食物中藻类毒素污染及暴露风险研究进展
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摘要
分析藻类毒素在不同食物中可能的污染类型、暴露水平、污染风险等。通过大量文献调研从藻类毒素的毒性、危害、污染情况、限量标准以及暴露风险等方面对不同食物中主要存在的藻毒素进行系统阐述和分析。目前报道显示海产品中藻毒素以贝类毒素的暴露频率较高,尤以软骨藻酸的暴露水平最大(> 1. 0×10~5μg/kg)。其他非海产食物中,微囊藻毒素的污染范围和污染程度最大,在淡水产品、农作物、藻类食品膳食补充剂等食品中都有相关报道;此外,藻类食品膳食补充剂中暴露频率相对较高的藻毒素是类毒素-a,最大暴露水平高于1×10~4μg/kg。该综述可以为不同食物中可能存在的藻类毒素及其暴露水平、污染风险等提供参考。
This review analyzed algal toxins regarding their toxicity,hazards,potential contaminated types,exposure levels,limit standards,and pollution risks in different foods. It was mainly found that the exposure frequency of shellfish toxins in seafoods are high,especially domoic acid,as it has the highest exposure level(> 1. 0 × 10~5μg/kg).On the other hand,other non-seafood products,such as freshwater products,crops,and algal dietary supplements etc. are contaminated by microcystins the most,resulting microcystins have the highest exposure level. In addition,a neurotoxin named anatoxin-a has relatively high exposure frequency in algal dietary supplements,and its maximum exposure level is above 1 × 10~4μg/kg. This review provides references for algal toxins and their exposure levels and contamination risks in different foods.
This review analyzed algal toxins regarding their toxicity,hazards,potential contaminated types,exposure levels,limit standards,and pollution risks in different foods. It was mainly found that the exposure frequency of shellfish toxins in seafoods are high,especially domoic acid,as it has the highest exposure level(> 1. 0 × 10~5μg/kg).On the other hand,other non-seafood products,such as freshwater products,crops,and algal dietary supplements etc. are contaminated by microcystins the most,resulting microcystins have the highest exposure level. In addition,a neurotoxin named anatoxin-a has relatively high exposure frequency in algal dietary supplements,and its maximum exposure level is above 1 × 10~4μg/kg. This review provides references for algal toxins and their exposure levels and contamination risks in different foods.
引文
[1]LIU C,ERSAN M S,PLEWA M J,et al.Formation of regulated and unregulated disinfection byproducts during chlorination of algal organic matter extracted from freshwater and marine algae[J].Water Research,2018,142:313-324.
[2]VILARIN'O N,LOUZAO M C,ABAL P,et al.Human poisoning from marine toxins:Unknowns for optimal consumer protection[J].Toxins,2018,10(8):324-362.
[3]PAPADIMITRIOU T,KAGALOU I,STALIKAS C,et al.Assessment of microcystin distribution and biomagnification in tissues of aquatic food web compartments from a shallow lake and evaluation of potential risks to public health[J].Ecotoxicology,2012,21(4):1 155-1 166.
[4]LARRAMENDY M L,SOLONESKI S.Cyanobacterial Toxins Emerging Contaminants in Soils:A Review of Sources,Fate and Impacts on Ecosystems,Plants and Animal and Human Health[M].Croatia:Bouaicha,Noureddine,2016:105-126.
[5]LEE J,LEE S,JIANG X.Cyanobacterial toxins in freshwater and food:Important sources of exposure to humans[J].Annual Review of Food Science and Technology,2017,8(1):281-304.
[6]TESTAI E,BURATTI F M,FUNARI E,et al.Review and analysis of occurrence,exposure and toxicity of cyanobacteria toxins in food[J].EFSA Supporting Publications,2016,13(2):998E.
[7]IARC Working Group.IARC monographs on the evaluation of carcinogenic risks to humans.Ingested nitrate and nitrite,and cyanobacterial peptide toxins[J].IARC monographs on the eualuation of carcinogenic risks to humans/World Health Organization,International Agencyfor Research on Cancer,2010,94(7):1-412.
[8]DEZ-QUIJADA L,GUZMN-GUILLN R,PRIETO OR-TEGA A I,et al.New method for simultaneous determination of microcystins and cylindrospermopsin in vegetable matrices by SPE-UPLC-MS/MS[J].Toxins,2018,10(10):406-416.
[9]BRAGA A C,CAMACHO C,MARQUES A,et al.Combined effects of warming and acidification on accumulation and elimination dynamics of paralytic shellfish toxins in mussels Mytilus galloprovincialis[J].Environmental Research,2018,164:647-654.
[10]PERL T M,BDARD L,KOSATSKY T,et al.An outbreak of toxic encephalopathy caused by eating mussels contaminated with domoic acid[J].New England Journal of Medicine,1990,322(25):1 775-1 780.
[11]ZI J,PAN X,MACISAAC H J,et al.Cyanobacteria blooms induce embryonic heart failure in an endangered fish species[J].Aquatic Toxicology,2018,194:78-85.
[12]MELLO F D,BRAIDY N,MARCAL H,et al.Mechanisms and effects posed by neurotoxic products of cyanobacteria/microbial eukaryotes/dinoflagellates in algae blooms:A review[J].Neurotoxicity Research,2018,33(1):153-167.
[13]CHAI Y,DENG W J,QIN X,et al.Occurrence of four species of algae in the marine water of Hong Kong[J].Marine Pollution Bulletin,2017,124(2):890-896.
[14]JIANG Tao,LIU Lei,LI Yang,et al.Occurrence of marine algal toxins in oyster and phytoplankton samples in Daya Bay,South China Sea[J].Chemosphere,2017,183:80-88.
[15]SONG Lirong,CHEN Wei,PENG Liang,et al.Distribution and bioaccumulation of microcystins in water columns:A systematic investigation into the environmental fate and the risks associated with microcystins in Meiliang Bay,Lake Taihu[J].Water Research,2007,41(13):2853-2 864.
[16]MGONJA J T,LUNING P,VAN DER VORST J G A J.Diagnostic model for assessing traceability system performance in fish processing plants[J].Journal of Food Engineering,2013,118(2):188-197.
[17]NICOLAS J,HOOGENBOOM R L A P,HENDRIKSENP J M,et al.Marine biotoxins and associated outbreaks following seafood consumption:Prevention and surveillance in the 21st century[J].Global Food Security,2017,15:11-21.
[18]吴多加,李凤琴.软骨藻酸与人类健康关系研究进展[J].卫生研究,2005,34(3):378-381.
[19]SAEED A F,AWAN S A,LING S,et al.Domoic acid:Attributes,exposure risks,innovative detection techniques and therapeutics[J].Algal Research,2017,24:97-110.
[20]ZIMBA P V,KHOO L,GAUNT P S,et al.Confirmation of catfish,Ictalurus punctatus,(Rafinesque),mortality from Microcystis toxins[J].Journal of Fish Diseases,2001,24(1):41-47.
[21]MAGALHES V F D,SOARES R M,AZEVEDO S M FO.Microcystin contamination in fish from the JacarepaguáLagoon(Rio de Janeiro,Brazil):Ecological implication and human health risk[J].Toxicon,2001,39(7):1 077-1 085.
[22]WILSON A E,GOSSIAUX D C,HK T O,et al.E-valuation of the human health threat associated with the hepatotoxin[J].Canadian Journal of Fisheries and Aquatic Sciences,2008,65(7):1 487-1 497.
[23]XIE L,XIE P,GUO L,et al.Organ distribution and bioaccumulation of microcystins in freshwater fish at different trophic levels from the eutrophic Lake Chaohu,China[J].Environmental Toxicology,2005,20(3):293-300.
[24]LIU Y,SONG L,LI X,et al.The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach,Misguruns mizolepis,Gunthe[J].Toxicon,2002,40(4):395-399.
[25]PAWLK-SKOWRON'SKA B,TOPOROWSKA M,RE-CHULICZ J.Simultaneous accumulation of anatoxin-a and microcystins in three fish species indigenous to lakes affected by cyanobacterial blooms[J].Oceanological and Hydrobiological Studies,2012,41(4):53-65.
[26]GUTIRREZ-PRAENA D,JOS,PICHARDO S,et al.Presence and bioaccumulation of microcystins and cylindrospermopsin in food and the effectiveness of some cooking techniques at decreasing their concentrations:A review[J].Food and Chemical Toxicology,2013,53(2):139-152.
[27]CORBEL S,MOUGIN C,BOUACHA N.Cyanobacterial toxins:Modes of actions,fate in aquatic and soil ecosystems,phytotoxicity and bioaccumulation in agricultural crops[J].Chemosphere,2014,96(2):1-15.
[28]CHEN Jian,HAN Fengxiang,WANG Fan,et al.Accumulation and phytotoxicity of microcystin-LR in rice(Oryza sativa)[J].Ecotoxicology and Environmental Safety,2012,76(2):193-199.
[29]SAQRANE S,OUAHID Y,ELGHAZALI I,et al.Physiological changes in Triticum durum,Zea mays,Pisum sativum and Lens esculenta cultivars,caused by irrigation with water contaminated with microcystins:A laboratory experimental approach.[J].Toxicon,2009,53(7-8):786-796.
[30]PEUTHERT A,CHAKRABARTI S,PFLUGMACHER S.Uptake of microcystins-LR and-LF(cyanobacterial toxins)in seedlings of several important agricultural plant species and the correlation with cellular damage(lipid peroxidation)[J].Environmental Toxicology,2007,22(4):436-442.
[31]ROMERO-OLIVA C S,CONTARDO-JARA V,BLOCKT,et al.Accumulation of microcystin congeners in different aquatic plants and crops--a case study from lake Amatitlan,Guatemala[J].Ecotoxicology and Environmental Safety,2014,102(1):121-128.
[32]MOHAMED Z A,AL SHEHRI A M.Microcystins in groundwater wells and their accumulation in vegetable plants irrigated with contaminated waters in Saudi Arabia[J].Journal of Hazardous Materials,2009,172(1):310-315.
[33]CHEN Jianzhou,DAI Jian,ZHANG Haiyang,et al.Bioaccumulation of microcystin and its oxidative stress in the apple(Malus pumila)[J].Ecotoxicology,2010,19(4):796-803.
[34]HEREMAN T C,BITTENCOURT-OLIVEIRA M C.Bioaccumulation of microcystins in lettuce[J].Journal of Phycology,2012,48(6):1 535-1 537.
[35]CORDEIRO-ARAU'JO M K,CHIA M A,DOCARMO-BITTENCOURT-OLIVEIRA M.Potential human health risk assessment of cylindrospermopsin accumulation and depuration in lettuce and arugula[J].Harmful Algae,2017,68:217-223.
[36]HUMPAGE A R,FALCONER I R.Oral toxicity of the cyanobacterial toxin cylindrospermopsin in male Swiss albino mice:Determination of no observed adverse effect level for deriving a drinking water guideline value[J].Environmental Toxicology,2003,18(2):94-103.
[37]RZYMSKI P,NIEDZIELSKI P,KACZMAREK N,et al.The multidisciplinary approach to safety and toxicity assessment of microalgae-based food supplements following clinical cases of poisoning[J].Harmful Algae,2015,46:34-42.
[38]ROY-LACHAPELLE A,SOLLIEC M,BOUCHARD MF,et al.Detection of cyanotoxins in algae dietary supplements[J].Toxins,2017,9(3):76-93.
[39]GILROY D J,KAUFFMAN K W,HALL R A,et al.Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements[J].Environmental Health Perspectives,2000,108(5):435-439.
[40]PARKER C H,STUTTS W L,DEGRASSE S L.Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of microcystins in blue-green algal dietary supplements[J].Journal of Agricultural and Food Chemistry,2015,63(47):10 303-10 312.
[41]VICHI S,LAVORINI P,FUNARI E,et al.Contamination by Microcystis and microcystins of blue-green algae food supplements(BGAS)on the Italian market and possible risk for the exposed population[J].Food and Chemical Toxicology,2012,50(12):4 493-4 499.
[42]VINOGRADOVA T,DANAHER M,BAXTER A,et al.Rapid surface plasmon resonance immunobiosensor assay for microcystin toxins in blue-green algae food supplements[J].Talanta,2011,84(3):638-643.
[43]HEUSSNER A H,MAZIJA L,FASTNER J,et al.Toxin content and cytotoxicity of algal dietary supplements[J].Toxicology&Applied Pharmacology,2012,265(2):263-271.
[44]ORTELLI D,EDDER P,COGNARD E,et al.Fast screening and quantitation of microcystins in microalgae dietary supplement products and water by liquid chromatography coupled to time of flight mass spectrometry[J].Analytica Chimica Acta,2008,617(1-2):230-237.
[45]YU F Y,LIU B H,CHOU H N,et al.Development of a sensitive ELISA for the determination of microcystins in Algae[J].Journal of Agricultural and Food Chemistry,2002,50(15):4 176-4 182.
[46]RELLAN S,OSSWALD J,SAKER M,et al.First detection of anatoxin-a in human and animal dietary supplements containing cyanobacteria[J].Food and Chemical Toxicology,2009,47(9):2 189-2 195.
[47]DRAISCI R,FERRETTI E,PALLESCHI L,et al.Identification of anatoxins in blue-green algae food supplements using liquid chromatography-tandem mass spectrometry[J].Food Additives and Contaminants,2001,18(6):525-531.
[48]MYHRE O,EIDE D M,KLEIVEN S,et al.Repeated five-day administration of L-BMAA,microcystin-LR,or as mixture,in adult C57BL/6 mice-lack of adverse cognitive effects[J].Scientific Reports,2018,8(1):2 308-2 322.
[2]VILARIN'O N,LOUZAO M C,ABAL P,et al.Human poisoning from marine toxins:Unknowns for optimal consumer protection[J].Toxins,2018,10(8):324-362.
[3]PAPADIMITRIOU T,KAGALOU I,STALIKAS C,et al.Assessment of microcystin distribution and biomagnification in tissues of aquatic food web compartments from a shallow lake and evaluation of potential risks to public health[J].Ecotoxicology,2012,21(4):1 155-1 166.
[4]LARRAMENDY M L,SOLONESKI S.Cyanobacterial Toxins Emerging Contaminants in Soils:A Review of Sources,Fate and Impacts on Ecosystems,Plants and Animal and Human Health[M].Croatia:Bouaicha,Noureddine,2016:105-126.
[5]LEE J,LEE S,JIANG X.Cyanobacterial toxins in freshwater and food:Important sources of exposure to humans[J].Annual Review of Food Science and Technology,2017,8(1):281-304.
[6]TESTAI E,BURATTI F M,FUNARI E,et al.Review and analysis of occurrence,exposure and toxicity of cyanobacteria toxins in food[J].EFSA Supporting Publications,2016,13(2):998E.
[7]IARC Working Group.IARC monographs on the evaluation of carcinogenic risks to humans.Ingested nitrate and nitrite,and cyanobacterial peptide toxins[J].IARC monographs on the eualuation of carcinogenic risks to humans/World Health Organization,International Agencyfor Research on Cancer,2010,94(7):1-412.
[8]DEZ-QUIJADA L,GUZMN-GUILLN R,PRIETO OR-TEGA A I,et al.New method for simultaneous determination of microcystins and cylindrospermopsin in vegetable matrices by SPE-UPLC-MS/MS[J].Toxins,2018,10(10):406-416.
[9]BRAGA A C,CAMACHO C,MARQUES A,et al.Combined effects of warming and acidification on accumulation and elimination dynamics of paralytic shellfish toxins in mussels Mytilus galloprovincialis[J].Environmental Research,2018,164:647-654.
[10]PERL T M,BDARD L,KOSATSKY T,et al.An outbreak of toxic encephalopathy caused by eating mussels contaminated with domoic acid[J].New England Journal of Medicine,1990,322(25):1 775-1 780.
[11]ZI J,PAN X,MACISAAC H J,et al.Cyanobacteria blooms induce embryonic heart failure in an endangered fish species[J].Aquatic Toxicology,2018,194:78-85.
[12]MELLO F D,BRAIDY N,MARCAL H,et al.Mechanisms and effects posed by neurotoxic products of cyanobacteria/microbial eukaryotes/dinoflagellates in algae blooms:A review[J].Neurotoxicity Research,2018,33(1):153-167.
[13]CHAI Y,DENG W J,QIN X,et al.Occurrence of four species of algae in the marine water of Hong Kong[J].Marine Pollution Bulletin,2017,124(2):890-896.
[14]JIANG Tao,LIU Lei,LI Yang,et al.Occurrence of marine algal toxins in oyster and phytoplankton samples in Daya Bay,South China Sea[J].Chemosphere,2017,183:80-88.
[15]SONG Lirong,CHEN Wei,PENG Liang,et al.Distribution and bioaccumulation of microcystins in water columns:A systematic investigation into the environmental fate and the risks associated with microcystins in Meiliang Bay,Lake Taihu[J].Water Research,2007,41(13):2853-2 864.
[16]MGONJA J T,LUNING P,VAN DER VORST J G A J.Diagnostic model for assessing traceability system performance in fish processing plants[J].Journal of Food Engineering,2013,118(2):188-197.
[17]NICOLAS J,HOOGENBOOM R L A P,HENDRIKSENP J M,et al.Marine biotoxins and associated outbreaks following seafood consumption:Prevention and surveillance in the 21st century[J].Global Food Security,2017,15:11-21.
[18]吴多加,李凤琴.软骨藻酸与人类健康关系研究进展[J].卫生研究,2005,34(3):378-381.
[19]SAEED A F,AWAN S A,LING S,et al.Domoic acid:Attributes,exposure risks,innovative detection techniques and therapeutics[J].Algal Research,2017,24:97-110.
[20]ZIMBA P V,KHOO L,GAUNT P S,et al.Confirmation of catfish,Ictalurus punctatus,(Rafinesque),mortality from Microcystis toxins[J].Journal of Fish Diseases,2001,24(1):41-47.
[21]MAGALHES V F D,SOARES R M,AZEVEDO S M FO.Microcystin contamination in fish from the JacarepaguáLagoon(Rio de Janeiro,Brazil):Ecological implication and human health risk[J].Toxicon,2001,39(7):1 077-1 085.
[22]WILSON A E,GOSSIAUX D C,HK T O,et al.E-valuation of the human health threat associated with the hepatotoxin[J].Canadian Journal of Fisheries and Aquatic Sciences,2008,65(7):1 487-1 497.
[23]XIE L,XIE P,GUO L,et al.Organ distribution and bioaccumulation of microcystins in freshwater fish at different trophic levels from the eutrophic Lake Chaohu,China[J].Environmental Toxicology,2005,20(3):293-300.
[24]LIU Y,SONG L,LI X,et al.The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach,Misguruns mizolepis,Gunthe[J].Toxicon,2002,40(4):395-399.
[25]PAWLK-SKOWRON'SKA B,TOPOROWSKA M,RE-CHULICZ J.Simultaneous accumulation of anatoxin-a and microcystins in three fish species indigenous to lakes affected by cyanobacterial blooms[J].Oceanological and Hydrobiological Studies,2012,41(4):53-65.
[26]GUTIRREZ-PRAENA D,JOS,PICHARDO S,et al.Presence and bioaccumulation of microcystins and cylindrospermopsin in food and the effectiveness of some cooking techniques at decreasing their concentrations:A review[J].Food and Chemical Toxicology,2013,53(2):139-152.
[27]CORBEL S,MOUGIN C,BOUACHA N.Cyanobacterial toxins:Modes of actions,fate in aquatic and soil ecosystems,phytotoxicity and bioaccumulation in agricultural crops[J].Chemosphere,2014,96(2):1-15.
[28]CHEN Jian,HAN Fengxiang,WANG Fan,et al.Accumulation and phytotoxicity of microcystin-LR in rice(Oryza sativa)[J].Ecotoxicology and Environmental Safety,2012,76(2):193-199.
[29]SAQRANE S,OUAHID Y,ELGHAZALI I,et al.Physiological changes in Triticum durum,Zea mays,Pisum sativum and Lens esculenta cultivars,caused by irrigation with water contaminated with microcystins:A laboratory experimental approach.[J].Toxicon,2009,53(7-8):786-796.
[30]PEUTHERT A,CHAKRABARTI S,PFLUGMACHER S.Uptake of microcystins-LR and-LF(cyanobacterial toxins)in seedlings of several important agricultural plant species and the correlation with cellular damage(lipid peroxidation)[J].Environmental Toxicology,2007,22(4):436-442.
[31]ROMERO-OLIVA C S,CONTARDO-JARA V,BLOCKT,et al.Accumulation of microcystin congeners in different aquatic plants and crops--a case study from lake Amatitlan,Guatemala[J].Ecotoxicology and Environmental Safety,2014,102(1):121-128.
[32]MOHAMED Z A,AL SHEHRI A M.Microcystins in groundwater wells and their accumulation in vegetable plants irrigated with contaminated waters in Saudi Arabia[J].Journal of Hazardous Materials,2009,172(1):310-315.
[33]CHEN Jianzhou,DAI Jian,ZHANG Haiyang,et al.Bioaccumulation of microcystin and its oxidative stress in the apple(Malus pumila)[J].Ecotoxicology,2010,19(4):796-803.
[34]HEREMAN T C,BITTENCOURT-OLIVEIRA M C.Bioaccumulation of microcystins in lettuce[J].Journal of Phycology,2012,48(6):1 535-1 537.
[35]CORDEIRO-ARAU'JO M K,CHIA M A,DOCARMO-BITTENCOURT-OLIVEIRA M.Potential human health risk assessment of cylindrospermopsin accumulation and depuration in lettuce and arugula[J].Harmful Algae,2017,68:217-223.
[36]HUMPAGE A R,FALCONER I R.Oral toxicity of the cyanobacterial toxin cylindrospermopsin in male Swiss albino mice:Determination of no observed adverse effect level for deriving a drinking water guideline value[J].Environmental Toxicology,2003,18(2):94-103.
[37]RZYMSKI P,NIEDZIELSKI P,KACZMAREK N,et al.The multidisciplinary approach to safety and toxicity assessment of microalgae-based food supplements following clinical cases of poisoning[J].Harmful Algae,2015,46:34-42.
[38]ROY-LACHAPELLE A,SOLLIEC M,BOUCHARD MF,et al.Detection of cyanotoxins in algae dietary supplements[J].Toxins,2017,9(3):76-93.
[39]GILROY D J,KAUFFMAN K W,HALL R A,et al.Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements[J].Environmental Health Perspectives,2000,108(5):435-439.
[40]PARKER C H,STUTTS W L,DEGRASSE S L.Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of microcystins in blue-green algal dietary supplements[J].Journal of Agricultural and Food Chemistry,2015,63(47):10 303-10 312.
[41]VICHI S,LAVORINI P,FUNARI E,et al.Contamination by Microcystis and microcystins of blue-green algae food supplements(BGAS)on the Italian market and possible risk for the exposed population[J].Food and Chemical Toxicology,2012,50(12):4 493-4 499.
[42]VINOGRADOVA T,DANAHER M,BAXTER A,et al.Rapid surface plasmon resonance immunobiosensor assay for microcystin toxins in blue-green algae food supplements[J].Talanta,2011,84(3):638-643.
[43]HEUSSNER A H,MAZIJA L,FASTNER J,et al.Toxin content and cytotoxicity of algal dietary supplements[J].Toxicology&Applied Pharmacology,2012,265(2):263-271.
[44]ORTELLI D,EDDER P,COGNARD E,et al.Fast screening and quantitation of microcystins in microalgae dietary supplement products and water by liquid chromatography coupled to time of flight mass spectrometry[J].Analytica Chimica Acta,2008,617(1-2):230-237.
[45]YU F Y,LIU B H,CHOU H N,et al.Development of a sensitive ELISA for the determination of microcystins in Algae[J].Journal of Agricultural and Food Chemistry,2002,50(15):4 176-4 182.
[46]RELLAN S,OSSWALD J,SAKER M,et al.First detection of anatoxin-a in human and animal dietary supplements containing cyanobacteria[J].Food and Chemical Toxicology,2009,47(9):2 189-2 195.
[47]DRAISCI R,FERRETTI E,PALLESCHI L,et al.Identification of anatoxins in blue-green algae food supplements using liquid chromatography-tandem mass spectrometry[J].Food Additives and Contaminants,2001,18(6):525-531.
[48]MYHRE O,EIDE D M,KLEIVEN S,et al.Repeated five-day administration of L-BMAA,microcystin-LR,or as mixture,in adult C57BL/6 mice-lack of adverse cognitive effects[J].Scientific Reports,2018,8(1):2 308-2 322.