C. K. Yap* & S. G. Tan

Transcription

1 Indian Journal of Marine Sciences Vol. 36 (3), September 2007, pp Iron (Fe) concentrations in the byssus and soft tissues of the green-lipped mussel Perna viridis (L.): Byssus as an excretion route of Fe and Fe bioavailability in the coastal waters C. K. Yap* & S. G. Tan Department of Biology, Faculty of Science, Universiti Putra Malaysia, UPM, Serdang, Selangor, Malaysia *[ / ] Received 29 May 2006, revised 13 June 2007 Iron (Fe) in the marine mussels has been reported to play an important role in the strength of attachment of the mussels since Fe is a major element in the attachment property on the mussel byssus. However, the distribution of Fe in the greenlipped mussel Perna viridis has not been reported in the literature. In this study, the distribution of Fe in different parts of the byssus and soft tissues of Perna viridis were investigated. It was found that the attachment plaque of the byssus accumulated the highest concentration of Fe, followed by the proximal, distal, stem and root. Iron levels were determined in the byssus and soft tissues of P. viridis collected from 12 geographical sites. It was found that the Fe levels in the byssus were the highest among all the soft tissues studied. The Fe levels could be about 3-17 times higher (P <0.05) in the byssus than in the different soft tissues of P. viridis. Since the byssus of blue mussel Mytilus edulis has been reported to be an excretion route for Fe, it is believed that the byssus of P. viridis is an excretion route for Fe. The Fe could be an essential metal for byssal formation and the byssus is a potential biomonitoring organ for Fe bioavailaibility in coastal waters. [Key words: Byssus, Fe, bioavailability, mussel, Perna viridis] Introduction Total iron (Fe) in oceans is estimated to be metric tones, contributed by geological processes and man-induced activities such as mining 1. It was also inferred that Fe concentrations in coastal waters is a function of freshwater input and is greatly influenced by riverine waters 2. In marine mussels, Fe concentration in the byssus was found to contribute to the strong attachment and byssus formation 3. The mussels have been found to secrete an adhesive that consist of a hardened matrix of proteins in the byssus. Fe found in the byssus is most likely to be assimilated in the soft tissues of the mussels with negligible direct adsorption from the seawater. Therefore, this may help to understand the chemistry of the glue for developing effective antifouling paints. Mytilus edulis uses Fe to produce its super-strong natural glue 3. Although information on the distribution of Fe in the byssus and soft tissue of marine mussels has been reported 4,5, the potential of the byssus of Perna viridis has not been discussed. *Corresponding author: Tel: Fax: , The uptake of Fe might be expected to be unusual since this metal normally occurs in seawater as a colloidal precipitate of hydrated ferric hydroxide 6 and readily accumulates to high concentrations in the common mussel M. edulis 7,8. The recommendation of using the byssus of P. viridis 9 as a biomonitoring material for Zn is based on the fact that it acts as an excretion route for Zn. This phenomenon is well supported by numerous studies that showed that the byssus of marine mussels could be used for biomonitoring purposes in coastal waters. Unlu & Fowler 13 reported that the byssus of M. galloprovincialis played an important role in the elimination of arsenic from the mussels. The byssus of M. edulis was shown to be a good biomonitoring tool for and Hg 5 and Cu 12. Hence, all the above reports suggested that the byssus can be a biomonitoring organ for toxic trace metals. Based on correlation analysis between Zn in the byssus and Zn geochemical fractions in the sediments, Yap et al. 14 confirmed that byssus was a good biomonitoring organ of Zn as a replacement for soft tissues since soft tissues had been shown to possess a Zn partial regulatory mechanism in P. viridis 9. This

2 228 INDIAN J. MAR. SCI., VOL. 35, NO. 3, SEPTEMBER 2007 brings into question the use of the total soft tissue of P. viridis in the biomonitoring of Fe and as an organ for Fe excretion. The objective of the present study was to determine the concentrations of Fe in the different soft tissues and byssus, and to discuss the potential of the byssus as an excretion route for Fe in P. viridis. Materials and Methods Perna viridis (L.) (Class Bivalvia; Order Mytiloida; Family Mytilidae) samples were collected between from 12 geographical sampling sites (with Fig. 1 Sampling sites along the coastal area of Peninsular Malaysia. Names of sampling sites represented by alphabets follow those in Table 1. three sites sampled twice in different years) along the west coast of Peninsular Malaysia (Fig. 1). Sampling dates, shell lengths, numbers of mussels analysed and site descriptions are given in Table 1. The mussels were collected from areas that are subjected to various types of human activities such as port, industrial, aquacultural, agricultural, domestic and recreational areas. After being transported to the laboratory, all the samples were stored at -10 C until analysis. Mussels of relatively similar sizes (shell lengths: mm) were analyzed individually from each sampling site. About 20 individuals from 9 populations were dissected into foot, mantle, gonad, crystalline style, gill, muscle and remaining soft tissues (visceral mass) in addition to byssus. All of these different tissues were pooled from the dissection of 20 individuals from each population. The dissection of the different parts of the soft tissues including byssus followed those reported by Yap et al. 9. Soft tissues and byssus were dried at 105 C for 72 hours in an oven to a constant dry weight. Two replicates of each tissue were digested in concentrated nitric acid (Ajax Chemicals, HNO 3 65%, Australia) in a hot-block digester first at low temperature (40 C) for 1 hour and then they were fully digested at 140 C for at least 3 hours. The prepared samples were determined for Fe levels by using an air-acetylene flame atomic absorption spectrophotometer (Perkin- Elmer Model 4100). The accuracy of the analysis was checked with a quality control sample made of a standard solution of Fe (1000 mg/l Fe 2 SO 4 ; MERCK). The recovery was %. In addition, the methodology of sample digestion was checked with Standard Reference Material 1577a, Bovine Liver Table 1 Dates of sampling, shell lengths and site descriptions No. Site Date of sampling Shell length (mean: mm) Site description A. P.Panjang-1 22 Sept ± 2.40 Urban and aquaculture A. P.Panjang-2 07 Oct ± 9.90 Urban and aquaculture B. Lukut 08 Aug ± 2.00 Aquaculture C. Telok Emas 09 April ± 6.90 Aquaculture D. Merlimau-1 19 April ± 2.10 Aquaculture D. Merlimau-2 09 April ± 1.15 Aquaculture E. Sebatu-1 17 April ± 2.10 Mussel aquaculture E. Sebatu-2 23 Sept ± 8.10 Mussel aquaculture F. Parit Jawa 08 April ± 45.9 Unknown G. Pontian 09 April ± 1.30 Unknown H. Pantai Lido 18 April ±26.0 Jetty and urban I. Kg. P. Puteh 18 April ± 32.1 Shipping, jetty, industries and urban J. Kuala Belungkor 18 April ± 1.06 Pristine area K. Kuala Pontian 08 April ± 26.6 Pristine area L. Nenasi 08 April ± 10.3 Pristine area

3 YAP & TAN: Fe CONCENTRATIONS IN MUSSELS 229 (National Bureau of Standard Certificate of Analysis) and the Fe recovery was 95.7% [measured: µg/g; certified: 194 ± 20 µg/g]. The Pearson s product moment correlation coefficient on the log 10 (mean + 1) transformed data 15 was applied to determine the strength of the relationships and the significance levels between any two variables by using the Statistical Analysis System (SAS) for Windows, Release 6.12 software. Results Based on the distribution of Fe in the different parts of the byssus, sampled from both the contaminated site at Kg. Pasir Puteh and the uncontaminated site at. Kuala Belungkor showed similar patterns of accumulation in which the attachment plaque was recorded to have the highest level of Fe followed by the proximal, distal and the rest of the byssal parts (Fig. 2). This result agrees with that reported by Yap et al. 16 in which the highest concentrations of Cd, Cu, Pb and Zn were found in the attachment plaque when compared to the proximal, distall, stem and root of the byssus of P. viridis collected from a relatively uncontaminated site. Samples from the contaminated site at Kg. Pasir Puteh accumulated higher levels of Fe in the attachment plaque, proximal, distal, stem and root of the byssus, when compared to similar parts in the byssus of mussels from the uncontaminated site at Kuala Belungkor Distribution in Fe different parts of the byssus would help in the understanding of the Fe accumulation pattern and binding affinity in the different parts of the byssus. However, the total byssus in the polluted Kg. Pasir Puteh samples was found to have a lower Fe level when compared those from Kuala Belungkor. Therefore, the Fe bioavailability at Kg. Pasir Puteh was lower when compared to Kuala Belungkor. This finding indicated that Fe was not an anthropogenic metal in the highly activity site, including a port and industries, at Kg. Pasir Puteh 9 and it was different from anthropogenic metals like Cd, Pb, Cu and Zn. The concentrations of Fe in the byssus and soft tissues of P. viridis collected from the west coast of Peninsular Malaysia are given in Fig. 3. From the 15 populations [12 stations], the Fe concentrations in the soft tissues of P. viridis ranged from 130 to 2700 µg/g dry weight (dw) while those in the mussel byssus ranged from 1200 to 3350 µg/g dw. The highest concentration of Fe was found in the byssus of Merlimau samples while that in the soft tissues was Fig. 2 Comparison of Fe concentrations (µg/g dry weight ± standard error), in the different parts of the byssus of Perna viridis samples from a polluted site at Kg. Pasir Puteh and an unpolluted site at Kuala Belungkor. for samples from Kuala Pontian. Since these two sampling sites had no observable human activities in their surroundings (Table 1), thus anthropogenic sources were unknown or not to be expected. The high bioavailability of Fe indicated naturally occurring Fe rather than due to pollution. In Table 2, the highest Fe level was found in the byssus, followed by the remaining soft tissues, gills, crystalline style, undigested algae in the digestive tract, mantle, gonad, muscle, foot, periostracum and total shell. In comparison to the different soft tissues (Table 3), the Fe level in the byssus was times higher than those in the foot and muscle, 9.6 time higher than that in the gonad, 6-7 times higher than those in the crystalline style and mantle, 203 times higher than those in the gills and remaining soft tissues. When compared to the hard tissues, the Fe level in the byssus was 25.5 and 30.7 times higher than those in the periostracum and total shell of P. viridis (Table 3). The Fe ranges found in the soft tissues were within the range reported for P. viridis from Dona Paula Bay 2, Goa, India ( µg/g dw). Kongkachuichai et al. 17 reported that steamed P. viridis from Thailand s coastal waters had 14.7 mg/100g (147 µg/g) of total Fe and 4.0 mg/100 g of heme Fe. Nicholson & Szefer 18 reported that Fe concentrations in the soft tissues and byssus of P. viridis of polluted and unpolluted sites of Hong Kong coastal waters were µg/g dry weight and µg/g dry weight, respectively. These wider ranges of Fe levels in the byssus than in the soft tissues agreed with those found in the present study.

4 230 INDIAN J. MAR. SCI., VOL. 35, NO. 3, SEPTEMBER 2007 Fig. 3 Distribution of Fe concentrations (µg/g dry weight) in the different soft tissues of Perna viridis collected from 9 sampling sites.

5 YAP & TAN: Fe CONCENTRATIONS IN MUSSELS 231 Table 2 Distribution of Fe concentrations (µg/g dry weight) in the different soft tissues of Perna viridis Mean ± SE Min-max N = 15 Shell length (mm) ± Total soft tissues 981 ± Byssus 2167 ± N = 9 Remaining soft tissues 1287 ± Muscle 167 ± Gills 1052 ± Crystalline style 581 ± Gonad 262 ± Mantle 456 ± Foot 157 ± Byssus 2191 ± N =2 Undigested algae 577 ± Periostracum 57.4 ± Total shell 47.7 ± Table 3 Ratios of Fe concentrations (µg/g dry weight) in the different soft tissues in relation to Fe concentrations in the byssus of Perna viridis Mean ± SE Min-max N = 9 Byssus/Remaining soft 3.43 ± tissues Byssus/Muscle ± Byssus/Gills 2.97 ± Byssus/Crystalline style 6.61 ± Byssus/Gonad 9.63 ± Byssus/Mantle 7.12 ± Byssus/Foot 16.6 ± Byssus/Byssus N = 2 Undigested algae 2.87 ± Periostracum 25.5 ± Total shell 30.7 ± Nicholson & Szefer 18 also found that the P. viridis samples collected from the polluted Victoria Harbour accumulated higher levels of Fe in their soft tissues and byssus than those from unpolluted areas. The Fe ratios of byssus/soft tissue for the field samples were found to be between 1.86 and 16.6 (Table 3). Szefer et al. 14 found the Fe ratios of byssus/soft tissue for M. galloprovincialis from the southern part of the Korean Peninsula to be between (byssus: µg/g dw and ST: µg/g dw). These Fe ratios were comparable Fig. 4 Concentrations (mean [µg/g] ± standard error) of Fe in the byssus and soft tissues of Perna viridis collected from 15 sites along the west coast of Peninsular Malaysia. to and higher than those (ratios of byssus/soft tissue as ) reported by Ikuta 10 from the same region. Szefer et al. 13 reported the Fe ratios of byssus/soft tissue in M. edulis trossulus from the Southern Baltic as being between The distributions of Fe in different soft tissues of Perna viridis collected from 9 sampling sites are presented in Fig. 4. Based on these results, three patterns can be found. First, tissue distribution of Fe was found in the different soft tissues of P. viridis. Second, the highest Fe concentration was found in the byssus among all the geographical populations (except for Nenasi) and this was always followed by the remaining soft tissues and the rest. Third, gills accumulated higher level of Fe when compared to foot, mantle, gonad, muscle and crystalline style (except for Sebatu-2). Those soft tissues (except for gills) which are less exposed to environmental seawater, more accurately indicate the metal bioavailability. Thus, the gills which are the first contact organ to environmental seawater with more surface area, accumulated more Fe and the Fe levels were even higher than in the remaining tissues of samples from Kuala Pontian. Pearson s correlation coefficient between Fe levels in byssus and soft tissues based on the field samples is positively correlated but not significant (R = 0.49; P >0.05). This low R value indicated Fe in the byssus is hardly related significantly (P >0.05) with that found in the soft tissue. Figure 5 shows that when the relationships between byssus and soft tissues were plotted against the shell length of P. viridis, it was found that Fe levels in the byssus and soft tissues were weakly and negatively correlated (R = to

6 232 INDIAN J. MAR. SCI., VOL. 35, NO. 3, SEPTEMBER 2007 Fig. 5 Relationship between shell length and Fe concentrations (mean [µg/g]) in the byssus and the soft tissues of Perna viridis (N = 15) ) with the shell length of P. viridis. These results indicated that the Fe concentrations accumulated in the byssus and soft tissues were negatively related to the size of the mussels but the correlations were not significant (P >0.05). Discussion The elevated levels of Fe found in the byssus of P. viridis could be due to a) excretion through the soft body of the mussel, b) adsorption of Fe available in its surrounding seawater onto the surfaces of byssal threads and c) it being an important metallic ion for the formation of byssus. The present field study showed that Fe levels in the byssus were higher than those in the total soft tissue. This indicated that the byssus could be more accumulative of Fe than the soft tissues of P. viridis. The narrow range (low max/min) of Zn found in the soft tissues of P. viridis indicated that the Zn level was strongly regulated in its body concentrations. When the present results are compared to the Zn partial regulation phenomenon reported by Yap et al. 12, the ratio of maximum to minimum was only less than 2.0 and this value is certainly much lower than the 7.5 recorded for Fe. However, the fact that Fe is a major element compared to Zn which could have caused some complications when these two different elements are compared or Are they comparable?. The weak relationship between Fe levels in the byssus and soft tissues did not indicate that the Fe concentrations found in the byssus of P. viridis could not be mainly due to the metabolic pathway, rather may be due to adsorption onto the byssus surface. Since Fe could be found in abundance in the coastal waters 1, the accumulation and elimination of Fe in the mussel soft tissue seem to be complicated and cannot be clearly shown by looking at the simple relationship between the byssus and soft tissues. Another possibility is that the Fe could be regulated at a much faster rate in the mussel soft tissue and this is shown in the high levels of Fe found in the byssus. Certainly, it is believed that the metabolic pathway could involve mechanisms such as a strong regulation through the byssus and detoxifying mechanisms by the protein binding sites found in the byssus. All these mechanisms would help to defend against the toxicity of Fe accumulated in the soft tissues of the mussel 2,30,31. However, the toxicity of Fe in marine bivalves was not reported in the literature. This could be due to its being a major and essential element required for the normal metabolic mechanisms and for byssal formation and attachment. Most interestingly, George et al. 21 found that a major proportion of Fe was excreted by transferring it to the byssus of M. edulis. Based on George s work, we believe that a similar phenomenon could have occurred for Fe in P. viridis although further validation is required. When compared to the byssus, the Fe levels in the total soft tissues and other soft tissues were at least 6 times lower than those in the byssus. This indicated that the byssus is a super-accumulator of Fe when compared to the total soft tissues. In fact, we also found that the Fe levels in the byssus were significantly higher than those in other soft tissues including undigested algae located in the digestive tract, foot, gonad, crystalline style, mantle, gill, muscle and visceral mass and in the hard tissues including the total shell and periostracum (Fig. 2). According to George et al. 21, M. edulis stored the highest level of Fe in the kidney among the different organs of the blue mussel. The biochemical function or physiological role of such a high concentration of Fe in marine mussels is largely unknown, although it may be involved in the tanning processes of the shell, foot, byssal gland and byssal threads 22. Therefore, this is an interesting subject for further studies. For gills and visceral mass, ingestion is by pinocytosis of the particles of ferric hydroxide, engulfment of the particle by the cell membrane being followed by pinching off to form membrane limited vesicles or sacs. Ferric Fe is bound to ligands characteristics of Class A ions when found

7 YAP & TAN: Fe CONCENTRATIONS IN MUSSELS 233 as hydroxo (phosphate) Fe III in the detoxified metabolic storage product ferritin, but it appears to bind (at least partly) to nitrogen in the Fe transport protein transferring, and in haemoglobin and cytochrome c 23. Storage of Fe in Mytilus probably occurs mainly in the kidney 21. According to Webb et al. 24, Fe deposits were found in the granules formed in the tissues of the freshwater mussel Velusunio angasi. With Perna, a significant proportion of the total Fe absorbed, is excreted via the byssal gland into the byssal threads. The absorbed Fe is transported to other tissues by amoebocytes in the haemolymph, the major proportion being deposited in the byssal threads. According to Goho et al. 3, Fe interacts with side chains on the protein molecules in the mussel byssus. The side chains, called dihydroxyphenylalanine (DOPA), have an affinity for Fe. As it turns out, a single Fe atom can bind to three DOPA side chains. The Fe-DOPA complex then reacts with oxygen, forming highly reactive chemical agents called radicals 3. Previous studies have shown that enzymes also stitch together the glue proteins. Their results indicated that the marine mussels pump Fe for the strength of attachment by using byssus This could help to explain the elevated level of Fe found in the byssus of P. viridis. Conclusion In conclusion, byssus of P. viridis could be an excretion route of Fe since the organ can readily accumulate significantly (P <0.05) higher levels of Fe than the different soft tissues of the mussel. Although Fe is not a common element from anthropogenic sources, the Fe levels found in the byssus and in the different soft tissues are interesting from the Fe excretion route point of view. Also, the byssus of P. viridis could be a potential biomonitoring organ for Fe bioavailaibility in coastal waters. 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