1H5. LEMNQLQGECAL AND macaw. szsmmmcs as mmwsa yam-w ORfiANIC mm m mm WATERS The“: In? “an Degree 05 M. 5. MICHIGAN STATE UNIVERSITY Eugene Howard Buck 1968 TH ESIS fl:- -v “I'- . — -r .k L I B R A P ”I." Michigan State a) University" j"! c .M—u... Tww "' " "— 4 _.__.__._f._.._._.. .. ABSTRACT THE LIMNOLOGICAL AND ECOLOGICAL SIGNIFICANCE OF DISSOLVED YELLOW ORGANIC ACIDS IN NATURAL WATERS by Eugene Howard Buck Yellow organic acids were extracted from natural waters and subsequently added to aquaria and experimental ponds. A quantitative procedure for the measurement of these com- pounds was developed using their characteristic fluorescence. This procedure is temperature sensitive and encounters inter- ference when detergents are also present. In natural water yellow organic acids may be allochthon- ous, mainly from runoff and leaf fall, or autochthonous, from the sediments and decay of aquatic vegetation, in origin. Loss to the environment was through a light-induced polymer- ization reaction and destruction by organisms as a source of energy or carbon. Diurnal and possibly annual cycles in acid concentration exist. Chemically and physically changes in the concentration of these compounds produced changes in pH, conductivity, alkalinity and optical density. The first three may be explained by a hypothetical union between these acids and calcium carbonate while optical density changes are a product Eugene Howard Buck of the characteristic light absorbance properties exhibited by the yellow organic acid molecule. Biologically these acids appeared to stimulate the growth of Navicula sp., Closterium Sp., Arthrodesmus sp. and Surirella sp. while large copepods and cladocera were adversely affected. This study indicates the significant role the yellow organic acids play in the chemical environment and the sub- stantial effects these compounds may exert in the eutrophi- cation process. THE LIMNOLOGICAL AND ECOLOGICAL SIGNIFICANCE OF DISSOLVED YELLOW ORGANIC ACIDS IN NATURAL WATERS BY Eugene Howard Buck A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1968 C.‘ ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. Robert C. Ball, not only for his continual guidance and advice during my work on this problem, but for the many chances to engage in research during my undergraduate work. This experience has been an invaluable part of my education. I also wish to acknowledge the assistance of Tom Hardgrove in the field portion of this problem along with all the other fellow graduate students whose comments were helpful in understanding and interpreting the results of this study. I am further grateful for financial assistance from the National Science Foundation under a National Science Foundation Graduate Fellowship. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 The problem . . . . . . . . . . . . . . . . . . . 2 METHODS. . . . . . . . . . . . . . . . . . . . . . . . 5 Acid recovery . . . . . . . . . . . . . . . . . . 5 [Acid measurement and addition . . . . . . . . . . 19 Temperature . . . . . . . . . . . . . . . . . . . 25 Alkalinity. . . . . . . . . . . . . . . . . . . . 26 Conductivity. . . . . . . . . . . . . . . . . . . 26 Spectrosc0py. . . . . . . . . . . . . . . . . . . 27 pH. . . . . . . . . . . . . . . . . . . . . . . . 27 Light penetration . . . . . . . . . . . . . . . . 28 Oxygen. . . . . . . . . . . . . . . . . . . . . . 51 Metal ions. . . . . . . . . . . . . . . . . . . . 31 Fungi and bacteria aquaria. . . . . . . . 32 Periphyton. . . . . . . . . . . . . . . . . . . 52 Centrifuged plankton. . . . . . . . . . . . . . . 55 Net plankton. . . . . . . . . . . . . . . . . . . 54 Other aquaria . . . . . . . . . . . . . . . . . . 35 Fate of acids in the ponds. . . . . . . . . . . . 55 Environmental acid profiles . . . . . . . . . . . 56 Aquaria contaminants. . . . . . . . . . . 56 RESULTS. . . . . . . . . . . . . . . . . . . . . . . . 58 Acid concentration--aquaria . . . . . . . . . . . 59 Acid concentration--ponds . . . . . . . . . . . . 42 pH—-aquaria . . . . . . . . . . . . . . . . . . . 45 pH--ponds . . . . . . . . . . . . . . . . . . . . 50 Conductivity--aquaria . . . . . . . . . . . . . . 51 Conductivity--ponds . . . . . . . . . . . . . . . 52 Optical density-~aquaria. . . . . . . . . . . . . 57 Optical density--ponds. . . . . . . . . . . . . . 61 Alkalinity--aquaria . . . . . . . . . . . . . . . 61 Alkalinity--ponds . . . . . . . . . . . . . . . . 66 Carbonate. . . . . . . . . . . . . . . . . . 66 Bicarbonate. . . . . . . . . . . . . . . . . 66 Total. . . . . . . . . . . . . . . . . . . . 67 iii TABLE OF CONTENTS - continued Temperature-—aquaria. . . . . . . . . . . . Temperature--ponds. . . . . . . . . . . . . Light penetration--ponds. . . . . . . . . . Oxygen—-ponds . . . . . . . . . . . . . . . Metal ions—-aquaria . . . . . . . . . . . . Metal ions--ponds . . . . . . . . . . . . . Fungi and bacteria--aquaria . . . . . . . . Fungi and bacteria--ponds . . . . . . . . . Centrifuged plankton--ponds . . . . . . . . Centrifuged plankton-~aquaria . . . . . . . Periphyton--ponds . . . . . . . . . . . . . Periphyton-—aquaria . . . . . . . . . . . . Net p1ankton--ponds . . . . . . . . . . . . Net plankton-~aquaria . . . . . . . . . . . Bottom organisms--ponds . . . . . . . . . . Bottom organisms--aquaria . . . . . . . . . Fish--ponds . . . . . . . . . . . . . . . . Fish--aquaria . . . . . . . . . . . . . Fate of the acids in the ponds. . . . . . Conditions in other natural bodies of water Cause of viscosity in the aquaria . . . . . DISCUSSION . . . . . . . . . . . , . . . . . . . Method of acid measurement. . . . . . . . . The origin and fate of the acids. . . . . . Effect of acids on chemical and physical features Effect of acids on biological communities . Implications of this study. . . . . . . . . SUMMARY. . . . . . . . . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . APPENDIX C O O O O O O O O O C O O O O O O O O 0 iv Page 67 67 68 69 7O 7O 7O 71 71 75 75 75 82 82 87 87 87 87 89 95 97 97 101 109 121 123 127 129 151 TABLE 1. 2. 5. LIST OF TABLES Page Summary of the acid addition to pond C. . . . . . 25 Summary of the acid additions to the experimental aquaria . . . . . . . . . . . . . . . . . . . . . 24 Significance of physical factors in the explana- tion of acid change . . . . . . . . . . . . . . . 44 Observed changes in acid concentration in light and dark bottles suspended for three weeks in pond C (Data in ppm.) . . . . . . . . . . . . . . 88 Calculated effects of different sources of acid change as determined by light and dark bottle experiments. (Data in ppm.). . . . . . . . . . . 9O Acid concentration of various selected bodies of water 0 O O O C O C O O O O O O O O O O O O O O O 96 Dynamics of acid change in a pond on a summer day 106 LIST OF FIGURES FIGURE 1. 10. 11. Acid extraction efficiency from natural water as monitored by fluorescence during column flow on Duolite A-4 anion exchange resin. . . . . . . .Relationship between fluorescence of several ~random acid concentrations in distilled water and temperature. . . . . . . . . . . . . . . . . Relationship between fluorometer units and weight of acids on the 5x scale. . . . . . . . . Relationship between fluorometer units on the 10x scale and concentration of acids computed from fluorescence readings on the 5x scale . . . Relationship between gram-calories per centi- meter square per thirty minutes as measured by pyrheliometer to units recorded by the transis- torized light meters . . . . . . . . . . . . . . Acid concentrations of the aquaria through the exper iment O O O O O O O O O O O O O O O O O O 0 Experimental relationship between pH and acid concentration determined in the aquaria. . . . . pH--acid concentration relationship in the indi- vidual aquaria with the theoretical trend of pH superimposed . . . . . . . . . . . . . . . . . . Experimental relationship between conductivity and acid concentration determined in the aquaria ,Conductivity-—acid concentration relationship in the individual aquaria with the theoretical trend in conductivity superimposed . . . . . . . Experimental relationship between Optical dens- ity and acid concentration determined in the aquaria. . . . . . . . . . . . . . . . . . . . . vi Page 14 18 21 50 41 47 49 54 56 60 LIST OF FIGURES — Continued FIGURE 12. Optical density--acid concentration relation- ship in the individual aquaria with the theo- retical trend in optical density superimposed . 15. Bicarbonate alkalinity in the fish aquaria pair during the study period . . . . . . . . . . . . 14. Index values for centrifuged plankton from the ponds . . . . . . . . . . . . . . . . . . . . . 15. Index values for centrifuged plankton from the aquaria . . . . . . . . . . . . . . . . . . . . 16. Index values for the dominant species found in the periphyton in the ponds . . . . . . . . . . 17. Index values for the dominant species found in the periphyton in the aquaria . . . . . . . . . 18. Net plankton index values for the ponds through the summer. . . . . . . . . . . . . . . . . . . 19. Net plankton index values for the designated aquaria pair. . . . . . . . . . . . . . . . . . 20. Acid concentration profiles of three lakes. . . 21. Acid concentration profile of the Clam River. . 22. Diurnal and annual acid cycles in a pond. . . . 25. Revised pH--acid concentration relationship . . .24. Revised conductivity--acid concentration rela- tionShj-p. O O O O O O O O O O O O O O O O O O 0 vii Page 65 65 74 77 79 81 84 86 92 94 108 112 116 INTRODUCT ION Investigators have long known of the distinct communi- ties which exist in highly colored bodies of water. A few have alluded to correlations of color with noticeable eco- logical changes in these systems (Transeau, 1905; Anthony and Hayes, 1964). It is only lately that interest has been directly focused on these colored compounds in an effort to explain their chemical and biological effects upon aquatic ecosystems. Most noteworthy in their attempts at chemical characterization of these compounds have been Shapiro (1957; 1958), Povoledo (1964), Povoledo and Gerletti (1964). Christman and Ghassemi (1966a;b) and Christman and Minear (1967). Shapiro (1964), Kent and Hooper (1965) and Christman (1967) have studied the interaction of these compounds with metallic ions in water while Anthony and Hayes (1964) have found color to be significant in quantitative expressions of bacterial standing crop. Qualitatively these colored molecules are a diverse mixture of acidic phenolic residues and multipolymeric chains of such units. Being so heterogeneous it is not pos— sible to set forth any definite chemical structure. Their derivation is probably from plant debris decomposition in the surrounding soil and within the aquatic system. It is the universality of distribution, the prominence of occurrence and the possible ecological significance of these compounds which influenced the choice of this problem. The problem It was decided that a general investigation into the ecological and limnological significance of these compounds could best be conducted with a field study involving the application of these acids to a natural ecosystem supple- mented by laboratory studies of aquaria containing the several distinct communities found in this system. The ob- jectives were to develop methods for the study of and to study the possible changes in the physical and chemical environment upon acid addition and to attempt to relate these changes to any ecological modifications. Quantifica- tion of these changes would also be attempted. The field work was conducted on two ponds, one experi- mental and one control, located at the Michigan State Univer- sity Agriculture Experiment Station at Lake City, Michigan. These ponds will be further designated as C, the experimental with a surface area of 0.17 acres, and D, the control with a surface area of 0.18 acres. Both ponds had an average depth of 5 to 4 feet and were situated adjacent to each other with a 10 to 15 foot wide dike as separation. They both had inlets from a common holding pond and so were prob- ably as similar in chemistry and biology as could be found for this purpose . Each pond was provided with connections to a constant monitoring system in the laboratory where temperature, oxygen and pH could be continuously recorded. Either pond could be connected to this system and the changeover be- tween ponds could be accomplished in 5 minutes. A Little Giant submersible pump, model 5E—12NR, was placed two feet beneath the surface near the center of each pond on a sub- merged screened platform and connected by i” I.D. tygon tub- ing buried three inches underground leading to the labora- tory° Water was forced into the laboratory at a rate of 670 gallons per hour where it flowed into a Sé-inch diameter cylindrical plexiglas monitoring site and came in contact with the measuring electrodes inserted through Specially constructed ports. Water left the laboratory and returned to the pond of origin along similar tubing. The aquaria studies were conducted in adjacent labora- tory facilities using water from pond D. Aquaria were set up with different community structure including bottom organisms, zooplankton, phytOplankton, periphyton, fungi and bacteria, and fish. Each aquaria was illuminated with a bank of two fluorescent lights about one foot above the water surface. Water lost by evaporation was replaced with dis- tilled water each week. Several independent experiments were planned and carried out in the effort to learn more of the mechanism of acid accumulation and disappearance from natural waters. Several lakes and a stream were also sampled to yield in- formation on the range and variability of acid occurrence in various environments. METHODS Acid recovery In the initial search for a readily available and easily recoverable organic compound to work with, I used a modification of the separation procedure outlined by Aronoff et al. (1947). This involved passing filtered lake water through first a column of Duolite C-5 cation exchange resin followed by passage through Duolite A-4 anion exchanger. After finding the most abundant organic sub- stances were the colored organic acids removed by the anion exchange column, the extraction process was considerably shortened. The final procedure as was used during the field work consisted of a 55 gallon oil drum with a plastic liner elevated atop a three foot stand to serve as a reservoir for gravity flow to the resin columns. This apparatus was located ten feet from the holding pond which served ponds C and D and was filled twice daily by either a Homelite gasoline pump or a Little Giant submersible pump. Water was siphoned out of the barrel through tygon i-inch I.D. tubing to porcelain filters holding glass wool at the level of the base of the barrel. The rate of flow was regulated in the tubing by adjustable hose clamps and the greater portion of the suspended particulate matter was removed by the filters. The glass wool was removed and replaced when- ever the filteration capacity was lowered appreciably so as to obstruct maximum column flow due to the accumulated material in the filter. The filtrate then flowed into the top of the resin column, passed through the column and discharged into the ground. A maximum of three columns with separate filters were maintained in operation during the summer. By adjust- ment for maximum flow, the columns were kept in operation around the clock. A total of 105 column-days (one column- day is equal to one column in Operation for one day) was the actual operation time of the apparatus. By comparison of fluorescence of the water in the barrel reservoir to that of the column effluent, these columns averaged 82% efficiency in the removal of acids from the water during the summer. Columns were removed from the barrel when this efficiency dropped in the range of 75% recovery which averaged about 15 days flow time with variation dependent upon flow rate through the column. The general pattern of acid recovery on the column is shown in Figure 1. By comparison with the breakthrough patterns of this type of resin (Duolite Ion Exchange Manual) the resin is filled to approximately 85% of exchange capacity. The columns were constructed of discarded water deionizer columns. The top was cut off just below the cap, the old resin removed and the plastic tube washed. As the .cammu mmCMSUxm coacm and muflaoso co 30am GESHOU mCHHDU mocmowmuosam ma UmHODACOE mm swam? amusumc Eoum mommaoflmmm :OHuomnuxm UHU< .fi musmflm 0 xx .19 u at 8 -° 0! X a: x K ' O X .70 x " a l -o x b 8 at” * X qg x I " x" O x ‘10 xx“ K x O X X ‘q. I 3“ O at ‘ qn “I X 0 ’0‘ at _N K x " .. -9 '0‘ x /I 1 1 1 J 1 O O O O O O O o 8 8 u. on 0 ¢ n NNO‘IOO 3H1 AB GENIVIEU 39N30538001$ 'IVIOJ. :10 ”030 83c! % OF TOTAL OPERATION TIME OF COLUMN F IGURE 1 A-4 anion exchanger was received from the company as a dry resin, it was necessary that it be soaked overnight in water before packing the columns. During this soaking the resin expanded 12% by volume. In the morning the water- resin slurry was poured into the column until about 50 cubic inches or 800 cubic centimeters of resin had been added. A water flow was then introduced at the base of the column which forced any trapped air out and arranged the resin particles within the column by size. Upon draining the water to within one inch of the surface of the resin, the column was ready for conditioning (Duolite Data Leaflet No. 5). First, two bed-volumes (about 1600 milliliters) of 1.5 N sodium hydroxide were introduced at the top of the column and the flow regulated for a passage time through the column of ten minutes. Next the column was rinsed with five bed- volumes of distilled water at the same flow rate. Then two bed-volumes of 2 N hydrochloric acid were passed through the column and it was rinsed again with five bed-volumes of distilled water. This entire cycle was repeated once more and was followed by a rinse with two bed-volumes of 95% ethanol to remove any non-polar impurities. After a final wash with five bed-volumes of distilled water, the column was ready for use. It was clamped into position beneath the filter of the collection apparatus and water was allowed to enter and flow through the column. 10 When the rate of acid extraction had decreased to the 75% efficiency range, the column was removed from the apparatus and taken into the laboratory for acid recovery and processing. The resin was removed from the column, placed in a large glass cylinder and one liter of 2 N sodium hydroxide was added. This mixture was stirred from time to time and allowed to stand for four to six hours. At this time the supernatant was poured off into an enamel pan and concentrated hydrochloric acid was stirred in until a distinct lightening of color occurred. This happened when the solution became acidic and the free acids were re- leased. The acidified supernatant was then evaporated to dryness in a drying oven held at a constant temperature of 56°C. .At this temperature no decomposition of the acid molecule should have occurred (Shapiro, 1957). Another liter of 2 N sodium hydroxide was added to the resin in the glass cylinder and this process repeated until little observable color could be extracted from the resin. When the combined extracts were completely dry, 100 ml of 95% ethanol were added to the pan. The mixture was stirred for maximum color extraction and this supernatant decanted into a small glass cylinder. This process was re- peated several times until only the white sodium chloride residue remained in the pan. The combined ethanol extracts were evaporated to dryness and extraction repeated on this dry residue with 95% ethanol until all color was removed and 11 only a white sodium chloride residue remained. The final product was a dark brown waxy amorphous substance with a penetrating odor similar to vanillin. This was then dissolved in 95% ethanol and stored in a refrigerator at 4OOF. in as saturated a solution as could be made, or a maximum of about 15 grams per 100 ml 95% ethanol. The observation by Shapiro (1957) of the intense fluor- escence produced by these compounds led to the attempt at quantitative measurement by fluorometric methods. Spectro- fluorometric analysis by Dr. Robert E. Phillips of G. K. Turner Associates found an intense fluorescence peaking at 4700 R with the maximum wavelength of activation at about 5550 A. This compares favorably with the fluorescence measured for organic color in water by Christman and Ghassemi (1966a;b). The emission peak varied with excitation wave- length which characterizes a heterogeneous molecular mixture. From these data it was decided to use the Turner Filter Fluorometer Model 111 with the primary filter #7-60 and the secondary filter combination of #2A and #48. The excitation by the mercury line at 5660 A with filter #7-60 was thought adequate for these compounds (R. E. Phillips, personal com- munication). .All readings were of samples in the standard 12 x 75 mm, 8 cc Pyrex cuvettes with sample fluorescence being recorded immediately when the maximum dial reading was reached, usually within 15 seconds. 12 When this combination was tried it was found to give variable results. Several physical and chemical factors were investigated for possible interference. Temperature was known to effect fluorescence (Udenfriend, 1962) so this was studied first. Erlenmeyer flasks were prepared with 100 ml of distilled water and several levels of acid concentration. Fluorescence was measured in arbitrary units as marked on the fluorometer while the temperature was adjusted through the range of 00 to 400 C by the application of ice or heat. .A standard of distilled water was used and a zero level was set as five units so that fluctuations of the standard could be more easily noticed and adjusted. The resultant plots are seen in Figure 2. From these it can be calculated that fluor- escence decreases 0.61% per degree Centigrade temperature increase. The expression for fluorescence corrected to 560 C., the temperature standard used in this study, is therefore: Fluorescence units = Fluorescence Zero 1 - 0.0061 (56.0 corrected to 56 C. units read — point - temperature) Christman and Ghassemi (1966a;b) report a direct re- lationship between pH and color intensity. This change in color was utilized in the preparation procedure to visually approximate acidification of the extracts, but closer in— vestigation showed that the variation in pH seen to affect color would not affect fluorescence to anywhere as 15 .musumummamu cam Hmpm3 pmdaaumflo CH mGOHumuucmocoo Uflom Eoocmu Hmum>mm mo mocmowmnosam cooBqu mflnmCOHpmHmm .N musmflm 14 g I5 1 I l l 30 35 4O 45 50 TEMPERATURE ’C 1 20 1 IO v n n N N - BTVOS XE NO ‘51an 83137'080015 AHVUIIGHV FIGURE 2 15 significant a degree under the conditions found in natural waters. In purified lignin solutions, Christman and Minear (1967) found that fluorescence was significantly decreased in both acid (pH less than 4.0) and alkaline solutions (pH above 7.0). When whole wastes were analyzed, little variation in fluorescence was found between pH 4.0 and pH 10.0 for low concentrations of the measured material. The pH effect does not appear significant enough in the range of natural waters to warrant correction of the measured values. This last mentioned study also found chloride ion con- centrations of up to 20,000 milligrams per liter had little effect upon fluorescence intensity. Fluorometer units, corrected to a standard 560 C., were then used to establish a quantitative measurement for the dissolved yellow organic acids. One-tenth of a milliliter was chosen for the basic volume measure for convenience. From the supply of concentrated acids in solution, 0.1 ml was diluted to 15 ml with distilled water. This dilution was read on the fluorometer, corrected to 560 C. and the arbitrary units designated as the fluorometer units of acids in 0.1 ml of the dilute solution. The total number of units in the diluted 15 ml solution would then be equal to the number of units originally in 0.1 ml of the concentrated stock solution or a dilution factor of 150. A powder paper was weighed on a Mettler analytical balance model H6T to 0.1 milligram accuracy and 10 ml of the 16 stock solution was placed in a depression formed by this paper in a plastic petrie dish. This was dried 24 hours at 560 C. in a drying oven. At that time the paper with the acid residue was weighed again. A blank of distilled water and three concentrations of acid in a wide range were measured on the fluorometer. The weight of the paper ini- tially was subtracted from the dried weight to obtain the uncorrected acid weight. This weight was then corrected for the change in the blank during drying resulting in the weight of the dried acids. The relationship between the total number of fluorometer units in 10 ml of concentrated acid and the weight of this quantity was then known allowing a standard curve to be drawn (Figure 5). The result was an average of 17020 fluorometer units per milligram of dried acids. The expression then for converting arbitrary fluorometer units previously corrected to 560 C. and measured on the 5x scale of the Turner model 111 fluor- ometer with the above stated filter combination into milli- grams per liter (ppm) of yellow organic acids dissolved in water is: . . (10000) (fluorgmeter units corrected Milligrams per = to 56 C. in 0.1 ml) liter of acids 17020 fluorometer units per milligram acids The factor of 10000 is necessary to convert the numerator to fluorometer units in one liter. Since the limit of sensitiv— ity on the fluorometer is one-half an arbitrary fluorometer Unit, sensitivity in this range is.i 0.5 ppm. 17 .mamom xm on» so mUHUm Mo uanOB cam mafia: umumfiouosam cmw3uon QHSmGOHumHOm .m muzmflm 18 l4x Io‘r I2 1: IO6 - O .92 01 1 I I 1 1 0 CD 00 CD 2 9 ”2 2 2 I K X X X 0 on on v- N 031038800 BTVOS XS NO suun UBIBNOHOITIJ 300 400 500 600 700 800 900 I000 MG ACID DISSOLVED IN IO ML ETHANOL 200 I00 FIGURE 5 17 .mamom xm map so moaom mo unmflwB paw muflc: Hmumeouosam cmwBDmQ QHSmCOHumHmm .m musmflm 18 Numer- nanos- ones!- amas- GHOS- 4xlos- 2xl06- 0 .92 01. 031038800 31VOS XE NO SLINn 8313NO8OI‘I'IJ 300 400 500 600 700 800 900 I000 IN IO ML ETHANOL 200 l00 MG ACID DISSOLVED FIGURE 5 19 By relating the readings on the other scales, 10x and 50x, to the 5x scale we can develop a conversion factor for these ranges. Temperature must still be corrected for in the same manner before conversion from fluorometer units to ppm. On the 10x scale the regression between ppm figured from 5x scale readings and the 10x scale fluorometer units (Figure 4) yields the expression: . . (10000) (fluoromeger units corrected M1lligrams per to 56 C.) liter acids = 64680 fluorometer units per milli- gram on 10x scale N = 15 r = 0.997 Sensitivity = i.0.08 ppm On the 50x scale the conversion likewise becomes: (10000) (fluorometer units corrected Milligrams per to 56 C.) liter acids = 185540 fluorometer units per milli- gram on 50x scale N = 6 r = 0.999 Sensitivity = 1.0.05 ppm or 50 ppb Acid measurement and addition Four water samples from each pond were taken twice weekly with a one liter Kemmerer water sampler at lfi-feet depth at randomly selected sampling points. These samples were placed in 120 ml polyethylene bottles and stored in a drying oven set at 560 C. to minimize possible error from temperature changes at the time of reading. When the water samples had reached the vicinity of 560 C., after about five hours in the oven, they were removed one at a time, their temperature recorded and a 5 ml aliquot transferred to the 20 .mamum xm on» no mmCHUmmH mocoommnosam Eoum Umusmaoo mnflom mo coapmupcmocoo paw wamom xoa map so muflcz HmumEouosam cmoBqu mangOHumamm .w musmam 21 1 1 x x d 4 1 - 4 I x x x }. d L_ 1 1 1 1 1 1 O O O O O O O O 2 01 O h 09 I0 1' n 0 .9E 01 031038800 31'03 XOI N0 SilNI'I 8313W0800'H I2 I4 I6 IO 1 MILLIGRAMS ACID PER LITER WATER (ppm) FIGURE 4 20 .mamum xm mnu so mmCHpmmH mocmowmnosam Eonm Umusmfioo wflflum mo coaumuucmucou Ucm mamom xoa mnu co muflcn Hmumaouozam cmmBqu QHLmCOHumamm .w musmflm 21 g/ l l l l I l o o o o o o a» o h o u) q- 'OOF 0.99 01 031038800 31V0$ XOI NO sunn 831351080013 30 I2 I4 I6 MILLIGRAMS ACID PER LITER WATER (ppm) IO FIGURE 4- 22 fluorometer cuvette. The Turner Filter Fluorometer model .111 with primary filter #7-60 and secondary filter combi— nation #2A and #48 was used on the 5x sensitivity scale for all normal measurements. Other scales were used in— frequently when the range of acid concentration made a dif- ferent scale more applicable. A distilled water blank was used to set a zero point at five fluorometer units and this calibration was rechecked between each sample and the next. 1A 120 ml polyethylene bottle was filled at mid-depth in each aquaria twice weekly and the identical procedure followed with these samples. Purified acids extracted from the local water source were added to pond C three times during the summer (Table 1). These acids were dissolved in 95% ethanol and distributed over the surface of the pond from the stern of a moving rowboat. The acid solution was added dropwise-and mixed by the action of the rowboat. Only on the third acid addition was an equal volume of ethanol added to the control pond. .Acids were added to the experimental aquaria four times during the summer (Table 2). One to three milliliters (depending on the aquaria volume) of the concentrated stock solution of acids were added at the water surface and mixed thoroughly. The aim was to raise the level of acid concen- tration several ppm each time although some of the aquaria were increased as much as 20 ppm after certain additions. An equal volume of ethanol was always added to the control aquaria at the same time. 25 Table 1. Summary of the acid additions to pond C ppm theoretical Date Time g acid m1 ethanol acid increase July 21 11:15 AM 5.91 109 0.008 July 28 8:00 PM 11.25 595 0.016 August 50 10:00 AM 70.46 1000 0.10 Table 2. Summary of 24 the acid additions to the experimental aquaria ppm acid con- centration Date‘ Time 9 acid ml ethanol increase July 26 10 AM - PM .045 - .100 1 - 5 5.0 — 5.5 August 4 2 PM - PM .110 - .115 1.9 - 2 5.7 — 6.9 August 10 11 AM - PM .144 2 4.8 - 9.0 August 17 .11 AM - PM .517 2 10.0 -20.0 25 Analysis of variance was performed on the pond data to determine if the acid additions in pond C were detectable. One set of samples was collected every hour for 25 hours and was tested by one-way analysis of variance for significant changes during a diurnal cycle. An attempt was made to analyze changes in the ponds attributable to physical factors such as rain, temperature, sunlight, etc. -Aquaria measurements were analyzed by computer for significant relationships with other chemical factors. Temperature Pond D was continuously monitored with a Taylor record— ing water thermograph with the sensing probe at a two foot depth. This probe was shaded so solar heating would not affect the readings. Both ponds C and D were measured by the monitoring system in the laboratory when that pond was connected to the system. Here temperature was measured with a Yellow Springs Instrument Tale-thermometer using a therm- istor probe and continuously recorded on the YSI model 80 laboratory recorder. Standardization of the instruments was checked every week. Temperatures of the aquaria were measured irregularly with a mercury thermometer. The col- lected data were analyzed to determine if the addition of these acids had any effect on the water temperature or the rate of heat exchange of the body of water. 26 Alkalinity Carbonate and bicarbonate alkalinity were measured us— ing the method described by Welch (1948). Commercially prepared and standardized N/50 sulfuric acid was used in all titrations. Duplicate samples were taken twice weekly from randomly selected sampling sites in both ponds with a one liter Kemmerer water sampler at a depth of lfi-feet. The water was transferred to 120 ml polyethylene bottles and titrated immediately on return to the laboratory. Analyses were performed on the differences in carbonate and bicarbonate between the ponds to determine if acid addition significantly affected this system. One set of aquaria, that with fish present, was ob- served for alkalinity changes. A 100 ml volumetric pipette was used to withdraw a sample at mid-depth twice weekly which was then transferred to a beaker and titrated. The change in bicarbonate (the only form of alkalinity recorded in the aquaria) between experimental and control was followed through increasing acid concentrations. Conductivity The water samples collected for acid concentration measurement were also used for conductivity measurements. Eighty milliliters of the water sample were placed in a 100 ml graduated cylinder and the probe of an Industrial Instru- ments type RC conductivity bridge inserted. The temperature of the sample, scale reading in micromhos per centimeter, 27 scale multiplier and probe constant were recorded and all Ineasurements were adjusted to micromhos per centimeter at 560 C. The temperature coefficient for the water from the ponds was experimentally determined to be 0.0157 per degree Centigrade or an increase in conductivity of 1.57% per degree Centigrade temperature increase. Aquaria samples were processed in the same manner as those from the ponds and all data were analyzed in the same manner as the acid concentration data. Spectrosc0py The water samples used for conductivity and acid concen- tration were also used for spectrosc0pic-0ptical density measurements. Ten ml of the sample were placed in the cuvette and optical density was read at 550 mu on a Bausch and Lomb Spectronic 20. Aquaria samples were treated in a like manner. Analysis of the data was in the same manner as the acid concentration data. pH Measurement of hydrogen ion concentration of the ponds was conducted in the laboratory at the monitoring site with .a Beckman EXpanded Scale pH Meter equipped with automatic temperature compensation. The first half of the summer no recorder was available so irregular readings were made during the day. During the later summer a Sargent Recorder Model SR was used and a continuous pH record made during the day. 28 Recording was done at a chart speed Of twelve inches per hour and a chart span from pH 5.0 to pH 10.0. pH was standardized once a week. Aquaria samples were taken and measured by the same instrument once a week. Analysis Of variance was performed on the pond data to determine if any significant changes in pH occurred which might have resulted from the acid additions. Aquaria measurements were used to define the relationship between chemical changes in the environment. Light penetration A Bristol pyrheliometer with chart recorder and digital read-out was in Operation each day from 4 AM to 8 PM tO measure the solar energy. The pyrheliometer bulb was lo- cated between the two ponds and about two feet above ground level. Also in Operation were two transistorized light sensing meters. One was positioned adjacent to the pyrheli— ometer bulb while the other was mounted at one foot beneath the water surface in one Of the ponds. This sensor was moved between ponds every three tO four days. A calibration run was made for both sensing meters against the standardized pyrheliometer so that the meter units could be converted intO gram-calories per centimeter square per time period. A time period Of thirty minutes was chosen for convenience (Figure 5). Using these conversions, the meter readings every day were expressed as gram—calories per centimeter square per thirty minutes and the percent Of 29 .mumumfi unmfia OONHHOumHmcmHu map ma OOOHOOOH muflcs Ou Hmumfioflamnnwm >9 Owusmmmfi mm mouscHE muuanu Mom mnmsqm umumE Iflucmo “on mOHuOHMOIEmum COOBDOQ mflnchAumHmm .m musmflm 50 OOON OONN OO 1 q — O._. ._.z mo III I U I 2 O 30 . o O I a 20 - a I l The pain" IndicaTe IndIvIduaI IOI ODOOIVOIIOI|0 while The line was coaeTruCTed Item The derlved “9"" 3° eauoTIOTI. Q 1 1 1 1 1 7.0 7.0 7.0 0. 2 0. 6 9 0 9H 70 :50. PERIPHYTON AQUARIA C E 2 50 Mean calcuIaTed pHi . 0.l7 2 ’5 e: 40 p. z u g 30 t l O u I 9 20- The pain" IndicOTe u I ind-vidual observer ( 5: T'Ione ehIle The line .0 b was coneTIucTed from The declved dquahon. Figuve 0e 0 A L L l 7.0 7.4 7.0 0.2 0.0 9.0 p” ’OI I ‘0!» IACTERIA AND FUNOI , AOIIAIIIA g 90- Mean caleuleTed 9H5 - e.oe .- 1 25 em 8 I» u 3 so . u I ‘3 20 E TIT. peInTe IedIcaTe 3 leelvlduel ebeerve- Tleae ehIle The line l0 - ' wee ceeelrueled Iran The deTIved F Iowa 80 .qucnfl.’ o 1 I I 1 A I 1 I 7.0 7.4 T. a 0.2 0.0 9 0 ,H E 70r- O Q 2 50.. PHYTOPLANKTON AOUARIA z 0 so I- : Mean calculated OHII 8.00 a I: p. 240” U U 2 ‘ ‘ I O 30 I. I.) O C 20 P a I l0 _ The pomTe IndIcaTe indIv-dual observanone while The line was consTrucTed Item The derived Figure ea eauOTlon. O 1 1 1 1 J 7.0 7,4 7.8 8.2 0.6 9.0 ON TOP £50. FISH AOUARIA B 2 z 50‘ Mean calculaTed pHi I 7.96 9 g. a 40- I: 5.. 2 U u SCI- 2 o u o 20*- 5 ‘1 The pom" IadicoTe Individual TOE observaTIoae while The line was cenenucTed TIOIII The «and “0"" 0T eauaTien. O 1 1 1 1 . 20 24 7.0 0.2 0.0 90 ”N F IGURE 8 50 theoretical line, pHi was taken as the average Of individual pHi's computed from each acid concentration-pH data set for that aquaria. pH-—ponds -Three-way analysis Of variance was performed on the pH data from the ponds divided by ponds (C and D), dates (before the first acid addition, between the first and second additions, between the second and third additions, after the third and last addition) and times (early morning, late morning, early afternoon, late afternoon) with a total Of 204 pH readings (see Appendix, Tables 6 and 7). A significant difference in times reflects the increase in pH through the day as carbon dioxide is removed from the water during photosynthesis. A significant interaction be- tween dates and ponds is a sign that the pH change was not the same in both ponds during the summer. Upon inSpection this interaction shows the pH in pond C increased through the summer as pond D decreased as compared tO expected values generated for each sampling period under the assumption that the effects Of time and Of pond upon pH were additive. As the mean pH Of pond C was 8.72, thus above the pH Of 8.02, this pond may have reacted in the manner predicted by the equation for pH change derived from the aquaria experiment (see page 45 and Figure 7--pH increases with an increase in acid concentration in waters with a pH above 8.02). 51 Conductivity--aquaria - The possible factors included for the explanation Of changes in conductivity were acid concentration, pH, acid concentration squared, the cross product Of acid concen- tration and conductivity, the cross product Of acid concen- tration and pH, the pH at the start Of the experiment, the acid concentration at the start of the experiment and the conductivity at the start Of the experiment. The parameter acid concentration squared was included tO incorporate changes which might have occurred if acid concentration affected conductivity through some intermediate mechanism and was thus magnified in effect. After solving the regres- sion equation, the remaining factors, all Of which had a probability Of greater than 99% significance in the explana— tion Of conductivity changes, were acid concentration, conductivity at the start Of the experiment, pH, the cross product Of acid concentration with conductivity and a con- stant. This equation was transformed so that conductivity at the start Of the experiment would be replaced by a factor equal to the theoretical conductivity when the acid concen- tration was 0.0 ppm and the entire expression simplified: Conduct;v:;y t -5.2814 (acid concentration) + C1 m$crom 0 cm a 1 - 0.02005 (acid concentration) 56 c.) where Ci (initial conductivity) indicates the theoretical conductivity when acid concentration is 0.0 ppm. This 52 expression was derived from measurements over the range Of acid concentration from 10.5 to 50.2 ppm and range of con- ductivity from 149 to 251 micromhos/cm at 560 C. (see Appendix, Table 8). In general this expression indicates a decrease in con- ductivity with increasing acid concentration below an initial conductivity (Ci) Of 164 micromhos/cm and an increase in conductivity with increasing acid concentration above this initial conductivity. A discontinuity exists around 50 ppm acids with the relationship Of conductivity tO acid concen- tration reversed at higher concentration levels (Figure 9). The transformed equation does not include pH since the re— gression coefficient Of this parameter approached the limit Of zero when Ci became an expression Of the theoretical conductivity when acid concentration equals 0.0 ppm. The individual experimental aquaria with the theoretical trend in conductivity change superimposed are presented in Figure 10. For this theoretical line, Ci was taken as the mean Of individual Ci's computed from each acid concentration- conductivity data pair for that aquaria since each aquaria would be expected tO have a characteristic Ci that could be found only by this method Of back-calculation. Conductivity--ponds Two-way analysis Of variance was performed on the con- ductivity data from the ponds with these data divided into 29 times (the roughly twice-weekly sampling times) and 55 .manmzvm 030 CH OmcflEHmumO coaumupcmocoo Oflum Ocm hufl>fluuswcoo OODBDDQ mwnmcoflumamu Hapcmeflummxm .m musmflm 54 m MMDOH .m “Down ._.< 20\ 00:20:02); >._._>_.PQDQZOO OON Om. Ow. ON. 00. On. 03 On. ON. 0: q d d .Eaaod m. coZchoocoo Boo cog: EoBOcE 08.5 om. Lo :2: x 19.6.30 0 055.53 cozotcoocoo Boo 053805 at: it, .2323 E OococOx .52. 0.0 2 529.2330 I G q 1 .Eaa 0.0 m. cozotceucoo x Boo cog: EuxmochLEE o! co 3.38228 a 0563.3 5282.3 Icoo BOO 95830:. 5:; x 53:02:50 S Oocozo 7‘ ‘11—,“ A A v‘ Co 33:05:00 O ofegmo cozozcoucoo Boo 053305 .2... £332.50 5 m m x O m x BOO cos; EuxmocfioLoE. om. .x x A x Oococo I o o. n. ON mm on mm NOI1V81N30N00 0IOV uIdd NI Figure 10. 55 Conductivity-acid concentration relationship in the individual aquaria with the theoretical trend in conductivity superimposed. Fig. 10a Fig. 10b Fig. 10c Fig. 10d Fig. 10e Fig. 10f ZOOplankton aquaria Bacteria and fungi aquaria Bottom organism aquaria Phytoplankton aquaria Periphyton aquaria Fish aquaria ACID CONCENTRATION ACID CONCENTRATION ACID CONCENTRATION 56 TO- 60- ZOOPLANKTON AQUARIA ”I“ Mean COICulaTed c.- no.3 «I ROI 1 l l ”I- ‘ . The eeihTe ledlceTe Indivldud To. eeeevveTthe ehIIe The llhe Dee ceheTTueTed he. The n..." .0. derlved eeueTlOR. I I I J T40 l00 T00 200 220 240 CONDUCTIVTTY IMICROMNOCICM AT SC'CI TOP ”I BOTTOM ORGANISM AQUARTA ”I Neon ceTeuleTed CI - 102.0 ADI- ' I 3°» 20. The eeleTe leeTeeTe w "I" eheeneTleee ehlle The The eee eeeeTreeTed he. The ”e". .0. defleed eeeeTTee. l A j # 140 T60 T00 200 220 240 CONDUCTIVITY IIICROINOO/CM AT “‘3 70- .0. PERIPHYTON AOUARTA °°’ ‘ I ' M000 eeleuIeTed CI - I000 .I ”I l x 20- ' The eehTe mm Ihdlvldud '0, ' , monum- eTIIM III. me wee eeeeTreeTed Toe-I The declved eeueTTeh. figure lOe n A A 41 1 'Mo l00 Ieo 200 220 240 CoedecTIvITy TIICIOTATTOSI cu AT 36%) 70F 2:0» BACTERIA AND PUNGI AOUARIA O E , 5°” Mean calculaTed C. - ITT.O 2 " I 2%- p. a 8 3 DOI- O U 9. tOI- 3 The poem IndicaTe Tedmdual .0. ‘ I ObserveTIaIe-Nle The line wee ceheTIuCTed Iran The “90'. TOT: derlved eauaTlen. (I I 1 1 1 TOO ICO I00 200 220 24C CONDUCTIVITT (MICROHTOSICM AT 36'C! T : PHYTOPLANKTON AOUARIA O E a ‘0’ Mean calcuIaTed CI . I66.0 .- t I- «- I 3 . . a a 30*- o o I 9 lol- I o n ‘ . The pelhTe IhdIceTe IhdIvIdueI Io. eheetveTIeee ehlle The line see eeReTTuCTed Item The o “9“" '0‘ deTlved eeueTlee. MO I00 IDO ZOO 220 240 CONDUCTIVITY (MICROMNOSICM AT 30' I 'OI T C O t I ACIO CONCENTRATION III ”In " 0 O V V 5 f 1) MO FISN AOUARIA Neeh calculeTed c. I I061 The noun IncheTe TRdTvIdueT eheerveTIeee while The The nee ceaeTTuCTed Tre- The TI . mm. '0' detleed eeue ee I00 200 220 CONDUCTIVITY (NICNONTIOSICN AT 00%) 1 I00 240 FIGURE 10 57 2 ponds (C and D) with a total Of 504 samples taken (see Appendix, Tables 9 and 10). A significant difference in times shows that conductiv- ity did not remain constant but changed (generally increased) through the summer in both ponds. Pond D (;'= 158.7) was significantly higher in conductivity than pond C (;'= 157.0). The significant interaction between ponds and times shows the conductivity change tO be different between the ponds during the sampling period. In this case, the conductivity in pond C decreased through the summer as pond D comparatively increased. The first acid addition was followed by a rise in conductivity in pond C with a gradual return to pre— addition levels. The second addition was also followed by an initial rise but soon conductivity decreased markedly. This conforms to the theoretical prediction from the aquaria results as the conductivity was below the 164 microth/cm value in pond C. At the third acid addition the conductivity Of pond C was slightly below 164 microth/cm and, as would be predicted, conductivity decreased very little even though more acid was added at this time than at the second addition. Optical density—-aquaria The possible factors included for the explanation Of changes in Optical density were acid concentration, pH, acid concentration squared, the cross product Of acid concentration and conductivity, the cross product Of acid concentration and pH, the cross product Of acid concentration and Optical 58 density, the Optical density at the start Of the experiment, the pH at the start Of the experiment, the acid concentra- tion at the start Of the experiment and the conductivity at the start Of the experiment. The parameter acid concentra- tion squared was included to incorporate changes which might have occurred if acid concentration affected Optical density through some intermediate mechanism and was thus magnified in effect. The regression solution was a compli- cated expression Of many factors showing that Optical density depends upon many different environmental conditions. Those parameters having a significance Of less than 0.01 were acid concentration, acid concentration squared, the cross product Of acid concentration with conductivity and the cross product Of acid concentration with optical density. Initial pH and initial acid concentration had a significance between 0.01 (and 0.05. The simplified expression became: 0.0046 (ppm acid) - 0.00017 (ppm acid)2 Optical density _ + 0.0000066 (ppm acid) x (conductivity) at 550 mu ‘ 1.0 - 0.0509 (ppm acid) over a range Of acid concentration Of 10.5 to 50.2 ppm, conductivity Of 149 to 251 micromhos/cm and Optical density Of 0.0511 to 0.2599 (see Appendix, Table 11). In general this expression shows increasing Optical density with an increase in acid concentration (Figure 11). Discontinuities exist in the regions Of 52 and 50 ppm acids. The individual experimental aquaria data with the theoretical trend in Optical density superimposed are presented in 59 .mflumsvm any CH Omcflaumumv coaumuucmocoo Uflom 0cm muflmcmv HOOHDQO QDDBDDQ mflnmcoflumamu Hmucmeaummxm .aa musmflm 60 .3“ mngh 3:03 2.4 5.523 .36.th ONO 9.0 3.0 0.6 00.0 No.0 u - d .6 an 0.0 a. cotatcoocoo Boa cons Eu \ 3:59.26 00. .6 33:03:00 6 9:63.30 3:323:09 Boo 9:39.05 :23 3.2.3 .6230 E «9.25 K x x\.Eaa 0.0 a. x\ 3:223:00 x\ Boo 5...: Eu \ 32.6326 00. .0 x x\ 23:02:30 a 9:833 co...o.;coucoo Boo 9:39.05 5.... 3.2.3 .323 c. eococo x\x d d e .52. 0.0 a. cozozcoocoo BOO so...) 60:29:22.: O! 1 .0 33:02:30 0 0563...... 5.335080 Boo 95332: 5.3 3.2.2. .323 509.210 o n V m o. a o O m n. 3 N I. a w. on m N m 3 u m on an 61 Figure 12. The same mean Ci used in the computation Of conductivity for each aquaria was used here in determining the conductivity values. Optical density--ponds Two-way analysis Of variance was performed on the Optical density data from the ponds as they were originally recorded--percent transmission. These data were divided into 29 sampling times and 2 ponds with a total Of 296 samples taken after the elimination Of those samples contain- ing visually large amounts Of suSpended plant material (see Appendix, Tables 12 and 15). Pond D had a significantly lower percent transmission mean (88.87) than did pond C (89.75). Both ponds showed significant changes (decreasing to mid-summer, then increas- ing) during the sampling period. The significant interaction again showed a difference between the way both ponds changed through the summer. The percent transmission Of pond C decreased during the summer as pond D comparatively in- creased. Such is as would be predicted from the theoretical model with increasing acid concentration. .Alkalinity--aquaria Methyl orange (bicarbonate) alkalinity was the only form present in the aquaria. Not enough data was collected for regression analysis although measurements from the fish aquaria pair indicate a decrease in bicarbonate alkalinity as acid concentration increases (Figure 15). -Figure 12. 62 Optical density-acid concentration relationship in the individual aquaria with the theoretical trend in Fig. 12a Fig. 12b Fig. 12c Fig. 12d Fig. 12e Fig. 12f Optical density superimposed. - ZOOplankton aquaria - Bacteria and fungi aquaria - Bottom organism aquaria - Phytoplankton aquaria - Periphyton aquaria - Fish aquaria 65 TO ‘00 ZOOPLANNTON AOUARIA NeeII COICIITOTed c, -l70.3 E 00 5 40+ 1. If. owl g 1 L 1 I e to I. o ‘ The pain" menu" IhdIvIdudl IO» ObeerveTIOhe INTI. The ”no eee COMTrucTed new The Flame ‘20 derIved equeTIOn. A I A A 4 I A I O 0.06 on 0.10 0.24 0.30 OPTICAL DENSITY AT 330 my 70 - E 6 ‘50. BOTTOM ORGANISM AQUARIA 5 Mean calcalaTed C. - was a 50 P 9 .- : a U l ‘2’ .wI , u l 1:: am: ' a l The palIITe ladICOTe Indiudual l0 . eDheTvaTlone ehITe The line eee coneTIucTed Tram The Flour: l2: denved eauaTIOII. A; I I J_ A 1 A J J I O 0.06 OI2 0 T0 024 O 30 OPTICAL DENSITY AT 330 II!" 70F E .50 PERIPHYTON AQUARIA a. > / 5 Mean calcuIaTed ci- l59.0 h I 2 50 I l 2 ”a” c ‘0 ’ .- a U ‘2’ 30 '- I o o I 2 20 . 8 . ‘ The ”Inn IRdICeTe Thdeud '0 _ ! abeerveTIeae IN" The IIhe eee eeReTIueTed ITONI The “can .2. detlved eeueTlee. 1— A L A A A A A O 0.06 0 l2 O.l0 0.24 0.30 OPTICAL OENsITv AT ”Own FIGURE 12 for 1.0. BACTERIA AND FUNOI AQUARIA Q : Neee eelculeTed q «770 - 00> ' l c p. a U u SOT z o l u e ZOI- I 2 I I The penal. lIIeIceTe TeerTduel Io__ 1 eeeerveTleee ehue The line eee ceeeTveCTed he. The T . F500" '20 «Mad eeue lee A l A I I A A I j o 0.06 0 l2 OJ. 0.24 0.30 OPTICAL DENSITY AT 300.) 70 _ Esol PHYTOPLANKTON AOUARIA O 5 Mean caTculaTed Ci-IDOJ 50 z ‘2 2 ¢ 40 e... z 30 o U I 9 20 I U l ‘ . The pain" IechaTe IedIeIdeeI IO eheereTIaae while The The In ceheTTucTed he. The derived eeueTlee. Frame IZd O 0.06 012 0J0 0.24 O. 30 OPTICAL DENSITY AT 330m; 70 E A50 FISH AQUARIA z 2 Neon celeuIeTed CI - l04.7 g 50 e. e c T- 40 2 Tel 0 a: 0 so u I a II 5 20 ' ‘ I l I The eeTRTe IedTeeTe IedTvIeeeT eheeneTleee ehITe The The ’0 eee eeeeTIueTed Toe. The Flame '2' derlved eeeeTlee. A_ A A A A L A A A A O 0.06 0.l2 O.l0 0.24 0.30 OPTICAL DENSITY AT 330" 64 .Uoflumm wvsum mflp mafiusc “Ham mflumsvm zmflw map CH muflcaamxam mumconumoflm .mfi munmflm 65 ma EDEN—“m mom. ”$2325 I 920 nTm mam on-m nub m_-w mIm mum wui. 2-5 EL. nip 1 . _ q . A . d . 1 in. Imm 29.2004 o.o< no 3.2.0“. .6» 4 xmocH .wfi musmflm 74 o w.“ Emu—”m mom. muzzam I up; Bum $8 A..-» 9.0 To 5;. 8.» ml. 2. W q 4 q q d d 000. \ h 20:53. 93 .8 32.8 b a s eeeeeee.eeeeeeeeeeeee eeeeeeeeeeee e0"./ .3 53:33.0 gall x .3 mg oIIIlo /o can “QEQUUObp-hb< Ocelo G@—°0°)—O> XIX / \ I 00w Eda 53.3565 e'|e d» flaw ele / \ ”.a' E:b*”°b:°fim .eeeeeheeee. .3» =°b°0°hfivk "0---- x // \\ H d» Iallmuld: .z 5 m o......... o .3 Hana eIIIIIe /\ I 000. O L EDWVA XBONI lNVNIWOO DONOd lNVNIWOO CI ONOd 75 Centrifuged plankton--aquaria The index values during the later portion of the summer are shown in Figure 15. The only possible significant points would be the dominance of Closterium sp. in the acid aquaria. All other prominent species do not differ between experimental and control. Periphyton--ponds Two-way analysis of variance was performed on the opti- cal density data from the periphyton-extracted chloroPhyll. A total of 81 samples were divided by two ponds and 9 two- week sample times (see Appendix, Tables 28 and 29). Pond D had a higher growth of periphyton as shown by the chlorophyll values while the significant interaction showed pond C to have been lower than expected in chlorophyll values for the later part of the summer. Both ponds decreased to mid-summer then increased to higher than starting chlorophyll values. The index values for the three dominant types of organ- isms found in the periphyton are shown in Figure 16. Growth of Navicula Sp. may have been promoted while that of Synedra sp. might have been inhibited in pond C after the addition of acids. Periphyton--aquaria The index values for the three dominant types of organ— isms found in the periphyton in the aquaria are shown in Figure 17. No trends appear significantly related to the 76 .MHHmsUm mnu Eoum GOstmam UmmDMHuucmo How mosam> xmocH .md musmflm 77 5-0 dm 0:2..5m d» 2.63.0855 do 9.323. d» oeuvcum d» E33320 .“ m.“ mngm muZZDm I wh xmocH .ma muzmam 79 m H mmDGHm 000. mus—20m I mh<0 9-0 0:0 NIm wNIm 070 N70 mum mNIh NNL. 9L. 0i. H 1 d u - q u - ~ 22:84 904 .._o 32.0.. a d d O/IWQ d» 2.37:5 eIIIIe d» mI_:..m_>Ioz x x d» oeuucxm olo .LNVNIWOO O ONOd 301VA XBONI iNVNlWOO O ONOd 80 .maumsqm map CH couwcmflumm mzu ca Ussom mmflommm ucmsHEoo map How mosam> mecH .ea musmflm b.“ MMBUHm 000. KNEE—am I wh<0 81 Tm «Ia 3-0 are «To n-» mu-“ «Ni. - u u d d u d u d -9. 3.2:: :o E o 0 .583 .3 33:82.6 22:84 93 .._o 326.. on m a. a; .IIII. « e h a m dm 2:262 xIIIIIx m dc Enocxm 0'0 ION . x o .o. s) o \ .. l/ \ O I \\ n (e V IR .2 m n W a 0 W Lou m V N l. WfllHVflDV 'IOHINOO BOWVA XBONI 'IVINBWIHEdXB 82 addition of acids. Net plankton--ponds The same type of index used for centrifuged plankton and periphyton was used again for net plankton. Figure 18 shows the change in this index for several organism types through the summer. Significance would be hard to define since variability could be introduced from many sources. The extremes would be the only possible significant feature. Here large copepods and cladocera seemed to be greatly re- duced ianominance in pond C during the middle of August. If this was a result of addition of acid its effect was indirect since no immediate response was seen at the time of addition. This may also be why no response was seen im- mediately in the week after the third and largest acid addition. Net plankton--aquaria Net plankton were of small number and only survived for several weeks after the start of the experiment in both aquaria. As both populations were going to extinction the scant data may have little significance in explaining the effect of acid addition though the large copepods and cladocera were observed to decline in dominance in the experimental prior to their decline in the control (Figure 19). 83 .umEEDm mfiu nmsousu mpcom mzu How mosam> xmosfl couxsmHm umz .mfi musmflm 84 FIG .nIo 3.302.; 88.3: 2323.0 uuoaoaoo :35 32.2.3 .30.. ¢NI0 m.“ mgmHm 000. mmZZDmIMP<0 870 070 onuo hm uh d u . 1 20:33 23. .6 32.0.. e 0 NIN SIN 00m 00m 00. 00. 00. .LNVNIWOU BO'IWI XBONI lNVNIWOO O ONOd 0 ONOd 85 .HHmm MHumsqm pmumcmflmmo mnu How mosam> xmosfl souxcmHm umz .mfi musmflm 86 m.“ EUHW mom. mmZZDm I wh40 GIG _I0 ONIN mmwu.h0¢ Dell. 4mm0u0440 xlllx mooauaoo 4442m 0330 m000u000 wom4I— 0'0 II I I I. I 20.....004 0.04 “.0 m...z.0n. 00 ON ON 0.? 00 .LNVNIWOO .LNVNIWOO WfllHVflOV TVLNBWIHBJXS 301VA XBONI WfllHVflOV 'IOHLNOO 87 Bottom organisms--ponds It was felt that a short term experiment such as this one, falling at the emergence time of many of the dominant bottom Species would not provide any significant data with the limited sampling time available. Bottom organisms--aquaria There was no large difference noticed in the survival of the introduced organisms. Several Odonata survived in each aquaria while all other forms perished. Both aquaria develOped the previously mentioned filamentous growth. Fish--ponds No dead fish were noticed in the ponds during the summer and all appeared to be in no distress. Fish-—aquaria The four green sunfish in both aquaria survived the entire experiment with no ill effects noted. None of the colorless filamentous material was observed in either of these aquaria. Fate of the acids in the ponds The total changes in ppm acid concentration in the light and dark bottles during the three week exposure in the pond are presented in Table 4. The change in the sample containing acids in distilled water showed that change which might be attributed to the effect of light on the acids. The difference between this 88 Table 4. Observed changes in acid concentration in light and dark bottles susPended for three weeks in pond C. (Data in ppm.) Distilled Filtered Centrifuged Untreated Bottle water and pond water pond water pond water type acids and acids and acids and acids Dark -4.6 +0.2 +2.5 -0.7 89 change and that observed for the 0.45 u filtered pond water indicated the magnitude of the effect of dissolved substances in the water. The difference between the filtered and the centrifuged samples likewise indicated the result of bacteria and ultraplankton activity. Any effect on the acid concen- tration by phytoplankton and zooplankton was revealed when the difference between the untreated and the centrifuged samples was found. Table 5 shows the calculated values at- tributed to these separate sources. A decrease in acid concentration measured by fluorescence indicates either destruction of the acid molecule or poly- merization of two acid groups (Christman and Minear, 1967). Conversely an increase in fluorescence would indicate acid production or a breakdown of polymerized acid grOUps. Conditions in other natural bodies of water In Figure 20 the acid concentration was seen to increase only near the bottom on those lakes which lack an established thermocline, Goose and Long Lakes. In Titus Lake, an oligo- trophic marl lake, acid concentration increased markedly as the thermocline was passed between 24 and 50 feet. Concen- tration remained relatively constant in the well-mixed upper water. ' On a stream such as the Clam River, Figure 21, changes were seen in acid level along the flow. The substantial rise near mile 1 can be traced to the effect of a sewage plant operated by the city of Cadillac and emptying into the river. 90 Table 5. Calculated effects of different sources of acid change as determined by light and dark bottle experiments. (Data in ppm.) Bacteria Phytoplankton Bottle Dissolved and ultra— and type Sunlight substances plankton zooplankton Light -1.5 -10.6 +0.5 -5.2 Dark -4.6 +4.8 +2.3 -5.2 91 Figure 20. Acid concentration profiles of three lakes. 92 j 0 x O x O - 4 x O x 0 - 8 . OOTTOM ! o GOOSE LAKE . T23N new $26 0 .. l2 bottom 0 LONG LAKE T23N R8W 'Al6 $26,27,34,35 e - 20 e - 24 e e [-28 e e ‘32 e e -'36 b II o om I TITUS LAKE (SANDS LAKE) T27N R9W 1 $26 I , l I l 2 4 6 8 IO l2 l4 ACID CONCENTRATION IN ppm FIGURE 20 IN FEET DEPTH 95 .nm>flm EMHU map 00 maflmoum soflumuucmocoo Uao4 .HN musmflm 94 >m4h00.m._. x mm>.m 2440 O mom—:00 04......040 mx4I. 200.... mm...2 ¢. N. dm HNSGHM 0. w 0 d 0. 4N Nn 0¢ 04 on NOIlVUlNSONOO 0 IOV wdd I" 95 Detergents fluoresce at approximately the same wavelength as the colored acids and interfere with accurate determin- ations. In the next four miles essentially all the material added by this plant and most of the acids entering the river from the lake origin have been removed as the stream flows through the Cadillac moraine. Table 6 presents measurements of acid concentration for various other bodies of water. Cause of viscosity in the aquaria Upon spraying the chromatogram run in the isopropanol: acetic acid:water solvent with sodium periodate reagent followed by benzidine, no Spots were found in the unhydrolyzed sample while one Spot, not corresponding to glucose, was found:h1the hydrolyzed sample. This Spot had an rf value of .75-.82. The same spot also had a reddish tint when sprayed with aniline-acid—oxalate reagent. This indicated the presence of aldopentose structure. No amines were present as no spots were seen following ninhydrin spray. In the n-butanolzacetic acidzwater solvent this same spot had an rf of .54. This compound consistently had a greater rf value than any of the other sugars co-chromatographed. Seliwanoff's test proved negative for ketose. Determinations of periodate consumed upon oxidation and formic acid produced were incon— clusive. The chemical causing the increased viscosity was most probably a polymonosaccharide of an uncommon sugar pro- duced by bacterial or fungal growth. 96 Table 6. Acid concentration of various selected bodies of water. M ======== LoCation ppm acids Remarks Lake Michigan 1. NE holland 0.68 at 77 meter depth 2. é-way across lake 0.62 at 157 meter depth 5. Racine 0.47 at 55 meter depth Lake Mendota 7.65 surface near shore Crawfish R., Columbus, Wis. 62.5 following heavy rain very turbid Charlotte R., Davenport, N. Y. 6.05 clear cool stream DISCUSSION Method of acid measurement Color, as measured by the comparison of samples of platinum—cobalt standards, was the only quantitative method widely used to measure the concentration of yellow organic acids in natural waters prior to the introduction of fluor— escence. When compared to fluorescence methods, the measurement of color by association with platinum-cobalt standards shows several disadvantages in the measurement of concentrations of these compounds. Color is more sensitive to pH change than is fluores- cence in the pH range of natural waters (Christman and Ghassemi, 1966a,b; Christman and Minear, 1967). In fact, differences in pH may nullify the comparative value of color when several bodies of water are investigated. Recourse may be made to an expression of color at a standard pH as .in Christman and Ghassemi (1966b) for streams in Western Washington, but this further complicates the methodology. The color to carbon ratios presented in this same previously mentioned study also indicate a high variability, most prob- ably arising from differing chemical structure. A modifica- tion used by Anthony and Hayes (1964) where measures of color and turbidity are separated Spectroscopically possesses these limitations also. 97 98 Shapiro (1957) arrived at a value of about 0.4 ppm acids per color unit at pH of 7.0 with acids from Linsley Pond. A comparison of both his and the values of Anthony and Hayes for Lake Mendota with my fluorescence measurements about ten years later shows a value of nearly 0.7 ppm acids per color unit. .It is possible that Lake Mendota has changed in the intervening years or seasonally though from the similarity of both the previous measurements, I would believe this not to be the case to this great an extent. As I have already mentioned, detergents may increase fluorescence in the same wavelengths as the acids. This may have been the case in Lake Mendota. Otherwise chemical differences in the acid molecules between Lake Mendota, Linsley Pond and my experimental ponds may be indicated. Assuming these acids to be 55.5% carbon as found by Shaprio and that the entire dissolved carbon is derived from these colored compounds, we can compare Christman and Ghassemi's Western Washington streams with values from 0.2 to 0.55 ppm acids per color unit. This wide range of values can be attributed to the poor quan- titative relationship between color and acid concentration probably arising from differences in the origin and chemistry of the acid molecules. Sensitivity of this method is about the same as that of fluorescence on the 5x scale or.i 0.5 ppm. Christman and Ghassemi (1966a) used fluorescence for quantification of these colored compounds by almost the same 99 method as I have presented. The one difference is their use of only filter 2A for a secondary (emission) filter before measurementcflffluorescence. This gives their method a wider response to all wavelengths above 415 mu whereas the method used in this study measures only those wavelengths around 460 mu. A product of these differences is a greater sensi— tivity (i 0.5 ppm on 1x scale) but less assurance of freedom from interfering substances for Christman and Ghassemi as Opposed to lesser sensitivity (1 0.5 ppm on 5x scale) and lessened interference from detergents by my method. Several problems do exist with the fluorescence method. A change in pH continues to cause a small change in fluores- cence although Christman and Minear point out that fluores— cence is insensitive to pH fluctuations over a broad range of pH values. pH was not found to be bothersome or necessary of adjustment for consistent reproducible results in this study. Christman and Minear also report a polymerization re- action which is accompanied by a decrease in fluorescence. The extent ofpolymerization of the acid molecules in a body of water in addition to other differences in chemical struc- ture or origin may also affect the accuracy of this method and its application to the comparison of different bodies of water. Detergents have been mentioned previously as possible sources of interference in fluorescence measurements. This 100 is most probably the cause of the extremely high values recorded below the sewage plant for two miles or more on the Clam River. Interference of this type would not be expected to affect the measurements from the ponds or aquaria in this study. When the presence of detergents is suspected, it might be well to use the methylene blue method (Standard Methods) to measure their concentration while at the same time preparing a standard curve for detergent concentration and fluorescence at the interfering wavelengths. Hence in this way one might be able to separate the two factors and gain considerably more confidence in what has actually been measured. Because of the high probability of unknown interference causing a loss of sensitivity in measurement, it would be best to measure fluorescepce over as small a range of wave- lengths as is possible. Correction should be made for deter- gents if they are present in quantity while pH may be ignored if changes are small and in the center of the range. The un- known effect of the extent of polymerization, different chemical structure or origin and the enhancement of fluores—) cence by certain salts must be merely acknowledged at this time as a possible source of error in the comparison of different waters until the magnitude of the differences aris- ing from these sources is better understood. It may be that with fluorescence we can actually measure the chemically or biologically active part of the molecule such that differences 101 in overall structure are of little consequence. If this is the case, instead of parts per million, some activity unit should be proposed. The origin and fate of the acids It has long been assumed that these yellow acids origin- ate from soil runoff and the decomposing vegetable material or humus. Shapiro (1957) supports this conclusion by the observation that soil extracts are very similar to the com- pounds isolated from lake water. Christman and Ghassemi (1966a) further clarify, based upon the chemical analysis of degradation products from these acids, that lignins, lignans and other wood phenolics are the most probable sources. It therefore seems that two general sources may exist for the colored acids found in water. Origin may be allo- chthonous from decomposing terrestrial vegetation and/or soil organic matter and this matter then carried into a body of water with runoff. This was not revealed to be a large source of acids in the ponds studied since rainfall had no significance in the explanation of acid changes measured and no increase of fluorescence was noticed following even heavy rains. I do not feel that subsurface seepage into the ponds accounts for an appreciable influx of acids since these molecules are highly adsorbed in the soil and may travel no more than a few inches from their point of release into the environment. 102 Autochthonous acids released from aquatic vegetation decomposition and extraction from bottom sediments of the ponds would be a second source. From the higher concen- tration of acids near the bottom of the three lakes measured, this second source would appear to be important. In Titus Lake, where a thermocline is present, these acids can be seen to build up higher concentrations relative to the upper waters than in the other two lakes where mixing occurs be— tween all strata. In streams such as the Clam River, allochthonous sources would be the more important. Between mile six and mile twelve, where acid concentration more than doubles, the stream flows through wooded land as opposed to Open farm land between mile twelve and mile fifteen where little change in acid concentration occurs. The slight increase below mile fifteen might be traced to input of acids from tributaries. From the variations seen in the lakes, ponds, and streams, this acid system is certainly not stable. In addition to the previously mentioned sources, autumn leaf fall and spring runoff may also be of importance. The loss of acids from a body of water is less under- stood than their source. Whipple (1927) has reported a lightening of colored waters which he attributed to "bleaching“ by the sun. When Christman and Minear found a rapid decrease in fluorescence during measurement, they proposed the concept of polymerization occurring between acid molecules which 105 reduces the number of available sites for a fluorescent re- action. I believe these two observations were of a similar phenomenon. The light and dark bottle experiment of this study was conceived to investigate this problem. A decrease of 1.5 ppm was measured in the light bottle having yellow organic acids dissolved in distilled water, but an even greater fall in concentration of 10.6 ppm was recorded in the light bottle containing acids dissolved in filtered pond water. Clearly some dissolved substance which occurs naturally in the pond water is necessary for this reaction. The decrease in the distilled water with acids was recorded for both light and dark bottles so this was probably not by the same mechanism. The concept of a polymerization seems to fit the cir- cumstances. Acid molecules may be joined together in a reaction catalyzed by sunlight and involving a dissolved sub- stance (ion; molecule ?) naturally occurring in the water. It is not known whether this dissolved material may act as a catalyst also or be involved directly in the reaction. It is my belief that this dissolved substance may be (a) doubly positive charged metal ion(s), mainly calcium. I support this by pointing out the relatively abundant nature of this type of ion in natural waters, the low color of hard water lakes, the fact that Christman and Minear were working with a 0.01 M calcium chloride solution of these acids when they observed this phenomenon and that Shapiro (1958) was able to produce 104 and associate bands on paper chromatograms with distinct acid-metal associations. One of Shapiro's possible explana- tions was that these zones may represent different salts or complexes of one or more acids. Through this polymerization reaction the molecular weight of the acid group would be increased. It would be theoretically possible for a point to be reached where the molecule would not be able to remain suspended or dissolved in the water column and would fall to the sediments. Mixing would tend to counteract this along with possible reactions breaking down the polymer bonding. The higher concentrations near the bottom of lakes could not be caused by this settling alone. This is especially true in stratified lakes where the bottom waters are exposed to little turbulence. In such undisturbed waters it would seem that settling would be more rapid in the absence of resuSpension by miXing afid'that zones of increased concentration would be less likely to occur. Precipitation is thought to be of minor importance in the loss of acids to a body of water. Other possible mechanisms causing changes in acid con- centration within a body of water, as inferred from the light and dark bottle experiment, might be a breakdown of polymer structure, thus an increase in measured fluorescence, when bacterial enzymes attack the acid molecule for use as a carbon and/or energy source; and also the destruction of the total molecule when used for the same purposes by higher organisms. 105 A diagram of acid balance in these ponds can now be con- structed to help visualize the dynamics of this system. Over the summer the ponds showed an average loss of about 0.05 ppm acids per day. A diurnal cycle was evident with a net loss for the daylight hours and a net gain during the hours of darkness. When the change for the light and dark bottle experiment was calculated, there was an average net loss of 0.71 ppm acids per day for the three weeks. Since the loss rate was so much greater in the bottles than the ponds, the source of acids that must have been replenishing the ponds was being excluded from the bottles. As this source was most likely the bottom sediments and vegetation decomposition in the ponds, we can assume an average net gain of 0.65 ppm acids per day from this area in order to achieve a balance between the two estimates of daily change. Table 7 shows the calculated movement of these acids in the experimental ponds on a typical summer day. Figure 22 presents an explanation of the diurnal and annual cycles. The effect of light polymerization is a net loss during the day while at night, when this reaction does not occur, acids continue their constant release from the sediments and through decomposition to create a net gain. In this same manner the annual cycle is created. A net loss is incurred in summer under conditions of high solar radi- ation. In winter the acids show a net increase when this loss through light-induced polymerization is reduced. 106 Table 7. Dynamics of acid change in a pond on a summer day. w —=:=—== 0.50 ppm per 0. 241: ppm per 0.71 ppm per 0.65 ppm per 0.014ppmpper 0.66 ppm per 0.71 ppm per 0.66 ppm per 0.05 ppm per day day day day day day day day day Loss of acids Light-induced polymerization Destruction by higher organisms Total loss per day Increase of acids Release from sediments and through decomposition All other sources (runoff, polymer dissociation) Total increase per day Total loss per day Total increase per day Net change (loss) observed per day 107 Figure 22. Diurnal and annual acid cycles in a pond. CHANGE IN ACID CONCENTRATION CHANGE IN ACID CONCENTRATION 108 DAILY CYCLE (SUMMER) loss l I I I2 MID. l2 NOON I2 MID. TIME OF DAY II-II-II-II- Release from sediments and decomposition oooefl Destruction by higher organisms -... Polymerization by solar radiation —— Average effect’of all causes giving change observed ANNUAL CYCLE loss L I JAN. JUNE DEC. TIME OF YEAR FIGURE 22 109 Effect of acids on chemical and physical features A basic assumption upon which all the data of this section rests is that the acids extracted, purified and added once again to the aquaria and ponds were not changed. in structure during the process. This assumption is prob- ably not completely met as 2 N sodium hydroxide was used in the extraction process and would tend to alter the molecular structure. This section must therefore be considered with certain reservations as to whether the observed changes can be normally attributed to the native acids or are the product of structural fragments not naturally occurring. It is my Ibelief that structural alteration was minimal as little change was noticed in the Spectra and in the chemical behavior of the acids after the recovery process. Also any fragments that might be produced may resemble smaller pOlymer units Which are normally present in the system. The use of a computer to solve for the relationship between different chemical parameters in the environment was helpful in understanding the effect of acids on these features though some problems were encountered. The asymptotes seen in all three theoretical equations are artifacts produced by the method used in solving for the parameter to be described. If higher power functions were included for all factors the asymptotes would be smoothed out to a more repre- sentative curve at these points. The equations described are alsc>completely symmetrical and certain portions of the 110 curves are not supported by the data. If these features can be taken into account, the meaning of these equations may be explained. The action of acids on pH seems to Show an interaction dependent upon the initial pH of the water. The partial correlation coefficient of acid concentration is negative showing generally decreasing pH with increasing acid concen- tration. I believe the general action of acid conCentration on pH actually to be a decrease in pH with rising acid concentration for waters with initial pH values below 8.02 while, for those waters with an initial pH above 8.02, a rise in pH proportionate to the extent the initial pH exceeds 8.02 until, in the region of 25 to 50 ppm acids, pH decreases with increasing acid concentration (Figure 25). This repre— sentation seems the more true to nature as no return approach to pH values near the initial pH was recorded for the aquaria at high acid concentrations. A clue to the possible cause for this strange pH be- havior may be taken from the value of the pivotal initial pH--8.02. The presence of increased acid is normally ex- pected to produce a decrease to a lower or more acidic pH as occurs at initial pH's below 8.02. The pH value of 8.02 is the approximate border pH where carbonate exists at higher pH's and bicarbonate only may be found at lower pH values. It could be that carbonate or calcium bonded as carbonate is joined in some way to the acid molecule preventing the 111 .QHSmCOADCHDH COHumuucmocoo UHUMIImm pomfl>mm .mm ousmflm 112 4— ONISVBUONI NOIIVHINSONOO OIOV I4 C) FIGURE 25 115 expression of acidity or releasing molecular fragments which are basic in pH. In turn, the carbonate so bonded may become untitratable so no expression of phenolphthalein alkalinity would be observed. This appears very possible when it is also recalled that alkalinity dropped markedly at high acid concentrations in the aquaria and that the polymerization reaction may involve calcium. So it may be said that the expression of pH under dif- ferent acid concentrations is dependent in some way upon the action of these acids on the carbonate-bicarbonate buffering system. As acid concentration becomes greater the capacity of the buffering system is evantually overcome (if the rate of acid increase is faster than the rate at which available insoluble calcium carbonate is dissolved and mobilized) and pH behaves in the generally accepted manner of decreasing as acids continue to rise. In the environment this relationship depends upon the rate of acid change in a body of water, the magnitude of the carbonate-bicarbonate system and the size and availability of the insoluble calcium carbonate reservoir. Through the rates of change of these three parameters it might be decided whether a lake might remain as hard water and relatively unproductive or become highly productive and more colored. Conductivity appears to be affected in a manner somewhat like that of pH. The asymptotes can again be excluded as products of the method of solving the expression. The action 114 of acid concentration on conductivity then is seen to be a mirror image of its action on pH. When an initial pH was above 8.02 with a rise in pH accompanying an increase in acid concentration, the initial conductivity for the same aquaria was usually near to or less than the pivotal initial conductivity value of 164 micromhos per centimeter. As acid concentration was increased, conductivity decreased as might happen if two molecules were brought into union as was sug- gested in the explanation of the rise in pH. As acid concen- tration continues to rise the fall in conductivity slows and then reverses direction to show a direct relationship between these two parameters at high acid levels. For water above an initial conductivity of 164 micromhos per centimeter, a steady rise in conductivity occurs when acids are increased (Figure 24). From the similar reaction of both pH and conductivity to a change in acid concentration it seems clearly evident that both these effects arise from the same source--an inter- action between these acids and calcium carbonate to form a complex. In the absence of calcium carbonate, at a pH below 8.02 or after all the available carbonate has been complexed, conductivity rises as it normally would when molecules capable of dissociation are added to water. Optical density, when the asymptotes are eliminated, shows an almost linear increase with increased acid concen- tration. This is what would be expected when a molecule 115 .mflszOADMHoH COHDCHDCOUCOU UHUCIIhuH>HuoDUCOU pomfl>om .wm ousmflm 116 4N HMDme 8.3 .2. Soxmorzomoéc >t>_5:ozoo cm. d A.— _ . A . x x . a \ I \ . \ 4 \\ \s \ \ \\ \\\ \ \\\I\ ‘4-— ONISVBUONI NOIIVHINBONOO OIOV 115 .mflnmCOHumHmu soflumnucoocoo UHUMII>DH>ADUDUCOU Umma>mm .«N musmflm 116 «N mmDGHm 3.8..” .2 Sexmorzomoi. 3.2.5.5200 om. . .— . . . . \ I. x x . z \ I x . \ s \\ \s \ \ \ \\ \\\ I\|\|\\\ -e-- SNISVBHONI NOIIVBINSONOO OIOV 117 absorbing light at 550 mu is measured at that wavelength and plotted against the concentration of that molecule. Before the ponds may be viewed in the light of these aquaria predictions, any possible sources of interference in the ponds must be evaluated. Since there was a large filamentous growth only in pond D during the later portion of the summer, this would be a source which might yield interaction between the ponds in the same manner as acid addition. This source of interference will have to be evalu— ated when the ponds are considered. The pH interaction in the ponds, pond C increased as compared to pond D, may have arisen from either acid addition in pond C, where the mean pH was 8.72, or from the decomposi- tion and carbon dioxide production of the filamentous growth in pond D. Either or both of these may have caused the ob- served interaction and the effects cannot be separated. Again with conductivity the causes of interaction cannot be separated. An increase in acid concentration in pond C, mean conductivity of 157 micromhos per centimeter, and the decomposition of vegetation in pond D would both produce interaction showing a decrease in conductivity in pond C relative to pond D. With Optical density it can be said with more confidence that the interaction recorded resulted from the addition of acids to pond C. The decomposition of a growth of filamentous material in pond D would be expected to decrease the percent transmission in this pond the same as would the addition of 118 acids to pond C. Interaction was shown to indicate a de- crease in percent transmission in pond C relative to pond D and would thus be a more likely indication of an observ- able effect of acid addition. .This might in turn be taken as proof that the effects of acid addition were observable in the ponds and that the interaction for pH and conductiv- ity probably do indicate, at least in part, the effect of acid addition. From the general decrease in bicarbonate alkalinity observed in the fish aquaria, some reaction must be taking place which involves the bicarbonate or calcium bicarbonate molecule. There are two possibilities for this reaction-- (1) bicarbonate combines with the acid molecule in the same manner as carbonate mentioned previously but at a slower and less preferential rate or (2) bicarbonate combines with the acid at an entirely different site causing different observable effects. Of these I believe the first to be the best explanation because it doesn't complicate the system through a set of assumptions for a new mechanism and the data can be fully explained by this same reaction. It may be that the action of acids with carbonate is so reactive that when carbon dioxide is removed from bicarbonate for photo- synthesis, the transitory calcium carbonate is complexed with the acid molecule before combination with a free carbon dioxide molecule can transform it back into bicarbonate. This would be where the slower or less preferential combi- nation would be observed depending upon the probability of 119 a chance encounter by a transitory calcium carbonate with an acid molecule before a free carbon dioxide molecule. In the ponds changes in alkalinity may be entirely explained by the extreme decomposition of vegetation in D. Carbonate alkalinity decreased in D relative to C the later half of the summer as would be expected with a high rate of decomposition in pond D and the subsequent combination of carbon dioxide produced with calcium carbonate to form bi- carbonate. This same process in pond D would produce the increase in bicarbonate relative to pond C. It is possible that the magnitude of these changes in pond D masked any noticeable effects of acid addition which may have occurred in pond D. Total alkalinity also did not Show any effects trace- able to acid addition. The lower total alkalinity in pond C at the end of the summer most likely indicates the excess of fixed carbon dioxide removed through photosnythesis to respired carbon dioxide replaced. In pond D the carbon dioxide released through decomposition held the total alkalin- ity at a high value throughout the summer. The temperature comparisons between ponds will not be evaluated as the calibration of the instruments was question- able. Light penetration, oxygen and metal ions failed to show any effects which might be traced to the addition of acids to the ponds. 120 In summary for this section, it might be said that very little chemical or physical effect was noted for the ponds that could be clearly called a response to the addition of yellow organic acids. It is only reasonable that this should be so Since the amount of acids added was in the magnitude of one—one hundredth of the total concentration of these acids naturally found in the ponds. Even the fluorescent measurement of acid concentration showed no significant in- crease in pond C until after the third addition. Soon after this time the observations were terminated before any other effects might have become noticeable. The process of extract- ing acids from the water for re-use in addition was under- estimated in planning and forced emphasis to be placed on the aquaria experiments. These aquaria experiments did turn out to be very reveal— ing. One reaction involving the complexing of carbonate salts, most likely calcium, to the acid molecules can be used to explain all the observed phenomena. This reaction may cause a rise in pH through the union with a part of the acid molecule blocking an acidic group. This union also shows a decrease in conductivity as two molecules capable of dissoci— ation are complexed so that at least a portion of the result— ant grouping does not dissociate. This reaction depletes the titratable carbonate alkalinity and may compete for bicarbonate when acid concentration is high. It may well be that this is the same mechanism that Christman and Minear described as light—induced polymerization. 121 Effect of acids on biological communities No effect of increased acid concentration was evident for bacteria and fungi, bottom organisms or fish. Centrifuged plankton from the ponds did appear to Show some significant changes which might be associated with acid addition. The use of the index value method appeared most adequate when the counts made were an average of twenty or more individuals per organism group. At counts less than this any differences in dominance were not evident unless the dominance was extreme. After the third acid addition the majority of organism groups exhibited greater dominance in pond D. Though this may be an indication of the adverse effect of the acids on growth, I would rather believe that it indicates a large dominance of one group, Arthrodesmus sp., in pond C whose growth may have been stimulated by the acid introduction. This same feature may be recognized in the aquaria centrifuged plankton, only here it is Closterium Sp. which exhibits the increased dominance in the acid aquarium. The interaction seen with the periphyton chlorophyll extracts cannot be separated between the promotion of peri- phyton growth in pond D after the filamentous bloom die—off and lower chlorophyll production in pond C from the change in dominance (If phytoplankton species. The growth of Navicula Sp. appears to have been stimulated by the addition of acid while the slower growth apparent for Synedra Sp. following the addition of acid may be only a lack of stimulation to the 122 extent of that expressed by Navicula sp. Little of signifi- cance can be seen in the aquaria studies of centrifuged plankters save a slight possibility of increased dominance of Surirella Sp. in the acid aquarium. The net plankton studies of the aquaria and ponds tend to show similar results. In these cases large copepods and cladocera appear much more dominant in the control aquarium and pond starting one to two weeks following the introduction of acid. These forms may be adversely affected by the in- creased acids in the experimental aquarium and pond C. Since the variability of this index value method has not been defined, the significance of all these results is uncertain. Many other factors may have varied between the ponds and aquaria to cause the observed values. If the observed effects were the result of acid addition they might be explained in several possible ways. Shapiro (1957) stimulated the growth of several algal cultures with the addition of purified yellow organic acids. Saunders (1957) has compiled a sizeable listing of algae capable of using organic compounds for energy or growth. The effect of growth stimulation may have been observed in this study with certain species of net plankters and in the periphyton. Observed changes in species as a lake becomes an acidic ‘bog may be the result of many environmental changes of which some are probably related to the increase of yellow organic acids. Patrick (1948;1954;1965) reports that certain species 125 of Navicula and Surirella tend to become-the characteristic algal Species under dystrophic conditions. Transeau (1905) reports for several Michigan bogs that “there have been marked variations within short periods of time in the color of the water and in the presence of such animals as Daphnia and Cyclops." Though no more can be added to the understanding of why these changes have been observed in this study, it might be said that more intense study should be made of certain Species which are known to show a response to acids or acid-related conditions. It appears that the causes cannot be entirely explained by large chemical and/or physical changes in the environment but more likely relate to (1) the increase in an essential nutrient or growth substance or (2) little under— stood or recognized physiological responses. Implications of this study From the scattered observations reported in this study, a relationship between acid concentration as measured by fluorescence and the character of a body of water can be seen. Oligotrophic lakes such as Titus and Lake Michigan are low in acids while the experimental ponds, Lake Mendota and other lakes measured are more eutrophic with higher acid levels. This seems a natural result as the more productive a body of water becomes, the more vegetation is produced which may in turn decompose with the release of acids. It therefore appears possible that there is a close relationship 124 between the process of eutrophication and the observed in- crease of dissolved yellow organic acids. If this is so the stage of eutrophication of a lake might be more easily determined through a simple observation of the acid concen— tration. What are the possible effects of this acid buildup as eutrophication progresses? The extreme chemical and physical conditions in dystrophic lakes can be explained through the mechanisms elucidated in this study. Low metal ions and low alkalinity can be explained by the formation of the calcium—acid complex and light—induced polymerization. .Low pH is explained by the high concentration of acids and little buffering action by the carbonate-bicarbonate system. Low conductivity most likely results from the fact that weak organic acids such as these being studied tend to dissociate most in dilute solution and remain largely undissociated in more concentrated solutions. Also large quantities of cal- cium and possibly other ions measured as conductivity may have been removed by these acids and precipitated. If these effects can be seen in dystrophic lakes, these acids must be able to exert an effect, although diminished, in waters where less acids are present. ~Some algal species might be influenced in the develop- ment of blooms by these compounds since they are capable of ~ growth stimulation. The mixing occurring after ice breaks up in the Spring and after the fall overturn may bring to the 125 upper waters those acids contained in the concentrated layers near the bottom where they have been released. These acids may help to promote the Spring and autumn growth pulses regularly observed in lakes in the temperate zones. -Stressful conditions in waters of high acid concentration may impose an added burden on the organism trying to survive in this environment. At these high acid levels, a small changein.acid concentration as might happen in the diurnal cycle causes a much greater change in the chemical and physi- cal parameters than a similar change at lower acid levels. Although the acid concentration in a body of water is important, the rate of change in this concentration through time should be an indication of the rate of eutrophication for this water. -A lake in which there is a balance between acids coming:h1and those lost should Show more stable condi- tions than a lake which shows a deficit in one direction. A method for measuring the direction and magnitude of acid change could be devised by measuring the acid concen- tration of a body of water several times during the year. .Along with this a light and dark bottle experiment could be conducted to evaluate the rate of change in acids from dif- ferent causes at different seasons. These rates would vary for different waters since the solar energy and the rate of availability of calcium carbonate would not necessarily be the same. A less involved but also less exact method would be the comparison of acid measurements taken at a standard 126 time of the year, such as after Spring ice breakup and com- plete circulation, over a period of several years. The better understanding of the dynamics of these acids in natural waters may lead to the development of better con- trol of environmental quality. Acid increase in,a lake might be halted or slowed by attacking one of two vital steps in the system-—(1) interference with the release of acids from the sediments and vegetation decomposition or, more likely, (2) encouragement of light-induced polymerization by supple- menting the available carbonate with fully dissolved calcium carbonate applied at the surface. It might be found that some other form of carbonate may work as well or better than calcium carbonate and would dissolve more readily. Other interesting sidelights to this problem are the possibilities of using the calculated conductivity and pH values at a theoretical acid concentration of 0.0 ppm in the characterization of bodies of water. These values must indi- cate the exchange balance of ions, excluding organic acids, within a basin or drainage system and would thus be of cer— tain limnological Significance. Other conditions of the environment in the aquatic system might be found to be related to these acids once their dynamics become better understood. S UMMARY Fluorometric measurement of yellow organic acid concen- tration in natural waters can be a sensitive quantitative procedure if fluorescence is adjusted to a standard tempera- ture and interference from such sources as detergents is minimized or taken into account. One must realize in the comparison between different bodies of water that the effect of differing chemical structure, the extent of polymeriza— tion and the action with salts on the fluorescence of the molecule has not been adequately defined. The origin of yellow organic acids found in waters may be either allochthonous or autochthonous with the later source, from the sediments and the decay of aquatic vegeta- tion, being the more important in the experimental ponds studied. These acids were primarily lost through a light- induced polymerization reaction with secondary loss attributed to their destruction by organisms as a source of energy or carbon. Diurnal and annual cycles were described which illustrate the integrated reSponse of a pond to these causes of change. The addition of yellow organic acids to natural waters produces changes in pH, conductivity, alkalinity and optical density. The first three may be explained by a hypothetical 127 128 union between these acids and calcium carbonate while optical density changes are a product of the characteristic light absorbance properties exhibited by the yellow organic acid molecule. Following the addition of acids the growth of Navicula sp., Closterium sp., Arthrodesmus Sp. and Surirella Sp. appeared to be stimulated while large copepods and cladocera were adversely affected. These changes in population domi- nance do not appear to be caused by large chemical or physical changes of the environment but more likely by subtle physio- logical responses to the acids. The concentration of these acids may indicate the stage of eutrophication. The direction and rate at which this process is moving may be easily calculated from a series of acid measurements through time. Many of the unique aspects of dystr0phic lakes may be explained through the interaction of these acids with the environment. The effect of low con- centrations of these acids in waters is not entirely under- stood at present though it is possible that many ecological relationships exist dependent upon the dynamics of yellow organic acids. LITERATURE CITED Anthony, E. H. and F. R. Hayes. 1964. Lake water and sedi- ment. VII. Chemical and Optical properties of water in relation to the bacterial counts in the sediments of twenty-five North American lakes. Limnol. Oceanog. 9:55-41. Aronoff, S., A. Benzon, W. Z. Hassid and M. Calvin. 1947. Distribution of C14 in photosynthesizing barley seed- lings. Science 105:664-665. Christman, Russell F. and Masood ghassemi. 1966a. The nature of organic color in water. Univ. Wash., College of Engineering, Dept. of Civil Eng. 45 pp. and Masood Ghassemi. 1966b. Chemical nature of organic color in water. J. Amer. Water Works Assoc. 58:725-741. and Roger A. Minear.- 1967. Fluorescence of lignin waste products. Univ. Wash., College of Eng., Dept. of Civil Eng. 22 pp.~ . 1967. The chemistry of rivers and lakes: The nature and properties of natural product organics and their role in metal ion transport. Environmental Sci. and Technol. .1:502-505. Clark, John M. Jr. (Ed.). 1964. Experimental Biochemistry. W. H. Freeman and CO., San Francisco. 228 pp. Diamond Alkali Company. 1960. Duolite ion exchange manual. 152 pp. . 1965. Duolite data leaflet no. 5, Duolite ion exchange resins. 2 pp. Kent, Fred and F. F. Hooper. 1965. Studies on iron-binding organic substances from Michigan waters. Papers of the Mich. Acad. of Sci., Arts and Letters. L:5-10. Patrick, Ruth. 1948. Factors affecting the distribution of diatoms. The Bot. Rev. 14:475-524. 129 150 Patrick, Ruth. 1954. The diatom flora of Bethany Bog. The J. of Protozoology. 1:54-57. 7. 1965. The structure of diatom communities under varying ecological conditions. Ann. of the N. Y. Acad. of Sci. 108:559-565. Phillips, Robert E., G. K. Turner Associates. Personal letter of June 5, 1966. Povoledo, Domenico. 1964. Some comparative physical and chemical studies on soil and lacustrine organic matter. Mem..Ist. Ital. Idrobiol., 17:21-52. and Marco Gerletti. .1964. Studies on the sedimen- tary, acid—soluble organic matter from Lake Maggiore (North Italy). I. Heterogeneity and chemical prOper— ties of a fraction precipitated by,barium ions. Mem. Ist. Ital. Idrobiol., 17:115-150. Saunders, George W. 1957. Interrelations of dissolved organic matter and phytoplankton. The Bot. Rev., 25:589-410. Shapiro, JOseph. 1957. «Chemical and biological studies on the yellow organic acids of lake water. Limnol. Oceanog. 2:161-179. . 1958. Yellow acid-cation complexes in lake water. Science 127:702-704. . .1964. Effect of yellow organic acids on iron and other metals in water. J. Amer. Water Works Ass., 56:1062-1082. "Standard Methods for the Examination of Water and Sewage," 1960, 11th ed., Amer. Pub. Health Assoc., New York. 626 pp. Transeau, Edgar Nelson. 1905-1906. The bogs and bog flora of the Huron River Valley. Bot. Gaz., 40, 41:551-575, 418-448. Udenfriend, Sidney. 1962. Fluorescence assay in biology and medicine. Academic Press, New York. pp. 106-108. Welch, Paul S. 1948. Limnological methods. McGraw-Hill Book CO., Inc., New York. 581 pp. Whipple, G. C. .1927. The microscopy of drinking water. 4th ed. Revised by G. M. Fair and M. C. Whipple. John Wiley and Sons, New York. 586 pp. APPEND IX 151 152 Table 1. Acid concentrations of the ponds through the experiment. Pond D Pond C Date N ppm N ppm Date average 7-19—1966 10 11.45 10 10.72 11.08 7-21 9 AM 11.46 10.65 11.05 7-21 5 PM 4 9.05 4 8.52 8.78 7-22 10 10.19 10 9.46 9.85 7-26 8:50 AM 4 10.80 4 10.06 10.45 7-26 9:50 AM 4 v10.21 4 9.91 10.06 7-26 7:50 PM 4 10.21 4 9.62 9.91 7-28 7:50 PM 4 15.07 4 11.75 12.41 7-28 9 PM 4 12.95 4 11.75 12.54 7-29 4 11.60 4 10.87 11.24 8-2 4 11.55 4 10.65 11.09 8-5 4 10.06 4 9.25 9.66 8-9 4 10.80 4 9.69 10.25 8-12 4 10.56 4 9.55 9.84 8-16 4 10.65 4 9.55 10.10 8-19 4 10.06 4 9.40 9.75 8-25 4 10.72 4 9.62 10.17 8-26 4 9.25 4 8.45 8.85 8-50 8:50 AM 4 9.25 4 8.81 9.05 8-50 11 AM 4 9.55 4 9.25 9.40 8-50 4 PM 4 9.11 4 8.67 8.89 9-2 4 9.11 4 8.67 8.89 9-6 4 9.40 4 8.74 9.07 9-9 4 9.40 4 8.89 9.14 Pond averages 10.47 9.72 10.10 Grand average 155 .Aoamom xmv COHHHHE mom munmm Op coamuo>coo ouomon .Oomm ou UODOOHHOO muflcs umpmaososam CH pump on» so Umfiuowuom mmB mammams4 ”ouoz mmmmm.mme mfim Hmboe mmmmfi. mmam.mm mmfi Honum do. swap mmoa mow.m omwmw. owomm.0fi mm coapomumucH do. away mmma mm.mmfi ¢m>m>.mm ammwm.mam mm mDEHB do. swap mmma mw.mmm N¢Omm.mm mwomm.mm a mpcom wuflaflnmnonm m moumsvm mDHMDUm EOOOOHM mouzom mo Eon cam: 00 85m mo mooumoa .mUcom DAD mo cowpmuucmocoo OHOC Mom magma mocmwnm> mo mammamc4 .N magma 154 Table 5. Acid concentration of pond D over a 25 hour period. Time N ppm 8-4-1966 5 PM 4 9.62 4 PM 4 9.62 5 PM 4 9.55 6 PM 4 9.47 7 PM 4 9.47 8 PM 4 11.09 9 PM 4 11.46 10 PM 4 11.60 11 PM 4 11.55 12 midnight 4 11.51 8-5-1966 1 AM 5 11.16 2 AM 4 11.55 5 AM 4 11.55 4 AM 4 11.58 5 AM 4 11.46 6 AM 4 11.24 7 AM 4 10.87 8 AM 4 10.87 9 AM 4 10.28 10 AM 4 10.06 11 AM 4 10.94 12 noon 4 10.94 1 PM 4 10.72 2 PM 4 10.21 5 PM 4 10.45 pond mean (8 PM through 5 PM only) 10.75 Note: From the start of the experiment through 7 PM a con- taminated standard was in use yielding the lower values. 155 .Amamum xm. COHHHHE Mom muumm Op coamum>coo muomon .0 own ou UODOOHHOO wuflcs Hmumfiouosam CH dump OSD so Umfiuomumm mmB mflmwamcd "Duoz >H>H.mmfi mm Hmuoe mmfimm. mum.wa w» Honnm a0. cmsu mmma mm.mm mmmmm.m wmm>.Nma «N muzom MDHHHQMQOHA._ m mmnmsvm moumsvm EOOOOHH monzom 00 Sam saw: 00 Sum mo mmmnmon .m 0cm 4 pmsms4 .mcHHmEMm H50: mm msflnsp 0 pcom CH mcoaumuucmocoo OHOM Mom magma OOCMHHC> mo mammamc4 .d OHQMB 156 Nmmmm.o mm x COHDCHDCOOCOU Odom mm¢o>.o mm HMSDHCS mm¢¢m.OI COHDCHOCDOCOD Odom mDCOHOHmmooo COHDOHOHHOO Hmauumm mmomo.o mumefiumm no sound Unaccmum mmmm.o u n OCDHOHMMOOO coaumaonuoo OHQHDHDE ooommomm.m 4w Hmuoa mwomwwoo.o smmOHmmm.o aw uouum do. cons whoa H>w>.NMH «mommdmm.o moannwmm.m m coammmummm .Qoum m monmswm moumsvm Eopmmum monsom 00 Sam coo: mo saw no mmoummn .maumsvm may ca.mm mo coaumcmmeO msu CH DSCOAMHcmHm muouomm mo mammamc4 .m magma 157 L I... I .P. mww.m u 0 HN>.m n U mammfi Ocom mmmuw>m Ucmnm mm mmo.m mfim.m som.m mas.m mo~.m mmmum>m CEDHOU mo.m H mm.m a Nm.s a mmm.n m >m0.m GOHDHOOC Ouflsu w>m.m oaa.m m Noo.m m ma>.m w mm4.m m waw.m ozp noum4 Nwm.m w Nww.m w Now.m w mum.w ma mwm.m mCOHuHOOm OHHQD 0cm Ocooom mmm.m Nna.m Ha mmm.m mN aam.m ma wwm.m on mo~.m COOBDOm mm.m H >m.m N IIIII I IIIII I m>m.m mCOHufiOOm Ocooom Ocm umuam mom.m «H.m N ma.m N mom.m w Nma.m m mom.m cooBumm mm.m m mH.m H mu.m N mm.m a- mwo.m COHDHOUC umuam fims.m 040.0 m mmh.m m mmm.m m mwm.m m Hoh.m whomom mcmoa mm 2. m0 2 m0 2 :0 Z m0 mama. coocuoumm coocuoumm mcacuoe mchHOE mmmuo>m mama mason mumq magma. 30m .DCOEHHOQNO map nmsounu mpcom onu mo mosam> mm .m OHQMB mm¢m¢>.mm mON Hmuoa 158 mHONOH.o HHHNHQ.>H msH Houum H. carp Amummum mma. amsmso.o msmssm.o m muobumm manna Haa Ho. swap mmOH Nmm.OH memno.H MNmmmN.m m mucom x mmuma H. cozy Houmoum >NH.H woomHH.o mmommo.H m mmEHu x mmpma H. cmsu umumoum NHo.N mMNmON.o memHm.o m moEHu x mpcom H. smnu Houmoum mam. mmOHmo.o HmNmmH.o m moumn Ho. cmnu mmmH mo>.ow HmwNmH.H Nmm>m¢.NH m mOEHB H. swap Hmummnm O>N.H mbhomH.o mnnomH.o H upcom muHHHQmQOHm. m mmumsvm mDHCDUWJl Eopwoum mousom MO San coo: mo 85m 00 momummn .mwcom on“ NO mm Mom OHQCD OOCMHHM> mo mHthmc4 .n OHQMB 159 omem.o huH>HuOCOCOO x COHumCquUCOO UHum MHNHH.OI mm NHmnm.o muH>HDOCOCOO HMHDHCH moo~m.0I COHDCCDCOOCOO OHOC mpCOHOHmHOOO COHumHOCHOO HmHuHmm NN>>.m OmeHumo mo COCHO UHmUCmum mmmm.o n H DCOHOHHHOOO COHumHOHCOO mHmHuHCE >5>m.Hmm0N HH Hmuoe mmNN.HH NomH.mmm OH Couum Ho. CmCu mmoH HHHm.mmm mmsm.0>om mumm.NmNON H COHmmmCmom MHHHHQmQOHm m woumsvm mmHmCWm EOOOOHM OOHCom mo Esm Cmmz mo 85m mo mmmumon CHCCCUC OCH CH muH>HuOCOCOU mo COHumCmmem OCH CH DCCOHMHcmHm muouomw mo mHmmHMCd .m OHQCB 140 Table 9. Conductivity of the ponds through the experiment. Pond D Pond C Date Date N umhos/cm N umhos/cm average 7-5-1966 10 .121.2 10 3156.6 128.9 7-8 10 172.5 10 188.4 180.55 7-12 10 161.2 10 .167.4 164.5 7-15 10 154.9 10 162.2 158.55 7-19 10 151.1 10 155.1 155.1 7-21 9 AM 4 140.25 144.25 142.25 7-21 5 PM 4 159.0 4 167.5 165.25 7-22 10 155.5 10 160.2 156.75 7-26 8:50 AM 4 146.25 4 150.0 148.125 7—26 9:50 AM 4 152.25 4 152.5 152.575 7-26 7:50 PM 4 148.5 4 151.25 149.875 7-28 7:50 PM 4 145.0 4 159.75 142.575 7-28 9 PM 4 145.5 4 144.5 144.0 7-29 4 144.0 4 141.5 142.75 8-2 4 144.25 4 142.5 145.575 8-5 4 152.25 4 148.75 150.5 8-9 4 148.75 4 147.5 148.125 8—12 4 157.5 149.0 155.25 8-16 4 162.75 150.25 156.5 8-19 4 166.25 151.75 159.0 8-25 4 176.75 4 154.5 165.625 8-26 4 179.25 4 162.25 170.75 8-50 8:50 AM 4 179.75 4 165.0 171.575 8-50 11 AM 4 179.0 4 161.75 170.575 8-50 4 PM 4 174.75 4 158.75 166.75 9-2 4 188.75 4 167.75 178.25 9-6 4 ~184.25 162.75 175.50 9-9 4 184.5 4 161.0 172.75 9~15 4 187.0 4 169.0 178.0 Pond averages 158.67 157.01 157.84 Grand average 141 HN¢.HHm>m mom Hmuoe Hmoo.H mN.mmm mHN COACH Ho. CmCu mmOH ¢.mm HNm¢.¢mm om>.mHOHH mN COHDOCCODCH Ho. COCO mmOH N.©mH w0H4.mme www.mOSmm NN onH6 H0. CCCD mmmH H.Nm www.mom mmm.NON H mOCom mpHHHQmQOCm m mmumsvm mmumswm EOUOOHH OOHCOm MO San CODE 00 saw no mmmumma .mUCom OCH Ho >DH>HuOCOCOO Com OOCmHHm> mo mHmemC4 .OH OHQMB 142 HmNmm.OI mm HCHuHCH «mmmm.OI COHumuuCOOCOO OHom HCHuHCH HNmmm.o muHmCOU HCOHumo x COHumHuCOOCOO OHOC mmHnH.o muH>HUOCUCOU x COHumHuCOOCOU UHUm nmem.OI pmumsvw COHumHuCOOCOU OHUm mwom>.o COHumHuCOOCOO UHOM muCOHOHmmOOU COHumHOCCOO HCHqum MNmoo.o OumEHumO mo COHHO UumpCmum mmmm.o u C CCOHUHHHOOO COHDMHOCCOO OHQHCHCE mmmmmNmH.o 44 Hmuoa mammoooo.o NHSMHHO0.0 Sm COHHM Ho. COCu mmmH mmmm.wmm HsmmmHNo.o meHmomH.o h COHmmOHmOm mpHHHQmCOCm . m mmumsvm moumsvm EOUOOCH OOCCOm mo Esm Cmoz HO 85m 00 moonmon OCu CH huHmCOU HCOHHQO mo COHumCmmeO OCC CH quOHmHCmHm muouomm mo mHmmHMCd .mHCmCUm .HH OHCCB 145 Table 12. Percent transmission of the pond water at 550 mu through the study. Pond D Pond C Date Date‘ ” ’ N % N average"' 7-5-1966 10 88.85 10 89.50 89.075 7-8 10 88.65 10 89.70 89.175 7—12 9 88.78 10 90.65 89.765 7-15 10 89.55 10 90.70 90.125 7-19 10 88.05 10 89.05 88.55 7-21 9 AM 88.125 89.50 88.812 7—21 5 PM 4 88.625 4 90.25 89.458 7-22 10 88.25 10 89.75 89.00 7-26 8:50 AM 4 87.875 4 89.00 88.458 7—26 9:50 AM 4 87.625 4 89.50 88.562 7-26 7:50 PM 4 89.125 4 90.575 89.75 7-28 7:50 PM 4 86.875 4 88.125 87.50 7-28 9 PM 4 87.50 4 88.125 87.812 7-29 4 87.575 4 88.875 88.125 8-2 4 87.875 4 88.50 88.188 8-5 4 89.625 4 90.625 90.125 8-9 5 88.667 5 88.855 88.75 8-12 5 88.667 4 89.50 89.145 8-16 4 88.25 5 89.555 88.714 8-19 4 89.875 4 90.0 89.958 8—25 4 88.625 4 90.125 89.575 8-26 4 90.0 4 90.75 90.575 8-50 8:50 AM 4 90.50 4 90.25 90.575 8-50 11 AM 4 90.575 5 89.855 90.145 8-50 4 PM 4 90.0 2 90.0 90.0 9-2 4 91.125 4 90.50 90.812 9—6 4 89.75 4 89.575 89.562 9—9 4 90.575 4 90.25 90.512 9—15 4 89.625 4 90.0 89.812 Pond averages 88.866 89.728 89.294 Grand average 144 HmNH.H>m mmN Hmuoe mmNH. ome.NOH NMN Conum Ho. COCu mmOH Hmm.N HHHN.H mem.wm mN COHCOOCOHCH Ho. COCu mmOH mm.wH omow.w HmmN.m>H mN OOEHB Ho. COCO mmOH mm.>NH mnmm.Hm mumm.dm H mpCom mpHHHCOCOCm m mOCOCOm OOCOCUO EOOOOCH Oousom mo ECO COOS Ho 85m 00 mOOHmOQ .mCOum3.OCom OCH mo COHOOHEOCOCD CCOOCOQ Com OOCOHHO> mo mHmeOCC .mH OHQOB 145 Table 14. Carbonate alkalinity of the ponds through the study. Pond D Pond C Date Date N ppm N ppm average” 7-11-1966 2 18.5 2 6.6 12.45 7-14 2 18.5 2 7.9 15.2 7-18 2 19.7 2 8.6 14.15 7-21 AM 2 21.0 2 10.9 15.95 7-21 PM 2 24.1 2 15.0 18.55 7-25 2 24.5 2 11.5 17.8 7-28 2 17.7 2 10.6 14.15 8-1 2 21.5 2 12.8 17.05 8-4 2 20.9 2 14.4 17.65 8-8 2 16.5 2 15.9 15.1 8-11 2 15.5 2 15.7 14.60 8-15 2 12.5 2 15.4 12.95 8-18 2 11.2 2 15.0 12.1 8—22 2 5.6 2 8.6 7.1 8-25 2 4.2 2 10.7 .7.45 8-29 2 9.8 2 17.1 15.45 9-1 2 4.0 2 9.6 6.8 9-9 2 2.0 2 7.4 4.7 9-12 2 5.0 2 8.2 5.6 Pond averages 14.20 11.14 12.52 Grand.average 146 mmmwNN.mHmN ms Hmuoa mNmow. om.om mm Conum Ho. COCO mmOH H.mw HoomHN.mm mn0¢m¢.mmm NH COHCOOCODCH Ho. COCC mmOH H.mm mHmmm.H> Homnmm.mHMH NH mOEHB Ho. COCu mmOH H.HNN mumHN.NSH mnmwN.me H mOCom MHHHHQOQOCC. m OOHOCOO OOHODUO EOUOOCH OOHCOm mo ECO COOS 00 Sam mo mOOCmOQ .OUCOQ OCu CH muHCHHOCHO OuOCOQHOO mo OOCOHHO> mo mHmmHOCC .mH OHAOB 147 Table 16. Bicarbonate alkalinity of the ponds through the study Pond D Pond C Date Date” N ppm N ppm averages 7-11-1966 2 48.75 2 65.05 55.90 7-14 2 48.65 2 59.55 54.00 7-18 2 42.05 2 55.70 47.875 7-21 AM 2 59.65 2 50.55 45.00 7-21 PM 2 55.90 2 48.20 42.05 7-25 2 51.05 2 45.50 58.275 7-28 2 56.60 2 45.65 41.125 8-1 2 54.5 2 41.70 58.00 8-4 2 54.85 2 58.55 56.60 8-8 2 45.50 2 42.5 42.90 8-11 2 40.65 2 58.15 59.40 8-15 2 45.00 2 58.55 41.775 8-18 2 45.85 2 58.60 42.225 8-22 2 57.65 2 45.10 50.575 8-25 2 61.25 2 42.4 51.825 8-29 2 49.50 2 57.95 45.625 9-1 2 54.20 2 58.00 46.10 9-9 2 67.40 2 50.55 58.875 9-12 2 69.90 2 48.40 59.15 Pond averages 46.65 45.47 46.056 Grand average 148 NNN>.NNNN Nb HOCOB SNNN.H ONNO.HN Nm Houum Ho. COCO mmOH mm.¢m NNOH.NNH ONNN.NNNN NH COHHOOCOCCH Ho. COCu OOOH >.NHH >>NH.OON mmmH.NONN NH mOEHB Ho. COCO mmOH >.NH. mNNN.NN mNNm.NN H OUCom muHHHCOCOCm m OOCOCOO mOCOCOO EOUOOHH OUHCON mo ECO COOS mo EON Ho wOOCmOQ .OOCOm OCC CH muHCHHOxHO OuOCOQCOOHQ mo OOCOHCO> mo OHOMHOC4 .HH OHQOB 149 OmOCO>O OCOHO HOSO.I mHm.I mmH.I mmmmum>m Ozom coHHHOOm mmmmaI Hem.I ms mso.+ mm OAHCH Amumm OAHCH mwmm.I mmm.I HOH omm.HI mmH 8cm Ocoomm COOBHOm UCOOOO HmOO.NI mHH.HI HH www.mI m cam HmAHH COOBHOm aoHHHOOO Hmmm.I mwm.I OOH «oo.+ ms HmAHH . OHOHOm OmOHO>O U 0Com z 0 0Com z UOHHOQ OOHHOm OEHB a .Hcsum OCH CmCOCCH mUCom OCH mo .CmOHmOEHOCH HOHOB HO 0 0Com mo OHCHOHOQEOH OCCHE OHHO mCHHCmOOE HO OHCHOHOQEOH UCOQV OOCOHOHHHU OHCHOHOQEOB .NH OHCOB 150 NOHNHN.ONOH HHN HOHOE NNHNSN.H NNHOONS.NHN HON COHHm Ho. COCH OOOH Nm.HN NNONHN.NN NHNHNNN.>OH m COHHOOHOHCH Ho. COCH mmOH NH.NH HMONNH.NN HOHNNH.NN m OOEHB OH. COCH COHOOCm NH.O NHNSNNN. NHNHNNN. H OUCom NHHHHCOCOHC. m OOHOCOO mOHOCvm EOOOOHH OOHCON Ho ECO COOS mo ECN Ho mOOHmOQ .OOCOQ OCH Ho ACQOHNOEHOCH HOHOB HO 0 0Com mo OHCHOHOQEOH OCCHE OHHm mCHHCmOOE HO OHCHOHOQEOH OCOQV OOCOHOHHHO OHCHOHOQEOH mo OOCOHHO> mo mHmmHOCC .NH OHCOB 151 NNNH. OOOHO>O OCOHO OONN. 0 NHNH. U mOmOHO>O 0Com mmmH. HHHH. mmmmum>m COOHHOQEOU NOHH. H NHSm. H 0 COHHHOOO NHNH. UHHCH SNBH. NH NHNN. NH O HOHHC mmNN. NH >OON. NH 0 UHHCH NNwH. NCO OCOOOO mNHH. HN NONH. HN 0 COO3HOm NNNN. H NNSN. H 0 OCOUOO HHNN. NCO HOHHH NONN. N NNNN. N O COOBHOm NSHN. H OHNN. H 0 COHHHOUO SNHN. HmHHH NNNN. N HNNH. m U OHOmOm OOOHO>O HOHOEOHHOCHNm z HOHOE HH4 z 0Com OOHHOm OOHCOm "HOHOBHOOCD "HOHOBHOOCD OEHB .NOCHO OCH COCOHCH mpCom OCH HO .OOOHHCO OH HOHOSHOOCCV OOHHOH COHHOHHOCOQ HCOHH .ON OHCOB 152 HNNNON.N NNH HOHOB HNSHNNHO. NNNOHHNN.H OHH HOHHm OH. COCH HOHOOHO NHN.O NNHNNNOO. NNNNNNOO. N OOHCH HH4 OH. COCH HOHOOCN NNN.H NNNNOHNO. NNNNHNNO. N mComHHOmEOO x OOEHB 0H. swan Cmummnm mom.o HmmoomHo. mmHmommo. m mmeHH x mwaom OH. COCH COHOOHO NHN.O NNNNNNOO. NNNNNNOO. H OCOOHHOQEOO x OOCom HO. COCH mmOH ON.N HNNOONOH. NNNNONHN. N OOEHB OH. COCH HOHOOHN NHN.H NNNNHNNO. NNNNHNNO. H mCOOHHOmEoo HO. COCH mmOH ON.N NNNNNNNH. NNNNNNNH. H mOCom NHHHHCOCOHA m OOHOCOO OOHOCOO EOUOOHH OOHCON Ho ECO COOE Ho EON HO OOOHOOQ .OOCOQ OCH HO .OOOHHCONHOHOBHOOCCV OOHHOH COHHOHHOCOC HCNHH Ho OOCOHHO> mo mHmmHOCC .HN OHCOB 155 Table 22. Mean change in ppm dissolved oxygen per hour be- tween 10 PM and 4 AM the following morning as a measure of total reSpiration in the ponds during the study. Time Pond C Pond D Period period N ppm/hr N ppm/hr average Before first 9 -.1187 7 -.1529 -.1557 addition Between first and 4 -.1742 5 -.1994 -.1850 second Between second and 19 —.1541 14 —.1575 -.1556 third After third 6 -.1484 2 -.1016 -.1567 addition Pond average -.1569 -.1460 -.1406 Grand average 154 ONHNNOOSN. NN HOHOB NNNNNHOO. NNNSNHNHN. NN COHEN OH. COCH HOHOOHO OmN.O NOHONNNOO. ONNHBBAOO. N COHHOOHOHCH OH. COCH HOHOOHO ONH.H HNHHSHNOO. NHNHNNNHO. N mOEHB 0H. COCH Ambmmum Hmm.o mmmmHmHoo. mmmmHmHoo. H mccom mHHHHCOCOHm m mOHOCOm mOHOCUm EOOOOHH OOHCON Ho ECO COOE HO EON Ho OOOHOOQ .mOCom OCH CH OCHCHOE OCHBOHHOH OCH 24 H OCO Em OH COO3HOC HDOC HOm COmwxo OO>HOOOHO E00 CH OOCOCU COOE mo OOCOHHO> HO mHmeOCd .NN OHQOB 155 Table 24. Ppm oxygen concentration at 10 PM as a measure of total photosynthetic oxygen production in the ponds. Time Pond C Pond D Period period N ppm N ‘ ppm mean Before first 9 6.776 7 7.750 7.195 addition Between first and 4 6.760 5 7.557 7.095 second Between second and 19 6.954 14 6.897 6.950 third After third 6 6.758 2 6.565 6.645 addition Pond mean 6.857 7.154 6.978 Grand mean 156 HNNHNN.NH NN HOHOB OnNmHn. NNNHNN.HH NN HOHHN OH. COCH COHOOCO NNN.H NNHNH.H HNmNmH.N N COHHOOHOHCH OH. swab AmummAm man. Hmmmm. OHmmmH.H m mmeHH OH. COCH HOHOOHO NHN.H SHNNNN.H >5NNNN.H H mOCom NHHHHCOCOH0 0 OOHOCUO OOHOCUO EOOOOHH OOHCON mo ECO COOE mo ECN mo mOOHmOQ .mOCo0 OCH CH 20 OH HO COHHOHHCOOCOO COmhxo E00 00 OOCOHHO> Ho mHthOCC .NN OHCOB 157 OCOOE OCOHO NH.N HN.H S.HH NH.N NN.H H.HH NN.N NN.H N.HH mCOOE Ocom NN.N NN.H HN.NN NN.N OH.H HN.HN N NN.> HO.N HH.NN N HHIN NH.N bN.H HN.NH ON.N HN.H NN.NH N ON.N NN.H NN.NH N SIN NH.H NO.N NH.HH NN.N HH.H HN.N N NO.N HN.N HN.HH N HNIN NH.> NH.H NN.N NN.H NN.H NH.NH N ON.N HN.H NN.H N 20 ONIN HN.> ON.H NH.NH NO.H NH.H NO.NH H NH.H HH.H HN.NH H 24 ONIN NH.N NN.H NN.> ON.N NO.H hm.N N NN.N NN.H HN.> H HNIN NN.> NN.H NO.N OO.> NN.N NO.H N NH.H NH.H NH.N N SHIN NN.N HH.H HN.N ON.N NN.N HH.N N ON.» NN.H EN.m N OHIN HH.N HH.N NH.N OO.N NN.N NN.N N NH.N HH.N NH.N N NIN ON.N NN.N O>.> ON.N NN.N ON.H N HH.N ON.N NN.H N HNIN OH.N NO.H HH.NH NN.H HN.H NN.NH N ON.N NN.N NH.NH N ONIS NH.N HN.N NN.N ON.N NH.N NO.H N NN.H HH.N HH.OH H NHIH NN.N NN.H HN.HH NO.N NN.N NO.NH H HN.N NH.N NN.N N NNNHINIH m2 O2 O0 m2 O2 O0 % m2 O2 OU % OHOQ Ii mCOOE OHOQ .NUDHO.OCH COCOHCH OUCO0 OCH CH OCOHHOHHCOOCOU COH HOHOE .E00v 0 0Com .E00. 0 0Com .NN OHQOB 158 NNNNNN.NHN NN HOHOB NHOOHO. OOOHHH.HO mm AOAAN OH. :mCH HOHOOAO ONH.H HHHOHO.H HmHmm.NH NH OOHuomAOHOH HO. cusp mmmH Ono.mH HOHNHO.HH HHNOOO.NHH NH mmaHH no. cash AOAOOAO ONO.H OHONON.H OHONON.H H mOcom .E00 CH OHOOV ECHOOCOOZ ONHOHHO.NO ON Hmboe OHONOHO. mHommH.NH NO COACH OH. Oman AOHOOHO NHN.H HmHNOHm.H HOHOOHN.OH NH OOHHOOAOHOH HO. COCH mmmH OHO.N mHmOOO.N HOHNOOO.HN NH mmsHe OH. COCH AOHOOAO mmm.o OOHHmOm. OOHHmOm. H mOcom .E00 CH OHOOV ECHOON NNNHN.HNNNN NN HOHOB NONNN.NNH NNNNN.NnNHH NN HOHHm OH. COCH HOHOOHO NHN.O ONONN.NHH HNNNN.NN>H NH COHHOOHOHCH HO. COCH OOOH N.OH NHNN>.HNON NNHOH.>NNHN NH OOEHB OH. COCH HOHOOCO NON.O NmNnH.NN NNNNH.NN H mOCo0 .HOHOEOHOC0 OEOHH Eoum COHOOHEOCOHH & CH OHOOV ECHOHOU NHHHHCOCoum 0 OOHOCOO OOHOCOO EOOOOHH OOHCON Ho ECO COOE Ho EON Ho mOOHmOC .OOCO0 OCH CH OCOH HOHOE How OOCOHHO> Ho mHmmHOCC .NN OHCOB 159 Table 28. Optical density data of extracted chlorophyll from periphyton from the ponds. Pond C Pond D Date Date N O.D. N O.D. mean 7-4 to 7-18 5 .020 4 .010 .0145 7—11 to 7-25 4 .0252 5 .0224 .0257 7-18 to 8-1 5 .0112 5 .0174 .0145 7-25 to 8-8 5 .0114 4 .0178 .0142 8-1 to 8-15 5 .0100 5 .0166 .0155 8-8 to 8-22 5 .0094 5 .0172 .0155 8-15 to 8-29 5 .0066 5 .0158 .0112 8-22 to 9~5 5 .0522 5 .0582 .0552 8-29 to 9-12 5 .0220 5 .0577 .0298 160 NHNNNNNOO. ON HOHOB NHNNHHOOOO. NNNHNNOOO. NN Houum HO. COCH mmOH NNN.N NONNNOOOO. NNONHnooo. N COHHOOHOHCH HO. COCH mmOH. NN.NH NHNNNNOOO. NNNNNNNOO. N mOEHB HO. COCH mmOH NH.NN NNNNNNOOO. NNNNNNOOO. H OUCom NHHHHCOCoum 0 OOHOOUm mOHOOUO EOOOOHH OOHOON HO EON COOE mo EON mo OOOHOOQ .OOCO0 OCH EOHH COHNC0HHO0 EOHH OOHOOHHxO HH>C0OHOHCO Com OHOO hHHmCOO HOOHH0O HON OOCOHHO> mo mHmeOCC .NN OHQOB ”'lililijlillillillllllllias