Evaluation of water quality during successive severe drought years within Microcystis blooms using fish embryo toxicity tests for the San Francisco Estuary, California

2018·0.67 MB·English

by  Tomofumi Kurobe

ScienceoftheTotalEnvironment610–611(2018)1029–1037
ContentslistsavailableatScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
Evaluation of water quality during successive severe drought years
within Microcystis blooms using fish embryo toxicity tests for the San
Francisco Estuary, California
TomofumiKurobea,⁎,PeggyW.Lehmanb,M.E.Haquec,TizianaSeddad,SarahLesmeistere,SweeTeha
aDepartmentofAnatomy,PhysiologyandCellBiology,SchoolofVeterinaryMedicine,UniversityofCalifornia,Davis,CA95616,USA
bCaliforniaDepartmentofFishandWildlife,2109ArchAirportRoad,Stockton,CA95206,USA
cDepartmentofZoology,FacultyofBiologicalSciences,JahangirnagarUniversity,Savar,Dhaka1342,Bangladesh
dDepartmentofVeterinaryMedicine,UniversityofSassari,ViaVienna2,07100Sassari,Italy
eCaliforniaDepartmentofWaterResources,3500IndustrialWay,WestSacramento,CA95691,USA
H I G H L I G H T S G R A P H I C A L A B S T R A C T
• Californiaexperiencedaseveremultiple
yeardroughtin2014and2015.
• Waterqualitywastestedbyfishembryo
toxicitytestsusingDeltaSmeltandMe-
daka.
• HighmortalitywasobservedinMedaka
embryoinconjunctionwiththegrowth
ofAeromonas.
• GrowthofAeromonaswasenhancedby
Microcystiscelllysate.
• Cyanobacterialbloomsmaybeacause
of waterquality deteriorationby en-
hancingbacterialgrowthinthewild.
a r t i c l e i n f o a b s t r a c t
Articlehistory: IntheSanFranciscoEstuary,California,thelargestestuaryonthePacificCoastofNorthAmerica,thefrequency
Received7April2017 andintensityofdroughtandassociatedcyanobacteriabloomsarepredictedtoincreasewithclimatechange.
Receivedinrevisedform28June2017 Toassesstheimpactofwaterqualityconditionsonestuarinefishhealthduringsuccessiveseveredrought
Accepted30July2017 yearswithMicrocystisblooms,weperformedfishembryotoxicitytestingwithDeltaSmeltandMedaka.Fishem-
Availableonline30August2017 bryoswereexposedtofilteredambientwatercollectedfromtheSanFranciscoEstuaryduringtheMicrocystis
bloomseasonin2014and2015,thethirdandfourthmostsevererecordeddroughtyearsinCalifornia.Medaka
Editor:HennerHollert
embryosincubatedinfilteredambientwatersexhibitedhighmortalityrates(N77%),whichwasmainlydueto
Keywords: bacterialgrowth.Medakamortalitydatawasnegativelycorrelatedwithchloride,andpositivelycorrelated
Fishembryotoxicitytesting withwatertemperature,totalanddissolvedorganiccarbon,andambientandnetchlorophyllaconcentration.
DeltaSmelt DeltaSmeltembryomortalityrateswerelower(b42%)andnoprominentseasonalorgeographictrendwasob-
Medaka served.TherewasnosignificantcorrelationbetweentheDeltaSmeltmortalitydataandwaterqualityparame-
Bacterialgrowth ters.AeromonaswasthedominantbacteriathatadverselyaffectedMedaka.ThegrowthofAeromonaswas
Developmentaldeformity suppressedwhensalinitywasgreaterthanorequalto1psuandresultedinasignificantreductioninmortality
Mortality
rate.BacterialgrowthtestdemonstratedthatthelysateofMicrocystiscellsenhancedthegrowthofAeromonas.
ToxinproductionbyMicrocystisisamajorenvironmentalconcern,however,weconcludethatdissolvedsub-
stancesreleasedfromMicrocystisbloomscouldresultinwaterqualitydeteriorationbypromotinggrowthofbac-
teria.Furthermore,adistinctivedevelopmentaldeformitywasobservedinMedakaduringthetoxicitytests;
⁎ Correspondingauthorat:1089VeterinaryMedicineDr.,VM3B,Room3202,SchoolofVeterinaryMedicine,UniversityofCalifornia,Davis,CA95616,USA.
E-mailaddress:[email protected](T.Kurobe).
http://dx.doi.org/10.1016/j.scitotenv.2017.07.267
0048-9697/©2017ElsevierB.V.Allrightsreserved.
1030 T.Kurobeetal./ScienceoftheTotalEnvironment610–611(2018)1029–1037
somiteformationwasinhibitedatthesametimethatcardiogenesisoccurredandthefunctionalheartwasob-
servedtobebeating.Theexactcauseoftheembryonicdevelopmentaldeformityisstillunknown.
©2017ElsevierB.V.Allrightsreserved.
1.Introduction ofaquaticspecies.TheStateofCaliforniaDepartmentofWaterRe-
sourcesestablishedaresearchprogramtoinvestigatethemagnitude,
FortheSanFranciscoEstuary(SFE)inthestateofCalifornia,the timing,distribution,andfoodwebimpactofMicrocystisintheupper
largestestuaryalongthePacificCoastofNorthAmerica,climatechange SFE,knownastheSacramentoandSanJoaquinDeltainthesummer
wasassociatedwithaseveremultipleyeardroughtthatbeganin2012 of2014and2015,thethirdandfourthdriestyearsonrecordinthe
andpersistedthrough2015(Cayanetal.,2009).The2012to2015 SFE.Asapartofthistwo-yearresearchprogram,thisstudyperformed
droughtinCaliforniawascharacterizedbybelowaverageprecipitation fishembryotoxicitytestingtodeterminethepotentialimpactofambi-
andaboveaverageatmospherictemperature(USEPA,2015).These entwaterqualityconditionsonthefisheryintheSFE.Weaddressedthe
atmosphericconditionsalsoproducedadecreaseinfreshwaterinflow hypothesisthatcontaminantsinthewatercolumnoriginatingfromnat-
and increase in water temperature, which favored the growth of uraland/oranthropogenicsourcesduringseveredroughtyearsleadto
cyanobacteria harmful algal blooms (CyanoHABs), particularly deteriorationinwaterqualitythatimpactthehealthoffishesintheSFE.
Microcystisspp.(Lehmanetal.,2017).Because,thefrequencyandinten-
sityofdroughtandassociatedcyanobacteriabloomsareexpectedtoin- 2.Materialsandmethods
crease with climate change, understanding their impact on fishery
productioninSFEiscriticallyimportant(IPCC,2007). 2.1.Sitedescription
Microcystisbloomsareamajorwaterqualityprobleminsurfacewa-
ters worldwide. Microcystis can produce cyanotoxins, including TheSFEcontains1100kmofwaterways,whichreceivefreshwater
microcystins,whichpromotelivercancerinhumansandwildlifefrom fromtheSacramentoRiveronthenorth,theSanJoaquinRiveronthe
freshwatertomarineecosystems(Zeguraetal.,2003;International south, and marine water from the San Francisco Bay on the west
Agency for Research on Cancer, 2006; Ibelings and Havens, 2008; (Fig.1).Thewaterintheestuaryisusedforagricultureanddrinking
Milleretal.,2010).Microcystisalsocontainslipopolysaccharideendo- waterandprovideshabitatforalargesuiteofaquaticorganismsinclud-
toxins,whichinhibitiontransportinfishgills,aswellasfishembryode- ingendangeredfishspecies(Nicholsetal.,1986;Brownetal.,2013).
velopment(Codd,2000).Microcystishasbeenassociatedwithnegative MicrocystisbloomsoccurinthesummerandfallintheSFE,butthe
impactstothehealthandsurvivaloffishexposedtomicrocystinsinthe length of the bloom season was longer during the drought years
dietandzooplanktonexposedtobothdissolvedmicrocystinsinthe (Lehman et al., 2017). Microcystis bloomsbegin in the SanJoaquin
waterandmicrocystinsinthediet(Geretal.,2009,2010;Acuñaetal., RiverandextendbothnorthwardandwestwardintotheSacramento
2012).Variouswaterqualityconditionsandenvironmentalfactors, RiverandSuisunBaywithflowandtide(Lehmanetal.,2005).The
suchaselevatedwatertemperature,highlightintensity,andincreased mainriverchannelsare12mdeepandarelinkedwithshallowwater
nutrients enhanced the growth of Microcystis and production of habitatsinfloodedislandsandfloodplainsthatareonlyafewmeters
microcystins(Davisetal.,2009;Pineda-Mendozaetal.,2016). deep.
Waterqualitydeteriorationduetoanthropogeniccontaminantscan
alsobeenhancedduringdroughtconditionsintheSFE.Forexample,re- 2.2.Fieldsampling
ducedfreshwaterinflowcanenhancepesticideconcentrationsinsur-
facewatersduetoconcentrationeffects,becausepesticideapplication Sampling was conducted bi-weekly at 10 stations during the
inagriculturallandsinCaliforniahasbeenconstantorslightlyincreas- Microcystisbloomseasonin2014(betweenJulyandDecember)and
ingsince2010(CADPR,2017).Herbicidesarealsosprayedtocontrolin- 2015(betweenAugustandNovember;Fig.1).Watertemperature,spe-
vasiveaquaticplants,suchaswaterhyacinth(Eichhorniacrassipes)and cificelectricalconductance,dissolvedoxygenconcentration,pH,and
EgeriadensaintheSFE(Santosetal.,2009).Controllingundesirableor- turbidityweremeasuredat0.3mdepthusinganYSI6600sonde(YSI,
ganismsiseconomicallyimportant,however,theincreasingconcentra- https://www.ysi.com/).Specificelectricalconductancedatawerecon-
tionsofanthropogenicchemicalsduringseveredroughtyearsraises vertedtosalinity.Waterwascollectedforwaterqualitymeasurements
environmentalconcernsonhealthofendemicaquaticorganismsin andfishembryotoxicitytestingwithavanDornsamplerat0.3mdepth
theSFE,wherefisheryproductionhasdeclinedsince2000(Sommer (subsurfacewatersamples)andimmediatelystoredonice.Waterfor
etal.,2007). chloride,ammonium,nitrateplusnitrite,silicateandsolublereactive
Toxicitystudiesusingfishembryosofferauniqueperspectiveon phosphorus measurements was filtered through Nucleopore filters
ecologicalhealthevaluation.Organismsattheembryonicstagearevul- (0.45μmporesize)andfrozenuntilanalysis(UnitedStatesEnviron-
nerabletocontaminantsbecausetheirtissuesandvitalbiologicalsys- mental Protection Agency, 1983; United States Geological Survey,
temsaredifferentiatinganddevelopingrapidly.Thusanydisruption 1985;AmericanPublicHealthAssociationetal.,1998).Waterfordis-
or impact at the embryonic stage can exert lifelong consequences, solved organic carbon (DOC) analysis was filtered through pre-
suchasdeformities(McKim,1977;Embryetal.,2010).Thecumulative combustedGF/Ffilters(poresize0.7μm)andkeptat−20°Cuntilanal-
effect of differentiation and development makes embryos an ideal ysis(AmericanPublicHealthAssociationetal.,1998).Unfilteredwater
modelforacuteandchronictoxicitystudiesaswell(Belangeretal., samplesfortotalandvolatilesuspendedsolids,totalorganiccarbon
2013;Wagneretal.,2017).Fishembryonictoxicitytestinghasbeen (TOC),andtotalphosphateanalyseswerekeptat4°Cuntilanalysis
widelyutilizedtoassessdevelopmentaltoxicityofvarioustypesof (AmericanPublicHealthAssociationetal.,1998).
chemicalcompounds,suchascyanotoxins,pesticides,andnanoparticles WatersamplesfordeterminationofMicrocystisbiovolume(N75μm
(Berryetal.,2007;Choetal.,2013;Alharbietal.,2016;Wagneretal., sizefraction)andchlorophyllaconcentrationwerealsocollectedfrom
2017). thesurfaceofthewatercolumnbyagentlehandtowofa0.3mdiam-
Despiteconcernsabouttheimpactsofdroughtonwaterqualityin eterplanktonnet(75μmmesh)overadistanceof30.5m.Thenet
theSFE,littleisknownabouthowCyanoHABandcontaminantsfrom wasfittedwithfloatsthatkepttheringjustbelowthesurface,making
anthropogenicsourcesduringdroughtyearscouldimpactthehealth thenettowanintegratedsampleofthe0.3msurfacelayer.Asurface
T.Kurobeetal./ScienceoftheTotalEnvironment610–611(2018)1029–1037 1031
Fig.1.MapoftheSanFranciscoEstuaryshowingthelocationofthesamplingstations.
nettowwasusedinordertogetarepresentativesampleofthelarge becauseitisahardyspeciesthatiseasytoculture,andimportantly
Microcystiscolonies,whichwerewidelydispersedacrossthesurface Medaka is an asynchronous spawner and lays eggs year-round
ofthewatercolumnandcanreach50,000μmindiameter.Microcystis underacontrolledlaboratory setting whileDeltaSmelteggsare
biovolume and chlorophyll a concentration in the net tow were available only in spring. Compared to other fish models such as
correctedtothetotalvolumeofwatersampledusingaGeneralOceanics Zebrafish, Medaka embryos have an extended post-fertilization
2030Rflowmeter. hatchtime,whichisidealforinvestigatingdevelopmentaleffects,
SamplesfordeterminationofMicrocystisbiovolumewerepreserved becausemoretimeisallowedfortoxicactiontooccur(Kinoshita
withLugol'ssolution.ThebiovolumeofMicrocystiscolonieswascom- etal.,2009;Wagneretal.,2017).
puted as an area based diameter using a FlowCAM digital imaging FertilizedDeltaSmelteggswereprovidedbyDr.Tien-ChiehHung
flowcytometer(FluidImagingTechnologies;Sierackietal.,1998).Cell attheFishConservationandCultureLaboratoryatUCDavis.Medaka
abundanceestimatesbasedonFlowCAMmeasurementswereclosely eggs were collected from in-house mass cultures at the Aquatic
correlatedwiththosedeterminedbymicroscopicanalyses(Lehman HealthProgram,UCDavis.Fishembryotoxicity testing wasper-
etal.,2017).Replicatewatersamplesforchlorophyllaandphaeophytin formedfollowingaprotocolproposedbyBraunbeckandLammer
pigmentanalysiswerefilteredthroughGF/Ffilters(0.7μmporesize), (2006) with some minor modifications. Briefly, fish eggs (b6 h
treatedwith1%magnesiumcarbonatesolutiontopreventacidity,and post-fertilization)werecollectedfromaquariums,cleanedinsaltso-
frozenuntilanalysis.Pigmentswereextractedin90%acetoneandquan- lutionfor10min(1%NaCl),incubatedinanembryorearingsolution
tifiedusingspectrophotometry(AmericanPublicHealthAssociation for30min(1gL−1NaCl,0.030gL−1KCl,0.040gL−1CaCl ·H O,
2 2
etal.,1998). 80 mg L−1 MgSO , and 1 mg L−1 methylene blue in distilled
4
Water samples for fish toxicity testing were transported to the water),andthensortedforviablefisheggs.Eggswereplacedin
AquaticHealthProgramat the UniversityofCalifornia (UC),Davis. 96-wellplatesfilledwithtwicefilteredambientwater(oneembryo
Watersampleswerefilteredtwice,firstbyglassmicrofiberfilter(GF/ perwell,200μLofambientwaterperwell).Atotalof32eggswere
F,poresize:1.5μm,FisherScientific,Waltham,MA),followedbyanad- usedperwatersampleperfishspecies.Forcontrolgroups,embryos
ditionalfiltrationwithapolyethersulfonesyringefilter(PESfilter,pore wereculturedinthefieldblank(DIwater)withadditionofminerals
size:0.22μm,GeneseeScientific,SanDiego,CA)tocompletelyremove forstabilizingpHandforfishosmoregulation(USEPA,2002).Fish
particulateorganicmatterandbacteria.FortoxicitytestingwiththeMe- embryoswerekeptinanenvironmentalchamber(PercivalScientif-
dakaembryos,exposuretotheambientwaterwasinitiatedwithin48h ic,Perry,IA)atoptimalgrowthtemperaturesforDeltaSmelt(16°C)
ofsamplecollection.ForDeltaSmeltembryotoxicitytesting,filtered andMedaka(25°C)ona16:8hlight:darkcycleduringtheexposure
water was stored in a −20 °C freezer, for up to five months, until period.At3dayspost-exposure,embryosthatfailedtodevelopdue
DeltaSmelteggswereavailableinJanuary. topooreggqualitywereremovedfromtheplates.Approximately
90%ofthewaterineachwellwaschangedevery48hfor14days
2.3.Fishembryotoxicitytesting oruntilhatching.Allembryoswereobserveddailyandrecorded
forsignsofabnormaldevelopment,mortality,andhatchingsuccess.
Two fish species were selected for the tests: Delta Smelt Embryoswithoutvisibleheartbeatswereconsidereddead.OnlyMe-
(Hypomesus transpacificus) and Medaka (Oryzias latipes). Delta dakafishembryotoxicitytestswereconductedatthepeakofthe
SmeltisapelagicfishspeciesendemictotheSFEandisanendan- Microcystis bloom in August and September of 2014. Both Delta
geredspeciesthatisconsideredtobeanindicatorofestuarinehealth Smelt and Medaka fish embryo toxicity tests were conducted
(Moyleetal.,2016).Medakawasusedasafishmodelforthisstudy throughoutthebloomseasonin2015.
1032 T.Kurobeetal./ScienceoftheTotalEnvironment610–611(2018)1029–1037
2.4.Taxonomicidentificationofbacteria control.Theexperimentwasperformedintriplicatewithatotalvolume
of3mLinasterilizedplastictube.Thebacterialdensitywasmeasured
Medaka embryos with active bacterial growth (Survey: August byplatecultureassay.Briefly,subsamplesofculturesolutionscontain-
2015;Samplingstations:FT,MI,andSJ;poolof3individualembryos ingbacteria(50μL)werecollectedatthebeginningandendoftheex-
perstation)wereusedforidentificationofbacteria.Medakaembryos periment(0and72hpost-inoculation),seriallydilutedby1:10with
withbacterialgrowthwerehomogenizedin1×phosphatebufferedsa- 1×PBS,mixedwith60%glycerol(finalconcentration:25%),andthen
line(PBS).Thehomogenateswereseriallydilutedwith1×PBS(1:10di- stored in −80 °C. The day after collecting the subsamples at 72 h
lutions)andtheninoculatedontoLuria-Bertani(LB)bacteriaculture post-inoculation,thesubsamplesofbacteriaculturesolutionswere
platestoobtainisolatedbacterialcolonies.At2dayspost-incubation slowlydefrostedonice,spreadontoLBcultureplates,andthenthe
atroomtemperature,bacterialcoloniesonthecultureplateswerevisu- plateswereincubatedatroomtemperatureuntilbacterialcoloniesbe-
allyexaminedandthedominantbacterialspeciesaccountingforover camevisible.Thenumberofbacterialcolonieswerecountedandthere-
90%oftheentirebacterialcolonieswassubjectedtotaxonomicidentifi- sultswerereportedasthenumberofcolonyformingunitspermilliliter
cationbydeterminingtheDNAsequenceforthe16SribosomalRNA ofculturesolution(cfumL−1).
gene(16SrDNA),amarkergenethatiswidelyusedforbacterialidenti-
fication(Santamariaetal.,2012;Salmanetal.,2013;Boydetal.,2015). 2.7.Statisticalanalysis
Aportionof16SrDNA,approximately1500basepairs,wasamplifiedby
apolymerasechainreactionwithagenericprimerset,EUBAandEUBB Spearman'srankcorrelationcoefficientwasusedtocalculatethe
withtheoptimizedamplificationcondition(Guntheretal.,2010).The correlationbetweenfishembryomortalityandwaterqualityparame-
amplifiedDNAfragmentwasextractedfromagelandsubmittedto ters(PRIMR-esoftwarever.6;http://www.primer-e.com/).Forthebac-
theUCDNASequencingFacilityforDNAsequencingreactions(http:// teriagrowthtestswithdifferentconcentrationsofMicrocystislysate,
dnaseq.ucdavis.edu/).TheDNAsequencesweresubjectedtoquality meansandstandarderror(S.E.)werecomputed,andthedifferences
trimmingusingGeneioussoftwarever.5.0.4,followedbyBLASTNfora among the treatment means were analyzed by one-way ANOVA,
DNAsequencesimilaritysearchandtaxonomicidentification(Kearse followedbypost-hoctestbyDunnett'stestusingapackage“multcomp”
etal.,2012;http://www.ncbi.nlm.nih.gov/). ver.1.4-6inRsoftwarever.3.3.0(Hothornetal.,2010;RCoreTeam,
2016).
2.5.Fishtoxicitytestingatdifferentsalinitylevels
3.Results
AnadditionalfishembryotoxicitytestwasconductedwithMedaka
todeterminehowincreasedsalinityaffectsmortality.Theexperimental 3.1.Waterqualitydata
solutionswerepreparedbyaddingInstantOceanSeaSaltsolutions(In-
stantOcean,Blacksburg,PA)toambientwatercollectedfromstationSJ Waterqualityparametersmeasuredinthisstudyareavailableinour
onAugust15th,2015toproducesalinityof1.0,2.5,and5.0psu.Thedi- previouspublication(Lehmanetal.,2017).In2014and2015,theDOC
lutionofambientwaterbysaltsolutionwasaconcern,thereforethe concentrationandwatertemperaturesvariedwithtimeandlocation
samevolumeoftheInstantOceanSeaSaltsolutionsatdifferentconcen- intheSFE(Fig.2).Amongthe10samplingstations,theDOCconcentra-
trations(2mL)wasspikedintotheambientwatertostandardizethe tionandwatertemperaturewereslightlyhigheratRRthantheotherlo-
experiment(38mLoftheambientwater,1:20dilution).Theambient cations (Fig. 2). The salinity at the confluence of the rivers was
waterfromSJwaschosenforthesalinitytestasitrepresentswater constantly above 1.5 psu (AT and CV; average: 3.7 psu) while the
quality in the South Delta where salinity was constantly low othersamplingstations(LowerSanJoaquinRiverandSouthDelta)
(b1.0psu)andMedakaexperiencedhighmortality(N90%).Theambi- hadlowersalinitylevelsthroughoutthesurveys(average:0.6psu,max-
entwaterspikedwithDIwaterwasusedasacontroltoensurethat imum:0.8psu).
highmortalityratewasreproducibleinthisexperiment.
3.2.Mortalityandadverseeffects
2.6.BacteriagrowthtestwithMicrocystislysate
Inboth2014and2015,Medakaembryosincubatedintheambient
Microcystiscelllysate,afluidcontainingcontentsoflysedMicrocystis waterfromthestationscontainingMicrocystisbloomsstartedtoexhibit
cells,waspreparedasfollows:Microcystis(StrainUTEXLB2386,non-
microcystinproducer)obtainedfromtheCultureCollectionofAlgaeat
theUniversityofTexas(https://utex.org/pages/about-us)wascultured
inCBmedia(500mL)withcontinuousaerationfor10daysinatemper-
aturecontrolledenvironmentalchamberat25°C(Shiraietal.,1989).
The algal cells were harvested by centrifugation at 2500 rpm for
10minatroomtemperature,followedby3rinseswithautoclavedDI
water and then resuspension in 30 mL of DI water. The algal cells
werelysedbyafreeze-thawmethod;thealgalcellswereplacedin
−80°Cfor20minandthenthawedslowlyatroomtemperature.The
freeze-thawcyclewasperformed3timesandthealgalcellswerevisu-
allyobservedunderalightmicroscope.Thealgallysateinthedissolved
fractionwascollectedbycentrifugation(2500rpmatroomtempera-
ture),followedbyfiltrationusingPESfilters(poresize:0.22μm,Gene-
seeScientific).TheDOCconcentrationwasmeasuredbysubmittinga
portionofthealgalcelllysatetotheStableIsotopeFacilityatUniversity
ofCalifornia,Davis(http://stableisotopefacility.ucdavis.edu/).Abacteria
growthtestwasperformedbyspikingAeromonasintothreedifferent
concentrationsoftheMicrocystislysatewithDOCconcentrationsof
27.7,14.7,and8.5mgL−1.TheAeromonasusedfortheexperiment
Fig.2.ScatterplotshowingtheDOCconcentrationandwatertemperaturerecordedfor
wasisolatedfromtheMedakatoxicitytesting.DIwaterwasusedasa the2014and2015surveys.
T.Kurobeetal./ScienceoftheTotalEnvironment610–611(2018)1029–1037 1033
growthofmotilebacteriaonthechorion(theouterenvelopeofthefish 3.4.BacteriagrowthtestwithMicrocystislysate
egg)oflivefishembryoswithin5daysofexposure(Fig.3a).Medaka
embryoswithbacteriadiedwithaportionoftheeggyolkortheentire ThebacterialgrowthtestdemonstratedthatMicrocystislysateen-
embryonicbodyextrudedfromthechorion(Fig.3bandc).In2014,the hancedthegrowthofAeromonas(Fig.4).ThedensitiesofAeromonasin-
Medakamortalityratesrangedbetween77.4and96.9%atthe8sam- creasedfrom6.3×104±1.4×102(Mean±S.E.)cfumL−1at0hpost-
plingstationslocatedintheLowerSanJoaquinRiverandSouthDelta inoculation(hpi)to1.46×106±1.25×105,2.87×106±1.07×106,
(BI,JP,MI,FT,SJ,OR,VCandRR),andbelow10%attheconfluenceof and7.40×105±5.44×105cfumL−1intheLysate1,2,and3treatment
theSacramentoandSanJoaquinrivers(ATandCV)forAugustandSep- attheendoftheexperiment(72hpi),respectively.Fewbacterialcolo-
tember(Table1a).Thehighmortalityratesofthefishembryoswereob- nieswereobservedinthecontrolgroupat72hpi.Thebacterialdensities
servedinconjunctionwiththegrowthofthebacteria,Aeromonas.In inLysate1and2werestatisticallysignificantfromthecontrolgroup.
2015,themortalityratesexceeded90%forallthesamplingstationsin
theLowerSanJoaquinRiverandSouthDeltainAugust,andthende- 4.Discussion
creasedinthefollowingmonths(Table1b).Growthofbacteriawas
stillobservedinembryosincubatedinOctoberandNovembersamples ThehighmortalityratesofMedakaembryoswereassociatedwith
fromthesamesamplingstations,howevernotallfishdied.Incontrast, thegrowthofmotilebacteria,Aeromonas.Aeromonasareubiquitously
attheconfluenceoftherivers(ATandCV),theMedakamortalityrates presentinnaturalwaterbodiesaswellasfishculturefacilities,and
remained10%orlowerthroughout2015.Bothin2014and2015,there cause disease, particularly when the abundance of the bacteria is
waslittlemortalityinthecontrolgroupsandgrowthofbacteriawas enhancedbyabioticfactors(e.g.,organicpollutants,watertemperature)
barelyobserved(b4%;Table1a,b;Fig.3e).TheMedakapercentmortal- orthefishimmunesystemiscompromisedbystressors(e.g.,inadequate
itywasnegativelycorrelatedwithchloride(Pearson'sr=−0.59,Pb waterquality,lowdissolvedoxygen)(JandaandAbbott,2010;White,
0.001),andwaspositivelycorrelatedwithMicrocystisbiovolume(r= 2013).MassmortalityeventscausedbyAeromonashavebeenwidelyre-
0.36,Pb0.05),ambientwatertemperature(r=0.58,Pb0.001),DOC portedinthewild,aswellasculturedfishpopulations(Harikrishnanand
(r=0.31,Pb0.05),TOC(r=0.47,Pb0.001),andsurfacechlorophyll Balasundaram,2005).TheadverseeffectsobservedinMedakaembryos,
aconcentration(r=0.34,Pb0.05)(Table2). suchasextrusionoftheeggyolkandfishbodyfromthechorion,were
TheDeltaSmeltmortalityratesremainedbelow20%inthemajority likelyduetoAeromonassinceathickbacteriallayerwasobservedon
ofthewatersamplestested(23/30or77%;Table1c).SimilartoMedaka, theaffectedfishembryos.AdultfishinfectedwithAeromonasexhibited
growthofmotilebacteriawasassociatedwithDeltaSmeltmortality. skinulcers,indicatingthatAeromonaserodesfishtissue(Harikrishnan
However,theDeltaSmeltmortalityrateswerelowerthanthatforMe- andBalasundaram,2005).Althoughspeculative,giventheeffectsof
daka,andrangedbetween3.1%(multiplestationsinSeptemberandNo- Aeromonasontheepidermaltissueofadultfish,itispossiblethatthead-
vember 2015) and 41.9% (JP in November 2015; Table 1c). Unlike verseeffectsobservedinthisstudywereduetoAeromonaserodingthe
Medaka,nocorrelationwasobservedbetweenDeltaSmeltmortality chorionlayerofthefishembryos.
dataandambientwaterqualityparameters(Table2). ItissuspectedthatAeromonasoriginatedfromthefishculturefacil-
Inaddition,adevelopmentaldeformity“heart-onlysyndrome”was ityandwereintroducedintothestudyfromtheembryos,becausethe
observedduringthetoxicitytestsforMedaka.Theterm,“heart-only ambientwatersampleswerefilteredtwice(1.7and0.22μm)priorto
syndrome”,wasusedtodescribethedistinctivesymptomthatoccurred theexposureteststocompletelyremoveanybacteria.AlthoughMeda-
wheninhibitionofsomiteformationco-occurredwithcardiogenesis kaeggsweretreatedwithsaltsolutiontolowerbacterialloadspriorto
andbeatingofafunctionalheart(Fig.3d).Thefrequencyofthedevelop- thefishembryotoxicitytesting,thecleaningprocessmaybeinsufficient
mentaldeformitywaslowatb0.5%ofthefishembryostests.Therewas incompletelyremovingbacteriaonfisheggs.However,becauseMeda-
noclearseasonalandgeographictrendintheoccurrenceofthedevel- kaembryosincontrolgroupsshowedlittletonomortality,weconclud-
opmentaldeformity,however,oneofthesamplinglocationsinwhich edthatthegrowthofAeromonaswasenhancedbydissolvedsubstances
thedevelopmentaldeformityobservedwaslocatedinthemiddleof intheambientwater.
largeagriculturalfields(VC).Thedeformitywasnotfoundineither AplausibleexplanationforthegrowthofAeromonasinMedakatests
thecontrolgroupsorthefishculturefacilities. isthepresenceofdissolvedsubstances,particularlytheorganiccarbon
releasedfromMicrocystiscells(e.g.cellorganelle,lipopolysaccharide,
3.3.SalinitytestwithMedakaembryos orcyanotoxins).TheMedakamortalitydatashowedapositivecorrela-
tionwithMicrocystisbiovolume,TOCorDOCconcentration,andother
ThereweresignificantreductionsintheMedakamortalityrates(≤ variablesthatarerelatedtoMicrocystisblooms,suchasambientand
13.3%)atsalinity≥1.0psu,comparedwiththesameambientwater net chlorophyll a concentration, and water temperature. We also
samplewithoutsaltaddition(96.7%;Table3).Growthofthemotilebac- foundthatthegrowthofAeromonaswassignificantlyenhancedbydis-
terialwasnotobservedatsalinity≥2.5psu.Nomortalitywasobserved solvedorganicsubstancesextractedfromMicrocystiscells.Dissolvedor-
inthecontrolgroup. ganic carbon is oftentheprimary substrate forbacterial growth in
Fig.3.ImagesofMedakaembryoincubatedintheambientwatersamplesshowinggrowthofbacteriaonthechorionasindicatedbytheredarrowhead(a),extrudedeggyolk(b)and
wholeembryonicbodyfromthechorion(c),inhibitionofsomiteformationwhileafunctionalheartwasobservedasindicatedbytheyellowarrowhead(d),andnormalMedakaembryo
incontrolgroupat8dayspost-fertilization(e).Scalebar:3mm.
1034 T.Kurobeetal./ScienceoftheTotalEnvironment610–611(2018)1029–1037
Table1
SummaryofthefishtoxicitytestingresultsforMedaka2014(a)and2015(b)andDeltaSmelt2015(c).
(a)
TreatmentNo. Station August September
Morta Totalb Mort(%)c Mort Total Mort(%)
1 AT 3 31 9.7 2 32 6.3
2 CV 1 32 3.1 1 31 3.2
3 BI 28 32 87.5 30 31 96.8
4 FT 27 32 84.4 28 32 87.5
5 JP 31 32 96.9 24 31 77.4
6 OR 30 32 93.8 30 31 96.8
7 MI 27 31 87.1 30 31 96.8
8 SJ 30 32 93.8 27 32 84.4
9 RR 28 31 90.3 25 32 78.1
10 VC 29 32 90.6 28 32 87.5
Control Fieldcontrol 0 32 0 1 32 3.1
(b)
TreatmentNo. Station August September October November
Mort Total Mort(%) Mort Total Mort(%) Mort Total Mort(%) Mort Total Mort(%)
1 AT 3 30 10.0 2 31 6.5 1 32 3.1 1 32 3.1
2 CV 2 32 6.3 1 31 3.2 1 32 3.1 0 32 0
3 BI 31 31 100 2 32 6.3 3 32 9.4 5 31 16.1
4 FT 32 32 100 7 32 21.9 9 32 28.1 5 31 16.1
5 JP 32 32 100 4 31 12.9 3 32 9.4 6 29 20.7
6 OR 32 32 100 3 31 9.7 14 32 43.8 1 27 3.7
7 MI 29 30 96.7 9 31 29.0 10 32 31.3 9 30 30.0
8 SJ 28 28 100 5 31 16.1 14 32 43.8 5 32 15.6
9 RR 27 29 93.1 2 32 6.3 19 32 59.4 9 32 28.1
10 VC 31 31 100 6 32 18.8 11 32 34.4 3 29 10.3
Replicate AT 2 32 6.3 1 31 3.2 3 32 9.4 0 30 0
Control Fieldcontrol 1 31 3.2 0 30 0 1 32 3.1 1 30 3.3
(c)
TreatmentNo. Station September October November
Morta Totalb Mort(%) Mort Total Mort(%) Mort Total Mort(%)
1 AT 4 32 12.5 4 32 12.5 3 32 9.4
2 CV 3 32 9.4 6 31 19.4 2 32 6.3
3 BI 3 32 9.4 8 32 25.0 1 32 3.1
4 FT 7 31 22.6 12 32 37.5 9 32 28.1
5 JP 4 32 12.5 2 32 6.3 13 31 41.9
6 OR 1 32 3.1 5 32 15.6 1 32 3.1
7 MI 6 32 18.8 6 32 18.8 5 32 15.6
8 SJ 6 32 18.8 7 32 21.9 5 32 15.6
9 RR 11 32 34.4 6 32 18.8 6 32 18.8
10 VC 1 32 3.1 6 32 18.8 4 32 12.5
Replicate AT 3 32 9.4 4 32 12.5 5 32 15.6
Control Fieldcontrol 3 32 9.4 6 32 18.8 5 32 15.6
a Numberofmortalities.
b Totalnumberofembryos.
c Percentageofmortality.
aquaticecosystems(Williamsonetal.,1999).Furthermore,DOCre- dissolvedorganiccarbonconcentrationwassignificantlycorrelated
leasedfromphytoplanktonandcyanobacteriaisconsideredtobehigh with the abundance of the cyanobacterium, Aphanizomenon
quality carbon for bacterial growth (Eiler et al., 2003; Bade et al., (Spearman'sr=0.33;Pb0.01,Lehmanetal.,2017).Alltheseresults
2007).Guillemetteetal.(2016)alsoreportedthatbacterialcommuni- suggestthatbloomsofMicrocystisandothercyanobacteriaspeciescan
tiesprefertoutilizealgalcarboneveninlakesthataredominatedbyter- contributetotheconcentrationoforganiccarboninambientwaters,
restrialcarbon. and therefore, will increase the potential growth of bacterial as
Bothallochthonousinputsofterrestrialmaterialsandautochtho- CyanoHABsbecomemoreintense.
nousinputsbyphytoplankton,benthicalgae,andaquaticmacrophytes Differenceinwatertemperaturemaypotentiallyexplainthehigh
allcontributetotheTOCandDOCinaquaticecosystems(Volketal., mortalityratesforMedakacomparedwiththelowmortalityratesfor
2002;Aitkenhead-Petersonetal.,2003;BertilssonandJones,2003; DeltaSmelt.IntheSFE,spawningofwildDeltaSmeltoccursinspring
Bade et al., 2007). However, the DOC in the SFE mainly originated whenthewatertemperatureisbetween7and15°C(Bennett,2005).
fromphytoplanktonorcyanobacteriaandnotterrestrialorganicmatter Duringtheexposuretests,DeltaSmeltembryosweremaintainedat
asdemonstratedbystableisotopicanalysis(Lehmanetal.,2015).In theoptimumtemperatureof16°Cfortheirnormaldevelopment.This
2014,thelargestbiomassofMicrocystiswasobservedsincetheblooms watertemperaturewassignificantlylowerthantheoptimumforthe
beganin1999,withmedianchlorophyllaconcentrationreachinglevels growthofAeromonasat28°C(Statneretal.,1988).RoufandRigney
thatwere13and9timesgreaterthaninpreviouswetanddryyears,re- (1971)reportedthatthegrowthofAeromonassignificantlydecreased
spectively(Lehmanetal.,2017).Inaddition,dissolvedorganicsub- at15°C.Incontrast,Medakatestswereperformedat25°C,whichis
stancescouldhavebeenreleasedfromothercyanobacteria.In2014, close to the optimum for Aeromonas growth. Although Aeromonas
T.Kurobeetal./ScienceoftheTotalEnvironment610–611(2018)1029–1037 1035
2 MK 0.33 TMaobrlteal3ityofMedakaembryosculturedinambientwateratdifferentsalinitylevels.
3 WT 0.11⁎⁎0.58 1TreatmentNo. SSJtdation bSa0l.i1nity(psu) 2M9orta T3o0talb 9M6o.7rt(%)c
4 DO 0.16⁎−0.27⁎⁎−0.57 234 SSSJJJ 125...050 400 333012 1003.3
Control Fieldcontrol b0.1 0 31 0
5 CL −0.26⁎⁎−0.59⁎−0.270.03 ab NToutmalbneurmofbmeroorftaelmitibesry.os.
c Percentageofmortality.
6 Turb −0.04⁎−0.35−0.14−0.07⁎⁎0.66 d AmbientwatercollectedatSJinAugust2015.
7 pH 0.01⁎0.390.12⁎⁎0.39⁎−0.36⁎⁎−0.45 geendroctwehitenhmtahtoedrtwiafflaeittreyernrtetestmuelmptsep.reartautrueruessewdafsonrothtetetswteodfiisnhtshpisesctieusdlyik,tehlyeadfiffefecrt--
⁎9⁎7 9 WeconsiderthehighMedakamortalityduetobacterialgrowthto
8 NH4 0.25−0.2−0.2⁎0.300.170.13−0.1 bcoenadpitoiotennstaianldlyaismsopcoirattaendtMfaicctroorcyasftfiescbtilnogomthseoinmfipsahctsopfefcuietusriendthroeuSgFhEt,
9 NO3 0.30−0.090.030.060.01−0.02−0.19⁎⁎0.77 p(Aalrotsicauslaaprildyisisnimtha)eafnredsThhwreaatedrfinreSghioadns(.DSopraowsonminagpeotfenAemnseer)icoacncuSrhbaed-
10 SRP 0.310.16⁎0.29−0.12−0.11−0.10−0.10⁎⁎0.60⁎⁎0.93 tnwumeebnelrasteofAyporuilnagndfisAhutghuasttrienmthaeinSianntJhoeaqfrueisnhRwivaeterrarnedg,ipornosdoufctehlearegse-
tuary until November (Walburg and Nichols, 1967; Feyrer et al.,
11 TP 0.320.09⁎0.26−0.09−0.07−0.08−0.13⁎⁎0.67⁎⁎0.94⁎⁎0.97 2im00p9a)c.tDedurbinygptahtehsoegmenoincthbas,cetemribarysouschanadseAaerrlyomlifoensatsa.gAemfisohngcotuhled1b0e
12 TOC 0.21⁎⁎0.47⁎⁎0.45⁎−0.24⁎⁎−0.56⁎⁎−0.450.050.22⁎⁎0.52⁎⁎0.68⁎⁎0.67 saoanthmdepwrlsianttaegtrisottenamsti(opFneirsga.tm2u)ro.enTsihtaoetrRweRdarwimneertehriwrsealsattteuirvdetyel,ymthhpieegrhDaetOruCtrhecason(nNtch2eo5nst°erCai)ntiaotnhndes
13 DOC 0.23⁎0.31⁎⁎0.35−0.15⁎⁎−0.48⁎⁎−0.410.08⁎0.26⁎⁎0.540.67⁎⁎0.67⁎⁎0.83 oythefeaAreDsr.OoCmCuorcrnoeannsctaleynntdthraaentrieyoinosstnh(oeNro4fb.fi5accimtaelgrdiaLal−tas1pr)eemcpioeigrsthdintughribanvigoedtehivneehsraseinvtyeceraednddgrraoobuwugnthh-t
14 DON 0.20−0.120.05−0.070.08−0.03−0.03⁎0.25⁎0.36⁎⁎0.33⁎0.36⁎0.27⁎0.27 dthaencdeoomfibnaacntetrsipaeicniesusrifnacfieshweatmerbirnyothteoxSiFcEit.yAetreosmtinogn,ahsowwaesvfoeru,ntdhitsorbee-
sultmaynotrepresentbacterialassemblagesinthefields.Furtherre-
15 TSS −0.16⁎⁎−0.55⁎−0.290.01⁎⁎0.79⁎⁎0.70⁎−0.30⁎0.240.04−0.12−0.08⁎⁎−0.59⁎⁎−0.510.12 sDeOaCrcohriigsinsatrtionnggflryomwaCryraannoteHdABtosoinnvtehsetiggraotwetthheofcpoantthroibgeuntiiocnbaocftetrhiea
inthefield.
meters. 16 TDS −0.25⁎⁎−0.58⁎−0.260.04⁎⁎0.99⁎⁎0.66⁎−0.360.170.02−0.10−0.06⁎⁎−0.56⁎⁎−0.480.08⁎⁎0.79 bryoTsh,e“hfienadritn-gonolfyasydnevderolompem”,eanltsaoldsuepfopromrtistythoebsimerpveodrtainncMeeodfaiknaveesmti--
a
qualitypar 17 Chl-a,sub 0.170.21−0.050.11⁎⁎−0.51⁎−0.400.09−0.100.080.110.11⁎⁎0.50⁎0.32⁎0.28⁎⁎−0.48⁎⁎−0.51 gDPlaeetvcitneolgonpnemmataue,nrCataalllaotntohdxriiaxcn,itatihnersdoaFpsiosscoghceeinarietcelldcaoiwnntictalhumdcieynatanhneottsaorixnreintshsteofrfaoemmmbObiersyncoitlnlwaitcaotdreiear-.,
er velopmentatmid-blastulastage,developmentofpericardialperitoneal
andwat 18 Chl-a 0.04⁎0.34−0.020.07⁎−0.35−0.21⁎0.28−0.18−0.110.00−0.05⁎0.230.180.09⁎−0.28⁎−0.35⁎⁎0.68
a
at b
d u
ortality 19 Phae,s −0.020.060.21−0.220.030.17−0.19⁎0.39⁎⁎0.48⁎⁎0.59⁎⁎0.62⁎⁎0.44⁎0.32⁎0.360.240.040.200.05
m
fiensh 20 MIC 0.10⁎0.36⁎0.28⁎−0.36−0.10−0.020.13−0.21−0.18−0.05−0.070.210.19⁎0.290.02−0.110.22⁎⁎0.420.09
e
w
mputedbet (DS)(MK)(WT)(DO)(CL)(Turb)(pH)(NH4)(NO3)(SRP)(TP)(TOC)(DOC)(DON)(TSS)(TDS)(Chl-a,sub)(Chl-a)(Phae,sub)(MIC) 5.
2fimancorrelationcoefcientsco DeltaSmeltmortalityMedakamortalityWatertemperatureDissolvedoxygenChlorideTurbiditypHAmmoniumNitrateSolublereactivephosphorusTotalphosphorusTotalorganiccarbonDissolvedorganiccarbonDissolvedorganicnitrogenTotalsuspendedsolidsTotaldissolvedsolidsSub-surfacechlorophyllaSurfacechlorophyllaSub-surfacephaeophytinMicrocystisbiovolume bumbersinboldindicateP0.0b0.05.b0.001. sFoiglu.4ti.oBna)rpculolttusrheodwwinigthabduifnfdearenncetocofAnecreonmtroantaiosnssp.o(fcoMloicnryocfoyrsmtisinlgysuanteit:mLyL−sa1teof1cu(lDtuOrCe
TableSpear 1234567891011121314151617181920 Then⁎P⁎⁎P caobnuncednatnrcaetiwona:sm27e.a7sumregdLa−t17)2,h2p(o1s4t.-7inmocgulaLt−io1n),.(a*nPdb30.0(85.,5**mPbg0L.−0011).).Thebacterial
1036 T.Kurobeetal./ScienceoftheTotalEnvironment610–611(2018)1029–1037
edemas,andinhibitionoforganogenesis(Berryetal.,2007).Similarly, Belanger,S.E.,Rawlings,J.M.,Carr,G.J.,2013.Useoffishembryotoxicitytestsforthepre-
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toprofenofos,anorganophosphateinsecticide,exhibitedvariousmor- FranciscoEstuary,California. 3.SanFranciscoEstuaryandWatershed, p.2.
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phologicaldeformitiesincludingyolksacedemaandmalformationsin
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