Influence of Bizerte city wastewater treatment plant (WWTP) on abundance and antibioresistance of culturable heterotrophic and fecal indicator bacteria of Bizerte Lagoon (Tunisia).

The waste water treatment plant (WWTP) of the city of Bizerte concentrates different types of chemical and biological pollutants in the Bizerte lagoon (Tunisia). Considering four upstream and downstream WWTP discharge stations, seventy nine, culturable bacterial strains were isolated and identified from water and sediment as fecal coliforms, fecal streptococci, pathogenic staphylococci and non-enterobacteriacea. Fecal coliforms were most abundant (2.5 105 bacteria/mg) in sediment of WWTP discharge. Leuconostoc spp (23.1%) and Chryseomonasluteola (23.1%) were the most prevalent culturable fecal indicator bacteria (FIB) isolated at the upstream discharge stations. However, Staphylococcus xylosus (13.9%) was the most prevalent culturable FIB isolated at the WWTP discharge stations. Moreover, high antibioticresistance phenotypes were present in all sampling stations, but especially in WWTP discharge station in both water and sediment. Resistance levels in water and sediment, respectively were amoxicillin (58.8%; 34.8%), penicillin (50%; 31.6%), oxacillin (60%; 33.3%), cefotaxim (55.2%; 39.1%), ceftazidim (66.7%; 50%), gentamycin (42.9%; 38.9%), tobramycin (50%; 25%), vancomycin (33.3; 71.4%), amikacin (66.7%; 0%) and ciprofloxacin (100%; 100%). Interestingly, ß-lactam antibiotic resistant FIB were mostly isolated from water as well as from sediments of upstream and WWTP discharge station. Canonical correspondence analysis CCA correlating antibiotic resistance profile with the abiotic data showed that, in water column, culturable bacterial strains isolated in upstream WWTP discharge stations were interestingly correlated with the resistance to amikacin, oxacillin, cefotaxim, ciprofloxacin and gentamycin, however, in sediment, they were correlated with the resistance to amoxicillin, oxacillin, céfotaxim and vancomycin. Serious ß-lactams and aminoglycosides acquired resistance appeared mainly in fecal streptococci and pathogen staphylococci groups.

Trophic restructuring (Wieser 1953) of free-living nematode in marine sediment experimentally enriched to increasing doses of pharmaceutical penicillin G.

Trophic structure of free living nematode from Bizerte lagoon was tested by a microcosmic study after 30 days of exposure with 5 increasing doses of pharmaceutical penicillin G (D1: 3 mg L(-1), D2: 30 mg L(-1), D3: 300 mg L(-1), D4: 600 mg L(-1), D5: 700 mg L(-1)). Results showed significant differences between nematode assemblages from undisturbed controls and those from penicillin G treatments. Selective deposit-feeders (1A) or nonselective deposit-feeders (1B), very abundant in the control microcosm, were significantly affected and their dominance declined significantly. Epistrate feeders (2A) were significantly gradual increase for all microcosms treated with penicillin G, appeared to be more tolerant to the antibiotic and to take advantage of the growing scarcity of other trophic groups. Compared to the control microcosms, omnivorous-carnivorous (2B) was found to be higher in all treated microcosms, with the exception of those treated with D5. Trophic index (Σθ(2)) was significantly reduced in all microcosms treated whereas trophic ratio 1B/2A appears to be insignificant.

Pesticides in Ichkeul Lake-Bizerta Lagoon Watershed in Tunisia: use, occurrence, and effects on bacteria and free-living marine nematodes.

This study aimed to identify the most commonly used agricultural pesticides around Ichkeul Lake-Bizerta Lagoon watershed. First survey of pesticide use on agricultural watershed was performed with farmers, Regional Commissioner for Agricultural Development, and pesticide dealers. Then, sediment contamination by pesticides and response of benthic communities (bacteria and free-living marine nematode) were investigated. The analysis of 22 active organochlorine pesticides in sediments was performed according to quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, biodiversity of indigenous bacterial community sediment was determined by terminal restriction fragment length polymorphism (T-RFLP), and free-living marine nematodes were counted. The results of the field survey showed that iodosulfuron, mesosulfuron, 2,4-dichlorophenoxyacetic acid (2,4 D), glyphosate, and fenoxaprops were the most used herbicides, tebuconazole and epoxiconazole the most used fungicides, and deltamethrin the most used insecticide. Sixteen organochlorine pesticide compounds among the 22 examined were detected in sediments up to 2 ppm in Ichkeul Lake, endrin, dieldrin, and hexachlorocyclohexane being the most detected molecules. The most pesticide-contaminated site in the lake presented the higher density of nematode, but when considering all sites, no clear correlation with organochlorine pesticide (OCP) content could be established. The bacterial community structure in the most contaminated site in the lake was characterized by the terminal restriction fragments (T-RFs) 97, 146, 258, 285, and 335 while the most contaminated site in the lagoon was characterized by the T-RFs 54, 263, 315, 403, and 428. Interestingly, T-RFs 38 and 143 were found in the most contaminated sites of both lake and lagoon ecosystems, indicating that they were resistant to OCPs and able to cope with environmental fluctuation of salinity. In contrast, the T-RFs 63, 100, 118, and 381 in the lake and the T-RFs 40, 60, 80, 158, 300, 321, and 357 in the lagoon were sensitive to OCPs. This study highlighted that the intensive use of pesticides in agriculture, through transfer to aquatic ecosystem, may disturb the benthic ecosystem functioning of the protected area. The free-living marine nematodes and bacterial communities represent useful proxy to follow the ecosystem health and its capacity of resilience.

Effects of increasing levels of pharmaceutical penicillin G contamination on structure of free living nematode communities in experimental microcosms.

A microcosm experiment was conducted to examine the effects of the pharmaceutical (penicillin G) on free living nematode communities of a Tunisian coastal zone (South-Western Mediterranean Sea). Sediments were contaminated with five penicillin G dose [D1 (3 mgL(-1)), D2 (30 mgL(-1)), D3 (300 mgL(-1)), D4 (600 mgL(-1)), D5 (700 mgL(-1))], and effects were examined after 30 days. Results showed significant differences between nematode assemblages from undisturbed controls and those from penicillin G treatments. Most univariate measures, including diversity (H'), species richness (d), equitability (J) and number of species (S) decreased significantly with increasing level of the antibiotic contamination. Results from multivariate analyses of the species abundance data demonstrated that responses of nematode species to the penicillin treatments were varied: Kraspedonema octogoniata and Paracomesoma dubium were eliminated at all the antibiotic doses tested and seemed to be intolerant species to penicillin G contamination; Oncholaimus campylocercoides although survived even the highest dose D5, showed definite reduction in its abundance and may be classified as "opportunistic" species at this dose, whereas, Nannolaimoides decoratus which showed a positive response with an increase in density even at highest concentration of contaminant, seems to be "penicillin G resistant" species.

Effects of 2-(4-Methoxyphenyl)-5, 6-trimethylene-4H-1, 3, 2-oxathiaphosphorine-2-sulfide on biomarkers of Mediterranean clams Ruditapes decussatus.

The effects of exposure to a novel synthetic organophosphorus compound, 2-(4-Methoxyphenyl)-5, 6-trimethylene-4H-1, 3, 2-oxathiaphosphorine-2-sulfide (OMTOS) concentrations (Control=0, C1=0.01, C2=0.1, C3=1 and C4=10µg/L) were investigated in the clam Ruditapes decussatus. Vitellogenin (Vg)-like protein levels in haemolymph from males and females were investigated. Concentrations of 1µg/L and 10µg/L significantly decreased Vg levels in male haemolymph after 7 days, whereas significant variations were only found in females treated with 10µg/L. On the other hand, superoxide dismutase (SOD), catalase (CAT) and acetylcholinesterase activities (AChE) in whole soft tissue were measured after 2, 4 and 7 days of exposure to the same series of concentrations. After 2 days of exposure, 0.1, 1, and 10µg/L of OMTOS increased SOD activity significantly, but this decreased with 10µg/L after 4 and 7 days. No changes in CAT activity were observed after 2 days compared to controls. OMTOS significantly reduced AChE activity after 4 and 7 days in treated clams with the highest concentration 10µg/L, but it did not induce significant variations at the other concentrations tested. Our study demonstrates that OMTOS alters biochemical parameters in R. decussatus, even at low concentrations, and suggests differing modes of action of the contaminant. Using clams is a powerful tool to provide valuable insights into possible mechanisms of environmental toxicity of novel synthetic organic products both in non-target organisms and the marine ecosystem. Additionally, our results highlight that biomarker responses facilitate elucidation of putative mechanisms of action of OMTOS in non-target species.

Effects of anthracene on filtration rates, antioxidant defense system, and redox proteomics in the Mediterranean clam Ruditapes decussatus (Mollusca: Bivalvia).

This study aimed at analyzing the impact of a toxic polyaromatic hydrocarbon (PAH), anthracene (ANT), on Ruditapes decussatus collected from a Tunisian coastal lagoon (Bizerte Lagoon). Filtration rates, several antioxidant enzymes--superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione transferase (GST)--as well as indices of protein oxidation status were determined in various tissues of this bivalve. Specimens were exposed to 100 µg/L of ANT for 2 days. ANT levels were evaluated using HPLC and were detected in the gill and digestive gland at different amounts. ANT exposure altered the behavior of bivalves by changing the siphon movement and decreasing filtration rate significantly. The enzymatic results indicated that ANT exposure affected the oxidative stress status of the gills of R. decussatus. In addition, modification of proteins was detected in the gills using redox proteomics after ANT treatment. Three protein spots were successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-MS). These proteins can be roughly related to muscle contraction function. In contrast, no significant modification of enzymatic and protein responses was detected in the digestive gland after ANT treatment. These data demonstrate that combined behavioral and biochemical analyses are a powerful tool to provide valuable insights into possible mechanisms of toxicity of anthracene in R. decussatus. Additionally, the results highlight the potential of the gill as a valuable candidate for investigating PAH toxicity.

Changes of benthic bacteria and meiofauna assemblages during bio-treatments of anthracene-contaminated sediments from Bizerta lagoon (Tunisia).

Sediments from Bizerta lagoon were used in an experimental microcosm setup involving three scenarios for the bioremediation of anthracene-polluted sediments, namely bioaugmentation, biostimulation, and a combination of both bioaugmentation and biostimulation. In order to investigate the effect of the biotreatments on the benthic biosphere, 16S rRNA gene-based T-RFLP bacterial community structure and the abundance and diversity of the meiofauna were determined throughout the experiment period. Addition of fresh anthracene drastically reduced the benthic bacterial and meiofaunal abundances. The treatment combining biostimulation and bioaugmentation was most efficient in eliminating anthracene, resulting in a less toxic sedimentary environment, which restored meiofaunal abundance and diversity. Furthermore, canonical correspondence analysis showed that the biostimulation treatment promoted a bacterial community favorable to the development of nematodes while the treatment combining biostimulation and bioaugmentation resulted in a bacterial community that advantaged the development of the other meiofauna taxa (copepods, oligochaetes, polychaetes, and other) restoring thus the meiofaunal structure. The results highlight the importance to take into account the bacteria/meiofauna interactions during the implementation of bioremediation treatment.

Responses of a free-living benthic marine nematode community to bioremediation of a PAH mixture.

The objectives of this study were (1) to assess the responses of benthic nematodes to a polycyclic aromatic hydrocarbon (PAH) contamination and (2) to test bioremediation techniques for their efficiency in PAH degradation and their effects on nematodes. Sediments with their natural nematofauna communities from Bizerte lagoon (Tunisia) were subjected to a PAH mixture (100 ppm) of phenanthrene, fluoranthene, and pyrene during 30 days. Nematode abundance and diversity significantly decreased, and the taxonomic structure was altered. Results from multivariate analyses of the species abundance data revealed that PAH treatments were significantly different from the control. Spirinia parasitifera became the dominant species (70 % relative abundance) and appeared to be an "opportunistic" species to PAH contamination while Oncholaimus campylocercoides and Neochromadora peocilosoma were strongly inhibited. Biostimulation (addition of mineral salt medium) and bioaugmentation (inoculation of a hydrocarbonoclastic bacterium) were used as bioremediation techniques. Bioremediation treatments enhanced degradation of all three PAHs, with up to 96 % degradation for phenanthrene resulting in a significant stimulation of nematode abundance relative to control microcosms. Nevertheless, these treatments, especially the biostimulation provoked a weak impact on the community structure and diversity index relative to the control microcosms suggesting their feasibility in biorestoration of contaminated sediments.

Effect of permethrin, anthracene and mixture exposure on shell components, enzymatic activities and proteins status in the Mediterranean clam Venerupis decussata.

Anthracene (ANT) and permethrin (PER) are two of the more toxic compounds reaching the marine environment. This study aimed to determine the impact of these molecules on Venerupis decussata, an economically important species cultured on the Tunisian coast. Shell structure and its possible transformation upon exposure to the two contaminants were studied by X-ray diffraction and gravimetric analyses. Results revealed a phase transition in shell composition from aragonite to calcite after PER exposure, to a mixture of PER and ANT (Mix) but not for ANT alone. Catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GST) activities were determined in digestive gland and gills after exposure to ANT, PER and Mix to assess the impact of the contamination on the oxidative status of V. decussata. Enzyme activities increased in the digestive gland after PER treatment and in the gills after ANT treatment. PER exposure significantly reduced the levels of free thiols and increased levels of carbonylated proteins in the digestive gland, as compared to controls. In contrast, ANT exposure significantly reduced free thiols and increased the number of carbonylated proteins in the gills. Mix induced additive effects as measured by both enzymatic and proteomic approaches. The present study suggests that PER has a strong effect on shell structure; that PER and ANT exposure generate compound-dependent oxidative stress in the tissues of V. decussata and that a mixture of the two compounds has synergistic effects on biochemical response.

More than 35 years of studies on marine nematodes from Tunisia: a checklist of species and their distribution.

This work provides an inventory of species of free-living nematodes from Tunisian waters, based on samples collected from September 1977 until March 2013. Sediment samples were taken from 8 ecosystems: the Lagoons of Bizerte, Ghar El Melh and Bou Ghrara, Northern and Southern Lakes of Tunis, Old Harbor of Bizerte, Bizerte Bay and Ichkeul Lake. A total of 31 families, 133 genera, and 249 species of marine nematodes are currently known from Tunisia. The Xyalidae (with 30 species), Cyatholaimidae (25), Chromadoridae and Linhomoeidae (each 21) and Comesomatidae (20) are the richest families. Among them, Daptonema (with 9 species), Pomponema and Sabatieria (with 7 each) are the most species-rich genera. Some species were recovered only from a specific type of ecosystem. In detail, 109 species were recovered from lagoons, 56 species from sandy beaches and 6 species from mudflats. The 78 remaining species were widely distributed in Tunisian waters and four of these (Terschellingia longicaudata, Oncholaimus campylocercoides, Sabatieria pulchra and Theristus flevensis) were present in most of the 8 ecosystems studied.

Biostimulation as an attractive technique to reduce phenanthrene toxicity for meiofauna and bacteria in lagoon sediment.

A microcosm experiment was setup to examine (1) the effect of phenanthrene contamination on meiofauna and bacteria communities and (2) the effects of different bioremediation strategies on phenanthrene degradation and on the community structure of free-living marine nematodes. Sediments from Bizerte lagoon were contaminated with (100 mg kg(-1)) phenanthrene and effects were examined after 20 days. Biostimulation (addition of nitrogen and phosphorus fertilizer or mineral salt medium) and bioaugmentation (inoculation of a hydrocarbonoclastic bacterium) were used as bioremediation treatments. Bacterial biomass was estimated using flow cytometry. Meiofauna was counted and identified at the higher taxon level using a stereomicroscope. Nematodes, comprising approximately two thirds of total meiofauna abundance, were identified to genus or species. Phenanthrene contamination had a severe impact on bacteria and meiofauna abundances with a strong decrease of nematodes with a complete disappearance of polychaetes and copepods. Bioremediation counter balanced the toxic effects of phenanthrene since meiofauna and bacteria abundances were significantly higher (p < 0.01) than those observed in phenanthrene contamination. Up to 98 % of phenanthrene removal was observed. In response to phenanthrene contamination, the nematode species had different behavior: Daptonema fallax was eliminated in contaminated microcosms, suggesting that it is an intolerant species to phenanthrene; Neochromadora peocilosoma, Spirinia parasitifera, and Odontophora n. sp., which significantly (p < 0.05) increased in contaminated microcosms, could be considered as "opportunistic" species to phenanthrene whereas Anticoma acuminata and Calomicrolaimus honestus increased in the treatment combining biostimulation and bioaugmentation. Phenanthrene had a significant effect on meiofaunal and bacterial abundances (p < 0.05), with a strong reduction of density and change in the nematode communities. Biostimulation using mineral salt medium strongly enhanced phenanthrene removal, leading to a decrease of its toxicity. This finding opens exciting axes for the future use of biostimulation to reduce toxic effects of PAHs for meiofauna and bacteria in lagoon sediment.

Effects of permethrin exposure on antioxidant enzymes and protein status in Mediterranean clams Ruditapes decussatus.

The effects of permethrin (PER) on a panel of antoxidant enzymes; superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) and indices of protein oxidation status (carbonylation and free thiols) were determined in digestive gland and gills of the clam Ruditapes decussatus. Animals were exposed to 100 ppb PER for 2 days. These enzyme activities increased significantly in digestive gland (p<0.05) after PER treatment and oxidative modification of proteins was detected in both gill and digestive gland extracts using redox proteomics. PER exposure significantly reduced the amount of protein free thiol groups in digestive gland rather than in gill, when compared to controls. Conversely, digestive gland showed significantly higher levels of carbonylated proteins than gill after PER exposure. Some proteins were successfully identified by mass spectrometry of tryptic peptides. Our data suggest that digestive gland of R. decussatus can be used as a model tissue for investigating environmental risk of PER contamination.

Impacts of bioremediation schemes for the mitigation of a low-dose anthracene contamination on free-living marine benthic nematodes.

A microcosm experiment was used to examine (1) the effects of different bioremediation schemes on degradation of anthracene and the structure of free-living marine nematodes in a lightly contaminated (4.5 µg g(-1)) sediment from Bizerte lagoon and (2) the responses of the nematode community upon an artificial spiking of a low dose anthracene (1 µg g(-1)). For that purpose sediment microcosms were incubated in laboratory for 40 days. Bioremediation techniques decreased the anthracene contamination, and interestingly, biodegradation were more efficient when anthracene was artificial supplied into the sediment suggesting that the addition of bioavailable anthracene stimulated the bacterial community to adjust towards a PAH-degrading community. Spiking with this low dose of anthracene provoked significant changes in the nematode community structure and abundance, with the elimination of specific species such as Mesacanthion diplechma, the decrease of the dominant species Oncholaimus campylocercoides and the increase in abundance of opportunistic species such as Spirinia parasitifera. This would suggest a low tolerance of the nematode community despite the presence of a weak anthracene contamination in the sediment that could have allow dominance of an anthracene tolerant nematode species. Anthracene toxicity was alleviated in biostimulation treatments, leading to a strong increase in nematode abundance, concomitantly with changes in the nematode community structure; Prochromadorella neapolitana became the most abundant species.

The roles of biological interactions and pollutant contamination in shaping microbial benthic community structure.

Biological interactions between metazoans and the microbial community play a major role in structuring food webs in aquatic sediments. Pollutants can also strongly affect the structure of meiofauna and microbial communities. This study aims investigating, in a non-contaminated sediment, the impact of meiofauna on bacteria facing contamination by a mixture of three PAHs (fluoranthene, phenanthrene and pyrene). Sediment microcosms were incubated in the presence or absence of meiofauna during 30 days. Bioremediation treatments, nutrient amendment and addition of a hydrocarbon-degrading bacterium, were also tested to enhance PAH biodegradation. Results clearly show the important role of meiofauna as structuring factor for bacterial communities with significant changes observed in the molecular fingerprints. However, these structural changes were not concomitant with changes in biomass or function. PAH contamination had a severe impact on total meiofaunal abundance with a strong decrease of nematodes and the complete disappearance of polychaetes and copepods. In contrast, correspondence analysis, based on T-RFLP fingerprints, showed that contamination by PAH resulted in small shifts in microbial composition, with or without meiofauna, suggesting a relative tolerance of bacteria to the PAH cocktail. The PAH bioremediation treatments were highly efficient with more than 95% biodegradation. No significant difference was observed in presence or absence of meiofauna. Nutrient addition strongly enhanced bacterial and meiofaunal abundances as compared to control and contaminated microcosms, as well as inducing important changes in the bacterial community structure. Nutrients thus were the main structural factor in shaping bacterial community composition, while the role of meiofauna was less evident.

Microbial community responses to bioremediation treatments for the mitigation of low-dose anthracene in marine coastal sediments of Bizerte lagoon (Tunisia).

PURPOSE: The main goals of this study were to investigate (1) the behavior of microbial communities in response to low-dose bioavailable anthracene addition in lightly contaminated sediment from Bizerte Lagoon and (2) the effects of bioremediation treatments on microbial biomass, activity, and community structure. METHODS: Sediment microcosms amended with 1 ppm anthracene were incubated in triplicate during 30 days. Biostimulation (addition of nitrogen and phosphorus fertilizer) and bioaugmentation (inoculation of a hydrocarbonoclastic bacterium) were used as bioremediation treatments. Bacterial biomass was estimated using flow cytometry. Sediment oxygen consumption was measured with oxygen microelectrodes. Bacterial community structure was assessed by molecular fingerprints (terminal restriction fragment length polymorphism; T-RFLP) analysis. RESULTS: Anthracene contamination resulted in a significant reduction of bacterial abundance with an impact on cell integrity. Concomitantly, sediment oxygen consumption was strongly inhibited. Correspondence analysis on T-RFLP data indicated that bacterial community structures from anthracene-contaminated microcosms were different from that of the control. Interestingly, the changes observed in microbial biomass, structure, and activities as a result of anthracene contamination were not alleviated even with the use of biostimulation and combination of biostimulation and bioaugmentation strategy for anthracene bioremediation. Nevertheless, both treatment methods resulted in different community structures relative to the contaminated and control microcosms with the appearance of distinct populations. CONCLUSION: Anthracene spiking severely affected microbial communities, suggesting dominance of nontolerant populations in this lightly-contaminated sediment. Although biostimulation and/or bioaugmentation treatments did not alleviate the anthracene toxic effects, the changes observed in microbial population and structure suggest that the proposed treatments might be promising to promote bacterial growth. Further works are still required to propose a more efficient strategy to stimulate biodegradation that takes into account the complex interactions between species for resource access.

Do trace metals (chromium, copper, and nickel) influence toxicity of diesel fuel for free-living marine nematodes?

The objective of this study was to test the hypotheses that (1) free-living marine nematodes respond in a differential way to diesel fuel if it is combined with three trace metals (chromium, copper, and nickel) used as smoke suppressants and that (2) the magnitude of toxicity of diesel fuel differs according to the level of trace metal mixture added. Nematodes from Sidi Salem beach (Tunisia) were subjected separately for 30 days to three doses of diesel fuel and three others of a trace metals mixture. Simultaneously, low-dose diesel was combined with three amounts of a trace metal mixture. Results from univariate and multivariate methods of data evaluation generally support our initial hypothesis that nematode assemblages exhibit various characteristic changes when exposed to different types of disturbances; the low dose of diesel fuel, discernibly non-toxic alone, became toxic when trace metals were added. For all types of treatments, biological disturbance caused severe specific changes in assemblage structure. For diesel fuel-treated microcosms, Marylynnia bellula and Chromaspirinia pontica were the best positive indicative species; their remarkable presence in given ecosystem may predict unsafe seafood. The powerful toxicity of the combination between diesel fuel and trace metals was expressed with only negative bioindicators, namely Trichotheristus mirabilis, Pomponema multipapillatum, Ditlevsenella murmanica, Desmodora longiseta, and Bathylaimus capacosus. Assemblages with high abundances of these species should be an index of healthy seafood. When nematodes were exposed to only trace metals, their response looks special with a distinction of a different list of indicative species; the high presence of seven species (T. mirabilis, P. multipapillatum, Leptonemella aphanothecae, D. murmanica, Viscosia cobbi, Gammanema conicauda, and Viscosia glabra) could indicate a good quality of seafood and that of another species (Oncholaimellus mediterraneus) appeared an index of the opposite situation.

Acute toxicities of cadmium and permethrin on the pre-spawning and post-spawning phases of Hexaplex trunculus from Bizerta Lagoon, Tunisia.

Marine gastropods, exposed to anthropogenic pollution, accumulate high chemical concentrations in their tissues, especially in the digestive glands. An evaluation of the impacts of cadmium (Cd) and permethrin (Perm), coupled with reproductive events (pre-spawning and post-spawning phase) throughout the year, was attempted by measuring catalase (CAT), a biomarker of defence, on Hexaplex trunculus experimentally exposed for 48 or 96 h. Specimens of gastropods were sampled from Bizerta Lagoon (Tunisia). The results show that CAT activity increased in gastropods exposed to the three cadmium concentrations (C1 Cd, 100 µg L(-1); C2 Cd, 200 µg L(-1); and C3 Cd, 300 µg L(-1)) and to the three permethrin doses (C1 Perm, 100 µg L(-1); C2 Perm, 150 µg L(-1); and C3 Perm, 200 µg L(-1)) tested. A decrease in CAT was noted in the digestive gland of the H. trunculus exposed to permethrin at the concentration of 200 µg L(-1) during the pre-spawning and post-spawning phases. H. trunculus in post-spawning was more sensitive to cadmium and permethrin than in the pre-spawning phase. The biochemical responses to pollutants (cadmium and permethrin) represented by CAT may act as a biomarker of exposure in this species.

An observational study on changes in biometry and generation time of Odontophora villoti (Nematoda, Axonolaimidae) related to petroleum pollution in Bizerte bay, Tunisia.

INTRODUCTION: We conducted a yearly polluted-reference sampling to assess the effects of petroleum pollution on life cycle characteristics of the meiobenthic nematode Odontophora villoti. Samples were taken every 15 days between 26 November 2004 and 25 November 2005 from two beaches of Bizerte bay (Tunisia), Rimel and Tunisian Refining Industries Company (TRIC). The latter site is located in front of the "Tunisian Refining Industries Company" runoff. DISCUSSION: When compared to the reference site, the mean body dimensions of O. villoti from the impacted site were significantly lower. The small size of affected nematodes was represented both by the length and width as a function of the life stage. It was also established that changes in lengths of body parts during molts were different between the two study sites. The low availability of oxygen from April to August seems to prevent the formation of embryos of O. villoti. Thus, two annual reproductive cycles with different durations were observed in Rimel and TRIC. Under stress, juvenile phase and egg production were generally shorter. Globally, the impact of petroleum pollution on O. villoti was expressed by a short egg-to-egg development time. CONCLUSION: Our study assessed the usefulness of life cycle characteristics (biometry and life stage durations) of O. villoti in biomonitoring, and the results are generally consistent suggesting that this species may be considered as an efficient bioindicator.

Organic matter compounds as source indicators and tracers for marine pollution in a western Mediterranean coastal zone.

INTRODUCTION: Complex organic compounds found in oil and sediments linked with a particular source (such as algae, bacteria or vascular plants) are defined as biomarkers and are useful dating indicators in organic geochemistry. METHODS AND RESULTS: This paper presents the composition of the organic matter (OM) on marine surface sediments from a degraded Tunisian coast analysed by pyrolysis and gas chromatography-mass spectrometry (GC-MS). High total OM contents (0.3-4.2%) were detected with high levels of saturated linear hydrocarbons. The aliphatic lipids had contributed with up to 11.7% of the total OM, and their distribution had consisted of resolved compounds (n-alkanes and fatty acid (FAs)) and an unresolved complex mixture. Hydrocarbons, primarily n-alkanes, were ranged from 368 to 3,886 µg g(-1). The FAs (674-2,568 µg g(-1)) were dominated by derived primary production, and the short chain FAs (C16 and C18) were the most abundant throughout. The ubiquitous presence of petroleum contamination, mainly from offshore oil exploration, discharge of pollutants from rivers, shipping activities and atmospheric deposition was found in all samples. The Gabès littoral seems to be quite to very polluted near the industrial zone of Ghannouch. The C/H ratio (generally around 5.9), the thermal analysis and GC-MS of n-alkanes and FAs showed that the OM in the studied area was composed of anthropogenic/petrogenic, marine and continental sources. CONCLUSIONS: Our study represents an innovative approach to assessing environmental pollution. The evaluation of organic matter by examination of sterols, alkanes and fatty acids allows the identification of source, both anthropogenic and natural.

Laboratory study on individual and combined effects of cobalt- and zinc-spiked sediment on meiobenthic nematodes.

Free-living nematodes are the most abundant taxa among the meiobenthos and the predominant prey for bottom-feeding fishes. They are able to accumulate toxicants from sediments which explain their use in this study as possible tools in nutritional quality assessment of fishes. Nematodes from sediments of Ghar El Melh lagoon (Tunisia) were subjected to cobalt and/or zinc enrichment in a microcosm experiment for 30 days. Three levels (low, medium, and high) of each treatment were used. Nematode abundance and diversity significantly decreased, and the taxonomic structure was altered. Results from multivariate analyses of the species abundance data revealed that all treatments were significantly different from the control. Both univariate and multivariate analyses of the data showed that the differential response occurred in all treatments, but the assemblages from microcosms contaminated with zinc alone were much more negatively affected compared with those exposed to cobalt alone. The presence of cobalt simultaneously with zinc seems to reduce its impact on nematode species composition. Such a result is suggestive of antagonistic interactions between these two metals. The responses of nematode species to the cobalt and zinc treatments were varied. Oncholaimellus mediterraneus, Oncholaimus campylocercoides, and Neochromadora trichophora were significantly affected with cobalt contamination but, they were not eliminated. Exposed to zinc, Hypodontolaimus colesi was eliminated and seemed to be an intolerant species versus this metal. Some of these species, "cobalt-sensitive" or "zinc-sensitive", were also affected by the metal combination even at low dose: O. mediterraneus, N. trichophora, and H. colesi. Differential sensitivity to cobalt and/or zinc may result in a subsequent competitive release of more tolerant species. A list of this kind of species was established to be used as a possible preventive tool versus contaminated fish. This was most evidently the case in Marylynnia stekhoveni and O. campylocercoides which increased at all doses of cobalt and zinc, respectively. These two resistant species have also the opportunity to dominate the nematode assemblage when the studied metals were added together. The level of health risk is probably higher for humans assimilating additional amount of cobalt and zinc, especially heavy smokers and/or patients using some medications including salts of these metals.