B­
1
+

M2+
M­
DOC
M­
Biotic
Ligand
MOH
MHCO
MCl
3
Organic
Matter
Complexation
Site
of
Action
Competing
Cations
Free
Metal
Ion
Inorganic
Ligand
Complexation
+

+
+
2+
Ca
+
H
Na+
Na
Biotic
Ligand
Model
Windows
Interface,
Version
2.0.0
User's
Guide
and
Reference
Manual
April
2003
HydroQual,
Inc.
1
Lethbridge
Plaza
Mahwah,
NJ
07430
U.
S.
A
B­
2
SECTION
1
1INTRODUCTION
TO
THE
BLM
1.1
INTRODUCTION
Metal
bioavailability
and
toxicity
have
long
been
recognized
to
be
a
function
of
water
chemistry
(
Sunda
and
Guillard
1976;
Sunda
and
Hansen
1979).
For
example,
formation
of
inorganic
and
organic
metal
complexes
and
sorption
on
particle
surfaces
can
reduce
metal
toxicity.
As
a
result,
metal
toxicity
can
be
highly
variable
and
dependent
on
ambient
water
chemistry
when
expressed
as
total
or
dissolved
metal
concentration.
In
contrast,
the
effects
of
water
chemistry
on
metal
toxicity
can
often
be
reduced
or
eliminated
when
metal
toxicity
is
related
to
free
metal
ion
concentrations
(
Sunda
and
Guillard
1976).
Allen
and
Hansen
(
1996)
have
shown
the
relationship
between
metal
speciation
and
toxicity
and
have
used
this
relationship
to
predict
the
range
of
effects
that
site­
specific
water
quality
characteristics
can
have
on
copper
toxicity.

1.2
BLM
FRAMEWORK
AND
CONCEPTUAL
MODEL
The
Biotic
Ligand
Model
(
BLM)
was
developed
to
incorporate
metal
speciation
and
the
protective
effects
of
competing
cations
into
predictions
of
metal
bioavailability
and
toxicity
(
Di
Toro
et
al.
2001).
A
formal
description
of
metal­
organism
interactions,
now
commonly
referred
to
as
the
Free
Ion
Activity
Model
(
FIAM),
was
described
by
Morel
(
1983a).
Pagenkopf
(
1983),
using
a
similar
approach,
applied
the
Gill
Surface
Interaction
Model
(
GSIM)
to
predict
metal
effect
levels
over
a
range
of
water
quality
characteristics.
The
BLM
is
founded
upon
the
principles
that
underlie
these
earlier
models.
The
BLM
incorporates
a
version
of
CHESS
(
Santore
and
Driscoll
1995)
that
has
recently
been
modified
to
include
the
chemical
and
electrostatic
interactions
described
in
WHAM
(
Tipping
1994).
The
BLM
includes
reactions
that
describe
the
chemical
interactions
of
copper
and
other
cations
to
physiologically
active
sites
(
or
"
biotic
ligands")
which
correspond
to
the
proximate
site
of
action
of
toxicity.
However,
inorganic
and
organic
ligands
can
also
bind
metal,
thereby
reducing
accumulation
at
the
biotic
ligand.
By
incorporating
the
biotic
ligand
into
a
chemical
equilibrium
framework
that
includes
aqueous
metal
complexation,
the
relation
between
free
metal
ion
concentrations
and
toxicity
is
an
inherent
feature
of
the
model.

The
BLM
framework
also
incorporates
the
competitive
effects
of
other
cations
that
interact
with
the
biotic
ligand
to
mitigate
toxicity.
For
example,
at
a
fixed
free
metal
concentration,
as
hardness
increases,
the
increased
Ca2+
competes
with
the
free
metal
for
binding
sites
at
the
biotic
ligand.
A
higher
free
metal
concentration
is
therefore
required
to
achieve
the
same
toxic
effect
in
the
presence
of
elevated
Ca2+
concentration.
The
BLM
uses
this
competitive
mechanism
to
simulate
the
reduction
in
metal
toxicity
due
to
elevated
hardness
concentrations.
Thus,
the
BLM
can
effectively
account
for
reduction
in
metal
toxicity
due
to
elevated
levels
of
hardness
cations
(
Meyer
et
al.
1999).

The
BLM
has
been
developed
using
published
information
on
metal
toxicity
and
biotic
ligand
accumulation
as
a
function
of
water
chemistry.
The
most
comprehensive
data
compiled
to
date
for
use
with
the
BLM
is
for
copper
toxicity
to
fathead
minnows
(
Pimephales
promelas).
Copper
accumulation
on
the
gill
has
been
associated
with
respiratory
distress
and
decreased
blood
plasma
Na
concentrations
due
to
interference
with
these
sites
(
Playle
et
al.
1992).
The
adsorption
of
copper
on
gill
surfaces
in
the
BLM
has
been
calibrated
to
measurements
of
copper
accumulation
on
the
gill
over
a
wide
range
of
water
quality
conditions
(
Playle
et
B­
3
al.
1992,
1993b).
Additionally,
MacRae
(
1994)
established
a
dose
response
relationship
necessary
to
determine
the
biotic
ligand
LC50
in
rainbow
trout.
In
the
BLM,
metal
toxicity
is
defined
as
the
amount
of
metal
necessary
to
result
in
accumulation
at
the
biotic
ligand
equal
to
the
biotic
ligand
LC50.
While
others
have
developed
models
capable
of
predicting
metal
bioaccumulation
on
the
gill
in
short
term
exposures
(
Playle
et
al.
1993a,
b),
the
BLM
is
the
first
that
includes
a
scheme
for
predicting
toxicity.
The
BLM
for
other
metals
and
organisms
is
based
on
a
similar
approach.

1.3
PREDICTION
MODE
The
BLM
interface
application
allows
the
user
to
run
the
BLM
either
in
toxicity
mode
or
in
the
speciation
mode.
When
run
in
the
toxicity
mode,
for
the
metal
and
organism
specified
by
the
user,
the
BLM
will
predict
the
amount
of
metal
required
to
cause
acute
mortality
in
the
water
specified
by
the
user.
However,
when
the
BLM
is
run
in
the
speciation
mode,
for
the
metal
concentration
specified
by
the
user,
the
BLM
will
predict
the
organic
and
the
inorganic
speciation
in
the
water
column.

1.4
BLM
APPLICATIONS
In
summary,
the
BLM
can
be
used
to
calculate
the
chemical
speciation
of
a
dissolved
metal
including
complexation
with
inorganic
and
organic
ligands,
and
the
biotic
ligand.
The
biotic
ligand
represents
a
discrete
receptor
or
site
of
action
on
an
organism
where
accumulation
of
metal
leads
to
acute
toxicity.
The
BLM
can
therefore
be
used
to
predict
the
amount
of
metal
accumulation
at
this
site
for
a
variety
of
chemical
conditions
and
metal
concentrations
(
i.
e.
the
inorganic,
organic,
and
biotic
speciation
of
metals
in
aquatic
settings).

According
to
the
conceptual
framework
of
the
BLM,
accumulation
of
metal
at
the
biotic
ligand
at
or
above
a
critical
threshold
concentration
leads
to
acute
toxicity.
This
critical
accumulation
on
the
biotic
ligand
is
also
termed
the
LA50,
the
Lethal
Accumulation
of
metal
on
the
biotic
ligand
that
results
in
50%
mortality
in
a
toxicological
exposure.
The
LA50
is
expressed
in
units
of
nmol/
g
wet
weight
of
the
biotic
ligand.
Since
the
BLM
includes
inorganic
and
organic
metal
speciation
and
competitive
complexation
with
the
biotic
ligand,
the
amount
of
dissolved
metal
required
to
reach
this
threshold
will
vary,
depending
on
the
water
chemistry.
Therefore,
in
addition
to
calculating
chemical
speciation,
the
BLM
can
also
be
used
to
predict
the
concentration
of
metal
that
would
result
in
acute
toxicity
within
a
given
aquatic
system.
B­
4
SECTION
2
2OVERVIEW
AND
HELP
FILE
LAYOUT
2.1
WHAT'S
NEW
IN
THIS
DISTRIBUTION?

Originally,
the
BLM
was
developed
as
an
MS­
DOS
based
program,
with
the
user
developing
the
BLM
input
files
using
an
external
spreadsheet
program
such
as
Microsoft
Excel,
running
the
BLM
in
the
MSDOS
environment,
and
then
analyzing
the
BLM
output
using
a
different
set
of
software
tools.
However,
in
order
to
facilitate
data­
entry,
model
simulations,
and
the
analysis
of
model
output
in
a
common
application
environment
and
in
a
more
efficient
and
user­
friendly
fashion,
a
graphical
user
interface
was
developed
for
the
BLM
and
first
distributed
as
BLM,
Windows
Interface
Version
1.0.0.
The
current
distribution,
Version
2.0.0,
is
an
updated
version
that
offers
additional
options
for
data
inputs
and
model
simulations.
The
new
functionalities
are
further
described
in
the
subsequent
sections.
The
BLM,
Windows
Interface
Version
2.0.0
incorporates
the
most
current
version
of
the
BLM,
Version
APE8.

Note
that
BLM
datafiles
created
using
the
older
version
of
the
BLM
Windows
Interface
can
be
used
directly
with
the
new
version.

2.2
HELP
FILE
LAYOUT
The
remainder
of
this
document
describes
the
hardware
and
software
requirements
for
installing
and
running
the
BLM
Windows
Interface,
the
data
requirements
of
the
BLM,
a
step­
by­
step
guide
to
using
the
various
functionalities
of
the
BLM
Windows
Interface
and
a
walk­
through
of
the
application
using
an
example
BLM
datafile.
B­
5
SECTION
3
3SETUP
AND
INSTALLATION
3.1
SYSTEM
REQUIREMENTS
The
BLM
Windows
Interface
is
designed
for
use
on
the
IBM
compatible
PC
family
of
microcomputers
running
Microsoft
Windows.
The
memory
requirements
of
the
BLM
Windows
Interface
are
modest
and
should
not
interfere
with
other
resident
programs.
The
minimum
hardware
and
software
requirements
and
the
recommended
system
configurations
are
described
below.

Minimum
System
Requirements
°
PC
Compatible,
Intel
Pentium
233
MHz
°
Microsoft
Windows
95
or
higher
°
32
MB
RAM
°
30
MB
free
disk
space
Recommended
System
Configuration
°
Intel
Pentium
3
or
higher,
500
MHz
or
faster
°
64
MB
RAM
°
100
MB
free
disk
space
Even
though
the
BLM
Windows
Interface
can
be
run
on
a
system
with
the
specified
minimum
requirements,
in
the
interest
of
computation
time,
the
recommended
system
configuration
or
a
higher
one
would
be
ideal.

0.1
INSTALLING
THE
BLM
WINDOWS
INTERFACE
°
Installing
from
a
disk
­
To
install
the
BLM
Windows
Interface
from
a
CD­
ROM,
insert
the
installation
disk
into
the
CD­
ROM
drive.
In
case
the
installation
does
not
start
up
automatically,
locate
and
run
the
program
"
setup.
exe"
located
in
the
main
directory
in
the
installation
disk
by
simply
double
clicking
on
the
file
name.

°
Installing
from
the
self­
extracting
(.
exe)
file
­
To
install
the
BLM
Windows
Interface
from
the
selfextracting
file
"
BLMWindowsInterface_
Version2.0.0.
exe"
simply
double
click
on
the
file
to
extract
its
contents
to
a
temporary
folder.
This
temporary
folder
can
be
deleted
once
the
installation
is
completed.
To
start
the
installation,
locate
and
run
the
program
"
setup.
exe"
located
in
the
temporary
folder
by
simply
double
clicking
on
the
file
name.

Note
that
on
PCs
running
Microsoft
Windows
2000
and
higher
or
any
version
of
Microsoft
Windows
NT,
the
user
may
have
to
be
logged
on
as
the
"
Administrator"
or
have
the
relevant
permissions
to
modify
the
"
System"
directory
in
order
to
install
the
necessary
files.

The
setup
program
will
guide
the
user
through
a
fairly
straightforward
installation
process,
querying
the
user
for
information
on
where
to
install
the
necessary
files.
During
the
installation,
a
shortcut
to
the
BLM
Windows
Interface
application
will
be
added
to
the
"
Programs"
sub­
menu
within
the
"
Start"
menu
on
the
B­
6
Microsoft
Windows
desktop.
In
addition,
the
BLM
Windows
Interface
application
will
also
be
registered
in
the
system
registry
so
that
the
BLM
datafiles
created
by
the
user
can
be
accessed
directly
by
just
double
clicking
on
the
file
name.
B­
7
SECTION
4
1DATA
REQUIREMENTS
The
BLM
predicts
metal
toxicity
and
speciation
for
a
particular
site
based
on
the
ambient
water
quality.
Therefore,
the
user
will
be
expected
to
provide
data
describing
the
physical
and
chemical
properties
of
the
site
water.
The
data
requirements
of
the
BLM
are
conventional
physical
and
chemical
parameters
that
are
easily
measurable
in
the
laboratory.
This
section
describes
the
general
physical
and
chemical
data
requirements
for
an
application
of
the
BLM
to
predict
metal
speciation
and
toxicity
in
aquatic
systems.

1.1
WATER
QUALITY
PARAMETERS
REQUIRED
The
ambient
water
quality
information
required
to
run
the
BLM
is
listed
below:

°
Temperature
°
pH
°
Dissolved
Organic
Carbon
°
Major
cations
(
Ca,
Mg,
Na,
and
K)
°
Major
anions
(
SO4
and
Cl)
°
Alkalinity
°
Sulfide
For
a
given
metal
some
of
these
chemical
inputs
have
an
important
effect
on
determining
metal
speciation,
while
other
chemical
inputs
have
only
minor
effects
on
BLM
predictions.
The
user
should
be
aware
of
the
relative
importance
of
each
of
the
chemical
inputs
to
decide
whether
adequate
information
is
available
for
a
meaningful
application
of
the
BLM.
The
guidelines
described
in
the
subsequent
sections
may
be
helpful
in
that
assessment.

Each
water
sample
has
to
be
fully
described
in
terms
of
the
above
water
quality
inputs
before
the
BLM
can
be
used.
However,
if
some
of
the
parameters
are
known
to
be
absent
in
the
water
sample,
a
nominal,
negligible
concentration
should
be
input
(
a
value
on
the
order
of
1E­
10
mg/
L
should
suffice
typically)
rather
than
a
zero
concentration.

0.0.1
Temperature
Temperature
measurements
are
typically
the
most
common
and
basic
of
all
water
quality
measurements
and
therefore
available
in
most
laboratory
characterizations
of
site­
water
chemistry.
Since
the
BLM
is
based
on
a
thermodynamic
chemical
equilibrium
modeling
framework,
temperature
measurements
are
important
to
determine
the
relevant
thermodynamic
reaction
rates.

0.0.2
pH
Accurate
pH
values
are
important
to
BLM
results
for
most
metals.
The
chemical
speciation
of
many
metals,
such
as
copper,
is
directly
affected
by
pH.
However,
pH
is
also
important
to
determine
the
metal
complexation
capacity
of
dissolved
organic
matter.
It
is
also
important
to
determine
the
speciation
of
inorganic
carbon,
which
relates
to
the
formation
of
metal
carbonate
complexes.
For
these
reasons,
pH
is
a
B­
8
required
chemical
input
to
the
BLM.
If
BLM
results
are
to
be
compared
to
laboratory
measurements
of
metal
toxicity,
then
it
is
preferable
that
the
pH
is
measured
within
the
test
chamber
during
the
exposure.

0.0.3
Dissolved
Organic
Carbon
Dissolved
organic
matter
plays
a
critical
role
in
determining
metal
speciation
and
bioavailability.
In
the
BLM,
the
presence
of
dissolved
organic
matter
is
specified
as
a
dissolved
organic
carbon
(
DOC)
concentration
in
mg/
L
and
is
a
required
input
for
the
BLM.
For
water
with
low
DOC
it
is
important
to
make
sure
that
analytical
detection
limits
are
sufficiently
low.
In
toxicity
studies,
the
test
organisms
themselves
may
be
a
significant
source
of
organic
matter
depending
on
the
number
of
organisms
and
the
volume
of
the
test
chamber.

Humic
Acid
Fraction
of
DOC
The
BLM
uses
a
description
of
organic
matter
chemistry
developed
for
the
Windermere
Humic
Aqueous
Model
(
WHAM,
Version
1.0),
which
characterizes
metal
complexation
with
both
humic
and
fulvic
organic
matter
sources.
It
is
therefore
necessary
to
specify
the
distribution
of
humic
and
fulvic
acids
in
the
organic
matter
present
in
a
given
water.
Unfortunately,
natural
organic
matter
composition
is
not
routinely
characterized
and
information
on
humic
and
fulvic
acid
content
is
not
likely
to
be
available.
In
the
absence
of
chemical
characterization,
a
value
of
10%
humic
acid
content
is
recommended
for
most
natural
waters.
The
variability
of
the
dissolved
organic
matter
content
in
diverse
water
sources
is
a
topic
of
current
study
by
BLM
investigators.

0.0.4
Metal
Concentrations
The
BLM
can
be
used
to
predict
the
speciation
and
bioaccumulation
of
metals
when
a
metal
concentration
is
provided
as
an
input.
When
the
model
is
used
in
metal
speciation
mode,
metal
concentrations
are
a
required
input.
However,
the
BLM
model
is
probably
most
useful
as
a
means
of
predicting
metal
toxicity
(
i.
e.,
a
concentration
associated
with
a
specific
toxicological
effect).
When
used
in
metal
toxicity
mode,
there
is
no
need
to
input
metal
concentrations.

0.0.5
Major
Cations
The
cations
Ca,
Mg,
Na,
and
K
are
all
necessary
inputs
to
the
BLM.
For
copper
and
silver,
Ca
and
Na
can
directly
compete
with
the
metal
at
biotic
ligand
sites
and
these
cations
will,
therefore,
have
a
direct
effect
on
predictions
of
metal
toxicity.
For
some
organisms,
Mg
may
play
a
critical
role
as
well.
These
cations,
therefore,
are
required
inputs
to
the
BLM.
On
the
other
hand,
K
currently
has
no
direct
effect
on
metal
toxicity
in
the
BLM
and
can
be
estimated
if
measurements
do
not
exist.

0.0.6
Major
Anions
The
anions
SO4
and
Cl
are
necessary
inputs
to
the
BLM
(
although
bicarbonate
is
also
an
important
anion,
it
is
discussed
separately
below).
In
freshwaters,
SO4
may
be
the
dominant
anion
and
is,
therefore,
important
for
determining
charge
balance
and
ionic
strength.
The
chemistry
of
metals
and
of
natural
organic
matter
is
dependent
to
varying
degrees
on
ionic
strength
and
so
SO4
has
some
importance
as
a
BLM
input.
However,
if
measurements
of
SO4
are
not
available,
the
concentrations
can
be
estimated.
For
copper
simulations,
Cl
is
only
important
as
a
contribution
to
ionic
strength,
but
for
silver
simulations
Cl
can
have
an
additional
importance
due
to
the
formation
of
silver­
chloride
complexes.
Therefore,
it
is
preferable
that
only
measured
Cl
concentrations
are
used
for
BLM
applications
involving
silver,
while
estimates
can
be
used
for
applications
involving
copper.
B­
9
0.0.7
Alkalinity
Inorganic
carbon
species
in
the
BLM
include
carbonate
(
CO3),
bicarbonate
(
HCO3),
and
carbonic
acid
(
H2CO3).
The
sum
of
these
species
is
called
dissolved
inorganic
carbon
(
DIC).
Bicarbonate
is
usually
the
most
important
DIC
species
in
natural
waters
since
it
is
the
dominant
species
between
pH
6.35
and
10.33.
Inorganic
carbon
is
a
critical
input
to
the
BLM
since
many
metals,
including
copper,
form
carbonate
complexes.
Silver,
on
the
other
hand,
does
not
form
carbonate
complexes,
and
so
DIC
is
not
a
critical
input
to
BLM
applications
for
silver.
Unfortunately,
measurements
of
DIC
are
not
often
made
in
natural
water
samples.
However,
if
it
can
be
reasonably
assumed
that
carbonate
alkalinity
is
the
dominant
source
of
the
measured
alkalinity,
the
DIC
can
be
estimated
from
alkalinity
and
pH
measurements
as
in
the
equation
below.

where
Alk.
=
alkalinity
in
equivalents/
L
=
2
x
10­
5
x
alkalinity
(
as
mg
CaCO3
/
L)
H
=
10­
pH
K1
=
10­
6.352
K2
=
10­
10.329
The
BLM
Windows
Interface
uses
this
expression
to
calculate
the
DIC
internally,
and
so
only
the
alkalinity
and
the
pH
need
to
be
specified.
Alkalinity
should
be
measured
on
filtered
samples
to
eliminate
potential
contribution
from
suspended
CaCO3
and
specified
in
units
of
mg/
L
of
CaCO3.
However,
depending
on
the
inorganic
carbon
option
selected,
the
user
may
also
opt
to
specify
DIC
concentrations
directly.
This
latter
option
would
be
preferred
generally,
and
especially
when
carbonate
alkalinity
is
not
the
dominant
source
of
measured
alkalinity,
but
must
depend
on
reliable
measurements
of
DIC.

0.0.8
Sulfide
Although
it
has
traditionally
been
assumed
that
sulfide
concentrations
are
negligible
in
aerated
waters,
recent
evidence
suggests
that
appreciable
sulfide
concentrations
persist
in
both
marine
and
freshwaters.
Waters
impacted
by
wastewater
treatment
plant
effluents
in
particular
can
have
elevated
sulfide
concentrations.
Sulfide
has
a
strong
affinity
for
many
metals
and
is
therefore
an
important
consideration
in
determining
metal
speciation
and
bioavailability.
If
it
is
present,
measured
sulfide
should
be
considered
a
required
input
to
the
BLM,
especially
when
sulfide
concentrations
are
similar
to
the
predicted
effect
levels
for
a
given
metal
and
organism.

At
the
present
time,
researchers
at
several
universities
are
still
looking
into
the
nature
of
sulfide­
metal
complexes
in
aqueous
systems.
The
persistence
of
sulfide
in
aerated
waters
may
be
linked
to
the
formation
of
stable
metal­
sulfide
clusters,
and
these
clusters
may
not
be
detected
by
traditional
sulfide
measurements.
Alternatively,
strong
metal
complexes
that
are
believed
to
be
due
to
sulfide
compounds
may
be
due
to
other
forms
of
reduced
sulfur
that
are
also
missed
by
traditional
sulfide
measurements.
Suitable
analytical
methods
that
measure
the
target
form
of
sulfide
and
which
do
not
measure
other
non­
reduced
forms
of
B­
10
sulfur,
are
under
development.
Also,
sulfide
levels
in
some
locations
may
be
known
to
be
low
and
well
below
the
effect
levels
of
interest
for
a
given
metal.
Therefore,
sulfide
measurements
may
not
be
critical
in
all
instances.
Since
these
research
questions
are
still
being
addressed,
metal­
sulfide
reactions
have
not
yet
been
incorporated
into
the
BLM.
The
sulfide
column
in
the
input
file
is
a
reminder
that
these
interactions
are
likely
to
be
added
to
a
subsequent
version
of
the
model.
Sulfide
concentrations
added
in
that
column
will
not
affect
the
BLM
calculation.
B­
11
SECTION
5
1STARTING
THE
APPLICATION
To
start
using
the
BLM
Windows
Interface,
select
the
application
using
"
Start
­­­­­>
Programs"
on
the
Microsoft
Windows
desktop.
The
user
will
be
presented
with
the
following
screen,
which
contains
the
user
input
areas
and
the
various
functions
implemented
in
this
version
of
the
BLM
Windows
Interface.

Figure
1:
Opening
Screen
for
the
BLM
Windows
Interface
Application
In
case
the
user
already
has
a
BLM
datafile
created
using
the
BLM
Windows
Interface,
the
file
can
be
opened
directly
by
just
double
clicking
on
the
file
name
through
a
file­
system
manager
such
as
Microsoft
Windows
Explorer.
B­
12
SECTION
6
2RUNNING
THE
APPLICATION
The
BLM
Windows
Interface
provides
access
to
the
BLM
in
its
full
suite
of
capabilities
(
i.
e.,
predicting
metal
speciation
and
toxicity,
predicting
Water
Effect
Ratios
(
WER),
comparison
to
laboratory
measurements
of
toxicity,
calibration
to
new
metals
and
organisms,
etc).
Providing
an
easy­
to­
use
interface
and
environment
for
developing
datasets
of
water
chemistry
information
and
applying
the
BLM
for
predictions
of
metal
speciation
and
toxicity
makes
the
process
of
BLM
development
more
efficient
and
productive.

The
following
sections
describe
the
various
functions
and
features
available
in
the
BLM
Windows
Interface
and
the
use
of
the
BLM
in
its
various
predictive
capabilities.

2.1
DESCRIPTION
OF
INTERFACE
Figure
2
shows
a
snapshot
of
the
BLM
Windows
Interface
application.
The
main
purpose
of
this
section
of
the
interface
application
is
to
provide
an
easy­
to­
use
editor
to
develop
input
files
containing
water
chemistry
information
for
the
BLM,
to
facilitate
checks
and
validate
the
user
inputs
for
the
various
parameters,
to
perform
checks
on
whether
the
values
entered
for
any
given
parameter
are
within
the
range
for
which
the
BLM
has
been
calibrated,
and
to
run
the
BLM
for
predictions
of
aquatic
speciation
or
toxicity
for
a
variety
of
metals
and
organisms.

Figure
2:
Snapshot
of
the
BLM
Windows
Interface
B­
13
As
shown
in
Figure
2,
the
interface
window
is
divided
into
seven
areas
broadly
based
on
their
functionality.
Each
of
these
is
described
in
the
subsequent
sections.

2.2
DATA
INPUTS
This
region
of
the
interface
window
contains
a
spreadsheet­
based
editor,
which
organizes
the
various
BLM
input
parameters
in
a
columnar
format
such
that
the
chemistry
for
each
discrete
water
sample
can
be
specified
on
a
separate
row.
Apart
from
the
water
chemistry
information,
two
additional
columns
are
also
provided
for
labeling
the
sites
and
the
samples
described
in
a
given
BLM
datafile.
Figure
3
shows
the
various
columns
typically
available
for
user
input.

Figure
3:
Columns
for
Data
Input
in
the
BLM
Windows
Interface
2.2.1
Site
Label
and
Sample
Label
Descriptors
The
first
column,
the
"
Site
Label,"
is
meant
to
contain
information
about
the
site
under
consideration.
For
example,
it
could
be
the
name
of
the
river
or
it
could
be
the
Mile
Point
along
a
river
if
the
same
file
contains
water
chemistry
data
for
more
than
one
location
along
a
particular
river.
The
information
contained
within
the
"
Sample
Label"
field
can
be
used
to
distinguish
the
various
water
chemistry
samples
available
for
a
particular
site.
For
instance,
at
a
given
site,
this
field
could
represent
the
date
and
time
at
which
the
site
water
samples
were
collected.
However,
for
both
the
site
and
the
sample
descriptor
fields,
there
is
an
upper
limit
of
20
characters
that
are
allowed
in
each
field.

2.2.2
Water
Chemistry
Inputs
The
subsequent
columns
contain
the
data
input
area
for
the
water
quality
parameters
described
under
Data
Requirements.
For
predictions
of
metal
toxicity,
metal
concentration
is
not
a
required
input,
since
the
BLM
will
predict
the
amount
of
metal
that
results
in
acute
toxicity
to
the
specified
organism.
However,
for
predictions
of
metal
speciation,
the
metal
concentration
is
a
required
input
and
if
no
metal
concentration
is
specified,
the
row
will
be
considered
incomplete
and
no
BLM
predictions
will
be
made
for
that
row.
For
all
other
water
quality
inputs,
any
row
with
a
missing
input
will
be
flagged
as
incomplete
and
no
BLM
predictions
will
be
made
for
that
row.

2.3
MENU
BAR
Located
at
the
very
top
of
the
interface
window,
the
menu
bar
provides
the
user
with
a
range
of
functions
and
features
including:

°
Managing
the
BLM
datafiles
°
Text
editing
functions
B­
14
°
Functions
to
select
between
various
units
for
data
inputs
°
A
help
function
These
features
are
described
below
in
further
detail.

0.0.1
File
Figure
4
shows
the
functions
available
under
this
menu
item.
Basic
file
management
utilities
to
create
a
new
BLM
datafile,
to
open
an
existing
BLM
datafile,
and
to
save
a
BLM
datafile
are
provided.

Figure
4:
Snapshot
of
File
Menu
Item
Shortcut
keys
(
shown
to
the
right
of
each
item)
are
also
implemented
for
all
the
different
functions
in
this
menu
item.

For
ease
of
access,
BLM
datafiles
can
also
be
opened
directly
by
double
clicking
on
the
BLM
datafile
in
a
file
system
manager
such
as
Microsoft
Windows
Explorer.
This
avoids
having
to
first
start
the
application
and
then
navigate
through
the
file
menu
to
locate
the
BLM
datafile
of
interest.

Note
that
the
BLM
datafiles
created
by
the
interface
application
are
given
a
".
BLM"
extension
by
default.
Even
though
the
BLM
datafile
created
by
the
interface
application
is
basically
an
ASCII
text
file,
it
is
recommended
that
the
user
not
modify
this
file
using
a
program
other
than
the
BLM
Windows
Interface
application.
Doing
so
may
result
in
the
BLM
datafile
getting
corrupted
and
if
this
happens,
the
next
time
the
user
tries
to
edit
that
BLM
datafile
using
the
BLM
Windows
Interface,
the
file
may
not
be
read
correctly
by
the
BLM
interface
application.

0.0.2
Edit
Figure
5
shows
the
editing
functions
available
in
the
BLM
Windows
Interface.
Basic
editing
functions
such
as
"
Cut,"
"
Copy,"
"
Paste,"
and
"
Delete"
are
implemented
in
the
interface
application.
B­
15
Figure
5:
Snapshot
of
Edit
Menu
Item
The
editing
functions
can
be
performed
on
a
single
cell
or
multiple
cells
selected
by
highlighting
the
cells
with
a
mouse
click
and
drag
operation
or
by
using
the
Shift
and
Arrow
functions
on
the
keyboard.
These
editing
functions
can
also
be
accessed
by
using
the
shortcut
keys
shown
to
the
right
of
each
item
or
by
clicking
the
right
mouse
over
the
selected
data
cells
and
then
selecting
the
editing
operation
from
the
editing
menu
that
is
displayed.
Note
that
it
is
also
possible
to
copy
and
paste
data
from
external
programs
such
as
a
spreadsheet
application
into
the
BLM
Windows
Interface.

0.0.3
View
This
feature
is
not
implemented
in
the
current
distribution
of
the
BLM
Windows
Interface
but
may
be
available
in
subsequent
versions.

0.0.4
Inputs
Measurements
of
the
water
quality
parameters
required
for
using
the
BLM
are
often
reported
with
varying
units.
In
order
to
provide
the
user
with
a
higher
degree
of
flexibility
to
develop
BLM
input
files,
the
BLM
interface
allows
data
inputs
in
several
different
units
by
means
of
this
menu
item,
as
shown
in
Figure
6.

Figure
6:
Snapshot
of
Inputs
Menu
Item
Units
B­
16
The
first
option,
"
Set
Units,"
allows
the
user
to
select
the
units
for
the
various
BLM
input
parameters,
as
shown
in
Figure
7.
For
each
parameter,
the
current
selected
units
are
highlighted
by
default
and
the
user
can
select
the
desired
units
from
the
list
of
options
shown.
When
changing
units
for
a
given
parameter,
data
already
input
for
that
parameter
is
converted
to
the
new
units
to
prevent
any
loss
of
data.

Figure
7:
View
of
a
Typical
"
Set
Units"
Screen
Inorganic
Carbon
The
second
option,
"
Inorganic
carbon,"
gives
the
user
the
option
to
select
between
various
options
for
specifying
the
inorganic
carbon
in
the
system.
As
mentioned
previously,
the
BLM
simulates
the
formation
of
metal­
carbonate
complexes
and
therefore
inorganic
carbon
is
a
required
input
for
BLM
simulations.
Inorganic
carbon
in
the
system
can
be
specified
in
one
of
two
ways
 
alkalinity
or
dissolved
inorganic
carbon.
Accordingly,
the
user
can
select
between
these
two
options
by
means
of
the
"
Inorganic
carbon"
feature,
as
shown
in
Figure
8.
B­
17
Figure
8:
View
of
Inorganic
Carbon
Input
Options
Screen
0.0.5
Help
Figure
9
shows
the
various
features
available
under
the
Help
menu
item.

Figure
9:
Snapshot
of
Help
Menu
Item
The
help
file
for
the
BLM
Windows
Interface
can
be
accessed
via
this
menu
item
and
can
be
browsed
by
its
contents,
by
a
keyword
index,
or
by
searching
for
a
particular
word
or
phrase.
In
addition,
under
the
"
Support"
sub­
item,
there
is
also
information
on
whom
to
contact
for
technical
support
and
sending
bug
reports,
etc.
A
short
description
of
the
BLM
can
be
found
under
the
sub­
item
"
About
BLM."

0.1
SHORTCUTS
MENU
This
group
of
icons
contains
shortcuts
to
some
of
the
menu
bar
items
and
some
additional
functions
that
are
not
available
on
the
menu
bar.
Figure
10
shows
the
various
icons
and
their
functions.

Figure
10:
Shortcut
Menu
Icons
0.1.1
Open
File
B­
18
This
is
a
shortcut
to
the
menu
bar
item
under
"
File
­­­­­>
Open"
and
is
provided
for
a
quick
mode
of
access
to
the
BLM
datafiles.
In
case
the
BLM
datafile
being
edited
by
the
user
has
changed
since
the
last
time
it
was
saved,
the
user
will
be
queried
for
a
confirmation
on
whether
to
proceed
to
open
another
datafile
with
or
without
saving
the
current
datafile.

0.1.2
Save
File
This
is
a
shortcut
to
the
menu
bar
item
under
"
File
­­­­­>
Save"
and
is
provided
for
a
quick
mode
of
saving
the
BLM
datafiles.
The
datafile
will
be
saved
under
the
same
name
it
was
last
saved
as.
In
case
the
user
wishes
to
save
the
file
under
a
different
file
name,
the
menu
bar
item
"
File
­­­­­>
Save
As"
should
be
chosen.

0.1.3
Metal/
Organism
Selection
As
mentioned
previously,
the
BLM
can
be
used
to
study
the
toxicity
and
speciation
for
a
variety
of
metals
and
organisms.
This
action
button
is
provided
to
allow
the
user
to
select
the
metal
and
the
organism
for
which
toxicity
or
speciation
has
to
be
predicted.
Clicking
on
this
icon
will
present
the
user
with
the
window
shown
in
Figure
11
and
the
user
can
choose
the
desired
metal
and
organism
for
the
BLM
predictions.
The
current
metal
and
organism
selections
are
displayed
in
the
Current
Selection
Display
area.

Figure
11:
Metal
and
Organism
Selection
Options
Metal
and
Organism
Options
Available
The
metal­
and
organism­
specific
parameter
files
that
are
distributed
along
with
the
current
distribution
of
the
BLM
Windows
Interface,
Version
2.0.0
are
indicated
by
the
options
that
are
not
grayed
out
in
Figure
11,
i.
e.,
the
combinations
available
for
the
user
to
choose
from.
Note
that
these
metal
and
organism
specific
parameter
files
are
part
of
an
ongoing
task
of
refining
the
calibration
and
application
of
the
BLM
and
may
therefore
undergo
revisions
from
time
to
time.
The
metal
and
organism
selections
made
by
the
user
are
also
saved
in
the
BLM
datafile
and
the
next
time
the
user
opens
the
BLM
datafile,
the
application
will
default
to
the
selections
made
by
the
user
at
the
time
the
file
was
saved.

It
is
advisable
to
develop
separate
BLM
datafiles
for
separate
metals
even
though
the
application
of
the
BLM
may
be
for
the
same
set
of
observations.
The
current
distribution
of
the
BLM
can
be
applied
to
only
B­
19
one
metal
at
a
time.
Since
the
input
metal
concentrations
are
specified
in
units
of
mg/
L,
the
interface
application
internally
converts
these
to
units
of
mols/
L
using
the
molecular
weight
for
the
metal
selected
by
the
user.
Changing
the
metal
for
the
BLM
application
within
an
existing
datafile
developed
for
a
different
metal
may
result
in
an
erroneous
conversion
from
units
of
mg/
L
to
mols/
L
when
the
user
saves
and
opens
the
datafile
the
next
time.

User
Defined
Normally,
when
run
in
the
toxicity
prediction
mode
for
a
given
organism
and
metal,
the
BLM
interface
application
will
derive
the
LA50
for
the
user
selected
organism
from
the
parameter
file
specific
to
that
particular
metal
and
organism.
The
BLM
will
then
predict
the
LC50
of
the
selected
metal
to
the
selected
organism
for
all
the
observations
with
a
complete
set
of
BLM
input
parameters.
However,
in
order
to
provide
additional
flexibility
in
operation,
the
BLM
can
be
run
for
a
given
metal
with
different
LA50s
for
different
rows
of
input.
That
is,
the
BLM
will
predict
LC50s
corresponding
to
different
LA50s
for
each
row.
This
is
accomplished
by
selecting
the
"
User
Defined"
option
shown
in
Figure
11
and
selecting
"
Ok."
This
will
add
an
extra
column
to
the
spreadsheet
editor
in
the
application
window
in
the
very
last
column
position,
to
the
extreme
right.
The
user
is
expected
to
populate
this
column
for
each
row
of
input,
with
the
desired
LA50.
Note
that
leaving
this
column
blank
for
any
line
of
input
can
result
in
the
BLM
treating
that
line
of
input
as
a
incomplete
input
and
will
result
in
failure
to
predict
toxicity.

User
Selected
In
addition
to
the
metal­
and
organism­
specific
parameter
files
that
are
distributed
along
with
the
current
distribution,
users
may
also
opt
to
develop
and
use
their
own
versions
of
these
files
for
BLM
predictions.
This
is
achieved
by
selecting
the
"
User
Selected"
option
shown
in
Figure
11
and
selecting
"
Ok."
The
user
will
then
be
queried
for
the
location
of
the
desired
parameter
file.
New
parameter
files
can
be
developed
by
the
user
along
the
lines
of
the
parameter
files
supplied
with
this
distribution
(
files
with
the
extension
".
DAT"
located
in
the
"
Model"
sub­
directory
within
the
BLM
home
directory).

0.1.4
Prediction
Mode
The
BLM
interface
application
allows
the
user
to
run
the
BLM
either
in
toxicity
mode
or
in
the
speciation
mode.
When
run
in
the
toxicity
mode,
for
the
metal
and
organism
specified
by
the
user,
the
BLM
will
predict
the
amount
of
metal
required
to
cause
acute
mortality
in
the
water
specified
by
the
user.
However,
when
the
BLM
is
run
in
the
speciation
mode,
for
the
metal
concentration
specified
by
the
user,
the
BLM
will
predict
the
organic
and
the
inorganic
speciation
in
the
water
column.

The
"
Prediction
Mode"
button
allows
the
user
to
toggle
between
the
speciation
and
toxicity
prediction
modes
in
the
BLM.
The
current
prediction
mode
is
also
displayed
in
the
Current
Selection
Display
area.
By
default,
the
BLM
interface
application
assumes
that
the
BLM
prediction
mode
is
the
toxicity
mode
unless
the
user
specifies
otherwise.
The
current
prediction
mode
is
also
saved
in
the
BLM
datafile
and
the
next
time
the
user
opens
up
the
BLM
datafile,
the
application
will
default
to
the
prediction
mode
at
the
time
the
file
was
saved.

0.1.5
Check
Inputs
After
creating
a
BLM
datafile,
the
user
may
wish
to
check
the
water
chemistry
inputs
to
verify
if
the
parameter
values
are
within
the
overall
range
for
which
the
BLM
has
been
calibrated
and
to
check
to
see
if
all
the
parameters
necessary
for
a
BLM
prediction
have
been
specified.
Clicking
on
this
icon
serves
to
generate
an
input
check
report
which
contains
information
on
what
parameters
are
out
of
range
(
too
high
or
B­
20
too
low
when
compared
to
range
for
which
the
BLM
has
been
calibrated)
and
what
parameters
are
missing
for
any
given
row
of
input.
The
range
of
parameter
values
for
which
the
BLM
has
been
calibrated
is
described
in
Input
Check
Range.
Figure
12
shows
an
example
of
such
an
input
check
report.

Figure
12:
An
Example
of
an
Input
Check
Report
Generated
by
the
Check
Inputs
Function
Note
that
a
similar
check
is
also
done
every
time
the
user
edits
the
contents
of
any
cell
in
the
water
chemistry
input
section.
However,
in
this
case
an
input
check
report
is
not
generated.
Instead,
the
out
of
range
parameter
value
is
highlighted
in
red
as
opposed
to
the
normal
text
color
of
black.

0.1.6
Run
BLM
This
icon
is
used
to
launch
the
BLM
program
to
predict
either
metal
toxicity
or
speciation
for
the
userspecified
selections
for
the
site
water
chemistry
described
in
the
BLM
datafile
currently
open
in
the
BLM
Windows
Interface.
In
case
the
BLM
datafile
has
been
edited
since
its
last
save,
the
user
is
queried
for
confirmation
on
whether
to
save
the
file
and
the
BLM
predictions
proceed
subsequently.

0.1.7
Help
This
feature
provides
a
point­
and­
click
help
functionality
for
several
features
of
the
interface
application.
To
use
this
feature,
simply
click
on
this
icon
and
point
and
click
on
the
icon
or
area
for
which
the
user
is
interested
in
finding
help/
additional
information.

0.2
CURRENT
SELECTION
DISPLAY
This
area
of
the
interface
window
displays
the
current
metal,
organism,
and
prediction
mode
selections
made
by
the
user.
For
the
example
shown
in
Figure
2
the
user
has
opted
to
predict
the
toxicity
of
copper
to
fathead
minnows
by
using
the
"
Shortcuts
Menu"
buttons
Prediction
Mode
and
Metal/
Organism
Selection.
B­
21
The
options
selected
by
the
user
are
saved
in
the
BLM
datafile
and
the
next
time
the
user
opens
the
BLM
datafile
the
application
defaults
to
the
selections
made
by
the
user
at
the
time
of
the
previous
file
save.

0.3
EDITING
CELL
This
area
shows
the
value
of
the
parameter
in
the
current
cell
as
it
is
being
edited.

0.4
DATAFILE
DESCRIPTION
This
area
is
provided
for
the
user
to
insert
comments
describing
the
BLM
datafile
which
will
then
be
saved
along
with
the
water
chemistry
parameters
input
by
the
user.
Though
it
is
not
of
critical
importance
to
the
use
of
the
BLM,
for
record
keeping
and
possibly
QA/
QC
purposes,
it
is
a
desirable
input.

0.5
ITEM
DESCRIPTION
Located
at
the
very
bottom
of
the
interface
window,
this
area
is
designed
to
show
a
brief
description
of
the
icon/
image/
area
the
mouse
cursor
is
currently
positioned
over.
For
the
case
shown
in
Figure
2,
the
mouse
cursor
is
positioned
over
the
"
Data
Inputs"
area.
Similar
messages
are
displayed
when
the
mouse
cursor
is
moved
over
other
areas
of
the
interface
window.

0.6
DESCRIPTION
OF
OUTPUT
FILES
When
run
in
the
metal
speciation
or
metal
toxicity
mode,
the
BLM
creates
two
output
files
within
the
directory
containing
the
BLM
input
file.
The
names
of
the
output
files
are
based
on
the
name
of
the
input
file.
For
example,
using
the
input
file
"
TEST.
BLM"
would
create
two
output
files,
"
TEST.
SIM"
(
the
simple
version
of
the
model
output),
and
"
TEST.
DET"
(
the
detailed
version).

The
detailed
version
of
the
model
output
contains
all
the
chemical
species
in
the
simulation.
Since
this
file
can
grow
quite
large,
the
more
useful
information
is
summarized
in
the
simple
version
of
output.
The
simple
version
of
the
model
output
contains
the
most
relevant
information
for
most
users.
Included
are
the
site
and
sample
labels,
the
mode
of
operation
(
i.
e.,
did
the
BLM
use
an
input
dissolved
metal
concentration
to
predict
metal
speciation
or
was
it
predicting
the
LC50?),
the
pH,
the
total
dissolved
metal
in
mol/
L
(
this
is
the
input
metal
concentration
in
the
speciation
mode
and
the
predicted
LC50
in
the
toxicity
prediction
mode),
the
free
metal
concentration
in
mol/
L,
the
activity­
corrected
free
metal
concentration
in
mol/
L,
concentration
of
metal
bound
to
DOC
in
mol/
L,
concentration
of
metal
and
metal
hydroxide
bound
to
DOC
in
mol/
L,
the
concentration
of
metal
on
the
biotic
ligand
in
nmol/
gwet
of
the
gill,
the
DOC
in
mg/
L,
the
percent
humic
acid
and
the
rest
of
the
input
water
chemistry
in
units
of
mol/
L.
B­
22
SECTION
7
1INPUT
CHECK
RANGE
In
order
to
provide
users
with
an
idea
of
the
range
of
water
chemistry
to
which
the
BLM
can
be
applied,
the
range
of
parameter
values
to
which
the
BLM
has
been
developed
and
calibrated
is
defined
in
the
BLM
interface
application.
The
users
can
check
to
verify
if
the
user
input
water
chemistry
parameter
values
are
within
this
range
to
which
the
BLM
has
been
calibrated.
This
is
done
by
using
the
"
Check
Inputs"
function.
The
ranges
prescribed
for
each
of
the
BLM
input
parameters
are
shown
below.

PARAMETER
LOWER
BOUND
UPPER
BOUND
Temperature
(
oC)
10
25
pH
4.9
9.2
DOC
(
mg/
L)
0.05
29.65
Humic
Acid
Content
(%)
10
60
Calcium
(
mg/
L)
0.204
120.24
Magnesium
(
mg/
L)
0.024
51.9
Sodium
(
mg/
L)
0.16
236.9
Potassium
(
mg/
L)
0.039
156
Sulfate
(
mg/
L)
0.096
278.4
Chloride
(
mg/
L)
0.32
279.72
Alkalinity
(
mg/
L)
1.99
360
DIC
(
mmol/
L)
0.056
44.92
Sulfide
(
mg/
L)
0
B­
23
SECTION
8
2EXAMPLE
APPLICATION
The
BLM
Windows
Interface
installation
also
contains
an
example
application
for
demonstration
purposes.
This
file
is
named
"
Kansas
River.
BLM"
and
is
installed
along
with
the
BLM
interface
application
and
is
located
in
the
"
Data"
directory
within
the
BLM
home
directory
on
the
user's
hard­
disk.
The
file
can
be
opened
directly,
by
double
clicking
on
the
file
name
through
a
file­
system
manager
such
as
Microsoft
Windows
Explorer
or
by
first
starting
the
BLM
Windows
Interface
application
and
selecting
the
file
through
the
"
File
­­­­­>
Open"
action.
This
example
datafile
contains
the
water
quality
observations
for
USGS
Station
6892350
on
the
Kansas
River
at
Desoto,
KS.
Although
in
this
case,
only
observations
with
a
complete
characterization
of
all
the
BLM
input
parameters
are
included
in
the
BLM
datafile,
it
is
recommended
that
all
the
available
water
quality
measurements
(
including
the
ones
without
a
complete
characterization
of
the
BLM
input
parameters)
be
included
in
the
BLM
datafile.

This
datafile
"
Kansas
River.
BLM"
can
be
used
to
predict
metal
speciation
using
the
input
metal
concentrations
or
to
predict
the
LC50
to
a
variety
of
metals
and
organisms.
However,
it
is
recommended
that
separate
BLM
datafiles
be
maintained
for
each
metal.
In
this
case,
the
datafile
contains
dissolved
copper
concentrations
and
the
BLM
can
be
used
to
predict
the
inorganic,
organic,
and
biotic
speciation
by
setting
the
BLM
prediction
mode
to
"
Speciation"
using
the
Shortcut
Menu
button
Prediction
Mode.
Metal
toxicity
for
the
specified
site
water
chemistry
can
also
be
predicted
by
setting
the
prediction
mode
to
"
Toxicity"
and
selecting
the
metal
and
organism
for
which
toxicity
is
to
be
predicted.
B­
24
SECTION
9
3CONTACT
INFORMATION
For
questions
or
problems,
including
bug
reports,
relating
to
the
use
and
application
of
the
Biotic
Ligand
Model
or
the
BLM
Windows
Interface,
please
contact
either:

Cindy
Roberts
U.
S.
EPA
1200
Pennsylvania
Ave,
NW
(
MC4304T)
Washington,
DC
20460
roberts.
cindy@
epa.
gov
or
Additional
information
including
support
details
can
be
found
online
at
http://
www.
hydroqual.
com/
blm.
B­
25
References
Allen,
H.
E.
and
D.
J.
Hansen.
1996.
The
importance
of
trace
metal
speciation
to
water
quality
criteria.
Water
Environ.
Res.
68:
42­
54.

Di
Toro,
D.
M.,
H.
E.
Allen,
H.
L.
Bergman,
J.
S.
Meyer,
P.
R.
Paquin
and
R.
C.
Santore,
2001.
A
Biotic
Ligand
Model
of
the
Acute
Toxicity
of
Metals.
I.
Technical
Basis,
Environmental
Toxicology
and
Chemistry.
20:
2383­
2396.

MacRae,
R.
K.,
December,
1994.
"
The
Copper
Binding
Affinity
of
Rainbow
Trout
(
Oncorhynchus
mykiss)
and
Brook
Trout
(
Salvelinus
fontinalis)
Gills,"
a
thesis
submitted
to
the
Department
of
Zoology
and
Physiology
and
The
Graduate
School
of
the
University
of
Wyoming
in
partial
fulfillment
of
the
requirements
for
the
degree
of
Master
of
Science
in
Zoology
and
Physiology.

Meyer,
J.
S.,
R.
C.
Santore,
J.
P.
Bobbitt,
L.
D.
DeBrey,
C.
J.
Boese,
P.
R.
Paquin,
H.
E.
Allen,
H.
L.
Bergman
and
D.
M.
Di
Toro.
1999.
Binding
of
nickel
and
copper
to
fish
gills
predicts
toxicity
when
water
hardness
varies,
but
free­
ion
activity
does
not.
Environ.
Sci.
Technol.
33:
913­
916.

Morel,
F.
M.,
1983a.
"
Complexation:
Trace
Metals
and
Microorganisms,"
in
Chapter
6
of
Principles
of
Aquatic
Chemistry,
Wiley
Interscience,
New
York,
pp.
301­
308.

Pagenkopf,
G.
K.
1983.
Gill
surface
interaction
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