MUTAGENISTIC TESTING OF INDUSTRIAL WASTES FROM
REPRESENTATIVE ORGANIC CHEMICAL INDUSTRIES
East Central Oklahoma State University
Ada, Oklahoma
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EPA-600/2-81-007
January 1981
MUTAGENISTIC TESTING OF INDUSTRIAL
WASTES FROM REPRESENTATIVE
ORGANIC CHEMICAL INDUSTRIES
by
Susan Stinnett, Don Noble,
Elmer Brown, and Harry Love
East Central Oklahoma State University
Ada, Oklahoma 74820
E.P.A. Grant No. R806557
Project Officer
John E. Matthews
Source Management Branch
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma 74820
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ADA, OKLAHOMA 74820
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TECHNICAL REPORT DATA
(Please read Imauctions on the reverse before completing)
1. REPORT NO.
2.
4. TITLE AND SUBTITLE
Mutagenistic Testing of Industrial Wastes from
Representative Organic Chemical Industries
5. REPORT DATE
January 1?31. Iss.uin^.-Da.te..
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Susan Stinnett, Don Noble, Elmer Brown, and Harry Love
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
East Central Oklahoma State University
Ada, Oklahoma 74820
10. PROGRAM ELEMENT NO.
1BB610/C33B1B
11. CONTRACT/GRANT NO.
Grant No. R806557
12. SPONSORING AGENCY NAME AND ADDRESS
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency
P. 0. Box 1198
Ada, OK 74820
13. TYPE OF, REPORT ANO PERIOD COVERED
Final/4-17-79 to 4-17-80
14. SPONSORING AGENCY CODE
600/15
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The general applicability of the Ames test for screening wastewater samples was
investigated. Application of the Ames test to raw and treated wastewaters from
representative organic chemical industries involved the:investigation of. several
problems: (1) the feasibility of. using-the Ames, test to detect mutagens in waste-
water samples; (2) the relative effectiveness of various waste treatment processes;
(3) the mechanics of establishing an Ames testing program; and (4) the economics of
using the test in routine environmental screening.
Sample results were analyzed on the basis of relative increases in revertant colonies
on test, plates as compared to control spontaneous reversion plates. For a sample to
be s-cored "positive," six replicate test plates gave an average count of at least
twice the control value. Of 46 samples provided, 6 were interpreted as positive,
22 were interpreted as negative, and 18 were not tested.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATi Field/Group
Water pollutid'n
Methodology
Carcinogens
Bioassay
Mutagens
Ames test
06F
3. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
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DISCLAIMER
This report has been reviewed by the Robert S. Kerr Environmental
Research Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
The Environmental Protection Agency was established to co-
ordinate administration of the major Federal programs designed
to protect the quality of our environment.
An important part of the Agency's effort involves the search
for information about environmental problems, management tech-
niques and new technologies through which optimum use of the
Nation's land and water resources can be assured and the threat
pollution poses to the welfare of the American people can be
minimized.
As one of these facilities, the Robert S. Kerr Environmental
Research Laboratory is responsible for the management of programs
to: (a) investigate the nature, transport, fate and management
of pollutants in groundwater; (b) develop and demonstrate methods
for treating wastewaters with soil and other natural systems;
(c) develop and demonstrate pollution control technologies for
irrigation return flows; (d) develop and demonstrate pollution
control technologies for animal production wastes; (e) develop
and demonstrate technologies to prevent, control or abate pollu-
tion from the petroleum refining and petrochemical -industries , '.
and (f) develop and- demonstrate technologies to manage pollution
resulting from combinations of industrial wastewaters or indus-
trial/municipal wastewaters.
The Ames test has been prominently mentioned as a method
for determining whether a substance exhibits mutagenic character-
istics. This report considers the possibility of using this
procedure to determine whether complex organic chemical waste-
waters exhibit mutagenic characteristics; and, if so, the
relative effectiveness of various waste treatment processes in
removing substances contributing these mutagenic effects.
Clinton W. Hall
Director
Robert S. Kerr Environmental Research Laboratory
iii
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ABSTRACT
The general applicability of the Ames test for screening wastewater
samples was investigated.* Application of the Ames test to raw and treated
wastewaters from representative organic chemical industries involved the
investigation of several problems: (1) the feasibility of using the Ames
test to detect mutagens in wastewater samples; (2) the relative effectiveness
of various waste treatment processes; (3) the mechanics of establishing an
Ames testing program; and (4) the economics of using the test in routine
environmental screening.
Samples were supplied in groups of influent, activated carbon-treated
influent, effluent, and activated carbon-treated effluent. Each was passed
through various types of glass filters to achieve sterility prior to testing.
Dose levels of filtered samples ranging from 0.5 microliter to 500 microliters
were incorporated into the test plates. Results were analyzed on the basis
of relative increases in revertant colonies on test plates as compared to
control spontaneous reversion plates. For a sample to be scored "positive",
six replicate test plates gave an average count of at least twice the control
value. Of 46 samples provided, 6 were interpreted as positive, 22 were
interpreted as negative, and 18 were not tested.
This report was submitted in fulfillment of Grant No. R806557 by
East Central University under the sponsorship of the U.S. Environmental
Protection Agency. This report covers a period from April 17, 1979 to
April 17, 1980, and work was completed as of December 31, 1979.
iv
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CONTENTS
Foreward
Abstract iv
Tables vi
Abbreviations and Symbols vii
Acknowledgment viii
1. Introduction 1
2. Conclusions 2
3. Recommendations 3
4. Materials and Methods 4
Media 4
Solutions 6
Sample Preparation 8
Bacterial Test Strains 9
Salmonella Checkouts 9
5. Experimental Procedures H
6. Results and Discussion 13
Potential for false positives 16
Potential for false negatives .... .17
References 18
Appendix
Average of Revertant Colonies in Ames Tests 19
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TABLES
Number Page
1. Samples Provided by EPA for Ames Testing 7
2. Standard Mutagens for Salmonella Checks 10
3. Dose Levels in a Typical Ames Test Procedure (In/al) 12
4. Spontaneous Reversion Rates 14
5. Spontaneous Reversion Rates Reported by Ames 14
6. Samples Whose Test Counts Averaged At Twice the Average
Spontaneous Reversion Rate 15
7. Samples Showing Negative Results in Ames Testing 15
8. Average Revertant Colonies in Ames Tests 20
Yi
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LIST OF ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
°C — degrees, Celsius
cm — centimeter
g — gram
yug — microgram
/il — microliter
/un — micrometer
/jmol — micromole
mg — milligram
ml — milliliter
mM — millimolar (or millimoles per liter)
ng — nanogram
psig — pounds per square inch, gauge
SYMBOLS
HC1 — hydrochloric acid
KC1 — potassium chloride
K^HPO^ — potassium monohydrogen phosphate
KH2P04 — potassium dihydrogen phosphate
MgCl2 — magnesium chloride
MgSO^ — magnesium sulfate
NADP — nicotinamide adenine dinucleotide phosphate
— sodium ammonium phosphate
— sodium monohydrogen phosphate
— sodium dihydrogen phosphate
Vii
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ACKNOWLEDGMENTS
For collecting and handling the samples, as well as for considerable
guidance and technical assistance, we wish to thank John Matthews, Project
Officer, and James McNabb, Robert S. Kerr Environmental Research Laboratory,
U.S. Environmental Protection Agency. We also appreciate the assistance of
Dr. James Vaughn, Brookhaven National Laboratory; Dr. Bruce Ames and
Ms. Dorothy Maron, Department of Biochemistry, University of California,
Berkeley.
For unprecedented kindness and competence, we acknowledge Van May,
Cynthia Norman, Pat Mason, and Mike Mason.
viii
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SECTION 1
INTRODUCTION
An increasing concern with the possible introduction of carcinogens into
the environment has resulted in a search for a simple, sensitive, and reliable
method for the detection of these chemicals. One such method has achieved
considerable attention among industries and governmental agencies interested
in routine screening for potential carcinogens in water supplies. The Ames
test, developed by Bruce Ames, University of California, Berkeley, is a
simple and relatively inexpensive test currently in use for this purpose.
The Ames test was originally designed to determine the ability of a
specific compound to cause mutations. As most carcinogens are also mutagens,
a correlation has been made between positive results in the test and potential
carcinogenicity. Test strains of bacteria, supplemented with extracts of rat
liver to simulate mammalian metabolism, respond readily to the presence of
most mutagens in minute quantities. The test is currently being investigated
as a possible method for detecting mutagens in industrial effluents.
In conducting this study, the general applicability .of ..the Ames test to ..
screening wastewater samples was investigated. The problems involved in
preparing samples for testing and in interpreting.the results are markedly .
different for environmental samples as opposed to pure compounds or extracts.
In applying the test to raw and treated industrial wastewaters, several
objectives were examined: (1) the determination of the feasibility of using
the Ames test to detect carcinogens in the environment; (2) the relative
effectiveness of various waste treatment procedures; (3) the mechanics of
establishing an Ames testing program; and (4) the economics of using the test
for screening. The controversial correlation between mutagenicity and
carcinogenicity was beyond the scope of this study.
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SECTION 2
CONCLUSION
On the basis of our experience and the data observed, we present the
following conclusions:
(1) It is possible to detect positive mutagenesis in at least some
samples with the procedure used in this project. Because the mutagenic
agents are so dilute in wastewater, negative results obtained from these
tests do not necessarily indicate the absence of mutagens.
(2) There is a potential for obtaining false positives using this
procedure.
(3) The effectiveness of waste treatment cannot fully be determined in
a screening program. That determination warrants a separate study.
(4) While the test does not appear to require much sophistication, it
requires a large laboratory used exclusively for Ames testing with a large
capacity automatic autoclave, considerable refrigerator space,"and a laminar-
flow hood designed for use with carcinogens. The laboratory will have a very
large demand placed on its electrical circuits.
(5) Preparation for an Ames test screening project requires a minimum
of 6 to 12 months. Some of the larger pieces of equipment needed are not
manufactured routinely, and there may be an unavoidable delay of several
months after the bids are accepted while a freezer or incubator is being
manufactured. To minimize this problem, equipment and supplies should be
ordered well in advance of the date Ames testing is scheduled to commence.
Other unavoidable delays will occur, as biochemicals such as nicotinamide
adenine dinucleotide phosphate (NADP) and S9 liver homogenate are often
back-ordered. As unforeseen events will arise during the course of a •
screening project, it is impossible to determine in advance the exact amounts
of such materials that will be consumed over a 6-12 month period, so they
will have to be reordered, perhaps several times.
(6) The initial expense of establishing a laboratory for screening
samples by Ames testing will be high. Once the laboratory is equipped and
staffed with people who have had time to perfect the necessary technique,
routine testing will be relatively inexpensive when compared to animal studies.
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SECTION 3
RECOMMENDATIONS
For each of the major goals of this project, we offer the following
recommendations for careful consideration:
(1) The wastewater samples provided for this study were carefully
tested according to Ames' procedures [1] and EPA's quality control guide-
lines [2]. The EPA should consider using the Anes test as only one of
several screening tests [3]. Before an industry is judged by EPA to release
either negative or positive effluents, a more thorough test should be applied;
including basic Ames testing replicated several times for each dose level and
including a wider range of dose levels.
(2) Sample preparation should be more thoroughly investigated. After
repeated filtering, bacteria remained in some samples. Although they did
not grow on minimal media, other samples might contain small organisms that
could grow on minimal .media and ruin the tests. A reliable filtration tech-
nique needs to be developed.
C3) To answer the statement that some mutagens might have been adsorbed
onto particulate matter that was removed by filtration, the contents ..trapped .
by these filters should also be analyzed. Some physical means of steriliza-
tion such as radiation or sonication might be employed to advantage here,
either along with or in place of filtration.
(4) When considering extensive Ames testing by small laboratories,
existing physical plants should be carefully observed. We recommend that
minimum equipment used for this type of screening should include 4 or 5 large
capacity heat blocks to be used in place of water baths, a Revco freezer,
a laminar-flow hood designed for work with carcinogens, two incubators used
only for Ames testing, and an automatic electronic colony counter. Without
this equipment it is very difficult to maintain a screening program that
tests up to 10 waste samples per week.
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SECTION 4
MATERIALS AND METHODS
MEDIA
Four media are required for Ames testing: Vogel-Bonner minimal medium
E (VB), top agar supplemented with traces of histidine and biotin (TA),
nutrient agar + 0.5% Nad (nutrient agar or NA), and nutrient broth + 0.5%
Nad (nutrient broth or NB). Nutrient agar and broth frequently contain
materials that alter the growth and spontaneous reversion of the tester
strains of Salmonella. This problem was experienced midway through this
study. Dorothy Maron of Ames' laboratory at Berkeley suggested that Oxoid
medium #2 be substituted for nutrient media. This substitution was not made
in this project since it would have nullified data already generated and
insufficient sample volumes were available to rerun all tests already
completed. Therefore, all data in this report were collected with nutrient
media; however, subsequent Ames testing should use Oxoid #2 from the
beginning. Oxoid medium #2 may be obtained from K. C. Biological, P. 0. Box
5441, Lenexa, Kansas, 66215.
The detailed work plan approved for this study included a time period
of 3-4 weeks during which all media and solutions were to have been prepared
and stored for later use. This proved impossible for several reasons out-
lined below.
VB medium consists of a salt solution to which agar and glucose are
added [4]. To prepare VB, a stock salt solution (5x strength) is first
prepared by dissolving in distilled water: 1.0 g magnesium sulfate (MgS04 •
7 H20) , 10.0 g citric acid (1130511507 • ^0) , 50.0 g potassium monohydrogen
phosphate (i^HPO^), and 17.5 g sodium ammonium phosphate (NaNH^HPO^ • 4 H20).
The salts are added individually to a quantity of distilled water in a
1000-ml volumetric flask, and they must be added in the order given, each one
being dissolved completely before the next is added. This prevents precipi-
tation of salt complexes which cannot be redissolved. The solution is brought
to a final volume of one liter by adding distilled water to the mark. This
stock 5x salt solution may be stored at 4°C for several days. When the actual
working medium is made, 200 ml of this stock solution were mixed with 15 g of
agar and 300 ml of distilled water. This mixture was autoclaved at 121°C for
20 minutes. It was held at 45°C until use, at which time 500 ml of warm
distilled water in which 20 g of glucose had been autoclaved were added to
the salt solution. The glucose solution has to be autoclaved separately to
prevent toxic complexes (inhibitory to bacterial growth) from being formed.
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The agar-salt solution can only be melted once, during sterilization, and
cannot be held at 45°C for even a few hours because the large concentration
of citrate chairs very easily and the medium darkens. Remelting solid VB is
not acceptable, so it is impossible to prepare it early and store it in large,
undispensed volumes as had been planned. Since each Ames test requires
approximately 100 plates of VB and each Salmonella check uses approximately
75 plates, the total number of VB plates used in this project was well above
the refrigerator capacity of the laboratory. As even brief storage at 4°C
produces condensate on the plates, it was more satisfactory to prepare enough
VB plates for 3 or 4 days testing (approximately 1000 plates) and store them
at room temperature for several days before use. This allowed the normal
condensation to dissipate somewhat and also allowed contaminated VB plates to
be discarded before use.
TA was to have been prepared, dispensed in tubes, autoclaved, then
stored at 4°C and remelted immediately prior to use. This technique yielded
control plates with no revertant colonies early in the testing. Although no
literature reference was found to explain the phenomenon, it appeared that
the histidine-biotin solution was heat-labile. Personal communications with
others involved in Ames testing supported this conclusion (James Vaughn,
Brookhaven National Laboratory, New York; Bill Stang, EPA, Denver; Dorothy
Maron, Univ, of Calif., Berkeley). TA was then prepared by dissolving 3.35 g
of Difco agar and 2.5 g of NaCl in distilled water to make 500 ml, autoclaving,
and storing at 4°C until use. The histidine-biotin supplement, containing
0.1048 g of L-histidine and 0.1221 g of biotin in distilled water to make
1000 ml, was filtered through a 0.45 micrometer (jam) Millipore membrane
filter into a sterile filter flask. It was then aseptically dispensed into
four sterile 250-ml Erlenmeyer flasks and refrigerated, 'On the day of TA
preparation, a 50-ml portion of the histidine-biotin solution was refiltered
through a Gelman acrodisc filter assembly.(0.'45 urn) into a large,- sterile
culture tube and was allowed to warm to room temperature. A 500-ml portion
of the agar solution was melted and cooled to 45°C in a water bath. A 50-ml
portion of warm sterile histidine-biotin solution was aseptically added to
the melted agar solution and swirled carefully to mix. Using a sterile 5-ml
Cornwall pipettor, the TA was then aseptically dispensed into pre-sterilized
tubes in 2-ml volumes. To keep contamination from entering the TA, the tip
of the pipettor was quickly flamed periodically. This made it necessary to
replace the valve assembly more frequently than usual.
TA dispensing was done in a large 45°C water bath, as cooling below 45°C
quickly solidifies TA and remelting inactivates the histidine-biotin supple-
ment. The water level had to be carefully regulated at just above the depth
of TA in the tubes; too much water tended to increase the contamination
problem, and too little water cooled the top agar so it solidified. Covers
over the baths allowed condensation to fall on the caps of the tubes and
caused contamination problems, so they were removed. This increased the
frequency of monitoring the bath levels and temperatures to four or five
checks per day, one of which was early in the morning and one late in the
evening.
Maintaining asepsis throughout TA preparation was the most difficult
technique to control, and some data compiled very ear17 in the testing
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schedule had to be discarded because of TA contamination. Screw-capped TA
tubes suggested by Ames were abandoned after a one-week trial because manipu-
lating the caps proved too difficult to allow aseptic conditions to be main-
tained. As each test represented considerable time, effort, and money,
subsequent batches of top agar were prepared no less than 24 hours prior to
use. After dispensing, each batch was tested for sterility by randomly
selecting several tubes, pouring each over a NA plate, and incubating over-
night at 37°C. This allowed contaminated batches to be identified and
discarded before use. Once the procedure was perfected, a responsible student
assistant who was proficient in microbiological techniques was able to perform
it routinely after several practice runs,
SOLUTIONS
S9 fraction of rat liver homogenate prepared from Aroclor-induced rats
[1] was obtained in 2-ml and 5-ml vials from Litton Bionetics, Kensington,
Maryland. It arrived frozen, packed in dry ice, and was immediately trans-
ferred to a Revco freezer for storage at -80°C.
S9 mix was prepared early in the morning of each day of testing. The
procedure outlined below was used for 5-ml vials of S9; it was cut propor-
tionally for 2-ml vials. A 5-ml vial of S9 allowed sufficient mix for three
Ames test.
S9 mix was prepared so that each ml of the final solution contained
0.07 ml of S9, 8 micromole Qumol) of magnesium chloride (MgCl2), 33^umol
of potassium chloride (KC1) , 5 ^ol of glucose-6-phosphate, 4/imol of NADP,
and 100 yumol of sodium phosphate buffer CpH 7.4). Stock salt solutions of
22.856 millimolar (mM) MgCl2 a.1616 g MgCl2 • 6 H20/ 250 ml distilled water),
128.562 mM KC1 C2.3763 g KC1/250 ml), 246,93 mM NaH2P04 (8:518 g NaH2P04/
250 ml), and 246.93 mM sodium monohydrogen phosphate (Na2HP04) C33.098 g
Na2HP04/250 ml) were prepared and stored at 3-5"C. A buffer solution CpH 7.4)
was prepared fresh each week by mixing appropriate amounts of the sodium
phosphate salt solutions. The buffer solution was stored in the refrigerator
for one week.
On the morning of the day that S9 mix was needed, a buffered solution
was prepared by mixing 25 ml of MgCl2 stock solution, 12.5 ml of KC1 stock,
28.93 ml of sodium phosphate buffer, 100.7 mg of glucose-6-phosphate (mono-
sodium salt) and 229 mg of NADP (sodium dihydrate). This solution was filter-
ed through a 0.45 ;um pore Gelman acrodisc disposable filter assembly into a
sterile 250-ml Erlenmeyer flask and placed on ice. A 5-ml vial of frozen S9
was hand-warmed on the outside until it could be poured into the flask. The
S9 was allowed to finish thawing in the flask, after which the flask was
gently swirled to thoroughly mix the contents. The S9 mix was kept on ice
throughout the 1-day testing procedures. Any excess was discarded at the
end of the working day.
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TABLE 1. SAMPLES PROVIDED BY EPA FOR AMES TESTING
Sample No,
1*
2
3*
4
5
6
7
8
9*
10
11
12
13*
14
15*
16
17*
18
19*
20
21*
22
Industrial
Category
Petrol. Ref.
n
it
11
Org. & Plas.
it
it
n
Pesticide
it
M
n
Rubber
M
tt
n
n
n
ti
it
Pharm .
it
Sample Sample No
Point
Inf.
Inf.
Eff.
Eff.
Inf.
Inf.
Eff.
Eff.
Inf.
Inf.
Eff.
Eff.
Inf.
Inf.
Eff.
Eff.
Inf.
Inf.
Eff.
Eff.
Inf.
Inf.
C-T
C-T
C-T
C-T
C-T
C-T
C-T
C-T
C-T •
..:
C-T
C-T
25*
26
29*
30
31*
32
33*
34
35*
36
37*
38
39
40
41*
42
43*
44*
'47*
48
49*
50
53
54
Industrial
Category
Pesticide
M
Pharm.
n
ii
n
Wood Pres .
n
n
ti
Org. & Plas.
n
n
it
Wood Pres .
ti
n
n
n
n
Pharm.
M
Org. & Plas.
M
Sample
Point
Inf.
Inf.
Inf.
Inf.
Eff.
Eff.
Inf.
Inf.
Eff.
Eff.
Inf.
Inf.
Eff.
Eff.
Inf.
Inf.
Eff.
Eff.
Eff.
Eff.
Inf.
Inf.
Eff.
Eff.
C-T
C-T
C-T
C-T
C-T
C-T
C-T
C-T
C-T
C-T
C-T
* No test conducted on these samples.
Abbreviations:
Petrol. Ref. - Petroleum refinery Pharm. r- Pharmaceuticals
Org. & Plas. - Organics & Plastics .Wood Pres. - Wood, preservatives
Inf. C-T - Influent, carbon-treated Eff. C-T - Effluent, carbon-treated
Inf. - Influent Eff. - Effluent
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SAMPLE PREPARATION
Samples collected by the Robert S. Kerr Environmental Research Laboratory,
Ada, Oklahoma, were pre-filtered through glass wool before transfer to the
investigators. A 30-ml volume of sample, numbered and designated influent,
carbon-treated influent, effluent, or carbon-treated effluent (Table 1) was
supplied for each of 46 different wastewaters. These were collected from 14
industrial sites involved in petroleum refining or production of organic
chemicals, pesticides, wood preservatives, rubber, or pharmaceutical products.
Samples were kept at 3-5°C throughout the grant period.
One-mi aliquots of each sample were initially tested for sterility by
spreading on NA plates. All were heavily contaminated with bacteria, making
Ames testing impossible. The usual membrane-filtration technique using
acetate filters was inappropriate for these samples, as the organic compounds
they contained might have reacted with or dissolved the filters. As an
alternative, Bush bacteriological fritted glass filters, pore diameter 0.9-1.4
yum (Corning #33992) were purchased from Sargent-Welch. The filters were
washed in dilute hydrochloric acid (HC1), rinsed in distilled water, wrapped
in heavy aluminum foil and sterilized in an oven at 260°C overnight before use.
Samples were filtered under pressure of 5-15 psig. The compressed air was
supplied by an Ametrol portable air tank equipped with a hand pump and
regulator.
Because many of the samples required several hours to pass through the
Bush filters (some required 48 hours or more), another alternative filtration
system was investigated. Gelman glass membrane filters, type A/E (pore size
0.45 yum) were used with a Millipore filtration apparatus to collect the
samples in sterile filter flasks. This technique failed to give sterile
samples, and because of limited time available for testing, it was abandoned
without further study.
After filtration, the samples were stored at 3-5°C. Each was checked
for sterility by plating 0.1-ml aliquots on two NA plates and two VB-TA plates.
After incubation for 24 hours at 37°C, most samples appeared to be sterile.
However, after 48 hours at 37°C, several showed heavy contamination with
micro-organisms that produced minute white colonies on NA and failed to grow
on VB-TA plates. These samples were refiltered, sometimes several times,
until the NA plates were negative after 48 hours.
Occasionally there was a delay of several days between sterility certi-
fication and Ames testing. In some samples the sample sterility controls
done with the Ames test showed contamination with the same type of colonies.
It appears that these samples were not actually sterilized, as. a few cells
passed through the filter (too few to be caught by the checking procedure).
Those cells apparently reproduced in the refrigerated samples. In some of
these cases, the tests were repeated on freshly-certified samples, and no
significant difference appeared in the data. Subsequent data were used for
all tests in which the VB-TA sterility controls were sterile.
8
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BACTERIAL TEST STRAINS
Four cultures of Salmonella typhimurium histidine auxotrophs, designated
TA 98, TA 100, TA 1535, and TA 1537 {1] were supplied by the laboratory of
Bruce Ames, Department of Biochemistry, University of California, Berkeley.
All four strains require histidine and biotin supplements for growth on
minimal media and are sensitive to ultraviolet irradiation and crystal
violet. TA 98 and TA 100 also carry a plasmid imparting resistance to ampi-
cillin, an antibiotic that kills the other strains.
Lyophilized cultures supplied by Ames were initially streaked on plates
of nutrient agar and were cultured in nutrient broth. Frozen permanent
reserve and working stock cultures were prepared by mixing 2 ml of the
initial broth bulture with 0.1 ml of spectrophotometric grade dimethylsul-
foxide (DMSO) in sterile glass vials. Each vial was immersed in acetone and
dry ice for quick freezing. The frozen permanent reserves were maintained
at -80°C in a Revco freezer, model ULT-185-AHG.
To avoid damage to the frozen working stocks, Ames suggested minimizing
thawing and refreezing by preparing stock NA plates once a month from the
frozen reserves. These plates, kept at 4°C in a refrigerator, served as the
source of inoculum for each set of working broth cultures.
Once each week, six 10-ml nutrient broth cultures and one NA slant
culture were inoculated from the stock plate for each strain. Broth cultures
were incubated overnight in a shaking 37°C water bath, and NA slant cultures
were incubated overnight at 37°C in a Precision Scientific incubator. Broth
culture cell .densities approximated 10 cells per ml as determined by com-
paring spontaneous- reversion rates with those of 10^ cells per ml cultures
from Ames' laboratory [1; personal communication, Dorothy Maron, 8-20-79].
One broth culture of each strain was randomly chosen for checkout procedures.
The other five were refrigerated and used later for testing.
SALMONELLA CHECKOUTS
Each randomly chosen broth culture was streaked on VB medium. Histidine
and biotin requirements were determined by streaking each strain on a VB
plate spread with 0.1 ml of 0.1 M histidine and on a VB plate spread with
0.1 ml of 0.5 mM biotin. When streaked directly from nutrient broth, enough
biotin was transferred from the broth to allow a few colonies to grow on the
histidine-supplemented plates. This was eliminated by making distilled water
suspensions of cells grown on NA slants and using these suspensions as sources
of inoculum for the VB-histidine-biotin checks. All tests described below
were performed on the broth cultures.
Sensitivity to ampicillin or crystal violet was determined by spreading
0.1 ml of culture on each of two NA plates. A filter paper disc impregnated
with either 10 micrograms C^g) crystal violet or 10 ;ug ampicillin was placed
in the center of each plate. Sensitivity was indicated by a clear zone of
inhibition around the disc following a 48-hour incubation at 37°C.
-------
Ultraviolet sensitivity was determined by spreading 0.1 ml of culture on
two NA plates and exposing one-half of each plate to a germicidal (254 nm)
Mineralight UV lamp, 110 volts and 0.16 amps. One plate for each strain was
exposed at a distance of 33 cm, the other at 19 cm. TA 98 and TA 100 were
exposed for 8 seconds, while TA 1535 and TA 1537 were exposed for 6 seconds.
After 48 hours at 37°C, sensitivity was determined by observing significantly
reduced or no growth on the irradiated side of the plate.
Positive mutagenesis was determined by plating 0.1 ml of culture in TA
(with histidine and biotin supplement), with or without 0.5 ml of S9 mix,
and spot testing with the suitable mutagen (Table 2). TA 1537 was very
difficult to mutate, and best results were obtained by incorporating the
mutagens into the TA before pouring. All work with mutagens was done wearing
gloves and protective clothing, in a hood vented to the outside. Plates
containing mutagens were incubated in an outdoor-vented 37°C Precision
Scientific incubator, counted under the hood, disinfected with amphyl, and
placed in bio-hazard bags for disposal (see section on waste disposal).
Mutagen solutions were purchased from Nanogens Inc., at concentrations of
100 ng//il. DMSO was the solvent used.
Spontaneous reversion rates for each strain were determined with and
without S9. Three to five plates of each strain were prepared by adding
0.1 ml of the overnight broth culture to TA and pouring over VB. An equal
number of plates containing 0.1 ml of culture and 0.5 ml of S9 mix were
prepared at the same time. These were incubated for 48 hours at 37°C.
_ TABLE 2. STANDARD MUTAGENS FOR SALMONELLA CHECKS _
Strain Mutagen .Used Concentration S9 Requirement
TA 98
TA 100
2-aminof luorine
N-methyl-N ' -nitro-
100 jal
+
N-nitrosoguanidine (MNNG) 25 /il
TA 1535 MNNG 25 /il
TA 1537 MNNG 25 ;ul
4-aminobiphenyl 100 /il
10
-------
SECTION 5
EXPERIMENTAL PROCEDURES
The procedures used in this project were modifications of the techniques
of Ames [1] and the quality control guidelines of the EPA [2]. It was essen-
tial to modify the techniques because pure compounds were not used and there
was a minimum amount of time for testing.
Each sample was prefiltered, checked for sterility, then refrigerated.
To minimize evaporation of volatile organics from the samples, plastic tape
was used to seal the containers. Each sample was diluted 1:20 by pipetting
5.0 ml of sample into a sterile 100-ml volumetric flask and diluting to the
mark with sterile distilled water. Sterility controls were done on the
unused dilution water, the diluted sample, and the concentrated sample
immediately following the dilution process. The diluted and undiluted samples
were also checked for sterility during and after the testing. Each sterility
check was done by plating 0.1 ml of sample on NA and 0.1 ml in TA on VB, then
incubating at 37°C for 48 hours.
A set of TA tubes for checking sterility of dilution water, samples and
agar was kept in the 45°C water bath along with a group of 80 TA tubes for -
Ames testing. These were arranged in four sets of 20 tubes, and were dosed
as shown in Table 3. Four tubes of each set received 10/il of the 1:20
dilution, a final dose level .of 0.5 ;ul. Four tubes of each set received
100 p.1 of the 1:20 dilution, a final dose level of 5/il. Four tubes of each
set received 20 jal of the undiluted sample, four received 100 /il of undiluted
sample, and the last four in each set received 500/il of undiluted sample.
No more than 500yul could be used for two reasons: a small volume of sample
was provided, and larger volumes when added to 0.5 ml S9 mix would have
diluted the top agar so it could not solidify. The latter was verified by
testing with water blanks instead of sample. All doses were delivered with
Oxford pipettors using sterile tips.-
Following the dose additions, the test was begun with set one containing
20 tubes. Ten of these (in the column on the right in set #1, Table 3) were
seeded with 0.1 ml each of a NB culture of TA 98, which had been previously
checked for genetic stability. Each tube was then mixed with a vortex mixer
and poured over a VB plate. The plates were immediately placed in a dark box
to prevent photorepair mechanisms from interfering with the detection of
mutations. When the top agar solidified, they were inverted and incubated at
37°C. The second group of ten tubes in set //I was then seeded as before with
TA 98. Before each tube was mixed, 0.5 ml of S9 mix was added. Within 20
seconds of the addition of S9 to a tube, it was mixed, poured, and placed in
11
-------
the dark. Speed was essential to prevent inactivation of the enzymes at 45 °C,
The above procedure was then repeated, substituting TA 100 in set #2,
TA 1535 in set #3, and TA 1537 in set #4. All plates were inverted and
incubated at 37°C for 48 hours, after which they were counted using a mechani-
cal register and a Quebec colony counter.
To complete a test, an experienced worker needs 2.5 hours. A worker
recording data needs 2-3 hours to count and record the data from one test.
As the data were recorded, the plates were autoclaved and discarded.
TABLE 3. DOSE LEVELS IN A TYPICAL AMES TEST PROCEDURE (IN
Set #1:
S9
500
500
100
100
20
20
5.0
5.0
0.5
0.5
TA 98
no S9
500
500
100
100
20
20
5.0
5.0
0.5
0.5
Set #2:
S9
500
500
100
100
20
20
5.0
5.0
0.5
0.5
TA 100
no S9
500
500
100
100
20
20
5.0
5.0
0.5
0.5
Set #3:
S9
500
500
100
100
20
20
5.0
5.0
0.5
0.5
TA 1535
no S9
500
500
100
100
20
20
5.0
5.0
0.5
0.5
. Set #4:
S9
500
500
100
100
20
20
5.0
5.0
0.5
0.5
TA 1537
no S9
500
500
100
100
20
20
5.0
5.0
0.5
0.5
12
-------
SECTION 6
RESULTS AND DISCUSSION
The Ames test was designed to determine whether a pure compound is able
to cause bacteria to mutate. The significance of this rather simple test
system lies in the correlation between the ability to cause bacterial muta-
tions and the ability to cause mutations in mammals, including humans [1].
Testing chemicals on animals is an expensive and time-consuming process.
It is hoped that the much simpler Ames test can give valid preliminary
information about mutagens. Our results suggest that the test may be useful
as one tool for the preliminary screening of wastewaters for potential
mutagenic activity.
The test works by inducing mutations in the areas of the bacterial
chromosome that have already been mutated. To appear on VB-TA plates, the
histidine-biotin deficient mutants must regain the ability to synthesize
these compounds. This is accomplished when the mutagenic compound reacts
with the genetic material, reversing the original mutations. Since this
phenomenon occurs with some regularity in the absence of mutagens, the
background "spontaneous reversion" rate must be determined before and during
Ames testing.
It is generally accepted 11,3] that a given amount of material is
considered to have mutagenic activity if test plates incorporating that dose
show at least twice as many colonies as the average number of spontaneous
revertants for the bacteria used. Data compiled for spontaneous reversions
are presented in Table 4. Three of the four strains showed lower rates than
expected from Ames1 data as summarized in Table 5. This is probably the
result of the media used.
A summary of the positive results obtained during this project appears
in Table 6. Complete data appear in the appendix. We initially tested each
influent and effluent using the procedure outlined previously. In each test,
we scored as "positive" plates of TA 98 with 46 or more colonies, plates of "
TA 100 with 224 or more colonies, plates of TA 1535 with 22 or more colonies,
and plates of TA 1537 with 20 or more colonies. All other plates were
scored as "negative". If an influent or an effluent showed a positive score
at any dose level for any of the test strains, corresponding dose levels of
the carbon-treated samples were tested. When an influent or an effluent
showed no positive scores, the corresponding carbon-treated samples were not
tested. Tests scored as positive were repeated; instead of averaging two
plates at the positive dose level, six plates were averaged. Only those dose
levels that scored positive as the average of six test plates appear in
13
-------
TABLE 4. SPONTANEOUS REVERSION RATES
Culture No. of tests x Range
TA 98 29 23 10-40
TA 100 30 114 86-192
TA 1535 34 11 8-16
TA 1537 31 10 4-18
TABLE 5. SPONTANEOUS REVERSION RATES REPORTED BY AMES II]
Culture x Range
TA 98 40 30-50
TA 100 160 120-200
TA 1535 20 10-35
TA 1537 7 3-15
14
-------
TABLE 6. SAMPLES WHOSE TEST COUNTS AVERAGED
AT LEAST TWICE THE AVERAGE SPONTANEOUS REVERSION RATE
Sample No.
6 (Infl.)
8 (Effl.)
18 (Infl.)
40 (Effl.)
42 (Infl.)
50 (Infl.)
Dose Level
5 /tl
100 jul
5 ,ul
500 >il
500 pi
5 /il
25 ;il
100 jul
100 jul
500 ;ul
Affected
Test Strain
TA 100
TA 100
TA 100
. TA 100
TA 1535
TA 100
TA 100
TA 100
TA 100
TA 100
S9
Requirement
+
-
+
-
+
-
•
-
+
"~
TABLE 7. SAMPLES SHOWING NEGATIVE RESULTS IN AMES TESTING
Sample No.
Sample No.
2
4
5
7
10
11
12
14
16
20
22
26
30
32
34
36
38
39
54
15
-------
Table 6. Table 7 lists the samples that yielded negative scores.
Positive results were anticipated because some of the molecules
generated by organic chemical industries have shown positive results in pure
compound testing. In industrial wastewaters, however, the exact chemicals
present and their individual concentrations are unknown. It is probable that
some mutagens may be present in a sample, but in concentrations below the
threshold of detection using these methods. Therefore, it is remarkable that
positive results were obtained in this study.
Some workers attempting to use the Ames test to screen wastewaters have
approached the concentration problem by extracting the samples with methylene
chloride {personal communication, Bill Stang, EPA, Denver], We chose not to
do this for two reasons: methylene chloride is a known carcinogen, and
handling it increases the hazard and the time needed in sample preparation;
also, because it is a mutagen, any residue of methylene chloride remaining in
the extracts could alter the results of the testing. By eliminating the
extraction procedure, we have eliminated these objections while raising
others. The lack of concentration in our samples may lead to negative scores
that might be positive if extracts were tested. Also, it is obvious that we
are testing the soluble phase of the samples; any mutagens in or on particu-
late matter have been removed prior to testing.
In reviewing the validity of using the Ames test as a screening
procedure, it is important to recognize that a potential exists for obtaining
false results, either negative or positive. The Ames test is a much more
complex bioassay than it appears; with such a large number of controls being
necessary, even the rigid maintenance of quality control standards allows a
margin of error to remain.
POTENTIAL FOR FALSE POSITIVES
(1) If the spontaneous reversion rate of a group of Salmonella cultures
is abnormally high, the data obtained in tests using those cultures will also
be high. When compared to the average spontaneous reversion rate, the data
may be high enough to receive a positive score, although the sample may not
be a true mutagen. ,
(2) Contamination of either the Salmonella culture or the sample could
yield abnormally high results. (It is probable, but not definite, that
contamination would be detectable and the data would be discarded.)
C3) An abnormally high population density in one culture could produce
abnormally high results.
Any of these possibilities should produce the .same effect in all tubes
of a test, regardless of the .dose level. In this project, we saw no
consistency of high results in all tubes of any test. We therefore conclude
that our positive results were not caused by these factors.
16
-------
POTENTIAL FOR FALSE NEGATIVES
(1) Mutagenic compounds usually exhibit a dose response curve in pure
compound tests. This means that below and above certain concentrations,
their mutagenic effect is not observed. It is quite probable that mutagens
may be present in wastewaters even though their concentrations are too small
to be detected in the assay.
(2) Many compounds require activation by mammalian enzymes before being
converted to a mutagenic state. It has been determined by McCann, et. al.
15] that the type of compound used to induce the rat liver enzyme production
is very significant in detecting mutagens. Some compounds in wastewater
might show negative results with Aroclor-induced S9, but positive results
with phenobarbitol-induced S9. Others might require induction by as yet
untried compounds.
(3) The amount of S9 used in the test system affects the results.
Some compounds lose their mutagenic effect in the presence of excessive S9,
although they require it for activation. Others require more than the usual
amount to show positive results [5]. This greatly complicates the problem
of interpreting data.
There is not enough evidence to indicate that one waste treatment system
sampled is more effective than another in removing potential mutagens from
wastewaters, although we found no positive results from carbon-treated
samples.
Because the potential for false positives is augmented.by experimental .
error in pipetting Salmonella into TA, and because the dose•levels are so
unusual, the positive results obtained in sample.#6 and sample #8 (Table 6)
are suspect. Five ;ul of sample is an extremely small volume to register
positive. Of all the samples tested, //42 seems to be most consistent.
The potentials for false positive or negative results are always present
in this type of bioassay; they cannot be totally eliminated. Any attempt at
minimizing these potentials will detract from the value of the test as a
rapid screening tool, but will certainly increase the confidence in the data.
It is apparent that the experimental design used in this project was suitable
only for preliminary screening of samples. It is not reasonable to classify
a sample as mutagenic or not mutagenic on the basis of this test alone.
Alternative bioassays, similar to the. Ames test,.have been developed for
detecting mutagenic activity of pure compounds. . These assays use yeasts,
other bacteria, or tissue culture cells as the test organisms. If the Ames
testing procedure outlined in this project is coupled with one or more of
these assays, it may prove to be very useful as a screening technique. Ames
testing should not be used as the only screening test for determining muta-
genic activity of wastewaters.
17
-------
REFERENCES
1. Ames, B.N., J. McCann, and E. Yamasaki. Methods for Detecting Carcino-
gens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity
Test. Mutagen Research, 31 (1975): 347-364, 1975.
2. Tracor Jitco, Inc. Quality Assurance Guidelines for Biological Testing.
EPA-600/4-78-043, U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Las Vegas, Nevada, 1975. 474 pp.
3. Brusick, D.J. Observations and Recommendations Regarding Routine Use of
Bacterial Mutagenesis Assays as Indicators of Potential Chemical Carcino-
gens. In: Strategies for Short-Term Testing for Mutagens/Carcinogens,
B.E. Butterworth, ed. CRC Press, West Palm Beach, Florida, 1979.
pp. 3-12.
4. Vogel, H.J. and D.M. Bonner. Acetylornithase of Escherichia coli:
Partial Purification and Some Properties. J. Biol. Chem. 218 (1956):
97-106, 1956.
5. McCann, J., E. Choi, E. Yamasaki, and B.N. Ames. Detection of Carcino-
gens as Mutagens in the Salmonella/Microsome Test: Assay of 300 Chem-
icals, Proc. Nat. Acad. Sci. U.S.A. 72: 5135-5139, 1975.
6. Weinstein, D. and T.M. Lewinson. A Statistical Treatment of the Ames
Mutagenicity Assay. Mutation Research 51 (1978): 433-434, 1978.
1.8
-------
APPENDIX
AVERAGE OF REVERTANT COLONIES IN AMES TESTS
The data contained in Table 8 represent a summary of the raw data
collected during this project.' Each value listed represents the mean value
for all plates tested. Unless otherwise noted, 2 plates were averaged to
obtain the mean. One asterisk C*) indicates an average of 4 plates, and
two asterisks (**) indicate an average of 6 plates.
The sample number designates the wastewater being tested. Dose level in
jil designates the amount of wastewater to which bacteria were exposed. Each
test strain received each dose level without and with the addition of S9.
To interpret the data, proceed as follows:
When O.S^ul of sample #2 was added to each of two tubes with approximate-
ly 10^ cells of TA 98 per tube, the mixtures (when plated on VB) yielded an
average of 17 revertant colonies per plate. When compared with the spontaneous
reversion rates appearing in Table 4, pg. 14, this shows less than twice the
average spontaneous reversion rate. When 0.5^il of sample #2 was added to
TA 98 and S9, the duplicate plates averaged 32 revertant colonies per plate.
This procedure is to be followed- throughout the reading of the table.
19
-------
TABLE 8. AVERAGE OF REVERTANT COLONIES IN AMES TESTS
Sample
No.
2 Infl.
4 Effl.
5 Infl.
C-T
6 Infl.
7 Effl.
C-T
8 Effl.
Dose
in
J*
0.5
5
25
100
500
0.5
5
25
100
500
5
100
0.5
5
25
100
500
0.5
5
25
100
500
0.5
5
25
100
500
98
no
S9
17
16
18
17
19
23
11
7
11
13
17
20
14
12
12
20
15
17
12
12
10
13
9
7
11
S9
32
27
37
27
30
30
31
33
32
26
37
44
47
40
29
44
27
30
18
32
31*
29*
25*
24*
27*
100
no
S9
107
109
105
107
122
103
110
97
125
155
70
90*
169**
181*
166**
192**
181**
134
. 52
116
138
123
165*
182*
170*
172*
178*
1535
S9
133
148
134
130
114
159
141
153
166
134
70
173*
230**
167*
94*
91*
207*
217
117
109*
119
176*
222**
195*
185*
160*
no
S9
12
15
11
13
9
18*
12
14
11
12
11*
11*
10*
12*
17**
10
6
5
9
13
15
13
16
19*
12
S9
11
13
15
12
16
10
12
15
12
14
5**
7**
12**
8**
13**
11
12
5
12
9
16
12
12
17
12
1537
no
S9
3
8
5
5
5
11
10
7
11
9
7
7
7
5
8
10
3
6
8
6
11
8
11
8
10
S9
6
17
13
11
9
18
11
11
10
13
8
8
11
8
12
11
10
9
7
6
15
10
8
9
12
(continued)
20
-------
TABLE 8 (continued)
Sample
No.
10 Infl.
11 Effl.
C-T
12 Effl.
14 Infl.
16 Effl.
18 Infl.
Dose
in
V1
0.5
5
25
100
500
0.5
5
25
100
500
0.5
5
25
100
500
0.5
5
25
100
500
0.5
5
25
100
500
0.5
5
25
100
500
91
no
S9
20
19
25
19
24
14
12
13
19
12
30
22
10
14
17
17*
18
29
33
23*
10
14
7
16
7
22*
17*
19*
17*
13*
8
S9
41 .
12
33
36
39
36
48
36
37
35
33
34
50
42
41
47
36
39*
44**
33
28
28
38
26
30
41**
44**
40**
41**
45**
100
no
S9
129
130
168**
183**
167**
168**
206**
214**
166**
178*
176*
183*
162**
154**
167*
68*
172*
118
146
196
136
100
133
152
118
172**
142**
138**
107**
191**
S9
134
128
225**
214**
193*
181**
206**
200**
185*
181
158*
154*
161*
172*
160*
163*
213*
203*
245*
248*
127
135
131
136
127
136*
176**
158**
165**
200**
153
no
S9
10
15
14
16*
15*
16
11
11
9
14
16
10
10
7
18
16
14*
8
7
11
10
13
11
12
16
11
12
17
- 17
15
15
S9
11
13
18
13
14
16*
9
16
16
14
15
10
11
15
12
7
10
10
13
12
15*
-9
10
14
8
15
15
15
16
16
15
no
S9
6
9
5
6
4
4
6
5
10
9
12
12
13
2
12
13
8
9
8
13
10
6
5
12
31
18
15
11
8
11
37
S9
. 10
11
11
10
10
13
13
14
8
9
12
12
8
10
19
13
7
9
9
7
21
5
8
8
6
13' .
12
10
8
10
Ccontinued)
21
-------
TABLE 8 (continued)
Sample
No.
20 Effl.
22 Infl.
26 Infl.
30 Infl.
32 Effl.
34 Infl.
Dose
in
/*
0.5
5
25
100
500
0.5
5
25
100
500
0.5
5
25
100
500
0.5
5
25
100
500
0.5
5
25
100
500
0.5
5
25
100
500
9f
no
S9
7
10
20
12
16
15
17
14
22
22
20
20
18
14
18
14*
16*
22*
22*
22*
11
11
13
11
16
9
14
24
12
5
3
S9
33
44
37
37
38
30
26
43
32
28
29
17
25
34
21
38*
36*
39*
42*
42*
30
37
26
28
27
21
20
31
25
54
100
no
S9
57
92
86
114
126
79*
69*
75*
98*
92*
123
156
129
166*
124*
128*
173*
182*
151*
167*
115
123
135
165
130
63*
81*
103*
122*
121*
S9
108
115
138
139
136
80
106
98
130
126
139
130
137
202
178
187*
195*
203*
201*
200*
137
149
147
139
160
82*
135*
114*
101*
139*
153
no
S9
9
8
11
11
6
5
8
11
3
10
14
11
12
12
8
14
12
13
11
11
9
16
13
8
10
5*
8*
10
8
12
;5
S9
14
6
11
14
14
9
11
7
10
10
8
10
10
11
16
10
12
15*
16
20*
25*
44*
13*
12
13
9
6
9
15
17
15
no
S9
5
8
8
8
8
6
9
7
9
10
7
8
6
5
8
15
7
12
15
10
11
6
5
59
5
2
6
8
4
6
37
S9
14
8
11
12
9
8
11
9
11
6
12
14
3
14
15
13
9
10
11
20
7
11
11
30
9
10
6
13
9
15
(continued)
22
-------
TABLE 8 (continued)
Sample
No.
36 Effl.
38 Infl.
Dose
in
^
0.5
5
25
100
500
0.5
5
25
100
500
9
no
S9
23
17
23
20
18
15
23
9
15
30
8
S9
37
32
31
41
37
27 .
34
44
35
27
100
no
S9
131
117
140
119
130
165*
163**
192**
167**
184**
S9
144
118
123
142
118
181*
173 *
201**
184
148
is:
no
S9
10
14
18
13
19
8
8
11
16
7
J5
S9
14
11
8
14
10
12
15
13
12
12
15
no
S9
12
11
10
12
9
10
9
6
6
15
37
S9
8
9
12
15
10
10
8
9
10
13
38 Infl. 500
C-T
40 Effl.
42 Infl.
48 Effl.
25
0.5
5
25
100
500
18*
13'
17
18
17
16
22
32
31
24
150*
151*
. 82*
82*
102*
80*
. 75*
101*
108*
96*
5
2
10
11
20**
. . 4
6
7
15.
22**
7
6
5
•9
9
8
4
13
8
9
0.5
5
25
100
500
5
7
9
9
6
34
36
46
39
35
177**
186**
205**
199**
203**
96*
169*
162*
173**
166*
6
15
14
8
9
8
10
13
15
15
10
7
8
12
7
12
10
14
17
8
0.5
5
25
100
500
31
16
16
24
17
45
35
36
31
34
93
97
95
87
106
128
115
125
132
111
9
12
8
8
7
5
10
10
9
8
20
6
8
3
7
9
12
8
11
12
(continued)
23
-------
TABLE 8 (continued)
Sample Dose
No. in
P1
50 Infl. 0.5
5
25
100
500
9
no
S9
26
27
25
43
50
8
S9
45
38
33"
33
46
100
no
S9
169*
185*
153*
173*
254**
S9
176**
205*
164*
158**
221**
is:
no
S9
18
13
12
10
15
35
S9
16*
6
11
14
15
15
no
S9
11
6
13
13
10
37
S9
12
11
11
4
12
54 Effl. 0.5 10 41 105 144 11 10 6 12
5 11 32 122 155 13 15 88
25 14 32 135 156 13 13 89
100 11 32 122 169 10 8 8 11
500 10 32 117 170 13 14 87
24
------- |