Application of a computational model for Michael addition reactivity in the prediction of toxicity to Tetrahymena pyriformis

A computational model to predict acute aquatic toxicity to the ciliate Tetrahymena pyriformis has been developed. A general prediction of toxicity can be based on three consecutive steps: 1. Identification of a potential reactive mechanism via structural alerts; 2. Confirmation and quantification of (bio)chemical reactivity; 3. Establishing a relationship between calculated reactivity and toxicity. The method described herein uses a combination of a reactive toxicity (RT) model, including computed kinetic rate constants for adduct formation (log k) via a Michael acceptor mechanism of action, and baseline toxicity (BT), modelled by hydrophobicity (octanol-water partition coefficient). The maximum of the RT and BT values defines acute toxicity for a particular compound. The reactive toxicity model is based on site-specific steric and quantum chemical ground state electronic properties. The performance of the model was examined in terms of predicting the toxicity of 106 potential Michael acceptor compounds covering several classes of compounds (aldehydes, ketones, esters, heterocycles). The advantages of the computational method are described. The method allows for a closer and more transparent mechanistic insight into the molecular initiating events of toxicological endpoints.     

Ecotoxicological evaluation of industrial port of Venice (Italy) sediment samples after a decontamination treatment

This work assesses the ecotoxicological effects of polluted sediment after a decontamination treatment process using a new sediment washing technique. Sediment samples were collected from four sites in Marghera Port industrial channels (Venice, Italy). Ecotoxicological evaluations were performed with Vibrio fischeri and Crassostrea gigas bioassays. Whole sediment and elutriate were deemed as the most suitable environmental matrices for this study. Toxicity scores developed in the Lagoon of Venice for V. fischeri on whole sediment and for C. gigas on elutriate were considered for the final ranking of samples. Ecotoxicological results showed that the treated sediment samples presented both acute and sub-chronic toxicities, which were mainly attributed to the presence of some remaining chemicals such as metals and polyaromatic hydrocarbons. The acute toxicity ranged from low to medium, while the sub-chronic one from absent to very high, suggesting that treated sediments could not be reused in direct contact with seawater.     

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.     

A model compound study: the ecotoxicological evaluation of five organic contaminants employing a battery of marine bioassays

This paper describes the ecotoxicological evaluation of five organic contaminants frequently detected in marine sediments (tributyltin, triphenyltin, benzo[a]pyrene, fluoranthene, and PCB 153) using three marine species (Vibrio fischeri, Tetraselmis suecica, and Tisbe battagliai). The sensitivity of each species varied for all compounds. The triorganotins were consistently the most toxic to all species. The applicability of each test system to assess the acute toxicity of environmental contaminants and their use in Toxicity Identification Evaluation (TIE) is discussed. Suitability of the Microtox and T. battagliai tests for employment in TIE studies were further assessed through spiking experiments with tributyltin. Results demonstrated that the most effective treatment to remove organotin toxicity from the sample was the C18 resin. The results of this study have important implications for risk assessment in estuarine and coastal waters in Ireland, where, at present the monitoring of sediment and water quality is predominantly reliant on chemical analysis alone.     

Converging hazard assessment of gold nanoparticles to aquatic organisms

The gold nanoparticles (Au-NPs) are being increasingly used because of their huge diversity of applications, and consequently, elevated levels in the environment are expected. However, due to their physico-chemical properties and functionalization a high variety of Au-NPs can be found, and complete toxicological information for each type of Au-NPs still lacks, and even, the toxicological information for the same species is sometimes contradictory. Therefore, hazard assessment should be done case by case. Hence, the objective of this study was to obtain ecotoxicological information of the same Au-NPs in aquatic organisms and to find a rationale for Au-NPs toxicity. For such a purpose, bare and hyaluronic acid capped Au-NPs (12.5 nm) along with Au-NPs bulk material were tested on freshwater algae, Daphnia and zebrafish. Results showed that while gold nanoparticles were found to be harmless to the tested organisms, the soluble gold showed to be toxic to algae and Daphnia, with an LC₅₀ between 1 and 2 mg L⁻¹. Comparing our results with those gathered in the literature, it appears that a common hazard assessment of Au-NPs on the studied organisms can be elucidated.     

Toxicity of mixtures of perfluorooctane sulphonic acid with chlorinated chemicals and lipid regulators

The toxicological interaction of perfluorooctane sulphonic acid (PFOS) with the chlorinated pollutants triclosan and 2,4,6-trichlorophenol and the lipid regulators gemfibrozil and bezafibrate was evaluated using the combination index-isobologram equation. The endpoint for bioassays was the growth rate inhibition of the green alga Pseudokirchneriella subcapitata. The results showed that most of the binary combinations assayed exhibited antagonism at all effect levels. The addition of a third component induced a less antagonistic or even synergistic behaviour. This was particularly marked for the ternary mixture of triclosan and 2,4,6-trichlorophenol with PFOS, for which synergism was very strong at all effect levels, with a combination index as low as 0.034 ± 0.002 at EC₅₀ for the mixture. The results obtained indicate that the evaluation of mixture toxicity from single component data using the concentration addition approach could severely underestimate combined toxicity.     

Toxicity testing with luminescent bacteria – Characterization of an automated method for the combined assessment of acute and chronic effects

The luminescent bacteria test according to EN ISO 11348 is frequently applied in (eco) toxicity testing and is applicable for a huge variety of environmental and industrial samples. A big disadvantage of this method is the very short exposure time, which is expressed in a low sensitivity in regard to substances with a delayed effect. Chronic effects, i.e. interference with cell growth, cannot be assessed with this conventional standard method. The goal of this research was to develop an automated testing system for long term toxicity towards the luminescent bacteria Vibrio fischeri by implementing microtitration-based instrumentation. The optimized method, hereinafter referred to as “kinetic luminescent bacteria test”, can be described as a miniaturized combination of the conventional short-term luminescence inhibition test according to EN ISO 11348 and the Photobacterium phosphoreum growth inhibition test (DIN 38412-37). The validation procedure included the evaluation of six reference compounds (3,4-Dichloroaniline, 3,5-Dichlorophenol, Chloramphenicol, Streptomycin sulfate, Potassium dichromate, Zinc sulfate heptahydrate) and three different endpoints that are acute luminescence inhibition (acute LI) after 30 min, chronic luminescence inhibition (chronic LI) after 24 h and growth inhibition (GI) after 14 h. The optimized method allows the assessment of acute and chronic effects within one test, by what a misinterpretation of the toxicity of substances with delayed bacterial toxicity can be prevented, without abandoning most of the advantages of the conventional short-term test. Therefore, the kinetic luminescent bacteria test is exceptional as an initial screening test for environmental samples or substances with unknown (eco) toxicological characteristics.     

Liver histopathology of the sharptooth catfish Clarias gariepinus as a biomarker of aquatic pollution

This paper reports on a comparative perspective of liver histopathological data of the sharptooth catfish Clarias gariepinus. The data was collected from a spectrum of relatively un-impacted and isolated, to polluted, eutrophic freshwater ecosystems. Results were compared between regional areas, by combining data from freshwater systems which has a similar pollution status and/or is located within the same geographical region. Measurements included necropsy observations, semi-quantitative liver histopathology (Liver Index), and selected biometrical indices. The aim was to establish whether the results of these measurements would differ between, and/or reflect the pollution status of, the different freshwater aquatic ecosystems. The histopathological analysis showed a higher prevalence of toxicopathic non-neoplastic, and pre-neoplastic alterations in C. gariepinus from the polluted sites. We also found a significant difference between the Liver Index, hepatosomatic index, and condition factor values of fish inhabiting impoundments known to be polluted, compared to the same species from the selected reference sites. Fish from polluted sites also had more macroscopic liver abnormalities. The results suggest that the liver histopathology of this bio-indicator fish species could be a useful biomarker of freshwater aquatic pollution.     

Application of biomarkers to assess the condition of European Marine Sites

A series of European Marine Sites has been designated as Special Areas of Conservation (SAC) in England. The aim of this study was to develop a practical methodology to assess the condition of SACs by applying a suite of biomarkers. Biomarkers were applied to the blue mussel Mytilus edulis and the shore crab Carcinus maenas from the Fal and Helford SAC (Cornwall). Individual biomarkers provided useful diagnostic information on the activity of certain classes of contaminants and an integrated Biomarker Response Index (BRI) was used to achieve a more holistic understanding of the condition of the SAC. The BRI indicated that the general health of both organisms was impacted in the upper part of the SAC (Fal Estuary) which correlated well with known chemical hotspots and sources of contamination. The BRI allows a pragmatic way to prioritise SAC sites that may require further investigative studies.     

Cyp35a2 gene expression is involved in toxicity of fenitrothion in the soil nematode Caenorhabditis elegans

In this study, the effect of organophosphorous (OP) pesticide, fenitrothion (FT), on the non-target organism was investigated using the soil nematode, Caenorhabditis elegans. Toxicity was investigated on multiple biological levels, from organism to molecular levels, such as, immoblity, growth, fertility, development, acetyl cholinesterase (AChE) activity and stress-response gene expressions. FT may provoke serious consequences on the C. elegans population, as it induced significant developmental disturbance. As expected, FT exposure inhibits AChE activity of C. elegans. The increased expression of the cytochrome p450 family protein 35A2 (cyp35a2) gene was also observed in FT exposed worms. To experimentally demonstrate the relationships between organism-level effects and the cyp35a2 gene expression in FT-exposed C. elegans, the integration of the gene expression with biochemical-, and organism level endpoints were attempted using a C. eleganscyp35a2 RNA interference (RNAi) and cyp35a2 mutant (gk317). The 24 h-EC50s of C. elegans on FT exposure were in the order of cyp35a2 RNAi in cyp35a2 mutant (gk317) > cyp35a2 mutant (gk317) > cyp35a2 RNAi in wildtype (N2) > wildtype (N2). The higher EC50 values of cyp35a2 RNAi and cyp35a2 mutant (gk317) compared to that of wildtypeC. elegans strongly supported that cyp35a2 gene plays an important role in the toxicity of FT towards C. elegans. The experiments with cyp35a2 RNAi also indicated that the development disturbance and decreased AChE activity, which were observed in FT exposed wildtype C. elegans were significantly rescued in the cyp35a2 RNAi C. elegans. Overall results suggest that the cyp35a2 may be an important gene for exerting FT toxicity in C. elegans.     

Evaluation of effectiveness of EDTA and sodium thiosulfate in removing metal toxicity toward sea urchin embryo-larval applying the TIE

Since the development of the TIE (Toxicity Identification and Evaluation) in 1988 it has been assumed that the capacity of EDTA and sodium thiosulfate to complex some metals, and thus remove their toxicity, can be applied to both freshwater and seawater ecotoxicological tests and the results subsequently interpreted. However, it is now known that there is a wide variability in the extent of this complexation. In this context, the removal of toxicity caused by the presence of Hg²⁺, Cd²⁺, Cu²⁺, Cr⁶⁺, Zn²⁺, Ni²⁺, Pb²⁺, Ag¹⁺ and Se²⁺, through metal complexation by EDTA and sodium thiosulfate, in relation to the performance of embryo-larval tests with the sea urchin Arbacia lixula was investigated. It was observed that EDTA was capable of removing the toxicity of Pb²⁺, Zn²⁺ and Cu²⁺ while sodium thiosulfate only reduced the toxicity of Ag¹⁺. Compared to the complexation observed in freshwater ecotoxicological tests, the complexing agents used in this study (EDTA and sodium thiosulfate) have a lower capacity to complex metals in the marine ecotoxicological test with A. lixula.     

Cytotoxicity assessment of four pharmaceutical compounds on the zebra mussel (Dreissena polymorpha) haemocytes, gill and digestive gland primary cell cultures

Pharmaceutical compounds are considered the new environmental pollutants but at present few studies have evaluated their ecotoxicity on aquatic invertebrates. This study was aimed to investigate the in vitro cytotoxicity of four common drugs, namely atenolol (ATL), carbamazepine (CBZ), diclofenac (DCF) and gemfibrozil (GEM), on three different cell typologies from the zebra mussel (Dreissena polymorpha): haemocytes, gill and digestive gland cells. Results obtained by the Trypan blue exclusion test revealed that exposure to increasing concentrations (0.001; 0.01; 0.1; 1 and 10 mg L⁻¹) of CBZ, DCF and GEM were able to significantly decrease the viability of each cell type, while the MTT (3(4,5-dimethyl-2thiazholyl)-2,5-diphenyl-2H-tetrazolium bromide) reduction assay highlighted only a slight reduction of mitochondrial activity of gill and digestive gland cells. Overall, DCF was the most cytotoxic drug for zebra mussel cells, followed by GEM, CBZ, while ATL has not a noteworthy toxic potential. Our preliminary results lay the groundwork for further in vitro evaluations, which will allow a better definition of the potential toxicity of these drugs.     

Toxicological evaluation of constructed wetland habitat sediments utilizing 10-day sediment toxicity test and bacterial bioluminescence

A toxicological evaluation was conducted on wetland habitats created as a result of run-off from agricultural areas. These temporary wetlands were created by using drop pipes as a means of reducing erosional cutting in agricultural fields. Toxicity bioassays utilizing bacterial bioluminescence and Hyalella azteca were used to assess sediment pore water and whole sediment, respectively. Inhibition of bacterial bioluminescence was initially used to determine relative toxicities of pore water from ten wetland sites. Constructed wetland sites were compared to the University of Mississippi Biological Field Station, a relatively pristine reference site. The H. azteca ten day sediment toxicity test was utilized to assess sediment from four selected sites using survival and growth as toxicological endpoints. Results from the toxicological evaluation, along with extensive ecological evaluations, were used to assess the best approach for implementation of temporary wetland habitats with existing agricultural practices.     

Inconsistency and comprehensiveness of risk assessments for heavy metals in urban surface sediments

Numerous indices have been developed to assess environmental risk of heavy metals in surface sediments, including the total content based geoaccumulation index (I_geo), exchangeable fraction based risk assessment code (RAC), and biological toxicity test based sediment quality guidelines (SQGs). In this study, the three indices were applied to freshwater surface sediments from 10 sections along an urbanization gradient of the Grand Canal, China to assess the environmental risks of heavy metals (Cu, Pb, Zn, Cd, and Cr) and to understand discrepancies of risk assessment indices and urbanization effects regarding heavy metal contamination. Results showed that Cd, Zn, and Pb were the most enriched metals in urban sections assessed by I_geo and over 95% of the samples exceeded the Zn and Pb thresholds of the effect range low (ERL) of SQGs. According to RAC, Cu, Zn, Cd, and Cr had high risks of adversely affecting the water quality of the Grand Canal due to their remarkable portions of exchangeable fraction in surface sediment. However, Pb showed a relative low risk, and was largely bounded to Fe/Mn oxides in the urban surface sediments. Obviously, the three assessment indices were not consistent with each other in terms of predicting environmental risks attributed to heavy metals in the freshwater surface sediments of this study. It is recommended that risk assessment by SQGs should be revised according to availability and site specificity. However, the combination of the three indices gave us a comprehensive understanding of heavy metal risks in the urban surface sediments of the Grand Canal.     

Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils

Five bioassays (inhibition of lettuce germination and growth, earthworm mortality, inhibition of springtail population growth, avoidance by springtails) were compared, using four coke factory soils contaminated by PAHs and trace elements, before and after biotreatment. For each bioassay, several endpoints were combined in an ‘ecoscore’, a measure of test sensitivity. Ecoscores pooled over all tested bioassays revealed that most organisms were highly sensitive to the concentration of 3-ring PAHs. When four soils were combined, behavioural tests using the springtail Folsomia candida showed higher ecoscores, i.e. they were most sensitive to soil contamination. However, despite overall higher sensitivity of behavioural tests, which could be used for cheap and rapid assessment of soil toxicity, especially at low levels of contamination, some test endpoints were more sensitive than others, and this may differ from a soil to another, pointing to the need for a battery of bioassays when more itemized results are expected.     

Spore release by the green alga Ulva: a quantitative assay to evaluate aquatic toxicants

A toxicity test using spore release of the aquatic green alga, Ulva, was developed and evaluated by assessing the toxicity of different organic and inorganic chemicals and elutriates of sewage or waste sludge. The toxic ranking of four metals was: Cu (EC50 of 0.040mgL(-1))>Cd (0.095mgL(-1))>Pb (0.489mgL(-1))>Zn (0.572mgL(-1)). The EC50 for TBTO ranged from 24 to 63microgL(-1). The most toxic VOC was formalin (EC50 of 0.788microlL(-1)) and the least toxic was acetone. Spore release was significantly inhibited in all elutriates; the greatest and least toxic effects were for industrial sewage (3.29%) and filtration bed (10.08%), respectively. The bioassay is simple, inexpensive and sensitive. The cosmopolitan distribution of Ulva means that the test would have a potential application worldwide.     

Environmental mutagenicity and toxicity caused by sodium metabisulfite in sea shrimp harvesting in Piauí, Brazil

Sodium metabisulfite is used in marine shrimp harvesting to prevent the occurrence of black spots. Shrimps are soaked in a sodium metabisulfite solution in ice, which is disposed of in sewages that run into marine canals, creating an environmental hazard. This study evaluates the toxicity and mutagenicity caused by sodium metabisulfite in sea waters and sediments collected in a shrimp farm in Cajueiro da Praia (Luis Correia), state of Piauí, Brazil, using the Allium cepa assay. Water and sediment samples were collected in the dry and in the rainy seasons, in three sites: upstream the shrimp farm (Site 1), at the point sodium metabisulfite is discharged (Site 2), and 100 m downstream the farm (Site 3). Three sample dilutions were used (50%, 25% and 10%) for all samples. A negative control (well water) and a positive control (copper sulfate 0.0006 mg mL⁻¹) were used in each experiment. At the end of the 72-h exposure period, onion roots were measured and removed. Mutagenicity analysis included the determination of mitotic index, chromosomal aberrations and the detection of micronuclei; analysis of root size and mitotic index were used as an index of toxicity. The A. cepa assay revealed that the water and sediments samples collected in the Piauí coast contaminated with sodium metabisulfite induce toxicity. The results demonstrate that the assay may be used as a regular tool in the analysis of water parameters in shrimp farms in the coast of Piauí state, and in strategies to preserve the region’s marine ecosystem.     

Comparative acute freshwater hazard assessment and preliminary PNEC development for eight fluorinated acids

Short-term 48, 72 and 96-h aquatic toxicity tests were conducted to evaluate the acute toxicity of eight fluorinated acids to the cladoceran, Daphnia magna, the green alga, Pseudokirchneriella subcapitata, and the rainbow trout, Oncorhynchus mykiss or the fathead minnow, Pimephales promelas. The eight fluorinated acids studied were tridecafluorohexyl ethanoic acid (6:2 FTCA), heptadecafluorooctyl ethanoic acid (8:2 FTCA), 2H-dodecafluoro-2-octenoic acid (6:2 FTUCA), 2H-hexadecafluoro-2-decenoic acid (8:2 FTUCA), 2H,2H,3H,3H-undecafluoro octanoic acid (5:3 acid), 2H,2H,3H,3H-pentadecafluoro decanoic acid (7:3 acid), n-perfluoropentanoic acid (PFPeA) and n-perfluorodecanoic acid (PFDA). The results of the acute toxicity tests conducted during this study suggest that the polyfluorinated acids, 8:2 FTCA, 8:2 FTUCA, 6:2 FTCA, 6:2 FTUCA, 7:3 acid and 5:3 acid, and the perfluorinated acids PFPeA and PFDA, are generally of low to medium concern based on evaluation of their acute freshwater toxicity (EC/LC50s typically between 1 and >100 mg L⁻¹) using the USEPA TSCA aquatic toxicity evaluation paradigm. For the polyfluorinated acids, aquatic toxicity generally decreased as the number of fluorinated carbons decreased and as the overall carbon chain length decreased from 12 to 8. Acute aquatic toxicity of the 5 and 10 carbon perfluorocarboxylic acids (EC/LC50s between 10.6 and >100 mg L⁻¹) was greater or similar to that of the 6-9 carbon perfluorocarboxylic acids (EC/LC50s > 96.5 mg L⁻¹). This study also provides the first report of the acute aquatic toxicity of the 5:3 acid (EC/LC50s of 22.5 to >103 mg L⁻¹) which demonstrated less aquatic toxicity than the 7:3 acid (EC/LC50s of 0.4-32 mg L⁻¹). The cladoceran, D. magna and the green alga, P. subcapitata had generally similar EC50 values for a given substance while fish were typically equally or less sensitive with the exception that PFPeA was most toxic to fish. Predicted no-effect concentrations (PNECs) were estimated using approaches consistent with REACH guidance and when compared with available environmental concentrations, these PNECs suggest that the fluorinated acids tested pose little risk for aquatic organisms.     

Toxicity profile of labile preservative bronopol in water: the role of more persistent and toxic transformation products

Transformation products usually differ in environmental behaviors and toxicological properties from the parent contaminants, and probably cause potential risks to the environment. Toxicity evolution of a labile preservative, bronopol, upon primary aquatic degradation processes was investigated. Bronopol rapidly hydrolyzed in natural waters, and primarily produced more stable 2-bromo-2-nitroethanol (BNE) and bromonitromethane (BNM). Light enhanced degradation of the targeted compounds with water site specific photoactivity. The bond order analysis theoretically revealed that the reversible retroaldol reactions were primary degradation routes for bronopol and BNE. Judging from toxicity assays and the relative pesticide toxicity index, these degradation products (i.e., BNE and BNM), more persistent and higher toxic than the parent, probably accumulated in natural waters and resulted in higher or prolonging adverse impacts. Therefore, these transformation products should be included into the assessment of ecological risks of non-persistent and low toxic chemicals such as the preservative bronopol.