Influence of pH on the toxicity of nitrophenols to Microtox and Spirotox tests

The toxicity of mononitrophenols and dinitrophenols (DNP) to luminescent bacteria Vibrio fischeri (Microtox test) and ciliated protozoan Spirostomum ambiguum (Spirotox test) was evaluated. Spirotox was more sensitive to the tested nitrophenols (NPs) than the Microtox test. 2,5-DNP was the most toxic and 2-NP was the least toxic to the both bioindicators. The toxicity depended greatly on the pH of the medium. The highest changes were observed for DNPs, where the toxicity decreased more than 20-times when the pH increased from 6 to 8. No significant decrease of the toxicity was found for NPs, when the pH increased from 6 to 7. Greater increase of the pH to 8 caused from 1.5 to 4-fold decrease of the toxicity.     

Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus

As the production of nanoparticles of ZnO, TiO2 and CuO is increasing, their (eco)toxicity to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus was studied with a special emphasis on product formulations (nano or bulk oxides) and solubilization of particles. Our innovative approach based on the combination of traditional ecotoxicology methods and metal-specific recombinant biosensors allowed to clearly differentiate the toxic effects of metal oxides per se and solubilized metal ions. Suspensions of nano and bulk TiO2 were not toxic even at 20 g l(-1). All Zn formulations were very toxic: L(E)C50 (mg l(-1)) for bulk ZnO, nanoZnO and ZnSO4.7H2O: 1.8, 1.9, 1.1 (V. fischeri); 8.8, 3.2, 6.1 (D. magna) and 0.24, 0.18, 0.98 (T. platyurus), respectively. The toxicity was due to solubilized Zn ions as proved with recombinant Zn-sensor bacteria. Differently from Zn compounds, Cu compounds had different toxicities: L(E)C50 (mg l(-1)) for bulk CuO, nano CuO and CuSO4: 3811, 79, 1.6 (V. fischeri), 165, 3.2, 0,17 (D. magna) and 95, 2.1, 0.11 (T. platyurus), respectively. Cu-sensor bacteria showed that toxicity to V. fischeri and T. platyurus was largely explained by soluble Cu ions. However, for Daphnia magna, nano and bulk CuO proved less bioavailable than for bacterial Cu-sensor. This is the first evaluation of ZnO, CuO and TiO2 toxicity to V. fischeri and T. platyurus. For nano ZnO and nano CuO this is also a first study for D. magna.     

Influence of organic solvents on the sensitivity of a bioluminescence toxicity test with Vibrio harveyi

The possibility of using organic solvents (OSs) to increase the susceptibility of bioluminescent microorganisms in a bioassay for assessing the toxicity of chemicals dissolved in water was investigated. To conduct the tests acetonitrile, dimethyl sulfoxide, ethanol, methanol, and isopropanol were used as OSs and Cd, Hg, and Zn as reference toxicants. The addition of OSs modified the toxicity of the three metals to Vibrio harveyi, according on the bioluminescence assay used. The sensitivity of the luminescence bioassay for Hg increased in the presence of the five OSs, thus indicating a greater toxic effect. However, the sensitivity of the assay for the other two metals, Cd and Zn, increased or decreased (lesser toxic effect) depending on the concentration at which the OSs were used. No correlation was observed between the concentration of the five OSs and the toxicity of the three reference toxicants. From this it can be deduced that none of these OSs could be recommended for increasing generically the sensitivity of toxicity biotests using V. harveyi.     

Acute toxicity of arsenic to Aliivibrio fischeri (Microtox® bioassay) as influenced by potential competitive–protective agents

In this study, we investigated the effect of some potential alleviative compounds against the acute toxicity of arsenic (As^V, As^III and DMA^V) on Aliivibrio fischeri (formerly Vibrio fischeri), a bioluminescent model bacterium, through the Microtox® bioassay. The compounds studied differed in their mechanism of action, and they included the following: phosphate and glycerol, as chemical analogues (and potential competitors) of As^V or As^III, respectively; citrate, a weak natural organic ligand; and the antioxidant ascorbic acid. Special attention was paid to phosphate effects, a widespread pollutant in natural environments. As^V was found to be more acutely toxic than As^III to A. fischeri, in accordance with its higher interaction with the bacteria. Both As^V and As^III were found to be much more acutely toxic than DMA^V, which was essentially non-acutely toxic even at very high concentrations. Phosphate presence (at equimolar P/As ratios or higher) resulted in the almost total suppression of bioluminescence inhibition, suggesting it exerts an alleviative effect against As^V acute toxicity on A. fischeri. Interestingly, the uptake and the percentage of extracellular As^V were not affected by the addition of phosphate, suggesting that such protective effect does not result from the competition for their common transporters. In contrast, the acute toxicity of As^III was essentially unaffected by phosphate. Glycerol did not decrease the acute toxicity or the uptake of As^III by A. fischeri, denoting the likely occurrence of an additional mechanism for As^III uptake in such bacteria. Similarly, citrate and ascorbic acid essentially did not caused alleviation of As^V or As^III acute toxicity. As for environmental and operational implications, P could beneficially protect aquatic microorganisms against acute detrimental effects of As^V, whilst its presence could mask the toxicity due to As^V when assessed using the Microtox® bioassay, thus leading to seriously underestimate the actual ecological and health risks.     

The toxicity of tri-substituted benzenes to the protozoan ciliate Spirostomum ambiguum

The Spirotox test utilises a large ciliate protozoan Spirostomum ambiguum as a test organism. The goal of the present study was to evaluate the toxicity of tri-substituted benzenes in the Spirotox test. Twenty-six organic compounds were tested in this study and included: dimethylphenols (DMPs), dichlorophenols (DCMs), trichlorobenzenes (TCBs), dichloroanilines (DCAs), dinitrophenols (DNPs), dinitroaniline (DNA), dinitrochlorobenzene (DNCB) and dinitrofluorobenzene (DNFB). The toxicity of the compounds tested varied almost four orders of a magnitude. DMPs and DCAs were the least toxic, whereas dinitro derivatives were the most toxic to S. ambiguum. When chlorine or fluorine atoms were replaced by amino or hydroxy substituents, the toxicity increased dramatically. The results of the Spirotox test were compared with three other bioassays that are widely used around the world: Microtox, Tetrahymena pyriformis and Daphnia magna. The Spirotox was less sensitive than these other bioassays for the majority of these compounds, with an exception found for the dinitro derivatives.     

Combined phytoremediation of metal-working fluids with maize plants inoculated with different microorganisms and toxicity assessment of the phytoremediated waste

The aim of this study was to validate the effectiveness of a phytoremediation procedure for metal-working fluids (MWFs) with maize plants growing in hydroponic culture in which the roots grow on esparto fibre and further improve bioremediation potential of the system with root beneficial bacteria, seeking a synergistic effect of the plant–microorganism combination. Chemical oxygen demand (COD), pH, total and type of hydrocarbons measured after phytoremediation indicated that the process with maize plants was successful, as demonstrated by the significant decrease in the parameters measured. This effect was mainly due to the plant although inoculated microorganisms had a relevant effect on the type of remaining hydrocarbons. The success of the phytoremediation process was further confirmed by two toxicity tests, one of them based on chlorophyll fluorescence measurements on maize plants and another one based on cyanobacteria, using a bioluminescent toxicity bioassay; both tests demonstrated that the phytoremediated waste was significantly less toxic than the initial non-phytoremediated MWFs.     

丙烯腈废水及几种处理工艺出水的毒性

分别利用小麦、发光菌毒性测试技术研究了模拟丙烯腈废水以及几种处理工艺出水对小麦芽伸长、根伸长、发芽率和发光菌发光度的毒性效应差异。结果表明,丙烯腈对小麦芽伸长的毒性影响最大;各类物质对小麦根伸长和发芽率并未造成毒性影响;其他物质对发光菌发光度的毒性最大。模拟废水对小麦芽伸长、根伸长、发芽率和发光菌发光度的毒性效应分别为:部分相加、部分相加、协同和拮抗作用。几种处理工艺出水对上述指标的毒性影响顺序依次是:模拟废水加成出水活性炭吸附出水Fenton法出水;Fenton法出水模拟废水加成出水活性炭吸附出水;Fenton法出水活性炭吸附出水模拟废水加成出水;活性炭吸附出水加成出水Fenton法出水模拟废水。比较几种处理工艺出水的毒性发现,几乎每种废水的毒性都有所降低,这说明几种处理工艺能有效降低模拟废水的毒性,具有深刻的实际指导意义。     

The use of bioluminescent biotests for study of natural and laboratory aquatic ecosystems

A set of bioluminescent tests was developed to monitor water quality in natural and laboratory ecosystems. It consisted of four bioluminescent systems: luminous bacteria, coupled enzyme system NADH:FMN-oxidoreductase-luciferase and triplet enzyme systems with alcohol dehydrogenase and trypsin. The set of biotests was applied for a small forest pond (Siberia, Russia), laboratory microecosystems polluted with benzoquinone and a batch culture of blue-green algae. Thereby effects of natural water compared to those of models of heavy pollution and “bloom” of blue-greens on the bioluminescent tests were revealed. The set of biotests was not affected by a natural seasonal variability of water quality in the unpolluted pond, but responded to the heavy pollution and the “bloom” of blue-greens. The set of biotests could be recommended as the alarm test to control the acute toxicity of natural water bodies.     

Toxicity of metals and organic chemicals evaluated with bioluminescence assays

The development of a bioluminescent sensor organism (Shk1) that was created for assessing wastewater toxicity was reported several years ago. In order to establish a test battery to better characterize wastewater toxicity, additional luminescent sensor organisms were later created. The present study focused on one promising candidate (PM6), a Pseudomonas spp. strain, because of its high level of luminescence compared to that of other newly created organisms. Using a batch toxicity testing protocol, the toxicity of 7 metals and 25 organic compounds was evaluated with the PM6 and Shk1 assays. Results indicated that the toxicity data of the PM6 and the Shk1 assays were correlated, and no assay appeared to be particularly more sensitive to a group of toxicants than the other assay. The results of the PM6 and Shk1 assays were further evaluated by comparing with the results of the Vibrio fischeri luminescence inhibition assay and activated sludge inhibition assays. Data suggested that PM6 and Shk1 more closely represented activated sludge organisms than V. fischeri. The suitability of using PM6 and Shk1 for assessing wastewater toxicity on activated sludge, both individually and in a test battery, was discussed.     

Anogenital distance and its application in environmental health research

Anogenital distance (AGD), a useful anthropometric measurement for genital development in both animals and humans, was originally found by reproductive toxicologists in rodent experiments. As an easy-to-measure and sensitive marker, AGD has become a bioassay of fetal androgen action and a well-established reproductive toxicity endpoint in animals. It is generally accepted that AGD is sexually dimorphic in many mammals, with males having longer AGD than females. Exposure to proposed endocrine disruptors may result in reduced AGD; thus, it has been used to measure health effects of compounds with endocrine-altering properties or endocrine-disrupting chemicals (EDCs) in environmental toxicology. Moreover, AGD is an important clinical measure to address endocrine-sensitive endpoints in the first year of life and to assess the adverse impact of in utero exposure to environmental EDCs. Recently, AGD has been identified as one of the endpoints in the US Environmental Protection Agency guidelines for reproductive toxicity studies in humans, but use of AGD in human studies is still rare, and the results remain mixed and inconclusive due to many reasons. In order to achieve a breakthrough, researchers are endeavoring to standardize the measurement of AGD, normalize age-specific population data in different ethnic groups, and conduct more in-depth human researches in this field.     

Passaic river sediment interstitial water phase I toxicity identification evaluation

A suite of tests was conducted to evaluate and identify the cause or causes of toxicity in Passaic River sediments. Sediment toxicity was measured with three types of bioassays: a whole sediment bioassay with the marine amphipod, Ampelisca abdita, and interstitial water bioassays with A. abdita and the bioluminescent bacterium Vibrio fisheri (Microtox((R))). In addition, a Phase I Toxicity Identification Evaluation (TIE) was conducted to elucidate the cause of observed toxicity. Analytical concentrations of selected residues in whole sediment and interstitial water from the five sampling stations were considered in conjunction with the conclusions drawn from the toxicity tests and Phase I TIE results. Finally, a toxic units approach was used to evaluate the predicted toxicity of measured interstitial water residue concentrations. There was a lack of toxic response in the short-term interstitial water bioassays, indicating that oxidants, soluble forms of metals, and dissolved phase neutral organics were not likely toxicants. However, there was significant toxicity indicated by the whole sediment A. abidita bioassays. After 10 days, there was complete or near complete mortality in amphipods exposed to all of the sediment samples tested. Removal of interstitial water toxicity by filtration was common to all four stations that exhibited measurable initial toxicity. The observed toxicity characteristics are consistent with particle associated neutral organics. This conclusion is supported by toxicity removal via filtration, lack of toxicity in the Microtox((R)) assays, and the fact that whole sediments were more toxic than was interstitial water.     

Toxicity to Microtox, micro-algae and duckweed of effluents from the Bai Bang paper company (BAPACO), a Vietnamese bleached kraft pulp and paper mill

Toxicity of the combined effluent as well as effluents from different production units of BAPACO, a Vietnamese bleached kraft integrated pulp and paper mill, was studied using three ecotoxicological tests: Microtox, the micro-alga, Selenastrum capricornutum, and the duckweed, Lemna aequinoctialis. Physico-chemical characteristics of the effluents were also analyzed. Due to unstable operating conditions of the mill, a number of samples taken at different monitoring periods from each effluent were tested, in order to get statistically reliable ranges and averages of toxicity characteristics. For the mill combined effluent, it was found that the micro-algae were the most sensitive followed by Microtox bacteria, while duckweed was not sensitive. Microtox tests showed that the bleaching filtrate from the chlorination stage (C-stage) was the most toxic among all effluents in the mill. The combined effluent from the bleaching plant contributed the largest TEF (Toxicity Emission Factor) to the toxicity balance of the whole BAPACO combined effluent. A less-than-additive mode of toxicity was obtained. In spite of high pollution loads, e.g. BOD, COD, SS, to the Red River, the toxicity of the effluent was considered to be low. However, during periods of low river water sublethal toxic effects on the biota may be expected.     

Assessing sediment toxicity and arsenite concentration with bacterial and traditional methods

Three sediment samples LP (pool where logs are stored), LF (brook through landfill area), KN (Kaskesniemi) which is in Lake Pyh?selk? downstream from the mill, were taken from an old sawmill area and one from the unpolluted Lake H?yti?inen. The arsenite concentration was measured by GFAAS and two arsenite biosensing bacterial strains Pseudomonas fluorescens OS8 (pTPT31) and Escherichia coli MC1061 (pTOO31). The toxicity of sediment and pore water samples was determined by using luminescent bacteria (Flash test) and, further, whole sediment toxicity was measured using 10 days growth test and 50 days emergency test with midges (Chironomus riparius). With the flash test a lowered EC50 value was found only in sediment LF (EC50=0.17 v/v%). The Flash test indicated that all sediment samples taken from the sawmill area were highly toxic to bacteria, whereas growth and the emergence of chironomids showed no effects in other samples than LF. The midges tolerate well the contaminated environment. In contrast, bioavailability of arsenite of sediment samples KN and LF was quite high determined using the biosensor-strains in a direct contact assay. The bioavailable fraction of sediment LP was 6-10% out of the total arsenite concentration obtained with GFAAS (0.46-0.77 microg g-1 dw). The results show that the choice of analysis method grossly affects the outcome without any of the method giving an incorrect result. Different methods measure different parameters of a toxic sample and can thus be used to complement each other.     

Comparison of bioluminescence and nitrification inhibition methods for assessing toxicity to municipal activated sludge

The aim of this study was to compare two alternative toxicity assessment methods to determine wastewater toxicity and predict treatment plant process upsets. The toxicity of two synthetic organic compounds (triclosan and 4-n-nonylphenol), which are commonly detected in municipal wastewater, and municipal and industrial wastewaters with different heavy metals content were evaluated by the nitrification inhibition assay and bioluminescence toxicity test. Comparison between both assays confirmed that Vibrio fischeri is generally more sensitive than autotrophic bacteria, and, if not calibrated, the bioluminescence method tends to overestimate toxic effects on activated sludge biomass. The nitrification inhibition assay appears to predict plant process upsets more accurately. Both methods showed a significant toxicity decrease through treatment that could be partially attributed to the significant heavy metals removal obtained by primary and secondary treatment. A good correlation for the two assays was obtained, as indicated by a high correlation coefficient (r2 = 0.80).     

Biodegradation and detoxification of organophosphate insecticide, malathion by Fusarium oxysporum f. sp. pisi cutinase

Efficiencies of two lypolytic enzymes (fungal cutinase and yeast esterase) in malathion degradation were investigated. Surprisingly, degradation rate of malathion by fungal cutinase was very high, i.e. almost 60% of initial malathion (500 mg l(-1)) was decomposed within 0.5 h, and nearly 50% of the degraded malathion disappeared within initial 15 min. With the yeast esterase, despite the same concentration, more than 65% of malathion remained even after 2-day treatment. During enzymatic degradation of malathion, two malathion-derived compounds were detected, and time-course changes in composition were also monitored. In the degradation by both fungal cutinase and yeast esterase, two additional organic chemicals were produced from malathion: malathion monoacid (MMA) and malathion diacid (MDA) by ester hydrolysis. Final chemical composition after 2 d was significantly dependent on the enzyme used. Fungal cutinase produced MDA as a major degradation compound. However in the malathion degradation by yeast esterase, an isomer of MMA was produced in abundance in addition to MDA. Toxic effects of malathion and its final degradation products were investigated using various recombinant bioluminescent bacteria. As a result, the degradation products (including MMA) by esterase severely caused membrane damage and inhibition of protein synthesis in bacterial cells, while in the fungal cutinase processes, malathion was significantly degraded to non-toxic MDA after the extended period (2 days).     

Toxicological properties of trialkyl phosphorothioate and dialkyl alkyl- and arylphosphonothioate esters

Impurities such as O,S,S-trimethyl phosphorodithioate (TMPD) and the S-methyl isomer of malathion (isomalathion) strongly potentiated the mammalian toxicity of malathion. In contrast, impurities present in the phosphoramidothioate insecticide acephate had an antagonizing effect on its mammalian toxicity. The potentiation of the toxicity of malathion was attributed to inhibition of mammalian liver and serum carboxylesterase. O,O,S-Trimethyl phosphorothioate (TMP), another impurity present in technical malathion and in other organophosphorus insecticides, proved to be highly toxic. Rats given a single oral dose of TMP at a level as low as 20 mg/kg died over a period of three weeks, with death occurring with non-cholinergic signs of poisoning. TMPD also caused similar delayed death in rats. O,O,O-Trimethyl phosphorothioate (TMP=S), also another impurity in technical malathion and a structural isomer of TMP, was a potent antagonist to the delayed toxicity of TMP. Examination of a number of related trialkyl phosphorothioate and dialkyl alkylphosphonothioate esters revealed several of these compounds to be highly toxic to rats.     

Effect directed analysis and mixture effects of estrogenic compounds in a sediment of the river Elbe

INTRODUCTION: Endocrine disrupting chemicals (EDCs) are present in the environment and can have serious effects on humans and wildlife. For the establishment of environmental quality guidelines and regulation of EDCs, a better understanding and knowledge of the occurrence and the behavior of environmental EDCs is necessary. The aim of the present study was to comprehensively identify substances that are responsible for the estrogenic effect of an environmental sediment sample taken from the river Elbe/Germany. DISCUSSION: The estrogenic effect of the organic sediment extract was determined using the yeast-estrogen-screen (YES). The sample was fractionated by liquid chromatography (LC) for effect directed analysis. The composition of estrogen-active fractions was further investigated by gas chromatography-mass spectrometry and high-resolution LC-MS analysis. The composition of the environmental sample was rebuilt with pure compounds in order to assess the partition of estrogenic activity caused by the identified compounds. The organic sediment extract showed an estrogenic potential of 1.9?±?0.4 ng/g ethinylestradiol equivalents in the sediment. The most prominent contaminants with an estrogenic potential were 17β-estradiol, estrone, and 4-iso-nonylphenols, but other xenoestrogens like bisphenol A and stigmasterol could be found as well. A rebuild of the sample was measured in the YES in order to investigate mixture effects. About 67 % of the observed estrogenic effect in the sediment extract could be explained by a mixture which contained all identified compounds. Chlorophene (o-benzyl-p-chlorophenol)-a widely used antiseptic that was also identified in the sediment extract-has xenoestrogenic properties in the YES that are in the range of other xenoestrogens like 4-n-nonylphenol. This is the first report on chlorophene acting as a xenoestrogen.     

Estimation of the environmental risk posed by landfills using chemical, microbiological and ecotoxicological testing of leachates

The leachates from 22 municipal solid waste (MSW) landfill sites in Southern Poland were characterized by evaluation of chemical, microbiological and ecotoxicological parameters. Chemical analyses were mainly focused on the identification of the priority hazardous substances according to Directive on Priority Substances, 2008/105/EC (a daughter directive of the WFD) in leachates. As showed, only five substances (Cd, Hg, hexachlorobutadiene, pentachlorobenzene and PAHs) were detected in the leachates. The compounds tested were absent or present at very low concentrations. Among them, only PAHs were found in all samples in the range from 0.057 to 77.2 μg L⁻¹. The leachates were contaminated with bacteria, including aerobic, psychrophilic and mesophilic bacteria, coliform and fecal coliforms, and spore-forming-bacteria, including Clostridium perfringens, and with filamentous fungi. From the analysis of specific microorganism groups (indicators of environmental pollution by pathogenic or opportunistic pathogenic organisms) it can be concluded that the landfill leachates showed sanitary and epidemiological hazard. In the ecotoxicological study, a battery of tests comprised of 5 bioassays, i.e. Microtox®, Spirotox, Rotoxkit F™, Thamnotoxkit F™ and Daphtoxkit F™ magna was applied. The leachate samples were classified as toxic in 13.6%, highly toxic in 54.6% and very highly toxic in 31.8%. The Spirotox test was the most sensitive bioassay used. The percentage of class weight score was very high - above 60%; these samples could definitely be considered seriously hazardous and acutely toxic to the fauna and microflora.No correlations were found between the toxicity values and chemical parameters. The toxicity of leachate samples cannot be explained by low levels of the priority pollutants. It seems that other kinds of xenobiotics present in the samples at subacute levels gave the high aggregate toxic effect.The chemical, ecotoxicological and microbiological parameters of the landfill leachates should be analyzed together to assess the environmental risk posed by landfill emissions.