**4. Discussion**

Much of the literature on sea turtles has worked with absolute concentrations of metals, which is appropriate for comparisons of very similar sample types such as different sea turtle tissues or the same tissue in different sea turtle species. In the present paper we used absolute concentrations to compare metals in tissues (kidney vs. liver) and to compare metal concentrations in different plant species. However in order to better understand the source of metals to turtles in this region, the profile of all metals combined was used as an environmentally acquired marker. For this objective, we removed the influence of concentration differences among samples by converting the data to percent contribution of each metal to the total metal signature of the individual sample. This approach enabled the comparison of metal profiles across greatly different samples and was more appropriate than comparisons of absolute concentrations alone. For example, a single plant species located in two different areas will accumulate metals using the same physiological mechanisms. Therefore, a difference in metal profiles of the plant species from two different locations is an indication of differences in the availability of the metals from the environment. However, differences in the absolute concentrations of metals in plants would not necessarily indicate environmental difference because other factors might also be at play (e.g. age of the plant).

#### **4.1 Comparison of metals in marine plant species**

Metal concentrations in marine flora are controlled by both the bioavailability of metals in the surrounding water and the uptake capacity of the particular plant species. Marine algae have the capacity to accumulate trace metals several thousand times higher than the concentration in seawater (Bryan and Langston, 1992; Sánchez-Rodríguez et al., 2001). Red algae, such as Gracilaria sp tend to reflect the environmental availability of metals but have higher bioaccumulation of Cadmium, Copper and Zinc than other macroalgal groups (Sánchez-Rodríguez et al., 2001; Roncarati, 2003). These same species are a major component of the green turtle diet along the Baja California Peninsula (Seminoff et al., 2002; López-Mendilaharsu et al., 2005), and we proposed previously (Gardner et al., 2006) that their foraging habits could account for the high metal concentrations found in this population. However, comparisons across plant species in the present study suggest that species differences in metal concentrations are minimal. The only significant difference detected between plant species was that Cadmium was higher in *Ruppia maritima* than all other species, and higher in *Gracilaria textorii* than *Codium amplivesiculatum*. *R. maritima* was encountered in only one of the sea turtle stomachs analyzed, contributing a relatively small percentage of the overall diet (8.7%) in this study, and was absent from the diet of 24 green turtles analyzed in previous work (López-Mendilaharsu et al., 2005). *Gracilaria textorii* made up a larger proportion of the turtles' stomach contents (16.5%), but was similar in Cadmium concentration to most other plant species ingested by the green turtles. The results of the PCA also support this conclusion since the bay-collected plant samples grouped separately from the samples in the stomach contents despite that both groups consisted of the same five plant species.

We found significant spatial and temporal variations in heavy metal concentrations in marine plants as previous spatial studies has shown in the region (Páez-Osuna et al., 2000; Sánchez-Rodríguez et al., 2001; Rodriguez-Castañeda et al., 2006, Rodríguez-Meza et al., 2008). The high concentration of Zinc and Fe in the upper region might be related to the isolation of the site (Rodríguez-Meza et al., 2008). Heavy metal concentration was, in some cases, in the levels of toxicity. Temporal variations in metal concentrations, such as high concentrations in Cadmium and other metals observed in April, may be related to local upwelling events. Surface water Cadmium concentrations have been strongly correlated with upwelling (Lares et al. 2002) which occurs during spring and early summer off the coast of Magdalena Bay (Zaytsev et al., 2003). These levels of Cadmium in seaweeds has not been observed in the Gulf of California studied populations but strong species and spatial variations where observed (Páez-Osuna et al., 2000; Sánchez-Rodríguez et al. 2001; Rodriguez-Castañeda et al., 2006). The differences in heavy metal concentrations that we found in the seaweeds did not generally correspond with patterns of those elements previously observed in the sediment from the same region or seaweed species (Rodríguez-Meza et al., 2008), contrary to the studied sites in the Gulf of California near a mine (Rodriguez-Castañeda et al., 2006) or near industrial ports (Páez-Osuna et al., 2000; Sánchez-Rodríguez et al., 2001; Rodriguez-Castañeda et al., 2006). This finding, together with the observed species differences, suggests that the metabolic condition and life cycle stage of the individual species might influence metal uptake and accumulation (Lobban and Wynne 1981). Similarly, Riget et al., (1995) found differences between seaweed species *Ascophyllum nodosum, Fucus vesiculosus*, and *Fucus distichus*. We found lower levels of Ni and Zinc in *H. johnstonii* than in the environment as reported by Rodríguez-Meza et al., (2008). Based on

our data, there are similarities between the composition and concentration of heavy metals between the plant species reviewed and the sediment; except in the case of Cu, Fe, and Mn (Rodríguez-Meza et al., 2008). All those elements are considered critical in the photosynthetic metabolism (Lobban and Wynne, 1981). We might assume that those elements are more easily assimilated by the plants because of their use in photosynthesis.

The role of seaweeds and seagrasses in coastal lagoons (like Banderitas or any other along the Baja California Peninsula) are relevant because they are feeding grounds for black turtles (*C. mydas*), loggerhead turtles (*Caretta caretta*), olive Ridley turtles (*Lepidochelys olivacea*), and hawksbill turtles (*Eretmochelys imbricata*) and migratory birds like Brant geese (*Branta bernicla*; Seminoff, 2000; Herzog and Sedinger, 2004). All of the species are included in the Mexican endangered species list (NOM ECOL 059) and on the red list in the UICN endangered species (www. uicnredlist.org). They are high productivity areas for fishing all kind of products (CONABIO, 2000; Carta Nacional, 2005). The fact that we found more significant variation in the spatial than temporal heavy metal concentrations in most of the species show that they might be constantly incorporated in the diet of many herbivorous animals (Gardner et al., 2006) with severe consequences in their health. Management strategies for these species should consider monitoring the levels of metals.

#### **4.2 Sea turtle tissue comparisons**

490 Health Management – Different Approaches and Solutions

Much of the literature on sea turtles has worked with absolute concentrations of metals, which is appropriate for comparisons of very similar sample types such as different sea turtle tissues or the same tissue in different sea turtle species. In the present paper we used absolute concentrations to compare metals in tissues (kidney vs. liver) and to compare metal concentrations in different plant species. However in order to better understand the source of metals to turtles in this region, the profile of all metals combined was used as an environmentally acquired marker. For this objective, we removed the influence of concentration differences among samples by converting the data to percent contribution of each metal to the total metal signature of the individual sample. This approach enabled the comparison of metal profiles across greatly different samples and was more appropriate than comparisons of absolute concentrations alone. For example, a single plant species located in two different areas will accumulate metals using the same physiological mechanisms. Therefore, a difference in metal profiles of the plant species from two different locations is an indication of differences in the availability of the metals from the environment. However, differences in the absolute concentrations of metals in plants would not necessarily indicate environmental difference because other factors might also be at play (e.g. age of the plant).

Metal concentrations in marine flora are controlled by both the bioavailability of metals in the surrounding water and the uptake capacity of the particular plant species. Marine algae have the capacity to accumulate trace metals several thousand times higher than the concentration in seawater (Bryan and Langston, 1992; Sánchez-Rodríguez et al., 2001). Red algae, such as Gracilaria sp tend to reflect the environmental availability of metals but have higher bioaccumulation of Cadmium, Copper and Zinc than other macroalgal groups (Sánchez-Rodríguez et al., 2001; Roncarati, 2003). These same species are a major component of the green turtle diet along the Baja California Peninsula (Seminoff et al., 2002; López-Mendilaharsu et al., 2005), and we proposed previously (Gardner et al., 2006) that their foraging habits could account for the high metal concentrations found in this population. However, comparisons across plant species in the present study suggest that species differences in metal concentrations are minimal. The only significant difference detected between plant species was that Cadmium was higher in *Ruppia maritima* than all other species, and higher in *Gracilaria textorii* than *Codium amplivesiculatum*. *R. maritima* was encountered in only one of the sea turtle stomachs analyzed, contributing a relatively small percentage of the overall diet (8.7%) in this study, and was absent from the diet of 24 green turtles analyzed in previous work (López-Mendilaharsu et al., 2005). *Gracilaria textorii* made up a larger proportion of the turtles' stomach contents (16.5%), but was similar in Cadmium concentration to most other plant species ingested by the green turtles. The results of the PCA also support this conclusion since the bay-collected plant samples grouped separately from the samples in the stomach contents despite that both groups consisted of the same

We found significant spatial and temporal variations in heavy metal concentrations in marine plants as previous spatial studies has shown in the region (Páez-Osuna et al., 2000; Sánchez-Rodríguez et al., 2001; Rodriguez-Castañeda et al., 2006, Rodríguez-Meza et al., 2008). The high concentration of Zinc and Fe in the upper region might be related to the isolation of the site (Rodríguez-Meza et al., 2008). Heavy metal concentration was, in some

**4. Discussion** 

five plant species.

**4.1 Comparison of metals in marine plant species** 

Pb, Cu and Mn concentrations in tissue from this study were within the range of those reported for sea turtles in other parts of the world (Lam et al., 2004; Storelli and Marcotrigiano, 2003). However, the average concentrations of Cadmium, Zinc and Ni in kidney of green turtles from Magdalena Bay were high compared to previously reports for sea turtle tissues (Sakai et al., 1995, 2000; Storelli and Marcotrigiano, 2003). Studies of loggerhead turtles (Maffucci et al., 2005) suggest that sea turtles can regulate Copper and Zinc concentrations through homeostatic processes but that Cadmium uptake is not controlled by active process and thus tissue concentrations of this metal reflect exposure. In agreement with these findings, we observed that Cadmium concentrations in green turtle liver were similar to their food and that the Cu concentration in sea turtle liver was greater

The Foragining Ecology of the Green Turtle in the Baja California Peninsula: Health Issues 493

components of regional biogeochemistry (Daesslé et al., 2000; Lares et al., 2002). Similar to the distribution of nutrients in the water column, metals such as Cadmium and Zinc are depleted in the surface and enriched in deeper water. Upwelling processes are an important mechanism that brings elevated concentrations of both nutrients and metals to the surface and thus available for marine floral accumulation. Therefore it is highly probable that the sea turtles collected within Magdalena Bay are utilizing foraging areas in an upwelling-rich coastal region outside of the Bay. Coastal lagoons of the Baja California Peninsula such as Magdalena Bay have been identified as priority areas for sea turtle conservation programs (Nichols et al., 2000). Long-term sea turtle monitoring studies have demonstrated high site fidelity to Estero Banderitas over time, and low emigration of sea turtles from Magdalena Bay to other coastal lagoons along the Baja California Peninsula (Grupo Tortuguero, unpublished data). Efforts to protect areas within Magdalena Bay have focused on the creation of a refuge in the mangrove channels of Estero Banderitas, in part, because of the perceived importance of this habitat for sea turtle foraging (Nichols and Arcas, 2001). However, data generated by our work suggest that sea turtles residing in Estero Banderitas are feeding in areas outside of the bay, most likely in coastal regions with high upwelling. These findings support those of López-Mendilaharsu et al. (2005) and indicate that green turtles utilize spatially distinct feeding habitats within coastal areas. Therefore, we recommend that sea turtle protected areas be designed with an appreciation of regional

The prescence of fibropapiloms are variable from 1.4% up to 90% of the population (Herbs *et al.,* 1999, Quackenbush et al., 2001, Chaloupka et al., 2009). The observed low proportion of the green turtles in Bahía Magdalena (less than 1%) agree with a well preserved environment and less stress situation for the animals. In the case of the epibionts we found a continuously prescence of cirripedia and balanus but not a diverse fauna like in the Atlantic

Conservation of threatened species, such as the green turtle (*Chelonia mydas*), is closely related to habitat quality. In particular there are issues related to heavy metals, the presence of epibionts, parasites and fibropapiloms who might play a crucial role in the species survivorship. 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 and their temporal or spatial variation and its influence in turtle health. The Baja California Peninsula, Mexico serves an important role for feeding and developing sea turtles. High concentrations of metals detected in food items (seaweeds and seagrasses) and in green turtles (*Chelonia mydas*) from Magdalena Bay prompted an investigation into the sources of metals in the region in relation to the health issues of the animals. We compared metal concentrations in sea turtle tissues in relation to plant species found in their stomach contents, and with the same species of plants collected inside a sea turtle refuge area known as Estero Banderitas and determine the health state of turtles based on our long term monitoring efforts. Our results showed that Iron, Copper, and Manganese were the most significant metals found in seagrasses, red, and green algae. We found significant more variation in temporal heavy metal concentrations in relation to the maximum abundance in

rather than local scales in order to protect broader foraging areas.

that even polychaetes has been reported (Lara Uc, 2011).

**4.4 Fibropapiloms and epibionts** 

**5. Conclusions** 

than in the stomach content. Similar relationships have been observed in green turtles from Japan (Anan et al., 2001). However, contrary to the findings of Maffucci et al., (2005), Zinc concentrations in the livers and kidneys of green turtles in our study were not significantly different from their stomach contents. The distribution of metals among organs is influenced by both duration and concentration of exposure. Liver is a major site of short-term Cadmium storage, whereas during long-term exposure, Cadmium is redistributed from the liver to the kidney where it is absorbed and concentrated (Thomas et al., 1994; Linder and Grillitsch, 2000; Rie et al., 2001). Therefore a significantly greater concentration of Cadmium in green turtle kidney than liver is often observed (Storelli and Marcotrigiano, 2003; Maffucci et al., 2005; Gardner et al., 2006) and likely results from years of accumulation in this long-lived species. While kidney Cadmium concentration may serve as a good indicator for assessments of sea turtle health, liver more closely reflects the concentration of this metal in the food and so analyses of liver may provide a better indication of recent environmental exposure. Accordingly, Cadmium concentrations in the livers analyzed in the present study were not different from the food in the sea turtles' stomachs. Concentrations of Fe and Zinc in liver were also similar to the stomach contents. Whereas, Plumb, Nickel and Manganese concentrations in liver were similar to kidney, but were lower than in the stomach contents, which may indicate metabolic processing of these metals. Alternatively, Copper concentration was higher in liver than in the turtles' food and appeared to be preferentially accumulated in liver over kidney.

#### **4.3 Metals in sea turtle stomach contents and marine plants from the bay**

Two principle components, PC(1) and PC(2), explained 68%of the total variance in the data.When plotted relative to PC(1) and PC(2), the plant samples collected in the bay formed a grouping at the left side of the plot while the green turtle tissue samples and the plants from the stomach contents plotted higher on PC(1) (Fig. 4A). Examination of the loadings plot for each of the metals confirmed that samples scoring high on PC1 had signatures dominated by Cadmium and Zinc (stomach contents and kidney) or Cu (liver) (Fig. 4B). This agrees with the observation that the plants in the stomach contents contained greater percent contributions of Cadmium and Zinc than the samples collected in the bay, while Pb and Mn contributed more to the metal profiles in the bay samples as shown in Fig. 2; a tendency that was consistent in all five plant species. The metal profiles in the sea turtle tissues more closely resembled the plants in the stomach contents than the same species of plants collected within Estero Banderitas. The fact that the concentrations of Cadmium, Fe and Zinc in green turtle liver were the same as the stomach contents but different from the plants collected in the bay suggests that sea turtles collected inside of Magdalena Bay use foraging resources outside of the Estero Banderitas region. Further support of this conclusion is provided by the fact that three algal species (*N. baileyi*, *P. capillacea* and *U. lactuca*) in the stomach contents were not found in Estero Banderitas. Franzellitti et al. (2004) proposed that tissue metal profiles can be used as "environmentally acquired markers" to determine sea turtle feeding areas. Similarly, principle component analyses have been applied previously to determine sources of metals in aquatic environments (Ruiz-Fernández et al., 2001). Comparison of the metal signature profiles in plants from the bay and the sea turtle stomach contents indicate that the plant species contained inside the sea turtle stomachs originated from a location outside of Estero Banderitas, in an area where Cadmium and Zinc concentrations dominate the metal profiles in the environment. Surface water metal concentrations have been strongly correlated with upwelling events and natural components of regional biogeochemistry (Daesslé et al., 2000; Lares et al., 2002). Similar to the distribution of nutrients in the water column, metals such as Cadmium and Zinc are depleted in the surface and enriched in deeper water. Upwelling processes are an important mechanism that brings elevated concentrations of both nutrients and metals to the surface and thus available for marine floral accumulation. Therefore it is highly probable that the sea turtles collected within Magdalena Bay are utilizing foraging areas in an upwelling-rich coastal region outside of the Bay. Coastal lagoons of the Baja California Peninsula such as Magdalena Bay have been identified as priority areas for sea turtle conservation programs (Nichols et al., 2000). Long-term sea turtle monitoring studies have demonstrated high site fidelity to Estero Banderitas over time, and low emigration of sea turtles from Magdalena Bay to other coastal lagoons along the Baja California Peninsula (Grupo Tortuguero, unpublished data). Efforts to protect areas within Magdalena Bay have focused on the creation of a refuge in the mangrove channels of Estero Banderitas, in part, because of the perceived importance of this habitat for sea turtle foraging (Nichols and Arcas, 2001). However, data generated by our work suggest that sea turtles residing in Estero Banderitas are feeding in areas outside of the bay, most likely in coastal regions with high upwelling. These findings support those of López-Mendilaharsu et al. (2005) and indicate that green turtles utilize spatially distinct feeding habitats within coastal areas. Therefore, we recommend that sea turtle protected areas be designed with an appreciation of regional rather than local scales in order to protect broader foraging areas.

#### **4.4 Fibropapiloms and epibionts**

The prescence of fibropapiloms are variable from 1.4% up to 90% of the population (Herbs *et al.,* 1999, Quackenbush et al., 2001, Chaloupka et al., 2009). The observed low proportion of the green turtles in Bahía Magdalena (less than 1%) agree with a well preserved environment and less stress situation for the animals. In the case of the epibionts we found a continuously prescence of cirripedia and balanus but not a diverse fauna like in the Atlantic that even polychaetes has been reported (Lara Uc, 2011).
