Short-term Toxicity Tests Research Articles

A candidate short-term toxicity test using Ampelisca brevicornis to assess sublethal responses to pharmaceuticals bound to marine sediments.

Lethal and sublethal responses related to different phases of metabolism (phases I and II enzymatic activities), neurotoxicity (acetylcholinesterase activity), oxidative stress (lipid peroxidation and antioxidant enzyme activities), and genetic damage (DNA strand breaks) were analysed to assess the possible adverse effects of pharmaceuticals bound to marine sediments. The crustacean amphipod Ampelisca brevicornis was chosen as the bioindicator species. Organisms were exposed for 10 days to sediment spiked with pharmaceutical compounds frequently used and previously detected in the environment: carbamazepine (CBZ), ibuprofen (IBP), fluoxetine (FX), 17α-ethynylestradiol (EE2), propranolol (PRO), and caffeine (CAF). Short-term bioassay to evaluate amphipod mortality was recommended to assess pollution by CBZ, FX, and PRO. IBP and PRO were metabolized by phases I and II detoxification enzymatic activities. Oxidative stress was caused by PRO and CAF. Contrary to expected results, DNA damage (strand breaks) decreased after the exposure of amphipods to sediment spiked with IBP, FX, EE2, PRO, and CAF (including environmental concentrations). FX was neurotoxic to amphipods. The battery of biomarkers tested allowed the assessment of bioavailability, oxidative stress, genotoxicity, and neurotoxicity of the pharmaceuticals analysed. The results of this study suggested that pharmaceutical products at concentrations currently found in the environment might cause a wide variety of adverse effects (based on laboratory studies). The results obtained here are useful for environmental risk assessment of marine sediments contaminated by pharmaceuticals. Nevertheless, more research is needed using field-based marine sediments.

Effects of mixtures of dissolved and particulate contaminants on phototrophic biofilms: new insights from a PICT approach combining toxicity tests with passive samplers and model substances.

Streams located in vineyard areas are particularly exposed to mixtures of dissolved and particulate contaminants such as metals and organic pesticides. In this context, phototrophic biofilms are increasingly used as indicators of river water contaminations through pollution-induced community tolerance (PICT) assessments based on short-term toxicity tests with individual or mixtures of toxicants. We conducted a laboratory experiment to evaluate the relative influence of the dissolved and particulate fractions on the effects of metals and pesticides on phototrophic biofilms in a context of contamination from a vineyard watershed. Three sets of artificial channels were supplied with (i) unfiltered water from a stream reference site, (ii) unfiltered water from a stream contaminated site, and (iii) filtered water (0.45μm) from the same contaminated site. Biofilm growth, diatom community structure, and dissolved toxicant concentrations differed slightly between channels supplied with unfiltered or filtered water from the contaminated site. However, PICT assessments with individual toxicants or mixtures of toxicants extracted from passive samplers suggested no significant difference in tolerance to metals and organic pesticides between phototrophic communities supplied with unfiltered or filtered contaminated water. Our results confirm the use of extracts from passive samplers as a promising approach in short-term toxicity tests to characterize impacts of contamination on aquatic communities.

The effect of glufosinate on nitrogen assimilation at the physiological, biochemical and molecular levels in Phaeodactylum tricornutum.

This study investigated the effects of glufosinate, a widely used herbicide, on the marine diatom Phaeodactylum tricornutum through short-term toxicity tests at the physiological and gene transcriptional levels. Glufosinate (4 mg L(-1)) decreased the amount of pigments but increased reactive oxygen species (ROS) and malondialdehyde levels. As a glutamine synthetase (GS) inhibitor, glufosinate affected the transcripts and activities of key enzymes related to nitrogen assimilation. Transcript levels of GS and nitrate reductase (NR) in P. tricornutum decreased to only 57 and 26 % of the control. However, transcript levels of nitrate transporter (NRT) and the small subunit of glutamate synthase (GltD) were 1.79 and 1.76 times higher than that of the control. The activities of NRT, GS and GOGAT were consistent with gene expression except for NR, which was regulated mainly by post-translational modification. Furthermore, the results of electron microscopy showed that chloroplast structure was disrupted in response to glufosinate exposure. These results demonstrated that glufosinate first disturbed nitrogen metabolism and caused a ROS burst, which disrupted chloroplast ultrastructure. Ultimately, the growth of P. tricornutum was greatly inhibited by glufosinate.

Linking community tolerance and structure with low metallic contamination: A field study on 13 biofilms sampled across the Seine river basin

It is difficult to assess the biological consequences of diffuse water contamination by micropollutants which are present in rivers at low, even sublethal levels. River biofilms, which respond quickly to changes of environmental parameters, are good candidates to acquire knowledge on the response of aquatic organisms to diffuse chemical contamination in the field. The study was designed as an attempt to link biofilm metal tolerance and metallic contamination in a field survey covering 13 different sampling sites in the Seine river basin (north of France) with low contamination levels. Cd and Zn tolerance of heterotrophic communities was assessed using a short-term toxicity test based on β-glucosidase activity. Metal tolerance levels varied between sites but there was no obvious correlation between tolerance and corresponding water contamination levels for Cd and Zn. Indeed, metallic contamination at the sampling sites remained subtle when compared to water quality standards (only two sampling sites had either Zn or both Cu and Zn concentrations exceeding the Environmental Quality Standards set by the EU Water Framework Directive). Yet, multivariate analysis of the data using Partial Least Squares Regression revealed that both metallic and environmental parameters were important variables explaining the variability of metal tolerance levels. Automated Ribosomal Intergenic Spacer Analysis (ARISA) was also performed on both bacterial and eukaryotic biofilm communities from the 13 sampling sites. Multivariate analysis of ARISA fingerprints revealed that biofilms with similar tolerance levels have similar ARISA profiles. Those results confirm that river biofilms are potential indicators of low, diffuse contamination levels of aquatic systems.

Efficacy Trial In An Acute Liver Trauma Animal Model Of Novel Infusible Hemostatic Product: Red Blood Cell-Derived Microparticles (RMP)

BackgroundIncreasing evidence indicates that natural circulating red cell microparticles (RMP) contribute significantly to hemostasis. Accordingly, we produced RMP in vitro by high pressure extrusion of washed RBCs. We previously reported the hemostatic activity of our RMP product in vitro as well as its efficacy in arresting ear bleeding in rabbits, suggesting the potential of RMP as a hemostatic agent (Jy et al Thromb. Haemost., in press). Here we studied its hemostatic efficacy in a rabbit model of acute liver injury, as well as its short-term toxicity. MethodsMale New Zealand White rabbits (mean weight 3.8kg) were randomly selected and assigned, one at a time, to an experimental (n = 9) or a control group (n = 10). All animals were sedated with 35mg/kg ketamine, 5mg/kg xylazine, and 0.01mg/kg glycopyrrolate 15 minutes before surgery. They were intubated and anesthetized with 2% isoflurane, and mechanically ventilated at approximately 20 breaths/min. The carotid artery and the jugular vein were canulated, and maintenance saline was administered at 20mL/hr. The abdomen was cleaned and shaved, and an incision was made from lower tip of the sternum to the bladder. A standardized injury of 9 incisions 30mm long by 4mm deep was inflicted to the liver, and two infusions of RMP (1.25x1011/kg) or saline were delivered via jugular catheter, first immediately post-incision, and again 10 minutes post-incision. Blood was collected in gauze pads at 10 min. intervals and weighed to calculate volume lost. Heart rate, blood pressure (BP), and temperature were continually monitored. The blood pressure (BP) of some of the animals fell very low (MAP<15) by 1 hr post-incision and resulted in death. Only those rabbits that survived 2 hr were used in analysis of blood loss and vital signs. Five of the 10 control rabbits survived 2 hr post-surgery (50%) compared to 5 of the 9 RMP-treated rabbits (56%). All surviving rabbits were euthanized with 0.2mL/kg Euthasol after 120 minutes. ResultsAt early time points (30 – 60 min post-injury) when bleeding was most rapid, administration of RMP had no significant effect: mean (± SD) blood loss at 30 min was 46.1 ± 20.6 mL in the control vs. 26.6 ± 9.0 mL (p=0.07) in the RMP group. At 60 min it was 67.2 ± 24.3 mL vs. 53.4 ± 6.9 mL (p=0.23), respectively. Although at these early time points there were not significant differences, a trend of reduced blood loss was observed.At the 90 min time point, blood loss was 78.6 ± 25.9 mL in the control group compared to 55.7 ± 5.6 mL in the RMP group (p<0.05), a relative difference of ≈ 30%. At 120 min, the corresponding values were 89.8 ± 29.3 mL and 57.7 ± 4.4 mL (p < 0.01), a relative difference of ≈ 36% .Vitals were not significantly different between the control and RMP groups at any time point. Drop in BP tended to be greater in controls than in RMP rabbits. The difference in BP was marginally significant at 120 min. post-injury: systolic BP fell by 39.6 ±17.2 mmHg in controls vs. 18.6 ±15.0 mmHg in RMP-treated (p=0.07). In a short-term toxicity test, quadruple dose (5.0x1011/kg of RMP) was administered and effects observed for three hours: no obvious adverse effects on heart rate, BP, or temperature were observed. Conclusion/DiscussionWe previously reported dose-dependent reduction by RMP infusion in ear bleeding time of thrombocytopenic rabbits. Efficacy of infusions of RMP is evident in this severe hemorrhagic liver injury model after 30 - 60 min of acute injury when rapid bleeding was substantially slowing down. At 90 and 120 min post-injury, significantly less blood loss was observed in the RMP group compared to the controls, with relative differences of 30% and 36%. Studies are in progress to improve the model and to optimize dosages and scheduling of RMP administration for severe bleeding. Disclosures:No relevant conflicts of interest to declare.

Do Biopesticides Affect the Demographic Traits of a Parasitoid Wasp and Its Biocontrol Services through Sublethal Effects?

Pesticide risk assessments are usually based on short-term acute toxicity tests, while longer-term population dynamic related traits, critical to the success of biological control and Integrated Pest Management (IPM) programs, are often overlooked. This is increasingly important with respect to new biopesticides that frequently cause no short-term acute effects, but that can induce multiple physiological and behavioral sublethal effects, leading to a decrease in population growth and ecosystem services. In this study we assessed the lethal and sublethal effects of six biopesticides [abamectin, azadirachtin, Bacillus thuringiensis, borax plus citrus oil (Prev-Am®), emamectin benzoate, and spinosad], used in tomato crops to control the invasive pest Tuta absoluta (Lepidoptera: Gelechiidae), on adults and pupae of the parasitoid Bracon nigricans (Hymenoptera: Braconidae). Data on female survival and production of female offspring were used to calculate population growth indexes as a measure of population recovery after pesticide exposure. Spinosad caused 100% and 80% mortality in exposed adults (even 10 d after the treatment) and pupae, respectively. Although most of the biopesticides had low levels of acute toxicity, multiple sublethal effects were observed. The biocontrol activity of both females that survived 1-h and 10-d old residues, and females that emerged from topically treated pupae was significantly affected by the application of the neurotoxic insecticides emamectin benzoate and abamectin. Furthermore, very low B. nigricans demographic growth indices were estimated for these two insecticides, indicating potential local extinction of the wasp populations. Among the tested products, Bt proved to be the safest for B. nigricans adults and pupae. Our findings emphasize that acute toxicity assessment alone cannot fully predict the actual impact of pesticides on non-target parasitoids. Thus, sublethal effects related to the species specific life-history variables must be carefully considered in order to assess pesticide risks and to incorporate new pesticides, including biopesticides, into IPM programmes.

Derivation of South African water quality guidelines for Roundup® using species sensitivity distribution

Glyphosate-based herbicides are among the leading products used in South Africa to control weeds and invading alien plant species. Although these herbicides ultimately find their way into aquatic ecosystems, South Africa has no water quality guideline based on indigenous species to protect the country's aquatic biota against these biocides. In this study, South African water quality guidelines (SAWQGs) for Roundup® based on species sensitivity distribution (SSD) using indigenous aquatic biota were developed. Short-term and long-term toxicity tests were conducted with eight different aquatic species belonging to five different taxonomic groups. Static non-renewal experimental methods were employed for short-term lethal tests (≤4 days), and static renewal for long-term sublethal tests (≥4 days ≤21 days). LC50 values for animal exposure and EC50 values for algae were calculated using probit analysis and linear regression of transformed herbicide concentration as natural logarithm data against percentage growth inhibition, respectively. No effect concentration (NEC) was determined based on the dynamic energy budget model, using survival data. The LC50, EC50 and NEC values were used to develop species sensitivity distribution (SSD) concentrations for Roundup®. Based on the SSD concentrations, the short-term and long-term SAWQGs for Roundup® were derived as 0.250 (0.106–0.589) mg/L, and 0.002 (0.000–0.021) mg/L, respectively. These WQGs may be useful in protecting South African aquatic life against transient or long-term exposure to glyphosate-based chemicals as part of integrated water resources management.

Mercury Induced Biochemical Alterations As Oxidative Stress In Mugil Cephalus In Short Term Toxicity Test

Mugil cephalus juveniles of size 2.5 ±0.6cm were exposed to mercury in short term chronic toxicity test through static renewal bioassay to detect the possible biochemical agent as biomarkers in aquatic pollution and in estuarine contamination as specific. Lipid peroxidation levels, glutathione S-transferase, catalase, reduced glutathione and acetylcholinesterase were studied as biochemical parameters.Increased thio-barbituric acid reactive substances levels were observed under exposure to mercury, leading to the oxidative damage resulting in lipid peroxidation. Decreased activities of antioxidants, catalase and increased glutathione-S-transferase under short term chronic exposures to mercury were more prominent in metal stress suggesting activation of physiological mechanism to scavenge the ROS produced. Decreased values of reduced glutathione on prolonged exposures to mercury indicate utilization of this antioxidant, either to scavenge oxy-radical or act in combination with other enzymes. The acetylcholinesterase activity was found to be decreased during mercury exposure. The results also suggest that mercury alters the active oxygen metabolism by modulating antioxidant enzyme activities, which can be used as biomarker to detect sub-lethal effects in aquatic pollution.

Safety evaluation of Morinda citrifolia (noni) leaves extract: assessment of genotoxicity, oral short term and subchronic toxicity

Morinda citrifolia L (noni) is an evergreen or small tree that grows in many tropical regions of the world. The use of the noni leaves has not been so studied however; there are reports of its pharmacological benefits. Aims: The objective of this investigation was to assess the genotoxicity, short-term, and subchronic oral toxicity of Morinda citrifolia L leaves aqueous extract. Methods: The genotoxicity of the M. citrifolia extract was investigated by measuring the frequency of micronuclei in mice bone marrow cells. The animals were treated with three doses of the extract (500, 1000, and 2000 mg/kg). For short-term toxicity, both sexes Wistar rats received 1000 mg/kg/day for 28 days. Animals were sacrificed for hematological and biochemical evaluation. For the subchronic study, Wistar rats were administered with three doses of M. citrifolia extract (100, 300, and 1000 mg/kg) by oral route for 90 days. Mortalities, clinical signs, body weight changes, food and water consumption, hematological and biochemical parameters, gross findings, organ weights, and histological examination were monitored during the study period. Results: Genotoxicity and short-term toxicity test resulted in absence of toxicity at doses between 500 and 2000 mg/kg. Significant differences were observed in hemoglobin, and differential leukocyte count after subchronic dosing of the extract. Histology evaluation did not reveal treatment-related abnormalities. Variations observed were within to normal range and reversible. Conclusions: In summary, 1000 mg/kg orally was the NOAEL for M. citrifolia extract for effects other than transient variations in some hematological parameters within normal range.

A New, Sensitive Marine Microalgal Recombinant Biosensor Using Luminescence Monitoring for Toxicity Testing of Antifouling Biocides

In this study, we propose the use of the marine green alga Ostreococcus tauri, the smallest free-living eukaryotic cell known to date, as a new luminescent biosensor for toxicity testing in the environment. Diuron and Irgarol 1051, two antifouling biocides commonly encountered in coastal waters, were chosen to test this new biosensor along with two degradation products of diuron. The effects of various concentrations of the antifoulants on four genetic constructs of O. tauri (based on genes involved in photosynthesis, cell cycle, and circadian clock) were compared using 96-well culture microplates and a luminometer to automatically measure luminescence over 3 days. This was compared to growth inhibition of O. tauri wild type under the same conditions. Luminescence appeared to be more sensitive than growth inhibition as an indicator of toxicity. Cyclin-dependent kinase (CDKA), a protein involved in the cell cycle, fused to luciferase (CDKA-Luc) was found to be the most sensitive of the biosensors, allowing an accurate determination of the 50% effective concentration (EC(50)) after only 2 days (diuron, 5.65 ± 0.44 μg/liter; Irgarol 1015, 0.76 ± 0.10 μg/liter). The effects of the antifoulants on the CDKA-Luc biosensor were then compared to growth inhibition in natural marine phytoplankton. The effective concentrations of diuron and Irgarol 1051 were found to be similar, indicating that this biosensor would be suitable as a reliable ecotoxicological test. The advantage of this biosensor over cell growth inhibition testing is that the process can be easily automated and could provide a high-throughput laboratory approach to perform short-term toxicity tests. The ability to genetically transform and culture recombinant O. tauri gives it huge potential for screening many other toxic compounds.

Assessment of Cyfluthrin commercial formulation on growth, photosynthesis and catalase activity of green algae

Aquatic environments of the pampasic region of Argentina are severely affected by agricultural contamination due to an increase in a glyphosate tolerant transgenic variety of soybean crops. The present study is aimed to determine the effects of a commonly used Cyfluthrin commercial formulation (CCF) on growth, some physiological and biochemical parameter of four species of green algae. Significant inhibition of algal growth was observed from 0.1mg Cyf/l. 96h IC50 were between 0.92 and 4.85mg Cyf/l. CCF caused algicidal effects. Photosynthesis was stimulated by 50% in Scenedesmus quadricauda cultures exposed to the lowest concentration (hormesis). Algal photosynthesis inhibition was observed at higher concentrations with IC50 values between 1.7 and 8.9mg Cyf/l. Similar toxicity endpoints were found as a consequence of applying the traditional methodology of short-term chronic toxicity test of 96h of exposition and the methodology developed using the Clark type photosynthetic oxygen evolution method. CAT activity was significantly increased between 23% and 33% considering the four species, at a lower concentration than those affecting algal growth and photosynthesis, indicating a potential biomarker. Taking into account that the extent of the soybean crops in the region is about fourteen million hectares, the improvement and extension of environmental tools for early detection of the action of pesticides on this essential group of organisms are discussed.

One-Hour Temperature-Based Toxicity Test with Daphnia magna

A 1-hour temperature-based toxicity test using Daphnia magna was developed and then compared with a 24-hour standard acute toxicity test. The 1-hour toxicity test was based on the observation that daphnids in high temperatures become weak and more susceptible to toxicants than daphnids at room temperature. Theorizing that this relationship could be utilized to produce a short-term toxicity test, we analyzed the experimental data and identified 36.5°C as the ideal temperature for a 1-hour temperature-based toxicity test. After the test was formulated, it was used to screen 15 toxicants. Eight heavy metals, 4 pesticides, 2 anions, phenol, and 2 industrial wastewaters were screened, using the 1-hour toxicity test and the 24-hour standard acute toxicity test. For the 1-hour toxicity test, vials containing 5 daphnids (<24 h), medium, and toxicant, all at room temperature, were prepared and placed in a water bath set at 36.5°C for 1 hour. After 1 hour, the numbers of live (no toxic effect) daphnids and immobilized (toxic effect) daphnids were counted, and the median effective concentrations (EC50) were determined. A comparison of the EC50 values of the 1-hour temperature-based toxicity test and those of the 24-hour standard acute toxicity test produced a correlation of 0.9999. When individual toxicants were compared, the 24-hour standard acute toxicity test was better at detecting lower test concentrations than the 1-hour temperature-based toxicity test. When both tests were used to screen industrial wastewater and their results were compared, the 1-hour toxicity test showed some promise. However, further testing will need to be done, because only 2 of the 10 samples collected initially contained significant amounts of toxicants for EC50 calculation. Finally, while the 1-hour toxicity test was effective in screening 15 tested toxicants and has the potential to be useful in screening industrial wastewater, this conclusion is limited to those 15 tested toxicants and 2 industrial wastewaters. Additional experiments will be needed for other toxicants. The 1-hour toxicity test presented in this study is the first temperature-based toxicity test developed for D. magna, and it will be an attractive short-term assay for its convenience, cost-efficiency, and replicability.

Pollution-induced community tolerance of freshwater biofilms: measuring heterotrophic tolerance to Pb using an enzymatic toxicity test

This study aims at investigating the impacts of Pb on freshwater biofilms with a pollution-induced community tolerance (PICT) approach using a recently developed short-term toxicity test based on β-glucosidase activity to measure biofilms' tolerance to Pb. We first investigated more closely the influence of the total suspended solids (TSS) concentrations of biofilm suspensions used for short-term toxicity tests performed to assess Pb tolerance. The Pb EC(50) values of four dilutions of the same biofilm suspension increased with their TSS concentrations. TSS-normalization allowed to obtain a unique measure of Pb tolerance, thus confirming that TSS-normalization of EC(50) values is a good means to estimate biofilm tolerance to Pb. The experiment was repeated with three different biofilm samples collected at different sites and dates. Second, biofilms were exposed to Pb (0, 1, 10 and 100 μg/L) for 3 weeks in microcosms to assess the impacts of Pb exposure on the communities. An increase in Pb tolerance was observed for the biofilm exposed to 100 μg/L. Automated Ribosomal Intergenic Spacer Analysis revealed modifications of bacterial and eukaryotic community structure with Pb exposure. Moreover, exposure to 100 μg/L Pb also led to an increase in Zn tolerance but not Cu tolerance. This study shows that tolerance acquisition to Pb can be detected after exposure to environmental concentrations of Pb using a PICT methodology and normalized EC(50) values as measures of Pb tolerance.

Short-term toxicity test: monitoring Klebsiella oxytoca bacterium respiration using a flow injection analysis/conductometric system

In this study, the Klebsiella oxytoca bacterium was used as a test organism in short-term toxicity evaluations, and Escherichia coli was used as reference. The inhibition of bacterial growth was quantified by flow injection analysis (FIA) via conductometric measurements of the CO2 produced during respiration. The results were expressed as effective concentration (EC50) values. K. oxytoca was more resistant than E. coli in respect to growth inhibition. The metal sensitivity order for K. oxytoca was found to be Hg2+ > Cd2+ > Cu2+ and Hg2+ > Cu2+ > Cd2+ for E. coli. The sensitivity to the Laborhex 2 detergent (active ingredient: chlorhexidine digluconate) was 1.55 ± 0.32 mg L-1 and 0.32 ± 0.10 mg L-1 for K. oxytoca and E. coli, respectively. The bacteria showed comparable sensitivities to the Riodeine Degermant detergent (active ingredient: polyvinyl pyrrolidone-iodine-PVP-I), of 11.0 ± 1.7 mg L-1 and 12.0 ± 2.0 mg L-1, for K. oxytoca and E. coli, respectively.

Cadmium affects toxicokinetics of pyrene in the collembolan Folsomia candida

Since toxicity is time dependent, short-term toxicity tests may overlook mixture effects, because chemical accumulation within an organism takes time. We therefore studied the effects of cadmium on the toxicokinetics of pyrene and its metabolites in the soil-dwelling collembolan Folsomia candida exposed through Lufa 2.2 soil. Single pyrene was rapidly taken up and steady state was reached within the 337-h exposure period. Simultaneous exposure to cadmium significantly decreased the pyrene uptake and elimination rate, resulting in a prolonged half life. Kinetics of the first phase metabolite OH-pyrene was also significantly influenced by cadmium. Cadmium increased the hydroxylation rate of pyrene but slowed down its further metabolization, again resulting in a prolonged half life. We showed that pyrene accumulation and metabolization are significantly influenced by the presence of cadmium. Our results suggest that mixture effects may be dependent on exposure time.

Assessment of diclofenac LC50 reference values in juvenile and embryonic stages of the zebrafish (Danio rerio)

The aim of the study was to compare the acute toxicity of diclofenac to juvenile and embryonic stages of the zebrafish (Danio rerio). Acute toxicity tests were performed on the aquarium fish Danio rerio, which is one of the model organisms most commonly used in toxicity testing. The tests were performed using a semi-static method according to OECD guideline No. 203 (Fish, acute toxicity test). Embryo toxicity tests were performed in zebrafish embryos (Danio rerio) in compliance with OECD No. 212 methodology (Fish, short-term toxicity test on embryo and sac-fry stages). The results were subjected to a probit analysis using the EKO-TOX 5.2 programme to determine 96hLC50 and 144hLC50 (median lethal concentration, 50% mortality after a 96 h or 144 h interval, respectively) values of diclofenac. The statistical significance of the difference between LC50 values in juvenile and embryonic stages of Danio rerio was tested using the Mann-Whitney non-parametric test implemented in the Unistat 5.1 programme. The LC50 mean value of diclofenac was 166.6 +/- 9.8 mg/L in juvenile Danio rerio, and 6.11 +/- 2.48 mg/L in embryonic stages of Danio rerio. The study demonstrated a statistically higher sensitivity to diclofenac (P < 0.05) in embryonic stages compared to the juvenile fish.

Adaptation of copper community tolerance levels after biofilm transplantation in an urban river

The Water Framework Directive requires the development of biological tools which can act as early-warning indicators of a sudden increase (accidental pollution) or decrease (recovery due to prevention) of the chemical status of aquatic systems. River biofilms, which respond quickly to modifications of environmental parameters and also play a key part in the functioning of aquatic ecosystems, are therefore good candidates to monitor an increase or a decrease of water pollution. In the present study, we investigated the biological response of biofilms transplanted either upstream (recovery) or downstream (deterioration of exposure levels) the urban area of Paris (France). Both modifications of Cu community tolerance levels and of global bacterial and eukaryotic community structure using automated ribosomal intergenic spacer analysis (ARISA) fingerprints were examined 15 and 30 days after the transplantation. Cu tolerance levels of the heterotrophic component of biofilms were assessed using a short-term toxicity test based on β-glucosidase (heterotrophic) activity. Cu tolerance increased for biofilms transplanted upstream to downstream Paris (5-fold increase on day 30) and conversely decreased for biofilms transplanted downstream to upstream (8-fold decrease on day 30). ARISA fingerprints revealed that bacterial and eukaryotic community structures of transplanted biofilms were closer to the structures of biofilms from the transplantation sites (or sites with similar contamination levels) than to biofilms from their sites of origin. Statistical analysis of the data confirmed that the key factor explaining biofilm Cu tolerance levels is the sampling site and not the site of origin. It also showed that Cu tolerance levels are related to the global urban contamination (both metals and nutrients). The study shows that biofilms adapt fast to modifications of their surroundings. In particular, community tolerance varies quickly and reflects the new exposure levels only 15 days after transplantation. Those results support the use of biofilms as reliable early-warning indicators of diffuse urban contamination.

A subchronic in situ exposure method for evaluating effects in small‐bodied fish at contaminated sites

In situ fish-caging studies at contaminated sites can provide information that is more realistic compared to traditional laboratory-based studies. However, few methods have been developed for exposing sentinel fish species for subchronic durations, and fewer still are optimized for exposing small-bodied fish while maintaining fish health and growth throughout the caging trial. Those methods typically lack a feeding regimen during the fish caging trial. While that may be acceptable or even appropriate for typical short-term toxicity testing, it does limit the duration of the exposure, and may not be suitable when post-caging trials or observations are necessary. Returning healthy fish to the lab following the in situ exposure would be important, for example, in studies designed to examine long-term or multigenerational effects following an in situ exposure. In this article we describe a subchronic method for caging small fish at contaminated sites while maintaining growth and reproductive development. Fathead minnow (Pimephales promelas) were caged in situ for 6 weeks, after which time they were returned to the lab where they were evaluated for health and reproductive performance. Growth and reproductive endpoints revealed no adverse effect on fish due to fish caging and related handling, demonstrating the suitability of our caging and feeding method for long-term caging studies.

Biological Safety Assessment of a Colored Zirconia Ceramic: Hemolysis and Short-Term Systemic Toxicity Tests

Objective: To evaluate the biological safety of a colored zirconia ceramic regarding its hemolytic activity and short-term systemic toxicity. Methods: Fresh anti-coagulating rabbit blood was mixed with test materials and the hemolytic activities detected spectrophotometrically. Suspensions of the ceramic were fed to rats for one week while monitoring food consumption and relative growth rate and with subsequent pathology examinations of vital organs and tissues. Results: The hemolytic activity of the ceramics was &lt;5%, and rats in all groups showed weekly relative growth rates of food utilization and body weight with no statistically significant differences. There were also no pathological changes observed among the various examined organs between the experimental and control groups. Conclusions: This colored ceramic showed good biocompatibility in terms of hemolytic activity and short-term systemic toxicity.

Biological Surface Coating and Molting Inhibition as Mechanisms of TiO2 Nanoparticle Toxicity in Daphnia magna

The production and use of nanoparticles (NP) has steadily increased within the last decade; however, knowledge about risks of NP to human health and ecosystems is still scarce. Common knowledge concerning NP effects on freshwater organisms is largely limited to standard short-term (≤48 h) toxicity tests, which lack both NP fate characterization and an understanding of the mechanisms underlying toxicity. Employing slightly longer exposure times (72 to 96 h), we found that suspensions of nanosized (∼100 nm initial mean diameter) titanium dioxide (nTiO2) led to toxicity in Daphnia magna at nominal concentrations of 3.8 (72-h EC50) and 0.73 mg/L (96-h EC50). However, nTiO2 disappeared quickly from the ISO-medium water phase, resulting in toxicity levels as low as 0.24 mg/L (96-h EC50) based on measured concentrations. Moreover, we showed that nTiO2 (∼100 nm) is significantly more toxic than non-nanosized TiO2 (∼200 nm) prepared from the same stock suspension. Most importantly, we hypothesized a mechanistic chain of events for nTiO2 toxicity in D. magna that involves the coating of the organism surface with nTiO2 combined with a molting disruption. Neonate D. magna (≤6 h) exposed to 2 mg/L nTiO2 exhibited a “biological surface coating” that disappeared within 36 h, during which the first molting was successfully managed by 100% of the exposed organisms. Continued exposure up to 96 h led to a renewed formation of the surface coating and significantly reduced the molting rate to 10%, resulting in 90% mortality. Because coating of aquatic organisms by manmade NP might be ubiquitous in nature, this form of physical NP toxicity might result in widespread negative impacts on environmental health.