Environmental implications of dental restorative materials on the zebrafish Danio rerio: Are dental chair drainage systems an emerging environmental threat?

This study aimed to evaluate the environmental impact of dental materials: commercial composite Tetric EvoCeram®, glass ionomer Equia Forte® HT Fil, laboratory-prepared composite, alkasite Cention® Forte, amalgam Amalcap® Plus, and samples from dental chair drainage systems (DCDS). Methacrylate monomers were detected in the eluates of experimental and commercials composites, and alkasite. In DCDS samples solely mercury was found at concentrations of 0.08-1.86 µg/L. The experimental composite (48 h incubation) exhibited the highest toxicity on zebrafish Danio rerio (LC50=0.70 g/L), followed by amalgam (LC50=8.27 g/L) < Tetric EvoCeram® (LC50=10.94 g/L) < Equia Forte® HT Fil (LC50=24.84 g/L) < Cention® Forte (LC50=32.22 g/L). Exposure of zebrafish to DCDS samples resulted in decreased larval body length and increased occurrences of edema and blood accumulation. The results obtained highlight the need for additional monitoring and further research on the release of unreacted monomers and mercury from dental materials and their environmental impact.

Combining short-term bioassays using fish and crustacean model organisms with ToxCast in vitro data and broad-spectrum chemical analysis for environmental risk assessment of the river water (Sava, Croatia).

This study focused on the short-term whole organism bioassays (WOBs) on fish (Danio rerio) and crustaceans (Gammarus fossarum and Daphnia magna) to assess the negative biological effects of water from the major European River Sava and the comparison of the obtained results with in vitro toxicity data (ToxCast database) and Risk Quotient (RQ) methodology. Pollution profiles of five sampling sites along the River Sava were assessed by simultaneous chemical analysis of 562 organic contaminants (OCs) of which 476 were detected. At each sampling site, pharmaceuticals/illicit drugs category was mostly represented by their cumulative concentration, followed by categories industrial chemicals, pesticides and hormones. An exposure-activity ratio (EAR) approach based on ToxCast data highlighted steroidal anti-inflammatory drugs, antibiotics, antiepileptics/neuroleptics, industrial chemicals and hormones as compounds with the highest biological potential. Summed EAR-based prediction of toxicity showed a good correlation with the estimated toxicity of assessed sampling sites using WOBs. WOBs did not exhibit increased mortality but caused various sub-lethal biological responses that were dependant relative to the sampling site pollution intensity as well as species sensitivity. Exposure of G. fossarum and D. magna to river water-induced lower feeding rates increased GST activity and TBARS levels. Zebrafish D. rerio embryo exhibited a significant decrease in heartbeat rate, failure in pigmentation formation, as well as inhibition of ABC transporters. Nuclear receptor activation was indicated as the biological target of greatest concern based on the EAR approach. A combined approach of short-term WOBs, with a special emphasis on sub-lethal endpoints, and chemical characterization of water samples compared against in vitro toxicity data from the ToxCast database and RQs can provide a comprehensive insight into the negative effect of pollutants on aquatic organisms.

Predictive Capability of QSAR Models Based on the CompTox Zebrafish Embryo Assays: An Imbalanced Classification Problem.

The CompTox Chemistry Dashboard (ToxCast) contains one of the largest public databases on Zebrafish (Danio rerio) developmental toxicity. The data consists of 19 toxicological endpoints on unique 1018 compounds measured in relatively low concentration ranges. The endpoints are related to developmental effects occurring in dechorionated zebrafish embryos for 120 hours post fertilization and monitored via gross malformations and mortality. We report the predictive capability of 209 quantitative structure-activity relationship (QSAR) models developed by machine learning methods using penalization techniques and diverse model quality metrics to cope with the imbalanced endpoints. All these QSAR models were generated to test how the imbalanced classification (toxic or non-toxic) endpoints could be predicted regardless which of three algorithms is used: logistic regression, multi-layer perceptron, or random forests. Additionally, QSAR toxicity models are developed starting from sets of classical molecular descriptors, structural fingerprints and their combinations. Only 8 out of 209 models passed the 0.20 Matthew's correlation coefficient value defined a priori as a threshold for acceptable model quality on the test sets. The best models were obtained for endpoints mortality (MORT), ActivityScore and JAW (deformation). The low predictability of the QSAR model developed from the zebrafish embryotoxicity data in the database is mainly due to a higher sensitivity of 19 measurements of endpoints carried out on dechorionated embryos at low concentrations.

Utilization of the zebrafish model to unravel the harmful effects of biomass burning during Amazonian wildfires.

Amazonian wildfires in 2019 have raised awareness about rainforest burning due to increased emissions of particulate matter and carbon. In the context of these emissions, by-products of lignin thermal degradation (i.e. methoxyphenols) are often neglected. Methoxyphenols entering the atmosphere may form intermediates with currently unknown reaction mechanisms and toxicity. This study for the first time provides a comprehensive insight into the impact of lignin degradation products [guaiacol, catechol], and their nitrated intermediates [4-nitrocatechol, 4,6-dinitroguaiacol, 5-nitroguaiacol] on zebrafish Danio rerio. Results revealed 4-nitrocatechol and catechol as the most toxic, followed by 4,6DNG > 5NG > GUA. The whole-organism bioassay integrated with molecular modeling emphasized the potential of methoxyphenols to inhibit tyrosinase, lipoxygenase, and carbonic anhydrase, consequently altering embryonic development (i.e. affected sensorial, skeletal, and physiological parameters, pigmentation formation failure, and non-hatching of larvae). The whole-organism bioassay integrated with in silico approach confirmed the harmful effects of lignin degradation products and their intermediates on aquatic organisms, emphasizing the need for their evaluation within ecotoxicity studies focused on aquatic compartments.

Toxicity prediction and effect characterization of 90 pharmaceuticals and illicit drugs measured in plasma of fish from a major European river (Sava, Croatia).

Chemical analysis of plasma samples of wild fish from the Sava River (Croatia) revealed the presence of 90 different pharmaceuticals/illicit drugs and their metabolites (PhACs/IDrgs). The concentrations of these PhACs/IDrgs in plasma were 10 to 1000 times higher than their concentrations in river water. Antibiotics, allergy/cold medications and analgesics were categories with the highest plasma concentrations. Fifty PhACs/IDrgs were identified as chemicals of concern based on the fish plasma model (FPM) effect ratios (ER) and their potential to activate evolutionary conserved biological targets. Chemicals of concern were also prioritized by calculating exposure-activity ratios (EARs) where plasma concentrations of chemicals were compared to their bioactivities in comprehensive ToxCast suite of in vitro assays. Overall, the applied prioritization methods indicated stimulants (nicotine, cotinine) and allergy/cold medications (prednisolone, dexamethasone) as having the highest potential biological impact on fish. The FPM model pointed to psychoactive substances (hallucinogens/stimulants and opioids) and psychotropic substances in the cannabinoids category (i.e. CBD and THC). EAR confirmed above and singled out additional chemicals of concern - anticholesteremic simvastatin and antiepileptic haloperidol. Present study demonstrates how the use of a combination of chemical analyses, and bio-effects based risk predictions with multiple criteria can help identify priority contaminants in freshwaters. The results reveal a widespread exposure of fish to complex mixtures of PhACs/IDrgs, which may target common molecular targets. While many of the prioritized chemicals occurred at low concentrations, their adverse effect on aquatic communities, due to continuous chronic exposure and additive effects, should not be neglected.

Adherence to Mediterranean Diet and Maternal Lifestyle during Pregnancy: Island-Mainland Differentiation in the CRIBS Birth Cohort.

Maternal nutrition and lifestyle in pregnancy are important modifiable factors for both maternal and offspring's health. Although the Mediterranean diet has beneficial effects on health, recent studies have shown low adherence in Europe. This study aimed to assess the Mediterranean diet adherence in 266 pregnant women from Dalmatia, Croatia and to investigate their lifestyle habits and regional differences. Adherence to the Mediterranean diet was assessed through two Mediterranean diet scores. Differences in maternal characteristics (diet, education, income, parity, smoking, pre-pregnancy body mass index (BMI), physical activity, contraception) with regards to location and dietary habits were analyzed using the non-parametric Mann-Whitney U test. The machine learning approach was used to reveal other potential non-linear relationships. The results showed that adherence to the Mediterranean diet was low to moderate among the pregnant women in this study, with no significant mainland-island differences. The highest adherence was observed among wealthier women with generally healthier lifestyle choices. The most significant mainland-island differences were observed for lifestyle and socioeconomic factors (income, education, physical activity). The machine learning approach confirmed the findings of the conventional statistical method. We can conclude that adverse socioeconomic and lifestyle conditions were more pronounced in the island population, which, together with the observed non-Mediterranean dietary pattern, calls for more effective intervention strategies.

Association of bacterial load in drinking water and allergic diseases in childhood.

BACKGROUND: Treatment of drinking water may decrease microbial exposure. OBJECTIVE: To investigate whether bacterial load in drinking water is associated with altered risk of allergic diseases. METHODS: We recruited 1,110 schoolchildren aged 6-16 years between 2011 and 2013 in Pozega-Slavonia County in Croatia, where we capitalized on a natural experiment whereby individuals receive drinking water through public mains supply or individual wells. We obtained data on microbial content of drinking water for all participants; 585 children were randomly selected for more detailed assessments, including skin prick testing. Since water supply was highly correlated with rural residence, we compared clinical outcomes across four groups (Rural/Individual, Rural/Public, Urban/Individual and Urban/Public). For each child, we derived quantitative index of microbial exposure (bacterial load in the drinking water measured during the child's first year of life). RESULTS: Cumulative bacterial load in drinking water was higher (median [IQR]: 6390 [4190-9550] vs 0 [0-0]; P < .0001), and lifetime prevalence of allergic diseases was significantly lower among children with individual supply (5.5% vs 2.3%, P = .01; 14.4% vs 6.7%, P < .001; 25.2% vs 15.1%, P < .001; asthma, atopic dermatitis [AD] and rhinitis, respectively). Compared with the reference group (Urban/Public), there was a significant reduction in the risk of ever asthma, AD and rhinitis amongst rural children with individual supply: OR [95% CI]: 0.14 [0.03,0.67], P = .013; 0.20 [0.09,0.43], P < .001; 0.17 [0.10,0.32], P < .001. Protection was also observed in the Rural/Public group, but the effect was consistently highest among Rural/Individual children. In the quantitative analysis, the risk of allergic diseases decreased significantly with increasing bacterial load in drinking water in the first year of life (0.79 [0.70,0.88], P < .001; 0.90 [0.83,0.99], P = .025; 0.80 [0.74,0.86], P < .001; current wheeze, AD and rhinitis). CONCLUSIONS AND CLINICAL RELEVANCE: High commensal bacterial content in drinking water may protect against allergic diseases.

Toxicity evaluation of olive oil mill wastewater and its polar fraction using multiple whole-organism bioassays.

Olive mill wastewater (OMW) as a by-product of olive oil extraction process has significant polluting properties mainly related to high organic load, increased COD/BOD ratio, high phenolic content and relatively acidic pH. Raw OMW from Slovenian Istria olive oil mill and its polar fraction were investigated in this study. Chemical characterization of OMW polar fraction identified tyrosol as the most abundant phenolic product, followed by catechol. Lethal and sub-lethal effects of OMW matrix and its polar fraction were tested using a battery of bioassays with model organisms: bacteria Vibrio fischeri, algae Chlorella vulgaris, water fleas Daphnia magna, zebrafish Danio rerio embryos, clover Trifolium repens and wheat Triticum aestivum. Raw OMW sample was the most toxic to V. fischeri (EC50 = 0.24% of OMW sample final concentration), followed by D. magna (EC50 = 1.43%), C. vulgaris (EC50 = 5.20%), D. rerio (EC50 = 7.05%), seeds T. repens (EC50 = 8.68%) and T. aestivum (EC50 = 11.58%). Similar toxicity trend was observed during exposure to OMW polar fraction, showing EC50 values 2.75-4.11 times lower comparing to raw OMW. Tested samples induced also sub-acute effects to clover and wheat (decreased roots, sprouts elongation); and to zebrafish embryos (increased mortality, higher abnormality rate, decreased hatching and pigmentation formation rate). A comprehensive approach using a battery of bioassays, like those used in this study should be applied during ecotoxicity monitoring of untreated and treated OMW.

Assessment of river sediment toxicity: Combining empirical zebrafish embryotoxicity testing with in silico toxicity characterization.

Quantitative chemical analyses of 428 organic contaminants (OCs) indicated the presence of 313 OCs in the sediment extracts from Sava River, Croatia. Pharmaceuticals were present in higher concentrations than pesticides thus confirming their increasing threat to freshwater ecosystems. Toxicity evaluation of the sediment extracts from four locations (Jesenice, Rugvica, Galdovo and Lukavec) using zebrafish embryotoxicity test (ZET) accompanied with semi-quantitative histopathological analyses exhibited correlation with cumulative number and concentrations of OCs at the investigated sites (10.05, 15.22, 1.25, and 9.13 µg/g respectively). Toxicity of sediment extracts and sediment was predicted using toxic unit (TU) approach and persistence, bioaccumulation and toxicity (PBT) ranking. Additionally, influential OCs and genes were identified by graph mining of the prior knowledge informed, site-specific chemical-gene interaction models. Predicted toxicity of sediment extracts (TUext) was similar to the results obtained by ZET and associated histopathology with Rugvica sediment being the most toxic, followed by Jesenice, Lukavec and Galdovo. Sediment TU (TUsed) favoured OCs with low octanol-water partition coefficients like herbicide glyphosate and antibiotics ciprofloxacin and sulfamethazine thus indicating locations containing higher concentrations of these OCs (Galdovo and Rugvica) as the most toxic. Results suggest that comprehensive in silico sediment toxicity predictions advocate providing equal attention to organic contaminants with either very low or very high log Kow.

Effects of CeO2 Nanoparticles on Terrestrial Isopod Porcellio scaber: Comparison of CeO2 Biological Potential with Other Nanoparticles.

Nano-sized cerium dioxide (CeO2) particles are emerging as an environmental issue due to their extensive use in automobile industries as fuel additives. Limited information is available on the potential toxicity of CeO2 nanoparticles (NPs) on terrestrial invertebrates through dietary exposure. In the present study, the toxic effects of CeO2 NPs on the model soil organism Porcellio scaber were evaluated. Nanotoxicity was assessed by monitoring the lipid peroxidation (LP) level and feeding rate after 14-days exposure to food amended with nano CeO2. The exposure concentration of 1000 µg of CeO2 NPs g-1 dry weight food for 14 days significantly increased both the feeding rate and LP. Thus, this exposure dose is considered the lowest observed effect dose. At higher exposure doses of 2000 and 5000 µg of CeO2 NPs g-1 dry weight food, NPs significantly decreased the feeding rate and increased the LP level. Comparative studies showed that CeO2 NPs are more biologically potent than TiO2 NPs, ZnO NPs, CuO NPs, CoFe2O4 NPs, and Ag NPs based on feeding rate using the same model organism and experimental setup. Based on comparative metal oxide NPs toxicities, the present results contribute to the knowledge related to the ecotoxicological effects of CeO2 NPs in terrestrial invertebrates exposed through feeding.

Genotoxicity monitoring of freshwater environments using caged crayfish (Astacus leptodactylus).

Genotoxicity of freshwater pollution was assessed by measuring DNA damage in haemocytes of caged freshwater crayfish Astacus leptodactylus by the means of Comet assay and micronucleus test, integrated with the measurements of physiological (total protein concentration) and immunological (total haemocyte count) haemolymph parameters as biomarkers of undergone stress. Crayfish were collected at the reference site (River Mreznica) and exposed in cages for 1 week at three polluted sites along the Sava River (Zagreb, Sisak, Krapje). The long term pollution status of these locations was confirmed by chemical analyses of sediments. Statistically significant increase in DNA damage measured by the Comet assay was observed at all three polluted sites comparing to the crayfish from reference site. In addition, native crayfish from the mildly polluted site (Krapje) cage-exposed on another polluted site (Zagreb) showed lower DNA damage than crayfish from the reference site exposed at the same location indicating adaptation and acclimatisation of crayfish to lower levels of pollution. Micronuclei induction showed similar gradient of DNA damage as Comet assay, but did not reach the statistical significance. Observed increase in total haemocyte count and total protein content in crayfish from polluted environments in the Sava River also confirmed stress caused by exposure to pollution. The results of this study have proved the applicability of caging exposure of freshwater crayfish A. leptodactylus in environmental genotoxicity monitoring using Comet assay and micronucleus test.

Genotoxic, physiological and immunological effects caused by temperature increase, air exposure or food deprivation in freshwater crayfish Astacus leptodactylus.

The aim of this research was to investigate influence of different environmental stressors, such as temperature increase, air exposure and food deprivation on DNA integrity of a bioindicator species, freshwater crayfish Astacus leptodactylus. DNA damage was measured in crayfish haemocytes using Comet assay and micronucleus test. Crayfish haemolymph was subsequentially sampled during their 7 days of exposure to increased temperatures (25 and 30 degrees C) and during 24 h of air exposure. Both groups were also monitored through the following 7 days of recovery period. Food deprived crayfish were monitored over a period of 2 weeks. Alterations of measured physiological and immunological haemolymph parameters (THC, lactate, glucose and protein concentration) indicated stress response in exposed crayfish. However, only the stress induced by increased temperature significantly increased DNA damage in freshwater crayfish while food deprivation or air exposure did not cause a significant genotoxic effect.