Daphnia Magna Assay Research Articles

Comparative toxicology of algal cell extracts and pure cyanotoxins: insights into toxic effects and mechanisms of harmful cyanobacteria Raphidiopsis raciborskii

Ongoing research on cyanotoxins, driven by the socioeconomic impact of harmful algal blooms, emphasizes the critical necessity of elucidating the toxicological profiles of algal cell extracts and pure toxins. This study comprehensively compares Raphidiopsis raciborskii dissolved extract (RDE) and cylindrospermopsin (CYN) based on Daphnia magna assays. Both RDE and CYN target vital organs and disrupt reproduction, development, and digestion, thereby causing acute and chronic toxicity. Disturbances in locomotion, reduced behavioral activity, and weakened swimming capability in D. magna have also been reported for both RDE and CYN, indicating the insufficiency of conventional toxicity evaluation parameters for distinguishing between the toxic effects of algal extracts and pure cyanotoxins. Additionally, chemical profiling revealed the presence of highly active tryptophan-, humic acid-, and fulvic acid-like fluorescence compounds in the RDE, along with the active constituents of CYN, within a 15-day period, demonstrating the chemical complexity and dynamics of the RDE. Transcriptomics was used to further elucidate the distinct molecular mechanisms of RDE and CYN. They act diversely in terms of cytotoxicity, involving oxidative stress and response, protein content, and energy metabolism, and demonstrate distinct modes of action in neurofunctions. In essence, this study underscores the distinct toxicity mechanisms of RDE and CYN and emphasizes the necessity for context- and objective-specific toxicity assessments, advocating nuanced approaches to evaluate the ecological and health implications of cyanotoxins, thereby contributing to the precision of environmental risk assessments.

Synthesis, Characterization and Cytotoxic Evaluation of New Pyrrolo[1,2-b]pyridazines Obtained via Mesoionic Oxazolo-Pyridazinones.

New pyrrolo[1,2-b]pyridazines were synthesized by 3 + 2 cycloaddition reaction between mesoionic oxazolo-pyridazinones and methyl/ethyl propiolate. The mesoionic compounds were generated in situ by action of acetic anhydride on 3(2H)pyridazinone acids obtained from corresponding esters by alkaline hydrolysis followed by acidification. The structures of the compounds were confirmed by elemental analyses and IR, 1H-NMR, 13C-NMR, and X-ray diffraction data. The regioselectivity of cycloaddition was evidenced by NMR spectroscopy and confirmed by X-ray analysis. The compounds were evaluated for their cytotoxicity on plant cells (Triticum aestivum L.) and crustacean animal cells (Artemia franciscana Kellogg and Daphnia magna Straus). The results indicated that the tested compounds exhibited low toxicity on the plant cell (IC50 values higher than 200 µM), while on Artemia nauplii no lethality was observed. Daphnia magna assay showed that pyrrolo[1,2-b]pyridazines 5a and 5c could exhibit toxic effects, whereas, for the other compounds, toxicity was low to moderate. Also, the cytotoxic effects of the compounds were tested on three human adenocarcinoma-derived adherent cell lines (colon LoVo, ovary SK-OV-3, breast MCF-7). The in vitro compound-mediated cytotoxicity assays, performed by the MTS technique, demonstrated dose- and time-dependent cytotoxic activity for several compounds, the highest anti-tumor activity being observed for 5a, 2c, and 5f, especially against colon cancer cells.

Ecotoxicological Assessment of Water and Sediment in Areas of Taal Lake with Heavy Aquaculture Practices Using Allium cepa and Daphnia magna Assay

Aquaculture is one of the main livelihoods of people living near Taal Lake. However, the quality of the lake is deteriorating due to the increasing number of fish cages and fish pens. In this study, ecotoxicological assessment of water and sediment in three areas (Gonzales, Bañaga, and Balakilong) of Taal Lake were conducted using Allium cepa and Daphnia magna assay. The result of A. cepa assay showed that areas with heavy aquaculture practices have a higher percentage of chromosomal aberrations (CAs) such as anaphase bridge, precocious chromosome, sticky chromosome, C-mitosis, and micronucleus (MN). This indicates the presence of cytotoxic and genotoxic compounds in the lake water. On the other hand, assessment of sediment samples using D. magna assay showed that the population exposed to samples from areas with heavy aquaculture practice showed a significantly lower number of offsprings compared to the control area. The result of our study shows the effectiveness of the two bioassays on the evaluation of ecotoxicity of water and sediment samples.

Chemical characterization and biopharmaceutical properties of three fruits from Côte d’Ivoire

The aim of the study was to characterize the fruit extracts of Ficus sur, Gardenia ternifolia and Uapaca togoensis prepared from water and methanol solvents. Metabolomic and antiradical properties were investigated. Enzyme inhibitory effects were also studied against acetylcholinesterase, butyrylcholinesterase, tyrosinase, α-amylase and α-glucosidase. Extracts’ toxicological properties were assayed through allelopathy, brine shrimp lethality and Daphnia magna assays. Extracts’ effects on human prostate cancer PC3 cell viability and isolated mouse colon specimens challenged with lipopolysaccharide were also evaluated. The gene expression of VEGFA, TNFα, HIF1α and COX-2 was measured in prostate tissue. Metabolomic analysis conducted on methanolic extracts identified 47 phytochemicals in U. togoensis, 45 in F. sur and 24 in G. ternifolia. The methanolic F. sur extract showed the highest antioxidant and enzyme inhibition properties compared to the other fruits. However, F. sur was also the most toxic as revealed by the higher grade of heart rate reduction, in the D. magna model, and by the increased viability of prostate cancer cells. Nevertheless, most of the tested extracts, including F. sur extracts, were effective in blunting LPS-induced gene expression of all tested biomarkers, in isolated prostate. Overall, the present toxicological screening suggests caution in the use of F. sur extracts.

New insights into transformation pathways of a mixture of cytostatic drugs using Polyester-TiO2 films: Identification of intermediates and toxicity assessment

The photocatalytic activity of two bio-based polymer photocatalysts [poly(ethylene terephthalate)-TiO2 (PET-TiO2) and poly(L-lactic acid)-graphene oxide-TiO2 (PLLA-GO-TiO2)] towards Tamoxifen (TAM), Cyclophosphamide (CP), Cytarabine (CYT) and 5-Fluorouracil (5-FLU) removal was explored and compared. The highest photocatalytic activity for the degradation of the cytostatic drugs was accomplished by PET-TiO2. Among the contaminants, TAM was the most easily removed, requiring 90 min for complete elimination, while CP showed the highest resistance to photocatalysis, not being completely removed after 6 h. Liquid chromatography coupled with high-resolution mass spectrometry analysis was employed for the identification of several transformation products (TPs) and potential pathways were proposed. A total of seventy (70) TPs including thirty-four (34) novel ones detected in AOPs were identified. The ecotoxicity of the mixture of the cytostatic drugs and TPs formed during the photocatalytic treatment was evaluated using Daphnia magna assay and was associated with the occurrence of specific TPs during the treatment process. The follow-up ECOSAR (Ecological Structure Activity Relationship) analysis further elucidated that only minor chemical transformations, such as the hydroxylation or the oxidative opening of an aromatic ring system, could hamper the adverse effects of cytostatic drugs in aquatic species. Such a comparative study on the mixture toxicity of cytostatics and their TPs is presented for the first time.

Transgenerational Effects of Toxicants: An Extension of the Daphnia 21-day Chronic Assay?

The assessment of transgenerational effects should be incorporated in standard chronic toxicity protocols for the sake of a realistic extrapolation of contaminant effects to the population level. We propose a simple add-on to the standard 21-day chronic Daphnia magna assay, allowing the assessment of the reproductive performance of the offspring (F1 generation) born from the first clutch of the parental (F0) generation. The extended generational assay was performed simultaneously with the standard reproduction assay. With this design, we evaluated the lethal, reproductive, and transgenerational effects of four widespread and extensively used substances: a biocide/anti-fouling (copper sulphate), an industrial oxidizing agent (potassium dichromate), a pharmaceutical (paracetamol), and a quaternary ammonium compound (benzalkonium chloride). Benzalkonium chloride was the most toxic in terms of lethality, whereas paracetamol, copper sulphate, and potassium dichromate caused deleterious effects in the reproductive performance of exposed D. magna. Adverse effects in the fitness of the daughter (F1) generation were observed in the case of maternal exposure to paracetamol and copper sulphate, although they were not very pronounced. These findings highlight the usefulness of our approach and reinforce the view-shared by other authors-of the need for a generalised formal assessment of the transgenerational effects of pollutants.

Combining physico-chemical analysis with a Daphnia magna bioassay to evaluate a recycling technology for drinking water treatment plant waste residuals

Recycling water treatment plant (WTP) waste residuals is considered to be a feasible method to enhance the efficiency of pollutant removal. This study also evaluated the safety and water quality of a pilot-DWTP waste residuals recycling technology by combining physical-chemistry analysis with a Daphnia magna assay. The water samples taken from each treatment step were extracted and concentrated by XAD-2 resin and were then analyzed for immobilization and enzyme activity with D. magna. The measured parameters, such as the dissolve organic carbon (DOC), UV254 and THM formation potential (THMFPs) of the recycling process, did not obviously increase over 15 days of continuous operation and were even lower than typical values from a conventional process. The extract concentration ranged from 0 to 2Leq/ml as measured on the 7th and 15th days and the immobilization of D. magna exposed to water treated by the recycling process was nearly equivalent to that of the conventional process. Both the superoxide dismutase (SOD) and the catalase (CAT) activity assay indicated that a lower dose of water extract (0.5, 1, 1.5Leq/ml) could stimulate the enzyme activity of D. magna, whereas a higher dose (2Leq/ml at the sampling point C3, R3, R4 ) inhibits the activity. Moreover, the SOD and CAT activity of D. magna with DOC and UV254 showed a strong concentration–effect relationship, where the concentration range of DOC and UV254 were 4.1–16.2mg/L and 0.071–4.382cm−1, respectively. The results showed that there was no statistically significant difference (p>0.05) between the conventional and recycling treatment processes and the toxicity of water samples in the recycling process did not increase during the 15-day continuous recycling trial.

Xanthone-photosensitized detoxification of the veterinary anthelmintic fenbendazole

Abstract Fenbendazole ( 1 ) is a common veterinary anthelmintic, toxic to water living microorganisms. Fluorescence quantum yields of 1 were found to be 0.11 in acetonitrile, 0.068 in methanol, 0.034 in cyclohexane, and 0.013 in water. The singlet excited state energy was ca . 96 kcal mol −1 in all solvents. The phosphorescence spectrum of 1 in ethanol at 77 K displayed a maximum at 450 nm, leading to a triplet energy of 75 kcal mol −1 . Experimental excited state energies agree well with the results of DFT calculations at the time-dependent B3LYP/6-311+G(d,p)//B3LYP/6-31G(d) level. Laser flash photolysis (LFP) of 1 at 266 nm led to transients absorbing in the 300–700 nm range, ascribed to radical cation 1 + , which were also observed upon 355 nm LFP of xanthone (XA) in the presence of 1 . Solar-simulated photolysis revealed XA-enhanced photodegradation of 1 and led to decreased toxicity, as shown by Daphnia magna assays.

Using Daphnia bio-sensor for random number generation

One of the most used bioassays for toxicity screening of chemicals and for toxicity monitoring of effluents and contaminated waters is the acute toxicity test performed with Daphnia Magna. Standard methods have been developed and gradually improved by national and international organizations dealing with toxicity testing procedure, in view of its application within a regulatory framework. As for all toxicity tests, the organisms used for the acute Daphnia magna assay have to be obtained from live stocks which are cultured in the laboratory on live food (micro-algae). The technical and biological problems inherent in year-round culturing and the culturing/maintenance costs of live stocks restricts its application to a limited number of highly specialized laboratories. This bottleneck in toxicity testing triggered investigations forward the concept of "microbiotests" or "small-scale" toxicity tests. This paper deals with the measurement of changes in the behavior of Daphniae using device BIOTOXINOMER, awarded by Ministry for Science and Technological Development of Republic Serbia with Diploma for best Innovation idea in year 2010 in category Medicine, Health, and Ecology. Actually, the main topic is digitalization of bio-monitoring results and random number generating on the base of Daphniae movement (distance). The on-line monitoring is available on site www.dundjer.co.rs/Daphniae and open-source software support is available on the same site.

Whole-cell bacterial biosensors for rapid and effective monitoring of heavy metals and inorganic pollutants in wastewater

The increasing number of potentially harmful pollutants in the wastewater effluent discharge necessitates the need for the development of fast and cost effective analytical techniques for extensive monitoring programmes to assess the effectiveness of the treatment process. This study compared the use of bacterial biosensors to the conventional Daphnia magna assay, Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) tests as well as chemical analysis, for monitoring the toxicity of wastewater. The bacterial biosensors constructed in this study, using S. sonnei and E. coli, were found to be sensitive to the toxicity of the wastewater effluents. A linear increase in bioluminescence with increasing concentration of heavy metals and inorganic pollutants in water was observed, with a correlation coefficient (r(2)) as high as 0.995 and 0.997, respectively. No notable correlation between biosensor toxicity and BOD and COD test results was observed. These bacterial biosensors could provide appropriate alternatives for a rapid, sensitive and cost effective detection of wastewater quality. However, the differences in sensitivity obtained for the different systems suggest that the use of a battery of toxicity assays may be required to provide a real ecotoxicological assessment of wastewater samples.

Use of a (Quantitative) Structure–Activity Relationship [(Q)Sar] Model to Predict the Toxicity of Naphthenic Acids

Naphthenic acids (NA) are a complex mixture of carboxylic acids that are natural constituents of oil sand found in north-eastern Alberta, Canada. NA are released and concentrated in the alkaline water used in the extraction of bitumen from oil sand sediment. NA have been identified as the principal toxic components of oil sands process-affected water (OSPW), and microbial degradation of lower molecular weight (MW) NA decreases the toxicity of NA mixtures in OSPW. Analysis by proton nuclear magnetic resonance spectroscopy indicated that larger, more cyclic NA contain greater carboxylic acid content, thereby decreasing their hydrophobicity and acute toxicity in comparison to lower MW NA. The relationship between the acute toxicity of NA and hydrophobicity suggests that narcosis is the probable mode of acute toxic action. The applicability of a (quantitative) structure–activity relationship [(Q)SAR] model to accurately predict the toxicity of NA-like surrogates was investigated. The U.S. Environmental Protection Agency (EPA) ECOSAR model predicted the toxicity of NA-like surrogates with acceptable accuracy in comparison to observed toxicity values from Vibrio fischeri and Daphnia magna assays, indicating that the model has potential to serve as a prioritization tool for identifying NA structures likely to produce an increased toxicity. Investigating NA of equal MW, the ECOSAR model predicted increased toxic potency for NA containing fewer carbon rings. Furthermore, NA structures with a linear grouping of carbon rings had a greater predicted toxic potency than structures containing carbon rings in a clustered grouping.

Age and exposure duration as a factor influencing Cu and Zn toxicity toward Daphnia magna

Standardized toxicity tests are generally performed with juvenile test organisms, e.g., in Daphnia magna assays neonates <24 h old are used. The purpose of this research was to investigate the influence of a delayed exposure to Cu and Zn on population parameters and toxicity values derived from these endpoints. Juveniles (< 24 h; T0) and 7-d old daphnids (T7) were exposed for 21 and 14 d, respectively. For Cu, juveniles were significantly more sensitive than 7 d old organisms following acute (48 h) as well as chronic (14 d) exposure. After 14 d of exposure to 130 μg/L Cu, mortality was 80% and 10% in T0 and T7, respectively. Juveniles per surviving female at this concentration decreased by 78% and 14% compared to the control. 14 d-NOEC and LOEC values (based on juveniles per surviving female) were 75 and 90 μg/L Cu for T0 and both>130 μg/L for T7. For Zn, survival in T0 and T7 was similar. Although T7 organisms produced significantly more offspring, 14 d-NOEC and LOEC values were equal to those of T0, i.e., 80 and 115 μg/L, respectively. For Cu as well as for Zn effect concentrations based on 14 and 21 d exposure were similar (results from T0). It can be concluded that acute and chronic toxicity data obtained from juvenile D. magna are more sensitive or equally sensitive than obtained from 7 d old organisms. As differences are observed between the two metals extrapolations of these conclusions to other toxicants and other aquatic species cannot be made without further investigation.

Sediment quality assessment of flowing waters in South-West Germany using acute and chronic bioassays

Four bioassays were selected to examine sediment-elutriates of three flowing waters in the Saarland (South-West Germany). The aims of this study were the determination of the most sensitive bioassay for a sediment assessment within routine screening procedures and the comparison of a recently developed long-term bacterial assay with other commonly used and standardized bioassays. Sediment samples were elutriated with water and also with dimethyl sulphoxide in order to extract organic compounds. An expected accumulation of toxicants in the sediments was checked by additional examination of surface water at respective sampling sites, applying bioassays as well as chemical analysis methods. Whereas the sensitivity of the Daphnia magna assay was relatively low, the Scenedesmus subspicatus chlorophyll fluorescence test denoted the presence of high loads of nutrients at all sites. The results obtained using the Vibrio fischeri short-term bioassay suggested that an ecotoxicological risk can be excluded. The long-term luminescent bacteria assay is a further development of the short-term V. fischeri-assay. Prolonging the test duration from 30 min to 24 h should track down subacute toxicities, thus leading to a greater degree of sensitivity. However, the prolongation caused severe problems in respect of an accurate interpretation of the results. The long-term assay is not suited for routine deployment without major improvements. The extractable content of metals, pesticides, polychlorinated biphenyls and chlorinated hydrocarbons remained below their threshold values by several orders of magnitude. The use of dimethyl sulphoxide as solvent caused a decrease of bioluminescence in both bacterial assays. The inhibition was caused by toxic metabolic products of dimethyl sulphoxide formed by autochthonous bacteria from the sediment samples during the elutriation procedure. Thus, the use of dimethyl sulphoxide is not recommended for examinations of environmental samples when applying a luminescent bacteria assay.

Sediment quality assessment of flowing waters in South-West Germany using acute and chronic bioassays

Abstract Four bioassays were selected to examine sediment-elutriates of three flowing waters in the Saarland (South-West Germany). The aims of this study were the determination of the most sensitive bioassay for a sediment assessment within routine screening procedures and the comparison of a recently developed long-term bacterial assay with other commonly used and standardized bioassays. Sediment samples were elutriated with water and also with dimethyl sulphoxide in order to extract organic compounds. An expected accumulation of toxicants in the sediments was checked by additional examination of surface water at respective sampling sites, applying bioassays as well as chemical analysis methods. Whereas the sensitivity of the Daphnia magna assay was relatively low, the Scenedesmus subspicatus chlorophyll fluorescence test denoted the presence of high loads of nutrients at all sites. The results obtained using the Vibrio fischeri short-term bioassay suggested that an ecotoxicological risk can be excluded. The long-term luminescent bacteria assay is a further development of the short-term V. fischeri-assay. Prolonging the test duration from 30 min to 24 h should track down subacute toxicities, thus leading to a greater degree of sensitivity. However, the prolongation caused severe problems in respect of an accurate interpretation of the results. The long-term assay is not suited for routine deployment without major improvements. The extractable content of metals, pesticides, polychlorinated biphenyls and chlorinated hydrocarbons remained below their threshold values by several orders of magnitude. The use of dimethyl sulphoxide as solvent caused a decrease of bioluminescence in both bacterial assays. The inhibition was caused by toxic metabolic products of dimethyl sulphoxide formed by autochthonous bacteria from the sediment samples during the elutriation procedure. Thus, the use of dimethyl sulphoxide is not recommended for examinations of environmental samples when applying a luminescent bacteria assay.