


Materials and Methods

Map 1. Locations from where samples were collected
(click to enlarge)
Tissues used in this study were derived from 17 different Mediterranean Monk Seal individuals. Samples were obtained from seals either found dead or that died during their treatment in the Seal Treatment and Rehabilitation Centre, Alonnisos. Information on the animals and the samples is provided in Table 1 locations of collection in Map 1. Localities were divided into six main geographic divisions (Table 1). Samples were analysed in the School of Ocean Sciences, Bangor, North Wales. Porcelain crucibles, previously washed with 10 % hydrochloric acid, were weighed and adequately labelled. Crucibles plus thawed sample tissues were weighed and placed in the oven at 60°C to desiccate. Samples were afterwards placed in a muffle furnace at a temperature of 540°C for five hours, until all the organic content was removed. Crucibles with samples were weighed again in order for ash weight to be calculated. 1% nitric acid was used to dissolve the ash and each sample was made up to 25ml solution in acid washed 25ml glass bottles sealed with polythene caps. Solutions were stored in acid washed plastic bottles until their analysis by Inductively Coupled Plasma–Atomic Emission Spectrometry (ICP-AES).

Table 1. Information of the Monk Seals’ samples used

Sample Code Date of Collection Area of Collection Geographic Division Stage of Development Sex State of Decomposition Tissue Type
T 94.1 29/10/1994 Skopelos SPORADES Pup M Medium Blubber+Skin
T 95.1 5/1/1995 Naxos CYCLADES Juvenile F Fresh Blubber
T 95.2 3/12/1995 N. Evia SPORADES Pup F Fresh Blubber
T 96.1 28/1/1996 Psara EAST AEGEAN Adult F Fresh Blubber
T 96.2 14/3/1996 Milos CYCLADES Juvenile M Medium Blubber+Skin
T 96.3 15/10/1996 Piperi SPORADES Pup F Medium Blubber+Skin
T 96.4 13/11/1996 Piperi SPORADES Pup F Fresh Blubber+Skin
T 96.5 12/12/1996 Fourni EAST AEGEAN Adult M Advanced Blubber+Skin
T 97.1 20/6/1997 Megara SARONIKOS Adult F Fresh Blubber
T 97.2 24/9/1997 Karpathos SOUTHEAST AEGEAN Adult M Fresh Blubber
T 97.3 26/12/1997 Ikaria EAST AEGEAN Pup F Fresh Blubber+Skin
T 98.1 28/11/1998 Nissyros SOUTHEAST AEGEAN Pup M Medium Blubber+Skin
T 98.2 9/12/1998 NE Evia SPORADES Pup M Advanced Blubber+Skin
T 99.1 15/4/1999 Chios EAST AEGEAN Adult F Medium Blubber
T 99.2 2/6/1999 Potidaea NORTH AEGEAN Adult F Medium Blubber
T 99.3 20/6/1999 Pteleos SPORADES Juvenile M Medium Blubber
T 99.4 22/8/1999 Milos CYCLADES Adult F Initial Blubber
F = female   M = male


All ICP-AES analyses were performed in the Institute of Environmental Science, Chemistry Tower, Bangor. Simultaneous semi-quantitative analysis of the 14 metals (Al, As, Cd, Co, Cr, Cu, Fe, Mg, Mn, Pb, Pt, Se, Si and Zn) was performed using an inductively coupled plasma-atomic emission spectrometer, ICP-AES (Jobin Yvon 138 ULTRACE). Copper and zinc were afterwards determined more accurately using a set of standard solutions in order to create calibration lines. 0, 2, 4, 6, 8 and 10 ppm Zn and 0, 0.05, 0.1, 0.2, 0.4, 0.6 ppm Cu were made up in 50 ml solutions with 1% nitric acid. The standard solutions produced values against which the concentrations of the two metals in the samples were determined quantitatively. The whole procedure was again performed in the ICP-AES.


Results

Parameters’ Influence on Metal Concentrations

Concentrations of metals in marine mammals are known to be affected by a number of parameters such as sex, age, location, tissue type, prey type as well as yearly changes in the environment. All the parameters, for which sufficient data were available, were taken into consideration in order to determine their influence in the concentrations of metals in the animals’ tissues. Principal Component Analysis (PCA) was carried out for the 12 of the examined metals in order to determine which of the above-mentioned parameters poses greater influences in the concentrations of the metals. The analysis resulted in 64% of the variability accounted to the first two components. Therefore plotting the scores for the values in the first component against those in the second component we could represent 64% of the available information (Figure 1). The plots of the first two components in relation to the different parameters indicate that tissue type and stage of development of the seals are responsible for the variability in the concentration values of the metals. The majority of the skin samples exhibited negative scores for the first principal component and most of them also have negative scores for the second. It could be implied therefore that skin samples are responsible for the highest concentrations in all metals. From the stage of development related plot it is clearly evident that samples derived from pups are those with the highest concentrations of all metals.

Figure 1. Plots of the scores of the first two principal components of PCA analysis of the 12 metals concentrations in relation to the different examined parameters (click on graphs to enlarge)


Copper and Zinc

The two methods of ICP-AES used for the determination of copper and zinc were investigated in terms of their resulted values in order to study the degree of difference, if any, between them. The semi-quantitative values were plotted against the quantitative ones for both copper and zinc and the equations of the fitted lines were calculated (Figure 2). Quantitative copper values were found to be greater than the values obtained from the semi-quantitative method by almost a factor of 2. Zinc values on the other hand did not differ between the two methods. It may be concluded therefore that the method of analysis could be important for some metals while it does not have any effect on others. The relationship between the quantitative values of the two metals was also investigated. The Pearson’s correlation coefficient was calculated to be 0.686 with a p-value of <0.001. Both variables though, were dependent and did not fit assumptions for single linear regression. Consequently copper and zinc concentrations were transformed to the logarithmic scale and the Geographic Mean Regression was utilized. Graphical presentation of the Log values for the two metals is presented in Figure 3 with a Pearson’s correlation coefficient of 0.815 (p-value <0.001) and the resulted equation for the geographic mean regression line:
LogCu (ug/g dry wt) = -0.736 + 0.882 x LogZn (ug/g dry wt)

Figure 2. Relationship of the semi-quantitative and quantitative results for copper and zinc (blue lines indicate the 100% precision between the two methods) and logarithmic plot of the relationship between quantitative copper and zinc concentrations (click on graphs to enlarge)


Discussion

For the first time the concentrations of metals in the blubber and skin of Monk Seals from the Eastern Mediterranean subpopulation and the Aegean in particular, were studied and analysed. Total heavy metal concentrations are by themselves insufficient to assess either the health status of the animals or the effective toxicity of the toxicant in question. Yet, they are important and necessary as they can provide a benchmark for what is to be regarded as a ‘normal’ or ‘abnormal’ heavy metal burden for animals (Wagemann & Muir, 1984). Extremely limited data regarding metal concentrations in monk seals are available (Borrell et al., 1997; Menchero et al., 1994). The former render this study very difficult to comment and evaluate and very important in terms of baseline information about the species and the area. Larger sample sizes and comparative data from polluted and unpolluted regions are required to help assess the possible contribution of pollution to the viability of Mediterranean Monk Seals.


Influence of Different Parameters on Monk Seal Metal Concentrations

Sex was not observed to account for any differences in the concentrations
Tissue type and stage of development accounted for differences in the concentrations for all metals studied with the highest concentrations observed in the skin of monk seal pups
No evident differences were found in the metals’ concentrations during the different years of collection. However, the differences in sample sizes and the very small number of samples obtained for some years, renders the comparisons difficult
The different locations of sample collection did not account for differences in the concentrations of metals


Copper and Zinc

The mean copper concentration obtained from our study was 5.173 ug/g dry weight which is towards the lower end of the ranges tentatively assigned (Law et al., 1991; Law, 1996)
Significant differences were observed between concentrations in blubber and skin with the greatest in the latter
Previous studies have reported a negative correlation between age and copper concentrations in Phoca hispida, Odobenus rosmarus rosmarus and Monodon monoceros (Wagemann, 1989; Wagemann & Stewart, 1994; Wagemann et al., 1983). The same was observed in our study as well, with pups having significantly higher concentrations than adult monk seals
Zinc exhibited higher concentrations in the skin, which is similar to what have been found in Phoca vitulina (Roberts et al., 1976), Phoca sibirica (Watanabe et al., 1996), Leptonychotes weddellii (Yamamoto et al., 1987) and Stenella coeruleoalba (Honda et al., 1982)
Zinc concentrations were significantly higher in pups compared to adult and juvenile monk seals
Copper and zinc exhibited a significant correlation. The same was the case in livers of Leptonychotes weddellii (Szefer et al., 1994). The mechanisms underlying the association of copper and zinc are largely unknown but it is speculated that this association is probably mediated by metallothionein, as these metals are inductors and constituents of this metalloprotein (Wagemann & Stewart, 1994)


Acknowledgements

Part of this study was submitted by A. Dosi for the fulfilment of the requirements of the M.Sc. Degree in Marine Biology at the University of Bangor, N. Whales. We would like to express our gratitude to Dr. Andy B. Yule for his advice and guidance throughout the completion of this thesis. We would also like to thank all authorities and individuals that contributed to this study, by reporting the strandings from where the samples were collected.


life logo This work has been partially funded by the European Commission, D.G. Environment, through the contracts, Life-Nature, B4-3200/96/500 and LIFE00NAT/GR/7248
This poster was presented at the 16th ECS (European Cetacean Society) Conference, "Marine Mammal Health: from Individuals to Populations", 7-11 April 2002, Liege, Belgium.


      

Copyright © 2002 A. Dosi, S. Adamantopoulou, P. Dendrinos, S. Kotomatas, E. Tounta, E. Androukaki, The Monachus Guardian. All Rights Reserved