Eduardo Teixeira Da Silva

Michael Ridd
email: michael.ridd@jcu.edu.au

David Klumpp
email: d.klumpp@aims.gov.au

The barnacle Balanus amphitrite

Keywords: Balanus amphitrite, biomonitor, Cadmium-109, uptake rate, assimilation efficiency

Introduction

A critical aspect when modelling ecotoxicological processes involving heavy metals is to obtain reliable estimates of the parameters that control metal uptake and accumulation in organisms. Manipulative experiments using radioisotopes are among the best methods to obtain these [START_REF] Wang | Assimilation efficiencies of chemical contaminants in aquatic invertebrates: A synthesis[END_REF], and there have been many studies determining assimilation efficiency (AE), influx and efflux rates 1 for Cd and other metals by organisms (Lee and Luoma, 1998;[START_REF] Wang | Assimilation efficiencies of chemical contaminants in aquatic invertebrates: A synthesis[END_REF]Wang, Qiu and Qian, 1999a;Wang, Qiu and Qian, 1999b;[START_REF] Qiu | Contribution of dietary bacteria to metal accumulation in the slipper limpet[END_REF][START_REF] Rainbow | Comparative assimilation of Cd, Cr, Se, and Zn by the barnacle Elminus modestus from phytoplankton and zooplankton diets[END_REF][START_REF] Ke | Trace metal ingestion and assimilation by the green mussel Perna viridis in a phytoplankton and sediment mixture[END_REF][START_REF] Rainbow | Effects of previous field-exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite[END_REF].

Current work on marine pollution has pointed out the importance of a systemic approach with a predictive capability to support environmental risk assessments for contaminants, especially for heavy metals [START_REF] Jørgensen | Fundamentals of ecological modelling[END_REF][START_REF] Poethke | Modelling effects of toxicants on pelagic food webs: Many problems -some solutions[END_REF][START_REF] Bradbury | Ecological risk assessment for chemical stressors: Challenges in predictive ecotoxicology research[END_REF][START_REF] Brock | Assessing chemical stress in aquatic ecosystems: remarks on the need of an integrative approach[END_REF][START_REF] Luoma | Metal uptake by phytoplankton during a bloom in South San Francisco Bay: Implications for metal cycling in estuaries[END_REF]Wang et al., 1999b). With this in mind, we carried out research addressing the need to understand how short-term variations in Cd concentrations in the environment determine its concentrations in the barnacle biomonitor Balanus amphitrite [START_REF] Da Silva | Relative contribution of food and water to the Cd burden in Balanus amphitrite in an urban tidal creek discharging into the Great Barrier Reef lagoon[END_REF][START_REF] Da Silva | Cadmium accumulation in the barnacle biomonitor Balanus amphitrite: Combining field and laboratory observations with modelling[END_REF].

Following on from these previous researches, a series of radiolabelled experiments was carried out in the present study, in order to ultimately develop a biodynamic ecotoxicological simulation model to study Cd accumulation in the barnacle biomonitor Balanus amphitrite in the natural environment (da Silva et al., in press). The Cd uptake rate constant from the dissolved phase, Cd AE from suspended particulate matter (SPM) and the efflux rate constant were obtained for Balanus amphitrite with experiments using 109 Cd. Even though some of these parameters have been published for Balanus amphitrite (e.g., Wang et al., 1999b;[START_REF] Rainbow | Effects of previous field-exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite[END_REF][START_REF] Rainbow | Acute dietary pre-exposure and trace metal bioavailability to the barnacle Balanus amphitrite[END_REF], in the present study the Cd concentrations used in the dissolved phase were more realistic for the environment being studied, where maximum dissolved Cd concentration is in the order of 400 ng L -1 . Moreover, quality of food may change the metal accumulation in an organism if there are different 1 Assimilation efficiency is the ingested material (or the influx of material into the organisms) less faeces, and it can be calculated as: [influx-faeces]/influx. Faeces elimination represents a rapid loss of some unassimilated metals that are purged from the gut (extracellular digestion). Efflux represents metabolic loss of assimilated metals [START_REF] Wang | Assimilation efficiencies of chemical contaminants in aquatic invertebrates: A synthesis[END_REF]. assimilation efficiencies between sources of food (Lee and Luoma, 1998;Wang et al., 1999a;Wang et al., 1999b;[START_REF] Ke | Trace metal ingestion and assimilation by the green mussel Perna viridis in a phytoplankton and sediment mixture[END_REF]. For example, bivalves have exhibited higher metal AE when suspended particles are enriched with phytoplankton (e.g., Lee and Luoma, 1998;[START_REF] Ke | Trace metal ingestion and assimilation by the green mussel Perna viridis in a phytoplankton and sediment mixture[END_REF][START_REF] Wang | Combined effects of food quantity and quality on Cd, Cr, and Zn assimilation to the green mussel, Perna viridis[END_REF]. Therefore, in the present study the Cd AE from SPM for Balanus amphitrite was determined working with the whole assemblage of diverse particles that comprise the natural environment of suspended particulate matter.

Methodology

Organism Collection and Acclimatization

Balanus amphitrite were collected, during the Austral Spring, from concrete bridge piers in Ross Creek, a tidal creek surrounded by the city of Townsville (19º15'S,146º50'E,Queensland,Australia). During the collection the water temperature and salinity were 26.2°C and 37.3, respectively. A complete description of the sampling area can be found in da [START_REF] Da Silva | Relative contribution of food and water to the Cd burden in Balanus amphitrite in an urban tidal creek discharging into the Great Barrier Reef lagoon[END_REF]2005). The barnacles were removed from the piers with stainless steel chisel.

Several of them were attached to dead barnacles or oysters. After collection, the organisms were directly transferred to a constant temperature room for preparation and the acclimatization period before the experiments. The barnacles were individualized when possible, cleaned and brushed to remove any other live organism. About 30 -40 barnacles, as individuals or groups of 2 -4 barnacles, were placed into 300-mL HDPE plastic chambers for acclimatization, without any substrate for attachment other than the walls and the bottom of the chambers, and fed with natural particles in seawater pumped from Ross Creek. A manually operated diaphragm pump was used to reduce any damage to the plankton community, thus keeping the food supply as close as possible to the one found in Ross Creek.

During the 17-day acclimatization period, the water temperature was 24 ± 1°C (mean ± range of variation), salinity was 37 ± 2 (mean ± range of variation), and barnacles were inspected daily for any epibiota and brushed if necessary. When salinity acclimatization was also needed, another 11-day acclimatization period was used. In this period, the organisms were acclimatized, with gradual changes of less than four units per day, to three salinities, 27.3, 32.3 and 37.3 (all ranging ± 2), which is in the range observed in Ross Creek. The lower salinities were obtained by mixing tap water with Ross Creek water in the appropriate proportion in a flow-through system, in order to achieve a required salinity and constant food supply.

ACCEPTED MANUSCRIPT

Study of Cd accumulation in the barnacle biomonitor Balanus amphitrite -Page 5 of 17

Radioisotope and Measurements

The radioisotope 109 Cd (half-life: t 1/2 = 462 d) was obtained from Amersham Biosciences UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, England) as CdCl 2 in 0.1 M hydrochloric acid with a total activity of 37.3 MBq in a volume of 1.26 mL containing 2.14 µg of 109 Cd. The activity of 109 Cd in the organisms was measured by a COBRA II -Auto-Gamma (Packard, Model D5002) gamma detector. The counting time was 2 minutes at an energy level of 15-30 keV, in order to maximize the number of counts per minute (CPM), produce propagated counting errors <5%, and keep counting time short enough to manage counting for the AE experiment. The measured radioactivity was transformed to 109 Cd mass in the organisms by comparison with standards of known 109 Cd mass and corrected for decay.

Cadmium Uptake Rate from the Dissolved Phase

The Cd uptake rate from the dissolved phase for Balanus amphitrite was determined under different Cd concentrations (referred to as the 'Cd concentration experiment') and salinities (referred to as the 'salinity experiment'). In the 'Cd concentration experiment' three acid leached 1-L HDPE Nalgene containing 500 mL of 0.45-µm filtered Ross Creek water (salinity 37.3) were prepared with final Cd concentrations of 106, 268 and 407 ng L -1 . These Cd concentrations were determined by Anodic Stripping Voltammetry (method presented elsewhere, da [START_REF] Da Silva | Relative contribution of food and water to the Cd burden in Balanus amphitrite in an urban tidal creek discharging into the Great Barrier Reef lagoon[END_REF]2005), 1 day after the medium preparation, to allow saturation of all adsorption sites in the bottles. These Cd concentrations were obtained by spiking the filtered Ross Creek water with 5 ng of 109 Cd (86.37 kBq). As the initial Cd concentration in the filtered Ross Creek water was 16 ng L -1 , the necessary amount of natural

Cd was added to it to obtain the desired concentrations. Seven organisms were placed into each bottle and kept for 24 hours, under 12:12 h light/dark cycle, at a temperature of 24 ± 1 °C (mean ± range of variation), salinity 37.3, and constant aeration. [START_REF] Rainbow | Effects of previous field-exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite[END_REF]2004) have shown that the Cd uptake rate from the dissolved phase by Balanus amphitrite is In the 'salinity experiment' a similar procedure as described for the 'Cd concentration experiment' was adopted, except that three acid leached 1-L Nalgene bottles were produced with 500 mL of salt water with salinities 27.3, 32.3 and 37.3 obtained by dilution with distilled water. Each batch presented a total Cd concentration of 369 ± 11 ng L -1 (mean ± range), which was obtained by adding 5 ng of 109 Cd (86.37 kBq) plus the necessary amount of natural Cd. Seven organisms were placed into each bottle and kept for 24 hours, under 12:12 h light/dark cycle, at a temperature of 24 ± 1 °C (mean ± range of variation), and constant aeration. The Cd uptake rate was calculated as indicated by Eq. 1.

Cadmium AE from SPM

In , at a temperature of 27 ± 1 °C (mean ± range of variation), salinity 36.6, and constant stirring. The radiolabelling procedure lasted five days, a time that the literature has identified as being enough to allow uniform radiolabelling of the particles (see, e.g., Wang et al., 1999b;[START_REF] Ke | Trace metal ingestion and assimilation by the green mussel Perna viridis in a phytoplankton and sediment mixture[END_REF][START_REF] Wang | Combined effects of food quantity and quality on Cd, Cr, and Zn assimilation to the green mussel, Perna viridis[END_REF][START_REF] Rainbow | Acute dietary pre-exposure and trace metal bioavailability to the barnacle Balanus amphitrite[END_REF]. During this period, SPM was removed daily from the medium by filtration (0.45µm Millipore filter), resuspended in fresh 500 mL of 0.45-µm filtered Ross Creek water and spiked with 25.5 ng of 109 Cd (4.4 kBq). This procedure was adopted instead of using culture mediums such as f/2 (e.g., [START_REF] Guillard | Culture of phytoplankton for feeding marine invertebrates[END_REF] to minimize any change in the particles assemblage, since the objective was to work with the natural SPM from the barnacle's environment.

After the radiolabelling procedure, each batch of radiolabelled SPM was filtered and transferred into 500 mL of fresh 0.45-µm filtered Ross Creek water, before being offered to the barnacles. This procedure was done twice, for each SPM batch, with 12-h intervals between them. This was done in order to guarantee that most of . These 109 Cd concentrations were obtained from a small amount of SPM of each batch retained on 0.45-µm filters and assayed for their activity.

The ´labelled´ phase

In the determination of the AE of Cd in Balanus amphitrite from SPM, was determined using the pulse-chase feeding technique (see, e.g., [START_REF] Wang | Assimilation efficiencies of chemical contaminants in aquatic invertebrates: A synthesis[END_REF]Wang et al., 1999b;[START_REF] Rice | Standard Methods for the Examination of Water and Wastewater[END_REF]. During the 'labelled' phase, 28 organisms were placed in two acid leached 500-mL HDPE plastic chambers, with each chamber receiving radiolabelled SPM in a recirculating system from each SPM batch (total volume 4 L in each), under light, at a temperature of 24 ± 1 °C (mean ± range of variation), salinity 36.6, and constant aeration.

Recirculation was enough to keep particles in suspension. At the end of the radiolabelling procedure, each SPM batch, originally obtained from site 1 and 2, presented a SPM mass concentration of 15.6 and 20.3 mg L -1 , and a chl a concentration of 9.2 and 9.4 µg L -1 , respectively. Chlorophyll-a was collected in duplicate from each SPM batch onto 25-mm Whatman GF/F filters, using a Sartorius mini filter holder adapted to a 60-ml plastic syringe.

Pigment determinations from acetone extracts of filters were done with a Turner Design AU-10 fluorometer following [START_REF] Arar | Vitro Determination of Chlorophyll-a and Phaeophytin-a in Marine and Freshwater Phytoplankton by Fluorescence[END_REF]. The organisms were kept starved for 2 days before the 'labelled' phase to maximize the feeding activity. After 30-45 min feeding, before egestion of radioactive faeces, individual barnacles were rinsed with non-radiolabelled seawater and their radioactivity counted. The seven most radioactive organisms from each feeding batch were selected to take part in the 'non-labelled' phase.

The ´non-labelled´ phase

In

Cadmium Efflux Rate

In the determination of the efflux rate constant, a similar procedure as for the determination of the AE was adopted. At the end of the course of the experiment for the AE determination, the four organisms that exhibited the highest radioactivity in the last counting (i.e., at time 68.2 h) from both SPM batches were selected to be kept in the depuration chamber allowing further depuration up to 144 h. The Cd efflux rate constant was determined by applying an exponential decay model to the temporal variation of the percentage of remained radioactivity in each organism from 22h onwards, which is the time when all labelled food has passed through the gut of Balanus amphitrite [START_REF] Rainbow | Acute dietary pre-exposure and trace metal bioavailability to the barnacle Balanus amphitrite[END_REF][START_REF] Rice | Standard Methods for the Examination of Water and Wastewater[END_REF]. No moulting was observed at this stage of the experiment.

Results

Cadmium Uptake Rate from the Dissolved Phase

The Cd uptake rates (ng g -1 h -1 , Fig. 1) at three different dissolved Cd concentrations were determined for 21 individual barnacles. A linear model has been used to represent variations in the uptake rate as a function of the dissolved Cd concentrations (e.g., [START_REF] Rainbow | Effects of previous field-exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite[END_REF][START_REF] Rainbow | Acute dietary pre-exposure and trace metal bioavailability to the barnacle Balanus amphitrite[END_REF]. The best-fit least squares linear regression model passing through zero was applied to Figure 1, and its yielded a Cd uptake rate constant (k u ) of 0.0072 L g -1 h -1 , which is the slope of this fitting.

In the 'salinity experiment' (tested salinities were 27.3, 32.3 and 37.3) from the Cd uptake rates (ng g -1 h -1 ) for a dissolved Cd concentration of 369 ± 11 ng L -1 (C w , mean ± range) did not vary significantly over the three salinities tested (linear regression, r 2 = 0.01 and p = 0.90, data not presented).

Cadmium AE from SPM with Different Chlorophyll Concentrations

The Cd AEs showed a bi-phasic depuration pattern for both low and high chl a-containing SPM (Fig. 3), the ratios chl a (µg L -1 ):SPM (mg L -1 ) being equal to 0.48 and 0.59, respectively. The results show initial rapid loss of ingested 109 Cd labeled SPM within the first 4 h and then a slower loss up to 68.2 h (Fig. 2). Cadmium AEs were obtained separately for both low and high chl a-containing SPM. Each AE was obtained by fitting a single exponential decay curve to the second stage of depuration (i.e. considering only the data points within 4 -68.2 h of experiment, as in Wang, 1999), for all data from all barnacles. The fittings yielded two AEs of 39.0 and 48.7% for low and high chl a containing SPM, respectively. In each curve fitting, the 14 barnacles used in each experiment were considered together.

Cadmium Efflux Rate Constant

An efflux rate constant of 0.0072 d -1 was obtained by multiplying the x-coefficient of the exponential decay model (Fig. 3) by 24, thus transforming the x-coefficient from an hour basis to daily basis. The curve fitting was obtained as a single best fit line considering the data points, from 22 h onwards, for four of the most 109 Cd active barnacles after 68.2 h of the beginning of the AE.

Discussion

Cadmium Uptake Rate Constant from the Dissolved Phase

The calculated Cd uptake rate constant (k w ) for Balanus amphitrite of 0.0072 L g -1 h

-1

compares well with literature values of 0.012 and 0.008 L g -1 h -1 [START_REF] Rainbow | Effects of previous field-exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite[END_REF]2004, respectively) despite their much higher dissolved Cd concentrations of between 500-50000 ng L -1 compared to 100-400 ng L -1 of this study, and the fact that barnacles were dissected before the analysis (therefore mantle was not included). This fact suggests that Cd uptake from the dissolved phase is a linear process for Balanus amphitrite and its Cd absorption sites are not saturated even by very high Cd concentrations, such as 50000 ng L -1

. Even though the Cd uptake rate may vary between populations of Balanus amphitrite [START_REF] Rainbow | Effects of previous field-exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite[END_REF], which justify repeating this experiments for the barnacles in Ross Creek, these authors did not observe correlation between uptake rate from solution and accumulated metal concentration, a surrogate measure of previous contaminant history. Thus, the rates determined in this work are suitable for Balanus amphitrite living along the total Cd gradient in Ross Creek.

Variations in salinity may affect accumulation of Cd by complex organisms such as barnacles [START_REF] Phillips | Toxicity and accumulation of cadmium in marine and estuarine biota[END_REF]. Salinity acts on chemical speciation, thus changing the metal bioavailability, and making metals more or less available for uptake from solution [START_REF] Sunda | Effect of chemical speciation on toxicity of cadmium to grass shrimp, Palaemonetes pugio: Importance of free cadmium ion[END_REF]. For example, [START_REF] Rainbow | Effects of previous field-exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite[END_REF] found a significantly higher Cd uptake for Balanus amphitrite exposed to salinity 15 compared to 33. Similar results were found by Lee et al.

(1998) for bivalves (Potamocorbula amurensis and Macoma balthica) when salinity ranged from 5 to 30, but minor changes were observed in Cd uptake from 20 to 30. In fact, small changes in activity of the free Cd ion are expected in such high salinities, when a relative complete complexation of dissolved Cd [START_REF] Phillips | Toxicity and accumulation of cadmium in marine and estuarine biota[END_REF] is expected. Therefore, the similar range of salinity in this study (from 27.3 to 37.3), which is in the range that Balanus amphitrite experiences in Ross Creek, had no-significant effect on the Cd uptake rate from solution.

The organism's size and its filtration rate, have been shown to affect the metal uptake rate constant from the dissolved phase in bivalves (e.g., [START_REF] Wang | Modeling the influence of body size on trace element accumulation in the mussel Mytilus edulis[END_REF]Lee et al., 1998).

We performed a comparison between uptake rates and individual barnacles' dry weight to test whether the barnacles' size affects its Cd uptake rate from the dissolved phase. Such a comparison was based on the fact that metal uptake rates for a species can be compared after allowance for the dissolved metal concentration [START_REF] Rainbow | Effects of previous field-exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite[END_REF]. Comparing the individual barnacles dry weight (ranging from 1.1 to 5.8 mg) against the Cd uptake rates obtained for them in both the 'Cd concentration experiment' and the 'salinity experiment' revealed a non-significant correlation (linear regression, r 2 = 0.01 and p = 0.81, data not presented). These results suggest that the barnacles' size does not affect the Cd uptake rate from the dissolved phase. Thus, the Cd uptake rate from solution obtained here can be used for a wider size range of Balanus amphitrite.

Cadmium AE from SPM with Different Chlorophyll Concentrations

The AE determined in this study are within the range of other studies of Balanus amphitrite feeding on different types of living particles (phytoplankton and zooplankton, Table 1).

However, on average, lower AEs were found in our study when the barnacles were fed on SPM poor in chl a. This suggests that non-living particles in SPM may reduce the Cd AE, or conversely that AE increases with higher proportions of phytoplankton (chl a) in SPM. In a previous study, da [START_REF] Da Silva | Cadmium accumulation in the barnacle biomonitor Balanus amphitrite: Combining field and laboratory observations with modelling[END_REF] comparing Cd accumulation in barnacles form two sites with a steady-state model found that the highest phytoplankton abundance in SPM resulted in a higher Cd AE. Similar patterns have been identified in bivalves that exhibited higher metal AE when suspended particles are enriched with phytoplankton (e.g., Lee and Luoma, 1998;[START_REF] Ke | Trace metal ingestion and assimilation by the green mussel Perna viridis in a phytoplankton and sediment mixture[END_REF][START_REF] Wang | Combined effects of food quantity and quality on Cd, Cr, and Zn assimilation to the green mussel, Perna viridis[END_REF].

The ratio between chl a mass concentration (µg L -1 ) and SPM mass abundance (mg L -1 ) used in the AE experiments were calculated for both SPM batches and compared to the AE determined in the experiments. The same procedure was applied to the data presented in da [START_REF] Da Silva | Cadmium accumulation in the barnacle biomonitor Balanus amphitrite: Combining field and laboratory observations with modelling[END_REF]. These two set of data presented the same general tendency of higher AE was observed when SPM is enriched with chl a (Fig. 4). The best-fit least squares linear ).

ACCEPTED MANUSCRIPT

Study of Cd accumulation in the barnacle biomonitor Balanus amphitrite -Page 12 of 17

As pointed out by Lee and Luoma (1998), the radiolabelling procedure can be critical in the interpretation of pulse-chase feeding experiments using mixed-particles assemblage, because a short equilibrium time may result in preferential partitioning of isotopes to easily exchangeable pools. Distribution coefficients as a function of time for Cd have exhibited rapid removal from solution within the first few hours, followed by a slower removal over a period of a few days (Turner and Millward, 1994;[START_REF] Ciffroy | Kinetic partitioning of Co, Mn, Cs, Fe, Ag, Zn and Cd in fresh waters (Loire) mixed with brackish waters (Loire estuary): experimental and modelling approaches[END_REF].The similarity between the AE determined in the radiolabelled experiments and those suggested by the steady-state model, presented in da [START_REF] Da Silva | Cadmium accumulation in the barnacle biomonitor Balanus amphitrite: Combining field and laboratory observations with modelling[END_REF], indicates that a five-day labeling was enough time to produce Cd-labeled particles that behaved similarly to those found in the environment (same trend for the points in Fig. 4).

Cadmium Efflux Rate Constant

The efflux rate constant determined in this study (ca. 0.0072 d -1 ) agrees with efflux rate constants reported in the literature for Balanus amphitrite (Wang et al., 1999b;[START_REF] Rainbow | Effects of previous field-exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite[END_REF], except for an unusual high value presented in [START_REF] Rainbow | Acute dietary pre-exposure and trace metal bioavailability to the barnacle Balanus amphitrite[END_REF]. The low efflux rate constant confirms that Balanus amphitrite has potential as a biomonitor for Cd, since this barnacle retains most of the metal that is assimilated. This fact makes the barnacle a more appropriate biomonitor for registering long term trends in Cd concentration in the environment rather than short term variations.

Community vs Single Species Approach

The quality of food may change the metal accumulation in an organism if there are different assimilation efficiencies between sources of food as seen in this study and elsewhere (Lee and Luoma, 1998;Wang et al., 1999a;Wang et al., 1999b;[START_REF] Ke | Trace metal ingestion and assimilation by the green mussel Perna viridis in a phytoplankton and sediment mixture[END_REF]. From our AE experiment, variations in the composition of the food particles (e.g., abundance of chl a)

affected the Cd accumulation in Balanus amphitrite, and it is therefore important to take this into account in kinetic models. In addition, when applying a systemic approach to understand Cd accumulation in aquatic organisms, it must be considered that: (i) different food particles may exhibit temporal and spatial variability that can change the relative Cd contribution to the organism's burden, and (ii) organisms can selectively eat different sources of food, which can present not only a specific Cd concentration but also different assimilation efficiencies. Thus, the variation in the quantity and quality of the ingested food may result in changes in Cd concentrations in organisms.

Therefore, the accuracy of ecotoxicological models for metal accumulation in organisms depends on how representative the selected food items are of the organism's natural diet [START_REF] Qiu | Contribution of dietary bacteria to metal accumulation in the slipper limpet[END_REF], because each item may present a specific AE. Usually, two feasible options can be adopted on the determination of AE from laboratory experiments: (i) to select a small number of species (Wang et al., 1999b;[START_REF] Wang | Dietary uptake of Cd, Cr, and Zn by the barnacle Balanus trionus: Influence of diet composition[END_REF][START_REF] Rainbow | Comparative assimilation of Cd, Cr, Se, and Zn by the barnacle Elminus modestus from phytoplankton and zooplankton diets[END_REF] or (ii) to work with the 'in situ' food (Lee and Luoma, 1998;[START_REF] Luoma | Metal uptake by phytoplankton during a bloom in South San Francisco Bay: Implications for metal cycling in estuaries[END_REF][START_REF] Ke | Trace metal ingestion and assimilation by the green mussel Perna viridis in a phytoplankton and sediment mixture[END_REF]. This paper presents the results of experiments designed to improve the parameterisation of an ecotoxicological model (da Silva et al., in press). Accordingly we decided to use a natural food source, including the whole array of particles usually present in SPM, which is expected to have a Cd AE similar to that for SPM in Ross Creek. e Wang et al. (1999b).

Figure Captions

f This study.

Table

  constant over time; therefore, a single exposure of 24 h was used in all experiments. At the end of the exposure period, the organisms were rinsed with non-labelled seawater, and the barnacle's soft tissue was removed from shells (i.e., all soft parts less the mantle -' bodies' in previous studies), rinsed with distilled water, dried to a constant weight in a fume cupboard, and assayed for the 109 Cd activity. The hourly Cd uptake rate from the dissolved phase (ng g -1 h -1 ) for each barnacle exposed to each tested dissolved Cd concentration was calculated as indicated below: b is the mass of 109 Cd in the barnacle's soft tissue (ng), C w is the natural Cd concentration in the dissolved phase (ng end of the 24-h exposure (ng L -1 ), and W b is the barnacle's soft tissue dry weight (g). Loss of Cd from the dissolved phase due to the organism's uptake, or due to adsorption to the container wall or organisms' shell, was insignificant (<2% between the beginning and the end of the experiment), ensuring an effectively constant Cd concentration during the experiment course.

  the determination of the AE of Cd in Balanus amphitrite natural suspended particulate matter (SPM, size-class of 0.45-50 µm, as defined in da Silva et al., 2004; da Silva et al., 2005) was used as 109 Cd-labeled food for Balanus amphitrite. Water for SPM collection was sampled from Ross Creek from two different locations, at which different phytoplankton abundances in SPM have been identified during previous sampling programs. SPM was obtained by passing • 4 L of 50-µm pre-filtered water through 0.45-µm Millipore filters and the retained material was resuspended in 500 mL of 0.45-µm filtered Ross Creek water. 2.4.1. Food Particles Radiolabelling Each SPM batch was radiolabelled in 500 mL of 0.45-µm filtered Ross Creek water (1-L Nalgene bottle) spiked with 25.5 ng of 109 Cd (4.4 kBq), under 14:10 h light/dark cycle under a light flux of the order of 100 µE m -2 s -1

109

  Cd was in SPM and not in solution. Low 109 Cd activities of <40 counts per minute (CPM) in 500 µL of 0.45-µm filtered medium (i.e., <1 ng 109 Cd L -1 ) were determined for each SPM batch just before being offered to the barnacles. The 109 Cd concentrations in each SPM batch were 5.3 and 5.5 ng( 109 Cd) g dry weight (SPM) -1

  the 'non-labelled' phase, the organisms were placed into new plastic chambers receiving water with SPM (0.45-50 µm) obtained from Ross Creek to promote depuration of the ingested radiolabelled SPM. The 'non-labelled' phase was run in a flow-through system, under 12:12 h light/dark cycle, at a temperature of 24 ± 1 °C (mean ± range of variation), salinity 36 ± 1 (mean ± range), and constant aeration. The 'non-labelled' phase for both batches lasted 68.2 h, in which the organisms were periodically removed from the depuration chambers, rinsed with non-radiolabelled seawater, their radioactivity counted, and placed back into the chambers. In addition, both depuration chambers were periodically rinsed with non-labeled seawater to remove any faeces thus avoiding any re-ingestion of egested labeled material.Counts for water from the depuration chambers presented an the dissolved phase. It was not possible to identify individual barnacles that had moulted and data for all moulters (<5% of the individuals for all experiments carried out) have been included in analyses.

  regression model was applied to Figure 4 yielding the following equation (r 2 = 0.77, p < 0.01, n = 6): y = 49.18 ⋅ x + 23.97, (2) where the y-coefficient represents the Cd AE from SPM for Balanus amphitrite (in %), and xcoefficient represents the chl a:SPM ratio (in µg chl a L -1 : mg SPM L -1

Figure 1 :

 1 Figure 1: The best-fit least squares linear regression model, passing through zero, between Cd uptake rates and dissolved Cd concentration obtained from Balanus amphitrite exposed to 109Cd for 24 h. The fitting yielded a Cd uptake rate constant (k u ) of 0.0072 L g -1 h -1 .

Figure 2 :

 2 Figure 2: The relationship of the percentage of retention of 109 Cd as a function of time for 14

Figure 3 :

 3 Figure 3: The relationship between the percentage of retention of 109 Cd as a function of time for the four most 109 Cd active barnacles after they were used in the experiment for the AE determination. Numbers in brackets denote the standard error of the parameter in the adjusted model. Different symbols denote each of the four barnacles. Marked white circles were not considered in the analysis because the barnacle was dead.

Figure 4 :

 4 Figure 4: The relationship between the Cd AE from SPM for Balanus amphitrite as a function of the ratio between chl a abundance (µg L -1 ) and SPM mass abundance (mg L -1) obtained from two independent measurements: 'estimated' from the steady-state model and field data (white circle), and 'measured' in the laboratory with 109 Cd and pulse chase feeding technique

a

  Activity remaining in the organism after a specific time had elapsed, compare to the initial activity. b Exponential decay model adjusted to the data of the temporal variation of the remained activity in the organism in a specific time interval. c[START_REF] Rainbow | Effects of previous field-exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite[END_REF].d[START_REF] Rainbow | Acute dietary pre-exposure and trace metal bioavailability to the barnacle Balanus amphitrite[END_REF].

Table 1 :

 1 The Cd AE from different food types for Balanus amphitrite.

	Food type	Time interval for AE determination	Mean AE ± 1 SD (%)	AE range (%) Source
	Phytoplankton
	Thalassiosira weissflogii	24 h a	35.2 ± 5.8	26.8-41.5	c
	Thalassiosira weissflogii	22 h a	59.6 ± 10.1	40.2-69.9	d
	Chaetoceros Muelleri	18-68 h b	34.8 ± 5.1		e
	Skeletonema costatum	18-68 h b	86.2 ± 8.2		e
	Zooplankton
	Artemia salina larvae	18-68 h b	63.4 ± 9.0	52.6-71.5	e
	Canthocalanus pauper	18-68 h b	71.6 ± 17.4		e
	Temora turbinata	18-68 h b	87.9 ± 8.

Acknowledgements

The authors thank Anna-Marie Babey and staff for their support on the experiments undertaken at the School of Biomedical and Veterinary Science -James Cook University, and Gillian Peck and staff from the Australian Nuclear and Technology Organization for the training and experience provided on the use of radioisotopes. E.T. da Silva acknowledges the receipt of a Scholarship to support this work from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).


Jaime Fernando González

Renate Reimschuessel

Badar Shaikh

Andrew S Kane

J F González
email: jfgonzalezma@unal.edu.co

Kinetics of hepatic phase I and II biotransformation reactions in 8 finfish species

Keywords: hepatic, phase I and II biotransformation reactions, finfish

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Phase I and II biotransformation reactions are of great importance to understand metabolism of endogenous molecules as well as transformation of xenobiotics and drugs in fish and other species [START_REF] Mansui | The great diversity of reactions catalyzed by cytochromes P450[END_REF][START_REF] Nelson | Metazoan cytochrome P450 evolution[END_REF][START_REF] Mortensen | Effects of 17 -ethynylestradiol on hormonal responses and xenobiotic biotransformation system of Atlantic salmon (Salmo salar)[END_REF][START_REF] Reynaud | Interactions between immune and biotransformation systems in fish: a review[END_REF][START_REF] Arukwe | Modulation of steroidogenesis and xenobiotic biotransformation responses in zebrafish (Dario rerio) exposed to water-soluble fraction of crude oil[END_REF]. However, biotransformation profiles are still unknown in several fish species of importance in aquaculture or as environmental bioindicators. Investigating these biotransformation profiles may help to understand capabilities, similarities and differences of these fish species to metabolize endogenous substrates, drugs and pollutants.

The substrates 7-ethoxycoumarin, 7-ethoxyresorufin, 7-pentoxyresorufin, 7-benzyloxyresorufin, resorufin and 1-chloro-2,4-dinitrobenzene are some of the model compounds used to investigate the kinetics of phase I and II reactions in fish. These substrates help to characterize the catalytic activity of various isoforms of phase I and II enzymes. The O-deethylation of 7 ethoxyresorufin (EROD activity) is a model reaction used to study the expression of CYP1A, an inducible cytochrome P450 isoform in fish [START_REF] Whyte | Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure[END_REF]. Other substrates and their respective phase I activities are used as model compounds as well (e.g. 7-ethoxycoumarin -ECOD-, 7pentoxyresoroufin -PROD -, 7-benzyloxyresoroufin -BROD -); however, the isoforms involved in such activities are either not determined yet in all fish species or represent the expression of different P450 isoforms depending on the species. Resorufin is a phase I metabolite obtained after EROD, that is used as substrate for phase II reactions such as glucuronidation and sulfation [START_REF] George | Enzymology and molecular biology of phase II xenobiotic-conjugating enzymes in fish[END_REF]). Glutathione-s-transferase activity, an important phase II activity performed to conjugate electrophilic compounds, is commonly assessed using reference substrates such as 1chloro-2,4-dinitrobenzene (CDNB) [START_REF] Habig | Glutathione S-transferases: the first enzymatic step in mercapturic acid formation[END_REF][START_REF] George | Enzymology and molecular biology of phase II xenobiotic-conjugating enzymes in fish[END_REF].

The purpose of this study was to compare the kinetics of specific phase I and II biotranformation reactions among 8 different finfish species of importance in the United States and around the world using the specific substrates aforementioned.

Materials and methods

Fish handling

Market-size specimens (n=8) of 8 fish species were obtained from commercial farms. After capture, the fish were sacrificed by cervical transection. The species used for this study were Ethoxyresorufin-o-dealkylation (EROD) (Modified from [START_REF] Eggens | Ethoxyresorufin-o-deethylase (EROD) activity in flatfish: fast determination with a fluoresecence plate reader[END_REF][START_REF] Haasch | Use of 7alkoxyphenoxazones, 7-alkyloxycoumarins and 7-alkoxyquinolines as fluorescent substrates for rainbow trout hepatic microsomes after treatment with various inducers[END_REF]. Reaction mixtures consisted of 50 L of buffer Tris-HCl (100 mM, pH=7.4), 25 L of microsomes (100 g of protein), 10 L of 7-ethoxyresorufin (range 0-2 M) and 25 L of NADPH (1 mM). EROD was monitored based on the RFUs of the resorufin produced by the reaction (excitation = 510 nm, emission = 580 nm). Pentoxyresorufin-(PROD) and benzyloxyresorufin-o-dealkylation (BROD) activities were assayed following the same protocol as for EROD. A 5 M substrate concentration was tested as the initial concentration point.

Phenobarbital-induced rat microsomes were used as a positive control. Glutathione-s-transferase (GST) kinetics was performed according to Habig & col. (1974). Reaction mixtures consisted of 165 L of 100 mM Tris-HCl buffer (pH=7.4), 7 L of 1-chloro-2,4-dinitrobenzene (CDNB) (0.1-2.0 mM range), 3.5 L of 60 mM reduced L-glutathione, and 10 g of cytosol. The rate of CDNB conjugation was monitored ( = 340 nm) for 5 minutes. For final calculations a molar absorption coefficient ( =9.6 mM -1 cm -1 ) was used adjusting the path length to the corresponding 96-well plate volume. UDP-glucuronosyltransferase (UDPGT) kinetics (Modified from [START_REF] Schell | Glucose and sulfate conjugation of phenolic compounds by the spiny lobster (Panulirus argus)[END_REF]. Reaction mixtures consisted of 50 L of phosphate buffer (pH= 7.4) with 0.1 % Triton X-100 and 10 mM MgCl 2 , 100 g of microsomal protein, 25 L of uridin 5'diphosphoglucuronic acid trisodium salt (UDPGA) and 10 uL of resorufin (1-50 M range).

Sulfotransferase (ST) kinetics (Modified from [START_REF] Schell | Glucose and sulfate conjugation of phenolic compounds by the spiny lobster (Panulirus argus)[END_REF][START_REF] Beckmann | Continuous fluorometric assay of phenol sulfotransferase[END_REF].

Reaction mixtures consisted of 50 L of phosphate buffer (pH= 7.4), 100 g of cytosolic protein, 10 L of resorufin (0.5-20 M range) and 25 L of adenosine 3'-phosphate 5'-phosphosulfate lithium salt (PAPS).

Calculation of kinetics parameters and statistical analysis

Results on velocity rates for all of the phase I and II enzymes were graphed as Michaelis-Menten (velocity vs. [substrate]) and Lineweaver-Burk (1/velocity vs 1/[substrate]) plots. Maximal velocity (V max ), Michaelis-Menten constant (K m ) and V max /K m ratio were derived by regression of the double-reciprocal plots [START_REF] Cornish-Bowden | Fundamentals of enzyme kinetics[END_REF]. Results are reported as means ± standard errors. Data sets were analyzed for normality and homogeneity of variances. Data being both normal and homogeneous were compared using one-way ANOVA test (comparison among species) followed by Tukey's mean separation test. SAS (Statistical Analysis Software, Cary, NC, USA) program was used for these data processing. Statistical significance was set at a significance level of = 0.05.

Results

ECOD and EROD kinetics. Channel catfish had the highest ECOD V max of all the species followed by Atlantic salmon, rainbow trout, hybrid striped bass and tilapia. This value was significantly different (p < 0.05) from all the others. K m was quite similar among Atlantic salmon, rainbow trout, channel catfish and tilapia. Hybrid striped bass K m was the highest value of all, being significantly different from the others (p < 0.05). V max /K m ratios in ECOD indicated that channel catfish and Atlantic salmon were the most efficient species of all (Table 1).

Rainbow trout and tilapia had lower V max /K m ratios and hybrid striped bass had the lowest V max /K m ratio showing the lowest ECOD efficiency of all the species. Bluegill microsomes did not present any response to 7-ethoxycoumarin. ECOD kinetics in striped bass and largemouth bass were not performed due to insufficient microsomal sample.

EROD kinetics parameters were done for five of the eight species examined in this study. Striped bass, hybrid striped bass and bluegill had very low velocity values (pmols resorufin/min/mg protein) at 1 M 7-ethoxyresorufin (4.0 ± 1.0, 2.8 ± 0.3, and 3.7 ± 1.0, respectively). Higher and lower substrate concentration values did not reveal changes in the velocity rate. Atlantic salmon (66.0 ± 7.0) and tilapia (74.0 ± 15.0) had the highest V max of all, followed by channel catfish (39.0 ± 7.0), rainbow trout (28.0 ± 8.0) and largemouth bass (27.0 ± 8.0). As far as K m is concerned, channel catfish (1.8 ± 0.5) and tilapia (2.1 ± 0.3) had higher values from those in largemouth bass (0.9 ± 0.1), rainbow trout (0.6 ± 0.07) and Atlantic salmon (0.2 ± 0.02) (p< 0.05). Atlantic salmon had the highest V max /K m ratio of this reaction as compared to tilapia, rainbow trout, largemouth bass and channel catfish (p < 0.05) (Table 1).

PROD and BROD kinetics. None of the fish species showed either PROD or BROD activities when a 5 M substrate concentration was tested for both alkoxycoumarins. Phenobarbial-induced rat microsomes that were used as positive controls, showed both PROD (23 pmols/ min / mg protein) and BROD (183 pmols / min / mg protein) activities.

Glutathione-s-transferase kinetics. The highest GST capacity was obtained for tilapia and Atlantic salmon, followed by rainbow trout, channel catfish and largemouth bass. The lowest V max values (p < 0.05) were found in striped bass, hybrid striped bass and bluegill. As for K m , channel catfish and striped bass showed the lowest values followed by bluegill, tilapia, and hybrid striped bass. The highest K m values were found in largemouth bass, rainbow trout and Atlantic salmon (Table 2). V max / K m ratios indicate that channel catfish and tilapia had the most efficient GST activity towards the substrate. Atlantic salmon and rainbow trout had lower ratios whereas striped bass, largemouth bass, bluegill and hybrid striped bass had the lowest GST efficiencies of all. 2).

For comparison purposes, Figure 1 shows the phase I and phase II reactions that were studied in the present work for the eight species.

Discussion

The present study was intended to compare the baseline kinetics of phase I and II biotransformation reactions among eight different fish species. Comparisons between present results and other studies were relatively difficult in some cases given that most of the investigations have dealt with catalytic activities at saturating conditions of substrates instead of full kinetics working a range of substrate concentrations.

ECOD and EROD kinetics. ECOD has been studied in mammals as a marker activity of CYP1A1, CYP1A2 CYP2B1, CYP2E1, and CYP2E6 [START_REF] Yun | Purification and characterization of human liver microsomal cytochrome P4502A6[END_REF][START_REF] Yamazaki | 7ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes[END_REF].

In fish, the catalysts of 7-ethoxycoumarin are still unknown; nevertheless, this reaction is apparently catalyzed in rainbow trout by the CYP1A [START_REF] Cravedi | Cytochrome P450-dependent metabolic pathways and glucuronidation in trout liver slices[END_REF]. Among the inducers of ECOD activity in fish species are Aroclor 1254 (a PCBs mixture) [START_REF] Elcombe | Induction and characterization of hemoproteins P450 and monooxygenation in rainbow trout, Salmo gairdneri[END_REF] and -naphthoflavone [START_REF] Haasch | Use of 7alkoxyphenoxazones, 7-alkyloxycoumarins and 7-alkoxyquinolines as fluorescent substrates for rainbow trout hepatic microsomes after treatment with various inducers[END_REF] in rainbow trout; and 3-methylcholanthrene and PCBs in tilapia (niloticus x aureus) [START_REF] Ueng | Comparative induction of cytochrome P450-dependent monooxygenases in the livers and gills of tilapia and carp[END_REF][START_REF] Ueng | Induction and purification of cytochrome P4501A1 from 3methylcholanthrene-treated tilapia, Oreochromis niloticus x Oreochromis aureus[END_REF]. Most of these compounds have also been found to be inducers of EROD activity. On the other hand, there is substantial ECOD activity in non-CYP1A induced channel catfish, suggesting and additional constitutive role [START_REF] Perkins | Characterization of the hepatic cytochrome P450 monooxygenase system and its role in the metabolism, toxicokinetics, and toxicity of aldicarb in channel catfish (Ictalurus punctatus)[END_REF][START_REF] Perkins | Isolation of a novel hepatic CYP2-related protein from channel catfish, Ictalurus punctatus[END_REF]. In the present study, channel catfish had the highest ECOD velocity and catalytic efficiency of all the species. This velocity doubled the values in Atlantic salmon, rainbow trout, tilapia, and hybrid striped bass. Despite these differences, all the values were quite similar to those in non-induced fish. V max in channel catfish (108.0 ± 6.9 pmols 7-hydroxycoumarin/min/mg protein) was quite similar to the value found by [START_REF] Perkins | Characterization of the hepatic cytochrome P450 monooxygenase system and its role in the metabolism, toxicokinetics, and toxicity of aldicarb in channel catfish (Ictalurus punctatus)[END_REF] (109.0 ± 3.0). ECOD for rainbow trout (42.1 pmols/min/mg protein) was lower than the one reported by [START_REF] Kleinow | A comparison of hepatic P450 induction in rat and trout (Oncorhynchus mykiss): delineation of the site of resistance of fish to phenobarbital-type inducers[END_REF] in the same species (80 pmols/min/mg protein).

The V max values found for tilapia (32.0 ± 0.9 pmols 7-hydroxycoumarin/min/mg protein) is below the ones reported in other studies. Ueng & col. (1992) and [START_REF] Ueng | Induction and purification of cytochrome P4501A1 from 3methylcholanthrene-treated tilapia, Oreochromis niloticus x Oreochromis aureus[END_REF] found 143.0 ± 27.0 and 311.0 ± 38.0 pmols 7-hydroxycoumarin/min/mg protein for ECOD activity in noninduced tilapia weighing 200-300 grams. These values are between 2 and 5-fold higher than the corresponding in the present work. These investigators performed ECOD assays at 37 o C while our incubations were performed at room temperature (20-21 o C). This factor may partially explain such differences between the studies. EROD activity has been studied in over 150 fish species. This activity is mainly examined to determine its suitability as a biomarker of pollution [START_REF] Whyte | Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure[END_REF]. In the present study, EROD V max values were quite similar among the species. Despite differences among some of them, all values were below 100 pmols/min/mg protein. Hybrid striped bass, striped bass and bluegill showed the lowest velocities. Changes in phase I reactions have been reported after drug use in aquacultured species. ECOD and EROD activities were significantly inhibited by enrofloxacin, a fluoroquinole antibiotic, after in vivo treatment in sea bass (Dicentrarchus labrax) [START_REF] Vaccaro | Inhibition of cytochrome P450 enzymes by enrofloxacin in the sea bass (Dicentrarchus labrax)[END_REF]. In a different study by our group, the inducing effects on EROD activity (2.2 -2.6 fold) was found after in vivo treatment of channel catfish with albendazole at therapeutic dosage (unpublished data, in revision). This suggests that knowing baseline alkoxyresorufins and alkoxycoumarins activities in fish species may help to predict effects due to some drug treatments in aquacultured finfish species.

PROD and BROD kinetics. The present results indicate that none of the fish species was reactive to either of these two alkoxyphenoxazones. Haasch and col. (1994) reported that sham-injected rainbow trout had no PROD activity and negligible BROD activity (1.1 pmols resorufin/min/mg protein). Although no P450 isoform(s) has been identified in fish as responsible for PROD and/or BROD activities, inducing effects such as the ones exerted by isosafrole, -NF and dexametasone in rainbow trout may help to differentiate between the expected baseline values, such as the ones found in our study, and a possible induction after exposure to contaminants or drugs.

Glutathion-s-transferase kinetics. GST activity is an indicator of conjugation of electrophilic compounds with glutathione (GSH), a cytosolic tripeptide in milimolar concentrations within the cell. These electrophilic compounds that are conjugated by GSH may exert adverse changes on macromolecules (e.g. proteins, lipids) within the cell structure [START_REF] Stephensen | Effects of redox cycling compounds on glutathione content and activity of glutathione-related enzymes in rainbow trout liver[END_REF]. V max , K m and V max /K m for CDNB conjugation were significantly different among the species in the present study. Interestingly, Atlantic salmon had one of the highest V max and K m values. On the other hand, channel catfish presented the lowest K m of all the species. Channel catfish and tilapia are among the species with the highest GST-mediated conjugation of CDNB. Juveniles of channel catfish kept in free-flowing tanks had high CDNB conjugation velocities ranging from 1325 [START_REF] Gallagher | Channel catfish glutathione-s-transferase isoenzyme activity toward (±)-anti-benzo[a]pyrene-trans-7,8dihydrodiol-9,10-epoxide[END_REF] to 1500 nmols/min/mg protein [START_REF] Gallagher | A comparison of glutathione-dependent enzymes in liver, gills and posterior kidney of channel catfish (Ictalurus punctatus)[END_REF]. [START_REF] Ankley | Comparative aspects of hepatic UDP-glucuronosyltransferases and glutathione-s-transferases in bluegill and channel catfish[END_REF] reported 827 nmols CDNB/min/mg protein in channel catfish that were maintained for 9 to 16 months in fiberglass tanks. These values are higher than the maximum velocities found in the present study (657 ± 39 nmols/min/mg protein). [START_REF] Pathiratne | Comparison of xenobiotic metabolizing enzymes of tilapia with those of other fish species and interspecies relationships between gene families[END_REF] reported high GST activity in tilapia (Oreochromis niloticus) (1900 ± 120 nmols/min/mg protein) using CDNB as a substrate. In the present study, tilapia had the highest V max of all the species (1508 ± 70 nmols/min/mg protein) and the second highest catalytic efficiency (V max /K m ) after channel catfish. GST activity has been considered of great importance in detoxification of electrophilic xenobiotics [START_REF] Shailaja | Evaluation of impact of PAH on a tropical fish, Oreochromis mossambicus using multiple biomarkers[END_REF]. Some authors have reported that a incidence of tumors after exposure to carcinogenic compounds such as PAH's, PCB's, and other environmental hepatocarcinogens in channel catfish may result from the notable GST activity found in this species [START_REF] Gallagher | Channel catfish glutathione-s-transferase isoenzyme activity toward (±)-anti-benzo[a]pyrene-trans-7,8dihydrodiol-9,10-epoxide[END_REF]. Tilapia has also been reported as a species with high GST-based conjugation [START_REF] Gadagbui | Species characteristics of hepatic biotransformation enzymes in two tropical freshwater teleosts, Tilapia (Oreochromis niloticus) and mudfish (Clarias anguillaris)[END_REF][START_REF] Shailaja | Evaluation of impact of PAH on a tropical fish, Oreochromis mossambicus using multiple biomarkers[END_REF]. Reports on CDNB conjugation in rainbow trout, largemouth bass, Atlantic salmon and bluegill indicate lower capacity as compared to channel catfish and tilapia. In rainbow trout, reports show a wide range of activity: 700 ± 80 nmols/min/mg protein [START_REF] Pérez-López | Characterization of hepatic and extrahepatic glutathione-s-transferases in rainbow trout (Oncorhynchus mykiss) and their induction by 3,3',4,4'-tetrachlorobiphenyl[END_REF], 348 ± 17 nmols/min/mg protein [START_REF] Laurén | Microsomal and cytosolic epoxide hydrolase and glutathione S-transferase activities in the gill, liver, and kidney of the rainbow trout, Salmo gairdneri. Baseline levels and optimization of assay conditions[END_REF], and 183 ± 17 nmols/min/mg protein [START_REF] Otto | Phase I and II enzymes and antioxidant responses in different tissues of brown bullheads from relatively polluted and non-polluted systems[END_REF]. Our study showed a V max value of 929 ± 65 (nmols/min/mg potein) which is higher than the one found by Pérez-López and col. (2000). Largemouth bass showed in the present study a V max value of 589 ± 52 nmols/min/mg protein which is lower than the one reported by Gallagher and collaborators (2000) in a full kinetic study (843 -1154 nmols/min/mg protein). GST activity has been of particular interest in this species given the high risk of exposure to pollutants that are detoxified through GSTs and its predatory habits [START_REF] Gallagher | In vitro kinetics of hepatic glutathione S-transferase conjugation in largemouth bass and brown bullheads[END_REF][START_REF] Pham | Conjugation of 4-hydroxynonenal by largemouth bass (Micropterus salmoides) glutathione S-transferases[END_REF].

Largemouth bass had in the study by Pham and collaborators (2002) one of the highest, GSTmediated 4-hydroxynonenal (4HNE) conjugation which was a good indicator of protection against peroxidation of polyunsaturated fatty acids in the liver. However, largemouth bass had in the present study a lower capacity towards CDNB than tilapia, channel catfish, and rainbow trout.

As for Atlantic salmon, a study in immature (~ 60 g in weigh) juveniles reports a CDNB conjugation of 290 ± 8 nmols/min/mg protein [START_REF] Nóvoa-Valiñas | Comparative study of purification and characterization of the cytosolic glutathione S-transferases from two salmonid species: Atlantic salmon (Salmo salar) and brown trout (Salmo trutta)[END_REF]. CDNB conjugation kinetics was studied in our research using Atlantic salmon ranging from 492 to 1000 g. Maximum velocity in this species was one of the highest of all (1349 ± 107 nmols/min/mg potein) but so was enzyme affinity (0.5 ± 0.10 mM), yielding a lower catalytic efficiency (2816 ± 329) than channel catfish and tilapia.

UDP-glucuronosyltransferase (UDPGT) kinetics. Glucuronidation is one of the major phase II pathways in fish and other species [START_REF] Kasper | Glucuronidation[END_REF][START_REF] Clarke | Glucuronidation in fish[END_REF]. Both endogenous (e.g., bilirubin, steroids, thyroid hormones, etc.) and exogenous compounds (e.g., PCBs, pesticides, drugs, etc.) are conjugated with glucuronic acid via UDPGTs [START_REF] George | Enzymology and molecular biology of phase II xenobiotic-conjugating enzymes in fish[END_REF].

Among the drugs and pesticides that are conjugated via UDPGTs are chloramphenicol et al., 1985), organophosphates [START_REF] Takimoto | Comparative metabolism of fenitrothion in aquatic organisms. I. Metabolism in the euryhaline fish, Oryzias latipes and Mugil cephalus[END_REF], and phenols [START_REF] Lech | Isolation and identification of 3-trifluoromethyl-4-nitrophenol glucuronide from bile of rainbow trout exposed to 3-fluromethyl-4-nitrophenol[END_REF][START_REF] Kane | UDPglucuronosyltransferase kinetics for 3-trifluoromethyl-4-nitrophenol (TFM) in fish[END_REF]. Aflatoxicol B1 glucuronide is also formed in rainbow trout exposed to this hepatocarcinogen [START_REF] Loveland | Glucuronides in bile of rainbow trout (Salmo gairdneri) injected with [ H] aflatoxin B1 and effects of dietary -naphthoflavone[END_REF]. Kinetics of UDPGT activity in the present study showed lower variability among the species as compared to the phase I reactions and the GSTmediated conjugation. With the exception of striped bass, largemouth bass and hybrid striped bass, the other species had quite similar results. In the past, differences in UDPGT -based conjugation have helped to understand distinct sensitivities among fish species to pollutants or drugs. [START_REF] Kane | UDPglucuronosyltransferase kinetics for 3-trifluoromethyl-4-nitrophenol (TFM) in fish[END_REF] found that the lower V max /K m ratio in sea lamprey (Petromyzon marinus) as compared to channel catfish, rainbow trout and bluegill helped to explain a high sensitivity to 3-trifluoromethyl-4-nitrophenol (TFM), due to lower UDPGT-based conjugation.

Sulfotransferase kinetics. Sulfation is catalyzed by sulfotransferases (ST), a group of cytosolic enzymes that have lower capacity than UDPGTs but a higher affinity for the substrates [START_REF] Parkinson | Biotransformation of xenobiotics[END_REF]. Some studies have identified ST activity towards thyroid hormones in plaice [START_REF] Osborn | Thyroxine metabolism in plaice, Pleuronectes platessa[END_REF] and rainbow trout (Finnson and Eales;[START_REF] Finnson | Glucuronidation of thyroxine and 3,5,3'-triiodothyronine by hepatic microsomes in rainbow trout, Oncorhynchus mykiss[END_REF], 1998). The effects of environmental estrogens and other endocrine disrupters on ST-mediated conjugation have been studied showing additional implications of these pollutants on biotransformation capabilities [START_REF] Nishiyama | Sulfation of environmental estrogens by cytosolic human sulfotransferases[END_REF][START_REF] Kirk | Environmental endocrine disrupters dysregulate estrogen metabolism and Ca 2+ homeostasis in fish and mammals via receptorindependent mechanisms[END_REF][START_REF] Ohkimoto | Sulfonation of environmental estrogens by zebrafish cytosolic sulfotransferases[END_REF]. The present work corroborated that STs are lower capacity / higher affinity enzymes in comparison to UDPGTs. V max values for ST in all the species were several times lower than the corresponding values for UDPGT. On the other hand, the low K m s indicated the higher binding affinities of ST in comparison to the high values for UDPGT (lower affinities).Interestingly, channel catfish microsomes were not reactive to 7-ethoxyresorufin for UDPGCT-based conjugation. However, cytosolic fractions of the same specimens responded to ST conjugation. ST-mediated conjugation of 7-ethoxyresorufin was one of the assays with the lowest variation among the species studied in this investigation. V max /K m ratios did not show significant differences amongst them. However, V max values were significantly higher for rainbow trout, tilapia, channel catfish, Atlantic salmon and largemouth bass. The lowest capacities were found in striped bass, hybrid striped bass and bluegill. Catalytic efficiencies were quite similar among the species due to higher binding affinities in the three species that had the lowest capacities (V max ).

Conclusions

Based on the catalytic probes that were used in this study to evaluate phase I and II biotransformation reactions, the 8 fish species that were investigated exhibited differences among them. Channel catfish, tilapia, rainbow trout and Atlantic salmon had the maximum capacities and the highest catalytic efficiencies for most of these catalytic probes. Hybrid striped bass, striped bass and bluegill displayed the lowest efficiencies. Largemouth bass was in between the two suggested groups, showing higher capabilities in some tests and lower in others.

In some cases, species that displayed low biotransformation capabilities (e.g. low V max ), had similar catalytic efficiencies than the ones with highest maximun velocities due to a higher binding affinity (e.g. low K m ) of their enzymatic systems. This was particularly evident for the UDPGT and ST reactions.

Major differences found in this study among the species with regard to GST-based biotransformation may help to understand their particular susceptibility or resistance to electrophilic compounds that exert deleterious effects on macromolecules within the cell.

Channel catfish and tilapia exhibited the highest capacity and efficiency in GST-mediated conjugation. Striped bass, hybrid striped bass and bluegill had very poor GST-based conjugation of CDNB.

Table 1. V max , K m and V max /K m ratios (means ± SEM from 3 to 8 samples per species) for ECOD and EROD kinetics in farm-raised fish (different letters in the same column denote statistically significant difference among the species, p < 0.05). ECOD velocity expressed as pmols 7 hydroxycoumarin/minute/mg protein. EROD velocity as pmols resorufin/minute/mg protein † microsomes did not show any activity towards 7-ethoxycoumarin -ECOD kinetics in striped bass and largemouth bass were nor performed due to insufficient microsomal sample. ‡: enzyme activity is reported only at 1 M substrate concentration due to very low constitutive levels of activity Table 2. V max , K m and V max /K m (means ± SEM of 3 -8 samples per species) for glutathione-stransferase (GST), UDP-glucuronosyltransferase (UDPGT) and sulfotransferase (ST) kinetics in farm-raised fish (different letters in the same column denote statistically significant difference among the species, p < 0.05) GST velocity expressed as nmols of CDNB/minute/mg protein; UDPGT and ST velocities expressed as pmols resorufin/minute/mg protein † NO UDPGT activity was detected using resorufin as substrate in channel catfish

ECOD EROD Fish species

Vmax K m V max /K m V max K m V max /
GST UDPGT ST Fish species V max K m V max /K m V max K m V max /K m V max K m V max /

  channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), red tilapia (Oreochromis sp.), largemouth bass (Micropterus salmoides), striped bass (Morone saxatilis), hybrid striped bass (Morone saxatilis x Morone crysops), and bluegill (Lepomis macrochuris). Atlantic salmon individuals were acclimated at the Center for Veterinary Medicine (FDA -Laurel, MD, USA). 2.2 Microsomes / cytosol preparation and phase I-II assays Microsomes and cytosol were prepared according to Vodicnik et al. (1981). Protein was measured using the bicinchoninic acid protein assay. All the incubations with either microsomes or cytosolic fractions were performed at room temperature (20 o C-22 o C). Ethoxycoumarin-odealkylation (ECOD) kinetics was based on Haasch & col (1994) and Schlenk & col. (2000) with some modifications. Reaction mixtures had 50 L of buffer Tris-HCl (100 mM, pH=7.4), 25 L of microsomes (100 g of protein), 10 L of 7-ethoxycoumarin (20-100 M range) and 25 L of NADPH tetrasodium salt (0.1 mM). ECOD activity was calculated based on the relative fluorescence units (RFUs) of the 7-hydroxycoumarin produced (excitacion = 380 nm, emission = 460 nm) in a microplate absorbance-fluorescence reader (TECAN, Genios TM , Austria).

  UDP-glucuronosyltransferase kinetics. UDPGT activity was expressed as pmols resorufin/min/ mg protein. Rainbow trout (930 ± 258) and Atlantic salmon (410 ± 86) had the highest UDPGT V max values of all the species. On the other hand, channel catfish microsomes did not have a UDPGT-based conjugation activity towards resorufin. Values for tilapia (368 ± 89), largemouth bass (273 ± 16), hybrid striped bass (271 ± 37), bluegill (263 ± 31) and striped bass (231 ± 29) were quite similar and not significantly different from each other (p > 0.05). K m ( M) values were quite similar among the 7 species tested, ranging from 17.5 ± 2.3 to 36.0 ± 7.0. As for V max / K m ratios, rainbow trout (30.4 ± 4.4) and Atlantic salmon (18.5 ± 3.3) had the highest values of all the species. Bluegill (16.3 ± 3.1), tilapia (15.0 ± 3.0) and largemouth bass (11.0 ± 1.0) had lower ratios than Atlantic salmon and rainbow trout. Striped bass (8.4 ± 1.0) and hybrid striped bass (8.2 ± 0.9) had the lowest efficiency ratios for this reaction (Table 2). Sulfotransferase kinetics. Values were expressed as pmols resorufin/min/mg protein. Tilapia (328.0 ± 17.0) and channel catfish (265.0 ± 27.0) had the highest conjugation velocities followed by Atlantic salmon (215.0 ± 14.0), rainbow trout (190.0 ± 20.0) and largemouth bass (147.0 ± 10.0). Bluegill (107.0 ± 23.0), hybrid striped bass (46.6 ± 4.0) and striped bass (45.0 ± 5.0) had again the lowest V max value of all the species. K m were very similar among all the species (range 0.3 ± 0.1 to 1.0 ± 0.2) with the exception of striped bass which had the lowest values of all (0.1 ± 0.03). V max /K m ratios for ST activity indicated that Atlantic salmon (435.5 ± 63.0) had the best enzymatic efficiency followed by channel catfish (388.0 ± 63.0), striped bass (384.0 ± 70.0), tilapia (354.0 ± 64.0), hybrid striped bass (309.9 ± 89.0), largemouth bass (300.0 ± 83.0) and rainbow trout (287.0 ± 18.8). Bluegill (167.0 ± 35.0) had the lowest ratio of all (Table

Figure 1 .

 1 Figure1. Relative V max /K m ratio (means ± SEM) for the phase I and II reactions tested in the eight species. Inter-species ratios are relative to each other and the Y-axis scale is provided for the purpose of species comparison only.

Acknowledgements

This work was funded by the Joint Institute of Food Safety and Nutrition (JIFSAN) -Contract # FDU001418. The authors thank Charlie Gieseker and Stanley Serfling for their help with the acclimation of Atlantic salmon at the Center for Veterinary Medicine (FDA).