Short-term microbial effects of a large-scale mine-tailing storage facility collapse on the local

25 Pages·2017·9.14 MB·English

RESEARCHARTICLE
Short-term microbial effects of a large-scale
mine-tailing storage facility collapse on the
local natural environment
HeathW.Garris1¤*,SusanA.Baldwin2,JonTaylor2,DavidB.Gurr2,DanielR.Denesiuk1,
JonathanD.VanHamme1,LauchlanH.Fraser1
1 DepartmentsofNaturalResourceSciences&BiologicalSciences,ThompsonRiversUniversity,
Kamloops,BritishColumbia,Canada,2 DepartmentofChemicalandBiologicalEngineering,Universityof
BritishColumbia,Vancouver,BritishColumbia,Canada
a1111111111
a1111111111
¤ Currentaddress:DepartmentofBiology,CovenantCollege,LookoutMountain,Georgia,UnitedStatesof
a1111111111 America
a1111111111 *[email protected]
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Abstract
WeinvestigatedtheimpactsoftheMountPolleytailingsimpoundmentfailureonchemical,
OPENACCESS
physical,andmicrobialpropertiesofsubstrateswithintheaffectedwatershed,comprisedof
Citation:GarrisHW,BaldwinSA,TaylorJ,Gurr 70hectaresofriparianwetlandsand40kmofstreamandlakeshore.Weestablishedabio-
DB,DenesiukDR,VanHammeJD,etal.(2018)
monitoringnetworkinOctoberof2014,twomonthsfollowingthedisturbance,andevaluated
Short-termmicrobialeffectsofalarge-scalemine-
tailingstoragefacilitycollapseonthelocalnatural riparianandwetlandsubstratesformicrobialcommunitycompositionandfunctionvia16S
environment.PLoSONE13(4):e0196032.https:// andfullmetagenomesequencing.Atotalof234sampleswerecollectedfromsubstratesat
doi.org/10.1371/journal.pone.0196032
3depthsand1,650,752sequenceswererecordedinageodatabaseframework.These
Editor:AndreaFranzetti,UniversitadegliStudidi datarevealedawealthofinformationregardingwatershed-scaledistributionofmicrobial
Milano-Bicocca,ITALY
communitymembers,aswellascommunitycomposition,structure,andresponsetodistur-
Received:October10,2017 bance.Substratesassociatedwiththeimpactzoneweredistinctchemicallyasindicatedby
Accepted:April5,2018 elevatedpH,nitrate,andsulphate.Themicrobialcommunityexhibitedelevatedmetabolic
capacityforselenateandsulfatereductionandanabundanceofchemolithoautotrophsin
Published:April25,2018
theThiobacillusthiophilus/T.denitrificans/T.thioparuscladethatmaycontributetonitrate
Copyright:©2018Garrisetal.Thisisanopen
attenuationwithintheaffectedwatershed.Themostimpactedarea(a6kmstreamconnect-
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which ingtwolakes)exhibited30%lowermicrobialdiversityrelativetotheremainingsites.The
permitsunrestricteduse,distribution,and tailingsimpoundmentfailureatMountPolleyMinehasprovidedauniqueopportunityto
reproductioninanymedium,providedtheoriginal
evaluatefunctionalandcompositionaldiversitysoonafteramajorcatastrophicdisturbance
authorandsourcearecredited.
toassessmetabolicpotentialforecosystemrecovery.
DataAvailabilityStatement:Allsequencedataare
availableathttps://www.ncbi.nlm.nih.gov/,project
numberPRJNA433688.Metadataareavailableat
http://www.datadryad.org/,accessionDOI:https://
doi.org/10.5061/dryad.d52df21.
Introduction
Funding:ThisprojectwassupportedbyaMitacs
Accelerate/ElevatePost-doctoralFellowshipfor
Miningproduceslargequantitiesofwastematerialannually,andcontainmentfailurespresent
HeathGarris,whereprofessionaldevelopment
challengestowaterqualityandwildlife.Contemporarymininginvolvesextensiverestructur-
seminars/trainingwereprovidedwithlittle/noinput
ingoflandscapeswherevegetationandnaturally-accretedsoilsareremoved,andcontiguous
intothecontentoftheresearchproposal/
execution.Thisprogramwasco-fundedbya bedrockisexposedandpartiallyconvertedtocoarseandfinewastes.Dependingontheparent
PLOSONE|https://doi.org/10.1371/journal.pone.0196032 April25,2018 1/25
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partnerindustry(MountPolleyMiningCorp)that materialcomposition,thesewastesarepronetoacceleratedchemicalweathering,constituting
contributedsubstantiallytothedevelopmentofthe afundamentallyalteredbiogeochemicalsettingbycomparisonwiththepre-existingecosystem
project(intheformofsiteaccess,feasibility
[1,2].Finewastes(oftentermed‘tailings’)typicallypresentthegreatestchallengesforcontain-
assessment,materialsupport,andmanuscript
mentastheyhavethegreatestpotentialforleaching(duetohighsurfaceareaforchemical
review).AdditionalfundingfromNSERCwas
weathering)andmobilityasdustorinfluidsuspensions.Forthisreason,currentbestpractices
awardedtosupportaninitial(pre-breach)water
qualityproject.NSERCmonitoredchangesinthe ofminingoperationsworldwidemanagetailingstobephysicallyandmetabolicallyisolated
proposedprojectscopebyrequestingreports,but fromthesurroundingwatershedthroughcontainmentinwasteimpoundments,suchastail-
didnotcontributetothecontentofthestudy ingsstoragefacilities[3].Overthedurationofamine’sactivelifetime,thequantityofmine
directly.Finally,GenomeBCandGenomeCanada
wastesaccumulatedonsitecanbecomeimmense.Forexample,beforetheimpoundmentfail-
supportedtheprojectthroughfundingand
ureattheMountPolleyMine(“theMine”),BritishColumbia,Canada,in2014,theMine’stail-
consultationonprojectscope/applicability/
suitabilitywithrespecttotheuseofgenomics ingsstoragefacilityhadthecapacitytocontain74millioncubicmetersofhydratedtailings
technologiesandaccomplishmentofprojectgoals, withinanimpoundmentoccupying2km2[4].
whichservedtoimproveprojectprecisions/ Despitebestpracticesforconstruction,inspectionandmaintenanceoftailingstoragefacili-
outcomes.Projectfundingwebreferences:Mitacs
ties,containmentfailureshaveoccurred[5].Underthesecircumstances,uncontrolleddeposi-
(https://www.mitacs.ca/en/impact/postdoctoral-
tionintopreviouslyprotectedsurroundingsdisturbsphysicalandchemicalproperties,and
fellows-share-program-benefits-mitacs-elevate-
theirassociatedabove-andbelow-groundecosystemswithinthesurroundinglandscape.
retreat);GenomeBC(https://www.genomecanada.
ca/en/metagenomics-assess-impacts-mount- Whilesurfaceerosionanddepositionareclearlyobserved,sub-surfacedisruptionsaremore
polley-mine-taillings-dam-breach-associated- challengingtoobserveand/orquantify.Suddenmajordisturbancesinthephysical,chemical
ecosystems);GenomeCanada(https://www. andbiologicalstructuresofnaturalsoilsandsedimentsarelikelytoimpactessentialecosystem
genomecanada.ca/en/metagenomics-assess-
functionssuchasnutrienttransport[6],weatheringanderosion,chemicaldetoxification,
impacts-mount-polley-mine-taillings-dam-breach-
waterqualitypreservation[7],supportofprimaryproductivity[8]andresiliencetoperturba-
associated-ecosystems);MountPolleyMine
(https://www.imperialmetals.com/assets/docs/mt- tions[9].Theseprocesseswilldeterminethepotentialforrecoveryinimpactedareas.Itfollows
polley/2015-02-23_MPFN.pdf). thatassessmentoftheaffectedsoilsandsedimentssoonafterthedisturbanceisrequiredto
evaluatethedegreetowhichtheseareasresistedtheimpactandinwhatwaystheirecosystem
Competinginterests:MountPolleyMineInc
providedmatchingfunds,siteaccess, functionsmayhavebeenaltered.
transportation,andmaterialassistancein Duetotheirtopographicpositionwithinlandscapes,wetlandsandriparianareasareoften
completingthisresearchproject(including thefirstrecipientsofminedsubstratesandchemicalswhencontainmentfailuresoccur.Owing
protectiveequipment,boatrentalandaccess,
totheirrelativelyhighbiologicalproductivityandaccumulationofsoilorganicmatter,these
SONDEwaterqualityprobes,andaccesstodata/
systemsarenotedfortheircapacitytoresisttoxicinputsandhaveevenbeenusedforpassive
schematicscollectedbymineemployees).This
remediationofmineeffluents[10,11].Minetailingsconsistofmineralsthatmightbecome
doesnotalterouradherencetoPLOSONEpolicies
onsharingdataandmaterials. sourcesofmetalandmetalloidcontaminantsdependingonthemineralogyoftheorebody,
mineralprocessingproceduresandenvironmentalconditionsinsidethetailings.Sequestration
anddetoxificationofmetalsandmetalloidsisfacilitatedthroughmetabolicprocessescarried
outbyspecifictypesofmicrobesthatinhabitnichesfoundinorganic-richsoilsandsediments.
Manymineremediationapproachescapitalizeonthesenaturalmetalsequestrationprocesses
byengenderingenvironmentalconditionsconducivetogrowthofbeneficialmicrobes,suchas
sulphate-reducingbacteria,Rhizobiaandmanyothermicrobeswiththecapacityformetal
immobilization[12–16].
Duringacatastrophicdisturbance,suchasaminetailingsimpoundmentfailure,theafore-
mentionedecosystemfunctionsofsoilandsedimentmicrobiomesarechallenged.Fewstudies
haveevaluatedtherolesoilmicrobialcommunitycompositionandalpha/betadiversityplayin
resiliencetodisturbance[17–20].Microbialcommunitiesmayrespondtosuddendisturbances
byexhibitingresilienceenablingrapidrecoverybacktopre-existingecosystemfunctions[21].
Theymaypersistinaperpetuallysuppressedstate,orexhibitshiftstoalternate,lessdesirable
statesthatprohibitrestorationorreducethebeneficialrelationshipsbetweenmicrobialdiver-
sity,soilandplantquality,andecosystemsustainability[22].Itisalsopossiblethatmicrobial
communitiesmayshiftinresponsetosuddenenvironmentalchangestowardsestablishment
ofalternatestructuralandfunctionalstatesthatpromoterestoration,toalbeitalternatebutsta-
bleecosystems[23].Depositionoftailingsmightresultinchronicdisturbancetosoiland
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sedimentmicrobiomes,whichisthoughttoleadtoreducedecosystemresiliencyshouldsub-
stratesbesubjectedtolargerstressorsinthefuture[24].
Forthisstudy,weaddresseddisturbanceofthestructureandfunctionofripariansoiland
sedimentmicrobialcommunitiesinthepathofdispersedtailingsandinnon-impactedsites
twomonthsfollowingthefailureofatailingsstoragefacilityatMountPolleyMine,(Likely,
BCCanada).Theimpoundmentfailureproducedawatershed-scaledisturbancegradient,
wheresoilswereerodedtobedrockandreplacedwithtailings(characteristicofHazeltine
Creek),whileothersexhibitedvaryingdegreesofmixingandburial(characteristicofPolley
Lake).Asaresult,MountPolleyisuniqueinthetimelinessofbaselinedatacollectedfollowing
thedisturbance,butsharesthedisadvantageofmanymajordisturbancestudiesinthelackof
dataquantifyingheterogeneityinsystemparametersbeforethedisturbanceoccurred.
TheMountPolleyMinetailingsstoragefacilityexperiencedafailureoffoundationmaterial
andsubsequentbreachatapproximately2:00am,Monday,August4,2014incentralBritish
Columbia,Canada[25].Foundationfailuresrepresent~6%ofthe147tailingsimpoundment
failuresreportedglobally[26].Overthefollowing2–3daysapproximately10.6millionm3of
mine-influenced,untreatedwaterandsedimentescapedintothenaturalwatershed[27],dis-
persingoverapproximately25hectaresoflake-associatedwetlandand45hectaresofriparian
area,anddepositingtogetherwithscourednaturalmaterialintoPolleyLakeandQuesnelLake
withtheriskoffurthertransportationdownstreamintotheQuesnelRiver[4].Impactsofthis
disturbanceonsoilandsedimentmicrobialpopulationsoftheaffectedriparianareaandlake-
associatedwetlandsweredeterminedthroughametagenomicsurveyalongthegradientoftail-
ingsdepositionawayfromtheimpoundmentfailureorigin.Thesemetagenomicrecordswere
combinedwithphysicalandchemicalcharacteristicsofthecollectedsubstratesandanalyzed
withinageodatabaseframeworktoassessmicrobialcommunityconnectionstoenvironmental
variablesincludingsoil,nutrient,andwaterstatus,andpassiveuptakeofcontaminants.
Materials&methods
Samplesitelocations
FieldsamplingwasconductedwithpermissionfromMountPolleyMineCorporation.Suitable
locationsforsamplecollectionweredeterminedbyperformingasurveyoftheaffectedwater-
shedviahighresolutionaerialphotographyandLandsatThematicMapperImagery[28].Sam-
plingconsistedof60establishedfieldsitesdistributedwithintheHazeltineCreekfloodplain
(19),PolleyLakefloodplain(20),QuesnelLakeFloodplain(9),andtheBootjackLakeFlood-
plain(12)(Fig1).
Shorelineecosystemsweretargetedsinceprimarymacrophyteproductivityishighestin
theseregionsandconsequentlytheyarelikelytoharbourdiversebelow-groundmicrobial
communitiesimportantforecosystemfunction.Forstreamandlakesidesamplesites,abase-
pointwasestablished(markedviaGPS(S1Table),surveyflagging,andaburiedgalvanized
steelpin).
Samplecollection
MonitoringsitesweresampledbetweenOctober7thand23rd,2014.Twotothreehundred
gramsofsubstratewereremovedfrom6locationsateachsiteandplacedintosterileWhirl-
Pak1bags.Sampleswerecollectedfromthreedistincthabitats(Fig3).
TheriparianandlakeareasaffectedbythetailingsimpoundmentfailureincludedHazeltine
Creek,PolleyLakeandQuesnelLake(Fig1).Theextentandamountoftailingsdepositedter-
restriallyandintothelakeshavebeenreportedindetailinthePostEventEnvironmental
ImpactAssessmentReportpublishedaftermuchsurveyworkwasconductedinthemonths
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Fig1.Biomonitoringnetworksamplesites.Sitesarelocatedwithintheaffectedwatershed,inhydricsoilsassociatedwithexistingwaterbodies.With
theexceptionofBootjackLake(referencearea)theselectedsitesreflectacontinuumofphysicalandchemicalimpactsfromtheevent.SubstrateCode
coloursreflectdesignationsdetailedinFig2.
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followingtheevent[4].Lessthan48hoursafterthefailure,short-waveinfraredreflectance
fromBootjackandPolleyLakeswasrecordedbyLandsat[28](USGS2011)andusedtodetect
solidssuspendedinPolleyLake(Fig4).
IncomparisontoBootjackLake,whichwasnotinthepathofthedepositedtailingssinceit
islocatedwestoftheminesite,higherloadingsofsuspendedsolidswereclearlyvisibleinPol-
leyLake.Basedontheintensityofthereflectance,theamountofsedimentattenuationinto
PolleyLakewasestimatedtofollowalogarithmicratelawwithdistanceawayfromthesouth-
eastshoreofPolleyLakewherethedepositedtailingsenteredtheLake(Fig4).Spacingofsam-
plinglocationsalongtheshoresofPolleyLakewasbasedonthisrelationship(Fig1).
Twosamplesweretakenfromthesurficiallayerofterrestrialsubstrate(0–5cmdepth)3m
apart1mparalleltothewater’sedge.Twosampleswerecollectedfromthemid-depthterres-
triallayer(25–30cmdepth)atthesamelocationsusingasoilprobeorEdelmanauger.For
BootjackandPolleyLakestheselattersamplesconstitutedclay-richhorizons.Twoadditional
sampleswereremovedfromthesub-aqueoussedimentlayerwithinthepermanentlysub-
mergedareaofthelittoralzone.Poreandsurfacewatersampleswerecollectedinseparate
bagsfromthesedimentlayer.Samplesforsulphateandsulfideanalysiswerepreservedin0.5%
zincacetate.Sampleswerefrozenondryiceimmediatelyaftersamplingandthenstoredat
-80˚Cuntilprocessing.PlotphotosweretakenforeachofthesamplelocationsusingaNikon
D300digitalSLRandincludedameterstickand1mX1mgridplacedinviewforreference.
Thesephotoswereusedtoqualitativelydescribesurfacemineralcomposition/grain-sizeand
amount/compositionofvegetationcover(Fig2).Habitatsvariedintheirestimateddegreeof
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Fig2.Sampleswerecollectedtorepresentthetypesofhabitatsaffectedbythespillincluding.(A)organic-richsoil
supportingadiversewetlandcommunityincludingliving,senescedanddecayedvegetation,(B)organic-dominated
lakeshorelackingextensivelivingvegetation,(C)minerallakeshoreincludingexposednativebedrock,cobbles,claysor
sands,(D)nativesubstrateswithevidenceoftailingsdeposition(visibleaccretionsoftailingssedimentsintermingled
withnativesubstrateorcoatingofplantstems/leaveswithfinetailings),(E)burialofnativesubstrateincoarsetailings
sands,(F)replacementofnativesubstratewithamixtureoffinetailingsandsands.Thetabletotherightpresents
meansurfacematerialestimates(n=5,1m2quadrats)forsamplesiteswithhabitatclassification(matchingcolors
fromfiguretotheleft).
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disturbancefromnovisibleimpactcharacteristicofBootjackLaketoerosionofallnativesub-
stratesandreplacementwithmaterialfromthetailingsimpoundment(characteristicofHazel-
tineCreek).Multiplequantitativeapproacheswereappliedtodeterminedistinctionsbetween
majorsubstratetypes(includingprevailingsurficialgrainsizesandorganiccontent).Inprac-
tice,freshlydepositedtailingswereeasilydistinguishedfromnativesubstratesbytheirlight
colorandmixtureofreddish-greysandsandfine,greysediments.
Physicalandchemicalparameters
Waterqualityparameters,pH,temperature,conductivity,dissolvedoxygen(DO),oxidation-
reductionpotential(ORP),andturbiditywererecordedinthefieldusingaYSIEXO2Multipa-
rameterWaterQualitySONDE(YSIInternational)forsurfacewaters(33 cmdepthdraft),
±3
adjacenttotheshorelinewhereterrestrialsampleswerecollectedandforporewaterimmedi-
atelyfollowingextractionfromauguredholes.Later,inthelaboratory,poreandsurfacewater
sampleswerethawedandanalyzedforsulphate(APHA[29]turbidimetricmethod4500-SO 2-
4
E),sulfide(methylenebluemethod4500-S2-D),ammonium(phenatemethod4500-NH F),
3
phosphate(ascorbicacidmethod4500-PE)andtotalnitrite/nitrate-N(cadmiumreduction
method4500-NO -Efollowedbycolorimetricmethod4500-NO -B).Physicalandchemical
3 2
metadataareavailablethroughDRYAD(datadryad.org;doi:10.5061/dryad.d52df21).
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Fig3.Cartooncross-sectionofanidealizedhydrosphererepresentinglayersthatweresampled.
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Microbialcommunityquantification
DNAextraction. JustpriortoDNAextraction,samplebagswithsolidssampleswere
thawedandthecontentshomogenizedmanually.Three250mgsubsamplesfromeachbag
weretakenforgenomicDNA(gDNA)extractionusingtheMoBioPowerSoil1DNAIsolation
kit(MoBioLaboratoriesInc.,CarlsbadCA,USA).ThethreegDNAextractionswerecom-
binedintoasingletube.TheconcentrationwasdeterminedusingaQubit13.0highsensitivity
fluorometricdoublestrandedDNAassay(ThermoFisherScientific,Waltham,MA,USA).
AmpliconPCR,librarypreparation,andsequencing. Smallsub-unitribosomalDNA
(SSUrDNA)ampliconswerepreparedforsequencingusingproceduresintheIllumina“16S
Fig4.Comparisonofshort-waveinfraredreflectance(representedinyellow-green)measuredinPolleyand
BootjackLakes48hourspost-damfailurevisually(inset)andasafunctionofdistanceawayfromthe
impoundmentfailure.
https://doi.org/10.1371/journal.pone.0196032.g004
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MetagenomicSequencingLibraryPreparationGuide”[30]withthefollowingmodifications:
PCRreactionsusedtheSsoFast™EvaGreen1Supermix(Bio-RadLaboratories(Canada)Ltd.,
Mississauga,ON,Canada),andprimersfortheV6-V8regionwereused:926f5’ AAACTYAA
AKGAATTGRCGGand1392r5’ ACGGGCGGTGTGTRC 3’.Afterrecordingtheconcentra-
tionineachindexed16SrDNAampliconpool,samplesweredilutedtoobtain4nMDNAin
20μLofDIH O.Paired-end300bpsequencingwasperformedonanIlluminaMiSeqatthe
2
UniversityofBritishColumbiaSequencingCentreintheFacultyofPharmaceuticalSciences
usingtheIlluminaMS-102-3003-MiSeqReagentKitv3(600cycles)andPhiXControlKitv3.
DNAformetagenomicwholegenomesequencingwaspurifiedfromthehighmolecular
weightbandon1%agarosegelusingexcisionandtheQIAquickGelExtractionKit(Qiagen)
andtheextractedgenomicDNAwasquantifiedusingQubitHSDNAassay(Thermo-Fisher
Scientific).Approximately1ngtotalDNAwasusedforlibrarypreparation.ADNAfragment
librarywithanindexateachendwasgeneratedforallsamplesusingtheNexteraXTDNA
samplepreparationkit(Illuminacatalogno.GA09115).Forqualitycontrolandsizeevalua-
tion,thesequencinglibrarywasanalyzedonaBioanalyzer2100(AgilentTechnologies).
Paired-endsequencingwasperformedasfortheampliconsequencing.Rawsequencefiles
frombothampliconandwholegenomesequencingweredepositedinthesequenceread
archive(SRA)onhttp://www.ncbi.nlm.nih.govunderprojectaccessionnumber
PRJNA433688.
Bioinformatics. SequencequalitywasinspectedusingFastQCversion0.10.1[31].Since
thequalityoftheforwardreadswasfarsuperiortothatforthereversereads,onlytheforward
readswereusedforfurtheranalysis.Therawforwardreadswerequalityfilteredusingthe
usearch[32]functionfastq_filtertotruncatetotheendsequencesifthePhredqualityscore
waslessthan15,removesequenceslessthan150basesinlength,andtodiscardreadswith
expectederrorsgreaterthan0.5andthosereadswithambiguousbasecalls.Highquality
sequenceswereclusteredinto97%sequencesimilaritycut-offoperationaltaxonomicunits
(OTUs)usingtheusearchfunctioncluster_otus.Taxonomicassignmenttorepresentative
sequencesfromeachOTUwasperformedwithMOTHURversion1.36.1[33]functionclas-
sify.seqsusingasthereferencetheSILVASSUdatabaseversion123[34].
WholegenomesequencereadswerepreprocessedtoremoveIlluminaandNexteraadapt-
ers,lowquality(Q<25)andshort(length<100bases)readsusingTrimGalore0.4.1(http://
www.bioinformatics.babraham.ac.uk/projects/trim_galore/).Highqualityreadswereassem-
bledwithvelvetandmeta-velvet[35].Openreadingframes(ORFs)werepredictedfromthe
assembledcontigsusingProdigalversion1.0.1,translatedtoaminoacidsequencesandanno-
tatedusingcustomdatabasesforrespiratorydenitrificationproteins(nitratereductase(NarG,
NapA),nitritereductasetoNO(NirK,NirS),nitritereductasetoammonia(NrfH)andnitrous
oxidereductase(NosZ)),selenatereductases(SerABC,SrdAandYgfK),dimethylsulfoxide
(DMSO)reductases[36],dissimilatorysulfitereductase(DsrAB)andtheproteindatabase
Uniref90.Allaminoacidsequencesforthecustomdatabases,excludingtheDMSOreductases,
weredownloadedfromtheUniprotrepository(http://www.uniprot.org/,accessed3July
2017).OpenreadingframepredictionandannotationwerecarriedoutusingtheMetapath-
waysversion2.5.1pipeline[37].Onlyhitswithbitscoresequaltoorgreaterthan80weretaken
intoconsideration.Consistencybetweenfunctionalannotationusingthedenitrification,sele-
natereductaseandsulfatereductasedatabasesandtheUniref90databasewasconfirmed.Since
selenatereductasesSerABandSrdAfromThaueraselenatisandBacillusselenatarsenatis,
respectively,belongtotheDMSOreductasefamilyofmolybdopterinoxidoreductases,ORFs
withhitstoSerABandSrdAaminoacidsequenceswerealsoqueriedagainstacustomDMSO
database[36]toconfirmtheirannotationtoselenatereductasewiththehighestbitscore.
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Microbialpopulationdiversity(richnessandevenness)ineachsamplewasassessedin
termsoftaxonomicrichness(#ofdetectedOTUs)andSimpson’sDiversity(1-D).TheOTU
tablewassubsampledwithMOTHURfunctionsub.sampleto3,000readspersampleandsplit
intothreeOTUtablesforeachlayerorhydrozone:surficial(0–5cm),deep(25–30)cmand
theaquaticsediments.
Beta-diversityofsampleswasexploredonnon-metricmultidimensionalscaling(NMDS)
ordinationplotsproducedusingBray-CurtisdissimilaritywiththeveganpackageoftoolsinR
version3.3.1.Statisticalcomparisonsofmicrobialpopulationcompositionswerecarriedout
usingUniFracdistances[38]withMOTHURfunctionsunifrac_weightedandunifrac_un-
weighted.Indicatorspeciesforeachbiome(BootjackLake,PolleyLake,QuesnelLakeand
HazeltonCreek)weredeterminedusingthemethodinDufrêneandLegendre[39]foreach
hydrozonelayer.
Results&discussion
Sitecharacteristics&chemistry
Siteswereclassifiedintodifferenthabitatsaccordingtothesurficialsubstrateappearance(Fig
2).HabitatsAtoCexhibitedanarrayofmacrophytes,organicdepositsandmineralsandlittle
tonoevidenceofthepresenceoftailings.Greytailingsandmagnetitesandspresentedadis-
tinctvisualcontrasttothedarkernativesubstrates,makingthemeasytodistinguishonthe
ground.TailingsatMountPolleyarederivedfromanalkalicporphyrycoppergolddeposit
wherecopperandcoppersulphidesareremovedpriortostorageinthetailingsimpoundment
[40].HabitatDexhibitedamixtureofdepositedtailingsandintactnativesubstrateswhilehab-
itatsEandFexhibitedextensivedepositedtailingsanddistinctionsweremadebasedonmate-
rialtexture(HabitatFexhibitingprimarilyfinegreytailings).Habitattypeswereassociated
closelywiththefourmajorwaterbodiesidentifiedinthisstudy.HazeltineCreekexhibitedthe
greatestimpactfromthedisturbance,withthedepthoferosionofsoilandparentmaterial
exceeding7minsomeareas,exposingbedrockanddepositingthick(~1-3m)tailingsand
associatedsandsinacorridorrangingfrom40–250meterswide.
TheshorelineofBootjackLakeconsistedofmostlyhabitatsAandBandlackeddeposited
tailings.TheLakeexhibitedextensivebackwaterdepressionsinitsterrestrialshorelinethat
wereorganicrich,under-layedwithanocclusivelayerofhighlyreducedclaybasedonclear
gleyingandlackofoxidizedrootingzones.Asaresult,BootjackLakesamplelayersweredis-
tinctlydifferentincomposition,wheresurficialsampleswerelargelycomprisedoforganic
material,andwiththeexceptionoftheoutlettoBootjackCreek,deepsampleswerecomposed
ofreducedclays.BootjackLakesedimentdepositswereprimarilyfinesedimentsimbeddedin
rockycobbles.HazeltineCreek,themainflowpathofthereleasedtailings,wasscouredand
almostentirelyreplacedbysedimentdepositedfromthetailingsoutwash(habitatF).Forboth
PolleyandQuesnelLakes,somesitesclosetothedepositionpathwerereplacedbytailings.
EventhoughalargeamountoftailingswaspushedintoPolleyLakeatthesouthend[4],there
werestillsomesiteswherenaturaldiversewetlandcommunitieswereintact.TheQuesnel
LakeshorelinesadjacenttotheHazeltineCreekdeltawheretailingsenteredthelakecontained
littlevegetationandcomprisedmostlydeepsanddepositswithexposedweatheredcobbles
(habitatsC&D).Onlysites20Aand21AonQuesnelLakewerevegetated,thoughonlythe
mostproximalsitestotheHazeltineoutflowexhibitedevidenceofimpactsfromthebreach
(depositedtailingsand/ordisplacedsediment).Themarkedlydifferentphysicalfeaturesofthe
QuesnelLakeshorelineversusthoseinBootjackLakeandatthenorthernendofPolleyLake
reflectconditiondifferencesthatexistedbeforethespill.QuesnelLake’sshorelineischaracter-
izedbyextensivewave-washedcobblesandlittlevegetationwithin10mofthelakeedge,even
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insitessampledatgreaterdistancesfromtheHazeltineCreekoutflow(site14Ais2.5kmfrom
theHazeltineCreekoutflowandexhibitedlittlevegetationcoverandnoevidenceofdeposited
tailings).
Soilmoistureandorganicmatter(SOM)contentsofthesamplescollectedasdetermined
byovendryingandloss-on-ignitiontestsvariedfrom1.7%to91.1%,and0to854g/Kgrespec-
tively,withSOMsignificantlyassociatedwithmoisturecontent(R2=0.74,F=401,P<0.001)
(S1Fig).Althoughtherewerenostatisticallysignificantdifferencesinmoisturecontentand
SOMbetweenlayersandhabitats,habitatAcontainedhigherSOMthantheotherhabitats,
withhabitatFsampleshavingthelowestamountsofSOM(S2Fig).WhencomparingSOMby
lake/stream,HazeltineCreeksurficialsamplescontainedsignificantlylowerSOMrelativeto
allPolleyLakesamplelayers(p =0.02,p =0.01,p =0.04)andBootjacksurficialsam-
surf deep sed
ples(p=0.02)basedonBonferroni-correctedMann-Whitneypairwisecomparisons(Kruskal-
WallisH=46.15,p<0.0001)(S3Fig).DeepsamplescollectedfromHazeltineCreekcontained
significantlylowerSOMthanPolleyLakedeep(p=0.02)andsurficialsamples(p=0.01).All
othercomparisonswerenotsignificant.
Pore-waterchemistrypropertiesweremeasuredinlakeshoresub-aqueoussediments(S2
TableandS4Fig).Temperaturevariationswereduetothetwodifferentsamplingperiods,6–8
and21–23October2014,duringwhichtheaverageporewatertemperatureswere13and9˚C,
respectively.PolleyandQuesnelLakesedimentshadsimilarcircum-neutralpHs(7.3 and
±0.7
7.8 ,respectively),whereasthepHinpore-waterfromBootjackLakewaslower(average
±0.3
6.5 ,p<0.01)andHazeltineCreekhigher(averageof8.9 ,p<0.001).Thesediments
±0.4 ±0.3
withinQuesnelLakewerethemostaerated(DO=7.7 mg/L)withthoseinBootjackLake
±1.4
havingthelowestoxygenconcentrations(DO=2.2 mg/L,p<0.001).Awiderangeofdis-
±0.8
solvedoxygenconcentrationsweremeasuredintheHazeltineCreekpore-waters.Conductivity
wasslightlyhigherinPolleyLakesedimentpore-waterscomparedwithBootjackandQuesnel
Lakes(p<0.05).NoconductivitydatawereobtainedforHazeltineCreek(sensormalfunction).
Theammonium-N,nitrate-NandphosphateconcentrationsinBootjackLakesediment
porewatersampleswereallbelow1mg/L(S2Table).Therewasmorenitrate-NintheQuesnel
Lakesediments(1.35 mg/L)withthehighestconcentrationfoundatsite12A(4.11
CI=0–2.92
mg/L),whichwasasitewheretailingsweredeposited.Nitrate-Nconcentrationswerealso
higherinHazeltineCreekthanintheotherlocations(averageof2.34 mg/L)withthe
CI=0–5.75
highestconcentrationfoundatsite36A(8.24mg/L).
InHazeltineCreek,theaveragesulphateconcentrationofthosesitesfromwhichpore
watercouldberecoveredwas251 mg/L.Onesitehadasulphateconcentrationas
CI=0–258
highas940mg/L(site36A)(S3Table),whichwasalsowherethehighestnitrate-Nconcentra-
tionwasmeasured.AtthePolleyLakesites,sulphateconcentrationswerehigherthanthosein
theotherLakes(BootjackandQuesnel),butnotashighinHazeltineCreek(averageof125
±56
mg/L).Siteswiththehighestsulphateconcentrationswereatlocationscontainingtailings
(sites58Aand60A).However,onefarsitethatwashighlyvegetated(site53A)alsohadahigh
sulphateconcentration.Sulphateconcentrationswerenotmeasuredintheporewaterssam-
pledfromBootjackLakeandQuesnelLake.SamplestakenfromQuesnelLakesincethe
impoundmentfailurefoundsulphatetobelessthan15mg/L[4].Takentogethersoilandpore
watercharacterizationtestsrevealedthatthedepositedtailingscreateddistincthabitatsand
environmentscomparedwithunaffectedsites.
Microbialcomposition
Diversityanddistribution. Atotalof1,650,752highqualitysequenceswithamean
lengthof227 baseswereobtainedfrom234sampleswithanaveragereadcountpersample
±40
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Fig5.Speciesrichness(sobs)anddiversity(Simpson’s1-D)ofmicrobialcommunitiesinthesurficial,deepandsedimentlayersinBootjackLake
(BJL),HazeltineCreek(HAZ),PolleyLake(POL)andQuesnelLake(QUE).Comparisonsarealsoshownforthesixhabitattypes(A-F)identifiedin
Fig2.ErrorBarsrepresentstandarderrorofthemean.
https://doi.org/10.1371/journal.pone.0196032.g005
of5,630 .Aftersubsamplingto3,000readspersample,15sampleswererejecteddueto
±2,839
insufficientsequencingdepth.Theimpactofthetailingsdepositionwasmostapparentfor
HazeltineCreekwherespeciesrichnesswasapproximatelyonethirdlessthanthatobserved
foralltheothersites.AlthoughHazeltineCreekwasscouredandmuchofitfilledwithtailings,
microorganismswerestilldetectedinalllayersofthosesitesthatyieldedenoughDNAfor
sequencing.Asexpected,theun-impactedreferencesite,BootjackLakehadthegreatestspe-
ciesrichness(observednumberofspecies(sobs)inFig5),especiallyinthesurficiallayers
thoughitonlydifferedsignificantlyfromHazeltineCreek(F =9.3,F =49.0,
3,21Sed 3,29Surf
F =17,p<0.0005).Aspre-eventmicrobialcommunitydataarenotavailable,the
3,26Deep
heighteneddiversityinBootjackLakemaybetheresultofpre-existingdifferencesamong
waterbodiesasBootjackexhibitedhighersoilorganicmatterreflectiveofelevated
productivity.
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