Agonistic and antagonistic toxic effects observed with miniaturized growth and luminescence inhibition assays

Kinetic data recordings with miniaturized growth and luminescence inhibition assays with prokaryotes (Pseudomonas putida and Vibrio fischeri) reveal typical sample and species specific growth and light emission patterns respectively. Growth inhibiting influences, based on toxicokinetic and toxicodynamic sample specificities appeared in each growth phase. Agonistic and antagonistic effects of combinations of heavy metals like Cd2+, Hg2+ and Pb2+ and other compounds were easily recognized after accurate curve discussions. Some of the influences that made it more difficult to evaluate toxicological results only by restriction on EC values (effective concentrations) were investigated. The findings may lead to a more comprehensive interpretation of toxicological data and a presumption on the composition of complex samples in terms of screening investigations. Therefore, the conclusions demonstrate the possibilities and limits of kinetic data recordings in chronic and acute toxicity testing.     

Two new growth inhibition tests with the filamentous algae Ceramium strictum and C. tenuicorne (Rhodophyta)

Two growth inhibition tests using the red marine macroalgae Ceramium strictum and the brackish water relative C. tenuicorne have been developed. Besides using phenol as a reference substance, the toxicity of a metal, a flame retardant and a complex effluent water were assayed. The two methods are reliable and repeatable bioassays for salinities between 4 and 30 per thousandth. The coefficients of variation (CV) for toxicity of the reference substance phenol were 15% for the Stereo Microscope Analysis test and between 24 and 51% for the Computer Image Analysis test (n=5). Ceramium spp. are common and important primary producers in temperate coastal waters and are thus relevant as test organisms. Both algae grow well in laboratorial conditions and tests can be performed all year around.     

Algal growth inhibition: effect of the choice of growth rate or biomass as endpoint on the classification and labelling of new substances notified in the EU

From the complete base set notifications of new substances currently available, we have investigated what effects the choice of using growth rate or biomass in the algal growth inhibition test has on the relative sensitivity of the three aquatic toxicity tests. Both parameters derived from the algal test were more sensitive than either fish or Daphnia tests. Changes in the classification of substances after the removal of either algae, Daphnia or fish data from the base set, when applying current legal practice, occur in 22.9%, 6.6% and 4.8% of the notifications, respectively. When always using growth rate as a parameter, these numbers change to 15.4%, 9.2% and 7.2%, respectively.     

Experimental studies and kinetic modeling of the growth of phenol-degrading bacteria in turbulent fluids

Understanding the interaction between microorganisms and fluid dynamics is important for aquatic ecosystems, though only sporadic attention has been focused on this topic in the past. In this study, particular attention was paid to the phenol-degrading bacterial strains Microbacterium oxydans LY1 and Alcaligenes faecalis LY2 subjected to controlled fluid flow under laboratory conditions. These two strains were found to be able to degrade phenols over a concentration range from 50 to 500 mg/L under different turbulence conditions ranging from 0 to 250 rpm. The time it took to reach total phenol degradation decreased when the turbulence was increased in both strains, with increasing energy dissipation rates ranging from 0.110 to 6.241 W/kg, corresponding to changes in the bacterial diffusive sublayer thickness (δ) and enhanced oxygen uptake. Moreover, the maximum specific growth rates of the two strains also increased with the enhancement of turbulence. A model integrating growth inhibition and fluid motion was proposed based on the self-inhibition Haldane model by introducing a turbulence parameter, α. The resulting modified Haldane model was designed to include fluid motion as a variable in the quantification of the physiological responses of microorganisms. This modified Haldane model could be considered a useful laboratory reference when modeling procedures for water environment bioremediation.

Cell nutrition uptake cartoon schematic diagram for M. oxydans LY1 under different turbulent condition (50 and 200 rpm).

     

活性污泥系统中自养菌浓度及生长动力学参数测定

利用批式呼吸计量法测定了实际污水处理厂厌氧/缺氧/好氧工艺和小试装置厌氧/好氧工艺好氧池内自养菌（X_A）的浓度及其最大比生长速率常数(μ_A)。结果表明,活性污泥系统中X_A与混合液挥发性悬浮固体的比例为1.60%～2.34%,μ_A实测值为0.69～0.76 d^-1,测定结果有较好的重现性,且与文献值非常接近,说明该方法具有较高的准确度和精密度。     

卡鲁塞尔氧化沟流速与水质组分分布的现场测试

通过实测卡鲁塞尔氧化沟不同位置处流速与水质组分,分析了流速对沟内污泥、溶解氧浓度的影响以及溶解性组分的微生物转化规律。结果表明,溶解氧与流速呈正相关、污泥浓度（MLSS）与流速呈负相关;水质组分在沟中不同区段呈现出各自特点,反映了COD、氨和磷酸盐的生物转化历程;流速也是影响溶解性组分混合的重要因素,改善进水口附近混合液的流场,提高局部流速有利于缩短进水组分均布的距离。     

A batch kinetic study on decolorization and inhibition of Reactive Black 5 and Direct Brown 2 in an anaerobic mixed culture

Decolorization and inhibition kinetic characteristics of two azo dyes namely Reactive Black 5 (RB 5) and Direct Brown 2 (DB 2) were investigated with partially granulated anaerobic mixed culture using glucose (3000 mg l(-1) COD) as carbon source and electron donor during batch incubation. Monod, zero-, first-, and second-order reaction kinetic models were tested in order to determine the most suitable rate model of substrate and color removal kinetic. The course of the decolorization and substrate removal process approximates to first-order kinetic model under batch conditions. Decolorization, and substrate removal were achieved effectively under test conditions but ultimate removal of azo dyes and substrate were not observed at high dye concentrations. Aromatic amine and volatile fatty acid accumulation were observed proportionally at a higher azo dye concentration. A competitive kinetic model that describes the anaerobic co-metabolism of increasing RB 5 and DB 2 dye concentrations with glucose as co-substrate has been developed based on the experimental data.     

Groundwater flow velocities in karst aquifers; importance of spatial observation scale and hydraulic testing for contaminant transport prediction

Environmental Science and Pollution Research - We review scale dependence of hydraulic conductivities and effective porosities for prediction of contaminant transport in four UK karst aquifers....     

Rhizosphere localized cationic peroxidase from barley roots is strongly activated by cadmium and correlated with root growth inhibition

The activity of Cd-induced POD isozyme isolated from the surface of intact barley roots growing under some abiotic stress conditions (toxic metals: Al, Co, Cu, Hg; drought, NaCl, extreme temperatures: heat, cold) and compounds activating (2,4-D) or inhibiting (SHAM) POD activity as well as H(2)O(2) and H(2)O(2) scavenger (DTT) was characterized. Strong Cd concentration-dependent accumulation of one cationic POD isozyme was observed on PAGE, which correlated with Cd- and other stress induced root growth inhibition. This isoPOD is basic with isoelectric point about 9 and its localization is restricted only to the apical part of the barley root tip.     

Root growth inhibition and induction of DNA damage in soybean (Glycine max) by chlorobenzenes in contaminated soil

The cytotoxic and genotoxic effects of 1,2,4-trichlorobenzene (TCB), chlorobenzene (CB), and hexachlorobenzene (HCB) on root growth and DNA strand breakage damage of soybean nuclei in the test soil were studied using the comet assay. Results indicated that the root growth was significantly inhibited, and DNA strand breaks and the comet tail in the root tip nuclei were both induced after 48 h exposure with TCB concentrations of 50, 100, 200, 300 microg g(-1) in the soil. DNA strand breakage was more sensitive to the TCB than the root growth. There was a significant dose-response relationship between the TCB exposure and DNA strand breakage in the soybean nuclei. Thus it is possible for DNA strand breakage to be used as a biomarker of soybean exposed to TCB contamination. Significant cytotoxic threshold concentration of the TCB exposure on the root growth inhibition was determined as 61 microg g(-1) in the soil. The toxicity of 100-1,000 microg g(-1) CB and HCB to the soybean seedlings in the soil were not observed after 48 h or longer exposure.     

Role of cation structure in the phytotoxicity of ionic liquids: growth inhibition and oxidative stress in spring barley and common radish

The present study determines the influence of three ionic liquids (ILs) containing cations with diversified structure on the growth and development of spring barley seedlings and common radish leaves. Increasing amounts of 1-butyl-1-methylpyrrolidinium hexafluorophosphate [Pyrrol][PF₆], 1-butyl-1-methylpiperidinium hexafluorophosphate [Piper][PF₆], and 1-butyl-4-methylpyridinium hexafluorophosphate [Pyrid][PF₆] were added to the soil on which both plants were cultivated. The results of this studies showed that the applied ILs were highly toxic for plants, demonstrated by the inhibition of length of plant shoots and roots, decrease of fresh mass, and increase of dry weight content. Common radish turned out to be the plant with higher resistance to the used ILs. The differences in the cation structure did not influence phytotoxity of ILs for spring barley. Furthermore, all ILs led to a decrease of photosynthetic pigments, which was directly followed by decreased primary production in plants. Oxidative stress in plants occurred due to the presence of ILs in the soil, which was demonstrated by the increase of malondialdehyde (MDA) content, changes in the H₂O₂ level, and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). The changes in the chlorophyll contents and the increase of POD activity turned out to be the most significant oxidative stress biomarkers in spring barley and common radish. Both spring barley and radish exposed to ILs accumulated a large amount of fluoride ion.

     

A reaction-based paradigm to model reactive chemical transport in groundwater with general kinetic and equilibrium reactions

This paper presents a reaction-based water quality transport model in subsurface flow systems. Transport of chemical species with a variety of chemical and physical processes is mathematically described by M partial differential equations (PDEs). Decomposition via Gauss-Jordan column reduction of the reaction network transforms M species reactive transport equations into two sets of equations: a set of thermodynamic equilibrium equations representing N(E) equilibrium reactions and a set of reactive transport equations of M-N(E) kinetic-variables involving no equilibrium reactions (a kinetic-variable is a linear combination of species). The elimination of equilibrium reactions from reactive transport equations allows robust and efficient numerical integration. The model solves the PDEs of kinetic-variables rather than individual chemical species, which reduces the number of reactive transport equations and simplifies the reaction terms in the equations. A variety of numerical methods are investigated for solving the coupled transport and reaction equations. Simulation comparisons with exact solutions were performed to verify numerical accuracy and assess the effectiveness of various numerical strategies to deal with different application circumstances. Two validation examples involving simulations of uranium transport in soil columns are presented to evaluate the ability of the model to simulate reactive transport with complex reaction networks involving both kinetic and equilibrium reactions.     

Modeling hydrocarbon biodegradation in tidal aquifers with water-saturation and heat inhibition effects.

A model is developed for hydrocarbon biodegradation, which includes saturated and unsaturated flow, multi-species transport, heat transport, and bacterial growth processes. Numerical accuracy of the model was tested against analytical solutions. The model was also verified against laboratory results for a saturated-flow problem and reasonable match was obtained. Expressions are proposed for inhibition due to water content and temperature fluctuations. Bioactivities under cyclic water content variation were studied under no-flow conditions. A quantitative approach was used to reconcile some of the apparent contradictory conclusions regarding the efficiency of biodegradation of soils under wetting and drying conditions. The efficiency depends on the nature of the oxygenation process. For cases involving the presence of dissolved oxygen and the absence of O2 vapor, subjecting the soil to constant water content close to its optimal value for degradation is most efficient. However, wetting and drying can enhance degradation if O2 is only provided through aeration or direct contact between air and the medium. Also presented are the results of a typical field application of the model and a discussion of the effects of tides, saturation inhibition, and heat inhibition. Other inhibition factors, such as pH or salinity, can be easily incorporated in the formulation. The quantitative approach developed here can be used in assessing bioremediation not only in tidal aquifers but also in areas where water-table or temperature effects are of significance. The approach can be useful in the design of remediation strategies under water-flow or no-flow conditions involving water content and temperature fluctuations.     

Sampling optimization, at site scale, in contamination monitoring with moss, pine and oak.

With the aim of optimizing protocols for sampling moss, pine and oak for biomonitoring of atmospheric contamination and also for inclusion in an Environmental Specimen Bank, 50 sampling units of each species were collected from the study area for individual analysis. Levels of Ca, Cu, Fe, Hg, Ni, and Zn in the plants were determined and the distributions of the concentrations studied. In moss samples, the concentrations of Cu, Ni and Zn, considered to be trace pollutants in this species, showed highly variable long-normal distributions; in pine and oak samples only Ni concentrations were log-normally distributed. In addition to analytical error, the two main source of error found to be associated with making a collective sample were: (1) not carrying out measurements on individual sampling units; and (2) the number of sampling units collected and the corresponding sources of variation (microspatial, age and interindividual). We recommend that a minimum of 30 sampling units are collected when contamination is suspected.     

Problems in testing and risk assessment of endocrine disrupting chemicals with regard to developmental toxicology.

Endocrine disrupting chemicals (EDCs) may affect mammalian development either indirectly (by impairing implantation, placental development, lactation, etc.) or directly, altering the maturation of target tissues. Current regulatory tests for reproductive/developmental toxicity should be carefully evaluated with regard to risk assessment of EDCs, considering hazard identification (are relevant endpoints being assessed?) and dose-response assessment (are sensitive NOEL/dose-response curves being provided?). Many in vitro and in vivo assays for sex steroid disruption are available; provided that the metabolic capacities of the assays are defined, they could be integrated in a sensitive battery for early detection of steroid-disrupting potentials. The screening battery should address further regulatory in vivo tests (e.g. what specific parameters have to be investigated). As regards dose-response, qualitative differences may be observed between lower and higher exposures, showing primary hormone-related effects and frank embryotoxicity, respectively. Other problems concern (a) the identification of critical developmental windows, according to hormone concentrations and/or receptor levels in the developing target tissues; (b) the potential for interactions between chemicals with common mechanism/target (e.g. xenoestrogens); (c) most important, besides sex steroids more attention should be given to other mechanisms of endocrine disruption, e.g., thyroid effects, which can be highly relevant to prenatal and postnatal development.     

Recombinant human AhR-mediated GUS reporter gene assays for PCB congeners in transgenic tobacco plants in comparison with recombinant mouse and guinea pig AhRs

Four expression plasmids for recombinant human aryl hydrocarbon receptor (hAhR) consisting of a ligand binding domain of hAhR, a DNA-binding domain of LexA and a transactivation domain of VP16 as well as β-glucuronidase (GUS) reporter genes were constructed. All the expression plasmids were transformed into tobacco plants. The selected transgenic tobacco plants were used to assay. PCB congeners showed GUS activity in a TEF-dependent manner. The selected transgenic tobacco plant XhD4V17 was compared with the transgenic tobacco plants XmD4V26 and XgD2V23 containing recombinant mouse (m) AhR-mediated GUS reporter gene expression cassette and recombinant guinea pig (g) AhR-mediated GUS reporter gene expression cassette for PCB congener-inducible GUS activity. The data revealed that the tobacco plant XgD2V23 was the most active in PCB congener-inducible GUS activity. In a 1:1 mixture of PCB126 and PCB80 a reduced PCB126-induced GUS activity was observed in plant XgD2V23, which could possibly be due to interaction between PCB126 and PCB80.     

Recombinant human AhR-mediated GUS reporter gene assays for PCB congeners in transgenic tobacco plants in comparison with recombinant mouse and guinea pig AhRs

Four expression plasmids for recombinant human aryl hydrocarbon receptor (hAhR) consisting of a ligand binding domain of hAhR, a DNA-binding domain of LexA and a transactivation domain of VP16 as well as β-glucuronidase (GUS) reporter genes were constructed. All the expression plasmids were transformed into tobacco plants. The selected transgenic tobacco plants were used to assay. PCB congeners showed GUS activity in a TEF-dependent manner. The selected transgenic tobacco plant XhD4V17 was compared with the transgenic tobacco plants XmD4V26 and XgD2V23 containing recombinant mouse (m) AhR-mediated GUS reporter gene expression cassette and recombinant guinea pig (g) AhR-mediated GUS reporter gene expression cassette for PCB congener-inducible GUS activity. The data revealed that the tobacco plant XgD2V23 was the most active in PCB congener-inducible GUS activity. In a 1:1 mixture of PCB126 and PCB80 a reduced PCB126-induced GUS activity was observed in plant XgD2V23, which could possibly be due to interaction between PCB126 and PCB80.     

Assessment of indoor air in Austrian apartments with and without visible mold growth

Fungal spores are transported across great distances in the outdoor air and are also regularly found indoors. Building conditions and behavior-related problems in apartments may lead to massive growth of mold within a very short period of time.The aim of this study was to evaluate whether the visible growth of mold indoors influences the concentration of fungal spores in the air as well as the variety of their species. Samples were collected from 66 households in Austria. For each sampling, the corresponding outdoor air was measured as reference value. The size of the visible mold growth was categorized in order to correlate the extent of mold growth with the concentration of airborne spores as well as the fungal genera. In order to determine fungal spore concentrations in the air, the one-stage MAS-100^® air sampler was used. Malt extract agar (MEA) and dichloran glycerol agar (DG18) plates were used as culture media. The total colony forming units (CFU) per m³ were determined. The fungi were identified from the isolated colonies.The results show that in apartments visibly affected by mold, the median values were significantly higher than those of apartments without visible mold growth. The extent of visible mold growth is significantly correlated with both concentration of fungal spores (p<0.001) as well as the predominance of Penicillium sp. and Aspergillus sp. (p<0.001) in indoor air. The total fungal concentration of Penicillium and Aspergillus in the air of apartments is recommended for assessing fungal exposure.     

Comparing growth development of <Emphasis Type="Italic">Myriophyllum</Emphasis> spp. in laboratory and field experiments for ecotoxicological testing

BACKGROUND, GOALS AND SCOPE: Risk assessment of herbicides and the evaluation of contaminated sediments based on algae and the macrophyte Lemna sp. alone may underestimate the potential hazard of certain compounds. Therefore, various test systems with Myriophyllum spp. have been developed recently to assess the phytotoxicity in surface waters and natural sediments. In the present study, experiments investigating the growth development of Myriophyllum spp. were performed in the laboratory under defined conditions and in mesocosms under environmentally realistic exposure conditions to evaluate the suitability of these species as potential standard test organisms in ecotoxicological testing. This study provides data on the endpoints biomass, plant length and root development. MATERIALS AND METHODS: Six independent experiments were performed to investigate the plant development of Myriophyllum spp. under control conditions. The main difference in the experiments was the complexity of the test systems ranging from simple laboratory experiments to complex outdoor mesocosm studies. At the start of each experiment, uniform cuttings of Myriophyllum spp. were placed in vessels with or without sediments to reduce variability between replicates. The endpoints considered in this investigation were biomass (fresh weight of the whole plant), length of the main shoot, length of the side shoots, total length of the plant (calculated from the length of the main and side shoots) and root formation. Root to shoot ratios were calculated as a further measure for plant development. Relative growth rates (RGR) based on plant length (RG(L)R) and on biomass (RG(B)R) were calculated. RESULTS: Despite the various experimental conditions, comparable growth was obtained in all test systems and the variability of endpoints, such as total length and biomass of plants, was low. It was observed that the RGR of M. spicatum in the simple laboratory test system with sediment were comparable to growth data obtained for M. verticillatum and M. spicatum grown in indoor and outdoor mesocosms, thus indicating that Myriophyllum growth tends to increase by the addition of sediment. High variability was determined for the endpoints length of the side shoots, total root length and biomass of roots. DISCUSSION: One challenge for a test design to investigate phytotoxicity on aquatic plants is to obtain good growth of the plants. From the results, it can be concluded that the experimental conditions in the various test systems were suitable to study the plant development of Myriophyllum spp. because obtained growth rates were comparable between laboratory and field investigations. Another challenge for developing a plant biotest system is the definition of sensitive endpoints. Low variability is preferred to detect minor effects of chemicals or polluted sediments on plant development. In our studies, the variability of the endpoints biomass and total length of plant was low and, therefore, they have much potential as endpoints for assessing toxicity. CONCLUSIONS: The methodologies presented in this study have applications within the risk assessment for aquatic plants and have the advantage of assessing effects taking into account the relevant exposure pathways via water and/or sediment for compounds under investigation. RECOMMENDATIONS AND PERSPECTIVES: Setting safe quality criteria for surface water and sediments is one of the challenges authorities are facing today. Myriophyllum spp. is recommended as suitable test species to investigate phytotoxicity in surface water and sediments. These results, thus, might serve as a basis for the compilation of a new harmonised guideline for ecotoxicological testing with aquatic macrophytes.