**2. Materials and methods**

#### **2.1 Bizerte and Ghar el Meleh lagoons**

In Tunisia, lagoon milieus cover a total space of 1100 km<sup>2</sup> and are distributed over the entire Mediterranean coastline [18]. These lagoons are of high ecological and economic importance but are experiencing rising anthropogenic pressure, being exposed to various types of environmental degradation resulting from agricultural, manufacturing, and touristic activities [19]. Among its many lagoons, two are the best known and are located in northern Tunisia (**Figure 1**):

The Ghar El Melh is a shallow coastal lagoon, which is isolated by a narrowvegetated sand strip from the Mediterranean Sea. It is located in the southern Mediterranean Sea on the northeastern coast of Tunisia (37°06–37°10 N and 10°08′–10°15 E) and is influenced by regional water circulation patterns [20]. It covers a surface area of about 3000 ha, including two small sub-lagoons, El Ouafi Lagoon to which it is permanently connected, and Sidi Ali El Meki Lagoon from which it is separated by embankments. The Ghar El Melh is linked to the Mediterranean Sea via a permanent channel. The lagoon displays different levels of salinity with the highest registered in the lagoon still areas. Freshwater inflows are cyclical, restricted in summer, and tall in winter, occasionally with the existence of exceptional floods generating a link between the lagoon and the Mejerda River. The benthic vegetation is dominated by *R. cirrhosa* that extensively covers (80–100%) the lagoon bottom in summer [21]. The benthic fauna has important biodiversity, such as the presence of molluscs, crustaceans, and fishes (26 species) [22].

*Marine Free-Living Nematodes as Tools for Environmental Pollution Assessment: A Special Focus… DOI: http://dx.doi.org/10.5772/intechopen.104721*

**Figure 1.** *Location of the Bizerte and Ghar ElMelh lagoons (Northern Tunisia).*

The Bizerte lagoon is situated in the north of Tunisia in the latitudinal and longitudinal extensions of 37°8′–37°16′N and 9°46′ and 9°56′E. It has a 150 km2 complete surface, an 8 m mean depth, and a 380 km2 catchment area. It is connected with the Mediterranean Sea through a channel 12 m deep and communicates in the south with the Lake of Ichkeul (140 km2 ) by the Tinja river. This ecosystem has great biodiversity (30 teleosts and eight elasmobranch fish species have been described) and its socio-commercial revenues in the animals' sale (mussel and oyster farming) [23]. Unfortunately, it receives freshwater inputs via eight rivers [24], urban (bounded by six cities) and industrial activity discharges as well as the products of agricultural activity. In addition, this lagoon is exposed to high biotic and abiotic variations [25].

#### **2.2 Experimental nematodes study**

Free-living marine nematodes are known to be well suited as bioindicators for monitoring studies in marine environments and bioassays (**Figure 2**) [26]. These worms are ubiquitous and occupy an important link in the food chain, feeding on microalgae and bacteria and, in turn, being preyed upon by macrobenthic predators, such as polychaetes, crabs, and fishes [27]. They are expected to be highly susceptible to sediment-associated pollutants because they live and feed on the sediment [8].

Technically, sediments containing meiofauna were collected from a coastal site in the Tunisian lagoon. Before being enriched with ECs, the sediment was arranged using the method of Austen et al. [28]. Then, large substrate particles (>63 mm) were removed by wet sieving, and appropriate concentrations of ECs were supplemented with sediment (100 g dry weight; dw). The microcosms used in this experiment were based on the original design of Austen et al. [28] and consisted of 570 mL glass bottles.

**Figure 2.**

*General organization of nematodes species"*Odontophora villoti" *(Axonolaimidae); A: male; B: gravid female.*

Treated microcosms were occupied by 300 g of homogenized sediments (200 g of natural sediment + 100 g of contaminated sediment) topped up with filtered natural water (0.1 mm). The control microcosm consisted of not treated and azoic sediments. Treatments were set up, each with minimum of three replications (control [(C)] and "treated by ECs" microcosm [29]. During the 30 days of the experiment, each microcosm was constantly aerated with an oxygen pump (**Figure 3**) [13].

At the end of the experiment, the sediment samples were fixed in a 4% buffered formalin solution. Nematodes were extracted by centrifuging with Ludox-TM three times and stained with Rose Bengal (0.2 g l−1) for one day [30]. The nematofauna taxa were counted on a stereomicroscope (50×, Wild Heerbrugg M5A Model), and a *Marine Free-Living Nematodes as Tools for Environmental Pollution Assessment: A Special Focus… DOI: http://dx.doi.org/10.5772/intechopen.104721*

#### **Figure 3.**

*The experimental design. [C]: control; [***EC***d1,* **EC***d2,* **EC***d3,* **EC***d4,* **EC***d5]: increasing doses of emerging contaminant.*

maximum of 100 individuals/replicates were randomly taken. Animals were slowly evaporated in anhydrous glycerol, mounted on slides under an oil immersion objective (100×). The Platt and [31–33] pictorial guides and NeMys database [34] were used to species identify, respectively.

#### **2.3 Biological parameters analysis**

Nematodes are the most diverse and numerically dominant metazoans in aquatic ecosystems, and, because of their rare ability to survive in extremely polluted conditions, they are usually the only persistent taxon in heavily polluted/disturbed habitats [35]. To assess the effect of chemical pollutants on the benthic fauna, the research work has focused on studying ecological indices of nematodes, such as the spatial or temporal diversity of a taxonomic group (Shannon–Wiener diversity (H')), the distribution of the relative species abundances (Pielou's evenness (J')), the species number present (Margalef's species richness (d)), Maturity Index [36] (MI; based on the ecological characteristics and reproductive strategies of nematodes), and Index of Trophic Diversity [37] (ITD; expressed in an index calculated on the basis of the percentage of each feeding type) were studied.

In addition, the functional and morphological attributes of each species (i.e., trophic diet, tail shape, amphid shape, and life history) were considered. Trophic diet was categorized based on the characteristics of the buccal cavity [38], as epigrowth feeders (2A), selective deposit feeders (1A), non-selective deposit feeders (1B), and omnivores/ predators (2B). The tail shape was illustrated into four types: conical (co), clavate/conicocylindrical (cla), short/round (s/r), and elongated/filiform (e/f) [39]. The Amphid shape was distinguished into eight categories based on the shape of the amphidial fovea, of which four categories were used in our study—circular (Cr), spiral (Sp), pocket (Pk), and indistinct (Id) [8]. The life strategy (c–p scale) was estimated on a scale of c–p=1 (good colonizers: short life cycle, great reproduction rates, resistant to various types of stress) to c–p=5 (good persisters: lengthy life cycles, limited offspring, sensitive to stress), analogous to K/r-strategists, following [36, 40, 41]. The adult length was assigned to four groups (<1 mm, 1–2 mm, 2–4 mm, and >4 mm) [10].

Supplementary studies were conducted for the analysis of bacterial abundance. Density (per gram) was calculated for each sediment sample. Aliquots of 100 μL

were successively diluted in PBS and then displaced on duplicate bacterial agar plates (Difco), which were later incubated at 37°C (overnight). Using the plaque method, 30 to 300 colonies were counted and the amounts were expressed as colony-forming units per gram of sediment (CFU g−1) [14].
