EPA-600/2-77-174
August 1977
Environmental Protection Technology Series
INDEX OF REFRACTORY ORGANICS
Robert S. Kerr Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Ada, Oklahoma 74820
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-77-174
August 1977
AN INDEX OF REFRACTORY ORGANICS
by
T.B. Helfgott, F.L. Hart and R.G. Bedard
Environmental Engineering Program
Civil Engineering Department
University of Connecticut
Storrs, Connecticut 06268
Grant No. R803231-01-05
Project Officer
Billy L. DePrater
Industrial Section, Source Management Branch
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma 74820
This study was conducted
in cooperation with
The University of Connecticut
Storrs, Connecticut 06268
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ADA, OKLAHOMA 74820
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DISCLAIMER
This report has been reviewed by the Robert S. Kerr Environmental
Research Laboratory, Office of Research and Development, 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.
ii
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FOREWORD
The Environmental Protection Agency was established to coordinate
administration of the major Federal programs designed to protect the quality
of our environment.
An important part of the agency's effort involves the search for in-
formation about environmental problems, management techniques and new techno-
logies through which optimum use of the nation's land and water resources can
be assured and the threat pollution poses to the welfare of the American people
can be minimized.
EPA's Office of Research and Development conducts this search through
a nationwide network of research facilities.
As one of these facilities, the Robert S. Kerr Environmental Research
Laboratory is responsible for the management of programs to: (a) investigate
the nature, transport, fate and management of pollutants in groundwater; (b)
develop and demonstrate methods for treating wastewaters with soil and other
natural systems; (c) develop and demonstrate pollution control technologies
for irrigation return flows; (d) develop and demonstrate pollution control
technologies for animal production wastes; (e) develop and demonstrate tech-
nologies to prevent, control or abate pollution from the petroleum refining
and petrochemical industries, and (f) develop and demonstrate technologies
to manage pollution resulting from combinations of industrial wastewaters or
industrial/municipal wastewaters.
This report contributes to the knowledge essential if the EPA is to meet
the requirements of environmental laws that it establish and enforce pollution
control standards which are reasonable, cost effective and provide adequate
protection for the American public.
W. C. Galegar
Director
Robert S. Kerr Environmental
Research Laboratory
111
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ABSTRACT
Refractory waterborne organics resist biodegradation, accumulate in the
environment and can inhibit life forms. This research develops laboratory
techniques for, and interpretations of, a Refractory Index (R.I.) to
assess quantitatively the persistency of refractory organics and uses the
R.I. determination to evaluate some 38 industrial, natural and combined or-
ganics. R.I. values close to 1.0 characterize readily biodegradable or-
ganics; intermediate R.I. values, approximately 0.3 to 0.7, indicate par-
tial degradation; R.I. values approaching zero indicate refractory organics;
negative R.I. values indicate inhibitors. The coefficient of variation for
R.I. values is 13%. Since negative R.I. values are of qualitative signifi-
cance only, a Biological Inhibition Value (B.I.V.) is developed and used to
assess quantitatively those organics found to interfere with the long-term
ultimate Warburg Respirometer determinations, which were the biochemical
tests used. In a few cases, confirmation of the R.I. interpretation was
performed by specific analysis using a model activated sludge unit. Cor-
relations between oxygen demand tests (BODs, BODy, TOD) and organic para-
meters (TOC, TN) are presented.
Suggestions for required pretreatments of industrial wastewaters before
allowing discharge into municipal sewage treatment plants are included as
an application of the Refractory Index criterion. The bibliography con-
tains 49 references.
This report was submitted in fulfillment of R803231 by the University of
Connecticut under the partial sponsorship of the U.S. Environmental Protection
Agency. This report covers a period from July 1, 1974 to June 1, 1976, and
work was completed as of May 1, 1977.
iv
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CONTENTS
Foreword i i i
Abstract iv
Figures vi
Tables vii
Abbreviations and Symbols viii
Acknowledgment ix
1. Introduction 1
2. Summary 3
3. Conclusions and Recommendations 6
4. Main Text 8
Objective 8
Background 8
Derivation and Laboratory Methods 10
Explanation of Terms 11
5. Refractory Index Data and Interpretations 14
General Explanations for Persistence 14
Presentation and Interpretation of R.I. Data for
Naturally Occurring Organic Compounds 15
Presentation and Interpretation of R.I. Data for
Synthetic and Industrial Organic Compounds 20
Presentation and Interpretation of R.I. Data for
Combinations of Organic Materials 26
Test Procedures: Interferences, Limitations,
Correlations and Confirmations 30
6. Closing Statement 39
References 40
Appendix 45
A. Derivation of UOD Measurement 46
B. Laboratory Methods and Materials 52
C. Warburg Respirometer 66
D. Suggested Pretreatment Requirements for Industrial
Waste 123
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FIGURES
Page
Figure 1. Laboratory scheme for determining
a refractory index 9
Figure 2. Metal interference with BOD5 data. ... 31
vi
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TABLES
Page
Table 1. List of Refractory Indices for Organic Materials
Tested 5
Table 2. Refractory Index Data for Naturally Occurring
Organics 16
Table 3. Refractory Index Data for Synthetic and Industrial
Compounds 21
Table 4. Chemicals Which Inhibit Respirometer Response 24
Table 5. Refractory Index Data for Combinations of Organic
Materials 27
Table 6. Color Analysis of Textile Dye 28
Table 7. Color Analysis of Industrial Dye Waste 29
Table 8. Models for Quick-Time Oxygen Demand Measurements of
Raw Sewage 34
Table 9. TOD Response for D-Glucose/Sewage Effluent 36
Table 10. TOD Response for 1,-Lysine/Sewage Effluent 37
Table 11. TOD Response for p-Chlorophenol/Sewage Effluent 38
Table A. Suggested Limits and Ranges for Industrial Wastewater
Pollutant Inputs to Municipal Sewage Treatment Plants . 126
Table B. Interpretation of Refractory Index Values 127
VII
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LIST OF ABBREVIATIONS AND SYMBOLS
B.I.V. - Biological Inhibition Value, Relative Decrease in Ultimate Biochemical
Oxygen Demand of Sewage due to presence of an inhibitory substance,
expressed as a fraction
- Ultimate Biochemical Oxygen Demand, mg/L as 02
- Ultimate Biochemical Oxygen Demand Measurement Without Nitrate
Corrections, mg/L as 02
AN03 - Nitrate Corrections Terms, mg/L as 02 Demand
|N02l - Concentration of Nitrites, mg/L as 02
|NO§| - Concentration of Nitrates, mg/L as 02
R.I. - Refractory Index, Normalized Value Indicating Degree of Degradation,
BODu
DOIT
TN - Total Nitrogen, Expressed as mg/L as 02
TOD - Total Oxygen Demand, mg/L as 02
TOxN - Total Oxidizable Nitrogen, mg/L as 02
UOD - Ultimate Oxygen Demand, mg/L as 02
s - Standard Deviation of a Population
x - Average of a Population
(s/x) - Coefficient of Variation
< - Less Than
> - Greater Than
Vlll
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ACKNOWLEDGMENTS
The authors of this Report acknowledge with gratitude the assistance
of the Institute of Water Resources at the University of Connecticut in
the preparation and editing of the manuscript.
In addition it should be noted that both the research and reporting
were supported in part by funds provided by the United States Department
of the Interior as authorized under the Water Resources Research Act of
1964, Public Law 88-379, as amended.
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SECTION 1
INTRODUCTION
The purpose of this research has been to develop and accumulate data
on a Refractory Index as a quantitative measure of persistence of organic
wastes especially those of industrial origin. The discharge of non-
biodegradable (refractory) organic wastes into receiving waters poses a
threat to the aquatic environment which is even greater than that of the
oxygen depletion realized when readily biodegradable materials are released
into the environment. Some refractory organics originate from industrial
waste either directly or via municipal wastewaters which receive waste
from industry. There are also, however, naturally occurring refractory
organics; some of these can be converted to harmful materials during such
processes as the chlorination of water supplies.
The subject of refractory organic materials as water pollutants is
a relatively new area of concern for environmental engineers, scientists
and administrators. Previously emphasis had been placed on immediate
effects of contamination such as oxygen depletion, toxicity responses and
general aesthetic properties. In contrast, the more recent concern is
with the long-range, perhaps more subtle, effects of bio-persistent organic
contamination. These materials which originate from point sources such as
industrial waste discharges and non-point sources such as agricultural
runoffs have been detected in waterways and potable water supplies after
extensive exposure to both the natural environment and controlled degrada-
tion through wastewater treatment facilities. This detection of bio-
persistence must be regarded as a warning to begin a control strategy
to prevent such organic material accumulations in water systems.
Because the classic five-day Biochemical Oxygen Demand (BODr) has been
a standard of wastewater quality, industries with high BOD values for waste-
water have tried to lower the BOD value of their discharge. A valid
approach to lowering BOD level is the use of effective treatment processes;
a non-appropriate approach to lowering the BOD level is to switch from
readily biodegradable organics to refractory organics which do not create an
immediate high biochemical oxygen demand because they resist degradation.
These substitutes, whose use is sometimes encouraged when regulatory agencies
rely solely on a five-day BOD criterion, can in fact be far more polluting
to the environment due to persistence, toxicity, effects on flora and fauna
and long-term depletion of oxygen levels in receiving waters. An example of
this is the switch by textile mills from starch to non-biodegradable sizers.
Certain industrial chemicals should be restricted from entering the
aquatic environment because of their persistence, the high energy expendi-
tures needed to decompose them by conventional methods (such as the activated
sludge bio-reactor), their accumulative degradation off products and/or
their inhibition to aquatic flora and fauna. The first step in organizing
an effective strategy is to develop a means of classifying materials with
respect to biopersistency in the aquatic environment. The index of
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refractory organics discussed and developed in this report is such a quanti-
tative parameter. The Refractory Index used here is essentially the ratio
of long-term aerobic biochemical oxygen demand under standard conditions to
the complete theoretical oxygen demand. This fraction expresses the amount
of the organic material that persists in receiving waters.
Ample evidence is presented here and in the literature to demonstrate
the existence of refractory organics (1) (3) (6) (13) (17) (18) (19) (22)
(24) (25) (27) (28) (33) (37) (43) (45) (48) (49). For those organics that
show a zero biodegradability plus-an inhibiton to the bacterial systems, an
additional analysis to measure refractoriness is needed; this is a bioassay
measure that quantifies the degree of interference with normal biochemical
oxygen demand reactions.
Even some relatively harmless organics, if they persist in the aquatic
environment, can be converted to harmful substances (e.g., humic acid to
chlorinated organics by means of disinfection practices). It is necessary
to lower the risk caused by the presence of harmful persistent organics from
industry and natural sources.
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SECTION 2
SUMMARY
AN INDEX OF REFRACTORY ORGANICS
The objective of this research has been to develop a Refractory Index
as a quantitative measure of persistence of organic residuals, industrial
and natural, that are frequently found in wastewaters and in aquatic envi-
ronments. The Refractory iJidex is formulated based on the ratio of long-
term ( > 20 days) ultimate (Total) Biochemical Oxygen Demand to real-time
measures of Theoretical (Total) Oxygen Demand and Total Oxidizable Nitrogen.
A method of evaluating the total quantity of oxygen needed for complete
oxidation to ultimate and products C02, H20, and N03- and termed Ultimate
Oxygen Demand (UOD) is derived. In addition, the Warburg Respirometer
Biochemical Oxygen Demand test, selected to predict optimum biodegradability
properties, and termed the Ultimate Biochemical Oxygen Demand (BOD 1 is
used. The ratio of these measurements (BOD to UOD) is called the"Refractory
Index (R. I.) and is offered as a parameterufor estimating biodegradability
and treatability of organic materials found in and entering into the
aquatic environment.
(R.I.) =600.,
UOD [1]
The R.I. data presented in this report have a statistically determined coef-
ficient of variation (s/x) of 0.13.
Lists of Refractory Index values for common industrial organics and
natural materials are presented along with a discussion on the significance
of the Refractory Index and its interpretation. Some materials, for example
glutamic acid and glucose, are readily degraded and have R.I. values
approaching 1.0; other materials, for example DDT, show negative R.I. values
interpreted as bacterial inhibition response. The most difficult R.I. values
to interpret are those between extremes, for example lysine, which has an
R.I. value of about 0.5; explanations for these intermediate values are
offered.
Organic materials, especially those originating from industry and
characterized as refractory (i.e., having R.I. values close to zero) are
considered persistent for stipulated environmental conditions. This per-
sistency is extrapolated to the bio-reactors of wastewater treatment and
the receiving aquatic environment. Certain materials, inhibitors, produce
a R.I. number of less than zero; this can be used as a bioassay parameter
for inhibitory organics and heavy metals of industrial wastes. A new term,
the Biological Interference Value (B.I.V.) is used to quantify inhibitor
responses; the greater the B.I.V., the greater the toxicity inhibition
response. Table 1 is a list of compounds tested with either the R.I. value
or, in the case of inhibitors, the B.I.V. value.
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Correlation models for different oxygen demand tests (TOD, BOD) and
tests for organics (TOG, IN and others) are presented with special attention
to real-time parameters (quickly determined measurements); however, great
caution should be exercised in using any such correlation for industrial and
variable wastes.
From the collected experimental evidence basic theoretical and empirical
reasons are offered for the cause of persistence in certain organics.
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TABLE 1.
LIST OF REFRACTORY INDICES FOR ORGANIC MATERIALS TESTED
Organics R.I. B.I.V.
Acetic Acid 0.6
Acetone 0.8
Adenine 0.1
Aniline 0.6
Antifreeze 1.0
Arginine 0.7
_L-Aspartic Acid 0.8
Benzene 0.2
Biphenyl 1.0
Bipyridine <0 0.6
Butyric Acid (Na Form) 0.8
Carboxymethyl Cellulose (CMS) 0.0
Chloroform <0 0.7
p-Chlorophenol 0.0
Cyanuric Acid <0 0.3
DDT <0 0.4
DDT with Carrier <0 0.3
Dichlorobiphenyl 0.2
Dichlorophenol <0 0.6
Ethylene Glycol 0.8
Ethylene Glycol with Inhibitor 0.0
Gasoline 0.2
D-Glucose 0.9
D-Glutamic Acid 1.0
L-Histidine 0.5
Humic Acid 0.0
Hydroquinone 0.5
L_-Lysine 0.5
Phenol 0.9
Propionic Acid (Na Form) 0.8
Propylene Glycol 0.8
Sevin (Carbaryl) with carrier 1.0
Sewage (Domestic) 0.7
Starch 0.6
Textile Dye 0.6
Textile Dye - Industrial Waste 0.1
L/Valine 0.9
Vinyl Chloride 0.0
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SECTION 3
CONCLUSIONS AND RECOWENDATIONS
Since there are non-biodegradable organics of industrial and natural
origin, new approaches to measuring, treating and otherwise managing these
refractory materials are necessary to avoid the hazards due to these per-
sistent contaminants in the aquatic environment. Quantitative determina-
tion of persistent contaminants is an essential step towards understanding
and dealing effectively with these materials.
This study develops and tests a method for determining the total amount
of theoretical oxidizable matter in wastewaters and expresses this amount
as Ultimate Oxygen Demand (UOD); for evaluating the total amount of aerobic
biodegradable matter present, an Ultimate Biochemical Oxygen Demand (BODu)
is used. Ultimate Oxygen Demand (UOD) is determined by combining the Total
Oxygen Demand (TOD) and Total Nitrogen (TN) measurements with each expressed
as mg/L 02- UOD defines the amount of oxygen needed for a complete oxi-
dation of organic matter to ambient aquatic environmental end products -
C02, H20 and N03-.
The Ultimate Biochemical Oxygen Demand (BODy) is determined by measuring
the total oxygen uptake of microorganisms originating from domestic sewage
over an extended period of time (> 20 days), while exposed to the specific
organic material being examined in a Warburg Respirometer apparatus.
Conditions for the test include: 20 C, buffered neutral pH test conditions,
absence of sunlight, sufficient nutrients, heterogenous bacterial popu-
lations and an aerobic environment. A nitrification correction term is
used to account for unequal nitrate production between a blank (raw sewage)
and test solution (raw sewage and test organic material); however, in most
cases this correction is small.
The ratio of BODy to UOD for a material indicates the fraction degraded.
This fractional ratio is expressed as a Refractory Index (R.I.) in a nor-
malized range of 1.0 to 0.0; however, negative values of R.I. which have
been noted (e.g., DDT) suggest that this test procedure could be useful as
a bioassay test to screen for inhibitors. For certain waste materials,
either organic or metallic, that inhibit biochemical degradation a quan-
titative term, the Biological Interference Value (B.I.V.) is used to ex-
press the interference in the long-term BOD test. Refractory Index values
of zero indicate no measurable degradation while Refractory Index values
of one imply complete degradation to the final end products. Organic
materials which either decompose to end products slowly or not at all are
known as bio-refractory.
Correlation coefficient models for different oxygen demand parameters
(BOD5, TOD) and between other measures of organics (TOC, TN, UV, Turbidity)
are offered since there is a need for relating real-time parameters to clas-
sic standards tests; however, great caution should be exercised in using
any such correlation for variable waste.
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The R.I. parameter is designed to assist engineering judgment in deter-
mining acceptability of a waste for discharge. It is useful in evaluating
degradability of specific industrial waste compounds, pesticides and natural
compounds. This information can be used to classify waste materials in
order to make decisions about the acceptability of discharge to the environ-
ment or to biological reactors, or to indicate when other treatment tech-
niques (such as activated carbon) are necessary.
There are water-borne organic constitutents, both natural and indus-
trial, that resist degradation. The R.I. parameter provides a means to
quantify the persistency of such refractory organics. It is recommended
that the procedures developed here be used to evaluate the nature of or-
ganic wastes by testing more materials for persistency, especially those
of industrial origin that waste energy by passing through bio-reactors
unaltered and that may end up as accumulated materials in the aquatic en-
vironment .
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SECTION 4
MAIN TEXT
A. OBJECTIVE
The primary objective of this study is to develop laboratory techniques
for a general parameter defining persistent organics. The parameter pre-
sented in this report is termed Refractory Index (R.I.) and is defined as
the ratio of biochemical oxidation to complete oxidation in the aquatic
environment. The principal uses for this parameter include:
1. Classification of specific compounds such as industrial by-products,
pesticides and natural compounds in terms of their ability to degrade.
2. Classification of industrial wastes for Sewer-Use-Ordinance s.
Wastes found to resist oxidation should be restricted from discharge to
biological treatment systems including the environment. Alternatives in-
clude providing pre-treatment or changing the industrial processes to eli-
minate the refractory materials.
3. Classification of waste discharges in terms of appropriate or
non-appropriate discharge to the environment. Materials which have been
found to resist biodegradation and, therefore, to accumulate in the environ-
ment can pose health and other dangers.
4. Preliminary evaluation of the degradability of a wastewater, waste
constituent or specific compound in a biological treatment system. After
receiving R.I. data, a complete treatable study can be planned.
B. BACKGROUND
1. Meaning of Refractory
Terms such as refractory, bio-refractory, stable, residual, recalci-
trant and persistent have been used to describe degradability characteris-
tics of organic materials found in wastewaters. Ludzack and Ettinger use
the term resistance for material that is "more difficult to destroy by
biological methods than domestic sewage..."(24). A. A. Rosen also applies
the term refractory to those substances resistant to biodegradation (38).
Rosen extends the meaning to include "organics which are not truly resis-
tant to oxidation but which persist sufficiently long in a receiving
water to exert a characteristic pollutional effect". G. C. McCallum also
uses refractory to describe materials "resistant to conventional treatment"
(30).
In this study refractory refers to a material and/or its breakdown
products which fail to degrade significantly in an aerobic environment with
organisms found in domestic sewage during a sufficiently long period of time.
8
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Fig. I LABORATORY SCHEME FOR DETERMINING A REFRACTORY
INDEX
QUICK-TIME MEASUREMENTS
UOD * TOD t TN
WARBURG RESPIROMETER
TEST*
ORGAN 1C + SEWAGE + NUTRIENTS
BLANK =
SEWAGE + NUTRIENTS
> BOD' = BOD,', - BOD;
UTEST UBLANK
NITRATE PROBE
A NO", = NO^ —
0 °BLANK
'TEST
Rl =
BODJj + AN03 BODy
UOD
UOD
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The key words in this definition are breakdown products, aerobic environment,
degrade significantly, sewage organisms and long period of time.
2. Recent Evidence of Refractory Materials
Since refractory materials persist in the biologically active environment
(such as biological treatment process* or receiving waters) they exert pol-
lutional effects longer than easily degraded materials (38). Examples of
such materials include chlorinated phenols and biphenols, pesticides, mer-
curic organic complexes and materials found in runoff waters such as humic
acids and motor oils (9) (23) (45). Although the impact of refractory sub-
stances is not completely understood, such properties as color, taste and
odor have been found characteristic of waters containing bio-resistant
materials. Refractory organics that exhibit color, taste and odor proper-
ties at high concentrations can also be responsible for public health dan-
gers and environmental deterioration at concentrations that do not affect per-
missible drinking water standards for sensory properties. In addition,
many residual organics that are not readily observable may be concentrated
in living tissue with adverse effects. Refractory organic materials in a
water system, regardless of their concentration, should be considered
potentially harmful. Possible effects include biological magnification,
toxicity, carcinogenicity and mutagenicity effects on man as well as on
various aquatic animals and plants. Other difficulties with refractory
materials include bio-treatment overloads from accumulations (29), fouling
of ion exchange resins and chelating trace metals (38) and, of course dangers
of overloading the ecological system and exposing people, plants and other
animals to excessive dosages of refractory materials.
Recent investigations have described accumulations of refractory
materials (48). The Mississippi River is reported to contain at least 34
different chemicals originating from industrial waste. Drinking water
supplies to Evansville, Indiana are reported to contain industrial con-
taminants from a discharge 150 miles upstream. Humic acids have been
reported to be converted to harmful materials by the standard water treat-
ment practice of chlorination. It is, therefore, becoming evident that
materials previously unrecognized, undetected or assessed as non-pollutants
(because of low BOD) can in fact present dangers.
C. DERIVATION AND LABORATORY METHODS
1. Derivation and Laboratory Procedure for UOD
The Ultimate Oxygen Demand Measurement (UOD) is designed to quantify
the total amount of oxygen needed to complete oxidation of a waterborn or-
ganic material to the end products of 002, H£0 and NO?. Since no single
analyzer is capable of yielding a direct measure of this value, the UOD
*Biological treatment implies biodegradation and, therefore, excludes the
flocculation, precipitation and sorption mechanisms found in activated
sludge and trickling filter treatment; however refractory material is found
in the sludges of such wastewater treatments.
10
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parameter is derived from a combination of Total Oxygen Demand (TOD) and
Total Nitrogen (TN) measurements. An examination of the TN and TOD measure-
ment capabilities along with a systematic derivation of the UOD parameter
with particular regards given to the assumptions and limitations charac-
teristic of this parameter is presented in Item A of the Appendix.
2. Laboratory Manual for UOD and BODy Evaluations
A working knowledge of the laboratory methods used to generate Refrac-
tory Index (R.I.) data is necessary in order to interpret R.I. evaluations
and to apply them correctly. Item B in the Appendix presents detailed step-
by-step laboratory procedures for R.I. evaluations. Explanations of the
reasons for each procedure are included. This Appendix is designed to
assist laboratory personnel to organize the analytical steps and to enable
the environmental engineer and scientist to develop an understanding of
R.I. data. Figure 1 illustrates schematically the procedures for deter-
mining R.I. values.
D. EXPLANATION OF TERMS
The following terms or expressions are used in this work:
1. Gross Parameter - A measurement that is a general evaluation and
therefore describes a number of entities collectively. Most environmental
engineering measurements (suspended solids, BOD, TOG, etc.) can be classi-
fied as gross parameters. A specific measurement, such as the concentra-
tion of oxalic acid, is not a gross parameter. Such specific analyses of
wastewaters cannot always be used because of their complex mixture of
transient forms of numerous organics at low concentrations. In addition,
knowledge of specific organic residuals does not always help in treatment
design.
2. mg/L as 02 - Concentration (weight per unit volume) of a material
expressed as molecule oxygen equivalence. This value is obtained by multi-
plying the material's concentration (mg/L) by the molecular weight of 02,
32 gm/g-mole, and dividing this product by the molecular weight of the ori-
ginal material. For example:
100 mg/L TOC as C = 100 x 32/12 = 250 mg/L TOC as 02 [2]
3. mg/L as 03 Demand Equivalence - Concentration of a material (weight
per unit volume) expressed as the amount of oxygen needed for biochemical
oxidation; i.e., one mole of ammonia (NHs) requires 2.0 moles of oxygen
(02) for complete conversion to H2) and HN03.
NH3 + 2.0 02 - > HNOs + H20
For example: 20 mg/L NHs = 20 x 32/17 x 2.0 = 75 mg/L NHs as 03 demand equi-
valence .
4. Refractory, Biorefractpry, Recalcitrant, Persistent - These terms
are used to define the biodegradability of organic materials in the aquatic
11
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environment. A material described as refractory is characterized by a resis-
tance to degradation to 002» H20 and N03 (end products of the ambient aquatic
oxidation).
5. TOD» Total Oxygen Demand - Determined on the Ionics Kfcdel 225 TOD
analyzer (mg/L as 02). This is a high temperature (900°C) catalyzed aerobic
oxidation.
6. TN, Total Nitrogen - Determined on the Dohrmann MCTS-10 nitrogen
analyzer "[mg/L as N). In this study TN is converted to mg/L as 03.
7. POD, Ultimate Oxygen Demand - Calculated from the TOD and TN measure-
ments. UOD defines the total quantity of oxygen needed to oxidize a material
to C02, t^O and N03~. This gross parameter is used to quantify the total
amount or material present.
8. TOxN, Total Oxidizable Nitrogen - The quantity of nitrogen present
that is theoretically capable of oxidization to N03". This is a term used
in the UOD derivation, not a measured quantity, and is expressed as oxygen
demand equivalence.
TOxN = 3/2 (Organic N + NH3) + 1/2 N02~ [4]
all expressed in mg/L as 02 demand equivalence.
9. BODu, ultimate Biochemical Oxygen Demand - Determined on the Warburg
Respirometer with a nitrification correction term. BODu defines the amount
of oxygen required to degrade organics under the specific test conditions.
This gross parameter is used to quantify the degradable organic material
present.
10. NO.V, Nitrification Correction - This term is used to correct for
unequal degrees of nitrification (nitrate production) in the Warburg test,
and is expressed in mg/L as 02 demand equivalence.
11. R. I... Refractory Index - A ratio of BOD /UOD, used to indicate the
bio-degradability property of an organic material. A Refractory Index of
1.0 indicates 100% degradation, while a Refractory Index of 0.0 indicates
no degradation. Intermediate values indicate partial degradation.
12. Warburg Respirometer Test Solutions - The BOD test procedure is
designed to determine the biochemical oxygen uptake response for known
chemical solutions of mixtures of unknown chemicals (e-g-» industrial wastes).
In the method adopted, the chemical is added to a solution of raw sewage plus
additional nitrogen and phosphorous nutrients if necessary. This mixture
(raw sewage, ammonia, phosphate buffer and test chemical) is referred to
as the Test Solution.
13. Warburg Blank Solution - A BOD ' value is determined by subtracting
the BOD ' value of raw sewage, ammonia Chloride and phosphate buffer from the
BOD ' or* the test solution. The raw sewage, ammonia chloride and phosphate
buf¥er solution is referred to as a Blank Solution.
12
-------
14. Biological Interference Value - Some of the compounds tested exhib-
ited an inhibition or interference response to the BODu process; that is,
there is more uptake for the sewage alone than for the sewage plus the tested
compound. In calculating the R.I. for such cases, the numerator of the ratio
BODu/UDD* is a negative number, thus also making the R.I. negative; this is
difficult to analyze quantitatively. For this reason the B'.I.V. (Biological
Interference Value) was developed. The B.I.V. represents the reduction in
BOD of the sewage due to the presence of the compounds
""*"*• sewage ~ sewage + compound-'' sewage *• ^
The range of B.I.V. is zero to one. Zero indicates complete or 100 percent
reduction of the sewage BOD, while 1.0 indicates no interference at all with
the BOD process.
[5]
13
-------
SECTION 5
REFRACTORY INDEX DATA AND INTERPRETATIONS
The Refractory Index data that follow are grouped into the categories
natural, synthetic, industrial and combinations because these categories
lend themselves to discussion; however, the distinction between categories
is to a large extent arbitrary. Along with the data presentation are offered
explanations and rationales for the biodegradability or refractoriness of
individual compounds and groups of compounds.
A. GENERAL EXPLANATION FOR PERSISTENCE
Specific reasons for the persistence of individual organic compounds are
presented with the data on refractory organics which follow. There are also
some general explanations of the phenomenon.
1. Insufficient time - The ultimate BOD test normally allows a 30 day
degradation period. This test time is extended if a measurable oxygen uptake
surge is noted. The time limit is used because the ultimate BOD test is
designed to have implications for actual treatment situations in which deten-
tion time is limited. This is not to be interpreted as stating that the 30
day plus time period can be extrapolated to indicate the ultimate fate (in
terms of aerobic degradation) of a material. Thus, it can be suggested that
the time limit on the R,I. classification determines persistency. On the
other hand, 30 days is far in excess of treatment times in wastewater treat-
ment plants.
2. Non-optimal environment - If a naturally occurring material is found
to resist degradation under the conditions provided in an experimental
system, the most obvious explanation for this persistency is that the en-
vironmental conditions of the test are inappropriate. In this case, the
environmental conditions include fixed temperature, nutrients, constant pH
(buffered), heterogenous bacterial seed, absence of light and sufficient
substrate. The interaction between time and this environment in the pre-
sence of microorganisms creates the biochemical environment, an enzymatic
system which whould degrade the compound of interest. These tests thus
take place under specific environmental conditions which may be non-optional
for the degradation of specific materials.
Although it is possible to conduct further study to determine what
conditions or series of conditions would accelerate the degradation pro-
cess, the information gained would not be directly applicable to the
existing biological wastewater treatment systems. It should be noted that
the main intent of this study is to determine persistence under conditions
which could be directly applied to existing systems. Therefore, exten-
sive acclimation procedures are not used since acclimated bacterial popu-
lations are difficult to maintain in wastewater treatment plants. There
is a reasonable degree of acclimation in the 30 day test period used.
14
-------
The environment of the BODu test was chosen to coincide with the
standard BOD technique; this approximates the optimum condition of a bio-
logical treatment process and is judged to be a generally optimum con-
dition for aerobic degradation.
3. Missing Link in Path of Degradation - Degradation is promoted by
enzymes produced by microorganisms. The microorganisms involved coexist
in a trophic dynamic balance with each other and within an entire eco-
system. If the balance normally found in the natural aquatic environment
of a lake or stream is critical to the degradation pathway for the material
being tested and is not present in the ultimate BOD test, the material
would be classified as refractory. It should be noted, however, that in
the BODu test sewage seed was used; the bacterial population found in
sewage is more heterogenous than that of most natural environments.
B. PRESENTATION AND INTERPRETATION OF R.I. DATA FOR NATURALLY OCCURRING
ORGANIC COMPOUNDS
Naturally occurring compounds are defined as those compounds commonly
found in metabolic pathways. This semi-arbitrary classification dis-
tinguishes these organic materials from the manufactured or process com-
pounds of industry. It is, of course, not possible nor correct to have
an exact distinction between natural and synthetic materials.
Table 2 lists compounds considered natural in this report; these
include simple substrates (e.g., glucose), building blocks of complex
life forms (e.g., amino acids), enzyme and co-enzyme intermediaries (e.g.,
adenine) and plant organic extracts (e.g., hydroquinone). Because these
compounds are naturally occurring, one might intuitively expect close to
complete degradation in the long-term BOD test as well as other aerobic
biological reactors.* R.I. values for readily degradable organics might
be expected to range above the 0.8 or 0.9 levels. Note that, since the
coefficient of variation (s/x) is 0.13 for the R.I. determination, for
a totally degraded material, ideal values of 0.87 to 1.13 would corres-
pond to a spread of one standard deviation.
Compounds such as glucose and glutamic acid do fall in this range.
However, many other natural materials fall short of this degree of degra-
dation, most notably the basic amino acids lysine and histidine, the co-
enzyme intermediary adenine and also acetic acid which is a product of
partial degradation.
*After all, it can be reasoned that we would be knee-deep in organic debris
if naturally occurring organics resist degradation. But there are, in fact,
persistent organic materials in the environment: life forms, petrochemi-
cals, humic materials. This brings into question the too often accepted
statement that natural organics are readily biodegradable. Thermodynami-
cally they are unstable but the pathway for degradation may not be avail-
able; inorganic nutrients can be limiting and inhibition can occur. Further,
can degradation of organic material occur without synthesis of the complex
organic material of the microorganisms involved with the decomposition?
15
-------
TABLE 2
REFRACTORY INDEX DATA FOR NATURALLY OCCURRING ORGANICS
Material
R.I.
BODu
TOD
TN
BOD5
TOC
Acetic Acid
Adenine
L-Aspartic Acid
L-Arginane
Butyrate (Na)
D-Glucose
D-Glutamic Acid
LrHistidine
Huniic Acids
Hydroquinone
L-Lysine
Propionate (Na)
Starch
L-Valine
0.61
0.12
0.81
0.65
0.84
0.93
0.98
0.52
0.0
0.41
0.47
0.80
0.63
0.93
mg as 02
100 mg
66
0
98
166
148
98
139
127
0
78
120
115
61
210
mg as 02
100 mg
125
207
97
196
175
107
121
181
240
190
208
143
127
198
mg as 02
100 mg
0
120
24
60
0
0
22
62
2
0
44
0
0
27
rag as 02
100 mg
71
12
69
90
135
77
86
69
0
2
82
108
50
185
mg as C
100 mg
47
44
36
45
55
40
41
47
64
66
49
49
44
51
mg as Q£ demand
10U mg
none
none
none
-71
none
-39
none
none
91
-19
2
none
11
none
-------
Table 2 presents the accumulated data for the naturally occurring or-
ganic group. This includes the R.I. values and normalized (per 100 mg of
tested organic) values for the ultimate Biochemical Oxygen Demand (BOD^) ,
the Total Oxygen Demand (TOD) and the Total Nitrogen (TN) content as well
as the nitrate correction value and related classic organic measurements:
5 -day Biochemical Oxygen Demand (BODs) and Total Organic Carbon (TOG) .
The graphical presentation of these data can be found in Part C of the
Appendix grouped alphabetically.
Certain individual compounds are described in the following para-
graphs with possible explanations for their R.I. values.
1 . Glucose and Glutamic Acid were found to degrade almost completely
(R.I. values of 0.93 and 0.98, respectively). Such a response is expected
since both compounds readily enter energy producing metabolic pathways,
are converted to C02 and F^O and do not usually form stable intermediaries.
If the energy produced in the degradation is not immediately released, it
is stored in other compounds (e.g., ATP) rather than as the compound itself
or a partially oxidized intermediate. The purpose for presenting R.I.
values for these readily degradable organics is to demonstrate the relia-
bility of the measurements.
L-Valine, (CH4)2CHCH(NH2)COOH, an acidic amino acid, was tested and
found to be readily degradable with an indicated R.I. of 0.93. Compounds
like glutamic acid and valine are known to be transformed to a -
ketoglutarate and ammonia by dehydrogenase enzymes during conditions that
favor amino acid oxidative degradation. The <* -ketoglutarate molecule
is an intermediate in the tricarboxylic acid cycle and consequently should
degrade almost completely to C02, H20 and
2. The relatively low R.I. value for the basic amino acids, lysine,
histidine and arginine concurs with previous research by Helfgott et_ al^ (19)
which identifies organic residuals from biological process effluents as:
carbonaceous, nitrogen bearing, cationic at high pH (electropositive at
pH = 9), dispersed, non-biodegradable and non-sorbable onto activated car-
bon. Possible explanations for intermediate R.I. values include the fol-
lowing which are not necessarily mutually exclusive.
a. A stable intermediary is produced that is more persistent than
the parent constituent. For lysine, for example, this might be the
decarboxylated compound cadaverine associated with decaying flesh and toxic
materials.
b. Some fraction of the material is converted to a refractory form
of the parent compound; e.g., lysine distributes itself between parent
L- lysine to a mixture of biodegradable I,- lysine and refractory D_- lysine;
Hence the R.I. value of about one half.
17
-------
c. The high solubility of certain organic constituents especially
small and charged species like lysine, causes a very slowly changing dis-
tribution between the aquatic phase and the bacterial mass where sorption
and degradation occur. The cationic nature of the basic amino acid may
limit the attachment to the bacterial cell mass. The portion of soluble
organic form in the water phase appears as persistent.
d. The eventual ecosystem developed in the bio-reactor used, the
Warburg Respirometer in this case, is an aquatic environment not adapted
to use all the lysine available as an energy source. The system is satis-
fied in this specific substrate requirement at some low concentration.
e. The basic amino acids as building blocks of protoplasm and cell
walls could be cycled in and out of the life forms without being degraded
to ultimate end products. Bacterial and algal cell wall structure, for
example, are known to persist in aerobic bio-reactors (H) (19) (49), and the
residual basic amino acids could reside there.
f. The basic amino acids are converted to polymers (perhaps the
end chain amino acid of cell wall structure of microorganisms). These
polymer forms of the amino acids appear as refractory.
3. Adenine, another basic nitrogen bearing organic (6 aminopurine),
is extremely refractory, R.I. = 0.12. Some of the same reasons for partial
stability of the basic amino acids apply here, but a strong case can be
made for conservation of the co-enzyme constituent by the bacteria that cy-
cle this chemical to support metabolisms of readily degradable substrates
like glucose and polymerize structural building materials like lysine.
Adenine is conserved in the microorganisms and not degraded as a substrate
energy source.
4. Certain other amino acids, valine, asparatic acid and glutamic acid
degraded almost completely (R.I.=0.93, 0.81, 0.98, respectively) in a pattern
anticipated for natural organics. These acidic amino acids can serve either
as energy substrate or as building material. In water of neutral pH these
amino acids are present as anions and can be readily sorbed by microorganisms
and metabolized.
5. Two organic acids, butyric and propionic, tested in sodium salt
forms showed ready degradation (0.84 and 0.87, respectively) as anticipated
for natural organic degradation intermediaries, but acetic acid shows only
partial degradation with an R.I. value of 0.61. It is thought that the
larger organic acids degrade to acetic acid (by means of a Beta oxidation
pathway), but acetic acid can either be further oxidized or enter into en-
zymatic pathways that build to larger organic materials of life. Like
lysine and adenine acetic acid is a relatively stable intermediary that is
conserved by cycling in and out of bacterial structures. (Synthesis of cellu-
lar material is a necessary half of the process of oxidative degradation.)
Acetic acid as a small highly soluble organic also leaves a large por-
tion in solution resisting adsorption into the biomass where degradation
and synthesis occurs. It is probably the protonated (un-ionized) form that
18
-------
absorbs and diffuses through bacterial cells by a hydrogen bonding'
mechanism. It is noted that effluents of sewage treatment plants con-
tain several mg/L of volatile organic acids especially acetic acid.
6. Starch is not a simple homogenous material but a polymer that
degrades first by hydrolysis to yield glucose, maltose and a high molecular
weight amylopectin which has a number of nonacetyl "ends"; thus, there are
branching and cross-linkages in natural starch in addition to linear glu-
cose polymers. That portion of the starch that yields simple sugars of
hydrolytic degradation undoubtedly degrades almost completely. The
unhydrolyzed and branched portion of starch appears relatively refractory.
This would account for the R.I. value of 0.63 showing partial degradation.
Starch is not hydrolyzed completely because of some branched linkage
that offers obstruction to enzyme attachment.
As a general rule for refractoriness, branching is an accepted empiri-
cal factor. The infamous case of "hard" (biorefractory) branched alkyl-
benzenesulfonate vs. "soft" (biodegradable) linear alkys sulfonates of
the detergent industry illustrates this point of branching limiting degra-
dation.
7. Hydroquinone (HO-Cfcfy-OH) is a natural material of plants that
also showed surprisingly high resistance to degradation (R.I. = 0.4).
This is difficult to explain but it is an empirical fact that symmetric
compounds (e.g., benzene) seem to show higher resistance to biodegradation
than unsymmetrical compounds (e.g., phenol).
8. Humic acids were found to resist degradation almost totally,
R.I. = 0. Such a result is consistent with known properties of this residual
found in natural waters. Humic acids, a non-chemically specific group of
large molecular weight that is soluble at low pH, are known for imparting
yellow color and for long-term persistence. In the ambient environment,
some humic acids probably disappear by adsorption, assimilation and de-
gradation.
It is interesting to speculate as to what causes the formation of
humic acid in the environment in order to obtain hints as to the persis-
tent nature of the material. The organic debris of plants and animals
enters into an aquatic environment that is generally nitrogen deficient.
Bacteria and algae utilize the organic nitrogen or mineralized nitrogen for
growth. The carbonaceous residual skeleton of decaying plants is thus a
localized environment that is nutrient unbalanced so that it no longer
constitutes a suitable substrate for microorganisms. Thus, it persists
for long periods of time. Some of the humic acids are the stable inter-
mediaries of biodegradation of resistant cellulose and natural starch
forms that were previously mentioned. They are high molecular weight,
condensed, aromatic compounds low in nitrogen but high in partially
oxidized intermediary groups (e.g., carboxyl groups). The size, branching
and resistance to hydrolysis fragmentation create a natural refractory
residual.
19
-------
C. PRESENTATION AND INTERPRETATION OF R.I. DATA FOR SYNIHETIC AND INDUSTRIAL
ORGANIC COMPOUNDS
The distinction between synthetic or industrial organic compounds and
natural compounds is to some extent arbitrary and is used here only to make
a convenient category for discussion. This category includes a wide variety
of compounds: common, simple organics of industry (e.g., phenols and benzene);
widely used materials that find their way into municipal treatment plants
(e.g., glycols and gasoline); the chemicals of agriculture (e.g., pesticides);
and some representative chlorinated organics (e.g., chloroform) that may be
produced during chlorination practices of water and wastewater treatment.
Materials found to fall into the refractory classification (low R.I.
values of <0.6; i.e., 1.0 - 3(s/x)) are not expected to degrade in an aerobic
wastewater treatment system such as an activated sludge process are expected
to .persist in natural water systems such as aquifers, rivers and lakes. The
practice of discharging such persistent industrial materials into the
environment should be discouraged.
Table 3 lists the compounds under this category for R.I. value, nitrate
correction and normalized parameters of oxygen demand and organic content.
Item C of the Appendix includes the graphic presentation of this information.
Many of the reasons for the occurrence of refractory natural organics
also apply to synthetic organic materials; in addition, much research and
development has been done by industry to create materials which.persist; i.e.,
do not degrade on the shelf, hold up under severe environmental conditions
and generally are not readily altered by bacterial actions. For example,
dyes are often selected that resist degradation by sweat, light, microorgan-
isms, severe cleaning conditions and are not resolubilized.
1. Acetone (dimethyl ketone) falls into the category of readily de-
gradable industrial organic materials. Acetone is a product of the anerobic
fermentation of material carbohydrate materials such as potato starch,
maize, etc. Acetone bodies are also found in sewage wastewaters as a by-
product of urine and the biodegradation of fats. The average R.I. value
of 0.8 (separate determinations of 0.93 and 0.71) for acetone demonstrates
its degradation to ultimate end products in the aerobic and nutrient rich
environment provided by the test.
There may have been volatile losses of acetone during the test procedure
but this common solvent of industry also has an easy entrance mechanism
through bacterial cell walls where oxidative degradation occurs.
2. Benzene and Phenol show disparate R.I. values that reflect findings
reported elsewhere (21) (39). Phenol undergoes biochemical oxidation in the
respirometer environment (R.I. = 0.87), while benzene resists degradation
(R. I. = 0.23). This presents an interesting example of how apparently similar
materials demonstrate very dissimilar degradation characteristics. Resonance
structure and symmetry are the factors imparting persistence to benzene; the
20
-------
Material
TABLE 3
REFRACTORY INDEX DATA FOR SYNTHETIC AND INDUSTRIAL COMPOUNDS
R.I. BODu TOD TN BODs TOG NOs"
mg as 02 mg as 02 mg as 02 mg as 02 mg as C mg as 0? demand
100 mg 100 mg 100 mg 100 mg
TOO mg
UO mg
Acetone 0.93
Aniline 0.58
Benzene 0.23
Biphenyl 1.14
Bipyridine < 0
Chloroform < 0
p-Chlorophenol 0
CMC 0
Cyahuric Acid < 0
DDT < 0
Dichlorophenol < 0
Ethylene Glycol 0.76
Gasoline 0.21
Phenol 0.87
Propylene Glycol 0.78
Sevin 1.0
Vinyl Chloride 0
220
208
69
198
-1
0
112
70
206
134
95
0
220
283
308
174
273
34
169
115
93
141
136
129
334
239
168
90
140
0
34
0
0
41
0
124
150
-86
64
-
-
0
0
74
0
0
0
0
0
0
5
0
-16
0
85
54
172
108
9
0
62
77
92
78
70
10
64
43
28
53
47
38
96
77
47
20
30
0
•18
-12
13
0
0
•57
-6
0
6
0
0
93
•44
0
0
0
-------
ability for the un-ionized (pK = 10) phenol to diffuse by a hydrogen bond
mechanism through microorganisms is thought to be the reason for the ready
degradation of phenol.
3. Aniline, in contrast to both refractory benzene and biodegradable
phenol, showed an intermediate refractory index of 0.58. Aniline is known
to be very sensitive to oxidation but is converted into a host of partial
oxidation products including azobenzene, azoxybenzene, nitrobenzene, qui-
none, dyestuff Aniline Black and several other intermediate end products
resulting from secondary products of condensation. The test method
developed for R.I. evaluation does not indicate intermediate hydrolysis
products or stable carbonaceous structures such as ring configurations.
Thus, those degradation products of aniline analogous to benzene appear
refractory and those analogous to phenol appear biodegradable. The hetero-
genous nature of the oxidation products of aniline results in an inter-
mediate indication of biodegradability.
4. Ethylene Glycol and Propylene Glycol with R.I. values of 0.76 and
0.78 fall into a category of compounds with high but not complete degrada-
tion. These are materials of commerce which are used in such large quanti-
ties for antifreeze and road de-icing that persistence in the environment
would be a problem. These materials are generally considered to degrade
well but not completely. This is probably a function of the high solu-
bility of these low molecular weight, double alcohol group materials. It
is not expected that these organics would be a serious problem except for
the oxygen demand.
5. Sevin (Carbaryl Sevin) is used as a pesticide, often replacing DDT
because sevin is known not to persist but rather to degrade almost totally.
The R.I. determined as 1.0 confirms this complete degradation. It is
interesting to compare this unchlorinated pesticide ((/j^H^^N) with the more
recalcitrant chlorinated pesticides.
6. Chlorinated organics have been found to fall into the category of
persistent organics by many current investigations (12)(15) (35) (38).
Of special current concern is the creation of persistent organics by
chlorination for disinfection of water supplies. The zero values of R.I.
for p-chlorophenol and vinyl chloride, indicating no degradation, as well
as the negative calculated values of R.I. for DDT, chloroform and dichloro-
phenol, indicating an inhibition of the bacterial system in the respiro-
meter, are of particular concern.
Materials in the chlorinated hydrocarbon category responded differently
to the ultimate BOD test than did the more readily degradable materials.
These chlorinated organics are found either to resist degradation or to
inhibit the bacterial populations in the sewage of the test solutions. The
ultimate BOD measurement is therefore found to have an application in
quantifying the degree of inhibition. For instance, the BODu for dichloro-
phenol plus sewage was evaluated at 260 mg/L 02, while the sewage alone had
22
-------
a BODu of 630 mg/L 02. This response not only indicates a resistance to
degradation by dichlorophenol but also an inhibition of sewage degradation
in the respirometer environment of roughly 60%. The R.I. calculation
from such data results in a negative number which, although indicative,
is difficult to interpret quantitatively; therefore, the S.i.v. is used to
quantify the response of the microorganisms in the respirometer environment
to these inhibitory organic materials.
Another interesting set of R.I. responses from chlorinated organics
is the negative oxygen uptake values (BOE^ of test lower than BODy of
blank) from DDT samples. Since, as noted, a negative R.I. value has
qualitative but not quantitative usefulness, a different way to express
the degree of problem of these organics is suggested. The relative loss
of ultimate BOD of the test organic material spiked into sewage of a deter-
mined BODy seems to be a more meaningful way of suggesting quantitative
inhibition. This calculation is termed the Biological Interference Value
(B.I.V.).
B.I.V. = (BODu2 - BODui)/BODu2 [6]
where BOD^ = BODy of sewage plus test organic
and BODu2 - EGD^ of sewage alone.
Table 4 gives the B.I.V. numbers for the limited number of inhibitory
organics tested.
7. Biphenyl and Bipyridine illustrate some interesting contrasts to
the chlorinated organics. Chlorinated biphenyls have been reported as
inhibitory to enzymatic systems; some are reported to be carcinogens as
well as refractory residuals. In these tests, the unchlorinated biphenyls
showed complete degradation with R.I. of 1.14 (considering the s/x of
0.13, this value is an indication of complete degradation). On the other
hand, biphenyls have been found in the natural environment (polar ice cap
cores). It should be stressed that the respirometer environment provides
a more suitable ecosystem for biological degradation than most natural eco-
systems. Natural environments, such as polar ice caps, tend to be nutrient
deficient whereas- the biochemical oxygen demand test conditions of the
respirometer provide nutrient excesses.
In bipyridine, one C on each phenolic ring is replaced with N. Bipyri-
dine is a refractory chemical that inhibits biochemical degradation of
sewage. The R.I. is negative since the ultimate BOD of the test chemical
bipyridine, is less than the ultimate BOD of the sewage into which it is
spiked. The B.I.V. number for bipyridine was found to be 0.56 as listed in
in Table 4.
To verify the results of high degradability for biphenyl, gas chroma-
tography analysis were conducted on the raw and bio-oxidized biphenyl
solutions. The gas chromatography data were collected to determine the
presence of biphenyl before and after biochemical oxidation under test
conditions. The biphenyl, which was assayed at 99% pure could not be
detected at the end of the test period.
23
-------
TABLE 4
CHEMICALS WHICH INHIBIT RESPIROMETER RESPONSE
calculated
Chemical B.I.V. R.I.
Bipyridine 0.56 -0.47
Chloroform 0.71 -0.27
Cyanuric Acid 0.26 -0.22
DDT 0.42 -0.21
DDT with carrier 0.34 -0.25
Dichlorophenol 0.59 -0.77
8. CMC (Carboxymethyl cellulose) is replacing starch as a sizer in the
cloth fabrication industry. In contrast to starch, which has the relatively
high R.I. value of 0.7 to 0.8, CMC has an R.I. value of 0, indicating a
refractory nature.
Users of sizers had been told to stop using starch because its biodegrad-
ability exerts a large biochemical oxygen demand on treatment plants and the
receiving aquatic environment. The industrial concerns, by switching to
CMC, avoided the oxygen demand problem but placed into the environment the
refractory CMC, which passes unaltered through treatment plants. This is a
clear-cut case of perversion of purpose which occurs when only one measure
of pollution (BOD) is used. A single measure of pollution can be manipulated
to show no apparent pollution, while the actual consequence to the environ-
ment is worse since the substitute persists, accumulates in the environment,
and eventually does harm. If, as advocated here, the ultimate BOD is com-
pared to the total oxygen demand, the pollutional effects of materials like
CMC become evident. Then alternative treatment processes can be selected
to remove if not reclaim this material.
OH OH OH
9- Cyanuric acid, N = C - N = C - N = C , is one of the few or-
ganic materials that appears to be more degradable in an anaerobic environ-
ment than in an aerobic environment. It is discussed here because of its
uniqueness (40).
24
-------
Cyanuric acid is a well-oxidized organic with a symmetrical ring
structure which implies high resistance to degradation. It can be
degraded more quickly by hydrolysis, a process which is accomplished both
in aerobic and anerobic environments. Since this material does not
serve well as an energy source, it has little oxidative potential.
Further, cyanuric acid is inhibitory to bacterial growth with a B.I.V.
number of 0.26.
In attempting to develop general rules for which organic materials
are degradable or persistent, there is the danger of overgeneralizing.
Cyanuric acid is an exception to the general rule that anaerobic degrada-
tion is quicker and more complete in reaching ultimate end products.
25
-------
D. PRESENTATION AND INTERPRETATION OF R.I. VALUES FOR COMBINATIONS OF
ORGANIC MATERIALS
This section of the report includes a discussion of data on organic
materials that are composites from municipal and industrial sources.
The absolute concentration of materials in composite wastewaters and
mixtures of organics is seldom known; therefore, the R.I. value as a nor-
malized ratio of ultimate biochemical oxygen demand to ultimate total
oxygen demand is particularly useful. The test technique can be applied
to composite wastes for which individual component assays are very diffi-
cult. Table 5 lists the data for combinations of materials.
1. Domestic sewage is a heterogenous mixture of numerous organics
in transient form tinder bacterial modification. The organics are of
natural, synthetic and industrial origin. This dynamic mixture of thou-
sands of different organics in dilute solution can contribute refractory
organics to the environment via the effluents and/or sludges of waste-
water treatment plants. Some refractory organics can even be created
during the treatment of wastewaters; for example, endogenous respiration
during activated sludge treatment contributes refractory bacterial cell
walls (11) (19) and chlorination produces refractory chlorinated hydro-
carbons .
The R.I. values shown in Table 5 are averages of 11 different
determinations in sets of at least three. These data illustrate the
relatively high average Refractory Index (R.I. = 0.7). The range is
also shown as an indication of sewage variability. Although raw sewage
is biodegradable, the R.I. value of 0.7 indicates the presence of some
refractory material.
2. Gasoline showed a low R.I. value of 0.21; it is known to be a
relatively persistent organic. The low solubility of gasoline is thought
to make it relatively unavailable for bacterial degradation in most
situations; however, a special procedure was used to disperse the gaso-
line in the respirometer for the BODu determination. The R.I. value is
for the soluble and micro-colloidal gasoline constituents.
When a commercial unleaded gasoline was tested, the BODu for the
sewage plus gasoline was slightly less than for sewage. The presence
of gasoline, however, completely inhibited the nitrification of the
nitrogen compounds in the sewage. When the AN03" correction was added
to the BODu1, an R.I. of 0.21 is calculated. Published data indicate that
gasoline degrades 22% in a BOD5 test. The nitrification problem is dis-
played in the Appendix; the typical nitrification hump exhibited for •
sewage Warburg Respirometer response did not occur for gasoline plus raw
sewage.
The gasoline was prepared for this test as follows: 50 ml gasoline
was added to 450 ml distilled water. This was stirred for several days,
then put in a separator funnel for approximately 30 days. The water was
carefully drawn off at the bottom in order not to include the lighter
gasoline fraction visible at the top. Tests of this mixture showed a TOC
26
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TABLE 5
REFRACTORY INDEX DATA FOR COMBINATIONS OF ORGANIC MATERIALS
Material R.I. BODu TOD TN BODs TOC
Antifreeze
DDT (with carrier)
Ethylene Glycol
(with inhibitor)
Gasoline
Humic Acid
Textile Dye -
Industrial Waste
Dye Material
Raw Sewage
Raw Sewage -Range
1.12
0
0
0.21
0.0
0.05
0.58
0.7
0.5-
0.9
mg as Q£
100 mg
208
0
0
70
0
mg/L 02
3.2
314
380
160-
650
mg as 02
100 mg
180
93
129
334
240
mg/L 02
527
468
470
180-
930
mg as 02
100 mg
0
0
nil
nil
2
mg/L 02
71
97
105
75-
190
mg as 02 mg as C mg as 02 demand
100 mg 100 mg
171 39
0
38 38
54 96
0 64
mg/L 02 mg/L C
0
-
220
130-
400
100 mg
0
16
_
93
30
mg/L 02
95
92
-
-------
of 76 mg/L C and a TOD of 270 mg/L 02- This is estimated to be 80 mg/L as
octane which yields a theoretical oxygen demand of 277 mg/L 03 and theo-
retical carbon of 67 mg/L C. In the test solution, 100 ml of raw sewage
were placed with 250 ml gasoline, and mixture aliquots of this were placed
in the Warburg flask.
It is particularly interesting to note that the presence of gasoline
seems completely to inhibit the nitrification of sewage. Gasoline is, of
course, the product of distillation of crude oil, a naturally occurring
refractory organic mixture.
I. Dyes represent typical refractory materials in that they resist
by design alteration in use. A dye was tested and found to have an R.I.
value of 0.56, indicating partial resistance to degradation in the test
used. The dye was most likely a combination or organics rather than a
single pure material. In the manufacture and application of dyes, inhibi-
tors are commonly used to impart greater stability.
From the same manufacturer that produced the dye discussed above, some
industrial wastewater was obtained and tested under the same procedures
described in this report. The R.I. value of this waste was found to be
0.05, indicating almost total refractoriness. This wastewater normally
passes through a rather elaborate treatment plant, primary sedimentation
and activated sludge, then into local rivers. It is interesting to note
that the dyes can be seen passing through these waste treatment steps and
into the receiving environment, clearly indicating the inappropriateness
of these treatment steps for this product.
Many of the organic constituents of this particular industry resist
biochemical degradation. Obviously, treatments other than bio-reactors
should be used for such wastes; in this case, activated carbon might be
more effective in protecting the receiving environment.
Color measurements were conducted on the initial and final test solu-
tions containing the individual dye in order to quantify the degree of
color removal due to long-term ( = 30 days) biodegradation procedures of
the Warburg Respirometer. Table 6 shows predominant wavelength, percent
luminance and percent colorimetric purity for the dye waste before and
after degradation in the Warburg Respirometer.
TABLE 6
COLOR ANALYSIS OF TEXTILE DYE
Before Oxidation After Oxidation
Predominant Wavelength (nm) 576.0 560.0
Luminance (%) . 60.2 84.0
Colorimetric Purity (%) 7.5 3.0
28
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This table shows that there was a 40% increase in total luminance and a
60% decrease in color at the predominant wavelength. This response
indicates a reduction in color influence during biological oxidation and
corroborates the R.I. evaluation.
. The industrial waste discharge of the manufacturer was also found to
resist biological degradation in the Warburg Respirometer (R.I. = 0.05).
Spectrophotometric color analysis of the filtered effluent, however,
shows a 200% increase of total luminance and a 70% decrease of color
purity, displayed in Table 7. These data indicate removal of color
during the biological degradation process and, therefore, disagree with
the R.I. evaluation. Such an apparent contradiction of results between
the R.I. value and the color loss can be explained by observing another
mechanism of pollution removal during the biological treatment (i.e., other
than biodegradation). At the end of the test, it was observed that the
biomass in the Warburg Respirometer contained color while the filtrate was
TABLE 7
COLOR ANALYSIS OF INDUSTRIAL DYE WASTE
Predominant Wavelength (nm)
Luminance (%)
Colorimetric Purity (%)
Before Oxidation
508
31.4
40
After Oxidation
500
92
12
relatively clear. The conclusion, therefore, is that dye constituents are
concentrated by biochemical or physical sorption. The implication of this
mechanism is that dye becomes a refractory constituent of sludge: i.e.,
the problem material is only transferred to another area, not degraded.
In fact, the writers have observed that dried activated sludge from a
municipal sewage treatment plant receiving industrial dye waste had a dye
color. Since this particular sludge is disposed of at a land fill,
leachate may contain pollutants from the dye.
4. Commercial grade antifreeze, largely ethylene glycol, was tested
and found to have an R.I. of essentially 1.0. (The actual value was 1.T2
but there is 13% relative error). Although the antifreeze did not appear
to be pure ethylene glycol, the UOD was the same as ethylene glycol.
undoubtedly, there were additives (or diluents) in the commercial anti-
freeze. Since the exact chemical composition of commercial products and
industrial waste is rarely known, the UOD measurements and comparisons are
particularly useful.
29
-------
6. Laboratory ethylene glycol listed as having an unspecified in-
hibitor additive had a negative R.I. value. This compares with a pure
ethylene glycol R.I. of 0.8 and an antifreeze R.I. of about 1.0. The con-
centration of the ethylene glycol and other additives in antifreeze were
not known. The presence of inhibitors in commercial composite materials
is a factor to be taken into account in wastewater treatment.
7. DDT in a carrier is reported on as a composite material. The
test also indicates an inhibitory response for the composite (B.I.V. =
0.34) which is close to that of DDT alone (B.I.V. = 0.42).
8. Humic acid data are repeated in Table 5 since this is not a
single material but a classification grouping developed by soil scientists
for refractory organics that are insoluble at low pH.
E. TEST PROCEDURES: INTERFERENCES, LIMITATIONS, CORRELATIONS AND
CONFIRMATIONS
1. Interferences
When industrial wastes include high concentrations of heavy metals or
sulfur, the R.I. technique must be interpreted carefully in evaluating the
persistence of organics. Heavy metals and sulfur can exert an apparent
high TOD and interfere with the long-term biochemical oxygen demand evalua-
tion. For example: Cd++ - » CdO and Organic S - » S02- The
sewage used for the tests reported here had low metal content (e.g.,
Cd < 20Ug/L) and low sulfur (organic sulfur < 1.1 mg/L; sulfides <
2 . 0 mg/L as a maximum) ; these values are well below the oxygen demand of
the tested solutions and are too low to have caused significant interfer-
ences .
In order to quantify the effect of metals on the test procedures, three
heavy metals, cadmium, mercury and lead, were tested. Figure 2 shows the
interference with BODs for increments of metals added. The amount by which
BOD5 is diminished by the metal is expressed as a fraction:
f = BODs with metal/BOD5 of sewage alone [7]
In order to normalize the metal concentration it is expressed as mg/L
CaC03 eq. (divide by 50 for meq/L). The BODs determinations were made
by the classic Standard Methods bottle BOD technique.
a. Cadmium - The interference from Cadmium is marginal although
present and seems to reverse itself at higher concentrations.
b. Lead - The interference from lead is pronounced and has high
scatter.
c. Mercury - The interference from mercury is the most pronounced
of the three metals, also with scatter.
30
-------
G-J
1.0
0.8
0.6
0.4
0.2
FIGURE 2
METAL INTERFERENCE WITH B005 DATA
1
10
mg/l METAL as CaC03 eq
20
-------
For inhibitory industrial wastes containing metals or toxic organics,
the calculated R.I. values will be negative, indicating a problem. Quan-
titatively, this can be expressed as a B.I.V. number. Specific analysis
would then be necessary to determine the interfering agent. The test pro-
cedure detects the presence of inhibitory materials.
The Appendix includes a set of curves for the Warburg Respirometer
BOD determinations for these metals.
2. Limitations
The limitations of the R.I. evaluation are discussed here in terms of
its purpose and applicability. The purpose of the R.I. is to determine if
an organic compound or a waste is biodegradable. The organic compound or
waste is subjected to bio- oxidation by sewage microorganisms. The total
amount of oxygen used in aerobic biodegradation (BODu) is measured by means
of a Warburg Respirometer and is compared to the total amount of oxygen that
would have been used had all of the compound been oxidized to the natural
end products of complete aerobic biodegradation (UOD) . The UOD can be
measured by using TOD and TN or it can be calculated from the compound
formula and quantity added to the sewage (if known). The R.I. is the ratio
of BOD to UOD.
a. UOD - The UOD determines the "strength" of the waste. For a
pure organic compound, the UOD can be calculated. For industrial wastes,
the "strength" depends on what compounds are present and in what concen-
trations. Since this specific information is not always known, it is
determined by use of the TOD and TN measurements. For these evaluations
to be accurate, the waste must have a relatively high TOD value; TOD loses
its sensitivity below 10 mg/L 02- All of the work done in this research
was performed in the 200 to 300 mg/L 02 range, the range of highest pre-
cision.
The TOD parameter cannot be used on wastes containing high levels of
certain oxidizable inorganic substances. Ammonia, the exception, is mea-
sured as part of the TN determination. At the high temperatures involved,
certain inorganics exhibit oxygen demand or release oxygen, casting doubt
on the TOD evaluation. In this category are included compounds that contain
sulfur, nitrate, nitrite and wastes containing metals in high concentrations.
In some cases, corrections can be made if detailed analyses of the waste-
water are done. In the tests conducted in this campaign, these interfer-
ences were minimal.
b. BODu - The BODy is determined by use of a Warburg Respiro-
meter. This instrument uses a manometer to measure partial pressure
changes due to the removal of oxygen from the atmosphere of the flask.
It is assumed that the only gas exchanged and measured is Q£ and that any
002 evolved from the biological reaction is converted to the carbonate-
bicarbonate system either in the test solution or the alkali solution in
the center well which contains KOH. Since the pressure is measured,
32
-------
wastes exhibiting significant volatilization, like chloroform, cannot be
evaluated for R.I. by use of the manometric technique. A bottle BOD series
is more appropriate for these.
The Warburg BODu is assumed to represent as complete a biodegradation
response as is possible for the specific compound being tested. Optimum
conditions are provided for nutrients, pH, temperature and oxygen tension.
It should be noted, however, that not all possible conditions are tested
for. Some compounds, like cyanuric acid, are more susceptible to anaerobic
biodegradation. Microorganisms not found in sewage that exist in nature
may be able to degrade the compound. Perhaps an acclimated population would
be best as in the case of CMC. For the purposes of waste treatment,
however, "special" conditions and "special" bacterial populations are hard
to maintain as they are easily imbalanced by variations of industrial
wastes in treatment plants.
One major disadvantage of the Warburg BODy test is time. Because of
the nature of the biodegradation process, and to assure as complete a reac-
tion as possible, the BODu test i-s extended to 30 days, more when the curve
is not on a plateau. When results from this test should be verified by
retesting, the time element becomes an increasing limitation. Correlations
are provided in the next section between quick- time measurements, TOD, TN
and TOC and the naturally slow biochemical oxygen demand tests.
It was mentioned earlier that wastes tested must be of high UOD classi-
fication for the purposes of TOD analysis. This also holds for BODy.
The combined response of the compound plus sewage must be significantly
above that of sewage alone. The BODu has an error associated with it of as
much as 11.5%. One way to insure the desired response is to put more
waste with the sewage, but then the size of the Warburg reaction flask
becomes a limiting factor. The total volume of liquid in the flask should
not exceed about 30 ml for 125 ml flasks.
These limitations, however, do not reduce the usefulness of the R.I.
to evaluate the biodegradability of organic wastes and compounds, provided
the limitations are known and understood. Inasmuch as these limitations
would exclude organic wastes exhibiting low oxygen demand (UOD) and com-
pounds exhibiting interference and high volatilization, all other water-
borne organic wastes, the vast majority, can be tested for R.I. with a high
degree of confidence.
3. Correlations
Correlations between bioassay measurement techniques, such as the
classic slow time BOD tests, and the real-time (quick determination)
of oxygen demand, TOD, or measure of organics, TOC and TOrganicN, as well
as oxidizible nitrogen, TOxN, for ammonia and organic nitrogen can supply
useful information for operational management of wastewaters, but must be
carefully considered.
33
-------
The technical paper "Quick-Time Instrumental Measurements of Organic
Characteristics" provides the derivation of correlation equations along
with the rationale and test procedures to arrive at the model equations
shown in Table 8. (18).
The correlation coefficients (r) demonstrate the reliance on the TN
determination in this work for estimating the organic content and oxygen
demand of wastewater along with the TOD value. TOG, while an effective
measure of carbonaceous organics, is not complete in determining an or-
ganic response to oxidation.
It must be cautioned and emphasized, however, that all correlations,
like crude analogies, are fraught with danger of hidden "lurking" variables
(e.g., the presence of refractory organics and/or inhibitors) and should
be used with great care in evaluating variable and industrial wastes. In
many cases, it is more appropriate to use the complete (total and gross)
measurements such as TOD, TN and TOG rather than the indirect, incomplete
biochemical response measurement such as BODg.
TABLE 8
MODELS FOR QUICK-TIME OXYGEN DEMAND MEASUREMENTS
OF RAW SEWAGE
Relationship Equation r
BODu = 0.90 (TOD + TN) - 32 0.98
BODs = 0.51 (TOD - TN/2) + 67 0.97
BODuc =0.48 (TOD - TN/2) + 151 0.57
BODs = °-58 (TOC) + 106 0.74
4. Correspondence of R.I. Value to Activated Sludge Treatment for
Selected Compounds" "
In order to evaluate the appropriateness of the R.I. value to sewage
treatment, three compounds were tested in a model activated sludge unit.
a. Experimental Procedure
The treatment system used in this experiment has the following
design characteristics (42):
Aeration Detention Time 6 hours
Sludge Recycle Rate 45%
34
-------
Primary Settling Detention 90 minutes
Secondary Settling Detention 105 minutes
Secondary Settling Overflow Rate 21,000 liters/day/m2
• Wastewater was pumped to this system at a constant rate (76/liters/
day) from a 60-liter holding tank. A previous study of this laboratory
scale treatment system showed TOD reduction in the range of 60 to 88%
with an average TOD reduction of 75%. An outline of the experimental
procedure is given below:
(1) The system is fed with domestic sewage for at least two
hours. Normal operation of this plant is to feed domestic sewage during
the day and rest the plant during evenings. The procedure of feeding raw
sewage for at least two hours prior to the experiment is to obtain suf-
ficient background data.
(2) A mixture of domestic sewage and chemical solution (50/50
percent by volume) is fed to the system at a total strength (TOD) approxi-
mately equal to the raw sewage. This precaution is followed to prevent an
organic overload on the biological processes. It should also be noted that
this mixture is similar to the solution used for the Warburg BOD analysis.
(3) TOD analyses are conducted on the secondary effluent
throughout the test to see if the TOD reduction is changed. An increase of
TOD in the effluent is interpreted as a negative process response to the
biological processes. No significant change in the TOD effluent is inter-
preted as a positive degradation response. Gas chromatography analyses of
the effluent were performed to determine if the added compound passes
through the biological process.
It should be noted that a thorough evaluation of the treatment response
phenomena for these chemicals would involve a number of runs and statis-
tical analyses of the results to verify that the biological treatment
variables, such as sludge age, settling characteristic of floe, and varia-
tion in removal capabilities, are independent of the final conclusion.
Such a procedure was not followed in this case because the data collected
are only intended to give a general concurrence with the R.I. values.
b. Glucose
Table 9 lists the secondary effluent TOD concentrations from time zero
(when glucose and raw sewage mixtures are first introduced) until the
holding tank contents are exhausted. The raw glucose/sewage mixture has
a TOD of 189 mg/L. The data show no pronounced change in TOD reduction
for the glucose/sewage mixture as compared to the sewage alone and, there-
fore, indicate a removal of glucose in the biological process. Because
glucose is classified in the very high R.I. range, the activated sludge
treatment response and the R.I. evaluation are in concurrence.
35
-------
TABLE 9
TOD RESPONSE FOR D-GLUCOSE/SEWAGE EFFLUENT
Secondary
Time, Effluent TOD I TOD
Hours (mg/L as 02) Removal
0 73 61
2 87 54
5 96 49
6 67 65
7 91 52
8 76 68
9 70 63
11 58 69
12 70 63
14 67 65
c. Lysine
Table 10 lists the secondary effluent TOD concentration from the
time of the lysine/sewage mixture injection to completion of the run.
The raw lysine/sewage solution has a TOD of 208 mg/L. The data show a
gradual decrease in the percent TOD reduction. Such a treatment response
concurs with the low R.I. characteristic of L-Lysine. The average R.I.
value of lysine is 0.54. This index value indicates a lower degradability
potential than domestic raw sewage (R.I. = 0.7). The increase of TOD after
biological treatment, therefore, is judged to be due to a decrease in bio-
degradation for the lysine/sewage mixture as compared with the raw sewage
alone.
36
-------
TABLE 10
TOD RESPONSE FOR L-LYSINE/SEWAGE EFFLUENT
Secondary
Time, Effluent TOD % TOD
Hours (mg/L as 0?) Removal
0 105 50
2.5 106 50
4.5 99 52
6.5 111 47
7.5 99 52
9.5 128 38
10.5 157 25
11.5 144 31
12.5 161 23
d. p-Chlorophenol
Table 11 lists the secondary effluent TOD concentration from time
zero to completion of the run for a mixture of sewage and p-chlorophenol.
The raw p-chlorophenol/sewage mixture has a TOD of 228 mg/L. Table 11
shows a reduction in TOD removal and, therefore, concurs with the negli-
gible R.I. evaluation of p-chlorophenol. Gas chromatography analysis of
the secondary effluent shows a gradual increase of p-chlorophenol from
0.0% at time zero to 571 at seven hours and 98% at twelve hours. Because
p-chlorophenol is identified as refractory, the expected treatment response
of this material is to pass through the process. An increase of secondary
effluent TOD is judged to indicate this type of response, i.e., the
presence of a refractory organic if an inhibitor of the activated sludge
process.
37
-------
TABLE 11
TOD RESPONSE FOR p-CHLOROPHENOL/SEWAGE EFFLUENT
Secondary
Time, Effluent TOD % TOD
Hours (mg/L as 02) Removal
0 117 48
2 117 48
4 . 125 45
6 174 23
7 165 27
8 171 25
12 156 31
38
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SECTION 6
CLOSING STATEMENT
This report presents quantitative evaluation techniques for deter-
mining if specific organic constituents, either singly or in combination,
are resistant to biodegradation or are inhibitory to the bacterial systems
used in wastewater treatment plants to process organic materials before
they enter the aquatic environment.
In addition to the measurement techniques, this report presents the
test results of a Refractory Index (R.I.) evaluation for over 38 waste
constituents. A Biological Inhibition Value (B.I.V.) was developed and
is used to assess six of the tested organic materials which were found to
interfere with the biochemical tests used (long-term Warburg Respirometer
BOD determinations). In addition, confirmation of Refractory Index values
has been made for three specific organic materials in at model (activated
sludge) bio-reactor. Correlations between the ox»/gen demand tests usisd
(BODs, BODu, TOD) and other measures of erganics (TOC, TN) are also
presented.
Industries should be encouraged to reclaim those raterials o£ value
from their effluent including organic constituents and the water itself
(36). If, however, the wastewaters are sent into municipal treatment plants,
there should be assurances that the specific wastes are treatable; i.e.,
degradable in the bio-reactors of the treatment plant and non-interfering
(non-inhibitory) with the available processes of the treatment plant.
A set of suggestions for pretreatment requirements for industrial waste is
included in the Appendix to the report as Item D.
The reference list which follows is partially annotated.
39
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REFERENCES (PARTIALLY ANNOTATED)
1. Alexander, M. Non-Biodegradable and Other Recalcitrant Molecules.
Biotechnology and Bioengineering, XV:611-47, 1973.
Alexander reasons that for a compound to be biodegradable the following
must occur: an organism having the potential for catabolizing the com-
pouid must exist; that microorganism must be present in the environment
or find its way there; the compound must be accessible to the potentially
active species and the bond requiring cleavage must be exposed; if the
enzymes involved in the initial stages of degradation (or any stage there-
of) are intracellular, the substrate must penetrate into the cell; if the
enzymes involved in the degradation are not constitutive, they must be
induced; the environment must allow for proliferation (growth) of the
requisite heterotrophs and for the functioning of the needed enzymes.
2. Arin, M.L. Monitoring with Carbon Analyzers. Environmental Science
and Technology, p 898, Oct. 1974.
Compares various TOD and TOG analyzers on the market. Describes
measurement capabilities and applications.
3. Balmat, J.L. Biochemical Oxidation of Various Particulate Fractions
of Sewage. Sewage and Industrial Wastes, 29(7):757-61, 1957.
4. Bunch, R.L. and C.W. Chambers. A Biodegradability Test for Organic
Compornds. J. Water Pollution Control Federation, 27(9): 1040-53, 1955.
5. Busch, A.W. and J.W. Lewis. BOD Progression in Soluble Substrates:
VTII. The Quantitative Error Due to Nitrate as a Nitrogen Source.
Proceedings of the 19th Purdue Industrial Waste Conference, Purdue
Uiiversity, West Lafayette, Indiana, pp 846-70, 1964.
6. Buzzell, J.C., R.H. Young and D.W. Rychman. Behavior of Organic
Chemicals in the Aquatic Environment, Part II. Washington, D.C.:
Manufacturing Chemists Association, 1968.
7. Caldwell, D.H. and W.F. Langelier. Manometric Measurement of the
Biochemical Oxygen Demand of Sewage. Sewage Works J., 20(2) :202-18,
1948.
8. Clifford, D.A. Automatic Measurement of Total Oxygen Demand. Pro-
ceedings of the 23rd Purdue Industrial Waste Conference, Purdue
Uhiversity, West Lafayette, Indiana, pp 999-99, 1968.
Introduces TOD analyser with specifications and response data for var-
ious materials; correlates data to COD and BOD data.
9. Davis, F.S. 2,4,5-T. Water-1972, AIChE Symposinn Series, 69(129):
269-78, 1973.
40
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10. Dawson, P. S. and S. H. Jenkins. The Oxygen Requirements of Activated
Sludge Determined by Manometric Methods II - Chemical Factors Affecting
Oxygen Uptake. Sewage and Industrial Wastes, 22(4):490-507, 1950.
11. Dean, R. B., S. Claesson, T. N. Gellersted and N. Boman. An Electron
Microscope Study of Colloids in Wastewater. Environmental Science and
Technology, 1(2):147, 1967.
12. Edwards, C. A. Persistent Pesticides in the Environment, CRC Press,
Division of the Chemical Rubber Co., Ohio, 1970.
13. Hart, F. L. and T. Helfgott. Bio-Refractory Index for Organics in
Water. Water Research, 9, Pergamon Press, 1975.
14. Hart, F. L. Measures of Biodegradability and Refractory Organics in
Wastewaters. Ph.D. Dissertation, University of Connecticut, 1974.
15. Hart, F. L., T. Helfgott and R. Bedard. An Evaluation of Persistency
for Water Borne Organics. Proceedings of the 30th Annual Purdue Con-
ference on Industrial Waste, May, 1975. Engineering Bulletin of Purdue
University, Engineering Extension Service Series 144, May 1970
(Forthcoming).
16. Helfgott, T. and U. Asrani. Analysis of Nitrogen in Waters. In:
Denitrification of Municipal Wastes, 1973 Proceedings, 48. Amherst:
University of Massachusetts Water Resources Research Center. In Press.
17. Helfgott, T. B. and H. Gomaa. One Pest into Another. Chemical Engin-
eering, p. 7, July 1969.
18. Helfgott, T. and F. L. Hart. Quick-Time Instrumental Measurements of
Wastewater Organic Characteristics. In Progress In Water Technology,
6, J. F. Andrews, R. Briggs § S. H. Jenkins, (Eds.), pp. 159-67.
Oxford: Pergamon Press, 1974.
19. Helfgott, T., Hunter, J. V. and R. Rickert. Analytic and Process
Classification of Effluents. J. San. Engg. Div. ASCE, 96(SA3):
779, 1970.
20. Heukelekian, H. Use of Direct Method of Oxygen Utilization in Waste
Treatment Studies. Sewage Works J., 19(5):875-82, 1947.
21. Heukelekian, H. and M. C. Rand. Biochemical Oxygen Demand of Pure
Organic Compounds. Sewage and Industrial Wastes, 27(9):1040-53, 1955.
A large data collection by various authors is presented.
41
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22. Hunter, J. V. and H. H. Heukelekian. Determination of Biodegradability
Using Warburg Respirometric Techniques. Proceedings of the 19th Purdue
Industrial Waste Conference, Purdue University, West Lafayette, Indiana,
1964, pp 616-27.
23. Hutzinger, 0., S. Safe and V. Zitko. Polychlorinated Biphenyls.
Analabs Research Notes, 12(2):1-15, 1972.
24. Klein, S. A. and D. Jenkins. Biodegradability of a Carboymelloxysuc-
cionate Detergent Builder. J. Water Pollution Control Federation,
p. 2107, Sept. 1974.
Studies the degree of CNNOS removal in a septic tank system. The
study found that 101 removal was experienced in the septic tanks
and approximately 90% was r.emoved in the leaching system.
25. Lamb, C. B. and G. F. Jenkins. BOD of Synthetic Organic Chemicals,
Proceedings of the 7th Purdue Industrial Waste Conference, Purdue
University, West Lafayette, Indiana, 1952.
Presents BOD bottle data for synthetic compounds such as ethylene
dichloride, acetone, acetic acid, isopropanol, with incubation times
exceeding 40 days. Concludes that synthetic organic responses to bio-
chemical attack differs from domestic raw sewage. The significant
test modification indicated by this finding is that acclimate seed with
more than five days' incubation time is needed in order reliably to
evaluate pollutional load.
26. LeBlanc, P. J. Review of Rapid BOD Test Methods. J. Water Pollution
Control Federation, p. 2202, Sept. 1974.
Reviews efforts to speed up BOD evaluations. Methods have included
increase in incubation temperatures and prefiltering to remove
predators on bacterial population.
27. Liv, D., P. T. A. Wong and B. J. Dutka. Studies of a Rapid NTA-
Utilizing Bacterial Mutant. J. Water Pollution Control Federation,
45(8):l729-35, August, 1973.
Studies the oxygen uptake response of a bacteria cultured in an NTA
substrate rich environment on various concentrations of NTA in a War-
burg Respirometer. The results indicated that the oxygen uptake was
directly related to the NTA concentrations and to temperature and that
the developed culture preferred NTA as a substrate over a domestic
wastewater.
28. Ludzack, F. J. and M. B. Ettinger. Chemical Structures Resistant to
Aerobic Biochemical Stabilization. J. Water Pollution Control Federa-
tion, 32(11):1173-1200, 1960.
42
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A literature review of studies that evaluate BOD responses for various
organic chemicals. Includes a number of BOD methods such as dilution
techniques and Warburg techniques with various seeds. In addition to
a large collection of data, an overall assessment of degradability
properties for groupings of chemicals (e.g., alcohols, phenols, alde-
hydes, etc.) is included.
29. Manka, J., A. Mandelbaum and A. Bortinger. Characterization of Organics
in Secondary Effluents. Environmental Science § Technology, 8(12):1017,
1974.
30. McCallum, G. E. Advanced Waste Treatment and Water Reuse. J. Water
Pollution Control Federation, 35(1):1-10, 1963.
31. McDermott, J. Sewage and Effluent Analyses university of Connecticut
Sewage Treatment Plant. Summary report submitted to the Environmental
Engineering Program, Civil Engineering Department, University of Con-
necticut, 1973.
32. McKinney, R. E. Microbiology for Sanitary Engineers. New YorkrMcGraw-
Hill, 1962.
33. Menzie, C. M. Metalolism of Pesticides. Bureau of Sport Fisheries
and Wildlife, Special Scientific Report -- Wildlife No. 127, U.S. Dept.
of Interior, July, 1969.
34. Neumann, K. E. Analysis of Residual Total Nitrogen in Wastewaters.
Master's Thesis, University of Connecticut, 1972.
35. Ongerth, H. J., D. P. Spath, J. Crook and A. E. Greenberg. Public Health
Aspects of Organics in Water. J. American Water Works Association,
July, 1973, pp. 495-497.
36. Rey, G., W. J. Lacy and A. Cywin. Industrial Water Reuse: Future
Pollution Solution. Environmental Science and Technology, 5(9):
760-65, 1971.
37. Rickert, D. A. and J. V. Hunter. General Nature of Soluble and Par-
ticulate Organics in Sewage and Secondary Effluent. Water Research,
5:421-36, 1971.
38. Rosen, A. A. Problems Associated with Refractory Organics. Paper
presented to Water Pollution Control Federation, Boston, Mass.,
Oct., 1970.
39. Ryckman, D. W., A. V. Prabhakara Rao and J. C. Buzzell. Behavior of
Organic Chemicals in the Aquatic Environment. Washington, D. C.:
Manufacturing Chemists Association, 1966.
Reviews measurement techniques and data from a literature review col-
lection of studies measuring degradation properties of specific organic
43
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compounds. In addition to oxygen uptake studies, this review
presents chemical analysis techniques and bioassay studies.
40. Saldick, J. Biodegradation of Cyanuric Acid. Applied Microbiology,
Dec., 1974, pp. 1004-08.
41. Schaffer, R. B., C. E. Van Hall, G. N. McDermott, D. Barth, V. A.
Stenger, S. J. Sebesta and S. H. Griggs. Application of a Carbon
Analyzer in Waste Treatment. J. Water Pollution Control Federation,
37(11):1545-66, 1965.
42. Shea, P. R. Physical Models of Current and Advanced Wastewater
Treatment Plants, Master's Thesis, University of Connecticut, 1973.
43. Shuval, H. I. and N. Gruener. Health Considerations in Renovating
Wastewaters for Domestic Use. Environmental Science § Technology,
7(7):600-5, 1973.
44. Taras, M. J. et al. (Ed.) Standard Methods for the Examination of
Water and Wastewater. New York: A.P.H.A., 1971 (13th Edition)
45. Tinker, J. PCBs at Maendy: Scare or Disaster? New Scientist
p. 760, June, 1973
46. Umbreit, W. W., R. H. Burr is and J. F. Stauffer. Manometric Tech-
niques. Burgess Publication Co., 4th edition, 1964.
47. Verstraete, W., J. P. Voets, and R. Vanloocke. Three-Step Measurement
by the Sapromat to Evaluate the BODs, the Mineral Imbalance and the
Toxicity of Water Samples. Water Research, 8:1077, Dec. 1974.
This paper presents oxygen uptake data of wastewater samples with
varying mineral contents. The Sapromat apparatus determines oxygen
uptake by measuring the quantity produced to maintain an equilibrium
02 environment in the biochemical reaction chamber.
48. Are You Drinking Biorefractories Too? Environmental Science and
Technology, 7(1):14-17, 1973.
49. Fate of Organic Pesticides in the Aquatic Environment. Advances in
Chemistry Series III, American Chemical Society, Washington, D. C.,
1972.
44
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APPENDIX
Item A Derivation of UOD Measurement
B Laboratory Methods and Materials
C Warburg Respirometer Curves for Ultimate
Biochemical Oxygen Demand and Refractory
Index Determination
D Suggested Pretreatment Requirements for
Industrial Waste Prerequisite to Discharge
into Municipal Sewage Treatment Plants
45
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ITEM A
Derivation of UOD Measurement
1. TOD MEASUREMENT RESPONSE
The Total Oxygen Demand Analyzer* reports (8) the amount of oxygen needed
to complete the following oxidation reactions for elements commonly found
in organic materials:
organic 2
2H+ + 1 /2 O
M + i /? n
Organic 1/Z °2
Q
"organic
S + 1/2 0
organic 2
~^ 2
Ho
- i /? o
•"•" — "* X/ *^ O
> so2
[9]
Oxyanions commonly found in wastewaters undergo the following reduction
reactions and therefore yield a negative TOD response (2) (8) (41) .
"W3
2
3
-* '~ "2(g)
\ xm 4. i /? n
\ cn + ' 1 /9 n
T oUo * » •!/ ^ U«
Z Z
LJ.^J
[14]
[15]
The TOD analyzer, therefore, yields a general measure directly related
to the organic materials present in water, but as with all gross measurements
found in environmental engineering laboratories, responses from other impuri-
ties can yield an output not related to the organic concentration. This
characteristic is considered during the UOD derivation and also must be noted
during recommended measurement application (14).
* Ionics Model 225 Total Oxygen Demand Analyzer
46
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2. TN MEASUREMENT RESPONSE
The Total Nitrogen Analyzer* reports all bounded nitrogen present in
water (20) but not_dinitrogen, N~. This includes ammonium (NH. ), nitrate
(NO,), nitrite (NO-) and organically bounded nitrogen. Because this measure-
ment does not separate nitrogen concentrations according to the oxidation
state (e.g., NH_ vs. N0_) ,adjustments must be made when applying the TN
measure for quantifying an oxygen demand parameter. This adjustment is
accomplished by deriving a Total Oxidizable Nitrogen (TOxN) expression (16)
(34).
3. DERIVATION OF UOD
UOD is defined as the amount of oxygen needed for complete oxidation
(end products of CO-, H-0 and N0_). The following assumptions are made to
generate this value:
a. Inorganic materials, such as heavy metals capable of TOD oxidation,
are negligible. This is especially true for non-industrial wastewaters.
b. The TOD Measurement is a response from 100% oxidation of organic
carbon (to CO-), 1001 oxidation of organic hydrogen (to H-0) and 33.3% oxi-
dation of organic nitrogen (to NO) (14).
c. Oxidation from organic sulfur is negligible. Average organic sul-
fur found in raw sewage at the University of Connecticut is 1.1 mg/L as S
(31). There is little if any industrial input here.
d.
present.
Total Nitrogen (TN) data report 100% of all nitrogen materials
e. The expression Total Oxidizable Nitrogen (TOxN) used in this deri-
vation defines the quantity of oxygen required to oxidize all nitrogen
(organic and inorganic) to N0_.
The general formula for Ultimate Oxygen Demand (all expressed in mg/L
as 02) is:
[UOD] = [TOD] + [1.12 N03] + [1/2 N0~] + 2/3 [TOxN] [16]
1.12 NO" is added to this expression because it is reduced to NCL 75 during
high temperature TOD combustion, yielding additional 1.12 0-. This was
experimentally determined. The NOl also reduces to NO yielding 1/2 0-.
These quantities, therefore, are added to the TOD value to arrive at a true
expression of oxidizable materials present. The resulting (TOD + 1.12 NO^
.+ 1/2 NO-) expression represents carbonaceous oxygen demand plus 1/3 nitrogen
oxygen demand. Because the UOD value also includes nitrogen oxygen derand,
two-thirds of the remaining oxidizable nitrogen is added (2/3 TOxN).
* Infratronics Model Dohrmann MCTS-10 Nitrogen Analyzer
47
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TOxN can be expressed as:
TOxN = 3/2 (Organic N + NH3) + 1/2 N0~ [17]
The 3/2 (N organic + NH..) + 1/2 N02 expression respresents the quantity
of oxygen required to completely oxidize all nitrogen forms to NO,. Noting
that total nitrogen equals organic nitrogen plus ammonia plus nitrate plus
nitrite (TN = N organic + NH + (NO" + NO" from total nitrogen (TN)). The
total oxidizable nitrogen value, therefore, is derived as:
TOxN = 3/2 (TN - NOl - NO") + 1/2 NOl El8]
*J ^ £
which simplifies to:
TOxN = 3/2 TN - 3/2 N0~ - N0~ [19]
Referring back to expression [10], UOD is now expressed as:
UOD = TOD + 1.12 NO" + 1/2 N0~ + 2/3 [3/2 TN - 3/2 NOJ - NO^] [20]
vdiich simplifies to:
UOD - TOD + TN + 0.12 NO^ - 1/6 NO^ [2l]
Average nitrate values for raw sewage at the University of Connecticut were
found to be 2.4 mg/L as N, while nitrite values were undetected (31). The
raw sewage entering the University of Connecticut Sewage Treatment Plant
is usually aerobic and little opportunity of denitrification (NO"-»- N-) occurs.
Therefore, assuming the quantities 0.12 NOl and 1/6 NOZ are negligible, a
final expression for Ultimate Oxygen Demand is given as:
UOD = TOD + TN as mg/L as 02 [22]
4. LABORATORY PROCEDURE FOR UOD
A complete step-by-step procedure for determining the UOD of a water
sample is presented in the laboratory manual section of this report. A gen-
eral outline of the steps needed to determine UOD is presented below:
48
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a. To evaluate the TOD concentration:
1. Calibrate TOD Analyzer with standard solutions of potassium
biphthalate.
2. Prepare sample (dilute for proper range, homogenize sample).
3. ' Determine at least three to ten TOD values and use average
for TOD concentration expressed in mg/L as 0~.
b. To evaluate the TN concentration:
1. Calibrate TN Analyzer with standard solutions of ammonia
chloride, potassium nitrate or urea.
2. Prepare sample (homogenize).
3. Determine at least three to ten TN values and use average for
TN concentration expressed in mg/L as N.
4. Change TN expression from mg/L as N to mg/L as 02 by the factor:
TN mg/L as 02 = TN mg/L as N x s"mole °2
1*8 N
g-mole
The UOD value is found by adding TOD and TN
UOD = TODX + TN (all expressed as 02) [23]
5. LABORATORY PROCEDURE FOR BODu MEASUREMENT
Ultimate Biochemical Oxygen Demand (BOD ) determines the amount of
oxygen required by a mixed and heterogeneousucommunity of bacteria as found
in domestic sewage under the following conditions (15) :
- Aerobic environment.
- 20°C.
- Neutral pH (approximately 7).
49
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- Mi, nutrient concentration at an approximate level where BOD,-/N
equals 100/5. (BOD5 - mg/L as 02; N - mg/L as N) . b
- P0| nutrient concentration at a sewage to buffer ratio as recommended
by Standard Methods for the dilution technique (44) .
- Trace nutrients supplied from raw sewage.
- Starting microorganism population from domestic sewage.
This test is designed to simulate the degradation process expected
to occur if the waste material in question is exposed to a municipal,
aerobic biological treatment system or a separate industrial waste bio-
logical treatment system using domestic sewage as the seed material.
Presented below is a discussion on the standard test conditions.
a. Temperature.
A 20°C temperature is chosen to remain consistent with standard
BODs test and to yield a conservative evaluation of biodegradability.
Higher temperatures (to a limit of about 35 to 40<>C) are expected to
accelerate the biological process (19) and, therefore, present an ecosystem
unrelated to the outside environment. Lower temperatures, on the other
hand, slow down the biodegradation process and yield overly conservative
results. It is expected that long-term BOD tests conducted at various
ambient temperatures above freezing result in the same absolute value pro-
vided proper bacterial seed nutrients and time are given.
b. Sewage Organisms.
Raw sewage is chosen as a seed because the large diversity of
organisms present provides the greatest possible starting point to develop
a microscopic ecosystem for utilizing the material being studied. Also,
the biosystem resulting from an exposure of raw sewage organisms and test
material during aerobic conditions for an extended period is expected to
parallel a municipal biological treatment biosystem. Refractory Index data,
therefore, are judged appropriate as indicators of biotreatment ability.
c. Acclimation Time.
Normal biodegradability or treatability studies, as conducted
by an environmental engineer, are started by acclimating an activated
sludge to the waste material being studied. If successful acclimation is
attained, the appropriate treatment coefficients for uptake rates,
removal rates, sludge production and sludge characteristics are found to
determine if the material is suitable for conventional biological treat-
ment (4) (7) (10) (16) (27) (32) (47) .
50
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The purpose of the BODu measurement, however, is not to derive
treatment coefficient parameters but is to evaluate the feasibility of
degrading the material through biological reaction. The period required
to degrade this material, is, of course, the primary concern for both
industrial and municipal aerobic biological treatment and natural stream
and lake purification reactions. The acclimation time, therefore, is
included to yield standardized results.
Acclimation studies have been demonstrated in laboratory and
even occasionally in treatment plants such as the activated sludge type.
However, industrial plants are also subject to bulking, toxic inputs and
wide variation in flow and concentrated input, and therefore have diffi-
culty in maintaining a reliable acclimated bacterial population.
d. Nutrients.
To assure that a proper nutrient supply is available for develop-
ment of the microorganism populations, ammonia chloride, phosphate buffer
and domestic sewage solutions are added to the standard BOD test solution.
BODU is found by subtracting oxygen uptake values of the blank
solution (raw sewage, ammonia chloride, phosphate buffer) from the test
solution (raw sewage, ammonia chloride, phosphate buffer and test material),
Nitrate readings of these solutions are taken at the completion of respira-
tion. In cases where different degrees of nitrification result, a nitri-
fication correction term is used. The resulting.general formula for
BODu, therefore, is:
BODu = BODu + AN03
Where: BOD = difference of oxygen uptake between test solution and blank
solution, and ANO;j = nitrification correction term.
The nitrification correction term is not applicable to cases
where nitrogen is present in the test material and the difference in nitri-
fication between the test solution and blank solution is less than the
quantity of possible nitrification from the nitrogen containing test
material.
51
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ITEM B
Laboratory Methods and Materials
1. METHODS AND MATERIALS
This section presents detailed instructions for arriving at Refractory
Index (R.I.) evaluations. Further details on the specific analyzers can
be found in the following references:
- Microcoulometric Titrating System, Instruction Manual, Dohrmann Envirotech,
Mountain View, California.
- Total Oxygen Demand Analyzer Operating Manual, Ionics Inc., Watertown, Mass.
- Orion Research, Analytical Methods Guide, Orion Research Inc., Cambridge,
Massachusetts.
- Umbreit, W. W., R. H. Burris and J. F. Stouffer, Manqmetric Techniques,
Burgess Publishing Co., Minnesota. 1964, 4th Edition (46).
2. PREPARATION OF SOLUTIONS
The procedure for R.I. evaluation requires preparation of TOD, NOZ, and
TN standard solutions; 201 KOH for the Warburg and "TOD analyzers; known
(quantitative and qualitative) solution of organic compounds for the BOD
analysis; and test solution for the Warburg analysis. u
Methods for preparation of these solutions follow:
a. TOD Standard Solution is made from potassium biphthalate
(KOCOC,H.-2-COCH). An 850.9 mg/L solution of potassium acid phthalate yields
a 1000 mg/L TOD as 02 solution.
1. Dry potassium biphthalate powder in a 104° C oven overnight.
2. Cool to room temperature in a desiccator.
3. Weigh chemical on balance to 0.1 mg.
4. Calculate TOD of solution by proportion, e.g., if weighed chemi-
cal is 90 mg and this is diluted to 1000 ml, the TOD (900 mg/L potassium
biphthalate) is:
900 x 1000 = 1050 mg/L TOD as 0.
850.9 L
52
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5. Dilutions of this standard are made to arrive at a series
of TOD standards within the anlayzer operating range; e.g., 200 mg/L,
100 mg/L, 50 mg/L and 20 mg/L as CL solutions.
6. Cover standards and store at 4°C.
b. Nitrate (NO,) Standard Solution is made from potassium nitrate
(KN03). . •*
1. Follow the same drying procedure as described above.
2. Prepare a 0.1 Molar solution of KN03 (10, 100 mg/L).
-2 -3 -4 -5
3. From this standard solution prepare 10 ,10 ,10 , 10
Molar solutions of KNO_.
4. Adjust pH of standard solution to correspond with the pH of
samples to be analyzed (pH = 4).
5. Cover standards and store at 4°C.
c. Total Nitrogen (TN) Standard Solutions are made from ammonia
chloride (NH.C1), potassium nitrate (KNOJ and urea [(NH?)?CO].
*T «J L* L*
1. Follow the same drying procedure as described above.
2. Prepare standard solutions of the compound and express in
mg/L of nitrogen. For example, a 100 mg/L solution of urea equals:
100 mg/L Urea x 2 moles N x 14g N/Mole N = 47 mg/L N as N
1 mole Urea 60g Urea/mole Urea
3. Cover and store standards at 4°C.
d. 20% KOH solution is made by dissolving 236 grams potassium hydrox-
ide (KOH) into one liter of solution.
1. A drying procedure is not required because the solution to be
made does not fall into a highly precise range (percent is parts per
hundred).
2. Add 236 grams KOH to a liter volumetric flask (Pyrex).
3. Fill the flask with distilled water. Pour cold water on the
flask during this step to avoid excess heating.
4. Store KOH in a plastic container. Avoid contact with skin and
eyes.
55
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e. Organic compound solutions for BOD test fall into four classi-
fications: water soluble, nonwater solubleuand compounds that may be
chemically or physically affected at 104°C. To make known solutions of
these organic materials, the following rules are followed:
1. For compounds that are not affected at a 104°C temperature,
follow the same drying procedure as discussed above.
2. For compounds that may be affected at a 104 C temperature
(i.e., amino acids), dry in a vacuum desiccator overnight.
3. Water soluble compounds are dissolved in distilled water, cov-
ered and stored at 4°C.
4. In some cases, nonwater soluble compounds (e.g., biphenyl)
are dissolved in acetone or another suitable solvent, placed in a volu-
metric flask, covered and stored at 4 C. After a measured volume of this
solution is placed in a Warburg flask, the acetone is volatized away by
placing the flask in a vacuum desiccator.
In another case the material was added as a dispersion in the best
manner possible, but while in this condition, only a fraction entered true
solution. Materials like DDT despite limited solubility showed an inhibitory
response.
f. Phosphate buffer solution is prepared as follows:
Dissolve 8.5 grams potassium dihydrogen phosphate (KH-PO.) > 21.75
grams dipotassium hydrogen phosphate (K-HPO.) and 33.4 grams disodium
hydrogen phosphate hepahydrate (Na-HPO. . 7H2^ into one liter of solution.
3. OPERATION OF TOD/TOC ANALYZER
This section describes routine operating procedure for the Ionics
Model 225 Total Oxygen Demand (TOD) and Total Organic Carbon (TOC)
Analyzer.
a. Standardization.
1. Prepare standard solutions of potassium acid phthalate as
described above.
2. Scrub the standard solution with nitrogen gas to remove
dissolved carbon dioxide. This procedure is followed to remove inorganic
carbon. The carbon analyzer, therefore, will report organic carbon.
3. Conduct TOD and TOC analyses on the standard solutions.
TOC (mg/L as C) values of the standard potassium biphthalate solution are
equal to 0.4 times the TOD (mg/L as 0-) concentration. Normal calibration
procedure is to analyze three or more standards with between three to ten
54
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TOD/TOC values conducted per standard.
4. Compute the TOD and TOG factors (Factor = Actual TOD/Scale
reading) and graph the TOD and TOC factor vs. the scale reading.
b. Sample Analysis.
1. Homogenize samples with particulate matter (e.g., raw sewage)
in a Waring'high speed blender.
2. Acidify and scrub sample as described in the above section,
3. Dilute the sample to fall within the TOD/TOC scale readings.
4. The TOD and TOC values are determined by averaging three to
ten outputs.
4. OPERATION OF TN ANALYZER
This section describes routine operating procedure for the Dohrmann
MCTS-10 Nitrogen System analyzer.
a. Standardization.
1. Prepare a TN standard solution as described in Section A.
2. Inject standard solutions at the expected unknown sample TN
concentraiton range. At least three injections are needed.
3. Calculate the TN recovery factor by the formula:
I Recovery = 1.74 (counts) (100%)
(luL) (mg/L N) (Range - ohms)
Factor = (1.74) TN standard [24]
% R (Range-ohms) x
b. TN Analysis.
1. The TN (mg/L as N) values for unknown standards are determined
by the formula:
TN = Counts x (1.74) Factor [25]
(uL Sample Volume) (Range-ohms)
55
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5. OPERATION OF NITRATE PROBE
This section describes routine operation of the Orion Nitrate probe.
a. Standardization.
1. Prepare series of 10 to 10 Molar concentration of potassium
nitrate as described in Section. 2b above.
2. Determine the electrode potential (+mv) direct readout for
each standard, and graph electrode potential vs. Molar concentration on a
5 cycle semi-log paper. (Molar concentration on the log scale and elec-
trode potential output on the arithmetic scale.) A straight line is
constructed from this data.
b. NO, Analysis.
1. Molar concentrations of nitrate are determined by reading
the electrode potential and using the standard graph.
2. mg/L NO., as N values are determined by multiplying the Molar
concentration by the molecular weight of nitrogen (14g).
6. OPERATION OF WARBURG APPARATUS
This section describes routine operating procedure for the Warburg
apparatus. Details on the BOD test procedure are given below;
a. Cleaning of Warburg Flasks.
1. Normal cleaning procedure:
- remove grease with paper towel.
- pour concentrated HC1 (technical grade) to about 1/3 of
flask and swirl.
- transfer HC1 to another flask.
- rinse at least five times with tap water and final rinse
with distilled water.
2. Special cleaning procedure (to be followed after about three
to four runs -- depending on condition of glassware):
- rinse flask with tap water to remove test solutions and KOH.
- soak flask overnight in Chromerage (made from concentrated
technical grade H-SO. and Chromerage).
56
-------
- remove flask from Chromerage and rinse at least five times
with tap water followed by a final rinsing with distilled
water.
b. Cleaning of Warburg manometers.
1. Normal cleaning procedure:
- remove manometer from support.
- remove manometer fluid and fluid reservoir.
- remove grease with paper towel.
- rinse manometer with tap water by hooking rubber pressure
line to manometer opening.
- dry in oven (104°C).
- install fluid reservoir so fluid rises to about the 5 cm
mark with no adjustment.
2. Special cleaning procedure (to be followed after three to four
runs -- depending on condition of glassware):
- remove manometer from support.
- remove manometer fluid and fluid reservoir.
- rinse residual manometer fluid.
- soak manometer overnight in Chromerage as above described.
- remove from Chromerage and rinse manometer with tap water.
- final rinse with distilled water.
- dry in oven (104°C).
- install manometer fluid reservoir as described above.
c. Preparation of Warburg Flasks for BODu Test.
- place absorbent filter paper into center KCH well (paper
is shaped as accordian).
- place light coating of grease to outside edge of KCH well
(this procedure is followed to prevent KCH from splashing
out to test solution).
57
-------
- add 0.5 ml of 20% KCH to center well.
- seal top of flask with cork.
d. Attaching manometer and flask to Warburg apparatus.
- place anhydrous lanolin on fitted manometer joint and flask
side-arm joint.
- attach springs to side-arm.
- add test solution to Warburg flask and attach to manometer
joint.
- attach springs to flask and manometer attachment.
- place flask into water bath and allow temperature inside
flask to reach water temperature before closing manometer
stopcock.
7. W3 DETERMINATION
Mi- is determined by the Kjeldahl distillation method. Further refer-
ence on this method can be found in Standard Methods. A step-by-step pro-
cedure is given below:
a. Add 50 ml sewage, 250 ml distilled water and 25 ml buffer solution
to an 800 ml Kjeldahl flask. Also prepare a blank solution by omitting the
sewage.
b. Distill this solution into a 100 ml Boric Acid solution until the
300 ml mark is reached.
c. Titrate 0.02 NH-SO. into the Boric Acid solution to the original pH.
d. Calculate the ammonia concentration by the formula:
JH_ = 28° (VV mg/L as N [26]
•> 50
V = titration of sample (ml)
VD = titration of blank (ml)
*Ah alternate method for ammonia determination is by means of ion selective
probes. These need to be calibrated frequently but are free of inter-
ferences and offer quick-time analysis.
58
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8. BODu TEST PROCEDURE
This section presents step-by-step analytical details used to arrive
at the BODu value.
a. Preparation of Test Solutions.
Preliminary information needed to prepare these solutions are TOD and
TN values of the raw sewage and test compounds or wastes, plus nitrogen
concentration of the raw sewage. The procedures followed for these evalua-
tions are discussed earlier.
1. Calculate the estimated BODs of raw sewage and the test com-
pound using the formula:
BOD5 = 0.51 (TOD - TN/2) + 67 [27]
2. Calculate UOD of the test compound or waste solution and
the raw sewage using the formula:
UOD = TOD + TN mg/L as 02 [23]
3. Determine the correct volume of test compound solution to be
added to the test solution by proportioning according to UOD:
(Xml test compound) (UOD test compound) = (a ml sewage) (UOD sewage)
4. Calculate the combined estimate BODs of the test compound plus
sewage using the formula:
unn T x- AI (a ml sewage) (BQDs sewage) + (b ml compound (BODs compound)
cui;5 icomoineaj a ml + b ml
[28]
Usually 15 to 20 ml raw sewage is chosen (a = 15 ml).
5. Calculate the required volume of NfyCl solution using the
proportion:
(ML NH4C1) (-mg/L as N) + (a ml sewage) (-mg/L NH3 as N sewage)
= (a ml + b ml) (required NH3 concentration) [29]
59
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The required NH_ concentration is calculated by using the ratio
BOTL conibined/N = ratio i 100/5.
6. Phosphate buffer solutions are added at the same sewage/buffer
ratio as recommended in Standard Methods;
- 1 ml buffer solution/Liter dilution water.
- 3 ml raw sewage/300 ml dilution water.
- Therefore sewage/buffer = 3/0.3x1 = 10/1.
- Because compound + sewage solution has a BOD,. = 2 x BOD,.
sewage, use a 5/1 ratio.
- When using 20 ml sewage, add 4 ml buffer solution.
At this point, the following information should be calculated for
each test solution:
- ml compound or waste required.
- ml raw sewage.
- ml NH^Cl solution required.
- ml phosphate buffer solution required.
Because one blank solution is used, all the test solutions are
proportioned equally except for the ml compound or waste added. Therefore,
the higher calculated values for M1.C1 and phosphate buffer solutions are
used in all the solutions tested.
b. Warburg Test Start Up.
1. Prepare batch solutions (total volume approximately 200 ml)
of the Warburg test solutions and Warburg blank solutions using the pro-
portions calculated in Section 1 above.
2. Add aliquot samples of these batch solutions to the Warburg
flasks so that the total volume in each flask equals the total volume
calculated in Section 1 above. Each test solution is evaluated in tripli-
cate.
3. Acidify the remaining test solution with H-SCL to pH = 4.0.
4. Determine the nitrate probe reading for the acidified solu-
tions.
60
-------
c. Data Collection.
'Manometer readings are taken according to the manometer fluid dis-
placement. Normal manometer reading is conducted by setting the right
side at 15 cm and reading the left side. During times when the manometer
fluid displacement is so large that it is impossible to set the right side
at 15, the following procedure is used:
1. Set right side at a point that will allow left side to be on
scale.
2. Read left side.
3. Set right side at a point that is twice as far from 15 as
from the previous setting (1).
4. Read left side.
5. Calculate where the fluid would be on the left side if the
right side could have been set at 15 through proportions.
Other information that is collected during each reading is the
time and the barometric pressure. A suggested order of data collection is
as follows:
- Set manometers at 15 (right side).
- Record time of reading.
- Read barometric pressure (mm Hg).
- Read left side of manometers.
- Open manometer stopcocks.
- Adjust manometer fluid.
- Close stopcocks.
The BOD test is conducted for at least 20 days. If a noticeable
oxygen uptake is noted after 20 days, the test is continued to 30 days.
After 30 days, the test is continued until oxygen uptake stops. At the
completion of oxygen uptake, the Warburg flask contents are transferred to
a vial and acidified to pH ^ 4 with H-SO..
Nitrate probe readings are then taken for each sample.
d. Data Analysis.
The BOD value mg/L 02 uptake is determined from the Warburg apparatus
by converting the change of pressure (found from manometer readings ) to
61
-------
loss of CU within the flask. A derivation of this conversion is arrived at
through tne following sequence:
- Calculate the ml of C^/ml of air within the flask at STP (gas con-
centrations at standard pressure and temperature are used in the derivation
to eliminate temperature corrections).
- Calculate the ml of 02/ml of fluid space at STP.
- The ml of 0- from time 1 to time 2 is then related to a change in
Warburg manometer pressure reading.
- ml
Details of
CL is then converted to mg 0- per liter sewage at test conditions.
' this derivation follow:
e. Derivation of Warburg Equations.
1. ml 0 /ml air space @ STP
PV/T = P'V'/T'
[prime signifies standard conditions]
P1 = 1 atm.
V - gas volume @ STP
T1 = 0° C = 273° K
T = 20° C = 293° K
V = volume of gas @ test condition = Vg
P = P-R = partial pressure of gas
273
V -=^- (P-R) minus water vapor pressure
yi _ o &"*>
I atm.
62
-------
2. ml 02/ml fluid
ml 02 _ Vf a (P R) - V"
ml fluid P0
Vf = volume fluid
a = Bunsen coef. (ml 02/ml water)
at 0° C, 1 atm. partial pressure
P-R = correction factor partial pressure
P0 of gas
3. Gas at start = V + V"
After uptake, the gas partial pressure will decrease by h.
h is measured as mm Brodi fluid. To keep consistent units Po will be
expressed as mm Brodi fluid. Proper conversion is as follows:
760 mm Hg 10,000 mm Brodi
1 atm x x = 10,000 mm Brodi fluid
1 atm 760 mm Hg
Gas at finish:
v' = V 273
2 g 293 (P-R-h)
10,000 mm Brodi
V2 = Vf a g"*ff
10,000
''
V = (V.. + VT ) - (V- + V-)
J. J_ Z Z
- « 273 h _
'g 293 10,000 vf a 10,000
4. v
V
293 £ 10,000
For oxygen, the following conversion is used:
63
-------
4. Conversion Factor for: ml 02/ml sgmple to mg 02/L sample
pV = nRT
n = 1 mole
V = x (volume in liters needed for
1 mole of 02 at the stated
conditions)
R = 0.0821 atm
mole °K
T = 293 °K (20 °C)
p = p (p = actual atmosphere
760 atm pressure, mm Hg)
V = nRT/p
V = volume of one mole = (1 mole) (0.0821 atm ) (293 °K)
mole "K
p/760 atm
- (1) (010821) (295) (760) (L/mole) = 18,282 L
p mole p
ml 07 x 1000 ml sample x L 02 x mole (p) x
ml sample L sample 1000 ml 02 18,282 L
52g 0 x 1000 mg = 52,000,000 p = 1.75 p mg 02
g.mole 02 1 g 18,282,000
/ 77^ \
mg 02 uptake = (Vg^| +V£ )h x 1.75 p [30]
10,000
64
-------
Dividing by vol. of sample = mg/L CL uptake.
Ihe accumulated oxygen uptake value for the test compound is determined
by subtracting the blank solution uptake from the test solution uptake.
t ' *
BOD (test compound) = BOD (test solution) - BODu (blank solution) [31]
, The nitrification correction (designated ANO,) is a term applied to the
BOD value to adjust for nitrification nonuniformities (5). A diagram of
theunitrification correction technique is illustrated in Figure 1.
BOD = BOD* + ANO" [32]
u LI O
ANO, equals the difference in nitrification occurring in raw sewage
plus test solution to the nitrification that has occurred in raw sewage
alone. In cases where nitrogen is present in the test material and more_
nitrification is noted in the solution containing this material, the ANO,
value cannot be used unless its value is larger than the total concentra-
tion of chemically bound nitrogen as 02 demand in?the test material. In
such cases, the difference is subtracted from BODU.
65
-------
ITEM C
Warburg Respirometer Curves for Ultimate
Biochemical Oxygen Demand and Refractory
Index Determination
(in Alphabetical Order)
66
-------
1200 r
1000
800
/
600
O
o
CD
400
200
10
1
.o-^row plus acetic acid
1
raw
15 20
Days
25
30
BODU- 456
75
BODU« 531
TOD * 867
TN * 0
UOD * 867
R.I. *0.6I
_J
35
Figure C-l OXYGEN UPTAKE FOR ACETIC ACID
-------
00
700
CSJ
0 500
(0
0
X.
e
0
0 300
CD
100
^^^ raw plus acetone
/
/ BO
/
/
JL ^— A^^ T N
/ — A- r<3W
/ Ar^-^^"A"-*"A U°
IS
•y R.I
r i i i i i i i
5 10 15 20 25 30 31
Days
u * 428
3" 9
BODU= 437
TOD « 618
0
UOD « 618
R.I. « 0.71
Figure C-2
OXYGEN UPTAKE FOR ACETONE
-------
700
VD
CM
O 500
to
o
o»
E
O
O 300
CD
100
s
10
1
raw plus acetone
o«
i
15 20
Days
25
30
BOD'
U
BODU
TOD
TN
UOD
R.I.
257
0
257
275
0
275
0.93
_J
35
Figure C-3 OXYGEN UPTAKE FOR ACETONE
-------
700 -
BOD;," -49
ANO§* 200
BODU» 154
row plus adenme
TOD = 767
TN » 486
Figure C-4 OXYGEN UPTAKE FOR ADENINE
-------
300
38
CM
O
g 200
^
o>
E
O
-
JO'
- /^
0 L /V
DQ 100 h 1*
//
L- ,
5
row plus adenine _
-,». 0 BODU-
^"^ A T°D *
""^ ^ fOW TN =
UOD = ;
R.I. s (
I 1 1 1 1 — J
10 15 20 25 30 35
114
!74
0.14
Days
Figure C-5 OXYGEN UPTAKE FOR ADENINE
-------
700
-vl
CO
1
1
10
15 20
Days
raw plus aniline
raw
25
30
u* 359
* -32
BODU * 326
TOD * 462
TN = 56
UOD = 518
R.I. * 0.63
J
35
Figure C-6 OXYGEN UPTAKE FOR ANILINE
-------
700
CM
O 500
row plus antifreeze
30
BODf,
'3
BODU
TOD
TN
301
0
301
267
I
268
1.12
35
Figure C-7 OXYGEN UPTAKE FOR ANTIFREEZE
-------
600 r
CJ
O
to
O
X
o»
E
O
O
00
100 -
o row plus arginine
i i
3 6 9 12
18
24
Days
30
40
BOD[, « 264
j= 168
BODU« 152
TOD = 180
TN = 55
UOD » 235
R.I. -0.65
50
Figure C-8 OXYGEN UPTAKE FOR ARGININE
-------
10001
BOD'U * 303
CM
O
8 -78
raw plus aspartic acid
o BODU - 303
o>
o
o
03
200
8
12 16
Days
raw
20
TOD = 299
TN * 75
UOD - 374
R.I. « 0.81
30
Figure C-9 OXYGEN UPTAKE FOR ASPARTIC ACID
-------
1000 r
BOD;,. 32
-12
M
O
w
O
-I
X.
0>
E
O
O
ffl
200-
row
plus benzene
BODU« 20
Figure C-IO OXYGEN UPTAKE FOR BENZENE
-------
1000
CM
O
o
_l
"X
0>
£
O
o
CQ
200
BOD
row plus biphenyl
BOD
U
TOD =
185
|3
198
174
TN * 0
UOD = 174
R.I. "1.14
3
6
9
12
18
24
Days
30
40 5
Figure C - 11
OXYGEN UPTAKE FOR BIPHENYL
-------
300 1-
00
CM
O
g200
N.
O»
E
o
O
CO
10
15 20
Days
-177
row plus bipyridine
BODU« -177
TOD - 320
54
TN
25
30
UOD - 374
R.I. * NEC.
B.I.V. « 0.56
_J
35
Figure C-12 OXYGEN UPTAKE FOR BIPYRIDINE
-------
500
155
0
cvi
O
w
O
O
O
OQ
100
Tow plus sodium butyrote BODU= 155
.A
R.I. « 0.84
Figure C-13 OXYGEN UPTAKE FOR SODIUM BUTYRATE
-------
300
CVJ
D
00
o
200
X.
0>
E
g 100
raw plus cmc
1
1
10
15 20
Days
25
30
« -3
= -31
j
BODyB -33
TOD « 460
TN * 0
UOD = 460
R.I. • 0
-J
35
Figure C-14 OXYGEN UPTAKE FOR CARBOXYMETHYL CELLULOSE
-------
ouu
CM
O
to 200
o
_J
o>
E
O
00 O
M CD
100
^^^ BOD* 8 -202
^X^row
^^ AN 03* 57
^^A— - """ BODU- -145
^^ TOD « 541
/ TN = 0
/ / row plus chloroform
/ /° R.I. s NEG
/ / i . 1 . 1 1 I8'1*'' °'71
10
15
20
25
30
35
Days
Figure C-15 OXYGEN UPTAKE FOR CHLOROFORM
-------
1000
(M
o
(ft
o
03
O
CD
200
raw plus pep
BOD'U = 24
BOD =-33
TOD « 72
TN
UOD « 72
R.I. = 0.0
3
6
9
12
18
24
Days
30
40 5
Figure C-16 OXYGEN UPTAKE FOR p-CHLOROPHENOL
-------
300 -
CM
O
00
CM
o
o
CO
100
raw plus cyanuric acid
12
18
24
Days
30
40
BODU* -89
TOD * 227
TN * 179
UOD = 406
R.I. = NEC.
B.I.V * 0.26
i
50
Figure C - 17 OXYGEN UPTAKE FOR CYANURIC ACID
-------
3001-
00
BODy- - 129
12
BODU« -117
TOD « 564
UOD • 564
Days
Figure C - 18
OXYGEN UPTAKE FOR DDT
-------
oo
en
OUQ
(VI
O
0
_j 200
X
E*
0
o
CO
100
—
^^.A— — '
^*t*^
c>r
s
-f~—
i
I
/
If ,
A-^^^ BODi-
^&****^
^ raw
^^ ANO~-
^^^ 3
^***
. -*r^"^ ^0 BODU-
.*r raw plus ddt T0o =
TN =
UOD =
R.I. =
B.I.V. »
1 1 1 1 i
-106
-54
-160
633
0
633
NEC.
0.34
10
15 20
Days
25
30
35
Figure C-19 OXYGEN UPTAKE FOR DDT WITH CARRIER
-------
00
ouo
200
CM
O
V)
o
0 100
0
CD
BOD A B
AN 0" s
row plus dichlorobiphenyl 3
^^0-0-— 0— 0- ° BODU.
.
-------
700 -
c»
-j
10
row plus dichlorophenol
15 20
Days
25
30
BOD'u«-372
ANOj* 113
BODu=-259
TOD = 338
TN
UOD
R.I.
B.I.V.
0
338
NEC.
0.59
35
Figure C-21 OXYGEN UPTAKE FOR DICHLOROPHENOL
-------
700
00
00
CM
O
g 500
o»
E
O
O
CD 300
row plus ethylene glycol
BODu » 207
0
BODU« 207
TOD « 271
TN
Figure C-22 OXYGEN UPTAKE FOR ETHYLENE GLYCOL
-------
00
<£>
700
CM
0
a, 500
o
_i
*s
M|
W1
E
o
o 30°
ffi
100
—
™*
^^-o-— —
o*^
- 1/^
ftr
1
I
1 1 1
5 10
raw
__ . _ A
^X^^ ^ BOD.1. --47
^** raw plus gasoline
^A^^ ANO^« 186
^*^ 3
BODU« 139
TOD « 675
TN « 0
UOD * 675
R.I. = 0.21
1 II 1 1
15 20 25 30 35
Days
Figure C - 23 OXYGEN UPTAKE FOR GASOLINE
-------
IUUU
CM
O
0
_J
-x
o»
E
0
o
CD
200
-
^^
' /
-/
o
j
/ ^^~
1 ^
7 ^
\r
V I I
4 8
Xrow plus glucose
^•»
^^**'^
^0** row
^~*~***^
lit i
12 16 20 30
Days
BOD^ >442
BODU-403
TOD -438
TN « 0
UOD -438
R.I. »0.93
Figure C-24 OXYGEN UPTAKE FOR GLUCOSE
-------
1000
BOD'U « 197
CVJ
O
M
O
o
O
CD
200
9 12
raw plus glutamic acid
18
24
Days
30
40
AN03= 95
BODU=292
TOD =247
TN « 43
UOD = 290
R.I. « 1.0
50
Figure C-25 OXYGEN UPTAKE FOR GLUTAMIC ACID
-------
1000 r
CM
O
' 10
O
E
o
O
m
200-
row plus glutomic acid
BODj, » 588
ANOj»-39
BODU * 588
TOD «462
TN « 138
UOD - 600
R.I. »0.98
Figure C - 26 OXYGEN UPTAKE FOR GLUTAMIC ACID
-------
5001
CM
o
10
o
<£>
O-J
O
O
m
100 -
raw
9 12
18
24
Days
30
40
raw plus histidine BOD
u
TOD
TN
» 116
= -104
= 116
= 166
* 57
UOD = 223
R.I. * 0.52
50
Figure C-27 OXYGEN UPTAKE FOR HISTIDINE
-------
700 -
vo
10
J_
15 20
Days
raw
raw plus humics
:. «-ioo
» 91
25
30
BODU « -8
TOD * 726
TN
UOD * 726
R.I.
35
Figure C-28 OXYGEN UPTAKE FOR HUMICS
-------
1000
BOD; * 70
CM
O
V)
O
-1
X.
o»
E
O
to O
200
raw plus hydroquinone
6 9 12
18
24
Days
30
40
-19
BODU * 51
TOD * 125
TN
UOD = 125
R.I. » 0.41
50
Figure C- 29 OXYGEN UPTAKE FOR HYDROQUINONE
-------
1000
10
ON
CM
O
to
O
o»
E
O
o
00
200
8
row plus lysine
12 16
Days
20
BOD'u * 106
J" 2
BODu = 108
TOD « 183
•O
'A TN = 39
UOD «222
R.I. -0.47
30
Figure C - 30 OXYGEN UPTAKE FOR LYSINE
-------
lOOOr
229
to
CM
O
CO
O
0>
E
O
O
m
200
8
raw plus lysine
12 16
Days
20
BODU • 235
TOD « 371
TN « 79
UOD • 450
R.I. -0.52
30
Figure C - 31 OXYGEN UPTAKE FOR LYSINE
-------
1000 r
OJ
O
M
O
_J
V,
o>
E
g
m
200-
8
row plus lysine
12 16
Days
20
BODj, • 365
« -12
BOD,, » 365
TOD « 612
TN
128
UOD «740
R.I. "0.49
30
Figure C-32 OXYGEN UPTAKE FOR LYSINE
-------
iooo r
CM
O
0»
E
O
O
m
200 -
row plus lysine
Figure C - 33
OXYGEN UPTAKE FOR LYSINE
-------
500 r
o
o
CM
O
to
O
E
O
o
00
100 -
raw plus phenol BODu»l64
raw
8
12
16
Days
20
« 44
BODU « 120
TOD »I39
TN « 0
UOD "139
R.I. -0.87
30
Figure C- 34 OXYGEN UPTAKE FOR PHENOL
-------
500 r
CM
O
W)
O
0»
E
O
O
CO
100 -
row plus sodium
propionate
O'
6 9 12
18
24
Days
30
141
40
BODU= 141
TOD * 175
TN = 0
UOD * 175
R.I. =0.80
50
Figure C-35 OXYGEN UPTAKE FOR SODIUM PROPIONATE
-------
700 r
i I
row plus propylene glycol
0 BODJ, « 326
raw
BODU« 326
TOD - 420
TN « 0
UOD • 420
R.I. • 0.78
12
18
24
Days
30
40
50
Figure C - 36 OXYGEN UPTAKE FOR PROPYLENE GLYCOL
-------
700 r
CM
o
M
O
O
O
CD
100
P row plus sevin
444
ANO:
BODU* 444
TOD * 420
TN « 22
UOD = 442
R.I. * 1.0
3 6 9 12
18
40
24 30
Days
Figure C-37 OXYGEN UPTAKE FOR SEVIN WITH CARRIER
50
-------
500 h
CM
o
to
o
o»
E
100-
8
12
16
Days
20
BODU • 300
A
30 32
Figure C - 38 OXYGEN UPTAKE FOR RAW SEWAGE
-------
700
CM
o
-------
300
o
ON
CM
O
* 200
X.
o»
E
Q
o
00 100
—
-
-
^«
tr
- S
.1
1 ,
BODU- 157
TOD « 204
TN « 75
UOD « 279
R.I. « 0.56
1 i 1 1 1 1
10
15 20
Days
25
30
35
Figure C-40 OXYGEN UPTAKE FOR RAW SEWAGE
-------
250 r-
CM
O
to
o
o>
E
o
o
CD
8
172
158
48
206
R.I. =0.84
Days
Figure C- 41 OXYGEN UPTAKE FOR RAW SEWAGE
-------
500 r-
o
00
CVI
O
tit
o
X
en
E
o
O
ffl
100 -,
8
12 16
Days
20
BOD
TOD
TN
UOD
R.I.
• 581
• 588
« 74
- 662
• 0.88
30
Figure C - 42 OXYGEN UPTAKE FOR RAW SEWAGE
-------
500r
o
VD
CM
O
to
o
o»
E
O
o
00
100
8
12 16
Days
20
30
BODU « 247
TOD « 180
TN « 91
UOD « 271
R.I. » 0.97
Figure C-43 OXYGEN UPTAKE FOR RAW SEWAGE
-------
700
CM
0 500
10
o
*
o
0 300
00
100
- ./ BOD
/TOD
.
/ UOD
/
' 1 1 1 1 1 1
5 10 15 20 25 30
« 629
« 578
» 123
« 701
•0.89
1
35
Figure C-44
Days
OXYGEN UPTAKE FOR RAW SEWAGE
-------
3001-
10
I
15 20
Days
25
BODU= 295
TOD * 470
TN « 107
UOD - 577
R.I. * 0.51
1
30
35
Figure C - 45 OXYGEN UPTAKE FOR RAW SEWAGE
-------
300 r-
csj
O
CO
O
200
X
0»
E
O
O
00
100-
10
1
1
15 20
Days
25
BODU- 312
TOD « 600
TN « 91
UOD - 691
R.I. • 0.45
J I
30
35
Figure C - 46 OXYGEN UPTAKE FOR RAW SEWAGE
-------
300 r-
0*200
co
o
o»
E
O
o 100
CD
£
/
10
1
15
20
Days
25
BODU* 305
TOD - 520
TN * 100
UOD = 620
R.I. * 0.49
1
30
J
35
Figure C-47 OXYGEN UPTAKE FOR RAW SEWAGE
-------
700 -
A»A— A
Fiaure C-48 OXYGEN UPTAKE FOR RAW SEWAGE
-------
1000
0*800
(0
o
O
O
00
600
400
200
o
10
1
1
15 20
Day $
raw plus starch BODU- 348
ANO"3= 191
25
30
BODU= 539
TOD « 997
TN
UOD - 997
R.I. « 0.54
35
Figure C-49 OXYGEN UPTAKE FOR STARCH
-------
I200r-
1000 -
CM
to
o
800-
600-
O
O
m
400 -
200
10
15 20
Days
raw
25
30
BODU'* 446
is
BODU « 464
TOD • 648
UOD « 648
R.I. " 0.72
35
Figure C - 50
OXYGEN UPTAKE FOR STARCH
-------
IZOO
1000
CM
0 800
M
O
_J
oi 600
E
O
O
00 400
200
BODj* 380
.x^raw plus starch ANO^* 37
^^^.^-0- — BODU « 417
0^-°— ° TOD * 648
- / ^^-A* — ' ^°W TN = 0
o ^^~*^**^
I A— -A— — *~* UOD *648
/$ R.I. * 0.64
//
r i i i i i i i
5 10 15 20 25 30 35
Days
Figure C-51 OXYGEN UPTAKE FOR STARCH WITH ADDITIONAL AMMONIA
-------
500 r-
00
i i
raw plus industrial waste
12
18
24
Days
30
40
Figure C-52 OXYGEN UPTAKE FOR INDUSTRIAL WASTE
TEXTILE DYE
BOD'u" -63
- 95
BODU« 32
TOD « 527
TN » 71
UOD * 598
R. I. « 0.05
_l
50
-------
IUUU
CM
O
o
_J
x.
o»
E
MB
0
GO
200
—
_
o<^^^
^^*
- or
/ \ ill i
3 6 9 12
BOD^ » 222
rowplutdy* _ ANQ-a 92
o— ""
XBODU* 314
^
A — —J^JJ "" TOD » 468
^^^ ^^^^
^-^* TN « 97
UOD = 565
R.I. * 0.56
ill I i
18 24 30 40 50
Days
Figure C - 53
OXYGEN UPTAKE FOR DYE , TEXTILE
-------
1000
N)
o
CM
O
(A
o
o»
O
O
CD
200 -
8
I
12
16
Days
raw plus valine
raw
20
30
• 392
- -46
BODU - 392
TOD « 371
TN -51
UOD • 422
R.I. - 0.93
Figure C -54
OXYGEN UPTAKE FOR VALINE
-------
300
CM
o
8 200
en
E
o
O
m 100
10
raw
raw plus v.c.
15
20
25
Days
-9
0
BODU« -9
TOD » 236
TN « 0
UOD • 236
R.I. « 0
30
35
Figure C - 55 OXYGEN UPTAKE FOR VINYL CHLORIDE
-------
400 r
M
o
to
o
N)
tv)
O
O
CD
100
row sewage - A
raw plus I mg/L Pb- •
raw plus 2mg/L Pb -A
raw plus 5mg/L Pb -o
10
15
20
25
30
37
Days
Figure C - 56
OXYGEN UPTAKE FOR LEAD
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ITEM D
SUGGESTED PRETREATMENT REQUIREMENTS
FOR INDUSTRIAL WASTEWATERS
PREREQUISITE TO DISCHARGE
INTO MUNICIPAL SEWAGE TREATMENT PLANTS
By
T. B. Helfgott
PhD., Environmental Sciences, B.Ch.E., M.Eng.
1. PREFACE
Before certain industrial wastes can be admitted into conventional
sewage treatment plants, they need, in most cases, to be pretreated. This
is especially true if the municipal treatment has a biological reactor
(trickling filters or activated sludge which is even more sensitive to
inhibitory materials) as part of the train of unit operations and pro-
cesses used to treat the wastewater to achieve required standards. The
various forms of biological treatment are easily disrupted. However,
fixed media bioreactors and completely mixed activated sludge are rela-
tively more stable.
When biological treatments do not operate at high efficiency, they act
as vehicles for the transport rather than the degradation of certain
refractory materials, discharging these pollutants into the receiving
aquatic environment. Not all wastes are amenable to treatment by any one
unit step. Usually a series of unit operations and processes are necessary
for managing the heterogenous constituents found in almost all wastewaters.
Two other.alternatives should be noted:
a. There are a few industrial wastewaters of relatively simple
and invariant composition, but these are the exceptions.
b. Physical-chemical treatment trains of unit steps can often
manage heterogenous wastewaters more effectively than conventional treat-
ment with its sensitive biological reactors.
Conventional plants are too often used as pipelines from the factory
to the receiving waters. Restraints based on the classical parameters
of BOD5 (5-day Biochemical Oxygen Demand, mg/L 02) and Suspended Solids
(mg of filterable solids per liter) can be easily evaded in some cases if
the idiosyncrasies of these analytic techniques are known. In addition,
charges to industry based on volumetric flow may not reflect the cost of
treating a difficult waste.
123
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The wastewaters that are most amenable to treatment by conventional
biological sewage treatment are characterized by readily settleable solids,
degradable and sorbable organics of not too high biochemical oxygen
demand, such as a typical household waste with roughly 200 to 400 mg/L
Biologically treatable wastes should have nutrient balance;
ciencies in nitrogen, phosphates, potassium or any of the 16 or so
essential nutrients will limit biological treatment effectiveness.
Normal municipal waste has an excess of nitrogenous and phosphate nutrients
beyond what is necessary to metabolize the degradable carbonaceous load of
the waste. Sometimes, therefore, the addition of carbonaceous BOD from
industrial wastes, such as food processing or those containing altered
hydrocarbons, brings the municipal wastewater into better nutrient balance.
This situation is, however, rarely realized with most industrial waste
discharges into municipal treatment plants.
2. CRITERIA
The industrial influent to a sewage treatment plant should
resemble municipal waste in having a BOD,, of 150 to 650 mg/L CL. That is,
it should be neither too dilute nor too concentrated in order not to alter
the concentration of BOD5 entering the plant to a value outside of this
range. If the receiving municipal plant has a biological treatment unit
process, the industrial wastewater should not contain refractory organics;
although nonbiodegradable organic constituents do not fully register as
BOD, they are serious environmental pollutants.
An industrial wastewater should not have a long-term biochemical oxygen
demand (BOD2Q days) in excess of twice the EGD^:
BOD2Q < 300-1200 mg/L
or the Total (Theoretical) Oxygen Demand (TOD) for carbonaceous material
should not be significantly greater than the extrapolated ultimate BOD by
a factor of 1.0 or 1.1:
TOD/BODultijnate < 1.1 (ideally 1.0)
The presence of certain inhibitory or toxic constituents (like heavy
metals, pesticides, certain chlorinated hydrocarbons) can make ordinarily
biodegradable organics refractory. That is, these constituents alter the
amenability of ordinary waste to biological treatment. Therefore, restraints
on the discharge of certain industrial pollutants are necessary.
Table A is a list of pollutional constituents that need to be con-
sidered. It includes suggested acceptable concentrations or ranges of
concentrations for each constituent listed. There are always extenuating
circumstances in individual cases for being above or below this listed
124
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concentration or range; standards are a guide, not a substitute for developed
j udgment.
It is nearly impossible to monitor for all possible inhibitors and
toxics, especially since synergistic and antagonistic responses are com-
plex. It is therefore, necessary to have a measure of toxicity of inhibi-
tion. Unfortunately, most of the toxicity tests now available take too
long to perform for real-time evaluation of wastes. These toxicity tests
are usually done on fish (two or more days) or by means of modified BOD
( 5 to 20 days) tests.
Most wastes contain, in addition to carbonaceous materials, nitro-
genous pollutants such as proteins, alkaloids, peptides, ammonia, nitrates
nitrites and/or cyanides. A ratio of the ultimate oxygen demand, expressed
as TOD plus oxidizible nitrogen, divided by the ultimate BOD (Nitrogenous
plus carbonaceous) is a more complete parameter for evaluating the total
environmental insult to oxygen resources.
(TOD + TN)/BODu = 1.1
Since bacterial growth is always associated with organic degradation,
the TOD and TN cannot equal the ultimate biochemical oxygen demand, there
always being a residual of bacteria, organic nutrients, refractory materials
and bacterial by-products.
Another criterion for evaluating industrial wastes is the refractory
index (R.I.), a quantitative measure of refractory organics. The R.I. is
defined as the ratio of ultimate BOD to ultimate TOD plus TN:
R.I. = BOD /(TOD + TN)
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TABLE A
SUGGESTED LIMITS AND RANGES
FOR INDUSTRIAL WASTEWATER POLLUTANT INPUTS
TO MUNICIPAL SEWAGE TREATMENT PLANTS
Constituent
Mercury
Manganese
Cadmium
Aluminum
Barium
Chromium
Copper
Lead
Nickel
Silver
Zinc
Total Metals
Pesticides
Free and Combined Chlorine
Cyanides
Fluorides
Total Dissolved Solids
Suggested Acceptable
Limit and Range
mg/L
0.0 - 0.05
1.0 - 2.0
0.1 - 0.2
0.2 - 0.5
1.0 - 2.0
0.05- 0.1
0.25- 0.5
0.2 - 0.5
0.05- 0.1
1.0 - 2.0
< 0.05
0.5 - 0.05
< 1.0
*
5 - 10
0.03- 0.1
< 20
<3000
*Varies with specific pesticide.
Note - Metal concentration should be based on analysis of filtered
waters. Many problem materials cannot be specified out of con-
text of the industrial waste and type of treatment plant (e.g.,
blood from diseased persons and formaldehyde).
126
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The interpretation of R.I. values is given in Table B:
TABLE B
INTERPRETATION OF REFRACTORY INDEX VALUES
R. I. Interpretation
1.0 Degradable
0.2 - 0.80 Stable Intermediates
0 Refractory
Negative* ( <0) Inhibitory
*Negative R.I. values provide only a quali-
tative indication of inhibition.
In the case of negative R.I. values, bioassays should be done as well as
detailed analyses for special organic or inorganic materials that inter-
fere with normal biodegradation.
The pH of industrial waste should be near neutral:
pH = 5.5 - 9.5
but, more important, it should be buffered in such a way that the treatment
response at the sewage plant is not altered. Caustic alkalinity and mineral
acidity should be decreased by neutralization processes.
Suspended solids offer buffering-like responses and impose a diffi-
cult loading and sludge management problem if applied to conventional
treatment plants in large concentrations; therefore, there should be
restrictions on industrial waste suspended solids input to conventional
plants.
Suspended Solids <<: 350 mg/L
Determination of settleable solids is both an easier test to perform
and a more intuitively logical parameter to understand. It can be used to
estimate precipitation loading for sludge management handling and is per-
formed quickly.
127
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There are many aesthetically offensive, obviously hazardous materials
and gross indirect parameters that are easy to spot which indicate that
restraints should be applied to the discharge of industrial wastewaters.
These include:
Visible Oil Sheen
Floating Solids
Color (Unusual)
Horrendous Odor (Phenols, H2S, CN~, etc.)
Dispersing Agents
Materials that can cause clogging of pipelines
Foam
Gasoline, benzene, naphtha, fuel oil and other flammable
or explosive materials
Viscous Solids
Aerosols and volatile materials
There are traditional restrictions on wastes:
Phenols ng range
Coliform Bacteria varies
Pathogenic Microorganisms -0-
Turbidity < 200 JTU
Dissolved Oxygen > 1.0 mg/L
Chlorine Residual < 10 mg/L
Temperature < ISQop
3. NON-QUANTITATIVE FACTORS
This section recommends certain practices that can help the opera-
tion of conventional treatment plants.
a. Monitoring. It is pointless to monitor for pollutants that are
known to be absent even if there is a standard test. On the other hand,
rare but troublesome pollutants such as cadmium should be monitored if an
industry has such a material in its inventory used in water-related pro-
cessing.
b. Real-time measurements and controls. Ion selective probes
such as nitrate detectors are real-time measurement techniques that, even
considering some losses of precision, accuracy and sensitivity, can be
more effective in controlling pollutantional output than slow time para-
meters such as BOD, toxicity and analyses for specific trace metals.
c. Pretreatment. If a waste does not meet the specifications,
then pretreatment or partial treatment can be required before discharge
into municipal treatment systems.
128
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d. Power. In the case of power failure, provision should be made
so that the burden of treatment is not shifted from the industry to the
municipal plant.
e. Flow. In order to optimize the operation of the sewage treat-
ment facility, flow should be designed to be continuous or supplied in
such a way as to supplement the normal diurnal variations of the plant.
Scheduling of industrial flow inputs can improve the operation of municipal
plants by enabling it to approach steady state performance. Changes in
flow or its characteristics should be reported by the industry and regulated
by the controlling agency. This may require monitoring.
f. Corrosive materials. These should be excluded from municipal
plants.
g. Odorous and explosive gases. Materials such as H?S, HCN,
^2' ^4 an<^ volatile solvents should be removed from industrial discharges
prior td municipal treatment plants.
h. Sequestered oils and metals. Because dispersing agents and
emulsifiers have a detrimental effect on the performance of biological
treatment, these should be removed by pretreatment.
i. Spills. Accidental or otherwise unavoidable spills should
be reported by the industry to the controlling agencies for the municipal
wastewater system.
j. Storm water runoff. Storm waters, especially the first
flush of water after a heavy rain can be as dirty or dirtier than muni-
cipal waste. It must enter the municipal system in a nondisruptive manner
and may require pretreatment or storage.
k. Industrial cooling water. The blowdown of cooling water
often contains particularly difficult pollutants such as chromium, algi-
cides and slime inhibitors and therefore should not enter municipal systems.
These wastewaters should be internally cleaned in the industrial plant
and recycled in the cooling system.
1. Radioactive materials. Radioactive materials with long half-
lives should neither be discharged into municipal wastewater systems nor
into any part of the ambient aquatic environment.
m. New and unknown wastes. Pilot plant tests can be used to
evaluate unknown waste from new complex industrial processes.
n. Overall environmental impact and secondary pollution problems.
The interrelation of industrial wastewaters, sludges, and air pollutants
must be evaluated so that pollution problems are not shifted from one pocket
of the environment to another. Dilution of pollutants is not the best
long-term environmental protection practice for many industrial wastes,
particularly since certain harmful refractory constituents can be recon-
centrated biologically in the receiving environment.
129
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4. Ultimate treatment
The ultimate treatment of wastewaters would consider them as
reusable resources. In-plant treatment to reclaim chemicals and water
is seen as the optimal goal. Since this goal is not always realizable,
this section has set forth some of the restrictions on the discharge of
industrial waste to municipal waste treatment plants.
130
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/2-77-174
3. RECIPIENT'S ACCESSION-NO.
TITLE AND SUBTITLE
An Index of Refractory Organics
5. REPORT DATE
August 1977 issuing date
6. PERFORMING ORGANIZATION CODE
AUTHOR(S)
T.B. Helfgott, F. L. Hart, and R.G. Bedard
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Engineering Program
University of Connecticut, Box U-37
Storrs, Connecticut 06268
10. PROGRAM ELEMENT NO.
1BB610
11. CONTRACT/GRANT NO.
R803231-01-05
2.SJEQ6ISORING AGENCY NAME AND ADDRESS
Robert S. Kerr Environmental Research Lab.-Ada, OK
Office of Research and Development
U.S. Environmental Protection Agency
Ada, Oklahoma 74820
13. TYPE OF REPORT AND PERIOD COVERED
Final - 7/74 - 6/76
14. SPONSORING AGENCY CODE
EPA/600/15
5. SUPPLEMENTARY NOTES
16. ABSTRACT
Refractory waterborne organics resist biodegradation, accumulate in the environ-
ment and can inhibit life forms. This research develops laboratory techniques for,
and interpretations of, a Refractory Index (R.I.) to quantitatively assess the per-
sistency of refractory organics and uses R.I. to evaluate some 38 industrial, natural
and combined organics. R.I. values close to 1.0 characterize readily biodegrada-
tion; R.I. values near zero indicate refractory organics; negative R.I. values indi-
cate inhibitors. The coefficient of variation for R.I. values is 13%. Since negative
R.I. values are of qualitative significance only, a Biological Inhibition Value
(B.I.V.) is developed and used to quantitatively assess those organics found to inter-
fere with the biochemical tests used, long-term ultimate Warburg Respirometer deter-
minations. In a few cases, confirmation of the R.I. interpretation was performed
using a model activated sludge unit and by specific analysis. Correlations between
oxygen demand tests (BODr, BODU, TOD) and organic parameters (TOG, TN) are presented.
Suggestions for required pretreatments of industrial wastewaters before allowing
discharge into municipal sewage treatment plants are included as an application of
the Refractory Index criterion. The bibliography contains 49 references.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Group
Activated sludge process
Chemical removal
Sewage disposal
Sludge digestion
Sewage treatment
Waste treatment
13B
07A
IS. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
unclassified
21. NO. OF PAGES
141
20. SECURITY CLASS (This page)
unclassified
22. PRICE
EPA Form 2220-1 (9-73)
131 -rirU.S. GOVERNMENT PRINTING OFFICE 1977-757-056/653't Region No. 5-1
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