The concentration of phthalates in toys and children's care items in Kosovo.

In this study, one hundred and twenty-eight toys and children's care items available in the Kosovo market were analyzed to determine the presence of phthalates and evaluate if the analyzed samples meet the national and EU standards. Toys and children's care items were imported from twelve countries, most of them from China. Phthalates were extracted and then analyzed using gas chromatography and mass spectrometry. Nine phthalates were analyzed in total, of them, benzyl butyl phthalate, di-isodecyl phthalate, diisobutyl phthalate and di-isononyl phthalate were the most frequently detected phthalates in the analyzed samples. Phthalates were not detected only in three toys and children's care items, others contained one or more phthalates, while none of them contained all (nine) analyzed phthalates. In total, 22 analyzed toys and children's care items did not fulfill at least one of the EU standards, of these 16 were toys and children's care items intended for children under age three and designed to be placed in the mouth. Overall, our work shows that toys and children's care items that exceed EU standards for phthalates are still present in the Kosovo market.

Commercial beers: A source of phthalates and di-ethylhexyl adipate.

Beer is one of the most consumed beverages worldwide. Different materials used along its production and packaging can result in human exposure to phthalates and adipates. The aim of this study was to assess simultaneously the levels of phthalates and di-ethylhexyl adipate (DEHA) in commercial beer samples (n = 66) with a method based on DLLME and detection with GC-MS/MS, and further evaluate human exposure. Six out of seven compounds studied were found in the beers analysed, with levels ranging from 1.77 to 205.40 µg/L. The most prevalent was DEHA at 205.40 µg/L, while dimethyl phthalate (DMP) was not present in any sample. Samples with 5-6 % alcohol, packed in aluminium cans and produced in an industrial environment presented the highest level of these contaminants. Despite low-risk exposure to phthalates and adipate with beer, it is important to remember the ubiquitous nature of these compounds, which can lead to cumulative exposure.

Lipophilic phthalic acid esters impair human sperm acrosomal reaction through the likely inhibition of phospholipase A2-signaling pathway.

Phthalic acid esters (PAEs) are recognized endocrine disruptors. Detection of PAEs in semen from idiopathic infertile males suggests possible direct mechanisms of sperm toxicity. In this study we aimed to correlate sperm function with semen levels of PAEs. Semen samples were obtained from 100 male patients attending the Unit of Andrology and Reproductive Medicine, University Hospital of Padova, (Italy), 22 of which having a recognized history of idiopathic infertility. Compared to fertile subjects, infertile patients showed reduced levels of acrosome reaction (AR), evaluated by CD46 staining upon progesterone (P4) triggering (p < 0.001). Subjects showing positive detection of PAEs in semen, evaluated by liquid chromatography-mass spectrometry (LC-MS), were significantly more represented in those reporting an history of infertility (13 out of 22), compared to fertile subjects (25 out of 78, P = 0.0266). In vitro sperm exposure to PAEs showed that lipophilic PAE representative Di-n-octyl phthalate (DNOP) had higher cell accumulation and inhibition of P4-induced AR than less lipophilic PAE representative Dibutyl phthalate (DBP). Computer-based binding analysis and fluorimetric inhibition assay, showed that both DNOP and DBP had similar Phospholipase-A2 (PLA2) inhibitory activity (respectively: 3.98 nM and 5.52 nM). However, only DNOP showed a significant inhibition of PLA2-mediated AR, triggered by A23187 calcium ionophore. Incubation with PLA2-related product arachidonic acid restored AR. Our data are suggestive of a novel mechanistic model of PAEs interference on sperm function, through the inhibition of PLA2-mediated signaling. According to this hypothesis, the inhibitory efficacy of the specific PAE is possibly linked to the corresponding cell accumulation.

Evaluation of occurrence, fate and removal of priority phthalate esters (PAEs) in wastewater and sewage sludge by advanced biological treatment, waste stabilization pond and constructed wetland.

Phthalate Esters (PAEs), detected in high concentrations generally in treated wastewater discharged from wastewater treatment plants (WWTPs), are important pollutants that restrict the reuse of wastewater. Investigating the fate of these endocrine-disrupting chemicals in WWTPs is crucial in order to protect both receiving environments and ecosystems. For this purpose, di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP) and benzyl butyl phthalate (BBP) in the group of PAEs were monitored in simultaneously both in wastewater and sludge lines of selected two nature-based WWTPs and one advanced biological WWTP. Although it was frequently stated that phthalates were significantly removed in WWTPs in many studies found in literature, negative removal efficiencies of selected phthalates in investigated WWTPs during the sampling period were observed generally in this study. One of the reasons for this concentration increase could be releasing of phthalates from microplastics in wastewater during the treatment process or the desorption of PAEs from treatment sludge. DNOP was the compound with the highest concentration increase at almost each treatment unit of the three WWTPs. On the other hand, total PAEs load was 1997 g d-1 in advanced biological WWTP and adsorption onto sludge of PAEs were determined as 90%. The side-stream total load returned from the decanter supernatant was 0.02% of the total PAEs load coming to advanced biological WWTP from the sewer system. As a result of detailed statistical analysis, the correlation between raw wastewater and primary clarifier (PC) effluent was determined as an increasing linear relation for DEHP and DNOP. On the other hand, moderate and strong correlations were observed both between septic tank and constructed wetland (CW) processes with raw wastewater. In the waste stabilization pond (WSP), while a significant correlation was not found between the sludge line data, homogeneous variance, strong and moderate correlations were obtained in the wastewater line data. However, while mean differences for all investigated PAEs were not significant (p > 0.05) in the wastewater line, mean differences of DEHP (p < 0.05) were significant in the sludge line according to ANOVA analysis.

Effect of DEHP and DnOP on mitochondrial damage and related pathways of Nrf2 and SIRT1/PGC-1α in HepG2 cells.

Di-2-ethylhexyl phthalate (DEHP) and Dioctyl phthalate (DnOP) are widely used as plasticizers in various industries for which the consequent health problems are of great concern. In this context, we treated HepG2 cells with DEHP or DnOP for 48 h. The results showed that DEHP and DnOP caused increase in oxygen species (ROS), malondialdehyde (MDA), Alanine aminotransferase (ALT) and Aspartate transaminase (AST). The proteins NF⁃E2-related factor 2 (Nrf2) and haemeoxygenase-1 (HO-1), were significantly down-regulated. Subsequently, the mitochondrial structure was disrupted, and the ATP content, the mitochondrial copy number as well as the expression of the corresponding mitochondrial genes were also reduced. The expression of sirtuin 1(SIRT1), PPAR gamma co-activator 1 alpha (PGC-1α), Nuclear respiratory factor 1(Nrf1), Mitochondrial transcription factor A (TFAM) on the SIRT1/PGC-1α pathway were significantly reduced. Finally, neither DEHP nor DnOP was found to induce apoptosis, but could significantly up-regulate Light chain 3 II (LC3II) levels. In conclusion, DEHP and DnOP could induce HepG2 cell damage via mitochondria, probably by causing oxidative stress, inhibiting the Nrf2 pathway and inhibiting the mitochondrial biogenesis pathway, which leads to excessive autophagy and cell death. DEHP and DnOP differ in the Nrf2 pathway, autophagic pathway and MAPK pathway, which may be structurally related.

Review of the existing maximum residue levels for meptyldinocap (DE-126) according to Article 12 of Regulation (EC) No 396/2005.

According to Article 12 of Regulation (EC) No 396/2005, EFSA has reviewed the maximum residue levels (MRLs) currently established at European level for the pesticide active substance meptyldinocap (DE-126). To assess the occurrence of meptyldinocap residues in plants, processed commodities, rotational crops and livestock, EFSA considered the conclusions derived in the framework of Regulation (EC) No 1107/2009, Commission Regulation (EU) No 188/2011 and the MRLs established by the Codex Alimentarius Commission as well as the European authorisations reported by Member States (including the supporting residues data). Based on the assessment of the available data, MRL proposals were derived and a consumer risk assessment was carried out. Although no apparent risk to consumers was identified, some information required by the regulatory framework was missing. Hence, the consumer risk assessment is considered indicative only and some MRL proposals derived by EFSA still requires further consideration by risk managers.

Early microRNA indicators of PPARα pathway activation in the liver.

MicroRNAs (miRNAs) are short non-coding RNA species that play key roles in post-transcriptional regulation of gene expression. MiRNAs also serve as a promising source of early biomarkers for different environmental exposures and health effects, although there is limited information linking miRNA changes to specific target pathways. In this study, we measured liver miRNAs in male B6C3F1 mice exposed to a known chemical activator of the peroxisome proliferator-activated receptor alpha (PPARα) pathway, di(2-ethylhexyl) phthalate (DEHP), for 7 and 28 days at concentrations of 0, 750, 1500, 3000, or 6000 ppm in feed. At the highest dose tested, DEHP altered 61 miRNAs after 7 days and 171 miRNAs after 28 days of exposure, with 48 overlapping miRNAs between timepoints. Analysis of these 48 common miRNAs indicated enrichment in PPARα-related targets and other pathways related to liver injury and cancer. Four of the 10 miRNAs exhibiting a clear dose trend were linked to the PPARα pathway: mmu-miRs-125a-5p, -182-5p, -20a-5p, and -378a-3p. mmu-miRs-182-5p and -378a-3p were subsequently measured using digital drop PCR across a dose range for DEHP and two related phthalates with weaker PPARα activity, di-n-octyl phthalate and n-butyl benzyl phthalate, following 7-day exposures. Analysis of mmu-miRs-182-5p and -378a-3p by transcriptional benchmark dose analysis correctly identified DEHP as having the greatest potency. However, benchmark dose estimates for DEHP based on these miRNAs (average 163; range 126-202 mg/kg-day) were higher on average than values for PPARα target genes (average 74; range 29-183 mg/kg-day). These findings identify putative miRNA biomarkers of PPARα pathway activity and suggest that early miRNA changes may be used to stratify chemical potency.

New insights into the risk of phthalates: Inhibition of UDP-glucuronosyltransferases.

Wide utilization of phthalates-containing products results in the significant exposure of humans to these compounds. Many adverse effects of phthalates have been documented in rodent models, but their effects in humans exposed to these chemicals remain unclear until more mechanistic studies on phthalate toxicities can be carried out. To provide new insights to predict the potential adverse effects of phthalates in humans, the recent study investigated the inhibition of representative phthalates di-n-octyl ortho-phthalate (DNOP) and diphenyl phthalate (DPhP) towards the important xenobiotic and endobiotic-metabolizing UDP-glucuronosyltransferases (UGTs). An in vitro UGTs incubation system was employed to study the inhibition of DNOP and DPhP towards UGT isoforms. DPhP and DNOP weakly inhibited the activities of UGT1A1, UGT1A7, and UGT1A8. 100 µM of DNOP inhibited the activities of UGT1A3, UGT1A9, and UGT2B7 by 41.8% (p < 0.01), 45.6% (p < 0.01), and 48.8% (p < 0.01), respectively. 100 µM of DPhP inhibited the activity of UGT1A3, UGT1A6, and UGT1A9 by 81.8 (p < 0.001), 49.1% (p < 0.05), and 76.4% (p < 0.001), respectively. In silico analysis was used to explain the stronger inhibition of DPhP than DNOP towards UGT1A3 activity. Kinetics studies were carried our to determine mechanism of inhibition of UGT1A3 by DPhP. Both Dixon and Lineweaver-Burk plots showed the competitive inhibition of DPhP towards UGT1A3. The inhibition kinetic parameter (Ki) was calculated to be 0.89 µM. Based on the [I]/Ki standard ([I]/Ki < 0.1, low possibility; 1>[I]/Ki > 0.1, medium possibility; [I]/Ki > 1, high possibility), these studies predicted in vivo drug-drug interaction might occur when the plasma concentration of DPhP was above 0.089 µM. Taken together, this study reveales the potential for adverse effects of phthalates DNOP and DPhP as a result of UGT inhibition.

Adverse effects of diisooctyl phthalate on the male rat reproductive development following prenatal exposure.

In a first study, rats were given diisooctyl phthalate (DIOP, CAS 27554-26-3) at 0, 0.1, 0.5, and 1g/kg/day, by gavage, on gestation days 6-20 (GD). There was a significant increase in resorptions at 1g/kg/day and a reduction in fetal weights at 0.5 and 1g/kg/day. Malpositioned testes were observed in fetuses at 1g/kg/day, and supernumerary lumbar ribs and ossification delay at 0.5 and 1g/kg/day. In a follow-up study, DIOP administered on GD 12-19 reduced fetal testicular testosterone at 0.1g/kg/day and above. Finally, postnatal reproductive assessment was conducted in adult male offspring prenatally exposed to DIOP on GD 12-21. Abnormalities of reproductive system (e.g. hypospadias, non scrotal testes, and hypospermatogenesis) were observed in a few adult males at 0.5g/kg/day, and with a high incidence at 1g/kg/day. Thus, DIOP displayed an antiandrogenic activity and disrupted the male reproductive development.

Biomonitoring of phthalate metabolites in the Canadian population through the Canadian Health Measures Survey (2007-2009).

Human exposure to phthalates occurs through multiple sources and pathways. In the Canadian Health Measures Survey 2007-2009, 11 phthalate metabolites, namely, MMP, MEP, MnBP, MBzP, MCHP, MCPP, MEHP, MEOHP, MEHHP, MnOP, and MiNP were measured in urine samples of 6-49 year old survey respondents (n=3236). The phthalate metabolites biomonitoring data from this nationally-representative Canadian survey are presented here. The metabolites MEP, MnBP, MBzP, MCPP, MEHP, MEOHP and MEHHP were detected in >90% of Canadians while MMP, MCHP, MnOP and MiNP were detected in <20% of the Canadian population. Step-wise regression analyses were carried out to identify important predictors of volumetric concentrations (µg/L) of the metabolites in the general population. Individual multiple regression models with covariates age, sex, creatinine, fasting status, and the interaction terms age×creatinine, age×sex and fasting status×creatinine were constructed for MEP, MnBP, MBzP, MCPP, MEHP, MEOHP and MEHHP. The least square geometric mean (LSGM) estimates for volumetric concentration (µg/L) of the metabolites derived from respective regression models were used to assess the patterns in the metabolite concentrations among population sub-groups. The results indicate that children had significantly higher urinary concentrations of MnBP, MBzP, MEHP, MEHHP, MEOHP and MCPP than adolescents and adults. Moreover, MEP, MBzP, MnBP and MEOHP concentrations in females were significantly higher than in males. We observed that fasting status significantly affects the concentrations of MEHP, MEHHP, MEOHP, and MCPP metabolites analyzed in this study. Moreover, our results indicate that the sampling time could affect the DEHP metabolite concentrations in the general Canadian population.