BrineShrimpLethalityAssayforFourDifferentMarine

Invertebrates

LaboratoryReport1

MarineSystems1(2803ENV)

GriffithSchoolofEnvironments
GriffithUniversity,GoldCoast

ReginaKimble

s5000977

06/04/2016


Introduction

Somemarineinvertebrateshavetoxiccompoundsandthiscanbemeasuredinashrimp
lethalityassay(Carballoetal.,2002).Ashrimplethalityassaytakesbrineshrimp,
Artemia
nauplii,andexposesthemtodifferentmarineinvertebratesinvaryingconcentrations.Itisa
widelyusedmethodtoassesstoxicityinmarineorpharmaceuticalplants(Carballoetal.,
2002).AnextractisdeemedtobehighlytoxicifithasaLD
,thelethaldosethatkills50%
50
ofasample,lessthan1mg/ml(GriffithSchoolofEnvironments,2016).

Forthisexperiment,fourmarineinvertebratesweretested,eachwasextractedinto3
differentsolutions:
A.)Candidaspongiaflabellata(0.26g)
B.)Polycarpaprocera(0.25g)
C.)Lissoclinumpatella(0.26g)
D.)Dysideaherbacea(0.24g)

Brineshrimpareagoodsubjecttotestonfortheyareanaccessibleandaffordablespecies
withanelementarylifeprocess(Meyeretal.,1982).Theycanbekeptinalabforlong
amountsoftimewithlittlemaintenance(Meyeretal.,1982).Theireggs(cysts)canbe
inducedtohatchwhenputinsalineconditionswherethebrineshrimpwillliveuptoayear
(Meyeretal.,1982).
Artemia
havebeentestedtobemostsensitivetotoxinsinpremature
growthstagesandafter48hoursofexposuretothetoxins(Carballoetal.,2002).

Method

Thedetailsoftheexperimentareasperthelaboratorymanual(GriffithSchoolof
Environments,2016).Howeverintheextraction,insteadofadding0.5mlofdimethyl
sulphoxide(DMSO)tosolution1,2mlwasadded.Intheserialdilutions,100l(solution1)
oftheextractwasaddedto300lofDMSO(solution2),makingatotalvolumeof400l.

Results

CalculationofConcentrationineachsolution(mg/ml):
Solution1 = Weightoforganism
V olumeofDMSO 1000

Solution2 = Solution1concentrationvolumetaken

TotalV olume
Solution2concentrationvolumetaken
Solution3 =

TotalV olume

Where:
VolumeofDMSO=2ml
VolumeTaken=100l
TotalVolume=400l

Candidaspongiaflabellata:
C oncentrationofSolution1 = 0.26g
2ml 1000 = 130mg/ml

C oncentrationofSolution2 = 130mg/mL100L
= 32.5mg/ml
400L
32.5mg/mL100l
C oncentrationofSolution3 =
= 8.125mg/ml
400l

Thiscalculationwasrepeatedforeachsolutionandsummarisedintable1.

Table1:Concentrationofeachsolution.

Solution1
(mg/ml)

130

125

130

120

Solution2
(mg/ml)

32.5

31.25

32.5

30

Solution3
(mg/ml)

8.125 7.8125 8.125 7.5

Next,theconcentrationofthesolutionineachwellwascalculated.Thisisdoneby
multiplyingtheconcentrationsabovebytheamountputin(0.005ml)togetthemassand
dividebythetotalvolume(0.255ml).

Solution1Firstwell: 130mg/mlx0.005ml
= 2.55mg/ml
0.255ml

Thisisdoneforeverywellandsummarisedinthetablebelow.

Table2:Concentrationineachcell.

Solution1:
Cells13

2.55mg/ml

2.45mg/ml

2.55mg/ml

2.67mg/ml

Solution2:
Cells46

0.64mg/ml

0.61mg/ml

0.64mg/ml

0.58mg/ml

Solution3:
Cells79

0.16mg/ml

0.15mg/ml

0.16mg/ml

0.15mg/ml

CalculationofMortality:
Deaths 100
M ortality(per100ofpopulation) = Population

Cell1hadaconcentrationof130mg/mlofCandidaspongiaflabellata.Therewere5brine
shrimpintotal4aliveand1dead.
M ortality(per1000ofpopulation) = 15 100 = 20%mortalityrate

Thiscalculationwasrepeatedforeachcellandsummarisedintable3.

Table3:Mortalityrate(%)ineachcell.Wherecells1to3containsolution1,cells4to6
containsolution2,andcells7to9containsolution3.Erepresentsthecontrolgroup.
LettersADrepresenttheircorrespondinginvertebratesstatedintheintroduction.

Cell1

20

10 0

50 0

Cell2

25 0

25 0

Cell3

18 0

Cell4

Cell5

Cell6

14

Cell7

20 0

Cell8

11

Cell9

1
1

CalculationofStandardErrorforEachExtract:

Themeaniscalculatedbytakingallthemortalityratesforonesolution,addingthemup,and
dividingby3.
M ean = (cellA + cellB + cellC)/3
Thenthemeanissubtractedfromeachindividualmortalityrate.Next,eachdeviationfrom
themeanissquared.Thentheyarealladdedtogether.Thesumisdividedbythesample
size(3)minus1.Thesquarerootofthatistakentogetstandarddeviation(SD).TheSDis
thendividedbythesamplesizetogetstandarderror.

CandidaspongiaflabellataSolution1:
M ean : (20 + 0 + 0)/3 = 6.7
C ell1StandardError : (20 6.7)2 = 176.89

C ell2StandardError : (0 6.7)2 = 44.89

C ell3StandardError : (0 6.7)2 = 44.89
176.89 + 44.89 + 44.89 = 266.67
266.67 = 133.335
31
SD : 133.335 = 11.547
StandardError : 11.547
= 3.85
3

Thisiscalculatedforeachsolutionandsummarisedintable4.

Table4:Standarderrorforeachsolution.

Solution1

3.85 4.19

5.66

Solution2

2.69

Solution3

3.85

2.12 2.12

GraphicalrepresentationofMortalityrate(%)versusconcentration(mg/ml):

Thereappearstobeaweak,positiveassociationbetweenmortalityrateandconcentration
(figure1).Thehighestconcentrationofcandidaspongiaflabellataproducedamortalityrate
2
of20%inonecell.Thereisamoderate(R
=0.25)exponentialassociation.

Figure1:Candidaspongiaflabellataextractionmortalityrateversusconcentration.

ThereappearstobelittleassociationbetweenconcentrationofPolycarpaproceraextract
andmortalityrate(figure2).ThelowestandhighestconcentrationofPolycarpaprocera
killedbrineshrimp,whilethemiddleamountofconcentrationkilledzerobrineshrimp.There
2
isaweak(R
=0.12752)linearassociation.Thestandarderrorbarsforsolution1and
solution3overlap.


Figure2:Polycarpaproceraextractionmortalityrateversusconcentration.

ThereappearstobeanegativeassociationbetweenconcentrationofLissoclinumpatella
andmortalityrate(figure3).ThemoreLissoclinumpatella,thelowerthebrineshrimp
mortalityrateis.Allcellsshoweda0%mortalityrate,exceptforonewiththelowest
2
concentrationofLissoclinumpatellainit,creatingthenegative,moderate(R
=0.25)
exponentialassociation.

Figure3:Lissoclinumpatellaextractionmortalityrateversusconcentration.

Thereappearstobeastrong,positiveassociationbetweenmortalityrateandconcentration
ofDysideaherbacea(figure4).AstheconcentrationofDysideaherbaceaincreases,so
2
doesthemortalityrateofbrineshrimp.Thereisastrong(R
=0.64967)exponential
association.Thestandarderrorbarsforsolution1somewhatoverlapswiththeothertwo
solutions.


Figure4:Dysideaherbaceaextractionmortalityrateversusconcentration.

CalculatingLC
50:

Themortalityrate(%)isconvertedtoaprobitnumber,usingaprobittable(0%mortality
receivesa2.67probitnumber),andtheconcentrationsarelogged.Probitisplottedagainst
thelogconcentration.Alinearregressiontrendlineisplottedanditsrelatedequationis
recorded.Theequationissettoequal5(y=5),tofindLC
.Aprobitnumberof5represents
50
50%mortalityrate.Theequationissolved,andthepositivexvalueisthelogconcentration.
Togettheconcentration,theinverselogistaken.

Figure5:Candidaspongiaflabellataprobitversuslogconcentration.

5 = 0.6351x + 3.0508
1.9492 = 0.6351x
x = 3.069

LC5o = (3.069) = 1172.20mg/ml

Thisiscalculatedforeachmarineinvertebrateandstatedbelowtheirrespectivegraphs.

Figure6:Polycarpaproceraprobitversuslogconcentration.

LC50 = 234963.28mg/ml

Figure7:Lissoclinumpatellaprobitversuslogconcentration.

LC50 = 0.00000037mg/ml


Figure8:Dysideaherbaceaprobitversuslogconcentration.

LC50 = 11.17mg/ml

Discussion

ThelowertheLC
,themoretoxicthemarineinvertebrateis.Bythisstandard,Lissoclinum
50
patellaisthemosttoxicmarineinvertebratetested(LC
of0.000000037mg/ml).Dysidea
50
herbaceaisthesecondmosttoxic(LC
of11.17mg/ml),candidaspongiaflabellataisthe
50
thirdmosttoxic(LC
of1172.20mg/ml),andpolycarpaproceraistheleasttoxic
50

invertebrate(LC
of234963.28mg/ml).Thecontrolgroup,immersedinasolutionentirely
50
madeupofDMSO,hada0%mortalityrate,indicatingithadnoeffectonthemortalityratein
thesolutionswiththemarineinvertebrateextractions.

LissoclinumpatellahadabigdifferenceinitsLC
concentrationfromtheotherinvertebrates
50
becauseallofitscells,butone,hada0%mortalityrate.Oneofthecellswiththelowest
concentration,hada11%mortalityrate,highlyskewingtheresults.Thishighvariationcould
beattributedtotheerrorscitedbelow.Duetothiserror,itslogicaltoassumethatdysidea
herbaceaisthemosttoxicinvertebratetested.

TheLC
valueswereunusuallyhighbecausetherewassignificantvariationinthemortality
50
rateofbrineshrimp,whichleadtoapoorfitforalinearregression.Gentlelinearslopes
causedlargeLC
values.Thevariationwithintrialscouldbeattributedtohumanerror.
50
Mistakescouldhavebeenmadewhilepipetting,leadingtoaninaccurateamountofDMSO
orsolutioninthecells.Theamountofbrineshrimpineachcellrangedfrom5to11,which
addedtothevariation.Mistakescouldhavebeenmadewhilecountingthebrineshrimp,as
differentfieldsofvisionwasntaccountedforwhilelookingthroughthemicroscopeaslive
brineshrimpfloattothetopwhiledeadonessink.Itsknownthatbrineshrimpshowmost
sensitivitytochemicalsafter48hours.Thebrineshrimpwereexposedtothesolutionsfor

lessthan24hours.Thisalsocouldaccountforthelowmortalityrateseeninthemajorityof
thecells..

Standarderrorbarsindicatewhetherthesolutionsmortalityratesarestatisticallydifferent
fromeachother.Generally,ifthestandarderrorbarsdonotoverlap,thetwovaluesare
statisticallydifferent(Cumming,FidlerandVaux,2007).Intheinvertebrateswherethis
appliesto(figures2&4),thestandarderrorbarssomewhatoverlap,alludingtotheideathat
themortalityratesforthedifferentsolutionsarenotsignificant.However,thismethodisnot
preciseandfurthertestingcanbedonetoaccuratelyprovestatisticaldifference.

Candidaspongiaflabellatahasfoundtocontaincytotoxicchemical,candidaspongiolides,
meaningitharmscancercells(Meragelmanetal.,2007).Thedatashowedithasalow
degreeoftoxicity,asthehighestconcentrationtestedhadamortalityrateof20%(figure1).
Itsmoderateexponentialassociationindicatesthedegreeoftoxicityincreaseswith
concentration.

PolycarpaproceraisanascidiantunicateunderthefamilyStyelidae(Sanamyan,2015).
Specificresearchonpolycarpaprocerahasnotbeenconducted,butitisknownthat
ascidianshavecytotoxicchemicalsusedfordefense(WattersandvandenBrenk,1993).
Thedatacollectedindicatedlittleassociation.Thiscouldmeantherewerevariationsor
humanerrorsinthebrineshrimpassayorthatpolycarpaproceraisingeneralatoxic
invertebrate.Itcontainedthesecondhighestmortalityrate(25%)atitshighest
concentration.

Research(Degnanetal.,1989)hasshownthattheascidianlissoclinumpatellahascytotoxic
cyclicpeptidesthatincreasesthelongevityofanimalswithtumors.Inthedata,there
appearedtobelittlenegativeassociationwiththeconcentrationoflissoclinumpatellaand
mortalityrates.Thiscouldbeduetothehumanerrorsmentionedpreviously.

Previousresearch(Handayanietal.,1997Sakaietal.,1997)hasshownthatdysidea
herbaceacontainshighlytoxicneurotoxins(LC
of0.96).Theneurotoxin,dysiherbaine,had
50
similarsymtompsonmiceasamnesicshellfishpoisoning,whichincludesvomiting,
headachesandshorttermmemoryloss(Sakaietal.,1997).Inthedata,dysideaherbacea
couldbeassessedasthemosttoxicasitshowedtohavethehighestmortalityrates(figure
4)andlowestLC
concentration.
50

Inconclusion,allmarineinvertebratestestedareknowntohavecytotoxicproperties.
Cancerouscellswerenottestedsothetruetoxicitypotentialwasnotfullyexplored.Human
errorcausedasignificantamountofvariationinthedata,whichleadtoaninaccurate
representationoftoxicityinthetestedinvertebratesandcontradictionswithpreviousstudies.
However,allinvertebratesdidshowalowormediumdegreeoftoxicity,supportingthe
evidencethattheyhavetoxicproperties.

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