Chemical and Biological Studies on the Yellow Organic Acids of Lake Water1

The yellow coloring matter of natural waters has been studied with particular emphasis on Lower Linsley Pond, Connecticut. The best method of isolation was by ethyl acetate extraction of acidified evaporation residues of filtered water. The material thus obtained was a mixture of colorless and colored carboxylic acids, the colored ones fluorescing under ultra‐violet light. Numerous yellow fractions were distinguished through paper chromatography, none of which showed maxima in either their visible or ultra‐violet absorption spectra. Chemical tests indicated a phenolic or enolic character with probable unsaturation. Elemental analyses showed 1.4% ash, no halogens or sulfur, and resulted in an empirical formula of (C 50 H 62 O 27 N) n . The nitrogen was believed to be an impurity, resulting in an empirical formula of C 21 H 26 O 11 on the basis of the determined mean molecular weight of 456. The mean equivalent weight of the mixture was 228. The salts of the acids were dialysable and the acids were amorphous as shown by x‐ray diffraction. Infra‐red spectroscopy of various derivatives indicated a mixture of hydroxy carboxylic acids and admitted the possibility of unsaturation and aromaticity, although the indications are that if aromaticity exists it is relatively inconsequential. The materials were very resistant to chemical oxidation. Lower Linsley Pond surface water contained between 4.5 and 9.0 milligrams of acids per liter, of which about 40% was estimated to be yellow. Raising the pH from 3 to 11 increased the color by 50%. Solutions of the salts obeyed Beer’s law in the range studied from 5 to 70 mg/L. The materials may affect organisms through alteration of light and by their buffering action. They were reasonably stable to biological decomposition over a period of 4½ months. Concentrations of up to 50 mg/L had no effect on pure cultures of Chlamydomonas Reinhardi, but Hematococcus pluvialis and Scenedesmus quadricauda were stimulated in their growth by concentrations of between 5 and 50 mg/L. The materials were able to retain iron in a filterable state at pH values up to 13.2. Eleven lakes showed very similar chromatographic patterns on the same date and on different dates. Evidence is presented for the hypothesis that some of the numerous colored fractions are different salts of a small number of acids. It is concluded that the materials originate as a result of decomposition in soil and possibly in sediments as well. The name ‘humolimnic acids’ is suggested for the materials to indicate their probable relation but improbable equivalence with so‐called ‘humic’ acids.