EPA-600/1-78-068
December 1978
EVALUATION OF TOXIC EFFECTS OF ORGANIC CONTAMINANTS IN RECYCLED WATER
by
Nachman Gruener
Gulf South Research Institute
New Orleans, Louisiana 70186
Contract No. 68-03-2464
Project Officer
Norman E. Kowal
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
/) PROTECTION
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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 commerical products constitute endorsement or recommendation for use.
ii
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FOREWORD
The U.S. Environmental Protection Agency was created because of
increasing 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 natural
environment. The complexity of that environment and the interplay among
its components require 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, and quantitate
harmful effects of pollutants that may result from exposure to chemical,
physical, or biological agents found in the environment. In addition to
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 physiological functions, and how
these functions are altered by low-level insults.
This report provides an assessment and discussion of the toxic effects
of water recycled for drinking purposes. With a better understanding of
the health effects, methods can be developed to produce recycled water
suitable for human consumption.
arner
Director
Health Effects Research Laboratory
111
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ABSTRACT
This report represents the results of a comprehensive series of
toxicological studies designed to evaluate the health effects of the
application of recycled water for drinking purposes. Water was prepared
in a highly advanced domestic sewerage pilot plant. Some 400,000 liters
of the finished water were concentrated down to a volume of 200 liters
with a total organic carbon content of 700 mg/liter. This concentrate
was incorporated into a gel-type diet which was fed to mice. A total of
900 animals was included in the experimental program, which extended to
150 days. The mice were tested for growth, food intake, mutagenicity,
mortality, blood physiology and biochemistry, and liver and nervous system
functions. Ten tissues were screened for pathological effects. Only
marginal changes were demonstrated in these tests.
In a second series of experiments, rodent and human cells were
tested in vitro for general toxicity, mutagenicity, and carcinogenicity.
Results for all three effects in the tissue cultures were positive.
These effects were significantly increased by the presence of a liver
activation system.
These results show that exposure for a limited time (20 percent of
a lifespan) to the concentrated, recycled water (about 100-1000 times
present human exposure) does not lead to physiological changes in mice.
On the other hand, the positive results from the mutagenicity and carcinogen-
icity studies in tissue culture indicate a need for more studies in this
area.
This report was submitted in fulfillment of Contract No. 68-03-2464 by
Gulf South Research Institute under the sponsorship of the U.S. Environmental
Protection Agency. This report covers a period from September 30, 1976
to May 31, 1978, and work was completed as of May 31, 1978.
IV
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CONTENTS
Foreword ill
Abstract iv
Figures v^
Tables vii
Acknowledgments xi
1. Introduction 1
2. Conclusions 3
3. Experimental Procedures 5
Preparation of the water samples 5
Food preparation 12
Toxicological tests 16
4. Results 30
Preparation of the raw water 30
Effluent concentration 33
Toxicological studies 35
5. Discussion 90
References. . 95
v
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FIGURES
Number Page
1 Stages 1 and 2 of reverse osmosis concentration system 6
2 Stage 3 of reverse osmosis concentration system 7
3 Effective processes used to isolate toxicological feed
sample 9
4 Closed-loop dialysis apparatus 11
5 Cation exchange system H
6 Filter assembly for final solution concentration 13
7 Blue Plains water treatment system 31
8 Cell counts and protein levels after exposure to recycled
water in the presence of S9 activation system 87
9 Direct exposure of WJ38 to concentrated recycled water 88
10 Effect of liver activation system on toxicity of concentrated
recycled water in WI38 cells 89
vi
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TABLES
Number Page
1 Basal Mixture for the lexicological Diet 14
2 Composition of Vitamin Mixture 14
3 Composition of Salt Mixture 15
4 Ions Required in Toxicological Diet 15
5 Chemical Analysis of the Concentrate 17
6 Amounts of Ions to Be Added to Toxicological Diets 18
7 Amounts of Salts to be Added to Toxicological Diets 18
8 Summary of Studies Performed 19
9 Toxicological Tests (In Vivo) 20
10 Number of Animals in the Different Studies 21
11 Tissues and Organs Examined in Gross Necropsy 27
12 Tissues for Microscopic Examinations 07
13 Design Data and Operating Conditions for Blue Plains Treatment
System 32
14 Organic Levels at Various Stages of Sample Preparation 34
15 Number of Mice used in In Vivo Studies 36
16 Average Food Consumption 35
17 Average Body Weight 37
18 Mice Body Weights 37
19 Mean Daily Food Consumption 37
Vll
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Tables, continued
Number Page
20 Body Weights and Food Consumption (Study I - Females) 39
21 Body Weights and Food Consumption (Study I - Males) 40
22 Hematological Indices for Mice Exposed to Concentrated Recycled
Water (Study I) 41
23 Hematological Indices for Mice Exposed to Concentrated Recycled
Water (Study II) 41
24 Blood Chemistry Results for Study I Males 43
25 Tissue Weights for Study I Males 44
26 Blood Chemistry Results for Study I Females 45
27 Tissue Weights for Study I Females 46
28 Blood Chemistry Results for Study II Males 47
29 Tissue Weights for Study II Males 48
30 Blood Chemistry Results for Study II Females 49
31 Tissue Weights for Study II Females 50
32 Blood Chemistry Results for Study III Males 51
33 Tissue Weights for Study III Males 52
34 Blood Chemistry Results for Study III Females 53
35 Tissue Weights for Study III Females 54
36 Blood Chemistry Results for Study IV Males 55
37 Tissue Weights for Study IV Males 56
38 Degree of Significance of Blood Chemistry Results for Study I
Males 57
39 Degree of Significance of Tissue Weights for Study I Males 58
40 Degree of Significance of Blood Chemistry Results for Study I
Females 59
41 Degree of Significance of Tissue Weights for Study I Females 60
viii
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Tables, continued
Number Page
42 Degree of Significance of Blood Chemistry Results for Study II
Males 61
43 Degree of Significance of Tissue Weights for Study II Males.
62
44 Degree of Significance of Blood Chemistry Results for Study II
Females 63
45 Degree of Significance of Tissue Weights for Study II Females 64
46 Degree of Significance of Blood Chemistry Results for Study III
Males 65
47 Degree of Significance of Tissue Weights for Study III Males 66
48 Degree of Significance of Blood Chemistry Results for Study III
Females 67
49 Degree of Significance of Tissue Weights for Study III Females 68
50 Degree of Significance of Blood Chemistry Results for Study IV
Males 69
51 Degree of Significance of Tissue Weights for Study IV Males 70
52 Blood Chemistry of 50 Mice Weighing 19-30 g 71
53 Sleeping Times in Mice after Exposure to Concentrated Recycled Water
(Study I) 73
54 Sleeping Times in Mice after Exposure to Concentrated Recycled Water
(Study II) 73
55 Motor Activity in Mice after Exposure to Concentrated Recycled Water
(Study I) 75
56 Motor Activity in Mice after Exposure to Concentrated Recycled Water
(Study II) 75
57 Dependence between Litter Size and Mean Body Weight 74
58 Litter Size and Mean Body Weights of Offspring Born to Exposed
Dams 76
59 Corrected Mean Body Weight. 77
60 Body Weights and Food Consumption of Pregnant Females 77
ix
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Tables, continued
Number Page
61 Research Plan 78
62 Effect of Administration of Concentrated Renovated Water on Survival
of Fetuses (Experiment I) 79
63 Effect of Administration of Concentrated Renovated Water on Survival
of Fetuses (Experiment II) 79
64 Effect of Administration of Concentrated Renovated Water on Survival
of Fetuses (Experiment III) 80
65 Salmonella Mutagenicity Test of Concentrated Reused Water 83
66 Direct Mutagenicity of Concentrated Recycled Water 83
67 Mutagenicity of Concentrated Recycled Water (first run) 84
68 Mutagenicity of Concentrated Recycled Water (second run) 84
69 Soft Agar Transformation 86
70 Effect of Water Concentrates on Cell Protein Level in Tissue
Culture 86
x
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ACKNOWLEDGMENTS
Mr. S. Lynch and Mr. J.K. Smith carried out and reported on the
concentration work. Dr. B. Buratto performed the histopathological
examinations, Mr. M. Lockwood participated in the in vitro studies, and
Ms. P. Uffelman assisted in the preparation of this manuscript.
XI
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SECTION 1
INTRODUCTION
Water used to be one of our most plentiful natural resources. In
recent years, however, we have discovered that water is no longer an
abundant commodity in many places in this country and other countries in
the world. Reasons for the reduced availability of water include popula-
tion growth, increases in average water consumption for domestic and
industrial purposes, and pollution. Although there are annual changes
in water reservoirs, long-term trends show that shortage of water will
become a problem in many places that are not now affected.
One approach to remedy this situation is conservation. Natural or
unintentional reuse by humans has been known for many years. But the
presence of hundreds of compounds in wastewater, among them known toxicants
and carcinogens, necessitates a more cautious approach to wide-scale
intentional reuse of treated wastewater. Treated wastewater can be used
for a variety of purposes, including agriculture, industry, recreation
and drinking purposes. Treated wastewater may be accessed for drinking
water in several ways, such as recharge of ground water or direct supply
with or without other sources of water. None of these approaches has
been dealt with in a practical way. Obviously, drinking water should
receive special toxicological consideration since it is a general daily
commodity used throughout the lifetime and by everyone in the population.
In spite of extensive work in this area and the interest of many
national and international bodies, relatively little effort has been
devoted to the question of the health effects of renovated drinking
water (1). Most of the work has been done on the technological aspects
of the problem, some on the chemistry and biology, and almost none in
the toxicological area. Although opinions of urgency and need for
priority of this subject have been expressed in conferences worldwide
(2,3), international collaboration is still occasional and unplanned.
Studies that have been performed can be considered preliminary (4,5) and
have been subject to inadequate water sample preparation, limitations on
the extent of the toxicological studies, or both.
When the U.S. Environmental Protection Agency first launched efforts
in this area, the necessity of finding a method to concentrate water was
recognized. Many concentration systems are inappropriate because of
selective removal or destruction of original compounds or addition of
contaminants. The concentration method must also be adaptable to large
scale operations. Reverse osmosis has been considered as the best choice in
these respects (6).
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When dealing with toxicological aspects of mixtures, two approaches
can be taken (7). The first approach involves chemical analysis of the
mixtures followed by individual toxicological assessment of each suspected
chemical. This methodology has been used in air pollution and food con-
tamination studies. In the second approach, the mixture is considered
as one test compound. This approach was selected for this project for
several reasons. (1) Hundreds of chemicals had already been identified
in water. (2) Long-term toxic effects of most of these chemicals are
unknown. (3) The unlimited possibilities of interactions of these
compounds in the body, both synergistically and antagonistically,
prevent the extrapolation of data from individual tests to the toxico-
logical assessment of the mixture (water).
The following factors are involved in the assessment of the toxico-
logical aspects of renovated water: (1) the presence of hundreds of
compounds, some of them known toxicants, (2) lack of toxicological data
on most of these compounds or at the low levels found in water (parts-
per-billion range), (3) the possibility of synergistic and antagonistic
interactions, and (4) general intake by the whole population from the
early days of life (and even before birth) until old age.
These considerations led us to set up an extensive project. Long-
term exposed mice were subjected to a battery of tests. The possible
effects on the fetuses and the newborns were also examined. Because of
special problems involved at present with mutagenic and carcinogenic
assays in the whole animals, these tests were done mainly in tissue
cultures.
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SECTION 2
CONCLUSIONS
1. Some 400,000 liters of finished water from an advanced wastewater
treatment plant was concentrated down to 200 liters with a final total
organic carbon (TOC) content of 700 mg/liter. The recovery of the
organic fraction was estimated to be only about 20 percent. A substantial
fraction of the inorganic ions were eliminated or exchanged with other
ions so the solution could be balanced according to animal nutritional
requirements.
2. A comprehensive, long-term toxicological study of this water
concentrate was run on mice. Nine hundred mice were included in different
studies which were extended up to 150 days. In these studies, observations
were made on the rate of growth, food intake, fertility, mutagenicity
and mortality, blood physiology and biochemistry, liver function test
behavior, and histcpathulogy. There were very few differences between
the experimental groups and the control animals. None of these changes
could be related to a clear pathological syndrome.
3. A mutagenicity test was performed on male and female mice
(dominant lethal mutation test). One experiment showed mutagenic proper-
ties of the water; the second was negative. Since a positive result
was found in testing different concentrated water in a previous study,
these results should be reviewed carefully.
4. Carcinogenicity and mutagenicity assays were carried out in
tissue cultures. The concentrated water was shown to be mutagenic in
hamster cells (V79). Human lung cells (WI38) were transformed by treat-
ment with the concentrated recycled water. They gained the ability to
grow on soft agar (anchorage independence) which is highly correlative
with the potential of malignancy. In studies run with bacterial systems
(salmonella/microsome assay), the number of revertants did not increase.
5. In vitro toxicity tests were done with human cells (WI38)
using cell protein as the biological indicator. Such a test is a poten-
tial candidate to serve as a biological monitoring system in the applica-
tion of reused water. Further studies are needed before conclusions are
drawn.
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6. The present study shows that exposure of mice to TOG levels
which are 100-1000 times the present levels of our water sources did not
cause significant changes in a large number of physiological and biochem-
ical parameters. On the other hand, mutagenicity and carcinogenicity of
the concentrated water were found in tissue culture assays.
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SECTION 3
EXPERIMENTAL PROCEDURES
PREPARATION OF THE WATER SAMPLES
Effluent from the Blue Plains (Washington, B.C.) pilot wastewater
treatment plant was concentrated in the field. A volume of approximately
400,000 liters was concentrated to approximately 800 liters over a
period of about two months (October - November 1976). The 800 liters
was further concentrated in the laboratory. The initial concentration
procedure was based on reverse osmosis technology. A flow schematic of
the reverse osmosis system is shown in Figures 1 and 2. The system,
which was housed in a mobile trailer, incorporated three somewhat repetitive
stages. Figure 1 shows Stages 1 and 2; Figure 2 represents Stage 3.
Each stage included a set of drums, each 208 liter capacity (55 gal);
high pressure pumps; reverse osmosis modules, and a back pressure valve.
Stage 3 included a deionization circuit in addition to these components.
Stage 1 incorporated acid addition to adjust system pH to 5.5 for optimum
operation of the cellulose acetate membrane system. Stage 3 had both
acid and base addition capabilities via the cation and anion exchangers.
Relay-logic circuitry controlled all stages so that the system ran
automatically.
Stage 1
Plant effluent or source water was pumped into Stage 1 via a sump
pump until drum la or Ib was filled. When the drum (drum la, for example)
was filled, the reverse osmosis system started processing the water in
that drum. No more fresh water entered the drum until it had finished
reverse osmosis processing. Meanwhile, the fill water had filled the
other drum of the pair (Ib) and was waiting for completion of the processing
of drum la. When drum la had finished processing, the reverse osmosis
system started operating on drum Ib while drum la filled for a second
time. This process was repeated as many times as necessary to complete
the total concentration.
Stage 1 was a single-pass system which split the process stream
into two parts. The fraction retained by the membrane was more concentrated
than the original feed water and was passed on to Stage 2. The water
permeating the membrane was run to sewer. A concentration of approximately
1.5-fold was achieved in this stage.
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Stage 2
The process drums of Stage 2 were filled alternately as they were
processed, as in Stage 1. The sequence was repeated as many times as
necessary. All valves and pumps were controlled by relay logic. Stage
2 was a recirculating system in which the concentrated stream from the
reverse osmosis modules was returned to the process drum. In this
manner, a 10-fold concentration could be achieved without exceeding a 50
percent recovery (2-fold concentration) within the cellulose acetate
module at any time. When the desired concentration had been reached,
the concentrate in the process drum was transferred from Stage 2 into
Stage 3 and the next batch for Stage 2 was started.
Stage 3
When the filling of a Stage 3 process drum was complete (after
several batches from Stage 2), Stage 3 processing started. While Stage
3 processed one drum the other was filling, as in Stages 1 and 2. In
Stage 3 processing, reverse osmosis concentration and ion exchange
deionization (Donnan system) circuits were run concurrently.
The deionization process had two objectives: to prevent inorganic
salt precipitation and to reduce the inorganic burden of the feed samples.
The deionization circuit consisted of a cation exchange membrane and an
anion exchange membrane in series. The concentrate from the process
drum circulated past the anion exchanger, where anions were exchanged
for OH, and then past a cation exchanger, where cations were exchanged
for H . It was anticipated that the mass transfer of the two exchangers
could be proportioned so that inorganic ion could in effect be exchanged
for water (HOH).
The major problem in preparing the sample after the completion of
field work was reducing the inorganic salt burden in the concentrated
sample while maintaining the organic level. Several variations of the
Donnan system were evaluated, but exchange rates from the anion system
were not sufficient, and the actual process rate threatened to produce
intolerable delays. Electrodialysis and closed-loop dialysis against
deionized water were considered as alternate procedures for sample
deionization. Although both methods removed significant amounts of the
salt, the closed-loop dialysis was chosen since it offered maximum
retention of organics and because process-size dialysis equipment was
available.
The toxicological sample was prepared using the closed-loop dialysis
method for overall inorganic reduction, as well as additional reverse
osmosis concentration; the cation exchange system was used occasionally
to balance cation levels. In addition, precipitative techniques were
used to reduce levels of SO, and Ca which were not easily handled by
the membrane systems. Figure 3 shows the sequence in which these processes
were applied to the sample.
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757 liters organic
concentrate
OH precipitation of divalent cations
Filtration
Dialysis (limited dialysate volume)
757 liters partially
deionized organic
concentrate
Reverse osmosis concentration
300 liters
deionized organic
concentrate
Cation exchange
Dialysis
Reverse osmosis concentration
204 liters
deionized
organic
concentrate
Precipitation of anionic SO.
Filtration (sterile)
Storage at 4°C
2-
204 liters
deionized
organic
concentrate
Figure 3. Effective processes used to isolate
toxicological feed sample.
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Hydroxide Precipitation of Divalent Cations
In early attempts to use Donnan dialysis to desalt the concentrate,
there were indications that membrane fouling was occurring due to the
formation of precipitates on the anion exchange membranes. These precipi-
tates were expecially bad in stagnant areas of the exchanger; portions
of the solids were soluble at low pH. These observations, coupled with
the fact that pumping was against a NaOH solution in the dialyzer,
indicated that the solids might be hydroxide precipitates of divalent
cations (e.g., Fe, Al, Ca, Mg). At this point, the concentrate was
adjusted to pH 14 with potassium hydroxide, and the alkali-insoluble
precipitates were filtered out. The pH adjustment prevented formation
of more of such precipitates and removed the possible source of fouling.
This adjustment did not substantially improve the Donnan dialysis process
but did remove some of the cationic burden in the sample. Analytical
data indicated nearly an 80 percent removal of those cations contributing
to the total hardness (Ca, Mg, etc.).
Closed-Loop Dialysis
Closed-loop dialysis removed most of the inorganics from the samples.
The equipment setup shown in Figure 4 was used to effect the deionization.
The Kill dialyzer is shown in schematic form only and the drawing does not
reveal the actual membrane configuration within the dialyzer. Prewashed
low-flux cellulose membrane was used in the dialyzer. The membrane area of
the dialyzer was approximately 0.5 m . With this system, 190 liters (50
gallons) of concentrate could be deionlzed sufficiently in about three days
to allow further concentration. System operation ensured optimum retention
of organics and prevented sample contaminantion. Solution flow was concur-
rent, with a positive differential membrane pressure of 0.34 atm (5 psi) on
the concentrate loop. This, coupled with the osmotic flow phenomenon,
resulted in a slight net flow of water into the concentrate loop.
Typical inorganic reductions with the system were greater than 80
percent. Retention or recovery was approximately 70 percent of organic
material based on TOC.
Cation Exchange
Early analyses of the concentration sample indicated the sodium level
was greater than acceptable for toxicological evaluation; the potassium
level was much below acceptable levels. The sodium level was reduced by
exchange with potassium via the system illustrated in Figure 5. The cationic
Donnan dialysis system worked well for initial removal of large quantities
of inorganics, but as the concentration gradient of inorganics across the
membranes decreased, so did the efficiency of inorganics removal. Sodium
and potassium levels were adjusted so that they would be acceptable for
feeding studies after the final concentration.
10
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y
A
— 7 —
B
-s
H
f— '
Deionized ^
•-later Loop
fir
r
Organic
Concentrate Loop
Kiil Dialyzer
A = XAD Absorption Resin Column
B = Mixed Bed Deionizer
Figure 4. Closed-loop dialysis apparatus.
Bath
Cation Exchange Hollow
Fiber Assembly
Organic Concentrate
Loop
Figure 5. Cation exchange system.
11
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Precipitation of Sulfate
Preliminary analysis of the final concentrate showed sulfate levels to
be nearly twice as high as desired. The preferred method for correcting
sulfate levels is use of an anion exchange system, exchanging the SO, for
an anion deficient in the organic concentrate. However, attempts with the
in-house anion exchange system, using PO, as an exchange anion, were
unsuccessful.
2-
The SO, was removed by taking advantage of the extremely limited
solubility of BaSO.. It was calculated from solubility data that if the
level of SO were reduced to 2000 mg/liter by the addition of barium, then
the maximum equilibrium concentration of barium allowable in solution would
be 7x10 mg/liter. Since this level was far below that of any anticipated
toxicological problem level, the precipitation was carried out. The barium
was added in the form of a mixture Ba(Cl)2'2H2° and Ba(OH)2«8H 0. The
total amount of barium added was calculated to be equal to that necessary
to reduce the SO, to 2000 mg/liter. The dichloride salt was chosen for
its solubility and its lack of effect on solution pH. Only a limited
amount of barium could be added as the chloride; the balance of the
required barium was added as the hydroxide. This necessitated pH readjustment
with H PO,. The BaSO, precipitate was filtered from the organic concentrate
mixture by sterile filtration.
Sterile Filtration
The final filtration of the concentrated water required a sterile
product for storage. The solution to be filtered contained BaSO, in sub-
stantial quantities and was moderately turbid. The BaSO, solids rapidly
settled to a 3/8-inch layer on the bottom of a 210-liter (55-gallon) drum.
The filter setup shown in Figure 6 was used to filter the solids and sterile
filter in one pass. The product was a clear, yellow-brown solution, col-
lected in sterilized one-gallon glass bottles. These bottles were kept at
4°C until used.
FOOD PREPARATION
The diet to be fed to the mice in the toxicological experiments was
composed of 50 percent solids and 50 percent water. The studies included
five groups: A-control, B-water sample diluted 1:8 with deionized water,
C-diluted 1:4, D-diluted 1:2, and E-undiluted concentrated water. The
undiluted concentrate contained about 700 mg/liter of total organic carbon
(TOG).
The solid portion of the mixture consisted of three parts: (1) a
basal mixture of the main nutritional ingredients (Table 1) , (2) the
vitamin mixture (Table 2), and (3) a salt mixture (Table 3). Mixtures (1)
and (2) were purchased from Teklad, Madison, Wisconsin. Wesson's modifica-
tion of Osborne-Mendel mix (8) was followed in preparing the salt mixture.
The composition of the total diet was:
12
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Pressure
Pot
D
5 um Nominal Filter
1 pm Nominal Filter
0.65 pra Absolute Filter
0.45 pm Absolute Filter
Sterile 0.22 ym
Absolute Filter
Sterile
One-Gallon
Receiving
Container
Figure 6. Filter assembly for final solution concentration.
13
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TABLE 1. BASAL MIXTURE FOR THE TOXICOLOGICAL DIET
Casein Vitamin Free Test
Sucrose
Dextrose, Hydrate, Technical
Dextrin, White, Technical
Corn Oil
(g/kg)
245.5
158.
214.
214.
167.4
1000.0
.5
,3
,3
TABLE 2. COMPOSITION OF VITAMIN MIXTURE
(g/kg)
P-Aminobenzoic Acid 0.1
Ascorbic Acid 0.2
Biotin 0.0005
Vitamin B-12 (0.1% trituation in mannitol) 0.05
Calcium Pantothenate 0.05
Choline Dihydrogen Citrate 0.7515
Folic Acid 0.002
Inositol 0.2
Niacinamide 0.05
Pyridoxine HC1 0.01
RiRiboflavin 0.02
Thiamine HC1 0.01
Dry Vitamin A-D (500,000 units of Vitamin A Acetate/g
and 50,000 units of Vitamin D-2/g) 0.006
Dry Vitamin E Acetate (500 U/g) 0.2
Menadione 0.005
14
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TABLE 3. COMPOSITION OF SALT MIXTURE
(percent)
Calcium carbonate, CaCO_
Calcium Phosphate Tribasic, Ca (PO.K
Cupric Sulfate, SuS04'5H20
Ferric Pyro Phosphate
Magnesium Sulfate, MgSo,
Manganese Sulfate, MnSO -H 0
Potassium Aluminum Sulfate, K Al? (SO ) , -24H20
Potassium Chloride, KC1
Potassium Iodide, Kl
Potassium Phosphate, Monobasic KH-PO,
Sodium Chloride, NaCl
Sodium Fluoride, NaF
21.00
14.90
0.04
1.47
9.00
0.02
0.01
12.00
0.01
31.00
10.50
0.06
. ,
TABLE 4. IONS REQUIRED IN TOXICOLOGICAL DIET
(mg/2kg food)
Cation Amount Anion Amount
Ca
Fe
Mn
Mg
K
Al
Na
Cu
5,677
218
2.9
720
6,075
0.4
1,663
4.0
Co.,
SO,
LL
P04
Cl
I
F
5,040
2,900
12,772
4,834
1.5
10.4
15
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Basal mixture
Vitamin mixture
Salt mixture
Agar
Water
Based on the figures in Table 3, the weights of the required ions for the
diet were calculated and are given in Table 4. The water analysis for
inorganic constituents is given in Table 5. Calculations based on the
figures in Tables 4 and 5 determine the amount of salts necessary for the
different dilutions of the concentrated water in the different groups
(Tables 6 and 7).
The diet was prepared by mixing an agar solution with the solids to
make a gelatinous food. One liter of water was heated to 70°C, and 30 g of
agar was slowly added while stirring until the agar was completely dissolved.
The basal mixture and the appropriate salt mixture were placed in a mechan-
ical stirrer and the agar solution was added and mixed thoroughly until the
temperature reached 45°C. The vitamin mix was added to this blend and
stirring was continued for another 10-15 minutes. This mixture was poured
into plastic jars and stored at 5°C. The diet was prepared each week and
food was replaced daily in the animals' cages.
TOXICOLOGICAL TESTS
In Vivo Studies
All the studies were carried out on mice-strain B6C3F1 purchased from
Charles River. Table 8 summarizes the different in vivo studies that were
included in the project and Table 9 gives the various experiments and tests
that were carried out. Table 10 provides a summary of the number of mice
used for each study. In Study P, mice aged 10 weeks were split into
cages; two females and one male in each cage. In Study I, mice aged 8
weeks were each placed in a separate cage. In these two studies, the mice
were quarantined for two weeks before the experiment started. Mice of
Studies II and III were born to the mice in Study P and were exposed through-
out gestation and lactation before the experiment started (immediately
after weaning). Study IV was exactly the same as Study I with the addition
of a 90-day recovery period. The mice in Studies P, I, II, III, and IV
were at the ages of 3, 5, 4, 6 and 6 months, respectively, when the studies
ended. Mice from Study P were checked for gross clinical or behavioral
changes, food consumption, body weights and lethality. These animals were
also used to produce the animals in Studies II and III. The first dominant
lethal mutation test and the first reproduction experiment test were done
on the animals of Study P.
16
-------
TABLE 5. CHEMICAL ANALYSIS OF THE CONCENTRATE
Parameter
As
Ba
Cu
Al
Cr
Zn
Pb
Ag
Hg
Se
Mn
Fe
Co
B
Si
N03-N
NO--N
F
mg/1
<0.01
<0.5
<0.10
0.40
<0.01
2.00
<0.01
<0.01
<0.01
<0.01
0.17
1.70
<0.20
<15
1.3
<0.1
<0.01
2.6
Parameter
Na
K
Ca
Mg
Cl
S°4
P°4
I
Total Hardness
(as CaCO )
Mg Hardness
(as CaCO )
Total Alkalinity
Total 132
Dissolved Solids
PH
Stability index
Saturating index
Odor Threshold
Turbidity
Color PCU
Total Organic
Carbon
mg/1
1300
4300
632
15
4400
2650
1500
2.9
1640
60
270
,000
5.5
7.3
0.9
4
3.2
260
704
17
-------
TABLE 6. AMOUNTS OF IONS ADDED TO TOXICOLOGICAL DIETS
(mg/kg)
Ion
Undiluted
1:2
1:4
Ca
Fe
Mn
K
Al
Na
CO
SO,
it
P°4
cr
i
F
5045
218
2
700
0
363
5126
250
11,460
400
1
8
.7
.0
.0
.0
5361
218
2.
700
0.
1013
5275
1575
12,022
2634
1.
9.
7
2
2
0
5522
218
2
700
0
1338
5275
2240
12,417
3734
1
10
.7
.3
.5
.0
5597
218
2.
700
0.
1500
5275
2570
12,590
4284
1.
10.
7
4
5
5
Salt
TABLE 7. AMOUNTS OF SALTS ADDED TO TOXICOLOGICAL DIETS
(mg/kg)
Undiluted
1:2
1:4
1:5
Control
CaCO
CaHPO
FePO
KH PQ
MgCl
/
MgSO^
MgCO
NaCO
KC1
Na SO,
NaF
KI
KA1SO
CuSO
MnSO^
NaCl
5666
9482
600
6300
535
312
1750
841
—
__
17
1.5
— —
15.6
8.0
6000
9482
600
7000
535
312
1750
1000
5533
2329
19
1.7
3.6
15.6
8.0
6000
9482
600
7500
535
312
1750
1200
7844
3313
21
2.0
3.6
15.6
8.0
—
6000
9482
600
8000
535
312
1750
1400
9000
3800
23
2.0
3.6
15.6
8.0
—
8400
5960
600
12,400
—
3600
—
—
4800
__
22.8
2.0
3.6
15.6
8.0
4200.0
18
-------
TABLE 8. SUMMARY OF STUDIES PERFORMED
Number of Animals
Study
P
I
II
Exposure Time
14 days
90 days
Gestation, Lactation
Males
50
200
100
Females
100
200
100
and another 90 days
III Gestation, Lactation 50 50
and another 150 days
IV 90 days and another 50
90 days on regular diet
In addition to the above-mentioned tests (in Study P), mice from
Studies I, II, and III were tested for hematology, blood chemistry, mixed
function oxidase activity, motor activity, and pathology. Another dominant
lethal mutation experiment was run on females and males from Study I, and a
second reproduction assay on Study III. Body weights were measured twice a
week. General observation and food consumption measurements were made
daily.
Hematology—
Mice were bled from their tails (after warming) and a blood sample was
taken with the aid of microcapillaries. Hemoglobin was measured by Coulter
Hemoglobinometer Model HGBR Serial No. 2090 which is an automatic rinsing
hemoglobinometer reading the color intensity of cyanomethemoglobin. Red
and white blood cells were counted with a Coulter Model FN No. 7356 based
on nonoptic measurement, one-by-one counting, and sizing particles suspended
in solution. The tests were run after 70 days of exposure, and animals for
the tests were chosen randomly using the table of random digits.
Blood Chemistry—
Each day approximately 50 animals from each group were tested. Animals
were fasted overnight before sacrifice. Blood was drawn by heart puncture
under anesthesia and was let stand for about 30 minutes at room temperature.
After this period, the blood was centrifuged in microtubes containing a
silicon material which after centrifugation seeks the interface between the
clot and the serum and forms a barrier between the two phases ("Microtainer",
Becton-Dickinson, New Jersey). The tubes were stored at 5°C and tested
after 24 hours. Serum was diluted with distilled water (1:1). This dilution
had no effect on the results. The samples were tested in a computer-
controlled Autoanalyzer (SMAC, Technicon, Tarrytown, New York). All daily
samples were run together at the same time, and a reference control was run
after every three samples. The following parameters were tested:
19
-------
TABLE 9. TOXICOLOGICAL TESTS (IN VIVO)
General Physiology
Food Consumption
Body Weight
Hematology
Hemoglobin
Red Blood Cell Count
White Blood Cell Count
Mixed Function Oxidase Activity
Motor Activity
Reproduction
Dominant Lethal Mutation
Pathology
Heart
Lungs
Spleen
Liver
Kidney
Adrenals
Brain
Tests
Ovaries
Microscopy and Tissue Weights
Blood Chemistry
Glucose
Chloresterol
Triglyceride
Total Protein
Albumin
Calcium
Phosphorus
Sodium
Potassium
Chloride
Carbon Dioxide
Urea Nitrogen
Uric Acid
Total Bilirubin
Creatinine
Alkaline Phosphate
LDH
GOT
GPT
CPK
20
-------
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W
M
O
H
LO
H
£3
W
W
Fn
PM
M
Q
W
p"{
H
53
M
CO
,-J
^r
M
^
CO O
CM CO CN rH
C* CT\ vO CTt
rH CO rH
O CTN rH O
CM CO CM rH
O CM CTi O
rH ~J rH rH
O 0*1 O> O
CM CO rH rH
O CM CT\ O
rH
-------
Glucose (Glu) Carbon dioxide (C02)
Cholesterol (Choi) Urea nitrogen (UN)
Triglycerides (Trig) Uric acid (UA)
Total protein (TP) Total bilirubin (Bill)
Albumin (Alb) Creatinine (Great)
Calcium (Ca) Alkaline phosphatase (AP)
Inorganic phosphorus (P) Lactate dehydrogenase (LDH)
Sodium (Na) Glutamic pyruvic transaminase (GPT)
Potassium (K) Glutamic oxaloacetic transaminase
(GOT)
Chloride (Cl) Creatine phosphokinase (CPK)
Glucose — This assay is based on the glucose oxidase-peroxidase procedure.
The specificity of glucose oxidase is combined with a peroxidase indicator
couple [3-methyl-2-benzothiazolinone hydrazone (MBTH) and N,N-dimethylaniline
(DMA)] to form a stable, intensely colored, water-soluble indamine dye,
which was read at 600 nm.
Glucose + 0. + H00 ^luc°se , Gluconic acid + H000 (1)
2 2 Oxidase 22
MBTH + DMA + H^ Peroxidases , indamine Dye + H20 + OH (2)
Cholesterol — The following mechanisms have been proposed for the
reaction of cholesterol with sulfuric acid.
Cholesterol + H_SO, - > bis-cholestadienyl monosulfonic acid (1)
bis-cholestadienyl monosulfonic acid + H SO, - >• bis-cholestadienyl
disulfonic acid (2)
The sample was added to a chilled (0°C) color reagent and the mixture
allowed to reach room temperature. The reaction is highly exothermic;
therefore, special care was taken to keep the optimum reaction temperature.
The absorbance was measured at 630 nm.
Triglycerides — Triglycerides are specifically hydrolyzed by lipase to
glycerol and free fatty acids. The glycerol product is phosphorylated to
glycerol phosphates by glycerol kinase which is then coupled to pyruvate
kinase to form pyruvate. This product enters the well-known NADH-NAD
reaction catalyzed by LDH. The whole reaction was followed by measuring
the absorbance at 340 nm. A control was subtracted from the test value to
correct for changes in the absorbance caused by endogenous serum inter-
ferences.
Triglycerides - ^ - >• Glycerol + Free fatty acids (1)
Glycerol + ATP ; Glycerol phosphate + ADP (2)
ADP + Phosphoenolpyruvate * ATP + pyruvate
T T)H
Pyruvate + NADH > Lactate + NAD
22
-------
Total Protein—Protein was determined by the biuret method. The
protein combines with the copper in the biuret reagent to form a purple
complex which was read at 550 nm.
Albumin—Bromocresol green (BCG) combines specifically with albumin to
form a stable complex. The albumin-BCG complex was read directly at 630
nm. A special reagent was added to the reaction mixture to minimize the
absorbance of the reaction blank to prevent turbidity and to provide linear-
Ity.
Calcium—The calcium method uses the metal complexing dye "Cresolphtha-
lein Complexone" which binds calcium ions in alkaline medium. The product
of this intereaction is a pink calcium dye complex with a maximum absorption
at 570 nm. First, serum was added to a diluted solution of HC1 containing
8-hydroxyquine which binds the free magnesium ions present in the serum. A
sample was mixed with Cresolphthalein Complexone containing 8-hydroxyquinino-
line. Upon the addition of diethylamine, a color complex is formed between
the calcium and the dye. The absorbance of the reaction product was measured
at 570 nm.
Inorganic phosphorus—The serum phosphorus is reacted with ammonium
molybdate. Instead of the popular method to reduce the complex phosphomolyb-
date with reducing agent, this assay is based on the fact that the unreduced
complex absorbs ultraviolet light. The absorbance was measured at 340 nm.
Sodium—The sodium method is a direct potentiometric procedure for the
quantitative measurement of sodium in serum by use of a sodium-selective
glass electrode. The sodium selective electrode responds to sodium ions
according to the Nernst equation.
Chloride—This is a colorimetric procedure:
Hg(SCN)2 + 2C1~ > HgCl2 + 2(SCN)~ (1)
3(SCN)~ + Fe+3 —-> Fe(SCN) (2)
The absorbance of the red complex Fe(SCN)« was measured at 480 nm.
Carbon dioxide—Carbon dioxide is present in the serum also as H-CO
and HCO-. CCk and H?CO« are present in serum in relatively small amounts.
All the species were in equilibrium with each other. The method used to
measure CO™ was based on the release of C02 by an acid. The released C02
is absorbed by an alkaline solution containing phenolphthalein, causing a
change in pH which results in a decrease in the abosrbance at 550 my.
Potassium—The potassium method, like the sodium method, is a direct
potentiometric procedure by means of a potassium ion selective electrode.
23
-------
Urea nitrogen — In a weak acid solution, diacetyl monoxime is hydrolyzed
to diacetyl which, in turn, reacts directly with urea in the presence of
acidic ferric ions. The presence of thiosemicarbazide intensifies the
color of the reaction. The absorbance was read at 520 nm.
Total bilirubin — The sample reacts with "Diazo" reagent to form azobil-
irubin complex. To this mixture a strong alkaline sodium potassium tartarate
buffer was added which solubilizes protein and eliminates the effect of
variation in sample pH. The colored complex was measured at 600 nm.
Creatinine — The creatinine method is based on the reaction of saturated
picric acid with creatinine in an alkaline medium to form a red color
chromogram which was measured at 505 nm.
Alkaline phosphatase — The method is based on the hydrolysis of p-
nitrophenyl phosphate. The product at alkaline pH gives a yellowish color.
P-nitrophenyl phosphate - Phosphataes > p_nitrophenol + H P0 + H 0
Mg ; 37°C; pH 10.25
The absorbance was read at 410 nm.
Lactate dehydrogenase — This enzyme catalyzes the following reaction.
1-Lactic acid + NAD > Pyruvic acid + NADH + H . NADH has an absorption
peak at 340 nm and the enzymatic activity is porportional to the amount of
NADH produced during a fixed time interval. Under specific conditions of
pH, temperature and substrate concentrations, the reaction obeys zero time
kinetics.
Glutamic oxaloacetic transaminase — The enzymatic reaction of GOT:
Aspartate + a-Ketoglutarate - >• oxaloactate 4- glutamate (1)
is coupled to malic dehydrogenase.
Oxaloacetate + NADH ^L>. NAD + Malate. (2)
All the reagents except ketoglutarate were mixed with the serum sample and
preincubated, followed by the addition of a-ketoglutarate which starts the
reaction, which was followed at 340 nm.
Glutamic pyruvic transaminase — The enzymatic reactions are as follows :
PPT
Alanine + a-ketoglutarate - > pyruvate + glutamate (1)
T DH
Pyruvate + NADH • - > + Lactate + NAD
Again, all the reagents were incubated except ketoglutarate, which is added
after the preincubation. The reaction is started by adding a-ketoglutarate.
24
-------
Creatinine phosphokinase (CPK)—The enzymatic reaction is as follows:
CPK
Creatine phosphate + ADP > Creatine + ATP
Cystain was added to ensure maximal activity. The reaction is stopped by
N-ethylmaleimide which also prevents the sulfhydryl groups from interfering
with the creatine coupling reaction. Diacetyl/orcinol reagent was added
and a condensation product was formed which developed a strong color upon
the addition of the sodium hydroxide solution and incubation at 45°C. The
presence of EDTA in the reagents prevents the precipitation of Mg(OH)_.
The color was measured at 520 nm.
Statistical Analyses—Statistical analyses were performed on a PDP-10
computer, using SPSS (Statistical Package for the Social Sciences). The
condescriptive procedure and the T-test subprogram were used (9). All groups
were compared statistically to the control group (A).
Liver Mixed Function Oxidase (MFO) Activity—
Sleeping times induced by sodium hexabarbital (100 mg/kg body weight)
were measured as a function of MFO activity. Latent time is defined as the
time between injection and the loss of righting reflex, and sleeping time
is the time between loss and gain of righting reflex.
Motor Activity Measurement—
This measurement was taken in a hexagonal box equipped with 2 perpen-
dicular light beams and 2 photoelectric cells connected to 2 independent
counters. Each animal was put in the box for 10 minutes and counts were
recorded after 5 and 10 minutes. The counts from the 2 counters were
combined at each time. All tests were run at the same time of the day in a
dimly lighted room. Animals were selected for the test by using the table
of random digits. Animals selected for the behavior test were excluded
from the mixed function oxidase test and vice versa.
Dominant Lethal Mutation Test—
Each selected male was mated with two unexposed females for 5 consecu-
tive weeks after ceasing the exposure. Each week, 2 new females were put
in the cage with the male and the mated females were left for another week,
and then sacrificed. The number of live and dead fetuses was counted. The
results were tested by nonparametric statistics (Mann-Whitney U test) (10, 11)
Data from all the experimental groups were combined for analysis since the
results did not differ significantly from group to group.
Pathology—
A board-certified or board-eligible veterinary pathologist with experi-
eace in laboratory animal pathology was responsible for all pathology
procedures, evaluations and reporting. Well-qualified laboratory technicians
performed post-mortem activities and slide preparations.
25
-------
All animals from this study were given a complete post-mortem examination.
The tissues and organs listed in Table 11 were examined in situ, then
removed and incised properly to ensure adequate fixation and placed in 10%
neutral buffered formalin. Following adequate fixation, the tissues listed
in Table 12 were trimmed and embedded in paraffin blocks sectioned according
to standard histological procedures and stained by the hematoxylin and
eosin method.
In Vitro Studies
Salmonella/Microsome Test—
The procedure followed that of Ames, et al_. (12) Tests were run on
strains TA1535, 1537, 1538, 98, and 100. Assays were performed with and
without activation because some compounds do not require conversion to
active forms by mixed function oxidases.
The activation system consisted of microsomal fractions prepared from
adult male Sprague-Dawley rats (150-200 g) injected with 500 mg/kg Aroclor
1254 five days prior to sacrifice. All food was removed 24 hours before
microsome preparation. The livers were excised, washed, weighed, and
homogenized in a Teflon homogenizer with three volumes of 0.15M KC1. Care
was taken throughout the procedure to maintain the tissue at 4°C. The
postmitochondrial supernatant (S9 fraction) was prepared by centrifugation
at 9000 x g for 10 minutes. The supernatant was frozen and stored at -80°C
in 1 ml aliquots.
The water sample was added directly to the molten top agar and poured
onto the plate together with the indicator test organism with or without
activation system. Negative controls included plates to measure the number
of spontaneous colonies, plates to check for the sterility of the microsomes,
and the water.
Mutagenicity Tests with Mammalian Cells—
Mutagenesis testing was performed according to the method described by
Kuroki, et al. (13) with slight modifications. V79 cells, derived from male
Chinese hamster lungs, were used for all mutagenesis tests. Cells were
cultured in Dulbecco's modified minimum essential medium (DMEM) supplemented
with 10% heat-inactivated (56°C, 30 min) fetal bovine serum (IFBS). For
subculture, cultures were trypsinized with 0.05 percent trypsin in 0.02
percent EDTA. All cultures were incubated at 37°C in a water-saturated, 10
percent CO,, atmosphere.
For mutagenesis testing, V79 cells were plated in 100-mm petri dishes
(Falcon) at a concentration of 6 x 10 /plate and were incubated overnight.
The cells were then incubated in 2 ml of the reaction mixture consisting of
0.3 ml S9 fraction (approximately 9 mg protein), 0.3 ml Sorensen-phosphate
buffer (0.055M, pH 7.4, containing 0.9 percent NaCl and 0.49 mg MgCl^H-O
per ml), 0.1 ml glucose-6-phosphate solution (13 mg/ml in PBS), 0.1 ml NADP
solution (6.3 mg/ml in PBS) per ml, and 0.2 ml PBS. The final concentrations
in the reaction mixture were 20 ymoles of inorganic phosphate, 1.53 ymoles
G-6-P, and 0.8 ymoles NADP.
26
-------
TABLE 11. TISSUES AND ORGANS EXAMINED IN GROSS NECROPSY
Tissue masses or suspect tumors
Regional lymph nodes
Skin
Mandibular lymph node
Mammary gland
Salivary gland
Larynx
Trachea
Lungs and bronchi
Heart
Thyroids
Parathyroids
Esophagus
Stomach
Duodenum
Jej unum
Ileum
Cecum
Colon
Rectum
Mesenteric lymph node
Liver
Thigh muscle
Sciatic nerve
Sternebrae, vertebrae;
or femur (plus marrow)
Costochondral junction, rib
Thymus
Gall bladder
Pancreas
Spleen
Kidneys
Adrenals
Bladder
Seminal vesicles
Prostate
Testes
Ovaries
Uterus
Nasal Cavity
Brain
Pituitary
Eyes
Spinal cord
TABLE 12. TISSUES FOR MICROSCOPIC EXAMINATIONS
Heart
Lungs and mainstem bronchi
Kidneys
Spleen
Liver
Mandibular lymph node
Brain (three sections including
cortex, midbrain and
cerebellum)
Testes (males)
Bladder and prostate
Ovaries (females)
Uterus (females)
27
-------
The concentrated water was combined 1:1 in 2x medium prior to addition
to the reaction mixture. Samples were added to plates containing the
reaction mixture and were incubated for 1 hr at 37°C. One pg/ml benzopyrene
was used as a positive control. Other controls include untreated cells and
cells exposed to the S9 mix without sample.
Following treatment, the cells were washed three times in PBS and
fresh medium was added. Cells were left for 2 hours. The cells were then
trypsinized, counted and replated for determination of the induced cytotox-
icity and mutagenicity. To measure cytotoxicity, 100 cells from each group
were plated in each of four 60-mm plates in 4 ml DMEM plus 10 percent IFBS.
After 8 days, these plates were fixed in absolute methanol and stained with
10 percent ,-Giemsa. Production of ouabain-resistant clones was measured by
plating 10 cells in each of 16 plates per point in 4 ml DMEM plus 10
percent IFBS. Forty-eight hours after plating, ouabain (final concentration
in each plate = 1 mM) was added to the mutagenesis plates. These cultures
were fixed and stained as above, 14 days after plating. The frequency of
ouabain-resistant colonies was calculated per 10 surviving cells, taking
into account the cytotoxicity and the number of cells plated.
The water was also treated for mutagenecity in the absence of the
activation system. V79 cells were seeded in 60-mm plates at concentration
of 10 and 10 cells per plate. After 24 hours, the medium was removed and
replaced by a final volume of 2 ml of medium and H_0 combined with 2X MEM
(1:1). MNNG (1 yg/ml) was used as positive control. All plates were
treated for 4 hours at 37°C, washed three times with PBS and 4 ml of fresh
DMEM 10 percent IFBS added. Ouabain was added 48 hours after treatment to
the plates containing 10 cells, as above. Toxicity plates were fixed and
stained as above 7 days, and the mutagenicity plates were fixed and stained
14 days after treatment.
Soft Agar Transformation Assay—
For the soft agar transformation assay, WI38,stock cells were trypsin-
ized and suspended at a concentration of 1.5 x 10 cells/ml in MEM + 10%
IFBS. This assay was performed with and without the Sq activation system
described earlier. Benzo(a)pyrene and MNNG were used as positive controls.
In samples containing the activation system, 1 ml of the Sq mix was added
to the 1-ml cell suspensions. The concentrated water and control compounds
were then added and all samples were incubated for 2 hours at 37°C in an
orbital shaker. After incubation, the cells were centrifuged at 50 x g for
10 min, and the supernatants containing the compound and microsomes were
discarded. Each pellet of cells was resuspended in 15 ml of growth medium
to which was added 1.25 ml of 2.5 percent agar solution. The final concen-
tration of agar was 0.3 percent. The suspension was quickly mixed and
layered, in 1.5 ml volumes, on each of ten 60 mm petri dishes containing 5
ml of MEM and 0.9 percent agar. The plates were allowed to gel and are
incubated at 37°C in a C00 incubator.
28
-------
Since there is evidence that transformation, at least in diploid
cells, is not a one-step process, and may be detectable in soft agar only
after several generations following treatment, replicate cultures were
seeded in 75 cm Falcon flasks and were maintained for subsequent assays.
Approximately every 2 weeks, these cells were assayed for soft agar transform-
ation by seeding them on agar plates as above. These cells received no
additional exposure to the compounds used in the initial treatment.
Soft agar plates were monitored for at least 3 weeks for the presence
of transformed colonies. Only those colonies exceeding 100 u diameter are
scored. Results were expressed as the number of transformed colonies per
10 survivors. Positive results must be 2.5 X untreated negative controls.
WI38 Toxicity Assay—
WI38 cells, derived from human embryonic lung, were seeded in culture
tubes in 2 ml MEM plus 10 percent 1FBS and placed on an orbital shaker at
37°C. After 24 hours, 3 tubes were washed 3 times with PBS, drained and
were frozen to determine base protein levels at zero time. Tubes were
exposed to water samples at either various concentrations or different
exposure times. In dose-response experiments, the tubes were treated for
72 hours. In kinetics experiments, the cells were exposed for the specified
time, washed and frozen. Controls were used for each point, including
untreated controls and controls for S9 toxicity in experiments employing an
activation system. The activation system consisted of Kuroki's mix described
above with a reduction in final S9 concentration to 25 yl per ml. Tubes
were assayed according to Lowry and were read at 660 nm. Both percent
inhibition and protein values were compared. Cells were counted in diluted
samples after trypsinization with the aid of hemocytometer.
29
-------
SECTION 4
RESULTS
PREPARATION OF THE RAW WATER
The water used in this study was concentrated from finished water
prepared in a treatment system located on the grounds of the Washington,
B.C., Blue Plains wastewater treatment facility. The treatment procedures
and the quality of the water obtained are described below. This information
is taken from EPA reports (14,15) concerning operation of the Blue Plains
pilot plant. The system degritted the municipal wastewater, using a screening
device to remove coarse materials, and included processes for lime clarifica-
tion, dispersed growth nitrification, denitrification, activated carbon
adsorption, alum treatment, and chlorination.
A diagram of the treatment system is presented in Figure 7, and design
and operating conditions are summarized in Table 13. The treatment system
was operated on a continuous basis with operators assigned to three 8-hour
shifts each day. The system was modular in design to permit additions and
modifications without affecting system integrity.
Performance of the Water Treatment System
Virus Removal—
Animal viruses in raw sewage samples ranged from 1750-17000 PFU/100
liters. No viruses were detected in the final effluent, even after concen-
tration. Sample volumes were in the range of 180-900 liters. A typical
concentration was from 380 liters down to 10-20 ml.
Metals—
Results of about 50 tests of effluent samples, each analyzing 14
metals, showed that none of the metal concentrations exceeded the levels
for drinking water cited in the EPA regulations (16). Other experiments
showed that lime treatment is primarily responsible for the reduction in
metal concentration.
General Organics—
The treatment system is capable of producing effluents with low levels
of total organic carbon. Some 230 samples showed a TOC level of 74.1 +_
11.0 rag/liter (M + S.D.) in the influent and only 2.79 + 1.35 mg/liter in
the effluent. The mean phenol concentration in 54 effluent samples was
3.66 ;+ 1.52 yg/liter, as compared to the 1962 drinking water standard of 1
pg/liter.
30
-------
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-------
TABLE 13. DESIGN DATA AND OPERATING CONDITIONS FOR
BLUE PLAINS TREATMENT SYSTEM
Raw Wastewater (Constant Flow)
Screening Device
Type
Size of Openings
(as CaCO )
Lime Clarification
Lime Dosage (pH 10.0)
Fed Dosage (as Fe)
Hydraulic Loading Rate
Detention Time
Sludge Wasting Rate
Percent Solids in Waste Sludge
Nitrification (Suspended Growth)
Detention Time
MLSS
SRT
Air Requirement
Clarifier Overflow Rate
Clarifier Detention Time
Denitrification (Fixed Film)
Media Size
Specific Surface Area
Hydraulic Loading Rate
Methanol/NO -N Ratio
Bed Depth
Detention Time (Empty Bed)
Operation
Granular Carbon Adsorption
Detention Time (Empty Bed)
Hydraulic Loading Rate
Columns in Series
Carbon Size (Filtrasorb 300)
Operation
Filtration with Alum and Polymer
Hydraulic Loading Rate
Dual Media
Coal (1.2-1.4 mm)
Sand (0.6-0.7 mm)
Alum
Disinfection with Chlorine
Detention Time
Residual
2.2 liters/second (35 gpm)
Bauer Hydrasive Model 552
0.040 inch screen
200 mg/1
15 mg/1
1050 gpd/ft
2.7 hours
2% to 3%
1.5% to 2.0%
3.5 hours
2000 mg/1
8 days
1450 ft /lb BOD
526 gpd/ft
2-3.6 hours
3 to 6 mm
245 ftZ/ft;J
5.9 gpm/ftZ
2:1 to 4:1
15 ft
9.5 min
Downflow Packed Bed
35 min
7 gpm/ft
4
8 x 30 Mesh
Downflow Packed Bed
3 gpm/ft'
2.0 ft
1.0 ft
5 mg/1
20 min
1 mg/1 Free Available
32
-------
Haloorganics—
Chloroform, bromodichloromethane, dibromochloromethane, bromoform,
carbon tetrachloride, and 1,2-dichloroethane were determined in the Influent
and effluent. Levels were on the low side of the concentration range
typically found in finished drinking water supplies.
Pesticides—
Twelve pesticides (from the DDT or parathion groups) were tested in
the effluent and none exceeded EPA standards for drinking water.
EFFLUENT CONCENTRATION
The concentration process was based on the reverse osmosis-Donnan
deionization technology as described in Section 3. The major problem in
preparing the sample was reducing the inorganic content of the water. Due
to the partial failure of the Donnan deionization system, alternate proce-
dures—electrodialysis and closed loop dialysis—were considered for sample
deionization. Electrodialysis was disadvantaged by an inconvenient tempera-
ture increase and associated control problem. Therefore, the sample was
prepared using the closed loop dialysis method for overall inorganic reduc-
tion. The cation exchange system was used occasionally to balance cation
levels, specifically, the exchange of Na for K . In addition, precipitative
techniques were used to reduce levels of SO and Ca which were not
easily handled by the membrane system.
Recovery of the Organic Soluble Fraction
The recovery calculation is based on TOC measurement. This estimation
may have sizable error because (1) low-level TOC measurements (around 1
ppm) usually are not very accurate, and (2) effluent TOC was not measured
throughout the concentration period, and values are based on those taken
periodically during the pilot plant operation. The pilot plant data suggest
a median value of about 2 mg/1, which was used for the recovery calculation.
The data in Table 14 reflect actual organic levels in the concentrate
at various stages of the sample preparation sequence. As shown, mass
retention was better in the final reverse osmosis concentration (94%) than
in the initial reverse osmosis stage (53%) , probably because most permeable
organics have been removed in early steps.
In summary, an aqueous organic concentrate was prepared from the Blue
Plains advanced waste treatment pilot plant effluent. Approximately 400,000
liters was concentrated to a final volume of 204 liters with a TOC level of
approximately 704 mg/liter. Because the Donnan dialysis system did not
function properly, several unanticipated process steps were necessary to
prepare the sample. All the additional steps were shown to decrease the
overall level of organics in the concentrate.
33
-------
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TOXICOLOGICAL STUDIES
In Vivo Studies
The toxicological study of the concentrated recycled water was performed
using CF1 mice. The main study was restricted to 90 days since the amount
of the concentrated water was limited. This schedule was based on preliminary
calculation of the daily water intake of the animal, minimum required
number of animals needed for statistical analysis, and the need to study
chronic exposure. Since the amount of the sample was limited, as many
experiments as possible were carried out on the same group of animals
without jeopardizing the individual experiments. All the in vivo toxicol-
ogical experiments performed were listed in Table 9. Because most of the
planned experiments had no precedent with regard to concentrated recycled
water, it was important to perform these experiments in duplicate.
The plan of the studies was given in Tables 8 and 10. Not every
animal in each study was used for all the experiments. The number of the
animals used in each experiment is shown in Table 15.
Palatability Test—
Before starting the experiments a palatability test was run. The food
was prepared as described in Section 3. The TOC level of the water was 292
mg/liter. Ten mice (B6C3F1), five males and five females, were fed the
test concentrate. A second group of each sex received the same diet food
containing deionizeu water; a third group received Wayne Meal. All food
and water were provided fresh daily.
Food consumption was measured daily by weighing the food remaining in
the bowl and then discarding it. Each animal was provided with an excess
of feed to assure no starvation occurred. All mice were observed for seven
days for signs of clinical effects. No evidence of toxicity from the test
food was observed. No resistance to consume the test food was noted. The
amount of food consumed by the mice on the three diets was approximately
equal (Table 16).
TABLE 16. AVERAGE FOOD CONSUMPTION (g/mouse/day)
Male Female
Wayne Meal
Control Gel
Test Gel
11.3
10.8
10.6
9.8
9.1
8.9
All animals were weighed at the time of the first feeding, and again
on days 3, 5 and 7. Results are given only for days 0 and 7. Weight gains
were about the same among the three groups (Table 17).
35
-------
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36
-------
TABLE 17. AVERAGE BODY WEIGHT (g)
Wayne Meal
Day 0 Day 7
Control Gel
Day 0 Day 7
Test Gel
Day 0 Day 7
Males
Females
12.8
11.6
19.8
17.8
13.0
12.0
17.8
17.8
12.6
11.6
18.6
16.6
Study P—
This preliminary experiment was carried out for 14 days. The purpose
of this study was to test for general health effects or lethality at several
TOC concentrations. The experiment was composed of 100 male mice and 200
female mice (aged 10 weeks) divided into 5 groups: A-control, B-water
sample diluted 1:8 with deionized water, C-diluted 1:4, D-diluted 1:2, and
E-undiluted concentrated water. One male and two females were put in each
cage. Food consumption and general condition were observed daily and body
weight on days 0, 3, 7, 10 and 14 recorded. Body weight and food consumption
are given in Tables 18 and 19.
TABLE 18. MICE BODY WEIGHTS (g)
Day
Group
A
B
C
D
E
Male
35.3+1.9
35.0+2.1
33.4+3.0
30.5+2.5
33.2+2.7
0
Female
22.9+2.1
22.1+1.8
22.0+1.6
20.8+2.9
21.1+1.4
Male
33.2+2.7
33.0+1.4
31.6+2.5
31 .4+1.6
33.0+3.2
7
Female
25.2+2.2
24.9+1.8
25 .8+1.1
25.1+2.1
23.1+1.8
14
Male
34.0+2.1
33.4+1.6
32.0+2.4
34.7+4.4
34.7+4.4
Female
29.0+3.5
30.7+3.2
31.9+2.9
29 .3+3.1
29 .3+3.1
TABLE 19. MEAN DAILY FOOD CONSUMPTION (g)
Group Male Number
Female
Number
A
B
C
n
E
7.8+0.7
7.2+0.9
7.2+0.5
7 , 5V ! . 2
/. y-i o.y
10
10
10
10
10
7.9+0.6
7.7+0.7
7.7+0.2
7.8+0.3
7.9+0.5
20
20
20
20
20
37
-------
Group B was exposed to about 10 mg of TOC per kilogram of body weight
per day and Group E to about 100 mg TOC/kg/day. Humans drinking water with
a TOC concentration of 1.0 mg/1 are exposed to 0.02-0.04 mg per kilogram of
body weight per day. Thus, the mice were exposed to 500-5000 times the
level humans are exposed to when drinking medium TOC water, and 50-500
times the exposure level for high-TOC drinking water (10 mg/1). No signif-
icant changes were observed in body weights or food intake for the mice
exposed to high TOC water for two weeks.
Studies I through IV—
Study I was started with 500 animals [part of these are classified as
Study IV (Table 8)]. As in Study P, these animals were quarantined for two
weeks after arrival. Animals of Study II were the offspring of the females
and males from Study P and were thus exposed to the concentrated water
throughout gestation and lactation.
Studies I and II were carried out for 90 days; Study I from the age of
five weeks and Study II from three weeks (weaning). Part of Study II was
extended to 150 days and was designated Study III. The results of the
tests performed during these studies are discussed in the following sections.
General Physiology—
Food consumption and clinical observations were recorded daily. Body
weight was measured twice a week. No difference in body weights was apparent
between the control animals and the experimental groups. A characteristic
picture of growth can be seen in Tables 20 and 21. The daily food consumption
(per kg body weight) decreased with age and was higher in females than in
males because of changes in body weight. This difference in exposure per
unit of body weight did not affect the toxicity, and no selective toxicity
could be shown in the young or in the females. Lethality among the animals
was low and could not be related to exposure of the experimental groups in
any of the studies.
Food consumption was in the range of 200-600 g/day/kg body weight,
which is equivalent to 100-300 g of water in the gel diet per day per kg
body weight. Based on 700 mg/1 TOC in the undiluted water, the mice in the
different groups were exposed to 12-140 mg TOC/day/kg body weight. Humans
might typically be exposed to water containing up to 10 mg/1 of TOC. Based
on daily water intake of 2 liters, human intake of TOC could be in the
range of 0.02-0.4 mg TOC/kg/day. Thus, the mice in these studies were
exposed to TOC doses 100-1000 times the expected human exposure.
Hematology—
Tables 22 and 23 list the data for hemoglobin, and red and white blood
cell counts in Studies I and II. No differences were noted between the
experimental groups and the controls.
Blood Chemistry and Tissue Weight—
Blood samples were taken by heart puncture from the mice on sacrifice
day. About 50 mice, including an equal number of females and males from
all groups, were sacrificed each day. The entire procedure took 2 weeks
38
-------
TABLE 20. BODY WEIGHTS AND FOOD CONSUMPTION
(Study I - Females)
Group
A
A
A
A
A
A
B
B
B
B
B
B
C
C
C
C
C
C
D
D
D
D
D
D
E
E
E
E
E
E
N
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
39
39
39
40
40
40
39
40
40
39
39
39
39
39
39
Day
3
17
31
45
65
85
3
17
31
45
65
85
3
17
31
45
65
85
3
17
31
45
65
85
3
17
31
45
65
85
Body Weight
(grams)
X S.D
20.0
22.3
22.8
25.6
28.7
27.9
20.3
23.0
23.7
25.9
28.9
27.5
21.3
23.2
23.6
25.3
27.6
27.0
20.1
21.0
23.2
24.1
26.3
26.6
20.0
21.7
23.6
25.1
25.9
27.3
2.1
2.2
3.0
2.6
3.2
3.8
2.3
2.0
1.8
2.3
3.2
3.6
1.8
1.5
1.5
1.8
3.7
3.6
1.9
2.3
2.7
1.8
4.0
4.6
1.8
1.6
1.7
1.9
2.2
3.3
Food Consumption
_ g/day/kg
X(g) S.D body weight
9.0
9.2
9.4
8.2
9.0
8.9
8.2
8.2
9.2
9.3
8.9
7.3
8.9
10.0
8.7
8.9
9.0
9.3
9.8
9.4
8.2
8.4
8.4
9.4
9.9
9.1
9.0
9.3
8.9
1.2
1.7
0.8
1.7
1.4
1.0
1.9
1.7
1.1
1.4
1.3
2.4
1.7
0.0
1.8
1.5
1.8
1.5
0.6
1.1
1.2
1.3
2.1
1.3
0.6
1.3
1.2
0.9
1.8
450.0
412.6
412.3
320.3
313.6
319.0
403.9
356.5
388.2
321.8
323.6
342.7
383.6
423.7
343.9
322.5
333.3
462.7
466.7
405.2
340.2
319.4
315.8
470.0
456.2
385.6
358.6
359.1
326.0
39
-------
TABLE 21. BODY WEIGHTS AND FOOD CONSUMPTION
(Study I - Males)
Group
A
A
A
A
A
A
B
B
B
B
B
B
C
C
C
C
C
C
D
D
D
D
D
D
E
E
E
E
E
E
N
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
39
39
40
40
40
40
39
39
Day
3
17
31
45
65
85
3
17
31
45
65
85
3
17
31
45
65
85
3
17
31
45
65
85
3
17
31
45
65
85
Body Weight
(grams)
X S.D
26.1
30.0
32.6
35.8
41.4
42.9
27.5
30.5
33.6
36.7
42.3
44.0
26.1
29.0
32.1
35.0
39.8
42.5
24.7
28.9
32.4
34.0
38.5
43.4
26.9
30.4
33.7
35.6
40.0
42.8
2.0
2.3
3.2
4.2
5.8
6.9
2.2
2.3
3.0
3.8
4.9
5.7
1.9
2.0
2.7
3.4
4.9
4.9
3.0
2.8
3.3
4.0
4.9
6.3
2.7
1.9
3.9
4.1
4.7
4.6
Mean Daily
Food Consumption
_ g/day/kg
X(g) S.D body weight
9.2
8.9
9.6
8.3
9.3
8.7
7.6
9.3
9.1
9.0
8.7
8.5
8.0
9.4
9.7
8.6
9.0
9.4
8.9
9.8
9.4
8.6
8.3
9.1
10.0
9.7
9.8
9.4
9.6
9.2
2.7
2.0
0.8
1.5
1.1
1.2
2.0
1.3
1.1
1.6
2.4
1.4
1.7
1.3
0.8
1.5
1.5
1.6
1.6
0.6
1.1
1.0
1.3
1.4
0.2
0.9
0.6
1.3
1.1
1.6
352.5
296.7
294.5
231.8
224.6
202.8
276.4
304.9
270.8
245.2
205.7
193.2
306.5
324.1
302.2
245.7
226.1
221.2
360.3
339.1
290.1
252.9
215.6
209.7
371.7
319.1
290.8
264.0
240.0
215.0
40
-------
TABLE 22. HEMATOLOGICAL INDICES FOR MICE EXPOSED TO CONCENTRATED
RECYCLED WATER (Study I)
Group Sex
A
A
B
B
C
C
D
D
E
E
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
N
17
19
16
16
16
17
18
19
17
15
Hemoglobin
(g/100 ml)
M
15.57
17.62
15.74
17.24
16.11
16.92
16.07
17.63
16.03
16.97
SD
0.97
1.68
0.97
1.77
1.32
1.63
0.99
1.49
1.36
2.82
Red Blood Cells
(106 cells/mm3)
M
8
9
9
9
9
9
9
9
9
9
.79
.67
.15
.59
.15
.39
.41
.96
.81
.23
SD
0.
0.
0.
1.
0.
1.
0.
0.
0.
1.
52
84
54
0
75
0
61
85
69
29
White Blood Cells
(103 cells/mm3)
M
10.
11.
12.
10.
13.
11.
11.
10.
12.
9.
75
66
13
36
3
33
93
45
8
93
SD
3.01
3.5
4.36
3.02
5.63
3.055
2.29
2.32
2.86
4.38
TABLE 23. HEMATOLOGICAL INDICES FOR MICE EXPOSED TO CONCENTRATED
RECYCLED WATER (Study II)
Group Sex
A
A
B
B
C
C
D
D
E
E
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
N
10
10
10
10
10
10
10
10
10
10
Hemoglobin
(g/100 ml)
M
16.23
17.96
15.83
17.05
15.55
16.8
15.97
16.84
16.1
17.07
SD
1.09
1.64
0.84
1.76
1.59
1.38
0.91
1.57
0.75
1.02
Red Blood Cells
(i o
(10 cells/mm )
9
9
9
9
8
9
8
8
9
9
M
.3
.98
.1
.37
.95
.47
.93
.98
.18
.69
SD
0.56
0.80
0.52
0.92
0.86
0.72
0.53
0.97
0.56
0.69
White Blood Cells
(103 cells/mm3)
M
12.
10.
12.
15.
14.
10.
12.
12.
13.
11.
23
67
74
32
79
15
95
01
13
0
SD
2.
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41
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for Study I and about a week for the other studies. Serum samples from the
mice were processed on an SMAC 20 Autoanalyzer (Technicon). Several prelim-
inary trials were run to verify the quality and dilution of the sample and
calibration of the instrument.
Results of the blood chemistry tests and the tissue weights for the
various experimental groups are presented in Tables 24 through 51. The
abbreviations for the different tests are explained in Section 3 (page 22).
Results for the experimental groups which were found to be significantly
different from the controls are marked by an asterisk (*). Tissue weight
relative to body weight is designated by the letter R. These values should
be multiplied by 0.001 to obtain the actual values. Tables 24 through 37
give the means, standard deviations, and number of individuals in each
measurement. Tables 38 through 51 give the range of the results and the
exact degree of significance (P value) for each test. Because of the large
number of tests it was arbitrarily decided to regard as significant only
those results which showed a P value <^ 0.05 in more than one experimental
group.
A general examination of the tables reveals that there are only a few
results for the experimental groups that differ significantly from those
for_the control group (P <^0.05). In Study I (males), Na , Cl~, K , and
HCO_ were higher in a few experimental groups than in the control group.
Such a combined change is logical, since the concentrations of these ions
usually depend on each other. However, this effect was not repeated in the
other studies. Males in Study II had lower urea values in the experimental
groups and higher values of serum proteins. Glucose, triglycerides, and
alkaline phosphatase were lower in the experimental groups than in the
control males in Study III. The females of this group had lower values of
cholesterol and urea but higher values of chloride. Almost none of these
limited significant results have been repeated in two different studies.
Results were similar for the tissue weight. Only a few significant
differences were noted. In Study I the males' brains were heavier in
Groups D and E than in the control group. The spleens in the females of
the Study I were smaller in the experimental groups than in the control.
The lungs were larger in the experimental groups of males from Study II.
In males and females from Study III, the spleens and adrenals were heavier
in the experimental groups than in the control. No significant pathological
results could be related to these tissue weight differences (see page 78 ).
There is very little back-up data available on the clinical chemistry
of laboratory animals for most of the parameters tested, primarily because
of sample volume limitations. Using the technology available today, a
volume of 0.5 ml serum is needed to perform all 20 tests. Approximately 1
ml of blood is needed to produce 0.5 ml of serum. We had more than 95
percent success in drawing this amount of blood from the mice. We were
able to find only one set of comparable data (for an unspecified strain of
mice), which had been tested using a similar instrument and included the
parameters examined in our study 'W.H. Baum, Scientific Associates,
Inc., St. Louis, Missouri 63123, personal communication). These results
are given in Table 52. Although no standard deviation is given, some
42
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TABLE 52. BLOOD CHEMISTRY OF 50 MICE WEIGHING 19-30 g*
(average of 100 trials)
Parameter
Glucose (mg/dl) 174
Albumin (g/dl) 3.4
Globulin (g/dl) 2.0
Total protein (g/dl) 4.0
CPK (IU/L) 111
Alkaline Phosphatase (IU/L) 200
LDH (IU/L) 210
SCOT (IU/L) 107
SGPT (IU/L) 82
Calcium (mg/dl) 9.3
Inorganic Phosphorus (mg/dl) 5.6
Sodium (mEq/L) 110
Potassium (mEq/L) 4.3
Chloride (mEq/L) 92
Iron (mcg/dl) 102
Cholesterol (mg/dl) 50
Triglyceride (mg/dl) 100
BUN (mg/dl) 10.5
Bilirubin (mg/dl) 0.05
Uric Acid (mg/dl) 0.6
Creatinine (mg/dl) 0.5
*Scientific Associates, Inc., 6200 S. Lindbergh, St. Louis, Mo. 63123
71
-------
comparison can be made. Average values were close for some parameters,
such as glucose, total protein, calcium, potassium, chloride, bilirubin,
and creatinine. Average values for other parameters were quite different.
For example, our mean value for cholesterol in the control group is
184.4 mg/dl, and the Scientific Associates value is 50 mg/dl. The data
for triglycerides show the opposite: our average is 35.5 mg/dl and the
Scientific Associates value, 100 mg/dl. CPK and LDH values measured in our
laboratory were 3 times the values measured in the other laboratory. Our
values for these enzymes are probably higher (and show the highest variability)
as a result of the blood drawing method (heart puncture). Strain differences
might also account for some of the variation between the two laboratories.
A more extensive data base is essential before conclusions are drawn from
such laboratory studies.
Liver Mixed Function Oxidase Activity—
The mixed function oxidase (MFO) system is located in the microsomes
of a number of tissues and metabolizes foreign compounds and certain endo-
genous derivatives. This enzymatic complex was once believed to act as a
detoxifying mechanism and in the last decade was found to be an activation
system for a large number of xenobiotics to their toxic derivatives. A few
hundred compounds are known to induce or increase the activity of the MFO,
and a smaller number of environmental pollutants are known to reduce or
inhibit the system. Among the inducers, some groupings are based on
chemical structure, such as the polyaromatic hydrocarbons (methylcholanthrene)
or the chloro-organic compounds (DDT) and many other unrelated comopounds
(such as phenobarbital). Inorganic or organic compounds, such as lead and
cannabinol compounds, can inhibit the MFO activity. The activity of this
system can be evaluated under in vitro or in vivo conditions. We chose to
test the activity in vivo by the determination of the sleeping times induced
by hexobarbital. Longer sleeping times will result from inhibition of the
MFO (slower metabolism of hexabarbital), and shorter sleeping times from
induction of the mixed function oxidases. Mice were injected with 100
mg/kg hexobarbital at certain times of the day, and the duration of loss of
righting reflex was measured. A sample of animals, chosen using the table
of random digits, was used for this test.
The results of Study I are given in Table 53 and those of Study II in
Table 54. Latent time is the time between injection and the loss of righting
reflex, and sleeping time is the time between loss and gain of the righting
reflex. Positive controls run on mice exposed to 500 mg/kg body weight of
Aroclor 1254 showed about a 50 percent shortening of sleeping time
induced by 100 mg/kg hexobarbital.
The variability of the results among the mice was quite large; however,
the only significant difference in relation to the controls was found in
Study I, where females in Group E (exposed to the highest TOC concentration)
had about 50 percent shorter sleeping time than the control. It should be
kept in mind that during the long exposure time, other factors in the
environment, including chemicals in food, water and air, could interfere
with these studies.
72
-------
TABLE 53. SLEEPING TIMES IN MICE AFTER EXPOSURE TO CONCENTRATED RECYCLED WATER
(Study I)
Latent Time (min)
Group
A
A
B
B
C
C
D
D
E
E
Sex
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
N
18
18
19
19
20
19
19
19
20
20
M
2.47
3.85
4.12
3.74
2.13
3.29
2.51
4.57
2.59
4.39
SD
0.69
2.57
5.17
1.59
0.79
0.91
1.07
2.40
0.81
2.66
Sleeping
M
59.30
34.28
48.49
40.80
49.95
31.65
56.11
41.57
31.91
31.61
Time (min)
SD
35.68
27.3
23.29
17.78
18.26
20.01
23.34
22.95
20.90
22.29
TABLE 54. SLEEPING TIMES IN MICE AFTER EXPOSURE TO CONCENTRATED RECYCLED WATER
(Study II)
Latent Time (min)
Group
A
A
B
B
C
C
D
D
E
E
Sex
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
N
10
10
10
10
9*
10
10
10
9**
10
M
2.516
1.966
2.666
2.416
2.379
2.125
3.740
2.216
3.190
2.358
SD
0.578
0.690
0.594
0.574
0.406
0.364
1.974
0.364
1.820
0.547
Sleeping
M
40.55
42.508
28.191
34.606
42.398
38.108
33.791
40.875
30.944
40.316
Time (min)
SD
15.210
17.800
13.487
14.723
14.034
18.285
25.970
15.227
17.449
8.509
*N172 did not lose righting reflex
**N243 did not lose righting reflex
73
-------
Motor Activity Test—
The well-known neurological and behavioral effects of wide-spread
pollutants such as pesticides and heavy metals bring into question the
possibility of subtle effects in those tissues caused by long, low-level
exposure to the toxins. Other areas of concern are: (1) the possible
interaction of several pollutants at low, submarginal level which increases
pathological effects and (2) exposure of especially sensitive groups in the
population, such as fetuses, newborns or the aged.
Behavioral studies have numerous drawbacks. In general, such studies
are expensive, long-term and difficult to interpret. Therefore, a short,
simple test was selected as an explorative study. The activity test measures
the number of movements per arbitrary time period. The measurement is
based on the interruption of a light beam which is sensed by a photoelectric
cell and translated to counts. Results of this test are presented in
Tables 55 and 56. There were no significant differences between any one of
the experimental groups and the control.
Reproduction—
The reproduction test was performed in Study P and Study III. Both
parents were exposed to concentrated water. The main difference between
these studies was that in Study III the mice were exposed throughout
gestation and lactation, while in Study P the mice were exposed from the
age of five weeks. In both studies, the offspring were exposed throughout
gestation.
There were 10 males and 20 females per group. Births started 21 days
after mating and ended by the 27th day. Analysis of the weights of the
newborns showed that both birth weight and weight gain are significantly
dependent on the size of the litter. A regression line was computed for
the relationship between litter size and mean body weight in the two
experiments. The coefficients derived from these lines are presented in
Table 57.
TABLE 57. DEPENDENCE BETWEEN LITTER SIZE AND MEAN BODY WEIGHT
Correlation Total Mean
Study N Coefficient (r) Slope Intercept Body Weight (g)
P
III
82
43
-0.82
-0.80
-1.49
-1.43
16.66
22.89
9.42
14.77
The results show that the correlation is negative (the higher the
litter size, the lower the mean body weight) and highly significant. Mean
body weights and litter size for the second experiment (study III) are
given in Table 58. Similar results were found in the first experiment
(study P). The corrected mean body weights (related to the litter size) at
day 28 for the two experiments are given in Table 59.
74
-------
TABLE 55. MOTOR ACTIVITY IN MICE AFTER EXPOSURE TO CONCENTRATE RECYCLED WATER
(Study I)
Group
A
A
B
B
C
C
D
D
E
E
Sex
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
N
16
18
14
14
17
14
15
15
14
15
Count per First
M
118.75
106.61
133.79
137.89
152.88
104.00
126.20
110.20
133.57
125.14
Five Minutes
SD
33.54
44.33
64.79
68.53
109.95
22.22
29.30
41.77
72.15
25.42
Count per
M
216.68
190.38
249.86
245.86
288.82
194.35
250.73
208.00
251.81
226.93
Ten Minutes
SD
67.25
68.78
118.53
67.14
207.37
43.54
56.62
71.53
115.10
60.50
TABLE 56. MOTOR ACTIVITY IN MICE AFTER EXPOSURE TO CONCENTRATE RECYCLED WATER
(Study II)
Group
A
A
B
B
C
C
D
D
E
E
Sex
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
N
10
10
10
10
10
10
10
10
10
10
Count per First
M
112.6
117.4
126.6
101.8
98.6
117.8
108.0
138.9
96.7
132.5
Five Minutes
SD
33.0
51.8
55.1
30.1
31.2
16.8
30.2
10.1
24.6
59.9
Count per
M
194.3
205.4
224.6
203.9
187.6
213.5
199.2
267.6
201.7
247.2
Ten Minutes
SD
52.0
85.2
88.3
54.4
51.0
28.6
52.0
131.4
50.5
115.8
75
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TABLE 59. CORRECTED MEAN BODY WEIGHT (Day 28)
Mean Body Weight
Group
Measured Calculated
Corrected
SD
Study P
Study III
A
B
C
D
E
A
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C
D
E
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3.9
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4.0
4.6
3.1
3.5
1.3
5.2
2.0
3.5
Mean body weight was corrected as follows. Calculated mean body
weight was found on the regression line at the corresponding mean litter
size for each group. The difference between the mean found in the data and
that computed from the regression line (the residue) was added to the total
mean body weight (9.4 in the first experiment and 14.8 in the second; see
Table 57). Standard deviations from the corrected mean body weights were
computed from the individual differences between the mean body weight of
each litter and its calculated number. These values were found to be very
close to the original standard deviations. No significant differences were
found among the different groups with relation to litter size or body
weight. Measurement taken on the pregnant mothers also showed no difference
in the mean of body weights or food consumption in the various experimental
groups (Table 60).
TABLE 60. BODY WEIGHTS AND FOOD CONSUMPTION OF PREGNANT FEMALES
Group Number
Body Weight
_ (g)
x S.D.
Food Consumption
(g)
x S.D.
A
B
C
D
E
20
20
20
20
20
29.0
30.7
31.9
31.9
29.3
3.5
3.2
2.9
3.8
3.1
8.4
8.2
8.2
8.2
8.4
1.4
1.5
1.3
1.4
1.5
77
-------
Dominant Lethal Mutation Test—
The dominant lethal mutation test has been used extensively to assess
the mutagenicity of a wide variety of substances. The standard method has
been to give adult male animals the chemical by a certain route and schedule
followed by mating each male weekly with two virgin females for 5-8 weeks.
Exposed females were mated with unexposed males for one week and sacrified
after another week (on day 15).
The test was run on two animal groups. The first group was 50 male
mice exposed for 14 days to concentrated, recycled water. The second group
consisted of 50 male and 50 female mice exposed for 90 days (since weaning).
Each group was subdivided into 5 groups: A (control) and E, D, C, and B,
exposed to undiluted concentrated renovated water (700 mg/1 TOG) and dilu-
tions of 1:2, 1:4, and 1:8, respectively. The summary of the study is
given in Table 61 and the results of experiments I, II and III are given in
Tables 62, 63, and 64.
TABLE 61. RESEARCH PLAN
Significance of the Effect
Exposure Time Number of Percentage of
Experiment (days) Sex Dead Fetuses Dead Fetuses
I
II
III
14
90
90
Male
Male
Female
p<0.05
p>0.3
p=0.07
p<0.05
p>0.3
p=0.07
In the first experiment (exposure time = 14 days), the mean number of
dead fetuses per pregnant female in all groups was double the number in the
control. This was found to be significant (p<0.05). In the second experi-
ment (exposure time = 90 days), there was no significant difference between
the groups. Although the mean number of fetuses in the experimental group
was higher in the second experiment than in the first, the control also
increased as much as twice; therefore, the difference was insignificant.
In the third study, females (exposed for 90 days) were mated with virgin
males and sacrificed after 14 days. Here, again, the mean number of dead
fetuses was more than double that in the control group, but because of the
relatively small number of animals, the p value (0.07) was only close to
the significance border line.
Pathology—
All animals from the first 90-day study were subjected to a complete
post-mortem examination. Microscopic slides prepared from groups A and E
included the following tissues: heart, lungs, kidney, spleen, liver,
brain, testes, ovaries, uterus, and mandibular lymph nodes. These slides
were examined histologically.
78
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CO LO **O -3" vO
**O LO LO CO O^
CO ^O **O LO ^O
o o o o o
r^ ^o in in in
cn
-------
The only gross abnormality observed at post-mortem was an infrequent
pale tan liver. Microscopic study of groups A and E revealed the tissues
to be within normal limits with the exception of some mild, non-specific,
reversible degenerative liver change occurring with a near equal frequency
in the male mice of both groups. Also observed were three benign lung
neoplasms (alveolar cell adenoma), a spontaneous lesion not uncommon in the
mouse lung.
The same protocol was followed for the second 90-day study, but only
half as many animals were involved. No microscopic abnormalities were
observed, with the exception of a small number of the mild degenerative
liver change as seen in the first study. The pathology findings in both
90-day studies are insignificant and reveal only the spontaneous changes
expected in a mouse population of similar size and age.
In Vitro Studies
Recycled water was found to be mutagenic in the in vivo test (dominant
lethal mutation test) in the study reported here and in a previous study (5).
A microbiological test using Salmonella strains and the mamalian mutagenicity
assays were instituted to verify the results of the dominant lethal mutation
test.
A related problem, the question of carcinogenicity, is even more
complex. Conventional carcinogenicity bioassays require prohibitive volumes
of water. Therefore, short i.n_ vitro assays, which demand only a small
amount of sample, were used to evaluate the potential carcinogenicity of
the reused water. Such tests are based on the following assumptions:
(1) Mutation is one of the first steps leading to the formation
of a tumor, and certain cellular changes (transformation) in vitro are
indicators of a progressive process leading to malignancy. Recent experi-
mental work has supported these assumptions. Several hundred compounds
(known carcinogens and noncarcinogens) were shown to give a very high
correlation in a bacterial mutagenicity test.
(2) There is a progression of changes with time transforming "normal
cells" to malignant cells.
(3) There is a relation between transformation and mutagenicity in
the same cell system.
(4) There are cases when chemically induced transformed cells (in
vitro) are capable of forming tumors in animals.
(5) Promoters can increase the rate of transformation or mutation
induced by chemicals in tissue culture systems.
Salmonella/Microsome Test—
The Salmonella/microsome test, developed by Ames and coworkers (12),
measures the reversion rate of several specially constructed Salmonella
strains unable to grow in the absence of histidine. Besides the histidine
mutation, the strains have undergone a mutation which causes loss of the
excision repair system and a mutation which results in loss of the lipopoly-
saccharide coating of the bacteria. These mutations render the bacteria
81
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more susceptible to mutagenesis. Strains TA1538, TA98, and TA1547 have
frame-shift mutations in the histidine operon, and strains TA100 and TA1535
have base-pair substitutions. Since many substances are not mutagenic (or
carcinogenic) prior to metabolic conversion, isolated mammalian liver
microsomes were included in the test systems to provide activation. The
results presented in Table 65 show that the water did not induce significant
mutation (i.e., 32 x control) in the bacteria. Additional attempts (not
shown) using strains 1538, 1557, and 1535 did not reveal any increase in
the mutation rates.
Mutagenicity Test with Mammalian Cells—
Mutagenicity tests using mammalian cells differ from chose using
bacteria in the gene structure and organization, in the enzyme repair
systems, and in the transport mechanisms of compounds through membranes.
In addition, the Salmonella test applies a backward mutation assay while
the mammalian test examines forward mutation. The same rat liver fraction
was used for activation in both assays.
A problem was encountered in the initial experiments with direct muta-
genicity. The cells that were exposed to the water samples still covered
the plates at the end of the experiments. It appeared that the inhibitor
ouabain was not as effective as in the controls. Although no further
studies were carried out to explain this phenomenon, it is probable that a
competitive inhibitor, such as potassium, which is present in this water at
a high concentration, could interfere with the action of ouabain. Such
interferences should be taken into account when testing environmental
samples which contain a variety of soluble materials. Two modifications
were made to solve the problem. First, the cells were trypsinized after
exposure and plated in new plates; second, the concentration of ouabain
was raised to 5 mM.
Table 66 shows the results of the mutagenicity test without activation,
and Tables 67 and 68 present the results of two different runs after
activation with rat liver fraction. While the results without activation
show a marginal effect, if any, those after activation show a clear mutagenic
effect in a dose-response relationship. Comparison of the plating efficiency
in Tables 67 and 68 shows that toxicity is increased substantially by the
liver activation system. This phenomenon is not specific to V79 cells and
was studied in more detail with human fibroblasts (WI38, see p. 85).
Soft Agar Transformation Assay—
Evidence derived from in vitro carcinogenic experiments indicates that
a progressive process leads to the development of a malignant cell (17).
One of the early noted changes is a morphological transformation, while at
a later stage the ability to grow on soft agar (anchorage independence) is
manifested. This anchorage independance is believed to be highly correlated
with cell malignancy (18). Based on the assumption that cellular transforma-
tion has a lag period and increases with time, the test was performed
repeatedly for an extended time after treatment.
82
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TABLE 65. SALMONELLA MUTAGENICITY TEST OF CONCENTRATED REUSED WATER*
S9
Control
Control +
0.1 ml H20
0.5 ml H20
0.1 ml H20 +
0.5 ml H20 +
BP (5ug) +
Number of Colonies per Plate
TA98 TA100
32,
55,
58,
56,
84,
90,
689,
37
57
45
46
64
78
761
140,
226,
216,
216,
340,
296,
1011,
155
236
236
230
316
326
1098
*Sq fraction is the supernatant obtained from liver homogenate after
centrifugation at 9000 g for 15 minutes. The TOC concentration in water
tested was 700 pg/ml. BP - Benzo(a)pyrene.
TABLE 66. DIRECT MUTAGENICITY OF CONCENTRATED RECYCLED WATER
Average Plating
Sample Efficiency (%)
Control
1 yg/ml MNNG3
0.1 ml H20b
0.2 ml H20
0.3 ml H20
0.4 ml H20
0.5 ml H20
91.0
48.5
85.0
82.8
77.5
76.2
71.2
Colonies
per 16 Plates
1
138
3
0
2
1
3
Mutants
per 10 Survivors
0.56
161.8
2.0
0
1.5
0.75
2.4
nNNG-N-Methyl-N-nitro-N-nitrosoguanidine
The concentration of TOC in water is 700 vig/ml.
83
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TABLE 67. MUTAGENICITY OF CONCENTRATED RECYCLED WATER (first run)3
Toxicity Colonies Oubain-Resistant
Sample (% Plating Efficiency) per 16 plates Mutants/10 Survivors
Control
Control + S9
0.1 ml H20+S9
0.2 ml H20+S9
0.3 ml H20+S9
0.5 yg BP+S9
1.0 yg BP+S9
88
78
44
46
24
55
38
1
0
1
4
6
54
104
0.5
0.0
1.0
3.8
10.9
43.8
142.2
o
H20 mixed 1:1 with 2 x MEM and added to each sample with 1 ml S9 mix plus
PBS to give 2 ml final volume. TOG level in water was 700 yg/ml. Cells
incubated with S9 alone have a plating efficiency of 70%. S9 is the rat liver
homogenate supernate after centrifugation at 9000 g. BP-Benzo(a)pyrene.
TABLE 68. MUTAGENICITY OF CONCENTRATED RECYCLED WATER (second run)a
Toxicity Colonies
Sample (% Plating Efficiency) per 16 plates
Control
Control + S9
0.1 ml H20+S9
0.2 ml H 0+S9
0.3 ml H20+S9
0.4 ml H20+S9
1.0 yg BP+S9
90
77
45
39
30
44
32
0
0
11
10
15
2
65
Oubain-Resistant
Mutants/ 10 Survivors
0
0.0
14.0
14.2
28.1
2.5
120.0
H?0 mixed 1:1 with 2 x MEM and added to each sample with 1 ml S9 mix plus
PBS to give 2 ml final volume. TOC level in water was 700 yg/ml. Cells
incubated with S9 only have a plating efficiency of 70%. S9 is the rat liver
homogenate supernate after centrifugation at 9000 g. BP-Benzo(a)pyrene.
84
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The cells were exposed for 2 hours and then assayed for growth on soft
agar after 2 weeks and every 2 weeks for 2 months. Table 69 shows the
results at post treatment passages 2 and 4 (FTP and FTP,). Again, while
without activation there was a marginal effect, a significant increase was
noted in the number of colonies on soft agar for cells incubated with liver
activation system. No dose-response relationship could be shown. The
spontaneous transformation increased with time as did the transformation of
the exposed plates. No colonies could be detected in cells incubated with
benzo(a)pyrene or MNNG up to 8 weeks after exposure.
W138 Toxicity Test—
If reused water is approved for potable use, continuous monitoring for
toxicity will probably be required. For this purpose a short, inexpensive
in yjLtr_£ assay using mammalian cells may be a feasible solution. Preliminary
studies using human lung fibroblasts (WI38) were initiated to assess this
possibility. In this assay, protein levels are used to indicate toxicity.
figure 8 shows thar changes in protein level reflect cell number. Figure 9
shows that toxicity increases with time and that in the first 24 hours the
effect is very small. The presence of the activation system dramatically
increased the toxic effect of the water (Figure 10). Table 70 shows a
close-response relationship between the amount of water and cell protein
level. Without activation, toxicity increased from 5.0 percent inhibition
at J7.5 )Jg TOG to 24,0 percent inhibition at 210 jjg TOG. In the presence
of the liver fraction, the inhibition was decreased with increased TOG
levels: from 87.4 percent at 17.5 yg TOG to 58.7 percent at 210 yg TOG.
This phenomenon hcts not been explained, but may result from inhibition of
the activation system by soluble chemicals in the water, or by interactions
of the compounds Ln the water.
85
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TABLE 69. SOFT AGAR TRANSFORMATION3
Average Number Colonies/Plate
Sample (PTP)2 (PTP>4
Control
0.1 ml HO
0.2 ml H20
0.2 ml H20 + Sg
0.3 ml HO
0.3 ml HO + S
6
13
10
40
11
22
9
15
17
62
12
51
Number Colonies/ 10 Cells
(PTP)2 (PTP)4
4.0
8.6
6.6
27.0
7.3
14.6
6.0
10.0
11.3
41.3
8.0
34.0
1.5 x 10 W138 cells treated for 2 hr in a final volume of 2 ml.
After treatment, cells were placed in a 75 cm flask. Soft agar growth
test 1.5 x 10 cells/dish x 10 dishes/point. TOG level in water is 700 yg/ml.
TABLE 70. THE EFFECT OF WATER CONCENTRATES ON CELL PROTEIN LEVEL IN
TISSUE CULTURE
Sample Sq
Zero time
Untreated control
Untreated control +
17.5 yg TOG
17.5 yg TOG +
35.0 yg TOG
35.0 yg TOG +
70.0 yg TOG
70.0 yg TOG +
140.0 yg TOG
140.0 yg TOG +
210.0 yg TOG
210.0 yg TOC +
Protein (yg)
19.0
53.0
50.0
51.5
22.5
48.0
25.5
45.5
29.0
46.5
31.5
45.0
33.0
Inhibition (%)
-
-
5.0
87.4
14.0
77.0
22.4
68.5
19.9
59.5
24.0
58.7
86
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350
D Control Protein
O Treated Protein
O Control Cell No.
A Treated Cell No.
40 60 80
Time (hrs)
100
120
Figure 8. Cell counts and protein levels after exposure to recycled
water in the presence of S9 activation system.
87
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O Control
Concentrated H20
Figure 9.
30 40 50 60
Time (hrs)
Direct exposure of WI38 to concentrated recycled water.
70
88
-------
160
140
120
100
80
o
i.
0.
60
40
20
T
T
T
T
O Control
D S9 Toxicity
• 89 and Concentrated H20
I
I
I
10 20 30 40
Time(hrs)
50 60
Figure 10. Effect of liver activation system on toxicity
of concentrated recycled water in WI38 cells.
89
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SECTION 5
DISCUSSION
The progress of wastewater treatment technology and increasing pressure
for new sources of water have brought the subject of reuse of recycled
water under intensive consideration. Direct reuse has been practiced in
Southwest Africa for about 10 years (19), and long-range plans are being
considered in the United States (20,21) and Israel (22).
Current wastewater treatment technology is designed to satisfy the
conventional quality criteria for drinking water. However, numerous organic
compounds existing in wastewater effluent cannot be removed using presently
available technology. Only a limited amount of work has been done in this
area, and rigorous toxicological and epidemiological studies are needed to
evaluate the possible long-term effects or safety of drinking recycled
water.
As a preliminary effort, this toxicological study was initiated to
evaluate the health aspects of renovated water prepared in an advanced
treatment plant. Three approaches were considered: (a) extensive analysis
of the water followed by toxicological assessment based on data for each
compound found in the water, (b) treatment of the water sample as a single
test compound, or (c) a compromise approach involving the testing of a
fraction of the water-soluble organic compounds.
The first approach requires and exhaustive analysis; several hundred
compounds are defined in water, and these compose only a small fraction of
the total organic content. In addition, toxicological data on these compounds
are limited, and long-term studies will be required to evaluate the produc-
tivity of such an approach. Such an extensive study disregards interactions
which could occur within the mixture (synergistic or antagonistic).
The holistic approach (water sample as one sample) has one major
drawback: It is difficult to make generalized predictions for other water
sources from the toxicological data obtained for a specific water sample.
Based on the relative merits and disadvantages of the three approaches, the
second approach was selected. A concentrated water sample was obtained
with the highest possible recoveries and was used for the toxicological
study.
90
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The concentration technology selected for the study was based on
reverse osmosis. Inorganic fractions were removed by techniques based on
the Donnan equilibrium principle. In small-scale laboratory experiments,
TOG recovery values in reverse osmosis concentrates were 80 to 90 percent
(6). However, for the present work, in which large volumes (400,000 liters)
were concentrated, the recovery percentage was much lower (Table 16).
There is no way to determine which compounds were lost during the process.
It is probable that most of the low molecular weight compounds (<150) were
lost, among them the volatile weak carcinogens.
The water concentrate was incorporated into a gel type diet for feeding
to the test mice. Ions contained in the concentrate were balanced according
to nutritional requirements. Although the feeding practice used in this
project demanded a significant effort, greater experimental control was
achieved. The diet used in the study (high content of agar) was found
adequate to the animals. The main characteristic of the diet was a low
fiber content. The modification of toxic effects by fibers has been studied
(23); however, information on this subject is limited, and the influence of
this factor in this project cannot be estimated. It should be noted that
any type of diet might influence the toxicity of the test sample.
Toxicological observations were made on rate of growth, food intake,
fertility, mutagenicity, mortality, blood physiology and biochemistry,
liver function, behavior, and general pathology. Most of the toxicol-
ogical tests did not reveal any significant changes in the exposed mice.
This was found true in general clinical and physiological assessment,
with tests such as growth and lethality rate, and with special assays for
evaluation of specific tissue fractions such as liver or the central nervous
system.
The most elaborate study was an examination of blood constituents.
Twenty-three parameters were checked, and only in a few cases were there
significant differences between the experimental groups and the control.
Although a number of significant differences were noted in two or more
groups in the same study, none were repeated in other studies. No specific
pathological syndrome could be related to any of the changes in the blood
serum. However, it is noteworthy that the number of changes in the 150-day
study was higher than in the 90-day studies. No significant changes were
found in animals that were subjected to the regular diet for 90 days after
an exposure time of 90 days. Although these differences are small, they
might indicate that an extended exposure period beyond 150 days (about 20
percent of the animal's lifetime), might reveal more significant pathological
cManges.
Likewise, the pathological results did not show any significant differ-
ences among the groups. The weights of the spleen and adrenals in the
various experimental groups were significantly different from the controls.
Attain, these differences were not accompanied by histopathological changes.
I}-"- M\lative tissue weight analysis did not provide any increase in sensi-
rs >y compared to that with absolute tissue weights. On the contrary,
iin: .• significant results were found with absolute tissue weights.
91
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Reproduction tests did not reveal changes in fertility or in the
weight or number of newborns. Results of chronic studies carried out on
mice born to exposed dams were not different from those for mice exposed
only after weaning (study I as compared to studies II and III). There are
several possible explanations for these observations: (a) The toxic chemicals
do not pass the placenta barrier or transfer through milk. (b) Doses of
the toxic materials are too low. (c) There are not toxic compounds in this
mixture. (d) The tests as performed cannot detect toxicity of these compounds.
Essentially no changes in the mixed function oxidase activity in the
liver were induced by exposure to concentrated water. Interpretation of
these negative results is difficult due to the complexity of the water
mixture and the length of the experiment.
General observations in previous experiments with rats exposed to
concentrated water indicated a resultant hyperactivity. Quantitative
experiments done in the present study failed to reproduce this result in
mice. This study should be considered a very preliminary behavioral assess-
ment. There are more complex behavioral tests which have more relevance to
human CNS toxicity.
Water concentrates were found to be mutagenic in previous studies
using the dominant lethal mutation assay (5). In the current studies, the
first experiment was positive the second was negative. The mean lethality
of the control fetuses in the second experiment was double that in the
first. Although the average fetal death in the experimental group was
higher in the second trial than in the first, the difference was not signif-
icant. Wide experience has shown that the dominant lethal mutation test
has several weaknesses. For example, studies performed on the same chemicals
in different laboratories are not consistent. In addition, there is not
always a dose-response relationship. Green and Springer mentioned that half
of the 24 chemicals they tested failed to show a dose-response relationship
(24). In an effort to resolve these conflicting results, mutagenicity
studies with cells in culture were included in the current experimental
program.
While short-term assays have been found useful for screening mutagenic
and carcinogenic compounds, a parallel in vitro assay for general toxicity
has not yet been established. Preliminary studies using human cells (WI38)
were initiated in this project for the purpose for formulating a protocol
for routine monitoring of water before dispatch. Water concentrates were
found quite toxic to the cells, especially in the presence of a liver
activation system. Surprisingly, the cells were more affected by the water
at smaller doses than at higher doses in the presence of the liver micro-
somes. This effect could be the result of antagonistic effects on the cells
of different compounds in the water. It is also possible that the micro-
somes are affected and indirectly affect the toxicity of the cells. The
limited experimental data on this subject does not permit a conclusive
explanation. Although several groups have tried to establish a biological
monitoring system for water toxicity (25), there has not yet been a sol-
ution to this important problem.
92
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Recently, several In vitro assays have been developed for the screening
of possible carcinogens. Such tests quickly provide accurate assessment of
the mutagenlc au,i carcinogenic potential of the chemicals tested (26).
Although there it> no mechanistic evidence that mutagenesis is an essential
part of carciiiogenesis, a substantial amount of circumstantial evidence has
been accumulated (13,27). Correlations were recently found between mutagenesis
arid transformation in the same tissue culture system (26,28). In the
present study, r;u Increase was noted in the number of revertant colonies
above background in ctie bacterial mutagenicity test after exposure to water
concentrate in Sue p; ,.>,-• ence or absence of the activation system; however, a
significant in-, re.-use in the number of ouabain-resistant mutants was found.
The results ,-j c --?uted in this study indicate that the concentrated
recycled water •_ u>ser increased rates of in vitro mutagenesis in mammalian
'ells, where-j^ •>< i ,j* aeeiii city was indicated in the bacterial system. This
apparent difference between bacterial and mammalian cells has been found
before. Ti can H,-< explained by differences in the genetic systems or other
basic ditferen <--. > P cell structure and function. Recently, studies have
been initiated *>v oLL,';;s l_o test the mutagenicity of organic fractions
isolated from '-/aifi '.-.'9) or purified compounds identified in water (30).
Simmon, et a'i . HO) found that 34 percent of 71 compounds which were identified
in water were shoxvii to be mutagenic in the microbiological assays. However,
.jince the selection c.f chemicals may have biased the results, they predicted
that 10 percenr o t!;t chemicals in drinking water would be found to be
ip.utagenlr or cjt.. .•.••^t'liic.
Soim pre-I h'siiKM •; work has been done in the past on the evaluation of
water fractions '" '•" ;-ussible carcinogenic effects in animals (31-33).
Contradictory .-.-.•suits were reported. In this respect, it is important to
mention the wort* done by Malaney, et _aJL. (34) who showed that activated
sludges faLLed : •-„• effect any significant removal of 27 proved (in animals)
carcinogens (sii-li ar-: ben?,o(a)pyrene or propionolactone) by oxidation mecha-
nisms within i.'uvu'. defection times.
Transformation of diploid cells in_ vitro is assumed to be a progressive
process which, ni.iv Uv?ct to the formation of malignant cells. One of the
Later events in this progression is the ability to grow on soft agar (35).
While many ro'i<-•!-• t '-ell cultures have been found to exhibit in vitro trans-
formation after exposure to chemicals (36), only limited success in trans-
forming human dip loir: cells has been reported. The water concentrate in
this study huiujed at, increase in the number of WI38 colonies able to grow
on soft agar, v'::!-^ potent carcinogens like benzo(a)pyrene and N-methyl-N-
ai tro-N-ui t ro.-:o>:,iian K! ine failed to transform the cells. This may be explained
by the presenci u'. promoting agents in the water. Promoters have recently
been shown to a,, i under in vitro conditions (37,38). These results illus-
trate that t!.
-------
An essential consideration is the relevance of such toxicological
assays to the problem of human health. At present, there is not a good
quantitative tool to relate experimental data to the human situation. It
is important to note that TOC levels of the water in this study were only
100 times the concentrations present in many drinking water supplies. Such
a difference between actual and experimental concentrations is considered
minimal in toxicological assays. Barnes and Denz (39) calculated the minimum
number of animals necessary to detect abnormal effects: To identify certain
effects in 5 percent of the animals within 95 percent certainty, 58 animals
must be studied; to identify effects in 1 percent of animals with the same
degree of confidence, 295 animals are needed (close to the number used in
this study). These pathological incidence rates, which represent the limit
of sensitivity of the present toxicological tests, would, of course, be
considered very high if found in the human population. It is generally
accepted that these odds can be improved by increasing the exposure dose,
as was done in this study, even though there is little data available on
the relationship between dose and effect at critical doses.
In summary, while the water concentrate did not cause serious tox-
icological effects in many of the tests performed on the animals, muta-
genicity and carcinogenicity properties of this water were demonstrated in
tissue cultures. More mutagenic and carcinogenic in vitro tests should be
performed on different water samples before these findings are extended to
large scale, long-term animal bioassay programs.
94
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1 -78-068
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Evaluation of Toxic Effects of Organic Contaminants
in Recycled Water
6. PERFORMING ORGANIZATION CODE
5. REPORT DATE
December 1978 issuing date
7 AUTHOR(S)
Dr. Nachman Gruener
8. PERFORMING ORGANIZATION REPORT NO.
9 PERFORMING ORGANIZATION NAME AND ADDRESS
Gulf South Research Institute
P. 0. Box 26518
New Orleans, Louisiana 70186
10. PROGRAM ELEMENT NO.
1BA607
11. CONTRACT/GRANT NO.
68-03-2464
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: Norman E. Kowal (513) 684-7477
16 ABSTRACT
This report represents the results of a comprehensive series of toxicological
studies designed to evaluate the health effects of the application of recycled water for
drinking purposes. Water was prepared in a highly advanced domestic sewerage pilot
plant. Some 400,000 lit?^s of the finished water were concentrated down to a volume of
200 liters with a total organic carbon content of 700 mg/1. This concentrate was incor-
porated into a gel-type diet which was fed to mice. A total of 900 animals was includec
in the experimental program, which extended to 150 days. The mice were tested for
growth, food intake, mutagenicity, mortality, blood physiology and biochemistry, and
liver and nervous system functions. Ten tissues were screened for pathological effects.
Only marginal changes were demonstrated in these areas.
In a second series of experiments, rodent and human cells were tested in vitro for
general toxicity, mutagenicity, and carcinogenicity. Results for all three effects in
the tissue cultures were positive. These effects were significantly increased by the
presence of a liver activation system.
These results show that exposure for a limited time (20% of a lifespan) to the
concentrated, recycled water (about 100-1000 times present human exposure) does not leac
to physiological changes in mice. On the other hand, the positive results from the
mutagenicity and carcinogenicity studies in tissue culture indicate a need for more
studies in this area.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Toxicity, Contaminants, Uastewater,
Sewage, Potable Water, Toxicology,
Mutagens, Carcinogens, Chemical Removal
b.IDENTIFIERS/OPEN ENDED TERMS
Advanced Wastewater
Treatment
COS AT I Field/Group
68G
57U
57Y
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
111
20 SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
99
*US GOVERNMEHT PRINTINGOFFICE 1978—657-060/1540
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