EPA-600/1-79-030
August 1979
SAFETY EVALUATION OF RENOVATED WASTEWATER
FROM A POULTRY PROCESSING PLANT
by
Julian B. Andelman
Graduate School of Public Health
University of Pittsburgh
Pittsburgh, Pennsylvania 15261
Grant Nos. R804286 & S803325
Project Officers
Jack L. Witherow
Industrial Pollution Control Division
Industrial Environmental Research Laboratory
Cincinnati, Ohio 452.68
and
Herbert R. Pahren
Field Studies Division
Health Effects Research Laboratory
Cincinnati, Ohio 45268
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory
and the Industrial Environmental Research Laboratory, U.S. Environmental
Protection Agency, and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies of the U.S.
Environmental Protection Agency, nor does mention of trade names or commercial
products constitute endorsement or recommendation for use.
11
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FOREWORD
The U.S. Environmental Protection Agency was created because of increas-
ing public and government concern about the dangers of pollution to the health
and welfare of the American people. Noxious air, foul water, and spoiled land
are tragic testimony to the deterioration of our national environment. The
complexity of that environment and the interplay between its components re-
quire a concentrated and integrated attack on the problem.
Research and development is that necessary first step in problem solution
and it involves defining the problem, measuring its impact, and searching for
solutions. To that end, the Industrial Environmental Research Laboratory
assists in developing and demonstrating new and improved methodologies that
will provide more efficient and economical pollution control methods. Con-
siderable effort is exerted in developing industrial waste recycle systems
which will reduce pollution and conserve our natural resources. The primary
mission of the Health Effects Research Laboratory is to provide a sound health
effects data base in support of the regulatory activities of the U.S. Environ-
mental Protection Agency. HERL conducts a research program to identify,
characterize, and quantitate harmful effects of pollutants that may result
from exposure to chemical, physical, or biological agents found in the environ-
ment. In addition to the valuable health information generated by these
activities, new research techniques and methods are being developed that con-
tribute to a better understanding of human biochemical and physiological
functions, and how these functions are altered by low-level insults.
This report describes a joint research effort by the two Laboratories.
A system was evaluated whereby a poultry processing plant could conserve water
and reduce stream pollution without detectable adverse effects on the poultry
product.
David G. Stephan
Director
Industrial Environmental
Research Laboratory
R. John Garner
Director
Health Effects Research Laboratory
iii
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PREFACE
Wastewater reuse is an important strategy in achieving the national
goal of limiting discharges into navigable waters. However, in such reuse
of water involving human consumption or exposure, such as in the food pro-
cessing industry, it is mandatory that the health of the consumer be pro-
tected. At the same time it is necessary for both the public and the
appropriate professions to adjust their views and judgments on both the
esthetic and technical aspects of such reuse. For example, the U,S, Depart-
ment of Agriculture requires that water used in most phases of food pro-
cessing be potable. Traditional standards of potability, currently being
revised, may not be sufficient to deal with the possible chemical modifica-
tions and build-up of concentrations of these materials that can occur in
recycle, particularly those for which health criteria may be unavailable.
The reuse of wastewater, involving potential risk from human exposure,
has been practiced in agriculture, such as in irrigation, the impoundment
of water for recreational purposes, groundwater recharge, and even for
domestic water supplies. The most notable example of the latter is
Windhoek, South Africa, at which sewage was reclaimed for direct potable
use starting in 1968. No such reuse of wastewater for potable purposes
is, however, currently being practiced in the United States.
The water reuse project reported here was designed to provide a safe
and economical supplemental supply of water at and to a poultry processing
plant, utilizing as its base an existing wastewater treatment system.
Although not intended for direct human consumption, the renovated water,
prior to its use in the poultry processing plant, must meet the highest
standards of safety. Because of the general lack of experience in practic-
ing and evaluating such water reuse, both existing standards of water
quality and, probably even more importantly, professional judgment are
required to assure that this important criterion is met.
IV
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ABSTRACT
A three-phase evaluation of reclaimed process wastewater for reuse was
undertaken at the Sterling Processing Corporation plant in Oakland, Maryland.
The main objective was to evaluate the safety for human consumption of poultry
exposed during processing to an average 50 percent mixture of treated well
water and reclaimed wastewater. To that end, a determination was made of the
ability and reliability of the water reclamation system to deliver satis-
factory quality water, and whether the processed poultry would have any excess
microbiological or chemical constituents, harmful to human health, as a result
of exposure to such water. After the renovation system was optimized (Phase
1), a two-month study (Phase 2) was instituted, which simulated recycle of
renovated water through the poultry plant. Chemical, physical, and micro-
biological analyses were performed on various water, wastewater and poultry
samples. An experimental chiller, filled with renovated water, was utilized
to compare the uptake of such constituents by the processed birds with that
resulting from exposure to the chiller in the processing plant using the
normally treated well water.
With only a few exceptions, the mean and even maximum concentrations of
the various measured constituents met existing U.S. standards for potable
water. In the rare cases when they did not, such as a maximum value margin-
ally exceeding a recommended limit for sulfate, even with direct consumption
of the water there would be no danger to human health. There were increased
concentrations of several chemical parameters compared to those in the
normally-treated well water. However, this is to be expected in a recycle
system, and the levels would not jeopardize the health and safety of the
consumers of the poultry in actual reuse. The gross organic concentrations
in the well water supply and the renovated water were high, but no harmful
concentrations of specific organic chemicals were found to be present. For
all comparable chemical constituents analyzed in the poultry exposed to the
two types of water, the mean concentrations of carcass washings were statistic-
ally indistinguishable. Also, it was determined that in most cases there was
a net leaching of chemicals from the carcasses to the chiller water, rather
than vice versa. An evaluation of the Phase 2 study, as well as other data,
leads to the conclusion that the safety of the consumers of the poultry would
not be jeopardized if the planned trial period of reuse (Phase 3) were insti-
tuted.
This report was submitted in fulfillment of Grant Nos. R-804286 and
S-803325 by the University of Pittsburgh and the Maryland Department of Health
and Mental Hygiene under sponsorship of the U.S. Environmental Protection
Agency. This report covers the period August 1974 to December 1977, and work
was completed as of January 1979.
v
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CONTENTS
Foreword iii
Preface iv
Abstract v
Figures viii
Tables ix
Abbreviations and Symbols xi
Acknowledgment xii
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. Overview 6
5. Methodology 11
6. Results and Interpretation 12
Introduction 12
Operation of renovation system 13
Physical and inorganic constituents 17
Statistical and other comparisons 25
Organic constituents 34
Microbiology and related 40
Steady-state considerations 48
7. Discussion 53
References 60
Appendices
A. 'Miscellaneous methodology 63
B. Calculation of steady-state in recycle 69
vii
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FIGURES
Number Page
1 Schematic diagram of wastewater treatment-renovation system
at poultry processing plant with water sampling points . . 7
2 NDV survival at 7 C in light in laboratory study 49
B-l Flow diagram for steady-state calculations 70
Vlll
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TABLES
Number Page
1 Phase 2 - Summary of Types of Samples Taken for Analyses
Each Week 9
2 Types of Analyses Performed Routinely 10
3 Phase 2 - Water Quality Characteristics and Macro Constituent
Concentrations in Z, A and E 18
4 Z, A and E Water Characteristics Related Primarily to Waste
Treatment Efficiency 20
5 Phase 2 - Trace Chemical Concentrations in Z, A and E . . . . 22
6 Phase 2 Analyses of Concentrations of Macro Constituents in
Water From Plant (PC) and Experimental Chillers (EC) ... 23
7 Phase 2 Analyses of Concentrations of Trace Constituents in
Water From Plant (PC) and Experimental Chillers (EC) ... 24
8 Phase 2 - Comparison of Carcass Analyses (Washings) From
Plant and Experimental Chillers - Macro Constituents ... 26
9 Phase 2 - Comparison of Carcass Analyses (Washings) From
Plant and Experimental Chillers 27
10 A Comparison of Physico-Chemical Constituents of Renovated
Water (E) in the Previous Study and Phase 2 28
11 Relationships Between Concentrations of Parameters in Phase 2
at Three Sampling Points (E, Z, and A) as Obtained From the
One-Tailed Statistical Tests (at Alpha 0.05) 30
12 Comparisons of Several Phase 2 Analyses of Carcasses
(Washing) in Plant and Experimental Chillers with Theoreti-
cal Concentrations Calculated From Their Respective Water
Sources, Z and E 32
13 Additional Comparisons of Phase 2 Analyses of Carcasses
(Washings) in Plant and Experimental Chillers with
Theoretical Concentrations Calculated From Their Respective
Water Sources, Z and E, as well as Chiller Waters, PC
and EC 33
ix
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Number Page
14 Phase 2 - Pesticide Analysis 36
15 Halogenated Methane Content of Selected Water Samples Taken
From November 16, 1976 to December 8, 1976, Inclusive ... 37
16 Organic Chemicals Identified Qualitatively by GC-MS Analysis
of Methylated (with Diazomethane) Methylene Chloride
Extracts of Water Samples 38
17 TOC Analysis of Selected Sites Subsequent to Phase 2 .... 39
18 Carbon Chloroform Extract Concentrations (CCE) ... 41
19 Salmonella Isolations in Phase 2 at Various Sample Sites
Using Selenite Brilliant Green Enrichment and Brilliant
Green Agar 44
20 MPN Indices of Salmonella for Water and Wastewater Samples
with TET Enrichment Using EGA with Sodium Sulfadiazine,
all Subsequent to Phase 2 46
21 Summary of NDV Laboratory Survival Experiment, Percent
Surviving After 5 Days 49
x
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ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
EGA —brilliant green agar
CCE —carbon chloroform extract
MPN —most probable number
NDV —Newcastle Disease Virus
SYMBOLS
Sampling Points
A Raw wastewater
C Lagoon system effluent,
chlorinated
C' Lagoon system effluent,
unchlorinated
D Micro-strainer effluent
DS River, downstream
E Renovated water
EB Carcasses from exptl.
chiller
EC Water from exptl. chiller
Miscellaneous
b Fraction remaining in
lagoons
c Fraction remaining in
renov. system
C Cone, in 50/50 renov.
mixture after n cycles
C Cone, in 50/50 renov.
g
mixture in steady-state
PFU —plaque forming units
SBG —selenite brilliant green
TET —tetrathionate
TOC —total organic carbon
L-l Lagoon 1
L-l-M Lagoon 1, middle
L-l-E Lagoon 1, effluent
L-2 Lagoon 2
PB Carcasses from plant
chiller
PC Water from plant chiller
R River, downstream
US River, upstream
X Effluent, sediment-basin
Y Well water, untreated
Z Well water, treated
C Cone, at x sample point
N Number of samples in statistical
calc.
r Ratio of C to C
s s E
S.D. Standard deviation
X Mean concentration
xi
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ACKNOWLEDGMENT
This study was a joint effort by the Maryland Department of Health and
Mental Hygiene (MHD), the Sterling Processing Company, Oakland, Maryland, and
the Graduate School of Public Health, University of Pittsburgh (GSPH). The
University of Pittsburgh wishes to acknowledge with thanks the cooperation of
the MHD and in particular James Clise, Director, Bureau of Sanitary Engineer-
ing and Col. Edward, S. Hopkins, Consulting Sanitary Engineer for the MHD,
who initiated and were the principal investigators for Phase 1 of the overall
study, funded by US EPA Grant S-803325, during which the water renovation
system was modified and optimized. They also prepared the portion of this
report concerning the operation of this system. The Cumberland and Central
Laboratories of the MHD performed many of the daily chemical and bacterio-
logical analyses for the facility; major responsibility being undertaken by
Mary Lynn Hotchkiss and Mary Elizabeth Malloy of the Cumberland Laboratory
and Muriel W. Trusheim of the Central Laboratory. Dan McGrail was in charge
of plant operation from May 1975 to August 1976, followed by Robert
Holtschneider from November 1976 to June 1977, both under the immediate super-
vision of Col. Hopkins. Thomas Sereno, MHD aide, was responsible for collec-
tion of samples submitted to the Cumberland Laboratory.
It is also a pleasure to acknowledge the enthusiastic cooperation and
persistence of Gilman Sylvester, the manager of the Sterling Processing Corp-
poration, as well as his staff. Several analyses, including those for pest-
icides and a few trace elements, were performed by the Analytical Services
Laboratory of the NUS Corporation, Cyrus Win. Rice Division, Pittsburgh, Pa.,
under the able direction of Ellen Gonter.
Faculty at the University of Pittsburgh who were particularly helpful
were John Armstrong and Robert Yee of the Microbiology Department of GSPH who
advised on the virology and bacteriology, respectively; also Iain Campbell of
the Biological Sciences Department of the College of Arts and Sciences who
advised and otherwise cooperated on the gas chromatography and mass spectro-
metry analyses.
Teresa Lester and Jan Wachter, members of the research staff of GSPH,
made substantial contributions on several aspects of this study, including
project planning, operation, data interpretation, and the writing of this
report. Other research staff or students working on this project were David
Capelli, Robert Lau, Kenneth Wasyczak, John Weaver, and Stephanie Wilson.
Finally, the University of Pittsburgh wishes to acknowledge with appre-
ciation the generous guidance, assistance, and patience of the EPA Project
Officers, Herbert Pahren and Jack Witherow.
xii
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SECTION 1
INTRODUCTION
In early 1970 a wastewater reclamation project was initiated at a poultry
processing plant in Oakland, Maryland located in the far western part of that
state near the junction of Maryland, West Virginia, and Pennsylvania. An
increase in production at the Sterling Processing Company was and is limited
by the lack of additional water of acceptable quality. The plant currently
slaughters and processes approximately 50,000 birds per day (usually chickens)
in an eight-hour operation, utilizing approximately 1,300,000 liters (350,000
gallons) per day of treated well water.
With substantial financial support, initially from the U.S. Department
of Interior in January 1971, and later the U.S. Environmental Protection
Agency (EPA), a joint project was developed by the Maryland State Department
of Health and Mental Hygiene and the Sterling Processing Corporation to
design, construct, and study the feasibility of using a wastewater renovation
system, utilizing as its raw water source the chlorinated effluent from the
second of two aerated lagoons. These lagoons were initially designed and
constructed in 1965-1966 to treat the plant wastewater prior to its discharge
into the adjacent Little Youghiogheny River.' The goal of this project was
to renovate the lagoon wastewater to the point that it could be mixed with
the well water, then undergo conventional water treatment and be used in the
Sterling plant to process the poultry, thereby providing a supplementary
source of water otherwise not available. Since the regulations of the U.S.
Department of Agriculture require that the water used to process poultry
"shall be ample, clean, and potable," it was deemed important that an exten-
sive study of the quality of the renovated water be instituted prior to its
reuse. In a report published by the EPA, Clise describes the initial project,
including the construction and operation of the renovation system, and the
evaluation of the quality of the renovated water (7).
The full reclamation system, as reported by Clise, begins with the two
aerated lagoons, followed by partial diversion of the chlorinated effluent
from the second to a microstrainer, then flocculation-sedimentation, chlor-
ination, and filtration. The initial study showed that the reclaimed water
met U.S. Public Health Service 1962 Drinking Water Standards for chemical,
microbiological and physical constituents without actual recycle through the
poultry processing plant. Nevertheless, there was concern that with actual
reuse there was the possibility that unmeasured constituents, such as patho-
genic microorganisms, heavy metals, pesticides and toxic organic chemicals,
might build up in recycle and be absorbed by the carcasses in the processing
plant.
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An additional project was proposed by the Maryland State Department of
Health and Mental Hygiene and funded by the EPA (Grant No. S803325) , the pur-
pose of which was to modify and optimize the reclamation system, to determine
the capability and reliability of the system for delivering satisfactory water
quality, and to evaluate the exposure of the processed carcasses to constit-
uents that could be harmful to human health. With separate funding (Grant
No. R804286), EPA supported the Graduate School of Public Health, University
of Pittsburgh, to design, supervise, and perform the sampling and analytical
part of the study, as well as evaluate the results from the points of view of
both the quality of the renovated water and the processed poultry possibly
affected by it. Three phases were planned in this study, the first two of
which have been completed. Phase 1 involved the operation of the reclamation
plant with a new sand filter, and measurement of those characteristics perti-
nent to optimizing the process. Phase 2 involved a study of a wide range of
physical, chemical and microbiological constituents, both at various points
in the reclamation system, as well as in process carcasses chilled with reno-
vated water, but without actual recycle through the plant. Phase 3 was to
involve recycle of the renovated water into the processing plant by mixing on
an average 50/50 basis with well water, the mixture then to undergo additional
full-scale conventional treatment. The carcasses were again to be measured,
as was the renovated water, and comparisons made of the levels of contaminants
with those in normal plant operation utilizing well water only in Phase 2.
Prior to proceeding to Phase 3, an evaluation was to be made of the
Phase 2 results by a committee consisting of representatives of the EPA, the
Maryland Health Department, the processing plant, the U.S. Department of
Agriculture, and the Graduate School of Public Health. The level of contam-
inants in the renovated wastewater and processed carcasses were to be con-
sidered as to their possible health significance. A similar evaluation was
to be made following Phase 3 so as to determine the safety of proceeding to
continuous operation with reclaimed wastewater. Finally, recommendations
were to be made on constituents to be monitored and corresponding procedures
needed to insure the reliability of the system and the protection of human
health.
This report describes and evaluates primarily the Phase 2 study of the
University of Pittsburgh. Additionally, a summary description of the modi-
fication and operation of the renovation system under a companion grant by
the EPA to the Maryland Department of Health and Mental Hygiene is included.
Phase 3, the trial three-month period of actual recycle of the renovated
water into the processing plant, has not been instituted because it has not
been approved by the appropriate federal agencies, in spite of the positive
recommendation by the above Committee, constituted for the purpose. Some of
the likely concerns which are the apparent basis for this lack of approval
will also be discussed.
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SECTION 2
CONCLUSIONS
Based on the regularly low or zero bacteriological counts in the renova-
ted water during the Phase 2 study, as well as at other times, the absence
of any avian virus that could cause human disease by the enteric route, and
the extensive wastewater and water treatment, including four points of chlor-
ination, it is highly unlikely that the contemplated reuse of water at the
Sterling Processing Corporation plant would pose a risk of disease from micro-
organisms to the consumers of the poultry.
The inorganic and physico-chemical characteristics of the renovated
water consistently met applicable standards of quality for potable water. A
few such parameters, and several non-health related ones, as expected were
high, but not at levels that would constitute a threat to human health, even
if the water were directly ingested. However, in actual reuse it would
receive additional treatment and would not be used as a drinking water supply.
A few non-health related chemical constituents were at concentrations that
could interfere with the optimal operation of the renovations system, and
should be adjusted. These include the low pH and high ammonia concentrations.
Some measurements of gross organic load, such as total organic carbon,
were high in both the treated well water and the renovated water, but there
were no statistical differences between them. None of the specific organic
chemicals that were found were at hazardous or unusually high concentrations,
and many are innocuous. There is a possible concern that unidentified toxic
organics could be present in the renovated water, including those that might
form as a result of reactions with chlorine. However, in the measurements
that might have uncovered them there was no evidence for their presence.
A comparison of carcasses exposed to the normally-treated well water
and to the renovated water in almost every instance showed no statistical
difference in the mean concentrations of measured constituents. For a few
chemicals, including calcium, ammonia, sulfate, and nitrate, there was an
apparent contribution to the carcasses by the chiller water. Calculations
indicate that for most of the chemicals there was much more leaching into
the chiller water from the carcasses than vice versa.
Mass-balance, steady-state calculations indicate that the Phase 2 re-
cycle simulation had probably reached steady-state, and the concentrations
measured in the renovated water could be used as a basis for predicting the
levels that would be encountered in actual reuse. For many of the constit-
uents such concentrations would be equal to or as much as one-third less than
those found in Phase 2.
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An analysis and review of all the measurements and operational charact-
eristics of the renovation system and Phase 2 results indicate that there is
no significant or discernible risk to the consumers of the poultry in the
planned reuse of the water. It is unlikely that in such reuse any constit-
uent limits for chemical or microorganisms for which there are current potable
water quality standards would be exceeded in the renovated water. Also it
is unlikely that these or any other constituents, already identified or not,
would be taken up by the carcasses in hazardous quantities as a result of
exposure to this water.
Low winter temperatures and lagoon turnovers create serious problems
for the renovated water system. Its operation should be suspended during
these periods.
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SECTION 3
RECOMMENDATIONS
The results of this and previous studies, of the wastewater and renova-
tion systems at the Sterling Processing Corporation plant indicate that there
are not any apparent concentrations of chemicals or microorganisms that did,
or in actual reuse would be likely to build up in the renovated water supply
to the point of jeopardizing the health of the consumers of the poultry pro-
cessed with that water. There are some steps that could and should be taken
to minimize and further reduce any possible risks or concerns. It is, there-
fore, recommended:
1. A trial period of reuse (Phase 3) should be instituted as soon as possible.
During this period there should be a full scale monitoring of the renova-
ted water and carcasses as originally planned, followed by a comprehen-
sive evaluation process prior to any permanent reuse.
2. Prior to and/or during the trial period of reuse more extensive organic
analyses of the renovated water and other pertinent samples should be
performed, with particular focus on chlorinated organics, especially
those that might form from the reactions of chlorine with waste products
from the poultry. Assessments should then be made of the possible health
significance of any identified and quantified organics, including a
determination of the likely impact of the quantities to which the con-
sumers of the poultry would be exposed.
3. Although not of primary concern, some effort should be expended on optimi-
zing the renovation system to minimize the unnecessarily high concentra-
tions of some chemicals measured in the renovated water. This includes
raising the pH and lowering the sulfate and ammonia levels in the renova-
ted water.
4. In addition to the routine monitoring of turbidity, pH and chlorine al-
ready in effect in the renovation system, other parameters should be
measured frequently on site to obtain an early indication of unusually
high levels of contaminants, at least during the trial period of reuse.
Examples are conducuivity and total organic carbon.
5. Some consideration and investigation should focus on the possibility of
reducing the organic content of the renovated water, either before or
after mixing with the wel-1 water. However, this must be evaluated, not
only in terms of the quality of the water itself, but also the impact on
the processed carcasses and the costs as well.
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SECTION 4
OVERVIEW
Figure 1 is a schematic of the poultry processing plant in relation to
its normal source of well water, the aerated lagoons discharging after chlor-
ination to the Little Youghiogheny River, and the diversion of a portion of
this discharge to the renovation system. Also shown are various sampling
points employed during the study, with their letter reference designations.
On occasion other sampling points were utilized, and these are identified on
page xi which contains a list of symbols, including those for the chiller
waters and the "dressed" poultry, referred to in this report as carcasses.
Poultry, usually chickens, are shipped by truck from the hatchery in
Delaware to the Sterling plant and are processed on the day of arrival. With-
in the plant the birds are slaughtered, scalded, picked, eviscerated, chilled,
cut-up and packaged. The poultry processing wastes resulting from the evis-
ceration step are treated by rotary screening for the removal of feathers and
viscera. The raw wastewater (sample point A, Figure 1) from the other poultry-
processing steps, the refrigeration drains and the plant clean-up passes to a
mechanically aerated primary lagoon which is equipped with a grease skimmer.
The effluent (sample point L-l-E) from the primary lagoon discharges through
a weir trough into a mechanically aerated secondary lagoon (sample point C'),
which also contains a grease skimmer. Each lagoon is about 1.8 m deep. The
first has a capacity of about 14,000 m^, and the second about 6,000 m . Their
combined retention time normally is two to three weeks. The effluent from
the secondary lagoon is chlorinated as it passes into a combination settling
unit and chlorine contact chamber. The chlorinated effluent (sample point C)
is discharged through an overflow weir trough to the Little Youghiogheny River.
As shown in Figure 1, a portion of the chlorinated lagoon effluent is
diverted to the renovation system, which in sequence consists of a micro-
strainer, flocculation with alum and lime in a basin, followed by filtration
through a sand filter and chlorination. During this study the effluent from
the renovation system, E, was returned to lagoon 1, as were the solids from
the micro-strainer. In actual reuse the renovated water would be mixed with
the well water Y, the mixture then to receive the normal treatment currently
utilized for the well water alone. This consists of pre-chlorination in the
mixing basin, alum-lime flocculation with final pH adjustment to precipitate
iron, settling, and filtration through two sand filters. Additional chlorine
for residual control is introduced into the main service line leading to the
processing plant.
In actual reuse the raw wastewater A, prior to its return to the poultry
processing plant, would receive four stages of chlorination: at the effluent
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roUGHIOGHENY RIVER
US
OS
COLLECTION BASIN
6
CHLORINE
CONTACT
CHAMBER
MICRO-
STRAINER
LAGOON 2
1
•E
\
A
- ^*
LAGOON l.£-^-
L-l-M
ROTARY
SCREENS
FLOCCULATION-
SEDIMENTATION BASINJ
SAND FILTER
CHLORINATOR
PRESSURE
STORAGE TANK
POULTRY
PROCESSING
PLANT
NORMAL
TREATMENT
MIXING
BASIN
WELL
WATER
Sample point identification
A, untreated wastewater; L-l-M, lagoon 1; L-l-E, lagoon 1 effluent; C',
lagoon 2 effluent, unchlorinated; C, lagoon 2 effluent, chlorinated; US,
river, upstream; DS, river, downstream; D, micro-strainer effluent; X, floccu-
lated, settled effluent; E, fully renovated water; Z, normally treated well
water.
Figure 1. Schematic diagram of wastewater treatment-renovation system at
poultry processing plant with water sampling points.
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from lagoon 2; subsequent to sand filtration in the renovation system; and
pre-chlorination and post-chlorination in the normal well water treatment
system. The renovation system was designed to treat about 50 percent of
the flow from the second lagoon effluent. In reuse the renovated water would
then be mixed 50/50 (on an equal basis) with the well water, the mixture then
receiving its normal treatment prior to use in the poultry processing plant.
An important difference between the Phase 2 study and the flow regime in
actual reuse is that in Phase 2 the flow through the renovation system was
about one-third of that through the processing plant, rather than the one-
half planned in actual reuse. Thus in Phase 2, since the renovated water was
discharged into the first lagoon, its combined flow with that of the waste-
water, A, is one-third higher than the normal waste flow or that expected
in actual reuse. This results in a higher dilution of any waste constituent
from A emitted to the lagoon system, but at the same time reduces the resid-
ence time within it.
The earlier study of Clise (7) indicated that the processing plant was
successful in reducing its water usage to about 26 1 per bird. For a typical
8-hour shift processing 50,000 birds, this amounts to 1.3 x 10^ 1 of water.
Of this total amount about half is used during processing and the remainder
for clean-up (7). Probably the most important water exposure of the carcasses
is that in the chiller prior to packaging. At the Sterling plant there are
two chillers operated in series, with fresh water flowing counter-current to
the movement of the carcasses. The typical water temperature in the first
is about 13 C, and about 2°C in the second, the total exposure or residence
time of a carcass in the combination of the two chillers being about 25 min-
utes. Regulations of the U.S. Department of Agriculture limit the water up-
take to a maximum of 12 percent of the carcass weight. At the Sterling plant
the typical uptake is 6 to 8 percent. Because the chiller constitutes such a
critical exposure to water in the plant, a large part of the study focused
on the chiller water quality, the possible influence on it of the constit-
uents in its finished water source, and the measurement of possible contamin-
ants in the carcasses subsequent to their exposures to the chiller system.
The sand filter in the renovation system was constructed to replace the
diatomite filter used in previous study (7), and the optimization of the
renovation system, Phase 1, began in the Fall of 1975 and continued inter-
mittently through the winter as weather conditions permitted. Preliminary
sampling for the Phase 2 study began in late February 1976 to optimize the
sampling and analytical methodology. This was followed by the full Phase 2
sampling program, approximately once a week for seven weeks from March 15
through May 3, 1976. On each of these days various water and wastewater
samples were collected. In addition, twenty-five carcasses were collected
for analysis from the plant chiller system. Finally, a small "experimental"
chiller was set up to simulate the plant chiller. Twenty-five carcasses
were taken from the plant, prior to exposure to the plant chiller, and placed
in the experimental chiller in such a way and for such a period as to simulate
the plant chiller. However, the experimental chiller was filled with renova-
ted water. The purpose was to analyze and compare the possible build-up of
contaminants in carcasses exposed to the plant chiller using normal, treated
well-water, versus those exposed to the experimental chiller using renovated
water. At no time during Phase 2 was renovated water used in the processing plant.
-------
The typical Phase 2 sampling points and the number of samples taken on
a typical sampling day are shown in Table 1. Not all of the sampling points
were subject to all the analyses. The types of analyses that were performed
are shown in Table 2. As shown there, Categories la, b, and c were routine-
ly performed only on water samples, while Id was to be done on carcass samples
as well. In fact, some la and Ib analyses were also performed on carcasses.
Category II and III analyses were done on both water and carcass samples.
Other sampling points were utilized and analyses not listed in Tables 1 and 2
occasionally performed.
Although analyses of viruses were not contemplated originally in this
study, the decision was made to attempt to measure an avian virus and use it
as an indicator or sentinel of the behavior of other viruses in the water
renovation system. Since it was reported that the chicken flocks were routine-
ly inoculated with live, attenuated Newcastle Disease virus (NDV), it was
decided to develop the methodology and sample the water and carcasses for NDV.
In addition, some laboratory die-off studies were performed using lagoon water
inoculated with NDV.
Finally, and subsequent to Phase 2, total organic carbon measurements
(TOC) were performed on the renovated water, the normally treated well water,
and that taken at other selected sampling points. In addition, a few samples
of the treated well and renovated water were analyzed by gas-chromatograph-
mass spectrometry (GC-MS) for some specific organics, other than the pesti-
cides of Category III. Also during one month subsequent to Phase 2, samples
were analyzed for trihalomethanes at several points in the water supply
system, lagoons, renovation system, and the receiving stream.
TABLE 1. PHASE 2 - SUMMARY OF TYPES OF
SAMPLES TAKEN FOR ANALYSES EACH WEEK
Water samples
Maximum number
Location per day
A - Raw waste 2
E - Renovated water 2
Z - Treated well water 2
PC- Plant chiller 2
EC- Experimental chiller 2
Birds (carcass samples)
PB- Plant chiller birds 5*
EB- Experimental chiller birds 5*
*50 carcasses were taken for analysis each sampling day, 25 PB and 25 EB. In
each case washings from 5 carcasses were composited or otherwise combined to
become a single carcass sample. Hence, 25 carcasses reduced to 5 samples.
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TABLE 2. TYPES OF ANALYSES PERFORMED ROUTINELY
Category I analyses
la - These analyses relate primarily to waste treatment efficiency and
none were performed on carcasses routinely
BOD, Suspended solids
Total solids
Grease .,,
Alkalinity
Organic nitrogen
Ammonia nitrogen
Ib and c - These analyses relate to waste treatment efficiency and renovated
water quality; none were performed on carcasses routinely
Ib l£
Turbidity CCE (Carbon chloroform extract)
Color
Total dissolved solids
Residual chlorine (total)
Residual chlorine (free)
Id - These analyses relate to waste treatment efficiency and water quality.
They were measured for water and carcass samples.
Total plant count Fecal coliform
Total coliform pH
Category II analyses
These analyses were performed on water and carcass samples
Salmonella Drug residual
Category III analyses
These chemical analyses were performed on water and carcass samples
Arsenic
Barium
Cadmium
Calcium
Chloride
Chromium
Copper
Cyanide
Fluoride
Hardness
Iron
Lead
Magnesium
Manganese
Mercury
MBAS
Nitrate
Potassium
Selenium
Silver
Sodium
Sulfate
Zinc
Pesticides
10
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SECTION 5
METHODOLOGY
Except where noted below all analytical methodology for chemicals and
microorganisms is consistent with U.S. Environmental Protection Agency (23,
24) or other standardized techniques (1). All necessary precautions for
transporting and preserving the integrity of the samples were taken. This
included adding chemical preservatives, storage in the cold, and the use of
various types of sample bottles and vials, depending on the nature of the
analysis. All water and wastewater samples were individual "grab" type (no
compositing was done).
The experimental chiller using renovated (E) water was operated on a
batch basis. That is, it was filled with renovated water and plant-made ice
was added to bring the temperature initially to about 13°C. The 25 carcasses
were then lowered into the bath within the drum and rotation begun. Addi-
tional ice to maintain 13°C was added. Approximately 15 minutes later more
ice was added to reduce the bath temperature to about 1°C, and chilling con-
tinued for another 10 minutes.
The carcasses were removed by handling with clean plastic gloves, and
both plant and experimental chiller carcasses were treated in the same fashion.
They were placed, after draining, in either clean or pre-sterilized (by auto-
claving) plastic bags and carried to the laboratory trailer. 1500 ml of
either distilled or distilled and sterile water was added to each such bag,
which was then shaken for one minute and the water contents poured for analy-
sis. This rinse sampling method has been used primarily for the detection of
bacteria in processed poultry (4, 6), but was employed in this study for both
microbiological and chemical analyses of the carcasses.
Trace metals were analyzed by atomic absorption spectrophotometry using
solvent extraction to increase the sensitivity. Reference samples to test
and improve, where necessary, the accuracy of the analyses were obtained from
the EPA and utilized for trace metals, several other inorganic constituents,
and pesticides.
A variety of non-standard microbiological methods were utilized and/or
modified for Salmonella and Newcastle Disease Virus, as well as the simulated
lagoon die-off rate studies for the latter, and these are described in
Appendix A, along with the methodology for the residual drug analysis. Simi-
larly, the details of the volatile and non-volatile organic chemical analyses
are presented, and the statistical methodology as well.
11
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SECTION 6
RESULTS AND INTERPRETATION
INTRODUCTION
The results presented in this section will focus primarily on Phase 2,
at least for the analyses of the inorganic constituents and physical para-
meters measured in the water, wastewater and carcasses. Additionally a
description will be presented of the operation of the renovation system,
along with some comparisons with the earlier study of Clise (7) and, where
appropriate, use of data from Phase 1 and post Phase 2. Data were also
obtained and are presented for many parameters in the chillers during Phase
2. Because of the large amount of data, for the most part the results will
be summarized, showing the number of analyses, the means, standard deviations,
and in some cases the maximum values. Most of the analyses in Phase 2 were
performed on samples taken on each of the seven sampling dates. In some
cases, however, and as originally planned, where the results were consis-
tently negative (below the level of sensitivity), the analyses were discon-
tinued.
Statistical analysis of the data is confined mostly to those inorganic
constituents and physical parameters for which there were sufficient numbers
to be meaningful and useful. In addition to comparing the Phase 2 results
with the earlier study of Clise (7), such comparisons have been made as are
helpful in determining any differences between the renovated (E), wastewater
(A) and treated well water (Z), thereby focusing on possible build-ups in
the renovation system or inefficiencies in treatment, as well as possible
health concerns. Of special interest also are the statistical analyses and
comparisons of carcasses exposed to the waters of the processing plant chiller
(PC) and experimental chiller (EC). Their possible differences were deter-
mined, and an analysis performed to assess the contributions of constituents
in the chiller water to those measured in the carcasses (washings).
Some organic parameters were measured routinely in Phase 2, but these
generally were the gross ones (rather than specific organic chemicals) which
are reported in tables showing several waste parameters. These include 6005
and organic nitrogen. Pesticides were analyzed for a few weeks in Phase 2,
but discontinued when the treated well and renovated water samples were all
negative. Several CCE results are reported for Phase 2 and other periods.
Subsequent to Phase 2 several samples were taken and analyzed at a number of
sampling points for TOC and specific organic chemicals, both volatiles
(trihalomethanes) as measured quantitatively by GC, and non-volatiles, quali-
tatively only by GC-MS. In general there were not sufficient organic analyses
12
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for statistical comparisons between Z and E water, although this could be
done in one or two cases. However, the organic measurements were of suffic-
ient scope to allow a reasonable assessment of their possible health impact
as a result of processing the chickens in the renovated water.
Although coliform, fecal coliform and standard plate count analyses
were performed on a variety of water, wastewater and carcass samples, it was
decided for the purpose of this report to focus primarily on a comparison of
the treated well and renovated waters as a means of evaluating and comparing
possible risks. Such bacterial levels in the chillers and carcasses would
most likely be unrelated to their respective water sources which are routine-
ly disinfected. In contrast, Salmonella was evaluated in a variety of samples
including carcasses, both in Phase 2 qualitatively, and subsequently for its
quantitative presence, after the appropriate methodology was developed. Al-
though for all practical purposes there is no basis for concern about avian
viruses with respect to human health in this project, Newcastle Disease
Virus (NDV) was selected for analysis as a possible sentinel or indicator
organism of viral behavior. NDV can cause mild conjunctivitis in humans, but
is not known to cause human disease via the gastro-intestinal route. The
results of attempts to isolate it in the carcasses and other samples during
Phase 2 are described. Additionally, the NDV die-off studies in the labora-
tory using lagoon water from the Sterling wastewater treatment system are
presented and evaluated. Although results were negative, the attempts to
assess the presence of antibiotic drugs in various samples using a bacterial
drug residual test are described.
Finally there will be presented briefly an analysis of the use of the
Phase 2 treatment and renovation scheme as a predictor of the behavior and
steady-state build-up of contamination in actual recycle. The detailed cal-
culations and basis for the analysis are described more fully in Appendix B.
OPERATION OF RENOVATION SYSTEM
Phase 1, the optimization of the renovation system, began in the Fall of
1975 and continued intermittently, depending in part on the winter weather
conditions, until February 1976 when preliminary sampling for Phase 2 was
instituted. Subsequent to Phase 2, starting in May 1976 and continuing to
the end of the project in June 1977, additional efforts were directed at
improving the operation and performance of the renovation system, such as by
pH adjustment and the use of powdered activation carbon.
More detailed information on the lagoon and renovation systems, as well
as characterizations of the wastewater, can be found in the report by Clise
(7). However, for orientation some of this information is included here.
The following material is a summary report of the pertinent aspects of the
operation of the renovation system.
Description of Lagoon and Renovation Systems
The wastewater treatment system consists of two lagoons totalling 2.75
acres (1.11 hectares) in area. Each lagoon is 140' (42.7 m) wide. The
13
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primary unit is 590' (177 m) long and the secondary lagoon is 230' (69 m)
long. Each pond is six feet (1.8 m) deep. Primary lagoon capacity is approx-
imately 3.75 million gallons (14.195 m^) and secondary capacity is 1.5 million
gallons (5,678 m^) providing holding capacity for 12 working days' flow.
The primary lagoon is equipped with 64 Link Belt circulators, a grease
skimmer, and an effluent wier trough discharging into the second lagoon.
Entering at the bottom of the circulators, wastewater is discharged at the
surface in one direction, creating a counter-clockwise surface flow. Air is
supplied to the circulators by three positive displacement blowers, each
powered by a 20 hp (14.9 kw) motor. The system provides 3,360 cfm (15.8 m-Vs)
of air at 2.8 psig (19.3 kN/m^). Air is distributed to the circulators
through a header pipe encircling the two lagoons. The secondary lagoon is
equipped with 40 Link Belt circulators, a grease skimmer, and a combination
settling unit and chlorine contact chamber with an overflow wier trough and
discharge line to the river.
Incoming raw wastewater has a BOD^ averaging 450 mg/1, amounting to a
loading approximating 400 Ibs/acre/day (448 kg/ha/day) with a 93% reduction
in the lagoon system. Raw wastewater suspended solids average 858 mg/1,
equaling a loading of 750 Ibs/acre/day (841 kg/ha/day) with an 88% reduction
in the system.
The advanced water treatment plant was designed to collect the chlorina-
ted effluent from the second lagoon at the point of discharge to the river.
Basic design of the advanced water treatment facility consists of a control
building; 70 micron microstrainer; a flash mix, flocculation and sedimenta-
tion basin; a gravity flow rapid sand filter; gas chlorinator; and 20,000
gallon (76 m-^) pressure storage tank. Supplemental equipment consists of a
3,000 gallon (11,360 liter) concrete pit used as a collection sump for the
lagoon effluent; sewage pump for delivery of effluent to the microstrainer;
a low head pump used for the application of alum and coagulant aid for de-
livery of microstrainer effluent to the flash mix unit; high head pump for
delivery to the sand filter; chlorine recorder; and electrical control panel.
All equipment is automatically controlled by the water height in each unit
and is rated at or above 300 gpm (19 liters/sec).
As originally operated, chlorination preceded storage and filtration as
a method of assuring breakpoint chlorination. In the later phases of the
project, piping changes were made to allow all physical treatment to occur
prior to chlorination. These changes were for the purpose of reducing as
far as possible the organic content of reclaimed water available for com-
bining with chlorine. Concurrent with this change, powdered activated carbon
was introduced in the flash mix for the removal of combined chlorine in the
sedimentation process.
Operating Procedures
During this study there were two pumping regimes, continuous and inter-
mittent. The initial pumping operation was to determine capability to meet
the demand under the adjusted pumping rate necessitated by balancing four
pumps in continous operation. Previous operations required only two units
14
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based on the original design. With the new adjustment the low head pump
established a rate for normal operation at 210 gpm (800 liters per min), com-
pared to the designed rate of 300 gpm (1140 liters per min). The system re-
mained under automatic control from pressure in the storage tank with con-
tinuous discharge into the No. 1 lagoon.
During Phase 2 the expected demand of 180 gpm (680 liter per min) by the
Sterling plant in actual reuse was simulated by discharging this volume of
filtered water from the storage tank to waste into the No. 1 lagoon. Normal
water requirement in the Sterling plant for a 16 hour day is 288,000 gallons
(1,130,000 liters). The new pumping rate in the water re-use plant will give
202,000 gallons (765,000 liters). Under this program the high lift pump was
in intermittent service as pressure varied in the storage tank between 55 and
70 psi. The pump cycle was 15 minutes in service and 12 minutes off with con-
tinuous flow through the sand filter. All raw water pumps in the Sterling
system are also on automatic control. The No. 3 pump at 175 gallons per
minute (656 liters per min) will be utilized with the re-use pump at 180 gpm
(675 liters per min). The re-use line is metered and proper adjustment can
be maintained. The other two Sterling pumps totaling 190 gpm (713 liters
per min) will remain in standby service. The re-use line will discharge into
the Sterling raw water sedimentation basin through a float valve thereby
maintaining a more or less controlled equal volume from the two systems. The
re-use water plant would be in continuous automatic service.
The sedimentation basin was drained and washed on a cycle of 2 million
gallons (7.6 million liters). This was usually done every other weekend when
the poultry processing plant was not operating. During the initial part of
the study the filter averaged 5.1 hours of service between backwashings at
8.4 feet (2.6 meters) of head loss. When simulating the reuse operation it
averaged 7.0 hours of service at 9.1 feet (3.4 meters) of head loss.
Chemical Treatment and Monitoring
In June 1976 (subsequent to Phase 2) an automatic turbidimeter was placed
in service to monitor the renovated water (E). During a period of 93 days the
hourly mean value was 1.08 Jackson turbidity units, and the maximum was 2.7.
Chlorine was added typically at a rate of 6.5 pounds (2950 grams) of
chlorine per hour, approximately 0.35 pounds (160 grams) per 1000 gallons or
44 mg/1 from January 6 to June 3, 1976, which included the Phase 2 period.
"Break point" chlorination was accomplished; the free residual chlorine in
the microstrainer effluent (243 tests) averaged 0.36 mg/1, with a total
residual of 0.84 mg/1, indicating that 0.48 mg/1 was present as combined
chlorine, probably chloramines. With subsequent use of carbon, the residual
chlorine was 0.2 mg/1 (243 tests). The post-chlorination dose averaged 1.7
mg/1, with a free chlorine residual of 0.22 mg/1 in the renovated water.
The automatic chlorine recorder was placed in service in June 1976.
Operating on post-chlorination, with 305 tests reported, the average free
residual in the filter effluent was 1.9 mg/1, with a minimum of 0.5. Die-
thyl-p-phenylene diamine (D.P.D.) replaced orthotolidine arsenite (O.T.A.)
as the procedure for residual chlorine on March 29, 1976. The automatic
15
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chlorine recorder gave results in agreement with the D.P.D. test.
To eliminate chlorine and chloramine fumes in the control house, acti-
vated carbon at 10 mg/1 was applied to the flash mixing tank beginning June
21, 1976. This was highly effective, as shown by the chlorine residual
measurements, which indicated that one-third of the measured values were
between 0.1 and 0.2 mg/1, and the remainder were below the sensitivity limit.
Under normal conditions the suspended solids in the lagoon system efflu-
ent varied from 12 to 88 mg/1 with a mean of 40 mg/1. Coagulant: dosage was
determined by daily or more frequent "jar tests." With normal raw lagoon
water it averaged 15.4 mg/1 alum, 5.3 mg/1 lime, 1 mg/1 polymer and 10 mg/1
carbon. Due to seasonal turnover of the lagoons from October 19 to December
13, 1976, the suspended solids of their effluent averaged 304 mg/1 with a
minimum of 114 mg/1.
A solid ice cap on the lagoons between December 13, 1976 arid March 8,
1977 prevented operation of the plant. From April 25 to June 8, 1977 a
second seasonal turnover occurred. In 1976 the dissolved oxygen was depleted
but the lagoons did not become anaerobic. However, in 1977 the lagoon be-
came anaerobic with resultant development of putrescible sludge and hydrogen
sulfide killing the organisms necessary to stabilize the deposited sludge.
During the period while the plant was out of service a series of jar
tests (147) was made to develop a coagulation procedure that would produce
compact, settleable floe. They indicated that a dosage of 22.2 mg/1 alum
per percent of settleable solids at a pH value of 6.3 would be practical.
However, during this operation screens would have to be removed from the
microstrainer and excessive backwashing of the sand filter on a 4 hour cycle
would be required. At normal pumpage this dosage is equivalent to 821 mg/1
of alum, 1382 mg/1 lime, 1 mg/1 of polymer and 10 mg/1 of carbon. These
dosages greatly exceeded the capacity of the 308 mg/1 feeding equipment, which
was designed for 7.8 mg/1 hexane soluble material, 58 mg/1 suspended solids
and 30 JTU turbidity. It was not possible to operate the plant during this
period. Supplementary chemicals such as hydrogen peroxide and excess chlo-
rine were also employed without appreciable effect. Based on this effort
and analysis, it was judged that operation of the reuse system should be
suspended during a period of lagoon turnover.
Hexane soluble material was found in the renovated water effluent at
concentrations varying from 0.2 to 6.0 mg/1. The average of 28 tests was
3.6 mg/1. Recent concentrations of hexane soluble material found in the
three Sterling wells ranged from 4.0 to 4.8 mg/1. These wells are in the
Pocono formation of the Deer Park anticline, which is generally sandstone.
They vary from 168 feet (53 meters) to 362 feet (112 meters) in depth. The
hexane soluble content of three other wells, ranging in distance from 15
meters to 5.5 kilometers from the Sterling plant, varied from 0.4 to 2.6
mg/1. There are abundant gas wells and coal seams in the vicinity at depths
which by lateral diffusion could readily influence the organic content of
the Sterling and other nearby wells.
16
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PHYSICAL AND INORGANIC CONSTITUENTS
Finished Water and Wastewater
The summary of the Phase 2 results for several physical and macro inor-
ganic constituents of Z, A and E waters is presented in Table 3, along with
the EPA Interim Primary and proposed Secondary Drinking Water Regulation
constituent limits (25, 26). In that table the former include only turbidity
and nitrate, the others being Secondary regulation limits. The nitrate max-
imum constituent limit was not exceeded in the E water in Phase 2, and the
mean of 3.5 mg/1 was considerably below that limit of 44 mg/1 (as NO^")•
Subsequent to Phase 2 an additional 24 samples of E water had a mean concen-
tration of 2.16 mg/1, lower than that in Phase 2.
The current EPA turbidity limit is one, rather than the five shown in
Table 3. However, five is permitted in certain instances, such as when it
does not interfere with disinfection or microbiological determinations. In
addition to the turbidity results for E in Phase 2 with a mean value of 1.6,
140 such analyses in Phase 1 had a mean of 2.9 units, and 175 analyses post
Phase 2, 2.2 units. It is apparent that, although generally higher than the
limit of one, they are less than five; also, as will be discussed subsequent-
ly, the disinfection processes were quite effective.
The pH and alkalinity in E, the renovated water, during Phase 2 were
significantly lower than that of Z, the treated well water, which for these
parameters was quite similar to A, the raw wastewater. These low values in
E can be attributed reasonably, at least in large part, to the extensive
chlorination, both of the secondary lagoon effluent and the renovation system,
as well as the addition of alum as a coagulant. The chlorine applied typi-
cally to the former was 25 mg/1 (7), and 44 mg/1 to the latter during Phase 2.
The application of this total amount of chlorine generates enough acid to
neutralize about 50 mg/1 alkalinity (calcium carbonate) and, at the same time
lowers the pH. In the previous study (7), as shown in Table 10, the average
pH was higher, namely 6.6, compared to 5.8 in Phase 2. Also, in about a one
year period subsequent to Phase 2 the average of 167 pH measurements was
6.06, with a standard deviation of 1.02. Although there is no direct health
risk from such relatively low values of pH, to the extent that this could
adversely affect treatment or cause corrosion, it should and could be recti-
fied relatively easily by the addition of soda ash. In actual reuse there
will be mitigation of this potential problem, in any event, due to the 50/50
mixing with well water.
As shown in Table 3, the average free chlorine residual during Phase 2
was 1.7 mg/1 in the renovated water. Subsequent to this period over 300
readings showed an average of 1.9 mg/1, and a minimum of 0.5. Such chlorine
levels, maintained consistently in conjunction with its application else-
where in the system, would be expected to provide a high degree of assurance
against risks from pathogenic microorganisms in actual reuse.
Of the remaining parameters the only one occasionally approaching a
recommended (esthetic or secondary) constituent limit in the renovated
water is sulfate. The mean concentration of 150 mg/1 is considerably below
17
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the recommended limit of 250 mg/1, although one value at 252 mg/1 exceeded
it marginally. Although not a health concern, the build-up could be reduced
by modification of the use of alum as a coagulant, a major contributor to the
sulfate in the renovation process.
The 389 mg/1 average for the total dissolved solids in the renovated
water during Phase 2, shown in Table 3, was considerably higher than that of
the treated well water, although less than the non-mandatory criterion value
of 500 mg/1. Also, as shown in Table 3, the sum of the mean values of the
concentrations of the cation and anion constituents is essentially identical
with that for the dissolved solids. One can thus conclude that it is unlike-
ly that any unknown or unidentified constituents are making substantial con-
tributions to the dissolved solids content of the renovated water. Neverthe-
less, it is apparent that there are large concentration increases for several
of these inorganic ion constituents in the renovated water compared to the
treated well water, even though at the levels encountered they do not pose a
health hazard. The listing in Table 3 of the various parameter statistics
for the wastewater, A, provides a perspective as to the possible sources of
constituents that are measured in E. Thus, for example, since the average
nitrate concentration of 3.5 mg/1 is substantially higher than the 0.15 mg/1
level in the raw wastewater, one can reasonably conclude that this is due to
biological nitrification in the lagoons of the various other nitrogen sources
in the waste effluent shown in Table 4.
The Phase 2 summary statistics in Z, A, and E for the parameters related
primarily to waste treatment are shown in Table 4. With the exception of
ammonia nitrogen, there is a considerable reduction in concentration in each
case in the E water compared to the raw wastewater, A. The relatively high
ammonia nitrogen concentration of 19 mg/1 (average) in E is of some interest
and possible concern. It probably results from biological decomposition of
organic nitrogen constituents in the lagoons, although some concentrations
measured in the raw waste are also high. The principal concern is that the
ammonia reacts with the chlorine disinfectant to form chloramines, which are
less effective disinfecting agents. However, because of this very reaction,
unless the samples are analyzed immediately, it is unlikely that with the
practice of breakpoint chlorination the ammonia should have been detected.
Subsequent to Phase 2 the ammonia in E decreased considerably, the average
concentration for 10 samples being 5.1 mg/1. This could have been a result
of an increase in nitrification in the lagoons during periods of warmer
temperature. It should be emphasized that the low bacterial counts in the
E water indicates that disinfection has not been affected. Also the pre-
sence of these concentrations of ammonia is not known to be a health hazard.
Finally, it is noteworthy that ammonia is sometimes added in municipal water
treatment plants in order to react with chlorine and form longer-lived chlor-
amines .
The organic nitrogen concentrations in the renovated water are sub-
stantially higher than in Z in Phase 2, although the average 3.4 mg/1 BOD
in E does not reflect this difference. For such measurements taken over an
18 month period, including and subsequent to Phase 2, the overall mean BOD
for E was 4.2 mg/1, still less than the 5.3 mean value for Z shown in Table
4.
19
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A summary of the results for the trace constituents in Z and E waters
during Phase 2 is shown in Table 5. It should be emphasized that the concen-
tration units here are micrograms per liter. All of the maximum concentra-
tions were well below the criterion values, except for lead in both E and Z
samples. They were not greatly different for the two waters, and the high
concentrations in each case were just about at the criterion limit. Only
in the case of iron and fluoride were the concentrations substantially higher
for E compared to Z. However, it is judged that for none of these six trace
elements were there any hazards in the renovated water, since, except for
the one high value for lead, they were well below the criterion limits.
As indicated in Table 5, all the measurements for silver, arsenic,
cadmium, chromium, and selenium were negative (below the sensitivity limits).
About half of the E samples were positive for cyanide at concentrations up
to 12 yg/1, but well below the health criterion value of 200 yg/1. It is
difficult to imagine any oxidizable cyanide being present in E because of
the large quantities of added chlorine. However, the analysis was for total
cyanide, so that the measurement may have detected such harmless combined
complexes as those involving iron, often used as an anti-caking agent, such
as in road salt. Several water samples were positive for mercury. These
occurred in three of the eight weeks, two of which were analyzed on the same
day. It is likely that in the latter cases there was contamination or analy-
tical error, perhaps as a result of the mercury preservative added to the
nitrate sampling bottle. On two subsequent weeks when this possibility was
removed, only one out of six E samples were positive (0.6 yg/1), and no Z
samples. In contrast, during the previous two weeks E, Z and all other
samples were positive and much higher. For the samples of the first four
weeks none of the E or Z samples were at concentrations higher than 0.2 yg/1,
the level of sensitivity of the method. In view of the fact that the highest
E sample was 1.7 yg/1, even though it was probably an erroneous reading due
to contamination, and the fact that the criterion value is 2 yg/1, it may be
concluded that there is not observed health hazard from mercury in the re-
novated water.
Chiller Water
Although a principal focus of this study is the possible difference in
the water quality of the renovated and treated well waters, it was recognized
that it would be useful also to analyze the plant and experimental chiller
waters during Phase 2. The results of these analyses for several macro and
trace constituents are presented in Tables 6 and 7, respectively. Although
the experimental chiller was operated, as described in Section 5, in such
a fashion as to simulate the carcass exposure to such water in the actual
plant operation, there was one important difference. In the poultry plant
the chiller was operated continuously and counter-currently, while the
experimental chiller was on a batch basis. Thus in the plant carcasses
continuously moved through the chiller at the same time that fresh water
was continuously added. In the experimental chiller a single group of 25
carcasses was added to a given volume of water. Thus the exposure of the
carcasses in these two situations and their uptake of chemicals or other
constituents would likely be somewhat different; similarly, the relative
quantities of such materials leaching from the carcasses into the chiller
water would also be expected to differ.
21
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TABLE 6. PHASE 2 ANALYSES OF CONCENTRATIONS (mg/1) OF MACRO CONSTITUENTS
IN WATER FROM PLANT (PC) AND EXPERIMENTAL CHILLERS (EC)
Parameter
Plant chiller (P_C_)_
N X S.D
X
S.D
Surfactants
Calcium
Magnesium
Potassium
Chloride
Sodium
Nitrate
Sulfate
12
12
12
12
11
12
6
6
0.22
39
3.4
37
120
80
0.89
14
0.14
9.3
4.9
11
32
26
0.96
6.8
7
9
9
9
9
9
5
5
0.047
58
3.0
25
197
95
2.7
167
0.023
7.8
0.22
6.9
89
43
1.0
53
Note
With the exception of sodium, the means for each parameter in PC and EC
are statistically different at alpha 0.05.
There are two important comparisons to make in evaluating and inter-
preting the chiller analyses. The first is that between the plant (PC) and
experimental chillers (EC), and the second between the chillers and their
corresponding water sources, Z and E. In the first case for the macro con-
stituents reported in Table 6 there are some obvious large differences in
mean concentrations, with, for example, calcium, chloride, potassium and
sulfate being much larger in EC than PC. At the same time a comparison of
these same parameters in their respective water sources, E and Z, shown in
Table 3, indicates that the calcium and sulfate concentrations are not very
different (EC vs. E, and PC vs. Z); in contrast, for chloride and potassium
the chiller waters are much higher. It is not unreasonable to conclude that
for these latter ions the carcasses themselves are probably contributing to
the chiller waters by a leaching or similar mechanism. This is confirmed by
the concentrations for these ions in the wastewater A, shown in Table 3, where
23
-------
TABLE 7. PHASE 2 ANALYSES OF CONCENTRATIONS (yg/1) OF TRACE CONSTITUENTS'
IN WATER FROM PLANT (PC) AND EXPERIMENTAL CHILLERS (EC)
Parameter
Copper
Iron
Lead
Fluoride
Zinc
Cyanide
Selenium
Plant
N
11
12
12
8
11
See
5
chiller
X
47
48
55
88
29
note b
200
(PC)
S.D
20
14
41
37
10
56
Exptl. chiller
N X
8 30
9 40
8 21
9 130
9 32
7 4.3
See note c
(EC)
S.D
11
24
7
59
24
2.7
Statistical comparisons of the means of all parameters in PC and EC show no
differences at alpha 0.05
l[n each case the analyses for the following trace elements were
below their respective sensitivity limits shown in parantheses in
mg/1. Arsenic (0.01); cadmium (0.01); chromium (0.03); silver
(0.02). Mercury: for PC, 2 of 10 samples were positive with con-
centrations of 0.4 and 1.5 yg/1; for EC, 2 of 16 were positive with
concentrations of 0.2 and 0.5 yg/1.
For cyanide in PC, 2 of 10 samples were positive with concentra-
tions of 1 and 2 yg/1.
c
For selenium in EC, 1 of 3 samples was positive with a concentra-
tion of 170 yg/1.
24
-------
they are substantially higher than those of the well water Z. These types
of interrelationships will be examined in more detail later in this section.
The trace constituents in the two chillers showed variable behavior, as
can be seen in Table 7. When comparing PC and EC, the mean concentrations
were not substantially different for iron and zinc. Copper and lead were
higher in PC, and fluoride higher in EC. Iron, fluoride and lead are much
higher in PC versus Z. Since the wastewater A is also much higher for these
elements than is Z, it is clear that the carcasses are contributing sub-
stantially to the chiller concentrations. However, of the three, this only
seems to apply to lead when making similar comparisons between EC and E.
Carcasses
Probably the most important results are those that assess any possible
differences between the quantities of constituents measurable in the washings
from chickens exposed to the normally used well water in the plant versus
those similarl> exposed to the renovated water in the experimental chiller.
These results are presented in Tables 8 and 9 for the former (PB) and the
latter (EB) for the macro constituents, waste type parameters and trace
elements. Most of the physical characteristics and inorganic constituents
measured for the Z and E water and shown in Tables 3, 4 and 5 were also
analyzed in the carcasses. The macro constituents shown in Table 8 were very
similar for PB and EB in every instance. The same applies to the waste type
characteristics of Table 9, with the possible exception of ammonia nitrogen,
which was somewhat higher in EB than PB. This could very well have been
influenced by the high ammonia concentrations in the renovated water, and
will be considered in more detail later in this section. Although five
trace constituent analyses are reported in Table 9, analyses were also per-
formed for silver, arsenic, cadmium, chromium, and selenium. However, for
each of the latter the results were below the limit of sensitivity. For
the five shown in Table 9 the mean values for PB and EB were quite similar,
with the possible exception of zinc, which was lower in EB. One can at this
point make the preliminary judgment that, for almost all the measured physical
and waste type parameters, as well as macro and trace inorganic constituents,
there are no important differences between the carcasses processed in the
plant and experimental chillers. This is, however, considered more fully
subsequently, where the statistical comparisons are discussed, as well as
the more detailed possible interrelationships of finished water, chiller
water and carcass constituents.
STATISTICAL AND OTHER COMPARISONS
Using the two-tail t-test described in Appendix A, comparisons were made
of the statistical differences of the means of many of the physical charact-
eristics, waste parameters and trace and macro inorganic constituents in Z
versus E, PC versus EC, and PB versus EB. Additionally these were also com-
pared, where appropriate, for this same set of parameters in the renovated
water during Phase 2 versus that in the earlier study of Clise (7) , and
these are shown in Table 10. Of the parameters listed there, the only means
that were statistically identical were those for total dissolved solids,
potassium, sodium and fluoride. Of the ten parameters whose means were
25
-------
TABLE 8. PHASE 2 - COMPARISON OF CAECASS ANALYSES (WASHINGS) FROM PLANT AND
EXPERIMENTAL CHILLERS - MACRO CONSTITUENTS (mg/l)
Plant birds (PB)
Experimental birds (EB)
Surfactants (MBAS)
Dissolved solids
Ca++
K+
Na+
Mg++
Cl"
wo3"
sou"
N
21
6
34
34
34
29
34
6
8
X
0.
220.
5-
17-
27-
0.
26.
0.
9-
081
3
3
9
4
57
5
20
53
S.D.
0.075
51.1
2.3
8.4
20.1
0.37
9-1
0.13
4.63
Max
0.27
320
12.0
40.6
78.6
1.5
46.4
0.44
15.0
N
16
6
34
34
34
26
34
6
12
X
0.
222.
5-
15-
24.
0-
24.
0.
8.
052
5
1
6
1
52
7
17
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0.
68.
2.
3.
16.
o.
^
0.
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032
7
1
9
9
44
1
075
43
Max
0.12
320
9-9
28.0
61.5
2.3
32.5
0.31
18.6
Note
Statistical comparisons of the means of all the above parameters in PB and EB
show no differences at alpha 0,05.
different, four were higher in Phase 2, namely calcium, sulfate, iron and
lead. There were a few differences in the nature and operation of the re-
novation system in the two studies. However, in neither study could any of
these parameters at the levels encountered be regarded as risks to health.
The results of the two-tail t-test comparions for Z versus E only in
Phase 2 are noted in Tables 3, 4 and 5. In Table 3, .for those parameters
which could be compared, only the means for turbidity, color and nitrate
were not statistically different. For those which were distinguishable,
only pH and alkalinity were lower in E compared to Z. The likely reasons for
these lower values in terms of chlorine addition were discussed earlier, as
were the sources of the other increments that would yield higher levels of
constituents in E. Of the seven waste characteristic type of parameters in
Table 4, suspended solids, BOD and grease were statistically indistinguish-
able for Z and E. The dissolved oxygen differences are not of great interest,
and the possible reasons for the others have been discussed. Of the six
trace elements shown in Table 5, fluoride, iron and manganese could be dis-
tinguished statistically, and in each case was higher in E than in Z. For
these three the higher mean concentrations in A compared to Z indicate that
the raw waste itself is contributing greatly to the increased levels in E.
26
-------
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-------
TABLE 10. A COMPARISON OF PHYSICO-CHEMICAL CONSTITUENTS OF RENOVATED WATER
(E) IN THE PREVIOUS STUDY AND PHASE 2 (mg/1)
Previous study (7 )
Constituent
Turbidity*
Color*
pH*
Dissolved solids
Alkalinity*
Potassium
Sodium
Calcium*
Chloride*
Nitrate*
Sulfate*
Fluoride
Copper*
Iron*
Lead*
Manganese*
N
54
15
207
158
101
20
19
26
162
89
23
23
26
27
8
10
X
3.5
90
6.6
335
104
11
21
46
117
31
13
0.21
0.06
0.27
0.01
0.02
S.D
1
5
1.4
129
57
9.5
15
12
53
8
5
0.13
0.01
0.19
0.01
0.02
N
35
36
30
16
15
15
14
16
12
8
8
14
14
14
15
14
Phase 2
X
1.6
3.7
5.8
389
35
15
30
54
89
3.5
150
0.15
0.038
0.057
0.024
0.0026
S.D
1.7
3.3
1.4
66
37
2.3
13
5.8
17
1.5
86
0.054
0.01
0.024
0.014
0.001
*The mean concentrations for these constituents were statistically differ-
ent at alpha 0.05; all others were indistinguishable.
Note
In both studies in almost all cases the concentrations for the following
constituents were below the sensitivity limit: cadmium, chromium, mercury,
selenium, silver, and pesticides. In Phase 2 only there were a few posi-
tive concentrations of cyanide up to 12 yg/1.
28
-------
The results of the one-tail t-test comparing Z, A and E for all the
relevant parameters listed in Tables 3, 4, and 5 are shown in Table 11. It
must be emphasized that this statistical test is a measure of which para-
meters are larger, not merely which are different. The first four relation-
ship categories listed in Table 11 correspond to waters with E parameters
larger than those for Z. However, among these categories the A concentra-
tions bear a different relationship to Z and E, reflecting, therefore,
different likely causes for the higher concentrations in E. Thus, for the
first two (A > E > Z and A = E > Z) where A is greater than or equal to Z,
the contributions from the raw waste are the probable source. For the third
(E > Z = A) other factors, involving incremental sources subsequent to the
raw waste emission, are responsible. For calcium, this can be attributed
to lime addition in the renovation system. For the fourth category (E > A >
Z) it is also apparent that there are incremental sources between A and Z.
These likely sources have been discussed previously for ammonia, chloride
and sulfate. Corrosion could account for the increase in iron.
The next four relationships, with the means for E equal to those for Z,
also each involve a different relationship to A. For the first (A > E = Z)
there is an incremental load in the waste which is reduced subsequently,
either in the lagoons or renovation system. This could also and probably
does apply to the next relationship (E = Z) for color and turbidity, but
data for A were not available. Copper was the only parameter indistinguish-
able in all three waters (A = E = Z), while nitrate was lower in A, but
equal in E and Z (A < E = Z). As discussed earlier, nitrification-denitri-
fication processes were probably in effect in the lagoon systems, and could
account for these nitrate relationships, along with decomposition of the
proteinaceous matter in the wastewater. The likely influences on pH (E < Z =
A) have been discussed.
As noted in Tables 6 and 7 for the chiller waters, PC and EC, the two-
tail t-test indicates that for the macro constituents the means were all
different, with the exception of sodium; in contrast, the five trace element
means were indistinguishable. Finally, and also as noted in Tables 8 and 9,
similar comparisons of the processed carcasses show no statistical differ-
ences between the mean concentrations of any of the macro or trace constit-
uents, with the exception of zinc, which was higher in the carcasses (PB)
processed in the plant chillers. Statistical comparisons were not similarly
made for the waste type parameters listed in Table 9, although ammonia is
higher in EC, and this has been discussed.
For the most part there are no significant differences, with the except-
ions noted, between the means of the various physical and inorganic chemical
parameters for the carcasses, PB and EB, processed'in the plant and experi-
mental chillers, in spite of such differences for the comparable constituents
in their respective water sources, Z and E, and chillers, PC and EC. The
question arises then as to why the latter exert very little, if any influence
on the constituents measured in the carcasses. It is possible that for some
such parameters any uptake by the carcasses is irreversible in that there
is sorption onto or into the flesh, without subsequent leaching in the
relatively short period in which the carcasses are analyzed by equilibration
(washing) with distilled water. To the extent that this may occur, this
29
-------
TABLE 11. RELATIONSHIPS BETWEEN CONCENTRATIONS OF
PARAMETERS IN PHASE 2 AT THREE SAMPLING
POINTS (E, Z, AND A) AS OBTAINED FROM THE
ONE-TAILED STATISTICAL TESTS (AT ALPHA 0.05)
Relationship Parameter
A >E >Z Dissolved solids, Magnesium, Manganese,
Organic-N, Potassium, Sodium, Total
solids
A = E >Z Fluoride
E >Z = A Calcium, Dissolved oxygen
E >A >Z Ammonia-N, Chloride, Iron,Sulfate
A >E = Z BOD5, Grease, Lead, Suspended solids
Zinc
E = Z *Color, *Turbidity
A = E = Z Copper
A< E = Z Nitrate
E< Z = A pH
*No data for raw wastewater, A, collected for this parameter
Note
A = Raw waste
E = Renovated water
Z = Treated well water
30
-------
could not be detected directly by the measurements performed in this study.
However, as shown in Table 11, only rarely, such as for nitrate, was the
concentration of a parameter in the raw wastewater, A, lower than that in
Z, the treated water source for the poultry processing plant. Thus, it is
reasonable to infer in most instances that instead of a net sorption there
is likely to be a net leaching from the carcasses to the chillers, and then
to the wastewater.
A more direct way to assess this question is through consideration of
the possible mass-balance relationships for the parameters analyzed in the
carcass washings relative to the chiller and related finished water sources.
By regulation of the Department of Agriculture the maximum allowable uptake
of water by the carcasses in the chiller is 12 percent. At the Sterling
plant a more typical uptake is 6 to 8 percent. The sampling procedure
involved shaking each carcass, after its exposure to the chiller, with 1500
ml of distilled water which presumably mixed or equilibrated with water taken
up by the carcass from the chiller. After the mixing, the washings were
collected for analysis. During Phase 2 the average carcass ("dressed")
weight was approximately 1.3 kg (about 2.9 Ibs). Assuming a maximum uptake
of 12 percent water in chiller, this corresponds to 156 ml (0.12 x 1300).
It can then be assumed that this 156 ml mixes completely with the 1500 ml
of the added distilled water, so that to the extent that any constituents
are present in the water taken up by the carcasses from the chillers, they
become diluted in the washings by a factor of 156/(1500 + 156) which equals
0.0942. If there is simply a dilution of the chiller water taken up direct-
ly by the carcasses, and no other factors are operating, then the carcass
washings PB and EB, should equal, for any parameter, the concentration in
their respective chiller, PC and EC, multiplied by 0.0942. Less likely, but
at least worthy of examination, is a similar possible relationship between
the finished waters and the respective carcass washings.
The results of such calculations based on this assumption of simple
dilution are shown in Tables 12 and 13. In the former, for several physical
characteristics, waste type parameters, and one metal, manganese, this was
done only for Z and E, not PC and EC, since the latter measurements were
not available. These calculated (theoretical) concentrations are shown in
Table 12 for the processing plant and experimental chiller. With the ex-
ception of ammonia in the experimental chiller, it is clear that in each
case the quantities measured in the carcasses (washings) were considerably
greater than those calculated from exposures to the respective water supplies
for the corresponding chillers. The ammonia in the carcasses in the experi-
mental chiller could indeed have been influenced by that in E water. Also,
if instead of the maximum allowable 12 percent water uptake, the more real-
istic 6 to 8 percent were used in the calculation, this would correspond to
an even larger dilution factor and lowering of the calculated value of 1.8
mg/1 for ammonia to 0.9 to 1.2 mg/1, very close to that actually measured
on the average in EB during Phase 2. In addition, the fact that for none
of the parameters listed in Table 12, including ammonia, were the mean con-
centrations for the carcasses significantly different, in spite of such
differences for five of the eight parameters in the Z and E waters, lends
further support to the conclusion that the latter waters were not making
significant contributions to the levels in the carcasses.
31
-------
TABLE 12. COMPARISONS OF SEVERAL PHASE 2 ANALYSES OF CARCASSES (WASHING)
IN PLANT AND EXPERIMENTAL CHILLERS WITH THEORETICAL CONCENTRA-
TIONS (mg/1) CALCULATED FROM THEIR RESPECTIVE WATER SOURCES,
Z AND E
Parameter
Total solids
Suspended solids
Dissolved solids
BOD
Grease
Organic-N
Ammonia-N
Manganese
(yg/D
Processing
plant
Calculated* Measured
from Z in PB
16 270
1.0 57
14 220
0.50 82
0.49 41
0.0013 5.7
0.0016 0.43
0.17 2.7
Experimental
chiller
Calculated* Measured
from E in EB
39 250
1.6 33
37 220
0.40 67
0.48 25
0.16 8.1
1.8 1.1
0.24 4.3
^'Assuming 12% water uptake by carcass in the chiller, followed by its
dilution with 1500 ml of distilled water in the measurement process. The
values thus calculated from Z and E are obtained by multiplying their
respective concentrations in Tables 3, 4, and 5 by 0.0942.
32
-------
TABLE 13. ADDITIONAL COMPARISONS OF PHASE 2 ANALYSES OF CARCASSES
(WASHINGS) IN PLANT AND EXPERIMENTAL CHILLERS WITH THEORETICAL
CONCENTRATIONS CALCULATED FROM THEIR RESPECTIVE WATER SOURCES,
Z AND E, AS WELL AS CHILLER WATERS, PC AND EC
Processing
plant
''"Calculated
Parameter
Surfactants
Calcium
Potassium
Sodium
Magnesium
Chloride
Nitrate
Sulfate
Copper
Iron
Lead
Zinc
0.
3.
0.
0.
0.
1.
0.
0.
3.
1.
2.
2.
Z
009
8
27
70
23
1
38
91
9
8
0
4
from
PC
0.
3.
3.
7.
0.
11
0.
1.
4.
4.
5.
2.
021
7
5
5
31
083
4
4
5
2
7
Experimental
chiller
^Calculated
Found
in PB
Concentrations
0.081
5.3
18
27
0.57
27
0.20
9.5
Concentrations
15
22
28
27
E
from
EC
Found
in EB
in mg/1
0
5
1
2
0
8
0
14
.004
.1
.4
.8
.29
.3
.33
0.
5.
2.
8.
0.
19
0.
16
04
5
4
9
28
25
0.
5.
16
24
0.
25
0.
8.
05
1
52
17
3
in yg/1
3
5
2
2
.7
.4
.2
.6
2.
3.
2.
3.
8
8
1
0
18
30
30
16
'''Assuming 12% water uptake by carcass in the chiller, followed by its
dilution with 1500 ml of distilled water in the measurement process. The
values thus calculated from Z, E, PC, and EC are obtained by multiplying
their respective concentrations in Tables 3, 5, 6 and 7 by 0.0942.
33
-------
This analytical approach can be made with greater assurance for the
parameters shown in Table 13 since for these, the chiller water analyses are
also available. Among all these macro and trace parameters, only the means
for zinc were statistically different in the carcasses (PB and EB); yet at
least 7 of the 12 were different between Z and E, as well as between PC and
EC. All of the theoretical values calculated for the four trace constituents
were considerably below those actually measured in the carcasses, both for
PB and EB. Among the nine macro constituents, when comparing the concentra-
tions calculated from Z, only calcium and nitrate were at levels close to
those found in PB; and only calcium, magnesium, nitrate, and sulfate were
comparable for E compared to EB. These same relationships apply when com-
paring the values calculated for PC to PB, and for EC to EB. Additionally
for the latter, surfactants are also comparable.
As was the case for ammonia in Table 12, the "calculated" sulfates of
Table 13 are about twice those actually found in EB, and this also could be
explained if the actual uptake of water from the chiller was the more real-
istic 6 percent than the maximum 12 percent permitted. The fact that the
values tabulated for calcium, magnesium, and possibly nitrate are relatively
constant in all columns would indicate that there is probably no major trans-
fer in either direction from carcass to chiller, either at the processing
plant or in the experimental chiller. For many of the other constituents
the gradual increase from calculated Z to PC, and then to that found in PB,
and the comparable increase in the experimental chiller system would lead
to the reasonable conclusion that there is a net flux or leaching from the
carcasses to the chiller, rather than the reverse. That is, these constit-
uents measured in the carcass washings are coming primarily from the car-
casses themselves, rather than the chillers; at the same time they are con-
tributing to the concentration levels in the chillers, and thereby increas-
ing them compared to those in their respective water supplies, Z and E.
Among all the constituents shown in Tables 12 and 13, perhaps only for
ammonia and sulfate in Phase 2 is there some evidence to conclude that the
transfer might have been from the water to the carcasses, at least for the
experimental chiller system. And yet, even for these parameters the PB and
EB mean concentrations were not statistically different, indicating that
these uptakes, if they were indeed occurring in the experimental chiller,
were not substantial.
ORGANIC CONSTITUENTS
A variety of measurements of gross organic parameters and specific
organic chemicals have been performed during and subsequent to Phase 2.
These include BOD^, organic nitrogen, CCE (carbon chloroform extract), halo-
genated methanes, MBAS (surfactants), pesticides, total organic carbon (TOG),
and specific organics extracted with methylene chloride and identified by
gas chromatography-mass spectrometry (GC-MS).
Analyses were performed for nine pesticides, six of which are speci-
fied in the E.P.A. Interim Primary Drinking Water Regulations (endrin,
lindane, methoxychlor, toxaphene, 2,4-D, and 2,4,5-T) and three others
(chlordane, heptachlor, and heptachlor epoxide). Samples for pesticide
analysis were collected on three separate days in a three-week period and
34
-------
the results are reported in Table 14. These analyses were discontinued when
all samples from the second and third days were negative. No positive values
were obtained for the E or Z samples, only for A, the raw waste, and PB and
PC, the carcasses and chiller water in the plant. Although some pesticides
obviously are present in the carcasses, probably as a result of their known
presence in the poultry feed, and do thereby contaminate the chiller water,
they were not detected in the renovated water and cannot be considered a
health risk were this water to be recycled into the poultry processing plant.
On four successive weeks in November and December 1976 volatile halo-
genated methanes were measured, the results being shown in Table 15. The
only organic found by this technique at quantifiable levels in the renovated
water, E, was chloroform, the maximum concentration being 3 micrograms per
liter. In some of the treated well water samples, Z, chloroform was measured
at concentrations of less than one microgram per liter, and one showed traces
of carbon tetrachloride and dibromochloromethane. For reference, the mean
chloroform concentration in finished U.S. public water supplies was reported
to be 21 micrograms per liter in the National Organics Reconnaissance Survey
(22). Although these renovated water samples are higher in chloroform com-
pared to the treated well water, they are still very low compared to the
concentrations found typically in public water supplies, and well below the
100 ppb (yg/1) limit proposed for total trihalomethanes by the EPA (26).
On two separate days subsequent to Phase 2 samples of E and Z water were
collected, extracted by methylene chloride, concentrated by distillation and
evaporation of the latter, and analyzed qualitatively by GC-MS. Although not
confirmed by comparison with knowns, the organics presumptively identified
are listed in Table 16. Another set of compounds was also found in both
types of water, namely halogenated and hydrohalogenated derivatives of cyclo-
hexene. However, research here and elsewhere has confirmed that this is an
artifact resulting from contamination of the highly purified methylene
chloride with cyclohexene. As is apparent, none of the compounds identified
and listed in Table 16 are chlorinated. The most likely source of the fatty
acids, at least in the renovated water, is the waste from the poultry, and,
therefore, is of no direct concern. In the renovated water dioctyl and
dibutyl phthalate were identified, but only the former in the treated well
water. Both of these compounds are used as plasticizers and have been widely
found in a variety of waters, including potable municipal supplies (20).
A summary of the total organic carbon (TOC) analyses of samples taken
on five successive weeks in November and December 1976 is shown in Table 17.
Each individual TOC value is a result of at least two measurements on the
same sample, the TOC being the difference between the total carbon and the
inorganic carbon analyses. Although the mean of the TOC values for the
renovated water samples at 20.0 mg/1 was higher than that of the treated
well water, 14.5 mg/1, they could not be considered different statistically
by the t-test at alpha 0.05. Nevertheless, it is apparent that there is an
increased TOC load in the system, as judged by the mean concentrations in
the first lagoon (L-^) , 50.5 mg/1; the second lagoon (C1 and C) , 35-39 mg/1;
and the microstrainer effluent (D), 39.8 mg/1. It appears that there is
some substantial reduction in the flocculation-sedimentation basin, since
its effluent (X) has a lower mean concentration of 22.4 mg/1, about equal
35
-------
TABLE 14. PHASE 2 - PESTICIDE ANALYSIS
Number of samples analyzed
Date
2/26
3/9
3/15
A
-)f
2
1
2
E
2
1
2
Z
-
1
2
PB
1
2
3
PC
1
1
-
EB
-
-
3
Samples which contained positive values for the pesticides analyzed
Pesticides Analyzed and Found (ug/l)
Pesticide
Chlordane
Endrin
Heptachlor
Kept. Epoxide
Lindane
Methoxychlor
Toxaphene
2,4-D
2,4,5-T
Sensitivity
Limit
0.2-1
0.1
0.06
0.1-0.2
0.06
0.5-1
3-6
0.05-1
0.5-1
•x-x-
Criterion Positive values
Value A PB PC
•X-X--X-
3 -
0.2 0.1 0.1
-V~ii -¥-
0.1 0.15 0.42 0.
-x-x-x-
0.1 -
k 0.06 0.14 0.
100 -
5 -
100 -
10 -
**E.P.A. National Interim Primary Drinking Water Regs. , except for:
***Proposed as above, but not adopted
to that of E. The samples from the three untreated wells, Yj, ^2 an^ Y3> are
also relatively high in TOC, as is the river water (R) downstream from the
effluent from the lagoon. Although the renovated water is relatively high
in TOC, it can be concluded that a substantial portion of the organic matter
responsible is already present in the well waters used as the water supply.
In addition, any incremental organic load is primarily due to the organic
matter from the chickens themselves, such as the fatty acids identified in
Table 16. Also as discussed previously with reference to Table 12, the
organic materials related to BOD and grease, as measured in the carcasses
PB and EB, are much higher than the values that are calculated to be contributed
36
-------
TABLE 15. HALOGENATED METHANE CONTENT OF SELECTED WATER SAMPLES
TAKEN FROM NOVEMBER 16, 1976 TO DECEMBER 8, 1976,
INCLUSIVE
Sample
C-l
C-2
C'-l
C'-2
D-l
D-2
E-l
E-2
X-l
X-2
-X--X-
Z-l
Z-2
Lagoon 1-1
Lagoon 1-2
River-1
River-2
Approximate
11/16/76
0
0
3* 2-3
1 0
<1 Trace
*
<1 0
<1 0
0
chloroform
11/23/76
0
0
2-3
Trace
0
0
0
0
0
concentration
11/30/76
0
0
Trace
0
Trace
0
0
0
(ppb)
12/8/76
Trace
0
Trace
1-2
0
0
0
0
^Denotes analysis performed by Calgon which acts as a means of
comparing the technique of the University of Pittsburgh with
that of an outside laboratory.
**This Z sample which was analyzed by Calgon showed traces of CC1. and
HCBrpClj none of the other samples analyzed by the laboratory showed
the presence of any halogenated methanes other than chloroform.
37
-------
TABLE 16. ORGANIC CHEMICALS IDENTIFIED QUALITATIVELY BY GC-MS ANALYSIS CF
METHYLATED (WITH DIAZOMETHANE) METHYLENE CHLORIDE EXTRACTS OF
WATER SAMPLES
Treated Renovated
Chemicals found* well water (Z) water (E)
Dibutyl phthalate NF +
Dioctyl phthalate + +
Methylated normal
fatty acids
C-10
C-ll
C-12
C-14
C-15
C-16
C-18
NF +
NF +
+ +
+ +
NF +
+ +
+ +
^Present (+); not found (NF)
=k]-n refers to an "n" carbon fatty acid
38
-------
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39
-------
from their respective water sources, Z and E. It is thus clear that the
substantial exposures of the carcasses to organic matter in the chillers are
mostly due to washings from carcasses previously moving through the counter-
current flow of water.
Several carbon chloroform extract (CCE) measurements were made, both
during Phase 2 and at other times. These measurements were done by the newer
miniaturized two-day sampling technique (5), for which a standard had been
proposed of 0.7 mg/1 compared to the 0.2 value for the older technique in
the 1962 PHS standards (28). In fact, it was shown in a comparison of the
two techniques that the newer method measures about 6.7 times as much CCE as
the older one (5). Over a period of approximately fifteen months, including
the Phase 2 period, the CCE analyses on the renovated water (16 samples)
shown in Table 18 averaged 0.6 mg/1, with a standard deviation of 0.4 mg/1.
For comparison, a single measurement of the normally treated well water also
had a CCE value of 0.6 mg/1. These results are consistent with the observa-
tion made above that, although apparently somewhat higher for E, there is no
statistical difference between the TOC averages for Z and E samples.
MICROBIOLOGY AND RELATED
Residual Drug Assay
The purpose of the residual drug assay was to measure antibiotic acti-
vity in the poultry, wastewater, and renovated water that could result from
the addition of such drugs in the feed of the poultry. It was ascertained
for example, that bacitracin is added at concentrations of about 5 g per
metric ton of feed.
Initially, samples were tested using Bacillus -subtilis as the test
organism and seed agar (antibiotic medium no. 1) as the test substrate. This
procedure was, however, modified to enable the detection of bacitracin and
sulfa drugs (incorporated in the feed of the chickens) by employing Bacillus
cereus as the test organism and Mueller-Hinton agar as the test substrate,
respectively. Only the samples collected on the last two sampling days in
Phase 2 were tested by the modified procedure. Samples were tested on six
sampling days in Phase 2 for wastewater, renovated water, treated well water,
experimental and plant chiller water, and carcasses. All samples were nega-
tive. That is, zones of inhibition, indicating the presence of drugs, were
not observed on the assay agar plates for any of the samples. The control
antibiotic discs showed zones of inhibition. Diameters of the zones of
inhibition ranged from 18 mm to 22 mm for 1 unit of penicillin-G per ml using
Bacillus subtilis as the test organisms, and from 29 mm to 30 mm for 1 mg of
streptomycin-sulfate per ml using Bacillus cereus as the test organism.
Subsequent to Phase 2 a variety of similar samples were tested using
Sarcina lutea, which is more sensitive to bacitracin (19) than is Bacillus
cereus. All such samples were similarly found to be negative. It may then
be concluded that either the bacitracin is metabolized and, hence, not pre-
sent in the carcass washings and wastewater, and/or this residual drug assay
is not sufficiently sensitive.
-------
TABLE 18. CARBON CHLOROFORM EXTRACT CONCENTRATIONS (CCE)
1976 week
sampled
3/8
3/153
3/29
4/19
5/18
6/9
6/15
6/23
7/2
7/16
7/28
CCE 1977 week
mg/1 sampled
Untreated well
0.58
Renovated water
0.96 4/14
1.2 6/16
1.5 6/28
0.18 6/30
0.59
0.36
0.44
0.22
0.61
0.58
0.61
CCE
mg/1
0.71
0.42
0.24
0.35
11/15
Renovated water
0.52
Arithmetic mean 0.59 mg/1
Standard deviation 0.36 mg/1
stored water
41
-------
Coliform, Fecal Coliform and Total Plate Count
The total coliform and fecal coliform results for the treated well water
(Z) were negative on five of the six sampling days tested (10 samples) during
Phase 2. The MPN results for these organisms as performed by the MHD Cumber-
land laboratory actually indicate that these were less than 2 organisms per
100 ml, and this will be cited here as negative. On one sampling day two
of the Z samples were reported as each having 2.2 organisms per 100 ml for
both total and fecal coliforms. This can occur occasionally and since it
was a single occurrence, it is not of particular concern.
The renovated water (E) was analyzed for bacteria on eight days during
and just prior to the Phase 2 period, 19 samples being taken. On six of the
eight days the total and fecal coliform results were less than 2 organisms
per 100 ml. On April 5 and 12 four E samples were analyzed. The reported
total coliform ranged from 15 to >240 organisms per 100 ml, and the fecal
coliform from 9 to >240. However, there is a strong likelihood that these
E sample results were confused with those of EC, the experimental chiller
water. The results for the latter for those two weeks were reported as neg-
ative for coliform and fecal coliform, a highly unlikely result compared to
all other EC and PC coliform analyses which were >240.
The total bacterial plate count analyses on the samples described above
indicated a maximum value of 25 organisms per ml for the Z samples and 15
for E, except for the four samples which are believed to be incorrect. For
these latter four, the values ranged from 1 to 2,000. A quite reasonable
total plate count for municipal water is 500 per ml.
For several months subsequent to Phase 2 samples of the renovated water
were taken and analyzed for coliforms and standard plate counts. Of the
59 such coliform analyses, 55 were negative (MPN <2 per 100 ml). The four
positive samples had MPN values of 3, 2.2, 3, and 8, respectively. Of the
54 samples analyzed for standard plate count, 28 were less than one per ml.
The remaining 26 had a mean standard plate count of 3.46 per ml, with a
standard deviation of 3.75.
In the earlier study of Clise (7), it was reported that for 352 samples
analyzed for bacteria, counts were obtained consistently as follows: <3
coliform per 100 ml; and <1 fecal strep per 100 ml. From this earlier study,
as well as that of Phase 2 and subsequently, it is clear that the renovated
water has low bacterial counts. The many samples consistently low in bacter-
ia also confirm the likelihood that the four high count samples in Phase 2
were a result of mislabeling of the samples, as discussed above. Also, as
will be discussed below, no renovated water samples have yielded any enteric
pathogens.
In addition, as emphasized throughout this report, in actual reuse the
renovated water would receive additional full-scale water treatment, includ-
ing disinfection with chlorine. It is thus apparent that in such reuse the
50/50 mixture of treated well and renovated water should be very low in
bacterial contamination and unlikely in that respect to constitute a risk to
human health.
42
-------
Salmonella Isolations
One of the microbiological constituents studied was Salmonella because
of its well-established occurrence in poultry and in wastewater from poultry
processing plants. Initially, using SBG enrichment and BGA media, Salmonella
organisms were isolated only from the untreated wastewater (A), the poultry
carcasses (PB), and the plant chiller water (PC) during Phase 2. Table 19
summarizes the data on the samples taken and those that were positive. A
total of 37 such isolates were identified as Salmonella and speciated as S.
enteritidis. None were isolated from E (the renovated water), Z (the treated
well water), the lagoons, C or C' (the unchlorinated and chlorinated efflu-
ents from lagoon 2 respectively), EC or EB (the experimental chiller water
and carcasses exposed therein, respectively), nor from any other point in the
water renovation system itself.
Quantitation of Salmonella by the MPN method was attempted for the
samples collected during Phase 2 using SBG as enrichment. However, even
though each morphologically different type of oxidase-negative, pink-colored
colony was picked from each BGA plate and identified, the majority of these
colonies were non-glucose-fermenters (i.e., false positive organisms), rather
than Salmonella. The large numbers of these false positive organisms made
the attempts to quantitate Salmonella by the MPN index using SBG unreliable
and irreproducible.
Another broth recommended by others (1, 9, 11) to support the growth
of Salmonella is TET enrichment. For inoculated water samples in our labor-
atory MPN indices for S_. typhimurium were significantly higher at the 95%
confidence level in the TET broth than in the SBG enrichment. Subsequent to
Phase 2 TET was, therefore, used to attempt to quantitate Salmonella. For
such water samples collected from the poultry plant the incidence of false
positive organisms was less than 50 percent. MPN indices of Salmonella
ranged from 0.04 to 7.30. For 14 of 17 Salmonella positive samples (82%),
MPN indices were <1 Salmonella per 100 ml, indicating relatively low recovery.
Although the still relatively high percentage of false positives precluded
accepting these MPN results with confidence, the sensitivity of the TET
enrichment was greater than that of SBG, as judged both by the laboratory
inoculation comparison and the fact that more samples were positive. Thus,
in contrast to the Phase 2 results with SBG shown in Table 19, using TET all
6 of the wastewater (A) samples were positive, as were 11 of 15 lagoon
samples (in comparison to 0 of 16 in Phase 2 with SBG). As would be expected,
all the finished well water (Z) or renovated water (E) samples were negative.
Salmonellae were isolated from the untreated wastewater and the first
lagoon, using BGA both with and without sodium sulfadiazine. However, they
were isolated from the second lagoon only on the medium supplemented with
sodium sulfadiazine. False positive organisms were never isolated from the
BGA medium containing sodium sulfadiazine. In contrast, 46 to 77% of the
organisms isolated from BGA not supplemented with sodium sulfadiazine were
identified as false positive organisms. These data indicate that sodium
sulfadiazine was effectively inhibiting the false positive organisms from
growing on the BGA plates.
43
-------
TABLE 19. SALMONELLA ISOLATIONS IN PHASE 2 AT
VARIOUS SAMPLE SITES USING SELENITE
BRILLIANT GREEN ENRICHMENT AND BRILLIANT
GREEN AGAR
Type of
sample
Carcass
Plant (PB)
Experimental (EB)
Chiller
Plant (PC)
Experimental (EC)
Wastewater
Raw (A)
Lagoon (various sites)
Finished Water
Well (Z)
Renovated (E)
No. of
weeks
sampled
4
4
3
3
6
3
3
7
Total
no. of
samples
13
14
3
3
12
16
12
26
Number
positive for
Salmonella*
5
0
1
0
7
0
0
0
*In each case identified by serotyping as Salmonella enteritidis,
either group B or C~.
MPN indices in 6 out of the 10 Salmonella positive samples were not
significantly different at the 95% confidence level using the EGA with and
without sodium sulfadiazine. The other four samples positive for Salmonella
had significantly higher MPN indices on the EGA medium with sodium sulfa-
diazine. It is likely that these higher MPN indices can be attributed to
the inhibition of the false positive organisms by sodium sulfadiazine, and
thus the unmasking of the Salmonella colonies of the EGA plates. These
results indicate that EGA medium containing sodium sulfadiazine is an effect-
ive plating medium both for supporting the growth of Salmonella organisms
and inhibiting the growth of false positives. Although more such data are
44
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required to prove this conclusively, it appears that this medium is effective
when used in the MF technique to determine MPN indices of Salmonella in water
and wastewater samples collected at poultry processing plants.
A summary of the results of the MPN indices for water and wastewater
samples using TET enrichment and EGA with sodium sulfadiazine is shown in
Table 20. Samples were taken on four dates subsequent to Phase 2, and none
were positive in the renovated (E), treated well (Z), or chlorinated lagoon
water (C). All of these latter samples had relatively large filtered volumes
ranging from 500 to 3000 ml. Although the MPN indices in Lagoon 1 were not
higher in each instance than those in the wastewater, there was a general
reduction from the wastewater to the unchlorinated effluent from Lagoon 2.
These results, along with those for the chlorinated lagoon effluent and
renovated water samples, both in Tables 19 and 20, indicate that protection
against Salmonella is highly effective in the overall renovation system.
Newcastle Disease Virus (NDV)
Although it is unlikely that viruses would survive the entensive chlor-
ination in the wastewater, renovation, and water treatment systems, it was
nevertheless considered important to have some evaluation of their possible
presence and survivability. In the earlier study of the renovated water at
the Sterling plant, Clise reported (7) that nine five-gallon composite samples
of the renovated water were examined for human enteric viruses and found to
be negative.
Because human wastes at the Sterling plant are segregated and not emitted
to the lagoons receiving wastewater from the poultry processing operations,
it is more likely that an avian virus could enter the renovation cycle. No
such viral diseases are known to be transmitted to humans by the gastro-
intestinal route. Nevertheless, it was decided initially to analyze carcass,
wastewater and renovated water samples for such an avian virus likely to be
present, in an attempt to assess the efficiency of the renovation process in
removing viruses.
Newcastle disease virus (NDV) was chosen. It produces an infectious,
highly contagious respiratory infection, mainly in chickens and turkeys,
although other poultry and man can contract the disease from infected birds
(3, 12, 16). Newcastle disease (or avian pneumoencephalitis) is character-
ized in birds by pneumonic and neurologic symptoms. However, in humans it
is manifested as conjunctivitis without corneal involvement, and usually
occurs only in poultry handlers and laboratory workers. Transmission (both
bird-to-bird and bird-to-man) of the virus is accomplished either through
the respiratory secretions or through the exudates, excreta and offal of
infected birds. The incidence of Newcastle disease in chickens is higher
in the fall and winter months, and the disease tends to disappear with
warmer weather. Birds recovering from infection can harbor and eliminate
NDV via the respiratory and gastro-intestinal tracts from 2 to 4 months.
Reinfection of chickens can occur with this virus. In order to prevent
transmission of this disease within a flock, the chickens are inoculated
intranasally with an attenuated, live NDV vaccine. Thus it could be detect-
able in the slaughtered carcasses or the wastewater treatment system.
45
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TABLE 20 . MPN INDICES OF SALMONELLA FOR WATER AND
WASTEWATER SAMPLES WITH TET ENRICHMENT
USING EGA WITH SODIUM SULFADIAZINE, ALL
SUBSEQUENT TO PHASE 2
MPN index per 100 ml
Lagoon 2
Sample Lagoon 1, effluent, ,
date Wastewater (A) center (L-l-M) unchloririatedCC )
11/11 4.25
3.71
11/24 0.28 0.07
12/1 21.6
40.9
12/8 1.10 0.29 0.04
0.18
Note
In addition, the following water samples on the indicated dates (and
the number of samples in parentheses) were negative for Salmonella:
Renovated (E): 11/17 (4); 12/1 (2); 12/8 (1).
Treated uell (Z): 11/11 (1); 12/1 (2).
Chlorinated lagoon (C): 12/8 (1)
NDV is a paramyxovirus that ranges in size from 70 to 120 mm. It is
somewhat resistant to adverse environmental conditions, e.g., pH (stable at
pH 2 to 12), temperature (stable at <_ 50°C), light and moisture. Conse-
quently, water can serve as a vehicle for transmission of the virus. NDV is
capable of hemagglutinating chicken, guinea pig or human type 0 red blood
cells. In the laboratory it is grown primarily in eggs by inoculation of
the allantoic cavity; however, it has been grown also in primary chick embryo
(CE) cells. Although NDV has also been grown in continuous primate cell
cultures, e.g., HeLa and monkey kidney, a lower titer of virus is produced
in the primate cells than in CE cells. The cytopathic effect (CPE) produced
by NDV in cell cultures is characterized by syncytium or giant cell forma-
tion. Infected cultures can also show hemadsorption when guinea pig or
chicken red blood cells are added.
NDV in Water and Carcass Samples—
Initially during Phase 2 attempts were made to detect NDV in the water
46
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and carcass samples by inoculating chick embryo cells and using a plaque
assay. However, other avian viruses that are capable of growing on primary
chick embryo cells and forming plaques could have been detected in the
samples. Several such samples collected on one day were inoculated on com-
plete monolayers of primary chick embryo cells. After 4 days of incubation,
cells were stained for plaques. Plaques were not observed on any of the
cells inoculated with the water and carcass samples. The positive control,
NDV at the 10~ dilution, produced too many plaques to count on the chick
embryo cells and thus had a titer of >^ 10" PFU/ml.
Since avian viruses were not detected in any of the water and carcass
samples by inoculating chick embryo cells, tissue specimens were subsequently
taken for analysis for the presence of avian viruses by a hemagglutination
test. The isolation and identification of NDV was attempted, since the
chickens were inoculated intranasally with an attenuated, live NDV vaccine.
If, however, it could not be isolated in the slaughtered chickens, it is
unlikely that it would be detected in the wastewater or renovation system.
Tissue extracts were prepared from 5 spleens, 5 livers and 5 lungs from
chickens obtained from the Sterling plant, and inoculated into the allantoic
cavity of eggs to permit avian viruses (if present in these tissues) to
replicate to a high titer. Serial 2-fold dilutions (to the 1:2048 dilution)
of the allantoic fluids were prepared and employed in hemagglutination tests.
No agglutination of the chicken red blood cells was observed with any of the
15 tissue extracts. The NDV positive control produced hemagglutination
titers of 4. The influenza-A virus controls produced hemagglutination titers
of 64 and 16. The decrease in the hemagglutination titer of influenza-A
virus was attributed to the virus undergoing one freeze-thaw between the two
tests which lowered the virus titer.
Although extensive testing was not performed, neither NDV nor other
avian viruses that would have been capable of growing on primary chick embryo
cells were thus found in the water, wastewater, or carcass samples, and NDV
was not detected in the tissue extracts. Since NDV is the most likely avian
virus of possible concern, however remote, in the renovation system, attempts
to isolate and identify viruses at the Sterling plant were discontinued.
NDV Survival in Lagoon Water—
Since NDV could not be detected in the carcasses, wastewater, nor at
any point in the wastewater treatment or renovation system, it was decided
to study its survival in lagoon water in the laboratory. The purpose was
to obtain an indication of the effectiveness of one important stage in the
overall treatment process not specifically designed to remove viruses, but
which would be expected to have an effect. The disinfection processes them-
selves would certainly further reduce the concentrations of any viruses.
Although the stability of NDV in the wastewater treatment lagoons would not
necessarily be indicative of the behavior of other viruses, if it were re-
latively unaffected by the lagoon, then this would indicate that disinfection
with chlorine and other stages of treatment in the renovation system might
have to be relied on to be confident of virus removal.
47
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The laboratory NDV survival experiments measured the possible effects
of light and temperature in lagoon, filtered lagoon, and distilled waters.
The flasks, inoculated with NDV, were exposed either to white fluorescent
light or kept in the dark, in each instance either at 25°C or 7°C. The
flasks were gently agitated and samples taken daily for several days and
assayed for NDV.
The initial results showed some differences, but not consistent, in
survivability in the light and dark, and a considerably higher rate of die-
off in lagoon water at 25°C compared to 7°C. More extensive experiments
confirmed that there is a temperature effect, although not quite as large,
and typical results are shown in Figure 2 and Table 21. In the former the
survival as a function of time is shown in the three types of water in light
and at 7°C only. The one-day titer for the lagoon water (LW) in Figure 2
is off scale because it is a few percent higher than the initial 100 percent.
This is likely due to analytical variability. Table 21 summarizes only the
5-day survival data.
Although the survival curves shown in Figure 2 are not smooth, and the
differences due to the experimental variables not consistent, some conclus-
ions, although not necessarily firm, can be made. There is no consistent
difference between the results in the light and dark, or in the filtered
and unfiltered lagoon waters. The survival, at least over the full five
days, is generally greater in distilled water than in either of the lagoon
waters. And finally, the survival is consistently greater, although of
varying relative magnitudes, at 7°C than at 25°C.
The time for 50 percent die-off in the lagoon waters varied from about
1 to 3 days at 25°C, and from 2^ to 5 days at 7°C. The survival curves,
plotted semi-logarithmically in Figure 2, are not generally linear and may
not follow first-order kinetics. Nevertheless, with the assumption that
they do, one can estimate NDV survival in the lagoon system. For the
approximately 15-day residence time in the two lagoons, the 5-day half-time
would correspond to a survival of 0.125, and the 1-day half-time to 0.00003.
To the extent that these laboratory experiments simulate accurately the NDV
behavior in the lagoons, it is apparent that there is significant die-off,
and that the disinfection and other stages in the water renovation process
may not have to be relied upon as the only protection against possible virus
contamination in the reuse of wastewater at the Sterling plant.
STEADY-STATE CONSIDERATIONS
An important question for any wastewater recycling process is the extent
to which the concentrations of waste constituents can build up. The steady-
state mass balance equations for such possible build-ups have been developed
for the Sterling Plant system in Appendix B, which also considers in some
detail the extent to which the Phase 2 flow regime, without actual recycle,
can be used to predict the steady-state concentrations that would occur with
recycle and reuse of the renovated water. As long as the process does not
involve a closed loop (no blow-down or partial diversion to receiving waters),
or there is some removal in treatment, mass balance considerations indicate
that a steady-state, rather than an infinite build-up will occur. In the
48
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TABLE 21. SUMMARY OF NDV LABORATORY SURVIVAL EXPERIMENT, PERCENT SURVIVING
AFTER 5 DAYS
Water type
Percent survival
7°C
Light Dark
25°C
Light Dark
Lagoon
Lagoon, filtered
Distilled
26 15
13 18
49 32
7 8
0 17
17 22
ioa
G
z
90
80
70
60
SO
30
z
UJ 2O
a
K
IU
a.
10
XLW
*LW-F
2 3
DAY
Figure 2. NDV survival at 7°C in light in laboratory study, (DW: distilled
water; LW: lagoon weter: LW-F: lagoon water, filtered)
49
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Sterling Plant partial removal in two modes can occur. The planned 50/50
mixing will result in at least half of the waste constituents generated in
any one cycle being emitted to receiving waters. The lagoon treatment and
the renovation system can also reduce the build-up. For this combination of
removals, and using mass balance, one can estimate the steady-state concen-
tration in the 50/50 renovated water, Cs, of the daily waste load, A, of a
parameter emitted from the plant. Such a relationship was developed in
Appendix B (Equation 5) and can be written as:
C = (A/V)(bc)/(2-bc) (1)
s
with V the daily volume of water used, and the fractional removals of the
constituent in question being (1-b) in the lagoons and (1-c) in the renova-
tion system. The ratio A/V is also simply the concentration of a constituent
in the waste, C^. The fractional concentration remaining in the renovated
water (prior to mixing with the well water) compared to the average concen-
tration in all waters discharging into the first lagoon is be. It should be
noted that Equation 1 assumes that the concentration of the constituent
emitted as a waste from the poultry processing plant is zero in the well
water supply, and is not added or removed in the normal water treatment
received by the 50/50 mixture of renovated and well water.
Aside from being able to calculate the steady-state concentration in
the water supply in actual recycle using Equation 1, the question arises
also as to whether the steady-state recycle of Phase 2 is a reasonable sim-
ulation of actual recycle into the poultry plant. As shown in Figure 1, in
Phase 2 the renovated water effluent was recycled into the first aerated
lagoon, whereas with reuse it would be mixed 50/50 with the untreated well
water, the mixture then to receive normal treatment. Also in Phase 2 the
flow through the lagoons was about one-third higher than it would be in
actual recycle, with the flow through the renovation system being about one-
third of that through the plant (compared to one-half in actual recycle).
This results in a higher dilution of waste constituents emitted to the lagoon
and cycled through the renovation system in Phase 2. However, in actual re-
cycle the 50/50 mixing with well water will tend to reduce its steady-state
concentration compared to that in Phase 2. Combining these factors in a mass
balance calculation permits a comparison of the steady-state concentrations
likely to be achieved in actual recycle (after 50/50 mixing with well water)
to that of the renovated water in Phase 2, the ratio of these two being rs.
In Appendix B Equation 20 expresses such a relationship for this ratio rg
as follows:
r = (l/3)(4-bc)/(2-bc) (2)
S
Thus, this ratio is highly dependent on the fractional removals in the
lagoon and renovation systems. With high degrees of removal (be approaching
zero) rs will approach 2/3. However, with very low fractional removal (be
not much less than unity), the ratio will approach 1.0. Thus, one can pre-
dict that, as a first approximation, the steady-state concentrations of the
50/50 renovated water mixture in actual recycle will, depending on the waste
constituent, be 0.67 to 1.0 times those observed in the renovated effluent
50
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in Phase 2. Other factors will modify the actual result, such as the fact
that the 50/50 mixture in actual recycle will undergo further treatment, or
the well water itself can contain a significant concentration of the con-
stituent in question. Nevertheless, the Phase 2 study may be taken as a
reasonable predictor for full recycle and reuse.
One could estimate be for a given parameter from a knowledge of its con-
centration in A and E during Phase 2, and this would be simply Cg/C^. How-
ever, because the flow regime in the lagoons in Phase 2 is different than it
would be in full recycle, this estimate could be incorrect and, therefore,
misleading. In Phase 2 the actual volume of flow into and through the
lagoons consists of three parts of waste A from the poultry plant and one
part of renovated water E, so that the effective concentration of a consti-
tuent discharged into the first lagoon is (3C^ + Cg)/4. Thus, during Phase 2
the actual fractional concentration of a constituent remaining in the renova-
ted water after moving first through the lagoons, and then the renovation
system, is simply G£ divided by this expression for the effective concentra-
tion, namely:
be = 4CE/(3CA + CE) (3)
A useful constituent to demonstrate these relationships is potassium,
since, as shown in the Phase 2 results of Table 3, it is quite low in the
treated well water, Z, it is found in much higher concentrations in A, and
is then significantly reduced in concentration as it appears in the renovated
water, E. For the purposes of this calculation we will neglect its concen-
tration in Z, and take C^ and CE as 32 and 15 mg/1, respectively. Using
Equation 3, the calculated value of be is 0.54. In contrast, the approximate
estimate of be, simply using CE/C^ is 0.47, about 13 percent lower than the
correct value. Having calculated be for potassium, one could then use
Equation 1 to calculate its expected steady-state concentration, after 50/50
mixture, in actual reuse, and this value, Cs, is 11.8 mg/1. Alternatively
one can use Equation 2 to calculate rs, the ratio of potassium expected in
the 50/50 mixture in actual reuse compared to that in E in Phase 2. Again
using the 0.54 value for be, rs is calculated to be 0.79. As a check on
the self-consistency of these equations, Cs, calculated from Equation 1,
divided by CE is 11.8/15 which equals 0.79.
These calculations can be modified readily, as discussed in Appendix B,
to meet a variety of circumstances, even for such a constituent as sulfate,
which, as shown in Table 3, seems to be generated in large concentrations
within the renovation system itself probably from alum. To the extent that
this is so, one can still estimate Cs for sulfate. Neglecting the contribu-
tions of sulfate from the treated well water and the poultry processing
plant, and assuming that there is no reduction of its concentration in the
lagoons, this is equivalent to the statement that be equals unity. Also, if
150 mg/1 sulfate were to be generated in the renovation system in actual
reuse, as we are assuming it is in Phase 2, this would result in Cs also
being 150 mg/1, since this concentration would ultimately return to the
renovation system, the total leaving it then becoming 300 mg/1. This then
would reduce to 150 mg/1 (Cs) after 50/50 mixing with well water. Thus,
51
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even where constituents are generated within the renovation system because
of chemical addition, and with no fractional removal elsewhere, there would
be only a finite build-up, and a predictable one.
Another important question concerning the Phase 2 study and actual re-
cycle is the rapidity with which steady-state is achieved. As discussed in
Appendix B, this is dependent principally upon the fractional removal in the
lagoon and renovation system, and, hence, be. Using Equation 15 of Appendix
B, one can calculate the concentration ratio in the 50/50 mixture achieved
after "n" cycles, Cn, compared to that in the steady-state, Cs, using the
flow regime contemplated in actual reuse. Doing this for 3 cycles (n=3),
one obtains values of C^/CS equal to 0.91 and 0.998 for be equal to 0.9 and
0.25, respectively. It is, therefore, apparent that, even over a wide range
of fractional removals, steady-state is approached rather rapidly. Although
this calculation does not apply exactly to Phase 2, which is a different flow
regime, it can be used as a first approximation for it. In the Sterling
system the residence time in the lagoons determines the time for a cycle.
This is normally approximately two to three weeks. Because of the increased
flow through the lagoons in Phase 2, this is probably reduced by about 25
percent, perhaps to two weeks. Phase 2 itself covered a period of seven
weeks, with a two-week preliminary sampling period. In addition, the pre-
ceding Phase 1 period involved several additional weeks with the same flow
regime. It can, therefore, be concluded that the Phase 2 study was in steady-
state, and its results can be used to estimate concentrations in the renovated
water in actual reuse.
52
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SECTION 7
DISCUSSION
The focus of this discussion will be whether the quality of the renova-
ted water, E, is sufficient to justify its reuse in processing poultry at the
Sterling plant, without risking the health of the consumers. It should be
emphasized that, prior to actual use in the plant, the renovated water will
be mixed 50/50 with the untreated well water, then receive the normal water
treatment that is currently utilized for it, including two stages of chlorina-
tion in addition to that received by the lagoon and renovation system efflu-
ents. Since the chiller is probably the single most important water exposure
of the birds in the processing plant, it is necessary to consider not only
whether there is a possible build-up of contaminants in the renovation system,
but also whether such concentrations cause a substantial transfer of such
materials to the "dressed" birds or carcasses; and, if so, does this con-
stitute a health hazard to the consumers of the chickens. Thus, not only
the quality of the renovated water, but also its intended use must be con-
sidered in making the judgment whether to proceed to a trial period of reuse
and, ultimately, to such reuse on a permanent basis.
Before reviewing the findings that would help answer these questions,
it is important to note that the analysis of the mass-balance steady-state
relationships indicates that the flow regime in Phase 2 is such that the
concentrations of constituents measured in the renovated water, E, can be
regarded as reasonable predictors of those that can be expected in the 50/50
treated mixture in the steady-state during actual recycle. For a given para-
meter the exact ratio, rg, between these concentrations in such reuse com-
pared to E in Phase 2 is dependent on several factors, not all of which have
been determined for every constituent. The likely maximum values of rg can
be estimated, and it is unlikely that any of the constituents studied could
build up to hazardous and unpredictable levels. Certainly the careful moni-
toring program of the proposed trial period of reuse would quickly discover
this unlikely event. Even if there were no removal in the lagoons or renova-
tion system, the maximum steady-state concentration, Cg, in the planned 50/50
reuse flow regime (with the waste from the processing plant itself being
the only source of a given constituent) would simply be equal to its concen-
tration in the plant wastewater without any recycling. Similarly, where
either the well water or renovation system were to be unique sources, Cs
would equal its respective concentration in the absence of any recycling.
This apparent lack of build-up is in fact due to the 50/50 dilution flow
regime. It indicates, for example, that if a contaminant were to get into
the well water source and not be removed subsequently at any stage of treat-
ment, it would never reach a concentration in the renovated 50/50 mixture,
53
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Cs, higher than that in the untreated well water itself. This would similar-
ly apply to concentrations generated in the water renovation system, such as
due to the addition of chemicals like alum. This possible situation, with
sulfate as the example, was discussed in Section 6.
It was also shown that although dependent on the removal factors, steady-
state is approached very rapidly. For the practical purpose of using Phase
2 as a predictor for reuse, and using the Phase 2 two-week residence time
in the lagoon system as the determinant of the time for a single cycle, it
was judged that steady-state was achieved.
The quality of the renovated water itself, and its comparison with the
treated well water and any appropriate water quality standards are of immed-
iate interest. Although the renovated water is not intended to be utilized as
a drinking water supply, it is required by the U.S. Department of Agriculture
to be potable and to be so certified by the appropriate governmental agency.
Prior to June 1977 the standards most commonly used for this purpose in the
United States were the 1962 U.S. Public Health Service (PHS) Drinking Water
Standards (28), which include both primary standards with mandatory, health
related constituent limits, and secondary ones with recommended limits,
principally related to esthetic concerns. The newer U.S. Environmental Pro-
tection Agency (EPA) Interim Primary Drinking Water Regulations, effective
in June 1977 (25) , and the proposed EPA Interim Secondary Drinking Water
Regulations (26) are very similar to, but not identical to the PHS standards.
These various standards address many constituents measured in this study and
both the treated well water and the renovated water quality were compared to
them.
With regard to such parameters the Phase 2 results indicate that in
almost every instance both the renovated (E) and treated well (Z) waters,
as shown in Tables 3 and 5, were well below the EPA and PHS criterion limits.
The newer EPA turbidity limit of one unit was frequently exceeded in both
of these waters during Phase 2, but their respective maximum values were
well below 5 units (the PHS limit), which is permitted by the EPA if there
is no interference with disinfection or microbiological determinations. The
fact that there were acceptably low bacterial levels indicates then that the
turbidity levels are also acceptable. It should also be noted that there
was no statistical difference between the mean values for turbidity in Z
and E water. Sulfate values in the renovated water were high, but only one
measurement marginally exceeded the recommended (not mandatory) secondary
criterion of 250 mg/1. The only other marginally high constituent was lead
which, however, had almost identical mean concentrations in the Z and E
waters, and both had maximum concentrations at the EPA criterion limit of
50 jJg/1. Because the mean concentrations of both waters were about one-half
of this limit, one can conclude that the lead levels cannot be regarded as
hazardous. In any event the much higher lead levels in the chiller water
in the plant, and in its effluent, along with the washings from the carcasses,
indicate that neither the Z nor the E waters in Phase 2 constituted a sub-
stantial source of lead to the processed birds. The renovated water quality
in the previous study (7), shown in Table 10, also met these various standards
with one or two exceptions. The turbidity was higher than in Phase 2, but
still acceptable on the basis discussed above. The color of the water was
54
-------
very much higher than in Phase 2, and well above the proposed EPA secondary
limits. Thus the Phase 2 results constitute an important improvement. The
iron levels in the renovated water were also much higher in the earlier study,
the mean concentration of 0.27 mg/1 approaching the EPA secondary limit of
0.3 mg/1. However, the Phase 2 mean concentration of 0.057 mg/1, although
higher than 0.019 mg/1 in Z, also represents a significant improvement. Also,
as with lead, the evaluation of iron levels in the carcasses showed that
there were no substantial contributions from the Z and E waters. One can
conclude, therefore, that for those macro and trace inorganic chemicals, as
well as other water characteristics for which drinking water standards are
available, the renovated water in the Phase 2 study, as well as the treated
well water, was of acceptable quality.
In addition to those constituents in the renovated and treated well
waters addressed by the standards previously discussed, other measured inor-
ganic constituents and waste parameters in many cases, as would be expected,
were substantially higher in the renovated water as compared to the treated
well water in Phase 2. Thus, the macro inorganic ions, other than nitrate,
all increased to the point of raising the total dissolved solids level in
E to an average concentration of 389 mg/1 (below the 500 mg/1 EPA secondary
guideline) compared to 143 in Z. However, this increase, as shown in Table
3, can be attributed essentially to the macro inorganic cations and anions,
and these are of no human health significance at the levels encountered. It
is, therefore, unlikely that any unidentified constituents are making sub-
stantial contributions to this increased quantity of total dissolved solids
in E. As noted in Section 6, the high average ammonia concentrations, 19
mg/1 in E during Phase 2, were substantially lower at 5.1 mg/1 subsequently.
Although the ammonia concentrations in the carcasses were somewhat higher
for those exposed to E versus Z, and the data suggest that indeed the ammonia
in E was probably responsible, the mean concentrations in PB versus EB were
not significantly different. It is highly unlikely that these levels con-
stitute a health hazard.
The organic chemical quality of the renovated water at the Sterling
plant remains perhaps the greatest area of possible concern, as it does for
the municipal water supply systems of the U.S., principally because of the
recent advances in the technology to identify and quantify trace organic
chemicals at very low concentrations, as well as their possible influence on
chronic disease in humans. The measured gross organic parameters, namely
BOD, CCE, TOG, and organic nitrogen, are of interest as indicators of speci-
fic organic constituents. The much higher organic nitrogen in the renovated
water most probably reflects the proteinaceous material and its breakdown
products from the poultry, but only those constituents that are not readily
biodegradable, since the BOD values for the renovated and treated well waters
were quite comparable. The mean BOD value of 3.4 mg/1 for the renovated
water is not untypical of many raw surface waters that are used for municipal
water supplies, such as Minneapolis and St. Cloud, Minnesota (17).
The mean TOC values for the renovated and treated well water, 20 and
14 mg/1, respectively, are high, but not uniquely so. The statewide average
for the major Minnesota rivers is 20 mg/1, with several large municipal
supplies utilizing them as raw water supplies (17). Similarly, most of the
55
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larger rivers there have concentrations of 15 to 30 mg/1. In a study of 80
municipal water supply systems of the U.S., non-volatile TOC concentrations
as high as 19 mg/1 were measured in the raw water and 12 mg/1 in the finished
water (22). About 98 percent of the latter were less than 5 mg/1. This
indicates that, in terms of a finished water supply, the renovated water
TOC values are undoubtedly high. However, after mixing 50/50 with the raw
well water and then full-scale treatment, the final mixture should not be
significantly different in TOC than the currently used treated well water.
The CCE measurements of the finished water show considerable variability.
The mean concentration in E over a 15 month period, 0.59 mg/1, is about equal
to the single value of 0.58 mg/1 measured in Z. However, time variations,
seasonal or otherwise, are also common in finished municipal water supplies
(5). Similarly, concentrations of 1.0 mg/1 of CCE are not uncommon in
finished water supplies, and concentrations considerably higher than this
have been found in rivers. For reference, 0.7 mg/1 has been proposed re-
cently (5) as a criterion value for public water supplies. Finally, it
should be noted that the gross organic parameters BOD and organic nitrogen
had very similar concentrations in the carcasses exposed, respectively, to
the normally treated well water and the renovated water. Also in both cases
the calculated contributions of these constituents to the levels actually
found in the carcasses were very small indeed, namely 2 percent or less.
Thus, there is no significant detectable increase in gross organic contamina-
tion of the carcasses or the renovated water itself in Phase 2, compared to
the treated well water and the carcasses exposed to it.
Several specific organic chemicals have been identified, and some quanti-
fied in this study. Pesticides were not found in either the renovated or
treated well water. Surfactants in the former were well below criterion
Levels. Several fatty acids were found in the renovated water, but also
Ln the treated well water. In any event, these constitute no human hazard.
The maximum concentration of the only halogenated methane found regularly
in the renovated water, chloroform, was three micrograms per liter, well
below the approximate median value of 20 found in the EPA National Organic
Reconnaissance Survey of U.S. public water supplies (22). Two phthalates
were found in the renovated water, and one of these in the treated well
water. As noted previously, both of these, widely used as plasticizers, have
been found in potable U.S. municipal water supplies, as well as many natural
waters.
It should be emphasized, however, that there have not been extensive
analyses of specific trace organics utilizing a variety of concentration,
separation and detection methodologies. Nevertheless, the measurements
that were performed on the water did not uncover any concentrations of
potentially toxic chemicals, at least at concentrations likely or known to
cause human disease. The specifically identified organic chemicals arising
from this waste renovation process do not constitute a human hazard were
this water to be used as contemplated in full recycle. One might neverthe-
less raise the question of the possible health hazard from organics not yet
identified. It is unlikely that, in terms of the reuse of this water for
processing poultry, such organics would be hazardous, since they arise pri-
marily and originally from the poultry wastes and are likely to be only
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natural materials and their degradation products. A possible question con-
cerns the reaction of chlorine with these materials to form hazardous by-
products. This concern is difficult to address. It should be pointed out,
however, that chlorination is practiced in certain food processing, and has
not been associated with any ill effects in humans.
Before leaving the question of the possible impact of various chemicals
on the carcasses, the findings on the possible relationships between their
analyses in Phase 2 relative to their respective water supplies and chiller
waters should be reviewed. Among all the inorganic constituents and physical
parameters measured, there were in almost every case no statistical differ-
ences between carcasses processed in the plant and experimental chillers.
Zinc was higher in the plant, and ammonia in the experimental chillers.
Furthermore, only for a few inorganics, and none of those with health related
criterion limits, did there seem to be any possible impact on the carcass
analyses. Such comparisons indicate, for the most part, that the movement
of such constituents in the chiller system is from the carcasses to the chiller,
rather than vice versa. This is certainly the reason for their presence in
the waste effluent from the plant at substantially higher concentrations
than in the treated well water. This additional perspective on the chemical
water quality must be emphasized because, in the last analysis, the critical
question is indeed, not whether the water meets particular standards, even
though it may and in this instance does, but whether it has any impact on
the quality of the processed chickens and the health of the consumer. A
careful analysis of the Phase 2 data and their interpretation indicates that
there is no basis for concern about the quantities of specific chemicals
identified in the carcasses. The only remaining uncertainty, however small,
is the trace levels of chlorinated organics, so far undetected, that might
be formed in the renovation system. However, this could be ascertained with
additional study, either prior to or during a Phase 3 trial period of reuse.
The low bacterial counts in the renovated water, as well as the measure-
ments showing the absence of specific pathogens such as Salmonella, demon-
strate that bacteria from the water supply do not constitute a risk. The
presence of a variety of such bacteria in the plant chiller water or car-
casses is certainly not unusual, and they have been shown to build-up rapid-
ly in the water as the poultry contact it (10). Newcastle Disease Virus
that might be expected in the poultry or wastewater could not be found in
either. The laboratory die-off experiments with this virus using lagoon
water from the Sterling plant indicated that one can expect substantial viral
removals in the aerated lagoon system. In view of the approximately two-
week's detention time in the aerated lagoons and the nature of the disin-
fection processes subsequent to them, which involve two stages of chlorina-
tion, this excellent microbiological quality is to be expected. With actual
recycle into the plant, this high quality and the additional treatment,
including disinfection, would insure, with a high degree of certainty, that
there would be no danger from pathogenic organisms in the reuse of this
renovated water.
An evaluation of many of these results was performed immediately follow-
ing completion of Phase 2 by a committee constituted to do so and make a
recommendation about proceeding to Phase 3, a three-month trial period of
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full recycling through the plant and water reuse. That committee did not at
the time have access to some measurements of organic water quality which
were done subsequent to Phase 2, including most of the CCE analyses, and all
of the TOG and specific organics, other than pesticides. The committee
recommended that there was no significant risk in proceeding to Phase 3. The
additional data on organic chemical quality should not modify that judgment.
Nevertheless, the final decision to proceed to Phase 3 was not and has
not been made as of this writing. The delay arose because of the require-
ment of the Department of Agriculture that the water to be reused in the
plant be designated as potable. There are differing opinions as to whether
it could be so regarded. The concerns center around two areas. First, is
the chemical and microbiological quality of the renovated water sufficient
to meet criteria of potability? In terms of meeting constituent limits
specified in drinking water standards or regulations, the answer is yes.
The gross organic load is high, but not much more so than the normally treated
well water. Nevertheless, a certification of potability has been made by
the legally authorized agency, the State of Maryland.
The second area of concern related to potability is the nature of the
raw water source. A long-standing concept, as stated in the 1962 Public
Health Service Drinking Water Standards (28), is the following:
"The water supply should be obtained from the most desirable
source which is feasible, and effort should be made to pre-
vent or control pollution of the source. If the source is
not adequately protected by natural means, the supply shall
be adequately protected by treatment."
In terms of the intended goal of this water renovation system, namely the
augmentation of the limited non-community well water source for the Sterling
plant, other possible available sources should be considered, using the above
concept. The local community, Oakland, will not and cannot provide additional
water to the Sterling plant. A detailed analysis on the specific current
water usages at Oakland, projected population growth, and water treatment
plant capacities, indicate that any substantial commitment of water to the
poultry processing plant could not be met (14). The only other possible
source is the Little Youghiogheny River, often not more than a small creek,
polluted immediately upstream by raw, municipal sewage from Oakland. The
renovated water, studied in Phase 2 meets the criterion of being the most
desirable, feasible raw water source, and will receive the additional full-
scale, normal water treatment during the Phase 3 trial period of reuse.
Another relevant and similar statement on the nature of the water source
may be found in Appendix A of the E.P.A. Interim Primary Drinking Water Re-
gulations, which discusses the concepts and rationale used in their develop-
ment (25):
"Production of water that poses no threat to the consumer's
health depends on continuous protection. Because of human
frailties associated with protection, priority should be
given to selection of the purest source. Polluted sources
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should not be used unless other sources are economically
unavailable, and then only when personnel, equipment, and
operating procedures can be depended on to purify and other-
wise continuously protect the drinking water supply."
In addition to the criterion of best available source already addressed, this
statement specifically focuses on economics and dependability. That this
renovation system is indeed economical has been shown in a previous report
(7). Sufficient data have been accumulated to demonstrate its dependability,
and experience with such seasonal problems as freezing and overturns of the
lagoons have indicated conditions when it should not be used to supplement
the well water supply.
Nevertheless, one can and perhaps should put aside the legal question
of potability and consider the following. If this renovated water were to
be recycled into the plant as described and used as intended, would there
be any significant, discernible risk to the consumers of the chickens pro-
cessed there? It may be concluded, reasonably, after weighing all the results
of this study and that which preceded it, that the Phase 3 trial period of
full recycle and reuse, if instituted, would not jeopardize the public health.
The basis for this judgement may be summarized as follows:
1) There are no apparent quantities of chemicals or micro-organisms in
the renovated water harmful to human health, even if the water were
directly consumed, which is not the intention.
2) The wastes generated within the chiller from the carcasses them-
selves, and to which subsequent ones are exposed, constitute a much
greater source of contaminant exposure than does the renovated
water.
3) The only possible likely source of exposure not identified that
might cause concern is the category of chlorinated organics genera-
ted by disinfection of the poultry wastes. Yet chlorination of
chiller water is commonly practiced and is not known to be hazard-
ous to humans.
The question of using judgement rather than regulations to make a dec-
ision in this instance is probably the central issue. The federal agencies
involved in the decision were rightly concerned about the possible precedent
in approving this system for reuse. Aside from questions about specific
chemicals that might be harmful, the probable underlying concern was that
approval would be a precedent for the direct use of wastewaters for reuse
before being able to fully develop criteria more stringent and elaborate
than we now have for our natural waters used as the raw water source. Never-
theless, in the absence of such criteria careful judgement can and should
allow Phase 3 to proceed. In view of the urgent need to conserve our water
resources, limit waste discharges, and improve water quality, the nation will
have to proceed to selective reuse of wastewater. Such a project as this is
a useful step in that direction.
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REFERENCES
1. American Public Health Association. Standard Methods for the Examina-
tion of Water and Wastewater, 14 th ed., Washington, D. C., 1976. 1193 pp.
2. Bellar, 1., and J. J. Lichtenberg. Determining Volatile Organics at
Microgram-per-litre Levels by Gas Chromatography. J. Amer. Water Works
Assoc., 66:739-744, 1974.
3. Blester, H. E. , and L. H. Schwarte. Newcastle Disease. In, Diseases
of Poultry. The Iowa State University Press, Iowa. 1959. pp. 464-503.
4. Blankenship, L. C., and N. A. Cox. Modified Water Rinse Sampling for
Sensitive, Non-adulterating Salmonellae Detection on Eviscerated
Broiler Carcasses. J. Milk and Food Technology, 39:680-681, 1976.
5. Buelow, R. W., Carswell, J. K., and J. M. Symons. An Improved Method
for Determining Organics by Activated Carbon Absorption and Solvent
Extraction - Part 1. J. Amer. Water Works Assoc., 65:57-72, 1973.
6. Carawan, R. E., W. M. Crosswhite, J. A. Macon, and B. K. Hawkins.
Water and Waste Management in Poultry Processing. EPA-660/2-74-031,
U. S. Environmental Protection Agency, Washington, D. C., 1974.
7. Clise, J. D. Poultry Processing Wastewater Treatment and Reuse.
Environmental Protection Technology Series, EPA-660/2-74-060.
U. S. Environmental Protection Agency, Washington, D. C., 1974. 52 pp.
8. Edwards, P. R., and W. H. Ewing. Identification of Enterobacteriaceae,
3rd ed. Burgess Publishing Co., Minneapolis, Minn., 1972. 362 pp.
9. Ewing, W. H., and W. J. Martin. Enterobacteriaceae. In E. H. Lennette,
E. H. Spaulding and J. P. Truant (ed.). Manual of Clinical Micro-
biology. American Society for Microbiology, Washington, D. C. 1974.
970 pp.
10. Hamza, A., S. Saad, and J. Witherow. Water Reuse in Poultry Processing.
In: Proceedings of the Eighth National Symposium on Food Processing
Wastes. EPA-600/2-77-184, U. S. Environmental Protection Agency,
Washington, D. C., 1977. 411-426 pp.
11. Harvey, R. W. S., and T. H. Price. Isolation of Salmonellas. Public
Health Lab Service Monograph Series No. 8. Her Majesty's Stationery
Office, London, Eng., 1974. pp. 1-52.
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12. Henle, W., and M. R. Hilleman. Newcastle Disease Virus. In E. H.
Lennette and N. J. Schmidt (ed.). Diagnostic Procedures for Viral
and Rickettsial Infections. American Public Health Assoc., Inc.,
New York, 1969. pp. 483-490.
13. Hofstad, M. S. A quantitative Study of Newcastle Disease Virus in
Tissues of Infected Chickens. Amer. J. Vet. Research, 12:334-339,
1951.
14. Hopkins, E. S. Availability of Water from the Oakland System for
Usage in the Sterling Processing Corporation Plant. Report to the
Maryland Department of Health and Mental Hygiene, Baltimore, Maryland,
August 1, 1977. 5 pp.
15. Huber, W. G., Carlson, M. B., and M. H. Lepper. Penicillin and Anti-
microbial Residues in Domestic Animals at Slaughter. J. Amer. Vet.
Med. Assoc., 154:1590-1595, 1969.
16. Hull, T. G. Newcastle Disease. In, Diseases Transmitted from Animals
to Man. Charles C. Thomas, Illinois, 1963. pp. 411-427.
17. Maier, W. J., and H. L. McConnell. Carbon Measurements in Water
Quality Monitoring. J. Water Pollution Control Feder. , 46:623-633,
1974.
18. McCollum, W. H., and C. A. Brandly. Hemolytic Activity of Newcastle
Disease Virus. Amer. J. Vet. Research, 16:584-592, 1955.
19. Read, R. B., Bradshaw, J. G., Swartzentruber, A. A., and A. R. Brazis.
Detection of Sulfa Drugs and Antibiotics in Milk. Applied Micro.,
21:806-808, 1971.
20. Shackelford, W. M., and L. H. Keith. Frequency of Organic Compounds
Identified in Water. Environmental Monitoring Series. EPA-600/4-76-
062. U. S. Environmental Protection Agency, Washington, D. C., 1976.
617 pp.
21. Steel, R. G. D., and J. H. Torrie. Principles and Procedures of
Biostatistics. McGraw-Hill Book Company, Inc., New York, New York,
1960. 481 pp.
22. Symons, J. M., Bellar, T. A., Carswell, J. K., DeMarco, J., Kropp,
K. L., Robeck, G. G., Seeger, D. R., Slocum, C. J., Smith, B. L., and
A. A. Stevens. National Organics Reconnaissance Survey for Halogenated
Organics. J. Amer. Water Works Assoc., 67:634-647 1975.
23. U. S. Environmental Protection Agency. Methods for Organic Pesticides
in Water and Wastewater. National Environmental Research Center,
Cincinnati. Ohio, 1971. 38 pp.
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24. U. S. Environmental Protection Agency. Methods for Chemical Analysis
of Waters and Wastes. EPA-625/6-74-003. Washington, D. C., 1974.
298 pp.
25. U. S. Environmental Protection Agency. National Interim Primary
Drinking Water Regulations. EPA-570/9-76-003. Washington, D. C.,
1976. 159 pp.
26. U.S. Environmental Protection Agency. Proposed National Secondary
Drinking Water Regulations. Federal Register, 40(62): 17143-17146,
March 31, 1977.
27. U.S. Environmental Protection Agency. Control of Organic Chemical
Contaminants in Drinking Water. Federal Register, 43(28):5756-5779,
Feb. 9, 1978.
28. U.S. Public Health Service. Drinking Water Standards. Publication
No. 956. U.S. Department of Health, Education and Welfare,
Washington, D.C., 1962. 61 pp.
29. Webb, R.G., A.W. Garrison, L.H. Keith, and J.M. McGuire. Current
Practice in GC-MS Analysis of Organics in Water. EPA-R2-73-277, U.S.
Environmental Protection Agency, Corvallis, Oregon, 1973. 91 pp.
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APPENDIX A
MISCELLANEOUS METHODOLOGY
STATISTICAL
Statistical analyses were performed to compare sets of measurements for
various parameters found in the water, wastewater, renovation system and
carcass washings. The t-test parameter was used to determine if two sets of
data came from the same population at given levels of confidence (21). Add-
itionally, in some cases similar analyses were performed to assess which mean
was greater after they were first determined to be different.
In using the t statistic the assumption is made that the tested para-
meters in the two groups are independent, normally distributed and have equal
variances. However, for relatively small samples (less than 30, which usually
was the case in this study) moderate departures from normality do not inter-
fere with the validity of the analysis.
In most instances the parameters being compared had no temporal relation-
ship to one another, even though the samples were taken on the same date. For
example, the time of travel for the wastewater (A) to pass through the lagoons
and appear as a renovated water sample (E) was about two weeks, the residence
time in the lagoons. Thus, it is not useful to compare only those samples
taken on a single day. For this reason principally, all comparisons were
made on grouped rather than paired data.
In each comparison the hypothesis tested initially was whether the pop-
ulation means were identical. The level of confidence selected for this
determination was 95 percent (a 5 percent level of significance, or alpha
equal to 0.05). In testing this hypothesis a two-tail test was performed.
In those cases where the hypothesis was also tested as to which population
mean was larger, a one-tail test was used, also at alpha equal to 0.05.
The conclusions drawn from the analyses discussed above are described
in the main body of this report in the following terms: when the t-test
parameter indicates that the population means are identical at alpha equal
to 0.05, it is concluded that the two sets of data for a given parameter,
such as in the renovated or treated well water, are statistically identical.
Similar language is used to refer to which of the groups have a larger pop-
ulation mean. For example, in Phase 2 for fluoride the two-tail t-test indic-
ates that the raw wastewater (A) and renovated water (E), with respective
means of 128 and 151 pg fluoride per liter, come from statistical indisting-
uishable populations at alpha 0.05. They are, however, different from the
treated well water (Z) with a mean of 58 yg per liter. In addition, the
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one-tail test confirms the fact that the means of A and E are larger than that
of Z. It is reported then that for fluoride A = E > Z, and it is stated that
the fluoride levels in A and E are statistically identical or equal, and
greater than that of Z.
ORGANIC CHEMICAL ANALYSIS
Volatile Halogenated Organics
The methodology for the volatile halogenated organics in water samples
was essentially an adaptation of that of Bellar and Lichtenberg, the purge
and trap technique coupled with gas chromatography (2). A commercial purge
and trap instrument was used, the Liquid Sample Concentrator, Model LSC-1
(Tekmar Company), along with a Packard 7400 Series gas chromatograph with a
tritium electron capture detector.
Usually 5 ml samples were concentrated and analyzed, following their
collection and sealing in serum vials. Both isothermal and temperature pro-
gramming was utilized, the GC column being packed with Chromosorb 101 and the
Liquid Sample Concentrator directly coupled to the gas chromatograph. The
system was calibrated with known concentrations of volatile halogenated
organics, such as chloroform, carbon tetrachloride, and methylene chloride,
using both peak heights and areas of the chromatograms. The detection limit
for chloroform, the only such compound detected by the GSPH laboratory, was
approximately 1 yg/1.
Non-volatile Organics
The general procedure utilized in this study for the identification of
non-volatile trace organics in water involved the extraction of these com-
pounds from samples into a suitable solvent, followed by concentration of the
extract into a volume acceptable for identification by GC-MS analysis. For
the most part the techniques were similar to or adaptations of those described
by Webb, Garrison, Keith, and McGuire (28).
All glassware was cleaned to remove trace organics, and samples were
collected and handled so as to contact only glass or Teflon. Samples were
typically 3000 ml and the pH was adjusted to 6 to 8 prior to extraction with
high purity methylene chloride. Samples were extracted with three volumes
of solvent, which were then subsequently combined. Sodium sulfate was used
to absorb water from any emulsions that were formed, and to dry the extracts.
Using a 3-ball Snyder column and Kuderna-Danish flask, the solvent was dis-
tilled from the extract at 72°C until about 1 ml of the latter remained.
The volume was then further reduced to any desired level by directing a stream
of clean, dry, inert gas over the surface of the tubes. The concentrated
extracts were then injected into the GC or GC-MS for analysis. In some cases
the concentrated extracts were first methylated using diazomethane (29). In
all instances double-distilled water was analyzed also in the extraction and
subsequent analysis as a control for possible contaminants introduced at any
stage of the concentration or analytical procedure.
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GC analyses were performed on a Packard Model 824 with flame ionization
detector. Glass columns were utilized, usually with 3% OV-17 packing on
Supelcoport 80-100 mesh and temperature programming. Similar or identical
conditions were then used on the LKB 9000 GC-MS instrument, which was inter-
faced to a Digital Equipment Corporation PDF 11 computer to normalize the
data and subtract background. Qualitative identifications (MS assignments)
were made with the aid of the Registry of Mass Spectral Data by Stenhagen,
Abrahanisson and McLafferty, but no concentration levels were established.
The identifications must be regarded as presumptive, since they were not con-
firmed by direct comparison using known compounds.
MICROBIOLOGICAL AND RELATED METHODS
Residual Drug Assay
A disc assay method modified from the procedure of Huber et al. (15) was
employed to detect residual drugs in the water and carcass samples. The first
method used in this laboratory employed Bacillus subtilis (Difco spore sus-
pension containing approximately 10 spores/ml) as the test organism and
seed agar (antibiotic medium no. 1) as the test substrate. On each day of
testing, assay agar plates (15 x 100mm) containing 21 ml of base agar (anti-
biotic medium no. 2) were prepared. After solidification of the base agar,
4 ml of seed agar containing 10 Bacillus subtilis spores per ml were evenly
poured on top of the base agar and allowed to solidify. Blank 0.5-inch filter
paper discs (three discs per sample) were impregnated with the water and car-
cass samples and immediately placed on the assay plates. Each plate contained
additionally one antibiotic control disc impregnated with 0.5 units of peni-
cillin-G per ml prepared by diluting a vial of penicillin-G powder with double
distilled water. After drying at room temperature, assay plates with the
impregnated discs were inverted and incubated at 37°C for 12 to 16h. The
sizes of the zones of inhibition on each plate were then measured.
This procedure was further modified according to the method of Read et
al. (19) in order to detect bacitracin and enhance the sensitivity of detect-
ing sulfa drugs and antibiotics in the water and carcass samples. Bacillus
cereus and Mueller-Hinton agar were thus employed as the test organism and
the test substrate, respectively. The control antibiotic discs were impreg-
nated with 1 mg of streptomycin-sulfate per ml instead of penicillin-G. The
assay agar plates were prepared as described previously with the above modi-
fications.
To further increase the sensitivity Sarcina lutea was also employed as
the test organism, since Read et al. (19) showed that this organism is more
sensitive to bacitracin, which is used in the feed of the chickens.
Salmonella Methodology
Isolation and Identification—
Salmonellae were isolated from the water and carcass samples using
standard Millipore membrane filtration (MF) techniques (1). Quantisation of
Salmonella was attempted using three 10-fold dilutions, five tubes per dilution,
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to determine the MPN index of organisms per 100 ml. The technique consisted
of filtering five-aliquot volumes per sample through 47 mm diameter, 0.45y
pore-sized Millipore membrane filters (Millipore Corp., cat. no. HAWG047SO).
Each filter was placed into 10 ml of enrichment broth to produce five tubes
in the first row of the MPN test. Tubes were agitated for 2 to 3 min to
remove organisms from the filters. Two 10-fold dilutions were made into
enrichment broth to complete the 15 tubes for the MPN test.
In an attempt to improve the recovery of Salmonella during this study,
either selenite brilliant green (SBG, Difco Laboratories) or tetrathionate
(TET, Baltimore Biological Laboratories) broths were employed as enrichment
media. After incubating the SBG tubes for 12 to 16h, or the TET tubes for
24h, both at 37°C, one loopful of enrichment broth from each tube was streaked
on brilliant green agar (EGA, BBL) plates, with or without the addition of
0.08 g of sodium sulfadiazine (Lederle Lab. Div., Am. Cyanamid Co., Pearl
River, N.Y.) per liter of agar. All plates were incubated at 37°C for 18 to
24h. The EGA plates were examined for pink-colored colonies, which were then
subjected to the oxidase test (8). Oxidase-negative, pink-colored colonies
were identified on the basis of their biochemical reactions in triple sugar
iron (TSI) agar (BBL), lysine iron agar (LIA, BBL), motility indole-ornithine
(MIO, Difco Laboratories) medium and an Enterotube (Roche Diagnostics). In
all of the latter tests incubation was at 37°C for 18 to 24h. Organisms that
produced typical Salmonella-like reactions (8) in TSI agar, LIA and MIO med-
ium, and were identified as Salmonella by the Encise system (Roche Diagnos-
tics) , were inoculated into sugar fermentation tubes containing purple broth
base medium (Difco Laboratories) and 1% of either raffinose, rhamnose or
trehalose. After incubation at 37°C for 12 to 18h, the Salmonella isolates
were speciated based on the sugars that were fermented (9).
Recovery Efficiency of MF Technique—
The recovery efficiency of the MF technique for S_. typhimurium was
determined as follows: five replicate 100-ml volumes from the Salmonella-
inoculated distilled water sample were filtered. The filters were placed
on trypticase soy agar (TSA, BBL) plates that were then incubated at 37°C
for 18 to 24h. Colonies were counted for each filter and the mean number
determined. The mean concentration of ^. typhimurium per ml in dilutions
of the TSB cultures (used to inoculate the distilled water) was calculated
from the colony counts on 10 replicate TSA plates inoculated per dilution.
The mean efficiency of recovery of the MF technique was 85%, calculated by
comparing the number of colonies counted on the filters with those deter-
mined from the dilutions of the TSB cultures.
The MPN Test with Different Enrichment Broths—
Experiments were performed to determine the relative efficiencies of
the SBG and TET enrichment broths, combined with EGA plates, for quantitating
_S. typhimurium by the MPN test. Also, single and mixed (with S_. typhimurium)
cultures of J£. cloacae were similarly studied in the enrichment broths to
determine the relative enrichment and inhibition of another organism commonly
isolated from the water samples, and its possible influence on Salmonella
quantitation. For the inoculated water samples MPN indices for ^. typhimurium
were significantly higher in the TET broth than in the SBG enrichment. MPN
indices in TET were also higher (in single cultures) for J5_. typhimurium than
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for _E. cloacae, indicating that TET broth could both support the growth of
Salmonella and repress that of Enterobacter. The results from these experi-
ments suggest that Salmonella organisms may grow to higher concentrations in
TET broth than in SBG enrichment, thereby facilitating quantitation by the
MPN method.
Viral Methodology
Virus Analysis Using Chick Embryo Cells—
Primary chick embryo (CE) cell cultures were used with a plaque assay to
detect the presence of avian viruses (e.g., NDV) in the water and carcass
samples. CE cells were prepared from trypsinized embryos. The volume of
cells was determined after centrifugation, and a 50% cell suspension was
prepared in minimum essential medium supplemented with calf serum, sodium
bicarbonate, antibiotics and mycostatin. CE cells were dispensed into tissue
culture plates which were then incubated at 37 C in a 5% CC^ atmosphere until
complete cell monolayers developed.
Water and carcass washing samples were filtered through 0.45p pore-size
Millipore Swinnex filters to remove any bacteria which might have been pre-
sent. Cell monolayers were inoculated with the undiluted and the lO"-"- dilu-
tion of the samples. Negative and positive controls included uninoculated
and NDV inoculated cell monolayers, respectively. After inoculation, plates
were incubated for 1 hr. at 37°C. To each plate, agar overlay medium was
added. Plates were reincubated at 37°C for 96 hr, then stained for plaques
using a solution of neutral red. Plaques were counted and plaque forming
units per ml (PFU/ml) were determined.
Presence of Avian Viruses in Tissue Extracts by Hemagglutination—
Spleens, livers and lungs were removed from chickens transported to the
laboratory on ice from the processing plant. Using a modification of the
procedure of Hofstad (13), 10% suspensions of the tissues were prepared in
nutrient broth and centrifuged. The supernatant fluids, filtered through
Millipore Swinnex filters to remove bacteria, were used to inoculate the
allantoic cavity of embyronated eggs. Inoculated eggs were incubated at
37°C in a moist environment and candled daily. Eggs which showed dead embryos
after 24 hr were chilled overnight at 4°C, after which allantoic fluids were
harvested and pooled.
Hemagglutination tests were performed on the allantoic fluids by a modi-
fied procedure of McCollum and Brandly (18). Serial 2-fold dilutions of the
fluids were prepared in saline, and each dilution was mixed equally with a
suspension of chicken red blood cells. NDV and influenza-A viruses were in-
cluded in the test as positive controls. The test was then read after 45
min of incubation at room temperature. The end point was the highest dilu-
tion of allantoic fluid that showed 1+ agglutination. The reciprocal of the
highest dilution was the hemagglutination titer.
NDV Survival—
Experiments were designed to study the effect of temperature and light
on the survival of NDV. Flasks of lagoon, filtered lagoon and distilled
(included as controls) water were inoculated with NDV and placed either in
67
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the light or the dark at 7 or 25°C. Flasks were gently agitated continuously
during the experiments to simulate the aeration of the lagoon water at the
poultry processing plant. Samples from each flask were removed at 0, 24, 48,
72, 96 and 120 hr post inoculation, then were inoculated onto primary CE
cells and assayed for plaques as described previously. Plaques were enumer-
ated and PFU/ml were determined.
68
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APPENDIX B
CALCULATION OF STEADY-STATE IN RECYCLE
There is often concern about the possible build-up of waste constituents
in water reuse and recycling. At first it might seem that such materials
would continue to accumulate during each recycle until, perhaps, they would
reach solubility limits. However, it can be shown readily that a steady-
state concentration, rather than an infinite build-up, will eventually be
attained, as long as some fraction of the parameter in question is lost in
recycle, whether through removal in treatment or the discarding of a portion
of the wastewater flow. In the latter case, the flow that is not utilized
would be replaced by a "fresh" source of water.
In the proposed recycle system at the Sterling Plant, 50 percent of the
lagoon effluent would be discarded to the river, and the remainder would
undergo renovation, be mixed 50/50 with the untreated well water, and then
the mixture would receive "normal" water treatment. For this system one can
readily determine the maximum possible steady-state concentration, even when
there is no reduction at any stage of treatment. For example, if a constit-
uent were added to the wastewater (as a result of processing the poultry) at
a rate of A grams per day, none of it was present in the well water, and
none added or removed at any stage of treatment, one half of the A grams
would still be lost to the receiving stream. In this case, it is obvious
that the maximum (steady-state) quantity of the constituent that would "leave"
the plant in the wastewater would be 2A grams per day. Half of this, or A
grams, would be emitted to the river. The other half would be returned with
the renovated water and added to the A grams generated at the plant, the max-
imum net of 2A grams per day thus appearing in the raw wastewater.
Where there is also partial removal at various stages of treatment, addi-
tion as a result of the use of chemicals in the treatment and renovation
systems, or the constituent is also present in the well water, the steady-
state calculation will be different from that resulting in the simple maxi-
mum factor of two shown above. It is useful to consider the effects of these
factors and, at the same time, evaluate the rate at which steady-state will
be achieved, i.e. the number of cycles required.
The following derivation for the steady-state equations in actual re-
cycle is based upon the specific design of the Sterling plant, although the
general principles can be applied to any reuse system. The derivation will
refer to the flow diagram for the treatment plant and renovation system
shown schematically in Figure B-l. In actual recycle the flows Zf, Cf, and
Ef, shown in Figure B-l, would each be 50% of that from the processing
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plant. (In Phase 2 they were 100, 100 and 33%, respectively, of the flow
from the processing plant, once steady-state was achieved.) It is assumed
for simplicity that there is zero concentration of parameter A in the un-
treated well water, and that A grams per day of this parameter are added to
the system from the processing of the poultry at the plant and emitted with
its liquid waste effluent. In addition to the 50 percent loss of parameter
A that occurs at the point where half of the chlorinated effluent from the
second lagoon is discarded to the river, there can be prior fractional removal
(1-b) in the two lagoons, as well as subsequent fractional removal in the
renovation system (1-c), including that due to normal treatment of the mix-
ture. It will be assumed that the fractional removal of parameter A in each
case will be independent of its concentration. The time for a complete cycle
(the period typically required for a waste constituent to be returned to the
poultry plant in the renovated water) is determined essentially by the re-
tention time in the lagoon system, approximately two weeks. Although one can
expect continuous changes in concentration of a waste constituent as it pro-
ceeds through a cycle, it is convenient for the calculations to consider
these after each such period.
At the start of the first cycle, the amount of waste parameter A that
is emitted in the plant effluent is A grams per day, since no additional
quantity has yet been added from the renovation system. At the end of the
first cycle, this material now returns to the plant, but is reduced in quanti-
ty by the three factors discussed above: its loss to the receiving stream,
reduction in the lagoons, and reduction in the renovation system. The net
effect is that the quantity returned (per day) to the plant at the end of
the first cycle is (A)(b)(c)/2. Thus, at the first day of the second cycle,
the amount leaving the plant will be increased by this quantity, and will,
therefore, be A + A(bc/2), or A(l + bc/2).
now OF
WELL WATER
BASIN
\
PROCESSING
PLANT
A grams/day
generated
LAGOONS
In
Phase 2 study
Path in
I actual recycle
I
RENOVATION
SYSTEM
now or
EFFLUENT TO
RIVER (C )
FLOW THROUGH
RENOVATION SYSTEM (I)
Figure B-l. Flow diagram for steady-state calculations.
70
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Similarly, at the end of the second cycle, the amount of A that is
returned to the plant will be A(l + bc/2) (bc/2) . Again, on the first day of
the second cycle this will be added to the A grams per day "generated" at
the plant, so that A + A(l + bc/2) (bc/2) will leave the plant, this quantity
being A{1 + (bc/2) + (bc/2)2}. After "n" cycles this becomes:
n n> (1)
where L is the amount (or load) of parameter A in grams leaving the plant
per day just after cycle "n". After an infinite number of cycles, and since
bc/2 is less than unity, this series converges and the quantity becomes:
L = A/{1 - (bc/2)} (2)
s
This then represents the amount of material, Ls, leaving the plant each day
in the steady-state.
One can similarly determine the concentration of A reaching the plant
in the renovated water in the steady-state, Cg (in effect is concentration
after mixing 50/50 with the well water, the total flow being V liters per
day through the plant) :
C = (L - A)/V (3)
S S
since the daily load coming into the plant is less than that going out in
the waste by A. With substitution of Equation 2 into 3:
C = [A/{1 - (bc/2)} - A]/V (4)
s
which can be rewritten as:
C = (A/V)(bc)/(2 - be) (5)
S
Equations 1 through 5 are useful in assessing the possible build-up of con-
centration of a waste-parameter in a renovation system, and relating this
to the pertinent parameter in the equation, namely the daily amount of waste
generated, A, and its fractional removal in the lagoons and renovation pro-
cess, (1-b) and (1-c), respectively. The equations can be used to estimate
steady-state concentrations without actually going to full recycle. That is,
if one can determine b and c removal parameters, as well as A, one can use
Equation 5 to estimate the concentration of A in steady-state in actual recycle.
Let us now consider an example which approximates conditions in the
Sterling plant study for total dissolved solids (TDS):
-Assume 820 mg/1 TDS in processing plant waste effluent in first cycle.
-Assume 370 mg/1 TDS in lagoon effluent in first cycle.
-Assume 335 mg/1 TDS in renovation system effluent in first cycle.
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-Thus, b = 370/820 =0.45 (55% removal); c = 335/370 =0.91 (9% removal;
and be = 0.41
Now one can calculate, using Equation 5, the expected steady-state concentra-
tion of IDS in the 50/50 mixture:
C = 820(0.41)/(2 - 0.41) (6)
s
C = 211 mg/1 IDS (7)
£s
Before 50/50 mixing with the well water, the steady-state concentration would
be twice this value, 422 mg/1, about 25 percent higher than that in the re-
novated water before 50/50 mixing at the end of the first cycle. As one can
see from this example, the amount of build-up is not necessarily high and may
be acceptable in many cases.
One can also estimate the number of cycles required to approach steady-
state. This can be important in doing a study where the time available and,
hence, the number of cycles is limited. One useful approach is to compare
Ln and Lg. The ratio of the two is a measure of the difference between the
daily waste emitted after n cycles and in the steady-state. On the first
day after the first cycle this ratio becomes:
L /L = (1 + bc/2)(l - bc/2) (8)
J- S
Using the data for TSD from the example above,
L /L = (1.205)(0.795) = 0.96 (9)
J- S
Thus, after only one cycle the daily load of waste emitted from the process-
ing plant is 96 percent of that in the steady-state.
By the same token, one can also calculate the renovated water concentra-
tion in the steady-state compared to that at the end of the first cycle. One
would obtain the same ratio for the renovated water either before or after
mixing 50/50 with the well water and subsequent full scale water treatment,
assuming the latter will have no effect on the parameter in question. (In
fact, this assumption may be incorrect for many parameters. However, except
for chemicals added in treatment, any additional effect would generally lower
rather than raise the steady-state concentrations). In the general case, and
using Equation 3:
C /C = (L - A)/(L - A) (10)
n s n s
At the end of the first cycle (n = 1), and using Equations 1 and 2, this
becomes:
C /C = (1 - bc/2) (11)
-L S
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For our IDS example, this ratio is 0.795, which indicates that at the end of
the first cycle the concentration of waste parameter A in the renovated water,
either before or after mixing with the well water, will be 79.5 percent of
that in the steady-state.
After two cycles, for the wastes leaving the plant:
L2/Lg = {1 + bc/2 + (bc/2)2}(l - bc/2) (12)
For the example at hand, this ratio is 0.991. The comparable ratio for the
water is:
CJC = {1 + bc/2}(l - bc/2) (13)
£ S
which is equal to 0.958. Thus, in this case, after only two cycles the TDS
concentrations in the wastewater effluent and the renovated water have reached
99 and 96 percent, respectively, of their steady-state concentrations. It
can also be shown readily that, the higher the fractional removal (the smaller
the factor be), the faster will be the attainment of steady-state. After "n"
cycles these ratios can be expressed as:
n
Ln/Lg = (1 - bc/2){n|0(bc/2)n> (14)
n-1
Cn/Cg = (1 - bc/2){n|0(bc/2)n} (15)
Another question of interest is whether the Phase 2 study can be expected
reasonably to simulate the build-up of waste constituents in the renovation
system that occurs in actual recycle. In Phase 2 the renovated water was
diverted into the lagoon system, rather than being mixed 50/50 with the well
water. These two paths are shown in Figure 1. Perhaps the most important
difference is that in Phase 2 the flow through the lagoons was about 1/3
higher than it would be in actual recycle (the flow through the renovation
system was about 1/3 of that through the plant, and both effluents were
emitted to the first lagoon). This results in a higher dilution of any waste
constituent emitted to the lagoon and cycled through the renovation system.
Equations for the flow regime in actual recycle thus have to be modified to
accurately reflect Phase 2. It can be shown readily that Equation 1 would
then take the form:
n
L = A{ Z_(bc/4)n} (16)
n n=(J
which becomes, in the steady-state,
L = A/{1 - (bc/4)} (17)
S
The factor 4 appears because one fourth of the effluent from the lagoons is
returned to the renovation system, the other three fourths being emitted to
the river. Similarly, one can show that:
73
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C = (L - A)/(V/3) (18)
S S
This is a direct analogy to Equation 3, but in this case the factor 3 appears
because the daily flow through the renovation system is one third of that
emitted from the poultry processing plant. With the insertion of Equation 17
into 18, the latter becomes:
C = (A)(3/V)(bc)/(4 - be) (19)
S
If one wishes now to compare the steady-state concentrations likely to
be attained for the Phase 2 and full recycle systems, one would simply take
the ratios of Equations 2 and 17 for the wastes, and 5 and 19 for the renova-
ted waters. For the latter case, and designating rs as the ratio of the steady-
state concentrations in the renovated water in actual recycle to that in Phase
2, one obtains:
r = (1/3)(4 - bc)/(2 - be) (20)
S
Thus, this ratio is highly dependent on the fractional removals in the lagoon
and renovation systems. With high degrees of removal (be approaching zero)
the ratio will approach 2/3. However, with very low removal (be not much
less than unity), the ratio will approach unity. Thus, one can estimate that
the steady-state concentrations in actual recycle should be 0.67 to 1.0 times
those observed in Phase 2.
There are, however, additional factors that should be considered. First,
in actual recycle there can be additional removal of a waste parameter after
the renovated water is mixed 50/50 with the well water and receives additional
"normal" water treatment. Thus, the "c" term might be lower in actual recycle.
Second, there was a higher flow through the lagoon system in Phase 2 compared
to that which would occur in actual recycle. Thus, in the latter case with
the lower flow, there would probably be greater degradation or loss for some
waste constituents, and, again, "b" would be lower. For those parameters
affected, the steady-state concentrations in actual recycle would be less
than those predicted by Equation 20.
Another factor of possible importance is the role of the well water as a
contributor of material to the renovation system. In the above derivations
it was assumed that any waste constituent generated at the poultry plant was
at zero concentration in the treated well water. One can modify this and
divide the daily generated waste in Phase 2, A, into A' generated at the
plant and A" contributed from the treated well water. Such a division has
no bearing on the Phase 2 calculations, since they do not distinguish between
these two possible sources of the waste parameter appearing in the plant
effluent. However, in actual recycle, A would often be smaller than it would
in Phase 2, since one source of it, A", would be reduced by half as a result
of the 50 percent usage of well water compared to Phase 2 during which the
well water flow was 100% of that of the effluent from the plant. For example,
if in the Phase 2 study a constituent, such as magnesium, had a concentration
in the treated well water 60 percent of that in the wastewater from the plant,
74
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then the Phase 2 value of A is made up of A', that generated at the plant,
and A" = 0.6A', that generated in the well water. Thus, in Phase 2, A = 1.6A".
That is, the total amount of this parameter emitted from the plant is 60 per-
cent higher than that generated at the plant, A'. However, with actual 50/50
recycle the well water would contribute only 0.3A', so that the sum of the
magnesium from the latter and that generated at the plant is A' + 0.3A' = 1,3A',
less than the 1.6A' emitted in the Phase 2 study.
The effect of this factor can be seen in the example given earlier for
TDS (total dissolved solids). The additional piece of information needed is
the TDS concentration in the treated well water, typically 155 mg/1. Thus,
using the concepts above, the load in actual 50/50 recycle generated at the
plant plus that due to the TDS in the renovated water would be 820 - (155/2) =
742 mg/1. This would directly reduce the steady-state concentration of TDS
in the renovated 50/50 mixture from 211 mg/1 (from Equation 6 and 7) to
211(742/820), or 191 mg/1, about 10 percent less. The greater the relative
concentration of a given parameter in the well water compared to the amount
generated in the plant, the greater is the need to make this modification.
In any event, it always operates in a direction to reduce the calculated
steady-state concentration of a waste parameter in the renovated water
delivered in actual recycle compared to that measured in Phase 2.
In considering all the factors, one can conclude that the Phase 2 study
can be used to predict reasonably well the steady-state concentrations that
can be expected in the 50/50 renovated water mixture in the actual recycle.
It is also apparent that there are additional factors that could play a role
and somewhat alter the conclusions. However, even in those circumstances
additional measurements can facilitate the use of modified steady-state equa-
tions, and thereby compensate for them.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/l-79-030
2.
4. TITLE AND SUBTITLE
Safety Evaluation of Renovated Wastewater
from a Poultry Processing Plant
6. PERFORMING ORGANIZATION CODE
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
August 1979 issuing date
7. AUTHOR(S)
J. B. Andelman
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
University of Pittsburgh
Graduate School of Public Health
Pittsburgh, Pennsylvania 15261
10. PROGRAM ELEMENT NO.
1BA607
11. CONTRACT/GRANT NO.
R804286 & S803325
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final Aug. 1974-Dec. 1977
14. SPONSORING AGENCY CODE
EPA/600/10 & EPA/600/12
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A three-phase evaluation of reclaimed process wastewater for reuse was under-
taken at the Sterling Processing Corporation plant in Oakland, Maryland. The main
objective was to evaluate the safety for human consumption of poultry exposed during
processing to an average 50 percent mixture of treated well water and reclaimed waste-
water. To that end, a determination was made of the ability and reliability of the
water reclamation system to deliver satisfactory quality water, and whether the pro-
cessed poultry would have any excess microbiological or chemical constituents, harmful
to human health, as a result of exposure to such water. After the renovation system
was optimized (Phase 1), a two-month study (Phase 2) was instituted, which simulated
recycle of renovated water through the poultry plant. Chemical, physical, and micro-
biological analyses were performed on various water, wastewater and poultry samples.
An experimental chiller, filled with renovated water, was utilized to compare the
uptake of such constituents by the processed birds with that resulting from exposure
to the chiller in the processing plant using the normally treated well water. An
evaluation of the Phase 2 study, as well as other data, leads to the conclusion that
the safety of the consumers of the poultry would not be jeopardized if the planned
trial period of reuse (Phase 3) were instituted.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
Public health, microbiology, water treat-
ment, potable water, water quality, water
reclamation, industrial wastes, industrial
water, industrial waste treatment, poultry,
ground water, organic wastes
Advanced waste treatment,
industrial reuse, reno-
vated water, drinking
water standards
57 U
18. DISTRIBUTION STATEMENT
Release to the public
19. SECURITY CLASS (This Report/
Unclassified
21. NO. OF PAGES
20. SECURITY CLASS (This page/
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77)
76
-., US GOVERNMENT PRINTING QfHCE 1979-657-060/5417
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