Relative importance of direct and trophic uranium exposures in the crayfish Orconectes limosus: Implication for predicting uranium bioaccumulation and its associated toxicity


  • Olivier Simon,

    Corresponding author
    1. Laboratoire de Radioécologie et d'Ecotoxicologie (LRE), Institut de Radioprotection et de Sûreté Nucléaire, Saint Paul Lez Durance, France
    • Laboratoire de Radioécologie et d'Ecotoxicologie (LRE), Institut de Radioprotection et de Sûreté Nucléaire, Saint Paul Lez Durance, France
    Search for more papers by this author
  • Magali Floriani,

    1. Laboratoire de Radioécologie et d'Ecotoxicologie (LRE), Institut de Radioprotection et de Sûreté Nucléaire, Saint Paul Lez Durance, France
    Search for more papers by this author
  • Virginie Camilleri,

    1. Laboratoire de Radioécologie et d'Ecotoxicologie (LRE), Institut de Radioprotection et de Sûreté Nucléaire, Saint Paul Lez Durance, France
    Search for more papers by this author
  • Rodolphe Gilbin,

    1. Laboratoire de Radioécologie et d'Ecotoxicologie (LRE), Institut de Radioprotection et de Sûreté Nucléaire, Saint Paul Lez Durance, France
    Search for more papers by this author
  • Sandrine Frelon

    1. Laboratoire de Radioécologie et d'Ecotoxicologie (LRE), Institut de Radioprotection et de Sûreté Nucléaire, Saint Paul Lez Durance, France
    Search for more papers by this author


Pollutants that occur at sublethal concentrations in the environment may lead to chronic exposure in aquatic organisms. If these pollutants bioaccumulate, then organisms higher in the food chain may also be at risk. Increased attention has thus been focused on the relative importance of dietary uptake, but additional knowledge of the cellular distribution of metals after dietary exposure is required to assess the potential toxicity. The authors address concerns relating to increasing uranium (U) concentrations (from 12 µg/L to 2 mg/L) in the freshwater ecosystem caused by anthropogenic activities. The objective of the present study is to compare uranium bioaccumulation levels in tissues and in the subcellular environment. The authors focused on the cytosol fraction and its microlocalization (TEM-EDX) in the gills and the hepatopancreas (HP) of the crayfish Orconectes limosus after 10 d of direct exposure (at concentrations of 20, 100, and 500 µg/L) and five trophic exposure treatments (at concentrations from 1 to 20 µg/g). Results indicated that adsorption of uranium on the cuticle represents the main contribution of total uranium accumulation to the animal. Accumulation in the gills should be considered only as a marker of waterborne uranium exposure. Accumulation in the HP after trophic environmental exposure conditions was higher (18.9 ± 3.8 µg/g) than after direct exposure. Moreover, no significant difference in the subcellular distribution of uranium (50%) in HP was observed between animals that had been exposed to both types of treatment. A potential toxic effect after uranium accumulation could therefore exist after trophic exposure. This confirms the need to focus further studies on the metal (uranium) risk assessment. Environ. Toxicol. Chem. 2013;32:410–416. © 2012 SETAC


Pollutants that occur at sublethal concentrations in water may lead to chronic exposure in aquatic organisms. These organisms can accumulate high metal concentrations, which may result in the trophic transfer of such pollution to predators. Trophic transfer can thus become a major source of exposure to metals 1–5. For this reason, attention has recently been focused on the relative importance of dietary uptake in assessing toxicity. A number of aquatic organisms, including fishes, can accumulate metals via aqueous and dietary exposure routes 6. Certain authors have emphasized the need for toxicological tests that focus on the trophic transfer of metals in aquatic animals, to assess the importance of food as a source of metal toxicity 7. Thus, awareness is growing among regulators concerning the suitability of water quality guidelines established for dissolved metals, and whether these are sufficiently protective 4, 5.

Ecological risk assessments often focus on the relationship between direct exposure and the associated effects, but many standard toxicology tests do not take trophic exposure into account, even though trophic exposure in controlled conditions is easy to reproduce and interpret as direct exposure standardized tests 8.

In parallel with the use of the biotic ligand model 9, the intention of the present study is to express the chronic toxic effects as a function of accumulated dose. This is referred to as the critical body residues approach 7, 10–14, which focuses on the characterization of metal distribution in organisms. Even if the relationship between bioaccumulation expressed by the metal-available fraction and toxicity remains complex 15–17, toxicity is now seen to depend partly on the biological model, the metal, and more accurately, chemical binding of the metal within cells 17, 18.

To adopt such an approach, additional knowledge about the cellular distribution of metals after dietary or direct exposures is required to assess potential toxic effects attributable to these two different routes of exposure.

Uranium is an element of interest: its concentration in freshwater ecosystems (from 12 µg/L to 2 mg/L) is currently increasing because of anthropogenic activities 19, 20. Although uranium is a radionuclide, its toxic effects and uptake are controlled by its chemical properties. The accumulation levels and toxic effects of this element have been evaluated in several biological models 21–24. In crayfish, uranium can impair mitochondrial function and induce oxidative stress 23. Very few studies have focused on its accumulation and associated toxic effects after trophic exposure 8, 25–27. The ecotoxicological profile of uranium is not yet fully understood, and the major pathway responsible for its accumulation levels has not yet been determined.

Various species of crayfish have been found to be suitable candidates for evaluating the importance of routes of exposure to uranium. These are often used as indicators of freshwater metal pollution, because they tend to rapidly accumulate metals and radionuclides (uranium) in their tissues via direct and trophic routes 23, 26, 28–32. Short-term exposure has been used to evaluate the transfer capability of direct and dietary exposure pathways. As prey for trophic exposure experiments, we selected the Asiatic clam, C. fluminea, which is well known for its high capacity for metal bioaccumulation 33–36.

The aim of the present study was to compare the uranium bioaccumulation rate at tissue and subcellular concentrations and its micro-localization in the gills and the hepatopancreas of the crayfish Orconectes limosus after 10 d. Two exposure treatments were adopted: direct exposure (with three uranium concentrations), and trophic exposure (with five treatments).


Exposure modalities

Intermoult crayfish O. limosus (12-month-old males) were collected in the field (Esparron Lake invaded by this species, France) and acclimated (in terms of water and food) for at least a month in laboratory conditions.

Direct exposure treatment

Crayfish (n = 10 per condition) were introduced into a 40-L experimental tank. Three nominal uranium concentrations in the exposure medium were chosen: 20 µg/L (low level; LL) comparable with concentrations measured in the discharges in the vicinity of old mine sites, 100 µg/L (medium level; ML), and 500 µg/L (high level; HL). The uranium exposure conditions were achieved by a constant flux, via a peristaltic pump from a feeding tank, to maintain a constant pH (6.5), temperature (20°C), and the ionic quality of the artificial soft water (in mg/L; Ca2+ = 11.5; Mg2+ = 8; Na+ = 11.6; K+ = 6.2; Cl = 13.5; NOmath image = 6.3; SOmath image = 8.1; HCOmath image = 71), as well as uranium concentrations, during the 10 d of exposure. The prevailing pH level, as well as the absence of added phosphate in the artificial medium, resulted in the maintenance of high levels of uranium bioavailability 23, 35–37. Crayfish received two uncontaminated bivalves as a meal, at 4 d and 8 d, over the 10-d exposure period. The uranium concentration in non-contaminated bivalves was below the detection threshold of the inductively coupled plasma atomic emission spectroscopy (ICP–AES) analytical procedure. Water samples for each exposure condition were collected at different times (at 0.5, 1, 2, 3, 5, 8, and 10 d) to measure the concentration of total uranium (by ICP–AES) and to manually adjust each nominal concentration on a daily basis.

Trophic exposure treatment

Crayfish from the same stock (n = 5 per condition) were introduced into individual chambers (cylinders made from plastic-coated netting: 0.7-cm, 20-cm diameter) inside a tank (150 L) containing sand (50 kg) and hard tap water (T = 20 ± 1°C, pH = 7.9, Ca2+ = 79 mg/L). Each chamber was constantly aerated by bubbling air. Two-thirds of the water column was removed per week to avoid direct exposure of crayfish. Each crayfish was fed a daily ration of one clam from a stock of five previously contaminated bivalves 26. These bivalves had been exposed to different treatments (in terms of pH, uranium in water, and exposure duration) the details of which are summarized in Table 1. Such differing exposure conditions resulted in differing levels of uranium content in the bivalves. The uranium concentrations in the whole body of the bivalves were as follows: LLTrophic (T): 0.9 ± 0.1; MTT: 10.7 ± 1; HLT1: 19.6 ± 7; HLT2: 20.2 ± 9; and HLT3: 20.4 ± 10 µg/g 26. Water samples were collected to measure uranium concentrations that had originated from two sources: that which had accumulated as a result of the digestion of uranium-contaminated bivalves, and that which had accumulated because of the eventual release of uranium after excretion by the crayfish. At the end of the 10-d trophic exposure, crayfish were prevented from feeding for 48 h before sampling. This period allowed for the clearing of the digestive tracts of the crayfish, as confirmed during tissue dissection.

Table 1. Exposure modalities (pH, uranium concentration in water [µg/L], and exposure duration [day] and accumulation in whole body, gills, and visceral mass [µg/g, fresh wt])
Exposure modalitiesAccumulation levels in bivalve
 Whole bodyGillsVisceral mass
pHUranium in water µg/LExposure duration (d)Average SDAverage SDAverage SD
  1. SD = standard deviation.

8.1100300.9 ± 0.11.03 ± 01.8 ± 0.4
71004210.7 ± 110.8 ± 119.8 ± 2.7
75001419.6 ± 767 ± 2213.6 ± 4.6
71500720.2 ± 948 ± 197.9 ± 4.6
6.55001020.4 ± 10.647 ± 3217.9 ± 13

Metal analysis at tissue levels and at subcellular levels

After the direct and trophic exposure periods, crayfish were collected and stored at –80°C. Tissues extracts from gills, the digestive gland/hepatopancreas (HP), green gland, muscle, stomach, intestine, carapace, and from the remaining tissue in the body were then subjected to uranium analysis, using ICP-AES.

Gills and hepatopancreas (n = 5 per condition) were selected as a measure of subcellular metal partitioning. The simplified technique 15 used in the present study comprised the following steps: (1) homogenizing with a Potter-Elvehjem glass homogenizer; (2) filtering the homogenate so as to isolate large cellular debris and portions of the gill cuticle (Ø = 20 µm); and (3) subcellular fractionation involving two successive differential centrifugations at 4°C (1,450 g for 15 min, then 100,000 g for 1.5 h). The purpose of this procedure was to obtain three types of cellular fractions: the C1 fraction containing nuclei and granules, the C2 fraction containing organelles, and the S2 fraction containing cytosol. The quantity of uranium in the cellular debris, pellets (C1 + C2), and cytosol fractions of both organs was then measured. The metal analysis in gills and HP was performed for LL and HL direct-exposure treatments and only in one trophic exposure condition (HLT3).

Each biological sample was digested in a polypropylene tube using nitric acid (3 ml, Merck, 65%) and then perchloric acid (2 ml, Merck, 33%) at 105°C for 3 h. Water and biological samples were diluted in 5 ml or 10 ml of ultrapure water that was acidified with 3.1 mM HNO3 and spiked with a known quantity of yttrium before analysis using a multi-component spectral fitting correction treatment, while taking into account the matrix effects on the ICP-AES signal. Uranium content was determined by averaging the data obtained at three different wavelengths (409, 417, and 424 nm). The detection limit was 3 and 10 µg/L for water and biological samples, respectively, but the limit for the carapace tissue was higher (detection limit = 50 µg/L). The accumulation level of U in the crayfish control was below our detection limit.

Transmission electron microscopy observations and X-ray analysis (TEM-EDX)

After the termination of direct and trophic exposures, fresh tissues of gills and hepatopancreas (n = 5) were routinely fixed with 2.5% glutaraldehyde in a sodium cacodylate buffer (pH 7.4) for 24 h at 4°C. Samples were then washed in a sodium cacodylate buffer and then postfixed in the same buffer to which 1% OsO4 had been added. Dehydration took place in a graded series of alcohol solutions, and Epon was used as the embedding medium 23. Ultrathin sections (110 nm) were observed with a scanning transmission electron microscope (TEM/STEM, tecnai; FEI company) equipped with a charge-coupled device camera, an energy dispersive X ray detector (EDX), and a beryllium low-background specimen holder. Quantitative analysis was carried out using the Genesis Spectrum TEM program (EDAX Inc.).

Statistical analysis

Results were expressed as a function of the mean and the standard deviation. Linear regression models, using Excel software, were set up between accumulation levels in gills or hepatopancreas and (1) all crayfish organs minus the carapace (i.e., the total sum of gills, hepatopancreas, green gland, muscle, stomach, intestine), and (2) gills of the bivalve, or water exposure conditions. Kruskal-Wallis tests were also performed between accumulation levels in gills and hepatopancreas after direct or trophic exposures. For all statistical results, a probability of p < 0.05 was considered significant.


No mortality occurred in any experimental treatment. For the three direct exposure routes, the concentration averages in water were very close (6%) to nominal concentrations. In terms of trophic exposure, the accumulation level of uranium in the water medium was lower than that of our detection limit.

Accumulation level in organs after direct and trophic exposures

The uranium accumulation concentrations (µg/g, fresh wt) in the gills and the HP for the direct and trophic exposure treatments, at the end of 10 d exposure (Fig. 1), were significantly different for the gills after the three direct exposures (p = 0.0012) and for HP after the five trophic exposures (p = 0.0018). High levels of uranium accumulation were measured in the gills (until 65 µg/g), although low concentrations were measured after trophic exposure from 0.3 to 1.1 µg/g. Higher uranium accumulation was measured in HP after trophic exposure (close to 18.9 ± 3.8 µg/g) than after that measured after direct exposure treatments (3.9 ± 3 µg/g), and a significant difference within the different trophic exposure modalities was noted.

Figure 1.

Uranium accumulation concentrations (µg/g, fresh wt) in the gills and the hepatopancreas (HP) for the direct (n = 10, A) and trophic (n = 5, B) exposure treatments at the end of the exposure duration (10d).

High accumulation concentrations were measured in the carapace (50 µg/g for HL) and the digestive tract (stomach + intestine) after direct exposures and after trophic exposures, respectively. These results have not been presented in the graph (Fig. 1).

The following step was performed to identify target organs as a function of exposure modalities and of accumulation measured in all organs of crayfish (i.e., in all organs of the crayfish except the carapace). Linear regression models (Table 2)—between accumulation levels in gills, in HP with the concentration of uranium in either the water or in bivalve gills, or in the whole body of crayfish minus the carapace—indicated several interesting trends. In the case of direct exposures, a linear relationship between gill crayfish concentration and uranium concentration in water (r2 = 0.72, n = 30) was indicated. Moreover, gill accumulation was linearly linked to the accumulation in the whole body of crayfish minus the carapace (r2 = 0.9, n = 30). In the case of trophic exposures, identical results were observed for the digestive gland (HP), which was the best indicator of the accumulation levels in the whole body of the crayfish minus the carapace (r2 = 0.88, n = 25). Moreover, the accumulation levels in HP could be linearly linked to the gill accumulation levels in bivalves (r2 = 0.68, n = 25). In both exposure conditions, no relationship was obtained between accumulation levels in gills and the HP.

Table 2. Linear relationships between accumulation concentrations in gills and in hepatopancras (HP) of crayfish as a function uranium (U) concentration in water, in bivalve gills for direct and trophic exposure routes and uranium concentration in whole body of the crayfish minus the carapace (organs)
 Linear relationSlope ± SD95% confidence intervalnR2
  1. SD = standard deviation.

Direct exposure[U] in gills vs [U] in water0.13 ± 0.010.09; 0.16300.72
[U] in gills vs [U] in organs9.8 ± 0.428.8; 10.7300.9
Trophic exposure[U] in HP vs [U] in bivalve gills0.25 ± 0.030.16; 0.34250.68
[U] in HP vs [U] in organs4.81 ± 0.274.23; 5.38250.88

Accumulation levels in subcellular fractions after direct and trophic exposure

The gills and the HP were the target organs for direct and trophic exposure, respectively. We selected these two organs for subcellular fractionation, as described previously. The distribution of uranium in the cellular debris of gills was high (27 ± 7% and 36 ± 8% after LL- and HL-direct exposure treatments, respectively). In HP, the accumulation in this fraction was not detected after all direct exposures, and reached 3.5 ± 1% in the HLT3 trophic treatments.

The uranium burden (µg) in the entire gill, as a function of the cytosol fraction after LL-20 µg/L and HL-500 µg/L direct exposure treatments (Fig. 2), accounted for 12% of the total accumulation in the gills. This was accumulated in the cytosol fraction (r2 = 0.99, n = 10). Uranium distribution in the cytosol fraction of HP for both exposure conditions (Fig. 2B) shows that the subcellular distribution in the HP was different from that measured in the gills, with 50% of uranium in the cytosol fraction (r2 = 0.69, n = 10) after the direct exposure route. In the case of the trophic exposure treatment, only one condition was analyzed, indicating that 56% of the uranium was accumulated in the HP cytosol fraction (r2 = 0.81, n = 5). High individual variation in the uranium burden could be observed, in particular, for the highest direct exposure levels.

Figure 2.

Total U amount (µg) measured in gills (A) as a function of cytosol fraction after direct exposure routes (LL-20 µg/L and HL-500 µg/L) and in HP (B) as function of cytosol fraction after direct exposure routes (LL-20 µg/L and HL-500 µg/L) and trophic exposure route (HLT3). Linear relationships were presented.

Microlocalization of uranium(TEM-EDX) in gills and HP of crayfish after direct and trophic exposures

Optic and electronic observations of uranium in the gills of the crayfish and associated with EDX spectra after direct exposure (HL-500 µg/L treatment), in the transverse section of gill filaments of podobranchs, show a biofilm (bacteria + algae + detritus) on the gill cuticle (Fig. 3).

Figure 3.

Histological observation (section transversal) of gills epithelium after 10 days of U exposure (HL 500 µg/L) and associated transmission electron micrographs of gills and hepatopancreas coupled with energy dispersive X-ray. O: oxygen, Os: osmium and Cu: copper. U: uranium and P: phosphorus. (Ex M) extra-cellular matrix, (C) cuticle, (G ep) gills epithelium, (R) absorptive cells. (B) secretory cells. (L) Lumen, (M) microvillous border. [Color figure can be seen in the online version of this article, available at]

The EDX analysis indicated the presence of uranium (+phosphorus) inside the cuticular biofilm. The quantity of biofilm on the gill surface varied according to the types of filament and different individuals. Moreover, the loss of the cuticular biofilm could be attributable to different steps in the chemical preparation of the biological samples for transmission electron microscopy observations. Neither cellular damage nor accumulation in the gill epithelium (that has a respiration and an excretory function) were observed when using this technique. We specifically focused on the nephrocytes—large cells close to the septa of the gill filaments—because their function is to filter the hemolymph. Unfortunately, no uranium was detected inside these cells.

The hepatopancreas absorbs, metabolizes, and stores nutrients, and synthesizes the digestive enzymes (Fig. 3). This organ is composed of several hundred blindly ended tubules that are bathed in hemolymph. Each hepatopancreatic tubule consists of a single-layered epithelium that includes four cell types, each with microvilli borders (embryonic [E] cells, resorptive [R] cells, fibrillar [F] cells, and blister-like [B] cells) and the midget (M) cells. Among these five cell types, which participate in digestion, uranium was only observed in the lumen of the tubules close to the microvillous border and inside the B cell vesicles. In the lumen, uranium was incorporated into an electron-dense deposit in the form of needles and was associated with phosphorus and iron. The quantity of uranium electron-dense deposits was low. No effect of cellular organization was observed.

After trophic exposure, neither cellular effect nor uranium deposits in HP was observed when using this technique.


Feeding behavior

In the 10-d periods of both exposure treatments, no mortality was observed, and despite the high accumulation level measured in HP, the appetite of trophic-exposed crayfish did not seem to be influenced by the presence of uranium in the diet. Further trophic exposure, performed during 69 d in identical exposure conditions, confirmed this result (Simon et al., unpublished data). Results indicated that the molt was the principal factor influencing the appetite of the crayfish. Feeding behavior, which could be used as a marker of exposure or effect 38, was thus not a sensitive endpoint for our exposure conditions. (Note that postmolted crayfish were eliminated from the experiments.) No effects on the predation behavior were observed in the fish Coregonus clupeaformis after 100 d trophic exposure (four doses of uranium in an artificial diet) 25 or in the fish Danio rerio after 20 d trophic exposure (two doses of uranium in an artificial diet: 50 and 500 µg/g) 8.

Transfer from water or trophic exposure

Taking into account these multiphysiological functions, gills are considered as the dominant uptake site of metals in fish and crustaceans, after direct exposure 39, 40. After direct exposure to acute Pb, Cr, or low methylmercury (CH3-Hg) waterborne exposures, accumulation levels in gills were higher than those in HP 2, 32, 41–43. Identical results were observed in this study and were also obtained after acute uranium exposures 23. Moreover, a linear relationship between the uranium concentration in water and in gills was observed. Thus, accumulation in gills could be used as a marker of uranium exposure. The combination of the optic observations, the EDX analysis, and the level of uranium accumulated in the cellular debris suggests a hypothesis that a high level of uranium had accumulated in the biofilm. Adsorption of uranium on the gill cuticle could represent the main contribution of total uranium accumulation. Gills accumulation should not be considered as a marker of potential uranium effects. Consequently, the molt will contribute to uranium depuration. In our experimental study, no uranium deposits in gill epithelium (respiration and excretory functions) was observed, and only 12% of total accumulation in gills was measured in the cytosol fraction, considered as the potential toxic fraction. However, the accumulation of uranium in gills led to significant molecular effects, that is, changes in gene expression after direct uranium exposure at low level (30 µg/L) in Procambarus clarkii 23.

Despite the high level of uranium in gills, the uranium microlocalization approach did not allow us to observe uranium in the gill epithelium, as observed in the gill bivalve 44. Large differences, in terms of metal distribution, were observed between these two model organisms.

The gills thus constituted an effective biological barrier after direct exposure but contributed to the HP contamination. In our experimental conditions, no linear relationship was observed between the quantities of uranium in gills and in the HP. This indicates that the kinetics—and mechanisms of transfer, uptake, excretion, and transport—are complex and need further investigation to model the accumulation transfer.

A significant accumulation of uranium in the HP was measured after all trophic exposures, values being much higher (×5) than those measured after the direct exposures. Hepatopancreas was the main target organ of the uranium accumulation after trophic transfer 26, as observed for other metals 32. Evidence was seen of trophic transfer of uranium to gills, and this was certainly linked to physiological properties (highly perfused organ) and participation in excretory metabolism 45. Accumulation in the gill after metal trophic transfer was observed in fish 8, 25, 46, and in the crustacean 2, 47, 48. The transport via hemolymph may, however, be low. Indeed, in the crab Carcinus maenas, gills are 10 times less able to accumulate Cd, which is less than gills that have been exposed to external Cd after internal Cd exposure 49.

In our experimental conditions, the HP distribution in the cytosol fraction was high (50%) after both direct and trophic exposures. The subcellular distribution (mainly organelles vs cytosol) was not dependent on exposure conditions. Taking into account the high concentration in HP, clearly uranium exposure could lead to significant effects in these organs, as demonstrated by Al Kaddissi et al. 23 for another crayfish species, P. clarkii. Trophic transfer therefore represents a significant source of uranium accumulation and could lead to the same biological effects as that for direct exposure, considering that a threshold of internalization is linked to toxic effects 11.

The measurement of metal transfer is currently based on the BCF (ratio between concentration in organism and concentration of direct exposure level), the trophic transfer factor (the ratio between quantity in organism and quantity provided by trophic exposure), and the assimilation efficiency (percentage) 14. A number of authors recommend that these parameters be treated with caution, because their behavior is affected by ambient concentrations 17. The characterization of uranium uptake can be assessed by taking into account the total uranium quantity provided to the target organ: in the gills after direct exposure 50 and the hepatopancreas (the food ration) after trophic exposure. High differences in uranium concentration in different organs were thus observed between both direct and trophic exposures, with results indicating 560 and 2800 µg uranium for the gills and only 150 µg uranium for the HP. Uranium accumulation in organisms can be expressed as the quantity of uranium responsible for the observed potential effect. Uranium is also noted to accumulate in the cytosol fraction 11, 14, and the cytosol fraction can be separated into several sub fractions (because of the presence of proteins that can be denatured by exposure to heat as well as heat-stable proteins) to better identify the toxic fraction 15, 51. Further developments based on the subcellular distribution in metal rich granules (MRG) or in proteins of the cytosolic fraction, could be addressed by incorporating newly acquired findings on uptake mechanisms and potential toxic effects.

We propose to calculate the metal bioavailable fraction (MBF) transfer, defined as the ratio between uranium burden in cytosol fraction and uranium quantity, provided by exposure. Metal bioavailable fraction transfer values were 10−5 and 10 × 10−5 for both direct exposure conditions (LL-20 µg/L, HL-500 µg/L) and 1,500 × 10−5 for trophic exposure, indicating a better transfer from food. These results confirm the need to incorporate trophic exposure in ecological risk assessments 5 and have also increased the interest in further investigating the critical-body-residues approach.


Accumulation concentrations in HP after trophic exposure were higher than those measured after direct exposure. No significant difference was observed in uranium subcellular distribution in the HPs that had been subjected to the two exposure treatments. Thus, a potential toxic effect after uranium accumulation could exist after trophic exposure. This result confirms that this latter route must be studied for a complete assessment of metal (uranium) risk.

Coupled analysis of microlocalization (TEM-EDX) and uranium distribution at low level in subcellular fractions (inductively coupled plasma–mass spectrometry) allows for a better understanding of metal accumulation in organisms.

The calculation of the BCF was found to be a good indicator of the metal accumulating to equilibrium. To perform a comparison of metal transfer rates between both exposures, two important aspects must be taken into account. First, the internal accumulation (organelles + granules and cytosol fractions), and second, the real uptake of metals in both biological barriers (gills and HP, for direct and trophic exposures, respectively) to assess the metal bioavailable fraction transfer.