ANNALE S • Ser. hist. nat. • 10 • 2000 • 1 (19) 
original scientific paper UD C 574:594.1 

THE INFLUENCE OF MARINE ANOXIA O N PRECIPITATION OF MYTILUS 
CALLOPROVINCIALiS SHELL CARBONATE IN THE COASTAL ZONE 
OF THE ROVINJ BAY (NORTHERN ADRIATIC) 

Tadej DOLENEC 
Department of Geology, Faculty of Natural Sciences and Engineering, University of Ljubljana, SI-1000 Ljubljana, Aškerčeva 12 and 
Jožef Stefan Institute, Si-1000 Ljubljana, Jamova 39 

Davorin MEDAKOVIČ 
Ruder Boškovič Institute, Center for Marine Research, HR-52210 Rovinj, Giordano Paliaga 5 
Sonja LOjEN 
Jožef Stefan Institute, SHGOO Ljubljana, Jamova 39 
ABSTRACT 
The results of this study suggest changes in shell growth structure and in the isotopic, as well as chemical composition of Mytiius galloprovincialis shells due to anoxia in the coastal zone of the Rovinj Bay, Northern Adriatic. The outer aragonitic "stress layer" of Mytiius galloprovincialis shells formed during pronounced summer anoxia due to intensive phytoplankton bloom in the years 1989 -1991 is massive, pink coloured and out of oxygen and carbon isotopic equilibria. It also exhibits higher C/O ratios relative to the uncoloured aragonitic layer precipitated by Mytiius galloprovincialis living in normal oxic conditions. 
Key words: Mytiius galloprovincialis, biomineralization, stable isotopes, anoxia, Rovinj Bay, Northern Adriatic 
INFLUENZA DELL'ANOSSIA MARIN A SULLA PRECIPITAZiONE CARBONATICA DELLA 
CONCHIGLI A Di MYTILUS GALLOPROVINCIALIS NELLA ZON A COSTIERA DELLA BAIA 
DI ROViGN O (ADRIATICO SETTENTRIONALE) 

SlNTESl 
I risultati di questo studio evidenziano variazioni nella struttura di crescita e nella composizione sia isotopica che chimica delle conchiglie di Mytiius galloprovincialis, dovute ad anossia nella zona costiera delta baia di Rovigno, Adriático settentrionale. Lo strato esterno di aragonite delle conchiglie di Mytiius galloprovincialis, formatosi durante una marcata anossia estiva dovuta ad un'intensiva fioritura fitoplanctonica negli anni 1988-1991, e massiccio, di colore rosa e non ríspetta I'equilibrio isotopico di ossigeno e carbonio. Questo strato eslbisce, inoltre, frazioni C/O piu alte rispetto a quelle dello strato di aragonite incolore, precipitato da Mytiius galloprovincialis in condizioni normali di ossigeno. 
Parole chiave: Mytiius galloprovincialis, biomineralizzazione, isotopi stabili, anossia, baia di Rovigno, Adriático settentrionale 
55 
ANNALES • Ser. hist. nat. • 10 • 2000 • 1 (19) 
Tadej DOL EN EC el si.-. THE iNFLUENC f O F MARIN E ANOXSA O N PRECIPITATION OF MYTILU5 CALLOPROV1NCIAUS.... .55-60 
INTRODUCTIO N 

Biomineralization is a complex combination of bio­chemical and physiological processes, depending on the endogenous activity of the organism and exogenous environmental influences. However, the influence and interaction between environmental factors, such as lack of oxygen and biomineralization, have been insuffici­ently investigated so far. This paper reports on the and ô 18 0 values measured in selected pink coloured shell layers taken from specimens of Mytilus gal­loprovincialis, which were found growing in the coastal zone of the Rovinj Bay, Northern Adriatic, during anoxic conditions caused by intensive phytoplankton bloom in the years 1989-1991. These data were used to make interpretations regarding alterations in biomineralization processes influenced by environmental changes. 
MATERIALS AND METHODS 

Determination of 5 1s O and 8 13 C was performed on separate shell layers using a Varian MAT 250 mass spectrometer. Organic matter was eliminated prior to the determination of 
ô 18 0 and 8 13 C by roasting the molluscan carbonate powder under vacuum for 1 hour at 380° C Thereupon the carbonate powder was reacted with > 100 % H3PO4 at 25°C (McCrea, 1950). C0 2 gas released during acid treatment was cryogenically cleaned and analyzed for O and C isotopic composi­tion. Data quality was maintained in part through fre­quent comparisons against internationally recognized isotopic standards and frequent processing of secondary laboratory standards. Before acid treatment, the minera­logical composition of the selected layer powder was checked by X-ray diffraction to determine the arago­nite/calcite ratio. Ail results reported here are for sam­ples of > 95 % aragonite or > 95 % calcite. Results are expressed relative to PDB for 6 18 0 and 5 13 C . The aver­age difference of duplicate analyses was about 0.1 %o for oxygen and 0.09 %ofor carbon. 
For characterization of the microstructure and com­position of Mytilus gailoprovincialis shells, SE M and EDS analyses were applied. A JEOL JS M 5800 SEM equipped with a Link ISIS 300 EDS was used for overall analyses. Quantitative analyses were performed using SE M Quant software and a virtual standard package-li­brary (VSP). Measured peak intensities in the spectra were corrected and quantified using a ZAP matrix cor­rection program. In the case of oxygen, semi-quantita­tive estimation is possible only by comparison between the peak areas of oxygen peaks and different spectra without exact quantification. The relative random errors of EDS were less than 3 % for major and selected minor oxides. 
Fig. 1; SEM photo of calcite fibres (C) and prismatic nacreous aragonite (A) layer in Mytilus gailoprovincialis shell. Si. 1: Elektronski (SEM) posnetek apnenčastih vlaken 
(C) 
in prizmatičnega biserovinastega aragonitnega sloja 

(A) 
pri lupinicah školjke Mytilus gailoprovincialis. 


Fig. 2: SEM photo of the visible difference in morpho­logy between the uncoloured (U) and pink (P) parts of the aragonite layer of a Mytilus gailoprovincialis shell structure. SI. 2: Elektronski (SEM) posnetek vidnih morfoloških razlik med neobarvanim (U) in rožnatim (P) delom aragonitnega sloja v sestavi lupinic školjke Mytilus gai­loprovincialis. 


RESULTS AN D DISCUSSION 
SE M analyses showed that Mytilus gailoprovincialis has an outer shell layer composed of calcite fibres and an inner nacreous shell layer, which is porcelaneous in appearance. Single fibres of the outer layer are ap­proximately 1.5 pm thick and are inclined towards the shell exterior (Fig. 1). The inner layer is composed of equidimensional, horizontally flattened aragonite prisms about 10-2 0 pm wide, with their long axes oriented perpendicular to the shell surface. In pink coloured 
56 


ANNALES • Scr. hist. nat • 10 • 2000 • 1 (19) 
Tadej DQLENE C etui.-. THE INFLUENCE Of; MARINE ANOX5A O N PRE Ci WATSO N O F MYTILUS GALLOPROVINCIAIIS..., 55-60 
sheHs, the inner layer is roughly divided into two sublayers. The inner uncoloured white sublayer consists of the same structure of flattened aragonite prisms, while the outer sublayer, up to 900 pm thick, is pink and composed of irregular aragonite grains and shows a massive structure (Fig. 2). These aragonite crystals are supposed to be precipitated during stress conditions and more rapidly than uncoloured ones. 
Comparison between peak areas of the oxygen and carbon peaks shows that the pink coloured aragonite contains more carbon than oxygen relative to the un­coloured aragonite or calcite (Figs. 3, 4). The C/O ratio of the pink layer is 1.4 and 2.3 while that of the uncol­oured aragonite from uncoloured and coloured shells varies between 0,9 and 1,2. Similar C/O values of 0.98 and 1 were also measured in calcite fibres. W e believe that the high C/O ratio of the pink aragonite layer indi­cates anoxic conditions during its precipitation. 
The Mytilus galloprovinciaiis shell carbonates show variation of 5 1s O in the range between + 0.07 and + 
2.21 % , and 5 13 C between + 0.18 and - 1.31 %o (Tab. 1). In uncoloured shells, the calcitic layer is either slightly enriched in the heavy oxygen isotope relative to the inner aragonite layer or contains more or less the 
.same quantity of heavy oxygen isotope as the aragonite one. Biogenic aragonite has been reported as having both 1s O depletion {Tarutani ei al., 1969; Horibe & Oba, 1972) and enrichment {Sornmer & Rye, 1978; Grossman, 1982) compared to biogenic calcite. The 
cps 
c o 
60-c/o = 1 C/ O = 0. 9 
MS magnitude of the 18 0 enrichment of aragonite relative to mollusc calcite was not temperature dependent (Grossman & Ku, 1986). In the coloured shell the uncol­oured aragonitic sublayer exhibits a higher S !e O value than the pink aragonitic sublayer and/or calcitic layer. The 1.22 %« enrichment in 8 1S 0 of the uncoloured aragonite sublayer relative to the pink layer would translate into a change of 4.5°C of seawater temperature using the Grossman & Ku (1986) equation. Such large changes in ambient seawater temperature most probably indicate a temperature-induced seasonal 5 ie O signal as well as isotopic disequilibrium. The pink aragonite layer was formed between July and August during seawater anoxia when the ambient water was warmer. 

The S 13 C of mussel carbonates vary between 0.18 and - 1.26 %o. In all investigated samples the calcite layer contained more light carbon ^ C isotope than the aragonite one. The isotopic difference between the calcite and aragonite layers most probably corresponds to different equilibrium fractionation in the caicite-water and aragonite-water systems. Aragonite was found to be enriched by 1 %0 in13C relative to coexisting calcite at the relevant temperatures (Grossman, 1984; Grossman & Ku, 1986)- In the coloured shell the pink aragonite sublayer was enriched (up to 0.91 %o) in light 1?-C rela­tive to the uncoloured aragonite sublayer. It is interest­ing to note that there is a negative correlation between S ,a O and S 13 C for the fibrous calcific layers and a posi­tive one for aragonitic layers (Fig. 5). 
cps 
e»­C/ O = 1.2 
14
2 
Energy (kcV) 

Fig. 3: Energy Dispersive Spectroscopy (EDS) spectra and C/O ratio of an uncoloured Mytilus galloprovinciaiis shell showing only small differences in the maxima of the oxygen and carbon peaks between the carbonate layers. 
(A) fibrous calcitic layer, (B) outer part of the aragonitic layer, (C) inner part of the aragonitic layer. SI. 3: EDS-spektri in C/O-razmerje neobarvane školjke Mytilus galloprovinciaiis kažejo le manjše razlike v največjih vsebnostih kisika in ogljika med dvema karbonatnima slojema. (A) vlaknati apnenčasti sloj, (B) zunanji del aragonitnega sloja, (C) notranji del aragonitnega sloja. 
57 

ANNALE S Ser. hist. nat. • 10 • 2000 1 (19) 
Tadej DOLENE C e i si: THE (NFi.UENCE O F MARINE ANOXi A O N PRECIPITATION Of MYTHUS GALLOPROVINCIALIS..., 55-60 
tps  CilS  tps «H  cps 60  
C/ O «  0.9 8  C/ O  =  1. 1  
c 9  

B 

Energy (keV) 

Fig. 4: Energy Dispersive Spectroscopy (EDS) spectra and C/O ratio of pink coloured Mytiius gelioprovincialis shell showing appreciable differences in the maxima of the oxygen and carbon peaks between different carbonate layers. (A) fibrous calcific layer, (B) uncobured aragonitic sublayer, (C) inner part of pink aragonitic sublayer, (D) outer part of pink aragonitic sublayer. SI. 4; EDS-spektri in C/O-razmerje rožnato obarvanih lupinic školjke Mytiius geliopro vine i a Iis kažejo na znatne razlike v največjih vsebnostih kisika in ogljika med dvema karbonatnima slojema. (A) vlaknati apnenčasti sloj, (B) neobarvani aragonitni podsloj, (C) notranji del rožnatega aragonitnega sloja, (D) zunanji del rožnatega aragonitnega sloja. 
Tab. 1. Stable oxygen and carbon isotope composition of Mytiius galloprovincialis shell carbonates. Tab. 1: Izotopska sestava kisika in ogljika v karbonatih lupinic školjke Mytiius galloprovincialis. 

Sample BuO (PD B %o) 513C (PDB %») Remarks 
R-202 a 1.65 - 1.04 calcite 
R-202 b 1.04 - 0.32 aragonite 
0-20 4 a 1.13 - 0.84 calcite 
0-204 b 1.39 0.18 aragonite (u) 
0-204 c 0.07 - 0.73 aragonite (p) 
M-202 a 1.01 - 1.31 calcite 
M-202 b 0.98 -0.14 aragonite (pk) 
S-1 a 2.21 - 1.26 calcite 
S-1 b 1.22 - 0.15 aragonite 
ST-1 a 0.33 - 0.77 calcite 
ST-1 b 0.81 -0.19 aragonite 
R-202, 5-1, ST-1 (uncoSoured Mytiius galloprovincialis shells), 0-204, M-202 (pinky to pink coloured Mytiius galloprovincialis shells), (u) - uncoloured, (p) -pink, (pk> -pinky 
R-202, S-1, ST-l (neobarvane iupinice školjke Mytiius gallo­provincialis), 0-204, M-202 (rahlo rožnato do rožnato obarvane Iupinice Školjke Mytiius galloprovincialis, (u) -neobarvane, (p) ­rožnate, (pk) - z rožnatim nadihom 
The water temperature, salinity, and the total dis­solved carbon (IDC ) content have been recognised as major factors controlling the oxygen and carbon isotope composition in shell carbonate of marine invertebrates (Anderson & Arthur, 1983; Rosenberg, 1980). Molluscs, in general, are believed to exert only a minimal vital ef­fect over their isotopic composition (jones, 1985), and thus their isotopic values are representative of environ­mental water conditions. However, calcium carbonate may not always be precipitated in equilibrium with the environment, and in such cases stable isotope analyses of the shell carbonate may be an unreliable technique for environmental reconstruction. Isotopic disequilib­rium may be in part due to metabolic effects or kinetic effects that are inherent in fast-growing shells, or areas of shell (Mitchell ef ai, 1994). Man y molluscs use aragonite to build their shell, but calcific molluscs and those with mixed mineralogy also are abundant. Be­cause aragonite is found to be enriched by 0.7 %«i n -O and by 1 %<> in 13 C relative to coexisting calcite at the relevant temperatures (Grossman, 1984; Grossm; i \ Ku, 1986) the calcific and aragonitic layers in moP . shells with mixed mineralogy must be sampled a;:d treated separately. In shells with mixed aragonite-ca'c composition, low temperatures are favourable i':r deposition of calcite, while warm waters favour the i'-r­mation of aragonite (Schifano & Censi, 1986). 
58 

ANNALES Ser. hist. nat. • 10 2000 • 1 (19) 
Tadej DOIENE C el d/.: THE INFLUENCE O F MARINE ANOXI A O N PRECIPITATION O F MYTHUS GALLOPROVINOAUS..., 55-60 
The calculated temperature for Mytilus galloprovin­
cialis shell calcite according to the equation of Craig 
(1965): 
T[°C] = 16.9 -4.2(SC - 8w) + 0.1 3(8r -8W )2 
(where 8 c is the oxygen isotopic composition of 
calcite vs. PDB and §vv !s oxygen isotopic com­
position of the water from which the calcite was pre­
cipitated vs. SMOW) , range from 11.8 to 19.8°C, while 
that of aragonite calculated according to Grossman & 
Ku (1986): 
T[°C] - 21.8 -4,69(S18 Oaragonit e -S18 Owater ) 
is between 20 and 26.2°C. Because no water sam­
ples were collected, the 5 1s O of seawater of + 1.0 %o had to be taken from the literature (Dolenec ef al, 2000). The cafculated values are generally regarded as typical of conditions in the investigated areas. The calculated temperature for Mytilus gailoprovinciaiis further implies that this mussel most probably calcifies shell carbonate ail the year round. During the colder -months of the year the mussel precipitated more calcite, : while during the summer this organism calcifies more 
-aragonite. 



Aragonitic line 
0.4 ] 


Y = 0.70 * X - 0.77 

N P 
o \ 

ST -0.4 
Q 

Calcific line 
-0.8 


Y s -0.21 * X - 0.78 

to 
-1.2 ­
-1.6­
0.5 1 1.5 2 2.5 



8!80 (PDB) % o 
FiS- 5. 8?s O vs. §13C diagram for Mytilus gailoprovinciaiis shelf carbonates showing a positive trend for aragonite (O) and a negative one for calcite (•). SI. 5: 8T80 vs. <b13C-diagram za karbonate luptnicŠkoljke Mytilus gailoprovinciaiis kaže pozitiven trend za aragonit (O) in negativen trend za kalcit C). 
Most marine carbonates reflect the S 13 C of total dis­solve d inorganic carbon (TDC) of the water in which they form (Anderson & Arthur, 1983), and this is prob­ably also the case with the Mytilus gailoprovinciaiis from the investigated area. Whereas many organisms display a vital carbon isotope fractionation effect, mol­luscs are considered to precipitate carbonate in or near isotopic equilibrium with ambient water (Wefer & Berger, 1991). Normally, when calcium carbonate pre­cipitates slowly from solution, equilibrium isotope parti­tioning is observed. However, faster precipitation may result in isotopic signatures, which are significantly out of equilibrium (Mitchell ef al., 1994). In the case of the pink coloured Mytilus gailoprovinciaiis shells we feel reasonably confident that the pink aragonite sublayer represents a stress layer formed during summer anoxia. The lower 8 i3 C of this layer thus could indicate the in­corporation of isotopically fight bicarbonate due to the decomposition of organic matter and/or kinetic effects due to the fast growth of the pink biogenic aragonite. This hypothesis is supported by field observations as well as by structural and chemical changes in the minor aragonitic layer, which are coincidental with the higher 12 C content. The pink aragonite sublayer is believed to form during pronounced summer anoxia. It contains a signature of stress conditions, which reflects the changes from a slow precipitation of uncoloured flattened aragonite prisms to irregular aragonite grains precipi­tated at high rates. Faster precipitation may result in isotopic signatures, which are significantly out of equi­librium. Kinetic effect have been described in both fast growing biogenic carbonates (McConnaughey, 1989) and in laboratory precipitated non-biological carbonates (Turner, 1982). 
CONCLUSIONS 
The impact of environmental factors such as marine anoxia in the coastal zone of the Northern Adriatic on the growth of Mytilus gailoprovinciaiis shells is indi­cated by changes in the growth structure of the outer part of the nacreous aragonitic layer, which is pink in colour and out of isotopic equilibrium with oxygen and carbon from the ambiental seawater. Isotopic disequili­brium most probably resulted from faster precipitation of aragonite during the summer period, as well as from lack of oxygen and higher concentrations of isotopically light organic derived CO j in the anoxic environment. The influence of higher concentrations of ambiental CO2 is also reflected in a higher C/O ratio of pink col­oured aragonite as compared to the uncoloured one. 

ACKNOWLEDGEMENTS 
The Ministry of Science and Technology, Republic of Slovenia, and Geoexp d.0.0 financially supported this study. To both these institutions w e express our sincere thanks. 
59 
ANNALES • Ser. hist. nat. • 10 • 2000 • 1 (19) 
Tadej DOlLNL C el ah THE INFLUENCE O F MARiNF ANOXI A O N PRECIPITATION O F MYTHUS GALLOPROVINCIAUS..., 55-60 

VPLIV MORSKE ANOKSljE NA IZLOČANJE KARBONATA V LUP1NICAH ŠKOLJKE MYTHUS GALLOPROVINCIAUS IZ PRIOBALNEGA DELA ROVINJSKEGA ZALIVA (SEVERNI JADRAN) 
Tadej DOLENEC 
Oddelek za geologijo, Naravoslovnotehniška fakulteta, Univerza v Ljubljani, SI-1000 Ljubljana, Aškerčeva 12 in Institut Jožef Siefan, SI-1000 Ljubljana, Jamova 39 
Davorin MEDAKOVIČ 
Inštitut Ruder ESoSkoviC, Center za raziskovanje morja, HR-52210 Rovinj, Giordano Paliaga 5 
Sonja LOJEN 
Inštitut Jožef Stefan, SI-1000 Ljubljana, Jamova 39 
POVZETEK 
Z raziskavo smo ugotovili vpliv morske anoksije v priobalnem delu Rovinjskega zaliva na zgradbo, izotopsko in kemično sestavo lupinic školjke Mytilus gailoprovincialis. Zunanji aragonitni del "stresne" plasti, ki je nastajal med močno poletno anoksijo zaradi intenzivnega cvetenja fitoplanktona v letih T 989-1991, je masiven in rožnat ter ni v izotopskem ravnotežju s kisikom in ogljikom. Poleg tega kaže tudi večje razmerje C/O v primerjavi z neobarvanim aragonitnim slojem pri školjkah, ki žive v normalnem oksidacijskem okolju. 
Ključne besede: Mytilus gailoprovincialis,  biomineralizacija, stabilni izotopi, anoksija, Rovinjskl zaliv, severni Jadran  
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