U.S. ENVIRONMENTAL.PROTECTION AGENCY
Region IX Laboratory
620 Central Avenue
Alameda, California 94501
SELECTED FIELD AND LABORATORY BIOLOGY METHODS
Table of Contents
I. Sample Collection
II. Sample Collection Forms
III. Algal Bioassays
A. Laboratory Bioassay Directions
B. Cell Mass Measurement
C. Measuring Dry Weight
D. Measuring Algal Chlorophyll
E. Maintaining Algal Cultures in the Laboratory
IV. Statistical Procedures
V. Fish Bioassays
VI. Use of Random Numbers
VII. Appendix
Tables of Useful Data
Prepared by Milton G. Tunzi, Ph.D., EPA Laboratory
620 Central Avenue, Alameda, California 94501
(Comments and corrections would be appreciated)
First Edition October 1973
Second Edition February 1974
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Page Intentionally Blank
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11758
U.S. ENVIRONMENTAL PROTECTION AGENCY
Region IX Laboratory
620 Central Avenue
Alameda, California 94501
SELECTED FIELD AMD LABORATORY BIOLOGY METHODS
Table of Contents
I. Sample Collection
II. Sample Collection Forms
III. Algal Bioassays
A. Laboratory Bioassay Directions
B. Cell Mass Measurement
C. Measuring Dry Weight
D. Measuring Algal Chlorophyll
E. Maintaining Algal Cultures in the Laboratory
IV. Statistical Procedures
V. Fish Bioassays
VI. Use of Random Numbers
VII. Appendix
Tables of Useful Data
Prepared by Milton G. Tunzi, Ph.D., EPA Laboratory
620 Central Avenue, Alameda, California 94501
(Comments and corrections would be appreciated)
First Edition October 1973
Second Edition February 1974
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I. Sample Collection
Representative samples from any body of water
are difficult to take. Directions can be given for a
completely statistically valid approach (e.g., random
sampling), but these would probably be beyond the
resources of most laboratories. Furthermore, the
approach should be determined in relation to the
purposes of the study. This may preclude a random-
sampling approach or make it unnecessary.
One of the best ways to assure that a sample is
representative of a site (whether that site be chosen
randomly as indicated above or arbitrarily as in this
section) is to composite 3 or 4 or more equal-volume
samples from each site. These can be put into a
plastic bucket, mixed, and a container filled from
this bucket.
A. Routine Sampling
1. Generally, the specific sampling sites are
chosen because they are accessible, equally
distant from each other, traditional
sampling sites, or in locations of importance
near dischargers or in areas of water use.
Samples may be taken above and below a dis-
charge pipe, or they may be taken in the
receiving water near the discharge pipe.
Many times the location selected is one
where a water quality standard may be
exceeded.
2. Rivers, streams, estuaries
Sites can be sampled from different depths,
from different locations around the sides
of a relatively-stationary large boat,
and from different locations if a small boat
is allowed to drift. Water moving past an
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anchored boat can be sampled every half-
minute, or longer time interval depending
on time limitations at the station. A
stream should also be sampled in this way
from the bank, i.e., with samples taken from
the stream throughout a given time period
and composited. A wide-month, liter,
plastic container attached to a pole can
be used to reach further from shore so that
flowing water can be more easily sampled,
or so that the moving part of a stream can be
reached. A wide stream which is not above
boot-top in depth can be sampled by sub-
samples from 4 or 5 locations in a transect
across the stream. The subsamples must be
taken upstream so that they will not be
contaminated by the stream bottom stirred
up from walking.
Compositing samples is suggested because
then fewer samples would have to be analyzed.
However, if variations within time or a
small space are desired, then the samples
could be kept discrete, i.e., not composited.
Spatial or temporal variations at one one or
more stations can then be used in statistical
comparisons between the stations.
3. Lakes, reservoirs, and ponds
If five individual samples for nitrate
analysis are to be taken from five-acre
Lake X whose water is being mixed thoroughly
(e.g., because of fall turnover), then one
might choose locations so that all parts of
the Lake would be represented. (Figure 1).
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Figure 1. Lake X, Divided into Sections,
Five Stations Shown.
In fact, one could take several samples in
the area where he was to sample the single
station and composite the several samples.
For example, four or five subsamples could
be taken in an area near sampling Station 1
(indicated on Figure 1 by circle), and the
composited sample would represent Station 1.
The compositing can be done from sample
taken at different lake depths. This com-
positing approach is very useful where little
time can be allotted to the execution of the
sampling or where the project does not
require a more sophisticated approach.
B. Random Sampling
Because of such variations as density, light
penetration differences and the like, most
waters would require a stratified random sampling
approach.
1. Random Sampling, Spatial Approach
First the water body is arbitrarily divided
into areas which are physically or geogra-
phically distinct. Then each area is divided
into a grid pattern, and each section is
numbered. The sections to be sampled in each
area are selected by using a random numbers
table. Only 2 or 3 locations are sampled in
each physically or geographically distinct
lake area. The same number of sections can
be sampled in each area if the areas are
approximately of the same size. The total
number of samples would depend on resources
and availability.
If a long channel is to be randomly sampled,
it can be divided into separate sections each
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4.
with its own subsections. The difficulty
with the subsection approach is that the
areas are hard to delimit on the water, as
there are no lines marked on the water surface.
2. Other random sampling approaches. As an
alternative to a spatial design, temporal
considerations also may be important. Time
intervals also can be selected randomly with
a new set of sections again selected by means
of a random number table each time samples
are taken.
A given approach may be suitable for one type
of measurement and not for another. Such
factors as water movement, animal migration,
and diurnal fluctuations cannot be overlooked
in designing proper sampling.
C. Tests for Random Distribution
There are several ways in which a biological
parameter can be tested to see if it is randomly
distributed. The simplest way would be to compare
the variance and means of samples taken from an
area. Table 1 shows the significance of three
such comparisons: S2 = X; S2^>X; s2
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5.
The main advantages of a random distribution in
a comparison of samples or in making statements
about parameters is that confidence limits can
be set which will delimit the true mean. However,
if just a "yes or no" answer is required about
whether there is a difference between samples,
then non-parametrical statistical approaches can
be used for both randomly and non-randomly
distributed parameters.
D. Non-parametric Statistics
These procedures are very useful because
no presumptions are made about sampling techniques,
analytical methods, time of collection and, of
course, distribution of the parameter. Further-
more, they are usually simpler tc calculate than
the parametric tests. At the selected probability
level, the results of the test give an answer
to the question of whether or not there is a
difference among the compared sampled means.
Confidence limits containing the true population
mean cannot be calculated using these tests.
This is one of their main drawbacks. Suggested
tests are below. The Kruskal-Wallis test is
given in the Statistical Section. The others
can be found in the references.
Test
Mann-Whitney U - Test
Kruskal-Wallis Test
An alternative to the t - test
Sample numbers do not have to
be equal
Comparable to a one-way analysis
of variance. Two or more
samples can be compared. As
above sample numbers do not
have to be equal
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6.
Wilcoxon's Signed Rank Test Use for detecting differences
in paired samples
Spearman Rank - Correlation This is the alternative to
Coefficient (rs) calculating the correlation
coefficient for bivariate
normal distributions (r)
References
Elliot, J. M., 1971. Some Methods for the Statistical
Analysis of samples of Benthic Invertebrates.
Scientific Publ. No. 25, Freshwater Biological
Association. Ambleside, Westmorland, England.
144 pp.
Snedecor, G. W. and W. G. Cochran. 1962. Statistical
Methods. Iowa State Univ. Press. Ames, Iowa. 534 pp.
Steel, R. G. D. and J. H. Torrie. Priciples and Procedures
of Statistics. McGraw-Hill Book Co., Inc., New York.
481 pp.
Woolf, C. M. 1968. Principles of Biometry. D. Van Nostrand
Co, Inc., Princeton, N. J. 359 pp.
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Table 1 Significance of the variance and mean.
measurement as indicated.
Transform by converting each X
Conditions of
the Samples
2 - (1)
= x
S2x
Distribution
Random (Poisson
Statistical
Approach
Use parametic
statistics
Uniform, Regular
(Underdispersion or
evenly spaced)
Contagious, Overdispersion
(clumped or aggregated)
Use non-parametric
statistics
Use non-parametric
statistics or
transform so than
S2 = x
f\ _
(1) S = x means approximately equal
Transformation
If numbers are low
transform each value
x = •/ x or x =
v/x + 1 if O's are
encountered
None used
Commonly encountered
x = logio x or
x = Iog10 (X + 1),
x is less than 1.0
If upon transformation of the data, the variance is approximately equal to the mean then
the data can be used in mormal statistical procedures such as t-tests, analysis of
variance, single and multiple regressions and correlations, analysis of covariance, etc.
S2 is the variance
x is the mean
S is the standard deviation
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II. Sample Collection Forms
On subsequent pages are sample collection forms,
including directions for sample preservation. The
sample preservation information is mostly from the
EPA "Methods for Chemical Analysis of Water and
Wastewater"; however, the determinations for which
the same preservatives are used are placed conti-
guously.
The forms are suggestions only and can be modi-
fied according to needs.
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B.
SAMPLE COLLECTION FORM
INSTRUCTIONS
Project Director - Indicate measurements, location, date,
sample points, sampler, time, samples to be taken and
whether composite or grab. Indicate composite frequency.
Check off these items on Sample Collection Form (one per
sample).
Sampler - On same form fill in field data, correct date,
and time of sampling if this information is different from
that entered by Project Director (I).
x x x x x x x
Location
Date
Time
Sample Point
Sampler(s)
Field Measurements
Flow
DO
Settleable Solids
Clarity
PH
Specific Conductance
Chlorophyll
(mis filtered)
,1.
BOD
COD
Coliform, Total
Nitrogen, Total
NH3-N
NO3-N
N02-N
Odor
Oil and Grease
Phenol
Phosphorus, Total
Cyanide
C
G
Composite
Grab
Remarks: I. Project Director
II. Sampler
Suspended Solids
Total Solids
Volatile Solids
Sulfide
Total Organic
Carbon
Turbidity
Pesticides
Oil Spill Sample
Fish Bioassays
Algal Bioassays
Benthic Sample
Heavy Metals
Arsenic
Chromium
Copper
Cadmium
Iron
Lead
Mercury
Nickel
Zinc
Specific
Conductance
Others
(See reverse side and attached pages for sample size, preservative and
container type)
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3.
Check Equipment to be Taken for Sampling
(Always take - distilled water or deionized water, jugs, cubi-
tainers, preservatives, rubber bulbs, Van Dorn Bottler or
Kemmerer Sampler, pipettes, squeeze bottles, plastic bucket,
rope, thermometer, towels, record book and/or Sample Collection
Forms.)
Flow - flow meter, weir apparatus
DO - DO meter, buret, reagents, thiosulfate soln.,
starch, beaker, BOD bottles
Specific conductance - meter, 2 cells (constants of
2x and lOx)
Clarity - Secchi disk and line
Benthic Samples - dredges, container, formalin
Chlorophyll - GF/C filters, filter flask, vacuum
pump (hand or electric), desiccant jars, styrofoam
container, ice
Algal count - container, formalin
Other Samples Number of Containers
Glass jugs
Cubitainers
Wide mouth plastic
jars
Preservatives, etc.
Styrofoam container and ice
Mailing container
H2SO4
10 N NaOH
HN03
CuS04 + H3P04
HgCl2 (Saturated solution)
2N Zn acetate
1:1 HNO3
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4.
Container*
NA
Glass
NA
Cub.
or
Gl.
Parameter
Dissolved
Oxygen
Dissolved
Oxygen
(by
titration)
pH, tempera-
ture, Settable
solids, clarity
Metals, Total
(one or all
can be analyzed
Preservative
Determine on
site
2 ml MnS04 +
2 ml ALK-I
Determine on
site
5 ml HNOs
per liter
Holding
Period
NA
4-8 hours
NA
6 months
Volume
Needed
NA
300 ml
NA
(For all
paramete
1 quart
Gl.
Gl.
Gl.
from same
sample)
Metals, Filtrate: 3 ml
Dissolved 1:1 HN03 per
liter
(With arsenic HN03 interferes with
reduction method; preserve arsenic
samples with HC1)
6 months
Total organic
carbon
2 ml H2S04 per
liter (pH 2)
Chemical Oxygen 2 ml H2SO4
Demand per leter
Oil and Grease
Petroleum
Products
2 ml H2S04
per liter-4°C
None required
individual
analyses
unless indi-
cated differ-
ently. One
gallon for
combinations
with same
preservative)
*Cub.
Gl.
NA
Cubitainer or polyethylene jar
glass
not applicable
7 days
7 days
24 hours
Bring to
Lab as soon
as possible
1 gallon
1 quart
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5.
Container
Gl.
Gl.
Gl.
Cub. or Gl.
Parameter
Pesticides,
PCB
Organo
Phosphates
Chlorinated
Hydrocarbons
Phenolics
Preservative
Maximum
Holding
Period
Volume
Needed
None required
(Put teflon or
aluminum foil
under cap)
2 gallons
12 hours
2 days
2 gallons
2 gallons
1 gallon
1.0 g CuS04/l + 24 hours
Cone. H3PO4 to
pH 4.0 - 4°C
(Use methyl orange indicator. At pH 4
it turns pink upon additions of H3PO4.
Use 2 drops indicator/100 ml of sample
or use pH meter)
Cyanide
Sulfide
Turbidity
Solids
Acidity-
Alkalinity,
Color, Thres-
hold Odor
Biochemical
Oxygen Demand
Sulfate, Odor
Fish Bioassays
2 ml ION NaOH/1 24 hours
7 days
1 gallon
2 ml 2N Zn
acetate per
liter
None
Available
Refrigerate at
4°C
Refrigerate at
4°C
Refrigerate at
4°C
Refrigerate at
4°C
Refrigerate at
4°C
7 days
7 days
24 hours
1 gallon
6 hours
7 days
6 hours
20 gallons of
sample. 20
gallons of
receiving
water
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6.
Container
Cub. or Gl,
Wide-mouth
Jar
Parameter
Preservative
Algal Bioassays Refrigerate
at 40°C
None Required
Chloride,
Hardnedd,
Specific
Conductance,
Fluoride,
Calcium
Algal Count 4% formalin
Benthic Sample 10% formalin
Kjeldahl
Nitrogen
Ammonia,
Nitrate-
Nitrite,
Phosphorus
1 ml/1 of sat
urated
4°C
Maximum
Holding
Period
12 hours
7 days
Indefinite
Indefinite
Unstable
7 days
Volume
Needed
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1.
III. Algal Bioassays
There are two general approaches in carrying out
algal bioassays: (1) Using indigenous algae found
naturally in a water sample (indigenous); or (2) adding
a laboratory-grown single culture of algae. It is
sufficient to say here that the use of indigenous
algae in a bioassay is much easier than adding laboratory
cultures. However, if the results of a bioassay are to be
expressed as the dry weight of algae, this parameter can
be more easily derived from single-specied bioassays.
(There are many more advantages and disadvantages, to
both approaches. These are discussed at length elsewhere
[Tunzi, 1972]).
Directions to follow in carrying out algal bioassays
will be divided into several sections:
Laboratory Bioassay directions
Cell Mass Measurement
Measuring Dry Weight
Measuring Algal Chlorophyll
Maintaining Algal Curtures in the Laboratory
A. Laboratory Bioassay Directions
(Bioassays utilizing Indigenous Algae)
Materials
Glass or polyethylene containers (e.g.,
cubitainers) for sample collection.
Ice chest, ice.
Filtration apparaters, vacuum pump.
Erlenmeyer flasks (250 or 500 ml each),
acid rinsed (0.1NH Cl), then rinsed with
tap water and distilled water; water
volume marks should be indicated on
side of flask.
Waterproof labeling pens; black ink
Examples: Sanford's Sharpie #49;
Scientific Products #P1226, Fine Tip
Marker.
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2.
Foam rubber stoppers for erlenmeyer flasks;
rubber stoppers for same.
Light box capable of 400 ft. candle
illumination at 20°C; check uniformity
of light with light meter.
Method
1. Collect samples in glass or polyethylene
containers. Collapsible cubitainers are the
most convenient. If the samples are from
eutrophic water, about 1 quart is sufficient.
Otherwise collect 1 gallon. If spiking of
samples with nutrients or effluent is antici-
pated, then collect 1 gallon.
2. Keep the samples out of the sunlight. If
necessary, surround samples by ice, but do not
freeze. It is usually not necessary to
ice if transport time is less than 1 hour.
3. If the samples are to be shipped a long dis-
tance, they can be put into styrofoam containers
and surrounded by ice. The algae in the samples
will remain cool and viable for about 12 hours
during transit.
4. At the laboratory filter part of each sample
for dry weight or chlorophyll determination
(50 to about 400 ml is needed for eutrophic
and 1 to 2 liters for oligotrophic waters).
5. Choose sample concentration. Suggested
additions of effluent to receiving water are
1%, 5%, 10%, 50% of total volume. When pre-
paring nutrients, prepare high concentrations
so that additions will be 5 ml/liter of
sample. Otherwise the distilled water used
to dissolve the nutrients will dilute the
sample so that comparisons with a control or
with other nutrient additions is difficult
(see Table 1, components of Macronutrient
. Medium for Algal Cultures, Section III-E.
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6. Prepare about 1 liter of each concentration
and mix well before adding the water to the
replicates.
7. Prepare at least 4 replicates per sample type.
Use black ink, waterproof pens for labeling.
8. Number the sample containing Erlenmeyer flasks
with a waterproof marker pen. Replicates
should be numbered; e.g., 1-1, 1-2, 1-3, 1-4;
2-1, etc. Number the flasks permanently on
the frosted parts. If a flask has consistent
erratic results compared to replicates of the
same series, discard it.
9. If samples are to be incubated without addi-
tions, the flasks can be filled directly from
the sample container. First shake sample
well; then put 125 ml into the 250 ml flasks
or 150 ml into 500-ml flask. Add the water
to the volume marks on flasks. Extreme
accuracy is not important.
10. Cover the flasks with foam rubber stoppers.
11. Take an initial cell-mass measurement on 2
of the 4 replicates (see Cell Mass Measure-
ments) .
12. Incubate the samples under 400 ft. candles
of light at 20°C. If higher temperatures
are used, the cultures grow too rapidly.
There does not appear to be much advantage
in intermittent lighting. The main point is
uniform light. A light meter should be used
to check that all areas of the incubation
shelf are receiving approximately equal
light ( + or -10%).
13. Measure the algal mass at about the same time
every day.
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4.
14. Before measuring the mass, plug the flask
with a rubber stopper and shake it vigorously.
This promotes aeration and lessens the possi-
bility of attached growth.
15. Expected growth curve, calculations, and
reporting forms are shown in Figures 1 and
2. A completed reporting form is shown in
the Statistical Section.
B. Cell Mass Measurement
Direct Cell Counting
Materials
Whipple micrometer reticule
Stage micrometer
At least 4 Sedgwick-Rafter Chambers
Pasteur pipette or automatic volume delivery
pipette.
Procedure
1. Calibrate the microscope and Whipple disc
(see Section 301 C, page 731, Standard
Methods, 13th Edition), using a stage micro-
meter.
2. Fill each Chamber with water from one of the
replicates by means of a Pasteur pipette or
automatic volume pipette. Let the chambers
settle for 5 minutes (Chamber volume is 1 ml).
3. Usually 2 strips are counted in each chamber
and a factor is used to convert the number of
cells counted to cells per ml for the sample.
Make two counts of the cells in each chamber
and record average.
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5.
4. Dilute aliquots from the flasks (with distilled
water) if the cell concentration becomes too
high. Serial dilutions may also be made to
check accuracy of counting technique.
Turbidimetry by Turbidity Meter
Materials
Hach 2100 turbidimeter or equivalent.,
Tubes for reading in Hach 2100.
Procedure
1. Calibrate the Hach Turbidimeter by means of
the standard (the one supplied with the machine
is adequate). The machine is set at the
value indicated on the standard tube (usually
50 - 80 JTU's).
2. Read turbidity in each sample.
3. Obtain an average reading by watching the
needle for 10-15 seconds. Fluctuations in
readings are to be expected.
4. Use the same sample tube for each of the
replicates of the same samples. It is not
necessary to rinse the tube with distilled
water between replicates of a single sample;
however, the same tube must be well-rinsed or
even washed between different samples.
5. When the maximum turbidity reading is reached
(after incubation of sample), combine the
water from the replicates, mix, and use for
dry weight measurements (see Section on
Weighing). (Maximum growth is reached when
readings are approximately the same for
2-3 days [see Statistical Procedures for
approach to evaluating differences in the
samples].)
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6.
6. The lower limits of the detectable turbidity
is about 2000 cells/ml, but 10 fold increases
changes turbidity only about 2 units.
Absorbance by Spectronic 20
Materials
B & L Spectronic 20; Spec 20 tubes
Procedures
1. Set the wave length at 600 nm.
2. Using special Spec 20 tubes, read absorbance
for each sample. Many tubes are required,
since the sub-samples have to be poured back;
generally 20-30 ml volume is utilized at
each reading, and discarding this would
deplete the incubating sample too drastically.
3. Take readings daily at about the same time.
4. When maximum value is reached and stabilized,
express terminal values as dry weight. The
water from the replicates can be combined to
give enough volume to yield weight differences
and the individual reading used for statistical
comparisons (see sections on Weighing and on
Statistical Treatment).
5. Depending on the size of the algal counts, the
Spectronic 20 is good starting at about the
100,000/ml level. It is an instrument rather
insensitive to any but large cell number changes.
In Vivo Fluorescence
Materials
1. Turner Model III Fluorometer (or equivalent) with
an ultra-violet light source F4T5, the red-
sensitive R-126 photomultiplier, Corning 5-60
-------
7.
primary .filter and 2-64 emission filter. The
general purpose photomultiplier can be used
for dense cultures (^104 cells/ml and up). The
R-126 photomultiplier is sensitive down to
1000/ml. In contrast to the turbidity meter,
10 fold increases in cell number changes
fluorescence 50-100 units.
Procedure
1. Zero the machine using the black plastic tube
which comes with the machine. Check the zero
calibration when changing from slit to slit
or after reading every 3 or 4 samples. There
are slits on the machine -IX, 3 X, 10X, 30X,
the latter allowing the most light to pass
through. Do not use the 1 X slit, as response
of the machine is not linear with this slit. On
top of the machine a dial reads from 0-100.
Record both slit and dial values for each
reading taken.
2. Establish a calibration factor for converting
readings from one scale to another.
3. Follow sample incubation, etc. under 1-14 of
the Laboratory Bioassay Directions, IIIA.
4. Shake the flasks thoroughly immediately
prior to reading as clumping of algae can
cause fluctuations in the readings.
5. Pour 5 ml of water directly into cuvette
and take reading. Each reading only takes
about 5 ml of sample, so that once the
aliquot is read the water used can be
thrown away.
6. Rinse tube as follows: for replicates of
the same sample, rinse the tube with a
subsample from the next replicate; between
different samples, rinse the tube with dis-
tilled water.
-------
8.
7. Wipe the outside of the cuvette dry before
inserting it in the holder.
8. Shake the next replicate, then take the
reading of the tube in the machine. This
should give about a 10-15 second period
between readings. It is important that the
time span be consistent.
9. When growth reaches a plateau (i.e., the
amount of fluorescence does not seem to
increase), combine the replicates for either
chlorophyll a or weight measurement. Since
algal chlorophyll fluorescence is the primary
cause of sample fluorescence, chlorophyll a
determination is the more reasonable one to
make (see Measuring Algal Chlorophyll, begin-
ning with Filtration, D-2). There is usually
not enough sample for both measurements.
C. Measuring Dry Weight
1. Wash 4.25 cm GF/C Whatman filters by placing
them in a pan of distilled water. Loose
fibers will separate from the filters.
2. Place filters on a towel to partially dry.
3. Place them separately on a sheet of aluminum
foil.
4. Dry them for three hours at 90°C. (Put
into a desiccator if filters are to be
stored for more than 5-10 minutes before
weighing.)
5. Number them lightly on their edges with
a soft lead pencil.
6. Weigh filters to nearest hundredth milligram.
Handle the filters with tweezers, grasping
the edges.
-------
9.
7. Put them into small envelopes with their
weights and number written on the outside
of the envelope.
8. When needed, filter as much sample as will
go through the filter in about two minutes at
low vacuum, less than 5 inches of mercury.
9. Record volume filtered in liters.
10. Double the filter, algal side inward.
11. Place filter on aluminum foil and dry for
at least three hours at 90°C.
12. Remove them from oven and, using forceps to
transfer, weigh them after they have cooled
for about five minutes. Cooling in a desiccator
may be desirable but appears to be of limited
advantage.
13. Subtract original weight of dried filter
from final weight and express results as mg/1
of dry weight.
D. Measuring Algal Chlorophyll
Introduction
There are two practical approaches to measuring
the concentration of indigenous algae in water.
They can be counted directly or may be enumerated
indirectly by determining the chlorophyll content
of a sample of water (or performing some other
mass measurement). The following method details
procedures for measuring chlorophyll concentration.
Materials and Equipment for Laboratory Analysis
.Whatman GF/C glass fiber filters, 4.25 cm
diameter
-------
10.
Filter-holding apparatus: either
Millipore or Gelman
Covered small glass jars containing desiccant
Freezer
Scissors
Tissue homogenizer with teflon pestle: either
Kontes Glass Co. No. 885-380-0023; or
A. H. Thomas Co. No. 4288B
Acetone, (90% acetone, 10% water) spectro-
photometric grade
Centrifuge tubes (if Kontes tissue homogenizer
not used)
Centrifuge adapters (necessary only if Kontes
tissue homogenizer used)
Pasteur pipettes
Beckman DU Spectrophotometer, or equivalent
Cuvettes (for Spectrophotometer), 1 cm or
small volume 5 cm ones
Hydrochloric acid, IN
Method
1. Sample Collection
It is best not to collect a single
grab sample. Instead an integrated sample
should be taken by collecting small (about
250 ml) equal-volume sub-samples at a given
site and depth over a time period, such as
10 minutes. These sub-samples should be mixed
together in a plastic busket and transferred
to a transport container (e.g., a 1-gallon
cubitainer).
Consult section on Sample Collection for further
considerations on representative samples.
2. Filtration
1. As soon as possible after collection
(the sooner the filtration the more
valid the data; e.g., a sample stored
in the dark on ice should be filtered
-------
11.
within 4-5 hours, if possible), filter
under low vacuum as much water as will
go through a Whatman GF/C filter within
about two minutes.
2. If possible, prepare 4-5 filtrations of
water from the same sample.
3. Record the water volume that has been
put through each filter.
4. Double the filters, algal side inward
and put them into a small jar with
dessicant and then into the freezer
for storage.
5. Extract for chlorophyll within three
weeks of filtration.
3. Extraction Methods
green
white
Figure 1. Folded Filter
Refer to Figure 1. Using scissors,
carefully trim off the white border to
the edge of the green algae-contining
section. Discard white section. Cut
each trimmed filter into smaller pieces,
putting these directly into tissue
grinder homogenizer.
2. Add 5 ml of acetone.
3. Grind filter with teflon pestle.
-------
12.
4. Ground-filter-and-acetone mixture should
only get slightly warm to touch during
the process. Move the tube slowly up and
down while grinding, ceasing the grinding
whenever the tube becomes warm to touch.
5. Keep ground-filter-and-acetone mixture
out of strong light by covering it with a
towel.
6. Pour the mixture into a centrifuge tube,
cover with parafilm (or other cover), and
shake well. If Kontes tissue grinder is
used, the container may be centrifuged
directly (if adapters are present), thus
making it unnecessary to transfer the
ground mixture to a centrifuge tube.
7. Let set in the dark for 20 minutes at
least.
8. Shake the centrifuge tube well again after
the 20-minute waiting period.
9. Centrifuge the tubes for 10 minutes at
2500 - 5000 RPM, preferably at the higher
RPM's. Tap the tubes to bring particulate
matter to the bottom. Recentrifuge.
10. By means of a Pasteur pipette, carefully
draw off enough of the supernatant to fill
a 1 cm cuvette (about 4 ml).
11. Take absorbance readings at 750 nm, blanking
against 90% acetone. If above 0.005-0.008
Optical Density (O.D.), re-centrifuge.
12. Take absorbancy at 663 nm, blanking against
90% acetone.
-------
13.
13. Add 2 drops of IN HC1 to each cuvette and
re-read absorbancy after 2 minutes at 663 nm.
One cm cuvettes do not have to be shaken to
disperse the acid, but it is necessary to
shake those of larger dimension.
14. If the absorbancy of the sample is too
high for the spectrophotometer scale,
the sample can be diluted. Keep an
accurate measure of the total amount of
acetone used, as this volume is necessary
for calculations.
4. Calculation of Results
1. The amount of chlorophyll a_ from algae
can be calculated as follows:
ug chl a per liter = 26.7 (ODb ~ ODa) x Ac
W x cm
where: ODa = optical density at 663 nm of extract
after acidification (step 13 of Extraction),
less the OD at 750 nm (step 11 of Extraction).
ODb = optical density of extract before
acidification (step 12), less OD at 750 nm
(step 11) .
Ac = volume of acetone in ml
cm = spectrophotometer cell path length in cm
W = volume of water in liters
2. Phaeophytin, a degradation product of chlorophyll,
may be calculated as follows:
>ig phaeophytin per liter = 26.7 [1.7 (ODa) - ODbl x Ac
W x cm
This value may be 0 or negative, indicating no phaeo-
phytin in sample.
3. Total chlorophyll a per liter in any sample is the
sum of the values obtained in 2 and 3.
-------
14.
4. If more than one filter was prepared, the chlorophyll
concentration values from the 4 or 5 filters can be
used to establish the standard deviation, standard
error, and 95% confidence limits of the chlorophyll
values for the sampling site (see Statistical
Procedures).
Discussion
As was mentioned in the introduction, there are numerous
ways to determine the amount of algae present in a sample.
These include direct counting, weight measurement (biomass),
trubidity determinations, and chlorophyll measurement.
Counting is a slow process. Its principal drawback
however is that algae vary greatly in size, so that to get an
estimate of the mass of algae in water each separate species
has to be measured and the total volume obtained by multi-
plying the number of each species times its volume. (This
value can be converted to mg/1 of algae by assuming a
specific gravity of about 1.0 for the algae.)
An extraction of algal chlorophyll is one of the
standard methods of estimating standing crops in water
(Strickland and Parsons, 1965). This is true because
chlorophyll is a necessary constituent of green plants,
serving as a catalyst in the initial carbon fixation process.
One problem in determining concentration, though, is that
the ratio of chlorophyll to cell mass can be changed,
especially by varying the light intensity. The chlorophyll
a_ to cell carbon ratios are in the range 1:40 to 1:100.
Biomass might also be determined. One disadvantage of
this procedure is that the volume of material available for
filtration is usually small so that the resulting weight of
the cells retained by the filter is not too much greater
than the weight of the filter itself. This causes a wide
variation in results. If one wishes to determine cell
weight, though, the method can be employed. Empirically it
has been observed that the cell dry weight is approximately
equal to two times the cell carbon (Maciolek, 1962).
A summary of the advantages of chlorophyll as a measure
of mass would include the following points:
-------
15.
1. The amount of chlorophyll is determined
spectrophotometrically. The precision of
this determination is greater than that for
any cell-count method.
2. Large volumes of water can be filtered to
determine chlorophyll. Only one ml at most
is used in direct counts (and hence is not
too representative).
3. The green chlorophyll color of algae is the
substance seen when one looks at algae in
water; therefore, measuring chlorophyll in
water usually is a direct way of quantifying
the size of an algal bloom.
E. Maintaining Algal Cultures in the Laboratory
The EPA Report Algal Assay Procedure -
Bottle Test (1971) available from Thomas Maloney,
EPA, NERC, Corvallis, Oregon gives useful infor-
mation on culturing algae. Pure cultures of
algae can be obtained from NERC, Corvallis or
from the Culture Collection of Algae, Dept, of
Botany, Indiana Univ., Bloomington, Indiana.
Direction are available in the Indiana University
listing for media for specific algae.
General Directions
These are applicable for Selenestrum, Scenedesmus and
mixtures.
1. Stock cultures can be kept viable for months if
they are kept out of direct light. They may be
stored at normal room temperature in a shelf of
the laboratory where the light is constantly
subdued or at least off at night. Cultures kept
under constantly high light will go through a
growth phase, exhaust nutrients, and usually die,
-------
16.
2. Use aseptic techniques for transferring uni-
algal cultures. Pasteur pipettes, flasks and
stoppers (or other covers) can be autoclaved or
heated to 90°C if an autoclave is not available.
3. Table 1 shows a simple mixture of nutrients
which will promote growth. Stock culture can
be kept in polyethylene or glass bottles. Micro-
nutrients are not needed as there appears to be
ample present as contaminants in the chemicals.
If they are desired, utilize those given in the
EPA Corvallis publication (or add to 1 liter
macronutrients 1 ml of a solution prepared by
adding a small amount of bouillon cube to 100
ml water) .
Table 1. Components of Macro-nutrient Medium for
Algal Cultures
Component Amount in g/1
NaN03 6 g/1
CaCl2 0.6
MgS04 1.8
NaCl 0.6
KH2PO4 0.875
K2HP04 0.375
NaHC03 10.0
Fe(SO4)2 (NH4)2 * 12 HOH 860 mg Dilute in
EDTA • 2 HOH 660 mg one liter
4. Add 10 ml of each chemical except the iron solution
above to a two liter flask and bring volume up to
1 liter with ion-free or distilled water.
5. Cover the flask with a beaker and heat to 90 °C or
autoclave for 20 minutes.
6. Autoclave the iron solution or heat to 90 °C. The
iron solution should be kept in a screw-cap flask.
Loosen caps when heating or autoclaving and tighten
when cool.
-------
17.
7. Add 1 ml of iron solution to liter of the macro-
nutrients when the latter has cooled.
8. The nutrient solution can then be dispensed to
sterile smaller flasks (250-ml ones are suitable)
9. Inoculate the small flasks with the stock algae.
Put under constant light of about 400 ft-candles.
Solutions should be densely green in about five
to seven days and ready for use.
References
Anonymous, 1971. Algal Assay Procedure. Bottle Test, NERC
Environmental Protection Agency. Corvallis. 82 pp.
Maciolek, J. A. , 1962. Limnological Organic Analyses by
Quantitative Dichromate Oxidation. Res. Rept. (50. U.S.
Fish and Wildlife Service. 61 pp.
Tunzi, M. G., 1972. Algal bioassays: Examples, advantages,
and limitations of current approaches, 173-197 pp. in
Proceedings of Seminar on Eutrophication and Biostimu-
lation. California Dept. of Water Resources. Sacramento.
229 pp.
Strickland, J. D. H., and T. R. Parsons. 1965. A Manual of
Sea Water Analysis. Fisheries Research Board of Canada.
Bull, No. 125. Ottawa, Canada.
-------
Algal Cell
Concentration
(any type of
measurement;
cell count,
optical
densityk
fluorescence,
chlorophyll
concentration)
10
Days
Algal Growth Data
Sheet Parameters
Initial chlorophyll
Concentration
Peak chlorophyll
Concentration
Increase in chlorophyll
Concentration
Days to reach peak
Maximum Growth rate
u, day ~l
Value from Figure
(A) 10
(E) 90
(E minus A) 80
4 days
J.^JL.) .i$)-i.»
t i
x0 - cell concentration at beginning of maximum growth
x, • cell concentration at end of maximum growth
t - time
n • maximum specific growth rate, (day "1)
Maximum growth rate is derived from the steepest part of the growth curve,
utilising the log of the cell concentration at the beginning and end of
the curve.
Fig. 1. Typical algal growth response. The values are the
means of the replicates, whose range are indicated by the
vertical lines.
-------
I
Sample
Location
-
-
?igure 2
ALGAL GROWTH 1
Average Initial
Chlorophyll
Concentration
L
DATA SHEET
jig Chi a/1
Average Increaae
In Chlorophyll
Concentration
»
Average Maximum
Chlorophyll
Concentration
i
Average Maximum
Growth Rate .
/\ . — -i
«!,» days 1
No. Of
Days to
Reach Peak
The results below connected by underlining are not different from each other at the 951 confidence level.
on the results of four replicates.
Average based
fiample Numbftr
Concentration
Increase jig Chi a/1
»le Humber
Concentration Maximum
jig Chi a/1
Nmhur
Maximum Growth Rate
Fig. 2. Data reporting sheet with multiple range section on the lower part,
for elaboration.
See statiscal section
-------
IV. Statistical Procedures
A. Introduction
Statistics is a scientific method involving
collection, analysis, and interpretation of
numerical data. An understanding of basic statis-
tical principles and procedures is helpful to both
field and laboratory workers. The mathematics
involved is simple except for advanced procedures
which are infrequently used.
The data collected for statistical treatment
are measurements or observations of a characteristic
of a population. The population can be the cells
in a series of flasks, the nitrate ions in a lake,
the oligochaetes in the sediments of a bay, etc.
Thus the population can have discrete physical
boundaries or ones which the planner sets himself.
Almost without exception we cannot make all
the desired measurements of a population, so that
instead we take a sample from the population. From
the sample mean, predictions can be made about the
same characteristic in the entire population. By
sample is meant a series of measurements, although
in reality we would have to take a separate sample
for each measurement.
Greek letters are used for population statis-
tical terms and English letters for sample: ones.
The measurements from a population are called
parameters and those of the sample called statistics.
Assuming that we have measurements from a
population, the above can be clarified by the following
table.
Population Sample
Parameter Statistic
Mean p (Mu) x
2 2
Variance & S
Standard
Deviation ©• (Sigma) S
-------
B,
2.
Definitions
1. Mean (x) . The average value calculated by
dividing the sum of the measurements by the
number (n) of measurements.
2. Variance (s2). The variability or spread of
the data about the mean (See Figure 1).
3.
4.
>, l
u
c
0)
3
cr
0)
Mean
Figure 1. Two sample measurements with
equal means but differing variances.
The standard deviation (s).
of the variance.
The square root
The standard error or the standard error of
the mean (S^). The standard deviation of the
sampling distribution of means.
5. Degrees of freedom - usually equal to i\ -1.
Calculations
The calculations for the above statistics
are very simple.
Given the data below collected from a
population with individual measurements listed
under x.
X
11
12
15
16
11
x - x
-2
-1
2
3
-2
(x - x)2
4
1
4
9
4
x2
121
144
225
256
121
65
22
867
-------
3.
(x-x)2 is the sum of the squared deviations which
is called the sum of the squares (SS).
x2 is calculated because it is used in the working
formula for the variance.
The mean being: n
£
x = 1
n
= 6_5_ = 13
5
The variance is:
n
s2 =
= 22 = 5.5
n-1
The working formula is simpler because the sum of
the squares does not have to be calculated.
n
,2 _
Ix2-
\
n
n-1
Standard deviation
.5
867 - (65)
_ 5
2.35
= 5.5
Standard error
x
By use of the standard deviation confidence limits
for the measurements can be set:
x + IS includes 68% of the sample measurement
etc. (see below).
Xr ........
<•
95
99
-3S -2S -IS
+1S +2S +35
This assumes that the sample measurements are
normally distributed.
-------
4 .
Confidence limits of the population are much
more important. That is we want to set limits
which bracket the true population mean or average
( Ai ).
This can be done by using the standard error S^
and t table values for the degrees of freedom
in our sample.
x + s~ will include the true population mean
(ja ) 68 out of 100 times.
x + (t Q QC) sx will include ;u 95 out of 100 times,
x + (t o.Ol) sx wiH include u 99 out of 100 times
The difference between sample confidence limits
and population confidence limits must be clearly
understood.
Using the last formula in the above data:
x + (4.60) (1.05) = 13± 4.83
The t(04Q5) and t/Q Q-J\ are found in a t table for
a range of n-1 values (a few values are given below)
Degrees of Freedom (n-1)
6 DF 5 DF 4 DF 2 DF 1 DF
t(0.05) 2.45 2.57 2.78 4.30 12.71
t(0.01) 3.71 4.03 4.60 9.93 63.66
By utilizing the t values for various degrees of
freedom, we can see the importance of high numbers
of replicates in sampling.
D. Group Comparison of Two populations
1. A comparison consist of two steps.
a. At test to determine if the sample means
come from one or two populations.
b. Confidence limits can be set for the sample
means if the t test is significant.
-------
5.
Special formulas for group comparisons
2
s
a. ;u = x + t (0.05) p
n
Note: If the t test is not significant then
both samples come from the same population.
The confidence limits for the population means
may overlap slightly even when the t test
is significant.
b. Pooled variance
n ;> / n \ „ n _ / n
2
V— ': _ / s— 2 S 2 _k~ x 2
2
"2
These formulas can be used whether or not
n1 = n2.
3 . Example of a group test
X
x2
X
V
X
32
31
52
44
159
6625
39.75
? 6625 -
X2
17
35
22
24
98
2574
24.50
(159)2 2574 - (98)2
4 + 4
(4-1) + (4-1)
-------
s 2 = 6625 - 6320 + 2574 - 2401
s 2 = 478 = 79.7
fr £
39.75 - 24.50 15.25
= 2.42
79.7(1/4+1/4) 39.85
For 6 degrees of freedom t[0.05] = 2.45. Therefore,
there is no difference between the set of data.
-------
7.
E. Comparison of Two Groups by Pairing
1. If two samples are not independent, then a pairing-
test can be used to compare them. Of course,
nl = n2- Generally, high values in one sample are
associated with high values in another.
2. Example of the data from a pairing test:
K! - x2
x± X2 difference d^
10 19
9 18
8 17
11 19
14 22
12 18
/_ 64 113
x 10.64 18.83
f n \
\ \2
l_ d
L =2401
. 1 1
Variance of the? dif
-9 81
-9 81
-9 81
-8 64
-8 64
-6 36
-49 407
xd= -8.17
n
*\~~ d2 = 407
1
/n\
21 d2 - E d 2
i \i/
2 n
:ference sd =
n - 1
407 _ (49)2 407 _ 2401
sd2 = 6 6 = 1.4
-------
8.
The standard error is:
s /
xd = / I-4 - ./0.233 = 0.48
V 6 v
t = I *d | = 8.17 = 17.0
s_ 0.48
xd
Since the t value of 17.0 is greater than the t
value for 5 degrees of freedom (0.05), there is a
significant difference between the sample means.
The 95% confidence limits for the difference
between the samples can be calculated by using the
following formula:
xd + t(0.05)
n
F. Comparison of data from more than two groups
1. Analysis of variance is the technique used to
compare one characteristic from three or more
populations.
Three or more populations cannot validly be
compared by the sequential use of a t test. It
is especially bad to single two groups out of a
large number and subject them to a t test to see
if they differ.
The completely randomized design is used for
comparing the means from three or more populations.
2. The following calculations for unequal sample size
can also be used when the same number of measurements
are made for all samples:
-------
9.
r
Sample
1
10
11
12
13
x 46
x2 534
n 4
x 11.50
(Zx)2 529
n
Correction
Total Sum
Treatment
2
6
12
14
32
376
3
10.67
341.3
(133)
3
10
12
10
9
14 _
55 )_ = 133
621 ^_= 1531
5 2 = 12
11.00
605 }_ = 1475.3
2
Factor =12 = 1474.1
of Squares = 1531 - 1474.1 = 56.9
Sum of Squares =
Analysis of
1475.3 - 1474.1 = 1.2
Variance Table
Source of Variation DF SS Mean Square
Total 11 56.9
Treatment 2 1.2 0.6 0.097
Error 9 55.7 6.2
Treatment mean square
F = Error mean square
F values for 2 degrees of freedom in the numerator
and 9 in the denominator for the 0.05 probability
level is 4.3, much higher than our F value. There-
fore, there is no significant difference between
our sample means.
-------
10.
3. If the F value is significant, a multiple range
test must be used to determine which samples
actually differ. Duncan's new multiple range
test for equal replication (p. 107) and unequal
replication (p 114) are good ones to use (Steel
and Torrie, 1960).
G. Non-parametric methods
These approaches are useful when it is not certain
that the normality of the population distribution and
its means and variance are the same as that of the sample,
One of the more useful tests is the Kruskal-Wallis
test which compares medians from 2 or more populations.,
An example of this ranking test is given below:
Data
Median =
Set I
6
8
8
12
14
30
23
12
Rank
2
3.5
3.5
6
7
11
10
m =7
Rn =43
Data Set II
21
15
4
11
Median = 13
Rank
9
8
1
5
n2
Ro
=4
= 23
Median-the middle value for odd number of values, and
the mean of the two middle values for even number of
values
11 + 15
= 2_6 = 13
2
An H value is then calculated where-
k = number of samples
T = total number of measurements in all sets
-------
11.
k-^ = degrees of freedom
R = sum of the rank
n = number of measurements
12 = a constant
H - T-rinr F- -g- "3
For the above data:
H - 12 432 + 232
H ~ 11 (12) —7— —4—
H = 0.035
The hypothesis made is that the populations are
identical. When the H value is greater that the chi-
square value for k-1 degrees at the 0.05 probability
level, then there is a difference between the sample
medians. For 1 degree of freedom this =3.84. So the
hypothesis is accepted. (Chi-square tables are found
in most mathematical handbooks and statistic books.)
-------
Sample 3/17/70
TABLE 1 - BIOASSAY DATA WITH MULTIPLE RANGE STATISTICAL PRESENTATION
ALQAL GROWTH DATA SHEET
V9 Chl a/1
Location
Redwood City Sewage
Treatment Plant
downstream location
•
San Francisco Bay
Number
12
1
2
3
4
5
6
15
Average Initial
Chlorophyll
Concentration
7.2
2.0
2.0
2.1
2.1
2.5 -_
2.8
3.0
Average Increase
In Chlorophyll
Concentration
2.0
33.6
0.0
66.6
69.8
40.3
26.1
6.8
Average Maximum
In Chlorophyll
Concentration
9.2
35.6
2.0
68.7
71.9
42.8
28.9
9.8
Average Maximum
^Growth Rate
Pb. days -1
0.13
'0.94
0.00
1.09
0.61
1.67
1.57
0.93
Ho. of
Days to
Reach Peak
6
6
0
3
3
3
3
2
The results below connected by underlining are not different from each other at the 95 percent confidence level. Average
based on the results of four replicates.
Sample Number
Concentration
Increase >ig Chl a/1
Sample Number
Concentration Maximum
jig Chl a/1
Sample Number
Maximum Growth Rate
ub, day'1
2
0.0
2
2.0
2
0.00
12
2.0
12
9.2
12
0.13
578 ITTI
15 6
978 287?
0715 0.93
1
3375
3575
1
7673
¥O
T~oc
3 4
5575 597ff
3 4
5877 71.9
6 5
> T757 T75T
-------
1.
V. Fish Bioassays
Sources of Fish
1. Collection
Test fish may be collected from a large body of
water using a seine; from a small slough, one can use
dip nets. Usually a collection permit is required
(get this from state fish and game departments).
Since collection of suitable and adequate number
of fish is somewhat uncertain, it should be done only
if time is of little importance, if the locations of
the desired fish are well-known, and if there is no
other way to obtain fish.
After collection, place fish in a suitable
transport container. A five-gallon plastic bucket
with a snap-on lid can hold 100 small (up to 2 inches)
fish for short distances.
If one collects his own fish, he must have
several good battery-operated aerators (e.g., the
Jorgensen portable aerator - $7.00; Lewis Air Pump -
$3.50). Take extra batteries and check at least
every hour to see that they are not run down. Aerate
fish on the way back to the laboratory.
Aeration is accomplished by connecting aerator
to a flexible line with an airstone at the end. The
airstone should be weighted or it will float to the
water surface. A large (No. 10) rubber stopper with
a hole in it (to put the tube through) will hold the
aerator under water.
2. Purchase
Commercial aquarium and fish stores generally
charge too much to make them a reasonable source of
fish. Names of dealers who supply fish for bioassay
are generally available from agencies such as State
Water Resources Control Boards and fish and game
agencies or from other persons who carry out fish
-------
2.
bioassays. The price per fish delivered is
usually 20-50 cents, depending upon the species.
This is normally the most economical way to get
fish. Be sure not to acquire more fish than
needed. It is usually easier to purchase fish in
lots as required than it is to maintain fish for
many weeks in an expectation that they might be
needed.
Possible sources of fish from agencies include:
(Normally the hatcheries supply fish only to other
public agencies)
Striped Bass
Bureau of Reclamation, Tracy
Phone 209-935-3122
Rainbow Trout
American River Hatchery
Phone 916-351-0314
Salmon, Steelhead Trout
Nimbus Fish Hatchery
Phone 916-351-0383
Black Bass, Blue Gill, Shad> Catfish
Elk Grove Hatchery Phone 916-685-9555
Two commercial fish dealers in the San
Francisco Bay area are:
William Putman
5449 Modoc St. Richmond, CA 94804
Alex Fish Company
2235 Juniperberry Drive
San Rafael, CA
A list of commercial fish dealers in California is
available from the California Fish and Game Department
-------
3.
Recommended Species
Ideally, the best fish to use are the most abundant
or economically significant young small ones found in the
receiving water area. However, this may be impractical or
impossible to carry out as they would be too difficult
to catch or only available during specific seasons.
A standard test species available throughout the
year would make comparisons between tests more meaning-
ful.* Fish most commonly used in California are:
euryhaline
3-spine stickleback Gasterosteus aculeatus
Threadfish shad Dorosoma petenese
Killifish Fundulus parvipinnis
Striped bass Roccus saxatilis
Fresh Water
Golden shiner Notemigonus chrysoleucas
Channel catfish Ictalurus punctatus
Maintenance of Fish
1. Disinfection
a. A new group of fish should be disinfected by
putting them (for about 5-10 minutes) in
water containing both .025 ml/1 of formalin and
0.05 mg/1 malachite green (Leteux and Meyer,
1972).
b. Fifty fish can be put into approximately two
gallons of water.
c. Watch them carefully, and remove them immediately
if they show signs of distress (floating up
slightly sideways).
2. Aeration
a. Before adding fish to water, aerate water for
12-24 hours.
*Table 3 lists animals suitable for bioassay in Hawaii
-------
4.
b. A twenty-gallon aquarium can hold 100 small fish
if it has two activated charcoal filters and
one to two airstones running constantly. Some-
times it is better to replace the water or part
of it every three or four days, but aerate the
water for 12-24 hours before adding it to the
tank.
c. One example of an activated charcoal filter
is the large-size Halvin which attaches on
the side of the aquarium. Examples of
electric air pumps are the Silent Giant
($15), Oscar and Star ($8). Activated
charcoal should be changed every two days.
The charcoal can be reused if fired in an
oven at 450°C for an hour. Less heat will
not destroy the organics absorbed in the
charcoal surfaces.
d. If one uses a compressor as an air source,
the air should first be passed through one
tube: the first half holding non-absorbant
cotton and the second half holding activated
charcoal. The cotton and charcoal should be
changed every month.
e. A large sand filter fiberglass system is shown
in Figure 1. This can be used for 200-300 fish.
The pump can be run constantly. Its size should
be sufficient to circulate the water in the tank
once per hour.
Back-flush the sand filter every 3 weeks. Turn
off the pump when feeding the fish.
3. Temperature for Fish Maintenance
a. Cold-water fish should be kept at 13-14°C in
order to remain disease-free. Warm-water fish
also are usually less apt to contact disease
when kept at these cool temperatures.
-------
5.
b. There are several ways to maintain these cool
temperatures. One of these is a water bath with
a refrigerant system. Another is a walk-in box
with a refrigerant system.
4. Feeding Fish During Maintenance Period
a. When feeding fish, turn off the aerators and
any filtration system (including activated
charcoal).
b. Throw in food slowly until fish cease eating;
this usually takes 10 to 15 minutes.
c. Look at the individual fish and remove any
that have any discoloration and, of course,
any dead ones. Generally only 1 or 2 fish
will die out of a hundred, and these in the
first days after delivery.
d. Fish should be fed 3 times a week; however,
they can do without feeding on the weekends.
e. Do not over feed.
f. Fish food may be purchased as pellets or in
frozen form. Fish food is available in bulk
in pellet form of various sizes. No. 2 is
suitable for small fish, but larger pellets
can be ground in a mortar if only one size is
available. Brine shrimp can be purchased
frozen. Chunks can be broken off as needed.
Put the frozen chunks into a beaker of water
until they melt apart. Stir them and let the
shrimp settle to the bottom. Pour off the
supernatant water, add more water and repeat the
process. In this way, less debris is added
along with the shrimp. (If one is feeding fish in
large tanks, the brine shrimp chunks can be
thrown in directly).
f. Fish are not to be fed 2 days before the
commencement of any test.
-------
6.
Holding and Dilution Water
Most fish are either marine or fresh-water, but
some fish can live in water of varying salinity.
These are termed euryhaline fish. If freshwater
discharges into a freshwater receiving water are being
tested for their toxicity, then a freshwater species
can be used and a marine species for saline discharges
into the ocean.
However, when low-salinity water is discharged
into an estuary or the ocean, then a euryhaline
species is the appropriate one to use. The euryhaline
species can be kept in a 1:1 mixture of marine and
tap water. It will withstand without great stress
transferral from this mixture into both the effluent
and sea water. These extremes in salinity would
be present in the test waters because the concentra-
tions used would include both sea water and effluent
and mixtures of the two.
Fresh water holding water and the dilution
water can be tap water that has been aged or
aerated for 12 hours. For some of the reasons
given above, there are usually two controls, one
the holding water and the other the dilution
water. If the fish are kept in the holding water
within the laboratory-maintained temperature range,
then the fish left in the holding water can be
considered controls.
When both the receiving water and the effluent
from the discharger are suspected to be toxic, a
double control can be made. Dilution water could be
river water upstream of the effluent in which a
double control should be used. Control 1 being
the river dilution water; control 2 the aged tap
water. Sometimes results will vary if you use
existing receiving water as a diluent instead of
-------
using tap water as diluent. For example, when
salts are high in receiving waters, this may have
a positive or negative effect on effluent toxicity.
If one has two controls (river water and tap water)
and there is mortality due to the receiving water
(river) rather than the effluent, use of the second
control, tap water, will make this obvious.
Bioassay Procedure
Materials Required
Bioassay containers. These may be five-gallon
(19-liter) aquaria, pickle jars or battery
jars which are available in sizes up to one
gallon; the size depends upon size of fish -
one gm fish per one liter water; fish nor-
mally require 10-15 liters per test sample.
Container-cleaning facilities (large thick rug;
garden hose).
Aluminum foil or lids for bioassay containers.
Temperature controllers. (Capable of maintaining
20°C + 2°C for warm-water fish and 15°C + 2°C
coldwater ones).
Aeration device
Dissolved oxygen meter. DO can be measured by
siphoning but then large-volumed containers
are required. See Fig. 2.
Thermometer. (Either a recording thermometer or
a small thermometer in a jar of water).
Optional: devices for measuring pH, conductivity,
turbidity, and hardness.
Data recording sheets (See attachment, Figure 3)
Bioassay organisms (e.g. fish) [fish must be held at
experimental temperatures for 10 days prior to
commencement of bioassay for legal purposes].
Method
Scrub bioassay glass containers clean and rinse
them well with tap water. If the containers are
large, it is safer to do this on a large thick
rug, using a light garden hose for rinsing the
jugs. This is best done out of doors on a cement
platform.
-------
8.
2. Let the containers drain for about one hour, then
let them air dry inside the laboratory. After
they are dry, cover them with aluminum foil or
lids to keep dust-free, or store upside down.
3. Normally ten fish are added to each container. The
weight of the fish cannot exceed 1 gram per liter of
water. If fish are too large for 1 container, put
five fish into each of two separate containers
containing the same sample solution.
4. There are several ways to increase the reliability of
the tests:
a. Increase the number of fish from 10 to 20 per
container (remaining consistent with the
weight to volume restriction above).
b. Prepare replicates of each concentration so
that there would be two or more of each test
solution (with 10 fish per container).
c. Prepare concentrations with closer increments of
toxicants e.g., instead of 10%, 20%, 30% there
would be 10%, 15%, 20% etc. additions.
5. a. Preparation of concentrations
The graph shown in Figure 4 is a standard plot
of log of concentration versus regular arithmetic
increments. This plot is based upon experimental
results which show that effect of a toxicant
upon an organism is logarithmic rather than
arithmetic. That is to say that, in general,
if one doubles the concentration one does
not double the mortality.
Actual additions of toxicant are in logarithmic
increments. An excerpt from Standard Methods
is given in 5b. It includes Table 1 which
shows some log increments; a more complete
-------
9.
range is expressed in Figure 4. Figure 4 shows
concentrations ranging from 100% to 10%. If
a wider range of concentrations were to be
employed, then several-cycle semilog paper
would be used - e.g., a range of 100% to
0.1% would require four-cycle semilog paper -
or divide values in Fig. 4 by 10 or multiples
of 10.
If possible, the actual concentrations chosen
would be based on a preliminary test of 12-24
hours with toxicants added in concentrations
covering a wide range of values. For an
unknown substance this might be 100%, 50%, 10%,
1%, and 0.1%. Regardless of the preliminary
results, if possible, always include a 100%
full strength test sample because many
toxicity standards are based on percent
survival in the pure test sample.
With experience and a preliminary test, the concen-
trations can be selected so that containers very close to
the TLso value will be the most numerous. A preliminary
test using 2-4 fish per concentration can be carried out
if the test material does not degrade. For example, if
the preliminary test using two fish per liter showed the
following results
Concentration of
Test Solution Survival
100% 0
50 0
25 1
10 2
1 2
Then the following concentrations could be set up:
100% (Optional)
56
32
24
18
10
-------
10.
Excerpt from Standard Methods, 13th Ed., p. 565
"Although a TLso may be determined by testing any
appropriate series of concentrations of the sub-
stance or waste assayed, the geometric series
of concentration values given in Table 1 is often
most convenient and has been widely used. These
values can represent concentrations expressed
as percent by volume or as milligrams per liter,
etc.; they may all be multiplied or divided, as
necessary, by any power
TABLE 1: GUIDE TO SELECTION OF EXPERIMENTAL
CONCENTRATIONS, BASED ON PROGRESSIVE BISECTION
OF INTERVALS ON LOGARITHMIC SCALE
Col. 1 Col. 2 Col. 3 Col. 4 Col. 5
10.0
8.7
7.5
6.5
5.6
4.9
4.2
3.7
3.2
2.8
2.4
2.1
1.8
1.55
1.35
1.15
1.0
of 10. For example, the two values in the first
column may be 10.0 and 1.0 as shown, or they may be
100 and 10, or 1.0 and 0.1, with the values in the
other columns changed accordingly. The values of
-------
11.
the series 10.0, 5.6, 3.2, 1.8, and 1.0 (i.e.,
Cols. 1-3), or 10.0, 7.5, 5.6, 4.2, 3.2, etc.
(Cols. 1 through 4), are evenly spaced when
plotted on a logarithmic scale."
6. At the beginning of the bioassay, measure dissolved
oxygen (DO) in each container. If it is below
4 mg/1, aerate that container until the DO is
above 4 mg/1.
7. Additional optional measurements (in order of
importance) include pH, conductivity, turbidity and
hardness (titration, expressed as EDTA as CaCO3).
Figure 3 shows a blank data sheet. Figure 5 shows a
typical data sheet with observations recorded.
8. Record the temperature daily (on Data Sheet,
Figure 5, range of temperature is recorded following
reading on 7-day recording thermometer).
9. Keep room semidark and do not let people wander need-
lessly in to frighten fish.
10. When transferring fish, do so gently so as not to
harm them.
11. Add fish in groups of two to the jugs. Random
placement of jugs and random addition of fish is
recommended (see section on Random Sampling).
12. Using data sheet, record mortality and D.O. at
least every 24 hours along with any other information
about the bioassay that may be subsequently of
interest. Remove dead fish as soon as they are
observed.
Calculation of Results
1. The TLso (concentration of toxicant killing 50% of
the fish) at 96 hours should be calculated by
plotting toxicant concentration on the ordinate
scale of semilog graph paper and survival on the
abscissa (normal scale axis).
-------
12.
For example, if the 96-hour results were obtained
from a toxicity test as below (in Table 2) the
TLso can be seen from Inset in Figure 5 to be
68%.
Table 2. Survival of Fish vs. Toxicant,
Typical Data
Survival
(per 10 fish total) % Toxicant
0 100
3 75
6 65
9 56
10 42
10 24
10 10
Statistical Treatment of Fish Bioassay Results
The TLso value can also be calculated by using the
Reed-Muench Method (Woolf, 1968). This method also
allows one to calculate the 95% confidence limits which
contain the true TLso value.
Utilizing the data given on the sample record
sheet for the "Northwest STP", the calculations are
given in Figure 6. Natural logs can be used in place
of logs to the base 10, if this is more convenient.
If the lowest dose in mg/1 or percent volume of
toxicant is less than one, multiply the dose values by
10 or 100 as logs values less than one are negative.
Then divide the resulting final values by the same
multiple. Express the TL values as whole numbers in
the example given.
REFERENCES
Leteux, F. and F. P. Meyer. The Progressive Fish
Culturist 34. 1972. "Mixtures of Malachite
Green and Formalin for Controlling Ichthyophthirius
and other Protozoan Parasites of Fish."
Woolf, C. M. 1968. Principles of Biometry. D. Van
Nostrand Company. Princeton, N. J. 359 pp.
-------
Table 3
TEST ORGANISM SUITABLE FOR THE STATE OF HAWAII
University of Hawaii at Manoa
i)(!,'nrlmont of Zoology
Edmiiiuisiiii Hall* 2538 The Mall
Honolulu, Hawaii 96622
Suggested Native Hawaiian Fauna
for Aquatic Bioassay
(John A. Maciolek - Associate Professor)
The following fresh and brackish waters animals are available on most islands
in Hawaii and generally can be kept without undue difficulty in aquaria and holding
tanks.
A. Freshwater species.
1. Shrimp: Atya bisulcata = opae kalaole, "mountain opae". Occurs in fast-
flowing streams to about 3,000' elevation. Very abundant in pristine streams,
but on Oahu, it is common only at higher elevations. Filter-feeds on stream
seston and epilithic algae. Normally completes its life cycle in freshwater
but larvae can tolerate salinity. Size: to about 2".
2. Fish: Awaous stamineus = o'opu nakea and Sicydium stimpsoni = o'opu nopili.
Doth species are abundent in the lower to middle reaches of perennial streams
on neighbor islands; much less common on Oahu. Larvae develop in ocean and
migrate upstream as post-larvae (hinana), often in great numbers, during
several months of the year. Juveniles and adults do not tolerate saline
water. Feed on benthic algae (especially nopili) and small invertebrates.
Size: hinana about 1"; nakea adult to 12"; nopili adult to 7".
B. Brackish water species: the following shrimp and fishes are broadly euryhaline
(freshwater to seawater).
1. Shrimp: Palaemon debilis = opae huna, "glass shrimp". Most common in estu-
aries and brackish shoreline ponds, but is also found in most protected
inshore marine areas. Omnivorous, feeds on plant materials, detritus, etc.
Can complete its life cycle in brackish water. Size: to 1.5".
2. Fish: Kuhlia sandvicensis = ahole, aholehole. Occur in estuaries and inshore
marine areas. Juveniles (to 3") invade lower reaches of streams. Carnivorous;
predaceous on invertebrates (shrimps, worms) and small fishes. Size: to 12".
3. Fish: Mugil cephalus = amaama, grey mullet. Habitat similar to Kuhlia, but
is herbivorous—feeding on phytoplankton, bottom sediments, etc. Fry and
small juveniles common in estuaries. Size: to at least 2 feet.
-------
Holes increase in size from middle to end
xPVC pipe 3/4" diam
2"
Sand
Filter
Hose 3/4" diam.
(doe O
.Spreader
PVC Pipe (bottom view)
Board at angle Water goes through holes in PVC
pipe onto spreader board.
" diam. PVC
ergency overflow
(in event of sand clogging)
Pump
i i/«" aiam vaive \ i R\
If- PVC (Y "II
II '
-< L-1
Fish Tanks with Filtering System
8" depth sand
(No. 12, White Monterey)
6" depth pea gravel
12" depth rock (l"-3" in size)
row of PVC collector pipe
i
3/4" diam.
Sand Filter, Details
(side view)
urface of sand in square feet
hould be approximately equal to
he flow in gallons per minute
cap
• ' t
^2" diam.
^outlet (2" diam.)
V
PVC Collector Pipes, Details
(viewed from above)
Each pipe has holes of 3/8" - 1/2" diam.
spaced regularly at 2" intervals along
length.
Figure 1. Diagram of Fish Tanks with Filtering System
(Details of Sand Filter and Collection Pipes Included)
-------
GLASS TUBING
FLEXIBLE TUBING
PINCH CLAMP
BUCKET
BOD BOTTLE
Figure 2 . The siphon is first filled with distilled water.
After putting the glass tubing into the test water, the pinch
clamp can be released and enough water siphoned into the
bucket to displace the distilled water by test water. Then
the end of the tube is put into the BOD bottle all the way to
the bottom. After overflowing the bottle about twice, slowly
withdraw the tubing, allowing the water to flow until the tube
is out of the bottle. Start with the control water and
proceed from the lower toxicant additions through the more
concentrated ones. Then the siphon can be utilized without
rinsing it with distilled water. Stopper the BOD bottles and
measure the dissolved oxygen by the routine Winkler method.
-------
Figure 3 - Data Recording Sheet
Source
Number and Kinds of Individuals
Collection Date
Bioassay Date
Temperature Range
Time
0
hour
i
t
24
hour
48
hour
72
hour
96
hour
Parameter
DO, mg/1
pH
EC, jumhos/cm
EDTA, as mg/1
CaCO3
JTU initial
1 hr
Survival
DO, mg/1
Survival
DO, mg/1
Survival
DO, mg/1
Survival
DO, mg/1
PH
EC , ^wmhos/cm
EDTA, as mg/1
CaCO-*
JTU
Control
Holding
Dilution
Waste Concentrations
i
-------
100
90
80 ' 87
60
50
40
30
20
10
f
Inset indicates determination of
from percent survival and concen-
trations given in Table 2. Plot 30%
survival at 75% concentration and
60% survival at 65% concentration
Draw straight line between points.
Line intercepts 50% survival at
42 point of 68% concentration.
(For Insejt Percent Survival cjf Fish in Experiment)
i
L i ijo I 2$- |3^L. y I sp ep 7^i.. __sp ;_S^L..
Regular Arithmetic Increments of the Log Scale
Figure 4. Guide to Fish Bioassay Concentration Selection
-------
Figure 5 - Completed Data Form
Source No^TKufit' STf* Collection Date
Number and Kinds of Individuals \Q ST) cKle b*cft / )•£
9£fc C.*f«
-------
Figure 6
CALCULATIONS FOR FISH BIOASSAY STATISTICAL CONFIDENCE LIMITS
Dose
18%
32
42
56
65
75
100
Log of
Dose
1.2553
1.5052
1.6232
1.7482
1.8129
1.8751
2.0000
No.
10
10
10
10
10
10
10
Number
Dead
0
0
3
10
10
10
10
Alive
10
10
7
0
0
0
0
Accumu!
Dead1
0
0
3
13
23
33
43
ated
Alive1
27
17
7
0
0
0
0
Total
27
17
10
13
23
33
43
Cumulated
% Mort.2
0
0
30
100
100
100
100
(S.E.) Standard error = /0.79 hR
n
h = interval between doses
R = interquartile range which is TL?s - TL25- If either of these TL50
values are not found, use either 2(TLso - TL25) or 2(TLys - TLso)
as the R value.
0.79 is a constant
n = number of organisms in each concentration (use mean number if variable)
h = 0.2499 + 0.1180 + 0.1250 + 0.647 + 0.0622 + 0.1249
h = 0.1241
TL2s = 1.5052 +
30
= 1.6035 = 40.1%
TL50 = 1.6232 + 20 (0.1250) = 1.6589 = 45.6%
70"
TLy5 = 1.6232 + 45 (0.1250) = 1.7036 = 50.5%
To"
R = 1.7036 - 1.6035 = .1001
SE = /0.79 x 0.1241 x .1001 = .0313
v/ !0
95% confidence limits equal:
TLso ±1.96 (SE)
1.6589-1.96 (.0313) = 1.5976 = 39.5%
1.6589+1.96 (.0313) = 1.7202 = 52.5%
95% CL = 40% - 52%
1. Accumulative dead are derived from adding downwards in the numbers
dead column and those alive by starting at the bottom of the numbers
alive column and adding upwards.
2. Cummulative % Mortality = Accumulative dead .1 total d^tf x 100
3. Interpolation to determine log value between 0 and 30% mortality.
-------
1.
VI. Use of Random Numbers
A. A table of random numbers is given in Table 1. This
listing can be used in randomization processes needed
for sample collection or experimental design.
For sample collection, the numbers selected would
be used to pick the locations to be sampled. It
would be essentially a process of limiting the
number of sample points, all points having an equal
probability of being selected. /
In experiments the random numbers are used to
assign positions of flasks, sequence of inoculation,
etc. The uses of random tables for the two purposes
will be explained below.
First it will be necessary to select the numbers
from the table. This consists of (1) selecting
the starting point and (2) listing sequentially
a sufficient amount of numbers.
1. Selecting the starting point
Table 1 has the columns and rows each
numbered 0-49. Without looking, put your eraser
or finger-tip on any location in the table.
Assume the point is at the intersection of
column 20 and row 30. The numbers there are
4113. Then we can start using numbers at
column 41 row 13. If the number selected is
too high, just move along the row until a number
under 50 is encountered or find another starting
location.
2. Listing the numbers
Using the above location, write down the
numbers. When getting to the end of the row start
back in the reverse direction in the next lower
-------
2.
B.
row. Group the numbers singly or in pairs
depending on whether more or less than 10
samples have to be randomized.
Assume that we have 15 samples to put
in random order, then starting at our above
location we would have: 93 15 11 80 45 81
51 41 80 16 57 42 87 53 95 65 36 etc.,
continuing until we have encountered numbers
1-15. In actual practice we would not write
down the numbers until one 15 or under was
encountered.
Use of the tables in experiments
Fish bioassays
In these experiments, we would like at least
to randomize the position of the jugs on the bench,
and add 2 fish per jug in the randomized order. For
example if we had the following jugs:
Control
1% 10% 25% 50% 75% 100%
Given
Numbers 1 234567
Utilizing the single sequence of numbers above,
we would have on the bench the jugs so:
10%
Control
50%
25%
75%
100% 1%
We could re-number the jugs as they appear now
on the bench 1, 2, 3 and utilize a new selected
sequence of numbers for adding 2 more fish per jug.
However, time considerations would probably preclude
this approach, although its advantage should not be
overlooked in a completely randomized experiment.
-------
3.
Variations to the above approach, will probably
be obvious.
Algal Bioassay Flasks
Randomization is possible in the sequence of
adding algae, placement of flasks on shelves, mass
measurement order, etc. The importance of randomi-
zation in bioassays probably should be secondary to
an orderly sequence which would minimize errors.
-------
Table 1
TEN THOUSAND RANDOM DIGITS
00
01
02
03
04
05
06
07
OS
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
4")
46
47
48
49
00-04
88758
35661
26335
60826
95044
83746
27998
82685
18386
21717
18446
66027
51420
27045
13094
92382
16215
09342
30148
23689
25407
25349
02322
15072
27002
66181
097 7'J
10791
74833
17583
45G01
60683
29!.>56
91713
85704
17921
13929
03248
50583
10636
43896
76714
22393
70942
92011
66456
96292
19680
07347
05-09
66605
42832
03771
74718
99896
47694
42562
32323
13862
13141
83052
75177
96779
62626
17725
62518
50809
14528
79001
19997
37726
69456
77491
33261
31036
83316
01822
07700
55767
24038
40977
33112
81169
84235
86588
26111
71341
18880
17972
46975
41278
80963
46719
92042
60326
00126
4434!!
07146
51142
95888 59255
10-14
33843
16240
46115
56527
13763
06143
63402
74625
10988
22707
31842
47398
54309
73159
14103
17752
49326
64727
03509
72382
73099
19693
56095
99219
85278
40386
45537
87481
31312
83701
39325
65995
18877
75296
82837
35373
80188
21667
12690
09449
42205
74907
15-19
43623
77410
88133
29508
31764
42741
10056
14510
04197
68165
08634
66423
87456
91149
00067
53163
77232
71403
79424
15247
51057
85568
03055
43307
74547
54316
13128
26107
76611
28570
09286
64203
15296
69875
20-24
62774
20686
40721
91975
93970
38338
81668
85927
18770
58440
11887
70160
78967
96509
68843
63852
90155
84156
39625
80205
68733
93876
37738
39239
84809
29505
51128
24857
67389
63561
41133
18070
94368
82414
67822 95963
86494
89827
48266
48277
01311 ! 61806
00452
45986
10425
16890
93766
34672
66560
15492
02083 62428 45177
22776
86346
45685
47761 13503
26738 OI9U3
67607 i 70796
25-29 30-34
25517
26656
06787
13695
60987
97694
48744
28017
72757
19187
86070
16232
79638
44204
63565
44840
69955
34083
73315
58090
75768
18661
18216
79712
36252
86032
82703
27805
04691
00098
34031
65437
16317
05197
35-39 40-44
09560 41880 85126
59698
95962
25215
14692
69300
08400
80588
71418
08421
08464
67343
68869
92237
93578
02592
93892
35613
18811
43804
77991
69018
81781
94753
09373
34563
75350
42710
39687
60784
94867
13624
34239
66596
83021 90732
01888
65735
07229 i 71953
80201 47889
16414
46916
59967
27489
57562
16037
04186
01889
20898 i 02227 76512 i 53185
53951 10935 ' 23333 76233
01212
63881
90139
06067
49243
30875
41388
98128
03057
13706
24536 60151 05498 64678 ! 87569
06898 99137
t
50871 j 81265 42223
t (
86241 13152
60841 j 91788
72237
71039
99864
83124
14756
81133
23872
20565
36205
49062
29969
24756
88572
70445
35670
86230
94548
72641
10332
32245
41450
69471
93204
25179
63471
13596
76098
1 1849
90896
031543
13083
32661
05315
16128
83052
27964
83117
73563
22287
31748
80754
03848
13599
61375
20502
65066
83303
06337
34165
19641
19896
54937
69503
03036
74390
50036
02196
49315
10814
03107
00906
10549
99682
82693
95386
83137
84081
30944
15606
72973
86104
08804
88730
84217
75171
80945
66081
46278
64751
79328
65074
31029
02766
53947
29875
19760
64278
47491
78354
93710
10760
45-49
60755
49187
86386
73439
21297
15083
18805
76379
44037
34208
36541
59411
55109
11804
15185
90169
57002
07468
828%
22799
70138
88257
18436
53912
77209
90760
40638
23455
86850
34997
57682
71987
12242
73498
83903
13367
28782
06023
28786
95218
79033
13056
05731
96012
14964
23974
26889
60405 ! 09745
17790
48694
55413
81953
-------
VII. APPENDIX
Contents Page
Equivalent Values 1
Physical Constants 4
Specific Conductance Conversion Figure 4
Mathematical Formulae 5
Oxygen Solubility and Nomograph 6
Interconversion Tables
Centegrade to Fahrenheit . 7
Meters to Feet 8
Plankton Neeting Aperture Size and Grades 9
Sediment Size Classification 9
Sieve Scales - Wentworth, Tyler, and U.S.
Sieve Series 10
Formula Weights 11
Atomic Weights 13
Relative Humidity 14
Stock Solutions 15
Composition of Commercial Acids and Bases 15
Exponential Arithmetic 16
Significant Figures 17
Use of Logarithms and Exponents 18
-------
-1-
.KO.UVAI.KNT VALUES
Depth
\ fathom = 6 feet
= 1.829 meters
Area
1 square incli — 6.42 s(jiuirc centimeters
1 s(|imrc foot -- 929.03 square centimeters
1 S(|ii;irc yard -- 0.836 square meter
1 acre = 43,560 square feet
= 4840 square yards
= 160 square rods
= 10 square chains (Guntcr's)
= 0.4047 hectare
1 section = 640 acres
= 1 square mile
1 square mile — 640 acres
= 259 hectares
= 2.59 square kilometers
1 square millimeter = 0.0015 square inch
1 square meter — 10.758 square feet
1 hectare = 10,000 square meters
= 2.5 acres (approximately)
Volume
1 cubic inch = 16.386 cubic centimeters
1 cubic foot = 28,316 cubic centimeters
— 7.48 gallons
= 0.0283 cubic meter
1 cubic yard — 0.7646 cubic meter
1 acre-foot = 325,850 gallons
1,000,000 cubic feet = 22.95 acre-feet
1 cubic centimeter = 0.061 cubic inch
1 cubic meter -- 35.314 cubic feet
-•= 1.308 cubic yards
Length
1 inch = 25.40 millimeters
= 2.54 centimeters
1 foot = 0.305 meter
= 30.5 centimeters
1 yard = 3 feet
= 0.914 meter
1 rod = 16.5 feet
= 5.5 yards
1 mile (statute) = 63,360 inches
= 5280 feet
= 1760 yards
= 320 rods
= 1609 meters
= 1.609 kilometers
= 0.867 geographic mile
1 millimeter = 0.0393 inch
1 centimeter = 0.393 inch
1 meter = 39.37 inches
= 3.281 feet
= 1.0936 yards
= 0.000621 mile
1 kilometer = 3281 feet
= 1000 meters
1 chain (Guntcr's) = 792 inches
= 66 feet
= 4 rods
= 0.0125 mile
1 link (Guntcr's) == 7.92 inches
= 0.04 rod
1 chain (engineer's) = 100 feet
1 link (engineer's) = 1 foot
-------
F.(»UIVAU-:.NT YAI.UKS
Capacity
1 U.S. pint = 473.IS cubic centimeters
1 U.S. quart = 2 pints
= 946 cubic centimeters
= 0.946 liicr
1 U.S. gallon = 231 cubic inches
= 4 quarts
= 3784 cubic centimeters
= 3.784 liters
1,000,000 gallons = 3.07 acre-feet
1 liter = 61.027 cubic inches
= 2.11 pints
= 1.0567 quarts
= 1000 cubic centimeters
Miscellaneous
1 atmosphere pressure = about 15 pounds per square inch
= about 1 ton per square foot
= about 1 kilo per square centimeter
Angles
1 circumference = 360 degrees
1 degree = 60 minutes
1 minute = 60 seconds
METRIC SYSTEM ENGLISH SYSTEM
Units of Length
Meier (in.) = 39.37 inches (in.) Yard = 0.914-1 in.
Centimeter (cm.) = 0.01 in. Inch (U.S.) = 2.51 cm. (Fig. 1-5)
Millimeter (mm.) = 0.001 ni.
Kilometer (km.) = 1000 in. Mile (U.S.) = 1.609 km.
Angstrom unit (A.U. or A) = 10~* cm.'
Units of Volume
Liter (I.) = volume of 1 kg. of water Liquid qunrt (U.S.) = 0.9163 I.
Milliliter (ml.) = 0.001 I. Cubic foot (U.S.) = 28.316 I.
Units of Weight
Gram (it.) = weight of 1 ml. of water Ounce (oz.)(avoirdupoi3) = 28.35 g.
Ql 'V \j
Milligram (ing.) = 0.001 g. Pound (Hi.) (avoirdupois) = 0.1336 kg.
Kilogrmii (kg.) = 1000 g. Ton (short) = 907.1«3 kfj.
Ton (metric) = 1000 kg. = 2201.62 Ib. Ton (long) = 221011). = 1.016 metric tons
-------
-3-
NUMKKICAL EQUIVALENTS
l.KNGTII
1 in. = 2.540cm
1 fl = 30.48 nil
1 in'i-- 1.609km
1 cm = 0.3937 in.
1 in =39.37 in.
1 km = 0.6214 mi
1 in = 3.28 ft
Sl'KKU
15mi/lir=22ft/sec
1 mi/lir= 1.467 ft/src
1 ini/lir — 44.7 cm/sec
1 km/hr = 27.78 cm/sec
FORCE
1 g.Xvt = 980 dynes
1 kg-wt = 2.205'lb
1 ox = 28.35 R-wt
1 llj = 453.6g-\vt
'1 lh = 4.448 X 10" dynes
1 II) = 4.448 newloiis
1 ncwton — 10" dynes
1 ncwton = 3.60 oz
PRESSURE
1 in. of mercury =
1 cm of mercury
1 cm of mercury
1 ft of water
1 in. of water
1 cm of water
1 cm of water
1 Ib/in.2
1 bar =
fl.ar
1 atmosphere
1 atmosphere
= 0.491 Ib/in.2
0.1934 Ib/in.2
0.0133 bar
0.433 Ib/in.2
0.0361 Ib/in.2
0.0142lb/in.2
0.980 millibar
0.0690 bar
= 10" dynes/em2
= 14.5 Ib/in.2
= 1.0132 bars
= 14.7lb
1 atmosphere = 1.058 tons/ft2
1 atmosphere = 76 cm ol mercury
WORK AND KNKKGY
1 joule = 107 ergs
1 joule = 0.738 fi-lb
1 joule = 0.000000278 kw-hr
1 joule = O.OOOU00373 hp-hr
1 joule = 0.239 cal
1 ft-Ib= 1.35 joules
1 ft-lb= 1.35 X 107ergs
1 ft-lb = 0.324 cal
1 fi-lb= 0.001286 Htu
1 cal = 4.18 joules
1 cal = 3.086 ft-lb
1 Bin = 252 cal
1 Btu= 778ft-lb
1 Btu = 1055 joules
1 kw-hr = 3.6 X 10(i joules
1 kw-hr =2.655 X 10(i ft-lb
1 kw-hr = 1.341 hp-hr
1 hp-hr = 1.98X 10" ft-lb
1 hp-hr = 2.68 X 10" joules
1 hp-hr = 0.746 kw-hr
POWER
1 hp = 746 watts
1 hp= 178cal/sec
1 Utu/hr = 0.293 walls
1 kw= 1.34 li|>
1 wall = 0.239 cal/sec
ELECTRICAL QUANTITIES
10 amp = 1 em unit
10 coulombs = 1 em unit
1 coulomb = 3 X 10" es units
300 volts = 1 es unit
1 microfarad = 9 X 10"' es units
1 millihenry = 10''em units
-------
-4-
ACCEPTED VALUES OF CERTAIN QUANTITIES
Velocity of light in vacuo
Gravitation constant
Electronic charge
Electronic charge
Number of molecules at 0° C atmospheric pressure
Number of molecules in 1 gram-molecular weight at
0° C atmospheric pressure (Avogadro's number)
Mass of hydrogen atom
Mass of electron
Mass of electron in atomic mass units
Mass of proton
Unit of atomic mass
Unit of atomic mass equivalent to
1 electron-volt
Planck's constant (h)
299,776 km/sec
6.670X 10~8cgsunit
4.80 X 10~10esunit
1.60X 10-'9coul
2.69 X 1019 per cm2
6.0233 X 1023
1.67 X 10~24g
9.11 X 10-28g
5.486 X 10~4 amu
1.67X 10-24g.
1.660X 10-24g
0.00146 erg .
1.60X 10-12erg
6.624 X 10~27 erg-sec
SOME GEOMETRICAL RELATIONS
TT= 3.1416, or 3f approximately
Circumference of a circle = 2 TTT
Area of a circle = irr2
Area of a sphere = 4 irr2
Volume of a sphere = 3 irr3
SOME TRIGONOMETRIC RELATIONS
sin 6 = rr F or 7 = R sin 0.
cos 6 — — • or x = R cos 0.
K
n y sin 6 n
tan u — •*- = 2» ory = x tan u.
x cos o
cos
e
n A cua i/ /i
cot 0 — - — -—a' or x —y cot u.
y sin a
l./U
1.60
1.50
1.40
o
& 1.30
*5
'•a 1.20
ir
3
2
1.10
1.00
0.90
nnn
\
>
\
\
\
\
V
\
\
Factors for converting specific
conductance of water to equiva-
lent values at 25 C (based on
0.01 A/ KC1 solution).
5 10 15 20 25
Temperature of Sample-°C
30
-------
A1 ATIiI-..NiATICAi. FORMULAS
Given
Sought
Formula
Triangle
1. Base (b) and
altitude (a')
2. Area (a) and base (/;)
or altitude (a')
3. Three sides (d, d', d")
4. Base (/;) and perpen-
dicular (•/)) of right-
angle triangle
5. Base (b) or perpen-
dicular (p) and hy-
potenuse (h) of right-
angle triangle
Trapezoni
(>. Sides (s and /) and
altitude (/)
Trapezium
7. Diagonal (d) and per-
pendiculars (p and
p') to diagonal drawn
from vertices of op-
posite angles
Circle
8. Radius (r)
9. Circumference (c)
10. Radius (r)
Sphere
11. Radius (/)
12. Radius (r)
Cylinder
13. Radius (r) and
altitude (a')
Cone
14. Radius (r) and
altitude (a')
15. Radius (r) and
slant height (/.')
Frustnnn of Cone
16. Areas of both bases
(b and />') and alti-
tude (a')
17. Circumferences (c
and c') and slant
height (b)
Area (rt)
Base (b), or
altitude (rt')
Area (a)
I lypotcnusc (A)
Base (/;), or per-
pendicular (p)
Area (a)
Area (a)
b
-------
Solubility of oxygen, from a wet atmosphere at a pressure of
760 mm. Hg, in mg. per liter, at temperatures from 0° to 35° C.
Temp.
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
0.0
14.16
13.77
13.40
13.05
12.70
12.37
12.06
11.76
11.47
11.19
10.92
10.67
10.43
10.20
9.98
9.76
9.56
9.37
9.18
9.01
8.84
8.68
8.53
8.38
8.25
8.11
7.99
7.86
7.75
7.64
7.53
7.42
7.32
7.22
7.13
7.04
0.1
14.12
13.74
13.37
13.01
12.67
12.34
12.03
11.73
11.44
11.16
10.90
10.65
10.40
10.17
9.95
9.74
9.54
9.35
9.17
8.99
8.83
8.67
8.52
8.37
8.23
8.10
7.97
7.85
7.74
7.62
7.52
7.41
7.31
7.21
7.12
7.03
0.2
14.08
13.70
13.33
12.98
12.64
12.31
12.00
11.70
11.41
11.14
10.87
10.62
10.38
10.15
9.93
9.72
9.52
9.33
9.15
8.98
8.81
8.65
8.50
8.36
8.22
8.09
7.96
7.84
7.72
7.61
7.51
7.40
7.30
7.20
7.11
7.02
0.3
14.04
13.66
13.30
12.94
12.60
12.28
11.97
11.67
11.38
11.11
10.85
10.60
10.36
10.13
9.91
9.70
9.50
9.31
9.13
8.96
8.79
8.64
8.49
8.34
8.21
8.07
7.95
7.83
7.71
7.60
7.50
7.39
7.29
7.20
7.10
7.01
0.4
14.00
13.63
13.26
12.91
12.57
12.25
11.94
11.64
11.36
11.08
10.82
10.57
10.34
10.11
9.89
9.68
9.48
9.30
9.12
8.94
8.78
8.62
8.47
8.33
8.19
8.06
7.94
7.82
7.70
7.59
7.43
7.38
7.28
7.19
7.09
7.00
0.5
13.97
13.59
13.22
12.87
12.54
12.22
11.91
11.61
11.33
11.06
10.80
10.55
10.31
10.09
9.87
9.66
9.46
9.28
9.10
8.93
8.76
8.61
8.46
8.32
8.18
8.05
7.92
7.81
7.69
7.58
7.47
7.37
7.27
7.18
7.08
6.99
0.6
13.93
13.55
13.19
12.84
12.51
12.18
11.88
11.58
11.30
11.03
10.77
10.53
10.29
10.06
9.85
9.64
9.45
9.26
9.08
8.91
8.75
8.59
8.44
8.30
8.17
8.04
7.91
7.79
7.68
7.57
7.46
7.36
7.26
7.17
7.07
6.98
0.7
13.89
13.51
1,3.15
12.81
12.47
12.15
11.85
11.55
11.27
11.00
10.75
10.50
10.27
10.04
9.83
9.62
9.43
9.24
9.06
8.89
8.73
8.58
8.43
8.29
8.15
8.02
7.90
7.78
7.67
7.56
7.45
7.35
7.25
7.16
7.06
6.97
0.8
13.85
13.48
13.12
12.77
12.44
12.12
11.82
11.52
11.25
10.98
10.72
10.48
10.24
10.02
9.81
9.60
9.41
9.22
9.04
8.88
8.71
8.56
8.41
8.27
8.14
8.01
7.89
7.77
7.66
7.55
7.44
7.34
7.24
7.15
7.05
6.96
0.9
13.81
13.44
13.08
12.74
ll41
12.09
11.79
11.50
11.22
10.95
10.70
10.45
10.22
10.00
9.78
9.58
9.39
9.20
9.03
8.86
8.70
8.55
8.40
8.26
8.13
8.00
7.88
7.76
7.65
7.54
7.43
7.33
7.23
7.14
7.05
6.95
n F jctt»t lor Otyftn
n «t Various A
Altitude
FW
0
110
6'j5
980
1310
1640
1970
2OT
2950
3260
3(10
3940
4270
4600
4910
5250
5WO
5910
6240
6560
6900
7?1'0
?yx>
Mrirei
0
ion
200
100
400
' 500
600
700
900
1000
1100
lAiO
1300
1400
T.OG
1(05
imO
1800
1'IUrt
?oco
?l«l
2?00
2(110
Plt\Mt
mm
760
750
741
732
721
714
705
696
687
679
671
661
6
-------
TEMPERATURES—CENTIGRADE TO FAHRENHEIT*
Ttwp. ° C. | 0
0
10
20
50
40
50
32.0
50.0
68.0
86.0
104.0
122.0
;
33.8
51.8
69.8
87.8
105.8
123.8
2
35.6
53.6
71.6
89.6
107.6
125.6
3
37.4
55.4
73.4
91.4
109.4
127.4
4
39.2
57.2
75.2
93.2
111.2
129.2
5
41.0
59.0
77.0
95.0
113.0
131.0
C
42.8
60.8
7.8.8
96.8
114.8
132.8
7
44.6
62.6
80.6
98.6
116.6
134.6
5
46.4
64.4
82.4
100.4
118.4
136.4
9
48.2
66.2
84.2
102.2
120.2
138.2
•Temperatures in degrees Centigrade expressed in left vertical column and in top horizontal row; corresponding temperatures in
degrees Fahrenheit in body of table.
TEMPERATURES—FAHRENHEIT TO CENTIGRADE*
Temp. ° /•'.
30
40
50
60
70
SO
90
100
0
- 1.11
4.44
10.00
15.56
21.11
26.67
32.22
37.78
/
- 0.56
5.00
10.56
16.11
21.67
27.22
32.78
38.33
•7
0.00
5.56
11.11
16.67
22.22
27.78
33.33
38.89
3
0.56
6.11
11.67
17.22
22.78
28.33
33.89
39.44
4
1.11
6.67
12.22
17.78
23.33
28.89
34.44
40.00
5
1.67
7.72
12.78
18.33
23.89
29.44
35.00
40.56
c
2.22
7.78
13.33
18.89
.24.44
30.00
35.56
41.11
-
2.78
8.33
13.89
19.44
25.00
30.56
36. !1
41.67
5
3.33
8.89
14.44
20.00
25.56
31.11
36.67
42.22
9
3.89
9.44
15. CO
20.56
26.11
31.67
37.22
L ? ~C
. _ . / \j
'Temperatures in degrees Fahrenheit expressed in left vertical column and in top horizontal rov.-; corresponding temperatures in
decrees Centigrade in bodv of table.
-------
METERS TO FEET*
Meters
0
111
20
30
41)
50
60
70
80
90
100
0
0.00
32.S1
65.62
98.43
131.24
164.04
196.85
229.66
262.47
295.28
328.09
;
3.28
36.09
68.90
101.71
1 34.52
167.33
200.13
232.94
265.75
298.56
331.37
2
6.56
39.37
72.18
104.99
137.80
170.61
203.42
236.22
269.03
391.84
334.65
3
9.84
42.65
75.46
108.27
141.08
173.89
206.70
239.51
272.31
305.12
337.93
4
13.12
45.93
7S.74
111.55
144.36
177.17-
209.98
242.79
275.60
308.40
341.21
5
16.40
49.21
82.02
114.83
147.64
1 80.45
213.26
246.07
278.88
3 1 1 .69
344.49
6
19.69
52.49
85.30
118.11
150.92
183.73
216.54
249.35
282.16
314.97
347.78
7
22.97
55.78
88.58
121.39
154.20
187.01
219.82
252.63
285.44
318.25
351.06
5
26.25
59.06
91.87
124.67
157.48
190.29
223.10
255.91
288.72
321.53
'354.34
9
29.53
62.34
95.15
127.96
160.76
193.57
226.38
259.19
292.00
324.81
357.62
"Length in meters expressed in left vertical column and in top horizontal row; corresponding lengths in feet in body of tal>lc.
I
CO
I
FEET TO METERS*
t'ett
0
10
20
30
40
50
60
70
80
90
100
0
0.000
3.048
6.036
9.144
12.192
15.239
18.287
21.335
24.383
27.431
30.479
;
0.305
3.353
6.401
9.449
12.496
15.544
18.592
21.640
24.688
27.736
30.784
2
0.610
3.658
6.706
9.753
12.801
15.849
18.897
21.945
24.993
28.041
31.089
i
0.914
3.962
7.010
10.058
13.106
16.154
19.202
22.250
25.298
28.346
31.394
•?
1.219
4.267
7.315
10.363
13.411
16.459
19.507
22.555
25.602
28.651
31.698
5
1.524
4.572
7.620
10.668
13.716
16.763
19.811
22.859
25.907
28.955
32.003
6
1.829
4.877
7.925
10.972
14.020
17.068
20.116
23.164
26.212
29.260
32.308
7
2.134
5.182
8.229
11.277
14.325
17.373
20.421
23.469
26.517
29.565
32.613
8
2. 438
5.486
. 8.534
1 1.5 8 2
14.630
17.678
20.726
23.774
26.822
29.870
32.918
9
2.743
5.791
8.839
11.8S7
14.935
17.983
21.031
24.079
27.126
30.174
33.222
'Length in feet expressed in left vertical column and in top horizontal row; corresponding lengths in meters in body of table.
-------
-9-
AU.U.\<;K A
01 SI.\M>AIU> (JKAUK ])KHU:K HOI.TIM; SII.K
Silk No.
0000
000
00
0
1
2
3
4. •
5
6
7
8
9
Mesbe*
per
Inch
IS
23
29
3S
•IS
54
58
62
66
74
82
86
97
,S/:c of
Aperture
(linn.)
1.364
1.024
0.752
0.569
0.417
0.366
0.333
0.318
0.282
0.239
0.224
0.203
0.168
Silk No.
10
11
12
13
14
15
16
17
18
20
21
25
/I/.'J/.'O
per
Inch
109
116
125
129
139
150
157
163
166
173
178
200
She of
Aperture
(•linn.)
0.158
0.145
0.119
0.112
0.099
0.094
0.08'6
0.081
0.079
0.076
0.069
0.064
GRADES AND Sl/K KAMil'.S OK SlLK Bol.TING Cl.OTII
Grade
Sundard
X quality
XX quality
XXX qunlitv
Grit gauze
XXX Grit gauze
Jtaiige of Sizes
Nos. 0000-25
Nos. 6-17
Nos. 0000-16
Nos. 6-18
Nos. 14-72
Nos. 14-72
WENTWORTH'S CLASSIFICATION OF COARSER SEDIMENTS BASED UPON SIZE
OF PARTICLES
Diameter of I'article
in win.
More than 256
256-64
64-4
4-2
2-1
1-0.5
0.5-0.25
0.25-0.125
0.125-0.062
0.062-0.004
Less than 0.004
Name Applied to
Particle
Boulder
Cobble
Pebble
Granule
Very coarse sand
Coarse sand
Medium sand
Fine sand
Very fine sand
Silt
Clay
-------
-10-
WEMAVOKTH GRADE SCAI.K, \/2 Sc.\i >. $2 SCAII:. COKHKSTONDING TYLKR
SIEVE OPKMM;S AND MESH, AND COKI-.HSI'ONDING MKSII OK U.S. SIEVE
SERIES
Wentwonh Grade
Scale
(iinn.)
4
Granule
2
Very coarse sand
1
Coarse sand
0.500 (%)
Medium sand
0.250(14)
Fine sand
0.125 (y8)
Very fine sand
0.062 ('/!„)
Silt
The Openings
Increase in the
Ratio of
\fior
1.414 mm.
4.00
2.83
2.00
1.41
1.00
0.707
0.500
0.354
0.250
0.177
0.125
O.OSS
0.062
•^/2 or
1.189wm.
4.00
3.36
2.83
2.38
2.00
1.68
1.41
1.19
1.00
0.840
0.707
0.595
0.500
0.420
0.354
0.297
0.250
0.210
0.177
0.149
0.125
0.105
0.08S
0.074
0.062
Tyler Screens
Mm.
3.96
3.33
2.79
2.36
1.98
1.65
1.40
1.17
0.991
0.833
0.701
0.589
0.495
0.417
0.351
0.295
0.246
0.208
0.175
0.147
0.124
0.104
O.OSS
0.074
0.061
Mesh
5
6
7
8
9
10
12
14
16
20
24
28
32
35
42
48
60
65
80
100
115
150
I/O
200
250
U.S. Sieve
Series,
Mesh
5
6
7
8
10
12
14
16
18
20
25
30
35
40
45
50
60
70
80
100
120
140
170
200
230
-------
11-
•;:; i q.
AgBr
AgBrOj
AgCNS
AgCI
Ag,CrO4
Agl
AglO,
AgNO,
Ag2O
Ag,PO4
Ag2S
Al,0,
AI(OH),
AI2(S04),
AsjO, _...
As205
As2S,
BaCO)
Ba(CNS)2
BaCI2
Ba(CIO«),
BaCrO4
BaO
BaO2
Ba(OH)z
Ba,(P04)2
BaSO4
Bi2S,
Cai(AsO4)2
CaBr,
CaCO,
CaC2O4
CaF,
Ca(IO,),
CaO
Ca(OH)2
Ca,(P04),
CaSO4
Ce02
Ce(S04)2
H,Ce(S04)4
(NH4),Ce(NO,)t
(NH,)2Ce(S04),-2H20
CO,
CO(NH2)2 (urea)
Cr,0,
CuCO,
Cul
CuO
Cu;O
Cu5O4-5H2O
187.80
235.80
165.96
143.34
231.77
. 234.79
282.79
169.89
231.76
418.62
247.83
101.96
78.00
342.16
. 197.82
229.82
246.02
197.37
253.53
208.27
336.27
253.37
153.36
169.36
171.38
602.03
233.43
514.20
398.06
199.91
100.09
128.10
78.08
389.90
56.08
74.10
310.19
136.15
172.13
332.26
528.42
548.26
500.44
44.01
60.06
1 52.02
123.55
190.45
79.54
143.08
249.69
CuS
Cu2S
FeCO,
Fe(Cr02)2
FeO
FeiOj __
Fe,04
Fe(OH),
Fc(OH),..
FeS2
FeSO.-7H2O
FeSO4-(NH4)2SO4-6H2O
Fe2(S04),
HBr
H2C2O4-2H2O (oxalic) ._
HC;H,O2 (acetic)
HC,H5O2 (benzoic)
HCI .
HCIO4
HNO,
HNH2SO, (sulfamic)
H202
H,P04
H,S
H2SO,
H2SO4
Hg(NO,)2
HgO
HgS
Hg2Br2
Hg2CI2
Hg2l2
KBr
KBrO,
KCN
KCNS
K2CO,
KCI
KCI03
KCIO4
K2Cr04
K2Cr20,
K,Fe(CN)t
K«Fc(CN)»
KHC2O4
KHC2O4-H2C2O4-2H2O
KHC4H4O4 (tartrate)
KrjC9H4O4 (phthalate)
KH(IO,)2
KH;P04
K,HPO4 f
159 11
1 1 5 •:,'
223 :.,'
71.8S
.159.7 '3
231 5S
8987
.10637
1199i
27803
392.16
399.9:)
80.92
126.07
60.05
122.12
36.46
100.46
63.02
97.10
34.02
98.00
34.03
82.03
98.08
324.63
21661
232.68
561.05
472.13
655.04
119.02
167.02
65.12
97.19
138.21
74.56
122.56
138.56
194.2!
294.2'.?
329.26
363.36
128.13
25-M'-1
183.1;;
20-5.2;
389. V;
137CV
175 1:
-------
-12-
no,
MO.
t '.V'O,
>->.,O.
••jo', .
» o
r.OH
K.PlCI,
>. so,
I. CO)
Id .. . ._....
i. .so,
VqCO,
Vg CIO<),
M(jNH,PO4
WqO
Vq OH),
Mq P,O,
MqSO.
M-iO,
Mn.O,
Mn:O,
MM 'OH),
V.o.P.Q,
No.AiO,
No.B.O,
NaBr
NoBrO,
NaC.H.O, .
NoCN
NoCNS
No;CO,
No.C;O,
NnCI
NoCIO
NuCIO,
NnHCO,
^J(ll
NoNO,
No O
-'•'O.O; .. . . ..'
f.'nOH
N» PO,
••o.S
'•!• SO,
•o.SO.
'• - S;O, 5H,O
NH,
NI'<,
ff-'i.KiO,
166.01
214.01
230.01
158.03
85.11
101. VI
94.20
56.11
486.03
174.27
73.89
. 42.40
109.95
84.33
223.23
137.34
40.32
58.34
222.59
120.39
86.94
157.88
228.82
88.96
283.83
191.88
201.26
102.91
150.91
82.03
49.01
81.08
106.00
134.01
58.45
74.45
90.45
84.02
149.90
85.00
61.98
. 77.98
40.00
163.95
78.05
126.05
142.05
248.19
17.03
32.05
124.10
^
r.
NH4CI
NH4NO,
NH4OH
(NH4)3PO4-12MoO,
(NH4)2PtCI6
(NH4)2S04 .
P2O5
PbCO,
PbC2O4
PbCrO4
Pbl2
.Pb(l03)2
PbMoO4
Pb(N03)2
PbO
PbO2
Pb304
Pb3(P04)2
PbSO4
Sb203
Sbz04
Sb2O5
Sb2S3
Si02
SnCI2
SnO2
SO,
S03
SrCO,
SrC2O4
SrO
Sr3(P04)2
SrSO4
TiO2
UF6
U03
U30,
V205
ZnBr2
ZnO
Zn2P2O7
ZnS
ZnSO4
Wafer for Hydrates:
1 H2O
2 H2O
3 H20
4 H2O
5 H2O
6 H2O
7 H2O
53.50
80.05
35.05
1876.50
443.91
132.15
141.95
267.22
295.23
323.22
461.03
557.03
367.16
331.23
223.21
239.21
685.63
811.58
303.27
291.52
307.52
323.52
339.72
60.09
189.61
150.70
64.07
80.07
147.64
175.65
103.63
452.84
183.70
79.90
352.07
286.07
842.21
181.90
225.21
81.38
304.71
97.45
161.45
18.02
36.03
54.04
72.06
90.08
108.10
126.11
-------
Element
Actinium
Aluminum
Americium
Antimony
Argon
Arsenic
Astatine
Barium
Berkclium
Beryllium
Bismuth
Boron
Bromine
Cadmium
Calcium
Californium
Carbon
Cerium
Cesium
Chlorine
Chromium
Cobalt
Columbium (see
Copper
Curium
Dysprosium
Einsteinium
Erbium
Europium
Fermium
Fluorine
Francium
Gadolinium
Gallium
Germanium
Gold
Hafnium
Helium
Holmium
Hydrogen
Indium
Iodine
Iridium
Iron
Krypton
Lanthanum
Lead
Lithium
Lutetium
Magnesium
Manganese
-
Svmbol
"M'^
Al
Am
Sb
A
As
At
Ba
Bk
Be
Bi
B
Br
Cd
Ca
Cf
C
Ce
Cs
Cl
Cr
Co
Niobium)
Cu
Cm
Dy
E
Er
Eu
Fm
F
Fr.
Gd
Ga
Ge
Au
Hf
He
Ho
H
In
1
Ir
Fe
Kr
La
Pb
Li
Lu
my
Mn
' ' Oj
At.
89
13
95
51
18
33
85
56
97
4
83
5
35
48
20
98
6
58
55
17
24
27
29
96
66
99
68
63
100
9
87
64
31
32
79
72
2
67
1
49
53
77
26
36
57
82
3
71
12
25
.*«•
uJ^-
No At.Wt.
227
2698
[2431
12176
39.944
74.91
[210|
137.36
[245|
9.013
209.00
10.82
79.916
112.41
40.08
|248|
12.011
140.13
132.91
35.457
52.01
58.94
63.54
[245]
162.51
[254|
167.27
152.0
[252|
19.00
[2331
157.26
69.72
72.60
1970
178.59
4.003
164.94
1.0030
114.82
12691
1922
5585
83 GO
13392
20/21
6 940
17499
243?
5474
:y)slXfi§
Element
... .. H-'levium
f.-tcuiy
11 •:)-bclenum
'.-odyinium
•,r on
•. j.timium
-. ,l.rl
'. rbium
•. irogen
Oi mium
Cuygcn
fcjiladium
Frospliorus
r.otinum
Plutonium
Polonium
Potassium •
Praseodymium
Promethium
Protoactinium
Radium
Radon
Rhenium
Rhodium
'.'uhidium
Ruthenium
Samarium
Scandium
Selenium
Silicon
Silver
Sodium
Strontium
Sulfur
Tantalum
Technetium
Tellurium
Terbium
Thallium
Thorium
Thulium
Tin
Titanium
Tungsten
Uranium
Vanadium
Xenon
Ytterbium
Yttrium
Zinc
Zirconium
Symbol
Mv
Hg
Mo
Nd
Ne
Np
Ni
Nb
N
Os
O
Pd
P
Pt
Pu
Po
K
Pr
Pm
Pa
Ra
Rn
Re
Rh
Rb
Ru
Sm
Sc
Se
Si
Ag
Na
Sr
S
Ta
Tc
Te
Tb
Tl
Th
Tm
Sn
Ti
W
U
V
Xe
Yb
Y
Zn
Zr
At.
101
80
42
60
10
93
28
41
7
76
8
46
15
78
94
84
19
59
61
91
88
86
75
45
37
44
62
21
34
14
47
11
38
16
73
43
52
65
81
90
69
50
22
74
92
23
54
70
39
30
40
NO At. Wt
[2561
200.61
95.95
144.27
20.183
[237]
58.71
92.91
14.008
190.2
16.
106.4
30.975
195.09
[242]
210.
39.100
140.92
[145]
231.
226,05
222.
186.22
102.91
8548
101.1
150.35
44.96
78.96
28.09
107.880
22.991
87.63
32.066
180.95
[99]
127.61
158.93
204.39
232.05
168.94
118.70
47.90
183.86
238.07
50.95
131.3
173.04
88.92
65.38
91.22
-------
RELATIVE HUMIDITY
Dry-Bulb Ther-
mometer: Degrees,
Fahrenheit
50
52
54
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
Difference between Dry-Bulb and Wet-Bulb Thermometers
1°
93
94
94
94
94
94
95
95
95
95
95
95
95
95
96
96
96
96
96
96
96
96
96
96
2°
87
87
88
S3
89
89
89
90
90
90
90
91
91
91
91
92
92
92
92
92
92
92
93
93
3°
80
81
82
82
83
84
84
85
85
85
86
86
86
87
87
87
88
88
88
88
88
89
*t
89
4°
74
75
76
77
78
78
79
79
80
81
81
82
82
82
83
83
84
84
84
85
85
85
86
86
5°
67
69
70
71
72
73
74
74
75
76
77
77
78
78
79
79
80
80
81
81
81
82
82
82
6°
61
63
64
65
67
68
69
70
71
71
72
73
74
74
75
75
76
77
77
77
78
78
79
79
7°
55
57
59
60
61
63
64
65
66
67
68
69
70
70
71
72
72
73
73
74
75
75
75
76
8°
50
51
53
55
56
58
59
60
61
63
64
65
66
66
67
68
69
69
70
71
71
72
72
73
9°
44
46
48
50
51
53
54
56
57
58
60
61
62
63
63
64
65
66
67
67
68
69
69
70
10°
38
40
43
44
46
48
50
51
53
54
55
57
58
59
60
61
62
63
63
64
65
65
66
67
11°
33
35
38
40
42
44
45
47
49
50
52
53
54
55
56
57
58
59
60
61
62
62
63
64
12°
27
30
32
35
37
39
41
43
45
46
48
49
50
52
53
54
55
56
57
58
59
59
60
61
13°
22
24
28
30
33
34
37
38
40
42
44
45
47
48
49
51
52
53
54
55
56
56
57
58
14°
16
20
23
25
28
30
32
34
36
38
40
42
43
45
46
47
48
49
51
52
53
54
54
55
15°
11
15
IS
21
24
26
28
30
32
34
36
38
40
41
43
44
45
46
48
49
50
51
52
53
16°
6
10
13
16
19
22
24
27
29
31
33
35
36
38
39
41
42
44
45
46
47
48
49
50
17°
1
5
8
12
15
18
20
23
25
27
29
31
33
35
36
38
39
41
42
43
44
45
46
47
18°
0
0
4
8
11
14
16
19
22
24
26
28
30
31
33
35
36
38
39
40
41
43
44
45
*>.
I
-------
-15-
Slock Solutions of Cations (SO mg. of cation per ml.)
Croup Ion Formula of Sail
I AR+ ABNO,
Pl>++ P1)(NO3),
Ilg,-1-1" Hg,(NO,),
II Pb++ P1>(NO,),
Bi+++- ]Ji(NO,),-5 H2O
Cu++ Cu(NO,V3 l(,0
Cd++ Cd(NO,),-4 11,0
Hg++ HgCli
As++t As4O<
Sb+++ SbCI,
Sn++ SnCli-2 H,O
Sn++++ SnCl4-3H20
III Co++ Co(NO,),-6 H,O
Ni++ Ni(NO,),-6 II 5O
Mn+* Mn(NO,)j-6 II,O
Fe*++ Fc.(NOi)j-9 HjO
Al+++ AI(NO,),-9 11,0
Cr+++ Cr(NO,),
Zn++ /n(NO,),
IV Ba++ BaCI,-2II,O
Sr++ Sr(NO,),
Ca++ Ca(NO,)s-41I20
V MB++ Mg(NO,),-6H,0
NH«+ NII«NO,
Na+ NaNO,
K+ KNO,
8.0
8.0
7.0
8.0
11.5
19.0
13.8
6.8
3.3
9.5
9.5
13".3
21.7
21.8
26.2
36.2
69.5
23.0
11.5
8.9
12.0
29.5
52.8
22 2
18.5
13.0
Crams />cr i(>0 ml. cij Solution
(dissolve in 0.6 M UNO,)
(dissolve in 3 M UNO,)
(heal in 50 ml. of 12 M HCI, then
tidd 50 nil. of wuler)
(dissolve in 6 M 1ICI, and dilute
with 2 M IIC1)
(dissolve in ,r,0 ml. of 12 M I1CI.
Dilute to 100 ml. with water.
Add a piece of tin metal)
(dissolve in 6 M 1IC1)
Composition of Commercial Acids and Bases
Arid or Base
Hydrochloric
Nitric
Sulfuric
Aortic
Atjucous ammonia
Specific
Gravity
1.19
1.42
1.84
1.05
0.90
I'ercenlage
by Weight
38
70
95
99
28
Molarily
12.4
15.8
17.8
17.3
14.8
Normality
12.4
15.8
35.6
17.3
14.8
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Exponential Arithmetic
-16-
In chemistry we use the exponential method of expressing very large and very
small numbers. These numbers arc expressed as a product of two numbers. Tin-
first number of the product is called the digit term. This term is usually a ninnlxT
not less than 1 and not greater than 10. The second number of the product i*
called the exponential term and is written as 10 with an exponent. Some example*
of the exponential method of expressing numbers are given below.
1000 = 1 X 103
100 = 1 X 10*
10 = 1 X 101
1 = 1 X 10°
0.1 = 1 X 10-'
0.01 = 1 X 10-2
0.001 = 1 X 10-»
2386 = 2.386 X 1000 = 2.386 X 10*
0.123 = 1.23 X .1 = 1.23 X 1f
10 and take the difference of the digit terms.
EXAMPLE. Subtract 4 X I0~7 from 5 X lO"6
SOLUTION. 4 X 10~7 = 0.1 X 10-"
(5 X lO"6) - (0.4 X 10 '6) = 4.6 X 10-'
3. Multiplication of Exponentials. Multiply the digit terms in the usual way
and add algebraically the exponents of the exponential terms.
EXAMPLE. Multiply 4.2 X 10~8 by 2 X 103
SOLUTION. 4.2 X 10~8
2 X 103
8.4 X 10-»
4. Division of Exponentials. Divide the digit term of the numerator by the
digit term of the denominator and subtract algebraically the exponents of the
exponential terms.
EXAMPLE. Divide 3.6 X 10~6 by 6 X 10~4
16x1 fl-~6
SOLUTION, '-j^p- - 0.6 X 10~l = 6 X 10~2
5. The Squaring of Exponentials. Square the digit term in the usual way and
multiply the exponent of the exponential term by 2.
ExAMPLK. Square the number 4 X 10~6
SOLUTION. (4 X l()-«)5 = 16 X 10-'- = 1.6 X 10"11
6. The Cubing of Exponentials. Cube the digit term in the usual way and
multiply the exponent of the exponential term by 3.
EXAMPLE. Cube the number 2XI03
SOLUTION. (2 X 103)3 = 2x2x2x109 = 8x H)9
7. Extraction of Square. Hoots of Exponentials. Decrease or increase the expo-
nential term so that the power of ten is evenly divisible by 2. Extract the square
root of the digit term by inspection or by logarithms and divide the exponential
term by 2.
EXAMPLE. Extract the square root of 1.6 X 10~7
SOLUTION. 1.6 x 10~7 = 16 x 10~8
VI6 X 10-" - VI6 X X/lF5 = 4 X 10-4
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-17-
Significant Figures
A bee keeper reports thai lie has 525,341 bees. The last three figures of the num-
ber are obviously inaccurate, for during the time the keeper was counting the
bees, some of them would have died and others would have hatched; this would
have made the exact number of bees quite difficult to determine. It would have
IKX-II more accurate if he had reported the number 525,000. In other words, the
last three figures arc not, significant, except to set the position of the decimal point.
Their exact values have no meaning.
In reporting any information in terms of numbers, only as many significant
figures should be used as are warranted by the accuracy of the measurement. The
accuracy of measurements is dependent upon the sensitivity of the measuring
instruments used. For example, if the weight of an object has been reported as
2.13 g., it is assumed thai the last figure (3) has been estimated and that the weight
lies between 2.125 g. and 2.135 g. The quantity 2.13 g. represents three signifi-
cant figures. The weight of this same object as determined by a more sensitive
balance may have been reported as 2.131 g. In this case one would assume the
correct weight to be between 2.1335 g. and 2.1315 g., and the quantity 2.134 g.
represents 4 significant figures. Note that the last figure is estimated and is also
considered as a significant figure.
A zero in a number may or may not be significant, depending upon the manner
in which it is used. When one or more zeros are used in locating a decimal point,
they are not significant. For example, the numbers 0.063, 0.0063, and 0.00063,
each have two significant figures. When zeros appear between digits in a number
they arc significant. For example, 1.008 g. has four significant figures. Likewise,
the zero in 12.50 is significant. However, the quantity 1370 cm. has four signifi-
cant, figures provided the accuracy of the measurement includes the zero as a sig-
nificant digit; if the digit 7 is estimated, then the number has only three significant
figures.
The importance of significant figures lies in their application to fundamental
compulation. When adding or subtracting, the last digit that is retained in the
sum or difference should correspond to the first doubtful decimal place (as indi-
cated by underscoring).
KXAMPLK. Add 4.383 g. and 0.0023 g.
SOLUTION. .4.383 g.
0.0023
4.385 g.
When multiplying or dividing, the product or quotient should contain no more
digits than the least number of significant figures in the numbers involved in the
computation.
EXAMPI.K. Multiply 0.6238.by 6.6
SOLUTION. 0.6238 X 6.6 = 4J.
In rounding off numbers, increase the last digit retained by one if the following
digit is five or more. Thus 26.5 becomes 27, and 26.4 becomes 26 in the rounding-
off process.
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-18-
The Use of Logarithms and Exponential Numbers
The common logarithm of a number is (lie power to which the number 10 must
be raised to equal that number. For example, the logarithm of 100 is 2 because the
number 10 must be raised to the second power to be equal to 100. Additional
examples are as follows:
Number
10,000
1,000
10
1
0.1
0.01
0.001
0.0001
Number Erpresaed
Exponentially
10«
10'
10'
10°
10-'
io-»
10-'
io-«
IjMjarithm
1
3
1
0
-I
9
-3
—I
What is the logarithm of 60? Because 60 lies between 10 and 100, which have
logarithms of 1 and 2, respectively, the logarithm of 60 must lie between 1 and 2.
The logarithm of 60 is 1.7782, i.e., 60 - 101-7785.
Every logarithm is made up of two parts, called the characteristic and the man-
tissa. The characteristic is that part of the logarithm which lies to the left of the
decimal point; thus the characteristic of the logarithm of 60 is 1. The mantissa
is that part of the logarithm which lies to I he right of the decimal point; thus
the mantissa of the logarithm of 60 is .7782. Tin- characteristic, of the logarithm
of a number greater than 1 is one less than the number of digits to the left of the
decimal point in the number.
Number
60
600
6000
52840
Clmraclerislic
1
2
3
4
Number
2.340
23.40
234.0
2340.0
Characteristic
0
1
2
3
The mantissa of the logarithm of a number is found in the logarithm table (see
Appendix B), and its value is independent of the position of the decimal point.
Thus 2.340, 23.40, 234.0, and 2340.0 all have the same mantissa. The logarithm
of 2.340 is 0.3692, that of 23.40 is 1.3692, that of 234.0 is 2.3692, and that of 2340.0
is 3.3692.
The meaning of the mantissa and characteristic can be better understood from
a consideration of their relationship to exponential numbers. For example, 2310
may be written 2.34 X 10s. The logarithm of (2.34 X 10') = the logarithm of
2.34 + the logarithm of 101. The logarithm of 2.34 is .3692 (mantissa) and the
logarithm of 10* is 3 (characteristic). Thus the logarithm of 2340 = 3 + .3692,
or 3.3692.
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-19-
The logarithm of a number less than 1 has a negative value, and a convenient
method of obtaining the logarithm of such a number is given below. For example,
we may obtain the logarithm of .00231 as follows: When expressed exponentially,
.00231 = 2.31 X 10~3. The logarithm of 2.31 X 10 ' = the logarithm of 2.31 + the
logarithm of I0~3. The logarithm of 2.31 is .3692 (mantissa) and the logarithm of
10-3 is -3 (characteristic). Thus the logarithm of .00231 = .3692 + (-3) = .3692 -
3 = -2.6208. The abbreviated form for the expression (.3692 - 3) is 3.3692.
Note that only the characteristic has a negative value in the logarithm 3.3692,
and that the mantissa is positive. The logarithm 3.3692 may also be written as
7.3692 - 10.
To multiply two numbers we add the logarithms of the numbers. For example,
suppose we multiply 412 by 353.
logarithm of 412 =2.6119
Logarithm of 353 = 2.5478
Logarithm of product = 5.1627
The number which corresponds to the logarithm 5.1627 is 145400 or 1.454 X 10s.
Thus 1.45 X I05 is (lie product of 412 and 353.
To divide two numbers we subtract the logarithms of the numbers. Suppose
we divide 412 by 353.
Logarithm of 412 = 2.6149
Logarithm of 353 = 2.5478
Logarithm of quotient = 0.0671
The number which corresponds to the logarithm 0.0671 is 1.17. Thus 412 divided
by 353 is 1.17.
Suppose we multiply 5132 by 0.3121. Add the logarithm of 0.3124 to that of
5432.
Logarithm of 5132 = 3.7350
Logarithm of 0.3124 = 1.4918
Logarithm of the product = 3.2298
The number which corresponds to the logarithm 3.2298 is 1697 or 1.697 X 10s.
Let us divide 5132 by 0.3121. Subtract the logarithm of 0.3124 from that of
5432.
Logarithm of 5432 = 3.7350
Logarithm of 0.3124 = 1.4948
Logarithm of the quotient = 4.2402
The number which corresponds to the logarithm 4.2102 is 17390 or 1.739 X 10*.
The extraction of roots of numbers by means of logarithms is a simple pro-
ccdure. For example, suppose we extract the cube root of 7235. The logarithm
of >X7235 or (7235)* is equal to % of the logarithm of 7235.
Logarithm of 7235 = 3.8594
£of 3.8591 = 1.2865
The number which corresponds to the logarithm 1.2865 is 19.34. Thus, 19.34
is the cube root of 7235.
•& GPO 7!XU2:t
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