Cadmium in the shore crab Carcinus maenas along the Norwegian coast: geographical and seasonal variation and correlation to physiological parameters

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1 Environ Monit Assess (2018) 190: Cdmium in the shore crb Crcinus mens long the Norwegin cost: geogrphicl nd sesonl vrition nd correltion to physiologicl prmeters Heidi Knutsen Amund Mge & Mrtin Wiech & Arne Duinker & Received: 29 November 2017 /Accepted: 7 Mrch 2018 /Published online: 27 Mrch 2018 # The Author(s) 2018 Abstrct Previously, high concentrtions of cdmium hve been found in the heptopncres of the edible or brown crb (Cncer pgurus) smpled from positions north of bout 67 N, compred to regions further south long the Norwegin cost, with no cler understnding why. In order to study similr orgnism in the sme ecosystem, the present study nlyzed 210 shore crbs (Crcinus mens) from four different loctions long the Norwegin cost, two in the North nd two in the South. The physiologicl vribles size, sex, molting stge, heptosomtic index, crpce color, nd gond mturtion were registered, in ttempt to explin the high interindividul vrition in cdmium levels in heptopncres. In contrst to the brown crbs, the shore crbs showed no cler geogrphicl differences in cdmium concentrtions. This indictes physiologicl differences between the two crb species. No cler nd consistent correltions were found between cdmium levels nd physiologicl prmeters, except for sex, where cdmium concentrtion in heptopncres ws twice s high in mles compred to femles. The cdmium levels lso vried with seson, with pproximtely 40 nd 60% lower cdmium Electronic supplementry mteril The online version of this rticle ( contins supplementry mteril, which is vilble to uthorized users. H. Knutsen (*): M. Wiech : A. Duinker : A. Mge Ntionl Institute of Nutrition nd Sefood Reserch, 5002 Bergen, Norwy e-mil: hknutsen10@gmil.com A. Mge University of Bergen, Allegt. 41, 2020 Bergen, Norwy concentrtion in April thn August for mle nd femle shore crbs, respectively. None of the nlyzed cdmium concentrtions in muscle met from clws exceeded EUs food sfety limit, nd low cdmium levels in soup prepred from shore crbs clerly indicted tht this dish is not problemtic regrding food sfety. Keywords Crcinus mens. Cdmium. Cncer pgurus. Shore crb soup. Sesonl vrition. Physiologicl prmeters Introduction It is well estblished tht mrine invertebrtes such s crustcens nd mollusks cn ccumulte cdmium (Jennings nd Rinbow 1979; Ry1984; Wright 1976). A comprison of typicl Europen foodstuffs reveled prticulrly high cdmium levels in crustcens (EFSA 2012). As such, cdmium in crustcens is of considerble interest both regrding toxic effects on the orgnisms itself (Weis 2012) s well s the suitbility s food for humns. To ensure food sfety, the Europen Commission hs estblished upper limits for cdmium in severl foods, where the limit for cdmium in clw met of crustcens is set t 0.5 mg Cd/kg wet weight (ww) (EU 2006). There is t present no legl limit for cdmium in the brown body met, commonly consumed from crbs (Mulvult et l. 2013), lthough it minly consists of heptopncres, where the mjority of cdmium is ccumulted (Bjerregrd 1990; Bjerregrd et l. 2005; Dviesetl.1981; Hutcheston1974;

2 253 Pge 2 of 13 Environ Monit Assess (2018) 190: 253 Wiech et l. 2017) nd gond. The brown crb Cncer pgurus is commercilly importnt, with n nnul ctch volume in Europe of pproximtely 50,000 t in totl (Bkketeig et l. 2016) nd rising to 10,000 t in Norwy lone (Norwegin Directorte of Fisheries 2016). Findings of cdmium levels bove the legl limit for clw met in the brown crb from Northern Norwy hd crucil negtive impct on the crb fisheries in this re. To investigte the cdmium levels in brown crbs long the whole Norwegin cost, cdmium levels in brown met nd muscle met from clws were mesured of in totl of 475 frozen nd cooked brown crbs smpled t 47 different sites long the Norwegin cost between July 2011 nd Jnury A pttern with significntly higher cdmium levels in brown crbs smpled from positions north of bout 67 N (round Sltfjorden) ws found compred to regions further south (Julshmn et l The verge cdmium concentrtions in brown met in frozen nd cooked brown crbs smpled from positions north of Sltfjorden vried from 6.7 to 25 mg/kg wet weight nd from 0.55 to 4.8 mg/kg wet weight in crbs smpled t positions south of Sltfjorden. In nother survey, cdmium ws mesured in brown crbs smpled t 20 loctions from Slten nd further north to Vesterålen (Frntzen et l. 2015). In greement with the survey from 2011 nd 2012, high levels of cdmium were found vrying from 2.4 to 17 mg/kg wet weight in brown met. No difference ws seen between smples from inner fjord nd outer cost loclities. Severl follow-up studies hve been performed in ttempt to explin the elevted cdmium levels in brown crbs from Northern Norwy compred to the rest of the Norwegin cost. However, no obvious point source from industry hs been determined responsible for the high cdmium levels found in the brown crbs (Flk 2012). Further, mesurements of cdmium in surfce wter, groundwter, soil, nd bedrock hve not displyed elevted cdmium levels in the Slten region (Finne 2013). Surveys hve shown reltively low cdmium levels in fish species nd blue mussels from Northern Norwy (Julshmn et l. 2013; Ørnsrud nd Måge 2012; Foldøy Tverdl 2012), nd no correltion to the elevted vlues in the brown crbs were found. As reported for the brown crb (Wiech et l. 2017), lso the shore crb Crcinus mens is ble to ccumulte high levels of cdmium in heptopncres (Rinbow et l. 1990). As these species re lso shring prts of the sme ecologicl niche, comprison of their cdmium levels is of considerble interest. The smller shore crb is found subtidlly s well s intertidlly on ll shores (Crothers 1968), while the brown crb is bundnt from the shllow sublittorl to depths of bout 100 m (Nel nd Wilson 2008). Shore crb is considered deliccy in Spin nd Portugl, with commercil fisheries yield of up to 900 t per yer for Frnce, Portugl, nd Spin together (Klssen nd Locke 2007). The culinry populrity is lso incresing in Norwy, especilly s bse for shore crb soup. In terms of food sfety, it is therefore importnt to study the cdmium levels in shore crbs. Comprison of brown nd shore crbs geogrphicl cdmium pttern would contribute to explining the high cdmium levels in brown crbs north of 67 N. In brown crbs, concluding studies re hmpered by lrge inter-individul vribility in cdmium between brown crbs from the sme geogrphicl res, with especilly high vrition in the heptopncres (Dvies et l. 1981; Mulvult et l. 2012; Wiech et l. 2017). Shore crbs hve lso shown to disply lrge vribility in their cdmium levels (Bjerregrd 1982, 1990, 1991; Bjerregrd nd Depledge 2002; Bondgrd et l. 2000; Nørum et l. 2003). The high vrition could be cused by biologicl fctors. Lbortory studies hve shown reltionships between cdmium levels nd physiologicl vribles such s sex (Bjerregrd et l. 2005), size (Bjerregrd nd Depledge 2002), molt stge (Bondgrd et l. 2000; Bondgrd nd Bjerregrd 2005; Nørum et l. 2005), ovrin mturtion (Bondgrd et l. 2000), nd vribles indicting the condition of the crb (Bjerregrd 1991), like wter content in tissues (Bjerregrd nd Depledge 2002). As observed by Bjerregrd et l. (2005), the tissue cdmium content my lso vry sesonlly. Cdmium bioccumultion increses with incresing temperture (Ry 1984), nd reduced slinity stimulte uptke of nionic cdmium species in brchyurn crbs (Wright 1977; Burke et l. 2003). In the present study, cdmium levels in shore crbs were investigted for comprison to the problemtic high levels in brown crbs, which hve mny prllels in physiology s well s similr ecologicl niche s the shore crb. Also, the cdmium levels in shore crbs were investigted due to food sfety resons. This pper describes geogrphicl (investigtion 1) nd sesonl (investigtion 2) vritions in cdmium levels in shore crbs smpled from four different sites long the Norwegin cost. In ddition, the study exmined effects of different physiologicl prmeters on individul cdmium levels (investigtion 3). Lstly, cdmium

3 Environ Monit Assess (2018) 190: 253 Pge 3 of concentrtions in shore crb soup (investigtion 4) re described for n evlution of food sfety. Mterils nd methods Smpling of biologicl mteril Mle nd femle shore crbs Crcinus mens with crpce width (CW) vrying from 29 to 88 mm were cught long the Norwegin cost in bited pots t pproximtely 1 5 m wter depth between Mrch nd August The smpling loctions (Fig. 1) were chosen ccording to erlier studies on cdmium in the brown crb (Julshmn et l. 2012; Wiech et l. 2017). Investigtion 1: geogrphicl vrition in cdmium Shore crbs of similr size (CW 62 ± 7 mm (men ± SD)) were collected from Kvitsøy (59 N), Sotr (60 N), Fleinvær (67 N), nd Vesterålen (68 N) during the spring of 2016 (Mrch My). From ech site, 30 shore crbs were collected with equl sex distribution, except from Fleinvær, with 27 mle nd three femle crbs. Although n effort ws mde to keep experimentl groups s uniform s possible, there ws some vrition in sizes of specimens, with the mle shore crbs from Fleinvær being significntly lrger (70 ± 3 mm) thn the other mles (64 ± 4 mm), nd the femles from Kvitsøy being significntly smller (50 ± 3 mm) thn the other femles (58 ± 4 mm). Investigtion 2: sesonl vrition in cdmium To investigte whether the cdmium content in shore crbs vries with seson, 30 shore crbs (61 ± 5 mm) were collected in the end of August 2016 (Sotr-August) in ddition to the 30 shore crbs (61 ± 5 mm) collected erlier from Sotr in the middle of April 2016 (Sotr- April). The sex distribution ws equl in both groups. Investigtion 3: physiologicl vribles nd their effect on cdmium To exmine the potentil effect of size on cdmium levels, in ddition to the 30 crbs from investigtion 1, Fig. 1 The Norwegin costline showing the four smpling res Kvitsøy, Sotr, Fleinvær, nd Vesterålen Vesterålen Fleinvær Sotr Kvitsøy

4 253 Pge 4 of 13 Environ Monit Assess (2018) 190: shore crbs were collected from Sotr nd Vesterålen during April My2016toobtinthelrgestsizernge possible (CW from 29 to 88 mm nd whole body weight from 6 to 170 g). To investigte the correltion of cdmium levels to further physiologicl vribles, severl individul physiologicl vribles were mesured in ll of the smpled shore crbs: After rrivl t the Ntionl Institute of Nutrition nd Sefood Reserch (NIFES), the crbs were scrificed following the guidelines in WHO/FAO (2012), by piercing the nerve gngli s described by Bker (1955) nd dissected freshly, s freezing nd boiling my ffect cdmium levels in crb tissues (Wiech et l. 2017). The sme observer recorded ll visully exmined mesures to minimize bis. For ech individul, crpce width, whole body wet weight, sex, dmge on the exoskeleton, nd missing legs nd/or clws were recorded. One of the following crpce colors ws ssigned: green, brown, blue/blck, ornge, or red. For femle shore crbs, the presence of sperm plug ws noted. Crbs were ssigned to one of four different molting stges (erly post molt, recent molt, intermolt, or degrded) by exmintion of crpce hrdness, levels of biofouling, nd visul indices ccording to Hig et l. (2016). To determine the heptosomtic index (HSI), n indiction of lipid stores, the heptopncres ws removed nd weighed, nd HSI ws clculted: m HP HSI ¼ 100% ð1þ m whole body m HP Where m HP nd m whole body re individul heptopncres nd whole body wet weights, respectively. Heptopncres smples were individully homogenized nd kept for nlysis. Gonds were stged s described in Hig et l. (2016) nd removed from mture femle crbs nd pooled for nlysis for ech loction seprtely. Pooled smples were lso prepred for muscle met for ech smpling re nd sex. After the wet weight ws obtined for ll smples, they were frozen nd subsequently freezedried (Freezone 18 l by Lbconco, Knss, USA) to determine the dry weight content. The wter content (WW%) ws obtined for ll smples: wet weight WW% ¼ 100% 100% dry weight ð2þ Where wet weight nd dry weight re smple weight before nd fter freeze drying, respectively. Investigtion 4: cdmium in shore crb soup To mesure possible cdmium exposure from shore crb soup, triplictes of soups were mde using crbs from Sotr nd Vesterålen, seprtely. From Sotr, 30 shore crbs were collected, with equl sex distribution (CW = 62 ± 6 nd 58 ± 4 mm for mle nd femle crbs, respectively). From Vesterålen, the selection ws limited to 15 mle crbs (CW = 74 ± 2 mm). Approximtely, the sme weight of shore crbs were used in ech triplicte (397 ± 13 nd 457 ± 1.0 g for Vesterålen nd Sotr, respectively). After the crbs were scrificed, they were cut in hlf nd fried while crushing with solid kitchen spoon in sucepn with heted vegetble oil with no slt. After bout 5 min, the crbs turned red, nd wter ws dded to cover the crbs (4 5 dl). After 30 min of boiling, the soup ws sifted off nd cooled for freeze-drying nd homogeniztion. Chemicl nlysis Freeze-dried tissue nd soup smples were homogenized nd prepred for metl nlysis using ICP-MS (icap Q) s described by Julshmn et l. (2007). The method ws ccredited ccording to NS-EN 17025, nd the qulity of the metl mesurements ws ssured by the use of the certified reference mterils (CRM), Tort- 3 (Lobster Heptopncres, Ntionl Reserch Council, Cnd) nd 1566b-O.T. (Oyster Tissue, Ntionl Institute of Stndrds nd Technology, Githersburg, USA). Averge vlues for ll metls were within 20% of the certified vlues, nd the dry weight (dw)-bsed quntifiction limit (LOQ dw ) for cdmium ws set to mg/kg with stndrd smple size (0.2 g). All individul smples were over the wet weight-bsed quntifiction limit (LOQ ww ), clculted s follows: LOQ ww ¼ LOQ dw wet weight smple dry weight smple Sttisticl nlysis ð3þ When necessry, the dt ws box-cox trnsformed to obtin normlity nd homogeneity of vrinces, tested for by norml plots nd Levene s F test,

5 Environ Monit Assess (2018) 190: 253 Pge 5 of respectively. Results were evluted using nlysis of vrince (ANOVA) followed by Tukey HSD post hoc test s the multiple comprison procedure. The significnce level ws Simple liner regression nlysis ws performed by using Person s liner correltion (STATISTICA v. 13.1, by Sttsoft, Tuls, USA). Results The men cdmium concentrtions in heptopncres ws 1.1 ± 1.2 mg/kg ww (men ± SD) corresponding to 3.4 ± 4.1 mg/kg dw (men ± SD) nd rnged from to 11 mg/kg ww corresponding to 0.13 to 39 mg/kg dw, underlining the high individul vribility. Furthermore, the cdmium concentrtions were higher for mle thn femle shore crbs for ll loctions with 1.3 ± 1.3 mg/ kg ww (men ± SD) corresponding to 4.3 ± 4.6 mg/ kg dw (men ± SD) nd 0.61 ± 0.79 mg/kg ww (men ± SD) corresponding to 2.0 ± 2.5 mg/kg dw (men ± SD) for mle nd femle shore crbs, respectively (Tble 1). Cdmium concentrtions in muscle met nd gonds were significntly lower thn in heptopncres for both sexes (p < ) with ± mg/kg ww (men ± SD) corresponding to ± mg/kg dw (men ± SD) for muscle met in clws, nd ± mg/kg ww (men ± SD) corresponding to ± mg/kg dw (men ± SD) for gonds, respectively (supplementry Tbles 1 nd 2). In muscle met, there ws no significnt difference between mles nd femles. For the nlyzed tissues, the cdmium distribution ws 99.7% in heptopncres nd 0.3% in muscle met from clws for the mle crbs. For the femle shore crbs, the cdmium distribution ws 92% in heptopncres, 0.1% in muscle met from clws, nd 7.9% in gonds. The wter content in these tissues ws 32 ± 5.3 nd 24 ± 2.1% for heptopncres nd muscle met for mle shore crbs; nd 31 ± 4.3, 24 ± 1.4, nd 42 ± 5.4% for for heptopncres, muscle, nd gonds for the femle shore crbs. More detiled vlues on weight, crpce width, dry mtter in heptopncres, nd heptosomtic index re presented in Tble 2. Investigtion 1: geogrphicl vrition in cdmium Cdmium concentrtions in heptopncres bsed on dry weight nd wet weight (Tble 1) did not vry significntly between the different smpling res for the femle shore crbs (Fig. 2). Mle crbs from Fleinvær however, hd significntly higher wet weight-bsed cdmium concentrtions in heptopncres compred to the mle crbs from Kvitsøy in Southern Norwy (p < 0.01), nd Vesterålen in Northern Norwy (p < 0.001). On dry weight bsis, cdmium concentrtions found in mle crbs from Fleinvær were in ddition higher thn in mle crbs from Sotr (p < 0.02). The mle crbs from Vesterålen did not hve higher cdmium levels compred to the crbs smpled further south (Fig. 2). There ws no significnt geogrphicl difference in cdmium levels in muscle nd gonds for neither mles nor femles (p > 0.05) (supplementry Tbles 1 nd 2). Tble 1 Cdmium concentrtions (mg/kg) bsed on wet weight (ww) nd dry weight (dw) in heptopncres of shore crbs (Crcinus mens) from the Norwegin cost Are Mle Femle N Men ± SD (ww) Rnge (ww) Men ± SD (dw) Rnge (dw) N Men ± SD (ww) Rnge (ww) Men ± SD (dw) Rnge (dw) Kvitsøy ± ± ± ± Sotr-April ± ± ± ± Fleinvær ± ± ± ± Vesterålen ± ± ± ± Sotr-August ± ± ± ± All res ± ± ± ± Men ± stndrd devition (SD) nd concentrtion rnges re given for ech group

6 253 Pge 6 of 13 Environ Monit Assess (2018) 190: 253 Tble 2 Weight (g), crpce width (CW mm), dry mtter content in heptopncres (DM, %), nd heptosomtic index (HSI, %) of shore crbs (Crcinus mens) from different sites long the Norwegin cost Are Mle Femle Weight (g) CW (mm) DM (%) HSI (%) Weight (g) CW (mm) DM (%) HSI (%) Kvitsøy 58 ± ± ± ± ± ± ± ± 0.99 Sotr-April 73 ± ± ± ± ± ± ± ± 2.4 Fleinvær 86 ± ± ± ± ± ± ± ± 0.89 Vesterålen 74 ± ± ± ± ± ± ± ± 1.6 Sotr-August 54 ± ± ± ± ± ± ± ± 1.0 All res 72 ± ± ± ± ± ± ± ± 2.1 Men ± stndrd devition (SD) nd concentrtion rnges re given for ech group Investigtion 2: sesonl vrition in cdmium Crbs smpled in August hd lower wet- nd dry weightbsed cdmium concentrtions in heptopncres thn in April, nd the difference ws significnt for the femle crbs (p < 0.03), while not significnt for the mle crbs (p > 0.05) (Fig. 3). The totl cdmium content in heptopncres ws not sttisticlly significntly different between August nd April (p > 0.1). However, there ws cler trend in mesured cdmium concentrtion, with bout two times lower concentrtions for both sexes in August thn in April. Sttisticlly, the difference ws probbly covered by the lrge vrition between individuls. There ws no significnt sesonl vrition in cdmium levels in muscle nd gonds (p >0.7). Investigtion 3: physiologicl vribles nd their effect on cdmium The size prmeters crpce width nd whole body weight were strongly correlted for both sexes (r 2 = 0.90, p < nd r 2 =0.88, p <0.001 for mle nd femle shore crbs, respectively (Supplementry Tble 3). Crpce width ws chosen s the min size prmeter for further exmintion, nd it ws positively correlted with wter content in heptopncres for both mle (r 2 = 0.25, p < ) nd femle (r 2 = 0.32, p < ) shore crbs. The heptosomtic index ws negtively correlted with crpce width for the mles (r 2 = 0.35, p < ), though not for femles (r 2 =0.026, p <0.2). Fig. 2 Geogrphicl vrition in cdmium concentrtions in heptopncres (mg/kg wet weight) of mle (left pnel, n = 72) nd femle (right pnel, n = 48) crbs collected from Kvitsøy nd Sotr in south, nd Fleinvær nd Vesterålen in north (medin ± 25% percentile is given, nd tringle symbols shows the men for ech loction; smpling points with no letters in common show sttisticlly significnt differences) Cd [mg/kg HP ww] Mle b b Femle 0.0 Kvitsøy Fleinvær Sotr Vesterålen Kvitsøy Fleinvær Sotr Vesterålen

7 Environ Monit Assess (2018) 190: 253 Pge 7 of Fig. 3 Cdmium concentrtions in heptopncres (mg/kg wet weight) of mle (left pnel, n = 30) nd femle (right pnel, n = 30) crbs collected from Sotr in April nd August (medin ± 25% percentile is given, nd tringle symbols shows the men for ech loction; smpling points with no letters in common show sttisticlly significnt differences) Cd [mg/kg HP ww] Mle Femle b April August April August Overll, size ws not correlted with cdmium concentrtions in heptopncres despite the reltively lrge size vrition for the shore crbs from Sotr nd Vesterålen (Fig. 4) difference of bout three times between mximum nd minimum crpce width. For the mle crbs from Vesterålen, there ws wek significntly positive correltion between crpce width nd cdmium concentrtions (r 2 =0.15,p <0.01onwet weight bsis nd r 2 = 0.29, p < on dry weight bsis). The correltion ws pronounced for crpce width nd totl cdmium content in heptopncres (r 2 =0.47,p < ). For mle crbs from Sotr, cdmium concentrtions were not significntly correlted with crpce width, but the totl cdmium content in Fig. 4 Reltionship between cdmium concentrtion in heptopncres (mg/kg wet weight) nd crpce width of mle (n =75)(, c) ndfemle (n =45)(b, d) shore crbs from Vesterålen in north (upper pnel) nd Sotr in south (lower pnel) Cd [mg/kg HP ww] Vesterålen Sotr A C B D Crpce width (cm)

8 253 Pge 8 of 13 Environ Monit Assess (2018) 190: 253 heptopncres showed wek correltion with crpce width (r 2 =0.13,p < 0.05). The cdmium concentrtions were not significntly correlted with crpce width for femle crbs from neither Vesterålen nor Sotr. However, the cdmium content ws wekly correlted with crpce width for the femles from Sotr (r 2 =0.15,p <0.05). Overll, there ws no cler correltion between cdmium concentrtion in heptopncres (mg/kg ww) nd the physiologicl vrible wter content. Only for mle shore crbs from Vesterålen wek significntly positive correltion (r 2 =0.20, p < 0.05) ws observed. On dry weight bsis (mg Cd/kg w), the correltion ws pronounced (r 2 =0.39, p < 0.001). In ddition, there ws significntly positive correltion between wternd cdmium content in heptopncres for the mle shore crbs from Vesterålen (r 2 =0.30,p <0.001). Mle shore crbs hd higher cdmium concentrtions nd higher totl mount of cdmium in heptopncres in ll experimentl groups (p < 0.003). Physiologicl vribles tht were significntly different between mle nd femle shore crbs might be of importnce for the different cdmium levels. The mle crbs were significntly lrger thn femles in both CW nd body weight (p < ), nd the femles hd significntly lower heptosomtic index (p < 0.02) (Tble 2). However, there ws no cler correltion between HSI nd cdmium levels when sexes were segregted, except for significnt negtive correltion between cdmium concentrtion on dry weight bsis nd HSI for the mle shore crbs from Vesterålen: r 2 = 0.27, p < 0.001). However, reltionship between cdmium concentrtion nd HSI ws indicted, s the femles hd significntly higher HSI nd lower cdmium levels in heptopncres. Additionlly, both sexes hd significntly higher HSI (p < ) nd lower cdmium concentrtion in August thn April t Sotr. For the femles from Sotr-April nd Sotr-August in totl, the correltion between cdmium concentrtion nd HSI ws significntly negtive (r 2 =0.24,p < 0.006), wheres there ws no significnt correltion between totl cdmium content in heptopncres nd HSI (p > 0.05). Femle crbs showed sesonl trend in dry mtter content in Sotr with lower dry mtter content in August (p = 0.066), while this ws not observed in mle crbs. In mle crbs, the dry mtter content incresed significntly from recent molt to inter-molt crbs, while this ws only visible s trend in femles (supplementry Fig. 1). The proportion of red femles ws lrger thn for mles (pproximtely 65 nd 50%, respectively), but no significnt correltion ws found between crpce color nd cdmium levels (p > 0.05) (supplementry Fig. 2). There were no cler nd consistent reltionships between cdmium nd other physiologicl vribles such s molt stge (supplementry Fig. 3), gond mturtion stge (supplementry Figs. 4 nd 5), nd the presence of sperm plugs, probbly linked to high vrition in Cd vlues nd low vrition in the mesurement of the prmeters. Investigtion 4: cdmium in shore crb soup The cdmium concentrtions in shore crb soups from Sotr nd Vesterålen rnged from 17 to 79 μg/kg ww, with verge vlues of 28 ± 11 nd 63 ± 14 μg/kg ww, respectively. In totl, the verge cdmium concentrtion ws 44 ± 22 μg/kg ww soup. Bsed on cdmium levels in heptopncres in shore crbs from Sotr nd Vesterålen (Tble 1), it ws clculted tht pproximtely 62% of the crbs cdmium levels were extrcted into the soup during the cooking process. Discussion Geogrphicl vrition in cdmium The min objective in the present study ws to investigte whether the shore crb shows s lrge differences in cdmium concentrtions in heptopncres s hve been found erlier in brown crbs from northern nd southern sites long the Norwegin cost. The cdmium concentrtions in the shore crbs in this study did not follow such cler geogrphicl pttern. Only mles from one of the two loctions in the North showed significntly higher concentrtions. This is in contrst to the brown crb, where concentrtions in the brown met were significntly higher in Northern Norwy, compred to Southern Norwy (Julshmn et l. 2012). Except for the reltively high cdmium concentrtions in mle shore crbs from Fleinvær, the cdmium levels in the nlyzed tissues in the present study correspond firly well to erlier published vlues for shore crbs from Denmrk nd Scotlnd (Depledge nd Bjerregrrd 2002; Bjerregrd 1982, 1990; Rinbow 1985). The mle shore crbs from Fleinvær were lrger thn the other mles, nd one reson for their high cdmium levels

9 Environ Monit Assess (2018) 190: 253 Pge 9 of my be tht they hve forged on different nd potentilly lrger nd older prey, with potentilly higher cdmium levels. It is lso possible tht the cdmium levels re higher in this re, lthough the cdmium concentrtions in the femles from Fleinvær were not correspondingly high. However, this my be due to the low smpling size of femle shore crbs from this re, with only three femles from Fleinvær. Brown crbs from the sme re did not show elevted cdmium levels compred to other smples in the North (Julshmn et l. 2013b; Julshmnetl.2012). The differences in geogrphicl cdmium pttern between shore crbs nd brown crbs from the Norwegin cost my be explined by severl cuses. As food presumbly is the most importnt cdmium source for crbs (Dvies et l. 1981; Bjerregrd et l. 2005), differences in diet might be of importnce for the different cdmium levels between shore crbs nd brown crbs smpled long the Norwegin cost. Compred to the cdmium levels in the brown crb (Julshmn et l. 2012), the levels in the shore crb in this study were low long the whole Norwegin costline. The brown crb generlly consumes lrger orgnisms (Mscr nd Seed 2001) with potentilly higher cdmium levels. Further, differences in diet might be result of their different distribution in the wter column, s the shore crb generlly lives in shllower wters (Crothers 1968; Nel nd Wilson 2008), where different prey species might be bundnt. Crbs lso ccumulte cdmium from the wter phse (Jennings nd Rinbow 1979; Weis2012; Dvies et l. 1981), nd it is possible tht differences in cdmium ccumultion from the wter is ssocited with the higher cdmium levels in brown crbs compred to shore crbs. As the brown crb is more bundnt in deeper wter, it is possible tht it is more exposed to the cdmium rich deep-wter (Flk nd Nøst 2013, Jnssen et l. 2014) thn the shore crb. However, the cdmium levels in brown met in brown crbs from Northern Norwy re shown to be higher in mles (Frntzen et l. 2015), even though femles generlly migrtes to greter extent to deeper wters where the cdmium concentrtions potentilly re higher (Ungfors et l. 2007; Flk nd Nøst 2013). Further, severl studies indicte tht the rtes of cdmium ccumultion from the wter phse to the heptopncres re too low to explin the high cdmium levels in heptopncres nd/ or brown met (Bjerregrd et l. 2005; Dvies et l. 1981; Jennings nd Rinbow 1979; Nørum et l. 2005). It might be n explntory feture tht the shore crb could probbly be better dpted to the cold climte in Northern Norwy compred to the brown crb. The wter temperture long the Norwegin cost generlly decreses with incresing ltitude, nd the men temperturefor2015to2017tsognesjøen(61 N),sttion close to our smpling sites in Southern Norwy, ws 10.0 C, rnging from 5.8 to 15.6 C t depth of 5 m (IMR 2018). At sttion in the proximity of our northernmost site, Eggum (68 N), men temperture of 8.3 C with, rnging from 4.7 to 12.3 C ws mesured for 2015 to 2017 t depth of 5 m(imr 2018). The shore crb is known to be very robust nd survives wide rnge of tempertures from pproximtely 0 to 35 C nd tolertes slinities from 4 to 52 (Klssen nd Locke 2007). This suggests tht the shore crb grows eqully good in northern nd Southern Norwy. There is evidence tht brown crbs do not feed t ll t tempertures below 5 C, s well s migrtionislimited(krlssonnd Christinsen 1996). Further, survey of brown crbs hs shownthttheymoltlessfrequentlyinnortherncompred to Southern Norwy (Snorre Bkke, personl communiction, Jnury 23, 2017). Consequently, the growth rte will be reltively lower for brown crbs from Northern Norwy. As such, brown crb of given size from Northern Norwy might hve hd longer time to ccumulte metls such s cdmium nd will consequently hve higher cdmium levels compred to brown crb of similr size smpled further south. This will probbly not pply for the shore crb, under the ssumption tht shore crbs hve similr growth rtes in the north nd south. Further, Bergey nd Weis (2007) hve suggested molting s mechnism for depurtion of led for the fiddler crb Uc pugnx. If molting is fesible mechnism for crbs to lso deprture cdmium, it is possible tht lower molting frequency for brown crbs from Northern Norwy results in less cdmium excretion nd thereby higher cdmium concentrtions compred to brown crbs from Southern Norwy. Sesonl vrition in cdmium In greement with findings from Bjerregrd et l. (2005), the results in the present study showed tht the cdmium concentrtions in heptopncres vry with seson. The concentrtion ws significntly lower for femle shore crbs smpled in August thn April. The sesonl vrition might be explined in terms of chnging biovilbility of the metl due to chnging

10 253 Pge 10 of 13 Environ Monit Assess (2018) 190: 253 physicochemicl conditions of the environment s well s chnging physiologicl stte of the individuls. With rising wter temperture, the crbs ctivity will probbly increse (Klssen nd Locke 2007; Griffen et l. 2012), which my led to incresed food intke. This is indicted by significntly higher heptosomtic index for both sexes in August thn April. With higher HSI, the lipid stores consequently increse, which seems to lower the cdmium concentrtion by dilution, s indicted by the significntly negtive correltion between HSI nd cdmium concentrtion for the femles smpled in August nd April in totl. Further, the higher concentrtions of cdmium in heptopncres in April could be explined by the tendency to higher wter content in heptopncres in the crbs smpled in August. This is consistent with the totl content of cdmium not differing between the 2 months. In ddition, the lower cdmium levels in August might be consequence of potentilly shorter biologicl hlf-life of cdmium s the temperture increses during summer, with subsequently higher ctivity mong the crbs, s discussed by Bjerregrd et l. (2005). Sesonl chnges in physiologicl vribles such s ovrin mturtion nd molt stge (Bondgrd et l. 2000: Bondgrd nd Bjerregrd 2005; Nørum et l. 2005) might lso influence the cdmium levels, but the spn in vrition for these prmeters ws too low to revel ny effects. In greement with the results in the present study, elevted cdmium levels re reported for the Americn oyster (Crssostre virginic) in April with decline throughout the summer (Frzier 1979). As the biovilbility of cdmium increse with temperture (Klssen nd Locke 2007; Ry 1984; Rinbow 1997; Burke et l. 2003), the cdmium ccumultion from the wter phse would probbly be higher for the shore crbs in August thn April. However, the cdmium levels were not elevted in August for the shore crbs in this study. Therefore, cdmium uptke from the wter phse does not explin the observed vritions. Physiologicl vribles nd their effect on cdmium For both sexes, the size prmeters were positively correlted with ech other nd with wter content in heptopncres, in greement with other studies (Bjerregrd nd Depledge 2002; Nissen et l.2005). For the mle shore crbs, the HSI ws negtively correlted with size, which indictes tht the reltive energy reserves decrese with incresing size. In concordnce to Nørum et l. (2003), we found trend towrds higher dry mtter content in heptopncres in intermolt crbs in comprison to recent molt crbs. This is most likely result of ctive forging. We found n indiction of cdmium ccumultion over the crb s lifetime, s the totl mount of cdmium ws correlted with size. However there ws no consistent effect of size on cdmium concentrtions even though the rnge in size ws reltively lrge. The increse of wter content with size might msk the increse of the wet weight-bsed concentrtion. Little or no correltion between size nd cdmium concentrtions hs been found for shore crbs from Denmrk (Bjerregrd nd Depledge 2002; Nissen et l.2005), king crbs (Pseudocrcinus gigs) from Austrli (Turoczy et l. 2001), nd brown crbs from Norwy (Julshmn et l. 2012). It is possible tht crbs excrete cdmium during molting (Bergey nd Weis 2007), which could explin why the cdmium concentrtions do not increse considerbly with size. However, this needs to be elucidted further. There ws cler difference between the sexes regrding cdmium concentrtions in heptopncres. The cdmium concentrtion ws more thn twice s high for the mle shore crbs. In correspondence with these results, Bjerregrd et l. (2005) lso found generlly lower cdmium concentrtion in femle heptpncres, compred to mle shore crbs. As food presumbly is the most importnt cdmium source for shore crbs (Bjerregrd et l. 2005; Pedersen et l. 2014), it is possible tht differences in forging strtegy between mle nd femle shore crbs my led to differences in ccumultion of cdmium. The sexes behve differently in the costl zone, where the femles generlly sty t deeper wters thn the mles (Reid et l. 1997). Furthermore, the mles re generlly lrger, with bigger nd presumbly stronger clws, which enbles forging on lrger orgnisms (Kiser et l. 1990) with potentilly higher cdmium levels. The cdmium concentrtion in muscle met from clws ws not significntly different between mle nd femle shore crbs. The mjority of the ccumulted cdmium ws mesured in heptopncres, nd the whole body cdmium distribution ws pproximtely 92, 7.9, nd 0.10% in heptopncres, femle gonds, nd muscle met from clws, respectively, for the nlyzed tissues. The cdmium distribution in the nlyzed tissues ws similr to other studies on both shore crbs nd brown crbs (Bondgrd nd Bjerregrd 2005;

11 Environ Monit Assess (2018) 190: 253 Pge 11 of Bjerregrd et l. 2005; Weis 2012; Frntzen et l. 2015; Wiech et l. 2017). Except for cler reltionship between cdmium concentrtions nd sex nd wek correltions between heptosomtic index nd crpce width nd cdmium, there ws no correltion between cdmium concentrtions nd the registered physiologicl vribles. Limited rnge in visully ssessed prmeters such s crpce color, number of legs nd clws, molting stge, presence of sperm plugs, nd gond mturtion might be the reson why no reltionships between cdmium levels nd these individul prmeters were found. Other studies hve shown higher cdmium ccumultion rtes for crbs in erly post-molt nd erly ovrin mturtion stges when exposed to cdmium in wter (Bondgrd et l. 2000; Bondgrd nd Bjerregrd 2005; Nørum et l. 2005), nd green shore crbs seems to ccumulte more cdmium thn red shore crbs (Nissen et l. 2005; Styrishve et l. 2000). Cdmium in shore crb soup Even though the mount of cdmium extrcted from the crbs to the soup ws reltively high, the cdmium concentrtions in the prepred shore crb soup were low. Therefore, shore crb soup ws considered to be sfe regrding cdmium exposure. Bsed on the highest mesured cdmium concentrtion of 79 μg Cd/kg ww, portion size of 100 g would constitute pproximtely 5% of the tolerble weekly intke (TWI) for person weighing 70 kg, bsed on the TWI of 2.5 μg Cd/kg body weight (EFSA 2009), set by the Europen Food Sfety Authority (EFSA). However, it is estimted tht the verge cdmium exposure from food is pproximtely 1.7 μg/kg body weight per week for n dult Norwegin person (VKM 2015). Tking this into considertion, the dditionl dietry cdmium exposure llocted to other dietry sources is 56 μg per week given body weight of 70 kg, which corresponds to pproximtely seven portions of shore crb soup per week. As such, shore crb soup is not considered problemtic regrding food sfety. Furthermore, cooking time of 30 min my be excessive s the soup my become bitter during the long cooking process (personl observtion). However, it ws chosen s worst-cse scenrio to ensure sufficient cdmium extrction. In ddition, ll the pooled smples were under the legl limit of 0.5 mg Cd/kg ww in clw met for humne consume, set by EU (EU 2006). Conclusion The cdmium concentrtions in shore crbs in this study were very low in muscle met from clws nd between mg Cd/kg ww nd 11 mg/kg ww in heptopncres. There ws no cler geogrphicl difference with ltitude s opposed to erlier findings in the brown crb. Possible explntions for this my be tht these species hve different feeding hbits or tht the shore crb is better dpted to the colder climte in Northern Norwy. Sex hd cler impct on the cdmium levels in heptopncres, s the mle shore crbs hd pproximtely twice s high cdmium levels compred to the femles. No cler nd consistent correltions were found between cdmium nd other registered individul vribles, but some minor reltionships were seen with n indiction of cdmium ccumultion over time s well s wek reltionship between cdmium concentrtions nd fluctuting wter contents of tissues. Cdmium concentrtions were lower in August thn in April. Most of the totl mount of cdmium ws llocted in the heptopncres while muscle met nd gonds of femles contributed together with less thn 10%. None of the mesured cdmium levels exceeded EUs legl limit of 0.5 mg Cd/ kg ww set for clw met for humn consumption. Low cdmium levels in shore crb soup mke it sfe food item regrding cdmium nd food sfety. Acknowledgements This work hs been supported by the Norwegin Food Sfety Authority. We lso wish to thnk Associte Professor Svein Are Mjøs t the University of Bergen for his dvice on this reserch nd on the sttisticl nlyzes of dt. Open Access This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License ( cretivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. References Bker, J. R. (1955). Experiments on the humne killing of crbs. Journl of the Mrine Biologicl Assocition of the United Kingdom, 34(1), Bkketeig, I. E., Huge, M., Kvmme, C., Sunnset, B. H., & Toft, K. Ø. R. (2016). Hvforskningsrpporten 2016: Tskekrbbe. In Fisken og hvet, 1, 186. Bergen: Institute of Mrine Reserch (IMR).

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