A review of toxicity testing protocols and endpoints with Artemia spp.

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EcologicalIndicators69(2016)35–49

Contents lists available atScienceDirect

Ecological

Indicators

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / e c o l i n d

A

review

of

toxicity

testing

protocols

and

endpoints

with

Artemia

spp.

G. Libralato

a,∗

,

E. Prato

b

_,

_L.

_Migliore

c

_,

_A.M.

_Cicero

d

_,

_L.

_Manfra

d,e

a_Department_of_{Environmental}_Sciences,_Informatics_and_Statistics,_University_Ca’_Foscari_Venice,_Via_Torino_155,₃₀₁₇₂_Mestre,_Venice,_Italy b_Institute_for_the_Coastal_Marine_Environment,_National_Research_Council_(CNR_IAMC),_Via_Roma_3,₇₄₁₀₀_Taranto,_Italy

c_Department_of_Biology,_University_Tor_Vergata,_Via_della_Ricerca_{Scientiﬁca,}₀₀₁₃₃_Rome,_Italy d_Italian_Institute_for_{Environmental}_Protection_and_Research,_Via_Vitaliano_Brancati_60,₀₀₁₄₄_Rome,_Italy e_Stazione_Zoologica_Anton_Dohrn,_Villa_Comunale,₈₀₁₂₁_Naples,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received6March2016

Receivedinrevisedform3April2016 Accepted5April2016

Keywords: Artemia

Toxicitytestmethods Hatching

Biomarker Behavioralendpoints

Immobilization/mortalityandsurvival

a

b

s

t

r

a

c

t

Artemiaspp.isanhistoricallypopularbiologicalmodelstillrequiringanofﬁcialinternationallybased standardization.Severalendpointsarecurrentlyavailable.Short-termacuteendpointsincludebiomarker (acetylcholinesterase;heatstressproteins;lipidperoxidation;thiobarbituricacidreactivesubstances; thioredoxinreductase;glutathione-peroxidase;glutathioneS-transferase;glutathionereductase;

alde-hydedehydrogenase;andadenylpyrophosphataseandFluotox),hatching(drybiomass,morphological

disordersandsize),behavioral(swimmingspeedandpathlength),teratogenicity(growth),and

immo-bilization(meaning mortality after 5–30s observation).Long-term chronictests focus on growth,

reproductionandsurvivalormortalityafter7–28dexposurefromlarvaltoadulthoodstage.Weanalyzed eachtestlookingatitsendpoint,toxicantandexperimentaldesignincludingreplicates,exposuretime, numberofexposedcystsororganismsandtheirrelativelifestage,exposureconditionsduring hatch-ingandtesting(salinity,pH,lightintensity,aerationdilutionmedia,andfoodsupply),typeoftesting chambers,andqualityassuranceandqualitycontrolcriteria.Similaritiesanddifferencesbetweenthe identiﬁedapproacheswerehighlighted.Resultsevidencedthathatching24hshort-termand14d long-termmortalityarethemostpromisingArtemiaspp.protocolsthatshouldgoforwardwithinternational standardization.

1. Introduction

TheadoptionandimplementationoftheEuropeanlegislation about the Registration, Evaluation, Authorization, and Restric-tionofChemicals(REACH)(EC,2006)requiredseveraladditional ecotoxicitydatapromotingthedecreaseofvertebratesusedin tox-icitytestingencouragingalternativestrategieswithinvertebrates, plantsaswellasorgan,tissue,andcellcultures(Dvoraketal.,2012). Duringthelast50years,variousinvertebrateswereassessedto investigatetheirsensitivitytomanyphysicalandchemicalagents fortheirpossibleuseaspre-screeningorscreeningmodels. Inter-nationally,Artemiaspp. brine shrimps (Crustacea, Branchiopoda, Anostraca),commonlyknownalsoasseamonkeys,areoneofthe mostfrequentlyusedspeciesfortoxicitytesting(VanSteertegem andPersoone,1993a,b).

Artemiaspp. is a major taxonin many hypersalinebiotypes throughout the world feeding primarilyon phytoplankton and

∗ Correspondingauthor.

E-mailaddress:giovanni.libralato@unive.it(G.Libralato).

beinganimportantprimaryconsumer(PersooneandSorgeloos, 1980;Vanhaeckeetal.,1987;Triantaphyllidisetal.,1998).Theyare ofeconomicalimportancebeingusedinaquacultureandin aquar-iology.Theyalsoactasanefﬁciencyandproductivitystimulator forsaltproductioninsolarsaltworks(Jonesetal.,1981;Migliore etal.,1997;Treece,2000).

Themainadvantagesofusingbrineshrimpsintoxicitytesting are:(i)rapidity(i.e.28–72hfromhatchingtotheﬁrstendpoint); (ii)cost-effectiveness;(iii)theavailabilityofnaupliihatchedfrom commercialdurablecysts(eggs)(i.e.homogeneityofthe popula-tion,availabilityallyear-roundwithoutthenecessityofculturing) (Nunesetal.,2006a;Manfraetal.,2012).Otheradvantagesare: (i)goodknowledgeofitsbiologyandecology;(ii)easy manipula-tionandmaintenanceunderlaboratoryconditions;(iii)smallbody sizeallowingaccommodationinsmallbeakersormicroplates;(iv) highadaptabilitytovarioustestingconditions(Nunesetal.,2006a; Kokkalietal.,2011).Conversely,severalcriticismsaboutArtemia spp.sensitivitywerepresentedbyalearning-by-doingapproach (Libralatoetal.,2010a,b;Libralato,2014).Forexample,thecysts’ productioncanreﬂecttheoccurrenceofgeneticvariationcaused bycrustaceans’geographicaloriginthatisrarelyknown(Migliore http://dx.doi.org/10.1016/j.ecolind.2016.04.017

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36 G.Libralatoetal./EcologicalIndicators69(2016)35–49 etal.,1997), althoughcertiﬁedcystsareusuallyutilizedin

tox-icitytesting.Theirorigincanhaveconsequencesonthegrowth, survival and reproduction of Artemia spp. specimens consider-ingespeciallysalinityandtemperature(VanhaeckeandSorgeloos, 1989;Triantaphyllidisetal.,1995).

Artemiaspp. naupliiwereusedtotest thetoxicity ofa wide rangeof chemicalssuchasarsenic(As) (Brix etal., 2003), cad-mium(Cd)(Kissa etal.,1984; Hadjispyrouetal., 2001;Sarabia et al., 1998a, 2002, 2006; Brix et al., 2006; Leis et al., 2014), chromium(Cr)(Hadjispyrouetal.,2001;Leisetal.,2014),cobalt (Kissa et al., 1984), copper(Cu) (Browne, 1980; Jorgensen and Jensen,1977;Brixetal.,2006),mercury(Hg)(Sarabiaetal.,1998b; Leisetal.,2014),nickel(Kissaetal.,1984),tin(Sn)(Hadjispyrou etal.,2001),zinc(Zn)(Brixetal.,2006;Garaventaetal.,2010), potassiumpermanganate,potassiumdichromate,andsilvernitrate (BooneandBaas-Becking,1931;Vanhaeckeetal.,1980),antibiotic drugs(Miglioreet al.,1993a,b,1997), engineerednanomaterials (Libralato,2014;Minettoetal.,2014;Corsietal.,2014;Callegaro etal.,2015),nano-sizedpolystyrene(Bergamietal.,2016),asbestos (StewartandSchurr,1980),phenoliccompounds(Guerra,2001), ethanolamines(Libralatoetal.,2010a)andtraceelements(Petrucci etal.,1995),triazineherbicides,insecticides,pesticides(Kuwabara etal.,1980;Varóetal.,1997,2002),acrylonitrile(Tongetal.,1996), carbammates(Barahona andSánchez-Fortún, 1999), phthalates, antifoulingagents(Grosch,1980;PersooneandCastritsi-Catharios, 1989a,b;Okamuraetal.,2000;Castritsi-Cathariosetal.,2007,2013, 2014;KoutsaftisandAoyama,2007), pharmaceuticals(Xuetal., 2015),anticorrosiveagents(Tornambèetal.,2012;Manfraetal., 2015a,2016),oil(Trieff,1980)andoildispersants(Zilliouxetal., 1973;Savorellietal.,2007),variousplantextracts(Cáceresetal., 1998),toxins(Granadeetal.,1976;Medlyn,1980;Vezieetal.,1996; Beattieet al., 2003) and environmentalmatrices suchas wood leachates(Libralatoetal.,2007),wastewater(Krishnakurmaretal., 2007;Libralatoetal.,2010b),seawaters(Manfraetal.,2011)and marinedischarges(Manfraetal.,2010).

Currently,varioustoxicitytestswithArtemiaspp.areavailable including short-term and long-term methods. Short-term toxi-city tests are more frequentlyused, some long-term protocols havebeen developed inthe last10 years, but noneof them is aninternationally standardisedmethod likeInternational Stan-dardOrganization(ISO),AmericanSocietyforTestingandMaterials (ASTM)orOrganizationforEconomicCo-operationand Develop-ment(OECD).Methodsfortestingimmobilization/mortalitywere standardisedonlyinItalybytheItalianAgencyforEnvironmental Protectionand ItalianInstitutefor WaterResearch(APAT IRSA-CNR)andItalianAgencyforStandardizationintheChemicalsector (Unichim).Despite thefrequentand widespreaduseofArtemia spp.intoxicitytesting,theharmonizationofprotocolsfollowedby internationalstandardizationactivitiesisstilllacking,and intercal-ibrationexercisesareurgentlynecessary(Libralato,2014).

Theaimofthisreviewpaperistocollect,organize,selectand discusstheexisting knowledgeaboutArtemiaspp. methods for toxicitytestingincludingbothshort-andlong-termbioassaysand organismhatchingandmaintenanceconditionsprovidingtipsfor protocolsdeﬁnition,implementationandstandardization.

2. Hatchingofcysts

Artemia spp. cyst hatching conditions can vary greatly as reportedinTableS1(n=42).Thiscanresultinadifferent evalu-ationofcysts/naupliisensitivity,althoughtheﬁrstfactorthatcan affectorganismsensitivityisthegeographicaloriginofcysts.Other speciesarecommerciallyavailable,buttheirsensitivitymustbe evaluatedonVanhaeckeetal. (1980)acase-by-case basisifno certiﬁcationortraceabilityisavailable(Guzzella,1997).

Thesecondkey point istostart thetoxicity testswith nau-pliibelongingtothesameclassofagebecausesomestagesare more sensitive than others (i.e. Instar I stage is less sensitive than Instar II–III stage) (Sorgeloos et al., 1978). Cyst hatching occurred24hbeforestarting thetoxicitytest (InstarIstage)in 10 papers (Vanhaecke et al., 1981; Barahona et al., 1994; Brix etal.,2003,2004;Caldwelletal.,2003;VenkateswaraRaoetal., 2007;Garaventaetal.,2010;Bustos-ObregonandVargas,2010) and30hin3papers(Persooneetal.,1993;Guerra,2001;Koutsaftis and Aoyama,2007).Mostauthorsused48hold larvaehatched at25±2◦C involvingduringthe exposurelarvae atInstar II–III (Guzzella, 1997; Hadjispyrou et al., 2001; APAT and IRSA-CNR, 2003;Favillaetal.,2006;Libralatoetal.,2007;Savorellietal.,2007; Pimenteletal.,2009;Manfraetal.,2011,2010;Kokkalietal.,2011; Pratoetal.,2011;Manfraetal.,2012;Tornambèetal.,2012;Leis etal.,2014;VeniandPushpanathan,2014;Manfraetal.,2015a,b, 2016;Rotinietal.,2015).

Eitherartiﬁcialornaturalseawatersareusedashatchingmedia. Artiﬁcialmediaaremorefrequentlyusedbecausesaltblendsto createtheidealsaltwaterarecommerciallyavailable(i.e.Crystal Sea®_Marinemix,_Forty_Fathoms®_,_Coral_Reef_Red_Sea_Salt®_,_Instant Ocean®₎_or_are_provided_along_with_toxkits_(Artoxkit,_2014)._The requiredseawatercanbeobtaineddissolvingthesesaltsindistilled anddeionizedwater.

Cystsareusually incubatedatbetween18–28◦C andlargely 35‰salinity.ThevaluesofpHvarybetween7.5–9.0andpHshould notbelower than7toobtaingood hatching(Vanhaecke etal., 1980).Duringthehatchingprocess,seawaterissometimesaerated byanairpumpbeingthemediumadequateaerationa prerequi-sitetoobtainasuccessfulhatching(Vanhaeckeetal.,1980).Manfra etal.(2016)proposedanoxygensaturationlevel>60%.The hatch-ingphaseoccurredinpresenceoflight(1000–4000lux)(Varóetal., 2002;Artoxkit,2014)orpartlyindarknesslikejust1hoflight dur-ing48hexposure(Guzzella,1997;Unichim,2012)or12–16hof lightduring24hexposure(Garaventaetal.,2010;Gambardella etal.,2014).

The hatching efﬁciency needs to be considered, but only Guzzella(1997)proposeda thresholdevaluatingcystshatching efﬁciencythatshouldbe>90%in≤32h.

Twomaincyst-hatchingprocedureswereidentiﬁedonthebasis of Artoxkit (2014)or Unichim(2012). Artoxkit (2014)suggests hatchingcysts30hbeforetoxicitytesting.Cystsaretransferredinto aPetridishwith9mLofseawaterpreparedwiththetoxkitsaltsand gentlyswirledtodistributethemevenly.ThePetridishisexposed toalightsource(1000–4000lux)for30h.AccordingtoUnichim (2012),cysthatchingstarts48hbeforethetoxicitytest.Seawater isusedashatchingmedium.Artemiacysts(100mg)aretransferred intoaPetridishwith12mLofseawaterexposedat1000–4000lux for1handfor23hindarknessat25±1◦C.After24h,hatched nau-pliiarepipettedtoanewPetridishformoultingcontainingnew freshseawaterthatisincubatedfor24hat25±1◦Cinthedark allowinglarvaetoreachIIorIIIInstarstage.

Sometimes,Artemiaembryosweresterilizedanddechorionated beforeuse,asintheprotocolsforaquaculturepurposes(Sorgeloos etal.,1977).DechorionatedcystsareArtemiaembryosenveloped onlybytheembryoniccuticleandtheoutercuticularmembrane (Légeretal.,1986).ThetechniquewassetupbyNakanishietal. (1962),whichusedachilleddilutedantiforminsolutiontodissolve thechorion.MorrisandAfzelius(1967)improvedthistechnique, whichallowsremovingtheouterpartoftheshellwithoutaffecting embryoviability.Thetemperatureofthemediummustbekept below40◦Ctomaintainthemaximalhatchingefﬁciency(Sorgeloos etal.,1977).Theuseofantibiotics(i.e.penicillin(50units/mL), streptomycin(50_␮g/mL)andsodiumtetraborate(0.2%w/v))was highlightedbyBagshawetal.(1986)andBrixetal.(2006).Embryos

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G.Libralatoetal./EcologicalIndicators69(2016)35–49 37 Short-term Hatching •Dry biomass •Morphological disorder •Size •Teratogenicity Biomarker •AChE •HSP •Fluotox •LP, TBARS, and TRed •GRed, GPx, and GST •ALDH and ATPases

Swimming •Speed •Path length

Immobilisaon •Mortality

Long-term

Growth •Body size •Weight •Morphological disorder

Reproducon •Mang •Reproducve rate •Oﬀspring

Immobilisaon •Mortality

Fig. 1. Summary of Artemia short- and long-term toxicity tests.

(AChE=AcetylCholinEsterase;HSP=heatstressproteins;LP=lipidperoxidation; TBARS=thiobarbituric acid reactive substances; TRed=thioredoxin reductase; GPx=glutathione-peroxidase;GST=glutathioneS-transferase;GRed=glutathione reductase;ALDH=aldehydedehydrogenase;andATPases=AdenylTriPhosphatase). werehydratedinsterileseawatercontainingantibioticspriorto toxicitytestinordertopreventpotentialbacterialinfections.

3. Toxicitytests

WedecidedtoclustertoxicitytestswithArtemiaspp.as short-termacuteandlong-termchronicbioassays(Fig.1)consideringthe lifespanofArtemiaspp.varyingbetween2–4monthsdependingon salinity,temperatureandspecies-speciﬁccharacteristics(Browne etal.,1991).Acutetoxicitytestsassesstheeffectsbasedon rel-ativelyhighexposureconcentrations(i.e.mg/L)fornomorethan 96h.Toxicityisgenerallyexpressedaslethalconcentrationcausing thedeathof50%ofthegroupoftestanimals(LC50),butalso hatch-ingandswimmingbehaviorimpairment.Theanalysiswascarried outconsideringsub-lethal(acutecysthatchingtest,biomarkers, teratogenicitytest,acutelarvaebehavioral test)andlethal end-points.

Chronictoxicitytestsinvestigatethelong-termexposurefrom weeks(e.g.2–4weeks)uptothewholelifecycleofArtemiaspp. atrelativelylowconcentrations(␮g/L)(Paneetal.,2012). Toxi-cityisexpressedasthenoobservedeffectconcentration(NOEC) andlowestobservedeffectconcentration(LOEC)considering sur-vival,growthandreproductionasendpoints(Brixetal.,2003,2004; Savorellietal.,2007;Manfraetal.,2012;Unichim,2012). 3.1. Short-termtoxicitytest

Acuteendpoints generally investigatetheextent oflethality (Persooneetal.,1993;Guzzella,1997;Artoxkit,2014)(TableS2)or othersigniﬁcantimpactsonArtemiaspp.suchashatching(Table1), growth(Miglioreetal.,1997;Sarabiaetal.,2008),andswimming (Garaventaetal.,2010;Gambardellaetal.,2014)(Table2).Inmost ofthecases,readingsarecarriedoutjustattheendoftheexposure period,keepingtoaminimumcontinuousand/orperiodic obser-vationsofsingleanimalsinanon-endpointperspective.Generally, long-termtoxicitytestsrequiretheperiodicalrecordingofdeath animalsdepictinghow contacttimemaycontributetotheﬁnal evolutionofeffects.

Artemia ‘death’ (i.e. mortality or lethality) presented vari-ousdeﬁnitionsgeneratingpotentialmisunderstandingaboutthe

observationofmoribundorreallydeadlarvae.Generally, mortal-ityevaluationiscarriedoutwithastereomicroscopebyeye,but computerbasedanalysissystemsareavailabletodecidewhether themonitoredArtemianaupliusisaliveordead(Garaventaetal., 2010;Alyuruketal.,2013).Widdows(1998)statedthatArtemia, andothercrustaceans,canlieonthebottomofthetestcontainer, butstilloccasionallymoveanappendage.‘Immobility’may there-forebeaneasierstatetodefine.Anyhow,differencesinhowthe immobilitystateisdefinedexist.Garaventaetal.(2010) consid-erednaupliiasdeadwhentheyarecompletelymotionlessordo notchangetheirbarycenterposition,ornoappendagemovement occursfor5s.Zulkiflietal.(2014)reportedthatnaupliiaredead iftheydonotshowanymovementafter10sobservation,while Alyuruketal.(2013)prolongedtheobservationperiodupto30s.

3.1.1. Acutecysthatchingtest

Since1980s’cystsandtheirembryonicdevelopmentwereused inecotoxicologyassummarizedinTable1.Thehatchingtoxicity testwasdevelopedtoassesstheeffectoftoxicantshaving differ-entcompositionandmodeofactionlikemetals(Bagshawetal., 1986;Brixet al.,2006;MacRaeand Pandey,1991;Rafieeetal., 1986;Jorgensenand Jensen,1977;Sarabiaetal.,1998a,b,2003, 2008), organiccompounds (Kuwabara etal.,1980; Alyürücand Çavas,2013;Rotinietal.,2015;Vismara,1998),antibioticdrugs (Miglioreetal.,1993a,b,1997)orcellextractsfrominvertebrates, macro-andmicroalgae(Caldwelletal.,2003;Carballoetal.,2002) as wellas stressevents generated by salinityand temperature (VanhaeckeandSorgeloos,1989).Hatchingtoxicitytestsarestatic orsemi-staticlastingbetween24hand96h.Theyinvestigatethe reducedemergenceofnaupliifromcysts/eggswhenexposed to toxicants compared tonegativecontrols.Results of‘cystbased’ toxicity testswithmetals,using differentprocedures,produced apparently inconsistentdata.Bagshawet al.(1986) tested fully hydratedArtemiacysts(20–30organismsperreplicate)at28◦C and36 PracticalSalinityUnit(PSU)underconstant illumination andshaking,evidencingthatencystedembryosaremoresensitive toCdandZnthanpre-naupliuslarvae.Cadmium(0.1␮M)retarded thehatchinganddevelopmentoflarvae,whilehigher concentra-tionsblockedthehatchingphasealmostcompletely.Zincwasless toxicthanCd,butitcausedsimilareffects.Bagshawetal.(1986) protocolwasusedbyRafieeet al.(1986)and Brixetal.(2006) confirmingboththetoxicity ofCdandCu(Artemiafranciscana), respectively.MacRaeandPandey(1991)showedthatArtemia fran-ciscanaCuandPb(0.1␮M)toxicitywassimilartoCdreducingthe rateandextentoflarvaldevelopment;ZnwaslesstoxicthanCd,Pb andNi.Sarabiaetal.(1998a,b,2003,2008),usinghydratedcystsof Artemiaparthenogenetica(10organismsperreplicate)didnot high-lightanyadverseeffectsofCd,HgandZntonaupliiemergence. The differencein Artemiaspp. sensitivity tometals was proba-blyduetothedifferencesintestingtemperatureandinterspecific species sensitivities. In 1931, Boon and Baas-Becking observed thattemperatureaffectedemergenceandhatching.Vanhaeckeand Sorgeloos(1989)demonstratedthattheincubationtemperature between25and37◦Ccansignificantlyaffectthehatching percent-ageofArtemiacystsevenwithingeographicalstrainsofbothA. franciscanaandA.partenogenetica.InArtemiaspp.,temperatureis knowntoprofoundlyinfluencechemicals’effects.Inparticular,low temperaturereducedtoxicityoforgano-metalliccompoundsinA. franciscana:CupyrithioneandDiurontoxicityat<25◦Cwere signif-icantlyreducedoreliminated,respectively(KoutsaftisandAoyama, 2008).Thustemperatureisavariableofextremeinteresttobe con-sideredwhilestandardizingfromhatching,larvaemaintenanceup totheendoftheexposuretotreatments.Theuseofafullrange oftemperatures,especiallyduringtoxicitytesting,maydrastically increasetheecologicalrelevanceofdata.

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38 G. Libralato et al. / Ecological Indicators 69 (2016) 35–49 Table1

Artemiaspp.behaviouraltoxicitytests.

References Test Toxicant Concentration (mg/L) Exposure (h) Cysts (mg) Endpoint T (◦C) Salinity (‰) pH Test light (lx) AereationDilution medium Food (cell/mL) Organisms per chamber

ReplicatesTestvessel (mL) TestmL QC Reference Toxi-cant NOEC/LOEC/LC50/EC50 Alyuruketal. (2013) S K2Cr2O7, p-coumaric acid

7-30-70-100 24 n.a. SWvelocityand pathscoveredby nauplii

n.a. 35 n.a. Low

light

n.a. Saline water

n.a. 5–8/20␮L n.a. MP(96)130␮L 0.2 n.a. K2Cr2O7 %survivalandvelocitiesfor

eachconcentrationandpaths

Davenportand Healy(2006)

S salinity 8.5–250 24 n.a. SWspeed 23

8.5-250

n.a. n.a. air-saturated

NSW Nofood n.a. n.a. Flat-faced transparent polystyrene canted-neck cultureﬂasks

50 n.a. n.a. n.a.

Gambardella etal.(2014)

S SnO2,CeO2and

Fe3O4NPs

10-100-1000 48 500 SWandM 20 37 n.a. 16h

light

n.a. ASW n.a. 10-15 9 25

compartment squarePetri dish

1 n.a. n.a. Nano-SnO2M<20%;

nano-CeO2:LC20=n.c.;%SW alteration<50%;nano-CeO2 EC20=80mg/L(70–90) Garaventaetal. (2010) S 1.zinc pyrithione;2. Macrotrol® MT-200;3. Eserine 1.0,1-1-10-100;2. 1.5-3-6-9-12;3. 0.1-1-10-100 24–48 500 SWandM 20 37 n.a. 16h light

n.a. ASW n.a. 10-15 9 Petridish 1 n.a. n.a. 1.LC50(48h)=35.74

(26.24-48.68); EC50(48h)=11.l9(6.56-19.09); LC50(24h)>100; EC50(24h)=19.57 (11.31-33.87);2. LC50(48h)=5.31(4.70-6.00); EC50(48h)=4.62(3.64-5.88); LC50(24h)=5.83(n.c.); EC50(24h)=6.39(5.72-7.15); 3)LC50(48h)>100; EC50(48h)=6.78(4.49-10.24); LC50(24h)>100; EC50(24h)=22.31(l 2.61-39.46). Huangetal. (2015) S 1.K2Cr2O7;2. Cd(NO3)2

1.0–100;2.0–1000 24 n.a. speedmovement 25 n.a. n.a. n.a. yes NSW n.a. n.a. n.a. n.a. n.a. n.a. n.a. LC50(K2Cr2O7)=32mg/L LC50(Cd(NO3)2)=626mg/L (speedmovement) Larsenetal. (2008) S Temperature, viscosity T=10–15–20◦C, viscosity 1.1-1.2-1.4-1.6 (×10–6m2_s−1)

n.a. n.a. SWvelocity n.a. n.a. n.a. n.a. n.a. NWS Rhodomonas

sp.

n.a. n.a. glassﬂasks n.a. n.a. n.a. InhibitionofSWvelocityfora 10◦Ctemperaturereduction Manfraetal. (2015b) S 1.CuSO4;2. SDS;3.DEG 1.2-4-8-16-32;2. 3-6-12-24-48;3. 10000-20000- 40000-80000-160000

48 20 SWandM 25±1 35±1 n.a. D n.a. ASW n.a. 10 3 MP(24) 1 control

mortal-ity≤10% 1. CuSO4 2.SDS 1.LC50(24h)=14.21±10.63; LC50(48h)=2.51±0.22;1. EC50(24h)=5.03±0.58 EC50(48h)=2.51±0.37;2. LC50(24h)=19.41±1.00; LC50(48h)=15.60±0.27;2. EC50(24h)=16.15±0.32; EC50(48h)=7.48±1.33;3. LC50(24h)>160000; LC50(48h)>160000;3. EC50(24h)=80370±3950; EC50(48h)=64560±3420 Venkateswara Raoetal. (2007) S 1.CPP;2.PF;3. MCP;4.ACEP 1.0.385±0.08;2. 7.71±1.48;3. 262.68±17.30;4. 2350.07±131 24 1000 nauplii SWspeed(cm/s) anddistance travelled(m/min)

n.a. n.a. n.a. n.a. n.a. ASW n.a. 25/each

expo-sure

n.a. MP(96) n.a. n.a. n.a. SWspeedinhibition(%): 1.89.00±3.32CPP2. 74.68±2.13PF3.62.48±1.87 MCP4.49.83±1.53ACEP Williams (1994a, 1994b)

S Notoxicant n.a. n.a. n.a. 1.newlyhatched

larvae(nauplii);2. larvaewithvisible limbbuds;3.aseries oflarvalstages identiﬁedbythe numberofactively beatingtrunk appendagespresent

21 35–40 n.a. n.a. yes Brine

solu-tion Yeast and green algae

n.a. n.a. n.a. n.a. n.a. n.a. Initialdevelopmentofnew limbsinArtemialarvaeis unimportantforpropulsion.

ACEP=acephate;ASW=artiﬁcialseawater;CPP=chlorpyriphos;D=darkeness;DEG=diethyleneglycol;EC50=effectconcentrationfor50%population;LC50=lethalconcentrationfor50%population;LOEC=lowestobserved

effectconcentration;M=mortality;MCP=monocrotophos;MP=multi-wellplate;n.a.=notavailable;NOEC=noobservedeffectconcentrations;NSW=naturalseawater;n.c.=notcalculable;NP=nanoparticles;PF=profenofos;

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G. Libralato et al. / Ecological Indicators 69 (2016) 35–49 39 Table2

Artemiaspp.short-termtoxicitytests.

References Test Toxicant Concentration (mg/L) Exposure (h) Cysts (mg) Endpoint T( ◦ C) Salinity (‰) pH Testlight (lx) Aeration Dilution medium Food (cell/mL) Organisms per chamber Replicates Test vessel (mL) TestmL QC Reference toxicant NOEC/LOEC/LC50/EC50

APATandIRSA-CNR (2003) S,A SDS, K2Cr2O7, CuSO4 SDS: 5-9-16-28-50; K2Cr2O7: 5-9-16-28-50; CuSO4: 1.5-2.7-4.8-8.4-15

24 n.a. M 25±2 n.a. 6.5-8.5 D n.a. ASPM,IO n.a. 10 3 MP24

wells 1 CM≤20% SDS, K2Cr2O7, CuSO4 Probitanalysis, Chi2 test: EC50(SDS) =23.2±6.5mg/L withASPMand 25.6±5.5mg/L withIO. EC50(K2Cr2O7) =16±8.4mg/Lwith ASPMand 15.1±9.6mg/L withIO. EC50(CuSO4) =4.5±2.0mg/L ASPMand 4.5±2.0mg/Lwith IO Artoxkit(2014) S,A _K2Cr2O7 10-18-32-56-100

24 n.a. M 25 35 n.a. D n.a. ASW no 10 3 MP24

wells

1 CM≤10% _K2Cr2O7 n.a.

Brixetal.(2004) S,A _SeO42− 42-56-75-100,-133

95 n.a. M 25±1 80-102 7.9–8.4 n.a. n.a. NSW daily

2mLof 5×105 cell/mL Platy-monas sp.

n.a. 4 Beakers 400 n.a. n.a. _{LC50 =}78(71–86)

mg/L Guzzella(1997) S,A SDS, K2Cr2O7, CuSO4 SDS: 5-9-16-28–50_K2Cr2O7: 5-9-16-28-50 CuSO4: 1.5-2.7-4.8-8.4-15

24 n.a. M 25±2 n.a. 6.5–8.5 D n.a. ASPM,IO n.a. 10 3 MP24

wells 1 CM≤20% SDS, K2Cr2O7, CuSO4 Probitanalysis, Chi2 test: EC50(SDS) =23.2±6.5mg/L withASPMand 25.6±5.5mg/L withIO. EC50(K2Cr2O7) =16±8.4mg/Lwith ASPMand 15.1±9.6mg/L withIO. EC50(CuSO4) =4.5±2.0mg/L ASPMand 4.5±2.0mg/Lwith IO Kissaetal.(1984) S,A 1._CdCl2 2. Na2CrO4 3. Ni(NO3)2 4. Co(NO3)3 1,2,30-200 4.0-250

24,48 n.a. M 24±0.5 n.a. n.a. n.a. n.a. NSW n.a. 20(3

daysold) n.a. Glass cuppels 50 n.a. n.a. _LC50(48h): 160mg/LforCd; 163mg/LforNi; 172mg/LforCo; 8mg/LforCr Libralatoetal. (2007) S,A Wood leachates 12-25-50-100% v/v

24 n.a. M 25 35 7.0–8.3 D n.a. IO n.a. 10-15 3 MP24

wells

2 CM≤10% _CuSO4 Trimmed

Spearman–Karber expressedas_LC50 andtransformed intoToxicityUnits (TU50):47.57 (41.65–54.75)for Quercusspp.and 78.21(75.58–84.09) forPiceaabies

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40 G. Libralato et al. / Ecological Indicators 69 (2016) 35–49 Table2(Continued)

References Test Toxicant Concentration (mg/L) Exposure (h) Cysts (mg) Endpoint T( ◦ C) Salinity (‰) pH Testlight (lx) Aeration Dilution medium Food (cell/mL) Organisms per chamber Replicates Test vessel (mL) TestmL QC Reference toxicant NOEC/LOEC/LC50/EC50 Manfraetal. (2015a,b)

S,A _CuSO4 n.a. 24 n.a. M 25±1 35±1 n.a. n.a. n.a. ASW n.a. 10 3 MP24

wells 1 CM≤10% _CuSO4 Trimmed Spearman–Karber: mean_EC50 between5.63and 23.31mg/L (CV<40%) Persooneetal. (1993)

S,A _CuSO4 n.a. 24 n.a. M n.a. n.a. n.a. n.a. n.a. ASW n.a. 10 n.a. MP24

wells

n.a. n.a. _CuSO4 MeanLC50

(Cu2+ mg/L):from 3.3to3.8;CV%: from32.8to50.7 PeterosandUy (2010) S,A crude extracts offour medicinal plants

10-100-1000 24 n.a. M n.a. n.a. n.a. L n.a. NSW n.a. 10 3 Vials 5 n.a. DMSO Probitanalisys,

LC50(24h)in mg/L=Brucea amarissima:37.7; Intsiabijuga:86.5; Laportea meyeniana:89.5; Pipturus arborescens:57.5

Solisetal.(1993) S,A 21active agents from plants

n.a. 24 n.a. M 22–29 n.a. n.a. D n.a. NSW n.a. n.a. 3 MP96

wells

0.1 n.a. DMSO Probitanalysis

mean_{LC50 value:} between 3.8×10−4±1.4×10−4 and>1000mg/L To˘gulga(1998) S,A SDS, K2Cr2O7

n.a. 24 n.a. M 25±1 n.a. 7.5±0.5 D yes NSW no n.a. n.a. Petridish 10 n.a. Litchﬁeldand

Wilcoxon’method, LC50:14.5SDS mg/L;34PDKmg/L. Bliss’method_LC50 =13.84±0.005SDS mg/L;_LC50 =32.84±0.007PDK mg/L Vanhaeckeetal. (1980)

S,A SDS n.a. 24 100 M 25±1 35 7.5 D n.a. Kinne

(1971)

no 10 8 Petridish 10 n.a. SDS Litchﬁeldand

Wilcoxon:InstarI mean_LC50 =33.9±5.2mg/L (CV=7.7%);Instar II–III:mean_LC50 =17.8±0.9mg/L (CV=5.1%) Vanhaeckeetal. (1981)

S,A SDS n.a. 24 100 M 25±1 35±1 8.0±0.5 D ContinuousIO n.a. 10 3 Glass

Petridish

10 CM≤10% SDS Litchﬁeldand Wilcoxon 13.3<_LC50(24 h-SDS)<19.9mg/L

A=acute;ASW=artiﬁcialseawater;CM=negativecontrolmortality;D=darkness;DMSO=dimethylsulfoxide;EC50=effectconcentrationfor50%population;LC50=lethalconcentrationfor50%population;LOEC=lowestobserved

effectconcentration;IO=InstantOcean®_ASW;_L₌_light;_M₌_mortality;_MP₌_multi-well_plate;_n.a.₌_not_available;_NOEC₌_no_observed_effect_{concentration;}_NSW₌_natural_seawater;_QC₌_quality_control;_S₌_static;_SDS₌_sodium

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G.Libralatoetal./EcologicalIndicators69(2016)35–49 41 Methanolandethanoltoxicitywasevaluated(Vismara,1998)by

exposinghydratedcystsfor48h.Thehatchingtestwasalso suc-cessfullyusedtoevaluatethetoxicityoftwobiocidesutilizedin antifoulingpaints(DiuronandIrgarol)(AlyürücandC¸avas,2013) byexposinghydratedcystsfor24h.Thehatchingtestwasalsoable toevaluate,inaconcentration-dependentway,thetoxicityofwater solublealgalextractsandcrude cellularextractsof Skeletonema costatumandNitzschiacommutata,andthediatom-derivedshort chainaldehydesdecadienalbyexposing20–30cystsperreplicate tofreshseawater(1mL)at15◦Cinmultiplatewells(Caldwelletal., 2003).

A quite different protocol using commercial brine shrimp hatcherwasappliedtoevaluatethetoxicity ofthreeantibiotics (Bacitracin, Flumequine and Sulfadimethoxine) (Migliore et al., 1993a,1997), two organiccompounds(diethyleneglycol(DEG) andsodiumdodecylsulphate(SDS))(Rotinietal.,2015),andthe bioactivity of extracts of marine invertebrates and macroalgae (isopropanolicextractsfromHyatellaspp.(sponge),Dysideaspp. (sponge),Paciﬁgorgiaadamsii(coral)andMuriceasp.(coral)already foundactiveagainsttwohumancelllines)(Carballoetal.,2002). Thismethodisaimedtoevaluatethedrybiomassofhatched nau-pliifrom1g(_∼200,000individuals)ofArtemiaspp.cystsincubated at25◦Cand35PSUunderconstantillumination(3000lx)and aer-ation(airbubbling,notcompulsory)for2–4days.Cystswereintact andnotdechorionated.Thefreenauplii,afterthesuccessful emer-gencefromthecystandhatching,aggregateoverasieveinthe centerofthehatcherduetotheirpositivephototacticactivity eas-ingtheircollection.Thiskindofapproachseemstobemoreviable fortheevaluationofthereducedemergenceand/orhatchingthan enumeratingone-by-onethecystsabletocompletetheir embry-onicdevelopmentwhenexposed toatoxicant(Kuwabaraetal., 1980;Rotinietal.,2015).Phototaxisisacost-effectiveandeasy wayofnaupliigroupingforcountingorbiomassproduction deter-mination.Anyhow,thismethoddoesnotallowdiscriminatingthe sub-lethaletiologyofthetreatmentexposure.Furtherinvestigation wouldbeinterestingaboutArtemiaspp.lightreception,nervous systemprocessing,motorfunctionaswellasdevelopmental mon-itoring.

Anothergroupof‘cystbasedtests’evaluatedthe morpholog-ical disorders and size of hatched nauplii (Raﬁee et al., 1986; Vismara,1998;Neumeyeretal.,2014).Bagshawetal.(1986)found reversiblearrest of developmentof pre-nauplii larvae emerged inCdspikedwaters(10␮MCdCl2)andmorphologicalalteration athigherconcentrations,consideringthephenotypicalterations anendpointwithinthehatchingtest.AlsointhecaseofHg,Go etal.(1990)describedthepresenceofmorphological abnormali-tiesanddelayeddevelopmentinexposedcystsduringemergence andhatchingphase.

Raﬁeeetal.(1986)incubatedhydratedcystsupto100hin pres-enceofCdandZnspikedseawater.Scanningelectronmicrograph andlightmicroscopyevidencedmorphologicalabnormalitiesand delayeddevelopment.Migliore etal.(1997)accordingtoRaﬁee etal.(1986)highlightedseverephenotypicalterationdueto Baci-tracinandchangesinpigmentationduetoFlumequine.Neumeyer etal.(2014)investigatedtheuseoffullyhydratedand dechori-onatedembryos(15–28perreplicate)incubatedat21–23◦Cand roomlightconditionobservinganddescribingbylightmicroscopy allArtemiadevelopmentphasesandrelativealterations consider-ingarangeofsalinity(20,25,30and35‰)anddensity(from1–3 embryo/mLtoapproximately350embryo/mL).Theyfoundthat (i)aberrantmorphologiesresultfromamismatchbetween devel-opmentand emergence;(ii)a smallsalinityincreaseorembryo abundancedecreasecanimpairtheemergencesuccesshavingonly moderateeffectsonthedevelopment;(iii)aberrantdevelopmental pathsproduceswimmingnaupliiandunhatchedcystsappearing normal in counts.Neumeyeret al. (2014)improvedthe design

of Artemiaspp,endpointassayofferingan alternativeapproach consideringthequantitativedevelopmentalprofileunderspecified conditions(22◦C,16:8or0:24light:darkcycle,20–30‰artificial seawater),evenifitcanbeaquitetimeconsumingactivity.

Some authors compared the sensitivity of hatching and acute/chronicmortalitytoxicitytest.JorgensenandJensen(1977) observed that when Artemia salina is exposed to Cu ions the hatchingrateismuchmoresusceptiblethanmortality,obtaining EC50valuesatleast100timesbelowtheanalogousLC50.Migliore et al. (1997) showedthat in A. franciscana both cystshatching andmortalityrespondedtoantibioticsexposureevidencing hatch-ingrateandlarvaemorphologicalalterations,andnaupliideath, respectively. Carballoetal. (2002)evidenced asimilar sensitiv-itybetweencysts’hatchabilityandnaupliimortalityexposed to variousorganicextracts,suggestingasimultaneousapplicationof bothassaystotestnaturalmarineproducts’pharmacological activ-ity.Sarabiaetal.(2003)conductedexperimentsbyexposingcysts andlarvaetoCd.Theydidnotobservetoxiceffectsoncysts (per-centage and timing of cyst hatching)and larvae (lifespan and reproductiveparameters).Caldwelletal.(2003)compared hatch-ing successand larvalmortality ofA. salina exposed to diatom extractsandaldehydes,showingthathatchabilityisalesssensitive endpoint.ThemostrecentpaperbyRotinietal.(2015)compared theresultsofhatching,acutemortalityandchronicmortalitytests onDEG and SDSranking theendpoints as follows: acute mor-tality<hatchability<chronicmortality.Theprotocoldescribedin Rotini et al.(2015)was intercalibratedatItalian level between 2006–2009involvingelevenlaboratoriestoringtoxicitytestswith A. franciscanausing CuSO4 as reference toxicant(Manfra etal., 2015b).

3.1.2. Biomarkers

Biomarkers are any measurablecharacteristics of an organ-ismreflectingaspecificorgeneralphysiologicalandhealthstate. Biomarkers are often compounds isolated from serum, hemo-cyanin, or other fluidsthat can be used asan indicator of the presenceorseverityofaparticulardiseasestate.Thebrineshrimp A.salinawasusedinfewscreeningsconsideringbiochemical mech-anisms (Nunes et al., 2006b). Espiritu et al. (1995) developed a 1henzymatic inhibition assay (Fluotox) withArtemianauplii usingafluorigenicenzymesubstratecomparingtheoutputwith 24and48hmortalitytest.Additionof4-methylumbelliferyl- ␤-d-galactoside(MUF)tothetestmediumdidnotinfluencethetoxicity ofCuSO4,CdCl2,K2Cr2O7,sodiumpentachlorophenate,SDS,and phenol.Uptake ofMUFand its subsequenthydrolysisform the 4-methylumbelliferonethatisstronglyfluorescentinalkaline solu-tionusinga UVlight. Theexposuretotoxicantsmayreduceor inhibitthisenzymaticpathwaythatcanbeusedasanearly mani-festationoftoxicity.

Varóetal.(2002)observedtheinhibitorypotentialof chlorpyri-fosanddichlorvosonacetylcholinesterase(AChE) activityusing A. salinaand A.parthenogenetica provokingtheaccumulationof acetylcholine(ChE)atneuromuscularjunctiondisruptingthe func-tionnerve(Peakall,1992).ResultshighlightedthetoleranceofA. salinatohighlevelsofChEinhibition(approximately80%)without lethaleffects.

Few data exist on the antioxidant defense mechanisms in Artemia spp. Nunes et al. (2006b) investigated the antioxidant skills of A. parthenogenetica to pharmaceuticals and personal care products. Benzodiazepine significantly inhibited ChE and glutathione-peroxidase (GPx) activities. Clofibrate and clofibric acidshowedsignificantdecreasesin Se-dependentGPx.In par-ticular, clofibrate caused a slight increase of lipid peroxidation (LP)(thiobarbituricacidreactivesubstances,TBARS)(Nunesetal., 2006b).Sodiumdodecylsulphate(SDS)widelyusedindetergents

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42 G.Libralatoetal./EcologicalIndicators69(2016)35–49 decreasedtheactivityofbothChEandglutathionereductase(GRed)

inA.parthenogenetica(Nunesetal.,2006b).

Nauplii of A. franciscana were selected as model organisms to assess the induction of acute oxidative stress of Aloe vera juice (Sirdaarta and Cock, 2008, 2010) showing 34%, 79% and 90% inhibition of thioredoxin reductase(TRed), GRed and GPx, respectively. Thus, an oxidative stress was induced after acute exposure(SirdaartaandCock,2008,2010)observingthatvitamin EcouldhelptopartiallyreducetheeffectsofA.verajuiceexposure (SirdaartaandCock,2010).

GlutathioneS-transferase(GST)activitywasinvestigatedduring theearlydevelopmentalstagesofArtemiaspp.hatchinginartificial seawaterpreparedwithmunicipalwastewatereffluent.Alterations oftheisoenzymeprofilewereevidencedwithamaximumeffect after48hfromhatching.Theisoenzymeprofiledependeduponthe organismageandcanbeaffectedbyenvironmentalfactorssuchas waterquality(Grammouetal.,2011).

Katranitsas et al. (2003) measured adenylpyrophosphatase (ATPase)andaldehyde dehydrogenase (ALDH)inbrine shrimps exposedtoCu-basedantifoulingpaintsevidencinganalterationof Artemiaosmoregulationuptodeath.AlsoCastritsi-Cathariosetal. (2012)suggestedthatALDHactivityinArtemianaupliicouldbea valuablebiomarkerfortheevaluationofantifoulingpaint.

Theheatstressproteins(HSPs)inArtemiawereobservedinearly developmentalstages,inencystedgastrulaembryosandnauplii. HSP26isundetectableinunstressedcells,appearinginpresence ofheatorsaltshock,cellcyclearrest,nitrogenorcarbon starva-tion,oxidativestressandlowpH.Theproteinp26isasmallheat shock/␣-crystallineproteinexhibitinganinvitromolecular chap-eroneactivityconferringathermo-tolerance(Thomas,2003).Its synthesisplaysacriticalroleinembryoencystment,diapauseand quiescence(Delinger,2002;Miahetal.,2010).

Proteomic approaches were considered to compare non-stressed(negativecontrol)andstressedorganisms(Miglioreetal., 2007)improvingtheunderstandingofphysiologicalmechanisms underlyingstressresponseand/ortoleranceandhighlighting puta-tivebiomarkers in the differentially expressedproteins (Rotini etal.,2013).Forexample,themicrotubuleproteomeencompasses tubulin and a group of proteins associated with tubulin upon microtubuleformationdeterminingmicrotubuleorganizationand functioning.Inpost-diapausedevelopment(0and12h)cellfree extractsof A. franciscana,tubulin assembly wasinvestigated in presenceandabsenceoftaxol(Paclitaxel,10␮M)(O’Connelletal., 2006)evidencingpoorlyformedmicrotubulesandabundantlow molecular mass proteins (Day et al., 2003). No proteins were presentin theabsenceof taxol.Withtaxol,tenof thefifty-five proteinsidentifiedin A. franciscanaproteomeappeared at both exposuretimes(0and12h):alldecreasedexceptforone.Fructose 1,6-bisphosphatealdolase,enolase,HSPsandATP-bindingprotein are representedin the undeveloped cysts asisoforms differing in both isoelectric point and molecular mass. In post-diapause A. franciscana,these proteinswerepresent in reduced amount. Becauseonlythe40SribosomalproteinS12increasedwithinthe microtubuleproteomeduringdevelopment(12h)furtherinvitro analysesarerequiredforphysiologicalverification.

3.1.3. Teratogenicitytest

Artemiaspp.larvalstagesgrowthinhibitioncanbealso consid-eredasateratogentestsystembasedondisturbanceofelongation development(relativetocontrolsraisedatthesametimeand con-ditions)from24to48hinanimalsculturedinmediumcontaininga presumptiveteratogen(Olson,1979;KersterandSchaeffer,1983). Eventhoughtheprotocolisnotsuitedtoparticulates,substances volatilizedfromwatersolutionat25◦Corwatersmayhavevery lowconcentrationsofteratogenssuchasmunicipalwastewatersor receivingwaters.Growthlengthinhibitioniscalculated

consider-ingmeasurementsbetweenthewell-pigmentedeyeandthepoorly deﬁnedendofthetailbycenteringananimalinthe stereomicro-scope’sﬁeld.Withtheexceptionofzinc,chemicalsinthe␮g/Lrange werenotteratogenic,whereaschemicalsbetween0.25and25mg/L wereteratogenic (Cd, Hg,Pb, Zn, bromoform,n-butylphthalate, 1,2-dichloroethane, nitrobenzene, tetrachloroethylene, toluene, 1,2,4-trichlorobenzeneand 1,1,3-thrichloroethane).Nevertheless thelimiteddata,KersterandSchaeffer(1983)statedthatthesystem isnotverysensitive.

3.1.4. Acutelarvaebehaviouraltest

DataaboutArtemiaspp.short-termacutebehavioral testare summarizedinTable1.Swimmingspeedisthemostfrequently usedbehavioralendpointofphysiologicalstatusforaquatic organ-isms (Faimali etal., 2006).Locomotion,likeswimming,is used asstressindicatorinseveralecotoxicologicalstudiesrepresenting asensitivemeasureoftoxicstressfor awiderange of environ-mental contaminants (Little and Finger, 1990). In the last ten years,theswimmingbehaviorwasstudiedasaresponseto inver-tebrates’ organicor inorganicexposure to pollutants including Artemia.Thereisstillagenerallackofappropriatebiocompatible automation,optoelectronicsensorsandalgorithmsforbehavioral dataanalysisthatcouldrepresentawaytosolvethemajor short-comingsnotyetaddressedinecotoxicology(e.g.toxicityteststo beperformedmanuallyandwithlow-throughput)(Huangetal., 2015).Williams(1994a,b)appliedaphysicalmodelregardingthe appendagesofArtemiatoempiricallydetermineforcecoefficients actingregimes relevanttodeveloping larvae. Thisfirstanalysis importedequationsexploringchangesinthemechanicsof swim-mingin animals too small for direct measurement. Larvae are interestingbecause,comparedtoadults,theycanundergo fluc-tuationsinsizeandshape.Artemiadevelopsfromasmallnauplius thatratchetsalongwithonepairofoarstoamulti-limbedadult glidingcontinuouslythroughthewater.Astheanimalsapproach theadultmorphologygrowingandaddinglimbsalongthetrunk, theiraverageswimmingspeedincreasesandthejerkyswimming ofhatchlingschangesintothesmoothglidingofadults.The grad-ualchangeinswimmingdoesnotcorrespondonlytothetrunk limbsincrease, butalsotoenergybudgetsupportingthe move-ment(Williams,1994b).DavenportandHealy(2006)assessedthe relationshipbetweenphysicalparameters(mediumsalinity,body density,andbuoyancy)andswimminginA.franciscanalarvae, find-ingthatthehorizontalswimmingspeedwasunaffectedbysalinity and viscosity(i.e.between8.5–100‰), buttheobserved differ-encesinverticalswimmingratesaresolelyduetotherelatively constant body density. Venkateswara Rao et al. (2007)studied thecovereddistanceandswimmingspeedofArtemianauplii sur-vivorsafter24hexposureto4pesticides(acephate,chlorpyrifos, monocrotophosand profenofos).Acomputerizedvideotracking system (Ethovision, Noldus Information Technology, Wagenin-gen,TheNetherlands)wasusedfortheautomationofbehavioral experiments.Twenty-fivenaupliiwereplacedindividuallyintoa 96 wellculture plateandtheirbehavior wasrecorded. Authors observed that theAChE enzyme activitymight beinhibited by pesticidesthusacetylcholineaccumulatedatneuromuscularlevel altering the locomotion behavior of organism interrupting the coordinationbetweenthenervousandmuscularjunctions.Larsen etal. (2008)analyzedtheeffect oftemperature-dependent vis-cosityofambientseawater(20‰)ontheswimming velocityof brineshrimpsusingvideo-microscoperecordingsatvarious tem-peratures (between 10◦C and 20◦C) and viscosities (between 1×10−6m2_/s_and_1.6_×₁₀−6_m2_/s)_(but_keeping_a_constant tem-peratureof20◦C).Theysuggestedthatmodificationsinswimming velocityof Artemiaweredue tochanges inkinematic viscosity. Recently,Garaventaetal.(2010)recordedtheswimmingspeed alterationofArtemiatogetherwithmortality,usingavideocamera

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G.Libralatoetal./EcologicalIndicators69(2016)35–49 43 withamacro-objectiveforrecordingthelarvalswimmingpaths.

Larvae were dark-adapted for 2minbefore thevideo start (i.e. theexperimentaltimerequired toreachsteady speed and uni-formspatialdistribution)andtheimageswereanalyzedusingan advancedimageprocessing softwaretoreconstructthe individ-ualpaths/tracks. Thissystemprovided a suitabletool todetect linearswimmingspeedofArtemia,sinceauthorsobtaineda veloc-ity(3.05mm/s)inaccordancewithotherstudiesusingthesame organism.Alterationsinswimmingspeedweredetectedfor vari-ouscompounds(Znpyrithione,Macrotrol®_MT-200,_and_eserine) <0.1–5%oftheirLC50 values.Gambardellaetal.(2014)usedthe samesysteminvestigatingthetoxicityofmetaloxide nanoparti-cles(MO-NPs)toA.salina,usingswimmingspeed,andenzymatic activity alterations, and mortality as endpoints thus including continuous orperiodic observation ofsingle animals in a non-endpointperspective.Althoughnomortalitywasfound,MO-NPs decreasedlarvaeswimmingspeedandbiochemicalresponses, evi-dencing the sensitivity of non-lethal endpoints. Alyuruk et al. (2013)proposedanothervideo-trackingmethodassessingwhether monitoredArtemiaaredeadoraliveobservingcolors/texturesof nauplii.AuthorsexposedA.salinanaupliiatvariousconcentrations between7and100mg/LofK2Cr2O7andp-coumaricacid. Consid-eringresultsbymicroscopyobservation,theproposedalgorithm tracksand countswererobustrelativetothenumber ofmotile nauplii.Thiskindofautomatedanalysiswasalsoabletoprovide additionalquantitativedatasuchascurvatureofpaths,numberof stopsandimmobiletime.Manfraetal.(2016)comparedthe swim-mingspeedalterationtohatchingandmortalityexposingArtemia toCuSO4,SDSandDEG.Thesystemformeasuringtheswimming speedproposedbyGaraventaetal.(2010)showedthatswimming speedwasmoresensitivethanmortality,withsensitivitysimilar andsometimeshigherthancysts’hatchability.

Recently, Huang et al. (2015) proposed a proof-of-concept lab-on-chipplatformcapableof performingfullyprogrammable time-lapseandvideo-microscopyofmultiplesamplesforrapidA. franciscanaecotoxicityanalysis.Thismethoddynamicallydetected sub-lethalbehavioralendpoints,suchaschangesinspeedof move-mentor distancetravelledbyeachanimal, observinghow they changed compared to the negative control exposureafter 24h contacttime.Neverthelesstheseendpointspresentpromising per-spectivescomparedtoArtoxkit(2014),furtherresearchactivityis stronglyrequiredtoidentifyclearcontaminant-response relation-ships.

3.1.5. Acutemortalitytest

Data about Artemia spp. short-term acute immobiliza-tion/mortality test are summarized in Table 2. The Artemia suitabilityforacutetoxicitystudiesiswelldocumentedinthe sci-entificliteratureoverthepast80years(BooneandBaas-Becking, 1931). The first general method for conducting toxicity tests withArtemiaspp.canbeattributedtoMichaeletal.(1956).This paper stimulated further research about the test development anddefinitionofmoredetailedtestingprocedures.In1975,this extensiveresearchledtothecreation oftheArtemia Reference Center(ARC-test)atGhentUniversity(Belgium)andtherelease ofthefirstshort-termtoxicityprotocol(i.e.mortality)withbrine shrimplarvae(Vanhaeckeetal.,1981;VanhaeckeandPersoone, 1981;VanhaeckeandPersoone,1984).Thebioassayiscarriedout in staticconditions for 24hat 25±1◦C in darknessin covered glass Petri dishes (60×12mm). A homogeneous population of InstarII–IIInaupliihatchedfromcysts(100mg)wasexposedto SDSasreferencetoxicant.Ifthetestmeetsthequalitycriteriaitis consideredasvalid:negativecontrols≤10%,and13.3mg/L<LC50 (SDS)<19.9mg/L, dissolved oxygen (DO) concentration after 24h>2mg/Linthelowestconcentrationwith100%mortalityof larvae(VanhaeckeandPersoone,1981).

AninternationalintercalibrationexercisefocusedonVanhaecke andPersoone(1981)protocolassesseditsreliability,accuracyand precisionconsideringK2Cr2O7andSDSasreferencetoxicantsfor intra-andinter-laboratoryexercises.Thestudyinvolved67 labo-ratories(59laboratoriesinEuropeand8inUS)(Vanhaeckeand Persoone,1984).ForSDS,theinterlaboratorycoefﬁcientof varia-tion(CV)was24.82%and25.25%forEuropeanandUSlaboratories, inthisorder.ConsideringK2Cr2O7asreferencetoxicant,theCVwas 34.89and18.49%forEuropeanandUS, respectively(Vanhaecke et al.,1981;Vanhaecke andPersoone, 1984).Resultsevidenced thatthetoxicitytestwascarriedoutwithlittledifﬁcultiesshowing satisfactoryandreliableresults.PersooneandCastritsi-Catharios (1989a,b)usedtheapproachoftheArtemiaReferenceCenter(ARC) (UniversityofGent,Belgium)testtoseta standardprotocolfor routinetoxicitytestingof antifoulingandotherpotentially haz-ardouspaints.Theeffectivenessofthistechniquewasassessedby furtherstudies(Castritsi-Cathariosetal.,2007,2013,2014).This protocolwasengineeredinatoxkitcalledArtoxkit(MicroBioTests Inc.,Mariakerke(Gent),Belgium)(Artoxkit,2014)includingabatch ofdormanteggs(cysts)ofA.franciscanatogenerate“ondemand” larvae eliminating the need for continuous culturing. In 1989, internationalintercalibrationexercisesinvolving129laboratories betweenEurope,USAandCanadainvestigated thereliabilityof ArtoxkitonCuSO4 asreferencetoxicant.Resultswere encourag-ingwithonlyfewparticipantsreportingproblems.Theprecision wasnotstrikingwithaCVequalto50%inEuropeandUSA,and 33%inCanada.Criticismsraisedabouttheinexperienceofsome participantsandtheinstabilityofCuSO4inseawaterdetermining a decreasein toxicityover thecourseoffew daysand creating somemisunderstandingaboutmoribundandreallydeadnauplii (Persooneetal.,1993).ManyauthorsusedtheArtoxkitsinceits commercialization(>180resultsinGoogleScholarsearchingfor “Artoxkit”18thJan2016)likeKoutsaftisandAoyama(2007).

Solisetal.(1993)suggestedamodiﬁcationoftheacute toxi-citytestdevelopinganewA.salinamicroplatecytotoxicityassay requiringasmallamountoftestingsample(0.6mg)upto1mg/mL inartiﬁcialseawater.Theuseof96-wellmicroplatesin100␮Lsea waterenabledtestingalargenumberofsamples.

A detailed procedure to perform acute test with Artemia spp. was reported by Guzzella (1997) and APAT and IRSA-CNR (2003) using nauplii at II–III Instar stage. They reported the outcome of an interlaboratory study (acute 24h mortal-ity)betweenﬁveItalian laboratoriesinvestigating SDS,K2Cr2O7 and CuSO4 as reference toxicants. Toxicity tests were car-ried out using as dilution water both ASPM artiﬁcialseawater (NaCl=26.4g, KCl=0.84g, CaCl2·H2O=1.67g, MgCl·H2O=4.6g, MgSO4·7H2O=5.58g,NaHCO3=0.17gandH3BO3=0.03gin1Lof ultrapurewater)andInstantOcean.TheCVwasbetween28%and 52%usingASPMandbetween21%and64%usingInstantOcean, suggestingthatASPMismoresuitablethanInstantOcean.

Between2006–2009,the24hmortalitytestwithA.franciscana wasintercalibratedtogetherhatchingassay(Manfraetal.,2015b). Threeinter-comparisonresultsconstitutedthedatasetfor calcu-lating the24hLC50 meanand therepeatability/reproducibility coefﬁcientsonCuSO4.TheCVvalueandtherandRvaluesindicated acceptableinter-laboratorytest-reliabilitysimilartoISO (1996). Guzzella(1997)andAPATandIRSA-CNR(2003)methodswereused forcystshatching.LarvaetransferfromcysthatchingPetridishto testchamberswasoperatedminimizingasmuchaspossiblethe dilutionof testmedium.Mortalitywasdeﬁnedoperationallyas totalabsenceofmovement(i.e.swimmingactivityormovement ofappendices)aftermechanicalstimulation(i.e.touchingthe lar-vaewiththetipofaglassPasteurpipette)forapproximately10s ofobservation.

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44 G. Libralato et al. / Ecological Indicators 69 (2016) 35–49 Table3

Artemiaspp.long-termtoxicitytests.

References Test Toxicant Concentrations (mg/L) Exposure (h) Cysts (mg) Endpoint T(◦C) Salinity (‰) pH Testlight (lx) Aeration Dilution medium Food (cell/mL) Organisms per chamber Replicates Test vessel (mL) Test mL QC Reference Toxicant NOEC/LOEC/LC50/EC50 Browneand Wanigasekera (2000)

n.a. Salinity n.a. 21 n.a. n.a. 15-24-30

60-120-180 n.a. Fluorescent light no ASW (renewal every3d (M)d7d (R) Yeast,and Dunaliella sp. 5/jar 10 jars 100 (M); 250 (R)

n.a. n.a. NOEC=8

(parentalSU) NOEC=56 (parentalG) NOEC=56 (parentalR) NOEC=56(F1 SU)NOEC=56 (F1G) Cunningham (1976) n.a. Dimilin 0-0.001- 0.002- 0.005-0.010

21-28 n.a. R 25±2 25 8.2 n.a. n.a. ASW Daily

renewal

15 5 n.a. n.a. n.a. n.a. n.a.

Nováková etal. (2007) n.a. CdCl2, ZnSO4 Cd: 5-10- 15-25-50-100-250; Zn: 50-100–250mg

10 n.a. M 20±1 n.a. n.a. n.a. n.a. NSW Glucose 10 n.a. polystyrene

Petri dishes

10 n.a. n.a. Synergisticor antagonistic effects Gebhardt (1976) n.a. Cd,Cu,Hg Cd: 0-1.0-3.3-33.0; Cu: 0-0.3- 0.03-0.003; Hg: 0-0.001- 0.003-0.05 25 n.a. G (length), R(time (d)until nauplii produc-tion) 27±2 150-320 n.a. 16h ﬂuo-rescent light n.a. NSW (renewal every3d) D.viridis (40–50 cells/mm3₎ every3d

5 n.a. 300 n.a. n.a. n.a. n.a.

Manfraetal. (2012) semi-S SDS 1.56- 3.12- 6.25-12.5-25 14 n.a. M 25±2 35±2 n.a. 900±100 and14h light n.a. ASW* D. tertiolecta (1×105 cells/mL)* 10 3 100 50 CM≤20% SDS LC50=8.0±5 Manfraetal. (2015a) semi-S DEG 12500- 25000- 50000- 100000-200000 14 n.a. M 25±2 35±2 n.a. 900±100 with14h light n.a. ASW* D. tertiolecta (1×105 cells/mL)* 10 3 100 50 CM≤15% n.a. NOEC(14 d)=25000 Manfraetal. (2015a,b) semi-S SDS 1.6-3.1- 6.2-12.5-25.0 14 n.a. M 25±2 35±2 n.a. 900±100 with14h light n.a. ASW* D. tertiolecta (1×105 cells/mL)* 10 3 100 50 CM≤15% K2Cr2O7 EC50(14 d)=8.03±1.11 (n=5);EC50(14 d)=8.50±3.34 (n=24) Savorelli etal. (2007) semi-S Dispersant 3.12- 6.25-12.5-25 7-14 n.a. M,G (carapace length) 25±1 35±1 n.a. 900±100 with14h light n.a. ASW* D. tertiolecta (1×105 cells/mL)* 10 3 100 50 CM≤20% SDS NOEC(7 d)=6.25(M, G);NOEC(14 d)=6.25(M andG,A. parthenogenet-ica);NOEC(14 d)=3.12(M,A. franciscana) NOEC(7d) dispersant=5 (M,A. franciscana)

ASW=artiﬁcialseawater;CM=negativecontrolmortality;DEG=diethyleneglycol;EC50=effectconcentrationfor50%population;G=growth;LC50=lethalconcentrationfor50%population;LOEC=lowestobservedeffect

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G.Libralatoetal./EcologicalIndicators69(2016)35–49 45 3.2. Long-termtoxicitytesting

Data about Artemiaspp. long-termimmobilization/mortality testaresummarizedinTable3.Mosteffortsindeveloping toxi-citytestswithArtemiaspp.havebeenfocusedonacuteendpoints duringthelast40years(Sorgeloosetal.,1978;Vanhaeckeetal., 1980,1981;Persooneand Castritsi-Catharios,1989a,b)andthus few(andstillnotinternationallystandardized)chronicprotocols exist.

Gebhardt(1976)evaluatedthechroniceffectsofCd,Cu,and HgonA.salina growthand reproductionusinga staticrenewal test withmedium and foodreplacement every 3days. Natural seawaterfromtheGreatSaltLake(Utah,USA)wasusedas test-ingmedium, and brineshrimp werefed withDunaliellaviridis. Adultswereinvestigatedforimmobilization,newlyhatched nau-pliiforgrowthandreproductioninhibitionandeggsforhatching experiments.CuandHgdidnotaffectgrowthandreproduction atconcentrationsbelowacutemortality.Matingoccurredbothin negativecontrols,andCuandHgspikedmediawhenbrineshrimps wereapproximately15daysold. Themaximumlengthreached wasofabout8mm.OnlyCdbetween1.0and33mg/Lsigniﬁcantly inhibitedgrowthandreproductionrate.Cddelayedmatingofabout twodayscomparedtothecontrols.Nomatingoccurredat33mg/L ofCd,andnonaupliiwereproducedduringtheexperimentatthis concentration.

Cunningham (1976) investigated the effects of Dimilin (TH 6040) on various brine shrimp life stages considering a static renewaltest.Artemiaculturesconsumedalgaepresentinthejars and were supplemented witha few dropsof yeast suspension daily.Brineshrimpreproductionwasevaluatedexposingpairsof organismsand monitoringthesubsequentnumberof produced nauplii.After21days,adultsurvivorshipinthenegativecontrol wasapproximately90%anddeclinedto70%after28days.After40 days,survivaldroppedbelow50%andallshrimpsweredeadafter 80days.

Browne and Wanigasekera (2000) measured reproductive performanceandsurvivalatninetemperature–salinity(T–S) com-binations(T=15,24and30◦C,andS=60,120and180‰)forfour sexual(A.franciscana,A.salina,ArtemiasinicaandArtemiapersimilis) andoneparthenogenetic(A.parthenogenetica)species.During sur-vivaltests,ArtemiawasfedwithyeastandDunaliellatertiolectaonce adayalternately.Thenumberofdeadindividualswascountedafter day1,2,3,5,7,14and21.Atsexualmaturity,surviving individu-alsweretransferredtojarscontainingbrineandalgalcultureand monitoredthroughouttheirlifecyclerecordingelevenlife-history traitsforeachspecies(i.e.numberofreproductivefemales,female pre-reproductiveandreproductiveperiods,life-spanfor reproduc-tivefemales,offspringperbrood,broodsperfemale,offspringper female,offspringperdayperfemale,inter-broodinterval, percent-ageofoffspringencysted,andmalelife-span).Allspeciesshowed thehighestreproductionrateat24◦Candat120‰salinityforA. parthenogenetica,A.sinicaandA.franciscana,andat180‰forA. salinaandA.persimilis.Onlyat24◦Cand120‰salinityallspecies completedtheirwholelifecycle.ResultsindicatedthatA. francis-canaisbotheuryhalineandeurythermalreproducingatfourT–S combinations.A.salinaappeared cold-adaptedand intolerantto lowsalinity(60‰)atanytemperature.Reproductiveperformance andsurvivalwereoptimalat24◦Cand180‰salinity.

Brix et al. (2003) determined the chronic (28 d) toxicity of As(Na3AsO4)toA.franciscanameasuringintriplicatetheeffects ongrowth,reproduction,and survival underintermittent ﬂow-throughconditionsconsideringafulllife-cycleapproach(Stephan etal.,1985).Thestudystartedconsidering24holdnauplii,and continuedlookingatthereproductionoftheparentalgeneration. CystswerehatchedinGreatSaltLake(Utah,USA)seawater(27◦C and28g/Lofsalts)thatwasusedalsoasdilutionwater.After11

d,brineshrimpsexuallymaturedandbeganpairingformating. Atthistime, arandomsubsample fromeachtest concentration wascollectedandweighed(dryweight).Sixadultpairsforeach test concentrationwerethen monitoredfor reproductionup to day28when survivingshrimps weremeasuredfordry weight. Adultsurvivalwasthemostsensitiveendpoint,withgrowthand reproductionslightlylesssensitivethansurvival.Thenoobserved effectconcentration(NOEC)forsurvivalwas8mg/L,andthelowest observedeffectconcentration(LOEC)was15mg/LofdissolvedAs. GrowthandreproductionLOECvalueswere>56mg/Lthatisthe highestconcentrationtested.Aftertheparentalgeneration expo-suretoAs,theF1generationacclimatedtoAsthuspresentinga signiﬁcantlowersensitivitythantheparentalgeneration.

Brixetal.(2004)investigatedtheuseofArtemiaforthe deriva-tionofachronicsite-specificwaterqualitystandardforSeinthe GreatSaltLake(Utah,USA).Themethodpreviouslydescribedin Brixetal.(2003)wasappliedtoA.franciscanaexposedto differ-entSenominalconcentrations(0,3.8,7.5,15,30,60mg/L).Growth oftheparentalgenerationonday11andreproductiononday21 werethetwomostsensitiveendpointsforArtemiawithrespectto theotherspeciesevaluated(i.e.brineflyandhypersalinealgae). BrineshrimpwastheGreatSaltLake’smostsensitivespecies res-ident,andthedirecteffectsofSeonresidentbiotawerenotthe criticalexposurepathwaysinderivingasite-specificwaterquality dischargelimitfortheGreatSaltLake.

Savorellietal.(2007)evaluatedtheconditionsfor7–14d bioas-say withthe brine shrimp Artemia, using A. franciscana and A. parthenogenetica.ExposurestoSDSand oildispersantwere car-riedinvestigatingsomaticgrowth(i.e.carapacelengthfromthe topfrontoftheheaduptothecaudalfurca)andmortality.Algal foodandtoxicantwererenewedthreetimesaweek.Testchambers wereincubatedat25±1◦C,900±100luxwitha14:10hlight/dark photoperiod.Savorellietal.(2007)proposedtheuseofII–IIIinstar stagenauplii,InstantOcean®_salt_mixture,_and_D._tertiolecta_as_food sourceforthe14dtoxicitytest(sensitivityincreasesbetween7d and14d)includinganegativecontrolsuccess≥80%.Mortalitywas selectedasendpointfortheproposedmethodbeingmoresensitive thangrowthforbothA.franciscanaandA.parthenogenetica.

Manfraetal.(2012)proposedavideopresentingona step-by-stepbasisthe14dtoxicitytestprotocolofSavorellietal.(2007) includingtestingmaterials(cysts,dilutionmedia,algalcultureand toxicant),cystactivation,preparationoftestingtoxicant(SDS) con-centrations,preparationoftestingmediaand foodrenewal.The video showedhowtotransfernauplii(n=10)fromPetridishes totestchambersinordertoreducedilutionoftestingsolutions. After14d,thenumberofsurvivinglarvaewascountedandLC50 calculated considering a threshold averagemortality≤20% and LC50(SDS)=8.0±5mg/L.Manfraetal.(2015a)considereda14d protocolwithA.franciscanatoinvestigatethetoxicityofDEGasan anticorrosiveagentfrequentlyusedinoffshoreoildrillingactivities. Manfraetal.(2016)publishedtheresultsoftheintercalibration exercisewithSDSconsideringthe14dtoxicitytest.Asfortheacute mortalitytest,elevenparticipatinglaboratoriescarriedoutthree intercomparison assays calculating LC50 values and repeatabil-ity/reproducibilitycoefﬁcients.ResultscompliedwithISO(1996).

4. Discussion

Aftermorethanﬁvedecadesofuseinecotoxicology,Artemia spp. demonstrated its ability mainly as pre-screening of toxic agents(Dvoraketal.,2012),thusArtemiaspp.endpointsseemto respondtothemarketneedoftoxicitytestingtools,eventhough nointernationallystandardisedtoxicitytestingprotocolscurrently existaccordingtoOECDandISO.

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46 G.Libralatoetal./EcologicalIndicators69(2016)35–49 Amongtheshort-termtoxicity tests,biomarkers and

terato-genicityarethelesspopularendpointswithjustfewpapersand citationsprobablyduetotheirlimitedsensitivity.Behavioral end-points, especially swimming inhibition, have been investigated onlyrecentlyandseemtohaveagreatpotentialinthenearfuture, mainlybecauseresultscanbeacquiredviaimageandvideo analy-sistakingintoaccountbothcontinuousandperiodicobservations ofsingleorgroupsofanimals.Hatchingandacutemortalityarethe mostcommonendpointswithadifferentlevelofon-going stan-dardizationprocess.Hatching(48hstatictest)wasintercalibrated atItalianlevel(Manfraetal.,2015b),whileacutemortality(24h statictest)wasintercalibratedonthebasisoftheavailable stan-dard(APATandIRSA-CNR,2003)atItalian(Manfraetal.,2015b) aswellasEuropeanlevel(Persooneetal.,1993).Bothprovided dataonCuSO4asreferencetoxicant.Amongthelong-termtoxicity tests,the14dstatic-renewalmortalitytestwasintercalibratedat Italianlevel(Manfraetal.,2015b)withSDSonthebasisofUnichim protocol(2012).

TomakeArtemiaspp.anofficialinternationallyrecognized stan-dard biological model in ecotoxicology and nanoecotoxicology, furthereffortsarenecessary.Thestepstowardsstandardization shouldinvolve(i)nationalmember(whothencontactsISO)upon arequestbyanindustrysectororgroupforastandard;(ii) cre-ationofagroupofexpertsfromallovertheworldnegotiatingall aspectsofthestandard,includingitsscope,keydefinitionsand con-tent;(iii)multi-stakeholderbrainstormingandreviewingprocess includingexpertsfromtherelevantindustry,consumer associa-tions,academia,nongovernmentalorganizationsandgovernment. Wehighlightsometaskstobeeasilyaccomplishedasacorrect practicetoamelioratethefeasibility,reliabilityandcomparability ofArtemiaspp.toxicity dataincludingallendpointsand poten-tiallycontributingtosupportofficialstandardization.Authorsdid notreportandreviewersdidnotrequireseveralkeyparameters. AccordingtoTablesS1,S2,1–3,frequentomissionsarepresent:pH, aerationconditions(DO),amountofhatchedcysts,feedingrate,test container,qualitycriteria(QC)fornegativecontrolsandreference toxicant.InTableS1,theacceptabilitythresholdforcysts hatcha-bilityisrepeatedlymissinglikeasthereferencetoxicant.Hatching toxicitytestswithArtemiaspp.neverprovidedsaltwaterpHvalues (TableS2),and,justinfewcases,dataaboutlightconditions dur-ingexposure,andpositiveandnegativecontrolsqualitycriteriafor acceptability.InTable1,thepositivecontrolisfrequentlyabsent likeasthedeterminationofEC50valuesfortheswimmingvelocity inhibition,thuslimitingthecomparabilitywithotherendpoints’ sensitivity.Withinthebehavioralendpoints,aharmonizationof protocolsisstronglyrequiredalsobecauseauthorsproposed time-by-timespecificapproaches,apparatuses,trackingandrecording devices.InTable2,thedatabaseisquitecompleteprobablybecause the24hshort-termtoxicitytestisthemostwidespread.Missing informationisgenerallyrelatedtotheamountofhatchedcystsand DOthatrepresentinterestingparameterstofullycircumscribethe generaltest(pre-)conditions. Similarly,itwasfoundforTable3 wheredataaboutreferencetoxicantsareveryscarcetoo.Finally,it isnotclearwhichisthecosteffectivenessofeachendpoint com-paredtoitsrelativesensitivityandcurrentreadinessforthemarket. In conclusion, we can provide the following order of easy-to-standardize toxicity tests: 24–48h mortality>14-28 d mortality>hatchingtest>behavioralendpoints>biomarkers.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.ecolind.2016. 04.017.

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