RESPONSE OF PERIPHYTON TO PHOSPHORUS iNTRODUCED INTO A MECHE’GAN TROUT WREAM Thesis {or flu Dayna of M. 5. MICHIGAN STATE UNIVERSETY Hugh F. Clifford £959 I\\\W\T\Y\\\N\W\\"\K\if\\ii\ii\\\\'\i\i\‘\\i'\f\j\\l L 3129310631620 LIBRARY Michigan State Univenky RESPONSE OF PERIPHYTON TO PHOSPHORUS INTRODUCED INTO A MICHIGAN TROUT STREAM By I” a HUGH F". CLIFFORD AN ABSTRACT Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1959 Approved ABSTRACT The summer program of 1958 for the West Branch of the Sturgeon River was divided into two closely related phases. For the second consecutive summer, phosphate in the form of an inorganic fertilizer was added directly to the river. No immediate positive response by the standing crop of the periphyton complex could be detected, although the periphyton mass increased in total phosphorus. When the amount of fertilizer was increased and the substrates were allowed to remain in the water a longer period of time, the standing crop of periphyton gave indications of increasing. Fast river currents appeared to affect the standing crop of periphyton adversely. Twenty three mi icuries of P32 was applied to the West Branch on August 5, 1958. The periphyton complex was initially responsible for the great amount of P32 retained in the experimental area. Downstream from the point of addition, the initial uptake of radiophosphorus decreased. Seven days after the addition of the isotope, the P32 of the periphyton was uniformly distributed in the experimental area. The P32 was believed rapidly lost from the peri- phyton; biologically to other organisms, and physically to the inanimate bottom complex and the current which transports it out of the system. H. F. C. RESPONSE OF PERIPHYTON TO PHOSPHORUS INTRODUCED INTO A MICHIGAN TROUT STREAM BY HUGH F. CLIFFORD A THESIS Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1959 6/057/ V'JL-go ACKNOWLEDGMENTS The writer would like to express his sincere gratitude to: Dr. Robert C. Ball for his guidance and assistance while super- vising the program; Dr. Frank F. Hooper for his guidance and assistance in many phases of the study while acting as co-investigator of the project; Dr. Philip J. Clark for statistical guidance; Dr. G. W. Prescott for guidance and assistance in the taxonomic study; William C. Bryant, David P. Borgeson, and Allen W. Knight with whose aid this study was carried out; Dr. W. Carl Latta and the staff of the Pigeon River Trout Research Station. This study was made possible by a graduate research assistantship from the Michigan State University Agriculture Experiment Station. ii TABLE OF CONTENTS INTRODUCTION ....................................... Description of the Study Area ...................... PRELIMINARY STUDY .................................. Sampling Stations ................................. Methods and Procedures ........................... Composition of Periphyton Communities on Artificial Substrates ...................... Fertilization ............. A .................. Periphyton ................................ Phosphorus Content of the Periphyton ......... Fluctuation and Water Temperature ........... Velocity of the River Water .................. Results and Discussion ............................. Composition of Periphyton Communities on Artificial Substrates ...................... Periphyton ................................. Phosphorus Content of the Periphyton ......... Velocity of the River Water ................. RADIOPHOSPHORUS STUDY ............................. Methods and Procedures ........................... 10 12 l3 13 16 16 ‘22 33 37 43 43 Page Description and Preparation of the Study Area . 43 Description and Distribution of the Isotope ..... 46 Field Sampling Methods. ..................... 47 Laboratory Procedures ..................... 48 Measurement of Activity ................... 49 Results and Discussion ............................ 52 Initial Uptake of P32 by Periphyton ........... 52 Accumulation of P32 in Periphyton ........... 57 Exchanged and Regenerated P32 .............. 62 Loss of P32 from Periphyton ................ 65 Accumulation of P32 in Rock Periphyton ....... 75 Position of Periphyton in Relation to Phosphorus 78 SUMMARY ............................................. 82 APPENDIX ............................................ 85 LITERATURE CITED .................................. 89 iv LIST OF TABLES Table Page 1. Relative frequency of diatoms counted in thirty microscopic fields selected from artificial substrates placed in the West Branch of the Sturgeon River July 11, 1958, and removed July 18, 1958 ................................... 17 2. A qualitative list of the algae identified in the West Branch of the Sturgeon River in the summers of 1958 and 1959 ........................ 21 3. Mean density of chlorophyll extracted from peri- phyton on weekly substrates from stations VII, 1-12--Expressed as Klett units ................... 23 4. Results of "t" tests calculated from mean chloro- phyll content of stations VII, 1-6, and stations VII, 7-12; for.the period of July l-8, 1958, and July 11—18, 1958 ............................... 28 5. Mean density of chlorophyll extracted from peri- phyton on weekly substrates from stations VII, 1-12, and bi-weekly substrates from the odd numbered stations below station VII--Klett units, 1958 .................................... 32. 6. Mean value of total phosphorus in the periphyton mass on weekly substrates from stations below permanent station VII--Expressed as p. p. b. , 1958 ....................................... 34 7. Result of "t" tests calculated from mean p. p. b. of total phosphorus in the periphyton of stations VII, 1-6 and 7-12, July 1-8, and July 11-18, 1958. . . 35 Table Page 8. Test of regression line computed from velocity of the river water and chlorophyll at four stations below permanent station VII on the West Branch of the Sturgeon River. 1958 ....................... 41 9. Initial uptake of P32 by periphyton four hours after the addition of the isotope to the West Branch of the Sturgeon River, August 5, 1958 ,,,,,, 53 10. Accumulation of P32 in periphyton of the'West Branch of the Sturgeon River for the third and seventh day after the release of the isotope, August 7, and August 11, 1958 ................... 53 11. Mean value of P32 accumulated in periphyton of the West Branch of the Sturgeon River per section for the first seven days after the addition of the isotope .............. 60 12. Accumulation of P32 (corrected counts per minute) in periphyton of the West Branch of the Sturgeon River for the thirteenth, nineteenth, and twenty- seventh day after the addition of the isotope, August 17, August 23, and August 30, 1958 ........ 61 13. Percent gain or loss of P32 activity in periphyton of of the West Branch of the Sturgeon River from August 5, 1958 to August 7, 1958; and from August 7, 1958 to August 11, 1958 ................ 73 14. Summary of weights, counts, and computation data per station (artificial substrates) of P32 in peri- phyton, August 5, and August 7, 1958 ,,,,,,,,,,,,, 86 15. Summary of weights, counts, and computation data per station (artificial substrates) of P32 in periphyton, August 11, to August 30, 1958 ,,,,,,,,, 87 16. Summary of weights, counts, and computation data per station (rocks) of P32 in periphyton, August 9, 1958. to August 23, 1958 ..................... 88 Vi Figure II. III. IV. VI. VII. VIII. LIST OF FIGURES Map of the West Branch of the Sturgeon River area, showing the stations used in the preliminary study , , Mean weekly water temperatures and daily staff gage readings from station VII on the West Branch of the Sturgeon River--July and August, 1958 ...... Relative frequency of the four major diatoms from artificial substrates placed in the West Branch of the Sturgeon River, July 11, 1958 and removed July 18, l958--Calculated in thirty fields from a . millipore filter pad ............................. Mean density of chlorophyll and total phosphorus from periphyton on weekly shingles of stations below permanent station VII--Expressed as Klett units and p. p. b. , 1958 ............................... 14s a“ Mean density of chlorophyll from weekly:hsni1bstrates of stations above permanent station VII, and bi-weekly substrates from stations below permanent station VII--Klett units, 1958 ........................... Comparison of the velocity of the surface water over the odd numbered stations below permanent station VII with the chlorophyll concentration on substrates for these stations, 1958 ............... Map of the West Branch of the Sturgeon River area, showing the stations used in the radiophosphorus study .......................................... Error in counts per minute for two minute counts, 95 percent confidence level. (After Kinsman, from Krumholz, 1954) ................................ Page 15 19 26 3O 39 45 51 Figure IX. XI. XII. XIII. XIV. Initial uptake of P32 by periphyton on artificial substrates four hours after the addition of the isotope for the entire experimental area of the West Branch of the Sturgeon River, August 5, 1958 ................................. Summary of P32 activity in periphyton of the West Branch of the Sturgeon River, 1958 .......... Mean loss of P32 activity in periphyton on artificial substrates for the entire experimental area during the first seven days after addition of isotope .................................... Percent gain or loss of P32 activity in periphyton from August 5, 1958 to August 7, 1958; and from August 7, 1958 to August 11, 1958 . .._ .................... Summary of activity of P32 in rock periphyton in the experimental area of the West Branch of the Sturgeon River for the summer of 1958 ................... Mean activity of radiophosphorus for the various biological levels sampled in the experimental area of the West Branch of the Sturgeon River during August, 1958 ............................. viii Page 55 64 67 71 77 80 INTRODUCTION In a relatively short span of years, the theories and practices of fish production in" our inland waters have passed from the uncomplicated ideas limited to legislation and stocking, to complex theories involving the entire environment of the fish. This environ- ment encompasses a vast array of biotic and abiotic factors. One of the primary abiotic factors is nutrients. Their translocation through the food chain makes them inseparably interrelated to the living organisms of a particular environment. The nutrient, phosphorus, and algae of the primary producers are two of these specific inseparable components that contribute to the food chain in a lotic environment. A better understanding of the relationship and interaction of these two components would contribute knowledge that could help formulate future theories and practices of fish production in our inland waters. In 1954 an experimental program was initiated on the West Branch of the Sturgeon River that was designed to evaluate the chemical, physical, and biological responses to the addition of nutrients in the form of inorganic fertilizer. The nutrients studied included both phosphorus and nitrogen; the biota of the food chain encompassed all trophic levels with the exception of converter organisms. This thesis contains part of the research performed on the West Branch during the fifth summer of the program, 1958, and is concerned with 1 to the nutrient, phosphorus. the response of the periphyton The experimental program of 1958, pertaining to peri- phyton, was divided into two closely related phases. The initial phase, from June 15 to July 28, was concerned with evaluation of periphyton responses to inorganic fertilizer, detection of total phosphorus in the periphyton mass, and measurement of certain physical factors that may be related to periphyton growth. These studies are treated in the initial section of the thesis. The remaining section of the thesis is concerned with the second phase of the summer's program, namely that of uptake, accumulation, and translocation of radioactive phosphorus in periphyton. Description of the Study Area The West Branch of the Sturgeon River is a hard-water stream which originates in Hoffman Lake, a marl lake located in Charlevoix county (T. 32N. , R. 4W, Sec. 26, 27, 34, and 35) Michigan. The river flows approximately thirteen miles through a narrow water- shed before its confluence with the Sturgeon River near Wolverine, 1Periphyton in this study follows closely that definition by Young (1945), pertaining to the assemblage of benthic or encrusting algae growing on free surfaces. It does not include animal materials as defined by Newcombe (1949). FIGURE 1. --Map of the West Branch of the Sturgeon River area, showing the stations used in the preliminary study. West Branch VI VII 0 7 lil VIII 3 5 ,1; 1 , ,f ’/ ’ 2 4 Fertilizer added N . 5 One half mile Cheyboygan county. The topography and general features of the river have been described by Grzenda (1955), Colby (1957), Keup (1958), and Carr (1959) and need not be elaborated on here. The experimental area for both the preliminary and iso- tope study of 1958 is a 2, 700 yard section of the river situated between permanent stations VI and VIII (Figure I). In the first 250 yards of the area, the stream is well sunlit and supports an abundant growth of Chara. Below this for the next 300 yards the gradient increases and the river is heavily shaded by cedars, hemlock, nine bark, and tag alder. Within this section is found the most impoverished growth of Chara for the entire experimental area (Knight, unpublished). From here to the terminus of the study area, 100 yards above Fulmer Creek (Station VII, 12), the gradient of the stream decreases and the stream in most places is well sunlit. Luxuriant growths of £11351 are to be found throughout this section, where they have developed on bars of sand and organic detritus. In this latter part of the experi- mental area, stream deflectors have created a habitat of pools alter- nating with swift-running areas. The entire experimental area is devoid of domiciliary and agricultural habitation as are all feeder streams, with the exception of a small run located approximately 600 yards above the terminus of the area which may deliver small amounts of extraneous nutrients to the river. Flow data indicatesauniform increase in water for the entire experimental area. Flow in cubic feet per second on July 7, 1959 for the beginning of the area, middle section (Station VII), and terminus of the area were 38. 17, 43. 48, and 49. 72 c. f. s. respectively. 2 2Courtesy of Carr and Vannote, 1959. PRELIMINARY STUD Y Sampling Stations Figure I shows the location of the sampling stations for the preliminary study of periphyton. The twelve stations in the upper area (stations VI, 1-12) are located at approximately 150 yard intervals. These stations served to evaluate data relating to the amount of peri- phyton that could be measured at the end of seven and fourteen day intervals at these specific localities. The twelve stations below station VII (VII, 1-12) were also located at approximately 150 yard intervals. Stations VII, 1-6 were above the point of fertilizer application while stations VII, 7-12 were subjected to the fertilizer effects. An attempt was made to establish all twelve stations in an environment that would be uniform to such physical factors as current velocity, light, and depth. Stations VII, 1-12 were used for the evaluation of fertilizer effect on periphyton growth and the amount of total phosphorus in the periphyton. Methods and Proc edure s Composition of Periphyton Communities on Artificial Substrates A study of the periphyton complex was started in the summer of 1958 and completed in the summer of 1959. Along with the study of the periphyton composition on plexiglass substrates, an attempt was made to identify the major filamentous and other algae of the West Branch. In 1958 the substrates taken from the stream were frozen immediately upon arrival at the field laboratory. Analysis was made the following winter. Since this method would preclude identification of algae other than the diatoms, during the summer of 1959 periphyton on artificial substrates was stored in a 6-3-1 algal preservative. The organisms were scraped from the substrates and filtered through a Millipore filter having a pore diameter of O. 45 microns. The density of organisms on a single substrate was such as to allow the entire periphyton complex of that shingle to be concen- trated in this way. Tabulation of frequency of occurrence of algae, and in some cases identifications, were made directly from the pad. The number of times an organism was observed in 30 fields was used as a measure of frequency of occurrence. Organisms that could not be identified with certainty on the filter pad were studied in water mounts . Fertilization To determine the response of periphyton to artificial enrichment, fertilizer in the form of di-ammonium phosphate was added to the river between stations VII, 6 and VII, 7. The growth of periphyton below this location (stations VII, 7-12) was compared with the growth in a section where the periphyton was not exposed to added nutrients (stations VII, 1—6). If an appreciably greater growth of periphyton occurred in the section of the river exposed to fertilizer (VII, 7-12) than in the unfertilized water (VII, 166) then fertilizer would be added at the point of isotope release in order to assure a rapid uptake of the radioactive phosphorus and to insure a measurable amount of periphyton. Eighty pounds of inorganic fertilizer was first added to the river between stations VII, 6 and VII, 7 on July 3, 1958. The apparatus used to distribute the fertilizer was essentially the same as that described by Correll (1958). Two 55 gallon drums were placed on the stream bank and the fertilizer mixed with river water in the drums. The siphoning apparatus was designed so that both drums would empty at a predetermined constant rate. Fertilization proceeded for a total of 48 hours. The rate of addition was not uniform. Mechanical failures in the sediment trap and particles clogging the jet were considered to be major factors in this erratic behavior of the fertilizer apparatus. Because of this non-uniformity in distribution of the fertilizer, no attempt was made to calculate the actual rate that the nutrients (phosphorus and nitrogen) entered the river. The 80 lbs. of fertilizer was distributed sometime within this 48 hour period. 10 The second application of fertilizer between stations VII, 6 and 7 was started on July 20, 1958 and continued intermittently until July 26, 1958, a period of six days. Between these dates, approximately 150 lbs. of fertilizer was added to the river. Periphyton The method used for collecting periphyton on artificial substrates in 1958, the fifth year of the program, was modified from those used the four previous years. Grzenda (op. cit. ), used both cedar shingles and bricks. The accrued periphyton was measured at thirty day intervals. Colby, in 1955, also used bricks and cedar shingles, measuring the changes in the periphyton mass at seven and thirty day intervals. Carr, in 1956, modified the schedule of removal of the cedar shingles so that measurements could be made at intervals of one, two, three, and four weeks. In that year, bricks and cedar shingles were evaluated at thirty day intervals. In 1957, Keup and Correll dispensed with the bricks and collected all samples pertaining to periphyton on cedar shingles. Substrates were collected at fourteen day intervals and a pigment analysis was made from periphyton on one third of the shingle. In 1958, the number of sampling stations was increased and the stations were concentrated in a much shorter area of the river (Figure I). The type of artificial substrate also differed from 11 previous years. Periphyton data were collected from "2 x "5 plexi- glass substrates. The substrates were placed six on a cross-bow; the cross-bow was supported by a steel stake and the whole assemblage was placed approximately 8 inches below the surface of the water. This program was coordinated with the isotope study that was to take place in August of 1958. It was decided to allow the plexiglass substrates], to remain in the river a period of seven days, instead of fourteen, for the first two collecting periods. During the first seven day period (July 1-8) the substrates at stations VII, 7-12 were subjected to fertilizer, while stations VII, 1-6 were above the point of fertilizer application. During the second seven day collection period none of the stations were exposed to fertilizer. If it was found that measurable (by weight) amounts of periphyton accrued in seven days, this time interval could be used for the isotope study and would make it possible to increase the number of sampling dates for radio- active periphyton. After the first two evaluations it was decided that weighable amounts of periphyton could not be accrued in seven days. The periphyton on the artificialsubstrates was then collected at the end of a fourteen day period. The procedure for all three evaluations was to pick up two substrates for pigment analysis at each station. The substrates were processed immediately upon return to the laboratory. All 12 macro-invertebrates, chiefly Trichoptera and Simuliidae larvae, were first picked off the substrates. The remaining plant material was washed with 95 percent ethyl alcohol and scraped off the substrates. This mixture was poured into a glass funnel and filtered through glass wool. The filtrate which contained the pigment was made up to a volume of fifty milliliters with additional alcohol. Final laboratory work consisted of measuring the density of pigment in each sample with a Klett-Summerson photoelectric colorimeter. A number 66 red filter was used in the colorimeter. The Klett unit was used for comparison of results. Phosphorus Content of the Periphyton The total phosphorus in the periphyton was determined by analyzing two substrates from each station. All macro-invertebrates were picked from the substrates to be analyzed. Total phosphorus was determined by digesting an unfiltered sample with sulfuric, nitric, and hydrochloric acids and determining the phosphate content by the molyfdate method as described by Ellis, Westfall, and Ellis (1948). Maximum color development was determined with a Klett-Summerson photoelectric colorimeter. Total phosphorus in p. p. b. was obtained from a graph based on known phosphorus standards. l3 §t_ream Flucfuation and Water Temperature Water flucruation in the West Branch of the Sturgeon River for July and August, 1958 was determined from a river staff gage located 10 yards above permanent station VII. Temperatures were also taken at station VII with a pocket thermometer. A composite of both weekly temperatures and daily gage readings are shown in Figure 11. Extensive water fluéXuations in the West Branch during July and August, 1958 were confined to three periods, July 4-8, July 9-13, and August l8-23. The greatest of these was only five tenths of a foot indicating the stable condition of the water level in the West Branch of the Sturgeon River. Figure 11 also indicates stable conditions in the river for the period preceeding the isotope release on August 5, 1958 and for the fourteen days immediately following the isotope addition. Since there was considerable variation in the time of day that temperatures were measured, only a general picture of fluciuations of the temperatures in the river water was obtained. Velocity of the River Water An attempt was made to measure surface velocity of the river water at the odd numbered stations below permanent station VII. A wooden float timed over a short distance of the stream was the method used and it was consistent enough to allow for comparative results. FIGURE 11. --Mean weekly water temperatures and daily staff gage readings from station VII on the West Branch of the Sturgeon River--July and August, 1958 14 00 0 V in in l l | N O \O \O L I 5 __52 5 4 4 20 10 5 ‘ 1/7 / .1 .4 1 a ‘13 '1 u .1 U) d 1 3° .1 <2 iii .1 q 1m 1 --—¢ 1— do or) .4 fit“ 'lN 1 a..( 1 .( O 4N .4 — >‘ 0—. ..I-n :3 F" "J .( —( J q -) .4 H I l m J J L J O O O O O O O [- o In Vt‘ m N v-c (100} 12 J0 qiuai suo) iqfitsq .IsAtx }O uoti'emontg 15 Gage Temperature 16 Results and Discussion Composition of Periphyton Communities on Artificial Substrates The results of the study of periphyton on the artificial substrates in the West Branch of the Sturgeon River indicate a community made up almost entirely of diatoms. Table 1 indicates that_S_yn_ec_lr_a 3111a; was the dominant species on the substrates at all stations inves- tigated during the period of July 11 to July 18, 1958. Although no attempt was made to establish patterns of community periodicity, substrates removed from the river in August of 1959 also showed a predominance of this species. Figure III shows the four major or- ganisms at the various stations for the period of July 11 to July 18, 1958. It is evident that at this time Synedra ulna accounts for the greatest portion of the periphyton complex on the artificial substrates. All stations were quite uniform as to community composition. Cymbella spp. , Navicula spp. , Eocconeis spp. , and Gomphonema spp. were the other principal diatoms making up the periphyton at this time. None of these, however, achieved a dominant position at any of the stations. When relating the periphyton community on the artificial substrates to the periphyton complex of the West Branch itself many factors must be considered. Colonization of the bare artificial areas 17 TABLE 1. --Relative frequency of diatoms counted in thirty micro- scopic fields selected from artificial substrates placed in the West Branch of the Sturgeon River July 11, 1958, and removed July 18, 1958. Relative Relative frequency frequency Station 1# Station 7# Sfliedra ulna 97 Sjrnedra ulna 93 Cymbella spp. 63 Navicula spp. 30 1111151131 spp. l7 Gomphonema spp. 27 Cocconeis spp. l7 Cymbella spp. l3 Gomphonema spp. 10 ELclgteiLa spp. l3 Cyclo_t_e__lla spp. 3 C_occoneis spp. 7 Station 2# Station 8# S_ynedra ulna 97 l Synedra ulna 77 Cymbella spp. 43 Cymbella spp. 13 Navicula spp. 23 Cgcconeis spp. 10 Cocconeis spp. 20 Fragilaria spp. 7 Gomphonema spp. 10 Navicula spp. 3 Cjclotella spp. 3 Oath-phonema spp. 3 Stephanodiscus spp. 3 Station 4# — Synedra ulna 87 Station 10# Cymbella spp. 34 Synedra ulna 83 Cocconeis spp. l7 Navicula spp. 17 Navicula spp. l3 Cocconeis spp. 7 Gomphonema spp. 7 GoWema spp. 7 Fragi_1aria spp. 3 Fragilampp. 3 Cyclotella spp. 3 Synedra spp. 3 Station 5# Station 11# Sinedra ulna 93 Synedra ulna 97 Navicula spp. 57 Cymbella spp. 37 Cymbe1___l_a spp. 43 man-{spp. 13 G3m_p__honem_a spp. 27 6353213 spp. 7 Cyclotell a spp. 3 'CISR‘SESHema spp. 7 Stephanodiscus spp. 3 FIGURE III. --Re1ative frequency of the four major diatoms from artificial substrates placed in the West Branch of the Sturgeon River July 11, 1958 and removed July 18, 1958 -- Calculated in thirty fields from a millipore filter pad. 18 .mmw MEMGOAQEOO dam .mZonflbwnu .mmm .350ng 9:5 dupmcrwm dam Edugdz .mmm 3280600 dam mica—E U as? duped m .nEm mwocooooO dam dadogmz .mmm MSMQE U «.53 mupocrwm damn 3980000 .93 3.90162 .Qmm 330,930 9:: mhpogm dam mfiocooooU dam madogdz .mmm .mSmQfltnnv 65.: dnpogm dam masconmfioo dam mwocooooU dam .mHSoTadZ and: dnpocrwm dam dfihdfiwmuh dam mwonooooU .mmm .mflonEU d5: dupoccwm dam .2395 0 Kim damcoamEoU .mmm mange/NZ 2:5 dupoc>w 100 I . r a _+ b _ _ _ P _ P r . h r _ T _ 0 0 0 O 0 0 0 O 0 0 O 0 0 0 O 0 0 9 8 7 6 5 4. 3 2 l O 9 8 7 6 5 4 3 m m $30G oaoomonoflh omv >ocoswonm nth/3.2mm ll 10 Stations 19 20 in relation to the time substrates are removed is one of the most important factors. The difference between periphyton on non-living and that on living substrates is another. Butcher (1932) could observe no differences in sessile algae on artificial substrates (glass slides) from that found on natural substrates in the river. Young (1945), however, failed to find any Gloeotrichia on rope or on glass slides placed within a clump of bullrushes bearing the blue-green algae. Periodicity of the various algae also must be known to establish the true picture of the periphyton complex. Blum (1957) reports that algae sampled at weekly intervals willoften reveal the complete disappearance within a period of only 6 to 10 days of an erstwhile conspicuous algae. Peters (1959) reports a marked seasonal periodicity of algal organisms which become attached to artificial substrates in the Red Cedar River, Michigan. Periodicity and living substrates were not investigated in the West Branch. To what degree the peri- phyton sampled on the substrates between July 11 and July 18, 1958 is related to the over-all periphyton complex in the West Branch can only be surmised. It is evident, however, that the community on the substrates is sufficiently uniform at all stations sampled to justify comparisons between stations as to chlorophyll content and uptake of phorphorus. Table 2 thows a list of the algae identified in the West Branch of the Sturgeon River during the summers of 1958 and 1959. 21 TABLE 2. --A qualitative list of the algae identified in the West Branch of the Sturgeon River in the summers of 1958 and 1959. Chlorophyta Clas s: Charophyceae Chara sp. Class: Chlorophyceae OedOjonium sp. Mougeotia sp. Dichotomosiphon sp. Rhodophyta Clas s: Rhodophyceae Etrachospermum moniliforme Ch r ys ophyta Clas s: Xanthophyc eae Egghefia sp. Class: Bacillariophyceae* Synedra ulna Gomphonema sphaerophorum Synedra sp. Fragilaria harrisonii Cocconeis placentula Meridion circulare Cocconeis sp. _ Amphora ovalis 675531-12 turgida Surirella biseriata Navicula spp. Stephanodiscus sp. Cyclotella sp. *The list of Bacillariophyceae is based on the taxonomic keys presented in Tiffany and Britton (1952). It represents only the species occuring on artificial substrates. 22 All the major fresh water phyla are represented with the exception of Cyanophyta. Plosila (1958) identified a number of blue-greens in the plankton samples of Hoffman Lake, the origin of the West Branch. Prescott (1951) reports that a cyanophycean flora is most frequently encountered in water that is eutrophic in type. The most conspicuous filamentous aglaes were Batrachospermum moniliforme and Oedogonium sp. B. moniliforme was especially abundant during June and July. By the middle of August this alga had almost completely disappeared from the river. Oedogonium sp. , on the other hand, reached its greatest numbers in August and early September. It was particularly abundant after heavy rains in August.‘ Between August 27 and August 30, 1959, a period immediately following a considerable rise in the water level of the West Branch, this alga covered practically every extending substrate in a 300 yard section immediately upstream from permanent station VII. Vaucheria sp. , although inconspicuous because its thallus is frequently covered by sand on the river bottom, was also found in considerable quantities. Dichotomosiphgn sp. and Mougeotia sp. were collected only rarely. Periphyton Investigations pertaining to primary producers in aquatic situations have an early root in limnological history. Early investi- gations oi the standing crop of primary producers included counting, 23 TABLE 3. --Mean density of chlorophyll extracted from periphyton on 7-day substrates from stations VII, 1-12. Expressed as Klett units. Station Date July 1-8 July 11-18 1 6 12 2 11 11 3 9 13 4 14 11 5 3 ll 6 6 9 7 4* 12 8 9* 10 9 6* 7 10 13* 15 11 6* 17 12 8* 12 *Stations exposed to 80 lbs. of fertilizer, July 3-5, 1958. 24 weighing, or determining the cell constituents. Because of the lack of universal acceptability and sometimes applicability, the diver- sity of such techniques offered only limited usefulness when compar- isons of production rates were made. In 1934, Harvey introduced a method which allowed quantitative estimates of chlorophyll to be made from extracted pigments. The chlorophyll method is still undergoing refinements, but has become a useful indicator of the standing crop. The swiftness of such a technique has made it a valuable tool for the limnologist. In this study the standing crop of periphyton is measured by the density of extracted pigments. Previous studies of the periphyton in the West Branch of the Sturgeon River indicate an increase in the standing crop following fertilization. In the first three years of the program (1954, 1955, and 1956) the fertilizer was applied to Hoffman Lake. Grzenda (op. cit. ) and Colby (op. cit. ) both found a positive response to the fertilizer by periphyton downstream from Hoffman Lake. Carr, in 1956, observed an increase in the standing crop after fertilization but also observed a natural increase in production at the control station. This natural increase may be a seasonal phenomenon. In 1957, when the fertilizer was applied directly to the river, Keup (o_p;_<_:_i_t.) found large increases of periphyton which he could not attribute to natural flucuation s . FIGURE IV. --Mean density of chlorophyll and total phos- phorus from periphyton on weekly shingles of stations be- low permanent station VII--Expressed as Klett units and p. p. b. , 1958. 25 12 ll 10 . , . . . . . — . . ) . 8 . 5 . 9 . i \ 6 \\ . \\ 1 3 8 ..\ L Y 5 I, .l. .8 Q/ I u 5 .1 I w 9 x L L .1 8.. d 1 A... m a . A. .1 . 1|. 1 fl 1 .I. J .n Y Y 1 ..l. e u u F J J 1 . . . T . L . L 4 1 J P p _ 0 0 0 O 6 6 2 8 4 1 1 . 1 1 33633 #1239330 A .n d .3 manosmmonm H.308 Stations 26 27 In 1958, the substrates remained in the water for seven days (July 1-8) and 80 lbs. of fertilizer was added between stations VII, 6 and VII, 7 for a period of forty eight hours between July 3 and July 5, 1958. No statistically significant difference in periphyton could be detected between the six stations (VII, 7-12) that were exposed to the fertilizer and the six stations (VII, 1-6) above the fertilizing point (Figure IV and Table 4, test 1). The substrates were then again placed at all stations in the river (VII, 1-12) for another seven day period (July 11-18). To ascertain if there might be a delayed response to the 80 lbs. of fertilizer already added, no additional fertilizer was added during this period. Again no statistically signi- ficant differences could be detected in the periphyton of stations VII, 7-12 when compared to stations VII, 1-6 (see test 2, Table 4). To determine if there might be a natural flucuation between stations VII, 7-12 and VII, 1-6 for the two sampling periods, July 1-8, and July 11-18, 1958, statistical comparisons were made between these two sections for the two different sampling periods. Tests 3, 4, 5, and 6, Table 4 indicate that no such flucuations occurred. Statistical analysis was not made for the five stations (VII, 1, 3, 5, and 9) used to evaluate the shingles exposed to fertilizer and removed after fourteen days (Figure V). Examination of these data (Table 5) indicates erratic results for stations 7 and 9, the two stations exposed to the fertilizer. It is believed that complete mixing of the added 28 TABLE 4. --Results of "t" tests calculated from mean chlorophyll content of stations VII, 1-6, and stations VII, 7-12; for the period of July 1-8, 1958, and July 11-18, 1958. Number Test Result 1 Means of stations 7-12 (July 1-8) is greater than means of stations 1-6 (July 1-8). t = 0.139 2 Means of stations 7-12 (July 11-18) is greater than means of stations 1-6 (July 11-18). t = O. 373 3 Means of stations 1-6 (July 1-8) is significantly different from means of stations 1-6 (July 11-18). t = l. 010 4. Means of stations 1-6 (July 1-8) is significantly different from means of stations 7-12 (July 11-18). t = l. 062 5 Means of stations 7-12 (July 1-8) is greater than means of stations 1-6 (July 11-18). t = l. 240 6 Means of stations 7-12 (July 1-8) is greater than means of stations 7-12 (July 11-18). t = l. 043 Degrees of freedom 2 10 l. 812 2.228 Critical value of "t" for tests 1, 2, 5, 6 Critical value of "t" for tests 3 and 4 All tests made at the five percent level. Hence no significant increase in chlorophyll content could be detected between the stations concerned for test 1, 2, 5, and 6; no significant differences in chloro- phyll content could be detected between the stations concerned for test 3 and 4. FIGURE V. --Mean density of chlorophyll from weekly sub- strates of stations above permanent station VII, and bi-weekly substrates from stations below permanent station VII--Klett units, 1958. 29 Chlorophyll (Klett units) 25 20 15 10 135 120 105 90 75 60 45 30 15 L__l I L l l J l—‘(r— 3 4 5 6 7 8 9 10 ll 12 Below permanent station VI--(week1y) ,,,-Below permanent station VlI--(bi-weekly) I Fertilized (180 lbs.) July 20-26, 1958 / I / / I / / \\ l \ I \ / \ \‘ § / “\\ / \\/ L I 1 4‘ L 1 l 3 5 .1. 7 9 Stations 30 31 fertilizer with the river water may not have occurred by the time the nutrients would be passing station 7, a distance of 75 yards from the point of application. An explanation of why the standing crop of periphyton did not increase following the first application of fertilizer involved many factors. The artificial substrates were only in the water seven days, a period believed to be insufficient for maximum growth of periphyton in the West Branch. The amount of fertilizer added (80 lbs.) was not as great as that added in 1957 (410 lbs. ). The micro- habitat of the plexiglass shingles may have varied from those of the cedar shingles used in 1957. Colby (op. cit. ) found a difference in the community complex between the wooden and brick substrates in 1955. The first two possibilities appear to be closer to the problem. When the substrates were exposed for fourteen days and a greater amount of fertilizer (150 lbs. ) was added, the few data available indicate a positive response of the periphyton to the fertilizer. The information obtained from stations VI, 1-12, where the plexiglass substrates were exposed for seven days without benefit of fertilizer (see Table 5 and Figure V), indicated that this section of the river should receive added nutrients in the form of fertilizer in order to insure that there would be a weighable amount of peri- phyton present when the isotope was to be released into the river. It was also decided to allow the plexiglass substrates to be in the 32 TABLE 5. --Mean density of chlorophyll extracted from periphyton on weekly substrates from stations VII, 1-12, and bi-weekly substrates from the odd numbered stations below station VII--Klett units, 1958. Station Date July 23-30 July 18-31 (Below VI) (Below VII) 1 12 63 2 12 - 3 10 35 4 9 ' - 5 13 29 6 9 _ 7 9 13* 8 8 - 9 27 141* 10 5 - 11 6 _ 12 10 - >1: Stations exposed to 150 lbs. of fertilizer, July 20-26, 1958. 33 water a period of fourteen days instead of seven, a procedure that would entail lengthening the sampling periods for radioactive periphyton. Phosphorus Content of the Periphyton After the first application of fertilizer to the West Branch (July 1-8), a significant increase in total phosphorus in the periphyton was detected in those stations (VII, 7-12) accessible to its effects (Tables 6 and 7). This increase of total phosphorus in periphyton did not carry over for the July 11 to 18 period, a period when no fer- tilizer was added to the river. An analysis of the first period (July 1-8) does not indicate progressively smaller amounts of phosphorus in the periphyton mass proceeding downstream from station 7 to station 12 (see Table 6 and Figure IV). This phenomenon, which did not occur, could be expected if enough phosphorus was lost, either biologically or physically, so as to dilute the amount of available phosphate as it passed each station. A possible reason why this did not occur in this study may lie in the tremendous amount of extraneous phosphate that would be available to the periphyton complex when fertilizer is added to the river. From the point of fertilization to station VII-12, a distance of 800 yards, it is possible that phosphate may have been available in such quantities as to allow the periphyton at these distances to all reach maximum accumulation values. It is also possible that the periphyton at these 34 TABLE 6. --Mean value of total phosphorus in the periphyton mass on weekly substrates from stations below permanent station VII-- Expressed as p. p. b. , 1958. Station Date July 1-8 July 11-18 1 107 56 2 50 79 3 117 60 4 48 44 5 76 60 6 89 35 7 125* 64 8 144* 111 9 94* 42 10 153* 47 11 119* 42 12 166* 47 *Stations exposed to 80 lbs. of fertilizer, July 3-5, 1958. 35 TABLE 7. "Result of ”t” tests calculated from mean p. p. b. of total phOSphorus in the periphyton of stations VII, 1-6, and 7-12, July 1-8, and July 11-18, 1958. Test Number Test Results 1 Means of stations 7-12 (July 1-8) is greater than means of stations 1-6 (July 1-8). t 1.911* 2 Means of stations 7—12 (July 11-18) is greater than means of stations 1-6 (July 11-18). t 0.146 3 Means of stations 1-6 (July 1-8) is significantly different from means of stations 1-6 (July 11-18). t 1.113 4 Means of stations 1-6 (July 1-8) is significantly different from means of stations 7-12 (July 11-18). t 0. 805 5 Means of stations 7-12 (July 1-8) is greater than means of stations 1-6 :1: (July 11-18). 3. 656 H H 6 Means of stations 7-12 (July 1-8) is greater than means of stations 7-12 (July 11-18). t 2, 825* Degree of freedom = 10. Critical value of "t" for tests 1, 2, 5, and 6 = 1. 812. Critical value of "t" for tests 3 and 4 = 2. 228. *Significant at the 5 percent level. o6 distances have simply satisfied a phosphorus debt that the complex may be subjected to in this river. That it may be just a case of satisfying a phosphorus debt is somewhat substantiated by the work of Rodhe (1948). Working with Scenedesmus spp. (a small green algae), he found that it could assimilate phosphorus very rapidly (one day) to satisfy a phosphate debt, but the accumulation of extra phosphorus required a fairly long time (seven days). In any case, a progressive decrease in the uptake of phosphorus by periphyton, proceeding down- stream from where it entered, would not be expected to endure long. Harvey 313.1. (1935), working with phytoplankton, found that only a fraction of the phosphate utilized by the plankton could be found as phosphorus compounds in the planktonic population. Goldberg 3521. (1951) also found that as much as 50 percent of the radioactive phos- phorus in cells containing more than the minimum requirements could be removed by washing with sea water free of radiophosphorus. This rapid loss of phosphorus from marine algae, noted by the above authors, appears to have occurred also for the periphyton in this river experiment (see test 6, Table 7). Since the actual amount of periphyton that was analyzed for total phosphorus could not be known with certainty, it may at first appear to be unwarranted to make such a conclusion from this test. A comparison of the chlorophyll content for each of these periods, however, indicates that stations VII, 7-12 for the second period (July ll-18) had an amount of periphyton 37 that was not significantly different from stations VII, 7-12 for the first period (July 1-8). (See test 6, Table 4.) If this is the actual case, the above inference from test 6 appears to be justified. That is, excessive amounts of phosphorus in the periphyton appear to be lost rapidly. With the methods employed in the preliminary study, it would be impossible to differentiate between naturally occurring phosphorus and that introduced extraneously. The actual amount loss and rate of loss could not be determined. It was decided to add artificial substrates to the river following the release of P32 in order to detect any re-accrual of the‘added P32, either from biological "feed back, " or from delayed eddy diffusion. Velocity of the River Water Many workers have observed that certain algae will grow more luxuriously in rapid water. Neel (1951) believes greater consumption of nutrients occurs in rapids than in pools. Whitford (1956) emphasizes the beneficial effect of current on algal communities. Ruttner (1953) is one of the chief proponents of rapid currents mani- festing beneficial effects on the quantity of organic production per unit area. He states: "In the rushing water of rapids the stones are thickly overgrown with mosses and algae and in addition there is richly developed animal life, such as one would not expect in an FIGURE VI. --Comparison of the velocity of the surface water over the odd numbered stations below permanent station VII with the chlorophyll concentration on substrates for these stations, 1958 38 12 8 2 6 0 4 8 R mu. m M 0 3 5 8 O 2 o O 0 O I o L 0— i2 - U A d . fl 41 4| m — . «I 4 a 9 J A q _ pcooom Mom Hoowllwfiuoaokr / L 1 .\\x m. .3 o. s. s o o s s .V C (A p F L _ _ I . .l _ L _ L _ r L e _ I L O 0 0 0 0 O 0 0 0 O 0 0 0 9 8 7 6 5 4 3 2 1 1 I. use: 52m- 4136830 Stations 39 40 oligotrophic mountain water. The stones of the lentic regions, on the other hand, exhibit a much smaller aufwuchs and usually fewer animals as well. ” Other investigators are inclined to believe the opposite is true; that there is more production in slower waters. Blum (1956) does not believe that there is enough evidence to indicate better growth in riffles. He cites an investigation in Brazil where it has been concluded that rapids are less productive than the quiet portions of rivers. In a recent paper by Douglas (1958), working with a diatom population, it was noted that an increase in the flow of water caused a marked decrease in the diatom population. Butcher (1932), using glass slides as artificial substrates, found the rate of increase of diatoms inversely proportional to the velocity of the current. Comparison was made in the West Branch between the crude surface velocity of the water over the odd numbered stations and the mean amount of periphyton found at these stations (Figure VI). There are indications that rapid current of the water may have a negative influence on the standing crop of periphyton on the plexi- glass substrates. To see if there was a correlation between the velocity of the river water and the standing crop of periphyton, a regression line was computed from chlorophyll and water velocity at four stations (3, 5, 7, 9) where measurements were made. A test was then performed to see if the regression line was significantly different from zero. The results (Table 8) indicate that the regression 41 TABLE 8. --Test of regression line computed from velocity of the river water and chlorophyll at four stations below permanent station VII on the West Branch of the Sturgeon River, 1958. Station X (velocity, c. f. s. ) Y (Klett units) 3 1. 60 119 5 l. 73 90 7 2. 31 71 9 l. 38 209 b = -126. 062 sy.x2 .—. 1840.640 a = 343. 489 Sb = 62. 361 t = -2. 021 with 2 degrees of freedom. -2. 920 at 10% level. Critical value of t -l. 886 at 20% level. Critical value of t b is only significantly different from 0 at the 20% level. 42 line was significantly different from zero only at the 80 percent level, not the 95 percent or 90 percent level. This further indicates that faster velocities may influence adversely the standing crop of peri- phyton. Observations made later, of periphyton collected for radio- active analysis, also indicates a greater biomass at stations almost lentic in nature. Since the periphyton complex consists almost entirely of diatoms (see Table 1), it is possible that this greater standing crop at lower velocities was due to a settling out of the potamoplankton on the artificial substrates. The smaller crop of periphyton in fast water does not mean that the production rate was lower here. Diatoms continually grow and break off in the'fast water. The rate of production would be governed by the turnover rate of diatoms, a phenomenon that was not investigated in this study. RADIOPHOSPHORUS STUDY Methods and Proc edur e s Description and Preparation of the Study Area The study area for measuring the response of periphyton to radioactive phosphorus encompassed stations both above and below permanent station VII (Figure VII). The stations for this phase of the program differed from those used during the preliminary study even though the same stretch of the river was used for both. The entire length of the area was 2, 700 yards long. Fourteen sampling stations were used for measurements of radioactive periphyton. Station 1 (control) was approximately 100 yards above the point of isotope addition. Stations 2 through 9 were at 150 yard intervals below the point of tracer entrance. Stations 10 through 14 were at 300 yard intervals. Preliminary investigation of this area revealed a low production of periphyton (see Table 5). An inorganic fertilizer, diammonium phosphate, was used to "prime" this area in order to insure a rapid uptake of the P32 and to provide a measurable amount of periphyton. The fertilizer was applied continuously for three days at the point where the isotope was to be released on August 1, 1958. 43 FIGURE VII. --Map of the West Branch of the Sturgeon River area, showing the stations used in the radiophos- phorus study. 44 i 9033.0. 8:05 05.955 11.9 / 44v .. . as x.» (es. (e 895225500 89685 m .3 m.m ..2 mm H 1.... 462.83%. . 1: Station Counts per minute per gram 1 0 2 1733 3 7972 4 ‘ 5611 5 4266 6 4222 7 4236 8 2222 9 1482 10 1481 11 3707 12 1390 13 1728 14 1934 * Counts are corrected for background and decay. FIGURE IX. --Initia1 uptake of P32 by periphyton on artificial substrates four hours after the addition of the isotope for the entire experimental area of the West Branch of the Sturgeon River, August 5, 1958. 54 7000 6000 5000 4000 3000 Activity in counts per minute 2000 1000 Stations 55 56 areas. In light of the taxonomic investigation (see Table 1), this possibility would not appear to be the case. A large amount of P32 was taken up initially by periphyton a distance of 2, 750 yards (station 14) downstream from the point of release. This indicates to some degree the amount of available P32 that must have remained in the water after passing through this distance of the river. Borgeson (1959) calculated that three percent of the isotope dosage passed through the study area of the West Branch of the Sturgeon River. Hutchinson (1950) observed the entire amount of radioactive phosphorus (35 millicuries) to be utilized by plankton in the epilimnion of a small eutrophic lake within minutes. Rigler (1956) found that over 95 percent of the radiophosphorus added to a lake was taken up by plankton within twenty minutes. Data indicate that in the West Branch,a lotic situation, a considerable amount of P32 is still available at the end of 108 minutes, the calculated time of movement of the initial pulse of P32 from the point of release to station 14 (Table 9). Whittaker (1953), working with an aquarium community, noted that P32 during the first few hours after tracer introduction, was rapidly absorbed by planktonic algae but after 15 hours plankton activity densities decreased while the P32 was more gradually taken up by bottom and side-wall algae. 1n the West Branch 1,11,” the primary producers are limited to periphyton andnaquatic plants, 57 there being no plankton in the strictest sense. It is therefore reasonable to suppose that the ability of the P32 to remain free in the water for longer periods of time than would be found in standing water series, is due to the lack of plankton in the community composition of the stream. Accumulation of P32 by Periphyton Since the amount of P32 that enters the periphyton after the initial pulse (uptake) could not be ascertained with certainty, it appears best to view the remainder of the data as values of accumulation (P32 that is present in the periphyton at a given time). Table 10 shows the amount of radiophosphorus accumulated in the periphyton for the third and seventh day after the isotope release, August 7 and August 11 respectively. The amount of P32 accumulated by the third day still shows indications of decreasing as one proceeds down- stream from the point of release, although the gradiation is not as striking as on the first day. By the seventh day, there is no evident decrease of the P32 in the periphyton proceeding downstream from station 3 to station 14. This is better illustrated if the stream is partitioned into sections and the mean value of P32 for each section is calculated. It can be seen (Table 11) that by the seventh day the P32 in the periphyton is quite evenly distributed in the entire experimental area. Davis and Foster (1958) showed that a radioactive 58 TABLE 10. --Accumulation of P32 in periphyton of the West Branch of the Sturgeon River for the third and seventh day after the release of the isotope, August 7, and August 11, 1958. Third day Seventh day Station CPM/ per gram* Station CPM/per gram* 1 O l O 2 2139 2 2178 3 2910 3 1167 4 5298 4 991 5 1427 5 440 6 1368 6 - 7 1202 7 350 8 3559 8 2191 10 1504 10 1943 11 3834 11 1491 12 1393 12 - 13 1107 13 910 14 182 14 1174 *Counts are corrected for background and decay. 59 element, such as P32, will eventually become uniform throughout the biota. This is because the isotope will be initially diluted with its stable form, first in solution, and eventually by exchange with its stable form which has not been in solution. Borgeson (op. cit. ) found that by August 15, this phenomenon also held for £113.33. sp. and Potamogeton pectinatus. Because the diatoms of the periphyton com- plex have a shorter retention time of P32 than the aquatic plants, it would be expected that the periphyton would first show a uniform distribution of the isotope. The retention time of an element, which is a function of the biochemistry of the particular element and components involved, is an especially important biological consideration when tracing the element through the various trophic levels and when considering values of accumulation. Davis and Foster (op. cit. ) have shown that retention is likely to be inversely related to the size of the "pool" or "reservoir" for the element in that trophic level. The element will remain for a longer period of time in the larger consumer organisms than in the smaller plants, although a major fraction of the element will at first be accumulated by the periphyton because of its relatively large total biomass. Odum 2L3} (1958) demonstrated that the ability of the biomass to accumulate large amounts of the element initially to be a function of the high surface to volume ratio of the small plants. 60 TABLE 11. --Mean value of P32 accumulated in periphyton of the West Branch of the Sturgeon River per section for the first seven days after the addition of the isotope. Section Counts per minute, per gram, corrected for background and decay August 5 August 7 August 11 0 ----- 700 yards 4761 2628 1194 700--1400 yards 2355 2021 1494 1400-2600 yards 2190 I 1629 , 1191 61 TABLE 12. —-Accumulation of P32 (corrected counts per minute) in periphyton of the West Branch of the Sturgeon River for the thirteenth, nineteenth, and twenty-seventh day after the addition of the isotope, August 17, August 23, and August 30, 1958. August 17 August 23 August 30 Station CPM Station CPM Station CPM per gram per gram per gram 1 O l O l 0 >3 :1: 3 1500 3 284 3 268 >1: 1 :1: 5 2718 5 263 5 364 37‘ 3:: :3 8 310 8 447 8 368 as . 11 616 11* 321 11* 357 :1: 14 1860 14 274 14 621 *Denotes substrates introduced to the water after the release of the isotope. 62 With the exception of station 14 on August 30, 1958, the uniformity in distribution of P32 in the periphyton continued for the remainder of the experiment. These remaining values (Table 12) are chiefly from substrates introduced after the application of P32 and will be treated separately from the above in a later section. The relatively large amount of radioactivity found in the periphyton at station 14 for August 30, 1958 may indicate that the accumulated "drift" of radioactivity is reaching a downstream station. Exchanged and Regenerated P32 Data from substrates placed in the water after the release of the isotope indicate that exchanged and/or regenerated P32 entered the system rapidly and in considerable amounts (Table 12). Since the substrates were not placed in the river until well after the initial pulse of P32 (the first post treatment shingles were not put in the river until two days after the release of the isotope), it is apparent that this re-accrual of P32 by periphyton could not have taken place from P32 held in situ, either by eddies or other entrapments. Figure X shows that for the thirteenth day of the experiment, August 17, approximately 33 percent of the total measured radiophos- phorus on the substrates was accumulated in the exchangeable and regenerated form. Substrates placed in the river August 23 (19 days after the release of the isotope) and collected August 30 still FIGURE X. --Summary of P32 activity in periphyton of the West Branch of the Sturgeon River, 1958. 63 ,_ . N _. . . M _ «L 2 ~36? ou poodponug woumuumndm mount was mcoflmum om umdwfixw mm umdwaoq S umsmdaw : «mums/w N. umdwsxw m “mamafl m z m w : mam .m . ,_L._.._...- -L. 1___‘L__. omouog mo c0363.”. Rooms mmouofi mo :oflflupd ouOmon noun? 5 moumuumndm Nhoh com 000 coma OOHN CONN comm unexS 19d ainutux 19d siunoo psioaxzog ooom Oomv 64 65 showed an appreciable amount of P32, most of which must have been previously adsorbed on, or incorporated into the periphyton (Table 12). In all cases a slight allowance must be made for the ”seeding on" of diatoms and other algae. These forms could be radioactive when they were seeded on, thus contributing activity which would not necessarily be exchanged or regenerated in form. In systems such as the West Branch, where the periphyton complex may be surviving perilously close to phosphorus deficient conditions, the re-utilization rate of phosphorus will depend to a great extent upon the retention time of phosphorus in the previous organism. When measurements are made from artificial substrates, the re-utilization of phosphorus may depend also on the age of the community on the substrate. It is possible that new substrates may pick up phosphorus faster than older ones. The substrates with the older community may be picking up only a replacement ration of phosphorus while those substrates that are new will be picking up phosphorus at a faster rate because of the "seeding on" and rapid initial growth of the community. Loss of P32 from Periphyton The loss of P32 from periphyton of the experimental area is determined on a relative basis. The assumption is made that the amount of P32 accumulated on each substrate when considered on a FIGURE XI. —-Mean loss of P32 activity in periphyton on artificial substrates for the entire experimental area during the first seven days after isotope addition, 1958. 66 w_-.‘.~w.n -- o- - 3000 2000 p 1000 P Edum “mm 3.958 you 3:300 E >fi>fio< Q! L». —~g-.~«D-.~".-L~'—.-.-——A-n.n J...- I .i}>s.ln I..- 1 ‘0 il‘lll‘ 11 10 1958 August, 67 68 weight basis, is equal to the amount accumulated for each of the other shingles for that particular station and date. Knight (op. cit. ) determined that the variation of activity within stations is not exten- sive for periphyton. Whittaker (SELF—it ) found that in his aquarium experiment periphyton did not show a loss of P32 until after the second week. His data were drawn from a periphyton complex consisting mainly of filamentous algae and a system offering continual exposure of the algae to the free P32. The mean value of P32 in periphyton of the entire experimental area of the West Branch declined very rapidly for the first seven days after the isotope release(Figure XI ). This would be expected since this is a uni-directional system and even with a rapid re-utilization of the regenerated and exchanged P32 the free marked atoms and Potamoplankton particles are continually and permanently moving out of the system. Because of this early and rapid loss of P32 from periphyton, it is unlikely that at any time a degree of equilibrium existed, where the uptake and loss of P32 would be constant and the resultant accumulation would be maximum and also constant. Davis and his associates (op. cit. ) found such a state existing in the Columbia River below the Hanford Works where the organisms are subject to continual or chronic exposure from radiomaterials. In systems such as these, concentration values can be calculated with a great deal of certainty. Krumholz (1954) 69 in White Oak Lake, Tennessee, found that the concentration factor for phosphorus in Spirogyra sp. to be approximately 850, 0.00 times the normal amount. Although there is a rapid loss of P32 from the periphyton of the experimental area as a whole, there is a somewhat different picture when individual stations are compared. Figure X shows the activity of periphyton for the stations sampled throughout the entire program. Indications are that stations in the lower half of the area gain considerable P32 from the upper area. When this is figured on a percentage basis, Figure XII indicates a large loss by the third day from the upper stations, with the exception of station 4, a slight gain for the middle stations and a loss for the last two stations. Between the third and seventh day of the experiment, station 10 continued to gain P32 while all other stations, with the exception of 14, lost P32. From this graph it can ge seen that station 14 showed the greatest loss (third day) and also the greatest gain (seventh day). It should be expected that much of the flucuation in the gain or loss of P32 for the various stations is due to variability inherent in the techniques of measurements and the method used for comparisons. Still, enough of a pattern persists to warrant further investigation as to the movement of P32 through the system. A prime possibility is that the isotope, once released into the system and taken up by the periphyton, is continually and constantly being lost to periphyton FIGURE XII. --Percent gain or loss of P32 activity in periphyton from August 5, 1958 to August 7, 1958; and from August 7, 1958 to August 11, 1958. 70 _P 540% T 3.66 0 Z 3.66 0 Z 4L 1 14 1L1 11 L 1 9 13 12 10 8 7 100 F 1 a _ .... _ 1 5 J 7 14 t t a. a L 1 g 1 g _ u u J J _ _ T L p a s _ p L _ _ F _ a a h _ 0 0 0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 0 0 0 8 6 4 2 2 4 6 8 0 8 6 4 Z Z 4 6 8 0 1|. 1 aid—U mmO‘H Cme mmOJ ”:80qu Stations 71 72 further downstream, with of course some being removed to a higher tropic level. If this is true, it would be unlikely that any station would show an appreciable gain of radiophosphorus after the initial pulse of P32. The quantity of P32 that is continually and constantly being lost from the periphyton in the upper reaches of the experimental area would never be available to the periphyton at the stations in the lower part of the area in amounts great enough for these stations to show a gain of P32. The periphyton in the lower area would also be continually and constantly losing the P32 initially incorporated into it. Another possibility is that the P32 moves through the system in more or less secondary pulse-like manners. The possibilities of secondary pulses are complicated not only by biological factors that contribute to the disappearance of P32 from the organisms involved, but also by physical factors of the system itself. Precipitation of P32 and sorption by silt particles will account for some loss of P32 (Foster, 1956). Hutchinson (1957) found that different algae varied in efficiency to utilize phosphorus. In a uniform periphyton complex, such as is found on the substrates in the West Branch, such a factor would have minimal affect on complicating a pulse like movement. Table 13 gives some indication that the P32 does move through the area in a manner that is not indicative of a constant and continued loss of the isotope from the system. During the study in the West Branch, the upper seven stations, which encompass the first 73 VTABLE 13. --Percent gain or loss of P32 activity in periphyton of the West Branch of the Sturgeon River from August 5, 1958 to August 7, 1958; and from August 7, 1958 to August 11, 1958. August 5 to August 7 August 7 to August 11 Station Percent Percent gain 10 s 5 gain los s 3 63 6O 4 6 81 5 67 69 6 68 - - 7 72 71 8 6O 38 10 2 29 11 3 9 12 1 - - 13 35 18 14 39 540 74 850 yards of the experimental area, did not show a gain of P32 after the initial pulse of the radioactive phosphorus (Figure XII). That is to say that stations 1-7 did not show an increase of P32 in relation to the time the substrates were removed from the river, it possibly being that the time of sampling was spaced at intervals too far apart to indicate if a gain of P32 occurred at any of these stations. The failure to capture an increase in P32 at these upper stations, in relation to the time samples were taken, would be expected if the turnover time for the periphyton is rapid. A rapid turnover of phos- phorus by plankton, including diatoms, was substantiated by Rigler (op. cit.) Goldberg 3531. (op. cit.'), and Rice (1953). Because some of the downstream stations did show a gain of P32 in the periphyton after the initial pulse, it does not necessarily indicate that secondary pulses of P32 occur in the system. Three sets of factors may be operating to determine the level of P32 in the periphyton. 1. Pure exchange in a kinetic sense. This could bring about a rapid loss of marked atoms when a considerable amount of marked atoms are present in the cells of the periphyton. Hence the loss would be greatest at the upstream stations which have the initial largest uptake. The downstream stations would not lose P32 as fast because the P32 level in water is higher due to drift etc. and because these 75 stations have a smaller initial proportion of marked to unmarked atoms. 2. Growth. Uptake rate of cells will vary with the physio- logical state of the community. Some of the colonies will be more active in accumulating phosphorus than others. In this respect the age of the community on the substrate must be considered. 3. Quantity of phosphorus in the cells. Starved cells will have a high P32 to total phosphorus ratio while cells subjected to fertilizer will have a low P32 to total phos- phorus ratio. If the ratio of P32 to total phosphorus is low, it will take longer to get rid of the marked atoms. Since fertilizer was added to the experimental area prior to the isotope release, this factor will be important in this system. Accumulation of P32 in Rock Periphyton P32 accumulation in periphyton collected on rocks followed a pattern similar to that of the artificial substrates. On the fifth day of the experiment, August 9, 1958, P32 values were greatest at station 3 and declined progressively at the downstream stations (Figure XIII). By the twelfth day of the experiment, August 16, the P32 in rock periphyton was quite evenly distributed throughout the FIGURE XIII. --Summary of activity of P32 in rock peri- phyton in the experimental area of the West Branch of the Sturgeon River for the summer of 1958. 76 Activity in counts per minute 1800 1500 1200 900 600 300 I August 23 August 16 Stations 77 11 78 entire experimental area. Because of the minute amount of marl and rock particles that could not be separated from the periphyton at the time of preparation, the rock periphyton never obtained as high specific activity as found on the artificial substrates. It is also believed that some of the P32 measured as activity of rock periphyton may in reality be P32 that has been encorporated into the marl and consequently lost permantly from the system. Position of Periphyton in Relation to Phosphorus Since P32 is biologically the same as its stable counter- part P31, its movement through the system will also indicate the movement of naturally occurring phosphorus. Figure XIV indicates to some degree the position the periphyton complex holds in relation to phosphorus entering the system. Because of the relatively large total biomass and the high surface to volume ratio possessed by the periphyton, it appears to be initially responsible for holding the majority of the phosphorus entering the system. The other producer organisms (aquatic plants including M sp. ) do not appear to be initially responsible in retaining much of the phosphorus entering the system. Hutchinson (op. cit. ) also found this phenomenon in the epilimnion of a small eutropic lake. He hypothesized that it is apparently the diatoms and other algae of FIGURE XIV. --Mean activity of radiophosphorus for the various biological levels sampled in the experimental area of the West Branch of the Sturgeon River during August, 1958. 79 Mean activity in counts per minute 3000‘ 200c( 1000 Periphyton Fish Aquatic insects Aquatic plants #7 11141111AJL11114144LJ11LL1J 5 10 15 20 25 30 August, 19 58 80 short life spans that make up the great reservoir of littoral phosphorus. Knight (op. cit. ) reports filamentous algae to be intermediate between the aquatic plants and the periphyton in regards to initial uptake of P32 in the West Branch. Although the initial uptake of phosphorus by periphyton is high, it also loses phosphorus much faster than the other biological levels sampled (see Figure XIV). The majority of the phosphorus concentrated by the primary consumers must come directly from that initially retained by the periphyton. The rapid loss of phosphorus from periphyton indicates that the permanent pool of phosphorus is small in periphyton in relation to aquatic insects and fish. 81 SUMMARY 1. The periphyton sampled on artificial substrates in the West Branch of the Sturgeon River was quite uniform in community composition. Diatoms made up the great majority of the algae on the substrates. A single species, Synedra ulna, accounted for the greater part of the diatom population on all artificial substrates. 2. No statistically significant increase in the standing crop of periphyton could be detected after the first application of eighty pounds of fertilizer to the West Branch of the Sturgeon River. The substrates were in the river a period of seven days. 3. Data available gave an indication of increase of periphyton after the second application of fertilizer. During this fertilization period; twice as much fertilizer was applied and the substrates were collected at the end of a fourteen day period. 4. A significant increase of total phosphorus in the periphyton mass was detected after the first application of fertilizer. 5. There are indications that rapid currents had a negative influence on the standing crop of periphyton on the plexiglass substrates. 6. The initial uptake of P32 by periphyton declined progres- sively downstream from the point of application. 82 83 7. The amount of P32 accumulated by periphyton three days after the isotope release into the area gave indications of decreasing progressively downstream from the point of addition. Seven days after the isotope was released into the system, the P32 was quite evenly distributed in the periphyton throughout the experimental area. 8. Data from substrates placed in the river after the addition of the isotope indicate that exchanged and/or regenerated P32 occurred in considerable amounts. 9. The re-utilization of phosphorus in systems such as the West Branch depend on the retention time of phosphorus in the pre- vious organism and the age of the community on the substrates sampled. -10. Loss of P32 from periphyton was rapid for the first seven days after the release of the isotope. 11. During the study, the first seven stations below the point of isotope addition did not show a gain of radiophosphorus after the initial pulse of P32. Seven days after the isotope addition, all of the seven lower stations, with the exception of station 13, had gained P32 from that measured four hours after the addition of the tracer. 12. Accumulation of radiophosphorus in rock periphyton followed a pattern similar to that accumulated on artificial substrates. Activity measurements of rock periphyton did not attain those of the artificial substrates. 84 13. The periphyton complex in the West Branch of the Sturgeon River appears to be more responsible initially for removing the P32 from the water mass than the aquatic plants and larger filamentous algae. 14. The permanent reservoir of phosphorus in the periphyton is smaller in relation to large consumer organisms. APPENDIX Summary of the computations performed in determining corrected counts per minute for the periphyton at each station during the summer of 1958. 85 86 TABLE 14. --Summary of weights, counts, and computation data per station (artificial substrates) of P32 in periphyton, August 5, and August 7, 1958. Date Decay Back- Sta- Raw Wet Corrected Corrected (1958) factor ground tion count weight CPM/gm. CPM/gm. (grams) plus decay August .9000 46 l 44 .025 - - 5 2 241 .125 1560 1733 3 2342 . 320 7175 7972 4 1056 . 200 5050 5611 5 545 .130 3829 4266 6 122 . 020 3800 4222 7 1285 .325 3812 4236 3 766 . 1560 2000 2222 3 453 .305 1334 1482 10 326 .210 1333 1481 11 463 .125 3336 3707 12 365 .255 1251 1390 13 1049 .645 1555 1728 14 664 .355 1741 1934 .August .8200 46 1 50 .184 - - 7 2 502 .260 1754 2139 3 702 .275 2386 2910 4 589 .125 4344 5298 5 549 . 430 1170 1427 6 377 .295 1122 1368 7 697 .660 986 1202 8 367 .110 2918 3559 10 379 .270 1233 1504 11 439 .125 3144 3834 12 280 .205 1142 1393 13 184 .152 908 1107 14 104 .390 149 182 87 TABLE 15. --Summary of weights, counts and computation data per station (artificial substrates) of P32 in periphyton, August 11, to August 30, 1958 Date Decay Back- Sta- Raw Wet Corrected Corrected (1958) factor ground tion count weight CPM/gm. CPM/gm. (grams) plus decay .August .5800 85 1 79 .063 - — 11 2 234 .118 1263 2178 3 263 .263 677 1167 4 268 .318 575 991 5 179 .368 255 440 6 - .. - - 7 299 1.050 203 350 8 174 .070 1271 2191 10 209 .110 1127 1943 11 232 .170 865 1491 12 - - - - 13 180 .180 528 910 14 166 .119 681 1174 August . 5000 52 1 61 . 010 - - 17 2 112 .170 353 706 3 217 .220 750 1500 5 211 .117 1359 2718 8: 135 .535 155 310 11 129 .250 308 616 14 159 .115 930 1860 .August .3800 54 1 59 .095 - - 23 3: 80 .240 108 284 5 119 .653 100 263 8: 82 .165 170 447 11 89 .286 122 321 14 80 .250 104 274 .August .2800 52 1* 55 .076 - - 30 3* 58 .080 75 268 5* 69 .167 102 364 8* 83 .300 103 368 11* 73 .210 100 357 14* 68 .092 174 621 *Denotes shingles introduced to experimental area after release of radio- active phosphorus. 88 TABLE 16. --Summary of weights, counts, and computation data per station (rocks) of P32 in periphyton, August 9, 1958 to August 23, 1958. Date Decay Back- Sta- Raw Wet Corrected Corrected (1958) factor ground tion count weight CPM/gm. CPM/ gm. (grams) plus decay August . 7800 47 1 51 . 195 - - 9 3 161 . 079 1443 1850 5 221 . 167 1042 1336 8 205 . 245 645 827 11 140 . 290 320 412 14 76 . 228 127 163 August . 5000 52 1 45 . 150 - - 16 3 154 . 395 258 516 5 156 . 300 347 694 8 154 . 370 276 552 11 114 . 315 197 394 14 99 . 440 107 214 August . 3700 54 1 55 . 290 - - 23 3 72 . 200 90 243 5 72 . 360 50 135 8 105 . 360 142 383 11 67 . 290 45 122 14 65 . 600 18 49 LITERATURE CITED Blum, J. L. 1956. The ecology of river water. Botanical Review, Vol. 22, No. 3, pp. 291-341. Borgeson, David P. 1959. The movement of radioactive phosphorus through a stream ecosystem. Master's thesis, Michigan State University. Butcher, R. W. 1932. Studies on the ecology of rivers. II. The microflora of rivers with special reference to the algae on the river bed. Ann. Bot. Vol. 46, pp. 813-161. Carr John F. 1959. Modification of trout stream ecology by fertilization. Master's thesis, Michigan State University. Colby, Peter J. 1957. Limnological effects of headwater fertilization on the West Branch of the Sturgeon River, Michigan. Master's thesis, Michigan State University. Correll, David L. 1958. Alteration of the productivity of a trout stream by the addition of phosphate. Master's thesis, Michigan State ‘ University. Davis, J. J. and R. R. Foster 1958. Bioaccumulation of radio-isotopes through aquatic food chains. Ecology, Vol. 39, pp. 530-535. Douglas, Barbara 1958. Observations on the ecology of attached diatoms in a small stony stream. Jour. of Ecology, Vol. 46, pp. 320-341. 89 9O Ellis, M. M., B. A. Westfall, and M. D. Ellis 1948. Determination of water quality. U. S. Dept. Inter. , Fish. and Wild. Ser., Research Rept. No. 9. Foster, R. F. 1958. Radioactive tracing of the movement of an essential element through an aquatic community with specific reference to radiophosphorus. (nmeeemekX Goldberg, E. D., T. J. Walker, and A. Whisenand. 1951. Phosphate utilization by diatoms. Woods Hole Oceano- graphic Inst., Biol. Bull. 101, pp. 274—284. Grzenda, Alfred R. 1956. The biological response of a trout stream to headwater fertilization. Master's thesis, Michigan State University. Harvey, H. W., L. H. N. Cooper, M. V. Lebour, and F. S. Russel. 1935. Plankton production and its control. Jour. Mar. Biol. Assoc. U. K., Vol. 20, pp. 407-422. Hutchinson, G. E. 1957. A treatise on limnology. Vol. 1, Geography, physics, and chemistry. 1015 pp. Wiley, New York. Hutchinson, G. E. and V. T. Bowen. 1950. Limnological studies in Connecticut. IX. A quantitative radio-chemical study of the phosphorus cycle in Linsley Pond. Ecology, Vol. 31, pp. 194-203. Keup, L. E. 1958. Biological responses of fertilization in a lake and stream. Master's thesis, Michigan State University. Krumholz, L. A. 1954. A summary of the finding of the ecological survey of White Oak Creek, Roane County, Tennessee, 1950-1953. U. S. Atomic Energy Commission, ORG-132. Neel, J. K. 1951. Interaction of certain physical and chemical features in a headwater limestone stream. Ecology, Vol. 32, pp. 368-391. 91 Newcombe, C. L. 1949. Attachment material in relation to water productivity. Trans. Amer. Micro. Soc., Vol. 68, pp. 355-361. Odum, E. P., E. J. Kuenzler, and Sister Marion Xavier Blunt. 1958. Uptake of P32 and primary productivity in marine organisms. Limno-Oceano. Vol. 3, No. 3, pp. 340-345. Peters, John C. 1959. An evaluation of the use of artificial substrates for determining primary production in flowing water. Master's thesis, Michigan State University. Plosila, Daniel S. 1958. An evaluation of lake production following artificial en- richment. Master's thesis, Michigan State University. Prescott, G. W. 1951. Algae of the Western Great Lakes area. Cranbrook Inst. of Sci. , Bull. No. 31, xiii + 946 pp. Rice, T. R. 1953. Phosphorus exchange in marine phytoplankton. Fishery Bull. 80, Vol. 54, pp. 77-89. Rigler, F. H. 1956. A tracer study of the phosphorus cycle in lake water. Ecology. Vol. 37, pp. 550-562. Rodhe, W. 1948. Environmental requirements of fresh-water plankton algae. Sym. Bot. Upsal., Vol. 10, 149 pp., Uppsala. Ruttner, Franz 1953. Fundamentals of limnology. (Translated by D. G. Frey and F. E. Fry) Univ. of Toronto Press, 242 pp. Tiffany, L. H. and M. E. Britton. 1952. The algae of Illinois. Univ. of Chicago Press, Chicago, Illinois. 92 Whittaker, R. H. 1953. Removal of radiophosphorus contaminant from the water in an aquarium community. Biology Re search Annual Report, 1952, U. S. Atomic Energy Commission Doc. HW 28636, pp. 14-19. Young, 0. W. 1945. A limnological investigation of periphyton in Douglas Lake, Michigan. Trans. Amer. Micr. Soc., Vol. 64, pp. 1-20. 1190M USE ONLY "I1111111111111111“