Exploring BPA alternatives - Environmental levels and toxicity review.

Bisphenol A alternatives are manufactured as potentially less harmful substitutes of bisphenol A (BPA) that offer similar functionality. These alternatives are already in the market, entering the environment and thus raising ecological concerns. However, it can be expected that levels of BPA alternatives will dominate in the future, they are limited information on their environmental safety. The EU PARC project highlights BPA alternatives as priority chemicals and consolidates information on BPA alternatives, with a focus on environmental relevance and on the identification of the research gaps. The review highlighted aspects and future perspectives. In brief, an extension of environmental monitoring is crucial, extending it to cover BPA alternatives to track their levels and facilitate the timely implementation of mitigation measures. The biological activity has been studied for BPA alternatives, but in a non-systematic way and prioritized a limited number of chemicals. For several BPA alternatives, the data has already provided substantial evidence regarding their potential harm to the environment. We stress the importance of conducting more comprehensive assessments that go beyond the traditional reproductive studies and focus on overlooked relevant endpoints. Future research should also consider mixture effects, realistic environmental concentrations, and the long-term consequences on biota and ecosystems.

A strategy for the investigation of toxic mechanisms and protection by efflux pumps using Schizosaccharomyces pombe strains: Application to rotenone.

Effects not related with the inhibition of complex I of the mitochondrial electron transport chain are studied in S. pombe, which lacks it. This study aims: First, the use of a strategy with S. pombe strains to investigate the toxicity, mechanisms of action, interactions and detoxication by efflux pumps. Second, to investigate the mechanisms of toxic action of rotenone. In the dose-response assessment, the yeast presented a good correlation with the toxicity in Daphnia magna for 15 chemicals. In the mechanistic study, the mph1Δ strain presented marked specificity to the interaction with microtubules by carbendazim. DNA damage caused by hydroxyurea, an inhibitor of deoxynucleotide synthesis, was identified with marked specificity with the rad3Δ strain. The sty1Δ strain was very sensitive to the oxidative and osmotic stress induced by hydrogen peroxide and potassium chloride, respectively, being more sensitive to oxidative stress than the pap1Δ strain. The protection by exclusion pumps was also evaluated. Rotenone presented low toxicity in S. pombe due to the lack of its main target, and the marked protection by the exclusion transporters Bfr1, Pmd1, Caf5 and Mfs1. Marked cellular stress was detected. Finally, the toxicity of rotenone could be potentiated by the fungicide carbendazim and the antimetabolite hydroxyurea. In conclusion, the use of S. pombe strains is a valid strategy to: a) assess global toxicity; b) investigate the main mechanisms of toxic action, particularly spindle and DNA interferences, and osmotic and oxidative stress not related to complex I inhibition; c) explore the detoxication by efflux pumps; and d) evaluate possible chemical interactions. Therefore, it should be useful for the investigation of adverse outcome pathways.

Gold(I) metallocyclophosphazenes with antibacterial potency and antitumor efficacy. Synergistic antibacterial action of a heterometallic gold and silver-cyclophosphazene.

One of the most important uses of phosphazenes today involves its biomedical applications. They can also be employed as scaffolds for the design and construction of a variety of ligands in order to coordinate them to metallic drugs. The coordination chemistry of the (amino)cyclotriphosphazene ligand, [N3P3(NHCy)6], towards gold(I) complexes has been studied. Neutral complexes, [N3P3(NHCy)6{AuX}n] (X = Cl or C6F5; n = 1 or 2) (1-4), cationic complexes, [N3P3(NHCy)6{Au(PR3)}n](NO3)n (PR3 = PPh3, PPh2Me, TPA; n = 1, 2 or 3) (6-12) [TPA = 1,3,5-triaza-7-phosphaadamantane] and a heterometallic compound [N3P3(NHCy)6{Au(PPh3)}2{Ag(PPh3)}](NO3)3 (13) have been obtained and characterized by various methods including single-crystal X-ray diffraction for 7, which confirms the coordination of gold atoms to the nitrogens of the phosphazene ring. Compounds 1, 4, 6-13 were screened for in vitro cytotoxic activity against two tumor human cell lines, MCF7 (breast adenocarcinoma) and HepG2 (hepatocellular carcinoma), and for antimicrobial activity against five bacterial species including Gram-positive, Gram-negative, and Mycobacteria. Both the median inhibitory concentration (IC50) and minimum inhibitory concentration (MIC) values are among the lowest found for any gold or silver derivatives against the cell lines and particularly against the Gram-positive (S. aureus) strain and the mycobacteria used in this work. Structure-activity relationships are discussed in order to determine the influence of ancillary ligands and the number and type of metal atoms (silver or gold). Compounds 4 and 8 showed not only maximal potency on human cells but also some tumour selectivity. Remarkably, compound 13, with both gold and silver atoms, showed outstanding activity against both Gram-positive and Gram-negative strains (nanomolar range), thus having a cooperative effect between gold and silver, with MIC values which are similar or lower than those of gentamicine, ciprofloxacin and rifampicine. The broad spectrum antimicrobial efficacy of all these metallophosphazenes and particularly of heterometallic compound 13 could be very useful to obtain materials for surfaces with antimicrobial properties that are increasingly in demand.

Sorption/Desorption and Kinetics of Atrazine, Chlorfenvinphos, Endosulfan Sulfate and Trifluralin on Agro-Industrial and Composted Organic Wastes.

The use of pesticides presents a risk to terrestrial and aquatic ecosystems. For this reason, the development of strategies to prevent and restore pollution is of the greatest interest, including the adsorption to organic matter. The aim of the present study was to investigate the sorption/desorption and kinetics of atrazine, chlorfenvinphos, endosulfan sulfate, and trifluralin onto several raw organic wastes by batch experiments. Three kinetic models were used to fit the obtained sorption kinetics data and two to fit the obtained adsorption isotherm data; both the Freundlich and pseudo-second-order kinetic models described the sorption isotherms well. The desorption study revealed hysteresis in all cases, showing strong, and not completely reversible, adsorption in most cases, with the exception of atrazine-sawdust and chlorfenvinphos-sawdust and chicken manure combinations, for which responses were weak and irreversible. The best kinetic, adsorption and desorption constants were achieved for the hydrophobic pesticides. With respect to sorption-desorption rates, orujillo was found to be the best adsorbent for atrazine, while composted urban solid waste was more suitable for trifluralin and endosulfan sulfate. Sorption constants and simple correlations indicated that, not only the organic matter content, but also the nature of the organic matter itself, and the pesticide and adsorbent properties, determine pesticide sorption-desorption. The use of wastes as efficient and cheap adsorbents for reducing the risk of pesticide pollution is proposed.

(Amino)cyclophosphazenes as Multisite Ligands for the Synthesis of Antitumoral and Antibacterial Silver(I) Complexes.

The reactivity of the multisite (amino)cyclotriphosphazene ligands, [N3P3(NHCy)6] and [N3P3(NHCy)3(NMe2)3], has been explored in order to obtain silver(I) metallophosphazene complexes. Two series of cationic silver(I) metallophosphazenes were obtained and characterized: [N3P3(NHCy)6{AgL}n](TfO)n [n = 2, L = PPh3 (2), PPh2Me (4); n = 3, L = PPh3 (3), PPh2Me (5), TPA (TPA = 1,3,5-triaza-7-phosphaadamantane, 6)] and nongem-trans-[N3P3(NHCy)3(NMe2)3{AgL}n](TfO)n [n = 2, L = PPh3 (7), PPh2Me (9); n = 3, L = PPh3 (8), PPh2Me (10)]. 5, 7, and 9 have also been characterized by single-crystal X-ray diffraction, thereby allowing key bonding information to be obtained. Compounds 2-6, 9, and 10 were screened for in vitro cytotoxic activity against two tumor human cell lines, MCF7 (breast adenocarcinoma) and HepG2 (hepatocellular carcinoma), and for antimicrobial activity against five bacterial species including Gram-positive, Gram-negative, and Mycobacteria strains. Both the IC50 and MIC values revealed excellent biological activity for these metal complexes, compared with their precursors and cisplatin and also AgNO3 and silver sulfadiazine, respectively. Both IC50 and MIC values are among the lowest values found for any silver derivatives against the cell lines and bacterial strains used in this work. The structure-activity relationships were clear. The most cytotoxic and antimicrobial derivatives were those with the triphenylphosphane and [N3P3(NHCy)6] ligands. A significant improvement in the activity was also observed upon a rise in the number of silver atoms linked to the phosphazene ring.

Investigation of mechanisms of toxicity and exclusion by transporters of the preservatives triclosan and propylparaben using batteries of Schizosaccharomyces pombe strains.

Triclosan (TCS) and propylparaben (PPB) are antimicrobials widely used. They present many similarities in their applications and also in their human and environmental health risks. In order to investigate the mechanisms of toxic action and the efflux pumps involved in their detoxication, we used a strategy with batteries of Schizosaccharomyces pombe yeast strains, either defective in cell signalling, in detoxification pumps, or in cell surveillance mechanisms. Yeast were exposed up to 20 h in solid medium or in liquid medium in 96-well plates. The mechanisms of action investigated were spindle defects (mph1), stress (pmk1), DNA interference (rad3) or diverse effects (MDR-sup). The efflux pumps investigated were Bfr1, Pmd1, Mfs1 and Caf5 or the Pap1 transcription factor. Here we show that TCS was 75 times more toxic than PPB in the wild type fission yeast. More oxidative stress and less protection by exclusion pumps were observed for TCS than for PPB. The cytotoxicity produced by TCS decreased from bfr1>mfs1>pmd1 > pap1 and caf5A deficient strains. In contrast, cytotoxic concentrations of PPB caused only a mild stress. The protection provided for PPB by the transporters was more marked than for TCS, decreasing from Pmd1, Caf5, Mfs1 and Bfr1. Furthermore, microtubule and DNA interferences were revealed for PPB, according to the cytotoxicity of mph1 and rad3 defective cells, respectively. As both compounds present complex adverse effects at concentrations close to exposure, and their combination clearly causes a strong potentiation, more exhaustive controls and regulations in their use should be considered.

Integration of fish cell cultures in the toxicological assessment of effluents.

The pollution by industrial and municipal effluents are major sources of concerns. Fish cell cultures were applied in different strategies of the evaluation of effluents, particularly whole toxicity, toxicity identification evaluation and mode of action studies based in adverse outcome pathways. Whole effluent toxicity was evaluated using a battery of five model systems from four trophic levels: Daphnia magna was the most sensitive system, followed by the hepatoma fish cell line PLHC-1, the bacterium Allivibrio fischeri, the fibroblastic fish cell line RTG-2 and the algae Chlorella vulgaris, detecting a risk of eutrofization. The uptake of neutral red was more sensitive than the content of protein assay. The main morphological alterations observed were cell loss, hydropic degeneration, and a general loss of lysosomes and of their perinuclear distribution. The toxicity was characterized in PLHC-1 cells through toxicity identification evaluation, in which a partial reduction with graduation at pH 11, filtration, aeration and addition of thiosulfate or EDTA was shown; on the other hand, a low sorption in solid phase extraction suggested that the main responsible were not organic compounds. Consequently, it was not necessary to apply an effect directed analysis HPLC fractionation. In the chemical identification phase, Zn, Cd, As, Cu and Pb were quantified in decreasing concentrations. In the toxicity confirmation phase, a reconstituted sample and individual solutions, presented decreasing toxicity: Zn > Pb > As+5 > Cd > Cu > As+3, the global toxicity being explained by response addition. In the last step, the mode of action was investigated using five specific biomarkers. While metallothionein and succinate dehydrogenase activity were increased, no changes occurred for lysosomal function, acetylcholinesterase and EROD activities, the responsibility of the toxicity for the elements found being confirmed.

Thermoluminescence as a complementary technique for the toxicological evaluation of chemicals in photosynthetic organisms.

Thermoluminescence is a simple technique very useful for studying electron transfer reactions on photosystem II (standard thermoluminescence) or the level of lipid peroxidation in membranes (high temperature thermoluminescence) in photosynthetic organisms. Both techniques were used to investigate the effects produced on Chlorella vulgaris cells by six compounds: the chemical intermediates bromobenzene and diethanolamine, the antioxidant propyl gallate, the semiconductor indium nitrate, the pesticide sodium monofluoroacetate and the antimalarial drug chloroquine. Electron transfer activity of the photosystem II significantly decreased after the exposure of Chlorella cells to all the six chemicals used. Lipid peroxidation was slightly decreased by the antioxidant propyl gallate, not changed by indium nitrate and very potently stimulated by diethanolamine, chloroquine, sodium monofluoroacetate and bromobenzene. For five of the chemicals studied (not bromobenzene) there is a very good correlation between the cytotoxic effects in Chlorella cells measured by the algal growth inhibition test, and the inhibition of photosystem II activity. The results suggest that one very important effect of these chemicals in Chlorella cells is the inhibition of photosynthetic metabolism by the blocking of photosystem II functionality. In the case of sodium monofluoroacetate, diethanolamine and chloroquine this inhibition seems to be related with the induction of high level of lipid peroxidation in cells that may alter the stability of photosystem II. The results obtained by both techniques supply information that can be used as a supplement to the growth inhibition test and allows a more complete assessment of the effects of a chemical in photosynthetic organisms of aquatic ecosystems.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment.

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Neutral red uptake assay for the estimation of cell viability/cytotoxicity.

The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.

Toxicological assessment of indium nitrate on aquatic organisms and investigation of the effects on the PLHC-1 fish cell line.

Indium nitrate is mainly used as a semiconductor in batteries, for plating and other chemical and medical applications. There is a lack of available information about the adverse effects of indium compounds on aquatic organisms. Therefore, the toxic effects on systems from four trophic levels of the aquatic ecosystem were investigated. Firstly, the bacterium Vibrio fischeri, the alga Chlorella vulgaris and the cladoceran Daphnia magna were used in the toxicological evaluation of indium nitrate. The most sensitive model was V. fischeri, with a NOAEL of 0.02 and an EC(50) of 0.04 mM at 15 min. Although indium nitrate should be classified as harmful to aquatic organisms, it is not expected to represent acute risk to the aquatic biota. Secondly, PLHC-1 fish cell line was employed to investigate the effects and mechanisms of toxicity. Although protein content, neutral red uptake, methylthiazol metabolization, lysosomal function and acetylcholinesterase activity were reduced in cells, stimulations were observed for metallothionein levels and succinate dehydrogenase and glucose-6-phosphate dehydrogenase activities. No changes were observed in ethoxyresorufin-O-deethylase activity. To clarify the main events in PLHC-1 cell death induced by indium nitrate, nine modulators were applied. They were related to oxidative stress (alpha-tocopherol succinate, mannitol and sodium benzoate), disruption of calcium homeostasis (BAPTA-AM and EGTA), thiol protection (1,4-dithiotreitol), iron chelation (deferoxiamine) or regulation of glutathione levels (2-oxothiazolidine-4-carboxylic acid and malic acid diethyl ester). The main morphological alterations were hydropic degeneration and loss of cells. At least, in partly, toxicity seems to be mediated by oxidative stress, and particularly by NADPH-dependent lipid peroxidation.

Ecotoxicological effects of the antioxidant additive propyl gallate in five aquatic systems.

Propyl gallate is an antioxidant widely used in foods, cosmetics and pharmaceuticals. The occurrence and fate of additives in the aquatic environment is an emerging issue in environmental chemistry. To date, there is little available information about the adverse effects of propyl gallate on aquatic organisms. Therefore, the toxic effects were investigated, using five model systems from four trophic levels. The most sensitive system was the hepatoma fish cell line PLHC-1 according to total protein content, with an EC(50) of 10 microM and a NOAEL of 1 microM at 72 h, followed by the immobilization of Daphnia magna, the inhibition of bioluminescence of Vibrio fischeri, the salmonid fish cell line RTG-2 and the inhibition of the growth of Chlorella vulgaris. Although protein content, neutral red uptake, methylthiazol metabolization and acetylcholinesterase activity were reduced in PLHC-1 cells, stimulations were observed for lysosomal function, succinate dehydrogenase, glucose-6-phosphate dehydrogenase and ethoxyresorufin-O-deethylase activities. No changes were observed in metallothionein levels. The main morphological observations were the loss of cells and the induction of cell death mainly by necrosis but also by apoptosis. The protective and toxic effects of propyl gallate were evaluated. General antioxidants and calcium chelators did not modify the toxicity of propyl gallate, but an iron-dependent lipid peroxidation inhibitor gave 22% protection. The results also suggest that propyl gallate cytotoxicity is dependent on glutathione levels, which were modulated by malic acid diethyl ester and 2-oxothiazolidine-4-carboxylic acid. According to the results, propyl gallate should be classified as toxic to aquatic organisms.

Ecotoxicological evaluation of sodium fluoroacetate on aquatic organisms and investigation of the effects on two fish cell lines.

Sodium monofluoroacetate (compound 1080) is one of the most potent pesticides. It is also a metabolite of many other fluorinated compounds, including anticancer agents, narcotic analgesics, pesticides or industrial chemicals. Other sources of water contamination are the atmospheric degradation of hydrofluorocarbons and hydrochlorofluorocarbons. However, there is little information available about the adverse effects of sodium fluoroacetate in aquatic organisms. Firstly, the bacterium Vibrio fischeri (decomposer), the alga Chlorella vulgaris (1st producer) and the cladoceran Daphnia magna (1st consumer) were used for the ecotoxicological evaluation of SMFA. The most sensitive models were C. vulgaris and D. magna, with a NOAEL of 0.1 and an EC50 of 0.5 mM at 72 h, respectively. According to the results after the acute exposure and due to its high biodegradation rate and low bioaccumulation potential, sodium fluoroacetate is most unlikely to produce deleterious effects to aquatic organisms. Secondly, two fish cell lines were employed to investigate the effects and mechanisms of toxicity in tissues from 2nd consumers. The hepatoma fish cell line PLHC-1 was more sensitive to SMFA than the fibroblast-like fish cell line RTG-2, being the uptake of neutral red the most sensitive bioindicator. Lysosomal function, succinate dehydrogenase and acetylcholinesterase activities were inhibited, glucose-6-phosphate dehydrogenase activity was particularly stimulated, and metallothionein and ethoxyresorufin-O-deethylase levels were not modified. Intense hydropic degeneration, macrovesicular steatosis and death mainly by necrosis but also by apoptosis were observed. Moreover, sulphydryl groups and oxidative stress could be involved in PLHC-1 cell death induced by SMFA more than changes in calcium homeostasis.

Toxicological effects of the lipid regulator gemfibrozil in four aquatic systems.

Gemfibrozil is a lipid-regulating agent widely used in patients at risk of coronary disease. Pharmaceutical products, such as gemfibrozil, are found in municipal effluents and represent a major source of contamination. To date, there is little available information about the adverse effects of gemfibrozil in aquatic organisms. For this reason, the toxic effects were investigated using model systems from four trophic levels. The most sensitive system was the immobilization of Daphnia magna, with a non-observed adverse effect level of 30 microM and a mean effective concentration of 120 microM after 72 h, followed by the inhibition of bioluminescence of Vibrio fischeri, the hepatoma fish cell line PLHC-1 line and the inhibition of the growth of Chlorella vulgaris. Although protein content, neutral red uptake, methylthiazol metabolization and lysosomal function were reduced in PLHC-1 cells, stimulations were observed for lysosomal function, metallothionein levels and succinate dehydrogenase, glucose-6-phosphate dehydrogenase and acetylcholinesterase activities. No changes were observed in ethoxyresorufin-O-deethylase activity. The main morphological alterations were hydropic degeneration and loss of cells. Modulation studies on gemfibrozil toxicity were also carried out. General antioxidants and calcium chelators did not modify the toxicity of gemfibrozil, whereas a Fe(III) chelator, a membrane permeable sulphydryl-protecting compound and glutathione level modifying agents did change the toxicity. One of the possible mechanisms of gemfibrozil toxicity seems to be the binding to sulphydryl groups, including those of glutathione. According to the result, gemfibrozil should be classified as harmful to aquatic organisms. However, comparing the concentrations in water and the toxicity quantified in the assayed systems, gemfibrozil is not expected to represent acute risk to the aquatic biota.

Ecotoxicological assessment of bromobenzene using a test battery with five model systems.

Bromobenzene (BrB) is used as a solvent for crystallization and as an additive to motor oils and may be released into the environment through various waste streams. However, there is limited available information about the toxic hazard of BrB in the aquatic environment. Consequently, the ecotoxicological effects induced by BrB were investigated using five model systems with representants from four trophic levels. The battery included bioluminescence inhibition of the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris and immobilization of the cladoceran Daphnia magna. Total protein content, neutral red uptake and MTS metabolization were reduced, while lysosomal function, succinate dehydrogenase activity, G6PDH activity and leakage, metallothionein levels and EROD activity were stimulated in PLHC-1 and RTG-2 fish cell lines. The most sensitive bioindicator was the bioluminiscence of V. fischeri, with an EC(50) of 0.04mM BrB at 15min and a non-observed adverse effect level of 0.02 mM BrB. There is a large difference in sensitivity to BrB among the model systems probably due to the metabolic capacity of the different species. PLHC-1 cells were more sensitive to BrB than RTG-2 cells. The most prominent morphological effects observed were hydropic degeneration, loss of cells and of the perinuclear pattern of distribution of lysosomes. Therefore, BrB should be classified as toxic to aquatic organisms.

Ecotoxicological evaluation of the antimalarial drug chloroquine.

There is limited information available about the potential environmental effects of chloroquine (CQ), a widely used antimalarial agent and a promising inexpensive drug in the management of HIV disease. The acute effects of CQ were studied using four ecotoxicological model systems. The most sensitive bioindicator was the immobilization of the cladoceran Daphnia magna, with an EC50 of 12 microM CQ at 72 h and a non-observed adverse effect level of 2.5 microM CQ, followed very closely by the decrease of the uptake of neutral red and the reduction of the lysosomal function in the fish cell line PLHC-1 derived from the top minnow Poeciliopsis lucida, probably due to the selective accumulation of the drug into the lysosomes. There was significant cellular stress as indicated by the increases on metallothionein and glucose-6P dehydrogenase levels after 24 h of exposure and succinate dehydrogenase activity mainly after 48 h. No changes were observed for ethoxyresorufin-O-deethylase (EROD) activity. The least sensitive model was the inhibition of bioluminescence in the bacterium Vibrio fischeri. An increase of more than five-fold in the toxicity from 24 to 72 h of exposure was observed for the inhibition of the growth in the alga Chlorella vulgaris and the content of total protein and MTS tetrazolium salt metabolization in PLHC-1 cells. At the morphological level, the most evident alterations in PLHC-1 cultures were hydropic degeneration from 25 microM CQ after 24h of exposure and the presence of many cells with pyknotic nuclei, condensed cytoplasm and apoptosis with concentrations higher than 50 microM CQ after 48 h of exposure. In conclusion, CQ should be classified as harmful to aquatic organisms.

Toxic cyanobacterial cells containing microcystins induce oxidative stress in exposed tilapia fish (Oreochromis sp.) under laboratory conditions.

The effects of microcystins from cyanobacterial cells on various oxidative stress biomarkers in liver, kidney and gill tissues in freshwater tilapia fish (Oreochromis sp.) were investigated under laboratory conditions. Microcystins are a family of cyclic peptide toxins produced by species of freshwater cyanobacteria (blue-green algae). Fish were exposed to the cyanobacterial cells in two ways: mixed with a commercial fish food or crushed into a commercial fish food so that the toxins were released. Two different exposure times were studied: 14 and 21 days. The oxidative status of fish was evaluated by analyzing the level of lipid peroxidation (LPO), as well as the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). The findings of the present investigation show that microcystins induce oxidative stress in a time-dependent manner and that the type of administration of the cyanobacterial cells influences the extent of these effects. Thus, the crushed cyanobacterial cells (released toxins) induced the antioxidant defences studied and increased the LPO level to a greater extent than the non-crushed cells. The liver was the most affected organ followed by kidney and gills. These results together with reports that fish can accumulate microcystins mean that cyanobacterial blooms are an important health, environmental and economic problem.

Ecotoxicological evaluation of the additive butylated hydroxyanisole using a battery with six model systems and eighteen endpoints.

The occurrence and fate of additives in the aquatic environment is an emerging issue in environmental chemistry. This paper describes the ecotoxicological effects of the commonly used additive butylated hydroxyanisole (BHA) using a test battery, comprising of several different organisms and in vitro test systems, representing a proportion of the different trophic levels. The most sensitive system to BHA was the inhibition of bioluminescence in Vibrio fischeri bacteria, which resulted in an acute low observed adverse effect concentration (LOAEC) of 0.28 microM. The next most sensitive system was the immobilization of the cladoceran Daphnia magna followed by: the inhibition of the growth of the unicellular alga Chlorella vulgaris; the endpoints evaluated in Vero (mammalian) cells (total protein content, LDH activity, neutral red uptake and MTT metabolization), mitotic index and root growth inhibition in the terrestrial plant Allium cepa, and finally, the endpoints used on the RTG-2 salmonid fish cell line (neutral red uptake, total protein content, MTS metabolization, lactate dehydrogenase leakage and activity, and glucose-6-phosphate dehydrogenase activity). Morphological alterations in RTG-2 cells were also assessed and these included loss of cells, induction of cellular pleomorphism, hydropic degeneration and induction of apoptosis at high concentrations. The results from this study also indicated that micronuclei were not induced in A.cepa exposed to BHA. The differences in sensitivity for the diverse systems that were used (EC50 ranged from 1.2 to >500 microM) suggest the importance for a test battery approach in the evaluation of the ecological consequences of chemicals. According to the results, the levels of BHA reported in industrial wastewater would elicit adverse effects in the environment. This, coupled with its potential to bioaccumulate, makes BHA a pollutant of concern not only for acute exposures, but also for the long-term.