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Using stable nitrogen isotopes in Patella sp. to trace sewage-derived material in coastal ecosystems
Ecological Indicators 36 (2014) 224–230
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Using stable nitrogen isotopes in Patella sp. to trace sewage-derived material in coastal ecosystems Petra Zˇ vab Roˇziˇc a,∗ , Tadej Dolenec a , Sonja Lojen b , Goran Kniewald c , Matej Dolenec a a
Department of Geology, Faculty of Natural Sciences and Engineering, Aˇskerˇceva 12, 1000 Ljubljana, Slovenia Joˇzef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia c Rud¯er Boˇskovi´c Institute, Bjieniˇcka 54, Zagreb, Croatia b
a r t i c l e
i n f o
Article history: Received 11 September 2012 Received in revised form 20 July 2013 Accepted 22 July 2013 Keywords: Patella caerulea Nitrogen stable isotope Bioindicator Organism tissues Organism size Coastal environment Adriatic Sea
a b s t r a c t Coastal environments are often exposed to different anthropogenic contaminants that can cause evident differences in coastal ecosystems. For this reason the use of various organisms as an indicator offers an important ecological study. In this survey the limpet Patella caerulea was examined as a potential anthropogenic bioindicator in coastal marine ecosystems using nitrogen isotope composition measurements. The results indicated generally significant variations between sampling sites. Lower ı15 N values were measured in less polluted areas, while at potentially polluted sites ı15 N enrichment was observed. The lowest ı15 N values were observed near a larger town where a well-regulated purification plant system was in operation and removing undesirable substances (i.e. nitrates) from the sewage. The results suggest that the limpets are useful indicators for tracing anthropogenically derived organic matter from coastal areas in marine ecosystems. Additionally, relatively small differences in ı15 N values were observed in different sizes of limpets, which suggest a rather uniform diet over the organism’s size spectrum. ı15 N variations between tissues in an individual organism were practically negligible. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Stable isotopes have been used effectively in ecological studies to trace the impact of different components, and consequently potential pollutants, on ecosystems, as well as to trace food webs. They provide a powerful tool for source determination and following anthropogenically derived material (from animal waste, septic systems, sewage treatment plants, etc.) from their source to different segments in the environment (Rau et al., 1981; Heaton, 1986; Tucker et al., 1999; Heikoop et al., 2000; Risk and Erdmann, 2000; Costanzo et al., 2001; Sigleo and Macko, 2002; Sarà et al., 2004; Vizzini and Mazzola, 2004; Vizzini et al., 2005; Fertig et al., 2009; Lassauque et al., 2010; Matsuo et al., 2010; Sherwood et al., 2010; Xu and Zhang, 2012). In these studies, various organisms (from primary producers to upper consumers) were used to understand the influence on the environment and determine the most applicable organism to function as an anthropogenic tracer. Dillon et al. (2005) found a ı15 N value of approximately −5.4 ± 2.6‰ in ammonium from rainwater sources, while wastewater ammonium had values ranging from +16 to +25‰ (Cifuentes et al., 1988; Desimone and Howes, 1996). The mean ı15 NO3 values from rainwater were −0.4 ± 2.1‰, while from municipal
∗ Corresponding author. Tel.: +386 1 4704 530. E-mail address: [email protected] (P. Zˇ vab Roˇziˇc). 1470-160X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ecolind.2013.07.023
wastewater the outfall was from +4.83 ± 4.52‰ (Dillon et al., 2005; Dillon and Chanton, 2008). Previous studies on nitrogen isotope composition (ı15 N) indicated significant differences between organisms exposed to anthropogenic sewage particles and naturally occurring materials (Tucker et al., 1999; Sarà et al., 2004, and references therein). It was established that nitrogen isotopic signatures of organisms (POM, macroalgae, seagrass, mussels, corals, fishes, etc.) from anthropogenically impacted areas were usually enriched compared to unpolluted sites (Tucker et al., 1999; Heikoop et al., 2000; Costanzo et al., 2001; Vizzini and Mazzola, 2004, 2006; Vizzini et al., 2005). Also ı15 N values indicate trophic relationships among organisms in diverse ecosystems (Hobson et al., 2002; Iken et al., 2005; Corbisier et al., 2006; Zˇ vab et al., 2010; Lemos Bisi et al., 2012). Furthermore, variations in ı15 N could also be related to size and/or age differences (Miniwaga and Wada, 1984; Wada et al., 1993; Jennings et al., 2002; Vizzini and Mazzola, 2002), differences in depth (Muscatine and Kaplan, 1994) and seasonal effects (Costanzo et al., 2001). Previous studies on the nitrogen isotope composition (ı15 N) of several marine organisms (particular organic matter, zooplankton, anemone, barnacles, sea grass, sponge, mussels) were used to trace anthropogenic pollution in the Adriatic Sea (Dolenec and Vokal, 2002; Dolenec et al., 2005, 2006a, 2006b, 2006c, 2007, 2011; Rogan et al., 2007; Zˇ vab et al., 2010). Our survey was focussed on the limpet Patella caerulea, which is a rather common organism, especially along the rocky eastern coast of the Adriatic Sea. Coastal
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Fig. 1. Position (marked by a box) of the study area in Southern Europe (a) and detailed sampling locations (1–14) along the Istrian coast (b).
areas are exposed to anthropogenic nitrogen inputs from untreated domestic and municipal waste from cities, marinas, tourist areas, and camping sites, as well as aquaculture activities. This paper presents the results of a survey utilising nitrogen stable isotope analyses to investigate the amount of discharge material (mainly municipal sewage) along the Istrian coast. The main purpose of study was to measure the nitrogen isotope composition of Patella sp. (P. caerulea) as a potential bioindicator for tracing anthropogenic pollution in the coastal marine environment. Furthermore, the objective was also to detect possible isotopic variations between limpets of different sizes or between different organism tissues. 2. Materials and methods 2.1. Study area Istria is the largest peninsula in the Adriatic Sea, the northwestsoutheast extending arm of the Mediterranean Sea. The Istria Peninsula is located in the northern Adriatic between the Gulf of Trieste in the north and the Bay of Kvarner in the south (Fig. 1a). The area of Istria lies predominately in Croatia, but is also shared with Slovenia and Italy. The coast of the Istria Peninsula is generally rocky with a tidal range of about 1.2 m. Samples of limpets were collected in the summer of 2008 at 14 locations between Savudrija in the north and the Raˇsa estuary in the south (Fig. 1b). Distances between sampling locations were between a few hundreds of metres (around some bays of cities) up to tens of km (between cities). Because the aim of the study was to observe the impact of different unnatural potential pollutants on limpets, sampling sites were exposed to different sources as well as amounts of anthropogenic inputs, including municipal and industrial sewage from cities, marinas, ports, and tourist facilities that are specially impacted in the summer.
2.2. Limpet Patella sp. Patella sp. is a limpet with a rounded to oval conical shell, a diameter of up to 60 mm and height around 10 mm. The shell can be greyish-white, smooth, granulated or coarse, with radial and concentric lines. The mantle edge overhangs a shallow groove, extended around the entire body, which houses the pallial gills. The muscle scar is horseshoe-shaped with an anterior opening (Harvey, 2009). It inhabits the medio-littoral zone, attaching to firm substrates including rocks and stones where dense populations form. The shape of the shells and its pedal muscle enable the limpet to live in this ecosystem, and to defy conditions like battering waves and tides. They are fixed on the substrate, and during moist conditions or high tides commonly move around to graze on algae and assorted debris covering the surrounding surface. During low tides they return to their primary spot, and inhabit exactly the same place by following the mucus trail that they deposit. Especially on calcareous rocks, they dig a depression that helps them attach and better shield the organism from dehydration and desiccation during periods of dryness (Fish and Fish, 1996; Hill, 2000; Nakhlé et al., 2006). Common limpets begin their life as males, becoming sexually mature at around 9 months of age. Most individuals undergo a sex change, typically becoming female at 2 or 3 years of age, although some remain as males. Spawning takes place once a year, usually from October to December. Fertilisation occurs externally; the larvae spend their first few days of life in the water column, after which time they settle on the shore. The lifespan varies, but is between 10 and 20 years (Fish and Fish, 1996; Hill, 2000). In our research, limpet P. caerulea were chosen for isotopic analyses. This species is one of the most abundant limpets in the Atlantic Sea, and inhabits the lower midlittoral (Simunovic, 1970; Della Santina et al., 1993; Santini and Chelazzi, 1995). Due to its limited inhabited area the diet also reflects the specific algal community typical of this zone (Della Santina et al., 1993).
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2.3. Sample collection and preparation Organisms were sampled by scuba diving from the sea at depth of approximately 1 m at 14 locations. Between 8 and 15 individuals were randomly sampled at each location, that approximately covered a 1 m × 1 m area. Selected samples from each sampling site were divided into four groups according to shell diameter (A < 3.0 cm, B = 3.0–3.5 cm, C = 3.5–4.0 cm, D > 4.0 cm). Size groups were separated with respect to the most common sizes of the sampled organism shells. Where more individuals of the same size were collected, at least three separate limpets were measured. Fresh limpet samples were placed in plastic bags and stored at −20 ◦ C until further processing. In the laboratory, the soft tissues of the organisms were dissected, freeze-dried for at least 72 h, and ground and homogenised to a fine powder using an agate mortar. Powder samples were packed into tin capsules for stable isotope analysis. 2.4. Stable isotope ratios The nitrogen stable isotope composition (ı15 N) was measured using a Europa 20-20 mass spectrometer with ANCA SL preparation module (PDZ Europa Ltd., U.K.). The results are expressed in permil (‰) as relative ı values in terms of ı15 N according to the following equation:
15
ı N=
Rsample Rstandard
− 1 × 1000
where R represents the 15 N/14 N ratio of the sample and standard (atmospheric nitrogen), respectively. Positive ı15 N values indicate enrichment with the heavy isotope 15 N, while negative values indicate a depletion of 15 N. The analytical precision (1 standard deviation) of triplicate analyses of IAEA N-1 and N-2 standards was better than ±0.16‰. Precision (1 standard deviation) of duplicate isotope analyses of samples was within ±0.2‰. 2.5. Statistics Statistical analysis was performed using Statistica 8.0 software. Calculated statistical results were presented in box-plot diagrams. On the diagrams the spatial distribution and variations between tissues of limpet individuals were presented. Spatial differences in ı15 N values between sampling locations of each size group of limpets were tested using one-way ANOVA. 3. Results and discussion 3.1. Spatial nitrogen isotope composition of limpet In order to find possible distinctions between sampling locations, the nitrogen isotope composition was measured in the muscle tissue of the limpet P. caerulea. The muscle tissue was chosen for comparison due to its lower turnover rate (Lorrain et al., 2002). It therefore indicates the isotopic characteristics of a longer time period and consequently reflects non-rapidly changing potential sources of pollution. The ı15 N values were significantly different between sampling sites, with a mean variation of up to 4‰ (Fig. 2 and Table 1). Generally, ı15 N values in more exposed coastal parts vary from 3.5 to 6.0‰, whereas in more restricted and consequently potentially more impacted areas such as bays, they varied from 4.5 to 7‰. The highest value of 8.1‰ was measured at sampling site 6 in the closed bay, which is affected due to untreated municipal waste from the surrounding town. The lowest ı15 N value of 2.8‰ was also observed near a larger town (about 60,000 residents) at location 10, which
Fig. 2. Box-plot diagram of nitrogen isotope composition in muscle tissue of Patella caerulea from separate sampling locations.
has a well-regulated purification plant system for the municipal and industrial sewage. Differences in ı15 N values in limpets with respect to location were calculated with one-way ANOVA statistical tests (Table 1). Statistically significant differences (p ≤ 0.05) between locations were generally found for the overall results as well as for separate size groups (see Section 3.2) of limpets. The exceptions were size groups D and E, where the results did not include all sampling locations. For this reason, the statistic comparison could not give representative results. Significant differences in nitrogen isotopic ratios in organisms between locations can reflect isotopic variations in the source of the food web, as well as the different diets of the consumers. Wastewater nutrients from septic systems are generally enriched in the heavy nitrogen isotope 15 N, which is the result of nitrogen transformation occurring in such waters before and after discharge (Heikoop et al., 2000). Consequently, enrichment in 15 N is usually observed in organisms related to this kind of anthropogenic matter. The difference in ı15 N values of the limpet P. caerulea between coastal locations is rather high, even when compared to other studies. The observed variability in ı15 N between locations also exceeds the transition shift between trophic levels (3.5‰; Miniwaga and Wada, 1984). Clearly higher ı15 N values of observed limpet were found in the coastal areas near larger towns, ports and marinas, representing potential sources of untreated organic matter from septic systems. The increasing ı15 N effect on different marine organisms, from primary producers to upper consumers, near sewage outfalls, port activities and other anthropogenic inputs, has been presented in previous studies (Costanzo et al., 2001; Piola et al., 2006; Pruell et al., 2006; Fertig et al., 2009; Lassauque et al., 2010). The ı15 N enrichment was also found in limpets exposed to organic matter from fish farms in the central Adriatic (average Table 1 Results of one-way ANOVA tests performed among sampling locations for particular limpet sizes (significant results are marked in bold). Size
F
p
A<3 B = 3–3.5 C = 3.5–4 D = 4–4.5 E = 4.5–5 All
6.67 6.87 5.06 1.11 0.14 11.47
0.00 0.00 0.04 0.62 0.77 0.00
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Fig. 3. Diagram of nitrogen isotope composition in muscle tissue of different sized limpets from separate sampling locations (1–14).
6.9‰). Aquaculture activities are also an important part of anthropogenic loads on coastal environments, mostly in the form of faecal and food waste that are enriched in 15 N and thus contribute to the elevated ı15 N levels in different organisms (Sarà et al., 2004; Vizzini and Mazzola, 2004; Dolenec et al., 2006c, 2007; Rogan et al., 2007). Samples of P. caerulea from sites further away from sewage disposal show lower ı15 N values. Also some previous studies indicated a 15 N decrease with distance from the potential pollution source, mainly an onshore-to-offshore decrease effect (Heikoop et al., 2000; Risk and Erdmann, 2000; Rogan et al., 2007). However, the lowest ı15 N concentrations were measured near Pula, the largest town on the peninsula, which could be explained by the action of purification plants that remove undesirable materials from municipal sewage. The latter presents one of the main anthropogenic nitrogen inputs in coastal ecosystems, and can cause considerable detrimental effects, mainly to organisms. The purification plants are therefore very important in reducing any ecological problems. The removal of nitrogen in the sewage treatment processes is effected by the biological oxidation of nitrogen from ammonia (NH3 ) to nitrite (NO2 − ), and then to nitrate (NO3 − ). Nitrate is later reduced to nitrogen gas, which is released to the atmosphere and thus removed from the sewage water. These nitrification and denitrification processes reduce the concentrations of heavy nitrogen 15 N in the products and consequently the isotopically lighter wastewater material inflow in the environment. ı15 N depletion was also observed in samples of particulate organic matter (POM) from cities with purification plant systems (Zˇ vab Roˇziˇc et al., submitted for publication). Furthermore, similar differences in the nitrogen isotope composition were observed at the same
locations and the same sampling time in POM, which represents one of the main food sources for benthic organisms at the base of the food structure (Zˇ vab Roˇziˇc et al., submitted for publication). The results showed that ı15 N values of limpet individuals were generally slightly lower than the POM. 3.2. ı15 N between different limpet size groups The sampled limpets covered a wide range of shell diameters ranging from <3.0 cm to >4.0 cm, and were divided into four groups based on shell diameter. In order to detect any variation between individuals of different sizes, the nitrogen isotope composition was measured in the muscle tissue of the limpets. Differences between size groups are presented in the diagrams in Fig. 3. Based on the results of all sampling locations shown in Fig. 3, we found a generally small difference in nitrogen isotope composition between the sizes of individual limpets. Generally, the difference in nitrogen isotope ratios between the smallest and largest individuals was 1.4‰, where the values were higher in the smallest individuals. In some locations, nitrogen isotope values were lower in the smallest individuals (group A), while in other locations the differences were practically negligible and were also lower in larger limpets (group D). The presented results suggested that isotopic differences might depend more on the location (isotopic composition) of their diet than organism size. The body size of the organisms has an important role in different processes such as the rate of respiration and production, energy requirements, mortality rates, patterns of predation, and vulnerability to mortality. Body size distribution is often used as a tool to
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determine and describe the trophic structure and energy flux in ecosystems (Jennings et al., 2002, and references therein). Some studies have shown variations in stable nitrogen (ı15 N) dependence upon the size and/or age of the individual (Rau et al., 1981; Miniwaga and Wada, 1984; Wada et al., 1993; Jennings et al., 2002; Vizzini and Mazzola, 2002). In contrast, Wilson et al. (2009) found no increase in nitrogen isotopic composition with increasing length in selected fish species. Rau et al. (1981) reported a positive correlation between the ı15 N of Dover sole and the weight of the organism. Similarly, Vizzini and Mazzola (2002) found a slight enrichment (of +1.7‰) in the nitrogen isotopic composition in sand smelt from the Mediterranean coastal area, which is comparable to our study. The differences were not in concordance with trophic level shifts, which for nitrogen ratios are +3.5‰ (Miniwaga and Wada, 1984), and are interpreted as an ontogenetic diet shift. Miniwaga and Wada (1984) reported that ı15 N of two marine mussels was found to be independent of their age, and may have varied due to fluctuations in the source material. The differences in ı15 N between marine mussels and fishes could be the result of the diverse diet of the fishes, which may change with age because of the development of organs for feeding, or also a result of changes in habitat due to their migratory nature. In contrast, many mussel species are sessile after their adhesion to the rock during the juvenile stage, and do not change their feeding habits (Miniwaga and Wada, 1984). Our results are the most comparable, and are consistent with this immobile lifestyle. The feeding habitats of the limpet P. caerulea are rather limited due to their specific living habits (as described in Section 2.2). This hypothesis may also explain the fact that despite a generally minor increase in ı15 N in larger individuals, at some individual locations a practically negligible or even a decrease in nitrogen isotope composition in the limpets with size was observed. Many studies have also explored the relationship between body size and trophic level using ı15 N (France et al., 1998; Jennings et al., 2002; Layman et al., 2005; Romanuk et al., 2010; Persaud et al., 2012), based mostly on fishes and some invertebrate communities. Generally the results of these investigations indicated a positive and significant relation between body mass and trophic level, but the use of body size in predicting the trophic position was found to be limited. In our case, the results do not provide enough evidence to interpret this relationship, due to a lack of results on potential limpet nutrition.
3.3. ı15 N in different tissues of limpet P. caerulea Previous studies found significant differences in nitrogen isotopic composition between the different tissues of an organism (Lorrain et al., 2002; Bodin et al., 2007; Deudero et al., 2009). It is known that isotopic variations among organs may reflect their different metabolic rates as well as their turnover times. Tissues with lower turnover times such as the muscle integrate diet isotopic signatures over a longer time period, over the whole life of the organism. In tissues with faster turnover rates, such as the digestive gland and gonads, isotope ratios reflect the diet over a shorter period (Lorrain et al., 2002; Piola et al., 2006). Therefore we also investigated the variability in nitrogen isotope composition within the individual organisms in our study. Limpets from two locations (7 and 14) were dissected, and different tissues of the body (muscle, digestive organs, head, mantle) were analysed separately. The variations in nitrogen isotope composition of the tissues are presented in Fig. 4. The results of the mean ı15 N values show fractionation in the following way: head (7.05‰), muscle (6.74‰), digestive organs (6.72‰) and mantle (6.41‰). Differences in mean ı15 N values between tissues within a single organism were below 0.7‰.
Fig. 4. Box-plot diagram of nitrogen isotope composition in different tissues of Patella caerulea.
Other studies found greater differences in isotopic composition between the tissues of the mussel species Pecten maximus (Lorrain et al., 2002) and Mytilus galloprovincialis (Deudero et al., 2009). There was documented enrichment in ı15 N values in the muscle compared to the digestive gland of 3.40‰ in P. maximus and 2.45‰ in M. galloprovincialis. Lorrain et al. (2002) interpreted such differences as being due to selective assimilation of different fractions of POM as one of the main bivalve diet constituents. Zˇ upan et al. (submitted for publication) and Ezgeta-Balic´ et al. (2013) also found a depletion in nitrogen isotopes in the digestive gland compared to muscle tissue in bivalves sampled on the south side of Paˇsman Island and in the Bay of Mali Ston in the central Adriatic Sea. The average fractionation between muscle tissue and digestive gland was −1.40, −2.35, −1.27 and 1.4–2.22‰ in M. galloprovincialis, Ostrea edulis, Mullus barbatus and Arca noae, respectively. Our results found an obviously lower fractionation between the tissues (no difference between muscle tissue and digestive organs), and as such differ from these earlier studies. 4. Conclusions Stable nitrogen isotope ratios were used in different ecological studies to trace the various natural and anthropogenic sources of nutrient inputs. The latter have an especially important role in the knowledge of and changes to coastal environments. Our survey therefore investigated the connections and differences in nitrogen isotope composition measured in limpet P. caerulea exposed to various sources of nutrient supply. The results of our ı15 N analysis on limpets indicated differences between sampling locations. We found clear 15 N-enrichment in limpets in areas near towns, ports, marinas and some other tourist facilities, most likely due to a nutrient supply principally from untreated sewage outflows. Lower ı15 N values were measured in less impacted areas, away from potential pollution sources. However the lowest ı15 N values were observed near a larger town, which we postulated was due to its well-regulated purification plant system that removes undesirable materials from the municipal sewage. These results suggest that the limpet P. caerulea can be a useful indicator for tracing anthropogenically derived organic matter from coastal areas that enter the marine ecosystem. We generally observed a slight increase in ı15 N values in larger individuals of limpets. Also, at some individual locations a practically negligible increase and even a decrease in the nitrogen isotope composition of limpets relative to size was determined. Our results,
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and similar studies on other organisms (mostly fish), suggest that more migratory organisms, and consequently more diverse diets between the growth stage and sizes of the organisms, play a notable role in ı15 N variability. Therefore the nitrogen isotope composition between different sizes of observed limpets can be the result of an extension of pollution in different growth stages of the organisms. The difference in ı15 N values between the tissues of individual organisms was less than 1‰, which was a smaller difference than found in previous studies. Acknowledgments The research was financially supported by the Ministry of Higher Education, Science and Technology, Republic of Slovenia (bilateral projects between Croatia and Slovenia 2001–2009), the Slovenian Research Agency (ARRS) and Geoexp, d.o.o., Trˇziˇc, Slovenia. I also thank my husband Boˇstjan Roˇziˇc for his technical and moral support. References Bodin, N., Le Loc’h, F., Hily, C., 2007. Effect of lipid removal on carbon and nitrogen stable isotope ratios in crustacean tissues. J. Exp. Mar. Biol. Ecol. 341, 168–175. Cifuentes, L.A., Sharp, J.H., Fogel, M.L., 1988. Stable carbon and nitrogen isotope biogeochemistry in the Dalaware Estuary. Limnol. Oceanogr. 33, 1102–1115. Corbisier, T.N., Soares, L.S.H., Petti, M.A.V., Muto, E.Y., Silva, M.H.C., McClelland, J., Valiela, I., 2006. Use of isotopic signatures to assess the food web in a tropical shallow marine ecosystem of Southeastern Brazil. Aquat. Ecol. 40, 381–390. Costanzo, S.D., Donohue, M.J., Dennison, W.C., Loneragan, N.R., Thomas, M., 2001. A new approach for detecting and mapping sewage impacts. Mar. Pollut. Bull. 42, 149–156. Della Santina, P., Sonni, C., Sartoni, G., Chelazzi, G., 1993. Food availability and diet composition of three coexisting Mediterranean limpets (Patella spp.). Mar. Biol. 116, 87–95. Desimone, L.A., Howes, B.L., 1996. Denitrification and nitrogen transport in a coastal aquifer receiving wastewater discharge. Environ. Sci. Technol. 30, 1152–1162. Deudero, S., Cabanellas, M., Blanco, A., Tejada, S., 2009. Stable isotope fractionation in the digestive gland, muscle and gills tissues of the marine mussel Mytilus galloprovincialis. J. Exp. Mar. Biol. Ecol. 368, 181–188. Dillon, K.S., Chanton, J.P., Smith, L., 2005. Nutrient transformations between rainfall and stormwater runoff in an urbanized coastal environment: Sarasota Bay, Florida. Limnol. Oceanogr. 50, 62–69. Dillon, K.S., Chanton, J.P., 2008. Nitrogen stable isotopes of macrophytes assess stormwater nitrogen inputs to an urbanized estuary. Estuaries Coasts 31, 360–370. Dolenec, T., Vokal, B., 2002. Isotopic composition of nitrogen and carbon in Anemonia sulcata as a possible indicator of anthropogenic impacts on marine coastal ecosystem of the Adriatic. Mater. Geoenviron. 49, 449–457. Dolenec, T., Vokal, B., Dolenec, M., 2005. Nitrogen-15 signals of anthropogenic nutrient loading in Anemonia sulcata as a possible indicator of human sewage impacts on marine coastal ecosystems: a case study of Pirovac Bay and the Murter Sea (Central Adriatic). Croat. Chem. Acta 78, 593–600. Dolenec, T., Lojen, S., Kniewald, G., Dolenec, M., Rogan, N., 2006a. ␦15 N of particulate organic matter and Anemonia sulcata as a tracer of sewage effluent transport in the marine costal ecosystem of Pirovac Bay and the Murter Sea (Central Adriatic). RMZ – Mater. Geoenviron. 53, 1–13. Dolenec, T., Lojen, S., Dolenec, M., Lambaˇsa, Zˇ ., Dobnikar, M., Rogan, N., 2006b. 15 N and 13 C enrichment in Balanus perforatus: tracers of municipal particulate waste in the Murter Sea (Central Adriatic, Croatia). Acta Chem. Slov. 53, 476–496. Dolenec, T., Lojen, S., Lambaˇsa, Zˇ ., Dolenec, M., 2006c. Effects of fish loading on sea grass Posidonia oceanica at Vrgada Island (Central Adriatic): a nitrogen stable isotope study. Isot. Environ. Health Stud. 42, 77–85. Dolenec, T., Lojen, S., Kniewald, G., Dolenec, M., Rogan, N., 2007. Nitrogen stable isotope composition as a tracer of fish farming in invertebrates Aplysina aerophoba, Balanus perforatus and Anemonia sulcata in central Adriatic. Aquaculture 262, 237–249. ´ G., Lambaˇsa Belak, Zˇ ., Lojen, S., Kniewald, G., Dolenec, Dolenec, M., Zˇ vab, P., Mihelˇcic, ˇ T., Rogan Smuc, N., 2011. Use of stable nitrogen isotope signatures of anthropogenic organic matter in the coastal environment: a case study of the Kosirina Bay (Murter Island, Croatia). Geol. Croat. 64, 143–152. ´ D., Dolenec, T., Dolenec, M., Zˇ vab Roˇziˇc, P., Lojen, S., Najdek, M., Peharda, Ezgeta-Balic, M., 2013. Seasonal differences of stable isotope composition in four bivalve species from the Adriatic Sea. Marine Biology Research, in press. Fertig, B., Carruthers, T.J.B., Dennison, W.C., Jones, A.B., Pantus, F., Longstaff, B., 2009. Oyster and macroalgae bioindicators detect elevated ␦15 N in Maryland’s coastal bays. Estuaries Coasts 32, 773–786. Fish, J.D., Fish, S., 1996. A Student’s Guide to the Seashore, second edition. Cambridge University Press, Cambridge.
229
France, R., Chandler, M., Peters, R., 1998. Mapping trophic continua of benthic foodwebs: body size–␦15 N relationship. Mar. Ecol. Prog. Ser. 174, 301–306. Harvey, B., 2009. Archipelagos wildlife library. The digital library of Greek flora and fauna [online 2012]. http://wildlife-archipelago.gr/wordpress/ Heaton, T.H.E., 1986. Isotopic studies of nitrogen pollution in the hydrosphere and atmosphere: a review. Chem. Geol. 59, 87–102. Heikoop, J.M., Risk, M.J., Lazier, A.V., Edinger, E.N., Jompa, J., Limmon, G.V., Dunn, J.J., Browne, D.R., Schwarcz, H.P., 2000. Nitrogen-15 signals of anthropogenic nutrient loading in reef corals. Mar. Pollut. Bull. 40, 628–636. Hill, J.M., 2000. Patella vulgata. Common limpet. Marine Life Information Network: Biology and Sensitivity Key Information Sub-programme [online]. Marine Biological Association of the United Kingdom, Plymouth [cited 06/08/02]. http://www.marlin.ac.uk/species/Patellavulgata.htm Hobson, K.A., Fisk, A., Karnovsky, N., Holst, M., Gagnon, J.M., Fortier, M., 2002. A stable isotope (␦13 C, ␦15 N) model for the North Water food web: implications for evaluating trophodynamics and the flow of energy and contaminants. Deep-Sea Res. II 49, 5131–5150. Iken, K., Bluhm, B.A., Gradinger, R., 2005. Food web structure in the high Arctic Canada Basin: evidence from ␦13 C and ␦15 N analysis. Polar Biol. 28, 238–249. Jennings, S., Pinnegar, J.K., Polunin, N.V.C., Warr, K.J., 2002. Linking size-based and trophic analyses of benthic community structure. Mar. Ecol. Prog. Ser. 226, 77–85. Lassauque, J., Lepoint, G., Thibaut, T., Francour, P., Meinesz, A., 2010. Tracing sewage and natural freshwater input in a Northwest Mediterranean bay: evidence obtained from isotopic ratios in marine organisms. Mar. Pollut. Bull. 60, 843–851. Layman, C.A., Winemiller, K.O., Arrington, D.A., Jepsen, D.B., 2005. Body size and trophic position in a diverse tropical food web. Ecology 86, 2530–2535. Lemos Bisi, T., Lepoint, G., de Freitas Azevedo, A., Dorneles, P.R., Flach, L., Das, K., Malm, O., Lailson-Brito, J., 2012. Trophic and mercury biomagnification in Brazilian tropical coastal food web. Ecol. Indic. 18, 291–302. Lorrain, A., Paulet, Y.-M., Chauvald, L., Savoye, N., Donval, A., Saout, C., 2002. Differential ␦13 C and ␦15 N signatures among scallop tissues: implications for ecology and physiology. J. Exp. Mar. Biol. Ecol. 275, 47–61. Matsuo, H., Matsuka, N., Omori, K., Koizumi, Y., Tekeuchi, I., 2010. Difference in stable nitrogen ratio of Sargassum piluliferum (Phaeophyceae: Fucales) associated with fish and pearl oyster aquaculture facilities. Fisheries Sci. 76, 147–153. Miniwaga, M., Wada, E., 1984. Stepwise enrichment of 15 N along food chains: further evidence and the relation between ␦15 N and animal age. Geochim. Cosmochim. Acta 48, 1135–1140. Muscatine, L., Kaplan, I.R., 1994. Resource partitioning by reef corals as determined from stable isotope composition. II. ı15 N of zooxantellae and animal tissue versus depth. Pacific Sci. 48, 304–312. Nakhlé, K.F., Cossa, D., Khalaf, G., Beliaeff, B., 2006. Brachidontes variabilis and Patella sp. as quantitative biological indicators for cadmium, lead and mercury in the Lebanese coastal waters. Environ. Pollut. 142, 73–82. Persaud, A.Q., Dillon, P.J., Molot, L.A., Hargan, K.E., 2012. Relationship between body size and trophic position of consumers in temperate freshwater lakes. Aquat. Sci. 74, 203–212. Piola, R.F., Moore, S.K., Suthers, I.M., 2006. Carbon and nitrogen stable isotope analysis of three types of oyster tissue in an impacted estuary. Estuar. Coast. Shelf Sci. 66, 255–266. Pruell, R.J., Taplin, B.K., Lake, J.L., Jayaraman, S., 2006. Nitrogen isotope ratios in estuarine biota collected along a nutrient in Narragansett Bay, Rhode Island, USA. Mar. Pollut. Bull. 52, 612–620. Rau, G.H., Sweeney, R.E., Kaplan, I.R., 1981. Differences in animal 13 C, 15 N and D abundance between a polluted and an unpolluted coastal site: likely indicators of sewage uptake by a marine food web. Estuar. Coast. Shelf Sci. 13, 701–707. Risk, M.J., Erdmann, M.V., 2000. Isotopic composition of nitrogen in Stomatopod (Crustacea) tissues as an indicator of human sewage impacts on Indonesian coral reefs. Mar. Pollut. Bull. 40, 50–58. Rogan, N., Dolenec, T., Lojen, S., Lambaˇsa, Zˇ ., Dolenec, M., 2007. 15 N signal of Aplysina aerophoba as a tracer of anthropogenic nitrogen in the Murter Sea and Pirovac Bay (Central Adriatic). RMZ – Mater. Geoenviron. 54, 63–76. Romanuk, T.N., Hayward, A., Hutching, J.A., 2010. Trophic level scales positively with the body size in fishes. Global Ecol. Biogeogr. 20, 231–240. Santini, G., Chelazzi, G., 1995. Glycogen content and rates of depletion in two limpets with different foraging regimes. Comp. Biochem. Physiol. 111A, 271–277. Sarà, G., Scilipoti, D., Mazzola, A., Modica, A., 2004. Effects of fish farming waste to sedimentary and particulate organic matter in a southern Mediterranean area (Gulf of Castellammare, Sicily): a multiple stable isotope study ␦13 C and ␦15 N. Aquaculture 234, 199–213. Sherwood, O.A., Lapointe, B.E., Risk, M.J., Jamieson, B.E., 2010. Nitrogen isotopic records of terrestrial pollution encoded in Floridian and Bahamian Gorgonian corals. Environ. Sci. Technol. 44, 874–880. Sigleo, A.C., Macko, S.A., 2002. Carbon and nitrogen isotopes in suspended particles and colloids, Chesapeake and San Francisco Estuaries. Estuar. Coast. Shelf Sci. 54, 701–711. Simunovic, A., 1970. Recherches écologiques sur les peuplements faunistiques de l’étage supralittoral des côtes rocheuses de l’Adriatique moyenne. Acta Adiatica 14, 3–39. Tucker, J., Sheats, N., Giblin, A.E., Hopkinson, C.S., Montoya, J.P., 1999. Using stable isotopes to trace sewage-derived material through Boston Harbor and Massachusetts Bay. Mar. Environ. Res. 48, 353–357. Vizzini, S., Mazzola, A., 2002. Stable carbon and nitrogen ratios in the sand smelt from a Mediterranean coastal area: feeding and effect of season and size. J. Fish Biol. 60, 1498–1510.
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Vizzini, S., Mazzola, A., 2004. Stable isotope evidence for the environmental impact of a land-based fish farm in the western Mediterranean. Mar. Pollut. Bull. 49, 61–70. Vizzini, S., Mazzola, A., 2006. The effects of anthropogenic organic matter inputs on stable carbon and nitrogen isotopes in organisms from different trophic levels in a southern Mediterranean coastal area. Sci. Total Environ. 368, 723–731. Vizzini, S., Benedetto, S., Thang, D.C., Mazzola, A., 2005. Spatial variability of stable carbon and nitrogen isotope ratios in a Mediterranean coastal lagoon. Hydrobiologia 550, 73–82. Wada, E., Kabaya, Y., Kurihara, Y., 1993. Stable isotopic structure of aquatic ecosystems. J. Biosci. 18, 483–499. Wilson, R.M., Chanton, J., Lewis, G., Nowacek, D., 2009. Isotopic variation (ı15 N, ı13 C and ı34 S) with body size in post-larval estuarine consumers. Estuar. Coast. Shelf Sci. 83, 307–312.
Xu, L., Zhang, M., 2012. Primary consumers as bioindicator of nutrient pollution in lake planktonic and benthic feed webs. Ecol. Indic. 14, 189–196. ´ Zˇ upan, I., Peharda, M., Dolenec, T., Dolenec, M., Zˇ vab Roˇziˇc, P., Lojen, S., Ezgeta-Balic, D., Arapov, J. Study of bivalve Noah’s Ark (Arca noae) cultured under experimental integrated aquaculture conditions, submitted for publication. Zˇ vab, P., Dolenec, T., Lojen, S., Kniewald, G., Vodopija, J., Lambaˇsa Belak, Zˇ ., Dolenec, M., 2010. Use of stable nitrogen (␦15 N) isotopes in food web of the Adriatic Sea, Croatia. RMZ – Mater. Geoenviron. 57, 41–52. Zˇ vab Roˇziˇc, P., Dolenec, T., Lojen, S., Kniewald, G., Dolenec, M. Variability in stable isotope composition of particulate organic matter in the marine coastal environment (Istra Peninsula, Northern Adriatic), submitted for publication.
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Ecological Indicators journal homepage: www.elsevier.com/locate/ecolind
Using stable nitrogen isotopes in Patella sp. to trace sewage-derived material in coastal ecosystems Petra Zˇ vab Roˇziˇc a,∗ , Tadej Dolenec a , Sonja Lojen b , Goran Kniewald c , Matej Dolenec a a
Department of Geology, Faculty of Natural Sciences and Engineering, Aˇskerˇceva 12, 1000 Ljubljana, Slovenia Joˇzef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia c Rud¯er Boˇskovi´c Institute, Bjieniˇcka 54, Zagreb, Croatia b
a r t i c l e
i n f o
Article history: Received 11 September 2012 Received in revised form 20 July 2013 Accepted 22 July 2013 Keywords: Patella caerulea Nitrogen stable isotope Bioindicator Organism tissues Organism size Coastal environment Adriatic Sea
a b s t r a c t Coastal environments are often exposed to different anthropogenic contaminants that can cause evident differences in coastal ecosystems. For this reason the use of various organisms as an indicator offers an important ecological study. In this survey the limpet Patella caerulea was examined as a potential anthropogenic bioindicator in coastal marine ecosystems using nitrogen isotope composition measurements. The results indicated generally significant variations between sampling sites. Lower ı15 N values were measured in less polluted areas, while at potentially polluted sites ı15 N enrichment was observed. The lowest ı15 N values were observed near a larger town where a well-regulated purification plant system was in operation and removing undesirable substances (i.e. nitrates) from the sewage. The results suggest that the limpets are useful indicators for tracing anthropogenically derived organic matter from coastal areas in marine ecosystems. Additionally, relatively small differences in ı15 N values were observed in different sizes of limpets, which suggest a rather uniform diet over the organism’s size spectrum. ı15 N variations between tissues in an individual organism were practically negligible. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Stable isotopes have been used effectively in ecological studies to trace the impact of different components, and consequently potential pollutants, on ecosystems, as well as to trace food webs. They provide a powerful tool for source determination and following anthropogenically derived material (from animal waste, septic systems, sewage treatment plants, etc.) from their source to different segments in the environment (Rau et al., 1981; Heaton, 1986; Tucker et al., 1999; Heikoop et al., 2000; Risk and Erdmann, 2000; Costanzo et al., 2001; Sigleo and Macko, 2002; Sarà et al., 2004; Vizzini and Mazzola, 2004; Vizzini et al., 2005; Fertig et al., 2009; Lassauque et al., 2010; Matsuo et al., 2010; Sherwood et al., 2010; Xu and Zhang, 2012). In these studies, various organisms (from primary producers to upper consumers) were used to understand the influence on the environment and determine the most applicable organism to function as an anthropogenic tracer. Dillon et al. (2005) found a ı15 N value of approximately −5.4 ± 2.6‰ in ammonium from rainwater sources, while wastewater ammonium had values ranging from +16 to +25‰ (Cifuentes et al., 1988; Desimone and Howes, 1996). The mean ı15 NO3 values from rainwater were −0.4 ± 2.1‰, while from municipal
∗ Corresponding author. Tel.: +386 1 4704 530. E-mail address: [email protected] (P. Zˇ vab Roˇziˇc). 1470-160X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ecolind.2013.07.023
wastewater the outfall was from +4.83 ± 4.52‰ (Dillon et al., 2005; Dillon and Chanton, 2008). Previous studies on nitrogen isotope composition (ı15 N) indicated significant differences between organisms exposed to anthropogenic sewage particles and naturally occurring materials (Tucker et al., 1999; Sarà et al., 2004, and references therein). It was established that nitrogen isotopic signatures of organisms (POM, macroalgae, seagrass, mussels, corals, fishes, etc.) from anthropogenically impacted areas were usually enriched compared to unpolluted sites (Tucker et al., 1999; Heikoop et al., 2000; Costanzo et al., 2001; Vizzini and Mazzola, 2004, 2006; Vizzini et al., 2005). Also ı15 N values indicate trophic relationships among organisms in diverse ecosystems (Hobson et al., 2002; Iken et al., 2005; Corbisier et al., 2006; Zˇ vab et al., 2010; Lemos Bisi et al., 2012). Furthermore, variations in ı15 N could also be related to size and/or age differences (Miniwaga and Wada, 1984; Wada et al., 1993; Jennings et al., 2002; Vizzini and Mazzola, 2002), differences in depth (Muscatine and Kaplan, 1994) and seasonal effects (Costanzo et al., 2001). Previous studies on the nitrogen isotope composition (ı15 N) of several marine organisms (particular organic matter, zooplankton, anemone, barnacles, sea grass, sponge, mussels) were used to trace anthropogenic pollution in the Adriatic Sea (Dolenec and Vokal, 2002; Dolenec et al., 2005, 2006a, 2006b, 2006c, 2007, 2011; Rogan et al., 2007; Zˇ vab et al., 2010). Our survey was focussed on the limpet Patella caerulea, which is a rather common organism, especially along the rocky eastern coast of the Adriatic Sea. Coastal
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Fig. 1. Position (marked by a box) of the study area in Southern Europe (a) and detailed sampling locations (1–14) along the Istrian coast (b).
areas are exposed to anthropogenic nitrogen inputs from untreated domestic and municipal waste from cities, marinas, tourist areas, and camping sites, as well as aquaculture activities. This paper presents the results of a survey utilising nitrogen stable isotope analyses to investigate the amount of discharge material (mainly municipal sewage) along the Istrian coast. The main purpose of study was to measure the nitrogen isotope composition of Patella sp. (P. caerulea) as a potential bioindicator for tracing anthropogenic pollution in the coastal marine environment. Furthermore, the objective was also to detect possible isotopic variations between limpets of different sizes or between different organism tissues. 2. Materials and methods 2.1. Study area Istria is the largest peninsula in the Adriatic Sea, the northwestsoutheast extending arm of the Mediterranean Sea. The Istria Peninsula is located in the northern Adriatic between the Gulf of Trieste in the north and the Bay of Kvarner in the south (Fig. 1a). The area of Istria lies predominately in Croatia, but is also shared with Slovenia and Italy. The coast of the Istria Peninsula is generally rocky with a tidal range of about 1.2 m. Samples of limpets were collected in the summer of 2008 at 14 locations between Savudrija in the north and the Raˇsa estuary in the south (Fig. 1b). Distances between sampling locations were between a few hundreds of metres (around some bays of cities) up to tens of km (between cities). Because the aim of the study was to observe the impact of different unnatural potential pollutants on limpets, sampling sites were exposed to different sources as well as amounts of anthropogenic inputs, including municipal and industrial sewage from cities, marinas, ports, and tourist facilities that are specially impacted in the summer.
2.2. Limpet Patella sp. Patella sp. is a limpet with a rounded to oval conical shell, a diameter of up to 60 mm and height around 10 mm. The shell can be greyish-white, smooth, granulated or coarse, with radial and concentric lines. The mantle edge overhangs a shallow groove, extended around the entire body, which houses the pallial gills. The muscle scar is horseshoe-shaped with an anterior opening (Harvey, 2009). It inhabits the medio-littoral zone, attaching to firm substrates including rocks and stones where dense populations form. The shape of the shells and its pedal muscle enable the limpet to live in this ecosystem, and to defy conditions like battering waves and tides. They are fixed on the substrate, and during moist conditions or high tides commonly move around to graze on algae and assorted debris covering the surrounding surface. During low tides they return to their primary spot, and inhabit exactly the same place by following the mucus trail that they deposit. Especially on calcareous rocks, they dig a depression that helps them attach and better shield the organism from dehydration and desiccation during periods of dryness (Fish and Fish, 1996; Hill, 2000; Nakhlé et al., 2006). Common limpets begin their life as males, becoming sexually mature at around 9 months of age. Most individuals undergo a sex change, typically becoming female at 2 or 3 years of age, although some remain as males. Spawning takes place once a year, usually from October to December. Fertilisation occurs externally; the larvae spend their first few days of life in the water column, after which time they settle on the shore. The lifespan varies, but is between 10 and 20 years (Fish and Fish, 1996; Hill, 2000). In our research, limpet P. caerulea were chosen for isotopic analyses. This species is one of the most abundant limpets in the Atlantic Sea, and inhabits the lower midlittoral (Simunovic, 1970; Della Santina et al., 1993; Santini and Chelazzi, 1995). Due to its limited inhabited area the diet also reflects the specific algal community typical of this zone (Della Santina et al., 1993).
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2.3. Sample collection and preparation Organisms were sampled by scuba diving from the sea at depth of approximately 1 m at 14 locations. Between 8 and 15 individuals were randomly sampled at each location, that approximately covered a 1 m × 1 m area. Selected samples from each sampling site were divided into four groups according to shell diameter (A < 3.0 cm, B = 3.0–3.5 cm, C = 3.5–4.0 cm, D > 4.0 cm). Size groups were separated with respect to the most common sizes of the sampled organism shells. Where more individuals of the same size were collected, at least three separate limpets were measured. Fresh limpet samples were placed in plastic bags and stored at −20 ◦ C until further processing. In the laboratory, the soft tissues of the organisms were dissected, freeze-dried for at least 72 h, and ground and homogenised to a fine powder using an agate mortar. Powder samples were packed into tin capsules for stable isotope analysis. 2.4. Stable isotope ratios The nitrogen stable isotope composition (ı15 N) was measured using a Europa 20-20 mass spectrometer with ANCA SL preparation module (PDZ Europa Ltd., U.K.). The results are expressed in permil (‰) as relative ı values in terms of ı15 N according to the following equation:
15
ı N=
Rsample Rstandard
− 1 × 1000
where R represents the 15 N/14 N ratio of the sample and standard (atmospheric nitrogen), respectively. Positive ı15 N values indicate enrichment with the heavy isotope 15 N, while negative values indicate a depletion of 15 N. The analytical precision (1 standard deviation) of triplicate analyses of IAEA N-1 and N-2 standards was better than ±0.16‰. Precision (1 standard deviation) of duplicate isotope analyses of samples was within ±0.2‰. 2.5. Statistics Statistical analysis was performed using Statistica 8.0 software. Calculated statistical results were presented in box-plot diagrams. On the diagrams the spatial distribution and variations between tissues of limpet individuals were presented. Spatial differences in ı15 N values between sampling locations of each size group of limpets were tested using one-way ANOVA. 3. Results and discussion 3.1. Spatial nitrogen isotope composition of limpet In order to find possible distinctions between sampling locations, the nitrogen isotope composition was measured in the muscle tissue of the limpet P. caerulea. The muscle tissue was chosen for comparison due to its lower turnover rate (Lorrain et al., 2002). It therefore indicates the isotopic characteristics of a longer time period and consequently reflects non-rapidly changing potential sources of pollution. The ı15 N values were significantly different between sampling sites, with a mean variation of up to 4‰ (Fig. 2 and Table 1). Generally, ı15 N values in more exposed coastal parts vary from 3.5 to 6.0‰, whereas in more restricted and consequently potentially more impacted areas such as bays, they varied from 4.5 to 7‰. The highest value of 8.1‰ was measured at sampling site 6 in the closed bay, which is affected due to untreated municipal waste from the surrounding town. The lowest ı15 N value of 2.8‰ was also observed near a larger town (about 60,000 residents) at location 10, which
Fig. 2. Box-plot diagram of nitrogen isotope composition in muscle tissue of Patella caerulea from separate sampling locations.
has a well-regulated purification plant system for the municipal and industrial sewage. Differences in ı15 N values in limpets with respect to location were calculated with one-way ANOVA statistical tests (Table 1). Statistically significant differences (p ≤ 0.05) between locations were generally found for the overall results as well as for separate size groups (see Section 3.2) of limpets. The exceptions were size groups D and E, where the results did not include all sampling locations. For this reason, the statistic comparison could not give representative results. Significant differences in nitrogen isotopic ratios in organisms between locations can reflect isotopic variations in the source of the food web, as well as the different diets of the consumers. Wastewater nutrients from septic systems are generally enriched in the heavy nitrogen isotope 15 N, which is the result of nitrogen transformation occurring in such waters before and after discharge (Heikoop et al., 2000). Consequently, enrichment in 15 N is usually observed in organisms related to this kind of anthropogenic matter. The difference in ı15 N values of the limpet P. caerulea between coastal locations is rather high, even when compared to other studies. The observed variability in ı15 N between locations also exceeds the transition shift between trophic levels (3.5‰; Miniwaga and Wada, 1984). Clearly higher ı15 N values of observed limpet were found in the coastal areas near larger towns, ports and marinas, representing potential sources of untreated organic matter from septic systems. The increasing ı15 N effect on different marine organisms, from primary producers to upper consumers, near sewage outfalls, port activities and other anthropogenic inputs, has been presented in previous studies (Costanzo et al., 2001; Piola et al., 2006; Pruell et al., 2006; Fertig et al., 2009; Lassauque et al., 2010). The ı15 N enrichment was also found in limpets exposed to organic matter from fish farms in the central Adriatic (average Table 1 Results of one-way ANOVA tests performed among sampling locations for particular limpet sizes (significant results are marked in bold). Size
F
p
A<3 B = 3–3.5 C = 3.5–4 D = 4–4.5 E = 4.5–5 All
6.67 6.87 5.06 1.11 0.14 11.47
0.00 0.00 0.04 0.62 0.77 0.00
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Fig. 3. Diagram of nitrogen isotope composition in muscle tissue of different sized limpets from separate sampling locations (1–14).
6.9‰). Aquaculture activities are also an important part of anthropogenic loads on coastal environments, mostly in the form of faecal and food waste that are enriched in 15 N and thus contribute to the elevated ı15 N levels in different organisms (Sarà et al., 2004; Vizzini and Mazzola, 2004; Dolenec et al., 2006c, 2007; Rogan et al., 2007). Samples of P. caerulea from sites further away from sewage disposal show lower ı15 N values. Also some previous studies indicated a 15 N decrease with distance from the potential pollution source, mainly an onshore-to-offshore decrease effect (Heikoop et al., 2000; Risk and Erdmann, 2000; Rogan et al., 2007). However, the lowest ı15 N concentrations were measured near Pula, the largest town on the peninsula, which could be explained by the action of purification plants that remove undesirable materials from municipal sewage. The latter presents one of the main anthropogenic nitrogen inputs in coastal ecosystems, and can cause considerable detrimental effects, mainly to organisms. The purification plants are therefore very important in reducing any ecological problems. The removal of nitrogen in the sewage treatment processes is effected by the biological oxidation of nitrogen from ammonia (NH3 ) to nitrite (NO2 − ), and then to nitrate (NO3 − ). Nitrate is later reduced to nitrogen gas, which is released to the atmosphere and thus removed from the sewage water. These nitrification and denitrification processes reduce the concentrations of heavy nitrogen 15 N in the products and consequently the isotopically lighter wastewater material inflow in the environment. ı15 N depletion was also observed in samples of particulate organic matter (POM) from cities with purification plant systems (Zˇ vab Roˇziˇc et al., submitted for publication). Furthermore, similar differences in the nitrogen isotope composition were observed at the same
locations and the same sampling time in POM, which represents one of the main food sources for benthic organisms at the base of the food structure (Zˇ vab Roˇziˇc et al., submitted for publication). The results showed that ı15 N values of limpet individuals were generally slightly lower than the POM. 3.2. ı15 N between different limpet size groups The sampled limpets covered a wide range of shell diameters ranging from <3.0 cm to >4.0 cm, and were divided into four groups based on shell diameter. In order to detect any variation between individuals of different sizes, the nitrogen isotope composition was measured in the muscle tissue of the limpets. Differences between size groups are presented in the diagrams in Fig. 3. Based on the results of all sampling locations shown in Fig. 3, we found a generally small difference in nitrogen isotope composition between the sizes of individual limpets. Generally, the difference in nitrogen isotope ratios between the smallest and largest individuals was 1.4‰, where the values were higher in the smallest individuals. In some locations, nitrogen isotope values were lower in the smallest individuals (group A), while in other locations the differences were practically negligible and were also lower in larger limpets (group D). The presented results suggested that isotopic differences might depend more on the location (isotopic composition) of their diet than organism size. The body size of the organisms has an important role in different processes such as the rate of respiration and production, energy requirements, mortality rates, patterns of predation, and vulnerability to mortality. Body size distribution is often used as a tool to
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determine and describe the trophic structure and energy flux in ecosystems (Jennings et al., 2002, and references therein). Some studies have shown variations in stable nitrogen (ı15 N) dependence upon the size and/or age of the individual (Rau et al., 1981; Miniwaga and Wada, 1984; Wada et al., 1993; Jennings et al., 2002; Vizzini and Mazzola, 2002). In contrast, Wilson et al. (2009) found no increase in nitrogen isotopic composition with increasing length in selected fish species. Rau et al. (1981) reported a positive correlation between the ı15 N of Dover sole and the weight of the organism. Similarly, Vizzini and Mazzola (2002) found a slight enrichment (of +1.7‰) in the nitrogen isotopic composition in sand smelt from the Mediterranean coastal area, which is comparable to our study. The differences were not in concordance with trophic level shifts, which for nitrogen ratios are +3.5‰ (Miniwaga and Wada, 1984), and are interpreted as an ontogenetic diet shift. Miniwaga and Wada (1984) reported that ı15 N of two marine mussels was found to be independent of their age, and may have varied due to fluctuations in the source material. The differences in ı15 N between marine mussels and fishes could be the result of the diverse diet of the fishes, which may change with age because of the development of organs for feeding, or also a result of changes in habitat due to their migratory nature. In contrast, many mussel species are sessile after their adhesion to the rock during the juvenile stage, and do not change their feeding habits (Miniwaga and Wada, 1984). Our results are the most comparable, and are consistent with this immobile lifestyle. The feeding habitats of the limpet P. caerulea are rather limited due to their specific living habits (as described in Section 2.2). This hypothesis may also explain the fact that despite a generally minor increase in ı15 N in larger individuals, at some individual locations a practically negligible or even a decrease in nitrogen isotope composition in the limpets with size was observed. Many studies have also explored the relationship between body size and trophic level using ı15 N (France et al., 1998; Jennings et al., 2002; Layman et al., 2005; Romanuk et al., 2010; Persaud et al., 2012), based mostly on fishes and some invertebrate communities. Generally the results of these investigations indicated a positive and significant relation between body mass and trophic level, but the use of body size in predicting the trophic position was found to be limited. In our case, the results do not provide enough evidence to interpret this relationship, due to a lack of results on potential limpet nutrition.
3.3. ı15 N in different tissues of limpet P. caerulea Previous studies found significant differences in nitrogen isotopic composition between the different tissues of an organism (Lorrain et al., 2002; Bodin et al., 2007; Deudero et al., 2009). It is known that isotopic variations among organs may reflect their different metabolic rates as well as their turnover times. Tissues with lower turnover times such as the muscle integrate diet isotopic signatures over a longer time period, over the whole life of the organism. In tissues with faster turnover rates, such as the digestive gland and gonads, isotope ratios reflect the diet over a shorter period (Lorrain et al., 2002; Piola et al., 2006). Therefore we also investigated the variability in nitrogen isotope composition within the individual organisms in our study. Limpets from two locations (7 and 14) were dissected, and different tissues of the body (muscle, digestive organs, head, mantle) were analysed separately. The variations in nitrogen isotope composition of the tissues are presented in Fig. 4. The results of the mean ı15 N values show fractionation in the following way: head (7.05‰), muscle (6.74‰), digestive organs (6.72‰) and mantle (6.41‰). Differences in mean ı15 N values between tissues within a single organism were below 0.7‰.
Fig. 4. Box-plot diagram of nitrogen isotope composition in different tissues of Patella caerulea.
Other studies found greater differences in isotopic composition between the tissues of the mussel species Pecten maximus (Lorrain et al., 2002) and Mytilus galloprovincialis (Deudero et al., 2009). There was documented enrichment in ı15 N values in the muscle compared to the digestive gland of 3.40‰ in P. maximus and 2.45‰ in M. galloprovincialis. Lorrain et al. (2002) interpreted such differences as being due to selective assimilation of different fractions of POM as one of the main bivalve diet constituents. Zˇ upan et al. (submitted for publication) and Ezgeta-Balic´ et al. (2013) also found a depletion in nitrogen isotopes in the digestive gland compared to muscle tissue in bivalves sampled on the south side of Paˇsman Island and in the Bay of Mali Ston in the central Adriatic Sea. The average fractionation between muscle tissue and digestive gland was −1.40, −2.35, −1.27 and 1.4–2.22‰ in M. galloprovincialis, Ostrea edulis, Mullus barbatus and Arca noae, respectively. Our results found an obviously lower fractionation between the tissues (no difference between muscle tissue and digestive organs), and as such differ from these earlier studies. 4. Conclusions Stable nitrogen isotope ratios were used in different ecological studies to trace the various natural and anthropogenic sources of nutrient inputs. The latter have an especially important role in the knowledge of and changes to coastal environments. Our survey therefore investigated the connections and differences in nitrogen isotope composition measured in limpet P. caerulea exposed to various sources of nutrient supply. The results of our ı15 N analysis on limpets indicated differences between sampling locations. We found clear 15 N-enrichment in limpets in areas near towns, ports, marinas and some other tourist facilities, most likely due to a nutrient supply principally from untreated sewage outflows. Lower ı15 N values were measured in less impacted areas, away from potential pollution sources. However the lowest ı15 N values were observed near a larger town, which we postulated was due to its well-regulated purification plant system that removes undesirable materials from the municipal sewage. These results suggest that the limpet P. caerulea can be a useful indicator for tracing anthropogenically derived organic matter from coastal areas that enter the marine ecosystem. We generally observed a slight increase in ı15 N values in larger individuals of limpets. Also, at some individual locations a practically negligible increase and even a decrease in the nitrogen isotope composition of limpets relative to size was determined. Our results,
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and similar studies on other organisms (mostly fish), suggest that more migratory organisms, and consequently more diverse diets between the growth stage and sizes of the organisms, play a notable role in ı15 N variability. Therefore the nitrogen isotope composition between different sizes of observed limpets can be the result of an extension of pollution in different growth stages of the organisms. The difference in ı15 N values between the tissues of individual organisms was less than 1‰, which was a smaller difference than found in previous studies. Acknowledgments The research was financially supported by the Ministry of Higher Education, Science and Technology, Republic of Slovenia (bilateral projects between Croatia and Slovenia 2001–2009), the Slovenian Research Agency (ARRS) and Geoexp, d.o.o., Trˇziˇc, Slovenia. I also thank my husband Boˇstjan Roˇziˇc for his technical and moral support. References Bodin, N., Le Loc’h, F., Hily, C., 2007. Effect of lipid removal on carbon and nitrogen stable isotope ratios in crustacean tissues. J. Exp. Mar. Biol. Ecol. 341, 168–175. Cifuentes, L.A., Sharp, J.H., Fogel, M.L., 1988. Stable carbon and nitrogen isotope biogeochemistry in the Dalaware Estuary. Limnol. Oceanogr. 33, 1102–1115. Corbisier, T.N., Soares, L.S.H., Petti, M.A.V., Muto, E.Y., Silva, M.H.C., McClelland, J., Valiela, I., 2006. Use of isotopic signatures to assess the food web in a tropical shallow marine ecosystem of Southeastern Brazil. Aquat. Ecol. 40, 381–390. Costanzo, S.D., Donohue, M.J., Dennison, W.C., Loneragan, N.R., Thomas, M., 2001. A new approach for detecting and mapping sewage impacts. Mar. Pollut. Bull. 42, 149–156. Della Santina, P., Sonni, C., Sartoni, G., Chelazzi, G., 1993. Food availability and diet composition of three coexisting Mediterranean limpets (Patella spp.). Mar. Biol. 116, 87–95. Desimone, L.A., Howes, B.L., 1996. Denitrification and nitrogen transport in a coastal aquifer receiving wastewater discharge. Environ. Sci. Technol. 30, 1152–1162. Deudero, S., Cabanellas, M., Blanco, A., Tejada, S., 2009. Stable isotope fractionation in the digestive gland, muscle and gills tissues of the marine mussel Mytilus galloprovincialis. J. Exp. Mar. Biol. Ecol. 368, 181–188. Dillon, K.S., Chanton, J.P., Smith, L., 2005. Nutrient transformations between rainfall and stormwater runoff in an urbanized coastal environment: Sarasota Bay, Florida. Limnol. Oceanogr. 50, 62–69. Dillon, K.S., Chanton, J.P., 2008. Nitrogen stable isotopes of macrophytes assess stormwater nitrogen inputs to an urbanized estuary. Estuaries Coasts 31, 360–370. Dolenec, T., Vokal, B., 2002. Isotopic composition of nitrogen and carbon in Anemonia sulcata as a possible indicator of anthropogenic impacts on marine coastal ecosystem of the Adriatic. Mater. Geoenviron. 49, 449–457. Dolenec, T., Vokal, B., Dolenec, M., 2005. Nitrogen-15 signals of anthropogenic nutrient loading in Anemonia sulcata as a possible indicator of human sewage impacts on marine coastal ecosystems: a case study of Pirovac Bay and the Murter Sea (Central Adriatic). Croat. Chem. Acta 78, 593–600. Dolenec, T., Lojen, S., Kniewald, G., Dolenec, M., Rogan, N., 2006a. ␦15 N of particulate organic matter and Anemonia sulcata as a tracer of sewage effluent transport in the marine costal ecosystem of Pirovac Bay and the Murter Sea (Central Adriatic). RMZ – Mater. Geoenviron. 53, 1–13. Dolenec, T., Lojen, S., Dolenec, M., Lambaˇsa, Zˇ ., Dobnikar, M., Rogan, N., 2006b. 15 N and 13 C enrichment in Balanus perforatus: tracers of municipal particulate waste in the Murter Sea (Central Adriatic, Croatia). Acta Chem. Slov. 53, 476–496. Dolenec, T., Lojen, S., Lambaˇsa, Zˇ ., Dolenec, M., 2006c. Effects of fish loading on sea grass Posidonia oceanica at Vrgada Island (Central Adriatic): a nitrogen stable isotope study. Isot. Environ. Health Stud. 42, 77–85. Dolenec, T., Lojen, S., Kniewald, G., Dolenec, M., Rogan, N., 2007. Nitrogen stable isotope composition as a tracer of fish farming in invertebrates Aplysina aerophoba, Balanus perforatus and Anemonia sulcata in central Adriatic. Aquaculture 262, 237–249. ´ G., Lambaˇsa Belak, Zˇ ., Lojen, S., Kniewald, G., Dolenec, Dolenec, M., Zˇ vab, P., Mihelˇcic, ˇ T., Rogan Smuc, N., 2011. Use of stable nitrogen isotope signatures of anthropogenic organic matter in the coastal environment: a case study of the Kosirina Bay (Murter Island, Croatia). Geol. Croat. 64, 143–152. ´ D., Dolenec, T., Dolenec, M., Zˇ vab Roˇziˇc, P., Lojen, S., Najdek, M., Peharda, Ezgeta-Balic, M., 2013. Seasonal differences of stable isotope composition in four bivalve species from the Adriatic Sea. Marine Biology Research, in press. Fertig, B., Carruthers, T.J.B., Dennison, W.C., Jones, A.B., Pantus, F., Longstaff, B., 2009. Oyster and macroalgae bioindicators detect elevated ␦15 N in Maryland’s coastal bays. Estuaries Coasts 32, 773–786. Fish, J.D., Fish, S., 1996. A Student’s Guide to the Seashore, second edition. Cambridge University Press, Cambridge.
229
France, R., Chandler, M., Peters, R., 1998. Mapping trophic continua of benthic foodwebs: body size–␦15 N relationship. Mar. Ecol. Prog. Ser. 174, 301–306. Harvey, B., 2009. Archipelagos wildlife library. The digital library of Greek flora and fauna [online 2012]. http://wildlife-archipelago.gr/wordpress/ Heaton, T.H.E., 1986. Isotopic studies of nitrogen pollution in the hydrosphere and atmosphere: a review. Chem. Geol. 59, 87–102. Heikoop, J.M., Risk, M.J., Lazier, A.V., Edinger, E.N., Jompa, J., Limmon, G.V., Dunn, J.J., Browne, D.R., Schwarcz, H.P., 2000. Nitrogen-15 signals of anthropogenic nutrient loading in reef corals. Mar. Pollut. Bull. 40, 628–636. Hill, J.M., 2000. Patella vulgata. Common limpet. Marine Life Information Network: Biology and Sensitivity Key Information Sub-programme [online]. Marine Biological Association of the United Kingdom, Plymouth [cited 06/08/02]. http://www.marlin.ac.uk/species/Patellavulgata.htm Hobson, K.A., Fisk, A., Karnovsky, N., Holst, M., Gagnon, J.M., Fortier, M., 2002. A stable isotope (␦13 C, ␦15 N) model for the North Water food web: implications for evaluating trophodynamics and the flow of energy and contaminants. Deep-Sea Res. II 49, 5131–5150. Iken, K., Bluhm, B.A., Gradinger, R., 2005. Food web structure in the high Arctic Canada Basin: evidence from ␦13 C and ␦15 N analysis. Polar Biol. 28, 238–249. Jennings, S., Pinnegar, J.K., Polunin, N.V.C., Warr, K.J., 2002. Linking size-based and trophic analyses of benthic community structure. Mar. Ecol. Prog. Ser. 226, 77–85. Lassauque, J., Lepoint, G., Thibaut, T., Francour, P., Meinesz, A., 2010. Tracing sewage and natural freshwater input in a Northwest Mediterranean bay: evidence obtained from isotopic ratios in marine organisms. Mar. Pollut. Bull. 60, 843–851. Layman, C.A., Winemiller, K.O., Arrington, D.A., Jepsen, D.B., 2005. Body size and trophic position in a diverse tropical food web. Ecology 86, 2530–2535. Lemos Bisi, T., Lepoint, G., de Freitas Azevedo, A., Dorneles, P.R., Flach, L., Das, K., Malm, O., Lailson-Brito, J., 2012. Trophic and mercury biomagnification in Brazilian tropical coastal food web. Ecol. Indic. 18, 291–302. Lorrain, A., Paulet, Y.-M., Chauvald, L., Savoye, N., Donval, A., Saout, C., 2002. Differential ␦13 C and ␦15 N signatures among scallop tissues: implications for ecology and physiology. J. Exp. Mar. Biol. Ecol. 275, 47–61. Matsuo, H., Matsuka, N., Omori, K., Koizumi, Y., Tekeuchi, I., 2010. Difference in stable nitrogen ratio of Sargassum piluliferum (Phaeophyceae: Fucales) associated with fish and pearl oyster aquaculture facilities. Fisheries Sci. 76, 147–153. Miniwaga, M., Wada, E., 1984. Stepwise enrichment of 15 N along food chains: further evidence and the relation between ␦15 N and animal age. Geochim. Cosmochim. Acta 48, 1135–1140. Muscatine, L., Kaplan, I.R., 1994. Resource partitioning by reef corals as determined from stable isotope composition. II. ı15 N of zooxantellae and animal tissue versus depth. Pacific Sci. 48, 304–312. Nakhlé, K.F., Cossa, D., Khalaf, G., Beliaeff, B., 2006. Brachidontes variabilis and Patella sp. as quantitative biological indicators for cadmium, lead and mercury in the Lebanese coastal waters. Environ. Pollut. 142, 73–82. Persaud, A.Q., Dillon, P.J., Molot, L.A., Hargan, K.E., 2012. Relationship between body size and trophic position of consumers in temperate freshwater lakes. Aquat. Sci. 74, 203–212. Piola, R.F., Moore, S.K., Suthers, I.M., 2006. Carbon and nitrogen stable isotope analysis of three types of oyster tissue in an impacted estuary. Estuar. Coast. Shelf Sci. 66, 255–266. Pruell, R.J., Taplin, B.K., Lake, J.L., Jayaraman, S., 2006. Nitrogen isotope ratios in estuarine biota collected along a nutrient in Narragansett Bay, Rhode Island, USA. Mar. Pollut. Bull. 52, 612–620. Rau, G.H., Sweeney, R.E., Kaplan, I.R., 1981. Differences in animal 13 C, 15 N and D abundance between a polluted and an unpolluted coastal site: likely indicators of sewage uptake by a marine food web. Estuar. Coast. Shelf Sci. 13, 701–707. Risk, M.J., Erdmann, M.V., 2000. Isotopic composition of nitrogen in Stomatopod (Crustacea) tissues as an indicator of human sewage impacts on Indonesian coral reefs. Mar. Pollut. Bull. 40, 50–58. Rogan, N., Dolenec, T., Lojen, S., Lambaˇsa, Zˇ ., Dolenec, M., 2007. 15 N signal of Aplysina aerophoba as a tracer of anthropogenic nitrogen in the Murter Sea and Pirovac Bay (Central Adriatic). RMZ – Mater. Geoenviron. 54, 63–76. Romanuk, T.N., Hayward, A., Hutching, J.A., 2010. Trophic level scales positively with the body size in fishes. Global Ecol. Biogeogr. 20, 231–240. Santini, G., Chelazzi, G., 1995. Glycogen content and rates of depletion in two limpets with different foraging regimes. Comp. Biochem. Physiol. 111A, 271–277. Sarà, G., Scilipoti, D., Mazzola, A., Modica, A., 2004. Effects of fish farming waste to sedimentary and particulate organic matter in a southern Mediterranean area (Gulf of Castellammare, Sicily): a multiple stable isotope study ␦13 C and ␦15 N. Aquaculture 234, 199–213. Sherwood, O.A., Lapointe, B.E., Risk, M.J., Jamieson, B.E., 2010. Nitrogen isotopic records of terrestrial pollution encoded in Floridian and Bahamian Gorgonian corals. Environ. Sci. Technol. 44, 874–880. Sigleo, A.C., Macko, S.A., 2002. Carbon and nitrogen isotopes in suspended particles and colloids, Chesapeake and San Francisco Estuaries. Estuar. Coast. Shelf Sci. 54, 701–711. Simunovic, A., 1970. Recherches écologiques sur les peuplements faunistiques de l’étage supralittoral des côtes rocheuses de l’Adriatique moyenne. Acta Adiatica 14, 3–39. Tucker, J., Sheats, N., Giblin, A.E., Hopkinson, C.S., Montoya, J.P., 1999. Using stable isotopes to trace sewage-derived material through Boston Harbor and Massachusetts Bay. Mar. Environ. Res. 48, 353–357. Vizzini, S., Mazzola, A., 2002. Stable carbon and nitrogen ratios in the sand smelt from a Mediterranean coastal area: feeding and effect of season and size. J. Fish Biol. 60, 1498–1510.
230
P. Zˇ vab Roˇziˇc et al. / Ecological Indicators 36 (2014) 224–230
Vizzini, S., Mazzola, A., 2004. Stable isotope evidence for the environmental impact of a land-based fish farm in the western Mediterranean. Mar. Pollut. Bull. 49, 61–70. Vizzini, S., Mazzola, A., 2006. The effects of anthropogenic organic matter inputs on stable carbon and nitrogen isotopes in organisms from different trophic levels in a southern Mediterranean coastal area. Sci. Total Environ. 368, 723–731. Vizzini, S., Benedetto, S., Thang, D.C., Mazzola, A., 2005. Spatial variability of stable carbon and nitrogen isotope ratios in a Mediterranean coastal lagoon. Hydrobiologia 550, 73–82. Wada, E., Kabaya, Y., Kurihara, Y., 1993. Stable isotopic structure of aquatic ecosystems. J. Biosci. 18, 483–499. Wilson, R.M., Chanton, J., Lewis, G., Nowacek, D., 2009. Isotopic variation (ı15 N, ı13 C and ı34 S) with body size in post-larval estuarine consumers. Estuar. Coast. Shelf Sci. 83, 307–312.
Xu, L., Zhang, M., 2012. Primary consumers as bioindicator of nutrient pollution in lake planktonic and benthic feed webs. Ecol. Indic. 14, 189–196. ´ Zˇ upan, I., Peharda, M., Dolenec, T., Dolenec, M., Zˇ vab Roˇziˇc, P., Lojen, S., Ezgeta-Balic, D., Arapov, J. Study of bivalve Noah’s Ark (Arca noae) cultured under experimental integrated aquaculture conditions, submitted for publication. Zˇ vab, P., Dolenec, T., Lojen, S., Kniewald, G., Vodopija, J., Lambaˇsa Belak, Zˇ ., Dolenec, M., 2010. Use of stable nitrogen (␦15 N) isotopes in food web of the Adriatic Sea, Croatia. RMZ – Mater. Geoenviron. 57, 41–52. Zˇ vab Roˇziˇc, P., Dolenec, T., Lojen, S., Kniewald, G., Dolenec, M. Variability in stable isotope composition of particulate organic matter in the marine coastal environment (Istra Peninsula, Northern Adriatic), submitted for publication.