WATER POLLUTION CONTROL RESEARCH SERIES
12020 EXG 03/72
THE EFFECT OF
CHLORINATION ON SELECTED
ORGANIC CHEMICALS
U.S. ENVIRONMENTAL PROTECTION AGENCY
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WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes the
results and progress in the control and abatement of pollution
in our Nation's waters. They provide a central source of
information on the research, development, and demonstration
activities in the water research program of the Environmental
Protection Agency, through in-house research and grants and
contracts with Federal, state, and local agencies, research
institutions, and industrial organizations.
Inquiries pertaining to Water Pollution Control Research Reports
should be directed to the Chief, Publications Branch (Water),
Research Information Division, R&M, Environmental Protection
Agency, Washington, D. C. 20460
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12020EXG0372
THE EFFECT OF CHLORINATION ON SELECTED ORGANIC CHEMICALS
by
The Manufacturing Chemists Association
1825 Connecticut Avenue, North West
Washington, B.C. 20009
for the
Office of Research and Monitoring
ENVIRONMENTAL PROTECTION AGENCY
Project #12020EXG
March 1972
LIBRARY
u. s. ENVIRO,W;;TAL PROTECTION AGENCY
E0, N. J, 08§1Z *
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.00
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EPA Review Notice
This report has been reviewed by the Environ-
mental Protection Agency, and approved for
publication. Approval does not signify that
the contents necessarily reflect the views
and policies of the Environmental Protection
Agency, nor does mention of trade names or
commercial products constitute endorsement
or recommendation for use.
11
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ABSTRACT
The results of this study indicated that chlorination of ef-
fluents containing certain organic chemicals can result in
the formation of stable reaction products, which may or may
not contain chlorine. It was further shown that there com-
pounds exercise a retardant influence on aquatic life.
Fourteen industrial organic chemicals were examined for their
persistence through biological treatment as the initial com-
pounds, or as degradation products. Semi-continuous activated
sludge systems were employed. The ability of each of the
chemicals to participate in reactions with free chlorine was
then determined in a series of batch experiments.
Certain of the test compounds formed persistent degradation
products during treatment. Five of the initial compounds re-
acted readily with chlorine, under conditions commonly em-
ployed in effluent chlorination.
Five of the chlorination products were further studied in res-
pirometer experiments to evaluate their persistence upon ,ex-
posure to a heterogeneous microbial population. Their toxi-
city to fish was determined using the static bioassay proce-
dure.
Finally, a series of bench scale, continuous flow ecosystems
was established for the evaluation of longer term effects of
three of the chlorination products. Several varieties of or-
ganisms, representing different levels in the food chain, were
studied.
This report was submitted in fulfillment of project #12020EXG
under the partial sponsorship of the Water Quality Office,
Environmental Protection Agency.
111
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CONTENTS
Section Page
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III INTRODUCTION 5
IV STUDY OBJECTIVES 7
V GENERAL DESCRIPTION OF PHASE I 9
VI EXPERIMENTAL METHODS - PHASE I 17
Analytical Procedures
Biological Degradation Studies 18
Chlorination Experiments 19
VII RESULTS OF PHASE I 21
Analytical Methods
Biological Degradation Studies 22
Acclimated Systems
Unacclimated Systems 31
Discussion of Results
Chlorination Experiments
Discussion of Results - Chlorination Studies 54
VIII RESPIROMETER STUDIES - PHASE II 65
Experimental Procedure
Study Results
IX STATIC BIOASSAYS - PHASE II 73
Series Number One
Series Number Two 74
Series Number Three
Summary 80
X PHASE THREE 81
Selection of Study Compounds
Flow Through bioassay Studies
XI RESULTS OF PHASE III 83
Fish
Vascular Plants 86
Benthic Macroinvertebrates 87
Microscopic Flora and Fauna 89
Summary 99
XII ACKNOWLEDGEMENTS 101
XIII REFERENCES 103
v
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FIGURES
Number Page
, SCHEMATIC REPRESENTATION OF BIOLOGICAL TREATMENT
PROCESS
2 BIOLOGICAL DEGRADATION OF ISOPROPANOL 24
3 BIOLOGICAL DEGRADATION OF ACETONE AND METHANOL 26
4 CHROMATOGRAMS ILLUSTRATING m-CRESOL BIODEGRADATION 27
5 BOD PROGRESSION OF BENZOIC ACID 29
6 BIOLOGICAL TREATMENT OF T-BUTANOL 30
7 VOLATILIZATION OF ETHYLBENZENE DURING CHLORINA- -.
TION EXPERIMENT
8 REACTION OF CHLORINE WITH PHENOL 35
9 MOLAR CHLORINE UPTAKE BY TEST COMPOUNDS 37
,n CHROMATOGRAMS OF PHENOL AND CHLORINATED PHENOL _.fi
SOLUTIONS
11 REACTION OF CHLORINE WITH m-CRESOL 40
19 CHROMATOGRAMS OF m-CRESOL AND CHLORINATED .,
m-CRESOL SOLUTIONS
13 REACTION OF CHLORINE WITH HYDROQUINONE 43
14 ULTRAVIOLET ABSORPTION SPECTRA-REACTION OF .,.
CHLORINE WITH HYDROQUINONE
15 REACTION OF CHLORINE WITH ANILINE 47
ULTRAVIOLET ABSORPTION SPECTRA OF CHLORINE
16 CONTACTED ANILINE SOLUTIONS: EFFECT OF 49
CHLORINE DOSAGE
ULTRAVIOLET ABSORPTION SPECTRA OF CHLORINE
17 CONTACTED ANILINE SOLUTIONS: EFFECT OF 50
CONTACT TIME
18 REACTION OF CHLORINE WITH DIMETHYLAMINE 53
, q EFFECT OF 2,4,6-TRICHLOROPHENOL ON A MIXED ,fi
MICROBIAL POPULATION
2n EFFECT OF 2,4,6-TRICHLOROPHENOL ON A MIXED fi7
MICROBIAL POPULATION
VI
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FIGURES
(continued)
Number Page
9, EFFECT OF 4-CHLORO-3-METHYLPHENOL ON A MIXED ,„
MICROBIAL POPULATION
22 EFFECT OF 4-CHLORO-3-METHYLPHENOL ON A MIXED fiq
MICROBIAL POPULATION
2_. EFFECT OF CHLORANIL ON A MIXED MICROBIAL __
POPULATION
-. EFFECT OF 2,4,6-TRICHLORANILINE ON A MIXED _,
MICROBIAL POPULATION L
25 RESULTS OF STATIC BIOASSAY SERIES NUMBER ONE 75
26 RESULTS OF STATIC BIOASSAY SERIES NUMBER ONE 76
27 RESULTS OF STATIC BIOASSAY SERIES NUMBER ONE 77
9fi MEDIAN TOLERANCE LIMIT DETERMINATIONS FOR _R
2,4,6-TRICHLOROPHENOL AND 4-CHLORO-3-METHYLPHENOL
29 RESULTS OF BIOASSAY NUMBER 3 - P-BENZOQUINONE 79
30 FATHEAD MINNOW POPULATIONS versus TIME 84
-., BACKGROUND MACRO IN VERTEBRATE POPULATIONS versus 00
31 TIME 88
_9 MAKE-UP OF AVERAGE FOUR WEEK MICROFAUNA
POPULATIONS
33 MAKE-UP OF AVERAGE TWO-WEEK MICROFAUNA POPULATIONS 91
34 TOTAL MICROFAUNA POPULATIONS 92
35 STALKED CILIATE POPULATIONS 93
36 MOTILE CILIATE POPULATIONS 94
37 DIATOM POPULATIONS 95
38 BLUE-GREEN ALGAE POPULATIONS 96
Vll
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TABLES
Number Page
1 ORGANIZATION OF THE PROJECT 8
,, CHEMICAL AND PHYSICAL CHARACTERISTICS OF TEST 10
COMPOUNDS 11
3 CHROMATOGRAPHIC ANALYSIS OF TEST COMPOUNDS ~,
RETENTION DATA
, ULTRAVIOLET ABSORPTION CHARACTERISTICS OF TEST ~9
COMPOUNDS
5 SUMMARY OF BIOLOGICAL DEGRADATION STUDIES 23
, SUMMARY OF COD DATA FROM BIOLOGICAL DEGRADATION
STUDIES
7 RESULTS OF PRELIMINARY CHLORINATION EXPERIMENTS 32
8 CHLORINATION OF PHENOL 36
9 CHLORINATION OF m-CRESOL 39
10 CHLORINATION OF HYDROQUINONE 42
11 CHLORINATION OF ANILINE 46
12 CHLORINATION OF DIMETHYLAMINE 52
13 PROBABLE PRODUCTS OF CHLORINATION 62
14 RESULTS OF STATIC BIOASSAYS 80
15 TABULAR SUMMARY OF FATHEAD MINNOW MORTALITIES 83
, CONCENTRATIONS (mg/1) OF SUBSTRATES DURING VARIOUS fic
TEST PERIODS
17 VASCULAR PLANTS GROWTH 86
Vlll
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SECTION I
CONCLUSIONS
Phase I; Chlorination Studies and Analytical Methods
1. Gas chromatographic analysis was successfully employed for
the detection of most of the initial test compounds in
aqueous solutions. The method was applied to the analysis
of effluents from bench scale biological systems, and was
used in monitoring the test chemicals during chlorination
experiments.
2. Ultraviolet absorption (UV) spectrophotometry demonstrated
applicability to the measurement of aromatic compounds in
relatively pure aqueous solutions. The method was used in
compound monitoring during the bioassay experiments, and
provided information on the nature of the products result-
ing from chlorination.
3. There is evidence to indicate the formation of degradation
products of several of the test compounds, during biologi-
cal treatment, in acclimated systems.
4. Five of the fourteen chemicals selected for study were ob-
served to participate in reactions with free chlorine,
under conditions encountered during conventional effluent
chlorination practice. The ability of these compounds to
react with chlorine can be related to the structural char-
acteristics of the chemicals.
5. Evidence indicates that chlorine reacts with these organic
chemicals by both substitution and oxidation, resulting in
a highly complex mixture of products. In some cases, it
was possible to identify the products of reaction.
6. The nature and distribution of the products of reaction
with chlorine are affected by a variety of parameters,
including concentrations and contact time.
Phase II: Chlorination Product Persistence and Toxicity
7. Several of the reaction products identified in Phase I
were examined in respirometer studies and found to be
resistant to degradation upon exposure to a heterogeneous
microbial population.
8. Toxicity to fish by these products was demonstrated in
static bioassay experiments, and 96-hour TL values were
established.
9. On the basis of the results developed in Phases I and II,
it is evident that chlorination of effluents containing
certain organic chemicals may result in the formation of
persistent and potentially deleterious reaction products.
- 1 -
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Phase III; Intermediate Bioassay Experiments
10. Considering the relative efforts involved, the intermed-
iate term, flow through bioassays as conducted did not
provide significantly better insight into the toxic ef-
fects of the test compounds, than did the routine four-
day static bioassays. The static test appears to pro-
vide a conservative toxicity limit with minimum effort.
11. Although some qualitative judgments of compound effects
on the test microorganism populations can be drawn, the
data were too variable to allow quantitative statistical
determinations of toxicity. Qualitative evidence was
suggestive of toxic or inhibitory effects on stalked
ciliates and diatoms for each of the three compounds at
the highest concentration levels tested.
12. A significant increase in effort, well above that practi-
cal for a project of this nature, would be needed to pro-
vide statistically definable determinations of toxicity
to microorganisms.
13. Of the two types of vascular plants tested, one exhibited
an erratic growth pattern and was unsuitable as an indi-
cator of toxicity. The second type was not erratic in
growth, but no toxicity was observed. The general out-
ward appearance of all plants was not adversely affected
by any of the compounds.
14. Stable macroinvertebrate populations could not be main-
tained in the model ecosystems for a time period of
suitable duration for compound testing.
15. On the level of experimentation performed, the energy and
food web interrelationships within the experimental eco-
systems could not be defined.
- 2 -
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SECTION II
RECOMMENDATIONS
1. The practice of routine chlorination of industrial efflu-
ents should be re-examined. In cases where it has been
demonstrated or suspected that organic chemicals are re-
acting with chlorine to produce undesirable products, an
alternative disinfection method should be considered.
2. Effluents known to contain chlorine reactive materials
should be monitored for those materials, and for unusual
increases in chlorine demand.
3. The present study should be extended to provide further
information regarding the nature and properties of the
products of reaction between chlorine and industrial
chemicals, which have escaped biological treatment.
4. Further studies should be undertaken to determine the
effects of these products of chlorination on biological
systems. These studies should include evaluations of
fish toxicities.
5. The intermediate term, flow through bioassay such as em-
ployed herein, is not recommended for future investiga-
tions of compound toxicity. By comparison, the routine
four-day static bioassay provides a simple method which
yields a conservative determination of toxicity to a test
organism, at considerable saving of effort.
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SECTION III
INTRODUCTION
The chlorination of raw or treated domestic wastewaters is
commonly practiced to achieve disinfection and deodorization.
In many municipalities, effluent chlorination is mandated by
public health codes. In recent years, this country has wit-
nessed a trend towards combined municipal—industrial waste
treatment. Additionally, an increasina number of industries
have been required to provide wastewater treatment facilities,
which often includes effluent chlorination. These developments
have raised serious questions regardina the impact of certain
industrial chemicals on wastewater treatment plant operation
in general, and on chlorination in particular. It is conceiv-
able that, under certain conditions, some organic compounds
may escape treatment or be only partially dearaded, such that
they are available for reaction with chlorine in the contact
chamber. Furthermore, the products of such a reaction could,
upon discharge, exercise a deleterious effect on the receiving
stream.
A typical example of such an undesirable reaction is the com-
bination of chlorine with phenol to produce the chlorophenols.
These materials are a source of obnoxious tastes and odors,
even at very low concentrations (yg/1).
Moreover, studies by Ingols and Jacobs have shown that the
chlorophenols are more resistant to biodearation than phenol
itself. Trichlorophenol was cited as beincr toxic to phenol—
(°)
adapted microorganisms. Chambers, et.al. also reported on
the increased resistance of the chlorinated products of both
phenol and m-cresol to biodegradation. In an extension of an
earlier work, Ingols, et.al. determined that the chlorophe-
nols were more toxic to fish than phenol.
The preceeding considerations clearlv indicate the need for a
re-examination of effluent chlorination practice. In addition,
research efforts should be directed towards identifying- those
chemicals which are present in wastewater effluents and which
will react with chlorine. Information should also be developed
on the impact of these materials on the ecoloov of a receivina
water.
In recognition of these problems, the Manufacturina Chemists'
Association, in cooperation with the Environmental Protection
Agency, initiated the present study for the purpose of gather-
ing information on the effect of chlorination on certain in-
dustrial organic compounds.
- 5 -
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SECTION IV
STUDY OBJECTIVES
The present study was undertaken in an attempt to develop in-
formation on the effects of chlorination on selected organic
chemicals. The need to develop testing procedures and. analy-
tical methods for the characterization of these effects was
judged to be of paramount importance. Specific project ob-
jectives included:
1. The examination of the influence of
selected organic compounds and/or their
degradation products on chlorine demand.
2. The identification of those chemicals
which form stable reaction products upon
contact with chlorine, under conventional
conditions of chlorination.
3. The characterization of those reaction
products in terms of their persistence
in biological systems, or their potential
to act as inhibitors or toxicants to such
svsterns .
4. The determination of the effects of
any persistent reaction products on the
ecolooy of a simulated receivina stream.
From an organizational standpoint, the study was arranged into
three distinct phases, as shown in Table 1. Each successive
phase was intended to represent a logical investiaative pro-
gression in the accomplishment of the objectives of the project
It was envisioned that some effort in each of the phases would.
proceed concurrently, since the nature of the studies necessi-
tated the refinement or development of experimental and analy-
tical methodoloaies. Consequently, subsequent descriptions of
the studies are not intended to be chronolocricallv consistent.
- 7 -
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SECTION V
GENERAL DESCRIPTION OF PH.ASF, I
Phase I was devoted to the examination of the influence of
chlorination on specific industrial chemicals, before and af-
ter biological treatment. Fourteen compounds, representing
several classes of organic chemicals, were chosen for these
initial investigations, as indicated in Table 1.
The selection of these compounds was made on the basis of
their industrial importance either as primary products or as
chemical intermediates. Each of the chemicals listed is known
to appear in industrial wastewater effluents.
Preliminary efforts during the first phase of the studv inclu-
ded the definition of chemical and phvsical characteristics of
each of the test compounds. A listing of these properties is
given in Table 2. This list was compiled in part from data
(4)
given in standard texts . Information on the response of
each of the compounds to BOD and COD analysis was established
experimentally.
The introduction of organic chemicals to a biological treat-
ment system, e.g., the activated sludge process, mav result in
either of two effects: (1) complete conversion of the starting
compounds to carbon dioxide, water, new microbial cells, and
possibly, some intermediate compounds; or (2) incomplete de-
gradation or no degradation, such that the starting compounds
appear in the effluent. In continuous svstems, some residual
organic material will always remain in solution as a result of
an equilibrium between the microbial cells and their liquor.
The compositions of treated effluents are hiahlv complex, and
have been characterized only in terms of general classes of
materials, e.g., carbohydrates, proteins, tannins, "fulvic"
and "humic" acids. The nature and distribution of specific
components is known to be affected by selection of treatment
parameters, as well as by the composition of the feed stock.
A well functioning continuous biological svstem, receiving a
feed stock of uniform composition, will achieve essentially
complete conversion of the organic material initially present.
Such a system is said to be acclimated, in contrast to a sys-
tem which is unacclimated. A biological process which is sub-
jected to intermittent or shock loads may be unacclimated to
certain components associated with these loads.
At this juncture, it is useful to review the conditions under
which a biological system achieves acclimatization. This may
be visualized by a consideration of the generalized biological
treatment process, as illustrated in Figure 1. It is noted
that the growth of a microbial population (sludge) progresses
through several phases upon exposure to a substrate: first, a
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lag phase, characterized by adaptation of the culture from
the previous environment to the present; second, a period of
maximum growth under conditions where unlimited food is
available; third, a period of declining growth where food
availability becomes a limiting condition; and finally, an
endogenous phase where, under severely limited food condi-
tions, cells die and are, in turn, consumed such that the
mass population is reduced. In heterogeneous systems, this
process occurs within the biological culture for each sub-
trate as a subset of the overall reaction. In a system
which is unacclimated to a particular compound, the lag
phase associated with that substrate may be prolonged, and
may result in the passage of that compound through the treat-
ment process, without any significant degradation.
In the evaluation of the effects of chlorine on organic chem-
icals subjected to biological treatment, two cases should be
considered: (1) an investigation of the possible reactions
of chlorine with the parent compounds which would persist
through unacclimated systems; and (2) the determination of
the probability of chlorine reactions with degradation pro-
ducts of the starting compounds, which would appear in the
effluents from acclimated biological systems.
The case of chlorine reaction with possible degradation pro-
ducts was examined first. Requisite to the evaluation of
potential effects of chlorination is the establishment of
the existence of significant degradation products which uni-
quely result from biological treatment of any of the test
compounds. In certain cases, the presence or absence of de-
gradation products can be inferred from a consideration of the
nature of the reactants and their known degradation character-
istics. A simple substrate, such as methanol, should undergo
virtually direct conversion to carbon dioxide, water, and
cellular material. For more complex organic molecules, degra-
dation may be accomplished by a series of reactions, involving
intermediate forms.
A series of experimental tests can be applied for the deter-
mination of intermediate product production. Ideally, the
detection of the intermediate product itself would be the
most direct approach. However, no single analytical techni-
que is universally applicable, and the selection of a method
must necessarily be based on some information about the ma-
terial being detected. In the absence of direct experimental
evidence, indirect procedures are often employed in the deter-
mination of the presence of degradation products. Monitoring
of the effluent for the disappearance of the parent compound
(by chromatographic or spectrophotometric procedures, for ex-
ample) and for a decrease in oxygen demand may provide some
useful information. If the rate of compound disappearance
closely parallels the rate of COD removal associated with
that compound, it may be generally asserted that no signifi-
cant degradation product buildup has occurred. On the other
- 13 -
-------
hand, if a net residual COD is observed, relative to a control
system, intermediate product formation mav be taking place.
The'lack of adherence to stoichiometry for oxygen utilization
may provide additional evidence for the production of inter-
mediates. If the complete disappearance of a parent compound
is accompanied by substantially less than theoretical oxvqen
consumption required to produce CO..,, water, and cells, there
is likelihood for the existence of degradation products.
Experience has shown that no single techniqxae, among those
described above, is conclusive in demonstrating the presence
or absence of significant intermediate products which are un-
iquely associated with the biological degradation of a speci-
fic compound. Indeed, even the application of all these pro-
cedures only leads to a "probable" result, due to the inherent
inadequacies of these experimental and analytical tests. The
following is a partial list of some of the conditions which
would tend to diminish the significance of the experimental
results:
1. partial or complete volatilization of
test compound or intermediate product
during biological treatment;
2. variable recovery (sensitivity) of the
COD test to parent compound or product;
3. direct chemical degradation (auto-oxida-
tion, photochemical reaction, etc.) of
compound (s) .
The nature of the biological treatment process itself is not
conducive to the maintenance of true steadv-state conditions.
Transient changes in microbial population dvnamics mav yield
erratic results. Background levels of residual byproduct ma-
terial from cellular lysis (endogenous respiration) may obscure
the presence of specific intermediate compounds.
Perhaps even more pertinent to the evaluation of the probabil-
ity of chlorine reactions with degradation products is the
fact that the nature and distribution of degradation products
may be controlled by the selection of treatment parameters.
As an example, ammonia (NEU) may be regarded as a byproduct of
organic nitrogen degradation. However, by appropriate adjust-
ment of loading conditions (food—to—microorganism ratio) , the
conversion of ammonia to oxidized nitrogen forms (NO- — NO_)
may be encouraged. Furthermore, it is known that ammonia will
participate in reactions with chlorine, leading to the forma-
tion of chloramines, but nitrate undergoes no reaction with
chlorine.
In summary, it may be concluded that:
- 14 -
-------
1. present methods for establishing the
existence of intermediate products
resulting from the biochemical de-
gradation of specific organic chemi-
cals lack general applicability and
are of questionable validity;
2. the nature and distribution of degra-
dation products may be drastically
altered by adjustment of treatment
parameters;
3. due to recognized experimental limi-
tations, the more practical case for
investigation is the ability of chlo-
rine to react with specific organic
chemicals, which have escaped biolo-
gical treatment.
In accordance with the considerations detailed above, the ma-
jor effort of Phase I was devoted to the examination of the
ability of chlorine to react with each of the initial test
compounds. Test conditions were selected to simulate those
that would be anticipated for full scale treatment plants:
dilute solutions of organic compounds, pH ranges near neutral-
ity, conventional applied chlorine dosages and contact times.
It should also be pointed out that all applied chlorine was
in the form of free chlorine, since ammonia—free solutions
were used. The principal criterion applied in the determina-
tion of chlorine—organic chemical reactions was that of the
chlorine demand, as derived from residual chlorine analysis.
Chromatographic techniques provided the means for monitoring
the concentrations of test compounds during the chlorination
experiments and, in some instances, facilitated the identifi-
cation of reaction products. Supplemental information was also
gained by the use of ultraviolet absorption spectrophotometry.
- 15 -
-------
SECTION VI
EXPERIMENTAL METHODS - PHASE I
Analytical Procedures
In view of the need for monitoring of the test compounds
throughout the experimental program, a substantial portion of
the effort in Phase I was devoted to the development of ana-
lytical techniques. It was recognized that any method worthy,
of consideration should have general applicability to the
range of selected organic chemicals and their derivatives, be
capable of selective identification of specific components in
complex systems, and be suitable for quantitative analysis at
low (mg/1) concentrations.
Liquid-gas chromatography was initially chosen for these in-
vestigations. The instrument used was a Perkin-Elmer Model
881 chromatograph with flame ionization detector and linear
temperature programing. Helium was employed as the carrier
gas, while hydrogen and air were supplied to the flame ioniza-
tion detector. Chromatograms were recorded on a Leeds and
Northrup Speedomax W recorder equipped with a Disc integrator.
Five columns were employed for chromatographic separations
during these studies, as listed below:
1. 61 x 1/8" (uncoated) Poropak Q,
100/120 mesh, packed in stainless
steel
2. 6' x 1/8" (uncoated) Poropak S,
150/200 mesh, packed in stainless
steel
3. 6' x 1/8" 15% K20M Carbowax TPA
on Chromosorb W HDMS, 80/100 mesh,
packed in stainless steel
4. 12' x 1/8" 15% K20M Carbowax TPA
on Chromosorb W HDMS, 60/80 mesh,
packed in glass
5. 6' x 1/8" Chromosorb 103, 80/100
mesh, packed in glass
In all cases, dual column systems were utilized to facilitate
baseline stability by compensating for column "bleeding" ef-
fects.
For purposes of parameter optimization and calibration, stan-
dard solutions of each of the test compounds were prepared in
distilled water. Where available, chromatographic or reagent
17 -
-------
grade chemicals were used. Injection of samples was accom-
plished with a Hamilton microliter syringe of suitable size
(1, 5, or 10 yl). Calibration curves were constructed by
plotting the product of the attenuation factor and area (rel-
ative Disc integrator units) as a function of sample weight.
Standardization was checked frequently durinq the experimental
program to insure quantitative reproducibility.
It became evident that chromatographic techniques, employing
direct aqueous injections of samples, were not applicable to
the separation and quantitative detection of some of the com-
pounds under investigation. It therefore was decided to ex-
plore the use of UV spectrophotometrv as an analvtical tool.
The instrument used for these studies was a Perkin-Elmer Model
202 double-beam ratio-recording spectrophotometer. Standard
solutions of each of the compounds of interest were prepared
in distilled water. Absorbance-wavelenqth recordinas were
obtained in the 190-350 nm region of the spectrum, using dis-
tilled water as the reference. Calibration curves were pre-
pared by plotting absorbance as a function of concentration
at each peak wavelength. Ultraviolet spectral scans for sam-
ples dissolved in a solvent other than distilled water em-
ployed the appropriate solvent in the reference cell.
Biological Degradation Studies
A series of semi-continuous activated sludae systems, each
receiving one of the selected compounds, in combination with
the necessary nutrients, was employed for these studies.
Each system consisted of a two-liter aeration chamber contain-
ing activated sludge which had been initially obtained from a
local domestic waste treatment plant. Sufficient dissolved
oxygen levels and mixing were provided by diffused air fed
into the bottom of each unit. Feedina was accomplished on a
daily schedule, according to the followinq procedure: (1) the
air supply was temporarily cut off and the biolocrical solids
allowed to settle; (2) a one-liter portion of supernatent was
removed by siphoning and set aside for analysis; (3) the ap-
propriate quantity of feed solution was dispensed into each
unit, and the final volume was re-adjusted to two liters with
tap water. In all cases, the feed consisted of 100 mg/1 of
each test compound, expressed as Theoretical Oxygen Demand
(TOD) , in combination with 100 mg/1 (as COD) of a svnthetic
"sewage" , containing a mixture of readilv decrradable organic
compounds and inorganic nutrients. A control unit, which re-
ceived 200 mg/1 (as COD) of the synthetic mixture was also
maintained during these studies. This synthetic media was
prepared at frequent intervals in accordance with the follow-
ing formula:
- 18 -
-------
SYNTHETIC DOMESTIC WASTE COMPOSITION FOR A COD OF 100,000 MG/L
__^ _ Ingredient _ grams/ liter
skim milk 48
peptone 48
gelatin 16
soluble starch 32
urea 8
disodium hydrogen phosphate 8
KC1 1.12
CaCl 1.12
0.80
)3 0.80
NH4C1 8
Following a two-week period of acclimatization, each of the
systems was monitored for reduction in soluble COD and for
disappearance of test substrate, where chromatographic techni-
ques were available. Analyses were performed immediately after
addition of test substrate and synthetic mixture, and at se-
lected intervals thereafter.
In an aerated biological reactor, substrate removal may also
occur by diffused air stripping of volatile components. To
investigate the significance of this phenomenon for each of
the test chemicals, a separate series of tests was conducted
using the same apparatus as previously described. In these
experiments, the biological culture was omitted, and an aque-
ous solution of each test chemical was aerated, using an air
flow rate similar to that employed in the degradation studies.
The concentration of each test compounds was monitored with
time, using chromatographic or spectrophotometric procedures.
Chlorination Experiments
To determine the effect of chlorine on each of the selected
compounds, two series of batch chlorination experiments were
conducted. The first set of tests were designed to identify,
in a qualitative sense, which of the selected chemicals were
capable of reacting with chlorine, under conventional treat-
ment conditions.
Aqueous solutions, containing approximately 10 mg/1 of each
compound, were prepared using distilled water and adjusted to
pH 7.4, with a phosphate (K2HP04— KH2P04 ) buffer. Where poss-
ible, the concentration of test chemical was checked using
chromatographic procedures. A stock chlorine solution, hav-
ing an approximate concentration of 1,000 mg C12/1, was pre-
pared from a commercial grade of sodium hypochlorite. This
solution was standardized frequently by iodimetric titration ,,-,
in accordance with the procedures described in Standard Methods
- 19 -
-------
Each test run consisted of four (4) 500 ml samples containina
the test compound, to which varying dosaaes of chlorine were
applied. Since a gradual loss of chlorine with time had been
demonstrated in preliminary tests, a control solution contain-
ing only 10 mg/1 of chlorine in phosphate-buffered distilled
water was included in each experiment. The four solutions of
test compound received nominal chlorine dosaqes of 0, 5, 10,
and 20 mg/1. Mixing was accomplished on a multiple stirring
("jar test") apparatus, using 1" x 3" stainless steel paddles
rotating at 80 rpm. Solution temperatures were maintained at
25°C ± 1°C.
Samples were withdrawn at 0.5, 1.0, 2.0, and 24 hours after
chlorine addition and immediately analyzed for free and com-
bined residual chlorine. The orthotolidine (OT) and ortho- ,_>
tolidine-arsenite procedures, as described in Standard Methods,
were followed in these determinations. Where chromatographic
calibrations were available, the samples were also analyzed
for the presence of the test compound. A sufficient quantity
of sodium thiosulfate was added to each sample prior to
chromatographic analysis to destroy the free chlorine resi-
dual. This step was taken to preclude the possibilitv of anv
extraneous compound—chlorine reactions during exposure to the
elevated temperature conditions of the chromatographic column.
Those compounds which had been observed to react with chlor-
ine in the preliminary chlorination experiments were subjected
to further examination in a series of detailed tests. The
experimental procedures were essentiallv the same as described
above. A wider range of applied chlorine dosaaes (up to 100
mg/1) was employed in these studies in an attempt to further
elucidate the stoichiometry of these reactions. Ultraviolet
absorption spectrophotometric procedures were used to provide
supplemental information on parent compound disappearance and
reaction product formation.
- 20 -
-------
SECTION VII
RESULTS OF PHASE I
Analytical Methods
On the basis of previous experience, chromatographic techni-
ques appeared to offer promise for the separation and quanti-
tative detection of the compounds selected for study. It was
envisioned that these procedures could also provide the means
for the identification of products of biological degradation
and/or chlorination.
Of the fourteen chemicals initially selected, reliable chro-
matographic techniques were developed for seven. These com-
pounds were amenable to chromatographic analysis at low (mg/1)
concentrations using the Poropak Q column. Peaks were well
separated from the water response and observed to be symmetri-
cal, with little or no tailing. Reasonable response times
were obtained for these materials, as summarized in Table 3.
Note that the relative retention times of these species are
sufficiently different, so as to allow the resolution of each
of the components in a complex mixture.
TABLE 3
CHROMATOGRAPHIC ANALYSIS OF TEST COMPOUNDS
RETENTION DATA
Column: 6' x 1/8" Poropak Q 100/120 Mesh (SS)
Compound
Methanol
Isopropanol
T-Butanol
Acetone
Benzene
Toluene
Ethylbenzene
Carrier Flow
frrt'] /rn n T~\ \
35
35
35
35
35
35
20
Column
Temperature
/ O /"i \
V '»-' /
120
150
150
150
160
190
225
Retention
Time
(TYI "i Ti ^
2.5
5.5
9.5
4.0
11.25
10.0
7.0
As a result of the lack of suitable gas chromatographic me-
thods for the analysis of certain of the test compounds, ul-
traviolet absorption spectrophotometric procedures were also
evaluated during the Phase I studies. The results of prelim-
inary tests confirmed the suitability of UV analysis for the
determinations of several of the test compounds, including
- 21 -
-------
phenol, m-cresol, nitrobenzene, aniline, and hydroguinone.
Dimethylamine was found to be inactive in the UV region.
The absorption characteristics of the compounds tested are
summarized in Table 4. Subsequent experiments also revealed
the utility of UV analysis for chlorinated product identifi-
cation and monitoring. These studies will be detailed in a
later section of the report.
TABLE 4
ULTRAVIOLET ABSORPTION CHARACTERISTICS OF TEST COMPOUNDS
Compound \ (nm) \ (nm) A (nm)
1 2 3
Phenol 207 235 288
m-Cresol 222 271 277
Hydroquinone 193 220 288
Aniline 199 230 280
Nitrobenzene 195 214 270
4-chloro-3-methylphenol* %200 279 287
2,4,6-trichlorophenol* ^214 245 312
2,4,6-trichloroaniline*
-------
Except in the case where the test substrate or its known de-
gradation products are directly measureable, a significant
determination problem results. This problem can, in most
cases, be surmounted by considering supplementary data on
COD removal, and degradation studies on the particular com-
pound. Employing a combination of the analytical information,
it is possible to examine each of the test compounds. Table
5 summarizes the study results.
TABLE 5
SUMMARY OF BIOLOGICAL DEGRADATION STUDIES
Results
Compound
, Complete loss of prTmary substrate(gas chroma-
" ^ tography). Identifiable intermediate acetone.
,, , Complete loss of primary substrate (gas chroma-
tography). No identifiable degradation product.
m-Cresol Biological oxidation of substrate.
Phenol Biological oxidation of substrate.
Complete loss of primary substrate (gas chroma-
tography). No identifiable degradation product.
Benzene Removal by stripping and biooxidation.
Toluene Removal by stripping and biooxidation.
Ethylbenzene Removal by stripping and biooxidation.
Presumptive evidence of complete loss of pri-
Benzoic Acid mary substrate with no degradation product for-
mation — biological oxidation.
TT -, . Possible substrate persistence or degradation
Hydroquinone , . ,. , . ^ ^
J ^ product formation.
Complete loss of primary substrate (gas chroma-
Nitrobenzene tograph). Possible formation of unidentified
degradation product.
Presumptive evidence for some primary substrate
Dimethylamine survival. Ammonia is a product of biological
oxidation.
, Partial survival of primary substrate, degrada-
tion product formation unknown.
.,. Possible survival of primary substrate suspec-
ted. Degradation product formation suspected.
Figure 2 presents batch study data illustrating the disappear-
ance of isopropanol with a corresponding increase in acetone,
- 23 -
-------
ISOPROPANOL AIR STRIPPING DATA-7 x
ACETONE INGROWTH
ISOPROPANOL DEGRADATION
234
TIME AFTER FEEDING-HOURS
FIGURE 2
BIOLOGICAL DEGRADATION OF ISOPROPANOL
-------
as determined by chromatographic analysis. A complete stoich-
iometric evaluation of the completeness of reaction was diffi-
cult because of acetone loss by stripping. No evidence of any
other product formation was uncovered. Acetone itself was
eventually lost from the system at longer aeration times (see
below).
Methanol disappearance, as measured by gas chromatography, and
COD disappearance data indicated that methanol is completely
degraded without the production of an intermediate product.
Comparison studies indicated that stripping is not a major
factor in removal, as shown in Figure 3.
Both phenol and m-cresol could be tracked by gas chromato-
graphy and COD analysis. A typical gas chromatographic output
for m-cresol is presented in Figure 4. In another experiment,
the phenol concentration was reduced from 33.7 mg/1 to 4.7
mg/1 after one hour. An analysis of residual COD data, as
shown in Table 5, indicated that no intermediate products were
present in detectable quantity.
Acetone was monitored by gas chromatography and showed com-
plete disappearance from the test units. COD data (refer to
Table 6) indicated no accumulation of an additional product.
Examination of the removal pathway indicated that a signifi-
cant portion of the acetone was removed by stripping, as evi-
dent from Figure 4. Approximately 50% of the removal was ac-
complished by biological oxidation.
TABLE 6
SUMMARY OF COD DATA FROM BIOLOGICAL DEGRADATION STUDIES
Compound The'
Acetone
Isopropanol
m-Cresol
Methanol
Phenol
Benzene
Ethylbenzene
Toluene
Benzoic Acid
Hydroquinone
Nitrobenzene
Dime thy lamine
T-Butanol
Aniline
Dretical Total
:OD Addedt
200
200
200
200
200
200
200
200
200
200
200
200
200
200
CONTROL 200
tlncludes 100 mg/1 synthetic sewage
"Calculated from Table 2.
Initial
:OD Recoverable
By Test*
151
175
150
193
195
104
101
104
200
197
106
100
164
152
200
(as COD) .
Residual
COD
(mg/1)
at 24-hr
Average
4T76
50.1
44.7
49.3
41.8
48.5
56.2
45.3
38.4
39.0
52.4
55.9
69.9
68.5
56.1
- 25 -
-------
TIME-HOURS
o
z:
<
STRIPPING ONLY
METHANOL
O-STRIPPING STUDY
-(--BIOLOGICAL
TREATMENT
AREA
TIME -HOURS
FIGURE 3
BIOLOGICAL DEGRADATION OF ACETONE AND METHANOL
-------
LU
CC
D
CD
CC
CD
UJ
Q
O
QQ
O
CO
LJ
cr
O
I
CD
o:
h-
co
CO
cr
CD
O
O
cr
x
o
-------
The removals of benzene, toluene, and ethylbenzene were moni-
tored by gas chromatography. Studies indicated that they
were, to a very significant degree, removed from the system
by stripping. If any biological degradation products were
formed by a small portion of these materials that were ac-
tually oxidized, they were below detection limits.
A primary substrate method for evaluating benzoic acid pre-
sence was not available. COD data (refer to Table 6) indi-
cated complete oxidation of the substrate with no apparent
degradation products. For supporting evidence, a sample was
studied for the regularity of oxygen use with both acclimated
and unacclimated seed. The results of this study are shown
in Figure 5. The regularity of both curves supports the con-
tention that the reaction is complete, with no significant
intermediates being formed. Although the evidence is not
totally conclusive, there is sufficient presumptive evidence
to infer the improbability of degradation product formation.
The lack of a totally satisfactory method of analysis made
determination of hydroquinone in low concentrations imprac-
tical. The substrate was substantially degraded, as shown
by the residual COD data in Table 6. However, indirect
oxygen utilization data suggested imcomplete oxidation, or
possible degradation product formation. Possible products
were not identified experimentally.
Gas chromatographic studies indicated that nitrobenzene dis-
appeared from solution during the study period. Analysis by
ultraviolet absorption indicated that removal by air strip-
ping was insignificant. COD and oxygen use data suggested
the formation of a degradation product of the reaction. The
product could not be identified experimentally in this study.
The product concentration was in the order of 10%-15% of the
original nitrobenzene as COD.
Although no satisfactory method of analysis was available
for low concentrations of dimethylamine, there is presump-
tive evidence that some primary substrate persists for a
long period during biological treatment. Ammonia was found
as a degradation product of oxidation.
T-butanol degrades very slowly and is detectable even after
exhaustive periods of biological oxidation. No degradation
product was identified, but considering the amount of the
t-butanol present, it cannot be positively concluded that a
product does not exist. Typical degradation data are shown
in Figure 6.
No suitable analytical method was available for low concentra-
tions of aniline. Based on COD data, (Table 6) it may be con-
cluded that either aniline or a degradation product remains,
even after substantial time. The analytical evidence is in-
sufficient to draw a valid conclusion. However, indirect oxy-
gen utilization studies suggested the persistence of aniline
through biological systems, particularly in the presence of
mixed substrates.
- 28 -
-------
500
THEORETICAL =430 MG/L
8
10 12 K
TIME-DAYS
16 18 20 22 24
FIGURE 5
BOD PROGRESSION OF BENZOIC ACID
29
-------
60
40
m 20
O-RUN I
•-RUN 2
0
24
TIME-HOURS
VOLATILIZATION OF T-BUTANOL
• -RUN
y-RUN 2
+-RUN 3
O-RUN 4
0
TIME-HOURS
BIOLOGICAL TREATMENT OF T-BUTANOL
FIGURE 6
30
-------
Unacclimated Systems
In an unacclimated system, the discharge may contain any of
the products found in the acclimated system, as well as the
original test substrate. The concentration and distribution
of products will depend on the actual conditions of discharge
and treatment.
Discussion of Results
The results of the biological degradation studies indicated
possible degradation product formation in acclimated systems,
for the following test substrates:
aniline
hydroquinone
nitrobenzene
t-butanol
isopropanol
dimethylamine
Ammonia was determined to be the product of dimethylamine de-
gradation, and acetone was identified as an intermediate in
isopropanol breakdown. Due to the limitations of the available
analytical methods, degradation products of the remaining ma-
terials could not be confirmed or identified. As a consequence,
it was decided to concentrate efforts on the determination of
problems associated wih chlorination of the original test
chemicals. This approach has direct application to "spill"
situations, in which a substrate passes through an unacclimated
biological system, without degradation. Such occurrences are
commonplace in industrial wastewater treatment practice.
Chlorination Experiments
The first series of chlorination experiments was designed to
determine which of the selected chemicals were capable of re-
acting with free chlorine under conditions commonly encountered
in conventional treatment plants. A portion of these prelimin-
ary investigations was devoted to the evaluation of the preci-
sion of the residual chlorine test and the stability of chlorine
in test solutions. A precision of 5% (relative standard devia-
tion) was obtained from a statistical treatment of thirteen rep-
licate chlorine residual determinations on control solutions
having nominal chlorine concentrations of 10 mg/1. In accord-
ance with this observation, a free chlorine demand of 10% of
the applied dosage was the criterion used to determine any sig-
nificant compound—chlorine reaction.
The results of the first series of compound chlorination exper-
iments are given in Table 7, which lists the chlorine demand as
a function of applied chlorine dosage and contact time for each
of the test chemicals. The data indicate that, under the test
conditions, no significant chlorine demand was exercised by the
following compounds:
- 31 -
-------
TABLE 7
RESULTS OF PRELIMINARY CHLORINATION EXPERIMENTS
ADolied Chlorine Demand (OTA) At Indicated
Compound (mg/1) chlorine Contact Time, hours*
(mg/1)
Methanol , 8
Isopropanol, 10
T-Butanol, 10
Acetone, 10
Acetone, 20
Benzene, 10
Toluene, 10
Ethylbenzene , 17.5
Benzoic Acid, 10
5
10
20
5
10
20
5
10
20
10
20
10
20
5
10
20
5
10
20
5
10
20
5
10
20
0.5
-0.3
-1.0
-0.6
-0.1
-0.6
-0.7
-0.2
-0.2
-0.2
0
-0.1
-0.7
-0.1
-0.1
0
-0.2
0
+ 0.4
-0.2
0
+ 0.1
0
-0.1
0
-0.2
1.0
-0.6
-0.7
-1.2
+ 0.1
-0.1
-0.8
0
-0.2
+0.2
0
-0.4
0
+0.2
-0.1
0
-0.3
+0.1
+ 0.3
-0.3
0
+ 0.2
0
0
+ 0.2
+ 0.2
2.0
-0.1
-1.2
-0.2
-0.4
-0.9
-0.7
0
0
-0.2
-0.4
-0.8
-0.7
+0.2
-0.2
-0.2
+ 0.3
-0.4
+ 0.3
-0.2
0
+ 0.2
0
+ 0.3
+ 0.2
-0.2
24.0
-0.5
0
-1.0
+0.3
+ 0.6
+ 0.8
+0.4
0
+ 0.6
*™
^
-0.6
-0.3
-0.7
-0.3
-0.4
-0.4
0
0
0
+ 0.7
0
+ 0.2
Phenol, 13.5
m-Cresol, 20
5 >4.0
10 >8.1
20 >16.2
5 >4.0
10 >8.1
20 >16.2
Hydroquinone,
10
5
10
20
>4.0
7.9
10.8
>4.0
>8.1
11.6
-
-
-
Aniline, 10
Dimethylamine, 20
Nitrobenzene, 10
*NOTE:Negative sign
solution than
5 >4.0
10 >8.1
20 >16'2
__ _2_£.
10 >8.1
20 10.7
5 +0.2
10 +0.2 -0,
20 +0.3 +0,
"indicates greater
in control.
+ 0.2
+ 0.2
+ 0.1
residual found
+0.3
+0.2
+0.8
in test
- 32 -
-------
methanol* benzene*
isopropanol* toluene*
t-butanol* ethyl-benzene*
ben zoic acid
acetone* nitrobenzene
Those compounds marked with an asterisk (*) were monitored by
gas chromatography and exhibited no sianificant net compound
loss (compound control minus test) durina the experiments.
Moreover, no secondary peaks were observed in any of the chro-
matograms to indicate the existence of any reaction products.
It should be noted, that benzene, toluene, and ethvlbenzene were
rapidly lost from both test and control solutions durinq mixing.
Volatilization was particularly evident in the case of ethyl-
benzene, as shown in Figure 7.
As evident from Table 7, chlorine was observed to react with
phenol, m-cresol, hydroquinone, aniline, and dimethvlamine.
Phenol (13.5 mg/1), m-Cresol (20 ma/1) and aniline (10 mq/1)
completely consumed all of the applied chlorine, up to the
highest dosage (16.2 mg/1) within a contact time of 0.5 hours.
Hydroquinone (10 mg/1) and dimethvlamine (20 mcr/1) completely
removed all of the applied chlorine at the two lower dosages
(4.0, 8.1 mg/1) after a 0.5 hour contact time.
Having identified those compounds which were capable of reac-
ting with chlorine, a more detailed experimental program was
initiated to provide supplemental information recrardinq their
chlorinating properties. These studies were designed to: (1)
further define the chlorine demands of each of the chemicals,
(2) establish the contact time required for reaction, and (3)
identify, where possible, the formation of specific products.
The experimental conditions of this series of experiments were
generally similar to those previously described (pH 7.4, 25°C,
etc.), however, a wider range of compound and applied chlorine
concentrations were utilized.
Phenol was examined at concentrations of 10 and 20 mg/1, with
applied chlorine dosages of 20, 50 and 100 mg/1. These data
are presented in Table 8 and Figure 8. It is apparent that
phenol undergoes a relatively rapid reaction with chlorine
during the first 15 minutes of contact, followed by a decreas-
ing rate up to 2 hours contact time. A comparison of control
and test chlorine residuals sucrcrests that the reaction is vir-
tually complete after two hours. It is noted that 20 mg/1 of
phenol completely consumed all of the applied chlorine (50 and
100 mg/1) after 1 hour, and that 10 ma/1 of phenol completely
exhausted 20 and 50 mg/1 of chlorine. For 10 mq/1 of phenol,
only the application of 100 rng/1 of chlorine resulted in a
measurable chlorine residual, after two hours. Based on this
latter test, it may be calculated that phenol exercises a
chlorine demand of 6.35 mg Cl0/ma phenol after two hours con-
tact time. This is equivalent to 8.4 moles of chlorine per
- 33 ~
-------
o
cc
LU
O
2
O
o
LU
2
LU
N
~Z.
LU
CD
I __
LU
•- 0 MG CL/L (CONTROL)
+- 5 MG CL/L
0-10 MGCL/L
0
CONTACT TIME, HOURS
FIGURE 7
VOLATILIZATION OF ETHYL3ENZENE DURING
CHLORINATION EXPERIMENT
-------
o
5
en
LJ
o:
UJ
zr
a:
o
x
o
t-
o
CONTACT TIME,HOURS
FIGURE 8
REACTION OF CHLORINE WITH PHENOL
- 35 -
-------
mole of phenol. The consumption of chlorine by phenol is
also shown in Figure 9, in which the data have been expressed
in terms of molar ratios (mmol chlorine per mmol phenol) .
TABLE 8
CHLORIMATION OF PHENOL
Phenol Applied Contact
Concentration Chlorine Time
(mg/1) (mg/1) (hr)
10
10
10
20
20
0
20 °
U 1
2
0
50 J
2
0
100 J
2
0
50 °
1
2
0
100 ^
2
.25
.5
.0
.0
.25
.5
.0
.0
.25
.5
.0
.0
.25
.5
.0
.0
.25
.5
.0
.0
(OTA) Net Chlorine
Chlorine Demand
Residue
(mg/1)
6.
1.
0
0
17.
8.
2.
0
50
44
39
36.
4.
0
0
0
12
2.
0
0
0
7
8
0
8
5
5
5
il mg/1
14
18. 3
>20
>20
32.2
42
47.2
_>50
50
56
61
63.5
45.5
>50
>50
>50
88
97. 5
>100
~100
mmol
ci2
nmol phenol
1
2
>2
>2
4
5
6
6
6
7
8
8
3
>3
>3
I3
5
6
>6
>6
.9
.4
.7
.7
.3
.6
.3
.6
.6
.4
.1
.4
.0
.3
.3
.3
.8
.5
.6
.6
An attempt was made to qualitatively identify the products
resulting from the reaction of chlorine with phenol, using
the gas chromatograph, equipped with dual chromosorb W (HDMS)
columns coated with K20M Carbowax TPA. In this experiment, a
solution containing 25 mg/1 of phenol and 25 mg/1 of chlorine
was prepared. This selection of concentrations was made to
provide an excess of phenol, relative to chlorine, in order to
minimize the possibility of direct chlorine oxidation of the
phenolic structure. After a contact time of 0.5 hours, a sam-
ple was withdrawn for residual chlorine and chromatoaraphic
analysis. As anticipated, no chlorine residual was detected.
The chromatogram obtained on the sample is shown in Figure 10,
which also includes the chromatoaraphic output for a pure
phenol solution for comparison. Several additional peaks are
evident in the chromatoqram of the chlorinated phenol solution,
- 36 -
-------
FIGURE
o
(GNnOdlAIOO
CO
Q
Z>
O
Q.
O
O
h-
CO
UJ
h-
LJ
v
3
QL
13
LU
QL
O
O
QL
O
-------
PHENOL SOLUTION (IOMG/L)
COLUMN- CHROMOSORB W HDMS
K 20M CARBOV/AX TPA
TEMPERATURE' 150°C
T
TIME (RELATIVE UNITS!
I |
CHLORINATED PHENOL SOLUTION
COLUMN-CHROMOSORB W HDMS
K 20M CARBOWAX TPA
TEMPERATURE PROGRAMMED
I40°-I65°C
-TIME (RELATIVE UNITS)
FIGURE 10
CHROMATOGRAMS OF PHENOL
AND
CHLORINATED PHENOL SOLUTIONS
-------
These were subsequently identified, on the basis of relative
retention data, as o-chlorophenol (1), 2,6 dichlorophenol
(3), 2,4 dichlorophenol (4), 2,4,6 trichlorophenol (5), and
p-chlorophenol (6).
The results of similar experiments, using m-cresol as the test
compound are shown in Table 9 and Figures 11 and 12. The re-
action of chlorine with m-cresol also proceeded quite rapidly
in the first 15 minutes, and was essentially complete after
two hours contact time. In the case of m-cresol (10 and 20
mg/1), the application of 50 and 100 mg/1 of chlorine produced
a free chlorine residual after two hours. The maximum observed
chlorine uptake (after 2 hours) was computed to be 3.84 mg Cl-/
mg m-cresol (5.9 moles Cl?/mole m-cresol), corresponding to
the solution which initially contained 10 mg/1 of m-cresol and
100 mg/1 of chlorine. The consumption of chlorine by m-cresol
is also shown in Figure 9, and indicates that m-cresol exer-
cises a lower chlorine demand than phenol, on a molar basis.
TABLE 9
CHLORINATION OF m-CRESOL
m-Cresol Applied Contact
Concentration Chlorine Time
(mg/1) (mg/1) (hr)
10
10
10
20
20
0.25
20 °'5
^U 1.0
2.0
0.25
50 °'5
DU 1.0
2.0
0.25
100 J;5
2.0
0.25
50 °'5
iu i.o
2.0
0.25
»» 2:»
2.0
(OTA)
Chlorine
Residual
(mg/1)
3.3
1.5
0.5
0.2
30.8
30.8
28.3
17.0
81.4
77.0
61.6
61.6
16.3
11.1
8.0
8.0
61.6
58.2
56.6
46.0
Net Chlorine
Demand
mg/1
T67T
18.5
19.5
19.8
19.2
19.2
21.7
33.0
18.6
23
38.4
38.4
33.7
38.9
42
42
38.4
41.8
43.4
54.0
mmol Cl~
mmol m-Cresol
2.5
2.8
3.0
3.0
2.9
2.9
3.3
5.0
2.8
3.5
5.9
5.9
2.6
3.0
3.2
3.2
2.9
3.2
3.3
4.1
- 39 -
-------
00
UJ
cc
cc
o
X
CJ
1001
80
60
40
20
0
20MG M-CRESOL/L
• -IOO MG/LJ. APPLIED
+ - 50 MG/LJ CHLORINE
CONTACT TIME , HOURS
FIGURE II
REACTION CF CHLORINE WITH M-CRESOL
-------
CO
UJ
>
LU
CC
UJ
co
2
o
Q-
00
UJ
cc
M-CRESOL SOLUrION(IOMG/L)
COLUMN-CHROMOSORB W HDMS
K20M CARBOWAX TPA
TEMPERATURE PROGRAMMED
I50°-I60°C
TIME(RELATIVE UNITS)
co
UJ
>
<
_)
LU
UJ
CO
z
o
Q_
CO
LJ
(T
CHLORINATED M-CRESOL SOLUTION
COLUMN CHROMOSORB W HDMS
K20M CARBOWAX TPA
TEMPERATURE' PROGRAMMED
135°- I65°C
— TIME (RELATIVE UNITS)
FIGURE 12
CHROMATOGRAMS OF M-CRESOL
AND
CHLORINATED M-CRESOL SOLUTIONS
-------
Chromatograms obtained for m-cresol and a chlorinated solution
of m-cresol are shown in Figure 12. Several additional peaks
are evident for the latter sample, reflecting the formation
of a complex mixture of products. None of these species could
be positively identified, due to the lack of commercially
available chlorine-substituted cresols. However, it is prob-
able that the products constitute a mixture of chloro-m-cresols
and oxidized forms.
The reaction of chlorine with hydroquinone was studied next,
using compound concentrations of 10 and 25 mg/1, with nominal
applied chlorine dosages of 10, 20, 25, and 50 mg/1. The
data for this experiment are presented in Table 10, and Figure
13. As in the case of phenol and m-cresol, a rapid initial
reaction was observed, followed by a declining rate of chlor-
ine uptake. The reaction appeared to be complete after two
hours. A maximum chlorine demand of 2.37 mg Cl~/mg hydroqui-
none (3.9 moles/mole) was observed under the test conditions.
(The molar consumption of chlorine is plotted in Figure 9,
and shows a lower chlorine demand by hydroquinone, relative
to phenol.)
TABLE 10
CHLORINATION OF HYDROQUINONE
Hydroquinone Applied Contact
Concentration Chlorine Time
(mg/1) (mg/1) (hr)
0.25
10 10.4 °*Q
2.0
0.25
10 20.8 °;jj
2.0
0.25
10 26 J'Q
2.0
0.25
10 52 J'jj
2.0
0.25
25 52 °'5
Zb ^ 1.0
2.0
(OTA)
Chlorine
Residual
(mg/1)
0
0
0
0
6.1
4.75
2.75
1.8
9.8
10.0
5.5
4.5
30.8
32.7
30.0
28.3
9.5
7.5
3.5
1.0
Net Chlorine
Demand
mg/1 mm°1 C12
mmol Hydroq.
>10.4
>10.4
>10.4
>10.4
14.7
16.05
18.05
19.0
16.2
16.0
20.5
21.5
21.2
19.3
22.0
23.7
42.5
44.5
48.5
51.0
^ 1 • 6
^ 1 • D
2.3
2.5
2.8
2.9
2.5
2.5
3.2
3.3
3.3
3.0
3.4
3.7
2.6
2.8
3.0
3.2
- 42 -
-------
+ - 52 MG/L
• -26 MG/L
O -20.8 MG/L
o
CONTACT TIME, HOURS
FIGURE 13
REACTION OF CHLORINE WITH HYDROQUINONE
-------
In the absence of an applicable chromatographic procedure, the
use of ultraviolet absorption spectrophotometry for reaction
product identification was explored. During the previous
studies, it was noted that the addition of chlorine to hydro-
quinone occasionally resulted in the production of orange
colored solutions. This behavior was particularly evident at
the higher reactant concentrations. Such phenomena are not
uncommon in aqueous solutions of aromatic organic chemicals,
and generally are the result of partial oxidation of the ring
structure. It was thus postulated that hydroquinone reacts
with chlorine, at least partially, to form p-benzoquinone as
the primary product:
OH
+ Chlorine
To test this hypothesis, a separate chlorination experiment
was conducted. Ultraviolet spectra were first obtained on
buffered (pH 7.4) solutions of both hydroquinone (10 and 20
mg/1), and p-benzoquinone (1, 2, and 5 mg/1), as shown in Fig-
ure 14. Note that hydroquinone exhibits absorption maxima
at ^220 nm and ^288 nm, whereas p-benzoquinone absorbs maxi-
mally at ^245 nm. The UV absorption characteristics of each
of the pure compounds are thus sufficiently different to al-
low their identification in a mixed sample.
A solution containing 10 mg/1 of hydroquinone and 20 mg/1
chlorine was next prepared. This corresponds to an applied
chlorine to hydroquinone molar ratio of approximately 3:1,
in correspondence to the previously observed chlorine demand.
As in previous studies, the pH was maintained at 7.4, using
the phosphate buffer. After a contact time of 10 minutes, a
sample was withdrawn for UV analysis. The resulting spectrum
is also shown in Figure 14, and demonstrates the rapid forma-
tion of p-benzoquinone, as indicated by the appearance of an
absorption maximum at ^245 nm. Correspondingly, the peak at
^288 nm is observed to diminish, reflecting a substantial de-
crease in the initial hydroquinone concentration. The peak
at ^245 nm (p-benzoquinone) was found to gradually diminish
during subsequent measurements over twenty-four hours. Cor-
respondingly, a broad "shoulder" in the 265 - 295 nm region
developed over this same period. The species associated with
this absorption was not identified.
Aniline was also re-examined in the series of detailed chlor-
ination experiments. Applied chlorine dosages of 18, 45, and
- 44 -
-------
0.0
o
\
UJ
o
0.5
m
o
^
UJ
O
GO
tr
8
CO
1.5
00
05
1.0
1.5
190
HYDROQUINONE
10 mg/l
p-BENZOQUINONE
5 mg/l
10 mg/l HYDROQUINONE
+ 20 mg/l CHLORINE
10 MINUTES CONTACT
240 290 340
WAVELENGTH - NANOMETERS
390
FIGURE 14
ULTRAVIOLET ABSORPTION SPECTRA-RE ACTION OF CHLORINE WITH
HYCROQUINONE
- )^ -
-------
90 mg/1 were added to solutions containing 10 and 20 mg/1 of
aniline. The data derived from these tests are given in Table
11 and Figure 15.
TABLE 11
CHLORINATION OF ANILINE
Aniline
Concentration
(mg/1)
10
10
10
20
20
Applied Contact
Chlorine Time
(mg/1) (hr)
0.25
0.5
18 1.0
2.0
3.0
0.25
0.5
45 1.0
2.0
3.0
0.25
0.5
90 1.0
2.0
3.0
0.25
0.5
45 1.0
2.0
3.0
0.25
0.5
90 1.0
2.0
3.0
(OTA)
Chlorine
Residual
(mg/1)
1.75
0.4
0.2
0.2
0.2
27.5
20
14
6
4
58
52
43
34
28
4.0
0.8
0.6
0.2
0.2
40
30
10
3.6
2.6
Net
mg/1
16.25
17.6
>17.8
"17.8
"17.8
17.5
25
31
39
41
32
38
47
56
62
41
44.2
>44.4
"44.8
"44.8
50
60
80
86.4
87.4
Chlorine
Demand
mmol Cl-
TniTif-\ 1 ZV n "i "1 i n ^
iLULIXJ _L fill_L J. -LI 1C
2.1
2.3
>2.3
"2.3
"2.3
2.3
3.3
4.1
5.1
5.4
4.2
5.0
6.2
7.3
8.1
2.7
2.9
>2.9
"2.9
>_2. 9
3.3
3.9
5.3
5.7
5.7
As previously observed for hydroquinone, the appearance of
orange colored solutions was evident at the higher applied
chlorine levels (50 and 100 mg/1). It may be noted from the
figure that aniline also initially undergoes a rapid reaction
with chlorine, followed by a declining rate of chlorine up-
take. Chlorine consumption was observed to continue over
contact times in excess of two hours. The maximum chlorine
uptake at three hours was calculated to be 6.2 mg Cl^/mg ani-
line (8.1 moles/mole).
- 46 -
-------
10 MG ANILINE/L
-90 MG/L
-45 MG/L
0-IR MG/L
CONTACT TIME,HOURS
FIGURE 15
REACTION OF CHLORINE WITH ANILINE
-------
In a separate experiment, an attempt was made to gather more
information pertaining to the nature of the reaction products
of chlorine and aniline. In the absence of reliable chroma-
tographic techniques, ultraviolet spectrophotometric methods
were employed for these tests. Solutions were prepared, in
accordance with previously described procedures, containing
10 mg/1 of aniline, to which chlorine dosages of 5, 10, and
20 mg/1 were added. Samples were withdrawn at varying inter-
vals of time for UV spectral scans. Recorded spectra from
this sequence of measurements are shown in Figures 16 and 17.
Figure 16 illustrates the effect of chlorine dosage on the
ultraviolet spectra of chlorine-contacted aniline solutions.
The scans shown were taken on samples after a contact time of
one hour. The spectrum indicated by a dashed line in Figure
16 is that of a 10 mg/1 aniline solution to which no chlorine
had been added, and is included for comparison. Aniline
alone exhibits fairly well resolved absorption maxima cen-
tered at 230 nm and 281 nm, with no significant absorption
above 310 nm. The addition of 5 mg/1 of chlorine has the
effect of shifting the peak absorbance to about 233 nm in a
broad band which extends to 330 nm, with evidence of unre-
solved inflections, and a shoulder in the region near 285 nm.
A dosage of 10 mg/1 of chlorine produces a further shift in
the absorption peak to about 252 nm. A broad band is again
evident which extends to 360 nm and retains the shoulder
around 285 nm. The highest chlorine addition (20 mg/1) was
observed to cause a further shift of the most prominent peak
to about 287 nm. The broadness of the absorption band is
also increased, extending to about 370 nm, and showing seve-
ral unresolved inflections between 220 and 270 nm.
Ultraviolet absorption spectra were also recorded at varying
time increments for the 10 mg/1 aniline + 10 mg/1 chlorine
solution. These res alts are illustrated in Figure 17.
The first scan was obtained after 10 minutes contact time and
shows a shift in the absorption peak, producing two poorly-
resolved maxima at about 253 nm and 260 nm, with a shoulder
centered around 286 nm. After a contact time of one hour,
there is no evidence of the dual peaks at 253 nm and 260 nm,
but a single broad absorption peak now appears at %252 nm.
Note also that the absorbance of the shoulder has diminished.
A final scan, taken at two hours, shows a further decrease in
the broad band maxim-am to about 246 nm. These results are
consistent with the data developed in the chlorine uptake
studies, and confirm the progressive nature of the aniline-
chlorine reaction over contact times in excess of two hours.
Spectra were obtained on dilute aqueous solutions of several
chlorine-substituted anilines. The absorption maxima of each
compound are as follows:
- 48 -
-------
LU
O
DO
DC
m
<
o
i
o
m
GC
o
in
m
<
UJ
o
•
CO
cc
o
C/5
CD
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
05
ANILINE'
~ ANILINE
15
ANILINE
190
10 mg/l ANILINE +
5 mg/l CHLORINE
I HOUR CONTACT
10 mg/l ANILINE +
10 mg/l CHLORINE
I HOUR CONTACT
10 mg/l ANILINE +
20 mg/ I CHLORINE
I HOUR CONTACT
240 290 340
WAVELENGTH - NANOMETERS
390
FIGURE 16
ULTRAVIOLET ABSORPTION SPECTRA OF CHLORINE CONTACTED
ANILINE SOLUTIONS: EFFECT OF CHLORINE DOSAGE
-------
10 rng/l AMIL'W f |
10 rng/i CHLORINE j
!0 MINUTES CONTACT 1
10 mg/l ANILINE +
10 mg/ I CHLORINE
HOUR CONTACT
10 mg/l ANILINE +
10 mg/l CHLORINE
2 HOUR CONTACT
240 290 340
WAVELENGTH-NANOMETERS
390
FIGURE 17
ULTRAVIOLET ABSORPTION SPECTRA OF CHLORINE CONTACTED
ANILINE SOLUTIONS: EFFECT OF CONTACT TIME
- 50 -
-------
Compound
aniline
o-chloroaniline
p-chloroaniline
2,4-dichloroaniline
2,6-dichloroaniline
2,4,6-trichloroaniline
A max
E-band(nm)
230
232
238.5
240
228-236
(broad)
242
A max
B-band (run)
281
286.5
293
298
291
304
It is evident that chlorine substitution on the aniline struc-
ture promotes a bathochromic shift in the wavelengths of maxi-
mum absorption. This effect is enhanced with increasing sub-
stitution and is more pronounced with para-substitution, than
with ortho-substitution. A comparison of Figure 16 with the
data given above reveals that none of the spectra obtained on
the pure chloroanilines can be precisely matched with those
recorded for the chlorinated aniline solutions. Although
there may be some indication of o-chloroaniline formation at
the 5 mg/1 level of applied chlorine (as evidenced by absorp-
tion at ^233 nm and o,285 nm) , it is apparent that a highly
variable and complex mixture of products is formed. The na-
ture and distribution of these reaction products are influ-
enced both by chlorine dosage, and by contact time. The pro-
gressive shift of the absorption maximum to higher wavelengths
and the appearance of a broad band with increasing chlorine
dosage suggests the formation of a mixture of chlorine-sub-
stituted anilines. In addition to the formation of chloro-
anilines, it is also possible that some degree of ring oxi-
dation proceeds simultaneously with ring substitution.
The final series of detailed chlorination experiments was
devoted to the re-examination of the chlorine-dimethylamine
reaction. Dimethylamine concentrations of 20 mg/1 and 100
mg/1 were employed in these tests, with nominal applied
chlorine levels ranging between 20 and 150 mg/1. Samples
were taken at varying time intervals for free residual chlo-
rine analysis (by the OTA procedure) and for total chlorine
residual (by iodimetric titration). The results of these in-
vestigations are given in Table 12 and Figure 18.
It is apparent from the data that: (1) the addition of chlo-
rine to dimethy1amine solutions results in a rapid consump-
tion of free chlorine; and (2) the total chlorine residual,
including a combined fraction formed in the initial stages of
the reaction, gradually diminishes with time. The initial
reaction appeared to be complete after a contact time of 15
minutes or less, whereas the loss of combined residual chlor-
ine was observed to proceed over several hours. The data
also indicate that the degree of chlorine uptake is variable,
- 51 -
-------
TABLE 12
CHLORINATION OF DIMETHYLAMINE
Concentration or^n^n Time
(mg/1) mg/1 mrao1 C12 (hr)
20
20
20
100
100
100
mmol DMA
0
0
21.3 .68 2
3
4
0
0
42.6 1.35 2
3
4
0
0
85.2 2.71 2
3
4
0
1
75 .48 2
3
5
0
1
112.5 .715 2
3
5
0
1
150 .95 2
3
5
.25
.5
.0
.0
.0
.0
.25
.5
.0
.0
.0
.0
.25
.5
.0
.0
.0
.0
.5
.0
.0
.0
.0
.5
.0
.0
.0
.0
.5
.0
.0
.0
.0
Chloi
Resic
(me
- Tot."
13
8
5
0
0
31
23
15
8
2
58
58
53
47
43
62
49
27
11
9
91
55
37
18
7
119
65
42
19
7
.3
-
.0
.3
.9
-
.9
.9
.0
.6
.5
-
.5
.2
.9
.9
.4
.4
.3
.7
.1
.0
.9
.7
.2
.8
.6
.0
.9
.5
.8
:ine
lual
J/D
Free
0.
0.
0.
0.
0.
0.
10
10
9
9
10
8
46
46
44
37
43
37
0
0
1
75
3
3
25
25
2
.0
.0
.5
.0
.0
.5
.5
.5
.5
.5
.5
.5
.1
0
0
-
—
.2
0
0
-
—
.2
.55
.2
-
—
Net Free Chlorine
Demand
mg/1 mmol C12
20.
21
21
21.
21.
21.
32.
32.
33.
33.
32.
34.
38.
38.
40.
44.
41.
44.
75
112
150
55"
05
05
1
6
6
1
6
6
1 ,
7 '
7
7
7
7
7 j
mmol DMA
•21 .67
•33.1 1.05
•41.5 1.32
.48
.5 .715
.95
- 52 -
-------
UJ
cr
o
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LU
o:
20MG DMA/L
0,» -852 MG/L T_APPLIED
-l-.x -42 6MG/L ICHLORIN
1234
CONTACT TIME -HOURS
150
a:
o
Q
O1
UJ
CC
100 MG DM A /L
-150 MG/L | APPLIED
O -112. 5 MG/L
234
CONTACT TIME - HO'JRS
FIGURE 18
REACTION OF CHLORINE WITH DIMETHYLAMINE
-------
and increases with increasing applied chlorine dosage. This
is evident from Table 12, which shows both the molar consump-
tion of chlorine per mole of DMA, and the applied chlorine to
DMA molar ratio. Note that dimethylamine readily consumes at
least 1 mole of chlorine per mole of DMA. At higher chlorine
applications, chlorine demands greater than unity were ob-
served, but a free chlorine residual was maintained. A max-
imum chlorine uptake of approximately 1.3 moles Cl^/mole DMA
was noted, at an applied chlorine to DMA molar ratio of about
2.7.
A rigorous characterization of the nature of the products of
the reaction of DMA with chlorine could not be made, due to
the lack of suitable analytical tests. In preliminary exper-
iments, it was found that DMA does not respond to the chemical
oxygen demand test. Although it was determined that DMA could
be detected in aqueous solutions by gas chromatography, the
response was found to be non-quantitative. Additionally, di-
methylamine was observed to be insensitive to ultraviolet
spectrophotometric analysis. These difficulties precluded
the positive identification of any products of the DMA-chlo-
rine reaction.
Discussion of Results - Chlorination Studies
One of the major objectives of the experimental program of
Phase I was the determination of the ability of each of the
selected chemicals to react with chlorine. A corollary, but
nonetheless vital requirement, was the establishment of test-
ing procedures by which this characterization could be
achieved. It was recognized, a priori, that virtually all of
the test compounds could, under the appropriate conditions,
react with chlorine. In fact, halogenation reactions are
among the most important in the synthesis of complex organic
chemicals. However, many of these reactions do not proceed
to any appreciable extent, unless rigorous chemical or physi-
cal driving forces are applied (elevated temperature, pres-
sure, catalysis, etc.). Obviously, such conditions would not
be encountered in conventional effluent chlorination practice.
The selection of experimental parameters was thus constrained
to a rather narrow range of ambient temperatures, pH values
near neutrality, and dilute aqueous solutions of reactants.
It was anticipated that some of the test compounds, which are
commonly employed in industrial halogenation processes, would
be essentially unaffected by the levels of chlorine commonly
applied to effluents. This assumption was confirmed by the
experimental data, which indicated that only five of the four-
teen original test chemicals were observed to react with chlo-
rine to any substantial degree.
None of the alcohols tested (methanol, isopropanol, t-butanol)
exhibited any tendency to exercise a chlorine demand. Al-
though many alcohols nay undergo halogenation, the reactant
- 54 -
-------
employed is usually a hydrogen halide (HX). Moreover, the
reaction is commonly achieved at elevated temperatures, in
the presence of a catalyst or strong acid, or in a nonaqueous
gas phase. As such, it is unlikely that a dilute aqueous sol-
ution of chlorine would affect any of the alcohols examined in
this study.
No significant chlorine uptake was observed in the case of
acetone. At first inspection, this result seemed anomalous,
as it is known that saturated ketones and aldehydes will
generally undergo halogenation in aqueous solutions. It has
been reported that the rate of halogenation of acetone is
dependent on the acetone concentration, and is significantly
increased by either acidic or alkaline catalysis, as repre-
sented by the following expression :
V = [(CH3)2CO][6 x 10~9 + 5.6 x ICT^H"1"] + 7 [OH~] ]
where V is the reaction rate in moles/liter/second, and the
bracketed terms correspond to the molar concentrations of the
respective species. Using this expression, the calculated
rate of chlorination_of 20 mg/1 of acetone at pH 7.4 is ap-
proximately 2.2 x 10 3 mmol/liter/hour, or about 0.6% per hour,
In the present study, the measurement of this low rate was
not compatible with the precision of the chromatographic
analysis of acetone nor the OTA method for chlorine. Conse-
quently, a chlorine uptake by acetone could not be observed,
under the test conditions.
Benzene, and its derivatives, toluene, ethyl benzene, and
benzoic acid, also showed no evidence of reactions with chlo-
rine. Again, halogenations of benzenes are rather common-
place in the chemical process industry, but a catalyst is us-
ually required to achieve this substitution.
It is thus unlikely that benzene or the alkyl benzenes could
react with chlorine in the dilute aqueous solutions consid-
ered in this study. Benzoic acid and nitrobenzene were con-
sidered to be even less reactive than benzene, due to the
electron-attracting and ring-deactivating effects of the
-COOH and -NO2 groups.
In contrast to benzene and the derivatives of benzene des-
cribed above, phenol was determined to be quite reactive to
chlorine, in dilute aqueous solution. This result is by no
means surprising, since the presence of taste and odor-causing
chlorophenols in certain chlorinated wastewater effluents has
(1 8)
been demonstrated by many investigators ' . The high reac-
tivity of phenol is attributable to the ring-activating elec-
tron-releasing properties of the -OH functional group.
- 55 -
-------
The nature of the activating group is such that halogen sub-
stitution in aqueous solution is preferentially favored in
the ortho- and para- positions, with respect to the -OH group.
This fact has also been confirmed by several researchers '
(9)
Burttschell et..a_l. proposed the following reaction scheme
to describe the chlorination of phenol:
OH OH
Cl
OH
Cl
Ring
Oxidation
Cl
Note that the reaction proceeds by the stepwise substitution
of the 2, 4, and 6 (ortho- and para-) positions of the aro-
matic ring. Ring oxidation follows the formation of 2,4,6—
trichlorophenol. It is probable that the reactions proceed
simultaneously, as well as sequentially, resulting in the for-
mation of a complex mixture of chlorophenols and the oxidation
products. The nature and distribution of these products are
doubtlessly affected by such parameters as reaction time, pro-
portions of reactants, pH, temperature, and other conditions.
Lee * ' has indicated that the maximum rate of chlorination
occurs between pH 7 and pH 9.
In the present study, the formation of all of the chlorophe-
nols mentioned above was confirmed using gas chromatographic
techniques. The appearance of several unidentified peaks in
the chromatograms also suggested the existence of oxidized
forms. Additional evidence for ring oxidation may be derived
from the fact that phenol exhibited a chlorine demand in ex-
cess of the amount required to completely form trichlorophenol.
A maximum chlorine uptake of 8.4 moles Cl^/mole phenol was
observed in this study, compared with a stoichiometric require-
ment of 3 moles/mole for trichlorophenol formation.
Meta-cresol, a methyl-substituted phenol, exhibited chlorinat-
ing properties similar to phenol. This is consistent with the
structural similarities (and therefore, reactivities) of the
two compounds. A moderately activating methyl group in the
meta position should serve to reinforce the pattern of chlorine
- 56 -
-------
substitution observed for phenol, i.e., by directing to the
ortho- and para- (2,4,6) positions. Upon the addition of
chlorine, it would therefore be expected that a complex mixture
of chlorine-substituted and oxidation products would form.
Evidence for this behavior is manifested by an observed chlo-
rine demand of 5.9 moles/mole, which is considerably in ex-
cess of the theoretical requirement to produce a tri-substi-
tuted m-Cresol, and by the appearance of at least eight peaks
in the chromatogram of a chlorinated m-Cresol solution. Un-
fortunately, nc qualitative identification of the products of
chlorination could be attempted, due to the lack of commer-
cially available chloro-m-Cresols.
In a study devoted to the oxidation of phenols by chlorine,
Eisenhauer suggested the following probable sequence of
reactions resulting from the chlorination of a substituted
phenol.
OH
OH
R
Cl
R'
COOH
COOH
Cl
COOH
COOH
Cl
O
(for m-Cresol, R is a methyl group).
The formation of non-aromatic oxidized products, such as car-
boxylic acids, at higher levels of applied chlorine, was pos-
tulated on the basis of observed shifts in ultraviolet absorp-
tion spectra. This hypothesis is consistent with the rela-
tively high chlorine demands noted for both m-Cresol and phe-
nol.
Hydroquinone is a dihydric phenol, containing -OH groups in
the 1,4 positions. This configuration of two highly activa-
ting substituents should even further magnify the destabili-
zation of the ring structure. Indeed, hydroquinone may be
readily converted to p-benzoquinone by a variety of oxidizing
agents.
As previously described, the oxidation of hydroquinone to p-
benzoquinone by chlorine was confirmed in this study, using
ultraviolet spectrophotometric procedures. Apparently, this
is not the only reaction, since a maximum chlorine demand of
3.7 moles Cl^/mole was observed, which is greater than the
«£
theoretical requirement for simple oxidation to p-benzoquinone,
Moreover, when a solution containing an applied chlorine to
hydroquinone molar ratio of about 1.5 was analyzed after ten
minutes contact time, it was found to contain approximately
one-half of concentration of p-benzoquinone that would have
- 57 -
-------
been expected if complete conversion had occurred. These
observations suggest the formation of other substituted or
oxidized products resulting from the reaction of chlorine
with hydroquinone.
During the preliminary experiments with hydroquinone, the
formation of a tetrasubstituted quinone, chloranil, was con-
sidered as a possibility:
0
Cl
O
0
However, attempts to isolate chloranil were unsuccessful, due
to its extremely low solubility in water. It is now believed
that chloranil is probably not a product of the hydroquinone-
aqueous chlorine reaction, since the usual method of prepara-
tion of chloranil involves the use of hydrogen chloride.
The possibility also exists for the formation of a charge-
transfer (IT) complex of p-benzoquinone and unreacted hydro-
quinone of the form:
HO
OH
0
O
The formation of this species is generally accompanied by the
appearance of a dark-colored solution and a bathochromic shift
- 58 -
-------
in the ultraviolet absorption spectrum . Phenomena of this
type were observed at the higher reactant (hydroquinone and
chlorine) concentrations, which could possibly explain the
non-stoichiometric production of p-benzoguinone, as described
above.
In summary, it appears that additions of chlorine to dilute
aqueous solutions of hydroquinone results in the formation of
p-benzoquinone as a primary product. This compound is be-
lieved to persist either as a separate species, as part of the
quinhydrone complex, or combinations of the two. Further addi-
tions of chlorine, resulting in chlorine uptakes of at least
3.7 moles Clp/mole are likely to produce further oxidized pro-
ducts, such as carboxylic acids.
Aniline, by virtue of a strongly activating functional group
(-NH0), should also be expected to possess chlorinating pro-
^
perties similar to phenol. It is known that bromine reacts
rapidly with aniline to form 2,4,6—tribromoaniline in high
yield, and it has been reported that, depending on the nature
of the oxidizing agent, aniline may be oxidized to either nit-
robenzene, p-benzoquinone, or other products of ring cleavage
It may thus be postulated, by analogy to phenol, that aniline
may undergo the following reaction steps when contacted with
aqueous chlorine solutions:
(7)
Cl
NH,
NH,
Cl
NH,
NH2
elf
~f
Cl
Cl
ci
Cl
ci
Ring
Oxidation
Indirect evidence for this reaction scheme is shown in Figure
9, in which the observed chlorine consumption by aniline, on
a molar basis, is noted to be quite similar to phenol.
It is also evident from the experimental data that the chlo-
rination of aniline results in the generation of highly com-
plex reaction products. Ultraviolet spectra taken on light-
ly chlorinated (0.5 mg Cl2Ang aniline) solutions (see Figure
16) suggest the production of a mixture of chloroanilines.
- 59 -
-------
Higher applied chlorine levels (1-2 mg Cl2/mg) seem to pro-
mote the formation of oxidized species. Note the UV absorp-
tion spectra obtained on a 10 mg/1 aniline solution, to which
10 mg/1 of chlorine had been added. The disappearance of
peaks in the region of 230 run and 280 run indicate a loss of
aromatic structure, normally associated with these absorp-
tions. Moreover, the appearance of a single broad band sug-
gests the formation of either a partially oxidized non-aro-
matic ring or a conjugated straight chain molecule that re-
sulted from ring scission.
There is also evidence for a continuing sequence of reactions
that proceeds even after a complete loss of free chlorine
residual. For the 1:1 solution described above, no free
chlorine residual was detected after a one-half-hour contact
time, yet the UV absorption pattern continued to change over
a two-hour period. This may be indicative of the production
of intermediate species, which may subsequently undergo struc-
tural rearrangement. The resulting broad band at 246 nm was
found to persist during UV scans taken over eighteen hours.
The position and the shape of this absorption is quite similar
to that observed for p-benzoquinone, a possible product of an-
iline oxidation.
Dimethylamine, a secondary aliphatic amine, is also known to
participate in halogen reactions, leading to the formation of
mono—N—haloamines.
In a previous study devoted to the rate of formation of chlor-
(12)
amines, Weil and Morris indicated that only a monochloro
derivative results from the reaction of chlorine and dimethyl-
amine, corresponding to the following stoichiometric represen-
tation:
H Cl
CH3-N-CH3 + C12 -> CH3-N-CH3 + HC1
It is evident from this reaction that the stoichiometric
chlorine requirement is one mole per mole of DMA. The results
from the present study generally confirm this stoichiometry,
however, in cases of higher applied chlorine doses, chlorine
demands greater than one mole per mole were observed.
(13)
Marks has stated that the monochloro derivative of di-
methylamine (N—chloro—DMA) should be amenable to analysis by
the acid iodometric titration procedure, which was utilized
in the supplementary DMA chlorination experiments. Indeed,
an iodometric chlorine residual in excess of the free chlo-
rine residual (OTA) was observed in the early stages of the
reaction, suggesting the initial formation of some chloro-DMA
compound, probably N-chloro—DMA. The subsequent loss of com-
bined chlorine residual indicates that the compound is either
unstable, or is lost from the system by volatilization.
- 60 -
-------
In summary, it has been shown that five of the fourteen ori-
ginal test chemicals were observed to participate in reac-
tions with free chlorine under the moderate conditions asso-
ciated with conventional effluent chlorination practice.
These compounds are: phenol, m-cresol, hydroquinone, aniline,
and dimethylamine. In some cases, it has been possible to
relate the experimentally determined chlorine-reactivity of
these species to their respective molecular structures. For
example, of the nine aromatic compounds examined in the study,
only those possessing "ring-activating" substituent groups
were noted to react with chlorine in dilute aqueous solutions.
These considerations should facilitate the preliminary char-
acterization of the potential of other similar species to
undergo chlorination reactions.
The information developed in this phase of the study clearly
indicates that the application of chlorine to dilute solutions
of any of the chlorine-reactive chemicals results in a highly
complex mixture of products. It is apparent that the nature
and distribution of these products is dependent on a variety
of parameters, which include reactant concentrations, pH, and
temperature. A list of the probable products of chlorination
has been assembled and is presented in Table 13. This tabula-
tion was compiled on the basis of the experimental data gene-
rated in this study, supplemented by various literature
sources.
One of the objectives of the experimental program of Phase I
was the selection of five products of chlorination for sub-
sequent studies. The chemicals initially chosen for further
examination were 2,4,6—trichlorophenol, 2,4,6—trichloroaniline,
4—chloro—3-methylphenol, N—chloro-JDMA, and chloranil. The
original selection was made on the basis of available infor-
mation relating to probability of formation, chemical stabil-
ity, and commercial availability, or ease of synthesis.
Specifically, 2,4,6—trichlorophenol was chosen since it had
been identified as a product of phenol chlorination, and rep-
resented the final reaction step prior to ring oxidation.
Its existence had also been demonstrated by previous investi-
gators, who indicated that it was difficult to biologically
degrade and was relatively toxic to aquatic organisms ' ' .
Trichloroaniline was selected for further study because of
the chemical similarity of aniline to phenol, as well as a
strong theoretical basis for its existence. Virtually no
information is available regarding the behavior of this com-
pound in biological systems.
The only commercially available chlorinated product of m-
Cresol was 4—chloro—3-methyl phenol, and as little is known
about the products of m-cresol chlorination, this compound
was carried into the Phase II studies.
- 61 -
-------
TABLE 13
PROBABLE PRODUCTS OF CHLORINATION
Phenol
o-chlorophenol
p-chlorophenol
2,4-dichlorophertol
2,6-dichlorophenol
2,4, 6-trichlorophenol
non-aromatic oxidation products
m-Cresol
2-chloro-3-methylphenol
4-chloro-3-methylphenol
6-chloro-3-methylphenol
2,4-dichloro-3-methylphenol
2,6-dichloro-3-methylphenol
4,6-dichloro-3-methyIphenol
2,4,6-trichloro-3-methylphenol
non-aromatic oxidation products
Hydroquinone
p-benzoquinone
non-aromatic oxidation products
Aniline
o-chloroaniline
p-chloroaniline
2,4-dichloroaniline
2,6-dichloroaniline
2,4, 6-trichloroaniline
non-aromatic oxidation products
Dimethylamine
N-chloro-DMA
oxidation products
Of the primary test chemicals considered, dimethylamine was
judged to be one of the more likely to survive biological
treatment intact. Since some evidence existed for the forma-
tion of the monochloro derivative, N—chloro—DMA was deemed
suitable for further study. Although N—chloro—DMA was not
available commercially, it was originally anticipated that
its synthesis could be achieved in the laboratory. However,
when subsequent studies indicated that N—chloro—DMA was
chemically unstable, this species was dropped from further
consideration.
- 62 -
-------
Chloranil, although not shown on the list of probable pro-
ducts of chlorination, was initially suspected to be a pro-
duct of hydroquinone chlorination. As such, it was origin-
ally chosen for examination in Phase II. When additional
experiments failed to establish the existence of chloranil
as a reaction product, p-benzoquinone, a confirmed product
of the hydroquinone-chlorine reaction was substituted in
the following studies.
- 63 -
-------
SECTION VIII
RESPIROMETER STUDIES - PHASE II
As a result of the Phase I studies, 5 compounds were selected
for further study in Phase II. The selected compounds were:
2,4,6—trichlorophenol
4—chloro—3-methyl phenol
chloranil
2,4,6—trichloroaniline
N—chlorodimethylamine
The first portion of Phase II was the conduction of respiro-
meter studies to examine the possible inhibition or toxicity
of the selected compounds to an operative biological system.
Experimental Procedure
The respirometer studies were conducted in a modified Warburg
apparatus, known as a "Gilson" respirometer. The system dif-
fered from a "Warburg" only in the method of pressure differ-
ence measurement. The control system was an inocculated syn-
thetic sewage of the composition described in a previous sec-
tion of the report. The test systems contained the same in-
occulum and sewage, plus the desired concentration of the
particular substrate. In most cases, a substrate concentra-
tion range of 1 mg/1 to 100 mg/1 was studied. For comparative
purposes, one experiment was conducted with copper as the test
substrate. The inhibitory influence of copper is well docu-
mented, and was included to provide a basis for comparison of
effects.
Study Results
The study results are presented graphically in Figures 19
through 24. Figure 19 shows that 2,4,6—trichlorophenol does
not inhibit reaction at 1 or 10 mg/1. In Figure 20, data for
50 mg/1 and 100 mg/1 2,4,6—trichlorophenol, and for 1 mg/1
and 10 mg/1 copper are presented. All reactions presented in
the plot show significant inhibition of the biological reac-
tion. Figures 21 and 22 provide information on 4—chloro—3—
methyl phenol. The substrate is mildly inhibitory at lower
concentration (10 mg/1), strongly inhibitory at 50 mg/1, and
apparently toxic at 100 mg/1. Figure 23 presents data for
chloranil, which is inhibitory at 10 mg/1. Higher concentra-
tions were not studied because of solubility limitations.
The 2,4,6—trichloroaniline data shown in Figure 24 suggests
the material is not inhibitory up to 10 mg/1. Solubility
considerations precluded investigation at higher concentra-
tions.
N—chlorodimethylamine was originally considered for study but
an investigation showed that the compound was unstable, and
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degraded at a rate sufficiently high to make its persistence
in an effluent improbable.
- 72 -
-------
SECTION IX
STATIC BIOASSAYS - PHASE II
Routine static bioassays were conducted to determine the
day median tolerance limits of an appropriate test organism to
the test compounds. For purposes of this study, the TL was
established to within one order of magnitude.
The four-day static bioassays were conducted according to
Standard Methods procedures.
Chlorine-free tap water which had been vigorously aerating for
two days prior to the start of testing, was used as dilution
water. Fathead minnows (P i mepha 1 es promelas) were used as test
organisms. Three-gallon glass aquaria, each holding ten liters
of water, were used as bioassay containers. Three separate
series of static bioassays were conducted.
Series Number One
Four compounds were tested at three different concentrations
each: 2,4,6—trichlorophenol (TCP) and 4—chloro—3-methyl phenol,
(CMP), both at 100, 10, and 1 mg/1; 2,4,6-trichloroaniline
(TCA), at 10, 1, and 0.1 mg/1; and chloranil at 1, 0.1, 0.01
rag/1.
The two phenolic compounds were added to the test containers as
aqueous solutions. Two containers, each holding five test fish,
received one concentration, bringing the total to six containers
and thirty test fish for each of the two compounds. In addi-
tion, two containers, each holding five fish in straight dilu-
ent water, served as controls.
The other two compounds, 2,4,6—trichloroaniline and chloranil,
because of their relative insolubility in water, were added to
the test containers as methanol solutions. The facilities for
testing each of the two compounds were exactly as described
for the two phenolic compounds. Additional contrpls had to be
set up to determine the effects of the methanol that was added
with the compounds. Since the chloranil in methanol solution
was of such strength as to result in a 400 mg/1 methanol con-
centration in the test containers when the chloranil concentra-
tion was 1 mg/1, two control containers, each holding five fish
in 400 ppm methanol in diluent, were provided. The 2,4,6—tri-
chloroaniline in methanol solution resulted in an 80 mg/1 meth-
anol concentration when the TCA concentration was 10 mg/1.
Therefore, two additional control containers were set up for 80
mg/1 methanol in diluent.
Over the four-day period of testing, the percent mortality of
all control fish was zero.
- 73 -
-------
The 96-hour median tolerance limits of fathead minnows to each
of the compounds tested were: 2,4,6-trichlorophenol <1.0 >.l
mg/1; 4-chloro-3-methyl phenol at <.l >.01 mg/1; 2,4,6-tri-
chloroaniline at <10 >1.0 mg/1; and chloranil at <1.0 >.l mg/1.
The test results are presented in Figures 25 through 28. The
chloranil test fish exhibited a somewhat unusual reaction pat-
tern because of the insoluble nature of the compound. Despite
precautions, the chloranil flaked out when the methanol solu-
tion mixed with the diluent water, and floated or sank to the
container bottom. Some of the test fish were seen ingesting
these solid particles, predominantly at the 1 mg/1 concentration
level, and exclusively at the start of testing. The remaining
fish did not do the same, and consequently, survived the 4-day
period. At the lower concentrations, only a few fish were af-
fected. More fish died at the 0.01 mg/1 concentration than at
the 0.1 mg/1 concentration, probably because more solid parti-
cles of chloranil were ingested by those fish.
Series Number Two
One compound, p-benzoquinone, was tested at three concentra-
tions, 10, 0.5, and 0.1 mg/1. The compound was dispensed to
the test containers as an aqueous solution. Exactly the same
facilities, including controls, were used for benzoquinone as
were used for each of the phenolic compounds in Series Number
One.
All of the test fish died within the first day of testing over
the entire concentration range, while the control fish survived.
However, the control fish mortality was too great after 4 days
to satisfy Standard Methods criteria for test validity. It was
thought that perhaps the benzoquinone vaporized from some of
the test containers and contaminated the control diluent waters.
Consequently, a third bioassay was set up to restudy benzoqui-
none. In this study, the control containers were well separated
from the test containers. Despite the unsatisfactory behavior
of the control fish, this test did yield valid information. The
reaction of benzoquinone was immediate and violent. It was de-
cided, therefore, to test the compound at lower concentration
levels in Bioassay Number Three, starting with 0.1 mg/1.
Series Number Three
Benzoquinone was tested at three concentrations, 0.1, 0.01, and
0.001 mg/1. The same procedures as used in Bioassay Number Two
were followed, except that four fish per container were used.
The results of Bioassay Number Three are presented in Fiaure
29.
All of the test fish in the 0.1 mg/1 benzoquinone water died
within the first two days of testing, while the lower concen-
tration levels had no significant effect. However, the control
- 74 -
-------
100
75
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TIME- DAYS
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TIME- DAYS
FIGURE 25
RESULTS OF STATIC BiOASSAY SERIES NO.!
- 75 -
-------
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FIGURE 26
RESULTS OF STATIC BIOASSAY SERIES NO. I
(CONTINUED) - 76 -
-------
100
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FIGURE 27
RESULTS OF STATIC BIOASSAY SERIES NO. I
(CONTINUED) - 77 -
-------
!0.0 r-
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96 MR.TLM
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PERCENT MORTALITY IN 96 HR
A
4-DAY MEDIAN TOLERANCE
LIMIT DETERMINATION FOR
2,4,6-TRICHLOROPHENOL
CMP
96HP.TLV
MG/L
1.0
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0 20 40 50 60 80 100
PERCENT MORTALITY IN 96 HR.
B
4-DAY MEDIAN TOLERANCE
LIMIT DETERMINATION FOR
4-CHLORO-3-METHYL PHENOL
FIGURE 28
- 76 -
-------
100
t 75
t-
CONTROLS
50
t-
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12.5
25
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TIME-DAYS
P-BENZOQUINONE'O.IMG/L
P-BENZOQUINONE'O.OI MG/L
100
87.5
t 75^
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£ 25
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TIME - DAYS
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TIME - DAYS
P- BENZOQUINONE- 0.01 MG/L
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75
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25
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TIME - DAYS
FIGURE 29
RESULTS OF BiOASSAY N0.3-P-BENZOQUINONE
- 79 •
-------
fish again exhibited high mortality after 4 days and this test
did not meet the Standard Methods criteria for validity ei-
ther. However, control losses were not as drastic as in the
previous bioassay, and the lower concentration levels of the
compound seemed non-toxic. The 96-hour TL probably has a
value of about 0.005 to 0.03 mg/1, as seen in Figure 29.
Summary
Five chlorinated organic compounds were tested for toxicity to
fathead minnows. Of the five, four 96-hour median tolerance
limits were determined. For the fifth compound, p-benzoquinone,
a probable toxic concentration was determined. These values
are presented in Table 14.
TABLE 14
RESULTS OF STATIC BIOASSAYS
Compound Tested
2,4,6—trichlorophenol
2,4,6—trichloroaniline
4—chloro—3—methyl phenol
chloranil
Compound Tested
p-benzoquinone
TL
96—hour m Range (mg/1)
1.0 - O.I
10 - 1.0
0.1 - .01
1. - 01.
Probable Toxic Concentration Level
(mg/1)
< 0.1
- 80 -
-------
SECTION X
PH7VSE THREE
Selection of Study Compounds
As a consequence of study limitations, it was required to
limit the Phase III studies to three compounds. 4—chloro—3—
methyl phenol was eliminated in view of the experimental data
indicating the greater probability of occurrence of the
2,4,6—trichlorophenol. Chloranil was eliminated in view of
significant question as to whether the compound can actually
be formed under treatment plant conditions. The substrates
selected for Phase III study were:
2,4,6—trichlorophenol
2,4,6—trichloroaniline
p-benzoquinone
Flow Through Bioassay Studies
Static bioassay investigations, such as performed in Phase
II of the study have long been undertaken to evaluate toxi-
cities of test substrates. The ease of performance and low
cost of such studies are attractive, but many questions have
arisen concerning the translation of such results to proto-
type conditions. Ideally, a broader study considering many
aspects of the food chain over a longer period of time would
be desirable. The cost and facilities required for conduct-
ing large scale bioassays has sharply curtailed investiga-
tions of this nature. This project chose to consider an in-
termediate scale bioassay, flow through ecosystem that could
be operated at a reasonable cost, but would provide better
information on environmental response at several trophic
levels.
Fish, vascular plants, macroinvertebrates, and microscopic
plants and animals were investigated. Twenty gallon aquaria
with flow through water systems were chosen as the study
systems, and study periods of approximately one month per
study conduction were considered.
- 81 -
-------
SECTION XI
RESULTS OF PHASE III
Fish
Fathead minnows obtained from a local hatchery were employed
as a test organism. Die-off of the test fish occurred in
most of the experimental units during the background acclima-
tion periods and in the control units during the bioassays,
complicating data interpretation.
Figure 30 is a graphic representation of the number of test
fish surviving versus time, for each experimental unit. In
Table 15, the duration of the study is broken down into con-
venient periods and the percent mortality is given for each-
period and for the whole study.
TABLE 15
TABULAR SUMMARY OF FATHEAD MINNOW MORTALITIES
Control Units
Unit
1
Unit #
Benzo.
4
5
TCP
6
7
TCA
2
3
Percent Mortality*
Oct. - Feb. Mar. April
6 0 25
6 0 18
May - June
10
0
Units Subjected to Addition of Compounds
Percent Mortality*
Background
6
13
0
17
10
20
Cone. #1 Cone. #2Cone. #3
4
19
30
0
22
11
24
14
24
0
0
100
100
100
100
Total
37
23
Total
100
100
100
100
30
27
*percent of the remaining population entering a particular
stage (Concentration #1, etc.,) that died during that stage.
The control fish populations were stable from the beginning of
the study until the middle of April. From this time until the
beginning of May, a high rate of die-off occurred, reaching 25%
in one case. This can only be attributed to disease, or some
natural cause, since no contamination by any of the compounds
was shown in spectrophotometric analyses.
- 83 -
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OCT NOV DEC JAN FEB MAR APR ! MAY I JUN
FIGURE 30
FATHEAD MINNOW POPULATIONS VS. TIME
-------
The total percent mortality of the control fish through the en-
tire duration of the experiment varied from 23% to 37%. There-
fore, no significant toxic effect may be attached to lower mor-
talities in any of the test units.
TCA showed no effects on the test fish at any concentration
level. This is consistent with the findings of the four-day
static bioassay, since the 96-hour TL as determined by that
test was not exceeded or even reached in the flow-through
studies.
Benzoquinone and TCP exhibited no significant effect at either
of the two lower concentration levels. Even at the second
concentrations, which compared with the 96-hour TL 's, the
mortality was less in these test units than in the controls.
This would seem to indicate either a greater resistance of the
test fish to both of these compounds when in a more natural
environment or the degradation or assimilation of the com-
pounds within the aquaria. Spectrophotometric measurements
indicated that some reduction in concentration did take place
in the units. A comparison between applied concentrations and
observed concentrations is presented in Table 16.
TABLE 16
CONCENTRATIONS (mg/1) OF SUBSTRATES DURING
VARIOUS TEST PERIODS
Period
SubstrateI2
Applied Measured Applied Measured Applied Measured
p-benzo- 0>01 (1) Q.108 0.006 1.15 0.23
quinone
2,4,6-
trichlor- _ _ 0.135 NA
oaniline
(TCA)
2,4,6-
0.06 (1) 0.47 0.25 4.15 1.75
J_
(TCP)
below measureable range.
When the third concentration levels of benzoquinone and TCP,
values ten times greater than their 96-hour TL 's, were dis-
m
pensed, the reaction of the test fish was immediate and vio-
lent. Within a few days after starting these bioassays, 100%
kill was effected in all four test units. Compound measure-
ments indicated that these lethal concentrations were some-
what less than half of the nominal levels delivered to the
aquaria.
- 85 -
-------
Vascular Plants
The growth of two vascular plants, Ludv i g i a and Anacha r i s ,
was studied in detail. The total length of all Ludv i g i a
stalks in each test aquarium was 35 to 40 inches at the be-
ginning of the study, and had increased to 75 to 100 inches
by the middle of May for the control, benzoquinone, and TCP
units. The plants in the TCA units had grown to over 100
inches by the end of May. None of the test compounds affected
the health and growth of Ludv i g i a at any concentration level.
The growth rates of Anacha r i s varied so extremely among the
aquaria during the background period of the study that compar-
ability of the units to one another was severely hampered. As
stated in Table 17, the average growth rates varied from less
than one inch per week to more than seven inches per week.
Prior to the start of the bioassays, the Anacha r i s plants were
re-distributed among the eight units so that each contained
approximately the same number of inches of total stalk length.
TABLE 17
VASCULAR PLANTS GROWTH
Average growth rate in Average growth rate in
inches per week* inches per week*
ni - over background period over bioassays periodt
Ludv i g i a Anacha r i s Ludv i g i a Anacharis
Control unit 1 1.1 0.9 3.5 2.4
Unit 8 0.9 1.7 3.8 5.7
Benzoquinone
Unit 4 1.0 7.1 4.9 1.8
Unit 5 0.9 4.5 3.0 3.0
TCP
Unit 6 0.8 3.2 3.4 5.4
Unit 7 0.8 5.6 4.1 6.3
TCA
Unit 2 1.6 3.2 5.7 6.9
Unit 3 1.3 4.3 4.7 5.1
*That is, the total number of inches of new growth contri-
buted by all six main stalks.
fFrom beginning of Cone. 1 to end of last Cone.
During the bioassays, the average growth rates of Anacha r i s in
the benzoquinone test aquaria were the slowest, showing a de-
crease from the background rates. The growth rates increased
in all other units. However, the control plants in Unit #1
- 86 -
-------
still exhibited very slow growth, less in fact than benzoqui-
none Unit #5. Also, benzoquinone Unit #4 suffered from the re-
distribution of plants, since it was given some of the slowest
growing stalks from Unit #1.
In summary, the inconsistancy of Anacha r i s growth proved to be
too great to use this plant as an indicator of compound effects.
It was observed, however, that none of the compounds affected
the general appearance of outward health of Anacha r i s.
Benthic Macroinvertebrates
The macroinvertebrate populations underwent so sharp a decrease
over the course of the background acclimation period as to ren-
der them unsuitable as test organisms. As shown in Figure 31,
out of 93 individuals (excluding Tub i fex) present in each aqua-
rium at the time of initial seeding, an average of only 15 per
aquarium could be observed in mid-March, a decrease of 84%.
The average Hyd ropsych i dae population decreased 90%, from 31
to 3 individuals; A s e 1 1 u s decreased 77%, from 13 to 3; Ectopr ia
69%, from 13 to 4; Gyraulus 79%, from 14 to 3; Helobdella 88%,
from 17 to 2; and Perr i ss i a 100%.
Several reasons for this drastic decline can be identified.
Some of the organisms avoid observation, either by burrowing
into the benthic sediments when observations were made, or by
burrowing into the sand matrix. Some of the organisms are
consumed by the test fish. The fish were not the only preda-
tory stress on the macroinvertebrates. Ase 11 us is a potential
predator on all of the other animals, and He 1obde 1 la preys
upon Gyraulus and Per riss}a. Since no effort was made to sep-
arate the various types of macroinverbrates from one another,
Helobde11 a and Ase11 us may have consumed some of the other
organisms.
Adequate food supplies may have been lacking. Specifically,
the microflora and fauna populations may have been too low to
support the macroinvertebrates during the background acclima-
tion period. The lack of nutrients in the diluent water may
have too severly limited the rapid growth of algae necessary
for a productive ecosystem.
Fish waste products and other detritus built up in the aquaria
with time, settling on the benthos in thickening sludge layers.
Perhaps the gases of decomposition released through bacterial
decomposition of this waste proved injurious to the macroinver-
tebrates. This bacterial activity also may have depleted the
dissolved oxygen resource at the benthos-water interface.
Finally, the marked drop in the caddis fly larva population was
influenced by a special factor unique to Hydropsych i dae, its
annual metamorphosis from larva to pupa to adult. Only the
larva may be used as a test organism, since it is active in
- 87 -
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water, while the pupa is immobile within its cocoon and the
adult is a terrestrial organism. In several instances from
February to March, immobile cocoons and adults struggling to
leave the water were observed. A significant portion of the
Hyd ropsych i dae population may have undergone metamorphosis
at this time.
Microscopic Flora and Fauna
A total of six sets of population counts were made, three of
two-week populations, and three of four-week populations.
The significance of each of these sets is listed below:
Four-week set No.l. — compares control
populations of April 5 to benzoquinone
and trichlorophenol populations devel-
oping at the end of the first concen-
tration levels of these compounds;
Four-week set No.2 — controls on May 5;
benzoquinone and TCP at the end of the
second concentration levels; and TCA near
the end of the first concentration level.
Four-week set No.3 — controls on June 1;
benzoquinone and TCP approximately one
week after the third concentration levels
ended; and TCA about mid-way through the
second concentration level;
Two-week set No.l — controls on April 20;
benzoquinone and TCP mid-way through the
second concentration levels; and TCA about
mid-way through the first concentration
level.
Two-week set No.2 — controls on May 20;
benzoquinone and TCP near the end of the
third concentration levels; and TCA at the
beginning of the second concentration level.
Two-week set No.3 — controls on June 15;
and TCA just after the second concentration
level ended.
The data from the microscopic observations program are summar-
ized in Figures 32 through 38. For each pair of parallel ex-
perimental units, four population counts were made during each
set of observations. The maximum, average, and minimum popula-
tions of each set were recorded.
Figures 32 and 33 are bargraphs of the average microfauna pop-
ations in each set of four-week and two-week observations, re-
spectively, and each population is broken down into its compo-
nents. Although the control populations exhibited considerable
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TOTAL MICROFAUNA POPULATIONS
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2 -WEEK STALKED
CILIATE POPULATIONS
\__
i
It
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v_
.2 SET NO. 3
4-WEEK STALKED
CILIATE POPULATIONS
X -CONTROL
0 -BENZCQUINOME
D -T C P
A-T C A
(SEE TABLE 16 FOR
^ CONCENTRATIONS)
N X
A
A
\ x Q p- — ^
2 SET NO, 3
FIGURE 35
STALKED CILIATE POPULATIONS
-------
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4-WEEK MOTILE
CiLIATE POPULATIONS
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(SEE TABLE 16 FOR CONCENTRATIONS)
X--
SET NO. 3
_M_AX. f~
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SET NO. 3
FIGURE 36
MOTILE CILIATE POPULATIONS
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DIATOM POPULATIONS
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BLUE-GREEN
ALGAE
POPULATIONS
SET NO 2
-
NO. 3
SET NO.I
SET NO. 2
SET NO. 3
FIGURE 33
BLUE-GREEN ALGAE POPULATIONS
- 96 -
-------
variation from one set to another, certain significant devia-
tions occurred in some of the test populations. Most notably,
both the four-week and the two-week benzoquinone populations
show a decline at the end of the second concentration level,
but especially during and after the third level, due primar-
ily to the decrease in stalked ciliates. The average four-
week control stalked ciliate population reaches a minimum of
50 individuals per square centimeter or 37% of the microfauna
population, while the benzoquinone population drops to 5 in-
dividuals or 12% in observation set No.3. In a similar fash-
ion, the average two-week benzoquinone stalked ciliate popu-
lation drops to 5 individuals or 5% in observation set No.2,
while the control minimum is 50 individuals, or 23%. It would
seem then, that benzoquinone has an inhibitory effect on this
organism at a concentration level of 1 mg/1 (concentration No.
3) and possibly at a concentration as low as 0.10 mg/1 (concen-
tration No.2).
While the decline in the TCP total microfauna population is not
as marked, the TCP stalked ciliate population drops to a mini-
mum of 10 individuals in four-week observation set No.3 and
two-week observation set No.2, being 12% and 8% of those mic-
rofauna populations, respectively. Thus, TCP seems to affect
this organism at a concentration of 4 ppm (concentration No.3).
The TCA microfauna populations compare favorably to the con-
trols although the four-week stalked ciliate population does
decline in set No.3 to 15 individuals or 14%, indicating a
possible inhibitory effect at the second concentration level
(1.4 ppm).
In contrast to the stalked ciliates, the control motile cili-
ate population is quite stable over the course of the bioassays,
Only one instance of significant deviation in the test motile
ciliate populations occurs. The four-week benzoquinone popula-
tion declines to a minimum average of 10 individuals per square
centimeter in observation set No.3, as compared to a control
minimum average of 30 individuals, indicating a possible inhib-
itory effect on this organism of the third concentration level.
The rotifer, amoeba, gastrotrich, and nematode populations fluc-
tuated to such a great degree and were so small in comparison
to the ciliate populations, as to make evaluation of possible
compound effects insignificant.
Figures 34, 35, and 36 plot the maximum, average, and minimum
populations of microfauna, stalked ciliates, and motile cili-
ates, respectively, for all observation sets. Note the decline
in microfauna and stalked ciliate populations in the TCP and
benzoquinone test units, and the general stability of the mo-
tile ciliate populations except for benzoquinone in four-week
observation set No.3. The data on microfauna populations would
- 97 -
-------
seem to indicate inhibitory effects of the compounds, espe-
cially with regard to stalked ciliates, at the following nom-
inal concentration levels:
benzoquinone at 1 ppm (concentration 3)
and possibly 1/10 ppm (concentration 2);
TCP at 4 ppm (concentration 3) ; and TCA
at 1.4 ppm (concentration 2).
There appeared to be no effects on the microfauna of any of
the compounds at lov/er concentration levels.
Four categories of microflora were observed in the experimen-
tal aquaria: diatoms, blue-green algae, green algae, and yel-
low-brown algae. Possible inhibitory effects were seen on two
of these, namely, the diatom populations and the blue-green
algae populations.
Figure 37 plots the maximum, average, and minimum diatom pop-
ulations in all observation sets. The four-week control pop-
ulations were exceptionally stable throughout the duration of
the bioassays, ranging from 17,000 to 65,000 cells per square
centimeter with, an average of 47,000. The four-week benzoqui-
none populations exhibited a steady decline, finally deviating
significantly in observation set No.3, with an average of only
9,000 cells and a maximum of 13,000. TCP exhibits a similar
trend, declining to an average of 8,000 and a maximum of 10,000.
TCA diatom populations remained somewhat higher than the control
populations in both sets of four-week data.
The two-week control diatom populations were also stable, with
a maximum of 40,000, a minimum of 9,000, and an average of
25,000 cells per square centimeter. In observation set No.2,
both the benzoquinone and the TCP diatom populations averaged
only 5,000 cells with maximum of 7,000. The TCA populations
were again somewhat higher than the controls, except in set
No.3, with an average and a maximum of 9,000 and 18,000, re-
spectively.
Thus, there is inconclusive evidence of inhibitory effects
on diatoms of benzoquinone at 1 ppm (concentration 3), and
possibly 1/10 ppm (concentration 2), TCP at 4 ppm (concentra-
tion 3), and TCA at 1.4 ppm (concentration 2).
The control populations of blue-green algae, as evident in
Figure 38, were extremely variable, reaching a maximum of
more than one million cells and a minimum of 25,000 cells in
four-week populations. The most significant observation
lies in the fact that, while tremendous blue-green algal
blooms occurred in the controls, in general the populations
remained low in the test units. It is not known, however, if
this was due to the action of the compounds, or to some phys-
ical vagary in the controls at the time of the blooms.
- 98 -
-------
In summary, the data from the microscopic observation program
indicate that all three compounds, especially benzoquinone and
TCP, may possibly inhibit diatoms and stalked ciliates at the
highest concentration levels of the compounds. Additional
evidence suggests that benzoquinone may inhibit these microor-
ganisms at its second concentration level. The variations in
basic population in the control unit were significant, and a
detailed statistical examination of the population variance
would be necessary to quantitatively define the impact of the
various test compounds. To carry out such a study would re-
quire the collection of a large number of observations, and
would be beyond economic practicality.
Summary
Substantial insight into the validity of the intermediate-term,
flow-through bioassay as a method for determining toxicity was
gained. Laboratory facilities incorporating model freshwater
ecosystems in a flow-through system were designed. Methods
for testing compounds and monitoring the ecosystems' response
to compounds were developed. Three chlorinated organic com-
pounds were investigated for toxicity to a variety of aquatic
test organisms.
It was found that the test fish, p i mepha1es promelas, was able
to tolerate higher concentrations of p-benzoquinone and 2,4,6-
trichlorophenol in the flow-through studies than in the four-
day static bioassays. As a result of its limited solubility
in water, 2,4,6-trichloroaniline could not be dispensed to the
model ecosystems at a concentration level as high as that de-
termined to be lethal in the static tests. The variation in
control fish populations during the flow-through bioassays
made it impossible to determine sub-lethal effects of the
compounds.
Qualitative analysis of microorganism population data suggested
inhibitory or toxic effects on diatoms and stalked ciliates of
the three compounds at the highest concentration levels tested.
However, the instability of the control microorganism popula-
tions prevented valid statistical treatment of the data. A
great increase in effort, well above that practical for a pro-
ject of this nature, would be necessary before strict defini-
tions of toxocity to microorganisms could be determined.
Of the three species of rooted vascular plants tested, only
L u d v i g i a exhibited an interpretable growth pattern. Compari-
sons between test and control plants indicated no toxicity of
any of the compounds tested. No adverse effects on the out-
ward appearance of any of the three species was observed.
The experimental ecosystems were unsuitable for the mainten-
ance of stable macroinvertebrate populations for a duration
sufficiently long for compounds testing.
- 99 -
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Although considerable information of a qualitative nature was
gathered, the flow-through studies did not yield reliable
quantitative data from which conclusive determinations of toxi-
city could be made.
Considering the relative efforts involved, the intermediate-
term, flow-through bioassay did not compare favorably to the
routine static bioassay as a reliable experimental method.
- 100 -
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SECTION XII
ACKNOWLEDGEMENTS
This project was undertaken by Hydroscience, Inc., under the
direction of Edwin L. Barnhart. The experimental studies
completed during the various phases of the project were con-
ducted under the immediate supervision of George R. Campbell,
Dr. George J. Kehrberger, and Michael J. Scherer. Dr. Charles
Wurtz served as consultant during the ecosystem studies.
The participation of the Manufacturing Chemists' Association
(MCA) Water Resources Committee, and the Water Quality Office
of the Environmental Protection Agency is gratefully acknow-
ledged.
The investigators are particularly indebted to Dr. Hend Gorchev
of the Environmental Protection Agency, Messrs. Best, Brown,
and Roznoy, and Doctors Marks and Freedman of the MCA Committee,
for their invaluable suggestions and assistance during the course
of the study.
This report is submitted in fulfillment of project number 12020
EXG, under the partial sponsorship of the Water Quality Office,
Environmental Protection Agency.
- 101 -
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SECTION XIII
REFERENCES
1. Ingols, R.S., and Jacobs, G.M., "BOD Reduction by Chlor-
ination of Phenol and Amino Acids," Sewage and Industrial
Wastes, 2£, No.3, pp.258-262 (1957).
2. Chambers, C.W., et.al., "Degradation of Aromatic Compounds
by Phenol-Adapted Bacteria", Jour. W.P.C.F., 35, No.12,
pp.1517-1527 (1963).
3. Ingols, R.S., et.al., "Biological Activity of Halophenols",
Jour. W.P.C.F., 38, No.4, pp.629-635 (1966).
4. Hodgman, C.D., et.al., (Editors), Handbook of_ Chemistry
and Physics, 42nd Edition, The Chemical Rubber Publishing
Company, Cleveland(19601.
5. Standard Methods for the Examination of_ Water and Waste-
Water , 13th Edition. A.P.H.A., New York, (197lTT~
6. Roberts, J.D., and Caserio, M.C., Basic Prinicples of Or-
ganic Chemistry, W.A. Benjamin Company, New York, (T9"65~T.
7. Ettinger, M.B., and Ruchoft, C.C., "Effect of Stepwise
Chlorination on Taste- and Odor- Producing Intensity of
Some Phenolic Compounds", Jour. A.W.W.A., 43, pp.651ff
(1951).
8. Ingols, R.S., and Ridenour, G.M., "The Elimination of
Phenolic Tastes by Chloro-Oxidation", Water and Sewage
Works, 95_, pp,187ff (1949).
9. Burttschell, R.H., et.al., "Chlorine Derivatives of
Phenol Causing Taste and Odor", Jour. A.W.W.A., 51, pp.
205-214, (1959).
10. Lee, G.F., "Kinetics of Reactions Between Chlorine and
Phenolic Compounds", in Principles and Applications of
Water Chemistry, Faust and Hunter (Editors),John WiTey
and Sons, New York, (1967).
11. Eisenhauer, H.R., "Oxidation of Phenolic Wastes", Jour.
W.P.C.F., 36, No.9, pp.1116-1128 (1964).
12. Weil, I., and Morris, J.C., "Kinetic Studies on the
Chloramines. I. The Rates of Formation of Monochloramine,
N—Chlormethylamine, and N—Chlorodimethylamine", Journal
A.C.S., 71., pp.1664-1671, (1949).
13. Marks, H., by private communication.
- 103 -
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
/. Report No. i
4. Title
EFFECT OF CHLORINATION ON SELECTED ORGANIC CHEMICALS
7. Author(s)
Barnhart, E.L.
rnr^Hg'T ~\ C-, . P
9. Organization
Hydroscience, Inc., Westwood, New Jersey 07675
12. Sponsoring Organization
3. Accession No.
w
5. Report Date
6.
8. Performing Organization
Report No.
10. Project No.
12020 EXG
11. Contract/Grant No.
Type of Report and
Period Covered
15. Supplementary Notes
Submitted to the Water Quality Office, Environmental Protection Agency,
by the Manufacturing Chemists' Association, Washington, D.C. 20009
16. Abstract
Fourteen industrial organic chemicals were examined for their persistence
through biological treatment, either as the initial compounds, or as
degradation products. Semi-continuous activated sludge systems were em-
ployed. The ability of each of the chemicals to participate in reactions
with free chlorine was then determined in a series of batch experiments.
It was found that certain of the test compounds formed persistent degrada-
tion products during treatment. Five of the initial compounds reacted
readily with chlorine, under conditions commonly employed in effluent
chlorination.
Five of the chlorination products were further studied in respirometer ex-
periments to evaluate their persistence in mixed microbial systems. Their
toxicity to fish was determined using the static bioassay procedure,
In the final phase of the study, a series of bench scale, continuous flow
ecosystems were established for the evaluation of longer term effects of
three of the chlorination products. Several varieties of organisms, rep-
resenting different levels in the food chain, were studied.
17a. Descriptors
*Chlorination, *Chemical Reactions, *Biodegradation, *Bioassay,
*Ecosystems, *Toxicity
17b. Identifiers
*0rganic Chemicals, *Activated Sludge, *Degradation Products,,
*Chlorination Products, *Respirometer Studies, *Continuous Flow
Ecosystem
17C.COWRR Field* Group
18. Availability
19. Security Class.
(Report)
20. Security Class.
(Page)
21. No. of
Pages
22. Price
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
US DEPARTMENT OF THE INTERIOR
WASHINGTON. D C 20240
Abstractor George R. Campbell
\institution Hydroscience, Inc.
WRSIC 102 (REV JUNE 1971)
9(3.2«|
OUS GOVERNMENT PRINTING OFFICE 1972 484-486/271 1-3
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