EPA-R3-73-010
FEBRUARY 1973 Ecological Research Series
Impairment
of the
Flavor of Fish
by Water Pollutants
,^°ST^
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington, D.C. 20460
-------
RESEARCH REPORTING SERIES
Research reports of the office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formation,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.
-------
EPA-R3-73-010
February 1973
IMPAIRMENT OF THE FLAVOR OF FISH
BY WATER POLLUTANTS
Dean L. Shumway
and
John R. Palensky
Oregon State University
Department of Fisheries and Wildlife
Corvallis, Oregon 97331
Project 18050 DDM
Project Officer
Dr. Gerald R. Bouck
Western Fish Toxicology Lab.
200 S.W. 35th Street
Corvallis, Oregon 97330
Prepared for
OFFICE OF RESEARCH AMD MONITORING
U.S. EHVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20U60
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402
Price $1.26 domestic postpaid or $1 OPO Bookstore
-------
EPA Review Notice
This report has been reviewed by the Environmental
Protection, and approved for publication. Approval
does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or
recommendation for use.
n
-------
ABSTRACT
Laboratory studies with fish were conducted to determine an appropriate
bioassay procedure for the examination of the flavor-imparting capacity
of wastes and waste components (organic compounds). In addition, the
flavor-imparting capacity and estimated threshold concentrations were
determined for a number of organic compounds and effluents. Flavor
evaluations were obtained through the use of taste panels.
Estimated threshold concentrations were determined for twenty two
organic compounds. The values ranged from 0.4 ppb (2,4-dichlorophenol)
to 95 ppm (formaldehyde). An additional twelve compounds were tested,
seven of which were not found to impair flavor at or near lethal levels.
Estimated threshold concentrations were determined for effluents from
kraft paper mills and a sulfite-base paper mill. The estimated threshold
concentrations for the effluents from the kraft and sulfite-base paper
mills were about 6 and 36 percent by volume.
The estimated threshold concentrations for primary, secondary, and
secondary chlorinated effluents from the Corvallis Sewage Treatment
Plant were determined to be 11-13, 21-23, and 20-26 percent by volume,
respectively.
This report was submitted in fullfillment of Project Number 18050 DDM
under the (partial) sponsorship of the U.S. Environmental Protection
Agency.
111
-------
CONTENTS
Section
I Conclusions 1
II Recommendations 3
III Introduction 5
IV Fish Facilities 7
Averill facilities 7
Oak Creek facilities 7
V Materials, Apparatus, and Methods 9
Experimental fish 9
Effluent tested 10
Chemicals tested 10
Experimental apparatus 11
Experimental procedures 13
VI Results and Interpretation 19
Initial exposure tests 19
Rates of flavor impairment 19
Clearing rates 22
Weight of fish per chamber 31
Dissolved oxygen concentration 31
Influence of pH 37
Influence of organic compounds on flavor 41
Paper processing effluents 41
Treated waste water 58
Interaction of compounds 62
Estimated threshold concentrations 66
VII Acknowledgments 73
VIII Literature Cited 75
IX List of Appendices 77
X Appendices 79
-------
FIGURES
Figure Page
1 Photograph of the facilities used for rearing rainbow trout 8
at the Averill Fisheries Laboratory located east of
Corvallis.
2 Drawing of the dilution apparatus used for the delivery of 12
solutions to the exposure chambers.
3 Photograph of the exposure apparatus showing the dilution 14
system, water control box, and the covered exposure
chambers.
4 Photographs showing a tray containing samples of fish being 16
served to judge seated in an isolation booth (upper
picture) and a judge evaluating samples of fish
(lower picture).
5 The influence of exposure time on the mean off-flavor 28
indices for trout exposed to three concentrations
("high," "medium," and "low") of 2,4-dichlorophenol
(Exper. U-7, U-8, and U-9) and butanethiol (Exper.
U-l, U-2, and U-3).
6 Mean off-flavor indices in relation to the length of time 30
trout were held in fresh water after 24 hour exposure
to 2,4-dichlorophenol concentrations of 100, 10, and
1 ppb (Exper. C-l, C-2, and C-3).
7 The relation between mean off-flavor indices and the pH 40
at which trout were held and exposed to 100 and 10 ppm
of pyridine (Exper. P-7 and P-8) and 100 and 10 ppb of
2,4-dichlorophenol (Exper. P-3 and P-4).
8 The influence of exposure to mixtures of two organic com- 65
pounds on mean off-flavor indices. The open plots
represent off-flavor indices for trout exposed to only
one compound; the closed plots (squares) represent
results of exposure to two compounds. The concentrations
of p_-chlorophenol and pyridine in the upper graph are in
ppb and ppm, respectively. The presentation is based on
data in Table 10.
9 Example of the procedure used in determining the estimated 67
threshold concentration (ETC). The curve was fitted to
the data by size. The LSD QC for 2,4,6-trichlorophenol
was 0.60 (Table 7, Exper. t-49).
VI
-------
TABLES
No. Page
1 Experimental conditions and results of tests with trout 20
exposed to chemicals for 96 hours.
2 Results and conditions of tests with trout exposed for 23
various periods of time to concentrations of pyridine,
butanethiol, 2,4-dichlorophenol, and p_-cresol.
3 Conditions and results of tests with trout exposed to 2,4- ^9
dichlorophenol and then placed in fresh water for various
periods of time.
4 Experimental conditions and results of tests in which different 32
weights of trout were exposed to p-chlorophenol, 2,4-
dichlorophenol, and pyridine. ~~
5 Results and conditions of tests with trout exposed to 2,4- 34
dichlorophenol and pyridine at various dissolved oxygen
concentrations.
6 Results and conditions of tests with trout exposed to 2,4- 38
dichlorophenol and pyridine at high, intermediate and
low pH values.
7 Results of tests in which fish were exposed to various con- 42
centrations of different organic compounds.
8 The influence of stabilized waste from sulfite and sulfate 5^
paper plants on the flavor of trout flesh.
9 The effect of primary and secondary treated waste water on 59
the flavor of trout flesh.
10 The results and experimental conditions of tests with trout 63
exposed to combinations of two chemicals.
11 Estimated threshold concentrations for the organic compounds 68
tested in this study.
12 Estimated threshold concentrations for paper process wastes 70
and primary and secondary treated waste waters.
vii
-------
SECTION I
CONCLUSIONS
(1) A standard bioassay procedure was developed and accepted for use in
laboratory studies on the flavor-imparting capacity of wastes and
waste components (organic compounds). The bioassay conditions
selected are: dissolved oxygen - near air-saturation, light level-
subdued or absent, pH - 7.0 to 8.0, temperature - 15°C for coldwater
fish and 20-25°C for warmwater fish, exposure period - 48 hours,
weight of fish per chamber - 150 to 1,000 grams in present apparatus,
and exposure system - flowing water.
(2) The flavor of trout exposed to 2,4-dichlorophenol, butanethiol,
o-cresol, and pyridine was found to reach maximum impairment in
less than 33.5 hours. After exposure to 2,4-dichlorophenol for
24 hours, trout lost the acquired off-flavor in about 33.5 hours.
(3) The influence of exposure to concentrations of two flavor-imparting
substances was examined. The observed off-flavor values were less
than additive.
(4) Estimated threshold concentrations were determined for twenty two
organic compounds. In addition, twelve organic compounds were
tested for threshold concentrations, seven of which were not found
to impair flavor at or very near lethal concentrations.
(5) Estimated threshold concentrations were determined for effluents
from two kraft process paper mills and a sulfite-base paper mill.
The estimated threshold concentration for the kraft effluents ranged
from 5 to 8 percent by volume. The estimated threshold concentra-
tion of the sulfite effluent was 36 percent by volume.
(6) Primary, secondary, and secondary chlorinated effluents from the
Corvallis Sewage Treatment Plant were evaluated. The estimated
threshold concentration values ranged from 11 to 13 percent by volume
for primary effluent, from 21 to 23 percent by volume for secondary
effluent, and from 20 to 26 percent by volume for secondary chlori-
nated effluent.
-------
SECTION II
RECOMMENDATIONS
(1) Fish are but one of a large number of important organisms that may
be contaminated by the discharge of tainting substance into the
aquatic environment. Studies should be initiated to determine the
impact of such materials on the quality of other organisms such as
crayfish, crabs, clams, oysters, lobsters, and other forms of
edible aquatic life, both fresh water and marine.
(2) With the methods employed in this study it would be possible to
determine the flavor-imparting capacity of a large number of
different types of domestic and industrial effluent. Such infor-
mation would be extremely valuable to state and federal agencies
responsible for establishing water quality and effluent standards.
(3) This program was limited to a preliminary examination of treated
waste water collected from one source (Corvallis). Effluents from
other treatment plants of the same general kind as well as other
kinds should be examined for their flavor-imparting capacities.
(4) Since many effluents contain numerous components capable of impair-
ing the flavor of fish, the interaction of combinations of these
compounds should receive further detailed study.
(5) More sophisticated and less laborious procedures should be
developed for the examination of tissue for impaired flavor. Taste
panels, although adequate for many needs, can neither determine
concentration of substances causing off-flavors nor examine small
samples of flesh. When taste panels are employed, at least fif-
teen trained judges should be employed. Panels comprised of
fewer than ten judges probably should be avoided.
(6) Information is needed on the relationship between the exposure
concentration of a compound and the resulting tissue concentration
and degree of flavor impairment.
-------
SECTION III
INTRODUCTION
The value of many recreationally and commercially important fisheries
is constantly being reduced by the introduction of fish-tainting sub-
stances into surface waters. While considerable effort has been directed
toward the sensory evaluation of foods and drinking water in recent
years, only a limited effort has been expended in determining the flavor-
imparting capacity of wastes and waste components (organic compounds)
commonly entering lakes and streams.
In only a few cases have the source and nature of substances causing
tainted fish problems been accurately identified (Westfall and Ellis,
1944; Tamura, Itazawa, and Morita, 1954; BSetius, 1954; Fetterolf, 1964;
Hasselrot, 1964; Nitta, et al., 1965; Shumway, 1966, Krishnaswami
and Kupchanko, 1969). Wastes discharged from kraft paper plants,
chemical plants producing pesticides, coal-tar processing, and oil
refineries have been identified, or strongly suggested, as sources of
fish tainting. Unfortunately, very little is known about the flavor-
imparting capacity of the waste components (specific organic compounds)
comprising these wastes.
Results of studies on the flavor-imparting capacity of wastes or waste
components (organic compounds) have been reported by Albersmeyer and
Erichsen (1959), Brandt (1955), BSetius (1954), Schultz (1961), Shumway
(1966), Shumway and Chadwick (1971), and Winston (1959). These investi-
gations, conducted under controlled laboratory conditions, dealt mainly
with phenolic compounds, although Shumway and Chadwick (1971) studied
treated and untreated kraft mill effluents.
A wide variety of organic compounds are capable of imparting objection-
able tastes and odors to the flesh of fish and other aquatic organisms.
In many cases these materials are capable of impairing flavor at con-
centrations far below levels otherwise considered detrimental to
aquatic organisms. To adequately protect our freshwater fisheries, both
commercial and sport, we must not only ensure that reproduction, growth,
migration, and other essential activities of fish will be protected, but
also ensure that the flavor of the flesh of fish will not be impaired
beyond acceptable limits. "Without this protection, otherwise productive
fish populations may become largely unutilized by man. With this in
mind, the research covered in this final report was planned and conducted.
Presented in this report are the results of a three-year laboratory
study on the influence of wastes and specific organic compounds on the
flavor of the flesh of freshwater fish. There were two objectives to the
three-year study. (1) To develop, through studies with freshwater
fish held in flowing water, appropriate standard procedures for the
evaluation of the flavor-imparting capacity of contaminants. The
-------
standardization of procedures was to include selection of specific
conditions for exposure of the fish and-,of methods of organoleptic
evaluation of the flesh. (2) To determine through studies using fresh-
water fish held in flowing water the flavor imparting capacity of a
substantial number of wastes and organic compounds commonly discharged
into surface waters. The research was conducted at the Oak Creek
Fisheries Laboratory of the Department of Fisheries and Wildlife, Oregon
State University, during the period of April 1, 1969 to March 31, 1972.
-------
SECTION IV
FISH FACILITIES
The design of the study and number of fish required necessitated the
construction of facilities at which relatively large numbers of fish
of several species could be held and reared. Facilities suitable for
the rearing of trout were constructed at the Averill Fisheries Labora-
tory located east of Corvallis. Facilities for holding warmwater fish
and facilities for temporary retention of trout were constructed at
the Oak Creek Fisheries Laboratory located west of Corvallis.
Averill Facilities: The Averill Fisheries Laboratory was selected for
holding and rearing trout primarily because it had a dependable and
plentiful year-round supply of well-water, with temperatures ranging
from 10°C in winter to 13°C in summer. In addition, problems with
fish diseases were nearly unknown at the location, as well as at other
laboratories located nearby.
Ten circular, self-cleaning, fish-holding tanks and one oval starter-
tank were assembled at the Averill site. The circular tanks were 2 ft.
deep and ranged in diameter from 5 to 8 ft. The oval tank was 1-ft.
deep, 2 ft. wide and 8 ft. long. The tanks, made of galvenized metal,
were originally intended as stock-watering tanks. Prior to installation,
the tanks were sandblasted and painted with epoxy paint. Each tank was
fitted with a self-cleaning standpipe system, metal-screen or nylon-
mesh covers to prevent fish from jumping out, and a water delivery system.
Five tanks were supplied with automatic feeding devices. A small metal
building, constructed on the site, was furnished with a work table,
sink, and freezer (storage of fish food) and has storage space for
maintenance supplies and tools. A 6-ft., metal-link fence protects the
fish and facilities from vandalism and unauthorized entry. A photograph
of the Averill site is presented in Figure 1.
Oak Creek Facilities: A second facility, capable of handling fish
collected from the wild or obtained from state owned hatcheries, was
constructed at the Oak Creek Fisheries Laboratory. This facility also
allowed the short-term retention of a limited number of fish reared at
the Averill site, thus facilitating fish handling requirements. Three
circular tanks of the type described above were assembled at the Oak
Creek site. Two 8-ft. tanks were used for warmwater fish and one 5-ft.
tank was used for temporary holding of Averill fish. The tanks were
covered with nylon-mesh nets and supplied with spring-water from the
water supply of the Oak Creek Fisheries Laboratory.
-------
\
oo
Figure 1. Photograph of the facilities used for rearing rainbow trout at the Averill Fisheries
Laboratory located east of Corvallis.
-------
SECTION V
MATERIALS, APPARATUS, AND METHODS
Experimental fish: Rainbow trout, Salmo gaivdneTi,, largemouth bass,
Micropterns saImoides, and bluegill, Lepomis maoroohirus, were used as
test fish in this investigation. Rainbow trout was the main test fish,
with bass and bluegill used only to a limited extent.
Two stocks of rainbow trout were selected initially and brought to the
Averill site. Kamloops trout (a variety of rainbow trout) eggs were
obtained from the Trout Lodge Springs Hatchery located in eastern Wash-
ington. The second stock of trout was obtained as eggs from Dr. L.
Donaldson's rainbow trout stock at the University of Washington. These
stocks were selected for consideration as the eventual test trout because
of their rapid growth characteristics and adaptability to hatchery opera-
tions. Reared under nearly identical conditions at the Averill site, the
Kamloops trout were found to grow somewhat more rapidly than did Dr.
Donaldson's variety; therefore, the Kamloops trout were selected as the
test stock.
During most of the first year of the study, rainbow trout reared at our
Averill site were too small for test purposes. During this stage of the
study, it was necessary to use rainbow trout obtained from the Oregon
State Game Commission's Roaring River Trout Hatchery located near Scio,
Oregon. These fish were transported from the hatchery to the Oak Creek
site in plastic-lined 55-gal. tanks. The hatchery trout were fed Oregon
Moist Pellets, a product obtained from a local dealer, both at the
hatchery and whole being held at the Oak Creek Laboratory.
Since trout eggs and alevins could not be adequately handled at the
Averill site, this was accomplished at an adjoining laboratory of the
Research Division of the Oregon State Game Commission, where facilities
were available. When ready to feed, the alevins were transported from
the laboratory to the Averill site in buckets and reared to the desired
size. The Oregon Moist Pellet was the only food provided the test
animals. Although a few fish experienced a mild bacterial infection of
the dorsal and caudal fins, no major disease problem developed during the
course of the three-year study.
Largemouth bass and bluegill were used as test fish in a few experiments.
The bass were obtained by seining from sloughs and ponds along the
Willamette River. Bluegill were captured from local ponds by angling.
After capture, the bass and bluegill were transported to the Oak Creek
site in plastic-lined 55-gal. tanks and held until needed. Food in the
form of live fish, angleworms, and Oregon Moist Pellets was provided the
bass and bluegill.
-------
Effluents tested: Kraft mill effluents (KME) were obtained from mills
located near Halsey and Albany, Oregon. Both mills process pulp and
paper from Douglas fir, Pseudotsuga mensiesii, treat their effluents in
large aerated stabilization lagoons and discharge treated effluents
directly into the Willamette River. The Albany and Halsey mills
process about 100 and 400 tons of pulp per day, respectively. The
water use rate for the Albany mill is about 10,000 gal. per ton; 35,000
gal. of water per ton is required by the Halsey mill. The effluent
discharged from the Albany mill has a BOD of about 50 to 75 ppm; the
BOD in the Halsey mill effluents is normally less than 20 ppm.
Effluent was obtained from an ammonia-base, sulfite mill located near
Lebanon, Oregon. The mill, which primarily produces liner board,
processes about 100 tons of pulp daily and uses 40,000 gal. of water
per ton. The effluent from this mill passes through an aerated stabili-
zation lagoon before being discharged into the South Santiam River.
BOD level of the effluent is normally at or below 125 ppm.
The Corvallis Sewage Treatment Plant was used as the source for primary,
secondary, and secondary chlorinated waste water. The input to the
Corvallis plant, which has a total capacity of about 15 million gallons
per day (MGD) is nearly all from domestic sources. The plant employs a
primary clarifier, two trickling filters, and a secondary clarifier.
Samples of primary treated waste water were collected at a point between
the primary clarifier and the trickling filters. Secondary treated
waste was collected at the point of discharge from the trickling filters.
In the Corvallis plant, chlorination of the effluent occurs between the
trickling filter and the secondary clarifier. Secondary, chlorinated
waste water was collected from the discharge of the secondary clarifier.
The treated waste water from the Corvallis plant is discharged directly
into a small creek a short distance above the confluence of the Willamette
River.
During the time experiments were being conducted, the plant discharge
rate and the BOD level of the inflow averaged 7.5 MGD and 168 ppm,
respectively. The ranges in discharge rate and inflow BOD were 4.4 to
12.0 MGD and .43 to 295 ppm, respectively. No attempt was made to
determine residual chlorine levels of the treated effluent after chlori-
nation and discharge from the secondary clarifier. Toxicity data
gathered during the tests strongly suggest that the chlorine level
fluctuated substantially and was probably quite high most of the time.
Chemicals tested: With only a few exceptions, the chemicals used in this
investigation were reagent grade. In the excepted cases, reagent grade
chemicals were not available and a chemical pure grade (exceeding U.S.
Pharmacopoeia and National Formulary) was utilized. All chemicals used
were obtained from either the J. T. Baker Chemical Company or the
Mallinckrodt Chemical Company.
10
-------
Experimental apparatus; The three experimental apparatus used in this
study were located in a 15°C constant temperature room which was
lighted continually with fluorescent lights. Figure 2 is a schematic
drawing of one of the three independently controlled, dilution apparatus.
One diluter supplied well-water to six exposure chambers; the other two
diluters supplied well-water to four chambers each. The flow of water
entered the 70-liter fiberglass exposure chambers at a rate of 250 ml/
min. In addition, each diluter was equipped with a temperature-control
unit, an oxygen supply, and a toxicant delivery system. A Geological
Survey analysis of the well-water from the source used in this study is
presented in Appendix 1.
Well-water entered the diluter system via a water-control box constructed
of wood, where it was continually circulated by a small submersible
pump and brought to the desired temperature by a thermostatically con-
trolled, stainless-steel immersion heater (Figure 2). From the control
box, the water passed into a tubular, plastic, manifold and was meted
into the glass mixing boxes through a small-diameter, adjustable
delivery tube. Each mixing box was fitted with a simple, constant-head
overflow tube and two adjustable delivery tubes. The larger of the two
delivery tubes discharged water to the exposure chamber and the other
delivery tube meted a small quantity of water into a lower mixing box.
The flow of water through the delivery tubes could be stopped by
rotating the discharge orifice into an up position. This was done with
the delivery tube leading to the lowest mixing box, through which only
control water was passed. Overflow water from the mixing boxes was dis-
charged from the system.
Contaminant solutions were introduced to the dilution at the uppermost
mixing box, where it was combined with an appropriate quantity of well-
water and passed on through the system in the manner previously
described. A simple, pivoting device was sometimes used at the point of
contaminant introduction as a safety precaution. If the flow of exchange
water slowed significantly or stopped, the safety device changed position
and diverted the flow of contaminant away from the mixing box, thus
preventing increases of concentration in the exposure chambers.
Each exposure chamber was provided with a cover to prevent fish from
jumping out and to reduce the light level, a centrally located, stainless-
steel standpipe for discharge of excess exchange water and for water-
level control, a relief tube for removal of water samples, a mercury
thermometer, inserted through the cover, and a water delivery port. The
exposure chambers could be quickly and easily drained by removal of
the standpipes.
Two types of contaminant control systems were employed. Twenty-liter
Mariotte bottles (constant-head bottles) were used for the introduction
of stock solutions of the organic compounds tested. Because of the
large volume needed, sewage treatment plant and paper process effluents
required a much more complicated delivery system. A 750-gal. fish
holding tank was placed in the constant-temperature room near the
11
-------
TOXICANT
SOLUTION
WATER
MANIFOLD
PIVOTING
SAFETY
TO AQUARIA
WATER CONTROL BOX
WATER
INLET
FLOAT
VALVE
PUMP
WASTE
Figure 2. Drawing of the dilution apparatus used for the delivery of solutions to
the exposure chambers.
-------
exposure apparatus and filled with effluent. A small, submersible
pump was placed on the tank and served to deliver a continuous supply of
effluent to a wooden box position above the first mixing box of the
largest diluter. The wooden box contained an overflow tube through
which excess effluent returned to the holding tank and several delivery
tubes similar to those described earlier. Through these tubes the
effluent was meted to the diluter. The effluent was not aerated while
in the large tank or wood head box.
Bottled gas, either air, oxygen, or an oxygen-nitrogen mixture was used
to maintain the desired dissolved oxygen concentrations in the exposure
chambers. The compressed gas passed from the gas cylinder through a
two-stage reduction valve, a gas manifold, ball-displacement, gas flow-
meters, and into the exposure chambers via gas dispersers. The intro-
duction of gas also served to ensure thorough mixing of the renewal
solution with that already present in that chamber. Although compressed
air and pure oxygen were used in some experiments, the introduction of
a mixture of 30% oxygen and 70% nitrogen proved highly satisfactory in
maintaining oxygen concentration at or near air-saturation without
excessive agitation of the water.
A photograph of one of the three exposure apparatus is present in
Figure 3.
Experimental procedures: The day prior to the start of each experiment,
the water and gas flows were adjusted to the desired levels and the
contaminant stock solution or effluent prepared for introduction. Dur-
ing the first day of the experiment, the exposure chambers were drained
of water, the contaminant flow started and adjusted, and the chambers
filled. Test fish were then introduced, the number of fish per chamber
depended on the experimental design, the size, and the species of fish
used. Observations and necessary adjustments of water and contaminant
flow rates and water temperature were made two to three times a day
during the experiment. Dissolved oxygen concentrations were normally
determined daily using the azide modification of the iodometric method;
pH values varied little and were determined only periodically.
When an experiment was terminated, the test fish were removed from each
chamber, killed, weighed and measured, cleaned (head and viscera
removed), placed in labeled plastic bags, and either held under
refrigeration at near 5°C for not more than 24 hours or frozen for a
few days until they could be prepared for flavor evaluation. After the
fish were removed, the flow of contaminant was stopped, the exposure
chambers drained, and the diluter and exposure chambers cleaned with
a mild detergent and thoroughly rinsed with acetone. Once cleaned,
the diluter and exposure chambers were reassembled and the flow of water
started once again.
The test fish were prepared for organoleptic evaluation by personnel of
the Sensory Evaluation Section of the Department of Food Science and
Technology, Oregon State University. The Sensory Evaluation Section,
13
-------
I
Figure 3. Photograph of the exposure apparatus showing the dilution
system, water control box, and the covered exposure
chambers.
14
-------
under the direction of Mrs. Lois S. McGill, Professor of Food Science
and Technology, also provided the facilities for the preparation and the
evaluation of the samples of fish. Each sample of cleaned fish was
wrapped in aluminum foil, placed in an individual pan, and cooked in
an oven at approximately 210°C until done (approximately 30 to 40 min).
No seasoning was added to the samples at any time. Each sample of fish
was then removed from the oven, skinned and boned, the flesh of all fish
within a sample lightly flaked and thoroughly mixed and a portion of
flesh placed in each of a number of small, coded paper cups. The number
of cups for each sample was determined by the number of judges that
would evaluate that sample. In most experiments, the control, or uncon-
taminated sample of fish, was divided between coded cups and cups marked
"reference." One cup from each group, including a cup from the group
marked "reference," was placed on each of a number of small trays (one
tray per judge). The tray of samples was then served immediately to the
judge who was seated in an individual isolation booth (Figure 4).
The judges, all of whom had previous experience in evaluating the flavor
of fish, were asked to smell the samples, and then to taste (masticate)
and score the samples on a 7-point word evaluation scale for intensity of
off-flavor (Appendix 2). They were asked to leave those samples with the
most intense odor to be rated last. They were told that the sample
marked "reference" contained flesh from the control fish. Judges were
not required to tast samples with extremely intense or obnoxious odors.
The word evaluation scale for off-flavor shown in Appendix 2 was converted
to a number scale of 0 to 6, with 0 representing the highest quality
(no off-flavor) and 6 the lowest quality (extreme off-flavor).
In addition to evaluating the sample of flesh for off-flavor, the
judges were asked to rate each sample for overall desirability. The
7-point hedonic scale presented in Appendix 2 was used. The ratings
ranged from very desirable (0) to very undesirable (6). The results of
the hedonic ratings, when compared with the results of the off-flavor
ratings, were found less instructive and are not included in this
report.
Once the samples were evaluated, their ratings were compiled and the
data treated statistically. A standard, two-way analysis of variance
program (ANOVA) written for the CDC 3300 in FORTRAN language was used
to test for experimental differences.
Near the completion of the first year, an attempt was made to develop an
evaluation system that utilized "odor" rather than "taste." This was
done because many judges strongly objected to the idea of masticating
the foul-tasting fish flesh that often resulted from the exposure tests.
In order to determine whether or not a change in procedure was advisable,
comparative tests were made for a number of test compounds. In these
tests, the judges were first asked to rate the samples on the basis of
smell only, record their judgments, and then to re-evaluate the samples
by the procedure normally utilized (tasting). The results showed that
with some chemicals the two methods were equally satisfactory, but with
15
-------
.^^HJPI^Hj
\i:''"
Figure 4. Photographs showing a tray containing samples of fish being
served to judge seated in an isolation booth (upper picture)
and a judge evaluating samples of fish (lower picture).
16
-------
other chemicals, the "odor" method proved far less sensitive and the
results more variable. Based on these findings, the "odor" method was
abandoned from further use.
Panel judges were selected on the basis of willingness to serve,
dependability, and of demonstrated ability to judge differences in the
flavor of fish flesh. Many of the panel judges that were used had
previous experience in evaluating the flavor of fish. This source of
judges was not sufficient to meet the needs of the project, however,
and a training program for new judges was initiated. Training sessions
were conducted at which a number of potential judges were asked to
evaluate a series of samples of contaminated fish flesh. The fish had
been previously exposed tg different concentrations of a chemical,
usually 2,4-dichlorophenol, the response to which had already been
determined. Prior to evaluating the samples, the group of potential
judges were carefully explained the procedures they must follow in
making their judgments. After each training session, the evaluations
made by each judge were compared with the average responses of the
group, as well as with the results from previous evaluations by
experienced panel judges. Those demonstrating the ability to make
reasonably reliable judgments were selected as judges. Normally, two
or more training sessions were required of a potential judge prior to
his selection as a new panel member.
To encourage participation by the judges in what was often a distasteful
experience (tasting), each judge was given a candy bar and $1 after
each series of flavor evaluations. The candy bar served as a mild
stimulant to return and provided a means of removing undesirable odors
or tastes from the mouth. The payment of $1 per test appeared to
encourage future participation, particularly with judges coming from the
student level.
17
-------
SECTION VI
RESULTS AND INTERPRETATIONS
As pointed out earlier, the first objective of this study was to develop
an appropriate standard procedure for the evaluation of the flavor-
imparting capacity of water contaminants. Five organic compounds,
£-cresol, 2,4-dichlorophenol, pyridine, n-butyl alcohol, and butanethiol,
were selected for use during the initial methodology-development phase.
During the initial phase, experiments were conducted to further under-
standing of the effects of various factors (exposure time, dissolved
oxygen concentration, pH, light, size or weight of fish, weight of fish
per chamber, rate of loss, etc.) on the flavor-imparting capacity of
organic compounds. After completion of the methodology period and the
selection of a standard procedure, routine testing of organic compounds
and effluents could commence.
Initial exposure tests: The first series of experiments were conducted
to determine the approximate flavor-imparting capacity of each of the
five selected compounds. In these experiments rainbow trout were exposed
to a wide range of concentrations (five levels and a control} for 96
hours at 15°C. The dissolved oxygen concentration was maintained near
air-saturation with compressed air; the pH of the water was 7.8. The
test fish were not shielded in any way from direct room illumination.
The results of the tests are presented in Table 1.
Of the five compounds tested, four produced significant impairment of
flavor (off-flavor) of the flesh at higher concentrations. Only n-
butyl alcohol failed to produce impaired flavor in the test trout,
even though high off-flavor index of 3.45 was obtained at the concentra-
tion of 100 ppm, the highest level tested. During the remainder of the
initial phase of the investigation, only the four compounds that demon-
strated strong flavor-imparting capabilities were utilized.
Rate of flavor impairment: One of the most crucial aspects of the
initial phase of the study was to determine the exposure time required
to produce maximum impairment of flavor. To evaluate this aspect, a
series of experiments were conducted on the rate of uptake (flavor
impairment) by trout exposed for various periods of time to p_-cresol,
2,4-dichlorophenol, butanethiol, and pyridine. Trout were exposed to
"high," "medium," and "low" concentrations of each chemical and a con-
trol for periods of 0.25, 1.27, 6.5, 33.5, and 168 hours (one week).
The "high" concentrations were well above previously estimated threshold
concentrations (level at which flavor impairment begins to be detected),
the "medium" concentrations were' close to the estimated threshold con*-
centrations, and the "low" concentrations were well below those levels.
At the end of each test period, trout were removed from each concentra-
tion and the control chamber and frozen for flavor evaluation at a later
date. The samples were evaluated (tasted) within a week after the last
19
-------
Table 1. Experimental conditions and results of tests in which trout
were exposed to various concentrations of chemicals for 96
hours at 15°C.
Mean
Exposure off -flavor „/
Exper. concentration index!/ LSLH
-------
Table 1. Continued
Mean
Exposure off -flavor
Exper. concentration index-
no . ppb
A- 6 Pyridine
0
0.1
1
10
100
1,000
A- 7 Pyridine
ppm (0-6)
July 24, 1969
August 7, 1969
0
0.01
0.1
1
10
100
Trout
1.50
2.05
2.20
1.55
2.20
2.45
Trout
1.75
2.20
2.20
1.50
1.55
4.20
2/ Standard
LSD-Q5 error of
the mean
10 Judgments
n.s. 0.32
0.42
0.44
0.34
0.47
0.64
10 Judgments
0.78 0.45
0.45
0.37
0.26
0.31
* 0.44
Fish per
chamber
grains
338
304
398
331
309
314
486
473
376
381
381
370
number
2
2
2
2
2
2
2
2
2
2
2
2
— Off-flavor index based on a scale of 0 (no off-flavor) to 6 (very
extreme off-flavor).
-/ The least significant difference at P=0.05 based on a two-way analysis
of variance. An asterisk (*) indicates a statistically significant
change in flavor from that of the control sample.
21
-------
samples were removed (168 hours).
Experimental conditions and results of the series of tests on the rate
of flavor impairment are presented in Table 2. Trout exposed to the
"high" concentration of each of the chemicals tested appeared to
attain maximum off-flavor in 33.5 hours or less. Additional exposure
time did not alter significantly the observed mean off-flavor indices.
At the "medium" and "low" concentrations of each of the test chemicals,
exposure time did not appear to influence the degree of flavor impair-
ment.
The results of experiments with butanethiol (U-l, U-2, and U-3) and
2,4-dichlorophenol (U-7, u-8, and U-9) are presented graphically in
Figure 5. As may be seen, rather substantial increases in off-flavor
occurred at the "high" concentration of each chemical after exposure
for only fifteen minutes (0.25 hour). Continued exposure for 1.27 hours
at the "high" concentration of both compounds resulted in the attainment
of maximum or near maximum flavor impairment. Additional exposure time
caused little or no change in the degree of flavor impairment. At 10
and 1 ppb of butanethiol, the mean off-flavor index appeared to increase
with exposure time, although the differences were not significant (Table
2).
Based on the results of the exposure-time tests, a standard exposure
period of 48 hours was adopted. It may well be that longer exposure
periods might produce a slightly higher degree of off-flavor; however,
our data strongly suggest that if this is so the exposure period required
would be longer than a week (168 hours), the maximum exposure period used
in our tests. Long-term exposure periods would surely cause many addi-
tional testing problems (i.e., increased mortality, need to provide food,
gear failures, disease, etc.).
Clearing rate: A series of experiments were conducted to determine the
relation between the degree of off-flavor and the length of time held in
fresh water after a 24-hour exposure to 100, 10, and 1 ppb of 2,4-
dichlorophenol. Samples of trout were removed from each group after 0.25,
1.27, 6.5, 33.5, and 168 hours exposure to fresh water and frozen for
later evaluation.
The results of the experiments on clearing rate are presented in Table 3
and Figure 6. Off-flavor values for trout exposed to 100 ppb of 2,4-
dichlorophenol were substantially reduced after 6.5 hours in fresh water.
After 33.5 hours in fresh water the flavor of the trout exposed at the 100
ppb level had returned to normal. Some reduction in off-flavor appeared
to occur in the first hour at 10 ppb of 2,4-dichlorophenol, and complete
loss had occurred after 6.5 hours of contact with fresh water. At the
lowest level tested (1 ppb), little or no change occurred in the off-
flavor index with exposure to fresh water. The changes in off-flavor
values observed at the 10 and 1 ppb levels were not statistically
significant (Table 2).
22
-------
Table 2. The experimental conditions and results of tests in which rainbow trout were exposed at
15°C for various periods of time to high, intermediate, and low concentrations of pyridine,
butanethiol, 2,4-dichlorophenol, and p_-cresol.
K)
Exposure
Exper. Concentration period
no . ppb
U-l Butanethiol
0
1
1
1
1
1
U-2 Butanethiol
0
10
10
10
10
10
U-3 Butanethiol
0
100
100
100
100
100
ppm (hrs . )
February 11, 1970
0.25
0.25
1.27
6.5
33.5
168
February 11, 1970
1.27
0.25
1.27
6.5
33.5
168
February 11, 1970
33.5
,0.25
1.27
6.5
33.5
168
Mean
off-flavor ?/
index-' LSIP-'
(0-6) '°5
10 Judgments
1.30 n.s.
1.05
1.55
1.80
1.95
1.80
10 Judgments
1.15 n.s.
1.45
1.80
2.20
2.65
1.90
10 Judgments
1.35 1.04
2.40 *
3.60 *
3.45 *
3.65 *
4.25 *
Standard
error of
the mean
0.26
0.14
0.23
0.28
0.28
0.47
0.21
0.39
0.45
0.34
0.48
0.26
0.26
0.46
0.43
0.46
0.45
0.37
Fish per
chamber
grams
145
110
97
153
196
170
188
191
158
192
200
114
164
143
140
161
131
165
number
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Dissolved
oxygen
(mg/1)
6.8
9.8
9.8
9.8
8.7
8.7
8.8
8.6
8.6
8.6
11.0
11.0
8.8
9.0
9.0
9.0
8.9
8.9
pH
7.8
7.8
7.8
7.8
7.7
7.7
7.8
7.8
7.8
7.8
8.0
8.0
7.8
7.8
7.8
7.8
7.8
7.8
-------
Table 2. Continued
to
Exper. Concentration
no
U-4
U-5
U-6
ppb ppm
0-Cresol December 3,
0
12.5
9.9
12.5
10.0
9.6
0-Cresol December 3
0
110
129
110
130
124
0-Cresol December 3
0
826
996
826
990
958
Exposure
period
(hrs.)
Mean
off -flavor 2/
index— LSD—'
(0-6)
1969 10 Judgments
1.29 1.15 n.s.
0.25
1.27
6.5
33.5
168
, 1969 10
6.5
0.25
1.27
6.5
33.5
168
, 1969 10
168
0.25
1.27
6.5
33.5
168
0.93
1.23
1.35
1.33
1.70
Judgments
1.03 n.s.
1.62
1.50
1.43
1.65
1.55
Judgments
1.18 n.s.
2.45
2.93
2.60
2.73
2.78
Standard
error of
the mean
0.14
0.15
0.15
0.26
0.20
0.30
0.17
0.23
0.18
0.31
0.24
0.29
0.20
0.27
0.24
0.40
0.30
0.33
Fish per
chamber
grams
306
471
291
243
248
251
275
490
274
252
257
256
443
495
236
260
268
235
number
2
4
2
2
2
2
2
4
2
2
2
2
4
4
2
2
2
2
Dissolved
oxygen
(mg/1)
12.5
10.6
10.0
10.6
10.0
10.6
9.3
10.4
13.0
10.4
13.0
10.4
6.9
8.0
13.0
8.0
13.0
8.0
PH
-
-
-
-
-
-
-
-
-
-
-
-
-
-
_
-------
Table 2. Continued
NJ
01
Exper. Concentration
no . ppb ppm
U-7 2,4-Dichlorophenol
0
0.
0.
0.
0.
0.
1
1
1
1
1
U-8 2,4-Dichlorophenol
0
10
10
10
10
10
U-9 2,4-Dichlorophenol
0
100
100
100
100
100
Exposure
period
(hrs . )
January 8,
0.25
0.25
1.27
6.5
33.5
168
January 8,
1.27
0.25
1.27
6.5
33.5
168
January 8,
168
0.25
1.27
6.5
33.5
168
Mean
off -flavor -,
index!/ LSEP/
(0-6)
-------
Table 2. Continued
N)
Exper. Concentration
- no. ppb ppm
U-10 Pyridine January
0
1
1
1
1
1
U-ll Pyridine January
0
10
10
10
10
10
U-12 Pyridine January
© 0
100
100
100
100
100
Mean
Exposure off- flavor 0/
. 1 / „ ~r,2/
period indexi/ LSD-v-
(hrs.) (0 -6)
22, 1970
6.5
0.25
1.27
6.5
33.5
168
22, 1970
33.5
0.25
1.27
6.5
33.5
168
22, 1970
168
0.25
1.27
6.5
33.5
168
10 Judgments
0.85 n.s.
0.95
1.35
1.00
1.40
1.50
10 Judgments
0.95 n.s.
1.05
0.75
1.25
1.45
1.45
10 Judgments
1.00 n.s.
2.30
2.60
3.30
3.80
3.25
Standard
error of
the mean
0.15
0.23
0.36
0.21
0.29
0.40
0.20
0.32
0.24
0.24
0.29
0.36
0.18
0.75
0.70
0.41
0.33
0.60
Fish per
chamber
grams
238
638
390
173
348
176
152
571
400
212
462
265
189
704
442
212
391
208
number
1
3
2
1
2
1
1
3
2
1
2
1
1
3
2
1
2
1
Dissolved
oxygen
(mg/D
11.0
10.3
10.3
10.3
8.8
9.1
11.0
9.8
9.8
9.8
9.9
9.2
10.3
9.9
9.9
9.9
8.9
9.2
pH
7.9
8.0
8.0
8.0
8.0
7.8
7.9
7.9
7.9
7.9
7.8
7.8
7.9
7.9
7.9
7.9
7.9
7.9
-------
Table 2. Continued
NJ
Exper
no.
U-13
U-14
U-15
Concentration
ppb ppm
2 , 4-Dichlorophenol
0
1
1
1
1
1
2, 4-Dichlorophenol
0
10
10
10
10
10
2 , 4-Dichlorophenol
0
100
100
100
100
100
Exposure
period
(hrs.)
October 6,
168
0.25
1.27
6.5
33.5
168
October 6,
6.5
0.25
1.27
6.5
33.5
168
October 6,
0.25
.25
1.27
6.5
33.5
168
Mean
off-flavor 2/
index!/ LSD— ^_
(0-6)
1970 9 Judgments
0.61 n.s.
1.11
1.61
2.00
1.05
0.94
1970 10
1.05
0.85
2.45
1.50
2.35
1.20
1970 10
0.45
1.45
2.50
2.70
1.50
1.65
Judgments
0.84
*
*
Judgments
0.79
*
*
*
*
*
Standard
error of
the mean
0.25
0.26
0.30
0.33
0.32
0.24
0.30
0.24
0.31
0.26
0.42
0.42
0.17
0.24
0.41
0.44
0.31
0.17
Fish per
chamber
grams number
335
322
350
348
355
340
361
355
470
372
360
302
342
316
465
449
323
299
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Dissolved
oxygen
(mg/1)
9.4
9.0
9.4
-
-
8.0
8.3
9.6
_
_
8.0
8.4
9.6
pH
-
-
-
-
-
-
-
_
_
-
_
_
/
Off-flavor index based on a scale of 0 (no off-flavor) to 6 (very extreme off-flavor).
The least significant difference at P=0.05 based on a two-way analysis of variance. An asterisk (*)
indicates a statistically significant change in flavor from that of the control sample.
-------
6
5
4
3
2,4-DICHLOROPHENOL
100 PPB
t 6
O
Z 5
LU .
BUTANETHIOL
100 PPB
33.5
168
TIME IN HOURS
Figure 5. The influence of exposure time on the mean off-flavor indices
for trout exposed to three concentrations ("high," "medium,"
and "low") of 2,4-dichlorophenol (Exper. U-7, U-8, and U-9)
and butanethiol (Exper. U-l, U-2, and U-3.}
28
-------
Table 3. Experimental conditions and results of tests in which trout
were exposed to 1, 10, and 100 ppb of 2,4-dichlorophenol for
24 hours, removed, and placed in fresh water for various periods
of time.
Concentration
(ppb)
Clearing
time
(hrs.)
Mean
off -flavor 2/
indexl/ LSD— ^
(0-6) -Ul
Standard
- error of
the mean
Fish per
chamber
grams number
Dissolved
oxygen
(mg/1)
C-l 2,4-Dichlorophenol February 5, 1970
100
100
100
100
100
0.25
1.27
6.5
33.5
168
3.90
3.60
2.40
1.10
1.70
1.18
*
*
0.44
0.42
0.56
0.33
0.34
170
126
154
115
148
1
1
1
1
1
11.7
11.7
11.7
9.9
10.8
C-2 2,4-Dichlorophenol February 5, 1970
10
10
10
10
10
0.25
1.27
6.5
33.5
168
2.40
2.15
1.20
1.40
1.20
n.s.
0.43
0.37
0.27
0.36
0.25
165
176
168
206
238
1
1
1
1
1
10.6
10.6
10.6
8.1
9.6
C-3 2,4-Dichlorophenol
1 0.25
1 1.27
1 6.5
1 33.5
1 168
February 5, 1970
,80
,55
,65
,20
n.s,
1.65
0.40
0.25
0.22
0.23
0.36
137
166
131
155
143
1
1
1
1
1
11.8
11.8
11.8
11.2
12.4
Off-flavor index based on a scale of 0 (no off-flavor) to 6 (very ex-
treme off-flavor). Values based on 10 judgments.
Least significant difference at P=0.05 based on a two-way analysis of
variance. An asterisk (*) indicates a statistical significant change
in flavor.
29
-------
6
X
LU
Q 5
I
U_
IL.
O
LU
0
2,4-DICHLOROPHENOL
100 PPB
IPPB
I
1
0.25
1.27 6.5
TIME IN HOURS
33.5
168
Figure 6. Mean off-flavor indices in relation to the length of time trout were held in fresh water
after 24 hour exposure to 2,4-dichlorophenol concentrations of 100, 10, and 1 ppb
(Exper. C-l, C-2, and C-3).
-------
The results of the experiment on clearing rate revealed that tainting of
the flesh of trout caused by exposure to 2,4-dichlorophenol is eliminated
rather rapidly once the fish is placed in fresh water. Trout appear to
obtain off-flavor from exposure to 2,4-dichlorophenol much more rapidly
than they lose it.
The rates at which taining substances are cleansed from the flesh of fish
may vary substantially. Korschgen, Baldwin, and Robinson (1970) reported
that the flavor of contaminated (tainted) carp failed to improve after
18 days retention in a clean-water pond. They also reported a study by
Leslie E. Whitesel, in which she reported that catfish transferred from
the Ohio River to clean water lost about one half of their off-flavor in
7 days, and nearly all in 21 days. Shumway (1966) reported results simi-
lar to those found in the study reported here in experiments with phenolic
wastes discharged from a plant producing pesticides. The major flavor
impairing substance in the waste was 2,4-dichlorophenol. Much remains
to be learned about the rate fish pick up and lose tainting substances.
Weight of fish per chamber: A series of experiments were conducted to
determine the weight of fish that could be held in the exposure chambers
without influencing the degree of flavor impairment. Experiments were
conducted with p_-chlorophenol, 2,4-dichlorophenol, and pyridine. In
each series of tests, trout of about the same size were selected from
the stock tanks and placed in the exposure chambers. The desired weight
of fish per chamber was attained by varying the number of fish rather
than the size of fish. In tests with p_-chlorophenol (1,000, 100, and
10 ppb) and 2,4-dichlorophenol (100 and 10 ppb) the weight of fish ranged
from about 135 g to somewhat over 1,000 g per chamber; the range of
weights used in the tests with pyridine was about 300 to 1,900 g, although
this varied from test to test. Each chamber held 70 liters of test
solution and was resupplied at a rate of 250 ml/minute. The trout were
exposed for 48 or 96 hours at 15°C. The results of the experiments
described above are presented in Table 4.
The off-flavor indices for trout exposed to the various concentrations
at the three organic compounds tested varied with concentration as
expected, but they did not vary with increasing weight of fish. The
results suggest that as much as 1,000 to 2,000 grams of fish could be
exposed in chambers without concern for the influences such relatively
large weights of fish might have on the degree of flavor impairment.
Dissolved oxygen concentration: A number of experiments were conducted
to determine the influence of moderate reduction of dissolved oxygen
concentration on the flavor-imparting capacity of 2,4-dichlorophenol
and pyridine. Trout were exposed to 100, 10, and 1 ppb of 2,4-
dichlorophenol and 100, 10, and 1 ppm of pyridine at three dissolved
oxygen concentrations ranging from near air-saturation to as low as
3.0 mg/liter in some experiments (Table 5). The trout were exposed for
48 hours at 15°C.
31
-------
Table 4. The experimental conditions and results of test in which different weights of rainbow trout
were exposed to selected concentrations of p_-chlorophenol, 2,4-dichlorophenol, and pyridine
at 15°C.
Exper. Concentration
no
W-l
W-2
W-3
W-4
ppb ppra
p_-Chlorophenol May 18,
10
10
10
10
p-Chlorophenol May 18,
100
100
100
100
Mean
off-flavor
index!/
(0-6)
1971 48-Hr.
1.20
1.20
1.30
0.80
1971 48 -Hr.
1.55
1.55
1.85
2.45
p_-Chlorophenol May 18, 1971 48-Hr.
1,000 2.40
1,000 1.95
1,000 3.50
1,000 3.10
2,4-Dichlorophenol July
10
10
10
10
Fish per
chamber
grams number
Exposure
138 1
258 2
602 4
1037 8
Exposure
132 1
233 2
588 4
1094 8
Exposure
133 1
239 2
513 4
1185 8
9, 1970 96 -Hr. Exposure
1.85 137 1
0.75
0.90
0.70
462 3
844 5
1418 8
„, Standard
LSD— ^j. error of
the mean
n.s. 0.52
0.52
0.31
0.39
n.s. 0.36
0.46
0.44
0.49
0.90 0.40
0.57
* 0.51
0.59
n.s. 0.42
0.28
0.28
0.20
Dissolved
oxygen
(mg/D
8.8
8.6
8.2
6.5
9.8
9.4
7.5
6.7
9.4
9.5
7.8
3.5
9.7
8.3
14.6
13.7
PH
7.7
7.9
7.4
7.7
8.0
7.8
7.8
7.8
8.0
7.7
7.5
7.4
_
_
_
-------
Table 4. Continued
Exper. Concentration
no.
W-5
W-6
W-7
W-8
ppb ppm
Mean
off-flavor
indexi/
(0-6)
2,4-Dichlorophenol July 9, 1970 96-Hr.
100 2.10
100 3.15
100 1.65
100
Pyridine June 3, 1970
1
1
1
Pyridine June 3, 1970
10
10
10
Pyridine June 3, 1970
100
100
100
2.40
48-Hr. Exposure
2.00
1.80
1.90
48-Hr. Exposure
2.30
1.85
1.60
48-Hr. Exposure
2.95
3.15
3.20
Fish per
chamber
g rams
llUlllUSSi
Exposure
133 1
402 3
708 5
1279
315
965
1876
397
1187
1688
309
976
1986
8
1
3
5
1
3
5
1
3
5
-, Standard
LSD— QS error of
the mean
n.s. 0.34
0.33
0.40
0.36
n.s. 0.33
0.40
0.30
n.s. 0.34
0.36
0.30
n.s. 0.35
0.32
0.55
Dissolved
oxygen
(mg/1) pH
9.8
8.9
10.4
11.9
9.2
6.4
4.0
7.3
5.3
4.3
9.7
5.8
3.0
Off-flavor index based on a scale of 0 (no off- flavor) to 6 (very extreme off-flavor).
based on 10 judgments.
Values are
2/
— The least significant difference at P=0.05 based on a two-way analysis of variance. An asterisk (*)
indicates a statistically significant difference in flavor.
-------
Table 5. The results and experimental conditions of tests with rainbow trout held at different dis-
solved oxygen concentrations and exposed for 48 hours concentration of 2,4-dichlorophenol
and pyridine. Water temperatures and pH values were near 15°C and 7.8-8.0, respectively.
04
Exper. Concentration
no
D-l
D-2
D-3
D-4
D-5
ppb ppm
2,4-Dichlorophenol August
10
10
10
2,4-Dichlorophenol August
100
100
100
2,4-Dichlorophenol March
1
1
1
2,4-Dichlorophenol March
10
10
10
2,4-Dichlorophenol March
100
100
100
Mean Mean
off-flavor dissolved
index!/ oxygen
(0-6) (mg/1)
12, 1970
1.75
0.80
0.80
12, 1970
3.85
3.75
2.60
10, 1971
0.55
1.00
0.60
10, 1971
1.85
2.35
1.35
10, 1971
3.60
3.80
3.55
10 Judgments
5.1
6.4
9.9
10 Judgments
5.8
6.0
10.4
10 Judgments
4.12
6.50
10.35
10 Judgments
3.92
6.90
10.40
10 Judgments
3.88
6.20
10.62
_, (Standard
LSD-Q error of
the mean
* 0
0
0.59 0
* 0
* 0
0.62 0
0
0
n.s. 0
0
0
n.s. 0
0
0
n.s. 0
.30
.34
.17
.32
.33
.27
.25
.30
.16
.39
.42
.25
.54
.33
.50
Fish per
chamber
grams
481
350
328
448
361
302
374
311
316
351
355
381
341
398
379
number
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
PH
_
-
-
-
-
-
_
-
_
_
-------
Table 5. Continued
cn
Mean
off-flavor
Exper. Concentration index!/
no. ppb ppm (0-6)
D-6 Pyridine August 15
10
10
10
D-7 Pyridine August 15
100
100
100
D-8 Pyridine March 18,
1
1
1
D-,9 Pyridine March 18,
10
10
10
, 1970 10 Judgments
0.50
1.20
0.70
, 1970 10 Judgments
3.70
2.50
3.40
Mean
dissolved
oxygen
(mg/1)
5.2
6.5
9.7
5.1
6.4
10.7
1971 Trout 11 Judgments
0.68 3.05
0.59
1.09
6.00
9.82
1971 Trout 11 Judgments
0.55 3.17
1.32
0.77
6.05
10.53
„. Standard
LSD— p.- error of
the mean
0.26
0.52
n.s. 0.21
0.44
0.43
n.s. 0.45
0.18
0.20
n.s. 0.52
0.22
0.45
a.s. 0.18
Fish per
chamber
grams
405
348
453
359
339
379
336
381
493
352
368
347
number
1
1
1
1
1
1
2
2
2
2
2
2
-------
Table 5. Continued
Exper.
no.
Concentration
ppb
ppm
Mean
off-flavor
index—
(0-6)
Mean
dissolved
oxygen
(mg/1)
LSDi05
Standard
error of
the mean
Fish per
chamber
gramsnumber
pH
D-10 Pyridine
March 18, 1971
100
100
100
Trout 11 Judgments
4.18 3.06
3.95 5.20
2.82 10.7
n.s.
0.44
0.56
0.58
355 2
370 2
308 2
—' Off-flavor index based on a 0 to 6 scale, with 0 representing no off-flavor and 6 very extreme off-
flavor.
2/
— Least significant difference (P=0.05) based on a two-way analysis of variance. An asterisk (*)
indicates a statistical difference in the flavor.
-------
In general, the results of the experiments with pyridine indicate that
moderately to substantially reduced dissolved oxygen concentrations
will not influence the degree of flavor impairment of the flesh of trout
held at concentrations of 100, 10, and 1 ppm. In some experiments with
2,4-dichlorophenol, however, fairly good correlation was noted between
reduction of dissolved oxygen concentration and the degree of flavor
impairment. In other experiments with 2,4-dichlorophenol, however,
exposure to moderately reduced dissolved oxygen concentrations had
little or no influence on the degree of flavor impairment.
In light of the conclusive results obtained in the experiments described
above with reduced dissolved oxygen concentrations, a firm conclusion
could not be reached concerning the influence of even moderate reductions
of dissolved oxygen on the degree of flavor impairment of the flesh of
trout. Since the dissolved oxygen concentration of the water in the
exposure chamber may influence the results of flavor experiments, and
since trout seem to withstand the stress of handling better at relatively
high dissolved oxygen concentrations, concentration in the exposure
chambers in subsequent testings was he'ld at or near air-saturation.
Influence of pH: The degree of ionization of an electrolyte is affected
by the pH of the media into which it is introduced. If the dissociation
product of a particular contaminant has a greater or lesser capacity to
impart off-flavor than the undissociated chemical, the pH of the
solution will be an important factor in determining the flavor-imparting
capacity of the chemical in question.
A series of experiments were conducted on the influence of pH on the
degree of flavor impairment of trout held at 100 and 10 ppb of 2,4-
dichlorophenol and 100 and 10 ppm of pyridine at "high" (8.8 to 9.3),
"normal" (7.6 to 8.0) and "low" (4.9 to 6.5) pH levels. The experiments
ranged in length from 24 to 96 hours. Sodium hydroxide and sulfuric
acid were used to adjust the pH of the incoming water. The experimental
condition and results of the experiments are presented in Table 6.
In the experiments with pyridine, a weak base, an increase of pH from
"normal" to about 9.3 resulted in little or no change in the degree of
flavor impairment at either test concentration. A decrease in pH from
"normal," however, caused a slight decrease in off-flavor at the 100 ppm
level, but the same decrease in pH caused a slight increase in off-
flavor at 10 ppm (See Figure 7). The divergent results, neither of
which proved significant at the 5 percent level (Table 6), suggest that
pH has no effect on the flavor-imparting capacity of pyridine.
The off-flavor index of trout exposed to 100 ppb of 2,4-dichlorophenol,
a weak acid, decreased with a change of pH from the "low" level to the
"high" level. The length of exposure time (24, 48, and 96 hours) did not
appear to alter the degree of flavor impairment. Changes in pH did
not appear to influence the degree of flavor-impairment at the lower
concentration of 2,4-dichlorophenol (10 ppb). The results of two tests
37
-------
oo
Table 6. Conditions and results of tests in which rainbow trout were exposed to a concentration of
2,4-dichlorophenol or pyridine maintained at a high, intermediate or low pH value and at a
temperature of 15°C.
Exper. Concentration
no. ppb ppm
P-l
P-2
P-3
P-4
P-5
2,4-Dichlorophenol July
10
10
10
2,4-Dichlorophenol July
100
100
100
2,4-Dichlorophenol July
10
10
10
2,4-Dichlorophenol July
100
100
100
2,4-Dichlorophenol July
10
10
10
Mean
off -flavor _,
index!/ Mean LSD^.
(0-6) pH >Ub
15, 1970
0.40
1.45
1.75
15, 1970
1.05
2.95
3.75
15, 1970
1.00
0.50
2.05
15, 1970
1.40
3.40
3.95
20, 1970
0.75
1.00
1.05
24-Hr. Exposure
8.88
7.79 n.s.
6.45
24-Hr. Exposure
8.87 *
7.66 0.74
6.26 *
48 -Hr. Exposure
8.90
8.01 n.s.
6.39
48-Hr. Exposure
8.88 *
7.83 0.92
6.28 *
96-Hr. Exposure
8.92 n.s.
7.93
6.21
Standard
error of
the mean
0.12
0.39
0.32
0.25
0.44
0.40
0.25
0.24
0.46
0.26
0.54
0.36
0.30
0.22
0.27
Fish per
chamber
grams
268
331
293
288
305
303
342
330
305
261
324
376
286
408
267
number
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Dissolved
oxygen
10.0
10.4
11.0
10.4
9.4
10.2
9.8
10.2
10.6
10.0
9.3
9.5
9.8
10.2
10.4
-------
Table 6. Continued
ID
Exper. Concentration
no.
P-6
P-7
P-8
ppb ppm
Mean
off- flavor
indexl/
(0-6)
2,4-Dichlorophenol July 20, 1970 96-Hr.
100 2.15
100
100
Pyridine August 3, 1970
10
10
10
Pyridine August 3, 1970
100
100
100
3.50
2.60
48-Hr. Exposure
0.75
0.75
1.55
48-Hr. Exposure
3.05
2.95
2.25
Mean LSD^/
IT * \JJ
pH
Exposure
8.88 *
7.97 0.87
6.11 *
9.3 n.s.
7.9
4.9
9.3 n.s.
7.8
5.7
Standard
error of
the mean
0.61
0.48
0.45
0.38
0.29
0.58
0.65
0.57
0.47
Fish per
chamber
grams
414
274
387
309
356
449
375
553
274
number
1
1
1
1
1
1
1
1
1
Dissolved
oxygen
9.9
9.7
10.5
10.1
10.2
10.3
10.4
9.3
10.5
— Off-flavor index based on a scale of 0 (no off-flavor) to 6 (very extreme off-flavor). In each
test the number of judgments was 10.
2/
— The least significant difference at P=0.05 based on a two-way analysis of variance. An asterisk
(*) indicates a statistically significant change in flavor.
-------
6
5
4
3
X 2
LU
Q
o:
1°
PYRIDINE
5.0
100 PPM
6.0
7.0
8.0
-O
-A
9.0
t 6
$ 4
3
2
0
2,4-DlCHLOROPHENOL
100 PPM
pH
Figure 7. The relation between mean off-flavor indices and the pH at
which trout were held and exposed to 100 and 10 ppm of
pyridine (Exper. P-7 and P-8) and 100 and 10 ppb of 2,4-dichlorophenol
(Exper. P-3 and P-4).
40
-------
with 2,4-dichlorophenol are presented graphically in Figure 7.
2,4-dichlorophenol, which is quite soluble in fat, partially dissociates
to 2,4-dichlorophenate, a compound readily soluble in water, but nearly
insoluble in fat. As the dissociation equilibrium shifts with increasing
pH, more^2,4-dichlorophenate is formed and less 2,4-dichlorophenol
remains in the solution for the fish to remove and concentrate in their
tissue.
pH is known to influence the toxicity of a number of compounds. The
toxicity of other material is unaltered by change in the pH. The same
appears to be true with the effect of pH on the flavor-imparting capacity
of organic compounds. In laboratory experiments designed to determine
threshold concentrations for compounds, the pH probably should be main-
tained between 7.0 and 8.0. Where the impact of a contaminant on a
particular body of water is under study, the pH of the receiving water
should be used.
Influence of organic compounds on flavor: Once a standard procedure
was selected for use, examination of the flavor imparting capacity of a
number of organic compounds and effluents could commence. Rainbow
trout and in a few cases, largemouth bass and bluegill, were exposed to
a range of concentration of a compound and a control for 48 hours. The
pH was maintained between 7.0 and 8.0, and the water temperature was held
at 15° C for trout and 20° C for bluegill and bass. Since most of the
contaminants tested had not been previously examined for their tainting
characteristic, the ranges of concentrations used were often selected
quite arbitrarily. In some cases, available toxicity data allowed the
setting of the upper limit of the range tested.
Presented in Table 7 are the experimental conditions and results of the
tests conducted with organic compounds. Also presented in Table 7 are
the statistical evaluations of the results of each test and other per-
tinent information. As may be seen in Table 7, experiments with many
organic compounds had to be repeated in order to obtain meaningful
results. Other experiments were duplicated using either bass or bluegill
as the test fish.
The number of judges used to evaluate the samples of fish proved to be
an important factor in obtaining meaningful results. Initially, the
flavor panels were comprised of 8 to 10 judges. This was later changed
to a minimum of fifteen judges. Because of the subjective nature of the
tests, the larger panels proved far more satisfactory than did the small
panels.
Paper processing effluents: Biologically stabilized effluents were
obtained from paper mills located near Halsey, Albany, and Lebanon,
Oregon, and evaluated for their flavor-imparting capacity. The experi-
mental conditions and results of the experiment are presented in Table 8.
41
-------
Table 7 . Experimental conditions and results of tests in which fish were exposed to various
concentrations of chemicals for 48 hours.
Exposure
Exper. concentration
No.
T-l
T-2
T-3
ppb ppm
Mean
off-flay,
index-^-
(0-6)
Acetone June 29, 1971 Trout
0 0.82
1
10
100
1,000
10,000
Acrylonitrile March
0
0.1
1.0
10
100
Acrylonitrile April
0
0.32
0.56
3.2
5.6
32.0
0.82
1.91
0.77
0.68
+
3, 1971
1.90
1.00
0.90
2.00
+
21, 1971
0.41
2.32
0.77
0.55
0.95
1.41
?r _, Standard
LSD— ^._ error of
the mean
11 Judgments
n.s. 0.22
0.22
0.50
0.24
0.23
Trout 10 Judgments
n.s. 0.49
0.31
0.31
0.50
-
Trout 11 Judgments
0.70 0.15
* 0.50
0.26
0.21
0.18
* 0.25
Fish per
chamber
grams
335
294
284
340
337
-
240
294
329
317
-
254
332
346
270
313
215
number
2
2
2
2
2
-
2
2
2
2
2
2
2
2
2
2
2
Mean .. Dissolved
temp.— oxygen
(C) (nig/1)
- _
-
_
-
-
11.0
10.0
10.2
10.0
10.2
9.0
9.9
10.0
10.6
10.0
10.1
pH
7.6
7.7
7.6
7.7
7.7
-
7.9
7.9
7.9
7.9
7.9
7.9
7.9
7.8
7.8
7.9
8.0
56.0
-------
Table 7
Continued
Mean
Exposure off-flavor
Exper. concentration index-^—
No.
T-4
T-5
T-6
T-7
PPb
ppm _
Amyl Acetate May 26
0
0
0.1
1
10
100
1,000
10,000
Aniline
Benzene
December 30,
0
0.1
1
10
100
February,?,
0
0.01
0.056
0.10
.56
1.00
5.60
n-Butanol September
0
0.1
1
10
100
(0-6)
. _ , Standard
' LSD05 error o£
the mean
Fish per Mean . ,
chamber temp.—
grams
number (C)
Dissolved
oxygen
(fflg/1)
PH
, 1971 Trout 11 Judgments
0.
1.
0.
0.
0.
0.
0.
1.
1970
0.
0.
0.
1.
1972
1.
0.
1.
0.
0.
1.
0.
36
04
41
59
91
86
95
36
n.s. 0
0
0
0
0
0
0
0
.14
.24
.16
.30
.31
.30
.18
.34
393
380
289
254
246
237
266
264
3
3
2
2
2
2
2
2
9
9
9
9
8
9
8
8
.4
.6
.2
.5
.6
.2
.7
.7
7.6
7.8
8.1
7.9
7.9
7.8
7.8
7.7
Trout 8 Judgments
69
88
69
63
+
Trout
06
62
53
75
56
47
78
13, 1971
0.
0.
0.
0.
0.
17
54
42
25
54
n.s . 0
0
0
0
16 Judgments
n.s. 0
0
0
0
0
0
0
.27
.25
.23
.25
-
.27
.13
.29
.17
.16
.30
.20
534
433
4 04
454
-
579
620
697
543
623
690
640
2
2
2
2
2
5
5
5
5
5
5
5
8.9
9.
10,
9.
10.
10.
9.
9.
8.
6.
7.
9.
,1
,0
8
5
4
0
4
8
0
6
8
-
_
_
-
7.7
7.6
7.9
7.8
7.9
7.5
7.6
Bluegill 12 Judgments
n.s. 0
0
0
0
0
.09
.23
.17
.12
.18
745
648
562
753
808
9
8
6
8
9
8.
8.
7.
10.
11.
3
5
7
2
0
7.3
7.4
7.1
7.2
7.2
-------
Table 7
Continued
Exposure
Exper. concentration
no.
T-8
T-9
T-10
T-ll
ppb ppra
Cresol September 20
0
0.005
0.05
0.5
5.0
Cresol September 28
0
0.005
0.05
0.5
5.0
m- Cresol September
0
0.005
0.05
0.5
5.0
m-Cresol September
0
0.005
0.05
0.5
5.0
Mean
off -flavor.
indexi£=-'
, 1971 Trout
0.70
0.80
0.83
2.33
4.33
, 1971 Trout
0.47
0.90
1.30
2.33
4.90
8, 1971 Trout
0.82
0.75
0.78
2.12
3.77
8, 1971 Trout
0.70
0.70
0.93
1.90
3.93
^i
Standard
error of
the mean
15 Judgments
0.71 0.21
*
*
0.27
0.21
0.33
0.31
15 Judgments
0.73 0.14
*
*
*
0.29
0.24
0.37
0.26
20 Judgments
0.63 0.18
*
*
0.22
0.13
0.33
0.31
15 Judgments
0.77 0.22
*
*
0.14
0.22
0.41
0.32
Fish per
chamber
grams
759
737
817
777
760
721
834
819
797
699
673
768
652
870
946
756
794
799
896
811
number
5
5
5
5
4
5
5
5
5
4
5
5
5
5
5
5
5
5
5
5
Mean . , Dissolved
temp.— oxygen
(C) (mg/1)
9.7
9.0
9.4
10.8
10.0
5.9
5.6
6.6
6.6
6.4
8.3
7.1
8.7
8.7
7.0
7.4
7.8
7.9
8.1
7.7
pH
7.2
7.1
7.2
7.1
7.0
7.2
7.2
7.4
7.3
7.3
7.4
7.3
7.4
7.1
7.2
7.3
7.3
7.2
7.2
7.0
-------
Table 7
Continued
Mean
Exposure off-flavor _,
Exper. concentration index!/!/ LSD^
no . ppb
ppm
(0-6)
T-12 £-Cresol September 20,
0 0
0.005 0
0.05 0
0.50 2
5.0
1971
.56
.87
.84
.91
T-13 p_-Cresol September 20, 1971
0
0.005
0.05
0.50
5.0
T-14 Dimethylamine August
T-15 Ethanethiol
0
1
10
100
1,000
T-16 Ethanethiol
1
0
0.56
5.6
56
October
October
0
1
10
100
,000
0
2
0
2
4,
0
0
0
1
5,
1
0
1
0
2
5,
3
0
0
1
2
.47
.17
.90
.20
+
1971
.08
.71
.66
.66
1971
.03
.70
.63
.73
.43
1971
.00
.97
.90
.37
.53
Trout 16
0.82
*
Trout 15
0.81
*
*
Trout 12
0.59
*
*
Trout 15
0.75
*
Trout 15
0.79
*
*
*
Standard
error of
the mean
Judgments
0.13
0.21
0.39
0.46
Judgments
0.12
0.30
0.37
0.40
-
Judgments
0.06
0.25
0.26
0.30
Judgments
0.29
0.17
0.38
0.22
0.43
Judgments
0.30
0.29
0.26
0.41
0.28
Fish per Mean . ,
chamber temp.—
grams
661
1067
898
727
854
788
745
1082
-
662
675
666
645
914
785
980
899
902
887
894
676
821
915
number (C)
4
5
5
4
5
5
5
5
-
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Dissolved
oxygen
(mg/D
9.9
9.3
9.6
9.4
9.1
8.9
10.
9.
6.
7.
8.
8.
6.
7.
7.
5.
5.
4.
5.
6.
5.
6.
3
5
9
7
9
0
1
9
1
7
9
9
1
7
5
7
pH
7
7
7
7
.5
.3
.4
.2
7.2
7.1
7.3
7.2
7.
7.
7.
9.
7.
7.
7.
7.
6.
7.
7.
7.
7.
7.
6
4
8
2
2
5
4
3
9
4
3
3
1
1
-------
Table 7. Continued
Exper .
no.
T-17
T-18
T-19
T-20
T-21
Mean
Exposure off -flavor _,
concentration indexlZ?/ LSD^r>
ppb ppm (0-6}
Ethylacrylate August 24, 1971 Trout 15
0 0.93 n.s.
1 1.13
10 1.20
100 0.87
Ethylacrylate August 24, 1971 Trout 21
0 0.71 0.53
1 0.83
10 0.69
100 1.81 *
Ethylacrylate September 28, 1971 Trout
0 0.81 0.68
0.01 0.86
0.1 0.83
1 1.17
10 3.25 *
Ethylacrylate September 28, 1971 Trout
0 1.24 0.65
0.01 1.06
0.10 1.00
1 1.97 *
10 3.15 *
Standard
error of
the mean
Judgments
0.24
0.31
0.45
0.19
Judgments
0.17
0.16
0.14
0.27
18 Judgments
0.22
0.18
0.22
0.24
0.38
17 Judgments
0.31
0.25
0.24
0.29
0.37
Fish per
chamber
grams
732
692
644
827
741
703
724
810
676
671
742
797
359
676
671
742
797
359
number
5
5
5
5
5
5
5
5
5
5
4
5
2
5
4
4
5
2
Mean .. Dissolved
temp.— oxygen
(C) (mg/1)
15.8 10.2
15.8 11.0
15.9 12.5
15.8 11.4
15.0
11.8
16.6
12.3
7.0
6.4
7.0
5.6
6.8
7.0
6.4
7.0
5.6
6.8
PH
7.5
7.6
7.6
7.4
7.6
7.6
7.5
7.7
7.3
7.4
7.6
7.5
7.2
7.3
7.4
7.6
7.5
7.2
Formaldehyde April 28, 1971 Trout 10 Judgments
0 0.75 n.s.
0.10 2.10
0.56 1.15
1.0 1.05
0.33
0.48
0.34
0.24
438
315
477
287
3
2
3
2
7.3
10.0
10.4
10.0
7.8
7.9
8.1
8.0
-------
Table 7. Continued
Exper
no.
T-22
T-23
T-24
T-25
Exposure
concentration
ppb ppm
Mean
off-flavor. . Standard
index-i^7 LSD^ error of
(0-6) ' the mean
Formaldehyde April 28, 1971
0
5.6
10
56
100
Formaldehyde May 12
0
32
100
320
B, 3'-dichlorodiethyl
0
.09
0.9
9
91
Methylamine January
0
0.001
0.01
0.10
1
10
0.55
0.80
0.55
0.50
2.05
, 1971
0.50
0.75
1.00
+
ether
0.40
0.45
1.40
2.90
+
5, 1971
1.06
1.00
1.50
1.06
1.38
1.69
Trout 10 Judgments
0.69 0.24
0.17
0.23
0.21
* 0.40
Trout 10 Judgments
n.s. 0.26
0.25
0.31
-
March 22, 1971 Trout
0.96 0.15
0.17
* 0.41
* 0.50
-
Trout 8 Judgments
n.s. 0.29
0.34
0.37
0.49
0.34
0.35
i Fish per Mean .
• chamber temp.—
grams number (C)
254
221
352
395
299
528
299
314
-
10 Judgments
547
351
396
362
-
558
355
390
505
556
473
2
2
2
3
o ™*
2
1
1
-
3
2
2
2
2
2
2
2
2
2
2
, Dissolved
oxygen
(mg/1)
9.2
9.8
9.7
9.4
9.9
9.2
10.3
10.2
-
8.6
9.3
8.1
9.3
9.8
8.2
9.4
10.1
10.2
10.0
10.6
PH
7.7
7.8
7.8
8.0
8.1
7.8
7.9
8.0
-
7.9
7.8
8.0
7.8
8.0
7.9
7.9
7.9
7.9
8.0
8.4
-------
Table 7.
Continued
oo
Mean
Exposure off-flavor
Exper. concentration indei£=/ LS
no.
T-26
T-27
T-28
T-29
ppb ppm (0-6)
Methylamine March 3, 1971 Trout
0 0.73 n
0.056 1.45
0.56 1.50
5.60 1.59
56.0 +
2-Ethyl-l-hexanol February 8, 1972
0 1.21 n
0.0056 0.83
0.056 0.83
0.56 1.17
5.6 1.21
2-Napthol February 21, 1972 Trout
0 1.20 0
0.01 0.63
0.032 1.33
0.10 0.87
0.32 1.53
1.0 2.40
3.2 +
Sodium pentachlorophenate November
0 1.03 n
0.2 0.97
2 0.96
20 0.83
200 +
_, Standard
D^ error of
the mean
11 Judgments
.s. 0.24
0.46
0.42
0.31
-
Trout 12 Judgments
.s. 0.43
0.24
0.29
0.29
0.27
15 Judgments
.68 0.40
0.21
0.21
0.25
0.45
* 0.52
22, 1971 Trout 15
.s. 0.22
0.19
0.19
0.26
-
Fish per Mean . ,
chamber temp.—
grams number (C)
321
326
348
315
-
713
736
781
870
668
644
841
620
693
961
584
-
Judgments
601
537
525
600
-
2
2
2
2
-
5
5
5
5 ^
O *"
5
5
5
5
5
5
-
6 15.0
5 15.6
5 15.6
5 15.6
-
Dissolved
oxygen
(mg/1)
9.0
9.2
10.0
10.0
10.0
10.2
10.4
10.4
9.6
9.6
11.6
11.4
10.6
10.4
10.2
11.4
-
7.8
7.0
6.6
5.4
5.2
PH
7.8
7.8
7.8
8.2
9.5
7.8
7.8
7.7
7.9
7.4
7.8
7.8
7.9
8.0
7.7
7.7
-
7.5
7.4
7.5
7.3
7.2
-------
Table 7, Continued
Exposure oi
Exper. concentration :
no. ppb ppm |
T-30 Phenol March 23, 1971
0
1.0
10
100
T-31 Phenol June 23, 1971
0
0.10
0.18
0.56
1.00
1.80
5.60
T-32 Phenol April 14, 1971
0
0.01
0.1
0.32
1.0
3.2
10.0
32
Mean
:f -flavor Standard
tndexi/-^ LSD^ error of
:0-6) >ui> the mean
Trout 10 Judgments
0.95 n.s.
1.55
1.30
0.90
Trout 12 Judgments
0.58 n.s.
0.46
1.33
1.08
1.29
0.75
1.08
Trout 10 Judgments
0.95 n.s.
0,35
0.55
0.80
0.50
1.05
+
+
T-33 2,3-Dichlorophenol March 1, 1972 Trout 15
0
0.01
0.032
0.10
0.32
1.0
0.63 0.64
0.70
0.77
1.43 *
3.20 *
5.13 *
0.22
0.66
0.41
0.29
0.32
0.18
0.49
0.31
0.27
0.20
0.45
0.19
0.13
0.20
0.32
0.26
0.25
-
-
Judgments
0.25
0.18
0.21
0.27
0.41
0.24
Fish per Mean ., Dissolved
chamber temp.— oxygen
grams
331
410
369
366
454
142
324
273
289
335
302
400
238
285
322
288
315
-
-
792
654
786
831
631
714
number (C)
2
2
2
2
2
1
2
2
2
2
2
3
2
2
2
2
2
_
-
5
5
5
5
5
5
(mg/1)
8.2
8.9
9.9
9.5
9.4
9.8
9.5
9.3
9.7
9.1
9.4
9.5
10.4
10.2
11.2
10.3
10.2
_
-
11.4
10.2
9.2
10.0
10.0
10.8
pH
7.9
7.7
7.7
8.0
7.9
8.1
7.8
8.0
7.8
7.9
8.0
7.9
7.5
8.1
8.0
7.9
7.7
_
-
7.7
7.6
7.7
7.4
7.5
7.5
-------
Table 7.
Continued
en
o
Exper
no.
T-34
T-35
T-36
T-37
Exposure
concentration
ppb ppm
2,4-Dichlorophenol
0
0.01
0.1
1
10
100
2 , 4-Dichlorophenol
0
0.10
1
10
100
2,5-Dichlorophenol
0
1
10
100
1,000
2 , 6-Dichlorophenol
0
1
10
100
1,000
Mean
off-flav
(0-6)
August 26
0.40
1.55
0.90
2.10
1.75
3.75
July 21,
0.55
0.50
0.55
1.00
3.50
February
0.93
0.90
1.13
2.80
4.83
February
0.73
0.47
0.80
2.03
4.0
27 37 st
1 LSDfs er
'Ub th
, 1970 Bass 10
0.88
*
*
*
*
1971 Bluegill
0.71
*
28, 1972 Trout
0.76
*
*
15, 1972 Trout
0.74
*
*
andard
ror of
Fish per
chamber
e mean grams number
Judgments
0.19
0.29
0.39
0.35
0.36
0.32
10 Judgments
0.24
0.21
0.22
0.34
0.38
802
546
509
477
472
520
446
478
446
401
419
15 Judgments
0.29 714
0.26
0.24
0.39
0.29
663
658
836
768
15 Judgments
0.21 670
0.12
0.19
0.33
0.44
651
544
608
602
2
2
2
2
2
2
6
6
6
6
6
6
5
5
5
5
5
5
5
5
5
Mean
temp
(C)
19.
19.
19.
19.
19.
18.
19.
19.
19.
19.
19.
-
-
-
-
-
-
_
-
;i/
8
8
8
7
3
7
8
9
9
8
9
Dissolved
oxygen
(mg/1)
8.
8.
9.
8.
9.
8.
8.
8.
8.
8.
8.
10.
10.
10.
10.
9.
9.
8.
9.
9.
5.
0
8
0
8
0
0
6
5
1
5
4
0
6
4
0
6
2
4
0
4
0
pH
-
-
-
-
-
7.8
7.8
7.8
7.9
7.9
7.5
7.7
7.6
7.6
7.4
7.6
7.7
7.8
7.6
7.5
-------
Table 7. Continued
Mean
Exposure off -flavor , .
Exper. concentration indexi^* LSD^
no.
T-38
T-39
T-40
T-41
ppb ppm (0-6)
m- Ch 1 oropheno 1
0
1
JL
10
XV
100
JL ^/ \^
1.000
J> » ^/ ^/ \f
1 r\ f\f\f\
10,000
m-Chlorophenol
0
1
10
100
1,000
o - Ch 1 or oph eno 1
0
1
10
100
1,000
o-Chlorophenol
0
1
10
100
1,000
April 21, 1971
1.15
1.50
0.90
0.80
2.15
+
August 17, 1971
0.50
0.92
0.50
0.23
0.62
August 11, 1971
2.23
0.77
0.15
1.23
5.08
August 11, 1971
1.30
0.37
0.57
1.77
4.56
Standard
error of
the mean
Fish per Mean ..
chamber temp.—
grams
number (C)
Dissolved
oxygen
pH
Trout 10 Judgments
n.s.
Bluegill
n.s.
Trout 13
0.78
*
*
*
*
Trout 15
0.65
*
*
*
0.45
0.35
0.23
0.24
0.53
13 Judgments
0.23
0.31
0.15
0.11
0.23
Judgments
0.41
0.32
0.09
0.21
0.33
Judgments
0.38
0.14
0.17
0.34
0.28
217
303
262
287
257
292
364
412
454
531
811
716
714
754
742
725
753
856
751
766
2
2
2
2
2
-
6 19.7
5 19.6
6 19.6
8 19.4
7 19.1
5
5
5
5
5
5
5
5
5
5
10.2
9.5
10.4
10.1
10.2
8.8
8.7
8.6
9.2
10.0
8.6
8.1
8.1
9.4
7.7
7.8
8.1
7.9
8.3
6.7
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
7.7
7.8
8.0
7.9
7.6
7.6
7.9
7.7
7.7
7.5
-------
Table 7. Continued
tn
Mean
Exposure off -flavor _,
Exper. concentration index!/.?/ LSD— ^P
no . ppb
T-42 p_-Chlorophenol
0
0.1
1
10
100
1,000
T-43 p_-Chlorophenol
0
2.1
4.5
21
45
210
450
T-44 p_-Chlorophenol
0
1
10
100
1,000
T-45 o-Phenylphenol
ppm (0-6)
February 10,
0.95
0.70
0.90
0.95
2.15
4.75
February 24,
0.70
0.85
0.75
1.10
1.85
2.30
3.60
July 14, 1971
1.0
0.55
1.45
1.25
2.85
September 8,
0 0.76
0.001 1.26
0.01 0.61
0.1 0.71
1 1.74
1971 Trout
0.69
*
*
1971 Trout
0.87
*
*
*
Bluegill
n.s.
1971 Trout
n. s.
Standard
error of
the mean
10 Judgments
0.24
0.27
0.32
0.27
0.27
0.31
10 Judgments
0.37
0.27
0.24
0.29
0.43
0.42
0.36
10 Judgments
0.41
0.22
0.38
0.43
0.42
17 Judgments
0.24
0.28
0.21
0.23
0.32
Fish per
chamber
grams number
614
559
446
751
657
500
293
250
344
286
369
312
304
457
269
501
502
453
608
830
728
834
813
2
2
2
2
2
2
2
2
2
2
2
2
2
6
6
6
6
6
5
5
5
5
5
Mean .. Dissolved
temp.— oxygen
(C) (mg/1)
8.3
7.7
10.6
9.8
9.6
10.0
10.4
10.7
11.5
10.9
11.0
11.0
10.0
19.6 7.9
19.6 7.8
19.6 8.1
19.7 8.0
19.6 7.8
10.3
6.6
8.3
8.0
7.0
pH
7.8
7.9
8.0
7.8
7.8
7.9
7.8
7.9
8.0
7.9
7.8
7.8
7.9
7.8
7.7
7.8
7.8
7.7
7.0
7.0
7.1
7.1
7.0
-------
Table 7. Continued
tn
Exposure
Exper. concentration
no. ppb ppm
Mean
off-flavor ,,
index!/!/ LSD^/
(0-6) 'Ub
Standard Fish per Mean 4 . Dissolved
error of chamber temp.— oxygen
the mean grams
T-46 o-Phenylphenol September 13, 1971 Trout 15 Judgments
0 1.20 n.s. 0.37 701
0.001
0.01
0.1
1
T-47 2,4,5-Trichlorophenol
0
0.1
1
32
100
T-48 2,4,5-Trichlorophenol
0
0.01
3.20
10
320
1,000
0.87
0.63
1.13
1.83
June 16, 1971 Trout
0.50 n.s.
0.70
0.70
0.50
0.70
June 16, 1971 Trout
0.55 n.s.
0.75
0.45
0.75
1.30
+
0.25 622
0.18 761
0.15 797
0.36 807
10 Judgments
0.21 480
0.26 305
0.30 291
0.25 432
0.25 272
10 Judgments
0.16 425
0.19 363
0.17 295
0.24 323
0.27 322
-
number (C)
5
5
5
5
5
3
2
2
2
2
2
2
2
2
2
-
(rng/1) pH
9.2 7.3
9.1 7.1
11.4 7.2
12.1 7.0
8.4 6.8
7.7
7.8
7.9
7.8
7.9
7.7
8.0
7.8
8.0
7.7
-
-------
Table 7. Continued
tn
Mean
Exposure off-flavor
Exper. concentration index1 ' ' \
no . ppb
ppm
(0-6)
_ , Standard
LSEP-Q5 error of
the mean
Fish per Mean ..
chamber temp.—
grams number (C)
T-49 2,4,6-Trichlorophenol May 12, 1971 Trout 10 Judgments
0 0.80 0.69 0.31 473
0.1
1
10
100
1,000
T-50 Pyridine August 3, 1971
0
T-51 Pyrocatechol
•1
10
100
January 26
0
0.32
1.0
3.2
10
32
0.60
0.50
1.00
1.60
3.50
Bluegill
0.90
0.40
0.65
3.60
0.31
0.15
0.24
* 0.31
* 0.34
10 Judgments
0.95 0.30
0.16
0.21
* 0.53
, 1972 Trout 15 Judgments
0.80 0.68 0.18
0.93
2.07
2.13
+
+
0.21
* 0.33
* 0.32
-
-
290
273
307
262
284
525
550
503
746
566
431
628
461
-
-
2
1
1
1
1
1
6 19.7
6 19.5
6 19.6
6 19.6
6
5
6
5
-
_
Dissolved
oxygen
Cmg/1)
9.2
9.3
9.4
9.8
9.8
9.8
8.0
8.2
8.9
8.8
7.4
13.4
8.0
13.8
-
_
PH
7.7
7.9
8.0
8.1
8.0
7.9
7.8
7.9
7.9
7.9
7.3
7.2
7.3
7.1
-
_
-------
Table 7. Continued
Exper
no.
T-52
Exposure
concentration
ppb ppm
Guaiacol April 25,
0
0.01
0.1
1
10
Mean
off-flavor
(0-6)
1972 Trout
0.83
1.40
1.00
1.90
4.10
_ , Standard
LSD— Q5 error of
the mean
15 Judgments
0.78 0.27
0.27
0.20
* 0.38
* 0.28
Fish per Mean .
chamber temp.— ;
grams
650
680
720
670
705
number (C)
5
5
5
5
5
f Dissolved
oxygen
(mg/1)
10.5
9.8
10.2
10.4
9.9
PH
7.8
7.6
7.7
7.9
7.8
Cn
tn
— Off-flavor index based on a scale of 0 (no off-flavor) to 6 (very extreme off-flavor)
21
— A plus sign (+) indicates all test fish died.
3/
—' The least significant difference at P=0.05 based on a two way analysis of variance. An asterisk
(*) indicates a statistically significant change in flavor from that of the control sample.
— Mean temperatures for an experiment are not listed when values were between 14.5 and 15.5°C.
-------
Table 8. Experimental conditions and results of tests in which rainbow trout were exposed for 48
hours to various concentrations of biologically stabilized waste from paper plants located
in Oregon.
tn
Mean
Waste off -flavor ,
Exper concentration index-=^-=/ LSD— {.,.
no. (% by vol.) (0-6)
E-l Kraft process, Albany December 7,
0
1.4
5.6
16.7
32.8
59.8
100
E-2 Sulfite process
0
1.1
20.7
33.5
50.7
66.5
E-3 Kraft process,
0
9.2
50.1
100
0.83
0.60
0.87
2.20
3.43
4.43
+
, Lebanon January
0.83
0.50
1.27
0.83
1.67
2.33
Halsey December 18
0.20
1.27
4.23
5.13
1971 15
0.70
*
*
*
12, 1972
0.57
*
*
, 1971
0.55
*
*
*
Standard
error of
Fish per
chamber
the mean grams
Judgments
0.17
0.17
0.19
0.40
0.43
0.37
-
15 Judgments
0.22
0.15
0.29
0.19
0.28
0.36
15 Judgments
0.08
0.27
0.29
0.28
652
703
684
677
597
641
-
640
471
653
427
659
664
538
473
494
410
number
5
5
5
5
5
5
6
5
5
5
5
5
5
5
5
4
Mean . , Dissolved
temp.— oxygen
(C) (mg/1) pH
15.2 7.5
13.0 7.7
10.2 7.4
7.8 7.6
7.4 7.2
6.0 7.1
6.0 7.2
7.3
7.3
7.3
7.3
7.1
6.9
14.9 - 7.3
14.8 - 7.4
14.0 - 7.3
13.1 - 7.4
-------
Table 8. Continued
Mean
Waste off-flavor .
Exper. concentration index-=^-=-' LSD-y._
no. (% by vol.) (0-6) '
E-4
Kraft process, Halsey,
0
1.3
8.1
20.3
33.0
50.0
65.9
January 18, 1972
0.80 0.68
1.07
1.20
2.13 *
3.80 *
4.47 *
4.93 *
Standard
error of
the mean
15 Judgments
0.20
0.18
0.33
0.29
0.38
0.34
0.30
Fish per Mean ^ ,
chamber temp.—
grams
735
648
463
566
597
604
524
number (C)
6
5
5
5
5
5
5
Dissolved
oxygen
(mg/1)
11.1
10.9
10.0
10.4
9.7
-
_
pH
7.3
7.6
7.3
7.2
7.1
7.1
7.2
C/l
Off-flavor index based on a scale of 0 (no off-flavor) to 6 (very extreme off-flavor).
21
— A plus sign (+) indicates all test fish died.
3/
— The least significant difference at P=0.05 based on a two-way analysis of variance. An asterisk
(*) indicates a statistically significant change in flavor from that of the control sample.
— Mean temperatures for an experiment are not listed when values were between 14.5 and 15.5°C.
-------
Biologically stabilized kraft mill effluent (KME) was found to produce
extreme high off-flavor at cpncentrations of 50 percent by volume and
above. At concentrations between 50 and 16 percent by volume, the off-
flavor indices were moderately high and ranged between 2.13 and 3.80.
Interestingly enough, the effluent from the mill at Albany, which is a
very old mill, produced nearly the same relationships between off-flavor
and KME concentration as did effluents from the newly constructed mill
at Halsey. The Halsey mill also uses about 3.5 times as much water per
ton of pulp as does the Albany mill.
Shumway and Chadwick (1971) reported that untreated KME from the Albany
mill caused impaired flavor in salmon at concentrations of about 1.0 to
2.0 percent by volume and above. They reported treated (biologically
stabilized) KME caused no flavor impairment at concentrations of about
3.0 percent by volume. In the study reported here concentrations of
stabilized KME of 1.0 to 8.1 percent by volume failed to impair flavor
of trout.
Biologically stabilized effluent from an ammonia-base, sulfite mill
located in Lebanon, Oregon, was found to impair the flavor of trout at
concentrations of 50.7 and 66.5 percent by volume. At lower concentra-
tions of the effluent (1.0, 20.7 and 33.7 percent by volume), no
impairment of the flavor of trout was noted. The results of the tests
with effluent from the sulfite mill are presented in Table 8.
Treated waste water (domestic): Experimental conditions and results of
experiments with primary, secondary, and secondary chlorinated treated
effluents from the Corvallis Sewage Treatment Facilities are shown in
Table 9. Two experiments were conducted with primary and secondary
effluents and three experiments with secondary chlorinated effluent.
The effluents were provided fresh each day during the experiments.
In general, waste water that received only primary treatment was fairly
toxic and impaired flavor of trout at concentrations of about 16 per-
cent by volume and above. Secondary treated (trickling filter) waste
water was non-toxic at concentrations of 100 percent by volume and pro-
duced impaired flavor in trout exposed at concentrations of 15 percent
by volume and above. The addition of chlorine to the secondary treated
waste water appeared to reduce the flavor-imparting capacity of the
effluent. Only chlorinated secondary effluent concentrations of 20 per-
cent by volume and above produced tainted fish. In one experiment with
secondary chlorinated effluent (S-5), no flavor impairment occurred at
33 percent by volume and the fish held at higher concentrations (60 and
100 percent by volume) all died.
Discharges from waste water treatment facilities contribute tremendous
volumes of treated water to lakes and streams across the nation. The
data presented in this report strongly suggest that these discharges are
potential hazards to the quality of the fish inhabiting the waters into
which they are discharged. Secondary treatment alone does not appear to
substantially reduce the flavor-imparting capacity of primary treated
58
-------
Table 9. Experimental conditions and results of tests in which rainbow trout were exposed for 48
hours to various concentrations of primary and secondary treated waste from the Corvallis,
Oregon Municipal Sewage Treatment Plant.
tn
to
Waste
Exper. concentration
no. (% by vol.)
S-l
S-2
S-3
Primary treatment
0
3
7
16
24
34
50
100
Primary treatment
0
4
7
25
SO
67
100
Mean
off -flavor ,, Standard
index-i^-' LSEP/ error of
(0-6) ' the mean
October 13, 1971
0.81 0
1.12
0.84
2.09
+
2.44
+
+
December 8, 1971
0.83 0
1.13
1.46
2.63
3.50
4.43
+
Secondary treatment October 18, 1971
0 0.90 0
5
6
15
32
60
100
0.90
1.03
1.57
1.43
3.00
4.30
16 Judgments
.62 0.21
0.39
0.27
* 0.39
-
* 0.33
-
-
15 Judgments
.70 0.17
0.20
0.24
* 0.33
* 0.47
* 0.37
-
15 Judgments
.74 0.22
0.30
0.30
0.37
0.30
* 0.35
* 0.37
Fish per Mean .
chamber temp.—
grams
1086
989
1037
1052
-
1148
-
-
406
411
447
485
416
434
-
962
1000
1072
1211
1054
959
1107
number (C)
5
5
5
5
_
5
-
-
5
5
5
5
5
5
-
4
5
5
5
5
5
O "•
, Dissolved
oxygen
(mg/1)
7.8
9.0
8.5
6.2
6.9
9.8
-
-
10.8
16.4
10.6
10.8
14.8
12.6
10.4
6.0
14.8
12.0
9.8
14.2
6.8
11.1
PH
6.8
7.3
6.8
7.1
6.7
7.0
7.0
7.0
7.5
7.5
7.6
7.5
7.5
7.4
7.5
7.2
7.3
7.4
7.3
7.3
7.3
7.2
-------
Table 9. Continued
ON
O
Waste
Exper. concentration
no. (% by vol.)
S-4 Secondary treatment
0
1
7.4
14.9
32.8
60.3
100
S-5 Secondary treatment,
0
1
7
10
17
25
33
60
100
S-6 Secondary treatment,
0
1
10
17
20
35
Mean
off-flavor
(0-6)
December 29,
0.78
0.81
0.62
0.69
2.44
3.43
4.06
chlorinated
1.19
0.63
0.83
0.94
0.84
1.52
1.72
+
+
chlorinated N
1.27
0.63
0.83
1.83
1.70
2.47
_ , Standard Fish per Mean .,
LSD— dp error of chamber temp.—
Dissolved
oxygen
the mean grams number (C) (mg/1)
1971 16 Judgments
0.64 0.18
0.18
0.15
0.18
* 0.27
* 0.32
* 0.33
October 22, 1971
n.s. 0.21
0.23
0.30
0.25
0.19
0.27
0.34
-
-
ovember 30, 197 1 15
0.66 0.30
0.22
0,22
0.38
0.35
* 0.34
555
672
496
606
638
664
774
16 Judgments
1171
1102
1241
981
1215
718
478
-
-
Judgments
642
561
513
668
602
664
6 15.1
6 14.9
6 14.8
7 14.7
6 14.6
6 14.3
7 13.7
5
5
5
4
5
3
2
-
_
5
5
5
5
5
5
12.0
11.2
13.2
13.6
14.6
12.4
12.2
12.4
12.0
6.0
13.4
7.6
13.2
13.3
_
-
7.5
8.0
9.6
7.0
7.0
8.5
pH
7.2
7.4
7.5
7.8
7.2
7.2
7.1
8.1
7.9
7.9
8.1
8.0
7.8
8.0
-
-
7.2
7.2
7.0
6.9
7.0
6.8
-------
Table 9. Continued
Waste
Exper. concentration
No. (% by vol.)
S-7
Secondary treatment,
0
1
19
31
49
65
.1
.7
.7
.2
.3
Mean
off-flavor 3 , Standard Fish per Mean 4 , Dissolved
indexl/^/ LSD— • error of chamber temp.— oxygen
(0-6) ' the mean
chlorinated
0.67
0.70
1.03
1.67
2.97
2.77
January 1, 1972
0.68 0.
0.
0.
* 0.
* 0.
* 0.
15
20
17
22
42
47
37
grams
Judgments
593
628
764
511
608
528
number (C)
8
6
5
6
5
5
(mg/D
11.
11.
7.
7.
11.
11.
0
8
6
4
2
8
pH
7.2
7
7
7
7
7
.4
.3
.3
.3
.4
o\
— Off-flavor index based on a scale of 0 (no off-flavor) to 6 (very extreme off-flavor).
2/
— A plus sign (+) indicates all test fish died.
— The least significant difference at P=0.05 based on a two-way analysis of variance. An
4/
asterisk (*) indicates a statistically significant change in flavor from that of the control
sample.
— Mean temperatures for an experiment are not listed when values were between 14.5 and 15.5°C.
-------
water; chlorination appears to provide some relief, however.
In a report on potential use of waste water, Thorslund (1971) stated that
"No tainting of fish flesh was noticed..." when fish were reared in large
experimental ponds receiving waste water from domestic sources. Obviously,
this observation disagrees with the results of the test reported here.
It may well be that other methods of waste water treatment (i.e., lagoons
and activated sludge) are better at reducing the tainting capacity of
domestic waste water than are trickling filters.
Interaction of compounds: Many experiments were conducted in the
investigation reported here to determine safe levels, or threshold con-
centrations, for specific organic compounds. It is rare, however, to
find an effluent that contains only one compound. More commonly,
effluents contain a relatively large number of different organic com-
pounds. Information was needed to determine whether or not tainting
substances acted individually on the flavor of fish or interacted in some
Kay.
Two series of experiments were conducted to evaluate the influate of
exposures to more than one tainting substance (Table 10). In the first
series, two experiments were conducted in which trout were exposed to con-
centrations of pyridine and p_-chlorophenol and combinations of both com-
pounds. In the second series of experiments, trout were exposed to con-
centrations of pyridine and 2,4-dichlorophenol and combinations of both
chemicals. The results of these experiments are presented in Figure 8.
In the experiments with pyridine and p_-chlorophenol (Exper. M-l and M-2),
trout were exposed to pyridine concentrations of 100, 28, and 10 ppm and
p_-chlorophenol concentrations of 1000, 130, and 10 ppb. Trout were also
exposed to the following combinations of pyridine (P) and p_-chlorophenol
(C): P = 18 ppm and C = 325 ppb, P = 28 ppm and C = 180 ppb, and P = 56
ppm and C = 32 ppb. As may be seen in Figure 8, exposure to the combina-
tions of the two compounds resulted in off-flavor indices below those that
might be expected. In fact, the off-flavor indices were very nearly mid-
way between the off-flavor concentration curves determined for trout
exposed to the two compounds tested (Fig. 8).
In the second series of experiments (Exper. M-3 and M-4), trout were
exposed to 100 and 10 ppb of 2,4-dichlorophenol, 100 ppm of pyridine and
a control. In addition, trout were exposed to 100, 10, and 1 ppb of 2,4-
dichlorophenol at a pyridine concentration of 100 ppm. The combination
of 100 ppm of both compounds resulted in an off-flavor index nearly the
same (slightly higher) as that obtained for pyridine at 100 ppm. The
additional exposure to the 100 ppb of 2,4-dichlorophenol appeared to have
little or no effect on the off-flavor index. The off-flavor index of
trout exposed to only pyridine, however, was considerably higher than
that for trout exposed to 100 ppb of 2,4-dichlorophenol. As the concen-
tration of 2,4-dichlorophenol decreased to 10 ppb and finally to 1 ppb in
the tests with combined compounds, the off-flavor index also decreased
and fell substantially below the off-flavor index for 100 ppm of
pyridine.
62
-------
Table 10. Experimental conditions and results of tests in which rainbow trout were exposed for 48
hours to concentrations of various chemicals and combinations of those chemicals at 15°C.
CKI
O
O
rn
cr> O>
II
CD
CL
Exposure
concentration
M
off
Exper. Chem A • Chem B i
no. (ppm) (ppb) (
M-l March 31
0
28
18
28
56
0
M-2 March 31
0
0
10
0
100
M-3 June 8,
0
100
100
100
, 1971 10
0
0
325
130
32
1000
, 1971 11
0
10
0
130
0
ean
-flavor „ ,
ndexi/ LSD-/
0-6) '
Judgments
0.
1
2
2
2
3
•
•
•
•
•
Judgments
0.
0
1
3
4
1971 9 Judgments,
0 1
1
10
100
2
3
4
.
•
•
•
, Pyridine
35 0.
75
45
40
55
30
, Pyridine
32 1.
50
00
09
64
Pyridine (A)
.11 1.
•
t
^
33
11
00
(A)
89
*
*
*
*
*
(A)
05
*
*
and
35
*
*
Standard Fish per
error of chamber
the mean grams number
and p_-chlorophenol (B)
0.13 366
0.41 373
0.49 387
0.50 365
0.54 387
>0.49 354
and p_-chlorophenol (B)
0.15 503
0.20 432
0.46 478
0.53 383
0.41 450
2,4-dichlorophenol (B)
0.48 271
0.63 314
0.68 314
0.54 267
2
2
2
2
2
2
3
2
2
2
2
2
2
2
2
Dissolved
oxygen
(mg/1)
10.
11.
10.
10.
10.
10.
8.
9.
9.
8.
10.
_
_
_
6
0
3
2
3
7
8
1
9
2
8
pH
8.0
7.
8.
7.
8.
7.
8.
7.
-
7.
_
_
_
9
0
9
0
8
0
9
6
-------
Table 10. Continued
Exposure
concentration o
Exper
no.
M-4
Chem A
(ppm)
June 8, 1971
0
0
0
100
Chem B
(ppb)
10 Judgment
0
10
100
0
Mean
ff-flavor
index!/ I
(0-6)
s , Pyridine
0.65
1.30
2.30
3.85
_, Standard
jSD— jl- error of
the mean
Fish per Dissolved
chamber oxygen
grams
(A) and 2,4-dichlorophenol (B)
1.12 0.22 282
0.28
* 0.40
* 0.54
297
268
288
number (mg/1)
2
2
2
2
pH
-
-
-
o\ — Off-flavor index based on a scale of 0 (no off-flavor) to 6 (very extreme off-flavor).
2J The least significant difference at P=.05 based on a two-way analysis of variance. An asterisk (*)
means a statistically significant difference in flavor from that of the control sample.
-------
X
LJ
Q
g
U.
I
u_
LL.
O
<
LJ
O
A
P-CHLOROPHENOL
PYRIDINE
BOTH CHEMICALS
CONTROL
PYRIDINE
i
ONTROL
10
61-
100
P-CHLOROPHENOL
1000
O
A
2,4-DCP IN PPB
PYRIDINE IN PPM
2,4-DCP PLUS 100 PPM PYRIDINE
CONTROL
I
10
100
£ CONCENTRATION
Figure 8. The influence of exposure to mixtures of two organic compounds
on mean off-flavor indices. The open plots represent off-
flavor indices for trout exposed to only one compound; the
closed plots (squares) represent results of exposure to two
compounds. The concentrations of p_-chlorophenol and pyridine
in the upper graph are in ppb and ppm, respectively. The
presentation is based on data in Table 10.
65
-------
The results of the test using combinations of two organic compounds
strongly suggest that the flavor imparting capacities of organic com-
pounds are not additive, although there does appear to be some inter-
action. Admittedly, the experimentation conducted in this investigation
on the question of interaction of compounds is far too meager for a con-
clusions statement. It does appear, however, that threshold concentrations
obtained for specific organic compounds should provide protection for
the quality of fish if they are not exceeded, even though several com-
pounds are involved. This may not be true, however, when compounds of
very similar structure are involved. Much additional study is needed on
this question.
Estimated threshold concentrations: Estimated threshold concentrations
(ETC) were determined for the organic compounds and effluents tested in
this study. The ETC is here defined as the highest estimated concen-
tration of a material that will not impair the flavor of the flesh of
exposed fish. In order to determine the ETC for a particular experi-
ment, the mean off-flavor indices obtained were plotted against exposure
concentration and a curve fitted by eye (See Figure 9). The curve need
not pass through the control point. After the curve was fitted to the
data, a mean off-flavor index was determined for the flat or independent
portion of the relationship and this value was then added to the LSD QS
value. In Figure 9 the off-flavor index for the curve was 0.58 and
the LSD Qg was 0.69. The sum of the two values is 1.27. This value was
then located on the ordinate scale and a horizontal line drawn across to
the eye-fitted curve. At the point of intercept, the line was extended
vertically to the abscissa in the manner shown in Figure 9 (dotted line).
As may be seen in Figure 9, the ETC for 2,4,6-trichlorophenol was deter-
mined as 52 ppb.
Estimated threshold concentrations were determined for the experiments
with organic compounds (Tables 1 and 7) and for the effluents (Tables 8
and 9). The ETC, the highest test concentration not impairing flavor,
and the lethal concentration (if determined) for the organic compounds
and effluents tested are presented in Table 11 and Table 12.
The ETC for organic compounds varied from 0.4 ppb for 2,4-dichlorophenol
to 95 ppm for formaldehyde. As a group the chlorinated phenols were
found to have the lowest ETC values. The location and number of the
chlorine ions appears important in determining the flavor-imparting
capacity of this group of compounds, with the latter being most important.
Another factor that may play an important role is the species of fish
tested. As may be seen in Table 11, ETC values were determined for
2,4-dichlorophenol using trout, bass, and bluegill. Trout and bass
produce similar ETC values (1.0 and 0.4 ppb), while the value determined
using bluegills was 14 ppb. For pyridine, however, trout and bluegill gave
very nearly the same ETC, 27 and 28 ppm, respectively.
The ETC for kraft process effluents ranges from 5 to 7 percent by
volume; the ETC for the sulfite-base effluent was 36 percent by volume.
The ranges of ETC for primary, secondary, and secondary chlorinated
treated waste water were 11 to 13 percent by volume, 21 to 22 percent
66
-------
X 6
UJ
Q
? 5
2,4,6-TRICHLOROPHENOL (T-49)
0.58 + 0.69 = 1.27
CONTROL
O.I I 10 52 100
CONCENTRATION IN PPB
1000
Figure 9. Example of the procedure used in determining the estimated threshold concentration (ETC).
The curve was fitted to the data by size. The 130,05 for 2,4,6-trichlorophenol was 0.69
(Table 7, Exper. T-49).
-------
oo
Table 11. The highest concentrations not causing impaired flavor, lethal concentrations, and estimated
threshold concentrations for the chemicals tested in this study. Data are presented for
96-hr and 48-hr tests.
Chemical
tested
Acetone
Acrylonitrile
Amyl Acetate
Aniline
Benzene
Butanethiol
n-Butanol
Cresol
m-cresol
o-cresol
p_-cresol
Dimethyl amine
Ethanethiol
Ethylacrylate
Formaldehyde
B,B'-Dichlorodiethyl
ether
Methyl amine
2, Ethyl- 1-hexanol
2-Napthol
Sodium pentachloro-
phenol
Phenol
2,3-Dichlorophenol
2,4-Dichlorophenol
2,4-Dichlorophenol
2,4-Dichlorophenol
Test
fish
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
trout
bass
bluegill
Estimated
threshold , ,
concentration—
ppb ppm
_
18
-
-
-
55
-
70
200
0.4
120
6.8
240
60
95
88
-
-
0.3
-
-
84
1
0.4
14
Highest
concentration not
impairing flavor
ppb
_
-
-
-
-
8
-
5
50
-
50
-
100
10
-
90
-
-
-
20
-
32
0.01
0.1
10
ppm
1,000
5.6
10
10
5.6
-
100
-
-
0.1
-
5.6
-
-
56
-
10
5.6
0.3
-
5.6
-
-
-
-
Lethal -.
concentration—
ppb ppm
10,000
56
-
100
-
-
_
-
-
100
5
_
-
-
320
91
56
-
3.2
200
10
-
-
-
-
Test
number
T-l
T-2,T-3
T-4
T-5
T-6
A- 2
T-7
T-8,T-9
T-10,T-11
A- 4
T-12,T-13
T-14
T-15
T-20
T-21,T-22,T-23
T-24
T-25,T-26
T-27
T-28
T-29
T-31,T-32
T-33
A- 5
T-34
T-35
-------
Table 11. Continued
Chemical
tested
2,5-Dichlorophenol
2,6-Dichlorophenol
m-Chlorophenol
m-Chlorophenol
£-Chlorophenol
£-Chlorophenol
£-Phenyl phenol
2,4,5-Trichlorophenol
2 , 4 , 6- Tr i ch 1 oroph eno 1
Pyridine
Pyridine
Pyrocatechol
Guaiacol
Test
fish
Trout
Trout
Trout
Bluegill
Trout
Trout
Trout
Trout
Trout
Trout
Bluegill
Trout
Trout
Estimated
threshold 1 ,
concentration—
ppb ppm
23
35
_
-
60
45
_
_
52
27
28
0.8
82
Highest
concentration not Lethal 0 ,
impairing
ppb
10
10
-
-
100
21
-
320
10
-
-
-
100
flavor concentration—
ppm ppb
-
-
1
1
-
-
1
1,000
-
10
10
0.3
-
ppm
-
-
10
-
-
-
-
-
-
-
-
10
-
Test
number
T-36
T-37
T-38
T-39
T-40,T-41
T-42
T-45,T-46
T-48
T-49
A- 7
T-50
T-51
T-52
— The estimated threshold concentrations were determined by methods described earlier in the text
and presented graphically in Figure 8.
21
— The lowest concentration tested at which 50 percent or more of the test fish died during the
exposure period.
-------
Table 12. Estimated threshold concentrations, highest concentrations not causing impaired flavor,
and lethal concentrations for treated waste water (Corvallis) and paper process wastes.
Description Estimated threshold
of concentrationi/
waste (% by vol)
Kraft process,
Albany
Kraft process
Halsey
Kraft process
Halsey
Sulfite process,
Lebanon
Primary treated
waste water
Primary treated
waste water
Secondary treated
waste water
Secondary treated
waste water
Secondary treated,
chlorinated waste water
Secondary treated,
chlorinated waste water
Secondary treated,
8
5
7
36
11
13
21
22
-
20
26
Highest concentration
not causing Lethal »,
impaired flavor concentration— Test
(% by vol) (% by vol) number
5.6
0
8.1
33.5
7
7
32
14.9
33
20
19.7
100 E-l
E-3
E-4
E-2
50 S-l
100 S-2
S-3
S-4
60 S-5
S-6
S-7
chlorinated waste water
— The estimated threshold concentrations were determined by methods described earlier in the text
, and presented graphically in Figure 8
— The lowest concentration tested at which 50 percent or more of the test fish died during the
exposure period.
-------
by volume, and 20 to 26 percent by volume, respectively. An ETC could
not be determined for one experiment with secondary chlorinated
effluent (Table 9, Experiment S-5), since no flavor impairment was
noted at a concentration of 33 percent by volume and all test fish died
at the next higher concentration of 60 percent by volume. Chlorination
of treated wastewater appears to play an important role in reducing the
flavor-imparting of secondary treated wastewater.
71
-------
SECTION VII
ACKNOWLEDGEMENTS
A large number of people have contributed to the completion and success
of this research project. The cooperation and suggestions of the
Environmental Protection Agency Project Officers, Drs. Donald A. Hilden
and Gerald R. Bouck were greatly appreciated. Professor Lois S. McGill
and her laboratory assistants, Mrs. Geraldine F. Starks and Melba M.
Carpenter, prepared and served the samples of fish to the panels of
judges. Messrs. Miles Potter, Steve Sasser, Lowell Moore, and Michael
Wirsing assisted with conduct of experiments and tended the stock of
experimental fish. Mrs. Marjorie A. Jackson typed the final manuscript.
Many students and staff members at Oregon State University served as
members of the panels that were used to evaluate the flavor of the samples
of fish. Without the concentration and the honest effort displayed by
these people, this project could not have been conducted. Messrs. D.
Carr, W. Liss, F. Bird, D. Wilmot, W. Wurtsbaugh, J. Lannan, J. Graybill,
S. Broderius, R. Coykendall, R. Reynolds, D. Higley, J. Richards, J.
Helle, T. Gharrett, M. Hough, K. Schoolcraft, B. Tolonen, R. Eddy, L.
Brake, P. Anderson, E. Krygier, E. Wilson, D. Kama, R. Kavanagh, D.
Borton, and L. Lamperti and Misses S. Johnson and K. Thorsen, and Mrs.
S. Olterman served faithfully and with considerable ability as panel
judges during the study. Many other people served as judges for shorter
periods of time. Special appreciation is extended to all of these
people.
73
-------
SECTION VIII
LITERATURE CITED
Albersmeyer W., and L. von Erichsen. 1959. Investigations of the
effects of tar constituents in waste waters. Parts II and IV.
Z. Fish. 8(1/3), 40-46.
Bandt, H. J. 1946. Damage to the taste of fish. Beitr. Wasser-Chem.
1, 36-39.
B5etius, J. 1954. Foul taste of fish and oysters caused by chlorophenol.
Meddr Danm. Fisk.-og Havunders. 1(4), 1-7.
Fetterolf, C. M. 1964. Taste and odor problems in fish from Michigan
waters. In: Proc. 18th Ind. Waste Conf. Engng. Bull., Purdue
Univ. (Engng. Ext. Series No. 115), 48(3), 174-182.
Hasselrot, T. B. 1964. Investigations with caged fish as an indication
of pollution from kraft pulp mills. Sartr. Vattenhyg. 2, 1-10.
Korschgen, B. M., R. E. Baldwin, and J. W. Robinson. 1970. Influence
of environment on palatability of carp. J. Food Science 35, 425-
428.
Krishnaswami, S. K., and E. E. Kupchanko. 1969. Relationship between
odor of petroleum refinery wastewater and occurrence of "oily" taste-
flavor in rainbow trout, Salmo ga-iTdnevi-L. J. Wat. Pollut. Control
Fed. 41(5), 189-196.
Nitta, T., K. Arakawa, K. Okubo, T. Okubo, and K. Tabata. 1965. Studies
on the problems of offensive odors in fish caused by wastes from
petroleum industries. Bull. Tokai Rec. Fish. Mes. Lab. 42, 23-37.
Schultze, E. 1961. The influence of phenol-containing effluents on the
taste of fish. Int. Reviges. Hydrobiol. 46(1), 84-90.
Shumway, D. L. 1966. Effects of effluents on flavor of salmon flesh.
Research Report, Agri. Experiment Station, Oregon State Univ.
Shumway, D. L., and G. G. Chadwick. 1971. Influence of kraft mill
effluent on the flavor of salmon flesh. Water Research 5,
997-1003.
Tamura T., Y. Itazawa, and Y. Morita. 1954. River water pollution by
sulfate paper mill wastes. Bull. Jap. Soc. Sci. Fish. 20, 344-349.
Thorslund, A. E. 1971. Potential uses of waste water and heated
effluents. EIFAC^ Occasional Paper No. 5. FAO, Rome, Italy. 23pp.
75
-------
Westfall, B. A., and M. M. Ellis. 1944. Pulp-mill pollution of the
Rainy River near International Falls, Minnesota. U.S. Fish Wild-
life Service, Spec. Scient. Rept. No. 7, 8pp.
Winston, A. W. 1959. Test for odor imparted to the flesh of fish.
Paper presented at the 2nd Seminar on Biological Problems in Water
Pollution, U.S. Public Health Service, Cinn., Ohio. Mimeographic
release by Dow Chem., Midland, Michigan.
76
-------
SECTION IX
LIST OF APPENDICES
Page
1 Geological Survey analysis of well water used in this study. 79
2 Ballot used in organoleptic evaluation of fish flesh. 80
77
-------
SECTION X
APPENDICES
Appendix 1. Geological Survey analysis of well water conducted
February 21, 1966. Sample of water was collected after
9-months steady pumping on well.
Specific conductance
(micromhos at 25 °C)
Dissolved solids
(evaporated at 180°C)
Hardness as CaCO,
Silica (Si02)
Magnesium (Mg)
Potassium (K)
Bicarbonate (HCO.^
Carbonate (CO,)
Iron (F)
259 ppm
165 ppm
0
32 ppm
9.9 ppm
0.3 ppm
162 ppm
0
0.08 ppm
PH
Color
Sodium (Na)
Nitrate (N03)
Fluoride (F)
Chloride (Cl)
Sulfate (S04)
7.5
Temperature (°C) 10
0
8.5 ppm
0.3 ppm
0.1 ppm
6.0 ppm
1.0
79
-------
Appendix 2. Ballot used in organoleptic evaluation of fish flesh.
Department of Food Science and Technology
Oregon State University
Name:
Date:
Test No.
Off-Flavor
None
Overall
Desirability
Slight
Moderate
Strong
Very Strong
Extremely Strong
Very Extremely Strong
Very Desirable
Moderately Desirable
Slightly Desirable
Neutral
Slightly Undesirable
Moderately Undesirable
Very Undesirable
U. S. GOVERNMENT PRINTING OFFICE : 1973—514-154/267
80
-------
1
Accession Number
w
5
r\ Subject Field & Group
05C
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
organization — — ^— ^^— — ^^^— ^— ^-^— — — — ^
Department of Fisheries and Wildlife
Title
Impairment of the Flavor of Fish by Water Pollutants
IQ Authors)
Shumway, Dean L. and
Palensky, John R.
16
21
Project Designation
EPA Program #18050
DDM
Note
22
Citation
Environmental Protection Agency report
number, EPA-R3-73-010, February 1973.
23
Descriptors (Starred First)
*Fish, *Taste, *0rganic Compounds, *Pulp Wastes, *Sewage Effluents, Water
Pollution, and Water Quality
25
Identifiers (Starred First)
*0rganoleptic Evaluation, Trout, Bass, Bluegill, and Laboratory Study
27 Abstract Laboratory studies were conducted with fish to determine an appropriate bio-
say procedure for the examination of the flavor-imparting capacity of wastes and waste
components (organic compounds). In addition, the flavor-imparting capacity and estimated
threshold concentrations were determined for a number of organic compounds and effluents.
Flavor evaluations were obtained through the use of taste panels.
Estimated threshold concentrations were determined for twenty two organic compounds. The
values ranged from 0.4 ppb (2,4-dichlorophenol) to 95 ppm (formaldehyde). An additional
twelve compounds were tested, seven of which were not found to impair flavor at or near
lethal levels.
Estimated threshold concentrations were determined for effluents from the Corvallis Sewage
Treatment Plant, kraft paper mills, and a sulfite-base paper mill. The estimated threshold
concentrations for primary, secondary, and secondary chlorinated effluents from the
Corvallis plant were determined to be 11-13, 21-23, and 20-26 percent by volume, respectiv-
ely. The estimated threshold concentrations for the effluents from the kraft and sulfite-
base paper mills were about 6 and 36 percent by volume, respectively (Shumway-OSU).
Abstractor
I. Shuim
Institution
Oregon StatA
mrc
WR:I02 (REV. JULY I9B»>
WRSIC
SEND, WITH COPY OF DOCUMENT, TO: WATER ftfetfOURC ES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 20240
* GPO! 1970—389-930
------- |