Municipal Environmental Research
Laboratory
Cincinnati OH 45268
EPA 600/9-79-034
—„-,. Delation
and Treatability of
Specific Pollutants
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US EPA-AWBERC LIBRARY
30701 100542890
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EPA-600/9-79-034
October 1979
Biodegradation and Treatability
of Specific Pollutants
by
Edwin F. Barth and Robert L. Bunch
Wastewater Research Division
AWBtRC. LIBRARY U.S.
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental
Research Laboratory, U.S. Environmental Protection Agency,
and approved for publication. Mention of trade names or
commercial products does not constitute endorsement or
recommendation for use.
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FOREWORD
The Environmental Protection Agency was created because of
increasing public and governmental concern about the dangers
of pollution to the health and welfare of the American people.
The complexity of the environment and the interplay between
its components requires a concentrated and integrated attack
on the problem.
Research and development is that necessary first step in problem
solving and it involves defining the problem, measuring its impact,
and searching for solutions. The Municipal Environmental Research
Laboratory develops new and improved technology and systems
for the prevention, treatment, and management of wastewater
and solid and hazardous waste pollutant discharges from municipal
and community sources. We are committed to the preservation
and treatment of public drinking water supplies, and to minimizing
the adverse economic, social, health, and aesthetic effects of
pollution. This publication is one of the products of MERL research
and, as such, is a most vital communications link between the
researcher and the user community.
This report describes biodegradation and treatability studies that
have been conducted over a period of 20 years by the Wastewater
Research Division.
Francis T. Mayo
Director
Municipal Environmental Research
Laboratory
in
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ABSTRACT
This report discusses fundamental and technical considerations
relating to laboratory studies of the fate and effects of organics and
metals in biological wastewater treatment processes.
The relationships between analytical control tests and biode-
gradation test methods are discussed.
Various laboratory apparatuses for both continuous flow and
static biodegradation methods are described.
Both aerobic and anaerobic test results are included in the report.
IV
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CONTENTS
Foreword iii
Abstract iv
Introduction vi
1. Biodegradation of Organic Compounds 1
Fundamental Considerations
Technical Considerations for Microbial Growth
Analytical Control for Biological Treatability Studies
Biodegradation Test Methods
2. Treatability of Specific Compounds 19
Anaerobic Studies
Aerobic Studies
3. Treatability of Metals by Activated Sludge Systems 46
Distribution of Metals through the Process
Effects of Metals on Aerobic Process
Effects on Anaerobic Digestion
Discussion
4. Bibliography 59
General
Treatability of Specific Organic Compounds
Metals
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INTRODUCTION
This compilation of biodegradation and treatability studies is
an overview of work conducted by the staff of EPA's Municipal
Environmental Research Laboratory over the last twenty years.
The report is not intended to be detailed, but rather discusses
approaches, considerations, and techniques evaluated during
laboratory studies of specific pollutants.
Several protocols are presented with a summary of the type
of results obtained for each. There is no intention at this time
of selecting one procedure as the best method for all environmental
decisions.
We intend that the report serve as a review and summary of
past investigations and provide the user with a framework for
deciding on directions to pursue when undertaking similar studies.
John J. Convery
Director
Wastewater Research Division
Municipal Environmental Research
Laboratory
VI
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Section 1. Biodegradation of
Organic Compounds
FUNDAMENTAL CONSIDERATIONS
Biodegradation, as a theoretical concept, refers to the process
by which an organic compound is converted to carbon dioxide,
water, and other inorganic constituents by the action of living
organisms. In practice, several qualifying terms are used to modify
this concept.
The operational definition of biodegradation can be divided
into three categories:
Ultimate Degradation: Conversion of the parent compound to
carbon dioxide, water, and inorganic compounds (if elements
other than carbon, hydrogen, and oxygen are present in the
parent compound).
Primary Degradation: A change in the parent compound such
that it no longer responds to the analytical measurement used
for detection.
Acceptable Degradation: Conversion of the parent compound
to the extent that undesirable properties are no longer manifest.
Because of the differences in duration, test procedures used,
endpoint criteria, and analytical methods selected, the demarcation
between categories is not clear-cut. Futher complicating this
situation is the fact that during biodegradation a certain amount
of the constituents of the parent compound will be assimilated
into the active biomass. Thus, it is impossible to do mass balances
for biodegradation studies on a routine basis.
Most of the published work on biodegradation deals with aerobic
systems. However, anaerobic degradation can be an important
environmental consideration and procedures for measurement
of this process are a necessary part of a fully developed set of
procedures for observing biodegradation. This is an area that
requires considerable developmental work.
The main consideration in biodegradability is whether or not
an organism exists that can break down, metabolize, or oxidize
the parent compound, either on first exposure to it or after a
suitable acclimation period. This acclimation period is important
and should be accounted for in all biodegradation studies concerned
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with environmental acceptability. Acclimation can allow organisms
to produce an induced enzyme in response to the presence of the
unfamiliar substrate; or provide time for population shifts to favor
an increase in organisms utilizing the specific substrate.
An academic distinction can be made depending on whether a
compound is degraded in a biological system where it is the sole
source of carbon; or whether degradation takes place in the
presence of multiple carbon sources (co-metabolism). From a
practical point of view, systems employing co-metabolism are
more similar to environmental situations and yield more realistic
estimates of biodegradation. Co-metabolism is also important
to the treatment of diverse industrial wastes, which are produced
when the manufacturer uses a variety of starting materials and
alternative reaction pathways. If biological treatment is one of
the possible means of disposing of the unusable or unsalable wastes
from an operation, the biodegradability of these mixed wastes
may be an important factor in the economics of process selection.
The above discussion did not include the factors of time and
extent of degradation. In real world situations, the extent of
degradation within a fixed time limit is a very important con-
sideration in any working definition of biodegradability. In
principle, laboratory testing should show the biodegradability
of the material tested as it would occur in the environment or in
the wastewater treatment plant. Obviously, most substances will
eventually be stabilized by microbial action, given enough time and
the right conditions. We must, therefore, consider the biodegra-
dation process as a rate process. For practical purposes related
to wastewater treatment, the substance under consideration should
degrade at a rate equal to or greater than the degradation of the
usual constituents of domestic waste, after the organisms have
become acclimated to the substance. A control test utilizing a
compound with known biodegradation characteristics, such as
linear alkylbenzene sulfonate, should be included in each
experiment.
Common experience has shown that if a specific assay is used
to monitor the extent of biodegradation, 100 percent degradation
is seldom achieved. This is due to the fact that the organisms
cannot derive energy by oxidizing compounds at very low
concentrations.
TECHNICAL CONSIDERATIONS FOR MICROBIAL GROWTH
The proper conditions must be maintained before any compound
can be biodegraded. Microorganisms cannot grow, and hence
organic compounds cannot be degraded, unless a number of
conditions are fulfilled. All necessary nutrients and growth factors
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must be present in the appropriate chemical and physical forms.
Solutions are more readily degraded than suspensions. An adequate
supply of water and sufficient hydrogen acceptors such as
molecular oxygen, nitrate, or carbon dioxide must be present.
Because many diverse species of microorganisms are involved with
environmental degradation, their growth can occur over a wide
range of temperature and pH values; however, for any one specific
case there may be optimal values. In general, the factors listed
below should be considered in aerobic testing.
PH
The culture medium should be buffered to maintain the pH
of the system between 6.5 and 8.5. If the medium contains nitro-
genous components and nitrification occurs, alkalinity can be
depleted. During the conversion of one part of ammonium nitrogen
to nitrate nitrogen, 7.1 parts of calcium carbonate (CaC03) alkali-
nity will be destroyed. Sodium bicarbonate is an excellent buffering
reagent for pH control.
Dissolved Oxygen
According to the culture method selected, oxygen can be supplied
by shaking, diffusion from the atmosphere, or aeration with
compressed air. The concentration of dissolved oxygen in the
culture medium should not be allowed to fall below about 1 mg/l.
Nutrient Balance
The proximate composition of protoplasm is C106H180045 N16Pi,
therefore, to ensure that synthesis of new cellular matter is not
hindered, the culture medium should have these elements avail-
able. In the process of degrading organics, carbon and hydrogen
are supplied by the parent compound and oxygen by substrates
such as water and air; however, nitrogen and phosphorus must
be externally supplemented. For each 1 mg/l of parent compound
carbon added to the culture medium, at least 0.2 mg/l of
ammonium nitrogen and 0.02 mg/l of phosphorus should be pro-
vided. Reagent grade phosphoric acid and ammonium hydroxide
can be used to supply these two elements.
Trace elements such as sulfur (S), potassium (K), calcium (Ca),
and magnesium (Mg) are required in such small amounts that
in all but the most unusual cases these will be present in sufficient
amounts as contaminants in the carriage water, chemicals used for
medium preparation, or inoculum. Preformed growth factors
such as thiamine or biotin are usually provided by the addition
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of yeast extract or domestic wastewater, depending on the medium
selected.
Temperature
Initial screening tests for biodegradation should be conducted
at 20°C ± 5°C. This allows experiments to be conducted at
ambient laboratory temperatures and is a reasonable temperature
for environmental assessment. More information at 30°C and
5°C can be developed if the situation warrants such detail.
Inoculum
Microorganisms to be used as the initial inoculum can be obtained
from a wide variety of sources, depending upon the infor-
mation desired, the culture medium used, and the choice of
culture method. For assessment of environmental biodegradation,
natural inocula rather than pure culture systems are preferred.
Natural inocula can be obtained from ambient air, forest or
garden soil, activated sludge or trickling filter biomass, natural
waters, settled wastewater or final effluent from biological pro-
cesses.
Toxicity
Controls must be included in biodegradation tests to ensure
that viable inoculum was used and that absence of degradation
is not the result of the initial toxicity of the compound.
If all the culture conditions cited above are controlled, a known
degradable compound, which is included as a control in a test
system, should show the expected degree of degradation. If not,
either the inoculum or the medium must be the source of trouble.
Since co-metabolism studies are recommended for environmental
degradation tests, the medium should contain readily degradable
materials from yeast extract or wastewater. Therefore, slight
microbial growth should be noted even in systems containing
non-degradable test compounds. If this growth is absent, it is an
indication the test compound is toxic. In that case, the test material
should be evaluated at lower concentrations and serial transfers
should be performed to accommodate acclimation of the micro-
organisms.
Medium Selection
Four types of media are commonly used:
Natural waters,
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Mineral salts plus yeast extract,
Mineral salts plus yeast extract and supplemental organic
materials, and
Settled domestic wastewater.
The first two are used mainly for determination of biodegradation
in screening tests. Because of their low organic content, oxygen
transfer problems are minimized and cell synthesis is minimal.
This latter point is important since the low cell yield precludes
concern with sorption of the test compounds on a massive surface.
These media are usually employed in batch tests.
The last two media are used for treatability studies. The higher
organic content supports luxuriant cell growth. Factors such
as sorption to cell mass and varying sludge age can be studied
with these media. These media can be utilized for either batch
or continuous flow systems.
ANALYTICAL CONTROL FOR BIOLOGICAL
TREATABILITY STUDIES
As in any chemical reaction, the process of biodegradation can be
followed by observing the disappearance of the reactants (organic
compounds and oxygen) or the appearance of products (cell matter,
carbon dioxide, and inorganic ions). The disappearance of some
specific organic compounds cannot, in many cases, be proof that
the compounds have been completely degraded. Most chemical
or physical tests depend on a particular functional group of the
organic compound. For example, in testing for a surfactant of the
alkylbenzene sulfonate type, several methods of assay could be
used. If the method were based on detection of the benzene ring,
then the benzene ring would have to be degraded for it not to be
detected. However, if the method were based on the formation
of sulfate, then the molecule would have to be so disintegrated
that the sulfonate group would split off the compound.
Specific Analyses
Almost any specific physical or chemical assay can be applied
to treatability studies providing the medium or metabolites do
not interfere. In many cases preliminary clean-up or concentration
steps may be necessary before the final analysis.
The possible assays cover the whole range of analytical tools
such as colorimetry, optical rotation, radionuclides, ultraviolet light
and infrared light absorption, liquid and gas chromatography,
and mass spectrograph.
In order to gauge ultimate degradation as compared with primary
degradation, it is necessary to use some type of analytical test
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that measures organic matter in conjunction with specific assay.
If a low concentration of total organics is measured after degra-
dation, it indicates that partially degraded end-products have
not accumulated.
Non-Specific Analyses
For compounds or unknown wastes for which a specific analysis
of sufficient sensitivity and applicability in the biological system
is not available, a non-specific analysis must be used. The most
commonly used methods have been the oxygen utilization or
biochemical oxygen demand (BOD), the chemical oxygen demand
(COD), and the total organic carbon (TOO. Unlike the methods
that depend on the observation of the disappearance of some
specific organic compound, these analyses are based on the dis-
appearance of oxygen and organic matter as the degradation pro-
ceeds, which is a general function of aerobic systems.
Of the three methods, only the carbon determination (TOC)
is a definitive measurement. The BOD and COD tests are empi-
rical tests that give results in terms of the amount of oxygen
utilized by matter under a prescribed analytical procedure. Because
they do not indicate the quantity of any particular constituent,
it is essential that they be conducted according to a standard
method.
The BOD Test -
The BOD of a sample is the weight of oxygen consumed by a
varying quantity of bacteria in contact with the sample at a spe-
cified temperature during a stated time period. The test does
not directly measure the amount of organic material present.
Phrases such as "BOD is rapidly removed" and "the conversion
of BOD to cellular material" should not be used since that type
of thinking can lead to incorrect assumptions about the amount
of oxygen required to stabilize organic matter under aerobic
conditions.
The test does not differentiate matter that is biologically oxi-
dized, from matter that is first synthesized into protoplasm and
then oxidized during the endogenous respiration of cellular
material. The BOD of a waste will always be less than the
theoretical ultimate BOD because the cellular material is relatively
resistant to microbial oxidation. Whether or not the BOD includes
the oxygen demand used in nitrification will depend on the sample,
inoculum, duration of the test, and addition of a nitrification
suppressor.
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The main limitation of the BOD test is the inapplicability of
test results to predicting what will take place when the waste is
actually oxidized in a different environment. The BOD test is a
measure of avidity for oxygen under specified conditions. It does
not necessarily follow that these same conditions exist in a waste
treatment plant or a stream. In spite of its imperfections, the
biochemical oxygen demand test is useful in measuring easily
oxidized wastes in samples requiring more than 3 mg/l of oxygen.
The COD Test -
The chemical oxygen demand is the amount of oxygen consumed
by a compound when it is digested with chromic and sulfuric
acids. With the proper catalyst, all but a few organic compounds
are oxidized to carbon dioxide and water under the conditions
of the standard test. The COD test does not measure the oxygen
required to oxidize ammonia or organic nitrogen.
The main limitation of the test is that the chemical oxidant
reacts with both stable and unstable organic matter. The test
does not differentiate between inert and biologically oxidizable
matter; neither does it provide any indication of the rate at which
the biologically active material would be stabilized under the
conditions of waste treatment or in streams.
The test has the advantages of requiring only a short time for
the determination and not being affected by toxic wastes. Used
in conjunction with the BOD test, it will give an indication of the
characteristics of wastes. For example, if the samples contain
substances that are difficult for microorganisms to oxidize, the
COD value will be much greater than the BOD value.
This procedure is used principally for samples that have a COD
value above 10 mg/l. It can also be used on samples containing
less than this amount to indicate an order of magnitude of or-
ganic content.
The TOO Test -
The most commonly used total organic carbon method depends
on the rapid combustion of a micro-sample in a stream of oxygen
in a heated tube. The carbon dioxide produced is measured with an
infrared analyzer at a wavelength specific for carbon dioxide.
This method was designed primarily for analysis of true solutions.
Unless the particles in samples are reduced to about 50 microns by
blending, the needle used for injection will act as a filter. The usable
sample size is 20 n\, so some difficulties may arise in obtaining
a representative sample. For this reason, in biodegradation studies
the dissolved organic carbon is usually determined. That is, the
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analysis is run on a filtered sample. The TOC apparatus is sentitive
to about 1 mg/l total carbon, but for quantitative determinations
it is desirable to have samples in the 2—3 mg/l range. In order to
measure only organic carbon, the inorganic carbon must first be
removed from the sample. In samples having a very small amount
of organic carbon, it is essential to eliminate completely any in-
organic carbon from the sample by first acidifying and then purging
with nitrogen. Some of the newer instruments permit the separate
determination of inorganic carbon. In that case, the decarbonation
step may be omitted as an external procedure.
The advantages of the method are that a definite substance is
measured in a very short time, and the results may be interpreted
in relation to the thoeretical ultimate oxygen demand. The dis-
advantage or limitation of the method is that all organic matter
is oxidized, which may or may not be the case in biological treat-
ment systems or streams. Many things can be oxidized at high
temperatures that would not be oxidized in a stream within a
reasonable time. For example, activated carbon is usually not
considered to exert a dissolved oxygen demand on a stream, yet
it can be oxidized at high temperatures.
BIODEGRADATION TEST METHODS
Over the years considerable research has been done on evaluating
microbial breakdown of organic compounds. Techniques have
ranged from Warburg respirometry used by microbiologists, to
BOD determinations and laboratory activated sludge units used by
environmental engineers.
The important features and limitations of various methods of
testing biodegradability are discussed below.
River Die-A way --
The river die-away method is very simple and has been used by
many investigators. The compound to be tested is added to a sam-
ple of river water at ambient room temperature. Periodically,
the solution is analyzed for the test compound to establish a
die-away or disappearance curve. The curve usually shows a lag
period of no degradation for several days and then a rapid removal.
The advantage of the river die-away test is that it requires very
little equipment and yet can indicate the rate of degradability
of the test material. However, the rate is only relative. If, after
the disappearance of the compound, an additional amount is
added, it will disappear in less time. This can be repeated several
times, resulting in a die-away curve with a saw-toothed appearance
(Fig. 1).
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100
LU
I-
LU
o
IT
50
10
20
30
DAYS
40
50
60
Figure 1, River die-away pattern.
A modification that can be used to reduce the lag time and to
speed the process is to add a small amount of activated sludge
to the river water.
The disadvantage of the test is that all river waters are not the
same. Some have more microorganisms than others, and the pH
and mineral constituents vary. To be able to use this procedure,
it is necessary to be able to analyze for the compound or some
constituent of the molecule that is a likely degradation product.
The method of assay will detect the original compound, but per-
haps not a slight modification of the compound. Thus, intermediate
products may go undetected. In spite of the inherent drawbacks
of the river die-away test, it is useful in obtaining preliminary
information on the ease of biodegradation. A large number of
compounds can be screened in a relatively short time.
Warburg Respirometer --
The disappearance of oxygen as degradation proceeds is a general
characteristic of aerobic biological systems. The Warburg respiro-
meter is one of the many apparatuses that permit oxygen uptake
rates to be determined for a biological system. The Warburg
respirometer is popular because it is commercially available, com-
pact, and permits a number of samples to be run simultaneously.
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There are a number of variables that can affect the results, but
the most important factor is the type and quantity of the micro-
organisms used. The seed (inoculum) used should be one that has
become adapted to the test compound or has at least been
subjected to an adaptation period.
Some of the advantages and disadvantages are:
Advantages:
1. Direct measurement of biological oxidation is possible.
2. This approach can be used for all compounds without
using a specific analytical test procedure. This is a decided
advantage in cases where no reliable analytical method
exists.
3. The results are available within a reasonable time.
4. Oxygen consumption, radioactive-carbon dioxide eval-
uation, and respiration assimilation balances can serve
as bases for measuring biodegradability.
5. Continuous observation of multiple samples over periods
of a few hours or several days is possible.
6. Lag periods and rate changes are readily determined.
Disadvantages:
1. Equipment is fairly expensive and requires a skilled tech-
nician,
2. The small sample size makes it difficult to obtain rep-
resentative samples of wastewater and industrial wastes.
3. All organic matter added to the system is subject to oxida-
tion. Therefore, pure organic compounds are needed
for fundamental studies unless a specific assay is included
in the study.
4. With extended runs, nitrification can occur, giving erro-
neous high results of oxygen uptake. Each part of
ammonium nitrogen converted to nitrate will consume 4.5
parts of oxygen.
5. Carbon dioxide evolved during respiration must be absorbed
from the gas phase. It has been reported that complete
absence of carbon dioxide can inhibit assimilation of
organic matter by bacteria.
6. Oxygen uptake results are difficult to interpret quanti-
tatively unless supplemented by other analyses.
Biochemical Oxygen Demand Test --
The biochemical oxygen demand test (BOD) is one of the older
methods for evaluating the biodegradability of substances. It was
first published as a standard by the British Royal Commission
for Sewage Disposal in 1912. For many years nothing better was
available for estimating the strength and controlling the treatment
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of wastewater. The sample is stored in a filled bottle for five days
in the dark at 20°C, then the amount of dissolved oxygen utilized
is measured. Instead of a single final value, a curve showing oxygen
uptake as a function of time can be developed if replicates of the
original sample are analyzed at different times.
With the development of reliable oxygen-sensing probes, it is
now possible to determine the oxygen content of a single sample,
at intervals or continuously, over the entire test period. Another
modification of the test apparatus is to determine the oxygen
uptake electronically based on the electrical current needed to
generate the oxygen required to maintain the pressure at equili-
brium in the closed flask.
The BOD test has numerous limitatioris relating to the appli-
cability of the test results to predicting what will take place in a
waste treatment plant or a stream. Only the limitations that are
pertinent to the utilization of the method for determining bio-
degradability will be discussed.
The advantages and disadvantages of the BOD test procedure are:
Advantages:
I.The apparatus and testing procedure are simple. The test
does not require a highly trained technician.
2. Extensive BOD data are available on compounds with
known waste treatability.
3. The small number of bacterial cells used and the organ-
ic-free BOD dilution water minimize interferences with
specific chemical analyses either for the parent compound
or intermediate breakdown products. •
4. Carbon dioxide is present throughout the test period
and does not become limiting as is possible in the Warburg
procedure.
5. Problems of aeration and oxygen transfer are not en-
countered if suitable initial concentrations are selected,
because all the oxygen required is present at the beginning
of the test.
Disadvantages:
1. Only a small amount of sample can be used because of the
limited solubility of oxygen in water.
2. The testing period is long. However, intermediate results
can be obtained in less time if oxygen depletion is meas-
ured periodically.
3. As with the Warburg procedure, nitrification and extraneous
organic matter can lead to erroneous conclusions.
4. The interpretation of the results has the same limitations
as other respirometer methods in that the results are not
directly convertible to a percentage of the compound
degraded.
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Flask Test Method -
Except for the activated sludge method, this is the most widely
used of all the laboratory procedures. There are many versions
of the technique, but most use a chemically defined medium.
The flasks may be aerated by continuous shaking on a mechanical
shaker, agitated with magnetic stirrers, aerated through a ceramic
filter, stirred with a paddle stirrer, or merely left quiescent. The
test mixture can be analyzed each day or at the end of a specified
time. A variety of inocula can be selected for this procedure.
If degradation is not evident or toxicity is suspected, the test
compound should be run at a lower concentration. Parallel runs
with known degradable compounds are advisable to confirm that
the conditions were correct. It is suggested that a compound
of similar structure and class serve as a reference standard.
The popularity of the flask test method is exemplified by the
fact that five leading countries (Austria, Canada, Germany,Sweden,
and England) have adopted some version of it as their screening
or presumptive test method for determining the biodegradability
of anionic detergents.
Some of the advantages and disadvantages of the flask test
method are:
Advantages:
I.The method does not require elaborate or specialized
laboratory equipment.
2. Reproducibility is generally good.
3. Sample size is not severely limited.
4. The test compound does not have to be pure.
5. Metabolic products can be identified without too much
interference if mineral salts are used and if the test com-
pound is the major carbon source.
Disadvantages:
1. The method requires a separate adapting process if adapta-
tion is not made a part of the test procedure.
2. Shaking on a mechanical shaker limits the number of
samples that can be run. If a static procedure is used,
sample concentration is limited owing to poor oxygen
transfer.
3. The test does not simulate any waste treatment process.
4. A chemical or physical analytical test method must be
available for each class of compounds tested.
5. Simple analytical procedures generally do not measure
partial degradation residues.
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Activated Sludge Method --
The activated sludge method is one of the most important waste-
water treatment processes. Laboratory scale models of the process
have been used for many years to study the treatability of various
municipal and industrial wastes. It was natural that the method
should become a major tool in biodegradability and treatability
testing.
The laboratory methods based on the activated sludge principle
vary in feed, residence time, and whether the feed is continuous
or batch operation. The continuous activated sludge system is
preferred because it corresponds more nearly to actual conditions.
A system that approximates the operation of a plug flow aeration
tank is known as the fill-and-draw or semicontinuous process.
In this method, the inoculum, the test material, and the feed are
placed in a vessel and aerated. The feed is usually a chemically
defined medium with organic supplements. The mixture is aerated,
usually for 23 hours. The air supply is then turned off and the
mixture allowed to settle (see Fig. 2). A portion of the su-
pernatant is removed and replaced with new feed and the test
compound. This sequence is then repeated for the duration of the
test. If the test lasts a long time, it is necessary to waste some
activated sludge periodically in order to maintain a reasonable
level of biomass. As in all aerobic biological processes, there is a
net increase in bacterial cells.
Although each cycle in the semicontinuous operation constitutes
a batch run, the method closely approaches plug flow conditions
in a conventional activated sludge plant. The aeration basin of the
activated sludge process is usually long and narrow. The recycled
sludge and the wastewater enter at one end of the aeration tank.
Ideally, the mixture moves as a plug along the entire length of
the tank. The organisms are subjected to all phases of the growth
curve from the log phase to the endogenous stage. That is, at the
head end of the tank, the organisms have an ample supply of
food. It is in this part of the tank that the organisms grow most
rapidly, and consequently the rate of removal of organic matter
is the greatest. As the plug moves along the tank, the food becomes
depleted, and if the retention period is correct, the bacteria will
start to enter the endogenous phase near the end of the aeration
basin.
Various designs for continuous flow activated sludge studies
have been reported in the literature. Figure 3 shows a continuous
flow, complete mix reactor of 300 ml capacity developed by
Monsanto Chemical Co. Figure 4 shows a 6 liter complete mix,
continuous flow unit developed by the U.S. Public Health Service.
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AERATION PERIOD (Al.
SETTLING PERIOD (B).
Figure 2. Four liter fill-and-draw reactors with two liters of test
mixture, in both the aeration period (A), and the
settling period (B).
14
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Figure 3. Continuous flow 300 ml reactor.
15
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Figure 19 shows a 750 liter per day continuous flow, plug flow
reactor.
Any material used for constructing equipment for biodegradation
or treatability studies on very low concentrations of test
compounds should be evaluated for sorption of the test compounds
on surfaces.
Small reactors of only several hundred milliliters can be used
to establish the biological characteristics of a test system, such as
degradation or sorption on the sludge surface. The larger units
of several liters capacity are useful to collect additional information
on the physical characteristics of the system, such as sludge
settleability. Finally, the large continuous flow pilot plants of
several hundred liters per day through-put capacity are necessary
to collect complete treatability and engineering design data. The
large pilot units can be used to track disproportionation of a
compound through the various unit processes and give guidance
on settler overflow rates, sludge production, aeration requirements
and detention time for the unit processes.
FEED LINE
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TOP VIEI
Separator made from a 10" section of 4"
Plexiglass tube cut in half longitudinally.
Bottom end cut at a 45°angle. Part of the
lower end is closed by a 1/8" Plexiglass plate
so that the two compartments are connected
by a 1/2" x 4" opening. Water proof "rubber
to metal" cement used to join Plexiglass to
glass.
Figure 4. Model activated sludge unit.
16
-------
The test apparatus selected should not be so small that sampling
would disturb the system. For biodegradability testing it is not
necessary to maintain a large unit. Small units are much more
desirable because of space, cost, and labor of servicing several
large units. In scaling down to very small units, there is some
difficulty in designing settling chambers. In most units the settling
chambers are an integral part of the aeration tank rather than
being separate units. Most of the laboratory units are the com-
pletely mixed type rather than the plug flow type.
A list of some of the advantages and limitations of the activated
sludge method follows:
Advantages:
I.The continuous and the semicontinuous methods using
high concentrations of biomass simulate in the laboratory
the treatment that wastewater normally receives.
2. There is considerable information on the characteristics
of these units since the method has been used for many
years.
3. Reproducibility is very good if the unit is operated until
steady state is achieved.
4. Sample size is not limited.
5. The test compound does not have to be pure. If mineral
salts medium is used, BOD or COD tests can be used
to calculate an oxygen material balance.
Disadvantages:
1. Continuous operating units require money, space, and
maintenance.
2. Single batch runs do not allow for acclimation unless the
test period is unduly prolonged.
3. Residence time of 23 hours in the fill-and-draw method
is not typical of most treatment plants, and therefore
relates only to extended aeration.
4. Most investigators feel that valid results cannot be obtained
in less than ten days. There are always unexplainable
fluctuations in the efficiency of the process that must
be averaged.
5. A chemical or physical analytical method must be available
for the analysis of each compound if wastewater is used
as feed.
Trickling Filter Method --
Many small towns use trickling filters as the means of secondary
treatment. The name is a misnomer to the extent that the filter is
not a filter in the ordinary chemical engineering connotation. The
principle of the method is that the supernatant from the primary
17
-------
settling tank is sprayed on a bed of crushed stones about 6 ft
deep. Recently, plastic trickling filter medium has come into
being. The wastewater trickles through the bed to under-drains
that lead to a final settling tank. The action of the filter consists
in mechanically removing the lighter solids not removed by sedi-
mentation and in the oxidation of the wastewater by contact with
the microbial film on the filter medium.
Experimental trickling filters have never been a popular device
for measuring biodegradability. Small units require small size
media that increase operational difficulties as a result of ponding,
air circulation, and side wall effects. If recycling is not practiced,
the most important dimension is the depth of the filter bed because
that determines the retention time. The bed should be at least 6
feet deep. The main limitation is the time required to develop,
acclimate, and equilibrate the biological film. It can be a matter of
months before steady state is reached. If a trickling filter is used
for biodegradability studies, the BOD or COD or TOC removals
also should be determined so that a comparison can be made be-
tween the percentage removal of the other constituents of the
wastewater and the test compound.
18
-------
Section 2. Treatability of Specific Compounds
ANAEROBIC STUDIES
Of all the biological treatment processes, anaerobic digestion is
one of the most susceptible to poisoning by toxic substances, espec-
ially heavy metals and chlorinated organic compounds. Anaerobic
digestion is two biological processes rolled into one. The first
group of bacteria degrade the starches, fats, and proteins to mostly
short-chain fatty acids. The second group, methane-producing
bacteria, continues the process of degradation to produce methane
as the end-product. This group is much more susceptible to inhibi-
tion than the first. The reduction of methane production signals
digester failure, which results in offensive sludge that is not well
digested. The toxic effect of chlorinated organic compounds at very
low concentrations is remarkable, and provides a warning that a
close watch must be kept on the disposal of the new chlorinated
hydrocarbons that come on the market. Toxic levels of a number of
organic substances are shown in Table 1.
The effects of metals on anaerobic digesters are dependent on
the amount of sulfides present. Sulfide precipitates heavy metals,
thus forming insoluble metal sulfides that are not toxic. Most
digesters contain enough sulfides that failures are not usually the
result of metals except for large slug dosages from spills. Some of
the more common metals and their acceptable concentrations in
digesters are shown in Table 2.
Although the resilience of bacterial populations has often been
stressed, this capability is more commonly observed in aerobic
processes than in anaerobic digestion. Once the performance of
an anaerobic digester has deteriorated, it is usually rather difficult
to reestablish.
Anaerobic tests for biodegradation should be managed so that
the test system has a hydrogen electrode (En) potential of about
-0.4 volts, dissolved oxygen is excluded, and methane gas is gen-
erated. Because anaerobic organisms have protracted generation
periods, time studies should extend to at least thirty days. If any
change is made to the system, the observations of the effects
should continue for at least three times the hydraulic detention
time of the system.
19
-------
TABLE 1. TOXICITY TO ANAEROBIC DIGESTERS
Concentration mg/l
Organic compound In wastewater In sludge
Acrylonitrile - 5
Carbon tetrachloride - 10
Chloroform 0.1
1, 2—Dichloroethane - <1
Methylene chloride 1
Di(5—chloro—2—hydroxypheny I (methane
(dichlorophen) 1
Pentachlorophenol 0.4
1,1, 1—Trichloroethane - 1
Trichlorofluoromethane 0.7
TABLE 2. TOXICITY OF METAL IONS TO ANAEROBIC
DIGESTION OF PRIMARY SLUDGE
Metal ion Concentration in wastewater, mg/l
Cadmium 1
Copper 10
Dichromate (as Cr) 50
Lead 50-70
Nickel 40
Tin(asSn) 9
Zinc 10
Sodium Salt of Nitrilotriacetic Acid
In 1971, the sodium salt of nitrilotriacetic acid was suggested as
a replacement for the phosphates in detergent formulations. The
material was found to be aerobically degradable, but reports of
anaerobic degradation were conflicting. To resolve this issue,
sodium nitrilotriacetic acid (NTA-Na3) was studied in thefill-and-
draw apparatus shown in Figure 5. At various times in the study
period, both primary sludge and waste-activated sludge served as
feed to the anaerobic digester.
20
-------
ATMOSPHERIC VENT
PRESSURE
SQUEEZE BULB
Figure 5. Digester apparatus.
The program was divided into three distinct study periods, the
first of which was a control period to document the digester per-
formance.
The second period concerned the degradability of NTA-Na3
in an anaerobic system receiving only primary sludge feed. To
initiate this study, 30 mg of NTA-Na3 were added directly to the
digester, yielding a concentration of 10 mg/l in the mixed contents.
For an interval of one week, the primary sludge feed also contained
10 mg/l of NTA-Na3. This was an acclimation period designed to
indicate whether the 10 mg/l concentration would have any dele-
terious effect on the digestion process. The gas production and pH
remained within normal limits. The NTA'Na3 concentration in
the daily feed sludge was then increased to 20 mg/l, and maintained
at that level for three months. Figure 6 shows the l\ITA'Na3
concentration in the mixed digester contents during this time
period. On day zero the concentration of NTA-Na3 was abruptly
increased to slightly over 10 mg/l as a result of the direct addition
of 30 mg of NTA-Na3 to the digester. The NTA-Na3 concentra-
tion gradually increased to the feed concentration 20 mg/l over a
120-day period. This would be predicted on the basis of the hy-
draulic detention time of 30 days. Over the 120 days of observation,
no appreciable degradation of the NTA-Na3 was noted in the
primary sludge digestion process. Table 3 shows that the digester
was performing satisfactorily with regard to organic loading, gas
production, and volatile solids destruction during this second
period. The conclusion was that NTA-Na3 was not degraded in
an anaerobic process operated entirely on primary sludge feed.
21
-------
Figure 6. NTA-Na3 concentration of digester, day 10 through 205.
TABLE 3. ANAEROBIC DIGESTER PERFORMANCE
Parameter
Study period
1
Volatile solids destroyed (%)
Volatile solids loading (g/l/day)
Gas produced from volatile
destroyed (l/g)
Gas produced from volatile
added (l/g)
Volatile solids in feed (%)
49 76 65
0.71 0.80 0.57
0.89
0.94
0.95
0.43 0.72 0.62
82 69 83
The third study period concerned the anaerobic digestion of
primary and waste-activated sludge. The digester was switched to
a daily feed of 80 percent primary sludge and 20 percent waste-
activated sludge, on a volume basis. The overall concentration of
NTA-Na3 was 20 mg/l. The waste-activated sludge was obtained
from an activated sludge plant that was acclimated to the degrada-
tion of NTA-Na3. The digestion process was monitored for two
weeks. Operation was satisfactory, and the concentration of
NTA«Na3 in the digester contents remained near 20 mg/l. The
percent of waste-activated sludge in the daily feed was then in-
creased to 50 percent by volume. Over a two-month period, the
22
-------
NTA-Na3 content of the mixed digester contents gradually de-
creased, until at the end of this period the concentration was near
zero (see Fig. 6). To verify that the anaerobic system was biologic-
ally degrading the NTA-Na3, a direct addition of NTA-Na3
(to yield about 20 mg/l in the mixed digester contents) was added
on day 190. The usual daily dose of 20 mg/l in the combined sludge
feed was continued. Figure 6 shows the response of the system as
measured by the NTA*Na3 content of the reactor contents. In
about 15 days (day 205), the residual NTA-Na3 was again near
zero.
Table 3 provides a summary of the digester's performance during
each of the three study periods. Typical anaerobic digester perform-
ance values were obtained in each period, a fact that indicates
good operational control of the reactor.
The results of this study support the conclusion that primary
sludge does not contain a microbial population that can murtiply
during anaerobic digestion and degrade NTA-Na3. However,
once acclimated organisms are established in the mixed digester
contents by means of an inoculum of facultative organisms from
waste-activated sludge, anaerobic degradation of NTA-Na3 can
proceed.
AEROBIC STUDIES
This section summarizes a variety of aerobic biodegradation test
methods that have been used with specific classes of compounds.
Bacterial Utilization of Lignans
Lignans are a family of methoxylated aromatic compounds of
known molecular composition that are structurally related to, but
less complex than, lignins. Lignins are more complex methoxylated
polymers that are extremely resistant to microbial action. Alpha-
conidendrin is a representative of the lignan type molecule.
Organisms capable of degrading this material were isolated from
natural sources using adaptation techniques.
A synthetic basal salts medium containing 0.1 to 1.0 percent
a-conidendrin as the sole carbon source was used for enrichment
cultures.
Since a-conidendrin is insoluble, the analytical procedure used to
detect degradation was determination of methoxyl groups on the
residual solids at various times. Alpha-conidendrin has a methoxyl
content of 17.5 percent. An adapted culture inoculated into an
aerated culture medium containing 200 mg/l a-conidendrin re-
duced this value to 0.8 percent in 20 days. Figure 7 shows the
daily progress of the degradation.
23
-------
PERCENT DEGRADATION OF
ALPHA - COWDENDRIN
METHOXYL CONTENT
RESIDUAL SOLIDS
INCUBATION TIME IN DAYS
Figure 7. Methoxyl content of residual solids and percentage
degradation of a-conidendrin.
The organisms adapted to the utilization of a-conidendrin
dissimilated vanillic, p-hydroxybenzoic, and protocatechuic acids
when grown on chemically defined media with each of these acids
as a sole carbon source. Cultures freshly isolated from a natural
source by streaking on a-conidendrin agar when incubated in a
medium with either vanillic, p-hydroxybenzoic, or protocatechuic
acid as the only carbon source showed turbidimetric measurements
indicating a limited amount of growth. However, when the same
culture was subjected to many successive transfers in a liquid
a-conidendrin medium, and was then inoculated into a medium
with any one of the above acids as the sole carbon source, the
amount of growth increased from 40- to 60-fold over that ob-
tained in the same acids before the culture was adapted.
Adaptation studies, together with chromatographic and spectro-
photometric analyses of the degradation products, indicate that the
metabolic pathway in the dissimulation of a-conidendrin by
pseudomonad strains may proceed from a-conidendrin —+ vanillic
acid —•» p-hydroxybenzoic acid — •» protocatechuic acid —* keto-
adipic acid.
24
-------
Degradation of Aromatic Compounds by Phenol-Adapted Cultures
A culture acclimated to 300 mg/l phenol was tested in Warburg
respirometers to determine its potential for degrading 104 aromatic
compounds. The washed culture was added to 0.067 M phosphate
buffer contained in the respirometers and the test compound was
introduced from the side arm to yield a 100 mg/l concentration.
Oxygen uptake was measured and chemical colorimetric analyses
were done for the specific aromatic compounds. Observations
were carried out for about 200 minutes, with the oxygen meas-
urements made at 10 minute intervals.
The various aromatic groups tested were phenols, benzyl alcohols,
hetrocyclics, cyclics, benzoic and other acids, benzaldehydes and
benzamides, and substituted benzenes. The results are shown in
Figures 8, 9, 10, 11, 12, and 13.
On the basis of the results for these organic compounds, there
appeared to be a relationship between molecular structure and
resistance to bacterial degradation. The results indicated significant
differences in resistance to oxidation within each of several well
defined groups of compounds. The relationship between molecular
structure and ease of degradation was also apparently affected by
the position of a group on the ring, the type of group, multiples
of the same or different substituents, and the size and complexity
of the substituent. The results indicated that there was a possible
relationship between molecular structure and ease of degradation
of some of the phenols. The presence of more than two hydroxyl
groups on the ring appeared to make a compound highly resistant
to oxidation, for example, phloroglucinol and pyrogallol. Dihydric
phenols seemed to be oxidized to about the same, or possibly a
somewhat lesser degree than phenol. The fact that none of the
nitrophenols tested, except o- and m-nitrophenol, and 2,4-dinitro-
phenol, showed any appreciable 02 uptake indicated that adding
a nitro group tended to increase resistance.
Chloro substitution showed that dichlorophenols were more
resistant to oxidation than monochlorophenols. The effect of the
methyl group was indicated by the relatively high level of activity
with cresols and with some dimethylphenols. The effect of position
of the substitution on the ring was illustrated with the cresols and
dimethylphenols. With cresols, substitution of the methyl group in
the para position resulted in a higher initial O2 uptake rate, but a
lower total O2 uptake, than was the case with o- and m-cresol.
The same effect for para-substituted compounds was observed with
dimethylphenols. High total 02 uptakes were noted with 2,4- and
3,4-dimethylphenol, whereas oxidative activity was lower with
dimethylphenols having methyl groups in other positions. All
monochloromethylphenols had a measurable 02 uptake while
25
-------
SUBSTRATE CONCENTRAT ION = 100 ppm
EXCEPT 2,4 DICHLOROPHENOL =60ppm
-2,6 DichloropHenolC'»JC'
_J 1 V I
2,4,6 Tfi
2,4 Dic
60 90 120 150 ISO 210
DURATION OF WARBURG RUN, MIN
Figure 8. Oxidation of hydroxy- and chlorophenols.
there was little activity with a dichloromethylphenol (4,6-dichloro-
m-cresol). This indicated that, as with the chlorophenols, dichloro
substitution increased the resistance of chloromethylphenols.
The presence of a methoxyl or phenoxyl group on the ring also
increased resistance to degradation.
Differences in O2 uptake related to molecular structure and sub-
stitution were also observed with benzoic acids. Activity with
benzoic acid was relatively higher than with its hydroxyl derivatives.
The latter in turn had greater activity than the trihydroxyl
derivatives. In general, the hydroxybenzoic acids were more sus-
ceptible to degradation than the other substituted benzoic acids.
The influence of position of substitution was evident in the case
of methylbenzoic acids because m-toluic acid was more readily
oxidized than the ortho and para derivatives. With methoxylated
26
-------
SUBSTRATE CONCENTRATION = 100 ppm
EXCEPT 2,4 DINITROPHENOL = 60 ppm
OH
350
O
-250
REPRESENT MAXIMUM ACTIVITY WITH
ANY OF THE NITROPHENOLS TESTED
30 6O 90 120 150 ISO 210
DURATION OF WARBURG RUN.MIN
Figure 9. Oxidation of methyl- and nitrophenols.
benzoic acids, increasing the number of methoxyl groups on the
ring apparently interfered with oxidation. Syringic acid was more
resistant to degradation than vanillic acid. The presence of two
carboxyl groups significantly reduced the rate of O2 uptake by
the benzoic acids. In the case of hydroxybenzoic acids and nitro-
benzoic acids, the number of compounds tested appeared to be
sufficient to provide some statistical basis for tentatively
concluding that the former were readily oxidized by the phenol-
adapted bacteria, whereas the latter were extremely resistant.
A high level of oxidation was observed with benzaldehyde and
its para hydroxyl derivative. Replacement of the hydroxyl by a
nitro group blocked the activity. This was demonstrated by the
27
-------
5 200 —
SUBSTRATE CONCENTRATION --100 PD™
EXCEPT 3,5 OINITROBENZOIC ACID = 60 pp
COOH
' 2,5 O'Mfoiybenzoic Acid (n°H
120 ISO 0 60 120 ISO 24O
DURATION OF WARBURG RUN, WIN
Figure 10. Oxidation of benzoic acids.
resistance of m- and p-nitrobenzaldehydes. Benzamide, a com-
pound in which an NH2 group replaces the hydrogen in the CHO
group of benzaldehyde, is also resistant. A measurable level of
activity was observed with vanillin.
Results obtained with benzene and its chloro derivatives, regard-
less of the number of position of the chloro substitutions, showed
that, in general, these compounds were resistant to degradation.
The effect of a nitro group was significant, as indicated by such
compounds as nitrobenzene, and m- and p-dinitrobenzene, which
had a low level of activity. In particular, the activity of the mono-
nitro-substituted benzene was below the endogenous level. The
28
-------
1 I 1 1
SUBSTRATE CONCENTRATION = 100 pp
60 90 120 ISO 180
DURATION OF WARBURG RUN, MINUTES
Figure 11. Oxidation of selected benzoic and amino acids compared
with that of tannic acid.
resistance of the nitrobenzenes to degradation seemed to decrease
as the number of nitro groups on the benzene ring increased.
Neither the methyl nor amino group appeared to interfere com-
pletely with the oxidation of benzene because both aniline and
toluene were oxidized to a limited extent. The position of the
nitro substitution may be important because there was evidence
of some oxidation of m-nitroaniline but little, if any, activity with
p-nitroaniline. Both 2,4-dinitrotoluene and 2,4,6-trinitrotoluene
(T.N.T.) appeared to be oxidized to some extent.
Studies of this type are useful for the preliminary screening of a
large number of compounds. The occurrence of oxidation under
these conditions indicates that the compound would be oxidized
29
-------
500
450 —
400 —
360
cfl
CE
o
IE
300 —
X
o
A - Benzamide U 0=CH
v /
• - p-Nitrobenzoldehyde (I
250 —
150 —
100 —
3O 60 9O I2O 150 180 2IO
DURATION OF WARBURG RUN, WIN
Figure 12. Oxidation of benzaldehydes and benzamides.
in municipal biological treatment plants. Failure of a compound
to be oxidized under these conditions is not conclusive since in
actual practice microbial population selection could occur.
Organic Compounds for Replacement of Phosphates in Detergent
Formulations -
The major function of phosphate in detergent formulations is
to prevent precipitation of calcium salts during laundering. As a
30
-------
5OO
450
400
350
a:
UJ
t 300
o
a:
u
250
200
150
100
50
SUBSTRATE CONCENTRATION = 100 ppm
OH
r Phi
-|_- J— ~~
Phenol
+ 1,3,5 Tnchlorobenzene Cl Cl
Representative of the Chlorobenzenes Tested
30 60 90 120 ISO 180
DURATION OF WARBURG RUN.MIN
Figure 13. Oxidation of benzenes.
result of the relationship between excessive phosphate discharges
and eutrophication, several organic compounds have been suggested
as substitutes for phosphates.
In 1967, the compounds shown in Figure 14 were candidates
for consideration as phosphate substitutes. These six materials were
tested for biodegradation using a basal salts medium with yeast
extract and an inoculum of settled wastewater. The compounds
were tested at concentrations of 5, 10, and 20 mg/l. The flasks
31
-------
NITRILOTRIACETIC ACID (NTA)
0
0 CH2-C-OH
HO-C-CH2-N
CH2-C-OH
0
HYDROXYETHYLIMINODIACETIC ACID (HEIDA)
0
H CH2-C-OH
HO-C-CH2-N
H CH2-C-OH
11
0
DIETHYLENETRIAMINEPENTAACETIC ACID (DTPA)
O
HO-C-CH,
0
11
CH2-C-OH
HO-C-CH2
11
0
.N-CH2-CH2-N-CH2-CH2-N
i
CH2
i
C-0
OH
Figure 14. Structural formulae of complexones.
32
CH2-C-OH
0
-------
ETHYLENEDIAMINETETRAACETIC ACID (EDTA)
0
HO-C-CH,
0
CH,-C-OH
HO-C-CH2'
ii
0
N-CH2-CH2-N
XCH2-C-OH
0
N-HYDROXYETHYLETHYLENEDIAMINETRIACETIC ACID (HEDTA)
0
0
HO-C-CH,
CHo-C-OH
HO-C-CH2-
11
0
N-CH2-CH2-N
H
C
i
H
1, 2-DIAMINOCYCLOHEXAIME-N, N' -TETRAACETIC ACID (DCTA)
0
HO-C-CH2
N -
,-N
0
CH,-C-OH
CH2-C-OH
0 0
Figure 14. (cont'd). Structural formulae of complexones.
33
-------
remained static and weekly serial transfers were made for three
consecutive weeks. After incubation for seven days, each flask
was analyzed for the test compound to detect the amount of
degradation. The chemical method used to determine the unde-
graded chelating agent was a complexation titration at pH 2.5
with copper sulfate using PAN (1-(2-pyridylazo)-2-naphthol) as
the indicator. Each compound listed was run on five different
occasions to eliminate any variability of the inoculum. The bio-
degradable compounds showed biodegradability in every test.
This method for testing for biodegradability provides an indica-
tion of the time required for adaptation, as well as a determination
of whether a compound is degraded during the test period.
Only NTA (nitrilotriacetic acid) and HEIDA (hydroxyethylimin-
odiacetic acid) lost their chelating ability under the test conditions.
The acclimation period for NTA was about two weeks; that for
HEIDA was slightly less. Both these compounds contain only one
nitrogen atom; the other four compounds, containing two nitrogen
atoms, did not degrade under these test conditions.
Although the analytical method only determined whether the
compound lost its chelating ability, it would be fairly safe to
assume that fragments of these molecules would be subject to
complete degradation. No manometric testing was done.
Another organic compound under consideration as a phosphate
replacement is carboxymethyl tartronate, more commonly re-
ferred to as GMT. This compound has the formula:
(Trisodium 20-oxa-1,1,3-propane Na02C CO2 Na
tricarboxylate) H-C-0-C-H
I I
Na02C H
The biodegradation of this compound at a concentration of 20
mg/l was studied in 300 ml continuous flow reactors as shown in
Figure 9, using a mineral salts medium with yeast extract and sup-
plemental organic materials. The results are shown in Figure 15.
The remarkable feature is that a period of about 12 weeks was
necessary for acclimation. Attempts to shorten the acclimation
period by external addition of forest and garden soil were not
successful. However, once acclimated, the culture could degrade
shock CMT doses, and could immediately degrade CMT after a
substrate starvation period.
During this study three analyses were used to monitor the CMT in
the reactor effluent: TOG, a non-specific colorimetric, and a specific
gas chromatographic procedure. After acclimation, the test reactor
and the control reactor effluent had about the same TOG concen-
tration. Figure 16 shows the comparison of the colorimetric and the
gas chromatographic assays during 18 weeks of the test period.
34
-------
0
Q
z
£
t-~
z
TOC, EXPERIMENTAL
REACTOR
CMT,EXPERIMENTAL
REACTOR (20mg/l)
CMT, CONTROL
REACTOR
50 mg/l CMT
O
6 8 10 12 14 16 18 20 22 24
TIME, PROGRESSIVE WEEKS
Figure 15. CMT and TOC of reactor effluents with synthetic feed.
25-
20-
15-
10-
5-
SYNTHETIC WASTEWATER
^
-------
Synthetic Detergents --
In the early 1960s great attention was focused on the biodegrada-
bility of synthetic detergents because of the occurrence of billows
of foam at wastewater treatment plants and along stretches of
rivers. Early production of detergents yielded compounds with
branching hydrocarbon chains that were relatively resistant to
biodegradation. Various alternative compounds were considered
for replacement.
Using a static mineral salts medium with added yeast extract,
many of these compounds were screened for biodegradability.
Provision for acclimation was a part of the protocol. Settled waste-
water was used to seed the medium. Tests on each compound were
repeated on five different occasions to determine the variability
of the seed. Table 4 gives the average results of these five replicate
tests for various synthetic detergents. Several phenolic compounds
with known degrees of degradability were included to verify the
method. The compounds were tested at a concentration of 10 mg/l.
The chemical analysis used to detect the undegraded alkyl
benzene sulfonates and alkyl sulfates was the methylene-blue
method. The cobalt-thiocyanate test procedure was used for the
nonionic surfactants. Details of this method can be found in
Reference 10. Indirect determinations also were made by judging
the foaming tendencies of the surfactants. The loss of surface
activity or foam does not necessarily mean complete degradation
of the molecule. The 4-amino-antipyrene method was used for the
assay of phenol, o-cresol, and the chlorinated phenols. The nitro
compounds were adjusted to pH 10.0 and determined spectro-
photometrically. As a further check on the phenolic compounds,
acid was added to all the terminal flasks, ether extracted, and the
ether examined for the benzene ring in the ultraviolet region of
the spectrum. No suitable solvent was found that would extract
polyethylene glycol 400 from the medium; therefore, it was
necessary first to dry the contents of the flasks at 80° C before
using the cobalt-thiocyanate procedure. The ether extraction
step was omitted and the complex formed directly with the
dried residue.
The results obtained demonstrate the general applicability of
this test. In addition to determining whether a compound is de-
graded during the test period, it provides an indication of the time
required for adaptation. This is very useful information because it
is not enough merely to know that a compound is degradable; the
rate at which it is degraded is equally important where water
quality depends on it. Such information is helpful in predicting
the behavior of a compound in a waste treatment plant and in sur-
face waters. For example, if microorganisms in a stream or waste
36
-------
TABLE 4. DEGRADABILITYOF SURFACTANTS
Percent
of compound degraded
Number of subculture
Name of compound
An ionic surfactants:
Linear alkyl (Ci2> benzene
sulfonate
Linear alkyl (Cio— Cis) ben-
zene sulfonate
Linear alkyl (Ci2— CH) ben-
zene sulfonate
Primary (Ci2) alcohol 3-mole
ethoxysulfate
Secondary (Cn— Cis ) alcohol
3-mole ethoxysulfate
Linear alkyl (Cu) benzene
sulfonate
Linear alkyl (Cn— CIB) phenol
5-mole ethoxysulfate
Branched alkyl (Cg) phenol
4-mole ethoxysulfate
N on ionic surfactants:
Linear primary (Cn) alcohol
8-mole ethoxylate
Linear primary (Ci2 ) alcohol
9-mole ethoxylate
Polyethylene glycol - 400
Branched alkyl (Cs) phenol
9-mole ethoxylate
Branched alkyl (Cg) phenol
1 5-mole ethoxylate
Branched (Cia) alcohol
9-mole ethoxylate
Phenol
o— Cresol
o— Nitrophenol
2,4— Dichlorophenol
m— Chlorophenol
2,4,6,— Trinitrophenol
Initial
84.5
90.5
93.5
99.5
96.0
61.0
39.5
9.5
98.5
99.5
52.0
0.0
12.5
1.5
99.5
99.5
81.0
8.0
0.0
0.0
1
98.5
90.5
97.0
99.5
97.0
92.5
12.5
9.0
99.0
99.5
42.0
0.0
6.5
0.0
99.5
99.5
84.5
47.0
0.0
0.0
2
99.0
92.5
97.0
99.5
97.0
96.0
11.5
3.5
99.0
99.5
63.5
0.0
0.0
2.0
99.5
99.5
93.5
91.5
0.0
0.0
3
99.0
92.0
97.0
99.5
97.5
96.0
12.0
7.0
99.0
99.5
61.0
0.0
0.0
2.0
99.5
99.5
98.5
98.5
0.0
0.0
37
-------
treatment plant were exposed continuously to a compound such
as 2,4-dichlorophenoI, the microorganisms probably would adapt
to degrade it. If, on the other hand, the exposure is intermittent,
such as a spill, one would not expect the compound to be degraded
in a waste treatment plant. The compound might persist in a
stream for days before the naturally occurring microorganisms
adapted to degrade it.
As a result of the public concern about nondegradable deter-
gents, leading manufacturers modified their production processes
to develop detergents with linear hydrocarbon chains. The market-
ing of these new materials began about 1965. Five treatment
plants located around the nation were monitored to determine
the change in influent wastewater surfactant quality. Monthly
raw wastewater samples were collected for 18 months. One liter
samples were aerated in 2-liter flasks for seven days after inocula-
tion with 40 mg of volatile solids from an activated sludge aeration
tank. The determination of the ratio of hard detergent (resistant
to degradation) to soft detergent (readily degraded) was calibrated
by experimental procedures using various mixtures of known lots of
hard and soft detergents. Methylene-blue active substance (MBAS)
was the analytical method used for determining degradation.
The data accumulated for one of the plants, in Richmond,
Indiana, are given in Figure 17. Similar results were noted at the
other four sites. It was concluded that a vigorous manufacturing
and marketing strategy had been deployed and the quality of
surfactants in influent wastewater rapidly changed from the
undesirable hard form to the acceptable soft form.
•Z. 80
LLJ
O
OC 70
(_• 60
z
111
O 50
or
LU
h- 40
111
D
Q 30
OC
J J A
19 6 5
YEAR AND MONTH
Figure 17. Percent hard detergent in a raw wastewater; Richmond,
Indiana.
38
-------
Steroid Hormones
The increased medical use of natural steroid hormones, coupled
with their recent use as oral contraceptives has raised concern
about their persistence in wastewater and the ability of conven-
tional waste treatment processes to remove these compounds.
The normal concentration of steroids in municipal wastewater in
the United States would not be expected to be more than a few
tenths of a milligram per liter. Nevertheless, since they are physio-
logically active in very small amounts, it is important to determine
to what extent the steroids are biodegraded in the normal course
of wastewater treatment and in the receiving bodies of water that
may eventually be used for water supplies.
There is little information on the biodegradation of steroid
hormones. The literature is almost void of studies that report ring
cleavage and complete oxidation of the molecules. A few studies
have been done on some of the natural steroids, but no biodegrada-
tion studies of the synthetic ovulation-inhibiting hormones have
been reported.
Hormones representing different groups of steroids were selected
for the biodegradability studies. The following is a list of represen-
tative hormone compounds from each of the classes of steroids
chosen for the study: (1) Sterols — cholesterol; (2) Adrenocortical
hormones — progesterone and pregnanediol; (3) Estrogenic
hormones — estrone, beta-estradiol, and estriol; (4) Androgenic
hormones — testosterone and androsterone; (5) Androgenic hor-
mone derivative — methyl testosterone; and (6) 17-ketosteroids
— estrone and androsterone. The most commonly used synthetic
sex hormones were used for these studies. They were: (1) Estrogens
— ethynyl estradiol and mestranol (ethynyl estradiol-3-methyl
ether) and (2) Progestins — norethynodrel (17-hydroxy-19-nor-
17a -pregn-5(10)-en-20-yn-3-one), norethindrone (17-hydroxy-19-
nor-17a-pregn-4-en-20-yn-3-one), dimethisterone (6a,21-dimethyl
17-hydroxy-pregn-4-en-20-yn-3-one), etyhnodiol diacetate (19-nor
-17a-pregn-4-en-20-yne-3(3,17-diol), norethindrone acetate, me-
droxyprogesterone acetate (17-hydroxy-6a-methyl-pregn-4-ene-
3,20-dione acetate) and chlormadinone acetate (6-chloro-17-
hydroxy-pregna-4,6-diene-3,20-dione).
The compounds were tested at a concentration of 20 mg/l in a
basal salts medium plus yeast extract, using activated sludge organ-
isms as inoculum. The flasks were aerated by shaking during the
entire observation period. Since these compounds are insoluble,
they were suspended in the medium by introducing the hormone
dissolved in acetone and warming the medium to evaporate the
acetone.
39
-------
Generic colorimetric assays for each group of steroids were used
to follow the progress of the degradation. The fate of the com-
pounds was also determined by spectrophotometric scanning to
determine ring cleavage, and use of ascending thin layer chroma-
tography for identification of residual products. Because the
materials are insoluble, both the supernatant and solids portion
of the medium had to be extracted with chloroform to account
for the total steroid content of the flask. Each compound was
evaluated three times in duplicate. Provision for acclimation was
made by transferring subcultures into fresh medium weekly. Table
5 shows the average percent degradation for the three test runs
on each steroid.
TABLE 5. AVERAGE PERCENT LOSS OF STEROIDS BY
WEEKLY SUBCULTURE ENRICHMENT
Percent degradation of compound
Number
Steroid compound
Androsterone
Cholesterol
Testosterone
Progesterone
Estrone
Beta-estradiol
Estriol
Pregnanediol
Methyl testosterone
Ethynyl estradiol
Norethynodrel
Norethindrone
Norethindrone acetate
Dimethisterone
Medroxyprogesterone acetate
Chlormadinone acetate
Ethynodiol diacetate
Mestranol
Initial
96
96
95
93
91
90
82
79
77
72
71
70
68
65
56
55
52
47
1
100
100
100
100
98
97
96
95
91
89
88
87
84
82
80
78
72
73
of subcultures
2
100
100
100
100
97
97
97
96
95
93
92
91
90
88
3
100
100
100
100
100
99
98
97
94
89
4
100
100
100
96
90
40
-------
The overall results of these studies show a variation in the relative
susceptibility of the natural hormones and the synthetic estrogen
and progestin components of oral contraceptives to biodegradation.
They indicate that in the microbial population found in activated
sludge, there are species capable of producing enzymes that will
degrade both natural and synthetic steroid hormones if conditions
for acclimation are provided.
Hazardous Organic Compounds
Benzidine (1,1'-biphenyl)-4,4'-diamine) a bicyclic arylamine, has
been demonstrated to be carcinogenic in experimental studies with
animals such as rats, mice, dogs, and hamsters. Literature citations
implicate many arylamines including benzidine as causative agents
of bladder carcinomas in humans. Because of this carcinogenic
property, benzidine has been included in the list of "14 Carcino-
gens" tabulated in the Federal Register. The Occupational Safety
and Health Agency has established standards to control worker
exposure to these compounds, which are used in various process
industries. Benzidine is used in hospitals, research laboratories,
and dyestuff industries.
Even though the use of materials such as benzidine is regulated, it
is advisable to ascertain the biodegradation of this compound in
conventional wastewater treatment, and to determine what protec-
tion a secondary facility could afford a receiving water in the event
that this material is discharged into a sewer system. Studies have
shown that the majority of carcinogenic compounds, because of
their strain-free condensed nuclear configuration, are resistant to
bio-oxidation by unacclimated microorganisms in activated sludge.
The purpose of this study was to find whether or not acclimation
to benzidine could be established in an activated sludge process
operated in a continuous feed mode within a specific range of feed
concentrations of benzidine under appropriate operating
conditions.
Continuous flow reactors of the type shown in Figure 4 were
used. The medium was settled municipal wastewater spiked with
selected concentrations of benzidine. The reactors were operated at
a hydraulic retention time of 24 hours and a solids retention time
of 30 days. Phase I concerned benzidine concentrations of 0.5, 1,
and 5 mg/l. Phase II studied benzidine concentrations of 10, 20,
and 30 mg/l. Biodegradation was followed by a specific colori-
metric assay of benzidine, utilizing oxidation with Chloroamine-T,
and residual chemical oxygen demand analysis.
Since the biological solids were in the range of 4,000 mg/l, the
sorption of benzidine to these active surfaces had to be evaluated.
The solids for the 0.5 mg/l benzidine reactor contained 0.003
41
-------
percent benzidine by weight. Solids from the 30 mg/l reactor con-
tained 0.3 percent sorbed benzidine. On a mass through-put basis,
both these determinations show that only about one percent of the
dosed material was sorbed to the biological floe.
The color of the reactor sludges during Phase II was remarkable.
The control unit mixed liquor was light tan in color during the
entire study. At the 6-week point the 10 mg/l, 20 mg/l, and 30 mg/l
dosed sludges were brown, dark brown, and brown-black, respec-
tively. The intermediate oxidation products and benzidine itself
are highly chromophoric compounds; and since these higher con-
centrations were not completely degraded, this visual observation
confirms the analytical results.
Figure 18 presents the observations collected during the 2 phases
of study.
100
90
o 50
0)
cr
ni(|
O - O 10 m
-------
Results from the biodegradation studies on the several concentra-
tions of benzidine show that acclimated extended aeration sludges
can degrade continuous doses of 1 mg/l benzidine. Less complete
degradation occurs at higher dosage levels and intermediate oxida-
tion products begin to accumulate in the system. Even at the
highest dose of benzidine, 30 mg/l, no marked interference with
carbonaceous removal efficiency was noted, since the increased
COD of this effluent could be related to the presence of undegraded
benzidine.
Concentrations of benzidine between 1 and 5 mg/l were not
studied, but because these studies employed an extended aeration
process and most secondary municipal facilities have far shorter
hydraulic retention times, it is recommended that influent benzi-
dine concentrations not exceed 1 mg/l. This limit would ensure
that unacclimated systems would not discharge significant concen-
trations of benzidine into receiving waters.
The work also suggests that ambient concentrations of benzidine
below 1 mg/l will be degraded by natural ecosystems, and the
material will not accumulate.
Two other compounds of environmental concern are acenaph-
thene (ANT) and dimethyl phthalate (DIVIP). Phthlate esters have
been recovered from many environmental samples. These com-
pounds are used as plastisizers and industrial intermediates. Some
studies have indicated that these compounds bioaccumulate in
plants and animals and subtle toxic effects have been noted. Naph-
thalene compounds have also been noted in a wide variety of
environmental samples. Recent studies have shown naphthalene
compounds to be toxic to aquatic invertebrates and fish. Know-
ledge of the biodegradation of these two compounds would be
helpful in determining the efficiency of municipal treatment
processes for control of the substances.
The compounds were studied in the continuous flow reactors
shown in Figure 3. Settled wastewater was used as feed and ANT
and DMP were added singly and in combination. Concentration
levels of 0.1 and 1.0 mg/l were selected. It was possible to study
ANT and DMP in combination because the compounds could be
resolved using a Perkin-Elmer # 900 gas chromatograph equipped
with a dual flame ionization detector and a Varian Series Number 4
computer data programmer.
The reactors were started with activated sludge and feed settled
wastewater for one week prior to the experimental run. Seven
reactors were operated at a hydraulic retention time of 6 hours
and a mixed liquor suspended solids concentration of 3,000 mg/l.
The ANT and DMP content of feeds and effluents was determined
by extraction of the samples with Freon-TF and evaporation of
the extract by means of the Kuderna-Danish evaporation pro-
cedure. The gas chromatograph could detect 5 /ug of ANT or
43
-------
DMP. Table 6 presents the results.
Reactors 1 and 2 were fed approximately 0.1 and 1.0 mg/l ANT,
respectively; Reactors 3 and 4, 0.1 and 1.0 mg/l DMP; Reactors 5
and 6, 0.1 and 1.0 of both ANT and DMP; Reactor 7 was a control.
ANT is insoluble in water and had to be dissolved in ethyl alcohol.
The proper amount of 2 percent alcoholic stock solution was added
to the wastewater feed to achieve the desired concentration.
Table 6 shows that after 48 hours of operation no ANT or DMP
could be detected in the final effluents of any of the seven reactors.
Trace amounts were noted in the first 24 hour sample. The TOC
contents of the feeds to Reactors 2 and 6 are elevated above the
other reactors because of the addition of the alcoholic solution to
achieve 1 mg/l ANT.
The test procedure was replicated once each week for five weeks.
In no case was either ANT or DMP noted in the effluents collected
after 48 hours of operation.
TABLE 6. BIODEGRADATION OF ANT AND DMP
Reactor number
Wastewater
feed, mg/l:
ANT
DMP
TOC
TKN
N03-N
Reactor effluent.
24 hr, mg/l:
ANT
DMP
TOC
TKN
N03-N
Reactor effluent,
48 hr, mg/l:
ANT
DMP
TOC
TKN
N03-N
1
0,13
-
19
13
0.3
0.00
-
3
1
10
0.00
-
5
0.8
10
2
0.97
-
35
13
0.1
0.02
-
7
1
9
0.00
-
7
1
8
3
0.08
18
12
0.1
-
0.00
3
1
11
-
0.00
3
1
10
4
-
0.89
17
14
0.1
-
0.01
5
2
13
-
0.00
3
0.8
13
5
0.10
0.10
20
12
0.1
0.00
0.00
6
1
10
0.00
0.00
4
0.9
10
6
0.93
0.85
38
11
0.1
0.01
0.01
5
0.09
9
0.00
0.00
7
0.8
8
Control
-
-
17
11
0.1
0.00
0.00
3
0.8
8
0.00
0.00
4
0.7
9
44
-------
To ensure that the aeration solids did not sorb the ANT or DMP,
after one test period the reactor contents were extracted with
Freon-TF, Insignificant sorption was noted. It is also unlikely that
the compounds were air-stripped from the reactors, on the basis of
the physical properties of ANT and DMP.
Table 6 shows that ANT and DMP singly or in combination at
concentrations of 0.1 or 1.0 mg/l do not interfere with the oxida-
tion efficiency of the microorganisms. The effluents contained low
residual TOC and nitrification was not inhibited.
45
-------
Section 3. Treatability of Metals by Activated
Sludge Systems
Extensive treatability studies on the interaction of metals with
activated sludge systems have been conducted at the U.S. Public
Health Service's Robert A. Taft Engineering Center in Cincinnati.
Continuous flow pilot plants with a flow capacity of 750 liters per
day were utilized. The design features of the pilot plants are shown
in Figure 19. This size pilot unit is sufficient to allow study of
chemical distribution, biological response, and engineering design
features.
These pilot units proved to be good simulants of wastewater
treatment processes. They were operated and maintained on a 24
hour, 7 day per week schedule, with sustained analytical super-
vision. Sufficient observations were made to establish statistically
valid evidence of performance in systems with metal input and
metal withdrawal in general working balance. Over a 5 year period,
chromium, copper, nickel, and zinc were studied individually and
in combination.
AIR HEADER
CAPACITY: 7.9 gal
DETENTION TIME: 2 hr
SURFACE OVERFLOW RATE:
102 gpd/ft2
PRIMARY
SETTLER
CAPACITY: 4.6 go!
DETENTION TIME: 1.2 hr
SURFACE OVERFLOW RATE: SPIRAL-FLOW
142 9pd ft2 AERATOR
CAPACITY: 23.6 gal
AERATION PERIOD: 6 hr
BOD LOADING: 0.5 TO 0.8 Ib 'day/lb
OF VOLATILE SOLIDS UNDER
AERATION
Figure 19. Plastic activated sludge pilot plant.
46
-------
The wastewater feed to the plant during the various studies was
either a weak supplemented domestic wastewater or a strong
nonsupplemented domestic wastewater. Both feeds give results
indistinguishable by the usual analytic measures.
DISTRIBUTION OF METALS THROUGH THE PROCESS
Complete material balances of the metals were made during each
study. Table 7 summarizes these studies. The table is based on the
amount of metal fed to a unit during a compositing period. Varia-
tion between compositing periods was common, as indicated by the
range of observations for the efficiency of the process in removing
metals. The percent metal unaccounted for in Table 7 is not a firm
figure, but represents the cumulative errors involved in sampling
sludges, flow measurements, and analytical methods.
Metal balances were performed for each selected concentration of
the metals studied, not just those shown in Table 7. Each metal was
studied in about five increments over the range of 1 to 20 mg/l. In
addition, four metals were simultaneously traced during a combina-
tion study. Over the concentration ranges studied, no great differ-
ence in the efficiency of the process in removing the metals was
noted. Zinc and copper, studied as the cyanide complexes, showed
the same overall removal as when studied as the sulfates.
Chromium, introduced to an activated sludge process as hexa-
valent chromate, can show wide variation in concentrations at the
various process outlets. Reducing substances in the raw sewage can
cause precipitation of trivalent chromium with the primary sludge.
Also, under anaerobic conditions, the organisms in the return
sludge entering the primary settler can utilize the oxygen of the
chromate radical and adsorb the trivalent chromium on the bio-
logical floe. Under these conditions chromium removal can reach
90 percent.
TABLE 7. DISTRIBUTION OF METALS THROUGH ACTIVATED
SLUDGE PROCESS WITH CONTINUOUS DOSAGE
% of metal fed
Outlet
Primary sludge
Excess activated sludge
Final effluent
Metal unaccounted for
Average efficiency of
process in removing
metal
Range of observations
Chromium (VI)
(15 mg/l)
2.4
27
56
15
44
18-58
Copper
(10 mg/l)
9
55
25
15
75
50-80
Nickel
(10 mg/l)
2.5
15
72
11
28
12-76
Zinc
(10 mg/l)
14
63
11
12
89
74-97
47
-------
Table 7 shows that a considerable portion of the metal introduced
is removed in the secondary sludge. The effects of the metals on the
mixed liquor are apparent even in the 1 to 2 mg/l range. During 5
years of study no bulking was encountered in a metal-fed system.
The floe in the final settler quickly settled. Control units frequently
bulked. Table 8 shows the effects of a combination of four metals
on the sludge density index and volatile solids content of mixed
liquor.
With the exception of zinc, the conventional activated sludge pro-
cess is not very efficient in the removal of metals from the influent
stream. The metal removed is concentrated at two points. In the
primary sludge, a maximum concentration would occur if all the
metal were removed with this sludge. Here the ratio of total flow
volume to primary sludge volume is a limiting factor. Another
point of concentration is in the secondary sludge. Since the volume
of secondary sludge removed from the process may be small com-
pared to the flow through the process, concentration may be high
at this point.
There is no net removal of metal if the primary and secondary
sludges containing the metals are not permanently removed from
the line of flow. For instance, an extended aeration plant passes
all the metal to the receiving stream unless secondary sludge is
removed.
The copper, chromium, and nickel discharged with the final ef-
fluent from an activated sludge plant receiving these metals are
predominantly in a soluble form. At an influent concentration of
10 mg/l, only a small amount of zinc is discharged, and this is an
insoluble zinc. At higher influent concentrations greater amounts
of zinc were discharged as soluble zinc.
EFFECTS OF METALS ON AEROBIC PROCESS
Many investigators of metal toxicity have employed batch opera-
tion or direct dosing of the metal to the aeration chamber. Data
from individual studies (Table 9) show that primary settling has two
TABLE 8. EFFECTS OF METALS ON MIXED LIQUOR SOLIDS
Analysis
Sludge density index
% Volatile solids
Control
unit
1.5
66.7
Mixed liquor from
Metal mixture
8.9 mg/l 4.9 mg/l 2.2 mg/l
3.2 3.4 2.4
57.9 61.8 63.8
48
-------
effects on the metals before entry into the aeration tank. First, the
total metal content of the primary effluent is less than that of the
influent because some metal is removed with the primary sludge.
Second, the chemical and physical characteristics of the wastewater
alter the form of the soluble metal introduced. This was especially
true in the case of zinc where 90 percent of the added soluble zinc
was converted to an insoluble form. The differentiation between
soluble and insoluble metal in all studies was made by filtration of
the sample through an 0.45 /xn membrane filter and by acid diges-
tion of the filtrate before analysis.
The procedure used to determine the concentration of metal in
the influent wastewater that would give a barely detectable reduc-
tion in efficiency during the aeration phase of treatment is indicated
by Figure 20, which shows the results of a study of copper.
During each run, data from an experimental pilot plant unit and
a control unit receiving no metal were compared. The metal was
added continuously to a constant wastewater feed of the pilot plant
unit. Two weeks of acclimation were allowed before data on the
quality of the final effluent were collected. This time interval is
also required for the metal content of the activated sludge to build
up to a condition of operating equilibrium. Final effluents from
both units were assayed daily for BOD, COD, suspended solids, and
turbidity. The run for any selected metal dosage was continued for
60 days to obtain sufficient data. The values for the two units were
then compared as frequency distribution curves. The parameter of
effluent quality in Figure 20 is COD; this is plotted as a frequency
distribution on arithmetic paper. As shown in the figure, copper
present continuously at 0.4 mg/l did not noticeably increase the
effluent COD of the experimental unit. A copper concentration of
1.2 mg/l however, caused a significant increase in effluent COD.
From this and the other parameters measured, it is concluded that
copper present continuously at 1 mg/l in the influent is the thres-
hold dose for the aeration phase.
TABLE 9. METALS IN PRIMARY EFFLUENTS
Soluble metal
introduced in Metal in primary
wastewater feed, effluent, mg/l
Metal
Chromium (VI)
Copper
Nickel
Zinc
mg/l
50
10
2.5
10
Total
47
9
2.0
9
Soluble
38
3.0
1.0
0.6
49
-------
200
150 -
~3i 100 -
Q
O
o
100
10 20 30 40 50 60 70 80 90
PERCENT OF OBSERVATIONS < STATED VALUE
Figure 20. Effect of copper, fed continuously as copper cyanide
complex, on COD of final effluents.
It is also useful to plot frequency distribution curves on proba-
bility paper. Readily available statistical measurements are given by
this type of presentation. If a-straight line is obtained with arith-
metic probability paper, normal distribution of data is verified. The
50 percent point is very close to the true arithmetic mean of the
observations, and the slope of the line is a measure of the standard
deviation. Figure 21 is such a plot of data collected during a study
of the effects of a mixture of four metals on the activated sludge
process. The need for extensive sampling is shown here. The control
unit had an average final effluent COD of 45 mg/l; however, contin-
uation of this point to the experimental unit shows that 12 percent
of the time the experimental unit's final effluent had a COD of 45
mg/l or less.
Copper and zinc are frequently used by the plating industry as
cyanide complexes. These two metals were studied in both the sol-
uble cation form (as sulfate) and as soluble cyanide complexes.
Results show that once the activated sludge acclimates to the
continuous presence of either form of the metal, there is no differ-
ence in effects on treatment efficiency. Figure 22A shows that
where turbidity of the final effluent was used as the measure of
treatment efficiency, after eight days the system receiving a 10 mg/l
50
-------
concentration of zinc as cyanide complex had acclimated to cyan-
ide and was producing effluent of stable turbidity. The cyanide
content of the effluent followed a similar pattern, with almost
complete removal of cyanide at the end of seven days. Figure 22B,
from a run with a 20 mg/l concentration of zinc as the sulfate,
showed no such acclimation. Direct comparison of the 10 mg/l
concentration of zinc sulfate and a 10 mg/l concentration of zinc
cyanide complex versus the same control unit is shown in Figure 23.
The BOD data collected after two weeks of acclimation, showed
no significant difference between the two forms of zinc fed.
METAL MIXTURE: Cr,Cu,
Ni, Zn; TOTALING 8.9 mg/liter
METAL
MIXTURE
^0 30 40 50 60 70 80 90 95 98 99
PERCENT OF OBSERVATIONS < STATED VALUE
Figure 21. COD of final effluents.
IOO
60
Zn (CN)T
COMPLEX, 10 mg/iiter
—1 1 1 1
B
ZnS04, 20 mg,|,ler
01 23456789 10 Ol 23456789 IO
TIME, days TIME, days
Figure 22. Comparison of acclimation to complexed zinc (A) and
zinc sulfate (B).
51
-------
Q
O
CD
I I
30 40 50 60
PERCENT OF OBSERVATIONS < STATED VALUE
Figure 23. Cumulative frequency data on quality of final effluents
with zinc concentration of 10 mg/l in wastewater feed.
The reaction pattern of the activated sludge process was the same
for each of the metals studied. A small dose of metal gives a signifi-
cant reduction in treatment efficiency, but substantially larger doses
do not further decrease the efficiency greatly. Figure 24 graphically
illustrates this situation. One to two milligrams per liter yield a de-
tectable response after which a plateau is reached, and complete
failure does not occur until one to two orders of magnitude are
reached.
Table 10 lists the concentrations of metals that give a significant
increase in the usual parameters applied to judging treatment effic-
iency. These may be considered threshold concentrations. It should
be borne in mind, however, that these limits were obtained under
carefully controlled laboratory operation. The significance of Figure
24 is that the threshold concentration is mainly of academic, rather
than practical interest and actual plant situations are concerned with
the plateau region of metal dosage and response.
52
-------
I-
LU
o
DC
o
z
UJ
u.
UJ
100
40
LU
or
20
in
tn
O
CONCENTRATION OF METAL IN INFLUENT SEWAGE
Figure 24. Response of system to metal dosage.
TABLE 10. CONTINUOUS DOSE OF METAL THAT Wl LL GIVE
SIGNIFICANT REDUCTION IN AEROBIC TREATMENT
EFFICIENCY
Metal
Concentration in
influent wastewater,
mg/l
Chromium (VI)
Copper
Nickel
Zinc
10
1
1-2.5
5-10
The results of these studies showed that the aeration phase of bio-
logical treatment can tolerate in the influent wastewater, chromium,
copper, nickel, and zinc up to a total heavy metal concentration of
10 mg/l, either singly or in combination, with about a 5 percent
reduction in overall plant efficiency.
Slug doses of metals to the activated sludge process were also
studied. The concentration of metal that constitutes a harmful slug
dose is determined by the waste volume, the volume and character-
istics of the dilution water, the specific form of the metal, and the
usage of the stream below the point of effluent discharge. For conven-
ience, only a single measure of effluent quality, such as an increase
in organic material passing through the plant, has been used to
53
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judge a harmful slug dose. As an example, in Figure 21, the control
unit has a COD of 70 mg/l or less 98 percent of the time; then a
harmful slug dose can be defined as that concentration of metal
that will yield an effluent COD in excess of this value for the sub-
sequent 24 hours of performance. The effects of slug doses were
observed on 4-hr metal doses to the influent wastewater.
Table 11 gives the results obtained. To fix these concentrations
more accurately would require an inordinate amount of time and
money. Table 11, therefore, is the best estimate of what concentra-
tion of metals causes an exceptional displacement of treatment
plant performance as the result of a slug dose.
Not reported in the table are the results of slug studies in which
the metals were added as cyanide complexes. In these cases, the
cyanide toxicity completely obscured the toxic effect of the metal.
In general, acclimation of the system to low concentrations of
metals or cyanide did not offer protection from slug doses.
The inhibition of nitrification by heavy metals has been previously
studied with regard to individual metals. A pilot plant that received
a combination of four metals also showed inhibition of nitrification.
There was no evidence of acclimation of the nitrifying organisms to
the metals. The oxygen requirement of this metal-loaded sludge was
less than that of the control unit because oxygen for the biological
transformation of ammonia to nitrate was not utilized. Figure 25
shows the nitrate content of the final effluents of a control and
metal-fed unit. Inhibition of nitrification is regarded as an important
effect of metal toxicity. A plant so affected would discharge all the
influent nitrogen in excess of that needed for synthesis, predomin-
antly in the form of ammonia. Such an effluent would require con-
siderable chlorine if downstream breakpoint chlorination were used,
and nitrification in the receiving stream would use large amounts of
oxygen.
TABLE 11. METAL CONCENTRATION IN 4-HR SLUG DOSE
THAT WILL PRODUCE HARMFUL SLUG, AS MEASURED
_ BY COD
Concentration
Metal influent wastewater, mg/l
Chromium (VI) >500
Copper 75
Nickel >50<200
Zinc 160
54
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FINAL EFFLUENT OF PILOT PLANT
CONTROL UNI T
FINAL EFFLUENT OF PILOT PLANT
UNIT RECEIVING METAL MIXTURE?
DO OF FINAL EFFLUENT
OF PILOT PLANT UNIT
RECEIVING METAL MIXTURE
I*-DO OF FINAL EFFLUENT OF
', PILOT PLANT CONTROL UNIT
o
Q
10 20 30 40 50 60 70 8O
TIME, DAYS
Figure 25. Nitrate nitrogen in final effluents.
EFFECTS ON ANAEROBIC DIGESTION
The metal-bearing sludges produced by the pilot plants were di-
gested in single stage non-mixed digesters. Organic loading was for
non-mixed operation. A small circulating pump was used once each
day to obtain representative samples of sludge for material balances.
In each metal study both primary sludge and combined primary and
secondary sludges were digested. The metal contents of the sludges
fed to the digesters during several of the runs are given in Table 12.
The primary sludges were about 2 percent solids, and the secondary
sludges, about 0.5 percent solids during these studies. On a percentage
-of-solids basis, the metals in the secondary sludge are concentrated
to a much greater extent than in the primary sludge.
A digester receiving combined sludges will contain more metal on
a percentage-of-solids basis than a digester receiving primary sludge,
when operated at the same influent metal concentration. Digester
failure as a result of heavy metals occurs at a lower influent metal
concentration in a combined sludge digester than in a primary
sludge digester. The maximum continuous influent sewage metal
concentrations for satisfactory anaerobic digestion are given in
Table 13.
55
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TABLE 12. METAL CONTENT OF SLUDGES FED TO
DIGESTERS
Metal
Chromium
(VI)
Copper
Nickel
Zinc
Continuous dose in
influent wastewater
mg/l
50
10
10
10
Excess activated
Primary sludge
mg/l
330
280
62
375
sludge
mg/l
530
160
89
328
TABLE 13. HIGHEST DOSE OF METAL IN CONTINUOUS
DOSAGE THAT Wl LL ALLOW SATISFACTORY
ANAEROBIC DIGESTION OF SLUDGES
Concentrations in
influent wastewater, mg/l
Metal
Chromium (VI)
Copper
Nickel
Zinc
Primary sludge
digestion
>50
10
>40
10
Combined sludge
digestion
>50a
5
>10a
10
a Higher dose not studied
The response of the anaerobic system to metal dosage does not ex-
hibit a plateau region as does the aeration phase; it is an all-or-none
reaction. Digestion either proceeds normally or ceases entirely.
This may be more apparent than real, however, because the analy-
tical measures used to assess digester performance are not as direct
as those for the aeration phase.
The results of these metal studies show that in the cases of
chromium, nickel, and zinc an influent metal concentration of 10
mg/l, either singly or combined, will not affect digestion. Copper
continuously present at 10 mg/l causes failure of combined sludge
digestion.
The prevailing conditions of anaerobic digestion are such that
soluble metal introduced with the feed sludges is efficiently con-
verted to an insoluble form. This is shown in Table 14.
56
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A few slug doses to anaerobic digesters were studied. The slugs to
the digesters were in conjunction with the aeration slugs. The sludges
produced by the activated sludge process during a metal slug were
collected and fed to satisfactorily operating digesters. In no case was
there any interruption of digestion caused by the metal-bearing
sludges. Concentrations of metals in the influent during these slug
studies are given in Table 15.
More detailed studies were not conducted because the logistics of
digester operation make it unlikely that an operating digester would
be upset by the sludges produced during a slug period. This belief is
based on the facts that a digester is not on the main flow stream and
only a small part of the total flow through the plant reaches it, and
the daily additions to a digester are only a fraction of the total
digester volume.
TABLE 14. SOLUBLE METAL CONTENT OF SLUDGES
COMPARED WITH TOTAL METAL CONTENT OF
DIGESTED SLUDGE
Soluble metal
Metal
Chromium (VI)
Copper
Nickel
Zinc
Concentration
in influent
waste water,
mg/l
50
10
10
10
Feed
Primary,
mg/l
38
2
10
0.3
sludges
Excess
activated,
mg/l
32
0.5
9
0.1
Digested
combined,
mg/l
3
0.7
1.6
0.1
Total metal
Digested
combined,
mg/l
420
196
70
341
TABLE 15. DIGESTERS FED COMBINED SLUDGES
PRODUCED DURING METAL SLUG TO
ACTIVATED SLUDGE PLANT
Metal
Chromium (VI)
Copper
Nickel
Concentration of metal in
wastewater feed, mg/l
500
410
200
Effect on
digestion
None
None
None
57
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DISCUSSION
The results of these studies show that for each phase of treatment,
aerobic or anaerobic, there are different bases for judging the con-
centrations of metals that are acceptable in the influent wastewater.
The plateau-type response of the aeration phase shows that concen-
trations of metal many times higher than the threshold concentration
can be received without greatly reducing efficiency. In a situation
in which removal of organic matter is not critical, the most sensitive
performance criterion may be the ability of the digester to handle
the sludge produced. Generally, biological secondary treatment
processes can tolerate up to 5 mg/l of the inorganic toxicants without
noticeable impairment of treatment efficiency. The composition of
municipal wastewater and the chemistry of the inorganic toxics is
such that disproportion of the inorganics occurs during treatment
and the materials are conservative in nature. Thus, if the inorganics
enter the treatment facilities at concentrations of 1 to 5 mg/l in the
raw water and removal occurs in the system, to yield low effluent
residuals, the inorganics will be found in concentrated side streams
such as primary sludge, waste biological sludge, digested sludge,
digester supernatant, or lagoon bottom sediment. Mainly, the
inorganics will exist as insoluble products in these side streams or
sludge deposits. Assessment of technology to enhance inorganic
toxics control must be based on the overall environmental trade-off
of low final effluent residuals versus concentration of the inorganics
in the sludge or sludge handling operations of municipal treatment
systems and their subsequent fate during ultimate disposal practices.
In other cases the amounts of metals passing through the plant to
the receiving stream may be the factor that determines the concen-
tration of metals permissible in the plant influent. Metals are not
destroyed in wastewater treatment processes, so management prac-
tices must be based on their distribution in various process streams.
A survey of four municipal treatment plants (Ref. 18, Chap. 10)
concerning the receipt of heavy metals, distribution of metals in the
process outlets, and effects of metals on treatment efficiency, has
shown a pattern of response similar to pilot plant studies.
58
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Section 4. Bibliography
GENERAL
1. Standard Methods Committee — Subcommittee on Biodegrad-
ability. 1967. Required characteristics and measurement of
biodegradability. Jour. Water Poll. Control Fed. 39(7): 1232-
1235.
2. Bunch, R. L. 1977. Criteria and assessment of waste treat-
ability. Proc. 5th U.S./Japan Conf. on Sewage Treatment
Technology, Tokyo. 17 pp.
3. Ludzack, F. J. 1960. Laboratory model activated sludge unit.
Jour. Water Poll. Control Fed. 32(6): 605-609.
4. Bunch, R. L., and C. W. Chambers. 1967. A biodegradability
test for organic compounds. Jour. Water Poll. Control Fed.
39(2): 181-187.
TREATABILITY OF SPECIFIC ORGANIC COMPOUNDS
5. Bunch, R. L. 1976. Effects and removal of toxic substances
on/by conventional biological treatment systems. EPA Toxic
Substances Seminar, Washington, D.C., October 1976. 10pp.
6. Moore, L., and E. F. Barth. 1976. Degradation of NTA acid
during anaerobic digestion. Jour. Water Poll. Control Fed.
48 (10): 2406-2409.
7. Tabak, H. H., C. W. Chambers, and P. W. Kabler. 1964.
Microbial metabolism of aromatic compounds. I. Decompo-
sition of phenolic compounds and aromatic hydrocarbons
by phenol-adapted bacteria. Jour. Bacteriol. 87(4):910-919.
8. Chambers, C. W., H. H. Tabak, and P. W. Kabler. 1963. De-
gradation of aromatic compounds by phenol-adapted bacteria.
Jour. Water Poll. Control Fed. 35(12): 1517-1528.
9. Tabak, H. H., C. W. Chambers, and P. W. Kabler. 1959.
Bacterial utilization of lignans. I. Metabolism of a-conidendrin.
Jour. Bacteriol. 78(4): 469-476.
10. Bunch, R. L., and M. B. Ettinger. 1968. Biodegradability of
potential organic substitutes for phosphates. Proc. 22nd
Ind. Waste Conf., Purdue Univ., Ext. Ser. No. 129:393.
59
-------
11. Barth, E. F., and M. B. Ettinger. 1967. Anionic detergents
in wastewater received by municipal treatment plants.
Jour. Water Poll. Control Fed. 39(5): 815-822.
12. Tabak, H. H., R. N. Bloomhuff, and R. L. Bunch. 1972.
Coprostanol: a positive tracer of fecal pollution. Develop.
Ind. Microbiol. 13: 296-307.
13. Tabak, H. H., and E. F. Barth. 1978. Biodegradability of
benzidine in aerobic suspended growth reactors. Jour. Water
Poll. Control Fed. 50: 552-558.
14. Tabak, H. H., and R. L. Bunch. 1970. Steroid hormones as
water pollutants. I. Metabolism of natural and synthetic
ovulation-inhibiting hormones by microorganisms of ac-
tivated sludge and primary settled sewage. Develop. Ind.
Microbiol. 11: 367-376.
15. Barth, E. F., et al. 1978. Biodegradation studies of carboxy-
methyl tartronate. Municipal Environmental Res. Lab.,
Cincinnati, Ohio, EPA Rept. No. EPA-600/2-78-115,
16. Bunch, R. L, and C. W, Chambers. 1967. A biodegrad-
ability test for organic compounds. Jour. Water Poll. Control
Fed., 39(2): 181-187.
17. Tabak, H. H., and E. F. Barth. 1979. The microbial degrada-
tion of naphthalene and phthalate ester compounds. In press.
METALS
18. U. S. Department of Health, Education, and Welfare, Public
Health Service, Water Supply and Pollution Control. 1965.
Interaction of heavy metals and biological sewage treatment
processes. Rept. 999-WP-22, Cincinnati, Ohio.
19. Barth, E. F,, M. B. Ettinger, B. V. Salotto, and G. N. Mc-
Dermott. 1965. Summary report on the effects of heavy
metals on the biological treatment processes. Jour. Water
Poll. Control Fed. 37:86.
20. Mulbarger, M. C., and J. A. Castelli. 1966. A versatile ac-
tivated sludge pilot plant—its design, construction and opera-
tion. Proc. 21st Ind. Waste Conf., Purdue Univ., Ext. Ser. No.
121:322.
60
T U.S. GOVERNMENT PRINTING OFFICE: 1979—657-060.
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