The Contribution of Acyl Amides to the Toxicity of the Harmful Alga Prymnesium parvum

The golden alga Prymnesium parvum has been implicated in fish and aquatic animal kills globally for over a century. In addition to widespread ecological impacts through the loss of entire fish populations within lakes, an economic burden is also felt by state and local agencies due to losses of fish raised for stocking lakes and the loss of fishing and recreational use of the affected water body. Multiple compounds have been implicated in P. parvum toxicity, but the unequivocal identification and characterization of all P. parvum toxins remains to be accomplished. In this work we isolated and structurally characterized toxic metabolites from P. parvum, examining uni-algal laboratory cultures and field collections of algal biomass from fish kill sites. An aggregate of saturated and unsaturated acyl amides are a previously undescribed class of P. parvum toxins. The amount of acyl amides present at multiple P. parvum bloom sites and reference sites was quantified. Further analysis of these compounds through selected bioassays demonstrated that acyl amides were cytotoxic, hemolytic, and ichthyotoxic, with the gill as the site of action. In addition, we demonstrated that the presence of divalent cations and increased pH significantly increased the toxicity of unsaturated acyl amides. Moreover, increased pH decreased the toxicity of fatty acids, a class of compounds previously implicated in P. parvum toxicity. Multiple abiotic and biotic factors are associated with P. parvum blooms and the toxicity of these blooms. We analyzed the effect of multiple physicochemical parameters on the toxicity of acyl amides using an artificial neural network and linear statistical tests. Our results demonstrated that increasing pH is significantly associated with an increase in the toxicity of acyl amides. Mixtures of environmentally relevant concentrations of unsaturated acyl amides at ecologically relevant pH levels resulted in a significant loss of viability of rainbow trout gill cells. Our results have identified a new toxin class from P. parvum. This is the first report to demonstrate that a toxin class implicated in P. parvum toxicity accumulates to lethal levels in the environment. We have developed a new method for analyzing complex mixtures that can be utilized to assess the effect of multiple chemical and physical factors on toxicity. These results highlight the potential role of physicochemical factors and their effect on algal toxins after they are released into the environment, illustrating the necessity to examine toxin chemistry in an environmentally relevant context

Bioaccumulation and histopathology of copper in Oreochromis mossambicus

Cu is one of the most toxic elements that affect fish populations when the fish are exposed to concentrations exceeding their tolerance. To investigate the effects of elementary Cu on aspects of bioconcentration, histology and behaviour, O. mossambicus were exposed to 0 and 0.75 ± 0.20 mg/l of Cu for 96 hours (short-term study), and 0, 0.11 ± 0.02, 0.29 ± 0.02, and 0.47 ± 0.04 mg/l of Cu for 64 days (longterm study) under controlled conditions in the laboratory. For the long-term study fish were sampled for gills, liver, and kidney Cu accumulation analysis after 1, 32 and 64 days of exposure and after 1, 2, 4, 16, 32, and 64 days for gills, liver and spleen histology analysis. Cu accumulation was concentration-duration dependent with the highest accumulation capacity in the liver. A multifactor linear model was developed for the relationship between exposure dose, exposure duration and Cu accumulation in the organs with the liver model: Log L = 3.35 + 0.85W + 0.31T (r² = 0.892) giving a better fit than the gills: G = −35.09 + 10.58W + 17.58T (r² = 0.632). Where L = Cu accumulation values in the liver, G = Cu accumulation values in the gills (both in μg/g dry mass); W = exposure dose in water (mg/l); and T = exposure time (days). Using this model Cu accumulation in organs can be estimated when exposure concentration and duration is known. This model should be tested under different conditions to determine the potential of the model in monitoring Cu toxicity in the environment. Lesions were observed in the liver, gills and spleen in all Cu treatments at all exposure concentration and exposure durations. However, the incidence and the degree of alteration was related to the concentration of Cu and duration of exposure. The sequential appearance of lesions in the order of, hepatic vacuolar degeneration, fatty degeneration and necrosis indicated a gradual increase in liver damage with larger duration of exposure time and increasing Cu concentration. The initial lesions in the gills were manifested as hypertrophy and hyperplasia of the gill epithelium causing increase in the thickness of the secondary lamellae, mucous cell hypertrophy and proliferation, mucous hypersecretion, proliferation of eosinophilic granule cells and hyperplasia of interlamellar cells. With increase in exposure time, necrosis of the eosinophilic granule cells, lamellar oedema, epithelial desquamation and increase in severity of lamellar hyperplasia were observed. These lesions indicated an initial defence mechanism of the fish against Cu toxicity followed by advanced histological changes that were related to Cu concentration and duration of exposure. Changes in the spleen were haemosiderosis, increase in the white pulp and macrophage centres, reduction in the red pulp, and necrosis suggesting that fish exposed to environmentally relevant levels of Cu may be histopathologically altered leading to anaemia and immunosuppression. Regression analysis was used to quantify the relationship between the total activity of the fish, and duration of exposure. There was a gradual decline in fish activity related to Cu concentration and duration of exposure before introducing food into the tanks. There was a constant activity after introducing food in the tanks at the control and 0.11 ± 0.02 mg/l Cu exposure levels irrespective of exposure time. Analysis of covariance (ANCOVA) was used to test for the difference in slopes between treatments. There was no significant difference (p > 0.05) between slopes of the control and 0.11 ± 0.02 mg/l Cu, and between 0.29 ± 0.02 and 47 ± 0.04 mg/l Cu before and after introducing food in the tanks. The slopes of both the control and 0.11 ± 0.02 mg/l Cu were significantly different from those of 0.29 ± 0.02 and 0.47 ± 0.04 mg/l Cu (p < 0.05). There were significant differences in the mean opercular movements per minute between treatments (p < 0.05). There was hyperventilation at 0.11 ± 0.02 mg/l Cu i.e. 87 ± 18 opercular movements per minute (mean ± standard deviation) and hypoventilation at 0.29 ± 0.02 and 0.47 ± 0.04 mg/l Cu i.e. 37 ± 34 and 13 ± 6 opercular movements per minute compared to the control. Hypo- and hyperventilation were related to the lesser and greater gill damage, respectively. In conclusion Cu accumulation and effects on histology of the liver, gills and were related to the concentration of Cu in the water and duration of exposure showing a gradual increase in incidence and intensity with larger duration of exposure time and increasing Cu concentration. The fish were initially able to homeostatically regulate and detoxify Cu. However, as the exposure continued, the homeostatic mechanism appears to have failed to cope with the increasing metal burden causing advanced histological changes

Bioaccumulation and histopathology of copper in Oreochromis mossambicus

Cu is one of the most toxic elements that affect fish populations when the fish are exposed to concentrations exceeding their tolerance. To investigate the effects of elementary Cu on aspects of bioconcentration, histology and behaviour, O. mossambicus were exposed to 0 and 0.75 ± 0.20 mg/l of Cu for 96 hours (short-term study), and 0, 0.11 ± 0.02, 0.29 ± 0.02, and 0.47 ± 0.04 mg/l of Cu for 64 days (longterm study) under controlled conditions in the laboratory. For the long-term study fish were sampled for gills, liver, and kidney Cu accumulation analysis after 1, 32 and 64 days of exposure and after 1, 2, 4, 16, 32, and 64 days for gills, liver and spleen histology analysis. Cu accumulation was concentration-duration dependent with the highest accumulation capacity in the liver. A multifactor linear model was developed for the relationship between exposure dose, exposure duration and Cu accumulation in the organs with the liver model: Log L = 3.35 + 0.85W + 0.31T (r² = 0.892) giving a better fit than the gills: G = −35.09 + 10.58W + 17.58T (r² = 0.632). Where L = Cu accumulation values in the liver, G = Cu accumulation values in the gills (both in μg/g dry mass); W = exposure dose in water (mg/l); and T = exposure time (days). Using this model Cu accumulation in organs can be estimated when exposure concentration and duration is known. This model should be tested under different conditions to determine the potential of the model in monitoring Cu toxicity in the environment. Lesions were observed in the liver, gills and spleen in all Cu treatments at all exposure concentration and exposure durations. However, the incidence and the degree of alteration was related to the concentration of Cu and duration of exposure. The sequential appearance of lesions in the order of, hepatic vacuolar degeneration, fatty degeneration and necrosis indicated a gradual increase in liver damage with larger duration of exposure time and increasing Cu concentration. The initial lesions in the gills were manifested as hypertrophy and hyperplasia of the gill epithelium causing increase in the thickness of the secondary lamellae, mucous cell hypertrophy and proliferation, mucous hypersecretion, proliferation of eosinophilic granule cells and hyperplasia of interlamellar cells. With increase in exposure time, necrosis of the eosinophilic granule cells, lamellar oedema, epithelial desquamation and increase in severity of lamellar hyperplasia were observed. These lesions indicated an initial defence mechanism of the fish against Cu toxicity followed by advanced histological changes that were related to Cu concentration and duration of exposure. Changes in the spleen were haemosiderosis, increase in the white pulp and macrophage centres, reduction in the red pulp, and necrosis suggesting that fish exposed to environmentally relevant levels of Cu may be histopathologically altered leading to anaemia and immunosuppression. Regression analysis was used to quantify the relationship between the total activity of the fish, and duration of exposure. There was a gradual decline in fish activity related to Cu concentration and duration of exposure before introducing food into the tanks. There was a constant activity after introducing food in the tanks at the control and 0.11 ± 0.02 mg/l Cu exposure levels irrespective of exposure time. Analysis of covariance (ANCOVA) was used to test for the difference in slopes between treatments. There was no significant difference (p > 0.05) between slopes of the control and 0.11 ± 0.02 mg/l Cu, and between 0.29 ± 0.02 and 47 ± 0.04 mg/l Cu before and after introducing food in the tanks. The slopes of both the control and 0.11 ± 0.02 mg/l Cu were significantly different from those of 0.29 ± 0.02 and 0.47 ± 0.04 mg/l Cu (p < 0.05). There were significant differences in the mean opercular movements per minute between treatments (p < 0.05). There was hyperventilation at 0.11 ± 0.02 mg/l Cu i.e. 87 ± 18 opercular movements per minute (mean ± standard deviation) and hypoventilation at 0.29 ± 0.02 and 0.47 ± 0.04 mg/l Cu i.e. 37 ± 34 and 13 ± 6 opercular movements per minute compared to the control. Hypo- and hyperventilation were related to the lesser and greater gill damage, respectively. In conclusion Cu accumulation and effects on histology of the liver, gills and were related to the concentration of Cu in the water and duration of exposure showing a gradual increase in incidence and intensity with larger duration of exposure time and increasing Cu concentration. The fish were initially able to homeostatically regulate and detoxify Cu. However, as the exposure continued, the homeostatic mechanism appears to have failed to cope with the increasing metal burden causing advanced histological changes

Copper Binding to Fish Gill Cell Lines: Towards Determination of Biotic Ligand Model Parameters

In aquatic environments, copper is a potentially toxic element that is also an essential nutrient, aiding in the growth and development of all aerobic organisms. In small quantities, copper acts as a micronutrient, playing a large role in many enzymatic reactions, however, at increased concentrations, copper is a potential aquatic pollutant that can cause metal toxicity. The biotic ligand model (BLM) is a useful tool that is often used for metals risk assessment and helps to establish protective water criteria guidelines. Previously, the BLM has been used to model metal complexation directly at the biotic ligand (e.g., gill) for both whole organisms and primary gill cells but has not yet been used to model metal complexation directly for gill cells from a continuous cell culture. Cell viability curves, using two fluorescence indicator dyes, Alamar Blue and CFDA-AM, were generated using both the RTgill-W1 and LSgill-e cell lines to determine what concentrations of copper (II) sulfate induces cell toxicity. These tests confirmed the titratable range for both cell lines in order to maintain viability was 0-300 µM in L-15, a specific culture media. For this study, graphite furnace atomic absorption spectroscopy (GFAAS) was used to directly characterize copper binding to both a rainbow trout gill cell line (RTgill-W1) and a lake sturgeon gill cell line (LSgill-e) in a monolayer, in suspension, and in cell culture inserts where copper titrations were performed at non-toxic concentrations. Both cell lines showed weaker binding than primary rainbow trout gill pavement cells and up to an order of magnitude less binding than primary rainbow trout gill mitochondrial rich cells. Results suggest that the interaction of copper with cells derived from a continuous cell line is much weaker than interactions with cells from a primary culture. Based on research performed, these results suggest the use of RTgill-W1, or LSgill-e cell lines would most likely not suffice as an alternative to whole organism and primary cell testing for viability and toxicity assays

An integrated approach to assess impact of environmental stress in carp, Cyprinus carpio L.: Biochemical, genotoxic, histopathological and individual level effects

Full version unavailable due to 3rd party copyright restrictions.Studies were undertaken to determine toxicological effects in a model species, Cyprinus carpio L. following hypoxic exposure either alone or in combination with representative heavy metal (i.e. copper; Cu) via a dietary route, at different levels of biological organisation (viz. biochemical, histological and individual level effects). Initially, the validation study of biological responses using a range of concentrations of dietary Cu as a relevant environmental contaminant was carried out (Chapter 3). The results showed a range of biological responses in exposed fish including significant genotoxic response as determined by induction of DNA strand breaks (i.e. the Comet assay) with bacterial enzymes Fpg and Endo-III (for detection of oxidative DNA damage) and reduction in growth rate suggesting the robustness of selected biomarkers. Subsequently, this approach was used initially to determine the biological responses following chronic hypoxic and hyperoxic exposure (Chapter 4). The results suggested that both hypoxic and hyperoxic conditions lead to a range of comparable biological responses. Following relative evaluation of chronic hypoxic and hyperoxic exposures, experiments were carried out to elucidate potential interactive effect of hypoxia in combination with dietary Cu (Chapter 5). The combined exposure of hypoxia and Cu induced a significantly higher level of DNA damage suggesting that DNA damage in fish can serve as a sensitive biomarker for changes in water quality as well as presence of genotoxic chemicals. The final sets of experiment were carried out to determine the biological responses in C. carpio following exposure to chronic hypoxic stress and subsequent recovery in normoxic condition for 7 days. Real-time PCR (qPCR) technology was used to examine the hypoxia inducible Factor-1 α (HIF-1α) gene expression pattern (Chapter 6). The results suggested that the expression levels of HIF-1α in response to hypoxia were significantly higher compared to normoxic controls, a high level of oxidative DNA damage under hypoxia and re-exposure to normoxic condition (i.e. recovery period). This will shed lights for development of adaptive response in higher vertebrates, which could also have significant clinical implications in human health.Ministry of Higher Education and Scientific Research/Republic of Ira

Histopathological effects of metal and metalic nanoparticles on the body systems of rainbow trout (Oncorhynchus mykiss)

Histopathology studies of metal nanoparticles (NPs) compared to traditional forms of metal in fish are scarce. Additionally, it is unclear whether metal nanoparticles cause greater or different pathologies compared to other forms of metal. The current study aimed to assess the pathological effects of Cu-NPs and TiO2 NPs on rainbow trout via various routes of exposure and, where appropriate, to compare them to either the equivalent dissolved metal salts or bulk powder forms. The first experiment showed that waterborne exposure to Cu-NPs and CuSO4 caused similar types of organ pathologies and alteration in the spleen content, however there were some material-type effects in the incidence injuries; with Cu-NPs in some organs by causing more injury in the intestine, liver, and brain when compared to effects caused by the equivalent concentration of CuSO4. Lowering water pH did have an effect on the toxicity of Cu-NPs and dissolved Cu in trout, and the results illustrated that both Cu treatments are more toxic at pH 5 than pH 7 by causing more physiological and pathological changes, although both CuSO4 and Cu-NP treatments showed similar types of organ lesions. Waterborne exposure to TiO2 NPs and bulk forms of TiO2 showed similar types of organ pathologies and alteration in the spleen contents, but there was a material-type effect in some organs (more injury with the bulk treatment than the NP form). After 96 h following intravenous injections of bulk or TiO2 NPs in trout, organs showed similar types of pathologies; except the spleen and kidney which showed a material-type effects (more injury with NPs than the bulk forms). This could be attributed to the highest Ti accumulation from the TiO2 NP treatment in the kidney and spleens, or to the role of these organs in filtrating the circulating blood. Overall, this thesis demonstrates that metal-NPs produced similar types of organ pathologies to traditional forms of metals through different routes of exposure, but there were some material-type effects on the incidence of injuries in some organs. The results have also added some understanding on the fate, and effects of NPs by identifying the target organs involved. Some of the nano-specific effects may need to be given extra consideration in environmental and human health risk assessments.Iragi Cultural Attache embass

Bioavailability and toxicity of nickel to freshwater organisms : a modeling approach

Water quality criteria setting for metals is hampered by the fact that the bioavailability and hence also the toxicity of many metals is largely influenced by physicochemical parameters such as water hardness, pH and dissolved organic matter. Consequently, a single water quality criterium may be too conservative for certain water bodies whereas it may not be protective enough for other water bodies. This metal-specific issue can be overcome with the development of mathematical models capable of predicting metal bioavailability and toxicity as a function of water chemistry. In the first part of this doctoral thesis it was investigated to what extent several physicochemical parameters (such as calcium, magnesium, pH, dissolved organic matter) affect the acute and chronic toxicity of nickel to freshwater organisms. For each testing organism (a unicellular green alga, an aquatic invertebrate and a fish) a bioavailability model was developed capable of accurately predicting nickel toxicity in both artificial and natural waters. An important point of concern was that bioavailability models such as those developed in the first part of this doctoral thesis may underestimate metal toxicity in waters with hardness far below the lower water hardness boundary for which these models were originally developed and validated using ‘standard’ test organisms. In the second part of this research it was therefore investigated whether this concern is scientifically justified for nickel. A series of toxicity tests with field-organisms (microalgae and water fleas) originating from soft and hard surface waters demonstrated that in most cases soft water organisms are equally sensitive to nickel as hard water organisms and that a single bioavailability model can be used to predict the water hardness-dependent toxicity of nickel to soft and hard water organisms. The data and models generated in this thesis were already used in the European risk assessment of nickel and nickel compounds. The pan-European water quality criterium for nickel will be based on the outcome of this risk assessment. Current developments at the international regulatory level feed the expectation that the application domain of this thesis will further expand

Etude des impacts toxiques des contaminants chimiques du Bassin d'Arcachon sur l'huitre cultivée Crassostrea gigas : Approche in-situ et expérimentale

The Arcachon Bay is a semi-enclosed lagoon and represents the core of strong economic stakes through the practice of oyster-farming. This activity has been affected for around thirty years by summer mortality events, and more recently by abnormally high death rates of juveniles. This work focused on the role of the Arcachon Bay’s main contaminants in this crisis, by studying their effects on the cupped oyster’s biology. Firstly, an in situ approach was adopted in order to identify the major pollutants of the bay: caged oysters were transplanted and sediments were sampled. The presence of contaminants in the samples was associated to biological responses. Secondly, the contaminants identified in situ were used in controlled conditions at the laboratory. Three experimentations were performed; the first dealt with the contamination pathways of tributyltin; the second focused on the biological effects of pesticides and copper; the third concerned the effects of PAH towards diploid and triploid oysters. Our results indicate that the inner stations present higher accumulation of metals and PAH, in accordance with the hydrodynamic features of the bay. The bioindicators used in situ exhibited seasonal trends related to the oysters’ gametogenesis. In the laboratory, most of the contaminants used at environmental levels induced an adaptive response of the exposed oysters. Our study highlights the importance of coupling in situ and laboratory approaches in order to understand the functioning of coastal ecosystems.Le bassin d’Arcachon est une lagune semi-fermée qui concentre de forts enjeux économiques grâce à la pratique de l’ostréiculture. Cette activité est affectée depuis une trentaine d’années par des phénomènes de mortalités estivales, et plus récemment par des surmortalités du naissain. Ces travaux se sont intéressés au rôle des polluants majeurs du bassin d’Arcachon dans ce contexte de crise en étudiant leurs effets sur la biologie de Crassostrea gigas. Une approche in situ a d’abord été adoptée afin d’identifier les contaminants les plus présents dans le milieu naturel. Des opérations de transplantation d’huîtres et des prélèvements de sédiments ont permis de quantifier divers contaminants et d’associer leur présence à des réponses biologiques. Les polluants ainsi identifiés ont ensuite été employés en conditions contrôlées au laboratoire. Trois expérimentations ont été réalisées : la première concerne l’étude des voies de contamination par le tributylétain ; la seconde concerne les effets des pesticides et du cuivre ; la troisième concerne l’effet des HAP sur des huîtres diploïdes et triploïdes. Nos résultats indiquent que les organismes transplantés au coeur de la lagune sont plus exposés aux polluants, en lien avec les caractéristiques hydrodynamiques du système. La plupart des paramètres biologiques étudiés sur le terrain ont par ailleurs montré une saisonnalité liée aux processus de gamétogenèse. Au laboratoire, la plupart des contaminants testés ont induit une réponse adaptative chez les huîtres exposées. Notre étude souligne l’importance de coupler approche de terrain et approche expérimentale pour comprendre le fonctionnement des écosystèmes côtiers