WATER POLLUTION CONTROL RESEARCH SERIES • 13O30 ELY O8/7i~9
REC-R2-7I-9
DWR NO.I74 -12
BIO-ENGINEERING ASPECTS OF AGRICULTURAL DRAINAGE
SAN JOAQUIN VALLEY, CALIFORNIA
THE EFFECTS OF AGRICULTURAL WASTE
WATER TREATMENT ON ALGAL BIO ASS AY
RESPONSE
AUGUST 1971
'."•M
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ENVIRONMENTAL PROTECTION AGENCY«RESEARCH AND MONITORING
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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 wastewaters 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 BIQASSAY 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.
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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 nale by tb.a Superintendent ot Documents, U.S. Government Printing Office, Washington, D.C. 2M02 - Price $1.00
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REVIEW NOTICE
This report has been reviewed 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.
.ii
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ABSTRACT
Laboratory bioassay experiments were performed to 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 water for bio-
assay. The algal growth throughout time was monitored by
chlorophyll fluorescence techniques. The fluorescence meas-
urements 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 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/l
or less.
Key words: Algal blooms - control, bioassay - algal,
chlorophyll, denitrification, fluorometry, tile
drainage.
iii
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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 I960,
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.
VI
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TABLE OF CONTENTS
ABSTRACT ill
BACKGROUND v
CHAPTER I - CONCLUSIONS 1
CHAPTER II - INTRODUCTION 3
CHAPTER III - METHODS AND PROCEDURES 5
Water Collection 5
Experimental Procedure 5
Experimental Design 8
CHAPTER IV - RESULTS 9
Chemical Analyses 9
San Joaquin River Water 9
Agricultural Drain Waste Water 9
Algal Bioassay Responses 13
Extracted Fluorescence Experiments 13
Direct Fluorescence Experiments 17
San Joaquin River Nitrate and Chlorophyll 25
CHAPTER V - DISCUSSION 29
APPENDIX A: RESULTS OF THE INDIVIDUAL EXPERIMENTS 33
APPENDIX B: ALGAL SPECIES AND NUMBERS 43
APPENDIX C: CELL COUNT AND CHLOROPHYLL
CONCENTRATION 45
APPENDIX D: BIOASSAY NITROGEN RESPONSES 47
ACKNOWLEDGEMENTS 53
LIST OF REFERENCES 54
PUBLICATIONS 55
VI1
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FIGURES
Number Page
1 Seasonal Chlorophyll Variations of San Joaquin
River Water 4
2 Flasks in Culture Box.
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
Cl 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
N02-N) 52
viii
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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 Growth Rate Responses,
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
ix.
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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 denitrification
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 growth 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-
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CHAPTER II - INTRODUCTION
Algal bioassay methods were used to evaluate nutrient
removal processes under study at the Interagency Agricultural
Waste Water Treatment Center (IAWWTC), 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 growth 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 water for that date. In this way,
results can be logically compared or summarized when 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
(ufc, 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-
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40
30
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Samples taken in 1966
atAntioch Bridge
1 - 1 - 1 - 1 -
JFMAMJJASO
MONTHS
Figure (.Seasonal Chlorophyll Variations in San Joaquin River
Water
AGRICULTURAL WASTE WATER STUDIES
SAN JOAQUIN VALLEYV CALIFORNIA
ENVIRONMENTAL PROTECTION AGENCY
REGION IX
SAN FRANCISCO,CALIFORNIA
-4-
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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, while the San Joaquin River
water was collected early the next day. Personnel from
the Interagency Agricultural Waste Water 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
(KN03 or KH2P04) were 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 blower and a system or ducts forced
-5-
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Figure 2. Flasks in Culture Box
-6-
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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. Fifty-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 NC>3
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 following
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-
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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
-8-
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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 1968 through November 1969.
Changes in most of the measured parameters appear
moderate. Total P has a value of 0.65 mg/1 for June, but
all other samples have concentrations between 0.07-0.16 mg
P/l. Changes in P04-P and organic N concentrations are
small. The highest total inorganic N values (NH3-N plus
NC-3-N) occur in the January and February samples, 1.10 and
0.95 mg N/l respectively, with low values of slightly
more than 0.10 mg N/l present during the summer months.
The highest total inorganic N to P04-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, NC-2-N,
PC-4-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/l or less in the algal pond and untreated agricultural
tile drain waste water and less than 1.5 mg N/l in the bacterial
filter water (Sword, 1970).
Untreated agricultural tile drainage is high in
NO3-N concentrations, 9.2 mg N/l to 22.5 mg N/l. 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/l produced by bacterial filter No. 19 on August 18,
1969. The measurement 9.2 mg NC-3-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/l) showing a high
efficiency of NC-3-N removal.
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TABLE 1. - CHEMICAL ANALYSIS OF SAN JOAQUIN RIVER WATER COLLECTED IN THE VICINITY OF ANTIOCH
1968 1969
Chemical
Alkalinity
CaC05,tng/l
P04-P,mgP/l
Total P,ragP/l
Org N,mg N/l
NH5,rog N/l
N03,mg N/l
Total inorganic
nitrogen, mg N/l
12/13 1/4 1/28 2/14 5/10 4/4 6/18 7/24 8/18 9/7 9/29 10/20 11/17
68 - 59 - 46 54 68 118 68 69 78
0.07 0.07 .02 0.08 0.07 0.07 0.07X 0.07 0.07 0.06 0.4 O.OS 0.06
0.07 - 0.09 0.11 0.08 - 0.65 0.12 0.13 0.10 0.12 0.14 0.16
0.54 0.46 0.47 - 0.37 0.51 0.42 0.26 0.35 0.41 0.32
0.15 0.17 0.28 0.08 0.15 0.12 0.09 0.10 <0.08 0.09 CO. 08 0.08 0.09
0.43 0.61 0.82 0.95 0.53 0.22 0.18 0.04 0.05 0.08 - 0.09 0.22
0.58 0.78 1.10 1.13 0.68 0.34 0.27 0.14 0.13 0.17 (0.08) 0.17 0.31
i
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1
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TABLE 2. - CHEMICAL ANALYSES OF AGRICULTURAL TILE DRAIN WASTE WATER
UTILIZED IN THE NUTRIENT RE-ADDITION EXPERIMENTS
Chemical
Date Anal yses
mc/1
12/13/69 NOg-N
Sol .Org-N
N05-N
N02-N
1/4/69 Sol. Org-N
P04-P
Total -P
N03-N
1/28/69 NOg-N
Sol. Org-N
P04-P
NOs-N
2/14/69 IK02-N
Sol .Org-N
P04-P
NOS-N
3/10/69 NOg-N
Sol .Org-N
P04-P
Untreated
Agricultural
Waste Water
20.3
^. 0.001
0.40
17.9
< 0.001
0.27
0.05
-£0.02
17.0
0.004
0.39
<0.02
19.6
0.005
0.36
0.10
18.6
0.01
0.25
0.04
Treated Agri
cultural Waste Water3
Bacterial Filter
No. 14
1.3
1.0
1.94
No. 11 No.
0.2 0.
-£0.001 0.
0.39 0.
No. 19
3.0
3.7
0.48
13 No. 14
6 0.4
01 0.04
70 0.89
-£0.02 -^0.02 <0.02
^0.02 <0.
02 <0.02
Algal "Pond Bacterial Filter
1.2
5.4
1.25
0.05
No.
1.1
14 No. 19
1.2
2.22 3.7
0.57 0.59
-£0.02 -3D. 02
No. 19 No. 20
3.2 0.36
2.80 1.44
1.60 1.48
0.03 0.12
8.0
0.04
1.17
0.02
No. 19 No. 20
2.7 <0.10
2.26 0.12
0.60 0.66
-£0.02 <0.02
a. Identification numbers refer to pilot test unit effluent not to bioassay
samples. Some of these units were not identified by numbers.
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TABLE 5. - CHEMICAL ANALYSIS OF AGRICULTURAL TILE DRAIN WASTE WATER UTILIZED IN
THE COMPARATIVE NITROGEN REMOVAL PROCESS EXPERIMENTS
Chemical Untreated Treated Agricultural Waste Water
Date Analyses Agricultural
_ (me/1) Waste Water Aleal Pond Bacterial Filter
NQ5-N
NOg-N
6/18/69 Soi.Org-N
POi-P
NQ3-N
7/24/69 NOg-N
Sol.Org-N
POi-P
NOs-N
8/18/69 NOg-N
Sol.Org-N
P04-P
NOs-N
W69 Sol**g:!j
P04-N
NOs-N
9/29/69 NOg-N
Sol.Org-N
POA-P
NOs-N
10/20/69 NOg-N
Sol.Org-N
POd-P
NOs-N
11/17/69 NOg-N
Sol.Org-N
POd-P
11.0
0.002
0.50
0.16
10.0
0.006
0.25
0.19
9.2
0.02
0.04
0.12
11.5
0.01
0.04
0.07
16.5
0.01
0.4
0.08
16.5
0.01
0.4
0.2
22.5
0.004
0.4
0.15
-£0.10
-------
Nitrite concentrations were unusually high in some
bacterial filter and algal pond samples taken before
June 18, 1969. For example, all of the bacterial filter
and algal pond samples collected on January 28, 1968,
contained more than 2 mg NO^-N/l and little more than 1 mg
N03-N/1. The agricultural tile drain waste water from which
the algal pond and bacterial filter samples were derived
contained 17.0 mg N03-N/1 but only 0.004 mg N02-N/1 indicating
that some of the effluent nitrite came from the biological
reduction of nitrate.
Inorganic PO.-P concentrations were usually less than
0.2 mg P/l in the untreated agricultural waste waters and
occasionally below the sensitivity of the analytical method
used.
ALGAL BIOASSAY RESPONSES
Extracted Fluorescence Experiments
a) Inorganic Phosphorus Additions:
The bioassay response of raw and treated agricultural
tile.drain waste water, with and without phosphorus
additions, is shown in Table 4. These extracted
fluorescence values are each the mean of two
replicate samples. Since these data are reported
in extracted fluorescence units it should be noted
that algal cellular chlorophyll concentration and
extracted fluorescence are highly correlated
(correlation coefficient = 0.99). The fluorescence
value in units is approximately 0.5 times the
concentration of chlorophyll a in micrograras per
liter.
The two samples of raw agricultural waste water
(12/13/68 and 1/4/69) had a marked response to
inorganic phosphorus additions. Extracted
fluorescence of the 12/13/68 sample increased from
220 to 1660 fluorescence units and from 330 to 750
fluorescence units in the 1/4/69 sample.
The bacterial filter effluent exhibited a lower
response than the raw waste water. The 1/4/69
samples for bacterial filter Nos. 11, 13, and 14
showed almost no growth relative to that of the
original waste water (1, 2, and 1 extracted
fluorescence units, respectively).
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TABLE 4. - MAXIMUM EXTRACTED FLOURESCENCE OF AGRICULTURAL TILE DRAIN WASTE
WATER BIQASSAYS BEFORE AND AFTER INORGANIC PHOSPHORUS ADDITIONS
Sample
100% Concentration
Untreated
Agricultural
Waste Water
Bacterial Filter
Effluent
No. 11
No. 13
No. 14
No. 14
Mb. 19
Date
12-13-68
1-4-69
1-4-69
1-4-69
12-13-68
1-4-69
12-13-68
Total
Soluble
mj N/l
20.70
18.17
0.59
1.31
4.24
1.33
7.18
Before P Addition
xng P04-P/1
0.2
<0.05
<0.02
<0.02
<0.02
<0.02
<0.02
Extracted
Flourescence
220
330
1
2
200
1
56
After P Addition
itg P04-P/1
1.2
<1.05*
<0.22 +
<0.22
<0.22
<0.22
<0.22
Esctracted
Flourescence
1660
750
1
10
232
33
600
* At least 1.00 ng PO.-P/l
+ At least 0.20 mg PO4-P/1
-------
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/l) 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-
-------
TABIJS s. - MAXIMUM Ejowcam FLOORESCBCE CF AGRJCUIOTRAL THE DRAIN
WATER BICASSAYS WITH NITROGEN ADDITICNS
SAMPLE AND DATE
1-28-69
Agricultural Waste Water
Treated AP (e)
Agricultural. BF(f)-14
Waste Water BF-19
2-14^69
Agricultural Waste Water
Treated AP-19
Agricultural AP-19
Haste Water AP-20
AP-20
3-10-69
Agricultural Waste Water
AP
Treated BF-19
Agricultural BF-19
Vfeste Mater BF-20
BF-20
% of (x)
DPAIN
10%
10%
10%
10%
10%
10%
30%
10%
30%
10%
100%
10%
30%
100%
30%
100%
P04-P/1
<0.02
0.02
<0.02
0.02
0.08
0.08
0.06
0.08
0.09
0.07
0.04
0.06
<0.06
0.02
<0.06
<0.02
Before NC^-N Addition
Total Solids
ng N/l
2.73
1.78
1.38
1.54
3.01
1.78
3.07
1.34
1.78
2.50
18.86
1.54
2.14
5.56
0.74
<0.88
Extracted
Flourescence
392
262
341
346
225
162
115
154
95
400
114
300
260
8
200
2
After 1/2 NO3-N Addition (d)
Total Solids
rag N/l
— —
10.28
9.88
10.04
__
11.58
12.87
11.14
11.58
,
10.84
11.44
14.86
10.06
10.18
Extracted
Flourescence
—
370
395
356
— — _
200
115
199
185
355
390
4
230
30
After 1 NC^-N Addition
Total Solids
mg N/l
— —
18.78
18.38
18.54
•.„•
21.38
22.67
20.94
21.38
__
___
20.14
20.74
24.16
19.34
19.48
Extracted
Flourescence
___
344
380
392
__
180
115
215
245
-__
440
380
7
205
1
on
I
**' Percentage of drain waste water of total sample equals drain water plus San Joaquin River water.
}d;The nitrogen was re-added to the treated waste water at concentrations equal to or half of that found in the tile drainage.
(e'AP = Algal Pond
= Bacterial Filter
-------
Direct Fluorescence Experiments
a) Cell Mass Changes;
The measured experimental cell mass changes were
the increases in chlorophyll and maximum chlorophyll
concentrations. These were derived by conversion
of direct fluorescence to chlorophyll a concentra-
tion for the three replicates per sampTe type.
Both variables were used because the samples for
comparison did not initially contain equal con-
centrations of algae; the amount depended mainly on
the dilution of the San Joaquin River water. The
test samples contained some algae, but most of the
algae were present in the San Joaquin River water.
The chemical analyses of the waste water used in the
direct fluorescence experiments are given in
Table 3. The waste water was added to San Joaquin
River water on each experiment date. The analyses
for the river water areishown in Table 1.
The complete experimental results from the algal
growth comparisons in Appendix A show increases
in chlorophyll and maximum chlorophyll concentrations.
b} Summary of Cell Mass Changes;
In five out of seven experiments, the untreated
agricultural tile drain waste water consistently
gave growth responses above those obtained for the
San Joaquin River control (0 percent concentration).
Usually the 10 percent and 20 percent concentrations
gave equal responses. However, the 1 percent addition
of treated agricultural waste water rarely gave growth
responses above those of the San Joaquin River control,
The results of the 10 percent and 20 percent bacterial
filter and algal pond additions can be understood by
relating the growth response to effluent nitrogen.
For example, in samples taken on August 18, 1969,
(Table 3), inorganic nitrogen in algal ponds
Nos. 14 and 15 was 3.61 and 1.78 mg N/l, respectively,
and 7.44 mg N/l in bacterial filter No. 19. In
contrast, bacterial filter No. 11 had only 0.50 mg
N/l of inorganic nitrogen. None of the samples
containing filter No. 11 effluent exceededjthe
control in algal response, whereas all of the other
-17-
-------
samples gave growth responses above the controls
at one or both of the 10 percent or 20 percent
additions (Table Al).
A few of the responses could not be explained on
the basis of the nitrogen content of the treated
effluents. Both of the treated agricultural
waste water samples of July 24, 1969 had a low
total inorganic nitrogen content (Table 3). The
algal pond effluent contained 0.17 mg N/l while
the bacterial filter contained 0.10 mg N/l. Yet
algal growth responses of the 20 percent bacterial
filter samples exceeded responses from the algal pond
(Appendix A)« Another example of responses not
clarified by the inorganic nitrogen concentrations
is shown by effluent samples from filters Nos. 10
and 15 of October 20, 1969. Sample No. 15 had a
total inorganic nitrogen content of 1.45 mg N/l,
while sample No. 10 totaled <0.15 mg N/l (Table 3).
Bioassay chlorophyll increases for the two
bacterial filters effluents, however, were equal
in the 10 percent additions. Sample No. 10 exceeded
No. 15 in the 20 percent additions by 20.4 to 12.6 mg
chlorophyll a_ per liter (chl a/1) .
Bacterial filter effluent contained up to 1.5 mg
NH3-N/1 during the summer months but normally
contained less than 1.0 mg KH3-N during other
periods (Sword, 1970). Since ammonia was analyzed
as part of the soluble organic nitrogen, it was not
included in the total inorganic nitrogen values.
In Figure 3, contrary to expectation, the bacterial
filter growth response drops below that of the
algal pond effluent response. For the same
concentrations, the algal pond and agricultural
tile drain waste water flasks show growth increasing
monatonically with waste water concentration.
c) Normalized Bioassay Chlorophyll Cell Mass Responses:
The variable algal growth in San Joaquin River
water (0%, without addition of waste water) indicated
that in order to summarize the results, normalization
of the data was more logical than utilization of the
absolute response values. Normalization was
accomplished by dividing the results for each
experiment by maximum bioassay growth response of
the control water used. Furthermore, bacterial
filter and algal pond values were not separated
but placed into four categories based on their
original N03-N plus N02-N concentrations:
-------
50
40
ID
CL
E
2 30
o>
o
20
X
rx
o
-------
0.13-0.17, 0.46-1.00, 1.45-1.86, and 2.94-7.44 mg
N/l. These categories were chosen arbitrarily
for statistical evaluation. The untreated
agricultural tile drain waste water responses were
reported together. Total inorganic nitrogen in
the tile drain ranged from 9.22 to 22.50 mg N/l
(Table 3). San Joaquin River water contained
from 0.13 to 0.27 mg N/l (Table 1). For comparison
purposes, the normalized responses for the San Joaquin
River water are 1.00. Agricultural tile drain
waste water comparisons are included in Table 6 but
not in Table 7. The ranges and means of inorganic
nitrogen in the original samples are listed under
each category.
It is best to utilize the mean value for the
inorganic nitrogen in the river water and the waste
water to calculate the mixed sample inorganic
nitrogen. Using this procedure, a sample made by
adding 10 percent treated waste water containing
2.94-7.44 mg N/l to San Joaquin River water would
have a final inorganic nitrogen concentration of
approximately 0.59 mg N/l. Utilizing the mean
values of the San Joaquin River water and of the
waste waters, the mean inorganic organic nitrogen
value of treated waste water is 4.12 mg N/l. Therefore,
100 ml of this water would contain 0.412 mg N,
and 900 ml of San Joaquin River water (0.20 mg
N/l, mean value) would contain 0.18 mg N. Adding
0.412 to 0.18 gives a concentration of 0.59 mg N/l
for the mixture. Other samples can be calculated
in the same manner, i.e. a 20 percent addition of the
same waste water to San Joaquin River water would
contain 0.98 mg N/l.
Another feature of Table 6 is the bracketing and
underlining of values which do not differ signifi-
cantly from each other at the 95 percent confidence
level. For the 1 percent additions, therefore, the
values 1.00 through 3.20 are connected by underlining.
This means that these responses are statistically
equal, regardless of their absolute value. The
number 6.63 is significantly higher in value than
the others.
Table 6 shows that the 10% and 20% additions of
the treated agricultural waste water containing
2.94 - 7.44 mg N/l were the only samples exceeding
San Joaquin River water in algal growth.
-20-
-------
TABLE 6. - NORMALIZED AIGAL BIOASSAY RESPONSE^) CF TREATED AND UNTREATED AGRICULTURAL WASTE WATERln)
mg N/l of NOa-N and NO?-N in Sample; Range and Mean
Normalized
Response (g)
Percent
Addition
1%
10%
20%
Combined 1%, 10%,
and 20% Additions
San Joaquin River Treated Agricultural Waste Water
Agricultural Waste Water
0.13-0.27 0.10-0.17 0.46-1.00 1.45-1.86 2.94-7.44 9.22-22.50
0.20 0.13 0.66 1.65 4.12 13.86
, 1.00 1.81 1.04 1.29 3.20 , 6.63
,1.00 2.70 1.88 3.91, 13.31 27.89
,1.00 5.94 3.94 9.36, ,20.98 29.55,
, 1.00 3.34 2.29 4.85 , 12.50 20.95
(g) Normalized Response = /«a*mun chlorophyll increases in treated water
e^ VMaxomum chlorophyll increases in San Joaquin River water
(h) Samples connected by underlining do not differ in the 95% confidence level
-------
Untreated tile drain waste water gave growth. re~
sponses above the undiluted San Joaquin River
water at all percentage additions. Growth
response was above the treated tile drainage
for all additions except the 20 percent addition,
where response equaled that of the 2.94-7.44 mg/1
nitrogen category.
Table 7 analyzes the data from the Table 6 column
"Treated Agricultural Waste Water." For example,
the 1%, 10%, and 20% data in the 0.10-0.17 mg/1
category in Table 6, (1.81, 2.70, and 5.94) are
located on the first horizontal data line in
Table 7. The number 1.00 is again included in
the analyses, because of the normalization technique.
When compared to Table 6, a finer distinction in
the responses is possible in Table 7 because of the
smaller variances associated with the treated
waste water as compared to the untreated tile
drainage.
Response differences at the 1% and 10% additions
in Table 7 are similar to those found in Table 6.
For the 20% additions of the treated waste water in
all nitrogen categories, however, bioassay
chlorophyll increases are greater than either the
1% additions or the San Joaquin River controls.
This response is unexpected since a 20% addition
of treated waste water (containing 0.46-1.00 mg
N/l) to San Joaquin River water would increase
sample inorganic nitrogen concentration to an
average of 0.36 mg N/l. Addition of the 0.10-0.17
mg N/l water would actually dilute the inorganic
nitrogen in the San Joaquin River water.
d) Normalized Growth Rates, MK:
i *"'
The normalized maximum growth rate data for two
experiments are listed in Table 8. The growth rate
values are based on two sample readings of the
sample fluorescences per day, rather than the usual
one-a-day recordings. Thus, they gave a better
estimate of the maximum growth rates in this experi-
mental series.
Growth responses for the untreated agricultural
tile drain waste water were the only ones exceeding
the San Joaquin River control (Table 8). Results
of all samples containing untreated agricultural
waste water (1%, 5%, 10%, and 20%) fell at the upper
end of the multiple range values, although they
did not significantly exceed all percentage addition
-22-
-------
TABLE 7. - NORMALIZED ALGAL BIOASSAY RESPONSE^
OF THE TREATED AGRICULTURAL WASTE
to
LO
1
NORMALIZED
RESPONSE (8>
NO3-N & NO2-N
in Sample
mg N/l
rH
(fl
ij 0.10-0.17
-p
rH
3
0 M
M-P 0.46-1.00
«
T3
-------
i
N>
TABLE 8. - NORMALIZED SUMMARY OF GROWTH RATE RESPONSES, g(fb) J
AP-10
SJR-0 BF-10
AP-20 BF-1 AP-1 BF-5 BF-20 AP-5 TD-1 TD-5 TD-20 TD-10
1.00 1.03 1.04 1.08 1.11
[
[
[
1.12 1.28 1.29 1.33 1.36
1
1
1
Code: SJR - San Joaquin River
AP - Algal Pond
BF - Bacterial Filter
TD - Agricultural Tile Drain Waste Water
j. The data are from two experiments in which the test waters were added to San Joaquin
River water to make 0, 1, 5, 10, and 20% of total volume. Values connected by under-
lining do not differ at the 95$ confidence level.
-------
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 'y/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 NC>3-N concentrations were the lowest of all those listed
(0.04 mg NC>3-N/1) . 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/l
respectively. Most of the bioassay maximum values for the
San Joaquin River water used in controls ranged from 30 to
45 jug chl. a/1. Maximum algal chlorophyll values appear
to b'e 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 NUTRIENT REMOVAL k
EFFICIENCY OF ALGAL POND AND BACTERIAL FILTER SYSTEMS
Algal Pond
Bacterial Filter
Number of
Observations
78
72
Normalized
Response i
Mean
6.30
6.83
F Value
0.12 l
Combined normalized data from inorganic nitrogen content
groupings in which both removal systems are represented.
The F value would have to be in excess of 3.91 before the
normalized response could be considered statistically dif-
ferent (F>0.05).
-26-
-------
TABLE 10. - SEASONAL CHLOROPHYLL AND NITRATE VARIATIONS IN THE
SAN JOAQUIN RIVER AT ANTtOCH BRIDGE
Chi, a/1 NOs-N. rog N/l
Date Initial Maximum Initial
Sample (Field) (Bioassay) (Field)
1968
11/14
12/13
1969
1/4
1/28
2/14
3/10
4/4
6/18
7/25
8/18
9/9
9/29
10/20
11/17
3.8
E.O
1.3
1.5
1.1
1.8
4.2
28.4
40.9
34.8
31.0
32.0
27.2
20.7
34.9
43.5
38.8
104.1
69.2
45.6
15.2
38.9
40.9
35.3
33.4
35.7
35.5
32.1
-
0.43
0.61
0.74
0.95
0.53
0.22
0.18
0.04
0.05
0.08
-
0.09
0.22
-27-
-------
CHAPTER V - DISCUSSION
Bioassays were used to evaluate algal growth-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 denitrification 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/l) showed growth
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 growth 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 N03~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/l would stimulate algal growth 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/l, gave responses
above the control. The 20% dilutions of treated waste water
in the category containing 0.10-0.17 mg N/l 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 denitrification 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 growth rates although the numbers obtained
are similar. The maximum specific growth rate data (u^) 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/l saturation coefficient (Ks) 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 CBain, 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 untreated 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 summer 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 /*g 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 same samples as shown in Table A2.a
are 80.4, 51.5 and 40.9 pg 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 jug chl. a/, respectively. The highest responses,
47.0 and 44.0 jug chl. a/, are statistically equal. Table
A2.a lists the maximum chlorophyll concentrations for the
same samples. The maximum values for the 10% and 20%
additions, 86.4 and 80.4 vg chl. a/, are statistically equal.
-33-
-------
TABLE Al. - BICftSSAY CHLOROPHYLL INCREASES (M)
(a.) PERCENTAGE COMPARISONS
t
u>
\. ug Chi. a/1
\% Additions
Experiment \
Date, 1969 \
1%
6/18 10%
100%
1%
7/24 10%
20%
1%
8/18 10%
20%
1%
9/8 10%
20%
1%
9/29 10%
20%
1%
10/20 10%
20%
1%
11/17 10%
20%
AGRICULTURAL
WASTE WATER
33.4
125.7
0.5
13.6
47.0
44.0
, 6.2
39.6
39.3
, 5.3
45.6
, 50.1
11.1
52.3
46.1
27.5
64.3
63.4
, 23.1
, 36.8
40.9
TREATED AGRICULTURAL WASTE WATER
Bacterial
t 19.9
, 27.3
16.5
, 1.5
, 3.0
18.1
No. 19
~^r
15.8
26.3
5.2
16.9
45.6 ,
No. 15
,~o~
, 7.6
, 15.6
No. 10
, 10.7
, 14.1
20.4
No. 6
2O"
36.5 ,
t 19.8
Filter W
No. 11
0.5
, 0.5
3.8
Covered Pond
3.8
, 2.4
, 2.4
No. 18
0.3
5.8 ,
15.8 j
No. 15
9.1,
10.1 ,
12.6
Algal Pond
14.6 ,
32.6 ,
61.4
No. 4
TT5,
1.5 ,
0.0
No. 15 No.14
~O~ ~CT[
5.2 , 8.5
i i
12.2 19.7
No. 6 No. 19
1.6 0.9 ,
3.8 , 31.9
1.4 , 36.5
No. 5
1.9^
39.7
34.6
No. 6
17.6
35.7
58.2
No.14
TO",
16.6
26.7 ,
Values connected by underlining do not differ at the 95 percent confidence level.
The Firebaugh unit numbers are given when available.
-------
TABLE Al. - BIOASSAY CHLOROPHYLL INCREASES (continued)
(b.) WATER TYPE COMPARISONS
EXPERIMENT
DATE, 1969
6/18
7/24
8/18
Q/Q
y/o
WATER TYPE
Agricultural Waste Water
Algal Pond
Bacterial Filter
Agricultural Waste Water
Algal Pond No. 4
Bacterial Filter
Agricultural Waste Water
Algal Pond No. 14
Algal Pond No. 15
Bacterial Filter No. 11
Bacterial Filter No. 19
Agricultural Waste Water
Algal Pond No. 6
Algal Pond No. 19
Bacterial Fi 1 t:p.r
Covered Pond
Bacterial Filter
0%
10.6
, 10.6
0%
, 10.6
0%
oTo
, 0.0
, 0.0
0.5
0.5
0.5
0.5
0.5
, 2.4
, 2.4
, 2.4
, 2.4
, 2.4
PERCENTAGE
1%
(jug Chi.
33.4
14.6 ,
100%
16.5 ,
1%
T376
1.5
1.5
6.2 j
4.4 ,
,
0.9
0.5
3.8 ,
5.3^
1.6
0.9 ,
3.8
5.2 ,
ADDITIONS
10%
a/1)
125.7
32.6
1%
19.9
10%
, 47.0
1.5
3.0 ,
4 39.6
8.5
i
5.2 ,
0.5
15.8
, 45.6
3.8
, 31.1
2.4
16.9
100%
0.5
61.4
10%
27.3
20%
44.0 ,
0.0 ,
18.1
39.3 ,
19.7
12.2
3.8 ,
26.3
50.1 ,
1.4 ,
36.5 ,
2.4 ,
45.6
I
U)
Ul
-------
TABLE Al. - BIOASSAY CHLOROPHYLL INCREASES - (continued)
(b.) WATER TYPE COMPARISONS - (continued)
EXPERIMENT
DATE, 1969
9/29
10/20
11/17
WATER TYPE
Agricultural Waste Water
Algal Pond No. 5
Bacterial Filter No. 15
Bacterial Filter No. 18
Agricultural Waste Water
Algal Pond No. 6
Bacterial Filter No. 10
Bacterial Filter No. 15
Agricultural Waste Water
Algal Pond No. 14
Bacterial Filter No. 6
PERCENTAGE ADDITIONS
1%
11.1
i 1.9
0.8
0.3
0%
8.3
8.3
i 8.3
. 8.3
10.5
10.5
0%
10.5
0%
3.7
3.7 ,
3.7
,3.7
1%
27.5
17.6
10.7 »
*
9.1
23.1
t 18.3
10%
36.5
10%
Chi. a/1)
52.3
39.7
7.6
5.8 t
10%
, 64.3
35.7
14.1
i
10.1
L36.8
16.6 ,
20%
, 19.8
20%
46.1
34.6
15.6
15.8
20%
63.4^
58.2
20.4
12.6 t
40.9 ,
26.7
1%
20.8 i
to
Oi
I
-------
TABLE A2. - BIOASSAY CHLOROPHYLL MAXIMA
(a.) PERCENTAGE COMPARISONS
Njg Chi. a/1
i Additions
experiment
Date, 1969
6/18
7/24
8/18
9/8
9/29
\
\
1%
10%
100%
1%
10%
20%
1%
10%
20%
1%
10%
20%
1%
10%
20%
AGRICULTURAL
WASTE WATER
60.1
155.6
, 24.2
51.5
86.4
80.4
Agricultural
Waste Water
,39.4
72.8
74.3
, 32.6
74.1
,78.9
43.5
83.5
75.3
TREATED AGRICULTURAL WASTE
Bacterial Filter (Q)
, 42.5
48.5
28.6 ,
, 42.5
, 42.5
51.5
Algal
Pond
No. 14
39T4~
L46.2
, 60.6
Bacterial
35.7
45.5
76.6 ,
No. 15
35.0
,38.2
, 45.6
Bacterial
Filter
No. 19
^779-
48.5 ,
59.1 ,
Filter
Covered Pond
31.1
i 31.9
,34.1
No. 18
t32TT-
36.3 ,
43.8 ,
Algal
Pond
No. 15
"3673"
i 40.2
47.8
No. 6
31.1
33.4 i
31.9 ,
No. 5
32TT ,
76.2
66.5
WATER
Algal Pond ®b
41.7 j
63.6
152.3
No. 4
1275",
40.9 .
40.9
Bacterial
Filter
No. 11
35TT".
32.6 i
35.7
Algal Pond
No. 19
31.9 i
60.5
65.0
w
^J
I
(p\
Values connected by underlining do not differ at the 95 percent confidence level.
Firebaugh unit nunbers are given when available.
-------
TABLE A2. - BIOASSAY CHLOROPHYLL MAXIMA (continued)
(a.) PERCENTAGE COMPARISONS (continued)
\ lug Chi. a/1
\% Additions
Experiment X
Date, 1969 \
10/20
11/17
1%
10%
20%
1%
10%
20%
AGRICULTURAL TREATED AGRICULTURAL WASTE WATER
WASTE V3ATER
Bacterial Filter (N) Algal. Pond
-------
TABLE A2. - BIQASSAY CHLOROPHYLL MAXIMA (continued)
(b.) WRITER TYPE CCMPARISCNS
EXPERIMENT
DATE, 1969
6/18
7/24
8/18
9/8
WATER TYPE
Agricultural Waste Water
Bacterial Filter
Algal Pond
Agricultural Waste Water
Bacterial Filter
Algal Pond No. 4
Agricultural Waste Water
Bacterial Filter No. 11
Bacterial Filter No. 19
Algal Pond No. 15
Algal Pond No. 14
Agricultural Waste Water
Bacterial Filter Covered Pond
Bacterial Filter
Algal Pond No. 6
Algal Pond No. 19
PERCENTAGE ADDITIONS
0%
38.9
, 38.9
, 38.9
0%
40.9
, 40.9
, 40.9
34.9
34.9
34.9
36.4
36.4
33.9
34.1
34.9
34.1
33.9
1%
(pg Chi. a
60.1
42.5 ,
,
41.7 ,
1%
51.5
42.5
42.5
39.4 ,
35.7
37.9 ,
36.4
39.4 ,
32.6 ,
31.1
35.7 ,
31.1
31.9 ,
10%
A)
155.6
48.5
63.6
10%
, 86.4
40.9
42.5 ,
, 72.8
32.6
48.5
40. 2 j
46.2
, 74.1
31.9
45.5
33.4
, 60.5
100%
24.2
28.6
152.3
20%
80.4 ,
40.9 ,
51.5
74.3 ,
35.7 ,
59.1
47.8
60.6
78.9 ,
34.1 ,
76.6
31.9 ,
65.0 ,
vo
-------
TABLE A2. - BIOASSAY CHLOROPHYLL MAXIMA (continued)
(b.) WATER TYPE COMPARISONS - (continued)
EXPERIMENT
DATE, 1969
9/29
10/20
11/17
WATER TYPE
Agricultural Waste Water
Bacterial Filter No. 15
Bacterial Filter No. 18
Algal Pond No. 5
Agricultural Waste Water
Bacterial Filter No. 10
Bacterial Filter No. 15
Algal Pond No. 6
Agricultural Waste Water
Algal Pond No. 14
Bacterial Filter No. 6
1%
43.5
, 35.0
32.1
32.1
0%
35.5
, 35.7
, 35.5
35.0
30.6
30.6
0%
30.6
PERCENTAGE
0%
(jug Chi
35.7
35.7
, 35.7
35.7
1%
54.6
36.5 ,
,
35.5
43.5
44.5
, 39.7
1%
( 42.2
ADDITIONS
10%
. a/1)
83.5
38.2 ,
36.3 ,
71.2
10%
i 90.7
39.9
36.5,
i
60.9
L58.2
38.1 ,
20%
41.8
20%
75.3
45.6
43.8
66.5
20%
88.8 ,
45.0
39.7
i
82.8
62.4 t
48.1
10%
58.6
I
*»
o
-------
100
80
v
I
a
O
ir
O
0% 100% San Joaquin River Water
0.04 mg- N03 - N/L
20
-O 20% Agricultural Watte Water
2.03 mg.N03- N/L
-a 20% Algal Pond Water
0 06 mg.N03- N/L
-A 20% Bacterial Filter Water
< 0-04 mg
-------
loo -
•r
Q
o
Q
. >
*
X
D-
x
•Q
0%
1%
10%
100% Son Joaquin River Water
Agricultural Waste Water
Agricultural Waste Water
-O 20% Agricultural Waste Water
-July ,24,1969-
234
ELAPSED TIME.DAYS
Figure A2 Algal Growth Response of San Joaquin River
Water with Added Agricultural Waste Water.
AGRICULTURAL WASTE WATER STUDIES
SAN JOAQUIN VALLEY, CALIFORNIA
ENVIRONMENTAL PROTECTION AGENCY
REGION IX
SAN FRANClSCO.CALIFORNlA
-42-
-------
APPENDIX B:
ALGAL SPECIES AND NUMBERS
Algae were identified and counted at the beginning and
end of several experiments. Personnel from the California
Department of Water Resources identified and counted algae
in Firebaugh water collected on September 9, 1969. Propor-
tional counts of the algal species were made by algal groups
(Table Bl). The proportional counts listed in Table Bl show
that diatoms comprised more than 74% of the species at the
beginning and end of the experiment in San Joaquin River
waters, in the bacterial filter, and in the pond water addi-
tions, with or without supplemental inorganic nitrogen.
The addition of phosphorus to any sample shifted the
population percentages from diatoms to green algae.
Inorganic nitrogen and phosphorus were also added to some
water samples.
-43-
-------
TABLE 81.- THE PROPORTIONAL COUNTS OF ALGAE FOUND INITIALLY AND AT THE TERMINATION OF THE BIOASSAY EXPERIMENTS
San Joaquin River
Algal Pond
Bacterial Filter
Agricultural
Waste Water
\ Percent of
X Total
N. No.
Alcal Grou^V.
Greens
Blue-greens
Diatoms
Flagellates
Initial
6
-
86
8
Terminal
6
15
75
6
6.7 wo
N/l
10
1
86
3
6.7 mg N/l r 256
6.7 mg P/l AP
56 2
3
23 89
21 6
556 AP
6.7 mg N/l
15
1
74
10
556 AP
6.7 mg N/l
3.3 me P/l
69
21
6
4
556 AP
6.7 mg N/l
13.2 me P/l
68
3
21
8
256
BF
1
13
84
Z
556 BF
6.7 mg N/l
3.3 TIE P/l
51
39
7
3
556 BF 1056
6.7 mg N/l
6.7 me P/l
53 12
12
28 83
7 5
556
13.2 mg P/l
55
1
35
9
r. These are additions in waste water concentrations.
-------
APPENDIX C:
CELL COUNT AND CHLOROPHYLL CONCENTRATION
Algal cells were counted and chlorophyll concentrations
were measured at the beginning and end of four experiments
conducted on June 18, 1969; July 24, 1969; August 18, 1969;
and November 17, 1969. Replicate flasks were combined and
a single count made of the algal cell concentrations numbers
in the combined samples. The correlation coefficient between
the algal numbers and the sample chlorophyll concentration
is 0.69, and is highly significant statistically (P<0.01).
The cell count versus chlorophyll data are shown in Figure
Cl.
-45-
-------
CTl
I
a>
2
a>
a.
io2
2
U
s
X
0.
o
a.
o
_i
r
L)
10
logy; 0.4094 logx + 0.2388
10* Z J 4 S 6 r 8 9|03 2 S 4 5 6 7 8 9,04 2 3
CELLS PER MILLIMETER
Figure Cl. Call Count vs Chlorophyll a Concentrations, Initial and Terminal Measurements.
-------
APPENDIX D
BIOASSAY NITROGEN RESPONSES
Nitrate determinations were made of each concentration
at the beginning and end of all experiments except the first
two (June 18 and July 24). It was expected that algal
growth would decrease the N03~N concentrations at the end of
the experiment period. This supposition proved true; most
samples had a terminal nitrate concentration of 0.10 mg N/l
or less (Table Dl).
Higher terminal NO3~N concentrations were found when the
initial concentration of nitrate was high. For example,
in the 20% agricultural waste water flasks of August 18, the
NO3-N was 1.88 mg N/l at the beginning and 1.00 mg N/l at
the end. Nitrate-nitrogen decreased 0.88 mg/1 for this
particular sample. In the 10% agricultural waste water
sample of September 29th the maximum decrease in NO3-N was
1.32 mg/1.
Slight increases in nitrate also occurred during some
experiments. For example, NO3-N for all concentrations
increased in the anaerobic covered pond of September 8th
although there was no detectable N03~N in the original
covered pond sample (Table 3). There was however 1.13 mg/1
of organic nitrogen found in the sample. Since no discern-
able growth of algae occurred using this water, N03-N
increases may have resulted from bacterial oxidation of the
organic nitrogen. In some of the experiments an increase in
nitrate may have resulted from oxidation of nitrite to
nitrate. Many of the samples listed in Table 2 had a high
concentration of nitrite-nitrogen.
The increase in chlorophyll and the decrease in NO3-N
are plotted in Figure Dl. The correlation coefficient
(r = 0.82) between the two variables is highly significant,
the regression line having the equation:
•^g N/l = 11.6x (ng chl. a/1) - 15.0
This formula gives a ratio of nitrate-nitrogen decrease to
chlorophyll increase of approximately 11:1. The ratio closely
agrees with the organic nitrogen-chlorophyll ratio of Yentsch
and Vaccaro (1958) for nitrogen enriched cultures, (7:1 to
10:1) and the ratio found by Manny (1969) which was 6:1 to
12:1. These ratios referred to fixed organic nitrogen while
ratios used in this project were based on the assumption
that the nitrate-nitrogen was metabolized into an organic
form.
-47-
-------
TABLE Dl. NITRATE CONCENTRATIONS IN TEST WATERS BEFORE AND AFTER BIOASSAY
N05-N
Experiment Dates- 8/18/69 to 8/25/69 Beginning
End
Decrease
San Joaquin River 100%
Agricultural
Waste Water
Algal
Pond No. 14
Hieh-rate
Algal
Pond No. 15
Low-rate
Bacterial
Filter No. 11
High-rate
Bacterial
Filter No. 19
low Rate
Experiment Dates
San Joaquin Ri
Agricultural
Waste Water
Algal
Pond No. 6
Alsal
Pond No. 19
Anaerobic
Covered
Pond
Bacterial
Filter
1%
10%
20%
1%
10%
20%
1%
10%
20%
1%
10%
20%
1%
10%
20%
- 9/8/69 to 9/16/69
ver 100%
\%
10%
20%
1%
1056
20%
1%
10%
2056
1%
10%
20%
1%
10%
20%
0.05
0.14
0.96
1.88
0.06
0.18
0.32
0.08
0.36
0.66
0.05
0.09
0.14
0.10
0.56
1.06
0.08
0.16
0.80
2.30
0.08
0.07
0.08
0.08
0.30
0.64
0.06
0.06
0.05
0.06
0.06
0.05
0,10
0.08
0.30
1.00
0.08
0.08
0.08
0.10
0.08
0.08
0.10
0.08
0.08
0.10
0.09
0.11
0.05
0.07
0.52
1.60
0.11
0.12
0.07
0.10
0.09
0.16
0.08
0.07
0.08
0.07
0.05
0.10
0
0.06
0.66
0.88
0
0.10
0.24
0
0.28
0.58
0
0.10
0.04
0
0.47
0.95
0.03
0.09
0.28
0.70
0
0
0.01
0
0.21
0.48
0
0
0
o 1
0.01
0 1
-48-
-------
TABLE Dl. NITRATE CONCENTRATIONS IN TEST WATERS BEFORE AND AFTER BIOASSAY - (continued)
mg/1 NOS-N
Experiment Dates - 9/29/69 to 10/6/69 Beginning
End
Decrease
San Joaquin River
Agricultural
Waste Water
Algal
Pond No. 5
Bacterial
Filter
No. 15
Bacterial
Filter
N°r 18
100$
1%
10$
20$
1%
10$
20$
1%
10$
20$
1$
10$
20$
0.03
0.18
2.1
3.8
0.05
0.47
0.84
0.03
0.08
0.20
0.03
0.08
0.11
0.03
0.03
0.78
2.7
0.03
0.03
0.14
0.04
0.04
0.03
0.03
0.03
0.03
0
0.15
1.32
1.1
0.02
0.45
0.70
0*
0.04
0.17
0
0.05
0.08
Experiment Dates - 10/20/69 to 10/27/69
San Joaquin River
Agricultural
Waste Water
Algal
Pond No. 6
Bacterial
Filter
No. 10
Bacterial
Filter
No. 15
100$
1$
10$
20$
1$
10$
20$
1$
10$
20$
1$
10$
20$
0.12
0.30
1.6
5.6
0.14
0.30
0.51
0.10
0.12
0.12
0.11
0.14
0.18
0.19
0.47
0.75
3.0
0.03
0.05
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0
0
0.85
0.6
0.11
0.27
0.48
0.07
0.09
0.09
0.08
0.11
0.15
Experiment Dates - 11/17/69 to 11/24/69
San Joaquin River
Agricultural
Waste Water
Algal
Pond No. 14
Bacterial
Filter No. 6
100$
1$
10$
£0$
1$
10$
20$
1$
10$
E0$
0.22
0.45
3.2
4.1
0.22
0.29
0.37
0.38
1.5
3.2
0.03
0.04
2.4
3.7
0.04
0.03
0.03
0.04
1.0
0.19
0.41
0.80
0.4
0.18
0.26
0.34
0.34
0.50
-49-
-------
1.5
UJ
en
UJ
oc
= i.o
E
o
UJ
o
O
ir
I
la
cc
H
Z
0.5
Y-O.OII6 X-O.OJ5
20 40 60 80 too 120
CHLOROPHYLL o ,(Mlcrogroms per Liter) INCREASE
140
Figure D I. Bioassoy Decrease in Nitrate-Nitrogen as .a Function of Chlorophyll
Increases..
-50-
-------
Figure D2 shows the log of the chlorophyll concentration
maximums as a function of the log of the original NO^-N con-
tained in the cultures. The correlation is highly significant
(r = 0.77) , the equation of the line being:
log (jLig chl. a/) = 0.2003 (log /*..g N/l) + 1.195
The ratio of total initial inorganic nitrogen to maximum
chlorophyll in the culture is approximately 16:1.
-51-
-------
iO
Ui
K>
I
o»
01
CX
E
o
o
L
o
10'
X
a.
o
£T
O
—i
X
o
S
z>
S
X
<
2
10'
logy-0.2003 log C X x !03) + M95
o o
ID'2 10-' 10°
INITIAL TOTAL INORGANIC NITROGEN, (Milligrams per Liter)
Figure 0 2. Bio assay Chlorophyll Maximum as a Function of Total Inorganic Nitrogen ( NO 3- N plus N02-N ),
10'
-------
ACKNOWLEDGEMENTS
The following list includes the personnel actively involved
in the algal bioassay project.
Firebaugh Project under the Direction of
Percy St. Amant Engineer/ Environmental
Protection Agency
Louis A. Beck Engineer, California
Dept. of Water Resources
Donald Swain Engineer/ U.S. Bureau of
Reclamation
Consultants to the Project
Dr. Perry L. McCarty .Stanford University,
Palo Alto
Dr. William J. Oswald University of California,
Berkeley
Dr. Clarence G. Golueke University of California,
Berkeley
Algal Bioassays of Agricultural Waste Water
Conducted by
Dr. Milton G. Tunzi Limnologist, Environmental
Protection Agency
Assisted by
Albert Katko Aquatic Biologist, EPA
Richard C. Bain/ Jr Sanitary Engineer, EPA
James E. Thoits Biological Technician, EPA
Ella M. McGehee Physical Science Aide, EPA
Dorothy M. White Physical Science Aide, EPA
Margaret Y. Chu Chemist, EPA
Mustafa M. Salma . .Chemist, EPA
David R. Wood Supervisory Chemist, EPA
Kathleen Shimmin Supervisory Microbiologist,
EPA
Gary Varney Biologist, California
Dept. of Water Resources
Report Prepared by
Dr. Milton G. Tunzi Limnologist, Environmental
Protection Agency
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LIST OF REFERENCES
Bain, R.C., Jr. 1969. Algal Growth Assessments by
Fluorescence Techniques: Proceedings of the
Eutrophication—Biostimulation Assessment Workshop,
Federal Water Quality Administration, Corvallis,
Oregon.
Bain, R.C., Jr., H.E. Pintler, A. Katko, and R.F. Minnehan.
1968. Nutrients and Biological Response: San Joaquin
Master Drain, Appendix, Part C, Federal Water Quality
Administration, Southwest Region, San Francisco, 117p.
FWPCA Methods for Chemical Analysis of Water and Wastes.
November 1969. Federal Water Quality Administration,
Analytical Quality Control Laboratory, Cincinnati,
Ohio, 280p.
Krone, R.B. 1966. Predicted Suspended Inflows to the
San Francisco Bay System, Prepared for the Federal Water
Pollution Control Administration, Southwest Region,
San Francisco.
Manny, B.A. 1969. The Relationship between Organic Nitrogen
and the Carotenoid to Chlorophyll A Ratio in Five
Freshwater Phytoplankton Species: Limnology and
Oceanography, V. 14, p. 69-79.
McGaughey, P.H., G.A. Rohlick, E.A Pearson, M. Tunzi,
A. Adinarayana, and E.J. Middlebrooks. 1968.
Eutrophication of Surface Waters-Lake Tahoe: Bioassay
of Nutrient Sources. LTAC, FWPCA Progress Report for
Grant No. WPD 48-01 (R 1), May.
Provisional Algal Assay Procedures. 1969. Joint Industry-
Government Task Force on Eutrophication, P.O. Box 3011,
Grand Central Station, New York, 43p.
Sword, B. R. 1970. Denitrification of Agricultural Tile
Drainage in Anaerobic Filters and Ponds, in this series.
Yentsch, C.S. and R.F. Vaccaro. 1958. Phytoplankton Nitrogen
in the Oceans: Limnology and Oceanography, V. 3,
p. 443-448.
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PUBLICATIONS
SAN JOAQUIN PROJECT, FIREBAUGH, CALIFORNIA
1968
"Is Treatment of Agricultural Waste Water Possible?"
Louis A. Beck and Percy P. St. Amant, Jr. Presented
at Fourth International Water Quality Symposium, San
Francisco, California, August 14, 1968; published
in the proceedings of the meeting.
1969
"Biological Denitrification of Wastewaters by Addition of
Organic Materials"
Perry L. McCarty, Beck, and St. Amant, Jr. Presented
at the 24th Annual Purdue Industrial Waste Conference,
Purdue University, Lafayette, Indiana. May 6, 1969,
"Comparison of Nitrate Removal Methods"
Beck, St. Amant, and Thomas A. Tamblyn. Presented at
Water Pollution Control Federation Meeting, Dallas,
Texas. October 9, 1969,
"Effect of Surface/Volume Relationship, COg Addition, Aeration,
and Mixing on Nitrate Utilization by Scenedesmus Cultures
in Subsurface Agricultural Waste Waters"
Randall L. Brown and James F. Arthur. Proceedings
of the Eutrophication-Biostimulation Assessment
Workshop, Berkeley, California. June 19-21, 1969.
"Nitrate Removal Studies at the iriteragency Agricultural
Waste Water Treatment Center, Firebaugh, California"
St. Amant and Beck. Presented at 1969 Conference,
California Water Pollution Control Association, Anaheim,
California, and published in the proceedings of the
meeting. May 9, 1969.
"The Anaerobic Filter for the Denitrification of Agricultural
Subsurface Drainage"
Tamblyn and B. R. Sword. Presented at the 24th
Annual Purdue Industrial Waste Conference, Lafayette,
Indiana. May 5-8, 1969.
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PUBLICATIONS (Continued)
1969 (Continued)
"Research on Methods of Removing Excess Plant Nutrients
from Water"
St. Amant, Beck. Presented at 158th National Meeting
and Chemical Exposition, American Chemical Society,
New York, New York. September 8, 1969.
"Nutrients in Agricultural Tile Drainage"
W. H. Pierce, Beck, and L. R. Glandon. Presented
at the 1969 Winter Meeting of the American Society of
Agricultural Engineers, Chicago, Illinois. December
9-12, 1969.
"Treatment of High Nitrate Waters"
St. Amant, McCarty. Presented at Annual Conference,
American Water Works Association, San Diego, California,.
May 21, 1969. American Water Works Association Journal,
Vol. 61, No. 12. December 1969, pp. 659-662.
The following papers were presented at the National Fall
Meeting of the American Geophysical Union, Hydrology Section,
San Francisco, California. December 15-18, 1969. They are
published in Collected Papers Regarding Nitrates in Agri-
cultural Waste Water. USDI, FWQA, #13030 ELY December 1969.
"The Effects of Nitrogen Removal on the Algal Growth
Potential of San Joaquin Valley Agricultural Tile Drainage
Effluents"
Brown, Richard C. Bain, Jr., and Milton G. Tunzi.
"Harvesting of Algae Grown in Agricultural Wastewaters"
Bruce A. Butterfield and James R. Jones.
"Monitoring Nutrients and Pesticides in Subsurface Agricultural
Drainage"
Glandon and Beck.
"Combined Nutrient Removal and Transport System for Tile
Drainage from the San Joaquin Valley"
Joel C. Goldman, Arthur, William J. Oswald, and Beck.
"Desalination of Irrigation Return Waters"
Bryan R. Sword.
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PUBLICATIONS (Continued)
"Bacterial Denitrification of Agricultural Tile Drainage"
Tamblyn, McCarty, and St. Amant.
"Algal Nutrient Responses in Agricultural Wastewater"
Arthur, Brown, Butterfield, and Goldman.
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1
5
Afcfxsioti fifumbcr
~ Subject Fii'ld & Croup
05D
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
Environmental Protection Agency, Region IX
Water Quality Office
100 California Street, San Francisco CA 94111
Title
The Effects of Agricultural Waste Water Treatment on Algal
Bioassay Response
J Q Authors)
TUNZI, Milton G.
16
21
Project Designation
13030 ELY
Available
from:
Environmental Protection Agency
Region IX, 100 California Street
San Francisco CA 94111
22
Citation
Agricultural Waste Water Studies
Report No. 13030 ELY 8/17-9
Pages: 59, Figures; 8, Tables; 14, References
Descriptors (Starred First)
23
* Eutrophication
* Bioassay
* Denitrification , Nitrates, Nitrogen
* Fluarometry
* Tile Drains
25
Identifiers (Starred First)
* Algal Blooms - control, bioassay - algal, chlorophyll,
denitrifica-tion, fluorometry, tile drainage
27
Abs'ract
Laboratory bioassay experiments were performed to test the effect
on algal growth of agricultural waste water befote 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 water* 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
alga] growth to that of the Delta water controls -as long as the
nitrates-nitrogen level in each removal system had been lowered to
approximately 2 mg N/l, or less.
Abstractor
Institution
V»f«:IOI (REV. JULY IS69I
WRSIC
I: WATER, RESOURCES SC'ENTIFIC IN FORM* TICN
.. U.S. DEPARTMCNT OF THE INTERIOR
WASHING TOW.' D, C. 90240
ft U, S, GOVERNMENT PRINTING OFFICE : 1972-514-l48/««
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