Toxicological & Environmental Chemistry最新文献

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QuEChERS applicability to measure land snail polycyclic aromatic hydrocarbons for risk assessment QuEChERS在测量蜗牛多环芳烃风险评估中的适用性
Toxicological & Environmental Chemistry Pub Date : 2020-06-25 DOI: 10.1080/02772248.2020.1781855
N. Morin-Crini, M. Louzon, C. Amiot, A. de Vaufleury
{"title":"QuEChERS applicability to measure land snail polycyclic aromatic hydrocarbons for risk assessment","authors":"N. Morin-Crini, M. Louzon, C. Amiot, A. de Vaufleury","doi":"10.1080/02772248.2020.1781855","DOIUrl":"https://doi.org/10.1080/02772248.2020.1781855","url":null,"abstract":"Abstract The methods conventionally used for the extraction of organic pollutants in different environmental matrices are not suitable for measuring low concentrations of polycyclic aromatic hydrocarbons bioaccumulated in terrestrial snails that are both indicator of soil quality and a food item of various consumers including human being. A protocol, adapted from the QuEChERS method, originally designed for the extraction of pesticides in fruits and vegetables, in combination with gas chromatography-triple quadrupole mass spectrometry was developed to assess the bioavailability of polycyclic aromatic hydrocarbon extraction at low levels in soils to the soil quality bioindicator Cantareus aspersus. Bioaccumulation was measured in snails exposed ex situ for 28 days under controlled conditions to soils lightly or highly contaminated (sum of 16 molecules in the range 3.32–140 mg kg−1 dry weight). The method was validated (low limits of detection, linear calibration curves, good precision and trueness) and found to be efficient to show that strongly contaminated soils were not necessarily those that presented the highest bioavailable organics concentrations. This methodology offers a tool to assess the environmental risk of contaminated soils and to prioritize the management of polycyclic aromatic hydrocarbons-contaminated soils.","PeriodicalId":23210,"journal":{"name":"Toxicological & Environmental Chemistry","volume":"61 1","pages":"209 - 223"},"PeriodicalIF":0.0,"publicationDate":"2020-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78322200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 4
SARS-CoV-2 epidemic: changes in air quality during the lockdown in Zagreb (Republic of Croatia) SARS-CoV-2疫情:萨格勒布(克罗地亚共和国)封锁期间空气质量的变化
Toxicological & Environmental Chemistry Pub Date : 2020-06-24 DOI: 10.1080/02772248.2020.1778703
I. Ljubenkov, S. Haddout, K. Priya, A. M. Hoguane
{"title":"SARS-CoV-2 epidemic: changes in air quality during the lockdown in Zagreb (Republic of Croatia)","authors":"I. Ljubenkov, S. Haddout, K. Priya, A. M. Hoguane","doi":"10.1080/02772248.2020.1778703","DOIUrl":"https://doi.org/10.1080/02772248.2020.1778703","url":null,"abstract":"","PeriodicalId":23210,"journal":{"name":"Toxicological & Environmental Chemistry","volume":"38 1","pages":"302 - 303"},"PeriodicalIF":0.0,"publicationDate":"2020-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73199890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Boron concentrations in tap water in many cities of Turkey 土耳其许多城市自来水中的硼浓度
Toxicological & Environmental Chemistry Pub Date : 2020-06-23 DOI: 10.1080/02772248.2020.1778701
R. Kuru, Şahin Yilmaz, O. Sacan, R. Yanardag, A. Yarat, F. Şahin
{"title":"Boron concentrations in tap water in many cities of Turkey","authors":"R. Kuru, Şahin Yilmaz, O. Sacan, R. Yanardag, A. Yarat, F. Şahin","doi":"10.1080/02772248.2020.1778701","DOIUrl":"https://doi.org/10.1080/02772248.2020.1778701","url":null,"abstract":"Abstract Boron concentrations in 335 tap water samples collected from 75 cities in Turkey have been determined by inductively coupled plasma-mass spectrometry. Boron levels ranged between 0.001 and 5.5 mg/L. The lowest boron levels were in Trabzon, the highest in Kayseri. Boron levels in tap water from cities of the Aegean, Eastern, and Central Anatolian Regions were higher than those from cities of the Marmara, Black Sea, South East, and Mediterranean Regions. The overall mean value was around 0.2 mg/L, lower than the limit levels given by the WHO and the European Union, and also the limit level established by Turkish regulations. In some cities, these limit values were exceeded, generally in the tap water of cities where the drinking water based on groundwater or of those located in the vicinity of hot springs, volcanic areas, or boron production regions. This is the most detailed study evaluating boron levels in the drinking water of Turkey. To conclusion, since the boron intake is being mainly with nutrients and drinking water it may effect public health. To know boron levels in drinking waters is also very important for epidemiologic studies and for establishment of database of boron level in drinking waters in Turkey. Graphical Abstract","PeriodicalId":23210,"journal":{"name":"Toxicological & Environmental Chemistry","volume":"46 1","pages":"240 - 249"},"PeriodicalIF":0.0,"publicationDate":"2020-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84950011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 6
Aqueous ractopamine exposure below 0.22 mg/L has no effect on mortality, malformation, or growth of developing Xenopus laevis tadpoles 莱克多巴胺低于0.22 mg/L的水暴露对非洲爪蟾蝌蚪的死亡率、畸形或生长没有影响
Toxicological & Environmental Chemistry Pub Date : 2020-06-23 DOI: 10.1080/02772248.2020.1778700
Melissa A. Sandoz, M. Lewis, Mike R Wages, E. Peterson, Sheree L. Clendening, Kimberly J. Wooten, Philip N. Smith
{"title":"Aqueous ractopamine exposure below 0.22 mg/L has no effect on mortality, malformation, or growth of developing Xenopus laevis tadpoles","authors":"Melissa A. Sandoz, M. Lewis, Mike R Wages, E. Peterson, Sheree L. Clendening, Kimberly J. Wooten, Philip N. Smith","doi":"10.1080/02772248.2020.1778700","DOIUrl":"https://doi.org/10.1080/02772248.2020.1778700","url":null,"abstract":"Abstract Ractopamine, a β-adrenergic receptor agonist, is commonly used as a repartitioning agent and growth promotor in animal agriculture. There is evidence that ractopamine can enter natural water sources from livestock wastes, at concentrations in the low ng/L range. Currently, aquatic toxicity data for ractopamine is scarce. Therefore, in two separate experiments Xenopus laevis tadpoles were exposed to ractopamine in tank water at concentrations ranging from 2 ng/L to 2.2 mg/L for up to 14 d utilizing FETAX methodology. No overt signs of toxicity were identified in measured endpoints including survival, growth, malformation, and developmental stage. Oral ractopamine exposure in mammals and fish is associated with cardiotoxicity, increased mortality, and physiological alterations; however, this study suggests that aqueous ractopamine is not a developmental toxicant or overtly toxic to amphibians at environmentally relevant concentrations. Graphical Abstract","PeriodicalId":23210,"journal":{"name":"Toxicological & Environmental Chemistry","volume":"80 1","pages":"261 - 271"},"PeriodicalIF":0.0,"publicationDate":"2020-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74495794","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Influence of creosote-polluted substrate on the bioremediation-potential microscopic fungi in the rhizosphere of plants 杂酚油污染基质对植物根际具有生物修复潜力的微真菌的影响
Toxicological & Environmental Chemistry Pub Date : 2020-06-23 DOI: 10.1080/02772248.2020.1778702
A. Maruška, D. Levisauskas, V. Snieškienė, A. Stankevičienė, Nicola Tiso, Jurgita Mikašauskaitė-Tiso, Tomas Drevinskas, M. Stankevičius, R. Mickienė, O. Ragažinskienė, T. Tekorius, Paulius Kaškonas, O. Kornyšova, V. Kaškonienė, K. Bimbiraitė-Survilienė, Gediminas Dūda, Vita Tumosaitė, S. Grigiškis, C. Polcaro, E. Galli, E. Donati, M. Zacchini
{"title":"Influence of creosote-polluted substrate on the bioremediation-potential microscopic fungi in the rhizosphere of plants","authors":"A. Maruška, D. Levisauskas, V. Snieškienė, A. Stankevičienė, Nicola Tiso, Jurgita Mikašauskaitė-Tiso, Tomas Drevinskas, M. Stankevičius, R. Mickienė, O. Ragažinskienė, T. Tekorius, Paulius Kaškonas, O. Kornyšova, V. Kaškonienė, K. Bimbiraitė-Survilienė, Gediminas Dūda, Vita Tumosaitė, S. Grigiškis, C. Polcaro, E. Galli, E. Donati, M. Zacchini","doi":"10.1080/02772248.2020.1778702","DOIUrl":"https://doi.org/10.1080/02772248.2020.1778702","url":null,"abstract":"Abstract Wooden sleepers used in railways are impregnated with preservatives with fungicidic and bactericidic properties, which contain a large amount of harmful polycyclic aromatic hydrocarbons (PAHs). The aim of this research was to reveal the type of fungi in the rhizosphere of plants, which tolerate pollutants and can be involved in bioremediation process of creosote-contaminated used wooden railway sleepers. Tagetes patula grown in the substrate with 12.5% creosote impregnated used wooden sleepers chips additive is characterized by the most intensive root development and the biggest population of active fungi, therefore it is recommended for further investigation. Number of colonies of fungi in the rhizosphere of investigated plants decreased in many cases by increasing the amount of sleepers chips additive in substrate, however it was greater in the substrates with creosote treated wooden sleepers chips additives (12.5, 25 and 37.5% wooden sleepers chips) than in the pure substrate and various substrates with additives of pure Pinus sylvestris chips. This is a pioneering study searching for the local fungi species in Lithuania, which can be further utilized for the bioremediation of PAHs.","PeriodicalId":23210,"journal":{"name":"Toxicological & Environmental Chemistry","volume":"9 1","pages":"224 - 239"},"PeriodicalIF":0.0,"publicationDate":"2020-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75253333","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Effects of CO2-driven acidification on the ostracod Cypridopsis vidua: what are its likely environmental consequences? 二氧化碳驱动的酸化对介形虫的影响:可能的环境后果是什么?
Toxicological & Environmental Chemistry Pub Date : 2020-06-23 DOI: 10.1080/02772248.2020.1779723
G. Parra, D. Espinoza-Villalobos
{"title":"Effects of CO2-driven acidification on the ostracod Cypridopsis vidua: what are its likely environmental consequences?","authors":"G. Parra, D. Espinoza-Villalobos","doi":"10.1080/02772248.2020.1779723","DOIUrl":"https://doi.org/10.1080/02772248.2020.1779723","url":null,"abstract":"Abstract If anthropogenic CO2 emissions are maintained at the current rate, the pH of freshwater ecosystems will drop by 0.2–0.3 pH units by the year 2100, which will have great effects on aquatic biota. The impact of acidification on the ostracod species Cypridopsis vidua as a model organism has been studied. To provide the best rearing conditions, a preliminary experiment with different food concentrations was performed. Cypridopsis vidua reached higher survival under the established experimental conditions when reared with 1.5 × 106 cells of Scenedesmus obliquus per ostracod. Two CO2-driven acidification experiments were designed: the first, an acute experiment (96 hours) pH 6.0, 6.5, 7.0 and 8.3 showed that at low pH the survival rate decreased by more than 90%. The second was a chronic experiment (21 days) pH 7.3, 7.8 and 8.3 with a sharp decrease (60%) in reproduction rate and effects on phototactic behaviour at low pH. Abbreviations: CCAP: culture collection of algae and protozoa (in the Scottish Association for Marine Science); CR1: chronic test at medium pH (7.78 ± 0.11); CR2: chronic test at low pH (7.30 ± 0.16); GLM: generalized linear model; IPCC: intergovernmental panel on climate change; LT: lethal time; TA: total alkalinity; pCO2: partial pressure of carbon dioxide; PI: phototactic index; R0: treatment in starvation (no food); R1: low food concentration treatment (7.5 × 105 cell per individual); R2: optimal food concentration treatment (1.5 × 106 cell per individual); R3: high food concentration treatment (3.0 × 106 cell per individual); UTM: universal transverse mercator (coordinate system); YSI: yellow spring instruments","PeriodicalId":23210,"journal":{"name":"Toxicological & Environmental Chemistry","volume":"1 1","pages":"284 - 301"},"PeriodicalIF":0.0,"publicationDate":"2020-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90217274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Synthesis of SnO2 quantum dots mediated by Camellia sinensis shoots for degradation of thiamethoxam 山茶芽介导氧化锡量子点合成降解噻虫嗪的研究
Toxicological & Environmental Chemistry Pub Date : 2020-04-20 DOI: 10.1080/02772248.2020.1776285
Jayanta Das, S. S. Dhar
{"title":"Synthesis of SnO2 quantum dots mediated by Camellia sinensis shoots for degradation of thiamethoxam","authors":"Jayanta Das, S. S. Dhar","doi":"10.1080/02772248.2020.1776285","DOIUrl":"https://doi.org/10.1080/02772248.2020.1776285","url":null,"abstract":"Abstract A novel method of synthesis of tin dioxide quantum dots employing Camellia sinensis shoots as reducing agent and stabilizer is presented. The quantum dots were characterized by UV spectroscopy, X-Ray diffraction analysis, Fourier transform infrared spectroscopy, scanning electron microscopy, energy-dispersive X-ray spectroscopy and transmission electron microscopy. The crystalline tin dioxide quantum dots with an average size of 4.3 nm were of flake like morphology capped by phenolic compounds of Camellia sinensis. The quantum dots were employed for the photocatalytic degradation of thiamethoxam resulting in 57% degradation within 45 mins. Graphical Abstract","PeriodicalId":23210,"journal":{"name":"Toxicological & Environmental Chemistry","volume":"59 1","pages":"186 - 196"},"PeriodicalIF":0.0,"publicationDate":"2020-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73658444","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 4
Extraction, chemical compositions, and cytotoxic activities of essential oils of Thevetia peruviana 紫檀精油的提取、化学成分及细胞毒活性研究
Toxicological & Environmental Chemistry Pub Date : 2020-04-20 DOI: 10.1080/02772248.2020.1770255
P. Khang, X. Mai, Hiep Hoang Phu, Sỹ Danh Thường, Shen Sida
{"title":"Extraction, chemical compositions, and cytotoxic activities of essential oils of Thevetia peruviana","authors":"P. Khang, X. Mai, Hiep Hoang Phu, Sỹ Danh Thường, Shen Sida","doi":"10.1080/02772248.2020.1770255","DOIUrl":"https://doi.org/10.1080/02772248.2020.1770255","url":null,"abstract":"Abstract In this study, The essential oil of flowers, fruits, and leaves of Thevetia peruviana, which were collected in Vietnam, were being reported for the first time. The essential oil of flowers was extracted by different methods: n-hexane extraction, distillation water, and supercritical CO2 extraction. The compositions of essential oil of different parts of Thevetia peruviana were analyzed by GC-FID and GC/MS systems. Major chemical compositions of essential oil were identified as monoterpenes, sesquiterpenes, terpenoids, and sterol. The activities of total essential oil extracts of the Thevetia peruviana exhibit inhibitory activities against five cancer cell lines.","PeriodicalId":23210,"journal":{"name":"Toxicological & Environmental Chemistry","volume":"17 1","pages":"124 - 131"},"PeriodicalIF":0.0,"publicationDate":"2020-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88130470","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Consumer safety evaluation after monitoring of endocrine disruptor pesticide residues: a case study of Thessaly, Central Greece 监测内分泌干扰物农药残留后的消费者安全评价:希腊中部色萨利的案例研究
Toxicological & Environmental Chemistry Pub Date : 2020-04-20 DOI: 10.1080/02772248.2020.1770256
E. Vlassi, E. Bempelou, K. Liapis, G. Arapis
{"title":"Consumer safety evaluation after monitoring of endocrine disruptor pesticide residues: a case study of Thessaly, Central Greece","authors":"E. Vlassi, E. Bempelou, K. Liapis, G. Arapis","doi":"10.1080/02772248.2020.1770256","DOIUrl":"https://doi.org/10.1080/02772248.2020.1770256","url":null,"abstract":"Abstract A large number of pesticides used in agricultural practice has been characterized or suspected to be Endocrine Disruptors, with dietary intake to be the major route of consumer’s exposure. In the present study the safety to consumer after the consumption of pome fruits and fruiting vegetables originated from the area of Thessaly, Central Greece was assessed. Objective samplings of 110 samples were conducted in local retail markets in an effort to quantify potential residues of targeted pesticides, previously characterized as endocrine disruptors. For the determination of residues, a QuEChERs-based multiresidue method coupled with liquid and gas chromatographic systems was applied and fully validated in accordance with the European Unions’s requirements in apples and tomatoes in three fortification levels. Acceptable results were obtained for all the validation parameters studied and the limit of quantification of 0.01 mg/kg and limit of detection of 0.0033 mg/kg were achieved. Based on the determined results, the most frequently determined pesticide was chlorpyrifos with a maximum concentration of 0.32 mg/kg in tomato samples. Cypermethrin and dimethoate were also determined in tomatoes and aubergines. The EFSA PRIMo rev.3.1 model was applied for all positive samples and acute risk for consumers was confirmed only in the case of determination of chlorpyrifos in tomatoes.","PeriodicalId":23210,"journal":{"name":"Toxicological & Environmental Chemistry","volume":"1 1","pages":"105 - 123"},"PeriodicalIF":0.0,"publicationDate":"2020-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77292244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Temporal dynamics of arsenic uptake and distribution: food and water risks in the Bengal basin 砷吸收和分布的时间动态:孟加拉盆地的食物和水风险
Toxicological & Environmental Chemistry Pub Date : 2020-04-20 DOI: 10.1080/02772248.2020.1767781
Sarath Pullyottum Kavil, Devanita Ghosh, Indira Pašić, J. Routh
{"title":"Temporal dynamics of arsenic uptake and distribution: food and water risks in the Bengal basin","authors":"Sarath Pullyottum Kavil, Devanita Ghosh, Indira Pašić, J. Routh","doi":"10.1080/02772248.2020.1767781","DOIUrl":"https://doi.org/10.1080/02772248.2020.1767781","url":null,"abstract":"Abstract Contaminated food chain is a serious contender for arsenic (As) uptake around the globe. In Nadia, West Bengal, we trace possible means of transfer of As from multiple sources reaching different trophic levels, and associated seasonal variability leading to chronic As uptake. This work considers possible sources-pathways of As transfer through food chain in rural community. Arsenic concentration in groundwater, soil, rice, and vegetable-samples collected detected in different harvest seasons of 2014 and 2016. Arsenic level in shallow groundwater samples ranged from 0.1 to 354 µg/L, with 75% of the sites above the prescribed limit by WHO (10 µg/L) during the boro harvest season. High soil As content (∼20.6 mg/kg), resulted in accumulation of As in food crops. A positive correlation in As conc. with increase over period in all sites indicating gradual As accumulation in topsoil. Unpolished rice samples showed high As content (∼1.75 mg/kg), polishing reduced 80% of As. Among vegetables, the plant family Poaceae with high irrigation requirements and Solanaceae retaining high moisture, have the highest levels of As. Contaminated animal fodder (Poaceae) and turf water for cattle are shown to contaminate milk (0.06 to 0.24 µg/L) and behoves strategies, practices to minimize As exposure.","PeriodicalId":23210,"journal":{"name":"Toxicological & Environmental Chemistry","volume":"2012 1","pages":"62 - 77"},"PeriodicalIF":0.0,"publicationDate":"2020-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87722024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 4
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