**2. Methodology**

#### **2.1 Study area**

Terminos Lagoon is the largest lagoon-estuarine ecosystem in Mexico by area and volume. The water body and immediately surrounding shorelands are fully

incorporated into a National Flora and Fauna Reserve comprised of 705,016 ha of open water and associated wetlands and upland. Terminos Lagoon consists of about 200,108 ha of open water including associated lagoons and channels, with an average depth of 4 m, surrounded by about 259,000 ha of mangrove and cattail marsh. Of the surrounding 180,000 ha of land that is in some productive use, 90% is cattle ranching, 6% is agricultural, and 4% is urbanized, principally the City of Carmen. It is separated from the Gulf of Mexico by the Carmen Island, a 37 km long, 4 km wide barrier island with two mouths, of 3.2 and 3.8 km located to the east and west, respectively.

Terminos Lagoon was declared as a Federal Flora and Fauna Protection Zone in 1994 and is considered a "critical habitat" by the Mexican Environmental Agency [http://www.paot.org.mx/centro/ine-semarnat/anp/AN19.pdf] due to its importance as a refuge for marine species, mangrove forests, sea grass and, associated fluvial lagoon delta system. Anthropogenic pressure mainly due to urban settlements, the disposal of wastewater in the lagoon and industrial activity based on the drilling and exploration of hydrocarbons, all these activities have been identified as a continuous threat to the quality of the ecosystems within the Terminos Lagoon.

This work summarizes the results of several investigations carried out in Terminos Lagoon Natural Protected Area where the content of heavy metals in a variety of aquatic organisms was analyzed. The sampling periods and collect sites are shown below, as well as the aquatic organisms used for the determination of heavy metals.

In 2009, during two sampling campaigns (rainy and dry seasons), the oyster (*Crassostrea virginica*) was collected at the mouth of three of the rivers that flow into the Terminos Lagoon: the Palizada River, the Chumpan River and the Candelaria River. At each site, three sampling points were established and 100 organisms were obtained from each one.

In 2013–2014 three different types of organisms were analyzed: the oyster (*Crassostrea virginica*) collected in two sites, Estero Pargo and Mouth of Atasta; shrimp (*Litopenaeus setiferus*) obtained by trawling in depths of less than 5 fathoms in the Terminos Lagoon; and the crab (*Callinectes sapidus*) collected at the mouth of the Palizada River. All the organisms were donated by the fishermen's cooperatives. 60 organisms of commercial size were obtained of each species.

In 2014, samples of three species of macrophytes (*Cyperus ligularis L., Lemna minor* and *Typha domingensis*) were collected and analyzed in the "Arroyo La Caleta", which is a natural water channel parallel to the coast that crosses Carmen City, with a variable extension between both banks. The main contribution of water enters through the west mouth of the Terminos Lagoon and does not present an outlet. Other contributions of water come from land and urban drainage. The system is 7.5 km long. In the case of *T. domingensis* and *C. ligularis L*., the complete plants were cut, stored in plastic bags, and placed in refrigeration for later analysis in the laboratory. The samples of *L. minor* were collected in plastic bags in which water from the "Arroyo La Caleta" was left and, like the previous macrophytes, they were stored in refrigeration.

In 2017, the clam *Rangia cuneata* was collected at four sampling points in the Atasta Lagoon, which is a lagoon that empties into Terminos Lagoon. In total, eight composed samples were analyzed for this study. In the same lagoon (Atasta) but in 2018, during two sampling campaigns (rainy and dry seasons), catfish (*Ariopsis felis*) was obtained by fishing with cast nets eight. 30 composed samples were analyzed.

**Table 1** summarizes information about of the analyzed organisms, their sampling location and year of collection, while **Figure 1** shows the Terminos Lagoon Natural Protected Area and the sampling locations of the organisms analyzed.

*Heavy Metal Contamination in a Protected Natural Area from Southeastern Mexico… DOI: http://dx.doi.org/10.5772/intechopen.95591*


#### **Table 1.**

*Organisms analyzed, their sampling location and year of collection.*

#### **Figure 1.**

*Terminos Lagoon natural protected area and sampling locations of the organisms analyzed: 1) Palizada River; 2) Chumpan River; 3) Candelaria River; 4) Pargo estuary; 5) Atasta mouth; 6) arroyo La Caleta; 7) Atasta lagoon.*

#### **2.2 Sample processing and analysis**

The methods used for the processing of tissues and extraction of heavy metals reported in the various studies considered for the evaluation in this work, have few variations or modifications according to "Official Mexican Standard" (NOM-117-SSA1–1994,*Test method for the determination of cadmium, arsenic, lead, copper, iron, zinc and mercury in food*) for food analysis that generally consisted of an acidic digestion of the tissues with a repetitive addition of concentrated HNO3 and H2O2.

**For bivalves**: before extracting the tissues, they were purged during a period of 24 hours in a system with a controlled salinity of 20 psu. By so doing, the bivalves eliminated all the organic matter from their intestines that could have interfered with the results. Finally, they were shucked manually. Organisms were dried through the process of lyophilization for 24 hours and then were homogenized. Subsequently, an acid digestion was carried out, according to the official Mexican standards, as mentioned at the beginning of this section.

Bivalves samples were analyzed by Plasma Emission Spectroscopy (ICP), Perkin Elmer model 400 instrument was used, and standard solutions (J. Baker). For the evaluation of the analytical quality, the samples of oyster tissues were treated in duplicate and were analyzed in parallel with the standard certificates of "Standard reference materials oyster tissue" (SRM-1566b), with a recuperation percentage of between 84 and 94%.

**For crustaceans and fish**: composite samples were used for which the edible part was extracted from the organisms of each species, the tissues were homogenized with a food processor and a final sample of (20 0.001 g) was taken. The digestion was carried out by adding 10 mL of HNO3 to the tissues and placed on a heating grill at a controlled temperature, after the total destruction of organic matter, 2 mL of H2O2 was added to each sample in 30% solution, concentrating them up to a volume of 1 mL, finally, the concentrate was filtered through Whatman No. filter paper 32, measuring to a final volume of 20 mL for subsequent analysis. The tissue samples were analyzed in an atomic flame absorption equipment adapted with a Thermo-Scientific brand graphite furnace.

**For macrophytes**: samples were dried in a drying oven at a temperature of 65° C for 96 hours. The dried samples were dissected at the root, stem, and leaf. The digestion was carried out as mentioned for crustaceans and fish, following the methodology of the official Mexican standard NOM-117-SSA1–1994 (Test method for the determination of cadmium, arsenic, lead, tin, copper, iron, zinc and mercury in food). Macrophytes samples were analyzed by Plasma Emission Spectroscopy (ICP).
