&EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
tPA 600 1-79 009
i ohm.irv 1979
Research and Development
Pyrogenic Activity of
Carbon-Filtered
Waters
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EPA-600/1-79-009
February 1979
PYROGENIC ACTIVITY OF CARBON-FILTERED WATERS
by
Harold W. Wolf
Bennie Joe Camp, and
Scott J. Hawkins
Texas A&M University
College Station, Texas 77843
and
James H. Jorgensen
University of Texas Health Science Center
San Antonio, Texas 78284
Grant No. R-804420
Project Officer
Herbert R. Pahren
Field Studies Division
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
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FOREWORD
The U.S. Environmental Protection Agency was created because of increas-
ing public and government concern about the dangers of pollution to the health
and welfare of the American people. Noxious air, foul water, and spoiled land
are tragic testimony to the deterioration of our national environment. The
complexity of that environment and the interplay between its components re-
quire a concentrated and integrated attack on the problem.
Research and development is that necessary first step in problem solution
and it involves defining the problem, measuring its impact, and searching for
solutions. The primary mission of the Health Effects Research Laboratory in
Cincinnati (HERL) is to provide a sound health effects data base in support of
the regulatory activities of the EPA. To this end, HURL conducts a research
program to identify, characterize, and quantitate harmful effects of pollutants
that may result from exposure to chemical, physical, or biological agents found
in the environment. In addition to the valuable health information generated
by these activities, new research techniques and methods are being developed
that contribute to a better understanding of human biochemical and physiologi-
cal functions, and how these functions are altered by low-level insults.
This report describes the endotoxin content and pyrogenic response found
in granular activated carbon filtered waters. With a better understanding of
any health effects, measures can be developed to reduce exposure to potentially
harmful materials.
R. J. Gainer
Director
Health Effects Research Laboratory
m
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ABSTRACT
The endotoxin content and pyrogenic response of granular activated carbon
(GAC) filtered waters were studied. GAC-filtered secondary effluent from an
activated sludge pilot plant contained free endotoxins in the range 6-250 yg/1
yielding positive pyrogenic responses in 18 of 20 trials. Samples obtained
from 27 different water supplies in the U.S. that utilize GAC adsorption con-
tained free endotoxin ranging from 1.2-25 yg/1 but none gave a pyrogenic re-
sponse. No relationship was discernible between endotoxin content and pyro-
genic response.
Small removals of total organic carbon (TOC) by GAC beds which had been
in operation in water treatment plants without regeneration for as long as
110 months were observed. However, 5 of 28 samples showed an increase in TOC
through GAC and 8 of 28 samples showed an increase in standard plate count.
One of 25 samples yielded pseudomonads, but none of the 28 samples contained
coli forms.
Good correlations were observed on non-disinfected AWT effluent samples
between standard plate count and total endotoxin (r = 0.945), standard plate
count and free endotoxin (r = 0.932), and total coliforms and free endotoxin
(r = 0.939). Lack of good correlations, however, were observed in assaying
AWT samples that had been subject to the disinfecting procedures of chlori-
nation, ozonation, pH or UV irradiation.
This report was submitted in fulfillment of Grant No. R-804420 by Texas
A&M University, under the sponsorship of the U.S. Environmental Protection
Agency. This report covers the period March 9, 1976 to July 31, 1978, and
work was completed as of August 29, 1978.
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CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
Acknowledgments ix
1. Introduction 1
2. Conclusions 2
3. Recommendations 10
4. Experimental Procedure 11
5. Analytical Procedures 16
6. AWT Plant Results 23
7. Water Treatment Plant Results 50
References 65
Appendices
A. AWT Effluent Samples' Analyses 67
B. Carbon Column Samples' Analyses 75
C. AWT Effluent Disinfection Samples 78
D. Water Treatment Plant Samples 85
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FIGURES
Number Page
1 Exterior view of sample kit 13
2 Interior views of sample kit arranged to hold
four 500-ml sample bottles 14
3 Flow diagram of the Demonstration Plant at the
Henry J. Graeser Environmental Research and
Training Facility, Dallas, Texas 24
4 Endotoxin equivalents vs. bacteriological assays ...... 28
5 Rabbit in stanchion 40
6 Rear of three stanchions shown mounted in inhalation
chamber which also shows plastic aerosol exit pipes .... 40
7 Two plastic aerosol entrance pipes below paperboard
deflector 42
8 Exhaust pipe connected to chemical hood 42
9 Aerosol chamber with three thermistors ready to
receive rabbits 43
10 Standard plate count after GAC vs^. change in TOC 53
11 TOC removal in mg/1 vs. months of operation of GAC 59
12 TOC removal in percent vs. months of operation
of GAC 60
13 Comparison of TOC removals observed with
Cincinnati pilot-plant data 61
14 Percent TOC removal vs. empty-bed contact time 63
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TABLES
Number Page
1 Endotoxin Content of Certain Municipal Drinking
Waters, Mississippi River Water, the Gulf of
Mexico, and Adjacent Bays 3
2 Results of Limulus Tests on Drinking Water Samples 5
3 Results of Limulus Tests onAWT Water Samples 6
4 Results of Standard Plate Counts, Total Coliform
Counts, and Endotoxin Determinations on 48 AWT
Samples 25
5 COD Concentrations and Endotoxin Activities of
AWT Waters Before, at the Midpoint, and After
GAC Filtration 30
6 Total and Free Endotoxin Activities of Different
Water Samples Subjected to Four Disinfection
Procedures 33
7 Increase in Total Endotoxin Activity of GAC
Product Water with Time 34
8 Effects of Shipment on Some of the Important
Variables of the Disinfection Process 35
9 Effects of Shipment on Bacteriological Qualities
of Water Samples 36
10 Coliphage and Coliform Assays of AWT Water Samples
After Receipt in College Station 38
11 Results of Ingestion Exposure 39
12 Hematological Assay Values of Individual Rabbits
Prior to I.V. Injection with Membrane-Filtered
AWT Samples 45
13 Hematological Assay Values of Individual Rabbits
Following I.V. Injection with Membrane-Filtered
AWT Samples 46
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14 Pyrogenic Response of Individual Rabbits Following
I.V. Injection with Membrane-Filtered AWT Samples 47
15 The Pyrogenic Results of Free Endotoxin-Containing
AWT Samples 49
16 Bacteriological Results of Water Treatment
Plant Samples Before and After GAC Filtration 51
17 TOC Concentrations and Endotoxin Activities of
Water Treatment Plant Samples Before and After
GAC Filtration 54
18 Operating Data for GAC in Water Treatment Plants
Sampled 56
19 TOC and Endotoxin Performance of GAC with Time 57
20 Monitoring Methods Utilized for GAC Control 64
VI 11
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ACKNOWLEDGMENTS
This report is the product of the coordinated efforts of many
individuals. Among those who deserve special recognition are Dr. Albert
C. Petrasek, Jr., who was Director of the Henry J. Graeser Environmental
Research and Training Facility in Dallas, Texas, and Mr. Allen L. .Messenger,
Environmental Science and Engineering, Inc., Austin, Texas, who as a,.
graduate student fulfilled several unanticipated roles. Our heartfelt
thanks to both.
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SECTION 1
INTRODUCTION
The efforts of U.S. Environmental Protection Agency (EPA) programs
relating to water pollution control and drinking water safety are resulting
in increased emphases on conservation of the water resource and its reuse--
but with an over-riding and dominant concern for protection of the public
health. Among the agents in water of health concern are some of the organic
chemicals -- particularly those commonly grouped as trihalomethanes (THMS)
and as synthetics (chemical products produced by man). The unit process
employed in water and waste water treatment that appears most promising in
providing for a greater degree of control of these unwanted organic chemical
substances is activated carbon adsorption.
Activated carbon has a long history of use in both water and waste
water treatment. It is available in powdered and granular forms. The
powdered form has found its greatest use in water treatment. Granular
activated carbon (GAC) has been the form most used in waste water treatment,
but this use has been largely restricted to pilot studies. Full-scale plant
use of GAC is rare in waste water treatment, Garland, Texas, being a notable
example.
Although the GAC function is primarily adsorptive, the observation of a
concomitant biological activity has long been observed. In fact, the obser-
vation of some denitrification occurring in GAC adsorption columns at the
Pomona, California, advanced waste treatment (AWTJ pilot plant gave rise to
successful experiments designed to enhance this biological process by pro-
viding a cheap carbon source via the addition of stoichiometric amounts of
methanol. The use of biological processes to treat waste water is common,
but the use of such processes to treat, drinking water (i.e., water for drink-
ing purposes) raises many apprehensions, A whole generation of engineers
schooled in the physical-chemical treatment, of water has been produced; few
have aquaintance witn the former use of slow sand filters in water treatment
which process is to a great extent biological.
In addition to this general apprehension, there is the caution express-
ed by Dr. Joshua Lederberg, Nobel laureate geneticist from Stanford Univer-
sity, who reminded the National Drinking Water Advisory Council of the EPA
that man still does not have a complete understanding of the carbon cycle.
Nevertheless, he also encouraged the thorough research and application of
adsorptive processes as the most promising tools available to obtain better
control of unwanted organic substances in our drinking water supplies.
The observation of substantial biological activity in GAC treatment of
waste water- does not prove that a similar degree of such activity will occur
1
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when treating drinking water. Source waters used for potable supplies have
considerably smaller amounts of biodegradable materials than do biologically
treated waste waters. Also, water treatment processes frequently employ
preliminary disinfection (prechlorination) as well as coagulation, floccula-
tion, and sedimentation techniques with the result that the waters that would
reach a GAG process would be of considerably higher quality than in a waste
water application. Nonetheless, some degree of biological development in a
water treatment application of GAC would be certain.
One of the main concerns about biological growth in GAC filters or beds
is the potential for generation of unwanted microorganisms arid/or their
toxic by-products. Among the microorganisms that might become established
are the pseudomonads. They are known for their ubiquity, growth in minimal
media, and their importance in denitrification (1). Also, some strains are
opportunistic pathogens of man.
Among the toxic by-products to be aware of are the gram-negative endo-
toxins. Gram-negative endotoxins are lipopolysaccharides which are all
thought to cause a pyrogenic response (fever) when injected into animals.
The rabbit is used in the standard United States Pharmacopeia (USP) pyrogen
test (2), but man can reportedly be 100-fold more sensitive to endotoxins
than is the rabbit (3).
Although some lipopolysaccharides will elicit a toxic response in some
individual animals when ingested in sufficient amounts, it is supposed to
occur only as a result of an increased permeability of the gastrointestinal
tract. Contention apparently exists about this point since it is reported
that the observation of absorption of endotoxin by the normal bowel is
disputed (4). Certainly, the normal bowel of most mammals contains a
generous supply of endotoxins (5).
Just as different animals possess different sensitivities to endotoxin,
so do different bacteria produce toxins of different toxicity. The recently
developed Limulus Amebocyte Lysate (LAL) assay for gram-negative endotoxins
includes within its measure lipopolysaccharides from organisms which are not
particularly endotoxic. Originally described by Levin and Bang (6), the
assay can detect as little as 1 nanogram of bacterial endotoxin per milli-
liter (or 1 yg/1) in a period of less than two hours. The test is reportedly
simple, specific, and rapid, and inexpensive when compared to the USP rabbit
pyrogen test (2). It has found use in detecting clinical endotoxemia as
well as for the detection of bacterial endotoxins (pyrogens) in biological
products and other drugs or fluids for parenteral administration to man
(7,8).
The LAL assay was first described in an environmental application by
DiLuzio and Friedmann (9). They had observed gram-negative endotoxin
contamination of their dionized water, then found endotoxins in New Orleans
tap water, and -- curiosity getting the better of them -- went on to examine
a host of different waters. Their results are reproduced in Table 1.
Additionally, they reported Mexico City tap water at 800 yg/ml, a commer-
cially bottled water from Mexico City as negative, random samples of beer,
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TABLE 1. ENDOTOXIN CONTENT OF CERTAIN MUNICIPAL DRINKING WATERS,
MISSISSIPPI RIVER WATER, THE GULF OF MEXICO, AND ADJACENT BAYS (9)
Sample tested
Drinking water
Baltimore, Md.
Chicago, 111.
Denver, Colo.
Galveston, Texas
Harrisburg, Pa.
Hazelton, Pa.
Kalamazoo, Mich.
Knoxville, Tenn.
Las Vegas, Nev.
Little Rock, Ark.
Los Angeles, Cal.
Memphis, Tenn.
Mobile, Ala.
Nashville, Tenn.
New Orleans, La.
Riverside, 111.
San Francisco, Cal
Washington, DC
Surface water
Water source
Endotoxin
Endo- concentration
toxin (ug ml'1)
Reservoirs
Lake Michigan
South Platt River and
streams from Bear
Creek
Artesian wells
Reservoir
Reservoir
Artesian well
Fort Loudoun Lake
Lake Mead (60%)
Wells (40%)
Lake Maumelle
Lake Winnoa
Wells (12%)
Colorado River (10%)
Owen's River aqueduct
from Mammouth Lake
(77.7%)
Artesian well
Reservoir
Cumberland River
Mississippi River
Chicago water supply
and 2 wells
Hetchy Reservoir
Potomac River
10
Barataria Bay, La.
Gulf of Mexico*
Little Dauphin Island Bay, Ala.
Mississippi River (surface levels)
Mississippi River (deep levels)
Mobile, Ala.(reservoir)
1
1
10
20
200
20
130
400
80
Samples were obtained between June and September 1972.
* Gulf of Mexico sample obtained off Grant Isle, La.
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cola drinks, and wine as negative, one brand of local commercial bottled
water as positive, another brand derived from a 610-M (2000-ft) deep
artesian well as negative, and milk at 30 to 130 ug/ml which rose sixteen-
fold after 24 hours at room temperature but which showed no rise when
refrigerated. The authors predicted value in use of the procedure for
monitoring water, milk and other beverages, and biological solutions.
Jorgensen, Lee, and Pahren (10) applied the LAL procedure to drinking
waters and to AWT effluents. Their tabular results are also reproduced
herein, drinking water samples in Table 2, and AWT water samples in Table 3.
Sodium thiosulfate was not added to any of the drinking water samples. The
lowest endotoxin activity shown for drinking water samples is from Miami
which also was the only ground water supply sampled.
All AWT samples were frozen prior to mailing. The only AWT samples in
Table 3 free of endotoxin are from Escondido which uses reverse osmosis as a
final treatment. Blue Plains uses breakpoint chlorination following GAG
filtration and had relatively low endotoxin levels. Samples from Pomona
treated by ozone did not differ substantially from samples treated by
"normal" chlorination., The highest endotoxin-containing samples -- both
from the Lake Tahoe plant -- were mailed to Cincinnati instead of to San
Antonio and because of the lengthy time in transit getting to the proper lab,
the results are not comparable with the other sample results.
The authors described variability in the potency of the amebocyte
lysate preparation, problems of sample shipment, failure to perform bacteria
plate counts, and failure to discriminate between bound and free endotoxin
as their greatest shortcomings. Bound endotoxin is described as endotoxin
remaining in association with the cell wall of viable bacteria, and free
endotoxin is endotoxin that has been solubilized without autolysis or
disruption of the cells. Further, the authors state that bound endotoxin
can be used as a means of quantifying the number of bacteria present. A
linear relationship exists between the number of cells and bound endotoxin
over the range 103 to 106 bacteria per ml. Two of the most recent papers
published utilize endotoxin methods to describe biomass and bacterial counts
(11,12).
The study reported herein attempts to fill in some of the shortcomings
while striving for additional objectives, vj_zj
1) To quantify pyrogenic activity of waters following GAG
filtration in terms of capacity to initiate a fever via
injection, ingestion, and inhalation in experimental animals,
and
2) To observe the relationships of pyrogenicity with bacterial
counts and the endotoxin assay.
3) To determine the effects of treatments on the pyrogenic or
endotoxin results and
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4) To observe potential effects of i.v. injection on the animals'
hernodynamic system.
The study commenced with AWT waters and then progressed to the exami-
nation of drinking water samples obtained from water treatment plants that
utilize GAC filtration.
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SECTION 2
CONCLUSIONS
1. The injection of drinking waters of varying endotoxin content (1.2 to 25
vig/1 assay) failed to demonstrate pyrogenic activity in any of the 28
rabbits tested.
2. The injection of AWT effluents of varying free-endotoxin content (6 to
250 pg/1 assay) into rabbits yielded positive pyrogenic results in 18 of
20 trials. There was, however, no discernible relationship between
endotoxin content and the increase in body temperature.
3. Although the ingestion of a membrane-filtered wastewater effluent (3000
yg/1 endotoxin activity) resulted in an elevated temperature in five of
eight animals tested, the increases were too small to satisfy the
requirements of the USP test for pyrogenicity.
4. The inhalation of a membrane-filtered waste water effluent aerosol (1560
yg/1 free-endotoxin activity) failed to show a pyrogenic response in 18
rabbits exposed.
5. Blood morphological changes as a result of the injection of pyrogen-
containing waters were observed, but the changes were within the normal
variations for the test animals.
6. That a potential exists for Pseudomonas proliferation in a GAC bed was
supported by one positive observation out of 25 examinations.
7. The numbers of bacteria found to be present in GAC product waters from
water treatment plants during this study were very few and certainly
would constitute no major health concern as long as a disinfection pro-
cess is subsequently applied.
8. The four disinfection processes employed (ultraviolet, chlorine, high
pH, and ozone) all appeared to decrease endotoxin content.
9. Good correlations were observed on non-disinfected AWT effluent samples
between standard plate count and total endotoxin (r=0.945), standard
plate count and free endotoxin (r=0.932), and total coliforms and free
endotoxin (r=0.939). Lack of good correlations, however, were observed
in assaying AWT samples that had been subject to the disinfecting pro-
cedures of chlorination, ozonation, high pH, or UV irradiation.
10. Free endotoxin appears refractory to GAC adsorption. Bound endotoxin
-------
appears removed via a filtration mechanism. Hence, total endotoxin is
reduced through GAC only by the amount of bound endotoxin filtered out.
11. Using two carbon columns in series on a filtered, nitrified activated
sludge effluent, the first column reduced COD 20 percent and total,
bound, and free endotoxins 64, 77, and 41 percent respectively. Addi-
tional reductions in COD, total, and bound endotoxin of 35, 20, and 42
percent, respectively, were observed in the second column (longer empty-
bed contact), but free endotoxin increased by 12 percent.
12. Small but substantial removals of TOC were observed after as long as
110 months of GAC operation. In five of 28 samples, however, an in-
creased TOC resulted. The lack of relationship of TOC removal to empty-
bed contact time suggests that adsorption kinetics are not descriptive
of the data collected.
13. The array of methods used to monitor the operation of GAC beds is
bewildering. The TOC procedure used only by a single plant and sug-
gested by the proposed regulations for the control of organic contami-
nants requires expensive, sophisticated instrumentation. The chemical
oxygen demand procedure (COD) can be used in lieu of TOC, yet not a
single use of COD was found in practice.
-------
SECTION 3
RECOMMENDATIONS
Pyrogenicity was demonstrated for AWT effluents but not for GAC-pro-
cessed drinking waters. Although endotoxin levels were generally lower for
drinking waters, many samples possessed higher endotoxin activity than some
of the AWT samples that resulted in a pyrogenic response. This suggests some
basic differences in water quality which must be further researched.
The drinking water samples were of higher microbiological quality prior
to GAC contact than were the AWT waters. Twenty-seven of the 28 drinking
water samples had been subjected to a prechlorination procedure., and the
microbiological mileau of the carbon beds could almost certainly be altered
as a consequence of this application. For example, coliforms were found to
be present in every sample of the GAC product water of the AWT application
whereas none were found in the drinking water samples. Such a difference in
microbiological mileau might explain the greater potency of AWT endotoxins
to elicit a pyrogenic response.
Since endotoxins appear to be refractory to adsorption processes but
possibly amenable to disinfection procedures, some kinetic studies of dis-
infection procedures on endotoxins are needed.
A general biological impairment of water by the GAC process was not
observed in this study with the possible exception of the Pseudomonas
detected in one sample. A different array of data might have been observed,
however, had prechlorination not been so broadly employed. Since prechlori-
nation practice might in the future be minimized to control trihalomethane
content, it is strongly recommended that studies of GAC and its impact on
the microbiological quality of processed waters be implemented on a variety
of waters when a prechlorination process is not employed.
A serious information gap appears to exist between the fields of
research and practice in the application of GAC in water treatment. The
suggestion is that training activities must be vastly improved if there is
to be any improvement in the nation's drinking water quality by virtue of
increased application of GAC.
10
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SECTION 4
EXPERIMENTAL PROCEDURE
AWT Plant and Operation
A detailed description of the Dallas Demonstration Plant has been
previously published (13). The unit processes involved in this study were:
1) No. 1 Activated Sludge Unit - this complete-mix unit treated
primary effluent from Dallas' White Rock Plant and was operated
to nitrify. An average COD value for the influent was about
270 mg/1, and aeration tank volume is about 560 M3 (6000 ft3).
Using a CODrBOD ratio of 2.2 for this waste water, the result-
ing loading/day on the unit in terms of BOD5 was about 0.5 kg
BOD5/M3 (31 lbs/1000/ft3). These values apply to a flow of
7.9 Ips (125 gpm). In the winter months, flow was dropped to
6.3 Ips (100 gpm) since nitrification proceeds more slowly at
colder temperatures. Average activated sludge effluent values
for COD were in the range 25-60 mg/1.
2) Activated sludge effluent was passed through a mixed-media
filter. The anthracite-sand-garnet filtration depth is 99 cm
(39 in), and it operated at a flow of 2.4 Ips (38 gpm) through-
out the study, the rate being approximately 175 M/day
(3 gpm/ft2). Effluent COD values were in the range 11-36 mg/1.
3) The water was lastly processed through two activated carbon
contactors operating in series. Flow through the first column
was consistently at 1.9 Ips (30 gpm) and through the second,
1.3 Ips (20 gpm). Both were charged with Calgon Filtrasorb
400, 8x30 mesh; the second column in the series had the newer
carbon. Empty-bed contact time was about 30 minutes for the
first column and about 45 minutes for the second column, a
total of approximately 75 minutes. Extreme COD values for
the product water ranged over 2.4-23 mg/1, with an average
value around 10 mg/1.
The microbiological quality of the final effluent waters were quite
high considering that the original water was sewage and that no disinfecting
agent was used. For example, the six samples assayed in August and September
on the first samples to be run at the Demonstration Plant showed the follow-
ing results:
11
-------
Assay
Total Plate Count/ml
Total Coliforms/100 ml
Coliphages, pfu/ml
Such microbiological qualities are more descriptive of a surface water than
of a non-disinfected waste water effluent.
AWT Sampling
Large samples of water were collected at the Demonstration Plant, mixed,
and divided into three smaller samples. One sample was retained at the plant
for characterization analyses (see Appendix pp A-l to A-8),and the other two
samples were shipped, one to the University of Texas Health Science Center
in San Antonio and the other to the Environmental Engineering laboratories
of Texas A&M University in College Station. Since Environmental Engineering
personnel had no previous experience with the LAL assay, a sub-contract was
made with the experienced personnel at the Health Science Center to perform
the LAL assays during the first year of effort.
The first samples were shipped wholly by bus. Straight-through bus
service from Dallas to San Antonio was fairly reliable. Samples to College
Station, however, had to change buses in Waco, and the delays sometimes
occasioned by this transfer (the next several buses might even be missed)
resulted in a change to air freight. Air freight, too, proved an unreliable
means of shipping samples. Only one set of samples shipped by air arrived
in the Environmental Engineering laboratories in College Station at the
proper temperature -- the very last samples to be shipped from Dallas.
Water Treatment^ Plant Sampling
The exasperating experiences with sample shipment over the relatively
short distance between Dallas and College Station demanded some rather
detailed planning if success was to be achieved with water samples shipped
from widely scattered points throughout the United States to College Station.
A minimum sample size was calculated to be 500 mis and two were required,
one before and one after GAC. Hence, two 500-ml glass bottles were placed
in a styrofoam insulated container (later, four 500-ml bottles were used.)
The container had cover latches on each end, but nylon web straps with
buckles and a handle were added for additional security (see Figure 1).
Gel paks were placed in a freezer overnight and inserted with the sample
bottles (see Figure 2). Such a kit was tested. The sample bottles were
filled with 27°C tap water at 7:30 in the morning. Three gel paks which
had been placed in a home freezer at 5:30 the previous evening were added.
The kit was closed and placed in the sun. Outdoor temperature reached 37°C.
The kit remained outdoors all through a warm night. The following morning
at 8:40 the kit was opened and the temperature of the sample bottles at
mid-depth was 4.5°C. Hence, we felt that if we could get 24-hour delivery,
12
-------
FIGURE 1. EXTERIOR VIEW OF SAMPLE KIT
13
-------
FIGURE 2. INTERIOR VIEWS OF SAMPLE KIT ARRANGED
TO HOLD FOUR 500-ML SAMPLE BOTTLES
14
-------
the samples would arrive in good condition.
The municipal supplies selected for sampling were obtained from a list
furnished by EPA. The list contained 45 supplies that utilize GAC filtration
either: a) in routine use, b) on a full plant scale, c) in experimental use,
or d) on a partial plant use basis. The supply we elected to sample was
contacted by telephone, the study, sampling, and analytical program explain-
ed, and their help sought. All supplies contacted agreed to help; there
were no turn-downs. Having agreed, the sampling kit was shipped to them.
It contained sterile sampling bottles, gel paks, sampling and shipping
instructions, a data sheet to be filled out,and a reimbursement statement
for $10.00 to help defray costs in getting the sample kits to and from air-
ports. Air freight was first utilized with disastrous results. The lid
clasps on the very first kit were knocked off and the kit was lost for 10
days. A repeat of this experience occurred with the second kit. Then Emery
Air Freight was utilized. This service commenced in excellent fashion,
providing 24-hour service. But for those locations where Emery had no
facilities, severe problems still occurred.
Laboratory personnel often wished to double-check an analysis and the
500-ml sample volumes were often inadequate. Hence, during the winter
months, the kit was shipped with four 500-ml sample bottles, fewer gel paks,
and "peanut styrofoam" filler. This was satisfactory but would not have
been so in warm weather.
Six of the 45 water treatment plants are located in a localized area
of New England. These plants were sampled last by project personnel. The
only dissolved oxygen values obtained during the study were from these six
plants.
15
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SECTION 5
ANALYTICAL PROCEDURES
The analytical procedures used in this study followed Standard Methods
(14) procedures unless otherwise noted. Additional details:
Chlorine residual - Method 409 E, DPD Ferrous Titrimetric Method.
Duplicate tests were run on each sample.
Dissolved oxygen - DO was measured using a YSI model 51 A oxygen meter
and a YSI 5720 A BOD probe. The DO meter was calibrated for temperature
and pressure before measurements were taken. BOD bottles (300 mis) were
filled with water, the probe inserted, and the DO read.
Standard plate count - One ml of sample was mixed with approximately
12 mis of Difco Plate Count Agar (at about 45°C) in 15x100 mm Pyrex petri
dishes. The plates were allowed to solidify, and were then inverted and
incubated at 35°C ±0.5° for 48 hours. Duplicate tests were run on each
sample. The number recorded was an average of the two counts.
Total coliforms - The MPN procedure (5-tube series) was used with
waste water samples and both MPN and MF procedures were used with drinking
water samples. The MPN procedure is Method 908 A, Standard Total Coliform
MPN Test. A dilution scheme of 10 ml, 1 ml, and 0.1 ml for drinking water
(10° to 10~5 for waste water, and 10° to 10~2 for disinfected waste water)
was used with Difco Lauryl Tryptose Broth in the presumptive test and Difco
Brilliant Green Bile Broth in the confirmed test. The MF procedure is
Method 909 A, Standard Total Coliform Membrane Filter Procedure. A 100-ml
sample was used with Mi Hi pore 0.45 ym filters and Difco Ends Agar.
Pseudomonas aeruginosa - Method (tentative) 914 E, Multiple-Tube
Techm'c for P-seudomonas aeruginosa.
Pseudomonas fluorescens - From a pour plate that had been incubated
for 24 hrs, 10 representative colonies were aseptically transferred to
sterile Pseudomonas Agar F agar slants. The slants were incubated for 48
hours at 35°C ± 0.5°. A green pigment formation would indicate the
presence of Pseudomonas fluorescens.
Gram stain - From a pour plate that had been incubated for 24 hrs,
10 representative colonies were aseptically transferred to individual
Corning Micro Slides. The colonies were dispersed with sterile dilution
water, fixed, and stained. The number of individual types of colonies
stained depended upon the ratio of each morphological group to the whole
16
-------
plate count. For example, if 4 colonies are similar among 20 colonies on
a plate, then 1 of the 4 colonies would be selected for staining.
Coliphages - To 10 mis of sample was added 0.5 ml of chloroform. The
solution was shaken 25 times and stored overnight in a refrigerator at
about 9°C. On the same day that the chloroform was added to the sample,
5 mis of tryptone was inoculated with Difco 15597 E_. co 1 i. The inoculated
solution was placed in an incubator (35°C ± 0.5°) overnight. The jE. coli
solution was then mixed on a vortex mixer and 0.1 ml was extracted and added
to 10 mis of tryptone broth. This solution provides the indicator cells for
the coliphage. Then 0.5 ml of the chloroformed sample was mixed with 2.0
mis of indicator solution and 2.5 mis of tryptone overlay agar (47°C) in a
sterile 16x125 mm pyrex test tube. The tube and contents were mixed on a
vortex mixer and poured onto a petri dish containing 20 mis of solidified
tryptone agar. The plate was swirled to evenly distribute the mixture over
the surface, and placed upright in an incubator (35°C ± 0.5°) for 24 hrs.
Duplicate tests were run for each sample and the number recorded is the
average titer (pfu/ml) calculated for each test.
Total organic carbon - This TOC procedure used Oceanography Interna-
tional (O.I.) equipment and methods. The procedure consists of two parts,
preparation of the ampule, and testing procedure.
Ampule preparation -A 5.0-ml sample is volumetrically pipetted into a
precombusted ampule covered with aluminum foil. The ampule with sample is
placed in a holder attached to the O.I. ampule sealing unit. The ampule
sealing unit consists of a purging unit in which purified oxygen is bubbled
through the sample and an oxygen-propane microburner that seals the ampules.
Then 0.25 ml of 6% phosphoric acid is added to the ampule with sample just
before purging. The sample is purged with purified 02 for 4 min. After 3
min. of purging, 1 ml of saturated persulfate solution is added to the
ampule. The ampule is sealed by the oxygen-propane microburner. A puri-
fied oxygen atmosphere is maintained inside the ampule during the sealing
process. After all the ampules have been sealed, they are placed in a
holding rack. The rack fits into a metal pressure vessel. Approximately
1 liter of distilled water is added to the pressure vessel. The vessel is
sealed by a metal top that bolts on. The pressure vessel is placed in an
oven at 170°C for 24 hours. The pressure vessel is allowed to cool to room
temperature before the ampules are removed. The ampules are stored at room
temperature until analyzed.
Testing procedure - The samples are analyzed on an O.I. ampule analyz-
ing unit. Standard TOC samples (10.0 ppm, 7.5 ppm, 5.0 ppm, and 2.5 ppm)
are run prior to the GAC samples. A linear curve is established relating
an integrated machine number with the respective TOC standard. Boiled
distilled water is used as dilution water for the TOC standards. The
dilution water is analyzed on the ampule analyzing unit and the integrated
machine number is subtracted from each of the TOC standards before the
linear curve is plotted. A minimum of 5 samples are analyzed to obtain an
average value. Once an average integrated machine number is found for a
GAC sample, the respective TOC value is taken from the standard TOC curve.
17
-------
Limulus amobocyte lysate assay - Two different sources of Limulus
lysate were used during the second half of the project. Prior to December
1977, lysate from Difco was used. After that time, lysate was obtained
from Associates of Cape Cod.
Difco procedure -
I. Standardization of Stock (stock in this description refers to
Difco Co. products)
A. Reconstitute the contents of the Pyrotrol Positive Control,
0.5 ng/.2 ml, with 4 mis pyrogen-free water.
B. Vortex contents for 1 min.
C. Preparation of dilutions.
1. Dilute positive control (0.5 ng/.2 ml) 1:1 giving
0.25 ng/.2 ml.
2. Vortex 1 min.
3. Draw 0.2 ml and add to Pyrotest vial. Swirl very
slightly. Do not agitate violently.
4. Dilute the 0.25 ng/.2 ml solution 1:1 giving 0.125
ng/0.2 ml and repeat steps 2 and 3.
5. Continue the 1:1 dilution scheme repeating steps
2 and 3 for sequential 1:1 dilutions of 0.0625 ng/
0.2 ml, 0.03125 ng/0.2 ml and 0.01565 ng/0.2 ml.
6. Add 0.2 ml of pure dilution water to a Pyrotest
vial as a control.
D. All prepared vials are incubated at 35°C for 75 min.
E. After incubation, the vials are examined for the forma-
tion of a gel and are graded from 1+ to 4+ (1+ is opaque
without any gel and 4+ is a solid gel.)
F. The sensitivity of the stock is the highest dilution of
the standard giving a positive Limulus test (3+ or 4+).
For example:
0.3125 ng/ml 0.156 ng/ml 0.0781 ng/ml
4+ 3+ 1+
The sensitivity of this stock would be 0.156 ng/ml.
II. Method of Testing
A. Agitate sample thoroughly for 1 min.
18
-------
B. Make dilutions
1. Dilute sample 1:100 and vortex for 1 min.
2. Draw 0.2 ml and add to one Pyrotest vial. Do not
agitate violently.
3. From the 1:100 dilution make a 1:1000 dilution
and vortex for 1 min.
4. Draw 0.2 ml of dilution and add to Pyrotest vial.
Do not agitate violently.
C. Incubate vials for 75 min. at 35°C. After incubation
record the formation of any gel as 1+ to 4+.
D. Once the range of endotoxin equivalents is found,
subsequent dilutions are made to find the approximate
endotoxin equivalent number. For example, if the
range is found to be above 1:100 but below 1:1000,
subsequent dilutions of 1:200, 1:400, and 1:800 can
be made and tested. If the end point turns out to be
1:400, the endotoxin equivalents would be 400 x 0.156
ng/ml (endotoxin sensitivity) = 62.4 ng/ml or 62 ng/ml
endotoxin equivalents.
III. Materials Used
A. Difco Pyrotest kit.
B. McGaw sterile water for irrigation or 0.9% sodium
chloride sterile irrigation solution.
C. Falcon 16x125 mm disposable tissue culture tubes.
D. Scientific Products disposable serological pipets;
1 and 10 ml, individually wrapped.
Associates of Cape Cod procedure -
A. Agitate sample thoroughly for 1 min.
B. Make dilutions.
1. Dilute sample 1:100 and vortex for 1 min.
2. Draw 0.1 ml and add to one pyrogen-free 10x75 mm
culture tube.
3. From the 1:100 dilution make a 1:1000 dilution
and vortex for 1 min.
4. Draw 0.1 ml of dilution and add to one pyrogen-
free 10x75 mm culture tube.
C. Remove LAL from freezer and add 1 ml of pyrogen-free
water to the vial and slowly swirl until all the LAL
19
-------
has gone into solution. Add 0.1 ml LAL to each pyrogen-
free culture tube with sample. Place the 10x75 mm culture
tubes in a 37°C water bath and incubate for 75 min. After
75 min. of incubation, slowly and carefully remove the
culture tubes singly, directly from the water bath. Invert
the culture tube 180°. A solution is considered positive
if the gel can be inverted twice without breaking. If
the gel breaks, the test is considered negative.
D. Once the range of endotoxin equivalents is found, subse-
quent dilutions are made to find the approximate endotoxin
equivalent number. The sensitivity of the LAL is deter-
mined by Associates of Cape Cod and each vial is labeled
with its sensitivity.
Pyrogenic assay -
The amount of bacterial pyrogen in an unknown sample can be
quantitatively determined by injecting the test sample into rabbits
and measuring changes in body temperature. The factors that influence
the rabbits response to pyrogens are normal temperature, body weight,
individual sensitivity and excitation (15).
Male New Zealand albino rabbits were selected for the assay.
Individual rabbits were selected on the basis of a normal temperature
between 37.8° and 39.5°C, and a weight of 2 to 4 kg. Each rabbit was
injected with 1 ml of pyrogen-free water under assay test conditions.
If their temperature response was greater than 0.3°C, they were
eliminated from the program. Seven out of fourteen rabbits, selected
at random, were given a known dose of pyrogens and checked for a
pyrogen response. All rabbits injected with the known quantity of
pyrogens had a fever response. To avoid the effect of tolerance the
rabbits were rested for two weeks between injections. At the end of
the project period, all rabbits were given a standard pyrogen dose of
2 ng/kg and monitored for a pyrogen response. All the rabbits in the
colony had a fever response to the 2 ng/kg dose.
Three rabbits were used for each assay. The rabbits were placed
in wooden stanchions one hour before being injected. Temperatures were
monitored using a YSI Tele-Thermometer and a small animal thermistor
probe. The probe was inserted 7-1Ocm into the rectum. Temperatures
were recorded every half-hour during the assay. The rabbits were
injected in the marginal vein with 1 ml 0.45 u - filtered sample.
Their temperatures were monitored for 2 hours or more after the
injection.
USP Pyrogen Test (15) -
The pyrogen test is designed to limit to an acceptable level the
risks of a febrile reaction in the patient to the administration, by
injection, of the product concerned. The dose specified for the test
20
-------
is related to that generally given to the patient, but for practical
reasons, it does not exceed 10 ml per kg. of body weight of the test
animal, injected in a brief period of time. For products that require
preliminary preparation or are subject to special conditions of admin-
istration, follow the additional directions given in the individual
monograph.
Apparatus - Render the syringes, needles, and glassware free from
pyrogens by heating at250°Cfor not less than 30 minutes or by any
other suitable method. Just prior to injecting it, warm the product
to be tested to approximately 37°C.
Test Animals - Use healthy, mature rabbits each weighing not less
than 1500 g. House the animals individually in an area of uniform
temperature [±3°C(±5°F)] and free from disturbances likely to excite
them. Before using an animal for the first time in a pyrogen test,
condition it by a sham test that includes all of the steps as directed
under Procedure except the injection of the test dose. Do not use
animals for pyrogen tests more frequently than once every 48 hours,
nor prior to 2 weeks following their having been given a test sample
that was adjudged pyrogenic.
Note - Perform the test under environmental conditions similar to
those under which the animals are housed. During the test, withhold
all food from the animals being used. Access to water may be allowed.
Temperature Recording - Use an accurate clinical thermometer for
which the time necessary to reach the maximum reading is known, or any
other temperature-recording device of equal sensitivity. Insert the
thermometer into the rectum of the test animal to a depth of not less
than 7.5 cm., and, after a period of time not less than that previously
determined as sufficient, record the animal's body temperature.
Procedure - Not more than 40 minutes prior to the injection of
the test dose, determine the "control temperature" of each animal;
this is the base for the determination of any temperature increase
resulting from the injection of a test solution. In any one test use
only those animals the control temperatures of which do not vary by
more than 1°C from each other, and do not use any animal with a
temperature exceeding 39.8°C.
Unless otherwise specified in the individual monograph, inject
into an ear vein of each of three rabbits 10 ml of the product per kg,
of body weight within 40 minutes. Record the temperature at 1, 2,
and 3 hours subsequent to the injection.
Interpretation and Retest - If no rabbit shows an individual
rise in temperature of 0.6°C ormore above its respective control
temperature, and if the sum of the three temperature rises does not
exceed 1.4°C,the product meets the requirements for the absence of
pyrogens. If one or two rabbits show a temperature rise of 0.6°C or
21
-------
more, or if the sum of the temperature rises exceeds 1.4°C,repeat the
test using five other rabbits. If not more than three of the eight
rabbits show individual rises in temperature of 0.6°C or more, and if
the sum of the eight temperature rises does not exceed 3.7°C,the
material under examination meets the requirements for the absence of
pyrogens.
22
-------
SECTION 6
AWT PLANT RESULTS
The Demonstration Plant of the Henry J. Graeser Environmental Research
and Training Facility, Dallas, Texas was operated for most of the first-year
study period as depicted in Figure 3. Large grab samples of product waters
were split into three subsamples; one was retained at the Demonstration
Plant and the other two were shipped to laboratories in San Antonio and
College Station, Texas. Analytical characterization of the samples was
performed at the Demonstration Plant; the data are in the Appendices, pp A-l
to A-l8. The University of Texas Health Science Center at San Antonio
directed its effort at in vitro approaches, separating the endotoxin
analyses into the bound and free forms, and observing the relationships of
all forms with microbiological analytical results. Texas A&M University in
College Station directed its effort at in vivo tests subjecting the test
animals to endotoxin administration via i.v. injection, ingestion, and
inhalation.
Comparison of Endotoxins with Viable Bacteriological Assays
A total of 48 AWT product water samples were examined for endotoxin,
standard plate count, and total coliforms (Table 4). Standard plate counts
varied from 0/ml to 5.5xlOVml plus several indeterminate values of TNTC
(too numerous to count). Total coliform counts by the membrane filter (MF)
method ranged from less than 2/100 ml to 1.4xl06/100 ml. The total endo-
toxin content (unfiltered samples) ranged from 6-600 pg/1 endotoxin equiva-
lents, free endotoxin levels (filtered samples) ranged from 3-480 yg/1
endotoxin equivalents, and the bound endotoxin content ranged from 0-250 yg/1
endotoxin equivalents. Note in Table 4 the apparent increase in endotoxin
content with time. The treatments of UV, Cl2, 0$, and pH are explained on
page 31.
The gross data for the finite values of Table 4 are displayed in Figure
4. Figure 4a compares the relationship between total endotoxin content and
total number of bacteria determined by the standard plate count procedures
(three replicates). The correlation coefficient was determined to be 0.726.
Figure 4b examines the relationship of bound endotoxin content to standard
plate count (correlation coefficient = 0.736). Figure 4c compares free
endotoxin content with the standard plate count (correlation coefficient =
0.620). Figure 4d compares total endotoxin content with total coliform
count (correlation coefficient = 0.822) and Figure 4e compares bound endo-
toxin content with total coliform count (correlation coefficient = 0.472).
Figure 4f relates the free endotoxin content to the total coliform count
(correlation coefficient ~ 0.525).
23
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A previous preliminary investigation demonstrated the feasibility of
testing water samples by the Limulus assay for the presence of endotoxin due
to gram-negative bacteria (10). However, in this present study using highly
treated waste waters which had been subjected to a biological process, the
results of Limulus assays did not correlate extremely well with the two
established techniques for assessing microbiological water quality, i.e.,
the standard plate count and total coliform count. Levels of total, bound,
or free endotoxin activities seemed unable to reliably predict the microbial
content of the water samples examined if disinfection treatments of Cl2» Oa,
high pH, or UV had been performed.
Examination of the first 24 samples from Table 4 which had not been
exposed to disinfection processes yielded mixed correlations: standard plate
count and total endotoxin (r = 0.945), standard plate count and free endo-
toxin (r = 0.932), and total coliforms and free endotoxin (r = 0.939), all
quite good. However, the correlations between standard plate count and
bound endotoxin (r = 0.745), total coliforms and total endotoxin (r = 0.822),
and total coliforms and bound endotoxin (r = 0.419) were less encouraging.
In the study by Evans, et al. (12), better correlations between endotoxin
content and bacterial numbers were achieved by use of the spectrophotometric
modification of the Limulus assay than by the clot formation end-point
method utilized in the present study. Evans, et al. found that the bound
endotoxin component (which was most predictive of bacterial numbers) was
decreased following chlorination procedures. However, it has also been
previously demonstrated that agitation of gram-negative bacteria in a liquid
environment serves to release additional amounts of cell-associated endo-
toxin (16). Thus, it is reasoned that the poor correlation between endo-
toxin content and densities of viable microorganisms observed in this study
result from a combination of partial destruction of Gram-negative bacteria
and varying degrees of solubilization of cell-associated endotoxin. There-
fore, the endotoxin content of highly treated waste water effluent, unlike
stream (12) or sea water (11), probably reflects the remnants of pre-
existant bacterial growth rather than continued proliferation of Gram-
negative microorganisms.
Endotoxin and Organic (COD) Removal
The operation of the two GAC columns in series afforded a comparative
observation of the endotoxin activity of the waters at different points in
the adsorption process. The points examined were: just prior to the first
contactor (influent), between the contactors (midpoint), and after passing
through the second contactor (effluent.) Perhaps more suitable terminology
would be expressed by: samples after 0, 30, and 75 minutes of empty-bed
carbon contact. These samples were coded and the type of sample being
analyzed was not known by laboratory personnel. Table 5 presents the
results of three such samples for both chemical oxygen demand (COD) and
endotoxin activity. The first column removed 6 mg/1 (26%) of COD, 286 yg/1
(64%) of total endotoxin activity, 67 j.ig/1 (41%) of bound endotoxin activity,
and 219 yg/1 (77%) of free endotoxin activity. The suggestion is that the
first column does better in removing free endotoxin (presumably by adsorp-
tion) than it does in removing the cells (as indicated by the bound
29
-------
TABLE 5. COD CONCENTRATIONS AND ENDOTOXIN ACTIVITIES OF AWT WATERS BEFORE,
AT THE MIDPOINT AND AFTER GAC FILTRATION
Sample Influent
No. (Before)
12-14
12-21
1-25
"X
% Removed
12-14
12-21
1-25
X"
% Removed
12-14
12-21
1-25
X"
% Removed
12-14
12-21
1-25
X"
% Removed
23.4
22.2
22.5
22.7
600
500
250
450
480
250
125
285
120
250
125
165
Midpoint
(Between)
COD,
17.7
17.8
14.7
16.7
26
Total Endotoxin
192
200
100
164
64
Free Endotoxin
48
100
50
66
77
Bound Endotoxin
144
100
50
98
41
Effluent
(After)
mg/1
9.4
14.3
9.0
10.9
35
Activity, yg/1
192
100
100
131
20
Activity, yg/1
96
50
75
74
+12
Activity, yg/1
96
50
25
57
42
Overall
52
71
74
65
30
-------
endotoxin). The second column removed an additional 5.8 mg/1 (35%) of COD,
33 yg/1 (20%) of total endotoxin activity, and 41 yg/1 (42%) of bound endo-
toxin activity, but produced an additional 8 yg/1 (12%) of free endotoxin
activity. A mass balance indicates that the first column in the series was
removing 0.96 kg/day (2.16 Ib/day) of COD, whereas the second column was
removing only 0.62 kg/day (1.39 Ibs/day). Since both columns contained the
same amount of carbon, the increased free endotoxin value in the effluent
from the second column is likely not attributable to an increased bacterial
growth (assuming the same biodegradability of the COD retained by both
columns), and therefore is more likely due to the death and lysis of cells
thus releasing more free endotoxin -- or to analytical insensitivity. Free
endotoxin is defined by Jorgensen (see page 4) as endotoxin that has been
solubilized without autolysis or disruption of the cells. However, in the
event of cell death and lysis, it would be impossible to separate the free
endotoxins into those coming from living cells and those coming from lysed
cells; both would be discernible only as free endotoxin.
Evaluation of Endotoxins with Four Disinfection Procedures
The endotoxin activity of samples subjected to four different dis-
infection procedures was determined in six sets of samples. The disinfection
procedures were conducted at the Demonstration Plant as follows:
UV - GAC product water was passed through a Kelly-Purdy UV
unit at a depth of 5 cm (2 in) and detention time of
3.9 minutes.
C12 - chlorine solution was added to a grab sample until a
free residual of approximately 1 mg/1 was achieved. It
was held for 30 minutes after which autoclaved sodium
thiosulfate was added.
pH - the pH of a grab sample was raised to approximately 11.5
by the addition of CaO and held for 2.5 hours. It was
then neutralized with the careful addition of a strong
acid. The CaO was not sterilized since plates of the
stock were negative.
Os - GAC product water at 1.58 Ips (25 gpm) was passed through
a Union Carbide injection ozone contactor which provided
an 03 dose of approximately 7.5 mg/1. The system provides
a contact time at the flow rate used of 1.6 minutes. The
sample was not "deozonated."
The chemical and microbiological assays and the actual disinfecting
parameters achieved for each sample are shown in the Appendix, pp A-12 to
A-18. The disinfecting procedures could not all be employed on the same
parcel of water at the same time; both UV and Os were in-line unit processes
and operated at different times, and the C12 and pH treatments were also
performed at different times on grab samples. Hence, the results of the
individual tests are not specifically comparable with each other.
31
-------
Table 6 shows the endotoxin activity of the individually disinfected
samples. Average values were determined for the measured total endotoxin
and free endotoxin assays, and from these averages the bound endotoxin values
were determined. The percent of the total endotoxin that was free and bound
are also shown. Differences between disinfection procedures cannot be
considered of significance because of the manner in which the tests were
conducted. The greater power of ozone to oxidize the free endotoxin as
compared with UV may be a valid observation, as might be the lesser effect
of ozone on the bound (cellular) endotoxin compared to the results of other
procedures.
The relatively high endotoxin values noted for the UV and C12 samples
of 2/8 should not be construed as errors. It had been observed that the
GAC product water quality had been increasing in endotoxin activity with
time. The decision was made -- wrongly it turns out -- to not run endotoxin
assays on samples prior to applying a disinfection procedure because it was
believed that sufficient such data were already available. However, Table 7
shows the rapidity of the general increase in product water endotoxin
activity with respect to time. Plotting the mean values on semi-log paper,
a minimum projected value for the period Jan 26 - Apr 18 when the disin-
fection samples were processed would be about 600 yg/1 -- somewhat lower for
the earlier samples and higher for the later samples. Hence, there is little
question but that the disinfection processes considerably reduce the endo-
toxin activity, but we cannot quantify the decrease nor can we prove it.
Impacts of Sample Shipment
Of the entire disinfection series, only three samples -- the last
three -- which were shipped by air arrived in a cool condition, and only
the last at a temperature which we had hoped to achieve for all. The more
important variables of the disinfection process are collected in Table 8 to
show the effects of shipment on these variables. The chlorine residuals
shown are for free chlorine, but the "after" values are likely the back-
ground scatter of the technique used. The high-pH treated samples showed
both the highest average turbidity and highest average COD.
Table 9 is a presentation of the bacteriological results on the various
disinfected samples, the before shipment values were determined before
samples were shipped from the Dallas site and the after shipment values were
determined after the samples were received at College Station. It should be
noted that the treatments were not quite as effective as was anticipated.
No coliforms were found in the ozonated or pH-treated waters before the
samples were shipped, but some were recovered after shipment. The chlori-
nation procedure used was not quite as effective as the ozone or pH
treatments in spite of the higher turbidity and COD of the high-pH treated
samples and the extremely short contact time for ozone (although the contact
continued in the sampling vessel). Rather extensive growth occurred during
shipment as determined by the plate count procedures. We had expected the
regrowth of the ozonated samples to be greater because ozone increases the
amount of biodegradable material present -- but the results appear to be of
the same order of magnitude as the other disinfected samples.
32
-------
TABLE 6. TOTAL AND FREE ENDOTOXIN ACTIVITIES
OF DIFFERENT WATER SAMPLES
SUBJECTED TO FOUR DISINFECTION PROCEDURES
Sample
Date
2/8
2/28
3/14
3/20
4/4
4/11
4/18
X"
% Free
UV
Endotoxin
320(320)
80(48)
30(22)
90(30)
30(22)
47(25)
100(78)
78
Bound
Endotoxin 22
(by difference,
ug/D
% Bound
22
Disinfection
C12
Equivalents,
160(80)
80(8)
30(22)
30(30)
47(25)
47(25)
66(32)
48
34
52
Procedure
PH
ng/1, Total
32(16)
80(8)
9(2)
12(6)
5(3)
47(25)
31(10)
32
21
68
03
(Free)
32(8)
80(8)
30(3)
15(12)
30(6)
30(6)
36(7)
19
29
81
33
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TABLE 9. EFFECTS OF SHIPMENT ON BACTERIOLOGICAL
QUALITIES OF WATER SAMPLES
Date of Standard Plate Count/ml
Sample
(1977) UV C12 pH 03
2/8
2/28
3/14
3/28
4/4
4/11
4/18
before 112
after 1,820
24
125
81
50
280
235
140
5,540
240
63
79
7
43
475
4
35,000
8
10
3
11
26
3,800
5
14
65
128
1
500
0
44,000
27
25
13
59
26
9,340
17
13
60
96
0
310
0
213
3
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3
379
3
11,800
8
0
50
8
Total Col i forms/1 00 ml
UV C12 pH 03
0
23
2
49
7
23
4
23
14
23
6
2
15
2
0
0
0
23
0
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0
0
0
0
0
2
0
0
0
0
0
2
0
0
0
0
0
0
0
0
_ Before
X Shipment
137
22
21
10
After
X Receipt 1,120 5,634 7,719 2,118
<1
21
0 0
1
0 =
-------
Coliphage assays were run on all samples after receipt of the samples
at College Station. Table 10 displays the individual coliphage results
along with the coliform MPN values for the various water samples. We cannot
explain the initial visibility of coliphages and subsequently the disappear-
ance.
Pyrogem'c Studies
The pyrogenic response of experimental animals exposed to various waste
water samples by ingestion, inhalation, and injection was studied. We did
not expect to find a pyrogenic response due to ingestion because the liter-
ature clearly indicates that there is no danger of endotoxin shock as long
as the intestinal wall is normal, i.e. does not have an increased perme-
ability (4), and the animals that were used in these studies were held under
veterinary care and consequently were quite healthy. We could not find many
references in the literature concerning the inhalation route of endotoxin
exposure. Several years ago, a federal office building in Dallas, Texas
was evacuated because of a high rate of illness among office workers, the
cause of which was suspected to be air borne. Extensive study failed to
reveal an etiologic agent and there was some speculation about the possi-
bility of air-borne endotoxins. Rylander ert ajL (18) published a clinical
investigation of sewage treatment plant workers and their exposure to
aerosols of sewage sludge and its dusts. They reported that the workers'
symptoms and the results of the blood studies performed were consistent with
the expected effects of exposure to endotoxins. However, air-borne endo-
toxins were not measured. Since there is such a void on this topic in the
literature and since we were set up to measure pyrogenic activity, a sub-
experiment was included to evaluate the potential for response to endotoxins
transmitted by the inhalation route. The main pyrogenic studies in this
report, however, concern the classical i.v. route of endotoxin administration.
Ingestion --
The test water was obtained from the College Station municipal waste
water treatment plant. This plant utilizes a trickling filter and a contact
stabilization unit operating in parallel. The combined product water prior
to clarification was sampled and after passage through a 0.45 ym membrane
assayed 3,000 yg/1 of endotoxin activity. Drinking water was withheld from
the animals for a prior period of 18-22 1/2 hours. Rectal temperatures were
determined and then each animal was provided 200 mis of test effluent from
which to drink freely over the exposure period. Then their temperatures
were again monitored. The exposure period and the before, after, and peak
temperatures observed are shown in Table 11. As can be seen, there were 5
temperature increases, 1 decrease, and 2 unchanged. None of the increases
satisfies the requirements of a 0.6°C increase according to the USP test for
pyrogenicity (15), but a pyrogenic response was nonetheless observed in a
majority of the test animals.
Inhalation --
A simple aerosol exposure chamber was built that was large enough to
hold three rabbits in individual stanchions -- Figure 5. The stanchions
37
-------
TABLE 10. COLIPHAGE AND COLIFORM ASSAYS OF AWT WATER
SAMPLES AFTER RECEIPT IN COLLEGE STATION
Sample
No.
8-12
8-19
9-9
9-14
9-21
9-28
10-5
10-12
10-19
10-26
11-9
11-16
11-23
12-6
12-14 A
12-14 B
12-14 C
12-21 A
12-21 B
12-21 C
1-25 A
1-25 B
1-25 C
Coliphages
pfu/ml
4
6
2
4
2
<2
<2
<2
2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
Total
Col i forms
MPN/10Q ml
.17 x 103
1.3 x 103
1.7 x 103
9.2 x 103
.49 x 103
.27 x 103
7.0 x TO3
.33 x 103
5.4 x 103
5.4 x 103
.33 x 103
.33 x 103
.79 x 103
3.5 x 103
350 x 103
2.2 x 103
.7 x 103
.17 x 103
3.5 x 103
49 x 103
92 x 103
7.9 x 103
3.3 x 103
Type of Sample
GAC product water
ii
n
11
M
n
n
n
n
n
n
n
n
n
GAC influent
GAC midpoint
GAC product water
GAC influent
GAC product water
GAC midpoint
GAC influent
GAC midpoint
GAC product water
38
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FIGURE 5. RABBIT IN STANCHION
FIGURE 6. REAR OF THREE STANCHIONS SHOWN MOUNTED
IN INHALATION CHAMBER WHICH ALSO SHOWS
PLASTIC AEROSOL EXIT PIPES
40
-------
were open in the rear Figure 6. Aerosols were generated by two DeVilbiss
No. 15 atomizers operating at 20 psi. Each atomizer outlet was directed
downward into a 500 ml beaker, each initially containing a reservoir of 300
mis of sample to be atomized. Atomizer suction lines were connected to
withdraw from these reservoirs. Each beaker was placed under a 3.8 cm (1-1/2
in) ID plastic pipe about 13 cm (5 in) long which exhausted into the chamber
-- Figure 7. A simple corrugated paper-board deflector was located between
the inlets and the rabbits. Three exhaust pipes of the same type as the
inlet pipes were connected to a box-manifold which was connected to the
chemical hood by a corrugated plastic pipe -- Figure 8. Figure 9 shows the
termistor probes in the chamber ready to receive rabbits.
A Climet 280 particle-size analyzer was used to evaluate an aerosol of
a membrane-filtered final clarifier effluent from the College Station waste
water treatment plant, since it was essential that respirable particles of
less than 5 ym be produced. In two tests, particle size numbers in the
0.3-0.5, 0.5-1.0, 1.0-3.0, and 3.0-5.0 urn ranges were observed. There was
an abundance of particles less than 3.0 ym but very few in the 3.0-5.0 ym
range, and the system was considered satisfactory for producing respirable-
sized particles.
Two aerosol sampling impingers, Ace Glass Incorporated -- 30 ml glass,
were used at a flow of 1 1pm with a sampling medium of 20 ml of 0.9% NaCl
irrigation solution which is pyrogen-free (McGaw Laboratories). One 1 pm
is the approximate breathing rate of a rabbit (19). Both sampling impingers
were used to determine the background endotoxin levels of the sampled chamber
aerosols when pyrogen-free distilled water was atomized as well as during
each test exposure. The 30-minute distilled water exposures demonstrated
that the background air was free of endotoxin down to the sensitivity of the
assay for the impinger fluid -- 0.06 yg/1.
All rabbits subsequently tested were first exposed to a pyrogen-free
aerosol control. We stipulated that we would not use an animal for test
if it showed an increase of 0.3°C or more in its rectal temperature after a
control exposure (17), but none showed such a response.
In the actual test exposures, which duplicated the control exposure
except for the substitution of endotoxin-containing water for the pyrogen-
free water, the animals were placed in the chamber one hour before exposure
and their temperatures recorded. They were then exposed for 30-minutes to
an aerosol of dilutions of 0.45 ym filtered final effluent from the College
Station plant which demonstrated an endotoxin activity of 1560 yg/1. The
content of endotoxin per liter of air to which the animals were exposed was
determined to be 0.6, 1.2, 1.25, and 25.0 ng/1 by the all-glass impingers.
The resulting calculated quantities of inhaled endotoxins was 18, 36, 37.5,
and 720 ng, respectively. Not a single animal of the 18 exposed demon-
strated a pyrogenic response. Since the last sample was undiluted final
effluent, it was considered not necessary to conduct any further tests.
Injection --
A total of 26 1-ml injections to 24 rabbits were made with carbon-fil-
tered waters from samples of 12-21 and 1-25 which were taken from different
41
-------
FIGURE 7. TWO PLASTIC AEROSOL ENTRANCE PIPES
BELOW PAPERBOARD DEFLECTOR
FIGURE 8. EXHAUST PIPE CONNECTED TO CHEMICAL HOOD
42
-------
FIGURE 9. AEROSOL CHAMBER WITH THREE
THERMISTORS READY TO RECEIVE
RABBITS
43
-------
points in the adsorption process (before, in the middle, and after) and of
2-8 and 3-1 which were samples that had been disinfected by UV, Os, C12, and
high pH. Initially, we had wanted to use guinea pigs along with rabbits to
determine the magnitude of pyrogenic response. However, the small blood
vessels of the guinea pigs proved to be too difficult for our student
researchers to locate and the animals were obviously uncomfortable with the
students' fumbling probes and consequently we obtained permission to
eliminate guinea pigs from the study.
Twenty rabbits were followed for changes in blood morphology that might
be relatable to the injection of samples. This was done because we desired
to use as few animals as possible in these studies and were concerned about
maintaining their good health. Table 12 presents the pre-injection blood
counts and Table 13 the post-injection blood counts for methematocrit (MHCT),
red blood cells (RBC), white blood cells (WBC), monocytes (MONO), basophiles
(BASO), eosinophles (EOSIN), lymphocytes (LYMPH), and neutrophiles (NEUTRO).
It appeared to us that there were some startling changes in the lymphocytes
and neutrophiles as well, perhaps, as in the white blood cells and monocytes.
Some of the changes were contrary to expectations, but when the literature
was checked it appeared that all observed changes are within normally
expected variations. After noting this, blood testing was discontinued.
The individual responses of the 26 injections are displayed in Table 14.
A pyrogenic response was observed following all 26 injections, ranging from
a minimum of 0.1°C to a maximum of 1.7°C and averaging 1.0°C. Of the 26
responses, three were of insufficient magnitude to qualify the injected
water as pyrogenic according to the USP test for pyrogenicity (15). Of these
three, one had not been disinfected, one had been treated by high pH, and one
by UV. Obviously, individual variability or sample variability are the
likely explanations.
Twenty of the 26 injection samples were analyzed for endotoxin content.
The temperature rise exhibited by each sample and its free endotoxin titer
are collected in Table 15. A plot (not included) of the two sets of data
showed that a relationship does not exist. It has been suggested that the
lack of a relationship should be expected because of the wide response in
individual animals (18).
The filtration of samples through 0.45 ytn membrane filters does not
positively exclude viruses. Hence, in order to check on viruses, coliphage
analyses were routinely conducted on all membrane-filtered samples as well
as on the samples prior to filtration but none was found.
44
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TABLE 15. THE PYROGENIC RESULTS OF FREE
ENDOTOXIN-CONTAINING AWT SAMPLES
Sample
12-21-A
II
B
ii
C
n
1-25-A
n
B
n
C
n
2-8-03
II
PH
n
C12
II
uv
II
Temp.
Rise
°C
0.8
0.7
1.3
1.2
1.0
1.1
1.3
1.2
0.4
1.4
1.2
1.2
1.0
0.8
0.1
0.7
1.3
1.2
1.7
1.0
Free
Endotoxin,
ng administered*
250
250
50
50
100
100
125
125
50
50
75
75
6
6
6
6
15
15
15
15
* ng/kg dose ^ ng administered v 3
49
-------
SECTION 7
WATER TREATMENT PLANT RESULTS
A total of 28 water treatment plant GAC systems were sampled. Several
plants whose samples were lost in shipment were resampled; one plant was
sampled and analyzed twice (Sample Nos. 11 and T4). The specific analytical
results and operational data relating to each sample are contained in the
Appendices, pp A-19 to A-70.
Microbiological and Endotoxin Observations
Table 16 is a compilation of the bacteriological results on each sample
pair -- one sample taken prior to GAC contact and the other after GAC contact.
The table shows only the standard plate count and total coliform results --
the latter by both MF and MPN procedures. Coliphage analyses were also run on
each sample; no coliphages were found either before or after GAC treatment.
In only two instances were coliforms found, and these were both before GAC
contact. They were detected by MF and MPN procedures and in both cases the
MPN procedure estimated a higher number than counted by MF. No coliforms
were found after GAC.
The standard plate count data of Table 16 show that 14 of the 28 samples
decreased thru GAC, 8 increased, and 6 were unchanged. Although the average
of the before-GAC samples of 18.9/nil increased to 61.4/ml after GAC, the
increase was caused by a minority of samples. If the two high after-GAC
sample values of 320 and 980 are deleted, the remaining average would be only
16.1 -- essentially unchanged from the before-GAC average.
The two highest after-GAC standard plate count values were from recently-
installed carbon beds (15 days of operation at time of sampling) at the same
plant. Sample 26 A-16 is also from that plant -- a third carbon bed. This
plant is experimenting with three different brands of activated carbon and
all three beds were placed in operation at the same time, were being back-
washed at the same frequency, etc. The disparate standard plate count results
are explained by the accidental loss of thiosulfate from the sample bottle of
26 A-16 during sampling. This does not, of course, explain the higher plate
count samples.
We should expect that the application of chlorine prior to GAC would
influence the microbiology of the water both prior to and following GAC.
However, only one of the 28 samples (No.33) in Table 16 came from a plant
that does not prechlorinate. Three samples showed no Cla residual (see data
in Appendices) prior to GAC even though prechlorination was practiced, these
being Nos. 24, 88, and T2. Sample 24 was one of two that contained coliforms
50
-------
TABLE 16. BACTERIOLOGICAL RESULTS OF WATER TREATMENT PLANT
SAMPLES BEFORE AND AFTER GAC FILTRATION
Sample
No.
77
22
11
55
33
44 A
44 B
88
66
99
12
14
16
18-4
18-5
24
26 A-12
26 A- 14
26 A-16
28
30 A-l
30 A-2
Tl
T2
T3
T4
T5
T6
"x
Standard
Plate
Count
per ml
74
18
25
0
39
0
0
88
32
0
14
86
15
1
3
48
0
0
0
0
1
64
3
8
1
3
3
2
18.9
Before GAC
Total Col
MF
0
0
0
0
0
0
0
0
9
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
0
0
0
i forms/100 ml
MPN
<2
<2
<2
<2
<2
<2
<2
<2
46
<2
<2
<2
<2
<2
<2
49
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
Standard
Plate
Count
per ml
34
11
5
0
4
0
1
8
2
0
1
20
0
0
1
5
320
980
0
0
64
241
3
11
2
1
1
3
61.4
After GAC
Total Col
MF
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
i forms/1 00 ml
MPN
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
<2
Standard Plate Count of 0 = <0.5/ml.
MF Coliform Value of 0 = <1.0/100 ml
51
-------
prior to GAC.
A most important relationship is the potential for impact of the organic
material (TOC) adsorbed onto the carbon to impact on the bacteriological
quality of the product water. Table 17 shows the TOC both before and after
carbon contact as well as the endotoxin results. The average of all TOC
analyses shows a removal through GAC from a level of 3.9 mg/1 before to 3.2
mg/1 after, a removal of 18 percent. Interestingly, the percent decrease in
total endotoxin through GAC also averages 18 percent.
A plot of the standard plate count of the after-GAC samples from Table
16 vs. the change in TOC through the GAC from Table 17 shows in Figure 10 a
rather scattered relationship favoring the hypothesis of an increased
standard plate count with an increased removal of TOC by the GAC bed. In
support of such a relationship is the nonparametric observation that the 7
plants with highest TOC removals include 5 of the 8 plants with the highest
plate counts:
TOC SPC after
Sample Removed Sample GAC
mg/1 #/ml
14 2.9 26 A-14 980
77 2.4 26 A-12 320
55 2.3 30 A-2 241
30 A-2 1.8 30 A-l 64
Tl 1.6 77 34
26 A-12 1.6 14 20
26 A-14 1.6 22 11
T2 11
Table 17 also shows that the averages of total endotoxin activity were
decreased through GAC from 21.2 to 17.3 pg/1, and the free endotoxin activity
also decreased but only slightly from an average of 9.0 to 8.7 yg/1.
By difference, the averages of bound endotoxin activity was decreased from
12.2 to 8.6 yg/1. The percent decreases for the three forms of endotoxin,
total, free, and bound, were 18, 3, and 30 percent, respectively. The
suggestion is that the carbon was functioning as a filter by removing bound
endotoxin and that essentially no adsorption of free endotoxin was occurring.
These results are compatible with the performance of the second (downstream)
GAC column in the AWT studies which show comparable removal values of 20,
+12, and 42 percent for the three forms of endotoxin, respectively. The
increased filtration ability of the AWT column should be expected since that
carbon was 3.05 M (10 ft) deep compared to the maximum of 1.5 M (5 ft) or
average of 0.76 M (30 in.) for the water treatment plant installations
(Table 18). Empty-bed contact time for the two applications differs consid-
erably also, averaging 8.75 minutes (Table 18) for the water treatment
applications vs. 45 minutes for the second GAC column in the AWT studies.
The increase in free endotoxin through the AWT carbon was discussed in
Section 6, and in context with the slight removal observed in the drinking
52
-------
1000
1
-)-)
o 100
o
QJ
+->
rd
Q.
-a
fO
-o
5 10
CO
1.0
0.1
1
»
....
,
A*
- ^
_
, 0_
,
-4 -3 -2 -1 0 +1 +2 +3 +4
mg TOC/1 Removed (+) or Added (-) by GAC
FIGURE 10. STANDARD PLATE COUNT AFTER GAC vs. CHANGE IN TOC
53
-------
TABLE 17. TOC CONCENTRATIONS AND ENDOTOXIN ACTIVITIES OF WATER
TREATMENT PLANT SAMPLES BEFORE AND AFTER GAC FILTRATION
Sample
No. T(
m
77 6
22 4
11 4
55 3
33 8
44 A 5
44 B 4
88 5
66 3
99 3
12 2
14 5
16 1
18-4 2
18-5 2
24 3
26 A-12 3
26 A-14 3
26 A-16 3
28 3
30 A-l 3
30 A-2 4
Tl 5
T2 2
T3 3
T4 3
T5 3
T6 3
X" 3
Before
DC Endotoxin
3/1 Total
.4 3.1
.4 3.1
.8 15.6
.6 12.5
.1 31.3
.3 6
.8 6
.9 120
.2 36
.0 6
.2 3.6
.2 60
.8 18
.7 5
.5 5
.9 37.5
.1 12.5
.1 12.5
.1 12.5
.5 25
.5 25
.5 25
.2 25
,9 12.5
.7 12.5
.5 25
.0 12.5
.3 25
.9 21.2
Equivalents, yg/1
Free Bound
3.1
1.6
3.1
6.3
12.5
2.4
2.4
24
18
0.6
0.06
12
12
1.3
1.3
5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
2.5
7.5
12.5
10
25
9.0
0.0
1.5
12.5
6.2
18.8
3.6
3.6
96
18
5.4
3.54
48
6
3.7
3.7
32.5
0
0
0
12.5
12.5
12.5
12.5
10
5
12.5
2.5
0
12.2
TOC
mg/1
4.0
3.8
3.3
1.3
7.5
4.4
4.0
6.4
2.9
2.3
1.2
2.3
1.7
2.1
2.2
3.3
1.5
1.5
1.6
2.4
4.5
2.7
3.6
3.7
3.5
2.0
3.7
3.2
After
Endotoxin Equivalents, yg/1
Total Free Bound
3.1
1.6
3.1
6.3
31.3
6
6
60
18
6
2.4
60
12
2.5
1.3
37.5
25
25
12.5
25
25
25
25
10
10
10
10
25
17.3
3.1
1.6
3.1
6.3
12.5
2.4
2.4
24
12
2.4
1.2
18
12
1.3
1.3
5
12.5
12.5
12.5
12,. 5
12.5
12.5
12.5
2.5
7.5
7.5
5
_25_
8.7
0
0
0
0
18.8
3.6
3.6
36
6
3.6
1.2
42
0
1.2
0
32.5
12.5
12.5
0
12.5
12.5
12.5
12.5
7.5
2.5
2.5
5
8.6
54
-------
water studies, supports the observation of a lack of carbon's ability to
adsorb free endotoxins.
Pseudomonas aeruginosa assays were conducted on 25 pair of before-and-
after GAC samples. Only 3 were positive. Plants 33 and 24 had 30 and 10
percent positives for P_. aeruginosa in before-GAC samples; both were negative
in the after-GAC samples. Only plant 14 yielded £. aeruginosa in the product
water after GAC, but this sample had a 70% yield. P_. fluorescens was deter-
mined on 21 pair of samples with completely negative results. All three
samples yielding pseudomonads prior to GAC showed total chlorine residual
values <0.05 mg/1.
Gram-stain results of 14 before-GAC samples showed from 22 to 100% to be
gram-negative organisms and of 14 after-GAC samples from 30 to 100% gram
negatives. Five samples of before-GAC water yielded 100% gram negative
organisms, and 10 samples of after-GAC water yielded 100% gram negatives.
Only 10 of the 14 samples were pairs taken from the same GAC beds.
Pyrogenic Observations
Every after-GAC water treatment plant sample was passed through a 0.45ym
membrane filter and one ml was injected into the marginal vein of rabbits.
The free endotoxin dose administered to each rabbit is shown in Table 17 and
ranged from 1.6 ng to 25 ng (1 yg/1 = 1 ng/ml). Since the weight of each
rabbit was close to the 3 kg average of all rabbits, the dose administered
ranged from approximately 0.5 ng/kg to 8.0 ng/kg. Not a single rabbit
developed a fever. At the end of the project, every rabbit used was given
an injection of a 2 ng/kg pyrogen standard and every one developed a fever.
These results are markedly different from those observed in the AWT studies
(Table 15) where all 20 rabbits injected showed a temperature rise, 18 of
which rises were sufficient by (JSP Pyrogen testing criteria to classify the
injected waters as pyrogenic. Although the free endotoxin levels were con-
siderably higher for the AWT waters, eight of the AWT samples contained free
endotoxins at. levels observed in the water treatment plant studies.
Operation of the GAC Beds
The water treatment, plants sampled yielded a considerable range in the
length of time that the GAC beds had been in operation. Plant No. 26,as has
been mentioned, had just installed three fresh beds of carbon -- each from a
different proprietor -- with the intention of comparing them. These three
GAC beds were the newest encountered in the study. Plant No. 22 had the
longest operational run of any -- 110 months! Based upon a single pair of
samples, it was still removing 14 percent of TOC and about 50% of the total
endotoxins at the time of .sampling. Table 19 shows the months of operation,
TOC removal in mg/1 and percent, and bound endotoxin removed in yg/1 for
each GAC bed sampled. Average removal of TOC for all data is 0.7 mg/1 and
percent removal, 16.4.
55
-------
TABLE 18. OPERATING DATA FOR GAC IN WATER
TREATMENT PLANTS SAMPLED
Sample
No.
77
22
11
55
33
44 A
44 B
88
G6
99
12
14
16
18-4
18-5
24
26 A-12
26 A-14
26 A-16
28
30 A-l
30 A-2
Tl
T2
T3
T4
T5
T6
Flow
Rate
5.5
2.0
2.5
2.0
2.0
2.8
2.8
3.0
2.0
2.3
1.5
2.0
2.8
2.4
2.4
1.5
2.2
2.2
2.2
1.9
4.0
4.0
0.75
0.64
1.6
2.7
3.0
2.8
Carbon
Depth
in.
36
24
30
18
15
48
48
30
12
16
31
14
30
30
30
18
33
33
33
28
60
60
48
11
48
33
11
10
Empty
Bed
Contact
min.
4.1
7.5
7.5
5.6
4.7
10.7
10.7
6.2
3.7
4.4
12.9
4.4
2.4
7.8
7.8
7.5
9.3
9.3
9.3
9.2
9.3
9.3
39.9
10.7
18.7
7.6
2.3
2.2
Instal-
lation
Date
10/70
9/68
9/71
8/71
10/71
Fall 74
Fall 74
1/76
4/77
1973
1/76
1972
1972
6/73
6/75
1971
3/78
3/78
3/78
1972
1962
1962
1973
9/74
3/77
9/71
1971
7/68
Last
Regener-
ation
Date
8/77
9/68
5/77
8/71
10/71
Fall 74
Fall 74
1/76
Mil
4/75
1/76
9/77
7/77
6/73
6/75
11/77
3/78
3/78
3/78
10/77
1962
12/77
7/76
9/74
3/77
5/77
6/77
3/75
Temp. C12 Residual
at Before GAC
Sampling mg/1
°C Free Total
23.3
17.2
11.7
11.1
5.0
5.0
5.0
4.4
1.0
2.0
2.2
6.1
1.7
2.8
2.8
1.1
4.0
4.0
4.0
14.0
16.7
16.7
13.0
13.0
10.0
12.0
10.0
11.0
2.4
0.2
0.0
<0.1
<0.1
0.1-0.4
3.2
0.7
0.4
1.0
0..2
0,.2
0.0
1.0
1.0
1.0
0.15
1.5
<0.1
0.35
0.0
0.5
0.1
0.15
1.0
4.1
0.8
0.3-0.8
0.0
0.1
0.1
0.2-0.6
3.6
0.8
1.7
0.35
0.35
0.0
1.25
1.25
1.25
0.4
1.7
0.1
0.0
0.3
1.0
0.7
0.45
8.75
56
-------
TABLE 19. TOC AND ENDOTOXIN PERFORMANCE OF GAC WITH TIME
Sample
No.
77
22
11
55
33
44 A
44 B
88
66
99
12
14
16
18-4
18-5
24
26 A-12
26 A-14
26 A-16
28
30 A-l
30 A-2
Tl
T2
T3
T4
T5
T6
JT
Month
Sampled
9/77
9/77
10/77
10/77
11/77
12/77
12/77
12/77
1/78
1/78
2/78
2/78
2/78
2/78
2/78
3/78
3/78
3/78
3/78
4/78
4/78
4/78
4/78
4/78
4/78
4/78
4/78
4/78
Months
of
Operation
1
110
5
74
73
38
38
23
9
33
25
5
7
56
32
4
0
0
0
6
12
4
21
43
13
11
10
37
TOC
Removed
mg/1
2.4
0.6
1.5
2.3
0.6
0.9
0.8
+0.5
0.3
0.7
1.0
2.9
0.1
0.6
0.3
0.6
1.6
1.6
1.5
1.1
+1.0
1.8
1.6
+0.8
0.2
1.5
+0.7
+3.4
0.7
TOC
Removed
%
38
14
31
64
7
17
17
+8
9
23
45
56
6
22
12
15
52
52
48
31
+29
40
31
+28
5
43
+23
+103
16.4
Bound
Endotoxin
Removed
uq/1
0
1.5
12.5
6.2
0
0
0
60
12
1.8
2.3
6
6
2.5
3.7
0
+12.5
+12.5
0
0
0
0
0
2.5
2.5
10
+2.5
0
57
-------
Sample Nos. 11 and T4 are from the same plant and GAC bed. The two
samplings yielded virtually the same results in spite of the carbon being
6-months older during the second sampling. Plant No. 44 had two identical
GAC beds in parallel operation; the sampling results showed them to be per-
forming essentially identically. Plant No. 18 had 2 GAC beds operated iden-
tically, but the carbon in one bed (18-4) was 24 months older than the carbon
in the other (18-5) bed. The data show the older bed to be removing more of
the TOC but less of the bound endotoxin. However, the TOC levels are quite
low; only two samples of the 28 contained lower TOC concentrations. Plant
No. 30 utilized two GAC beds in series operation. Water passed through 30A-1
(the primary bed) first which contained spent carbon and then passed through
30 A-2 (the secondary bed) which contained the newer carbon. Of interest is
that the primary bed was the second sample that we observed up to that time
which showed an increase in TOC through the GAC bed. And it was a substantial
increase of 29 percent vs. the other sample's increase of only 8 percent.
Three more samplings among the last five samples taken during the project
were also to yield substantial increases. However, in the case of Plant No.
30, the secondary bed removed 40 percent of the applied TOC so that an overall
removal of TOC occurred through the plant. Endotoxin levels were unchanged
through both beds.
The decreases (or increases) of TOC through the GAC beds in terms of
mg/1 and percent are plotted vs. months of operation in Figures 11 and 12,
respectively. Figure 13 shows a curve of percent TOC removals with time
through a GAC bed observed in a pilot-scale study by EPA in Cincinnati, Ohio
using Ohio River water (20). Empty-bed contact time was almost, the same,
approximately 9 minutes for the pilot plant vs. our 8.75-minute average. The
percent reductions observed in our study for those beds in operation for less
than 10 months are plotted as dots on the same figure. The data seem to show
that better removals were consistently observed for Ohio River water, so
subsequently the individual empty-bed contact time in minutes for each dot
was added to the figure. Viewed along with the contact time, the data do not
seem to be as disparate with the main exception being the three clustered
data points at one-half month which are from the three beds (samples 26 A-12,
26 A-14, and 26 A-16) in the plant that was previously mentioned as experi-
menting with 3 different carbons.
The relatively common occurrence of an increase in TOC through a GAC bed
-- 5 times out of 28 samplings -- might give cause for some concern. However,
it must be remembered that these results are based upon single samples, and
on that basis some dispersion of results should be expected. We wondered
about the likelihood of a temperature-change effect in going from winter to
spring. The average temperature at the time of sampling (Table 18) for all
samples collected in the winter months -- December through March -- was
3.92°C and for all samples collected in the one spring month April -- was
12.93°C. The increase of 9°C might well be sufficient to stir some biological
activity. Endotoxin data are of no help in estimating this likelihood
(Table 17), but this should be expected in view of the AWT-water findings
(Section 6) that endotoxins appear unable to reliably predict the microbial
content of water samples which have been subjected to disinfection (C12,
03, high pH, or UV) treatments. All but one of the water treatment plants
practice prechlorination.
58
-------
o
CM
O
o
o
oo
o
oo
o
it!
S_
0)
CL
O
O
CO
CD
u_
_i o
z
o o
c_> uj
O Q-
I O
O
o
c\j
oo
CvJ
OO
O)
CO
fO
O)
S-
CJ
O)
O en
H- E
O)
co
rO 3
O) S- (_3
S- -C
-------
o
CvJ
O
O
o
<
CD
O
O)
O
O
OJ
oo
fO 3
OJS-0
s--c <
O -M C5
60
-------
Reduction
in TOC,
100
80
60
40
20
Curve from Symons (20) showing
pilot-plant data
(7*5)0*2)
(7.5)
(2.4)
(3.7)
I i
0123
4567
Months
9 10
FIGURE 13. COMPARISON OF TOC REMOVALS OBSERVED
WITH CINCINNATI PILOT-PLANT DATA
61
-------
The last six carbon beds were sampled by study-team members and dis-
solved oxygen (DO) determinations were therefore made. The before-and
after-GAC values determined were:
Sample DO content, mg/1
No. Before GAC After GAC
Tl 9.4 9.3
T2 10.8 10.7
T3 11.0 10.6
T4 10.9 10.7
T5 11.3 11.2
T6 11.8 11.8
10.87 10.72
The very slight drop reinforces the observation of only a very slight
degree of biological activity in the water treatment plant applications of
GAC.
Figure 14 is a plot of percent TOC removed vs. empty-bed contact time
in minutes. Negative removals, that is, data showing increases of TOC
through GAC, were not included in this plot. Obviously, a near vertical
line is apparent which suggests that the two parameters are unrelated. The
months of operation of the GAC associated with each data point is also shown
in the figure, and once again a non-parametric observation reveals that the
average time of operation of the 11 highest (% TOC removed) data points is
13.3 months whereas the average time of operation of the 11 lowest % removed
points is 37.7 months. The water treatment plant data assembled in this
report, therefore, are better described by the duration of operation of the
carbon than by empty-bed contact time. We believe this simply reflects that
the adsorptive capacity of the carbon beds which processed the waters sampled
in this study was quite generally exhausted, and that the removals -- or
increases -- were functions primarily of a low-level of residual adsorptive
activity in the beds coupled with an occasional mechanical sloughing.
The project inquired of all locations what tests were used to evaluate
the effectiveness of GAC operation. Table 20 lists those tests that were
cited by two or more plants. Not listed are single votes for TOC, standard
plate count, Fe, Mn, purgeable organic carbon, non-purgeable organic carbon,
alkalinity, hardness, COz, phenol no., fluoride, core samples, DO, and
abrasion no. The scatter strongly suggests the need for a rather massive
training effort to accompany any program designed to promote more widespread
application of GAC.
62
-------
60
74
50
40
« 0.5
0.5
25
11
4
TOC
Removed, JO
59
21
20
10
33
56
38
4
110
32
'73
13
10 20 30
Empty-Bed Contact, min.
FIGURE 14. PERCENT TOC REMOVED vs..
EMPTY-BED CONTACT TIME
40
63
-------
TABLE 20. MONITORING METHODS UTILIZED FOR GAC CONTROL
4-* 2^5
p-» jj
^ **""
i- -P -P i- -r-
(D
-------
REFERENCES
1. Alexander, M. Introduction to Soil Microbiology. John Wiley & Sons,
Inc., N.Y., 1961, p. 300.
2. Wachtel, R.E. and Tsuji, K. Comparison of Limulus amebocyte lysates
and correlation with the United States Pharmacopeia! pyrogen test.
Appl. Environm. Microbiol. 33_, 1265 (1977).
3. Greisman, S.E. eit al_. Mechanisms of endotoxin tolerance in man.
Bacterial Endotoxins, Ed. Landy, M. and Braun, W. Rutgers, New
Brunswick, N.J. (1964).
4. Braude, A.I. Absorption, distribution, and elimination of endotoxins
and their derivatives. Bacterial Endotoxins, Ed. Landy, M. and Braun,
W. Rutgers, New Brunswick, N.J.(1964).
5. Bennett, I.L. Approaches to the mechanisms of endotoxin action.
Bacterial Endotoxins, Ed. Landy, M. and Braun, W. Rutgers, New
Brunswick, N.J. (1964).
6. Levin, J. and Bang, F.B. Clottable protein in Limulus: Its locali-
zation and kinetics of its coagulation by endotoxin. Thromb. Diath.
Haemorrh. 19, 186 (1968).
7. U.S. Dept. Health, Education, and Welfare. Limulus amebocyte lysate.
Fed. Reg. 38, 26130 (1973).
8. Cooper, J.F., Levin, J., and Wagner, H.N. Quantitative comparison of
in vitro and in vivo methods for the detection of endotoxin. J. Lab.
Clin. Med. 78, 138 (1971).
9. DiLuzio, N.R. and Friedmann, T.J. Bacterial endotoxins in the
environment. Nature (London) 244, 49 (1973).
10. Jorgensen, J.H., Lee, J.C., and Pahren, H.R. Rapid detection of
bacterial endotoxins in drinking water and renovated wastewater.
Appl. Environm. Microbiol. 32_, 347 (1976).
11. Watson, S.W., Novitsky, T.J., Quinby, H.L., and Valois, F.W. Deter-
mination of bacterial number and biomass in the marine environment.
Appl. Environm. Microbiol. 33^, 940 (1977).
65
-------
12. Evans, T.M., Schillinger, J.E., and Stuart, D.G. Rapid determination
of bacteriological water quality by using Limulus lysate. Appl.
Environm. Microbiol. ^35_, 376 (1978).
13. Petrasek, A.C., Jr. Wastewater Characterization and Process Reli-
ability for Potable Wastewater Reclamation. EPA-600/2-77-210,
Municipal Environmental Research Lab, Cincinnati, Ohio, (Nov.1977), p.12.
14. American Public Health Association. Standard Methods for the Exami-
nation^ of Hater and Wastewater, 14th Edition.Published jointly by
APHA, AWWA, and WPCF (1976).
15. The United States Pharmacopeia, 19th Revision. The United States
Pharmacopeia Convention, Inc. Mack Printing Co., Easton, Pa.
(1975) p. 613.
16. Jorgensen, J.H., and Smith, R.F. Measurement of bound and free
endotoxin by the Limulus assay. Proc. Soc. Exp. Biol. Med. 146.
1024 (1974).
17. Tennent, D.M., and Ott, W.H. Quantitative assay of pyrogens by the
febrile response in rabbits. Intern. Congr. Anal. Chem. 77, 643
(Nov. 1952).
18. Rylander, R. et al_. Sewage workers syndrome. The Lancet, 478
(Aug. 28, 197eTy.
19. Guyton, A.C. Measurement of respiratory volumes of lab animals.
Am. J. Physio!. 150. 70 (1947).
20. Symons, J.M. Interim Treatment Guide for Controlling Organic
Contaminants in Drinking Water Using Granular Activated Carbon.
U.S. Environmental Protection Agency, Municipal Environmental Research
Lab, Cincinnati, Ohio,(Nov. 1977),p. 74.
66
-------
APPENDIX A
AWT EFFLUENT SAMPLES,
BEFORE SHIPMENT
Sample No. 10-5
pH, units 6.8
Spec. cond., ymhos at 25°C 660
Total alkalinity, CaC03 73
Total hardness, CaC03 120
NH3 -N as N 0.4
Org-N as N Q.2
N03 as N 12.6
N02 as N .007
Total P 5.8
Cl" as NaCl 69.7
COD 10.6
Turbidity, NTU .31
Color, units o
TSS ' i
TDS 466
Standard plate count per ml 29 x 10
Total coliforms per 100 ml 3 x 10
All values are in mg/1 unless otherwise noted,
67
-------
AWT EFFLUENT SAMPLES,
BEFORE SHIPMENT
Sample 10-12
pH, units 6.7
Spec. cond., pmhos at 25°C 650
Total alkalinity, CaC03 46
Total hardness, CaC03 120
NH3-N as N 0
Org-N as N 0.4
N03 as N 12.8
N02 as N .07
Total P 5.6
Cl" as NaCl 67.2
COD 10.0
Turbidity, NTU 0.5
Color, units 0
TSS 0
TDS 496
Standard plate count per ml 33 x 10
Total coliforms per 100 ml 8x10
All values are in mg/1 unless otherwise noted.
68
-------
AWT EFFLUENT SAMPLES,
BEFORE SHIPMENT
Sample No. 10-19
pH, units 6.6
Spec. cond., nmhos at 25°C 630
Total alkalinity, CaC03 49
Total hardness, CaC03 150
NH3 -N as N 0
Org-N as N 1.1
N03 as N 12.7
NO2 as N 0.0
Total P 5.6
Cl" as NaCl 85.0
COD 9.1
Turbidity, NTU 0.5
Color, units 0
TSS 0
TDS 502
Standard plate count per ml 63 x 10
Total coliforms per 100 ml 47 x 10
All values are in mg/1 unless otherwise noted.
69
-------
AWT EFFLUENT SAMPLES,
BEFORE SHIPMENT
Sample No. 10-26
pH, units 6.8
Spec, cond., umhos at 25°C 590
Total alkalinity, CaC03 51
Total hardness, CaC03 120
NH3 -N as N 0
Org-N as N 0.7
N03 as N 12.0
N02 as N 0.04
Total P 5.6
Cl" as NaCl 72.0
COD 7.0
Turbidity, NTU 0.5
Color, units 0
TSS 0
TDS 488
Standard plate count per ml 79 x 10
Total coliforms per 100 ml 40 x 10
All values are in mg/1 unless otherwise noted
70
-------
AWT EFFLUENT SAMPLES,
BEFORE SHIPMENT
Sample No. 11-9
pH, units 6.9
Spec. cond., ymhos at 25°C 530
Total alkalinity, CaC03 79
Total hardness, CaC03 136
NH3 -N as N 0.0
Org-N as N 1.1
N03 as N 13.2
N02 as N 0.07
Total P 5.8
Cl" as Nad 72.5
COD 11.4
Turbidity, NTU 0.4
Color, units 5
TSS 1
TDS 492
Standard plate count per ml 29 x 10
Total coliforms per 100 ml 75 x 101
All values are in mg/1 unless otherwise noted.
71
-------
AWT EFFLUENT SAMPLES,
BEFORE SHIPMENT
Sample No. 11-16
pH, units 6.5
Spec, cond., pmhos at 25°C 520
Total alkalinity, CaC03 66
Total hardness, CaC03 120
NH3 -N as N 0
Org-N as N 0.4
N03 as N 18.2
N02 as N 0.7
Total P 4.4
Cl" as NaCI 69.6
COD 6.5
Turbidity, NTU 0.4
Color, units 0
TSS 1
TDS 464
2
Standard plate count per ml 82 x 10
2
Total coliforms per ml 49 x 10
All values are in mg/1 unless otherwise noted.
72
-------
AWT EFFLUENT SAMPLES,
BEFORE SHIPMENT
Sample No. 11-23
pH, units 6.7
Spec. cond., ymhos at 25°C 610
Total alkalinity, CaC03 53
Total hardness, CaC03 118
NH3 -N as N 0.0
Org-N as N 0.6
N03 as N 15.4
N02 as N 0.01
Total P 5.8
Cl" as NaCl 76.8
COD 11.6
Turbidity, NTU 0.4
Color, units 5
TSS 0
TDS 496
9
Standard plate count per ml 62 x 10
Total coliforms per 100 ml 49 x 10
All values are in mg/1 unless otherwise noted.
73
-------
AWT EFFLUENT SAMPLES,
BEFORE SHIPMENT
Sample No. 12-7
pH, units 6.8
Spec. cond., pmhos at 25°C 550
Total alkalinity, CaC03 89
Total hardness, CaC03 154
NH3 -N as N 0.0
Org-N as N 1.0
N03 as N 14.7
N02 as N 0.1
Total P 7.0
Cl" as NaCl 96.2
COD 9.5
Turbidity, NTU 0.5
Color, units 0
TSS 0
TDS 479
2
Standard plate count per ml 35 x 10
Total coliforms per 100 ml 36 x 10
All values are in mg/1 unless otherwise noted
74
-------
APPENDIX B
CARBON COLUMN SAMPLES,
BEFORE SHIPMENT
Sample No. 12-14
pH, units
Spec. cond., ymhos at 25°C
Total alkalinity, CaC03
Total hardness, CaC03
NH3 -N as N
Org-N as N
N03 as N
N02 as N
Total P
Cl" as NaCl
COD
Turbidity, NTU
Color, units
TSS
TDS
Standard plate count per ml
Total coliforms per 100 ml
Influent
7.1
670
108
172
0
2.0
11.9
0
5.3
89.0
23.4
1.5
30
4
490
Midpoint
7.0
670
98
170
0
1.5
12.4
0
4.9
89.0
17.7
.68
10
1
478
Effluent
6.0
650
94
168
0
1.3
12.8
0
4.8
91.4
9.4
.62
5
1
460
22x10^
eoxio'
29x10^
49x10'
1
34x10
1
All values are in mg/1 unless otherwise noted.
75
-------
CARBON COLUMN SAMPLES,
BEFORE SHIPMENT
Sample No. 12-21
pH, units
Spec, cond., ymhos at 25°C
Total alkalinity, CaC03
Total hardness, CaC03
NH3-N as N
Org-N as N
N03 as N
N02 as N
Total P
Cl" as NaCl
COD
Turbidity, NTU
Color, units
TSS
TDS
Standard plate count per ml
Total coliforms per 100 ml
Influent
6.8
670
92
150
0
2.9
14.2
0
5.3
74.4
22.2
1.2
50
2
562
34xl03
60x1 O4
Midpoint
6.7
650
87
146
0
2.2
14.0
0
4.8
72
17.8
0.5
15
1
498
24x1 0?
90x1 O2
Effluent
6.9
650
92
156
0
1.4
14.8
0
4.8
72
14.3
0.4
10
0
520
75xl02
96x1 O2
All values are in mg/1 unless otherwise noted.
76
-------
CARBON COLUMN SAMPLES,
BEFORE SHIPMENT
Sample No. 1-25
pH, units
Spec. cond., ymhos at 25°C
Total alkalinity, CaC03
Total hardness, CaC03
NH3-N as N
Org-N as N
N03 as N
N02 as N
Total P
Cl" as NaCl
COD
Turbidity, NTU
Color, units
TSS
TDS
Standard plate count per ml
Total coliforms per 100 ml
Influent
7.1
105
146
0
2.0
6.8
.00
4.3
69.7
22.5
2.0
30
8
540
38x1 O3
49x1 O4
Midpoint
7.0
79
98
0
1.4
5.9
0
3.2
55.3
14.7
1.0
20
1
562
21xl02
14xl02
Effluent
6.8
93
142
0
0.7
9.6
.01
4.4
72.1
9.0
0.6
5
1
498
39xl02
64xlO]
All values are in mg/1 unless otherwise noted.
77
-------
APPENDIX C
AWT EFFLUENT DISINFECTION SAMPLES,
BEFORE SHIPMENT
Sample No. 2-8 pH UV 03 C12
pH, units 6.9 7.3 7.4 7.3
Spec, cond., prnhos at 25°C
Total alkalinity, CaC03 32 116 105 117
Total hardness, CaC03 700 180 156 180
NH3-N as N 0000
Org-N as N .84 .56 .35 0
N03 as N 5.8 6.1 6.0 6.4
N02 as N .01 .01 .00 .01
Total P .02 3.6 4.0 4.0
Cl" as NaCl 348 55.3 65 288
COD 23.2 12.0 9.4 11.7
Turbidity, NTU .23 .35 .25 .35
Color, units 5 ' 5 5 5
TSS 0000
TDS 1322 444 382 440
Standard plate count per IT
Total coliforms per 100 ml
Standard plate count per ml 1x10° 112x10° 43x10° 0x10°
All values in mg/1 unless otherwise noted.
C12 = free residual chlorine, 0.9 mg/1.
03 - ozone dose applied, 8.4 mg/1.
pH = raised to 11.56, then neutralized to 6.9.
78
-------
AWT EFFLUENT DISINFECTION SAMPLES,
BEFORE SHIPMENT
Sample No. 2-28 pH UV 03 C12
pH, units 6.9 7.2 7.0 7.0
Spec, cond., Mmhos at 25°C
Total alkalinity, CaC03 49 95 88 89
Total hardness, CaC03 300 142 140 132
NH3-N as N 0000
Org-N as N 2.3 1.5 1.6 1.5
N03 as N 5.8 6.0 7.8 5.8
N02 as N .02 .01 0 .01
Total P 1.1 3.2 3.0 3.2
Cl" as NaCl 69.7 75.0 65 67
COD 11.8 11.2 9.3
Turbidity, NTU 0.5 0.4 0.4 0.4
Color, units 55 05
TSS 4001
TDS 1240 490 508 504
Standard plate count per ml 0 0 0 4x10°
Total coliforms per 100 ml 0 2x10° 0 0
All values in mg/1 unless otherwise noted.
C12 = free residual chlorine, 1.02 mg/1.
03 = ozone dose applied, 8.3 mg/1.
pH = raised to -11.53, then neutralized to 6.9.
79
-------
AWT EFFLUENT DISINFECTION SAMPLES,
BEFORE SHIPMENT
Sample No. 3-14 pH UV 03 C12
pH, units 6.9 7.0 7.0 6.7
Spec, cond., umhos at 25°C
Total alkalinity, CaC03 149 120 97 97
Total hardness, CaC03 356 204 194 200
NH3-N as N 0000
Org-N as N 1.1 2.0 0.6 1.2
N03 as N 9.6 5.6 4.4 4.8
N03 as N 0.03 0.11 0.01 0.04
Total P .62 4.0 3.9 4.2
Cl" as NaCl 84 77 75
COD 13.3 12.6 2.6
Turbidity, NTU 1.8 .65 .45 .65
Color, units 10 15 5 10
TSS 2101
TDS 1400 450 420 480
Standard plate count per ml 27x10° 81x10° 3x10° 8x10°
Total coliforms per 100 ml 0 7x10° 0 0
All values in mg/1 unless otherwise noted.
Cl2 = free residual chlorine, 1.2 mg/1.
03 = ozone dose applied, 7.5 mg/1.
pH = raised to 11.4, then neutralized to 6.9.
80
-------
AWT EFFLUENT DISINFECTION SAMPLES,
BEFORE SHIPMENT
Sample No. 3-28 pH UV 03 C12
pH, units 6.2 7.0 7.0 6.9
Spec. cond., ymhos at 25°C
Total alkalinity, CaC03 29 119 112 100
Total hardness, CaC03 398 210 192 160
NH3-N as N 0000
Org-N as N 01.50 .08
N03 as N 7.2 7.2 7.1 7.0
N02 as N 0.008 0.008 0.002 0.002
Total P 2.6 2.4 2.6 3.4
Cl" as NaCl 48.1 89.0 86.6 96.2
COD 7.0 7.8 7.3 11.0
Turbidity, NTU 1.4 0.28 0.37 0.48
Color, units 55 55
TSS 0100
TDS 914 386 448 408
Standard plate count per ml 13x10° 280x10° 3x10° 3x10°
Total coliforms per 100 0x10° 4x10° 1x10° 0x10°
All values in mg/1 unless otherwise noted.
C12 = free residual chlorine = 1.4 mg/1.
03 = ozone dose applied, 4.9 mg/1.
pH = raised to 11.8, the neutralized to 6.2.
81
-------
AWT EFFLUENT DISINFECTION SAMPLES,
BEFORE SHIPMENT
Sample No. 4-4 pH UV 03 C12
pH, units 7.1 7.2 7.2 7.0
Spec, cond., ymhos at 25°C - - -
Total alkalinity, CaC03 49 149 141 141
Total hardness, CaC03 416 204 198 204
NH3-N as N 0000
Org-N as N 0.9 0.8 0.8 0.7
N03 as N 7.8 6.4 5.6 6.0
N02 as N 0.06 0.02 0.03 0.02
Total P 1.5 3.7 3.3 3.8
Cl" as NaCl 84 98 94 106
COD 8.6 9 9.2 9.4
Turbidity, NTU 2.0 .45 .35 0.45
Color, units 5 ' 10 5 5
TSS 2110
TDS 814 506 480 586
Standard plate count per ml 26 140 3 26
Total coliforms per 100 ml 0 14 0 0
All values in mg/1 unless otherwise noted.
C12 = free residual chlorine, 1.5 mg/1.
03 = ozone dose applied, 8.9 mg/1.
pH = achieved, 12.5.
82
-------
AWT EFFLUENT DISINFECTION SAMPLES,
BEFORE SHIPMENT
He No. 4-11
pH, units
Spec. cond. , ymhos at 25°C
Total alkalinity, CaC03
Total hardness, CaC03
NH3-N as N
Org-N ad N
N03 as N
N02 as N
Total P
Cl" as NaCl
COD
Turbidity, NTU
Color, units
TSS
TDS
Standard plate count per ml
Total col i forms per 100 ml
PH
8.3
-
39
354
0
-
15.2
-
-
90
18
0.85
20
8
724
17
0
UV
7.3
-
91
192
0
-
10.8
-
-
84
10.7
0.3
10
3
562
240
6
03
7.3
-
68
154
0
-
14.4
-
-
54
9.1
0.25
5
4
462
8
0
C12
7.2
-
71
196
0
-
15.2
-
-
62
12
0.3
10
5
476
5
1
All values in mg/1 unless otherwise noted.
Cl2 = free residual chlorine, 2.0 mg/1 .
O3 = ozone dose applied, 5.58 mg/1 .
pH = achieved, 11.5.
83
-------
AWT EFFLUENT DISINFECTION SAMPLES,
BEFORE SHIPMENT
Sample No. 4-18 pH UV 03 C12
pH, units 7.9 6.9 7.1 7.3
Spec, cond., umhos at 25°C - - -
Total alkalinity, CaC03 63 140 136 138
Total hardness, CaC03 258 230 218 232
NH3-N as N 0000
Org-N as N 1.5 0.6 0.6 1.3
N03 as N 9.6 9.6 9.2 9.3
N02 as N - -
Total P 0.3 5.2 4.2 4.6
Cl" as NaCl 84 66 74 82
COD 11.1 10.4 11.9 12
Turbidity, NTU 0.6 0.5 0.3 0.4
Color, units 15 25 10 15
TSS 2160
TDS 748 540 474 456
Standard plate count per ml 60 79 50 65
Total coliforms per 100 ml 0 15 0 0
All values in mg/1 unless otherwise noted.
C12 = free residual chlorine, 1.6 mg/1.
03 = ozone dose applied, 9. mg/1.
pH = achieved, 11.3.
84
-------
APPENDIX D
WATER TREATMENT PLANT SAMPLES
Sample # 77
Treatment prior to GAC:
Prechlorination, Alum-lime Coagulation, Sedimentation
GAC installation date: Oct. 1970
Last regeneration: New carbon Sept. 1977
Depth of GAC, in.: 36
Flow rate, gpm/ft2: 5.5
Empty bed contact time, min: 4.1
C12 residual before GAC, mg/1:
0.0 mg/1 free, 1.0 mg/1 total
pH value
TOC, mg/1
Endotoxins (LAL) Unfiltered, ng/ml
Endotoxins (LAL) Filtered, ng/ml
Endotoxins (Rabbit Assay) Filtered, ng/ml
Chlorine Residual, mg/1
Temperature at sampling, °C
Temperature on arrival, °C
Dissolved oxygen, mg/1
Total plate count organisms per ml 74
Col i forms per 100 ml, Mi Hi pore 0
Coliforms per 100 ml, MPN <2
Fecal coliforms per 100 ml, MPN <2
Coliphage, Pfu/ml <2
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa
Percent of total plate count
P. fluorescens
**
Before-*
6.7
6.4
3.1
3.1
<2
<0.05
23.3
10.0
After-*
6.6
4.0
3.1
3.1
<2
<0.05
23.3
12.0
34
0
<2
<2
<2
* GAC Filtration.
** Lower detection limit.
85
-------
WATER TREATMENT PLANT SAMPLES
Sample # 22
Treatment prior to GAC:
Prechlorination,Lime-softening, Sedimentation
GAC installation date: September 1968
Last regeneration: None
Depth of GAC, in.: 24
Flow rate, gpm/ft2: 2
Empty bed contact time, min: 7.5
C12 residual before GAC, mg/1:
2.4 mg/1 free, 4.1 mg/1 total
Before-* After-*
pH value 7.7 7.7
TOC, mg/1 4.4 3.8
Endotoxins (LAL) Unfiltered, ng/ml 3-1 1-6
Endotoxins (LAL) Filtered, ng/ml !-6 I-6
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C M.2. 17.2
Temperature on arrival, °C H-° H-°
Dissolved oxygen, mg/1
Total plate count organisms per ml 18 H
Coliforms per 100 ml, Millipore 0 °
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa
Percent of total plate count
P. fluorescens
* GAC Filtration.
** Lower detection limit.
86
-------
WATER TREATMENT PLANT SAMPLES
Sample # 11
Treatment prior to GAC:
Prechlorination, Alum-lime Coagulation, Sedimentation
GAC installation date: 1971
Last regeneration: 24 inches Virgin carbon, May 1977
Depth of GAC, in.: 30
Flow rate, gpm/ft2: 2.5
Empty bed contact time, min: 7.5
C12 residual before GAC, mg/1:
0.20 mg/1 free, 0.80 mg/1 total
Before-* After-*
pH value 6.7 6.6
TOC, mg/1
Endotoxins (LAL) Unfiltered, ng/ml 15.6 3.1
Endotoxins (LAL) Filtered, ng/ml 3.1 3.1
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 11.7 11.7
Temperature on arrival, °C 9.0 9.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 25 5
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa
Percent of total plate count
P. fluorescens
* GAC Filtration.
** Lower detection limit.
87
-------
WATER TREATMENT PLANT SAMPLES
Sample # 55
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: 1971
Last regeneration: None
Depth of GAC, in.: 18
Flow rate, gpm/ft2: 1.75
Empty bed contact time, min: 6.4
C12 residual before GAC, mg/1:
0.0 mg/1 free, 0.3-0.8 mg/1 total
Before-* After-*
pH value 6.7 6.7
TOC, mg/1 3.6 1.3
Endotoxins (LAL) Unfiltered, ng/ml 12.5 6.3
Endotoxins (LAL) Filtered, ng/ml 6.3 6.3
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2.0 <2.0
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 11.1 11-1
Temperature on arrival, °C 8.0 8.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 00
Col i forms per 100 ml, Mi Hi pore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa
Percent of total plate count
P. fluorescens
* GAC Filtration.
** Lower detection limit.
88
-------
WATER TREATMENT PLANT SAMPLES
Sample # 33
Treatment prior to GAC:
Powdered GAC, Aeration, Alum-Nalco 607 Coagulation, Sedimentation
GAC installation date: 1971
Last regeneration: None
Depth of GAC, in.: 15
Flow rate, gpm/ft2: 2
Empty bed contact time, min: 4.7
C12 residual before GAC, mg/1:
0.0 mg/1 free,0.0 mg/1 total
(no prechlorination)
Before-* After-*
pH value 7.2 7.2
TOC, mg/1
Endotoxins (LAL) Unfiltered, ng/ml 31.3 31.3
Endotoxins (LAL) Filtered, ng/ml 12.5 12.5
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 5.0 5.0
Temperature on arrival, °C 13-0 13.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 39 4
Col i forms per 100 ml, Mi Hi pore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 90 100
Percent of total plate count
Pseudomonas aeruginosa 30 0
Percent of total plate count
P. fluorescens
* GAC Filtration.
** Lower detection limit-
89
-------
WATER TREATMENT PLANT SAMPLES
Sample # 44 A
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation, Sand Filtration
GAC installation date: Fall 1974
Last regeneration: None
Depth of GAC, in.: 48
Flow rate, gpm/ft2: 2.8
Empty bed contact time, min: 10.7
C12 residual before GAC, mg/1:
<0.1 mg/1 free, 0.1 mg/1 total
Before-* After-*
pH value 6.6 6.7
TOC, mg/1 5.3 4.4
Endotoxins (LAL) Unfiltered, ng/ml 6.0 6.0
Endotoxins (LAL) Filtered, ng/ml 2.4 2.4
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 5.0 5.0
Temperature on arrival, °C 6.0 6.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 00
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens
* GAC Filtration.
** Lower detection limit.
90
-------
WATER TREATMENT PLANT SAMPLES
Sample # 44 B
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation, Sand Filtration
GAC installation date: Fall 1974
Last regeneration: None
Depth of GAC, in.: 48
Flow rate, gpm/ft2: 2.8
Empty bed contact time, min: 10.7
C12 residual before GAC, mg/1:
<0.1 mg/1 free, 0.1 mg/1 total
Before-* After-*
pH value
TOC, mg/1 4.8 4.0
Endotoxins (LAL) Unfiltered, ng/ml 6.0 6.0
Endotoxins (LAL) Filtered, ng/ml 2.4 2.4
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 5.0 5.0
Temperature on arrival, °C 6.0 6.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 0 1
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens
* GAC Filtration.
** Lower detection limit-
91
-------
WATER TREATMENT PLANT SAMPLES
Sample # 88
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: January 1976
Last regeneration: None
Depth of GAC, in.: 30
Flow rate, gpm/ft2: 3
Empty bed contact time, min: 6.2
C12 residual before GAC, mg/1:
0.0 mg/1 free,0.0mg/l total
Before-* After-*
pH value 7.9 7.9
TOC, mg/1 5.9 6.4
Endotoxins (LAL) Unfiltered, ng/ml 120 60
Endotoxins (LAL) Filtered, ng/ml 24 24
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 4.4 4.4
Temperature on arrival, °C 10.0 9.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 88 8
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 22 100
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens " 0 0
* GAC Filtration.
** Lower detection limit.
92
-------
WATER TREATMENT PLANT SAMPLES
Sample # 66
Treatment prior to GAC:
Prechlorination, Coagulation, Sedimentation
GAC installation date: April 1977
Last regeneration: None
Depth of GAC, in.: 12
Flow rate, gpm/ft2: 2
Empty bed contact time, min: 3.7
C12 residual before GAC, mg/1:
0.1-0.4 mg/1 free, 0.2-0.6 mg/1 total
Before-* After-*
pH value 7.8 7.5
TOC, mg/1 3.2 2.9
Endotoxins (LAL) Unfiltered, ng/ml 36 18
Endotoxins (LAL) Filtered, ng/ml 18 12
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 1-0 1.0
Temperature on arrival, °C 3.5 3.5
Dissolved oxygen, mg/1
Total plate count organisms per ml 32 2
Coliforms per 100 ml, Mi 11ipore 9 0
Coliforms per 100 ml, MPN 46 <2
Fecal coliforms per 100 ml, MPN 12 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 30
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit.
93
-------
WATER TREATMENT PLANT SAMPLES
Sample # 99
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: 1973
Last regeneration: April 1975
Depth of GAC, in.: 16
Flow rate, gpm/ft2: 2.29
Empty bed contact time, min: 4.4
C12 residual before GAC, mg/1:
3.25 mg/1 free, 3.60 mg/1 total
Before-* After-*
pH value 8.0 8.0
TOC, mg/1 3.0 2.3
Endotoxins (LAL) Unfiltered, ng/ml 6.0 6.0
Endotoxins (LAL) Filtered, ng/ml 0.6 2.4
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 1-75 <0.05
Temperature at sampling, °C 2.0 2.0
Temperature on arrival, °C 5.0 6.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 00
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit.
94
-------
WATER TREATMENT PLANT SAMPLES
Sample # 12
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: January 1976
Last regeneration: None
Depth of GAC, in.: 31
Flow rate, gpm/ft2: 1.5
Empty bed contact time, min: 12.9
C12 residual before GAC, mg/1:
0.67 mg/1 free, 0.82 mg/1 total
Before-* After-*
pH value 7.2 7.2
TOC, mg/1 2.2 1.2
Endotoxins (LAL) Unfiltered, ng/ml 3.6 2.4
Endotoxins (LAL) Filtered, ng/ml 0.06 1.2
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 2.2 2.2
Temperature on arrival, °C 7.0 7.0
Dissolved oxygen, mg/1
Total plate count 'organisms per ml 14 1
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 40
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit.
95
-------
WATER TREATMENT PLANT SAMPLES
Sample # 14
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: 1972
Last regeneration: Sept. 1977
Depth of GAC, in.: 14
Flow rate, gpm/ft2: 2
Empty bed contact time, min: 4.4
C12 residual before GAC, mg/1:
0.4 mg/1 free, - mg/1 total
Before-* After-*
pH value 7.7 7.7
TOC, mg/1 5.2 2.3
Endotoxins (LAL) Unfiltered, ng/ml 60 60
Endotoxins LAL) Filtered, ng/ml 12 18
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 6.1 6.1
Temperature on arrival, °C 7.0 7.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 86 20
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 50 30
Percent of total plate count
Pseudomonas aeruginosa 0 70
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration .
** Lower detection limit.
96
-------
WATER TREATMENT PLANT SAMPLES
Sample # 16
Treatment prior to GAC:
Prechlorination, Ferric Sulfate Coagulation, Sedimentation
GAC installation date: 1972
Last regeneration: July 1977
Depth of GAC, in.: 30
Flow rate, gpm/ft2: 2.8
Empty bed contact time, min: 2.44
C12 residual before GAC, mg/1:
1.0 mg/1 free, 1.7 mg/1 total
Before-* After-*
pH value
TOC, mg/1
Endotoxins (LAL) Unfiltered, ng/ml
Endotoxins (LAL) Filtered, ng/ml
Endotoxins (Rabbit Assay) Filtered, ng/ml**
Chlorine Residual, mg/1
Temperature at sampling, °C
Temperature on arrival, °C
Dissolved oxygen, mg/1
Total plate count organisms per ml
Coliforms per 100 ml, Millipore
Coliforms per 100 ml, MPN
Fecal coliforms per 100 ml, MPN
Coliphage, Pfu/ml
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa
Percent of total plate count
P. fluorescens
7.5
1.8
18
12
<2
<0.05
1.7
9.0
15
0
<2
<2
<2
60
0
0
7.4
1.7
12
12
<2
<0.05
1.7
9.0
0
0
<2
<2
<2
0
0
* GAC Filtration.
** Lower detection limit
97
-------
WATER TREATMENT PLANT SAMPLES
Sample # 18-4
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: June 1973
Last regeneration: None
Depth of GAC, in.: 30
Flow rate, gpm/ft2: 2.4
Empty bed contact time, min: 7.79
C12 residual before GAC, mg/1:
0.2 mg/1 free, 0.35 mg/1 total
Before-* After-*
pH value 6.4 6.4
TOC, mg/1 2.7 2.1
Endotoxins (LAL) Unfiltered, ng/ml 5.0 2.4
Endotoxins (LAL) Filtered, ng/ml 1.3 1.3
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 2.8 2.8
Temperature on arrival, °C 13.0 13.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 1 0
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 100
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit.
98
-------
WATER TREATMENT PLANT SAMPLES
Sample # 18-5
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: June 1975
Last regeneration: None
Depth of GAC, in.: 30
Flow rate, gpm/ft2: 2.4
Empty bed contact time, min: 7.79
C12 residual before GAC, mg/1:
0.2 mg/1 free, 0.35 mg/1 total
Before-* After-*
pH value 6.9 6.9
TOC, mg/1 2.5 2.2
Endotoxins (LAL) Unfiltered, ng/ml 5.0 1.3
Endotoxins (LAL) Filtered, ng/ml 1.3 1.3
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 2.8 2.8
Temperature on arrival, °C 13.0 13.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 3 1
Col i forms per 100 ml, Mi Hi pore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 100 100
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit.
99
-------
WATER TREATMENT PLANT SAMPLES
Sample # 24
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: 1971
Last regeneration: Nov. 1977
Depth of GAC, in.: 18
Flow rate, gpm/ft2: 1.5
Empty bed contact time, min: 7.48
C12 residual before GAC, mg/1:
0.0 mg/1 free, 0.0 mg/1 total
Before-* After-*
pH value 6.9 6.8
TOC, mg/1 3.9 3.3
Endotoxins (LAL) Unfiltered, ng/ml 37.5 37.5
Endotoxins (LAL) Filtered, ng/ml 5.0 5.0
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 1.1 1,1
Temperature on arrival, °C 8.0 8.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 48 5
Coliforms per 100 ml, Millipore 4 0
Coliforms per 100 ml, MPN 49 <2
Fecal coliforms per 100 ml, MPN 2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 90 100
Percent of total plate count
Pseudomonas aeruginosa 10 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit.
100
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WATER TREATMENT PLANT SAMPLES
Sample # 26 A-12
Treatment prior to GAC: Prechlorination, Precaustic (NaOH),
Alum-coagulation, Sedimentation
GAC installation date: March 1978 (15-days operation)
Last regeneration: None
Depth of GAC, in.: 33
Flow rate, gpm/ft2: 2.15
Empty bed contact time, min: 9.35
C12 residual before GAC, mg/1:
1.0 mg/1 free, 1.25 mg/1 total
Before-* After-*
pH value 6.4 6.4
TOC, mg/1 3.1 1.5
Endotoxins (LAL) Unfiltered, ng/ml 12.5 25.0
Endotoxins (LAL) Filtered, ng/ml 12.5 12.5
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 4.0 4.0
Temperature on arrival, °C 9.0 9.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 0 320
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative - 100
Percent of total plate count
Pseudomonas aeruginosa - 0
Percent of total plate count
P. fluorescens - 0
* GAC Filtration.
** Lower detection limit.
101
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WATER TREATMENT PLANT SAMPLES
Sample # 26 A-14
Treatment prior to GAC: Prechlorination, Precaustic (NaOH),
Alum-coagulation, Sedimentation
GAC installation date: March 1978 (15 days-operation)
Last regeneration: None
Depth of GAC, in.: 33
Flow rate, gpm/ft2: 2.15
Empty bed contact time, min: 9.35
C12 residual before GAC, mg/1:
1.0 mg/1 free, 1.25 mg/1 total
pH value
TOC, mg/1
Endotoxins (LAL) Unfiltered, ng/ml
Endotoxins (LAL) Filtered, ng/ml
Endotoxins (Rabbit Assay) Filtered, ng/ml**
Chlorine Residual, mg/1
Temperature at sampling, °C
Temperature on arrival, °C
Dissolved oxygen, mg/1
Total plate count organisms per ml
Coliforms per 100 ml, Millipore
Coliforms per 100 ml, MPN
Fecal coliforms per 100 ml, MPN
Coliphage, Pfu/ml
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa
Percent of total plate count
P. fluorescens
Before-*
6.4
3.1
12.5
12.5
<2
<0-05
4.0
9.0
After-*
6.4
1.5
25.0
12.5
<2
<0.05
4.0
9.0
0
0
<2
<2
<2
980
0
<2
<2
<2
100
* GAC Filtration.
** Lower detection limit.
102
-------
WATER TREATMENT PLANT SAMPLES
Sample # 26 A-16
Treatment prior to GAC: Prechlorination, Precaustic (NaOH),
Alum-coagulation, Sedimentation
GAC installation date: March 1978 (15-days operation)
Last regeneration: None
Depth of GAC, in.: 33
Flow rate, gpm/ft2: 2.15
Empty bed contact time, min: 9.35
C12 residual before GAC, mg/1:
1.0 mg/1 free, 1.25 mg/1 total
Before-* After-*
pH value 6.4 6.4
TOC, mg/1 3.1 1.4
Endotoxins (LAL) Unfiltered, ng/ml 12.5 12.5
Endotoxins (LAL) Filtered, ng/ml 12.5 12.5
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 4.0 4.0
Temperature on arrival, °C 9.0 9.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 00
Coliforms per 100 ml, Mi Hi pore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa
Percent of total plate count
P. fluorescens
* GAC Filtration.
** Lower detection limit.
103
-------
WATER TREATMENT PLANT SAMPLES
Sample # 28
Treatment prior to GAC:
Prechlorination, Powered-carbon, Potassium Permanganate
GAC installation date: 1972
Last regeneration: Oct. 1977
Depth of GAC, in.: 24
Flow rate, gpm/ft2: 1-9
Empty bed contact time, min: 9.18
C12 residual before GAC, mg/1:
0.15 mg/1 free, 0.4 mg/1 total
Before-* After-*
pH value 7.6 7.6
TOC, mg/1 3.5 2.4
Endotoxins (LAL) Unfiltered, ng/ml 25.0 25.0
Endotoxins (LAL) Filtered, ng/ml 12.5 12.5
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 14.0 14.0
Temperature on arrival, °C 7.0 7.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 00
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa
Percent of total plate count
P. fluorescens
* GAC Filtration.
** Lower detection limit-
104
-------
WATER TREATMENT PLANT SAMPLES
Sample # 30 Primary GAC
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: Dec. 1977
Last regeneration: None
Depth of GAC, in.: 60
Flow rate, gpm/ft2: 4.0
Empty bed contact time, min: 9.35
C12 residual before GAC, mg/1:
1.5 mg/1 free, 1.7 mg/1 total (spent carbon)
Before-* After-*
pH value 6.8 6.8
TOC, mg/1 3.5 4.5
Endotoxins (LAL) Unfiltered, ng/ml 25.0 25.0
Endotoxins (LAL) Filtered, ng/ml 12.5 12.5
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 16.7 16.7
Temperature on arrival, °C 12.0 12.0
Dissolved oxygen, mg/1
Total plate count organisms per ml 1 64
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative - 100
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. f1uorescens 0 0
* GAC Filtration.
** Lower detection limit.
105
-------
WATER TREATMENT PLANT SAMPLES
Sample # 30 Secondary GAC
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: Dec. 1977
Last regeneration: None
Depth of GAC, in.: 60
Flow rate, gpm/ft2: 4.0
Empty bed contact time, min: 9.35
C12 residual before GAC, mg/1:
<0.1 mg/1 free, 0.1 mg/1 total (virgin carbon)
Before-* After-*
pH value 6.8 6.8
TOC, mg/1 4.5 2.7
Endotoxins (LAL) Unfiltered, ng/ml 25.0 25.0
Endotoxins (LAL) Filtered, ng/ml 12.5 12.5
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 16.7 16.7
Temperature on arrival, °C 12.0 12.0
Dissolved oxygen, mg/1 64 241
Total plate count organisms per ml
Col i forms per 100 ml, Mi Hi pore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 100 90
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit.
106
-------
WATER TREATMENT PLANT SAMPLES
Sample # Tl
Treatment prior to GAC:
Prechlorination, Precaustic(NaOH), Alum-coagulation, Sedimentation
GAC installation date: 1973
Last regeneration: July 1976
Depth of GAC, in.: 48
Flow rate, gpm/ft2: 0.75
Empty bed contact time, min: 39.89
C12 residual before GAC, mg/1:
0.35 mg/1 free, - total
Before-* After-*
pH value 5.8 5.8
TOC, mg/1 5.2 3.6
Endotoxins (LAL) Unfiltered, ng/ml 25.0 25.0
Endotoxins (LAL) Filtered, ng/ml 12.5 12.5
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0,05
Temperature at sampling, °C 13.0 13.0
Temperature on arrival, °C 7.0 7.0
Dissolved oxygen, mg/1 9.4 9.3
Total plate count organisms per ml 33
Coliforms per 100 ml, Millipore 0 °
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 50 50
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit'
107
-------
WATER TREATMENT PLANT SAMPLES
Sample # T2
Treatment prior to GAC: Prechlorination, Precaustic (NaOH),
Diatomatious Earth, Alum-coagulation, Sedimentation
GAC installation date: Sept. 1974
Last regeneration: None
Depth of GAC, in.: 11
Flow rate, gpm/ft2: 0.64
Empty bed contact time, min: 10.71
C12 residual before GAC, mg/1:
- mg/1 free, 0.0 mg/1 total
Before-* After-*
pH value
TOC, mg/1
Endotoxins (LAL) Unfiltered, ng/ml
Endotoxins (LAL) Filtered, ng/ml
Endotoxins (Rabbit Assay) Filtered, ng/ml**
Chlorine Residual, mg/1
Temperature at sampling, °C
Temperature on arrival, °C
Dissolved oxygen, mg/1
Total plate count organisms per ml
Coliforms per 100 ml, Millipore
Coliforms per 100 ml, MPN
Fecal coliforms per 100 ml, MPN
Coliphage, Pfu/ml
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa
Percent of total plate count
P. fluorescens
5.9
2.9
12.5
2.5
<2
<0.05
13.0
8.0
10.8
8
0
<2
<2
<2
100
0
0
5.9
3.7
10.0
2.5
<2
<0.05
13.0
8.0
10.7
11
0
<2
<2
<2
100
0
0
* GAC Filtration.
** Lower detection limit.
108
-------
WATER TREATMENT PLANT SAMPLES
Sample # T3
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: March 1977
Last regeneration: None
Depth of GAC, in.: 48
Flow rate, gpm/ft2: 1.6
Empty bed contact time, min: 18.70
C12 residual before GAC, mg/1:
- mg/1 free, 0.3 mg/1 total
Before-* After-*
pH value 5.9 6.1
TOC, mg/1 3.7 3.5
Endotoxins (LAL) Unfiltered, ng/ml 12.5 10,0
Endotoxins (LAL) Filtered, ng/ml 7.5 7.5
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 10.0 10.0
Temperature on arrival, °C 8.0 8.0
Dissolved oxygen, mg/1 11.0 10.6
Total plate count organisms per ml 1 2
Coliforms per 100 ml, Millipore 0 0
Col 1 forms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 100 100
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit.
109
-------
WATER TREATMENT PLANT SAMPLES
Sample # T4
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: Sept. 1971
Last regeneration: May 1977
Depth of GAC, in.: 33
Flow rate, gpm/ft2: 2.7
Empty bed contact time, min: 7.62
C12 residual before GAC, mg/1:
0.5 mg/1 free, 1.0 mg.l total
Before-* After-*
pH value 6.4 6.0
TOC, mg/1 3.5 2.0
Endotoxins (LAL) Unfiltered, ng/ml 25.0 10.0
Endotoxins (LAL) Filtered, ng/ml 12.5 7.5
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 12.0 12.0
Temperature on arrival, °C 6.0 6.0
Dissolved oxygen, mg/1 10.9 10.7
Total plate count organisms per ml 3 1
Col i forms per 100 ml, Mi Hi pore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit-
110
-------
WATER TREATMENT PLANT SAMPLES
Sample # T5
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation
GAC installation date: 1971
Last regeneration: June 1977
Depth of GAC, in.: 11
Flow rate, gpm/ft2: 3.0
Empty bed contact time, min: 2.29
C12 residual before GAC, mg/1:
0.1 mg/1 free, 0.7 mg.l total
Before-* After-*
pH value 6.9 7.0
TOC, mg/1 3.0 3.7
Endotoxins (LAL) Unfiltered, ng/ml 12.5 10.0
Endotoxins (LAL) Filtered, ng/ml 10.0 5.0
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 10.0 10.0
Temperature on arrival, °C 7.0 7.0
Dissolved oxygen, mg/1 11.3 11.2
Total plate count organisms per ml 3 1
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative 50 50
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit
111
-------
WATER TREATMENT PLANT SAMPLES
Sample # T6
Treatment prior to GAC:
Prechlorination, Alum-coagulation, Sedimentation, Sand Filtration
GAC installation date: July 1968
Last regeneration: March 1975
Depth of GAC, in.: 10
Flow rate, gpm/ft2: 2.8
Empty bed contact time, min: 2.2
C12 residual before GAC, mg/1:
0.15 mg/1 free, 0.45 mg/1 total
Before-* After-*
pH value 5.6 5.6
TOC, mg/1 3.3 6.7
Endotoxins (LAL) Unfiltered, ng/ml 25.0 25.0
Endotoxins (LAL) Filtered, ng/ml 25.0 25.0
Endotoxins (Rabbit Assay) Filtered, ng/ml** <2 <2
Chlorine Residual, mg/1 <0.05 <0.05
Temperature at sampling, °C 11.0 11.0
Temperature on arrival, °C 6.0 6.0
Dissolved oxygen, mg/1 11.8 11.8
Total plate count organisms per ml 23
Coliforms per 100 ml, Millipore 0 0
Coliforms per 100 ml, MPN <2 <2
Fecal coliforms per 100 ml, MPN <2 <2
Coliphage, Pfu/ml <2 <2
Percent of total plate count
Gram negative - 100
Percent of total plate count
Pseudomonas aeruginosa 0 0
Percent of total plate count
P. fluorescens 0 0
* GAC Filtration.
** Lower detection limit.
112
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-79-009
2.
RECIPIENT'S ACCESSION NO.
4, TITLE AND SUBTITLE
5. REPORT DATE
February 1979 issuing date
Pyrogenic Activity of Carbon-Filtered Waters
6. PERFORMING ORGANIZATION CODE
7 AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Harold W. Wolf, Bennie Joe Camp, Scott J. Hawkins
and AJames H. Jorqensen ...
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Texas A&M University
College Station, Texas 77843
and ^University of Texas Health Science Center
San Antonio, Texas 78284
10. PROGRAM ELEMENT NO.
1CC614
11. CONTRACT/GRANT NO.
R-804420
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory - Cinn, OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final Report
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
Project Officer: Herbert R. Pahren (513)684-7217
is. ABSTRACT y^ encj0tox-jn content and pyrogenic response of granular activated carbon
(GAC) filtered waters were studied. GAC-filtered secondary effluent from an activated
sludge pilot plant contained free endotoxins in the range 6-250 yg/1 yielding positive
pyrogenic responses in 18 of 20 trials. Samples obtained from 27 different water
supplies in the U.S. that utilize GAC adsorption contained free endotoxin ranging from
1.2-25 yg/1 but none gave a pyrogenic response. No relationship was discernible
between endotoxin content and pyrogenic response.
Small removals of total organic carbon (TOC) by GAC beds which had been in oper-
ation in water treatment plants without regeneration for as long as 110 months were
observed. However, 5 of 28 samples showed an increase in TOC through GAC and 8 of 28
samples showed an increase in standard plate count. One of 25 samples yielded pseudo-
monads, but none of the 28 samples contained coliforms.
Good correlations were observed on non-disinfected AWT effluent samples between
standard plate count and total endotoxin (r = 0.945), standard plate count and free
endotoxin (r = 0.932), and total coliforms and free endotoxin (r = 0.939). Lack of
good correlations, however, were observed in assaying AWT samples that had been subject
to the disinfecting procedures of chlorination, ozonation, pH or UV irradiation.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Lipopolysaccharides, Pyrogens, Water
Treatment, Carbon, Potable Water, Water
Quality, Water Reclamation
b.IDENTIFIERS/OPEN ENDED TERMS
Endotoxin
Granular Activated Carbo
Advanced Waste Treatment
COSATl Field/Group
57 U
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
123
2O SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE! -j o
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