Chronic toxicity of uranium to three benthic organisms in laboratory spiked sediment.

Due to mining activities, concentration of uranium (U) in the environment nearby former and operating sites can be higher than in other areas. The derivation of quality criteria for U in freshwater ecosystems, rivers and lakes includes the consideration of contaminated sediments and the associated risk to the benthic life. Therefore, the derivation of a quality criteria for sediment has been viewed as a logical and necessary extension of the work already done to establish water quality criteria. In order to contribute to the determination of a Quality Standard for sediment (QSsediment) according to the European recommendations, this study focuses on the acquisition of a new toxicity dataset, to enrich the few rare existing data, most often unsuitable. A basic set of organisms, including three complementary benthic organisms (Chironomus riparius, Hyalella azteca, Myriophyllum aquaticum), was chronically exposed to U spiked to a standard laboratory-formulated sediment, according to the related bioassay guidelines (ISO/FDIS16303, OECD 218/9, ISO/DIS 16191). We looked to determine when possible both NOEC and EC10 values for each organism. For C. riparius, a NOEC (emergence rate) value was estimated at 62 mgU, kg-1, dm and the EC10 value reached 188 mgU, kg-1, dm (CI95% 40-885 mgU kg-1, dm). For H. azteca, a NOEC (survival rate) value of 40 mgU kg-1, dm was observed while the EC10 value at 296 mgU kg-1, dm (CI95% = 155-436 mgU kg-1, dm) was slightly higher than for growth at 199 mgU kg-1, dm (CI95% = 107-291 mgU kg-1 dm). Finally, the less sensitive organism seemed to be the plant, M. aquaticum, for which we determined a NOEC value of 100 mgU kg-1, dm. Results obtained regarding the toxicity of U made it possible to suggest a preliminary QSsediment value of 4 mgU kg-1, dry mass. This value was shown conservative compared to U sediment quality criteria derived by other jurisdictions.

Uranium transfer and accumulation in organs of Danio rerio after waterborne exposure alone or combined with diet-borne exposure.

Uranium (U) toxicity patterns for fish have been mainly determined under laboratory-controlled waterborne exposure conditions. Because fish can take up metals from water and diet under in situ exposure conditions, a waterborne U exposure experiment (20 µg L-1 , 20 d) was conducted in the laboratory to investigate transfer efficiency and target organ distribution in zebrafish Danio rerio compared with combined waterborne exposure (20 µg L-1 ) and diet-borne exposure (10.7 µg g-1 ). 233 Uranium was used as a specific U isotope tracer for diet-borne exposure. Bioaccumulation was examined in the gills, liver, kidneys, intestine, and gonads of D. rerio. Concentrations in the organs after waterborne exposure were approximately 500 ng g-1 fresh weight, except in the intestine (> 10 µg g-1 fresh wt) and the kidneys (200 ng g-1 fresh wt). No significant difference was observed between waterborne and diet-borne conditions. Trophic U transfer in organs was found but at a low level (< 10 ng g-1 fresh wt). Surprisingly, the intestine appeared to be the main target organ after both tested exposure modalities. The gonads (57% at 20 d) and the liver (41% at 20 d) showed the highest accumulated relative U burdens. Environ Toxicol Chem 2019;38:90-98. © 2018 SETAC.

Dynamic energy-based modeling of uranium and cadmium joint toxicity to Caenorhabditis elegans.

Toxicokinetic - toxicodynamic energy-based models offer new alternatives to the commonly used approaches for the analysis of mixture toxicity data. Based on the Dynamic Energy Budget theory, DEBtox models enable the description of several endpoints over time simultaneously under the same framework. However, such model still has to be faced with experimental data in a multi-contamination context. In this study, the predictive capacities of a DEBtox model to describe the uranium and cadmium joint toxicity over the entire growth and reproduction period of the soil nematode Caenorhabditis elegans was examined. The two reference additivity approaches, Concentration Addition and Response addition, implemented in the DEBtox model were tested. Assuming no interaction between the two toxicants through Response addition, the DEBtox model allowed a rather accurate fit of the U and Cd joint effects on the growth and reproduction of C. elegans: an interaction between the two metals at the toxicokinetic or toxicodynamic level seems thus unlikely or has only minor consequences. Interestingly, this study underlines that even if the compounds of a mixture share the same DEBtox physiological mode of action (in this case a decrease in assimilation), the Response addition approach may provide a better fit of joint toxicity data than the Concentration addition approach. Moreover, the present work highlighted limitations in the model predictions which are related to the simplifications of the DEBtox framework and its adaptations to the physiology of C. elegans and which lead to an overestimation of the U and Cd joint toxicity in some cases.

Assessment of co-contaminant effects on uranium and thorium speciation in freshwater using geochemical modelling.

Speciation modelling of uranium (as uranyl) and thorium, in four freshwaters impacted by mining activities, was used to evaluate (i) the influence of the co-contaminants present on the predicted speciation, and (ii) the influence of using nine different model/database combinations on the predictions. Generally, co-contaminants were found to have no significant effects on speciation, with the exception of Fe(III) in one system, where formation of hydrous ferric oxide and adsorption of uranyl to its surface impacted the predicted speciation. Model and database choice on the other hand clearly influenced speciation prediction. Complexes with dissolved organic matter, which could be simulated by three of the nine model/database combinations, were predicted to be important in a slightly acidic, soft water. Model prediction of uranyl and thorium speciation needs to take account of database comprehensiveness and cohesiveness, including the capability of the model and database to simulate interactions with dissolved organic matter. Measurement of speciation in natural waters is needed to provide data that may be used to assess and improve model capabilities and to better constrain the type of predictive modelling work presented here.

Nested interactions in the combined toxicity of uranium and cadmium to the nematode Caenorhabditis elegans.

Uranium is a natural, ubiquitous radioactive element for which elevated concentrations can be found in the vicinity of some nuclear fuel cycle facilities or intensive farming areas, and most often in mixtures with other contaminants such as cadmium, due to co-occurrence in geological ores (e.g. U- or P-ore). The study of their combined effects on ecosystems is of interest to better characterize such multi-metallic polluted sites. In the present study, the toxicity of binary mixture of U and Cd on physiological parameters of the soil nematode Caenorhabditis elegans was assessed over time. Descriptive modeling using concentration and response addition reference models was applied to compare observed and expected combined effects and identify possible synergistic or antagonistic interactions. A strong antagonism between U and Cd was identified for length increase and brood size endpoints. The study revealed that the combined effects might be explained by two nested antagonistic interactions. We demonstrate that the first interaction occurred in the exposure medium. We also identified a significant second antagonistic interaction which occurred either during the toxicokinetic or toxicodynamic steps. These findings underline the complexity of interactions that may take place between chemicals and thus, highlight the importance of studying mixtures at various levels to fully understand underlying mechanisms.

Use of diatom assemblages as biomonitor of the impact of treated uranium mining effluent discharge on a stream: case study of the Ritord watershed (Center-West France).

The rehabilitation of French former uranium mining sites has not prevented the contamination of the surrounding aquatic ecosystems with metal elements. This study assesses the impact of the discharge of treated uranium mining effluents on periphytic diatom communities to evaluate their potential of bioindication. A 7-month survey was conducted on the Ritord watercourse to measure the environmental conditions of microalgae, the non-taxonomic attributes of periphyton (photosynthesis and biomass) and to determine the specific composition of diatom assemblages grown on artificial substrates. The environmental conditions were altered by the mine waters, that contaminate the watercourse with uranium and with chemicals used in the pit-water treatment plants (BaCl2 and Al2(SO4)3). The biomass and photosynthetic activity of periphyton seemed not to respond to the stress induced by the treated mining effluents whereas the altered environmental conditions clearly impacted the composition of diatom communities. Downstream the discharges, the communities tended to be characterized by indicator species belonging to the genera Fragilaria, Eunotia and Brachysira and were highly similar to assemblages at acid mine drainage sites. The species Eunotia pectinalis var. undulata, Psammothidium rechtensis, Gomphonema lagenula and Pinnularia major were found to be sensitive to uranium effluents whereas Neidium alpinum and several species of Gomphonema tolerated this contamination. The relevance of diatoms as ecological indicator was illustrated through the changes in structure of communities induced by the discharge of uranium mining effluents and creates prospects for development of a bioindicator tool for this kind of impairment of water quality.

Sensitivity evaluation of the green alga Chlamydomonas reinhardtii to uranium by pulse amplitude modulated (PAM) fluorometry.

Although ecotoxicological studies tend to address the toxicity thresholds of uranium in freshwaters, there is a lack of information on the effects of the metal on physiological processes, particularly in aquatic plants. Knowing that uranium alters photosynthesis via impairment of the water photo-oxidation process, we determined whether pulse amplitude modulated (PAM) fluorometry was a relevant tool for assessing the impact of uranium on the green alga Chlamydomonas reinhardtii and investigated how and to what extent uranium hampered photosynthetic performance. Photosynthetic activity and quenching were assessed from fluorescence induction curves generated by PAM fluorometry, after 1 and 5h of uranium exposure in controlled conditions. The oxygen-evolving complex (OEC) of PSII was identified as the primary action site of uranium, through alteration of the water photo-oxidation process as revealed by F0/Fv. Limiting re-oxidation of the plastoquinone pool, uranium impaired the electron flux between the photosystems until almost complete inhibition of the PSII quantum efficiency ( [Formula: see text] , EC50=303 ± 64 µg UL(-1) after 5h of exposure) was observed. Non-photochemical quenching (qN) was identified as the most sensitive fluorescence parameter (EC50=142 ± 98 µg UL(-1) after 5h of exposure), indicating that light energy not used in photochemistry was dissipated in non-radiative processes. It was shown that parameters which stemmed from fluorescence induction kinetics are valuable indicators for evaluating the impact of uranium on PSII in green algae. PAM fluorometry provided a rapid and reasonably sensitive method for assessing stress response to uranium in microalgae.

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

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.

Are antioxidant and transcriptional responses useful for discriminating between chemo- and radiotoxicity of uranium in the crayfish Procambarus clarkii?

The main objectives of this study were to evaluate uranium (U) toxicity in the crayfish Procambarus clarkii at a low dose of exposure and to discriminate between the chemotoxicity and radiotoxicity of U. We conducted two sets of experiments using either 30 µg L(-1) of depleted uranium (DU) or (233)U, which differ from each other only in their specific activity (DU=1.7×10(4)Bqg(-1), (233)U=3.57×10(8)Bqg(-1)). The endpoints were oxidative stress responses and mitochondrial functioning in the gills and hepatopancreas, which were measured in terms of enzyme activities and gene expression levels. U accumulation levels were measured in different organs (gills, hepatopancreas, stomach, intestine, green gland, muscles, and carapace), and internal dose rates in the hepatopancreas were compared after DU and (233)U exposures. Significant U accumulation occurred in the organs of P. clarkii, and mitochondrial damage and antioxidant responses were detected. Despite the huge difference (21,000×) in the specific activities of DU and (233)U, few significant differences in biological responses were detected in P. clarkii exposed to these two pollutants. This finding indicates that the radiotoxicity was low compared to the chemotoxicity under our exposure conditions. Finally, genes expression levels were more sensitive markers of U toxicity than enzyme activities.

Effects of uranium on crayfish Procambarus clarkii mitochondria and antioxidants responses after chronic exposure: what have we learned?

We examined the impacts of Uranium (U) on mitochondria and on the response of antioxidants in the gills and the hepatopancreas of crayfish Procambarus clarkii after long-term exposure (30 and 60 days) to an environmentally relevant concentration (30 µg U/L). The expression of mitochondrial genes (12s, atp6, and cox1), as well as the genes involved in oxidative stress responses (sod(Mn) and mt) were evaluated. The activities of antioxidant enzymes (SOD, CAT, GPX and GST) were also studied. U accumulation in organs induced changes in genes' expression. The evolution of these transcriptional responses and differences between gene expression levels at high and low doses of exposure were also discussed. This study demonstrated that, after long-term exposure, U caused a decrease in antioxidant activities and induced oxidative stress. A possible ROS-mediated U cytotoxic mechanism is proposed. Expression levels of the investigated genes can possibly be used as a tool to evaluate U toxicity and seem to be more sensitive than the enzymatic activities. However a multiple biomarker approach is recommended as the perturbed pathways and the mode of action of this pollutant are not completely understood.

Effects of uranium uptake on transcriptional responses, histological structures and survival rate of the crayfish Procambarus clarkii.

This work aims to investigate the accumulation levels and effects (transcriptional responses, histopathology and survival rate) associated with a wide range of dissolved uranium (U) concentrations (0, 0.03, 0.6, 4 and 8 mg/L of U) on adult male crayfish Procambarus clarkii during 4 (T4) and 10 (T10) days of exposure. The follow-up of the crayfish mortality showed that P. clarkii was highly resistant to U. Increasing waterborne U concentrations led to increasing bioaccumulation in key crayfish organs and increasing histological damages. U distribution in tissues was also evaluated using transmission electron microscopy and showed the presence of a detoxified form of U in the gill's epithelium in the shape of flakes. Expression levels of mitochondrial genes (cox1, atp6 and 12S gene) and genes involved in oxidative stress (sod(Mn) and mt) were examined together with the housekeeping gene 18S. atp6 and mt genes of P. clarkii were cloned and sequenced before analysis. Significant correlations were observed between U bioaccumulation and the down-regulation of both cox1 and sod(Mn) genes. This work provides a first U toxicogenomic and histopathological pattern of P. clarkii, identify U biomarkers and associate gene expression endpoints to accumulation levels. It also provides new insights into the mechanisms involved in U stress.

Internal distribution of uranium and associated genotoxic damages in the chronically exposed bivalve Corbicula fluminea.

Uranium (U) internal distribution and involved effects in the bivalve Corbicula fluminea have been studied after direct chronic exposure (90 d, 10 µg.L-1). U distribution was assessed at the subcellular level (Metal Rich Granules -MRG-, pellets and cytosol fractions) in two main organs of the bivalve (gills and visceral mass). Micro-localisation was investigated by TEM-EDX analysis in the gills epithelium. DNA damage in gill and hemolymph samples was measured by the Comet assay. The 90-d exposure period led to a significant increase of U concentration in gills over time (× 5) and a large U quantity in subcellular granules in gills. Finally, a significant increase (× 2) in DNA damage was noted in exposed gills and haemocytes. This study shows that the accumulation levels and consequently the potential toxicity cannot be successfully predicted only on the basis of concentration in water or in tissues and subcellular fractions after chronic exposure.

Biology-based modeling to analyze uranium toxicity data on Daphnia magna in a multigeneration study.

Recent studies have investigated chronic toxicity of waterborne depleted uranium on the life cycle and physiology of Daphnia magna. In particular, a reduction in food assimilation was observed. Our aims here were to examine whether this reduction could fully account for observed effects on both growth and reproduction, for three successive generations, and to investigate through microscope analyses whether this reduction resulted from direct damage to the intestinal epithelium. We analyzed data obtained by exposing Daphnia magna to uranium over three successive generations. We used energy-based models, which are both able to fit simultaneously growth and reproduction and are biologically relevant. Two possible modes of action were compared - decrease in food assimilation rate and increase in maintenance costs. In our models, effects were related either to internal concentration or to exposure concentration. The model that fitted the data best represented a decrease in food assimilation related to exposure concentration. Furthermore, observations of consequent histological damage to the intestinal epithelium, together with uranium precipitates in the epithelial cells, supported the assumption that uranium has direct effects on the digestive tract. We were able to model the data in all generations and showed that sensitivity increased from one generation to the next, in particular through a significant increase of the intensity of effect, once the threshold for appearance of effects was exceeded.

Transfer modelling and toxicity evaluation of the effluent from an installation of cleansing and uranium recovery using a battery of bioassays.

On July 7, 2008, a leak of effluent from an Installation of Cleansing and Uranium Recovery (Tricastin, France) led to the spillage of uranium in a stream. The acute toxicity of the effluent was evaluated, and compared to the toxicity of uranium nitrate in bioassays using several organisms: Chlamydomonas reinhardtii, Daphnia magna, Chironomus riparius and Danio rerio. A sediment bioassay was also performed on C. riparius using water and sediment sampled along the river. Results showed that effluent EC(50) 72 h was 0.65 mg U/l for algae and LC(50) 48 h was 1.67 mg U/l for daphnia, while values obtained for uranium nitrate were higher. The LC(50) 96 h of effluent to C. riparius was 22.7 mg U/l, similar to value for uranium nitrate; the sediment collected was not toxic to C. riparius larvae. The LOEC of effluent and uranium nitrate on HT(50) of D. rerio were similar (0.03 mg U/l), but larvae were more sensitive to uranium nitrate than to effluent. Our results suggest that other substances contained in the effluent could potentially be toxic to wildlife in association with uranium. In parallel, the modelling of the transfers based on uranium measurements in the surface water was used to fill data gaps and assess the impact along the river. These results provided an estimate of exposure conditions that occurred along the river. This approach allowed us to see that the risk to ecosystem during this incident was certainly low and concerned a short period of time, but it could have existed at least for some species.

Effects of chronic uranium exposure on life history and physiology of Daphnia magna over three successive generations.

Daphnia magna was exposed to waterborne uranium (U) at concentrations ranging from 10 to 75 microgL(-1) over three successive generations (F0, F1 and F2). Progeny was either exposed to the same concentration as mothers to test whether susceptibility to this radioelement might vary across generations or returned to a clean medium to examine their capacity to recover after parental exposure. Maximum body burdens of 17, 32 and 54 ng U daphnid(-1) were measured in the different exposure conditions and converted to corresponding internal alpha dose rates. Low values of 5, 12 and 20 microGy h(-1) suggested that radiotoxicity was negligible compared to chemotoxicity. An increasing sensitivity to toxicity was shown across exposed generations with significant effects observed on life history traits and physiology as low as 10 microgL(-1) and a capacity to recover partially in a clean medium after parental exposure to

A probabilistic assessment of the chemical and radiological risks of chronic exposure to uranium in freshwater ecosystems.

Uranium (U) presents a unique challenge for ecological risk assessments (ERA) because it induces both chemical and radiological toxicity, and the relative importance of these two toxicities differs among the various U source terms (i.e., natural, enriched, depleted). We present a method for the conversion between chemical concentrations microg L(-1)) and radiological dose rates (microGy h(-1)) for a defined set of reference organisms, and apply this conversion method to previously derived chemical and radiological benchmarks to determine the extent to which these benchmarks ensure radiological and chemical protection, respectively, for U in freshwater ecosystems. Results show that the percentage of species radiologically protected by the chemical benchmark decreases with increasing degrees of U enrichment and with increasing periods of radioactive decay. In contrast, the freshwater ecosystem is almost never chemically protected by the radiological benchmark, regardless of the source term or decay period considered, confirming that the risks to the environment from uranium's chemical toxicity generally outweigh those of its radiological toxicity. These results are relevant to developing water quality criteria that protect freshwater ecosystems from the various risks associated with the nuclear applications of U exploitation, and highlight the need for (1) further research on the speciation, bioavailability, and toxicity of U-series radionuclides under different environmental conditions, and (2) the adoption of both chemical and radiological benchmarks for coherent ERAs to be conducted in U-contaminated freshwater ecosystems.

The effects of waterborne uranium on the hatching success, development, and survival of early life stages of zebrafish (Danio rerio).

In this study, we investigated the effects of the radioactive metal uranium (U) on the embryonic development, hatching success, growth rate, and survival of juvenile zebrafish (Danio rerio). We studied the effects of depleted uranium (20-500 microg L(-1) of DU), inducing mainly chemical toxicity due to its low specific activity, and the combined effects of chemical and radiological toxicity by using a higher specific activity uranium isotope (20 and 100 microg L(-1) of (233)U). Results showed that early life stages are significantly affected by uranium exposure through both chemical and combined (chemical and radiological) toxicity. Experiments showed significant effects of U on hatching success starting at the concentration of 250 microg L(-1) of DU, causing a 42% delay in median hatching times relative to control. Furthermore, a reduction of growth (decrease in body length and weight) was observed followed by a high mortality of pro-larvae stage (up to 100% at DU concentrations of 250 microgL(-1) upon a 15 day exposure). Bioaccumulation measurements highlighted that U was mainly localised in the chorion but penetrated in the embryo inside eggs at a higher concentration. The effects differed depending on the isotopic composition of the uranium: sublethal defects in the tail detachment process were more pronounced for (233)U than DU exposure, while the presence of (233)U specifically affected embryo development and led to higher mortality rates of the prolarvae. The results from this study showed that the early life stages of zebrafish seems to be more sensitive to uranium contamination than more mature stages, and underline the importance of including pro-larval stages into toxicity tests in order to improve the relevancy for environmental risk assessments.

Effects of waterborne uranium on survival, growth, reproduction and physiological processes of the freshwater cladoceran Daphnia magna.

Acute uranium toxicity (48 h immobilisation test) for Daphnia magna was determined in two different exposure media, differing in pH and alkalinity. LC(50) varied strongly between media, from 390+/-40 microgL(-1)U at pH 7 to 7.8+/-3.2 mgL(-1)U at pH 8. According to the free ion activity model uranium toxicity varies as a function of free uranyl concentration. This assumption was examined by calculating uranium speciation in our water conditions and in those reported in the literature. Predicted changes in free uranyl concentration could not solely explain observed differences in toxicity, which might be due to a competition or a non-competitive inhibition of H(+) for uranium transport and/or the involvement of other bioavailable chemical species of uranium. Chronic effects of uranium at pH 7 on mortality, ingestion and respiration, fecundity and dry mass of females, eggs and neonates were investigated during 21-day exposure experiments. A mortality of 10% was observed at 100 microgL(-1)U and EC(10) for reproduction was 14+/-7 microgL(-1)U. Scope for growth was affected through a reduction in feeding activity and an increase in oxygen consumption at 25 microgL(-1)U after 7 days of exposure. This had strong consequences for somatic growth and reproduction, which decreased, respectively, by 50% and 65% at 50 microgL(-1)U after 7 days and at 25 microgL(-1)U after 21 days. Uranium bioaccumulation was quantified and associated internal alpha dose rates from 2.1 to 13 microGyh(-1) were estimated. Compared to the toxicity of other alpha-emitting radionuclides and stable trace metals, our results confirmed the general assumption that uranium chemical toxicity predominates over its radiotoxicity.

Effect of selenate on growth and photosynthesis of Chlamydomonas reinhardtii.

Algal communities play a crucial role in aquatic food webs by facilitating the transfer of dissolved inorganic selenium (both an essential trace element and a toxic compound for a wide variety of organisms) to higher trophic levels. The dominant inorganic chemical species of selenium in freshwaters are selenite (SeO(3)(2-)) and selenate (SeO(4)(2-)). At environmental concentrations, selenite is not likely to have direct toxic effects on phytoplankton growth [Morlon, H., Fortin, C., Floriani, M., Adam, C., Garnier-Laplace, J., Boudou, A., 2005a. Toxicity of selenite in the unicellular green alga Chlamydomonas reinharditii: comparison between effects at the population and sub-cellular level. Aquat. Toxicol. 73(1), 65-78]. The effects of selenate, on the other hand, are poorly documented. We studied the effects of selenate on Chlamydomonas reinhardtii growth (a common parameter in phytotoxicity tests). Growth inhibition (96-h IC(50)) was observed at 4.5+/-0.2 microM selenate (p<0.001), an effective concentration which is low compared to environmental concentrations. Growth inhibition at high selenium concentrations may result from impaired photosynthesis. This is why we also studied the effects of selenate on the photosynthetic process (not previously assessed in this species to our knowledge) as well as selenate's effects on cell ultrastructure. The observed ultrastructural damage (chloroplast alterations, loss of appressed domains) confirmed that chloroplasts are important targets in the mechanism of selenium toxicity. Furthermore, the inhibition of photosynthetic electron transport evaluated by chlorophyll fluorescence induction confirmed this hypothesis and demonstrated that selenate disrupts the photosynthetic electron chain. Compared to the classical 'growth inhibition' parameter used in phytotoxicity tests, cell diameter and operational photosynthetic yield were more sensitive and may be convenient tools for selenate toxicity assessment in non-target plants.