**3. Results**

individual bag, and then the animals were sorted by hand. Twenty minutes were devoted to each quadrat. Humidity and temperature of air and soil were measured *in situ* at each site. At the laboratory, amphipod specimens were preserved in 70% ethanol. Then, they were identified, counted and sexed. The identification of these species was carried out under Leica MS 5

**Stations Wetland type Governorate GPS Sediment type** 105. Bnt Jedidi Dam Nabeul 36°25′09″N/010°27′26″E Sandy-clay-silt 106. Ermal wadi Dam Sousse 36°19′50″N/010°21′29″E Sandy loam 107. Jneyhiya Dam Siliana 36°12′25″N/009°44′20″E Sandy loam 108. Siliana Dam Siliana 36°07′57″N/009°21′14″E Loamy sand 109. Lakhmas Dam Siliana 35°59′55″N/009°28′15″E Sandy-clay-silt

110. El Gattar Dam Siliana 36°01′47″N/009°15′56″E Sandy 111. Cheikh El Maïz Dam Siliana 36°01′15″N/009°15′8″E Sandy 112. El Kharroub wadi Dam Siliana 36°01′43″N/009°15′8″E Sandy 113. Mchaker wadi Dam Siliana 35°58′57″N/009°10′20″E Sandy loam 114. Ermal Dam Siliana 35°49′21″N/009°07′33″E Loamy sand 115. Mallègue Dam Kef 36°18′48″N/008°42′21″E Sandy loam 116. Kasseb Dam Béja 36°45′36″N/009°0′5″E Sandy 117. Ermal Dam Siliana 36°23′54″N/010°04′52″E Loamy sand

The particle size, organic matter and heavy metals of soil samples taken from 117 stations were analysed. Grain size distribution of these composite samples was analysed using differ-

A subsample was brought to the inductively coupled plasma-mass spectrometry (ICP-MS) laboratory at University of Kiel and sieved to obtain the <250-μm grain size fraction which was then dried and milled [39]. Heavy metals were extracted from a 250-mg sample of powder with 10 mL 7 N nitric acid on a hot plate at 80°C (2.5 h). The solution was made up to 20 mL, centrifuged at 3500 rpm for 15 min, and the supernatant transferred to a 20-mL sample vial. The metals vanadium (V), chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), cadmium (Cd), tin (Sn), thallium (Tl), lead (Pb), lithium (Li), rubidium (Rb) and strontium (Sr) were analysed by inductively coupled plasma-mass spectrometry (ICP-MS). Average analytical reproducibility was estimated from replicate analyses of some samples and was found to be better than 2% Relative Standard Deviation (RSD) (1 sigma relative standard deviation) for all elements. The accuracy of analytical results was monitored by analysing certified reference materials (CRM): GSMS-2 (marine sediment; Chinese Academy of Geological Sciences, PR China) and Reference material, coastal sediment (PACS-1) (coastal sediment;

binocular microscope, using the key of Ruffo [38].

**Table 1.** Localization of the studied stations.

ent sieves in descending order (from 2 to 25 μm).

**2.3. Soil analysis**

106 Selected Studies in Biodiversity

#### **3.1. Temperature, humidity, organic matter and grain size**

Temperature (°C) and humidity (%) were measured *in situ* in different wetland types. The mean values for these two parameters varied between 22.453 ± 2.797°C in dams, 27.387 ± 5.289°C in sebkhas, 51.243 ± 18.627% in sebkhas and 65.50 ± 12.388% in lagoons (**Figure 1A** and **B**).

The percentage of organic matter differs between and within wetland types (**Figure 1C**). The highest values were observed in the banks of Bizerte lagoon (9.46%), Majin Chitane (12.23%), Halk Menzel (16.13%), Bargou wadi (20.66%), Ouled Ali (17.62%) and Kasseb (12.64%) (**Figure 1C**).

An heterogeneity in grain size nature was observed between stations ranging from sandy substrates, loamy sand, sandy loam, sandy silt, sandy-clay, silty clay, clay-loam, sandy-clay-silt, sandy-clay-loam, sandy-silt-loam, fine silt to clay substrates (**Table 1**).

#### **3.2. Heavy metals**

In the lagoon, the highest concentrations for the majority of heavy metals, vanadium, nickel, zinc, arsenic, cadmium, thallium and lead were recorded in the northern lagoon of Tunis. The bank of Bizerte lagoon was characterized by the highest concentrations of chromium (26.393 ppm) and manganese (281.748 ppm). While the highest copper content (39.098 ppm) was observed in El Bcherliya. The Korba lagoon revealed the highest concentration in cobalt and rubidium with 8.311 and 15.814 ppm, respectively. Bhiret El Biben was characterized by the highest concentration of lithium (29.087 ppm), strontium (2101.549 ppm) and tin (7.340 ppm). In addition, the lowest concentration for all the heavy metals studied was recorded in the bank of Sidi Ali Mekki lagoon. The different metals analysed in these lagoons do not exceed the maximum tolerate values [42] except lead that exceeds 100 ppm in the northern lagoon of Tunis (133.556 ppm).

In wadis, the highest concentrations of vanadium (44.619 ppm), chromium (40.413 ppm), zinc (147.822 ppm) and lead (303.910 ppm) were recorded in Lahmam wadi. The highest concentration of cobalt (19,723 ppm), nickel (29,283 ppm), rubidium (27,016 ppm), and thallium (0.183 ppm) were found in Zit wadi. Whereas for lithium, manganese, copper, arsenic, strontium, cadmium and tin, the highest concentrations were, respectively, recorded in Soufi wadi (26.527 ppm), Mazbla wadi (644.069 ppm), Joumin wadi (19.448 ppm), Lanj wadi (9.380 ppm), Khniss wadi (1410.100 ppm), Bargou wadi (1.412 ppm) and El Korsi (1.019 ppm). According to Henin [42], the different prospected wadis were not polluted except for Joumin, Lassoued and Bargou wadis, which were considered as polluted with cadmium whose percentage exceeds the maximum tolerated value (0.7 ppm) as well as Lahmam wadi in which a lead concentra-

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Sixteen hill lakes and 17 dams belonging to different bioclimatic stages were prospected. In

Concerning heavy metals, in hill lakes, the highest concentrations of vanadium (46.795 ppm), cobalt (14.661 ppm), nickel (30.362 ppm), copper (16.611 ppm) and lead (23.047 ppm) were observed in Sidi Daoued hill lake. The highest concentration of zinc (88.804 ppm), arsenic (4.590 ppm), cadmium (3.031 ppm), thallium (0.176 ppm) was found in Zrab wadi hill lake. The khlifa wadi hill lake was characterized by the important content of lithium (24.891 ppm) and strontium (930.812 ppm) and it was rather Ouled Ali hill lake that presented the highest concentration of rubidium (16.526 ppm) and tin (0.629 ppm). Hill lakes of Khalsi and Ksayir Hamdoun were characterized by the highest concentration of chromium (43.394 ppm) and manganese (530.039 ppm). This analysis of heavy metals revealed that only Ain Ben Ali, Zad and Khalsi hills lakes were contaminated by the cadmium. Concerning dams, our results showed that Sidi Barrak dam was characterized by the highest concentration of majority of heavy metals, namely manganese (1060.291 ppm), cobalt (14.085 ppm), copper (22.840 ppm), zinc (151.90 ppm), arsenic (6.246 ppm), thallium (0.544 ppm) and lead (166.067 ppm); while Kasseb dam showed the highest concentration of vanadium (37.377 ppm), chromium (41.476 ppm), nickel (32.579 ppm) and rubidium (18.714 ppm). The highest concentration in lithium (15.013 ppm), cadmium, (5.426 ppm) and tin (0.137 ppm) was found in Gattar dam;

Eight species of amphipoda Talitridae, namely *Orchestia montagui* Audouin, 1826, *Orchestia mediterranea* Costa, 1853, *Orchestia gammarellus* (Pallas, 1766), *Orchestia stephensen*i Cecchini, 1928, *Orchestia cavimana* Heller, 1865, *Platorchestia platensis* (Kroyer, 1845), *Deshayesorchestia deshayesii* (Audouin, 1826) and *Talitrus saltator* (Montagu, 1808) were collected in different

Species richness (S) varied between stations of the same as well as the different types of

In lagoons, species richness varied between one species in El Bcherliya and eight species in the bank of Bizerte lagoon near Menzel Jmil. The differences observed between lagoons were

the banks of these closed and artificial ecosystems, no amphipod was found.

while that of strontium (731.645 ppm) was observed in Jneyhiya dam.

tion exceeded 100 ppm.

**3.3. Species richness**

wetlands.

wetlands.

**Figure 1.** Environmental factors (A: Temperature (°C), B: Humidity (%), C: Organic matter (%)) measured at each wetland types.

In the second type of wetlands, lakes, the highest contents of manganese (1806 ppm), zinc (131.955 ppm), arsenic (4.211 ppm), cadmium (0.678 ppm), thallium (0.170 ppm) and lead (47.060) were observed in Ichkeul lake. Furthermore, Bouhnach lake was characterized by the important contents of lithium (7.735 ppm), vanadium (23.893 ppm), chromium (15.711 ppm), cobalt (7.497 ppm), nickel (16.567 ppm), strontium (643.783 ppm) and tin (0.140 ppm) and it was rather Majin Chitan lake that presented the highest concentration of copper (15.577 ppm) and rubidium (11.632 ppm). According to Henin [42], these stations were not contaminated since the content of heavy metals does not exceed the maximum tolerated values.

Concerning sebkhas, the highest concentrations in vanadium, rubidium and thallium were recorded in Sebkha Halk Menzel (36.357, 19.239 and 0.140 ppm, respectively). For chromium, manganese and tin, the highest concentrations were recorded in sebkhas of Sidi Khlifa (554.628 ppm), Moknine (387.880 ppm) and Sousse (2.793 ppm), respectively. The highest concentration of cobalt, nickel, strontium and cadmium was, respectively, observed in the Halk Menzel (10.460 ppm), Raoued (23.106 ppm), Sidi El Hani (3305.249 ppm) and Golla (0.422 ppm). Concerning copper, arsenic and lithium, the highest concentration was, respectively, recorded in sebkha Ariana (23.238 ppm), sebkha Golla (14.507 ppm) and sebkha Kairouan (41.861 ppm), and it was rather the sebkha Monastir that showed the highest concentration of zinc (86.453 ppm) and lead (48.741 ppm). According to these results and taking into account the tolerance thresholds, no sebkha was considered polluted with the exception of sebkha Sidi Khlifa that was considered as polluted by chromium which exceeds the maximum tolerated value (150 ppm) [42].

In wadis, the highest concentrations of vanadium (44.619 ppm), chromium (40.413 ppm), zinc (147.822 ppm) and lead (303.910 ppm) were recorded in Lahmam wadi. The highest concentration of cobalt (19,723 ppm), nickel (29,283 ppm), rubidium (27,016 ppm), and thallium (0.183 ppm) were found in Zit wadi. Whereas for lithium, manganese, copper, arsenic, strontium, cadmium and tin, the highest concentrations were, respectively, recorded in Soufi wadi (26.527 ppm), Mazbla wadi (644.069 ppm), Joumin wadi (19.448 ppm), Lanj wadi (9.380 ppm), Khniss wadi (1410.100 ppm), Bargou wadi (1.412 ppm) and El Korsi (1.019 ppm). According to Henin [42], the different prospected wadis were not polluted except for Joumin, Lassoued and Bargou wadis, which were considered as polluted with cadmium whose percentage exceeds the maximum tolerated value (0.7 ppm) as well as Lahmam wadi in which a lead concentration exceeded 100 ppm.

Sixteen hill lakes and 17 dams belonging to different bioclimatic stages were prospected. In the banks of these closed and artificial ecosystems, no amphipod was found.

Concerning heavy metals, in hill lakes, the highest concentrations of vanadium (46.795 ppm), cobalt (14.661 ppm), nickel (30.362 ppm), copper (16.611 ppm) and lead (23.047 ppm) were observed in Sidi Daoued hill lake. The highest concentration of zinc (88.804 ppm), arsenic (4.590 ppm), cadmium (3.031 ppm), thallium (0.176 ppm) was found in Zrab wadi hill lake. The khlifa wadi hill lake was characterized by the important content of lithium (24.891 ppm) and strontium (930.812 ppm) and it was rather Ouled Ali hill lake that presented the highest concentration of rubidium (16.526 ppm) and tin (0.629 ppm). Hill lakes of Khalsi and Ksayir Hamdoun were characterized by the highest concentration of chromium (43.394 ppm) and manganese (530.039 ppm). This analysis of heavy metals revealed that only Ain Ben Ali, Zad and Khalsi hills lakes were contaminated by the cadmium. Concerning dams, our results showed that Sidi Barrak dam was characterized by the highest concentration of majority of heavy metals, namely manganese (1060.291 ppm), cobalt (14.085 ppm), copper (22.840 ppm), zinc (151.90 ppm), arsenic (6.246 ppm), thallium (0.544 ppm) and lead (166.067 ppm); while Kasseb dam showed the highest concentration of vanadium (37.377 ppm), chromium (41.476 ppm), nickel (32.579 ppm) and rubidium (18.714 ppm). The highest concentration in lithium (15.013 ppm), cadmium, (5.426 ppm) and tin (0.137 ppm) was found in Gattar dam; while that of strontium (731.645 ppm) was observed in Jneyhiya dam.

#### **3.3. Species richness**

In the second type of wetlands, lakes, the highest contents of manganese (1806 ppm), zinc (131.955 ppm), arsenic (4.211 ppm), cadmium (0.678 ppm), thallium (0.170 ppm) and lead (47.060) were observed in Ichkeul lake. Furthermore, Bouhnach lake was characterized by the important contents of lithium (7.735 ppm), vanadium (23.893 ppm), chromium (15.711 ppm), cobalt (7.497 ppm), nickel (16.567 ppm), strontium (643.783 ppm) and tin (0.140 ppm) and it was rather Majin Chitan lake that presented the highest concentration of copper (15.577 ppm) and rubidium (11.632 ppm). According to Henin [42], these stations were not contaminated since the content of heavy metals does not exceed the maximum tolerated values.

**Figure 1.** Environmental factors (A: Temperature (°C), B: Humidity (%), C: Organic matter (%)) measured at each

**C** 

**A**

**0**

**50**

**Humidity (%)**

**100**

**B** 

**Lagoon Lake Sebkhas Oueds Hill Lakes Dams**

**Lagoon Lake Sebkhas Oueds Hill lakes dam**

**Lagoon Lake Sebkhas Oueds Hill Lakes Dams**

Concerning sebkhas, the highest concentrations in vanadium, rubidium and thallium were recorded in Sebkha Halk Menzel (36.357, 19.239 and 0.140 ppm, respectively). For chromium, manganese and tin, the highest concentrations were recorded in sebkhas of Sidi Khlifa (554.628 ppm), Moknine (387.880 ppm) and Sousse (2.793 ppm), respectively. The highest concentration of cobalt, nickel, strontium and cadmium was, respectively, observed in the Halk Menzel (10.460 ppm), Raoued (23.106 ppm), Sidi El Hani (3305.249 ppm) and Golla (0.422 ppm). Concerning copper, arsenic and lithium, the highest concentration was, respectively, recorded in sebkha Ariana (23.238 ppm), sebkha Golla (14.507 ppm) and sebkha Kairouan (41.861 ppm), and it was rather the sebkha Monastir that showed the highest concentration of zinc (86.453 ppm) and lead (48.741 ppm). According to these results and taking into account the tolerance thresholds, no sebkha was considered polluted with the exception of sebkha Sidi Khlifa that was considered as polluted by chromium which exceeds the maximum tolerated

value (150 ppm) [42].

**A** 

**45**

108 Selected Studies in Biodiversity

**30**

**0**

wetland types.

**10**

**Organic matter (%)**

**20**

**30**

**0**

**15**

**Temperature (°C)**

Eight species of amphipoda Talitridae, namely *Orchestia montagui* Audouin, 1826, *Orchestia mediterranea* Costa, 1853, *Orchestia gammarellus* (Pallas, 1766), *Orchestia stephensen*i Cecchini, 1928, *Orchestia cavimana* Heller, 1865, *Platorchestia platensis* (Kroyer, 1845), *Deshayesorchestia deshayesii* (Audouin, 1826) and *Talitrus saltator* (Montagu, 1808) were collected in different wetlands.

Species richness (S) varied between stations of the same as well as the different types of wetlands.

In lagoons, species richness varied between one species in El Bcherliya and eight species in the bank of Bizerte lagoon near Menzel Jmil. The differences observed between lagoons were highly significant (*F* = 5.317; *df* = 9; *p* < 0.0001). In the bank of lakes, amphipods were collected only in Ichkeul lake (*S* = 5).

*Orchestia gammarellus* in sebkha Ouafi (0.5 ind.m−2); this density became more pronounced in sebkha Moknine and Gargour with, respectively, 32.5 and 36 ind.m−2. In these two last sta-

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Concerning wadis, 558 individuals were found. The most important global mean density was observed in the bank of El Korsi. *Orchestia mediterranea*, species living in allopatry in Khniss, Laakarit and El Fared wadis showed a density, respectively, equal to 49.5, 47.5 and 0.5 ind.m−2; whereas, where it was in sympatry, its density was equal to 16 (El Korsi) and 7.5 ind.m−2 (Tinja). Furthermore, *Talitrus saltator*, which was the only amphipod collected in Lebna wadi estuary (67 ind.m−2), showed a relatively lower density in El Korsi (13 ind.m−2)

According to the Simpson index (Is), the most important diversity was observed in the Bizerte lagoon where we noted the highest value which tends towards the specific richness (6.059)

In Ghar El Melh old harbour, we obtained the lowest diversity compared to that observed in Boughaz. This result could be explained by the fact that this index does not consider rare

The Shannon-Weaver (H') index ranged from 1.287 in the bank of Sidi Ali Mekki lagoon to 2.771 in the bank of Bizerte lagoon where the diversity was relatively significant. This index, which takes into account the rare species, was often accompanied by the equitability index, which was more or less insensitive to specific richness. It ranged from 0.812 (Sidi Ali Mekki)

In the banks of different sebkhas, we did not observed a great diversity, so the analysis of

In wadis, results showed that the Simpson index varies between 1 in the Lebna wadi estuary, Khniss, Laakarit and El Fared wadis and 5.78 in El Korsi station where we found the highest species richness (*S* = 6). The Shannon-Weaver index confirmed the previous index showing that the most important diversity was observed in El Korsi station (*H*′ = 2.56). Moreover, the study of the equitability index showed that the community was more balanced in this station

**3.6. Amphipod distribution according to environmental factors and wetland types**

To better understand the species distribution in the different wetland types and to elucidate the parameters involved in their distribution, a canonical correspondence analysis was carried out (**Figure 2**). The first three axes, F1, F2 and F3 extract, respectively, 71.43, 20.23 and 5.55% of the variance. The two species, *Orchestia mediterranea* and *Orchestia gammarellus* that dominated the majority of lagoons and sebkhas were positively correlated with the strontium content and negatively with concentrations of vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, rubidium, cadmium, thallium and lead. However,

tions, *Orchestia mediterranea* had a density of 28.5 and 52 ind.m−2, respectively.

and Tinja (6 ind.m−2).

species into account.

diversity was not carried out.

and the community was more balanced in Boughaz.

to 0.996 (Boughaz) where the community was more balanced.

(*J*′ = 0.99) where species were equitably distributed.

**3.5. Diversity**

Concerning sebkhas, among 30 sebkhas studied, talitrids were found in only four sebkhas namely: El Ouafi, Maâmoura, Moknine and Gargour. Species richness was equal to one species in the bank of sebkhas El Ouafi and Maâmoura namely *Orchestia gammarellus* and *Talitrus saltator*, respectively. In two other sebkhas, *Orchestia gammarellus* and *Orchestia mediterranea* were collected.

For wadis, individuals were collected only in six wadis among the 41 stations prospected. Species richness varies from one (El Fared wadi, Laakarit wadi, Khniss wadi and Lebna wadi) to six species (El Korsi). In hill lakes and dams, no species were collected.

#### **3.4. Relative abundance and density**

A total of 340 specimens of amphipoda Talitridae were collected in lagoons. The bank of Bizerte lagoon revealed statistically the most important relative abundance of amphipod community (36.04%) (Anova test: *F* = 5.330, *df* = 9, *p* < 0.0001). Moreover, in this station, *Orchestia mediterranea* was the most abundant species (25.7%). However, in the banks of El Bcherliya, the Ghar El Melh old harbour, Tunis north and south lagoons, it was rather *Orchestia gammarellus* that dominated. These two species have the same relative abundance (46.7%) in bank of Sidi Ali Mekki lagoon. In Bhiret El Biben lagoon, *Orchestia montagui* was the most abundant species (28.3%). The Anova test revealed that differences between the different lagoons were highly significant (*F* = 7.922; *df* = 7; *p* < 0.0001). The mean community density varied between 0.5 ind.m−2 in the bank of El Bcherliya and 241.5 ind.m−2 in that of Bizerte lagoon. Furthermore, our results showed that *Orchestia mediterranea* presented the most important density in the bank of Bizerte lagoon (62 ind.m−2). Whereas, in the bank of El Bcherliya, Ghar El Melh old harbour, Tunis north and south lagoons, it was *Orchestia gammarellus* that exhibited the largest density with, respectively, 0.5, 19, 34 and 34.5 ind.m−2. These two species were recorded with the same mean density in the bank of Sidi Ali Mekki lagoon (3.5 ind.m−2). In lakes, 170 individuals were collected where *Orchestia mediterranea* presented the highest abundance (26.5%) and density (22.5 ind.m−2).

In sebkhas, 352 specimens of amphipods were collected. Sebkha Gargour revealed the highest relative abundance (50%) followed by sebkha Moknine which abundance was equal to 34.66%. However, in the bank of the two other sebkhas, the abundance was relatively low in Mâamoura with 15.06% and very low in sebkha El Ouafi with 0.28%. The Anova test revealed a highly significant difference in relative abundance between these sebkhas (*F* = 8.288, *df* = 29, *p* < 0.0001). The relative abundance of *Orchestia gammarellus* and *Talitrus saltator* were maximal (100%), respectively, in sebkha El Ouafi and Mâamoura since each sebkha harbours only one species. In Moknine, abundance was greater for *Orchestia gammarellus* (53.3%) than in *Orchestia mediterranea* (46.7%) and inversely in the sebkha Gargour where the highest abundance was recorded for *Orchestia mediterranea* with 59.1%. In addition, no significant difference in relative abundance between species was found (Anova test: *F* = 1.461, *df* = 2, *p* = 0.233). The global mean density oscillated between 0.5 ind.m−2 in sebkha El Ouafi and 88 ind.m−2 in sebkha Gargour. The study of the mean density per species showed a very low density of *Orchestia gammarellus* in sebkha Ouafi (0.5 ind.m−2); this density became more pronounced in sebkha Moknine and Gargour with, respectively, 32.5 and 36 ind.m−2. In these two last stations, *Orchestia mediterranea* had a density of 28.5 and 52 ind.m−2, respectively.

Concerning wadis, 558 individuals were found. The most important global mean density was observed in the bank of El Korsi. *Orchestia mediterranea*, species living in allopatry in Khniss, Laakarit and El Fared wadis showed a density, respectively, equal to 49.5, 47.5 and 0.5 ind.m−2; whereas, where it was in sympatry, its density was equal to 16 (El Korsi) and 7.5 ind.m−2 (Tinja). Furthermore, *Talitrus saltator*, which was the only amphipod collected in Lebna wadi estuary (67 ind.m−2), showed a relatively lower density in El Korsi (13 ind.m−2) and Tinja (6 ind.m−2).
