Michela Mariottini

Interactive effects of n-TiO2 and 2,3,7,8-TCDD on the marine bivalve Mytilus galloprovincialis

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. The bivalve Mytilus sp, largely utilized as a sentinel for marine contamination, has been shown to represent a significant target for different types of NP, including n-TiO2, one of the most widespread in use. In this work, the possible interactive effects of n-TiO2 and 2,3,7,8-TCDD, chosen as models of NP and organic contaminant, respectively, were investigated in Mytilus galloprovincialis. In vitro experiments with n-TiO2 and TCDD, alone and in combination, were carried out in different conditions (concentrations and times of exposure), depending on the target (hemocytes, gill cells and biopsies) and the endpoint measured. Mussels were also exposed in vivo to n-TiO2 (100 μg L(-1)) or to TCDD (0.25 μg L(-1)), alone and in combination, for 96 h. A wide range of biomarkers, from molecular to tissue level, were measured: lysosomal membrane stability and phagocytosis in hemocytes, ATP-binding cassette efflux transporters in gills (gene transcription and efflux activity), several biomarkers of genotoxicity in gill and digestive cells (DNA damage, random amplified polymorphic DNA-RAPD changes), lysosomal biomarkers and transcription of selected genes in the digestive gland. The results demonstrate that n-TiO2 and TCDD can exert synergistic or antagonistic effects, depending on experimental condition, cell/tissue and type of measured response. Some of these interactions may result from a significant increase in TCDD accumulation in whole mussel organisms in the presence of n-TiO2, indicating a Trojan horse effect. The results represent the most extensive data obtained so far on the sub-lethal effects of NPs and organic contaminants in aquatic organisms. Moreover, these data extend the knowledge on the molecular and cellular targets of NPs in bivalves.

Cytochrome P450, acetylcholinesterase and gonadal histology for evaluating contaminant exposure levels in fishes from a highly eutrophic brackish ecosystem: the Orbetello Lagoon, Italy

Biochemical markers and ovarian histology were investigated in prespawning females of grass goby (Zosterisessor ophiocephalus) and grey mullet (Mugil cephalus) collected, respectively, in late spring and summer 2000 in four sites of a highly eutrophic brackish ecosystem of central Italy, the Orbetello Lagoon. Exposure to chlorinated and aromatic hydrocarbons was evaluated in fish livers by the somatic liver index (SLI) and by measuring 7-ethoxyresorufin-O-deethylase (EROD) and benzo(a)pyrene monooxygenase (BaPMO) activities. Acetylcholinesterase (AChE) activity was measured in brain and gills to evaluate exposure to organophosphates (OPs) and carbamates (CBs). The gonad somatic index (GSI) was used to confirm ovarian maturation and ovarian histology was investigated as a potential biomarker for environmental effects. Samples from the Western Basin, near a sewage treatment plant (STP) off the town of Orbetello, showed higher SLI values and higher EROD and BaPMO activities than those collected from the Ansedonia Canal (AC) in the Eastern Basin (p<0.05) and respect to those from reference sites: the Albegna River (AR) Delta for grass goby and the Nassa Canal (NC), connected with the sea, for grey mullet both located in the Western Basin as well. Low brain AChE activity was observed in both species from the reference sites (AR and NC) in association with the presence of anomalies in developing oocytes: unexpectedly small in grass goby and irregular disintegrated cytoplasm in grey mullet. The results indicate that the Western Basin is more polluted than the Eastern Basin particularly in the Orbetello where the sewage treatment plant may be a source of aromatic and chlorinated compounds while the Albegna River and the Nassa Canal may be sources of OPs and CBs.

Fish as bioindicators of brackish ecosystem health: integrating biomarker responses and target pollutant concentrations

Abstract Biological responses and contaminant levels in biological tissues were investigated in fish specimens of grass goby (Zosterisessor ophiocephalus) collected in spring 2000 in a moderately polluted brackish ecosystem, the Orbetello lagoon (southern coast of Tuscany, Italy). The complexity of harmful effects that contaminants may cause in fish over time was assessed. General condition indices (somatic liver index, and gonadal somatic index), cytochrome P450 enzymes, acetylcholinesterase activity and ovarian morphology were integrated with the content of organochlorines (OCs—hexachlorobenzene, DDTs and polychlorinated biphenyls) in liver and ovaries and p-nonylphenol (NP) and lower ethoxylate (NPE1–2) content in muscle and ovaries as well. Samples collected from the western basin of the lagoon near the Orbetello sewage treatment plant had somatic liver index values and two P450 activities significantly higher than those collected from the same basin at the Albegna river delta and in the Ansedonia canal in the eastern basin (P PCBs > HCB) were particularly lower in samples from STP compared to levels detected in sites AC and AR. Light microscopy of ovaries revealed that although the gonads were sexually mature, the diameter of the oocytes and eggs was smaller than that commonly reported. High levels of OCs and NPs were, in fact, observed in grass goby ovaries, and the differences observed between acetylcholinesterase activities among investigated sites suggest exposure to insecticides as well. The integration of biological responses and contaminant tissue content reveals that certain areas of the lagoon are selectively impacted by various human activities and helps in distinguishing relationships between biological responses and contaminant load in the lagoon environment.

Congener profile and toxicity assessment of polychlorinated biphenyls in human adipose tissue of Italians and Chileans

Abstract Human exposure to polychlorobiphenyls (PCBs) in humans was determined by analyzing adipose tissue samples collected in 1996–1997 from two different localities: Siena (Italy) and Concepcion (Chile). ΣPCBs was higher in Italian samples than that from Chile (493 and 53 ng/g wet wt., respectively). Thirty-seven different PCB congeners were identified in all samples. The prevailing PCB congeners in both groups were 22′44′5 pentachlorobiphenyl (IUPAC no. PCB 118), 22′344′5′ (PCB-138) and 22′44′55′ (PCB-153) hexachlorobiphenyls and 22′33′44′5 (PCB-170), 22′344′55′ (PCB-180) and 22′34′55′6 (PCB-187) heptachlorobiphenyls. PCB-153 accounted for more than 20% of the total PCB residue in both groups, while the remaining six congeners accounted for approximately 70%. Hexachlorobiphenyls were the most abundant congeners in all samples, with 42% of total residue in those from Italy and 43% in the Chilean samples, followed by heptachlorobiphenyls with 41 and 36% in Italian and Chilean samples, respectively. Average concentrations of non- ortho substituted coplanar congeners were below 1 pg/g wet wt. In the samples from Siena no noticeable differences were observed between the three average coplanar concentrations, while in those from Concepcion 33′44′tetrachlorobiphenyl (PCB-77) was much higher than 33′44′pentachlorobiphenyl (PCB-126) and 33′44′55′hexachlorobiphenyl (PCB-169). For each sample the contribution to the total toxic equivalent values (ΣTEQs) of each non- ortho , mono- ortho and di- ortho substituted PCB congeners was assessed. The overall TEQs calculated for the monitored PCBs, were 10.16 pg/g wet wt. in Italian samples and 1.09 pg/g wet wt. in Chileans ones. In both groups the main contribution to ΣTEQs were the di- ortho substituted PCB congeners (Siena: 6.17 pg/g wet wt.; Concepcion: 0.56 pg/g wet wt.) and the mono- ortho substituted PCB congeners (Siena: 3.97 pg/g wet wt.; Concepcion: 0.50 pg/g wet wt.).

Levels of Persistent Organic Pollutants (POPs) in sediments from Lenga estuary, central Chile

Superficial sediments from Lenga estuary in the VIII region of central Chile were analysed for Polychlorinated Biphenyls (PCBs) and Hexachlorobenzene (HCB). Organic carbon (OC) analysis was also performed and the statistical correlations (Pearson correlations) between compounds were also tested. Results showed the total PCB concentrations (ng g(-)(1)d.w.) fluctuated from ∼ 20 to 10,000. These levels are higher than those reported in other coastal sediments of the world. The PCB profile was dominated by middle to highly chlorinated congeners (6-Cl=40% and 7-Cl=30%). HCB concentrations (ng g(-)(1)d.w.), were lower than the PCBs and, fluctuated between 1 (at L1) and 870 (at L4). OC values ranged from 1% (L1) to 7% (L4) showing a significant positive correlations between OC% and PCBs (r=0.86; p<0.05) and for HCB (r=0.71). These results are an important contribution to knowledge of levels of POPs levels in coastal Chilean environments.

Persistent Organic Pollutants in Human Milk from Central Italy: Levels and Time Trends

Persistent organic pollutants (POPs) such as HCB, p,p′-DDE, and PCBs were measured in Italian breast milk. This work is part of a study on human milk, adipose tissues, and food carried out in the same area over the last 20 years. The results showed the prevalence of p,p′-DDE and PCBs over HCB. Comparison of our results with those of previous studies carried out in the same area showed that concentrations are decreasing. No statistically significant differences in organochlorine levels were found when the samples were divided into maternal age classes and into the categories “primiparae” and “multiparae”. In order to quantify the amount of the molecules of interest transmitted by mother to child during breast feeding, we estimated the daily intake of each class of compounds: our results indicated that HCB and p,p′-DDE were several times lower than the safety thresholds.

Anthropogenic impacts on the Orbetello lagoon ecosystem

The aim of the present study was to assess the environmental quality of Orbetello lagoon (Tyrrhenian coast, Italy), using a biomonitoring method based on measuring organochlorinated pollutants in the Anguilla anguilla, chosen as sentinel organism, and remote sensing data obtained by elaboration of Landsat 7 Enhanced Thematic Mapper (ETM) data of 2002. Polychlorinated biphenyls and organochlorine pesticides were determined by gas chromatography/mass spectrometry in muscle of European eels captured in seven sites having different levels of pollution in Orbetello lagoon. Remote sensing data and chemical analysis showed the Albegna river as a probable vehicle of pesticides (hexachlorocyclohexanes).

Ecotoxicological Assessment of Vlora Bay (Albania) by a Biomonitoring Study Using an Integrated Approach of Sublethal Toxicological Effects and Contaminant Levels in Bioindicator Species

Abstract An integrated ecotoxicological study based on bioaccumulation and biomarker responses in sea urchin, mussel, and fish was conducted in Vlora Bay (Albania). Despite several past industrial activities and current increase of anthropogenic impact characterising Vlora Bay, no ecotoxicological data have been published so far, underlining the need to investigate the area and to validate the ecotoxicological approach. Heavy metals, polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides (OC), and polychlorinated biphenyls (PCBs) were measured in native sea urchin (Arbacia lixula), Mediterranean mussel (Mytilus galloprovincialis), and red mullet (Mullus barbatus). Several biomarkers such as expression of heat-shock proteins (HSP-70) in sea urchin (PCR), detoxification enzyme activities and apoptosis in mussels, and acetylcholinesterase (AChE) activity in mussels and red mullet were investigated. Overall, ecotoxicological data suggest an environmental quality of Vlora Bay resembling a marine protected area except for levels of Cd, Ni, and Hg in mussels and red mullet gonads; these results are in agreement with past industrial activities. OC pesticides were found to be below the detection limit in sea urchin. PCB levels were very low (4.13–13.87 ngg−1 fresh weight) both in mussels and fish, while PAHs were similar to those reported for moderately impacted areas. No exposure to neurotoxic pesticides (physiological AChE activities) or to cytotoxic contaminants (low apoptosis frequency: <1%) can be inferred both in mussels and fish. . Detoxification enzyme activities in mussels were highly similar compared with those reported for low-impact areas. The results of the present ecotoxicological study on Vlora Bay also support its suitability in environmental quality assessment of marine coastal areas.

Occurrence of PCDD/PCDFs and PCBs in soil and comparison with CYP1A response in PLHC-1 cell line

The responsiveness of CYP1A (gene transcription and EROD enzyme activity) in the cell line Poeciliopsis lucida hepatoma (PLHC-1) upon exposure to extracts of contaminated soil samples was investigated and compared to levels of PCDD/PCDFs and PCBs including non-ortho obtained by GC/MS analysis. Soil samples A and B were collected in sites A and B. Two fractions, not purified (np) and purified (p), were obtained from each sample and analyzed for PCDD/PCDF and PCB content by GC/MS; in parallel they were tested for 24 h with PLHC-1. CYP1A response was investigated at gene (RT-qPCR) level and as 7-ethoxyresorufin-O-deethylase (EROD) enzyme activity. Chem-TEQs and Bio-TEQs were then calculated. ∑TEQ calculated for PCDD/Fs and PCBs was 0.081 pg/g and 20.32 pg/g for samples A and B, respectively. PLHC-1 showed less up-regulation of cyp1a gene on exposure to the two purified fractions (Ap 2.1-fold and Bp 1.8-fold) than to non-purified fractions (up to 15-fold for Anp and 13-fold for Bnp). EROD was also induced 2.38- and 9.44-fold in the two purified fractions (Ap and Bp) compared to model inducer 2,3,7,8-TCDD, and up to 16.03-fold for non-purified Anp and 33.79-fold for Bnp. The combination of CYP1A response, obtained in a PLHC-1 cell-based bioassay, with contaminant residue analysis provided a better description of the presence and toxicity of dioxin-like compounds in an environmental matrix.

PCB Muscle Content and Liver EROD Activity in the European EEL (Anguilla Anguilla) Treated with Aroclor 1254

The European eel (Anguilla anguilla) is a euryaline fish suitable for investigating exposure, accumulation patterns and biological effects of lipophilic pollutants such as polychlorinated biphenyls (PCBs). Eels were collected from the Orbetello Lagoon (Tyrrhenian coast of Italy) and injected with increasing doses of Aroclor 1254 to better characterise EROD activity as biomarker in eels from Mediterranean brackish environments. Fish muscle was analysed for PCB content and EROD activity was measured in liver microsomiaI fraction. Significant differences of PCB levels were observed in exposed fish (p < 0.05) while EROD activity increased significantly reaching the highest values at the maximum dose of 50mg/Kg (p < 0.001). Highly positive correlation (r = 0.82; p < 0.01) was found between PCB content and EROD activity. Isomer profiles showed decreases in hexa-CBs. The European eel is confirmed to be a useful sentinel species for assessing pollution in brackish environments whereas EROD activity is characterised as a sensitive biomarker of exposure for PCB mixtures.