Florfenicol induces malformations of embryos and causes altered lipid profile, oxidative damage, neurotoxicity, and histological effects on gonads of adult sea urchin, Paracentrotus lividus.

The frequent occurrence of antibiotics in the aquatic environment has engendered negative impacts on non-target organisms. The effects of the veterinary antibiotic florfenicol (FLO) during the embryo-larval development of the sea urchin, Paracentrotus lividus was assessed using four increasing concentrations (1, 2, 5 and 10 mg/L). Furthermore, FLO toxicity to adults was investigated through the analysis of oxidative damage, histopathological alterations, lipid metabolism and acetylcholinesterase activity following an exposure period of 96 h. FLO induced embryotoxicity with estimated EC50 values of 5.75, 7.56 and 3.29 mg/L after 12 h, 24 h and 48 h, respectively. It generated oxidative stress assessed as lipid peroxidation in gonads despite the increased antioxidant activity of catalase (CAT). Neurotoxicity was also evident since the AChE activity significantly decreased. Moreover, FLO affected the lipid metabolism by increasing saturated fatty acid (SFA) and monounsaturated fatty acid proportions (MUFA), except in the group exposed to 5 mg/L. The increase in polyunsaturated fatty acid (PUFA) levels and docosahexaenoic acid (DHA, C22:6n-3) proportions were noted with all FLO concentrations. Eicosapentaenoic acid (EPA, C20:5n-3) decreased, while arachidonic acid (ARA, C20:4n-6) increased in sea urchins exposed to 5 and 10 mg/L FLO. Histopathological alterations of gonadal tissues represent an additional confirmation about the toxicity of this antibiotic that might decrease the reproductive performance of this species. Nevertheless, even if reproduction of sea urchins would be partially successful, the embryotoxicity would compromise the normal development of the embryos with consequences on the population.

Bioremediation of petroleum refinery wastewater by fungal stains isolated from the fishing harbour of Bizerte (Mediterranean Sea).

The study was conducted in order to explore the potential of fungi isolated from surface and bottom seawater collected from the fishing harbour of Bizerte on the bioremediation of industrial effluent (IE) contaminated by petroleum hydrocarbon. Among the 128 fungal isolates, 11 were isolated from surface seawater and 7 from bottom seawater, representing 18 taxa in total. The gas chromatography mass spectrometry (GC-MS) was used for the determination of hydrocarbon compounds in IE. An initial screening of fungal growth using six concentrations ranged between 20 and 70% (v/v) IE has allowed the identification of the optimal concentration for fungal growth as well as selection of species able to tolerate high amounts of hydrocarbon. Colorimetric test employing 2,6-dichlorophenol indophenol and gravimetric method was applied for the assessment of fungal growth using 20% EI. By checking the phylogenetic affiliation of the high-performing stains as identified using ITSr DNA sequence, a dominance of Ascomycetes was detected. Indeed, Aspergillus terreus and Penicillium expansum may degrade 82.07 and 81.76% of residual total petroleum hydrocarbon (TPH), respectively. Both species were collected from surface seawater. While, Aspergillus niger, Colletotrichum sp and Fusarium annulatum displayed comparable degradation rates 40.43%, 41.3%, and 42.03%, respectively. The lowest rate of degradation 33.62% was detected in Emericellopsis phycophila. All those species were isolated from bottom seawater, excepting A. niger isolated from surface water. This work highlighted the importance of exploring the potential of fungi isolated from the natural environment on the bioremediation of industrial effluent. Our results promoted the investigation of the potential of the high-performing isolates A. terreus and P. expansum on the bioremediation of IE at pilot-scale and then in situ.

Effects of dietary Pb and Cd and their combination on lysosomal and tissue-level biomarkers and histopathology in digestive gland of the land snail, Cantareus apertus (Born, 1778).

The present study was aimed at determining cell and tissue-level biomarkers and histopathological alterations in the green garden snail, Cantareus apertus (Born, 1778), exposed to different nominal dietary concentrations of Pb (25, 100 and 2500 mg Pb/kg), Cd (5, 10 and 100 mg Cd/kg) and their combination (25 mg Pb + 5 mg Cd/Kg, 100 mg Pb + 10 mg Cd/kg and 2500 mg Pb + 100 mg Cd/ kg) for 1 and 8 weeks. Lead and Cd exerted histopathological effects on the digestive gland in a dose-dependent manner and related to lysosomal and tissue-level biomarkers. The biological responses observed included digestive cell vacuolisation and numerical atrophy, calcium cell hydropic degeneration, excretory cell hypertrophy, inflammatory responses, blood vessel congestion, and disruption of the blood vessel wall and the interstitial connective tissue. Lysosomal enlargement and transient intracellular accumulation of neutral lipids and lipofuscins were also observed, together with alterations in the cell type composition and thinning of the digestive gland epithelium and with diverticular distortion. This response profile fits well with the biological effects reported after metal exposure in gastropods from other regions, as well as with data obtained in parallel studies dealing with metal bioaccumulation and intralysosomal accumulation, mortality, feeding, growth, oxidative stress and neurotoxicity exerted elicited by Pb, Cd and their mixture in green garden snails under the present experimental conditions. Consequently, C. apertus seems to be a suitable model species for the biomarker-based assessment of the biological effects of Pb and Cd, alone or in combinations, thus providing a challenging opportunity to advance in identifying suitable sentinel species for metal pollution biomonitoring and ecosystem health assessment in soil ecosystems in Northern Africa.

Green garden snail, Cantareus apertus, as biomonitor and sentinel for integrative metal pollution assessment in roadside soils.

The present investigation was conceived to study, in a small scale field study, the potential of the green garden snail, Cantareus apertus, as biomonitor and sentinel for integrative metal pollution assessment in soils. For this purpose, we investigated the association between the trace metal (Cd, Pb, As, Fe, Cr, Cu, Ni, and Zn) concentrations in soil, plants (Trifolium repens), and C. apertus depending on the distance (20, 150, and 700 m) from a main roadside in Tunisia as well as between metal concentrations and biomarkers of oxidative stress, oxidative damage, and neurotoxicity in C. apertus. Results revealed a clear association between the concentration of metals such as Ni, Cu, and Zn in snail digestive gland, both amongst them and with oxidative stress and neurotoxicity biomarkers recorded in the same organ. Interestingly, Ni, Pb, and Zn occurred at the highest concentration in soil, plant, and snails and the association appeared related to the immediacy of the roadside and the concentration of these three metals tended to decrease with distance from the roadside in the soil-plant-snail system. Conversely, Cd and Cu were bioaccumulated in plants and snails but their concentrations in soil were not high and did not show a decline in concentration with distance from the roadside. After PCA analysis, PC-01 (56% of the variance) represented metal bioaccumulation and associated toxic effects in snails in the presence of high levels of metal pollution (nearby the roadside) while PC-02 (35% of the variance) represented stress induced by moderate levels of metal pollution (at intermediate distances from the roadside). The four studied sites were clearly discriminated one from each other, depending on how they are affected by traffic pollution. In summary, this field study reveals that (a) C. apertus can be used as biomonitor for metal pollution in roadside soils and as sentinel for pollution effects assessment based on biochemical biomarkers; and (b) that oxidative stress and neurotoxicity biomarkers endow with a powerful biological tool for metal pollution biomonitoring in soils, especially in combination with chemical analysis of the soil-plant-snail transfer system. Moreover, this study provides some baseline data for future impact assessments concerning trace metal pollution in Tunisia.

Bioaccumulation and tissue distribution of Pb and Cd and growth effects in the green garden snail, Cantareus apertus (Born, 1778), after dietary exposure to the metals alone and in combination.

The present study was aimed at determining bioaccumulation and cell and tissue distribution of Pb and Cd in the green garden snail, Cantareus apertus (Born, 1778), exposed to different nominal dietary concentrations of Pb (25, 100 and 2500 mg Pb/kg), Cd (5, 10 and 100 mg Cd/kg) and their combination (25mg Pb+10 mg Cd/kg and 2500 mg Pb+100 mg Cd/kg) for 1 and 8 wk. Pb and Cd were bioaccumulated in the digestive gland in a dose-dependent manner and the degree of effects on growth was related to the level of exposure, though metal-metal interactions were observed after treatment with mixtures of Pb and Cd. The present results are absolutely comparable to those obtained in other terrestrial pulmonates in other regions and therefore they absolutely support that C. apertus is suitable as biomonitor for the assessment of the Pb and Cd levels and their biological effects in soil ecosystems in Northern Africa.

Effects of Dietary Pb and Cd and Their Combination on Glutathion-S-Transferase and Catalase Enzyme Activities in Digestive Gland and Foot of the Green Garden Snail, Cantareus apertus (Born, 1778).

The present study was focused on the assessment of glutathion-S-transferase (GST) and catalase (CAT) activities in the digestive gland and foot of the land snail, Cantareus apertus (Born, 1778), exposed to different nominal dietary concentrations of Pb (25 and 2500 mg Pb/Kg), Cd (5 and 100 mg Cd/Kg) and their combination (25 mg Pb + 5 mg Cd/Kg and 2500 mg Pb + 100 mg Cd/Kg) for 7 and 60 days. GST activity was significantly increased after 7 and 60 days exposure to the highest concentration of Pb, Cd and their combination. The levels of CAT activity were different in the two studied organs but in both cases it resulted increased after 7 and 60 days of exposure, which varied significantly between metals and dietary concentrations. Therefore, it can be concluded that GST and CAT enzymes in digestive gland and foot of C. apertus are responsive to Cd, Pb and their combination, whereby they are suitable to be included in a battery of biomarkers for ecosystem health assessment in metal polluted soils using this species as sentinel.

Copper, zinc, and cadmium body concentrations in Hexaplex trunculus collected from the Tunisian coast.

Copper, Zn, and Cd were analyzed in the soft tissues of the edible gastropod Hexaplex trunculus collected from seven localities along the Tunisian coast. The body concentration of these metals was significantly different depending on the gender and site. Copper and Zn varied, respectively, from 47.70 to 343.64 µg/g dry weight (dw) and 149.46 to 530.44 µg/g dw, being higher in males. Cadmium varied from 0.22 to 18.95 µg/g dw and was always higher in females. Overall, the highest concentrations of the three metals were all recorded in Gabès fishing harbor. Comparison of metal concentrations with the European standards compiled by the Food and Agriculture Organization showed that values exceeded standards in several localities. Imposex had been previously recorded in H. trunculus along the Tunisian coast; however, it does not seem to be related with Cu, Zn, and Cd pollution because the body concentration of these metals is not correlated with imposex degree. Nevertheless, interactive effects resulting from the combination of different pollutants and other stressors cannot be disregarded.

Shell disturbances and butyltins burden in commercial bivalves collected from the Bizerta lagoon (northern Tunisia).

Shell disturbances and soft tissues butyltin burden were investigated in commercial bivalves Lithophaga lithophaga, Mytilus galloprovincialis, Solen marginatus and Crassostrea gigas from the Bizerta lagoon. Shell disturbances were found in all bivalves, being scarce in S. marginatus. In the internal valve of L. lithophaga, burrowing annelids and sipunculids living inside galleries were observed, while in the external valve, brown-blackish or white stains were found. In M. galloprovincialis, a yellowish mass located at the shell anterior side was found fixed firmly to the pearly layer by a hard brownish structure covering some annelid elliptic eggs. In the internal shell layer of some specimens collected in April, embryos belonging to tubiculous annelids at various developmental stages were observed. In C. gigas, shell thickening was revealed in some specimens corresponding to white doughy deposits at the internal valve and between shell layers. In S. marginatus, only one specimen showing a cavity at the posterior site was found. Total butyltin concentrations in the studied bivalves varied between 30 and 245 ng/g dry weight with tributyltin (TBT) being the predominant compound. The highest concentration was recorded in L. lithophaga collected from the Bizerta Bay and the lowest concentration in S. marginatus from Maghraoua. This study provided baseline data that could serve for long-term monitoring of TBT pollution in Tunisia, since legislation to reduce the use of TBT-based antifouling paints has not been introduced yet.

Monitoring of organotin pollution in Bizerta channel (northern Tunisia): temporal trend from 2002 to 2010.

Imposex and tributyltin (TBT) body burden were quantified in the gastropod Hexaplex trunculus collected from the Bizerta channel between 2002 and 2010. Except for the imposex frequency that remained maximal (100%), all the other imposex indices decreased throughout the study period. Similarly, TBT levels also decreased over time, being the less frequent compound among butyltins, with a proportion of 22.2%, against 42.9% for dibutyltin (DBT) and 34.9% for monobutyltin (MBT). These findings reflect the effectiveness of new generation of TBT-free antifouling paint introduced in the Tunisian market and global ban of TBT on reducing the environmental impact of this biocide.

First record of butyltin body burden and imposex status in Hexaplex trunculus (L.) along the Tunisian coast.

We have assessed for the first time the current status of tributyltin (TBT) contamination in Hexaplex trunculus along Tunisian coastal waters. Two sampling campaigns have been performed in July 2004 and July 2007 at seven different sites. The snails were analyzed for imposex status, i.e. Imposex incidence, Relative Penis Length Index (RPLI) and Vas Deferens Sequence Index (VDSI), followed by the quantification of butyltins in their tissues. Imposex was detected in six populations from the 2004 samples and in five populations from the 2007 samples. The imposex incidence ranged from 0 (no imposex) to 100%, the RPLI from 0 to 56.7 and the VDSI from 0 to 4.4. TBT was also detected in six populations in 2004 and five populations in 2007. The concentrations ranged from 1.5 to 73.7 ng Sn g(-1) dw in 2004 samples and from 1.2 to 23.9 ng Sn g(-1) dw in 2007 samples. Temporal comparison of the data indicates some evidence of imposex recovery in most affected populations. Improvements in reduced body concentrations of TBT in both sexes were also seen in most stations. These TBT concentrations were only significantly correlated to imposex indices in samples from 2007 (r > 0.8, n = 7, p < 0.05). The present results will serve as a reference for long-term monitoring of butyltin contamination in the Tunisian coast, where TBT restrictions were not yet implemented.