Nitrogen stable isotope ratio in the manila clam, Ruditapes philippinarum, reflects eutrophication levels in tidal flats

Marine Pollution Bulletin 58 (2009) 1447–1453

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Nitrogen stable isotope ratio in the manila clam, Ruditapes philippinarum, reflects eutrophication levels in tidal flats Satoshi Watanabe a,*, Masashi Kodama b, Masaaki Fukuda c a

Japan International Research Center for Agricultural Sciences, 1-1 Owashi, Tsukuba, Ibaraki 305-8686, Japan National Research Institute of Fisheries Science, 2-12-4 Fukuura, Kanazawa, Yokohama, Kanagawa 236-8648, Japan c Hokkaido National Fisheries Research Institute, 116 Katsurakoi, Kushiro, Hokkaido 085-0802, Japan b

a r t i c l e

i n f o

Keywords: Eutrophication Nitrogen stable isotope ratio Dissolved inorganic nitrogen Particulate organic matter Manila clam, conchiolin

a b s t r a c t Understanding the effects of anthropogenic eutrophication on coastal fisheries may help in the enhancement of fishery production by effective utilization of sewage effluents, as well as in the consequent reduction of eutrophication. In this study, it was revealed that the nitrogen stable isotope ratio (d15N) in the soft tissues of the manila clam, Ruditapes philippinarum, can be used as an indicator of anthropogenic eutrophication levels in tidal flat environments by investigation of d15N in dissolved inorganic nitrogen (DIN), particulate organic matter (POM), sedimentary organic matter (SOM) and soft tissues of the clam in five tidal flats in Japan with different levels of DIN concentration. In addition, it was found that the acid insoluble fraction of the shell organic matrix, conchiolin, can be used as a proxy for the soft tissues in d15N analyses. This will contribute in easier storage handling and the expansion of chances for sample acquisition. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Human activities have led to increases in nutrients, nitrogen and phosphorus, in aquatic environments, resulting in eutrophication of coastal waters (Nixon, 1995). Anthropogenic nutrient enrichments have ecological impacts, including enhanced primary production and changes in community structure (Paerl, 1997; Sommer et al., 2002). While adverse effects of eutrophication, such as the occurrence of toxic red tides and consequent hypoxia are often emphasized, moderate eutrophication may also have positive effects upon coastal fisheries (Yamamoto, 2003). It may become unnecessary, for instance for farmers to apply fertilizers for Porphyra aquaculture to prevent bleaching of thalli due to depletion of nitrogenous nutrients (Nishikawa et al., 2007) if effective use of anthropogenic waste in the watersheds is achieved. Appropriate control of nutrient load to coastal waters through sewage and riverine water may lead to not only enhanced coastal fisheries in algae and animals but also the consequent effective reduction of eutrophication. Fishery production of the manila clam, Ruditapes philippinarum, has markedly declined for the last two decades due to stock depletion in tidal flats in Japan. While the stock depletion is considered

* Corresponding author. Tel.: +81 29 838 6609; fax: +81 29 838 6655. E-mail address: [email protected] (S. Watanabe). 0025-326X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2009.06.018

to be attributable to factors, such as reclamation of tidal flats, environmental deteriorations and poor fishery management (Ishii et al., 2001; Kakino, 1986; Toba, 2004), local fishermen in some parts of Japan anecdotally claim that the depletion is due partly to the diminished food supply for R. philippinarum by reduced nutrient loads to coastal waters. Understanding the effects of anthropogenic nutrient loads to stock conditions of R. philippinarum may help improve fishery management; however, there is little information available about it. Sewage treated water contains dissolved inorganic nitrogen (DIN) with significantly high nitrogen stable isotope ratio (d15N) due to denitrification during the treatments (Macko and Ostrom, 1994). Applications of nitrogenous fertilizer to agricultural farmlands lead to an enhancement of soil denitrification and increase in d15N in groundwater (Ogawa et al., 2001). The d15N of nitrate has higher values in human and animal wastes (10–20‰) than in atmospheric deposition (2–8‰) and N fixation by cyanobacteria (2 to 0‰) (McClelland et al., 1997; Oowada et al., 2003). Thus, groundwater with an elevated d15N appears to act as an indicator of the level of anthropogenic nitrogen loads to coastal waters (McClelland et al., 1997), and the d15N delivered to the coastal waters has a pervasive influence on nitrogen of primary producers living in the coastal waters (Costanzo et al., 2005; McClelland and Valiela, 1998). Some benthic animals (zebra mussels, amphipods and snails) were found to preferentially assimilate sewage-derived particulate

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organic matter (DeBruyn and Rasmussen, 2002) and consequently have higher d15N values. The d15N of animals including polychaetes, bivalves, shrimps and teleost fishes are suggested to be higher in the food web originated from primary producers assimilating sewage effluent (Hadwen and Arthington, 2007; McKinney et al., 2001), and therefore, d15N of these animals may be used as an indicator of nitrogen sources to coastal waters. However, these studies did not directly compare DIN concentrations in ambient water and d15N in the animals. In this study, concentration and d15N in DIN were measured in various tidal flats in Japan to confirm the relationship between the level of anthropogenic eutrophication and 15N enrichment. The d15N of particulate organic matter in the bottom water, sedimentary organic matter and soft tissues of R. philippinarum were measured to study the mutual relationships to determine whether d15N in R. philippinarum can be used as an indicator of anthropogenic eutrophication levels in the tidal flat environment. In addition, the feasibility of using shell organic matrix was also tested as a substitute for soft tissues in R. philippinarum for easier storage handling and the possibility of expansion of sample acquisition.

GF/D filters soaked with 25 ll 2 M H2SO4 and bound by a Teflon membrane by floating the filter on the sample water in a sealed container. After the DIN recovery, the membrane was removed and the filter was concealed in a tin container, and d15N was measured immediately. 2.3. Surface sediment analyses The top 1 cm sediment of the tidal flat was collected (n = 5 for each site) with a core sampler made of a 50 ml injection syringe (inner diameter of 29 mm) by cutting off the needle end. The sediment sample was placed in a 50 ml centrifugal tube. Distilled water was added ad libitum to the tube and sonicated in a cold water bath to collect particulate sedimentary organic matter (SOM) that accumulated on the sample surface with a Pasteur pipette. The SOM sample was stored in a 2 ml micro test tube, decarbonated with 1 N HCl, rinsed three times with distilled water, oven-dried at 60 °C overnight, and concealed in a tin container for d15N measurement (n = 5 for each site). 2.4. Clam analyses

2. Materials and methods 2.1. Sample collection Bottom water, surface sediment and R. philippinarum were sampled at tidal flats in Kanzanji (34°460 N, 137°370 E), Sakume (34°470 N, 137°360 E) and Washizu (34°430 N, 137°330 E) in Lake Hamana, Shizuoka Prefecture on August 10, 2005, Wajiro (33°410 N, 130°260 E) in Hakata Bay, Fukuoka Prefecture on December 8, 2005, and Seaside Park of Yokohama (35°200 N, 139°380 E) in Tokyo Bay, Kanagawa Prefecture on December 12, 2005. These sites were selected in an attempt to obtain data from varied eutrophication levels. Lake Hamana was expected to be less eutrophied as compared to heavily populated Hakata Bay and Tokyo Bay. Sewage treatment plants were located near Washizu, Wajiro and Seaside Park of Yokohama. Kanzanji was located in the vicinity of a river mouth where household effluent water was discharged. Sakume was close to neither a sewage treatment plant nor household effluent and therefore expected to be the most oligotrophic among all the sites.

The whole soft tissue samples of R. philippinarum (n = 20 for each site) were collected by gently scraping with forceps; the samples were then lyophilized and ground to a fine powder. Since lipids can fluctuate in amount and have a lighter carbon stable isotope ratio (d13C) than other tissue fractions (DeNiro and Epstein, 1981; Tieszen et al., 1983), samples for stable isotopic analyses are usually defatted. A fraction of powdered samples was defatted by the conventional Folch method (Folch et al., 1957) using methanol and chloroform. The defatted samples were then dried and concealed in a tin container for d15N and d13C measurement. The acid insoluble fraction of shell organic matrix (i.e. conchiolin, Fremy, 1855) was obtained by decalcification of the shells. The shells were carefully cleaned of remaining soft tissues, broken into small pieces and placed in a 15 ml centrifugal tube. Concentrated HCl was added to the tube drop by drop until CaCO3, the main inorganic component of the shell, was completely removed as bubbles (CaCO3 + 2HCl ? CaCl2 + H2O + CO2"). The decalcified samples were then collected into a 1.5 ml micro test tube, rinsed and centrifuged with distilled water three times and oven-dried at 60 °C overnight. The conchiolin obtained was concealed in a tin container for d15N and d13C measurements.

2.2. Bottom water analyses 2.5. Stable isotopic analyses The bottom water samples were collected with a vampire sampling suction pump (The Fluid Life Corporation) through an opening of PVC pipe that was fixed to the tidal flat with a metal plate (500 ml, n = 5 for each site except for Seaside Park of Yokohama where n = 3). A small portion of the water samples (approx 10 ml) were immediately filtered through 0.45 lm Millipore HV filters (Millipore Corporation) into acid washed polypropylene tubes and was frozen at 20 °C until laboratory analysis for concentration of dissolved inorganic nitrogen, [DIN] (n = 5). Concen  trations of ammonia ([NHþ 4 ]), nitrate ([NO3 ]) and nitrite ([NO2 ]) were measured according to the standard method using a TRAACS 800 auto analyzer (Buran Lubbe Co.). The rest of the water samples were filtered with pre-combusted Whatman GF/F filters (GE Healthcare). Particulate organic matter (POM) collected on the filter was kept overnight in a glass jar saturated with HCl fume to remove CaCO3. The POM sample was then scraped off with a spatula and concealed in a tin container for d15N analysis (n = 5 except for Seaside Park of Yokohama where n = 3). The modified ammonia diffusion method (Holmes et al., 1998; Sigman et al., 1997) was employed to collect DIN from the filtered bottom water (n = 3 for each site). DIN was recovered in Whatman

The d15N and d13C of the prepared samples were analyzed using an EA-1108 elemental analyzer (Carlo Erba) coupled with an isotope ratio mass spectrometer (Finnigan Mat ConFlo II, Mat 252; Toyokawa, 2001). The isotope ratios were expressed as a per mil (‰) deviation from international standards (i.e. fossil calcium carbonate for C and air for N): d13C, d15N = (Rsample/Rstandatrd  1)  1000, where R is 13C/12C and 15N/14N. Instrumental precision was 0.2‰ (Toyokawa, 2001). Atomic organic carbon: nitrogen (C/N) ratios of the soft tissues and conchiolin in R. philippinarum were analyzed at the same time. The isotopic fractionation factor (a) of d15N was estimated expediently based on the results of the measurements. The values for a were calculated as the difference between two samples; for example, a for DIN to SOM was obtained as 1 + (d15NSOM  d15NDIN)/ 1000, assuming that total DIN is used for primary production in the SOM. The coefficient of variation (CV) was calculated as standard deviation/mean (%) for comparison of variance among the a  þ values. In these estimations, the ratio of [NO 3 ], [NO2 ] and [NH4 ] in [total DIN] and constituents of POM and SOM are ignored for simplification.

S. Watanabe et al. / Marine Pollution Bulletin 58 (2009) 1447–1453

respectively. The mean [total DIN] had a significant positive logarithmic correlation with the mean d15N in DIN (Fig. 1C, R2 = 0.91, P < 0.05).

3. Results 3.1. Concentrations and d15N of DIN in bottom water The mean [total DIN] in bottom water had two orders of magnitude difference among the sampling sites: 3.8 ± 0.44 lM (±standard error), 1.2 ± 0.22 lM, 4.5 ± 0.24 lM, 53 ± 4.0 lM and 32 ± 0.20 lM for Kanzanji, Sakume, Washizu, Wajiro and Seaside Park of Yokohama, respectively (Fig. 1A). All the differences were statistically significant, except among the three sites in Lake Hamana (ANOVA: P < 0.0001, Tukey: P < 0.0001, P > 0.08 in Lake Hamana). The proportion of [NHþ 4 ] in [total DIN] was high in Sakume and Washizu (80% and 77%, respectively), and [NO 3 ] was predominant, occupying more than 60% in the other sampling sites (Fig. 1B). Proportion of [NO 2 ] was low in all the five sites, ranging from 2.2% to 8.1%. The mean d15N value in total DIN had a wide range: 4.8 ± 2.6‰, 2.0 ± 0.42‰, 0.13 ± 0.87‰, 13 ± 1.3‰ and 10 ± 0.27‰ for Kanzanji, Sakume, Washizu, Wajiro and Seaside Park of Yokohama,

b

100

[total DIN] (log μM)

A

10

c

a

a a

1 KZ

SA

WZ

WJ

SY

1

B

NO3-

0.8 Ratio

NO2-

0.6

Particulate organic matters and total DIN in the bottom water had relatively lower d15N values in Lake Hamana and higher values in Hakata Bay. The mean d15N in POM in the bottom water ranged from 7.3 ± 0.071‰ in Sakume to 11 ± 0.030‰ in Wajiro, and the mean d15N of SOM ranged from 4.7 ± 0.18‰ in Sakume to 10 ± 0.19‰ in Wajiro (Table 1). The mean d15N in total DIN ranged from 2.0 ± 0.42‰ in Sakume to 13 ± 1.3‰ in Wajiro (Table 1), and it had a significant positive linear correlation with the mean d15N in POM (Fig. 2A, R2 = 0.81, P < 0.05) and SOM (Fig. 2B, R2 = 0.87, P < 0.05). The slope and the y-intercept of the two regression lines significantly differed from 1 and 0, respectively (t-test, P < 0.01). 3.3. d15N of clam and the relationship with DIN and particulate organic matters The mean d15N in the soft tissues of R. philippinarum varied at different sampling sites: 8.6 ± 0.052‰, 8.1 ± 0.089‰, 9.8 ± 0.077‰, 14.5 ± 0.12‰ and 12.0 ± 0.11‰ for Kanzanji, Sakume, Washizu, Wajiro and Seaside Park of Yokohama, respectively. All the differences were statistically significant (ANOVA: P < 0.0001, Tukey: P < 0.01). The mean d15N in R. philippinarum soft tissues had a significant positive linear correlation with the mean d15N in POM (Fig. 3A, R2 = 0.98, P < 0.01), SOM (Fig. 3B, R2 = 0.98, P < 0.01) and total DIN (Fig. 3C, R2 = 0.78, P < 0.05). The y-intercept (6.9) of the regression line between d15N in R. philippinarum and POM differed significantly from 3.5 (i.e. the median value for d15N fractionation per trophic level Minagawa and Wada, 1984; t-test, P < 0.01), and the slope (2.0) differed significantly from 1 (t-test, P < 0.01). The y-intercept (2.3) of the regression line between d15N in R. philippinarum and SOM did not differ significantly from 3.5 (t-test, P > 0.2), and the slope (1.2) did not differ significantly from 1 (t-test, P > 0.1). The slope (0.38) of the regression line between d15N in R. philippinarum and total DIN differed significantly from 1 (t-test, P < 0.01). 3.4. Relationship between DIN, particulate organic matters and clam

04 0.4

0 KZ

SA

WZ

WJ

SY

15

δ15Ntotal DIN (‰)

3.2. d15N of DIN and d15N of particulate organic matters

NH4+

0.2

C

1449

10 5 R2 = 0.91 P < 0.05

0

-5 1

10

100

[total DIN] (log μM)

The mean [total DIN] in the bottom water was positively correlated to the mean d15N in R. philippinarum soft tissues (Fig. 4A, R2 = 0.91, P < 0.05), POM (Fig. 4B, R2 = 0.95, P < 0.01) and SOM (Fig. 4C, R2 = 0.97, P < 0.01). Ballpark estimates of isotopic fractionation factor (a) of d15N based on the results of this study were summarized in Table 2. The a values for assimilation of DIN by SOM had a wide range from 0.9977 (i.e. 2.3‰) to 1.0066 (i.e. +6.6‰) and the mean was 1.0020 ± 0.0019. The mean a for the assimilation of total DIN by POM and by R. philippinarum was 1.0036 ± 0.0023 and 1.0055 ± 0.0018, respectively. The mean a for the assimilation of SOM and POM by R. philippinarum was 1.0035 ± 0.0002 and 1.0019 ± 0.0006, respectively. The coefficient of variation (CV) had relatively high values for the a for assimilation of DIN by SOM (42.0%), POM (50.5%) and R. philippinarum (40.6%) as compared to those for the assimilation of SOM (5.4%) and POM (13.6%) by R. philippinarum. 3.5. Stable isotope ratios in shell conchiolin

Fig. 1. Mean concentrations (±SE) in total DIN (A, B) and the relationship between mean concentration and mean d15N in total DIN (C) in tidal flats. KZ: Kanzanji, SA: Sakume and WZ: Washizu in Lake Hamana, WJ: Wajiro in Hakata Bay and SY: Seaside Park of Yokohama in Tokyo Bay. d15Ntotal DIN = 3.8 ln [total DIN]  2.7. Letters (a, b and c) indicate significant difference (Tukey P < 0.05).

The recovery rate of conchiolin from shell fractions was approximately 7% (wt/wt). The mean C/N ratio of the conchiolin and the defatted soft tissues was 3.5 ± 0.020 and 3.5 ± 0.031, respectively,

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Table 1 Mean (±SE) d15N (‰) in particulate organic matter (POM), sedimentary organic matter (SOM) and total dissolved inorganic nitrogen (DIN) collected in Kanzanji (KZ), Sakume (SA), Washizu (WZ), Wajiro (WJ) and Seaside Park of Yokohama (SY). Site

d15NPom

n

d15Nsom

n

d15Ndin

KZ SA WZ WJ SY

7.9 ± 0.11 7.3 ± 0.07 8.5 ± 0.10 10.6 ± 0.03 9.5 ± 0.28

5 5 5 5 5

5.8 ± 0.13 4.7 ± 0.18 6.2 ± 0.08 10.2 ± 0.19 8.5 ± 0.28

5 5 5 5 5

4.8 ± 2.58 2.0 ± 0.42 0.1 ± 0.87 12.5 ± 1.29 10.1 ± 0.27

n

n

3 3 3 3 3

d15Npom

SA

WZ

WJ

SY

d15Nsom

SA

WZ

WJ

SY

d15Ndin

SA

WZ

WJ

SY

KZ SA WZ WJ

ns – – –

ns

ns ns ns –

***

KZ SA WZ WJ

**

ns

***

***

KZ SA WZ WJ

*

ns ns – –

**

ns

***

***

* **

***

– –

*** *** ***

– – –

***

***

***

– –

***

***

–

ns

– – –

***

**

–

ns

P < 0.05. P < 0.01. P < 0.001.

12 10

B

10

8 6

2

R = 0.81 P < 0.05

-5

12

0

5

10

15

15

δ Ntotal DIN (‰)

15 δ NSOM (‰)

A δ15NPOM(‰)

***

8 R2 = 0.87 P < 0.05

6 4

-5

0

5 10 δ15Ntotal DIN (‰)

15

Fig. 2. Relationship between mean (±SE) d15N in total DIN and POM in the bottom water (A) and SOM in the tidal flat surface (B). d15Ntotal d15Ntotal DIN = 0.34  d15NSOM + 5.4.

and the difference was statistically significant (paired t-test, P < 0.05). The d15N in the conchiolin (6.1–13.9‰, mean: 9.4 ± 0.30‰) and the defatted soft tissues (7.4–15.3‰, mean: 10.5 ± 0.32‰) had a strong positive linear correlation (Fig. 5A, R2 = 0.96, P < 0.0001): d15Nconchiolin = 0.91  d15Nsoft tissues  0.17. The y-intercept (0.17) of the regression line did not differ significantly from 0 (t-test, P > 0.5), and the slope (0.91) differed significantly from 1 (t-test, P < 0.001). The d13C value of the conchiolin (16.9‰ to 12.3‰, mean: 15.0 ± 0.13‰) and the defatted soft tissues (16.5‰ to 13.7‰, mean: 15.0 ± 0.07‰) had a weak positive linear correlation (Fig. 5B, R2 = 0.22, P < 0.01): d13Cconchiolin = 0.83  d13Csoft tissues  2.6. The y-intercept (2.6) and the slope (0.83) did not differ significantly from 0 and 1, respectively (t-test, P > 0.1). 4. Discussion The mean [total DIN] in the bottom water of the studied tidal flats increased in the following order: Lake Hamana (Kanzanji, Sakume and Washizu), Tokyo Bay (Seaside park of Yokohama) and Hakata Bay (Wajiro). This result is in agreement with the data for chemical oxygen demand (COD) reported for these areas: 1.3 mg/l, 2.5 mg/l and 2.9 mg/l for Lake Hamana, Tokyo Bay off the coast of Seaside Park of Yokohama and eastern Hakata Bay, respectively, in 2005 (Ministry of Environment, Government of Japan, 2006). The coastlines of Hakata Bay and Tokyo Bay are heavily populated, and Wajiro is located in closed-off section of the bay where water circulation is poor. Thus, it seems that the observed DIN concentrations properly represent the eutrophication level in the study sites. The ratio of [NHþ 4 ] in [total DIN] was higher in Sakume and Washizu than in other sites. Since NHþ 4 is gradually reacted into  NO 2 and NO3 in the wild (Billen, 1975; Middelburg and Nie-

DIN

= 0.19  d15NPOM + 7.8;

uwenhuize, 2001) and DIN is usually released in the form of NO 3 in sewage effluent, high [NHþ 4 ] is generally thought to indicate high decomposition activity of organic materials in the water and sediment (Kuwae et al., 2003). The sample collections in Lake Hamana were conducted in summer, when decomposition is expected to be more active than in winter in which sample collections were conducted for Hakata Bay and Tokyo Bay. Although effects of propor  tional pattern of [NHþ 4 ], [NO2 ] and [NO3 ] were not examined, [total DIN] had a strong positive correlation with d15N in total DIN. These results endorse previous studies which have shown that anthropogenic eutrophication increases d15N in DIN in environmental waters (Macko and Ostrom, 1994; McClelland et al., 1997; Ogawa et al., 2001; Oowada et al., 2003). Sedimentary organic matter in tidal flats may mainly contain microalgae, microfauna, algal and animal detritus and terrestrial detritus, of which the microalgae are considered to take up and assimilate DIN in the bottom water. Since isotopic fractionation (a) of both d15N and d13C are negligible during decomposition (Haines, 1977; Macko and Ostrom, 1994), algal detritus produced in the area is also considered to reflect the stable isotopic values of the DIN in the bottom water. The a for DIN assimilation by algae are reported to be 1.001–1.019, 0.991–1.004 and 0.993–1.020 for  þ NO 3 , NO2 and NH4 (Montoya and McCarthy, 1995; Wada et al., 1975; Waser et al., 1998). Although the components of particulate organic matters were not analyzed in this study, the rough estimates of the mean a values for the uptake of total DIN by POM (1.0036) and SOM (1.0020) were within the reported ranges. The y-intercept (offset) for the linear regression lines between d15N in total DIN and POM (+7.8, a = 1.0078) and between total DIN and SOM (+5.4‰, a = 1.0054) were also within the reported ranges. Although the slopes significantly differed from 1 in these regressions, indicating that d15N in POM and SOM may not be directly

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S. Watanabe et al. / Marine Pollution Bulletin 58 (2009) 1447–1453

A

A

16

14

12 10 2

R = 0.98 P < 0.01

8

15 d Nclam (‰)

15 d Nclam (‰)

14

15 d Nclam (‰)

8

10 15N (‰) d POM

1

12 10 R 2 = 0.98 P < 0.01

B

12

10

6 6

8

10

2

1

C

10 R2= 0.78 P < 0.05

6 -5

0

5

10

15

15 d Ntotal DIN(‰)

15 d NSOM(‰)

d15 Nclam (‰)

14 12

R = 0.95 P < 0.01

6

12

16

8

100

8

d15 NSOM(‰)

C

10

[total DIN] (log mM)

14

4

2

R = 0.91 P < 0.05

6

12

16

8

10

15 d NPOM (‰)

6

12

8

6

B

16

10 [total DIN] (log mM)

100

12 10 8 6

R 2= 0.97 P < 0.01

4 1

10

100

[total DIN] (log mM)

Fig. 3. Relationship between mean (±SE) d15N in Ruditapes philippinarum soft tissues and POM in the bottom water (A), SOM in the tidal flat surface (B) and total DIN in the bottom water (C). d15Nclam = 2.0  d15NPOM + 6.9; d15Nclam = 1.2  d15NSOM + 2.3; d15Nclam = 0.38  d15Ntotal DIN + 8.7. The dashed lines indicate Y = X + 3.5.

Fig. 4. Relationship between mean concentration (±SE) in total DIN and d15N in Ruditapes philippinarum soft tissues (A) and POM in the bottom water (B) and SOM in the tidal flat surface (C). d15Nclam = 1.6 ln [total DIN] + 7.3; d15NPOM = 0.81 ln [total DIN] + 7.1; d15NSOM = 1.4 ln [total DIN] + 4.2.

proportional to that in total DIN in the bottom water, the mean d15N in POM and SOM and total DIN was in a significant positive  þ linear correlation. While d15N in NO 3 , NO2 and NH4 should be analyzed separately for more precise understanding of nitrogen pathways, it seems reasonable to conclude that d15N in POM and SOM represents that in total DIN, since almost all algae can grow on  þ NO 3 , NO2 or NH4 , and in most instances, comparable growth occurs on all three sources (Syrett, 1981). R. philippinarum is held to feed mainly on marine organic particles, including phytoplankton (Yokoyama et al., 2005), microphytobenthos (Kanaya et al., 2005; Yamaguchi et al., 2004) and their detritus (Numaguchi, 2001). Stable isotopic analyses in this study revealed a strong positive linear correlation in d15N between R. philippinarum and POM and between R. philippinarum and SOM. The y-intercept of the regression between R. philippinarum and SOM (2.3) was not significantly different from 3.5 (a = 1.0035), the median value of a for d15N per trophic level increase (i.e. between 1.003 and 1.004, Minagawa and Wada, 1984), but in contrast the y-intercept for R. philippinarum vs. POM regression (6.9) was far below and significantly different from 3.5. While the slope for regression between R. philippinarum and SOM (1.2)

was not significantly different from 1, the slope for R. philippinarum vs. POM (2.0) was significantly larger than 1. The rough estimation of a for assimilation of nitrogen in SOM by R. philippinarum was 1.0035, which was within the previously reported range of a for d15N per trophic level increase; however, the a for POM assimilation by R. philippinarum (1.0019) was outside the reported range. Thus, although both POM and SOM are significantly correlated to R. philippinarum, SOM seems to be more directly linked to R. philippinarum. Higher CV for the a for POM assimilation by R. philippinarum (13.6%) than SOM assimilation (5.4%) also suggests relatively weaker linkage between POM and R. philippinarum. This may be explained by POM in the water column tending to be more widely mixed and dispersed in tidal flats as well as offshore waters by tidal currents as compared to SOM which can be a d15N indicator for a smaller area of R. philippinarum habitat. The correlation of d15N between total DIN and R. philippinarum was positively significant; therefore, d15N is thought to be a valid tracer of nitrogen in a tidal flat from dissolved inorganic form to the soft tissues of the filter feeding bivalve, supposedly linked through primary production and consumption. Although R. philippinarum is not considered to directly utilize riverine particulate organic matter (Kasai et al.,

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Table 2 Isotopic fractionation factor (a) in d15N of particulate organic matter (POM), sedimentary organic matter (SOM), total dissolved inorganic nitrogen (DIN) and R. philippinarum (Clam) collected in Kanzanji (KZ), Sakume (SA), Washizu (WZ), Wajiro (WJ) and Seaside Park of Yokohama (SY). Site

DIN to SOM

DIN to POM

SOM to clam

POM to clam

DIN to clam

KZ SA WZ WJ SY

1.0010 1.0066 1.0061 0.9977 0.9984

1.0031 1.0092 1.0084 0.9980 0.9995

1.0028 1.0035 1.0036 1.0043 1.0035

1.0007 1.0009 1.0013 1.0040 1.0025

1.0038 1.0101 1.0097 1.0020 1.0019

Mean SE CV (%)

1.0020 0.0019 42.0

1.0036 0.0023 50.5

1.0035 0.0002 5.4

1.0019 0.0006 13.6

1.0055 0.0018 40.6

A

16

B

-10

15 δ Nconchiolin(‰)

14

13 δ Cconchiolin(‰)

SE: standard error. CV: coefficient of variation.

-12

12 10 8 6

R2 = 0.96 P < 0.0001

6

8

10

12

14

16

-14 -16 -18

R2 = 0.22 P < 0.01

-17

-16

-15

-14

-13

13 δ Csoft tissues (‰)

15 δ Nsoft tissues (‰)

Fig. 5. Relationship of d15N (A) and d13C (B) between soft tissues and conchiolin in Ruditapes philippinarum. d15Nconchiolin = 0.91  d15Nsoft 0.83  d13Csoft tissue  2.6. The dashed lines indicate Y = X.

2004), it seems to contribute in the removal of anthropogenic DIN through the food chain in tidal flat assemblages. The mean [total DIN] had a significant positive correlation with the mean d15N in POM, SOM and R. philippinarum. Therefore, anthropogenic eutrophication is considered to increase the d15N in particulate organic matters in the tidal flats and consequently R. philippinarum that feeds on these particles. On the basis of these results, it is surmised that the d15N in R. philippinarum soft tissues can be used as an indicator of nitrogenous eutrophication level in a tidal flat, and this method can be used to study the effects of eutrophication on R. philippinarum stock conditions in fishing grounds. Effective use of anthropogenic inorganic nitrogen sources for coastal fisheries may become possible by regulation of river and sewage flow (Yamamoto, 2003) or nitrogen load based on the monitoring of the d15N in R. philippinarum. Stable isotopic analyses of animals are usually performed in soft tissues, which must be frozen, freeze-dried or oven-dried for storage since formalin and ethanol preservations alter isotopic signatures of the samples (Kaehler and Pakhomov, 2001). In this study, the feasibility of using shell organic matrix (conchiolin) in stable isotopic analyses was tested for easier storage handling and the possibility of expansion of sample acquisition. As the results of comparisons of d15N and d13C between the defatted whole soft tissues and conchiolin of R. philippinarum, the d15N in the conchiolin was almost directly proportional to that in the soft body. The slope of the regression line (0.91) was not significantly different from 1, and although the y-intercept (0.17) was significantly different from 0, the difference may be negligible for the instrumental precision is 0.2‰. Therefore, d15N can be considered almost equal between the conchiolin and the defatted soft body of R. philippinarum. The correlation of d13C between the conchiolin and the soft tissues was weaker than that in d15N. Although the y-intercept (0.72) and the slope (4.3) of the regression line did not differ significantly from 0 and 1, respectively, direct proportionality between these two factors seems too meager to be conclusive due to the

tissue

 0.17; d15Nconchiolin =

low correlation. Although there are studies on the relationship between d13C in inorganic fraction of mollusc shells and ambient environmental factors, such as d13C in inorganic carbon, CO2 concentration and salinity (Colonese et al., 2007; Gillikin et al., 2006; Lecuyer et al., 2004; Mook and Vogel, 1968), there are few studies on the shell organic matrix. Stott (2002) reported that d13C was different by as much as 5‰ between the conchiolin and soft tissues of a land snail, Helix aspersa, and d13C in the conchiolin did not track the snail diet. Since it is not known whether this is true for aquatic bivalve shells, more careful study seems necessary. The C/N ratio was significantly higher in conchiolin than in defatted soft tissues, indicating the conchiolin containing additional carbon rich compounds, such as lipids and mucopolysaccharides (Beedham, 1958; Meenakshi et al., 1969). It may be necessary to elucidate the effects of carbon rich compounds on d13C, as well as isotopic fractionation associated with shell matrix secretion by the mantle. This study showed that the shell conchiolin could be a proxy of the soft tissues in d15N analyses in R. philippinarum. Not only does the use of shell conchiolin make the storage handling of samples easier, it may also extend applicability of the stable isotopic method to old museum samples and fossil shells. It is assumed that changes in stable isotope ratios are negligible during decomposition and diagenesis (Haines, 1977; Macko and Ostrom, 1994), and a palaeological study indicated that the original isotopic compositions of bulk organic matter in soils from late Miocene age (i.e. 5 million years ago) are conserved to date (Cerling et al., 1989). Although further examination of time course change in d15N is necessary, d15N in shell conchiolin may be used as a useful tool to study eutrophication history of the coastal waters. 5. Conclusions The d15N in the soft tissues of R. philippinarum was positively correlated to total DIN concentration in the bottom water in tidal flats. The higher d15N in R. philippinarum seemed to be attributable to higher d15N in the food particles, especially those in sediment

S. Watanabe et al. / Marine Pollution Bulletin 58 (2009) 1447–1453

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