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Selection of target mussel tissue for application of cellular energy allocation as a physiological biomarker in native mussels Mytilus galloprovincialis (Lamarck, 1819).

ABSTRACT Three selected mussel tissues (digestive gland, mantle, and gills) were studied to determine which was the most suitable for the potential use of the cellular energy allocation (CEA) methodology in indigenous mussels Mytilus galloprovincialis. In addition, the applicability of CEA in the assessment of natural stress caused by salinity fluctuations in stratified estuary was tested in selected tissues. It was important to identify the mussel gender to reliably assess the changes in organism energy budget. CEA value was calculated as a ratio between available energy ([E.sub.a]) and energy consumption ([E.sub.c]). Mantle tissue was under the strongest influence of the differences in protein and lipid content between male and female mussels, and therefore reflected physiological changes in the organism itself, rather than those caused by natural environmental stress. CEA in gills had lower values than in mantle and digestive gland, and was similar at two selected sampling sites, so the changes in CEA caused by natural stress could not be detected in the gill tissue. Greater [E.sub.c] in mussels from the estuarine site than from the coastal site was detected only in the digestive gland tissue, and can probably be attributed to the energetically costly maintenance of osmotic balance. Last, using digestive gland tissue in CEA analysis demonstrated a clear difference between coastal and estuarine sampling sites, providing the measure of the natural stress posed by variations in salinity.

KEY WORDS: cellular energy allocation, mussel Mytilus galloprovineialis, digestive gland tissue, mantle tissue, gill tissue

INTRODUCTION

The cellular energy allocation (CEA) methodology has been developed (De Coen & Janssen 1997) as a biochemical alternative to the physiological scope for growth (Widdows & Johnson 1988). It is based on the metabolic cost hypothesis, which suggests that toxic stress induces metabolic changes in an organism, which might lead to a depletion of its energy reserves, resulting in adverse effects on growth and reproduction (Calow & Sibly 1990). The concept of the CEA approach is to quantify the available energy reserves ([E.sub.a]) and energy consumption ([E.sub.c]) at a cellular level of biological organization (biochemically) and to integrate them into a general stress indicator. Energy consumption is estimated by measuring the electron transport activity (ETS) at the mitochondrial level, whereas the energy reserve available for metabolism is assessed by measuring the total lipid, protein, and carbohydrate content of the test organism. The different energy reserve fractions were transformed into energetic equivalents using their respective energy of combustion, whereas the quantity of oxygen consumed, as derived from the ETS data, was transformed into energetic equivalents using the oxyenthalpic equivalents for an average lipid, protein, and carbohydrate mixture (Gnaiger 1983).

The CEA approach has proved to be ecologically relevant because cellular effects have been linked to higher levels of biological organization (De Coen & Janssen 2003, Smolders et al. 2004). Although this technique was developed and validated initially with Daphnia magna, the CEA methodology can also be used with other invertebrates and vertebrates. So far, CEA has been applied in laboratory and field studies in different ecosystems (e.g., freshwater, estuarine, and marine ecosystems) using different organisms that cover a variety of animal taxa. A brief overview of these studies is given in Table 1. From the practical side, the CEA methodology is very convenient when studying small invertebrates (e.g., amphipods, mysids, cladocerans), because a small amount of sample is needed to perform all the required biochemical analyses. On the other hand, when studying mussels (Mytilus sp.), the differences in biochemical composition of distinct animal tissues have to be taken into consideration. Because the CEA approach handles the biochemical parameters in terms of energy, different organs (e.g., mantle, digestive gland, gills, and muscles) contribute in a different extent to the energy budget of the total organism. Besides, biochemical composition of bivalves varies seasonally depending on the latitude at which they are found and is strongly related to water temperature, food availability, and the gametogenic cycle of the animal (Okumus & Stirling 1998). A close relationship has also been reported between the gametogenic cycle, condition index, and the storage-consumption cycle of reserves, particularly glycogen (Gabbott 1975).

In this study we examined three mussel organs--gills, digestive gland, and mantle---which constitute the bulk of the soft tissue. The main goal of the current study was to determine which mussel tissue is the most suitable for the potential use of the CEA methodology as a biomarker in the indigenous mussel Mytilus galloprovineialis. In addition, we tested the applicability of the CEA methodology to the assessment of natural stress caused by strong fluctuations in salinity in the stratified estuary.

MATERIALS AND METHODS

Sampling of Mussels

Indigenous mussels (M. galloprovincialis) were collected from coastal rocks or concrete embankment structures between 0.5 m and 1 m below the sea surface. The sampling was performed in November 2008. Two sampling sites were selected in the Krka River estuary based on their differences in abiotic factors, primarily in terms of salinity. These sites were characterized as the coastal site (Zablace) with fewer salinity/ temperature (S/T) variations and the estuarine site (Martinska) with more S/T variations (Erk et al. 2011). The estuary of the karstic river Krka is a salt-wedge, highly stratified estuary, located in the central part of the eastern Adriatic coast in Croatia (Fig. 1). This estuary is regarded as fairly unpolluted (Omanovic et al. 2006, Cukrov et al. 2008).

Biometric measurements, sex determination, and dissection of digestive gland, mantle, and gills were performed immediately after collection at the marine station located at Martinska (Fig. 1). The tissues were stored in liquid nitrogen and transported to the laboratory in Zagreb for further analysis.

Sex Determination

The method used to determine sex of the mussels involved heating a piece of mantle tissue (20-50 mg wet weight) in a solution of trichloroacetic acid (20% w/v; 2 mL) with a thiobarbituric acid (0.75% w/v; 0.5 mL) (Jabbar & Davies, 1987). The presence of a yellow or pink color, determined visually, was used to identify male and female animals, respectively.

Cellular Energy Allocation Measurements

Measurements of lipid, carbohydrate, and protein energy content, and ETS activity were performed in 10 individuals in each selected tissue and for each sampling site. At sampling site Zablace, 3 male and 7 female individuals were analyzed, whereas at site Martinska, 5 male and 5 female individuals were taken for biochemical analyses. Each individual sample was measured in replicate.

[FIGURE 1 OMITTED]

CEA was measured according to Verslycke and Janssen (2002) with minor modifications. Lipids were extracted following the method of Bligh and Dyer (1959), and lipid concentrations were calculated by reference to standards of tripalmitin in chloroform. Protein content was measured by the method described by Bradford (1976) using bovine serum albumin as a standard. Carbohydrate content was analyzed with the phenolsulfuric acid method (Dubois et al. 1956), using glucose as a standard. The different energy reserve fractions (lipid, protein, carbohydrate = available energy, [E.sub.a]) were transformed into energetic equivalents using their respective energy of combustion (39,500 mJ/mg lipid, 24,000 mJ/mg protein, 17,500 mJ/mg glycogen) (Gnaiger 1983). Energy consumption ([E.sub.c]) was estimated by measuring the activity of the mitochondrial ETS according to Owens and King (1975). The quantity of oxygen consumed, as derived from the ETS data, was transformed into energetic equivalents using the oxyenthalpic equivalents for an average lipid, protein, and carbohydrate mixture (484 kJ/mol [O.sub.2]) (Gnaiger 1983).

The [E.sub.a], [E.sub.c] and CEA value were calculated as described by Verslycke and Janssen (2002):

[E.sub.a] (available energy) = [E.sub.carbohydrate] + [E.sub.lipid] + [E.sub.protein] (mJ/mg ww)

[E.sub.c] (energy consumption) = ETS activity (mJ/mg ww/h)

CEA (cellular energy allocation) = [E.sub.a]/[E.sub.c]

From this, it is evident that a decrease of CEA indicates either a reduction in available energy or higher energy expenditure, both resulting in a lower amount of energy available for growth or reproduction.

Statistical Analysis

Statistical analyses were performed with the software packages SigmaStat for Windows version 3.5 (Systat Software, Inc., Chicago, IL) and Statistica 8.0 (StatSoft, Inc., Tulsa, OK). Differences in lipid, protein, carbohydrate energy contents, and differences in ETS activity between sampling sites were tested using the t-test. Differences between male and female in all measured parameters mentioned earlier were detected using the t-test. Differences between 3 organs in the parameters mentioned earlier were tested using 1-way ANOVA followed by pairwise multiple comparisons (Tukey test). All tests were performed at a probability level of 0.05, or the probability levels are indicated in the figures. To assess the degree of association between variables and to gain insight into the separation of individuals between the sampling locations according to their energetic parameters, a correlation-based principal component analysis (PCA) was performed using a data matrix of 6 parameters ([E.sub.carbohydrates], [E.sub.proteins], [E.sub.lipids], [E.sub.a], [E.sub.c], and CEA) and two sampling locations.

RESULTS

By transforming the measured concentrations of total carbohydrates, proteins, and lipids into their energetic equivalents, it was possible to compare the relative contribution of carbohydrate, lipid, and protein contents of digestive gland, mantle, and gills to the energy budget of the respective organ. The energy contents of total carbohydrates, proteins, and lipids measured in different organs are presented in Figure 2A-C. The lowest values in energy content of all measured Ea components--carbohydrates, proteins, and lipids--were evident in the gills (Fig. 2A-C; differences between the tissues are represented by different letters in the bars on the graph). These differences were significant in all cases between digestive gland and gills, but not always between mantle and gills (Fig. 2A-C).

[FIGURE 2 OMITTED]

In this study, the biochemical measurements were performed on both female and male mussels, and the respective results were analyzed independently. With regard to energy content of total carbohydrates, there were no significant differences between male and female mussels in any of the analyzed tissues at both sampling sites (Fig. 2A). At the coastal site Zablace, females had greater energy content of total proteins in all 3 organs analyzed, whereas at the estuarine site the differences in the energy content of total proteins between male and female were not significant, except in the mantle (Fig. 2B; respective differences are marked bellow the bars on the graph). In digestive gland and gills, there were no significant differences in the energy content of total lipids between male and female; but in the mantle, females had greater energy content of total lipids at both sampling sites (Fig. 2C). Similar to the lipids, no significant differences in total Ea between male and female were found in digestive gland and gills; but in the mantle, female had greater [E.sub.a] at both sampling sites (Fig. 2D). No significant differences were found in [E.sub.c] in the gills between male and female (Fig. 2E). However, in digestive gland males had greater [E.sub.c] than females at both sampling sites (Fig. 2E). In contrast, [E.sub.c] in mantle tissue was higher in female mussels from coastal site Zablace (Fig. 2E).

When considering the differences in all measured parameters between the 2 sampling sites, they were significant in most cases in digestive gland (Fig. 2A-C). Significant differences between the 2 sampling sites were found only in digestive gland--in particular, in [E.sub.c] (Fig. 2E) and in the calculated CEA values (Fig. 2F).

The PCA performed on the measured parameters in digestive gland showed that the first 2 axes accounted for 80.27% of variability between the mussels (Fig. 3A). The first axis explained ~42% of total variance and displayed markedly positive loadings with the energy content of total carbohydrates ([E.sub.carbohydrates]), total [E.sub.a], and CEA values (Fig. 3A). The energy content lipids ([E.sub.lipids]) and proteins ([E.sub.proteins]) also showed a positive association with this axis, whereas an opposite relationship was observed for [E.sub.c] (Fig. 3A). This first axis separated individuals between the sampling sites (Fig. 3B) mainly according to the values of CEA and [E.sub.c], which were inversely related to each other (Fig. 3A). Thus, the right side of the first axis grouped the mussels from the coastal site Zablace (Fig. 3B) because they had high CEA values and a lower level of [E.sub.c]. Most individuals from the estuarine site Martinska were grouped on the left side of the first axis (Fig. 3B) because these mussels had significantly higher [E.sub.c] and lower CEA values compared with those from the coastal site (Fig. 2E, F). The second axis had high loads with the energy content of lipids, total [E.sub.a], and [E.sub.c] (Fig. 3A). The observed variable association pattern is mainly related to 2 individuals at the site Martinska, which had a very high content of lipids and total [E.sub.a], but also high [E.sub.c]. The PCA performed on the other 2 studied tissues (mantle and gills) did not show a clear separation of individuals between the coastal and estuarine sampling sites according to their CEA and [E.sub.c] values (data not shown).

[FIGURE 3 OMITTED]

DISCUSSION

The stratified estuary represents a variable environment that affects significantly the metabolism of marine molluscs because of sometimes abrupt changes in salinity, which can cause important changes in the energy metabolism of animals. Organisms that live in such extreme environmental conditions might fail to reproduce, or may die as a result of natural environmental stress (e.g., thermal stress, salinity stress). Nevertheless, mussels (Mytilus sp.) are able to cope with changes in abiotic factors (salinity, temperature, dissolved oxygen) as a result of their ability to adapt to a wide range of salinities, and their efficient respiratory physiology (Zandee et al. 1986, Hawkins & Bayne 1992). When organisms live in suboptimal environments, there is a cost of dealing with stress in terms of metabolic resources. The energy available for growth, based on energy budget analysis rather than on direct measurement of growth itself, therefore provides a sensitive measure of stress in an organism. To give reliable answers about the extent of changes in an organism's energy budget, the calculated CEA value should reflect the physiological status of the organism caused by external stress, not stress resulting from the normal reproductive cycle or gender differences. It has been shown that various aspects of the relationship exist between gametogenesis and the utilization of glycogen and protein reserves in the mantle tissue of M. edulis (Bayne et al. 1982). Furthermore, concerning the gender differences, Zaba and Davies (1980) found that during the spawning period, mantle tissue slices from female mussels metabolized glucose twice as rapidly as those from males per unit tissue weight. Livingstone (1981) demonstrated that the increase in glucose-6-phosphate dehydrogenase activity that occurred in the mantle tissue during the winter months was confined to female mussels. Therefore, it was important in the current study to identify the gender of mussels and take it into account to assess reliably the changes in CEA caused by natural stress.

Our results show that measuring the CEA in different organs of mussels can give different information considering the same environmental conditions (Fig. 2F). The question was raised about the most appropriate target organ that could be applied for this purpose.

Bivalves of the genus Mytilus have a specific storage tissue, the mantle (containing two complementary types of cells), which undergoes seasonal variations in biochemical composition and in its cellular structure in relation to the reproductive cycle (Mathieu & Lubet 1993). Because of the high weight contribution of mantle tissue when the gonads are developed within it, and its prominent variability in biochemical composition depending on the phase of the reproductive cycle, the changes in CEA caused by natural stress could be masked. In our study, the mantle tissue was under the strongest influence of the differences in protein and lipid content between male and female mussels (Fig. 2B, C), and therefore reflected, in the first place, physiological changes in the organism itself rather than those caused by environmental stress. The trends of total Ea (Fig. 2D), which is a sum of energy content of carbohydrates, lipids, and proteins, actually reflected the trends of lipids (Fig. 2C), because lipid content gives the greatest contribution in energy equivalents (39,500 mJ/mg). Because CEA is calculated as a ratio between Ea and [E.sub.c], it strongly influences the final CEA value.

In mussels, gill filament consists mainly of a single layer of various types of epithelial cells (ciliated and nonciliated columnar cells, and mucous cells) and endothelial cells surrounding a central lumen and resting on a basement membrane (Dumouhtsidou & Dimitriadis 2004) that are deprived in terms of lipid content. Scarce lipid content in gill tissue was also found in the current study (Fig. 2C), leading to the low Ea values. In addition, the differences in [E.sub.c] between sampling sites were not detected in gills (Fig. 2E). As a consequence, the calculated CEA values in gills showed lower values than in mantle and digestive gland, with similar values at both sampling sites (Fig. 2F). Therefore, in this case, the changes in CEA caused by natural stress could not be detected.

With regard to the digestive gland, the breakdown of the digestive epithelium appears to be a generalized stress response, resulting not only from exposure to a wide range of contaminants, but also to physiological extremes such as increased salinity and starvation (Livingstone & Pipe 1992). It is known that pollutant exposure may induce alterations in cell-type ratios in the digestive epithelium (basophilic cells become more abundant than digestive cells), and therefore the cellular composition of the digestive epithelium was examined as a marker of the general condition of the digestive gland (Cajaraville et al. 1990). Thus, the digestive gland appeared to be a good candidate for the target tissue for application of CEA as a physiological biomarker.

The correlation-based PCA was performed for each studied tissue to detect for which tissue all measured variables gave the distinction between the estuarine site and the coastal site. The clear separation of individuals between the sampling locations according to their energetic parameters by PCA (Fig. 3B) was obtained only for digestive gland. Indeed, in this study, CEA calculated from the measured biochemical parameters in digestive gland tissue demonstrated a clear difference between coastal and estuarine sampling sites (Fig. 2F), providing the measure of the natural stress posed by variations in salinity. Furthermore, the decisive energy component that contributes to the calculation of the CEA value was energy consumption ([E.sub.c]). Although significant differences in [E.sub.c] values were observed between male and female mussels, they were congruously higher in males from both sampling sites (Fig. 2E). This difference pointed to the importance of identifying mussel gender to assess reliably the changes in organism energy budget. [E.sub.c] was significantly greater in mussels living at the estuarine site Martinska than at the coastal site Zablace in both genders only when measurements were performed in digestive gland (Fig. 2E). Mean [E.sub.c] was approximately 50% greater at the estuarine site than at the coastal site (Fig. 2E). Because Martinska was the site with high salinity fluctuations, mussels living at this site were exposed to a more demanding environment. To help reduce the rate of associated changes in cell volume, mussels respond immediately by closing the shell (Bayne et al. 1976). As osmoconformers, mussels maintain their internal salinity such that it is always equal to the surrounding seawater. They maintain the volume of cells relatively constant by actively regulating their internal concentration of free amino acids and ions to match the osmolarity of the environment (Lange 1972). All these regulatory processes are costly energetically. Thus, greater [E.sub.c] in mussels from the estuarine site than from the coastal site may be caused by the energetically costly maintenance of osmotic balance, and this distinction was detected only in digestive gland tissue.

In a field study with mussels caged along a pollution gradient in the Statfjord oilfield (Smolders et al. 2006), the use of digestive gland was shown to have the highest values of CEA at the reference station, but statistically significant differences were masked by the high SD. In the same study in the pollution gradient in the German Bight, the measurement of CEA was performed on whole mussel tissue, and the CEA value recorded at the reference station was similar to the CEA values at 2 stations along the pollution gradient. Thus, although we can only speculate, the possible reason for lack of ability to detect the differences in CEA values between the stations in the German Bight may lie in the choice of whole mussel tissue studied.

As a concluding remark, we note that using digestive gland tissue in CEA analysis as a physiological biomarker can have an advantage over using other mussel tissues or the whole soft tissue of mussels when detecting changes caused by environmental stress.

ACKNOWLEDGMENTS

The financial support by the Ministry of Science, Education and Sport of Republic Croatia (project no. 098-0982934-2721 and national monitoring project "Adriatic") is acknowledged.

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MARIJANA ERK, * DUSICA IVANKOVIC AND ZELJKA STRIZAK

Ruder Boskovic Institute, Division for Marine and Environmental Research, Bijenicka c. 54, PO Box 180, HR-10002 Zagreb, Croatia

* Corresponding author. E-mail: erk@irb.hr

DOI: 10.2983/035.031.0108
TABLE 1.
Species and respective tissues/organs in which the cellular energy
allocation methodology has been applied.

Zoological Group               Species               Tissue/Organ

Cladocera             Daphnia magna               Whole organism

                      Daphnia magna               Whole organism

                      Daphnia magna               Whole organism

                      Daphnia magna               Whole organism

Amphipods             Gammarus setosus            Whole organism

                      Onisimus litoralis          Whole organism

                      Onisimus litoralis          Whole organism

                      Gammarus wilkitzkii         Whole organism
                                                  (ovigerous females)

Mysidacea             Neomysis integer            Whole organism

                      Neomysis integer            Whole organism

                      Neomysis integer            Whole organism

                      Neomysis integer            Whole organism

                      Neomysis integer            Whole organism

                      Neomysis integer            Whole organism

Polychaeta            Hediste diversicolor        Whole organism

Gastropods            Melanoides tuberculata      Whole organism

                      Helisoma duryi              Whole organism

Bivalves              Dreissena polymorpha        Whole organism

                      Liocyma fluctuosa           Whole organism

                      Mytilus edulis              Whole organism,
                                                  digestive gland

                      Mytilus galloprovincialis   Digestive gland

Actinopterygii        Solea senegalensis          Liver, muscle
  Pleuronectiformes

Actinopterygii        Boreogadus saida            Liver
  Gadiformes

Actinopterygii        Gadus morhua                Liver
  Gadiformes

Fish larvae           --                          Batches of pooled
  zooplankton                                     individuals

Insecta Hemiptera     Brachynema germarii         Whole organism

Zoological Group               Species                Condition

Cladocera             Daphnia magna               Laboratory

                      Daphnia magna               Laboratory

                      Daphnia magna               Field/Laboratory

                      Daphnia magna               Laboratory

Amphipods             Gammarus setosus            Laboratory

                      Onisimus litoralis          Laboratory

                      Onisimus litoralis          Field

                      Gammarus wilkitzkii         Laboratory

Mysidacea             Neomysis integer            Laboratory

                      Neomysis integer            Laboratory

                      Neomysis integer            Laboratory

                      Neomysis integer            Field

                      Neomysis integer            Laboratory

                      Neomysis integer            Laboratory

Polychaeta            Hediste diversicolor        Laboratory

Gastropods            Melanoides tuberculata      Laboratory

                      Helisoma duryi              Laboratory

Bivalves              Dreissena polymorpha        Field (caged
                                                  mussels)

                      Liocyma fluctuosa           Laboratory

                      Mytilus edulis              Field (caged
                                                  mussels)

                      Mytilus galloprovincialis   Field

Actinopterygii        Solea senegalensis          Laboratory
  Pleuronectiformes

Actinopterygii        Boreogadus saida            Field
  Gadiformes

Actinopterygii        Gadus morhua                Field (caged fish)
  Gadiformes

Fish larvae           --                          Field (wild
  zooplankton                                       populations)

Insecta Hemiptera     Brachynema germarii         Laboratory

Zoological Group               Species                  Stressor

Cladocera             Daphnia magna               Lindane,
                                                  Hg[Cl.sub.2]

                      Daphnia magna               Zn

                      Daphnia magna               Zn

                      Daphnia magna               Ni, binary mixtures
                                                  Ni-Pb and Ni-Cd

Amphipods             Gammarus setosus            Oil-related
                                                  compounds

                      Onisimus litoralis          Oil-related
                                                  compounds

                      Onisimus litoralis          Seasonality in the
                                                  high Arctic

                      Gammarus wilkitzkii         Water soluble
                                                  fraction of oil

Mysidacea             Neomysis integer            Effect of abiotic
                                                  factors (different
                                                  combinations of
                                                  temperature,
                                                  salinity, and
                                                  dissolved oxygen)

                      Neomysis integer            Tributyltin

                      Neomysis integer            Chlorpyrifos

                      Neomysis integer            Polluted Scheldt
                                                  estuary

                      Neomysis integer            Endocrine disruptors
                                                  (testosterone,
                                                  flutamide,
                                                  ethinylestradiol,
                                                  precocene,
                                                  nonylphenol,
                                                  fenoxycarb,
                                                  methoprene)

                      Neomysis integer            Cd, salinity

Polychaeta            Hediste diversicolor        Tributyltin,
                                                  perfluorononanoic
                                                  acid

Gastropods            Melanoides tuberculata      Cd, Zn, Cd/Zn
                                                  mixture

                      Helisoma duryi              Cd, Zn, Cd/Zn
                                                  mixture

Bivalves              Dreissena polymorpha        Pollution gradient
                                                  in an
                                                  effluent-dominated
                                                  stream

                      Liocyma fluctuosa           Oil-related
                                                  compounds

                      Mytilus edulis              Pollution gradient
                                                  in the German

                                                  Bight and Statfjord
                                                  oilfield (Norway)

                      Mytilus galloprovincialis   Effect of salinity

Actinopterygii        Solea senegalensis          Effect of 4
  Pleuronectiformes                               different
                                                  isonitrogenous diets

Actinopterygii        Boreogadus saida            Seasons in the
  Gadiformes                                      Arctic region

Actinopterygii        Gadus morhua                Pollution gradient
  Gadiformes                                      in the German

                                                  Bight and Statfjord
                                                  oilfield (Norway)

Fish larvae           --                          Pollution gradient
  zooplankton                                     in the German

                                                  Bight and Statfjord
                                                  oilfield (Norway)

Insecta Hemiptera     Brachynema germarii         Pyriproxyfen
                                                  (insecticide)

Zoological Group               Species                 Reference

Cladocera             Daphnia magna               De Coen and Janssen
                                                  (1997)

                      Daphnia magna               Muyssen and Janssen
                                                  (2001)

                      Daphnia magna               Muyssen et al.
                                                  (2002)

                      Daphnia magna               Vandenbrouck et al.
                                                  (2009)

Amphipods             Gammarus setosus            Olsen et al. (2007)

                      Onisimus litoralis          Olsen et al. (2007)

                      Onisimus litoralis          Nygard et al. (2010)

                      Gammarus wilkitzkii         Olsen et al. (2008)

Mysidacea             Neomysis integer            Verslycke and
                                                  Janssen (2002)

                      Neomysis integer            Verslycke et al.
                                                  (2003)

                      Neomysis integer            Verslycke et al.
                                                  (2004c)

                      Neomysis integer            Verslycke et al.
                                                  (2004a)

                      Neomysis integer            Verslycke et al.
                                                  (2004b)

                      Neomysis integer            Erk et al. (2008)

Polychaeta            Hediste diversicolor        Stomperudhaugen et
                                                  al. (2009)

Gastropods            Melanoides tuberculata      Moolman et al.
                                                  (2007)

                      Helisoma duryi              Moolman et al.
                                                  (2007)

Bivalves              Dreissena polymorpha        Smolders et al.
                                                  (2004)

                      Liocyma fluctuosa           Olsen et al. (2007)

                      Mytilus edulis              Smolders et al.
                                                  (2006)

                      Mytilus galloprovincialis   Erk et al. (2011)

Actinopterygii        Solea senegalensis          Rueda-Jasso et al.
  Pleuronectiformes                               (2004)

Actinopterygii        Boreogadus saida            Nahrgang et al.
  Gadiformes                                      (2008)

Actinopterygii        Gadus morhua                Smolders et al.
  Gadiformes                                      (2006)

Fish larvae           --                          Smolders et al.
  zooplankton                                     (2006)

Insecta Hemiptera     Brachynema germarii         Bagheri et al.
                                                  (2010)
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Author:Erk, Marijana; Ivankovic, Dusica; Strizak, Zeljka
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:4EXCR
Date:Apr 1, 2012
Words:5611
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