Anwendung einer enzymatischen Methode zur Charakterisierung von Huminstoffen Enzymatic Characterization of Humic Substances

A method is described for the specific hydrolysis of amino acids from humic substances by using the proteolytic enzyme “Pronase E”. The amino acids are analyzed by a fluorescence detector after precolumn derivatization and HPLC-separation. The pattern and the concentration of amino acids after the enzymatic hydrolysis are set up for humic substances of different origin. Due to the results the enzymatic hydrolysis is suitable for the characterization of humic substances with a nitrogen content higher than 3%. As shown in a kinetic study of the hydrolysis of a fulvic acid the influence of humic substances on the enzyme activity increases in case of lower nitrogen content. Due to the specificity of the proteolytic enzyme the operationally defined method is limited as shown by a protein of a known amino acid content. The amino acids leucine, valine, tyrosine and histidine are released to the highest extent. The determined total extent for the hydrolysis of Insulin B is 26.34% and for Casein 38.37%. By comparison of the enzymatic hydrolysis with the classic acid hydrolysis the advantages of the biochemical method are the simple experimental handling and the mild reaction conditions. Amino acids which are very unstable like glutamine and asparagine can only be analyzed by the enzymatic hydrolysis.     

Influence of IHSS Standard and Reference Materials on Copper and Mercury Toxicity to Vibrio fischeri

The reduction in light emission of the marine bacterium Vibrio fischeri used in the standard Microtox^® bioassay was measured for the metals copper and mercury. The concentration at which the light emission was reduced by 50% (EC₅₀) was determined to be (3.43 ± 0.83) μmol/L for Cu²⁺ and (0.66 ± 0.01) μmol/L for Hg²⁺. The reduction of the toxicity of these metals by humic and fulvic acids were studied using IHSS Standard and Reference Materials. Copper toxicity was reduced 17...20% by the soil and peat fulvic acids and 9...20% by the aquatic fulvic acids. While there appeared to be little difference in the reduction of Cu toxicity by fulvic acids from soils, peats, or aquatic systems, Hg toxicity was reduced 3.6...7.3% by the soils and peats, while aquatic fulvic acids reduced Hg toxicity 14...16%. Soil fulvic acids appear to have significantly less capacity to reduce Hg toxicity than Cu toxicity. Humic acids had much higher reductions of Cu toxicity (44...124%) compared to the fulvic acids, with little difference between aquatic and soil or peat humic acids, 44...124% and 67...100%, respectively. However, humic and fulvic acids, regardless of origin, had approximately the same effect on Hg toxicity with 3.5...16% reduction by fulvic acids and 8...20% reduction by humic acids. Unlike the fulvic acids, no clear trend was observed relative to origin of the humic acids. There was no correlation between percent reduction of Cu or Hg toxicity by the organic compounds and copper binding capacity (CuBC), C/N ratio, or carboxyl content of the materials. Examination of natural organic matter (NOM) isolated by reverse osmosis techniques from three water sources had reductions of both Cu and Hg toxicity that were most similar to the Suwannee River and Nordic fulvic acids.     

Association of Humic Substances: Verification of Lambert‐Beer Law

The precision UV/Vis‐absorption spectra of a naturally occurring aquatic humic substance were measured in the range of concentration 0.003 to 1.4 g/L DOC (dissolved organic carbon). Lambert‐Beer law is well obeyed at low concentrations whereas deviations have been observed for DOC concentrations above 0.1 g/L: they are interpreted in terms of self‐solvation and formation of micelles.     

Die gaschromatographische Bestimmung von Diethylentriaminpentaessigsäure in Oberflächenwasser

Gaschromatographic Determination of Diethylenetriaminepentaacetic Acid in Surface Water. A method for the determination of diethylenetriaminepentaacetic acid (DTPA) is reported. The determination is based on the preconcentration on a strong base anion exchange resin, esterification with n-propanol and following gaschromatographic separation using a N-selective detector. The sensitivity of the method, including enrichment, is 1 μg/L in river water. The identity of DTPA in river water was proved by mass spectrography. The method can be used also for the quantitative determination of nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA) and ethyleneglycolbis-(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). The method was used to examine river water. An investigation along the river Rhine, taking grab samples from January and february 1992 showed that DTPA was present in some sampling places in similiar concentrations as NTA and EDTA.     

Adsorbed and Bound Residues in Fulvic Acid Fractions of a Contaminated Groundwater – Isolation:, Chromatographic and Spectroscopic Characterization

Humic substances (HS) were isolated from two contaminated groundwater samples (B22 and B53) from a site of a former gas facility. The isolation yielded almost only the fulvic acid fractions (FA). For characterization spectroscopic (UV, fluorescence) and chromatographic techniques (hydrophobic interaction chromatography – HIC as well as size-exclusion chromatography – SEC) were applied. The sample designated B22 FA was collected from the contamination plume whereas the sample B53 FA was collected downstream. Distinct differences were exhibited by these samples. The UV and fluorescence spectra as well as the HIC and SEC chromatograms of the B53 FA sample resemble those of the FA fraction obtained from natural water (groundwater, bog). The HIC and SEC chromatograms reveal the presence of organic compounds in B22 FA which can be derived from coal tar contaminants or their metabolites. Some of the compounds can be extracted from the FA fraction with non-polar organic solvents indicating adsorptive forces between the contaminants and the FA fraction.     

Entwicklung von Enzymimmunoassays zum Nachweis von Phenoxycarbonsäure-Herbiziden in Trink- und Grundwässern Development of Enzyme Immunoassays for the Detection of Phenoxycarboxylic Acid Herbicides in Drinking Water and Groundwater

Competitive solid phase enzyme immunoassays using polyclonal antibodies were developed for the detection of the phenoxycaboxylic acids MCPB [4-(4-chloro-2-methylphenoxy)butyric acid], Mecoprop [2-(4-chloro-2-methylphenoxy)propionic acid], and 2,4-D [(2,4-dichloro-phenoxy)acetic acid] in drinking water and ground water. The carrier protein for the immunization was bovine serum albumin, horseradish peroxidase conjugates were employed as enzyme tracer. For the three antisera, the optimization of detection limits and test sensitivities was our first consideration. For the mecoprop and 2,4-D antisera, the strongest influences were the pH value and the ionic strength, as much as the use of enzyme tracers with lower affinities. The MCPB antiserum reacted with 2,4-DB[4-(2,4-dichlorophenoxy)butyric acid] with equal specificity, either could be detected at 0.02 μg/L (80% B/B₀), middle of the test (50 B/B₀) lying at 0.1 μg/L. The detection limit with mecoprop antiserum was optimized to 0.02 μg/L, the prescribed limit for drinking water of 0.1 μg/L ling at 60 % B/B₀. The strongest cross-reactivity was found for mecoprop methyl ester. MCPB. 2,4-DB, and dichloroprop have crossreactivities of 50%, 6.7%, and 6.3%, respectively. The 2,4-D antiserum reacts less sensitively with 2,4-D, the detection limit being 0.4 μg/L. The 2,4-D isooctyl ester and 2,4-D methyl ester demonstrate as cross-reacting compounds high cross-reactivites of 3630% and 2230%. The cross-reactions of the compounds 2,4-DB, MCPB, and MCPA [(4-chloro-2-methylphenoxy)acetic acid] lie at 52%, 69%, and 41%. 100 ground water tests were spiked within laboratory. All positive samples were correctly identified. Falsely negative results did not appear.     

Spectroscopic Characterization of Fulvic Acid Fractions of a Contaminated Groundwater

Two fulvic acid (FA) samples taken from a former gas production facility in the Southwest of Germany were characterized using advanced fluorescence techniques. Steady-state fluorescence (fluorescence excitation, synchronous fluorescence) as well as time-resolved fluorescence were applied. Distinct differences between the sample B22 FA taken within the contamination plume and the sample B53 FA taken downstream were found. Comparison with a model compound for metabolites and humic substances revealed that due to the downstream passage the characteristics of the dissolved organic matter became more humic-like. The assignment of single classes of compounds in the sample B22 FA is discussed in terms of their synchronous fluorescence spectra and fluorescence decay time distribution.     

Removal of Various Phenoxyalkanoic Acid Herbicides from Water by Organo‐clays

Dodecylammonium bentonite (DB) and dodecylammonium sepiolite (DS) were used as sorbents for phenoxyalkanoic acid herbicides 2,4‐D ((2,4‐dichlorophenoxy)acetic acid), 2,4‐DP ((RS)‐2‐(2,4‐dichlorophenoxy)propionic acid), 2,4‐DB (4‐(2,4‐dichlorophenoxy)butyric acid), 2,4,5‐T ((2,4,5‐trichlorophenoxy)acetic acid), and MCPA ((4‐chloro‐2‐methylphenoxy)acetic acid). Langmuir, Freundlich, and the linear Henry’s Law isotherm adsorption parameters were calculated from the adsorption isotherms. Langmuir equation showed poor fit for both adsorbents. According to the evaluation using the Freundlich equation, the DS sample showed much higher and stronger sorption capacity than DB. Similar behaviour was also observed in the case of the linear Henry’s Law isotherm. The adsorption of the herbicides on both DB and DS decreased in the order of 2,4‐DB > 2,4,5‐T > 2,4‐DP > 2,4‐D > MCPA.