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Uptake and depuration of 131I from labelled diatoms (Skeletonema costatum) to the edible periwinkle (Littorina littorea)
Journal of Environmental Radioactivity 96 (2007) 75e84 www.elsevier.com/locate/jenvrad
Uptake and depuration of 131I from labelled diatoms (Skeletonema costatum) to the edible periwinkle (Littorina littorea) R.C. Wilson a,*, J. Vives i Batlle a, S.J. Watts a, P. McDonald a, T.G. Parker b a
Westlakes Scientific Consulting Ltd., The Princess Royal Building, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3LN, UK b British Nuclear Group, Sellafield, Cumbria CA20 1PG, UK Accepted 15 January 2007 Available online 18 April 2007
Abstract Uptake and depuration of 131I into winkles through consumption of the diatom Skeletonema costatum is described. The work follows on from previous studies that investigated the uptake of iodine into winkles from seawater and seaweed. Incorporation of 131I in S. costatum from labelled seawater followed linear first-order kinetics with an uptake half-time of 0.40 days. Iodine uptake in winkles from labelled S. costatum also followed linear first-order kinetics, with a calculated equilibrium concentration (CN) of 42 Bq kg1 and a transfer factor (TF) of 1.1 104 with respect to labelled diatom food. This TF is lower than that observed for uptake of 131 I in winkles from labelled seaweed. For the depuration stage, a biphasic sequence with biological half-lives of 1.3 and 255 days was determined. The first phase is biokinetically important, given that winkles can lose two-thirds of their activity during that period. This study shows that, whilst winkles can obtain radioactive iodine from phytoplankton consumption, they do not retain the majority of that activity for very long. Hence, compared with other exposure
* Corresponding author. Fax: þ44 (0) 1946 514091. E-mail address: [email protected] (R.C. Wilson). 0265-931X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvrad.2007.01.018
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pathways, such as uptake from seawater and macroalgae, incorporation from phytoplankton is a relatively minor exposure route. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Winkle; Phytoplankton; Skeletonema costatum; Biokinetic; Uptake; Depuration radioiodine; Iodine
1. Introduction There is a growing interest in biokinetic modelling, which has many benefits over the simple concentration factor approach currently in widespread use in dose assessments. In a recent questionnaire commissioned by the International Union of Radioecology (IUR) on environmental protection, it was suggested that ‘biokinetic modelling was the way forward’. It also showed that there is a requirement for a better understanding of the transfer of radionuclides through the environment, with a benefit inherent to this type of modelling (IUR, 2006). Over the past few years a number of experiments have been conducted to investigate biokinetic parameters for iodine uptake in the edible periwinkle, Littorina littorea (henceforth referred to as the winkle). These have included uptake from seawater (Vives i Batlle et al., 2005) and through the consumption of the seaweed Chondrus crispus (Wilson et al., 2005). Winkles are a species of interest because, prior to these recent studies, little was known about iodine accumulation and uptake characteristics in these organisms. A number of earlier studies reported the concentration factor (CF) for radioiodine, not for winkles, but for other mollusc species, not specifically from the Irish Sea. These species included mussels (Shunhua et al., 1997; Sombrito et al., 1982; Whitehead et al., 1988) and clams (Cuvin-Aralar and Umaly, 1988, 1991; Mayr et al., 1988). Information on radioiodine depuration kinetics is even more scant: for mussels it is suggested that turnover is fast, with biological half-lives of 2e3 days and elimination following a simple exponential fall (Cuvin-Aralar and Umaly, 1988, 1991; Shunhua et al., 1997; Sombrito et al., 1982). The above knowledge gap notwithstanding, the dose to the critical group consumers of seafood in the vicinity of Sellafield, UK, contains a winkle component, of which 15% can be attributed to 129I, released under authorisation from the Sellafield site (BNGSL, 2006). Therefore a better understanding of the uptake characteristics should result in a more realistic assessment of the radiological impact of present and future discharges of radioiodine. To complete our investigations of iodine uptake pathways in winkles, this study determines the biokinetic parameters associated with the consumption of phytoplankton, in particular diatoms, given that winkles are known to eat microalgae (such as benthic diatoms and dinoflagellates) as well as macroalgae (Edwards and Davies, 2002). The marine diatom Skeletonema costatum was chosen for this study as it is a common species found in many marine systems, including the Irish Sea (McKinney et al., 1997). It is hypothesised that, in the natural environment, S. costatum can incorporate 129I with a relatively short biological half-time (of the order of 1 day or less, typical of passive adsorption onto the cell walls), and that, subsequently, L. littorea can assimilate this radioiodine through feeding on diatoms, whilst depurating in a manner consistent with that observed from the consumption of the seaweed C. crispus (Wilson et al., 2005). A laboratory experiment using 131I as a tracer was designed to test this hypothesis. 131I was chosen due to the ease of measurement compared to that of 129I, which is released under licence from the Sellafield site, and the fact that it is not environmentally available to any significant
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extent. As far as the authors are aware, there is no evidence to support the ability of winkles to isotopically fractionate radioiodine and therefore we believe that 131I is a suitable surrogate for 129I. 2. Materials and methods The investigation was designed to consist of two phases. The first phase involved labelling cultures of S. costatum with 131I. This was expected to yield information regarding the uptake of 131I into phytoplankton. The second phase was the feeding of the diatoms to the winkles, allowing uptake and depuration of 131I associated with the consumption of diatoms, to be studied within the winkles. 2.1. Winkles As in the two previous studies, winkles were collected from Nethertown on the Cumbrian coast. Following removal of any encrusting organisms from their shells, the winkles were allowed to acclimatise in 9 l glass aquarium tanks containing 5 l of local seawater. Three sets of 10 winkles were each kept in small labelled plastic cages to aid identification throughout the experiments. Cages were chosen that enabled thorough mixing of water within the cages with that of the bulk solution. With the tank containing only 5 l of seawater, the option of being above the water level was available to the winkles (during the uptake phase only). The tanks were maintained at 10e15 C which is consistent with temperatures in the Irish Sea (Kershaw et al., 1992) and bubbled with air to ensure that conditions within the tank did not become anoxic. 2.2. Diatoms The marine diatom S. costatum was chosen for this study as it is a common species found in many marine systems, including the Irish Sea (McKinney et al., 1997). It is also a species suitable for culturing within a laboratory environment. Cultures of the cells were supplied by Culture Collection of Algae and Protozoa (CCAP), Oban, in f/2 þ sodium metasilicate media (CCAP 1077/5). 2.2.1. Media All cultures were grown in f/2 þ sodium metasilicate media. The seawater used for the media was collected at Nethertown and filtered using a 0.2-mm cellulose nitrate filter paper. All stocks were stored at 5 C in the dark prior to use. 2.2.2. Growth conditions Cells were inoculated into 5 l glass conical flasks, which had been autoclaved at 120 C prior to use. Each flask contained 3 l of media. A cotton wool bung was placed in the opening of the flask to permit gaseous exchange. Inoculated flasks were then placed within an orbital-shaking incubator, where the flasks were shaken at 100 rpm and maintained at 15 C. Again, this temperature is representative of that in the Irish Sea (Kershaw et al., 1992). Finally, cultures were subjected to a 16 h light/8 h dark light regime. 2.2.3. Cell counting The number of cells present within each culture was measured using a haemocytometer and an optical microscope. 2.3. Diatom uptake of
131
I
Three 5 l conical flasks containing 3 l of media were inoculated with cells of S. costatum. Sufficient I tracer (Amersham Biosciences, supplied in 0.02 M NaOH) was then added to the culture to obtain an activity concentration of 2 Bq ml1. The initial contact time between the 131I in seawater and the diatoms before feeding the winkles was in excess of 60 h. Thereafter, diatoms were extracted at intervals of 24 h from the same solution during the 3-day period covering the duration of the experiment. 131
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Samples were taken daily to measure the activity in the algae as well as the activity in the culture medium. The latter was necessary to ensure the algal cells were subjected to a constant activity due to the short half-life of the 131I being used (8.04 days). With such a short half-life, loss of the tracer through decay will have been far greater than loss through adsorption and evaporation/volatilisation. The activity in diatom cultures was maintained at the specified level after counting the diatoms and the culture medium each day. The two components were separated using a syringe-driven 25 mm diameter 0.45 mm cellulose nitrate filter. Ten millilitres of suspension was passed through the filter and both the filter paper and the filtrate were measured for the emission of the characteristic g-rays from 131I at 364.5 keV. Daily diatom cell counts were also made and recorded. This operational sequence was repeated each day for a period of 10 days. 2.4. Winkle uptake of
131
I
Prior to feeding, each batch of 10 winkles was weighed and placed in a calibrated counting geometry to be analysed for 131I, using g-ray spectroscopy. Next, each batch was fed S. costatum separately. Six hundred millilitres of labelled culture suspension was passed through a 110-mm GF/C filter. The S. costatum were retained on the filter paper and the winkles were then placed upon it and allowed to graze on the cells for a period of 3 h (Vives i Batlle et al., 2006). This procedure was performed in separate containers and monitored to ensure that the food source was available to the winkles at all times. Immediately after feeding, the winkles were analysed for 131I. The fraction of diatoms not eaten was not measured. During the analysis for 131I, the tank seawater was replenished with fresh seawater. When analysis was complete, the winkles were returned to the plastic cages in the tank. This feeding process was repeated four times over a 3-day period. 2.5. Winkle depuration of
131
I
Following the uptake phase of the experiment, the release of iodine from the winkles was observed. Due to the low levels of iodine present within the winkles, it was decided to bulk the three batches into one at this stage. This had two benefits: (a) it reduced the daily count time required to obtain a statistically acceptable count area at 364.5 keV, and (b) it also allowed the release to be observed over a longer period of time before the levels of iodine became undetectable. After the final feed, the winkles were placed into the tank containing fresh seawater. At various intervals over an 11-day period the winkles were removed, weighed and analysed for 131I. They were then placed back into the tank in which the seawater had been replenished. Gamma emissions from 131I were counted using a high purity germanium crystal and the data were processed by Ortec Maestro software. Spectra were analysed with FitzPeaks32 Gamma Analysis and Calibration Software Version 3.48.
3. Results and discussion 3.1.
131
I uptake experiment
3.1.1. Diatoms (S. costatum) Concentrations of 131I increased from less than 2.5 mBq g1 to 280 Bq g1 during the 10 days of the uptake experiment (Fig. 1). Previous studies of iodine uptake into organisms have shown that the uptake trend follows first-order kinetics as shown in Eq. (1) (Vives i Batlle et al., 2005; Wilson et al., 2005): CðtÞ ¼ Ceq 1 eat
ð1Þ
Mean Activity in S. costatum (Bq g-1)
R.C. Wilson et al. / J. Environ. Radioactivity 96 (2007) 75e84
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450 400 350 300 250 200 150 100 50 0 0
50
100
150
200
250
Time (hours) Fig. 1. Uptake curve of
131
I in S. costatum (error bars indicate 2s counting uncertainty, n ¼ 3).
where C(t) is the concentration of 131I in S. costatum (Bq g1), Ceq is the equilibrium concentration, t is time and a is the time constant (ln2/Tu1/2, where Tu1/2 is the uptake half-time) which relates to loss during uptake (Whicker and Schultz, 1982). The uptake half-time was calculated to be 0.40 days, which is consistent with the expected short uptake period. It is known that certain metals bind very quickly to the surface of single cell organisms, within the order of 30 min, followed by a much slower intracellular uptake (Xue et al., 1988). From the data generated in this study, it is believed that rapid binding occurs and that the intracellular uptake is insignificant compared to the surface binding; hence the data fit a single-phase uptake model. A concentration factor (CF) of 141 l kg1 was also derived. This is eight times lower than the average 1100 l kg1 derived from the literature (Bowen, 1979; Coughtrey et al., 1984; IAEA, 2004; Kuenzler, 1967). However, the latter is a generic value for phytoplankton whereas the present experimentally-derived CF is species-specific. It is not obvious that the large difference between the present data and literature data can be attributed to differences in phytoplankton species only, although this is entirely possible. 3.1.2. Winkles Following optimised grazing times of 3 h on radiolabelled S. costatum (Vives i Batlle et al., 2006), there was a measurable increase of 131I within the winkles, confirming that uptake had occurred. The daily feeding cycle produced a serrated pattern of uptake/release cycles within each 24-h period, as seen in Fig. 2. Clearly, the separation between the upper and lower data points of this serrated pattern is statistically significant at the 2s level. 3.1.3. Equilibrium concentration To calculate the transfer factor of 131I from diatoms to winkles, a reference equilibrium concentration (Ceq) must be calculated. A detailed description of the calculation can be found in Wilson et al. (2005). The main assumption to be made is that the uptake half-time (Tu1/2 ¼ ln2/a) equates to the early-phase biological half-time (Tb1/2). This necessary ‘‘best guess’’ is not unreasonable as an approximation. The model for winkles developed by Vives i Batlle et al.
R.C. Wilson et al. / J. Environ. Radioactivity 96 (2007) 75e84
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A
120
Activity (Bq kg-1)
100 80 60 40 20 0 0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
50.0
60.0
70.0
80.0
50.0
60.0
70.0
80.0
Time (hours)
B
120
Activity (Bq kg-1)
100 80 60 40 20 0 0.0
10.0
20.0
30.0
40.0
Time (hours)
C
350
Activity (Bq kg-1)
300 250 200 150 100 50 0 0.0
10.0
20.0
30.0
40.0
Time (hours) Fig. 2. Uptake of 131I in winkles from grazing on labelled S. costatum, batches A, B and C, each containing 10 winkles (error bars indicate 2s counting uncertainty).
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(2006) shows that, following optimisation of the model parameter set, uptake half-times and biological half-lives for different components of the winkle (shell, soft tissue, storage organs), though not identical, are still reasonably similar. Individual Ceq values were calculated using Eq. (2): CðtÞ zCn þ Ceq aðt tn Þ
ð2Þ
where Cn and tn are the concentration of 131I and time of the n-th daily cycle. This approach produces an average equilibrium concentration from the three batches of winkles of 42 Bq kg1 fresh weight. 3.1.4. Transfer factor The transfer factor was calculated using Eq. (3). This gives an indication as to the proportion of radioactivity transferred from the diatoms to the winkles. TF ¼
Ceq Csc
ð3Þ
In Eq. (3), Csc is the activity in S. costatum in Bq kg1 fresh weight. When winkles were fed with the diatoms, it was not possible to obtain an accurate fresh weight mass for the diatoms. However, plankton cell counts can be determined using a haemocytometer. These results, used in conjunction with the mass of an individual cell (Brown, 1991), were used to estimate the fresh mass of algae being consumed. The average transfer factor from the three batches was calculated as 1.1 104 (this is a dimensionless quantity). This is considerably lower than the TF derived by Wilson et al. (2005) of 0.07 for the uptake from seaweed. Such a low TF value indicates that the uptake of iodine into winkles from the consumption of S. costatum is not the most significant bioaccumulation pathway. 3.2.
131
I depuration from winkles
During depuration, the activity concentration within the winkles fell by 84% from 93 Bq kg1 to 15 Bq kg1 over an 11-day period (Fig. 3). Such a fall is clearly significant within the 2s uncertainties reported. Previous studies (Vives i Batlle et al., 2005; Wilson et al., 2005) have shown that depuration follows first-order kinetics which can be represented by Eq. (4). CðtÞ ¼ C0 eat
ð4Þ
Previous work also showed that depuration can occur in a number of distinct phases. To investigate what number of release phases was most appropriate for this experiment, ln(C/C0) was plotted against time (Fig. 4) where C is the activity at time t and C0 is the starting activity. A biphasic release model (Fig. 4) generates biological half-lives of 1.3 and 255 days, with coefficients of determination (r2) being 1 (n ¼ 2) and 0.71, respectively. The shallow exponential fit relating to the 255-day biological half-life is compounded by the low statistical power of the data set, as exemplified by the relatively large error bars in Fig. 3. However, the trend is clearly discernible from the graph. The variance here may be considered the sum of two terms, one due to the analytical method and one due to possible fluctuations in the uptake by the winkle. The error bars in the data refer to the analytical method (they are, predominantly,
R.C. Wilson et al. / J. Environ. Radioactivity 96 (2007) 75e84
82 120
Activity (Bg kg-1)
100
80
60
40
20
0 0
50
150
100
200
250
300
Time (hours) Fig. 3. Release of
131
I from winkles (single batch of 30) (error bars indicate 2s counting uncertainty).
counting errors). The variance due to the winkle would, on the basis of our analysis, be relatively small, as reflected by the p-value of less than 0.0005, implying a very low probability that the exponential fit observed occurred by chance. The first biological half-life of 1.3 days is comparable with that reported by Wilson et al. (2005) of 0.9 days from the consumption of C. crispus. However, with phytoplankton, this initial phase spans a much shorter timescale than with C. crispus. This is illustrated by the percentage loss 2 h after feeding. Winkles fed on seaweed lost just 5% of the activity within 2 h compared to winkles fed on diatoms, which lost 72%. This marked difference could be due to one of two factors, or even a combination of these. First, that the diatoms are less 0
Bi-phasic - Phase 1
Ln(C/C0)
-0.5
R2 = 1, Tb1/2 = 1.3 days (n=2)
-1
Monophasic -----R2 = 0.44 (n =13, p
0.01)
-1.5
-2 B-phasic - Phase 2 R2 = 0.71,Tb1/2 = 255 days (n=12, p
0.0005)
-2.5 0
50
100
150
200
Time (hours) Fig. 4. Biphasic release of
131
I from winkles.
250
300
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digestible than seaweed and therefore have a short transit time through the digestive tract, reducing the time available for uptake. Second, that iodine is predominantly incorporated on the diatom cell surface, from where it dissolves relatively quickly into the digestive juices. The smaller fraction eliminated with a much longer biological half-life (255 days for S. costatum compared to 24 days for C. crispus) could represent an intracellular component which is actively absorbed by the gut and eliminated much more slowly. With the evidence at hand, it is not possible to reach any firm conclusions on this matter. To provide a clearer answer for this marked difference in loss characteristics would require a more physiologically driven study, investigating the excretion process for the winkle and, particularly, the physico-chemical form of the excreted iodine. Such an undertaking lies beyond the scope of the present paper but does signal a direction for future studies. 4. Conclusions Uptake and depuration experiments were undertaken successfully in the laboratory on winkles from the Cumbrian coast near Sellafield, that were previously fed with labelled S. costatum. The 131I concentrations in S. costatum during labelling followed linear first-order kinetics with a mono-phasic pattern generating an uptake half-time of 0.40 days. The CF was calculated as being 141 l kg1. This is lower than CF values in the literature for iodine in phytoplankton. It should be noted that these literature values are for the generic phytoplankton phylum whereas the value reported in this study is species-specific for S. costatum. The uptake of 131I from labelled S. costatum in winkles also followed linear first-order kinetics producing a calculated equilibrium concentration (CN) of 42 Bq kg1, resulting in a transfer factor of 1.1 104 with respect to the labelled diatoms used as food. For depuration, a biphasic sequence with biological half-lives of 1.3 and 255 days was determined. The first phase is consistent with that seen following the consumption of C. crispus. However the second phase is considerably longer. This indicates that following the consumption of labelled S. costatum, the majority of iodine is processed relatively quickly, but any iodine incorporated into the system is processed (and eventually released) at a much slower rate. This study demonstrates that whilst winkles have the ability to obtain radioactive iodine from the consumption of phytoplankton, they do not retain that activity for very long. It is therefore concluded that compared with other exposure pathways such as uptake from seawater and macroalgae, incorporation of radioiodine from phytoplankton is a relatively minor exposure route. From this study alone, it has not been possible to deduce if 131I is excreted as part of the diatom or whether 131I is released from the diatom surfaces in the digestive system of the winkles and excreted separately. The answer depends critically on two factors: first, did the labelling of the diatoms result in surface sorption, or in a predominantly intracellular fraction of 131I in the microalgae? Second, are diatoms excreted whole or does the digestive process manage to break down cells and release their contents? This is an area of research worth considering, signalling the direction for future investigations. References BNGSL, 2006. Monitoring our environment. Discharges and monitoring in the UK. Annual Report 2005. British Nuclear Group Sellafield Ltd. Bowen, H.J.M., 1979. Environmental Chemistry of the Elements. Academic Press.
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Brown, M.R., 1991. The amino-acid and sugar composition of 16 species of microalgae used in mariculture. Journal of Experimental Marine Biology and Ecology 145 (1), 79e99. Coughtrey, P.J., Jackson, D.J., Jones, C.H., Thorne, M.C., 1984. Radionuclide Distribution and Transport in Terrestrial and Aquatic Ecosystems e A Critical Review of Data, vols. 1e6. A.A.Balkema, Rotterdam. Cuvin-Aralar, M.L.A., Umaly, R.C., 1988. Uptake and elimination of iodine-131 by the freshwater clam Corbicula manilensis Philippi from water. Natural and Applied Science Bulletin 40, 141e158. Cuvin-Aralar, M.L.A., Umaly, R.C., 1991. Accumulation and tissue distribution of radioiodine (131I) from algal phytoplankton by the fresh-water clam Corbicula manilensis. Bulletin of Environmental Contamination and Toxicology 47 (6), 896e903. Edwards, M., Davies, M.S., 2002. Functional and ecological aspects of the mucus trails of the intertidal prosobranch gastropod Littorina littorea. Marine Ecology Progress Series 239, 129e137. IAEA, 2004. Sediment distribution coefficients and concentration factors for biota in the marine environment, Technical Reports Series No. 422, Vienna, International Atomic Energy Agency. IUR, 2006. IUR web based questionnaire results for environmental protection. Research, Facilities and Scientific Priorities. International Union of Radioecology, IUR Report 5. Kershaw, P.J., Pentreath, R.J., Woodhead, D.S., Hunt, G.J., 1992. A review of radioactivity in the Irish Sea. A Report prepared for the Marine Pollution Monitoring Group, Lowestoft, UK, Ministry of Agriculture, Fisheries and Food Aquatic Environment Monitoring Report 32. MAFF, Directorate of Fisheries Research. Kuenzler, E.J., 1967. Elimination of iodine, cobalt, iron, and zinc by marine zooplankton. In: Nelson, D.J., Evans, F.C. (Eds.), Proc. 2nd National Symposium on Radioecology. Ann Arbor, Michigan, May 15e17, 1967. USAEC Conf. No. 670503, pp. 462e473. Mayr, L., Moraes, R., Lopes, M.A., Vicente, C., Mauro, J.N., 1988. Transit and absorption of nuclear industry derivatives by marine biota (Transito e absorc¸ao de radionuclı´deos na biota marinha derivados de industria nuclear). In: Proc. 2. General Congress of Nuclear Energy. Rio de Janerio (Brazil), pp. 281e293. McKinney, E.S.A., Gibson, C.E., Stewart, B.M., 1997. Planktonic diatoms in the north-west Irish Sea: a study by automatic sampler. Biology and Environment: Proceedings of the Royal Irish Academy 97B (3), 197e202. Shunhua, C., Qiong, S., Xiaokui, K., 1997. Effects of body size on accumulation and distribution of 125I in the green mussel (Perna viridis). Beijing, China, China Nuclear Information Centre. Report CNIC-01210. Sombrito, E.Z., Banzon, R.B., de la Mines, A.S., Bautista, E., 1982. Uptake of iodine-131 in mussel (Mytilus smaragdinus) and algae (Caulerpa racemosa). The Nucleus (Journal of the Radioisotope Society of Philippines) 22 (1), 83e89. Vives i Batlle, J., Wilson, R.C., McDonald, P., Parker, T.G., 2006. A biokinetic model for the uptake and release of radioiodine by the edible periwinkle Littorina littorea. In: Povinec, P.P., Sa´nchez-Cabeza, J.A. (Eds.), Radionuclides in the Environment, vol. 8. Elsevier, pp. 449e462. Vives i Batlle, J., Wilson, R.C., McDonald, P., Parker, T.G., 2005. Uptake and depuration of 131I by the edible winkle Littorina littorea: uptake from seawater. Journal of Environmental Radioactivity 78 (1), 51e67. Whicker, F.W., Schultz, V., 1982. Radioecology: Nuclear Energy and the Environment, vol. 2. CRC Press. Whitehead, N.E., Ballestra, S., Holm, E., Huynhngoc, L., 1988. Chernobyl radionuclides in shellfish. Journal of Environmental Radioactivity 7 (2), 107e121. Wilson, R.C., Vives i Batlle, J., McDonald, P., Parker, T.G., 2005. Uptake and depuration of 131I by the edible periwinkle Littorina littorea: uptake from labelled seaweed (Chondrus crispus). Journal of Environmental Radioactivity 80 (3), 259e271. Xue, H.B., Stumm, W., Sigg, L., 1988. The binding of heavy-metals to algal surfaces. Water Research 22 (7), 917e926.
Uptake and depuration of 131I from labelled diatoms (Skeletonema costatum) to the edible periwinkle (Littorina littorea) R.C. Wilson a,*, J. Vives i Batlle a, S.J. Watts a, P. McDonald a, T.G. Parker b a
Westlakes Scientific Consulting Ltd., The Princess Royal Building, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3LN, UK b British Nuclear Group, Sellafield, Cumbria CA20 1PG, UK Accepted 15 January 2007 Available online 18 April 2007
Abstract Uptake and depuration of 131I into winkles through consumption of the diatom Skeletonema costatum is described. The work follows on from previous studies that investigated the uptake of iodine into winkles from seawater and seaweed. Incorporation of 131I in S. costatum from labelled seawater followed linear first-order kinetics with an uptake half-time of 0.40 days. Iodine uptake in winkles from labelled S. costatum also followed linear first-order kinetics, with a calculated equilibrium concentration (CN) of 42 Bq kg1 and a transfer factor (TF) of 1.1 104 with respect to labelled diatom food. This TF is lower than that observed for uptake of 131 I in winkles from labelled seaweed. For the depuration stage, a biphasic sequence with biological half-lives of 1.3 and 255 days was determined. The first phase is biokinetically important, given that winkles can lose two-thirds of their activity during that period. This study shows that, whilst winkles can obtain radioactive iodine from phytoplankton consumption, they do not retain the majority of that activity for very long. Hence, compared with other exposure
* Corresponding author. Fax: þ44 (0) 1946 514091. E-mail address: [email protected] (R.C. Wilson). 0265-931X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvrad.2007.01.018
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pathways, such as uptake from seawater and macroalgae, incorporation from phytoplankton is a relatively minor exposure route. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Winkle; Phytoplankton; Skeletonema costatum; Biokinetic; Uptake; Depuration radioiodine; Iodine
1. Introduction There is a growing interest in biokinetic modelling, which has many benefits over the simple concentration factor approach currently in widespread use in dose assessments. In a recent questionnaire commissioned by the International Union of Radioecology (IUR) on environmental protection, it was suggested that ‘biokinetic modelling was the way forward’. It also showed that there is a requirement for a better understanding of the transfer of radionuclides through the environment, with a benefit inherent to this type of modelling (IUR, 2006). Over the past few years a number of experiments have been conducted to investigate biokinetic parameters for iodine uptake in the edible periwinkle, Littorina littorea (henceforth referred to as the winkle). These have included uptake from seawater (Vives i Batlle et al., 2005) and through the consumption of the seaweed Chondrus crispus (Wilson et al., 2005). Winkles are a species of interest because, prior to these recent studies, little was known about iodine accumulation and uptake characteristics in these organisms. A number of earlier studies reported the concentration factor (CF) for radioiodine, not for winkles, but for other mollusc species, not specifically from the Irish Sea. These species included mussels (Shunhua et al., 1997; Sombrito et al., 1982; Whitehead et al., 1988) and clams (Cuvin-Aralar and Umaly, 1988, 1991; Mayr et al., 1988). Information on radioiodine depuration kinetics is even more scant: for mussels it is suggested that turnover is fast, with biological half-lives of 2e3 days and elimination following a simple exponential fall (Cuvin-Aralar and Umaly, 1988, 1991; Shunhua et al., 1997; Sombrito et al., 1982). The above knowledge gap notwithstanding, the dose to the critical group consumers of seafood in the vicinity of Sellafield, UK, contains a winkle component, of which 15% can be attributed to 129I, released under authorisation from the Sellafield site (BNGSL, 2006). Therefore a better understanding of the uptake characteristics should result in a more realistic assessment of the radiological impact of present and future discharges of radioiodine. To complete our investigations of iodine uptake pathways in winkles, this study determines the biokinetic parameters associated with the consumption of phytoplankton, in particular diatoms, given that winkles are known to eat microalgae (such as benthic diatoms and dinoflagellates) as well as macroalgae (Edwards and Davies, 2002). The marine diatom Skeletonema costatum was chosen for this study as it is a common species found in many marine systems, including the Irish Sea (McKinney et al., 1997). It is hypothesised that, in the natural environment, S. costatum can incorporate 129I with a relatively short biological half-time (of the order of 1 day or less, typical of passive adsorption onto the cell walls), and that, subsequently, L. littorea can assimilate this radioiodine through feeding on diatoms, whilst depurating in a manner consistent with that observed from the consumption of the seaweed C. crispus (Wilson et al., 2005). A laboratory experiment using 131I as a tracer was designed to test this hypothesis. 131I was chosen due to the ease of measurement compared to that of 129I, which is released under licence from the Sellafield site, and the fact that it is not environmentally available to any significant
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extent. As far as the authors are aware, there is no evidence to support the ability of winkles to isotopically fractionate radioiodine and therefore we believe that 131I is a suitable surrogate for 129I. 2. Materials and methods The investigation was designed to consist of two phases. The first phase involved labelling cultures of S. costatum with 131I. This was expected to yield information regarding the uptake of 131I into phytoplankton. The second phase was the feeding of the diatoms to the winkles, allowing uptake and depuration of 131I associated with the consumption of diatoms, to be studied within the winkles. 2.1. Winkles As in the two previous studies, winkles were collected from Nethertown on the Cumbrian coast. Following removal of any encrusting organisms from their shells, the winkles were allowed to acclimatise in 9 l glass aquarium tanks containing 5 l of local seawater. Three sets of 10 winkles were each kept in small labelled plastic cages to aid identification throughout the experiments. Cages were chosen that enabled thorough mixing of water within the cages with that of the bulk solution. With the tank containing only 5 l of seawater, the option of being above the water level was available to the winkles (during the uptake phase only). The tanks were maintained at 10e15 C which is consistent with temperatures in the Irish Sea (Kershaw et al., 1992) and bubbled with air to ensure that conditions within the tank did not become anoxic. 2.2. Diatoms The marine diatom S. costatum was chosen for this study as it is a common species found in many marine systems, including the Irish Sea (McKinney et al., 1997). It is also a species suitable for culturing within a laboratory environment. Cultures of the cells were supplied by Culture Collection of Algae and Protozoa (CCAP), Oban, in f/2 þ sodium metasilicate media (CCAP 1077/5). 2.2.1. Media All cultures were grown in f/2 þ sodium metasilicate media. The seawater used for the media was collected at Nethertown and filtered using a 0.2-mm cellulose nitrate filter paper. All stocks were stored at 5 C in the dark prior to use. 2.2.2. Growth conditions Cells were inoculated into 5 l glass conical flasks, which had been autoclaved at 120 C prior to use. Each flask contained 3 l of media. A cotton wool bung was placed in the opening of the flask to permit gaseous exchange. Inoculated flasks were then placed within an orbital-shaking incubator, where the flasks were shaken at 100 rpm and maintained at 15 C. Again, this temperature is representative of that in the Irish Sea (Kershaw et al., 1992). Finally, cultures were subjected to a 16 h light/8 h dark light regime. 2.2.3. Cell counting The number of cells present within each culture was measured using a haemocytometer and an optical microscope. 2.3. Diatom uptake of
131
I
Three 5 l conical flasks containing 3 l of media were inoculated with cells of S. costatum. Sufficient I tracer (Amersham Biosciences, supplied in 0.02 M NaOH) was then added to the culture to obtain an activity concentration of 2 Bq ml1. The initial contact time between the 131I in seawater and the diatoms before feeding the winkles was in excess of 60 h. Thereafter, diatoms were extracted at intervals of 24 h from the same solution during the 3-day period covering the duration of the experiment. 131
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Samples were taken daily to measure the activity in the algae as well as the activity in the culture medium. The latter was necessary to ensure the algal cells were subjected to a constant activity due to the short half-life of the 131I being used (8.04 days). With such a short half-life, loss of the tracer through decay will have been far greater than loss through adsorption and evaporation/volatilisation. The activity in diatom cultures was maintained at the specified level after counting the diatoms and the culture medium each day. The two components were separated using a syringe-driven 25 mm diameter 0.45 mm cellulose nitrate filter. Ten millilitres of suspension was passed through the filter and both the filter paper and the filtrate were measured for the emission of the characteristic g-rays from 131I at 364.5 keV. Daily diatom cell counts were also made and recorded. This operational sequence was repeated each day for a period of 10 days. 2.4. Winkle uptake of
131
I
Prior to feeding, each batch of 10 winkles was weighed and placed in a calibrated counting geometry to be analysed for 131I, using g-ray spectroscopy. Next, each batch was fed S. costatum separately. Six hundred millilitres of labelled culture suspension was passed through a 110-mm GF/C filter. The S. costatum were retained on the filter paper and the winkles were then placed upon it and allowed to graze on the cells for a period of 3 h (Vives i Batlle et al., 2006). This procedure was performed in separate containers and monitored to ensure that the food source was available to the winkles at all times. Immediately after feeding, the winkles were analysed for 131I. The fraction of diatoms not eaten was not measured. During the analysis for 131I, the tank seawater was replenished with fresh seawater. When analysis was complete, the winkles were returned to the plastic cages in the tank. This feeding process was repeated four times over a 3-day period. 2.5. Winkle depuration of
131
I
Following the uptake phase of the experiment, the release of iodine from the winkles was observed. Due to the low levels of iodine present within the winkles, it was decided to bulk the three batches into one at this stage. This had two benefits: (a) it reduced the daily count time required to obtain a statistically acceptable count area at 364.5 keV, and (b) it also allowed the release to be observed over a longer period of time before the levels of iodine became undetectable. After the final feed, the winkles were placed into the tank containing fresh seawater. At various intervals over an 11-day period the winkles were removed, weighed and analysed for 131I. They were then placed back into the tank in which the seawater had been replenished. Gamma emissions from 131I were counted using a high purity germanium crystal and the data were processed by Ortec Maestro software. Spectra were analysed with FitzPeaks32 Gamma Analysis and Calibration Software Version 3.48.
3. Results and discussion 3.1.
131
I uptake experiment
3.1.1. Diatoms (S. costatum) Concentrations of 131I increased from less than 2.5 mBq g1 to 280 Bq g1 during the 10 days of the uptake experiment (Fig. 1). Previous studies of iodine uptake into organisms have shown that the uptake trend follows first-order kinetics as shown in Eq. (1) (Vives i Batlle et al., 2005; Wilson et al., 2005): CðtÞ ¼ Ceq 1 eat
ð1Þ
Mean Activity in S. costatum (Bq g-1)
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450 400 350 300 250 200 150 100 50 0 0
50
100
150
200
250
Time (hours) Fig. 1. Uptake curve of
131
I in S. costatum (error bars indicate 2s counting uncertainty, n ¼ 3).
where C(t) is the concentration of 131I in S. costatum (Bq g1), Ceq is the equilibrium concentration, t is time and a is the time constant (ln2/Tu1/2, where Tu1/2 is the uptake half-time) which relates to loss during uptake (Whicker and Schultz, 1982). The uptake half-time was calculated to be 0.40 days, which is consistent with the expected short uptake period. It is known that certain metals bind very quickly to the surface of single cell organisms, within the order of 30 min, followed by a much slower intracellular uptake (Xue et al., 1988). From the data generated in this study, it is believed that rapid binding occurs and that the intracellular uptake is insignificant compared to the surface binding; hence the data fit a single-phase uptake model. A concentration factor (CF) of 141 l kg1 was also derived. This is eight times lower than the average 1100 l kg1 derived from the literature (Bowen, 1979; Coughtrey et al., 1984; IAEA, 2004; Kuenzler, 1967). However, the latter is a generic value for phytoplankton whereas the present experimentally-derived CF is species-specific. It is not obvious that the large difference between the present data and literature data can be attributed to differences in phytoplankton species only, although this is entirely possible. 3.1.2. Winkles Following optimised grazing times of 3 h on radiolabelled S. costatum (Vives i Batlle et al., 2006), there was a measurable increase of 131I within the winkles, confirming that uptake had occurred. The daily feeding cycle produced a serrated pattern of uptake/release cycles within each 24-h period, as seen in Fig. 2. Clearly, the separation between the upper and lower data points of this serrated pattern is statistically significant at the 2s level. 3.1.3. Equilibrium concentration To calculate the transfer factor of 131I from diatoms to winkles, a reference equilibrium concentration (Ceq) must be calculated. A detailed description of the calculation can be found in Wilson et al. (2005). The main assumption to be made is that the uptake half-time (Tu1/2 ¼ ln2/a) equates to the early-phase biological half-time (Tb1/2). This necessary ‘‘best guess’’ is not unreasonable as an approximation. The model for winkles developed by Vives i Batlle et al.
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A
120
Activity (Bq kg-1)
100 80 60 40 20 0 0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
50.0
60.0
70.0
80.0
50.0
60.0
70.0
80.0
Time (hours)
B
120
Activity (Bq kg-1)
100 80 60 40 20 0 0.0
10.0
20.0
30.0
40.0
Time (hours)
C
350
Activity (Bq kg-1)
300 250 200 150 100 50 0 0.0
10.0
20.0
30.0
40.0
Time (hours) Fig. 2. Uptake of 131I in winkles from grazing on labelled S. costatum, batches A, B and C, each containing 10 winkles (error bars indicate 2s counting uncertainty).
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(2006) shows that, following optimisation of the model parameter set, uptake half-times and biological half-lives for different components of the winkle (shell, soft tissue, storage organs), though not identical, are still reasonably similar. Individual Ceq values were calculated using Eq. (2): CðtÞ zCn þ Ceq aðt tn Þ
ð2Þ
where Cn and tn are the concentration of 131I and time of the n-th daily cycle. This approach produces an average equilibrium concentration from the three batches of winkles of 42 Bq kg1 fresh weight. 3.1.4. Transfer factor The transfer factor was calculated using Eq. (3). This gives an indication as to the proportion of radioactivity transferred from the diatoms to the winkles. TF ¼
Ceq Csc
ð3Þ
In Eq. (3), Csc is the activity in S. costatum in Bq kg1 fresh weight. When winkles were fed with the diatoms, it was not possible to obtain an accurate fresh weight mass for the diatoms. However, plankton cell counts can be determined using a haemocytometer. These results, used in conjunction with the mass of an individual cell (Brown, 1991), were used to estimate the fresh mass of algae being consumed. The average transfer factor from the three batches was calculated as 1.1 104 (this is a dimensionless quantity). This is considerably lower than the TF derived by Wilson et al. (2005) of 0.07 for the uptake from seaweed. Such a low TF value indicates that the uptake of iodine into winkles from the consumption of S. costatum is not the most significant bioaccumulation pathway. 3.2.
131
I depuration from winkles
During depuration, the activity concentration within the winkles fell by 84% from 93 Bq kg1 to 15 Bq kg1 over an 11-day period (Fig. 3). Such a fall is clearly significant within the 2s uncertainties reported. Previous studies (Vives i Batlle et al., 2005; Wilson et al., 2005) have shown that depuration follows first-order kinetics which can be represented by Eq. (4). CðtÞ ¼ C0 eat
ð4Þ
Previous work also showed that depuration can occur in a number of distinct phases. To investigate what number of release phases was most appropriate for this experiment, ln(C/C0) was plotted against time (Fig. 4) where C is the activity at time t and C0 is the starting activity. A biphasic release model (Fig. 4) generates biological half-lives of 1.3 and 255 days, with coefficients of determination (r2) being 1 (n ¼ 2) and 0.71, respectively. The shallow exponential fit relating to the 255-day biological half-life is compounded by the low statistical power of the data set, as exemplified by the relatively large error bars in Fig. 3. However, the trend is clearly discernible from the graph. The variance here may be considered the sum of two terms, one due to the analytical method and one due to possible fluctuations in the uptake by the winkle. The error bars in the data refer to the analytical method (they are, predominantly,
R.C. Wilson et al. / J. Environ. Radioactivity 96 (2007) 75e84
82 120
Activity (Bg kg-1)
100
80
60
40
20
0 0
50
150
100
200
250
300
Time (hours) Fig. 3. Release of
131
I from winkles (single batch of 30) (error bars indicate 2s counting uncertainty).
counting errors). The variance due to the winkle would, on the basis of our analysis, be relatively small, as reflected by the p-value of less than 0.0005, implying a very low probability that the exponential fit observed occurred by chance. The first biological half-life of 1.3 days is comparable with that reported by Wilson et al. (2005) of 0.9 days from the consumption of C. crispus. However, with phytoplankton, this initial phase spans a much shorter timescale than with C. crispus. This is illustrated by the percentage loss 2 h after feeding. Winkles fed on seaweed lost just 5% of the activity within 2 h compared to winkles fed on diatoms, which lost 72%. This marked difference could be due to one of two factors, or even a combination of these. First, that the diatoms are less 0
Bi-phasic - Phase 1
Ln(C/C0)
-0.5
R2 = 1, Tb1/2 = 1.3 days (n=2)
-1
Monophasic -----R2 = 0.44 (n =13, p
0.01)
-1.5
-2 B-phasic - Phase 2 R2 = 0.71,Tb1/2 = 255 days (n=12, p
0.0005)
-2.5 0
50
100
150
200
Time (hours) Fig. 4. Biphasic release of
131
I from winkles.
250
300
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digestible than seaweed and therefore have a short transit time through the digestive tract, reducing the time available for uptake. Second, that iodine is predominantly incorporated on the diatom cell surface, from where it dissolves relatively quickly into the digestive juices. The smaller fraction eliminated with a much longer biological half-life (255 days for S. costatum compared to 24 days for C. crispus) could represent an intracellular component which is actively absorbed by the gut and eliminated much more slowly. With the evidence at hand, it is not possible to reach any firm conclusions on this matter. To provide a clearer answer for this marked difference in loss characteristics would require a more physiologically driven study, investigating the excretion process for the winkle and, particularly, the physico-chemical form of the excreted iodine. Such an undertaking lies beyond the scope of the present paper but does signal a direction for future studies. 4. Conclusions Uptake and depuration experiments were undertaken successfully in the laboratory on winkles from the Cumbrian coast near Sellafield, that were previously fed with labelled S. costatum. The 131I concentrations in S. costatum during labelling followed linear first-order kinetics with a mono-phasic pattern generating an uptake half-time of 0.40 days. The CF was calculated as being 141 l kg1. This is lower than CF values in the literature for iodine in phytoplankton. It should be noted that these literature values are for the generic phytoplankton phylum whereas the value reported in this study is species-specific for S. costatum. The uptake of 131I from labelled S. costatum in winkles also followed linear first-order kinetics producing a calculated equilibrium concentration (CN) of 42 Bq kg1, resulting in a transfer factor of 1.1 104 with respect to the labelled diatoms used as food. For depuration, a biphasic sequence with biological half-lives of 1.3 and 255 days was determined. The first phase is consistent with that seen following the consumption of C. crispus. However the second phase is considerably longer. This indicates that following the consumption of labelled S. costatum, the majority of iodine is processed relatively quickly, but any iodine incorporated into the system is processed (and eventually released) at a much slower rate. This study demonstrates that whilst winkles have the ability to obtain radioactive iodine from the consumption of phytoplankton, they do not retain that activity for very long. It is therefore concluded that compared with other exposure pathways such as uptake from seawater and macroalgae, incorporation of radioiodine from phytoplankton is a relatively minor exposure route. From this study alone, it has not been possible to deduce if 131I is excreted as part of the diatom or whether 131I is released from the diatom surfaces in the digestive system of the winkles and excreted separately. The answer depends critically on two factors: first, did the labelling of the diatoms result in surface sorption, or in a predominantly intracellular fraction of 131I in the microalgae? Second, are diatoms excreted whole or does the digestive process manage to break down cells and release their contents? This is an area of research worth considering, signalling the direction for future investigations. References BNGSL, 2006. Monitoring our environment. Discharges and monitoring in the UK. Annual Report 2005. British Nuclear Group Sellafield Ltd. Bowen, H.J.M., 1979. Environmental Chemistry of the Elements. Academic Press.
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Brown, M.R., 1991. The amino-acid and sugar composition of 16 species of microalgae used in mariculture. Journal of Experimental Marine Biology and Ecology 145 (1), 79e99. Coughtrey, P.J., Jackson, D.J., Jones, C.H., Thorne, M.C., 1984. Radionuclide Distribution and Transport in Terrestrial and Aquatic Ecosystems e A Critical Review of Data, vols. 1e6. A.A.Balkema, Rotterdam. Cuvin-Aralar, M.L.A., Umaly, R.C., 1988. Uptake and elimination of iodine-131 by the freshwater clam Corbicula manilensis Philippi from water. Natural and Applied Science Bulletin 40, 141e158. Cuvin-Aralar, M.L.A., Umaly, R.C., 1991. Accumulation and tissue distribution of radioiodine (131I) from algal phytoplankton by the fresh-water clam Corbicula manilensis. Bulletin of Environmental Contamination and Toxicology 47 (6), 896e903. Edwards, M., Davies, M.S., 2002. Functional and ecological aspects of the mucus trails of the intertidal prosobranch gastropod Littorina littorea. Marine Ecology Progress Series 239, 129e137. IAEA, 2004. Sediment distribution coefficients and concentration factors for biota in the marine environment, Technical Reports Series No. 422, Vienna, International Atomic Energy Agency. IUR, 2006. IUR web based questionnaire results for environmental protection. Research, Facilities and Scientific Priorities. International Union of Radioecology, IUR Report 5. Kershaw, P.J., Pentreath, R.J., Woodhead, D.S., Hunt, G.J., 1992. A review of radioactivity in the Irish Sea. A Report prepared for the Marine Pollution Monitoring Group, Lowestoft, UK, Ministry of Agriculture, Fisheries and Food Aquatic Environment Monitoring Report 32. MAFF, Directorate of Fisheries Research. Kuenzler, E.J., 1967. Elimination of iodine, cobalt, iron, and zinc by marine zooplankton. In: Nelson, D.J., Evans, F.C. (Eds.), Proc. 2nd National Symposium on Radioecology. Ann Arbor, Michigan, May 15e17, 1967. USAEC Conf. No. 670503, pp. 462e473. Mayr, L., Moraes, R., Lopes, M.A., Vicente, C., Mauro, J.N., 1988. Transit and absorption of nuclear industry derivatives by marine biota (Transito e absorc¸ao de radionuclı´deos na biota marinha derivados de industria nuclear). In: Proc. 2. General Congress of Nuclear Energy. Rio de Janerio (Brazil), pp. 281e293. McKinney, E.S.A., Gibson, C.E., Stewart, B.M., 1997. Planktonic diatoms in the north-west Irish Sea: a study by automatic sampler. Biology and Environment: Proceedings of the Royal Irish Academy 97B (3), 197e202. Shunhua, C., Qiong, S., Xiaokui, K., 1997. Effects of body size on accumulation and distribution of 125I in the green mussel (Perna viridis). Beijing, China, China Nuclear Information Centre. Report CNIC-01210. Sombrito, E.Z., Banzon, R.B., de la Mines, A.S., Bautista, E., 1982. Uptake of iodine-131 in mussel (Mytilus smaragdinus) and algae (Caulerpa racemosa). The Nucleus (Journal of the Radioisotope Society of Philippines) 22 (1), 83e89. Vives i Batlle, J., Wilson, R.C., McDonald, P., Parker, T.G., 2006. A biokinetic model for the uptake and release of radioiodine by the edible periwinkle Littorina littorea. In: Povinec, P.P., Sa´nchez-Cabeza, J.A. (Eds.), Radionuclides in the Environment, vol. 8. Elsevier, pp. 449e462. Vives i Batlle, J., Wilson, R.C., McDonald, P., Parker, T.G., 2005. Uptake and depuration of 131I by the edible winkle Littorina littorea: uptake from seawater. Journal of Environmental Radioactivity 78 (1), 51e67. Whicker, F.W., Schultz, V., 1982. Radioecology: Nuclear Energy and the Environment, vol. 2. CRC Press. Whitehead, N.E., Ballestra, S., Holm, E., Huynhngoc, L., 1988. Chernobyl radionuclides in shellfish. Journal of Environmental Radioactivity 7 (2), 107e121. Wilson, R.C., Vives i Batlle, J., McDonald, P., Parker, T.G., 2005. Uptake and depuration of 131I by the edible periwinkle Littorina littorea: uptake from labelled seaweed (Chondrus crispus). Journal of Environmental Radioactivity 80 (3), 259e271. Xue, H.B., Stumm, W., Sigg, L., 1988. The binding of heavy-metals to algal surfaces. Water Research 22 (7), 917e926.