Ethylenediaminetetraacetic Acid Extraction Research Articles

Changes of metal forms in sewage sludge after EDTA washing

Heavy metals in sewage sludge can be classified as difficult solid waste with potential hazards for environment. This report examined how an aqueous solution of EDTA (ethylenediaminetetraacetic acid) washing changes selected heavy metals’ (Zn, Cu, Cd, Ni, Pb) fractionation in sewage sludge. The analyzed sludge was dried and homogenized sewage sludge was obtained from a municipal treatment plant. The samples of sewage sludge were extracted with 0.1M EDTA solution for 24 h. Primary as well as extracted sewage sludge was examined for the total content of heavy metals and speciation of mentioned heavy metals using BCR sequential extraction procedure. Analyzed sewage sludge samples were quite significantly contaminated with heavy metals. The total content of zinc, copper, cadmium, nickel, and lead in initial sewage sludge was equal to: 2306.5, 263.7, 9.0, 118.0, and 117.0 mg kg−1 dM, respectively. After EDTA extraction: 1514.5, 253.5, 7.5, 83.0, and 58.0 mg kg−1 dM, respectively. In sewage sludge before extraction, the percentage of metals in fractions I (exchangeable and carbonates), II (Fe and Mn oxides), III (organic matter and sulfides), and IV (residual) was: for Zn 21, 47, 31, 1%, for Cu 0, 0, 99, 1%, Cd 0, 32, 68,0%, Ni 28, 22, 42, 8%, and Pb 6, 3, 34, 57%, respectively. In sludge after extraction, the percentage of metals in fractions was for Zn 17, 47, 33, 2%, Cu 0, 0, 98, 2%, Cd 0, 30, 70, 0%, Ni 13, 20, 46, 21%, and Pb 4, 5, 24, 67%, respectively. EDTA washing resulted in decreasing of nickel and lead percentage in mobile fractions and an increase in fractions strongly bounded to sludge matrix. The effect was most evident for Ni. Efficiency of EDTA solution for zinc, copper, and cadmium fractionation changes was practically negligible. EDTA extraction influenced the content of metals in municipal sewage sludge fractions. The process caused a decrease in each fraction; however, a strong chelating agent removed metals mostly from a dominant fraction.

Effect of regrinding conditions on pyrite flotation in the presence of copper ions

In this study, the effect of regrinding conditions on copper activation on the pyrite surface and subsequent pyrite flotation in the cleaner stage was investigated. Pyrite was activated by copper ions during primary grinding, and the rougher flotation concentrate was reground in a tumbling mill prior to the cleaner flotation. The regrinding condition was manipulated by two types of grinding media, stainless steel and mild steel. It was found that pyrite flotation in the cleaner stage was depressed especially when mild steel media was used during regrinding. An additional amount of collector improved pyrite flotation especially when stainless steel media was used during regrinding. An additional amount of copper ions further increased pyrite flotation after regrinding with mild steel media but decreased pyrite flotation after regrinding with stainless steel media. XPS (X-ray photoelectron spectroscopy) analyses and EDTA (ethylene diamine tetraacetic acid) extraction revealed that the different response of pyrite flotation in the cleaner stage to stainless steel and mild steel media used during regrinding was associated with the modified property of the Cu-activated pyrite surface carried from the rougher flotation concentrate and the new pyrite surface created during regrinding.

Comparison of Extraction Procedures for Assessing Soil Metal Bioavailability of to Wheat Grains

AbstractAgricultural soils have been analyzed to elucidate whether the trace metal distribution changes in relation to agricultural activities and to predict environmental risk. In addition to the extractability of Cu, Mn, Ni, Pb, and Zn in soils was compared by single extraction (hydrochloric acid (HCl), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), calcium chloride (CaCl2), water (H2O)), and sequential extraction procedures. The modified Community Bureau of Reference (BCR) sequential extraction procedure (three‐step) used to extraction of metals in soil samples. The extraction capacity of the analyzed metals was found by using single extraction procedures in the order: HCl > EDTA > DTPA > CaCl2 > H2O. A single correlation analysis was used to investigate the relationship between extractable metal concentrations in soil solutions and metal accumulation in wheat grains. Simple correlation analyses indicated that the extractable Pb and Ni of soils by HCl, EDTA, and DTPA single extraction procedures were significantly correlated with the metal contents of wheat grains. For CaCl2, H2O, and BCR extraction procedures there was a relatively poor correlation between the extractable Cu, Mn, Ni, Pb, and Zn of soils and metal contents of wheat grains.

The concentration of polysaccharides and proteins in EPS of Pseudomonas putida and Aureobasidum pullulans as revealed by 13C CPMAS NMR spectroscopy

Extracellular polymeric substances were extracted from the bacterial strain Pseudomonas putida and the fungal species Aureobasidium pullulans using three different methods (formaldehyde-NaOH, ethylenediaminetetraacetic acid (EDTA) and cation-exchange-resin). The composition of the extracellular polymeric substances (EPS) was analysed by biochemical and high-resolution solid state 13C nuclear magnetic resonance (NMR) spectroscopic methods. The EPS yield was strongly dependent on the extraction method, with the formaldehyde-NaOH method showing the best extraction efficiency. The NMR method revealed that when using the EDTA extraction method, about 40% of the EDTA accumulated in the EPS and that was responsible for the apparent high extraction yields. EPS protein content determined by the NMR method was up to 30% higher than the protein content determined using the biochemical (Lowry) method for P. putida and for A. pullulans. The average protein carbon content determined by the NMR method was approximately 70% of the total carbon content. NMR results could be supported by elemental analysis, which showed a high nitrogen content (approximately 10%) in the EPS. The carbohydrate carbon content detected with both methods in the cell aggregates and the EPS was approximately 20% in each. In this study, quantitative 13C cross-polarisation magic angle spinning NMR spectroscopy was conducted on unlabeled cell strains, and EPS and could be used to quantify protein and carbohydrate of different samples.

Experimental Assessment of Using Soluble Humic Substances for Remediation of Heavy Metal Polluted Soils

Since heavy metals are nondegradable and strongly bonded in soils, remediation of heavy metal polluted soils by extraction is difficult and current extraction techniques require harsh chemicals such as ethylenediaminetetraacetic acid (EDTA). However, use of EDTA is environmentally problematic because of costs, persistence, toxicity and deterioration of soil structure. Therefore, the potential of soluble natural humic substances (HS) to extract heavy metals from contaminated soils is tested as an environmentally friendly substitute for EDTA. A strongly polluted, calcareous urban soil (CRC soil) and a moderately polluted agricultural soil (CUP soil) were extracted at neutral pH in batch mode by three HS solutions from beech and Norway spruce litter (Beech-HS and Spruce-HS) and processed cow slurry (Bio-HS), all containing 25 mM dissolved organic carbon (DOC). After 10 extractions with a solution to soil ratio of 5:1 (L/kg), 8% to 39% of the total Cd, Cu, Ni and Pb soil contents, lowest for Ni and highest for Cu/Pb, were extracted. Natural and processed HS samples had comparable capacities to extract the heavy metals. A comparison of 100 mM DOC of Bio-HS and EDTA as extractants for Cu from the CRC soil showed extraction of 67% by EDTA and 41% by Bio-HS, indicating somewhat higher efficiency of EDTA than of HS. Sequential extraction of the CRC soil after Bio-HS and EDTA extraction showed removal of exchangeable, carbonate- and metal oxide-bound Cu but also of some residual Cu. It is therefore concluded that HS appears to be an attractive and promising alternative to EDTA as remediation agent for heavy metal polluted soils provided cheap HS of good quality is easily available.

More than inorganic copper is bioavailable to aquatic mosses at environmentally relevant concentrations

The present study investigates how dissolved organic matter (DOM) alters copper bioavailability at environmentally relevant concentrations (1-5 microg/L of dissolved copper, 1-4 mg/L of dissolved organic copper). A methodology combining two biological endpoints (short-term and steady-state bioaccumulation of copper by the aquatic moss Fontinalis antipyretica) and a sampling of labile copper with diffusion gradient in thin films (DGT) is proposed for batch experiments conducted with mineral water and various DOM, ethylenediaminetetra-acetic acid (EDTA), humic acid, and natural Seine River (France) extracts (hydrophobic and transphilic fractions). All types of DOM reduce the bioavailability of copper to aquatic mosses, and this reduction was more pronounced for the short-term biological endpoint, which was taken as being representative for environmental exposure. Labile copper sampled with DGT made it possible to estimate short-term bioaccumulation in the case of EDTA and natural Seine River extracts. With humic acid solutions, however, labile copper was lower than bioavailable copper. This result suggests that at realistic metal concentrations and with certain types of natural DOM, bioavailable copper might comprise not only inorganic copper but also some weak organic complexes. Hence, labile copper, in situ sampled with DGT, might not systematically overestimate bioavailable copper, as suggested previously on the basis of in vitro toxicity studies.

A critical evaluation of the 0.05 M EDTA extraction of Pb from forest soils

Among many single-step extraction procedures proposed, 0.05 M EDTA (ethylenediaminetetraacetic acid) extraction is widely used. Although it has been often criticised, this protocol remains an effective and simple approach for a fast determination of the potential availability of several metals (e.g., Pb, Cd, Cu, Zn, etc.). However, other metallic elements present in soils at high concentrations such as Ca and Fe can possibly influence the extraction of the target metal due to competition for the EDTA ligand. The aim of this study was to evaluate the role of these metals during the 0.05 M EDTA extraction procedure. Furthermore, sequential extraction and Pb isotope analyses (206/207Pb) were used in order to obtain more detailed information. The results of this study showed that especially the concentration and crystallinity of Fe play a very important role during the extraction of the target metals from low to moderately contaminated soils and this fact should be taken into account during result interpretation.

Effect of Organic Matter and Salinity on Ethylenediaminetetraacetic Acid–Extractable and Solution Species of Cadmium and Lead in Three Agricultural Soils

A study was conducted to investigate the chemical speciation of added cadmium (Cd) and lead (Pb) and their availability as influenced by fresh organic matter (OM) and sodium chloride (NaCl) in three agricultural soils. The soils were treated with 20 mg Cd/kg as cadmium nitrate [Cd(NO3)2 · 4H2O], 150 mg Pb/kg as lead nitrate [Pb(NO3)2], 20 g/kg alfalfa powder, and 50 mmol/kg of NaCl and then incubated for 3 months at 60% water‐holding capacity (WHC) and constant temperature (25 °C). Subsamples were taken after 1, 3, 6, and 12 weeks of incubation, and electrical conductivity (EC), pH, dissolved organic carbon (DOC), and concentrations of cations and anions were determined in the 1:2.5 soil/water extract. Available Cd and Pb were determined in 0.05 M ethylenediaminetetraacetic acid (EDTA) extract. Concentrations of organic and inorganic species of Cd and Pb in soil solution were also predicted using Visual Minteq speciation program. The most prevalent species of dissolved Pb and Cd in the soils were Pb‐DOC and Cd2+ species, respectively. Salinity application increased the available and soluble Cd significantly in the acid and calcareous soils. It, however, had little effect on soluble Pb and no effect on available Pb. Organic‐matter application decreased availability of added Pb significantly in all soils. In contrast, it raised soluble Pb in all soils except for the acid one and approximated gradually to the added Pb with time. Impact of OM on available Cd was somewhat similar to that of Pb. Soluble Cd increased by OM application in the calcareous soil, whereas it decreased initially and then increased with time in the other soils.

Trace elements in wild grasses: a phytoavailability study on a remediated field

There have been significant efforts to establish a widely usable method for the prediction of trace element bioavailability in soil. In this work, we used extraction with 0.01 M CaCl2 and 0.05 M ethylenediaminetetraacetic acid (EDTA) to estimate bioavailable concentrations of As, Cd, Cu, Pb, and Zn in a soil moderately contaminated with trace elements 1 and 2 years after the application of three amendments. The experiment took place in a field plot of a soil affected by the toxic spill of the Aznalcóllar mine. Four treatments were established: three with amendments (biosolid compost, sugar beet lime, and a combination of leonardite plus sugar beet lime) and a control without amendment. Trace element concentrations of two representative species in each year (Lamarckia aurea and Poa annua in 2004 and Lamarckia aurea and Bromus rubens in 2005) were analyzed. The results showed a positive effect of the amendments both on soil and vegetation. Trace element concentrations in plants growing in the amended subplots were lower than those in plants from nonamended subplots. As a rule, concentrations of CaCl2-soluble Cd, Cu, and Zn in soil were positively correlated with trace elements in plants, whereas EDTA extraction was scarcely correlated with plant concentration. For species of grasses, especially L. aurea, CaCl2 seems to be a more suitable extractant to predict trace element bioavailability in this contaminated soil.

Enriched stable isotopes for determining the isotopically exchangeable element content in soils

SummaryThe proportion of metals in soils in equilibrium with soil solution can be determined using isotopic dilution. For this purpose, an isotope dilution mass spectrometric (IDMS) technique has been applied for the elements Cd, Cr, Cu, Mo, Ni, Pb, Tl and Zn. Conventionally, sorbed amounts of heavy metals in soils are analysed by ethylenediamine tetra‐acetic acid (EDTA) extraction. The IDMS technique and EDTA extraction were both applied to 115 soil samples and compared. For Cd, Cu, Ni, Pb and Zn, the results of the IDMS technique correlated well with the results of EDTA extractions (rs(Cd) = 0.965, rs(Cu) = 0.916, rs(Ni) = 0.878, rs(Pb) = 0.922, rs(Zn) = 0.962; all at P < 0.001). For Cd and Zn, no significant differences between the results of both methods could be observed, which suggests that EDTA and IDMS determined the same pool. EDTA extracted more Cu, Ni and Pb than was determined by IDMS (7, 26 and 13%, respectively). The correlation between EDTA extraction and IDMS for Cr was significant but weak (rs(Cr) = 0.361). For Tl and Mo, EDTA extraction and IDMS did not correlate, and IDMS yielded larger concentrations than EDTA. This can be explained by the fact that Tl and Mo do not form stable EDTA complexes, which are essential for the EDTA technique. Recovery experiments demonstrated that added Cd, Cu, Mo, Ni, Pb, Tl and Zn could be recovered successfully by IDMS analysis (mean recovery = 103 ± 9%). Adsorption isotherms for soil samples were determined for Tl, thereby demonstrating that IDMS gave a better estimation of the native content of sorbed Tl in soils than EDTA extraction.

Development of Mild Extraction Methods for the Analysis of Natural Dyes in Textiles of Historical Interest Using LC-Diode Array Detector-MS

Analysis of dyes extracted from textiles of historical interest can give valuable information as to where, when, and how the textiles were made. The most widely used method for extraction of colorants involves heating with HCl, which frequently decomposes glycosidic dye components to their parent aglycons, with consequent loss of information about the source of the dye. This is particularly true for flavonoid dyes, many of which are glycosides. We have developed or improved upon two mild textile extraction methods that use ethylenediaminetetraacetic acid (EDTA) and formic acid and are efficient in extracting dyes, but preserve glycosidic linkages. The relative efficiencies of the HCl, EDTA, and formic acid extraction methods are compared by analyzing extracts of dyed samples of silk using HPLC coupled with diode array and mass spectrometric detection. HPLC profiles of EDTA or formic acid extracts of silk dyed, for example, with pagoda tree buds and onionskins are clearly distinguishable as to the plant material used, whereas profiles of HCl extracts are not. Thus, extraction of textiles with EDTA or formic acid reagents can yield significantly more information about the original dyestuff than can extraction with a strong acid.

Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation

Although 14-day-old mouse embryonic calvarial cells cultured in plastic culture dishes in the presence of 1alpha,25-dihydroxyvitamin D3[1alpha,25-(OH)2D3] for 7 days could barely resorb bone slices, the same calvarial cells cultured with an ethylenediaminetetraacetic acid (EDTA) extract from bovine bone powder under the same conditions stimulated pit formation on bone slices in a dose-dependent manner. Therefore, the present study was conducted to purify and characterize this osteoclast maturation-inducing factor(s) from the bone matrix. The protein having osteoclast maturation-inducing activity in the EDTA extract was purified by gel filtration over Superdex 75 preparation grade and chromatography on hydroxyapatite, Mono Q, and C8 reversed-phase HPLC by monitoring the ability of the eluted fractions to elicit pit formation on bone slices. The molecular weight of the purified protein estimated by high-resolution polyacrylamide gel electrophoresis was 5.7 kDa and 6.8 kDa in the respective absence and presence of 2-mercaptoethanol. The sequence of the 30-amino-acid purified protein corresponded to the 7th to 36th residues of bovine osteocalcin. The osteocalcin fragment, missing the initial 6 residues at the N-terminal region, exhibited higher osteoclast maturation-inducing ability than bovine intact osteocalcin on a per weight basis. The osteocalcin fragment had no effect on the expression of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and osteoprotegerin (OPG) genes in calvarial cells, nor did it enhance the bone-resorbing activity of mature osteoclasts. When the osteocalcin fragment was added to late-stage (days 4-7) or to early-stage (days 0-3) cultures of calvarial cells pretreated with 1alpha,25-(OH)2D3, its stimulatory effect was observed in the late-stage cultures rather than in the early-stage ones. In addition, the osteocalcin fragment directly enhanced the formation of osteoclasts with bone-resorbing ability from Mac-1+ c-Fms+ cells in the presence of macrophage colony-stimulating factor (MCSF) and RANKL. These results suggest that the osteocalcin fragment in bone matrix is involved in osteoclast maturation, especially at a late stage of osteoclast differentiation.

Factors affecting EDTA extraction of lead from lead-contaminated soils

The effects of solution:soil ratio, major cations present in soils, and the ethylenediaminetetraacetic acid (EDTA):lead stoichiometric ratio on the extraction of lead using EDTA were studied for three different Superfund site soils, one rifle range soil, and one artificially lead-contaminated soil. Extraction of lead from the lead-contaminated soils was not affected by a solution:soil ratio as low as 3:1 but instead was dependent on the quantity of EDTA present. Results of the experiments showed that the extraction efficiencies were different for each soil. If sufficiently large amount of EDTA was applied (EDTA–Pb stoichiometric ratio greater than 10), most of the lead were extracted for all soils tested except for a Superfund site soil from a lead mining area. The differences in extraction efficiencies may be due to the major cations present in soils which may compete with lead for active sites on EDTA. For example, iron ions most probably competed strongly with lead for EDTA ligand sites for pH less than 6. In addition, copper and zinc may potentially compete with lead for EDTA ligand sites. Experimental results showed that addition of EDTA to the soil resulted in a very large increase in metals solubility. The total molar concentrations of major cations extracted were as much as 20 times the added molar concentration of EDTA. For some of the soils tested, lead may have been occluded in the iron oxides present in the soil which may affect lead extraction. While major cations present in the soil may be one of the factors affecting lead extraction efficiency, the type of lead species present also play a role.

Toxicity identification of metals: Development of toxicity identification fingerprints

The ecotoxicological and chemical response of five metals (Cd, Cr, Cu, Ni, and Zn) to selective fractionation techniques was assessed using the organism Daphnia magna. The metals Cu and Zn revealed similar toxicity patterns, showing an increased toxicity when lowering the pH, a marked reduction in toxicity after addition of ethylenediaminetetraacetic acid (EDTA). Similar toxicity reductions were obtained by passing the metal solutions over ion exchangers, activated charcoal, and filtration/solid-phase extraction units at neutral and basic pHs. The discrimination between Cu- and Zn-toxicity occurred in the oxidant reduction test, i.e., Zn toxicity was not affected by the addition of Na2S2O3, while Cu toxicity was strongly reduced. A second cluster was formed by Cd and Ni. The toxicity of both metals markedly decreased in the EDTA, ion exchangers, activated charcoal, filtration, and solid-phase extraction tests at high pHs. Discrimination between the toxicity of both metals was accomplished with the graduated pH test, in which a pH-independent toxicity response was found for Ni while the toxicity of Cd increased at lower pHs. Compared with the other metals, a very distinct toxicity pattern for Cr was observed. From the applied fractionation techniques, only the graduated pH and the anion resin tests resulted in a marked change in Cr toxicity. The produced toxicity patterns for each individual metal could therefore be used as fingerprints (i.e., toxicity identification fingerprints) in order to identify the toxicity caused by individual metals for D. magna.

Serogroups of the beer spoilage bacterium Megasphaera cerevisiae correlate with the molecular weight of the major EDTA-extractable surface protein.

Megasphaera cerevisiae is a Gram-negative obligate anaerobe that causes turbidity and off-flavour and aroma in beer. Seven isolates of M. cerevisiae were obtained worldwide, and their extractable surface antigens were focused upon to determine if there is more than one serogroup of this bacterium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of ethylenediaminetetraacetic acid (EDTA) bacterial extracts revealed a predominant protein with apparent molecular weights of 46,000, 45,000, and 43,000 for three, two, and two isolates, respectively. When mouse anti-serum generated against any of the EDTA extracts was reacted with denatured bacterial proteins in immunoblots, all bacterial isolates exhibited extensive cross-reactivity involving three antigens, one being the major EDTA-extractable protein. In contrast, when the sera were tested for surface reactivity with intact bacteria, three cross-reactivity groups were observed, with the groups individually comprised of bacteria having the same size major EDTA-extractable surface protein. When BALB/c mice immunized with a bacterium from each of the three serogroups were used for monoclonal antibody (Mab) hybridoma production, bacterial surface-reactive Mabs were obtained whose reactivities parallel the three polyclonal antibody-defined serogroups. Through combining these surface-reactive Mabs, it will be possible to rapidly detect and identify beer contamination by M. cerevisiae belonging to any serogroup.

EDTA Extraction of Heavy Metals from Different Soil Fractions and Synthetic Soils

A laboratory-prepared contaminated soil was partitioned into four fractions, namely carbonate, Fe/Mn oxides, organic matter and clay mineral, according to the form in which the heavy metal bound with soil constituents. Individual contaminated soil fractions and synthetic soils were prepared for the study of soil extraction using ethylenediaminetetraacetic acid (EDTA). The effect of contact time and EDTA concentration were evaluated for both individual soil fractions and synthetic soils. The extraction reached equilibrium rapidly, after about 30 min. A 0.01 M EDTA solution was less effective than a 0.05 M or a 0.10 M EDTA. EDTA was proved to be effective for metal removal from the four individual soil fractions and synthetic soils. In general, approximately 90% of metals were removed from synthetic soils by 0.10 M EDTA. EDTA extraction of Pb from a contaminated carbonate fraction was thought to be affected by the formation of lead carbonates. A simple equation based on the sum of the released heavy metal from the individual components is used to check if there are interactions among the different soil components when mixed. The estimated values agreed well with the experimentally measured results only for the 0.10 M EDTA system.

Remediation of Lead Contaminated Soil by EDTA. I. Batch and Column Studies

Extraction using ethylenediaminetetraacetic acid (EDTA), and other chelates has been demonstrated to be an effective method of removal of Pb from many contaminated soils. However, column leaching of Pb from alkaline soils with EDTA has been problematic due to extremely low soil permeability. The first purpose of this study was to develop batch extraction procedures and methods of analysis of batch extraction data to provide Pb solubility information which can be used to model the column extraction of Pb from soils. The second purpose was to determine the effect of the addition of KOH and CaCl2 to K2H2EDTA extract solution on both hydraulic conductivity and Pb removal. A Pb-contaminated soil sample was collected from an abandoned battery recycling facility. Both batch shaker extractions and column leaching experiments were completed using 5 different EDTA extract solutions. When only CaCl2 was added to EDTA no change in the amount of Pb removed by batch extraction was observed. As expected, lead solubility was observed to decrease as pH was increased by the addition of KOH. However, Pb solubility was only slightly decreased by the addition of both CaCl2 and KOH. The amount of time required to leach 6.0 L of extraction solution through the soil columns varied from 2 to 33 days. The addition of CaCl2 and/or KOH resulted in increased soil hydraulic conductivity relative to the EDTA-only solution. The hydraulic conductivity was related to residual calcium carbonate content, suggesting that dissolution of CaCO3 and subsequent production of CO2 gas in the soil pores was partially responsible for the observed reductions in soil permeability. However, Pb removal was diminished with the addition of CaCl2 and KOH because of the decreased Pb solubility and also kinetic limitations associated with the shorter residence time of the extract solution in the column.

Evaluation of EDTA and fish skin extract in primary culture of fish liver cells

The isolation, primary culture and attachment of liver cells to the substratum from a tropical catfish (Heteropneustes fossilis) using ethylenediaminetetraacetic acid (EDTA) as isolating agent of liver cells and skin extract (SE) from fish as attachment substrate for the primary culture of liver cells has been standardised. A suitable temperature for such cultures has also been determined. Attachment efficiency of the liver cells in culture and their intracellular lactate dehydrogenase (LDH) activity have been taken as parameters for characterization of the primary culture. Disaggregation of liver cells with EDTA is very potent to isolate substantial rumber of cells from the liver of H. fossilis. An ideal concentration of EDTA for liver cell isolation has been standardized. Matrix prepared from carp and catfish skin at different pH (2.0, 4.0, 6.0 and 8.0) were also evaluated for liver cell culture by considering the attachment efficiency of the cells over the substratum as well as retention of intracellular LDH enzyme after 48 hours of seeding. Matrix of carp skin was compared with that of catfish as suitable substrate for primary culture of fish liver cells. It has been found that the SE prepared at pH 4.0, both from carp and catfish skin, performed better than those at other pHs. At the same time, the matrix of carp skin was found to be better than that of catfish skin. Cultures were incubated separately at 17 and 23 °C in air atmosphere. Incubation temperature at 23 °C was found to be more suitable than that at 17 °C. The percent of detached/unattached cells showed only marginal variation between two temperatures but LDH-activity recorded drastic reduction (between 50 to 75%) depending upon the pH of the matrix during preparation. Our finding establishes despensibility of enzyme (collagenase/trypsin) for cell isolation in catfish. Our studies also exhibit that carp skin extract performs better than catfish skin extract in terms of attachment efficiency as well as intracellular LDH activity. This study indicates that no species/generic barrier exists in matrix between catfish and carp.

Distribution of Zinc, Manganese, Copper, Cobalt, and Nickel in Andosols Profiles

Abstract The distribution of zinc, manganese, copper, cobalt, and nickel in Andosols was investigated. Sixty nine soil samples were collected from different horizons of an Andosols profile in Miyakonojo Basin in south Kyushu, Japan, The total contents of heavy metals were determined by digestion and four extraction solutions, 1 M NH4Ac (ammonium acetate) pH 4.5, 0.1 M HCl, 0.01 M EDTA (ethylenediaminetetraacetic acid) pH 6.5, and 0.005 M DTPA (diethylenetri-aminepentaacetic acid) pH 7.3 were used to determine the contents of available Zn, Mn, Cu, Co, and Ni in Andosols in relation to the organic carbon content. The results of the extraction analysis showed that by the use of 0.1 M H Cl high value of extracted heavy metals in the upper layers of the humus horizons were obtained while EDTA extraction yielded a large amount of the above mentioned metals in the high humus horizons. The extractable heavy metals contents were high and these metals closely related to the organic carbon content mostly in the humus horizons in the profile. Where, biocycling process may play an important role in the concentration of heavy metals. Based on the study, it was found that the total content of Zn increased towards the C horizons or pumice layers in the soil profile. Such a trend was also found in the case of the Mn content. While the Cu content in the humus horizons was much higher in the upper part of each humus horizon. According to this study the distribution of heavy metals, Cu (organic matter complexes) in the Andosols profile was more stable than that of Zn (organic matter complexes) in soils. It was shown that Zn in the surface humus horizon was enriched but that some amount was leached under buried conditions. The same phenomenon was also observed in the distribution of Mn in the profile. The movement of Co and Ni in the soil profile was limited, as evidenced by the sharp reduction in the concentrations of these two metals in buried soils. Hence, it is concluded that the distribution of Zn, Mn, Cu, Co, and Ni was considerably higher in the humus horizons of the Andosols profiles.

Oxidation capacity of aquifer sediments

A laboratory extraction method (Ti 3+ -EDTA extraction) for the determination of the oxidation (electron accepting) capacity related to oxides and hydroxides of aquifer sediments was developed. At room temperature, titanous ions (0.008 M Ti 3+ ) in a solution of 0.05 M ethylenediaminetetraacetic acid (EDTA) reduced oxidized aquifer species. This operationally defined oxidation capacity (ORC) was determined as μmoles of electrons accepted per gram of sediment sample. Well-described oxidized iron and manganese minerals were reduced (ferrihydrite 98%, akageneite 100%, goethite and hematite 93%,magnetite 9%, pyrolmite 99%) whereas organic matter in the sediments was not reduced significantly