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Colloidal complexed silver and silver nanoparticles in extrapallial fluid of Mytilus edulis
Marine Environmental Research 71 (2011) 17e21
Contents lists available at ScienceDirect
Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev
Colloidal complexed silver and silver nanoparticles in extrapallial fluid of Mytilus edulis Michael Zuykov*, Emilien Pelletier, Serge Demers Institut des sciences de la mer de Rimouski (ISMER), Université du Québec à Rimouski, Rimouski, 310 allée des Ursulines, QC G5L 3A1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 June 2010 Received in revised form 14 August 2010 Accepted 19 September 2010
Metal transport in mollusk extrapallial fluid (EPF) that acts as a “bridge” between soft tissues and shell has surprisingly received little attention until now. Using ultrafiltration and radiotracer techniques we determined silver concentrations and speciation in the EPF of the blue mussel Mytilus edulis after shortterm uptake and depuration laboratory experiments. Radiolabelled silver (110mAg) was used in dissolved or nanoparticulate phases (AgNPs < 40 nm), with a similar low Ag concentration (total radioactive and cold Ag w0.7 mg/L) in a way that mussels could uptake radiotracers only from seawater. Our results indicated that silver nanoparticles were transported to the EPF of blue mussels at a level similar to the Ag ionic form. Bulk activity of radiolabelled silver in the EPF represented only up to 7% of the bulk activity measured in the whole mussels. The EPF extracted from mussels exposed to both treatments exhibited an Ag colloidal complexed form based on EPF ultrafiltration through a 3 kDa filter. This original study brings new insights to internal circulation of nanoparticles in living organisms and contributes to the international effort in studying the potential impacts of engineered nanomaterials on marine bivalves which play an essential role in coastal ecosystems, and are important contributors to human food supply from the sea. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Mussels Mytilus edulis Extrapallial fluid Bioavailability Metals Silver Nanoparticles Toxicity
1. Introduction Heavy metals will continue to be introduced into the aquatic environment in the present century, but sources may change as metal engineered nanomaterials (ENMs) are developing at an accelerated rate and their introduction into various environmental compartments is imminent. Although initial knowledge of the ecotoxicological properties of some nano-sized chemicals can be found in papers published in 1960s (e.g. AuNPs in Bevelander and Nakahara, 1966), specific attention to this problem has only increased during the last decade (Moore, 2006). Only some sparse data on the potential toxicity of nano-silver (AgNPs) to bivalves (e.g. their effect on embryonic development of oysters) are already available (Ringwood et al., 2010). As a contribution to this field, bioaccumulation of AgNPs in the extrapallial fluid (EPF) of the blue mussel Mytilus edulis has been examined in the present paper. There is no doubt that EPF represents a large volume among the other internal liquids within an adult M. edulis as it could reach around 700e800 mL. The EPF fills the extrapallial cavity (EC) between the mantle and the shell valve. Closed from the
* Corresponding author. Tel.: þ1 418 723 1986x1570. E-mail address: [email protected] (M. Zuykov). 0141-1136/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2010.09.004
surrounding seawater environment, the EPF can be defined as an aqueous microenvironment that contains a complex mixture of proteins, carbohydrates, glycoproteins, amino acids, peptides, organic acids, lipids and inorganic components (Crenshaw, 1972; Misogianes and Chasteen, 1979; Yin et al., 2005). The direct relationship between growing nacre and the bathing EPF suggests a specific action of this fluid and organic compounds therein on the mineralization process. However, mineralizing ability is not exclusively controlled by the EPF as calcified structures (mineral concretions) were also found in some internal tissues bathed by the hemolymph of some bivalves (Moura et al., 2000). Bivalves can build prismatic and nacreous layers mainly by precipitation from two separate reservoirs, referred to as the marginal and central EPF, respectively (Fig. 1). In the present work only the central EPF reservoir was considered. Although EPF is an important bridge between soft tissues and inner shell, measurements of pollutants (trace metals or organic compounds) into the EPF have been rarely reported in the literature, and a special study on their transport to the EPF has never been undertaken. The purpose of this study was to compare translocation of radiolabelled silver (110mAg) from soft tissues to the central EPF of M. edulis after an uptake experiment where Ag was in added to seawater in a free-ionic phase (mostly as AgCl2 4 seawater) and as silver nanoparticles (AgNPs). In particular, we
18
M. Zuykov et al. / Marine Environmental Research 71 (2011) 17e21
Fig. 1. Mytilus edulis (A, B) Shell with sampling port for extraction of extrapallial fluid: (A) exterior view; (B) schematic drawing through the near shell edge; (C) Internal shell surface, prismatic layer and muscle attachment covered with paraffin; (D) SEM micrograph of nacreous layer. Scale bar: A, C-5 mm; D-20 mm.
examined whether: the speciation of accumulated Ag changed in the EPF in comparison with its initial form, the uptake and depuration kinetics of free-ionic Ag and AgNPs in the EPF differed after short-term experiments, and the amount of Ag incorporated onto the internal shell surface from the EPF differed for both treatments. In addition, an autoradiographic technique was used to illustrate the distribution of AgNPs within soft tissues of M. edulis.
geometry used for sample measurements. Counting time was adjusted from 2 to 10 min, and the propagated counting error was below 10%. Five mussels were used as replicates in each treatment. The filtered seawater (0.22 mm) was spiked with 110mAg or 110mAgNPs as appropriate, and allowed to equilibrate for 15 h prior to the
2. Materials and methods Experimental blue mussels of a large size (w55e60 mm) were grown under laboratory conditions at the Institut des Sciences de la Mer de Rimouski (ISMEReUQAR, Quebec, Canada). For repeated EPF extraction, two sampling ports were installed onto the valves of each mussel (see Fig. 1 A, B) following the method developed by the authors and described elsewhere (Zuykov et al., in press). Prior to the uptake experiment, mussels were acclimated for two days without being fed. No food was added to aquariums during experiments. All exposures to silver were conducted for each animal in an individual Teflon container to minimize wall adsorption of the metal and with 500 ml of 0.22-mm filtered seawater. The AgNPs used for these studies were produced using a protocol modified from Sardar et al. (2007) and adapted by our laboratory for the radioactive isotope of silver (Al-Sid-Cheikh M. and E. Pelletier, unpublished results). Briefly, 200 mg of 110mAgNO3 was added to 4 mL of Milli-Q water, with added poly(allyl)amine (PAAm). The solution was refluxed for 1 h 30 min and then centrifuged for 1 h to 25 000 g separate ionic and particulate silver. The solid silver was resuspended in pure water and centrifuged again. Finally, radiolabelled nanoparticles were dispersed in pure water and stored at 7 C in the dark until further use. The size of NP was monitored by UVevisible absorption spectroscopy. Based on transmission electron microscopy the average particle size was about 20e35 nm in diameter (Fig. 2). The radioactivity of 110mAg in all fluids and solid samples (including ultrafiltration membranes) was determined using a NaI (Tl) gamma spectrometer (Canberra) and spectral analysis software (MultiCalc). The gamma detector was calibrated with standard solutions of 110mAg in the same
Fig. 2. TEM observation of silver nanoparticles (<40 nm) obtained by the optimized method of Sardar et al. (2007). Scale bar: 250 nm.
M. Zuykov et al. / Marine Environmental Research 71 (2011) 17e21 Table 1 Concentration of
110m
19
Ag (in Bq/ml) and pH in water solutions in ten (10) experimental aquariums before and after uptake experiments.
Treatment
Ag-dissolved
Experimental aquarium
1
2
3
15.54
19.74
18.22
Starting solution 110m Ag in water (not fractioned) 110m Ag in fraction <3 kDa 110m Ag in fraction >3 kDa pH After uptake experiment (3 h 30 min) 110m Ag in water (not fractioned) 110m Ag in fraction<3 kDa 110m Ag in fraction >3 kDa pH
7.41
AgNPs (< 40 nm)
7.69
4
5
17.50 1.25 8.03
18.28 1.40 8.05
10.00 2.12 7.82
8.72 1.72 7.92
7.35
3. Results and discussion The use of low silver concentrations in both treatments with a stable pH around 8 avoided any mortality among experimental animals. Subsequent daily inspections of the mussels within one month following the end of the depuration experiment revealed normal physiological conditions. A mass balance calculation revealed that after the uptake experiment about 90% of starting radioactivity in each experimental container was associated with mussels, 2e7% of AgNPs and 10e18% of free-ionic silver were associated with the container walls and with the plastic aeration tubing. The studied mussels accumulated >60% of Ag in their soft tissues in both treatments (Table 2), a result quite similar to that of
110m
7
8
12.66
13.24
14.40
5.29
experiments. For comparative purpose, we prepared a similar radioactive silver concentration (total radioactive and cold silver: w0.7 mg/L) in all experimental containers for both treatments (Table 1). After an uptake exposure of 3 h 30 min, mussels were removed from radioactive solution, rinsed with non-labeled seawater and dabbed with filter paper. The bulk radioactivity of each mussel and extracted EPF samples was measured. After gamma counting all mussels were transferred to clean individual containers containing 500 ml of 0.22-mm filtered and non-labeled seawater for a 72 h-depuration phase with subsequent measurements of silver activity in EPF and on the interior shell surface, as well as for autoradiography of soft tissues.
Table 2 Concentration and bulk activity of
6
5.86
9
10
16.43 1.57 7.78
16.46 0.94 7.79
10.66 1.13 7.63
10.80 2.03 7.71
8.88
Guo et al. (2002) who determined silver in low and high molecular weight treatments in oysters (Crassostrea virginica). After a 72-h depuration period, four mussels (two from each treatment) were used for whole-body autoradiography according to the method described by Rouleau et al. (2003). Analysis of 50-mm-thick serial sections of soft tissues, which were exposed on screens sensitive to gamma emitting 110mAg, revealed a similar silver distribution between animals from both treatments, with maximum concentrations located in the digestive organs (Fig. 3). This observation is in agreement with data reported for other metals added to experimental aquariums both in dissolved and in particulate phases (George et al., 1986; Berthet et al., 1992; Erk et al., 2005; Gagné et al., 2008; Peyrot et al., 2009). Although, free-ionic silver appeared to accumulate in the whole mussel at a higher levels than in the EC, the EPF Ag concentration (and its depuration pattern) was similar in both treatments (ManneWhitney Test, P ¼ 0.841). Based on our previous examination of the EPF of same sized mussels, we estimated the Ag bulk activity in the EPF using a total volume of 700 mL (Table 2). Doing so, we observed a low bulk activity of radiolabelled Ag in the EPF ranging from 0.07% to 7% of total Ag activity detected in the mussel. More than 90% of radiolabelled free-ionic Ag and AgNPs found in the EPF after the uptake experiment and depuration period were measured in the colloidal fraction retained by an ultrafiltration membrane with a cutoff at 3 kDa. However, silver found in the seawater in experimental containers after uptake and depuration experiments
Ag in experimental solutions, mussels and extrapallial fluid (EPF) after uptake and depuration experiments.
Treatment
Ag-dissolved
Mollusk
1
2
3
4
5
6
7
8
9
10
Weight of alive mollusk (g) Weight of shell (g) After uptake experiment (3 h 30 min) Bulk activity 110mAg (Bq) in: Whole mollusk (wet weight) EPF (in 700 ml) Concentration 110mAg (Bq/ml) in: EPF (not fractioned) EPF, in fraction <3 kDa EPF, in fraction >3 kDa After depuration experiment (72 h) Bulk activity 110mAg (Bq) in: Whole mollusk (wet weight) Shell EPF (in 700 ml) Concentration 110mAg (Bq/ml) in: EPF (not fractioned) EPF, in fraction <3 kDa EPF, in fraction >3 kDa HC1, after leaching of internal shell surface
n/d n/d
24.46 9.72
32.10 12.11
23.19 n/d
22.93 n/d
n/d n/d
28.85 12.16
22.77 7.85
18.90 n/d
21.17 n/d
n/d n/d
4221 21
4204 26
2816 16
4225 3
n/d n/d
3373 45
2242 47
2273 12
2586 11
n/d-Not determined.
AgNPs (< 40 nm)
56.11 n/d Below detection limit n/d 29.89
n/d
n/d
n/d
27.46
n/d
n/d
n/d
n/d
37.14
22.68
4.64
n/d
65.54
67.54
17.75
15.63
n/d n/d n/d
1639 285 108
441 n/d 9
992 n/d 4
n/d n/d n/d
1058 283 3
684 204 28
665 n/d 11
629 n/d 2
n/d
n/d
n/d
7.25
n/d
n/d
n/d
n/d
154.44 0.43
12.22 n/d
6.27 n/d
n/d n/d
3.63 0.32
39.33 0.44
15.98 n/d
3.36 n/d
655 178 5
5.28 n/d Below detection limit n/d 6.98 n/d 0.63
20
M. Zuykov et al. / Marine Environmental Research 71 (2011) 17e21
Fig. 3. Whole-body autoradiography of Mytilus edulis after uptake experiment with free-ionic silver (A, B) and silver nanoparticles (C, D). The large greenish mass in B and D is the digestive gland of the mussel. Scale bar: 5 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
with both treatments did not tend to aggregate and passed through the 3 kDa membrane which suggests that complexation of silver forms by high molecular weight organic molecules (possibly proteins and carbohydrates) is occurring in the EPF. As clearly indicated from the literature (see references in Zuykov et al., 2009), metals found in the EPF can be incorporated into the bivalve interior shell surface in much lower quantities than those found in its exterior surface (in the periostracum). In the present study we attempted to find Ag on the interior shell surface (nacreous and prismatic layers) of all available shells with LA-ICPMS technique (Laser Ablation Induced Coupled Plasma Mass Spectrometry), but it was below detectable levels (< 1 ppm). In an attempt to confirm the presence of Ag incorporated into the surface of the nacreous layer, it was leached for 5 min using hydrochloric acid (10%) and subsequently the radioactivity of the leached Ag was measured in the resulting acidic solution. Radiolabelled silver was found in a very low concentration for shells from both treatments (Table 2). 4. Conclusion Our results show that although silver bioavailability in M. edulis may be related to its speciation (free-ionic or particulate form), the distribution in soft tissues and the transport of Ag to the EPF seems not to be related to one particular chemical form. Measurement of radioactivity confirmed a low amount of Ag in EPF even during a period of maximal body burden (first hours after uptake). We suggest hemocytes may play an important role in silver translocation to EC.
Acknowledgements The authors are grateful to G.A. Kolyuchkina for helpful discussion, D. Chasteen, L. Guo, W. Fisher for important support in writing the paper. We gratefully acknowledge the preparation of the radioactive nanoparticles by Isabelle Desbiens (ISMER). This work was supported by NSERC Discovery grants (E.P. and S. D).
References Berthet, B., Amiard, J.C., Amiard-Triquet, C., Martoja, M., Jeantet, A.Y., 1992. Bioaccumulation, toxicity and physico-chemical speciation of silver in bivalve molluscs: ecotoxicological and health consequences. The Science of the Total Environment 125, 97e122. Bevelander, G., Nakahara, H., 1966. Correlation of lysosomal activity and ingestion by the mantle epithelium. Biological Bulletin 131, 76e82. Crenshaw, M.A., 1972. The inorganic composition of molluscan extrapallial fluid. Biological Bulletin 143, 506e512. Erk, M., Ruus, A., Ingebrigtsen, K., Hylland, K., 2005. Cadmium accumulation and Cd-binding proteins in marine invertebrates e a radiotracer study. Chemosphere 61, 1651e1664. Gagné, F., Auclair, J., Turcotte, P., Fournier, M., Gagnon, C., Sauvé, S., Blaise, C., 2008. Ecotoxicity of CdTe quantum dots to freshwater mussels: impacts on immune system, oxidative stress and genotoxicity. Aquatic Toxicology 86, 333e340. George, S.G., Pirie, B.J.S., Calabrese, A., Nelson, D.A., 1986. Biochemical and ultrastructural observations of long-term silver accumulation in the mussel, Mytilus edulis. Marine Environmental Research 18 (4), 255e265. Guo, L., Santschi, P.H., Ray, S.M., 2002. Metal partitioning between colloidal and dissolved phases and its relation with bioavailability to American oysters. Marine Environmental Research 54, 49e64. Misogianes, M.J., Chasteen, N.D., 1979. A chemical and spectral characterization of the extrapallial fluid of Mytilus edulis. Analytical Biochemistry 100, 324e334. Moore, M.N., 2006. Do nanoparticles present ecotoxicological risks for the health of the aquatic environment? Environmental International 32, 967e976.
M. Zuykov et al. / Marine Environmental Research 71 (2011) 17e21 Moura, G., Vilarinho, L., Santos, A.C., Machado, J., 2000. Organic compounds in the extrapallial fluid and haemolymph of Anodonta cygnea (L.) with emphasis on the seasonal biomineralization process. Comparative Biochemistry and Physiology, Part B 125, 293e306. Peyrot, C., Gagnon, C., Gagné, F., Willkinson, K.J., Turcotte, P., Sauvé, S., 2009. Effects on cadmium telluride quantum dots on cadmium bioaccumulation and metallothionein production to the freshwater mussel, Elliptio complanata. Comparative Biochemistry and Physiology, Part C 150, 246e251. Ringwood, A.H., McCarthy, M., Bates, T.C., Carroll, D.L., 2010. The effects of silver nanoparticles on oysters embryos. Marine Environmental Research 69, S49eS51. Rouleau, C., Xiong, Z.-H., Pacepavicius, G., Huang, G.-L., 2003. Uptake of waterborne tributyltin in the brain of fish: axonal transport as a proposed mechanism. Environmental Science and Technology 37 (15), 3298e3302.
21
Sardar, R., Park, J.-W., Shumaker-Parry, J.S., 2007. Polymer-induced synthesis of stable gold and silver nanoparticles and subsequent ligand exchange in water. Langmuir 23 (23), 11883e11889. Yin, Y., Huang, J., Paine, M.L., Reinhold, V.N., Chasteen, N.D., 2005. Structural characterization of the major extrapallial fluid protein of the mollusk Mytilus edulis: implications for function. Biochemistry 44, 10720e10731. Zuykov, M.A., Pelletier, E., Demers, S., 2010. Method for repeated extrapallial fluid extraction from bivalve mollusks. Journal of Molluscan Study, doi:10.1093/ mollus/eyq029. Zuykov, M.A., Pelletier, E., Rouleau, C., Popov, L., Fowler, S.W., Orlova, M., 2009. Autoradiographic study of 241Am distribution in the shell of the freshwater zebra mussel Dreissena polymorpha exposed under laboratory conditions. Microchimica Acta 167, 173e178.
Contents lists available at ScienceDirect
Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev
Colloidal complexed silver and silver nanoparticles in extrapallial fluid of Mytilus edulis Michael Zuykov*, Emilien Pelletier, Serge Demers Institut des sciences de la mer de Rimouski (ISMER), Université du Québec à Rimouski, Rimouski, 310 allée des Ursulines, QC G5L 3A1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 June 2010 Received in revised form 14 August 2010 Accepted 19 September 2010
Metal transport in mollusk extrapallial fluid (EPF) that acts as a “bridge” between soft tissues and shell has surprisingly received little attention until now. Using ultrafiltration and radiotracer techniques we determined silver concentrations and speciation in the EPF of the blue mussel Mytilus edulis after shortterm uptake and depuration laboratory experiments. Radiolabelled silver (110mAg) was used in dissolved or nanoparticulate phases (AgNPs < 40 nm), with a similar low Ag concentration (total radioactive and cold Ag w0.7 mg/L) in a way that mussels could uptake radiotracers only from seawater. Our results indicated that silver nanoparticles were transported to the EPF of blue mussels at a level similar to the Ag ionic form. Bulk activity of radiolabelled silver in the EPF represented only up to 7% of the bulk activity measured in the whole mussels. The EPF extracted from mussels exposed to both treatments exhibited an Ag colloidal complexed form based on EPF ultrafiltration through a 3 kDa filter. This original study brings new insights to internal circulation of nanoparticles in living organisms and contributes to the international effort in studying the potential impacts of engineered nanomaterials on marine bivalves which play an essential role in coastal ecosystems, and are important contributors to human food supply from the sea. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Mussels Mytilus edulis Extrapallial fluid Bioavailability Metals Silver Nanoparticles Toxicity
1. Introduction Heavy metals will continue to be introduced into the aquatic environment in the present century, but sources may change as metal engineered nanomaterials (ENMs) are developing at an accelerated rate and their introduction into various environmental compartments is imminent. Although initial knowledge of the ecotoxicological properties of some nano-sized chemicals can be found in papers published in 1960s (e.g. AuNPs in Bevelander and Nakahara, 1966), specific attention to this problem has only increased during the last decade (Moore, 2006). Only some sparse data on the potential toxicity of nano-silver (AgNPs) to bivalves (e.g. their effect on embryonic development of oysters) are already available (Ringwood et al., 2010). As a contribution to this field, bioaccumulation of AgNPs in the extrapallial fluid (EPF) of the blue mussel Mytilus edulis has been examined in the present paper. There is no doubt that EPF represents a large volume among the other internal liquids within an adult M. edulis as it could reach around 700e800 mL. The EPF fills the extrapallial cavity (EC) between the mantle and the shell valve. Closed from the
* Corresponding author. Tel.: þ1 418 723 1986x1570. E-mail address: [email protected] (M. Zuykov). 0141-1136/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2010.09.004
surrounding seawater environment, the EPF can be defined as an aqueous microenvironment that contains a complex mixture of proteins, carbohydrates, glycoproteins, amino acids, peptides, organic acids, lipids and inorganic components (Crenshaw, 1972; Misogianes and Chasteen, 1979; Yin et al., 2005). The direct relationship between growing nacre and the bathing EPF suggests a specific action of this fluid and organic compounds therein on the mineralization process. However, mineralizing ability is not exclusively controlled by the EPF as calcified structures (mineral concretions) were also found in some internal tissues bathed by the hemolymph of some bivalves (Moura et al., 2000). Bivalves can build prismatic and nacreous layers mainly by precipitation from two separate reservoirs, referred to as the marginal and central EPF, respectively (Fig. 1). In the present work only the central EPF reservoir was considered. Although EPF is an important bridge between soft tissues and inner shell, measurements of pollutants (trace metals or organic compounds) into the EPF have been rarely reported in the literature, and a special study on their transport to the EPF has never been undertaken. The purpose of this study was to compare translocation of radiolabelled silver (110mAg) from soft tissues to the central EPF of M. edulis after an uptake experiment where Ag was in added to seawater in a free-ionic phase (mostly as AgCl2 4 seawater) and as silver nanoparticles (AgNPs). In particular, we
18
M. Zuykov et al. / Marine Environmental Research 71 (2011) 17e21
Fig. 1. Mytilus edulis (A, B) Shell with sampling port for extraction of extrapallial fluid: (A) exterior view; (B) schematic drawing through the near shell edge; (C) Internal shell surface, prismatic layer and muscle attachment covered with paraffin; (D) SEM micrograph of nacreous layer. Scale bar: A, C-5 mm; D-20 mm.
examined whether: the speciation of accumulated Ag changed in the EPF in comparison with its initial form, the uptake and depuration kinetics of free-ionic Ag and AgNPs in the EPF differed after short-term experiments, and the amount of Ag incorporated onto the internal shell surface from the EPF differed for both treatments. In addition, an autoradiographic technique was used to illustrate the distribution of AgNPs within soft tissues of M. edulis.
geometry used for sample measurements. Counting time was adjusted from 2 to 10 min, and the propagated counting error was below 10%. Five mussels were used as replicates in each treatment. The filtered seawater (0.22 mm) was spiked with 110mAg or 110mAgNPs as appropriate, and allowed to equilibrate for 15 h prior to the
2. Materials and methods Experimental blue mussels of a large size (w55e60 mm) were grown under laboratory conditions at the Institut des Sciences de la Mer de Rimouski (ISMEReUQAR, Quebec, Canada). For repeated EPF extraction, two sampling ports were installed onto the valves of each mussel (see Fig. 1 A, B) following the method developed by the authors and described elsewhere (Zuykov et al., in press). Prior to the uptake experiment, mussels were acclimated for two days without being fed. No food was added to aquariums during experiments. All exposures to silver were conducted for each animal in an individual Teflon container to minimize wall adsorption of the metal and with 500 ml of 0.22-mm filtered seawater. The AgNPs used for these studies were produced using a protocol modified from Sardar et al. (2007) and adapted by our laboratory for the radioactive isotope of silver (Al-Sid-Cheikh M. and E. Pelletier, unpublished results). Briefly, 200 mg of 110mAgNO3 was added to 4 mL of Milli-Q water, with added poly(allyl)amine (PAAm). The solution was refluxed for 1 h 30 min and then centrifuged for 1 h to 25 000 g separate ionic and particulate silver. The solid silver was resuspended in pure water and centrifuged again. Finally, radiolabelled nanoparticles were dispersed in pure water and stored at 7 C in the dark until further use. The size of NP was monitored by UVevisible absorption spectroscopy. Based on transmission electron microscopy the average particle size was about 20e35 nm in diameter (Fig. 2). The radioactivity of 110mAg in all fluids and solid samples (including ultrafiltration membranes) was determined using a NaI (Tl) gamma spectrometer (Canberra) and spectral analysis software (MultiCalc). The gamma detector was calibrated with standard solutions of 110mAg in the same
Fig. 2. TEM observation of silver nanoparticles (<40 nm) obtained by the optimized method of Sardar et al. (2007). Scale bar: 250 nm.
M. Zuykov et al. / Marine Environmental Research 71 (2011) 17e21 Table 1 Concentration of
110m
19
Ag (in Bq/ml) and pH in water solutions in ten (10) experimental aquariums before and after uptake experiments.
Treatment
Ag-dissolved
Experimental aquarium
1
2
3
15.54
19.74
18.22
Starting solution 110m Ag in water (not fractioned) 110m Ag in fraction <3 kDa 110m Ag in fraction >3 kDa pH After uptake experiment (3 h 30 min) 110m Ag in water (not fractioned) 110m Ag in fraction<3 kDa 110m Ag in fraction >3 kDa pH
7.41
AgNPs (< 40 nm)
7.69
4
5
17.50 1.25 8.03
18.28 1.40 8.05
10.00 2.12 7.82
8.72 1.72 7.92
7.35
3. Results and discussion The use of low silver concentrations in both treatments with a stable pH around 8 avoided any mortality among experimental animals. Subsequent daily inspections of the mussels within one month following the end of the depuration experiment revealed normal physiological conditions. A mass balance calculation revealed that after the uptake experiment about 90% of starting radioactivity in each experimental container was associated with mussels, 2e7% of AgNPs and 10e18% of free-ionic silver were associated with the container walls and with the plastic aeration tubing. The studied mussels accumulated >60% of Ag in their soft tissues in both treatments (Table 2), a result quite similar to that of
110m
7
8
12.66
13.24
14.40
5.29
experiments. For comparative purpose, we prepared a similar radioactive silver concentration (total radioactive and cold silver: w0.7 mg/L) in all experimental containers for both treatments (Table 1). After an uptake exposure of 3 h 30 min, mussels were removed from radioactive solution, rinsed with non-labeled seawater and dabbed with filter paper. The bulk radioactivity of each mussel and extracted EPF samples was measured. After gamma counting all mussels were transferred to clean individual containers containing 500 ml of 0.22-mm filtered and non-labeled seawater for a 72 h-depuration phase with subsequent measurements of silver activity in EPF and on the interior shell surface, as well as for autoradiography of soft tissues.
Table 2 Concentration and bulk activity of
6
5.86
9
10
16.43 1.57 7.78
16.46 0.94 7.79
10.66 1.13 7.63
10.80 2.03 7.71
8.88
Guo et al. (2002) who determined silver in low and high molecular weight treatments in oysters (Crassostrea virginica). After a 72-h depuration period, four mussels (two from each treatment) were used for whole-body autoradiography according to the method described by Rouleau et al. (2003). Analysis of 50-mm-thick serial sections of soft tissues, which were exposed on screens sensitive to gamma emitting 110mAg, revealed a similar silver distribution between animals from both treatments, with maximum concentrations located in the digestive organs (Fig. 3). This observation is in agreement with data reported for other metals added to experimental aquariums both in dissolved and in particulate phases (George et al., 1986; Berthet et al., 1992; Erk et al., 2005; Gagné et al., 2008; Peyrot et al., 2009). Although, free-ionic silver appeared to accumulate in the whole mussel at a higher levels than in the EC, the EPF Ag concentration (and its depuration pattern) was similar in both treatments (ManneWhitney Test, P ¼ 0.841). Based on our previous examination of the EPF of same sized mussels, we estimated the Ag bulk activity in the EPF using a total volume of 700 mL (Table 2). Doing so, we observed a low bulk activity of radiolabelled Ag in the EPF ranging from 0.07% to 7% of total Ag activity detected in the mussel. More than 90% of radiolabelled free-ionic Ag and AgNPs found in the EPF after the uptake experiment and depuration period were measured in the colloidal fraction retained by an ultrafiltration membrane with a cutoff at 3 kDa. However, silver found in the seawater in experimental containers after uptake and depuration experiments
Ag in experimental solutions, mussels and extrapallial fluid (EPF) after uptake and depuration experiments.
Treatment
Ag-dissolved
Mollusk
1
2
3
4
5
6
7
8
9
10
Weight of alive mollusk (g) Weight of shell (g) After uptake experiment (3 h 30 min) Bulk activity 110mAg (Bq) in: Whole mollusk (wet weight) EPF (in 700 ml) Concentration 110mAg (Bq/ml) in: EPF (not fractioned) EPF, in fraction <3 kDa EPF, in fraction >3 kDa After depuration experiment (72 h) Bulk activity 110mAg (Bq) in: Whole mollusk (wet weight) Shell EPF (in 700 ml) Concentration 110mAg (Bq/ml) in: EPF (not fractioned) EPF, in fraction <3 kDa EPF, in fraction >3 kDa HC1, after leaching of internal shell surface
n/d n/d
24.46 9.72
32.10 12.11
23.19 n/d
22.93 n/d
n/d n/d
28.85 12.16
22.77 7.85
18.90 n/d
21.17 n/d
n/d n/d
4221 21
4204 26
2816 16
4225 3
n/d n/d
3373 45
2242 47
2273 12
2586 11
n/d-Not determined.
AgNPs (< 40 nm)
56.11 n/d Below detection limit n/d 29.89
n/d
n/d
n/d
27.46
n/d
n/d
n/d
n/d
37.14
22.68
4.64
n/d
65.54
67.54
17.75
15.63
n/d n/d n/d
1639 285 108
441 n/d 9
992 n/d 4
n/d n/d n/d
1058 283 3
684 204 28
665 n/d 11
629 n/d 2
n/d
n/d
n/d
7.25
n/d
n/d
n/d
n/d
154.44 0.43
12.22 n/d
6.27 n/d
n/d n/d
3.63 0.32
39.33 0.44
15.98 n/d
3.36 n/d
655 178 5
5.28 n/d Below detection limit n/d 6.98 n/d 0.63
20
M. Zuykov et al. / Marine Environmental Research 71 (2011) 17e21
Fig. 3. Whole-body autoradiography of Mytilus edulis after uptake experiment with free-ionic silver (A, B) and silver nanoparticles (C, D). The large greenish mass in B and D is the digestive gland of the mussel. Scale bar: 5 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
with both treatments did not tend to aggregate and passed through the 3 kDa membrane which suggests that complexation of silver forms by high molecular weight organic molecules (possibly proteins and carbohydrates) is occurring in the EPF. As clearly indicated from the literature (see references in Zuykov et al., 2009), metals found in the EPF can be incorporated into the bivalve interior shell surface in much lower quantities than those found in its exterior surface (in the periostracum). In the present study we attempted to find Ag on the interior shell surface (nacreous and prismatic layers) of all available shells with LA-ICPMS technique (Laser Ablation Induced Coupled Plasma Mass Spectrometry), but it was below detectable levels (< 1 ppm). In an attempt to confirm the presence of Ag incorporated into the surface of the nacreous layer, it was leached for 5 min using hydrochloric acid (10%) and subsequently the radioactivity of the leached Ag was measured in the resulting acidic solution. Radiolabelled silver was found in a very low concentration for shells from both treatments (Table 2). 4. Conclusion Our results show that although silver bioavailability in M. edulis may be related to its speciation (free-ionic or particulate form), the distribution in soft tissues and the transport of Ag to the EPF seems not to be related to one particular chemical form. Measurement of radioactivity confirmed a low amount of Ag in EPF even during a period of maximal body burden (first hours after uptake). We suggest hemocytes may play an important role in silver translocation to EC.
Acknowledgements The authors are grateful to G.A. Kolyuchkina for helpful discussion, D. Chasteen, L. Guo, W. Fisher for important support in writing the paper. We gratefully acknowledge the preparation of the radioactive nanoparticles by Isabelle Desbiens (ISMER). This work was supported by NSERC Discovery grants (E.P. and S. D).
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