**2. Materials and methods**

#### **2.1 Study area**

478 Health Management – Different Approaches and Solutions

adults, green turtles forage largely on marine algae and seagrasses with variation in the diet due to the relative availability of food types over geographic and temporal scales (Garnett et al., 1985; Brand-Gardner et al., 1999; Seminoff et al., 2002). In the process of metal bioaccumulation in marine food chains is poorly understood because very little data is available on metal concentration at different trophic levels (de la Lanza et al. 1989; Talavera-Saenz et al. 2007) or their temporal (Abdallah et al., 2006; Rodriguez-Castañeda et al., 2006) or spatial variation (Kalesh and Nair, 2006) and their effects on the photosynthetic process (Catriona et al. 2002). High concentrations of heavy metals have been found in sea turtles from many regions of the world (Storelli and Marcotrigiano, 2003). Although metal concentrations vary greatly by region and tissue type, green turtles (*Chelonia mydas*) have been found to have exceptionally high kidney cadmium concentrations. Elevated Cadmium levels have been measured in green turtles from around the world including Japan (Sakai et al., 2000; Anan et al., 2001), China (Lam et al., 2004), Europe (Caurant et al., 1999), Australia (Gordon et al., 1998) and the Arabian Sea (Bicho et al., 2006). Gordon et al. (1998) found that Cadmium concentrations in green turtles from Australia were up to three times higher than the levels reported in commercial seafood products. The presence of epibionts, parasites (internal and external) might occasionally cause the death of some marine turtles and being predecessors of fibropapiloms (Aguirre y Lutz, 2004; Work, 2000, Work *et al*., 2005). The presence of fibropapiloms in Hawaiian waters was related with the presence of hirudineans (Díaz, *et al.,* 1992). This kind of infections are might be related with their foraging habitat and its conservation condition, their health condition to escape predators and, for the females, the fecundity reduction (Gámez et al., 2006; Alfaro, et al., 2006; Badillo, 2007). The Baja California Peninsula serves an important role as foraging grounds for five of the world's seven sea turtle species (Gardner and Nichols, 2001). Although much of the peninsula is considered pristine, exploitation of mineral deposits has occurred since the 19th Century and concentrations of Cadmium, Zinc, Copper and Plumb in sediment and marine fauna have been observed above those in more industrialized regions (Gutiérrez-Galindo et al., 1999; Shumilin et al., 2000). In the mid 1970's, Martin and Broenkow (1975) reported that concentrations of Cadmium along the coast of the Baja California Peninsula were remarkably elevated as compared to other regions of the eastern Pacific. Sources of heavy metals in Baja California have been generally attributed to natural factors related to upwelling and the biogeochemistry of the region, however, the potential contribution from anthropogenic sources (e.g. mining and urbanization) cannot be entirely dismissed (Martin and Broenkow, 1975; Sañudo-Wihelmy and Flegal, 1996; Méndez-Rodríguez et al., 1998; Gutiérrez- Galindo et al., 1999; Shumilin et al., 2001). Rodríguez-Meza et al. (2008) developed an extensive evaluation of the heavy metals in sediments and seaweeds along ten sites in the bay. They suggested that the high levels of some heavy metals are related to terrigenous input from the arroyos and biogenic origin by the upwelling. In order to better understand the sources of heavy metals to marine species, more information is needed on metal concentrations in primary producers that make up the base of the food chain. However, few (Riosmena-Rodriguez et al., 2010) papers have approached the study of natural levels of heavy metals in seaweed communities and their temporal and spatial variation. Previous studies in Magdalena Bay, Mexico (Méndez et al., 2002; Gardner et al., 2006) have found high concentrations of metals in marine vertebrates, despite the lack of obvious anthropogenic sources. For example, Cadmium, Zinc, and Iron concentrations in the herbivorous green turtle, *Chelonia mydas*, were the highest ever reported in sea turtles globally (Gardner et al., 2006). In Magdalena Bay, like other regions of the Baja California

Magdalena Bay is located on the Pacific coast of the Baja California Peninsula, Mexico between 24° 15′ N and 25° 20′ N, and 111° 30′ W and 112° 15′ W. It is a shallow lagoon protected from the Pacific by barrier islands, with high productivity resulting from seasonal marine upwelling along the coast. Diverse marine habitats within the bay include sandy bottoms and rocky margins, extensive beds of the seagrass *Zostera marina* and a diverse assemblage of macroalgae. A sea turtle refuge area known as Estero Banderitas is located within the mangrove channels in the northwest region of the Bay where green turtles reside year-round (Fig. 1). Because of the perceived importance of this area for green turtle foraging, its protection has been identified as a priority for conservation efforts (Arriaga et al., 1998; Nichols et al., 2000). Rodríguez-Meza et al. (2008) has found that the presence of heavy metals in the bay is heavily influenced by sediment type, organic material, and carbonates and concluded that there was no evidence of human impacts.

Fig. 1. Study area in Estero Banderitas (24º 50´ - 25º 00´ N and 112º08´ W) located in Bahía Magdalena, Baja California Sur, Mexico.

The Foragining Ecology of the Green Turtle in the Baja California Peninsula: Health Issues 481

total metal signature of the individual sample. Fe was removed from these analyses because of its high Concentration and dominance of the metal signature profile. Principal Components Analysis (PCA) of the percent contribution of the metals in plants and turtle tissues. Additionally, factorial analysis was used to determine trends in the presence of heavy metals in the seaweed samples and the relative spatial and/or temporal variation. All analysis was conducted using the Statgraphics Plus software program (Version 5, Rockville,

Monthly sampling has been develop in the Estero Banderitas and more recently in Estero San Buto as part of the monitoring efforts in was the prescence of fibropapiloms and epibionts by a physical inspection of each animal by region as head, neck, carapace, front or back fins, anus or tail. Comparative analysis was done of the proportion of animals with fibropapiloms and epibionts using the database and literature described in Lara-Uc (2011) in relation to the Bahía

Based on our analysis, we found temporal and spatial variations in the concentration in several of heavy metals in seaweeds and seagrasses. In comparisons between the profiles of heavy metals in major plant groups, we found that Nickel differed significantly between the major groups (P=0.01), wherein seagrasses had lower concentrations (Tables 1 and 2). Analyzing all of the species (all sites combined), we found significant seasonal differences in the heavy metal concentrations with the exception of Zinc (P=0.53). Samples collected in April had a higher concentration of Cadmium (P<0.001) and Iron (P=0.002) and a lower concentration of Plumb (P<0.001) and Nickel (P=0.002) than the other months. Manganese was highest in November (P=0.049) and Copper was higher in November compared to February (P=0.01). In comparisons of the metal concentrations between plant species, the only significant differences were detected for Cadmium (p=0.009) in *Ruppia maritima* than all other species. In the case of the analysis of green algae alone, using all species combined, we

In the case of other metals, we found significantly temporal differences in Plumb (Pb) concentration in *G. vermiculophylla* (P=0.02) in November but this species also had the highest concentration of Ni (P=0.03) in relation to the other species. Also, there were significant differences in the concentrations of Cadmium (P=0.001), Iron (P=0.01), and Nickel (P=0.002), while Plumb (P<0.001) and Copper (P=0.03) were significantly different than the same metals in November. In the same month, highest Nickel concentrations were recorded in *Codium amplivesiculatum*, while in April, *C. amplivesiculatum*, *Codium cuneatum*, and *Caulerpa sertularioides* from the middle region had the highest concentrations of Copper (7.3μg g−1 dw), Ni (11μg g−1 dw), and Mn (61.4μg g−1 dw), respectively. In February, like November, we had the highest Iron concentration and several species were responsible for this difference (in *H. johnstonii*; 567.5μg g−1 dw) and Zinc concentration (in *G. textorii*; 46.8μg g−1 dw). However, the lower zone had the highest concentrations of Cadmium (in *G. textorii*; 4.4μg g−1 dw) and Ni (*L. pacifica* and *Chondria nidifica*; 13.3 and 13.3μg g−1 dw). Copper (in *L. pacifica*; 2.9μg g−1 dw) and Plumb concentrations were highest in *G. andersonii* 

Madalena population information (Hinojosa-Arango unpublished data).

found temporal significant differences of Cadmium in April (P=0.01).

from the middle zone (3.8μg g−1 dw).

**3.1 Temporal and spatial variation of metal concentration in plant species** 

MD).

**3. Results** 

**2.7 Prescence of fibropapiloms and epibionts** 

#### **2.2 Marine plant collection**

Three separate sampling trips were made in Estero Banderitas (November 2004, February, 2005 and April, 2005) in order to collect marine plants available during different seasons. Algae and seagrass samples were collected along the length of the mangrove channel using 16 transects of 30 m length. Every 6 m along the transects, plants were manually collected within a 25 cm2 to 1 m2 area, depending on the density of the flora at that location, for a total of 80 samples per trip. The samples were stored in labeled plastic bags and contents were separated by species using taxonomic keys (Riosmena Rodríguez, 1999). Samples were sun-dried in the field and then pressed to further remove moisture.
