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Race-specific associations of urinary phenols and parabens with adipokines in midlife women: The Study of Women's Health Across the Nation (SWAN) النص الكامل
2022
Lee, Seulbi | Karvonen-Gutierrez, Carrie | Mukherjee, Bhramar | Herman, William H. | Park, Sung Kyun
Adipokines, cytokines secreted by adipose tissue, may contribute to obesity-related metabolic disease. The role of environmental phenols and parabens in racial difference in metabolic disease burden has been suggested, but there is limited evidence. We examined the cross-sectional associations of urinary phenols and parabens with adipokines and effect modification by race. Urinary concentrations of 6 phenols (bisphenol-A, bisphenol-F, 2,4-diclorophenol, 2,5-diclorophenol, triclosan, benzophenone-3) and 4 parabens (methyl-paraben, ethyl-paraben, propyl-paraben, butyl-paraben) were measured in 2002–2003 among 1200 women (mean age = 52.6) enrolled in the Study of Women's Health Across the Nation Multi-Pollutant Study. Serum adipokines included adiponectin, high molecular weight (HMW)-adiponectin, leptin, soluble leptin receptor (sOB-R). Linear regression models were used to estimate the adjusted percentage change in adipokines per inter-quantile range (IQR) increase in standardized and log-transformed levels of individual urinary phenols and parabens. Bayesian kernel machine regression (BKMR) was used to evaluate the joint effect of urinary phenols and parabens as mixtures. Participants included white (52.5%), black (19.3%), and Asian (28.1%) women. Urinary 2,4-dichlorophenol was associated with 6.02% (95% CI: 1.20%, 10.83%) higher HMW-adiponectin and urinary bisphenol-F was associated with 2.60% (0.48%, 4.71%) higher sOB-R. Urinary methyl-paraben was associated with lower leptin in all women but this association differed by race: 8.58% (−13.99%, −3.18%) lower leptin in white women but 11.68% (3.52%, 19.84%) higher leptin in black women (P interaction = 0.001). No significant associations were observed in Asian women. Additionally, we observed a significant positive overall effect of urinary phenols and parabens mixtures in relation to leptin levels in black, but not in white or Asian women. Urinary bisphenol-F, 2,4-dichlorophenol and methyl-paraben may be associated with favorable profiles of adipokines, but methyl-paraben, widely used in hair and personal care products, was associated with unfavorable leptin levels in black women. Future studies are needed to confirm this racial difference.
اظهر المزيد [+] اقل [-]Multigenerational study of the obesogen effects of bisphenol S after a perinatal exposure in C57BL6/J mice fed a high fat diet النص الكامل
2021
Brulport, Axelle | Le Corre, Ludovic | Maquart, Guillaume | Barbet, Virginie | Dastugue, Aurélie | Severin, Isabelle | Vaiman, Daniel | Chagnon, Marie-Christine
Bisphenol S is an endocrine disruptor exhibiting metabolic disturbances, especially following perinatal exposures. To date, no data are available on the obesogen effects of BPS in a mutligenerational issue.We investigated obesogen effects of BPS in a multigenerational study by focusing on body weight, adipose tissue and plasma parameters in male and female mice.Pregnant C57BL6/J mice were exposed to BPS (1.5 μg/kg bw/day ie a human equivalent dose of 0.12 μg/kg bw/day) by drinking water from gestational day 0 to post natal day 21. All offsprings were fed with a high fat diet during 15 weeks. Body weight was monitored weekly and fat mass was measured before euthanasia. At euthanasia, blood glucose, insuline, triglyceride, cholesterol and no esterified fatty acid plasma levels were determined and gene expressions in visceral adipose tissue were assessed. F1 males and females were mated to obtain the F2 generation. Likewise, the F2 mice were cross-bred to obtain F3. The same analyses were performed.In F1 BPS induced an overweight in male mice associated to lipolysis gene expressions upregulation. In F1 females, dyslipidemia was observed. In F2, BPS exposure was associated to an increase in body weight, fat and VAT masses in males and females. Several plasma parameters were increased but with a sex related pattern (blood glucose, triglycerides and cholesterol in males and NEFA in females). We observed a down-regulation in mRNA expression of gene involved in lipogenesis and in lipolysis for females but only in the lipogenesis for males. In F3, a decrease in VAT mass and an upregulation of lipogenesis gene expression occurred only in females.BPS perinatal exposure induced sex-dependent obesogen multigenerational effects, the F2 generation being the most impacted. Transgenerational disturbances persisted only in females.
اظهر المزيد [+] اقل [-]Hazardous pollutants in the environment: Fish host-parasite interactions and bioaccumulation of polychlorinated biphenyls النص الكامل
2021
Brázová, Tímea | Miklisová, Dana | Barčák, Daniel | Uhrovič, Dalibor | Šalamún, Peter | Orosová, Martina | Oros, Mikuláš
The present paper reports on the interrelationships of fish, parasites and the bioaccumulation of hazardous organic compounds in the Zemplínska Šírava water reservoir in eastern Slovakia, which is heavily polluted with polychlorinated biphenyls (PCBs). The concentrations of these contaminants were measured in various fish matrices (dorsal and abdominal muscle tissues, hepatopancreas, intestine wall and adipose tissue) of the freshwater bream, Abramis brama (Cyprinidae), and in its intestinal parasite Caryophyllaeus laticeps (Cestoda), which was used for the first time as a model for a PCB bioaccumulation study. Regarding the fish, the highest concentrations of PCBs were found in the intestine, followed by hepatopancreas and muscle tissues. The amounts of PCBs were higher in abdominal muscles than in their dorsal parts. Concentrations of ∑PCBs above the limits set by European regulations were detected in both muscle parts in the fish, confirming the persistent unfavorable conditions in this locality and high risk for biota and humans. Based on bioconcentration factor values (BCFs), PCBs reached much higher levels in cestodes compared to bream matrices. Some significant differences in PCB amounts between infected and uninfected bream were determined. Fulton's condition factor (CF) significantly differed in infected and non-infected fish (p ˂ 0.05), with CF values surprisingly lower in fish free of parasites compared to parasitized fish, which suggests a “mutualistic” relationship between the parasite and its host.
اظهر المزيد [+] اقل [-]Associations between persistent organic pollutants and endometriosis: A multipollutant assessment using machine learning algorithms النص الكامل
2020
Associations between persistent organic pollutants and endometriosis: A multipollutant assessment using machine learning algorithms النص الكامل
2020
Endometriosis is a gynaecological disease characterised by the presence of endometriotic tissue outside of the uterus impacting a significant fraction of women of childbearing age. Evidence from epidemiological studies suggests a relationship between risk of endometriosis and exposure to some organochlorine persistent organic pollutants (POPs). However, these chemicals are numerous and occur in complex and highly correlated mixtures, and to date, most studies have not accounted for this simultaneous exposure. Linear and logistic regression models are constrained to adjusting for multiple exposures when variables are highly intercorrelated, resulting in unstable coefficients and arbitrary findings. Advanced machine learning models, of emerging use in epidemiology, today appear as a promising option to address these limitations. In this study, different machine learning techniques were compared on a dataset from a case-control study conducted in France to explore associations between mixtures of POPs and deep endometriosis. The battery of models encompassed regularised logistic regression, artificial neural network, support vector machine, adaptive boosting, and partial least-squares discriminant analysis with some additional sparsity constraints. These techniques were applied to identify the biomarkers of internal exposure in adipose tissue most associated with endometriosis and to compare model classification performance. The five tested models revealed a consistent selection of most associated POPs with deep endometriosis, including octachlorodibenzofuran, cis-heptachlor epoxide, polychlorinated biphenyl 77 or trans-nonachlor, among others. The high classification performance of all five models confirmed that machine learning may be a promising complementary approach in modelling highly correlated exposure biomarkers and their associations with health outcomes. Regularised logistic regression provided a good compromise between the interpretability of traditional statistical approaches and the classification capacity of machine learning approaches. Applying a battery of complementary algorithms may be a strategic approach to decipher complex exposome-health associations when the underlying structure is unknown.
اظهر المزيد [+] اقل [-]Associations between persistent organic pollutants and endometriosis: A multipollutant assessment using machine learning algorithms النص الكامل
2020
Matta, Komodo | Vigneau, Evelyne | Cariou, Véronique | Mouret, Delphine | Ploteau, Stéphane | Le Bizec, Bruno | Antignac, Jean-Philippe | Cano-Sancho, Germán | Laboratoire d'étude des Résidus et Contaminants dans les Aliments (LABERCA) ; École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE) | Statistique, Sensométrie et Chimiométrie (StatSC) ; École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE) | Centre hospitalier universitaire de Nantes (CHU Nantes)
International audience
اظهر المزيد [+] اقل [-]Exposure to air pollution interacts with obesogenic nutrition to induce tissue-specific response patterns النص الكامل
2018
Pardo, Michal | Kuperman, Yael | Levin, Liron | Rudich, Assaf | Haim, Yulia | Schauer, James J. | Chen, Alon | Rudich, Yinon
Obesity and exposure to particular matter (PM) have become two leading global threats to public health. However, the exact mechanisms and tissue-specificity of their health effects are largely unknown. Here we investigate whether a metabolic challenge (early nutritional obesity) synergistically interacts with an environmental challenge (PM exposure) to alter genes representing key response pathways, in a tissue-specific manner. Mice subjected to 7 weeks obesogenic nutrition were exposed every other day during the final week and a half to aqueous extracts of PM collected in the city of London (UK). The expression of 61 selected genes representing key response pathways were investigated in lung, liver, white and brown adipose tissues. Principal component analysis (PCA) revealed distinct patterns of expression changes between the 4 tissues, particularly in the lungs and the liver. Surprisingly, the lung responded to the nutrition challenge. The response of these organs to the PM challenge displayed opposite patterns for some key genes, in particular, those related to the Nrf2 pathway. While the contribution to the variance in gene expression changes in mice exposed to the combined challenge were largely similar among the tissues in PCA1, PCA2 exhibited predominant contribution of inflammatory and oxidative stress responses to the variance in the lungs, and a greater contribution of autophagy genes and MAP kinases in adipose tissues. Possible involvement of alterations in DNA methylation was demonstrated by cell-type-specific responses to a methylation inhibitor. Correspondingly, the DNA methyltransferase Dnmt3a2 increased in the lungs but decreased in the liver, demonstrating potential tissue-differential synergism between nutritional and PM exposure. The results suggest that urban PM, containing dissolved metals, interacts with obesogenic nutrition to regulate diverse response pathways including inflammation and oxidative stress, in a tissue-specific manner. Tissue-differential effects on DNA methylation may underlie tissue-specific responses to key stress-response genes such as catalase and Nrf2.
اظهر المزيد [+] اقل [-]Organobromine compound profiling in human adipose: Assessment of sources of bromophenol النص الكامل
2015
Gao, Shixiong | Wan, Yi | Zheng, Guomao | Luo, Kai | Kannan, Kurunthachalam | Giesy, John P. | Lam, Michael H.W. | Hu, Jianying
Bromophenols (BRPs) have been widely detected in human tissues, however, relative proportions from natural products and/or anthropogenic flame retardants are not clear. 21 polybrominated diphenyl ethers (PBDEs), 15 MeO/OH-PBDEs, and 10 BRPs were simultaneously quantified in adipose collected from people from New York City, USA. An in vitro assay utilizing human liver microsomes was performed for detected predominant organobromine. High concentrations of 2,4,6-triBRP and PBDEs were observed, and extremely low concentrations of naturally occurring MeO/OH-PBDEs were detected. Similar biotransformatioin rates of BRPs and MeO/OH-PBDEs indicated that the relative high concentration of 2,4,6-triBRP in humans was not of natural origin. Significant correlation observed between concentrations of 2,4,6-triBRP and BDE-209 suggested that the two chemicals may share a common source. Both 2,4,6-triBRP and BDE-209 were detected in commercial ABS resins, suggesting that plastic products made from ABS resins could be potential sources of co-exposure of the two compounds for humans.
اظهر المزيد [+] اقل [-]Discovery and widespread occurrence of polyhalogenated 1,1'-dimethyl-2,2'-bipyrroles (PDBPs) in marine biota النص الكامل
2013
Hauler, Carolin | Martin, René | Knölker, Hans-Joachim | Gaus, Caroline | Mueller, Jochen F. | Vetter, Walter
Polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles (PDBPs) are halogenated natural products (HNPs) previously shown to bioaccumulate in marine mammals and birds. Since their discovery in 1999, six hexahalogenated and a few lesser halogenated congeners have been identified in diverse marine mammal samples. Here we report the identification of 17 additional hexahalogenated PDBPs in the blubber extract of a humpback dolphin (Sousa chinensis) from Queensland, Australia. Thirteen of these new PDBPs were also detected in an Australian sea cucumber (Holothuria sp.). Additional samples were also tested positive on several new PDBPs, including an Australian venus tuskfish (Choerodon venustus) as well as a white whale (Delphinapterus leucas) and a sperm whale (Physeter macrocephalus) from the Northern Hemisphere. GC/ECNI-MS-SIM quantification of the molecular ions was carried out with the help of synthesized standards. The sum concentration of PDBPs was 1.1 mg/kg lipid in the humpback dolphin and 0.48 mg/kg lipid in the sea cucumber.
اظهر المزيد [+] اقل [-]Organohalogen compounds in blubber of Indo-Pacific bottlenose dolphin (Tursiops aduncus) and spinner dolphin (Stenella longirostris) from Zanzibar, Tanzania النص الكامل
2010
Mwevura, Haji | Amir, Omar A. | Kishimba, Michael | Berggren, P. | Kylin, Henrik
Blubber samples of Indo-Pacific bottlenose (Tursiops aduncus) and spinner (Stenella longirostris) dolphins from Zanzibar, East Africa, were analyzed for a wide range of organohalogen compounds. Methoxylated polybrominated diphenyl ethers (MeO-BDEs), presumably biogenic, were found at higher concentrations than anthropogenic organochlorine pesticides (OCPs). Only traces of industrial pollutants, such as polychlorinated biphenyls, were detected. The OCP levels found off Zanzibar were lower than those reported from other regions while MeO-BDE levels were higher. The relative composition of the OCPs indicated recent use of lindane (γ-hexachlorocyclohexane) and aged residues of DDT and technical HCH. Placental transfer was estimated to 2.5% and 0.5% of the total burden of OCPs and MeO-BDEs, respectively. Overall transfer from mother to calf in Indo-Pacific bottlenose dolphins was estimated to 72% and 85% for the OCPs and MeO-BDEs burdens, respectively. Health effects of MeO-BDEs are not known, but structural similarities with well-known environmental toxins are cause for concern.
اظهر المزيد [+] اقل [-]Socio-demographic, lifestyle, and dietary determinants of essential and possibly-essential trace element levels in adipose tissue from an adult cohort النص الكامل
2018
Rodríguez-Pérez, Celia | Vrhovnik, Petra | González-Alzaga, Beatriz | Fernández, Mariana F. | Martin-Olmedo, Piedad | Olea, Nicolás | Fiket, Željka | Kniewald, Goran | Arrebola, Juan P.
Socio-demographic, lifestyle, and dietary determinants of essential and possibly-essential trace element levels in adipose tissue from an adult cohort النص الكامل
2018
Rodríguez-Pérez, Celia | Vrhovnik, Petra | González-Alzaga, Beatriz | Fernández, Mariana F. | Martin-Olmedo, Piedad | Olea, Nicolás | Fiket, Željka | Kniewald, Goran | Arrebola, Juan P.
There is increasing evidence linking levels of trace elements (TEs) in adipose tissue with certain chronic conditions (e.g., diabetes or obesity). The objectives of this study were to assess concentrations of a selection of nine essential and possibly-essential TEs in adipose tissue samples from an adult cohort and to explore their socio-demographic, dietary, and lifestyle determinants. Adipose tissue samples were intraoperatively collected from 226 volunteers recruited in two public hospitals from Granada province. Trace elements (Co, Cr, Cu, Fe, Mn, Mo, Se, V, and Zn) were analyzed in adipose tissue by high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). Data were collected on socio-demographic characteristics, lifestyle, diet, and health status by face-to-face interview. Predictors of TE concentrations were assessed by using multivariable linear and logistic regression. All TEs were detected in all samples with the exception of Se (53.50%). Iron, zinc, and copper showed the highest concentrations (42.60 mg/kg, 9.80 mg/kg, and 0.68 mg/kg, respectively). Diet was the main predictor of Cr, Fe, Mo, and Se concentrations. Body mass index was negatively associated with all TEs (β coefficients = −0.018 to −0.593, p = 0.001–0.090) except for Mn and V. Age showed a borderline-significant positive correlation with Cu (β = 0.004, p = 0.089). Residence in a rural or semi-rural area was associated with increased Co, Cr, Fe, Mo, Mn, V and Zn concentrations and with β coefficients ranging from 0.196 to 0.544 (p < 0.05). Furthermore, individuals with higher educational level showed increased Cr, Co, Fe and V concentrations (β coefficients = 0.276–0.368, p = 0.022–0.071). This is the first report on the distribution of these TEs in adipose tissue and on their determinants in a human cohort and might serve as an initial step in the elucidation of their clinical relevance.
اظهر المزيد [+] اقل [-]Socio-demographic, lifestyle, and dietary determinants of essential and possibly-essential trace element levels in adipose tissue from an adult cohort النص الكامل
2018
Rodríguez-Pérez, Celia | Vrhovnik, Petra | González-Alzaga, Beatriz | Fernández Cabrera, Mariana Fátima | Martín Olmedo, Piedad | Olea Serrano, Nicolás | Zeljka, Fiket | Kniewald, Goran | Arrebola Moreno, Juan Pedro
There is increasing evidence linking levels of trace elements (TEs) in adipose tissue with certain chronic conditions (e.g., diabetes or obesity). The objectives of this study were to assess concentrations of a selection of nine essential and possibly-essential TEs in adipose tissue samples from an adult cohort and to explore their socio-demographic, dietary, and lifestyle determinants. Adipose tissue samples were intraoperatively collected from 226 volunteers recruited in two public hospitals from Granada province. Trace elements (Co, Cr, Cu, Fe, Mn, Mo, Se, V, and Zn) were analyzed in adipose tissue by high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). Data were collected on socio-demographic characteristics, lifestyle, diet, and health status by face-to-face interview. Predictors of TE concentrations were assessed by using multivariable linear and logistic regression. All TEs were detected in all samples with the exception of Se (53.50%). Iron, zinc, and copper showed the highest concentrations (42.60 mg/kg, 9.80 mg/kg, and 0.68 mg/kg, respectively). Diet was the main predictor of Cr, Fe, Mo, and Se concentrations. Body mass index was negatively associated with all TEs (β coefficients = −0.018 to −0.593, p = 0.001–0.090) except for Mn and V. Age showed a borderline-significant positive correlation with Cu (β = 0.004, p = 0.089). Residence in a rural or semi-rural area was associated with increased Co, Cr, Fe, Mo, Mn, V and Zn concentrations and with β coefficients ranging from 0.196 to 0.544 (p < 0.05). Furthermore, individuals with higher educational level showed increased Cr, Co, Fe and V concentrations (β coefficients = 0.276–0.368, p = 0.022–0.071). This is the first report on the distribution of these TEs in adipose tissue and on their determinants in a human cohort and might serve as an initial step in the elucidation of their clinical relevance.
اظهر المزيد [+] اقل [-]Diet and metabolic state are the main factors determining concentrations of perfluoroalkyl substances in female polar bears from Svalbard النص الكامل
2017
Tartu, Sabrina | Bourgeon, Sophie | Aars, Jon | Andersen, Magnus | Lone, Karen | Jenssen, Bjørn Munro | Polder, Anuschka | Thiemann, Gregory W. | Torget, Vidar | Welker, Jeffrey M. | Routti, Heli
Diet and metabolic state are the main factors determining concentrations of perfluoroalkyl substances in female polar bears from Svalbard النص الكامل
2017
Tartu, Sabrina | Bourgeon, Sophie | Aars, Jon | Andersen, Magnus | Lone, Karen | Jenssen, Bjørn Munro | Polder, Anuschka | Thiemann, Gregory W. | Torget, Vidar | Welker, Jeffrey M. | Routti, Heli
Perfluoroalkyl substances (PFASs) have been detected in organisms worldwide, including Polar Regions. The polar bear (Ursus maritimus), the top predator of Arctic marine ecosystems, accumulates high concentrations of PFASs, which may be harmful to their health. The aim of this study was to investigate which factors (habitat quality, season, year, diet, metabolic state [i.e. feeding/fasting], breeding status and age) predict PFAS concentrations in female polar bears captured on Svalbard (Norway). We analysed two perfluoroalkyl sulfonates (PFSAs: PFHxS and PFOS) and C8-C13 perfluoroalkyl carboxylates (PFCAs) in 112 plasma samples obtained in April and September 2012–2013. Nitrogen and carbon stable isotope ratios (δ15N, δ13C) in red blood cells and plasma, and fatty acid profiles in adipose tissue were used as proxies for diet. We determined habitat quality based on movement patterns, capture position and resource selection functions, which are models that predict the probability of use of a resource unit. Plasma urea to creatinine ratios were used as proxies for metabolic state (i.e. feeding or fasting state). Results were obtained from a conditional model averaging of 42 general linear mixed models. Diet was the most important predictor of PFAS concentrations. PFAS concentrations were positively related to trophic level and marine diet input. High PFAS concentrations in females feeding on the eastern part of Svalbard, where the habitat quality was higher than on the western coast, were likely related to diet and possibly to abiotic factors. Concentrations of PFSAs and C8-C10 PFCAs were higher in fasting than in feeding polar bears and PFOS was higher in females with cubs of the year than in solitary females. Our findings suggest that female polar bears that are exposed to the highest levels of PFAS are those 1) feeding on high trophic level sea ice-associated prey, 2) fasting and 3) with small cubs.
اظهر المزيد [+] اقل [-]Diet and metabolic state are the main factors determining concentrations of perfluoroalkyl substances in female polar bears from Svalbard النص الكامل
2017
Diet and metabolic state are the main factors determining concentrations of perfluoroalkyl substances in female polar bears from Svalbard النص الكامل
2017
Tartu, Sabrina | Bourgeon, Sophie | Aars, Jon | Andersen, Magnus | Lone, Karen | Jenssen, Bjørn Munro | Polder, Anuschka | Thiemann, Gregory W. | Torget, Vidar | Welker, Jeffrey Martin | Routti, Heli Anna Irmeli
Accepted manuscript version. Published version available in <a href=https://doi.org/10.1016/j.envpol.2017.04.100> Environmental Pollution 2017, 229 :146-158. </a> | Perfluoroalkyl substances (PFASs) have been detected in organisms worldwide, including Polar Regions. The polar bear (Ursus maritimus), the top predator of Arctic marine ecosystems, accumulates high concentrations of PFASs, which may be harmful to their health. The aim of this study was to investigate which factors (habitat quality, season, year, diet, metabolic state [i.e. feeding/fasting], breeding status and age) predict PFAS concentrations in female polar bears captured on Svalbard (Norway). We analysed two perfluoroalkyl sulfonates (PFSAs: PFHxS and PFOS) and C<sub>8</sub>-C<sub>13</sub> perfluoroalkyl carboxylates (PFCAs) in 112 plasma samples obtained in April and September 2012–2013. Nitrogen and carbon stable isotope ratios (δ<sup>15</sup>N, δ<sup>13</sup>C) in red blood cells and plasma, and fatty acid profiles in adipose tissue were used as proxies for diet. We determined habitat quality based on movement patterns, capture position and resource selection functions, which are models that predict the probability of use of a resource unit. Plasma urea to creatinine ratios were used as proxies for metabolic state (i.e. feeding or fasting state). Results were obtained from a conditional model averaging of 42 general linear mixed models. Diet was the most important predictor of PFAS concentrations. PFAS concentrations were positively related to trophic level and marine diet input. High PFAS concentrations in females feeding on the eastern part of Svalbard, where the habitat quality was higher than on the western coast, were likely related to diet and possibly to abiotic factors. Concentrations of PFSAs and C<sub>8</sub>-C<sub>10</sub> PFCAs were higher in fasting than in feeding polar bears and PFOS was higher in females with cubs of the year than in solitary females. Our findings suggest that female polar bears that are exposed to the highest levels of PFAS are those 1) feeding on high trophic level sea ice-associated prey, 2) fasting and 3) with small cubs.
اظهر المزيد [+] اقل [-]