Seasonal antioxidant responses in the sea urchin

Marine Pollution Bulletin 122 (2017) 392–402

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Seasonal antioxidant responses in the sea urchin Paracentrotus lividus (Lamarck 1816) used as a bioindicator of the environmental contamination in the South-East Mediterranean

MARK

Sandra Amria, Mohamed-Faouzi Samarb, Fériel Sellemc, Kheireddine Oualid,⁎ a Laboratory of Environmental Biosurveillance, Department of Biology, Faculty of Natural Sciences and Life and Earth Sciences and the Universe, University 08 Mai 1945, Guelma, Algeria b Department of Agronomy, Faculty of Natural Sciences and Life, University of Chadli Benjedid El Tarf, Algeria c Laboratoire resources marines vivantes, Institut National des Sciences et Technologies de la Mer Salammbo, Tunisia d Laboratory of Environmental Biosurveillance, Department of Biology, Faculty of Sciences, Badji Mokhtar University, BP 12, El hadjar, Annaba 23000, Algeria

A R T I C L E I N F O

A B S T R A C T

Keywords: Sea urchin Oxidative stress Biomarkers Mediterranean Sea Pesticides Gulf of Annaba Reproduction

In this study, sea urchin Paracentrotus lividus were sampled seasonally at three stations during 2012 in the coastal areas of the Gulf of Annaba (southeast Mediterranean). For all sea urchins, the gonad index was calculated to determine sea urchin reproductive status. Moreover, a set of biochemical parameters, including biomarkers and oxidative stress parameters, was measured in gonads. The pesticides and physiochemical parameters were measured and dosed in sea water. The results obtained highlighted that the levels of pesticide were generally low and below those commonly applied by environmental quality standards (EQS), indicating that no alarm state is currently present in the Gulf of Annaba. In addition to pollution, seasonal change is an important factor influencing biomarker activity, and the significant increases in biomarker levels in spring are a major observed trend. This activity may also be related to reproductive status. Seasonal variability was confirmed by the significant results of the Kruskal-Wallis test and by the high degree of divergence between seasons in PCA, with a total of 83.83% of variance explained. These results indicate that environmental factors that vary seasonally may affect the antioxidant status of the sea urchin Paracentrotus lividus.

1. Introduction Pollution of the marine environment is a global concern due to the devastating effects of contaminants, which are reaching increasingly alarming levels. According to the European Marine Strategy Framework Directive (2008/56/EC), a marine habitat with good environmental status has contaminant concentrations that do not give rise to pollutant effects (European Commission, 2008a). The presence of pesticides in different coastal and marine habitats around the world has already been detected on the coast of Hong Kong in China (Xu et al., 2015b), the Antarctic coast of New Zealand (Emnet et al., 2015), the bay of Vilaine in France (Caquet et al., 2013), the Caribbean and Pacific coasts of the United States (Menzies et al., 2013), the northern Adriatic sea of Italy (Loos et al., 2013), the Baltic sea of Germany (Nödler et al., 2013), the Mar Menor lagoon in Spain (Moreno-González et al., 2013), the Thermaikos Gulf in the Northern Aegean sea of Greece (Arditsoglou and

Voutsa, 2012), the Mi-Black sea Coast in Turkey (Binnur Kurt and Boke Ozkoc, 2004) and even at polar latitudes (Chernyak et al., 1996; Hoekstra et al., 2002; Sobek and Gustafsson, 2004). The presence of pesticides can pose a serious problem and their accumulation, bioamplification and transformation in aquatic ecosystems is a real risk for human health, fauna and the environment (Navarro et al., 2000). The works of Navarro et al. (2004); Maund et al. (1997); Sundaram (1997); Hadfield et al. (1993); Ramesh et al. (1990); Mhlanga and Madziva (1990); Rico et al. (1989); Najdek and Bazulic (1988); Rivera et al. (1986) have revealed ecological damage caused in distinct aquatic ecosystems following the generalized use of agricultural pesticides. The exposure of aquatic organisms to pesticides can lead to the production of reactive oxygen species (ROS) (Üner et al., 2006; Ahmad et al., 2000), causing an imbalance between the production of ROS and endogenous antioxidant activity (Kamat et al., 2000). The physiological response of marine organisms is strongly dependent on fluctuations of

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Corresponding author. E-mail addresses: [email protected] (S. Amri), [email protected] (M.-F. Samar), [email protected] (F. Sellem), [email protected] (K. Ouali). http://dx.doi.org/10.1016/j.marpolbul.2017.06.079 Received 14 November 2016; Received in revised form 18 June 2017; Accepted 27 June 2017 Available online 11 July 2017 0025-326X/ © 2017 Elsevier Ltd. All rights reserved.


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(February), spring (April), summer (July) and autumn (October). Three sampling stations were selected in the Gulf of Annaba (Algeria) to implement an integrated monitoring strategy. Fig. 1. The Gulf is a large bay located on the eastern Algerian coast, 600 km from Algiers and 100 km from the Tunisian border, open to the Mediterranean Sea to the north, and extending 40 km between Cap Rosa in the east and Cap Garde in the west (Belabed et al., 2013). The characteristics of the stations selected are shown in Table 1.

biotic and abiotic factors such as salinity, oxygen concentration, and temperature and food availability, resulting in difficulties in interpreting the biological effects exerted by xenobiotics (Camus et al., 2004; Manduzio et al., 2004). Changes in biomarker levels may simply be a natural part of the annual physiological cycle of the species and quite unrelated to changes in exposure to chemical pollution (Sheehan and Power, 1999). The Gulf of Annaba is constantly threatened due to its proximity to port activities, runoff, urban and industrial discharge and the most active agricultural regions of eastern Algeria. The benthic fauna and flora living in close contact are appropriate representative samples of great importance for explaining the link between the health of aquatic organisms and contamination levels. Until now, the pollutants detected most often have been heavy metals and nutrient salts. The main objective of this study was to investigate whether seasonal variation may influence the biochemical parameters of the sea urchin. An integrated biological approach was chosen that implements a combination of physical, chemical and biological measures in the framework of a relatively comprehensive study using the marine invertebrate belonging to the phylum Echinodomata. The sea urchin Paracentrotus lividus is an edible echinoid found in great abundance on the Mediterranean coast (Tejada et al., 2013). Its gonads make it a highly appreciated sea food in various countries including France, Spain, Italy (Fernández-Boán et al., 2013) and Japan (Powell et al., 2014). Due to its value as a luxury food (Cook and Kelly, 2007), it has also shown itself to be an excellent bioindicator of pollution in the marine environment due to its sedentary habits and known sensitivity to pollutants (Soualili et al., 2008). It has been used in several studies as a bioindicator of local pollution (Angioni et al., 2014; Bayed et al., 2005; Demnati et al., 2002; Coteur et al., 2003). In recent years it has acquired importance as a model organism in marine ecosystem biomonitoring programs (Fabbrocini et al., 2010; Bellas and Paredes, 2001).

2.2. Environmental characterization During sampling, temperature, pH, salinity, turbidity and dissolved oxygen were measured in situ with field multi-parameters. Water samples were collected in plastic containers that were sealed and transferred to the laboratory in ice packs to determine the following chemical parameters: nitrate (NO3−), nitrite (NO2−), ammonia nitrogen (NH4+) and orthophosphate (PO43 −) using the manual colorimetric methods of Aminot and Kérouel (2004b). For pesticide determination, freshwater samples were collected in amber glass bottles and kept at low temperature in the laboratory, where the water was filtered and stored. Five pesticides were analyzed with ELISA kits (Atrazine, Diuron, and glyphosate from Abraxis; Nonylphenol from Tokiwa Chemical Industries). Each kit consisted of eight separate 12well immunoreader strips precoated with specific antibodies. The assay was performed following the manufacturer's guidelines and the readings were performed using an MR – 96 A automatic Elisa plate reader from Mindray. 2.3. Animal sampling and sample preparation Specimens of Paracentrotus lividus 50 mm in diameter (market size) were collected from three stations in the Gulf of Annaba. The samples were transported in a cooler with oxygenated seawater. The specimens from each station were measured and dissected in the laboratory. The gonads were weighed, immediately removed and homogenized in 20 mM Tris buffer (pH 7.6) containing 1 mM EDTA, 0.5 M sucrose, 0.15 M KCl and 1 mM DTT. The homogenate was centrifuged at 9000g for 20 min at 4 °C. The supernatant was stored at − 40 °C until analysis.

2. Materials and methods 2.1. Sampling station The study was carried out over four seasons in 2012: winter

Fig. 1. Map of the three sampling stations (S.1, S.2 and S.3) and of the main processes governing the marine dynamic inside the Annaba Gulf (Algeria).

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Table 1 Characterization of sampling stations at Annaba Gulf. Position of sampling stations

Geographic coordinates

Depth (cm)

Soil

Pollution

Associated species encountered

Cap grade (S.1)

36° 58′ 04. 03″ N 07° 47′ 29. 81″ E 36° 56′ 00. 95″ N 07° 45′ 57. 59″ E 36° 52′ 30. 62″ N 08° 02′ 56. 77″ E

74.16 ± 17.81

Rocky

Unpolluted

56.25 ± 20.35

Sandy

Urban

59.16 ± 19.75

Sandy

Agricultural and industrial

Arbacia lixula, Sphaerechinus granularis, Corallina sp. and Posidonia oceanica. Arbacia lixula, Sphaerechinus granularis, Corallina sp., Posidonia oceanica and Ulva sp. Arbacia lixula, Sphaerechinus granularis, Cladophora sp., Corallina sp., Ulva sp. and Posidonia oceanica.

Lacaroub beach (S.2) Draouch beach (S.3)

Superoxide anion (O2−%) production was measured using the method of Hassoun and Ray (2003) based on SOD - sensitive cytochrome c reduction. Absorbance was measured at 550 nm and the results were expressed in nmol of reduced cytochrome c/min/g of tissue using an extinction coefficient of 2.1 × 104 M− 1 cm− 1. For lipid peroxidation, malondialdehyde (MDA) was measured using the method of Uchiyama and Mihara (1978). Absorbance was measured spectrophotometrically at 532 nm, and MDA contents were calculated using 1, 1, 3, 3 tetra ethoxypropane as a standard with the results expressed as nmol of malondialdehyde/mg of tissue.

2.4. Gonadal index To determine the physiological state of the organism, we calculated the gonad index (GI) proposed by Semroud and Kada (1987) using the following formula: GI (mg/mm3) = (Dry weight of gonads in mg) / (Test diameter in mm)3.

2.5. Biochemical analysis Reduced glutathione (GSH) was determined using the method of Weckbecker and Cory (1988). This method is based on the appearance of a yellowish colour when 5, 5′-dithiobis-(2 - nitrobenzoic acid) (DTNB) is added to compounds containing sulfhydryl groups. Absorbance was measured at 412 nm and GSH concentration was calculated using reduced glutathione as a standard, and the results were expressed as μmol reduced GSH/mg protein. Protein content was determined at 595 nm according to the Bradford colorimetric method (Bradford, 1976) using bovine serum albumin (BSA) as a standard. Glutathione-S transferase (GST) activity was measured spectrophotometrically at 340 nm and 37 °C using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate according to the method of Habig et al. (1974). Enzyme activity was calculated as nmol CDNB conjugate formed/min/ mg protein using a molar extinction coefficient of 9.6 mM− 1 cm− 1. Glutathione peroxidase (GPx) activity was measured using the method of Flohé and Günzler (1984), and absorbance was recorded at 420 nm. Enzyme activity was calculated as μmol of disappeared glutathione/ min/mg protein. Superoxide dismutase (SOD) activity was measured spectrophotometrically using the method of Beauchamp and Fridovich (1971) with slight modifications, focusing on the reduction of nitroblue tetrazolium (NBT) by the xanthine oxidase/hypoxanthine system at 560 nm. A 50% reduction of NBT was considered one unit of enzyme activity. Catalase (CAT) activity was determined by measuring the rate of hydrogen peroxide reduction at 240 nm using the method of SaintDenis et al. (1998). CAT activity was calculated using a molar extinction coefficient of − 0.04 mM− 1 cm− 1. The results were expressed in μmol of H2O2 reduced/min/mg protein.

2.6. Statistical analysis The data are expressed in mean values ± standard deviation of the mean. Statistical analysis of the data was performed using XL STAT 2014 software, and the normal distribution was verified by applying the Shapiro-Wilk test, making it possible to choose non-parametric methods for the statistical analysis. Analysis of variance (Kruskal-Wallis test) was used to compare seasons. Principal component analysis (PCA) was used to discriminate the seasons as a function of environmental parameters and biological responses. Tests were performed at a 0.05 level of significance. 3. Results 3.1. Environmental characterization The seasonal variations in the physicochemical parameters of the water are represented in Table 2. The results show that changes in the physicochemical parameters are directly related to seasonal rhythm; the lowest temperatures are recorded during the winter, whereas during the spring the temperatures show an appreciable increase, with a maximum in summer. The pH is constant and generally alkaline throughout the sampling period, with a slight increase in winter. The salinity is relatively constant, with the exception of winter and a value of 24‰ recorded at station S.3; the turbidity of the sea water showed a

Table 2 Seasonal variations of physicochemical parameters analyzed at the 3 sampling stations (S.1, S.2 and S.3) of Annaba Gulf from the winter to autumn 2012. Seasons

Stations

Temperature (°C)

pH

Winter

S.1 S.2 S.3 S.1 S.2 S.3 S.1 S.2 S.3 S.1 S.2 S.3

13.90 14.06 18.00 16.83 17.66 18.86 26.66 26.03 29.43 25.63 27.40 24.03

9.01 9.21 9.36 9.07 8.90 8.48 8.38 8.02 8.14 8.36 8.62 8.46

Spring

Summer

Autumn

± ± ± ± ± ± ± ± ± ± ± ±

0.36 0.15 0.34 0.05 0.15 0.04 0.05 0.05 0.32 0.35 0.30 0.05

± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.02 0.02 0.07 0.01 0.04 0.02 0.03 0.03 0.05 0.02 0.03

Salinity (‰)

Dissolved oxygen (mg/L)

Turbidity (NTU)

NO3− (μM)

NO2− (μM)

NH4+ (μM)

PO43 − (μM)

35.36 35.93 24.00 35.66 35.30 35.36 35.86 35.76 35.26 36.26 36.26 35.43

02.06 ± 0.06 02.68 ± 0.05 11.11 ± 1.89 09.46 ± 0.15 03.01 ± 0.03 10.12 ± 0.02 04.50 ± 0.10 0.00 ± 0.00 02.7 ± 0.052 05.14 ± 0.01 01.83 ± 0.02 02.06 ± 0.07

01.04 ± 0.02 02.52 ± 0.15 36.46 ± 0.61 0.98 ± 0.01 02.96 ± 0.05 02.62 ± 0.10 02.78 ± 0.19 06.03 ± 0.06 02.77 ± 0.03 01.34 ± 0.04 02.25 ± 0.04 15.23 ± 0.20

15.91 29.13 12.37 10.24 16.18 11.01 14.08 11.94 08.36 09.00 03.33 01.40

0.10 ± 0.01 04.55 ± 3.38 0.67 ± 0.03 0.34 ± 0.01 0.18 ± 3.38 0.02 ± 0.00 0.07 ± 0.03 0.18 ± 0.02 0.08 ± 0.04 0.11 ± 0.02 0.15 ± 0.03 0.03 ± 0.02

10.63 13.28 106.7 04.55 07.38 06.92 03.96 12.25 03.62 20.37 24.47 16.96

0.68 ± 0.01 12.18 ± 0.03 0.95 ± 0.27 01.49 ± 0.01 0.58 ± 0.03 02.40 ± 0.06 0.45 ± 0.03 0.36 ± 0.06 0.60 ± 0.03 0.72 ± 0.04 0.97 ± 0.05 01.47 ± 0.13

± ± ± ± ± ± ± ± ± ± ± ±

0.32 0.11 0.10 0.05 0.26 0.09 0.05 0.05 0.15 0.25 0.58 0.05

Limit of detection: NO3− = 0.01 μM - NO2− = 0.01 μM - NH4+ = 0.05 μM - PO4− 3 = 0.02 μM.

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± ± ± ± ± ± ± ± ± ± ± ±

1.48 1.50 1.75 1.48 1.50 0.83 0.39 0.39 0.39 0.33 0.58 0.33

± ± ± ± ± ± ± ± ± ± ± ±

0.39 0.25 79.1 0.39 0.25 1.39 1.41 3.94 0.53 7.73 2.56 0.64


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Table 3 Seasonal variations of pesticides analyzed at the 3 sampling stations (S.1, S.2 and S.3) of Annaba Gulf from the winter to autumn 2012 (Mean values ± DS; n = 3). Seasons

Stations

Nonylphenol (μg/L)

Diuron (μg/L)

Glyphosate (μg/L)

Atrazine (μg/L)

Winter

S.1 S.2 S.3 S.1 S.2 S.3 S.1 S.2 S.3 S.1 S.2 S.3

0.40 0.06 0.76 1.28 1.38 1.01 0.15 0.28 0.20 2.11 1.67 0.53

0.05 ± 0.11 ± 0.23 ± 0.11 ± 0.28 ± 0.06 ± 01.0 ± 0.39 ± 0.34 ± < 0.03 < 0.03 < 0.03

0.63 ± 0.080 15.83 ± 0.06 16.23 ± 0.10 01.51 ± 0.42 01.31 ± 0.19 07.83 ± 0.57 0.62 ± 0.026 01.34 ± 0.01 0.70 ± 0.017 0.74 ± 0.010 0.63 ± 0.010 0.65 ± 0.020

< 40 < 40 < 40 < 40 0.10 ± 0.014 0.08 ± 0.006 < 40 < 40 < 40 < 40 < 40 < 40

Spring

Summer

Autumn

± ± ± ± ± ± ± ± ± ± ± ±

0.005 0.002 0.333 0.017 0.005 0.010 0.007 0.005 0.005 0.006 0.002 0.003

0.005 0.008 0.002 0.001 0.005 0.002 0.008 0.001 0.001

Limit of detection: Nonylphenol = not indicated - Diuron = 0.03 μg/L – Glyphosate = 0.05 μg/L - Atrazine = 40 ng/L.

3.3. Biochemical parameters

minimum in spring and a maximum in winter. The dissolved oxygen ranges from a maximum in cold seasons and a minimum in hot seasons. The waters of the gulf of Annaba are highly charged with nutritional salts, and the dominant character of the gulf of Annaba is its highest level for NH4+ in winter; the oxidized forms of nitrogen (NO3− and NO2−) and PO4 −3 always occur at a low fraction compared to the NH4+. The concentrations of pesticides differ among seasons (Table 3). The highest level of contamination, both in terms of the number of detected compounds and concentrations was observed at stations S.2 and S.3. Glyphosate and nonylphenol are detected at 100% in samples, suggesting chronic exposure to these substances. Atrazine is quantified in spring only; the results obtained suggest that contamination by this compound may also exhibit seasonality. Diuron was detected in nine of the 12 samples (75%) and the concentration was ≤ 1.00 μg/L. The application of the Kruskal-Wallis test for comparing the seasonal variation of environmental parameters reveals the existence of a significant difference between seasons for pH (p = 0.028), temperature (p = 0.03), nitrate (p = 0.03) and diuron (p = 0.03).

The response of the sea urchin Paracentrotus lividus to various environmental parameters by season is shown in Fig. 3 (A: GSH; B: GST; C: GPx; D: SOD; E: CAT; F: O2−%; G: MDA). The sea urchin appears to be more sensitive to fluctuations of environmental parameters during winter periods than other seasons. The result shows a significant decrease in GSH, GPX and CAT in winter. However, GST and SOD activities were lower in summer. It is also noteworthy that the levels of biomarkers dosed in the gonads are extremely high in spring compared to other seasons. However, the oxidative stress parameters present higher values for MDA and O2−% in winter, followed by summer and autumn. The application of the Kruskal-Wallis test for comparing the seasonal variation of biochemical parameters reveal a significant difference between seasons for GSH (p = 0.02), GST (p = 0.04) and GPX (p = 0.03).

3.4. Principal component analysis Using principal component analysis (PCA) as a preliminary and exploratory approach made it possible to visualize the structure of the seasonal variation in the Gulf of Annaba using 21 variables. Moreover, it permitted seeking similarities between different seasons. It should be underlined that the PCA was performed on standard normal distributions (normed PCA), whose results are summarized in Fig. 4. The PCA results clearly showed that the first two factorial axes together explain 83.83% of the total variation. Axis 1 explains 42.22% of the total variability; this axis is positively correlated with the variables glyphosate (r = 0.99; cos2 = 0.99), dissolved oxygen (r = 0.97; cos2 = 0.94) and pH (r = 0.95; cos2 = 0.91). The latter values contributed considerably to its construction, in addition to other positive correlations observed for nitrite (r = 0.81; cos2 = 0.90), orthophosphate (r = 0.72; cos2 = 0.85) and nitrate (r = 0.57; cos2 = 0.75), which contributed less to the construction of this axis. In addition, this axis was negatively correlated with temperature (r = −0.98; cos2 = 0.97), diuron (r = − 0.91; cos2 = 0.84) and to a lesser degree salinity (r = −0.86; cos2 = 0.75) and catalase (r = −0.74; cos2 = 0.54). Furthermore, axis 2 explained 41.61% of the total variation, highlighting the specificity of the spring and winter seasons in contrast with the other two seasons (summer and autumn). It was mainly built with the variables turbidity (r = −0.99; cos2 = 0.99), atrazine (r = 0.96; cos2 = 0.93), SOD (r = 0.95; cos2 = 0.91), GPX (r = 0.83; cos2 = 0.70), GSH (r = 0.77; cos2 = 0.60), MDA (r = −0.99; cos2 = 0.99), O2−% (r = − 0.92; cos2 = 0.86) and the gonad index (r = − 0.73; cos2 = 0.53), which contributed considerably to the construction of this axis.

3.2. Gonadal index The seasonal variation of the gonad index of the sea urchin Paracentrotus lividus is shown in Fig. 2. The calculation of the gonadal index led us to observe that the highest values were found in spring for the three stations; the very high increase in the gonadal index corresponded to maximum maturity, the significant reduction of the GI signified the release of sexual materials. The application of the KruskalWallis test for comparison of seasonal variation of gonad index revealed a significant difference between seasons (p = 0.04).

Fig. 2. Seasonal variations the gonad index in the sea urchin Paracentrotus lividus at 3 sampling stations (S.1, S.2 and S.3) of the Annaba Gulf from the winter to autumn 2012 (Mean values ± DS; n = 3).

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4. Discussion

closest to the plumes of the El-Mafrag estuary, and during heavy rainfall a very large quantity of fresh water is discharged following the flooding of the region. Our results are supported by those of many authors, such as Ounissi et al. (2014) and Ziouch (2014) who report the existence of similar fluctuations between 23 and 37.9‰. The fluctuation of oxygen during the study period is highly variable, and the high concentrations causing saturation in the winter and spring season can be explained by phytoplankton flares that can cause a temporary endogenous oxygen supply in coastal environment (Aminot and Kérouel, 2004a). However, the low quantities recorded in summer can be explained by the rise in temperature, which limits the solubility of oxygen, urban and agricultural discharges, and the hydrodynamic calmness illustrated by the drop (Lacaze, 1996). To this is added the bacterial degradation of detritus, which consumes an enormous amount of oxygen. This consumption is more important with increased temperature (Belaud, 1996). The variation in turbidity is approximately 0.3 to 3 NTU in coastal waters (Aminot and Kérouel, 1997), and the elevation of turbidity during the cold season could be related to the continental contributions of solid material and the remittance in suspension of sedimentary deposits by swells and currents (Aminot and Kérouel, 2004a). Under the conditions most commonly encountered in marine environments, nutrients are not directly toxic to the species that live there, but they may be the cause of indirect nuisances such as eutrophication and anoxia in the marine environment (Le Pape et al., 1996). The seawater at the stations contains high mineral salts, especially in winter, as explained by intense agricultural activity and the absence of the wastewater plants (Falfushynska et al., 2009). Inputs of suspended matter through freshwater river discharge, agricultural runoff and/or the microbial regeneration of organic compounds of both marine and terrestrial origins are commonly referred as the main sources (Newton et al., 2003). In the Gulf of Annaba, anthropogenic influence seems to also depend on the hydrodynamic and hydrologic

4.1. Environmental characterization The deterioration of the water at the stations studied mainly resulted from discharge and the lack of appropriate wastewater treatment plants in most of the towns' regions. The fluctuation of measured and dosed parameters reveal a period of disturbance characteristic of winter, when very high values are reached at stations S.2 and S.3, coinciding with a period of submersion affecting the region during February 2012 when 169.4 mm of rainwater was recorded on the west coast of the Gulf of Annaba (meteorological station 603600) and 221.76 mm on the east coast (meteorological station 603670). In addition, the human population is concentrated along the coast, where a large number of its activities are concentrated. Whatever permanent or seasonal, industrial activity is the chief source of pollution (ANPEA, 1987), discharges occur either directly into the gulf or into the rivers and wadis. The introduction of pollutants into the Gulf via the main rivers has already been characterized by Bougherira et al. (2015); Keblouti et al. (2015); and Chaoui et al. (2013). The seasonal variability in physicochemical parameters is strongly influenced by seasonal events; the temperature depends clearly on climatic conditions, and the temperatures measured reflect the influence of the moderate Mediterranean climate and show the existence of two distinct periods: warm and cold. The pH influences many chemical or biological processes (Hinga, 2002). According to Barnabé (1991), the pH of Mediterranean coastal waters varies between 7.9 and 8.3. Fluctuations in pH can be affected by processes such as: mixing with freshwater, accumulation of organic matter of continental origin and contamination by agricultural, urban or industrial discharges (Aminot and Kérouel, 2004a). The salinity of the Mediterranean sea is 38–39‰, and the low value recorded during the winter period seems to be due to a dilution phenomenon due to the intrusion of fresh water. Station S.3 is

Fig. 3. Seasonal variations the biochemical parameters in the sea urchin Paracentrotus lividus at the 3 sampling stations (S.1, S.2 and S.3) of the Annaba Gulf from the winter to autumn 2012 (Mean values ± DS; n = 3).

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Fig. 3. (continued)

paint for boats smaller than 25 m (Giacomazzi and Cochet, 2004). The concentrations of diuron measured in this study were above the standard fixed by the Environmental Quality Standards (EQS) of 1.8 μg/L. In light of our results, we suggest that the coastal waters of Annaba have not yet reached an alarming state compared to the coastal waters of western Japan (0.03–3.05 μg/L) (Okamura et al., 2003), the coast of the united Kingdom (0.01–6.74 μg/L) (Thomas et al., 2001), the Spanish Mediterranean coast (2 μg/L) (Martinez et al., 2000), the Dutch coast (1.13 μg/L) (Lamoree et al., 2002) and the bay des Veys in France (0.02–0.254 μg/L) (Buisson et al., 2008). This was also the case for atrazine, an herbicide of the triazine group (Pick et al., 1992), which has been classified as a carcinogenic chemical

sedimentary conditions that govern the gulf (Grimes et al., 2010). The pesticide concentrations were high, with the highest values found in winter at the stations closest to the outlets of the main drainage networks. This drainage appears to be the main source of herbicides; the runoff water carried by the two rivers, the Bounamoussa and El Kebir, which flow into the Gulf via the El-Mafrag estuary, contains discharged pollutants of different origins. The watersheds of these rivers are exploited by intensive farming Khélifi-Touhami et al. (2006). Diuron is an antifouling agent derived from urea (Thomas et al., 2002) whose use was totally forbidden in 2004. It was recognized by the European Commission as a priority hazardous substance (Malato et al., 2002). Some European countries have limited its use as an antifouling

Fig. 4. Principal component analysis based on the seasonal variation of the Gulf of Annaba. Factorial plane: D1: 42.22%; D2: 41.61%. (A): Correlation circle of variables assayed with the first two principal axes. (B): Projection of seasons on the first two principal axes.

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results are consistent with those already found by Amri et al. (2017) in the southeast Mediterranean; Guettaf et al. (2000) in the southwest Mediterranean; Bayed et al. (2005) in the northern Moroccan Atlantic coast and Lozano et al. (1995) in the northwest Mediterranean.

substance and endocrine disruptor by the United States Environmental Protection Agency (US-EPA, 1990). Despite its total prohibition by the European Union in 2004 (European Commission, 2004), it has been detected in many countries around the world, in the Spanish Mediterranean (0.2–1.5 ng/L) (Bueno et al., 2009), in the northern Adriatic near to Italy (1.3–5.9 ng/L) (Carafa et al., 2007), in the Amvrakikos Gulf of Grèce (nd – 800 ng/L) (Readman et al., 1993), and in the Baltic Sea near Germany (0–2.1 ng/L) (Nödler et al., 2013); in the Gulf of Annaba it remained below the maximum admissible concentration fixed at 2 μg/L proposed by the EQS for coastal waters (European Commission, 2008b). Another compound that also causes environmental concern due to its effect on the endocrine system is nonylphenol (NP), which is extremely toxic to aquatic and marine organisms (Lozano et al., 2012). Produced by the primary degradation of alkylphenolethoxylates (APEs) (Giger et al., 1984), it can imitate natural hormones by reacting with oestrogen receptors (Jobling et al., 1996). Its presence in aquatic habitats is mainly correlated to wastewater discharges and anthropic activity (Soares et al., 2008) and has already been detected in marine ecosystems by numerous studies, such as those of Jackson and Sutton (2011) in California (San Francisco Bay, 0.024–6.25 μg/L); Arditsoglou and Voutsa (2008) in Greece (Thessaloniki coast, 0.181–0.915 μg/L); Li et al. (2005) in Korea (Saemangeum Bay, 298 μg/L); and Petrovic et al., (2002) in the Mediterranean (Spanish coast, 0.3–4.1 μg/L). Regarding the sampling site, the NP content was always lower than the maximum admissible concentration proposed by the EQS, which has adopted a value of 2 μg/L (European Commission, 2008b). Nonetheless, in autumn a value of 2.11 μg/L was recorded; this slight increase can be explained by the rise in temperature during this period. The works of Xu et al. (2015a) explain that high temperature favours bacterial activity, which degrades alkylphenols. Glyphosate is an organophosphorous herbicide frequently used in agriculture due to its low toxicity and high efficiency (Kolpin et al., 2006); it is included in the list of authorized substances according to Appendix I of Directive 91/414/CEE (European Commission, 1991). It is not usually taken into account in most marine monitoring programs even though it is one of the most widely used herbicides in the world (Stachowski-Haberkorn et al., 2008). Glyphosate remains particularly poorly documented for marine environments (Munaron, 2004). The Australian Guideline recommended values of 370–2000 μg/L in freshwater (Anzecc and Armcanz, 2000) that can be applied in the absence of recommended marine values (Mercurio et al., 2014). The results obtained are generally lower, but this does not mean that the situation is not worrisome. Indeed, several studies by Le et al. (2010); Modesto and Martinez (2010); and Sandrini et al. (2013) have shown that glyphosate is also toxic to animals, causing alterations in haematological parameters, antioxidant enzyme activities and genetic expression. The most probable explanation for the high concentrations of glyphosate is its application just before samples were taken and the flood that struck the region. The same phenomenon was observed in a Spanish river where a concentration of 137 μg/L was detected three days after the application of glyphosate to control riparian vegetation (Puértolas et al., 2010). In Algeria, glyphosate is used as an active ingredient in 16 commercial herbicides (Cekufosate, Diss, Glypan, Ground-Up, Herbocol 360, Kalach 360 SL, Mamaba, Ouragan Système 4, Pilarsato, Ridasata, Round-Up, Round Up Ultra, Soumax, Sykosto, Tiller, Yamasate) intended to combat weeds, aquatic plants, cuscuta (dodder) and adventitious citrus shoots, perennials, dicotyledonous plants and grasses (DPVCT, 2003).

4.3. Biochemical parameters The oxidative stress state is also a seasonal phenomenon; the impact of environmental factors on enzyme activity has been reported in many studies (Madeira et al., 2015; Barda et al., 2014). Oxidative stress is known to be high in winter (Manduzio et al., 2004) because intense rainfall may increase drainage of the surrounding agricultural soil and increases the impact of pesticides and fertilisers (Caricato et al., 2010) which have major impacts on animal physiology. However, the elevated antioxidant enzyme levels observed in spring may be due to the reproduction period and/or associated with the competing occurrence of algae, which synthesize toxic or repellent secondary metabolites (Lemée et al., 1996) that are a possible source of reactive oxygen species (ROS) in marine organisms (Box et al., 2008). The obtained results by Tejada et al. (2013) indicated that antioxidant enzyme activities have increased significantly in sea urchin Paracentrotus lividus fed with these algae. Reduced glutathione is involved in phase II metabolism and plays a crucial role in intracellular protection against reactive oxygen species, essentially via the enzymatic activities of GST and GPx (Anderson, 1997). The low GSH content in winter can be explained by the combined effect of pesticides and environmental parameters. These observations agree with previous studies by Benali et al. (2015); Contardo-Jara et al. (2009); Glusczak et al. (2007); Leiniö and Lehtonen (2005), indicating that they can stimulate the expression of various biomarkers. However, the rapid and unexpected increase in GSH in spring coincided with the reproduction period of the sea urchin Paracentrotus lividus in the Gulf of Annaba. It appears that the physiological state of these animals is strongly disturbed during the reproduction period. Indeed, the reproduction cycle appears to affect their defensive capacities. Delaporte et al. (2006) showed that the concentration of circulating haemocytes decreases during gametogenesis. In addition, their phagocytic activity and adhesion capacity are inhibited during this period, with the possible involvement of reproductive enzymes. Thus, to cope with stress conditions, aquatic organisms develop an adaptive process by maintaining their antioxidant systems at high levels (Wilhelm Filho et al., 2005). These results corroborate the works of Solé et al. (1995) obtained in the mussel Mytilus galloprovincialis, for which egg-laying leads to an increase in antioxidant activities followed by a progressive decrease despite the availability of food and higher temperatures. In living organisms, GST plays a crucial role in protecting cells against toxic compounds and has been used as a biomarker of exposure to anthropic compounds (Park et al., 2009), catalysing the addition of nucleophiles of the thiol function of reduced glutathione to a wide range of electrophile compounds such as pesticides and heavy metals (Hazarika and Sarkar, 2001). The increase in GST activity observed suggests activation of detoxification processes, likely reflecting high stress (Louiz et al., 2016). Similar results were found in Paracentrotus lividus exposed to polycyclic aromatic hydrocarbons (Cunha et al., 2005). Furthermore, we notice that the levels of enzyme activity declined after spring. A weak response by GST may be due to acclimatization to habitat conditions so that toxicity levels to which they are continuously exposed and are not high enough to elicit a response (Wepender et al., 2008). Glutathione peroxidase reduces H2O2 and lipid hydroperoxide to function and uses reduced glutathione as a cofactor, via which it transfers oxygen and transforms it into oxidized glutathione (Goudable and Favier, 1997). Several authors have described seasonal variability of GPX activity (Vidal-Liñán et al., 2010). The results obtained indicated a weak response of GPX in winter and autumn compared to the spring

4.2. Gonad index The estimation of the gonad index helps to determine the breeding period; high GI represents the genital maturation stage and the lowest GI values represent the post-spawning period (Schmidt et al., 2013). In the Gulf of Annaba, reproduction takes place in the spring, and our 398


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season into three clusters. The first corresponds to winter, characterized by increased stress oxidative in sea urchin Paracentrotus lividus and high content of MDA and O2−%. The second cluster is the spring, corresponding to the period of gametogenesis of the sea urchin in the Gulf of Annaba, characterized by high levels of enzymatic activity. The third cluster groups the summer and autumn and is characterized by a period of reconstitution and the degenerative process of the gonads following gametogenesis.

and summer. In spring, a large amount of the energy obtained from nutrition is stored to cover metabolic needs, especially those needed for reproduction (Houtteville and Lubet, 1974). During winter, when food is scarce and adverse environmental conditions are present, the sea urchin implements a strategy favouring enzymes that consume little energy. This has already been observed by Guderley et al. (2003), who measured decreased GPx activity in Gadus morhua L. deprived of food resources, because glutathione peroxidase consumed GSH and NADPH (Janssens et al., 2000). Superoxide dismutase is a key enzyme for the dismutation of the superoxide anion (O2−%), which constitutes the first radical form capable of attacking cell components (Afonso et al., 2007). Superoxide dismutase activity was higher during winter and spring. In winter this coincided with high lipid peroxidation values, suggesting an increase in superoxide anion formation (Braghirolli et al., 2016). However, in spring, this coincides with lower lipid peroxidation values, suggesting the metabolism of the superoxide anion by SOD. After a period of intense reproductive activity to apparently conduct maintenance of oxidative status, the SOD activity and superoxide anion remains more or less constant during summer and autumn. This is explained by a series of endogenous factors that must be considered with caution, such as weight loss outside the reproduction period (Amiard and Amiard Triquet, 2008) and the degenerative process of the gonads following gametogenesis. Furthermore, the collective role of high temperature and low dissolved oxygen content appears to be responsible for high levels of superoxide anion, as also observed by Paital and Chainy (2013) who reported that these two abiotic parameters could cause oxidative stress in all marine organisms. Many scientists consider catalase a sensitive and important biomarker of oxidative stress superior to SOD, revealing the biological effects on the redox state of marine organisms (Regoli et al., 2002a). The elimination of H2O2 is a key strategy of marine organisms against oxidative stress (Regoli et al., 2002b). The lowest CAT activity was found in the winter despite the unfavourable conditions; this inhibition has been suggested to be a transitory response against pollution (Regoli and Principato, 1995) before the occurrence of a compensatory effect (Regoli et al., 1998). Catalase activity was higher in spring and could be linked to the degree of superoxide dismutase activity, as these enzymes operate sequentially, and the increase in CAT activity is induced by the hydrogen peroxide potentially derived from the SOD reaction. Lipoperoxidation (LPO) is currently used as an oxidative stress response marker in aquatic animals. The imbalance between the production and elimination of ROS can lead to oxidative stress and then to lipoperoxydation (Sun et al., 2008). Malondialdehyde is the final product of LPO and is often used as a biomarker to evaluate the level of oxidative stress resulting from xenobiotics (Shi et al., 2005). In the present study, the sea urchin indicated increased markers of antioxidant defences but without significant effects on lipid peroxidation. Similar results were obtained by Freitas et al. (2012); the enzymatic activities and LPO of Diopatra neapolitana did not reflect the metallic contamination of sediments, since other factors such as food availability, the state of reproduction and respiratory intensity could also modify the state of lipid peroxidation (Geracitano et al., 2004). In spring, the lower levels of LPO may reflect reduced activity or more efficient action of the antioxidant system, particularly antioxidant enzymes (Braghirolli et al., 2016). Several studies reported that sea urchins gonads possess significant antioxidant activity (Qin et al., 2011; Sheean et al., 2007). PCA proved useful to highlight the existence of seasonal variation along factor 1, where environmental physicochemical parameters and pesticides were the primary contributor to discrimination between the warm seasons with high temperatures (summer and autumn) and the cold seasons with heavy rainfall (winter and spring). We could also observe along factor 2 that the biomarkers and GI were the main contributor of this axis and allowed for the observation of a certain degree of seasonal difference. The results obtained permitted grouping of the

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