2  

Tak  til…  .........................................................................................................................................................  4  

Forord  ............................................................................................................................................................  5  

Introduktion  ................................................................................................................................................  6  

Tungmetaller  i  det  arktiske  Grønland  ..........................................................................................................  7  Den  arktiske  tidevandszone  .............................................................................................................................  9  Blåmuslingen  Mytilus  edulis  ..........................................................................................................................  12  Blåmuslingens  økologi  og  biogeografi  ......................................................................................................................  12  Blåmuslingen  som  miljøindikator  ...............................................................................................................................  13  Blåmuslingens  kuldetolerance  .....................................................................................................................................  14  Blykontaminerede  blåmuslinger  i  Arktis  .................................................................................................................  15  

Cellemembraner  hos  ektotermer  stresses  af  kulde  og  bly  .................................................................  16  Cellemembranen  .................................................................................................................................................................  16  Cellemembranens  temperatursensitivitet  ..............................................................................................................  18  Kulde  stresser  membranen  hos  ektoterme  organismer  ...................................................................................  18  Peroxidering  af  cellemembranen  ................................................................................................................................  19  Bly  øger  peroxidering  og  stresser  membranen  ....................................................................................................  20  

Synergetisk  stressinteraktion  mellem  kulde  og  tungmetal  ...............................................................  21  Aktuelt  studie  .....................................................................................................................................................  23  

Manuscript  ................................................................................................................................................  27  

Abstract  ................................................................................................................................................................  29  1.  Introduction  ....................................................................................................................................................  30  2.  Materials  and  Methods  ................................................................................................................................  33  2.1  Animals  and  holding  conditions  ...........................................................................................................................  33  2.2  Experimental  design  ..................................................................................................................................................  33  2.3  Pb  analysis  .....................................................................................................................................................................  34  2.4  PLFA  analysis  ................................................................................................................................................................  34  2.5  Statistical  analysis  ......................................................................................................................................................  35  

3.  Results  ..............................................................................................................................................................  35  3.1  Pb  content  in  mussels  ...............................................................................................................................  35  3.2  Effects  of  Pb  on  PLFAs  ..............................................................................................................................................  36  3.3  Effects  of  sub-­‐zero  temperatures  and  Pb  on  survival  .................................................................................  37  

4.  Discussion  .......................................................................................................................................................  39  4.1  Internal  lead  ..................................................................................................................................................................  39  

  3  

4.2  Pb  effect  on  PLFA  composition  and  survival  ..................................................................................................  40  4.3  Implications  for  risk-­‐assessments  .......................................................................................................................  41  

Acknowledgements  ..........................................................................................................................................  42  References  ...........................................................................................................................................................  43  

  4  

Tak til… Jeg vil gerne takke min vejleder, Mikael Kristian Sejr, Seniorforsker ved Arktisk

Forskningscenter, Institut for Bioscience, Aarhus Universitet, for god og brugbar vejledning og en

stor villighed til at give mig de bedste rammer for mit speciale. Jeg vil også takke Jakob Thyrring,

Ph.d. studerende ved Arktisk Forskningscenter, Institut for Bioscience, Aarhus Universitet, som har

været medvejleder og en stabil, hjælpsom og inspirerende støtte og samarbejdspartner igennem hele

projektet. Endvidere vil jeg takke Martin Holmstrup, Professor ved Jordfaunaøkologi og

Økotoksikologi, Institut for Bioscience, Aarhus Universitet, for vejledning og hjælp til

gennemførelse af og forståelse for laboratoriearbejdet. I denne forbindelse vil jeg også takke Lise

Lauridsen, Stine Slotsbo og Ana Silva Patricio, som ligeledes har været meget behjælpelige og

imødekommende i forbindelse med mit laboratoriearbejde. Jeg vil gerne takke Matilde Rasmussen

og Siri Elmegaard for inspirerende feedback og mine kontormakkere Ane Middelbo og Sofie

Mathiessen for mange gode timer på kontoret. Afslutningsvis takker jeg Bo Michelsen samt familie

og venner for støtte og opmuntring igennem hele processen.

  5  

Forord Hypotesen i dette specialestudie er, at der er synergi imellem effekterne af bly i økologisk

realistiske koncentrationer og realistisk temperatur på blåmuslingens kuldetolerance i Arktis. Som

mulig mekanistisk forklaring herpå undersøges det, hvorvidt den potentielle synergetiske

interaktion på kuldetolerancen skyldes, at bly resulterer i membranskader via ændringer i

cellemembranens fedtsyrekomposition. Specialet består af en introduktion til emnet, hvor den

teoretiske baggrund for hypotesen gennemgås. Efterfølgende forefindes manuskript til en artikel

omhandlende selve studiet med henblik på udgivelse i det videnskabelige tidsskrift Aquatic

Toxicology.

Introduktionen giver først et overblik over den grønlandske minehistorie og hvordan

tungmetalforurening, såsom bly, i visse marine områder udgør en risiko for den arktiske flora og

fauna. Herefter følger en kort beskrivelse af den arktiske tidevandszone, hvor arktiske organismer

udsættes for ekstrem kulde. Det tredje afsnit omhandler specialets forsøgsorganisme, blåmuslingen,

som er en dominerende art i den arktiske tidevandszone og en hyppigt anvendt marin miljøindikator

for tungmetaller. Fjerde afsnit er et teoretisk overblik over cellemembranens fysiske og fysiologiske

egenskaber og vil belyse hvorledes denne stresses af både kulde og tungmetal. Dette afsnit danner

således baggrund for vores hypotese om den mulige synergetiske stresseffekt af disse to faktorer på

blåmuslingens kuldetolerance. Afslutningsvis opsummeres det teoretiske ræsonnement for det

aktuelle studie, som manuskriptet omhandler.

  6  

Introduktion

Test  af  mulige  synergetiske  

interaktioner  mellem  frysegrader  og  bly  (Pb)  kontaminering  i  grønlandske  blåmuslinger    

(Mytilus  edulis)  

 

 

   

  7  

Tungmetaller  i  det  arktiske  Grønland   Tidligere blev havet opfattet som et enormt reservoir for sikker bortskaffelse af forurenende

stoffer såsom tungmetaller (Ansari et al. 2004, Torres et al. 2008). I dag er marin forurening med

tungmetaller af antropogen oprindelse en global bekymring (AMAP 2005, Reichelt-Brushett 2012).

Dette skyldes tungmetallers bioakkumulering i, og mulige toksiske effekter på, marine organismer

(Josefson et al. 2008, Torres et al. 2008, Søndergaard 2013). Den primære tungmetalforurening af

Arktis sker via atmosfæren. Dette, samt minedriften i Arktis, har i de seneste årtier medført en

tiltagende bekymring for forurening af det arktiske marine økosystem (AMAP 2010). I Arktis har

cadmium (Cd) og kviksølv (Hg) størst økotoksikologisk betydning. Disse metaller findes i så høje

koncentrationer i arktisk flora og fauna, at det har sundhedsmæssige konsekvenser for organismerne

selv, men også for de mennesker der indtager dem (AMAP 2005). Endvidere er også bly fortsat en

kilde til bekymring i Arktis på trods af, at det atmosfæriske indhold af bly er faldet signifikant efter

forbuddet imod blyholdigt benzin i den vestlige verden (AMAP 2005). Særligt i visse marine

områder nær minedrift findes der fortsat høje blykoncentrationer, hvilket uddybes nedenfor.

Minedrift har fundet sted i Grønland siden 1840’erne og landet er fortsat særdeles rigt på mange

ressourcer såsom petroleum, gas, olie og forskellige mineraler, eksempelvis bly (Pb), zink (Zn), jern

(Fe), kobber (Cu) og guld (Au) (Figur 1) (Rosing et al. 2014). Vedtagelsen af Grønlands

Selvstyrelov i 2009 betød, at Grønland fik rettighederne over landets ressourcerigdom, inklusive

underjordiske mineralressourcer. Dette har medført en politisk og økonomisk vilje til i langt højere

grad at udnytte ressourcerne ved blandt andet minedrift, så landet kan opnå sin målsætning om at

blive en selvstændig økonomi (Rosing et al. 2014). Eksempelvis har Grønland i de seneste år solgt

et stigende antal minerettigheder til udenlandske minedriftskompagnier (Nuttall 2014) og minedrift

forventes at bidrage betydeligt til den grønlandske økonomi allerede indenfor de næste 5-10 år

(Rosing et al. 2014).

  8  

 Figur   1   viser   de   størst   kendte  tungmetal-­‐   og   mineralreserver   i  Grønland.  Man   forventer   en  markant  øget   minedrift   på   mineraler   i  Grønland   de   kommende   år.   Efter  Rosing  et  al.  (2014).  

  9  

Udledning af mineaffald har betydet en markant tungmetalforurening af nogle grønlandske

fjorde. Den forhenværende bly- og zinkmine Black Angel i Maarmorilik, Vestgrønland, lukkede

årligt 500.000 tons mineaffald direkte ud i det omkringliggende fjordsystem indtil den lukkede i

1990 efter 17 års drift (Loring & Asmund 1989b). Da minedriften var på sit højeste, målte man

blyindhold i blåmuslinger (Mytilus edulis) på op til 800 µg/g (baggrundsindholdet var 3 µg/g) og

man observerede bly- og zinkkontaminerede organismer op til 50 km fra minen (Loring & Asmund

1989b). Regelmæssig monitering fra minens åbning og frem til 2009 har vist, at koncentrationen af

opløst bly og zink i havvandet faldt kraftigt ved minens lukning (0,1% i 1995 i forhold til 1988

(Schiedek et al. 2009)). Dog er indholdet af bly og zink i fjordsystemets organismer og sediment

stadig kraftigt forhøjet 20 år efter minens lukning og dette forhøjede indhold ses op til 12 km fra

minen (Søndergaard et al. 2011). Man har således for nylig fundet blåmuslinger ved Maarmorilik

med blyindhold på op imod 3000 µg/g (Bach, 2014, pers. komm.) og man forventer at se forhøjede

niveauer af zink og bly i fjordsystemets planter og dyr de næste 50 år (Søndergaard et al. 2011).

Rettighederne til Black Angel minen blev solgt i 2008 og bly-, zink- og sølvudvinding forventes

genoptaget inden for de næste par år (Råstofstyrelsen 2013).

Lignende eksempler på tungmetalforurening af grønlandske fjorde fra minedrift er fra en

olivinmine i Fiskefjorden i Sydvestgrønland (Søndergaard 2013) samt tungmetaludledning fra en

kryolitmine i Ivittuut i Sydgrønland. I sidstnævnte eksempel måltes i 2007, 20 år efter

kryolitminens lukning, et blyindhold i blåmuslinger på op til 200-500 gange baggrundsniveau (0,7-

1,7 µg/g) (Johansen et al. 2008b). Tungmetalforurening har således langvarige konsekvenser for

den arktiske flora og fauna i Grønland og dette kan forblive et økologisk problem mange år endnu -

både på grund af bioakkumulering i arktiske organismer og på grund af den forventede øgede

mineaktivitet i Grønland.

Den  arktiske  tidevandszone   Arktis kan defineres som området nord for polarcirklen (66°32′ N), men i denne introduktion

defineres Arktis ud fra en isoterm med en maksimal gennemsnitlig lufttemperatur på 10°C i årets

varmeste måned, hvilket økologisk set svarer til de 4,8% af jordens overflade nord for trægrænsen

(Figur 2) (AMAP 1998, CAFF 2013).

  10  

Arktis rummer nogle af de mest ekstreme

habitater på jorden kendetegnet ved

permanent eller midlertidigt isdække samt

op til 6 måneders kontinuerligt vintermørke,

hvor primærproduktionen stort set ophører

(Sejr, pers. komm.). Temperaturen kan

komme ned på -25°C i vintermånederne, og

der er store temperatursvingninger både

dagligt og over året (Blicher et al. 2013).

Der er endvidere store stedslige variationer i

temperaturen imellem de enkelte

mikrohabitater. Dette fremgår eksempelvis

af figur 3, som viser, at   alger   har   en  

markant   isolerende   effekt   relativt   til  

eksponerede   klipper   i   både   den   øvre   og  

midterste   tidevandszone   (Sejr et al.,

upubliceret).

Figur   2   viser   forskellige   definitioner   på   Arktis.   Den  stiplede   blå   linie   angiver   polarcirklen,   imens   den   røde  linie  angiver  en  isoterm  med  en  maksimal  gennemsnitlig  lufttemperatur  på  10°C  i  årets  varmeste  måned.  Når  der  i  denne  introduktion  tales  om  Arktis  menes  der  området  nord  for  denne  isoterm.  

Figur  3  viser  temperaturen  i  tidevandszonen  over  året  2011-­‐2012  i  Nuuk,  Grønland.  Temperaturen  vises   i   (A)  den  øvre  tidevandszone,  (B)  den  midterste  tidevandszone  og   (C)  den  nedre   tidevandszone.  Temperaturen  vises   både  på  eksponerede  klipper  (rød  graf)  og  under  alger  (sort  graf).  Efter  Sejr  et  al.  upubliceret.  

  11  

Tidevandszonen er den del af kysten, der skiftevis dækkes og blotlægges af havvand som følge

af tidevandets cyklus (Figur 4). I økotoksikologisk sammenhæng er tidevandszonen interessant, da

denne zone ofte er særligt udsat for antropogen forurening (eksempelvis olieudslip og minedrift).

Desuden giver zonen let adgang til indsamling af marine organismer til økotoksikologiske studier.

Blotlægningen af havbunden i tidevandszonen eksponerer (semi)-sessile organismer for luft. I

Arktis betyder det, at zonens organismer skal kunne klare lufttemperaturer ned til -25°C (Blicher et

al. 2013), hvorfor lufttemperatur er vigtigt for hyppigheden og distributionen af organismer i den

arktiske tidevandszone. Endvidere er andre abiotiske stresspåvirkninger som eksempelvis bølger og

is også af betydning herfor (Aarset 1982, Aunaas et al. 1988). Scrosati og Eckersley (2007)

undersøgte kanten af landisens, den såkaldte ”isfods”, effekt i tidevandszonen i St. Lawrence

Bugten i det østlige Canada, der har lufttemperaturer tilsvarende i Arktis. Studiet viste, at isfoden

har en isolerende effekt, da lufttemperaturer ned til -20°C aldrig gav en lavere temperatur under

isfoden end -7°C. Studiet indikerer, at isfoden yder langsigtet beskyttelse til zonens organismer

imod meget lave frysegrader (Scrosati & Eckersley 2007).

Figur  4  viser  en  skematisk  oversigt  over  tidevandszonen  (”intertidal”)  og  de  zoner,  der  grænser  op  her  til.  

  12  

Blåmuslingen  Mytilus  edulis   Organismer i tidevandszonen er som

nævnt interessante i økotoksikologiske

studier, da tidevandszonen ofte er særligt

udsat for forurening. I sådanne studier

anvendes blåmuslingen (Mytilus edulis)

ofte, da den er en ideel miljøindikator

(Figur 5), hvilket blandt andet vil blive

uddybet i dette afsnit.

Blåmuslingen M. edulis er en del af et

artskompleks med Mytilus trossulus og

Mytilus galloprovincialis. Arterne er

morfologisk set ens, men de adskiller sig

genetisk (Mcdonald & Koehn 1988, Westfall & Gardner 2010). I Grønland er der kun identificeret

M. edulis (Wanamaker et al. 2007, Versteegh et al. 2012). Når ”blåmuslingen” i denne introduktion

benævnes, refereres der til M. edulis, med mindre andet er angivet.

Blåmuslingens  økologi  og  biogeografi  

Blåmuslingen er en bentisk, semisessil filtrator (Bayne et al. 1976, Seed 1976, Gosling 2003).

Arten kan tilpasse sig relativt store fluktuationer i abiotiske forhold (som temperatur, salinitet,

udtørring og ilt) og kan derfor leve i en bred vifte af habitater (Seed & Suchanek 1992).

Eksempelvis findes M. edulis fra tidevandszonen og ned til 40 m, i både brakvand og havvand samt

under både milde og ekstreme temperaturforhold (Seed & Suchanek 1992)(FAO 2014a). Den er

fremherskende på stejle, bølgeudsatte kyster og dominerer både i tidevandszonen og kystzonen

(”subtidal” på figur 4). I den arktiske tidevandszone er lufttemperatur samt lokal fysisk stress fra

skurrende isflager afgørende for blåmuslingens øvre udbredelsesgrænse (Gosling 2003, Blicher et

al. 2013). Arten danner store banker ved hjælp af fasthæftning med byssustråde til substrat og til

hinanden. Blåmuslingen er økologisk set en vigtig art, da den danner fødegrundlag for mange

organismer såsom fugle, fisk og invertebrater. De tætte muslingebanker fungerer endvidere som

habitat for et artsrigt alge- og dyresamfund (Seed 1976, Seed & Suchanek 1992). Blåmuslingen er

desuden af stor kommerciel betydning (FAO 2014b).

Figur   5.   Blåmuslinger   vokser   i   tætte   banker   og   filtrerer  vandet  for  fødepartikler  med  gællerne.  

  13  

Temperatur er afgørende for blåmuslingens biogeografi og artens nordlige grænse afgøres af

minimum lufttemperatur, hvilket eventuelt kan modificeres af en isfod (Seed 1976, Scrosati &

Eckersley 2007, Jones et al. 2010). Blåmuslingen er særligt udbredt på den nordlige halvkugle

(Seed & Suchanek 1992) og den findes i arktiske områder i Norge, Rusland, Canada og Grønland i

både tidevandszonen og kystzonen (Figur 4) (Blicher et al. 2013). I de seneste år er blåmuslingen

endvidere blevet observeret på både Svalbard og i Qaanaaq i det nordvestlige Grønland. Hvorvidt

muslingerne gyder i disse områder eller fragtes hertil i larvestadiet med havstrømme vides endnu

ikke (AMAP 2012, Thyrring, pers. komm.).

Blåmuslingen  som  miljøindikator  

Organismers optag af tungmetaller er oftest proportionelt med mængden af metallet i

omgivelserne (Livingstone & Pipe 1992). En miljøindikator for tungmetal kan defineres som en art,

der akkumulerer tungmetaller i sit væv. Herved fås et mål for biotilgængelighed af tungmetal i det

pågældende miljø, det vil sige hvor stor en andel af den totale tungmetalmængde, der er tilgængelig

for dyr og planter og således økotoksikologisk relevant (Rainbow 1995). Dette mål afhænger af

miljøindikatorens optagelses- og udskillelsesrate af et pågældende metal (Søndergaard et al. 2014).

Miljøindikatorer kan således anvendes til at analysere geografiske og temporale variationer i

biotilgængeligheden af tungmetal i marine økosystemer (Rainbow 1995).

Udover at opfylde kravet om akkumulering af tungmetaller i vævet, så er ideelle miljøindikatorer

sessile, hyppigt forekommende, nemme at identificere, robuste overfor skiftende abiotiske forhold

og de har en relativt lang levetid (Søndergaard et al. 2014). Blåmuslingen lever oftest op til de fleste

af disse kriterier. Som filtrator optager blåmuslingen både tungmetal opløst i vandet og bundet til

partikulært materiale (Rainbow 1995), hvilket gør blåmuslingen særligt attraktiv til risikovurdering

af tungmetaller i akvatiske miljøer, også af bly (Schulz-Baldes 1974, Phillips 1976, Simpson 1979,

Bourgoin 1990, Widdows & Donkin 1992, Mubiana et al. 2005). Blåmuslingen er særdeles velegnet

til risikovurdering af kystnære områder nær minedrift, da organismen er en dominerende art langs

klippekyster (Rainbow 1995), og den har da også hyppigt været anvendt til dette formål i Grønland

(Loring & Asmund 1989b, Riget et al. 1997, Johansen et al. 2008b, Søndergaard et al. 2011,

Zimmer et al. 2011, Søndergaard 2013, Søndergaard et al. 2014).

  14  

Blåmuslingens  kuldetolerance  

Blåmuslingen er en af de mest kuldetolerante organismer i den arktiske tidevandszone (Aarset

1982). Kuldestress og -mortalitet er normalt i den arktiske region, og kuldetolerance er således af

afgørende betydning for blåmuslingens fitness og distribution (Aarset 1982, Bourget 1983). Når

blåmuslingen udsættes for kold luft i tidevandszonen lukkes skallen i, hjerteraten og cilieaktiviteten

nedsættes og anaerob metabolisme indtræffer (Aarset 1982). Blåmuslingens kuldetolerance består i,

at den tåler frysning af ekstracellulærvæsken, ligesom mange andre kuldetolerante organismer

(Aarset 1982). Supercooling, en proces hvor en væske holdes flydende under dets frysepunkt, er

ifølge ældre studier ikke observeret hos blåmuslingen (Williams 1970, Aarset 1982), men det er for

nylig observeret, at blåmuslinger først fryser ved -5 til -8°C afhængig af størrelse (Sejr, upub. data).

Så længe cellerne ikke fryser intracellulært, kan de potentielt overleve perioder med lave

frysegrader (Mazur 1977, Odintsova & Boroda 2012). Når ekstracellulærvæsken fryser, mindskes

molfraktionen af frit vand, og intracellulært vand bevæger sig da ud i det ekstracellulære rum ved

osmose, hvilket fører til cellulær dehydrering (Aarset 1982). Cellernes kuldetolerance afgøres af en

maksimal tolerance overfor dehydrering og/eller et minimum tolereret cellevolumen. Williams

(1970) har påvist, at blåmuslingen kan overleve frysning af op til 64% af dens kropsvæske.

Kuldeskade indtræffer således, når en vis mængde af kropsvæsken fryser (Williams 1970, Aarset

1982). En del af forklaringen herpå kan være, at intracellulære proteiner ofte er hydrerede, hvilket

stabiliserer proteinstrukturen. Ved cellulær dehydrering kan det bundne vand ekstraheres ved

osmose, hvormed proteinerne kan denaturere med kronisk celleskade som følge. Dehydrering af

cellen kan desuden medføre membranskader (Karow & Webb 1965, Meryman 1971). Irreversible

kuldeskader ved kuldestress kan resultere i en øget mortalitet hos blåmuslingen, der først indtræffer

flere uger efter stresspåvirkningen (Aarset 1982).

En afgørende del af blåmuslingens kuldetolerance menes at bestå i, at arten kan regulere den

intracellulære koncentration af frie aminosyrer. Ved at øge koncentrationen kan cellen sænke

intracellulærvæskens frysepunkt samt opretholde et vist osmotisk potentiale, der muliggør

regulering af cellevolumen. Cellen kan således undgå en letal grad af dehydrering ved kuldestress

(Williams 1970). I overensstemmelse med denne teori er det observeret, at blåmuslinger

akklimatiseret til høj salinitet har en højere kuldetolerance (Williams 1970). Det kan skyldes, at det

hyperosmotiske saline miljø får cellerne til at dehydrere. Det resulterer i et cellulært respons, der

øger mængden af frie aminosyrer. Herved er cellerne bedre rustet til at modstå efterfølgende

  15  

kuldestress. Desuden medfører den høje salinitet en mindre isdannelse ekstracellulært og deraf en

lavere grad af cellulær dehydrering (Lange 1963, Williams 1970).

Udover fysiologisk baseret kuldetolerance kan blåmuslingen også beskyttes via artens tendens til

at vokse i banker, under alger og i klippesprækker, hvilket kan beskytte dem imod kulde, is og

bølger (Williams 1970). Det er endvidere muligt, at blåmuslingens kappehule fungerer som et

internt varme-reservoir, når blåmuslingen ved lavvande fryser i den arktiske tidevandszone.

Kappehulen rummer cirka 10 gange så meget vand som vævet. Havvand i kappehulen fryser før

vævet. Isdannelsen er en ektoterm proces, der frigiver latent varme til vævet således, at

vævsfrysning forsinkes. Frysning af kappehulen kan dermed forkorte den periode, hvor vævet er

frosset ved lavvande. Dette bidrager til blåmuslingens kuldetolerance, eftersom blåmuslingens

mortalitet ved kulde i høj grad afgøres af, hvor længe vævet eksponeres for frysning (Williams

1970, Aarset 1982). Store individer er mere kuldetolerante end små individer (Bourget 1983),

sandsynligvis fordi deres kappehule indeholder mere vand og fordi de har et mindre

overflade/volumen forhold, hvilket også giver en langsommere afkøling.

Blåmuslingen adapteres til kulde ved naturlig selektion. Bourget (1983) fandt, at LT50

(temperatur, hvor 50% dør) for blåmuslinger i tidevandszonen i den canadiske St. Lawrence Bugt

var -17,5°C for store muslinger (> 3cm) ved 24 timers stresseksponering. Ved sammenligning med

et tidligere studie, der viste en lavere kuldetolerance hos blåmuslinger fra et mildere klima (-10°C i

Massachusetts, New England, (Williams 1970)), konkluderede Bourget (1983), at der hos

blåmuslingen selekteres for kuldetolerance i arktisk klima.

Det enkelte individ kan endvidere akklimatisere sig til kulde ved at øge sin kuldetolerance over

tid. Gællevæv fra blåmuslingen øger kuldetolerancen efter 2-3 uger ved overførsel fra 15 til 5°C,

hvorimod kuldetolerancen hos kulde-akklimatiserede dyr allerede mindskes igen efter 1 uge ved

overførsel til højere temperatur (Theede 1972).

Blykontaminerede  blåmuslinger  i  Arktis  

Blåmuslingen bioakkumulerer tungmetaller opløst i havvandet samt via føden. Artens optag af

tungmetal opløst i havvand er ikke en temperaturafhængig proces (Baines et al. 2006), hvorimod

artens tungmetaloptag fra føden er langt mere effektiv ved lav temperatur (2°C) end ved høj

temperatur (12°C) (Baines et al. 2005). Forskellen i assimileringseffektiviteten var 122-945%

(afhængig af metallet) højere ved lav temperatur, hvilket skyldes blåmuslingens øgede

  16  

fødeassimilationsrate ved lav temperatur (Baines et al. 2005). Endvidere var udskillelsesraten lavere

ved 2°C (50-80%) end ved 12°C på grund af nedsat respiration (Baines et al. 2005). Disse

temperatureffekter kan have stor indflydelse på organismers bioakkumulering af tungmetal fra føde

og bør inddrages som faktor, når man vurderer udledte tungmetallers toksicitet for flora og fauna i

forskellige miljøer (Baines et al. 2005).

Nogle organismer er aktivt i stand til at udskille tungmetaller fra vævet, men det er muslinger

generelt ikke (Depledge & Rainbow 1990). Fordøjelseskirtlen (hepatopankreas) er hos M.

galloprovincialis vist at optage og binde tungmetaller, hvormed stofferne uskadeliggøres. Organet

spiller derved en vigtig rolle i tungmetalmetabolismen (Viarengo et al. 1981), hvilket sandsynligvis

også gør sig gældende hos M. edulis. Flere transplantationsstudier i Grønland har vist, at

blåmuslingen frigiver bly langsomt efter kontaminering (Riget et al. 1997, Søndergaard et al. 2011,

Zimmer et al. 2011), hvilket kan skyldes langvarig akkumulering i fordøjelseskirtlen. Riget et al.

(1997) viste eksempelvis, at blykontaminerede blåmuslinger fra Maarmorilik overført til et

ukontamineret område i løbet af det første år nedsatte blyindholdet fra 744 til 400 µg/g tørvægt,

hvorefter koncentrationen forblev konstant de næste fire år. Blyindholdet i det enkelte individ kan

således kun mindskes ved fortynding og/eller fysiologisk udskiftning af væv, efterhånden som

muslingen vokser (Zimmer et al. 2011). Dette kan øge de langsigtede økologiske konsekvenser af

tungmetalforurening for blåmuslingen.

Cellemembraner  hos  ektotermer  stresses  af  kulde  og  bly   Dette afsnit er et teoretisk overblik over cellemembranens fysiske og fysiologiske egenskaber og

hvorledes denne stresses af både tungmetal og kulde, hvilket lægger til grund for studiets hypotese

om, at den mulige synergi imellem effekterne af kulde og bly på blåmuslingens kuldetolerance

skyldes ændringer i membranens fedtsyrekomposition.

 

Cellemembranen  

Alle celler er omgivet af en semipermeabel membran, der virker som barriere til det omgivende

miljø. Membranens funktionalitet er afgørende for cellens (og organismens) overlevelse, da

cellemembranen gør det muligt for cellen at kontrollere sit indre miljø (Singer & Nicolson 1972,

Hazel & Williams 1990). En cellemembran er en dynamisk todimensionel lagdelt struktur

bestående af orienterede lipider med indlejrede transportproteiner, der tillader effektiv transport af

  17  

udvalgte molekyler og ioner (Figur 6) (Singer & Nicolson 1972). I cellemembranen findes primært

tre lipidtyper; kolesterol, glykolipider og fosfolipider, hvoraf den sidstnævnte er dominerende (Berg

et al. 2007). Et fosfolipid består typisk af glycerol esterbundet til to fosfolipidfedtsyrer (PLFAs)

samt en fosfatgruppe. På fosfatgruppen er der endvidere esterbundet en alkohol. PLFAs er

hydrerede kulstofkæder af varierende længde med en terminal carboxylsyregruppe. Fosfolipider er

således amfipatiske molekyler med en upolær hale bestående af PLFAs samt et polært hoved.

Denne amfipatiske struktur er afgørende for fosfolipiders spontane formation til en membran i et

hydrofilt miljø, hvor PLFAs pakkes tæt sammen via nonkovalente hydrofobe bindinger (Figur 6)

(Singer & Nicolson 1972, Berg et al. 2007).

 

 

 

       

 

 

 

         

 

 

Figur  6  viser  strukturen  af  et  fosfolipid-­‐molekyle  og  cellemembranen.  (a)  Et  fosfolipidmolekyle  består  af  en   upolær   hale   i   form   af   to   fedtsyrer   (PLFAs)   samt   et   polært   hoved,   hvilket   giver   molekylet   dets  amfipatiske   natur.   PLFAs  er   lange   carbonkæder  mættet   eller   umættet  med  hydrogen.   Dobbeltbindinger  imellem  carbon-­‐atomerne  giver  PLFA  et  knæk.  PLFAs  er  esterbundet  til  et  polært  hoved,  der  består  af  et  glycerolmolekyle  esterbundet   til  en   fosfatgruppe,  som   igen  er   esterbundet   til  en  alkohol,  her   cholin.  (b)  Fosfolipidernes   amfipatiske   natur   gør,   at   de   spontant   samler   sig   til   en   bimolekylær   struktur   med   et  hydrofilt   ydre   og   et   hydrofobt   indre.   Indlejrede   transportproteiner   er   ikke   vist.   Fra  http://chemistry.tutorvista.com/biochemistry/phospholipids.html      

  18  

Cellemembranens  temperatursensitivitet  

Cellemembranens funktionalitet, herunder membranens fluiditet og fasetilstand, er sensitiv

overfor temperaturændringer (Hazel & Williams 1990). Temperaturen, hvor membranen abrupt går

fra en funktionel flydende-krystallin fase til en dysfunktionel stiv gelfase kaldes

fasetransitionstemperaturen (Tm). Fasetransitionen ændrer i høj grad membranens fysiske

organisering og således dens fysiologiske egenskaber (Hazel & Williams 1990).

Det mest observerede cellulære respons til temperaturændringer hos ektotermer er regulering af

cellemembranen (Hazel & Williams 1990). Dette sker på flere måder. Kolesterol regulerer

fluiditeten i animalske membraner og herved også Tm (Crockett 1998, Berg et al. 2007). Et øget

kolesterolindhold giver rigiditet til membranen og mindsker endvidere risikoen for transition til

gelfase, der ellers kan medføre en række forstyrrelser af membranen (Seelig 1978, Quinn 1985,

Hazel 1995). Sammenligninger af kolesterolindhold i akklimatiserede (og akklimerede) ektotermer

viser et stigende kolesterolindhold ved stigende temperatur (Crockett 1998). Endvidere afhænger

Tm af egenskaber ved PLFAs. PLFAs er en divers gruppe af molekyler med meget forskellige

kemiske og fysiske egenskaber (Hazel & Williams 1990, Berg et al. 2007). Både længde samt antal

og placering af cis carbon-carbon dobbeltbindinger er afgørende for de enkelte PLFA’s smeltepunkt

og således for membranens overordnede Tm (Figur 6). Lange PLFAs interagerer kraftigere med

hinanden end korte, hvorfor lange PLFAs relativt til korte øger Tm. Ligeledes øger mættede PLFAs

Tm, da disse er lige molekyler, der pakkes helt tæt og interagerer effektivt med nabomolekyler.

Modsat sænker umættede PLFAs Tm, særligt flerumættede fedtsyrer (PUFAs), da dobbeltbindinger

giver PLFAs et knæk, hvormed pakningen af PLFAs bliver mere løs og dermed relativt svagere

(Wang et al. 1999, Berg et al. 2007). Således korresponderer højere Tm værdier med højere grad af

orden i pakningen af PLFAs (Separovic & Gawrisch 1996).

Kulde  stresser  membranen  hos  ektoterme  organismer  

Ektoterme organismer har samme kropstemperatur som omgivelserne og har derfor varierende

kropstemperatur over året og døgnet. Omgivelsernes temperatur er således af afgørende fysiologisk

og økologisk betydning for disse organismer (Cairns et al. 1975, Hazel & Williams 1990, Pernet et

al. 2007). I den arktiske tidevandszone indtræffer daglige temperaturfluktuationer som følge af

tidevandets cyklus. Her når ektoterme organismers kropstemperatur ned på lave frysegrader. Kulde

stresser biologiske membraner, som er det primære sted for kuldeskade hos invertebrater (Aarset

1982, Hazel & Williams 1990). Kuldechok, hvor organismer kortvarigt udsættes for lav temperatur,

  19  

kan enten have umiddelbar død eller kroniske skader på muskler og nervesystem som følge. Det

menes at være resultatet af, at membranen overgår til den dysfunktionelle gelfase, hvor membranen

eksempelvis ikke kan opretholde vitale iongradienter (Quinn 1985, Kelty et al. 1996).

Inden for de genetiske rammer er det muligt for et individ at akklimatisere sig til foranderlige

temperaturforhold (Aarset 1982, Heugens et al. 2001). Mange ektoterme dyr er endvidere adapteret

til temperaturvariationer, således at fysiologiske processer kan forløbe effektivt over et relativt bredt

temperaturspektrum (Crockett 1998). Den mest almindelige tilpasning til temperaturændringer er

regulering af cellemembranen. Dette fænomen kaldes homeoviskøs adaption. Ektoterme dyr

regulerer primært membranen ved at regulere membranens PLFA komposition og dermed

membranens grad af orden (Hazel & Williams 1990, Hazel 1995). Denne membranregulering som

respons på temperaturændringer er observeret hos flere ektoterme tidevandsarter (Hall et al. 2002,

Pernet et al. 2006, Pernet et al. 2007), endda indenfor få timer hos M. californianus samlet øverst i

tidevandszonen (Williams & Somero 1996).

Når ektoterme organismer udsættes for kulde øges graden af umættethed (primært PUFAs) i

membranen for at sænke Tm og dermed opretholde en funktionel fluiditet ved lav temperatur (Hazel

1995). Dette er eksempelvis påvist hos gællevæv fra henholdsvis blåmusling og østers (Crassostrea

virginica). Begge arter viste en signifikant relation imellem den overordnede grad af umættethed i

membranen og akklimatiseringstemperatur (Pernet et al. 2007). En øget grad af umættethed øger

overlevelsen ved kolde temperaturer (Hazel & Williams 1990, Hazel 1995) og særligt et forøget

PUFA indhold ses i de mest kuldetolerante organismer (Hazel 1988). I henhold til dette observerede

Pernet (2007), at blåmuslingen havde en højere grad af umættethed og PUFA i sine cellemembraner

end østers, der er en mindre kuldetolerant art.

 

Peroxidering  af  cellemembranen  

Som ovenfor beskrevet stresses cellemembranen hos ektoterme dyr af kulde. Alle organismer er

endvidere kontinuerligt udsat for oxidativt stress forårsaget af reaktive oxygenarter (ROS). ROS

skabes ved en ufuldstændig reduktion af ilt (O2) (Livingstone et al. 1990). Som forsvar har alle

organismer et antioxidantsystem, der nedbryder ROS (Sukhotin et al. 2002).

Peroxidering betyder oxidering af lipider og er en skadelig proces, hvor ROS oxiderer og

degraderer membranlipiderne, hvilket resulterer i effekter varierende fra lokale ændringer i

membranfluiditeten til celledød (Marcelo 2004, Valko et al. 2005). Den mest almindelige ROS til

  20  

peroxidering anses for at være OH- (Nyska & Kohen 2002, Marcelo 2004). Peroxidering initieres

ved, at OH- abstraherer H+ fra en methylengruppe ved siden af en dobbeltbinding i en umættet

PLFA. Herved dannes en fedtsyreradikal (Figur 7.1). PUFAs er meget følsomme overfor

peroxidering, da C=C

dobbeltbindingen gør C-H

bindingen i methylengruppen

svag og derfor mere følsom

overfor oxidering (Nyska &

Kohen 2002, Valko et al. 2005).

Den dannede fedtsyreradikal

reagerer med omgivende O2 og

danner en lipoperoxyl radikal

(Figur 7.2). Denne er ligeledes

meget reaktiv og kan reagere

med et nabo-PUFA molekyle,

hvormed der dannes en

lipidhydroperoxid. Samtidig

omdannes nabo-PUFA til en

fedtsyreradikal, der kan indgå i

samme reaktionskæde (Figur

7.3). Lipidhydroperoxid reagerer

eventuelt med Fe2+, men

undergår under alle

omstændigheder degradering

(Figur 7.4 og 7.5). Peroxidering af én PUFA via ROS igangsætter altså en autokatalytisk proces i

membranen, hvor PUFAs nedbrydes, indtil der ikke er flere eller indtil en antioxidant bremser

kædereaktionen (Nyska & Kohen 2002, Marcelo 2004, Valko et al. 2005).

Bly  øger  peroxidering  og  stresser  membranen  

Det er velkendt, at mange tungmetaller kan inducere produktionen af ROS og øge peroxidering

(Christie & Costa 1984, Viarengo et al. 1990, Valko et al. 2005). Flere studier har vist, at bly øger

peroxidering i både planter og dyr (Gerber et al. 1978, Shafiq Ur 1984, Lawton & Donaldson 1991,

Figur   7   viser   peroxidering   af   flerumættede   fedtsyrer   (PUFAs)   ved  eksponering   til   reaktive   oxygenarter.   Se   tilhørende   tekst   for   detaljer.  Efter  Marcelo  (2004)  

  21  

Stohs & Bagchi 1995, Verma & Dubey 2003). Blykontaminering er generelt forbundet med en

dosisafhængig stigning i peroxidering og mange af blys toksiske effekter synes at være forbundet

med ændringer i membranens PLFAs (Donaldson & Knowles 1993).

Geret et al. (2002) har vist, at flere tungmetaller (Ag, Cd og Hg) signifikant øger peroxidering i

blåmuslingen. I henhold til dette viste studiet endvidere, at tungmetaleksponeringen inducerede

produktionen af proteinet metallothionein (MT) (Geret et al. 2002). MT binder overskydende

metalioner i vævet (sekvestrering) og regulerer således indirekte peroxidering (Geret et al. 2002).

Desuden har MT en direkte antioxidant virkning, da proteinet binder og uskadeliggør OH-

(Thornalley & Vasak 1985). Af disse grunde kan MT anvendes som indikator på biologiske effekter

af tungmetalkontaminering (Schiedek et al. 2009). Blåmuslinger fra Maarmorilik kontamineret med

høje blykoncentrationer (>100 x baggrundsniveau) er ligeledes vist at have et øget MT indhold

(Schiedek et al. 2009).

Viarengo et al. (1991) har vist, at blåmuslingen har et lavere antioxidantniveau om vinteren.

Dette kan skyldes den ektoterme organismes nedsatte metabolisme eller at fysiologisk

antioxidantproduktion kræver energi. Blåmuslingen i Arktis er ofte begrænset af

fødetilgængelighed, særligt i de mørke vintermåneder, hvor produktionen af fytoplankton stort set

går i stå på grund af lysmangel (Sejr, pers. komm.). Den observerede reduktion i

antioxidantniveauet blev tilsvaret af en øget peroxidering af cellemembranerne. Længerevarende

lave vintertemperaturer øger ifølge dette studie således indirekte peroxidering og mindsker dermed

potentielt mængden af PUFA. (Viarengo et al. 1991).

Synergetisk  stressinteraktion  mellem  kulde  og  tungmetal   I 1984 fremsatte Environmental Protection Agency (EPA) risikovurderingsprocedurer til at

vurdere og minimere risikoen af forskellige giftige stoffer i miljøet (EPA 1984). Disse danner

baggrund for undersøgelser af forskellige stoffers toksicitet og letale koncentration ud fra

standardiserede økotoksikologiske laboratorietests, hvor alle andre parametre end

stofkoncentrationen holdes konstante (og typisk favorable) for testorganismen (Løkke et al. 2013).

Det kan dog være problematisk at ekstrapolere resultaterne fra laboratoriet til det naturlige miljø,

hvor organismer sjældent oplever optimale forhold og oftest er udsat for flere stressfaktorer ad

gangen (Holmstrup et al. 2010).

  22  

Når flere former for stress interagerer og

simultant påvirker en organisme, kan det resultere

i synergetisk stress (figur 8). Det betyder, at den

kombinerede stresseffekt er større end de

adderede effekter af de enkelte stressfaktorer. Der

tages ikke højde for synergetiske interaktioner

med andre stressfaktorer eller for subletale

effekter, hvis man vurderer toksiciteten af et stof i

et som ovenfor beskrevet standard-

laboratorieforsøg (Holmstrup et al. 2010).

Eftersom den omgivende temperatur hos

ektoterme organismer påvirker visse fysiologiske

processer, forventes en synergetisk interaktion

med et toksisk stof, der påvirker de samme

fysiologiske processer (Heugens et al. 2001). I

henhold til dette kan metalkontaminering mindske

organismens tolerance overfor ekstreme

temperaturer og omvendt (Ansari et al. 2004),

eftersom både tungmetal og kulde stresser

cellemembraner. Synergetisk stresspåvirkning af

kulde og tungmetal er ikke et velstuderet område,

men fokus på interaktioner imellem kemisk og

fysisk stress er dog øget i det seneste årti, primært på grund af klimaforandringer (EPA 2003, Noyes

et al. 2009, Hooper et al. 2013, Løkke et al. 2013). Synergi imellem kulde og tungmetal er blevet

undersøgt hos nogle få terrestriske arter, men ikke marine arter. Bindesbøl et al. (2009a) viste

eksempelvis, at både nikkel (Ni) og kviksølv (Hg) interagerede synergetisk med kuldestress (-6°C)

på frysetolerancen hos regnorm (Dendrobaena octaedra). Bly havde derimod ingen interaktion

med kuldestress i dette studie, sandsynligvis på grund af for lave anvendte blykoncentrationer

(ingen mortalitet af bly i kontroller uden fryseeksponering) (Bindesbøl et al. 2009a). Holmstrup et

al. (2008) har som et andet eksempel vist en signifikant synergetisk interaktion imellem kviksølv og

kuldechok på springhaler (Folsomia candida). Studiet viste, at LT50 under kontrolforhold (ingen

Hg) var -6°C. Med stigende subletale kviksølvkoncentrationer øgedes LT50 gradvist til -2°C.

Figur   8   viser   et   skematisk   eksempel   på   en  synergetisk   interaktion   imellem   en   kemisk   og   en  fysisk   (”natural”)   stressfaktor.   (A)   viser,   at   en  organismes   performance   falder,   når   et   kritisk  stressniveau   nås,   altså   hvor   organismen   ikke  længere  kan  kompensere  for  de  negative  effekter  af  en   naturlig   stressfaktor.   (B)   viser,   at   ved  tilstedeværelsen   af   en   kemisk   stressfaktor  indtræffer  dette  kritiske  stressniveau  ved  en  lavere  grad  af  fysisk  stress.  Efter  Løkke  (2013).  

  23  

Omvendt øgede kuldechok toksiciteten af kviksølv, således at kviksølvkoncentrationer, der under

kontrolforsøg med optimale temperaturforhold ikke havde haft letale effekter, fik det. På baggrund

af deres resultater påpegede Holmstrup et al. (2008) risikoen for at undervurdere tungmetallers

toksicitet med laboratorieforsøg, hvor organismer kun udsættes for én stresspåvirkning ad gangen

(Holmstrup et al. 2008). Også kobber (Cu) er vist at interagere synergetisk med kuldestress på

frysetolerance hos flere arter (Holmstrup et al. 1998, Bossen 2001). Bindesbøl et al. (2005) viste

eksempelvis en signifikant synergetisk interaktion imellem subletale kobberkoncentrationer og

kuldestress hos regnorm (D. octaedra) fra Grønland og konkluderede derfor, at kobberforurening er

af betydning for regnormens biogeografi (Bindesbøl et al. 2005). Et senere studie af samme gruppe

har vist, at kobbers skadelige effekt på regnormens kuldetolerance skyldes tungmetallets toksiske

effekt på cellemembranerne, da gruppen fandt et signifikant fald i PUFAs samt et signifikant øget

peroxideringsniveau ved kobbereksponering (Bindesbøl et al. 2009b). Flere andre studier viser

ligeledes, at den synergetiske interaktion imellem tungmetal og kuldestress på kuldetolerance

(delvist) skyldes membranskader (se Holmstrup 2010 for review).

Aktuelt  studie   Dette afsnit opsummerer, hvorledes den ovenstående teoretiske introduktion danner baggrund

for det aktuelle videnskabelige studie, som manuskriptet omhandler.

I de seneste årtier er der kommet øget fokus på tungmetalforurening af marine miljøer. Store

mængder af tungmetal er udledt til havet, og dette forventes at fortsætte i fremtiden. Udledte

tungmetaller vækker stor bekymring grundet deres akkumulering i sediment og i marine organismer

samt stoffernes potentielle toksiske (sub-)letale effekter (Ansari et al. 2004). Særligt marine

områder nær minedrift er kraftigt kontaminerede med tungmetaller såsom bly.

Blåmuslinger i den arktiske tidevandszone udsættes for ekstremt lave lufttemperaturer i

vintermånederne (Scrosati & Eckersley 2007, Blicher et al. 2013). I kontaminerede grønlandske

fjordsystemer er de samtidig udsat for høje blykoncentrationer (Johansen et al. 2008, Bach, pers.

komm.). Lave temperaturer udøver stress på cellemembranerne hos ektoterme organismer, da

membranen kræver en vis fluiditet for at være fuldt funktionel. Kommer temperaturen under

fasetransitionstemperaturen Tm går membranen i en dysfunktionel stiv gelfase (Hazel & Williams

1990). Bly stresser cellemembranen ved at øge peroxidering, som kan mindske mængden af

  24  

PUFAs. Færre PUFAs øger generelt Tm, da membranen bliver mere rigid og derfor kræver en

højere temperatur for at holdes flydende.

Eftersom både kulde og bly stresser biologiske membraner, kan bly potentielt gøre organismer

mere sårbare overfor kulde og omvendt (Heugens et al. 2001). En sådan synergetisk interaktion af

tungmetal og kulde er tidligere påvist hos flere terrestriske organismer, men ikke hos marine arter

(Bindesbøl et al. 2005, Holmstrup et al. 2008, Bindesbøl et al. 2009a, b). Disse synergetiske

stressinteraktioner er generelt vigtige at forstå, før man ekstrapolerer resultater fra laboratoriet til

naturen. En subletal blykoncentration i laboratoriet kan have letale konsekvenser i det naturlige

økosystem, hvor flere stressfaktorer simultant interagerer på organismens fitness (Heugens et al.

2001).

Det er således hypotesen i dette studie, at der er en synergetisk interaktion mellem effekterne af

henholdsvis bly i økologisk realistiske koncentrationer og økologisk realistisk kulde på

blåmuslingens kuldetolerance i Arktis. Dette undersøges i et fuld-faktorielt forsøg, hvor

blåmuslinger indsamlet i tidevandszonen i Nuuk, Grønland (maj 2014), udsættes for 5

blykoncentrationer og 6 temperaturer (figur 9-11). Som mulig forklaring herpå undersøges det,

hvorvidt den potentielle synergetiske interaktion skyldes, at bly skader membranen via ændringer i

PLFA kompositionen grundet øget peroxidering.

Figur   9.   Blåmuslinger   samlet   i   Nuuk,  Grønland,   kontamineres   med   forskellige  blykoncentrationer  i  en  uge.  

Figur  10.  100  blåmuslinger  tørres  enkeltvist  inden  blyanalyse  af  vævet.  

  25  

Blåmuslingen anvendes som testorganisme, da den er en

velstuderet og hyppigt anvendt miljøindikator. Der findes

således en baggrundsviden om denne art, også fra arktiske

lokaliteter. Bly undersøges af flere grunde: (1) bly er en

kilde til bekymring i arktiske marine miljøer, eftersom bly

fortsat findes i høje koncentrationer i visse arktiske

fjordsystemer; (2) bly har toksiske effekter ved både letale

og subletale koncentrationer (AMAP 2005, Liu et al. 2011,

Ouyang et al. 2012, Liu et al. 2014) og (3) bly

bioakkumuleres og øger cellulær peroxidering. Desuden

forventer man flere blyminer i Grønland i fremtiden (Rosing

et al. 2014), som kan forårsage yderligere blyforurening. Bly

og kuldes mulige synergetiske interaktion på blåmuslingens

frysetolerance er yderst relevant at undersøge, da et positivt resultat kan indebære, at man ikke kan

vurdere blys toksicitet alene ud fra standardiserede økotoksikologiske laboratorietests. Yderligere

bør man i så fald overveje, om man kan overføre europæiske grænseværdier1 for bly i marine

miljøer til Arktis, da arktiske organismer udsættes for ekstrem kulde. Synergetiske interaktioner

angående kuldetolerance gør sig potentielt gældende for en række andre arter og tungmetaller. Det

er kritisk at forstå disse toksiske stoffers interaktion med andre former for stress (i Arktis særligt

kuldestress) for at kunne vurdere risikoen for det arktiske marine økosystem i forbindelse med de

antropogene tungmetaludledninger, som vi forventer vil fortsætte i fremtiden.

   

 

 

         

                                                                                                               1  Grænseværdien  for  bly  i  marine  vande  er  i  EU  er  fastsat  til  0,34  μL/L  (Miljøministeriet  2010).  

Figur  11.  PLFA  komposition  af  100  blåmuslinger  analyseres.  

  26  

                                                                                   

  27  

Manuscript

A  test  of  possible  synergetic  

interactions  between  sub-­‐zero  temperatures  and  lead  (Pb)  contamination  in  Greenland  blue  mussel  

(Mytilus  edulis).  

  28  

Authors and addresses

Bodil Klein Juhl1, Jakob Thyrring1*, Martin Holmstrup1,2, Mikael K. Sejr1,3

1Department of Bioscience, Aarhus University, Arctic Research Centre, C. F. Møllers Allé 8,

building 1110, DK-8000 Aarhus C, Denmark 2Department of Bioscience, Aarhus University, Soil Fauna Ecology and Ecotoxicology, Vejlsøvej

25, DK-8600 Silkeborg, Denmark 3Greenland Climate Research Centre, Greenland Institute of Natural Resources, Nuuk, Greenland

*Corresponding author: thyrring@bios.au.dk; phone: +45 87156697

Keywords

Arctic, Freeze tolerance, Freezing, Mining, membrane lipids; PLFA, Risk assessment

 

 

       

       

  29  

Abstract     In their natural habitat, organisms are exposed to multiple stressors. Previous studies have

shown, that interactions between chemical and physical stressors can lead to increased mortality.

Heavy metal contamination stresses the cell membrane due to increased peroxidation. Likewise,

sub-zero temperatures also potentially reduce membrane functionality. We conducted a full

factorial experiment with 5 Pb concentrations between 0 and 6.50 mg Pb/L and 6 temperatures from

0 to -17ºC to test the hypothesis that environmentally realistic levels of Pb may interact

synergetically with realistic sub-zero temperatures on the freezing mortality of the blue mussel

(Mytilus edulis) collected in Nuuk, Greenland. We further investigated whether these interactions

are due to Pb-induced alterations of PLFA composition and membrane damage. We found a highly

significant interaction between temperature and mortality, however no effect of lead was seen. Nor

a significant interaction between temperature and Pb on mortality of blue mussels was found, but a

trend was observed at intermediate sub-zero temperatures with increased freezing mortality with

increasing Pb concentrations. At low temperature, ectothermic organisms will regulate the

composition of phospholipid fatty acids (PLFAs) typically by increasing the amount of

polyunsaturated fatty acids (PUFAs) of the cell membrane to sustain a proper fluidity and

functionality. Pb had a significant effect on the molar percentage of PUFAs, which was reduced

from 72.3% (control) to 70.8% in mussels exposed to the highest Pb concentration. Also a

significant increase in monounsaturated fatty acids (MUFAs) was observed, in particular due to an

increase in 18:1. Further, blue mussels exposed to the highest Pb concentrations (4.50 and 6.50 mg

Pb/L) had a significantly higher !"#$!"#$

ratio compared to controls and mussels exposed to the lowest

Pb concentration (0.50 mg Pb/L). The overall unsaturation index was not significantly related to Pb

concentration. Thus, this study revealed significant effects of sub-lethal concentrations of the

hazardous heavy metal Pb on a cellular level in blue mussels. A biochemical background for a

decreased freezing tolerance over time is possibly observed as Pb significantly decreases PUFA

content. This and other studies indicate that synergetic interactions between physical and chemical

stressors should be considered in future risk assessments of heavy metal contamination of natural

habitats.

  30  

1.  Introduction Declining sea ice cover and political and economical interests in the vast potential of oil and

hard mineral reserves in the Greenlandic underground are all drivers for Greenland’s aspirations

concerning the future exploitation of mineral recourses, including iron (Fe), gold (Au), zinc (Zn),

lead (Pb), gas and oil (Nuttall 2014, Rosing et al. 2014).

In Greenland, mining activities have been conducted for over a century with well-documented

long-term environmental impacts, including heavy metal contamination of fjords and organisms

(Søndergaard et al. 2011, Søndergaard et al. 2014). For example, at an abandoned mining site in

South-Greenland, Johansen et al. (2008) found Pb concentrations in blue mussels (Mytilus edulis)

elevated to 200-500 times above background concentration, and Loring & Asmund (1989) reported

Pb tissue concentrations exceeding 800 µg/g (background 3 µg/g) in mussels collected close to the

Maarmorilik mine in West Greenland. Because of previous environmental contamination, recent oil

and mineral exploration in the Arctic region have lead to growing concerns about uncontainable

pollution of the Arctic marine ecosystems (AMAP 2010). Heavy metal waste from increasing

mining activities is an environmental threat due to potential pollution and contamination of the

sensitive marine flora and fauna (Reichelt-Brushett 2012, Søndergaard 2013).

In the Arctic, contaminated organisms simultaneously get exposed to multiple stressors, both

chemical and physical. Chemical contamination may under some conditions reduce the cold

tolerance of invertebrates, leading to increased mortality when low temperature is combined with

contamination (Holmstrup et al. 2008, Bindesbøl et al. 2009a, b). Still, in traditional

ecotoxicological laboratory studies organisms are usually exposed to a single chemical, while held

at otherwise optimal conditions. Risk-assessment and environmental monitoring methods often

consist of point sampling and transplant studies with the focus of estimating uptake/release rates of

contaminants, mortality and metal tissue content (Søndergaard et al. 2011, Zimmer et al. 2011).

However, such studies have limitations, as they do not consider synergetic stress interactions of

multiple stressors acting simultaneously on organisms in natural environments. Both physical and

chemical stressors can interact synergetically and in the Arctic, extreme temperatures, increasingly

acidic oceans (AMAP 2013), additive effects of multiple contaminants, food scarcity during winter

and other relevant stressors are factors worth considering when conducting risk assessment in this

region. Also, such traditional studies often do not include sub-lethal effects. Sub-lethal

concentrations of metals may affect growth, and enzyme activities of plants and animals (Naimo

1995, Ouyang et al. 2012). Consequently, as both chemical and physical induced stress is known to

  31  

have chronic and (sub)-lethal effects, multi-factorial studies investigating synergetic interactions

between multiple stressors are crucial to incorporate in future risk-assessments of chemicals in the

Arctic (Heugens et al. 2001).

In marine environments a number of species are frequently included when conducting risk-

assessments (Søndergaard et al. 2014). One key indicator organism is the blue mussel (M. edulis)

because it is relatively long-lived, sessile and abundant with potential for concentrating and

assimilating metals directly from seawater and suspended particles (Zimmer et al. 2011,

Søndergaard et al. 2014). In subarctic Greenland, intertidal blue mussels are widely distributed

(Blicher et al. 2013). Here they create unique habitats with a rich associated fauna and they are an

important food source for higher trophic levels such as fish, sea birds and mammals (Gosling 2003).

In the intertidal zone, sub-zero air temperatures are a primary factor influencing blue mussels

distributions-patterns (Blicher et al. 2013). With minimum winter temperatures down to -25ºC

(Blicher et al. 2013), cold stress and freezing mortality is common among intertidal blue mussels in

the Arctic region (Bourget 1983). When acclimatizing to sub-zero temperatures, ectotherms modify

the lipid composition of their cell membrane. The phase behaviour and physical properties of

membrane phospholipid fatty acids (PLFAs) are extremely sensitive to temperature changes (Hazel

& Williams 1990). Fully functional membranes are in a liquid-crystalline phase, but if membranes

are cooled to a threshold temperature [the phase-transition-temperature (Tm)], the membrane enters

a dysfunctional rigid gel phase (Hazel 1995). To lower Tm and thereby ensure the proper fluidity at

low temperatures, ectotherms regulate cholesterol content and the proportion of unsaturated fatty

acids, mainly they up-regulate the content of polyunsaturated fatty acids (PUFAs) (Cossins &

Raynard 1987, Hazel 1995). Such changes in cell membrane PLFA composition are probably

important for freeze tolerance in cold adapted ectothermic animals (Hazel 1995, Kostal et al. 2003).

When blue mussels are exposed to sub-zero temperatures (approximately -7°C depending on body

size (Sejr, unpublished data)), ice forms in extracellular fluids. Since only water molecules form the

ice, the concentration of extracellular solutes increases. This causes cells to dehydrate as

intracellular water moves down the osmotic gradient into the extracellular space. Excessive cell

dehydration is lethal as it disrupts membrane function and protein structures (Meryman 1971). To

increase freezing tolerance, blue mussels change the intracellular free amino acid concentration,

altering the freezing point of intracellular fluids and preventing an intolerable degree of dehydration

(Williams 1970, Aarset 1982).

  32  

When the hazardous heavy metal lead (Pb) is released into marine environments, it may be

assimilated into marine organisms. Filter-feeding bivalves are exposed to large quantities of water

during feeding and respiration, and in this process they accumulate Pb in both soft tissues and the

shell (Sericano 2000). On a cellular level, Pb contamination has been shown to cause mitochondrial

swelling, deformation and decreased membrane permeability (Goyer & Krall 1969, Raghavan et al.

1981). Heavy metal contamination with various metals has been demonstrated to increase reactive

oxygen species (ROS) production, which accelerates lipid peroxidation, destabilises cell

membranes and alternate PLFA composition (Christie & Costa 1984, Gallego et al. 1996, Valko et

al. 2005). Increased ROS production has also been demonstrated with Pb contamination (Lawton &

Donaldson 1991, Verma & Dubey 2003). ROS affects both monounsaturated (MUFAs) and

polyunsaturated fatty acids, but PUFAs are particularly susceptible to oxidation by ROS (Nyska &

Kohen 2002). PUFAs are vulnerable to peroxidation since double bonds weaken the adjacent

monoallylic C-H bonds and facilitate the reaction of –H with an oxidizing agent, resulting in lipid

radicals and eventually lipid chain termination (Marcelo 2004). Lipid peroxidation will hereby alter

the overall degree of unsaturation from a highly polyunsaturated cell membrane to a more ordered

and rigid membrane structure. This would in theory increase the phase-transition-temperature (Tm)

of the membrane, which leads to a local reduction in membrane fluidity (Hazel & Williams 1990).

If ambient temperature decreases to Tm it can lead to a loss of vital functions and selective

properties (Hazel & Williams 1990). Since both sub-zero temperatures and Pb possibly stress cell

membranes by making it inadequately fluid for full functioning, a synergetic effect of these

stressors can possibly exist.

In the present study we test the hypothesis that synergetic interactions between Pb at ecological

relevant tissue concentrations and realistic sub-zero air temperatures will increase mortality due to

freezing in the arctic blue mussel. Further, to understand the importance of altering cell membrane

fluidity in the freezing tolerance strategy of blue mussels, we test the possibility that Pb

contamination destabilizes the cell membrane by modifying the PLFA composition, and hereby

cause blue mussels to become more vulnerable to the sub-zero temperatures of the arctic intertidal

zone.

  33  

2.  Materials  and  Methods  

2.1  Animals  and  holding  conditions  

We used blue mussels (Mytilus edulis) of similar size (~3 cm) collected in the intertidal zone of

Kobbefjord near Nuuk, subarctic Greenland (64°N) in May 2014. Collected animals were

immediately transferred to aerated tanks before transported to holding facilities at Aarhus

University, Denmark. Blue mussels were kept in 40 L aquariums of constant salinity (27 PSU) and

temperature (1ºC). Animals were allowed to acclimate to laboratory conditions for seven days

before experiments were conducted. Mussels were fed with commercially available phytoplankton

mix (Reed Mariculture, Shellfish Diet – 1800 Formula, Campbell, CA, USA). Half the water

volume was daily exchanged with fresh filtrated seawater to minimise build-up of pseudofeces and

excretion products.

2.2  Experimental  design  

A full factorial experimental design was used to determine survival after exposure to six sub-

zero air temperatures (0, -7, -8, -11, -14, -17ºC) and five Pb concentrations (0, 0.50, 1.80, 4.50 and

6.50 mg Pb/L seawater), giving a total of 30 treatments including control groups.

Pb contaminated seawater was made using a stock solution (10 g Pb/L) made from the analytical

salt lead(II)nitrate [Pb(NO3)2] and deionised water. Mussels were exposed to water concentrations

of 0, 0.50, 1.80, 4.50 and 6.50 mg Pb/L seawater for seven days at 5ºC prior to the freezing

experiment. Water was aerated by air pumps and changed every 24 hours. After seven days of

exposure, 15 mussels were randomly allocated to every experimental combination of Pb and

temperature (a total of 450 mussels). To simulate aerial exposure in the arctic intertidal zone,

mussels from each Pb treatment were transferred to a freezing cabinet (WTB Binder Labortechnik,

Tuttlingen, Germany) programmed to gradually lower the temperature from 5ºC to experimental

temperature over 45 minutes. Mussels were kept at the target temperature (±0.5ºC) for 60 minutes

before a gradual increase of temperature to 5ºC over 45 minutes was initiated. All mussels were

then returned to holding aquariums (5ºC) to recover.

Survival rates were assessed after 1 and 24 hours, respectively, using tactile stimuli and cardio

activity as indicators of survival (using infrared sensors) (Aarset 1982, Burnett et al. 2013).

Mussels were considered alive if they reacted to tactile stimuli and had cardio activity. The mussels

  34  

were finally frozen and stored at -80ºC until further analysis of tissue Pb content and phospholipid

fatty acid (PLFA) composition.

2.3  Pb  analysis    

We randomly sampled 20 mussels from each Pb treatment for analysis of Pb tissue content and

PLFA composition. Approximately 10 mg (fresh weight) of gill tissue was dissected out and stored

at -80ºC for PLFA analysis. Remaining tissue was dried for 24 h (60ºC) and acid-digested in 65%

nitric acid at increasing temperatures (80-110ºC) until all fluid had evaporated. Tissue was then re-

dissolved in 6 mL 2% nitric acid and whirly mixed until clear. Pb content was analysed using flame

atomic absorption spectrometry (AAS) (Perkin-Elmer 4100, Ueberlingen, Germany). Certified

reference material (lobster hepatopancreas from National Research Council Canada) was used to

verify the analytical quality, resulting in a measured concentration of 139% (± 7.83% SE, N = 5) of

the certified value.

2.4  PLFA  analysis  

All gill tissue (see above) was transferred to centrifuge glass tubes and extracted with a mixture

of 1.5 mL chloroform, 1.25 mL buffer (50 mM K2HPO4, pH 7.4) and 3 mL methanol (Waagner et

al. 2013). The chloroform phase containing lipids was transferred to a test tube and evaporated

under nitrogen flow. Lipid extract was dissolved in 300 µL chloroform and fractionated on

prepacked columns with 100 mg of silic acid (Bond Elut Extraction Cartridges, Varian US). PLFAs

were extracted from the column using 1.5 mL methanol and dried under nitrogen flow. Collected

PLFAs were trans esterified by mild alkaline methanolysis (Dowling et al. 1986). Heptane, ELGA

water, and acetic acid (1 M) (1:1:0.15 v/v/v) were added. The biphasic mixture was whirly mixed

and centrifuged for 5 min at 1500 g. The heptane phase containing the fatty acid methyl esters

(FAMEs) was collected. Extraction with heptane was repeated. Combined heptane fractions were

dried under nitrogen flow. Resulting FAMEs were re-dissolved in heptane for gas chromatographic

analysis coupled with mass spectrometry on a Shimadzu GCMS-QP2010 Plus. Identification of

individual FAMEs was based on mass spectra of known FAME standards (Nu-check Prep, Elysian,

MN, USA). Identified fatty acids were designated as X:Y, where X is the number of carbon atoms

and Y is the number of double bonds. Areas of identified peaks were quantified using external

standards, and mol% distributions were calculated (Waagner et al. 2013).

  35  

2.5  Statistical  analysis    

We used one-way analysis of variance (ANOVA) to test for differences in the relative amounts

of polyunsaturated fatty acids (PUFAs), monounsaturated fatty acids (MUFAs) and the !"#$!"#$

relationship among Pb treatments. Post-hoc pair-wise (Tukey HSD) tests were used to compare

significant treatment effects (p < 0.05). Finally, generalized linear models (GLM) were used to

model the effects of temperature and Pb and any interactions between the two on survival.

Prior to any analysis, data exploration was carried out following the protocol of Zuur et al. (2010).

Relationships between covariates were assessed using Pearson correlation coefficients (Zuur et al.

2010). Cook’s plot and boxplots were used to identify outliers and to investigate relationships

between variables. PUFA, MUFA and !"#$!"#$

data showed homogeneity and normality of

distribution. For survival we found no indication of zero-inflation or over-dispersion, and we

decided to use GLM with a binomial distribution for the survival model. Once we had identified

valid models we re-examined the residuals to ensure model assumptions were acceptable. Sample

size is denoted n.

3.  Results  

3.1  Pb  content  in  mussels   We found a positive relationship between internal Pb concentration and Pb concentration in

seawater. Mussel tissue Pb content increased with increasing Pb concentration of the seawater,

except at the highest concentration (Fig. 1). The highest tissue concentration was found in mussels

exposed to Pb concentrations of 4.50 mg/L with a mean tissue concentration of 3516 µg Pb/g dry

weight (dw). Control mussels had an average Pb content of 39.9 µg Pb/g dw.

  36  

Figure 1. Internal lead (Pb) concentrations (µg/g dw) in blue mussels (Mytilus edulis) exposed to five different lead

concentrations (mg Pb/L seawater) for seven days at 5ºC. Error bars indicate standard error. Sample size, n = 100.

3.2  Effects  of  Pb  on  PLFAs  

Nine different phospholipid fatty acids (PLFAs) were identified (Table 1). The overall

unsaturation index was not significantly related to Pb concentration.

Polyunsaturated fatty acids (PUFAs) were the most abundant fatty acids found in the cellular

membranes of gill tissue (Table 1). Three predominant PUFAs; 20:2, 20:4 and 20:5 each accounted

for at least 14% of the total PLFAs. Pb had a significant effect on the molar percentage of PUFAs,

which was reduced from 72.3% (control) to 70.8% in mussels exposed to 6.50 mg Pb/L (Fig. 2a;

ANOVA, p = 0.0115). Of the individual fatty acids, 18:1 responded significantly to Pb exposure

(data not shown) (ANOVA, p < 0.0001). The monounsaturated fatty acids (MUFAs); 18:1 and 20:1,

accounted for approximately 13% of the total PLFAs. MUFA mol% increased significantly with Pb

concentrations (Fig. 2b; ANOVA, p = 0.0016). Finally, we found a highly significant increase in

the ratio between MUFAs and PUFAs (Fig. 2c; ANOVA, p < 0.0001), indicating Pb contamination

induced a change in the PLFA composition of the cell membrane, with relatively more MUFAs

compared to PUFAs. Tukey HSD tests revealed that mussels exposed to Pb concentrations of 4.50

and 6.50 mg Pb/L had a significantly higher !"#$!"#$

ratio compared to control and mussels exposed to

the lowest Pb concentration (0.50 mg Pb/L) (Fig. 2c; Tukey HSD, all p < 0.05).

0 1 2 3 4 5 6 7

0

500

1000

1500

2000

2500

3000

3500

4000

Water Pb concentration (mg/L)

Inte

rnal

Pb

conc

entr

atio

n (µ

g/g

dw)

  37  

 

3.3  Effects  of  sub-­‐zero  temperatures  and  Pb  on  survival    

Temperature had a highly significant effect on the survival of blue mussels (Table 2; p <

0.0001). However, we found no effects of lead (Table 2; p = 0.5322) nor a synergetic interaction

between temperature and Pb on survival of blue mussels was found (Table 2; p = 0.8852). The

survival rate declined significantly from 100% at 0ºC to 0% in mussels exposed to -17ºC, with the

exception of mussels exposed to 1.80 mg Pb/L, where 20% survived (Table 2). Although not

significant, Pb exposed mussels showed a trend of being less tolerant to freezing at intermediate

sub-zero temperatures (-8 and -11ºC) (Table 2).

PLFA 0 0.50 1.80 4.50 6.50

16:0 7.9 ± 0.2 8.1 ± 0.4 7.9 ± 0.2 8.3 ± 0.3 8.4 ± 0.3

18:0 7.4 ± 0.2 6.8 ± 0.2 7.1 ± 0.2 7.3 ± 0.2 7.3 ± 0.2

18:1 2.5 ± 0.1 2.6 ± 0.2 3.4 ± 0.2 3.5 ± 0.2 3.3 ± 0.2

20:1 9.9 ± 0.3 9.8 ± 0.2 9.9 ± 0.2 10.0 ± 0.2 10.1 ± 0.2

20:2 16.5 ± 0.6 15.1 ± 0.7 15.0 ± 0.6 15.3 ± 0.6 14.6 ± 0.6

20:3 3.2 ± 0.1 3.0 ± 0.1 2.8 ± 0.1 3.0 ± 0.1 2.9 ± 0.1

20:4 16.0 ± 0.7 15.5 ± 0.8 15.4 ± 0.8 15.1 ± 0.7 15.4 ± 0.9

20:5 29.6 ± 0.8 32.2 ± 0.8 31.8 ± 0.6 30.7 ± 0.9 31.3 ± 0.8

22:2 7.0 ± 0.3 7.1 ± 0.3 6.7 ± 0.2 6.9 ± 0.4 6.6 ± 0.2

MUFAs 12.4 ± 0.3 12.4 ± 0.2 13.3 ± 0.3 13.5 ± 0.3 13.5 ± 0.2

PUFAs 72.3 ± 0.4 72.8 ± 0.4 71.7 ± 0.5 70.9 ± 0.5 70.8 ± 0.5

MUFA/PUFA 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0

Table 1. Molar percentage distribution (mean ± standard error) of phospholipid fatty acids (PLFAs)

among five different lead (Pb) contamination levels (mg Pb/L). Total percentage of monounsaturated

fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs) and ratio of MUFAs versus PUFAs are

presented in the bottom of the table. Sample size, n = 87.

  38  

0 1 2 3 4 5 6 7

70

71

72

73

74

PU

FA (M

olar

pro

cent

age) ab

a

ab

b b

0 1 2 3 4 5 6 7

12.0

12.5

13.0

13.5

14.0

MU

FA (M

olar

pro

cent

age)

aababc

c

bc

0 1 2 3 4 5 6 7

0.16

0.17

0.18

0.19

0.20

Water Pb concentration (mg/L)

MU

FA/P

UFA

a

b

a

b

ab

a)

b)

c)

Figure 2. Molar percentage of phospholipid fatty acids (PLFA) in blue mussels (Mytilus edulis)

exposed to five different lead (Pb) concentrations (mg Pb/L) for seven days at 5ºC. a) The molar

percentage of monounsaturated fatty acids (MUFA). b) The molar percentage of polyunsaturated fatty

acids (PUFA). c) The ratio of MUFA versus PUFA. Different letters indicate a significant difference (p

< 0.05) among lead concentrations. Error bars indicate standard error. Sample size, n = 87.

 

  39  

Lead concentration (mg/L)

Temperature 0 0.50 1.80 4.50 6.50

0 100 100 100 100 100

-7 93 87 100 93 93

-8 33 67 47 27 20

-11 20 20 13 7 0

-14 0 7 0 7 7

-17 0 0 20 0 0

 

 

4.  Discussion   Investigating sub-lethal effects and potential interactions among multiple stressors is important

in order to understand and evaluate environmental impacts of heavy metal contamination in natural

environments. This study reveals significant effects of sub-lethal concentrations of the hazardous

heavy metal lead (Pb) on a cellular level in blue mussels (Mytilus edulis), a commonly used

biological indicator species for marine risk-assessment.

4.1  Internal  lead  

Blue mussels were exposed to multiple Pb concentrations, which resulted in a net increase of the

Pb concentration in whole-body extracts. With the exception of mussels exposed to 6.50 mg Pb/L,

tissue concentration increased correspondingly to water concentration. In general, mussels cannot

actively excrete heavy metals (Depledge & Rainbow 1990), but when exposed to high Pb

concentrations (i.e. 6.50 mg Pb/L) mussels may close valves or reduce filtration rates to reduce

heavy metal uptake (Kramer et al. 1989, Liu et al. 2014). This is consistent with our observation

that mussels of the 6.50 mg Pb/L treatment often had valves closed (data not shown). Internal Pb

content ranged from approximately 40 - 3500 µg/g dw, which is within tissue concentrations found

in blue mussels collected at contaminated fjords near mining operations, where it can exceed 3000

µg/g dw (Ansari et al. 2004, Bach unpublished data).

Table 2. Percentage survival of Mytilus edulis after 7 days of Pb exposure followed by various

sub-zero temperatures. Survival was evaluated after 24 hours. Sample size, n = 450.

  40  

4.2  Pb  effect  on  PLFA  composition  and  survival    

We found that Pb tissue content induced a significant increase in molar percentage of

monounsaturated fatty acids (MUFAs), while the molar percentage of polyunsaturated fatty acids

(PUFAs) overall decreased. This caused a net shift to a relatively higher proportion of MUFAs

compared to PUFAs. This is consistent with the more general observation that contaminants

influence fatty acid molar composition of cell membranes (Valko et al. 2005, Bindesbøl et al.

2009b). However, this is the first study to show that heavy metal exposure alternate phospholipid

fatty acid (PLFA) composition in a marine invertebrate, and only few studies have investigated

heavy metal effects on fatty acids in terrestrial animals. For example, Donaldson & Knowles (1993)

concluded that Pb toxicosis can result from altered fatty acid composition, and Bindesbøl et al.

(2009b) showed how copper can change PLFA composition to a lesser degree of unsaturation and

increase freezing mortality in earthworms (Dendrobaena octaedra). These results correspond with

the observed reduction in PUFA content in blue mussels due to Pb contamination. PUFAs are

particular susceptible to oxidation by ROS, which previously has been shown accelerated by Pb

(Lawton & Donaldson 1991, Nyska & Kohen 2002, Verma & Dubey 2003). That the relative

amount of MUFAs will increase as PUFAs are terminated by lipid peroxidation corresponds with

our present findings. The membrane hereby changes from a highly polyunsaturated cell membrane

to a more ordered and rigid membrane structure. Theoretically, this increases the phase-transition-

temperature (Tm) of the membrane and leads to a local reduction in membrane fluidity (Hazel &

Williams 1990). If ambient temperature decreases to Tm and the gel phase og the membrane is

entered, it can lead to a diminished membrane function (Hazel 1995).

As functional membranes with a high degree of unsaturation (especially PUFAs) are important

for ectothermic animals to survive sub-zero temperatures, we hypothesised a synergetic interaction

between effects of Pb and sub-zero temperatures on freezing tolerance of the blue mussel, since Pb

increases peroxidation. However, we did not find any significant interactions on survival among

such stressors, although a trend was seen at -8ºC and -11ºC with higher mortalities in Pb exposed

groups. A biochemical background for a decreased freezing tolerance over time is possibly

observed as Pb significantly decreases PUFA content. We therefore envision a long-term study to

fully understand if any interactions of temperature and Pb are present in naturally exposed

populations as a supplement to this short-term experiment, where mussels were exposed to Pb for

only seven days. Long-term contamination (e.g. months or years) could result in more

comprehensive changes of the cellular PLFA composition and thereby enhance the observed effects

  41  

of Pb. In this regard, replicating our study on resident blue mussel from a contaminated site (e.g.

near mining operations) would improve basic understanding of chronic Pb toxicosis and

vulnerability to freezing. Furthermore, this study was conducted in spring acclimated mussels

(collected late May), which were well-fed and in good energetic condition. Viarengo et al. (1991

showed a seasonal variation in antioxidant enzyme systems in blue mussels. The study showed a

lower antioxidant content during winter, presumably due to an altered metabolic state and scarce

food availability. The enzyme systems (e.g. superoxide dismutase, catalase) protect cells against

lipid peroxidation. The reduction in antioxidant system during cold and food scarcity is of special

concern in the Arctic where blue mussels are exposed to low temperatures most of the year and

food scarcity during winter. In the cold Arctic climate, lipid peroxidation could therefore have a

significant effect on survival as antioxidant defence systems are reduced, making oxidative stress

and cellular damage more likely. Thus, it must be well described how the energetic status of an

individual influences its vulnerability to contaminants. We therefore argue that future studies, in

general, should regard seasonal variation in the use of bio-monitoring species (see also Nahrgang et

al. 2013).

4.3  Implications  for  risk-­‐assessments  

Besides altered PLFA composition and potentially reduced freezing tolerance, heavy metals like

Pb may impair cellular structures and functions. Heavy metals and oil spills are known to cause

multiple sub-lethal effects, e.g. mitochondria swelling, membrane leakage, gill degradation and

reduced fecundity (Johnson 1977, Donaldson & Knowles 1993, Liu et al. 2014). Considering both

sub-lethal effects of toxicants and possible synergy between multiple stressors is important to

develop thorough ecotoxicological risk assessments of pollutants dispersed from mining and oil

activities and hereby assess the environmental impact of bioavailable elements and, if needed,

address measures to reduce it. Today, most risk assessments are based on studies where only

bioaccumulation, lethal concentrations or single factor experiments are being assessed. The results

of this study and previous work (Bindesbøl et al. 2005, Holmstrup et al. 2008, Bindesbøl et al.

2009a, Bindesbøl et al. 2009b) indicate that assessment of pollutants by such traditional methods

may be inadequate to estimate the impact of pollutant on the resident biota under natural conditions,

where organisms are exposed to multiple stressors. Especially in the Arctic, where future large-

scale mining and oil explorations are expected, it is crucial that future risk measures and

environmental assessments also consider local abiotic (e.g. low temperature, increasingly acidic

  42  

oceans, ice cover, decreasing salinity) and biotic (food scarcity) stressors and their interactions with

various contaminants in this region.

 

Acknowledgements     The authors thank Lise Lauridsen for technical support and Christian Damgaard for statistical

advice. We also thank Kattegatcentret, Denmark, for providing filtrated seawater. JT was supported

by a grant from ‘Selskabet for Arktisk Forskning og Teknologi (SAFT)’.

  43  

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