Common and inter-specific toxic effects in three wild fish species after chronic gamma irradiation of early stages.

The objective of this study was to investigate the effects of gamma irradiation on the aquatic environment. We used three wild fish species to compare phenotypic responses with a fish model such as Danio rerio. We focused on embryonic development, a sensitive life stage to stressors like ionizing radiation, to evaluate the effects of exposure to 0.5 and 5 mGy h-1 on Arctic char, trout and stickleback embryos from fertilization to free-swimming larvae. Irradiation did not cause mortality but induced an acceleration of hatching in the three species. These new data on wild species, obtained under comparable irradiation conditions, did not go against the threshold values for the protection of freshwater aquatic ecosystems. Moreover, irradiation caused inter-specific sublethal effects, such as an increase in non-eyed egg proportion in Arctic char, an increase in the incubation period in trout and an acceleration of larval mortality in stickleback. The consequences of these early effects on the adult stage remain to be studied.

Physiological effects of gamma irradiation in the honeybee, Apis mellifera.

Terrestrial ecosystems are exposed to various kinds of pollutants, including radionuclides. The honeybee, Apis mellifera, is commonly used in ecotoxicology as a model species for evaluating the effects of pollutants. In the present study, honeybees were irradiated right after birth for 14 days with gamma rays at dose rates ranging between 4.38 × 10-3 and 588 mGy/d. Biological tissues (head, intestine and abdomen) were sampled at D3, D10 and D14. Ten different physiological markers involved in nervous (acetylcholinesterase (AChE)), antioxidative (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST)), immune system (phenoloxidase (PO)) and metabolism (carboxylesterases (CaEs) and alkaline phosphatase (ALP)) were measured. Univariate analyses were conducted to determine whether each individual biomarker response was positively or negatively correlated with the dose rate. Then, multivariate analyses were applied to investigate the relationships between all the biomarker responses. Although no mortality occurred during the experiment, several biomarkers varied significantly in relation to the dose rate. Globally, the biomarkers of antioxidant and immune systems decreased as the dose rate increased. Reversible effects on the indicator of the neural system were found. Concerning indicators of metabolism (carboxylesterases), variations occurred but no clear pattern was found. Taken altogether, these results help better understand the effects of ionizing radiation on bees by identifying relevant physiological markers of effects. These results could improve the assessment of the environmental risk due to ionizing radiation in terrestrial ecosystems.

Uptake, depuration, dose estimation and effects in zebrafish exposed to Am-241 via dietary route.

Zebrafish were chronically exposed to Am-241, an alpha-emitting radionuclide via daily consumption of an enriched artificial diet. Am-241 uptake was quantified in Danio rerio after 5 and 21 days of exposure via daily Am-spiked food ingestion and after 21 days of exposure followed by 5 days of depuration. Americium accumulates mostly in digestive tract, muscle, rest of the body but the accumulation levels and trophic transfer rate (0.033-0.013%) were low. Corresponding cumulative doses were calculated for the whole body (9 mGy) and for the digestive tract (42 mGy) with internal alpha radiation contributing to more than 99% of the total dose. Genotoxic effects (gamma-H2AX assay) and differential gene expressions of main biological functions were examined. Although fish were exposed to a low dose rate of 13 µGy h-1, DNA integrity and gene expression linked to oxidative stress, hormonal signaling and spermatogenesis were altered after 21 days of Am-241 exposure. These results underline the higher toxicity of alpha emitter Am-241, as compared to other studies on gamma radiation exposure.

Effects of in situ exposure to tritiated natural environments: A multi-biomarker approach using the fathead minnow, Pimephales promelas.

Aquatic ecosystems are chronically exposed to radionuclides as well as other pollutants. Increased concentrations of pollutants in aquatic environments can present a risk to exposed organisms, including fish. The goal of this study was to characterize the effects of tritium, in the context of natural environments, on the health of fathead minnow, Pimephales promelas. Fish were exposed to tritium (activity concentrations ranging from 2 to 23,000Bq/L) and also to various concentrations of several metals to replicate multiple-stressor environments. Fish were exposed for 60days, then transferred to the tritium background site where they stayed for another 60days. Tritium, in the forms of tritiated water (HTO) and organically bound tritium (OBT), and a series of fish health indicators were measured in fish tissues at seven time points throughout the 120days required to complete the exposure and the depuration phases. Results showed effects of environmental exposure following the increase of tritium activity and metals concentrations in water. The internal dose rates of tritium, estimated from tissue HTO and OBT activity concentrations, were consistently low (maximum of 0.2µGy/h) compared to levels at which population effects may be expected (>100µGy/h) and no effects were observed on survival, fish condition, gonado-somatic, hepato-somatic, spleno-somatic and metabolic indices (RNA/DNA, proteins/DNA and protein carbonylation (in gonads and kidneys)). Using multivariate analyses, we showed that several biomarkers (DNA damages, MN frequency, gamma-H2AX, SFA/MUFA ratios, lysosomal membrane integrity, AChE, SOD, phagocytosis and esterase activities) were exclusively correlated with fish tritium internal dose rate, showing that tritium induced genotoxicity, DNA repair activity, changes in fatty acid composition, and immune, neural and antioxidant responses. Some biomarkers were responding to the presence of metals, but overall, more biomarkers were linked to internalized tritium. The results are discussed in the context of multiple stressors involving metals and tritium.

External gamma irradiation-induced effects in early-life stages of zebrafish, Danio rerio.

In the general context of validation of tools useful for the characterization of ecological risk linked to ionizing radiation, the effects of an external gamma irradiation were studied in zebrafish larvae irradiated for 96 h with two dose rates: 0.8 mGy/d, which is close to the level recommended to protect ecosystems from adverse effects of ionizing radiation (0.24 mGy/d) and a higher dose rate of 570 mGy/d. Several endpoints were investigated, such as mortality, hatching, and some parameters of embryo-larval development, immunotoxicity, apoptosis, genotoxicity, neurotoxicity and histological alterations. Results showed that an exposure to gamma rays induced an acceleration of hatching for both doses and a decrease of yolk bag diameter for the highest dose, which could indicate an increase of global metabolism. AChE activity decreased with the low dose rate of gamma irradiation and alterations were also shown in muscles of irradiated larvae. These results suggest that gamma irradiation can induce damages on larval neurotransmission, which could have repercussions on locomotion. DNA damages, basal ROS production and apoptosis were also induced by irradiation, while ROS stimulation index and EROD biotransformation activity were decreased and gene expression of acetylcholinesterase, choline acetyltransferase, cytochrome p450 and myeloperoxidase increased. These results showed that ionizing radiation induced an oxidative stress conducting to DNA damages. This study characterized further the modes of action of ionizing radiation in fish.

Detection of immunotoxic effects of estrogenic and androgenic endocrine disrupting compounds using splenic immune cells of the female three-spined stickleback, Gasterosteus aculeatus (L.).

Today, the list of endocrine disrupting compounds (EDCs) in freshwater and marine environments that mimic or block endogenous hormones is expanding at an alarming rate. As immune and reproductive systems may interact in a bidirectional way, some authors proposed the immune capacities as attractive markers to evaluate the hormonal potential of environmental samples. Thus, the present work proposed to gain more knowledge on direct biological effects of natural and EDCs on female fish splenic leucocyte non-specific immune activities by using ex vivo assays. After determining the optimal required conditions to analyze splenic immune responses, seven different EDCs were tested ex vivo at 0.01, 1 and 100nM over 12h on the leucocyte functions of female three-spined stickleback, Gasterosteus aculeatus. In summary, we found that natural hormones acted as immunostimulants, whilst EDCs were immunosuppressive.

Effects of low-dose exposure to pesticide mixture on physiological responses of the Pacific oyster, Crassostrea gigas.

This study investigated the effects on the physiology of Pacific oyster, Crassostrea gigas, of a mixture of pesticides containing 0.8 µg L(-1) alachlor, 0.6 µg L(-1) metolachlor, 0.7 µg L(-1) atrazine, 0.6 µg L(-1) terbuthylazine, 0.5 µg L(-1) diuron, 0.6 µg L(-1) fosetyl aluminum, 0.05 µg L(-1) carbaryl, and 0.7 µg L(-1) glyphosate for a total concentration of 4.55 µg L(-1) . The total nominal concentration of pesticides mixture corresponds to the pesticide concentrations in the shellfish culture area of the Marennes-Oleron basin. Two varieties of C. gigas were selected on the foreshore, based on their characteristics in terms of resistance to summer mortality, to assess the effects of the pesticide mixture after 7 days of exposure under controlled conditions. The early effects of the mixture were assessed using enzyme biomarkers of nitrogen metabolism (GS, glutamine synthetase), detoxification metabolism (GST, glutathione S-transferase), and oxidative stress (CAT, catalase). Sublethal effects on hemocyte parameters (phagocytosis and esterase activity) and DNA damages (DNA adducts) were also measured. Changes in metabolic activities were characterized by increases in GS, GST, and CAT levels on the first day of exposure for the "resistant" oysters and after 3-7 days of exposure for the "susceptible" oysters. The formation of DNA adducts was detected after 7 days of exposure. The percentage of hemocyte esterase-positive cells was reduced in the resistant oysters, as was the hemocyte phagocytic capacity in both oyster varieties after 7 days of exposure to the pesticide mixture. This study highlights the need to consider the low doses and the mixture of pesticides to evaluate the effects of these molecules on organisms.

Developmental energetics of zebrafish, Danio rerio.

Using zebrafish (Danio rerio) as a case study, we show that the maturity concept of Dynamic Energy Budget (DEB) theory is a useful metric for developmental state. Maturity does not depend on food or temperature contrary to age and to some extent length. We compile the maturity levels for each developmental milestone recorded in staging atlases. The analysis of feeding, growth, reproduction and aging patterns throughout the embryo, juvenile and adult life stages are well-captured by a simple extension of the standard DEB model and reveals that embryo development is slow relative to adults. A threefold acceleration of development occurs during the larval period. Moreover we demonstrate that growth and reproduction depend on food in predictable ways and their simultaneous observation is necessary to estimate parameters. We used data on diverse aspects of the energy budget simultaneously for parameter estimation using the covariation method. The lowest mean food intake level to initiate reproduction was found to be as high as 0.6 times the maximum level. The digestion efficiency for Tetramin™ was around 0.5, growth efficiency was just 0.7 and the value for the allocation fraction to soma (0.44) was close to the one that maximizes ultimate reproduction.

Pollutant effects on Pacific oyster, Crassostrea gigas (Thunberg), hemocytes: screening of 23 molecules using flow cytometry.

The shellfish industry is an important economic activity in France, occurring mostly in estuarine zones subject to pollution due to anthropogenic activities. The harmful effects of pollutants on species inhabiting these estuarine zones are not well known. Among marine species, bivalve mollusks--particularly Pacific oyster, Crassostrea gigas--may serve a model of interest. The species is sedentary and filter-feeding, which favors bioaccumulation of pollutants in their tissues. Oysters may be suitable for studies on disturbance by pollutants of physiological activities, among which defense mechanisms are poorly documented in bivalves. In this study, effects of pollutants on hemocyte functions were monitored in Pacific oyster, C. gigas. Hemocytes were exposed in vitro to selected pollutants. The strategy for investigating the effects of pollutants on hemocyte functions is based on several biomarkers, which is more relevant than that of published papers based on single-endpoint experiments. Pollutants belonging to the most important groups of xenobiotics (PAHs, PCBs, and pesticides) were selected and their effect on hemocyte activities was analyzed using flow cytometry. Twenty-three pollutants were tested and eight of them showed significant modulation of hemocyte activities. PAHs and PCB 77 induced a decrease of hemocyte activity after an incubation periods of 4 and 24 h at 200 micro mol/L. Three pesticides (2,4D, paraoxon, and chlorothalonil) modulated hemocyte activities. A mixture of eight pesticides also decreased phagocytotic activity. This study is one of the first to investigate the effects of so many pollutants on hemocyte functions at the same time and therefore allows a real comparison of different pollutant effects.

A flow cytometric approach to study intracellular-free Ca2+ in Crassostrea gigas haemocytes.

Bivalve haemocytes are essential in defence mechanisms including phagocytosis. They also produce molecules including hydrolytic enzymes and antimicrobial peptides that contribute to pathogen destruction. Although haemocyte activities have been extensively studied, relatively little is known about the intracellular signalling pathways that are evoked during haemocyte activation and especially the role of calcium. Flow cytometry has been used for the first time to define the effect of cell incubation in haemolymph and artificial sea water (ASW) on Pacific oyster, Crassostrea gigas, haemocytes. Cell viability, enzymatic activities (esterases and aminopeptidases), phagocytosis and granulocyte percentage were analysed. Viability and some activities were different in haemolymph and ASW. Cytoplasmic-free calcium in circulating haemocytes was then investigated by flow cytometry in both media using a calcium probe (Fluo-3/AM). To explore calcium homeostasis, different calcium modulators were tested. The calcium chelator Bapta/AM (10 microM) reduced significantly the percentage of Fluo-3-positive cells in ASW. In addition, ryanodine (5 microM) induced a significant enhancement of the percentage of Fluo-3 positive cells in haemolymph and in ASW. Flow cytometry may be used to study calcium movements in C. gigas haemocytes, but several haemocyte incubation media need to be tested in order to confirm results. The objective of the study should be considered before selecting a particular experimental medium.

In vitro effects of cadmium and mercury on Pacific oyster, Crassostrea gigas (Thunberg), haemocytes.

In the past decades, shellfish culture has developed in a significant way around the world. However, culture areas are often subject to recurring anthropic pollution. The recrudescent presence of industrial wastes is a source of heavy metals and results in pollutant transfer towards the aquatic environment in estuarine areas. Because of their mode of life, bivalves, including mussels and oysters, are suggested as ideal indicator organisms. The development of techniques allowing the analysis of the effects of pollutants on bivalve biology may lead to the monitoring of pollutant transfer in estuarine areas. In this context, the effects of cadmium and mercury on defence mechanisms were analysed in Pacific oysters, Crassostrea gigas. Pollutant effects were tested in vitro on oyster haemocytes. Cell viability and enzymatic activities (esterase, peroxidase, aminopeptidase, phagocytosis activities) were monitored by flow cytometry. Enzymatic phenoloxidase-like activity was also evaluated by spectrophotometry. High pollutant concentrations were used in order to detect the acute effect and to approach real pollutant concentrations existing in animal tissues. Cadmium induced no effect on oyster haemocytes under the tested conditions. On the contrary, mercury caused a significant haemocyte mortality after a 24 h in vitro incubation. Aminopeptidase positive cell percentage was enhanced by the pollutant, and phenoloxidase-like activity was inhibited. These in vitro results show that mercury may be expected to have an impact on bivalve immune functions in contaminated areas.

Study of atrazine effects on Pacific oyster, Crassostrea gigas, haemocytes.

Shellfish farming is an important economic activity around the world. This activity often takes place in areas subjected to various recurring pollutions. The recrudescent use of herbicides in agriculture including atrazine implies pollutant transfer towards aquatic environment in estuarine areas. Harmful effects of such substances on animals in marine environment, particularly on cultured bivalves, are poorly documented. Bivalve molluscs such as mussels and oysters have been postulated as ideal indicator organisms because of their way of life. They filter large volumes of seawater and may therefore accumulate and concentrate contaminants within their tissues. Moreover, development of techniques allowing effect analysis of such compounds on bivalve biology may lead to the development of diagnosis tools adapted to analyze pollutant transfer towards estuarine areas. In this context, influence of atrazine on defence mechanisms was analyzed in Pacific oysters, Crassostrea gigas. Atrazine was tested in vitro and in vivo on oyster haemocytes, and its effects were analyzed by flow cytometry. Haemocyte viability, cell cycle and cellular activities were monitored. Atrazine induced no significant effect in oyster under tested conditions except for peroxidase activity.