3-11-95
Derivation of Conversion Factors for the Calculation
of Dissolved Freshwater Aquatic Life Criteria for Metals
Charles E. Stephan
Environmental Research Laboratory - Duluth
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804

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NOTICES
This document has NOT been reviewed in accordance with U.S.
Environmental Protection Agency policy and approved for
publication.
This document does not constitute rulemaking by the Agency and
may not be relied on to create a substantive or procedural right
enforceable by any other person. The Government may take action
that is at variance with information stated in this document.
This document does not establish a binding norm or prohibit
alternatives not included in the document. It is not finally
determinative of the issues addressed. Agency decisions in any
particular case will be made by applying the law and regulations
on the basis of specific facts when regulations are promulgated
or permits are issued.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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ABSTRACT
This document presents information concerning the percent of the
total recoverable metal that was dissolved in toxicity tests with
freshwater organisms. Under contract with the U.S. EPA, the
University of Wisconsin at Superior simulated conditions that
existed in important aquatic toxicity tests with metals. The
final report by UWS presents a detailed description of the
methodology used, the raw data, and some summary statistics. The
data from these simulation tests are analyzed and interpreted
herein. For the metals for which simulation tests were
conducted, the recommended conversion factors range from 0.316
for the CMC for chromium(III) to 1.000 for the CMC and the CCC
for arsenic(III). The results of these simulation tests should
provide a basis for making reasonable decisions concerning
conversion factors for deriving dissolved criteria from total
recoverable criteria for aquatic life in fresh water.
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CONTENTS
Notices	ii
Abstract	iii
Appendices 	 v
Acknowledgments 	 vi
1.	Introduction 	 1
2.	Design of the Project	3
3 . Results			20
4.	Discussion		29
5.	References	35
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APPENDICES
A.	Copper		A-l
B.	Zinc			B-l
C.	Chromium (I II) 		C-l
D.	Lead 		D-1
E.	Arsenic(III)	E-l
F.	Chromium (VI)	1	F-l
G.	Selenium		G-l
H.	Nickel	H-l
I.	Cadmium	1-1
J.	Calculation of Time-Weighted Averages 	 J-l
K.	Calculation of Conversion Factors 	 K-l
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ACKNOWLEDGMENTS
Russ Erickson provided much help with the design of the project
and the interpretation of the data. Bob Spehar was the project
officer for the contract with the University of Wisconsin at
Superior (UWS) and provided coordination and oversight. Jim
Fiandt of the U.S. EPA performed the analyses for Total Organic
Carbon. Gary Chapman, Dave Hansen, Margarete Heber, Suzanne
Lussier, and Bob Spehar aided in formulating the project and
reviewing this report. Larry Brooke and Thomas Markee of UWS
provided many useful comments on drafts of this report.
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1. INTRODUCTION
On October 1, 1993, it became the policy of the Office of Water
of the U.S. EPA that "the use of dissolved metal to set and
measure compliance with water quality standards is the
recommended approach, because dissolved metal more closely
approximates the bioavailable fraction of metal in the water
column than does total recoverable metal" (Prothro 19 93).
Previously, aquatic life criteria for metals had been expressed
only in terms of the total recoverable measurement or the acid-
soluble measurement. These measurements had been used because
the results of most aquatic toxicity tests on metals were
expressed either in terms of nominal concentrations (i.e.,
intended concentrations) or- in terms of concentrations that had
been measured using the total recoverable method or other methods
that are expected to give equivalent concentrations in toxicity
tests. (When the concentrations were not measured, the initial
total recoverable concentration was assumed to equal the nominal
concentration.) This expected equivalency between a variety of
methods when used to measure concentrations in many toxicity
tests is the rationale for the decision that a factor of 1.0
should be used to convert aquatic life criteria for metals that
are expressed on the basis of the acid-soluble measurement to
criteria expressed on the basis of the total recoverable
measurement (Attachment #2 of Prothro 1993).
The new policy recommending use of dissolved metal created the
problem of deciding how aquatic life criteria should be derived
for dissolved metal. (The term "metal" is used herein to include
both "metals" and "metalloids".) The concentration of dissolved
metal has been measured in very few toxicity tests with aquatic
organisms, and so dissolved criteria for aquatic life cannot be
derived directly from the results of toxicity tests on metals.
One alternative was to assume that all of the metal was dissolved
in the toxicity tests that were most important in the derivation
of the aquatic life criteria. Although this is likely to be true
for some metals, application of this assumption to some other
metals would result in criteria that would be underprotective by
an unknown amount. In addition, this approach probably would
discourage generation of data concerning the percent of metal in
toxicity tests that is dissolved and discourage measurement of
dissolved metal in future toxicity tests.
Another alternative was to repeat all of the toxicity tests that
were most important in the derivation of the criteria and measure
the metal using both the total recoverable and dissolved methods
(and possibly other methods). This approach would allow
derivation of dissolved criteria and would increase confidence in
the resulting criteria. Substantial resources would be required,
however, to conduct the tests, especially to obtain the necessary
test organisms. Also, this approach might make it difficult to
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use the Recalculation Procedure based on species that "occur at
the site" if dissolved data are not available for the most
sensitive species in the site-specific dataset.
As a third alternative, EPA decided to use the following equation
to convert total recoverable criteria to dissolved criteria:
Dissolved criterion = (CF) (Total recoverable criterion)
where CF = Conversion Factor. The CF would correspond to the
percent of the total recoverable metal that is dissolved. A
literature search was conducted and the data that were found and
considered possibly useful were presented in Attachment #2 of
Prothro (1993). It was concluded that these data were not
sufficient to allow derivation of the CFs and that pertinent high
quality data should be generated for the metals of concern. One
option was to determine the percent dissolved at the
concentrations that are specified in the criteria; a second
option was to determine the percent dissolved at the
concentrations that occurred in the toxicity tests that were most
important in the derivation of the criteria.
Because the criteria are derived from the results of toxicity
tests and the percent dissolved for a metal might depend on such
factors as the type of the test, it was decided that "simulation
tests" should be conducted using solutions simulating those used
in the toxicity tests that were most important in the derivation
of aquatic life criteria for each metal. The intent was to mimic
the way criteria would have been derived if dissolved metal had
been measured in each of the toxicity tests.
The CF would be the fraction corresponding to the percent of the
total recoverable metal that was dissolved in the toxicity tests
that were most important in the derivation of the criterion for
the metal. It would probably be appropriate to apply these CFs
not only to national criteria but also to recalculated criteria
if the recalculated criteria were not too different from the
national criteria to which the factors were derived to apply. It
was possible, of course, that the simulation tests might show
that it is not appropriate to use the same CF for both acute and
chronic tests or even for all acute tests. Similarly, separate
CFs might be necessary for fresh and salt water for some or all
metals; only freshwater simulations are discussed herein.
A work assignment was written to have the University of Wisconsin
at Superior conduct freshwater "simulation tests" that were
designed by the U.S. EPA. The purpose of this report is to
describe the design of the project and present the analysis and
interpretation of the data resulting from these simulation tests.
The results presented herein are based on the report titled
"Results of Freshwater Simulation Tests concerning Dissolved
Metal" (Brooke 1995) .
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2. DESIGN OF THE PROJECT
The metals that were selected to be the subjects of freshwater
simulation tests were arsenic(III), cadmium, chromium(III),
chromium(VI), copper, lead, mercury(II), nickel, selenium(IV),
silver, and zinc, but they were not tested in alphabetical order.
Except for minor refinements, the initial methodology was used
with copper, zinc, chromium(III), lead, arsenic(III), and
chromium(VI). Some major refinements affected the planned
simulations for selenium(IV), nickel, cadmium, mercury, and
silver. Due to resource limitations, no simulations were
conducted with either mercury or silver. The metals are
addressed in the appendices in the order in which they were
tested to facilitate the description of the impact of the
refinements on the individual metals.
Selection of Simulations to be Performed
The following decisions were made concerning the work to be done:
A.	The conditions most likely to substantially affect the percent
of the total recoverable metal that is dissolved in toxicity
tests are:
1.	the test species;
2.	the presence or absence of food, and if present, the kind
and amount of food;
3.	the technique used (static, renewal, or flow-through);
4.	the duration of the test;
5.	the hardness, pH, alkalinity, and temperature of the
dilution water;
6.	the tested concentrations of the metal; and
7.	the acidity of the stock solution used.
B.	The two processes that are most likely to cause the percent
dissolved to be less than 100 percent are precipitation and
sorption.
1.	Both the probability and the amount of precipitation
increase as the concentration of metal increases; thus
precipitation might cause the percent dissolved to be lower
in acute tests than in chronic tests with the same test
organisms.
2.	The amount of sorption increases as the concentration of
total suspended solids increases; also the importance of
sorption increases as the concentration of metal decreases.
Thus sorption might cause the percent dissolved to be lower
in chronic tests than in acute tests.
C.	Both precipitation and sorption might take several hours to
reach equilibrium for some metals.
D.	It is not necessary to simulate all pertinent conditions; it
is only necessary to determine whether the percent dissolved
is sufficiently similar for a metal under sufficient relevant
test conditions that it is reasonable to derive a freshwater
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dissolved Criterion Maximum Concentration (CMC) and/or
Criterion Continuous Concentration (CCC) for a metal by-
multiplying the freshwater total recoverable CMC and/or CCC by
conversion factors that are appropriate for the metal.
E.	The purpose of simulation tests should be to determine whether
food, organisms, hardness, duration, and concentration of the
metal make a substantial difference in the percent of the
metal that is dissolved.
F.	Simulations should be conducted at hardnesses of 50 and 200
mg/L for each metal whose criterion is hardness-dependent;
alkalinity and pH should be consistent with hardness.
G.	Only 48- and 96-hr static simulations should be performed
initially; other simulations should be performed later if
necessary.
H.	Both dissolved and total recoverable metal should be measured
at 1 and 48 hours in 48-hour static simulations and at 1, 48,
and 96 hours in 96-hour static simulations.
I.	Selection of the kinds of simulations and the concentrations
with which simulations should be performed with a metal should
be based on the toxicity tests that were most important in the
derivation of the criterion for that metal. In freshwater
criteria documents, all chronic tests are fed tests, and
virtually all acute tests are unfed tests. Thus in fresh
water, the range of concentrations of metal in unfed
simulations should usually cover both the lowest four GMAVs
and the acute values used in the derivation of freshwater
acute-chronic ratios (ACRs) that were used in the derivation
of the criterion. Similarly, the range of concentrations of
metal in fed simulations should cover the chronic values used
in the derivation of the Final Chronic Value; these will
usually be the chronic values used in the derivation of the
Final Acute-Chronic Ratio (FACR).
J. At least two concentrations should be used for each type of
simulation performed with each metal to determine whether the
percent dissolved depends on the concentration of the metal.
Simulations should be performed with three concentrations if
the range is greater than a factor of ten.
K. The same concentrations should not necessarily be used for
different simulations. The goal should be to determine
whether the same percent dissolved can be used across a
variety of conditions, not to determine the impact of specific
conditions on the percent dissolved. If two test
concentrations are similar, however, it is desirable to have
them be the same.
L. Extrapolation of the results can be based on the following
assumptions:
1.	The percent dissolved at one hour in an unfed static
simulation is a useful approximation of the percent
dissolved in a comparable unfed flow-through simulation for
the same metal; the same is assumed for fed simulations.
2.	The percent dissolved in a flow-through chronic test does
not change substantially from two days to, for example,
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thirty days if the flow rate is acceptably high, the tanks
are cleaned regularly, etc.
3.	The percent of a metal that is dissolved is similar in
comparable static, renewal, and flow-through toxicity tests
if duration does not make a substantial difference in a
static simulation.
4.	The percent of a metal that is dissolved is similar in
unfed tests with all species if the percent dissolved is
sufficiently similar in unfed simulations with Daphnia
magna and fathead minnows; this is even more likely to be
true if the percent dissolved is also similar in a fed
simulation test.
5.	The percent of a metal that is dissolved is similar in a
daphnid chronic test and in chronic tests in which fish are
fed trout chow, if the daphnid food contains trout chow.
Although these five assumptions seem reasonable, it would have
been desirable to have had time and resources to test them.
An examination of the tests that were most important in the
derivation of the freshwater criterion for each individual metal
resulted in the following:
Arsenic(III)
1.	The FACR was based on three freshwater ACRs and a saltwater
ACR for mysids.
2.	Freshwater simulations should cover the following:
Unfed: 874 to 14,400 ug/L.
Fed: 914 to 3,026 ug/L.
3.	The freshwater criterion is not hardness-dependent.
Cadmium
1.	A freshwater FACR was not used; the freshwater CCC was
based on the four lowest chronic values.
2.	Freshwater simulations should cover the following:
Unfed: 2.4 to 30.0 ug/L.
Fed: 0.13 to 3.9 ug/L.
3.	The freshwater criterion is hardness-dependent.
Chromium(III)
1.	A freshwater FACR was not used. The freshwater CCC was
based on a chronic test with rainbow trout by Stevens and
Chapman (1984), for which they reported that 83 to 89
percent of the metal was dissolved at the CCC.
2.	Freshwater simulations should cover the following:
Unfed: 2221 to 8684 ug/L.
3.	The freshwater criterion is hardness-dependent.
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Chromium(VI)
1.	The freshwater FACR was based on four chronic tests with
daphnids.
2.	Freshwater simulations should cover the following:
Unfed: 29 to 67 ug/L.
Fed:	6 to 40 ug/L.
3.	The freshwater criterion is not hardness-dependent.
Copper
1.	The FACR was based on four freshwater chronic tests, three
of which were daphnid tests for which Chapman (1993a)
reported values for percent dissolved.
2.	Freshwater simulations should cover the following:
Unfed: 17 to 69 ug/L.
Fed:	6 to 29 ug/L.
3.	The freshwater criterion is hardness-dependent.
Lead
1.	The FACR was based on five freshwater ACRs and a saltwater
ACR for mysids; three of the five freshwater chronic tests
were daphnid tests for which Chapman (1993a) reported
values for percent dissolved.
2.	Freshwater simulations should cover the following:
Unfed: 143 to 4100 ug/L.
Fed:	12 to 128 ug/L.
3.	The freshwater criterion is hardness-dependent.
Mercury
1.	The national freshwater CCC is based on bioaccumulation and
an FDA action level. A conversion factor for the CCC is
probably inappropriate because both particulate and
dissolved mercury might be methylated.
2.	The GLI CCC is based on the chronic toxicity of mercury to
aquatic organisms.
3.	Freshwater simulations should cover the following:
Unfed: 2.6 to 20 ug/L.
4.	The freshwater criterion is not hardness-dependent.
Nickel
1.	The FACR was based on five freshwater ACRs and a saltwater
ACR for mysids; three of the five freshwater chronic tests
were daphnid tests for which Chapman (1993a) reported
values for percent dissolved.
2.	Freshwater simulations should cover the following:
Unfed: 1500 to 28,000 ug/L.
Fed:	15 to 526 ug/L.
3.	The freshwater criterion is hardness-dependent.
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Selenium
1.	The freshwater criterion was based on data concerning
Belews Lake and on a FACR of 8.314. A conversion factor
for the freshwater CCC should be based on the percent of
total recoverable selenium that was dissolved in Belews
Lake.
2.	The FACR was based on a variety of freshwater and saltwater
chronics. The major purpose of the freshwater simulations
should be to determine whether the dissolved FACR should be
the same as the total recoverable FACR. It would also be
desirable for the simulations to cover the four lowest
GMAVs.
3.	Freshwater simulations should cover the following:
Unfed: 776 to 47,000 ug/L.
Fed:	92 to 2,891 ug/L.
4.	The freshwater criterion is not hardness-dependent.
Silver
1. Freshwater simulations
Unfed: 0.39 to 30
Unfed: 4.8 to 270
should cover the following:
ug/L (hardness = 30 to 100 mg/L)
ug/L (hardness = 150 to 280 mg/L)
Zinc
1.	The freshwater FACR was based on five freshwater ACRs and a
saltwater ACR for mysids. Three of the five freshwater
chronic tests were daphnid tests for which Chapman (1993a)
reported values for percent dissolved.
2.	Freshwater simulations should cover the following:
Unfed: 94 to 701 ug/L.
Fed: 47 to 371 ug/L.
3.	The freshwater criterion is hardness-dependent.
It was decided that the following five types of simulation tests
would be used to obtain data concerning the percent dissolved
metal in freshwater toxicity tests:
1.	A 96-hr static unfed exposure of fathead minnows in Lake
Superior water, with samples taken at 1, 48, and 96 hours for
metal analyses.
2.	A 48-hr static fed exposure of daphnids in Lake Superior
water, with samples taken at 1 and 48 hours for metal
analyses. The daphnids would be fed a yeast-trout chow-cereal
leaf (YTC) mixture at a concentration that is typically used
in life-cycle tests with daphnids.
3.	A 48-hr static fed exposure of fathead minnows in Lake
Superior water, with samples taken at 1 and 4 8 hours for metal
analyses. The fish would be fed live newly hatched brine
shrimp nauplii at a concentration that is typically used in
early life-stage tests. (This simulation would to be
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performed with two representative metals to which fish are
sensitive.)
4.	A 48-hr static fed exposure of daphnids in Lake Superior water
to which sufficient calcium sulfate had been added to raise
the hardness to 200 ± 10 mg/L, with samples taken at 1 and 48
hours for metal analyses.
5.	A 96-hr static unfed exposure of fathead minnows in Lake
Superior water to which sufficient calcium sulfate had been
added to raise the hardness to 200 ± 10 mg/L, with samples
taken at 1, 48, and 96 hours for metal analyses.
When the simulation tests were conducted, the solutions
containing fathead minnows (i.e., simulation types 3 and 5) were
aerated, but the others were not.
For each metal whose criterion was hardness-dependent, either the
fourth or fifth type of simulation would be used to determine
whether hardness (and the related alkalinity and/or pH) caused a
measurable difference in the percent dissolved. The fourth type
of simulation would be performed if daphnids were more sensitive
to the metal, whereas the fifth type of simulation would be
performed if fish were more sensitive to the metal. (Except for
simulations with copper, whenever calcium sulfate was added to
increase hardness to 200 ± 10 mg/L, sufficient sodium bicarbonate
was also added to increase alkalinity to 130 ± 10 mg/L.)
Each type of simulation would be performed only with the metals
for which it was relevant; the simulations deemed necessary were:
Metal
Type of
Simulation
Concentrat ion
(uq/L)	
Arsenic(III)
1
2
1,000; 10,000
1,000; 3,000
Cadmium
1
2
3
4
3; 30
0.3; 3
0.3; 3
0.3; 3
Chromium(III)
1
5
2,000; 9,000
2,000; 9,000
Chromium(VI)
1
2
30; 70
5; 3 0
Copper
1
2
3
4
20; 70
5; 20
5; 20
5; 20
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Lead
1
2
4
130; 1,000; 4,100
10; 13 0
10; 13 0
Mercury
1
2.5; 2 0
Nickel
1
2
4
2,000; 20,000
15; 100; 500
15; 100; 500
Selenium(IV)
1
2
700; 7,000; 47,000
90; 700; 2,900
Silver
1
5
0.3; 3; 3 0
3; 30; 300
Zinc
1
2
4
90; 700
40; 4 00
40; 4 00
The above plans were based on the national aquatic life criteria,
but the GLI CCC for mercury is quite different from the national
CCC for mercury. Whereas the national CCC is based on
bioaccumulation and an FDA action level, the GLI CCC is based on
chronic toxicity to aquatic life. In addition, it is stated on
page 5 of Attachment #2 of Prothro (1993) that it is not
appropriate to adjust the CCC for mercury or the CMC and CCC for
selenium because these are bioaccumulative chemicals. Regardless
of whether these metals bioaccumulate, the important
consideration is the exposure that relates to the effect on which
the CMC or CCC is based. The CMC is based on acute toxicity and
so a relevant consideration is the bioavailability of the metal
in the water column; therefore, a total recoverable CMC may be
converted to a dissolved CMC if an appropriate CF is used and if
there are no overriding risk management considerations. For a
CCC, the exposure can be from the water column and from the food,
with the food chain consisting of some organisms whose primary
exposure is to pollutants in the water column and other organisms
whose primary exposure is to pollutants in the sediment. It
appears that exposure to the sediment contributes substantially
to the concentration of mercury in the food chain, but does not
contribute substantially to the concentration of selenium in the
food chain. Therefore, it is as acceptable to convert the total
recoverable CCC for selenium to a dissolved CCC as it is to
convert, for example, the total recoverable CCC for copper to a
dissolved CCC. In contrast, it is not acceptable to convert a
total recoverable CCC for mercury to a dissolved CCC if the CCC
for mercury is based on mercury residues in aquatic organisms.
It can, however, be acceptable to convert a CCC for mercury to a
dissolved CCC if the CCC is based on the toxicity of mercury to
aquatic organisms because the important exposure is through the
water column; as before, a dissolved criterion may be derived if
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an appropriate CF is used and if there are no overriding risk
management considerations.
Design of the Simulations
For each type of simulation to be conducted with a metal,
simulations were conducted in three test chambers at each of the
desired concentrations; i.e., there were three experimental units
for each treatment. Except for selenium(IV), nickel, and
cadmium, the test chambers were conditioned before the
simulations began. Each test chamber contained the organisms
specified at a density recommended in ASTM Standard E729
concerning acute toxicity tests. If oversized chambers were
used, they contained more than ten organisms per test chamber.
When placed in the test solutions, the fathead minnows were
between 20 and 35 days old, whereas the daphnids were between 1
and 48 hours old. When fed simulations were conducted, the
organisms were fed as described above. Test chambers containing
fathead minnows were maintained in a temperature-controlled water
bath, whereas those containing daphnids were maintained in a
temperature-controlled chamber.
The solutions used in the simulations and in the checks of the
analytical methods (see below) were made using stock solutions
that were prepared by dissolving one of the following salts in
metal-free deionized and/or distilled water:
Arsenic(III): sodium arsenite
Cadmium:	cadmium chloride or sulfate
Chromium(III): chromic chloride or nitrate or chromium
potassium sulfate
Chromium(VI): potassium chromate or dichromate or sodium
chromate or dichromate
Copper:	cupric chloride, nitrate, or sulfate
Lead:	lead chloride or nitrate
Mercury:	mercuric chloride or sulfate
Nickel:	nickelous chloride, nitrate, or sulfate
Selenium(IV): sodium selenite
Silver:	silver nitrate
Zinc:	zinc chloride, nitrate, or sulfate
If the pH of Lake Superior water (LSW) was changed by more than
0.1 pH unit when it was spiked at the highest concentration
specified for a simulation with that metal, the pH of the stock
solution was adjusted using the minimum necessary amount of
either sodium hydroxide or sulfuric acid. The exact
concentration of metal used in a simulation was not considered
critical, but each concentration was to be within ± 30 percent of
that specified.
For some metals, a plastic screen was used to exclude daphnids,
brine shrimp nauplii, and brine shrimp cases from samples taken
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from simulation solutions. For two simulations containing
copper, this was accomplished by pouring the solution through the
plastic screen, with the initial solution being used to condition
the screen. For some other simulations containing copper and for
several subsequent metals, the samples were sucked through a
sampling apparatus that contained the plastic screen. When this
sampling apparatus was used for metals other than copper, it was
used to obtain samples from all solutions used in pre-simulation
tests and all simulation solutions. For selenium(IV), nickel,
and cadmium, neither the plastic screen nor the sampling
apparatus was used; daphnids were kept out of the samples by-
careful sampling, but some samples might have contained brine
shrimp nauplii and/or cases.
Immediately before any samples were taken from a test chamber,
the solution in the chamber was starred to ensure that settling
of particles did not result in a gradient in the concentration of
the metal at the time of sampling. For most metals, the sampling
procedure consisted of stirring the simulation solution, sucking
the solution through the sampling apparatus to condition the
apparatus and the plastic screen, taking a sample for analysis
using the acidification method, taking a sample to be used to
condition the membrane filter and the filter holder, and taking a
sample for analysis using the dissolved method. For
selenium(IV), nickel, and cadmium, however, the simulation
solution was stirred before each individual sample was taken,
i.e., the solution was stirred before a sample was taken for
analysis using the acidification method and it was stirred again
before a sample was taken for analysis using the dissolved
method. (As stated above, samples for these three metals were
not put through the plastic screen.) Each time a set of three
test chambers was sampled, two were sampled once for each
analytical method and the third was sampled twice for each
analytical method. When the duplicate sampling was performed,
the whole sampling procedure was repeated beginning with the
stirring of the simulation solution and using a sampling
apparatus that was freshly acid-washed. Duplicate filtrations of
the same solution were performed using different filters just as
if two different solutions were being filtered.
In addition to the specified metal measurements, the following
were also completed:
a.	Hardness, alkalinity, and total suspended solids were measured
once during the simulation in one test chamber in each set of
three.
b.	Temperature and pH were measured at 1 hour and at the end of
the simulation in each test chamber.
c.	A sample of at least 50 mL for measurement of total organic
carbon (TOC) was collected from one test chamber in each set
of three, acidified within one hour to pH s 2 with HCl or
H2S04, and delivered to the U.S. EPA.
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During simulations with some of the metals, the concentrations
measured by the acidification and dissolved methods decreased
substantially from 1 to 48 and/or 96 hours. In an attempt to
determine whether this might have been due to sorption onto the
test chamber, after the last sample was taken from simulations
for selenium(IV), nickel, and cadmium, the solution remaining in
the test chamber was acidified, mixed, and sampled after 15 to 20
minutes for analysis for the metal. When solutions were to be
acidified at the end of the simulations, the test chambers were
not conditioned before the beginning of the simulations.
Analytical methodology
To help ensure adequate accuracy and precision, both (a) the
detection limit for each metal and (b) the concentrations of
metals in blanks were required to be less than 10 % of the lowest
concentration with which a simulation was to be performed with
the metal; 33 % was used for silver because of the existing
analytical capabilities of the contractor. Clean techniques,
solvent extraction, ion exchange columns, etc., would have been
used if necessary to satisfy these requirements. The detection
limit for each metal was determined using the procedure described
in the Federal Register (1984). (Because the metal was
concentrated by a factor of two during the total recoverable
sample preparation procedure, the detection limit of the total
recoverable method was a factor of two lower than those of the
acidification and dissolved methods. For convenience, the
detection limit reported herein is that of the acidification and
dissolved methods.)
The procedures described by U.S. EPA (1991) were used for
preparing and analyzing samples for the total recoverable and
dissolved methods, except that any necessary procedures that were
not described in U.S. EPA (1991) were obtained from.U.S. EPA
(1983). Although the purpose of this project concerned the total
recoverable and dissolved methods, it was considered desirable to
also use a method that consisted only of acidification of the
sample (with no digestion and no filtration) for three reasons:
1.	Samples obtained during many of the toxicity tests used in
criteria documents for metals were analyzed using various
versions of the "acidification method".
2.	For routine analyses, the acidification method is easier to
perform than the total recoverable method.
3.	It is better to check the dissolved method by comparing it
with the acidification method than by comparing it with the
total recoverable method because the acidification method is
less subject to contamination and to loss of metal.
The "acidification method" was defined to consist of (a)
acidification of the sample the same as the filtrate is acidified
in the dissolved method, followed by (b) mixing the acidified
12

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sample and allowing it to equilibrate for at least 4 hours before
analysis. No filtration was performed before or after
acidification.
Sample preparation was the only difference between the three
analytical methods. Whenever data were to be compared for
different sample preparation methods, quantification for all of
the methods was performed using the same analytical technique.
To account for possible matrix effects on standard curves for
copper, three sets of standards were prepared: the first
contained 1 % nitric acid and was used with acidified LSW; the
second contained 2 % nitric acid and 1 % hydrochloric acid and
was used with solutions resulting from the total recoverable
digestion; the third contained 0.05 N sodium bicarbonate and 1 %
nitric and was used with acidified bicarbonate solutions.
Filtrations for determining dissolved metal were performed with
polycarbonate filters that were (a) effective to 0.40 um, (b) 47
mm in diameter, and (c) held in an all-plastic filtration
apparatus. The polycarbonate membrane filters had individual
holes of relatively uniform diameter of 0.40 um (~ ± 20 %) and
retained particles only on the surface (Horowitz et al. 1992).
When the concentration of dissolved metal in a solution was
measured more than once, each filtration was performed using a
different 0.40-um polycarbonate membrane filter and the filter
apparatus was cleaned and conditioned as it would have been
between two different solutions.
To minimize contamination and loss, the filter apparatus and
membrane filter were cleaned by rinsing with and/or dipping in
metal-free acidified (to pH s 2) distilled and/or deionized water
and then rinsing with metal-free distilled and/or deionized
water; they were then conditioned by filtering and discarding
about 100 mL of the solution to be filtered. The concentration
of the metal of concern in the acidified solution used for the
acid washing was less than 10 % (33 % for silver) of the lowest
concentration with which a simulation was to be performed with
that metal; the concentration of metal in the acidified solution
was measured using either the acidification method or the total
recoverable method.
Regarding silver, U.S. EPA (1991) says "Store in amber container"
on page 135; this was interpreted to mean that "all standards and
samples for silver must be in amber containers, except that
samples may be in clear containers if they are analyzed within
eight hours and are not in bright light during that time".
Before any simulations were conducted with a metal, the following
comparison of the acidification and total recoverable methods was
performed to determine whether these two methods would give
comparable results if used to analyze simulation solutions:
13

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A.	Sufficiently large volumes of the following solutions'were
prepared as necessary for each metal:
11.	Metal-free distilled or deionized water. "Metal-free
water" was defined as "water that contains less than 10 %
(33 % for silver) of the lowest concentration of the metal
with which a simulation is to be performed with that
metal, as measured by either the acidification method or
the total recoverable method".
12.	Lake Superior water (with no added food) spiked with the
lowest concentration (± 30 %) of the metal with which a
simulation was to be performed with that metal.
13.	Lake Superior water with food added the same as it would
be added in fed simulations with daphnids (simulation type
2 above, but without daphnids). The food used was a
yeast-trout chow-cereal leaf (YTC) mixture and was added
at a rate of 6.5 mL per 800 mL; the change in volume was
not taken into account in the calculation of the results.
14.	Lake Superior water with daphnid food added the same as
for solution 13 above, and spiked with the metal the same
as for solution 12 above.
15.	Lake Superior water with food added the same as it would
be added in fed simulations with fathead minnows
(simulation type 3 above, but without fathead minnows).
16.	Lake Superior water with fathead minnow food added the
same as for solution 15 above, and spiked with the metal
the same as for solution 12 above.
17.	Lake Superior water with no added food and no added metal.
(Solution 17 was used only with selenium(IV), nickel, and
cadmium.)
When such solutions were used, the same amount of metal was .
added to solutions 12, 14, 16; the same amount of daphnid food
was added to solutions 13 and 14; and the same amount of
fathead minnow food was added to solutions 15 and 16. The
Lake Superior water used to prepare solutions 12, 13, 14, 15,
16, and/or 17 (as needed for a particular metal) was from the
same batch so that the background concentration of the metal
would be the same for each solution. The Lake Superior water
was obtained from a pipe that extended about 1000 feet into
Lake Superior at Superior, Wisconsin. It was expected that
the concentrations in unspiked solutions (i.e., solutions 11,
13, 15, and 17) would be below the detection limit for most
metals.
B.	Solutions 11 and 12 were used for all metals. Solutions 13
and 14 were used only for metals for which fed simulations
were to be performed with daphnids (simulation types 2 and 4
above). Solutions 15 and 16 were used only for metals for
which fed simulations were to be performed with fathead
minnows (simulation type 3 above). Solution 17 was used with
selenium(IV) , nickel, cadmium, mercury; it would have been
used with silver if the comparison had been conducted with
silver. Thus for each metal the solutions used in the
14

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comparison of the acidification and total recoverable methods
were:
Arsenic(III)	11,12,13,14
Cadmium	11,12,13,14,15,16,17
Chromium(III)	11,12
Chromium(VI)	11,12,13,14
Copper	11,12,13,14,15,16
Lead	11,12,13,14
Mercury	11,12,17
Nickel	11,12,13,14,17
Selenium(IV)	11,12,13,14,17
Silver	11,12,17
Zinc	11,12,13,14
This comparison was performed with mercury, but not silver.
C.	Each solution was mixed and then equilibrated at least
overnight.
D.	The following twelve analyses were performed on the first
solution, and then on the second solution, and then on the
third solution, etc., until the twelve analyses had been
performed on each solution prepared for the metal:
A sample was prepared for analysis using the total
recoverable method and then a sample was prepared for
analysis using the acidification method. This sequence
was repeated six times so that six samples were prepared
alternately for analysis by the total recoverable method
and six samples for the acidification method.
For some metals, each solution was stirred just before each
total recoverable sample was taken, but was not stirred just
before a sample was taken for the acidification method. For
the last few metals (i.e., selenium, nickel, and cadmium),
however, the solution was stirred just before each total
recoverable sample was taken and was stirred again just before
each acidification sample was taken.
Until use of solution 17 was begun, the concentration of the
metal was measured in one or more separate batches of Lake
Superior water for all metals except chromium(VI). The value
reported for chromium(III) would have included any chromium(VI)
that was in the water.
Also before any simulations were conducted with a metal, the
following evaluation of the dissolved method was performed to
determine whether the dissolved methodology worked with that
metal:
A. Sufficiently large volumes of the following four solutions
were prepared for each metal:
21.	Metal-free distilled or deionized water.
22.	A 0.05 N sodium bicarbonate solution (prepared by
dissolving metal-free sodium bicarbonate in the same
metal-free water used as solution 21 above). "Metal-free
sodium bicarbonate" was defined as sodium bicarbonate that
contains a sufficiently low amount of the metal that at
least 90 percent of the analyses of a 0.05 N sodium
15

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bicarbonate solution using the acidification method will
be less than 10 % (33 % for silver) of the lowest
concentration with which a simulation was to be performed
with that metal. (For cadmium, a 0.001 N sodium
bicarbonate solution with added calcium sulfate was also
used.)
23.	A 0.05 N sodium bicarbonate solution (prepared the same
way as solution 22 above) and spiked with the lowest
concentration (± 30 %) of the metal with which a
simulation was to be performed with that metal. (For
cadmium, a 0.001 N sodium bicarbonate solution with added
calcium sulfate was also used.)
24.	Lake Superior water spiked with the metal the same as for
solution 23 above.
Each of these four solutions was used only to compare results
obtained using the two analytical methods on each solution
individually. These solutions were not used to check
recovery, and so it was not necessary that solutions 23 and 24
be spiked identically or that the concentration of metal be .
measured by each method in unspiked LSW.
B.	Each solution was mixed and then equilibrated at least
overnight.
C.	The following twenty analyses were performed on the first
solution, and then on the second solution, and then on the
third solution, and then on the fourth solution:
A sample was prepared for analysis using the acidification
method and then a sample was prepared for analysis using
the dissolved method. This sequence was repeated ten
times so that ten samples were prepared alternately for
analysis by the acidification method and ten samples for
the dissolved method.
For some metals, each solution was stirred just before each
sample was taken for the acidification method, but was not
stirred just before the sample was taken for the dissolved
method. For the last few metals (i.e., selenium, nickel, and
cadmium), however, the solution was stirred just before each
acidification sample was taken and was stirred again just
before each dissolved sample was taken.
D.	Simulations were not - performed with a metal unless the
following requirements were satisfied:
1.	For the metal-free water (solution 21), at least 90 percent
of the results obtained using the acidification method and
at least 90 percent of the results obtained using the
dissolved method were required to be less than 10 % (33 %
for silver) of the lowest concentration with which a
simulation was to be performed with that metal.
2.	For the unspiked sodium bicarbonate solution (solution 22),
at least 90 percent of the results obtained using the
acidification method and at least 90 percent of the results
obtained using the dissolved method were required to be
less than 10 % (33 % for silver) of the lowest
16

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concentration with which a simulation was to be performed
with that metal.
3.	For the spiked sodium bicarbonate solution (solution 23),
the quotient of the dissolved mean divided by the
acidification mean was required to be between 0.9 and 1.1.
[At the concentrations at which this comparison was to be
performed, 100 percent of each metal was expected to be
dissolved in metal-free bicarbonate solution (MFBS) and so
the mean results obtained by the two methods were expected
to agree well.]
4.	For the spiked Lake Superior water (solution 24) the
quotient of the mean obtained using the dissolved method
divided by the mean obtained using the acidification method
was required to be less than 1.1. (In LSW, some of the
metal might not be dissolved and so the quotient might be
less than 0.9, but it should not be greater than 1.1.)
5.	For the four sets of results obtained by analyzing the two
spiked solutions (solutions 23 and 24) using the two
analytical methods, all four coefficients of variation were
required to be less than 20 percent.
As a check on the analyses performed for the simulations, the
following two solutions were prepared and then analyzed using
both the acidification and dissolved methods at the beginning and
end of each group of samples, and in the middle of any group
containing more than 12 samples:
a.	A 0.05 N sodium bicarbonate solution (prepared by dissolving
metal-free sodium bicarbonate in metal-free deionized and/or
distilled water, letting the solution equilibrate overnight or
longer after dissolution of the sodium bicarbonate, and
maintaining it in a closed bottle). For cadmium, a 0.001 N
sodium bicarbonate solution with added calcium sulfate was
used.
b.	A 0.05 N sodium bicarbonate solution (prepared using sodium
bicarbonate and water from the same batches as above, spiked
with the metal of interest to the lowest concentration with
which a simulation was to be performed with that metal,
equilibrated as above, and maintained in a closed bottle).
For cadmium, the spike was added to a 0.001 N sodium
bicarbonate solution to which calcium sulfate had been added.
In addition, recoveries were performed regularly during the
analyses on samples from simulation solutions and/or bicarbonate
solutions.
The labware, apparatus, and procedures used for collecting,
handling, preparing, storing, and analyzing samples for
measurements using the acidification and dissolved methods were
the same for all such analyses performed during the comparisons
of the methods and for the analyses of the simulation solutions.
Special attention was given to conditioning of apparatus,
labware, and sample containers to prevent bias of the results:
17

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1.	Acid-cleaned plastic, such as polyethylene or a fluoroplastic,
was the only material that ever contacted a sample, except
during the digestions used in the total recoverable method.
2.	Because acid-cleaned plastic and glass sorb some metals from
unacidified solutions, but not from acidified solutions, all
sampling apparatus, etc., was conditioned if it contacted a
sample only before the sample was acidified, but it was not
conditioned if it contacted the sample after the sample was
acidified:
a.	If the sample was acidified in the sample container, the
sample container was not conditioned.
b.	If the sample was not acidified in the sample container,
the sample container was conditioned.
3.	Because samples for the dissolved method are not acidified
until after filtration, all sampling apparatus, sample
containers, labware, filter holders, membrane filters, etc.,
that contacted the sample before or during filtration were
conditioned by rinsing with a portion of the sample and
discarding the portion.
4.	For the total recoverable method:
a.	The sampling apparatus was conditioned because the sample
was not acidified until it was in a sample container.
b.	Sample containers were not conditioned because the samples
were acidified in the sample containers.
5.	If the total recoverable and dissolved measurements were
performed on the same sample (rather than on two different
samples from the same solution), all of the apparatus and
labware, including the sample container, was conditioned
before the sample was placed in the sample container; then an
aliquot was removed for analysis using the total recoverable
method (and acidified, digested, etc.) and an aliquot was
removed for analysis using the dissolved method (and filtered,
acidified, etc.)
a. If a different procedure had been used, bias of the results
could have occurred when working with metals that sorb from
unacidified solutions and then desorb when the sample is
acidified. For example, if a container had been
conditioned and filled with sample and an aliquot was
removed for the dissolved measurement and then the solution
in the container was acidified before removal of an aliquot
for the total recoverable measurement, the resulting
measured total recoverable concentration might have been
biased high because the acidification might have desorbed
metal that had been sorbed onto the walls of the sample
container. The amount of bias would have depended on the
relative volumes involved and the amount of sorption and
desorption.
For selenium(IV), nickel, and cadmium, some procedures were
changed so that conditioning of apparatus, measurement of pH,
etc., were performed in such a way that they could not cause
changes in pH of solutions or in the concentrations of metals in
18

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solutions. Specifically, if apparatus or labware was conditioned
in simulation solution, it was conditioned in a sample of the
simulation solution that had been poured into another container;
it was not conditioned in the simulation solution in the test
chamber. For selenium(IV), nickel, and cadmium, samples were
taken from test chambers using a rigid tube that remained in the
chamber throughout the simulation; this tube was also used to mix
the solution just before each individual sample was taken.
19

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3. RESULTS
Analytical methods
Data concerning several important properties of the three
analytical methods can be compared across metals. One such
property is the mean COV, using data obtained in the pre-
simulation comparisons of the methods:
Metal		Mean COV

Total
Recoverable
Acidification
(T.R.) (Diss . )
Dissolved
Copper
4 .18
%
1.38 %
1.55 %
3 .00
%
Zinc
5.45
%
3 . 35 %
4.40 %
4 . 65
%
Chromium(III)
1. 00
%
0.90 %
2.00 %
10 . 85
%*
Lead
3 . 60
%
6.65 %**
6.05 %
6 . 70
%
Arsenic(III)
1. 85
%
1.35 %
1.90%
2 .10
%
Chromium(VI)
4 . 75
%
5.05 %
4.35%
6 . 25
%
Selenium(IV)
3 . 50
%
1.95 %
2.40 %
2 . 10
%
Selenium(IV)
5.45
%
2.70 %



Nickel
2 . 15
%
4 . 75 %
5.30 %
4 . 70
%
Cadmium
2 . 77
%
4 .40 %
3 . 05 %
3 . 80
%
Cadmium



2.75 %
2 . 65
%
* This value is the average of 2.5
% (which was
determined
using spiked MFBS) and 19.2 % (which was determined using
spiked LSW). The MFBS and the LSW were spiked at about
the same concentration, but all of the chromium(III) was
dissolved in the spiked MFBS, whereas only about one-half
was dissolved in the spiked LSW.
** This value is the mean of 10.7 and 2.6 %.
These data do not allow a direct comparison the means COVs for
the three methods because data from which COVs can be calculated
were not generated using all three methods to analyze the same
solutions. Mean COVs for the total recoverable and acidification
methods can be directly compared, as can mean COVs for the
dissolved and acidification methods. In addition, the two COVs
determined for the acidification method using different solutions
can be compared.
The duplicate mean COVs obtained with selenium and cadmium in
replicate solutions show considerable variation for the total
recoverable and dissolved methods, but much better agreement for
the acidification method. In addition, the pair of mean COVs
obtained for the acidification method show reasonable agreement
for most metals. The acidification method definitely gave the
lowest COVs with copper and zinc and the highest with nickel; the
results obtained with the other metals are mixed.
20

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The agreement between replicate analyses of a simulation solution
can also be compared across metals. The data used in this
comparison are the results of the duplicate analyses that were
performed on the simulation solution in one of the three test
chambers for each treatment in the simulation tests. These are
analyses of duplicate samples, not duplicate analyses of one
sample. Each datum is the quotient of the higher of the
duplicate values divided by the lower of the two values; "N" is
the number of quotients (i.e., the number of solutions that were
analyzed twice). Except for one solution containing
chromium(VI), the same solutions were analyzed using both the
acidification and dissolved methods. Such data are not available
for the total recoverable method because it was not used to
analyze simulation solutions.
Metal	Acidification Method	Dissolved Method

N
Ranae
Mean
_N
Ranae
Mean
Copper
18
0.91-1.00
0 . 96
18
0.87-1. 00
0.95
Zinc
14
0.90-1.00
0 . 97
14
0.91-1.00
0 . 97
Chromium(III)
12
0.82-0.99
0 . 94
12
0.38-0 . 84
0 .63
Lead
17
0.87-1.00
0 . 95
17
0.36-1.00
0.86
Arsenic(III)
10
0 . 93-1. 00
0 . 97
10
0.95-1. 00
0.98
Chromium(VI)
9
0 . 96-1.00
0 . 98
10
0.90-1.00
0 . 98
Selenium(IV)
15
0.90-1.00
0 . 98
15
0.93-1 . 00
0.98
Nickel
18
0.97-1.00
0 . 99
18
0.96-1.00
0 . 98
Cadmium
14
0 . 91-1.00
0 . 98
14
0.95-1. 00
0 . 99
The reproducibility was good for all of the analyses except for
the measurement of dissolved chromium(III) and lead; these two
also gave the poorest reproducibility with the acidification
method. Interestingly, all of the conversion factors that are
less than 0.8 5 are for chromium(III) and lead.
A third property that can be compared is the percent recovery.
Most of the data concerning recovery was obtained during analyses
of simulation solutions, and such recovery data are only
available for the acidification and dissolved methods. These
recoveries were performed by spiking into solutions to which acid
had been added; the spikes were added after the solutions had
been acidified for the acidification method and after the
filtrate had been acidified for the dissolved method.
Metal
Acidification
Method
Dissolved
Method
N
Ranae
Mean
N
Ranae

Mean
Copper
9
98-114 %
103 %
7
90-102
%
98 %
Zinc
8
92-106 %
97 %
6
90-101
%
97 %
Chromium(III)
6
78-107 %
95 %
6
91-111
%
100 %
Lead
7
97-119 %
108 %
10
81-121
%
101 %
Arsenic(III)
4
88-105 %
98 %
5
101-111
%
106 %
21

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Chromium(VI)	5	80- 98 %	90 %	5	83-102 %	95	%
Selenium(IV)	7	95-122 %	99 %	7	76-113 %	96	%
Nickel	11	83-118 %	99 %	9	88-124 %	101	%
Cadmium	6	91-112 %	98 %	8	82-124 %	98	%
Because the spikes were added to solutions that contained acid,
it is surprising that so many of the recoveries were below 95 %
or above 105 %; it is even more surprising that some of the means
are outside this range.
Another useful property is the mean blanks that were obtained in
the pre-simulation comparisons of the methods. Two means are
available for the acidification method because it was separately
compared with the total recoverable and dissolved methods.
Metal
Mean Blank (ua/L)

Total
Recoverable
Acidification
(T.R.) (Diss.)
Dissolvi
Copper
<
0 . 37
<
0 .31
<
0 .31
<
0 . 31
Zinc
<
3 . 0
<
3 . 3
<
3 . 0
<
3 . 1
Chromium(III)
<
100
<
100
<
100
<
100
Lead
<
1 . 0
<
1. 0
<
1 . 0
<
1 . 0
Arsenic(III)
<
2 . 0
<
2 . 0
<
2 . 0
<
2 . 0
Chromium(VI)
<
0 . 14
<
0 . 14
<
0 .15
<
0 . 15
Selenium(IV)
<
6 . 0
<
6 . 0
<
6 . 0
<
6 . 0
Nickel
<
1 . 0
<
1 . 0
<
1. 0
<
1 . 0
Cadmium
<
0 . 034
<
0 . 03
<
0 . 03
<
0 . 03
There were only a few small differences between the mean blanks.
The easiest comparison of the total recoverable and acidification
methods is the comparison of the mean concentrations obtained by
replicate analyses of a sample of Lake Superior water that was
spiked with the metal (i.e., "solution 12" used in the pre-
simulation comparisons of the methods).
Metal		Mean Concentration (uq/L)	 Ratio

Total Recoverable
Acidification

Copper
4 .81
4 .46
1 . 08
Zinc
36 . 04
40 . 53
0 . 89
Chromium(III)
2062
2070
1. 00
Lead
9.291
9.639
0 . 96
Arsenic(III)
1040
1061
0 . 98
Chromium(VI)
5 .601
5 .154
1 . 09
Selenium(IV)
64 . 98
88 . 92
0 . 73
Selenium(IV)
55.20
69 .31
0 . 80
Nickel
14 . 10
12 . 54
1 . 12
Cadmium
0.280
0 .195
1 .43
22

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The ratio given is the total, recoverable mean divided by the
acidification mean. Each mean is the result of six measurements,
and each ratio is expected to be 1. The differences between the
methods cannot be explained on the basis of the differences in
the blanks given above. The total recoverable method is expected
to be more subject to contamination and loss than the
acidification method; many of the ratios are greater than 1, but
the ratio for zinc seems low, whereas those for selenium(VI) are
very low. Volatilization of selenium during the digestion step
in the total recoverable procedure is a possibility.
The best comparison of the dissolved and acidification methods is
the comparison of the mean concentrations obtained by replicate
analyses of MFBS that was spiked with the metal (i.e., "solution
23" used in the pre-simulation comparisons of the methods).
Metal		Mean Concentration (ua/L)	 Ratio

Dissolved
Acidification

Copper
3 . 866
3 . 982
0 . 97
Zinc
40.24
38 .80
1 . 04
Chromium(III)
1997
2008
0 . 99
Lead
8 .397
9 . 025
0 . 93
Arsenic(III)
997 .4
975.6
1 . 02
Chromium(VI)
4 .483
4 . 314
1 . 04
Selenium(IV)
88.61
85 . 73
1 . 03
Nickel
11.70
11. 66
1 . 00
Cadmium
0 .3107
0 . 3591
0 . 87
Cadmium
0 .3072
0 . 3057
1. 00
The ratio given is the dissolved mean divided by the
acidification mean, and each mean is the result of ten
measurements. At the concentrations used, all of each metal is
expected to be dissolved in MFBS, and so each ratio is expected
to be 1. All of the ratios are close to 1, except for the first
ratio for cadmium. The differences between the methods cannot be
explained on the -basis of the differences in the blanks given
above.
Simulation tests
The results of measurements of water quality characteristics are
given for each simulation by Brooke (1995) . The measured
concentration of dissolved oxygen was acceptable in all
simulations. The measured value of pH was acceptable, except
that the values measured at the end of two simulations with zinc
were unreasonably low; the results of these simulations were not
used. All except two of the measured concentrations of TSS were
below 5 mg/L. Some of the measured concentrations of TOC were
23

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below 5 mg/L; the concentration of TOC was not consistently-
higher in simulation solutions containing YTC.
The raw data from each simulation are also given by Brooke
(1994), but the calculated results for each metal are presented
in the appendices to this document. All of the planned tests
were completed except for mercury and silver. For mercury, the
pre-simulation tests were completed, but no simulation tests were
completed. For silver, neither pre-simulation tests nor
simulation tests were completed. No results are presented herein
for mercury or silver.
Because the methodology was refined at various times during the
project, the individual metals are addressed in the appendices in
the order in which the simulation tests were performed. To
clearly identify how the various refinements affected the work
with each individual metal, the following items are addressed in
the appendix for each individual metal:
a.	How was the concentration of metal determined in Lake Superior
water (LSW)?
b.	Was alkalinity increased if hardness was increased?
c.	What sodium bicarbonate solution was used?
d.	Were the test chambers conditioned with simulation solution
before the simulations were begun?
e.	If samples were passed through plastic screen, how was this
accomplished and how was the plastic screen conditioned?
f.	When samples were taken from a solution, was the solution
stirred only once before a pair of samples was taken or was
the solution stirred just before the first sample was taken
and stirred again just before the second sample was taken.
g.	Were the solutions remaining in the test chambers at the end
of the simulations acidified to see if there was an indication
of sorption of metal onto the test chambers?
The appendices consider three kinds of results for each metal:
A. Pre-simulation test results.
1.	The detection limit for each metal was required to be less
than 10 % (except 33 % for silver) of the lowest
concentration with which a simulation test was to be
performed with that metal.
2.	Use of the acidification method with each metal was
evaluated by comparison with the total recoverable method
based on replicate analyses of specified solutions. The
number and compositions of the solutions specified for this
comparison depended on the simulation tests that were to be
performed with that particular metal. Each of two or more
specified solutions was analyzed six times alternately by
the two methods. For each metal, one of the specified
solutions was metal-free water so that blanks for the two
methods could be compared. The other solutions were
prepared by adding the metal, calcium sulfate, sodium
bicarbonate, daphnid food, and/or fathead minnow food to
24

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LSW, depending on the simulations that were to be conducted
with that metal. For a particular metal, if any of these
was added to more than one solution, the same amount was
added to each solution to which it was added.
3. Use of the dissolved method was evaluated by comparison
with the acidification method based on replicate analyses
of the following four solutions:
Solution 21: Metal-free water.
Solution 22: Metal-free bicarbonate solution (MFBS).
Solution 23: MFBS spiked with the metal.
Solution 24: LSW spiked with the metal.
Each of the four solutions was analyzed ten times
alternately by the dissolved and acidification methods.
a.	For each of the four sets of ten blanks that were
obtained by analyzing solutions 21 and 22 by both of the
methods, at least 90 percent of the values in each set
of ten blanks was required to be less than 10 % (33 %
for silver) of the lowest concentration with which a
simulation was to be performed with that metal .
b.	For the replicate analyses of solution 23 by the two
methods, the quotient of the dissolved mean divided by
the acidification mean was required to be between 0.9
and 1.1.
c.	For the replicate analyses of solution 24 by the two
methods, the quotient of the dissolved mean divided by
the acidification mean was required to be less than 1.1.
d.	For the replicate analyses of solutions 23 and 24 by the
two methods, each of the four coefficients of variation
(COVs) was required to be less than 20 percent.
B.	Results of checks of the acidification and dissolved methods
during analyses of the samples from the simulation tests.
These included blanks, replicate analyses of a standard
(MFBS spiked with the metal), duplicate analyses of
simulation solutions, and spike recoveries in MFBS and/or
in simulation solutions.
C.	Results of the simulation tests.
The mean of the results obtained using the acidification
method and the mean of the results obtained using the
dissolved method, as well as the resulting values for
percent dissolved, were calculated for each sampling time
in each simulation.
For selenium(IV), nickel, and cadmium (i.e., the last three
metals), some data are given concerning whether there might have
been sorption of metal onto the walls of the test chamber.
25

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Conversion factors
The purpose of the simulation tests was to obtain data that could
be used to derive conversion factors (CFs). The way that CFs are
derived from the data must take into account the data on which
the existing total recoverable (or acid soluble) criteria are
based and the way that dissolved criteria would be derived if all
the toxicity tests were repeated and the concentrations of
dissolved metal were measured. This is not simply a matter of
determining the percent dissolved at one or more points in time
during a simulation test.
The simulations were intended to mimic things that happened
during the toxicity tests, such as sorption onto test chambers,
uptake by test organisms, precipitar.ion, and conversion of one
oxidation state to another for arsenic, chromium, and selenium.
The concept was that these would have occurred during toxicity
tests if they occurred during simulation tests.
Interpretation of the results of simulation tests should take
into account how results of toxicity tests should be calculated
for the purposes of the derivation of water quality criteria.
a.	The '85 guidelines specified that results of static and
renewal tests should be based on initial concentrations, but
that nominal concentrations are acceptable for most test
materials if measured concentrations are not available (page
30 of U.S. EPA 1985). Unless otherwise noted, all
concentrations reported in criteria documents for metals are
expected to be essentially equivalent to acid-soluble
concentrations and to total recoverable concentrations.
b.	It is the consensus of the Aquatic Life Criteria Guideline
Committee (Chapman, Delos, Erickson, Hansen, and Stephan) that
if a dissolved LC50 is calculated from a static or renewal
toxicity test, it should be calculated on the basis of the
time-weighted average (TWA) of the measured dissolved
concentrations of the metal; calculation of a TWA is explained
in Appendix J. This is how LC50s and EC50s would have been
calculated if the toxicity tests had been repeated and the
dissolved concentrations had been measured.
The "TWA percent dissolved" obtained from a static simulation
test can be calculated either by (1) calculating the TWA of the
measured dissolved concentrations and then dividing by the
initial concentration measured by the acidification method or (2)
dividing each dissolved concentration by the initial
concentration measured by the acidification method and then
calculating the TWA of the quotients. These two procedures will
give the same value for the "TWA percent dissolved".
In the appendix for each metal, as many of the following three
kinds of TWAs as possible were calculated from the results of
each simulation:
26

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1.	For use with flow-through tests, the 1-hour percent dissolved
was calculated from all simulations.
2.	For use with 48-hr static tests and tests renewed at 48 hours,
the 48-hr TWA percent dissolved was calculated from all
simulations.
3.	For use with 96-hr static tests, the 96-hr TWA percent
dissolved was calculated from all simulations that lasted 96
hours.
The equations used to calculate the TWAs are given in Appendix K.
The average value of each of the three TWAs was calculated for
each metal. How these three TWAs were used to calculate the
recommended Conversion Factors (CFs) for the CMC and/or CCC
depended on the data that were important in the derivation of the
CMC, CCC, and/or FACR for that metal, as explained in each
appendix. The equations used to calculate the CFs are also given
in Appendix K.
The following recommended conversion factors are derived in the
appendices:
Metal	Recommended Conversion Factors
CMC	CCC
Arsenic(III)	1.000	1.000
Cadmium3
Hardness = 50 mg/L	0.973	0.938
Hardness = 100 mg/L	0.944	0.909
Hardness = 200 mg/L	0.915	0.880
Chromium(III)	0.316	0.860b
Chromium(VI)	0.982	0.962
Copper	0.960	0.960
Lead3
Hardness = 50 mg/L	0.892	0.892
Hardness = 100 mg/L	0.791	0.791
Hardness = 200 mg/L	0.690	0.690
Nickel	0.998	0.997
Selenium	0.922	0.922
Zinc	0.978	0.986
3 The recommended conversion factors (CFs) for any hardness can
be calculated using the following equations:
27

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Cadmium
CMC: CF = 1.136672 - [(In hardness)(0 . 041838)]
CCC: CF = 1.101672 - [(In hardness)(0 . 041838)]
Lead (CMC and CCC): CF = 1.46203 - [(In Hardness)(0.145712)]
where:
(In hardness) = natural logarithm of the hardness.
b This CF applies only if the CCC is based on the test by Stevens
and Chapman (1984). If the CCC is based on other chronic
tests, it is likely that the CF should be 0.590, 0.376, or the
average of these two values (see Appendix G).
The recommended CFs are given to three decimal places because
they are intermediate values in the calculation of dissolved
criteria.
28

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4. DISCUSSION
Analytical methods
The "acidification method" was used here merely to facilitate the
project. This is not an official EPA method and there are no
plans to consider it for adoption as an official EPA method.
This project certainly could have been completed without using
the acidification method, but it was decided by EPA that it would
be advantageous to use the acidification method because it is
less complex and less subject to contamination and loss of metal
than the total recoverable method. In addition, versions of the
acidification method have been used to measure metals in many
toxicity tests with aquatic organisms.
It is not necessary that only EPA approved methods be used in
this kind of project. The "acidification method" is somewhat
similar to the "acid-soluble method" that was addressed by EPA in
several publications. The two methods differ mostly in the kind
and amount of acid used, the minimum duration between the adding
of acid and the analyzing of the solution, and whether the sample
is filtered. Use of the acidification method in this project
should not be interpreted to constitute a change in EPA policy as
to how metals should be measured for regulatory purposes such as
permit monitoring, ambient monitoring, establishing water effect
ratios, etc.
For each metal, it was expected that the COV for the
acidification method would be lower than the COVs for the total
recoverable and dissolved methods because the acidification
method is less complex and less subject to contamination and loss
of metal. Although the acidification method gave the lowest COVs
for copper and zinc, it gave the highest for nickel; the results
were mixed for the other metals. The COV was especially high
(19.2 %) for the measurement of dissolved chromium(III) in Lake
Superior water; this was not a sampling problem because the COV
obtained using the acidification method to analyze the same
solution was only 1.3 %.
These COVs were calculated to evaluate the acceptability of the
methodology. Additional COVs could have been calculated from
duplicate analyses of the solution in one test chamber and from
the analyses of the triplicate test chambers. These additional
COVs could have been compared with the COVs that were calculated,
but this would not have aided in the interpretation and use of
the data.
The variation between replicate analyses of simulation solutions
was low, except that it was higher for the measurement of
dissolved lead and was quite high for the measurement of
dissolved chromium(III). A number of the recoveries were
29

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surprisingly high or low, considering that they were performed by
spiking into acidified solution. Almost all of the blanks were
very low for all three methods. The acidification and total
recoverable methods produced similar results for all metals
except selenium when they were added to Lake Superior water.
All of the results discussed above, except for the data
concerning reproducibility of duplicate analyses, were obtained
during studies of the analytical methods that were performed
before the simulations were begun. As expected, QA/QC checks
during the analyses of simulation solutions were not as good and
produced some high blanks and low recoveries.
Two observations seem appropriate:
1.	The COV of an analytical method should be less that 10 %.
2.	If possible, the COV of the dissolved method should be checked
using a solution in which about 50 % of the metal of concern
is dissolved.
Simulation tests
Although it would have been desirable to conduct many more
simulation tests and to test some or all of the five assumptions
given in item L on page 4, the additional cost would have made
the project prohibitively expensive. A regulatory authority
might decide, therefore, to continue using total recoverable
criteria until additional questions are answered concerning
appropriate conversion factors.
After the simulations were begun, it was decided that some
refinements in the methodology would be beneficial.
a.	Solution 17 was added to the comparison of the total
recoverable and acidification methods to provide a comparison
at a low concentration and to aid in the interpretation of the
analyses of spiked Lake Superior water.
b.	Alkalinity was increased whenever hardness was increased to
maintain a reasonable relationship between the two in
simulations in which the hardness was 200 mg/L.
c.	The intent was for the sodium bicarbonate solution to have
about the same alkalinity as Lake Superior water. The
solution to be used, however, was initially specified to be
0.05 N sodium bicarbonate, rather than 0.001 N sodium
bicarbonate that has the appropriate alkalinity.
d.	After the concentrations measured using the acidification
method were observed to decrease during simulation tests with
several metals, the addition of acid to the test chambers at
the end of the test was instituted to determine whether the
decrease was likely to be due to sorption onto the walls of
the test chamber. This also made it inappropriate to
condition the test chambers before the beginning of a simulation.
30

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e.	A sampling apparatus was used during several simulations to
keep daphnids, brine shrimp nauplii, and brine shrimp cases
out of the samples and was conditioned in the test chambers
before samples were taken. This might have been the cause of
the low pHs in two of the simulations with zinc. This
apparatus was not used for the last few metals.
f.	Stirring of the solution before each sample was taken, rather
than stirring before each pair of samples, was deemed
desirable to reduce the possibility of a gradient in
precipitate when the second sample was taken.
Although these refinements were considered desirable, no studies
were conducted to determine whether they actually made a
difference in the results.
Although it might be argued that loss of metal to the walls of
the test chamber should not be taken into account the calculation
of conversion factors, it was taken into account here because it
probably also occurs in actual toxicity tests.
Two observations seem appropriate:
1.	Equipment to sample test solutions or make measurements on
test solutions should not be placed in the solutions during
the test; one alternative is to leave a sampling tube, for
example, in the test chamber from the beginning to the end of
the test.
2.	Measurement of such water quality characteristics as pH at the
end of a test not only characterize the test solution but also
might detect problems that occurred during the test.
Conversion factors
Determination and use of a CF for the CMC is relatively
straightforward because the CMC is calculated from the results of
acute toxicity tests. Determination and use of a CF for the CCC
is not as straightforward for some metals because the CCC is
calculated by dividing the FAV by the FACR. The following
example illustrates, however, that conversion of the FAV and FACR
results in the same dissolved CCC as does conversion of the CCC.
Assume:
CF for the CMC = 0.70
CF for the CCC =0.90
Based on total recoverable measurements:
FAV = 84 ug/L
FACR = (90 ug/L)/(30 ug/L) = 3
31

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One calculation approach is to calculate the total recoverable
CMC and CCC and then convert them to dissolved:
CMC = FAV/2 = (84 ug/L)/2 = 42 ug/L
CCC = FAV/FACR = (84 ug/L)/3 = 28 ug/L
Conversion to dissolved:
CMC = (0.70)(42 ug/L) =29.4 ug/L
CCC = (0.90) (28 ug/L) =25.2 ug/L
A second calculation approach is to convert the total
recoverable FAV and FACR to dissolved and then calculate the
CMC and CCC:
FAV = (0.70) (84 ug/L) = 58.8 ug/L
FACR = (0-70) (90 ug/L) = 63 ug/L = 2.33333
(0.90) (30 ug/L) 27 ug/L
CMC = FAV/2 = (58.8 ug/L)/2 = 2 9.4 ug/L
CCC = FAV/FACR = (58.8 ug/L)/2.33333 =25.2 ug/L
The two approaches give the same results.
The situation is more complicated when the CFs are hardness
dependent and the FACR was calculated from ACRs that were
determined at different hardnesses. An even more difficult
situation might involve metals for which the FACR was based on
both freshwater and saltwater ACRs (i.e., arsenic, lead, nickel,
and zinc). These are complicated if the CFs for the CMC and the
CCC are not the same in fresh and salt water.
Although additional testing is probably desirable, the data
contained herein should be sufficient to justify deriving and
using CFs to convert total recoverable criteria to dissolved
criteria when use of dissolved criteria are otherwise desirable.
One of the reasons a regulatory authority might decide to
continue using total recoverable criteria is to wait until
additional questions are answered concerning appropriate
conversion factors.
Some of the values for percent dissolved in Attachment 2 of
Prothro (1993) are substantially lower than the values obtained
in this project, possibly because some of the values in
Attachment 2 were determined at concentrations that are
substantially different from the relevant CMC or CCC. Some of
the differences might be due to differences in such water quality
32

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characteristics as pH. Few of the values contained in Attachment
2 are supported by the validation and QA studies that were
conducted as part of the work performed by UWS and some reports
do not present such information as the type of filter used. A
particular source of concern is sorption of metal by the filter
and the filter holder, which could result in the reported
dissolved concentrations being inappropriately low.
Some values for percent dissolved have been reported for toxicity
tests that were important in the derivation of criteria; in
particular, Chapman (1993) reported percent dissolved for acute
and chronic tests on copper, lead, nickel, and zinc. One of the
problems with these data is the lack of QA/QC data for the
dissolved measurements; another is the inability to use these
data in the determination of time-weighted averages. These same
issues can be raised concerning the percent dissolved data
reported by Stevens and Chapman (1984) for the chronic toxicity
test on chromium(III); one difference is that the CCC for
chromium(III) was based on this test, whereas the CCCs for
copper, lead, nickel, and zinc were based on several ACRs for
each metal. Also, the test on chromium(III) was a flow-through
test and so the percent dissolved probably did not change
substantially during the test. In contrast, the life-cycle tests
with daphnids were renewal tests and the percent dissolved might
have changed from the beginning to the end of the test.
For cadmium, chromium(III), and lead (i.e., for the metals for
which the concentrations measured by the acidification method
decreased by more than 20 percent in more than one simulation
test), pertinent references in the criteria documents were
searched for information concerning whether the test
concentrations decreased during freshwater static and renewal
tests. The references considered pertinent were those concerning
either renewal chronic tests or static and renewal acute tests in
which the test concentrations were measured and the species was
in one of the four genera that were most sensitive to the metal.
Only two of the references provided relevant information. Canton
and Slooff (1982) reported substantial declines in the
concentration of cadmium, but the methodology used was not
described in detail. Chapman (1993b) reported that total copper
concentrations decreased about 25 to 3 0 percent in 2 or 3 days,
but the measurements were of total copper in the water column;
dissolved concentrations appeared to be more stable than total
concentrations.
The COVs that were calculated and the additional COVs that could
be calculated could have been used in a hypothesis test to
determine whether the conversion factors are significantly
different from 1. This might be used as an rationale for setting
any CF that is not significantly different form 1 to be equal to
1. This was not done because there is no rationale for believing
that the CF should be 1. An alternative would be to determine
33

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whether the CF is significantly different from 0.90 or 0.80, and
set the CF equal to the lowest value from which it is not
significantly different.
Some people might feel that conversion factors should be 1.0
because conservatism is built into other portions of the process
used to derive permit limits. It is inappropriate to be
unnecessarily underprotective (or overprotective) in one portion
of a program just because another portion of the program is or is
assumed to be overprotective (or underprotective). The correct
approach is to try to make each portion of the program as
appropriate as possible.
Conversion factors were derived here for selenium even though
Prothro (1993) stated that it is not appropriate to adjust the
CMC and CCC for selenium or the CCC for mercury because these are
bioaccumulative chemicals. Regardless of whether.these metals
bioaccumulate, the important consideration is the exposure that
relates to the effect on which the CMC or CCC is based. As
explained earlier, if an appropriate CF is used and if there are
no overriding management considerations, dissolved CMCs may be
derived for selenium and mercury and a dissolved CCC may be
derived for selenium. Under the same circumstances, a dissolved
CCC may be derived for mercury if the total recoverable CCC is
based on toxicity to aquatic organisms, but not if the total
recoverable CCC is based on mercury residues in aquatic
organisms. Although a conversion factor cannot be used to derive
a dissolved CCC for mercury if the CCC is based on mercury
residues in aquatic organisms, this does not necessarily mean
that losses due to fate and transport processes, such as
volatility, cannot be taken into account in the derivation of a
permit limit.
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5. REFERENCES
ASTM. 1994. Guide for Conducting Acute Toxicity Tests with
Fishes, Macroinvertebrates, and Amphibians. Standard E72 9. In:
Volume 11.04 of the 1994 Annual Book of ASTM Standards. American
Society for Testing and Materials, Philadelphia, PA.
Brooke, L.T. 1995. Percent Dissolved Metal in Freshwater
Simulation Tests. University of Wisconsin - Superior. Superior,
WI.
Canton, J.H., and W. Slooff. 1982. Toxicity and Accumulation
Studies of Cadmium (Cd2+) with Freshwater Organisms of Different
Trophic Levels. Ecotoxicol. Environ. Safety 6:113-128.
Chapman, G.A. 1993a. Memorandum to C. Stephan. June 4.
Chapman, G.A. 1993b. Memorandum to C. Stephan. February 9.
Cumbie, P.M. 1978. Belews Lake Environmental Study Report:
Selenium and Arsenic Accumulation. Technical Report Series No.
78-04. Duke Power Company, Charlotte, North Carolina.
Federal Register. 1984. Vol. 49, No. 209, pp. 198-199.
Horowitz, A.J., K.A. Elrick, and M.R. Colberg. 1992. The Effect
of Membrane Filtration Artifacts on Dissolved Trace Element
Concentrations. Water Res. 26:753-763.
Prothro, M. 1993. Memorandum concerning "Office of Water Policy
and Technical Guidance on Interpretation and Implementation of
Aquatic Life Metals Criteria". October 1.
U.S. EPA. 1985. Guidelines for Deriving Numerical National
Water Quality Criteria for the Protection of Aquatic Organisms
and Their Uses. PB85-227049. National Technical Information
Service, Springfield, VA.
Stevens, D.G., and G.A. Chapman. 1984. Toxicity of Trivalent
Chromium to Early Life Stages of Steelhead Trout. Environ.
Toxicol. Chem. 3:125-133.
U.S. EPA. 1983. Methods for Chemical Analysis of Water and
Wastes. EPA-600/4-79-020. Sections 4.1.1, 4.1.3 and 4.1.4.
National Technical Information Service, Springfield, VA.
U.S. EPA. 1991. Methods for the Determination of Metals in
Environmental Samples. EPA-600/4-91-010. National Technical
Information Service, Springfield, VA.
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Appendix A: Copper
The three kinds of results described in Section 3 are presented
below for copper; the methodology used to obtain these results is
described in Section 2. The refinements made in the methodology
during the project affected the work with copper as follows:
a.	The concentration of copper in Lake Superior water (LSW) was
measured in the three batches of LSW that were used in these
tests with copper.
b.	Although the hardness of LSW was increased for some
simulations with copper, alkalinity was not increased for any
simulation with copper.
c.	The sodium bicarbonate solution was 0.05 N.
d.	Each test chamber was conditioned with simulation solution
before the simulation was begun.
e.	Samples from solutions 11, 12, 13, and 14 were not passed
through the plastic screen. Samples from solutions 15 and 16
were poured through the screen; the screen was conditioned by
pouring a portion of the solution through the screen and
discarding this portion of the solution. Samples from
solutions 21, 22, 23, and 24 and from all of the simulation
solutions were passed through the plastic screen by using the
sampling apparatus that was described in Section 2; the
apparatus was conditioned in the solution to be sampled.
f.	Each solution that was sampled was stirred just before a pair
of samples was taken; solutions were not stirred just before
each individual sample was taken. If a total recoverable
sample was in the pair, it was taken before the acidification
sample; if a dissolved sample was in the pair, it was taken
after the acidification sample.
g.	Solutions remaining in the test chambers at the end of the
simulations were not acidified to see if there was an
indication of sorption onto the test chambers.
Pre-simulation test results
1.	The detection limit for copper was determined to be 0.31
ug/L. The lowest concentration with which a simulation was
to be performed with copper was 5 ug/L, and so the
detection limit was less than ten percent of the lowest
concentration.
2.	The acidification method was compared with the total
recoverable method by analyzing each of the following
solutions six times by each method:
Soln 11: Metal-free water.
Soln 12: LSW plus copper.
Soln 13: LSW plus daphnid food.
Soln 14: LSW plus daphnid food and copper.
A-1

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Soln 15: LSW plus fathead minnow food.
Soln 16: LSW plus fathead minnow food and copper.
The mean measured concentrations (in ug/L, with the
standard deviations in parentheses) were:
Method		Solution

11
12
13
14
15
16
Acidification
<0.31
4 .46
2 . 36
4 . 62
2 .40
4 . 64


(0.08)
(0.04)
(0.06)
(0.03)
(0.05)
Total Recoverable
<0.37
4 . 81
2 .60
4 . 64
2 . 53
4 . 65


(0.14)
(0.16)
(0.25)
(0.09)
(0.14)
Analyses of three batches of LSW (to which food had not
been added) using the acidification method indicated that
the concentration of copper was 2.3, 2.4, and 2.5 ug/L.
The mean of these three concentrations is about the same as
the mean concentrations (2.36 and 2.40 ug/L) that were
obtained when LSW plus food was analyzed using the
acidification method, but is lower than the two mean
concentrations (2.60 and 2.53 ug/L) that were obtained when
LSW plus food was analyzed using the total recoverable
method.
a.	Solution 11 was used to determine method blanks. All
six blanks for the acidification method were less than
0.31 ug/L; of the six blanks for the total recoverable
method, three were less than 0.31 ug/L and the other
three ranged from 0.3 5 to 0.58 ug/L.
b.	The concentrations measured in solution 13 and in
solution 15 were higher using the total recoverable
method than the acidification method.
c.	Taking into account the blanks and standard deviations,
the agreement between the results obtained using the two
methods was reasonably good for solutions 12, 14, and
16 .
d.	For all six of the solutions, the total recoverable
method gave higher mean concentrations than did the
acidification method, although the difference was very
small for solutions 14 and 16.
e.	Comparison of the results for solutions 14 and 16 with
those for solution 12 indicates that both foods possibly
increased the concentration of copper measured using the
acidification method, but possibly decreased the
concentration measured using the total recoverable
method.
f.	The following differences should all be the same if
there was no effect of food or analytical method:
A- 2

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Method
#14 - #13
#16 - #15
Acidification	2.26 ug/L	2.24 ug/L
Total recoverable	2.04 ug/L	2.12 ug/L
The results might indicate a difference between the two
methods, but the number of comparisons is small and the
differences are comparable to the standard deviations,
g. The five coefficients of variation (COVs) obtained using
the acidification method to analyze solutions 12 through
16 ranged from 1.0 to 1.7 %, whereas the five COVs
obtained using the total recoverable method to analyze
the same solutions ranged from 2.8 to 6.2 %.
3.	The dissolved method was compared with the acidification
method by analyzing each of the following solutions ten
times by each method:
Soln 21: Metal-free water.
Soln 22: Metal-free bicarbonate solution (MFBS).
Soln 23: MFBS plus copper.
Soln 24: LSW plus copper.
a.	For the replicate analyses of solutions 21 and 22 using
both methods, all forty blanks were less than 0.31 ug/L.
The blanks were sufficiently low.
b.	For the replicate analyses of solution 23, the quotient
of the dissolved mean divided by the acidification mean
was 0.97, which was acceptable.
c.	For the replicate analyses of solution 24, the quotient
of the dissolved mean divided by the acidification mean
was 0.98, which was acceptable.
d.	For the replicate analyses of solutions 23 and 24, the
COVs were 1.8 and 1.3 % for the acidification method and
3.3 and 2.7 % for the dissolved method. Each COV was
less than 20 % and was therefore acceptable.
4.	The COVs obtained with the three methods were:
Method
Number
Range
Mean

of COVs
(%)
(%)
Total Recoverable
5
2.8 to 6.2
4 . 18
Dissolved
2
2.7 to 3.3
3 . 00
Acidification
7
1.0 to 1.8
1.43
All of the COVs for the acidification method were lower
than all of the COVs for the other two methods.
These results indicate that the analytical methods were
performed acceptably before the simulation tests were begun.
A- 3

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Checks of the acidification and dissolved methods during analyses
of samples from the simulation tests
1. Two sets of QA samples were analyzed because the samples
from the simulation tests were analyzed in two batches.
a.	All blanks were less than 0.31 ug/L for both methods and
were therefore acceptable.
b.	The results of replicate analyses of MFBS spiked with
copper were:
Method
Number
Range
(ug/L)
Mean
(ug/L)
COV
(%)
Acidification
8
3.6 to 4.5
4.19
7.3

4
3.7 to 4.3
4 . 02
6 . 5
Dissolved
8
3.8 to 4.3
3 . 97
4 . 8

4
3.6' to 4.0
3 .80
4.2
The quotients of the dissolved mean divided by the
acidification mean were 0.95 and 0.94. A quotient of
0.97 was reported above for spiked MFBS, and a quotient
of 0.98 was reported above for spiked LSW. The
dissolved method gave lower COVs than did the
acidification method, and the COVs for both methods were
higher than the pre-simulation test mean COVs.
c. No spike recoveries were performed with the QA samples.
2.	For each pair of duplicate analyses of a simulation
solution, the lower value was divided by the higher value;
the resulting quotients were:
Method		Number	Range		Mean
Acidification	18	0.91 to 1.00	0.96
Dissolved	18	0.87 to 1.00	0.95
3.	The results of recoveries in simulation solutions were:
Method		Number	Range	Mean
Acidification	9	98 to 114 %	103 %
Dissolved	7	90 to 102 %	98 %
These recoveries were performed by spiking into acidified
solutions; i.e., for the dissolved method the spike was
added after the solution was filtered and acidified.
These results indicate that the analytical methods were
performed acceptably during the analyses of the simulation
solutions.
A-4

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Results of the simulation tests
Type Time Hard Sp. Food Acidifica. Dissolved	%	%
of
Sim.
(hr)
(a)
(b)
(c)
Method
(d)
Method
(d)
Diss.
(e)
Diss.
(f)
1
1
50
FM
No
19 . 34

19 .10

98 . 8
98.8
1
48
50
FM
No
17 . 90(
-7%)
17 . 20
(-10%)
96 .1
88. 9
1
96
50
FM
NO
15.95(
-18%)
14 . 88
(-22%)
93 .3
76. 9
1
1
50
FM
No
70 .32

69.65

99.0
99.0
1
48
50
FM
No
64 . 50 (
-8%)
61.53
(-12%)
95.4
87.5
1
96
50
FM
No
59.85 (
-15%)
61.28
* (-12%)
102 . 4
87. 1
2
1
50
DM
YTC
4 . 968

4 . 841

97.4
97.4
2
48
50
DM
YTC
4.908 (
-1%)
4 . 965
( + 3%)
101. 2
99 . 9
2
1
50
DM
YTC
19 . 77

17 . 91

90.6
90 . 6
2
48
50
DM
YTC
18.49(
-6%)
18 . 64
( + 4%)
100 . 8
94 . 3
3
1
50
FM
BS
4 .793

4 . 728

98 . 6
98 . 6
3
48
50
FM
BS
4.536(
-5%)
4 . 022
(-15%)
88. 7
83 . 9
3
1
50
FM
BS
18.59

19.44

104. 6
104 . 6
3
48
50
FM
BS
17.23 (
-7%)
15.43
(-21%)
89.5
83 . 0
4
1
200
DM
YTC
5 .000

4 . 947

98.9
98. 9
4
48
200
DM
YTC
4.743 (
-5%)
4 . 549
(-8%)
95.9
91. 0
4
1
200
DM
YTC
18 . 76

18 . 06

96.3
96.3
4
48
200
DM
YTC
17.16 {
-9%)
16 .95
(-6%)
98 . 8
90 .4
* = One value was considered an outlier and was not used,
a = mg/L.
b = Species (FM = fathead minnow; DM = Daphnia magna).
c = Food (YTC = yeast-trout chow-cereal leaf; BS = brine
shrimp nauplii).
d = ug/L; the numbers in parentheses are percent change from
the concentrations at one hour,
e = These values for percent dissolved were calculated based
on the concentrations at the same time measured using the
acidification method,
f = These values for percent dissolved were calculated based
on the concentrations at one hour measured using the
acidification method.
The concentrations at 4 8 and 96 hours were lower than those at
1 hour for all ten cases for the acidification method and for
eight of ten cases for the dissolved method. The decrease in
the concentrations from 1 hour to 4 8 and 96 hours might have
A- 5

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been due to precipitation, uptake by test organisms, and/or
sorption onto test chambers. Precipitation could cause the
dissolved measurements to be lower if equilibrium had not been
achieved within the first hour and could cause the
acidification measurements to be lower if the stirring did not
resuspend all of the precipitate and/or did not keep it
suspended during the sampling. Uptake is not likely important
because fishes and daphnids do not bioconcentrate substantial
amounts of copper. The chambers were conditioned before the
simulation tests began, but this conditioning might not have
been sufficient to prevent sorption. Data obtained with
nickel and cadmium indicate that sorption onto the test
chambers was probably substantial and was probably greater in
simulations containing daphnids and YTC.
The 36 values for percent dissolved ranged from 76.9 to 104.6
percent.
Interpretation of the Results
The interpretation of the results is described in Section 3.
Based on the initial concentration measured by the
acidification method, the time-weighted averages (TWAs)
obtained for percent dissolved for each simulation were:
Type of
Hard.
Species
Food
Initial
TWA
% dissolved
Simulation
(mcr/L)
(a)
(b)
Cone.c
l-hrd
4 8 - hre
96 -hr
1
50
FM
No
19 . 34
98 . 8
93 . 8
88.4
1
50
FM
No
70 . 32
99 . 0
93 . 2
90 .3
2
50
DM
YTC
4 . 968
97 .4
98 . 6
	
2
50
DM
YTC
19 . 77
90 . 6
92 .4
	
3
50
FM
BS
4 . 793
98 .6
91 . 2
	
3
50
FM
BS
18 . 59
104 . 6
93 . 8
	
4
200
DM
YTC
5 . 000
98 . 9
95 . 0
	
4
200
DM
YTC
18 . 76
96 .3
93 . 4
	



Mean TWA
o
00
CTl
93 . 9
89.4
a Species (FM = fathead minnow; DM = Daphnia magna).
b Food (YTC = yeast-trout chow-cereal leaf; BS = brine shrimp
nauplii).
c Initial concentration (ug/L) measured by the acidification
method.
d This is the percent dissolved at 1 hour.
e This TWA was calculated as the average of the percent
dissolved values at 1 and 48 hours (see Section 3).
f This TWA was calculated by giving the value for percent
dissolved at 48 hours twice the weight as the values for
percent dissolved at 1 and 96 hours (see Section 3).
A-6

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The 18 TWAs ranged from 88.4 to 104.6 percent. The value of
104.6 is obviously high, but the replicates are similar and
the other TWA from that simulation seems reasonable. The one-
hour mean TWA would be 97.1 if the TWA of 104.6 was not used.
The mean TWA decreased as the duration of the simulation
increased. For each duration, the range of the TWAs was
relatively small and did not seem to depend on species, food,
or hardness.
The acute toxicity tests that were important in the derivation
of the freshwater criterion for copper were flow-through tests
and 48-hr static tests. Therefore, the recommended conversion
factor for the CMC is 0.960, which is the average of 0.980 and
0.93 9.	1
Of the four freshwater chronic toxicity tests that were used
in the derivation of the criterion, three were renewal tests
and one was a flow-through test. Therefore, the recommended
conversion factor for the CCC is 0.960, which is the average
of 0.980 and 0.939.
Many of the percent dissolved values given for copper in
Attachment 2 of Prothro (1993) are reasonably close to 96.0
percent, but many are substantially lower.
A- 7

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Appendix B: Zinc
The three kinds of results described in Section 3 are presented
below for zinc; the methodology used to obtain these results is
described in Section 2. The refinements made in the methodology
during the project affected the work with zinc as follows:
a.	The concentration of zinc in Lake Superior water (LSW) was not
measured in the batch of LSW used to obtain the results
reported below; the concentration was measured in a different
batch of LSW.
b.	Whenever the hardness of LSW was increased for a simulation
with zinc, the alkalinity was also increased.
c.	The sodium bicarbonate solution was 0.05 N.
d.	Each test chamber was conditioned with simulation solution
before the simulation was begun.
e.	Samples for measurement of zinc were passed through a plastic
screen by using the sampling apparatus that was described in
Section 2; the apparatus was conditioned in the solution to be
sampled.
f.	Each solution that was sampled was stirred just before a pair
of samples was taken; solutions were not stirred just before
each individual sample was taken. If a total recoverable
sample was in the pair, it was taken before the acidification
sample; if a dissolved sample was in the pair, it was taken
after the acidification sample.
g.	The solutions remaining in the test chambers at the end of the
simulations were not acidified to see if there was an
indication of sorption onto the test chambers.
Pre-simulation test results
1.	The detection limit for zinc was determined to be 3.0 ug/L.
The lowest concentration with which a simulation was to be
performed with zinc was 40 ug/L, and so the detection limit
was less than ten percent of the lowest concentration.
2.	The acidification method was compared with the total
recoverable method by analyzing each of the following
solutions six times by each method:
Soln 11:	Metal-free water.
Soln 12:	LSW plus zinc.
Soln 13:	LSW plus daphnid food.
Soln 14:	LSW plus daphnid food and zinc.
The mean measured concentrations (in ug/L, with the
standard deviations in parentheses) were:
B-l

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Method
Solution
11
12
13
14
Acidification
<3.3 40.53
(1.1)
<3.7 36.14
(1.4)
Total Recoverable
< 3.0 36.04
(1-5)
<3.0 37.48
(2.5)
Analysis of a different batch of LSW (without daphnid food)
using the acidification method found that the concentration
of zinc was less than 3.0 ug/L.
a.	Solution 11 was used to determine method blanks. The
six blanks for the acidification method ranged from <
3.0 to 4.3 ug/L; all six blanks for the total
recoverable method were less than 3.0 ug/L.
b.	The total recoverable method did not detect zinc in
solution 13, whereas the acidification method did.
c.	Taking into account the blanks and standard deviations,
the agreement between the results obtained using the two
methods was reasonably good for solution 12 and very
good for solution 14.
d.	The results obtained with solutions 12 and 14 indicate
that the daphnid food had negligible effect on results
obtained using both analytical methods. Surprisingly,
the results of the analyses of solution 12 using the
acidification method are higher than the other three
results.
e.	The two coefficients of variation (COVs) obtained using
the acidification method to analyze solutions 12 and 14
were 2.7 and 4.0 %, whereas the two COVs obtained using
the total recoverable method to analyze the same
solutions were 4.2 and 6.7 %.
3. The dissolved method was compared with the acidification
method by analyzing each of the following solutions ten
times by each method:
Soln 21: Metal-free water.
Soln 22: Metal-free bicarbonate solution (MFBS).
Soln 23: MFBS plus zinc.
Soln 24: LSW plus zinc.
a.	For the replicate analyses of solutions 21 and 22, all
twenty blanks for the acidification method and nineteen
of the twenty blanks for the dissolved method were less
than 3.0 ug/L; the other one was 5 ug/L. The blanks
were sufficiently low.
b.	For the replicate analyses of solution 23, the quotient
of the dissolved mean divided by the acidification mean
was 1.04, which was acceptable.
B-2

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c.	For the replicate analyses of solution 24, the quotient
of the dissolved mean divided by the acidification mean
was 1.05, which was acceptable.
d.	For the replicate analyses of solutions 23 and 24, the
COVs were 4.5 and 4.3 % for the acidification method and
4.6 and 4.7 % for the dissolved method. Each COV was
less than 20 % and was therefore acceptable.
4. The COVs obtained with the three methods were ¦.
Method
Number
Range
Mean

of COVs
(%)
(%)
Total Recoverable
2
4.2 to 6.7
5.45
Dissolved
2
4.6 to 4.7
4 . 65
Acidification
4
2.7 to 4.5
3 . 88
The COVs for the three methods were similar.
These results indicate that the analytical methods were
performed acceptably before the simulation tests were begun.
Checks of the acidification and dissolved methods during analyses
of samples from the simulation tests
1. Two sets of QA samples were analyzed because the samples
from the simulation tests were analyzed in two batches.
a.	All blanks were less than 3.0 ug/L for both methods and
were therefore acceptable.
b.	The results of replicate analyses of MFBS spiked with
zinc were:
Method
Number
Range
Mean
COV


(ucr/L)
(ua/L)
(%>
Acidification
8
35.6 to 42 .4
37 . 6
5.4

3*
29.5 to 34 .0
32 . 5
5 . 0
Dissolved
8
33.4 to 43 .6
36 . 8
6.3

4
29.1 to 34.0
31. 6
6.4
* = One value was considered an outlier and was not
used.
The quotients of the dissolved mean divided by the
acidification mean were 0.98 and 0.97. A quotient of
1.03 was reported above for spiked MFBS and a quotient
of 1.05 was reported above for spiked LSW. The
acidification method gave slightly lower COVs than did
the dissolved method, and the COVs for both methods were
higher than the pre-simulation test mean COVs.
B- 3

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c. Recoveries in MFBS for the acidification method gave
96.6, 88.4, and 103.3 %; recoveries in MFBS for the
dissolved method gave 100.0 and 110.4 %. These
recoveries were performed by spiking into acidified
solutions; i.e., for the dissolved method the spike was
added after the solution was filtered and acidified.
For each pair of duplicate analyses of a simulation
solution, the lower value was divided by the higher value;
the resulting quotients were:
Method
Number
Range
Mean
Acidification
Dissolved
14
14
0.90 to 1.00
0.91 to 1.00
0 . 97
0 . 97
3. The results of recoveries in simulation solutions were
Method
Number
Range
Mean
Acidification
Dissolved
8
6
92 to 106 %
90 to 101 %
97 %
97 %
These recoveries were performed by spiking into acidified
solutions; i.e., for the dissolved method the spike was
added after the solution was filtered and acidified.
These results indicate that the analytical methods were
performed acceptably during the analyses of the simulation
solutions.
Results of the simulation tests
Type Time Hard Sp. Food Acidifica. Dissolved %	%
of (hr) (a) (b) (c) Method	Method Diss. Diss.
Sim.	(d)	(d)	(e) (f)
98 .3
98 .3
92.4
99.8
94 .3
92 .3
100	.5
101	.8#
101.2
103 .1#
1
1
50
FM
NO
92 .66

91.06

98 .3
1
48
50
FM
No
92 . 76
(0%)
91. 07
(0%)
98 . 2
1
96
50
FM
NO
84 . 97
(-8%)
85 . 60
(-6%)
100 . 7
1
1
50
FM
No
674 . 8

673 .3

99 . 8
1
48
50
FM
No
645 . 2
(-4%)
636 . 6
(-5%)
98 .7
1
96
50
FM
No
625 . 2
(-7%)
623 . 0
(-7%)
99 .6
2
1
50
DM
YTC
38 .79

38 . 97

100 . 5
2
48
50
DM
YTC
38 .22*(-1%)
39.47
( + 1%)
103 .3#
2
1
50
DM
YTC
364 . 3

368 . 5

101 . 2
2
48
50
DM
YTC
368 .4
( + 1%)
375 . 6
( + 2%)
102.0#
B-4

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4
4
1 200 DM YTC 36.60
48 200 DM YTC 35.20*(-4%)
36 . 03
34.68 (-4%)
98.5 98.5
98.5 94.8
4
4
1 200 DM YTC 323.6
48 200 DM YTC 313.0*(-3%)
318 . 7
317.4*(0%)
98.5 98.5
101.4 98.1
*	= One value was considered an outlier and was not used.
#	= This value was not used because the pH of the simulation
solution was unreasonably low at the end of the
simulation.
a = mg/L.
b = Species (FM = fathead minnow; DM = Daphnia magna).
c = Food (YTC = yeast-trout chow-cereal leaf).
d = ug/L; the numbers in parentheses are percent change from
the concentrations at one hour,
e = These values for percent dissolved were calculated based
on the concentrations at the same time measured using the
acidification method,
f = These values for percent dissolved were calculated based
on the concentrations at one hour measured using the
acidification method.
The concentrations at 48 and 96 hours were lower than those at
1 hour for six of eight cases for the acidification method,
but they were lower for only four of eight cases for the
dissolved method. The decrease in the concentrations from 1
hour to 48 and 96 hours might have been due to precipitation,
uptake by test organisms, and/or sorption onto test chambers.
Precipitation could cause the dissolved measurements to be
lower if equilibrium had not been achieved within the first
hour and could cause the acidification measurements to be
lower if the stirring did not resuspend all of the precipitate
and/or did not keep it suspended during the sampling. Uptake
is not likely important because fishes and daphnids do not
bioconcentrate substantial amounts of zinc. The chambers were
conditioned before the simulation tests began, but this
conditioning might have not been sufficient to prevent
sorption. Data obtained with nickel and cadmium indicate that
sorption onto the test chambers was probably substantial and
was probably greater in simulations containing daphnids and
YTC.
The 28 values for percent dissolved only ranged from 92.3 to
103.3 percent. For the percent dissolved based on the
acidification measurement made at the same time, in four of
the cases, the highest percent dissolved occurred at the end
of the simulation and in all four of these cases, the percent
dissolved at the end was greater than 100 percent; the other
two cases gave very similar results. For two of these, the
percent dissolved was greater than 100 percent at the end of
B-5

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the simulation even when the percentage was calculated based
on the initial concentration measured by the acidification
method.
Interpretation of the Results
The interpretation of the results is described in Section 3.
Based on the initial concentration measured by the
acidification method, the time-weighted averages (TWAs)
obtained for percent dissolved for each simulation were:
Type of Hard. Species Food Initial TWA % dissolved
Simulation (mq/L) (a)	(b) Cone .c l-hrd 48 -hre 96-hrf
1	50	FM	No 92.66	98.3	98.3	96.8
1	50	FM	No	674.8	99.8	97.0	95.2
2	50	DM	YTC 38.79	100.5 			
2	50	DM	YTC	364.3	101.2 			
4	200	DM	YTC 36.60	98.5	96.6		
4	200	DM	YTC	323.6	98.5	98.3		
Mean TWA	99.5 97.8 96.0
a Species (FM = fathead minnow; DM = Daphnia magna).
b Food (YTC = yeast-trout chow-cereal leaf).
c Initial concentration (ug/L) measured by the acidification
method.
d This is the percent dissolved at 1 hour.
e This TWA was calculated as the average of the percent
dissolved values at 1 and 48 hours (see Section 3).
f This TWA was calculated by giving the value for percent
dissolved at 48 hours twice the weight as the values for
percent dissolved at 1 and 96 hours (see Section 3).
The 14 TWAs ranged from 95.2 to 102.2 percent. The mean TWA
decreased slightly as the duration of the simulation
increased. For each duration, the range of the TWAs was small
and did not seem to depend on species, food, or hardness.
The acute toxicity tests that were important in the derivation
of the freshwater criterion for zinc were flow-through,
renewal, 48-hr static, and 96-hr static tests. Therefore, the
recommended conversion factor for the CMC is 0.978, which is
the average of 0.995, 0.978, and 0.960.
Of the five freshwater chronic toxicity tests that were used
in the derivation of the criterion, three were renewal tests
and two were flow-through tests. Therefore, the recommended
B-6

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conversion factor for the CCC is 0.986, which is the average
of 0.995 and 0.978.
Many of the percent dissolved values given for zinc in
Attachment 2 of Prothro (1993) are reasonably close to 98.1
and 99.2 percent, but many are substantially lower.
B-7

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Appendix C: Chromium(III)
The three kinds of results described in Section 3 are presented
below for chromium(III); the methodology used to obtain these
results is described in Section 2. The refinements made in the
methodology during the project affected the work with
chromium(III) as follows:
a.	The concentration of chromium(III), in Lake Superior water
(LSW) was not measured in the batch of LSW used to obtain the
results reported below; the concentration was measured in a
different batch of LSW.
b.	Whenever the hardness of LSW was increased for a simulation
with chromium{III), the alkalinity was also increased.
c.	The sodium bicarbonate solution was 0.05 N.
d.	Each test chamber was conditioned with simulation solution
before the simulation was begun.
e.	Samples for measurement of chromium(III) were passed through a
plastic screen by using the sampling apparatus that was
described in Section 2; the apparatus was conditioned in the
solution to be sampled.
f.	Each solution that was sampled was stirred just before a pair
of samples was taken; solutions were not stirred just before
each individual sample was taken. If a total recoverable
sample was in the pair, it was taken before the acidification
sample; if a dissolved sample was in the pair, it was taken
after the acidification sample.
g.	The solutions remaining in the test chambers at the end of the
simulations were not acidified to see if there was an
indication of sorption onto the test chambers.
Because the analytical method used to measure chromium(III) could
not differentiate between chromium(III) and chromium(VI) , it is
appropriate to report some information in terms of chromium
rather than chromium(III).
Pre-simulation test results
1.	The detection limit for chromium(III) was determined to be
100 ug/L. The lowest concentration with which a simulation
was to be performed with chromium(III) was 2000 ug/L, and
so the detection limit was less than ten percent of the
lowest concentration.
2.	The acidification method was compared with the total
recoverable method by analyzing each of the following
solutions six times by each method:
Soln 11: Metal-free water.
Soln 12: LSW plus chromium(III).
The mean measured concentrations (in ug/L, with the
standard deviations in parentheses) were:
C-l

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Method
Soln 11
Soln 12
Acidification
< 100
2070
(19)
Total Recoverable
< 100
2062
(21)
Analysis of a different batch of LSW using the
acidification method found that the concentration of
chromium was less than 100 ug/L.
a.	Solution 11 was used to determine method blanks. All
blanks for both methods were less than 100 ug/L.
b.	Taking into account the blanks and standard deviations,
the agreement between the results obtained using the two
methods was very good for solution 12.
c.	The coefficient of variation (COV) obtained using the
acidification method to analyze solution 12 was 0.9 %,
whereas the COV obtained using the total recoverable
method to analyze the same solution was 1.0 %.
3. The dissolved method was compared with the acidification
method by analyzing each of the following solutions ten
times by each method:
Soln 21: Metal-free water.
Soln 22: Metal-free bicarbonate solution (MFBS).
Soln 23: MFBS plus chromium(III) .
Soln 24: LSW plus chromium(III) .
a.	For the replicate analyses of solutions 21 and 22 using
both methods, all forty blanks were less than 100 ug/L.
The blanks were sufficiently low.
b.	For the replicate analyses of solution 23, the quotient
of the dissolved mean divided by the acidification mean
was 0.99, which was acceptable.
c.	For the replicate analyses of solution 24, the quotient
of the dissolved mean divided by the acidification mean
was 0.45, which was acceptable.
d.	For the replicate analyses of solutions 23 and 24, the
COVs were 2.7 and 1.3 % for the acidification method and
2.5 and 19.2 % for the dissolved method. Each COV was
less than 20 % and was therefore acceptable, but the COV
for analysis of solution 24 (i.e., spiked LSW) using the
dissolved method was borderline. The COV for the
dissolved method was low for spiked MFBS, in which all
of the chromium(III) was dissolved, but was high for
spiked LSW, in which about one-half of the chromium(III)
was dissolved. The low COV for the acidification method
for solution 24 indicated that the high COV for the
dissolved method was not due to sampling variation.
C-2

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4. The COVs obtained with the three methods were:
Method Number	Range	Mean
	 of COVa m (%)
Total Recoverable	l			1.00
Dissolved	2	2.5 to 19.2	10.85
Acidification	3	0.9 to 2.7	1.63
The high COV obtained for the dissolved method with spiked
LSW (i.e, solution 24) is a concern.
These results indicate that the analytical methods were
performed acceptably before the simulation tests were begun,
although one COV for the dissolved method is high.
Checks of the acidification and dissolved methods during analyses
of samples from the simulation tests
1. Only one set of QA samples was analyzed because all of the
samples from the simulation tests were analyzed at the same
time.
a.	All blanks were less than 100 ug/L for both methods and
were therefore acceptable.
b.	The results of replicate analyses of MFBS spiked with
chromium(III) were:
Method Number Range	Mean COV
	 	 (ug/L) (uq/L) (%)
Acidification	9	1902 to 2092	1981 3.1
Dissolved	9	1902 to 2017	1946 2.3
The quotient of the dissolved mean divided by the
acidification mean was 0.98. A quotient of 0.99 was
reported above for spiked MFBS, and a quotient of 0.45
was reported above for spiked LSW. The COVs were
similar to the pre-simulation test COVs for spiked MFBS.
c. Recoveries in MFBS for the acidification method gave
92.6 and 98.9 %; the only recovery in MFBS for the
dissolved method gave 95.5 %. These recoveries were
performed by spiking into acidified solutions; i.e., for
the dissolved method the spike was added after the
solution was filtered and acidified.
2. For each pair of duplicate analyses of a simulation
solution, the lower value was divided by the higher value;
the resulting quotients were:
C- 3

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Method
Number
Range
Mean
Acidification	12	0.82 to 0.99	0.94
Dissolved	12	0.38 to 0.84	0.63
The values for the acidification method are acceptably
high; for the dissolved method, however, both the highest
value of 0.84 and the mean of 0.6 3 are low values for
duplicates. These low values are consistent with the high
pre-simulation test COV of 19.2 % that was obtained when
spiked LSW (i.e., solution 24) was analyzed using the
dissolved method.
3. The results of recoveries in simulation solutions were:
Method	 Number	Range	Mean
Acidification	6	78 to 107 %	95 %
Dissolved	6	91 to 111 %	100 £
It is surprising that the range of these recoveries is so
great considering that they were performed by spiking into
acidified solutions; i.e., for the dissolved method the
spike was added after the solution was filtered and
acidified.
These results indicate that the analytical methods were
performed acceptably during the analyses of the simulation
solutions. The agreement between the duplicate analyses was
not very good for the dissolved method, which agrees with the
high COV reported in the pre-simulation test results.
Results of the simulation tests
Type
Time
Hard
Sp.
Food
Acidifica.
Dissolved
%
%
of
(hr)
(a)
(b)

Method
Method
Diss.
Diss.
Sim.




(c)
(c)
(d)
(e)
1
1
50
FM
No
2166 .
1540
71.1
71. 1
1
48
50
FM
No
1468(-32%)
384(-75%)
26 . 2
17 . 7
1
96
50
FM
No
1066(-51%)
274(-82%)
25 . 7
12 . 7
1
1
50
FM
No
9866
5874
59 . 5
59 . 5
1
48
50
FM
No
9258(-6%)
598 (-90%)
6 . 5
6 . 1
1
96
50
FM
No
7089 (-28%)
961 (-84%)
14 . 6
9 . 7
5
1
200
FM
No
2331
1467
62 . 9
62 . 9
5
48
200
FM
No
1234 (-47%)
403 (-73%)
32 . 7
17 . 3
5
96
200
FM
No
960(-59%)
345 (-76%)
35 . 9
14 . 8
C-4

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5
1
200
FM
No
9647
4105
42 .6
42 .6
5
48
200
FM
NO
6528 (-32%)
2253 (-45%)
34 . 5
23.4
5
96
200
FM
NO
5289 (-45%)
848 (-79%)
16 . 0
8.8
a = mg/L.
b = Species (FM = fathead minnow).
c = ug/L; the numbers in parentheses are percent change from
the concentrations at one hour within each simulation,
d = These values for percent dissolved were calculated based
on the concentrations at the same time measured using the
acidification method,
e = These values for percent dissolved were calculated based
on the concentrations at one hour measured using the
acidification method.
For both analytical methods, all concentrations at 48 and 96
hours were substantially lower than those at 1 hour. The
decrease in the concentrations from 1 hour to 48 and 96 hours
might have been due to precipitation, uptake by test
organisms, and/or sorption onto test chambers. Precipitation
could cause the dissolved measurements to be lower if
equilibrium had not been achieved within the first hour and
could cause the acidification measurements to be lower if the
stirring did not resuspend all of the precipitate and/or did
not keep it suspended during the sampling. Uptake is not
likely important because fishes and daphnids do not
bioconcentrate substantial amounts of chromium(III). The
chambers were conditioned before the simulation tests began,
but this conditioning might have not been sufficient to
prevent sorption. Data obtained with nickel and cadmium
indicate that sorption onto the test chambers was probably
substantial and was probably greater in simulations containing
daphnids and YTC.
The range of the values for percent dissolved was 6 to 71.
Interpretation of the Results
The interpretation of the results is described in Section 3.
Based on the initial concentration measured by the
acidification method, the time-weighted averages (TWAs)
obtained for percent dissolved for each simulation were:
Type of
Hard.
Species
Food
Initial
TWA
% dissolved
Simulation
(mcr/L)
(a)

Cone.b
l-hrc
00
1
tr
a
96-hr1
1
50
FM
No
2166
71. 1
44 .4
29 . 8
1
50
FM
No
9866
59 . 5
32 . 8
20.4
5
200
FM
No
2331
62 . 9
40 .1
28 .1
5
200
FM
No
9647
42 . 6
33 . 0
24 . 6
C-5

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Mean TWA
Hard. = 50
Hard. = 200
All
65.3 38.6 25.1
52.8 36.6 26.4
59.0 37.6 25.7
a Species (FM = fathead minnow).
b Initial concentration (ug/L) measured by the acidification
method.
c This is the percent dissolved at 1 hour.
d This TWA was calculated as the average of the percent
dissolved values at 1 and 48 hours (see Section 3).
e This TWA was calculated by giving the value for percent
dissolved at 48 hours twice the weight as the values for
percent dissolved at 1 and 96 hours (see Section 3).
The 12 TWAs ranged from 20.4 to 71.1 percent. The percent
dissolved decreased as the initial concentration of
chromium(III) increased, probably because the percent
dissolved was controlled more by precipitation than sorption.
The mean TWA decreased as the duration of the simulation
increased. The relation between percent dissolved and
hardness was much different at 96 hours than it was at one
hour.
The acute toxicity tests that were important in the derivation
of the freshwater criterion for chromium(III) were 48-hr and
96-hr static tests. Therefore, the recommended conversion
factor for the CMC is 0.316, which is the average of 0.376 and
0.257 .
The CCC for chromium(III) was based on a test by Stevens and
Chapman (1984), who reported the percent dissolved to be in
the range of 83 to 89 percent. Therefore, the recommended
conversion factor for the CCC for chromium(III) is 0.860. If,
however, the CCC for chromium(III) is based on other chronic
tests, it is. likely that the CF should be 0.590 or 0.376 or
the average of the two.
The percent dissolved values given for chromium(III) in
Attachment 2 of Prothro (1993) tend to show a lower percent
dissolved at higher concentrations, which is consistent with a
precipitation mechanism.
C-6

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Appendix D: Lead
The three kinds of results described in Section 3 are presented
below for lead; the methodology used to obtain these results is
described in Section 2. The refinements made in the methodology
during the project affected the work with lead as follows:
a.	The concentration of lead in Lake Superior water (LSW) was not
measured in the batch of LSW used to obtain the results
reported below; the concentration was measured in a different
batch of LSW.
b.	Whenever the hardness of LSW was increased for a simulation
with lead, the alkalinity was also increased.
c.	The sodium bicarbonate solution was 0.05 N.
d.	Each test chamber was conditioned with simulation solution
before the simulation was begun.
e.	Samples for measurement of lead were passed through a plastic
screen by using the sampling apparatus that was described in
Section 2; the apparatus was conditioned in the solution to be
sampled.
f.	Each solution that was sampled was stirred just before a pair
of samples was taken; solutions were not stirred just before
each individual sample was taken. If a total recoverable
sample was in the pair, it was taken before the acidification
sample; if a dissolved sample was in the pair, it was taken
after the acidification sample.
g.	The solutions remaining in the test chambers at the end of the
simulations were not acidified to see if there was an
indication of sorption onto the test chambers.
Pre-simulation test results
1.	The detection limit for lead was determined to be 1.0 ug/L.
The lowest concentration with which a simulation was to be
performed with lead was 10 ug/L, and so the detection limit
was ten percent of the lowest concentration.
2.	The acidification method was compared with the total
recoverable method by analyzing each of the following
solutions six times by each method:
Soln	11: Metal-free water.
Soln	12: LSW plus lead.
Soln	13: LSW plus daphnid food.
Soln	14: LSW plus daphnid food and lead.
The mean measured concentrations (in ug/L, with the
standard deviations in parentheses) were:
D-l

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Method
Solution
11
12
13
14
Acidification
<1.0 9.639 <1.0
(1 - 03)
7 . 875
(0.21)
Total Recoverable ' <1.0 9.291 < 1.0
(0.23)
8 . 552
(0.40)
Analysis of a different batch of LSW (without daphnid food)
using the acidification method found that the concentration
of lead was less than 1.0 ug/L.
a.	Solution 11 was used to determine method blanks. All
blanks for both methods were less than 1.0 ug/L.
b.	Neither method detected lead in solution 13.
c.	Taking into account the blanks and standard deviations,
the agreement between the results obtained using the two
methods was reasonably good for solution 12 and for
solution 14.
d.	The results obtained with solutions 12 and 14 indicate
that the food might have reduced the concentration of
lead or caused an interference with both methods.
e.	The two coefficients of variation (COVs) obtained using
the acidification method to analyze solutions 12 and 14
were 10.7 and 2.6 %, whereas the two COVs obtained using
the total recoverable method to analyze the same
solutions were 2.5 and 4.7 %.
3. The dissolved method was compared with the acidification
method by analyzing each of the following solutions ten
times by each method:
Soln 21: Metal-free water.
Soln 22: Metal-free bicarbonate solution (MFBS).
Soln 23: MFBS plus lead.
Soln 24: LSW plus lead.
a.	For the replicate analyses of solutions 21 and 22 using
both methods, all forty blanks were less than 1.0 ug/L.
The blanks were sufficiently low.
b.	For the replicate analyses of solution 23, the quotient
of the dissolved mean divided by the acidification mean
was 0.93, which was acceptable.
c.	For the replicate analyses of solution 24, the quotient
of the dissolved mean divided by the acidification mean
was 0.90, which was acceptable.
d.	For the replicate analyses of solutions 23 and 24, the
COVs were 7.7 and 4.4 % for the acidification method and
9.2 and 4.2 % for the dissolved method. Each COV was
less that 20 % and was therefore acceptable.
D-2

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4. The COVs obtained with the three methods were:
Method
Number
of COVs
Range
<%)
Mean
(%)
Total Recoverable
2
2 . 5 to 4.7
3 .60
Dissolved
2
4.2 to 9.2
6 .70
Acidification
4
2.6 to 10.7
6.35
The COVs for the three methods overlapped, but the mean for
the total recoverable method was lower than the other two
means.
These results indicate that the analytical methods were
performed acceptably before the simulation tests were, begun.
Checks of the acidification and dissolved methods during analyses
of samples from the simulation tests
1. Two set of QA samples were analyzed because the samples
from the simulation tests were analyzed in two batches.
a.	All blanks were less than 1.0 ug/L for both methods and
were therefore acceptable.
b.	The results of replicate analyses of MFBS spiked with
lead were:
Method
Number
Range
(ucr/L)
Mean
(ua/L)
COV
(%)
Acidification
8
9.1 to 10.5
9.889
4 . 7

4
8.4 to 9.6
9.136
5.0
Dissolved
8
9.3 to 10.3
9 .706
3.4

4
8.2 to 9.3
8.829
5.0
The quotients of the dissolved mean divided by the
acidification mean were 0.98 and 0.97. A quotient of
0.93 was reported above for spiked MFBS, and a quotient
of 0.90 was reported above for spiked LSW. The COVs
were similar and were lower than the pre-simulation test
mean COVs.
c. Recoveries in MFBS for the acidification method gave
83.9, 90.1, and 77.9 %, which are low; recoveries in
MFBS for the dissolved method gave 98.8 and 97.9 %. It
is surprising that some of these recoveries are so low
considering that they were performed by spiking into
acidified solutions; i.e., for the dissolved method the
spike was added after the solution was filtered and
acidified.
D-3

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For each pair of duplicate analyses of a simulation
solution, the lower value was divided by the higher value;
the resulting quotients were:
Method
Number
Range
Mean
Acidification
Dissolved
17
17
0.87 to 1.00
0.36 to 1.00
0 . 95
0 . 86
For the dissolved method, 4 of the 17 values ranged from
0.36 to 0.77, which seems very low.
3. The results of recoveries in simulation solutions were:
Method
Number
Range
Mean
Acidification
Dissolved
7
10
97 to 119 %
81 to 121 %
108 %
101 %
It is surprising that the range of the recoveries was so
large and that 5 of the 17 were above 113 %, considering
that these recoveries were performed by spiking into
acidified solutions;. i.e., for the dissolved method the
spike was added after the solution was filtered and
acidified.
These results indicate that the analytical methods were
performed acceptably during the analyses of the simulation
solutions.
Results of the simulation tests
Type Time Hard Sp. Food Acidifica.	Dissolved %	%
of (hr) (a) (b) (c) Method	Method Diss. Diss.
Sim.	(d)	(d)	(e)	(f)
1
1
50
FM
No
139 . 7

140 . 6

100 . 6
100
1
48
50
FM
No
117 . 2
(-16%)
109.1 (
-22%)
93 . 1
78
1
96
50
FM
No
102 . 0
(-27%)
92 . 2 (
-34%)
90 .4
66
1
1
50
FM
No
1093

1141

104 .4
104
1
48
50
FM
No
799.4
(-2.7%)
664.9(
-42%)
83 .2
60
1
96
50
FM
No
856 . 0
(-22%)
759 . 3 (
-33%)
88 . 7
69
1
1
50
FM
No
4417

4556

103 .1
103
1
48
50
FM
No
3595
(-19%)
3139 (
-31%)
87.3
71
1
96
50
FM
No
3630
(-18%)
3102 (
-32%)
85.5
70
2
1
50
DM
YTC
10.44

9 . 717

93 .1
93
2
48
50
DM
YTC
8 . 70
(-17%)
7.220 (
-26%)
83 . 0
69
1
1
D-4

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2
1
50
DM
YTC
136 . 7

131. 6

96 . 3
96.3
2
48
50
DM
YTC
115 . 5
(-16%)
107. 5
(-18%)
93 . 0
78 . 6
4
1
200
DM
YTC
11.26

7 . 852

69 . 7
69 . 7
4
48
200
DM
YTC
9. 54
(-15%)
5 . 569
(-29%)
58 . 3
49 . 5
4
1
200
DM
YTC
133 . 0

101.7

76 . 5
76 . 5
4
48
200
DM
YTC
112 . 5
(-15%)
84 . 7
(-17%)
75 . 3
63 . 7

mg/L.









b = Species (FM = fathead minnow; DM = Daphnia magna).
c = Food (YTC = yeast-trout chow-cereal leaf) .
d = ug/L; the numbers in parentheses are percent change from
the concentrations at one hour,
e = These values for percent dissolved were calculated based
on the concentrations at the same time measured using the
acidification method,
f = These values for percent dissolved were calculated based
on the concentrations at one hour measured using the
acidification method.
For both analytical methods, all concentrations at 48 and 96
hours were substantially lower than those at 1 hour. The
decrease in the concentrations from 1 hour to 48 and 96 hours
might have been due to precipitation, uptake by test
organisms, and/or sorption onto test chambers. Precipitation
could cause the dissolved measurements to be lower if
equilibrium had not been achieved within the first hour and
could cause the acidification measurements to be lower if the
stirring did not resuspend all of the precipitate and/or did
not keep it suspended during the sampling. Uptake is not
likely important because fishes and daphnids do not
bioconcentrate substantial amounts of lead. The chambers were
conditioned before the simulation tests began, but this
conditioning might have not been sufficient to prevent
sorption. Data obtained with nickel and cadmium indicate that
sorption onto the test chambers was probably substantial and
was probably greater in simulations containing daphnids and
YTC.
The 34 values for percent dissolved ranged from 4 9.5 to 104.4
percent.
Interpretation of the Results
The interpretation of the results is described in Section 3.
Based on the initial concentration measured by the
acidification method, the time-weighted averages (TWAs)
obtained for percent dissolved for each simulation were:
D-5

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Type of
Hard.
Species Food
Initial
TWA
% dissolved
Simulation
(mcr/L)
(a)
(b)
Cone.c
l-hrd
4 8 -hre
96 -hrf
1
• 50
FM
No
139.7
100 . 6
89.4
80 . 7
1
50
FM
No
1093
104 .4
82 . 6
73 . 9
1
50
FM
No
4417
103 .1
87 . 1
78 . 9
2
50
DM
YTC
10 .44
93 .1
81. 2
	
2
50
DM
YTC
136 . 7
96 . 3
87 . 4
	
4
200
DM
YTC
11. 26
69 . 7
59 . 6
	
4
200
DM
YTC
133 . 0
76 . 5
70 . 1
	


Mean TWA







AH

92 . 0
79 . 6
77 . 8



No food







Hard =
50 mg/L
102 . 7
86.4
77 . 8



YTC







Hard =
50 mg/L
94 . 7
84 . 3
	



Hard =
200 mg/L
73 .1
64 . 8
	
a Species (FM = fathead minnow; DM = Daphnia magna).
b Food (YTC = yeast-trout chow-cereal leaf).
c Initial concentration (ug/L) measured by the acidification
method.
d This is the percent dissolved at 1 hour.
e This TWA was calculated as the average of the percent
dissolved values at 1 and 48 hours (see Section 3).
f This TWA was calculated by giving the value for percent
dissolved at 48 hours twice the weight as the values for
percent dissolved at 1 and 96 hours (see Section 3).
The 17 TWAs ranged from 59.6 to 104.4 percent. The mean TWA
decreased substantially as the duration of the simulation
increased. The 1-hr TWA depended on food and hardness,
whereas the 4 8-hr TWA depended on hardness, but not food. All
three of the 1-hr TWAs for simulations with no food were
greater than 100 percent.
The acute toxicity tests that were important in the derivation
of the freshwater criterion for lead were flow-through,
renewal, 48-hr static, and 96-hr static tests. Because all of
the acute tests used in the derivation of the criterion were
unfed tests and the percent dissolved is higher in unfed
tests, the recommended conversion factor (CF) for the CMC is
0.890, which is the average of 102.7, 86.4, and 77.8. All of
these values were determined at hardness = 5 0 mg/L.
Of the five freshwater chronic toxicity tests that were used
in the derivation of the criterion, three were renewal tests
and two were flow-through tests; all of these were fed tests.
Therefore, the recommended CF for the CCC at hardness = 50
mg/L is 0.895, which is the average of 0.947 and 0.84, and
D-6

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0.690 at hardness = 200 mg/L, which is the average of 0.731
and 0.648.
Because the CFs for the CMC and CCC are similar at hardness =
50 mg/L, but the CF for the CCC at hardness = 200 mg/L is
quite different, the CFs for the CMC and CCC will be assumed
to be 0.892 at hardness = 50 mg/L, and will be assumed to be
0.690 at hardness = 200 mg/L. In addition, it will be assumed
that the following equation can be used to calculate the CF at
any hardness:
CF = 1.46203 - [ (In Hardness) (0.145712)]
where:
(In hardness) = the natural logarithm of the hardness.
Many of the percent dissolved values given for lead in
Attachment 2 of Prothro (1993) are reasonably close to 89.2
and 69.0 percent, but many.are substantially lower.
D-7

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Appendix E: Arsenic(III)
The three kinds of results described in Section 3 are presented
below for arsenic(III); the methodology used to obtain these
results is described in Section 2. The refinements made in the
methodology during the project affected the work with
arsenic(III) as follows:
a.	The concentration of arsenic in Lake Superior water (LSW) was
not measured in the batch of LSW used to obtain the results
reported below; the concentration was measured in a different
batch of LSW.
b.	Neither the hardness nor the alkalinity of LSW was increased
for any simulation with arsenic(III) because the criterion for
arsenic is not hardness-dependent.
c.	The sodium bicarbonate solution was 0.05 N.
d.	Each test chamber was conditioned with simulation solution
before the simulation was begun.
e.	Samples for measurement of arsenic(III) were passed through a
plastic screen by using the sampling apparatus that was
described in Section 2; the apparatus was conditioned in the
solution to be sampled.
f.	Each solution that was sampled was stirred just before a pair
of samples was taken; solutions were not stirred just before
each individual sample was taken. If a total recoverable
sample was in the pair, it was taken before the acidification
sample; if a dissolved sample was in the pair, it was taken
after the acidification sample.
g.	The solutions remaining in the test chambers at the end of the
simulations were not acidified to see if there was an
indication of sorption onto the test chambers.
Because the analytical method used to measure arsenic(III) could
not differentiate between arsenic(III) and arsenic(V), it is
appropriate to report some information in terms of arsenic rather
than arsenic(III) .
Pre-simulation test results
1.	The detection limit for arsenic(III) was determined to be 2
ug/L. The lowest concentration with which a simulation was
to be performed with arsenic(III) was 1000 ug/L, and so the
detection limit was less than ten percent of the lowest
concentration.
2.	The acidification method was compared with the total
recoverable method by analyzing each of the following
solutions six times by each method:
Soln 11: Metal-free water.
Soln 12: LSW plus arsenic(III) .
Soln 13: LSW plus daphnid food.
Soln 14: LSW plus daphnid food and arsenic(III).
E-l

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The mean measured concentrations (in ug/L, with the
standard deviations in parentheses) were:
Method
Solution
11
12
13
14
Acidification
< 2.0 1061 < 2.0
(10)
1025
(19)
Total Recoverable
<2.0 1040 < 2.0
(19)
1033
(19)
Analysis of a different batch of LSW (without daphnid food)
using the acidification method found that the concentration
of arsenic was less than 2.0 ug/L.
a.	Solution 11 was used to determine method blanks. All
blanks for both methods were less than 2.0 ug/L.
b.	Neither method detected arsenic in solution 13.
c.	Taking into account the blanks and standard deviations,
the agreement between the results obtained using the two
methods was good for solution 12 and very good for
solution 14.
d.	Both methods gave lower concentrations of arsenic in
solution 14 than in solution 12; the reduction was
greater for the acidification method than for the total
recoverable method, because the acidification method
measured more arsenic in solution 12 than did the total
recoverable method.
e.	The two coefficients of variation (COVs) obtained using
the acidification method to analyze solutions 12 and 14
were 0.9 and 1.8 %, whereas the two COVs obtained using
the total recoverable method to analyze the same
solutions were 1.9 and 1.8 %.
3. The dissolved method was compared with the acidification
method by analyzing each of the following solutions ten
times by each method:
Soln 21: Metal-free water.
Soln 22: Metal-free bicarbonate solution (MFBS).
Soln 23: MFBS plus arsenic(III).
Soln 24: LSW plus arsenic(III).
a.	For replicate analyses of solutions 21 and 22 using both
methods, all forty blanks were less than 2.0 ug/L. The
blanks were sufficiently low.
b.	For the replicate analyses of solution 23, the quotient
of the dissolved mean divided by the acidification mean
was 1.02, which was acceptable.
c.	For the replicate analyses of solution 24, the quotient
of the dissolved mean divided by the acidification mean
was 1.00, which was acceptable.
E-2

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d. For the replicate analyses of solutions 23 and 24, the
COVs were 2.1 and 1.7 % for the acidification method and
3.1 and 1.1 % for the dissolved method. Each COV was
less than 20 % and was therefore acceptable.
4. The COVs obtained with the three methods were:
Method
Number
of COVs
Range
(%)
Mean
(%)
Total Recoverable
Dissolved
Acidification
2
2
4
1.8 to 1.9
1.1 to 3.1
0.9 to 2.1
1	. 85
2	. 10
1 . 62
The COVs for the three methods were all low and similar.
These results indicate that the analytical methods were
performed acceptably before the simulation tests were begun.
Checks of the acidification and dissolved methods during analyses
of samples from the simulation tests
1. Only one set of QA samples was analyzed because all of the
samples from the simulation tests were analyzed at the same
time.
a.	All blanks were less than 2.0 ug/L for both methods and
were therefore acceptable.
b.	The results of replicate analyses of MFBS spiked with
arsenic(III) were:
Method
Number
Range
(ucr/L)
Mean
(ucr/L)
COV
(%)
Acidification
Dissolved
8
8
932 to 1007
934 to 1030
970
972
2	. 9
3	.2
The quotient of the dissolved mean divided by the
acidification mean was 1.00. A quotient of 1.02 was
reported above for spiked MFBS, and a quotient of 1.00
was reported above for spiked LSW. The COVs for both
methods were low, but were higher than the pre-
simulation test mean COVs.
c. The only recovery in MFBS for the acidification method
gave 94.6 %; recoveries in MFBS for the dissolved method
gave 94.2 and 100.9 %. These recoveries were performed
by spiking into acidified solutions; i.e., for the
dissolved method the spike was added after the solution
was filtered and acidified.
E-3

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2.	For each pair of duplicate analyses of a simulation
solution, the lower value was divided by the higher value;
the resulting quotients were:
Method	 Number	Range		Mean
Acidification	10	0.93 to 1.00	0.97
Dissolved	10	0.95 to 1.00	0.98
3.	The results of recoveries in simulation solutions were:
Method		Number	Range ^	Mean
Acidification	4	88 to 105 %	98 %
Dissolved	5	101 to 111 %	106 %
These recoveries were performed by spiking into acidified
solutions; i.e., for the dissolved method the spike was
added after the solution was filtered and acidified.
These results indicate that the analytical methods were
performed acceptably during the analyses of the simulation
solutions.
Results of the simulation tests
Type
Time
Hard
Sp.
Food
Acidifica.
Dissolved
%
%
of
(hr)
(a)
(b)
(c)
Method
Method
Diss.
Diss.
Sim.




(d)
(d)
(e)
(f)
1
1
50
FM
No
1006

1009

100.3
100.3
1
48
50
FM
No
1022
( + 2%)
1022
(+1%)
100.0
101.6
1
96
50
FM
No
1018
( + 1%)
968
(-4%)
95.1
96 .2
1
1
50
FM
No
9650

9559

99.1
99.1
1
48
50
FM
NO
9943
(+3%)
9886
(+3%)
99.4
102 .4
1
96
50
FM
NO
9559
(-1%)
9569
(0%)
100 .1
99.2
2
1
50
DM
YTC
1033

1038

100.5
100.5
2
48
50
DM
YTC
1065
(+3%)
1041
(0%)
97.7
100 . 8
2
1
50
DM
YTC
2811

2874

102.2
102.2
2
48
50
DM
YTC
2833
( + 1%)
2672
(-7%)
94 .3
95.1
a = mg/L.
b = Species (FM = fathead minnow; DM = Daphnia magna).
c = Food (YTC = yeast-trout chow-cereal leaf).
d = ug/L; the numbers in parentheses are percent change from
the concentrations at one hour.
E-4

-------
e = These values for percent	dissolved were calculated based
on the concentrations at	the same time measured using the
acidification method,
f = These values for percent	dissolved were calculated based
on the concentrations at	one hour measured using the
acidification method.
For both analytical methods, the concentrations at 48 and 96
hours seemed to be randomly higher and lower than those at one
hour, with the largest amount of change being 7 percent; the
changes for the two methods did not seem to be correlated.
The 20 values for percent dissolved only ranged from 94.3 to
102.4 percent.
Interpretation of the Results
The interpretation of the results is described in Section 3.
Based on the initial concentration measured by the
acidification method, the time-weighted averages (TWAs)
obtained for percent dissolved for each simulation were:
Type of
Hard.
Species
Food
Initial
TWA
% dissolved
Simulation
(ma/L)
(a)
(b)
Cone.c
1 -hrd
48 - hre
96 - hrf
1
50
FM
No
1006
100 . 3
101. 0
99 . 9
1
50
FM
No
9650
99 .1
100 . 8
100 . 8
2
50
DM
YTC
1033
100 . 5
100 . 6

2
50
DM
YTC
2811
102 . 2
98 . 6




Mean
TWA
100 .5
100 . 2
100 .4
a Species (FM = fathead minnow; DM = Daphnia magna).
b Food (YTC = yeast-trout chow-cereal leaf) .
c Initial concentration (ug/L) measured by the acidification
method.
d This is the percent dissolved at 1 hour.
e This TWA was calculated as the average of the percent
dissolved values at 1 and 4 8 hours (see Section 3).
f This TWA was calculated by giving the value for percent
dissolved at 48 hours twice the weight as the values for
percent dissolved at 1 and 96 hours (see Section 3).
The 10 TWAs ranged from 98.6 to 102.2 percent. The mean TWA
did not depend on the duration of the simulation. For each
duration, the range of the TWAs was very small and did not
seem to depend on species or food. Because arsenic(III) is an
oxyion, it is not expected to sorb or precipitate in toxicity
tests with aquatic organisms, and the percent dissolved is not
E-5

-------
expected to depend on hardness; the criterion for arsenic is
not hardness-dependent.
The acute toxicity tests that were important in the derivation
of the freshwater criterion for arsenic were flow-through
tests and 48-hr static tests. Therefore, the recommended
conversion factor for the CMC would be 1.0035, which is the
average of 1.005 and 1.002. Because the conversion factor
cannot be greater than 1.0, the recommended conversion factor
is 1.000.
Of the three freshwater chronic toxicity tests that were used
in the derivation of the criterion, one was a renewal test and
two were flow-through tests. Therefore, the recommended
conversion factor for the CCC would be 1.0035, which is the
average of 1.005 and 1.002. Because the conversion factor
cannot be greater than 1.0, the recommended conversion factor
is 1.000 .
The percent dissolved values given for arsenic in Attachment 2
of Prothro (1993) are close to 100 percent.
E-6

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Appendix F: Chromium(VI)
The three kinds of results described in Section 3 are presented
below for chromium(VI); the methodology used to obtain these
results is described in Section 2. The refinements made in the
methodology during the project affected the work with
chromium(VI) as follows:
a.	The concentration of chromium(VI) in Lake Superior water (LSW)
was not measured.
b.	Neither the hardness nor the alkalinity of LSW was increased
for any simulation with chromium(VI) because the criterion for
chromium(VI) is not hardness-dependent.
c.	The sodium bicarbonate solution was 0.05 N.
d.	Each test chamber was conditioned with the simulation solution
before the simulation was begun.
e.	Samples for measurement of chromium(VI) were passed through a
plastic screen by using the sampling apparatus that was
described in Section 2; the apparatus was conditioned in the
solution to be sampled.
f.	Each solution that was sampled was stirred just before a pair
of samples was taken; solutions were not stirred just before
each individual sample was taken. If a total recoverable
sample was in the pair, it was taken before the acidification
sample; if a dissolved sample was in the pair, it was taken
after the acidification sample.
g.	The solutions remaining in the test chambers at the end of the
simulations were not acidified to see if there was an
indication of sorption onto the test chambers.
Because the analytical method used to measure chromium(VI) could
not differentiate between chromium(III) and chromium(VI), it is
appropriate to report some information in terms of chromium
rather than chromium(VI).
Pre-simulation test results
1.	The detection limit for chromium(VI) was determined to be
0.14 ug/L. The lowest concentration with which a
simulation was to be performed with chromium(VI) was 5
ug/L, and so the detection .limit was less than ten percent
of the lowest concentration.
2.	The acidification method was compared with the total
recoverable method by analyzing each of the following
solutions six times by each method:
Soln	11: Metal-free water.
Soln	12: LSW plus chromium(VI).
Soln	13: LSW plus daphnid food.
Soln	14: LSW plus daphnid food and chromium(VI).
F-l

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The mean measured concentrations (in ug/L, with the
standard deviations in parentheses) were:
Method
Solution
11
12
13
14
Acidification
< 0.14 5.154
(0.31)
< 0.14 5.515
(0.23)
Total Recoverable
< 0.14 5.601
(0.30)
< 0.14 5.326
(0.22)
LSW was not analyzed for chromium(VI).
a.	Solution 11 was used to determine method blanks. All
blanks for both methods were less than 0.14 ug/L.
b.	Neither method detected chromium in solution 13.
c.	Taking into account the blanks and standard deviations,
the agreement between the results obtained using the two
methods was reasonably good for solution 12 and for
solution 14.
d.	The addition of food to LSW did not result in a
measurable amount of chromium in solution 13 using
either the acidification method or the total recoverable
method. For the acidification method, there was more
chromium in solution 14 than in solution 12, but for the
total recoverable method, the concentration in solution
14 was less than the concentration in solution 12. For
each method, the difference is comparable to the
standard deviation.
e.	The two coefficients of variation (COVs) obtained using
the acidification method to analyze solutions 12 and 14
were 6.0 and 4.1 %, whereas the two COVs obtained using
the total recoverable method to analyze the same
solutions were 5.3 and 4.2 %.
3. The dissolved method was compared with the acidification
method by analyzing each of the following solutions ten
times by each method:
Soln 21: Metal-free water.
Soln 22: Metal-free bicarbonate solution (MFBS).
Soln 23: MFBS plus chromium(VI).
Soln 24: LSW plus chromium(VI).
a. For the replicate analyses of solutions 21 and 22, one
blank for the acidification method was 0.25 ug/L, and
the other 19 blanks for the acidification method were
all less than 0.14 ug/L. Three of the blanks for the
dissolved method were 0.25, 0.25, and 0.15 ug/L, and the
other 17 blanks for the dissolved method were all less
than 0.14 ug/L. Because the lowest concentration with
which a simulation was to be conducted was 5 ug/L, the
F-2

-------
blanks were sufficiently low, even though four of the
blanks were somewhat above the detection limit.
b.	For the replicate analyses of solution 23, the quotient
of the dissolved mean divided by the acidification mean
was 1.04, which was acceptable.
c.	For the replicate analyses of solution 24, the quotient
of the dissolved mean divided by the acidification mean
was 0.99, which was acceptable.
d.	For the replicate analyses of solutions 23 and 24, the
COVs were 3.3 and 5.4 % for the acidification method and
6.6 and 5.9 % for the dissolved method. Each COV was
less than 20 % and was therefore acceptable.
4. The COVs obtained with the three methods were:
Method
Number
Range
Mean

of COVs
(%>
<%)
Total Recoverable
2
4.2 to 5.3
4 . 75
Dissolved
2
5.9 to 6.6
6 .25
Acidification
4
3.3 to 6.0
4 . 70
The mean COV for the dissolved method was higher than those
for the total recoverable and acidification methods.
These results indicate that the analytical methods were
performed acceptably before the simulation tests were begun.
Checks of the acidification and dissolved methods during analyses
of samples from the simulation tests
1. Two sets of QA samples were analyzed because the samples
from the simulation tests were analyzed in two batches.
a.	Of the ten blanks for the acidification method, six were
less than 0.14 ug/L, but the other four were 0.34, 9.53,
1.33, and 0.84 ug/L. Of the ten blanks for the
dissolved method, eight were less than 0.14 ug/L, but
the other two were 0.38 and 0.20 ug/L. Six of the
twenty blanks seem high and two of these were very high.
b.	The results of replicate analyses of MFBS spiked with
chromium(VI) were:
Method
Number
Range
(uq/L)
Mean
(ua/L)
COV
(%)
Acidification
4
4.8 to 5.2
4 . 95
3 . 0

6
4.6 to 5.7
5.08
9 . 2
Dissolved
4
4.8 to 5.3
4 . 98
4 . 9

6
4.6 to 5.2
4 . 92
6 . 2
F-3

-------
The quotients of the dissolved mean divided by the
acidification mean were 1.01 and 0.97. A quotient of
1.04 was reported above for spiked MFBS and a quotient
of 0.99 was reported above for spiked LSW. The COVs
were similar and were similar to the pre-simulation test
mean COVs.
c. The only recovery in MFBS for the acidification method
gave 97.6 %; recoveries in MFBS for the dissolved method
gave 108.0 and 102.7 %. These recoveries were performed
by spiking into acidified solutions; i.e., for the
dissolved method the spike was added after the solution
was filtered and acidified.
2. For each pair of duplicate analyses of a simulation
solution, the lower value was divided by the higher value;
the resulting quotients were:
Method	
Acidification
Dissolved
3. The results of
Method	
Acidification
Dissolved
Number
9
10
recoveries
Number
5
5
Range	
0.96 to 1.00
0.90 to 1.00
Range
80 to 98 %
83 to 102 %
Mean
0. 98
0 . 98
Mean
90 %
95 %
in simulation solutions were:
The mean recovery for the acidification method seems low
considering that the recoveries were performed by spiking
into acidified solutions; i.e., for the dissolved method
the spike was added after the solution was filtered and
acidified. The recoveries reported in section l.c above
are higher.
These results indicate that the analytical methods were
performed acceptably during the analyses of the simulation
solutions.
Results of the simulation tests
Type Time Hard Sp. Food Acidifica.	Dissolved	%	%
of (hr) (a) (b) (c) Method	Method	Diss.	Diss.
Sim. 	 	 	 	 	[dj	 	£d_)		(e)	(f)
1
1
50
FM
No
29 .71

29.84

100 . 4
100 .4
1
48
50
FM
No
28.72
(-3%)
28 .21
(-5%)
98 . 2
95 . 0
1
96
50
FM
No
27 . 59
(-7%)
27 . 88
( -7%)
101. 1
93 . 8
F-4

-------
1
1
50
FM
NO
68 . 97

69 . 63

101. 0
101 . 0
1
48
50
FM
No
66 . 18
(-4%)
68.35
(-2%)
103 .3
99 .1
1
96
50
FM
No
70 . 10
(+2%)
69 .12
(-1%)
98 . 6
100 .2
2
1
50
DM
YTC
4 . 710

4 . 825

102 .4
102 .4
2
48
50
DM
YTC
4 . 645
(-1%)
4 . 321
(-10%)
93 . 0
91. 7
2
1
50
DM
YTC
26 . 90

26 .19

97.3
97 . 3
2
48
50
DM
YTC
22 . 66
(-16%)
22 .40
(-14%)
98 . 9
83 . 3
a = mg/L.
b = Species (FM = fathead minnow; DM = Daphnia magna).
c = Food (YTC = yeast-trout chow-cereal leaf).
d = ug/L; the numbers in parentheses are percent change from
the concentrations at one hour,
e = These values for percent dissolved were calculated based
on the concentrations at the same time measured using the
acidification method,
f = These values for percent dissolved were calculated based
on the concentrations at one hour measured using the
acidification method.
The raw data from the simulation tests did not show the
irregularities that occurred in the pre-simulation test
results and in the checks during the analyses.
The 20 values for percent dissolved ranged from 83.3 to 103.3
percent.
The concentrations at 48 and 96 hours were lower than those at
1 hour for five of six cases for the acidification method and
for all six cases for the dissolved method. The decreases
were less than eight percent for the solutions that contained
neither organisms nor food. Three of the four cases for
solutions containing daphnids and daphnid food showed
decreases of 10 to 14 percent, and the fourth showed a
decrease of 1 percent.
The decrease in the concentrations from 1 hour to 48 and 96
hours might have been due to precipitation, uptake by test
organisms, and/or sorption onto test chambers. Precipitation
could cause the dissolved measurements to be lower if
equilibrium had not been achieved within the first hour and
could cause the acidification measurements to be lower if the
stirring did not resuspend all of the precipitate and/or did
not keep it suspended during the sampling. Uptake is not
likely important because fishes and daphnids do not
bioconcentrate substantial amounts of chromium(VI). The
chambers were conditioned before the simulation tests began,
but this conditioning might not have been sufficient to
prevent sorption. Data obtained with nickel and cadmium
F-5

-------
indicate that sorption onto the test chambers was probably-
substantial and was probably greater in simulations containing
daphnids and YTC.
Interpretation of the Results
The interpretation of the results is described in Section 3.
Based on the initial concentration measured by the
acidification method, the time-weighted averages (TWAs)
obtained for percent dissolved for each simulation were:
Type of
Hard.
Species
Food
Initial
TWA
% dissolved
Simulation
(mcr/L)
(a)
(b)
Cone.c
l-hrd
48 - hre
96 -hrf
1
50
FM
No
29 . 71
100 .4
97. 7
96 . 0
1
50
FM
No
68 . 97
101. 0
100 . 0
99 . 8
2
50
DM
YTC
4 . 710
102 .4
97 . 0
	
2
50
DM
YTC
26 . 90
97 . 3
90 . 3
	
Mean TWA	100.3 96.2 97.9
a Species (FM = fathead minnow; DM = Daphnia magna).
b Food (YTC = yeast-trout chow-cereal leaf).
c Initial concentration (ug/L) measured by the acidification
method.
d This is the percent dissolved at 1 hour.
e This TWA was calculated as the average of the percent
dissolved values at 1 and 48 hours (see Section 3).
f This TWA was calculated by giving the value for percent
dissolved at 4 8 hours twice the weight as the values for
percent dissolved at 1 and 96 hours (see Section 3).
Nine of the ten TWAs are between 96.0 and 102.4 percent; the
other one was 90.3 percent. In each simulation the mean TWA
decreased as the duration of the simulation increased. For
each duration, the range of the TWAs was small and did not
seem to depend on species or food. Because chromium(VI) is an
oxyion, it is not expected to sorb or precipitate in toxicity
tests with aquatic organisms, and the percent dissolved is not
expected to depend on hardness; the criterion for chromium(VI)
is not hardness-dependent.
The acute toxicity tests that were important in the derivation
of the freshwater criterion for chromium(VI) were flow-through
tests and renewal tests. Therefore, the recommended
conversion factor for the CMC is 0.982, which is the average
of 1.003 and 0.962.
All four of the freshwater chronic toxicity tests that were
used in the derivation of the criterion were renewal tests.
F-6

-------
Therefore, the recommended conversion factor for the CCC is
0.962 .
The percent dissolved values given for chromium(VI) in
Attachment 2 of Prothro (1993) are close to 98.2 and 96.2
percent, but were determined at very high concentrations of
chromium(VI).
F-7

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Appendix G: Selenium
The three kinds of results described in Section 3 are presented
below for selenium(IV); the methodology used to obtain these
results is described in Section 2. The refinements made in the
methodology during the project affected the work with
selenium(IV) as follows:
a.	The concentration of selenium(IV) in Lake Superior water (LSW)
was measured in the batch of LSW used to obtain the results
reported below.
b.	Neither the hardness nor the alkalinity of LSW was increased
for any simulation with selenium(IV) because the criterion for
selenium(IV) is not hardness-dependent.
c.	The sodium bicarbonate solution was 0.05 N.
d.	Test chambers were not conditioned with simulation solution
before simulations were begun.
e.	Samples for measurement of selenium(IV) were not passed
through a plastic screen.
f.	Each solution that was sampled was stirred just before each
individual sample was taken, e.g., the solution was stirred
before a sample was taken for analysis using the acidification
method and was stirred again before a sample was taken for
analysis using the dissolved method.
g.	The solutions remaining in the test chambers at the end of the
simulations were acidified to see if there was an indication
of sorption onto the test chambers.
Because the analytical method used to measure selenium(IV) could
not differentiate between selenium(IV) and selenium(VI), it is
appropriate to report some information in terms of selenium
rather than selenium(IV).
Pre-simulation test results
1.	The detection limit for selenium(IV) was determined to be
6.0 ug/L. The lowest concentration with which a simulation
was to be performed with selenium(IV) was 90 ug/L, and so
the detection limit was less than ten percent of the lowest
concentration.
2.	The acidification method was compared with the total
recoverable method by analyzing each of the following
solutions six times by each method:
Soln 11: Metal-free water.
Soln 12: LSW plus selenium(IV).
Soln 13: LSW plus daphnid food.
Soln 14: LSW plus daphnid food and selenium(IV).
Soln 17: LSW (with nothing added).
The mean measured concentrations (in ug/L, with the
standard deviations in parentheses were:
G-l

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Method
	Solution	
11	12	13	14 	17
Acidification	< 6.0 88.92 < 6.0 88.59 < 6.0
(2.2)	(1.2)
Total Recoverable < 6.0 64.98 < 6.0 62.18 < 6.0
(1.4)	(3.0)
Because the concentrations obtained using the total
recoverable method were so much lower than those obtained
using the acidification method, this comparison was
repeated after solutions 12, 13, 14, and 17 were prepared
again:
Method		Solution	
		11	12	13	14	17
Acidification	< 6.0 69.31 < 6.0 68.37 < 6.0
(1.7)	(2.0)
Total Recoverable < 6.0 55.20 < 6.0 33.46 < 6.0
(4.6)	(0.9)
The results obtained using the total recoverable method
were again much lower than those obtained using the
acidification method. It is possible that this was due to
volatilization during the digestion step of the total
recoverable procedure.
a.	Solution 11 was used to determine method blanks. All
blanks for both methods were less than 6.0 ug/L.
b.	Neither method detected selenium in solution 13 or in
solution 17.
c.	Taking into account the blanks and standard deviations,
the agreement between the results obtained using the two
methods was very poor for solution 12 and for solution
14,- the total recoverable method gave much lower mean
concentrations for solutions 12 and 14 than did the
acidification method.
d.	The results obtained with solutions 12 and 14 indicate
that the daphnid food had negligible effect on results
obtained using the acidification method". The food might
have had a small effect on the results obtained using
the total recoverable method the first time; food had a
large effect on the results obtained using the total
recoverable method the second time.
e.	The four coefficients of variation (COVs) obtained using
the acidification method to analyze solutions 12 and 14
ranged from 1.4 to 2.9 %, whereas the four COVs obtained
using the total recoverable method to analyze the same
solutions ranged from 2.1 to 8.3 %.
G-2

-------
3.	The dissolved method was compared with the acidification
method by analyzing each of the following solutions ten
times by each method:
Soln 21: Metal-free water.
Soln 22: Metal-free bicarbonate solution (MFBS).
Soln 23: MFBS plus selenium(IV).
Soln 24: LSW plus selenium(IV).
a.	For the replicate analyses of solutions 21 and 22 using
both methods, all forty blanks were less than 6.0 ug/L.
The blanks were sufficiently low.
b.	For the replicate analyses of solution 23, the quotient
of the dissolved mean divided by the acidification mean
was 1.03, which was acceptable.
c.	For the replicate analyses of solution 24, the quotient
of the dissolved mean divided by the acidification mean
was 0.96, which was acceptable.
d.	For the replicate analyses of solutions 23 and 24, the
COVs were 2.2 and 2.6 % for the acidification method and
1.8 and 2.4 % for the dissolved method. Each COV was
less than 20 % and was therefore acceptable.
4.	The COVs obtained with the three methods were:
Method
Number
Range
Mean

of COVs
<%)
(%)
Total Recoverable
4
2.1 to 8.3
4 .48
Dissolved
2
1.8 to 2.4
2 .10
Acidification
6
1.4 to 2.9
2 . 35
The COVs for the total recoverable method were somewhat
higher than those for the dissolved and acidification
methods.
These results indicate that the analytical methods were
performed acceptably before the simulation tests were begun.
Checks of the acidification and dissolved methods during analyses
of samples from the simulation tests
1. Two sets of QA samples were analyzed because the samples
from the simulation tests were analyzed in two batches.
a.	All blanks were less than 6.0 ug/L for both methods and
were therefore acceptable.
b.	The results of replicate analyses of MFBS spiked with
selenium(IV) were:
G-3

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Method Number	Range	Mean	COV
	 	 (ua/L) (ua/L) (%)
Acidification
Dissolved
6
4
6
4
50.28 to 77.10	59.83	18.9
51.96 to 59.22	54.78	6.2
50.77 to 81.07	61.66	21.4
54.41 to 57 .84	55.87	2.8
In both sets of six values, two of the values might have
been a pair of outliers.
The quotients of the dissolved means divided by the
acidification means were 1.03 and 1.02. A quotient of
1.03 was reported above for spiked MFBS, and a quotient
of 0.96 was reported above for spiked LSW. The first
COV determined for each method was substantially higher
than the second; the second COV for each method was
similar to the pre-simulation test mean COV for the
method.
c. Recoveries in MFBS for the acidification method gave
78.3, 59.7, and 72.0 %; recoveries in MFBS for the
dissolved method gave 55.5, 58.4, 56.0, and 55.1 %. It
is surprising that these recoveries are so low
considering that they were performed by spiking into
acidified solutions; i.e., for the dissolved method the
spike was added after the solution was filtered and
acidified. The recoveries reported below are
substantially higher.
2. For each pair of duplicate analyses of a simulation
solution, the lower value was divided by the higher value;
the resulting quotients were:
Method
Acidification
Dissolved
Number
15
15
Range
0.90 to 1.00
0.93 to 1.00
Mean
0 . 98
0 . 98
3. The results of recoveries in simulation solutions were:
Method
Acidification
Dissolved
Number
7
7
Range
95 to 112 %
76 to 113 %
Mean
99 %
96 %
These recoveries were performed by spiking into acidified
solutions; i.e., for the dissolved method the spike was
added after the solution was filtered and acidified. These
recoveries in simulation solutions were much higher than
those reported above for MFBS.
G-4

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These results indicate that the analytical methods were
performed acceptably during the analyses of the simulation
solutions.
Results of the simulation tests
Type
Time
Hard
Sp.
Food
Acidifica.
Dissolved
%
.%
of
(hr)
(a)
(b)
(c)
Method
Method
Diss.
Diss.
Sim.




(d)

(d)
(e)
(f)
1
1
50
FM
No
672 .3

674 . 8
100 .4
100.4
1
48
50
FM
No
672.7
(0)
678.1 (0)
100.8
100.9
1
96
50
FM
No
628 .4
(-7)
621.5 (-8)
98.9
92.4
1
1
50
FM
No
7432

7469
100 .5
100.5
1
48
50
FM
No
7098 (
-4)
7296 (-2)
102 .8
98 .2
1
96
50
FM
No
7466 (0)
7477 (0)
100 .2
100.6
1
1
50
FM
No
7180

7110
99.0
99.0
1
48
50
FM
No
7078 (
-1)
7034 (-1)
99 .4
98.0
1
96
. 50
FM
No
7395 (+3)
7348 (+3)
99 .4
102.3
2
1
50
DM
YTC
60 . 05

56.18
93 .6
93.6
2
48
50
DM
YTC
61. 11
(+2)
60.18 (+7)
98.5
100.2
2
1
50
DM
YTC
573 .2

587.3 (-2)
102 .5
102.5
2
48
50
DM
YTC
579 . 0
( + 1)
578.4 (0)
99.9
100.9
2
1
50
DM
YTC
2779

2859
102 . 9
102 . 9
2
48
50
DM
YTC
2709 (
-3)
2690 (-6)
99 .3
96.8
a = mg/L.
b = Species (FM = fathead minnow; DM = Daohnia magna).
c = Food (YTC = yeast-trout chow-cereal leaf).
d = ug/L; the numbers in parentheses are percent change from
the concentrations at one hour,
e = These values for percent dissolved were calculated based
on the concentrations at the same time measured using the
acidification method,
f = These values for percent dissolved were calculated based
on the concentrations at one hour measured using the
acidification method.
The 3 0 values for percent dissolved only ranged from 92.4 to
102.9 percent.
For both analytical methods, the concentrations at 48 and 96
hours seemed to be randomly higher and lower than those at one
G-5

-------
hour, with the largest amount of change being 8 percent. The
changes for the two methods seemed to be correlated.
To obtain an indication of whether sorption occurred, the test
chambers were not conditioned before the simulations began and
acid was added to the solutions remaining in the test chambers
after the end of the simulations. The acidified solutions
were mixed for 15 to 3 0 minutes and sampled. For each
simulation (i.e., for each set of three test chambers), the
ratio of the mean concentration of selenium in the acidified
solution to the last mean concentration measured using the
acidification method was calculated:
Type of Food Mean Concentration3 Ratiob
Simulation 	 Before	After 	
1
No
628.4
679 . 5
1 . 08
1
No
7466 .
7365 .
0 . 99
1
No
7395 .
7354 .
0 . 99
2
YTC
61.11
56 . 74*
0 . 93
2
YTC
579 . 0
594 . 0
1. 03
2
YTC
2709 .
2736 .
1 . 01
* One value was considered an outlier and was not used.
a ug/L; n = 3 for each mean.
b The "after" mean concentration divided by the "before"
mean concentration.
The volumes of the solutions remaining in the different test
chambers were not measured and so it is not known how similar
the volumes were. Thus the ratios can only give a qualitative
comparison of the metal that was "desorbed" by adding acid to
the remaining solution while it was still in the test chamber.
The addition of the acid caused a substantial change in the
concentration of selenium in the remaining solution for only
one of the six simulations.
Interpretation of the Results
The interpretation of the results is described in Section 3.
Based on the initial concentration measured by the
acidification method, the time-weighted averages (TWAs)
obtained for percent dissolved for each simulation were:
G-6

-------
Type of
Hard.
Species
Food
Initial
TWA
% dissolved
Simulation
(ma/L)
(a)
(b)
Cone.c
l-hrd
48 -hre
96-hrf
1
50
FM
No
672.3
100 .4
100 .6
98 .6
1
50
FM
No
7432
100 .5
99.4
99.4
1
50
FM
No
7180
99.0
98.5
99.3
2
50
DM
YTC
60.05
93 .6
96.9

2
50
DM
YTC
573 .2
102 .5
101.7
	
2
50
DM
YTC
2779
102.9
99.8
	



Mean TWA
99 . 8
99.5
99 .1
a Species (FM = fathead minnow; DM = Daphnia magna).
b Food (YTC = yeast-trout chow-cereal leaf).
° Initial concentration (ug/L) measured by the acidification
method.
d This is the percent dissolved at 1 hour.
e This TWA was calculated as the average of the percent
dissolved values at 1 and 48 hours (see Section 3).
f This TWA was calculated by giving the value for percent
dissolved at 48 hours twice the weight as the values for
percent dissolved at 1 and 96 hours {see Section 3).
The 15 TWAs ranged from 93.6 to 102.9 percent. The mean TWA
decreased slightly as the duration of the simulation
increased. For each duration, the range of the TWAs was small
and did not seem to depend on species or food. Because
selenium(IV) is an oxyion, it is not expected to sorb or
precipitate in toxicity tests with aquatic organisms and the
percent dissolved is not expected to depend on hardness; the
criterion for selenium(IV) is not hardness-dependent.
The purpose of the simulations with selenium was to determine
whether the dissolved FACR should be the same as the total
recoverable FACR, i.e., dc>es the percent dissolved depend on
(1) the concentration of selenium and/or (2) whether food is
present.
a.	The acute toxicity tests that were used in the derivation
of the freshwater FACR for selenium(IV) were flow-through
tests and 48-hr static tests. Therefore, the recommended
conversion factor for the numerator of the FACR is 0.996,
which is the average of 0.998 and 0.995.
b.	Of the six freshwater chronic toxicity tests that were used
in the derivation of the FACR, three were flow-through
tests and three were renewal tests. Therefore, the
recommended conversion factor for the denominator of the
FACR is 0.996, which is the average of 0.998 and 0.995.
Thus the dissolved FACR equals the total recoverable FACR, and
the conversion factor for the CMC equals the conversion factor
for the CCC.
G- 7

-------
The freshwater CCC was based on field data from Belews Lake,
and so conversion of the CCC from total recoverable to
dissolved should also be based on data from Belews Lake. Data
for the four samples presented by Cumbie (1978) in Table 11
indicate that an average of 92.2 percent of the selenium in
the water column in Belews Lake was dissolved. Thus the
recommended conversion factor for the CCC is 0.922. This
applies to the selenium in the water column in Belews Lake; it
is expected that both selenium(IV) and selenium(VI) were
present.
No values for the percent dissolved are given for selenium(IV)
in Attachment 2 of Prothro (1993).
G-8

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Appendix H: Nickel
The three kinds of results described in Section 3 are presented
below for nickel; the methodology used to obtain these results is
described in Section 2. The refinements made in the methodology
during the project affected the work with nickel as follows:
a.	The concentration of nickel in Lake Superior water (LSW) was
measured in the batch of LSW used to obtain the results
reported below.
b.	Whenever the hardness of LSW was increased for a simulation
with nickel, the alkalinity was also increased.
c.	The sodium bicarbonate solution was 0.05 N.
d.	Test chambers were not conditioned with simulation solution
before simulations were begun.
e.	Samples for measurement of nickel were not passed through a
plastic screen.
f.	Each solution that was sampled was stirred just before each
individual sample was taken, e.g., the solution was stirred
before a sample was taken for analysis using the acidification
method and was stirred again before a sample was taken for
analysis using the dissolved method.
g.	The solutions remaining in the test chambers at the end of the
simulations were acidified to see if there was an indication
of sorption onto the test chambers.
Pre-simulation test results
1.	The detection limit for nickel was determined to be 1.0
ug/L. The lowest concentration with which a simulation was
to be performed with nickel was 15 ug/L, and so the
detection limit was less than ten percent of the lowest
concentration.
2.	The acidification method was compared with the total
recoverable method by analyzing each of the following
solutions six times by each method:
Soln 11: Metal-free water.
Soln 12: LSW plus nickel.
Soln 13: LSW plus daphnid food.
Soln 14: LSW plus daphnid food and nickel.
Soln 17: LSW (with nothing added).
The mean measured concentrations (in ug/L, with the
standard deviations in parentheses) were:
H-1

-------
Method
Solution
11	12
13 	14
17
Acidification
<1.0 12.54
(0.50)
< 1.0 12.26
(0.68)
< 1.0
Total Recoverable
<1.0 14.10
(0.29)
< 1.0 14.42 < 1.0
(0.33)
a.	Solution 11 was used to determine method blanks. All
blanks for both methods were less than 1.0 ug/L.
b.	Neither method detected nickel in solution 13 or in
solution 17.
c.	Taking into account the blanks and standard deviations,
the agreement between the results obtained using the two
methods was not very good; the total recoverable method
gave higher mean concentrations for solutions 12 and 14
than did the acidification method.
d.	The results obtained with solutions 12 and 14 indicate
that the daphnid food had negligible effect on results
obtained using both methods.
e.	The two coefficients of variation (COVs) obtained using
the acidification method to analyze solutions 12 and 14
were 4.0 and 5.5 %, whereas the two COVs obtained using
the total recoverable method to analyze the same
solutions were 2.0 and 2.3 %.
The dissolved method was compared with the acidification
method by analyzing each of the following ^solutions ten
times by each method:
Soln 21: Metal-free water.
Soln 22: Metal-free bicarbonate solution (MFBS).
Soln 23: MFBS plus nickel.
Soln 24: LSW plus nickel.
a.	For the replicate analyses of solutions 21 and 22 using
both methods, all forty blanks were less than 1.0 ug/L.
The blanks were sufficiently low.
b.	For the replicate analyses of solution 23, the quotient
of the dissolved mean divided by the acidification mean
was 1.00, which was acceptable.
c.	For the replicate analyses of solution 24, the quotient
of the dissolved mean divided by the acidification mean
was 0.94, which was acceptable.
d.	For the replicate analyses of solutions 23 and 24, the
COVs were 4.9 and 5.7 % for the acidification method and
6.2 and 3.2 % for the dissolved method. Each COV was
less than 20 % and was therefore acceptable.
The COVs obtained with the three methods were:
H-2

-------
Method	Number	Range	Mean
		of COVs	(%)	(%)
Total Recoverable 2	2.0 to 2.3	2.15
Dissolved 2	3.2 to 6.2	4.70
Acidification 4	4.0 to 5.7	5.02
Each of the COVs for the total recoverable method was lower
than those for the dissolved and acidification methods.
These results indicate that the analytical methods were
performed acceptably before the simulation tests were begun.
Checks of the acidification and dissolved methods during analyses
of samples from the simulation tests
1. Two sets of QA samples were analyzed' because the samples
from the simulation tests were analyzed in two batches.
a.	All blanks were less than 1.0 ug/L for both methods and
were therefore acceptable.
b.	The results of replicate analyses of MFBS spiked with
nickel were:
Method
Number
Range
Mean
COV


(uq/L)
(ua/L)
(%)
Acidification
8
12.11 to 13.18
12 . 58
2 . 9

4
13.45 to 14.37
13 . 99
3 . 2
Dissolved
8
11.18 to 13.18
12 .41
5 . 0

4
13.29 to 14.69
14 .11
7 . 1
The quotients of the dissolved mean divided by the
acidification mean were 0.99 and 1.01. A quotient of
1.00 was reported above for spiked MFBS and a quotient
of 0.94 was reported above for spiked LSW. The
acidification method gave slightly lower COVs than did
the dissolved method, and the COVs were similar to the
pre-simulation test mean COVs.
c. Recoveries in MFBS for the acidification method gave
99.4 and 80.1 %; recoveries in MFBS for the dissolved
method gave 95.9, 87.5, 98.7, 110.8, and 104.5 %. These
recoveries were performed by spiking into acidified
solutions; i.e., for the dissolved method the spike was
added after the solution was filtered and acidified.
2. For each pair of duplicate analyses of a simulation
solution, the lower value was divided by the higher value;
the resulting quotients were:
H-3

-------
Method
Number
Range
Mean
Acidification	18	0.97 to 1.00	0.99
Dissolved	18	0.96 to 1.00	0.98
3. The results of recoveries in simulation solutions were:
Method		Number	Range	Mean
Acidification	11	83 to 118 %	99 %
Dissolved	9	88 to 124 %	101 %
It is surprising that the range of these recoveries is so
great considering that they were performed by spiking into
acidified solutions; i.e., for the dissolved method the
spike was added after the solution was filtered and
acidified. The means, however, are close to 100 %. The
range here is greater than that reported in section l.c
above.
These results indicate that the analytical methods were
performed acceptably during the analyses of the simulation
solutions.
Results of the simulation tests
Type
Time
Hard
Sp.
Food
Acidifica.
Dissolved
%
%
of
(hr)
(a)
(b)
(c)
Method
Method
Diss.
Diss .
Sim.




(d)
(d)
(e)
(f)
1
1
50
FM
No
2146
2152
100 . 3
100 . 3
1
48
50
FM
No
2215 (+3)
2164 (+1)
97 . 7
100 . 8
1
96
50
FM
No
2187 (+2)
2178 (+1)
99 . 6
101. 5
1
1
50
FM
No
21774
22648
104 . 0
104 . 0
1
48
50
FM
No
21130 (-3)
21207 (-6)
100 .4
97.4
1
96
50
FM
No
22054 (+1)
21511 (-5)
97 . 5
98 . 8
2
1
50
DM
YTC
14.96
15.32
102 .4
102 .4
2
48
50
DM
YTC
12.94 (-14)
13.16 (-14)
101. 7
88 . 0
2
1
50
DM
YTC
101. 6
100 .1
98.5
98 . 5
2
48
50
DM
YTC
101.5 (0)
101.0 (+1)
99 . 5
99.4
2
1
50
DM
YTC
490 .4
486 . 2
99 .1
99 . 1
2
48
50
DM
YTC
520.2 (+6)
518.8 (+7)
99.7
105 . 8
4
1
200
DM
YTC
17.06
17 . 00
99 . 6
99 . 6
4
48
200
DM
YTC
16.55 (-3)
16.72 (-2)
101. 0
98 . 0
H-4

-------
4 1 200 DM YTC 113.1	112.0	99.0 99.0
4 48 200 DM YTC 109.2 (-3) 109.9 (-2) 100.6 97.2
4 1 200 DM YTC 491.4	482.6	98.2 98.2
4 48 200 DM YTC 500.5 (+2) 498.2 (+3) 99.5 101.4
a = mg/L.
b = Species (FM = fathead minnow; DM = Daphnia magna).
c = Food (YTC = yeast-trout chow-cereal leaf).
d = ug/L; the numbers in parentheses are percent change from
the concentrations at one hour,
e = These values for percent dissolved were calculated based
on the concentrations at the same time measured using the
acidification method,
f = These values for percent dissolved were calculated based
on. the concentrations at one hour measured using the
acidification method.
The 36 values for percent dissolved ranged from 88.0 to 105.8
percent.
For both analytical methods, the concentrations at 4 8 and 96
hours seemed to be randomly higher and lower than those at one
hour, with the largest amount of change being 14 percent; the
changes for the two methods seemed to be correlated.
To obtain an indication of whether sorption occurred, the test
chambers were not conditioned before the simulations began and
acid was added to the solutions remaining in the test chambers
after the end of the simulations. The acidified solutions
were mixed for 15 to 3 0 minutes and sampled. For each
simulation (i.e., for each set of three test chambers), the
ratio of the mean concentration of nickel in the acidified
solution to the last mean concentration measured using the
acidification method was calculated:
Type of Food Mean Concentration3 Ratio15
Simulation 	 Before	After 	
1
No
2187 .
2204 .
1.01
1
NO
22054.
21630.
0 . 98
2
YTC
12 . 94
15 . 81
1 . 22
2
YTC
101. 5
114 . 4
1 . 13
2
YTC
520 . 2
565 . 0
1 . 09
4
YTC
16 . 55
19 .39*
1. 17
4
YTC
109 . 2
117 . 2
1 . 07
4
YTC
500 . 5
523 . 6
1 . 05
* One value was considered an outlier and was not used.
H-5

-------
a ug/L; n = 3 for each mean.
b The "after" mean concentration divided by the "before"
mean concentration.
The volumes of the solutions remaining in the different test
chambers were not measured and so it is not known how similar
the volumes were. Thus the ratios can only give a qualitative
comparison of the metal that was "desorbed" by adding acid to
the remaining solution while it was still in the test chamber.
The ratios are higher for the solutions containing YTC, and
they decrease as the concentration increases. The ratios were
1.09 and 1.05 for the two YTC simulations that contained the
highest concentration of nickel, both of which showed an
increase, not a decrease, by both analytical methods from 1 to
48 hours. Apparently, sorption occurred within the first hour
of the simulation test, desorption occurred between 1 and 48
hours, and additional desorption occurred when acid was added
to the solution remaining in the test chamber after the end of
the simulation test.
Interpretation of the Results
The interpretation of the results is described in Section 3.
Based on the initial concentration measured by the
acidification method, the time-weighted averages (TWAs)
obtained for percent dissolved for each simulation were:
Type of Hard. Species Food Initial
TWA % dissolved
lation
(mq/L)
(a)
(b)
Cone.c
1 -hrd
1
50
FM
No
2146
100 . 3
1
50
FM
No
21774
104 . 0
2
50
DM
YTC
14 . 96
102 .4
2
50
DM
YTC
101 . 6
98 . 5
2
50
DM
YTC
490 .4
99.1
4
200
DM
YTC
17 . 06
99.6
4
200
DM
YTC
113 . 1
99 . 0
4
200
DM
YTC
491.4
98 .2



Mean
TWA
100 . 1
100. 6
100 . 7
95 . 2
99 . 0
102 .4
98 . 8
98	. 1
99	. 8
100 . 8
99 .4
99.3 100.1
Species (FM = fathead minnow; DM = Daphnia magna).
Food (YTC = yeast-trout chow-cereal leaf).
Initial concentration (ug/L) measured by the acidification
method.
This is the percent dissolved at 1 hour.
This TWA was calculated as the average of the percent
dissolved values at 1 and 48 hours (see Section 3).
H-6

-------
f This TWA was calculated by giving the value for percent
dissolved at 48 hours twice the weight as the values for
percent dissolved at 1 and 96 hours (see Section 3).
The 18 TWAs ranged from 95.2 to. 104.0 percent. The mean TWA
did not seem to depend on the duration of the simulation. For
each duration, the range of the TWAs was small and did not
seem to depend on hardness, but did seem to depend slightly on
food and/or species.
The acute toxicity tests that were important in the derivation
of the freshwater criterion for nickel were flow-through, 48-
hr static, and 96-hr static tests. Therefore, the recommended
conversion factor for the CMC is 0.998, which is the average
of 1.001, 0.993, and l.OOl.
Of the five freshwater chronic toxicity tests that were used
in the derivation of the criterion, three were renewal tests
and two were flow-through tests. Therefore, the recommended
conversion factor for the CCC is 0.997, which is the average
of 1.001 and 0.993.
One of the percent dissolved values given for nickel in
Attachment 2 of Prothro (1993) is 100 percent, but the other
seven range from 76 to 93 percent.
H-7

-------
Appendix I: Cadmium
The three kinds of results described in Section 3 are presented
below for cadmium; the methodology used to obtain these results
is described in Section 2. The refinements made in the
methodology during the project affected the work with cadmium as
follows:
a.	The concentration of cadmium in Lake Superior water (LSW) was
measured in the batch of LSW used to obtain the results
reported below.
b.	Whenever the hardness of LSW was increased for a simulation
with cadmium, the alkalinity was also increased.
c.	Two sodium bicarbonate solutions were used. One was 0.05 N;
the other was 0.001 N with sufficient calcium sulfate added to
give a hardness of 50 mg/L.
d.	Test chambers were not conditioned with simulation solution
before simulations were begun.
e.	Samples for measurement of cadmium were not passed through a
plastic screen.
f.	Each solution that was sampled was stirred just before each
individual sample was taken, e.g., the solution was stirred
before a sample was taken for analysis using the acidification
method and was stirred again before a sample was taken for
analysis using the dissolved method.
g.	The solutions remaining in the test chambers at the end of the
simulations were acidified to see if there was an indication
of sorption onto the test chambers.
Pre-simulation test results
1. The detection limit for cadmium was determined to be 0.03
ug/L. The lowest concentration with which a simulation was
to be performed with cadmium was 0.3 ug/L, and so the
detection limit was ten percent of the lowest
concentration.
The acidification method was compared with the total
recoverable method by analyzing each of the following
solutions six times by each method:
Soln 11: Metal-free water.
Soln 12: LSW plus cadmium.
Soln 13: LSW plus daphnid food.
Soln 14: LSW plus daphnid food and cadmium.
Soln 15: LSW plus fathead minnow food.
Soln 16: LSW plus fathead minnow food and cadmium.
Soln 17: LSW (with nothing added).
The mean measured concentrations (in ug/L, with the
standard deviations in parentheses) were:
1-1

-------
Method		Solution	
	 11	12	13	14	15 . 16	17
Acidifica. <0.03 0.195 <0.03 0.192 <0.03 0.190 <0.03
(0.01)	(0.01)	(0.01
Tot. Reco. <0.03) 0.280 <0.03 0.245 <0.03 0.277 <0.03
(0.01)	(0.01)	(0.01)
a.	Solution 11 was used to determine method blanks. Eight
blanks were less than 0.03 ug/L; one acidification blank
was 0.06 ug/L, and three total recoverable blanks were
between 0.05 and 0.09 ug/L.
b.	For solutions 13, 15, and 17, three of 18 acidification
measurements and two of 18 total recoverable
measurements were between 0.03 and 0.09 ug/L; the other
31 measurements were < 0.03 ug/L.
c.	Taking into account the blanks and standard deviations,
the total recoverable method consistently gave higher
mean concentrations for solutions 12, 14, and 16 than
did the acidification method.
d.	The results obtained with solutions 12, 14, and 16
indicate that both foods had negligible effect on
results obtained using both methods.
e.	The three coefficients of variation (COVs) obtained
using the acidification method to analyze solutions 12,
14, and 16 were 6.5, 3.7, and 3.0 %, whereas the three
COVs obtained using the total recoverable method to
analyze the same solutions were 3.4, 2.3, and 2.6 %.
3. The dissolved method was compared with the acidification
method by analyzing each of the following solutions ten
times by each method:
Soln 21: Metal-free water.
Soln 22: Metal-free bicarbonate solution (MFBS).
Soln 23: MFBS plus cadmium.
Soln 24: LSW plus 'cadmium.
The MFBS was 0.05 N sodium bicarbonate.
a.	For the replicate analyses of solutions 21 and 22 using
both methods, all forty blanks were less than 0.03 ug/L.
The blanks were sufficiently low.
b.	For the replicate analyses of solution 23, the quotient
of the dissolved mean divided by the acidification mean
was 0.87, which was too low to be acceptable.
c.	For the replicate analyses of solution 24, the quotient
of the dissolved mean divided by the acidification mean
was 0.87, which was acceptable.
d.	For the replicate analyses of solutions 23 and 24, the
COVs were 3.9 and 2.2 % for the acidification method and
3.6 and 4.0 % for the dissolved method. Each COV was
less than 20 % and was therefore acceptable.
1-2

-------
4.	The dissolved method was again compared with the
acidification method by analyzing each of the following
solutions ten times by each method:
Soln 21: Metal-free water.
Soln 22: Metal-free bicarbonate solution (MFBS).
Soln 23: MFBS plus cadmium.
Soln 24: LSW plus cadmium.
This time the MFBS was 0.001 N sodium bicarbonate with
sufficient calcium sulfate to give a hardness of 50 mg/L.
a.	For the replicate analyses of solutions 21 and 22 using
both methods, all forty blanks were less than 0.03 ug/L.
The blanks were sufficiently low.
b.	For the replicate analyses of solution 23, the quotient
of the dissolved mean divided by the acidification mean
was 1.00, which was acceptable.
c.	For the replicate analyses of solution 24, the quotient
of the dissolved mean divided by the acidification mean
was 1.00, which was acceptable.
d.	For the replicate analyses of solutions 23 and 24, the
COVs were 2.5 and 3.0 % for the acidification method and
2.4 and 2.9 % for the dissolved method. Each COV was
less than 20 % and was therefore acceptable.
Although the quotient of the dissolved mean divided by the
acidification mean changed from 0.87 for 0.05 N sodium
bicarbonate to 1.00 for 0.001 N sodium bicarbonate plus
calcium sulfate, the quotient for Lake Superior water
changed from 0.87 to 1.00 at the same time.
5.	The COVs obtained with the three methods were:
Method
Number
Range
Mean

of COVs
(%)
(%)
Total Recoverable
3
2.3 to 3.4
2 . 77
Dissolved
4
2.4 to 4.0
3 . 22
Acidification
7
2.2 to 6.5
3 . 54
The COVs for the three methods were similar.
These results indicate that the analytical methods were
performed acceptably before the simulation tests were begun.
Checks of the acidification and dissolved methods during analyses
of samples from the simulation tests
1. Three sets of QA samples were analyzed because the samples
from the simulation tests were analyzed in three batches,
a. One blank was 0.4 ug/L and the other 33 blanks were less
than 0.03 ug/L and were therefore acceptable for both
methods.
1-3

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b. The results of replicate analyses of MFBS spiked with
cadmium were:
Method
Number
Range
Mean
COV


(ug/L)
(ug/L)
(%)
Acidification
8
0.235 to 0.336
0.293
14 .4

4
0.320 to 0.336
0.324
2.6

5
0.269 to 0.303
0.296
5.0
Dissolved
8
0.235 to 0.320
0.288
11. 3

4
0.286 to 0.320
0.307
5.3

5
0.269 to 0.332
0.295
8 . 9
The MFBS used was 0.001 N sodium bicarbonate with
sufficient calcium sulfate added to give a hardness of
50 mg/L. The quotients of the dissolved mean divided by
the acidification mean were 0.98, 0.94, and 1.00. A
quotient of 1.00 was reported above for spiked 0.001 N
MFBS and quotients of 0.87 and 1.00 were reported above
for spiked LSW. The two methods gave similar COVs,
which were slightly higher than the pre-simulation test
mean COVs.
c. Recoveries in MFBS for the acidification method gave
98.2 and 105.9 %; the only recovery in MFBS for the
dissolved method gave 112.7 %. These recoveries were
performed by spiking into acidified solutions; i.e., for
the dissolved method the spike was added after the
solution was filtered and acidified.
2. For each pair of duplicate analyses of a simulation
solution, the lower value was divided by the higher value;
the resulting quotients were:
Method		Number	Range		Mean
Acidification	14	0.91 to 1.00	0.98
Dissolved	14	0.95 to 1.00	0.99
3. The results of recoveries in simulation solutions were:
Method		Number	Range	Mean
Acidification	6	91 to 112 %	98 %
Dissolved	8	82 to 124 %	98 %
It is surprising that the range of these recoveries is so
great considering that•they were performed by spiking into
acidified solutions; i.e., for the dissolved method the
spike was added after the solution was filtered and
acidified. The means, however, are close to 100 %. The
1-4

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range here is greater than that reported in section l.c
above.
These results indicate that the analytical methods were
performed acceptably during the analyses of the simulation
solutions.
Results of the simulation tests
Type
Time
Hard
Sp.
Food
Acidifica.
Dissolved
%
%
of
(hr)
(a)
(b)
(c)
Method
Method
Diss.
Diss .
Sim.




(d)
(d)
(e)
(f)
1
1
50
FM
No
3 . 501
3 . 534
100 . 9
100 . 9
1
48
50
FM
No
3.343 (-5)
3.254* (-8)
97 . 3
92 . 9
1
96
50
FM
No
2.722 (-22)
2.647 (-25)
97 . 2
75 . 6
1
1
50
FM
No
31. 15
31.26
100 . 3
100 . 3
1
48
50
FM
No
30.26 (-3)
29.96 (-4)
99 . 0
96 . 2
1
96
50
FM
No
30.38 (-2)
29.31 (-6)
96 . 5
94 . 1
2
1
50
DM
YTC
0.3280
0.3251
99 . 1
99 . 1
2
48
50
DM
YTC
0 . 2264 (-31)
0.2180 (-33)
96 . 3
66 . 5
2
1
50
DM
YTC
2 . 669
2.467*
92 .4
92 .4
2
48
50
DM
YTC
2.675 (0)
2.624 (+6)
98 . 1
98 . 3
3
1
50
FM
BS
0 .3304
0.3202
96 . 9
96 . 9
3
48
50
FM
BS
0.3079 (-7)
0.3031 (-5)
98 . 5
91.7
3
1 ¦
50
FM
BS
3 . 722
3 . 737
100 . 4
100 .4
3
48
50
FM
BS
2.870 (-22)
2.881 (-23)
100.4
77.4
4
1
200
DM
YTC
0.2724
0 . 2635*
96 . 7
96 . 7
4
48
200
DM
YTC
0.1709(-37)
0.1449 (-45)
84 . 8
53 . 2
4
1
200
DM
YTC
3 .340
3 . 031
90 . 8
90 . 8
4
48
200
DM
YTC
2.744 (-18)
2.615 (-14)
95 . 3
78 . 3
* =
One value
was
considered an outlier and was not used,

a = mg/L.
b = Species (FM = fathead minnow; DM = Daphnia magna).
c = Food (YTC = yeast-trout chow-cereal leaf; BS = brine
shrimp nauplii) .
d = ug/L; the numbers in parentheses are percent change from
the concentrations at one hour,
e = These values for percent dissolved were calculated based
on the concentrations at the same time measured using the
acidification method.
1-5

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f = These values for percent dissolved were calculated based
on the concentrations at one hour measured using the
acidification method.
The 36 values for percent dissolved ranged from 53.2 to 100.9
percent. The concentrations at 48 and 96 hours were lower
than those at 1 hour for all except one case for both
analytical methods. These decreases might have been due to
precipitation, uptake by test organisms, and/or sorption onto
test chambers. Precipitation could cause the dissolved
measurements to be lower if equilibrium had not been achieved
within the first hour and could cause the acidification
measurements to be lower if the stirring did not resuspend all
of the precipitate and/or did not keep it suspended during the
sampling. Uptake is not likely important because fishes and
daphnids do not bioconcentrate substantial amounts of cadmium.
To obtain an indication of whether sorption occurred, the test
chambers were not conditioned before the simulations began and
acid was added to the solutions remaining in the test chambers
after the end of the simulations. The acidified solutions
were mixed for 15 to 3 0 minutes and sampled. For each
simulation (i.e., for each set of three test chambers), the
ratio of the mean concentration of cadmium in the acidified
solution to the last mean concentration measured using the
acidification method was calculated:
Type of Food Mean Concentration3 Ratiob
Simulation 	 Before	 After 	
1
No
2 .722
3 .596
1.32
1
No
30.38
32 .31
1. 06
2
YTC
0 . 2264
0.4675
2 . 06
2
YTC
2 . 675
4.099
1. 53
3
BS
0 . 3079
0.3792
1. 23
3
BS
2 . 870
4 .019
1.40
4
YTC
0 .1709
0.3591*
2 .10
4
YTC
2 . 744
4 .200
1.53
* One value was considered an outlier and was not used.
a ug/L; n = 3 for each mean.
b
The "after" mean concentration divided by the "before"
mean concentration.
1-6

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The volumes of the solutions remaining in the different test
chambers were not measured and so it is not known how similar
the volumes were. Thus the ratios can only give a qualitative
comparison of the metal that was "desorbed" by adding acid to
the remaining solution while it was still in the test chamber.
The ratios are higher for the solutions containing daphnids
and YTC, and the ratios decrease as the concentration
increases for three of the four types of simulations. Both of
these trends seem to be related to the loss of cadmium from
the beginning to the end of the simulation tests.
Interpretation of the Results
The interpretation of the results is described in Section 3.
Based on the initial concentration measured by the
acidification method, the time-weighted averages (TWAs)
obtained for percent dissolved for each simulation were:
Type of
Hard.
Species
Food
Initial
TWA
% dissolved
Simulation
(mcr/L)
(a)

(b)
Cone.c
l-hrd
00
tr
o
96 - hr
1
50
FM

No
3 . 501
100 . 9
96 . 9
90 . 6
1
50
FM

No
31. 15
100 .3
98 . 2
96 . 7
2
50
DM

YTC
0.3280
99 .1
82 . 8
	
2
50
DM

YTC
2 . 669
92 .4
95.4
	
3
50
FM

BS
0.3304
96 . 9
94 . 3
	
3
50
FM

BS
3 . 722
100 .4
88 . 9
	
4
200
DM

YTC
0.2724
96 . 7
75 . 0
	
4
200
DM

YTC
3 . 340
90 . 8
84 . 6
	


Mean
TWA







All

97 . 2
89 . 5
93 . 6



No
food







Hard =
50 mg/L
100 . 6
97 . 6
93 . 6



BS








Hard =
50 mg/L
98 . 6
91. 6
	



YTC







Hard =
50 mg/L
95 . 8
89 . 1
	



Hard =
200 mg/L
93 . 8
79 . 8
	
a Species (FM = fathead minnow; DM = Daphnia magna).
b Food (YTC = yeast-trout chow-cereal leaf; BS = brine shrimp
nauplii).
c Initial concentration (ug/L) measured by the acidification
method.
d This is the percent dissolved at 1 hour.
e This TWA was calculated as the average of the percent
dissolved values at 1 and 48 hours (see Section 3).
1-7

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f This TWA was calculated by giving the value for percent
dissolved at 48 hours twice the weight as the values for
percent dissolved at 1 and 96 hours (see Section 3).
The 18 TWAs ranged from 75.0 to 100.9 percent. For all except
one simulation, the TWA decreased as the duration of the
simulation increased. The TWA also depended on food,
hardness, and the concentration of cadmium. In four out of
five comparisons at 48 and 96 hours, the TWA increased with
the concentration of cadmium; at 1 hour, the opposite occurred
for three out of four comparisons. The two exceptions
concerning the concentration of cadmium both occurred with
brine shrimp.
The acute toxicity tests that were important in the derivation
of the freshwater criterion for cadmium were flow-through,
renewal, 48-hr static, and 96-hr static tests. Because all of
these are unfed tests and the percent dissolved is higher in
unfed tests, the recommended conversion factor (CF) for the
CMC is 0.973, which is the average of 1.006, 0.976, and 0.936.
All of these were determined at hardness = 50 mg/L.
The chronic toxicity tests that were important in the
derivation of the criterion were renewal and flow-through
tests. They were all fed tests, but some were fed brine
shrimp and some were fed YTC. The percent dissolved depended
on the food used and on the hardness:
Mean TWAs	Average
Hardness = 50 mg/L
BS 0.986, 0.916	0.951
YTC 0.958, 0.891	0.924
Hardness = 2 00 mg/L
YTC	0.938, 0.798	0.868
BS (predicted)	0.893*
* [0.951][(0.868)/(0.924)] = 0.893
Therefore, the recommended CF for the CCC at hardness = 50
mg/L is 0.938, which is the average of 0.951 and 0.924, and at
hardness = 200 mg/L is 0.880, which is the average of 0.868
and 0.893. It will be assumed that the following equation can
be used to calculate the CF for the CCC at any hardness:
«
CF for the CCC = 1.101672 - [(In hardness)(0.041838)]
where:
(In hardness) = the natural logarithm of the hardness.
1-8

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It will also be assumed that the same slope describes the
relationship between the CF for the CMC and hardness, so that
the following equation can be used to calculate the CF for the
CMC at any hardness:
CF for the CMC = 1.136672 - [(In hardness)(0.041838)]
Two of the values for percent dissolved given for cadmium in
Attachment 2 of Prothro (1993) are 41 and 59 percent, but the
other ten range from 75 to 96 percent and are within the range
of the TWAs reported here.
1-9

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Appendix J: Calculation of Time-Weighted Averages
If a sampling plan (e.g., for measuring metal in a treatment in a
toxicity test) is designed so that a series of values are
obtained over time in such a way that each value contains the
same amount of information (i.e., represents the same amount of
time), then the most meaningful average is the arithmetic
average. In most cases, however, when a series of values is
obtained over time, some values contain more information than
others; in these cases the most meaningful average is a time-
weighted average (TWA). If each value contains the same amount
of information, the arithmetic average will equal the TWA.
A TWA is obtained by multiplying each value by a weight and then
dividing the sum of the products by the sum of the weights. The
simplest approach is to let each weight be the duration of time
that the sample represents. Except for the first and last
samples, the period of time represented by a sample starts
halfway to the previous sample and ends halfway to the next
sample. The period of time represented by the first sample
starts at the beginning of the test, and the period of time
represented by the last sample ends at the end of the test. Thus
for a 96-hr toxicity test, the sum of the weights will be 96 hr.
The following are hypothetical examples of grab samples taken
from 96-hr flow-through tests for two common sampling regimes:
Sampling
Cone. Weight
Product
Time-weighted average
time (hr)
(ma/L)
(hr)
(hr)(ma/L)
(ma/L)
0
12
48
576

96
14
48
672



96
1248
1248/96 = 13.00
0
8
12
96

24
6
24
144

48
7
24
168

72
9
24
216

96
8
12
96



96
720
720/96 = 7.500
When all
the weights
are the same, the
arithmetic average equal;
the TWA.
Similarly,
if only one sample
is taken, both the
arithmetic average and the
TWA equal the value of that sample.
The calculations are more complex for composite samples and for
samples from renewal tests. In all cases, however, the sampling
plan can be designed so that the TWA equals the arithmetic
average.
J-l

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Appendix K: Calculation of Conversion Factors
The three values for Percent Dissolved (PD) are calculated as:
Dissolved Concentration at 1 hour
Acidification Concentration at 1 Hour
Dissolved Concentration at 48 hours
Acidification Concentration at 1 Hour
Dissolved Concentration at 96 hours
Acidification Concentration at 1 Hour
PD (1)
PD(48)
PD(96)
The three Time-Weighted Averages (TWAs) are calculated as:
TWA(1) = PD(1)
TWA (48) = PD(1) +PP(48)
TWA(96) - PD'1' * 2 ' PD(48> - PD(96)
4
The equations for TWA(48) and TWA(96) are not exact because they
both treat PD(1) as if it were PD(0). In the calculation of
TWA(96), twice as much weight is given to PD(48) as to either
PD(1) or PD(96).
The Conversion Factors (CFs) are calculated as unweighted means
of various combinations of the three TWAs, depending on the kinds
of toxicity tests that were important in the derivation of the
CMC or CCC in the criteria document:
1.	48-hr static tests only:
CF = TWA (4 8) = PD(1) ^PE>(48)
2.	96-hr static tests only:
CF = TWA(96) = FPU) * 2-PD(48) + PD(96)
Flow-through tests only:
CF = TWA (1) = PD (1)
K-l

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48-hr static tests and 96-hr static tests:
Cp = TWA (48) + TWA (96)
PD (1) + PD(48) ^ PD(1) + 2 " PD(48) +PD(96)
2	4
2
3 * PD(1) + 4 • PD(48) + PD(96)
8
48-hr static tests and flow-through tests:
Cp = TWA (48) + TWA (1)
PD (1) + PD (48) 4. on M ^
2
2
6 • PD (1) + 2•PD(48)
8
96-hr static tests and flow-through tests
cp _ TWA (96) + TWA (1)
2
PD(1) + 2 • PD(48) + PD(96)
+ PD(1)
2
5 • PD(1) + 2•PD (48) + PD(96)
8
48-hr and 96-hr static tests and flow-through tests:
Cp = TWA (48) + TWA (96) + TWA(l)
PD(1) + PD(48) ^ PD (1) + 2 • PD (48) + PD(96) „ Dn M ^
2	4
3
7 • PD (1) + 4 * PD(48) + PD(96)
12
K-2

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