Duckweed (Lemna gibba) growth inhibition bioassay for evaluating the toxicity of olive mill wastes before and during composting

Two-phase olive mill waste (TPOMW) is considered the main problem confronting the modern oil extraction and processing industry. Composting has been recently proposed as a suitable method to treat TPOMW so that it is suitable for use in agriculture. In the work reported here, the Lemna gibba bioassay was tested to assess the toxicity of TPOMW before and during the composting process. The method was compared with the Lepidium sativum bioassay and with other chemical maturity indices traditionally reported in the literature. The L. gibba test proved to be a simple, sensitive, and accurate method to evaluate toxicity before and during the composting of TPOMW. Plant growth response was measured by two methods: counting the number of fronds (leaves) and measuring total frond area (TFA) with image analysis software. Compared to the counting of fronds (L. gibba) or seeds (L. sativum), the use of area-measuring software permitted a very rapid, unbiased and easy way of analysing the toxicity of TPOMW before and during composting. Although the accuracy of the frond count method was similar to the traditional cress seed test, data analysis showed that the TFA measurement method was statistically more accurate (significantly lower variance) than the frond count approach. Highly significant correlations were found between TFA and some important maturation indices commonly reported in literature indicating that the L. gibba bioassay can be a useful tool to determine the degree of maturity of TPOMW composts.     

Evaluation and refinement of a continuous seed germination and early seedling growth test for the use in the ecotoxicological assessment of soils.

Four higher plant species (Avena sativa L., oat; Lepidium sativum L., cress; Brassica rapa Metzg., turnip; and Phaseolus vulgaris L., bush bean) and 15 soils including five mineral oil-contaminated soils were tested to assess a soil-based continuous seed germination and early seedling growth bioassay. Seed germination was recorded and the shoot biomass of 14-day seedlings measured. An automatic self-watering system was compared with the daily hand watering. Results obtained from this study show that the self-watering system had minor effects on both seed germination and early seedling growth, and that soil nutrient status rather than soil texture significantly affected both seedling emergence and shoot biomass. Proportional dilution of suspicious samples from primary tests with an appropriate control soil is recommended for secondary tests. Shoot biomass is a more sensitive measurement endpoint than seed germination. Bush bean should not be recommended for use due to its low sensitivity to mineral oil and poor germination in soil.     

Evaluation of lipid peroxidation as a toxicity bioassay for plants exposed to copper

Agricultural soils may contain toxic levels of copper (Cu) due to sewage sludge spreading or industrial pollution but chemical analyses may not be representative of Cu bioavailability, defined as the soil Cu fraction that plants can actually absorb (i.e. Cu fraction which is not strongly adsorbed to soil components). Lipid peroxidation caused by Cu in plants was investigated as a relevant bioassay of toxicity. Seven-day-old rapeseed plantlets were grown on Cu-supplemented medium in controlled conditions. Lipid-peroxidation was assessed by measuring: (1) the 2-thiobarbituric acid (TBA)-reactive substances; (2) the hydroperoxy acids by HPLC analysis; and (3) the alkane outputs by gas chromatography. We first verified the correlation between the results obtained by each method and then discussed their advantages and disadvantages within the context of a bioassay, showing that the volatile alkane output measurement is the most precise and easy to perform method for this purpose.     

Optimisation of a microbial bioassay for contaminated soil monitoring: bacterial inoculum standardisation and comparison with Microtox assay

This work represents the first step to set up a toxicity testing procedure and to evaluate the sensitivity of the test microorganism to several classes of environmental pollutants. First, three different techniques were employed to standardise the microbial inoculum, then two different toxicity assessment protocols have been compared: Microtox and a dehydrogenase (DHase) activity inhibition test. The main goal was the optimisation of a microbial bioassay based on the dehydrogenase activity (DHase) inhibition in Pseudomonas fluorescens bacterial strain ATCC 13525. Triphenyl tetrazolium chloride (TTC) was used as electron acceptor and its reduction produces Triphenyl formazane (TPF). The P. fluorescens DHase inhibition bioassay was investigated for being a reliable and rapid method for assessing toxicity. The optimisation of the operating conditions resulted in a repeatable bioassay. Then, P. fluorescens and Vibrio fischeri sensitivity were firstly compared by testing Zn++, one of the reference compounds for Microtox test. In addition, other compounds (Ni++, Cd++, Cu++, phenol) were also tested with both bioassays. A high statistical significance of data was obtained with the logistic curve. The present work has demonstrated that P. fluorescens is as sensitive as Microtox culture (V. fischeri), for some of the metal ions. With reference to organic compounds, the lower sensitivity of P. fluorescens to phenol makes its use difficult in organic polluted samples.     

Cr(VI) reduction into Cr(III) as a mechanism to explain the low sensitivity of Vibrio fischeri bioassay to detect chromium pollution

Vibrio fischeri bacteria, used as a biological target in either acute or chronic toxicity tests, display a low sensitivity to Cr(VI). This phenomenon could be due to the capacity of these bacteria to reduce Cr(VI) into Cr(III). This reducing capacity was found to depend on culture medium composition, pH value, incubation time and the presence of a carbon source. It also depends on the nature of the carbon source, glucose being more efficient than glycerol. This is probably related to differences in bacterial metabolism when given either glucose or glycerol. The thermostable Cr(VI)-reducing activity found in the supernatants of V. fischeri cultures grown on glucose suggests that, under these conditions, the bacteria release non-proteic reducing substances which have not been identified yet.     

Safety Evaluation for a Biodiesel Process Using Prion-Contaminated Animal Fat as a Source (6 pp)

Background Due to the bovine spongiform encephalopathy (BSE), specified risk material (SRM) as well as animal meat and bone meal (MBM) are banned from the food and feed chain because of a possible infection with pathogenic prions (PrPSc). Nowadays, prions are widely accepted to be responsible for TSE(transmissible spongiform encephalopathies)-caused illnesses like BSE and scrapie, and especially for the occurrence of the new variant of CJD in humans. Presently, SRM and MBM are burnt under high temperatures to avoid any hazards for humans, animals or the environment. The aim of this study was to evaluate a method using animal fat separated from Category I material which includes SRM and the carcasses of TSE-infected animals, or animals suspected of being infected with TSE, as a source for producing biodiesel by transesterification, analogous to the biodiesel process using vegetable oil. Methods For this purpose, animal fat was spiked with scrapie-infected hamster brain equivalents – as representative for a TSE-infected animal – and the biodiesel manufacturing process was downscaled and performed under lab-scale conditions. Results and Discussion The results analysed by Western blotting showed clearly that almost each single step of the process leads to a significant reduction of the concentration of the pathogenic prion protein (PrPSc) in the main and side-products. Conclusion The data revealed that the biodiesel production, even from material with a high concentration of pathogenic prions, can be considered as safe. Recommendations and Outlook The obtained results indicated that biodiesel produced from prion-contaminated fat was safe under the tested process conditions. However, it has to be pointed out that the results cannot be generalized because a different process control using other conditions may lead to different results and then has to be analysed independently. It is clear that the production of biodiesel from high risk material represents a more economic usage than the combustion of such material.     

Evaluation of Saccharomyces cerevisiae strains as probiotic agent with aflatoxin B(1) adsorption ability for use in poultry feedstuffs

In this study the aflatoxin B(1) (AFB(1)) removal capacity, the tolerance to salivary and gastrointestinal conditions, autoaggregation and coaggregation with pathogenic bacteria of Saccharomyces cerevisiae strains isolated from broiler feces, were evaluated. Only four of twelve isolated strains were identified as Saccharomyces cerevisiae using molecular techniques. The results obtained in AFB(1) binding studies indicated that the amount of AFB(1) removed was both strain and mycotoxin-concentration dependent. Therefore, a theoretical model was applied in order to select the most efficient strain to remove AFB(1) in a wide range of mycotoxin concentration. The results indicated that S. cerevisiae 08 and S. cerevisiae 01 strains were the most efficient microorganisms in the mycotoxin removal. Viability on simulated salivary and gastrointestinal conditions was investigated and S. cerevisiae 08 strain showed the best results, achieving 98% of total survival whereas S. cerevisiae 01 reached only 75%. Autoaggregation and coaggregation assays showed S. cerevisiae 08 as the most appropriate strain, mainly because it was the unique strain able to coaggregate with the four bacterial pathogens assayed. Consequently, S. cerevisiae 08 is the best candidate for future in vivo studies useful to prevent aflatoxicosis. Further quantitative in vitro and in vivo studies are required to evaluate the real impact of yeast-binding activity on the bioavailability of AFB(1) in poultry. However, this study could be useful in selecting efficient strains in terms of AFB(1) binding and provide an important contribution to research into microorganisms with potential probiotic effects on the host.     

Development of an optimal bacterial medium based on the growth inhibition assay with Vibrio fischeri.

Chronic toxicity level of chemicals to bacteria can depend on composition and concentration of medium ingredients. This was demonstrated by means of a growth rate inhibition test with the marine bacterium V. fischeri. In a minimal medium (following the validity criterion of achieving a least cell multiplication rate) containing only yeast extract as organic nutrient component, V. fischeri was to Cu2+ 11.9 times, to Hg2+ 3 times and to Zn2+ 2.8 times more sensitive than in the complete defined medium. Obviously yeast extract did not interfere greatly in the complexation mechanisms. The detection limits of the toxicity of Cd2+, 3,5-dichorophenol and nitrobenzene were not influenced by the substrate.     

Assessing the quality of marine coastal environments: comparison of scope for growth and Microtox bioassay results of pollution gradient areas in eastern Mediterranean (Greece)

Methods for assessing the quality of marine coastal environments are numerous and rapidly evolving. The integration of physiological parameters termed as Scope for Growth (SfG) and the luminescent bioassay Microtox were applied in several sites of Saronikos and Amvrakikos Gulfs (Greece) to assess the environmental quality and compare the results of the two methods. Each site in the two areas undergoes different types and levels of pollution. Both methods could identify a pollution gradient reflecting the quality of each site. A very good linear correlation was noticed between the two methods (r = 0.98). The stress order, which was similar in both methods, exhibited the expected pollution gradient according to the pressure that each site undergoes. Results suggest that for screening and fast isolation of polluted sites before further analysis, the rapid and simple Microtox assay could operate as an alternative to the more complex SfG method.     

Determination of 2,3,7,8-TCDD toxic equivalent factors (TEFs): support for the use of the in vitro AHH induction assay

The in vitro induction of the cytochrome P₁-450-dependent monooxygenases, aryl hydrocarbon hydroxylase (AHH) or ethoxyresorufin O-deethylase (EROD) by 2,3,7,8-TCDD and related toxic halogenated aryl hydrocarbons in rat hepatoma H-4-II E cells has been developed as a short term quantitative bioassay for these toxic chemicals. There was a linear correlation between the -log EC₅₀ (in vitro) AHH induction vs the -log ED₅₀ (in vivo) for body weight loss, thymic atrophy, hepatic AHH and EROD induction in the rat for several polychlorinated biphenyl, dibenzo-p-dioxin and dibenzofuran congeners and mixtures. These data clearly support the utility of the in vitro AHH induction assay as a short term test system for quantitating the “toxic or 2,3,7,8-TCDD equivalents” in an extract containing toxic halogenated aromatics. The bioassay method is rapid, relatively accurate and much more cost effective than conventional analytical methods such as gas-chromatography-mass spectrometry from which it is difficult to determine the levels of 2,3,7,8-TCDD equivalents in specific analytes.     

Potential of recycling gamma-irradiated sewage sludge for use as a fertilizer: a study on chickpea (Cicer arietinum)

The effects of gamma-irradiated sludge on the growth and yield of chickpea (Cicer arietinum) in pot cultures have been studied. Compared to plants grown only in soil, root length, fresh weight and dry weight of plants grown in soil supplemented with unirradiated sludge were found to be significantly reduced. This inhibition in growth was found to be nullified when plants were grown in soil supplemented with gamma-irradiated sludge, suggesting that gamma radiation induced inactivation of toxic substance(s) in sludge. The protein content of plants grown in soil supplemented with irradiated sludge was also found to be significantly increased compared to those grown with unirradiated or no sludge, after 45 days. There was no significant effect of gamma irradiated sludge on shoot length, total soluble sugars, starch content and yield of chickpea plants. The results obtained suggest that the sludge tested, and obtained from the digester of a conventional domestic sewage treatment plant, is inhibitory to several growth parameters. Gamma irradiation of sewage resulted in removal of this inhibition. This suggests a possibility of beneficial and safe recycling of gamma-irradiated sludge for agricultural uses.     

Characterisation of a bioassay using the marine alga Dunaliella tertiolecta associated with spectroscopic (visible and infrared) detection

This paper describes an ecotoxicological test using the alga Dunaliella tertiolecta associated with visible (VIS) and infrared (FTIR) spectroscopic detection. The estimation of toxicity is performed by measuring the cell density at 432 nm instead of 665 nm the results are comparable with those obtained by conventional visual or automatic estimation of celle density. FTIR spectroscopy gives an insight to molecular modifications in cell of Dunaliella tertiolecta caused by pollutants. A relevant result obtained by FTIR is the high similarity observed between the molecular modifications caused by toxic effect and the molecular modifications caused by samples with a stimulation (i.e., hormetic) effect.     

Engineered silica nanoparticles alleviate the detrimental effects of Na+ stress on germination and growth of common bean (Phaseolus vulgaris)

Environmental Science and Pollution Research - During the past 10 years, exploiting engineered nanoparticles in agricultural sector has been rapidly increased. Nanoparticles are used to...     

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.     

离子液体对三种农作物发芽和生长的毒性研究

为了考察离子液体的生态毒性,研究了5种离子液体及其浓度变化对3种农作物发芽及生长状况的影响。结果表明：就种子发芽这一生态毒理指标而言,不同作物对离子液体毒性的敏感程度不同,其次序为黄瓜>玉米>白菜;在一定浓度下,离子液体对白菜、黄瓜和玉米均具有一定的毒害作用;对于所考察的5种离子液体,阴离子为卤素的［EMIM］Br和［BMIM］Cl对植物发芽和生长的抑制作用远大于阴离子为烷基硫酸酯和烷基磷酸酯的同类离子液体 ［EMIM］［ES］、［EMIM］［DEP］和［MMIM］［DMP］。这种毒性顺序可能与卤素离子对细胞膜较强的穿透能力和由于离子液体的稳定性所造成的阴阳“离子对”的共迁移有关。     

Development of a sandwich enzyme-linked immunosorbant assay for the quantification of vitellogeinin in Bullfrog (Rana catesbeiana)

A sandwich enzyme-linked immunosorbant assay (ELISA) was developed to quantitatively detect Bullfrog (Rana catesbeiana) vitellogenin (VTG) levels. This procedure involved a sandwich ELISA using monoclonal antibodies of BVmA1 and BVmA2 against Bullfrog-VTG, and BVmA1 conjugated to horseradish peroxidase as the detection antibody. The assay range was between 9.4 ng/ml and 1200 ng/ml and the recovery of the VTG added to Bullfrog control male serum was 92.0-108.8%. Male Bullfrog was induced by injection 17beta-estradiol (E2) for four weeks and Bullfrog-VTG levels were measured each week. Histological analysis was performed for investigating the correlation of the effect to male reproduction and Bullfrog-VTG level variation depending on E2 dose. After two weeks of E2 exposure, the induced Bullfrog-VTG level was significantly higher than Bullfrog control female (p<0.05). After four weeks of E2 exposure, the rupture and fusion of seminiferous tubule in the testes of male Bullfrog were shown and provided direct evidence that the reproduction of male Bullfrog was affected by estrogenic compounds. Bullfrog-VTG bioassay, using the sandwich ELISA, could be a sensitive and useful tool for quantification of estrogenic principles in aquatic environments.     

Inhibitory effect of ragweed (Ambrosia artemisiifolia L)-inflorescence extract on the germination of Amaranthus hypochondriacus L and growth of two soil algae

In this study the ecological importance of common ragweed (Ambrosia artemisiifolia L.; Asteraceae) was investigated in in vitro bioassays as it is one of the most widespread annual weeds occurring on arable land in Hungary. Eight fractions were derived from ragweed inflorescence water extract by low vacuum column chromatography and their biological activity was investigated by Amaranthus hypochondriacus L. cv. Edit (Amaranthaceae) germination bioassay. As a result of the germination bioassay the chromatographic method was modified to harvest the most effective fractions in only one fraction, which was used in three concentration (10, 50, 100 mg x kg(-1)) for algal growth inhibition bioassay.Results showed significant growth inhibition in case of both applied algal species.     

Evaluation of polypropylene and poly (butylmethacrylate-co-hydroxyethylmethacrylate) nonwoven material as oil absorbent

Polypropylene (PP) and poly(butylmethacrylate-co-hydroxyethylmethacrylate) (PBMA-co-HEMA) nonwoven materials as oil absorbents have been fabricated for the first time via melt blown method. As-prepared nonwovens were investigated in terms of mass per unit area, density, air permeability, contact angle, and morphology observations for fiber diameter distribution and single fiber surface by a field emission scanning electron microscope. The nonwovens are demonstrated as fast and efficient absorbents for various kinds of oils with oil absorbency up to seven to ten times their own weight. The nonwovens show excellent water repulsion but superoleophilic properties. The measured contact angles for water and toluene are more than 127° and ca. 0°, respectively. The addition of PBMA-co-HEMA makes the nonwoven surface more hydrophobic while conserving superoleophilicity. Compared with PP nonwoven, broad diameter distribution of the blend nonwoven is attributed to poor melt fluidity of PBMA-co-HEMA. In terms of single fiber, coarse surface and the presence of point-like convexities lead to the fibers being more readily wetted by oil. More interesting, oil–water separation and oil recovery can be easily carried out by filter and absorption–desorption process, the recovered materials contained hardly any oil droplet and could be reused for next cycles.     

Some issues relating to the use of perfluorooctanesulfonate (PFOS) samples as reference standards

Samples of potassium perfluorooctanesulfonate (PFOSK) from three suppliers were analyzed by LC-ESI-MS/MS for purity and by LC-ESI-MS for the percentage of linear isomer present. Our data indicated that the purity ranged from 80% to 98% and the percentages of linear isomer from 67% to 79%. The proportion of branched isomers present in the samples was also estimated using (19)F NMR. These results agreed quite closely with those found by LC-ESI-MS indicating that there is essentially no difference in overall SIM response factor for the branched isomers vs. that of the linear isomer. Several further observations relevant to the use of standards when analyzing for PFOS were encountered during this study. It appears unlikely that matrix effects attributable to the cation (sodium or potassium) present in PFOSNa or PFOSK internal standards is an issue. In seeking potential matrix effects, it was found that the chromatography was improved substantially when the standard was injected as a solution in 80:20 methanol/water rather than 100% methanol. Notably, in concert with the improvement in chromatography, an increase of about 10% in response was observed. In some closely related studies, when (18)O(2) mass-labeled perfluorohexanesulfonate was used as an internal standard, the actual and theoretical concentration ratios matched closely those for related native sulfonates as long as they did not co-elute. However, when they did co-elute, the peak intensities of the native species were enhanced by about 5%, while those of the labeled compound were suppressed by a similar amount. If this effect were not taken into account, the concentration of the native would be inflated by 10%.     

Development and validation of a new fluorescence-based bioassay for aquatic macrophyte species

Bioassays with unicellular algae are frequently used as ecotoxicological test systems to evaluate the toxicity of contaminated environmental samples or chemicals. In contrast, aquatic macrophyte test systems are still rarely used as they are laborious to handle because species exhibit distinct ecological requirements. The aim of this study was to establish a fast and reproducible measuring system for aquatic macrophyte species to overcome those limitations for use. Thus, a newly developed pulse-amplitude modulated chlorophyll fluorometer (Imaging-PAM) was applied as an effect detection in short-term bioassays with aquatic macrophyte species. This multiwell-plate-based measuring device enables the incubation and measurement of up to 24 samples in parallel. The Imaging-PAM was used (i) to establish and validate the sensitivity of the test systems to three Photosystem II (PSII) inhibitors (atrazine, prometryn, isoproturon), (ii) to compare the test systems with established biotests for macrophytes and (iii) to define necessary time scales in aquatic macrophyte testing. The results showed that fluorescence-based measurements with the Imaging-PAM allow rapid and parallel analysis of large amounts of aquatic macrophyte samples and of toxicants effects of the PSII inhibitors tested on aquatic macrophytes. Measurements revealed a good correlation between obtained median effective concentrations (EC50s) for the new and the established biotest systems. Hence, the Imaging-PAM measuring device is a promising tool to allow fast chemical effect screening for high amounts of samples with little time and material and thus offers scope for high-throughput biotesting using aquatic macrophyte species.