Aqueous Eluates Research Articles

Ecotoxicity of Caffeine as a Bio-Protective Component of Flax-Fiber-Reinforced Epoxy-Composite Building Material.

Caffeine is a verified bio-protective substance in the fight against the biodegradation of cellulose materials, but its ecotoxicity in this context has not yet been studied. For this reason, the ecotoxicity of flax-fiber-reinforced epoxy composite with or without caffeine was tested in the present study. Prepared samples of the composite material were tested on freshwater green algal species (Hematococcus pluvialis), yeasts (Saccharomyces cerevisae), and crustacean species (Daphnia magna). Aqueous eluates were prepared from the studied material (with caffeine addition (12%) and without caffeine and pure flax fibers), which were subjected to chemical analysis for the residues of caffeine or metals. The results indicate the presence of caffeine up to 0.001 mg/L. The eluate of the studied material was fully toxic for daphnids and partially for algae and yeasts, but the presence of caffeine did not increase its toxicity statistically significantly, in all cases. The final negative biological effects were probably caused by the mix of heavy metal residues and organic substances based on epoxy resins released directly from the tested composite material.

Assessment of acute and chronic ecotoxicological effects of aqueous eluates of stone wool insulation materials

BackgroundStone wool is an inorganic mineral insulation material increasingly used to reduce the climate impact of buildings. The acute and chronic ecotoxicological potential of stone wool eluates have been studied in a battery of standardized laboratory ecotoxicological tests. The experiments were conducted with stone wool test materials in fibrous and milled form, with and without the presence of organic binder. For the preparation of eluates, the OECD protocol on the transformation/dissolution of metals and metal compounds was applied. The resulting eluates were used in acute tests, i.e., bioluminescence test with Aliivibrio fischeri (DIN EN ISO 11348-1:2009), algae growth test with Desmodesmus subspicatus (OECD No. 201) and immobilization test with Daphnia magna (OECD No. 202), as well as chronic tests, i.e., the Daphnia magna reproduction test (OECD No. 211) and the nematode growth and reproduction test with Caenorhabditis elegans (ISO 10872:2010).ResultsWhile no acute or chronic ecotoxicological effects of the eluates were observed for fibrous stone wool material, the milled test materials showed some chronic effects on aquatic invertebrates. Depending on the test materials and concentrations of milled stone wool used in the eluate preparation, these chronic effects included significant stimulation or inhibition of daphnid reproduction and nematode growth. The chemical analysis conducted in parallel to the ecotoxicological assessment indicated no leaching of organic substances from the applied binder or mineral oils and no formation of nanoparticles by the milling of stone wool. Furthermore, ICP-MS and ICP-OES analysis of eighteen elements revealed that only aluminum and nickel could be quantified in the eluates, at concentrations of approximately 750 µg/L and 7 µg/L, respectively.ConclusionsBased on the present ecotoxicological assessment, eluates from stone wool fibers cannot be considered as chemically hazardous to the aquatic environment. However, additional investigations of the ecotoxicological potential of the milled material and the environmental exposure of stone wool products are necessary for a complete evaluation of potentially negative effects of stone wool insulation materials.

Metal contamination and toxicity of soils and river sediments from the world's largest platinum mining area

Mining activities in the world's largest platinum mining area in South Africa have resulted in environmental contamination with Pt (e.g., the Hex River's vicinity). The present study compared a Pt mining area with a non-mining area along this river in terms of (1) metal concentrations in different grain size fractions from soils and aquatic sediments; (2) the toxicological potential of aquatic sediments based on the Consensus-Based Sediment Quality Guideline (CBSQG); and (3) the chronic toxicity of aqueous eluates from soils and sediments to Caenorhabditis elegans. Platinum concentrations were higher in the mining area than in the non-mining area. For most metals, the sediment silt and clay fraction contained the highest metal concentrations. Based on the CBSQG, most sampling sites exhibited a high toxicological potential, driven by Cr and Ni. Eluate toxicity testing revealed that C. elegans growth, fertility, and reproduction inhibition were not dependent on mining activities or the CBSQG predictions. Toxicity was instead likely due to Cd, Fe, Mn, Ni, Pt, and Pb. In conclusion, the investigated region is loaded with a high geogenic background resulting in high reproduction inhibition. The mining activities lead to additional environmental metal contamination (particularly Pt), contributing to environmental soil and sediment toxicity.

An alternate methodology for studying diffusion and elution kinetics of dimethacrylate monomers through dentinal tubules

ObjectiveEthoxylated bisphenol A dimethacrylate (bisEMA) is a base monomer in several dental resin composites. It was the main aim of the present study to determine if bisEMA can reach the dental pulp by generally passive diffusion through the coronal dentinal tubules stimulated via eluent liquids surrounding the root structures only. MethodsIn 20 human third molar teeth, standard Class-I occlusal cavities were prepared and provided either with an adhesive system alone or additionally with a composite restoration, according to the instructions of the manufacturer. The teeth were placed in an elution chamber such that the elution media only came into contact with the tooth root/tooth base where they were incubated at 37 °C for up to 7 d. Samples were taken after 1, 2, 4 and 7 d. Gas chromatography/mass spectrometry was used to identify bisEMA and other monomers in ethanol/water (3:1) and aqueous eluates. ResultsbisEMA was only found in ethanol/water eluates, where the teeth had received a composite restoration. Traces of bisEMA with up to three ethylene oxide units could be detected in these eluates. Depending on the dentin thickness, different elution kinetics of bisEMA were determined. Regardless of the treatment of teeth, triethylene glycol dimethacrylate (TEGDMA) and tetraethylene glycol dimethacrylate (TEEGDMA) were found in ethanolic/aqueous eluates in equal amounts. Most TEGDMA and TEEGDMA diffused through the dentin within the first 24 h. SignificanceDepending on the dentin layer thickness, bisEMA was released for varied time periods, resulting in varied concentrations and exposure times for the different cells of the dental pulp. The concentrations of TEGDMA and TEEGDMA were greatest for cells of the dental pulp within the first 24 h.

Water-soluble factors eluated from surface pre-reacted glass-ionomer filler promote osteoblastic differentiation of human mesenchymal stem cells.

Surface pre-reacted glass-ionomer (S-PRG)-containing dental materials, including composite and coating resins have been used for the restoration and/or prevention of dental cavities. S-PRG is known to have the ability to release aluminum, boron, fluorine, silicon, and strontium ions. Aluminum ions are known to be inhibitors whereas boron, fluorine, silicon, and strontium ions are known to be promoters of mineralization, via osteoblasts. However, it remains to be clarified how an aqueous eluate obtained from S-PRG containing these ions affects the ability of mesenchymal stem cells (MSCs), which are known to be present in dental pulp and bone marrow, to differentiate into osteogenic cell types. The present study demonstrated that 200- to 1,000-fold-diluted aqueous eluates obtained from S-PRG significantly upregulated the mRNA expression level of the osteogenic differentiation marker alkaline phosphatase in human MSCs (hMSCs) without exhibiting the cytotoxic effect. In addition, the 500- to 1,000-fold-diluted aqueous eluates obtained from S-PRG significantly and clearly promoted mineralization of the extracellular matrix of hMSCs. It was additionally demonstrated that hMSCs cultured on the cured resin composites containing S-PRG fillers exhibited osteogenic differentiation in direct correlation with the weight percent of S-PRG fillers. These results strongly suggested that aqueous eluates of S-PRG fillers promoted hard tissue formation by hMSCs, implicating that resins containing S-PRG may act as a useful biomaterial to cover accidental exposure of dental pulp.

Study of the removal mechanism of aquatic emergent pollutants by new bio-based chars.

This work is dedicated to study the potential application of char byproducts obtained in the gasification of rice husk (RG char) and rice husk blended with corn cob (RCG char) as removal agents of two emergent aquatic contaminants: tetracycline and caffeine. The chars presented high ash contents (59.5-81.5%), being their mineral content mainly composed of silicon (as silica) and potassium. The samples presented a strong basic character, which was related to its higher mineral oxides content. RCG char presented better textural properties with a higher apparent surface area (144m2g-1) and higher micropore content (V micro=0.05cm3g-1). The alkaline character of both chars promoted high ecotoxicity levels on their aqueous eluates; however, the ecotoxic behaviour was eliminated after pH correction. Adsorption experiments showed that RG char presented higher uptake capacity for both tetracycline (12.9mgg-1) and caffeine (8.0mgg-1), indicating that textural properties did not play a major role in the adsorption process. For tetracycline, the underlying adsorption mechanism was complexation or ion exchange reactions with the mineral elements of chars. The higher affinity of RG char to caffeine was associated with the higher alkaline character presented by this char.

Comparison of Stir Bar Sorptive Extraction (SBSE) and Solid Phase Microextraction (SPME) for the Analysis of Polycyclic Aromatic Hydrocarbons (PAH) in Complex Aqueous Soil Leachates

Stir bar sorptive extraction (SBSE) and solid phase microextraction (SPME) are well-established sample preparation methods for the analysis of polycyclic aromatic hydrocarbons in aqueous samples. However, complex matrices especially characterized by slurry particles and dissolved organic matter (DOM) can hamper the extraction of PAH with both SBSE and SPME and lead to different results. Thus, we produced aqueous eluates from PAH-contaminated soils differing in particle size distribution and organic matter content and determined the PAH concentration in the eluates with both SBSE and SPME. Furthermore, we tested the influence of filtration on the PAH analysis. The excess finding of PAH with SBSE compared to SPME ranged from −16.6 to 24.5 %. The differences increased after filtration. We found a strong positive correlation of the excess finding to the total organic carbon content (TOC) and a negative one to the pH value. The results indicate that SBSE is less affected by complex matrices than SPME.

Biological and Chemical Characterizations of Allelopathic Potential of Diverse Accessions of the Cover Crop Sunn Hemp

There has been increasing interest in recent years in sunn hemp (Crotalaria juncea), as a leguminous cover crop and green manure, for weed and pest management and improving soil health. Aqueous extracts and ground shoot tissue have previously been demonstrated to be phytotoxic. To further explore its allelopathic potential, bioassays and chemical characterization of water-soluble eluates of sunn hemp were undertaken. Lettuce (Lactuca sativa) radicle growth inhibition was conducted with aqueous eluates from thinly sliced sunn hemp leaves, stems, and seeds, and all three tissues exhibited the inhibitory potential. Fourteen accessions originating from the United States, India, Brazil, South Africa, Pakistan, and Nigeria had water-soluble allelochemicals in leaves, suggesting that allelopathic potential is widely distributed in this species. The highest level of inhibitory potential was found in accession IN-86. Further characterization of IN-86 leaf eluates indicated that the inhibitory compound(s) was/were not soluble in chloroform, but was/were stable when boiled for 15 minutes and resistant to 1 n HCl. Binding and elution from AG-1(OH−) ion-exchange resin also were observed. An analysis of leaf eluates of IN-86 using high-performance liquid chromatography (HPLC) followed by mass spectrometry (MS) showed the presence of a compound with a mass-to-charge ratio of 148, consistent with the spectrum for hydroxynorleucine, a phytotoxic nonprotein amino acid previously reported in seeds of C. juncea. However, its low concentration (<1 μg·mL−1) suggested that other components of the eluate were responsible for the observed allelopathic effect. The results indicate the feasibility for development of weed control strategies using allelochemicals derived from sunn hemp biomass of select genotypes IN-86, NG-71, and BR-20 from India, Nigeria, and Brazil, respectively.

Predicting acute and chronic effects of wood preservative products in daphnia magna and pseudokirchneriella subcapitata based on the concept of concentration addition

The current European legislation requires that combined effects of the active substances and any substance of concern contained in biocidal products are taken into account in environmental risk assessment. The hypothesis whether the consideration of active substances together with all formulation additives that are labeled as presenting an environmental hazard is sufficient for a reliable environmental risk assessment was tested in the present study by investigating 3 wood preservative products. Relevant single substances in the products, some of their generic mixtures, the biocidal products themselves, and aqueous eluates prepared from the products (representing potential environmental mixtures) were tested for effects on algal growth and Daphnia acute immobilization as well as reproduction. Predictions for the products and the eluates were based on the concept of concentration addition and were mostly found to provide reliable or at least protective estimates for the observed acute and chronic toxicity of the mixtures. The mixture toxicity considerations also indicated that the toxicity of each product was dominated by just 1 of the components, and that assessments based only on the dominating substance would be similarly protective as a full-mixture risk assessment. Yet, there remained uncertainty in some cases that could be related to the toxicity of transformation products, the impact of unidentified formulation additives, or synergistic interaction between active substances and formulation additives.

Characterization of chromium-containing wastes and soils affected by the production of chromium tanning agents

Wastes of unknown composition derived from the production of trivalent chromium (Cr(III)) salts used as tanning agents are deposited in the area of Kanpur, India. The questions of whether these samples are chromite ore processing residue (COPR) and whether Cr occurs in its toxic hexavalent form (Cr(VI)) arise. Twenty-one samples from two disposal sites and surrounding soils were analyzed, specifically examining their elemental and mineralogical composition. Additionally, aqueous eluates with different liquid-to-solid ratios were performed and analyzed for Cr(VI). The samples were classified in accordance to the sum of silicon and aluminum and the sum of calcium and Cr contents: uncontaminated, moderately contaminated, and highly contaminated material. Highly contaminated material exhibited extremely alkaline pH values up to 12.5 and total Cr contents ranging from 65.7 to 110 g/kg, whereas uncontaminated material had comparatively moderate pH values and Cr contents <1 g/kg. In total, seven crystalline phases commonly found in COPR were identified in the contaminated samples, of which five phases (brownmillerite, hydrocalumite, hydrogarnet, magnesiochromite, and periclase) are known to be able to accommodate Cr whereas hydrogarnet and hydrocalumite are the main host phases for Cr(VI). Batch tests showed that dissolution controlled the Cr(VI) concentrations in the eluates. Six samples were clearly identified as highly Cr-contaminated COPR. Leaching of Cr(VI) and resulting contamination of soils and water bodies is a key environmental risk arising from these COPR sites especially during the monsoon season. This situation is of particular concern as the local population use the highly Cr(VI)-contaminated water not only for the needs of livestock and irrigation but also as drinking water due to the absence of alternative water resources.

Comparison of stir bar sorptive extraction (SBSE) and liquid–liquid extraction (LLE) for the analysis of polycyclic aromatic hydrocarbons (PAH) in complex aqueous matrices

Stir bar sorptive extraction (SBSE) is an equilibrium extraction method used amongst others for the analysis of polycyclic aromatic hydrocarbons (PAH) in aqueous samples. We compared SBSE to liquid–liquid extraction (LLE) for aqueous eluates obtained from batch and column tests of PAH contaminated soils to check whether SBSE might be considered as an alternative sample preparation method. We used soils with different particle size distribution, organic matter content, and resulting eluate turbidity to test the sample preparation methods on eluates with matrices of varying complexity. Furthermore, we studied the influence of diluted organic matter (DOM) on the PAH sorption process to the polymer coating of the stir bar during SBSE. In the majority of cases, we found higher PAH concentrations (up to 288%) with SBSE than with LLE. The turbidity correlates with the difference in PAH results, i.e. the greater the turbidity in the eluate, the more PAH we found with SBSE compared to LLE. We observed no similar trend regarding the amount of total organic carbon (TOC). The presence of DOM in the eluate seems to hamper the SBSE slightly, the PAH recovery varied between 82 and 104%.

Eluted substances from unpolymerized and polymerized dental restorative materials and their Nernst partition coefficient

Components released from resin-based dental materials are important factors in the assessment of the biocompatibility of these materials. The study was performed to investigate the elution of substances from unpolymerized and polymerized composites (Tetric=TET; Arabesk=ARA), ormoceres (Admira)=ADM; Definite=DEF) and the compomere (Hytac=HYT). Specimens were polymerized and immersed in either water or methanol. Besides the unpolymerized specimens were dissolved in methanol. Eluted substances were detected by gas chromatography/mass spectroscopy. The Nernst partition coefficient (DC) of 64 substances, eluted from various resin-based dental materials was determined. Only in methanolic and aqueous eluates from unpolymerized and polymerized specimens of DEF and ADM, the comonomere 1,2/1,3-glycerol dimethacrylate was measured. Triethylene glycol dimethacrylate was eluted into the aqueous and methanolic phase from polymerized specimens from TET and ARA. 2-Hydroxyethyl methacrylate was eluted into the methanolic phase from polymerized specimen from TET and HYT. Residuals of monomer synthesis like triphenyl stibane were found in unpolymerized specimens of TET and ARA. Camphorquinone was eluted into the methanolic eluate from polymerized and unpolymerized specimens from all tested dental materials. Highest DC of 20.8 was found for 1/2-cyclohexene methacrylate. DC of urethane dimethacrylate and bisphenol-A-glycidyldimethacrylate varies from different manufacturers. Impurities from manufacturing process were found in some resin-based materials. Various well-known substances causing allergic reactions were found in aqueous or methanolic elutes. Patients, dental and manufacturing personnel are exposed to these substances.

Phytotoxic effects of sewage sludge extracts on the germination of three plant species

In order to evaluate the ability of three types of extracts to explain the ecotoxicological risk of treated municipal sewage sludges, the OECD 208A germination test was applied using three plants (Lolium perenne L., Brassica rapa L., and Trifolium pratense L.). Three equivalent batches of sludge, remained as dewatered sludge, composted with plant remains and thermally dried, from an anaerobic waste water treatment plant were separated. Samples from these three batches were extracted in water, methanol, and dichloromethane. Plant bioassays were performed and the Germination Index (GI) for the three plants was evaluated once after a period of 10 days. Germination in extracts was always lower than the respective controls. The germination in composted sludge (GI 40.9-86.2) was higher than the dewatered (GI 2.9-45.8), or thermally dried sludges (GI 24.6-64.4). A comparison of the germination between types of extracts showed differences for dewatered sludge with the three plants, where the water and methanol extracts had significantly lower germination than the dichloromethane extract. A higher half maximal effective concentration (EC50) in composted extracts was established, mainly in the water extract (EC50 431-490 g kg(-1)). On the contrary, the germination was strongly inhibited in the water extract of the dewatered sludge (EC50 14 g kg(-1)). The germination was positively correlated with the degree of organic matter stability of the parent sludge, and an inverse correlation was detected for total nitrogen, hydrolysable nitrogen and ammonium content. It is concluded that the phytotoxic effect of the water extract is more closely related to hydrophilic substances rather than lipophilic ones, and care must be taken with dewatered sludge application, especially with their aqueous eluates. Results obtained in this work show the suitability of the use of sludge extracts in ecotoxic assays and emphasize the relevance of sewage sludge stabilization by post-treatment processes.

Influence of petroleum hydrocarbon contamination on microalgae and microbial activities in a long-term contaminated soil.

Petroleum hydrocarbons are widespread environmental pollutants. Although biodegradation of petroleum hydrocarbons has been the subject of numerous investigations, information on their toxicity to microorganisms in soil is limited, with virtually no work conducted on soil algae. We carried out a screening experiment for total petroleum hydrocarbons (TPH) and their toxicity to soil algal populations, microbial biomass, and soil enzymes (dehydrogenase and urease) in a long-term TPH-polluted site with reference to an adjacent unpolluted site. Microbial biomass, soil enzyme activity, and microalgae declined in medium to high-level (5,200-21,430 mg kg(-1) soil) TPH-polluted soils, whereas low-level (<2,120 mg kg(-1) soil) pollution stimulated the algal populations and showed no effect on microbial biomass and enzymes. However, inhibition of all the tested parameters was more severe in soil considered to have medium-level pollution than in soils that were highly polluted. This result could not be explained by chemical analysis alone. Of particular interest was an observed shift in the species composition of algae in polluted soils with elimination of sensitive species in the medium to high polluted soils. Also, an algal growth inhibition test carried out using aqueous eluates prepared from polluted soils supported these results. Given the sensitivity of algae to synthetic pollutants, alteration in the algal species composition can serve as a useful bioindicator of pollution. The results of this experiment suggest that chemical analysis alone is not adequate for toxicological estimations and should be used in conjunction with bioassays. Furthermore, changes in species composition of algae proved to be more sensitive than microbial biomass and soil enzyme activity measurements.

Can fingernail creatinine concentrations be used to predict duration of azotemia?

Clinical use of fingernail creatinine estimation to predict duration of azotemia is yet to be validated. We studied the fingernail creatinine concentrations in 48 subjects: seven controls, nine with acute renal failure, five with rapidly progressive glomerulonephritis, 12 with chronic renal failure and 15 with end-stage renal failure on maintenance hemodialysis. The creatinine concentration in aqueous eluates of powdered nail clippings was determined by the alkaline picrate reaction. The mean fingernail creatinine concentration was significantly higher in patients with chronic renal failure (93.7 ± 83.7 µg/g), and end-stage renal disease on maintenance hemodialysis (118.4 ± 46.8 µg/g) as compared to those with acute renal failure (36.6 ± 23.7 µg/g) and rapidly progressive glomeru lonephritis (35.8 ± 20.6 µg/g). The creatinine concentrations did not differ significantly between normal subjects (27.2 ± 28.7 µg/g) and those with acute renal failure and rapidly progressive glomerulonephritis. However because of large variability in the values of fingernail creatinine concentrations within each group, the test lacked specificity. Therefore, this investigation is an unreliable indicator of duration of azotemia in individual patients and is not likely to be of much clinical use.

Isolation and characterization of an alkaliphilic bacterium capable of growing on 2,4‐dichlorophenoxyacetic acid and 4‐chloro‐2‐methylphenoxyacetic acid

AbstractA bacterial strain, called P4a, was isolated from debris of concrete samples of a demolished herbicide factory. The samples were contaminated with chlorinated and methylated phenoxyalkanoic acids. Strain P4a was able to utilize 2,4‐dichloro‐ (2,4‐D) and 4‐chloro‐2‐methylphenoxyacetic acid (MCPA) as the sole source of carbon and energy; degradation of 2,4‐dichlorinated and 4‐chloro‐2‐methylated phenoxypropionic acid and ‐butyric acid derivatives was not found. Growth on 2,4‐D was observed from a pH of 5.6 up to a pH of about 10, with optimum growth at around 8.5. No supplements were found to be required for growth on 2,4‐D, but the presence of yeast extract increased the growth rate from less than 0.05 h−1 to 0.2 h−1. The strain was metabolically active up to pH values of 12, which corresponded to the pH of aqueous eluates from such material. It was able to degrade 2,4‐D under these conditions up to an initial concentration of 400 mg/1 and in fact did degrade 1,600 mg/l of 2,4‐D at an initial pH value of 11. Strain P4a was tentatively identified as Comamonas acidovorans on the basis of the substrate utilization pattern (BIOLOG), fatty acid profile (MIS) and G+C content.

The promotive effect of smoke derived from burnt native vegetation on seed germination of Western Australian plants.

Exposure of dormant seed to cold smoke derived from burnt native vegetation had a positive influence on germination in one or more seed provenances in 45 out of 94 species of native Western Australian plants that are normally hard to germinate. When tested under controlled conditions some species showed earlier germination in smoke treatments than controls; in others smoke-treated seeds continued to germinate for several weeks after controls had achieved full germination. In the remainder, treated and control seeds germinated to similar time schedules. A group of 23 species which responded positively had previously been recorded as extremely difficult or impossible to germinate using conventional techniques. These included members of the genera Geleznowia (Rutaceae), Hibbertia (Dilleniaceae), Stirlingia (Proteaceae), Verticordia (Myrtaceae), Actinostrobus (Cupressaceae) and Pimelea (Thymelaeaceae). Both large- and small-seeded species were encountered amongst the positively responding taxa, which encompassed representatives of 15 families and 26 genera of dicotyledons, 5 families and 8 genera of monocotyledons and the gymnosperm Actinostrobus acuminatus. Sowing seeds on smoke-fumigated filter papers or watering with aqueous eluates of smoke elicited similar degrees of stimulation of germination, as did exposure to gaseous smoke in a readily germinating species Anigozanthos manglesii (Haemodoraceae) and the normally intractable species Lysinema ciliatum (Epacridaceae). Exposing recently burnt and unburnt natural bushland sites to smoke, smoked water or smoked dry sand elicited a significant germination response in 15 species. Over one third of the species sampled in the burnt site exhibited germination additional to that caused by the fire. Data are discussed in relation to previous germination studies on Australian and other taxa.

Enzyme immunoassay for detection of Giardia lamblia cyst antigens in formalin-fixed and unfixed human stool.

An antigen-capture enzyme-linked immunosorbent assay employing rabbit and mouse antisera to Giardia lamblia cyst antigens was developed for the diagnosis of Giardia infection through detection of G. lamblia-specific stool antigens in cell-free aqueous eluates of human stool. This is the first report of the use of anti-cyst antibodies in an enzyme immunoassay for G. lamblia. The assay gave a positive result with 54 of 59 stools from patients with symptomatic, clinically diagnosed giardiasis, giving the test a sensitivity of 91.5%. A negative reading was obtained with all of 25 stools from G. lamblia-negative control patients. The assay could detect as few as 20 sonicated cysts added to control stool eluate. The assay was more sensitive to cyst-derived antigens than to trophozoite-derived antigens. With two exceptions, the assay gave a negative result with stools from patients infected with Entamoeba histolytica (seven), Cryptosporidium sp. (four), or Blastocystis hominis (seven) and thus appears to be specific for G. lamblia antigens. Storage of stool eluates for more than 6 months at 4 degrees C as unpreserved aqueous eluates or as formalinized eluates did not affect the ability of the assay to detect the giardial antigens. The enzyme-linked immunosorbent assay proved useful for monitoring the levels of G. lamblia-specific stool antigens in the stool of patients undergoing antigiardial chemotherapy.

Ethylene as a toxin synergist in Verticillium wilt of tomato

Culture filtrates of Verticillium albo-atrum produced two major components toxigenic to tomato shoots. Aqueous extracts of acetone powders derived from 14-day culture filtrates were chromatographed on Bio-Gel P60 polyacrylamide gel and separated into a protein peak possessing strong polygalacturonase (PG) and pectin transeliminase (PTE) activities. A second peak consisted entirely of carbohydrate and was tentatively identified as a keto-polysaccharide, probably a fructosan mol. wt 3000. Petioles and leaves of healthy tomato plants cv. Craigella susceptible (S) and resistant (R) to V. albo-atrum treated with aqueous eluates of both components only developed symptoms with the protein, pectolytic enzyme fractions. The sequence of loss of turgor, chlorosis and necrosis was identical with the natural infection syndrome in older leaves. Leaves of the S isoline responded initially more severely than R, but by 60 h no differences between isolines could be observed. Leaves treated with the polysaccharide fractions remained symptomless up to 72 h. Endogenous ethylene production from detached leaves was increased only in leaves treated with fractions 6 to 15 containing protein and pectolytic enzymes. S leaves produced more in the initial (0 to 20 h) period but thereafter, from 45 to 92 h R leaves, produced variable but higher levels. At 140 h ethylene output fell in R leaves as tissue became severely necrotic. Pre-treatment of leaves with 10·0 parts/10 6 ethylene prior to uptake of the culture filtrate fractions resulted in increased wilting and necrosis in leaves treated with fractions 6 to 15, compared with non-ethylene-treated leaves in similar fractions. Symptoms appeared by 18 h and were severe by 42 h. Leakage experiments with 86Rb revealed no differences between healthy, ethylene-treated and control, S and R leaves. Polysaccharide-treated leaves previously exposed to ethylene showed a marked increase in ionic permeability comparable to the response of ungassed leaves to the protein, pectolytic enzyme component. The hypothesis is advanced for the rôle of ethylene as a toxin synergist in V. albo-atrum wilt of tomato. The implications of the in vivo interacton of ethylene and undetectable concentrations of fungal toxins in the early stages of pathogenesis are discussed in relation to the interpretation of symptoms.

The metabolism of tritiated dopamine in regions of the rat brain in vivo. I. The separation of catecholamines and their metabolites.

Abstract— A simple combination of acid‐ and base‐exchange resin columns enabled a more complete separation of amine, acid and neutral metabolites of catecholamines from individual small brain samples to be made. Fractions containing individual catecholamines or metabolites were obtained in aqueous eluates suitable for fluorimetric or radioisotopic analysis. With consistent intraventricular injection and brain dissection techniques, this separation method enables a study of the metabolism of catecholamines in regions of the rat brain and the effects of drugs on this metabolism.