P G Cardoso
email: gcardoso@ci.uc.pt

A I Lillebø

E Pereira

A C Duarte

M A Pardal

Different mercury bioaccumulation kinetics by two macrobenthic species: the bivalve Scrobicularia plana and the polychaete Hediste diversicolor

Keywords: Mercury, bioaccumulation, kinetics, Hediste diversicolor, Scrobicularia plana

Mercury is a global priority pollutant and given its huge relevance in terms of environmental damage and a threat to human health, it is important to study the bioaccumulation processes at the level of macrobenthic organisms and evaluate possible consequences for the trophic chain. In this work we evaluate the mercury accumulation rates of two distinct and economically important macrobenthic species, the polychaete Hediste diversicolor and the bivalve Scrobicularia plana, through a mesocosms laboratory experiment.

The present experiment demonstrated different bioaccumulation kinetics for each species: while S. plana has a rapid accumulation after 48 hours of exposure to contaminated sediments and then reaches a steady state, H. diversicolor has a linear mercury accumulation throughout the experiment time (31 days). The different patterns observed can be related to different feeding strategies and assimilation efficiencies. In addition, we can conclude that independently of the mercury concentration in the sediments, the two studied species in both contaminated sediments reached the same percentage (25%) of mercury accumulated at the end of the experiment when compared to the maximum concentrations recorded in the field. This experiment highlights that the mercury bioaccumulation rate by these two economically and ecologically important macrobenthic species is a reasonably rapid process which can promote serious consequences for the higher trophic levels, constituting a severe risk to the natural environment and ultimately to human health.

INTRODUCTION

Sediments represent, quantitatively, the major compartment for metal storage in aquatic environments. Although the total metal concentrations in sediments give a convenient measure of their metal contamination, such measures do not necessarily predict the toxicity of these contaminants to aquatic organisms. The ecotoxicological risk posed by contaminated sediments will depend on metal availability as well as of the ability of living organisms to assimilate metals [START_REF] Literature Cited Amiard | Relashionship between the lability of sediment-bound metals (Cd, Cu, Zn) and their bioaccumulation in benthic invertebrates[END_REF]. Bioaccumulation at organism level is the result of uptake mechanisms, which control the entry of metals through the biological barriers separating individuals from their surrounding environment, and excretion mechanisms, which give rise to transfers into the external environment [START_REF] Inza | Dynamics of cadmium and mercury compounds (inorganic mercury or methilmercury): uptake and depuration in Corbicula fluminea. Effects of temperature and pH[END_REF].

Over the past 10 years, a considerable interest has been generated regarding the kinetic of metal bioaccumulation in aquatic invertebrates, mainly bivalves. However, much information is lacking, specifically about the bioaccumulation rates of an important trace metal, mercury, by different economically important macrobenthic species.

In the specific case of mercury, a highly deleterious chemical element that has both natural and anthropogenic sources, contamination problems are enhanced when it enters the food web via biomethylation and bioaccumulation processes. The sources, fate and bioaccumulation of mercury have received increasing attention (US EPA 1997, Regulation EC 466/2001) due to the impact of fish consumption on human health. Knowledge of the concentration of heavy metals in biological tissues is required, particularly in benthic fauna, in order to understand the bioconcentration in the food web.

The relationship between metal accumulation and the feeding behaviour of the benthic organisms is an important factor in terms of bioavailability of different metals in the aquatic environment [START_REF] Monperrus | Determination of metal and organometal trophic bioaccumulation in the benthic macrofauna[END_REF]. The common ragworm, Hediste diversicolor, is one of the few polychaete species to have a commercial interest because of its use as bait in recreational fishing and as food in aquaculture (Scaps, 2002). H. diversicolor is omnivorous but is also an active predator. It may behave as a deposit-feeder collecting food near its burrow opening (Nielsen et al., 1995, Scaps, 2002). As a burrowing species, this worm lives in close contact with sediments and, as a consequence, may be appointed as an appropriate indicator of contamination. Scrobicularia plana is a long-lived deposit feeding bivalve species living in muddy-sandy sediments. It has a wide geographical distribution (Verdelhos et al., 2005) and has been previously used in biomonitoring programs and toxicity tests [START_REF] Coelho | Pattern and annual rates of Scrobicularia plana mercury bioaccumulation in a human induced mercury gradient (Ria de Aveiro, Portugal)[END_REF].

Considering that the above mentioned macrofaunal species have high economic relevance and represent an important position in the food web, constituting the food source for higher trophic levels, the main goals of the present study were (1) to determine the kinetics of mercury bioaccumulation in those two distinct macrobenthic species (Hediste diversicolor and Scrobicularia plana) and (2) to evaluate the influence of mercury sediment concentration on the mercury accumulation rate of these two species.

MATERIALS AND METHODS

Study sites description

Sediments were collected in October 2007 in two different temperate estuarine systems, the Mondego Estuary (40º 08' N, 8º 50' W) (Figure 1A) and the Ria de Aveiro (40º 38' N, 8º 44' W) (Figure 1B) located both on the Atlantic coast of Portugal. The Mondego Estuary is located 60 km south from the Ria de Aveiro. Station 1 at the Mondego Estuary has been used as a reference condition since no local source of mercury contamination is known (Coelho et al., 2006 and references therein). Ria de Aveiro was contaminated by effluents coming from a mercury cell chloralkali plant from the 1950s until 1994. The discharges resulted in an accumulation of about 33 ton of mercury in the lagoon, much of which (about 27 ton) are known to be trapped in the sediments of the basin named Laranjo [START_REF] Cardoso | Influence of bioturbation by Hediste diversicolor on mercury fluxes from estuarine sediments: a mesocosms laboratory experiment[END_REF]. In the last decade, the industrial mercury discharge decreased considerably, being nowadays within the regulatory levels (50 µg L -1 , the limit value for discharges from chlor-alkali electrolysis industry, in accordance with Directive 82/176/EEC). In this Laranjo basin two sampling sites were chosen: one characterized by strongly contaminated sediments (Laranjo -station 3), collected approximately 450 m away from the mercury point source, and the other one corresponding to a moderately contaminated sediments (Chegado -station 2), collected at the opposite end of the basin, approximately 1250 m away from the mercury point source towards Chegado Quay (referred along the text as Chegado) (Figure 1B). The three sediment environments can be considered as similar in terms of physical characteristics (muddy sediments) and representing a gradient of mercury contamination. Furthermore, only the superficial layer of the sediments (10 cm depth) was considered relevant in this study.

Invertebrates

The bivalve Scrobicularia plana and the polychaete Hediste diversicolor were collected from the Mondego station (reference condition). In the laboratory, the polychaete organisms were measured (first setiger width) and all the organisms used in the experiment belonged to the class < 1 mm, corresponding to an average burrow surface area of 1 cm 2 (Mortimer et al., 1999).

Scrobicularia plana individuals were, also, measured in terms of total length. All the individuals used in the experiment belonged to the class 2-3 cm, corresponding to 2+ year old individuals (Verdelhos et al., 2005). The organisms used in the experiment were left in clean seawater at 20ºC during four days to acclimate and depurate. Oxic conditions were ensured by air-bubbling the water.

Experimental set-up

The experiments were performed in acid-washed glass containers of 3 litres (Ø: 14 cm). To reduce the natural heterogeneity and to achieve similar starting conditions, all the sediments were homogenised before use and their macrofauna was removed. The water used in the experiment was collected in the Mondego Estuary (reference condition) and filtered through 0.45 µm pore size Millipore filters, in order to retain particles present in the water. Sediment mesocosms were established by transferring 2000 g (wet weight-wwt) of homogenised and fauna-free sediment to each glass container. Then, 1.0 L of filtered (0.45µm) estuarine water (salinity -30) from the Mondego Estuary was added to each container, always maintained under oxic conditions. The glass containers were randomly distributed in a dark room to avoid primary production and kept at 20ºC. The containers were maintained in these conditions during 48 hours to stabilize before introducing the organisms. In each of the glass containers 5 individuals of Hediste diversicolor and 3 individuals of Scrobicularia plana were introduced, according to the mean densities observed in the field [START_REF] Cardoso | Polychaetes as indicators of habitat recovery in a temperate estuary under eutrophication[END_REF], Verdelhos et al., 2005). At defined times, the organisms (Hediste diversicolor and Scrobicularia plana) were removed from the glass containers, rinsed with seawater and then placed in constantly aerated clean seawater at 20°C for 48 hours to allow depuration, being afterwards dissected in the case of S. plana, and then freeze-dried for later mercury analysis.

For each of the three conditions (reference, low and high contamination) four different sampling times were chosen (t1 -2 days; t2 -5 days; t3 -9 days; t4 -31 days). At each sampling condition and occasion three replicates were carried out. Therefore, the experimental set-up included thirty six (3x4x3) sub-experiments.

To evaluate the total mercury accumulation ability of the studied organisms in the field, the two species under study were collected at each sampling site. In the laboratory, the individuals were subjected to depuration, following the same procedure as described above.

Analytical methodologies

Sediment samples

Sediment samples of each site were analysed for water and organic matter contents, grain size, total carbon, mercury, iron and manganese. The physico-chemical properties of the sediments were analysed to evaluate if there were any differences that could influence the availability and the accumulation of mercury and to achieve the degree of metal contamination in each site.

The sediments were previously homogenised, freeze-dried and manually sieved through a 1 mm mesh to remove coarser material and debris.

The water content of the sediments was determined by subtracting the value of dry weight from the wet weight of a sediment sample. Organic matter content of the sediments was evaluated as weight loss on ignition (% LOI) at 450ºC during 8 hours. Fine particles (less than 63 µm) were determined by wet sieving of a dried sample using a 63 µm mesh size sieve.

Sediment samples were analysed for total mercury by thermal decomposition coupled to atomic absorption spectrometry after gold amalgamation, using a LECO AMA-254 (Advanced Mercury Analyser). Analytical quality control was ensured by using certified reference material (CRM). The CRM used were MESS-3 (marine sediments) and PACS-2 (polluted marine sediments). Measured and certified values for total mercury in the CRMs used were in agreement, with recoveries between 88 and 109% and standard deviations always less than 5%.

Total iron and manganese concentrations were determined on approximately 100 mg of freeze-dried sediment added into an acid-washed Teflon reactor with further addition of 1 mL of aqua regia and 6 mL of concentrated hydrofluoric acid. The Teflon reactors were sealed and placed in an oven for 1 hour at 100ºC. After cooling, the solution was quantitatively transferred into acid cleaned 100 mL volumetric flasks that contained 5.6 g of boric acid (Rantala and Loring, 1977).

The volume was completed with Milli-Q water and mixed to promote the dissolution of the boric acid, and the solution was transferred to plastic bottles and stored at 4ºC. Afterwards, iron and manganese were determined by Flame Atomic Absorption Spectrophotometry (Perkin Elmer, model AAnalyst 100).

A partial digestion using a hydroxylamine hydrochloride-acetic acid mixture (HAA) was used to evaluate the content of iron and manganese hydrous oxides of the sediments. This extraction was performed using approximately 1g of freeze-dried sediment, to which 20 mL of 0.04 mol L -1 hydroxylamine hydrochloride in 25% glacial acetic acid was added. The selective extractions with HAA were conducted in centrifuge tubes, which were subjected to continuous agitation during 6 hours [START_REF] Canário | Mercury in contaminated sediments and pore waters enriched in sulphate (Tagus estuary, Portugal)[END_REF]. After extraction, solid-solution separation was obtained by centrifuging (B. Braun, model Sigma 4-10) at 6000 rpm for 30 minutes. The supernatant was filtered through 0.45 µm pore size Millipore filters and analysed by using Flame Atomic Absorption Spectrophotometry (Perkin Elmer, model AAnalyst 100).

Biological samples

Biological samples were analysed for total mercury also by thermal decomposition atomic absorption spectrometry with gold amalgamation, using a LECO AMA-254. Analytical quality control was performed by using TORT-2 as certified reference material. The recovery values obtained for TORT-2 analysis ranged from 102-118% and standard deviations always less than 3%.

Data analysis

The model based on a Michaelis-Menten equation applied to the bivalve Scrobicularia plana was performed using the GraphPad Prism software package, version 5.

RESULTS

Sediment characterization and mercury concentrations

The percentage of organic matter (% LOI) of the sediments of the three studied sites was similar, with Laranjo showing slightly higher organic matter content than Chegado and Mondego Estuary sites (Table 1). The percentage of water content of the sediments of the three areas was also similar, ranging between 42% for the Mondego Estuary and 50% for Chegado and Laranjo (Table 1). In terms of percentage grain size < 63 µm, the sediment samples were quite similar in the three sites (around 50%).

Mercury concentrations in the sediments from the three sites clearly show the existence of a strong contamination gradient. The sediments from the Laranjo contain more mercury than those from the Chegado and both sites have high levels when compared to the reference station, the Mondego Estuary (Table 1). Concentrations of total iron were higher in Mondego Estuary (27133±30 g g -1 ) and Laranjo (29156±909 g g -1 ) than in Chegado (23378±270 g g -1 ) site (Table 1). Concentrations of total manganese were higher in the Mondego Estuary (236 ±1.3 g g -1 ) than in Chegado (177±4.7 g g -1 )

and Laranjo (126±2.3 g g -1 ) sites (Table 1).

The highest concentrations of iron hydrous oxides occurred in Chegado followed by Mondego Estuary and Laranjo. In Chegado, concentrations of iron hydrous oxides were 2918±101 g g -1 (12%), while Mondego Estuary and Laranjo presented similar concentrations, around 2770±325 g g -1 (10%) for the first one while the latter recorded 2564±135 g g -1 (9%) (Table 1).

Regarding the concentrations of manganese hydrous oxides, this metal presented a different pattern compared to iron, since it was more abundant in the Mondego Estuary, 36.2±0.8 g g -1 (15%) than in the Chegado, 18.8±0.7 g g -1 (11%). Laranjo presented the lowest concentrations of manganese hydrous oxides, around 10.3±0.5 g g -1 (8%) (Table 1).

Mercury bioaccumulation rates

Field mercury concentrations of the studied macrobenthic species (S. plana and H. diversicolor), as expected, presented lower mercury concentrations in the Mondego Estuary, followed by the Chegado and finally the Laranjo (Table 1). In the case of S. plana, this bivalve exceeded the maximum mercury levels allowed for human consumption (0.5 g g -1 wet weight, Regulation EC 466/2001). This species reached higher mercury concentrations than the polychaete H. diversicolor, which may be due in part to its longer life span [START_REF] Coelho | Mercury contamination in invertebrate biota in a temperate coastal lagoon (Ria de Aveiro, Portugal)[END_REF]) (Table 1).

Concerning the total mercury concentration accumulated by the studied species during the experimental period, it can be observed that the two macrobenthic species showed different kinetics. In the reference condition (Mondego Estuary), S. plana and H. diversicolor did not show significant differences in terms of mercury concentration between the beginning and the end of the experiment (t-test, S. plana: t 4 =0.22, p>0.05; H. diversicolor: t 4 =-0.7, p>0.05).

On the other hand, in Chegado and Laranjo, the bivalve presented a model based on a Michaelis-Menten equation (Chegado, R 2 =0.897; Laranjo, R 2 =0.786). In both contaminated areas, S. plana reached higher mercury concentrations throughout the first 48 hours, stabilizing during the rest of the experimental period. After 48 hours of exposition to contaminated sediments, the bivalves attained 25% of mercury accumulated in relation to the mercury concentrations recorded in the field (Figures 2A-C).

For the polychaete H. diversicolor, its body burdens of total mercury increased all over the experimental period, for both contaminated sediments, Chegado and Laranjo (Chegado R 2 =0.994; Laranjo R 2 =0.980) (Figures 2D-F). At the end of the experiment, the ragworm reached the same percentage of mercury accumulated (25%) in relation to the values recorded in the field. A steady state did not appear to occur in H. diversicolor during the 31-day accumulation period for total mercury. According to the regression equations (Fig. 2E,F), the time to achieve the steady state for total mercury in H. diversicolor was estimated to be approximately 120-180 days, respectively, in Laranjo and Chegado.

A comparison of mercury body burdens for both macrobenthic species in the high and intermediate contaminated sediments showed that accumulation was higher in the Laranjo (high contamination) than in the Chegado (intermediate contamination) sediments.

Analysing the mercury accumulation rate per day for the two studied species, we can observe that for S. plana there was a huge accumulation of mercury after the first 2 days (equivalent to 0.026 g g -1 per day). However, after 5 days of exposure, the increment on mercury concentrations in S. plana declined abruptly (0.0009 g g -1 per day, approximately two orders of magnitude). Since then and until the end of the experiment, the mercury accumulation rate was even lower. On the other hand, for H. diversicolor, the mercury accumulation was linear, with a regular increment (0.0006 g g -1 per day) throughout the experiment (Figures 3A,B).

DISCUSSION

This study showed that total mercury concentrations in Scrobicularia plana and Hediste diversicolor correlate well with total mercury levels in sediments, which is in accordance with other works [START_REF] Coelho | Mercury contamination in invertebrate biota in a temperate coastal lagoon (Ria de Aveiro, Portugal)[END_REF], Zizek et al., 2007). On the other hand, the bivalve S. plana recorded higher levels of total mercury than the polychaete H. diversicolor, which can be related in part to their life spans and their bioaccumulation rates. S. plana is a long lived bivalve species with a lifespan of 5 years (Verdelhos et al., 2005), while for the polychaete H. diversicolor its lifespan ranges between 12 and 15 months (Abrantes et al., 1999). Therefore, a possible explanation for the higher levels of mercury measured in the bivalve Scrobicularia plana could be related to the fact that during its longer life period it accumulates more mercury than the polychaete H. diversicolor, even though bioaccumulation rates could be different for the two species.

The mesocosms experiment also demonstrated that these two different macrobenthic species revealed distinct behaviours concerning the mercury accumulation kinetics. S. plana exhibits a model similar to a Michaelis-Menten like kinetics, reaching a maximum accumulation rate after 48 hours of exposure and then accumulation stabilises until the end of the experiment. The polychaete H. diversicolor shows a linear accumulation rate throughout the experiment, which means that the steady state is reached later relative to the bivalve S. plana. According to the observed model, we can estimate that H. diversicolor will take approximately 120 to 180 days to reach the field mercury concentrations, respectively in Laranjo and Chegado. A possible explanation for the distinct bioaccumulation kinetics of mercury by the two studied species may be due to a combination of factors, such as different feeding strategies, ingestion rates and assimilation efficiencies. H. diversicolor is ominivorous but it is also an active predator. However, it can also behave as a deposit-feeder collecting food near its burrow opening (Scaps, 2002). On the other hand, S. plana is a facultative surface deposit-feeder. It is able to take its food from the surface of the sediment and out of the water column (as filter feeder) [START_REF] Kamermans | Similarity in food source and timing of feeding in deposit-and suspensionfeeding bivalves[END_REF], Chícharo et al., 2002). Considering that deposit/filter feeder species, like S. plana feeds essentially on sediment particles to which mercury is associated [START_REF] Monperrus | Determination of metal and organometal trophic bioaccumulation in the benthic macrofauna[END_REF] it is possible that this species accumulates more mercury than H. diversicolor. Besides feeding behaviour, species-specific physiological properties of organisms, such as efficiency of assimilation, metal excretory rates and biochemical properties, including the number of metal binding ligands may also influence the accumulation of metals in organisms' tissues [START_REF] Ernst | The effect of feeding behavior on Hg accumulation in the ecophysiologically different earthworms Lumbricus terrestris and Octolaseon cyaneum: A microcosm experiment[END_REF]. Therefore, all these processes together may explain the higher mercury accumulation by the bivalve S. plana.

From this experiment we conclude that independently of the mercury concentration in the sediments, the two studied species in both contaminated sites, Chegado and Laranjo, reached the same percentage (25%) of mercury accumulated at the end of the experiment compared to the concentrations recorded in organisms collected in the field and living in each site. However, the mercury accumulation rate for both species at the two sites was different. For S. plana, the accumulation rate was 3 times higher in Laranjo than in Chegado, while for H. diversicolor it was 2 times higher. This can be explained by the higher mercury concentrations of the Laranjo sediments and also the feeding strategy and assimilation efficiency of Scrobicularia plana.

It would be expected that S. plana as a filter feeder, could also accumulate mercury from the water column (dissolved fraction); however, according to a previous study by [START_REF] Cardoso | Influence of bioturbation by Hediste diversicolor on mercury fluxes from estuarine sediments: a mesocosms laboratory experiment[END_REF], it was possible to conclude that the mercury concentration in the water caused by the bioturbation activity of H. diversicolor was very low (10-15 ng.L -1 ). So, the water column should not be considered as a mercury source. This phenomenon can be related to the sediments' characteristics from the Ria de Aveiro, since these sediments are rich in organic matter and in iron/manganese hydrous oxides compared to other worldwide systems (see [START_REF] Cardoso | Influence of bioturbation by Hediste diversicolor on mercury fluxes from estuarine sediments: a mesocosms laboratory experiment[END_REF]. These physicochemical characteristics of the sediments may explain, in part, the high retention of mercury in sediments of this system.

To our knowledge there are only a few studies (e.g. [START_REF] Inza | Dynamics of cadmium and mercury compounds (inorganic mercury or methilmercury): uptake and depuration in Corbicula fluminea. Effects of temperature and pH[END_REF][START_REF] Burton | Bioaccumulation of total mercury and monomethilmercury in the earthworm Eisenia fetida[END_REF] concerning the kinetics of mercury bioaccumulation by different macroinvertebrate species, despite the enormous importance of this trace metal to the biota. Most of the published studies (e.g. Pan andWang, 2004, Baudrimont et al., 2005) used different species of bivalves as biomonitor species, such as mussels, cockles and other species of clams. Very few studies examined the bioaccumulation rates of mercury in worms. Despite the different habitat occupied by earthworms, our results are in agreement with [START_REF] Burton | Bioaccumulation of total mercury and monomethilmercury in the earthworm Eisenia fetida[END_REF]: the earthworm Eisenia fetida had the same behaviour as H. diversicolor in our study, showing a linear accumulation rate of mercury over the experimental period and also the accumulation increased with increasing concentrations of mercury in the sediments.

Regarding other studies using bivalves as biomonitor species, they revealed a different behaviour from the one observed with S. plana in the present study. According to [START_REF] Inza | Dynamics of cadmium and mercury compounds (inorganic mercury or methilmercury): uptake and depuration in Corbicula fluminea. Effects of temperature and pH[END_REF], the bivalve Corbicula fluminea demonstrated a linear trend for mercury accumulation.

In conclusion, the bioaccumulation rate of mercury by these two economically and ecologically important macrobenthic species is a quite rapid process that can promote serious consequences for the higher trophic levels (e.g. fishes and birds) which use these species as food resources. Therefore, the mercury biomagnification process can be very fast, constituting a severe risk to the human health.

  of the Adour estuary coastal zone (SW France, Bay of Biscay). Journal of Environmental Monitoring 7, 693-700. Mortimer, R.J.G., Davey, J.T., Krom, M.D., Watson P. G, Frickers P. E, Clifton R.J., 1999. The effect of macrofauna on porewater profiles and nutrient fluxes in the intertidal zone of the Humber estuary. Estuarine Coastal and Shelf Science 48, 683-699. Nielsen, A.M., Eriksen, N.T., Iversen, J.J.L., Riisgard, H.U., 1995. Feeding, growth and respiration in the polychaetes Nereis diversicolor (facultative filter-feeder) and N. virens (omnivorous)a comparative study. Marine Ecology Progress Series 125, 149-158. Pan, J-F, Wang, W-X, 2004. Uptake of Hg (II) methylmercury by the green mussel Perna viridis under different organic carbon conditions. Marine Ecology Progress Series 276, 125-136. Pereira, M.E., Lillebø, A.I., Pato, P., Válega, M., Coelho, J.P., Lopes, C.B., Rodrigues, S., Cachada, A., Otero, M.,•Pardal M. A., Duarte A.C. 2008. Mercury pollution in Ria de Aveiro (Portugal): a review of the system assessment Environmental Monitoring Assessment DOI 10.1007/s10661-008-0416-1 Rantala, R.T.T., Loring, D.H., 1977. A Rapid Determination of 10 Elements in Marine Suspended Particulate Matter by Atomic Absorption. Atomic Absorption News 16, 51-52. Scaps, P., 2002. A review of the biology, ecology and potential use of the common ragworm Hediste diversicolor (O.F. Müller) (Annelida: Polychaeta) Hydrobiologia 470, 203-218. www.epa.gov/mercury/report.htm Verdelhos, T., Neto, J.M., Marques, J.C., Pardal, M.A., 2005. The effect of eutrophication abatement on the bivalve Scrobicularia plana. Estuarine Coastal and Shelf Science 63, 261-268.Žižek, S., Milena, Horvat, M., Gibi ar, D., Fajon, V., Toman, M.J., 2007. Bioaccumulation of mercury in benthic communities of a river ecosystem affected by mercury mining.

Figure 1 -

 1 Figure 1 -Sediment sampling sites: A -Station 1 -Mondego Estuary; B -Ria de Aveiro (Station 2

Figure 2 -

 2 Figure 2 -Mercury concentrations (µg g -1 ) accumulated by the two species during the experimental

Figure 3 -

 3 Figure 3 -Mercury accumulation per day (µg g -1 /day) in the two species, during the experimental

Figueira

  Figueira da Foz Harbour

Table 1 -

 1 Physico-chemical characterization of the sediments and mercury content of S. plana and

		PORTUGAL
		North Arm
	Gala Bridge
	Murraceira Island	Connection
		of
		2 arms
		1 Km
	Armazéns Channel	Pranto River
	Intertidal Areas

H. diversicolor in the field conditions for the three sampling sites (* -Standard deviation; ** -Standard error.

A

ACKNOWLEDGEMENTS

This work was supported by FCT (Fundação para a Ciência e Tecnologia) through a Pos-Doc grant attributed to P. G. Cardoso (SFRH/BPD/22176/2005). The authors are indebted to all the colleagues, especially those from the Chemistry laboratory from the Aveiro University that assisted in the field and laboratory work.


Annelie Hilvarsson
email: annelie.hilvarsson@marecol.gu.se

Cecilia Ohlauson

Hans Blanck

Åke Granmo

Åke Granmo

Bioaccumulation of the new antifoulant medetomidine in marine organisms

Keywords: medetomidine, antifouling substance, bioaccumulation, elimination, uptake, periphyton, benthos

Biofouling is a huge problem globally and new alternative antifoulants are presently being investigated. One candidate is medetomidine, a commonly used sedative in veterinary medicine, which has been shown to effectively prevent settlement of barnacles.

The purpose of this study was to measure uptake, elimination and bioconcentration of medetomidine in Mytilus edulis, Abra nitida, Crangon crangon and periphyton communities to evaluate the risk of bioaccumulation in the marine environment.

Bioconcentration factors (BCF) and bioaccumulation factors (BAF) were used to assess the bioaccumulation. The calculations of these factors were based on the distribution of the radiolabelled medetomidine. BCF for Crangon crangon was 2.8 while Mytilus edulis had a BCF of 134 and the periphyton communities' BCF was 1195 l/kg fresh weight (FW). The concentration of medetomidine in the animals reached steady state after 24 to 48 hours for all test systems except for Abra nitida, which never stabilised enough to calculate a bioaccumulation factor (BAF). Elimination from the organism's tissues was rapid for three of the test systems with half-lives between 1 and 24 h. Abra nitida had a half-life of 96-120 h. The present study demonstrates that the bioconcentration and bioaccumulation of medetomidine differs between aquatic organisms and that microalgal communities in the form of periphyton have the highest bioconcentration factor of the organisms tested.

Introduction

Medetomidine (4-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole) is commonly used as a sedative in veterinary medicine and functions as an α 2 -adrenoceptor agonist in mammals.

It is presently under development for use as an antifouling compound and has been shown to successfully prevent settlement of barnacles at nanomolar concentrations [START_REF] Dahlström | Surface active adrenoceptor compounds prevent the settlement of cyprid larvae of Balanus improvisus[END_REF]. Much is known about mammalian effects [START_REF] Macdonald | Behavioural and neurochemical effects of medetomidine, a novel veterinary sedative[END_REF][START_REF] Scheinin | Medetomidine -a novel α 2 -adrenoceptor agonist: A review of its pharmacodynamic effects[END_REF][START_REF] Vainio | Cardiovascular and respiratory effects of medetomidine in dogs and influence of anticholinergics[END_REF][START_REF] Kuusela | Clinical effects and pharmacokinetics of medetomidine and its enantiomers in dogs[END_REF] and pharmacokinetics [START_REF] Salonen | Pharmacokinetics of medetomidine[END_REF][START_REF] Kuusela | Clinical effects and pharmacokinetics of medetomidine and its enantiomers in dogs[END_REF][START_REF] Ranheim | Medetomidine and atipamezole in sheep: disposition and clinical effects[END_REF] of medetomidine. Effect studies have also been conducted on fish [START_REF] Bellas | Sublethal effects of a new antifouling candidate on lumpfish (Cyclopterus lumpus L.) and Atlantic cod (Gadus morhua L.) larvae[END_REF][START_REF] Ruuskanen | Expression and function of α 2 -adrenoceptors in zebrafish: drug effects, mRNA and receptor distributions[END_REF][START_REF] Lennquist | Alteration of CYP1A activity in fish as a response to medetomidine exposure[END_REF][START_REF] Hilvarsson | Medetomidine as a candidate antifoulant: Sublethal effects on juvenile turbot (Psetta maxima L.)[END_REF][START_REF] Lennquist | Effects of medetomidine on hepatic EROD activity in three species of fish[END_REF] and invertebrates [START_REF] Bellas | Effects of medetomidine, a novel antifouling agent, on the burrowing bivalve Abra nitida (Müller)[END_REF][START_REF] Krång | Effects of a candidate antifouling compound (medetomidine) on pheromone induced mate search in the amphipod Corophium volutator[END_REF]. However, to our knowledge very little is known about bioaccumulation processes in aquatic organisms.

Information on bioaccumulation and the risk of biomagnification of a chemical is essential when conducting a risk assessment. Accumulation of a contaminant in the aquatic environment is determined by its physical and chemical properties as well as availability and persistence in water and the type of food chains exposed. The amount of bioaccumulation is often correlated to the octanol-water partitioning coefficient (K ow ) with a linear relationship between bioaccumulation and K ow for most substances below log K ow 6. Highly hydrophobic substances (log K ow > 6) do not display this correlation since bioaccumulation is limited by molecule size.

The indicators traditionally used for bioaccumulation are the bioconcentration factor (BCF) or the bioaccumulation factor (BAF). The factors are used as a measure of how much of a certain contaminant an organism can absorb from the environment. BCF is defined as the ratio of the contaminant in the tissue of an organism to the concentration in the water, and BAF as the ratio of the contaminant in the tissue to the concentrations in any compartments (such as water, sediment, food) relevant for the main uptake routes from the environment. Both BCF and BAF are measured at steady-state conditions [START_REF] Spacie | Bioaccumulation and bioavailability in multiphase systems[END_REF] and most commonly presented as wet weight with the unit l/kg [START_REF] Arnot | A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms[END_REF] which is also used throughout this article. The criteria set by the European Chemicals Bureau for BCF states that a substance with a value above 2000 is bioaccumulative (Technical Guidance Document on Risk Assessment EUR 20418 EN/2).

However, there are many more assessment endpoints from regulatory agencies such as the US Environmental Protection Agency, Environment Canada and the United Nations with BCF values ranging from 1000 to 5000 [START_REF] Arnot | A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms[END_REF]. These values do however only concern wet weight that causes some problems when working with systems like periphyton communities that are commonly measured as dry weight due to a high proportion of interstitial water. A BCF based on dry weight gives extremely high values and cannot be compared with fresh weight based BCFs. One way to normalise this difference is to express all values as lipid weight based concentrations since lipid content is one of the most important factors determining uptake of lipophilic compounds. Other factors like the amount of organic carbon in the organisms can however also influence accumulation, especially for primary producers. Normalisation to lipid content would then cause an overestimation of the bioaccumulation so to choose a normalisation method that suite all test species or systems can be difficult [START_REF] Berglund | Persistent organic pollutants in river food webs: influence of trophic position and degree of heterotrophy[END_REF]. In this study no normalisations were performed.

Medetomidine has a QSPR-estimated log K ow of 3.13 (pH 8, QSPR (Quantitative Structure Pharmacokinetic Relationship)). It is intermediately hydrophobic and may bioaccumulate (log K ow > 3) according to the Technical Guidance Document on Risk Assessment (EUR 20418 EN/2). Because of the imidazole group in medetomidine, a substantial amount of the compound is likely to be adsorbed to sediment particles [START_REF] Sundberg | Interactions of histidine and other imidazole derivatives with transition metal ions in chemical and biological systems[END_REF][START_REF] Handa | Antifouling agent release from marine coatings-ion pair formation/dissolution for controlled release[END_REF] and might therefore constitute a risk especially to sediment-living and sediment-eating organisms. However, a log K ow below 4.5 is not enough to classify a substance to be bioaccumulative based on K ow values alone (Technical Guidance Document on Risk Assessment EUR 20418 EN/2).

The present study aims to investigate the bioaccumulation of medetomidine in a selection of marine aquatic species to increase the knowledge of the substance behaviour. The different test organisms are chosen to get a broad understanding on the potential accumulation in the marine environment, from microbiota in periphyton communities, to filter-(blue mussel, Mytilus edulis) and sediment-feeders (Abra nitida). A crustacean, the brown shrimp (Crangon crangon), was also included since medetomidine is known to have effects on other crustaceans, i.e. barnacles. They were also selected because they are key species in their individual habitats and very abundant [START_REF] Wikander | Biometry and behaviour in Abra nitida (Müller) and A. longicallus (Scacchi) (Bivalvia, Tellinacea)[END_REF][START_REF] Pihl | Production, abundance, and biomass of mobile epibenthic marine fauna in shallow waters, Western Sweden[END_REF][START_REF] Gosling | The mussel Mytilus: Ecology, physiology, genetics and culture[END_REF]).

This study is part of a larger risk assessment study of medetomidine conducted within the research programme Marine Paint funded by the Swedish Foundation for Environmental Research (MISTRA).

Materials & methods

Biological material

Periphyton growing on glass discs (1.5 cm 2 ) mounted on polyethylene holders [START_REF] Blanck | Validity of an ecotoxicological test system -shortterm and long-term effects of arsenate on marine periphyton communities in laboratory systems[END_REF] were sampled from the coastal environment. The glass discs were gently cleaned with paper tissues on all sides except the colonized one and sorted to achieve a homogenous set of glass discs for uptake and elimination studies.

Blue mussels, Mytilus edulis, with an average length of 30 mm (± 5 mm) were collected by hand at a depth of approximately 0.5-1 m. Brown shrimps, Crangon crangon, with an average length of 30 mm (± 5 mm) were collected using a push-net at a depth of approximately 1 m.

Periphyton, Mytilus edulis and Crangon crangon were collected from bays nearby Sven Lovén Centre for marine sciences (formerly Kristineberg Marine Research Station) at the Gullmarsfjord, on the west coast of Sweden. Abra nitida with an average length of 13 mm (± 1 mm) and sediment (silty clay >85% <4µm, TOC 4%) were collected using a benthic sledge (Warén) from a depth of approximately 100 m in the Gullmarsfjord.

Experimental solutions

Medetomidine was 14 C-labelled in the methyl group directly bound to the chiral carbon.

The specific activity was 0.51 GBq/mmol. It was synthesised using 14 C-labelled alkyl substituted bromobenzene as starting material to prepare a Grignard reagent and supplied by the Marine Paint Formulation project. The medetomidine stock solution was dissolved in ethanol and experimental solutions were made by dissolving the stock solution in filtered seawater (GF/F for periphyton, 12 µm for the other test species) to a nominal concentration of 20 µg/l (100 nM). The concentration was chosen to achieve a 14 C-level above the detection limit but below a medetomidine concentration that cause physiological effects in the test organisms. The concentration is not related to predicted environmental concentrations (PEC), it is approximately 5000 times higher than PEC values derived by modeling (unpublished data, Ohlauson C).

Spiking of sediment

Sediment was spiked by mixing 1.5 kg (wet weight) of sieved sediment (0.5 mm mesh size) with 1.5 l of experimental medetomidine solution. The mixture was rotated in darkness (15°C) for 24 h in a tumbler (Rotary tumbler Model QT66, Lortone, inc., Seattle, WA) before start of the experiment.

Exposure and sampling

Exposure of periphyton communities to 14 C-labelled medetomidine was performed in glass containers (10x15x5 cm) with 300 ml of test medium placed on a shaker (60 rpm) at 25 µmol photons x m -2 x s -1 with a 13:9 light dark regime and a constant temperature of 18°C). Five replicate containers were used each with 20 discs. After 48 hours of uptake the discs were rinsed and transferred to clean containers with uncontaminated water for another 48 hours to study elimination. To avoid reuptake of 14 C-labelled medetomidine the water was changed again after 30 minutes. All solutions were changed every 24 hours. Samples were taken after 30 s, 1, 16 and 32 min (n=3) and at 1, 4, 8, 16, 24 and 48 hours (n=5), both during exposure to 14 C-medetomidine and uncontaminated water. The sampled disc was immediately rinsed by quickly dipping it in water three times to eliminate 14 C-medetomidine trapped in the interstitial water. The disc was then put into a 20 ml glass scintillation vial with the periphyton-colonised side upwards. Since fresh weight is difficult to estimate with good precision due to trapped interstitial water periphyton dry weight was measured at time 0 and after 48 hours in uncontaminated water (n=3). Discs were dried to constant weight (± 1%) at 70°C and weighed. The periphyton biofilm was dissolved by incubating each disc in 0.5 ml HNO 3 at 100°C for 30 minutes. When the samples reached room temperature 0.5 ml H 2 O 2 was added and the discs were incubated at 100°C for 30-60 minutes. The clean glass discs were rinsed in deionised water, dried in a fume hood and weighed again. When needed, periphyton dry weight was transformed to wet weight by multiplying with a factor of 12.5 [START_REF] Sladecek | Relationship between wet weight and dry weight of the periphyton[END_REF][START_REF] Sladecek | Determination of the periphyton production by means of the glass slide method[END_REF].

For the Crangon, Mytilus and Abra experiments, three replicates were used as well as controls without medetomidine to control for mortality throughout the experimental period. Mytilus edulis and Crangon crangon were exposed to 14 C-labelled medetomidine for 8 days in glass or plexiglass aquaria containing 2.5 l of experimental solution. The shrimps were individually separated in order to avoid cannibalism. No sediment or bottom gravel was added. Following exposure, they were kept in uncontaminated filtered seawater for an elimination period of 5 days. Solutions were changed every 24 hours.

Temperature was kept constant at 15°C and a 14:10 light dark regime was used. Samples were taken at 0, 1 day, 2, 4, 6, and 8 days of exposure and following 6 and 12 h, 1 day, 2, 3 and 5 days in uncontaminated water for the Mytilus experiment and at 0, 1 day, 2, 4, 6, and 8 days of exposure and following 6 h, 1 day, 2, 3 and 5 days in uncontaminated water for the Crangon experiment. Abra nitida was exposed to 14 C-labelled medetomidine for 2 days in glass aquaria containing one third of the spiked water-sediment mixture (0.5 kg sediment and 0.5 l experimental solution). Following exposure they were kept in uncontaminated filtered seawater and sediment for an elimination period of 5 days.

Experimental solutions were not changed during the exposure period of 48 hours but during the elimination phase water was changed every 24 hours. Temperature was kept constant at 10°C and a 14:10 light dark regime was used. Samples were taken at 0, 3, 6 and 12 h, 1 and 2 days of exposure and following 3, 6 and 12 h, 1, 2, 3, 4 and 5 days in uncontaminated water. One specimen was taken from each replicate at each sampling occasion.

Tissue preparation

Periphyton samples were prepared for scintillation counting in a similar way as for photosynthesis measurements [START_REF] Blanck | Validity of an ecotoxicological test system -shortterm and long-term effects of arsenate on marine periphyton communities in laboratory systems[END_REF] i.e. dissolved with 1 ml of dimethylsulfoxide and left for at least 10 min before adding 9 ml of scintillation cocktail (Ready Safe™, Beckman Instruments). Vials were shaken vigorously to obtain homogeneous samples prior to scintillation counting the next day. For brown shrimp and Abra nitida whole animals, without exoskeleton or shells, were used. The soft tissue from blue mussel was homogenised and a subsample of approximately 150 mg from each mussel was used. The tissue of the experimental animals was placed in scintillation vials and 2 ml of Soluene® (PerkinElmer) was added to each sample. All vials were placed in an incubator (50°C, Heidolph incubator 1000 polymax 1040) and gently revolved until the tissue was completely dissolved. 10 ml of scintillation cocktail (Hionic-Flour, PerkinElmer) were added, samples were shaken and allowed to stand over night before scintillation counting.

Water and sediment samples

From the periphyton experiment water samples were taken for scintillation counting (0.5 ml) at 1, 16, 32 min and 1, 4, 8, 16, 24 and 48 hours (n=1), both during exposure to 14 Cmedetomidine and uncontaminated water. 9 ml of scintillation cocktail (Ready Safe™, Beckman Instruments) was then added to each sample.

Water samples (2 ml) for later scintillation counting were also taken from the Mytilus and Crangon experiments at the start of the experiment and after 1 day (before solution change), 1 day (after solution change), 2, 3, 4, 5, 6, 7 and 8 days (n=3) and from the Abra experiment, at the start of the experiment and after 1 and 2 days (n=3). Sediment samples (100 mg) were taken at the start of the experiment and after 1 and 2 days (n=3). 10 ml of scintillation cocktail (Insta-gel Plus, Chemical Instruments) were added to water and sediment samples. All samples were shaken and allowed to stand over night before scintillation counting.

Results

The concentration of 14 C-labelled medetomidine reached steady state in the periphyton communities after 48 hours with a rapid uptake during the first 8 hours of exposure followed by a stabilisation after 24 hours (Figure 1). After the first 8 hours the concentration in periphyton was 15.5 µg/g fresh weight (FW) (77.2 nmoles/g) and at steady state the concentration was 22.7 µg/g FW (113 nmoles/g). Mean water concentration was 19.0 µg/l (94.6 nM) during the uptake phase of the experiment, giving a bioconcentration factor of 1195 and 14941 if presented as l/kg dry weight (Table 1 andFigure 5). Elimination was much faster than the uptake with a half-life of less than 1 hour. The steady state level reached was 2.80 µg/g FW (14.0 nmoles/g) in periphyton giving a BCF of 722 at the end of the elimination period.

Steady state was reached within 24 hours of exposure for both Mytilus edulis (Figure 2) and Crangon crangon (Figure 3). The bioconcentration factor at the end of exposure is calculated to 134 and 2.8 respectively (Table 1). BCF did not change significantly over time (24 hours till 8 days) for either Mytilus or Crangon (Figure 6). Elimination of medetomidine was also rapid with a half-life of less than 6 h in Mytilus and 6-24 h in Crangon. The mean water concentration was 19.2 µg/l (95.8 nM) in the Mytilus experiment and 16.0 µg/l (80.0 nM) in the Crangon experiment. The tissue concentration in Crangon is lower on a few sampling occasions (48 and 96 h) compared to after 24 h exposure because of a lower water concentration than expected, but increased as soon as the water concentration increased (Figure 3). However, BCF values were stable between 1.5 and 2.8 (Figure 6). The concentration of medetomidine in the tissue of Abra nitida has probably not reached steady state after the exposure period of 48 hours (Figure 4 and 5), thus a correct BAF cannot be calculated. However, a ratio of the concentration in tissue after 48 hours and the concentration in all compartments (sediment and water) gives a factor of 2.6. This might be an underestimation compared to a true BAF for Abra nitida. The elimination half-life of medetomidine is approximately 96-120 h in Abra (Table 1). The mean water concentration in the Abra experiment was 0.55 µg/l (2.75 nM) and the mean sediment concentration 18.1 µg/kg (90.6 nmoles/g) demonstrating medetomidine's ability to adsorb to sediment particles.

No significant mortality (< 5%) and no difference between controls and treatments were observed in any of the experiments.

Discussion

Medetomidine is shown to have a maximal bioconcentration factor of 1195, which occurred in periphyton at steady state. The rate of uptake is quite high in periphyton communities compared to other test species. This is probably due to periphyton's large surface area and high lipid content [START_REF] Wang | The response of a laboratory stream system to PCB exposure: Study of periphytic and sediment accumulation patterns[END_REF]. Whether medetomidine is rapidly mobile in and out of the essentially unicellular periphyton organisms, or only adsorbed to the surface cannot be conclusively clarified in the present study. However, the fit to a two-compartment bioconcentration model [START_REF] Newman | Quantitative methods in aquatic ecotoxicology[END_REF] is consistent with a surface adsorption with a rapid first phase followed by a secondary slower accumulation (data not shown). The rapid elimination (t 1/2 < 1 h) implies as well that most of the medetomidine is adsorbed and only a small fraction is taken up by the algal cells since the concentration decreased immediately when exposure ended. The medetomidine remaining in periphyton after 48 hours is 79 nmoles/g dry weight corresponding to 2.3 pmoles per square centimeter.

For brown shrimp a considerably lower bioconcentration factor (2.8) is found compared to blue mussel (BCF 134). A similar result has previously been observed in these two species when exposed to nonylphenol [START_REF] Ekelund | Bioaccumulation of 4nonylphenol in marine animals -a re-evaluation[END_REF], demonstrating again that bioaccumulation is species dependent, with a BCF of 100 for brown shrimp and 3400 for blue mussel. As a consequence, bioaccumulation should always be studied in several species to get the full range of possible bioconcentration and bioaccumulation factors.

The experiment with Abra nitida suggests that it may take longer time to reach a steadystate condition in sediment systems and the elimination of medetomidine also seems to be slower. One possible explanation for the slower uptake of medetomidine in A. nitida, compared to the other species tested, might be because of medetomidine's ability to interact with surrounding molecules, e.g. metals in the sediment [START_REF] Sundberg | Interactions of histidine and other imidazole derivatives with transition metal ions in chemical and biological systems[END_REF], and might therefore be transported through the gut of the organisms without being readily absorbed by the tissues. Further experiments with longer exposure periods and other sediment-eating organisms are needed to validate the results from this study which indicates that only minor uptake of medetomidine is through the sediment. However, the concentration used in the present study (20 µg/l, 100 nM) may have been high enough to impair the bivalves feeding behaviour thus decreasing the uptake of medetomidine.

Earlier studies have shown a decreased sediment reworking activity already at 10 nM but the amount of faeces produced were however not affected up to 375 nM indicating that the bivalves fed normally, but at a different layer in the sediment, when exposed to medetomidine [START_REF] Bellas | Effects of medetomidine, a novel antifouling agent, on the burrowing bivalve Abra nitida (Müller)[END_REF]. The animals used in the present study buried into the sediment and stayed buried throughout the experiment. However, feeding activity was not measured and the sediment was not removed from the gut prior to analysis.

Our results show, in agreement with mammalian data, a rapid uptake and elimination of the 14 C-labelled molecule in all test organisms, except for Abra. Medetomidine is biotransformed in the hepatic tissue in mammals and not more than 5 % is excreted as unchanged medetomidine [START_REF] Salonen | Pharmacokinetics of medetomidine[END_REF]. In vitro studies in rat show that medetomidine only have one major metabolite, hydroxymedetomidine, while three major metabolites and some minor metabolites were found in vivo. Two different pathways are suggested after hydroxylation and they depend on the concentration of medetomidine. None of the major metabolites have α -receptor activity, thus this transformation removes the active molecule [START_REF] Salonen | Biotransformation of medetomidine in the rat[END_REF]. However, the study indicates that methylation might occur to a very minor extent which could produce a metabolite that would be slowly eliminated and thus accumulate in fatty tissues during long time exposure [START_REF] Salonen | Biotransformation of medetomidine in the rat[END_REF]. Metabolites are not analysed in the present study and it cannot be entirely excluded that a small amount of the N-methylated conjugate is formed.

Further studies with collection of excretion products and analysis of metabolites are needed to investigate whether this potentially bioaccumulating metabolite is produced in invertebrates as well. The medetomidine used in this experiment was also only 14 Clabelled in one position, which makes it impossible to distinguish between intact medetomidine and metabolites containing the labelled carbon. For further studies medetomidine with several 14 C-labels will be used.

A comparison of medetomidine to other antifouling substances regarding bioaccumulative potential displayed varying results. Tributyltin, TBT, which was prohibited for use in 2008 due to the environmental damages caused by the substance [START_REF] Champ | Economic and environmental impacts on ports and harbors from the convention to ban harmful marine anti-fouling systems[END_REF], has a octanol-water partitioning coefficient in the same range as medetomidine with log K ow of 3.74 [START_REF] Laughlin | Tributyltin in seawater: speciation and octanol-water partitioning coefficient[END_REF]. BCF for TBT was also in the same range as for medetomidine regarding algae with values above 1000. For filtrating molluscs the BCF for TBT was however much higher, with values above 100 000 reported (Ranke and Jastorff 2000 and references therein). Studies have also demonstrated that log K ow is not a good predictor of bioconcentration or bioaccumulation for TBT with experiments resulting in much higher BCF or BAF than predicted [START_REF] Huang | Effects on tributyltin chloride on marine bivalve mussels[END_REF]. Another antifouling biocide, Irgarol 1051 , have log K ow values between 2.8 and 3.95 recorded. Like TBT and medetomidine Irgarol also has a rather high affinity for algae and water plants with BCFs of at least 1000 published. Other organism groups have lower BCFs for Irgarol, with values around 200 reported for fish (Ranke and Jastorff, 2000 and references therein). Both TBT and Irgarol are good examples that log K ow does not display the full risk of bioaccumulation of a substance.

Experimental studies of several organism groups are necessary to separate substances with a high potential to accumulate from the other non-accumulative substances with similar octanol-water partitioning coefficients.

Conclusion

Medetomidine seems to be a new antifouling compound with rapid elimination and a BCF below the bioaccumulative classification level (BCF<2000) set by the European Chemicals Bureau (Technical Guidance Document on Risk Assessment EUR 20418 EN/2). However, further studies on degradation and metabolites are necessary before a complete risk assessment can be produced.

ACCEPTED MANUSCRIPT

  modelling. The research was funded through the research programme Marine Paint, by the Swedish Foundation for Strategic Environmental Research, MISTRA. Additional grants were received from Birgit and Birger Wåhlström foundation.

  Figure captions

Figure 2

 2 Figure 2Bioaccumulation in Mytilus edulis exposed to a nominal concentration of 20 µ g/l (100

Figure 3

 3 Figure 3Bioaccumulation in Crangon crangon exposed to a nominal concentration of 20 µ g/l (100

Figure 4

 4 Figure 4Bioaccumulation in Abra nitida exposed to a nominal concentration of 20 µ g/l (100 nM)

Figure 5

 5 Figure 5

Figure 6

 6 Figure 6

  Figure 1

Table 1

 1 Bioconcentration factors in organisms exposed to medetomidine. Mytilus edulis and Crangon crangon steady-state was reached within the exposure period but probably not for Abra nitida

	Parameter	Community/Species
		Periphyton Mytilus edulis Abra nitida Crangon crangon
	Mean medetomidine
	conc. in water (g/l)	19.0	19.2	0.55	16.0
	conc. in water (nM)	95	96	2.75	80
	Mean medetomidine conc. in sediment (g/kg)	-	-	18.1	-
	Conc. of medetomidine in
	tissue at end of exposure a	22.66	2.54	0.049	0.044
	(g/g FW)
	Bioconcentration factor (l/kg FW)	1195 (14941) b	134	2.6 c	2.8
	Estimated half-life t ½ (h) <1	<6	96-120	6-24
	b based on dry weight
	c bioaccumulation factor calculated without steady-state reached

a for periphyton communities,

Table 1

 1

Acknowledgements

We wish to thank Magnus Nydén and Paul Handa in the Marine Paint Formulation project for providing us with medetomidine. Emma Nohrén and Linda Svanberg are acknowledged for technical assistance and Thomas Backhaus for periphyton curve