Aryl hydrocarbon reporter gene bioassay for screening polyhalogenated dibenzo-p-dioxins/furans and dioxin-like polychlorinated biphenyls in hydrochar and sewage sludge.

The suitability of the AhR reporter gene bioassays to screen the presence of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) in sewage sludge (SL) and related hydrochar (HC) was here investigated. Samples of SL obtained from six WWTPs were processed by hydrothermal carbonization to obtain the resultant HCs and both tested with DR-CALUX® bioassay. Levels of PCDD/Fs and dl-PCBs were also determined analytically in the same samples by GC-MS/MS. Bioanalytical Toxicity Equivalent values (BEQ) resulted in one order of magnitude higher in HC compared to SL samples and those obtained from the dl-PCBs fraction higher than those from PCDD/Fs. BEQ and TEQWHO values, the latter obtained by GC-MS/MS analysis on the same matrices, were highly correlated showing also a similar trend in the six WWTPs (RS= 0.8252, p < 0.001; Pearson's R RP =0.8029, p < 0.01). The suitability of AhR bioassays and in particular of the DR-CALUX® to screen the presence and biological activity of legacy organohalogen compounds in both SL and HC matrices was demonstrated for the first time which support their usage for the assessment of potential risks associated with their further environmental applications.

Eco-Interactions of Engineered Nanomaterials in the Marine Environment: Towards an Eco-Design Framework.

Marine nano-ecotoxicology has emerged with the purpose to assess the environmental risks associated with engineered nanomaterials (ENMs) among contaminants of emerging concerns entering the marine environment. ENMs' massive production and integration in everyday life applications, associated with their peculiar physical chemical features, including high biological reactivity, have imposed a pressing need to shed light on risk for humans and the environment. Environmental safety assessment, known as ecosafety, has thus become mandatory with the perspective to develop a more holistic exposure scenario and understand biological effects. Here, we review the current knowledge on behavior and impact of ENMs which end up in the marine environment. A focus on titanium dioxide (n-TiO2) and silver nanoparticles (AgNPs), among metal-based ENMs massively used in commercial products, and polymeric NPs as polystyrene (PS), largely adopted as proxy for nanoplastics, is made. ENMs eco-interactions with chemical molecules including (bio)natural ones and anthropogenic pollutants, forming eco- and bio-coronas and link with their uptake and toxicity in marine organisms are discussed. An ecologically based design strategy (eco-design) is proposed to support the development of new ENMs, including those for environmental applications (e.g., nanoremediation), by balancing their effectiveness with no associated risk for marine organisms and humans.

Occurrence and spatial distribution of dioxin and dioxin-like compounds in topsoil of Taranto (Apulia, Italy) by GC-MS analysis and DR-CALUX® bioassay.

The aim of the present study was to assess the occurrence and spatial distribution of PCDD/Fs and dioxin-like compounds in topsoils of Taranto (Apulia Region), one of the most heavily industrialized and contaminated area of Southern Italy. A combined approach of chemical analysis by GC-MS/MS and AhR reporter gene bioassay was applied in a subset of topsoil samples (n = 20) collected in 2017-18 from ten sites embracing three levels of risk (from high to low) in the framework of a large survey inside Taranto municipality. TCDD-BEQs and GC-MS/MS TEQWHO and TEQTHEORETICAL revealed a decreasing trend with the distance from main industrial settings and landfill areas. A strong correlation between TCDD-BEQs and TEQWHO values (R2 = 0.85) and TEQTHEORETICAL (R2 = 0.88) was also found. In 3 out of 10 topsoil investigated, BEQs and TEQWHO/THEORETICAL resulted above Italian National Regulatory Limits for ∑PCDD/Fs in green, private and recreational used soils (10 ng TEQ/kg d.w. D.Lgs 152/2006) and for ∑PCDD/F/dl-PCBs in agricultural and farming soil (6 ng TEQ/kg d.w. D.M. 46/2019). GC-MS/MS pattern revealed the highest prevalence of dl-PCBs in 6 out of 10 sites, followed by PCDFs and PCDDs. Those sites are all located in proximity of main industrial steel and iron ore sinter plant, steel plant's landfills and illegal dumping sites. An update on occurrence and spatial distribution of PCDD/Fs and dl-PCBs contamination of Taranto urban soils was obtained and the DR-CALUX® bioassay was further recommended as a suitable screening tool for environmental and human risk assessment.

First Attempt to Couple Proteomics with the AhR Reporter Gene Bioassay in Soil Pollution Monitoring and Assessment.

A topsoil sample obtained from a highly industrialized area (Taranto, Italy) was tested on the DR-CALUX® cell line and the exposed cells processed with proteomic and bioinformatics analyses. The presence of polyhalogenated compounds in the topsoil extracts was confirmed by GC-MS/MS analysis. Proteomic analysis of the cells exposed to the topsoil extracts identified 43 differential proteins. Enrichment analysis highlighted biological processes, such as the cellular response to a chemical stimulus, stress, and inorganic substances; regulation of translation; regulation of apoptotic process; and the response to organonitrogen compounds in light of particular drugs and compounds, extrapolated by bioinformatics all linked to the identified protein modifications. Our results confirm and reflect the complex epidemiological situation occurring among Taranto inhabitants and underline the need to further investigate the presence and sources of inferred chemicals in soils. The combination of bioassays and proteomics reveals a more complex scenario of chemicals able to affect cellular pathways and leading to toxicities rather than those identified by only bioassays and related chemical analysis. This combined approach turns out to be a promising tool for soil risk assessment and deserves further investigation and developments for soil monitoring and risk assessment.

Exposure to a nanosilver-enabled consumer product results in similar accumulation and toxicity of silver nanoparticles in the marine mussel Mytilus galloprovincialis.

The incorporation of silver nanoparticles (AgNPs) in commercial products is increasing rapidly. The consequent release of AgNPs into domestic and industrial wastewater raises environmental concerns due to their anti-microbial properties and toxicity to non-target aquatic organisms. The aim of the present study was to investigate the effects of nanArgen™ (Nanotek S.A.), a AgNP-enabled consumer product, in the marine bivalve Mytilus galloprovincialis. Two environmentally relevant concentrations of nanArgen™ (1 and 10 µg/L) were tested in vivo for 96 h, and Ag was quantified in mussel soft tissue and natural seawater (NSW). nanArgen™ suspensions were characterized via TEM, SEM, EDS, DLS, and UV-vis optical analysis. Several molecular and biochemical responses were investigated in exposed mussels: lysosomal membrane stability by Neutral Red Retention Time (NRRT) assay; micronucleus (MN) frequency in hemocytes; metallothionein (MT) protein content and gene expression (mt10 and mt20); catalase (CAT) and glutathione-S-transferase (GST) activities; malondialdehyde (MDA) accumulation in digestive glands; and efflux activity of ATP-binding cassette transport proteins (ABC) in gill biopsies. SEM, TEM and DLS analyses confirmed the presence of well-defined AgNPs in nanArgen™ which were roughly spherical with an average particle size of approx. 30 ± 10 nm. DLS analysis revealed the formation of AgNP aggregates in nanArgen™ suspension in NSW (Z-average of 547.80 ± 90.23 nm; PDI of 0.044). A significant concentration-dependent accumulation of Ag was found in mussels' whole soft tissue in agreement with a concentration-dependent decrease in NRRT and an increase of MN frequency in hemocytes and GST activities in digestive glands. A significant increase in MDA levels and MT via both molecular and biochemical tests, were also observed but only at the highest nanArgen™ concentration (10 µg/L). No changes were observed in CAT activities. ABC efflux activities in gill biopsies showed a significant decrease (p < 0.05) only at the lowest concentration (1 µg/L). On such basis, nanArgen™ is shown to be able to induce toxicity and Ag accumulation in marine mussels similarly to AgNPs and in short-term exposure conditions at environmentally relevant concentrations. AgNP-enabled products, instead of pristine AgNPs, should be the focus of future ecotoxicity studies in order to address any risks associated to their widespread use, disposal and uncontrolled release into the aquatic environment for non target species.

Amino-modified polystyrene nanoparticles affect signalling pathways of the sea urchin (Paracentrotus lividus) embryos.

Polystyrene nanoparticles have been shown to pose serious risk to marine organisms including sea urchin embryos based on their surface properties and consequently behaviour in natural sea water. The aim of this study is to investigate the toxicity pathways of amino polystyrene nanoparticles (PS-NH2, 50 nm) in Paracentrotus lividus embryos in terms of development and signalling at both protein and gene levels. Two sub-lethal concentrations of 3 and 4 µg/mL of PS-NH2 were used to expose sea urchin embryos in natural sea water (PS-NH2 as aggregates of 143 ± 5 nm). At 24 and 48 h post-fertilisation (hpf) embryonic development was monitored and variations in the levels of key proteins involved in stress response and development (Hsp70, Hsp60, MnSOD, Phospho-p38 Mapk) as well as the modulation of target genes (Pl-Hsp70, Pl-Hsp60, Pl-Cytochrome b, Pl-p38 Mapk, Pl-Caspase 8, Pl-Univin) were measured. At 48 hpf various striking teratogenic effects were observed such as the occurrence of cells/masses randomly distributed, severe skeletal defects and delayed development. At 24 hpf a significant up-regulation of Pl-Hsp70, Pl-p38 Mapk, Pl-Univin and Pl-Cas8 genes was found, while at 48 hpf only for Pl-Univin was observed. Protein profile showed different patterns as a significant increase of Hsp70 and Hsp60 only after 48 hpf compared to controls. Conversely, P-p38 Mapk protein significantly increased at 24 hpf and decreased at 48 hpf. Our findings highlight that PS-NH2 are able to disrupt sea urchin embryos development by modulating protein and gene profile providing new understandings into the signalling pathways involved.

Nano-sized polystyrene affects feeding, behavior and physiology of brine shrimp Artemia franciscana larvae.

Nano-sized polymers as polystyrene (PS) constitute one of the main challenges for marine ecosystems, since they can distribute along the whole water column affecting planktonic species and consequently disrupting the energy flow of marine ecosystems. Nowadays very little knowledge is available on the impact of nano-sized plastics on marine organisms. Therefore, the present study aims to evaluate the effects of 40nm anionic carboxylated (PS-COOH) and 50nm cationic amino (PS-NH2) polystyrene nanoparticles (PS NPs) on brine shrimp Artemia franciscana larvae. No signs of mortality were observed at 48h of exposure for both PS NPs at naplius stage but several sub-lethal effects were evident. PS-COOH (5-100µg/ml) resulted massively sequestered inside the gut lumen of larvae (48h) probably limiting food intake. Some of them were lately excreted as fecal pellets but not a full release was observed. Likewise, PS-NH2 (5-100µg/ml) accumulated in larvae (48h) but also adsorbed at the surface of sensorial antennules and appendages probably hampering larvae motility. In addition, larvae exposed to PS-NH2 undergo multiple molting events during 48h of exposure compared to controls. The activation of a defense mechanism based on a physiological process able to release toxic cationic NPs (PS-NH2) from the body can be hypothesized. The general observed accumulation of PS NPs within the gut during the 48h of exposure indicates a continuous bioavailability of nano-sized PS for planktonic species as well as a potential transfer along the trophic web. Therefore, nano-sized PS might be able to impair food uptake (feeding), behavior (motility) and physiology (multiple molting) of brine shrimp larvae with consequences not only at organism and population level but on the overall ecosystem based on the key role of zooplankton on marine food webs.

Combination effects of nano-TiO2 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on biotransformation gene expression in the liver of European sea bass Dicentrarchus labrax.

The aim of present study was to investigate the influence of titanium dioxide nanoparticles (nano-TiO2, Aeroxide® P25) on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dependent biotransformation gene expression in liver of juvenile European sea bass Dicentrarchus labrax. An in vivo 7day waterborne exposure was performed with nano-TiO2 (1mg/L) and 2,3,7,8-TCDD (46pg/L), singly and in combination. The mRNA expression of aryl hydrocarbon receptor repressor (Ahrr), estrogen receptor (erß2), ABC transport proteins as Abcb1, Abcc1-c2-g2, cytochrome P450 (cyp1a), glutathione-s-transferase (gsta), glutathione reductase (gr) and engulfment and motility (ELMO) domain-containing protein 2 (elmod2) was investigated. Ahrr, erß2, abcc1 and abcg2 resulted down-regulated with respect to controls in all experimental groups. Co-exposure to nano-TiO2 and 2,3,7,8-TCDD caused a further significant down regulation of ahrr, erß2, Abcb1 and Abcc2 compared to single chemical exposure (nano-TiO2 or 2,3,7,8-TCDD alone). No effects were observed for 2,3,7,8-TCDD and nano-TiO2 alone in abcb1 gene, while abcc2 was down-regulated by nano-TiO2 alone. Cyp1a, gst and elmod2 genes were up-regulated by 2,3,7,8-TCDD and to a similar extent after co-exposure. Overall the results indicate that nano-TiO2 is unlikely to interfere with 2,3,7,8-TCDD-dependent biotransformation gene expression in the liver of European sea bass, although the effects of co-exposure observed in ABC transport mRNAs might suggest an impact on xenobiotic metabolite disposition and transport in European sea bass liver.

Influence of titanium dioxide nanoparticles on 2,3,7,8-tetrachlorodibenzo-p-dioxin bioconcentration and toxicity in the marine fish European sea bass (Dicentrarchus labrax).

The present study investigated the influence of nano-TiO(2) (1 mg L(-1)) on 2,3,7,8-tetrachlorodibenzo-p-dioxin(2,3,7,8-TCDD) (46 pg L(-1)) bioconcentration and toxicity in the European sea bass (Dicentrarchus labrax) during 7 days in vivo exposure. A multimarkers approach was applied in different organs: detoxification in liver; innate immunity and pro-inflammatory response and adaptive immunity in gills and spleen; genotoxicity in peripheral erythrocytes and muscle. Bioconcentration of 2,3,7,8-TCDD in presence of nano-TiO2 was investigated in liver, skin and muscle as well as interaction between nano-TiO2 and organic pollutants in artificial sea water (ASW). Nano-TiO2 negatively influenced immune response induced by 2,3,7,8-TCDD in spleen but not in gills and reduced the DNA damage induced by 2,3,7,8-TCDD in erythrocytes. nano-TiO2 did not interfere with 2,3,7,8-TCDD detoxification and bioconcentration according to the observed no interaction of the nano-TiO2 with organic pollutants in ASW.

Induction of CYP1A and ABC transporters in European sea bass (Dicentrarchus labrax) upon 2,3,7,8-TCDD waterborne exposure.

The aim of this study was to characterize the responsiveness of CYP1A and ABC transport proteins in European Sea bass (Dicentrarchus labrax) waterborne exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) (46 pg/L) for 24 h and 7 days. Genes modulation (abcb1, abcc1-2, cyp1a), EROD activity were investigated in liver and 2,3,7,8-TCDD bioconcentration in liver and muscle. TCDD induced significantly cyp1a gene expression and EROD activity at 24 h and 7 d. A significant up-regulation of abcb1 was also observed but only after 7 days. No modulation of abcc1 and abcc2 genes was observed. Waterborne TCDD exposure was able to induce CYP1A and abcb1 encoding for P-glycoprotein in juvenile of European sea bass.