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WP"5" Deliverable"5.3"

Innovative monitoring techniques

D5- 3 Report on the application of non- invasive biosensors

Dissemination level

Public"

LEAD"CONTRACTOR" Akvaplan8niva"(APN)"

AUTHORS" 1,4 " "1,2 1,3 "2 "2" Hector"Andrade"(APN) , Jean8Charles"Massabuau"(CNRS) ," Sabine"Cochrane"(APN) ,"Pierre"Ciret"(CNRS) ,"Damien"Tran"(CNRS) ," Mohamedou"Sow"(CNRS)"2,""Lionel"Camus""(APN)4"" " Contributions*as*follows:** 1"Writing"of"the"report" 2"Development"of"the"technology"(in"the"field"since"2005)"and"system"maintenance" 3"Wider"applications"for"environmental"assessments" 4"Application"of"the"method"to"the"Arctic" "

APN:"Akvaplan8niva" CNRS:"Centre"National"de"la"Recherche"Scientifique"

SUBMISSION"DATE" 28|"10|"2104

1" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

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Contents

1." Foreword"and"acknowledgements" 5"

2." Executive"summary" 5"

3." Introduction" 6" 3.1" Brief"outline"of"the"concept,"hypotheses"and"report"aims"...... "6" 3.2" Background"for"bivalve"molluscs"as"bio8sensors"...... "8" 3.3" About"the"Icelandic"scallop"Chlamys*islandica"...... "9"

4." Materials"and"Methods" 10" 4.1" Study"site"and"biosensor"deployment"...... "10" 4.2" Valvometry"and"behavior"analysis"using"HFNI"...... "12" 4.3" Environmental"abiotic"factors"...... "14" 4.4" Determination"of"biological"cycles"...... "15" 4.5" Growth"cycles"...... "16" 4.6" Mortalities"...... "16"

5." Results" 16" 5.1" General"...... "16" 5.2" Recording"baseline"biological"rhythms:"Gaping"behavior"in"relation"to"environmental"parameters"...... "16" 5.3" Gaping"behavior"and"environmental"cycles"...... "17" 5.4" Mortality"...... "18" 5.5" Growth"...... "19"

6." Discussion" 21" 6.1" Discussion"of"results"obtained"so"far"...... "21" 6.2" Laboratory"applications"and"validation"of"animal"health"...... "21" 6.3" Evaluation"of"the"system"performance"...... "22" 6.4" Applicability"of"the"method"for"MSFD"monitoring"...... "23" 6.5" Operational"strategy"and"costs"of"the"system"...... "24" 6.6" Outreach"...... "26"

7." Conclusions"and"future"directions" 26"

8." References" 27" "

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3" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

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Tables

Figure"1."The"Icelandic"scallop"Chlamys*islandica."Source:"www.naturalhistory.museumwales.ac.uk.""""...... "9" Figure"2."Location"of"Kongsfjorden"in"Svalbard/Spitsbergen,"where"biosensors"have"been"deployed"...... "11" Figure"3."The"old"pier"at"Kongsfjorden,"Svalbard"and"deployment"of"biosensors"during"the"polar"night"(date)."...... "12" Figure"4."Chlamys*islandica"with"electrodes"glued"on"each"valve"(arrow"on"right"picture)."...... "12" Figure"5."Schematic"representation"of"a"typical"bivalve"biosensor"setup."...... "13" Figure"6."Photoperiod"measured"as"hours"of"light"from"January"(day"0)"to"December"(day"365)"in"2012.."...... "14" Figure"7."Tidal"activities"in"Ny"Ålesund"from"1th"of"January"2012"to"the"31th"December"2012."...... "15" Figure"8."Valvometry"recordings"(gaping"behaviour)"of"the"Icelandic"scallop"Chlamys*islandica."...... "17" Figure"9."Actograms"of"valvometry"for"two"individual"scallops"in"Ny"Ålesund"during"216"days"...... "18" Figure"10."Valvometry"recordings"of"maximum"daily"valve"opening"of"Icelandic"scallop"Chlamys*islandica."...... "19" Figure"11."Continuous"growth"measurements"for"C.*islandica"deployed"in"Ny"Ålesund"2012"to"2014."...... "20"

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1. Foreword and acknowledgements Deliverable"583"of"the"DEVOTES"project"documents"the"method"and"results"obtained"through"one"of"the" innovative"monitoring"techniques"proposed"within"WP5,"Task"5.1.1:"Applying"remote"sensing"to"assess" marine"biodiversity"(from"species"distribution"to"habitat"types"and"their"heterogeneity)."

The"task"description"is"as"follows:"High"frequency,"non8invasive"biosensors"to"measure"movement"and" shell"growth"in"bivalves"with"powerful"mathematical"analysis"and"recent"technological"advances"in"real" time" data" transfer" and" analysis" will" be" applied" on" Arctic" bivalves" at" three" different" locations" in" the" Barents"Sea"for"three"years"(note:"of"the"three"sites,"data"from"one"locality"is"dedicated"to"the"DEVOTES" project)."The"main"goals"will"be"to"(i)"further"understanding"of"bivalve"behaviour"and"shell"growth"in"a" remote"ecosystem"such"as"the"Arctic,(ii)"study"the"chronobiology"of"the"animal"in"geographical"locations" where" the" solar" rhythm" (dark" winter," 24" hr" light" in" the" summer)" and" food" availability" differ" from" temperate" regions," (iii)" generate" data" during" the" winter" season" of" the" Arctic" which" is" poorly" studied" owing"to"difficult"access"and"limited"logistics,"(iv)"to"study"the"impact"of"climate"on"animal"behaviour,"as" a"proxy"for"detecting"changes"in"water"quality."These"northern"locations"represent"areas"where"climate" change"is"a"fact,"and"therefore"give"reference"point"for"the"already"established"network"of""molluscan" eye""biosensors"elsewhere"across"Europe"(see"web"site"map).""

We"thank"collaborators"at"the"University"Centre"on"Svalbard"(UNIS)"and"the"research"facility"at"King's" Bay,"Ny"Ålesund"for"field"opportunities."We"further"thank"the"DEVOTES"community"for"lively"discussions" on"the""talking"clam""concept."In"particular,"we"thank"Mike"Elliott"(University"of"Hull)"for"debate"on"the" technology," Maria" C." Uyarra" (AZTI)" for" critical" comments" to" the" manuscript" and" Melanie" Austin" (Plymouth"Marine"Laboratory)"for"discussions"on"future"cost8benefit"assessments"of"the"technique."

2. Executive summary This" report" outlines" the" application" of" high" frequency," non8invasive" biosensors" (HFNI)" to" measure" biological" rhythms" and" shell" growth" in" bivalves" using" real" time" data" transfer" and" analysis" (http://molluscan8eye.epoc.u8bordeaux1.fr/index.php?lang=en&page=accueil&wid=)." The" method" is" being"applied"on"Arctic"bivalves"at"three"different"locations"in"the"Barents"Sea"over"a"period"of"three" years"and"beyond."This"report"outlines"the"method,"its"applications"and"the"results"achieved"from"so"far" after"2.5"years"of"deployment,"at"one"of"these"locations,"the"data"from"which"are"fully"dedicated"to"the" DEVOTES"project."The"ultimate"aim"of"this"activity"within"DEVOTES"is"to"determine"the"potential"utility"of" the"method"for"long8term"water"quality"monitoring,"in"the"context"of"the"Marine"strategy"Framework" Directive"(MSFD)."

The" main" scientific" goals" of" the" application" are" to" (i)" further" understanding" of" bivalve" behaviour" and" shell" growth" in" a" remote" ecosystem" such" as" the" Arctic,(ii)" study" the" chronobiology" of" the" animal" in" geographical" locations" where" the" solar" rhythm" (dark" winter," 24" hr" light" in" the" summer)" and" food" availability" differ" from" temperate" regions," (iii)" generate" data" during" the" winter" season" of" the" Arctic" which"is"poorly"studied"owing"to"difficult"access"and"limited"logistics,"(iv)"to"study"the"impact"of"climate" on" animal" behaviour," as" a" proxy" for" detecting" changes" in" water" quality." These" northern" locations"

5" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

" represent" areas" where" climate" change" is" a" fact," and" therefore" give" reference" point" for" the" already" established"network"of""molluscan"eye""biosensors"elsewhere"across"Europe"(see"web"site"map).""

This"report"had"the"following"aims:"

• to"describe"the"background"for"using"non8invasive"bio8sensors"on"bivalve"molluscs;" • to"describe"the"technology"and"operating"procedures;" • to"document"and"interpret"the"results"achieved"so"far,"and" • to"discuss"the"utility"of"the"method"within"DEVOTES"and"the"MSFD."

The"non8invasive"biosensor"system"presented"here"has"recorded"valve"gaping"and"shell"growth"over"a" period"of"2.5"years."Both"the"growth"and"the"behaviour"patterns"were"strongly"linked"to"environmental" cycles"such"as"photoperiod"(light"conditions)"and"productivity,"both"of"which"are"strong"environmental" drivers"in"the"Arctic,"as"well"as"tidal"cycles."Because"this"system"was"tested"in"a"pristine"environment,"the" aim"of"confirming"the"strong"relationships"between"behaviour"and"growth"with"environmental"factors" was"achieved."We"have"successfully"tested"the"current"system"in"a"challenging"Arctic"environment,"and" supplemented" with" the" experiences" elsewhere" across" Europe," are" confident" of" high" success" in" deployment"in"almost"any"other"environment."

We"have"identified"a"number"of"potential"uses"of"the"system"for"monitoring"and"early8warning"alerts"of" changes"in"water"quality,"such"as"temperature,"releases"of"contaminants"to"toxic"algal"blooms."Potential" relevant"applications"for"the"MSFD"include"their"use"in"bathing"beaches"and"large"harbours,"petroleum" installations"and"aquaculture"sites."""

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3. Introduction

3.1 Brief outline of the concept, hypotheses and report aims This"report"outlines"the"application"of"high"frequency"non8invasive"(HFNI)"bio8sensors,"as"a"potential"tool" for" marine" monitoring" and" assessments." In" this" case," we" use" bivalve" molluscs" as" our" "monitoring" assistants"."The"principle"of"the"method"is"based"on"the"regular"gaping"behaviour"(closing"and"opening" of"the"valves)"of"bivalve"molluscs"(clams)"and"the"fact"that"physical"or"chemical"stressors"disrupt"that" gaping"pattern."A"number"of"organisms"are"placed"in"cages,"in*situ,"and"microsensors"(electrodes)"are" attached"to"the"valves"to"measure"the"distance"between"them."The"data"are"recorded"live,"in"real"time" and"displayed"online"on"the"internet."The"basic"concept"is"that"when"a"bivalve"is"facing"a"stressor,"such" as"a"pollutant,"its"gaping"behaviour"changes,"indicating"a"change"in"water"quality."The"method"employed" here"monitors"bivalve"gaping"behaviour"in"the"natural"environment,"remotely,"and"over"a"time"period"of" years,"requirements"that"must"be"fulfilled"if"bivalve"behaviour"is"to"be"a"useful"biomonitoring"tool."We" detect"abrupt"changes"but"also"smoothed"changes"associated"to"silent"pollution."

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Wherever"the"valvometer"is"installed,"the"data"are"automatically"and"continuously"transmitted"on"a"real8 time"basis"safely"from"the"acquisition"place"to"the"data"processing"center"in"Arcachon,"using"cellular"and" internet" networks." Precise" interpretation" of" the" data" is" possible" at" any" time," including" a* posteriori" analysis"to"reconstruct"gaping"history"in"any"particular"situation"of"interest,"e.g."under"a"pollution"event," or" under" climate" stress." These" data" and" scientific" interpretation" can" be" used" for" technical" and" administrative"investigations.""

The" bio8sensor" system" also" acts" as" a" public8friendly" communication" and" outreach" tool." Signals" are" available" to" the" public" through" the" MolluscanEye" website" (http://molluscan8eye.epoc.u8 bordeaux1.fr/index.php?lang=en&page=enregis&wid)." The" system" does" not" introduce" non8indigenous" species"as"the"bivalves"used"are"collected"from"the"local"populations.""

The" innovation" of" the" method" lies" in" the" extremely" small" sensors" which" do" not" restrict" the" natural" behaviour" of" the" organism" and" which" can" be" deployed" intervention8free" for" years," the" fact" that" the" system" also" measures" shell" growth" and" finally" that" the" data" are" streamed" online" in" real8time" and" displayed"on"a"web"site"where"it"can"be"easily"read"and"is"freely"accessible"to"managers"and"general" public."

The" technology" already" is," or" has" been," tested" and" deployed" in" a" number" of" marine" locations" across" Europe"(France,"Spain,"Scotland),"in"the"Tropics"(New"Caledonia,"French"Polynesia"in"discussion)"and"the" Arctic" (Norway," Russia)" (http://molluscan8eye.epoc.u8bordeaux1.fr/" )." The" locations" across" Europe" comprise"both"water"bodies"which"are"directly"pressured,"such"as"harbours,"but"also"others"which"are" more"remote."Data"from"the"bivalves"placed"in"a"harbour"setting"have"demonstrated"episodic"incidences" of" disturbance," but" it" is" not" always" possible" to" pinpoint" directly" the" exact" cause" of" that" disturbance." When"this"occurs,"the"technology"reveals"the"problem"becoming"the"starting"point"of"an"enquiry."In"New" Caledonia,"the"same"technology"deployed"on"giant"clams"Hippopus*hippopus"has"shown"cyclical"patterns" in"gaping"behaviour"driven"by"seasonal"temperature"variations"(Schwartzmann"et"al."2011)."This"opens" up"the"possibility"that"the"method"could"be"used"in"further"research"both"on"the"impacts"of"climatic" warming" on" marine" organisms" but" also" as" an" indicator" of" such" change." Of" course" continuous" measurements" of" water" quality" will" detect" changes," but" what" is" interesting" for" biological" effects" monitoring"is"to"determine"the"point"at"which"the"changes"begin"to"impact"the"function"of"the"organisms" in"that"system."

In"DEVOTES,"we"further"explore"the"relationship"between"changes"in"water"quality"and"the"behaviour" and" function" of" organisms," in" this" case" bivalve" molluscs." To" do" this," we" have" established" an" Arctic"" location" at" approximately" 79" °N," where" there" is" almost" no" direct" human" influence," but" marked" environmental"fluctuations"in"water"quality,"both"seasonal"(light,"temperature,"salinity,"productivity"etc.)" and" also" due" to" the" already" very" apparent" ongoing" climatic" changes." Even" in" the" last" decade," areas" which" previously" contained" Arctic" water" now" have" an" increasing" influence" of" Atlantic" water." Our" hypothesis" was" that" chronobiology" and" growth" rate," and" their" potential" alteration" by" environmental" stressors"can"be"used"as"early"and"non8invasive"signal"of"environmental"change"and"its"impact"on"marine" organisms."From"this"basis,"we"can"determine"the"robustness"and"efficiency"of"the"biosensors"as"a"tool" for"detecting"biologically"significant"changes"in"water"quality"also"elsewhere,"where"the"nature"of"those" changes"may"not"be"so"apparent."" 7" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

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This"report"has"the"following"aims:"

• to"describe"the"background"for"using"non8invasive"bio8sensors"on"bivalve"molluscs;" • to"describe"the"technology"and"operating"procedures;" • to"document"and"interpret"the"results"achieved"so"far,"and" • to" discuss" the" utility" of" the" method" within" DEVOTES" and" the" Marine" Strategy" Framework" Directive"(MSFD)"

3.2 Background for bivalve molluscs as bio-sensors Marine"ecosystems"all"over"the"globe"are"facing"multiple"simultaneous"stressors"including"rapid"climatic" change"and"increased"resource"exploitation,"such"as"fishing,"petroleum"exploration"and"shipping."The" interactions"between"multiple"human"stressors"often"are"poorly"understood,"such"that"the"ecosystem" effects"of"cumulative"pressures"may"be"more"complex"than"we"are"yet"aware"of"(see"Micheli"et"al.,"2013," Crain"et"al.,"2008"and"references"therein)."

In" the" Arctic," cohesive" studies" of" temporal" variation" in" ecosystem" structure" and" function," and" the" impacts"of"human"activities"such"as"resource"extraction,"have"been"constrained"by"both"political"factors" and" the" logistical" challenges" involved" in" carrying" out" fieldwork" (Blicher" et" al." 2010," Wassmann" &" Reigstad"2011).""Critical"knowledge"gaps"include"the"study"of"basic"biological"processes"and"the"lack"of" time"series"to"study"temporal"variations"(Wassmann"&"Reigstad"2011,"Kuklinski"et"al."2013,"Nahrgang"et" al."2013),"hampering"our"understanding"of"ecosystem"functioning."For"example,"until"recently,"there"was" a"common"acceptance"that"biological"activity"at"all"trophic"levels"and"biomass"was"low"during"the"polar" night."New"evidence"suggest"however,"that"key"trophic"linkages"are"maintained"under"the"almost"no8 light" conditions," from" the" dial" vertical" migrations" of" bio8luminescent" zooplankton" to" the" continuous" foraging" of" seabirds" (Berge" et" al." 2012," Kuklinski" et" al." 2013," Johnsen" et" al." 2014)." Advancements" in" technology,"especially"in"the"fields"of"underwater"vehicles"and"optical"sensors"are"facilitating"ecosystem" studies"during"the"polar"night"(e.g."Johnsen"et"al."2014)."However,"such"methodology"still"requires"the" repeated" mobilization" of" researchers" to" remote" sampling" locations" that" might" be" inaccessible" during" harsh" winters" or" prohibited" expensive" to" reach." A" solution" is" to" develop" unmanned" systems," able" to" work"continuously"without"human"intervention"over"longer"time"periods.""

Gaping"behaviour"of"molluscan"bivalves"i.e."the"opening"and"closing"of"their"valves"has"been"proven"as"a" successful"tool"to"infer"how"environmental"stimuli"drive"the"life"history"of"several"species"allowing"for" understating" drivers" of" ecosystem" processes." Gaping" activity" of" many" species" is" closely" related" to" physiological" process" such" as" breathing" and" nutrition" which" responds" to" environmental" conditions" following" rhythmic" cycles" (Garcia8March"et"al."2008)."This"behaviour"can"be"measured"by"using"high8 frequency,"non8invasive"valvometers"(HFNI"valvometers)"that"are"glued"to"the"animals"and"deployed"in" the" field" for" long" periods" of" time." For" example," the" gaping" behaviour" of" 15" giant" clams" Hippopus* hippopus"monitored"in"New"Caledonia"during"a"1"year"period"revealed"that"patterns"of"growth"and"daily" behaviour"are"related"to"light"availability"and"changes"in"water"temperature"(Schwartzmann"et"al."2011)." A" similar" study" revealed" that" although" light" is" a" significant" driver" of" biological" rhythm" for" the" oyster" Crassostrea*gigas,"the"tide"cycle"is"the"main"driver"affecting"species"behaviour"(Tran"et"al."2011).""

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In"the"present"study"the"HFNI"technology"was"adapted"and"tested"in"Arctic"conditions."The"goal"was"to" study" the" gaping" behaviour" and" growth" patterns" of" the" Icelandic" scallop" Chlamys* islandica" in" Kongsfjorden" (Ny" Ålesund," Svalbard)" and" relate" it" to" environmental" parameters" to" identify" drivers" of" biological" activity." Here," we" present" data" comprising" more" than" 2.5" years" of" recordings" and" online" analysis" that" was" published" daily" on" a" dedicated" website" (http://molluscan8eye.epoc.u8 bordeaux1.fr/index.php?rubrique=accueil&lang=en"""or""google""molluscan"eye)."In"the"high"Arctic,"the" most"obvious"driver"of"marine"systems"is"the"marked"seasonality,"which"fluctuates"between"the"polar" night"in"winter,"where"marine"organisms"receive"no"light"exposure,"to"the"opposite"scenario"in"summer," where" there" is" no" darkness." Productivity" and" water" quality" (such" as" temperature" and" salinity)" are" markedly" affected" by" this" seasonality," and" thus" we" also" expected" to" see" clear" links" between" these" physical"drivers"and"organism"function"in"bivalves.""

3.3 About the Icelandic scallop Chlamys islandica The"chosen"species"for"the"bio8sensor"deployment"was"the"Icelandic"scallop,"Chlamys*islandica"(Figure" 1)."This"bivalve"is"widely"distributed"at"high"latitudes,"ranging"from"77.5°N"to"41.7°N;"70.7°W"to"20°W" (Rosenberg"et"al."2013),"and"they"are"harvested"for"human"consumption."In"the"Northeast"Atlantic"this" species"is"found"in"the"Kara"Sea,"White"Sea,"Svalbard,"Barents"Sea,"Bjørnøya,"Iceland,"Jan"Mayen,"Novaya" Zemlya"and"the"western"coast"of"Norway"(Garcia"2006,"Beuchel"&"Gulliksen"2008).""

" " Figure* 1.* The* Icelandic* scallop* Chlamys" islandica* Source:* www.naturalhistory.museumwales.ac.uk.* Left:* upper* valve;*dead*specimen*and*Right:*live*specimen*showing*normal*gaping*behaviour.*Photo:*Erling*Svensen.**

Individuals"of"this"species"inhabits"the"hard"bottom"of"the"ocean,"between"10"to"110"m"deep,"and"exhibit" a"temperature"preference"around"284"°C."In"the"Barents"Sea,"Chlamys*islandica"spawns"in"the"summer," in" June/July," with" a" planktonic" larvae" stage" lasting" several" weeks." The" average" size" of" the" Svalbard" population"is"around"60880"mm"and"lives"up"to"10"years"(Brand"2006,"Strand"&"Parsons"2006)."Its"growth" is" optimal" during" the" six" first" years" of" its" life." As" any" living" organism," C.* islandica,"presents"biological" rhythms" which" are" controlled" by" environmental" factors" including" light" availability," food" quantity" and" quality,"and"temperature"(Blicher"et"a."2010).""

9" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

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BOX"1."DEFINITION"OF"TERMS"

______"

Actogram" diagram"depicting"the"activity"patterns"of"organisms."Usually,"activity"on"a"time"base"of"24h"is" plotted"on"horizontal"lines"below"each"other"for"consecutive"days;"

Biosensor"" analytical"device"that"combines"a"biological"component"with"a"physicochemical"detector;"

HFNI" high"frequency,"non8invasive"(as"applied"to"biosensors);"

MSFD"" Marine"Strategy"Framework"Directive"

Valvometry"the"opening"and"closing"activities"of"bivalves,"also"referred"to"as"the"gaping"activity."

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4. Materials and Methods

4.1 Study site and biosensor deployment In"this"project,"16"individuals"of"Chlamys*islandica*were"placed"in"Kongsfjorden,"a"26"km"long"open"(i.e." no"sill"at"entrance)"glacial8fjord"system,"situated"at"approximately"79°"latitude"on"the"western"coast"of" Spitsbergen"(Figure"2)."Despite"its"high"Arctic"location,"the"fjord"system"is"influenced"by"both"Atlantic" and"Arctic"water"masses,"due"to"the"increasing"northward"penetration"of"the"northern"branch"of"the" Gulf"stream,"which"flows"along"the"west"coast"of"Svalbard."Physical"processes"that"control"the"circulation" patterns"are"mainly"the"tides"and"wind"circulation"but"also"the"freshwater"input"brought"about"by"glacier" ablation,"snowmelt,"summer"rainfall"and"ice"calving"(Hop"et"al."2012,"Svendsen"et"al."2002,"Wlodarska8 Kowalczuk"et"al."2005).""

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Figure* 2.* Location* of* Kongsfjorden* (red* rectangle)* in* Svalbard/Spitsbergen,* where* the* biosensors* have* been* deployed.* Main* currents* along* Norway* and* Svalbard* are* also* shown.* Source:* Hop* et* al.* (2002)* and* www.barentswatch.no/!

During" the" field" deployment," 16" adult" individuals" of" C.* islandica" within" the" same" size" class" were" equipped"with"the"HFNI"valvometry"technology"and"placed"in"a"cage"prior"to"immersion,"to"protect"them" from"predators."The"biosensors"were"first"deployed"during"the"winter"of"2011"(this"1st"installation,"along" the"pier,"was"destroyed"by"an"iceberg)"and"then"a"new"one"on"May"20th,"2012"under"the"old"pier"at" Kongsfjorden" (Figure" 3)" between" the" pillars," to" test" whether" the" HFNI" valvometry" could" effectively* record" behavioural" alterations" driven" by" natural" environmental" cycles." " Once" the" deployment" was" conducted,"no"more"fieldwork"was"needed,"and"the"device"is"still"running"today."

11" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

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Figure*3.*The*old*pier*at*Kongsfjorden,*Svalbard*and*deployment*of*biosensors*during*the*polar*night,**2011.*

4.2 Valvometry and behavior analysis using HFNI To"monitor"valve"movement"behaviour"of"bivalves"directly"in"the"field,"a"high"frequency"(10"Hertz),"non8invasive" (HFNI)"valvometer"technology"developed"for"laboratory"analysis"(Tran"et"al.,"2003,"2007)"was"adapted"for"the"field" conditions"of"Kongsfjorden."A"pair"of"light"electrodes"was"glued"on"each"half"shell"(Figure"4).""

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Figure*4.*Chlamys*islandica*with*electrodes*glued*on*each*valve*(arrow*on*right*picture).*

The" electrodes," designed" to" minimize" disturbance" to" bivalve’s" behavior," were" made" up" of" two" resin8coated" electromagnets" (50" mg" each)." The" total" weight" of" the" electrode" (resin" +magnet)" was" approximately" 0.5–1.0" g." Between"the"electrodes,"an"electromagnetic"current"was"generated,"which"allowed"measurement"of"the"amount" of"valve"opening"and"closing."Each"pair"of"electrodes"was"connected"by"a"floating"cable"to"an"analogical"electronic" card"that"managed"the"electrodes"and"was"housed"in"a"waterproof"box"next"to"the"animals."The"first"electronic" card"was"connected"to"a"second"located"on"the"pier,"which"constituted"the"processor"that"saves"and"digitizes"the" data"for"transfer"to"the"laboratory"workstation."A"Linux"operating"system"drives"the"first"card"and"processes"the" initial"analysis"of"the"data"(Figure"5).""

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Figure*5.*Schematic*representation*of*a*typical*bivalve*biosensor*setup.*

The"system"is"built"to"sample"data"at"10"Hz"from"16"animals"in"a"sequential"order."Every"0.1"s,"three" packets"of"information"are"produced:"distance"between"valves"at"the"electrode"level,"sampling"time,"and" animal" number." Thus," as" a" whole," a" total" of" 3" ×" 864,000" pieces" of" information/day" describe" the" behaviour"of"a"whole"group"of"16"animals"(2,592,000"data"points)."At"the"individual"level,"it"means"that" the"system"performs"a"measurement"of"the"opening"status"every"1.6"s,"as"there"are"16"animals,"and"that" a"total"of"54,000"data"points"characterize"the"gaping/closing"behaviour"of"any"individual"every"day."The" raw"data"is"transferred"from"the"field"to"the"laboratory"using"either"an"Ethernet"network"socket"(the" situation"at"Ny"Ålesund)"or"a"cellular"telephone"network"(GPRS;"General"Packet"Radio"Service),"with"a" mobile"phone"module"embedded"in"the"2nd"electronic"card."In"both"cases,"data"are"transferred"daily"at" 00:00"GMT"to"a"laboratory"workstation"(DELL"precision"690"using"both"Bash"Linux"second"mathematical" codes"written"in"R,"(http://CRAN.R8project.org/doc/FAQ.RFAQ.html)"that"is"designed"to"capture,"process"and" distribute"information"derived"from"the"data"(Tran"et"al."2011)."

After"finishing"the"installation"process"in"fieldwork,"the"data"is"processed"and"analysed"on"a"daily"basis," and"easy8to8read"graphs"are"instantly"produced."In"this"manner"daily,"weekly"or"monthly"trends"can"be" identified" quickly." The" metadata" is" also" stored" providing" a" background" allowing" for"gaping" behaviour" comparisons" when" any" change," subtle," overt" or" violent" occurs" over" time." Records" are" available" since" February"2006"thanks"to"the"data"stocking"policy."

Registered"users"have"access"to"all"the"records"from"the"individuals"deployed"at"their"site"using"the"PRO" website"version."Here"dynamic"graphs"deploy"simultaneously"gaping"behaviour"of"1,"4"or"16"bivalves.""A"

13" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

" basic"idea"is"to"produce"a"single"mathematical"equation""(click"on"hyperlink)"for"each"animal"for"each" day,"then"to"apply" these"equations"so"that"the"maximum"possible"amount"of""digested""data"can"be" obtained"graphically."The"PRO"version"also"allows"inter8site"comparisons."

In"the"pages"of"MolluSCAN"eye"PRO,"professionals"can"integrate"various"types"of"information"and"derive" graphs"describing"the"various"aspect"of"the"bivalves"ethology,"chronobiology"or"physiology."The"graphs" allow" the" relevance" of" single" observations" to" be" placed" in" a" broader" context" of" comprehensive" behaviour." Four" key" examples" of" the" types" of" data" produced" are" shown" on" the" website" (http://molluscan8eye.epoc.u8bordeaux1.fr/index.php?rubrique=contenu_sitePro&lang=en):" biological" rhythms,"growth"rates"and"spawning"or"death"records"

4.3 Environmental abiotic factors All" the" biological" rhythms" resulted" from" the" scallops" activities" are" then" compared" to" local" abiotic" oceanographic8phenomena,"such"as"the"solar"cycle"(light"availability)"and"the"tide."""

The*solar*cycle**

A" particularity" strong" environmental" seasonality" in" the" Svalbard" archipelago" affecting" life" cycles" of" species" is" the" high" photoperiod" variation" between" winter" and" summer" (Figure" 6)." By" comparison," in" temperate"regions,"the"variation"of"the"photoperiod"during"the"year"is"low"with"a"minimal"photoperiod" of"9"hours"and"a"maximal"one"of"16."

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16 time+(h) 12

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4 DurÈe de la photophase (h) photophase la de DurÈe Photoperiod 0 0 50 100 150 200 250 300 350 Time+(Joursdays) "

Figure*6.*Photoperiod*measured*as*hours*of*light*from*January*(day*0)*to*December*(day*365)*in*2012.*The*black* line* represents* the* total* time* of* light* period* received* in* Svalbard.* The* black* dotted* line* shows* for* comparison*the*photoperiod*in*the*Bay*of*Arcachon.*The*red*dotted*line*corresponds*to*the*photoperiod* at*the*equinox.!

The*tidal*cycle**

Tidal"activities"in"Ny"Ålesund"present"a"semi"diurnal"cycle"comprising"two"tides"a"day"with"a"maximum"amplitude"" of"about"1.5"m"(Figure"7)."

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! 200

180

160

140

120 (cm) 100

level 80

60 Water! Hauteur d'eau (en cm) (en d'eau Hauteur 40

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0 2000 4000 6000 8000 Time!(Heureshours ) "

Figure*7.*Tidal*activities*in*Ny*Ålesund*from*1th*of*January*2012*to*the*31th*December*2012.!

4.4 Determination of biological cycles After" the" collection" of" individual" opening" amplitude" data" over" time," then" biological" rhythms" were" characterized"according"to"the"different"seasonal"patterns"created"by"the"software"Times"Series"Analysis" Seriel" Cosinor" 6.3" using" the" Cosinor" model" (http://www.euroestech.com)." To" validate" the" biological" rhythm," first" the" data" are" statistically" controlled" and" then" if" the" quality" is" correct," the" potential" periodicity"can"be"investigated."If"a"period"is"statistically"demonstrated,"then"the"rhythm"is"defined"using" the"Cosinor"model."For"the"deployment"in"Ny"Ålesund,"3"different"phases"were"differentiated"for"the" recording"period:"The"polar"day,"the"alternation"of"day/night,"and"the"polar"night."All"data"analyses"are" performed"for"the"whole"population"and"for"each"individual"scallop.""

Actograms*

Actograms," which" depict" activity" patterns" in" organisms," are" produced" using" the" software" Chronos8Fit" 1.06"and"on"each"line,"the"activities"of"two"consecutive"days"are"represented."Every"day,"the"average" valve"activity"is"calculated"and"then"all"the"data"higher"than"this"average"are"plot"on"dark"and"the"lower" ones"on"white."It"is"then"an"easy"way"to"summarize"and"have"an"overview"of"the"valvometry"pattern" overtime.""

Environmental*factors*

All" the" biological" rhythms" resulted" from" the" scallops" activities" are" then" compared" to" local" abiotic" oceanographic8phenomena,"such"as"the"photoperiod"and"the"tide."These" factors" were" obtained" from" the" website" of" IMCCE" (Institut" de" Mécanique" Céleste" et" de" Calcul" des" Ephémérides," http://www.imcce.fr/fr/ephemerides/phenomenes/rts/rts.php).ext." The" relations" between" environmental" factors" and" gaping" behaviour" have" been" analysed" thoroughly" by" Picot" (2013)" in" the" period"20.05.11"to"31.12.12;"comprising"266"days"of"records."These"results"are"presented"here"but"also"

15" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

" expanded" to" document" growth" until" September" 2014." Data" comprises" thus" more" than" 2.5" years" of" records."

4.5 Growth cycles To"obtain"the"growth"history"on"a"daily"scale,"we"took"advantage"of"the"fact"that,"in"bivalve"molluscs," calcification"takes"place"in"the"mantle"cavity,"all"over"the"shell’s"internal"surface."Consequently,"if"daily" growth"layers"are"produced,"the"minimal"distance"between"electrodes"will"increase"assuming"that"valves" become"closed"at"least"once"(Schwartzmann"et"al."2011)"or"at"least"comes"back"to"a"steady"resting"value" that"is"the"case"in"Chlamys*sp."To"obtain"a"growth8rate"index,"these"daily"values"are"isolated"and"plotted" as" a" function" of" time" since" the" 20th" of" May," 2012" to" the" 5th" of" September" 2014" and" related" to" the" photoperiod"cycle.""

4.6 Mortalities The" same" operation" can" be" done" with" the" maximum" daily" opening" values," giving" complementary" behavioral" information." Indeed," the" maximal" opening" status" is" an" index" of" a" clam’s" welfare," as" a" decrease"of"valve"opening"is"a"major"way"to"protect"the"soft"body"in"endangered"molluscan"bivalves."It" also"allows"identifying"and"dating"mortality"events."When"a"bivalve"dies,"valves"become"widely"open"and" inert."Comparisons"to"typical"records"allow"us"to"establish"the"exact"time"at"which"the"adductor"muscle" ceases"to"contract.""

5. Results

5.1 General The"following"section"provides"an"insight"in"the"results"obtained"from"the"20th"of"May,"2012"to"the"5th"of" September" 2014." Results" presented" here" include" the" relation" between" gaping" behaviour," tides" and" photoperiod"from"May"2012"to"December"2012"(Picot"2013)"and"the"relation"between"growth"and"light" availability"brought"about"the"polar"night/day"interface"from"the"May"20th"2012"to"September"5th"2014."" However,"other"aspects"of"the"species"life"history"and"its"relation"to"the"environment"are"being"studied" and" will" be" published" in" 2015." These" include" among" others," detection" of" spawning" events" and" the" effects"of"temperature"on"gaping"behaviour"and"growth."It"is"important"to"mention"that"after"finishing" the" deployment" in" 2012," no" fieldwork" has" been" required" as" the" system" continues" to" work" properly" demonstrating"its"effectiveness"in"the"challenging"Arctic"conditions.""""

5.2 Recording baseline biological rhythms: Gaping behavior in relation to environmental parameters Deployment" of" the" biosensor" device" in" Ny" Ålesund" was" completed" successfully" in" May"2012" and" allowed" for" a" general"understanding"of"the"challenges"working"with"the"biosensor"system"at"higher"latitudes,"the"species"and"its" biological" rhythm." Recordings"started"on"the"20th" of"May"2012"showing"the"species"gaping" behaviour."A"typical" recording"revealed"times"of"openings"and"closures"(Figure"8A)"that"varied"with"the"time"of"the"day"and"season."For" example,"on"the"29th"of"August,"animal"4"closed"at"09:45"(Figure"8B).""

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Figure* 8.* Valvometry* recordings* (gaping* behaviour)* of* the* Icelandic* scallop* Chlamys* islandica,* animal* #4* in* Ny* Ålesund,* Svalbard* from* august* the* 25th* to* the* 31st* (A)* and* during* a* one* hour* interval* for* the* same* animal*on*the*29th*of*August*2014*(B).**The*axis*distance*refers*to*the*space*measured*(mm)*between*the* electrodes*glued*in*each*valve.*

5.3 Gaping behavior and environmental cycles The" actograms" illustrating" the" valvometry" of" Chlamys* islandica" in" Ny" Ålesund" indicated" different" patterns" of" rhythm"according"to"the"season"(Figure"9)."During"the"alternation"between"day/light,"from"the"27th"of"August"to" the"21th"of"October"2012,"the"valvometry"was"synchronized"with"the"circadian"cycle,"and"opening"periods"occurred" during"the"night"while"closing"time"was"observed"during"the"day"(green"circles)."This"indicates"that"C.*islandica"is" more"active"during"the"night"suggesting"relative"higher"filtration"rates."However"during"the"polar"day"or"the"polar" night," valve" activity" becomes" non8synchronous" as" each" scallop" exhibit" a" particular" rhythm" that" varies" greatly" between"individuals."This"implies"that"when"the"alteration"of"day"and"night"disappears,"the"gaping"behaviour"at"the" population"level"is"not"cued"by"any"other"environmental"drivers"(Picot"2013).""

17" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

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Figure* 9.* Actograms* of* valvometry* for* two* individual* scallops* in* Ny* Ålesund* during* 216* days* showing* the* scotophase*in*dark*and*the*photophase*in*white*during*48*hours.!The*photoperiod*indicates*the*hours*of* light*(white)*and*darkness*(black)*during*the*recording*period.*The*green*ovals*indicate*a*period*with* high*synchronization*during*the*alternation*between*day/light*

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5.4 Mortality The"maximum"daily"valve"opening"can"be"used"to"trace"bivalves"overall"well8being"(Schwartzmann"et"al." 2011)" and" by" plotting" these" values," mortality" events" can" be" easily" distinguished" as" valves" become" completely"open."In"this"regard,"2"individuals"died,"one"on"the"26th"of"March"and"another"in"the"6th"of" May"2014"(Figure"10)."The"fact"that"mortality"can"be"recorded"is"important"for"traceability."When"several" animals"die"at"the"same"time"and"date,"one"can"investigate"the"causes"which"might"be"brought"about" pollution"events,"environmental"stress,"etc.""

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Figure* 10.* Valvometry* recordings* of* maximum* daily* valve* opening* of* Icelandic* scallop* Chlamys* islandica,* in* Ny* Ålesund,*Svalbard*from*January*to*July*2014*showing*dead*events*of*2*individuals*(red*circles)*on*the*26th* of*March*and*6*of*May*2014.***

5.5 Growth The"long8term"series"of"data"(May"20th,"2012"to"September"5th,"2014"was"analysed"in"September"2014"revealing" cumulative"growth"patterns"for"the"species"which"varied"greatly"between"individuals"(Figure"11)."Most"of"the"clams" have"at"least"double"their"size,"presenting"seasonal"variations"in"growth"rate"intensity.""

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19" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

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Figure*11.*Continuous*shell*growth*measurements*for*C.*islandica*deployed*in*Ny*Ålesund*from*May*20th*2012*to* September*5th*2014.*Blue*dotted*lines*represent*growth*patterns*of*individual*scallops*

During"the"polar"day,"clam"growth"increased"at"an"exponential"rate"until"the"alternation"of"night/"day"at"the"end"of" August."However,"during"the"polar"night,"the"growth"rate"lowered"until"the"alternation"of"day/night"at"the"end"of" February." " From" about" May," under" polar" day" light" conditions," growth" rate" again" increased" abruptly" probably" coinciding" with" the" phytoplankton" spring" bloom" that" usually" occurs" in" Kongsfjorden" during" the" same" month" (Hegseth"&"Tverberg"2013)."""

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6. Discussion

6.1 Discussion of results obtained so far This"study"has"been"conducted"in"order"to"develop"a"new"tool"for"the"early"detection"of"environmental" stress"using"valvometry"in"scallops."Arctic"ecosystems"are"currently"in"focus"due"to"the"increased"interest" for"petroleum"exploitation,"and"native"Icelandic"scallop"populations"can"potentially"be"impacted"after"an" oil" spill," thus" the" choice" of" study" organism" is" timely." For" this" purpose," the" natural" chronobiology" of" Chlamys* Islandica" has" been" studied" in* situ" and" correlated" to" natural" environmental" rhythms" such" as" photoperiod"and"tidal"cycles."

The"valvometry"data"showed"that"for"C.*islandica,"the"classic"alternation"of"valve"opening"and"closing" during"24h"in"other"species"was"not"observed,"and"that"only"the"change"in"the"opening"amplitude"can"be" used"to"characterize"the"rhythm."These"scallops"presented"a"well"synchronized"circadian"cycle"(24h)"with" the"photophase"and"the"scotophase"during"alternation"of"day"and"night."However,"during"the"polar"night" and"day"periods,"this"cycle"is"lost"at"the"population"level,"with"each"individual"presenting"highly"variable" cycles." The" different" conditions" of" the" polar" night" have" been" analyzed" at" the" population" level" and" revealed"that"the"astronomical"sunrise/sunset"condition"was"significant."However,"no"significant"model" has"been"found"and"no"evidence"of"relationship"between"the"percentage"of"circadian"individuals"and"the" light"intensity"was"found"during"the"different"polar"night."

The" other" abiotic" factor" used" for" the" creation" of" actogram" was" the" tidal" rhythm." None" of" the" 3" conditions"analyzed"for"Ny"Ålesund,"i.e.,"polar"day,"alternation"of"day/night"and"polar"night"exhibit"a" tidal"cycle"at"the"individual"or"population"level."This"can"be"explained"by"the"fact"that"the"tidal"amplitude" through"the"year"is"relatively"low"in"Svalbard"(about"1"metre)"and"that,"below"the"old"pier,"it"was"not" strong"enough"to"induce"a"biological"cycle"in"scallops."

6.2 Laboratory applications and validation of animal health The"HFNI"sensors"described"in"this"report"have"been"used"and"tested"in"the"laboratory"for"many"years" prior" to" using" them" in" the" field." Already" a" decade" ago," the" sensors" were" used" to" investigate" the" protective" valve8closure" strategy" adopted" by" the" Asiatic" freshwater" clam" (Corbicula* fluminea)" when" encountering"cadmium"contamination"(Tran"et"al,"2003)."In"this"study,"potential"stress"to"the"organisms" due" to" the" technique" was" studied" and" responses" in" organisms" compared" between" those" in" stressed" versus" non8stressed" conditions." Having" determined" that" the" sensors" do" not" cause" any" measureable" changes" in" the" behaviour" of" the" animals," and" thus" assuming" that" their" function" is" maintained," experimental"studies"on"responses"to"specific"contaminants"and"interactions"with"environmental"drivers" have" continued." The" sensitivity" threshold" to" 3" other" metals" was" studied:" copper" (Tran" et" al," 2004);" uranium," (Fournier" et" al," 2004)," and" mercury" (Tran" et" al," 2007)" Interestingly" in" the" present" global" context,"the"sensitivity"of"the"valve"closure"response"to"uranium"exposure"in"C.*fluminea"was"found"to" be" influenced" by" the" pH" of" the" water" (Fournier" et" al.," 2004)" and" CO2" (Tran" et" al." 2003)." The" oyster" Crassostrea*gigas"further"was"found"to"have"a"characteristic"and"measureable"response"to"exposure"to" the" parasitic" worm" Polydora* sp" (Chambon" et" al;" 2007)" and" to" harmful" toxic" algae" (Tran" et" al.," 2010;" Haberkorn" et" al." 2011;" Mat" et" al." 2013))." Finally" the" relationship" between" raw" oil" concentration" and" gaping"behavior"has"also"been"studied"(in"prep)." 21" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

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Because"the"system"uses"live"individuals,"and"the"aim"is"for"those"organisms"to"function"as"biosensors" for"a"long"time,"it"is"not"practicable"in*situ"to"analyse"the"physiological"status"of"the"organisms"relative"to" those""free"ranging""in"the"field."We"work"on"the"hypothesis"that"since"the"behaviour"and"growth"of"our" sensor"organisms"was"strongly"correlated"to"the"environmental"drivers,"and"showed"no"aberrations"over" time"(except"the"presumably"natural"death"of"two"animals),"we"assume"that"the"sensors"are"not"causing" stress" to" the" organisms." This" is" further" ensured" by" the" lack" of" human" intervention" since" initial" deployment.""

A" useful" check" would" be" to" conduct" sclerochronology" on" the" shells" of" the" two" dead" animals," and" compare"the"growth"rings"patterns"with"those"harvested"nearby"in"the"field."

6.3 Evaluation of the system performance In" this" study," the" HFNI" valvometry" biosensor" was" successfully" applied" to" the" Arctic" scallop" Chlamys* islandica."This"has"allowed"us"to"perform"a"long8term"study"of"the"organisms'"biological"rhythms."The" system" is" still" fully" operational" after" 2.5" years" of" deployment," sending" data" on" a" continuous" basis" to" CNRS"in"Arcachon,"despite"the"challenging"Arctic"conditions."Further,"no"maintenance"of"the"biosensors" has" been" required," such" that" fieldwork" and" installation" costs" have" been" kept" to" a" minimum." The" equipment"originally"has"been"developed"for"laboratory"experiments,"and"we"therefore"now"consider"it" approved"and"reliable"for"in8situ"locations.""

The"use"of"non8invasive"bio8sensors"to"record"molluscan"gaping"behaviour,"for"the"purposes"of"water" quality" assessment" is" not" new" in" principle" (see" for" example" http://www.mosselmonitor.nl/," although" other"systems"also"exist)."However,"our"method"differs"from"others"in"a"number"of"significant"ways:"

• The" sensors" are" the" most" lightweight" developed" to" date," causing" minimal" disturbance" to" the" animals;" • The"animals"are"kept"in"as"close"to"their"natural"habitat"as"possible"–"in"hanging"cages"and/or"in" suitable"substrates;" • Once"installed,"the"sensors"are"long8term"maintenance8free,"and"we"have"proved"this"by"having" some" systems" already" running" for" several" years" as" demonstrated" by" the" 2.5" years" record" presented"in"this"report."No"other"system"has"operated"for"several"years"even"in"the"laboratory;" • Clams"clean"their"shells,"and"the"system"is"designed"to"utilise"this"function,"resulting"in"a"built"in" anti8fouling"function;"" • The"data"are"delivered"cable8free,"automatically"streamed"to"a"mobile"phone"line"or"an"internet" socket,"and"uploaded"onto"the"website,"and"automatically"treated,"in"real"time,"where"they"can" be"viewed"continuously;" • The"system"works"with"both"marine"and"freshwater"bivalve"species."To"date"the"team"has"used" the"system"on"blue"and"brown"mussels"(Mytilus*edulis*and*Perna*perna),"giant"tropical"clams" (Hippopus* hippopus)," scallops" (Chlamys* islandica)," oysters" (Crassostrea* gigas)," and" the" Asiatic" clam"(Corbicula*fluminea);"

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• The" system" is" the" only" one" which" allows" for" simultaneously" recordings" of" clam" growth" and" gaping"behaviour;" • The"developed"software"and"algorithm"allows"the"simultaneous"real8time"treatment"of"the"data" produced"by"up"to"16"animals"per"system,"daily"to"quickly"identify"the"key"biological"rhythms"of" the"animal"and"the"environmental"drivers;" • The"system"allows"to"identify"very"precisely,"i.e."up"to"the"minute,"when"animals"died;"" • The"system"allows"to"identify"very"clearly"when"the"animal"is"spawning"and"to"actually"predict" the" spawning" time" with" accuracy" (oysters" and" arctic" scallops)." This" attribute" may" have" an" application"in"commercial"mussel"farms."It"is"a"sign"of"clam"welfare"and"good"water"quality."

6.4 Applicability of the method for MSFD monitoring The"current"set8up"of"the"non8invasive"sensor"system"is"applicable"to"the"monitoring"phase"of"the"MSFD." The"method"is"most"suitable"for"long8term,"inasitu"monitoring"of"changes"in"water"quality"–"not"in"terms" of" actual" values" of" components," but" in" terms" of" arising" disturbances," either" acute" or" gradual." If" the" continuous" data" feeds" do" not" show" any" abnormalities," then" one" may" assume" the" water" quality" is" as" usual" (according" to" previously" measured" levels)." If" there" is" an" abrupt" change," then" it" will" provide" an" early8warning"that"a"change"has"occurred,"and"a"more"detailed"water"quality"measurement"can"be"done," should"this"be"the"appropriate"action."An"alarm"system"would"make"monitoring"of"the"deployed"systems" efficient"in"terms"of"human"effort."

Some"relevant"applications"are"described"below"(in"alphabetical"order):"

Aquaculture!sites."At"aquaculture"sites"where"it"is"not"possible"to"locate"the"cage"groups"in"exposed"or" deep" water," a" form" of" online" monitoring" alerting" to" changes" in" water" quality" can" contribute" to" maintaining" efficient" fish" health" and" thus" growth." Especially" in" areas" where" seasonal" upwelling" is" prominent,"an"early8warning"of"deterioration"in"water"quality,"usually"as"a"result"of"over8enriched"sea8 floor"conditions,"could"in"the"most"extreme"case"prevent"mass"mortalities"due"to"oxygen"depletion"If"the" mussel"sensors"indicate"stressful"conditions,"then"quantitative"water"measurements"can"immediately"be" carried"out."Temporary"transfer"of"cages"to"a"less"exposed"area"could"be"a"remedial"action"from"such"a" warning."

Additionally," as" mentioned" above," the" sensors" allow" the" detection" and" possibly" also" prediction" of" spawning"behaviour,"which"may"have"considerable"application"to"mussel"farms"(using"the"same"species" as"a"bio8sensor"as"is"being"cultured)."Pre8spawned"mussels"are"required"for"human"consumption,"and" thus"the"mussel"sensor"could"contribute"to"determining"optimal"harvesting"times."If"there"is"a"reliable" link"between"the"behavior"of"the"bio8sensors"and"the"culture"organisms,"then"the"system"could"help"the" producer"provide"a"quality"quarantee"(i.e."not"8spawned)"for"the"harvested"produce.""

Bathing!beaches!and!harbors.*Recordings"of"molluscan"gaping"behavior"at"bathing"beaches"or"harbors" will" allow" detection" of" change" in" water" quality." A" working" hypothesis" would" be" that" if" the" mussels" behave"as"normal,"we"may"assume"no"adverse"change"has"occurred."If"a"sudden"change"is"episodic,"it" could"be"linked"with"a"single"event,"but"if"the"aberrant"behavior"persists,"then"quantitative"monitoring"of" water" quality" should" be" implemented." Coupled" with" the" actual" measurements" of" toxic" algal" blooms,"

23" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

" bacterial"content"and"other"contaminants,"this"would"provide"an"efficient"system"to"safeguard"human" safety," even" during" periods" where" daily" physical" measurements" are" not" being" carried" out" by" the" municipality."The"system"would"have"a"further"public"appeal,"because"the"information"is"made"available" on"an"openly"accessible"web"site."

Climate6related!or!other!changes!in!hydrographic!properties:"Changes"in"gaping"behaviour"and"growth" rate"can"be"related"to"climate8induced"stress."For"example,"the"gaping"behaviour"of"the"giant"clam"H.* hippopus"becomes"erratic"at"increased"temperatures"and"solar"irradiance"(Schwartzmann"et"al."2011)."" Moreover,"bivalve" populations" at" the" edge" of" their" thermal" maxima" (temperature"above" which" most" individuals" respond" with" unorganized" locomotion," subjecting" the" animal" to" likely" death)" can" present" massive"mortalities"due"to"effects"of"increased"temperature"(Jonasson"et"al."2004,"Jonasson"et"al."2007" and"references"therein)"and"this"can"be"measured"with"the"HFNI"system"as"explained"above.""

Contaminant! detection.*The"HFNI"system"can"be"used"to"detect"acute"pollution"in"case"of"accidental" contamination," but" it" has" a" specific" added8value" for" the" detection" of" chronic" pollutions" and" “silent”" pollutions"or"transitory"pollutions"which"are"difficult"or"impossible"to"detect"otherwise,"due"to"its"24/7" monitoring" capabilities" over" very" long" periods" of" times." Cumulative" effects" of" toxicity" can" also" be" assessed.""

Petroleum! installations."Produced"water"(the"water"which"is"produced"as"a"by8product"of"oil"and"gas" extraction)is"notoriously"difficult"to"monitor,"due"to"dilution"in"water"currents"etc."Deploying"this"system" at" appropriate" locations" around" oil" and" gas" production" units" will" allow" the" continuous" detection" of" water"quality"at"biologically8relevant"levels."This"would"cover"both"intentional"discharges"but"would"also" give"early8warning"of"unplanned"leakages"to"the"water"column."A"parallel"system"set"up"at"the"sea"floor" would"also"have"an"application"to"detection"of"unintentional"discharges"at"the"sea"floor,"for"example" from"pipelines"or"sub8sea"production"templates."

Toxic!algae!alerts.*In"areas"where"toxic"algae"may"prevent"a"risk"to"human"or"culture"organism"health," remote"bio8sensors"could"function"as"an"early"warning"system"due"to"their"documented"changes"in"the" gaping"behaviour,"in"reaction"to"toxic"alga,"to"alert"when"physical"sampling"is"needed"(Tran"et"al."2010)." This"would"help"to"reduce"the"total"costs"of"algal"monitoring"schemes,"or"supplement"with"data"from" areas" where" routine" physical" sampling" is" not" carried" out." See" also" under" aquaculture" and" bathing" beaches."

6.5 Operational strategy and costs of the system At"present,"the"hardware"and"software"is"not"sold"to"clients;"it"remains"the"property"of"the"specialists," but" the" data" outputs" become" the" property" of" the" client." The" concept" thus" essentially" is" a" service" supplied" on" commission," comprising" the" combined" expertise" of" a" team" comprising" biologists" and" electronics" specialists," mathematicians" from" CNRS" and" the" University" of" Bordeaux" and" Akvaplan8niva" (Norway),"as"required.""

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The" cost" for" one8year" deployment" ranges" from" 40,000" –" 60,000" €," depending" on" installation" requirements" the" number" of" units" required" to" be" in" operation," and" disassembly." The" energy" consumption"of"the"electronics"is"very"low"(1"W),"and"it"can"work"for"up"to"2"years"or"more"without"any" human"intervention."Extending"the"deployment"beyond"one"year"requires"mostly"only"personnel"costs."

The"costs"cover"the"installation"of"the"system,"the"daily"publication"of"the"data"and"metadata"(as"easy"to" read"graphs)"over"a"one"year"period"on"the"MolluSCAN"eye"website"(automatic"update),"the"production" of" a" dedicated" webpage" with" pictures" and" a" text" plus" a" hotline" with" the" specialists." The" volume" of" streamed"data"requires"the"use"of"a"super8computer;"this"is"why"the"data"are"handled"centrally."

The" reduction" of" human" intervention" is" also" a" positive" factor" for" both" safety" and" environmental" concerns." These" advantages" come" handy" when" working" in" fragile" ecosystems" or" under" very" harsh" environmental" conditions" such" as" in" the" Arctic" and" other" extreme" cold" places." Valvometry" is" non8 hazardous,"does"not"need"any"specific"authorization"for"field"implementation"and"is"not"restricted"under" international"legislations."However,"local"restrictions"to"placement"of"scientific"instruments"may"apply."

The"perspective"of"costs"and"cost8efficiency"will"have"a"different"meaning,"depending"on"the"target"user8 group."An"annual"sum"40"–"60"k€"per"annum"may"be"a"very"worthwhile"investment"for"a"larger"industry" where" very" large" financial" resources" depend" on" maintaining" a" stable" water" quality," or" where" timely" intervention"thanks"to"an"early"warning"system"could"prevent"far"greater"losses."For"such"purposes"the" 'cost"of"doing'"may"be"far"less"than"the"potential"'cost"of"not"doing."

Smaller"enterprises"may"not"be"able"to"justify"the"costs"for"such"technology"and"data"processing."There" are"other,"less"expensive"technology"available"on"the"open"market."These"are,"however,"based"on"less" advanced" technology" and" do" not" have" real8time" data" feeds" (the" data" usually" need" to" be" periodically" downloaded"using"a"cable)."Further,"such"systems"often"need"up"to"weekly"maintenance"(which"in"areas" with"difficult"environmental"conditions,"such"the"Artic,"this"may"not"be"possible),"including"cleaning"of" the" sensors," and" the" organisms" typically" need" to" be" replaced" after" a" period" of" several" months." Such" regular"handling"may"or"may"not"affect"the"organisms,"and"the"maintenance"commitments"mean"that" the"system"is"restricted"to"operations"in"areas"under"regular"scrutiny."This"type"of"simpler"system"may" be"more"suitable"for"smaller,"short8term"applications,"but"the"current"technology"excels"for"larger8scale" and/or" long8term" use" and" where" a" maintenance8free" sensor" is" needed." Thus" operational" limitations" need"to"be"weighed"against"financial"limitations."

During" the" present" phase" of" testing" and" determining" the" applicability" of" these" high" frequency," non8 invasive"biosensors"as"a"potential"new"innovative"tool"for"monitoring,"commercialisation"plans"are"still" under"development,"but"the"system"is"being"considered"for"use"by"some"industries,"and"we"propose"the" method"as"also"being"highly"applicable"for"MSFD"long8term"monitoring"of"changes"in"water"quality"(see" Sow"et"al.,"2011).""

"

25" Deliverable"5.3"Report"on"the"application"of"non8invasive"biosensors"in"bivalves"

"

6.6 Outreach The"activities"developed"during"the"project"and"results"achieved"so"far"have"been"disseminated"though" exhibitions,"social"media,"newspaper"articles,"student"thesis"and"researcher"presentations."In"Tromsø," Norway,"the"local"aquarium"(www.polaria.no)"is"hosting"an"exhibition"that"has"been"running"since"the" 18th"of"November"2012"with"the"following"items:"

•" A"tank"with"scallops"connected"to"a"biosensor"device" •" TV"screens"with"tutorial"presentations"in"3"languages"(Norwegian,"English"and"French)." •" Four"high"quality"posters"covering"sclerochronology"studies"using"bivalves"" •" Samples"of"bivalves"that"scientist"use"for"research"purposes" It"is"estimated"that"about"7,200"people"visited"the"exhibition"between"20128"2013.""During"the"opening," the"local"newspaper""Tromsø""and"the"national"newspaper""Aftenposten""covered"the"event."A"similar" exhibition"was"hosted"from"the"21.06826.06.2014"in"the"festival""Science"in"the"City","Carlsberg"Byen," Denmark" (http://scienceinthecity.dk/en/event/arctic8knowhow8talking8clams84" )" which" received" about" 5,000" visitors." Social" media" outreach" includes" the" creation" of" a" web" site" on" Facebook:" (www.facebook.com/TalkingClams)" which" can" redirect" the" public" to" live" data" from" the" Ny" Ålesund" deployment"at:"" http://molluscan8eye.epoc.u8 bordeaux1.fr/index.php?rubrique=enregistrement&lang=en&site=NYALESUND""

On" the" website" http://molluscan8eye.epoc.u8bordeaux1.fr/" data" regarding" gaping" activity" of" oysters," blue" mussels" and" other" scallops" deployed" at" several" European" localities" can" be" seen." This" data" is" processed"on"a"daily"basis"and"published"on"the"website"after"one"day.""

7. Conclusions and future directions In" conclusion," the" non8invasive" biosensor" system" presented" here" has" continuously" recorded" valve" gaping"and"shell"growth"over"a"period"of"2.5"years."Both"the"growth"and"the"behaviour"patterns"were" strongly"linked"to"environmental"cycles"such"as"photoperiod"(light"conditions)"and"productivity,"both"of" which"are"strong"environmental"drivers"in"the"Arctic,"as"well"as"tidal"cycles."Because"this"system"was" tested"in"a"pristine"environment,"the"aim"of"confirming"the"strong"relationships"between"behaviour"and" growth"with"environmental"factors"was"achieved."We"have"successfully"tested"the"current"system"in"a" challenging"Arctic"environment,"and"supplemented"with"the"experiences"elsewhere"across"Europe,"are" confident"of"high"success"in"deployment"in"almost"any"other"environment."

We"have"identified"a"number"of"potential"uses"of"the"system"for"monitoring"and"early8warning"alerts"of" changes" in" water" quality," such" as" temperature," releases" of" contaminants" to" toxic" algal" blooms," all" of" which"are"documented"experimentally,"and"most"also"in"the"field."Potential"relevant"applications"for"the" MSFD"include"bathing"beaches"and"harbours,"petroleum"installations"and"aquaculture"sites."""

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This"study"will"continue"for"the"remaining"of"the"DEVOTES"project."Upcoming"activities"include"a"March" 2015"visit"to"Ny"Ålesund"to"retrieve"the"biosensor"and"to"update"the"HFNI"valve"system."This"will"allow" us"to"install"new"generation"technology"which"is"more"energy"efficient"with"improved"communication" capabilities."A"final"DEVOTES"peer8reviewed"publication"will"contain"a"multiyear"analysis"of"gaping"data" for" this" site,"and"compared"with"data"from"two"other" sites" in" the" Barents" Sea" and/or" elsewhere," as" appropriate."Further,"we"will"prepare"an"application"to"the"call"for"proposals"for"Infrastructure"for"the" Svalbard"Integrated"Arctic"Earth"Observing"System"(SIOS)"in"late"Autumn,"2014."See"http://www.sios8 svalbard.org."

8. References Berge"J,"Båtnes"AS,"Johnsen"G,"Blackwell"SM,"Moline"MA"(2012)"Bioluminescence"in"the"high"Arctic"during"the"polar" night."Marine"Biology"159:2318237"

Beuchel" F," Gulliksen" B" (2008)" Temporal" patterns" of" benthic" community" development" in" an" Arctic" fjord" (Kongsfjorden,"Svalbard):"results"of"a"248year"manipulation"study."Polar"Biology"31:9138924"

Blicher" M," Rysgaard" S," Sejr" M" (2010)." Seasonal" growth" variation" in" Chlamys* islandica" (Bivalvia)" from" sub8Arctic" Greenland"is"linked"to"food"availability"and"temperature."Marine"Ecology"Progress"Series"407:"71886"

Brand" AR" (2006)" Chapter" 12" Scallop" ecology:" Distributions" and" behaviour." In:" Sandra" ES," Parsons" GJ" (eds)" Developments"in"Aquaculture"and"Fisheries"Science,"Book"Volume"35."Elsevier""

Chambon,"C.,"Legeay,"A.,"Durrieu,"G.,"Gonzalez,"P."Ciret,"P.,"Massabuau,"JC."(2007)"Influence"of"the"parasite"worm" Polydora" sp." on" the" behaviour" of" the" oyster" Crassostrea" gigas:" a" study" of" the" respiratory" impact" and" associated"oxidative"stress."Mar."Biol."152:"3298338"

Crain"CM,"Kroeker"K,"Halpern"BS"(2008)"Interactive"and"cumulative"effects"of"multiple"human"stressors"in"marine" systems."Ecol"Lett"11:130481315"

"Fournier," E.," Tran," D.," Denison," F.," Massabuau," J.8C." and" J" Garnier8Laplace" (2004)." Valve" closure" response" to" uranium" exposure" for" a" freshwater" bivalve" Corbicula" fluminea" :" quantification" of" the" influence" of" pH." Envir."Tox."Chem"23:110881111"

Garcia8March" JR," Solsona" MAS," Garcia8Carrascosa" AM" (2008)" Shell" gaping" behaviour" of" Pinna" nobilis" L.," 1758:" circadian"and"circalunar"rhythms"revealed"by"in"situ"monitoring."Marine"Biology"153:6898698"

Garcia"EG"(2006)"The"fishery"for"Iceland"scallop"(Chlamys"islandica)"in"the"Northeast"Atlantic."In:"Southward"AJ," Sims" DW" (eds)" Advances" in" Marine" Biology," Vol" 51," Book" 51." Academic" Press" Ltd8Elsevier" Science" Ltd," London""

Haberkorn," H.," Tran," D.," Massabuau," J.C.," Ciret," P.," Soudant," P." (2011)" Relationship" between" valve" activity," microalgae"concentration"in"the"water"and"toxin"accumulation"in"the"digestive"gland"of"the"Pacific"oyster" Crassostrea*gigas"exposed"to"Alexandrium*minutum."Mar."Poll."Bull."62:"119181197"

Hop"H,"Pearson"T,"Hegseth"EN,"Kovacs"KM,"Wiencke"C,"Kwasniewski"S,"Eiane"K,"Mehlum"F,"Gulliksen"B,"Wlodarska8 Kowalczuk"M,"Lydersen"C,"Weslawski"JM,"Cochrane"S,"Gabrielsen"GW,"Leakey"RJG,"Lonne"OJ,"Zajaczkowski" M," Falk8Petersen" S," Kendall" M," Wangberg" SA," Bischof" K," Voronkov" AY," Kovaltchouk" NA," Wiktor" J," Poltermann"M,"di"Prisco"G,"Papucci"C,"Gerland"S"(2002)"The"marine"ecosystem"of"Kongsfjorden,"Svalbard." Polar"Res"21:1678208"

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Kuklinski"P,"Berge"J,"McFadden"L,"Dmoch"K,"Zajaczkowski"M,"Nygard"H,"Piwosz"K,"Tatarek"A"(2013)"Seasonality"of" occurrence" and" recruitment" of" Arctic" marine" benthic" invertebrate" larvae" in" relation" to" environmental" variables."Polar"Biology"36:5498560"

Johnsen"G,"Candeloro"M,"Berge"J,"Moline"M"(2014)"Glowing"in"the"dark:"discriminating"patterns"of"bioluminescence" from"different"taxa"during"the"Arctic"polar"night."Polar"Biology"37:7078713"

Jonasson" JP," Thorarinsdottir" GG," Eiriksson" H," Marteinsdottir" G" (2004)" Temperature" tolerance" of" Iceland" scallop," Chlamys"islandica"(OF"Muller)"under"controlled"experimental"conditions."Aquac"Res"35:140581414"

Jonasson" JP," Thorarinsdottir" G," Eiriksson" H," Solmundsson" J," Marteinsdottir" G" (2007)" Collapse" of" the" fishery" for" Iceland"scallop"(Chlamys"islandica)"in"Breidafjordur,"West"Iceland."ICES"J"Mar"Sci"64:2988308""

Mat,"A,"Haberkorn"H,"JP"Bourdineaud,"Massabuau"JC,"Tran"D"(2013)"Genetic"and"genotoxic"impacts"in"the"oyster" Crassostrea"gigas"exposed"to"the"harmful"alga"Alexandrium"minutum."Aquatic"Toxicology."1408141:"4588 465"

Micheli"F,"Halpern"BS,"Walbridge"S,"Ciriaco"S,"Ferretti"F,"Fraschetti"S,"Lewison"R,"Nykjaer"L,"Rosenberg"AA"(2013)" Cumulative" Human" Impacts" on" Mediterranean" and" Black" Sea" Marine" Ecosystems:" Assessing" Current" Pressures"and"Opportunities."PLoS"One"8"

Nahrgang"J,"Brooks"SJ,"Evenset"A,"Camus"L,"Jonsson"M,"Smith"TJ,"Lukina"J,"Frantzen"M,"Giarratano"E,"Renaud"PE" (2013)" Seasonal" variation" in" biomarkers" in" blue" mussel" (Mytilus" edulis)," Icelandic" scallop" (Chlamys" islandica)" and" Atlantic" cod" (Gadus" morhua)—Implications" for" environmental" monitoring" in" the" Barents" Sea."Aquatic"Toxicology"127:21835"

Picot"S"(2013)"Etude"chronobiologique"du"comportement"valvaire"du"pecten"arctique,"Chlamys"islandica"implanté" in" situ" à" Ny8Alesund," Svalbard." MSc" Sciences" biologiques" marines," Université" de" Bretagne" occidentale," France."

Schwartzmann" C," Durrieu" G," Sow" M," Ciret" P," Lazareth" CE," Massabuau" JC" (2011)" In" situ" giant" clam" growth" rate" behavior"in"relation"to"temperature:"A"one8year"coupled"study"of"high8frequency"noninvasive"valvometry" and"sclerochronology."Limnol"Oceanogr"56:194081951""

Sow"M;"G"Durrieu,"L"Briollais,"P"Ciret;"JC"Massabuau"(2011)"Water"quality"assessment"by"means"of"valvometry"and" high8frequency"data"modeling."Envir"Monitor"Assess."182:1558170Strand"Ø,"Parsons"GJ"(2006)"Chapter"21" Scandinavia." In:" Sandra" ES," Parsons" GJ" (eds)" Developments" in" Aquaculture" and" Fisheries" Science," Book" Volume"35."Elsevier"

Svendsen"H,"Beszczynska8Moller"A,"Hagen"JO,"Lefauconnier"B,"Tverberg"V,"Gerland"S,"Orbaek"JB,"Bischof"K,"Papucci" C," Zajaczkowski" M," Azzolini" R," Bruland" O," Wiencke" C," Winther" JG," Dallmann" W" (2002)" The" physical" environment"of"Kongsfjorden8Krossfjorden,"an"Arctic"fjord"system"in"Svalbard."Polar"Res"21:1338166"

Tran"D,"Nadau"A,"Durrieu"G,"Ciret"P,"Parisot"JP,"Massabuau"JC"(2011)"Field"Chronobiology"of"a"Molluscan"Bivalve:" How" the" Moon" and" Sun" Cycles" Interact" to" Drive" Oyster" Activity" Rhythms." Chronobiology" International" 28:3078317"

Tran" D," Ciret" P," Ciutat" A," Durrieu" G," Massabuau" JC" (2003)" Estimation" of" potential" and" limits" of" bivalve" closure" response"to"detect"contaminants:"Application"to"cadmium."Environ"Toxicol"Chem"22:9148920"

Tran"D,"Fournier"E,"Durrieu"G,"Massabuau"J8C"(2007)"Inorganic"Mercury"Detection"By"Valve"Closure"Response"in"the" Freshwater" Clam" Corbicula" Fluminea:" Integration" of" Time" and" Water" Metal" Concentration" Changes." Environ"Toxicol"Chem"26:154581551""

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Tran,"D.,"Haberkorn,"H.,"Soudant,"P.,"Ciret,"P."and"Massabuau,"J.C."(2010)"Behavioral"responses"of"Crassostrea*gigas" exposed"to"the"harmful"algae"Alexandrium"minutum."Aquaculture.""298:"3388345"

Tran,"D.,"Massabuau"J8C""and"J"Garnier8Laplace."(2003)."The"effect"of"carbon"dioxide"on"uranium"bioaccumulation" in"the"freshwater"clam"Corbicula"fluminea"Envir."Tox."Chem."23:"7398747"Wassmann"P,"Reigstad"M"(2011)" Future" Arctic" Ocean" Seasonal" Ice" Zones" and" Implications" for" Pelagic8Benthic" Coupling." Oceanography" 24:2208231"

Wlodarska8Kowalczuk"M,"Pearson"TH,"Kendall"MA"(2005)"Benthic"response"to"chronic"natural"physical"disturbance" by"glacial"sedimentation"in"an"Arctic"fjord."Marine"Ecology"Progress"Series"303:31841"

Website"links:"

DEVOTES"link"project"website:"http://devotes8project.eu/" MEECE"link"project"website:"http://www.meece.eu/" MEECE"Atlas"link:"http://www.meeceatlas.eu/Menu/" MolluSCAN"eye"website:"http://molluscan8eye.epoc.u8bordeaux1.fr/index.php?rubrique=accueil&lang=en" "

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