u fl. n o — p n ir r. X n 3 O i' ?^ D. VI 'X a V, w O 3 m o ft o p D' o "o. e. e- G. m z 3D 30 TJ < 0) C 00 (D c o; o -♦» -*> (D 00 z m > Q) □3 CO X Z H > t- C _^ WJ "0 ^ w O) 1^ 3} ^ 3' 0> I c (A $ r* m 0) 5' •< Q> 3 H CO Z o> t > CD 0' V) 3 m <-* Z -< S- tL m z < 30 -n O O Z CO " i ? I -o o o S -H m m (/) O 5 ^ > en m Z o -< PHYSICAL SCI. LIB. _:leai WATER POLLUTION CONTROL RESEARCH SERIES • | 3030 ELY 08/ 71 -9 REC-R2-7I-9 ^ DWRNO. 174-12 JN ? 'yiD BIO-ENGINEERING ASPECTS OF AGRICULTURAL DRAINAGE SAN JOAQUIN VALLEY, CALIFORNIA THE EFFECTS OF AGRICULTURAL WASTE WATER TREATMENT ON ALGAL BIOASSAY RESPONSE VIRONMENTAL PROTECTION AGENCY»RESEARCH AND MONITORING BIO-ENGINEERING ASPECTS OF AGRICULTURAL DRAINAGE SAN JOAQUIN VALLEY, CALIFORNIA The Bio-Engineering Aspects of Agricultural Drainage reports describe the results of a unique interagency study of the occurrence of nitrogen and nitrogen removal treatment of subsurface agricultural wastev/aters of the San Joaquin Valley, California. The three principal agencies involved in the study are the Water Quality Office of the Environmental Protection Agency, the United States Bureau of Reclamation, and the California Department of Water Resources. Inquiries pertaining to the Bio-Engineering Aspects of Agricultural Drainage reports should be directed to the author agency, but may be directed to any one of the three principal agencies. THE REPORTS It is planned that a series of twelve reports will be issued describing the results of the interagency study. There will be a summary report covering all phases of the study. A group of four reports will be prepared on the phase of the study related to predictions of subsurface agricultural wastewater quality ~ one report by each of the three agencies, and a summary of the three reports. A group of three reports will be prepared to include: (1) the techniques to remove nitrogen in drainage effluent during transport, (2) the possibilities of reducing nitrogen in drainage water by on-farm practices, and (3) desalination of subsurface agricultural waste waters. This report, "THE EFFECTS OF AGRICULTURAL WASTE WATER_ TREAT- MENT ON ALGAL BIOASSAY RESPONSE," has been prepared with three other reports on the treatment methods studied and on the biostimulatory testing of the treatment plant effluent. A summary of the three basic reports will also be prepared. BIO-ENGINEERING ASPECTS OF AGRICULTURAL DRAINAGE SAN JOAQUIN VALLEY, CALIFORNIA THE EFFECTS OF AGRICULTURAL WASTE WATER TREATMENT ON ALGAL BIOASSAY RESPONSE Prepared by the Environmental Protection Agency William D. Ruckelshaus, Administrator The agricultural drainage study was conducted under the direction of: Paul De Falco, Jr., Regional Administrator, Region 9 ENVIRONMENTAL PROTECTION AGENCY 100 California Street, San Francisco, California 94111 Robert J. Pafford, Jr. , Regional Director, Region 2 UNITED STATES BUREAU OF RECLAMATION 2800 Cottage Way, Sacramento, California 95825 John R. Teerink, Deputy Director CALIFORNIA DEPARTMENT OF WATER RESOURCES 1416 Ninth Street, Sacramento, California 95814 REC-R2-71-9 DWR No. 174-12 13030 ELY 08/71-9 August 1971 For sale by the Superintendent of Documents, U.S. Oovemment Printing Office, Washington, D.C. 20402 - Price $1.00 REVIEW NOTICE This report has been reviev/ed by the U.S. Bureau of Reclamation and the California Department of Water Resources, and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Bureau of Reclamation, or the California Department of Water Resources. The mention of trade names or commercial products does not constitute endorsement or recommendation for use by either of the two federal agencies or the California Department of Water Resources. ABSTRACT Laboratory bioassay experiments were performed to test the effect on algal grov/th of agricultural waste water before and after the v;aste water had been subjected to two different nitrogen removal processes. The v/aste v/aters v;ere added in various percentages to San Joaquin River Delta v/ater for bio- assay. The algal growth throughout time was monitored by chlorophyll fluorescence techniques. The fluorescence meas- urements shov/ed logarithmic growth similar to the type usually observed in the Delta water over the vernal growth period. The laboratory experiments gave positive statistical evidence that the untreated agricultural waste water would promote substantial algal growth above that of the San Joaquin River controls. Both nitrogen removal processes v/ere equally effective in lowering the algal growth to that of the Delta water controls as long as the nitrate-nitrogen level in each removal system had been lowered to approximately 2 mg N/1 or less. Key words: Algal blooms - control, bioassay - algal, chlorophyll, denitrif ication , f luorometry , tile drainage. BACKGROUND This report is one of a series which presents the findings of intensive interagency investigations of practical means to control the nitrate concentration in subsurface agricultural waste water prior to its discharge into other water. The primary participants in the program are the Water Quality Office of the Environmental Protection Agency, the United States Bureau of Reclamation, and the California Department of Water Resources, but several other agencies also are cooperating in the program. These three agencies initiated the program because they are responsible for providing a system for disposing of subsurface agricultural waste water from the San Joaquin Valley of California and protecting water quality in California's water bodies. Other agencies cooperated in the program by providing particular knowledge pertaining to specific parts of the overall task. The need to ultimately provide subsurface drainage for large areas of agricultural land in the western and southern San Joaquin Valley has been recognized for some time. In 1954, the Bureau of Reclamation included a drain in its feasibility report of the San Luis Unit. In 1957, the California Department of Water Resources initiated an investigation to assess the extent of salinity and high ground water problems and to develop plans for drainage and export facilities. The Burns-Porter Act, in 1960, authorized San Joaquin Valley drainage facilities as part of the State Water Facilities. The authorizing legislation for the San Luis Unit of the Bureau of Reclamation's Central Valley Project, Public Law 86-488, passed in June 1960, included drainage facilities to serve project lands. This Act required that the Secretary of Interior either provide for constructing the San Luis Drain to the Delta or receive satisfactory assurance that the State of California would provide a master drain for the San Joaquin Valley that would adequately serve the San Luis Unit. Investigations by the Bureau of Reclamation and the Department of Water Resources revealed that serious drainage problems already exist and that areas requiring subsurface drainage would probably exceed 1,000,000 acres by the year 2020. Disposal of the drainage into the Sacramento-San Joaquin Delta near Antioch, California, was found to be the least costly alternative plan. Preliminary data indicated the drainage water would be relatively high in nitrogen. The then Federal Water Quality Administration conducted a study to determine the effect of discharging such drainage water on the quality of water in the San Francisco Bay and Delta. Upon completion of this study in 1967, the Administration's report concluded that the nitrogen content of untreated drainage waters could have significant adverse effects upon the fish and recreation values of the receiving waters . The report recommended a three-year research program to establish the economic feasibility of nitrate-nitrogen removal. As a consequence, the three agencies formed the Interagency Agricultural Waste Water Study Group and developed a three-year cooperative research program which assigned specific areas of responsibility to each of the agencies. The scope of the investigation included an inventory of nitrogen conditions in the potential drainage areas, possible control of nitrates at the source, prediction of drainage quality, changes in nitrogen in transit, and methods of nitrogen removal from drain waters including biological- chemical processes and desalination. TABLE OF CONTENTS Page ABSTRACT BACKGROUND CHAPTER I - CONCLUSIONS CHAPTER II - INTRODUCTION CHAPTER III - METHODS AND PROCEDURES Water Collection , Experimental Procedure, Experimental Design..., CHAPTER IV - RESULTS Chemical Analyses San Joaquin River Water , Agricultural Drain Waste Water. Algal Bioassay Responses 13 Extracted Fluorescence Experiments Direct Fluorescence Experiments San Joaquin River Nitrate and Chlorophyll 13 17 25 CHAPTER V - DISCUSSION, APPENDIX A APPENDIX B APPENDIX C APPENDIX D: ACKNOWLEDGEMENTS . . LIST OF REFERENCES PUBLICATIONS RESULTS OF THE INDIVIDUAL EXPERIMENTS ALGAL SPECIES AND NUMBERS CELL COUNT AND CHLOROPHYLL CONCENTRATION BIOASSAY NITROGEN RESPONSES 29 33 43 45 47 53 54 55 FIGURES Number Page 1 Seasonal Chlorophyll Variations of San Joaquin River Water 4 2 Flasks in Culture Box 6 3 Growth Responses Measured by Culture Chlorophyll a 19 Al Algal Growth Response of San Joaquin River Water and Mixed Samples 41 A2 Algal Growth Response of San Joaquin River Water with Added Agricultural Waste Water 42 CI Cell Count vs Chlorophyll a_ Concentrations, Initial and Terminal Measurements 46 Dl Bioassay Decrease in Nitrate-Nitrogen as a Function of Chlorophyll Increases 50 D2 Bioassay Chlorophyll Maximum as a Function of Total Inorganic Nitrogen (NO3-N plus NO2-N) 52 TABLES Number Page 1 Chemical Analysis of San Joaquin River Water Collected in the Vicinity of Antioch 10 2 Chemical Analyses of Agricultural Tile Drain Waste Water Utilized in the Nutrient Re-addition Experiments 11 3 Chemical Analysis of Agricultural Tile Drain Waste Water Utilized in the Comparative Nitrogen Removal Process Experiments 12 4 Maximum Extracted Fluorescence of Agricultural Tile Drain Waste Water Bioassays Before and After Inorganic Phosphorus Additions 14 5 Maximum Extracted Fluorescence of Agricultural Tile Drain Waste Water Bioassays with Nitrogen Additions 16 6 Normalized Algal Bioassay Response of Treated and Untreated Agricultural Waste Water 21 7 Normalized Algal Bioassay Response of the Treated Agricultural Waste Water 23 8 Normalized Summary of Grov/th Rate Responses, Ab 24 9 Bioassay Comparison of the Nutrient Removal Efficiency of Algal Pond and Bacterial Filter Systems 26 10 Seasonal Chlorophyll and Nitrate Variations in the San Joaquin River at Antioch Bridge 27 Al Bioassay Chlorophyll Increases 34 A2 Bioassay Chlorophyll Maxima 37 Bl The Proportional Counts of Algae Found Initially and at the Termination of the Bioassay Experiments 44 Dl Nitrate Concentrations in Test Waters Before and After Bioassay 48 CHAPTER I - CONCLUSIONS 1. San Joaquin River algal bioassay responses were related to the nitrate and algal content. Summer and early fall bioassay samples had a high initial algal growth and a low nitrate concentration; little growth occurred in the laboratory. Winter and spring samples had a low initial algal population and a high nitrate concentration; large algal growth occurred during the course of the bioassays. 2. Mixtures of San Joaquin River water and untreated agricultural drainage consistently stimulated algal growth, regardless of the season. 3. The bioassay responses of mixtures of treated agricultural drainage with San Joaquin River water showed that nitrogen removal from agricultural drainage is definitely effect- ive in reducing biostimulation. 4. Effluents from algal pond and anaerobic denitrif ication treatment systems produced statistically equal bioassay responses when compared on the basis of the nitrate and nitrite remaining in the effluent. In general, algal bioassay responses varied inversely with the efficiency of nitrogen removal. High terminal chlorophyll bioassay concentrations were associated with inefficient removal of nitrogen from the waste water. 5. When inorganic nitrogen was below 2 mg/1 in treated drainage, algal grov/th responses of the mixture of treated drainage and San Joaquin River water produced responses similar to those of San Joaquin River controls. 6. Algal growth responses observed in untreated agricultural waste water were reproduced when nitrate-nitrogen was re-added to the treated waste waters bringing the levels back to those found in the original waste water. -1- CHAPTER II - INTRODUCTION Algal bioassay methods were used to evaluate nutrient removal processes under study at the Interagency Agricultural Waste Water Treatment Center (lAWIJTC) , Firebaugh , California. This study was conducted as part of a project to reduce algal growth when agricultural waste water is mixed with San Joaquin Delta v;ater. Upon adding waste water or nitrogen and phospho- rus to San Joaquin River water, analyses were made comparing bioassay algal growth responses in treated and untreated agricultural waste water. The experimental growth responses were similar to algal gro\;th observed in the San Joaquin River (Figure 1) . The proposed agricultural tile drain will empty into the San Joaquin River near Antioch. The first part of this report is devoted to the chemical analyses of both the San Joaquin River water collected near Antioch and the treated and untreated agricultural waste water. This section is followed by the algal bioassay responses to nutrient chemical addition and to agricultural waste water. Much of the data has been normalized to overcome the variable growth response of the control medium, the San Joaquin River water. Normalization consisted of dividing all of the data from each bioassay by the bioassay value for the San Joaquin River v\7ater for that date. In this way, results can be logically compared or summarized v;hen the responses for the basic medium are equalized for all bioassays This normalization technique was applied to the waste water treated chlorophyll growth responses and growth rates (ub, day" ) and to the algal pond and bacterial filter bioassay efficiency comparisons. The final part of the main report relates seasonal variations in chlorophyll and nitrate concentrations in the San Joaquin River water. The detailed analyses of all the experiments utilizing bacterial filter, algal pond, and untreated agricultural waste water are given in the appendices along with sections reporting bioassay algal species changes, and bioassay correlations between cell count, chlorophyll, and nitrate concentrations . -3- *o Co 30 o o o o So o <9 20 o o oo o o o og o o o© 10 o - ■* 8 ° ° 1 — o^ 1—0 — r ^ Samples taken in 1966 at Antiocti Bridge \ 1 1 1 1- JFMAMJJASO MONTHS Figure I. Seasonal Chlorophyll Variations in San Joaquin River Water AGRICULTURAL WASTE WATER STUDIES SAN JOAQUIN VALLEY, CALIFORNIA ENVIRONMENTAL PROTECTION AGENCY REGION IX SAN FRANCISCO, CALIFORNIA -4- CHAPTER III - METHODS AND PROCEDURES WATER COLLECTION The agricultural waste water was collected in polyethylene containers on the afternoon prior to the beginning of each bioassay, v/hile the San Joaquin River water v/as collected early the next day. Personnel from the Interagency Agricultural Waste '-later Treatment Center stopped at Antioch, California to collect San Joaquin River water on their way to the EPA laboratory at Alameda, California. Usually the Antioch samples were collected approximately four hours before the bioassay was started. The algal pond waste water was prefiltered through GFA glass filter pads at Firebaugh immediately after collection, reducing algal concentration to that found in the bacterial filter and anaerobic pond waters. This filtration also corresponded to the algal harvesting from the pond waters which is an integral part of the algal stripping process. EXPERIMENTAL PROCEDURE Untreated agricultural drainage, algal pond, anaerobic pond, or bacterial filter waters were added separately to San Joaquin River water in dilutions ranging from 1 percent to 50 percent of the total sample volume. These percentage addi- tions were chosen because they approximated the probable future concentrations of waste water in the San Joaquin River. Undiluted waste water was used in some initial experiments to test for specific nutrient deficiencies in these waters. Each sample for bioassay was prepared in triplicate. A sample of 300 ml was placed in a 500 ml Erlenmeyer flask which was then plugged with a foam rubber stopper. An aliquot was saved for nitrate analysis. In another series of bioassays , inorganic nutrients (KNO3 or KH2PO4) v/ere added to the San Joaquin River water to determine the results of nitrate or phosphate, alone or combined, on the algal growth. Before any assay was started, chlorophyll a concentrations were measured. Incubation of the cultures was in a 20° C + 1° C constant temperature walk-in refrigerator. The flasks were put on plate glass shelves with cool-white fluorescent lights mounted one inch below each shelf (Figure 2) . Approximately 600 ft-candles of light reached the bottom of the flasks. Flasks were incubated from 5 to 15 days until algae reached maximum growth. A blov/er and a system or ducts forced -5- Figure 2, Flasks in Culture Box -6- air constantly through the space between the fluorescent tubes and the plate glass and through the area above the glass shelves. (Without this forced-air system, the fluorescent tubes cause a slight warming of the plate glass shelves.) Since the blower was running continuously, the air temperature was kept nearly constant. In the first series of bioassays (12/13/68-4/4/69) , algal growth was monitored by extracted chlorophyll fluorescence. The cultures were subsampled on the day they were set up and on alternate days thereafter. Fif ty-milliliter subsamples were taken from each culture. Following filtration through GFC glass fiber pads, they were macerated in a cell homogenizer, and chlorophyll was extracted by 90 percent acetone. The method is described in detail by Bain (1969) . Algal growth in the second series of bioassays (6/18/69 - 11/17/69) was measured daily, except for weekends, by direct fluorescence of a small subsample ( 10 ml) taken from each flask being incubated. The foam rubber stoppers were replaced by ones of hard rubber and the flasks were shaken. Readings were taken on a Turner Fluorometer equipped with a Corning CS 5-60 primary filter and a Corning CS 2-60 secondary filter. Normally the 30x scale was used. The subsamples were discarded after fluorescence readings since the flasks contained enough volume for many readings with- out appreciable change in the surface-to-volume ratio. The readings were taken until algal fluorescence had reached a plateau or had started to decline, which usually occurred in 5 to 7 days. At the termination of the experiment, the water from the replicate flasks was combined and used for NO3 determinations . Direct fluorescence readings were converted to chlorophyll concentrations for each sample. These readings were followed by filtration and 90 percent-acetone extraction of larger volumes of water from 1-liter flasks containing the same media as the smaller flasks. Extracted samples were measured for optical densities at specific wavelengths follov;ing chlorophyll determination procedures recommended by Strickland and Parsons (1965). Analyses for nitrate - nitrogen, nitrite-nitrogen, organic nitrogen, ammonia, inorganic phosphorus, and total phosphorus followed the procedures listed in the FWPCA Methods for Chemical Analyses of Water and Wastes, (1969) . -7- EXPERIMENTAL DESIGN The experimental design was factorial, permitting an analysis of variance of the data followed by a multiple range test, if the variance was significant. The multiple range test used was that of Student-Neuman-Keuls (Steel and Torrie , 1960). Three growth parameters were derived from the flask culture growth curves: (1) increase in chlorophyll concentration, (2) maximum chlorophyll concentration, and (3) maximum observed growth rate (pj-, , day" ) , -8- CHAPTER IV - RESULTS CHEMICAL ANALYSES San Joaquin River Water Chemical analyses for the San Joaquin River water used as the basic medium in the algal bioassays are given in Table 1. The collection dates correspond to bioassay experiment dates and cover a period of one year, from December 196 8 through November 19 69. Changes in most of the measured parameters appear moderate. Total P has a value of 0.6 5 mg/1 for June, but all other samples have concentrations between 0.07-0.16 mg P/1. Changes in PO4-P and organic N concentrations are small. The highest total inorganic N values (NH3-N plus NO3-N) occur in the January and February samples, 1.10 and 0.95 mg N/1 respectively, with low values of slightly more than 0.10 m.g N/1 present during the summer months. The highest total inorganic N to PO4-P ratio was approximately 11:1 in the January 4 sample; the lowest was 2:1 in July and August. Agricultural Drain Waste Water The chemical analyses of agricultural waste water samples added to San Joaquin River water are listed in Tables 2 and 3. The determinations include NO3-N, NO2-N, PO4-P, and soluble organic nitrogen. Any NH3-N in the samples was reported as part of the soluble organic nitrogen. On the basis of many determinations, the NH3-N level is 0.10 mg N/1 or less in the algal pond and untreated agricultural tile drain waste water and less than 1.5 mg N/1 in the bacterial filter water (Sword, 1970) . Untreated agricultural tile drainage is high in NO3-N concentrations, 9 . 2 mg N/1 to 22.5 mg N/1. Nitrate- nitrogen concentrations also vary in the treated agricultural waste water because samples showed different levels of nitrogen removal. The highest effluent nitrate value was 5.1 mg N/1 produced by bacterial filter No. 19 on August 18, 1969. The measurement 9 . 2 mg NO3-N in the original untreated waste water indicated that less than half of the nitrate was removed. Some of the treated samples had trace concentrations of nitrate (<0.10 mg N/1) showing a high efficiency of NO3-N removal. -9- X o o 8 6 cc z Ul ^ ^ « ■'I' _i 00 a> c^ a> o •^ o o •-H CD « • • • o o o d o o N U3 00 oo GO to 'S< to o 1 o d d d d V o (£> o to 03 00 c- 00 o M o o •— * • '-^ d o O o o o c^ to CV2 00 lo to 00 o ■* o o i-H «D • • • • • d o O o V o o c~- C\2 ^_, o Tl< •<* •^ o to o •-H m d o o o o o UJ f- - «o o 5D to o ^H C\2 ^ • • • • • d O o o o o c^ CV2 w •<* o C\2 to ' • o ' ' d • o d 00 C\2 a a> c- 00 w o 1 o o ■<1< N 00 •—* ,__, • • • • d o o o ^-« ^ c- >< o •H W rH di fa .H a ^ * LD + CM CM CN CN CM ■P ■^ CM O CM CM CM CN CN s 2 rH iH CD CD O d d V V V V V V ^ 0) g ?§ ^ &^ O O iH CM O i-t WD -H H cu oj cn O in 4-1 -H ^^ (N n CN rrt w o Oj rH Cm tH Q) \ V4 P^ o 1 LT) CM CM CM CM CM iw; ■-* (N O O O O O o fSi " K o o d d O d d V V V V V V ^ O [^ cri rH •^ ro 00 iH r~ <-i in m CM n T-i o c» d rH «* rH t> ^c55H^ CN rH 00 CT» (T» (T> CO CTi OD 1^ VO 1X> 1X> <^ VD vo ii 1 1 1 1 1 1 1 to iH 1 1 1 rH 1 rH Q 1 <-i CM rH r-i 1 CM rH rH C ■H 1^ rH OJ S ^§ n3 +J •H P SIS .s§ m rH rH en rH u5 u o as 4J MH U W i g i g g o ^ I I 22 o o o CM •-{ o -p ■P HI HI iH !51 rH •-{ -P -p -< <: -14- Additions of phosphorus to the bacterial filter effluent brought dramatic algal response only in the No. 19 sample of 12/13/68 (Table 4), where fluorescence increased from 56 to 600 units. This filter had the highest remaining concentration of total soluble nitrogen (7.18 mg N/1) of any listed for 12/13/68 or 1/4/69. Phosphorous limited samples (N:P>300) gave the highest growth responses upon the additions of phosphorus . b) Nitrate-Nitrogen Additions : Nitrate was added to effluent from algal ponds and bacterial filters to determine whether nitrate re-addition would increase algal growth to values comparable to those of untreated waste water (Table 5) . For all of the responses listed, algal growth in the treated controls was less than that in raw waste water. The addition of nitrate-nitrogen to the bacterial filter and algal pond samples usually increased algal chlorophyll fluorescence above the level found in the original bacterial filter and algal pond effluents. For example, the 10 percent algal pond sample of 1/28/69 had a fluorescent bioassay response of 262 units. Nitrogen additions increased the fluorescence to 370 and 344 units but did not increase fluorescent values to those of the original untreated waste water. The sample containing 10 per- cent untreated agricultural waste water had a direct fluorescence of 392 units. Based on the ratios of nitrogen-to-phosphorus , it was noted that nitrogen did not appear to be lacking in the cultures. Phosphorous values include only inorganic phosphorous values , and not organic phosphorus which could have been used by the algae. Increases in fluorescence did not occur after each nitrogen re-addition to the treated waste waters . In the 100 percent bacterial filter samples, as shown by the bacterial filter No. 19 samples of 3/10/69, algal bioassay responses, both with and without nitrogen additions, were very low and almost equal to each other. One probable reason for the lack of response was the few algae contained in the 100 percent bacterial filter effluent. -15- 5 5e r- m in (N 00 o o o^ o 00 y CM 3 2 a u in o in in in 00 i-H rH »» i-i iH CM r>) CO r~ T oo n vD (Ti ro CO r^ ^ VD 00 1 I 00 ^ 00 O i-< O TT O O 00 O n Ol IT) in (N 00 vo CO in Ol 1 • vo l~ OV d 04 rH CM CN -\ 4 rH ON oJ •^ r- .g O CM o o o o 1 ■ o o o o m o g •-\ rA rH rH fH , 1-3 d 1 s OP C O •8 ^t N (1) ■H in § dV * dP dP "d Sa rH O i-l O fM ffl (JP ss as c o •rl " ■H W^ ^ ^ VD 00 « o CN (Tl O •rH CM 3 cr (0 *r> c (0 CO m. +J &? O 00 r-\ rH O r- 00 a\ ro CO nH . • • • c rg i-i n n o „ rH •H 4J -H Xf tJ < 0) en (0 +J iH •^ o> O c fe^ 00 o fM CM M « 2 H D O O O o o o o O o « • • • • o r-l rH rH rH 1^ — 1 02 nH \ r~ o ■>* 2 r-{ O 00 •"a- ^_^ 2 tr o <-t r-i r^ DC 1 B 1 1 1 1 ■ — ' (N 0) O v^ ID r^ Q W O -H rH TT ■>3< W W 2 a • • • • ts] 2 O O rH CM H O i^ S J cu CO rtj CO 2 o 2 1 c O 2 ja:}.i ?M 9^se M T Ejn:;xnoTji DV ps^^B ajj, -23- I C k. D -O (0 3 O to UJ CO 95 UJ cc § Q UJ M Zj <: s cc s .-H ^ lO I I I o. u_ u. •< 03 CO 0) 0) — C U_ flj — . -O I- ^ ^ n '^ o" o nj -H (0 Q- — ^ O I- D -3 ^ « O «j -H" •— c on o k. (ij -H nj on CO U) u • (0 -1-' .->«. C ^ O 0) * Cvi "O C T) «♦- — . C C (0 O c o 'b^ 0) ^H LO E 05 o o E «J t^ O E "- (0 4) -f O (0 (0 X) -H » nJ on ■T3 L. c 4) >^4 O > c ^ — • <-4 h-QC '-• o -o o o -24- I values of the algal pond and bacterial filter water. The line does not include the tile drain 1% addition (1.28) indicating that even this small percentage of untreated waste water gives higher algal growth rates than the San Joaquin River control. A substantial overlapping of the multiple range confidence lines occurs between the growth values of the various samples of the treated waste water and the untreated tile drainage. Furthermore, the underlining for the normalized San Joaquin River water (1.00) extends to the algal pond 5% addition response (1.12), signifying no statistical difference in the responses included by the under- lining (Table 8) . e) Comparative Algal Pond and Bacterial Filter Nutrient Removal Efficiencies! A comparison was made of the normalized growth responses of the two nitrogen removal systems . The only growth responses utilized had comparable concentrations of inorganic nitrogen and the same sample dilution for both systems. In Table 9 the normalized response values 6.30 and 6.83 are averages of the ratio of maximum chlorophyll increase in the treated waste water samples to that of the San Joaquin River water. The response values 6.30 and 6.83 are not statistically different (P< 0.01) . San Joaquin River Nitrate and Chlorophyll Table 10 presents initial nitrate concentrations and seasonal chlorophyll concentrations both initially and following incubation. Initial field chlorophylls for the San Joaquin River samples were low from November through May. They reached a peak of 40.9 jU/g chl. a/1 in July. Little growth occurred during incubation in the laboratory for the samples taken in the summer; no bioassay response was recorded for the July sample when NO3-N concentrations were the lowest of all those listed (0.04 mg NO3-N/I). Highest chlorophyll increases were recorded for the 1/28/69 and 2/14/69 samples. These contained the highest initial NO^-N concentrations, 0.74 and 0.95 mg N/1 respectively. Most of the bioassay maximum values for the San Joaquin River water used in controls ranged from 30 to 45 Mg chl. a/1. Maximum algal chlorophyll values appear to be equal for many of the sampling months, regardless of whether results were obtained from field or laboratory data. -25- TABLE 9. - BIOASSAY COMPARISON OF THE NLfTRIENT REMOVAL ^ EFFICIENCY OF ALGAL POND AND BACTERIAL FILTER SYSTEMS Number of Observations Nornnalized Response S Mean F Value Algal Pond 78 6.30 0.12 ^ Bacterial Filter 72 6.83 k. Combined normalized data from inorganic nitrogen content groupings in which both removal systems are represented. 1. The F value would have to be in excess of 3.91 before the normalized response could be considered statistically dif- ferent (P>0.05). -26- TABLE 10. - SEASONAL CHLOROPHYLL AND NITRATE VARIATIONS IN THE SAN JOAQUIN RIVER AT ANTIOCH BRIDGE jjs Chi, a/1 NOst-N. me N/l Date Initial Maximum Initial Sample (Field) (Bioassay) (Field) 1968 11/14 3.8 34.9 > 12/13 1969 1/4 2.0 1.3 43.5 38.8 0.43 0.61 1/28 1.5 104.1 0.74 2/14 1.1 69.2 0.95 3/10 1.8 45.6 0.53 4/4 4.2 15.2 0.22 6/18 28.4 38.9 0.18 7/25 40.9 40.9 0.04 8/18 34.8 35.3 0.05 9/9 31.0 53.4 0.08 9/29 32.0 55.7 - 10/20 27.2 35.5 0.09 11/17 20.7 32.1 0.22 -27- CHAPTER V - DISCUSSION Bioassays were used to evaluate algal grov/th-promoting properties of treated and untreated agricultural waste water. Samples were diluted by adding them to San Joaquin River water at 1%, 10%, and 20% by volume. The algal growth bioassays were evaluated by measuring chlorophyll concen- tration maxima and increases. San Joaquin River water collected in the summer contained a high initial concentration of algae and so growth was minimal , thus termi- nating with only a small increase in chlorophyll concentration, Maximum chlorophyll concentration gives a measure of the total algal mass which can be supported by the sample regard- less of whether the conversion of nutrients has occurred in the receiving waters before or after laboratory incubation. Increase in chlorophyll indicates the amount of algal mass formed during the bioassay, however, bioassays that ended with highest increases in chlorophyll also had the highest maximum concentrations of chlorophyll. Bioassay responses can be referred to without specifying either chlorophyll maxima or increases. The agricultural waste water treated by either the algal pond or anaerobic denitrif ication systems showed small algal growth responses in the 1% dilution samples. In 10% or 20% dilution samples treated waters promoted algal growth when the removal systems were not performing well. Treated waste water with nitrate concentrations at the lower limit of chemical detectability (<0.10 mg N/1) showed grov/th responses in the 10% and 20% dilution samples similar to those of the San Joaquin River controls. Instances of substantial algal response occurred when nitrate concentrations were low, indicating other forms of available nitrogen: ammonia and nitrite-nitrogen were present in some samples. There were also examples of little growth in spite of the high nitrate-nitrogen content of the algal pond and bacterial filter samples, suggesting that these systems might have removed other essential plant nutrients or added toxic materials to their effluents. Samples from the nutrient removal systems almost always gave growth responses below those of untreated agricultural tile drain waste water during the same series of algal assays. This did not necessarily hold true for 1% additions to San Joaquin River water since such small grov7th differences between samples are more difficult to detect than those at the higher percentage additions. -29- Normalization of the assay values shov;s algal response of treated waste water above that of the San Joaquin River control. These data were also grouped to compare experimental responses on the basis of the residual effluent NO3-N and NO2-N in the treated tile drain waste water. Statistical evaluation indicates that an effluent nitrogen concentration of less than 2 mg N/1 would stimulate algal grov\7th response equal to that of the San Joaquin River control. However, all of the 20% dilutions of treated waste water (Table 7) , even those containing less than 2 mg N/1, gave responses above the control. The 20% dilutions of treated waste water in the category containing 0.10-0.17 mg N/1 were statistically equivalent to the river water and hence should not increase the bioassay response. There could be several explanations for the anomalous responses in Table 7: 1. The response might be due to limitation by nutrients other than nitrogen, 2. The ammonia in the bacterial denitrif ication system effluent, which was not differentiated from the organic nitrogen by the analysis, probably con- tributed significantly to the algal growth. 3. Interpretation of algal growth as based solely upon waste water inorganic nitrogen content might not take all factors into account since the algal pond system obviously would remove algal nutrients other than inorganic nitrogen. Some of these nutrients might be potentially limiting in river/waste water mixtures. Normalization techniques were also used on growth rate data. These normalized growth rates should not be confused with the absolute grov;th rates although the numbers obtained are similar. The maximum specific grov;th rate data (uj-^) is significant in evaluating residence time effects on algal growth. Doubling of the growth rate may more than double the algal mass. This may be critical in areas of short residence time, such as in the transport canals and Carquinez Straits. Observed growth rates during treated waste water bioassays were not statistically different from San Joaquin water. Samples containing untreated agricultural waste water showed growth rates above those of the San Joaquin River controls . The high and variable nitrate concentration found in the San Joaquin River water which was used for sample dilution might have caused observed growth rate differences in different effluents. Most samples of river water exceeded the 0.06 mg N/1 saturation coefficient (Kg) found in -30- earlier studies (McGauhey, et al., 1968). Since the detention time of Delta water is sufficient to allow present algal crops to reach their peak (Bain, et al, 1968), nutrient content is presently more important in the San Joaquin Delta than is growth rate. If sediment concen- trations become lower and thereby increase light penetration in the Delta-San Francisco Bay System (Krone, 1966), the high bioassay growth rate presently found in San Joaquin River water would indicate the possibility of frequent algal bloom. Initial bioassays were carried out to determine the effects of inorganic nitrogen and phosphorus compound additions upon the waste water bioassay responses. The addition of nitrate-nitrogen to the algal pond or bacterial filter sample effluents stimulated algal responses so that they equaled the lantreated tile drain waste water six out of eight times in samples of comparable dilution. Additions of inorganic phosphorus to treated waste water effluents gave mixed responses. Inadequate algal bioassays were made to provide a statistical evaluation of the results. Two samples of untreated tile drain water responded to the addition of PO4-P, as did one sample out of five of the bacterial filter systems when PO^-P was added. The concomitant use of effluents from two distinct methods of nutrient removal in algal bioassays suggested probable comparison of their efficiency for nutrient removal. Only bioassay data in which both systems had similar con- centrations of inorganic nitrogen were compared. It was found that normalized bioassay responses for both systems are almost equal and their differences not statistically significant. Growth rate data comparisons (Table 10) indicated no differences in the algal pond and bacterial filter responses. The algal bioassay growth of the San Joaquin River water was related to its NO3-N. Summer and early fall samples exhibited little or no growth between the initial and terminal values made over a period of a week or more. This -31- lack of algal growth suggests nutrient exhaustion in the Delta waters sampled during the suiraner and early fall. By contrast, samples from other seasons of the year showed large differences between initial and terminal chlorophyll values. This condition was probably due to available nitrate. Most of the maximum bioassay values recorded were between 30 and 45 pg chl. a/1. This would corres- pond to approximately 6 - lOmg algae/1 dry weight. -32- APPENDIX A: RESULTS OF THE INDIVIDUAL EXPERIMENTS Results from the seven experiments utilizing waters collected from June 18 to November 17, 1969, are listed in Tables Al and A2 . The data from each experiment are divided into two sections. One compares all water types for a single percentage dilution (Al.a, A2.a) and the other compares all of the percentage dilutions for a specific water type (Al . b , A2 . b ) . In comparing the data, underlining was used to indicate values similar to each other at the 95% confidence level. This procedure was allowable because the analyses of variance F values were highly significant for water types, percentages and interaction. In these experiments "interaction" means that increasing the percentage of test waters in San Joaquin River water did not yield comparable increases or decreases in the algal growth for the different test waters. For example, the values in Tables Al and A2 should be read in conjunction with the responses for the July 24 experiments in Figures Al and A2 . Both figures show algal chlorophyll concentration changes throughout the conduct of the experiment. It can be seen in Figure Al that initial chlorophyll values for the different water samples at the beginning of the experiment are not equal and that both the control and the 20% algal pond flasks had no growth. Chlorophyll concentrations decreased from their initial values for these latter two samples. In Table Al.a, the July 24 experiment shows increases in chlorophyll for the 20% agricultural waste water, bacterial filter, and algal pond samples; 44.0, 18.1 and 0.0 Mg chl. a/, respectively. Because each value is statistically different, underlining does not connect them. The maximum chlorophyll concentrations for the Scune samples as shown in Table A2.a are 80.4, 51.5 and 40.9 //g chl. a/. All values are statistically different at the 95"% confidence level. The algal growth responses for all percentage additions of untreated waste water are graphed in Figure A2 . Increases in chlorophyll concentrations, shown in Table Al.b, for the 0, 1, 10 and 20% waste water additions are 0.0, 13.6, 47.0 and 44.0 ^g chl. a/, respectively. The highest responses, 47.0 and 44.0 /ug chl. a/, are statistically equal. Table A2.a lists the maximum chlorophyll concentrations for the same samples. 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(N CM •-{ a R < \ \ \ jQ W P '° Agricultural Waste Water Studies Report No. 13030 ELY 8/17-9 Pages: 59, Figures: 8, Tables: 14, References: 9 s CSfarred First) * Eutrophication * Bioassay * Denitrif ication * Fluorometry * Tile Drains Nitrates, l«iitrogen W red Fust) Algal Blooms - control, bioassay - algal, chlorophyll, denitrif ica-tion , fluorometry, tile drainage U' Laboratory bioassay experiments were performed tc test the effect on algal growth of agricultural waste water before and after the waste water had been subjected to two different nitrogen removal processes. The waste waters were added in various percentages to San Joaquin River Delta v;ater* for bioassay.' The algal growth throughout time was monitored by chlorophyll fluorescence techniques. The fluorescence measurements showed logarithmic growth similar to the type usually observed in the Delta Water over the vernal growth period. The laboratory experiments gave positive statistical evidence that" the untreated agricultural waste water would promote substantial algal growth above that of the San Joaquin River controls. Both nitrogen removal processes were equally effective in lowering the algaJ growth to that of the Delta water controls as long as the nitrate-nitrogen level in each removal system had been lowered to approximately 2 mg N/1, or less. ir^si^ ■ e ^j ' 1 p ■ c TOM. n. c 2'ii* tX]. S. GOVERNMENT PRINTING OFFICE: 1972-514-148/66 1961- 351- J3 i "■^ ft> M *-< rt t— o* £ "> o ►^ c 2. «< Ji •< < o o n J* c &. S u d. c. n o 1 »5 S 3 n ° ?; a ;3 « _ ra g& 1 - 3J D B 3 tng 11 O Dg s- s t3 »-f - C w • C; r> -g. tr 5. 2 p re . 3 M 3 J- w re « s- o " K' S- S f? re S »" » 3 E. o- "^ o o »rf* u < n Jl, re s s tr r. tj cr cr o re (t 3 2 3 3) •V < tt (D O c c 3) O o (D* (D o' z 3^ 00 Q> s * m z O 00 o o 3 (A a Z H > c ^ W -D w O o> 31 5" " O" O (D o X c H (A (0 5' •< 5' m O a) 3 H lo o CO O o $ (D O Z > (D o' o (0 3 m Z o -< ^^ 7 64 m ■1 z < 3) "0 o o z (0 m > z o H m > > r- z "0 o 3) o -n m H m m O w H -0 O > z o > o m Z o THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW BOOKS REQUESTED BY ANOTHER BORROWER ARE SUBJECT TO RECALL AFTER ONE WEEK. RENEWED BOOKS ARE SUBJECT TO IMMEDIATE RECALL IW14'»t ^^Ih f'^C-D JAN 1333 5RNIA, DAVIS ^Book Slip-Series 458 1175 00499 7766 TC C2 A2 California. Bulletin. 9'/J Dept. of Water Reoo\u:ces, PHYSICAL SCIENCES LIBRARY