United States
                   Environmental Protection
                   Agency
 Robert S. Kerr Environmental
 Research Laboratory
 Ada OK 74820
                    Research and Development
 EPA-600/S2-83-007 Mar. 1983
&EPA         Project  Summary

                   Advanced  Biological
                   Treatment  of  Municipal
                   Wastewater Through
                   Aquaculture
                   Dempsey H. Hall and Joel E. Shelton
                     This research project was initiated
                    with the overall objectives of: (a) assess-
                    ing the potential of aquaculture as a
                    suitable means of  treating municipal
                    sewage in a mid-temperate latitude on
                    an annual basis, (b) providing a set of
                    design criteria for  implementation of
                    aquaculture as an advanced wastewater
                    treatment method, and (c) achieving an
                    effluent quality amenable to PL 92-500
                    and the 1977, 1983. and 1985 stan-
                    dards and goals.
                     Two four-celled raceways construc-
                    ted in a series adjacent to a primary
                    wastewater stabilization pond were
                    used in the studies. One raceway series
                    functioned as the experimental system,
                    while the other served as a control. The
                    first experimental phase used a source
                    of wastewater from the primary waste-
                    water stabilization  pond. The  second
                    experimental phase used a source of
                    wastewater from the primary clarif ier of
                    an activated sludge treatment plant.
                    The primary clarifier also provided the
                    source of wastewater to the primary
                    wastewater  stabilization pond.  Under
                    both experimental conditions the experi-
                    mental raceway was stocked  with a
                    native Oklahoma  fish, Pimephales
                    promelas  Raf., at two stocking densi-
                    ties, one for each experimental phase.
                     An analysis of the wastewater quality
                    data assembled during the two experi-
                    mental phases revealed  moderate re-
                    ductions in suspended solids during the
                    first experimental phase which could
                    have been attributed to the presence of
                    the fish. No distinguishable reductions
                    in five-day biochemical oxygen demand
(BOD5) were statistically supported that
could be attributed to the fish stock.
Analyses of nutrient parameters also
indicated no distinguishable reductions
due to fish populations. During the
second experimental phase, high  mor-
tality  due primarily to oxygen stress
revealed no observable impact on the
quality of wastewater that could be
attributed to the fish. Retention time of
the wastewater within each cell of the
raceway appeared to play a strong role
in the observed percentage reductions
in most of the regulatory and nutrient
parameters.
  A marked reduction in fecal coliform
organisms was attributed to the reten-
tion time of the wastewater rather than
due to influences of the fish present in
the cells.
  Biological studies of fish growth and
reproductive capabilities revealed mod-
erate successes with respect to repro-
duction, while the analysis of growth
revealed exceptional potentials for pro-
duction of biomass over a short period
of time.
  The full project report covers the
period of March 1977 through August
1979  and describes the experiments
and results in detail.
  This Project Summary was developed
by EPA's Robert S. Kerr Environmental
Research Laboratory, Ada, OK, to an-
nounce key findings of  the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).

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Introduction

  The  current  demand for advanced
wastewater treatment practices, arising
from the enactment of PL 92-500, the
Federal Water Pollution Control Act of
1972, coupled with a demand for energy
conservation and the underlying econom-
ic implications,  is rapidly creating  a
dilemma in the field of wastewater treat-
ment. In Oklahoma alone, approximately
80 percent of the communities are cur-
rently utilizing wastewater stabilization
ponds as a means of  treating municipal
sewage.  Such conventional treatment
practices have not adequately produced
effluents of a quality amenable to estab-
lished standards provided by PL 92-500.
  The  problem that currently exists in
finding an equitable solution arises not
from a lack of advanced waste treatment
technology  but  from a cost-effective
implementation  of advanced physical-
chemical treatment processes in  areas
where the serviced population is inade-
quate  to finance and maintain  such
facilities. Such a  problem establishes a
strong premise for  investigating the
potentials of utilizing existing conven-
tional treatment methods and supplement-
ing physico-chemical and biological treat-
ment processes with extended biological
activity in the form of aquaculture.
  The  primary objectives of this  study
were to: (a) assess the potential of  aqua-
culture as a suitable means of treating
municipal sewage in a mid-temperate
latitude on an annual basis, (b) provide  a
set of design criteria for implementation
of aquaculture as a wastewate treatment
method, and (c) achieve an effluent quality
amenable to PL 92-500 and the  1977,
1983,  and  1985 standards and  goals.
Subordinate objectives included a quanti-
tative  determination of the degree  of
sewage treatment during major seasons,
and  an evaluation of practical uses and
economic potentials of biological products
of the system.
  The scope of this study was somewhat
broad in context, yet limited in application
due to the limited background information
available and the physico-chemical nature
of the wastewater environment.  Such
limitations necessitated the use of certain
criteria in selecting a suitable test orga-
nism, namely: (a) a species of fish  native
to Oklahoma waters,  (b) a species known
to have a high tolerance for unstable
conditions which exist at various intervals
in wastewater stabilization ponds, primar-
ily  dissolved  oxygen (D.O.),  and (c)  a
species of fish readily available and easily
handled. For these reasons the fathead
minnow  (Pimephales promelas Raf.),
although not the  most efficient filter
feeder, was selected as the test organism.
The fathead minnow has been classified
as an opportunistic feeder, utilizing small
invertebrate organisms as well as algae,
and its long intestinal tract suggests an
ability to  function efficiently as a herbi-
vore.

Conclusions

   The analysis of the regulatory param-
 eter suspended solids, under the waste-
 water and stocking conditions employed
 in the first experimental phase, revealed
 an apparent  contribution of the fish
 present  within  the  test cells.  Such  a
 contribution was reflected in a  compar-
 ison between the concentration of sus-
 pended solids within the effluent in the
 final cell of the experimental versus
 control  series  raceways. Unlike  the
 suspended  solids parameter,  another
 regulatory parameter, BOD5, showed no
 statistically significant contrast between
 experimental and control cells,  with re-
 spect to concentrations monitored. Such
 results suggest that the direct reduction
 in suspended solids was insignificant to
 indirectly stimulate a reduction in BOD5.
 The observed  percentage reduction in
 BOD5 from influent to effluent of each
 raceway (control and experimental) was
 apparently related directly to the retention
 time of  wastewater rather than  the
 presence of the fish within the waste-
 water.
   The analyses of regulatory parameters
 (BOD5and suspended solids), and nutrient
 parameters, under wastewater and stock-
 ing conditions in the second experimental
 phase revealed no apparent contribution
 of the fish to a  reduction in concentra-
 tions.
   Considering the nature of the waste-
 water tested under the second experi-
 mental phase  (wastewater  of  primary
 treatment quality), it is  apparent that  if
 cultures of fathead minnows are to be
 implemented as a cost-effective means of
 supplemental wastewater treatment, they
 should be considered as an adjunct to the
 wastewater stabilization pond and not as
 a substitute for secondary treatment
 afforded by the wastewater stabilization
 pond processes.
   Under the wastewater conditions pro-
 vided in the first experimental phase,
 wastewater from a primary wastewater
 stabilization pond, the fathead  minnow
 exhibited the ability to live and reproduce
 successfully with only limited threat from
 disease.
  Under the wastewater conditions pro-
vided in the second experimental phase,
wastewater from primary treatment qual-
ity, the fathead minnow  was  able  to
survive. However, under extreme stresses
initiated by wastewater with a high oxy-
gen demand, such an existence was extremely
limited and prohibited reproductive rigor
and success.
  In summary, the wastewater community
provides an extremely opportunistic envi-
ronment  which, if  managed and moni-
tored closely, may provide a very cost-
effective  approach to the  utilization  of
nutrients that conventionally are carried
away by receiving stream waters and very
often end up overloading and upsetting
the balance  of nutrients provided for m
the natural stream  environment. Finfish
within the wastewater environment pro-
vide an opportunity for recycling nutrients,
capturing fundamental  elements within
protein-rich products usable in numerous
ways by man. Such an  approach can  be
feasibly implemented as a valuable water
reuse technique.

Recommendations

   A definite potential exists for  the inte-
 gration of aquaculture techniques and
 wastewater treatment. If such a  goal is to
 be implemented, further studies are
 necessary to more clearly define the intri-
 cate food web that exists within the waste
 stabilization pond. Such a  definition
 would provide a stronger basis for selec-
 ting the  appropriate species of fish to be
 cultured.
   The unstable nature of  raw and even
 primarily treated municipal wastewater
 limits the  potential of introducing a
 population of finfish due to the high level
 of competition for  oxygen; therefore, if
 finfish culture in the wastewater environ-
 ment is to be a successful integration of
 productivity and wastewater treatment,
 such operations should focus on supple-
 menting the secondary treatment mode.
   Due to the-wide range of diurnal fluctu-
 ations in oxygen and temperature within
 the wastewater stabilization pond, finfish
 culture  practices  should  include: (1) a
 source of emergency aeration to provide a
 means of quickly stabilizing septic con-
 ditions  often  experienced during  the
 year-round operation of the lagoon, partic-
 ularly during late summer and early spring
 seasons, (2) adequate manpower and
 equipment for handling fish stock to limit
 the stresses imposed on the fish  during
 handling, (3) disease control protocol to
 ensure minimal stock  loss, and (4) alter-
 native planning for wastewater flows,

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including multiple raceway designs,
which  allow  the  operator a means  of
alternating flow and thus eliminate prob-
lems with overloading.
  The successful implementation of aqua-
culture practices with wastewater treat-
ment will rely heavily on interdisciplinary
skills of biologists  and wastewater treat-
ment specialists particularly in organiza-
tion and establishment of such an
operation. Proper initial organization will
ensure a  smooth operation which should
require only minimal skills to maintain
after installation.

 Facilities,
 Operation, and Results

   The study was conducted  at the
 Bethany-Warr  Acres sewage treatment
 facility which  serves a  portion of the
 northwest Oklahoma City municipal com-
 plex. The treatment phases of the facility
 consisted of: (a) screening, (b) grinding
 (comminutor),  (c)  aeration,  (d) primary
 and secondary clarification, and (e) sludge
 digestion. Following these treatment
 phases the effluent was discharged to
 primary and secondary wastewater stabil-
 ization ponds, where the wastewater
 underwent biological oxidation processes
 before being discharged into the adjacent
 receiving stream  (Bluff  Creek, a  first-
 order stream location on the Cottonwood
 Creek drainage basin).
   In order to obtain the  necessary con-
 trols, with respect to flow and retention of
 wastewater,  a separate small-scaled
 series of wastewater stabilization ponds
 were constructed adjacent to the existing
 primary  wastewater stabilization pond.
 This facility, as  shown in  Figure 1,
 consisted of eight earthen cells, arranged
 in two series  with four cells in  each
 series. Each  cell  had a surface area of
 approximately  0.1 hectare  (0.25 acres).
 The two series of cells were constructed
 in parallel, with each individual cell within
 one series corresponding to the same
 numbered cell within the adjacent series,
 with respect to  sequence,  flow,  and
 retention time. This design allowed  for
 one series to serve as an experimental
 control for the other series. All cells
 within a single series were constructed
 as identically as possible to its correspond-
 ing cell  of the adjacent series,  and all
 ponds were  designed  with  the same
 general  specifications with respect to
 depth, area, and distribution receptacles.
 At the end of the two series of treatment
 cells, one large cell was constructed to
 receive flows from both series of  cells.
 The contents of this cell was periodically
used for irrigation  purposes  or  was
pumped back into the secondary waste-
water stabilization pond of the permanent
treatment facility.
  The  experimental  facilities were de-
signed to maintain maximum achieveable
control on flow of wastewater  through
use of distribution receptacles equipped
with 30° V-notch weirs. The arrangement
of these  receptacles was designed to
allow the investigator an alternate means
of distributing flow of wastewate through-
out the entire series of experimental cells.
Due to the nature of the wastewater
received by the experimental system, the
serial flow pattern was selected as the
most appropriate design for experimenta-
tion, as it allowed for optimum retention
time which in turn produced a quality of
wastewater suitable for fish culture.
  During  the course  of the  study, the
existing treatmentfacility was reconstruc-
ted and modified. This necessitated the
utilization of two  experimental phases
with two separate sets of conditions. The
two sets  of conditions differed primarily
with respect to wastewater characteris-
tics  and  stocking  density of fish. The
physical characteristics of flow and reten-
tion were the same for both experimental
phases.
  During  the first experimental phase,
from October 19, 1977 through  May 17,
1978,  primary lagoon effluent was pro-
vided as  the source of wastewater for
experimentation. Each of  the four  cells
within the test series were stocked with
Pimephales promelas Raf.  at a density of
approximately 38 kilograms (kg)/0.1
hectare (83 pounds (lb)/0.25 acres).
  During the second experimental phase,
from August 2,  1978 through  May 9,
1979,  the wastewater source was sup-
plied directly from the primary clarifier of
the existing treatment facility, by means
of a submersible pump transmitting waste-
water to  the centrally located collection
point and distributing the wastewater to
the two series raceways. Due to the high
suspended solids and low D.O. content of
the wastewater received from the pri mary
clarifier,  it was necessary to  retain the
wastewater, allowing time for stabiliza-
tion  and  production of phytoplankton.
This  was accomplished by eliminating
fish  stock from the first cell in the
experimental series, which provided a
more stable environment for the f inf ish in
the remaining cells of this series. The
remaining three cells were stocked at a
density of 151  kg/0.1  hectare  (334
lb/0.25 acres).
  Under both experimental phases, waste-
water was received by a centrally located
distribution receptable (Figure 1) and was
distributed to each  of  the two series
raceways at an  average flow of  0.95
liters/second (I/sec)  (15 gallons per
minute [gprn]). The flow rate varied from
0.63  I/sec (10 gpm) to  1.26 I/sec (20
gpm) depending on several uncontrollable
factors inherent to gravitational distribu-
tion. This variation in flow necessitated
daily monitoring and correction by adjust-
ment of the V-notch weirs in the distri-
bution receptacles of the first cell in each
series. The flow between each cell within
each  series varied somewhat from the
flow  to  the  first cell in each  series,
depending on the evaporation rate and
the amount of rainfall. Without compen-
sating for input from rainfall or loss from
evaporation, the retention time of waste-
water in each cell was approximately 15-
20 days.
   For  purposes of identifying sample
locations, each of the cells in the two
series was assigned numbers sequenti-
ally through the series. Along with the
numbered location, subscripts were as-
signed to designate the control series and
thetest series; A- designating the control
series with no fish and B -designating the
experimental series which contained fish
(Figure 1).
   During  this study, under both phases,
wastewater samples were collected
weekly from the discharge point at each
cell within the two series and a sample
was collected from the initial wastewater
supplied to the system. All samples were
collected on the same weekday at approxi-
mately the same time of day (1100-1200
hour). Along with the wastewater  sam-
ples, temperature and dissolved  oxygen
data were collected at each  sampling
location.   Several  water quality param-
eters were analyzed including BOD5,
suspended solids, and nutrients.
  The performance of the system under
two operational modes was evaluated by
determining the mean effluent levels from
each cell for each  of the selected param-
eters  and by comparing changes in the
character of the flow as it moved through
the experimental series of cells with the
character of the flow at the corresponding
points in the control series. In addition,
overall performance of each series was
determined  by comparing  the effluent
concentration from the last cell in each
series with the level of that parameter in
the influent to the series.
  In order to minimize the variability due
to the day-to-day changes in the character
of the influent and variability due to
seasonal  influences on temperature, sta-
tistical comparison of each cell  in the

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experimental series with the correspond-
ing cell in the control series was con-
ducted utilizing a t-test of observations
paired according to sample  date. This
permitted the use of an alpha  level of
0.05.
  Due to the observed critical periods of
low oxygen concentrations encountered
during the study, a  means  of  supple-
menting  photosynthetic oxygen  produc-
tion was provided using forced air blowers
and perforated distribution lines for  dif-
f users. The supplemental aeration supply
was designed to provide aeration to
experimental and control cells  concur-
rently to  minimize bias to the experi-
mentation.  Most  of  the supplemental
aeration was necessaryduring the winter
months when  extended periods of  ice
cover jeopardized the existence of suf-
ficient dissolved oxygen concentrations.
  In addition to the monitoring of physico-
chemical  parameters, biological activity
of fish was monitored visually and through
analysis of growth patterns. Factors moni-
tored visually included reproductive activ-
ity, movement and congregation of fish
due to oxygen stress,  mortality, disease
and periodic inventory of fish  stock.
  During  the first experimental phase,
the statistical analysis of BOD5 data
(Table 1)  indicated a  significantly lower
concentration in  the first  experimental
cell than in the first control cell, at both
the 0.1  and 0.05 alpha levels. The only
other cells that maintained any significant
differences were the second  cells of the
raceway series, which displayed signifi-
cant differences  at the 0.1  level only.
Considering the real differences in mean
effluent concentrations and the relatively
narrow range of percent reduction values,
it was assumed that  no real  differences
existed between the control cell effluent
BOD5 levels and the  experimental  cell
BOD5 levels.
  Under the second experimental phase,
the results of the BOD5 analyses indicated
no statistically significant differences in
effluent  concentrations for each of the
experimental and control cells of the first
three stages in the raceway  series.  The
last  experimental cell containing fish,
displayed  significantly higher  effluent
BOD5 than its corresponding control cell.
Also, the observed percentage reduction
was much higher for the control series
than for the experimental series contain-
ing fish.
  Statistical  analysis  of the  suspended
solids data during the first experimental
phase revealed  a  somewhat different
trend from that of BODs concentration
within the first two  experimental cells
Return Effluent —Secondary Lagoon f f

Holding
Cell
I .
Pt/mp-llj 	

™" * "* ™" 1
.;,«, j-{fa, H
i
i *
4 4B j-i i 3B j-H


N X \
\ X ^
. x x
Surface Irrigation * j— -
\ \ \
\ \ \
\ \ \
\ \ \
T 2A FT T 1A I
1 ' - 1 ^ f
1
i i
El IT1 1
* 2B J-| * !B j
• , . ,. ,. • t
30°V-Notch Weir
Drain
(. Inlet
Primary
Lagoon
Figure  1.    Diagram of Oklahoma State  Department of Health, Aquaculture research
            facilities located at the Bethany-Warr Acres Municipal Wastewater Treatment Plant.
Table  1.
Analytical Results for Regulatory Parameter.  BODs. Sampled During First and
Second Experimental Phases
                                   First Phase
                                             Effluent
Stat.
Para,
x mg/l
S
N
x mg/l
S
N
Infl. Cell
57.8
34.0 Contr
Exp.
t (paired observations)

x mg/l
S
N
x mg/l
S
N

60.9
41.7 Contr.
31
Exp.
1
24.6
12.6
250
19.9
11.6
24.0
a.b

428
23.8
33.0
47.2
28.3
33.0
2
27.0
17.2
25.0
21.5
14.1
25.0
b
Second Phase
32.5
17.8
33.0
31.7
21.3
32 O
3
27.5
15.9
24.0
24.6
14.7
24.0


30.3
20.4
33.Q
28.4
15.7
32.0
t (paired observations)
4 Red.
22.4 61
13.2
24.0
21.5 63
11 1
22.0


26.7 41C
14.6
33.0
30. 1 33C
14.0
33.0
a.b
"Indicates statistical significance at X = 0.05.
'"Indicates statistical significance at X = 0.10.
cComposite of cell 1 (x = 15.0, S - 26.1. N = 66) was used to calculate % reduction.
                                     4

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(Table 2). No significant difference exists
for effluent suspended solids concentra-
tion between test cells and corresponding
control cells. The third experimental cell
showed a significantly lower concentra-
tion of suspended solids at an alpha level
of 0.1 than its corresponding control and
the fourth experimental  cell  showed a
significantly lower concentration  at the
0.1 and the .05 alpha levels. Comparison
of the  overall 20  percent reduction in
suspended solids from the point of intro-
duction of influent into the raceway to the
effluentfrom the numberfour experimen-
tal cell, and the 11 percent increase in
suspended solids  observed  across the
control cells indicates that a real  differ-
ence exists. This difference can be attrib-
uted to the high finfish population present
in the third and fourth experimental cells
and  their  role in  reducing  suspended
solids.  Also, the absence of significant
reductions in  the  first two cells  of the
series likely resulted from the low finfish
population levels.
  Concentrations  of  suspended  solids
during the second experimental  phase
tend to reflect the  same pattern. Experi-
mental cells three and four were found to
contain significantly  higher concentra-
tions of suspended solids than the corre-
sponding control  cells. In  fact, a  94
percent increase in suspended solids was
observed from the  influent into the race-
way series to the  effluent of the  fourth
experimental  cell,  and  a four percent
increase was  observed over the control
series. Such results support the view that
a large proportion of the suspended solids
present in the primary  clarifier  waste
stream were not suitable for consumption
by the finfish and their population levels
contributed to the suspended solids load.
  Experimental results for nutrient  par-
ameters revealed a somewhat contradic-
tory pattern.  Observed  differences in
concentrations between experimental
cells and corresponding controls failed to
reveal any significance on which to base
a sound conclusion about a reduction in
nutrients due to fish living within the
cells.
  During the course of study,  visual
inspection of the general condition of the
fish within the wastewater environment
revealed  a  healthy and reproductively
viable population with only a moderate
amount of disease, apparently initiated by
handling during seining  operations. Al-
though  spawning  was observed during
the study, the apparent success of such
reproductive activity was low as reflected
by standing crop  harvested during the
second experimental phase. Low oxygen
Table 2.
Analytical Results for Regulatory Parameter, Suspended Solids. Sampled During
First and Second Experimental Phases.
                                   First Phase
                                            Effluent
Stat.
Para.
x mg/l
S
N
x mg/l
S
N
Infl.
32.8
19.2
25

Cell
Contr.
Exp.
1
24.2
21.7
24.0
22.5
17.0
26.0
2
34.3
20.8
26.0
30.3
23.8
25.0
3
34.1
20.5
25.0
26.3
11.7
25.0
4
36.5
23.4
26.0
26.4
8.5
25.0
%
Red.
-11
20
t (paired observations)
x mg/l      45.1
  S         31.3
  N         32

x mg/l
  S
  N

t (paired observations)
          Contr.
           Exp.
30.1
21.2
32.0

20.3
14.2
34.0
Second Phase

       26.5
       15.5
       34.0

       28.6
       19.2
       33.0
29.6
23.1
34.0

37.8
20.8
34.0

a.b
                                                              a,b
28.6
13.0
34.0

57.5
28.4
34.0

a.b
                                                            -94
"Indicates statistical significance atX- 0.05.
^Indicates statistical significance at X = 0.10.
"Composite of cell 1 (x = 29.7, S = 17.9, N = 66) was used to calculate % reduction.
concentrations experienced - throughout
the course of the study probably contri-
buted the single most detrimental impact
on the ability of the young minnows to
survive the embryonic stages of develop-
ment.
  Although the research described in this
article has been funded wholly or in part
                              by the U.S. Environmental Protection
                              Agency through grant number R803703
                              to Oklahoma State Department of Health,
                              it has not been subjected to the Agency's
                              required peer and policy  review and
                              therefore does not necessarily reflect the
                              views  of the  Agency and  no official
                              endorsement should be inferred.
   Dempsey H. Hall and Joel £. She/ton are with the Oklahoma State Department of
     Health. Oklahoma City. OK 73152.
   William R. Duffer is the EPA Project Officer (see below).
   The complete report, entitled "Advanced Biological Treatment of Municipal Waste-
     water ThroughAquaculture."(OrderNo. PB83-159319;Cost:$11.50. subject
     to change) will be available only from:
          National Technical Information Service
          5285 Port Royal Road
          Springfield, MA 22161
          Telephone: 703-487-4650
   The EPA Project Officer can be contacted at:
          Robert S. Kerr Environmental Research Laboratory
          U.S. Environmental Protection Agency
          P.O. Box 1198
          Ada, OK 74820
                                                                                  U. S. GOVERNMENT PRINTING OFFICE: 1983/659-095/1914

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Environmental Protection
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Information
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