Biological effects of palytoxin-like compounds from Ostreopsis cf. ovata: A multibiomarkers approach with mussels Mytilus galloprovincialis

Massive blooms of the harmful benthic dinoflagellate Ostreopsis cf. ovata are of growing environmental concern in the Mediterranean, having recently caused adverse effects on benthic invertebrates and also some intoxication episodes to humans.The toxicological potential of produced palytoxin-like compounds was investigated in the present study on a typical marine sentinel species, the mussel Mytilus galloprovincialis. Organisms were sampled during various phases of a O. cf. ovata bloom, in two differently impacted sites. The presence of the algal toxins was indirectly assessed in mussels tissues (mouse test and hemolysis neutralization assay), while biological and toxicological effects were evaluated through the measurement of osmoregulatory and neurotoxic alterations (Na⁺/K⁺-ATPase and acetylcholinesterase activities), oxidative stress responses (antioxidant defences and total oxyradical scavenging capacity), lipid peroxidation processes (level of malondialdehyde), peroxisomal proliferation, organelle dysfunctions (lysosomal membrane stability, accumulation of lipofuscin and neutral lipids), immunological impairment (granulocytes percentage).Obtained results demonstrated a significant accumulation of algal toxins in mussels exposed to O. cf. ovata. These organisms exhibited a marked inhibition of the Na⁺/K⁺-ATPase activity and alterations of immunological, lysosomal and neurotoxic responses. Markers of oxidative stress showed more limited variations suggesting that toxicity of the O. cf. ovata toxins is not primarily mediated by an over production of reactive oxygen species. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess biological alterations and toxicological events associated to blooms of O. cf. ovata in marine organisms.     

Decrease in catalase activity of Folsomia candida fed a Bt rice diet

Here we report the effects of three Bt-rice varieties and their non-Bt conventional isolines on biological traits including survival, reproduction, and the activities of three antioxidant enzymes superoxide dismutase, catalase and peroxidase, in the Collembolan, Folsomia candida. The reproduction was significantly lower when fed Kemingdao and Huahui1 than those feeding on their non-GM near-isogenic varieties Xiushui and Minghui63 respectively, this can be explained by the differences of plant compositions depended on variety of rice. The catalase activity of F. candida was significantly lower when fed the Bt-rice variety Kemingdao compared to the near-isogenic non-Bt-rice variety Xiushui. This suggests that some Bt-rice varieties may impose environmental stress to collembolans. We emphasize that changes in activity of antioxidant enzymes of non-target organisms are important in understanding the ecological consequences for organisms inhabiting transgenic Bt-rice plantations.     

Combined effects of soil moisture and carbaryl to earthworms and plants: simulation of flood and drought scenarios

Studying tolerance limits in organisms exposed to climatic variations is key to understanding effects on behaviour and physiology. The presence of pollutants may influence these tolerance limits, by altering the toxicity or bioavailability of the chemical. In this work, the plant species Brassica rapa and Triticum aestivum and the earthworm Eisenia andrei were exposed to different levels of soil moisture and carbaryl, as natural and chemical stressors, respectively. Both stress factors were tested individually, as well as in combination. Acute and chronic tests were performed and results were discussed in order to evaluate the responses of organisms to the combination of stressors. When possible, data was fitted to widely employed models for describing chemical mixture responses. Synergistic interactions were observed in earthworms exposed to carbaryl and drought conditions, while antagonistic interactions were more representative for plants, especially in relation to biomass loss under flood-simulation conditions.     

Transcriptome analysis provides insights for understanding the adverse effects of endosulfan in Drosophila melanogaster

Indiscriminate use of agrochemicals worldwide, particularly, persistent organic pollutants (POPs), is of concern. Endosulfan, a POP, is used by various developing/developed nations and is known to adversely affect the development and the hormonal profiles of humans and animals. However, little is known about the molecular players/pathways underlying the adverse effects of endosulfan. We therefore analyzed the global gene expression changes and subsequent adverse effects of endosulfan using Drosophila. We used Drosophila melanogaster keeping in view of its well annotated genome and the wealth of genetic/molecular reagents available for this model organism. We exposed third instar larvae of D. melanogaster to endosulfan (2.0 μg mL⁻¹) for 24 h and using microarray, we identified differential expression of 256 genes in exposed organisms compared to controls. These genes are associated with cellular processes such as development, stress and immune response and metabolism. Microarray results were validated through quantitative PCR and biochemical assay on a subset of genes/proteins. Taking cues from microarray data, we analyzed the effect of endosulfan on development, emergence and survival of the organism. In exposed organisms, we observed deformities in hind-legs, reminiscent of those observed in higher organisms exposed to endosulfan. In addition, we observed delayed and/or reduced emergence in exposed organisms when compared to their respective controls. Together, our studies not only highlight the adverse effects of endosulfan on the organism but also provide an insight into the possible genetic perturbations underlying these effects, which might have potential implications to higher organisms.     

Environmental conditions enhance toxicant effects in larvae of the ground beetle Pterostichus oblongopunctatus (Coleoptera: Carabidae)

The wide geographical distribution of ground beetles Pterostichus oblongopunctatus makes them very likely to be exposed to several environmental stressors at the same time. These could include both climatic stress and exposure to chemicals. Our previous studies demonstrated that the combined effect of nickel (Ni) and chlorpyrifos (CHP) was temperature (T)-dependent in adult P. oblongopunctatus. Frequently the different developmental stages of an organism are differently sensitive to single stressors, and for a number of reasons, such as differences in exposure routes, their interactions may also take different forms. Because of this, we studied the effects of the same factors on the beetle larvae. The results showed that all factors, as well as their interactions, influenced larvae survival. The synergistic effect of Ni and CPF was temperature-dependent and the effect of Ni × T interaction on the proportion of emerged imagines indicated stronger toxicity of Ni at 25 °C than at 10 °C.     

Ecotoxicological responses of the earthworm Eisenia fetida exposed to soil contaminated with HHCB

Although polycyclic musks have been shown to cause lethal and sub-lethal effects on organisms, their biochemical toxicity to earthworms is not well understood. In the current study, we investigated the responses of antioxidant systems and lipid peroxidation after exposing Eisenia fetida to soil contaminated with 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-γ-2-benzopyran (HHCB). Significant increase in lipid peroxidation level was observed on day 14 at two high concentrations, 50 and 100 mg kg⁻¹. Among antioxidant enzymes, the primary response to chronic HHCB exposure can be attributed to superoxide dismutase (SOD) and catalase (CAT). Of the two enzymes, SOD exhibited more sensitive response to HHCB stress. In addition, these two enzymes could have a combined effect on fighting damage by reactive oxygen species, evidenced by a marked relationship between lipid peroxidation and enzyme activity. On the other hand, dose-dependent inhibition of peroxidase (POD) activity has been observed throughout the test. The results suggest that the variations in investigated parameters of E. fetida could be used as responsive biomarkers for oxidative stress caused by HHCB in a soil environment.     

Short and long-term effects of clofibric acid and diclofenac on certain biochemical and ionoregulatory responses in an Indian major carp, Cirrhinus mrigala

Extensive use of pharmaceuticals in human and veterinary medicine and aquaculture practices pose a serious threat to aquatic organisms. In the present investigation, Cirrhinus mrigala an Indian major carp was exposed to different concentrations of clofibric acid (CA) and diclofenac (DCF) and certain biochemical and ionoregulatory responses were assessed under short and long term exposures. During short-term (96 h) exposure period, plasma glucose and sodium (Na⁺) levels were increased at all concentrations (1, 10 and 100 μg L⁻¹) of CA and DCF treated fish. Plasma protein and chloride (Cl⁻) levels were found to be decreased at all concentrations of CA and DCF exposed fish comparatively to control groups. Meanwhile an increase in plasma potassium (K⁺) level was noted in fish exposed to CA treatments alone and in DCF treatments it was decreased. In long-term exposure (35 d), plasma Na⁺ and Cl⁻ levels were found to be significantly increased at all concentration of CA and DCF. However, a biphasic trend was observed in plasma glucose, protein and K⁺ levels. In both the treatments, a significant (P < 0.01 and P < 0.05) changes were observed in all parameters measured in fish exposed to different concentrations of CA and DCF. The results of the present investigation indicate that both the drugs caused significant changes in biochemical and ionoregulatory responses of fish at all concentrations. The alterations of these parameters can be useful in monitoring of pharmaceutical residues present in aquatic environment.     

Biochemical biomarkers in Nile tilapia (Oreochromis niloticus) after short-term exposure to diesel oil, pure biodiesel and biodiesel blends

Fossil fuels such as diesel are being gradually replaced by biodiesel, a renewable energy source, cheaper and less polluting. However, little is known about the toxic effects of this new energy source on aquatic organisms. Thus, we evaluated biochemical biomarkers related to oxidative stress in Nile tilapia (Oreochromis niloticus) after two and seven exposure days to diesel and pure biodiesel (B100) and blends B5 and B20 at concentrations of 0.01 and 0.1 mL L⁻¹. The hepatic ethoxyresorufin-O-deethylase activity was highly induced in all groups, except for those animals exposed to B100. There was an increase in lipid peroxidation in liver and gills in the group exposed to the higher concentration of B5. All treatments caused a significant increase in the levels of 1-hydroxypyrene excreted in the bile after 2 and 7 d, except for those fish exposed to B100. The hepatic glutathione-S-transferase increased after 7 d in animals exposed to the higher concentration of diesel and in the gill of fish exposed to the higher concentration of pure diesel and B5, but decreased for the two tested concentrations of B100. Superoxide dismutase, catalase and glutathione peroxidase also presented significant changes according to the treatments for all groups, including B100. Biodiesel B20 in the conditions tested had fewer adverse effects than diesel and B5 for the Nile tilapia, and can be suggested as a less harmful fuel in substitution to diesel. However, even B100 could activate biochemical responses in fish, at the experimental conditions tested, indicating that this fuel can also represent a risk to the aquatic biota.     

Effects of organic contaminants in reactive oxygen species, protein carbonylation and DNA damage on digestive gland and haemolymph of land snails

The present study focused on early responses of land snails Eobania vermiculata to organic environmental contaminants, by investigating the use of a newly-established method for the measurement of protein carbonylation as a new biomarker of terrestrial pollution, as well as by measuring the ROS production and the DNA damage. Land snails were exposed to different concentrations of chlorpyrifos, parathion-methyl or PAHs in vivo or in vitro in the laboratory. The susceptibility of exposed snails was increased in relation to oxidative stress induced by contaminants tested. A statistically significant increase in ROS production, protein carbonylation and DNA damage was revealed in the snails treated with pollutants, compared to the untreated ones. The results indicated the effectiveness of measuring ROS production and DNA damage and reinforce the application of the present ELISA method in organic terrestrial pollution biomonitoring studies.     

Behavioural early warning responses to polluted water

Behavioural early warning responses to polluted surface water from an industrial effluent have been measured in two freshwater macroinvertebrate species using the four electrode impedance conversion technique. Specimens ofGammarus pulex (L.) andHydropsycho angustipennis (Curtis) were caught from reference streams and exposed to water from above and below the factory and to reference water for about 1 h with different kinds of behaviour being registered on-line every 10 min, such as time spent on locomotion, number of “high swimming peaks”, number of activity phases, cleaning, time spent on ventilation, ventilation frequency and netspinning. Additionally, G.pulex was exposedin situ above and 100 m, 500 m and 1000 m below the factory for 24 h. G.pulex reacted to water pollution within 1 h with less time spent on locomotion, fewer “high swimming peaks” and lower number of activity phases (p < 0.01). The same results were found after thein situ exposure (p < 0.05) and the pollution gradient mirrored survival and behavioural performance of the organisms. H. angustipennis reacted to water pollution within 1 h with decreased time spent on ventilation (p < 0.001), no change in locomotion and netspinning during daytime. During the night, exposure to water pollution resulted in increased locomotion (p < 0.0001). Simultaneous multispecies on-line biomonitoring of industrial effluents is recommended for reliable risk assessment.     

Screening of Cd tolerant genotypes and isolation of metallothionein genes in alfalfa (Medicago sativa L.)

In order to evaluate Cd tolerance in wide-ranging sources of alfalfa (Medicago sativa) and to identify Cd tolerant genotypes which may potentially be useful for restoring Cd-contaminated environments, thirty-six accessions of alfalfa were screened under hydroponic culture. Our results showed that the relative root growth rate varied from 0.48 to 1.0, which indicated that different alfalfa accessions had various responses to Cd stress. The candidate fragments derived from differentially expressed metallothionein (MT) genes were cloned from leaves of two Cd tolerant genotypes, YE and LZ. DNA sequence and the deduced protein sequence showed that MsMT2a and MsMT2b had high similarity to those in leguminous plants. DDRT-PCR analysis showed that MsMT2a expressed in both YE and LZ plants under control and Cd stress treatment, but MsMT2b only expressed under Cd stress treatment. This suggested that MsMT2a was universally expressed in leaves of alfalfa but expression of MsMT2b was Cadmium (Cd) inducible.     

In situ studies on growth,oxidative stress responses,and gene expression of juvenile bighead carp (Hypophthalmichthys nobilis) to eutrophic lake water dominated by cyanobacterial blooms

Cyanobacterial blooms have received increasing attention as a public biohazard for human and animal health. To assess the effect of cyanobacteria-dominant lake water on juvenile fish, we measured the responses of specific growth rate, condition factor, body weight and body length, oxidative stress, and related gene expression of juvenile bighead carp Hypophthalmichthys nobilis exposed to in situ eutrophic lake (Chl a was around 7.0 μg L⁻¹). Results showed in situ cyanobacteria-dominant lake water had no effect on the growth performance, but significantly elevated the contents of malondialdehyde, the expression of heat shock protein 70, and the activity of superoxide dismutase, indicating that oxidative stress occurred. Meanwhile in situ lake water significantly decreased the expression of catalase and glutathione S-transferase genes. We conclude that in situ cyanobacteria-dominated lake water was harmful to juvenile bighead carp based on the oxidative stress and changes in the related gene expression levels.     

Cd-tolerant Suillus luteus: A fungal insurance for pines exposed to Cd

Soil metal pollution can trigger evolutionary adaptation in soil-borne organisms. An in vitro screening test showed cadmium adaptation in populations of Suillus luteus (L.: Fr.) Roussel, an ectomycorrhizal fungus of pine trees. Cadmium stress was subsequently investigated in Scots pine (Pinus sylvestris L.) seedlings inoculated with a Cd-tolerant S. luteus, isolated from a heavy metal contaminated site, and compared to plants inoculated with a Cd-sensitive isolate from a non-polluted area. A dose-response experiment with mycorrhizal pines showed better plant protection by a Cd-adapted fungus: more fungal biomass and a higher nutrient uptake at high Cd exposure. In addition, less Cd was transferred to aboveground plant parts. Because of the key role of the ectomycorrhizal symbiosis for tree fitness, the evolution of Cd tolerance in an ectomycorrhizal partner such as S. luteus can be of major importance for the establishment of pine forests on Cd-contaminated soils.     

Monitoring behavioural responses to metals in gammarus pulex (L.) (Crustacea) with impedance conversion

An impedance conversion technique was used to study the behaviour of Gammarus pulex (L.) exposed to acutely toxic concentrations of Pb (0.01, 0.05, 0.1 and 0.5 mg Pb 1^-1) and to field concentrations of Cu (≤ 0.05 mg Cu 1^-1). Initial stress responses were studied during short-term exposure (1 h), and sublethal toxic effects were monitored during 7 (Pb) and 35 days (Cu), respectively. Exposure to Pb caused 30 % mortality and resulted in a bioconcentration factor (BCF) of 2700 at 0.5 mg Pb l^-1 after 168 h. Exposure to Cu polluted stream water caused no mortality within 35 days, and uptake was low (BCF 5.8). Gammarus pulex reacted with initial stress responses to metal exposure within 30 min (Cu) or 1 h (Pb). The reactions consisted of increased ventilation and decreased locomotion. Sublethal concentrations of Pb and Cu caused toxic effects on the behaviour of G. pulex after several days of exposure, consisting of increased ventilation and decreased locomotion. Impedance conversion is an appropriate method for detecting stress responses to metals and can be used in “early warning” biomonitoring systems as well as for acute and chronic behavioural toxicity testing.     

Ammonia emissions from two mechanically ventilated UK livestock buildings

Ammonia emission rates from livestock buildings are required to construct an accurate emission inventory for the UK. Ventilation and ammonia emission rates from a fattening pig unit and a broiler house, both mechanically ventilated, were estimated using fan wheel anemometers and thermal converters with a chemiluminescence NO_x-analyser to measure the ventilation rate and the ammonia concentration, respectively. The estimated ammonia emission factors were 46.9 and 16.6 kg lu^-1 a^-1 for the fattening pig unit and the broiler house, respectively. Both emission factors were within the range reported in the literature. A tracer gas (CO) method, based on a constant tracer release rate, was validated for measuring ventilation rates from naturally ventilated livestock buildings. Air inlets and outlets were identified using the air temperature or tracer concentration in the opening. Tracer concentration was found to be a more suitable criterion than temperature. In both houses, a significant correlation between the estimated ventilation rate using the tracer method and the measured ventilation rate using fan wheel anemometers was found. The ventilation rate was underestimated by 12 and 6% for the piggery and broiler house, respectively. The instantaneous ammonia emission derived from the tracer gas method was lower than the ammonia emission derived from the fan wheel anemometer method by 14 and 16% for the piggery and broiler house, respectively. The ventilation and ammonia emission estimates using the tracer method were within acceptable range from the ventilation and emission rates measured using measuring fans, but because of its accuracy and simplicity the fan wheel anemometer method is preferred for long-term measurements of ventilation rate in mechanically ventilated buildings.     

Effect of water quality and confounding factors on digestive enzyme activities in Gammarus fossarum

The feeding activity and subsequent assimilation of the products resulting from food digestion allow organisms to obtain energy for growth, maintenance and reproduction. Among these biological parameters, we studied digestive enzymes (amylase, cellulase and trypsin) in Gammarus fossarum to assess the impact of contaminants on their access to energy resources. However, to enable objective assessment of a toxic effect of decreased water quality on an organisms’ digestive capacity, it is necessary to establish reference values based on its natural variability as a function of changing biotic and abiotic factors. To limit the confounding influence of biotic factors, a caging approach with calibrated male organisms from the same population was used. This study applied an in situ deployment at 23 sites of the Rhone basin rivers, complemented by a laboratory experiment assessing the influence of two abiotic factors (temperature and conductivity). The results showed a small effect of conductivity on cellulase activity and a significant effect of temperature on digestive enzyme activity but only at the lowest temperature (7 °C). The experimental conditions allowed us to define an environmental reference value for digestive enzyme activities to select sites where the quality of the water impacted the digestive capacity of the organisms. In addition to the feeding rate, this study showed the relevance of digestive enzymes as biomarkers to be used as an early warning tool to reflect organisms’ health and the chemical quality of aquatic ecosystems.     

Carbamazepine, carbamazepine epoxide and dihydroxycarbamazepine sorption to soil and occurrence in a wastewater reuse site in Tunisia

Although a number of manufactured nanoparticles are applied for the medical and clinical purposes, the understanding of interaction between nanomaterials and biological systems are still insufficient. Using nematode Caenorhabditis elegans model organism, we here investigated the in vivo toxicity or safety of hydroxylated fullerene nanoparticles known to detoxify anti-cancer drug-induced oxidative damages in mammals. The survival ratio of C. elegans rapidly decreased by the uptake of nanoparticles from their L4 larval stage with resulting in shortened lifespan (20 d). Both reproduction rate and body size of C. elegans were also reduced after exposure to 100 μg mL⁻¹ of fullerol. We found ectopic cell corpses caused by apoptotic cell death in the adult worms grown with fullerol nanoparticles. By the mutation of core pro-apoptotic regulator genes, ced-3 and ced-4, these nanoparticle-induced cell death were significantly suppressed, and the viability of animals consequently increased despite of nanoparticle uptake. The apoptosis-mediated toxicity of nanoparticles particularly led to the disorder of digestion system in the animals containing a large number of undigested foods in their intestine. These results demonstrated that the water-soluble fullerol nanoparticles widely used in medicinal applications have a potential for inducing apoptotic cell death in multicellular organisms despite of their antioxidative detoxifying property.     

Growth, photosynthesis and antioxidant responses of two microalgal species, Chlorella vulgaris and Selenastrum capricornutum, to nonylphenol stress

The effect of nonylphenol (NP) on growth, photochemistry and biochemistry of two green microalgae, Chlorella vulgaris and Selenanstrum capricornutum, and their ability to degrade NP were compared. The 96 h EC₅₀ of C. vulgaris and S. capricornutum were greater than 4.0 and 1.0 mg L⁻¹ NP, respectively, suggesting that the former species was more tolerant to NP. Both microalgae acclimated to NP stress through down-regulating their photosynthetic activities, including antenna size (chlorophyll a content), maximal photochemistry (Fv/Fm) and the light absorbed by PSII (ABS/CS₀), but the dissipation of energy from reaction centres (DI₀/RC) increased with the increase of NP concentrations. In C. vulgaris, the changes of these parameters were more significant than in S. capricornutum and recovered completely after a 96 h exposure. The antioxidant responses, such as GSH content, CAT and POD activities in C. vulgaris increased with the increase of NP concentrations after a 24 h exposure, but these changes disappeared with exposure time and recovered to the control levels after 96 h. In S. capricornutum, although GSH content, CAT and POD activities also increased when exposed to low- to moderate-NP concentrations, these values were significantly reduced at a high concentration (4 mg L⁻¹) even after a 96 h exposure, indicating its antioxidant responses were significantly delayed. It is clear that the more NP-tolerant species, C. vulgaris, acclimated better with a faster recovery of its photosynthetic activity from the NP-induced damage, and exhibited more efficient and rapid responses to NP-induced oxidative stress. C. vulgaris also had a higher NP degradation ability than S. capricornutum.