Methylmercury and total mercury distribution in tissues of gray whales (Eschrichtius robustus) and spinner dolphins (Stenella longirostris) stranded along the lower Gulf of California, Mexico

Distribución de metilmercurio y mercurio en tejidos de ballenas grises (Eschrichtius robustus) y delfines tornillo (Stenella longirostris) varados en el bajo Golfo de California, México

Jorge R. Ruelas-Inzunza¹, Milena Horvat², Héctor Pérez-Cortés³ and Federico Páez-Osuna^4*

¹ Technological Institute of the Sea Graduate Program on Marine Sciences and Limnology Institute of Marine Sciences and Limnology P.O. Box 757 Mazatlán, Mexico.

³ National Program on Research and Conservation of Marine Mammals La Paz, Baja California Sur, México.

⁴ Institute of Marine Sciences and Limnology National Autonomous University of Mexico P.O. Box 811, Mazatlán, Mexico. *E-mail: paezos@servidor.unam.mx

Recibido en julio de 2002;
aceptado en octubre de 2002.

Abstract

Four gray whales Eschrichtius robustus and eleven spinner dolphins Stenella longirostris were found stranded along the coasts of the lower Gulf of California, Mexico. Muscle, kidney and liver samples from all the specimens were used for mercury (Hg) analysis. Mean concentrations of total Hg (THg) were significantly different (P < 0.05) in all the tissues of spinner dolphins, resulting in a decreasing order liver>kidney>muscle. Mean values of THg in the three tissues of gray whales were not statistically different. Similarly, methylmercury (MeHg) levels in the analyzed tissues of dolphins and whales were not statistically different. Considering species-related differences, it seems that THg and MeHg levels in the studied marine mammals did not play a key role in the stranding of the specimens, though more studies about the implications of Hg compounds in stranding events are necessary.

Key words: mercury, methylmercury, Stenella longirostris, Eschrichtius robustus, Gulf of California (Mexico).

Resumen

Se encontraron varadas cuatro ballenas grises Eschrichtius robustus y once delfines tornillo Stenella longirostris a lo largo de las costas del bajo Golfo de California, México. Se utilizaron muestras de músculo, riñón e hígado de todos los especímenes para los análisis de mercurio (Hg). Las concentraciones medias de Hg total fueron significativamente diferentes (P < 0.05) en todos los tejidos de los delfines tornillo, resultando en un orden decreciente de concentraciones hígado>riñón>músculo. Los valores medios de Hg total en los tres tejidos de las ballenas grises no fueron estadísticamente diferentes. De manera similar, los niveles de metilmercurio (MeHg) en los tejidos analizados de los delfines y de las ballenas no fueron estadísticamente diferentes. Considerando que existen diferencias a nivel de especies, al parecer los niveles de Hg total y de MeHg en los mamíferos marinos estudiados no jugaron un papel decisivo en el varamiento de los organismos, aunque se requieren más estudios acerca de las implicaciones de los compuestos de Hg durante los eventos de varamiento.

Palabras clave: mercurio, metilmercurio, Stenella longirostris, Eschrichtius robustus, Golfo de California (México).

Introduction

Mercury may occur naturally in the environment (mineral deposits, volcanoes, forest fires, oceanic emission, and crust degassing) or be released by human activities such as mining, mineral processing, chloroalkaly production, and combustion of fossil fuels (Renzoni et al., 1998). The contamination of the marine environment by Hg compounds is the result of natural phenomena in conjunction with anthropogenic discharge, especially near the coastline; anthropogenic discharges contribute to an increase of the Hg flux and to the alteration of the chemical forms and species of this trace element present in the water column and sediments (André et al., 1990). Methylmercury is formed in aquatic sediments by bacterial methylation of inorganic Hg. Most of the MeHg so formed enters biological tissue due to its high affinity for sulfhydryl groups and lipids. As a result, an accumulation of Hg (mainly as MeHg) occurs in aquatic organisms, with concurrent biomagnification phenomena throughout the trophic chain. These findings have raised the concerns that marine mammals transiently residing in urban-influenced waters may also experience adverse effects induced by chemical contaminants (Varanasi et al., 1994).

Increasing Hg concentrations are found at higher trophic levels due to the process of biomagnification (Palmisano et al., 1995); however, such metal levels are not the same in all the tissues and organs of specimens. In marine mammals, MeHg is by far the most predominant form of Hg present in the muscle tissue, while the liver content is only a small percent of the total (Palmisano et al., 1995).

Marine mammals sometimes are affected by exposure to metallic pollutants (Szefer et al., 1995). These organisms, with a long life span, can be considered as strong candidates for ass-esing cumulative and biomagnified pollutants like Hg, and such results can be extrapolated to the human population. Establishing databases on contaminants in marine mammals will help to understand the role of contaminants in mortality events and provide a basis to investigate, predict and mitigate these events (Beck et al., 1997).

The gray whale Eschrichtius robustus lives in the northern Pacific and is represented by two populations, an almost extinct asiatic population, and a numerous american population. American specimens migrate from the Bering and Beaufort seas, where they feed during the summer, to the Gulf of California for their reproductive activities (Rice and Wolman, 1971). Eschrichtius robustus feeds mainly on small crustaceans (amphipods) by filtering the bottom sediments (Nerini, 1984). The spinner dolphin Stenella longirostris is distributed in tropical and subtropical waters all over the world. They feed mainly on squid and fish (Perrin, 1975).

Methods

The stranded mammals

Four gray whales (Eschrichtius robustus Lilljeborg, 1861) were found stranded along the eastern coast of the Gulf of California between February 17 and March 18, 1999 (fig. 1a, b and d). Muscle, kidney and liver samples were taken from every specimen for analysis. In the case of spinner dolphins (Stenella longirostris Gray, 1828), eleven stranded specimens were found dead in the Bay of La Paz (fig. 1c), in the lower Gulf of California in August 7, 1993. Specimens were identified, measured and weighed, prior to dissection and separation of every sample. The time elapsed between their stranding and sampling, was from 1 to 5 days depending of the accesibility of the stranding sites. Samples were kept frozen until the laboratory work was initiated. In the laboratory, portions of the different tissues were freeze-dried (Labconco freeze-drying system) for 48 hours at 40-133 x 10^-3 mBar and -49°C; dried samples were ground with an automatic agate mortar (Retsch) for 10 minutes.

Mercury analysis

Duplicates of every sample were used for THg and MeHg analysis. The digestion of tissues (0.25-g aliquots) for THg analysis was made by using concentrated HNO₃ (5 ml) with a microwave digestion system (CEM-MDS, 2000) in order to decompose the samples, and oxidize and convert any organic forms of Hg into inorganic Hg (MESL, 1997). Determination of THg was made by means of a vapor generator coupled to an atomic absorption spectrophotometer (VGA-AAS); inorganic Hg was reduced to its elemental form with SnCl₂, cold Hg vapor was then passed through a quartz absorption cell of an atomic absorption spectrophotometer (AAS) in which its concentration was measured (MESL, 1997).

In the case of MeHg, the pretreatment of samples consisted on acid leaching with 6M HCl, centrifugation and separation of organic and inorganic Hg on an ion-exchange resin (Dowex 1x8, 100-200 mesh size). After the separation of Hg forms, samples were subjected to UV radiation for two days (MESL, 1997). Determination of Hg was made by VGA-AAS following the same steps as for THg. In order to assure the quality of the analysis dogfish liver certified reference material (DOLT-2; National Research Council of Canada) was analyzed. The agreement between the analytical results for the reference material and their certified values was satisfactory; i.e. recoveries were 98.0% for THg and 92.8% for MeHg, and precision varied ±0.05% for THg and ±2.6% for MeHg. To check for contamination, blanks were also analyzed using this procedure after every 6-8 samples were analyzed.

The normality of concentration data was assessed by a Kolmogorov-Smirnoff test. Mean concentrations of THg and MeHg in the different tissues were compared (one-way ANOVA) in order to define significant differences. The statistical analysis was conducted using GraphPad Prism 2.01 (Graph Pad Software Inc., San Diego CA).

Results and discussion

Biological data

From the morphometric and age data in table 1, all the analyzed specimens were considered as adults. Gray whales were not aged though only morphometric data were provided. Perrin (1975) reported that newborn spinner dolphins S. longirostris from the eastern Pacific are 80 cm in length and can reach a total length of 200 cm when adults. In this study the mean length (181 cm) and weight (46.9 kg) were within the ranges reported. In the case of the gray whales E. robustus, the reported mean length ranges from 14.5 m in adults to 4.5 m in newborns (Urbán-Ramírez et al., 1997); the mean length of the stranded whales was 9.5 m. In Cetacea, sexual maturity is established by gross and microscopic examination of the gonads, and where possible, the reproductive organs. Sexual maturity does not always correspond to the beginning of reproduction (Bryden, 1972).

Hg and MeHg concentrations

Mean concentrations of THg were significantly different (P < 0.05) in all the tissues of spinner dolphins, resulting in a decreasing order liver>kidney>muscle. The total Hg values in the three tissues of gray whales were not statistically different. Methylmercury levels in the analyzed tissues of dolphins and whales were not statistically different.

In gray whales, the mean THg values presented in table 2 were highest in kidney, followed by liver and muscle. Regarding MeHg, the order was muscle>kidney>liver. The mean percentage of MeHg was higher in the muscle (75.2%) than in liver (22.7%) and kidney (18.4%). Comparing with other heavy metals examined in these same gray whales from the same study area (Ruelas-Inzunza and Páez-Osuna, 2002) it is evident that Cd is accumulated in the same tissue order than THg, but Cu, Mn, Pb and Zn had a contrasting pattern.

In the case of the spinner dolphins, the mean levels of MeHg were highest in the liver, followed by muscle and kidney. From the mean percents of MeHg, figures for muscle (74.3%) were higher than for kidney (7.6%) and for liver (1.6%) (table 2).

Working with aquatic organisms and specially with large whales, often poses several problems: firstly, it is not possible to determine whether the concentrations found are a result of natural environmental levels or are affected to some extent by anthropogenic activities; secondly, the influence of biological parameters on the dynamics of metals in whales has not been well established and it is unknown how biochemical and physiological processes during aging could affect metal concentrations, not to mention antagonistic or synergistic effects among different metals (Sanpera et al., 1996).

It is well known that MeHg is harmful for marine mammals, even though kidney and liver acumulate a high proportion of it and no apparent damage occurs. In this sense, most of the Hg accumulated in the marine mammal liver, being inorganic, suggests that this organ is a demethylation site (Caurant et al., 1996). As suggested by Bryan (1984), the demethylation mechanism has evolved in response to biomagnification of MeHg either in species at high food chain levels or in individuals of the same species with highest levels of Hg.

A comparison of our data shows that the dolphins analyzed had lower levels of THg in the liver tissue than specimens from other sites, such as Stenella coeruleoalba (Frodello et al., 2000), Delphinus delphius (Law et al., 1992), Lagenorhynchus acutus (Law et al., 1991) and Tursiops truncatus (Storelli and Marcotrigiano, 2000). Regarding gray whales, similarly, our data results were lower than the values reported for the same species (Varanasi et al.. 1994) and Mesoplodon densirostris (Law et al., 1997). It is necessary to point out that except for Delphinus delphius all the specimens were found stranded. Regardless the levels found, it is particularly difficult to demonstrate a causal link between pollution and the strandings of marine mammals given the lack of a sufficient number of tissue samples from both healthy and stranded animals, and the inability to conduct controlled laboratory studies with live animals, particularly with large marine mammals (Varanasi et al., 1994).

Law et al. (1992) studied the levels of certain metals (including Hg) in marine mammals and found low levels that were unlikely to have contributed to mortality. Additionaly, several considerations should be taken into account: firstly, it is not possible to determine whether the concentrations found are a result of natural environmental levels or are affected to some extent by anthropogenic activities (Sanpera et al., 1996); secondly, the concentrations of trace elements have been shown to vary substantially from group to group (Caurant et al. ,1996); and lastly, a combined effect of some kind of pathogen and an impairment of the immunological system might account for such mortality events.

Considering the water content for the different tissues and the reported units, MeHg levels for this study were lower to the values reported elsewhere (Caurant et al., 1996; Storelli et al., 1998; 1999). The limit of tolerance for Hg in mammalian hepatic tissue seems to be within the range 100-400 mg/kg wet weight (Wagemann and Muir, 1984).

In marine mammals, MeHg is by far the most predominant form of Hg present in muscle tissues, while in liver its content is only a few percent of the total (Palmisano et al., 1995). In muscle, MeHg is firmly bond by carbon-mercury and sulphydryl linkages, which could account for the high ratio of organic Hg in this tissue, while in the liver the low percentage of organic Hg observed supports the hypothesis of a demethy-lating activity in this organ (Storelli et al., 1999).

Acknowledgements

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